The Scientific World Journal: Cell Biology The latest articles from Hindawi Publishing Corporation © 2015 , Hindawi Publishing Corporation . All rights reserved. Innovative Targeting Strategies in Drug Therapy for Inflammatory Diseases: Mechanistic Approaches Mon, 30 Mar 2015 07:43:27 +0000 Duen-Suey Chou, Joen-Rong Sheu, Philip A. Thomas, and Muniyan Sakthivel Copyright © 2015 Duen-Suey Chou et al. All rights reserved. VEGF Correlates with Inflammation and Fibrosis in Tuberculous Pleural Effusion Thu, 26 Mar 2015 07:42:23 +0000 Objective. To investigate the relationship among angiogenic cytokines, inflammatory markers, and fibrinolytic activity in tuberculous pleural effusion (TBPE) and their clinical importance. Methods. Forty-two patients diagnosed with TBPE were studied. Based on chest ultrasonography, there were 26 loculated and 16 nonloculated TBPE patients. The effusion size radiological scores and effusion vascular endothelial growth factor (VEGF), interleukin- (IL-) 8, plasminogen activator inhibitor type-1 (PAI-1), and tissue type plasminogen activator (tPA) were measured. Treatment outcome and pleural fibrosis, defined as radiological residual pleural thickening (RPT), were assessed at 6-month follow-up. Results. The effusion size and effusion lactate dehydrogenase (LDH), VEGF, IL-8, PAI-1, and PAI-1/tPA ratio were significantly higher, while effusion glucose, pH value, and tPA were significantly lower, in loculated than in nonloculated TBPE. VEGF and IL-8 correlated positively with LDH and PAI-1/tPA ratio and negatively with tPA in both loculated and nonloculated TBPE. Patients with higher VEGF or greater effusion size were prone to develop RPT (; VEGF, odds ratio 1.28, ; effusion size, odds ratio 1.01, ), and VEGF was an independent predictor of RPT in TBPE (receiver operating characteristic curve , ). Conclusions. Effusion VEGF correlates with pleural inflammation and fibrosis and may be targeted for adjunct therapy for TBPE. Mauo-Ying Bien, Ming-Ping Wu, Wei-Lin Chen, and Chi-Li Chung Copyright © 2015 Mauo-Ying Bien et al. All rights reserved. Static Magnetic Field Attenuates Lipopolysaccharide-Induced Inflammation in Pulp Cells by Affecting Cell Membrane Stability Thu, 26 Mar 2015 07:27:14 +0000 One of the causes of dental pulpitis is lipopolysaccharide- (LPS-) induced inflammatory response. Following pulp tissue inflammation, odontoblasts, dental pulp cells (DPCs), and dental pulp stem cells (DPSCs) will activate and repair damaged tissue to maintain homeostasis. However, when LPS infection is too serious, dental repair is impossible and disease may progress to irreversible pulpitis. Therefore, the aim of this study was to examine whether static magnetic field (SMF) can attenuate inflammatory response of dental pulp cells challenged with LPS. In methodology, dental pulp cells were isolated from extracted teeth. The population of DPSCs in the cultured DPCs was identified by phenotypes and multilineage differentiation. The effects of 0.4 T SMF on DPCs were observed through MTT assay and fluorescent anisotropy assay. Our results showed that the SMF exposure had no effect on surface markers or multilineage differentiation capability. However, SMF exposure increases cell viability by 15%. In addition, SMF increased cell membrane rigidity which is directly related to higher fluorescent anisotropy. In the LPS-challenged condition, DPCs treated with SMF demonstrated a higher tolerance to LPS-induced inflammatory response when compared to untreated controls. According to these results, we suggest that 0.4 T SMF attenuates LPS-induced inflammatory response to DPCs by changing cell membrane stability. Sung-Chih Hsieh, Jeng-Ting Tsao, Wei-Zhen Lew, Ya-Hui Chan, Lin-Wen Lee, Che-Tong Lin, Yung-Kai Huang, and Haw-Ming Huang Copyright © 2015 Sung-Chih Hsieh et al. All rights reserved. Change of Scaling-Induced Proinflammatory Cytokine on the Clinical Efficacy of Periodontitis Treatment Wed, 25 Mar 2015 06:58:54 +0000 Proinflammatory cytokines are key inflammatory mediators in periodontitis. This study aimed to investigate the relationship between proinflammatory cytokines in saliva and periodontal status. To investigate the usefulness of cytokines in the therapeutic approach for periodontal disease, the relationship between stimulated cytokine changes and the periodontitis treatment outcome was investigated in this study. Saliva was obtained from 22 patients diagnosed by dentists as having chronic periodontitis. The proinflammatory cytokine (interleukin-1 (IL-1), interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor (TNF-), and tumor necrosis factor (TNF-)) levels were determined using a commercially available kit. The IL-1 and IL-6 levels increased, whereas the TNF- levels decreased with the severity of periodontitis (4 mm pocket percentage). Poststimulation IL-1, IL-6, and IL-8 levels were higher in patients who had an improved treatment outcome. The differences of IL-6 levels (cut point: 0.05 μg/g) yielded a sensitivity and specificity of 90.0% and 81.82%, respectively, for predicting the periodontitis treatment outcome. Among the proinflammatory cytokines, stimulated IL-6 was an excellent marker for predicting the periodontitis treatment outcome. Kou-Gi Shyu, Cheuk-Sing Choy, Daniel Chung-Lang Wang, Wei-Chen Huang, Shyuan-Yow Chen, Chien-Hsun Chen, Che-Tong Lin, Chao-Chien Chang, and Yung-Kai Huang Copyright © 2015 Kou-Gi Shyu et al. All rights reserved. Intrinsic Apoptosis Pathway in Fallopian Tube Epithelial Cells Induced by Cladribine Wed, 05 Nov 2014 07:16:39 +0000 Cladribine is a purine nucleoside analog which initiates the apoptotic mechanism within cells. Moreover, the available data confirms that cladribine, with the participation of the p53 protein, as well as the proapoptotic proteins from the Bcl-2 family, also induces the activation of the intrinsic apoptosis pathway. However, while there has been a lot of research devoted to the effect of cladribine on lymphatic system cells, little is known about the impact of cladribine on the reproductive system. The aim of our study was to evaluate apoptosis in oviduct epithelial cells sourced from 15 different female rats. In so doing, the sections were stained with caspases 3, 9, and 8. Results suggest that cladribine also induces apoptosis in the oviduct epithelial cells by way of the intrinsic pathway. Indeed, the discontinuing of the administration of cladribine leads to a reduction in the amount of apoptotic cells in the oviduct epithelium. Ewelina Wawryk-Gawda, Patrycja Chylińska-Wrzos, Marta Lis-Sochocka, Kamila Bulak, and Barbara Jodłowska-Jędrych Copyright © 2014 Ewelina Wawryk-Gawda et al. All rights reserved. Characterisation of Drosophila UbxCPTI000601 and hthCPTI000378 Protein Trap Lines Wed, 15 Oct 2014 08:27:07 +0000 In Drosophila, protein trap strategies provide powerful approaches for the generation of tagged proteins expressed under endogenous control. Here, we describe expression and functional analysis to evaluate new Ubx and hth protein trap lines generated by the Cambridge Protein Trap project. Both protein traps exhibit spatial and temporal expression patterns consistent with the reported endogenous pattern in the embryo. In imaginal discs, Ubx-YFP is expressed throughout the haltere and 3rd leg imaginal discs, while Hth-YFP is expressed in the proximal regions of haltere and wing discs but not in the pouch region. The UbxCPTI000601 line is semilethal as a homozygote. No T3/A1 to T2 transformations were observed in the embryonic cuticle or the developing midgut. The homozygous survivors, however, exhibit a weak haltere phenotype with a few wing-like marginal bristles on the haltere capitellum. Although hthCPTI000378 is completely lethal as a homozygote, the hthCPTI000378/hthC1 genotype is viable. Using a hth deletion (Df(3R)BSC479) we show that hthCPTI000378/Df(3R)BSC479 adults are phenotypically normal. No transformations were observed in hthCPTI000378, hthCPTI000378/hthC1, or hthCPTI000378/Df(3R)BSC479 embryonic cuticles. We have successfully characterised the Ubx-YFP and Hth-YFP protein trap lines demonstrating that the tagged proteins show appropriate expression patterns and produce at least partially functional proteins. Siew Woh Choo, Ching Yew Beh, Steven Russell, and Robert White Copyright © 2014 Siew Woh Choo et al. All rights reserved. Effect of Antrodia camphorata on Inflammatory Arterial Thrombosis-Mediated Platelet Activation: The Pivotal Role of Protein Kinase C Tue, 14 Oct 2014 12:00:40 +0000 Antrodia camphorata is a rare Taiwanese medicinal mushroom. Antrodia camphorata extract has been reported to exhibit antioxidant, anti-inflammation, antimetastasis, and anticancer activities and plays a role in liver fibrosis, vasorelaxation, and immunomodulation. Critical vascular inflammation leads to vascular dysfunction and cardiovascular diseases, including abdominal aortic aneurysms, hypertension, and atherosclerosis. Platelet activation plays a crucial role in intravascular thrombosis, which is involved in a wide variety of cardiovascular diseases. However, the effect of Antrodia camphorata on platelet activation remains unclear. We examined the effects of Antrodia camphorata on platelet activation. In the present study, Antrodia camphorata treatment (56–224 μg/mL) inhibited platelet aggregation induced by collagen, but not U46619, an analogue of thromboxane A2, thrombin, and arachidonic acid. Antrodia camphorata inhibited collagen-induced calcium () mobilization and phosphorylation of protein kinase C (PKC) and Akt. In addition, Antrodia camphorata significantly reduced the aggregation and phosphorylation of PKC in phorbol-12, 13-dibutyrate (PDBu) activated platelets. In conclusion, Antrodia camphorata may inhibit platelet activation by inhibiting of and PKC cascade and the Akt pathway. Our study suggests that Antrodia camphorata may be a potential therapeutic agent for preventing or treating thromboembolic disorders. Wan-Jung Lu, Shih-Chang Lin, Chang-Chou Lan, Tzu-Yin Lee, Chih-Hsuan Hsia, Yung-Kai Huang, Hsiu-Chuan Lee, and Joen-Rong Sheu Copyright © 2014 Wan-Jung Lu et al. All rights reserved. Extract of Antrodia camphorata Exerts Neuroprotection against Embolic Stroke in Rats without Causing the Risk of Hemorrhagic Incidence Mon, 21 Jul 2014 06:26:31 +0000 In this study, the neuroprotective effect of an extract of Antrodia camphorata (A. camphorata), a fungus commonly used in Chinese folk medicine for treatment of viral hepatitis and cancer, alone or in combination with aspirin was investigated in a rat embolic stroke model. An ischemic stroke was induced in rats by a selective occlusion of the middle cerebral artery (MCA) with whole blood clots and then orally treated with A. camphorata (0.25 and 0.75 g/kg/day) alone and combined with aspirin (5 mg/kg/day). Sixty days later, the brains were removed, sectioned, and stained with triphenyltetrazolium chloride and analysed by a commercial image processing software program. Brain infarct volume, neurobehavioral score, cerebral blood perfusion, and subarachnoid and intracerebral hemorrhage incidence were perceived. In addition, potential bleeding side effect of the combinative therapy was assessed by measuring hemoglobin (Hb) content during intracerebral hemorrhage and gastric bleeding, prothrombin time (PT), and occlusion time (OT) after oral administration. Posttreatment with high dose A. camphorata significantly reduced infarct volume and improved neurobehavioral score (P < 0.05). Since A. camphorata alone or with aspirin did not alter the Hb level, this treatment is safe and does not cause hemorrhagic incident. Remarkably, the combination of A. camphorata and aspirin did not show a significant effect on the bleeding time, PT and OT increase suggesting that A. camphorata may have the neuroprotective effect without the prolongation of bleeding time or coagulation time. From these observations, we suggest that combinative therapy of A. camphorata and aspirin might offer enhanced neuroprotective efficacies without increasing side effects. Ye-Ming Lee, Chiu-Yun Chang, Ting-Lin Yen, Pitchairaj Geraldine, Chang-Chou Lan, Joen-Rong Sheu, and Jie-Jen Lee Copyright © 2014 Ye-Ming Lee et al. All rights reserved. Antihepatoma Activity of Artocarpus communis Is Higher in Fractions with High Artocarpin Content Mon, 14 Jul 2014 11:11:28 +0000 Extracts from natural plants have been used in traditional medicine for many centuries worldwide. Artocarpus communis is one such plant that has been used to treat liver cirrhosis, hypertension, and diabetes. To our knowledge, this study is the first to investigate the antihepatoma activity of A. communis toward HepG2 and PLC/PRF/5 cells and the first to explore the relationship between antihepatoma activity and the active compound artocarpin content in different fractions of A. communis. A. communis methanol extract and fractions induced dose-dependent reduction of tumor cell viability. DNA laddering analysis revealed that A. communis extract and fractions did not induce apoptosis in HepG2 and PLC/PRF/5 cells. Instead, acridine orange staining revealed that A. communis triggered autophagic cell death in a dose-dependent manner. The antihepatoma activity of A. communis is attributable to artocarpin. The fractions with the highest artocarpin content were also the fractions with the highest antihepatoma activity in the following order: dichloromethane fraction > methanol extract > ethyl acetate fraction > n-butanol fraction > n-hexane fraction. Taken together, A. communis showed antihepatoma activity through autophagic cell death. The effect was related to artocarpin content. Artocarpin could be considered an indicator of the anticancer potential of A. communis extract. Cheng-Wei Tzeng, Feng-Lin Yen, Liang-Tzung Lin, Chiang-Wen Lee, Ming-Hong Yen, Wen-Sheng Tzeng, and Chun-Ching Lin Copyright © 2014 Cheng-Wei Tzeng et al. All rights reserved. Andrographolide Inhibits Nuclear Factor-B Activation through JNK-Akt-p65 Signaling Cascade in Tumor Necrosis Factor-α-Stimulated Vascular Smooth Muscle Cells Thu, 10 Jul 2014 07:55:20 +0000 Critical vascular inflammation leads to vascular dysfunction and cardiovascular diseases, including abdominal aortic aneurysms, hypertension, and atherosclerosis. Andrographolide is the most active and critical constituent isolated from the leaves of Andrographis paniculata, a herbal medicine widely used for treating anti-inflammation in Asia. In this study, we investigated the mechanisms of the inhibitory effects of andrographolide in vascular smooth muscle cells (VSMCs) exposed to a proinflammatory stimulus, tumor necrosis factor-α (TNF-α). Treating TNF-α-stimulated VSMCs with andrographolide suppressed the expression of inducible nitric oxide synthase in a concentration-dependent manner. A reduction in TNF-α-induced c-Jun N-terminal kinase (JNK), Akt, and p65 phosphorylation was observed in andrographolide-treated VSMCs. However, andrographolide affected neither IκBα degradation nor p38 mitogen-activated protein kinase or extracellular signal-regulated kinase 1/2 phosphorylation under these conditions. Both treatment with LY294002, a phosphatidylinositol 3-kinase/Akt inhibitor, and treatment with SP600125, a JNK inhibitor, markedly reversed the andrographolide-mediated inhibition of p65 phosphorylation. In addition, LY294002 and SP600125 both diminished Akt phosphorylation, whereas LY294002 had no effects on JNK phosphorylation. These results collectively suggest that therapeutic interventions using andrographolide can benefit the treatment of vascular inflammatory diseases, and andrographolide-mediated inhibition of NF-κB activity in TNF-α-stimulated VSMCs occurs through the JNK-Akt-p65 signaling cascade, an IκBα-independent mechanism. Yu-Ying Chen, Ming-Jen Hsu, Cheng-Ying Hsieh, Lin-Wen Lee, Zhih-Cherng Chen, and Joen-Rong Sheu Copyright © 2014 Yu-Ying Chen et al. All rights reserved. The p85 Regulatory Subunit of PI3K Mediates cAMP-PKA and Insulin Biological Effects on MCF-7 Cell Growth and Motility Wed, 09 Jul 2014 09:47:28 +0000 Recent studies have shown that hyperinsulinemia may increase the cancer risk. Moreover, many tumors demonstrate an increased activation of IR signaling pathways. Phosphatidylinositol 3-kinase (PI3K) is necessary for insulin action. In epithelial cells, which do not express GLUT4 and gluconeogenic enzymes, insulin-mediated PI3K activation regulates cell survival, growth, and motility. Although the involvement of the regulatory subunit of PI3K () in insulin signal transduction has been extensively studied, the function of its N-terminus remains elusive. It has been identified as a serine (S83) in the that is phosphorylated by protein kinase A (PKA). To determine the molecular mechanism linking PKA to insulin-mediated PI3K activation, we used mutated forms to prevent phosphorylation (p85A) or to mimic the phosphorylated residue (p85D). We demonstrated that phosphorylation of S83 modulates the formation of the complex and its subcellular localization influencing the kinetics of the insulin signaling both on MAPK-ERK and AKT pathways. Furthermore, the S83 phosphorylation plays a central role in the control of insulin-mediated cell proliferation, cell migration, and adhesion. This study highlights the S83 role as a key regulator of cell proliferation and motility induced by insulin in MCF-7 cells breast cancer model. E. Di Zazzo, A. Feola, C. Zuchegna, A. Romano, C. F. Donini, S. Bartollino, C. Costagliola, R. Frunzio, P. Laccetti, M. Di Domenico, and A. Porcellini Copyright © 2014 E. Di Zazzo et al. All rights reserved. Cadmium Treatment Alters the Expression of Five Genes at the Cda1 Locus in Two Soybean Cultivars [Glycine Max (L.) Merr] Mon, 02 Jun 2014 11:06:53 +0000 Westag 97 has larger capacity of Cd accumulation in roots which prevents Cd from translocating into stems and leaves; conversely, AC Hime has smaller capacity of Cd accumulation in roots; more Cd is transported into stems and leaves. The different capacity of Cd in roots between Westag 97 and AC Hime causes the different Cd concentration in seeds. Meanwhile, according to the different expression levels of RSTK, ISCP, and H+-ATPase between Westag 97 and AC Hime, RSTK may be involved in transporting Cd into stems and leaves; H+-ATPase may be correlated to the capacity of Cd accumulation in roots; and Cd caused some changes of fundamental life process which leaded to the different expression patterns of ISCP between Westag 97 and AC Hime. Yi Wang, Xue Xiao, Tiequan Zhang, Houyang Kang, Jian Zeng, Xing Fan, Lina Sha, Haiqin Zhang, Kangfu Yu, and Yonghong Zhou Copyright © 2014 Yi Wang et al. All rights reserved. Sanguis draconis, a Dragon’s Blood Resin, Attenuates High Glucose-Induced Oxidative Stress and Endothelial Dysfunction in Human Umbilical Vein Endothelial Cells Mon, 02 Jun 2014 09:10:38 +0000 Hyperglycaemia, a characteristic feature of diabetes mellitus, induces endothelial dysfunction and vascular complications by limiting the proliferative potential of these cells. Here we aimed to investigate the effect of an ethanolic extract of Sanguis draconis (SD), a kind of dragon’s blood resin that is obtained from Daemonorops draco (Palmae), on human umbilical vein endothelial cells (HUVEC) under high-glucose (HG) stimulation and its underlying mechanism. Concentration-dependent (0–50 μg/mL) assessment of cell viability showed that SD does not affect cell viability with a similar trend up to 48 h. Remarkably, SD (10–50 μg/mL) significantly attenuated the high-glucose (25 and 50 mM) induced cell toxicity in a concentration-dependent manner. SD inhibited high glucose-induced nitrite (NO) and lipid peroxidation (MDA) production and reactive oxygen species (ROS) formation in HUVEC. Western blot analysis revealed that SD treatments abolished HG-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2), nuclear transcription factor, κB (NF-κB), VCAM-1, and E-selectin, and it also blocked the breakdown of PARP-116 kDa protein in a dose-dependent manner. Furthermore, we found that SD increased the expression of Bcl-2 and decreased Bax protein expression in HG-stimulated HUVEC. Thus, these results of this study demonstrate for the first time that SD inhibits glucose induced oxidative stress and vascular inflammation in HUVEC by inhibiting the ERK/NF-κB/PARP-1/Bax signaling cascade followed by suppressing the activation of VCAM-1 and E-selectin. These data suggest that SD may have a therapeutic potential in vascular inflammation due to the decreased levels of oxidative stress, apoptosis, and PARP-1 activation. Yi Chang, Ting-Chen Chang, Jie-Jen Lee, Nen-Chung