Journal of Biophysics

Analysis of the REJ Module of Polycystin-1 Using Molecular Modeling and Force-Spectroscopy Techniques

Sun, 26 May 2013 16:18:25 +0000

Polycystin-1 is a large transmembrane protein, which, when mutated, causes autosomal dominant polycystic kidney disease, one of the most common life-threatening genetic diseases that is a leading cause of kidney failure. The REJ (receptor for egg lelly) module is a major component of PC1 ectodomain that extends to about 1000 amino acids. Many missense disease-causing mutations map to this module; however, very little is known about the structure or function of this region. We used a combination of homology molecular modeling, protein engineering, steered molecular dynamics (SMD) simulations, and single-molecule force spectroscopy (SMFS) to analyze the conformation and mechanical stability of the first ~420 amino acids of REJ. Homology molecular modeling analysis revealed that this region may contain structural elements that have an FNIII-like structure, which we named REJd1, REJd2, REJd3, and REJd4. We found that REJd1 has a higher mechanical stability than REJd2 (~190 pN and 60 pN, resp.). Our data suggest that the putative domains REJd3 and REJd4 likely do not form mechanically stable folds. Our experimental approach opens a new way to systematically study the effects of disease-causing mutations on the structure and mechanical properties of the REJ module of PC1.

Comparative Trace Elemental Analysis in Cancerous and Noncancerous Human Tissues Using PIXE

Stephen Juma Mulware — Thu, 16 May 2013 15:37:24 +0000

The effect of high or low levels of trace metals in human tissues has been studied widely. There have been detectable significant variations in the concentrations of trace metals in normal and cancerous tissues suggesting that these variations could be a causative factor to various cancers. Even though essential trace metals play an important role such as stabilizers, enzyme cofactors, elements of structure, and essential elements for normal hormonal functions, their imbalanced toxic effects contribute to the rate of the reactive oxygen species (ROS) and formation of complexities in the body cells which may lead to DNA damage. The induction of oxidative-induced DNA damage by ROS may lead to isolated base lesions or single-strand breaks, complex lesions like double-strand breaks, and some oxidative generated clustered DNA lesions (OCDLs) which are linked to cell apoptosis and mutagenesis. The difference in published works on the level of variations of trace metals in different cancer tissues can be attributed to the accuracy of the analytical techniques, sample preparation methods, and inability of taking uniform samples from the affected tissues. This paper reviews comparative trace elemental concentrations of cancerous and noncancerous tissues using PIXE that has been reported in the published literature.

Reduced Dynamic Models in Epithelial Transport

Julio A. Hernández — Thu, 28 Feb 2013 09:18:59 +0000

Most models developed to represent transport across epithelia assume that the cell interior constitutes a homogeneous compartment, characterized by a single concentration value of the transported species. This conception differs significantly from the current view, in which the cellular compartment is regarded as a highly crowded media of marked structural heterogeneity. Can the finding of relatively simple dynamic properties of transport processes in epithelia be compatible with this complex structural conception of the cell interior? The purpose of this work is to contribute with one simple theoretical approach to answer this question. For this, the techniques of model reduction are utilized to obtain a two-state reduced model from more complex linear models of transcellular transport with a larger number of intermediate states. In these complex models, each state corresponds to the solute concentration in an intermediate intracellular compartment. In addition, the numerical studies reveal that it is possible to approximate a general two-state model under conditions where strict reduction of the complex models cannot be performed. These results contribute with arguments to reconcile the current conception of the cell interior as a highly complex medium with the finding of relatively simple dynamic properties of transport across epithelial cells.

Potassium Current Is Not Affected by Long-Term Exposure to Ghrelin or GHRP-6 in Somatotropes GC Cells

Sun, 24 Feb 2013 15:30:54 +0000

Ghrelin is a growth hormone (GH) secretagogue (GHS) and GHRP-6 is a synthetic peptide analogue; both act through the GHS receptor. GH secretion depends directly on the intracellular concentration of Ca2+; this is determined from the intracellular reserves and by the entrance of Ca2+ through the voltage-dependent calcium channels, which are activated by the membrane depolarization. Membrane potential is mainly determined by K+ channels. In the present work, we investigated the effect of ghrelin (10 nM) or GHRP-6 (100 nM) for 96 h on functional expression of voltage-dependent K+ channels in rat somatotropes: GC cell line. Physiological patch-clamp whole-cell recording was used to register the K+ currents. With Cd2+ (1 mM) and tetrodotoxin (1 μm) in the bath solution recording, three types of currents were characterized on the basis of their biophysical and pharmacological properties. GC cells showed a K+ current with a transitory component sensitive to 4-aminopyridine, which represents ~40% of the total outgoing current; a sustained component named delayed rectifier , sensitive to tetraethylammonium; and a third type of K+ current was recorded at potentials more negative than −80 mV, permitting the entrance of K+ named inward rectifier (KIR). Chronic treatment with ghrelin or GHRP-6 did not modify the functional expression of K+ channels, without significant changes () in the amplitudes of the three currents observed; in addition, there were no modifications in their biophysical properties and kinetic activation or inactivation.

