Showing papers in "Cell Biochemistry and Biophysics in 2008"

Vascular Adaptation and Mechanical Homeostasis at Tissue, Cellular, and Sub-cellular Levels

Abstract: Blood vessels exhibit a remarkable ability to adapt throughout life that depends upon genetic programming and well-orchestrated biochemical processes. Findings over the past four decades demonstrate, however, that the mechanical environment experienced by these vessels similarly plays a critical role in governing their adaptive responses. This article briefly reviews, as illustrative examples, six cases of tissue level growth and remodeling, and then reviews general observations at cell-matrix, cellular, and sub-cellular levels, which collectively point to the existence of a "mechanical homeostasis" across multiple length and time scales that is mediated primarily by endothelial cells, vascular smooth muscle cells, and fibroblasts. In particular, responses to altered blood flow, blood pressure, and axial extension, disease processes such as cerebral aneurysms and vasospasm, and diverse experimental manipulations and clinical treatments suggest that arteries seek to maintain constant a preferred (homeostatic) mechanical state. Experiments on isolated microvessels, cell-seeded collagen gels, and adherent cells isolated in culture suggest that vascular cells and sub-cellular structures such as stress fibers and focal adhesions likewise seek to maintain constant a preferred mechanical state. Although much is known about mechanical homeostasis in the vasculature, there remains a pressing need for more quantitative data that will enable the formulation of an integrative mathematical theory that describes and eventually predicts vascular adaptations in response to diverse stimuli. Such a theory promises to deepen our understanding of vascular biology as well as to enable the design of improved clinical interventions and implantable medical devices.

Keep your fingers off my DNA: Protein-protein interactions mediated by C2H2 zinc finger domains

Abstract: Cys2-His2 (C2H2) zinc finger domains (ZFs) were originally identified as DNA-binding domains, and uncharacterized domains are typically assumed to function in DNA binding. However, a growing body of evidence suggests an important and widespread role for these domains in protein binding. There are even examples of zinc fingers that support both DNA and protein interactions, which can be found in well-known DNA-binding proteins such as Sp1, Zif268, and Ying Yang 1 (YY1). C2H2 protein-protein interactions (PPIs) are proving to be more abundant than previously appreciated, more plastic than their DNA-binding counterparts, and more variable and complex in their interactions surfaces. Here we review the current knowledge of over 100 C2H2 zinc finger-mediated PPIs, focusing on what is known about the binding surface, contributions of individual fingers to the interaction, and function. An accurate understanding of zinc finger biology will likely require greater insights into the potential protein interaction capabilities of C2H2 ZFs.

Recent Applications of Kirkwood–Buff Theory to Biological Systems

Abstract: The effect of cosolvents on biomolecular equilibria has traditionally been rationalized using simple binding models. More recently, a renewed interest in the use of Kirkwood–Buff (KB) theory to analyze solution mixtures has provided new information on the effects of osmolytes and denaturants and their interactions with biomolecules. Here we review the status of KB theory as applied to biological systems. In particular, the existing models of denaturation are analyzed in terms of KB theory, and the use of KB theory to interpret computer simulation data for these systems is discussed.

Structure, Function, and Modification of the Voltage Sensor in Voltage-Gated Ion Channels

Abstract: Voltage-gated ion channels are crucial for both neuronal and cardiac excitability Decades of research have begun to unravel the intriguing machinery behind voltage sensitivity Although the details regarding the arrangement and movement in the voltage-sensor domain are still debated, consensus is slowly emerging There are three competing conceptual models: the helical-screw, the transporter, and the paddle model In this review we explore the structure of the activated voltage-sensor domain based on the recent X-ray structure of a chimera between Kv12 and Kv21 We also present a model for the closed state From this we conclude that upon depolarization the voltage sensor S4 moves ~13 A outwards and rotates ~180°, thus consistent with the helical-screw model S4 also moves relative to S3b which is not consistent with the paddle model One interesting feature of the voltage sensor is that it partially faces the lipid bilayer and therefore can interact both with the membrane itself and with physiological and pharmacological molecules reaching the channel from the membrane This type of channel modulation is discussed together with other mechanisms for how voltage-sensitivity is modified Small effects on voltage-sensitivity can have profound effects on excitability Therefore, medical drugs designed to alter the voltage dependence offer an interesting way to regulate excitability

Polyunsaturated fatty acid modulation of voltage-gated ion channels.

