Search PubMed⌕ Search

Biomedical subjects

C Pasternak

Publications and source records attributed to C Pasternak.

16 recordsLinked to original sources
PubMed1999

Thioredoxin is involved in oxygen-regulated formation of the photosynthetic apparatus of Rhodobacter sphaeroides.

Thioredoxin, a redox active protein, has been previously demonstrated to be essential for growth of the anoxygenic photosynthetic bacterium Rhodobacter sphaeroides. In the present study, the involvement of thioredoxin in the formation of the photosynthetic apparatus of R. sphaeroides WS8 was investigated by construction and analysis of a mutant strain disrupted for the chromosomal trxA copy and carrying a plasmid-borne copy of trxA under the control of the hybrid ptrc promoter inducible by IPTG (isopropyl-beta-D-thiogalactopyranoside). This strain was viable in the absence of IPTG but was affected in pigmentation. When shifted from high to low oxygen tension conditions, the trxA mutant showed a reduced bacteriochlorophyll content in comparison to that of the wild type. Although thioredoxin is able to regulate aminolevulinic acid (ALA) synthase (the first enzyme of the tetrapyrrole biosynthetic pathway) activity by a dithiol-disulfide exchange, our mutant strain exhibited a level of ALA synthase activity identical to that of the wild type, suggesting that thioredoxin is involved in other steps to regulate the synthesis of the photosynthetic apparatus. Accordingly, we showed that the trxA mutation affects the oxygen-regulated expression of the puf operon encoding the pigment-binding proteins of the light-harvesting and reaction center complexes. Upon transition from aerobic to semiaerobic growth conditions, the maximal puf mRNA level was found to be 40 to 50% lower in the mutant strain than in the wild type. The stability of the puf transcripts was identical in both strains grown under low oxygen tension, indicating that the role of thioredoxin in regulating puf expression occurs at the transcriptional level.

5-Aminolevulinate Synthetase↗
PubMed1999

Forces in cell locomotion.

The molecular mechanisms that drive animal cell locomotion are partially characterized, but not definitively understood. It seems likely that actin polymerization contributes to the forward protrusion of the leading edge of a migrating cell. Both myosin-dependent contractile forces and selective detachment of adhesive interactions with the substratum seem to contribute to release of the posterior of an extended cell. It is probable, but not certain, that a separate 'traction' force advances the cell body towards the forward anchorage sites formed by the advancing lamellipodium. The molecular mechanism of this force is unknown. Determining the role of traction forces in migrating fibroblasts and keratocytes is complicated by the fact that the primary functions of the relatively strong forces exerted on the substratum by these cells may be to establish tissue 'tone' and to remodel tissue matrices, rather than to drive locomotion. In accordance with this notion, rapidly moving cells such as neutrophils and Dictyostelium amoebae exert weaker forces on the substratum as they migrate. The traction force in cell migration may be distinct from traction forces with tissue functions. Ultimately, the mechanism may be revealed by using molecular genetics to disrupt the motors that provide this force. Reconstituted tissues provide systems in which to investigate the regulation of cell forces and their contribution to tissue mechanical properties and development.

Actins↗
PubMed1998

Molecular cloning and expression analysis of the Rhodobacter capsulatus sodB gene, encoding an iron superoxide dismutase.

Genetic complementation of a sodA sodB Escherichia coli mutant strain was used to clone Rhodobacter capsulatus genes involved in detoxification of superoxide radicals. After sequence analysis, 1 of the 16 identical clones obtained by this selection procedure was shown to contain an open reading frame with sequence similarity to that coding for Fe-containing superoxide dismutases (SodB). The R. capsulatus sodB gene was expressed in E. coli, and the nature of the metal ligand was confirmed by inhibitor sensitivity assays with lysates from both bacterial species. Activity staining of cleared Rhodobacter lysates resolved by polyacrylamide gel electrophoresis indicated that SodB was the only superoxide dismutase present in this phototrophic organism. The sodB gene was expressed at low levels in R. capsulatus cells grown under anaerobic or semiaerobic conditions, but expression was strongly induced upon exposure of the bacteria to air or to methyl viologen. Attempts to construct a sodB mutant in this organism by allelic exchange of the chromosomal copy of the gene with a suicide plasmid containing a mutated sodB gene were unsuccessful, strongly suggesting that the encoded superoxide dismutase is essential for viability of R. capsulatus in aerobic cultures.