Thermal Aggregation of Recombinant Protective Antigen: Aggregate Morphology and Growth Rate

Daniel J. Belton and Aline F. Miller — Wed, 13 Feb 2013 15:49:35 +0000

The thermal aggregation of the biopharmaceutical protein recombinant protective antigen (rPA) has been explored, and the associated kinetics and thermodynamic parameters have been extracted using optical and environmental scanning electron microscopies (ESEMs) and ultraviolet light scattering spectroscopy (UV-LSS). Visual observations and turbidity measurements provided an overall picture of the aggregation process, suggesting a two-step mechanism. Microscopy was used to examine the structure of aggregates, revealing an open morphology formed by the clustering of the microscopic aggregate particles. UV-LSS was used and developed to elucidate the growth rate of these particles, which formed in the first stage of the aggregation process. Their growth rate is observed to be high initially, before falling to converge on a final size that correlates with the ESEM data. The results suggest that the particle growth rate is limited by rPA monomer concentration, and by obtaining data over a range of incubation temperatures, an approach was developed to model the aggregation kinetics and extract the rate constants and the temperature dependence of aggregation. In doing so, we quantified the susceptibility of rPA aggregation under different temperature and environmental conditions and moreover demonstrated a novel use of UV spectrometry to monitor the particle aggregation quantitatively, in situ, in a nondestructive and time-resolved manner.

Cytoskeletal Strains in Modeled Optohydrodynamically Stressed Healthy and Diseased Biological Cells

Sean S. Kohles, Yu Liang, and Asit K. Saha — Wed, 05 Dec 2012 08:58:57 +0000

Controlled external chemomechanical stimuli have been shown to influence cellular and tissue regeneration/degeneration, especially with regards to distinct disease sequelae or health maintenance. Recently, a unique three-dimensional stress state was mathematically derived to describe the experimental stresses applied to isolated living cells suspended in an optohydrodynamic trap (optical tweezers combined with microfluidics). These formulae were previously developed in two and three dimensions from the fundamental equations describing creeping flows past a suspended sphere. The objective of the current study is to determine the full-field cellular strain response due to the applied three-dimensional stress environment through a multiphysics computational simulation. In this investigation, the multiscale cytoskeletal structures are modeled as homogeneous, isotropic, and linearly elastic. The resulting computational biophysics can be directly compared with experimental strain measurements, other modeling interpretations of cellular mechanics including the liquid drop theory, and biokinetic models of biomolecule dynamics. The described multiphysics computational framework will facilitate more realistic cytoskeletal model interpretations, whose intracellular structures can be distinctly defined, including the cellular membrane substructures, nucleus, and organelles.

Redox Regulation of Calcium Signaling in Cancer Cells by Ascorbic Acid Involving the Mitochondrial Electron Transport Chain

Sun, 25 Nov 2012 09:15:08 +0000

Previously, we have reported that ascorbic acid regulates calcium signaling in human larynx carcinoma HEp-2 cells. To evaluate the precise mechanism of Ca2+ release by ascorbic acid, the effects of specific inhibitors of the electron transport chain components on mitochondrial reactive oxygen species (ROS) production and Ca2+ mobilization in HEp-2 cells were investigated. It was revealed that the mitochondrial complex III inhibitor (antimycin A) amplifies ascorbate-induced Ca2+ release from intracellular stores. The mitochondrial complex I inhibitor (rotenone) decreases Ca2+ release from intracellular stores in HEp-2 cells caused by ascorbic acid and antimycin A. In the presence of rotenone, antimycin A stimulates ROS production by mitochondria. Ascorbate-induced Ca2+ release in HEp-2 cells is shown to be unaffected by catalase. The results obtained suggest that Ca2+ release in HEp-2 cells caused by ascorbic acid is associated with induced mitochondrial ROS production. The data obtained are in line with the concept of redox signaling that explains oxidant action by compartmentalization of ROS production and oxidant targets.