Chang, Yung-Kai Huang, Cheuk-Sing Choy, and Thanasekaran Jayakumar Copyright © 2014 Yi Chang et al. All rights reserved. RHBDL2 Is a Critical Membrane Protease for Anoikis Resistance in Human Malignant Epithelial Cells Wed, 28 May 2014 13:14:47 +0000 Anoikis resistance allows metastatic tumor cells to survive in a homeless environment. Activation of epithelial growth factor receptor (EGFR) signaling is one of the key mechanisms for metastatic tumor cells to resist anoikis, yet the regulation mechanisms of homeless-triggered EGFR activation in metastatic tumor cells remain unclear. Rhomboid-like-2 (RHBDL2), an evolutionally conserved intramembrane serine protease, can cleave the EGF ligand and thus trigger EGFR activation. Herein, we demonstrated that RHBDL2 overexpression in human epithelial cells resulted in promotion of cell proliferation, reduction of cell adhesion, and suppression of anoikis. During long-term suspension cultures, increased RHBDL2 was only detected in aggressive tumor cell lines. Treatment with the rhomboid protease inhibitor or RHBDL2 shRNA increased cleaved caspase 3, a marker of apoptosis. Finally, inhibition of EGFR activation increased the cleaved caspase 3 and attenuated the detachment-induced focal adhesion kinase phosphorylation. Taken together, these findings provide evidence for the first time that RHBDL2 is a critical molecule in anoikis resistance of malignant epithelial cells, possibly through the EGFR-mediated signaling. Our study demonstrates RHBDL2 as a new therapeutic target for cancer metastasis. Tsung-Lin Cheng, Chao-Han Lai, Shinn-Jong Jiang, Jui-Hsiang Hung, Shi-Kai Liu, Bi-Ing Chang, Guey-Yueh Shi, and Hua-Lin Wu Copyright © 2014 Tsung-Lin Cheng et al. All rights reserved. An Improved Quantitative Analysis Method for Plant Cortical Microtubules Mon, 10 Mar 2014 09:45:21 +0000 The arrangement of plant cortical microtubules can reflect the physiological state of cells. However, little attention has been paid to the image quantitative analysis of plant cortical microtubules so far. In this paper, Bidimensional Empirical Mode Decomposition (BEMD) algorithm was applied in the image preprocessing of the original microtubule image. And then Intrinsic Mode Function 1 (IMF1) image obtained by decomposition was selected to do the texture analysis based on Grey-Level Cooccurrence Matrix (GLCM) algorithm. Meanwhile, in order to further verify its reliability, the proposed texture analysis method was utilized to distinguish different images of Arabidopsis microtubules. The results showed that the effect of BEMD algorithm on edge preserving accompanied with noise reduction was positive, and the geometrical characteristic of the texture was obvious. Four texture parameters extracted by GLCM perfectly reflected the different arrangements between the two images of cortical microtubules. In summary, the results indicate that this method is feasible and effective for the image quantitative analysis of plant cortical microtubules. It not only provides a new quantitative approach for the comprehensive study of the role played by microtubules in cell life activities but also supplies references for other similar studies. Yi Lu, Chenyang Huang, Jia Wang, and Peng Shang Copyright © 2014 Yi Lu et al. All rights reserved. p104 Binds to Rac1 and Reduces Its Activity during Myotube Differentiation of C2C12 Cell Mon, 27 Jan 2014 08:01:59 +0000 The p104 protein inhibits cellular proliferation when overexpressed in NIH3T3 cells and has been shown to associate with p85, Grb2, and PLC1. In order to isolate other proteins that interact with p104, yeast two-hybrid screening was performed. Rac1 was identified as a binding partner of p104 and the interaction between p104 and Rac1 was confirmed by immunoprecipitation. Using a glutathione S-transferase (GST) pull-down assay with various p104 fragments, the 814–848 amino acid residue at the carboxyl-terminal region of p104 was identified as the key component to interact with Rac1. The CrkII which is involved in the Rac1-mediated cellular response was also found to interact with p104 protein. NIH3T3 cells which overexpressed p104 showed a decrease of Rac1 activity. However, neither the proline-rich domain mutant, which is unable to interact with CrkII, nor the carboxy-terminal deletion mutant could attenuate Rac1 activity. During the differentiation of myoblasts, the amount of p104 protein as well as transcript level was increased. The overexpression of p104 enhanced myotube differentiation, whereas siRNA of p104 reversed this process. In this process, more Rac1 and CrkII were bound to increased p104. Based on these results, we conclude that p104 is involved in muscle cell differentiation by modulating the Rac1 activity. Ki Young Choi, Min Sup Lee, Young Jun Cho, Myong Ho Jeong, Seung Jin Han, and Seung Hwan Hong Copyright © 2014 Ki Young Choi et al. All rights reserved. Intranuclear Crosstalk between Extracellular Regulated Kinase1/2 and Signal Transducer and Activator of Transcription 3 Regulates JEG-3 Choriocarcinoma Cell Invasion and Proliferation Wed, 30 Oct 2013 10:42:23 +0000 Invasiveness of trophoblast and choriocarcinoma cells is in part mediated via leukemia inhibitory factor- (LIF-) induced activation of signal transducer and activator of transcription 3 (STAT3). The regulation of STAT3 phosphorylation at its ser727 binding site, possible crosstalk with intracellular MAPK signaling, and their functional implications are the object of the present investigation. JEG-3 choriocarcinoma cells were cultured in presence/absence of LIF and the specific ERK1/2 inhibitor (U0126). Phosphorylation of signaling molecules (p-STAT3 (ser727 and tyr705) and p-ERK1/2 (thr 202/tyr 204)) was assessed per Western blot. Immunocytochemistry confirmed results, but also pinpointed the location of phosphorylated signaling molecules. STAT3 DNA-binding capacity was studied with a colorimetric ELISA-based assay. Cell viability and invasion capability were assessed by MTS and Matrigel assays. Our results demonstrate that LIF-induced phosphorylation of STAT3 (tyr705 and ser727) is significantly increased after blocking ERK1/2. STAT3 DNA-binding capacity and cell invasiveness are enhanced after LIF stimulation and ERK1/2 blockage. In contrast, proliferation is enhanced by LIF but reduced after ERK1/2 inhibition. The findings herein show that blocking ERK1/2 increases LIF-induced STAT3 phosphorylation and STAT3 DNA-binding capacity by an intranuclear crosstalk, which leads to enhanced invasiveness and reduced proliferation. Diana M. Morales-Prieto, Stephanie Ospina-Prieto, Wittaya Chaiwangyen, Maja Weber, Sebastian Hölters, Ekkehard Schleussner, Justine S. Fitzgerald, and Udo R. Markert Copyright © 2013 Diana M. Morales-Prieto et al. All rights reserved. Oncogene- and Oxidative Stress-Induced Cellular Senescence Shows Distinct Expression Patterns of Proinflammatory Cytokines in Vascular Endothelial Cells Mon, 30 Sep 2013 14:33:30 +0000 Senescent cells are metabolically active and produce a variety of proinflammatory cytokines. It was previously reported that atherosclerotic plaques contain senescent cells, suggesting that senescence may contribute to the progression of atherosclerosis. In this study, we induced cellular senescence in vascular endothelial cells (VECs) using hydrogen peroxide (H2O2) or an adenovirus that expresses a constitutively active mutant of Ras (AdRas12V) and studied the expression of cytokines. Both H2O2 treatment and AdRas12V infection induced senescence in VECs, as assessed by senescence-associated -Gal activity and the expression of proteins such as . In addition, both treatments induced the expression of a variety of cytokines, including interleukin-1 (IL-1) and nerve growth factor (NGF). AdRas12V infection induced IL-1 expression more significantly than H2O2 treatment, whereas both treatments induced comparable mRNA and protein expression levels of NGF. These results suggest that senescent cells express different patterns of proinflammatory cytokines, depending on the trigger that induced senescence. It is therefore possible that senescent cells can differentially induce inflammation in the surrounding tissues, depending on the cause of senescence. Etsu Suzuki, Masao Takahashi, Shigeyoshi Oba, and Hiroaki Nishimatsu Copyright © 2013 Etsu Suzuki et al. All rights reserved. Biobanking in a Constantly Developing Medical World Mon, 23 Sep 2013 14:02:18 +0000 Biobank is a very sophisticated system that consists of a programmed storage of biological material and corresponding data. Biobanks are created to be used in medical research, in clinical and translational medicine, and in healthcare. In the past 20 years, a large number of biobanks have been set up around the world, to support the modern research directions in medicine such as omix and personalized medicine. More recently, embryonic and adult stem cell banks have been developed. Stem cell banking was reported to be required for medical research as well as clinical transplant applications. The quality of the samples stored in a biobank is very important. The standardization is also important; the biological material stored in a biobank must be processed in a manner that allows compatibility with other biobanks that preserve samples in the same field. In this paper, we review some issues related to biobanks purposes, quality, harmonization, and their financial and ethical aspects. Stefan-Alexandru Artene, Marius Eugen Ciurea, Stefana Oana Purcaru, Daniela Elise Tache, Ligia Gabriela Tataranu, Mihaela Lupu, and Anica Dricu Copyright © 2013 Stefan-Alexandru Artene et al. All rights reserved. Hypoxic Culture Conditions as a Solution for Mesenchymal Stem Cell Based Regenerative Therapy Tue, 27 Aug 2013 08:20:29 +0000 Cell-based regenerative therapies, based on in vitro propagation of stem cells, offer tremendous hope to many individuals suffering from degenerative diseases that were previously deemed untreatable. Due to the self-renewal capacity, multilineage potential, and immunosuppressive property, mesenchymal stem cells (MSCs) are considered as an attractive source of stem cells for regenerative therapies. However, poor growth kinetics, early senescence, and genetic instability during in vitro expansion and poor engraftment after transplantation are considered to be among the major disadvantages of MSC-based regenerative therapies. A number of complex inter- and intracellular interactive signaling systems control growth, multiplication, and differentiation of MSCs in their niche. Common laboratory conditions for stem cell culture involve ambient O2 concentration (20%) in contrast to their niche where they usually reside in 2–9% O2. Notably, O2 plays an important role in maintaining stem cell fate in terms of proliferation and differentiation, by regulating hypoxia-inducible factor-1 (HIF-1) mediated expression of different genes. This paper aims to describe and compare the role of normoxia (20% O2) and hypoxia (2–9% O2) on the biology of MSCs. Finally it is concluded that a hypoxic environment can greatly improve growth kinetics, genetic stability, and expression of chemokine receptors during in vitro expansion and eventually can increase efficiency of MSC-based regenerative therapies. Nazmul Haque, Mohammad Tariqur Rahman, Noor Hayaty Abu Kasim, and Aied Mohammed Alabsi Copyright © 2013 Nazmul Haque et al. All rights reserved. RhoA Regulation of Cardiomyocyte Differentiation Thu, 27 Jun 2013 18:01:26 +0000 Earlier findings from our laboratory implicated RhoA in heart developmental processes. To investigate factors that potentially regulate RhoA expression, RhoA gene organisation and promoter activity were analysed. Comparative analysis indicated strict conservation of both gene organisation and coding sequence of the chick, mouse, and human RhoA genes. Bioinformatics analysis of the derived promoter region of mouse RhoA identified putative consensus sequence binding sites for several transcription factors involved in heart formation and organogenesis generally. Using luciferase reporter assays, RhoA promoter activity was shown to increase in mouse-derived P19CL6 cells that were induced to differentiate into cardiomyocytes. Overexpression of a dominant negative mutant of mouse RhoA (mRhoAN19) blocked this cardiomyocyte differentiation of P19CL6 cells and led to the accumulation of the cardiac transcription factors SRF and GATA4 and the early cardiac marker cardiac α-actin. Taken together, these findings indicate a fundamental role for RhoA in the differentiation of cardiomyocytes. Mari Kaarbø, Denis I. Crane, and Wayne G. Murrell Copyright © 2013 Mari Kaarbø et al. All rights reserved. Transcriptional Regulation of Fucosyltransferase 1 Gene Expression in Colon Cancer Cells Sun, 03 Mar 2013 15:25:49 +0000 The α1,2-fucosyltransferase I (FUT1) enzyme is important for the biosynthesis of H antigens, Lewis B, and Lewis Y. In this study, we clarified the transcriptional regulation of FUT1 in the DLD-1 colon cancer cell line, which has high expression of Lewis B and Lewis Y antigens, expresses the FUT1 gene, and shows α1,2-fucosyltransferase (FUT) activity. 5′-rapid amplification of cDNA ends revealed a FUT1 transcriptional start site −10 nucleotides upstream of the site registered at NM_000148 in the DataBase of Human Transcription Start Sites (DBTSS). Using the dual luciferase assay, FUT1 gene expression was shown to be regulated at the region −91 to −81 nt to the transcriptional start site, which contains the Elk-1 binding site. Site-directed mutagenesis of this region revealed the Elk-1 binding site to be essential for FUT1 transcription. Furthermore, transfection of the dominant negative Elk-1 gene, and the chromatin immunoprecipitation (CHIp) assay, supported Elk-1-dependent transcriptional regulation of FUT1 gene expression in DLD-1 cells. These results suggest that a defined region in the 5′-flanking region of FUT1 is critical for FUT1 transcription and that constitutive gene expression of FUT1 is regulated by Elk-1 in DLD-1 cells. Fumiko Taniuchi, Koji Higai, Tomomi Tanaka, Yutaro Azuma, and Kojiro Matsumoto Copyright © 2013 Fumiko Taniuchi et al. All rights reserved. BrdU Pulse Labelling In Vivo to Characterise Cell Proliferation during Regeneration and Repair following Injury to the Airway Wall in Sheep Thu, 28 Feb 2013 11:52:37 +0000 The response of S-phase cells labelled with bromodeoxyuridine (BrdU) in sheep airways undergoing repair in response to endobronchial brush biopsy was investigated in this study. Separate sites within the airway tree of anaesthetised sheep were biopsied at intervals prior to pulse labelling with BrdU, which was administered one hour prior to euthanasia. Both brushed and spatially disparate unbrushed (control) sites were carefully mapped, dissected, and processed to facilitate histological analysis of BrdU labelling. Our study indicated that the number and location of BrdU-labelled cells varied according to the age of the repairing injury. There was little evidence of cell proliferation in either control airway tissues or airway tissues examined six hours after injury. However, by days 1 and 3, BrdU-labelled cells were increased in number in the airway wall, both at the damaged site and in the regions flanking either side of the injury. Thereafter, cell proliferative activity largely declined by day 7 after injury, when consistent evidence of remodelling in the airway wall could be appreciated. This study successfully demonstrated the effectiveness of in vivo pulse labelling in tracking cell proliferation during repair which has a potential value in exploring the therapeutic utility of stem cell approaches in relevant lung disease models. B. Yahaya, G. McLachlan, and D. D. S. Collie Copyright © 2013 B. Yahaya et al. All rights reserved. Proteins Reprogramming: Present and Future Thu, 22 Nov 2012 11:53:34 +0000 Induced pluripotent stem cells (iPSCs) are of great clinical interest for they are derived from one’s own somatic cells and have the potential of committed differentiation without immunological rejection after autografting. However, the use of viral and other modified vectors may still cause tumorigenesis due to chromosome insertion mutation, leading to limited practical use. iPSCs generated by reprogramming proteins overcome the potential safety risk and complicated manipulation procedures, thus they own better application prospective, yet some technical difficulties need to be studied and resolved, for instance, low reprogramming efficiency, unclear transduction, and reprogramming mechanism. In this paper, we summarize the current progress of proteins reprogramming technology for generation of iPSCs and discuss the promising efficiency-improved reprogramming methods by proteins plus other kinds of chemical compounds. Yang Yang, Bin Liu, Jianwen Dong, Liangming Zhang, Mao Pang, and Limin Rong Copyright © 2012 Yang Yang et al. All rights reserved. Comparison of Oogenesis and Sex Steroid Profiles between Twice and Once Annually Spawning of Rainbow Trout Females (Oncorhynchus mykiss) Mon, 19 Nov 2012 11:04:48 +0000 This study compares the gonadosomatic index (GSI), oocyte growth (OG), gonadal histology, and plasma level concentrations of sex hormones (estradiol-17β (E2) and vitellogenin (V)) of twice-spawning (T-SP) and once-spawning (O-SP) females of rainbow trout throughout the additional and the normal reproductive cycle, respectively. In T-SP, the GSI values rapidly increase from May to November, in contrast to O-SP, which showed low and constant GSI values (1.19 to 14.5 and 1.19 to 0.63, resp.). T-SP exhibited a marked increase of OG in the same period, reaching a maximum diameter of 4,900 ± 141.42 μm, in contrast to O-SP, which presented a slow OG. The gonadal histology of T-SP agreed with the general pattern of ovogenesis observed for O-SP (vitellogenesis, ovulation, and recrudescence); however, this process was nonsynchronous between the two breeder groups. Plasma steroid levels showed significant variation during oogenesis, which agreed with the GSI, OG, and gonadal histology patterns. The level of E2 increased to a maximum value of 26.2 ng/mL and 36.0 ng/mL in O-SP and T-SP, respectively, one or two months before the spawning event where vitellogenesis was fully active. The V concentrations followed a pattern similar to those of E2. Francisco Estay, Nelson Colihueque, and Cristian Araneda Copyright © 2012 Francisco Estay et al. All rights reserved. Genomic Profiling of a Human Leukemic Monocytic Cell-Line (THP-1) Exposed to Alpha Particle Radiation Thu, 11 Oct 2012 13:20:51 +0000 This study examined alpha (α-) particle radiation effects on global changes in gene expression in human leukemic monocytic cells (THP-1) for the purposes of mining for candidate biomarkers that could be used for the development of a biological assessment tool. THP-1 cells were exposed to α-particle radiation at a dose range of 0 to 1.5 Gy. Twenty-four hours and three days after exposure gene expression was monitored using microarray technology. A total of 16 genes were dose responsive and classified as early onset due to their expression 24 h after exposure. Forty-eight transcripts were dose responsive and classified as late-onset as they were expressed 72 h after exposure. Among these genes, 6 genes were time and dose responsive and validated further using alternate technology. These transcripts were upregulated and associated with biological processes related to immune function, organelle stability and cell signalling/communication. This panel of genes merits further validation to determine if they are strong candidate biomarkers indicative of α-particle exposure. Vinita Chauhan and Matthew Howland Copyright © 2012 Vinita Chauhan and Matthew Howland. All rights reserved. Erratum to “Fusion Pore Diameter Regulation by Cations Modulating Local Membrane Anisotropy” Tue, 31 Jul 2012 10:29:25 +0000 Doron Kabaso, Ana I. Calejo, Jernej Jorgačevski, Marko Kreft, Robert Zorec, and Aleš Iglič Copyright © 2012 Doron Kabaso et al. All rights reserved. Biology and Biotechnology of Follicle Development Tue, 22 May 2012 15:53:19 +0000 Growth and development of ovarian follicles require a series of coordinated events that induce morphological and functional changes within the follicle, leading to cell differentiation and oocyte development. The preantral early antral follicle transition is the stage of follicular development during which gonadotropin dependence is obtained and the progression into growing or atresia of the follicle is made. Follicular growth during this period is tightly regulated by oocyte-granulosatheca cell