Abstract: Arachidonic acid (AA) was found to inhibit the function of whole-cell voltage-gated (VG) calcium currents nearly 16 years ago. There are now numerous examples demonstrating that AA and other polyunsaturated fatty acids (PUFAs) modulate the function of VG ion channels, primarily in neurons and muscle cells. We will review and extract some common features about the modulation by PUFAs of VG calcium, sodium, and potassium channels and discuss the impact of this modulation on the excitability of neurons and cardiac myocytes. We will describe the fatty acid nature of the membrane, how fatty acids become available to function as modulators of VG channels, and the physiologic importance of this type of modulation. We will review the evidence for molecular mechanisms and assess our current understanding of the structural basis for modulation. With guidance from research on the structure of fatty acid binding proteins, the role of lipids in gating mechanosensitive (MS) channels, and the impact of membrane lipid composition on membrane-embedded proteins, we will highlight some avenues for future investigations.

The Protein-Binding Potential of C2H2 Zinc Finger Domains

Abstract: There are over 10,000 C2H2-type zinc finger (ZF) domains distributed among more than 1,000 ZF proteins in the human genome. These domains are frequently observed to be involved in sequence-specific DNA binding, and uncharacterized domains are typically assumed to facilitate DNA interactions. However, some ZFs also facilitate binding to proteins or RNA. Over 100 Cys2-His2 (C2H2) ZF-protein interactions have been described. We initially attempted a bioinformatics analysis to identify sequence features that would predict a DNA- or protein-binding function. These efforts were complicated by several issues, including uncertainties about the full functional capabilities of the ZFs. We therefore applied an unbiased approach to directly examine the potential for ZFs to facilitate DNA or protein interactions. The human OLF-1/EBF associated zinc finger (OAZ) protein was used as a model. The human O/E-1-associated zinc finger protein (hOAZ) contains 30 ZFs in 6 clusters, some of which have been previously indicated in DNA or protein interactions. DNA binding was assessed using a target site selection (CAST) assay, and protein binding was assessed using a yeast two-hybrid assay. We observed that clusters known to bind DNA could facilitate specific protein interactions, but clusters known to bind protein did not facilitate specific DNA interactions. Our primary conclusion is that DNA binding is a more restricted function of ZFs, and that their potential for mediating protein interactions is likely greater. These results suggest that the role of C2H2 ZF domains in protein interactions has probably been underestimated. The implication of these findings for the prediction of ZF function is discussed.

Functional mapping of bluetongue virus proteins and their interactions with host proteins during virus replication.

Abstract: Bluetongue virus (BTV) is a double-stranded RNA (dsRNA) virus which is transmitted by blood-feeding gnats to wild and domestic ruminants, causing high morbidity and often high mortality. Partly due to this BTV has been in the forefront of molecular studies for last three decades and now represents one of the best understood viruses at the molecular and structural levels. BTV, like the other members of the Reoviridae family is a complex non-enveloped virus with seven structural proteins and a RNA genome consisting of 10 dsRNA segments of different sizes. In virus infected cells, three other virus encoded nonstructural proteins are synthesized. Significant recent advances have been made in understanding the structure-function relationships of BTV proteins and their interactions during virus assembly. By combining structural and molecular data it has been possible to make progress on the fundamental mechanisms used by the virus to invade, replicate in, and escape from, susceptible host cells. Data obtained from studies over a number of years have defined the key players in BTV entry, replication, assembly and egress. Specifically, it has been possible to determine the complex nature of the virion through three dimensional structure reconstructions; atomic structure of proteins and the internal capsid; the definition of the virus encoded enzymes required for RNA replication; the ordered assembly of the capsid shell and the protein sequestration required for it; and the role of three NS proteins in virus replication, assembly and release. Overall, this review demonstrates that the integration of structural, biochemical and molecular data is necessary to fully understand the assembly and replication of this complex RNA virus.

Calponin in non-muscle cells.

Abstract: Calponin is an actin filament-associated regulatory protein expressed in smooth muscle and non-muscle cells. Calponin is an inhibitor of the actin-activated myosin ATPase. Three isoforms of calponin have been found in the vertebrates. Whereas the role of calponin in regulating smooth muscle contractility has been extensively investigated, the function and regulation of calponin in non-muscle cells is much less understood. Based on recent progresses in the field, this review focuses on the studies of calponin in non-muscle cells, especially its regulation by cytoskeleton tension and function in cell motility. The ongoing research has demonstrated that calponin plays a regulatory role in non-muscle cell motility. Therefore, non-muscle calponin is an attractive target for the control of cell proliferation, migration and phagocytosis, and the treatment of cancer metastasis.