A Molecular Dynamics Approach to Ligand-Receptor Interaction in the Aspirin-Human Serum Albumin Complex

H. Ariel Alvarez, Andrés N. McCarthy, and J. Raúl Grigera — Wed, 21 Nov 2012 11:23:00 +0000

In this work, we present a study of the interaction between human serum albumin (HSA) and acetylsalicylic acid (ASA, C9H8O4) by molecular dynamics simulations (MD). Starting from an experimentally resolved structure of the complex, we performed the extraction of the ligand by means of the application of an external force. After stabilization of the system, we quantified the force used to remove the ASA from its specific site of binding to HSA and calculated the mechanical nonequilibrium external work done during this process. We obtain a reasonable value for the upper boundary of the Gibbs free energy difference (an equilibrium thermodynamic potential) between the complexed and noncomplexed states. To achieve this goal, we used the finite sampling estimator of the average work, calculated from the Jarzynski Equality. To evaluate the effect of the solvent, we calculated the so-called “viscous work,” that is, the work done to move the aspirin in the same trajectory through the solvent in absence of the protein, so as to assess the relevance of its contribution to the total work. The results are in good agreement with the available experimental data for the albumin affinity constant for aspirin, obtained through quenching fluorescence methods.

The Aggregation of Huntingtin and α-Synuclein

María Elena Chánez-Cárdenas and Edgar Vázquez-Contreras — Thu, 26 Jul 2012 13:01:58 +0000

Huntington’s and Parkinson’s diseases are neurodegenerative disorders associated with unusual protein interactions. Although the origin and evolution of these diseases are completely different, characteristic deposits of protein aggregates (huntingtin and 𝛼-synuclein resp.), are a common feature in both diseases. After these observations, many studies are performed with both proteins. Some of them try to understand the nature and driving forces of the aggregation process; others try to find a correlation between the genetic and failure in protein function. Finally with the combination of both approaches, it was proposed that possible strategies deal with pathologic aggregation. Unfortunately, if protein aggregation is a cause or a consequence of the neurodegeneration observed in these pathologies, it is still debatable. This paper describes the process of aggregation of two proteins: huntingtin and α synuclein. The characteristics of the aggregation reaction of these proteins have been followed with novel methods both in vivo and in vitro; these studies include both the combination with other proteins and the presence of various chemical compounds. The ultimate goal of this study was to summarize recent findings on protein aggregation and its possible role as a therapeutic target in neurodegenerative diseases and their role in biomaterial science.

pH-Dependent Interaction between C-Peptide and Phospholipid Bicelles

Sofia Unnerståle and Lena Mäler — Mon, 16 Jul 2012 15:00:32 +0000

C-peptide is the connecting peptide between the A and B chains of insulin in proinsulin. In this paper, we investigate the interaction between C-peptide and phospholipid bicelles, by circular dichroism and nuclear magnetic resonance spectroscopy, and in particular the pH dependence of this interaction. The results demonstrate that C-peptide is largely unstructured independent of pH, but that a weak structural induction towards a short stretch of β-sheet is induced at low pH, corresponding to the isoelectric point of the peptide. Furthermore, it is demonstrated that C-peptide associates with neutral phospholipid bicelles as well as acidic phospholipid bicelles at this low pH. C-peptide does not undergo a large structural rearrangement as a consequence of lipid interaction, which indicates that the folding and binding are uncoupled. In vivo, local variations in environment, including pH, may cause C-peptide to associate with lipids, which may affect the aggregation state of the peptide.

Inhibitory Effects of Arginine on the Aggregation of Bovine Insulin

Michael M. Varughese and Jay Newman — Mon, 09 Jul 2012 11:35:02 +0000

Static and dynamic light scattering were used to investigate the effects of L-arginine, commonly used to inhibit protein aggregation, on the initial aggregation kinetics of solutions of bovine insulin in 20% acetic acid and 0.1 M NaCl as a model system for amyloidosis. Measurements were made as a function of insulin concentration (0.5–2.0 mM), quench temperature (60–85°C), and arginine concentration (10–500 mM). Aggregation kinetics under all conditions had a lag phase, whose duration decreased with increasing temperature and with increasing insulin concentration but which increased by up to a factor of 8 with increasing added arginine. Further, the initial growth rate after the lag phase also slowed by up to a factor of about 20 in the presence of increasing concentrations of arginine. From the temperature dependence of the lag phase duration, we find that the nucleation activation energy doubles from 17±5 to 36±3 kcal/mol in the presence of 500 mM arginine.

