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R Lal

Publications and source records attributed to R Lal.

At least 91 records · Page 5Linked to original sources
PubMed1995

Heart gap junction preparations reveal hemiplaques by atomic force microscopy.

Current structural models of gap junctions indicate two apposed plasma membranes with hexagonally packed hemichannels in each membrane aligning end to end. These channels connect the cytoplasms of contacting cells. Images of isolated rat heart gap junctions have been made with the atomic force microscope in aqueous media. We show that native cardiac gap junctions have a thickness of 25 +/- 0.6 nm. This decreases to 17 nm when they are treated with trypsin, which is known to remove some cytoplasmic components of connexin 43. Imaging shows subunits with a center to center spacing of approximately 9-10 nm and long range hexagonal packing, measurements in agreement with studies using freeze-fracture and negative-stain electron microscopy. In addition to gap junctions, we imaged structures that had all the characteristics of native gap junctions except their thickness was limited to 9-11 nm. They also show long range hexagonal packing and center to center spacing of 9-10 nm. These structures decrease in thickness, to 6-9 nm, when treated with trypsin. We have called these structures hemiplaques. They appear to be present endogenously in the preparation, as we have ruled out their being an artifact of imaging by AFM. However, it remains to be determined if they are a consequence of the procedure used in isolating gap junctions or a possible intermediary in gap junction formation.

Animals↗
PubMed1995

Imaging real-time neurite outgrowth and cytoskeletal reorganization with an atomic force microscope.

An atomic force microscope was used to image the morphology and structural reorganization of rat NIH/3T3 fibroblasts and PC-12 cells growing in petri dishes. NIH/3T3 fibroblasts had a uniform morphology and an extensive cytoskeletal network. Cell thickness varied from approximately 2-3 microns above the nucleus to approximately 20-30 nm over the distal processes, and cytoskeletal fibers as small as 30 nm wide were observed. Imaging over an extended period of time showed a limited degree of cytoskeletal reorganization. Localized force dissection did not induce significant retraction of cellular processes and immediate cell death. Differentiating PC-12 cells with a neuronal phenotype had a nonuniform morphology, abundant cytoskeletal elements, neuritic processes, and growth cones. The cell thickness varied from approximately 5-8 microns over the nucleus to approximately 100-500 nm over the neuritic processes; growth cones approximately 50-700 nm wide and end structures approximately 30-150 nm wide were visible. Repeated imaging showed reorganization of the growth cone, especially the appearance and disappearance of beadlike features and fibrous organization. Thus an atomic force microscope can be used for high-resolution real-time studies of the dynamic subcellular mechanisms that drive cell behavior.

3T3 Cells↗
PubMed1995

Dynamic micromechanical properties of cultured rat atrial myocytes measured by atomic force microscopy.

The atomic force microscope (AFM) was used to quantify micromechanical properties (i.e., localized to an area of approximately 0.015 microns 2) of cultured rat atrial myocytes. Quiescent cells in calcium-free solution were quite compressible over the nuclear region, e.g., a force of 3-4 nN produced 180-225 nm cell indentation. Transverse stiffness of quiescent cells increased by approximately 2-fold after an increase in extracellular calcium from 0 to 5 mM and by approximately 16-fold after fixation with Formalin. There was five- to eightfold variation in stiffness of quiescent cells over the cell surface, such that stiffness was lowest over the nuclear region, and it increased toward the cell periphery. These regional variations correlated with the cytoskeletal heterogeneity as revealed by the AFM and fluorescence imaging. Localized contractile activity of beating cells could be monitored in terms of the surface deformation with high transverse spatial (approximately 1-3 nm) and temporal (60-100 microseconds) resolutions. Alterations in cell contractile activity with physiological perturbations and dynamic changes in cell stiffness during a single contraction could be observed. These results demonstrate the feasibility of AFM-based characterization of highly localized cellular micromechanical properties. Relationships among localized cell mechanical behavior and the underlying biochemical and/or structural environment, a crucial aspect in understanding cellular (dys)function, can now be directly examined.

Animals↗
PubMed1995

Subcellular distribution of shear stress at the surface of flow-aligned and nonaligned endothelial monolayers.

The stresses acting on the luminal surface of endothelial cells due to shear flow were determined on a subcellular scale. Atomic force microscopy was used to measure the surface topography of confluent endothelial monolayers cultured under no-flow conditions or exposed to steady shear stress (12 dyn/cm2 for 24 h). Flow over these surface geometries was simulated by computational fluid dynamics, and the distribution of shear stress on the cell surface was calculated. Flow perturbations due to the undulating surface produced cell-scale variations of shear stress magnitude and hence large shear stress gradients. Reorganization of the endothelial surface in response to prolonged exposure to steady flow resulted in significant reductions in the peak shear stresses and shear stress gradients. From the relationship between surface geometry and the resulting shear stress distribution, we have defined a hydrodynamic shape factor that characterizes the three-dimensional morphological response of endothelial cells to flow. The analysis provides a complete description of the spatial distribution of stresses on individual endothelial cells within a confluent monolayer on a scale relevant to the study of physical mechanisms of mechanotransduction.