The Role of the Biochemical and Biophysical Environment in Chondrogenic Stem Cell Differentiation Assays and Cartilage Tissue Engineering

Abstract: The field of regenerative medicine offers hope for the development of a cell-based therapy for the repair of articular cartilage (AC). Yet, the greatest challenge in the use of stem cells for tissue repair, is understanding how the cells respond to stimuli and using that knowledge to direct cell fate. Novel methods that utilize stem cells in cartilage regeneration will require specific spatio-temporal controls of the biochemical and biophysical signaling environments. Current chondrogenic differentiation research focuses on the roles of biochemical stimuli like growth factors, hormones, and small molecules, and the role of the physical environment and mechanical stimuli, such as compression and shear stress, which likely act through mechanical receptors. Numerous signals are associated with chondrogenic-like activity of cells in different systems, however many variables for a controlled method still need to be optimized; e.g., spatial and temporal application of the stimuli, and time of transplantation of an engineered construct. Understanding the necessary microenvironmental signals for cell differentiation will advance cell therapy for cartilage repair.

Biomolecular Self-assembly and its Relevance in Silica Biomineralization

Abstract: Biomineralization, which means the formation of inorganic materials by biological processes, currently finds increasing research interest. It involves the synthesis of calcium-based minerals such as bones and teeth in vertebrates, and of shells. Silica biomineralization occurs, for example, in diatoms and silica sponges. Usually, biominerals are made up of amorphous compounds or small microcrystalline domains embedded into an amorphous matrix. Nevertheless, they exhibit very regular shapes and, as in the case of diatoms, intricate nanopatterns of amazing beauty. It is, therefore, commonly assumed that biominerals are formed under the structure-directing influence of templates. However, single molecules are by far too small to direct the formation of the observed shapes and patterns. Instead, supramolecular aggregates are shown to be involved in the formation of templating superstructures relevant in biomineralization. Specific biomolecules were identified in both diatoms and silica sponges, which elegantly combine two indispensable functions: on the one hand, the molecules are capable of inducing silica precipitation from precursor compounds. On the other hand, these molecules are capable of self-assembling into larger, structure-directing template aggregates. Such molecules are the silaffins in the case of diatoms and the silicateins in sponges. Long-chain polyamines of similar composition have meanwhile been discovered in both organisms. The present review is especially devoted to the discussion of the self-assembly behavior of these molecules. Physico-chemical studies on a model compound, poly(allylamine), are discussed in detail in order to elucidate the nature of the interactions responsible for self-assembly of long-chain polyamines and the parameters controlling this process. Numerous biomimetic silica synthesis experiments are discussed and evaluated with respect to the observations made on the aforementioned “natural” biomolecules.

Disordered proteins : Biological membranes as two-dimensional aggregation matrices

Abstract: Aberrant folded proteins and peptides are hallmarks of amyloidogenic diseases. However, the molecular processes that cause these proteins to adopt non-native structures in vivo and become cytotoxic ...

Control of Nucleosome Positions by DNA Sequence and Remodeling Machines

Abstract: Recent studies have shown that promoter nucleosomes frequently adopt specific positions, and indicate that these positions functionally regulate transcription factor binding. Other studies indicate that DNA sequence has a major role in establishing these nucleosome positions, suggesting that evolution has selected for specific, default arrangements of promoter nucleosomes. Finally, recent studies indicate that ATP-dependent chromatin remodeling complexes move nucleosomes away from default positioning sequences, either to complex-preferred locations or to establish a complex-preferred spacing between nucleosomes. Here we will review these recent findings, and consider how combinations of promoter sequence and specific remodeling complexes may act to switch chromatin between permissive and repressive conformations.

