BioMed Research International: Biochemistry The latest articles from Hindawi Publishing Corporation © 2014 , Hindawi Publishing Corporation . All rights reserved. Neuroimaging to Investigate Multisystem Involvement and Provide Biomarkers in Amyotrophic Lateral Sclerosis Thu, 17 Apr 2014 13:29:05 +0000 Neuroimaging allows investigating the extent of neurological systems degeneration in amyotrophic lateral sclerosis (ALS). Advanced MRI methods can detect changes related to the degeneration of upper motor neurons but have also demonstrated the participation of other systems such as the sensory system or basal ganglia, demonstrating in vivo that ALS is a multisystem disorder. Structural and functional imaging also allows studying dysfunction of brain areas associated with cognitive signs. From a biomarker perspective, numerous studies using diffusion tensor imaging showed a decrease of fractional anisotropy in the intracranial portion of the corticospinal tract but its diagnostic value at the individual level remains limited. A multiparametric approach will be required to use MRI in the diagnostic workup of ALS. A promising avenue is the new methodological developments of spinal cord imaging that has the advantage to investigate the two motor system components that are involved in ALS, that is, the lower and upper motor neuron. For all neuroimaging modalities, due to the intrinsic heterogeneity of ALS, larger pooled banks of images with standardized image acquisition and analysis procedures are needed. In this paper, we will review the main findings obtained with MRI, PET, SPECT, and nuclear magnetic resonance spectroscopy in ALS. Pierre-François Pradat and Mohamed-Mounir El Mendili Copyright © 2014 Pierre-François Pradat and Mohamed-Mounir El Mendili. All rights reserved. Genetics of Type 2 Diabetes: Insights into the Pathogenesis and Its Clinical Application Thu, 17 Apr 2014 06:30:37 +0000 With rapidly increasing prevalence, diabetes has become one of the major causes of mortality worldwide. According to the latest studies, genetic information makes substantial contributions towards the prediction of diabetes risk and individualized antidiabetic treatment. To date, approximately 70 susceptibility genes have been identified as being associated with type 2 diabetes (T2D) at a genome-wide significant level (). However, all the genetic loci identified so far account for only about 10% of the overall heritability of T2D. In addition, how these novel susceptibility loci correlate with the pathophysiology of the disease remains largely unknown. This review covers the major genetic studies on the risk of T2D based on ethnicity and briefly discusses the potential mechanisms and clinical utility of the genetic information underlying T2D. Xue Sun, Weihui Yu, and Cheng Hu Copyright © 2014 Xue Sun et al. All rights reserved. Detection of Herpes Simplex and Varicella-Zoster Virus in Clinical Specimens by Multiplex Real-Time PCR and Melting Curve Analysis Wed, 16 Apr 2014 15:54:38 +0000 Herpes simplex viruses types 1 and 2 (HSV-1 and HSV-2), and varicella-zoster virus (VZV) are common agents resulting in various forms of clinical manifestation from skin vesicle to disseminated viral infection. The aim of the present study was to develop a real-time PCR and melting curve analysis which detect and differentiate HSV-1, HSV-2, and VZV, to compare with PCR-RFLP using clinical specimens, and to introduce the 4-year experience in the clinical laboratory. Three pairs of primers for HSV-1, HSV-2, and VZV were designed. Primers for human endogenous retrovirus-3 (HERV-3), an internal control, were adopted. A hundred selected specimens and many clinical specimens were tested for methods comparison and assay validation. Increased sensitivity and specificity were obtained from real-time PCR. In review of results of clinical specimens submitted to clinical laboratory, a total of 46 of 3,513 specimens were positive in cerebrospinal fluids, blood, skin vesicles, genital swabs, aqueous humor, and ear discharge. Thus, this method could be a rapid and accurate alternative to virus culture and other molecular tests for detection and typing of HSV-1, HSV-2, and VZV. Yun Ji Hong, Mi Suk Lim, Sang Mee Hwang, Taek Soo Kim, Kyoung Un Park, Junghan Song, and Eui Chong Kim Copyright © 2014 Yun Ji Hong et al. All rights reserved. Effect of the Combination of Gelam Honey and Ginger on Oxidative Stress and Metabolic Profile in Streptozotocin-Induced Diabetic Sprague-Dawley Rats Wed, 16 Apr 2014 13:58:52 +0000 Diabetic complications occur as a result of increased reactive oxygen species (ROS) due to long term hyperglycaemia. Honey and ginger have been shown to exhibit antioxidant activity which can scavenge ROS. The main aim of this study was to evaluate the antioxidant and antidiabetic effects of gelam honey, ginger, and their combination. Sprague-Dawley rats were divided into 2 major groups which consisted of diabetic and nondiabetic rats. Diabetes was induced with streptozotocin intramuscularly (55 mg/kg body weight). Each group was further divided into 4 smaller groups according to the supplements administered: distilled water, honey (2 g/kg body weight), ginger (60 mg/kg body weight), and honey + ginger. Body weight and glucose levels were recorded weekly, while blood from the orbital sinus was obtained after 3 weeks of supplementation for the estimation of metabolic profile: glucose, triglyceride (TG), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH): oxidized glutathione (GSSG), and malondialdehyde (MDA). The combination of gelam honey and ginger did not show hypoglycaemic potential; however, the combination treatment reduced significantly () SOD and CAT activities as well as MDA level, while GSH level and GSH/GSSG ratio were significantly elevated () in STZ-induced diabetic rats compared to diabetic control rats. Nur Fathiah Abdul Sani, Levin Kesu Belani, Chong Pui Sin, Siti Nor Amilah Abdul Rahman, Srijit Das, Thent Zar Chi, Suzana Makpol, and Yasmin Anum Mohd Yusof Copyright © 2014 Nur Fathiah Abdul Sani et al. All rights reserved. Plastic Changes in the Spinal Cord in Motor Neuron Disease Wed, 16 Apr 2014 08:56:03 +0000 In the present paper, we analyze the cell number within lamina X at the end stage of disease in a G93A mouse model of ALS; the effects induced by lithium; the stem-cell like phenotype of lamina X cells during ALS; the differentiation of these cells towards either a glial or neuronal phenotype. In summary we found that G93A mouse model of ALS produces an increase in lamina X cells which is further augmented by lithium administration. In the absence of lithium these nestin positive stem-like cells preferentially differentiate into glia (GFAP positive), while in the presence of lithium these cells differentiate towards a neuron-like phenotype (βIII-tubulin, NeuN, and calbindin-D28K positive). These effects of lithium are observed concomitantly with attenuation in disease progression and are reminiscent of neurogenetic effects induced by lithium in the subependymal ventricular zone of the hippocampus. Francesco Fornai, Michela Ferrucci, Paola Lenzi, Alessandra Falleni, Francesca Biagioni, Marina Flaibani, Gabriele Siciliano, Francesco Giannessi, and Antonio Paparelli Copyright © 2014 Francesco Fornai et al. All rights reserved. Metabolomic Prediction of Pregnancy Viability in Superovulated Cattle Embryos and Recipients with Fourier Transform Infrared Spectroscopy Tue, 15 Apr 2014 00:00:00 +0000 We analyzed embryo culture medium (CM) and recipient blood plasma using Fourier transform infrared spectroscopy (FTIR) metabolomics to identify spectral models predictive of pregnancy outcome. Embryos collected on Day 6 from superovulated cows in 2 countries were individually cultured in synthetic oviduct fluid medium with BSA for 24 h before embryo transfer. Spent CM, blank controls, and plasma samples (Day 0 and Day 7) were evaluated using FTIR. The spectra obtained were analyzed. The discrimination capability of the classifiers was assessed for accuracy, sensitivity (pregnancy), specificity (nonpregnancy), and area under the ROC curve (AUC). Endpoints considered were Day 60 pregnancy and birth. High AUC was obtained for Day 60 pregnancy in CM within individual laboratories (France , Spain ), while cumulative data decreased the AUC (). Predictions for CM at birth were lower than Day 60 pregnancy. Predictions with plasma at birth improved cumulative over individual results (Day 0: France ; Spain ; cumulative ). Plasma generally predicted pregnancy and birth better than CM. These first results show that FTIR metabolomics could allow the identification of embryos and recipients with improved pregnancy viability, which may contribute to increasing the efficiency of selection schemes based on ET. Marta Muñoz, Asli Uyar, Eva Correia, Claire Ponsart, Catherine Guyader-Joly, Daniel Martínez-Bello, Brigitte Marquant-Le Guienne, Alfonso Fernandez-Gonzalez, Carmen Díez, Jose Nestor Caamaño, Beatriz Trigal, Patrice Humblot, Susana Carrocera, David Martin, Emre Seli, and Enrique Gomez Copyright © 2014 Marta Muñoz et al. All rights reserved. Regulation of p63 Protein Stability via Ubiquitin-Proteasome Pathway Tue, 15 Apr 2014 00:00:00 +0000 The p53-related p63 gene encodes multiple protein isoforms, which are involved in a variety of biological activities. p63 protein stability is mainly regulated by the ubiquitin-dependent proteasomal degradation pathway. Several ubiquitin E3 ligases have been identified and some protein kinases as well as other kinds of proteins are involved in regulation of p63 protein stability. These regulators are responsive to diverse extracellular signaling, resulting in changes of the p63 protein levels and impacting different biological processes. Chenghua Li and Zhi-Xiong Xiao Copyright © 2014 Chenghua Li and Zhi-Xiong Xiao. All rights reserved. Castanea sativa Mill. Flowers amongst the Most Powerful Antioxidant Matrices: A Phytochemical Approach in Decoctions and Infusions Mon, 14 Apr 2014 17:22:12 +0000 Infusions and decoction of chestnut tree flowers have been used for different medical purposes, but their phytochemical profile and antioxidant activity are still mostly unknown. Herein, decoctions and infusions of flowers from the two most appreciated chestnut cultivars (longal and judia) in Trás-os-Montes, Portugal, were prepared and characterized with regard to their content in free sugars, organic acids, and phenolic compounds, such as flavonoids and hydrolyzable tannins, and their antioxidant activity. Overall, the decoction of the cultivar judia was the sample with both the highest quantity of flavonoids and antioxidant activity. The phenolic compound with the highest abundance in all samples was trigalloyl-HHDP-glucoside, followed by pentagalloyl glucoside. The sample with the highest quantity of total phenolic compounds was judia infusion, closely followed by longal decoction, which also gave the highest quantities of ellagitannins. Regarding sugars and organic acids, the profiles were more similar. These results corroborate ancestral claims of the health benefits of infusions and decoctions of chestnut flowers. Márcio Carocho, Lillian Barros, Albino Bento, Celestino Santos-Buelga, Patricia Morales, and Isabel C. F. R. Ferreira Copyright © 2014 Márcio Carocho et al. All rights reserved. Preliminary Phytochemical Screening and In Vitro Antioxidant Activities of Parkinsonia aculeata Linn. Sun, 13 Apr 2014 11:12:47 +0000 Butanol and hexane leaves extracts of Parkinsonia aculeata L. (Fabaceae) were assessed for its antioxidant potential by in vitro methods. Phytochemical analysis and antioxidant activity of plant extracts were studied using different in vitro assays. UPLC analysis of extracts was carried out for the identification of chemical constituents. The total phenolic contents of the butanol and hexane leaf extract were 42 mgGAE/g and 34 mgGAE/g whereas flavonoid contents of these extracts were found to be 0.044 mgRE/g and 0.005 mgRE/g, respectively. Among both extracts, butanol extract shows maximum inhibition (%) of 93.88%, 80.02%, 52.06%, 94.68%, and 69.37% in DPPH, non-site-specific and site-specific, FTC, and TBA assays and absorbance of 0.852 and 0.522 in reducing power and CUPRAC assay at the highest concentration tested. The FRAP and TAC values of butanol extract were found to be 678 μM Fe(II)/g and 36 mgAAE/100 mg. UPLC analysis of extracts revealed the presence of various polyphenols. The tested plant extracts were found to possess potent antioxidant and free radical scavenging activity which may be due to the presence of flavonoids and polyphenols. Sonia Sharma and Adarsh Pal Vig Copyright © 2014 Sonia Sharma and Adarsh Pal Vig. All rights reserved. A Paradoxical Chemoresistance and Tumor Suppressive Role of Antioxidant in Solid Cancer Cells: A Strange Case of Dr. Jekyll and Mr. Hyde Thu, 03 Apr 2014 08:23:03 +0000 Modulation of intracellular antioxidant concentration is a double-edged sword, with both sides exploited for potential therapeutic benefits. While antioxidants may hamper the efficacy of chemotherapy by scavenging reactive oxygen species and free radicals, it is also possible that antioxidants alleviate unwanted chemotherapy-induced toxicity, thus allowing for increased chemotherapy doses. Under normoxic environment, antioxidants neutralize toxic oxidants, such as reactive oxygen species (ROS), maintaining them within narrow boundaries level. This redox balance is achieved by various scavenging systems such as enzymatic system (e.g., superoxide dismutases, catalase, and peroxiredoxins), nonenzymatic systems (e.g., glutathione, cysteine, and thioredoxin), and metal-binding proteins (e.g., ferritin, metallothionein, and ceruloplasmin) that sequester prooxidant metals inhibiting their participation in redox reactions. On the other hand, therapeutic strategies that promote oxidative stress and eventually tumor cells apoptosis have been explored based on availability of chemotherapy agents that inhibit ROS-scavenging systems. These contradictory assertions suggest that antioxidant supplementation during chemotherapy treatment can have varied outcomes depending on the tumor cellular context. Therefore, understanding the antioxidant-driven molecular pathways might be crucial to design new therapeutic strategies to fight cancer progression. Jolie Kiemlian Kwee Copyright © 2014 Jolie Kiemlian Kwee. All rights reserved. Effects of Chlorophenoxy Herbicides and Their Main Transformation Products on DNA Damage and Acetylcholinesterase Activity Thu, 27 Mar 2014 14:17:26 +0000 Persistent pesticide transformation products (TPs) are increasingly being detected among different environmental compartments, including groundwater and surface water. However, there is no sufficient experimental data on their toxicological potential to assess the risk associated with TPs, even if their occurrence is known. In this study, the interaction of chlorophenoxy herbicides (MCPA, mecoprop, 2,4-D and dichlorprop) and their main transformation products with calf thymus DNA by UV-visible absorption spectroscopy has been assessed. Additionally, the toxicity of the chlorophenoxy herbicides and TPs was also assessed evaluating the inhibition of acetylcholinesterase activity. On the basis of the results found, it seems that AChE is not the main target of chlorophenoxy herbicides and their TPs. However, the results found showed that the transformation products displayed a higher inhibitory activity when compared with the parent herbicides. The results obtained in the DNA interaction studies showed, in general, a slight effect on the stability of the double helix. However, the data found for 4-chloro-2-methyl-6-nitrophenol suggest that this transformation product can interact with DNA through a noncovalent mode. Sofia Benfeito, Tiago Silva, Jorge Garrido, Paula B. Andrade, M. J. Sottomayor, Fernanda Borges, and E. Manuela Garrido Copyright © 2014 Sofia Benfeito et al. All rights reserved. Significance of Lewis Phenotyping Using Saliva and Gastric Tissue: Comparison with the Lewis Phenotype Inferred from Lewis and Secretor Genotypes Mon, 24 Mar 2014 12:11:12 +0000 Lewis phenotypes using various types of specimen were compared with the Lewis phenotype predicted from Lewis and Secretor genotypes. This is the first logical step in explaining the association between the Lewis expression and Helicobacter pylori. We performed a study of the followings on 209 patients who underwent routine gastroscopy: erythrocyte and saliva Lewis phenotyping, gastric Lewis phenotyping by the tissue array, and the Lewis and Secretor genes genotyping. The results of phenotyping were as follows [Le(a−b−), Le(a+b−), Le(a−b+), and Le(a+b+), respectively, in order]: erythrocyte (12.4%, 25.8%, 61.2%, and 0.5%); saliva (2.4%, 27.3%, 70.3%, and 0.0%); gastric mucosa (8.1%, 6.7%, 45.5%, and 39.7%). The frequency of Le, , , and alleles was 74.6%, 21.3%, 3.1%, and 1.0%, respectively, among 418 alleles. The saliva Lewis phenotype was completely consistent with the Lewis phenotype inferred from Lewis and Secretor genotypes, but that of gastric mucosa could not be predicted from genotypes. Lewis phenotyping using erythrocytes is only adequate for transfusion needs. Saliva testing for the Lewis phenotype is a more reliable method for determining the peripheral Lewis phenotype of an individual and the gastric Lewis phenotype must be used for the study on the association between Helicobacter pylori and the Lewis phenotype. Yun Ji Hong, Sang Mee Hwang, Taek Soo Kim, Eun Young Song, Kyoung Un Park, Junghan Song, and Kyou-Sup Han Copyright © 2014 Yun Ji Hong et al. All rights reserved. Lack of Association between TLR4 Genetic Polymorphisms and Diabetic Nephropathy in a Chinese Population Sun, 23 Mar 2014 12:37:36 +0000 Objective. Toll-like receptor 4 (TLR4) plays a central role in innate immunity. Activation of innate immune response and subsequent chronic low-grade inflammation are thought to be involved in the pathogenesis of diabetic nephropathy. In this study, we aimed to investigate whether TLR4 variants are associated with diabetic nephropathy in the Chinese population. Methods. Seven tagging single nucleotide polymorphisms (SNPs) of TLR4 based on HapMap Chinese data were genotyped in 1,455 Chinese type 2 diabetic patients. Of these patients, 622 were diagnosed with diabetic nephropathy and 833 were patients with diabetes for over 5 years but without diabetic nephropathy. Results. None of the SNPs and haplotypes showed significant association to diabetic nephropathy in our study. No association between the SNPs and quantitative traits was observed either. Conclusion. We concluded that common variants within TLR4 genes were not associated with diabetic nephropathy in the Chinese type 2 diabetes patients. Danfeng Peng, Jie Wang, Jiemin Pan, Rong Zhang, Shanshan Tang, Feng Jiang, Miao Chen, Jing Yan, Xue Sun, Tao Wang, Shiyun Wang, Yuqian Bao, and Weiping Jia Copyright © 2014 Danfeng Peng et al. All rights reserved. A Short-Term Incubation with High Glucose Impairs VASP Phosphorylation at Serine 239 in response to the Nitric Oxide/cGMP Pathway in Vascular Smooth Muscle Cells: Role of Oxidative Stress Sun, 23 Mar 2014 08:57:26 +0000 A reduction of the nitric oxide (NO) action in vascular smooth muscle cells (VSMC) could play a role in the vascular damage induced by the glycaemic excursions occurring in diabetic patients; in this study, we aimed to clarify whether a short-term incubation of cultured VSMC with high glucose reduces the NO ability to increase cGMP and the cGMP ability to phosphorylate VASP at Ser-239. We observed that a 180 min incubation of rat VSMC with 25 mmol/L glucose does not impair the NO-induced cGMP increase but reduces VASP phosphorylation in response to both NO and cGMP with a mechanism blunted by antioxidants. We further demonstrated that high glucose increases radical oxygen species (ROS) production and that this phenomenon is prevented by the PKC inhibitor chelerythrine and the NADPH oxidase inhibitor apocynin. The following sequence of events is supported by these results: (i) in VSMC high glucose activates PKC; (ii) PKC activates NADPH oxidase; (iii) NADPH oxidase induces oxidative stress; (iv) ROS impair the signalling of cGMP, which is involved in the antiatherogenic actions of NO. Thus, high glucose, via oxidative stress, can reduce the cardiovascular protection conferred by the NO/cGMP pathway via phosphorylation of the cytoskeleton protein VASP in VSMC. Isabella Russo, Michela Viretto, Gabriella Doronzo, Cristina Barale, Luigi Mattiello, Giovanni Anfossi, and Mariella Trovati Copyright © 2014 Isabella Russo et al. All rights reserved. Role of Plasma Membrane Caveolae/Lipid Rafts in VEGF-Induced Redox Signaling in Human Leukemia Cells Tue, 11 Mar 2014 09:24:57 +0000 Caveolae/lipid rafts are membrane-rich cholesterol domains endowed with several functions in signal transduction and caveolin-1 (Cav-1) has been reported to be implicated in regulating multiple cancer-associated processes, ranging from tumor growth to multidrug resistance and angiogenesis. Vascular endothelial growth factor receptor-2 (VEGFR-2) and Cav-1 are frequently colocalized, suggesting an important role played by this interaction on cancer cell survival and proliferation. Thus, our attention was directed to a leukemia cell line (B1647) that constitutively produces VEGF and expresses the tyrosine-kinase receptor VEGFR-2. We investigated the presence of VEGFR-2 in caveolae/lipid rafts, focusing on the correlation between reactive oxygen species (ROS) production and glucose transport modulation induced by VEGF, peculiar features of tumor proliferation. In order to better understand the involvement of VEGF/VEGFR-2 in the redox signal transduction, we evaluated the effect of different compounds able to inhibit VEGF interaction with its receptor by different mechanisms, corroborating the obtained results by immunoprecipitation and fluorescence techniques. Results here reported showed that, in B1647 leukemia cells, VEGFR-2 is present in caveolae through association with Cav-1, demonstrating that caveolae/lipid rafts act as platforms for negative modulation of VEGF redox signal transduction cascades leading to glucose uptake and cell proliferation, suggesting therefore novel potential targets. Cristiana Caliceti, Laura Zambonin, Benedetta Rizzo, Diana Fiorentini, Francesco Vieceli Dalla Sega, Silvana Hrelia, and Cecilia Prata Copyright © 2014 Cristiana Caliceti et al. All rights reserved. Role of G Protein-Coupled Receptors in Control of Dendritic Cell Migration Mon, 10 Mar 2014 13:55:50 +0000 Dendritic cells (DCs) are highly efficient antigen-presenting cells. The migratory properties of DCs give them the capacity to be a sentinel of the body and the vital role in the induction and regulation of adaptive immune responses. Therefore, it is important to understand the mechanisms in control of migration of DCs to lymphoid and nonlymphoid tissues. This may provide us novel insight into the clinical treatment of diseases such as autoimmune disease, infectious disease, and tumor. The chemotactic G protein-coupled receptors (GPCR) play a vital role in control of DCs migration. Here, we reviewed the recent advances regarding the role of GPCR in control of migration of subsets of DCs, with a focus on the chemokine receptors. Understanding subsets of DCs migration could provide a rational basis for the design of novel therapies in various clinical conditions. Yuan Liu and Guixiu Shi Copyright © 2014 Yuan Liu and Guixiu Shi. All rights reserved. Role of Methylglyoxal in Alzheimer’s Disease Sun, 09 Mar 2014 11:29:36 +0000 Alzheimer’s disease is the most common and lethal neurodegenerative disorder. The major hallmarks of Alzheimer’s disease are extracellular aggregation of amyloid β peptides and, the presence of intracellular neurofibrillary tangles formed by precipitation/aggregation of hyperphosphorylated tau protein. The etiology of Alzheimer’s disease is multifactorial and a full understanding of its pathogenesis remains elusive. Some years ago, it has been suggested that glycation may contribute to both extensive protein cross-linking and oxidative stress in Alzheimer’s disease. Glycation is an endogenous process that leads to the production of a class of compounds known as advanced glycation end products (AGEs). Interestingly, increased levels of AGEs have been observed in brains of Alzheimer’s disease patients. Methylglyoxal, a reactive intermediate of cellular metabolism, is the most potent precursor of AGEs and is strictly correlated with an increase of oxidative stress in Alzheimer’s disease. Many studies are showing that methylglyoxal and methylglyoxal-derived AGEs play a key role in the etiopathogenesis of Alzheimer's disease. Cristina Angeloni, Laura Zambonin, and Silvana Hrelia Copyright © 2014 Cristina Angeloni et al. All rights reserved. PPAR-γ Impairment Alters Peroxisome Functionality in Primary Astrocyte Cell Cultures Tue, 04 Mar 2014 10:03:46 +0000 Peroxisomes provide glial cells with protective functions against the harmful effects of H2O2 on neurons and peroxisome impairment results in nervous lesions. Agonists of the γ-subtype of the Peroxisome-Proliferator-Activated-Receptors (PPAR) have been proposed as neuroprotective agents in neurodegenerative disorders. Nevertheless, the role of PPAR-γ alterations in pathophysiological mechanisms and the relevance of peroxisome functions in the PPAR-γ effects are not yet clear. In a primary cell culture of rat astrocytes, the irreversible PPAR-γ antagonist GW9662 concentration-dependently decreased the activity of catalase, the most important antioxidant defense enzyme in peroxisomes. Catalase functionality recovered in a few days and the PPAR-γ agonist rosiglitazone promoted reversal of enzymatic damage. The reversible antagonist G3335 reduced both the activity and expression of catalase in a rosiglitazone-prevented manner. G3335 reduced also the glutathione reductase expression, indicating that enzyme involved in glutathione regeneration was compromised. Neither the PPAR-α target gene Acyl-Coenzyme-A-oxidase-1 nor the mitochondrial detoxifying enzyme NADH:ubiquinone-oxidoreductase (NDFUS3) was altered by PPAR-γ inhibition. In conclusion, PPAR-γ inhibition induced impairment of catalase in astrocytes. A general decrease of the antioxidant defenses of the cell suggests that a PPAR-γ hypofunction could participate in neurodegenerative mechanisms through peroxisomal damage. This series of experiments could be a useful model for studying compounds able to restore peroxisome functionality. Lorenzo Di Cesare Mannelli, Matteo Zanardelli, Laura Micheli, and Carla Ghelardini Copyright © 2014 Lorenzo Di Cesare Mannelli et al. All rights reserved. Hepatitis B Virus X Upregulates HuR Protein Level to Stabilize HER2 Expression in Hepatocellular Carcinoma Cells Thu, 27 Feb 2014 13:48:52 +0000 Hepatitis B virus- (HBV-) associated hepatocellular carcinoma (HCC) is the most common type of liver cancer. However, the underlying mechanism of HCC tumorigenesis is very complicated and HBV-encoded X protein (HBx) has been reported to play the most important role in this process. Activation of downstream signal pathways of epidermal growth factor receptor (EGFR) family is known to mediate HBx-dependent HCC tumor progression. Interestingly, HER2 (also known as ErbB2/Neu/EGFR2) is frequently overexpressed in HBx-expressing HCC patients and is associated with their poor prognosis. However, it remains unclear whether and how HBx regulates HER2 expression. In this study, our data showed that HBx expression increased HER2 protein level via enhancing its mRNA stability. The induction of RNA-binding protein HuR expression by HBx mediated the HER2 mRNA stabilization. Finally, the upregulated HER2 expression promoted the migration ability of HBx-expressing HCC cells. These findings deciphered the molecular mechanism of HBx-mediated HER2 upregulation in HBV-associated HCC. Chao-Ming Hung, Wei-Chien Huang, Hsiao-Lin Pan, Pei-Hsuan Chien, Chih-Wen Lin, Lei-Chin Chen, Yu-Fong Chien, Ching-Chiao Lin, Kar-Hee Leow, Wen-Shu Chen, Jhen-Yu Chen, Chien-Yi Ho, Pao-Sheng Hou, and Yun-Ju Chen Copyright © 2014 Chao-Ming Hung et al. All rights reserved. Nuclear Nox4-Derived Reactive Oxygen Species in Myelodysplastic Syndromes Wed, 26 Feb 2014 07:31:48 +0000 A role for intracellular ROS production has been recently implicated in the pathogenesis and progression of a wide variety of neoplasias. ROS sources, such as NAD(P)H oxidase (Nox) complexes, are frequently activated in AML (acute myeloid leukemia) blasts and strongly contribute to their proliferation, survival, and drug resistance. Myelodysplastic syndromes (MDS) comprise a heterogeneous group of disorders characterized by ineffective hematopoiesis, with an increased propensity to develop AML. The molecular basis for MDS progression is unknown, but a key element in MDS disease progression is the genomic instability. NADPH oxidases are now recognized to have specific subcellular localizations, this targeting to specific compartments for localized ROS production. Local Nox-dependent ROS production in the nucleus may contribute to the regulation of redox-dependent cell growth, differentiation, senescence, DNA damage, and apoptosis. We observed that Nox1, 2, and 4 isoforms and p22phox and Rac1 subunits are expressed in MDS/AML cell lines and MDS samples, also in the nuclear fractions. Interestingly, Nox4 interacts with ERK and Akt1 within nuclear speckle domain, suggesting that Nox4 could be involved in regulating gene expression and splicing factor activity. These data contribute to the elucidation of the molecular mechanisms used by nuclear ROS to drive MDS evolution to AML. Marianna Guida, Tullia Maraldi, Francesca Beretti, Matilde Y. Follo, Lucia Manzoli, and Anto De Pol Copyright © 2014 Marianna Guida et al. All rights reserved. Altered Nitrogen Balance and Decreased Urea Excretion in Male Rats Fed Cafeteria Diet Are Related to Arginine Availability Mon, 24 Feb 2014 09:02:07 +0000 Hyperlipidic diets limit glucose oxidation and favor amino acid preservation, hampering the elimination of excess dietary nitrogen and the catabolic utilization of amino acids. We analyzed whether reduced urea excretion was a consequence of higher ; (nitrite, nitrate, and other derivatives) availability caused by increased nitric oxide production in metabolic syndrome. Rats fed a cafeteria diet for 30 days had a higher intake and accumulation of amino acid nitrogen and lower urea excretion. There were no differences in plasma nitrate or nitrite. and creatinine excretion accounted for only a small part of total nitrogen excretion. Rats fed a cafeteria diet had higher plasma levels of glutamine, serine, threonine, glycine, and ornithine when compared with controls, whereas arginine was lower. Liver carbamoyl-phosphate synthetase I activity was higher in cafeteria diet-fed rats, but arginase I was lower. The high carbamoyl-phosphate synthetase activity and ornithine levels suggest activation of the urea cycle in cafeteria diet-fed rats, but low arginine levels point to a block in the urea cycle between ornithine and arginine, thereby preventing the elimination of excess nitrogen as urea. The ultimate consequence of this paradoxical block in the urea cycle seems to be the limitation of arginine production and/or availability. David Sabater, Silvia Agnelli, Sofía Arriarán, José-Antonio Fernández-López, María del Mar Romero, Marià Alemany, and Xavier Remesar Copyright © 2014 David Sabater et al. All rights reserved. A Novel Mutation in Leptin Gene Is Associated with Severe Obesity in Chinese Individuals Sun, 23 Feb 2014 11:30:33 +0000 Obesity is a clinical syndrome which is driven by interactions between multiple genetic and environmental factors. Monogenic obesity is a rare type of obesity which is caused by a mutation in a single gene. Patients with monogenic obesity may develop early onset of obesity and severe metabolic abnormalities. In this study, we screened mutations of LEP in a total of 135 Chinese individuals including 35 obese patients whose BMI ≥32 kg/m2 and 100 controls with BMI <25 kg/m2. Moreover, detailed information and clinical measurements of the participants were also collected. Finally, we identified a novel nonsynonymous mutation H118L in exon 3 of LEP in one patient with BMI 46.0 kg/m2. This mutation was not identified in the controls. We speculated that the mutation H118L in LEP might be associated with severe obesity in Chinese subjects. However, the substantial mechanism should be further investigated. Yue Zhao, Nanchao Hong, Xiao Liu, Beibei Wu, Shanshan Tang, Jianjun Yang, Cheng Hu, and Weiping Jia Copyright © 2014 Yue Zhao et al. All rights reserved. Trichostatin A Suppresses EGFR Expression through Induction of MicroRNA-7 in an HDAC-Independent Manner in Lapatinib-Treated Cells Sun, 23 Feb 2014 00:00:00 +0000 Lapatinib, a dual EGFR/HER2 tyrosine kinase inhibitor, has been shown to improve the survival rate of patients with advanced HER2-positive breast cancers. However, the off-target activity of lapatinib in inducing EGFR expression without tyrosine kinase activity was demonstrated to render HER2-negative breast cancer cells more metastatic, suggesting a limitation to the therapeutic effectiveness of this dual inhibitor in HER2-heterogeneous tumors. Therefore, targeting EGFR expression may be a feasible approach to improve the anticancer efficiency of lapatinib-based therapy. Inhibition of HDAC has been previously reported to epigenetically suppress EGFR protein expression. In this study, however, our data indicated that treatment with HDAC inhibitors trichostatin A (TSA), but not suberoylanilide hydroxamic acid (SAHA) or HDAC siRNA, can attenuate both protein and mRNA expressions of EGFR in lapatinib-treated triple-negative breast cancer cells, suggesting that TSA may suppress EGFR expression independently of HDAC inhibition. Nevertheless, TSA reduced EGFR 3′UTR activity and induced the gene expression of microRNA-7, a known EGFR-targeting microRNA. Furthermore, treatment with microRNA-7 inhibitor attenuated TSA-mediated EGFR suppression. These results suggest that TSA induced microRNA-7 expression to downregulate EGFR expression in an HDAC-independent manner. Chih-Yen Tu, Chia-Hung Chen, Te-Chun Hsia, Min-Hsiang Hsu, Ya-Ling Wei, Meng-Chieh Yu, Wen-Shu Chen, Ke-Wei Hsu, Ming-Hsin Yeh, Liang-Chih Liu, Yun-Ju Chen, and Wei-Chien Huang Copyright © 2014 Chih-Yen Tu et al. All rights reserved. Redox Signaling as a Therapeutic Target to Inhibit Myofibroblast Activation in Degenerative Fibrotic Disease Thu, 20 Feb 2014 10:53:29 +0000 Degenerative fibrotic diseases encompass numerous systemic and organ-specific disorders. Despite their associated significant morbidity and mortality, there is currently no effective antifibrotic treatment. Fibrosis is characterized by the development and persistence of myofibroblasts, whose unregulated deposition of extracellular matrix components disrupts signaling cascades and normal tissue architecture leading to organ failure and death. The profibrotic cytokine transforming growth factor beta (TGFβ) is considered the foremost inducer of fibrosis, driving myofibroblast differentiation in diverse tissues. This review summarizes recent in vitro and in vivo data demonstrating that TGFβ-induced myofibroblast differentiation is driven by a prooxidant shift in redox homeostasis. Elevated NADPH oxidase 4 (NOX4)-derived hydrogen peroxide (H2O2) supported by concomitant decreases in nitric oxide (NO) signaling and reactive oxygen species scavengers are central factors in the molecular pathogenesis of fibrosis in numerous tissues and organs. Moreover, complex interplay between NOX4-derived H2O2 and NO signaling regulates myofibroblast differentiation. Restoring redox homeostasis via antioxidants