interactions. A cluster of early expressed genes is required for normal folliculogenesis. Granulosa cell factors stimulate the recruitment of theca cells from cortical stromal cells. Thecal factors promote granulosa cell proliferation and suppress granulosa cell apoptosis. Cell-cell and cell-extracellular matrix interactions influence the production of growth factors in the different follicular compartments (oocyte, granulosa, and theca cells). Several autocrine and paracrine factors are involved in follicular growth and differentiation; their activity is present even at the time of ovulation, decreasing the gap junction communication, and stimulating the theca cell proliferation. In addition, the identification of the factors that promote follicular growth from the preantral stage to the small antral stage may provide important information for the identification for assisted reproduction techniques. Gustavo Adolfo Palma, Martin Eduardo Argañaraz, Antonio Daniel Barrera, Daniela Rodler, Adrian Ángel Mutto, and Fred Sinowatz Copyright © 2012 Gustavo Adolfo Palma et al. All rights reserved. Increased NTPDase Activity in Lymphocytes during Experimental Sepsis Thu, 03 May 2012 09:10:48 +0000 We investigated in rats induced to sepsis the activity of ectonucleoside triphosphate diphosphohydrolase (NTPDase; CD39; E.C., an enzyme involved in the modulation of immune responses. After 12 hours of surgery, lymphocytes were isolated from blood and NTPDase activity was determined. It was also performed the histology of kidney, liver, and lung. The results demonstrated an increase in the hydrolysis of adenosine-5-triphosphate (ATP) (𝑃<0.01), but no changes regarding adenosine-5-monophosphate (ADP) hydrolysis (𝑃>0.05). Histological analysis showed several morphological changes in the septic group, such as vascular congestion, necrosis, and infiltration of mononuclear cells. It is known that the intracellular milieu contains much more ATP nucleotides than the extracellular. In this context, the increased ATPasic activity was probably induced as a dynamic response to clean up the elevated ATP levels resulting from cellular death. Claudia de Mello Bertoncheli, Carine Eloise Prestes Zimmermann, Jeandre Augusto dos Santos Jaques, Cláudio Alberto Martins Leal, Jader Betsch Ruchel, Bruna Cipolatto Rocha, Kelly de Vargas Pinheiro, Viviane do Carmo Gonçalves Souza, Daniel Roulim Stainki, Sônia Cristina Almeida Luz, Maria Rosa Chitolina Schetinger, and Daniela Bitencourt Rosa Leal Copyright © 2012 Claudia de Mello Bertoncheli et al. All rights reserved. Antioxidant Protection against Curative and Palliative Doses of Ionizing Irradiation in Human Blood Decreases with Aging Wed, 02 May 2012 19:30:08 +0000 Reactive oxygen species (ROS) are independently recognized to play a significant role in radiation-induced damage on healthy tissue and in aging process. However, an age-related alteration of antioxidant (AO) system in radiation response in humans is poorly investigated. The aim of this paper was to evaluate the irradiation effects on the activities and expression of AO system in the blood of healthy women during aging. Blood samples were irradiated with curative and palliative doses of 2 Gy or 9 Gy γ-rays. AO capacity for detoxification of O2•− and H2O2 in response to 2 Gy γ-irradiation decreases in women above 58 years, while in response to 9 Gy shows signs of weakening after 45 years of age. Due to reduction of AO capacity during aging, cytotoxic effects of curative and palliative doses of irradiation, mediated by ROS, may significantly increase in older subjects, while removal of H2O2 excess could reduce them. Jelena Kasapović, Vesna Stojiljković, Ljubica Gavrilović, Nataša Popović, and Zorka Milićević Copyright © 2012 Jelena Kasapović et al. All rights reserved. Mechanistic Framework for Establishment, Maintenance, and Alteration of Cell Polarity in Plants Wed, 02 May 2012 17:03:01 +0000 Cell polarity establishment, maintenance, and alteration are central to the developmental and response programs of nearly all organisms and are often implicated in abnormalities ranging from patterning defects to cancer. By residing at the distinct plasma membrane domains polar cargoes mark the identities of those domains, and execute localized functions. Polar cargoes are recruited to the specialized membrane domains by directional secretion and/or directional endocytic recycling. In plants, auxin efflux carrier PIN proteins display polar localizations in various cell types and play major roles in directional cell-to-cell transport of signaling molecule auxin that is vital for plant patterning and response programs. Recent advanced microscopy studies applied to single cells in intact plants reveal subcellular PIN dynamics. They uncover the PIN polarity generation mechanism and identified important roles of AGC kinases for polar PIN localization. AGC kinase family members PINOID, WAG1, and WAG2, belonging to the AGC-3 subclass predominantly influence the polar localization of PINs. The emerging mechanism for AGC-3 kinases action suggests that kinases phosphorylate PINs mainly at the plasma membrane after initial symmetric PIN secretion for eventual PIN internalization and PIN sorting into distinct ARF-GEF-regulated polar recycling pathways. Thus phosphorylation status directs PIN translocation to different cell sides. Based on these findings a mechanistic framework evolves that suggests existence of cell side-specific recycling pathways in plants and implicates AGC3 kinases for differential PIN recruitment among them for eventual PIN polarity establishment, maintenance, and alteration. Pankaj Dhonukshe Copyright © 2012 Pankaj Dhonukshe. All rights reserved. Characterization of Mononucleated Human Peripheral Blood Cells Wed, 02 May 2012 13:35:23 +0000 Unspecialized cells that can renew themselves and give rise to multiple differentiated cell types are termed stem cells. The objective of this study was to characterize and investigate, through molecular and biochemical analyses, the stemness of cells derived from isolated mononucleated cells that originated from peripheral blood. The isolated mononucleated cells were separated according to their physical characteristics (adherent and suspension), after 4 to 7 days into a 14-day culturing period in complete medium. Our results revealed that adherent and suspension cells were positive for mesenchymal stem cell (MSC) and hematopoietic stem cell (HSC) markers, respectively. Differentiation of adherent cells into osteoblasts was associated with expression of the OPN gene and increasing ALP enzyme activity, while differentiation of suspension cells into osteoclasts was associated with expression of the TRAP gene and increasing TRAP enzyme activity. In conclusion, molecular and biochemical analyses showed that mononucleated cells consist of MSC (adherent) and HSC (suspension), and both cell types are able to differentiate into specialized cells from their respective lineage: osteoblast (MSC) and osteoclast (HSC). Ruzanna Ab Kadir, Shahrul Hisham Zainal Ariffin, Rohaya Megat Abdul Wahab, Shabnam Kermani, and Sahidan Senafi Copyright © 2012 Ruzanna Ab Kadir et al. All rights reserved. Oxidative Stress and Heart Failure in Altered Thyroid States Wed, 02 May 2012 11:59:51 +0000 Increased or reduced action of thyroid hormone on certain molecular pathways in the heart and vasculature causes relevant cardiovascular derangements. It is well established that hyperthyroidism induces a hyperdynamic cardiovascular state, which is associated with a faster heart rate, enhanced left ventricular systolic and diastolic function whereas hypothyroidism is characterized by the opposite changes. Hyperthyroidism and hypothyroidism represent opposite clinical conditions, albeit not mirror images. Recent experimental and clinical studies have suggested the involvement of ROS tissue damage under altered thyroid status. Altered-thyroid state-linked changes in heart modify their susceptibility to oxidants and the extent of the oxidative damage they suffer following oxidative challenge. Chronic increase in the cellular levels of ROS can lead to a catastrophic cycle of DNA damage, mitochondrial dysfunction, further ROS generation and cellular injury. Thus, these cellular events might play an important role in the development and progression of myocardial remodeling and heart failure in altered thyroid states (hypo- and hyper-thyroidism). The present review aims at elucidating the various signaling pathways mediated via ROS and their modulation under altered thyroid state and the possibility of antioxidant therapy. Pallavi Mishra and Luna Samanta Copyright © 2012 Pallavi Mishra and Luna Samanta. All rights reserved. Blood Cell Profiles of the Tadpoles of the Dubois's Tree Frog, Polypedates teraiensis Dubois, 1986 (Anura: Rhacophoridae) Wed, 02 May 2012 11:49:05 +0000 The present paper describes a sequential study of the leukocyte profiles and the changes in morphometry and morphology of erythrocytes in the tadpoles of Polypedates teraiensis during their development and metamorphosis, that is, transfer from an aquatic mode to a terrestrial mode of life. Blood smears of 21 different stages (Gosner stage 26 to 46) of tadpoles were investigated. Population of erythrocytes was heterogeneous in population represented by various forms (oval, elliptical or rounded cells, comma shaped, teardrop shaped, schistocytes, senile erythrocytes, crenulated RBCs). Correlation between various morphometric values of erythrocytes was determined with different developing stages of tadpoles. Amongst the leucocytes, the lymphocytes were the most abundant cells followed by neutrophils. Neutrophils and monocytes showed varied morphologic forms. The percentage of lymphocytes and neutrophils showed a negative whereas percentage of eosinophil, basophil, and monocytes showed a positive correlation with the developmental stages of tadpoles. Blood platelets were also observed, which were rounded in shape and found in aggregates. Madhusmita Das and Pravati Kumari Mahapatra Copyright © 2012 Madhusmita Das and Pravati Kumari Mahapatra. All rights reserved. Freezing of Oocytes and Its Effect on the Displacement of the Meiotic Spindle: Short Communication Wed, 02 May 2012 11:08:10 +0000 Our investigations focused on spindle dynamics/displacement in frozen-thawed human oocytes. In each oocyte, prior to freezing and after thawing and culturing, the presence/location of the spindle was determined with the Polscope technique. A total of 259 oocytes have been thawed with a survival rate of 81.1%. From the 210 survived oocytes, 165 were fertilized (78.6%) and 89.1% of them cleaved. A total of 143 embryos were transferred into 63 patients resulting in 11 clinical pregnancies (17.5%), 7 of which resulted in live birth of 8 babies (1 twin pregnancy). We were able to detect the spindle in 221 of 259 oocytes (85.3%). After thawing and culturing the oocytes, we were able to visualize the spindle in 177 of 210 oocytes (84.3%). In 83 of these 177 oocytes, the spindle was observed to be in the same location as it was before cryopreservation (46.9%). However, in 94 of these 177 oocytes (53.1%), the spindle reformed in a different position/location relative to the polar body. Our results show that after thawing and culture in half of the spindle-positive oocytes the spindle was detected in a new location, indicating that the spindle and the polar body move relative to each other. János Konc, Katalin Kanyo, Rita Kriston, József Zeke, and Sándor Cseh Copyright © 2012 János Konc et al. All rights reserved. The Golgi in Cell Migration: Regulation by Signal Transduction and Its Implications for Cancer Cell Metastasis Tue, 01 May 2012 16:00:33 +0000 Migration and invasion are fundamental features of metastatic cancer cells. The Golgi apparatus, an organelle involved in posttranslational modification and sorting of proteins, is widely accepted to regulate directional cell migration. In addition, mounting evidence suggests that the Golgi is a hub for different signaling pathways. In this paper we will give an overview on how polarized secretion and microtubule nucleation at the Golgi regulate directional cell migration. We will review different signaling pathways that signal to and from the Golgi. Finally, we will discuss how these signaling pathways regulate the role of the Golgi in cell migration and invasion. We propose that by identifying regulators of the Golgi, we might be able to uncover unappreciated modulators of cell migration. Uncovering the regulatory network that orchestrates cell migration is of fundamental importance for the development of new therapeutic strategies against cancer cell metastasis. Valentina Millarte and Hesso Farhan Copyright © 2012 Valentina Millarte and Hesso Farhan. All rights reserved. Autophagic Cell Death Is Induced by Acetone and Ethyl Acetate Extracts from Eupatorium odoratum In Vitro: Effects on MCF-7 and Vero Cell Lines Mon, 30 Apr 2012 14:27:42 +0000 Eupatorium odoratum (EO) contains many biologically active compounds, the anticancer effects of which are not well documented. This study evaluates the cytotoxic effects and mechanism of action of EO extracts on MCF-7 and Vero cell lines. Evaluation of the cytotoxic activity using MTT assay, morphological alterations, and apoptosis were carried out. Autophagy was evaluated by LC3-A protein expression. Cytotoxic activity, membrane blebbing and ballooning at 24 hours, replacement by mass vacuolation, and double membrane vesicles mimicking autophagy and cell death were observed in the cancer cells. No apoptosis was observed by DNA fragmentation assay. Overexpression of LC3-A protein indicated autophagic cell death. Cell cycle analysis showed G0 and G2/M arrest. The Vero cells did not show significant cell death at concentrations <100 μg/mL. These results thus suggest that acetone and ethyl acetate extracts of EO induce cell death through induction of autophagy and hold potential for development as potential anticancer drugs. Faizah Bt. Harun, Syed Mohsin Syed Sahil Jamalullail, Khoo Boon Yin, Zulkhairi Othman, Anita Tilwari, and Prabha Balaram Copyright © 2012 Faizah Bt. Harun et al. All rights reserved. The Comet Assay for the Evaluation of Genotoxic Potential of Landfill Leachate Mon, 30 Apr 2012 14:25:20 +0000 Genotoxic assessment of landfill leachate before and after biological treatment was conducted with two human cell lines (Me45 and NHDF) and Daphnia magna somatic cells. The alkali version of comet assay was used to examine genotoxicity of leachate by DNA strand breaks analysis and its repair dynamics. The leachate samples were collected from Zabrze landfill, situated in the Upper Silesian Industrial District, Poland. Statistically significant differences (Kruskal-Wallice ANOVA rank model) were observed between DNA strand breaks in cells incubated with leachate before and after treatment (𝑃<0.001). Nonparametric Friedman ANOVA confirmed time-reliable and concentration-reliable cells response to leachate concentration. Examinations of chemical properties showed a marked decrease in leachate parameters after treatment which correlate to reduced genotoxicity towards tested cells. Obtained results demonstrate that biological cotreatment of leachate together with municipal wastewater is an efficient method for its genotoxic potential reduction; however, treated leachate still possessed genotoxic character. Kamila Widziewicz, Joanna Kalka, Magdalena Skonieczna, and Paweł Madej Copyright © 2012 Kamila Widziewicz et al. All rights reserved. Monocytes Do Not Transdifferentiate into Proper Osteoblasts Mon, 30 Apr 2012 11:37:21 +0000 Recent publications suggested that monocytes might be an attractive cell type to transdifferentiate into various cellular phenotypes. Aim was, therefore, to evaluate the potential of blood monocytes to transdifferentiate into osteoblasts. Monocytes isolated from peripheral blood were subjected to two previously published treatments to obtain unique, multipotent cell fractions, named programmable cells of monocytic origin (PCMOs) and monocyte-derived mesenchymal progenitor cells (MOMPs). Subsequently, MOMPs and PCMOs were treated with osteogenic differentiation medium (including either vitamin D or dexamethasone) for 14 days. Regarding a variety of surface markers, no differences between MOMPs, PCMOs, and primary monocytes could be detected. The treatment with osteogenic medium neither resulted in loss of hematopoietic markers nor in adoption of mesenchymal phenotype in all cell types. No significant effect was observed regarding the expression of osteogenic transcription factors, bone-related genes, or production of mineralized matrix. Osteogenic medium resulted in activation of monocytes and appearance of osteoclasts. In conclusion, none of the investigated monocyte cell types showed any transdifferentiation characteristics under the tested circumstances. Based on our data, we rather see an activation and maturation of monocytes towards macrophages and osteoclasts. Andreas Schmitt, Sabrina Ehnert, Lilianna Schyschka, Peter Buschner, Andreas Kühnl, Stefan Döbele, Sebastian Siebenlist, Martin Lucke, Ulrich Stöckle, and Andreas K. Nussler Copyright © 2012 Andreas Schmitt et al. All rights reserved. Fusion Pore Diameter Regulation by Cations Modulating Local Membrane Anisotropy Mon, 12 Mar 2012 14:48:42 +0000 The fusion pore is an aqueous channel that is formed upon the fusion of the vesicle membrane with the plasma membrane. Once the pore is open, it may close again (transient fusion) or widen completely (full fusion) to permit vesicle cargo discharge. While repetitive transient fusion pore openings of the vesicle with the plasma membrane have been observed in the absence of stimulation, their frequency can be further increased using a cAMP-increasing agent that drives the opening of nonspecific cation channels. Our model hypothesis is that the openings and closings of the fusion pore are driven by changes in the local concentration of cations in the connected vesicle. The proposed mechanism of fusion pore dynamics is considered as follows: when the fusion pore is closed or is extremely narrow, the accumulation of cations in the vesicle (increased cation concentration) likely leads to lipid demixing at the fusion pore. This process may affect local membrane anisotropy, which reduces the spontaneous curvature and thus leads to the opening of the fusion pore. Based on the theory of membrane elasticity, we used a continuum model to explain the rhythmic opening and closing of the fusion pore. Doron Kabaso, Ana I. Calejo, Jernej Jorgačevski, Marko Kreft, Robert Zorec, and Aleš Iglič Copyright © 2012 Doron Kabaso et al. All rights reserved. Mechanisms Underlying the Osteo- and Adipo-Differentiation of Human Mesenchymal Stem Cells Mon, 12 Mar 2012 13:51:15 +0000 Human mesenchymal stem cells (hMSCs) are considered a promising cell source for regenerative medicine, because they have the potential to differentiate into a variety of lineages among which the mesoderm-derived lineages such adipo- or osteogenesis are investigated best. Human MSCs can be harvested in reasonable to large amounts from several parts of the patient’s body and due to this possible autologous origin, allorecognition can be avoided. In addition, even in allogenic origin-derived donor cells, hMSCs generate a local immunosuppressive microenvironment, causing only a weak immune reaction. There is an increasing need for bone replacement in patients from all ages, due to a variety of reasons such as a new recreational behavior in young adults or age-related diseases. Adipogenic differentiation is another interesting lineage, because fat tissue is considered to be a major factor triggering atherosclerosis that ultimately leads to cardiovascular diseases, the main cause of death in industrialized countries. However, understanding the differentiation process in detail is obligatory to achieve a tight control of the process for future clinical applications to avoid undesired side effects. In this review, the current findings for adipo- and osteo-differentiation are summarized together with a brief statement on first clinical trials. Yu Zhang, Dilaware Khan, Julia Delling, and Edda Tobiasch Copyright © 2012 Yu Zhang et al. All rights reserved. Technological Progress in Generation of Induced Pluripotent Stem Cells for Clinical Applications Mon, 12 Mar 2012 13:22:36 +0000 Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is achieved by viral-mediated transduction of defined transcription factors. Generation of iPSCs is of great medical interest as they have the