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Biomedical subjects

H Shuman

Publications and source records attributed to H Shuman.

At least 19 recordsLinked to original sources
PubMed1992

Saturable binding of halothane to rat brain synaptosomes.

The hypothesis that volatile anesthetics act directly on or bind specifically to membrane proteins remains controversial. In earlier in situ electron probe microanalysis studies in cardiac muscle we showed preferential partitioning of halothane into mitochondria. To determine whether partitioning represents saturable binding or simple solubility, a photoaffinity labeling method was developed for halothane to examine binding in rat brain synaptosomes. Radioligand binding assays were then used to determine binding parameters for this important inhalational anesthetic. UV-light exposure of synaptosomes incubated with clinical concentrations of [14C]halothane resulted in sufficient labeling to allow characterization of binding sites. Analysis of saturation and competition curves showed that greater than 60% of [14C]halothane photolysis product binding to synaptosomes was specific, with low affinity (Kd = 0.49 +/- 0.16 mM) and high binding site concentration (Bmax = 1.87 +/- 0.75 nmol/mg of protein). Halothane photoaffinity labeling was partially inhibited by isoflurane (20%), chloroform (44%), 2-bromotrifluoroethane (20%), and dichlorotrifluoroethane (20%) but not by ethanol. The Kd measured with this photoaffinity approach is similar to the concentration of halothane required to produce anesthesia in rats.

Affinity Labels↗
PubMed1992

Incorporation of fluorescently labeled contractile proteins into freshly isolated living adult cardiac myocytes.

When fluorescently labeled contractile proteins are injected into embryonic muscle cells, they become incorporated into the cells' myofibrils. In order to determine if this exchange of proteins is unique to the embryonic stage of development, we isolated adult cardiac myocytes and microinjected them with fluorescently labeled actin, myosin light chains, alpha-actinin, and vinculin. Each of these proteins was incorporated into the adult cardiomyocytes and was colocalized with the cells' native proteins, despite the fact that the labeled proteins were prepared from noncardiac tissues. Within 10 min of injection, alpha-actinin was incorporated into Z-bands surrounding the site of injection. Similarly, 30 sec after injection, actin was incorporated into the entire I-bands at the site of injection. Following a 3-h incubation, increased actin fluorescence was noted at the intercalated disc. Vinculin exchange was seen in the intercalated discs, as well as in the Z-bands throughout the cells. Myosin light chains required 4-6 h after injection to become incorporated into the A-bands of the adult muscle. Nonspecific proteins, such as fluorescent BSA, showed no association with the myofibrils or the former intercalated discs. When adult cells were maintained in culture for 10 days, they retain the ability to incorporate these contractile proteins into their myofibrils. T-tubules and the sarcoplasmic reticulum could be detected in periodic arrays in the freshly isolated cells using the membrane dye WW781 and DiOC6[3], respectively. In conclusion, the myofibrils in adult, as in embryonic, muscle cells are dynamic structures, permitting isoform transitions without dismantling of the myofibrils.

Actinin↗
PubMed1991

Energy-loss near-edge fine structure and compositional profiles of cryomilled oxide-dispersion-strengthened aluminum.

Spatially resolved electron energy-loss spectroscopy (SREELS) makes possible the identification of small second-phase particles in an oxide-dispersion-strengthened aluminum alloy produced by mechanical alloying in a liquid-nitrogen slurry. Near-edge fine structure profiles based on aluminum L23 features that are characteristic of Al-Al or Al-O bonding yield the metal/oxide fraction as a function of probe position. Profiles of nitrogen fraction are derived from linear sequences of spectra so that detection limits can be analyzed at each point in a scan. Quantitative analysis of these linear profiles shows that particles with diameters in the range 10 to 30 nm are rich in nitrogen. A combination of statistical analysis of background removal results with visual inspection of derivative spectra is used to estimate detection limits.

Aluminum↗
PubMed1990

Cloning of cDNA for proteinase 3: a serine protease, antibiotic, and autoantigen from human neutrophils.

Closely similar but nonidentical NH2-terminal amino acid sequences have been reported for a protein or proteins in human neutrophils whose bioactivities is/are diverse (as a serine protease, antibiotic, and Wegener's granulomatosis autoantigen) but that share(s) several features: localization in the azurophil granules, a molecular mass of approximately 29 kD, reactivity with diisopropylfluorophosphate, and the ability to degrade elastin. We previously purified one such entity, termed p29b. Using a monospecific antibody, we have cloned from human bone marrow a cDNA encoding the complete p29b protein in its mature form, along with pre- and pro-sequences. The predicted amino acid sequence agrees closely with the NH2-terminal sequence obtained previously from purified p29b, as well as with sequences newly obtained from CNBr fragments. The primary structure is highly homologous to elastase, cathepsin G, T cell granzymes, and other serine proteases, and shares both the catalytic triad and substrate binding pocket of elastase. Hybridization of the full-length cDNA with restriction enzyme digests of human genomic DNA revealed only one fragment. This suggests that the closely related species described previously are the same, and can be subsumed by the term used for the first-described activity, proteinase 3. Proteinase 3 is more abundant in neutrophils than elastase and has a similar proteolytic profile and specific activity. Thus, proteinase 3 may share the role previously attributed to neutrophil elastase in tissue damage, and has the potential to function as an antimicrobial agent.