or NOX4 inactivation as well as by enhancing NO signaling via activation of soluble guanylyl cyclases or inhibition of phosphodiesterases can inhibit and reverse myofibroblast differentiation. Thus, dysregulated redox signaling represents a potential therapeutic target for the treatment of wide variety of different degenerative fibrotic disorders. Natalie Sampson, Peter Berger, and Christoph Zenzmaier Copyright © 2014 Natalie Sampson et al. All rights reserved. Effects of Dietary Cholesterol and Its Oxidation Products on Pathological Lesions and Cholesterol and Lipid Oxidation in the Rabbit Liver Thu, 20 Feb 2014 09:28:10 +0000 This study was conducted to determine the effects of dietary cholesterol (CHO) and cholesterol oxidation products (COPs) on the induction of pathological lesions in rabbit liver tissues. Liver lesions were induced only when the levels of CHO and COPs in the diet were very high. The amount of CHO measured in the liver increased when dietary CHO was increased; by comparison, dietary COPs affected liver CHO amounts to a lesser extent. The TBARS (thiobarbituric acid reactive substances) value measured for the liver samples also increased when dietary CHO and COP levels were elevated, and the TBARS value was more strongly affected by the amount of COPs in the diet than by the amount of CHO. At 6 and 12 weeks, COP levels were the highest in the group that received 1.2 g CHO + 0.8 g COPs, followed by the 0.5 g CHO + 0.5 g COPs and 1.6 g CHO + 0.4 g COPs groups; the control (0 g) group showed the lowest COP levels among all groups. In this study, we found that not only dietary CHO but also COPs were involved in hypercholesterolemia induced liver lesions when the amount of CHO and COPs was high. Sun Jin Hur, Ki Chang Nam, Byungrok Min, Min Du, Kwon Il Seo, and Dong Uk Ahn Copyright © 2014 Sun Jin Hur et al. All rights reserved. Characterization of -Treated Rice Husk Adsorbent and Adsorption of Copper(II) from Aqueous Solution Tue, 11 Feb 2014 09:38:39 +0000 Rice husk, a surplus agricultural byproduct, was applied to the sorption of copper from aqueous solutions. Chemical modifications by treating rice husk with H3PO4 increased the sorption ability of rice husk for Cu(II). This work investigated the sorption characteristics for Cu(II) and examined the optimum conditions of the sorption processes. The elemental compositions of native rice husk and H3PO4-treated rice husk were determined by X-ray fluorescence (XRF) analysis. The scanning electron microscopic (SEM) analysis was carried out for structural and morphological characteristics of H3PO4-treated rice husk. The surface functional groups (i.e., carbonyl, carboxyl, and hydroxyl) of adsorbent were examined by Fourier Transform Infrared Technique (FT-IR) and contributed to the adsorption for Cu(II). Adsorption isotherm experiments were carried out at room temperature and the data obtained from batch studies fitted well with the Langmuir and Freundlich models with of 0.999 and 0.9303, respectively. The maximum sorption amount was 17.0358 mg/g at a dosage of 2 g/L after 180 min. The results showed that optimum pH was attained at pH 4.0. The equilibrium data was well represented by the pseudo-second-order kinetics. The percentage removal for Cu(II) approached equilibrium at 180 min with 88.9% removal. Ying Zhang, Ru Zheng, Jiaying Zhao, Fang Ma, Yingchao Zhang, and Qingjuan Meng Copyright © 2014 Ying Zhang et al. All rights reserved. ROS, Notch, and Wnt Signaling Pathways: Crosstalk between Three Major Regulators of Cardiovascular Biology Tue, 04 Feb 2014 09:41:44 +0000 Reactive oxygen species (ROS), traditionally viewed as toxic by-products that cause damage to biomolecules, now are clearly recognized as key modulators in a variety of biological processes and pathological states. The development and regulation of the cardiovascular system require orchestrated activities; Notch and Wnt/β-catenin signaling pathways are implicated in many aspects of them, including cardiomyocytes and smooth muscle cells survival, angiogenesis, progenitor cells recruitment and differentiation, arteriovenous specification, vascular cell migration, and cardiac remodelling. Several novel findings regarding the role of ROS in Notch and Wnt/β-catenin modulation prompted us to review their emerging function in the cardiovascular system during embryogenesis and postnatally. C. Caliceti, P. Nigro, P. Rizzo, and R. Ferrari Copyright © 2014 C. Caliceti et al. All rights reserved. Evaluation of Buspirone on Streptozotocin Induced Type 1 Diabetes and Its Associated Complications Mon, 20 Jan 2014 11:27:01 +0000 We have evaluated the effect of buspirone (1.5 mg/kg/day, p.o.) type 1 diabetes induced cardiovascular complications induced by streptozotocin (STZ, 45 mg/kg, i.v.) in Wistar rats. Various biochemical, cardiovascular, and hemodynamic parameters were measured at the end of 8 weeks of treatment. STZ produced significant hyperglycaemia, hypoinsulinemia, and dyslipidemia, which was prevented by buspirone treatment. STZ produced increase in serum creatinine, urea, lactate dehydrogenase, creatinine kinase, and C-reactive protein levels and treatment with buspirone produced reduction in these levels. STZ produced increase in cardiac and LV hypertrophy index, LV/RV ratio, and LV collagen, which were decreased by buspirone treatment. Buspirone also prevented STZ induced hemodynamic alterations and oxidative stress. These results were further supported by histopathological studies in which buspirone showed marked reduction in fibrosis and cardiac fiber disarray. In conclusion, our data suggests that buspirone is beneficial as an antidiabetic agent in type 1 diabetes mellitus and also prevents its cardiac complications. Suchi Raghunathan, Pratik Tank, Shraddha Bhadada, and Bhoomika Patel Copyright © 2014 Suchi Raghunathan et al. All rights reserved. PLP-Dependent Enzymes Wed, 15 Jan 2014 09:53:51 +0000 Alessandro Paiardini, Roberto Contestabile, Ashley M. Buckle, and Barbara Cellini Copyright © 2014 Alessandro Paiardini et al. All rights reserved. The CCN Family Proteins: Modulators of Bone Development and Novel Targets in Bone-Associated Tumors Tue, 14 Jan 2014 14:20:27 +0000 The CCN family of proteins is composed of six extracellular matrix-associated proteins that play crucial roles in skeletal development, wound healing, fibrosis, and cancer. Members of the CCN family share four conserved cysteine-rich modular domains that trigger signal transduction in cell adhesion, migration, proliferation, differentiation, and survival through direct binding to specific integrin receptors and heparan sulfate proteoglycans. In the present review, we discuss the roles of the CCN family proteins in regulating resident cells of the bone microenvironment. In vertebrate development, the CCN family plays a critical role in osteo/chondrogenesis and vasculo/angiogenesis. These effects are regulated through signaling via integrins, bone morphogenetic protein, vascular endothelial growth factor, Wnt, and Notch via direct binding to CCN family proteins. Due to the important roles of CCN family proteins in skeletal development, abnormal expression of CCN proteins is related to the tumorigenesis of primary bone tumors such as osteosarcoma, Ewing sarcoma, and chondrosarcoma. Additionally, emerging studies have suggested that CCN proteins may affect progression of secondary metastatic bone tumors by moderating the bone microenvironment. CCN proteins could therefore serve as potential therapeutic targets for drug development against primary and metastatic bone tumors. Po-Chun Chen, Hsu-Chen Cheng, Shun-Fa Yang, Chiao-Wen Lin, and Chih-Hsin Tang Copyright © 2014 Po-Chun Chen et al. All rights reserved. Oxidative Stress and Bone Resorption Interplay as a Possible Trigger for Postmenopausal Osteoporosis Sun, 12 Jan 2014 10:15:40 +0000 The underlying mechanism in postmenopausal osteoporosis (PO) is an imbalance between bone resorption and formation. This study was conducted to investigate whether oxidative stress (OxS) might have a role in this derangement of bone homeostasis. In a sample of 167 postmenopausal women, we found that increased serum levels of a lipid peroxidation marker, hydroperoxides, were negatively and independently associated with decreased bone mineral density (BMD) in total body (, ), lumbar spine (, ), and total hip (, ), as well as with increased bone resorption rate (, ), as assessed by the serum concentration of C-terminal telopeptide of type I collagen (CTX-1). On the contrary, the OxS marker failed to be correlated with the serum levels of bone-specific alkaline phosphatase (BAP), that is, elective marker of bone formation. Importantly, multiple regression analysis revealed that hydroperoxides is a determinant factor for the statistical association between lumbar spine BMD and CTX-1 levels. Taken together, our data suggest that OxS might mediate, by enhancing bone resorption, the uncoupling of bone turnover that underlies PO development. Carlo Cervellati, Gloria Bonaccorsi, Eleonora Cremonini, Arianna Romani, Enrica Fila, Maria Cristina Castaldini, Stefania Ferrazzini, Melchiorre Giganti, and Leo Massari Copyright © 2014 Carlo Cervellati et al. All rights reserved. Systemic Oxidative Stress and Conversion to Dementia of Elderly Patients with Mild Cognitive Impairment Sun, 12 Jan 2014 10:14:06 +0000 Mild cognitive impairment (MCI) is regarded as a prodromal phase of late onset Alzheimer’s disease (LOAD). It has been proposed that oxidative stress (OxS) might be implicated in the pathogenesis of LOAD. The aim of this study was to investigate whether a redox imbalance measured as serum level of hydroperoxides (i.e., by-products of lipid peroxidation) and/or serum antioxidant capacity might be predictive of the clinical progression of MCI to LOAD. The levels of these two markers were measured in 111 patients with MCI (follow-up: years), 105 patients with LOAD, and 118 nondemented healthy controls. Multivariate analysis adjusted for potential confounding factors, including age, gender, smoking, and comorbidities, showed a significant increase () in baseline levels of OxS in MCI and LOAD as compared to cognitive healthy controls. No differences in either of OxS markers were found by comparing MCI patients who converted () or not converted () to LOAD. Overall, these results suggest that systemic OxS might be a precocious feature of MCI and LOAD. However, the role of OxS as an early prognostic marker of progression to LOAD needs further investigations. Carlo Cervellati, Arianna Romani, Davide Seripa, Eleonora Cremonini, Cristina Bosi, Stefania Magon, Carlo M. Bergamini, Giuseppe Valacchi, Alberto Pilotto, and Giovanni Zuliani Copyright © 2014 Carlo Cervellati et al. All rights reserved. Vitamin B6-Dependent Enzymes in the Human Malaria Parasite Plasmodium falciparum: A Druggable Target? Thu, 09 Jan 2014 09:57:21 +0000 Malaria is a deadly infectious disease which affects millions of people each year in tropical regions. There is no effective vaccine available and the treatment is based on drugs which are currently facing an emergence of drug resistance and in this sense the search for new drug targets is indispensable. It is well established that vitamin biosynthetic pathways, such as the vitamin B6 de novo synthesis present in Plasmodium, are excellent drug targets. The active form of vitamin B6, pyridoxal 5-phosphate, is, besides its antioxidative properties, a cofactor for a variety of essential enzymes present in the malaria parasite which includes the ornithine decarboxylase (ODC, synthesis of polyamines), the aspartate aminotransferase (AspAT, involved in the protein biosynthesis), and the serine hydroxymethyltransferase (SHMT, a key enzyme within the folate metabolism). Thales Kronenberger, Jasmin Lindner, Kamila A. Meissner, Flávia M. Zimbres, Monika A. Coronado, Frank M. Sauer, Isolmar Schettert, and Carsten Wrenger Copyright © 2014 Thales Kronenberger et al. All rights reserved. Physalis alkekengi Carotenoidic Extract Inhibitor of Soybean Lipoxygenase-1 Activity Thu, 09 Jan 2014 08:21:28 +0000 The aim of this study was to evaluate the effect of the carotenoidic saponified extract of Physalis alkekengi sepals (PA) towards the lipoxygenase (LOX) oxidation of linoleic acid. Lipoxygenase activity in the presence of carotenoids, standard and from extract, was followed by its kinetic behaviour determining the changes in absorption at 234 nm. The standard carotenoids used were -carotene (-car), lutein (Lut), and zeaxanthin (Zea). The calculated enzymatic specific activity (ESA) after 600 s of reaction proves that PA carotenoidic extract has inhibitory effect on LOX oxidation of linoleic acid. A longer polyenic chain of carotenoid structure gives a higher ESA during the first reaction seconds. This situation is not available after 600 s of reaction and may be due to a destruction of this structure by cooxidation of carotenoids, besides the classical LOX reaction. The PA carotenoidic extract inhibiting the LOX-1 reaction can be considered a source of lipoxygenase inhibitors. Veronica Sanda Chedea, Adela Pintea, Andrea Bunea, Cornelia Braicu, Andreea Stanila, and Carmen Socaciu Copyright © 2014 Veronica Sanda Chedea et al. All rights reserved. Chlorotoxin-Fc Fusion Inhibits Release of MMP-2 from Pancreatic Cancer Cells Wed, 08 Jan 2014 10:05:30 +0000 Chlorotoxin (CTX) is a 36-amino acid peptide derived from Leiurus quinquestriatus (scorpion) venom, which inhibits low-conductance chloride channels in colonic epithelial cells. It has been reported that CTX also binds to matrix metalloproteinase-2 (MMP-2), membrane type-1 MMP, and tissue inhibitor of metalloproteinase-2, as well as CLC-3 chloride ion channels and other proteins. Pancreatic cancer cells require the activation of MMP-2 during invasion and migration. In this study, the fusion protein was generated by joining the CTX peptide to the amino terminus of the human IgG-Fc domain without a hinge domain, the monomeric form of chlorotoxin (M-CTX-Fc). The resulting fusion protein was then used to target pancreatic cancer cells (PANC-1) in vitro. M-CTX-Fc decreased MMP-2 release into the media of PANC-1 cells in a dose-dependent manner. M-CTX-Fc internalization into PANC-1 cells was observed. When the cells were treated with chlorpromazine (CPZ), the internalization of the fusion protein was reduced, implicating a clathrin-dependent internalization mechanism of M-CTX-Fc in PANC-1 cells. Furthermore, M-CTX-Fc clearly exhibited the inhibition of the migration depending on the concentration, but human IgG, as negative control of Fc, was not affected. The M-CTX-Fc may be an effective instrument for targeting pancreatic cancer. Samah El-Ghlban, Tomonari Kasai, Tsukasa Shigehiro, Hong Xia Yin, Sreeja Sekhar, Mikiko Ida, Anna Sanchez, Akifumi Mizutani, Takayuki Kudoh, Hiroshi Murakami, and Masaharu Seno Copyright © 2014 Samah El-Ghlban et al. All rights reserved. Food-Derived Bioactive Peptides on Inflammation and Oxidative Stress Thu, 02 Jan 2014 11:32:14 +0000 Chronic diseases such as atherosclerosis and cancer are now the leading causes of morbidity and mortality worldwide. Inflammatory processes and oxidative stress underlie the pathogenesis of these pathological conditions. Bioactive peptides derived from food proteins have been evaluated for various beneficial effects, including anti-inflammatory and antioxidant properties. In this review, we summarize the roles of various food-derived bioactive peptides in inflammation and oxidative stress and discuss the potential benefits and limitations of using these compounds against the burden of chronic diseases. Subhadeep Chakrabarti, Forough Jahandideh, and Jianping Wu Copyright © 2014 Subhadeep Chakrabarti et al. All rights reserved. Novel Strategies for the Treatment of Chondrosarcomas: Targeting Integrins Mon, 30 Dec 2013 14:03:59 +0000 Chondrosarcomas are a heterogeneous group of malignant bone tumors that are characterized by the production of cartilaginous extracellular matrix. They are the second most frequently occurring type of bone malignancy. Surgical resection remains the primary mode of treatment for chondrosarcomas, since conventional chemotherapy and radiotherapy are largely ineffective. Treatment of patients with high-grade chondrosarcomas is particularly challenging, owing to the lack of effective adjuvant therapies. Integrins are cell surface adhesion molecules that regulate a variety of cellular functions. They have been implicated in the initiation, progression, and metastasis of solid tumors. Deregulation of integrin expression and/or signaling has been identified in many chondrosarcomas. Therefore, the development of new drugs that can selectively target regulators of integrin gene expression and ligand-integrin signaling might hold great promise for the treatment of these cancers. In this review, we provide an overview of the current understanding of how growth factors, chemokines/cytokines, and other inflammation-related molecules can control the expression of specific integrins to promote cell migration. We also review the roles of specific subtypes of integrins and their signaling mechanisms, and discuss how these might be involved in tumor growth and metastasis. Finally, novel therapeutic strategies for targeting these molecules will be discussed. Jui-Chieh Chen, Yi-Chin Fong, and Chih-Hsin Tang Copyright © 2013 Jui-Chieh Chen et al. All rights reserved. Poly-S-Nitrosated Albumin as a Safe and Effective Multifunctional Antitumor Agent: Characterization, Biochemistry and Possible Future Therapeutic Applications Mon, 30 Dec 2013 08:07:59 +0000 Nitric oxide (NO) is a ubiquitous molecule involved in multiple cellular functions. Inappropriate production of NO may lead to disease states. To date, pharmacologically active compounds that release NO within the body, such as organic nitrates, have been used as therapeutic agents, but their efficacy is significantly limited by unwanted side effects. Therefore, novel NO donors with better pharmacological and pharmacokinetic properties are highly desirable. The S-nitrosothiol fraction in plasma is largely composed of endogenous S-nitrosated human serum albumin (Mono-SNO-HSA), and that is why we are testing whether this albumin form can be therapeutically useful. Recently, we developed SNO-HSA analogs such as SNO-HSA with many conjugated SNO groups (Poly-SNO-HSA) which were prepared using chemical modification. Unexpectedly, we found striking inverse effects between Poly-SNO-HSA and Mono-SNO-HSA. Despite the fact that Mono-SNO-HSA inhibits apoptosis, Poly-SNO-HSA possesses very strong proapoptotic effects against tumor cells. Furthermore, Poly-SNO-HSA can reduce or perhaps completely eliminate the multidrug resistance often developed by cancer cells. In this review, we forward the possibility that Poly-SNO-HSA can be used as a safe and effective multifunctional antitumor agent. Yu Ishima, Ulrich Kragh-Hansen, Toru Maruyama, and Masaki Otagiri Copyright © 2013 Yu Ishima et al. All rights reserved. Heavy Water Reduces GFP Expression in Prokaryotic Cell-Free Assays at the Translation Level While Stimulating Its Transcription Wed, 25 Dec 2013 13:39:15 +0000 The in vitro proliferation of prokaryotic and eukaryotic cells is remarkably hampered in the presence of heavy water (D2O). Impairment of gene expression at the transcription or translation level can be the base for this effect. However, insights into the underlying mechanisms are lacking. Here, we employ a cell-free expression system for the quantitative analysis of the effect of increasing percentages of D2O on the kinetics of in-vitro GFP expression. Experiments are designed to discriminate the rates of transcription, translation, and protein folding using pDNA and mRNA vectors, respectively. We find that D2O significantly stimulates GFP expression at the transcription level but acts as a suppressor at translation and maturation (folding) in a linear dose-dependent manner. At a D2O concentration of 60%, the GFP expression rate was reduced to 40% of an undisturbed sample. We observed a similar inhibition of GFP expression by D2O in a recombinant Escherichia coli strain, although the inhibitory effect is less pronounced. These results demonstrate the suitability of cell-free systems for quantifying the impact of heavy water on gene expression and establish a platform to further assess the potential therapeutic use of heavy water as antiproliferative agent. Luisa S. Hohlefelder, Tobias Stögbauer, Madeleine Opitz, Thomas M. Bayerl, and Joachim O. Rädler Copyright © 2013 Luisa S. Hohlefelder et al. All rights reserved. Antibacterial Activity of Nanocomposites of Copper and Cellulose Tue, 24 Dec 2013 17:00:05 +0000 The design of cheap and safe antibacterial materials for widespread use has been a challenge in materials science. The use of copper nanostructures combined with abundant biopolymers such as cellulose offers a potential approach to achieve such materials though this has been less investigated as compared to other composites. Here, nanocomposites comprising copper nanofillers in cellulose matrices have been prepared by in situ and ex situ methods. Two cellulose matrices (vegetable and bacterial) were investigated together with morphological distinct copper particulates (nanoparticles and nanowires). A study on the antibacterial activity of these nanocomposites was carried out for Staphylococcus aureus and Klebsiella pneumoniae, as pathogen microorganisms. The results showed that the chemical nature and morphology of the nanofillers have great effect on the antibacterial activity, with an increase in the antibacterial activity with increasing copper content in the composites. The cellulosic matrices also show an effect on the antibacterial efficiency of the nanocomposites, with vegetal cellulose fibers acting as the most effective substrate. Regarding the results obtained, we anticipate the development of new approaches to prepare cellulose/copper based nanocomposites thereby producing a wide range of interesting antibacterial materials with potential use in diverse applications such as packaging or paper coatings. Ricardo J. B. Pinto, Sara Daina, Patrizia Sadocco, Carlos Pascoal Neto, and Tito Trindade Copyright © 2013 Ricardo J. B. Pinto et al. All rights reserved. Gemifloxacin, a Fluoroquinolone Antimicrobial Drug, Inhibits Migration and Invasion of Human Colon Cancer Cells Tue, 10 Dec 2013 17:58:00 +0000 Gemifloxacin (GMF) is an orally administered broad-spectrum fluoroquinolone antimicrobial agent used to treat acute bacterial exacerbation of pneumonia and bronchitis. Although fluoroquinolone antibiotics have also been found to have anti-inflammatory and anticancer effects, studies on the effect of GMF on treating colon cancer have been relatively rare. To the best of our knowledge, this is the first report to describe the antimetastasis activities of GMF in colon cancer and the possible mechanisms involved. Results have shown that GMF inhibits the migration and invasion of colon cancer SW620 and LoVo cells and causes epithelial mesenchymal transition (EMT). In addition, GMF suppresses the activation of NF-κB and cell migration and invasion induced by TNF-α and inhibits the TAK1/TAB2 interaction, resulting in decreased IκB phosphorylation and NF-κB nuclear translocation in SW620 cells. Furthermore, Snail, a critical transcriptional factor of EMT, was downregulated after GMF treatment. Overexpression of Snail by cDNA transfection significantly decreases the inhibitory effect of GMF on EMT and cell migration and invasion. In conclusion, GMF may be a novel anticancer agent for the treatment of metastasis in colon cancer. Jung-Yu Kan, Ya-Ling Hsu, Yen-Hsu Chen, Tun-Chieh Chen, Jaw-Yuan Wang, and Po-Lin Kuo Copyright © 2013 Jung-Yu Kan et al. All rights reserved. Analysis of the Anticancer Phytochemicals in Andrographis paniculata Nees. under Salinity Stress Tue, 26 Nov 2013 13:18:22 +0000 Salinity causes the adverse effects in all physiological processes of plants. The present study aimed to investigate the potential of salt stress to enhance the accumulation of the anticancer phytochemicals in Andrographis paniculata accessions. For this purpose, 70-day-old plants were grown in different salinity levels (0.18, 4, 8, 12, and 16 dSm−1) on sand medium. After inducing a period of 30-day salinity stress and before flowering, all plants were harvested and the data on morphological traits, proline content and the three anticancer phytochemicals, including andrographolide (AG), neoandrographolide (NAG), and 14-deoxy-11,12-didehydroandrographolide (DDAG), were measured. The results indicated that salinity had a significant effect on the aforementioned three anticancer phytochemicals. In addition, the salt tolerance index (STI) was significantly decreased, while, except for DDAG, the content of proline, the AG, and NAG was significantly increased (). Furthermore, it was revealed that significant differences among accessions could happen based on the total dry weight, STI, AG, and NAG. Finally, we noticed that the salinity at 12 dSm−1 led to the maximum increase in the quantities of AG, NAG, and DDAG. In other words, under salinity stress, the tolerant accessions were capable of accumulating the higher amounts of proline, AG, and NAG than the sensitive accessions. Daryush Talei, Alireza Valdiani, Mahmood Maziah, Sreenivasa Rao Sagineedu, and Mohd Said Saad Copyright © 2013 Daryush Talei et al. All rights reserved. Association of Genetic Variants of BMP4 with Type 2 Diabetes Mellitus and Clinical Traits in a Chinese Han Population Mon, 18 Nov 2013 18:22:15 +0000 BMP4 is one of the transforming growth factor-β superfamily, which can participate in adipogenesis. Gene encoding BMP4 is acknowledged as a convincing candidate that may contribute to both glucose and lipid metabolism. In this paper, we aimed to test the impacts of BMP4 variants on type 2 diabetes in a large sample of Chinese population. We genotyped 10 tagging single nucleotide polymorphisms within the BMP4 region in 6822 participants and acquired detailed clinical investigations and biochemistry measurements. We found that BMP4 rs8014363 showed nominal association towards type 2 diabetes, with the T allele conferring a high risk of type 2 diabetes (, 95%CI 0.999–1.229, for allele; , 95%CI 1.000–1.231, for genotype), but it was no longer statistically significant after adjusting for multiple testing (empirical for allele based on 10,000 permutations). Moreover, we observed a significant association of rs8014363 with triglyceride level and a trend towards association with high-density lipoprotein cholesterol after adjusting for age, gender, and BMI ( and 0.068, resp.). Our data suggested that the genetic variants of BMP4 may not play a dominant role in glucose metabolism in Chinese Han population, but a minor effect cannot be ignored. Shanshan Tang, Rong Zhang, Weihui Yu, Feng Jiang, Jie Wang, Miao Chen, Danfeng Peng, Jing Yan, Yuqian Bao, and Weiping Jia Copyright © 2013 Shanshan Tang et al. All rights reserved. Parthenium hysterophorus: A Probable Source of Anticancer, Antioxidant and Anti-HIV Agents Sun, 17 Nov 2013 11:15:26 +0000 The present work reports the anticancer, antioxidant, lipo-protective, and anti-HIV activities of phytoconstituents present in P. hysterophorus leaf. Dried leaf samples were sequentially extracted with nonpolar and polar solvents. Ethanol fraction showed noticeable cytotoxic activity (81–85%) in SRB assay against MCF-7 and THP-1 cancer cell lines at 100 μg/ml concentration, while lower activity was observed with DU-145 cell line. The same extract exhibited 17–98% growth inhibition of HL-60 cancer cell lines in MTT assay, showing concentration dependent response. Ethanol extract caused 12% reduction in mitochondrial membrane potential and 10% increment in sub G1 population of HL-60 cell lines. Several leaf fractions, namely, ethyl acetate, ethanol, and aqueous fractions exhibited considerable reducing capability at higher concentrations. Most of the extracts demonstrated appreciable (>75%) metal ion chelating and hydroxyl radical scavenging activities at 200 µg/ml. All the extracts except aqueous fraction accounted for about 70–80% inhibition of lipid peroxidation in rat liver homogenate indicating protective response against membrane damage. About 40% inhibition of reverse transcriptase (RT) activity was observed in hexane fraction in anti-HIV assay at 6.0 µg/ml concentration. The study showed that phytochemicals present in P. hysterophorus leaf have considerable potential as cytotoxic and antioxidant agents with low to moderate anti-HIV activity. Shashank Kumar, Gousia Chashoo, Ajit K. Saxena, and Abhay K. Pandey Copyright © 2013 Shashank Kumar et al. All rights reserved. Depletion of Luminal Pyridine Nucleotides in the Endoplasmic Reticulum Activates Autophagy with the Involvement of mTOR Pathway Sun, 17 Nov 2013 09:00:58 +0000 It has been recently shown that redox imbalance of luminal pyridine nucleotides in the endoplasmic reticulum (ER) together with oxidative stress results in the activation of autophagy. In the present study we demonstrated that decrease of luminal NADPH/NADP+ ratio alone by metyrapone was sufficient to promote the mechanism of “self-eating” detected by the activation of LC3. Depletion of luminal NADPH had also significant effect on the key proteins of mTOR pathway, which got inactivated by dephosphorylation. These findings were also confirmed by silencing the proteins (glucose-6-phosphate transporter and hexose-6-phosphate dehydrogenase) responsible for NADPH generation in the ER lumen. However, silencing the key components and addition of metyrapone had different effects on downstream substrates 4EBP1 and p70S6K of mTOR. The applied treatments did not compromise the viability of the cells. Our data suggest that ER stress caused by luminal NADPH depletion activates a pro-survival autophagic mechanism firmly coupled to the activation of mTOR pathway. Orsolya Kapuy and Gábor Bánhegyi Copyright © 2013 Orsolya Kapuy and Gábor Bánhegyi. All rights reserved. Allicin Attenuates Inflammation and Suppresses HLA-B27 Protein Expression in Ankylosing Spondylitis Mice Wed, 13 Nov 2013 13:27:29 +0000 Here we aimed to determine the therapeutic effect of allicin on ankylosing spondylitis (AS) and explore the mechanism(s) of action. AS mouse model was constructed by transferring the HLA-B2704 gene into Kunming mice and verified by RT-PCR and CT imaging. Verified AS mice were randomly divided into model group () and allicin-treated groups (50, 100, and 200 mg/kg, resp., , p.o., for 2 months). Wild type mice were used as control (). The levels of AS-related inflammatory factors were measured by ELISA. mRNA and protein expressions of HLA-B27 were checked by RT-PCR and western blotting. As the results, the mouse model of AS was successfully established, and high-dose allicin could markedly alleviate spine inflammatory injury possibly via reducing the secretion of the inflammatory factors (IL-6, IL-8, and TNF-α) sharply in AS mice. Moreover, allicin significantly inhibited HLA-B27 protein translation but failed to suppress HLA-B27 gene transcription in AS mice, indicating a posttranscriptional mechanism of this modulation. In conclusion, allicin has potential to be used for AS treatment as an anti-inflammatory nutraceutical. Xin Gu, Haishan Wu, and Peiliang Fu Copyright © 2013 Xin Gu et al. All rights reserved. Thermal and Chemical Stability of Two Homologous POZ/BTB Domains of KCTD Proteins Characterized by a Different Oligomeric Organization Wed, 06 Nov 2013 13:39:22 +0000 POZ/BTB domains are widespread modules detected in a variety of different biological contexts. Here, we report a biophysical characterization of the POZ/BTB of KCTD6, a protein that is involved in the turnover of the muscle small ankyrin-1 isoform 5 and, in combination with KCTD11, in the ubiquitination and degradation of HDAC1. The analyses show that the domain is a tetramer made up by subunits with the expected α/ structure. A detailed investigation of its stability, carried out in comparison with the homologous pentameric POZ/BTB domain isolated from KCTD5, highlights a number of interesting features, which are shared by the two domains despite their different organization. Their thermal/chemical denaturation curves are characterized by a single and sharp inflection point, suggesting that the denaturation of the two domains is a cooperative two-state process. Furthermore, both domains present a significant content of secondary structure in their denatured state and a reversible denaturation process. We suggest that the ability of these domains to fold and unfold reversibly, a property that is somewhat unexpected for these oligomeric assemblies, may have important implications for their biological function. Indeed, these properties likely favor the formation of heteromeric associations that may be essential for the intricate regulation of the processes in which these proteins are involved. Luciano Pirone, Carla Esposito, Stefania Correale, Giuseppe Graziano, Sonia Di Gaetano, Luigi Vitagliano, and Emilia Pedone Copyright © 2013 Luciano Pirone et al. All rights reserved. Exploring Different Virtual Screening Strategies for Acetylcholinesterase Inhibitors Mon, 04 Nov 2013 10:16:08 +0000 The virtual screening problems associated with acetylcholinesterase (AChE) inhibitors were explored using multiple shape, and structure-based modeling strategies. The employed strategies include molecular docking, similarity search, and pharmacophore modeling. A subset from directory of useful decoys (DUD) related to AChE inhibitors was considered, which consists of 105 known inhibitors and 3732 decoys. Statistical quality of the models was evaluated by enrichment factor (EF) metrics and receiver operating curve (ROC) analysis. The results revealed that electrostatic similarity search protocol using EON (ET_combo) outperformed all other protocols with outstanding enrichment of 95% in top 1% and 2% of the dataset with an AUC of 0.958. Satisfactory performance was also observed for shape-based similarity search protocol using ROCS and PHASE. In contrast, the molecular docking protocol performed poorly with enrichment factors 30% in all cases. The shape- and electrostatic-based similarity search protocol emerged as a plausible solution for virtual screening of AChE inhibitors. Nibha Mishra and Arijit Basu Copyright © 2013 Nibha Mishra and Arijit Basu. All rights reserved. The Role of Red Blood Cells in Enhancing or Preventing HIV Infection and Other Diseases Thu, 10 Oct 2013 18:14:19 +0000 Aim. To highlight the apparently neglected role of erythrocyte antigens in the epidemiology of infectious diseases, especially HIV, with the prime objective of stimulating research in this area. Method. A literature search was performed on the PubMed for relevant papers from 1984 to 2013, the era covering active HIV research. This was achieved by using the phrases “erythrocyte blood groups HIV” (81 papers) or “red cell antigen, blood groups, and HIV” (60 papers). A manual Google Scholar search was done and supplemented by original papers referenced by various authors. However, the review was limited by the relative scarcity of papers on the subject, and only papers written in English were reviewed during the period October 2012 to September 2013. Results. Many communicable and noncommunicable diseases are associated with specific blood groups. Examples of these diseases are discussed in detail. HIV has been shown to bind to erythrocytes, and candidate erythrocyte-binding molecules and mechanisms are also discussed. Moreover, erythrocyte-HIV binding is associated with increased viral infectivity, thus, underscoring the need to study this phenomenon and its implications for HIV epidemiology. Conclusion. Erythrocyte antigens may be important in the pathogenesis