Force-Velocity Curves of Motor Proteins Cooperating In Vivo

Abstract: Motor proteins convert chemical energy into work, thereby generating persistent motion of cellular and subcellular objects. The velocities of motor proteins as a function of opposing loads have been previously determined in vitro for single motors. These single molecule “force–velocity curves” have been useful for elucidating motor kinetics and for estimating motor performance under physiological loads due to, for example, the cytoplasmic drag force on transported organelles. Here we report force–velocity curves for single and multiple motors measured in vivo. Using motion enhanced differential interference contrast (MEDIC) movies of living NT2 (neuron-committed teratocarcinoma) cells at 37°C, three parameters were measured—velocity (v), radius (a), and effective cytoplasmic viscosity (η′)—as they applied to moving vesicles. These parameters were combined in Stokes’ equation, F = 6πaη′v, to determine the force, F, required to transport a single intracellular particle at velocity, v. In addition, the number of active motors was inferred from the multimodal pattern seen in a normalized velocity histogram. Using this inference, the resulting in vivo force–velocity curve for a single motor agrees with previously reported in vitro single motor force–velocity curves. Interestingly, however, the curves for two and three motors lie significantly higher in both measured velocity and computed force, which suggests that motors can work cooperatively to attain higher transport forces and velocities.

Lung ischemia: a model for endothelial mechanotransduction.

Abstract: Endothelial cells in vivo are constantly exposed to shear associated with blood flow and altered shear stress elicits cellular responses (mechanotransduction). This review describes the role of shear sensors and signal transducers in these events. The major focus is the response to removal of shear as occurs when blood flow is compromised (i.e., ischemia). Pulmonary ischemia studied with the isolated murine lung or flow adapted pulmonary microvascular endothelial cells in vitro results in endothelial generation of reactive oxygen species (ROS) and NO. The response requires caveolae and is initiated by endothelial cell depolarization via KATP channel closure followed by activation of NADPH oxidase (NOX2) and NO synthase (eNOS), signaling through MAP kinases, and endothelial cell proliferation. These physiological mediators can promote vasodilation and angiogenesis as compensation for decreased tissue perfusion.

Adaptive changes in cardiac fibroblast morphology and collagen organization as a result of mechanical environment.

Abstract: There is a growing body of work in the literature that demonstrates the significant differences between 2D versus 3D environments in cell morphologies, spatial organization, cell-ECM interactions, and cell signaling. The 3D environments are generally considered more realistic tissue models both because they offer cells a surrounding environment rather than just a planar surface with which to interact, and because they provide the potential for more diverse mechanical environments. Many studies have examined cellular-mediated contraction of 3D matrices; however, because the 3D environment is much more complex and the scale more difficult to study, little is known regarding how mechanical environment, cell and collagen architecture, and collagen remodeling are linked. In the current work, we examine the spatial arrangement of neonatal cardiac fibroblasts and the associated collagen organization in constrained and unconstrained collagen gels over a 24 h period. Collagen gels that are constrained by their physical attachment to a mold and similar gels, which have been detached (unconstrained) from the mold and subsequently contract, offer two simple mechanical models by which the mechanisms of tissue homeostasis and wound repair might be examined. Our observations suggest the presence of two mechanical regimes in the unconstrained gels: an outer ring where cells orient circumferentially and local collagen aligns with the elongated cells; and a central region where unaligned stellate/bipolar cells are radially surrounded by collagen, similar to that seen throughout constrained gels. The evolving organization of cell alignment and surrounding collagen organization suggests that cellular response may be due to the cellular perception of the apparent stiffness of local physical environment.

Extremely high frequency electromagnetic radiation enforces bacterial effects of inhibitors and antibiotics.

Abstract: The coherent electromagnetic radiation (EMR) of the frequency of 51.8 and 53 GHz with low intensity (the power flux density of 0.06 mW/cm2) affected the growth of Escherichia coli K12(λ) under fermentation conditions: the lowering of the growth specific rate was considerably (~2-fold) increased with exposure duration of 30–60 min; a significant decrease in the number of viable cells was also shown. Moreover, the enforced effects of the N,N′-dicyclohexylcarbodiimide (DCCD), inhibitor of H+-transporting F0F1-ATPase, on energy-dependent H+ efflux by whole cells and of antibiotics like tetracycline and chloramphenicol on the following bacterial growth and survival were also determined after radiation. In addition, the lowering in DCCD-inhibited ATPase activity of membrane vesicles from exposed cells was defined. The results confirmed the input of membranous changes in bacterial action of low intensity extremely high frequency EMR, when the F0F1-ATPase is probably playing a key role. The radiation of bacteria might lead to changed metabolic pathways and to antibiotic resistance. It may also give bacteria with a specific role in biosphere.