Amino Acid Sequence↗
PubMed1990

Aggregation of dispersed human cytotrophoblastic cells: lessons relevant to the morphogenesis of the placenta.

The syncytial trophoblast of the human placenta forms by the fusion of mononuclear cytotrophoblast cells. Cytotrophoblast cells only fuse with other trophoblastic cells, indicating a specificity to this interaction. To explore the cellular aggregation which precedes fusion, we examined the association of cytotrophoblast cells isolated from term placentae and JEG-3 choriocarcinoma cells, a cytotrophoblast-like cell line, in suspension culture. Cytotrophoblast cells were isolated by dispersion of chorionic villi in trypsin-DNase in Ca2+/Mg2(+)-free medium. JEG-3 cells were released from culture flasks by trypsinization in Versene-EDTA buffer. In suspension culture, each cell type aggregated forming tissue-like masses over a 24-hr period. Transmission electron microscope analysis demonstrated the formation of numerous desmosomes between the aggregated cells. In outgrowth culture, the aggregates created in suspension were maintained as microvilli-covered multicellular structures with hollow cores. The extent of aggregation was dependent upon the concentration of cells in the incubations with greater aggregation occurring with higher cell densities. Aggregation of both cytotrophoblast cells and JEG-3 cells progressed rapidly during the initial 10 hr of incubation and then continued at a slower rate. Aggregation took place in serum-containing and serum-free medium, but was impeded in Ca2+/Mg2(+)-free medium. Incubation of JEG-3 and cytotrophoblast cells in the presence of the protein synthesis inhibitor, cycloheximide, prevented aggregation, whereas the inhibitor of N-linked glycosylation, tunicamycin, did not. The inhibitor of RNA synthesis, actinomycin D, had no effect on the aggregation of the cells during the initial 6 hr of aggregation. These findings suggest that trypsin treatment in Ca2+/Mg2(+)-poor medium removed a protein(s) from the trophoblast cell surface which must be resynthesized for cell-cell association to take place.

Cell Aggregation↗
PubMed1990

Subcellular distribution of an inhalational anesthetic in situ.

To better understand the mechanisms and sites of anesthetic action, we determined the subcellular partitioning of halothane in a tissue model. A method was found to fix the in vivo distribution of halothane in rat atrial tissue for subsequent electron microscopy and x-ray microanalysis. Atrial strips were exposed to various concentrations of halothane, rapidly frozen, cryo-sectioned, and cryo-transferred into an electron microscope. Irradiation of the hydrated cryosections with the electron beam caused halothane radiolysis, which allowed retention of the halogen-containing fragments after dehydration of the sections. The bromine from halothane was detected and quantified with x-ray microanalysis in various microregions of atrial myocytes. Halothane (bromine) partitioned largely to mitochondria, with progressively lower concentrations in sarcolemma, nuclear membrane, cytoplasm, sarcomere, and nucleus. Partitioning could not be explained solely by distribution of cellular lipid, suggesting significant and differential physicochemical solubility in protein. However, we found no saturable compartment in atrial myocytes within the clinical concentration range, which implies little specific protein binding.

Animals↗
PubMed1989

Genetics and molecular pathogenesis of Legionella pneumophila, an intracellular parasite of macrophages.

In addition to providing a powerful approach for identifying bacterial factors required for full infectivity and disease production, genetic analysis of Legionella pathogenesis should also lend critical insight into the biology of the macrophage and into the pathogenesis of other intracellular parasites. The interaction between L. pneumophila and the macrophage exhibits many features found in a wide variety of prokaryotic and eukaryotic intracellular human pathogens. For example, binding to complement receptors has been shown to occur for Mycobacterium tuberculosis, M. leprae, Leishmania donovani, Leishmania major and Histoplasma capsulatum. Coiling phagocytosis has been observed during entry of L. donovani. Phagosomes that contain Toxoplasma gondii or M. tuberculosis fail to fuse with lysosomes and, in the case of T. gondii, have been shown to remain close to neutral pH. Although the molecular bases for these phenomena are unknown, their functional similarities to the L. pneumophila-macrophage interaction provide optimism that generally applicable principles are involved. The genetic techniques reviewed here will provide the molecular tools with which such questions of a general biologic nature can be framed and eventually answered. Together with more traditional methods in biochemistry, microbiology and cell biology, molecular genetics offers a robust means toward identifying and understanding the bacterial factors involved in the pathogenesis of Legionnaires' disease. Molecular studies of L. pneumophila can also help address questions concerning the epidemiology, diagnosis and prevention of disease. For example, the distribution of virulence factors might help explain and predict the attack rates of different L. pneumophila strains or Legionella species. Moreover, bacterial genes/factors that are shown to be conserved in Legionella strains could be used to develop such diagnostic tools as DNA probes. Novel types of vaccines consisting of genetically constructed, avirulent L. pneumophila strains or subunit vaccines based on the molecular characterization of virulence factors might be developed and tested as protective immunogens. In this way, the capacity to analyze and to manipulate L. pneumophila genetically may facilitate the use of Legionnaires' disease as a model infection for studying protective cell-mediated immunity. Apart from its clinical significance as the etiologic agent of Legionnaires' disease, L. pneumophila may be a key to broader understandings in microbial pathogenesis and human cell biology and immunology. Although the extremely complex processes of bacterial infection and virulence are best understood when a variety of experimental approaches are employed, we believe that the evolving molecular genetic techniques reviewed here will be critical elements in many important breakthroughs in the future.