Electronic and Spatial Structures of Water-Soluble Dinitrosyl Iron Complexes with Thiol-Containing Ligands Underlying Their Ability to Act as Nitric Oxide and Nitrosonium Ion Donors

Anatoly F. Vanin and Dosymzhan Sh. Burbaev — Tue, 14 Feb 2012 12:14:42 +0000

The ability of mononuclear dinitrosyl iron commplexes (M-DNICs) with thiolate ligands to act as NO donors and to trigger S-nitrosation of thiols can be explain only in the paradigm of the model of the [Fe+(NO+)2] core ({Fe(NO)2}7 according to the Enemark-Feltham classification). Similarly, the {(RS−)2Fe+(NO+)2}+ structure describing the distribution of unpaired electron density in M-DNIC corresponds to the low-spin (𝑆=1/2) state with a d7 electron configuration of the iron atom and predominant localization of the unpaired electron on MO(dz2) and the square planar structure of M-DNIC. On the other side, the formation of molecular orbitals of M-DNIC including orbitals of the iron atom, thiolate and nitrosyl ligands results in a transfer of electron density from sulfur atoms to the iron atom and nitrosyl ligands. Under these conditions, the positive charge on the nitrosyl ligands diminishes appreciably, the interaction of the ligands with hydroxyl ions or with thiols slows down and the hydrolysis of nitrosyl ligands and the S-nitrosating effect of the latter are not manifested. Most probably, the S-nitrosating effect of nitrosyl ligands is a result of weak binding of thiolate ligands to the iron atom under conditions favoring destabilization of M-DNIC.

Quantitative Reappraisal of the Helmholtz-Guyton Resonance Theory of Frequency Tuning in the Cochlea

Charles F. Babbs — Wed, 19 Oct 2011 11:34:54 +0000

To explore the fundamental biomechanics of sound frequency transduction in the cochlea, a two-dimensional analytical model of the basilar membrane was constructed from first principles. Quantitative analysis showed that axial forces along the membrane are negligible, condensing the problem to a set of ordered one-dimensional models in the radial dimension, for which all parameters can be specified from experimental data. Solutions of the radial models for asymmetrical boundary conditions produce realistic deformation patterns. The resulting second-order differential equations, based on the original concepts of Helmholtz and Guyton, and including viscoelastic restoring forces, predict a frequency map and amplitudes of deflections that are consistent with classical observations. They also predict the effects of an observation hole drilled in the surrounding bone, the effects of curvature of the cochlear spiral, as well as apparent traveling waves under a variety of experimental conditions. A quantitative rendition of the classical Helmholtz-Guyton model captures the essence of cochlear mechanics and unifies the competing resonance and traveling wave theories.

CsmA Protein is Associated with BChl a in the Baseplate Subantenna of Chlorosomes of the Photosynthetic Green Filamentous Bacterium Oscillochloris trichoides belonging to the Family Oscillochloridaceae

Thu, 15 Sep 2011 10:21:01 +0000

The baseplate subantenna in chlorosomes of green anoxygenic photosynthetic bacteria, belonging to the families Chloroflexaceae and Chlorobiaceae, is known to represent a complex of bacteriochlorophyll (BChl) a with the ~6 kDa CsmA proteins. Earlier, we showed the existence of a similar BChl a subantenna in chlorosomes of the photosynthetic green bacterium Oscillochloris trichoides, member of Oscillochloridaceae, the third family of green photosynthetic bacteria. However, this BChl a subantenna was not visually identified in absorption spectra of isolated Osc. trichoides chlorosomes in contrast to those of Chloroflexaceae and Chlorobiaceae. In this work, using room and low-temperature absorbance and fluorescence spectroscopy and sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of alkaline-treated and untreated chlorosomes of Osc. trichoides, we showed that the baseplate BChl a subantenna does exist in Oscillochloridaceae chlorosomes as a complex of BChl a with the 5.7 kDa CsmA protein. The present results support the idea that the baseplate subantenna, representing a complex of BChl a with a ~6 kDa CsmA protein, is a universal interface between the BChl c subantenna of chlorosomes and the nearest light-harvesting BChl a subantenna in all three known families of green anoxygenic photosynthetic bacteria.

F-Ratio Test and Hypothesis Weighting: A Methodology to Optimize Feature Vector Size

R. M. Dünki and M. Dressel — Wed, 17 Aug 2011 09:56:38 +0000

Reducing a feature vector to an optimized dimensionality is a common problem in biomedical signal analysis. This analysis retrieves the characteristics of the time series and its associated measures with an adequate methodology followed by an appropriate statistical assessment of these measures (e.g., spectral power or fractal dimension). As a step towards such a statistical assessment, we present a data resampling approach. The techniques allow estimating 𝜎2(𝐹), that is, the variance of an F-value from variance analysis. Three test statistics are derived from the so-called F-ratio 𝜎2(𝐹)/𝐹2. A Bayesian formalism assigns weights to hypotheses and their corresponding measures considered (hypothesis weighting). This leads to complete, partial, or noninclusion of these measures into an optimized feature vector. We thus distinguished the EEG of healthy probands from the EEG of patients diagnosed as schizophrenic. A reliable discriminance performance of 81% based on Taken's χ, 𝛼-, and 𝛿-power was found.

