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S Fleischer

Publications and source records attributed to S Fleischer.

At least 199 records · Page 11Linked to original sources
PubMed1983

Expression of an mRNA coding gene under the control of an RNA polymerase I promoter.

We have placed a 225-bp fragment from the 5' end of the mouse rDNA transcription unit (from -169 to +56) in front of the SV40 tumor antigen coding sequence. After microinjection of this chimeric plasmid into nuclei of mouse L-cells expression of SV40 large T antigen has been observed. The expression of T antigen was dependent on the correct orientation of the rDNA fragment relative to the T antigen-coding region and was seen only in mouse cells. This indicates that the 225-bp rDNA fragment contains the sequence information required for pre-rRNA transcription and demonstrates for the first time that a protein-coding gene can be transcribed and expressed under the control of an RNA polymerase I promoter.

Animals↗
PubMed1983

Study of neurotrophism in normal/dystrophic parabiotic mice.

The trophic influences of nerve and muscle on one another were studied in normal and dystrophic littermates of C57BL/6J dy2J mice parabiosed at 20 to 23 days after birth. Each parabiont had a soleus muscle cross-reinnervated by a tibial nerve of its partner. Ultrastructural abnormalities of muscle and endplate were quantified and compared 6 to 7 months postoperatively. The dystrophic nerve degenerated despite reinnervation to a normal muscle. The normal muscle did not prevent the dystrophic nerve from degenerating, and the dystrophic nerve induced degenerative changes in the reinnervated normal muscle. Normal nerve did not retard the genetically programmed degeneration of the dystrophic muscle. The dystrophic muscle, however, did not appear to cause normal nerve terminals to degenerate. We conclude that both nerve and muscle cells in dystrophic mice express characteristics of muscular dystrophy. Muscle fibers of a few motor units further suffer from abnormal neurotrophic influence because of the degeneration of the motor neurons. Myotrophic influence on nerve was not observed.

Animals↗
PubMed1983

Induced Ca2+ release in skeletal muscle sarcoplasmic reticulum by sulfhydryl reagents and chlorpromazine.

Sarcoplasmic reticulum is a specialized membrane system in muscle involved in the energized uptake, storage, and release of Ca2+. The sulfhydryl content of normal and reconstituted sarcoplasmic reticulum was measured using Ellman reagent. For both preparations, we find 17 and 26 mol sulfhydryls per mole calcium pump protein assayed in the absence and presence of sodium dodecyl sulfate. The release of Ca2+ from sarcoplasmic reticulum, which triggers muscle contraction, likely involves the regulation of a channel. This report deals with an experimental approach to studying the Ca2+ release in isolated sarcoplasmic reticulum. We find that sulfhydryl agents of which water-soluble mercurials were most effective induce Ca2+ release. Chlorpromazine acts synergistically with the sulfhydryl reagents. Ca2+ release under optimal conditions is very rapid compared with calcium leakage from preloaded but untreated sarcoplasmic reticulum. The imposed rapid release of Ca2+ is suggestive of the opening of a channel. Ca2+ release by mercurials is retained in reconstituted sarcoplasmic reticulum membrane vesicles.

Animals↗
PubMed1983

Ultrastructural changes in muscle and motor end-plate of the dystrophic mouse.

A comparative study of dystrophic mice (C57BL/6J-dy2J) and normal littermates, 6 to 9 months old, has revealed numerous ultrastructural changes in the dystrophic soleus. Vacuoles, swollen mitochondria, vesicular aggregates, membranous bodies, Z-line degradation, localized hypercontraction, myofibrillar disorientation, and focal necrosis were common. We report here new observations. Discontinuous, dense bands (240 A wide) were observed rarely between inner and outer membranes in some nuclei. Closely associated with infoldings of nuclear membrane were paracrystalline, vesicular structures, actin-like filaments, and autophagic vacuoles. With the degeneration of the nuclear membrane, actin-like filaments were observed within the nucleus. The primary synaptic clefts were widened and contained osmophilic-dense granules (diameter 0.2 to 0.7 micrometer). There was loss of secondary synaptic folds. Discontinuity of pre- and postsynaptic membranes could be observed, though rarely. Lysosome-like dense bodies were present among myofilaments. The myeloid bodies and autophagic vacuoles that were associated with nuclei, sarcoplasmic reticulum, and mitochondria gave a positive acid phosphatase reaction. Such degeneration changes were not observed in the normal soleus.