Composition-driven surface domain structuring mediated by sphingolipids and membrane-active proteins. Above the nano- but under the micro-scale: mesoscopic biochemical/structural cross-talk in biomembranes.

Abstract: Biomembranes contain a wide variety of lipids and proteins within an essentially two-dimensional structure. The coexistence of such a large number of molecular species causes local tensions that frequently relax into a phase or compositional immiscibility along the lateral and transverse planes of the interface. As a consequence, a substantial microheterogeneity of the surface topography develops and that depends not only on the lipid-protein composition, but also on the lateral and transverse tensions generated as a consequence of molecular interactions. The presence of proteins, and immiscibility among lipids, constitute major perturbing factors for the membrane sculpturing both in terms of its surface topography and dynamics. In this work, we will summarize some recent evidences for the involvement of membrane-associated, both extrinsic and amphitropic, proteins as well as membrane-active phosphohydrolytic enzymes and sphingolipids in driving lateral segregation of phase domains thus determining long-range surface topography.

Copper (II) Ions Affect Escherichia coli Membrane Vesicles’ SH-Groups and a Disulfide-Dithiol Interchange Between Membrane Proteins

Abstract: The SH-groups in Escherichia coli membrane vesicles, prepared from cells grown in fermentation conditions on glucose at slightly alkaline pH, have a role in the F0F1-ATPase operation. The changes in the number of these groups by ATP are observed under certain conditions. In this study, copper ions (Cu2+) in concentration of 0.1 mM were shown to increase the number of SH-groups in 1.5- to 1.6-fold independent from K+ ions, and the suppression of the increased level of SH-groups by ATP was determined for Cu2+ in the presence of K+. Moreover, the increase in the number of SH-groups by Cu2+ was absent as well as the inhibition in ATP-dependent increasing SH-groups number by Cu2+ lacked when vesicles were treated with N-ethylmaleimide (NEM), specific thiol-reagent. Such an effect was not observed with zinc (Zn2+), cobalt (Co2+), or Cu+ ions. The increased level of SH-groups was observed in the hycE or hyfR mutants with defects in hydrogenases 3 or 4, whereas the ATP-dependent increase in the number of these groups was determined in hycE not in hyfR mutants. Both changes in SH-groups number disappeared in the atp or hyc mutants deleted for the F0F1-ATPase or hydrogenase 3 (no activity of hydrogenase 4 was detected in the hyc mutant used). A direct effect of Cu2+ but not Cu+ on the F0F1-ATPase is suggested to lead to conformational changes or damaging consequences, increasing accessible SH-groups number and disturbing disulfide-dithiol interchange within a protein–protein complex, where this ATPase works with K+ uptake system or hydrogenase 4 (Hyd-4); breaks in disulfides are not ruled out.

Many commercially available antibodies for detection of CHOP expression as a marker of endoplasmic reticulum stress fail specificity evaluation.

Abstract: Endoplasmic reticulum (ER) stress contributes to beta cell death in type 2 diabetes (T2DM). ER stress is characterized by increased level of ER stress markers such as C/EBP homologous protein (CHOP). Activation of CHOP leads to its translocation into the nucleus, where it induces cell death. We previously reported nuclear CHOP in pancreatic sections from T2DM, but not T1DM, and in human islet amyloid polypeptide (IAPP) transgenic rodent pancreatic sections. These studies underscore the importance of studying nuclear CHOP. We have observed inconsistency in the detection of CHOP antibodies reported in the literature and also in our own experiments. To investigate the specificity of CHOP antibodies, we first induced ER stress by tunicamycin in rat insulinoma (INS) cells and prepared nuclear and cytoplasmic fractions. Then we examined CHOP expression by Western blotting and immunocytochemistry using seven commercially available CHOP antibodies in INS cells and human IAPP (h-IAPP) transgenic rodent pancreatic tissue. These studies show that three commercially available CHOP antibodies out of seven tested were non-specific. In conclusion, we give recommendations for CHOP antibody selection and methods to verify CHOP antibody specificity. Also, we propose that the authors report the catalog and lot numbers of the CHOP antibodies used.