potential to be a source of patient-specific cells. For the eventual goal of clinical application, it is necessary to overcome the limitations of low reprogramming efficiency and chromosomal abnormalities due to viral DNA integration. In this paper, we summarize the current state of reprogramming technology for generation of iPSCs and also discuss potential approaches to the development of safe iPSCs for personalized cell-based replacement therapy. Seung-Ick Oh, Chang Kyu Lee, Kyung Jin Cho, Kyung-Ok Lee, Ssang-Goo Cho, and Sunghoi Hong Copyright © 2012 Seung-Ick Oh et al. All rights reserved. Yeast as a Tool to Study Signaling Pathways in Mitochondrial Stress Response and Cytoprotection Thu, 02 Feb 2012 08:20:13 +0000 Cell homeostasis results from the balance between cell capability to adapt or succumb to environmental stress. Mitochondria, in addition to supplying cellular energy, are involved in a range of processes deciding about cellular life or death. The crucial role of mitochondria in cell death is well recognized. Mitochondrial dysfunction has been associated with the death process and the onset of numerous diseases. Yet, mitochondrial involvement in cellular adaptation to stress is still largely unexplored. Strong interest exists in pharmacological manipulation of mitochondrial metabolism and signaling. The yeast Saccharomyces cerevisiae has proven a valuable model organism in which several intracellular processes have been characterized in great detail, including the retrograde response to mitochondrial dysfunction and, more recently, programmed cell death. In this paper we review experimental evidences of mitochondrial involvement in cytoprotection and propose yeast as a model system to investigate the role of mitochondria in the cross-talk between prosurvival and prodeath pathways. Maša Ždralević, Nicoletta Guaragnella, Lucia Antonacci, Ersilia Marra, and Sergio Giannattasio Copyright © 2012 Maša Ždralević et al. All rights reserved. Autophagy Regulates the Post-Translational Cleavage of BCL-2 and Promotes Neuronal Survival Mon, 01 Jan 1900 00:00:00 +0000 B-cell lymphoma 2 protein (BCL-2) is one of the more widely investigated anti-apoptotic protein in mammals, and its levels are critical for protecting from programmed cell death. We report here that the cellular content of BCL-2 is regulated at post-translational level along the autophagy/lysosome pathways in organotypic cultures of post-natal mouse cerebellar cortex. Specifically this mechanism appears to be effective in the cerebellar granule cells (CGCs) that are known to undergo massive programmed cell death (apoptosis) during post-natal maturation. By the use of specific agonists/antagonist of calcium channels at the endoplasmic reticulum it was possible to understand the pivotal role of calcium release from intracellular stores in CGC neuroprotection. The more general significance of these findings is supported by a very recent study Niemann-Pick transgenic mice. Laura Lossi, Graziana Gambino, Chiara Salio, and Adalberto Merighi Copyright © 2010 Laura Lossi et al. All rights reserved. Delta Helicase Corresponds to KIAA0221: A Link Between DNA Polymerase Delta and RNA Surveillance Complex Mon, 01 Jan 1900 00:00:00 +0000 L.M. Carastro, C.k. Tan, A.G. So, and K.M. Downey Copyright © 2001 L.M. Carastro et al. All rights reserved. NITRIC OXIDE PROTECTS MOUSE THYMOCYTES FROM APOPTOSIS INDUCED BY -IRRADIATION Mon, 01 Jan 1900 00:00:00 +0000 Yue Chen, Ala Stanford, Rosie Hoffman, and Henri Ford Copyright © 2001 Yue Chen et al. All rights reserved. Lack of Obvious Influence of PLLA Nanofibers on the Gene Expression of BMP-2 and VEGF during Growth and Differentiation of Human Mesenchymal Stem Cells Mon, 01 Jan 1900 00:00:00 +0000 Growth factors like bone morphogenetic protein 2 (BMP-2) and vascular endothelial growth factor (VEGF) play an important role in bone remodeling and fracture repair. Therefore, with respect to tissue engineering, an artificial graft should have no negative impact on the expression of these factors. In this context, the aim of this study was to analyze the impact of poly(L-lactic acid) (PLLA) nanofibers on VEGF and BMP-2 gene expression during the time course of human mesenchymal stem cell (hMSC) differentiation towards osteoblasts. PLLA matrices were seeded with hMSCs and cultivated over a period of 22 days under growth and osteoinductive conditions, and analyzed during the course of culture, with respect to gene expression of VEGF and BMP-2. Furthermore, BMP-2–enwoven PLLA nanofibers were used in order to elucidate whether initial down-regulation of growth factor expression could be compensated. Although there was a great interpatient variability with respect to the expression of VEGF and BMP-2, PLLA nanofibers tend to result in a down-regulation in BMP-2 expression during the early phase of cultivation. This effect was diminished in the case of VEGF gene expression. The initial down-regulation was overcome when BMP-2 was directly incorporated into the PLLA nanofibers by electrospinning. Furthermore, the incorporation of BMP-2 into the PLLA nanofibers resulted in an increase in VEGF gene expression. Summarized, the results indicate that the PLLA nanofibers have little effect on growth factor production. An enhancement in gene expression of BMP-2 and VEGF can be achieved by an incorporation of BMP-2 into the PLLA nanofibers. Markus D. Schofer, S. Fuchs-Winkelmann, C. Wack, M. Rudisile, R. Dersch, I. Leifeld, J. Wendorff, A. Greiner, J. R. J. Paletta, and U. Boudriot Copyright © 2009 Markus Schofer et al. All rights reserved. Thiol Redox Transitions in Cell Signaling: a Lesson from N-Acetylcysteine Mon, 01 Jan 1900 00:00:00 +0000 The functional status of cells is under the control of external stimuli affecting the function of critical proteins and eventually gene expression. Signal sensing and transduction by messengers to specific effectors operate by post-translational modification of proteins, among which thiol redox switches play a fundamental role that is just beginning to be understood. The maintenance of the redox status is, indeed, crucial for cellular homeostasis and its dysregulation towards a more oxidized intracellular environment is associated with aberrant proliferation, ultimately related to diseases such as cancer, cardiovascular disease, and diabetes. Redox transitions occur in sensitive cysteine residues of regulatory proteins relevant to signaling, their evolution to metastable disulfides accounting for the functional redox switch. N-acetylcysteine (NAC) is a thiol-containing compound that is able to interfere with redox transitions of thiols and, thus, in principle, able to modulate redox signaling. We here review the redox chemistry of NAC, then screen possible mechanisms to explain the effects observed in NAC-treated normal and cancer cells; such effects involve a modification of global gene expression, thus of functions and morphology, with a leitmotif of a switch from proliferation to terminal differentiation. The regulation of thiol redox transitions in cell signaling is, therefore, proposed as a new tool, holding promise not only for a deeper explanation of mechanisms, but indeed for innovative pharmacological interventions. Tiziana Parasassi, Roberto Brunelli, Graziella Costa, Marco De Spirito, Ewa Krasnowska, Thomas Lundeberg, Eugenia Pittaluga, and Fulvio Ursini Copyright © 2010 Tiziana Parasassi et al. All rights reserved. Protein Repair and Degradation during Aging Mon, 01 Jan 1900 00:00:00 +0000 Cellular aging is characterized by a build-up of oxidatively modified proteins. The steady-state level of oxidized proteins depends on the balance between the rate of protein oxidative damage and the rates of protein degradation and repair. Therefore, the accumulation of oxidized protein with age can be due to increased protein damage, decreased oxidized protein degradation and repair, or the combination of both mechanisms. The proteasomal system is the major intracellular proteolytic pathway implicated in the degradation of oxidized protein, and the peptide methionine sulfoxide reductase catalyzes the reduction of methionine sulfoxide (i.e., oxidized methionine) to methionine within proteins. A short summary on protein oxidative damage and oxidized protein degradation is given, and evidence for a decline of proteasome function with age is presented. Arguments for the implication of peptide methionine sulfoxide reductase in the age-related accumulation of oxidized protein are also discussed. Bertrand Friguet Copyright © 2002 Bertrand�Friguet. All rights reserved. Separation of Membrane Vesicles and Cytosol from Yeast, Cultured Cells, and Bacteria in a Small Volume Self-Generated Gradient in a Fixed-Angle Rotor Mon, 01 Jan 1900 00:00:00 +0000 There are many situations when it is necessary to separate rapidly and efficiently a cytosolic and a membrane vesicle fraction from yeast, cultured cells, or from bacteria. This Protocol Article describes the flotation of the vesicles through a self-generated gradient from a dense sample zone using the low-viscosity medium iodixanol. As the sample is exposed to the gmax the tendency of the proteins to sediment overcomes any diffusion in the opposite direction and are therefore completely separated from the vesicles. John Graham Copyright © 2002 John Graham. All rights reserved. Making the Final Cut — Mechanisms Mediating the Abscission Step of Cytokinesis Mon, 01 Jan 1900 00:00:00 +0000 Cytokinesis is the final stage of mitotic cell division that results in a physical separation of two daughter cells. Cytokinesis begins in the early stages of anaphase after the positioning of the cleavage plane and after the chromosomes segregate. This involves the recruitment and assembly of an actomyosin contractile ring, which constricts the plasma membrane and compacts midzone microtubules to form an electron-dense region, termed the midbody, located within an intracellular bridge. The resolution of this intracellular bridge, known as abscission, is the last step in cytokinesis that separates the two daughter cells. While much research has been done to delineate the mechanisms mediating actomyosin ring formation and contraction, the machinery that is responsible for abscission remains largely unclear. Recent work from several laboratories has demonstrated that dramatic changes occur in cytoskeleton and endosome dynamics, and are a prerequisite for abscission. However, the mechanistic details that regulate the final plasma membrane fusion during abscission are only beginning to emerge and are the subject of considerable controversy. Here we review recent studies within this field and discuss the proposed models of cell abscission. John A. Schiel and Rytis Prekeris Copyright © 2010 John A. Schiel and Rytis Prekeris. All rights reserved. OptiPrep™ Density Gradient Solutions for Mammalian Organelles Mon, 01 Jan 1900 00:00:00 +0000 Any density gradient for the isolation of mammalian organelles should ideally only expose the sedimenting biological particles to an increasing concentration of the gradient solute. Thus they will experience only an increasing density and viscosity, other parameters such as osmolality, pH, ionic strength and the concentration of important additives (such as EDTA and DTT) should remain as close to constant as possible. This Protocol Article describes the strategies for the dilution of OptiPrep™ in order to prepare such solutions for mammalian organelles and membranes. John Graham Copyright © 2002 John Graham. All rights reserved. Fractionation of Golgi, Endoplasmic Reticulum, and Plasma Membrane from Cultured Cells in a Preformed Continuous Iodixanol Gradient Mon, 01 Jan 1900 00:00:00 +0000 A continuous iodixanol gradient within the range 0-30% (w/v) iodixanol can resolve the major membrane compartments of the endoplasmic reticulum, Golgi membranes, and plasma membrane from a postnuclear supernatant prepared from a cultured cell homogenate. The precise density range of the gradient and the centrifugation conditions (100,000-200,000 g for 2-16 h) vary with the type of cell and the requirements of the separation. The strategy is widely used to study the processing of proteins within cells. John Graham Copyright © 2002 John Graham. All rights reserved. The Fas/FasL System in Reproduction: Survival and Apoptosis Mon, 01 Jan 1900 00:00:00 +0000 For centuries, the question of “whether there is life after death” has intrigued the mind of philosophers, and the same question fascinates researchers in the field of apoptosis today. The death of a cell is by no means the end of the story. On the contrary, growing evidence suggests that the clearance of apoptotic bodies by macrophages is an important regulatory component in tissue renewal. Without death by apoptosis, the life of reproductive tissues and their function would not be possible. The survival signals that counteract cell death also prepare the cells for apoptosis, and dead cells are important stimuli for tissue survival. The Fas/FasL system is an important mediator in apoptosis and is and excellent example of this apparently contradictory phenomenon. Gil Mor, Shawn Straszewski, and Marijke Kamsteeg Copyright © 2002 Gil Mor et al. All rights reserved. Electron Microscopic Radioautographic Study on Mitochondrial DNA Synthesis in Adrenal Cortical Cells of Developing and Aging Mice Mon, 01 Jan 1900 00:00:00 +0000 In order to study the aging changes of intramitochondrial DNA synthesis of mouse adrenal cortical cells, eight groups of developing mice, each consisting of three individuals (total 24), from fetal day 19 to postnatal newborn at days 1, 3, 9, 14, to adult at months 1, 2, and 6, were injected with 3H-thymidine, sacrificed 1 h later, and the adrenal tissues were fixed and processed for electron microscopic (EM) radioautography. On EM radioautograms obtained from each animal, the number of mitochondria and the mitochondrial labeling index labeled with 3H-thymidine showing DNA synthesis in each adrenal cortical cell, in three zones, were counted and the results in respective developing groups were compared. From the results, it was demonstrated that the numbers of mitochondria in the three zones, the zona glomerulosa, fasciculata, and reticularis, of mice at various ages increased from fetal day 19 to postnatal month 6 due to development and aging of animals, respectively, while the number of labeled mitochondria and the labeling index of intramitochondrial DNA syntheses incorporating 3H-thymidine increased from fetal day 19 to postnatal month 2, reaching the maxima, and decreased to month 6. It was shown that the activity of intramitochondrial DNA synthesis in the adrenal cortical cells in developing and aging mice changed due to aging. Tetsuji Nagata Copyright © 2008 Tetsuji Nagata. All rights reserved. Role of Stromal Cells in Protecting Young and Aged B-Lineage Precursors from Dexamethasone-Induced Apoptosis Mon, 01 Jan 1900 00:00:00 +0000 Jeannette M. Pifer, Robert P. Stephan, Deborah A. Lill-Elghanian, and Pamela L. Witte Copyright © 2001 Jeannette M. Pifer et al. All rights reserved. Founding Editorial – Bone Biology Mon, 01 Jan 1900 00:00:00 +0000 Felix Bronner Copyright © 2002 Felix Bronner. All rights reserved. Apoptosis Gene Hunting Using Retroviral Expression Cloning: Identification of Vacuolar ATPase Subunit E Mon, 01 Jan 1900 00:00:00 +0000 Over the past 10-15 years there has been an explosion of interest in apoptosis. The delayed realisation that cell death is an essential part of life for any multicellular organism has meant that, despite the recent and rapid developments of the last decade, the precise biochemical pathways involved in apoptosis remain incomplete and potentially novel genes may, as yet, remain undiscovered. The hunt is therefore on to bridge the remaining gaps in our knowledge. Our contribution to this research effort utilises a functional cloning approach to isolate important regulatory genes involved in apoptosis. This mini-review focuses on the use and advantages of a retroviral expression cloning strategy and describes the isolation and identification of one such potential apoptosis regulatory gene, namely that encoding vacuolar ATPase subunit E. Claire L. Anderson and Gwyn T. Williams Copyright © 2003 Claire L. Anderson and Gwyn T. Williams. All rights reserved. Homogenization of Mammalian Cultured Cells Mon, 01 Jan 1900 00:00:00 +0000 Satisfactory homogenization of cultured cells is a necessary prerequisite to any fractionation schedule. Protocols are given for homogenization in iso-osmotic (A) and hypo-osmotic (B) media that should be broadly applicable to any cell type and to any subsequent fractionation procedure. Alternative procedures are also summarized in the Notes section, but detailed operation of some of the automated devices is beyond the scope of this short Protocol Article. John Graham Copyright © 2002 John Graham. All rights reserved. The TGF-β Family: Signaling Pathways, Developmental Roles, and Tumor Suppressor Activities Mon, 01 Jan 1900 00:00:00 +0000 Intercellular communication is a critical process for all multicellular organisms, and communication among cells is required for proper embryonic development and adult physiology. Members of the Transforming Growth Factor-β (TGF-β) family of secreted proteins communicate information between cells via a complex signaling pathway, and family members are capable of inducing a wide range of cellular responses. The purpose of this review is to provide the reader with a broad introduction to our current understanding of three aspects of the TGF-β family. These are the molecular mechanisms utilized by TGF-β signaling pathways, the developmental roles played by TGF-β family members in a variety of species, and the growing list of cancers in which various TGF-β signaling pathways display tumor suppressor activity. Aaron N. Johnson and Stuart J. Newfeld Copyright © 2002 Aaron N. Johnson and Stuart J. Newfeld. All rights reserved. TRANSGENIC STUDIES ON THE CGRP-RECEPTOR COMPONENT PROTEIN (RCP) Mon, 01 Jan 1900 00:00:00 +0000 The CGRP-receptor component protein (RCP) is a 17-kDa intracellular peripheral membrane protein that is required for signal transduction by the calcitonin receptor-like receptor (CRLR). We have demonstrated that RCP is expressed in tissues and cell lines containing CGRP receptors, and that RCP expression is regulated in parallel with the biological efficacy of CGRP in vivo. We have made stable cell lines which express an antisense RCP cDNA, and have shown that loss of RCP protein correlated with loss of CGRP signal transduction, but not CGRP binding. RCP thus represents a new class of protein required for CGRP-mediated signal transduction. We are generating RCP-deficient mice using homologous recombination to extend these cell culture studies into an in vivo. model. These RCP-null mice will have important uses for development of therapeutic strategies targeted at migraine, hypertension, and chronic pain. Laila O. Mnayer, Mark I. Rosenblatt, and Ian M. Dickerson Copyright © 2001 Laila O. Mnayer et al. All rights reserved. Receptor Tyrosine Kinases and Inhibitors in Lung Cancer Mon, 01 Jan 1900 00:00:00 +0000 Lung cancer is a deadly disease with high mortality and morbidity. Most cases of lung cancer are due to non-small cell carcinoma, with 16% of cases being small cell carcinoma. The biology at a cellular level is of interest at many levels. Knowing cellular pathways helps to further enhance our knowledge of how lung cancer cells survive, proliferate, and metastasize. The receptor tyrosine kinases (RTKs) located at the cellular membrane are becoming of great interest as sites for targeted therapies for lung cancers. This review will discuss the RTKs that are involved in lung cancers and the newer therapies that are being tested. We will specifically discuss receptors such as epidermal growth factor receptor, c-Kit receptor, VEGF receptor, c-Met receptor, insulin growth factor receptor, and Eph receptor. The inhibitors against the specific RTKs are in various preclinical and clinical trials, and this will be detailed. Evan Pisick, Simha Jagadeesh, and Ravi Salgia Copyright © 2004 Evan Pisick et al. All rights reserved. STRESS AND THE CONTROL OF APOPTOSIS Mon, 01 Jan 1900 00:00:00 +0000 Douglas R. Green Copyright © 2001 Douglas R. Green. All rights reserved. IMMUNOHISTOCHEMICAL LOCALIZATION OF CALCITONIN RECEPTOR-LIKE RECEPTOR AND RECEPTOR ACTIVITY MODIFYING PROTEINS IN HUMAN CEREBRAL AND CRANIAL VASCULATURE Mon, 01 Jan 1900 00:00:00 +0000 Calcitonin gene-related peptide and adrenomedullin are both members of a structurally related