and epidemiology of many diseases, including HIV. Modisa S. Motswaledi, Ishmael Kasvosve, and Oluwafemi O. Oguntibeju Copyright © 2013 Modisa S. Motswaledi et al. All rights reserved. Effect of Jatropha curcas Peptide Fractions on the Angiotensin I-Converting Enzyme Inhibitory Activity Thu, 10 Oct 2013 18:13:27 +0000 Hypertension is one of the most common worldwide diseases in humans. Angiotensin I-converting enzyme (ACE) plays an important role in regulating blood pressure and hypertension. An evaluation was done on the effect of Alcalase hydrolysis of defatted Jatropha curcas kernel meal on ACE inhibitory activity in the resulting hydrolysate and its purified fractions. Alcalase exhibited broad specificity and produced a protein hydrolysate with a 21.35% degree of hydrolysis and 34.87% ACE inhibition. Ultrafiltration of the hydrolysate produced peptide fractions with increased biological activity (24.46–61.41%). Hydrophobic residues contributed substantially to the peptides’ inhibitory potency. The 5–10 and <1 kDa fractions were selected for further fractionation by gel filtration chromatography. ACE inhibitory activity (%) ranged from 22.66 to 45.96% with the 5–10 kDa ultrafiltered fraction and from 36.91 to 55.83% with the <1 kDa ultrafiltered fraction. The highest ACE inhibitory activity was observed in F2 ( μg/mL) from the 5–10 kDa fraction and F1 ( μg/mL) from the <1 kDa fraction. ACE inhibitory fractions from Jatropha kernel have potential applications in alternative hypertension therapies, adding a new application for the Jatropha plant protein fraction and improving the financial viability and sustainability of a Jatropha-based biodiesel industry. Maira R. Segura-Campos, Fanny Peralta-González, Arturo Castellanos-Ruelas, Luis A. Chel-Guerrero, and David A. Betancur-Ancona Copyright © 2013 Maira R. Segura-Campos et al. All rights reserved. Fullerenols as a New Therapeutic Approach in Nanomedicine Mon, 07 Oct 2013 17:29:13 +0000 Recently, much attention has been paid to the bioactive properties of water-soluble fullerene derivatives: fullerenols, with emphasis on their pro- and antioxidative properties. Due to their hydrophilic properties and the ability to scavenge free radicals, fullerenols may, in the future, provide a serious alternative to the currently used pharmacological methods in chemotherapy, treatment of neurodegenerative diseases, and radiobiology. Some of the most widely used drugs in chemotherapy are anthracycline antibiotics. Anthracycline therapy, in spite of its effective antitumor activity, induces systemic oxidative stress, which interferes with the effectiveness of the treatment and results in serious side effects. Fullerenols may counteract the harmful effects of anthracyclines by scavenging free radicals and thereby improve the effects of chemotherapy. Additionally, due to the hollow spherical shape, fullerenols may be used as drug carriers. Moreover, because of the existence of the currently ineffective ways for neurodegenerative diseases treatment, alternative compounds, which could prevent the negative effects of oxidative stress in the brain, are still sought. In the search of alternative methods of treatment and diagnosis, today’s science is increasingly reaching for tools in the field of nanomedicine, for example, fullerenes and their water-soluble derivatives, which is addressed in the present paper. Jacek Grebowski, Paulina Kazmierska, and Anita Krokosz Copyright © 2013 Jacek Grebowski et al. All rights reserved. Frataxin mRNA Isoforms in FRDA Patients and Normal Subjects: Effect of Tocotrienol Supplementation Mon, 23 Sep 2013 08:50:23 +0000 Friedreich’s ataxia (FRDA) is caused by deficient expression of the mitochondrial protein frataxin involved in the formation of iron-sulphur complexes and by consequent oxidative stress. We analysed low-dose tocotrienol supplementation effects on the expression of the three splice variant isoforms (FXN-1, FXN-2, and FXN-3) in mononuclear blood cells of FRDA patients and healthy subjects. In FRDA patients, tocotrienol leads to a specific and significant increase of FXN-3 expression while not affecting FXN-1 and FXN-2 expression. Since no structural and functional details were available for FNX-2 and FXN-3, 3D models were built. FXN-1, the canonical isoform, was then docked on the human iron-sulphur complex, and functional interactions were computed; when FXN-1 was replaced by FXN-2 or FNX-3, we found that the interactions were maintained, thus suggesting a possible biological role for both isoforms in human cells. Finally, in order to evaluate whether tocotrienol enhancement of FXN-3 was mediated by an increase in peroxisome proliferator-activated receptor-γ (PPARG), PPARG expression was evaluated. At a low dose of tocotrienol, the increase of FXN-3 expression appeared to be independent of PPARG expression. Our data show that it is possible to modulate the mRNA expression of the minor frataxin isoforms and that they may have a functional role. Provvidenza Maria Abruzzo, Marina Marini, Alessandra Bolotta, Gemma Malisardi, Stefano Manfredini, Alessandro Ghezzo, Antonella Pini, Gianluca Tasco, and Rita Casadio Copyright © 2013 Provvidenza Maria Abruzzo et al. All rights reserved. The Pyridoxal 5′-Phosphate (PLP)-Dependent Enzyme Serine Palmitoyltransferase (SPT): Effects of the Small Subunits and Insights from Bacterial Mimics of Human hLCB2a HSAN1 Mutations Mon, 23 Sep 2013 08:05:23 +0000 The pyridoxal 5′-phosphate (PLP)-dependent enzyme serine palmitoyltransferase (SPT) catalyses the first step of de novo sphingolipid biosynthesis. The core human enzyme is a membrane-bound heterodimer composed of two subunits (hLCB1 and hLCB2a/b), and mutations in both hLCB1 (e.g., C133W and C133Y) and hLCB2a (e.g., V359M, G382V, and I504F) have been identified in patients with hereditary sensory and autonomic neuropathy type I (HSAN1), an inherited disorder that affects sensory and autonomic neurons. These mutations result in substrate promiscuity, leading to formation of neurotoxic deoxysphingolipids found in affected individuals. Here we measure the activities of the hLCB2a mutants in the presence of ssSPTa and ssSPTb and find that all decrease enzyme activity. High resolution structural data of the homodimeric SPT enzyme from the bacterium Sphingomonas paucimobilis (Sp SPT) provides a model to understand the impact of the hLCB2a mutations on the mechanism of SPT. The three human hLCB2a HSAN1 mutations map onto Sp SPT (V246M, G268V, and G385F), and these mutant mimics reveal that the amino acid changes have varying impacts; they perturb the PLP cofactor binding, reduce the affinity for both substrates, decrease the enzyme activity, and, in the most severe case, cause the protein to be expressed in an insoluble form. Ashley E. Beattie, Sita D. Gupta, Lenka Frankova, Agne Kazlauskaite, Jeffrey M. Harmon, Teresa M. Dunn, and Dominic J. Campopiano Copyright © 2013 Ashley E. Beattie et al. All rights reserved. Bioactive Compounds and Antioxidant Activity of Fresh and Processed White Cauliflower Sun, 22 Sep 2013 15:05:03 +0000 Brassica species are very rich in health-promoting phytochemicals, including phenolic compounds, vitamin C, and minerals. The objective of this study was to investigate the effect of different blanching (i.e., water and steam) and cooking (i.e., water boiling, steam boiling, microwaving, and stir-frying) methods on the nutrient components, phytochemical contents (i.e., polyphenols, carotenoids, flavonoid, and ascorbic acid), antioxidant activity measured by DPPH assay, and phenolic profiles of white cauliflower. Results showed that water boiling and water blanching processes had a great effect on the nutrient components and caused significant losses of dry matter, protein, and mineral and phytochemical contents. However, steam treatments (blanching and cooking), stir-frying, and microwaving presented the lowest reductions. Methanolic extract of fresh cauliflower had significantly the highest antioxidant activity (68.91%) followed by the extracts of steam-blanched, steam-boiled, stir-fried, and microwaved cauliflower 61.83%, 59.15%, 58.93%, and 58.24%, respectively. HPLC analysis revealed that the predominant phenolics of raw cauliflower were protocatechuic acid (192.45), quercetin (202.4), pyrogallol (18.9), vanillic acid (11.90), coumaric acid (6.94), and kaempferol (25.91) mg/100 g DW, respectively. Fouad A. Ahmed and Rehab F. M. Ali Copyright © 2013 Fouad A. Ahmed and Rehab F. M. Ali. All rights reserved. Evaluation of Fatty Acid and Amino Acid Compositions in Okra (Abelmoschus esculentus) Grown in Different Geographical Locations Sun, 22 Sep 2013 10:37:30 +0000 Okra has different uses as a food and a remedy in traditional medicine. Since it produces many seeds, distribution of the plant is also quite easy. Although seed oil yield is low (4.7%), since the linoleic acid composition of the seed oil is quiet high (67.5%), it can still be used as a source of (UNSAT) unsaturated fatty acids. In this study, samples of okra grown in four different locations were analyzed to measure fatty acid and amino acid compositions. The content of the lipid extraction ranged from 4.34% to 4.52% on a dry weight basis. Quantitatively, the main okra fatty acids were palmitic acid (29.18–43.26%), linoleic acid (32.22–43.07%), linolenic acid (6.79–12.34%), stearic acid (6.36–7.73%), oleic acid (4.31–6.98%), arachidic acid (ND–3.48%), margaric acid (1.44–2.16%), pentadecylic acid (0.63–0.92%), and myristic acid (0.21–0.49%). Aspartic acid, proline, and glutamic acids were the main amino acids in okra pods, while cysteine and tyrosine were the minor amino acids. Statistical methods revealed how the fatty acid and amino acid contents in okra may be affected by the sampling location. Rokayya Sami, Jiang Lianzhou, Li Yang, Ying Ma, and Jing Jing Copyright © 2013 Rokayya Sami et al. All rights reserved. Novel Spectrophotometric Method for the Quantitation of Urinary Xanthurenic Acid and Its Application in Identifying Individuals with Hyperhomocysteinemia Associated with Vitamin Deficiency Mon, 16 Sep 2013 15:01:02 +0000 A novel spectrophotometric method for the quantification of urinary xanthurenic acid (XA) is described. The direct acid ferric reduction (DAFR) procedure was used to quantify XA after it was purified by a solid-phase extraction column. The linearity of proposed method extends from 2.5 to 100.0 mg/L. The method is precise, yielding day-to-day CVs for two pooled controls of 3.5% and 4.6%, respectively. Correlation studies with an established HPLC method and a fluorometric procedure showed correlation coefficients of 0.98 and 0.98, respectively. Interference from various urinary metabolites was insignificant. In a small-scale screening of elderly conducted at Penghu county in Taiwan (), we were able to identify a group of twenty individuals having hyperhomocysteinemia (>15 μmole/L). Three of them were found to be positive for XA as analyzed by the proposed method, which correlated excellently with the results of the activation coefficient method for RBC’s AST/B6 functional test. These data confirm the usefulness of the proposed method for identifying urinary XA as an indicator of vitamin B6 deficiency-associated hyperhomocysteinemic condition. Chi-Fen Chen, Tsan-Zon Liu, Wu-Hsiang Lan, Li-An Wu, Chin-Hung Tsai, Jeng-Fong Chiou, and Li-Yu Tsai Copyright © 2013 Chi-Fen Chen et al. All rights reserved. -3 PUFAs in the Prevention and Cure of Inflammatory, Degenerative, and Neoplastic Diseases Mon, 16 Sep 2013 09:02:24 +0000 Achille Cittadini, Gabriella Calviello, Hui-Min Su, and Karsten Weylandt Copyright © 2013 Achille Cittadini et al. All rights reserved. 7,8-Dihydroxyflavone Suppresses Oxidative Stress-Induced Base Modification in DNA via Induction of the Repair Enzyme 8-Oxoguanine DNA Glycosylase-1 Sat, 14 Sep 2013 14:03:32 +0000 The modified guanine base 8-oxoguanine (8-oxoG) is abundantly produced by oxidative stress, can contribute to carcinogenesis, and can be removed from DNA by 8-oxoguanine DNA glycosylase-1 (OGG1), which acts as an 8-oxoG glycosylase and endonuclease. This study investigated the mechanism by which 7,8-dihydroxyflavone (DHF) inhibits oxidative stress-induced 8-oxoG formation in hamster lung fibroblasts (V79-4). DHF significantly reduced the amount of 8-oxoG induced by hydrogen peroxide (H2O2) and elevated the levels of OGG1 mRNA and protein. DHF increased the binding of nuclear factor erythroid 2-related factor 2 (Nrf2) to antioxidant response element sequences in the upstream promoter region of OGG1. Moreover, DHF increased the nuclear levels of Nrf2, small Maf proteins, and the Nrf2/small Maf complex, all of which are decreased by H2O2 treatment. Likewise, the level of phosphorylated Akt, which activates Nrf2, was decreased by H2O2 treatment but restored by DHF treatment. The levels of OGG1 and nuclear translocation of Nrf2 protein were decreased upon treatment with PI3K inhibitor or Akt inhibitor, and DHF treatment did not restore OGG1 and nuclear Nrf2 levels in these inhibitor-treated cells. Furthermore, PI3K and Akt inhibitors abolished the protective effects of DHF in cells undergoing oxidative stress. These data indicate that DHF induces OGG1 expression via the PI3K-Akt pathway and protects cells against oxidative DNA base damage by activating DNA repair systems. Ki Cheon Kim, In Kyung Lee, Kyoung Ah Kang, Ji Won Cha, Suk Ju Cho, Soo Young Na, Sungwook Chae, Hye Sun Kim, Suhkmann Kim, and Jin Won Hyun Copyright © 2013 Ki Cheon Kim et al. All rights reserved. Characterization of C-S Lyase from C. diphtheriae: A Possible Target for New Antimicrobial Drugs Wed, 11 Sep 2013 11:03:16 +0000 The emergence of antibiotic resistance in microbial pathogens requires the identification of new antibacterial drugs. The biosynthesis of methionine is an attractive target because of its central importance in cellular metabolism. Moreover, most of the steps in methionine biosynthesis pathway are absent in mammals, lowering the probability of unwanted side effects. Herein, detailed biochemical characterization of one enzyme required for methionine biosynthesis, a pyridoxal-5′-phosphate (PLP-) dependent C-S lyase from Corynebacterium diphtheriae, a pathogenic bacterium that causes diphtheria, has been performed. We overexpressed the protein in E. coli and analyzed substrate specificity, pH dependence of steady state kinetic parameters, and ligand-induced spectral transitions of the protein. Structural comparison of the enzyme with cystalysin from Treponema denticola indicates a similarity in overall folding. We used site-directed mutagenesis to highlight the importance of active site residues Tyr55, Tyr114, and Arg351, analyzing the effects of amino acid replacement on catalytic properties of enzyme. Better understanding of the active site of C. diphtheriae C-S lyase and the determinants of substrate and reaction specificity from this work will facilitate the design of novel inhibitors as antibacterial therapeutics. Alessandra Astegno, Alejandro Giorgetti, Alessandra Allegrini, Barbara Cellini, and Paola Dominici Copyright © 2013 Alessandra Astegno et al. All rights reserved. Acetylsalicylic Acid Reduces the Severity of Dextran Sodium Sulfate-Induced Colitis and Increases the Formation of Anti-Inflammatory Lipid Mediators Sun, 08 Sep 2013 14:29:40 +0000 The role of non-steroidal anti-inflammatory drugs in inflammatory bowel disease is controversial, as they have been implicated in disease aggravation. Different from other cyclooxygenase inhibitors, acetylsalicylic acid (ASA) enhances the formation of anti-inflammatory and proresolution lipoxins derived from arachidonic acid as well as resolvins from omega-3 polyunsaturated fatty acids such as docosahexaenoic acid (DHA). In this study, we examined the effect of ASA on murine dextran sodium sulfate colitis. A mouse magnetic resonance imaging (MRI) protocol and post mortem assessment were used to assess disease severity, and lipid metabolites were measured using liquid chromatography-coupled tandem mass spectrometry. Decreased colitis activity was demonstrated by phenotype and MRI assessment in mice treated with ASA, and confirmed in postmortem analysis. Analysis of lipid mediators showed sustained formation of lipoxin A4 and an increase of DHA-derived 17-hydroxydocosahexaenoic acid (17-HDHA) after treatment with ASA. Furthermore, in vitro experiments in RAW264.7 murine macrophages demonstrated significantly increased phagocytosis activity after incubation with 17-HDHA, supporting its proresolution effect. These results show a protective effect of ASA in a murine colitis model and could give a rationale for a careful reassessment of ASA therapy in patients with inflammatory bowel disease and particularly ulcerative colitis, possibly combined with DHA supplementation. Thomas Köhnke, Beate Gomolka, Süleyman Bilal, Xiangzhi Zhou, Yanping Sun, Michael Rothe, Daniel C. Baumgart, and Karsten H. Weylandt Copyright © 2013 Thomas Köhnke et al. All rights reserved. Responses of Growth Performance and Proinflammatory Cytokines Expression to Fish Oil Supplementation in Lactation Sows’ and/or Weaned Piglets’ Diets Sun, 01 Sep 2013 11:35:01 +0000 The study was conducted to investigate whether dietary fish oil could influence growth of piglets via regulating the expression of proinflammatory cytokines. A split-plot experimental design was used with sow diet effect in the main plots and differing piglet diet effect in the subplot. The results showed that suckling piglets from fish oil fed dams grew rapidly () than control. It was also observed that these piglets had higher ADG, feed intake, and final body weight () during postweaning than those piglets from lard fed dams. Furthermore, there was a significant decrease () in the expression of interleukin 6 and tumor necrosis factor-α in longissimus dorsi muscle. In contrast, there was a tendency () towards lower ADG and higher feed : gain in weaned piglets receiving fish oil compared with those receiving lard. Meanwhile, splenic proinflammatory cytokines expression was increased () in piglets receiving fish oil during postweaning period. The results suggested that 7% fish oil addition to sows' diets alleviated inflammatory response via decreasing the proinflammatory cytokines expression in skeletal muscle and accelerated piglet growth. However, 7% fish oil addition to weaned piglets' diets might decrease piglet growth via increasing splenic proinflammatory cytokines expression. Jie Luo, Feiruo Huang, Chenglin Xiao, Zhengfeng Fang, Jian Peng, and Siwen Jiang Copyright © 2013 Jie Luo et al. All rights reserved. Polyphenolic Contents and Antioxidant Properties of Different Grape (V. vinifera, V. labrusca, and V. hybrid) Cultivars Wed, 21 Aug 2013 10:04:09 +0000 The polyphenolic contents and the antioxidant activity of the skins and pulps of different grape cultivars were estimated using HPLC and DPPH antioxidant assay, respectively. The phenolics and flavonoids identified were quercetin, kaempferol, caffeic acid, p-coumaric acid, cinnamic acid, and (−)-epicatechin. The total phenolic contents were found to be the highest in the grape skin of Flouxa (>400 mg/100 g), followed by Campbell Early and Tamnara (>300 mg/100 g), and then by Red Globe and Ruby Seedless (>250 mg/100 g), and the total phenolic content was the lowest in Italia and Delaware (<60 mg/100 g). The antioxidant activities of the grape extracts varied from 12.5% (Ruby Seedless) to 60.2% (Hongiseul) for skins, whereas the antioxidant activities of the grape extracts varied from 35.4% (Campbell Early) to 84.5% (Hongiseul) for pulps. The grape pulps have stronger antioxidant activities than those of the grape skins. Our results suggest that the phenolic and flavonoid contents in extracts of grape skins and pulps showed statistically significant correlations with the free radical scavenging activity. Shivraj Hariram Nile, S. H. Kim, Eun Young Ko, and Se Won Park Copyright © 2013 Shivraj Hariram Nile et al. All rights reserved. An Active C-Terminally Truncated Form of Ca2+/Calmodulin-Dependent Protein Kinase Phosphatase-N (CaMKP-N/PPM1E) Wed, 07 Aug 2013 13:26:54 +0000 Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) and its nuclear homolog CaMKP-N (PPM1E) are Ser/Thr protein phosphatases that belong to the PPM family. CaMKP-N is expressed in the brain and undergoes proteolytic processing to yield a C-terminally truncated form. The physiological significance of this processing, however, is not fully understood. Using a wheat-embryo cell-free protein expression system, we prepared human CaMKP-N (hCaMKP-N(WT)) and the truncated form, hCaMKP-N(1–559), to compare their enzymatic properties using a phosphopeptide substrate. The hCaMKP-N(1–559) exhibited a much higher value than the hCaMKP-N(WT) did, suggesting that the processing may be a regulatory mechanism to generate a more active species. The active form, hCaMKP-N(1–559), showed Mn2+ or Mg2+-dependent phosphatase activity with a strong preference for phospho-Thr residues and was severely inhibited by NaF, but not by okadaic acid, calyculin A, or 1-amino-8-naphthol-2,4-disulfonic acid, a specific inhibitor of CaMKP. It could bind to postsynaptic density and dephosphorylate the autophosphorylated Ca2+/calmodulin-dependent protein kinase II. Furthermore, it was inactivated by H2O2 treatment, and the inactivation was completely reversed by treatment with DTT, implying that this process is reversibly regulated by oxidation/reduction. The truncated CaMKP-N may play an important physiological role in neuronal cells. Atsuhiko Ishida, Kumiko Tsumura, Megu Oue, Yasuhiro Takenaka, Yasushi Shigeri, Naoki Goshima, Yasuhiro Ishihara, Tetsuo Hirano, Hiromi Baba, Noriyuki Sueyoshi, Isamu Kameshita, and Takeshi Yamazaki Copyright © 2013 Atsuhiko Ishida et al. All rights reserved. Metabolomics in Plants and Humans: Applications in the Prevention and Diagnosis of Diseases Tue, 06 Aug 2013 10:55:50 +0000 In the recent years, there has been an increase in the number of metabolomic approaches used, in parallel with proteomic and functional genomic studies. The wide variety of chemical types of metabolites available has also accelerated the use of different techniques in the investigation of the metabolome. At present, metabolomics is applied to investigate several human diseases, to improve their diagnosis and prevention, and to design better therapeutic strategies. In addition, metabolomic studies are also being carried out in areas such as toxicology and pharmacology, crop breeding, and plant biotechnology. In this review, we emphasize the use and application of metabolomics in human diseases and plant research to improve human health. Diego F. Gomez-Casati, Maria I. Zanor, and María V. Busi Copyright © 2013 Diego F. Gomez-Casati et al. All rights reserved. Cloning and Characterization of EF-Tu and EF-Ts from Pseudomonas aeruginosa Mon, 05 Aug 2013 09:01:17 +0000 We have cloned genes encoding elongation factors EF-Tu and EF-Ts from Pseudomonas aeruginosa and expressed and purified the proteins to greater than 95% homogeneity. Sequence analysis indicated that P. aeruginosa EF-Tu and EF-Ts are 84% and 55% identical to E. coli counterparts, respectively. P. aeruginosa EF-Tu was active when assayed in GDP exchange assays. Kinetic parameters for the interaction of EF-Tu with GDP in the absence of EF-Ts were observed to be = 33 μM, = 0.003 s−1, and the specificity constant was  s−1 μM−1. In the presence of EF-Ts, these values were shifted to = 2 μM, = 0.005 s−1, and the specificity constant was  s−1 μM−1. The equilibrium dissociation constants governing the binding of EF-Tu to GDP () were 30–75 nM and to GTP () were 125–200 nM. EF-Ts stimulated the exchange of GDP by EF-Tu 10-fold. P. aeruginosa EF-Tu was active in forming a ternary complex with GTP and aminoacylated tRNA and was functional in poly(U)-dependent binding of Phe-tRNAPhe at the A-site of P. aeruginosa ribosomes. P. aeruginosa EF-Tu was active in poly(U)-programmed polyphenylalanine protein synthesis system composed of all P. aeruginosa components. Stephanie O. Palmer, Edna Y. Rangel, Alberto E. Montalvo, Alexis T. Tran, Kate C. Ferguson, and James M. Bullard Copyright © 2013 Stephanie O. Palmer et al. All rights reserved. Preparation, Physicochemical Characterization, and Cell Viability Evaluation of Long-Circulating and pH-Sensitive Liposomes Containing Ursolic Acid Sun, 04 Aug 2013 10:52:09 +0000 Cancer is one of the leading causes of death worldwide. Although several drugs are used clinically, some tumors either do not respond or are resistant to the existing pharmacotherapy, thus justifying the search for new drugs. Ursolic acid (UA) is a triterpene found in different plant species that has been shown to possess significant antitumor activity. However, UA presents a low solubility in aqueous medium, which presents a barrier to its biological applications. In this context, the use of liposomes presents a promising strategy to deliver UA and allow for its intravenous administration. In this work, long-circulating and pH-sensitive liposomes containing UA (SpHL-UA) were developed, and their chemical and physicochemical properties were evaluated. SpHL-UA presented adequate properties, including a mean diameter of 191.1 ± 6.4 nm, a zeta potential of 1.2 ± 1.4 mV, and a UA entrapment of 0.77 ± 0.01 mg/mL. Moreover, this formulation showed a good stability after having been stored for 2 months at 4°C. The viability studies on breast (MDA-MB-231) and prostate (LNCaP) cancer cell lines demonstrated that SpHL-UA treatment significantly inhibited cancer cell proliferation. Therefore, the results of the present work suggest the applicability of SpHL-UA as a new and promising anticancer formulation. Sávia Caldeira de Araújo Lopes, Marcus Vinícius Melo Novais, Cláudia Salviano Teixeira, Kinulpe Honorato-Sampaio, Márcio Tadeu Pereira, Lucas Antônio Miranda Ferreira, Fernão Castro Braga, and Mônica Cristina Oliveira Copyright © 2013 Sávia Caldeira de Araújo Lopes et al. All rights reserved. Efficiency of Barley Bran and Oat Bran in Ameliorating Blood Lipid Profile and the Adverse Histological Changes in Hypercholesterolemic Male Rats Thu, 01 Aug 2013 12:24:25 +0000 The efficiency of oat bran and barley bran in lowering the induced hyperlipidemia and hypercholesterolemia in blood of male Albino rats (Rattus rattus) was studied. Twenty rats were divided into four groups each consisted of five rats and fed the specified test diets for eight weeks. The first group (G1) is the negative group which was fed basal diet, the second group (G2) was fed 1.0% cholesterol, was the third group (G3) fed 1.0% cholesterol and 10% oats bran, and the fourth group (G4) was fed 1.0% cholesterol and 10% barley bran. Feeding rats on 1% cholesterol significantly increased serum total cholesterol, low density lipoprotein, and very low density lipoprotein and triglyceride and decreased serum high density lipoprotein. Furthermore, enzyme activity of alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase was increased, and lipid peroxide was increased, whereas catalase and glutathione-S-transferase were decreased. Kidney functions parameters in the cholesterol supplemented group were elevated compared with the negative control. In addition, histological alteration in kidney, liver, heart, and testes was observed, compared with the negative control. Hypercholesterolemic rats supplemented with oat bran and barley bran showed significant decrease in lipid parameters, significant increase in high density lipoprotein-cholesterol, improved antioxidant enzyme, and improved histopathology of kidney, liver, heart, and testes. In conclusion, both oat bran and barley bran had protective effects against induced hyperlipidemia and improved histological alterations. Oat bran appeared more efficient than barley bran in lowering the lipid profile levels in hypercholesterolemic rats. Haddad A. El Rabey, Madeha N. Al-Seeni, and Hanan M. Amer Copyright © 2013 Haddad A. El Rabey et al. All rights reserved. Asymmetry of the Active Site Loop Conformation between Subunits of Glutamate-1-semialdehyde Aminomutase in Solution Wed, 31 Jul 2013 12:55:44 +0000 Glutamate-1-semialdehyde aminomutase (GSAM) is a dimeric, pyridoxal 5′-phosphate (PLP)- dependent enzyme catalysing in plants and some bacteria the isomerization of L-glutamate-1-semialdehyde to 5-aminolevulinate, a common precursor of chlorophyll, haem, coenzyme B12, and other tetrapyrrolic compounds. During the catalytic cycle, the coenzyme undergoes conversion from pyridoxamine 5′-phosphate (PMP) to PLP. The entrance of the catalytic site is protected by a loop that is believed to switch from an open to a closed conformation during catalysis. Crystallographic studies indicated that the structure of the mobile loop is related to the form of the cofactor bound to the active site, allowing for asymmetry within the dimer. Since no information on structural and functional asymmetry of the enzyme in solution is available in the literature, we investigated the active site accessibility by determining the cofactor fluorescence quenching of PMP- and PLP-GSAM forms. PLP-GSAM is partially quenched by potassium iodide, suggesting that at least one catalytic site is accessible to the anionic quencher and therefore confirming the asymmetry observed in the crystal structure. Iodide induces release of the cofactor from PMP-GSAM, apparently from only one catalytic site, therefore suggesting an asymmetry also in this form of the enzyme in solution, in contrast with the crystallographic data. Barbara Campanini, Stefano Bettati, Martino Luigi di Salvo, Andrea Mozzarelli, and Roberto Contestabile Copyright © 2013 Barbara Campanini et al. All rights reserved. Nutritional Properties of Dietary Omega-3-Enriched Phospholipids Wed, 31 Jul 2013 08:58:22 +0000 Dietary fatty acids regulate several physiological functions. However, to exert their properties, they have to be present in the diet in an optimal balance. Particular attention has been focused on tissue highly polyunsaturated fatty acids (HPUFAs) n-6/n-3 ratio, influenced by the type and the esterified form of dietary fatty acids. Dietary EPA and DHA when esterified to phospholipids (PLs) are more efficiently incorporated into tissue PLs and seem to possess peculiar properties through specific mechanism(s) of action, such as the capacity to affect endocannabinoid biosynthesis at much lower doses than EPA and DHA in triglyceride form, probably because of the above mentioned higher incorporation into tissue PLs. Downregulation of the endocannabinoid system seems to mediate the positive effects exerted by omega-3-enriched PLs on several parameters of metabolic syndrome. PLs are one of the major dietary forms of EPA and DHA we are exposed to with the everyday diet; therefore, it is not surprising that it guarantees an effective EPA and DHA nutritional activity. Future studies should address whether EPA and DHA in PL form are also more effective than other formulations in ameliorating other pathological conditions where n-3 HPUFAs seem to exert beneficial activities such as cancer and psychiatric disorders. Elisabetta Murru, Sebastiano Banni, and Gianfranca Carta Copyright © 2013 Elisabetta Murru et al. All rights reserved. High Glucose-Induced Oxidative Stress Increases the Copy Number of Mitochondrial DNA in Human Mesangial Cells Tue, 30 Jul 2013 08:21:14 +0000 Oxidative damage to mitochondrial DNA (mtDNA) has been linked to the pathogenicity of diabetic nephropathy. We tested the hypothesis that mtDNA copy number may be increased in human mesangial cells in response to high glucose-induced reactive oxygen species (ROS) to compensate for damaged mtDNA. The effect of manganese superoxide dismutase mimetic (MnTBAP) on glucose-induced mtDNA copy number was also examined. The copy number of mtDNA was determined by real-time PCR in human mesangial cells cultured in 5 mM glucose, 25 mM glucose, and mannitol (osmotic control), as well as in cells cultured in 25 mM glucose in the presence and absence of 200 μM MnTBAP. Intracellular ROS was assessed by confocal microscopy and flow cytometry in human mesangial cells. The copy number of mtDNA was significantly increased when human mesangial cells were incubated with 25 mM glucose compared to 5 mM glucose and mannitol. In addition, 25 mM glucose rapidly generated ROS in the cells, which was not detected in 5 mM glucose. Furthermore, mtDNA copy number was significantly decreased and maintained to normal following treatment of cells with 25 mM glucose and MnTBAP compared to 25 mM glucose alone. Inclusion of MnTBAP during 25 mM glucose incubation inhibited mitochondrial superoxide in human mesangial cells. Increased mtDNA copy number in human mesangial cells by high glucose could contribute to increased mitochondrial superoxide, and prevention of mtDNA copy number could have potential in retarding the development of diabetic nephropathy. Ghada Al-Kafaji and Jamal Golbahar Copyright © 2013 Ghada Al-Kafaji and Jamal Golbahar. All rights reserved. Spectroscopic Studies on Unfolding Processes of Apo-Neuroglobin Induced by Guanidine Hydrochloride and Urea Wed, 24 Jul 2013 11:13:53 +0000 Neuroglobin (Ngb), a recently discovered globin, is predominantly expressed in the brain, retina, and other nerve tissues of vertebrates. The unfolding processes of apo-neuroglobin (apoNgb) induced by guanidine hydrochloride (GdnHCl) and urea were investigated by spectroscopic methods. In the unfolding processes, apoNgb's tertiary structural transition was monitored by the changes of intrinsic fluorescence emission spectra, and its secondary structural transition was measured by the changes of far-ultraviolet circular dichroism (CD) spectra. In addition, 8-anilino-1-naphthalenesulfonic acid (ANS), a hydrophobic cluster binding dye, was also used to monitor the unfolding process of apoNgb and to explore its intermediates. Results showed that GdnHCl-induced unfolding of apoNgb was via a three-state pathway, that is, Native state → Intermediate state → Unfolded state , during which the intermediate was inferred by an increase in fluorescence intensity and the change of CD value. Gibbs free energy changes are 10.2 kJ·mol−1 for the first unfolding transition and 14.0 kJ·mol−1 for the second transition. However, urea-induced unfolding of apoNgb only underwent a two-state transition: Native state → Partially unfolded state . The result showed that GdnHCl can efficiently affect the conformational states of apoNgb compared with those of urea. The work will benefit to have an understanding of the unfolding mechanism of apoNgb induced by GdnHCl and urea. Cui Zhang, Chaohui Gao, Jianshuai Mu, Zhanglei Qiu, and Lianzhi Li Copyright © 2013 Cui Zhang et al. All rights reserved. Age-Related Changes in Hepatic Activity and Expression of Detoxification Enzymes in Male Rats Mon, 22 Jul 2013 08:45:12 +0000 Process of aging is accompanied by changes in the biotransformation of xenobiotics and impairment of normal cellular functions by free radicals. Therefore, this study was designed to determine age-related differences in the activities and/or expressions of selected drug-metabolizing and antioxidant enzymes in young and old rats. Specific activities of 8 drug-metabolizing enzymes and 4 antioxidant enzymes were assessed in hepatic subcellular fractions of 6-week-old and 21-month-old male Wistar rats. Protein expressions of carbonyl reductase 1 (CBR1) and glutathione S-transferase (GST) were determined using immunoblotting. Remarkable age-related decrease in specific activities of CYP2B, CYP3A, and UDP-glucuronosyl transferase was observed, whereas no changes in activities of CYP1A2, flavine monooxygenase, aldo-keto reductase 1C, and antioxidant enzymes with advancing age were found. On the other hand, specific activity of CBR1 and GST was 2.4 folds and 5.6 folds higher in the senescent