PubMed Health⌕ Search

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↗
PubMed1979

Lysosomal composition in cultured vascular smooth muscle cells: electron probe analysis.

Spherical electron-dense organelles in the perinuclear region of cultured guinea pig aortic smooth muscle cells were identified as lysosomes by their ability to accumulate acridine orange and by cytochemical demonstration of their acid phosphatase content. The number and size of lysosomes increased in subcultured cells. The elemental composition of the lysosomes was quantitated by electron probe analysis of whole freeze-dried cells and of cryosections. In lysosomes at this stage in their development, the sulfur concentration was higher than that in the cytoplasm and the K/Na concentration ratio was similar to that in the cytoplasm.

Acid Phosphatase↗
PubMed1979

Electron probe analysis of vascular smooth muscle. Composition of mitochondria, nuclei, and cytoplasm.

Electron probe analysis of dry cryosections was used to determine the composition of the cytoplasm and organelles of rabbit portal-anterior mesenteric vein (PAMV) smooth muscle. All analytical values given are in mmol/kg wt +/- SEM. Cytoplasmic concentrations in normal, resting muscles were: K, 611 +/- 1.7; Na, 167 +/- 2.7; Cl, 278 +/- 1.0; Mg, 36 +/- 1.1; Ca, 1.9 +/- 0.5; and P, 247 +/- 1.1. Hence, the sum of intracellular Na + K exceeded cytoplasmic Cl by 500 mmol/kg dry wt, while the calculated total, nondiffusible solute was approximately 50 mmol/kg. Cytoplasmic K and Cl were increased in smooth muscles incubated in solutions containing an excess (80 mM) of KCl. Nuclear and cytoplasmic Na and Ca concentrations were not significantly different. The mitochondrial Ca content in normal fibers was low, 0.8 +/- 0.5, and there was no evidence of mitochondrial Ca sequestration in muscles frozen after a K contracture lasint 30 min. Transmitochondrial gradients of K, Na, and Cl were small (0.9--1.2). In damaged fibers, massive mitochondrial Ca accumulation of up to 2 mol/kg dry wt in granule form and associated with P could be demonstrated. Our findings suggest (a) that the nonDonnan distribution of Cl in smooth muscle is not caused by sequestration in organelles, and that considerations of osmotic equilibrium and electroneutrality suggest the existence of unidentified nondiffusible anions in smooth muscle, (b) that nuclei do not contain concentrations of Na or Ca in excess of cytoplasmic levels, (c) that mitochondria in PAMV smooth muscle do not play a major role in regulating cytoplasmic Ca during physiological levels of contraction but can be massively Ca loaded in damaged cells, and (d) that the in situ transmitochondrial gradients of K, Na, and Cl do not show these ions to be distributed according to a large electromotive Donnan force.

Animals↗
PubMed1979

Electron probe analysis of muscle and X-ray mapping of biological specimens with a field emission gun.

Recent electron probe analytic studies of freeze-dried cryosections of vascular smooth and vertebrate striated muscle are reviewed. The results show that the sarcoplasmic reticulum of striated muscle is not in ionic communication with the extracellular space. Vacuolation by hypertonic solutions and fatigue involves the T-tubule system. The high calcium content of the terminal cisternae of the resting muscle has been quantitated in situ. In smooth muscle, the high Cl content is distributed in the cytoplasm, and mitochondria in rabbit portal vein smooth muscle cells do not contain high concentrations of calcium. Mitochondrial calcium loading in the form of granules is generally due to fiber damage. Nuclear and mitochondrial composition in situ has been quantitated and compared to the composition of the cytoplasm of the same cells. Preliminary phosphorus x-ray maps of smooth muscle show the feasibility of this approach in defining the composition of organelles in thin cryosections. The use of x-ray maps at intermediate resolution is illustrated with tropomyosin paracrystals labelled with Hg-containing dye at the thiol residues. Mercury x-ray maps of such paracrystals show the 40nm periodicity of the thiol groups and their Fourier transforms contain information to a spatial resolution of 10-20nm.

Animals↗
PubMed1978

Composition of vacuoles and sarcoplasmic reticulum in fatigued muscle: electron probe analysis.