neuropeptide family. Both are potent vasodilators and known to be expressed in the trigeminovascular system. Central to functional binding of these neuropeptides is calcitonin receptor-like receptor (CRLR), a G-protein coupled receptor whose cell surface expression and pharmacology is determined by co-expression of a receptor activity-modifying protein (RAMP). CRLR combined with RAMP1 binds CGRP with high affinity, whilst CRLR coexpression with either RAMP2 or RAMP3 confers high affinity binding of adrenomedullin. We sought to investigate expression of these receptor components in human cerebral and cranial vasculature to further characterize CGRP receptor family content and potential function(s) in this vascular system. Antisera specific for CRLR, RAMP1, RAMP2, and RAMP3 were raised, characterized, and applied to tissue sections utilizing modern immunohistochemical techniques and confocal microscopy. Localization data demonstrated for the first time, the presence of these receptor component proteins in human cerebrovasculature, and suggest that both CGRP and adrenomedullin receptors may arise from coassembly of these accessory proteins with CRLR. These novel data advance our understanding of the function of the trigeminovascular system and the normal and potential pathophysiological role of CGRP family neuropeptides. A. Wainwright, L. Edvinsson, and K.R. Oliver Copyright © 2001 A. Wainwright et al. All rights reserved. Isolation of Human Polymorphonuclear Leukocytes (Granulocytes) from a Leukocyte-Rich Fraction Mon, 01 Jan 1900 00:00:00 +0000 Human peripheral blood polymorphonuclear leukocytes (PMNs) or granulocytes from a leukocyte-rich plasma (LRP) are banded at an interface between two layers of iodixanol. If the denser layer of iodixanol is omitted the PMNs may alternatively be pelleted. The procedure can be adapted to blood from other species by small changes to the density of the two iodixanol layers. The method works optimally with EDTA- or citrate-anticoagulated blood. John Graham Copyright © 2002 John Graham. All rights reserved. Dental Tissue — New Source for Stem Cells Mon, 01 Jan 1900 00:00:00 +0000 Stem cells have been isolated from many tissues and organs, including dental tissue. Five types of dental stem cells have been established: dental pulp stem cells, stem cells from exfoliated deciduous teeth, stem cells from apical papilla, periodontal ligament stem cells, and dental follicle progenitor cells. The main characteristics of dental stem cells are their potential for multilineage differentiation and self-renewal capacity. Dental stem cells can differentiate into odontoblasts, adipocytes, neuronal-like cells, glial cells, osteoblasts, chondrocytes, melanocytes, myotubes, and endothelial cells. Possible application of these cells in various fields of medicine makes them good candidates for future research as a new, powerful tool for therapy. Although the possible use of these cells in therapeutic purposes and tooth tissue engineering is still in the beginning stages, the results are promising. The efforts made in the research of dental stem cells have clarified many mechanisms underlying the biological processes in which these cells are involved. This review will focus on the new findings in the field of dental stem cell research and on their potential use in the therapy of various disorders. Vladimir Petrovic and Vladisav Stefanovic Copyright © 2009 Vladimir Petrovic and Vladisav Stefanovic. All rights reserved. THE OXIDATIVE STRESS RESPONSE GENE P66 shc AND THE TUMOR SUPPRESSOR GENE P53 INDUCE MITOCHONDRIAL DNA DAMAGES Mon, 01 Jan 1900 00:00:00 +0000 Mirella Trinei, Marco Giorgio, Sara Barozzi, and Pier Giuseppe Pelicci Copyright © 2001 Mirella Trinei et al. All rights reserved. Cyclin-Dependent Kinase Inhibitors Induce Apoptosis in Plant Cells Mon, 01 Jan 1900 00:00:00 +0000 M. Strnad, L. Havlia, P. Binarova, V. Kryscaron, J. Hanuscaron, V. Siglerova, L. Boumlgre, and E. Heberle-Bors Copyright © 2001 M. Strnad et al. All rights reserved. Initiation of apoptosis by photodynamic therapy on MCF7 cells Mon, 01 Jan 1900 00:00:00 +0000 Hana Kolacaronova, Kamila Reblova, Miroslav Strnad, and R. Lenobel Copyright © 2001 Hana Kolacaronova et al. All rights reserved. Mitochondria: Regulators of Cell Death and Survival Mon, 01 Jan 1900 00:00:00 +0000 The past 5 years has seen an intense surge in research devoted toward understanding the critical role of mitochondria in the regulation of cell death. Apoptosis can be initiated by a wide array of stimuli, inducing multiple signaling pathways that, for the most part, converge at the mitochondrion. Although classically considered the powerhouses of the cell, it is now understood that mitochondria are also “gatekeepers” that ultimately determine the fate of the cell. The mitochondrial decision as to whether a cell lives or dies is complex, involving protein-protein interactions, ionic changes, reactive oxygen species, and other mechanisms that require further elucidation. Once the death process is initiated, mitochondria undergo conformational changes, resulting in the release of cytochrome c (cyt c), caspases, endonucleases, and other factors leading to the onset and execution of apoptosis. The present review attempts to outline the complex milieu of events regulating the mitochondrial commitment to and processes involved in the implementation of the executioner phase of apoptotic cell death. David J. Granville and Roberta A. Gottlieb Copyright © 2002 David J. Granville and Roberta A. Gottlieb. All rights reserved. REGULATION OF CELL PROLIFERATION: ROLE OF A CHROMOSOME-MATRIX PROTEIN Mon, 01 Jan 1900 00:00:00 +0000 Miryam A. Fragoso, Mariana Salas-Vega, Viviana Fernandez, Hassan Tahi, Jean-Marie Parel, and Dora E. Vega-Salas Copyright © 2001 Miryam A. Fragoso et al. All rights reserved. Isolation of Human Platelets (Thrombocytes) Mon, 01 Jan 1900 00:00:00 +0000 Platelets from human blood can be isolated in high yield by centrifugation of whole blood over an iodixanol density barrier of 1.063 g/ml. The separation from all of the blood cells (which form a pellet) is based on the slower sedimentation velocity of the smaller platelets. John Graham Copyright © 2002 John Graham. All rights reserved. Vesicles Generated during Storage of Red Blood Cells Enhance the Generation of Radical Oxygen Speciesin Activated Neutrophils Mon, 01 Jan 1900 00:00:00 +0000 Erythrocytes are known to shed vesicles in vivo, under various conditions in vitro, and, with impact for transfusion medicine, during storage of red blood cell concentrates (Vsto vesicles). Vsto vesicles of blood transfusions have been shown to deliver glycosylphosphatidylinositol-linked proteins to recipient erythrocytes, to display prothrombotic activity, and to have an inhibitory effect on macrophages. The interaction of Vsto vesicles with and their effect on neutrophilic granulocytes has not yet been studied in detail. Fluorescentlylabeled Vsto and calcium-induced vesicles were preparedin order to study the uptake of labeled vesicular components by neutrophils as compared to the process of phagocytosis of zymosan using flow cytometry and confocal microscopy. The activating effect of Vsto vesicles on neutrophils was addressed by a luminometric assay for stimulated radical oxygen species (ROS) generation. Coincubation of vesicles and neutrophils results in a transfer of vesicular components to the cells. This uptake is different from a phagocytotic process and is enhanced upon interference with the cellular actin cytoskeleton. Preincubation of neutrophils with Vsto vesicles results in an enhanced ROS generation by neutrophils, which is further increased upon fMLP stimulation and during zymosan phagocytosis. The activating effect of Vsto vesicles on neutrophils might be due to the specific accumulation of lysophospholipidsin Vsto vesicles and should be considered as a possible contributor to the pathogenesis of transfusion-related acute lung injury. Herbert Jank and Ulrich Salzer Copyright © 2011 Herbert Jank and Ulrich Salzer. All rights reserved. Insulin/IGF-I and Related Signaling Pathways Regulate Aging in Nondividing Cells: from Yeast to the Mammalian Brain Mon, 01 Jan 1900 00:00:00 +0000 Mutations that reduce glucose or insulin/insulin-like growth factor-I (IGF-I) signaling increase longevity in organisms ranging from yeast to mammals. Over the past 10 years, several studies confirmed this conserved molecular strategy of longevity regulation, and many more have been added to the complex mosaic that links stress resistance and aging. In this review, we will analyze the similarities that have emerged over the last decade between longevity regulatory pathways in organisms ranging from yeast, nematodes, and fruit flies to mice. We will focus on the role of yeast signal transduction proteins Ras, Tor, Sch9, Sir2, their homologs in higher organisms, and their association to oxidative stress and protective systems. We will discuss how the “molecular strategy” responsible for life span extension in response to dietary and genetic manipulations appears to be remarkably conserved in various organisms and cells, including neuronal cells in different organisms. Taken together, these studies indicate that simple model systems will contribute to our comprehension of aging of the mammalian nervous system and will stimulate novel neurotherapeutic strategies in humans. Edoardo Parrella and Valter D. Longo Copyright © 2010 Edoardo Parrella and Valter D. Longo. All rights reserved. OptiPrep? Density Gradient Solutions for Macromolecules and Macromolecular Complexes Mon, 01 Jan 1900 00:00:00 +0000 Any density gradient for the isolation of mammalian cells should ideally only expose the sedimenting particles to an increasing concentration of the gradient solute. Thus they will experience only an increasing density and viscosity, other parameters such as osmolality, pH, ionic strength and the concentration of important additives (such as EDTA or divalent cations) should remain as close to constant as possible. This Protocol Article describes the strategies for the dilution of OptiPrep™ in order to prepare such solutions for mammalian cells. John Graham Copyright © 2002 John Graham. All rights reserved. Purification of Peroxisomes in a Preformed Iodixanol Gradient in a Fixed-Angle Rotor Mon, 01 Jan 1900 00:00:00 +0000 In iodixanol, peroxisomes are the densest organelle in the light mitochondrial fraction and are therefore easily separated from the other components (lysosomes, mitochondria, etc.) in a preformed isosmotic continuous gradient. The resolution of the peroxisomes is far superior than that in sucrose and, unlike in Percoll® there is no contamination from endoplasmic reticulum. John Graham Copyright © 2002 John Graham. All rights reserved. Investigation of the Proliferation, Apoptosis/Necrosis, and Cell Cycle Phases in Several Human Multiple Myeloma Cell Lines. Comparison of Viscum album QuFrF Extract with Vincristine in an In Vitro Model Mon, 01 Jan 1900 00:00:00 +0000 Multiple myeloma is a haematological disorder of malignant plasma cells. Interleukin-6 (IL-6) is a potent growth factor for the proliferation of these cells. Vincristine as a chemotherapeutic agent is used mainly in combination with other chemotherapeutic substances in the treatment of different haematological disorders. Viscum album QuFrF (VAQuFrF) extract is an experimental drug that is not used in the treatment in tumour patients. It contains 2000 ng lectin and 10 µg viscotoxin in 10 mg extract. In this study, the effects of VAQuFrF extract were compared with those of vincristine in six human multiple myeloma cell lines (Molp-8, LP-1, RPMI-8226, OPM-2, Colo-677, and KMS-12-BM) using an in vitro model. As parameters, the IL-6 production, proliferation, apoptosis/necrosis, and cell cycle phases of the cells were taken. To measure the IL-6 production, apoptosis/necrosis, and cell cycle phases, the substances were tested in dose ranges of 10, 50, and 100 µg/106 cells. To measure the proliferation of the cells, the substances were tested in dose ranges of 1, 5, and 10 µg/105 cells. The profile of the antitumour effects of the two substances is identical. (1) Neither VAQuFrF extract nor vincristine produced IL-6 in any cell line. (2) Both substances inhibited the proliferation of the cells (cytostatic effect), arrested the cell cycle phases, and increased the number of apoptotic/necrotic cells (cytocidal effect). At a dose of 10 µg/105 cells, VAQuFrF more effectively inhibited the proliferation than vincristine (p < 0.01) in the cell lines Molp-8, LP-1, and RPMI-8226. (3) VAQuFrF affected the tumour cells mainly via cytostatic effect. Vincristine had a clear cytocidal effect. These findings indicate that VAQuFrF extract could be a novel drug in the treatment of multiple myeloma. Eva Kovacs Copyright © 2010 Eva Kovacs. All rights reserved. The Urodele Limb Regeneration Blastema: The Cell Potential Mon, 01 Jan 1900 00:00:00 +0000 The developmental potential of the limb regeneration blastema, a mass of mesenchymal cells of mixed origins, was once considered as being pluripotent, capable of forming all cell types. Now evidence asserts that the blastema is a heterogeneous mixture of progenitor cells derived from tissues of the amputation site, with limited developmental potential, plus various stem cells with multipotent abilities. Many specialized cells, bone, cartilage, muscle, and Schwann cells, at the injury site undergo dedifferentiation to a progenitor state and maintain their cell lineage as they redifferentiate in the regenerate. Muscle satellite reserve stem cells that are active in repair of injured muscle may also dedifferentiate and contribute new muscle cells to the limb blastema. Other cells from the dermis act as multipotent stem cells that replenish dermal fibroblasts and differentiate into cartilage. The blastema primordium is a self-organized, equipotential system, but at the cellular level can compensate for specific cell loss. It is able to induce dedifferentiation of introduced exogenous cells and such cells may be transformed into new cell types. Indigenous cells of the blastema associated with amputated tissues may also transform or possibly transdifferentiate into new cell types. The blastema is a microenvironment that enables dedifferentiation, redifferentiation, transdifferentiation, and stem cell activation, leading to progenitor cells of the limb regenerate. Kenyon S. Tweedell Copyright © 2010 Kenyon S. Tweedell. All rights reserved. Molecular Analysis of TTP, a Key Regulator of TNF Activity Mon, 01 Jan 1900 00:00:00 +0000 Barbra A. Johnson, Michael Yaffe, and T. Keith Blackwell Copyright © 2001 Barbra A. Johnson et al. All rights reserved. The Podocyte in Diabetic Kidney Disease Mon, 01 Jan 1900 00:00:00 +0000 Approaching epidemic levels, diabetic kidney disease (DKD) is now the leading cause of end-stage renal disease (ESRD). Microalbuminuria is an early clinical marker of DKD that results from damage to the glomerular filtration barrier at the level of the highly differentiated glomerular podocyte cells. Injury to these epithelial cells, podocytopathies, includes cellular hypertrophy, foot process effacement, detachment from the glomerular basement membrane, and apoptosis. Here we review the role of a number of recently identified factors that contribute to podocytopathies in DKD. These factors include members of the renin-angiotensin system (RAS), including angiotensin-converting enzyme (ACE) types 1 and 2, prorenin and its receptor, reactive oxygen species (ROS), prostanoids, peroxisome proliferator-activated receptors (PPAR), advanced glycation end-products (AGEs) and their receptors (RAGE), adiponectin, and microRNAs. As the number of therapeutic options that slow, but do not halt, the progression of DKD to ESRD remains limited, a more comprehensive understanding of the signaling events that contribute to this increasingly prevalent disease may identify novel avenues for treatment and prevention. Erin Stitt-Cavanagh, Laura MacLoed, and Chris R.J. Kennedy Copyright © 2009 Erin Stitt-Cavanagh et al. All rights reserved. Electron Microscopic Radioautographic Study on Protein Synthesis in Mitochondria of Binucleate Hepatocytes of Aging Mice Mon, 01 Jan 1900 00:00:00 +0000 In order to study the aging changes of intramitochondrial protein synthesis in mouse hepatocytes, 10 groups of aging mice, each consisting of three individuals, total 30, from fetal day 19 to postnatal year 2, were injected with 3H-leucine, a protein precursor, sacrificed 1 h later, and the liver tissues processed for electron microscopic radioautography. On electron microscopic radioautograms obtained from each animal, the numbers of mitochondria, the numbers of labeled mitochondria, and the mitochondrial labeling index labeled with 3H-leucine that showed protein synthesis in each hepatocyte, both mononucleate and binucleate cells, were counted and the averages in respective aging groups were compared. From the results, it was demonstrated that the numbers of mitochondria, the numbers of labeled mitochondria, and the labeling indices of intramitochondrial protein syntheses in both mononucleate and binucleate hepatocytes of mice at various ages increased due to development of animals. The numbers of mitochondria, the numbers of labeled mitochondria, and the labeling indices of intramitochondrial protein synthesis in binucleate hepatocytes were more than those of mononucleate hepatocytes at the same aging stages. Tetsuji Nagata Copyright © 2007 Tetsuji Nagata. All rights reserved. Programmed Cell Death of Human Skin Fibroblast Induced by Benzimidazole Mon, 01 Jan 1900 00:00:00 +0000 T. Ravikumar, N. Shanmugasundaram, and Mary Babu Copyright © 2001 T. Ravikumar et al. All rights reserved. Encoding Selectivity of a Transmembrane Channel Mon, 01 Jan 1900 00:00:00 +0000 Robert M. Stroud, Peter Nollert, Larry J.W. Miercke, William E.C. Harries, and Joe O'Connell Copyright © 2002 Robert M. Stroud et al. All rights reserved. Secondary Necrosis of Apoptotic Neutrophils Contributes to Inflammatory Lung Injury in vivo Mon, 01 Jan 1900 00:00:00 +0000 Djordje Medan, Liying Wang, and Yon Rojanasakul Copyright © 2001 Djordje Medan et al. All rights reserved. Cigarette Smoke Extract Promotes Human Vascular Smooth Muscle Cell Proliferation and Survival through ERK1/2- and NF-κB–Dependent Pathways Mon, 01 Jan 1900 00:00:00 +0000 Tobacco use is one of the major risk factors of cardiovascular disease. The underlying molecular mechanisms that link cigarette smoke to cardiovascular disease remain unclear. The present study was designed to examine the effects of dimethyl sulfoxide (DMSO)–soluble smoke particles (DSPs) on human aortic smooth muscle cell (HASMC) cultures, and to explore the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) and nuclear factor-κB (NF-κB) signal mechanisms involved. Serum-starved HASMCs were treated with DSPs for up to 48 h. DSPs promoted cell proliferation in a concentration-dependent manner from 0.05 to 0.2 ?l/ml. Activation of ERK1/2 and NF-κB was seen after exposure to DSPs. This occurred in parallel with the increase in cell population, bromodeoxyuridine incorporation, and cyclinD1/cyclin-dependent kinase 4 expression. Blocking phosphorylation of ERK1/2 by MAPK inhibitors U0126 and PD98059, and inhibiting activation of NF-κB by IκB (IκB) kinase inhibitors wedelolactone or IMD-0354, abolished the DSP effects. However, either a p38 inhibitor (SB203580) or an inhibitor of lipopolysaccharide (polymyxin B), or nicotinic receptor blockers (mecamylamine and α-bungarotoxin), did not inhibit a DSP-induced increase in the cell population. DSPs increased the expression of intercellular adhesion molecule 1 and the release of interleukin-6 in HASMCs, both of which were inhibited by ERK1/2 or NF-κB pathway inhibitors. Furthermore, cell apoptosis and necrosis were found in serum-starved HASMCs. DSPs decreased cell death and increased B-cell leukemia/lymphoma 2 expression. Blocking phosphorylation of ERK1/2 or NF-κB attenuated DSP-induced cell death inhibition. Cigarette smoke particles stimulate HASMC proliferation and inhibit cell death. The intracellular signal mechanisms behind this involve activation of ERK1/2 and NF-κB pathways. Qing-wen Chen, Lars Edvinsson, and Cang-Bao Xu Copyright © 2010 Qing-wen Chen et al. All rights reserved. TROPHIC FACTOR WITHDRAWAL INDUCES A NOVEL PATHWAY: p38 MAPK ACTIVATES NHE1 RESULTING IN INTRACELLULAR ALKALINIZATION, AN EARLY STEP IN APOPTOSIS Mon, 01 Jan 1900 00:00:00 +0000 Annette R. Khaled, Kyungjae Kim, Kathrin Muegge, Craig Thompson, Larry Fliegel, and