Aerobiosis↗
PubMed1996

Cloning, nucleotide sequence and characterization of the rpoD gene encoding the primary sigma factor of Rhodobacter capsulatus.

A synthetic oligodeoxynucleotide probe based on a highly conserved region of the sigma factors was used to identify and clone the rpoD gene encoding the principal sigma factor of R. capsulatus. The deduced polypeptide contains 674 amino acids and has a predicted molecular mass of 75,942 Da. The deduced amino acid sequence of R. capsulatus RpoD protein exhibits 46.2% and 45.7% identity with housekeeping sigma factors of Pseudomonas and E. coli, respectively. Unsuccessful attempts to inactivate the single chromosomal rpoD gene of R. capsulatus by gene replacement technique indicate that this gene is essential for cell survival, as expected for the primary sigma factor. The rpoD transcript 5'-end was mapped by primer extension analysis, 74 bp upstream of the initiation codon and DNA sequence analysis has identified a motif resembling the delta 70 E. coli consensus promoter sequences at the expected distance from this proposed transcription start site. rpoD gene expression, as measured by the activity of the phi (rpoD'-lacZ+) translational fusion, was found to be constant throughout exponential and early plateau phases, but significantly increased at later times of the stationary phase.

Amino Acid Sequence↗
PubMed1996

Expression of the thioredoxin gene (trxA) in Rhodobacter sphaeroides Y is regulated by oxygen.

The structural gene (trxA) coding for thioredoxin in the photosynthetic bacterium Rhodobacter sphaeroides has been cloned and sequenced previously. In the present study, the role of oxygen in trxA expression in R. sphaeroides Y was investigated using mRNA analyses and plasmid-borne trxA'-lacZ+ translational and transcriptional fusions. Northern analysis revealed a trxA-specific transcript of approximately 420-460 nucleotides, indicating that trxA is transcribed as a single gene. By studying the beta-galactosidase activity in strains harboring various phi(trxA'-lacZ+) fusion constructs, the promoter region of the trxA gene was localized within a 64-bp region located 97 nucleotides upstream of the trxA initiator codon. A single trxA transcription initiation site was mapped by primer extension, 27 bp upstream of the trxA gene. Based on these results and the DNA sequence analysis, we propose that a sigma70 consensus sequence serves as a trxA promoter. Results from oxygen shift experiments, as deduced from both mRNA analysis and fusions of the trxA promoter region to lacZ indicate that transcription of the R. sphaeroides trxA gene is regulated by high oxygen tension. DNA sequences involved in this oxygen regulation were also localized in the 64-bp region containing the trxA promoter. Based on our findings the hypothetical biological function of thioredoxin from R. sphaeroides is discussed.

Base Sequence↗
PubMed1995

Mechanical function of dystrophin in muscle cells.