Mechanosensitive Channels: Insights from Continuum-Based Simulations

Abstract: Mechanotransduction plays an important role in regulating cell functions and it is an active topic of research in biophysics. Despite recent advances in experimental and numerical techniques, the intrinsic multiscale nature imposes tremendous challenges for revealing the working mechanisms of mechanosensitive channels. Recently, a continuum-mechanics-based hierarchical modeling and simulation framework has been established and applied to study the mechanical responses and gating behaviors of a prototypical mechanosensitive channel, the mechanosensitive channel of large conductance (MscL) in bacteria Escherichia coli (E. coli), from which several putative gating mechanisms have been tested and new insights are deduced. This article reviews these latest findings using the continuum mechanics framework and suggests possible improvements for future simulation studies. This computationally efficient and versatile continuum-mechanics-based protocol is poised to make contributions to the study of a variety of mechanobiology problems.

Endothelial injury induces vascular smooth muscle cell proliferation in highly localized regions of a direct contact co-culture system.

Abstract: Though previous studies have indicated a relationship between the proliferation of endothelial cells and vascular smooth muscle cells (VSMCs) in co-culture, the results have been contradictory and the signaling mechanism poorly understood. In this transmembrane co-culture study, VSMCs and endothelial cells were grown to confluence on opposite sides of a microporous membrane to mimic the intima/media border of vessels. The endothelial layer was injured, and then cultured for 3 days, resulting in partial re-endothelialization. VSMC proliferation across from the injured/partially recovered endothelial region was significantly higher than across from the de-endothelialized region (a sevenfold increase) and the uninjured region (a threefold increase). ELISA indicated that PDGF, which was undetectable in uninjured co-culture and homotypic controls, increased after injury and the addition of a piperazinyl-quinazoline carboxamide PDGF receptor inhibitor blocked VSMC proliferation across from the injured/partially recovered region. We conclude that co-culture signaling initiated by endothelial cell injury locally stimulates VSMC proliferation and that this signaling could be mediated by PDGF-BB.

MRGing chromatin dynamics and cellular senescence.

Abstract: Normal primary cells have a finite ability to divide in culture and after a number of population doublings enter a state of irreversible cell cycle arrest known as replicative senescence. Several cellular stresses have been shown to induce a senescence-like growth arrest including shortened telomeres, DNA-damaging stresses, and drastic changes in chromatin structure, for example, through histone deacetylase (HDAC) induction. Histones are core components of chromatin which are subject to a number of chemical modifications that influence the dynamic state of chromatin structure. Proper chromatin structure formation is crucial for most DNA-dependent processes including transcription, replication, and repair which have a profound impact on cellular proliferation and senescence. Several genes important for chromatin remodeling such as the tumor suppressors p53 and retinoblastoma (Rb) affect cellular senescence by mediating changes in chromatin structure and gene expression. The Morf4-Related Gene (MRG) family of transcription factors forms stable interactions with chromatin-modifying complexes including histone acetyltransferase (HAT) and HDAC complexes and interact with Rb. Further, the MRG family was founded by a gene, Mortality Factor on Chromosome 4, capable of inducing senescence in immortalized cell lines. In this paper, we review the role of the MRG family of proteins in chromatin dynamics and cellular senescence.

Exchange of Microtubule Molecular Motors During Melanosome Transport in Xenopus laevis Melanophores is Triggered by Collisions with Intracellular Obstacles

Abstract: The observation that several cargoes move bidirectionally along microtubules in vivo raised the question regarding how molecular motors with opposed polarity coordinate during transport. In this work, we analyzed the switch of microtubule motors during the transport of melanosomes in Xenopus melanophores by registering trajectories of these organelles moving along microtubules using a fast and precise tracking method. We analyzed in detail the intervals of trajectories showing reversions in the original direction of transport and processive motion in the opposite direction for at least 250 nm. In most of the cases, the speed of the melanosome before the reversion slowly decreases with time approaching zero then, the organelle returns over the same path moving initially at a very high speed and slowing down with time. These results could be explained according to a model in which reversions are triggered by an elastic collision of the cargo with obstacles in the cytosol. This interaction generates a force opposed to the movement of the motor-driven organelle increasing the probability of detaching the active motors from the track. The model can explain reversions in melanosome trajectories as well as other characteristics of in vivo transport along microtubules observed by other authors. Our results suggest that the crowded cytoplasm plays a key role in regulating the coordination of microtubules-dependent motors.