Scott K. Durum Copyright © 2001 Annette R. Khaled et al. All rights reserved. Muc4\sialomucin Complex, A Specific Intramembrane Modulator of ERBB2/HER2/NEU, Potentiates Primary Tumor Growth and Suppresses Apoptosis in A Xenotransplanted Melanoma Mon, 01 Jan 1900 00:00:00 +0000 Coralie Carothers Carraway, Masanobu Komatsu, Scott Jepson, Maria Arango, and Kermit Carraway Copyright © 2001 Coralie Carothers Carraway et al. All rights reserved. Increased Apoptosis and Proliferative Capacity are Early Events in Cyst Formation in Autosomal-Dominant, Polycystic Kidney Disease Mon, 01 Jan 1900 00:00:00 +0000 Previous studies have highlighted epithelial proliferation and apoptosis in the cyst lining as common features in animal models of cystic disease. In this study, we sought to evaluate the timing and extent of these changes in renal tissue obtained from patients with autosomal-dominant, polycystic kidney disease (ADPKD) subjected for nephrectomy for a variety of clinical indications. Cell proliferation was assessed using an antibody to proliferating cell nuclear antigen (PCNA), and apoptosis was evaluated by the use of terminal deoxynucleotidyl transferase (TdT) digoxigenin-deoxyuridine (dUTP) nick end-labeling technique (ApopTag®). The origin of cystic structures was evaluated using antibodies to epithelial membrane antigen (EMA). The lineage of interstitial mononuclear cells was assessed by anti CD 45 and CD 68 monoclonal antibodies. We found an increased rate of proliferation within the epithelium, not only of cystic, but also of noncystic, tubules that was significantly higher than the corresponding values from normal kidney (p ≤ 0.0001). Apoptotic index values were significantly increased within the epithelium lining noncystic and cystic structures (p < 0.001). In the interstitium, increased proliferation and apoptosis rates were also noted. Interstitial infiltrates were dense and consisted mainly of CD 68–positive macrophages and CD 45–positive lymphocytes. The present study demonstrated that changes in cell turnover are early events in cyst formation. The observation of mild proportionate elevation of both proliferation and apoptosis values of the epithelium lining cysts explains the lack of increase risk of renal cell carcinoma in ADPKD. The development of heavy interstitial inflammation could contribute to progressive tubulointerstitial scarring, leading to progressive renal failure. Salwa Ibrahim Copyright © 2007 Salwa Ibrahim. All rights reserved. B709 MITOCHONDRIAL CONTROL OF CELL DEATH Mon, 01 Jan 1900 00:00:00 +0000 Guido Kroemer Copyright © 2001 Guido Kroemer. All rights reserved. Computer-Assisted Reconstruction and Motion Analysis of the Three-Dimensional Cell Mon, 01 Jan 1900 00:00:00 +0000 Even though several microscopic techniques provide three-dimensional (3D) information on fixed and living cells, the perception persists that cells are two-dimensional (2D). Cells are, in fact, 3D and their behavior, including the extension of pseudopods, includes an important 3D component. Although treating the cell as a 2D entity has proven effective in understanding how cells locomote, and in identifying defects in a variety of mutant and abnormal cells, there are cases in which 3D reconstruction and analysis are essential. Here, we describe advanced computer-assisted 3D reconstruction and motion analysis programs for both individual live, crawling cells and developing embryos. These systems (3D-DIAS, 3D-DIASemb) can be used to reconstruct and motion analyze at short time intervals the nucleus and pseudopodia as well as the entire surface of a single migrating cell, or every cell and nucleus in a developing embryo. Because all images are converted to mathematical representations, a variety of motility and dynamic morphology parameters can be computed that have proven quite valuable in the identification of mutant behaviors. We also describe examples of mutant behaviors in Dictyostelium that were revealed through 3D analysis. David R. Soll, Deborah Wessels, Paul J. Heid, and Edward Voss Copyright © 2003 David R. Soll et al. All rights reserved. Blood Cell Membrane Fluidity and Intracellular Ca2+ Changes in Antiretroviral-Naïve and -Treated HIV-1–Infected Patients Mon, 01 Jan 1900 00:00:00 +0000 We previously showed that lymphocytes and erythrocytes of HIV-1–infected patients, prior to antiretroviral therapy, presented significant changes in intracellular calcium concentration ([Ca2+]int) and membrane fluidity. The present study evaluates the same parameters after response to highly active antiretroviral therapy (HAART). Blood samples were collected from patients prior to and after antiretroviral therapy, and from control subjects. Membrane fluidity and [Ca2+]int were assessed by fluorescence spectroscopy measurements, using three different probes: TMA-DPH and DPH for membrane fluidity, and fura-2 for Ca2+. When compared with the control group, both untreated and treated patients presented increased lymphocyte [Ca2+]int and decreased lymphocyte membrane fluidity, without significant differences between the two groups of patients. On the contrary, the therapy reversed the membrane fluidity variations observed in erythrocytes. The decreased erythrocyte [Ca2+]int of untreated patients was not reversed by HAART. AIDS patients present changes in lymphocyte (mostly noninfected) and erythrocyte properties, partially reversed by HAART, consistent with a process of facilitated propagation of the infection to new cells, stimulation of virion production, and maintenance of a reservoir of erythrocyte-bound infectious virus. These observations can be related with the action of the HIV Nef protein in the cell's proteins and lipid composition, as well as with the recently observed cell infection by HIV-1 via endocytosis. Nuno C. Santos, J. Martins e Silva, Teresa Freitas, M. Doroana, N. Duarte, L. Tavares, F. Antunes, and Carlota Saldanha Copyright © 2010 Nuno C. Santos et al. All rights reserved. MAPK/SAPK SIGNALING IN APOPTOSIS AND bFGF-MEDIATED SURVIVAL Mon, 01 Jan 1900 00:00:00 +0000 Chantal Schamberger and Christa Cerni Copyright © 2001 Chantal Schamberger and Christa Cerni. All rights reserved. Anti-Inflammatory Properties of Dendrimers per se Mon, 01 Jan 1900 00:00:00 +0000 Dendrimers are polybranched and polyfunctionalized tree-like polymers. Unlike linear polymers, they have perfectly defined structure and molecular weight, due to their iterative step-by-step synthesis. Their multivalent structure and supramolecular properties have made them attractive nanotools for applications, particularly in biology and medicine. Among the different biological and medical properties of dendrimers that have been developed over the past decades, the anti-inflammatory properties of several groups of dendrimers are the most recently discovered. Thereof, dendrimers emerge as promising, although heretical, drug candidates for the treatment of still-uncured chronic inflammatory disorders. This mini-review is based on the five main scientific articles giving an overview of what can be the spectrum of anti-inflammatory characteristics displayed by dendrimers. Myriam Hayder, Séverine Fruchon, Jean-Jacques Fournié, Mary Poupot, and Rémy Poupot Copyright © 2011 Myriam Hayder et al. All rights reserved. HIV-Envelope–Dependent Cell-Cell Fusion: Quantitative Studies Mon, 01 Jan 1900 00:00:00 +0000 Interaction in vitro between cells infected with human immunodeficiency virus (HIV) and surrounding, uninfected, target cells often leads to cell fusion and the formation of multinucleated cells, called syncytia. The presence in HIV-infected individuals of virus strains able to induce syncytia in cultures of T cells is associated with disease progression and AIDS. Even in the asymptomatic stage of infection, multinucleated cells have been observed in different organs, indicating that fused cells may be generated and remain viable in the tissues of patients. We used lymphocytic cells transfected for the expression of the HIV-envelope (Env) glycoproteins to develop a method for the direct quantification of fusion events by flow cytometry (Huerta et al., 2006, J. Virol. Methods 138, 17–23; López-Balderas et al., 2007, Virus Res. 123, 138–146). The method involves the staining of fusion partners with lipophilic probes and the use of fluorescence resonance energy transfer (FRET) to distinguish between fused and aggregated cells. We have shown that such a flow-cytometry assay is appropriate for the screening of compounds that have the potential to modulate HIV-Env–mediated cell fusion. Even those syncytia that are small or few in numbers can be detected. Quantitative analysis of the fusion products was performed with this technique; the results indicated that the time of reaction and initial proportion of fusion partners determine the number, relative size, and average cellular composition of syncytia. Heterogeneity of syncytia generated by HIV-Env–mediated cell-cell fusion may result in a variety of possible outcomes that, in turn, may influence the biological properties of the syncytia and surrounding cells, as well as replication of virus. Given the myriad immune abnormalities leading to AIDS, the full understanding of the extent, diverse composition, and role of fused cells in the pathogenesis of, and immune response to, HIV infection is an important, pending issue. Leonor Huerta, Nayali López-Balderas, Evelyn Rivera-Toledo, Guadalupe Sandoval, Guillermo Gómez-Icazbalceta, Carlos Villarreal, Edmundo Lamoyi, and Carlos Larralde Copyright © 2009 Leonor Huerta et al. All rights reserved. Proteomic Analysis of Rough and Smooth Endoplasmic Reticulum Mon, 01 Jan 1900 00:00:00 +0000 Jacques Paiement, Line Roy, Annalyn Gilchrist, Alex Bell, Rob Kearney, David Y. Thomas, and John J.M. Bergeron Copyright © 2002 Jacques Paiement et al. All rights reserved. Preparation of Crude Subcellular Fractions by Differential Centrifugation Mon, 01 Jan 1900 00:00:00 +0000 The employment of differential centrifugation to prepare crude fractions of subcellular particles from homogenates is often a necessary first step to a subsequent purification of one or more particles on a density gradient. Buoyant density gradient purification of peroxisomes or lysosomes for example is almost invariably carried out on a light mitochondrial fraction so as to eliminate smaller particles that may have similar densities. Unless they are first removed, large rapidly sedimenting particles in homogenates may also disturb shallow gradients designed to fractionate small low-density microsomes. John Graham Copyright © 2002 John Graham. All rights reserved. Cellular Senescence: Many Roads, One Final Destination Mon, 01 Jan 1900 00:00:00 +0000 Cellular senescence is a tumor-suppressor mechanism that has been shown to occur in response to multiple signals, including oncogenic stress, DNA damage, oxidative stress, telomere shortening, and other tumor-promoting insults. Over the past decade, much has been uncovered regarding the phenotype of this tumor-suppressor response and the underlying pathways necessary for its establishment. However, we have also learned that the intricate details of signaling pathways underlying senescence as a tumor-suppressor response are very much context dependent. In addition, cross-talk among pathways, and negative and positive feedback loops, all complicate our understanding of this process. This short review attempts to summarize what is known to date regarding senescence in tumor suppression, both in vitro and in vivo. Further insights into pathways necessary for senescence will hopefully identify appropriate targets for interventions to not only induce senescence as a treatment of cancerous lesions, but also to maintain this state in premalignant lesions in an effort to prevent progression to cancer. Raya Saab Copyright © 2010 Raya Saab. All rights reserved. Rabs, Rips, FIPs, and Endocytic Membrane Traffic Mon, 01 Jan 1900 00:00:00 +0000 Rab GTPases, proteins belonging to the Ras-like small GTP-binding protein superfamily, have emerged as master regulators of cellular membrane transport. Rab11 GTPase, a member of the Rab protein family, plays a role in regulating various cellular functions, including plasma membrane recycling, phagocytosis, and cytokinesis. Rab11 acts by forming mutually exclusive complexes with Rab11-family binding proteins, known as FIPs. Rab11-FIP complexes serve a role of �targeting complexes� by recruiting various membrane traffic factors to cellular membranes. Recent studies have identified several Rab11-FIP complex-binding proteins that regulate distinct membrane traffic pathways. Rytis Prekeris Copyright © 2003 Rytis Prekeris. All rights reserved. OptiPrepTM Density Gradient Solutions for Nonmammalian Organelles Mon, 01 Jan 1900 00:00:00 +0000 Any density gradient for the isolation of nonmammalian organelles should ideally only expose the sedimenting biological particles to an increasing concentration of the gradient solute. Thus they will experience only an increasing density and viscosity, other parameters such as osmolality, pH, ionic strength and the concentration of important additives (such as EDTA and DTT) should remain as close to constant as possible. This Protocol Article describes the strategies for the dilution of OptiPrep™ in order to prepare such solutions for organelles and membranes from nonmammalian sources such as yeast. John M. Graham Copyright © 2002 John M. Graham. All rights reserved. Cell Cycle Dynamics of the Nuclear Envelope Mon, 01 Jan 1900 00:00:00 +0000 The nuclear envelope (NE) consists of an inner and an outer membrane, nuclear pore complexes, and the underlying nuclear lamina, a filamentous scaffold structure formed by lamins. The inner membrane is linked to the lamina and chromatin by its integral membrane proteins, such as lamin B receptor (LBR), emerin, and various isoforms of lamina-associated polypeptides (LAP) 1 and 2, which bind lamins and/or chromatin. During mitosis, the NE is disassembled upon phosphorylation of its core components, and the NE is torn apart by a dynein-driven microtubule-dependent mechanism. Nuclear reassembly after sister chromatid separation requires a timely coordinated and dephosphorylation-dependent association of lamin-binding proteins and lamins with chromosomal proteins and targeting of membranes to specific sites on chromosomes. Various chromatin-binding domains in lamina proteins, such as the LEM domain, present in all LAP2 isoforms and in emerin, as well as unique regions in lamina proteins and in specific LAP2 isoforms have been implicated in defined steps of NE reformation. Furthermore, novel mechanisms of membrane fusion involving Ran GTPase are just beginning to emerge. Roland Foisner Copyright © 2003 Roland�Foisner. All rights reserved. DELAYING AGING WITH MITOCHONDRIAL MICRONUTRIENTS AND ANTIOXIDANTS Mon, 01 Jan 1900 00:00:00 +0000 Hani Atamna, Bruce N. Ames, and Jiankang Liu Copyright © 2001 Hani Atamna et al. All rights reserved. Isolation of Rat and Human Hippocampal Neuron Fractions in a Discontinuous Density Gradient Mon, 01 Jan 1900 00:00:00 +0000 The plating efficiency of neurons in culture is highly dependent on the concentration of cells used to establish the monolayer. A discontinuous iodixanol gradient permits both the production of a viable concentrated suspension of neurons and purification from other brain tissue elements. The gradient that is described in this Protocol Article is applicable to brain tissue from rat and also from human biopsy specimens. John Graham Copyright © 2002 John Graham. All rights reserved. Stress-activated protein kinases (SAPKs) in IGF-I mediated cell survival Mon, 01 Jan 1900 00:00:00 +0000 Darren Krause, Anthony Lyons, Patrick Walsh, and Rosemary OConnor Copyright © 2001 Darren Krause et al. All rights reserved. Recent Insights into the Implication of Nitric Oxide in Osteoblast Differentiation and Proliferation during Bone Development Mon, 01 Jan 1900 00:00:00 +0000 Bone tissue renovation is a dynamic event in which osteoblasts and osteoclasts are responsible for the turnover between bone formation and bone resorption, respectively. During bone development, extracellular matrix remodeling is required for osteoblast differentiation and the process is largely mediated by the proteolytic activity of extracellular matrix metalloproteinases (MMPs), which play a fundamental role in osteoblast migration, unmineralized matrix degradation, and cell invasion. The recent advances towards investigation in osteogenesis have provided significant information about the transcriptional regulation of several genes, including MMPs, by the expression of crucial transcription factors like NFAT, ATF4, osterix, TAZ, and Cbfa-1–responsive elements. Evidence from gene knock-out studies have shown that bone formation is, at least in part, mediated by nitric oxide (NO), since mice deficient in endothelial nitric oxide synthase (eNOS) and mice deficient in the eNOS downstream effector (cGMP)-dependent protein kinase (PKG) show bone abnormalities, while inducible NOS (iNOS) null mice also show imbalances in bone osteogenesis and abnormalities in bone healing. Recently, in vitro data showed that Cbfa-1 and the MAPK pathways were crucial for osteoblastic cell differentiation, and NO was found to play a significant role. This article sheds light on some of the mechanisms that may influence NO-mediated actions in bone development. Marta Saura, Carlos Tarin, and Carlos Zaragoza Copyright © 2010 Marta Saura et al. All rights reserved. Fas-Induced Apoptosis of Human Neutrophils is Promoted by Phosphatidyl Inositol 3-Kinase but Suppressed by P38-MAPK Activity Mon, 01 Jan 1900 00:00:00 +0000 Maria Alvarado-Kristensson, M. Isabella Poumlrn-Ares, Simone Grethe, David Smith, Limin Zheng, and Tommy Andersson Copyright © 2001 Maria Alvarado-Kristensson et al. All rights reserved. Expression of Heme Oxygenase-1 Mediated by A Protein Transduction Domain Protects Insulin Producing Cells from Cytokine- Induced Cytotoxicity Mon, 01 Jan 1900 00:00:00 +0000 Melina Ribeiro, Dagmar Klein, Antonello Pileggi, R. Damaris Molano, CHristopher Fraker, Camillo Ricordi, and Luca Inverardi Copyright © 2002 Melina Ribeiro et al. All rights reserved. Homogenization of Mammalian Tissues Mon, 01 Jan 1900 00:00:00 +0000 Satisfactory homogenization of a tissue is a necessary prerequisite to any fractionation schedule. A detailed protocol is given for rat liver because of the widespread use of this tissue. Although this technique should be broadly applicable to any soft tissue and to any subsequent fractionation procedure, there are certain tissues and applications that require either minor or extensive modification. Some of these points are addressed in the Notes section. John Graham Copyright © 2002 John Graham. All rights reserved. Purification of Gastric Mucosal ECL Cells from a Crude Elutriation Fraction Mon, 01 Jan 1900 00:00:00 +0000 Acid-secreting parietal cells from the gastric mucosa are widely studied as a model in studies on ion transport and the endocrine/paracrine ECL cells effectively control parietal cell function. Discontinuous gradients of iodixanol for the purification of ECL cells were subsequently simplified to the use of a density barrier. This technique is now commonly used following initial centrifugal elutriation. John Graham Copyright © 2002 John Graham. All rights reserved. Aged yeast mother cells show markers of apoptosis Mon, 01 Jan 1900 00:00:00 +0000 Peter Laun, Alena Pichova, Frank Madeo, Adolf Ellinger, Sepp D. Kohlwein, Kai-Uwe Froumlhlich, Ian Dawes, and Michael Breitenbach Copyright © 2001 Peter Laun et al. All rights reserved. ACTIVATION OF THE JNK/SAPK AND P38 MITOGEN-ACTIVATED PROTEIN KINASE SIGNALING PATHWAYS SENSITIZE TUMOR CELLS TO CISPLATIN-INDUCED APOPTOSIS Mon, 01 Jan 1900 00:00:00 +0000 Abdel Mansouri, Lon D. Ridgway, Qingxiu Zhang, and Francois X. Claret Copyright © 2001 Abdel Mansouri et al. All rights reserved. MICROSCOPIC PHOTOSENSITIZATION WITH CHLOROMETHYL-X-ROSAMINE ON A SUBPOPULATION OF MITOCHONDRIA INDUCES APOPTOSIS IN HUMAN OSTEOSARCOMA CELLS Mon, 01 Jan 1900 00:00:00 +0000 Maggie Lum, Tetsuhiro Minamikawa, and Phillip Nagley Copyright © 2001 Maggie Lum et al. All rights reserved. Pan-Caspase Inhibitor Prevents Anti-CD3 Mediated T-Lymphocyte Proliferation Mon, 01 Jan 1900 00:00:00 +0000 Raja S. Mahidhara, R. Hoffman, R.L. Simmons, and T.R. Billiar Copyright © 2001 Raja S. Mahidhara et al. All rights reserved. Molecular Regulation of Compromised B Lymphopoeisis in Aged Mice Mon, 01 Jan 1900 00:00:00 +0000 Bonnie B. Blomberg, Diep Nguyen, Erin M. Sherwood, Karen Kamm, Marta