Mechanisms of Cellular Uptake of Cell-Penetrating Peptides

Thu, 07 Apr 2011 15:41:37 +0000

Recently, much attention has been given to the problem of drug delivery through the cell-membrane in order to treat and manage several diseases. The discovery of cell penetrating peptides (CPPs) represents a major breakthrough for the transport of large-cargo molecules that may be useful in clinical applications. CPPs are rich in basic amino acids such as arginine and lysine and are able to translocate over membranes and gain access to the cell interior. They can deliver large-cargo molecules, such as oligonucleotides, into cells. Endocytosis and direct penetration have been suggested as the two major uptake mechanisms, a subject still under debate. Unresolved questions include the detailed molecular uptake mechanism(s), reasons for cell toxicity, and the delivery efficiency of CPPs for different cargoes. Here, we give a review focused on uptake mechanisms used by CPPs for membrane translocation and certain experimental factors that affect the mechanism(s).

Examinations of tRNA Range of Motion Using Simulations of Cryo-EM Microscopy and X-Ray Data

Thomas R. Caulfield, Batsal Devkota, and Geoffrey C. Rollins — Mon, 28 Mar 2011 11:37:27 +0000

We examined tRNA flexibility using a combination of steered and unbiased molecular dynamics simulations. Using Maxwell's demon algorithm, molecular dynamics was used to steer X-ray structure data toward that from an alternative state obtained from cryogenic-electron microscopy density maps. Thus, we were able to fit X-ray structures of tRNA onto cryogenic-electron microscopy density maps for hybrid states of tRNA. Additionally, we employed both Maxwell's demon molecular dynamics simulations and unbiased simulation methods to identify possible ribosome-tRNA contact areas where the ribosome may discriminate tRNAs during translation. Herein, we collected >500 ns of simulation data to assess the global range of motion for tRNAs. Biased simulations can be used to steer between known conformational stop points, while unbiased simulations allow for a general testing of conformational space previously unexplored. The unbiased molecular dynamics data describes the global conformational changes of tRNA on a sub-microsecond time scale for comparison with steered data. Additionally, the unbiased molecular dynamics data was used to identify putative contacts between tRNA and the ribosome during the accommodation step of translation. We found that the primary contact regions were H71 and H92 of the 50S subunit and ribosomal proteins L14 and L16.

EI of the Phosphotransferase System of Escherichia coli: Mathematical Modeling Approach to Analysis of Its Kinetic Properties

T. A. Karelina, H. Ma, I. Goryanin, and O. V. Demin — Sun, 20 Mar 2011 13:49:24 +0000

The mathematical model of the operation of the first enzyme of the Escherichia coli phosphotransferase system, EI, is proposed. Parameters of the kinetic model describing the operation of EI under different conditions are identified on the basis of a large amount of known experimental data. The verified model is employed to predict modes of operation of EI under both in vivo physiological conditions and in vitro nonphysiological conditions. The model predicts that under in vivo physiological conditions, the rate of phosphotransfer from EI to the second protein of the phosphotransferase system HPr by the dimer is much higher than by the monomer. A hypothesis is proposed on the basis of calculations that the transfer by a monomer plays a role in the regulation of chemotaxis. At submicromolar pyruvate concentration, the model predicts nonmonotonic dependence of the phosphotransfer rate on the substrate (PEP) concentration.

Exploring the Membrane Mechanism of the Bioactive Peptaibol Ampullosporin A Using Lipid Monolayers and Supported Biomimetic Membranes

Thu, 17 Feb 2011 10:39:31 +0000

Ampullosporin A is an antimicrobial, neuroleptic peptaibol, the behavior of which was investigated in different membrane mimetic environments made of egg yolk L-α-phosphatidylcholine. In monolayers, the peptaibol adopted a mixed α/310-helical structure with an in-plane orientation. The binding step was followed by the peptide insertion into the lipid monolayer core. The relevance of the inner lipid leaflet nature was studied by comparing ampullosporin binding on a hybrid bilayer, in which this leaflet was a rigid alkane layer, and on supported fluid lipid bilayers. The membrane binding was examined by surface plasmon resonance spectroscopy and the effect on lipid dynamics was explored using fluorescence recovery after photobleaching. In the absence of voltage and at low concentration, ampullosporin A substantially adsorbed onto lipid surfaces and its interaction with biomimetic models was strongly modified depending on the inner leaflet structure. At high concentration, ampullosporin A addition led to the lipid bilayers disruption.