Animals↗
PubMed1988

Atrial-specific granules in situ have high calcium content, are acidic, and maintain anion gradients.

The composition and pH of atrial-specific granules of rat heart were determined by electron probe x-ray microanalysis and fluorescence microscopy, respectively. The high (75 mmol/kg of dry weight) calcium content and higher than cytoplasmic concentration of chloride in atrial-specific granules were visualized in high-resolution x-ray maps. The Cl- content of granules and cytoplasm decreased and a bromide gradient (granule greater than cytoplasm) was established during incubation in low-chloride, NaBr-containing solutions. Scanning confocal fluorescence light microscopy of live atria incubated with acridine orange demonstrated dye accumulation, indicative of low intragranular pH. We conclude that the granules represent a hitherto unrecognized intracellular store of cardiac calcium and can develop and maintain an anion gradient, presumably through cotransport by means of a proton-pumping ATPase.

Animals↗
PubMed1988

Contrast in confocal scanning microscopy with a finite detector.

The optical properties of a general scanning microscope are determined within the framework of Fourier imaging theory. For a simple model optical system, with Gaussian lens and detector apertures, the contrast transfer function can be expressed in terms of elementary functions. The theory predicts that spatial resolution and depth discrimination vary continuously with detector aperture and that defocus phase contrast is present in transmission images obtained with a symmetric objective, collector lens confocal microscope.

Fourier Analysis↗
PubMed1987

Electron energy loss analysis of near-trace-element concentrations of calcium.

The quantitation of near-trace-element concentrations of calcium (25 ppm atomic fraction) with electron energy loss spectroscopy (EELS) is demonstrated. The data collection, with an energy-stabilized parallel recording spectrometer, subsequent signal processing, and quantitation procedures are described. The quantitative results obtained with EELS, in the biologically relevant range of 1 to 100 mmol/kg, are directly compared with simultaneously collected and previously validated energy-dispersive X-ray spectroscopy (EPMA). The experimentally determined sensitivity of EELS for Ca detection is five-fold better than for EPMA, and the theoretically attainable sensitivity of EELS is ten-fold better than for EPMA. However, the attainment of this sensitivity with EELS is technically more difficult and limited by specimen thickness. The sensitivity of EELS experimentally demonstrated in this study permits the detection of three calcium atoms in a 10 nm diameter spot of an organic matrix, with a field-emission-gun-equipped scanning transmission electron microscope.

Calcium↗
PubMed1986

High spatial resolution analysis using parallel detection eels.

The application of parallel detection electron energy loss spectroscopy to the characterization of small particles or interfaces is demonstrated. Data are presented that show variations of low energy loss plasmons, Al L23 edge near edge fine structure, and oxygen concentration with spatial resolution better than 10 nm. The use of this technique for chemical and electronic structure measurements from very small specimen volumes is discussed.

Microscopy, Electron↗
PubMed1986

Elemental imaging and resolution in energy-filtered conventional electron microscopy.

Energy-filtered transmission electron microscopy (EFEM) was used to image the distributions of uranium and carbon in uranyl acetate stained catalase crystals. The spatial resolution obtained from inelastic C K-edge and U O4,5-edge images, determined from the highest-order reflection in the computed diffraction pattern, was 3.4 nm for both carbon and uranium. The resolution limit imposed by the delocalization of inelastic scattering was estimated from cross-section measurements to be 0.6 nm for U and 0.2 nm for C. Considering both delocalization and the effects of microscope aberrations, for an objective lens chromatic aberration coefficient of 2.8 mm and 10 eV energy window, the calculated resolutions are 2.0 nm for C and 1.2 nm for U. The effects of plural inelastic and elastic-inelastic scattering were sufficiently large to show crystalline structure in unprocessed pre-edge inelastic images. Previously suggested methods for eliminating these artifacts were applied to obtain the compositional information in the catalase EFEM images.

Carbon↗