rats compared with the young ones, respectively. Interindividual variability in CBR1 activity increased significantly with rising age. We suppose that elevated activities of GST and CBR1 may protect senescent rats against xenobiotic as well as eobiotic electrophiles and reactive carbonyls, but they may alter metabolism of drugs, which are CBR1 and especially GSTs substrates. Erika Vyskočilová, Barbora Szotáková, Lenka Skálová, Hana Bártíková, Jitka Hlaváčová, and Iva Boušová Copyright © 2013 Erika Vyskočilová et al. All rights reserved. A Comparative Study for the Evaluation of Two Doses of Ellagic Acid on Hepatic Drug Metabolizing and Antioxidant Enzymes in the Rat Thu, 18 Jul 2013 11:32:10 +0000 The present study was designed to evaluate different doses of ellagic acid (EA) in vivo in rats for its potential to modulate hepatic phases I, II, and antioxidant enzymes. EA (10 or 30 mg/kg/day, intragastrically) was administered for 14 consecutive days, and activity, protein, and mRNA levels were determined. Although the cytochrome P450 (CYP) 2B and CYP2E enzyme activities were decreased significantly, the activities of all other enzymes were unchanged with the 10 mg/kg/day EA. In addition, western-blot and qRT-PCR results clearly corroborated the above enzyme expressions. On the other hand, while the NAD(P)H:quinone oxidoreductase 1 (NQO1), catalase (CAT), glutathione peroxidase (GPX), and glutathione S-transferase (GST) activities were increased significantly, CYP1A, 2B, 2C, 2E, and 19 enzyme activities were reduced significantly with 30 mg/kg/day EA. In addition, CYP2B, 2C6, 2E1, and 19 protein and mRNA levels were substantially decreased by the 30 mg/kg/day dose of EA, but the CYP1A protein, and mRNA levels were not changed. CYP3A enzyme activity, protein and mRNA levels were not altered by neither 10 nor 30 mg/kg/day ellagic acid. These results indicate that EA exerts a dose-dependent impact on the metabolism of chemical carcinogens and drugs by affecting the enzymes involved in xenobiotics activation/detoxification and antioxidant pathways. Gurbet Celik, Aslı Semiz, Serdar Karakurt, Sevki Arslan, Orhan Adali, and Alaattin Sen Copyright © 2013 Gurbet Celik et al. All rights reserved. Protein Homeostasis Defects of Alanine-Glyoxylate Aminotransferase: New Therapeutic Strategies in Primary Hyperoxaluria Type I Tue, 16 Jul 2013 12:47:03 +0000 Alanine-glyoxylate aminotransferase catalyzes the transamination between L-alanine and glyoxylate to produce pyruvate and glycine using pyridoxal 5′-phosphate (PLP) as cofactor. Human alanine-glyoxylate aminotransferase is a peroxisomal enzyme expressed in the hepatocytes, the main site of glyoxylate detoxification. Its deficit causes primary hyperoxaluria type I, a rare but severe inborn error of metabolism. Single amino acid changes are the main type of mutation causing this disease, and considerable effort has been dedicated to the understanding of the molecular consequences of such missense mutations. In this review, we summarize the role of protein homeostasis in the basic mechanisms of primary hyperoxaluria. Intrinsic physicochemical properties of polypeptide chains such as thermodynamic stability, folding, unfolding, and misfolding rates as well as the interaction of different folding states with protein homeostasis networks are essential to understand this disease. The view presented has important implications for the development of new therapeutic strategies based on targeting specific elements of alanine-glyoxylate aminotransferase homeostasis. Angel L. Pey, Armando Albert, and Eduardo Salido Copyright © 2013 Angel L. Pey et al. All rights reserved. The Glycosylation of AGP and Its Associations with the Binding to Methadone Mon, 15 Jul 2013 13:55:50 +0000 Methadone remains the most common form of pharmacological therapy for opioid dependence; however, there is a lack of explanation for the reports of its relatively low success rate in achieving complete abstinence. One hypothesis is that in vivo binding of methadone to the plasma glycoprotein alpha-1-acid glycoprotein (AGP), to a degree dependent on the molecular structure, may render the drug inactive. This study sought to determine whether alterations present in the glycosylation pattern of AGP in patients undergoing various stages of methadone therapy (titration < two weeks, harm reduction < one year, long-term > one and a half years) could affect the affinity of the glycoprotein to bind methadone. The composition of AGP glycosylation was determined using high pH anion exchange chromatography (HPAEC) and intrinsic fluorescence analysed to determine the extent of binding to methadone. The monosaccharides galactose and N-acetyl-glucosamine were elevated in all methadone treatment groups indicating alterations in AGP glycosylation. AGP from all patients receiving methadone therapy exhibited a greater degree of binding than the normal population. This suggests that analysing the glycosylation of AGP in patients receiving methadone may aid in determining whether the therapy is likely to be effective. Jennifer L. Behan, Yvonne E. Cruickshank, Gerri Matthews-Smith, Malcolm Bruce, and Kevin D. Smith Copyright © 2013 Jennifer L. Behan et al. All rights reserved. Structure-Based Mechanism for Early PLP-Mediated Steps of Rabbit Cytosolic Serine Hydroxymethyltransferase Reaction Mon, 15 Jul 2013 11:30:51 +0000 Serine hydroxymethyltransferase catalyzes the reversible interconversion of L-serine and glycine with transfer of one-carbon groups to and from tetrahydrofolate. Active site residue Thr254 is known to be involved in the transaldimination reaction, a crucial step in the catalytic mechanism of all pyridoxal 5′-phosphate- (PLP-) dependent enzymes, which determines binding of substrates and release of products. In order to better understand the role of Thr254, we have expressed, characterized, and determined the crystal structures of rabbit cytosolic serine hydroxymethyltransferase T254A and T254C mutant forms, in the absence and presence of substrates. These mutants accumulate a kinetically stable gem-diamine intermediate, and their crystal structures show differences in the active site with respect to wild type. The kinetic and crystallographic data acquired with mutant enzymes permit us to infer that conversion of gem-diamine to external aldimine is significantly slowed because intermediates are trapped into an anomalous position by a misorientation of the PLP ring, and a new energy barrier hampers the transaldimination reaction. This barrier likely arises from the loss of the stabilizing hydrogen bond between the hydroxymethyl group of Thr254 and the ε-amino group of active site Lys257, which stabilizes the external aldimine intermediate in wild type SHMTs. Martino L. Di Salvo, J. Neel Scarsdale, Galina Kazanina, Roberto Contestabile, Verne Schirch, and H. Tonie Wright Copyright © 2013 Martino L. Di Salvo et al. All rights reserved. Enhanced EGFP Fluorescence Emission in Presence of PEG Aqueous Solutions and -- Copolymer Vesicles Wed, 10 Jul 2013 08:48:45 +0000 An EGFP construct interacting with the PIB1000-PEG6000-PIB1000 vesicles surface reported a ~2-fold fluorescence emission enhancement. Because of the constructs nature with the amphiphilic peptide inserted into the PIB core, EGFP is expected to experience a “pure” PEG environment. To unravel this phenomenon PEG/water solutions at different molecular weights and concentrations were used. Already at ~1 : 10 protein/PEG molar ratio the increase in fluorescence emission is observed reaching a plateau correlating with the PEG molecular weight. Parallel experiments in presence of glycerol aqueous solutions did show a slight fluorescence enhancement however starting at much higher concentrations. Molecular dynamics simulations of EGFP in neat water, glycerol, and PEG aqueous solutions were performed showing that PEG molecules tend to “wrap” the protein creating a microenvironment where the local PEG concentration is higher compared to its bulk concentration. Because the fluorescent emission can be perturbed by the refractive index surrounding the protein, the clustering of PEG molecules induces an enhanced fluorescence emission already at extremely low concentrations. These findings can be important when related to the use of EGFP as reported in molecular biology experiments. Noor Muhammad, Nadezda Kryuchkova, Tamara Dworeck, Francisco Rodríguez-Ropero, and Marco Fioroni Copyright © 2013 Noor Muhammad et al. All rights reserved. Clinical Application Neutrophil Gelatinase-Associated Lipocalin and Kidney Injury Molecule-1 as Indicators of Inflammation Persistence and Acute Kidney Injury in Children with Urinary Tract Infection Tue, 09 Jul 2013 13:38:19 +0000 Background. The aim of this study was to examine the novel renal biomarkers neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) to assist pediatricians in the assessment of longer duration of inflammation and acute kidney injury (AKI) development during urinary tract infection (UTI). Methods. The patients enrolled in the study comprised 50 children (mean age was 6 months) with UTI. NGAL in serum and urine (sNGAL and uNGAL, resp.) and KIM-1 in urine were measured by enzyme-linked immunosorbent assays. Results. uNGAL levels in subjects with longer duration of inflammation were higher (115.37 ng/mL) than uNGAL levels in subjects with shorter duration of inflammation (67.87 ng/mL, ). Difference in sNGAL and KIM-1 levels was not significant ( and , resp.). Significant difference was seen in KIM-1 excretion among groups with and without AKI (). KIM-1 was not able to discriminate between subjects with and without AKI (area under the curves (AUC) = 0.620, ). Conclusions. uNGAL cannot be used for screening of the duration of inflammation during UTI. Accuracy of KIM-1 in screening of AKI development in children with UTI is low. We suggest larger studies to check the negative predictive value of KIM-1 for the development of AKI. Stanislava Petrovic, Natasa Bogavac-Stanojevic, Amira Peco-Antic, Ivana Ivanisevic, Jelena Kotur-Stevuljevic, Dusan Paripovic, Miron Sopic, and Zorana Jelic-Ivanovic Copyright © 2013 Stanislava Petrovic et al. All rights reserved. The Role of Magnetic Nanoparticles in the Localization and Treatment of Breast Cancer Tue, 09 Jul 2013 10:53:03 +0000 The role of magnetic nanoparticles (MNPs) in medical applications is rapidly developing. Advances in nanotechnology are bringing us closer to the development of dual and multifunctional nanoparticles that are challenging the traditional distinction between diagnostic and treatment agents. The current use of MNPs in breast cancer falls into four main groups: (1) imaging of primary and metastatic disease, (2) sentinel lymph node biopsy (SLNB), (3) drug delivery systems, and (4) magnetic hyperthermia. The current evidence for the use of MNPs in these fields is mounting, and potential cutting-edge clinical applications, particularly with relevance to the fields of breast oncological surgery, are emerging. M. Ahmed and M. Douek Copyright © 2013 M. Ahmed and M. Douek. All rights reserved. Cytotoxicity of Biologically Synthesized Silver Nanoparticles in MDA-MB-231 Human Breast Cancer Cells Mon, 08 Jul 2013 15:04:47 +0000 Silver nanoparticles (AgNPs) have been used as an antimicrobial and disinfectant agents. However, there is limited information about antitumor potential. Therefore, this study focused on determining cytotoxic effects of AgNPs on MDA-MB-231 breast cancer cells and its mechanism of cell death. Herein, we developed a green method for synthesis of AgNPs using culture supernatant of Bacillus funiculus, and synthesized AgNPs were characterized by various analytical techniques such as UV-visible spectrophotometer, particle size analyzer, and transmission electron microscopy (TEM). The toxicity was evaluated using cell viability, metabolic activity, and oxidative stress. MDA-MB-231 breast cancer cells were treated with various concentrations of AgNPs (5 to 25 μg/mL) for 24 h. We found that AgNPs inhibited the growth in a dose-dependent manner using MTT assay. AgNPs showed dose-dependent cytotoxicity against MDA-MB-231 cells through activation of the lactate dehydrogenase (LDH), caspase-3, reactive oxygen species (ROS) generation, eventually leading to induction of apoptosis which was further confirmed through resulting nuclear fragmentation. The present results showed that AgNPs might be a potential alternative agent for human breast cancer therapy. Sangiliyandi Gurunathan, Jae Woong Han, Vasuki Eppakayala, Muniyandi Jeyaraj, and Jin-Hoi Kim Copyright © 2013 Sangiliyandi Gurunathan et al. All rights reserved. Metabolomic Analysis of Differential Changes in Metabolites during ATP Oscillations in Chondrogenesis Wed, 26 Jun 2013 08:46:29 +0000 Prechondrogenic condensation is a critical step for skeletal pattern formation. Recent studies reported that ATP oscillations play an essential role in prechondrogenic condensation. However, the molecular mechanism to underlie ATP oscillations remains poorly understood. In the present study, it was investigated how changes in metabolites are implicated in ATP oscillations during chondrogenesis by using capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS). CE-TOF-MS detected 93 cationic and 109 anionic compounds derived from known metabolic pathways. 15 cationic and 18 anionic compounds revealed significant change between peak and trough of ATP oscillations. These results implicate that glycolysis, mitochondrial respiration and uronic acid pathway oscillate in phase with ATP oscillations, while PPRP and nucleotides synthesis pathways oscillate in antiphase with ATP oscillations. This suggests that the ATP-producing glycolysis and mitochondrial respiration oscillate in antiphase with the ATP-consuming PPRP/nucleotide synthesis pathway during chondrogenesis. Hyuck Joon Kwon and Yoshihiro Ohmiya Copyright © 2013 Hyuck Joon Kwon and Yoshihiro Ohmiya. All rights reserved. Omega-3 Fatty Acids Inhibit Tumor Growth in a Rat Model of Bladder Cancer Thu, 20 Jun 2013 08:23:45 +0000 Omega-3 (-3) fatty acids have been tested on prevention and treatment of several cancer types, but the efficacy on “in vivo” bladder cancer has not been analyzed yet. This study aimed at evaluating the chemopreventive efficacy of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) mixture in an animal model of bladder cancer. Forty-four male Wistar rats were divided into 4 groups during a 20-week protocol: control; carcinogen—N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN); -3 (DHA + EPA); and -3 + BBN. BBN and -3 were given during the initial 8 weeks. At week 20 blood and bladder were collected and checked for the presence of urothelium lesions and tumors, markers of inflammation, proliferation, and redox status. Incidence of bladder carcinoma was, control (0%), -3 (0%), BBN (65%), and -3 + BBN (62.5%). The -3 + BBN group had no infiltrative tumors or carcinoma in situ, and tumor volume was significantly reduced compared to the BBN (0.9 ± 0.1 mm3 versus 112.5 ± 6.4 mm3). Also, it showed a reduced MDA/TAS ratio and BBN-induced serum CRP, TGF-β1, and CD31 were prevented. In conclusion, omega-3 fatty acids inhibit the development of premalignant and malignant lesions in a rat model of bladder cancer, which might be due to anti-inflammatory, antioxidant, anti-proliferative, and anti-angiogenic properties. Belmiro Parada, Flávio Reis, Raquel Cerejo, Patrícia Garrido, José Sereno, Maria Xavier-Cunha, Paula Neto, Alfredo Mota, Arnaldo Figueiredo, and Frederico Teixeira Copyright © 2013 Belmiro Parada et al. All rights reserved. Recent Developments in Nanoparticle-Based siRNA Delivery for Cancer Therapy Mon, 17 Jun 2013 18:44:47 +0000 RNA interference (RNAi) is a gene regulation mechanism initiated by RNA molecules that enables sequence-specific gene silencing by promoting degradation of specific mRNAs. Molecular therapy using small interfering RNA (siRNA) has shown great therapeutic potential for diseases caused by abnormal gene overexpression or mutation. The major challenges to application of siRNA therapeutics include the stability and effective delivery of siRNA in vivo. Important progress in nanotechnology has led to the development of efficient siRNA delivery systems. In this review, the authors discuss recent advances in nanoparticle-mediated siRNA delivery and the application of siRNA in clinical trials for cancer therapy. This review will also offer perspectives on future applications of siRNA therapeutics. Jong-Min Lee, Tae-Jong Yoon, and Young-Seok Cho Copyright © 2013 Jong-Min Lee et al. All rights reserved. Extremophilic SHMTs: From Structure to Biotechnology Thu, 13 Jun 2013 13:05:10 +0000 Recent advances in molecular and structural biology have improved the availability of virtually any biocatalyst in large quantity and have also provided an insight into the detailed structure-function relationships of many of them. These results allowed the rational exploitation of biocatalysts for use in organic synthesis. In this context, extremophilic enzymes are extensively studied for their potential interest for many biotechnological and industrial applications, as they offer increased rates of reactions, higher substrate solubility, and/or longer enzyme half-lives at the conditions of industrial processes. Serine hydroxymethyltransferase (SHMT), for its ubiquitous nature, represents a suitable model for analyzing enzyme adaptation to extreme environments. In fact, many SHMT sequences from Eukarya, Eubacteria and Archaea are available in data banks as well as several crystal structures. In addition, SHMT is structurally conserved because of its critical metabolic role; consequently, very few structural changes have occurred during evolution. Our research group analyzed the molecular basis of SHMT adaptation to high and low temperatures, using experimental and comparative in silico approaches. These structural and functional studies of SHMTs purified from extremophilic organisms can help to understand the peculiarities of the enzyme activity at extreme temperatures, indicating possible strategies for rational enzyme engineering. Sebastiana Angelaccio Copyright © 2013 Sebastiana Angelaccio. All rights reserved. The Omega-3 Polyunsaturated Fatty Acid DHA Induces Simultaneous Apoptosis and Autophagy via Mitochondrial ROS-Mediated Akt-mTOR Signaling in Prostate Cancer Cells Expressing Mutant p53 Mon, 10 Jun 2013 08:27:54 +0000 Docosahexaenoic acid (DHA) induces autophagy-associated apoptotic cell death in wild-type p53 cancer cells via regulation of p53. The present study investigated the effects of DHA on PC3 and DU145 prostate cancer cell lines harboring mutant p53. Results show that, in addition to apoptosis, DHA increased the expression levels of lipidated form LC3B and potently stimulated the autophagic flux, suggesting that DHA induces both autophagy and apoptosis in cancer cells expressing mutant p53. DHA led to the generation of mitochondrial reactive oxygen species (ROS), as shown by the mitochondrial ROS-specific probe mitoSOX. Similarly, pretreatment with the antioxidant N-acetyl-cysteine (NAC) markedly inhibited both the autophagy and the apoptosis triggered by DHA, indicating that mitochondrial ROS mediate the cytotoxicity of DHA in mutant p53 cells. Further, DHA reduced the levels of phospho-Akt and phospho-mTOR in a concentration-dependent manner, while NAC almost completely blocked that effect. Collectively, these findings present a novel mechanism of ROS-regulated apoptosis and autophagy that involves Akt-mTOR signaling in prostate cancer cells with mutant p53 exposed to DHA. Soyeon Shin, Kaipeng Jing, Soyeon Jeong, Nayeong Kim, Kyoung-Sub Song, Jun-Young Heo, Ji-Hoon Park, Kang-Sik Seo, Jeongsu Han, Jong-Il Park, Gi-Ryang Kweon, Seung-Kiel Park, Tong Wu, Byung-Doo Hwang, and Kyu Lim Copyright © 2013 Soyeon Shin et al. All rights reserved. Advances in Molecular Diagnostics Mon, 27 May 2013 09:43:28 +0000 Tavan Janvilisri, Arun K. Bhunia, and Joy Scaria Copyright © 2013 Tavan Janvilisri et al. All rights reserved. Interaction of Human Dopa Decarboxylase with L-Dopa: Spectroscopic and Kinetic Studies as a Function of pH Sun, 26 May 2013 10:15:16 +0000 Human Dopa decarboxylase (hDDC), a pyridoxal 5′-phosphate (PLP) enzyme, displays maxima at 420 and 335 nm and emits fluorescence at 384 and 504 nm upon excitation at 335 nm and at 504 nm when excited at 420 nm. Absorbance and fluorescence titrations of hDDC-bound coenzyme identify a single of ~7.2. This could not represent the ionization of a functional group on the Schiff base but that of an enzymic residue governing the equilibrium between the low- and the high-pH forms of the internal aldimine. During the reaction of hDDC with L-Dopa, monitored by stopped-flow spectrophotometry, a 420 nm band attributed to the 4′-N-protonated external aldimine first appears, and its decrease parallels the emergence of a 390 nm peak, assigned to the 4′-N-unprotonated external aldimine. The pH profile of the spectral change at 390 nm displays a pK of 6.4, a value similar to that (~6.3) observed in both and profiles. This suggests that this pK represents the ESH+ → ES catalytic step. The assignment of the pKs of 7.9 and 8.3 observed on the basic side of and the PLP binding affinity profiles, respectively, is also analyzed and discussed. Riccardo Montioli, Barbara Cellini, Mirco Dindo, Elisa Oppici, and Carla Borri Voltattorni Copyright © 2013 Riccardo Montioli et al. All rights reserved. Mechanisms of Omega-3 Polyunsaturated Fatty Acids in Prostate Cancer Prevention Thu, 23 May 2013 16:27:22 +0000 This review focuses on several key areas where progress has been made recently to highlight the role of omega-3 polyunsaturated fatty acid in prostate cancer prevention. Zhennan Gu, Janel Suburu, Haiqin Chen, and Yong Q. Chen Copyright © 2013 Zhennan Gu et al. All rights reserved. Dietary ω-3 Polyunsaturated Fatty Acid DHA: A Potential Adjuvant in the Treatment of Cancer Thu, 23 May 2013 15:52:33 +0000 ω-3 Polyunsaturated fatty acids (PUFAs), mainly present in fish oil, are part of the human diet. Among PUFAs, docosahexaenoic acid (DHA) has received particular attention for its anti-inflammatory, antiproliferative, proapoptotic, antiangiogenetic, anti-invasion, and antimetastatic properties. These data suggest that DHA can exert antitumor activity potentially representing an effective adjuvant in cancer chemotherapy. This review is focused on current knowledge supporting the potential use of DHA for the enhancement of the efficacy of anticancer treatments in relation to its ability to enhance the uptake of anticancer drugs, regulate the oxidative status of tumor cells, and inhibit tumor cell invasion and metastasis. Nicolò Merendino, Lara Costantini, Laura Manzi, Romina Molinari, Donatella D'Eliseo, and Francesca Velotti Copyright © 2013 Nicolò Merendino et al. All rights reserved. Laboratory Medicine Tue, 14 May 2013 10:38:31 +0000 Mina Hur, Andrew St. John, and Antonio La Gioia Copyright © 2013 Mina Hur et al. All rights reserved. Oxidation of Marine Omega-3 Supplements and Human Health Tue, 30 Apr 2013 16:26:57 +0000 Marine omega-3 rich oils are used by more than a third of American adults for a wide range of purported benefits including prevention of cardiovascular disease. These oils are highly prone to oxidation to lipid peroxides and other secondary oxidation products. Oxidized oils may have altered biological activity making them ineffective or harmful, though there is also evidence that some beneficial effects of marine oils could be mediated through lipid peroxides. To date, human clinical trials have not reported the oxidative status of the trial oil. This makes it impossible to understand the importance of oxidation to efficacy or harm. However, animal studies show that oxidized lipid products can cause harm. Oxidation of trial oils may be responsible for the conflicting omega-3 trial literature, including the prevention of cardiovascular disease. The oxidative state of an oil can be simply determined by the peroxide value and anisidine value assays. We recommend that all clinical trials investigating omega-3 harms or benefits report the results of these assays; this will enable better understanding of the benefits and harms of omega-3 and the clinical importance of oxidized supplements. Benjamin B. Albert, David Cameron-Smith, Paul L. Hofman, and Wayne S. Cutfield Copyright © 2013 Benjamin B. Albert et al. All rights reserved. Effect of Tea (Camellia sinensis) and Olive (Olea europaea L.) Leaves Extracts on Male Mice Exposed to Diazinon Thu, 18 Apr 2013 08:44:58 +0000 The present study was aimed to evaluate the effects of tea and olive leaves extracts and their combination in male mice intoxicated with a sublethal concentration of diazinon. Exposure of mice to 6.5 mg/kg body weight of diazinon for seven weeks resulted in statistical increases of serum alanine aminotransferase, aspartate aminotransferase, gamma glutamyl transferase, alkaline phosphatase, creatine kinase, creatinine, glucose, triglycerides, and cholesterol, while the value of serum total protein was declined. Treating diazinon-intoxicated mice with tea and olive leaves extracts or their combination significantly attenuated the severe alterations in these hematobiochemical parameters. Moreover, the results indicated that the supplementation with combination of tea and olive leaves extracts led to more attenuation effect against diazinon toxicity. Additionally, these new findings suggest that the effect of tea and olive leaves extracts and their combination against toxicity of diazinon may be due to antioxidant properties of their chemical constituents. Finally, the present study indicated that the extracts of tea and olive leaves and their combination can be considered as promising therapeutic agents against hepatotoxicity, cardiotoxicity, nephrotoxicity, and metabolic disorders induced by diazinon and maybe by other toxicants and pathogenic factors. Atef M. Al-Attar and Isam M. Abu Zeid Copyright © 2013 Atef M. Al-Attar and Isam M. Abu Zeid. All rights reserved. Experimental Evidence of -3 Polyunsaturated Fatty Acid Modulation of Inflammatory Cytokines and Bioactive Lipid Mediators: Their Potential Role in Inflammatory, Neurodegenerative, and Neoplastic Diseases Wed, 17 Apr 2013 20:19:08 +0000 A large body of evidence has emerged over the past years to show the critical role played by inflammation in the pathogenesis of several diseases including some cardiovascular, neoplastic, and neurodegenerative diseases, previously not considered inflammation-related. The anti-inflammatory action of -3 polyunsaturated fatty acids (PUFAs), as well as their potential healthy effects against the development and progression of the same diseases, has been widely studied by our and others’ laboratories. As a result, a rethinking is taking place on the possible mechanisms underlying the beneficial effects of -3 PUFAs against these disorders, and, in particular, on the influence that they may exert on the molecular pathways involved in inflammatory process, including the production of inflammatory cytokines and lipid mediators active in the resolving phase of inflammation. In the present review we will summarize and discuss the current knowledge regarding the modulating effects of -3 PUFAs on the production of inflammatory cytokines and proresolving or protective lipid mediators in the context of inflammatory, metabolic, neurodegenerative, and neoplastic diseases. Gabriella Calviello, Hui-Min Su, Karsten H. Weylandt, Elena Fasano, Simona Serini, and Achille Cittadini Copyright © 2013 Gabriella Calviello et al. All rights reserved. Chemical Composition, Nutritive Value, and Toxicological Evaluation of Bauhinia cheilantha Seeds: A Legume from Semiarid Regions Widely Used in Folk Medicine Wed, 17 Apr 2013 11:42:45 +0000 Among the Bauhinia species, B. cheilantha stands out for its seed protein content. However, there is no record of its nutritional value, being used in a nonsustainable way in the folk medicine and for large-scale extraction of timber. The aim of this study was to investigate the food potential of B. cheilantha seeds with emphasis on its protein quality to provide support for flora conservation and use as raw material or as prototype for the development of bioproducts with high added socioeconomic value. B. cheilantha seeds show high protein content (35.9%), reasonable essential amino acids profile, low levels of antinutritional compounds, and nutritional parameters comparable to those of legumes widely used such as soybean and cowpea. The heat treatment of the seeds as well as the protein extraction process (to obtain the protein concentrate) increased the acceptance of diets by about 100% when compared to that of raw Bc diet. These wild legume seeds can be promising alternative source of food to overcome the malnutrition problem faced by low income people adding socioeconomic value to the species. Daniel Câmara Teixeira, Davi Felipe Farias, Ana Fontenele Urano Carvalho, Mariana Reis Arantes, José Tadeu Abreu Oliveira, Daniele Oliveira Bezerra Sousa, Mirella Leite Pereira, Hermogenes David Oliveira, Manoel Andrade-Neto, and Ilka Maria Vasconcelos Copyright © 2013 Daniel Câmara Teixeira et al. All rights reserved. Cardioprotective Effects of ω-3 PUFAs in Chronic Kidney Disease Thu, 04 Apr 2013 10:00:37 +0000 The prevalence rate of chronic kidney disease (CKD) is increasing worldwide, and cardiovascular disease (CVD) is a main cause of death in patients with CKD. The high incidence of CVD in CKD patients is related to chronic inflammation, dyslipidemia, malnutrition, atherosclerosis, and vascular calcification. Omega-3 polyunsaturated fatty acids (ω-3 PUFAs) have been shown to reduce the risk of CVD. In this paper, we review the beneficial effects of ω-3 PUFAs on CVD and the possible cardioprotective mechanisms of ω-3 PUFAs in CKD patients by determining the effect of ω-3 PUFAs in the general population. ω-3 PUFAs have several cardioprotective benefits, such as reducing inflammation, decreasing oxidative stress, inhibiting platelet activity, exerting antiarrhythmic effects, and improving triglyceride levels, in the general population and patients with CKD. Modifications of erythrocyte membrane fatty acid content, including an increased ω-3 index and decreased oleic acid, after ω-3 PUFAs supplementation are important changes related to CVD risk reduction in the general population and patients with CKD. Further basic and clinical studies are essential to confirm the effects of ω-3 PUFAs on vitamin D activation, vascular calcification prevention, cardiovascular events, and mortality in CKD patients. Su Mi Lee and Won Suk An Copyright © 2013 Su Mi Lee and Won Suk An. All rights reserved. Amylin Uncovered: A Review on the Polypeptide Responsible for Type II Diabetes Sun, 31 Mar 2013 10:18:19 +0000 Amylin is primarily responsible for classifying type II diabetes as an amyloid (protein misfolding) disease as it has great potential to aggregate into toxic nanoparticles, thereby resulting in loss of pancreatic -cells. Although type II diabetes is on the increase each year, possibly due to bad eating habits of modern society, research on the culprit for this disease is still in its early days. In addition, unlike the culprit for Alzheimer’s disease, amyloid -peptide, amylin has failed to receive attention worthy of being featured in an abundance of review articles. Thus, the aim of this paper is to shine the spotlight on amylin in an attempt to put it onto the top of researchers’ to-do list since the secondary complications of type II diabetes have far-reaching and severe consequences on public health both in developing and fully developed countries alike. This paper will cover characteristics of the amylin aggregates, mechanisms of toxicity, and a particular focus on inhibitors of toxicity and techniques used to assess these inhibitors. Karen Pillay and Patrick Govender Copyright © 2013 Karen Pillay and Patrick Govender. All rights reserved. Mean Platelet Volume Reflect Hematopoietic Potency and Correlated Blood Group O in Cord Blood from Healthy Newborn Wed, 27 Mar 2013 18:25:21 +0000 We evaluated the relationship between mean platelet volume (MPV) and characteristics of 10,577 cord blood (CB) units in a public CB bank in Korea. Blood group O has the highest MPV ( = 0.002). MPV correlated with CB volume (), Hb (), WBC (), TNCs (), CD34+ cell (), CD34+ cells/TNCs (), gestational age ( = −0.102), and birth weight (); ( in all). MPV may be one of the useful decision parameters of process priority in CB bank. Hye Ryun Lee, Jeong Su Park, Sue Shin, Eun Youn Roh, Jong Hyun Yoon, Eun Young Song, Byung Jae Kim, and Ju Young Chang Copyright © 2013 Hye Ryun Lee et al. All rights reserved. NGAL and Metabolomics: The Single Biomarker to Reveal the Metabolome Alterations in Kidney Injury Wed, 27 Mar 2013 10:55:22 +0000 Conditions affecting kidney structure and function can be considered acute or chronic, depending on their duration. Acute kidney injury (AKI) is one of a number of acute kidney diseases and consists of an abrupt decline in kidney function after an injury leading to functional and structural changes. The widespread availability of enabling technologies has accelerated the rate of novel biomarker discovery for kidney injury. The introduction of novel biomarkers in clinical practice will lead to better preventative and therapeutic interventions and to improve outcomes of critically ill patients. A number of biomarkers of functional change and cellular damage are under evaluation for early diagnosis, risk assessment, and prognosis of AKI. Neutrophil gelatinase-associated lipocalin (NGAL) has emerged as the most promising biomarker of kidney injury; this protein can be measured by commercially available methods in whole blood, plasma, serum, and urine. Concomitantly, metabolomics appears to be a snapshot of the chemical fingerprints identifying specific cellular processes. In this paper, we describe the role of NGAL for managing AKI and the potential benefits deriving from the combined clinical use of urine NGAL and metabolomics in kidney disease. A. Noto, F. Cibecchini, V. Fanos, and M. Mussap Copyright © 2013 A. Noto et al. All rights reserved. The Associated Ion between the VDR Gene Polymorphisms and Susceptibility to Hepatocellular Carcinoma and the Clinicopathological Features in Subjects Infected with HBV Sat, 23 Mar 2013 17:48:29 +0000 Aim. To evaluate the possible association between the vitamin D receptor (VDR), single-nucleotide polymorphisms (SNPs), and hepatocellular carcinoma (HCC) in patients with chronic hepatitis B virus (HBV) infection. Method. 