Perez, and Richard L. Riley Copyright © 2001 Bonnie B. Blomberg et al. All rights reserved. The Multifaceted Role of the Inflammasome in Inflammatory Bowel Diseases Mon, 01 Jan 1900 00:00:00 +0000 Inflammasomes are intracellular multiprotein complexes that coordinate the maturation of interleukin (IL)-1β and IL-18 in response to pathogens and metabolic danger. Both cytokines have been linked to intestinal inflammation. However, recently evolving concepts ascribe a major role to the inflammasome in maintaining intestinal homeostasis. This review recapitulates its position in the development of inflammatory bowel disease, thereby outlining a model in which hypo- as well as hyperfunctionality can lead to an imbalance of the system, depending on the specific cell population affected. In the epithelium, the inflammasome is essential for regulation of permeability and epithelial regeneration through sensing of commensal microbes, while excessive inflammasome activation within the lamina propria contributes to severe intestinal inflammation. Donata Lissner and Britta Siegmund Copyright © 2011 Donata Lissner and Britta Siegmund. All rights reserved. CELLULAR SENESCENCE, AGING AND CANCER Mon, 01 Jan 1900 00:00:00 +0000 Judith Campisi Copyright © 2001 Judith Campisi. All rights reserved. Purification of Parietal and Chief Cells from the Gastric Mucosa Mon, 01 Jan 1900 00:00:00 +0000 Acid-secreting parietal cells from the gastric mucosa are widely studied as a model in studies on ion transport. A discontinuous gradient of iodixanol has been found to be superior to earlier protocols using Nycodenz® and this method, which removes a significant amount of contaminating cells and mucus is a very useful prelude to further purification by elutriation. John Graham Copyright © 2002 John Graham. All rights reserved. Separation of Monocytes from Whole Human Blood Mon, 01 Jan 1900 00:00:00 +0000 Human peripheral blood monocytes are isolated by flotation from whole blood through a single low-density barrier prepared from OptiPrep™ at 4°C. The separation from lymphocytes depends on the more rapid rate of flotation of the monocytes because of their slightly lower density and larger size. The method works optimally only with fresh (within 2 h of drawing) EDTA-anticoagulated blood. Preliminary evidence suggests that this technique may be applicable to blood from rats. John Graham Copyright © 2002 John Graham. All rights reserved. Formation of Neural Precursor Cell Populations by Differentiation of Embryonic Stem Cells In Vitro Mon, 01 Jan 1900 00:00:00 +0000 Recent interest in the generation of neural lineages by differentiation of embryonic stem cells arises from the opportunities represented by a developmentally normal, unlimited source of material that can be manipulated genetically with precision. Several experimental approaches, which differ conceptually, in the route of differentiation and the characteristics of the resulting cell population have been reported. In this review we undertake a comparative analysis of these approaches and their suitability for experimental investigation or implantation. Joy Rathjen and Peter D. Rathjen Copyright © 2002 Joy Rathjen and Peter D. Rathjen. All rights reserved. Microscopic Photosensitization: A New Tool to Investigate the Role of Mitochondria in Cell Death Mon, 01 Jan 1900 00:00:00 +0000 Active involvement of mitochondria in cell death has been well-documented, but local apoptotic signaling between subsets of mitochondria has been poorly explored to date. Using mitochondrially localized CMXRos as a photosensitizer coupled to laser irradiation by confocal laser scanning microscopy, we demonstrate that partial irradiation of about half the mitochondria in human 143B TK– cells induces rapid loss of mitochondrial membrane potential (ΔΨm) in nonirradiated mitochondria. Cells so partially irradiated show apoptotic indications, including mobilization of cytochrome c and binding of annexin V within 2 h following irradiation. The loss of ΔΨm in nonirradiated mitochondria did not occur in cells photoirradiated in the absence of CMXRos. Increasing the proportion of irradiated mitochondria in each cell (up to about 50%) generated a correspondingly greater percentage of cells in which nonirradiated mitochondria lost ΔΨm and which also showed apoptotic indications. Only at the highest level of irradiation (global for all mitochondria in one cell) were signs of necrosis evident (judged by uptake of propidium iodide). Because laser irradiation is specific to the subpopulation of mitochondria targeted, the data imply that a signal emanating from irradiated mitochondria is processed by their nonirradiated counterparts. We conclude that intermitochondrial signaling occurs in the subcellular response to induction of apoptosis. May-Ghee Lum, Tetsuhiro Minamikawa, and Phillip Nagley Copyright © 2002 May-Ghee Lum et al. All rights reserved. MORPHOLOGICAL AND BIOCHEMICAL CHARACTERIZATION OF A CELL DEATH PROCESS INDUCED BY E3, A NEW SYNTHETIC DIARYLSULFONILUREA ANALOGUE Mon, 01 Jan 1900 00:00:00 +0000 M. Alonso, M. Miglaccio, I. Encio, A. Asumendi, V. Martinez-Merino, E. Hilario, and M. Garcia Copyright © 2001 M. Alonso et al. All rights reserved. Electron Tomography: Towards Visualizing Supramolecular Architecture Inside Cells Mon, 01 Jan 1900 00:00:00 +0000 W. Baumeister, O. Medalia, J. Boehm, S. St. Nickell, and S. A. Frangakis Copyright © 2002 W. Baumeister et al. All rights reserved. Holistic Medicine IV: Principles of Existential Holistic Group Therapy and the Holistic Process of Healing in a Group Setting Mon, 01 Jan 1900 00:00:00 +0000 In existential holistic group therapy, the whole person heals in accordance with the holistic process theory and the life mission theory. Existential group psychotherapy addresses the emotional aspect of the human mind related to death, freedom, isolation, and meaninglessness, while existential holistic group therapy addresses the state of the person�s wholeness. This includes the body, the person�s philosophy of life, and often also love, purpose of life, and the spiritual dimension, to the same extent as it addresses the emotional psyche and sexuality, and it is thus much broader than traditional psychotherapy.Where existential psychotherapy is rather depressing concerning the fundamental human condition, existential holistic therapy conceives life to be basically good. The fundamentals in existential holistic therapy are that everybody has the potential for healing themselves to become loving, joyful, sexually attractive, strong, and gifted, which is a message that most patients welcome. While the patient is suffering and fighting to get through life, the most important job for the holistic therapist is to keep a positive perspective of life. In accordance with these fundamentals, many participants in holistic group therapy will have positive emotional experiences, often of an unknown intensity, and these experiences appear to transform their lives within only a few days or weeks of therapy.An important idea of the course is Bohm�s concept of �holo-movement� in the group, resulting from intense coherence between the group members. When the group comes together, the individual will be linked to the totality and the great movement forward towards love, consciousness, and happiness will happen collectively � if it happens at all. This gives the individual the feeling that everything that happens is right, important, and valuable for all the participants at the same time. Native Americans and other premodern people refer to this experience as �the spiritual design�. This design is actually an underlying regulation that appears when people, through their feelings and engagement for each other, tie the group together and engage their complex emotional intelligence. Practically, this means that all participants are sunk in the same information matrix, so that everybody learns from each other. Everything that happens in the perception of each trainee has immediate and developing relevance for him.Spontaneous healing happens far more effectively in a group setting, where all the participants stand together and support each other, than it does in the clinic, where the therapist is alone with the patient. A 5-day course in personal development can be compatible to a half year of holistic individual therapy. Soren Ventegodt, Niels Jorgen Andersen, and Joav Merrick Copyright © 2003 Soren Ventegodt et al. All rights reserved. Isolation of Peripheral Blood Mononuclear Cells from Macaques on a Density Barrier Mon, 01 Jan 1900 00:00:00 +0000 The standard techniques for the isolation of human peripheral blood mononuclear cells (PBMCs) using commercial “lymphocyte isolation media” cannot be satisfactorily extended to experimental animals without manipulating either the density or the osmolality of the medium. PBMCs from Macaques can also be isolated from whole blood by sedimentation on to a density barrier containing approx. 10% iodixanol, polysucrose (Ficoll) with a density of approx 1.074 g/ml. John Graham Copyright © 2002 John Graham. All rights reserved. Extracellular and Intracellular Regulation of Calcium Homeostasis Mon, 01 Jan 1900 00:00:00 +0000 An organism with an internal skeleton must accumulate calcium while maintaining body fluids at a well-regulated, constant calcium concentration. Neither calcium absorption nor excretion plays a significant regulatory role. Instead, isoionic calcium uptake and release by bone surfaces causes plasma calcium to be well regulated. Very rapid shape changes of osteoblasts and osteoclasts, in response to hormonal signals, modulate the available bone surfaces so that plasma calcium can increase when more low-affinity bone calcium binding sites are made available and can decrease when more high-affinity binding sites are exposed. The intracellular free calcium concentration of body cells is also regulated, but because cells are bathed by fluids with vastly higher calcium concentration, their major regulatory mechanism is severe entry restriction. All cells have a calcium-sensing receptor that modulates cell function via its response to extracellular calcium. In duodenal cells, the apical calcium entry structure functions as both transporter and a vitamin D�responsive channel. The channel upregulates calcium entry, with intracellular transport mediated by the mobile, vitamin D–dependent buffer, calbindin D9K, which binds and transports more than 90% of the transcellular calcium flux. Fixed intracellular calcium binding sites can, like the body's skeleton, take up and release calcium that has entered the cell, but the principal regulatory tool of the cell is restricted entry. Felix Bronner Copyright © 2001 Felix Bronner. All rights reserved. Chronic vs. Acute Interactions between Supraoptic Oxytocin Neurons and Astrocytes during Lactation: Role of Glial Fibrillary Acidic Protein Plasticity Mon, 01 Jan 1900 00:00:00 +0000 In this article, we review studies of astrocytic-neuronal interactions and their effects on the activity of oxytocin (OXT) neurons within the magnocellular hypothalamo-neurohypophysial system. Previous work over several decades has shown that withdrawal of astrocyte processes increases OXT neuron excitability in the hypothalamic supraoptic nucleus (SON) during lactation. However, chronically disabling astrocyte withdrawal does not significantly affect the functioning of OXT neurons during suckling. Nevertheless, acute changes in a cytoskeletal element of astrocytes, glial fibrillary acidic protein (GFAP), occur in concert with changes in OXT neuronal activity during suckling. Here, we compare these changes in GFAP and related proteins with chronic changes that persist throughout lactation. During lactation, a decrease in GFAP levels accompanies retraction of astrocyte processes surrounding OXT neurons in the SON, resulting from high extracellular levels of OXT. During the initial stage of suckling, acute increases in OXT levels further strengthen this GFAP reduction and facilitate the retraction of astrocyte processes. This change, in turn, facilitates burst discharges of OXT neurons and leads to a transient increase in excitatory neurochemicals. This transient neurochemical surge acts to reverse GFAP expression and results in postburst inhibition of OXT neurons. The acute changes in astrocyte GFAP levels seen during suckling likely recur periodically, accompanied by rhythmic changes in glutamate metabolism, water transport, gliotransmitter release, and spatial relationships between astrocytes and OXT neurons. In the neurohypophysis, astrocyte retraction and reversal with accompanying GFAP plasticity also likely occur during lactation and suckling, which facilitates OXT release coordinated with its action in the SON. These studies of the dynamic interactions that occur between astrocytes and OXT neurons mediated by GFAP extend our understanding of astrocyte functions within the central nervous system. Yu-Feng Wang and Kathryn A. Hamilton Copyright © 2009 Yu-Feng Wang and Kathryn Hamilton. All rights reserved. A MITOCHONDRIA-INITIATED APOPTOTIC PATHWAY Mon, 01 Jan 1900 00:00:00 +0000 Xiaodong Wang Copyright © 2001 Xiaodong Wang. All rights reserved. Septins: New Microtubule Interacting Partners Mon, 01 Jan 1900 00:00:00 +0000 Originally characterized as regulators of cytokinesis, septins were later implicated in other cellular processes. Recent studies show that septins have a broader role in microtubule-dependent processes, such as karyokinesis, exocytosis, and maintenance of cell shape. Many members of the septin family have been shown to colocalize or interact with the microtubule cytoskeleton, suggesting that these might be general properties of septins. Septins could play an important role in regulating microtubule dynamics by interacting with microtubule-associated proteins (MAPs) that modulate microtubule stability. Being able to associate with both microtubules and actin, septins can play an important role as adaptors between the two cytoskeletons and as regulators of processes in which both actin and microtubules are involved. As septins are associated with various neurodegenerative diseases and cancer, a better understanding of the biology of septins and their interactions with microtubules is important in order to develop possible therapeutic strategies for these diseases. Rosalind Silverman-Gavrila and Lorelei Silverman-Gavrila Copyright © 2008 Rosalind Silverman-Gavrila and Lorelei Silverman-Gavrila. All rights reserved. Evaluating the Role of Wnt Signal Transduction in Promoting the Development of the Heart Mon, 01 Jan 1900 00:00:00 +0000 Wnts are a family of secreted signaling proteins that are encoded by 19 distinct genes in the vertebrate genome. These molecules initiate several signal transduction pathways: the canonical Wnt, Wnt/Ca2+, and Wnt/planar cell polarity pathways. Wnt proteins have major impact on embryonic development, tumor progression, and stem cell differentiation. Wnt signal transduction also influences the formation of the heart, yet many issues concerning the involvement of Wnt regulation in initiating cardiac development remain unresolved. In this review, we will examine the published record to discern (a) what has been shown by experimental studies on the participation of Wnt signaling in cardiogenesis, and (b) what are the important questions that need to be addressed to understand the importance and function of Wnt signal transduction in facilitating the development of the heart. Leonard M. Eisenberg and Carol A. Eisenberg Copyright © 2007 Leonard M. Eisenberg and Carol A. Eisenberg. All rights reserved. THE ROLE OF CGRP-RECEPTOR COMPONENT PROTEIN (RCP) IN CGRP-MEDIATED SIGNAL TRANSDUCTION Mon, 01 Jan 1900 00:00:00 +0000 The calcitonin gene-related peptide (CGRP)-receptor component protein (RCP) is a 17-kDa intracellular peripheral membrane protein required for signal transduction at CGRP receptors. To determine the role of RCP in CGRP-mediated signal transduction, RCP was depleted from NIH3T3 cells using antisense strategy. Loss of RCP protein correlated with loss of cAMP production by CGRP in the antisense cells. In contrast, loss of RCP had no effect on CGRP-mediated binding; therefore RCP is not acting as a chaperone for the CGRP receptor. Instead, RCP is a novel signal transduction molecule that couples the CGRP receptor to the cellular signal transduction machinery. RCP thus represents a prototype for a new class of signal transduction proteins that are required for regulation of G protein-coupled receptors. M.A. Prado, B. Evans-Bain, S.L. Santi, and I.M. Dickerson Copyright © 2001 M.A. Prado et al. All rights reserved. Identification and Characterization of a Minimal Apoptotic Domain from p53 Mon, 01 Jan 1900 00:00:00 +0000 Kevin M. Ryan and Karen H. Vousden Copyright © 2001 Kevin M. Ryan and Karen H. Vousden. All rights reserved. Regulation of T-Cell Apoptosis by Sequences Encoded at the Luca-15 Candidate Tumour Suppressor Locus Mon, 01 Jan 1900 00:00:00 +0000 Mirna Mourtada-Maarabouni, Leslie C. Sutherland, Jeremy P. Clark, Colin S. Cooper, and Gwyn T. Williams Copyright © 2001 Mirna Mourtada-Maarabouni et al. All rights reserved. STRUCTURAL DETERMINANTS IN THE CALCITONIN RECEPTOR-LIKE RECEPTOR (CRLR) IMPORTANT FOR CGRP AND ADRENOMEDULLIN (AM) RECEPTOR FUNCTION OF CRLR/RECEPTOR-ACTIVITY-MODIFYING PROTEIN (RAMP) 1 AND CRLR/RAMP2 HETERODIMERS Mon, 01 Jan 1900 00:00:00 +0000 Cell surface protein cross-linking, coimmmunoprecipitation, and confocal microscopy identified CRLR/RAMP1-, CRLR/RAMP2-, and calcitonin receptor isotype 2 (CTR2)/RAMP1 heterodimers as CGRP-, AM-, and CGRP/amylin receptors, linked to cAMP production. Along these lines, effects of structural alterations in the N-terminal extracellular domain of the human CRLR on cell surface expression as well as the association with RAMP and CGRP or AM have been investigated. W. Born, K. Leuthauser, R. Gujer, R. Muff, and J.A. Fischer Copyright © 2001 W. Born et al. All rights reserved. Human Mesenchymal Stem Cells as Mediators of Breast Carcinoma Tumorigenesis and Progression Mon, 01 Jan 1900 00:00:00 +0000 Lyndsay V. Rhodes and Matthew E. Burow Copyright © 2010 Lyndsay V. Rhodes and Matthew E. Burow. All rights reserved. A Role for the Cytoskeleton in Heart Looping Mon, 01 Jan 1900 00:00:00 +0000 Over the past 10 years, key genes involved in specification of left-right laterality pathways in the embryo have been defined. The read-out for misexpression of laterality genes is usually the direction of heart looping. The question of how dextral looping direction occurred mechanistically and how the heart tube bends remains unknown. It is becoming clear from our experiments and those of others that left-right differences in cell proliferation in the second heart field (anterior heart field) drives the dextral direction. Evidence is accumulating that the cytoskeleton is at the center of laterality, and the bending and rotational forces associated with heart looping. If laterality pathways are modulated upstream, the cytoskeleton, including nonmuscle myosin II (NMHC-II), is altered downstream within the cardiomyocytes, leading to looping abnormalities. The cytoskeleton is associated with important mechanosensing and signaling pathways in cell biology and development. The initiation of blood flow during the looping period and the inherent stresses associated with increasing volumes of blood flowing into the heart may help to potentiate the process. In recent years, the steps involved in this central and complex process of heart development that is the basis of numerous congenital heart defects are being unraveled. Kersti K. Linask and Michael VanAuker Copyright © 2007 Kersti K. Linask and Michael VanAuker. All rights reserved. SNAREs: Could They be the Answer to an Energy Landscape Riddle in Exocytosis? Mon, 01 Jan 1900 00:00:00 +0000 During exocytosis, chemical transmitters stored in secretory vesicles can be released upon fusion of these intracellular organelles to the plasma membrane. In this process, SNARE proteins that form a ternary core complex play a central role. This complex could provide the means for generation/storage of energy necessary for driving the fusion of vesicular and plasma membranes. Recently, the amount of energy for (dis)assembly of the ternary complex has been measured using various experimental approaches, including atomic force microscopy, the surface force apparatus, and isothermal titration calorimetry. The obtained measurements are in good agreement with the calculated energy required for membrane fusion achieved by theoretical modeling