Animals↗
PubMed1995

Rifamycins: strain improvement program.

Rifamycins are primarily produced by Gram-positive bacterium Amycolatopsis mediterranei, which belongs to the order Actinomycetales. These antibiotics, apart from their application against pathogens of tuberculosis and leprosy, have also been found to be effective against several other pathogens including Mycobacterium avium and Pneumococcus. Because of the importance of rifamycin, the producer strain A. mediterranei has been genetically manipulated since 1957 in order to develop a strain that can either produce larger amounts of rifamycin or derivatives of rifamycin. In this article, the importance of the producer strain, traditional methods (mutations and recombination) of strain improvement, their limitations, and the development of a cloning vector and transformation methods that have made recombinant DNA techniques accessible for genetic manipulations of A mediterranei are discussed.

Actinobacteria↗
PubMed1995

Hepatic artery aneurysm.

A 25-year-old man who presented with recurrent attacks of acute abdominal pain was diagnosed to have recurrent attacks of acute pancreatitis with pseudocyst formation. Laparotomy revealed aneurysm of the hepatic artery which was successfully managed with endoaneurysmorraphy.

Acute Disease↗
PubMed1994

Recent trends in rifamycin research.

Rifamycin is a clinically useful macrolide antibiotic produced by the gram positive bacterium Amycolatopsis mediterranei. This antibiotic is primarily used against Mycobacterium tuberculosis and Mycobacterium leprae, causative agents of tuberculosis and leprosy, respectively. In these bacteria, rifamycin treatment specifically inhibits the initiation of RNA synthesis by binding to beta-subunit of RNA polymerase. Apart from its activity against the bacteria, rifamycin has also been reported to inhibit reverse transcriptase (RT) of certain RNA viruses. Recently, rifamycin derivatives have been discovered that are effective against Mycobacterium avium, which is associated with the AIDS complex. Consequently, the importance of and demand for rifamycin has increased tremendously, the world over. In this article, recent trends in rifamycin research and accessibility of recombinant DNA techniques to increase rifamycin production are reviewed.

AIDS-Related Opportunistic Infections↗
PubMed1994

Atomic force microscopy of freeze-fracture replicas of rat atrial tissue.

Atomic force microscopy (AFM) has provided three-dimensional (3-D) surface images of many biological specimens at molecular resolution. In the absence of spectroscopic capability for AFM, it is often difficult to distinguish individual components if the specimen contains a population of mixed structures such as in a cellular membrane. In an effort to understand the AFM images better, a correlative study between AFM and the well-established technique of transmission electron microscopy (TEM) was performed. Freeze-fractured replicas of adult rat atrial tissue were examined by both TEM and AFM. The same replicas were analysed and the same details were identified, which allowed a critical comparison of surface topography by both techniques. AFM images of large-scale subcellular structures (nuclei, mitochondria, granules) correlated well with TEM images. AFM images of smaller features and surface textures appeared somewhat different from the TEM images. This presumably reflects the difference in the surface sensitivity of AFM versus TEM, as well as the nature of images in AFM (3-D surface contour) and TEM (2-D projection). AFM images also provided new information about the replica itself. Unlike TEM, it was possible to examine both sides of the replica with AFM; the resolution on one side was significantly greater compared with the other side. It was also possible to obtain quantitative height information which is not readily available with TEM.

Animals↗
PubMed1994

Biological applications of atomic force microscopy.

The newly developed atomic force microscope (AFM) provides a unique window to the microworld of cells, subcellular structures, and biomolecules. The AFM can image the three-dimensional structure of biological specimens in a physiological environment. This enables real-time biochemical and physiological processes to be monitored at a resolution similar to that obtained for the electron microscope. The process of image acquisition is such that the AFM can also measure forces at the molecular level. In addition, the AFM can interact with the sample, thereby manipulating the molecules in a defined manner--nanomanipulation! The AFM has been used to image living cells and the underlying cytoskeleton, chromatin and plasmids, ion channels, and a variety of membranes. Dynamic processes such as crystal growth and the polymerization of fibrinogen and physicochemical properties such as elasticity and viscosity in living cells have been studied. Nanomanipulations, including dissection of DNA, plasma membranes, and cells, and transfer of synthetic structures have been achieved. This review describes the operating principles, accomplishments, and the future promise of the AFM.

Animals↗
PubMed1994

Shear stress-induced reorganization of the surface topography of living endothelial cells imaged by atomic force microscopy.