Animals↗
PubMed1983

Purification of morphologically intact triad structures from skeletal muscle.

A procedure has been devised for isolation of triads (t-tubule/sarcoplasmic reticulum (SR) junctional complexes) from rabbit skeletal muscle. The procedure consists of preparation of a heavy microsomal fraction followed by two sequential 90-min sucrose gradient centrifugations to enrich the triads. A pyrophosphate/phosphate/magnesium buffer system was introduced to decrease aggregation in order to achieve effective separation. The preparation time is 12 h. Some differences between purified triads isolated by two variants of this method are noted. The purity of the triad fractions has been estimated by particle counting to be in the vicinity of 50%. There is good retention of morphology and Ca++-loading activity and enrichment in Na+,K+-ATPase and adenylate cyclase. The triads are practically devoid of contractile elements, mitochondria, and free plasmalemma, and low in content of light SR. The method for obtaining enriched triads is reproducible, and sufficient yields are obtained for structural, biochemical, and functional characterization.

Adenylyl Cyclases↗
PubMed1983

Morphology of isolated triads.

The triad is the junctional association of transverse tubule with sarcoplasmic reticulum terminal cisternae. A procedure for the isolation of highly enriched triads from skeletal muscle has been described in the previous paper. In the present study, the structural features of isolated triads have been examined by thin-section, negative-staining, and freeze-fracture electron microscopy. In isolated triads, key features of the structure observed in situ have been retained, including the osmiophilic "feet," junctional structures between the transverse tubule and terminal cisternae. New insight into triad structure is obtained by negative staining, which also enables visualization of feet at the junctional face of the terminal cisternae, whereas smaller surface particles, characteristic of calcium pump protein, are not visualized there. Therefore, the junctional face is different from the remainder of the sarcoplasmic reticulum membrane. Junctional feet as viewed by thin section or negative staining have similar periodicity and extend approximately 100 A from the surface of the membrane. Freeze-fracture of isolated triads reveals blocklike structures associated with the membrane of the terminal cisternae at the junctional face, interjunctional connections between the terminal cisternae and t-tubule, and intragap particles. The intragap particles can be observed to be closely associated with the t-tubule. The structure of isolated triads is susceptible to osmotic and salt perturbation, and examples are given regarding differential effects on transverse tubules and terminal cisternae. Conditions that adversely affect morphology must be considered in experimentation with triads as well as in their preparation and handling.

Animals↗
PubMed1983

Preparation of representative samples of subcellular fractions for electron microscopy by filtration with dextran.

A simple method is described for obtaining a representative field of subcellular fractions for thin section electron microscopy. The prefixed sample is admixed together with high molecular weight dextran, filtered onto a Millipore filter, and embedded in polymer. Filtration serves to uniformly distribute structures of different sizes and densities. The dextran serves as a nonosmophilic spacer, increasing the space between individual structures and preventing sample compression during filtration onto Millipore filters. Sample aggregation can also readily be assessed by the procedure.

Dextrans↗
PubMed1982

Isolation of plasma membrane vesicles from rabbit skeletal muscle and their use in ion transport studies.