968 chronic HBV infection patients were enrolled, of which 436 patients were diagnosed HCC patients, and 532 were non-HCC patients. The clinicopathological characteristics of HCC were evaluated. The genotypes of VDR gene at FokI, BsmI, ApaI, and TaqI were determined. Results. The genotype frequencies of VDR FokI C>T polymorphism were significantly different between HCC and non-HCC groups. HCC patients had a higher prevalence of FokI TT genotype than non-HCC subjects. With FokI CC as reference, the TT carriage had a significantly higher risk for development of HCC after adjustments with age, sex, HBV infection time, α-fetoprotein, smoking status, and alcohol intake. In addition, we also found that the TT genotype carriage of FokI polymorphisms were associated with advanced tumor stage, presence of cirrhosis, and lymph node metastasis. The SNP at BsmI, ApaI, and TaqI did not show positive association with the risk and clinicopathological features of HCC. Conclusion. The FokI C>T polymorphisms may be used as a molecular marker to predict the risk and to evaluate the disease severity of HCC in those infected with HBV. Xing Yao, Huazong Zeng, Guolei Zhang, Weimin Zhou, Qiang Yan, Licheng Dai, and Xiang Wang Copyright © 2013 Xing Yao et al. All rights reserved. Evaluation of Multiplex PCR with Enhanced Spore Germination for Detection of Clostridium difficile from Stool Samples of the Hospitalized Patients Sun, 17 Mar 2013 15:37:04 +0000 Clostridium difficile poses as the most common etiologic agent of nosocomial diarrhea. Although there are many diagnostic methods to detect C. difficile directly from stool samples, the nucleic acid-based approach has been largely performed in several laboratories due to its high sensitivity and specificity as well as rapid turnaround time. In this study, a multiplex PCR was newly designed with recent accumulated nucleotide sequences. The PCR testing with various C. difficile ribotypes, other Clostridium spp., and non-Clostridium strains revealed 100% specificity with the ability to detect as low as ~22 genomic copy number per PCR reaction. Different combinations of sample processing were evaluated prior to multiplex PCR for the detection of C. difficile in fecal samples from hospitalized patients. The most optimal condition was the non-selective enrichment at 37∘C for 1 h in brain heart infusion broth supplemented with taurocholate, followed by the multiplex PCR. The detection limit after sample processing was shown as being 5 spores per gram of fecal sample. Two hundred and thirty-eight fecal samples collected from the University affiliated hospital were analyzed by the enrichment multiplex PCR procedure. The results suggested that the combination of sample processing with the high-performance detection method would be applicable for routine diagnostic use in clinical setting. Surang Chankhamhaengdecha, Piyapong Hadpanus, Amornrat Aroonnual, Puriya Ngamwongsatit, Darunee Chotiprasitsakul, Piriyaporn Chongtrakool, and Tavan Janvilisri Copyright © 2013 Surang Chankhamhaengdecha et al. All rights reserved. Aldose Reductase Inhibitory Activity of Compounds from  Zea mays L. Sun, 17 Mar 2013 15:29:38 +0000 Aldose reductase (AR) inhibitors have a considerable therapeutic potential against diabetes complications and do not increase the risk of hypoglycemia. Through bioassay-guided fractionation of an EtOH extract of the kernel from purple corn (Zea mays L.), 7 nonanthocyanin phenolic compounds (compound 1–7) and 5 anthocyanins (compound 8–12) were isolated. These compounds were investigated by rat lens aldose reductase (RLAR) inhibitory assays. Kinetic analyses of recombinant human aldose reductase (rhAR) were performed, and intracellular galactitol levels were measured. Hirsutrin, one of 12 isolated compounds, showed the most potent RLAR inhibitory activity (IC50, 4.78 μM). In the kinetic analyses using Lineweaver-Burk plots of 1/velocity and 1/substrate concentration, hirsutrin showed competitive inhibition against rhAR. Furthermore, hirsutrin inhibited galactitol formation in rat lens and erythrocytes sample incubated with a high concentration of galactose; this finding indicates that hirsutrin may effectively prevent osmotic stress in hyperglycemia. Therefore, hirsutrin derived from Zea mays L. may be a potential therapeutic agent against diabetes complications. Tae Hyeon Kim, Jin Kyu Kim, Young-Hee Kang, Jae-Yong Lee, Il Jun Kang, and Soon Sung Lim Copyright © 2013 Tae Hyeon Kim et al. All rights reserved. An Effective Degumming Enzyme from Bacillus sp. Y1 and Synergistic Action of Hydrogen Peroxide and Protease on Enzymatic Degumming of Ramie Fibers Sun, 17 Mar 2013 15:21:46 +0000 Enzymatic degumming, as an alternative to chemical processing, has attracted wide attention. However, to date, little information about other enzyme components with effective degumming except pectinase has been reported, and there is no report about the effect of bleaching agent (H2O2) on enzymatic degumming and combining enzymatic degumming and H2O2 bleaching process. In this study, we found that the crude enzyme of wild-type Bacillus sp. Y1 had a powerful and fast degumming ability. Its PGL activity was the highest at pH 9.6–10.0 and and stable at pH 7–10.5 and 30–, having a wide scope of pH and temperature. Its PGL also had a high H2O2 tolerance, and the gum loss and brightness of fibers could be significantly improved when H2O2 was added into it for degumming. The synergistic action was also found between it and H2O2 on the degumming and bleaching of ramie fibers. All showed that it was very suitable for a joint process of enzymatic degumming and H2O2 bleaching. It also contained more proteins compared with a control pectinase, and its high protease content was further substantiated as a factor for effective degumming. Protease and pectinase also had a synergistic action on degumming. Fenfen Guo, Mouyong Zou, Xuezhi Li, Jian Zhao, and Yinbo Qu Copyright © 2013 Fenfen Guo et al. All rights reserved. Structures and Properties of Naturally Occurring Polyether Antibiotics Sun, 17 Mar 2013 15:14:23 +0000 Polyether ionophores represent a large group of natural, biologically active substances produced by Streptomyces spp. They are lipid soluble and able to transport metal cations across cell membranes. Several of polyether ionophores are widely used as growth promoters in veterinary. Polyether antibiotics show a broad spectrum of bioactivity ranging from antibacterial, antifungal, antiparasitic, antiviral, and tumour cell cytotoxicity. Recently, it has been shown that some of these compounds are able to selectively kill cancer stem cells and multidrug-resistant cancer cells. Thus, they are recognized as new potential anticancer drugs. The biological activity of polyether ionophores is strictly connected with their molecular structure; therefore, the purpose of this paper is to present an overview of their formula, molecular structure, and properties. Jacek Rutkowski and Bogumil Brzezinski Copyright © 2013 Jacek Rutkowski and Bogumil Brzezinski. All rights reserved. Development of a Broad-Range 23S rDNA Real-Time PCR Assay for the Detection and Quantification of Pathogenic Bacteria in Human Whole Blood and Plasma Specimens Sun, 17 Mar 2013 14:16:01 +0000 Molecular methods are important tools in the diagnosis of bloodstream bacterial infections, in particular in patients treated with antimicrobial therapy, due to their quick turn-around time. Here we describe a new broad-range real-time PCR targeting the 23S rDNA gene and capable to detect as low as 10 plasmid copies per reaction of targeted bacterial 23S rDNA gene. Two commercially available DNA extraction kits were evaluated to assess their efficiency for the extraction of plasma and whole blood samples spiked with different amount of either Staphylococcus aureus or Escherichia coli, in order to find the optimal extraction method to be used. Manual QIAmp extraction method with enzyme pre-treatment resulted the most sensitive for detection of bacterial load. Sensitivity of this novel assay ranged between 10 and 103 CFU per PCR reaction for E. coli and S. aureus in human whole blood samples depending on the extraction methods used. Analysis of plasma samples showed a 10- to 100-fold reduction of bacterial 23S rDNA in comparison to the corresponding whole blood specimens, thus indicating that whole blood is the preferential sample type to be used in this real-time PCR protocol. Our results thus show that the 23S rDNA gene represents an optimal target for bacteria quantification in human whole blood. Paolo Gaibani, Mara Mariconti, Gloria Bua, Sonia Bonora, Davide Sassera, Maria Paola Landini, Patrizia Mulatto, Stefano Novati, Claudio Bandi, and Vittorio Sambri Copyright © 2013 Paolo Gaibani et al. All rights reserved. Detection of Mycobacterium tuberculosis by Using Loop-Mediated Isothermal Amplification Combined with a Lateral Flow Dipstick in Clinical Samples Tue, 05 Mar 2013 08:06:58 +0000 Tuberculosis (TB) is a communicable disease caused by the bacterium Mycobacterium tuberculosis (MTB) and is a persistent problem in the developing countries. Loop-mediated isothermal amplification (LAMP) allows DNA to be amplified rapidly at a constant temperature. Here, a LAMP method was combined with a chromatographic lateral-flow dipstick (LFD) to detect IS6110 gene of M. tuberculosis specifically and rapidly. The reaction was optimized at 63°C for 60 min, and the amplified DNA hybridized to an FITC-labeled oligonucleotide probe for 5 min was detected at the LFD test line 5 min after application. Excluding the step of DNA extraction, the test results could be generated approximately within 1 h. In addition to the advantage of short assay time, this technique could avoid the contact of carcinogenic ethidium bromide due to the exclusion of the electrophoresis analysis step. Furthermore, the data indicated that LAMP-LFD could detect M. tuberculosis genomic DNA as little as 5 pg. The technique showed a significant specificity since no cross-hybridization to M. intracellulare (MIC), M. fortuitum (MFT), M. avium (MAV), M. kansasii (MKS), and M. gordonae (MGD) genomic DNAs was observed. In the clinical unknown samples test, the sensitivity of LAMP-LFD was 98.92 % and the specificity was 100 % compared to those of the standard culture assay. Based on its sensitivity, specificity, rapidity, low cost, and convenience, LAMP-LFD could be applicable for use in both laboratories and epidemiological surveys of MTB. Thongchai Kaewphinit, Narong Arunrut, Wansika Kiatpathomchai, Somchai Santiwatanakul, Pornpun Jaratsing, and Kosum Chansiri Copyright © 2013 Thongchai Kaewphinit et al. All rights reserved. A Review of Haptoglobin Typing Methods for Disease Association Study and Preventing Anaphylactic Transfusion Reaction Thu, 28 Feb 2013 19:05:50 +0000 Haptoglobin, the product of the gene, is a glycoprotein involved in the scavenging of free hemoglobin. Haptoglobin levels increase or decrease in response to various acquired conditions, and they are also influenced by genetic predisposition. There were 2 major alleles, and , and 1 minor allele, . Many researchers have attempted to study the haptoglobin types and their association with disease; however, no definitive conclusions have been reached yet. It is reported that patients who are genetically deficient in haptoglobin are at risk of anaphylaxis against blood components containing haptoglobin. Haptoglobin genotypes also affect the reference intervals of haptoglobin levels. Many studies have attempted to establish simple and accurate typing methods. In this paper, we have broadly reviewed several methods for haptoglobin typing—phenotyping, Southern blotting, conventional PCR, real-time PCR, and loop-mediated isothermal amplification. We discuss their characteristics, clinical applications, and limitations. The phenotyping methods are time consuming and labor intensive and not designed to detect patients harboring . The rapid and robust haptoglobin genotyping may help in preventing fatal anaphylactic reactions and in establishing the relationships between the haptoglobin phenotypes and diseases. Dae-Hyun Ko, Ho Eun Chang, Taek Soo Kim, Eun Young Song, Kyoung Un Park, Junghan Song, and Kyou Sup Han Copyright © 2013 Dae-Hyun Ko et al. All rights reserved. Potential Use of Atlantic Cod Trypsin in Biomedicine Thu, 28 Feb 2013 16:12:34 +0000 Surface proteins of viruses and bacteria used for cell attachment and invasion are candidates for degradation by proteases. Trypsin from Atlantic cod (Gadus morhua) was previously demonstrated to have efficacy against influenza viruses in vitro and on skin. In this paper, cod trypsin is shown to be 3–12 times more effective in degrading large native proteins than its mesophilic analogue, bovine trypsin. This is in agreement with previous findings where cod trypsin was found to be the most active among twelve different proteases in cleaving various cytokines and pathological proteins. Furthermore, our results show that cod trypsin has high efficacy against herpes simplex virus type 1 (HSV-1) and the respiratory syncytial virus (RSV) in vitro. The results on the antipathogenic properties of cod trypsin are important because rhinovirus, RSV, and influenza are the most predominant pathogenic viruses in upper respiratory tract infections. Results from a clinical study presented in this paper show that a specific formulation containing cod trypsin was preferred for wound healing over other methods used in the study. Apparently, the high digestive ability of the cold-adapted cod trypsin towards large native proteins plays a role in its efficacy against pathogens and its positive effects on wounds. Ágústa Gudmundsdóttir, Hilmar Hilmarsson, and Bjarki Stefansson Copyright © 2013 Ágústa Gudmundsdóttir et al. All rights reserved. Development of a Prognostic Score Using the Complete Blood Cell Count for Survival Prediction in Unselected Critically Ill Patients Thu, 28 Feb 2013 15:58:05 +0000 Objective. The purpose of this study was to develop a new prognostic scoring system for critically ill patients using the simple complete blood cell count (CBC). Methods. CBC measurements in samples from 306 patients in an intensive care unit were conducted with automated analyzers, including levels of neutrophils, lymphocytes, erythrocytes, hemoglobin, and platelets. The time of sampling and the time of death were recorded. values were calculated according to the measured values, reference mean values, and standard deviations. The prognostic score was equivalent to the median of the value of each of the measured parameters. Results. There was a significant correlation between survival time and neutrophil, lymphocyte, and platelet levels (). Prognostic scores were calculated from the value of these three parameters. Survival times decreased as the prognostic score increased. Conclusions. This study suggests that a model that uses levels of neutrophils, lymphocytes, and platelets is potentially useful in the objective evaluation of survival time or disease severity in unselected critically ill patients. Fang Chongliang, Li Yuzhong, Shi Qian, Liu Xiliang, and Liu Hui Copyright © 2013 Fang Chongliang et al. All rights reserved. Validation of New Allele-Specific Real-Time PCR System for Thiopurine Methyltransferase Genotyping in Korean Population Thu, 28 Feb 2013 11:22:01 +0000 Introduction. Thiopurine drugs are metabolized via S-methylation and catalyzed by thiopurine S-methyltransferase (TPMT). Patients with very low TPMT activity are at high risk of fatal bone marrow toxicity when standard doses of thiopurine drugs are administered. TPMT genotyping can predict TPMT activity and is not affected by transfusion or red blood cell defects. Here, we report a new allele-specific real-time polymerase chain reaction (PCR) system for thiopurine methyltransferase genotyping that is validated in Korean population. Materials and Methods. Three major TPMT single-nucleotide polymorphisms (TPMT*2, *3B, and *3C) were genotyped using real-time PCR with the allele-specific primers and probes. Internal positive controls were included in each well, and an automatic interpretative algorithm was applied. This system was validated using 244 clinical samples and 2 commercial DNA samples that had been previously genotyped using PCR-direct sequencing. Results. All of the obtained results are concordant with those of the reference method. All of the internal positive control reactions were successful. The allele frequency of TPMT*3C was 2.05% (10 of 488 alleles). All of the patients with variant alleles were heterozygotes, and no homozygotes were detected. No TPMT*2, *3A, or *3B alleles were observed in this Korean population. Conclusion. This rapid, accurate, and user-friendly genotyping system can be readily used to improve the efficacy and safety of thiopurine treatments in clinical practice. Sollip Kim, Hye Won Lee, Woochang Lee, Sail Chun, and Won-Ki Min Copyright © 2013 Sollip Kim et al. All rights reserved. Total 25-Hydroxyvitamin D Determination by an Entry Level Triple Quadrupole Instrument: Comparison between Two Commercial Kits Thu, 28 Feb 2013 11:20:23 +0000 Objective. 25-hydroxyvitamin D2/D3 (25-OHD2/D3) determination is a reliable biomarker for vitamin D status. Liquid chromatography-tandem mass spectrometry was recently proposed as a reference method for vitamin D status evaluation. The aim of this work is to compare two commercial kits (Chromsystems and PerkinElmer) for 25-OHD2/D3 determination by our entry level LC-MS/MS. Design and Methods. Chromsystems kit adds an online trap column to an HPLC column and provides atmospheric pressure chemical ionization, isotopically labeled internal standard, and 4 calibrator points. PerkinElmer kit uses a solvent extraction and protein precipitation method. This kit can be used with or without derivatization with, respectively, electrospray and atmospheric pressure chemical ionization. For each analyte, there are isotopically labeled internal standards and 7 deuterated calibrator points. Results. Performance characteristics are acceptable for both methods. Mean bias between methods calculated on 70 samples was 1.9 ng/mL. Linear regression analysis gave an of 0.94. 25-OHD2 is detectable only with PerkinElmer kit in derivatized assay option. Conclusion. Both methods are suitable for routine. Chromsystems kit minimizes manual sample preparation, requiring only protein precipitation, but, with our system, 25-OHD2 is not detectable. PerkinElmer kit without derivatization does not guarantee acceptable performance with our LC-MS/MS system, as sample is not purified online. Derivatization provides sufficient sensitivity for 25-OHD2 detection. Jacopo Gervasoni, Andrea Cocci, Cecilia Zuppi, and Silvia Persichilli Copyright © 2013 Jacopo Gervasoni et al. All rights reserved. Biomarkers of Hypochromia: The Contemporary Assessment of Iron Status and Erythropoiesis Thu, 28 Feb 2013 10:51:44 +0000 Iron status is the result of the balance between the rate of erythropoiesis and the amount of the iron stores. Direct consequence of an imbalance between the erythroid marrow iron requirements and the actual supply is a reduction of red cell hemoglobin content, which causes hypochromic mature red cells and reticulocytes. The diagnosis of iron deficiency is particularly challenging in patients with acute or chronic inflammatory conditions because most of the biochemical markers for iron metabolism (serum ferritin and transferrin ) are affected by acute phase reaction. For these reasons, interest has been generated in the use of erythrocyte and reticulocyte parameters, available on the modern hematology analyzers. Reported during blood analysis routinely performed on the instrument, these parameters can assist in early detection of clinical conditions (iron deficiency, absolute, or functional; ineffective erythropoiesis, including iron restricted or thalassemia), without additional cost. Technological progress has meant that in recent years modern analyzers report new parameters that provide further information from the traditional count. Nevertheless these new parameters are exclusive of each manufacturer, and they are patented. This is an update of these new laboratory test biomarkers of hypochromia reported by different manufactures, their meaning, and clinical utility on daily practice. Eloísa Urrechaga, Luís Borque, and Jesús F. Escanero Copyright © 2013 Eloísa Urrechaga et al. All rights reserved. Coagulation Proteins Influencing Global Coagulation Assays in Cirrhosis: Hypercoagulability in Cirrhosis Assessed by Thrombomodulin-Induced Thrombin Generation Assay Thu, 21 Feb 2013 09:54:59 +0000 Background. Liver disease is accompanied by profound hemostatic disturbances. We investigated the influences of pro- and anticoagulation factors on global coagulation tests including prothrombin time (PT), activated partial thromboplastin time (aPTT), and thrombin generation assay (TGA) in cirrhosis. We also investigated whether cirrhotic patients exhibit hypo- or hypercoagulability using the TGA. Methods. The TGA was performed on a calibrated automated thrombogram, given lag time, endogenous thrombin potential (ETP), and peak thrombin in 156 cirrhotic patients and 73 controls. Results. PT was determined according to the factor (F) II, FV, FVII, FIX, and protein C levels. We observed that aPTT was dependent on FII, FIX, and FX levels. The ETP was dependent on FII, antithrombin, and protein C with 5 pM tissue factor (TF) stimulation, and FIX and protein C at 1 pM TF. The ETP ratio with 1 pM TF increased significantly in cirrhosis, indicating hypercoagulability, whereas that with 5 pM TF did not increase in cirrhosis. Conclusion. PT and the TGA are sensitive to protein C levels. Even with prolonged PT, the TGA can detect hypercoagulability in cirrhosis. Further studies should evaluate global coagulation status in cirrhosis patients using the newly devised TGA system. Nam Youngwon, Ji-Eun Kim, Hae Sook Lim, Kyou-Sup Han, and Hyun Kyung Kim Copyright © 2013 Nam Youngwon et al. All rights reserved. Two Classifiers Based on Serum Peptide Pattern for Prediction of HBV-Induced Liver Cirrhosis Using MALDI-TOF MS Tue, 19 Feb 2013 08:52:36 +0000 Chronic infection with hepatitis B virus (HBV) is associated with the majority of cases of liver cirrhosis (LC) in China. Although liver biopsy is the reference method for evaluation of cirrhosis, it is an invasive procedure with inherent risk. The aim of this study is to discover novel noninvasive specific serum biomarkers for the diagnosis of HBV-induced LC. We performed bead fractionation/MALDI-TOF MS analysis on sera from patients with LC. Thirteen feature peaks which had optimal discriminatory performance were obtained by using support-vector-machine-(SVM-) based strategy. Based on the previous results, five supervised machine learning methods were employed to construct classifiers that discriminated proteomic spectra of patients with HBV-induced LC from those of controls. Here, we describe two novel methods for prediction of HBV-induced LC, termed LC-NB and LC-MLP, respectively. We obtained a sensitivity of 90.9%, a specificity of 94.9%, and overall accuracy of 93.8% on an independent test set. Comparisons with the existing methods showed that LC-NB and LC-MLP held better accuracy. Our study suggests that potential serum biomarkers can be determined for discriminating LC and non-LC cohorts by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. These two classifiers could be used for clinical practice in HBV-induced LC assessment. Yuan Cao, Kun He, Ming Cheng, Hai-Yan Si, He-Lin Zhang, Wei Song, Ai-Ling Li, Cheng-Jin Hu, and Na Wang Copyright © 2013 Yuan Cao et al. All rights reserved. Development of a Generic Microfluidic Device for Simultaneous Detection of Antibodies and Nucleic Acids in Oral Fluids Thu, 14 Feb 2013 18:18:08 +0000 A prototype dual-path microfluidic device (Rheonix CARD) capable of performing simultaneously screening (antigen or antibody) and confirmatory (nucleic acid) detection of pathogens is described. The device fully integrates sample processing, antigen or antibody detection, and nucleic acid amplification and detection, demonstrating rapid and inexpensive “sample-to-result” diagnosis with performance comparable to benchtop analysis. For the chip design, a modular approach was followed allowing the optimization of individual steps in the sample processing process. This modular design provides great versatility accommodating different disease targets independently of the production method. In the detection module, a lateral flow (LF) protocol utilizing upconverting phosphor (UCP) reporters was employed. The nucleic acid (NA) module incorporates a generic microtube containing dry reagents. Lateral flow strips and PCR primers determine the target or disease that is diagnosed. Diagnosis of HIV infection was used as a model to investigate the simultaneous detection of both human antibodies against the virus and viral RNA. The serological result is available in less than 30 min, and the confirmation by RNA amplification takes another 60 min. This approach combines a core serological portable diagnostic with a nucleic acid-based confirmatory test. Zongyuan Chen, William R. Abrams, Eran Geva, Claudia J. de Dood, Jesús M. González, Hans J. Tanke, R. Sam Niedbala, Peng Zhou, Daniel Malamud, and Paul L. A. M. Corstjens Copyright © 2013 Zongyuan Chen et al. All rights reserved. Inorganic Phosphate Modulates the Expression of the NaPi-2a Transporter in the trans-Golgi Network and the Interaction with PIST in the Proximal Tubule Thu, 14 Feb 2013 09:06:32 +0000 Inorganic phosphate (Pi) homeostasis is maintained by the tight regulation of renal Pi excretion versus reabsorption rates that are in turn modulated by adjusting the number of Pi transporters (mainly NaPi-2a) in the proximal tubules. In response to some hormones and a high dietary Pi content, NaPi-2a is endocytosed and degraded in the lysosomes; however, we show here that some NaPi-2a molecules are targeted to the trans-Golgi network (TGN) during the endocytosis. In the TGN, NaPi-2a interacts with PIST (PDZ-domain protein interacting specifically with TC10), a TGN-resident PDZ-domain-containing protein. The extension of the interaction is proportional to the expression of NaPi-2a in the TGN, and, consistent with that, it is increased with a high Pi diet. When overexpressed in opossum kidney (OK) cells, PIST retains NaPi-2a in the TGN and inhibits Na-dependent Pi transport. Overexpression of PIST also prevents the adaptation of OK cells to a low Pi culture medium. Our data supports the view that NaPi-2a is subjected to retrograde trafficking from the plasma membrane to the TGN using one of the machineries involved in endosomal transport and explains the reported expression of NaPi-2a in the TGN. Miguel A. Lanaspa, Yupanqui A. Caldas, Sophia Y. Breusegem, Ana Andrés-Hernando, Christina Cicerchi, Moshe Levi, and Victor Sorribas Copyright © 2013 Miguel A. Lanaspa et al. All rights reserved. A Pentaplex PCR Assay for the Detection and Differentiation of Shigella Species Wed, 13 Feb 2013 16:23:03 +0000 The magnitude of shigellosis in developing countries is largely unknown because an affordable detection method is not available. Current laboratory diagnosis of Shigella spp. is laborious and time consuming and has low sensitivity. Hence, in the present study, a molecular-based diagnostic assay which amplifies simultaneously four specific genes to identify invC for Shigella genus, rfc for S. flexneri, wbgZ for S. sonnei, and rfpB for S. dysenteriae, as well as one internal control (ompA) gene, was developed in a single reaction to detect and differentiate Shigella spp. Validation with 120 Shigella strains and 37 non-Shigella strains yielded 100% specificity. The sensitivity of the PCR was 100 pg of genomic DNA, 5.4 × 104 CFU/ml, or approximately 120 CFU per reaction mixture of bacteria. The sensitivity of the pentaplex PCR assay was further improved following preincubation of the stool samples in Gram-negative broth. A preliminary study with 30 diarrhoeal specimens resulted in no cross-reaction with other non-Shigella strains tested. We conclude that the developed pentaplex PCR assay is robust and can provide information about the four target genes that are essential for the identification of the Shigella genus and the three Shigella species responsible for the majority of shigellosis cases. Suvash Chandra Ojha, Chan Yean Yean, Asma Ismail, and Kirnpal-Kaur Banga Singh Copyright © 2013 Suvash Chandra Ojha et al. All rights reserved. Urine Cell-Free DNA Integrity as a Marker for Early Prostate Cancer Diagnosis: A Pilot Study Wed, 13 Feb 2013 11:40:32 +0000 Circulating cell-free DNA has been recognized as an accurate marker for the diagnosis of prostate cancer, whereas the role of urine cell-free DNA (UCF DNA) has never been evaluated in this setting. It is known that normal apoptotic cells produce highly fragmented DNA while cancer cells release longer DNA. We thus verified the potential role of UCF DNA integrity for early prostate cancer diagnosis. UCF DNA was isolated from 29 prostate cancer patients and 25 healthy volunteers. Sequences longer than 250 bp (c-Myc, BCAS1, and HER2) were quantified by real-time PCR to verify UCF DNA integrity. Receiver operating characteristic (ROC) curve analysis revealed an area under the curve of 0.7959 (95% CI 0.6729–0.9188). At the best cut-off value of 0.04 ng/μL, UCF DNA integrity analysis showed a sensitivity of 0.79 (95% CI 0.62–0.90) and a specificity of 0.84 (95% CI 0.65–0.94). These preliminary findings indicate that UCF DNA integrity could be a promising noninvasive marker for the early diagnosis of prostate cancer and pave the way for further research into this area. Valentina Casadio, Daniele Calistri, Samanta Salvi, Roberta Gunelli, Elisa Carretta, Dino Amadori, Rosella Silvestrini, and Wainer Zoli Copyright © 2013 Valentina Casadio et al. All rights reserved. Development of a Novel System for Mass Spectrometric Analysis of Cancer-Associated Fucosylation in Plasma α1-Acid Glycoprotein Wed, 13 Feb 2013 09:00:03 +0000 Human plasma α1-acid glycoprotein (AGP) from cancer patients and healthy volunteers was purified by sequential application of ion-exchange columns, and N-linked glycans enzymatically released from AGP were labeled and applied to a mass spectrometer. Additionally, a novel software system for use in combination with a mass spectrometer to determine N-linked glycans in AGP was developed. A database with 607 glycans including 453 different glycan structures that were theoretically predicted to be present in AGP was prepared for designing the software called AGPAS. This AGPAS was applied to determine relative abundance of each glycan in the AGP molecules based on mass spectra. It was found that the relative abundance of fucosylated glycans in tri- and tetra-antennary structures (FUCAGP) was significantly higher in cancer patients as compared with the healthy group (). Furthermore, extremely elevated levels of FUCAGP were found specifically in patients with a poor prognosis but not in patients with a good prognosis. In conclusion, the present software system allowed rapid determination of the primary structures of AGP glycans. The fucosylated glycans as novel tumor markers have clinical relevance in the diagnosis and assessment of cancer progression as well as patient prognosis. Takayuki Asao, Shin Yazawa, Toyo Nishimura, Takashi Hayashi, Hideyuki Shimaoka, Abby R. Saniabadi, and Hiroyuki Kuwano Copyright © 2013 Takayuki Asao et al. All rights reserved. Human Platelet Antigen Genotyping and Expression of CD109 (Human Platelet Antigen 15) mRNA in Various Human Cell Types Tue, 12 Feb 2013 14:07:50 +0000 CD109 gene encodes a glycosylphosphatidylinositol-linked glycoprotein found in a subset of platelets and endothelial cell, and human platelet antigen (HPA) 15 is found on CD109. We evaluated the HPA genotype and/or the CD109 mRNA expression on two peripheral blood stem cells (PBSC), two peripheral bloods (PB), 12 granulocyte products, natural killer (NK)-92, B-lymphocyte (CO88BV59-1), K-562 leukemia cell line, human embryonic stem cell (hESC), and human fibroblasts (HF). HPA genotyping was performed by SNaPshot assay and CD109 mRNA expression was evaluated by real-time PCR with SYBR green and melting curve analysis. Genotype HPA-15a/-15a was found in PBSC#1 and two granulocyte products, and HPA-15a/-15b was found in PBSC#2, eight granulocyte products, NK-92, K-562, hESC, and HF, and HPA-15b/-15b was found in two granulocyte products. CD109 mRNA expression was highly increased in HF and increased in CD34+ and CD34− PBSCs and some granulocyte products, compared to the PB. However, the increase of expression level varied among the PBSC and granulocyte products. The CD109 mRNA expression of NK-92, K-562, hESC, and CO 88BV59-1 was not detected. HPA genotype was evaluated in various cells and the expression of CD109, which contains HPA 15, was different among cell lines and high in HF and PBSCs. Sang Mee Hwang, Mi Jung Kim, Ho Eun Chang, Yun Ji Hong, Taek Soo Kim, Eun Young Song, Kyoung Un Park, Junghan Song, and Kyou-Sup Han Copyright © 2013 Sang Mee Hwang et al. All rights reserved. Differential Expression of Na+/H+-Exchanger (NHE-1, 2, and 4) Proteins and mRNA in Rodent’s Uterus under Sex Steroid Effect and at Different Phases of the Oestrous Cycle Mon, 11 Feb 2013 10:11:37 +0000 Precise uterine fluid pH regulation may involve the Na+/H+-exchanger (NHE). We hypothesized that NHE isoforms are differentially expressed under different sex steroid treatment and at different oestrous cycle phases which may explain the uterine fluid pH changes observed under these conditions. Method. Oestrous cycle phases of intact WKY rats were identified by vaginal smear. Another group of rats was ovariectomized and treated with 0.2 μg 17β-oestradiol (E), 4 mg progesterone (P), and E followed by P (E + P). The animals were then sacrificed and the uteri were removed for mRNA and protein expression analyses by real-time PCR and western blotting, respectively. NHE isoforms distribution was detected by immunohistochemistry (IHC). Results. NHE-1 mRNA and protein were upregulated at diestrus (Ds) and following P treatment. Meanwhile, NHE-2 and NHE-4 proteins and mRNA were upregulated at proestrus (Ps) and estrus (Es) and following E treatment. NHE-1 was found predominantly at the apical membrane, while NHE-2 and NHE-4 were found at the apical and basolateral membranes of the luminal epithelia. NHE-4 is the main isoform upregulated by E. Conclusion. Differential expressions of uterine NHE isoforms 1, 2, and 4 could explain the observed changes in the uterine fluid pH under these conditions. Khadijeh Gholami, Sekaran Muniandy, and Naguib Salleh Copyright © 2013 Khadijeh Gholami et al. All rights reserved. Microsphere Suspension Array Assays for Detection and Differentiation of Hendra and Nipah Viruses Wed, 06 Feb 2013 13:11:18 +0000 Microsphere suspension array systems enable the simultaneous fluorescent identification of multiple separate nucleotide targets in a single reaction. We have utilized commercially available oligo-tagged microspheres (Luminex MagPlex-TAG) to construct and evaluate multiplexed assays for the detection and differentiation of Hendra virus (HeV) and Nipah virus (NiV). Both these agents are bat-borne zoonotic paramyxoviruses of increasing concern for veterinary and human health. Assays were developed targeting multiple sites within the nucleoprotein (N) and phosphoprotein (P) encoding genes. The relative specificities and sensitivities of the assays were determined using reference isolates of each virus type, samples from experimentally infected horses, and archival veterinary diagnostic submissions. Results were assessed in direct comparison with an established qPCR. The microsphere array assays achieved unequivocal differentiation of HeV and NiV and the sensitivity of HeV detection was comparable to qPCR, indicating high analytical and diagnostic specificity and sensitivity. Adam J. Foord, John R. White, Axel Colling, and Hans G. Heine Copyright © 2013 Adam J. Foord et al. All rights reserved. Steroids and Related Compounds: Basic and Clinical Aspects Sun, 03 Feb 2013 07:18:44 +0000 Fátima Regina Mena Barreto Silva, Leila Zanatta, Rozangela Curi Pedrosa, and Ming-Zhu Fang Copyright © 2013 Fátima Regina Mena Barreto Silva et al. All rights reserved. Gene Silencing of 4-1BB by RNA Interference Inhibits Acute Rejection in Rats with Liver Transplantation Thu, 31 Jan 2013 10:08:31 +0000 The 4-1BB signal pathway plays a key role in organ transplantation tolerance. In this study, we have investigated the effect of gene silencing of 4-1BB by RNA interference (RNAi) on the acute rejection in rats with liver transplantation. The recombination vector of lentivirus that contains shRNA targeting the 4-1BB gene (LV-sh4-1BB) was constructed. The liver transplantation was performed using the two-cuff technique. Brown-Norway (BN) recipient rats were infected by the