Noninvasive Method for Simultaneously Measuring the Thermophysical Properties and Blood Perfusion in Cylindrically Shaped Living Tissues

Abstract: An easy-to-use noninvasive method was developed to simultaneously measure the thermophysical parameters and blood perfusion in cylindrically shaped living tissues. This method is based on a two-dimensional mathematical model which requires temperature measurements at only three separate points along the axial direction on the cylinder surface. A sensitivity analysis has shown that the key thermophysical parameters, such as the thermal conductivity, volumetric heat capacity, and blood perfusion can be estimated simultaneously with high accuracy. Genetic algorithm (GA) selection, crossover, and mutation operators were developed to solve this multi-parameter optimization problem. This three-point method was validated by measuring the properties of a dynamic tissue-equivalent phantom with known thermal parameters. The method has also been applied to measure the thermophysical parameters and blood perfusion in human forearms with measured results agreeing well with the literature values.

Profile of Exoglycosidases in Synovial Cell Cultures Derived from Human Synovial Membrane

Abstract: Objective Determining the activity of lysosomal exoglycosidases in tissue cultures of synoviocytes derived from the knee joints of patients with injured anterior cruciate ligaments (ACL), juvenile idiopathic arthritis (JIA), and rheumatoid arthritis (RA) Methods The following exoglycosidases in cultured synoviocytes were analyzed with p-nitrophenyl derivatives of appropriate sugars as substrates: hexosaminidase (HEX) and its isoenzyme A (HEX-A), β-glucuronidase (GluA), β-galactosidase (GAL), α-mannosidase (MAN), and α-fucosidase (FUC) Results In our cell cultures, fibroblast-like synovial cells (FLS) dominated In the group of patients with ACL-injuries, and in the groups of patients with JIA and RA, the activity of the investigated exoglycosidases was significantly higher in the intra- rather than in the extracellular compartment Hexosaminidase was the predominant exoglycosidase Stimulation of synoviocytes by IL-1β in cell cultures significantly increased the activity of HEX, HEX-A, and GluA in both compartments, as well as of GAL, MAN, and FUC in the intracellular compartment Stimulation by IL-1β rheumatoidal synoviocytes increased by 128–201% the activity of HEX and HEX A in intracellular compartments and 33–72% in extracellular compartment Conclusions The profile of lysosomal exoglycosidases in a cell culture of human synoviocytes is similar, but not identical, to those in the knee joint Hexosaminidase is the dominant glycosidase in cultured unstimulated and IL-1β-stimulated human synoviocytes The HEX inhibitors may be new drugs for the treatment of inflamed knee joints

Clinically relevant concentration determination of inhaled anesthetics (halothane, isoflurane, sevoflurane, and desflurane) by 19F NMR.

Abstract: Biophysical studies of protein–anesthetic interactions using nuclear magnetic resonance (NMR) spectroscopy are often conducted by the addition of micro amounts of neat inhaled anesthetic which yields much higher than clinically relevant (0.2–0.5 mM) anesthetic concentrations. We report a 19F NMR technique to measure clinically relevant inhaled anesthetic concentrations from saturated aqueous solutions of these anesthetics (halothane, isoflurane, sevoflurane, and desflurane). We use a setup with a 3-mm NMR tube (containing trifluoroacetic acid as standard), coaxially inserted in a 5-mm NMR tube containing anesthetic solution under investigation. All experiments are conducted in a 5-mm NMR probe. We also have provided standard curves for four inhaled anesthetics using NMR technique. The standard curve for each of these anesthetics is helpful in determining the prerequisite amount of aqueous anesthetic solution required to prepare clinically relevant concentrations for protein–anesthetic interaction studies.

The Effect of N -Ethylmaleimide on Permeability Transition as Induced by Carboxyatractyloside, Agaric Acid, and Oleate

Abstract: In this work, we studied the effect of N-ethylmaleimide on permeability transition. The findings indicate that the amine inhibited the effects of carboxyatractyloside and agaric acid. It is known that these reagents interact with the adenine nucleotide carrier through the cytosolic side. When oleate, which interacts through the matrix side, was used it was found that the amine amplified the effects of oleate on permeability transition. The results also show that N-ethylmaleimide strengthened the inhibition induced by carboxyatractyloside, agaric acid, and oleate on ADP exchange. Furthermore, it was also found that oleate improved the binding of eosin-5-maleimide on the adenine nucleotide translocase.