A method has been developed for the isolation of sealed plasma membrane vesicles from rabbit white skeletal muscle. The final preparation was highly purified as indicated by enrichment of plasma membrane marker enzymes (i.e. ouabain-sensitive (Na+,K+)-ATPase, adenylate cyclase, and acetylcholinesterase). The absence of sarcoplasmic reticulum and mitochondria as contaminants was indicated by the low specific activity of marker enzymes, i.e. Ca2+-ATPase, succinate-cytochrome c reductase, and monoamine oxidase. Thin section and negative staining electron microscopy confirmed the absence of sarcoplasmic reticulum and mitochondrial contamination. The plasma membrane preparation consisted largely of sealed vesicles as observed by electron microscopy and as also demonstrated by latency of enzymic activities, which were unmasked by preincubation with detergent (sodium dodecyl sulfate). Membrane sidedness was estimated from latency of ouabain-sensitive (Na+,K+)-ATPase activity and acetylcholinesterase activity. The latency studies suggest that most of the vesicles are oriented inside out with respect to the orientation of the sarcolemma membrane in the muscle fiber. The inside-out plasma membrane vesicles actively accumulated sodium ions upon addition of ATP. The sodium ions were concentrated greater than 8-fold inside the vesicles and were released upon addition of the ionophore monensin. The sodium ions were taken up in the presence of K+ or NH4+ but not of choline. Uptake was inhibited by low concentrations of vanadate or digitoxin. The Na+ uptake was concomitant with Rb+ efflux. Therefore, the sodium ion transport and the resulting gradients formed appear to have been generated by the ouabain-sensitive (Na+,K+)-ATPase. Batrachotoxin, which opens Na+ channels in excitable tissues, prevents most of the Na+ uptake, suggesting the presence of toxin-activated Na+ channels in these plasma membrane vesicles.

Acetylcholinesterase↗
PubMed1982

Reactivation of D-beta-hydroxybutyrate dehydrogenase with short-chain lecithins: stoichiometry and kinetic mechanism.

D-beta-Hydroxybutyrate dehydrogenase (BDH), purified as soluble, lipid-free apoenzyme (inactive) from either beef heart or rat liver mitochondria, can be reactivated by short-chain lecithins in the monomeric state. The enzyme was reactivated with dihexanoyl- [PC(6:0)], diheptanoyl- [PC(7:0)], and dioctanoyllecithins [PC(8:0)]. The titration curves of enzyme activity as a function of the phospholipid concentration are consistent with a model in which the enzyme contains two identical, noninteracting lecithin binding sites. The simultaneous occupation of these sites (via an equilibrium random mechanism) is required to activate the apoenzyme. Similar results were obtained with both rat liver and beef heart apoenzymes. The maximal velocities obtained with the different lecithins were similar [110-140 mumol of NAD+ reduced min-1 (mg of protein)-1]. The KL values (the apparent dissociation constants of the lecithin-site complexes) were 1.2 X 10(-4) M [PC(8:0)], 1.5 X 10(-3) M [PC(7:0)], and 4.5 X 10(-3) M [PC(6:0)] at 37 degrees C. This was confirmed by using phospholipase A2 to compete with the dehydrogenase for the lecithin monomers. Comparison of the delta G degrees values for complex formation with the different lecithins shows an average contribution of approximately 2.4 kJ/mol (0.9RT) per CH2 group. The interaction of the apolar moiety of lecithin with the protein seems to be essential for effective binding of phosphatidylcholine to apoBDH. The delta G degrees values, when combined with the estimated delta H degrees values, suggest that the binding of lecithin to the apoenzyme is approximately 60% enthalpy and approximately 40% entropy driven.

Animals↗
PubMed1982

Rat liver L-glutamate dehydrogenase, malate dehydrogenase, D-beta-hydroxybutyrate dehydrogenase, and sulfite oxidase are each synthesized as larger precursors by cytoplasmic free polysomes.

Four cytoplasmically synthesized rat liver mitochondrial enzymes, located either as soluble enzymes in the mitochondrial matrix (L-glutamate dehydrogenase and malate dehydrogenase or in the intermembrane space (sulfite oxidase) or as an integral membrane protein located on the matrix face of the inner mitochondrial membrane (D-beta-hydroxybutyrate dehydrogenase), were all shown to be synthesized as precursors larger than their mature counterparts by 1000-6000 daltons. These larger forms were detected in vitro, in a cell-free protein synthesizing system programmed with either total rat liver RNA or with RNA isolated from free polysomes or with free polysomes, and in vivo, in the two cases that were investigated (L-glutamate dehydrogenase and D-beta-hydroxybutyrate dehydrogenase), by pulse labeling of Buffalo rat liver cells in culture. The intracellular site of synthesis of all four mitochondrial enzymes was shown to be primarily on free polysomes and not on membrane-bound polysomes.