recombinant LVs. The results showed that gene silencing of 4-1BB by RNAi downregulated the 4-1BB gene expression of the splenic lymphocytes in vitro, and the splenic lymphocytes isolated from the rats with liver transplantation. LV-sh4-1BB decreased the plasma levels of liver injury markers including AST, ALT, and BIL and also decreased the level of plasma IL-2 and IFN-γ in recipient rats with liver transplantation. Lentivirus-mediated delivery of shRNA targeting 4-1BB gene prolonged the survival time of recipient and alleviated the injury of liver morphology in recipient rats with liver transplantation. In conclusion, our results demonstrate that gene silencing of 4-1BB by RNA interference inhibits the acute rejection in rats with liver transplantation. Yang Shi, Shuqun Hu, Qingwei Song, Shengcai Yu, Xiaojun Zhou, Jun Yin, Lei Qin, and Haixin Qian Copyright © 2013 Yang Shi et al. All rights reserved. Effect of Linseed Oil Dietary Supplementation on Fatty Acid Composition and Gene Expression in Adipose Tissue of Growing Goats Sun, 27 Jan 2013 08:24:33 +0000 This study was conducted to determine the effects of feeding oil palm frond silage based diets with added linseed oil (LO) containing high α-linolenic acid (C18:3n-3), namely, high LO (HLO), low LO (LLO), and without LO as the control group (CON) on the fatty acid (FA) composition of subcutaneous adipose tissue and the gene expression of peroxisome proliferator-activated receptor (PPAR)α, PPAR-γ, and stearoyl-CoA desaturase (SCD) in Boer goats. The proportion of C18:3n-3 in subcutaneous adipose tissue was increased () by increasing the LO in the diet, suggesting that the FA from HLO might have escaped ruminal biohydrogenation. Animals fed HLO diets had lower proportions of C18:1 trans-11, C18:2n-6, CLA cis-9 trans-11, and C20:4n-6 and higher proportions of C18:3n-3, C22:5n-3, and C22:6n-3 in the subcutaneous adipose tissue than animals fed the CON diets, resulting in a decreased n-6:n-3 fatty acid ratio (FAR) in the tissue. In addition, feeding the HLO diet upregulated the expression of PPAR-γ () but downregulated the expression of SCD () in the adipose tissue. The results of the present study show that LO can be safely incorporated in the diets of goats to enrich goat meat with potential health beneficial FA (i.e., n-3 FA). M. Ebrahimi, M. A. Rajion, Y. M. Goh, A. Q. Sazili, and J. T. Schonewille Copyright © 2013 M. Ebrahimi et al. All rights reserved. Rapid Nongenomic Action of Aldosterone on Protein Expressions of Hsp90(α and β) and pc-Src in Rat Kidney Tue, 22 Jan 2013 13:31:08 +0000 Previous in vitro studies indicated that aldosterone nongenomically phosphorylates epidermal growth factor receptor (EGFR) through activation of upstream signals, heat shock protein 90β (Hsp90β), and cytosolic (c)-Src kinase. We demonstrated that aldosterone rapidly elevates EGFR phosphorylation in rat kidney. There are no in vivo data regarding renal Hsp90(α and β) and phosphorylated (p)c-Src protein expressions. The present study further investigates the expressions of these proteins. Male Wistar rats were intraperitoneally injected with normal saline solution or aldosterone (Aldo: 150 μg/kg BW). After 30 minutes, abundances and localizations of these proteins were determined. Aldosterone enhanced renal Hsp90β protein abundance (), but Hsp90α and pc-Src protein levels remained unaltered. Expression of Hsp90(α and β) was induced prominently in the proximal convoluted tubules (PCTs). Activation of Hsp90α was observed in vascular and outer medulla regions, whereas Hsp90β was induced in the cortex. Immunoreactivity of pc-Src was elevated in PCT with obvious staining at the luminal membrane. This in vivo study is the first to demonstrate that aldosterone nongenomically elevates Hsp90(α and β) protein expressions in rat kidney. Aldosterone had no effect on pc-Src protein levels but modulated localization. These results indicate that aldosterone regulates upstream mediators of EGFR transactivation in vivo. Somchit Eiam-Ong, Kittisak Sinphitukkul, Krissanapong Manotham, and Somchai Eiam-Ong Copyright © 2013 Somchit Eiam-Ong et al. All rights reserved. Progesterone and Related Compounds in Hepatocellular Carcinoma: Basic and Clinical Aspects Wed, 16 Jan 2013 13:40:19 +0000 Primary liver cancer is the fifth most common cancer worldwide and the third most common cause of cancer mortality. Hepatocellular carcinoma (HCC) accounts for 85% to 90% of primary liver cancers. Major risk factors for HCC include infection with HBV or HCV, alcoholic liver disease, and most probably nonalcoholic fatty liver disease. In general, men are two to four times more often associated with HCC than women. It can be suggested that sex hormones including progesterone may play some roles in HCC. Rather, very limited information discusses its potential involvement in HCC. This paper thus collects some recent studies of the potential involvement of progesterone and related compounds in HCC from basic and clinical aspects. In addition, two synthetic progestins, megestrol acetate (MA) and medroxyprogesterone acetate (MPA), will be discussed thoroughly. It is noted that progesterone can also serve as the precursor for androgens and estrogens produced by the gonadal and adrenal cortical tissues, while men have a higher incidence of HCC than women might be due to the stimulatory effects of androgen and the protective effects of estrogen. Eventually, this paper suggests a new insight on the associations of progesterone and related compounds with HCC development and treatment. Yao-Tsung Yeh, Chien-Wei Chang, Ren-Jie Wei, and Shen-Nien Wang Copyright © 2013 Yao-Tsung Yeh et al. All rights reserved. Microvesicles as Potential Ovarian Cancer Biomarkers Tue, 08 Jan 2013 14:49:05 +0000 Although the incidence of ovarian cancer is low (i.e., less than 5% in European countries), it is the most lethal gynecologic malignancy and typically has a poor prognosis. To ensure optimal survival, it is important to diagnose this condition when the pathology is confined to the ovary. However, this is difficult to achieve because the first specific symptoms appear only during advanced disease stages. To date, the biomarker mainly used for the diagnosis and prognosis of ovarian cancer is CA125; however, this marker has a low sensitivity and specificity and is associated with several other physiological and pathological conditions. No other serum ovarian cancer markers appear to be able to replace or complement CA125, and the current challenge is therefore to identify novel markers for the early diagnosis of this disease. For this purpose, studies have focused on the microvesicles (MVs) released from tumor cells. MVs may represent an ideal biomarker because they can be easily isolated from blood, and they have particular features (mainly regarding microRNA profiles) that strongly correlate with ovarian cancer stage and may be effective for early diagnosis. Ilaria Giusti, Sandra D’Ascenzo, and Vincenza Dolo Copyright © 2013 Ilaria Giusti et al. All rights reserved. The Steady-State Serum Concentration of Genistein Aglycone Is Affected by Formulation: A Bioequivalence Study of Bone Products Mon, 31 Dec 2012 18:53:16 +0000 An FDA-regulated, prescription medical food (Fosteum; 27 mg natural genistein, 200 IU cholecalciferol, 20 mg citrated zinc bisglycinate (4 mg elemental zinc) per capsule) and an over-the-counter (OTC) supplement (Citracal Plus Bone Density Builder; 27 mg synthetic genistein, 600 mg elemental calcium (calcium citrate), 400 IU vitamin D3, 50 mg magnesium, 7.5 mg zinc, 1 mg copper, 75 μg molybdenum, 250 μg boron per two tablets) were compared to a clinically proven bone formulation (27 mg natural genistein, 400 IU cholecalciferol, 500 mg elemental calcium (calcium carbonate) per tablet; the Squadrito formulation) in an 8-day steady-state pharmacokinetic (PK) study of healthy postmenopausal women () randomized to receive 54 mg of genistein per day. Trough serum samples were obtained before the final dose on the morning of the ninth day followed by sampling at 1, 2, 4, 6, 8, 10, 12, 24, 36, 48, 72, and 96 hrs. Total serum genistein, after β-glucuronidase/sulfatase digestion, was measured by time-resolved fluorometric assay. Maximal time (), concentration (), half-life (), and area under the curve (AUC) were determined for genistein in each formulation. Fosteum and the Squadrito study formulation were equivalent for genistein (2 hrs), (0.7 M), ( versus  hrs), and AUC ( versus  ng·hr/mL). The OTC supplement’s synthetically derived genistein, however, showed altered (6 hrs), (0.57 μM), ( hrs), and AUC ( ng·hr/mL). Differences in uptake may be due to multiple ingredients in the OTC supplement which interfere with genistein absorption. Alessandra Bitto, Bruce P. Burnett, Francesca Polito, Silvia Russo, Rosario D'Anna, Lakshmi Pillai, Francesco Squadrito, Domenica Altavilla, and Robert M. Levy Copyright © 2013 Alessandra Bitto et al. All rights reserved. Glucocorticoid-Induced Osteoporosis in Children with 21-Hydroxylase Deficiency Sat, 29 Dec 2012 13:05:42 +0000 21-Hydroxylase deficiency (21-OHD) is the most common cause of congenital adrenal hyperplasia (CAH), resulting from deletions or mutations of the P450 21-hydroxylase gene (CYP21A2). Children with 21-OHD need chronic glucocorticoid (cGC) therapy, both to replace congenital deficit in cortisol synthesis and to reduce androgen secretion by adrenal cortex. GC-induced osteoporosis (GIO) is the most common form of secondary osteoporosis that results in an early, transient increase in bone resorption accompanied by a decrease in bone formation, maintained for the duration of GC therapy. Despite the conflicting results in the literature about the bone status on GC-treated patients with 21-OHD, many reports consider these subjects to be at risk for osteoporosis and fractures. In bone cells, at the molecular level, GCs regulate various functions including osteoblastogenesis, osteoclastogenesis, and the apoptosis of osteoblasts and osteocytes. In this paper, we focus on the physiology and biosynthesis of endogenous steroid hormones as well as on the effects of GCs on bone cells, highlighting the pathogenetic mechanism of GIO in children with 21-OHD. Annamaria Ventura, Giacomina Brunetti, Silvia Colucci, Angela Oranger, Filomena Ladisa, Luciano Cavallo, Maria Grano, and Maria Felicia Faienza Copyright © 2013 Annamaria Ventura et al. All rights reserved. Simultaneous Quantification of Flavonol Glycosides, Terpene Lactones, Biflavones, Proanthocyanidins, and Ginkgolic Acids in Ginkgo biloba Leaves from Fruit Cultivars by Ultrahigh-Performance Liquid Chromatography Coupled with Triple Quadrupole Mass Spectrometry Thu, 27 Dec 2012 19:01:47 +0000 On the basis of liquid chromatography coupled with triple quadrupole mass spectrometry working in multiple reaction monitoring mode, an analytical method has been established to simultaneously determine flavonol glycosides, terpene lactones, biflavones, proanthocyanidins, and ginkgolic acids in Ginkgo biloba leaves. Chromatographic separation was carried out on an Acquity BEH C18 column (100 mm × 2.1 mm, 1.7 μm) with gradient elution of acetonitrile and 0.10% formic acid (v/v) at a flow rate of 0.4 mL/min, and column temperature 30°C. The developed method was validated in terms of linearity, accuracy, precision, stability, and sensitivity. The optimized method was successfully applied to analyze twenty-two G. biloba leaf samples of fruit cultivars collected from different places in China. Furthermore, hierarchical clustering analysis (HCA) was performed to evaluate and classify the samples according to the contents of the twenty-four chemical constituents. All of the results demonstrated that the developed method was useful for the overall evaluation of the quality of G. biloba leaves, and this study was also helpful for the comprehensive utilization and development of G. biloba resources. Xin Yao, Gui-Sheng Zhou, Yu-Ping Tang, Ye-Fei Qian, Han-Liang Guan, Hanqing Pang, Shaoqing Zhu, Xuan Mo, Shu-Lan Su, Chun Jin, Yong Qin, Da-Wei Qian, and Jin-Ao Duan Copyright © 2013 Xin Yao et al. All rights reserved. Characterization of Sera with Discordant Results from Reverse Sequence Screening for Syphilis Thu, 27 Dec 2012 13:15:46 +0000 Reverse sequence screening for syphilis (RSSS) (screening with treponemal tests, followed by confirmation with nontreponemal tests) has been increasingly adopted. CDC recommends confirmation of discordant results (reactive EIA/CIA and nonreactive nontreponemal test) with Treponema pallidum particle agglutination assay (TP-PA). We characterized sera with discordant results from RSSS with Architect Syphilis TP CIA. Among 15,713 screening tests using Architect Syphilis TP at Seoul National University Gangnam Center between October 2010 and May 2011, 260 (1.7%) showed reactive results. Rapid plasma reagin (RPR) and TP-PA were performed on 153 available sera among them. On sera with discordant results between Architect Syphilis TP and TP-PA, INNO-LIA Syphilis Score and FTA-ABS were performed. Among 153 sera, RPR was nonreactive in 126 (82.4%). Among them, TP-PA was positive in 103 (81.7%), indeterminate (±) in 7 (5.6%), and negative in 16 (12.7%). Out of 16 CIA(+)/RPR(−)/TP-PA(−) sera, INNO-LIA Syphilis Score and/or FTA-ABS were negative on 14 sera. Out of 7 CIA(+)/RPR(−)/TP-PA(±) sera, INNO-LIA Syphilis Score and FTA-ABS were positive/reactive in 6 sera. RSSS with confirmation by TP-PA on sera with discordant results between Architect Syphilis TP and RPR effectively delineated those discordant results and could be successfully adopted for routine checkup for syphilis. Kyunghoon Lee, Hyewon Park, Eun Youn Roh, Sue Shin, Kyoung Un Park, Myoung Hee Park, and Eun Young Song Copyright © 2013 Kyunghoon Lee et al. All rights reserved. Optimisation of the Enzymatic Hydrolysis of Blood Cells with a Neutral Protease Thu, 27 Dec 2012 10:59:13 +0000 For utilizing the blood cells (BCs) effectively, enzymatic hydrolysis was applied to produce the enzymatically hydrolyzed blood cells (EHBCs) by using a neutral protease as a catalyst. The results of the single-factor experiments showed optimal substrate concentration, enzyme to substrate ratio (E/S), pH, temperature, and incubation period were 1.00%, 0.10, 7.00, 50.00°C, and 12.00 h, respectively. The optimized hydrolysis conditions from response surface methodology (RSM) were pH 6.50, E/S 0.11, temperature 45.00°C, and incubation period 12.00 h. Under these conditions (substrate concentration 1.00%), the degree of hydrolysis (DH) was 35.06%. The free amino acids (FAAs) content of the EHBCs (35.24%) was 40.46 times higher than BCs while the total amino acids (TAAs) content was lower than BCs. The scores of lysine (human 0.87; pig 0.97), valine (human 1.42; pig 1.38), leucine (human 1.50; pig 1.90), tyrosine (human 0.84; pig 1.09), and histidine (human 2.17; pig 2.50) indicated that the EHBCs basically fulfilled the adult human and pig nutritional requirements. The calculated protein efficiency ratios (C-PERs) of the EHBCs were 3.94, 6.19, 21.73, and 2.04. In summary, the EHBCs were produced successfully with optimized conditions and could be a novel protein source for humans and pigs. Yanbin Zheng, Qiushi Chen, Anshan Shan, and Hao Zhang Copyright © 2013 Yanbin Zheng et al. All rights reserved. Three New Steroidal Glycosides from the Roots of Cynanchum stauntonii Wed, 26 Dec 2012 15:25:35 +0000 Three new steroidal glycosides, named as stauntosides L, M, and N (1–3), along with one known steroidal glycoside, anhydrohirundigenin monothevetoside (4), were isolated from the 95% ethanol extract of the roots of Cynanchum stauntonii. The structures of these new compounds were elucidated on the basis of extensive spectroscopic analyses, mainly 1D and 2D NMR, HRESI-MS, and chemical methods. Jin-Qian Yu, Zhi-Hui Zhang, An-Jun Deng, and Hai-Lin Qin Copyright © 2013 Jin-Qian Yu et al. All rights reserved. MDR Gene Expression Analysis of Six Drug-Resistant Ovarian Cancer Cell Lines Wed, 26 Dec 2012 09:04:03 +0000 Ovarian cancer is the leading cause of death among gynaecological malignancies. Multiple drug resistance makes cancer cells insensitive to chemotherapy. In this study, we developed six primary ovarian cancer cell lines (W1MR, W1CR, W1DR, W1VR, W1TR, and W1PR) resistant to drugs such as methotrexate, cisplatin, doxorubicin, vincristine, topotecan, and paclitaxel. A chemosensitivity assay MTT test was performed to assess drug cross-resistance. Quantitative real-time polymerase chain reaction and Western blot were also performed to determine mRNA and protein expression of genes involved in chemoresistance. We observed high cross-resistance to doxorubicin, vincristine, and paclitaxel in the cell lines resistant to these agents. We also found a significant correlation between resistance to these drugs and increased expression of P-gp. Two different mechanisms of topotecan resistance were observed in the W1TR and W1PR cell lines. We did not observe any correlation between MRP2 transcript and protein levels. Cell lines resistant to agents used in ovarian cancer treatment remained sensitive to methotrexate. The main mechanisms of drug resistance were due to P-gp expression in the doxorubicin, vincristine, and paclitaxel resistant cell lines and BCRP expression in the topotecan resistant cell line. Radosław Januchowski, Karolina Wojtowicz, Patrycja Sujka-Kordowska, Małgorzata Andrzejewska, and Maciej Zabel Copyright © 2013 Radosław Januchowski et al. All rights reserved. Multimolecular Salivary Mucin Complex Is Altered in Saliva of Cigarette Smokers: Detection of Disulfide Bridges by Raman Spectroscopy Wed, 26 Dec 2012 08:47:23 +0000 Saliva contains mucins, which protect epithelial cells. We showed a smaller amount of salivary mucin, both MG1 and MG2, in the premenopausal female smokers than in their nonsmoking counterparts. Smokers' MG1, which contains almost 2% cysteine/half cystine in its amino acid residues, turned out to be chemically altered in the nonsmoker’s saliva. The smaller acidic glycoprotein bands were detectable only in smoker’s saliva in the range of 20–25 kDa and at 45 kDa, suggesting that degradation, at least in part, caused the reduction of MG1 mucin. This is in agreement with the previous finding that free radicals in cigarette smoke modify mucins in both sugar and protein moieties. Moreover, proteins such as amylase and albumin are bound to other proteins through disulfide bonds and are identifiable only after reduction with DTT. Confocal laser Raman microspectroscopy identified a disulfide stretch band of significantly stronger intensity per protein in the stimulated saliva of smokers alone. We conclude that the saliva of smokers, especially stimulated saliva, contains significantly more oxidized form of proteins with increased disulfide bridges, that reduces protection for oral epithelium. Raman microspectroscopy can be used for an easy detection of the damaged salivary proteins. Motoe Taniguchi, Junko Iizuka, Yukari Murata, Yumi Ito, Mariko Iwamiya, Hiroshi Mori, Yukio Hirata, Yoshiharu Mukai, and Yuko Mikuni-Takagaki Copyright © 2013 Motoe Taniguchi et al. All rights reserved. Comparison of International Normalized Ratio Measurement between CoaguChek XS Plus and STA-R Coagulation Analyzers Mon, 24 Dec 2012 10:48:30 +0000 Background. Point-of-care testing (POCT) coagulometers are increasingly being used in the hospital setting. We investigated whether the prothrombin time international normalized ratio (INR) results by CoaguChek XS Plus (Roche Diagnostics GmbH, Mannheim, Germany) can be used reliably without being confirmed with the INR results by STA-R system (Diagnostica Stago S.A.S, Asnières sur Seine, France). Methods. A total of 118 INR measurements by CoaguChek XS Plus and STA-R were compared using Passing/Bablok regression analysis and Bland-Altman plot. Agreement of the INR measurements was further assessed in relation to dosing decision. Results. The correlation of INR measurements between CoaguChek XS Plus and STA-R was excellent (correlation coefficient = 0.964). The mean difference tended to increase as INR results increased and was 0.25 INR in the therapeutic range (2.0-3.0 INR). The overall agreement was fair to good (kappa = 0.679), and 21/118 (17.8%) INR measurements showed a difference in dosing decision. Conclusion. The positive bias of CoaguChek XS Plus may be obvious even in the therapeutic INR range, and dosing decision based on the CoaguChek XS Plus INR results would be different from that based on the STA-R results. The INR measurements by POCT coagulometers still need to be confirmed with the laboratory INR measurements. Mina Hur, Hanah Kim, Chul Min Park, Antonio La Gioia, Sang-Gyu Choi, Ju-Hee Choi, Hee-Won Moon, and Yeo-Min Yun Copyright © 2013 Mina Hur et al. All rights reserved. Role of Sex Steroid Hormones in Bacterial-Host Interactions Mon, 24 Dec 2012 07:42:38 +0000 Sex steroid hormones play important physiological roles in reproductive and nonreproductive tissues, including immune cells. These hormones exert their functions by binding to either specific intracellular receptors that act as ligand-dependent transcription factors or membrane receptors that stimulate several signal transduction pathways. The elevated susceptibility of males to bacterial infections can be related to the usually lower immune responses presented in males as compared to females. This dimorphic sex difference is mainly due to the differential modulation of the immune system by sex steroid hormones through the control of proinflammatory and anti-inflammatory cytokines expression, as well as Toll-like receptors (TLRs) expression and antibody production. Besides, sex hormones can also affect the metabolism, growth, or virulence of pathogenic bacteria. In turn, pathogenic, microbiota, and environmental bacteria are able to metabolize and degrade steroid hormones and their related compounds. All these data suggest that sex steroid hormones play a key role in the modulation of bacterial-host interactions. Elizabeth García-Gómez, Bertha González-Pedrajo, and Ignacio Camacho-Arroyo Copyright © 2013 Elizabeth García-Gómez et al. All rights reserved. Comparison of Three Multiple Allergen Simultaneous Tests: RIDA Allergy Screen, MAST Optigen, and Polycheck Allergy Mon, 24 Dec 2012 07:35:41 +0000 We compared the performances of 3 Multiple Allergen Simultaneous Test (MAST) assays: RIDA Allergy Screen (R-Biopharm, Darmstadt, Germany), MAST Optigen allergy system (Hitachi Chemical Diagnostics, Mountain View, CA), and Polycheck Allergy (Biocheck GmbH, Munster, Germany). Forty sera that tested positive with the RIDA Allergy Screen (20 for food and 20 for inhalant panel) were subjected to MAST Optigen and Polycheck Allergy. For 26 available sera with discrepant results, 62 ImmunoCAP allergen-specific IgE tests (Pharmacia Diagnostics, Uppsala, Sweden) were performed. Percent agreements (kappa value) were 87.6% (0.59) and 91.3% (0.60) between RIDA and MAST; 89.9% (0.55) and 88.3% (0.46) between RIDA and Polycheck; and 86.8% (0.51) and 90.6% (0.61) between MAST and Polycheck. Compared with ImmunoCAP, agreements (kappa value) of inhalant and food panels were 51.7% (0.04) and 33.3% (−0.38) for RIDA; 60.7% (0.27) and 81.8% (0.59) for MAST; and 65.5% (0.26) and 45.5% (0.07) for Polycheck. The agreements between RIDA, MAST, and Polycheck and ImmunoCAP-positivity were 45.7%, 88.2%, and 28.6%, respectively, and the agreements for ImmunoCAP-negativity were 37.0%, 51.9%, and 88.9%. MAST Optigen showed better agreement with ImmunoCAP than other assays in the food panel. Better sensitivity of MAST Optigen and better specificity of Polycheck Allergy were suspected. Minje Han, Sue Shin, Hyewon Park, Kyoung Un Park, Myoung Hee Park, and Eun Young Song Copyright © 2013 Minje Han et al. All rights reserved. Fermentative Production of Value-Added Products from Lignocellulosic Biomass Mon, 26 Nov 2012 13:56:33 +0000 Silvio S. da Silva, Anuj K. Chandel, S. Ranil Wickramasinghe, and José M. G. Domínguez Copyright © 2012 Silvio S. da Silva et al. All rights reserved. Bioconversion of Sugarcane Biomass into Ethanol: An Overview about Composition, Pretreatment Methods, Detoxification of Hydrolysates, Enzymatic Saccharification, and Ethanol Fermentation Mon, 26 Nov 2012 08:44:31 +0000 Depleted supplies of fossil fuel, regular price hikes of gasoline, and environmental damage have necessitated the search for economic and eco-benign alternative of gasoline. Ethanol is produced from food/feed-based substrates (grains, sugars, and molasses), and its application as an energy source does not seem fit for long term due to the increasing fuel, food, feed, and other needs. These concerns have enforced to explore the alternative means of cost competitive and sustainable supply of biofuel. Sugarcane residues, sugarcane bagasse (SB), and straw (SS) could be the ideal feedstock for the second-generation (2G) ethanol production. These raw materials are rich in carbohydrates and renewable and do not compete with food/feed demands. However, the efficient bioconversion of SB/SS (efficient pretreatment technology, depolymerization of cellulose, and fermentation of released sugars) remains challenging to commercialize the cellulosic ethanol. Among the technological challenges, robust pretreatment and development of efficient bioconversion process (implicating suitable ethanol producing strains converting pentose and hexose sugars) have a key role to play. This paper aims to review the compositional profile of SB and SS, pretreatment methods of cane biomass, detoxification methods for the purification of hydrolysates, enzymatic hydrolysis, and the fermentation of released sugars for ethanol production. Larissa Canilha, Anuj Kumar Chandel, Thais Suzane dos Santos Milessi, Felipe Antônio Fernandes Antunes, Wagner Luiz da Costa Freitas, Maria das Graças Almeida Felipe, and Silvio Silvério da Silva Copyright © 2012 Larissa Canilha et al. All rights reserved. Plasminogen Receptors Tue, 20 Nov 2012 08:49:22 +0000 Lindsey A. Miles, Edward F. Plow, David M. Waisman, and Robert J. Parmer Copyright © 2012 Lindsey A. Miles et al. All rights reserved. Ectonucleotidases in Cancer and Inflammation Tue, 16 Oct 2012 12:33:24 +0000 John Stagg, Linda F. Thompson, and Karen M. Dwyer Copyright © 2012 John Stagg et al. All rights reserved. Potential Role of Kringle-Integrin Interaction in Plasmin and uPA Actions (A Hypothesis) Tue, 16 Oct 2012 11:21:21 +0000 We previously showed that the kringle domains of plasmin and angiostatin, the N-terminal four kringles (K1–4) of plasminogen, directly bind to integrins. Angiostatin blocks tumor-mediated angiogenesis and has great therapeutic potential. Angiostatin binding to integrins may be related to the antiinflammatory action of angiostatin. We reported that plasmin induces signals through protease-activated receptor (PAR-1), and plasmin-integrin interaction may be required for enhancing plasmin concentration on the cell surface, and enhances its signaling function. Angiostatin binding to integrin does not seem to induce proliferative signals. One possible mechanism of angiostatin's inhibitory action is that angiostatin suppresses plasmin-induced PAR-1 activation by competing with plasmin for binding to integrins. Interestingly, plasminogen did not interact with 𝛼v𝛽3, suggesting that the 𝛼v𝛽3-binding sites in the kringle domains of plasminogen are cryptic. The kringle domain of urokinase-type plasminogen activator (uPA) also binds to integrins. The uPA-integrin interaction enhances uPA concentrations on the cell surface and enhances plasminogen activation on the cell surface. It is likely that integrins bind to the kringle domain, and uPAR binds to the growth factor-like domain (GFD) of uPA simultaneously, making the uPAR-uPA-integrin ternary complex. We present a docking model of the ternary complex. Yoshikazu Takada Copyright © 2012 Yoshikazu Takada. All rights reserved. CD73-Generated Adenosine: Orchestrating the Tumor-Stroma Interplay to Promote Cancer Growth Tue, 16 Oct 2012 09:30:45 +0000 Despite the coming of age of cancer immunotherapy, clinical benefits are still modest. An important barrier to successful cancer immunotherapy is that tumors employ a number of mechanisms to facilitate immune escape, including the production of anti-inflammatory cytokines, the recruitment of regulatory immune subsets, and the production of immunosuppressive metabolites. Significant therapeutic opportunity exists in targeting these immunosuppressive pathways. One such immunosuppressive pathway is the production of extracellular adenosine by CD73, an ectonucleotidase overexpressed in various types of cancer. We hereafter review the biology of CD73 and its role in cancer progression and metastasis. We describe the role of extracellular adenosine in promoting tumor growth through paracrine and autocrine action on tumor cells, endothelial cells, and immune cells. Bertrand Allard, Martin Turcotte, and John Stagg Copyright © 2012 Bertrand Allard et al. All rights reserved. Ectonucleotidases in Solid Organ and Allogeneic Hematopoietic Cell Transplantation Tue, 16 Oct 2012 09:23:38 +0000 Extracellular nucleotides are ubiquitous signalling molecules which modulate distinct physiological and pathological processes. Nucleotide concentrations in the extracellular space are strictly regulated by cell surface enzymes, called ectonucleotidases, which hydrolyze nucleotides to the respective nucleosides. Recent studies suggest that ectonucleotidases play a significant role in inflammation by adjusting the balance between ATP, a widely distributed proinflammatory danger signal, and the anti-inflammatory mediator adenosine. There is increasing evidence for a central role of adenosine in alloantigen-mediated diseases such as solid organ graft rejection and acute graft-versus-host disease (GvHD). Solid organ and hematopoietic cell transplantation are established treatment modalities for a broad spectrum of benign and malignant diseases. Immunological complications based on the recognition of nonself-antigens between donor and recipient like transplant rejection and GvHD are still major challenges which limit the long-term success of transplantation. Studies in the past two decades indicate that purinergic signalling influences the severity of alloimmune responses. This paper focuses on the impact of ectonucleotidases, in particular, NTPDase1/CD39 and ecto-5′-nucleotidase/CD73, on allograft rejection, acute GvHD, and graft-versus-leukemia effect, and on possible clinical implications for the modulation of purinergic signalling after transplantation. Petya Chernogorova and Robert Zeiser Copyright © 2012 Petya Chernogorova and Robert Zeiser. All rights reserved. Plasminogen Binding Proteins and Plasmin Generation on the Surface of Leptospira spp.: The Contribution to the Bacteria-Host Interactions Mon, 15 Oct 2012 14:28:05 +0000 Leptospirosis is considered a neglected infectious disease of human and veterinary concern. Although extensive investigations on host-pathogen interactions have been pursued by several research groups, mechanisms of infection, invasion and persistence of pathogenic Leptospira spp. remain to be elucidated. We have reported the ability of leptospires to bind human plasminogen (PLG) and to generate enzimatically active plasmin (PLA) on the bacteria surface. PLA-coated Leptospira can degrade immobilized ECM molecules, an activity with implications in host tissue penetration. Moreover, we have identified and characterized several proteins that may act as PLG-binding receptors, each of them competent to generate active plasmin. The PLA activity associated to the outer surface of Leptospira could hamper the host immune attack by conferring the bacteria some benefit during infection. The PLA-coated leptospires obstruct complement C3b and IgG depositions on the bacterial surface, most probably through degradation. The decrease of leptospiral opsonization might be an important aspect of the immune evasion strategy. We believe that the presence of PLA on the leptospiral surface may (i) facilitate host tissue penetration, (ii) help the bacteria to evade the immune system and, as a consequence, (iii) permit Leptospira to reach secondary sites of infection. Monica L. Vieira, Marina V. Atzingen, Rosane Oliveira, Renata S. Mendes, Renan F. Domingos, Silvio A. Vasconcellos, and Ana L. T. O. Nascimento Copyright © 2012 Monica L. Vieira et al. All rights reserved. Production of Ethanol from Sugars and Lignocellulosic Biomass by Thermoanaerobacter J1 Isolated from a Hot Spring in Iceland Sun, 14 Oct 2012 14:58:21 +0000 Thermophilic bacteria have gained increased attention as candidates for bioethanol production from lignocellulosic biomass. This study investigated ethanol production by Thermoanaerobacter strain J1 from hydrolysates made from lignocellulosic biomass in batch cultures. The effect of increased initial glucose concentration and the partial pressure of hydrogen on end product formation were examined. The strain showed a broad substrate spectrum, and high ethanol yields were observed on glucose (1.70 mol/mol) and xylose (1.25 mol/mol). Ethanol yields were, however, dramatically lowered by adding thiosulfate or by cocultivating strain J1 with a hydrogenotrophic methanogen with acetate becoming the major end product. Ethanol production from 4.5 g/L of lignocellulosic biomass hydrolysates (grass, hemp stem, wheat straw, newspaper, and cellulose) pretreated with acid or alkali and the enzymes Celluclast and Novozymes 188 was investigated. The highest ethanol yields were obtained on cellulose (7.5 mM·g−1) but the lowest on straw (0.8 mM·g−1). Chemical pretreatment increased ethanol yields substantially from lignocellulosic biomass but not from cellulose. The largest increase was on straw hydrolysates where ethanol production increased from 0.8 mM·g−1 to 3.3 mM·g−1 using alkali-pretreated biomass. The highest ethanol yields on lignocellulosic hydrolysates were observed with hemp hydrolysates pretreated with acid, 4.2 mM·g−1. Jan Eric Jessen and Johann Orlygsson Copyright © 2012 Jan Eric Jessen and Johann Orlygsson. All rights reserved. Bacterial Plasminogen Receptors: Mediators of a Multifaceted Relationship Sun, 14 Oct 2012 14:54:21 +0000 Multiple species of bacteria are able to sequester the host zymogen plasminogen to the cell surface. Once localised to the bacterial surface, plasminogen can act as a cofactor in adhesion, or, following activation to plasmin, provide a source of potent proteolytic activity. Numerous bacterial plasminogen receptors have been identified, and the mechanisms by which they interact with plasminogen are diverse. Here we provide an overview of bacterial plasminogen receptors and discuss the diverse role bacterial plasminogen acquisition plays in the relationship between bacteria and the host. Martina L. Sanderson-Smith, David M. P. De Oliveira, Marie Ranson, and Jason D. McArthur Copyright © 2012 Martina L. Sanderson-Smith et al. All rights reserved. The Biochemistry and Regulation of S100A10: A Multifunctional Plasminogen Receptor Involved in Oncogenesis Sun, 14 Oct 2012 14:49:48 +0000 The plasminogen receptors mediate the production and localization to the cell surface of the broad spectrum proteinase, plasmin. S100A10 is a key regulator of cellular plasmin production and may account for as much as 50% of cellular plasmin generation. In parallel to plasminogen, the plasminogen-binding site on S100A10 is highly conserved from mammals to fish. S100A10 is constitutively expressed in many cells and is also induced by many diverse factors and physiological stimuli including dexamethasone, epidermal growth factor, transforming growth factor-α, interferon-γ, nerve growth factor, keratinocyte growth factor, retinoic acid, and thrombin. Therefore, S100A10 is utilized by cells to regulate plasmin proteolytic activity