We have directly measured the contribution of dystrophin to the cortical stiffness of living muscle cells and have demonstrated that lack of dystrophin causes a substantial reduction in stiffness. The inferred molecular structure of dystrophin, its preferential localization underlying the cell surface, and the apparent fragility of muscle cells which lack this protein suggest that dystrophin stabilizes the sarcolemma and protects the myofiber from disruption during contraction. Lacking dystrophin, the muscle cells of persons with Duchenne muscular dystrophy (DMD) are abnormally vulnerable. These facts suggest that muscle cells with dystrophin should be stiffer than similar cells which lack this protein. We have tested this hypothesis by measuring the local stiffness of the membrane skeleton of myotubes cultured from mdx mice and normal controls. Like humans with DMD mdx mice lack dystrophin due to an x-linked mutation and provide a good model for the human disease. Deformability was measured as the resistance to indentation of a small area of the cell surface (to a depth of 1 micron) by a glass probe 1 micron in radius. The stiffness of the membrane skeleton was evaluated as the increment of force (mdyne) per micron of indentation. Normal myotubes with an average stiffness value of 1.23 +/- 0.04 (SE) mdyne/micron were about fourfold stiffer than myotubes cultured from mdx mice (0.34 +/- 0.014 mdyne/micron). We verified by immunofluorescence that both normal and mdx myotubes, which were at a similar developmental stage, expressed sarcomeric myosin, and that dystrophin was detected, diffusely distributed, only in normal, not in mdx myotubes. These results confirm that dystrophin and its associated proteins can reinforce the myotube membrane skeleton by increasing its stiffness and that dystrophin function and, therefore, the efficiency of therapeutic restoration of dystrophin can be assayed through its mechanical effects on muscle cells.

Animals↗
PubMed1993

Studies of mechanical aspects of amoeboid locomotion.

When a cell crawls over a surface, it exerts forces which both change its shape and deformability and propel it forward. The mechanisms involved are poorly understood. They can best be studied by combining biochemical and molecular genetic methods with direct, quantitative measurements of mechanical properties. Measurements of cellular deformability provide indications of contractile tension developed within the cell and of cytoskeletal reorganizations which influence local cellular viscoelasticity. An example is the capping of cross-linked cell surface proteins, which occurs on cells as diverse as mammalian lymphocytes and the unicellular amoeba, Dictyostelium discoideum. Deformability measurements show that cells stiffen as they cap. Measurements on wild-type Dictyostelium cells and on cells engineered to lack conventional myosin (myosin II) demonstrate that capping requires myosin II and that the concurrent cellular stiffening results from a myosin-II-dependent contractile force. Measurements of the systematic transport of beads rearward over the surfaces of cells characterize a mechanism of movement which could be used to drive the cell forward. Capping is one such mechanism. A distinct myosin-II-independent form of rearward transport is revealed in measurements of fluorescent beads on the Dictyostelium cells which lack this protein. In addition to studies of cell locomotion, measurements of cellular mechanical properties can provide quantitative assays of the functions of cytoskeletal components. Such studies are motivated by the nature of cytoskeletal proteins whose function, in contrast to enzymes, are mechanical rather that catalytic.

Animals↗
PubMed1990

Activation of mechanical responses in leukocytes.

Different kinds of leukocytes undergo cytoskeleton-dependent mechanical responses associated with their specific physiological functions. We have investigated cellular stiffening of several types of leukocytes using a method which measures the force resisting cellular indentation. We have found that lymphocytes stiffen in response to crosslinking cell surface antigens in a process associated with the much studied capping and patching processes. Further studies of myosin-deficient mutants of the ameba Dictyostelium discoideum suggest that this stiffening process results from a myosin dependent contractile process. Rat basophilic leukemia cells and pancreatic islet cells stiffen when triggered to secrete. The function of these cytoskeleton dependent processes is now unknown, but, at least in the islet cells, may be related to a regulation of the rate of secretion. Primary neutrophils stiffen in response to the chemotactic agent, fMet-Leu-Phe. This stiffening may be responsible for retention of these cells in the pulmonary microcirculation during response to inflammation. These observations pose the challenge of determining the structural basis, mechanism, and physiological function of each of these cellular responses.

Animals↗
PubMed1990

Protection against membrane-mediated cytotoxicity by calcium and zinc.

Injection of S. aureus alpha toxin or ultraviolet-inactivated Sendai virus into the mammary gland of lactating mice leads to cytopathic changes in the alveolar cells compatible with a breakdown of their permeability barrier. Simultaneous administration of Ca2+ or Zn2+ prevents such changes, as it does when added to alveolar cells in vitro. It is concluded that increased levels of Zn2+ (and under certain conditions of Ca2+) may exert a protective role that is clinically significant.