Computational Laser Spectroscopy in a Biological Tissue

M. Gantri, H. Trabelsi, E. Sediki, and R. Ben Salah — Wed, 07 Apr 2010 15:41:22 +0000

We present a numerical spectroscopic study of visible and infrared laser radiation in a biological tissue. We derive a solution of a general two-dimensional time dependent radiative transfer equation in a tissue-like medium. The used model is suitable for many situations especially when the external source is time-dependent or continuous. We use a control volume-discrete ordinate method associated with an implicit three-level second-order time differencing scheme. We consider a very thin rectangular biological-tissue-like medium submitted to a visible or a near infrared light sources. The RTE is solved for a set of different wavelength source. All sources are assumed to be monochromatic and collimated. The energetic fluence rate is computed at a set of detector points on the boundaries. According to the source type, we investigate either the steady-state or transient response of the medium. The used model is validated in the case of a heterogeneous tissue-like medium using referencing experimental results from the literature. Also, the developed model is used to study changes on transmitted light in a rat-liver tissue-like medium. Optical properties depend on the source wavelength and they are taken from the literature. In particular, light-transmission in the medium is studied for continuous wave and for short pulse.

DSC Study of Collagen in Disc Disease

S. Skrzyński, A. Sionkowska, and A. Marciniak — Tue, 09 Feb 2010 14:21:50 +0000

Differential scanning calorimetry (DSC) has been used to estimate the effect of disc disease on the collagen helix-coil transition and morphology for tissue extracted from patients during surgical operation. Forty discs were obtained from patients with degenerative disc disease undergoing surgery for low back pain. The patients were in the age between 20 and 70 years old. The specimens were kept wet during DSC experiment. The data allow the comparison between thermal stability of collagen tissue from healthy patients and from patients suffering from disc disease. In the paper the comparison between thermal helix-coil transition for collagen fibers from patients suffering from disc disease and collagen fibers from healthy organisms has been discussed. The heating rate has an influence on the position on denaturation temperatures of collagen in disc tissues. Higher helix-coil transition temperature of collagen in degenerated disc suggests that additional intermolecular cross linking of collagen fibers occurs. Denaturation temperatures of collagen in degenerated male disc possess smaller values than in female ones. Disc disease induces changes in collagen structure and leads to formation of additional crosslinks between collagen fibers.

An Overview of the Importance of Conformational Flexibility in Gene Regulation by the Transcription Factors

Shagufta H. Khan and Raj Kumar — Thu, 04 Feb 2010 11:34:34 +0000

A number of proteins with intrinsically disordered (ID) regions/domains are reported to be found disproportionately higher in transcription factors. Available evidences suggest that presence of ID region/domain within a transcription factor plays an important role in its biological functions. These ID sequences provide large flexible surfaces that can allow them to make more efficient physical and functional interactions with their target partners. Since transcription factors regulate expression of target genes by interacting with specific coregulatory proteins, these ID regions/domains can be used as a platform for such large macromolecular interactions, and may represent a mechanism for regulation of cellular processes. The precise structural basis for the function of these ID regions/domains of the transcription factors remains to be determined. In the recent years there has been growing evidence suggesting that an induced fit-like process leads to imposition of folded functional structure in these ID domains on which large multiprotein complexes are built. These multiprotein complexes may eventually dictate the final outcome of the gene regulation by the transcription factors.

Navigation by Induction-Based Magnetoreception in Elasmobranch Fishes

T. C. A. Molteno and W. L. Kennedy — Sun, 18 Oct 2009 09:28:34 +0000

A quantitative frequency-domain model of induction-based magnetoreception is presented for elasmobranch fishes. We show that orientation with respect to the geomagnetic field can be determined by synchronous detection of electrosensory signals at harmonics of the vestibular frequency. The sensitivity required for this compass-sense mechanism is shown to be less than that known from behavioral experiments. Recent attached-magnet experiments have called into doubt the induction-based mechanism for magnetoreception. We show that the use of attached magnets would interfere with an induction-based mechanism unless relative movement between the electrosensory system and the attached magnet is less than 100 𝜇m. This suggests that further experiments may be required to eliminate induction as a basis for magnetoreception.