in response to a wide diversity of physiological stimuli. The expression of the oncogenes, PML-RARα and KRas, also stimulates the levels of S100A10, suggesting a role for S100A10 in pathophysiological processes such as in the oncogenic-mediated increases in plasmin production. The S100A10-null mouse model system has established the critical role that S100A10 plays as a regulator of fibrinolysis and oncogenesis. S100A10 plays two major roles in oncogenesis, first as a regulator of cancer cell invasion and metastasis and secondly as a regulator of the recruitment of tumor-associated cells, such as macrophages, to the tumor site. Patricia A. Madureira, Paul A. O'Connell, Alexi P. Surette, Victoria A. Miller, and David M. Waisman Copyright © 2012 Patricia A. Madureira et al. All rights reserved. The Serine Protease Plasmin Triggers Expression of the CC-Chemokine Ligand 20 in Dendritic Cells via Akt/NF-κB-Dependent Pathways Sun, 14 Oct 2012 14:47:44 +0000 The number of dendritic cells is increased in advanced atherosclerotic lesions. In addition, plasmin, which might stimulate dendritic cells, is generated in atherosclerotic lesions. Here, we investigated cytokine and chemokine induction by plasmin in human dendritic cells. In human atherosclerotic vessel sections, plasmin colocalized with dendritic cells and the CC-chemokine ligand 20 (CCL20, MIP-3α), which is important for homing of lymphocytes and dendritic cells to sites of inflammation. Stimulation of human dendritic cells with plasmin, but not with catalytically inactivated plasmin, induced transcriptional regulation of CCL20. By contrast, proinflammatory cytokines such as TNF-α, IL-1α, and IL-1β were not induced. The plasmin-mediated CCL20 expression was preceded by activation of Akt and MAP kinases followed by activation of the transcription factor NF-κB as shown by phosphorylation of its inhibitor IκBα, by nuclear localization of p65, its phosphorylation, and binding to NF-κB consensus sequences. The plasmin-induced CCL20 expression was dependent on Akt- and ERK1/2-mediated phosphorylation of IκBα on Ser32/36 and of p65 on Ser276, whereas p38 MAPK appeared to be dispensable. Thus, plasmin triggers release of the chemokine CCL20 from dendritic cells, which might facilitate accumulation of CCR6+ immune cells in areas of plasmin generation such as inflamed tissues including atherosclerotic lesions. Xuehua Li, Tatiana Syrovets, and Thomas Simmet Copyright © 2012 Xuehua Li et al. All rights reserved. Characterization of Plasminogen Binding to NB4 Promyelocytic Cells Using Monoclonal Antibodies against Receptor-Induced Binding Sites in Cell-Bound Plasminogen Sun, 14 Oct 2012 13:55:36 +0000 The NB4 promyelocytic cell line exhibits many of the characteristics of acute promyelocytic leukemia blast cells, including the translocation (15 : 17) that fuses the PML gene on chromosome 15 to the RARα gene on chromosome 17. These cells have a very high fibrinolytic capacity. In addition to a high secretion of urokinase, NB4 cells exhibit a 10-fold higher plasminogen binding capacity compared with other leukemic cell lines. When tissue-type plasminogen activator was added to acid-treated cells, plasmin generation was 20–26-fold higher than that generated by U937 cells or peripheral blood neutrophils, respectively. We found that plasminogen bound to these cells can be detected by fluorescence-activated cell sorting using an antiplasminogen monoclonal antibody that specifically reacts with this antigen when it is bound to cell surfaces. All-trans retinoid acid treatment of NB4 cells markedly decreased the binding of this monoclonal antibody. This cell line constitutes a unique model to explore plasminogen binding and activation on cell surfaces that can be modulated by all-trans retinoid acid treatment. Mercè Jardí, Pere Fàbregas, María Sagarra-Tió, María José Pérez-Lucena, and Jordi Félez Copyright © 2012 Mercè Jardí et al. All rights reserved. Cell Surface Remodeling by Plasmin: A New Function for an Old Enzyme Sun, 14 Oct 2012 13:32:04 +0000 Plasmin, one of the most potent and reactive serine proteases, is involved in various physiological processes, including embryo development, thrombolysis, wound healing and cancer progression. The proteolytic activity of plasmin is tightly regulated through activation of its precursor, plasminogen, only at specific times and in defined locales as well as through inhibition of active plasmin by its abundant natural inhibitors. By exploiting the plasminogen activating system and overexpressing distinct components of the plasminogen activation cascade, such as pro-uPA, uPAR and plasminogen receptors, malignant cells can enhance the generation of plasmin which in turn, modifies the tumor microenvironment to sustain cancer progression. While plasmin-mediated degradation and modification of extracellular matrix proteins, release of growth factors and cytokines from the stroma as well as activation of several matrix metalloproteinase zymogens, all have been a focus of cancer research studies for decades, the ability of plasmin to cleave transmembrane molecules and thereby to generate functionally important cleaved products which induce outside-in signal transduction, has just begun to receive sufficient attention. Herein, we highlight this relatively understudied, but important function of the plasmin enzyme as it is generated de novo at the interface between cross-talking cancer and host cells. Elena I. Deryugina and James P. Quigley Copyright © 2012 Elena I. Deryugina and James P. Quigley. All rights reserved. Production of Adenosine by Ectonucleotidases: A Key Factor in Tumor Immunoescape Sun, 14 Oct 2012 13:07:39 +0000 It is now well known that tumor immunosurveillance contributes to the control of cancer growth. Many mechanisms can be used by cancer cells to avoid the antitumor immune response. One such mechanism relies on the capacity of cancer cells or more generally of the tumor microenvironment to generate adenosine, a major molecule involved in antitumor T cell response suppression. Adenosine is generated by the dephosphorylation of extracellular ATP released by dying tumor cells. The conversion of ATP into adenosine is mediated by ectonucleotidase molecules, namely, CD73 and CD39. These molecules are frequently expressed in the tumor bed by a wide range of cells including tumor cells, regulatory T cells, Th17 cells, myeloid cells, and stromal cells. Recent evidence suggests that targeting adenosine by inhibiting ectonucleotidases may restore the resident antitumor immune response or enhance the efficacy of antitumor therapies. This paper will underline the impact of adenosine and ectonucleotidases on the antitumor response. François Ghiringhelli, Mélanie Bruchard, Fanny Chalmin, and Cédric Rébé Copyright © 2012 François Ghiringhelli et al. All rights reserved. Analysis of Casein Biopolymers Adsorption to Lignocellulosic Biomass as a Potential Cellulase Stabilizer Sun, 14 Oct 2012 13:06:09 +0000 Although lignocellulosic materials have a good potential to substitute current feedstocks used for ethanol production, conversion of these materials to fermentable sugars is still not economical through enzymatic hydrolysis. High cost of cellulase has prompted research to explore techniques that can prevent from enzyme deactivation. Colloidal proteins of casein can form monolayers on hydrophobic surfaces that alleviate the de-activation of protein of interest. Scanning electron microscope (SEM), fourier transform infrared spectroscopy (FT-IR), capillary electrophoresis (CE), and Kjeldahl and BSA protein assays were used to investigate the unknown mechanism of action of induced cellulase activity during hydrolysis of casein-treated biomass. Adsorption of casein to biomass was observed with all of the analytical techniques used and varied depending on the pretreatment techniques of biomass. FT-IR analysis of amides I and II suggested that the substructure of protein from casein or skim milk were deformed at the time of contact with biomass. With no additive, the majority of one of the cellulase mono-component, 97.1 ± 1.1, was adsorbed to CS within 24 h, this adsorption was irreversible and increased by 2% after 72 h. However, biomass treatment with skim-milk and casein reduced the adsorption to 32.9% ± 6.0 and 82.8% ± 6.0, respectively. Anahita Dehkhoda Eckard, Kasiviswanathan Muthukumarappan, and William Gibbons Copyright © 2012 Anahita Dehkhoda Eckard et al. All rights reserved. PHA Productivity and Yield of Ralstonia eutropha When Intermittently or Continuously Fed a Mixture of Short Chain Fatty Acids Sun, 14 Oct 2012 13:02:03 +0000 The research described in this present study was part of a larger effort focused on developing a dual substrate, dual fermentation process to produce Polyhydroxyalkanoate (PHA). The focus of this study was developing and optimizing a strategy for feeding a mixture of SCFAs (simulated ARF) and maximizing PHA production in a cost-effective way. Three different feeding strategies were examined in this study. The substrate evaluated in this study for the growth phase of R. eutropha was condensed corn solubles, a low-value byproduct of the dry-mill, corn ethanol industry. The culture was grown to high cell densities in nitrogen-supplemented condensed corn solubles media in 5 L bioreactors. The overall growth rate of R. eutropha was 0.2 h−1. The 20 mL ARF feeding every 3 h from 48 to 109 h strategy gave the best results in terms of PHA production. PHA productivity (0.0697 g L−1 h−1), PHA concentration (8.37 g L−1), and PHA content (39.52%) were the highest when ARF was fed every 3 h for 61 h. This study proved that condensed corn solubles can be potentially used as a growth medium to boost PHA production by R. eutropha thus reducing the overall cost of biopolymer production. Panchali Chakraborty, Kasiviswanathan Muthukumarappan, and William R. Gibbons Copyright © 2012 Panchali Chakraborty et al. All rights reserved. Changes in the Material Characteristics of Maize Straw during the Pretreatment Process of Methanation Sun, 14 Oct 2012 13:00:52 +0000 Pretreatment technology is important to the direct methanation of straw. This study used fresh water, four bacterium agents (stem rot agent, “result” microbe decomposition agent, straw pretreatment composite bacterium agent, and complex microorganism agent), biogas slurry, and two chemical reagents (sodium hydroxide and urea) as pretreatment promoters. Different treatments were performed, and the changes in the straw pH value, temperature, total solid (TS), volatile solid (VS), and carbon-nitrogen ratio (C/N ratio) under different pretreatment conditions were analyzed. The results showed that chemical promoters were more efficient than biological promoters in straw maturity. Pretreatment using sodium hydroxide induced the highest degree of straw maturity. However, its C/N ratio had to be reduced during fermentation. In contrast, the C/N ratio of the urea-pretreated straw was low and was easy to regulate when used as anaerobic digestion material. The biogas slurry pretreatment was followed by pretreatments using four different bacterium agents, among which the effect of the complex microorganism agent (BA4) was more efficient than the others. The current study is significant to the direct and efficient methanation of straw. Yongzhong Feng, Xiaoling Zhao, Yan Guo, Gaihe Yang, Jianchao Xi, and Guangxin Ren Copyright © 2012 Yongzhong Feng et al. All rights reserved. The Potential of Cellulosic Ethanol Production from Grasses in Thailand Sun, 14 Oct 2012 12:53:07 +0000 The grasses in Thailand were analyzed for the potentiality as the alternative energy crops for cellulosic ethanol production by biological process. The average percentage composition of cellulose, hemicellulose, and lignin in the samples of 18 types of grasses from various provinces was determined as 31.85–38.51, 31.13–42.61, and 3.10–5.64, respectively. The samples were initially pretreated with alkaline peroxide followed by enzymatic hydrolysis to investigate the enzymatic saccharification. The total reducing sugars in most grasses ranging from 500–600 mg/g grasses (70–80% yield) were obtained. Subsequently, 11 types of grasses were selected as feedstocks for the ethanol production by simultaneous saccharification and cofermentation (SSCF). The enzymes, cellulase and xylanase, were utilized for hydrolysis and the yeasts, Saccharomyces cerevisiae and Pichia stipitis, were applied for cofermentation at 35°C for 7 days. From the results, the highest yield of ethanol, 1.14 g/L or 0.14 g/g substrate equivalent to 32.72% of the theoretical values was obtained from Sri Lanka ecotype vetiver grass. When the yields of dry matter were included in the calculations, Sri Lanka ecotype vetiver grass gave the yield of ethanol at 1,091.84 L/ha/year, whereas the leaves of dwarf napier grass showed the maximum yield of 2,720.55 L/ha/year (0.98 g/L or 0.12 g/g substrate equivalent to 30.60% of the theoretical values). Jinaporn Wongwatanapaiboon, Kunn Kangvansaichol, Vorakan Burapatana, Ratanavalee Inochanon, Pakorn Winayanuwattikun, Tikamporn Yongvanich, and Warawut Chulalaksananukul Copyright © 2012 Jinaporn Wongwatanapaiboon et al. All rights reserved. So Many Plasminogen Receptors: Why? Sun, 14 Oct 2012 12:51:10 +0000 Plasminogen and plasmin tether to cell surfaces through ubiquitously expressed and structurally quite dissimilar family of proteins, as well as some nonproteins, that are collectively referred to as plasminogen receptors. Of the more than one dozen plasminogen receptors that have been identified, many have been shown to facilitate plasminogen activation to plasmin and to protect bound plasmin from inactivation by inhibitors. The generation of such localized and sustained protease activity is utilized to facilitate numerous cellular responses, including responses that depend on cellular migration. However, many cells express multiple plasminogen receptors and numerous plasminogen receptors are expressed on many different cell types. Furthermore, several different plasminogen receptors can be used to support the same cellular response, such as inflammatory cell migration. Here, we discuss the perplexing issue: why are there so many different Plg-Rs? Edward F. Plow, Loic Doeuvre, and Riku Das Copyright © 2012 Edward F. Plow et al. All rights reserved. The Plasminogen Receptor, Plg-RKT, and Macrophage Function Sun, 14 Oct 2012 12:49:00 +0000 When plasminogen binds to cells its activation to plasmin is markedly enhanced compared to the reaction in solution. Thus, cells become armed with the broad spectrum proteolytic activity of plasmin. Cell-surface plasmin plays a key role in macrophage recruitment during the inflammatory response. Proteins exposing basic residues on the cell surface promote plasminogen activation on eukaryotic cells. We have used a proteomics approach combining targeted proteolysis with carboxypeptidase B and multidimensional protein identification technology, MudPIT, and a monocyte progenitor cell line to identify a novel transmembrane protein, the plasminogen receptor, Plg-RKT. Plg-RKT exposes a C-terminal lysine on the cell surface in an orientation to bind plasminogen and promote plasminogen activation. Here we review the characteristics of this new protein, with regard to membrane topology, conservation of sequence across species, the role of its C-terminus in plasminogen binding, its function in plasminogen activation, cell migration, and its role in macrophage recruitment in the inflammatory response. Lindsey A. Miles, Shahrzad Lighvani, Nagyung Baik, Nicholas M. Andronicos, Emily I. Chen, Caitlin M. Parmer, Sophia Khaldoyanidi, Jenna E. Diggs, William B. Kiosses, Mark P. Kamps, John R. Yates III, and Robert J. Parmer Copyright © 2012 Lindsey A. Miles et al. All rights reserved. The Plasminogen Activation System and the Regulation of Catecholaminergic Function Sun, 14 Oct 2012 12:14:42 +0000 The local environment of neurosecretory cells contains the major components of the plasminogen activation system, including the plasminogen activators, tissue plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), as well as binding sites for t-PA, the receptor for u-PA (uPAR), and also the plasminogen activator inhibitor, PAI-1. Furthermore, these cells express specific binding sites for plasminogen, which is available in the circulation and in interstitial fluid. Colocalization of plasminogen and its activators on cell surfaces provides a mechanism for promoting local plasminogen activation. Plasmin is retained on the cell surface where it is protected from its inhibitor, 𝛼2-antiplasmin. In neurosecretory cells, localized plasmin activity provides a mechanism for extracellular processing of secreted hormones. Neurotransmitter release from catecholaminergic cells is negatively regulated by cleavage products formed by plasmin-mediated proteolysis. Recently, we have identified a major plasminogen receptor, Plg-RKT. We have found that Plg-RKT is highly expressed in chromaffin cells of the adrenal medulla as well as in other catecholaminergic cells and tissues. Plg-RKT-dependent plasminogen activation plays a key role in regulating catecholaminergic neurosecretory cell function. Hongdong Bai, Samir Nangia, and Robert J. Parmer Copyright © 2012 Hongdong Bai et al. All rights reserved. The CD39-Adenosinergic Axis in the Pathogenesis of Immune and Nonimmune Diabetes Sun, 14 Oct 2012 11:47:13 +0000 Diabetes mellitus encompasses two distinct disease processes: autoimmune Type 1 (T1D) and nonimmune Type 2 (T2D) diabetes. Despite the disparate aetiologies, the disease phenotype of hyperglycemia and the associated complications are similar. In this paper, we discuss the role of the CD39-adenosinergic axis in the pathogenesis of both T1D and T2D, with particular emphasis on the role of CD39 and CD73. Joanne S. J. Chia, Jennifer L. McRae, Peter J. Cowan, and Karen M. Dwyer Copyright © 2012 Joanne S. J. Chia et al. All rights reserved. The Annexin A2/S100A10 System in Health and Disease: Emerging Paradigms Sun, 14 Oct 2012 11:32:18 +0000 Since its discovery as a src kinase substrate more than three decades ago, appreciation for the physiologic functions of annexin A2 and its associated proteins has increased dramatically. With its binding partner S100A10 (p11), A2 forms a cell surface complex that regulates generation of the primary fibrinolytic protease, plasmin, and is dynamically regulated in settings of hemostasis and thrombosis. In addition, the complex is transcriptionally upregulated in hypoxia and promotes pathologic neoangiogenesis in the tissues such as the retina. Dysregulation of both A2 and p11 has been reported in examples of rodent and human cancer. Intracellularly, A2 plays a critical role in endosomal repair in postarthroplastic osteolysis, and intracellular p11 regulates serotonin receptor activity in psychiatric mood disorders. In human studies, the A2 system contributes to the coagulopathy of acute promyelocytic leukemia, and is a target of high-titer autoantibodies in patients with antiphospholipid syndrome, cerebral thrombosis, and possibly preeclampsia. Polymorphisms in the human ANXA2 gene have been associated with stroke and avascular osteonecrosis of bone, two severe complications of sickle cell disease. Together, these new findings suggest that manipulation of the annexin A2/S100A10 system may offer promising new avenues for treatment of a spectrum of human disorders. Nadia Hedhli, Domenick J. Falcone, Bihui Huang, Gabriela Cesarman-Maus, Rosemary Kraemer, Haiyan Zhai, Stella E. Tsirka, Laura Santambrogio, and Katherine A. Hajjar Copyright © 2012 Nadia Hedhli et al. All rights reserved. Factors Affecting Poly(3-hydroxybutyrate) Production from Oil Palm Frond Juice by Cupriavidus necator (CCUG52238T) Sun, 14 Oct 2012 10:37:01 +0000 Factors influencing poly(3-hydroxybutyrate) P(3HB) production by  Cupriavidus necator  CCUG52238T utilizing oil palm frond (OPF) juice were clarified in this study. Effects of initial medium pH, agitation speed, and ammonium sulfate (NH4)2SO4 concentration on the production of P(3HB) were investigated in shake flasks experiments using OPF juice as the sole carbon source. The highest P(3HB) content was recorded at pH 7.0, agitation speed of 220 rpm, and (NH4)2SO4 concentration at 0.5 g/L. By culturing the wild-type strain of C. necator under the aforementioned conditions, the cell dry weight (CDW) and P(3HB) content obtained were 9.31 ± 0.13 g/L and 45 ± 1.5 wt.%, respectively. This accounted for 40% increment of P(3HB) content compared to the nonoptimized condition. In the meanwhile, the effect of dissolved oxygen tension (DOT) on P(3HB) production was investigated in a 2-L bioreactor. Highest CDW (11.37 g/L) and P(3HB) content (44 wt.%) were achieved when DOT level was set at 30%. P(3HB) produced from OPF juice had a tensile strength of 40 MPa and elongation at break of 8% demonstrated that P(3HB) produced from renewable and cheap carbon source is comparable to those produced from commercial substrate. Mior Ahmad Khushairi Mohd Zahari, Hidayah Ariffin, Mohd Noriznan Mokhtar, Jailani Salihon, Yoshihito Shirai, and Mohd Ali Hassan Copyright © 2012 Mior Ahmad Khushairi Mohd Zahari et al. All rights reserved. Lovastatin Production by Aspergillus terreus Using Agro-Biomass as Substrate in Solid State Fermentation Sun, 14 Oct 2012 10:35:00 +0000 Ability of two strains of Aspergillus terreus (ATCC 74135 and ATCC 20542) for production of lovastatin in solid state fermentation (SSF) using rice straw (RS) and oil palm frond (OPF) was investigated. Results showed that RS is a better substrate for production of lovastatin in SSF. Maximum production of lovastatin has been obtained using A. terreus ATCC 74135 and RS as substrate without additional nitrogen source (157.07 mg/kg dry matter (DM)). Although additional nitrogen source has no benefit effect on enhancing the lovastatin production using RS substrate, it improved the lovastatin production using OPF with maximum production of 70.17 and 63.76 mg/kg DM for A. terreus ATCC 20542 and A. terreus ATCC 74135, respectively (soybean meal as nitrogen source). Incubation temperature, moisture content, and particle size had shown significant effect on lovastatin production (𝑃<0.01) and inoculums size and pH had no significant effect on lovastatin production (𝑃>0.05). Results also have shown that pH 6, 25°C incubation temperature, 1.4 to 2 mm particle size, 50% initial moisture content, and 8 days fermentation time are the best conditions for lovastatin production in SSF. Maximum production of lovastatin using optimized condition was 175.85 and 260.85 mg/kg DM for A. terreus ATCC 20542 and ATCC 74135, respectively, using RS as substrate. Mohammad Faseleh Jahromi, Juan Boo Liang, Yin Wan Ho, Rosfarizan Mohamad, Yong Meng Goh, and Parisa Shokryazdan Copyright © 2012 Mohammad Faseleh Jahromi et al. All rights reserved. The Plasminogen System in Regulating Stem Cell Mobilization Sun, 14 Oct 2012 09:26:46 +0000 The treatment of patients with hematopoietic progenitor and stem cells (HPSCs) to reconstitute hematopoiesis after myeloablative therapy or to repair ischemia after myocardial infarction has significantly improved clinical outcomes. Successful blood or bone marrow transplants require a sufficient number of HPSCs capable of homing to the injured site to regenerate tissue. Granulocyte-colony stimulating factor (G-CSF) is widely used clinically for stem cell mobilization. However, in some patients the response is poor, thus a better understanding of the mechanisms underlying G-CSF-regulated stem cell mobilization is needed. The pasminogen (Plg) system is the primary fibrinolytic pathway responsible for clot dissolution after thrombosis. Recent evidence suggests that Plg plays a pivotal role in stem cell mobilization from the bone marrow to the peripheral circulation, particularly in HPSC mobilization in response to G-CSF. This paper will discuss the potential mechanisms by which the Plg system regulates stem cell mobilization, focusing on stepwise proteolysis and signal transduction during HPSC egress from their bone marrow niche. Clear elucidation of the underlying mechanisms may lead to the development of new Plg-based therapeutic strategies to improve stem cell mobilization in treating hematological and cardiovascular diseases. Yanqing Gong and Jane Hoover-Plow Copyright © 2012 Yanqing Gong and Jane Hoover-Plow. All rights reserved. Ocriplasmin for Vitreoretinal Diseases Sun, 14 Oct 2012 09:25:13 +0000 Fibronectin and laminin are clinically relevant plasmin receptors in the eye. Located at the vitreoretinal interface, they are cleaved by ocriplasmin (Microplasmin, ThromboGenics, Iselin, NJ), a novel ophthalmic medication. A series of clinical trials to study ocriplasmin for the treatment of vitreoretinal diseases such as vitreomacular traction, macular hole, and exudative age-related macular degeneration are underway. The results are promising and may impact patient care. Irena Tsui, Carolyn K. Pan, Ehsan Rahimy, and Steven D. Schwartz Copyright © 2012 Irena Tsui et al. All rights reserved. Ectonucleotidases in Tumor Cells and Tumor-Associated Immune Cells: An Overview Sun, 14 Oct 2012 09:20:36 +0000 Increasing evidence points out that genetic alteration does not guarantee the development of a tumor and indicates that complex interactions of tumor cells with the microenvironment are fundamental to tumorigenesis. Among the pathological alterations that give tumor cells invasive potential, disruption of inflammatory response and the purinergic signaling are emerging as an important component of cancer progression. Nucleotide/nucleoside receptor-mediated cell communication is orchestrated by ectonucleotidases, which efficiently hydrolyze ATP, ADP, and AMP to adenosine. ATP can act as danger signaling whereas adenosine, acts as a negative feedback mechanism to limit inflammation. Many tumors exhibit alterations in ATP-metabolizing enzymes, which may contribute to the pathological events observed in solid cancer. In this paper, the main changes occurring in the expression and activity of ectonucleotidases in tumor cells as well as in tumor-associated immune cells are discussed. Furthermore, we focus on the understanding of the purinergic signaling primarily as exemplified by research done by the group on gliomas. Letícia Scussel Bergamin, Elizandra Braganhol, Rafael Fernandes Zanin, Maria Isabel Albano Edelweiss, and Ana Maria Oliveira Battastini Copyright © 2012 Letícia Scussel Bergamin et al. All rights reserved. A Novel Partially Biobased PAN-Lignin Blend as a Potential Carbon Fiber Precursor Sun, 14 Oct 2012 09:19:51 +0000 Blends of polyacrylonitrile (PAN) and lignin were prepared with three different lignin types by solution blending and solution casting. Among three types of lignin, one type was chosen and different blend concentrations were prepared and casted. The casted blend films were characterized chemically with fourier transform infrared spectroscopy (FTIR), and thermally with thermogravimetric analysis (TGA). The mechanical properties of the blends were measured using dynamic mechanical analysis (DMA). FTIR analysis shows an excellent interaction of PAN and lignin. The interaction of the lignins and PAN was confirmed by TGA analysis. The DMA results reveal that the lignin enhance the mechanical properties of PAN at room temperature and elevated temperatures. The blend structure and morphology were observed using scanning electron microscopy (SEM). SEM images show that excellent polymer blends were prepared. The results show that it is possible to develop a new precursor material with a blend of lignin and PAN. These studies show that the side product of paper and cellulosic bioethanol industries, namely, lignin can be used for new application areas. M. Özgür Seydibeyoğlu Copyright © 2012 M. Özgür Seydibeyoğlu. All rights reserved. CD73 Is Critical for the Resolution of Murine Colonic Inflammation Sun, 14 Oct 2012 09:16:04 +0000 CD73 is a glycosyl-phosphatidylinositol-(GPI-) linked membrane protein that catalyzes the extracellular dephosphorylation of adenosine monophosphate (AMP) to adenosine. Adenosine is a negative regulator of inflammation and prevents excessive cellular damage. We investigated the role of extracellular adenosine in the intestinal mucosa during the development of Dextran-Sulfate-Sodium-(DSS-)salt-induced colitis in mice that lack CD73 (CD73−/−) and are unable to synthesize extracellular adenosine. We have found that, compared to wild-type (WT) mice, CD73−/− mice are highly susceptible to DSS-induced colitis. CD73−/− mice exhibit pronounced weight loss, slower weight recovery, an increase in gut permeability, a decrease in expression of tight junctional adhesion molecules, as well as unresolved inflammation following the removal of DSS. Moreover, colonic epithelia in CD73−/− mice exhibited increased TLR9 expression, high levels of IL-1β and TNF-α, and constitutive activation of NF-κB. We conclude that CD73 expression in the colon is critical for regulating the magnitude and the resolution of colonic immune responses. Margaret S. Bynoe, Adam T. Waickman, Deeqa A. Mahamed, Cynthia Mueller, Jeffrey H. Mills, and Agnieszka Czopik Copyright © 2012 Margaret S. Bynoe et al. All rights reserved. α-Enolase, a Multifunctional Protein: Its Role on Pathophysiological Situations Sun, 14 Oct 2012 09:15:13 +0000 α-Enolase is a key glycolytic enzyme in the cytoplasm of prokaryotic and eukaryotic cells and is considered a multifunctional protein. α-enolase is expressed on the surface of several cell types, where it acts as a plasminogen receptor, concentrating proteolytic plasmin activity on the cell surface. In addition to glycolytic enzyme and plasminogen receptor functions, α-Enolase appears to have other cellular functions and subcellular localizations that are distinct from its well-established function in glycolysis. Furthermore, differential expression of α-enolase has been related to several pathologies, such as cancer, Alzheimer's disease, and rheumatoid arthritis, among others. We have identified α-enolase as a plasminogen receptor in several cell types. In particular, we have analyzed its role in myogenesis, as an example of extracellular remodelling process. We have shown that α-enolase is expressed on the cell surface of differentiating myocytes, and that inhibitors of α-enolase/plasminogen binding block myogenic fusion in vitro and skeletal muscle regeneration in mice. α-Enolase could be considered as a marker of pathological stress in a high number of diseases, performing several of its multiple functions, mainly as plasminogen receptor. This paper is focused on the multiple roles of the α-enolase/plasminogen axis, related to several pathologies. Àngels Díaz-Ramos, Anna Roig-Borrellas, Ana García-Melero, and Roser López-Alemany Copyright © 2012 Àngels Díaz-Ramos et al. All rights reserved. Bacterial Plasminogen Receptors Utilize Host Plasminogen System for Effective Invasion and Dissemination Sun, 14 Oct 2012 09:14:49 +0000 In order for invasive pathogens to migrate beyond the site of infection, host physiological barriers such as the extracellular matrix, the basement membrane, and encapsulating fibrin network must be degraded. To circumvent these impediments, proteolytic enzymes facilitate the dissemination of the microorganism. Recruitment of host proteases to the bacterial surface represents a particularly effective mechanism for enhancing invasiveness. Plasmin is a broad spectrum serine protease that degrades fibrin, extracellular matrices, and connective tissue. A large number of pathogens express plasminogen receptors which immobilize plasmin(ogen) on the bacterial surface. Surface-bound plasminogen is then activated by plasminogen activators to plasmin through limited proteolysis thus triggering the development of a proteolytic surface on the bacteria and eventually assisting the spread of bacteria. The host hemostatic system plays an important role in systemic infection. The interplay between hemostatic processes such as coagulation and fibrinolysis and the inflammatory response constitutes essential components of host defense and bacterial invasion. The goal of this paper is to highlight mechanisms whereby pathogenic bacteria, by engaging surface receptors, utilize and exploit the host plasminogen and fibrinolytic system for the successful dissemination within the host. Sarbani Bhattacharya, Victoria A. Ploplis, and Francis J. Castellino Copyright © 2012 Sarbani Bhattacharya et al. All rights reserved. The Role of Nephritis-Associated Plasmin Receptor (NAPlr) in Glomerulonephritis Associated with Streptococcal Infection Sun, 14 Oct 2012 09:13:51 +0000 It is well known that glomerulonephritis can occur after streptococcal infection, which is classically referred to as acute poststreptococcal glomerulonephritis (APSGN). The pathogenic mechanism of APSGN has been described by so-called immune complex theory, which involves glomerular deposition of nephritogenic streptococcal antigen and subsequent formation of immune complexes in situ and/or the deposition of circulating antigen-antibody complexes. However, the exact entity of the causative antigen has remained a matter of debate. We isolated a nephritogenic antigen for APSGN from the cytoplasmic fractions of group A streptococcus (GAS) depending on the affinity for IgG of APSGN patients. The amino acid and the nucleotide sequences of the isolated protein revealed to be highly identical to those of reported plasmin(ogen) receptor of GAS. Thus, we termed this antigen nephritis-associated plasmin receptor (NAPlr). Immunofluorescence staining of the renal biopsy tissues with anti-NAPlr antibody revealed glomerular NAPlr deposition in essentially all patients with early-phase APSGN. Furthermore, glomerular plasmin activity was detected by in situ zymography in the distribution almost identical to NAPlr deposition in renal biopsy tissues of APSGN patients. These data suggest that NAPlr has a direct, nonimmunologic function as a plasmin receptor and may contribute to the pathogenesis of APSGN by maintaining plasmin activity. Takashi Oda, Nobuyuki Yoshizawa, Kazuo Yamakami, Yutaka Sakurai, Hanako Takechi, Kojiro Yamamoto, Naoki Oshima, and Hiroo Kumagai Copyright © 2012 Takashi Oda et al. All rights reserved. Enzymatic Saccharification and Ethanol Fermentation of Reed Pretreated with Liquid Hot Water Sun, 14 Oct 2012 09:13:02 +0000 Reed is a widespread-growing, inexpensive, and readily available lignocellulosic material source in northeast China. The objective of this study is to evaluate the liquid hot water (LHW) pretreatment efficiency of reed based on the enzymatic digestibility and ethanol fermentability of water-insoluble solids (WISs) from reed after the LHW pretreatment. Several variables in the LHW pretreatment and enzymatic hydrolysis process were optimized. The conversion of glucan to glucose and glucose concentrations are considered as response variables in different conditions. The optimum conditions for the LHW pretreatment of reed area temperature of 180°C for 20min and a solid-to-liquid ratio of 1 : 10. These optimum conditions for the LHW pretreatment of reed resulted in a cellulose conversion rate of 82.59% in the subsequent enzymatic hydrolysis at 50°C for 72 h with a cellulase loading of 30 filter paper unit per gram of oven-dried WIS. Increasing the pretreatment temperature resulted in a higher enzymatic digestibility of the WIS from reed. Separate hydrolysis and fermentation of WIS showed that the conversion of glucan to ethanol reached 99.5% of the theoretical yield. The LHW pretreatment of reed is a suitable method to acquire a high recovery of fermentable sugars and high ethanol conversion yield. Jie Lu, XueZhi Li, Jian Zhao, and Yinbo Qu Copyright © 2012 Jie Lu et al. All rights reserved. Accelerated Fibrinolysis