Animals↗
PubMed1989

Capping of surface receptors and concomitant cortical tension are generated by conventional myosin.

We have investigated the role of cytoskeletal contraction in the capping of surface proteins crosslinked by concanavalin A on mutant Dictyostelium cells lacking conventional myosin. Measurements of cellular deformability to indicate the development of cortical tension show that cells of the wild-type parental strain, AX4, stiffen early during capping and relax back towards the softer resting state as the process is completed. Mutant cells lacking myosin (mhcA-) have a lower resting-state stiffness, and fail to stiffen and to cap crosslinked proteins on binding concanavalin A. Hence conventional myosin is essential both for capping and for the concomitant increase in cell stiffness. Furthermore, depletion of cellular ATP by azide causes a 'rigor' contraction in AX4 cells which makes them stiffen and become spherical. By contrast, the mhcA- cells fail to respond in these ways. These measurements of cortical tension in non-muscle cells can thus be directly correlated with the presence of conventional myosin, demonstrating that contractile tension generated by myosin can drive both a change of cell shape and the capping of crosslinked surface receptors.

Concanavalin A↗
PubMed1989

Intermolecular versus intramolecular interactions of Dictyostelium myosin: possible regulation by heavy chain phosphorylation.

Dictyostelium myosin has been examined under conditions that reveal intramolecular and intermolecular interactions that may be important in the process of assembly and its regulation. Rotary shadowed myosin molecules exhibit primarily two configurations under these conditions: straight parallel dimers and folded monomers. All of the monomers bend in a specific region of the 1860-A-long tail that is 1200 A from the head-tail junction. Molecules in parallel dimers are staggered by 140 A, which is a periodicity in the packing of myosin molecules originally observed in native thick filaments of muscle. The most common region for interaction in the dimers is a segment of the tail about 200-A-long, extending from 900 to 1100 A from the head-tail junction. Parallel dimers form tetramers by way of antiparallel interactions in their tail regions with overlaps in multiples of 140 A. The folded configuration of the myosin molecules is promoted by phosphorylation of the heavy chain by Dictyostelium myosin heavy chain kinase. It appears that the bent monomers are excluded from filaments formed upon addition of salt while the dimeric molecules assemble. These results may provide the structural basis for primary steps in myosin filament assembly and its regulation by heavy chain phosphorylation.

Dictyostelium↗
PubMed1985

Lymphocyte mechanical response triggered by cross-linking surface receptors.

Using a recently developed method (Petersen, N. O., W. B. McConnaughey, and E. L. Elson, 1982, Proc. Natl. Acad. Sci. USA., 79:5327-5331), we have measured changes in the deformability of lymphocytes triggered by cross-linking cell surface proteins. Our study was motivated by two previously demonstrated phenomena: the redistribution ("capping") of cross-linked surface immunoglobulin (sIg) on B lymphocytes and the inhibition of capping and lateral diffusion ("anchorage modulation") of sIg by the tetravalent lectin Concanavalin A (Con A). Both capping and anchorage modulation are initiated by cross-linking cell surface proteins and both require participation of the cytoskeleton. We have shown that the resistance of lymphocytes to deformation strongly increased when sIg or Con A acceptors were cross-linked. We have measured changes in deformability in terms of an empirical "stiffness" parameter, defined as the rate at which the force of cellular compression increases with the extent of compression. For untreated cells the stiffness was approximately 0.15 mdyn/micron; for cells treated with antibodies against sIg or with Con A the stiffness increased to approximately 0.6 or 0.4 mdyn/micron, respectively. The stiffness decreased after completion of the capping of sIg. The increases in stiffness could be reversed to various extents by cytochalasin D and by colchicine. The need for cross-linking was demonstrated by the failure both of monovalent Fab' fragments of the antibodies against sIg and of succinylated Con A (a poor cross-linker) to cause an increase in stiffness. We conclude that capping and anchorage modulation involve changes in the lymphocyte cytoskeleton and possibly other cytoplasmic properties, which increase the cellular viscoelastic resistance to deformation. Similar increases in cell stiffness could be produced by exposing cells to hypertonic medium, azide ions, and to a calcium ionophore in the presence of calcium ions. These results shed new light on the capabilities of the lymphocyte cytoskeleton and its role in capping and anchorage modulation. They also demonstrate that measurements of cellular deformability can characterize changes in cytoskeletal functions initiated by signals originating at the cell surface.