Parvalbumin Isoforms for Enhancing Cardiac Diastolic Function

Abstract: Diastolic heart failure (DHF), characterized by depressed myocardial relaxation performance and poor ventricular filling, is a distinct form of heart failure accounting for nearly half of the heart failure patients with otherwise normal systolic performance. Defective intracellular calcium (Ca2+) cycling is an important mechanism underlying impaired relaxation in DHF. Recently, genetic manipulation of Ca2+ handling proteins in cardiac myocytes has been explored for its potential therapeutic application in DHF. Specifically, ectopic expression of the skeletal muscle Ca2+ binding protein parvalbumin (Parv) has been shown to accelerate myocardial relaxation in vitro and in vivo. Parv acts as a unique “delayed” Ca2+ buffer during diastole by promoting Ca2+ transient decay and sequestration and corrects diastolic dysfunction in an energy-independent manner. This brief review summarizes the rationale and development of Parv gene transfer approaches for DHF, and in particular, discusses the divergent effects of Parv isoforms on cardiac myocyte Ca2+ handling and contractile function with the long-range goal of alleviating diastolic dysfunction in DHF.

Novel α-KTx Sites in the BK Channel and Comparative Sequence Analysis Reveal Distinguishing Features of the BK and KV Channel Outer Pore

Abstract: The α-KTx peptide toxins inhibit different types of potassium channels by occluding the outer channel pore composed of four identical α subunits. The large-conductance, calcium-activated (BK or Slo1) and voltage-dependent (KV) potassium channels differ in their specificity for the different α-KTx subfamilies. While many different α-KTx subfamilies of different sizes inhibit KV1 channels with high affinity, only one subfamily, α-KTx 1.x, inhibits BK channels with high affinity. Two solvent-exposed regions of the outer pore that influence α-KTx binding, the turret and loop, display high sequence variability among different potassium channels and may contribute to differences in α-KTx specificity. While these α-KTx domains have been studied in KV1 channels, little is known about the corresponding BK α-KTx domains. To define α-KTx sites in the BK outer pore, we examined the effect of 19 outer pore mutations on specific binding of 125I-labeled iberiotoxion (IbTX or α-KTx 1.3) and on their cell-surface expression. Similar to α-KTx sites in the Shaker KV1 loop, site-directed mutations in the BK loop disrupted specific IbTX binding. In contrast, mutations in the BK turret region revealed three novel α-KTx sites, Q267, N268, and L272, which are distinct from α-KTx sites in the KV1 turret. The BK turret region shows no sequence identity with KV1 and MthK turrets of known 3D structure. To define the BK turret, we used secondary structure prediction methods that incorporated information from sequence alignment of 30 different Slo1 and Slo3 turret sequences from 5 of the 7 major animal phyla representing 27 different species. Results of this analysis suggest that the BK turret contains 18 amino acids and is defined by a cluster of strictly conserved polar residues at the N-terminal side of the turret. Thus, the BK turret is predicted to have six more amino acids than the KV1 turret. Results of this work suggest that BK and KV1 outer pores have a similar α-KTx domain in the loop preceding the inner helix, but that the BK turret comprises a unique α-KTx interaction surface that likely contributes to the exclusive selectivity of BK channels for α-KTx1.x toxins.

Dynamic Effects of Hg2+-induced Changes in Cell Volume

Abstract: Using a microfluidic volume sensor, we studied the dynamic effects of Hg2+ on hypotonic stress-induced volume changes in CHO cells. A hypotonic challenge to control cells caused them to swell but did not evoke a significant regulatory volume decrease (RVD). Treatment with 100 μM HgCl2 caused a substantial increase in the steady-state volume following osmotic stress. Continuous hypotonic challenge following a single 10-min exposure to HgCl2 produced a biphasic volume increase with a steady-state volume 100% larger than control cells. Repeated hypotonic challenges to cells exposed once to Hg2+ resulted in a sequential approach to the same steady-state volume. Stimulation after reaching steady state caused a reduction in peak cell volume. Repeated stimulation was different than continuous stimulation resulting in a more rapid approach to steady state. Substituting extracellular Na+ with impermeant NMDG+ in the hypotonic solution produced a rapid RVD-like volume decrease and eliminated the Hg2+-induced excess swelling. The volume decrease in the presence of Hg2+ was inhibited by tetraethylammonium and 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid disodium, blockers of K+ and Cl− channels, respectively, suggesting that part of the Hg2+ effect was increasing NaCl influx over KCl efflux. The presence of multiple phases of steady-state volume and their sensitivity to the stimulation history suggests that factors beyond solute fluxes, such as modification of mechanical stress within the cytoskeleton also plays a role in the response to hypotonic stress.