and Its Propagation on Vascular Endothelial Cells by Secreted and Retained tPA Sun, 14 Oct 2012 09:12:34 +0000 We successfully visualized the secretory dynamics of tissue-type plasminogen activator (tPA) tagged by green fluorescent protein (tPA-GFP) from cultured vascular endothelial cells (VECs) using total internal reflection fluorescence (TIRF) microscopy and demonstrated that tPA-GFP secreted from VECs was retained on cell surfaces in a heavy-chain-dependent manner. Progressive binding of Alexa568-labeled Glu-plasminogen was also observed on the surface of active tPA-GFP expressing cells via lysine binding sites (LBS), which was not observed on inactive mutant tPA-GFP expressing cells. These results suggest that retained tPA on VECs effectively activated plasminogen to plasmin, which then facilitated the binding of additional plasminogen on the cell surface by proteolytically cleaving surface-associated proteins and exposing their C-terminal lysine residues. Thus prolonged retention of tPA appeared to play an important role in initiating and amplifying plasmin generation on VECs. LBS-dependent binding of plasminogen was also observed as a narrow band at the lytic front of the fibrin mesh formed on active tPA-GFP expressing cells, which expanded outward as the lytic area increased. This binding was not observed on inactive mutant tPA-GFP expressing cells or in the presence of aprotinin. The binding of plasminogen to partially digested fibrin appears to be indispensable for spontaneous fibrinolysis. Tetsumei Urano and Yuko Suzuki Copyright © 2012 Tetsumei Urano and Yuko Suzuki. All rights reserved. Stable Plastid Transformation for High-Level Recombinant Protein Expression: Promises and Challenges Mon, 08 Oct 2012 16:19:29 +0000 Plants are a promising expression system for the production of recombinant proteins. However, low protein productivity remains a major obstacle that limits extensive commercialization of whole plant and plant cell bioproduction platform. Plastid genetic engineering offers several advantages, including high levels of transgenic expression, transgenic containment via maternal inheritance, and multigene expression in a single transformation event. In recent years, the development of optimized expression strategies has given a huge boost to the exploitation of plastids in molecular farming. The driving forces behind the high expression level of plastid bioreactors include codon optimization, promoters and UTRs, genotypic modifications, endogenous enhancer and regulatory elements, posttranslational modification, and proteolysis. Exciting progress of the high expression level has been made with the plastid-based production of two particularly important classes of pharmaceuticals: vaccine antigens, therapeutic proteins, and antibiotics and enzymes. Approaches to overcome and solve the associated challenges of this culture system that include low transformation frequencies, the formation of inclusion bodies, and purification of recombinant proteins will also be discussed. Meili Gao, Yongfei Li, Xiaochang Xue, Xianfeng Wang, and Jiangang Long Copyright © 2012 Meili Gao et al. All rights reserved. Lysine Acetylation: Elucidating the Components of an Emerging Global Signaling Pathway in Trypanosomes Wed, 03 Oct 2012 10:45:35 +0000 In the past ten years the number of acetylated proteins reported in literature grew exponentially. Several authors have proposed that acetylation might be a key component in most eukaryotic signaling pathways, as important as phosphorylation. The enzymes involved in this process are starting to emerge; acetyltransferases and deacetylases are found inside and outside the nuclear compartment and have different regulatory functions. In trypanosomatids several of these enzymes have been described and are postulated to be novel antiparasitic targets for the rational design of drugs. In this paper we overview the most important known acetylated proteins and the advances made in the identification of new acetylated proteins using high-resolution mass spectrometry. Also, we summarize what is known so far about the acetyltransferases and deacetylases in eukaryotes, focusing on trypanosomes and their potential use as chemotherapeutic targets. Victoria Lucia Alonso and Esteban Carlos Serra Copyright © 2012 Victoria Lucia Alonso and Esteban Carlos Serra. All rights reserved. Generation of a Chinese Hamster Ovary Cell Line Producing Recombinant Human Glucocerebrosidase Wed, 03 Oct 2012 10:35:47 +0000 Impaired activity of the lysosomal enzyme glucocerebrosidase (GCR) results in the inherited metabolic disorder known as Gaucher disease. Current treatment consists of enzyme replacement therapy by administration of exogenous GCR. Although effective, it is exceptionally expensive, and patients worldwide have a limited access to this medicine. In Brazil, the public healthcare system provides the drug free of charge for all Gaucher’s patients, which reaches the order of $ 84 million per year. However, the production of GCR by public institutions in Brazil would reduce significantly the therapy costs. Here, we describe a robust protocol for the generation of a cell line producing recombinant human GCR. The protein was expressed in CHO-DXB11 (dhfr−) cells after stable transfection and gene amplification with methotrexate. As expected, glycosylated GCR was detected by immunoblotting assay both as cell-associated (~64 and 59 kDa) and secreted (63–69 kDa) form. Analysis of subclones allowed the selection of stable CHO cells producing a secreted functional enzyme, with a calculated productivity of 5.14 pg/cell/day for the highest producer. Although being laborious, traditional methods of screening high-producing recombinant cells may represent a valuable alternative to generate expensive biopharmaceuticals in countries with limited resources. Juliana Branco Novo, Ligia Morganti, Ana Maria Moro, Adriana Franco Paes Leme, Solange Maria de Toledo Serrano, Isaias Raw, and Paulo Lee Ho Copyright © 2012 Juliana Branco Novo et al. All rights reserved. Antilithiasic and Hypolipidaemic Effects of Raphanus sativus L. var. niger on Mice Fed with a Lithogenic Diet Wed, 03 Oct 2012 09:34:28 +0000 In Mexico, Raphanus sativus L. var. niger (black radish) has uses for the treatment of gallstones and for decreasing lipids serum levels. We evaluate the effect of juice squeezed from black radish root in cholesterol gallstones and serum lipids of mice. The toxicity of juice was analyzed according to the OECD guidelines. We used female C57BL/6 mice fed with a lithogenic diet. We performed histopathological studies of gallbladder and liver, and measured concentrations of cholesterol, HDL cholesterol and triglycerides. The juice can be considered bioactive and non-toxic; the lithogenic diet significantly induced cholesterol gallstones; increased cholesterol and triglycerides levels, and decreased HDL levels; gallbladder wall thickness increased markedly, showing epithelial hyperplasia and increased liver weight. After treatment with juice for 6 days, cholesterol gallstones were eradicated significantly in the gallbladder of mice; cholesterol and triglycerides levels decreased too, and there was also an increase in levels of HDL (). Gallbladder tissue continued to show epithelial hyperplasia and granulocyte infiltration; liver tissue showed vacuolar degeneration. The juice of black radish root has properties for treatment of cholesterol gallstones and for decreasing serum lipids levels; therefore, we confirm in a preclinical study the utility that people give it in traditional medicine. Ibrahim Guillermo Castro-Torres, Elia Brosla Naranjo-Rodríguez, Miguel Ángel Domínguez-Ortíz, Janeth Gallegos-Estudillo, and Margarita Virginia Saavedra-Vélez Copyright © 2012 Ibrahim Guillermo Castro-Torres et al. All rights reserved. Serum Peptidome Patterns of Colorectal Cancer Based on Magnetic Bead Separation and MALDI-TOF Mass Spectrometry Analysis Wed, 03 Oct 2012 08:05:32 +0000 Background. Colorectal cancer (CRC) is one of the most common cancers in the world, identification of biomarkers for early detection of CRC represents a relevant target. The present study aims to determine serum peptidome patterns for CRC diagnosis. Methods. The present work focused on serum proteomic analysis of 32 health volunteers and 38 CRC by ClinProt Kit combined with mass spectrometry. This approach allowed the construction of a peptide patterns able to differentiate the studied populations. An independent group of serum (including 33 health volunteers, 34 CRC, 16 colorectal adenoma, 36 esophageal carcinoma, and 31 gastric carcinoma samples) was used to verify the diagnostic and differential diagnostic capability of the peptidome patterns blindly. An immunoassay method was used to determine serum CEA of CRC and controls. Results. A quick classifier algorithm was used to construct the peptidome patterns for identification of CRC from controls. Two of the identified peaks at m/z 741 and 7772 were used to construct peptidome patterns, achieving an accuracy close to 100% (>CEA, 𝑃<0.05). Furthermore, the peptidome patterns could differentiate validation group with high accuracy. Conclusions. These results suggest that the ClinProt Kit combined with mass spectrometry yields significantly higher accuracy for the diagnosis and differential diagnosis of CRC. Nai-Jun Fan, Chun-Fang Gao, Xiu-Li Wang, Guang Zhao, Qing-Yin Liu, Yuan-Yao Zhang, and Bao-Guo Cheng Copyright © 2012 Nai-Jun Fan et al. All rights reserved. Protegrin-1 Inhibits Dengue NS2B-NS3 Serine Protease and Viral Replication in MK2 Cells Tue, 02 Oct 2012 14:52:26 +0000 Dengue diseases have an economic as well as social burden worldwide. In this study, the antiviral activity of protegrin-1 (PG-1, RGGRLCYCRRRFCVCVGR) peptide towards dengue NS2B-NS3pro and viral replication in Rhesus monkey kidney (MK2) cells was investigated. The peptide PG-1 was synthesized by solid-phase peptide synthesis, and disulphide bonds formation followed by peptide purification was confirmed by LC-MS and RPHPLC. Dengue NS2B-NS3pro was produced as a single-chain recombinant protein in E. coli. The NS2B-NS3pro assay was carried out by measuring the florescence emission of catalyzed substrate. Real-time PCR was used to evaluate the inhibition potential of PG-1 towards dengue serotype-2 (DENV-2) replication in MK2 cells. The results showed that PG-1 inhibited dengue NS2B-NS3pro at IC50 of 11.7 μM. The graded concentrations of PG-1 at nontoxic range were able to reduce viral replication significantly () at 24, 48, and 72 hrs after viral infection. However, the percentage of inhibition was significantly () higher at 24 hrs compared to 48 and 72 hrs. These data show promising therapeutic potential of PG-1 against dengue infection, hence it warrants further analysis and improvement of the peptide features as a prospective starting point for consideration in designing attractive dengue virus inhibitors. Hussin A. Rothan, Ammar Y. Abdulrahman, Pottayil G. Sasikumer, Shatrah Othman, Noorsaadah Abd Rahman, and Rohana Yusof Copyright © 2012 Hussin A. Rothan et al. All rights reserved. Persistent Dystrophin Protein Restoration 90 Days after a Course of Intraperitoneally Administered Naked 2′OMePS AON and ZM2 NP-AON Complexes in mdx Mice Tue, 02 Oct 2012 13:46:15 +0000 In Duchenne muscular dystrophy, the exon-skipping approach has obtained proof of concept in animal models, myogenic cell cultures, and following local and systemic administration in Duchenne patients. Indeed, we have previously demonstrated that low doses (7.5 mg/Kg/week) of 2-O-methyl-phosphorothioate antisense oligoribonucleotides (AONs) adsorbed onto ZM2 nanoparticles provoke widespread dystrophin restoration 7 days after intraperitoneal treatment in mdx mice. In this study, we went on to test whether this dystrophin restoration was still measurable 90 days from the end of the same treatment. Interestingly, we found that both western blot and immunohistochemical analysis (up to 7% positive fibres) were still able to detect dystrophin protein in the skeletal muscles of ZM2-AON-treated mice at this time, and the level of exon-23 skipping could still be assessed by RT real-time PCR (up to 10% of skipping percentage). In contrast, the protein was undetectable by western blot analysis in the skeletal muscles of mdx mice treated with an identical dose of naked AON, and the percentage of dystrophin-positive fibres and exon-23 skipping were reminiscent of those of untreated mdx mice. Our data therefore demonstrate the long-term residual efficacy of this systemic low-dose treatment and confirm the protective effect nanoparticles exert on AON molecules. Elena Bassi, Sofia Falzarano, Marina Fabris, Francesca Gualandi, Luciano Merlini, Gaetano Vattemi, Daniela Perrone, Elena Marchesi, Patrizia Sabatelli, Katia Sparnacci, Michele Laus, Paolo Bonaldo, Paola Rimessi, Paola Braghetta, and Alessandra Ferlini Copyright © 2012 Elena Bassi et al. All rights reserved. Cloning of Acyl-ACP Thioesterase FatA from Arachis hypogaea L. and Its Expression in Escherichia coli Tue, 02 Oct 2012 13:21:28 +0000 In this study, a full-length cDNA of the acyl-ACP thioesterase, AhFatA, was cloned from developing seeds of Arachis hypogaea L. by 3′-RACE. Sequence analysis showed that the open reading frame encodes a peptide of 372 amino acids and has 50–70% identity with FatA from other plants. Real-time quantitative PCR analysis revealed that AhFatA was expressed in all tissues of A. hypogaea L., but most strongly in the immature seeds harvested at 60 days after pegging. Heterologous expression of AhFatA in Escherichia coli affected bacterial growth and changed the fatty acid profiles of the membrane lipid, resulting in directed accumulation towards palmitoleic acid and oleic acid. These results indicate that AhFatA is at least partially responsible for determining the high palmitoleic acid and oleic acid composition of E. coli. Gao Chen, Zhen-ying Peng, Lei Shan, Ning Xuan, Gui-ying Tang, Yan Zhang, Lan Li, Qing-fang He, and Yu-ping Bi Copyright © 2012 Gao Chen et al. All rights reserved. Proteomic Profiling of the Dioxin-Degrading Bacterium Sphingomonas wittichii RW1 Tue, 02 Oct 2012 12:12:14 +0000 Sphingomonas wittichii RW1 is a bacterium of interest due to its ability to degrade polychlorinated dioxins, which represent priority pollutants in the USA and worldwide. Although its genome has been fully sequenced, many questions exist regarding changes in protein expression of S. wittichii RW1 in response to dioxin metabolism. We used difference gel electrophoresis (DIGE) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to identify proteomic changes induced by growth on dibenzofuran, a surrogate for dioxin, as compared to acetate. Approximately 10% of the entire putative proteome of RW1 could be observed. Several components of the dioxin and dibenzofuran degradation pathway were shown to be upregulated, thereby highlighting the utility of using proteomic analyses for studying bioremediation agents. This is the first global protein analysis of a microorganism capable of utilizing the carbon backbone of both polychlorinated dioxins and dibenzofurans as the sole source for carbon and energy. David R. Colquhoun, Erica M. Hartmann, and Rolf U. Halden Copyright © 2012 David R. Colquhoun et al. All rights reserved. Pigment Epithelium-Derived Factor: Chemistry, Structure, Biology, and Applications Wed, 05 Sep 2012 15:23:28 +0000 S. Patricia Becerra, Crispin R. Dass, Takeshi Yabe, and Susan E. Crawford Copyright © 2012 S. Patricia Becerra et al. All rights reserved. Enhancement of Lipid Extraction from Marine Microalga, Scenedesmus Associated with High-Pressure Homogenization Process Sun, 26 Aug 2012 10:00:35 +0000 Marine microalga, Scenedesmus sp., which is known to be suitable for biodiesel production because of its high lipid content, was subjected to the conventional Folch method of lipid extraction combined with high-pressure homogenization pretreatment process at 1200 psi and 35°C. Algal lipid yield was about 24.9% through this process, whereas only 19.8% lipid can be obtained by following a conventional lipid extraction procedure using the solvent, chloroform : methanol (2 : 1, v/v). Present approach requires 30 min process time and a moderate working temperature of 35°C as compared to the conventional extraction method which usually requires >5 hrs and 65°C temperature. It was found that this combined extraction process followed second-order reaction kinetics, which means most of the cellular lipids were extracted during initial periods of extraction, mostly within 30 min. In contrast, during the conventional extraction process, the cellular lipids were slowly and continuously extracted for >5 hrs by following first-order kinetics. Confocal and scanning electron microscopy revealed altered texture of algal biomass pretreated with high-pressure homogenization. These results clearly demonstrate that the Folch method coupled with high-pressure homogenization pretreatment can easily destruct the rigid cell walls of microalgae and release the intact lipids, with minimized extraction time and temperature, both of which are essential for maintaining good quality of the lipids for biodiesel production. Seok-Cheol Cho, Woon-Yong Choi, Sung-Ho Oh, Choon-Geun Lee, Yong-Chang Seo, Ji-Seon Kim, Chi-Ho Song, Ga-Vin Kim, Shin-Young Lee, Do-Hyung Kang, and Hyeon-Yong Lee Copyright © 2012 Seok-Cheol Cho et al. All rights reserved. Soluble Host-Defense Lectins Thu, 26 Jul 2012 10:39:24 +0000 Misao Matsushita, David C. Kilpatrick, Bok Luel Lee, and Nobutaka Wakamiya Copyright © 2012 Misao Matsushita et al. All rights reserved. Heterologous Expression of Plant Cell Wall Degrading Enzymes for Effective Production of Cellulosic Biofuels Sun, 15 Jul 2012 13:30:46 +0000 A major technical challenge in the cost-effective production of cellulosic biofuel is the need to lower the cost of plant cell wall degrading enzymes (PCDE), which is required for the production of sugars from biomass. Several competitive, low-cost technologies have been developed to produce PCDE in different host organisms such as Escherichia coli, Zymomonas mobilis, and plant. Selection of an ideal host organism is very important, because each host organism has its own unique features. Synthetic biology-aided tools enable heterologous expression of PCDE in recombinant E. coli or Z. mobilis and allow successful consolidated bioprocessing (CBP) in these microorganisms. In-planta expression provides an opportunity to simplify the process of enzyme production and plant biomass processing and leads to self-deconstruction of plant cell walls. Although the future of currently available technologies is difficult to predict, a complete and viable platform will most likely be available through the integration of the existing approaches with the development of breakthrough technologies. Sang-Kyu Jung, Vinuselvi Parisutham, Seong Hun Jeong, and Sung Kuk Lee Copyright © 2012 Sang-Kyu Jung et al. All rights reserved. Clinical Value of CD24 Expression in Retinoblastoma Thu, 14 Jun 2012 14:41:23 +0000 Background. The expression of CD24 has been detected in a wide variety of human malignancies. Downregulation of CD24 inhibits proliferation and induces apoptosis in tumor cells, whereas its upregulation increases tumor growth and metastasis. However, no data on CD24 protein levels in retinoblastoma are available, and the mechanism of CD24 involvement in retinoblastoma progress has not been elucidated. The aim of this study was to explore the expression profile of CD24 in the retinoblastoma tumor samples and to correlate with clinicopathological parameters. Methods. Immunohistochemistry was performed for CD24 on the archival paraffin sections of retinoblastoma and correlated with clinicopathological features. Western blotting was performed to confirm immunoreactivity results. Results. CD24 immunoreactivity was observed in 72.0% (36/50) of the retinoblastoma specimens. Among the 35 low-risk tumors, CD24 was expressed in 62.9% (22/35) tumors and among the 15 high-risk tumors, CD24 was expressed in 93.3% (14/15) tumors. High-risk tumors showed significantly increased expression of CD24 compared to tumors with low-risk (𝑃<0.05). Conclusions. This is the first correlation between CD24 expression and histopathology in human retinoblastoma. Our study showed increased expression of CD24 in high risk tumors compared to low risk tumors. Further functional studies are required to explore the role of CD24 in retinoblastoma. Jia Li, Changqing Li, Hongfeng Yuan, and Fang Gong Copyright © 2012 Jia Li et al. All rights reserved. Quantitation of Pyrrole-Imidazole Polyamide in Rat Plasma by High-Performance Liquid Chromatography Coupled with UV Detection Wed, 13 Jun 2012 11:29:05 +0000 A simple and robust method using high-performance liquid chromatography with UV detection was developed and validated for the determination of six pyrrole-imidazole (PI) polyamides (HN.49, TGF-β1f, TGF-β1t, HN.50f, HN.50t, and LOX-1) in rat plasma. After the plasma proteins were precipitated with methanol containing phenacetin as an internal standard, the analytes were separated on a Luna C18 (2) (5 μm, 4.6×150 mm). Calibration curves were linear over the range of 0.5 to 200 μg/mL for HN.49, 0.25 to 200 μg/mL for TGF-β1f, TGF-β1t, HN.50t, and LOX-1, 1 to 200 μg/mL for HN.50f in rat plasma. The inter- and intraday precision were below 15%, and the accuracy was within 15% at the quality controls. The validated method was successfully applied to sample analysis for the pharmacokinetic study. Tomonori Kamei, Takahiko Aoyama, Chihiro Tanaka, Takafumi Nagashima, Yukio Aoyama, Hiroyuki Hayashi, Hiroki Nagase, Takahiro Ueno, Noboru Fukuda, and Yoshiaki Matsumoto Copyright © 2012 Tomonori Kamei et al. All rights reserved. Identification of Pigment Epithelium-Derived Factor Protein Forms with Distinct Activities on Tumor Cell Lines Mon, 04 Jun 2012 08:30:52 +0000 Purpose. Pigment epithelium-derived factor (PEDF) is a multifunctional serpin. The purpose of this study is to identify PEDF protein forms and investigate their biological activities on tumor cell lines. Methods. Recombinant human PEDF proteins were purified by cation- and anion-exchange column chromatography. They were subjected to SDS-PAGE, IEF, deglycosylation, heparin affinity chromatography, and limited proteolysis. Cell viability, real-time electrical impedance of cells, and wound healing assays were performed using bladder and breast cancer cell lines, rat retinal R28, and human ARPE-19 cells. Results. Two PEDF protein peaks were identified after anion-exchange column chromatography: PEDF-1 eluting with lower ionic strength than PEDF-2. PEDF-1 had higher pI value and lower apparent molecular weight than PEDF-2. Both PEDF forms were glycosylated, bound to heparin, and had identical patterns by limited proteolysis. However, PEDF-2 emerged as being highly potent in lowering cell viability in all tumor cell lines tested, and in inhibiting tumor and ARPE-19 cell migration. In contrast, PEDF-1 minimally affected tumor cell viability and cell migration but protected R28 cells against death caused by serum starvation. Conclusion. Two distinct biochemical forms of PEDF varying in overall charge have distinct biological effects on tumor cell viability and migration. The existence of PEDF forms may explain the multifunctional modality of PEDF. P. Subramanian, M. Deshpande, S. Locatelli-Hoops, S. Moghaddam-Taaheri, D. Gutierrez, D. P. Fitzgerald, S. Guerrier, M. Rapp, V. Notario, and S. P. Becerra Copyright © 2012 P. Subramanian et al. All rights reserved. Efficacy of Continuously Administered PEDF-Derived Synthetic Peptides against Osteosarcoma Growth and Metastasis Wed, 30 May 2012 10:23:06 +0000 The potent antiangiogenic pigment epithelium-derived factor (PEDF) has shown promise against osteosarcoma, a tumour that originates in the bone and metastasises to the lungs. Neurotrophic, antiangiogenic, antiproliferative, and antimetastatic properties of PEDF have been attributed to a number of functional epitopes on the PEDF glycoprotein. StVOrth-2 (residues 78–102) and StVOrth-3 (residues 90–114) are two PEDF-derived peptides based on these functional epitopes. StVOrth-2 has previously been shown to inhibit osteosarcoma cell proliferation, while StVOrth-3 increased osteosarcoma cell adhesion to collagen I in vitro. In this paper, we have evaluated systemically and continuously delivered StVOrth-2 and StVOrth-3 using a clinically relevant murine model of osteosarcoma with spontaneous metastasis. Treatment with StVOrth-2 or StVOrth-3 with microosmotic pumps was initiated after primary osteosarcoma was established in the tibia. While treatment with StVOrth-2 and StVOrth-3 did not appear to affect local tumour invasion, tumour necrosis or apoptosis, StVOrth-2 predominantly restricted the growth of primary tumours, while StVOrth-3 restricted the burden of pulmonary metastatic disease. No peptide caused gross toxicity in mouse tissues as assessed by measuring weight of animals, serum biochemistry, and gross tissue observation. The differential effects exhibited by StVOrth-2 and StVOrth-3 in this orthotopic model of osteosarcoma may be related to the functional epitopes on the PEDF glycoprotein that they represent. Matthew L. Broadhead, Peter F. M. Choong, and Crispin R. Dass Copyright © 2012 Matthew L. Broadhead et al. All rights reserved. Cell and Molecular Biology Underpinning the Effects of PEDF on Cancers in General and Osteosarcoma in Particular Mon, 28 May 2012 09:56:48 +0000 Cancer is becoming an increasingly common disease in which abnormal cells aggressively grow, invade, and metastasize. In this paper, we review the biological functions of PEDF (pigmented epithelium-derived factor) against cancer, with a focus on a particular type of bone cancer called osteosarcoma. PEDF is a 50 kDa glycoprotein and is a potent inhibitor of angiogenesis, via its ability to decrease proliferation and migration of endothelial cells. This paper critically examines the anticancer activities of PEDF via its role in antiangiogenesis, apoptosis-mediated tumor suppression, and increased tumor cell differentiation. Recently, an orthotopic model of osteosarcoma was used to show that treatment with PEDF had the greatest impact on metastases, warranting an evaluation of PEDF efficacy in other types of cancers. Vijay Chandolu and Crispin R. Dass Copyright © 2012 Vijay Chandolu and Crispin R. Dass. All rights reserved. Role of Pigment Epithelium-Derived Factor in Stem/Progenitor Cell-Associated Neovascularization Tue, 22 May 2012 15:40:39 +0000 Pigment epithelium-derived factor (PEDF) was first identified in retinal pigment epithelium cells. It is an endogenously produced protein that is widely expressed throughout the human body such as in the eyes, liver, heart, and adipose tissue; it exhibits multiple and varied biological activities. PEDF is a multifunctional protein with antiangiogenic, antitumorigenic, antioxidant, anti-inflammatory, antithrombotic, neurotrophic, and neuroprotective properties. More recently, PEDF has been shown to be the most potent inhibitor of stem/progenitor cell-associated neovascularization. Neovascularization is a complex process regulated by a large, interacting network of molecules from stem/progenitor cells. PEDF is also involved in the pathogenesis of angiogenic eye disease, tumor growth, and cardiovascular disease. Novel antiangiogenic agents with tolerable side effects are desired for the treatment of patients with various diseases. Here, we review the value of PEDF as an important endogenous antiangiogenic molecule; we focus on the recently identified role of PEDF as a possible new target molecule to influence stem/progenitor cell-related neovascularization. Jung-Tung Liu, Yuh-Lien Chen, Wen-Chi Chen, Huey-Yi Chen, Yi-Wen Lin, Shu-Huei Wang, Kee-Ming Man, Hui-Min Wan, Wei-Hsian Yin, Po-Len Liu, and Yung-Hsiang Chen Copyright © 2012 Jung-Tung Liu et al. All rights reserved. Diverse Functions of Pulmonary Collectins in Host Defense of the Lung Sun, 20 May 2012 10:17:23 +0000 Pulmonary surfactant is a mixture of lipids and proteins that covers alveolar surfaces and keeps alveoli from collapsing. Four specific proteins have been identified in surfactant. Among them, two C-type lectins, surfactant proteins A and D (SP-A and SP-D), are known to be implicated in host defense and regulation of inflammatory responses of the lung. These host defense lectins are structurally characterized by N-terminal collagen-like domains and lectin domains and are called pulmonary collectins. They prevent dissemination of infectious microbes by their biological activities including agglutination and growth inhibition. They also promote clearance of microbes by enhancing phagocytosis in macrophages. In addition, they interact with the other pattern-recognition molecules, including Toll-like receptors (TLRs) and TLR-associated molecules, CD14 and MD-2, and regulate inflammatory responses. Furthermore, recent studies have demonstrated that these collectins modulate functions of neutrophil-derived innate immune molecules by interacting with them. These findings indicate that pulmonary collectins play critical roles in host defense of the lung. Shigeru Ariki, Chiaki Nishitani, and Yoshio Kuroki Copyright © 2012 Shigeru Ariki et al. All rights reserved. The Emerging Role of PEDF in Stem Cell Biology Thu, 17 May 2012 09:34:41 +0000 Encoded by a single gene, PEDF is a 50 kDa glycoprotein that is highly conserved and is widely expressed among many tissues. Most secreted PEDF deposits within the extracellular matrix, with cell-type-specific functions. While traditionally PEDF is known as a strong antiangiogenic factor, more recently, as this paper highlights, PEDF has been linked with stem cell biology, and there is now accumulating evidence demonstrating the effects of PEDF in a variety of stem cells, mainly in supporting stem cell survival and maintaining multipotency. Mina Elahy, Swati Baindur-Hudson, and Crispin R. Dass Copyright © 2012 Mina Elahy et al. All rights reserved. Investigating Potential Mechanisms of Obesity by Metabolomics Wed, 16 May 2012 14:57:10 +0000 Obesity is a serious health problem with an increased risk of several common diseases including diabetes, cardiovascular disease, and cancer. Metabolomics is an emerging analytical technique for systemic determination of metabolite profiles, which is useful for understanding the biochemical changes in obesity or related diseases both in individual organs and at the organism level. Increasingly, this technology has been applied to the study of obesity, complementing transcriptomics and/or proteomics analyses. Indeed, the alterations of metabolites in biofluids/tissues are direct indicators of variations in physiology or pathology. In this paper, we will examine the obesity-related alterations in significant metabolites that have been identified by metabolomics as well as their metabolic pathway associations. Issues concerning the screening of biologically significant metabolites related to obesity will also be discussed. Baogang Xie, Michael J. Waters, and Horst Joachim Schirra Copyright © 2012 Baogang Xie et al. All rights reserved. Soluble Host Defense Lectins in Innate Immunity to Influenza Virus Wed, 16 May 2012 08:17:45 +0000 Host defenses against viral infections depend on a complex interplay of innate (nonspecific) and adaptive (specific) components. In the early stages of infection, innate mechanisms represent the main line of host defense, acting to limit the spread of virus in host tissues prior to the induction of the adaptive immune response. Serum and lung fluids contain a range of lectins capable of recognizing and destroying influenza A viruses (IAV). Herein, we review the mechanisms by which soluble endogenous lectins mediate anti-IAV activity, including their role in modulating IAV-induced inflammation and disease and their potential as prophylactic and/or therapeutic treatments during severe IAV-induced disease. Wy Ching Ng, Michelle D. Tate, Andrew G. Brooks, and Patrick C. Reading Copyright © 2012 Wy Ching Ng et al. All rights reserved. Biochemical and Molecular Analysis of Some Commercial Samples of Chilli Peppers from Mexico Mon, 14 May 2012 14:30:35 +0000 The genus Capsicum provides antioxidant compounds, such as phenolics and carotenoids, into the diet. In Mexico, there is a wide diversity of species and varieties of chilli peppers, a fruit which has local cultural and gastronomic importance. In the present study, the relationship of the carotenoid and phenolic profiles with the RAPD fingerprint of three different commercial cultivars of chilli peppers of seven regions of Mexico was investigated. Through RAPD, the species of chilli were differentiated by means of different primers (OPE-18, MFG-17, MFG-18, C51, and C52). The genetic distance found with OPE 18 was in the order of 2.6. The observed differences were maintained when the chromatographic profile of carotenoids, and the molecular markers were analyzed, which suggest a close relationship between carotenoids and the genetic profile. While the chromatographic profile of phenols and the molecular markers were unable to differentiate between genotypes of chilli peppers. In addition, by using infrared spectroscopy and statistical PCA, differences explained by geographic origin were found. Thus, this method could be an alternative for identification of chilli species with respect to their geographic origin. Ivonne Guadalupe Troconis-Torres, Marlon Rojas-López, César Hernández-Rodríguez, Lourdes Villa-Tanaca, Ignacio Eduardo Maldonado-Mendoza, Lidia Dorantes-Álvarez, Darío Tellez-Medina, and María Eugenia Jaramillo-Flores Copyright © 2012 Ivonne Guadalupe Troconis-Torres et al. All rights reserved. New Protein Vector ApE1 for Targeted Delivery of Anticancer Drugs Wed, 09 May 2012 10:22:28 +0000 A new chimeric gene ApE1 encoding the receptor-binding domain of the human alpha-fetoprotein fused to a sequence of 22 glutamic acid residues was constructed. A new bacterial producer strain E. coli SHExT7 ApE1 was selected for ApE1 production in a soluble state. A simplified method was developed to purify ApE1 from bacterial biomass. It was shown that the new vector protein selectively interacts with AFP receptors on the tumor cell surface and can be efficiently accumulated in tumor cells. In addition, ApE1 was shown to be stable in storage and during its chemical modification. An increased number of carboxyl groups in the molecule allows the production of cytotoxic compound conjugates with higher drug-loading capacity and enhanced tumor targeting potential. N. V. Pozdniakova, N. V. Gorokhovets, N. V. Gukasova, A. V. Bereznikova, and E. S. Severin Copyright © 2012 N. V. Pozdniakova et al. All rights reserved. Biological Functions of the Novel Collectins CL-L1, CL-K1, and CL-P1 Wed, 11 Apr 2012 16:05:56 +0000 Collectins are characterized by a collagen-like sequence and a carbohydrate recognition domain and are members of the vertebrate C-type lectin superfamily. Recently, “novel collectins”, different from “classical collectins” consisting of mannan-binding lectin (MBL) and surfactant proteins A and D (SP-A and SP-D), have been found by reverse genetics. These “novel collectins” consist of collectin liver 1 (CL-L1), collectin kidney 1 (CL-K1), and collectin placenta 1 (CL-P1) and are encoded by three separate genes. Experimental findings on human and animal collectins have shown that both novel collectins and classical collectins play an important role in innate immunity. Based on our recent results and those of others, in this paper, we summarize the new biological functions of these novel collectins in embryonic morphogenesis and development. Katsuki Ohtani, Yasuhiko Suzuki, and Nobutaka Wakamiya Copyright © 2012 Katsuki Ohtani et al. All rights reserved.