Animals↗
PubMed1982

Normal function in sarcoplasmic reticulum from mice with muscular dystrophy.

A rapid, gentle technique has been developed for the isolation of sarcoplasmic reticulum (SR) from small amounts of skeletal muscle from normal and dystrophic mice. Assays for mitochondrial and surface membrane marker enzymes revealed only low levels of contamination in the isolated fractions. A small amount of calcium-insensitive ("basal") ATPase in the normal preparation and a higher value in the dystrophic were shown to be due to contamination by a lighter membrane fraction of probable surface membrane origin. Isolated SR from normal and dystrophic mice were indistinguishable by thin section and freeze-fracture electron microscopy. Only small differences in calcium loading rates and capacity, and in calcium-stimulated ATPase activity, were present. These were attributable to small differences in purity. We conclude that there is no difference in SR from normal and dystrophic mice in the properties measured.

Adenosine Triphosphatases↗
PubMed1982

Proton NMR T1, T2, and T1 rho relaxation studies of native and reconstituted sarcoplasmic reticulum and phospholipid vesicles.

The phospholipids protons of native and reconstituted sarcoplasmic reticulum (SR) membrane vesicles yield well-resolved nuclear magnetic resonance (NMR) spectra. Resonance area measurements, guided by the line shape theory of Bloom and co-workers, imply that we are observing a large fraction of the lipid intensity and that the protein does not appear to reduce the percent of the signal that is well resolved. We have measured the spin-lattice (T1) and spin-spin (T2) relaxation rates of the choline, methylene, and terminal methyl protons at 360 MHz and the spin-lattice relaxation rate in the rotating frame (T1 rho) at 100 MHz. Both the T1 and T2 relaxation rates are single exponential processes for all of the resonances if the residual water proton signal is thoroughly eliminated by selective saturation. The T1 and T2 relaxation rates increase as the protein concentration increases, and T2 rate decrease with increasing temperature. This implies that the protein is reducing both high frequency (e.g., trans-gauche methylene isomerizations) and low frequency (e.g., large amplitude, chain wagging) lipid motions, from the center of the bilayer to the surface. It is possible that spin diffusion contributes to the effect of protein on lipid T1's although some of the protein-induced T1 change is due to motional effects. The T2 relaxation times are observed to be near 1 ms for the membranes with highest protein concentration and approximately 10 ms for the lipids devoid of protein. This result, combined with the observation that the T2 rates are monophasic, suggests that at least two lipid environments exist in the presence of protein, and that the lipids are exchanging between these environments at a rate greater than 1/T2 or 10(3) s-1. The choline resonance yields single exponential T1 rho relaxation in the presence and absence of protein, whereas the other resonances measured exhibit biexponential relaxation. Protein significantly increases the single T1 rho relaxation rate of the choline peak while primarily increasing the T1 rho relaxation rate of the more slowly relaxing component of the methylene and methyl resonances.

Animals↗
PubMed1982

Static and time-resolved structural studies of the Ca2+-ATPase of isolated sarcoplasmic reticulum.

X-ray and neutron diffraction studies of oriented multilayers of isolated light sarcoplasmic reticulum (SR) have provided the separate profile structures of the lipid bilayer and the Ca2+-ATPase molecule within the membrane profile to approximately 10 A resolution. These studies utilized biosynthetically deuterated SR phospholipids incorporated isomorphously into the isolated SR membranes via exchange proteins. Time-resolved x-ray diffraction studies of these oriented SR membrane multilayers have indicated that significant changes occur in the membrane profile structure within a single turnover of the Ca2+-transport cycle. These studies utilized the flash photolysis of caged ATP to effectively synchronize the ensemble of Ca2+-ATPase molecules in the multilayer, synchrotron x-radiation to provide 100- to 500-millisecond data collection times, and double-beam spectrophotometry to monitor Ca2+ transport in the oriented SR membrane multilayer.

Animals↗