Tropomyosin Period 3 Is Essential for Enhancement of Isometric Tension in Thin Filament-Reconstituted Bovine Myocardium

Tue, 13 Oct 2009 11:51:00 +0000

Tropomyosin (Tm) consists of 7 quasiequivalent repeats known as “periods,” and its specific function may be associated with these periods. To test the hypothesis that either period 2 or 3 promotes force generation by inducing a positive allosteric effect on actin, we reconstituted the thin filament with mutant Tm in which either period 2 (Δ2Tm) or period 3 (Δ3Tm) was deleted. We then studied: isometric tension, stiffness, 6 kinetic constants, and the pCa-tension relationship. N-terminal acetylation of Tm did not cause any differences. The isometric tension in Δ2Tm remained unchanged, and was reduced to ∼60% in Δ3Tm. Although the kinetic constants underwent small changes, the occupancy of strongly attached cross-bridges was not much different. The Hill factor (cooperativity) did not differ significantly between Δ2Tm (1.79 ± 0.19) and the control (1.73 ± 0.21), or Δ3Tm (1.35 ± 0.22) and the control. In contrast, pCa50 decreased slightly in Δ2Tm (5.11 ± 0.07), and increased significantly in Δ3Tm (5.57 ± 0.09) compared to the control (5.28 ± 0.04). These results demonstrate that, when ions are present at physiological concentrations in the muscle fiber system, period 3 (but not period 2) is essential for the positive allosteric effect that enhances the interaction between actin and myosin, and increases isometric force of each cross-bridge.

Crystal Structural and Functional Analysis of the Putative Dipeptidase from Pyrococcus horikoshii OT3

Sun, 28 Jun 2009 15:03:54 +0000

The crystal structure of a putative dipeptidase (Phdpd) from Pyrococcus horikoshii OT3 was solved using X-ray data at 2.4 Å resolution. The protein is folded into two distinct entities. The N-terminal domain consists of the general topology of the 𝛼/𝛽 fold, and the C-terminal domain consists of five long mixed strands, four helices, and two 310 helices. The structure of Phdpd is quite similar to reported structures of prolidases from P. furiosus (Zn-Pfprol) and P. horikoshii (Zn-Phdpd), where Zn ions are observed in the active site resulting in an inactive form. However, Phdpd did not contain metals in the crystal structure and showed prolidase activity in the absence of additional Co ions, whereas the specific activities increased by 5 times in the presence of a sufficient concentration (1.2 mM) of Co ions. The substrate specificities (X-Pro) of Phdpd were broad compared with those of Zn-Phdpd in the presence of Co ions, whose relative activities are 10% or less for substrates other than Met-Pro, which is the most favorable substrate. The binding constants of Zn-Phdpd with three metals (Zn, Co, and Mn) were higher than those of Phdpd and that with Zn was higher by greater than 2 orders, which were determined by DSC experiments. From the structural comparison of both forms and the above experimental results, it could be elucidated why the protein with Zn2+ ions is inactive.

Role of the Endothelium during Tumor Cell Metastasis: Is the Endothelium a Barrier or a Promoter for Cell Invasion and Metastasis?

Claudia Tanja Mierke — Thu, 05 Mar 2009 14:43:36 +0000

The malignancy of cancer disease depends on the ability of the primary tumor to metastasize to distant organs. The process of the metastasis formation has largely been analyzed, but still main pathways regarding the extravasation step at the end of the metastasis formation process are controversially discussed. An agreement has been reached about the importance of the endothelium to promote metastasis formation either by enhancing the growth of the primary tumor or by homing (targeting) the tumor cells to blood or lymph vessels. The mechanical properties of the invading tumor cells become the focus of several studies, but the endothelial cell mechanical properties are still elusive. This paper describes the different roles of the endothelium in the process of metastasis formation and focuses on a novel role of the endothelium in promoting tumor cell invasion. It discusses how novel biophysical tools and in vivo animal models help to determine the role of the endothelium in the process of tumor cell invasion. Evidence is provided that cell mechanical properties, for example, contractile force generation of tumor cells, are involved in the process of tumor cell invasion.

Roots of Diversity Relations

Peter Würtz and Arto Annila — Thu, 11 Dec 2008 10:46:52 +0000

The species-area relationship is one of the central generalizations in ecology; however, its origin has remained a puzzle. Since ecosystems are understood as energy transduction systems, the regularities in species richness are considered to result from ubiquitous imperatives in energy transduction. From a thermodynamic point of view, organisms are transduction mechanisms that distribute an influx of energy down along the steepest gradients to the ecosystem's diverse repositories of chemical energy, that is, populations of species. Transduction machineries, that is, ecosystems assembled from numerous species, may emerge and evolve toward high efficiency on large areas that hold more matter than small ones. This results in the well-known logistic-like relationship between the area and the number of species. The species-area relationship is understood, in terms of thermodynamics, to be the skewed cumulative curve of chemical energy distribution that is commonly known as the species-abundance relationship.