Electron probe analysis, cryo-ultramicrotomy, and freeze-substitution were used to determine the nature of vacuolation and the subcellular composition in fatigued frog skeletal muscle fibers. The vacuoles caused by fatigue were part of the T-tubule system and contained high concentrations of NaCl. The calcium concentration in the terminal cisternae was higher than previously measured normal resting values. Mitochondrial calcium content was relatively low (mean +/- SEM, 2 +/- 2 mmol/kg dry weight). Fiber NaCl was increased. It is concluded that fatigue is not due to the depletion of calcium stores from the terminal cisternae or to uncoupling of mitochondria due to calcium loading but may be caused by multiple mechanisms including failure of the T-tubule action potential.

Animals↗
PubMed1977

Bragg diffraction imaging of defects at crystal surfaces.

Scanning Bragg diffraction images of lattice defects are calculated for a medium energy electron beam at grazing incidence. A stacking fault intersecting the surface introduces additional dynamical beams which interfere to produce a band of fringes near the fault. A dislocation emerging at the surface perturbs the local lattice orientation and therefore changes the reflected beam intensities. It is shown that for a probe size of 10 A the contrast for these defects is about 50 percent while the reflected intensity is 1-10 percent of the incident intensity.

Crystallography↗
PubMed1977

Elemental distribution in striated muscle and the effects of hypertonicity. Electron probe analysis of cryo sections.

A method of rapid freezing in supercooled Freon 22 (monochlorodifluoromethane) followed by cryoultramicrotomy is described and shown to yield ultrathin sections in which both the cellular ultrastructure and the distribution of diffusible ions across the cell membrane are preserved and intracellular compartmentalization of diffusabler ions can be quantitated. Quantitative electron probe analysis (Shuman, H., A.V. Somlyo, and A.P. Somlyo. 1976. Ultramicros. 1:317-339.) of freeze-dried ultrathin cryto sections was found to provide a valid measure of the composition of cells and cellular organelles and was used to determine the ionic composition of the in situ terminal cisternae of the sarcoplasmic reticulum (SR), the distribution of CI in skeletal muscle, and the effects of hypertonic solutions on the subcellular composition if striated muscle. There was no evidence of sequestered CI in the terminal cisternae of resting muscles, although calcium (66mmol/kg dry wt +/- 4.6 SE) was detected. The values of [C1](i) determined with small (50-100 nm) diameter probes over cytoplasm excluding organelles over nuclei or terminal cisternae were not significantly different. Mitochondria partially excluded C1, with a cytoplasmic/ mitochondrial Ci ratio of 2.4 +/- 0.88 SD. The elemental concentrations (mmol/kg dry wt +/- SD) of muscle fibers measured with 0.5-9-mum diameter electron probes in normal frog striated muscle were: P, 302 +/- 4.3; S, 189 +/- 2.9;C1, 24 +/- 1.1;K, 404 +/- 4.3, and Mg, 39 +/- 2.1. It is concluded that: (a) in normal muscle the "excess CI" measured with previous bulk chemical analyses and flux studies is not compartmentalized in the SR or in other cellular organelles, and (b) the cytoplasmic C1 in low [K](0) solutions exceeds that predicted by a passive electrochemical distribution. Hypertonic 2.2 X NaCl, 2.5 X sucrose, or 2.2 X Na isethionate produced: (a) swollen vacuoles, frequently paired, adjacent to the Z lines and containing significantly higher than cytoplasmic concentrations of Na and Cl or S (isethionate), but no detectable Ca, and (b) granules of Ca, Mg, and P = approximately (6 Ca + 1 Mg)/6P in the longitudinal SR. It is concluded that hypertonicity produces compartmentalized domains of extracellular solutes within the muscle fibers and translocates Ca into the longitudinal tubules.

Animals↗
PubMed1976

Electron probe x-ray analysis of single ferritin molecules.

Single molecules and groups of two or three ferritin molecules were subjected to electron probe x-ray microanalysis in a transmission electron microscope equipped with a liquid nitrogen cooled stage. Significant Fe Kalpha peaks were generated during 100-sec counts when single ferritin molecules were excited with a probe current of 0.35 nA/60 nm spot, less than the maximal current available in a thermionic gun. There was a linear relationship between the number of ferritin molecules analyzed and count rates. The experimental results are compared to the theoretically calculated Fe Kalpha yields and to the results of Isaacson and Johnson [(1975) Ultramicroscopy I, 33-52] with electron energy loss analysis. We conclude that current state of the art electron probe x-ray analysis can realize the theoretically predicted sensitivity of the method, and estimate 0.9 X 10(-19) g of Fe as the minimal mass detectable with maximal (thermionic) probe current during a 100-sec count and with 95% confidence.

Ferritins↗