approaches. Whether the energy expenditure to form the ternary SNARE complex can be utilized towards membrane fusion and/or docking/tethering of vesicles to the plasma membrane still remains one of the key contemporary issues in biophysics and neuroscience. Wei Liu and Vladimir Parpura Copyright © 2010 Wei Liu and Vladimir Parpura. All rights reserved. Mesenchymal Stem Cells: “Repair Cells” that Serve Wounds and Cancer? Mon, 01 Jan 1900 00:00:00 +0000 Jürgen Dittmer Copyright © 2010 Jürgen Dittmer. All rights reserved. Wound Repair Capability in EDS Fibroblasts can be Retrieved by Exogenous Type V Collagen Mon, 01 Jan 1900 00:00:00 +0000 Simona Viglio, Nicoletta Zoppi, Antonella Sangalli, Angelo Gallanti, Sergio Barlati, Monica Mottes, Marina Colombi, and Maurizia Valli Copyright © 2008 Simona Viglio et al. All rights reserved. Early Contact Stage of Apoptosis: Its Morphological Features and Function Mon, 01 Jan 1900 00:00:00 +0000 Apoptosis has been a biological phenomenon of intense interest for 20 years, but the earlier morphological features of apoptosis have not been determined hitherto. Using the methods of semi- and ultrathin sections, the livers of intact embryos and young rats have been studied under the effect of cycloheximide to determine morphological features of an early stage of apoptosis. It is discovered that both in hepatoblasts and hepatocytes, apoptosis, besides the well-known stages, also includes an early contact stage, distinguishing features of which are agglutination of bound ribosomes (breaking of translation), elimination of the nucleolus, reduction of free polysomes (and in hepatocytes, reduction of cisterns of rough endoplasmic reticulum), formation of cytoplasmic excrescences, and cell shape changes. The early stage of apoptosis is characterized by close contact with neighboring cells. At a certain phase of the contact stage of apoptosis, the nucleolus reappears in the nucleus and the number of free polysomes in the cytoplasm increases, which suggests the renewal of synthesis of new RNA and proteins. Close contact of differentiating and mitotic hepatoblasts with apoptotic cells indicates a certain functional relationship between these cells that is realized not only by micropinocytosis, but through gap junctions as well. We assume that the apoptotic cell, besides proteolytic products, can contain newly synthesized, low-molecular substances, the relocation of which from apoptotic to neighboring cells may contribute to both functional activity and proliferation of adjacent hepatoblasts and, therefore, the function of apoptosis may not be limited only to the elimination of harmful, damaged, and unwanted cells. Etheri Mikadze and Teo Mamatsashvili Copyright © 2006 Etheri Mikadze and Teo Mamatsashvili. All rights reserved. CONVERSION FROM A TYPE I TO A TYPE II DEATH RECEPTOR-MEDIATED PATHWAY THROUGH A PARTIAL INHIBITION OF CASPASE-8 Mon, 01 Jan 1900 00:00:00 +0000 Bryan W. Johnson, Enrique Cepero, and Lawrence H. Boise Copyright © 2001 Bryan W. Johnson et al. All rights reserved. Analysis of Mitochondrial Function in Antigen Specific na ve, Effector and MEmory CD8 + T Cells Mon, 01 Jan 1900 00:00:00 +0000 Jason M. Grayson, J. Gibson Lanier, Laurie E. Harrington, John D. Altman, and Rafi Ahmed Copyright © 2001 Jason M. Grayson et al. All rights reserved. Neural Induction, Neural Fate Stabilization, and Neural Stem Cells Mon, 01 Jan 1900 00:00:00 +0000 The promise of stem cell therapy is expected to greatly benefit the treatment of neurodegenerative diseases. An underlying biological reason for the progressive functional losses associated with these diseases is the extremely low natural rate of self-repair in the nervous system. Although the mature CNS harbors a limited number of self-renewing stem cells, these make a significant contribution to only a few areas of brain. Therefore, it is particularly important to understand how to manipulate embryonic stem cells and adult neural stem cells so their descendants can repopulate and functionally repair damaged brain regions. A large knowledge base has been gathered about the normal processes of neural development. The time has come for this information to be applied to the problems of obtaining sufficient, neurally committed stem cells for clinical use. In this article we review the process of neural induction, by which the embryonic ectodermal cells are directed to form the neural plate, and the process of neural�fate stabilization, by which neural plate cells expand in number and consolidate their neural fate. We will present the current knowledge of the transcription factors and signaling molecules that are known to be involved in these processes. We will discuss how these factors may be relevant to manipulating embryonic stem cells to express a neural fate and to produce large numbers of neurally committed, yet undifferentiated, stem cells for transplantation therapies. Sally A. Moody and Hyun-Soo Je Copyright © 2002 Sally A. Moody and Hyun-Soo Je. All rights reserved. COORDINATED ACTIVATION OF CASPASES AND APOPTOSIS IN ALLOGENEIC ANTIGEN STIMULATED HUMAN T CELLS Mon, 01 Jan 1900 00:00:00 +0000 Sundararajan Jayaraman and John N. Barrett Copyright © 2001 Sundararajan Jayaraman and John N. Barrett. All rights reserved. Isolation of a Mouse Motoneuron-Enriched Fraction from Mouse Spinal Cord on a Density Barrier Mon, 01 Jan 1900 00:00:00 +0000 After a combined enzymic and mechanical disruption of the spinal cord tissue, the low-density motoneurons band at the interface of a 1.06-g/ml barrier through which other contaminating cells sediment. John Graham Copyright © 2002 John Graham. All rights reserved. Stem Cell Quandary Quashes Research, Smithsonian Secretary Backs Down from CRC Closure - and Welcome Back to Star Wars Mon, 01 Jan 1900 00:00:00 +0000 Nature leads this week with a story about how questions surrounding stem cell research have thrown a funding agency and the German government into the boxing ring. The contested overhaul of the U.S. Smithsonian Institution tops the news this week in Science. Shauna M. Haley Copyright © 2001 Shauna M. Haley. All rights reserved. Dynamic Analysis of DNA Damage by Flow Cytometry and FISH Mon, 01 Jan 1900 00:00:00 +0000 The micronucleus assay, developed to assess DNA damage induced by noxious agents, supplies information on whether the damage is due to clastogenic or aneugenic action. Although it is the test that can be used to assess agents' toxicity, it cannot provide information on the molecular events that result in the induction of micronuclei. To study the molecular events, the combination of both microscopic and analytical techniques is required. Flow-sorting induced micronuclei, based on their DNA content, in combination with chromosomal FISH and other molecular techniques, may provide information on these events. Demetrios P. Matthopoulos Copyright © 2006 Demetrios P. Matthopoulos. All rights reserved. AGING ENHANCES THE ACTIVATION OF THE PERMEABILITY TRANSITION PORE IN MITOCHONDRIA Mon, 01 Jan 1900 00:00:00 +0000 Hagai Rottenberg and Michael Mather Copyright © 2001 Hagai Rottenberg and Michael Mather. All rights reserved. REGULATION OF cIAP1 AND XIAP EXPRESSION BY NITRIC OXIDE, CYTOKINES, AND ITS RELATION TO APOTOSIS INDUCTION IN RAT MESANGIAL CELLS AND RAW 264.7 MACROPHAGES Mon, 01 Jan 1900 00:00:00 +0000 Markus Manderscheid, Udo K. Meszligmer, and Josef Pfeilschifter Copyright © 2001 Markus Manderscheid et al. All rights reserved. Trastuzumab Resistance: Role for Notch Signaling Mon, 01 Jan 1900 00:00:00 +0000 Epidermal growth factor receptor-2 (ErbB-2/HER2) is a potent breast oncogene that has been shown to be amplified in 20% of breast cancers. Overexpression of ErbB-2 predicts for aggressive tumor behavior, resistance to some cytotoxic and antihormonal therapies, and poor overall survival. Trastuzumab, the humanized, monoclonal antibody directed against ErbB-2 has shown tremendous efficacy and improved overall survival for women when combined with a taxane-based chemotherapy. However, resistance to trastuzumab remains a major concern, most notably in women with metastatic breast cancer. Numerous mechanisms that include overexpression of alternate receptor tyrosine kinases and/or loss of critical tumor suppressors have been proposed in the last several years to elucidate trastuzumab resistance. Here we review the many possible mechanisms of action that could contribute to resistance, and novel therapies to prevent or reverse the resistant phenotype. Moreover, we provide a critical role for Notch signaling cross-talk with overlapping or new signaling networks in trastuzumab-resistant breast. Kinnari Mehta and Clodia Osipo Copyright © 2009 Kinnari Mehta and Clodia Osipo. All rights reserved. Purification of Islets of Langerhans from Porcine Pancreas Mon, 01 Jan 1900 00:00:00 +0000 Flotation through a slightly hyperosmotic discontinuous gradient iodixanol achieves a much higher recovery of islets of an improved viability than the customary method using sedimentation on to a diatrizoate/polysaccharide barrier. Flotation techniques achieve an enhanced separation of the islets from any residual digestive enzymes and from acinar cells. The method has been extended to human pancreas. John Graham Copyright © 2002 John Graham. All rights reserved. Ca2+ Signaling in B Cells Mon, 01 Jan 1900 00:00:00 +0000 An increase in intracellular Ca2+ concentration is one of the major initial steps in B-cell activation that occurs within minutes after antigen receptor (BCR) engagement. In recent years, significant advances have been made in characterizing molecular mechanisms of Ca2+ signaling in lymphocytes, although the majority of work was done on T cells. This mini-review discusses several underexplored areas of Ca2+ signaling in B cells: (1) Ca2+ signaling in immune synapse and multifaceted Ca2+ responses within a single cell, (2) source of Ca2+ involved in Ca2+-dependent protein phosphorylation events and the role of store-operated influx, (3) role of BCR coreceptors in Ca2+ signaling, and (4) Ca2+ signaling and maintenance of B-cell tolerance and clinical significance of Ca2+ signaling alterations. Taras Lyubchenko Copyright © 2010 Taras Lyubchenko. All rights reserved. SEX HORMONES, APOPTOSIS AND THE FAS/FAS LIGAND SYSTEM IN NORMAL ENDOMETRIAL TISSUE REMODELING Mon, 01 Jan 1900 00:00:00 +0000 Gil Mor, Sarit Aschkenazi, and Joon Song Copyright © 2001 Gil Mor et al. All rights reserved. Purification of Intact Plant Protoplasts by Flotation at 1g Mon, 01 Jan 1900 00:00:00 +0000 From a standard plant tissue digest adjusted to a density of 1.07 g/ml, protoplasts can be harvested by flotation through a low density barrier (1.03 g/ml). The delicate nature of these bodies is suited to this flotation strategy which can be carried out at 1g. John Graham Copyright © 2002 John Graham. All rights reserved. SEQUENTIAL EVENTS DURING NUCLEAR APOPTOSIS Mon, 01 Jan 1900 00:00:00 +0000 Madeleine Kihlmark, Gabriela Imreh, and Einar Hallberg Copyright © 2001 Madeleine Kihlmark et al. All rights reserved. CONTROL OF CYTOTOXIC LYMPHOCYTE SURVIVAL IN VIVO BY MEMBERS OF THE TNF-R FAMILY Mon, 01 Jan 1900 00:00:00 +0000 Eckhard R. Podack Copyright © 2001 Eckhard R. Podack. All rights reserved. ANTICANCER DRUG-INDUCED APOPTOSIS AND CYTOTOXICITY IN PROSTATE CANCER CELLS ARE MODULATED BY ORGAN-SPECIFIC STROMAL CELL FACTORS Mon, 01 Jan 1900 00:00:00 +0000 Bal L. Lokeshwar, Kamalaveni R. Prabhakar, Tie Yan Shang, Zafiria Mourelatos, and De-Quan Li Copyright © 2001 Bal L. Lokeshwar et al. All rights reserved. CHANGES IN INTRACELLULAR ADP:ATP RATIOS AS A MARKER OF APOPTOSIS Mon, 01 Jan 1900 00:00:00 +0000 Tracy D. Simmons, Kevin J. Slater, and Sharon P.M. Crouch Copyright © 2001 Tracy D. Simmons et al. All rights reserved. THE INSULIN SIGNALING PATHWAY AND AGING IN DROSOPHILA Mon, 01 Jan 1900 00:00:00 +0000 Linda Partridge Copyright © 2001 Linda Partridge. All rights reserved. Cell Death and Ageing – A Question of Cell Type Mon, 01 Jan 1900 00:00:00 +0000 Replicative senescence of human cells in primary culture is a widely accepted model for studying the molecular mechanisms of human ageing. The standard model used for studying human ageing consists of fibroblasts explanted from the skin and grown into in vitro senescence. From this model, we have learned much about molecular mechanisms underlying the human ageing process; however, the model presents clear limitations. In particular, a long-standing dogma holds that replicative senescence involves resistance to apoptosis, a belief that has led to considerable confusion concerning the role of apoptosis during human ageing. While there are data suggesting that apoptotic cell death plays a key role for ageing in vitro and in the pathogenesis of various age-associated diseases, this is not reflected in the current literature on in vitro senescence. In this article, I summarize key findings concerning the relationship between apoptosis and ageing in vivo and also review the literature concerning the role of apoptosis during in vitro senescence. Recent experimental findings, summarized in this article, suggest that apoptotic cell death (and probably other forms of cell death) are important features of the ageing process that can also be recapitulated in tissue culture systems to some extent. Another important lesson to learn from these studies is that mechanisms of in vivo senescence differ considerably between various histotypes. Pidder Jansen-Dürr Copyright © 2002 Pidder Jansen-Durr. All rights reserved. Current Status of Human Adipose–Derived Stem Cells: Differentiation into Hepatocyte-Like Cells Mon, 01 Jan 1900 00:00:00 +0000 The shortage of human organ donors and the low cell quality of available liver tissues represent major obstacles for the clinical application of orthotropic liver transplantation and hepatocyte transplantation, respectively. Therefore, worldwide research groups are investigating alternative extrahepatic cell sources. Recent in vitro studies have demonstrated that mesenchymal stem cells (MSCs) from various sources, including human bone marrow, adipose tissue, and umbilical cord, can be differentiated into hepatocyte-like cells when appropriate conditions are used. In particular, interest exists for human adipose–derived stems cells (hASCs) as an attractive cell source for generating hepatocyte-like cells. The hASCs are multipotent MSCs that reside in adipose tissue, with the ability to self-renew and differentiate into multiple cell lineages. Moreover, these cells can secrete multiple growth factors and cytokines that exert beneficial effects on organ or tissue injury. In this review, we will not only present recent data regarding hASC biology, their isolation, and differentiation capability towards hepatocytes, but also the potential application of hASC-derived hepatocytes to study drug toxicity. Additionally, this review will discuss the therapeutic potential of hASCs as undifferentiated cells in liver regeneration. Feras Al Battah, Joery De Kock, Tamara Vanhaecke, and Vera Rogiers Copyright © 2011 Feras Al Battah et al. All rights reserved. New Perspectives in Chondrogenic Differentiation of Stem Cells for Cartilage Repair Mon, 01 Jan 1900 00:00:00 +0000 Wei Seong Toh, Zheng Yang, Boon Chin Heng, and Tong Cao Copyright © 2006 Wei Seong Toh et al. All rights reserved. Calpain is a Major Regulator of Neutrophils Apoptosis Mon, 01 Jan 1900 00:00:00 +0000 Frank Altznauer and Hans-Uwe Simon Copyright © 2001 Frank Altznauer and Hans-Uwe Simon. All rights reserved. Separation of Human Monocytes from a Leukocyte-Rich Plasma Mon, 01 Jan 1900 00:00:00 +0000 Human peripheral blood monocytes are isolated by flotation from a dense leukocyte-rich plasma (LRP) through two lower-density barriers prepared from OptiPrep™. The separation from lymphocytes depends on the more rapid rate of flotation of the monocytes because of their slightly lower density and larger size. The method works optimally only with fresh (within 2 h of drawing) EDTA-anticoagulated blood. John Graham Copyright © 2002 John Graham. All rights reserved. Cannabinoids on the Brain Mon, 01 Jan 1900 00:00:00 +0000 Cannabis has a long history of consumption both for recreational and medicinal uses. Recently there have been significant advances in our understanding of how cannabis and related compounds (cannabinoids) affect the brain and this review addresses the current state of knowledge of these effects. Cannabinoids act primarily via two types of receptor, CB1 and CB2, with CB1 receptors mediating most of the central actions of cannabinoids. The presence of a new type of brain cannabinoid receptor is also indicated. Important advances have been made in our understanding of cannabinoid receptor signaling pathways, their modulation of synaptic transmission and plasticity, the cellular targets of cannabinoids in different central nervous system (CNS) regions and, in particular, the role of the endogenous brain cannabinoid (endocannabinoid) system. Cannabinoids have widespread actions in the brain: in the hippocampus they influence learning and memory; in the basal ganglia they modulate locomotor activity and reward pathways; in the hypothalamus they have a role in the control of appetite. Cannabinoids may also be protective against neurodegeneration and brain damage and exhibit anticonvulsant activity. Some of the analgesic effects of cannabinoids also appear to involve sites within the brain. These advances in our understanding of the actions of cannabinoids and the brain endocannabinoid system have led to important new insights into neuronal function which are likely to result in the development of new therapeutic strategies for the treatment of a number of key CNS disorders. Andrew J. Irving, Mark G. Rae, and Angela A. Coutts Copyright © 2002 Andrew J. Irving et al. All rights reserved. The Two Thrombospondin Type I Repeat Domains of HB-GAM Display a Cooperative Function in N-syndecan Binding and Regulation of Synaptic Plasticity Mon, 01 Jan 1900 00:00:00 +0000 Erkki Raulo, Sarka Tumova, Ivan Pavlov, Anni Hienola, Sari Lauri, Tomi Taira, and Heikki Rauvala Copyright © 2006 Erkki Raulo et al. All rights reserved. Mitochondrial Depolarization During TNF-induced Apoptosis is Caspase-dependent and Independent of Bcl-x L and the Permeability Transition Pore Mon, 01 Jan 1900 00:00:00 +0000 E. Cepero, B.W. Johnson, and L.H. Boise Copyright © 2001 E. Cepero et al. All rights reserved. Purification of Peroxisomes in a Self-Generated Gradient Mon, 01 Jan 1900 00:00:00 +0000 In iodixanol, peroxisomes are the densest organelle in the light mitochondrial fraction and are therefore easily separated from the other components (lysosomes, mitochondria, etc.) in a self-generated gradient. Self-generated gradients make sample handling very easy and are highly reproducible but need to be formed in either a vertical, near-vertical, or small volume high-performance fixed-angle rotor. The resolution of the peroxisomes is far superior than that in sucrose and, unlike in PercollⓇ there is no contamination from endoplasmic reticulum. John Graham Copyright © 2002 John Graham. All rights reserved. Newt Opportunities for Understanding the Dedifferentiation Process Mon, 01 Jan 1900 00:00:00 +0000 Urodele amphibians, such as the newt Notophthalmus viridescens, have the unique ability to regenerate limbs, spinal cord, eye structures, and many vital organs through a process called epimorphic regeneration. Although the cellular basis of regeneration has been studied in detail, we know relatively little about the molecular controls of the process. This review provides an overview of forelimb regeneration in the newt, addressing what we know about cellular and molecular aspects. Particular focus is placed on the dedifferentiation process, which yields a population of embryonic-like pluripotent cells that will eventually reform the lost structure. This cellular plasticity seems to be the key to regenerative ability. We discuss the dedifferentiation process in newt forelimb regeneration and outline the various studies that have revealed that mammalian cells also have the ability to dedifferentiate if given the appropriate triggers. Ziad Y. Chaar and Catherine Tsilfidis Copyright © 2006 Ziad Y. Chaar and Catherine Tsilfidis. All rights reserved. Transduction of TAT/PTD Antiapoptotic Fusion Proteins in Pancreatic Islets Mon, 01 Jan 1900 00:00:00 +0000 Jennifer Embury, Melina Ribeiro, Dagmar Klein, Antonello Pileggi, R. Damaris Molano, CHristopher Fraker, Norma Kenyon, Camillo Ricordi, Luca Inverardi, and Ricardo L. Pastori Copyright © 2001 Jennifer Embury et al. All rights reserved.