Selective Detection of NADPH Oxidase in Polymorphonuclear Cells by Means of NAD(P)H-Based Fluorescence Lifetime Imaging

R. Niesner, P. Narang, H. Spiecker, V. Andresen, K.-H. Gericke, and M. Gunzer — Sun, 16 Nov 2008 15:16:14 +0000

NADPH oxidase (NOX2) is a multisubunit membrane-bound enzyme complex that, upon assembly in activated cells, catalyses the reduction of free oxygen to its superoxide anion, which further leads to reactive oxygen species (ROS) that are toxic to invading pathogens, for example, the fungus Aspergillus fumigatus. Polymorphonuclear cells (PMNs) employ both nonoxidative and oxidative mechanisms to clear this fungus from the lung. The oxidative mechanisms mainly depend on the proper assembly and function of NOX2. We identified for the first time the NAD(P)H-dependent enzymes involved in such oxidative mechanisms by means of biexponential NAD(P)H-fluorescence lifetime imaging (FLIM). A specific fluorescence lifetime of 3670±140 picoseconds as compared to 1870 picoseconds for NAD(P)H bound to mitochondrial enzymes could be associated with NADPH bound to oxidative enzymes in activated PMNs. Due to its predominance in PMNs and due to the use of selective activators and inhibitors, we strongly believe that this specific lifetime mainly originates from NOX2. Our experiments also revealed the high site specificity of the NOX2 assembly and, thus, of the ROS production as well as the dynamic nature of these phenomena. On the example of NADPH oxidase, we demonstrate the potential of NAD(P)H-based FLIM in selectively investigating enzymes during their cellular function.

Flexibility of the Cytoplasmic Domain of the Phototaxis Transducer II from Natronomonas pharaonis

Thu, 16 Oct 2008 10:41:06 +0000

Chemo- and phototaxis systems in bacteria and archaea serve as models for more complex signal transduction mechanisms in higher eukaryotes. Previous studies of the cytoplasmic fragment of the phototaxis transducer (pHtrII-cyt) from the halophilic archaeon Natronomonas pharaonis showed that it takes the shape of a monomeric or dimeric rod under low or high salt conditions, respectively. CD spectra revealed only approximately 24% helical structure, even in 4 M KCl, leaving it an open question how the rod-like shape is achieved. Here, we conducted CD, FTIR, and NMR spectroscopic studies under different conditions to address this question. We provide evidence that pHtrII-cyt is highly dynamic with strong helical propensity, which allows it to change from monomeric to dimeric helical coiled-coil states without undergoing dramatic shape changes. A statistical analysis of predicted disorder for homologous sequences suggests that structural flexibility is evolutionarily conserved within the methyl-accepting chemotaxis protein family.

Cell Volume and Sodium Content in Rat Kidney Collecting Duct Principal Cells During Hypotonic Shock

Evgeny I. Solenov — Sun, 27 Jul 2008 00:00:00 +0000

The purpose of this study was to investigate the time course of the volume-regulatory response and intracellular sodium concentration ([Na+]i) in the principal cells of rat kidney outer medulla collecting duct (OMCD) epithelia during acute swelling in hypotonic medium. Hypotonic shock was created by PBS diluted with 50% of water. Changes in cell volume were measured with calcein quenching method. Intracellular sodium concentration was studied with fluorescence dye Sodium Green. Principal cells of microdissected OMCD fragments swelled very fast. The characteristic time of swelling (𝜏1) was 0.65±0.05 seconds, and the volume increased more than 60% (92.9±5.6 and 151.3±9.8 𝜇m3 control and peak volumes correspondently, 𝑃<.01). After cell volume reached the peak of swelling, the RVD began without lag period. The characteristic time of volume decreasing to new steady-state level (𝜏2) was 8.9±1.1 seconds. In hypoosmotic medium, cell volume stabilized on higher level in comparison with control (110.3±8.3 𝜇m3, 𝑃<.01). After restoration of the medium osmolality to normotonic, cell volume stabilized on significantly low level in comparison with control level (71.4±6.1 𝜇m3, 𝑃<.01). During the hypoosmotic shock, [Na+]i decreased from control level in isotonic PBS to the low level in hypoosmotic solution (27.7±1.4 and 5.8±0.23 mM, 𝑃<.01). Calculation of sodium content per cell has shown the significant sodium entry into the cells, which caused a temporary increase correlated with the peak of cell volume caused by swelling. The conclusion is made that in our model of hypoosmotic shock, swelling activates transporters with high permeability for Na+ that provides sodium flux into the cells.