Animals↗
PubMed1984

Cross-linking surface immunoglobulin increases the stiffness of lymphocytes.

Cross-linking surface immunoglobulin (sIg) on B-lymphocytes causes a substantial increase in the mechanical stiffness of the cells. This has been demonstrated using a new method for measuring cellular deformability. The method is based on a device, the "Cell Poker", which we use to determine the force required slightly to indent or compress a cell adherent to a rigid substrate in culture. Cross-linking of sIg by bivalent anti-sIg antibodies is necessary to elicit the increase in stiffness; binding of monovalent Fab fragments is insufficient. The increase in stiffness is partially reversed by cytochalasin D and by completion of the capping of the cross-linked sIg. The modulation of cellular deformability and the induction of cellular dynamic processes such as capping are similar in their requirements for cross-linking sIg and in their sensitivity to cytochalasins. This suggests that both kinds of responses stem from similar cellular processes and structures. These results emphasize the mechanical capability of lymphocytes and suggest that the physiological functions of these cells are likely to employ this capability.

Animals↗
PubMed1982

Increase in lipid microviscosity of unilamellar vesicles upon the creation of transmembrane potential.

Diffusion potential of potassium ions was found in unilamellar vesicles of phosphatidyl choline. The vesicles, which included potassium sulfate buffered with potassium phosphate were diluted into an analogous salt solution made of sodium sulfate and sodium phosphate. The diffusion potential was created by the addition of the potassium-ionophore, valinomycin. The change in lipid microviscosity, ensuing the formation of membrane potential, was measured by the conventional method of fluorescence depolarization with 1,6-diphenyl-1,3,5-hexatriene as a probe. Lipid microviscosity was found to increase with membrane potential in a nonlinear manner, irrespective of the potential direction. Two tentative interpretations are proposed for this observation. The first assumes that the membrane potential imposes an energy barrier on the lipid flow which can be treated in terms of Boltzmann-distribution. The other interpretation assumes a decrease in lipid-free volume due to the pressure induced by the electrical potential. Since increase in lipid viscosity can reduce lateral and rotational motions, as well as increase exposure of functional membrane proteins, physiological effects induced by transmembrane potential could be associated with such dynamic changes.

Liposomes↗
PubMed1978

Bacteriorhodopsin: lipid environment and conformational changes.

The polar lipids of the purple membrane were exchanged for different phosphatidylcholine species. The resulting complexes had the same protein to lipid-phosphorus ratio as the natural membrane, but only about 0.5-1.0 mole of original lipid was still present per mole of bacteriorhodopsin. In such complexes the bacteriorhodopsin photocycle is slowed down 10-20 times, but the strong protein-protein interaction is not abolished. Due to the slow rate of the photocycle we were able to measure in the light the ratio between net proton release and net accumulation of the last intermediate of the photocycle, the unprotonated M412. This ratio was not constant and equal to 1.0, as expected for a single deprotonation reaction, but varied with pH from 1.5 to 0.4. The variable ratio suggests that light-induced conformational changes occur in the nonchromophore part of the protein, which shift the pKa values of unidentified groups so as to cause binding or release of additional protons. A similar conclusion was drawn from experiments on the kinetics of proton transfer by bacteriorhodopsin in subbacterial particles of Halobacterium halobium and in reconstituted bacteriorhodopsin proteoliposomes. However, in this case light-induced association and dissociation of additional protons occurs simultaneously on different sides of the membrane.

Bacteriorhodopsins↗