We report the first topographical data of the surface of living endothelial cells at sub-light-microscopic resolution, measurements essential for a detailed understanding of force distribution in the endothelium subjected to flow. Atomic force microscopy was used to observe the surface topography of living endothelial cells in confluent monolayers maintained in static culture or subjected to unidirectional shear stress in laminar flow (12 dyne/cm2 for 24 hours). The surface of polygonal unsheared cells was smooth, with mean excursion of surface undulation between peak height (over the nucleus) and minima (at intercellular junctions) of 3.4 +/- 0.7 microns (mean +/- SD); the mean height to length ratio was 0.11 +/- 0.02. In cells that were aligned in the direction of flow after a 24-hour exposure to laminar shear stress, height differentials were significantly reduced (mean, 1.8 +/- 0.5 micron), and the mean height to length ratio was 0.045 +/- 0.009. Calculation of maximum shear stress and maximum gradient of shear stress (delta tau/delta x, where tau is shear stress at the cell surface) at constant flow velocity revealed substantial streamling of aligned cells that reduced delta tau/delta x by more than 50% at a nominal shear stress of 10 dyne/cm2. Aligned cells exhibited ridges extending in the direction of flow that represented imprints of submembranous F-actin stress-fiber bundles mechanically coupled to the cell membrane. The surface ridges, approximately 50 nm in height and 200 to 1000 nm in width, were particularly prominent in the periphery of the aligned cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Atomic force microscopy of cloned nicotinic acetylcholine receptor expressed in Xenopus oocytes.

The nicotinic acetylcholine receptor (AChR) was expressed in Xenopus oocytes from in vitro transcribed mRNA and was imaged by atomic force microscopy. A characteristic pentameric structure of AChR was readily observed on the extracellular face of the cell membrane, with a central pore surrounded by protruding AChR subunits. These structures were seen only in mRNA-injected oocytes that also gave acetylcholine-induced membrane currents. The size of individual AChR channels, the angles between subunits, and the interchannel spacing were all compatible with the current model of AChR. In addition, localized patches of microscopic AChR clustering were observed, with packing density approaching that at the neuromuscular junction. These findings show the potential of studying cloned membrane proteins in oocytes for both their surface topography and their structure-function relationship in native membrane without the need for crystallization.

Animals↗
PubMed1993

Antibody perturbation analysis of gap-junction permeability in rat cardiac myocytes.

We have used site-directed antibodies against various segments of the connexin43 (Cx43) gap-junction protein in an attempt to explore the role of different portions of this molecule in regulating junctional permeability. The antibodies used in the present study were raised against epitopes exposed at the cytoplasmic face of the junctions, specifically the amino (AT-2) and carboxy (CT-360) termini and the cytoplasmic loop (CL-100) of Cx43. Neonatal rat cardiac myocytes, which are known to express Cx43, were microinjected with a series of anti-Cx43 antibodies, followed by Lucifer yellow. The extent of cell coupling was quantified as the percentage of instances of intercellular transfer of the dye. The effectiveness of the AT-2 and CT-360 antibodies varied strongly and differentially with the external calcium concentration. In the absence of antibody, the dye permeability was unaffected by calcium. In medium containing physiological concentrations of calcium, the antibodies inhibited dye transfer to different degrees: AT-2 and CT-360 antibodies inhibited well while the CL-100 antibody had very little effect on dye permeability. Our results indicate that several highly conserved cytoplasmic domain of Cx43 could be involved in regulating junctional permeability, and that calcium modulates the effect of antibodies on junctional permeability.

Animals↗
PubMed1993

Imaging of reconstituted biological channels at molecular resolution by atomic force microscopy.

Using atomic force microscopy (AFM), we obtained high-resolution surface images of the bacterial outer membrane channels Escherichia coli OmpF porin and Bordetella pertussis porin that were reconstituted in artificial bilayer membranes as two-dimensional crystalline arrays. These porins were chosen because they are among the most extensively studied proteins of this type and are known for their well-defined crystalline nature in the native membrane. Such reconstituted membrane proteins are ideal specimens to assess the suitability and resolution of AFM for imaging biomembranes and associated proteins. Although OmpF porin often showed a mixed pattern of rectangular and hexagonal arrays with approximately 8.4 x 9.8- and approximately 7.2-nm-spacings, respectively, B. pertussis porin showed mostly a rectangular pattern with an approximately 7.9 x 13.8-nm spacing. The packing patterns of the E. coli OmpF porin in the membrane are very close to those found in electron-microscopic studies. When B. pertussis porin was imaged in a buffer solution, its trimeric subunits were apparently resolved, and the surface of each monomer revealed beadlike structures. This is the first report of such a high-resolution structural analysis of B. pertussis porin by any imaging method. We also imaged the lipid bilayer itself as an internal control for imaging and to further ascertain the resolution. Individual polar head groups of bilayer lipid molecules were resolved, suggesting the intrinsic resolution of AFM for bioimaging.

Bordetella pertussis↗