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N R Kramarcy

Publications and source records attributed to N R Kramarcy.

At least 19 recordsLinked to original sources
PubMed1992

The 30 S lobster skeletal muscle Ca2+ release channel (ryanodine receptor) has functional properties distinct from the mammalian channel proteins.

The 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps)-solubilized ryanodine receptor (RyR) of lobster skeletal muscle has been isolated by rate density centrifugation as a 30 S protein complex. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the purified 30 S receptor revealed a single high molecular weight protein band with a mobility intermediate between those of the mammalian skeletal and cardiac M(r) 565,000 RyR polypeptides. Immunoblot analysis showed no or only minimal cross-reactivity with the rabbit skeletal and canine cardiac RyR polypeptides. By immunofluorescence the lobster RyR was localized to the junctions of the A-I bands. Following planar lipid bilayer reconstitution of the purified 30 S lobster RyR, single channel K+ and Ca2+ currents were observed which were modified by ryanodine and optimally activated by millimolar concentrations of cis (cytoplasmic) Ca2+. Vesicle-45Ca2+ flux measurements also indicated an optimal activation of the lobster Ca2+ channel by millimolar Ca2+, whereas 45Ca2+ efflux from mammalian skeletal and cardiac muscle sarcoplasmic reticulum (SR) vesicles is optimally activated by micromolar Ca2+. Further, mammalian muscle SR Ca2+ release activity is modulated by Mg2+ and ATP, whereas neither ligand appreciably affected 45Ca2+ efflux from lobster SR vesicles. These results suggested that lobster and mammalian muscle express immunologically and functionally distinct SR Ca2+ release channel protein complexes.

Animals↗
PubMed1992

Association of the Mr 58,000 postsynaptic protein of electric tissue with Torpedo dystrophin and the Mr 87,000 postsynaptic protein.

Dystrophin was purified by immunoaffinity chromatography from detergent-solubilized Torpedo electric organ postsynaptic membranes using monoclonal antibodies. A major doublet of proteins at Mr 58,000 and minor proteins at Mr 87,000, Mr 45,000, and Mr 30,000 reproducibly copurified with dystrophin. The Mr 58,000 and Mr 87,000 proteins were identical to previously described peripheral membrane proteins (Mr 58,000 protein and 87,000 protein) whose muscle homologs are associated with the sarcolemma (Froehner, S. C., Murnane, A. A., Tobler, M., Peng, H. B., and Sealock, R. (1987) J. Cell Biol. 104, 1633-1646; Carr, C., Fischbach, G. D., and Cohen, J. B. (1989) J. Cell Biol. 109, 1753-1764). The copurification of dystrophin and Mr 58,000 protein was shown to be specific, since dystrophin was also captured with a monoclonal antibody against the Mr 58,000 protein but not by several control antibodies. The Mr 87,000 protein was a major component (along with the Mr 58,000 protein) in material purified on anti-58,000 columns, suggesting that the Mr 58,000 protein forms a distinct complex with the Mr 87,000 protein, as well as with dystrophin. Immunofluorescence staining of skeletal and cardiac muscle from the dystrophin-minus mdx mouse with the anti-58,000 antibody was confined to the sarcolemma as in normal muscle but was much reduced in intensity, even though immunoblotting demonstrated that the contents of Mr 58,000 protein in normal and mdx muscle were comparable. Thus, the Mr 58,000 protein appears to associate inefficiently with the sarcolemmal membrane in the absence of dystrophin. This deficiency may contribute to the membrane abnormalities that lead to muscle necrosis in dystrophic muscle.

Animals↗
PubMed1992

Amphibian ryanodine receptor isoforms are related to those of mammalian skeletal or cardiac muscle.

The ryanodine receptor (RyR)-Ca2+ release channels of frog skeletal muscle have been purified as 30S protein complexes comprised of two high molecular weight polypeptides. The upper and lower bands of the frog doublet comigrated on sodium dodecyl sulfate polyacylamide gels with the mammalian skeletal and cardiac RyR polypeptides, respectively. Immunoblot analysis showed that a polyclonal antiserum to the rat skeletal RyR preferentially cross-reacted with the upper band, whereas monoclonal antibodies to the canine cardiac RyR preferentially cross-reacted with the lower band of the frog receptor doublet. Immunoprecipitation studies indicated the presence of two homooligomer 30S RyR complexes comprised of either the lower or upper polypeptide band of the frog doublet, and immunocytochemical staining revealed their colocalization in frog gastrocnemius muscle. After planar lipid bilayer reconstitution of the 30S frog RyR, single-channel currents were observed that exhibited a Na+ and Ca2+ conductance and pharmacological characteristics similar to those of the mammalian skeletal and cardiac Ca2+ release channels. These results suggest that amphibian skeletal muscle expresses two distinct RyR isoforms that share epitopes in common with the mammalian skeletal or cardiac RyR.

Animals↗
PubMed1991

Localization of dystrophin relative to acetylcholine receptor domains in electric tissue and adult and cultured skeletal muscle.

Two high-affinity mAbs were prepared against Torpedo dystrophin, an electric organ protein that is closely similar to human dystrophin, the gene product of the Duchenne muscular dystrophy locus. The antibodies were used to localize dystrophin relative to acetylcholine receptors (AChR) in electric organ and in skeletal muscle, and to show identity between Torpedo dystrophin and the previously described 270/300-kD Torpedo postsynaptic protein. Dystrophin was found in both AChR-rich and AChR-poor regions of the innervated face of the electroplaque. Immunogold experiments showed that AChR and dystrophin were closely intermingled in the AChR domains. In contrast, dystrophin appeared to be absent from many or all AChR-rich domains of the rat neuromuscular junction and of AChR clusters in cultured muscle (Xenopus laevis). It was present, however, in the immediately surrounding membrane (deep regions of the junctional folds, membrane domains interdigitating with and surrounding AChR domains within clusters). These results suggest that dystrophin may have a role in organization of AChR in electric tissue. Dystrophin is not, however, an obligatory component of AChR domains in muscle and, at the neuromuscular junction, its roles may be more related to organization of the junctional folds.

Animals↗
PubMed1991

Commercial preparations of colloidal gold-antibody complexes frequently contain free active antibody.

Using a simple fluorescence test, we show that commercially prepared colloidal gold complexes with goat second antibodies often contain free active antibody. Because such antibodies will compete with antibody-colloidal gold particles for antigen binding sites, labeling intensity at the ultrastructural level must necessarily be submaximal to an unknown degree with such preparations. A survey of five preparations suggests that the problem may be widespread. We recommend that a test of the sort described be incorporated routinely into protocols with all colloidal gold products.

Animals↗
PubMed1990

Dystrophin as a focal adhesion protein. Collocalization with talin and the Mr 48,000 sarcolemmal protein in cultured Xenopus muscle.

Monoclonal antibodies against dystrophin and the postsynaptic 58 kDa protein from Torpedo electric organ were used to localize homologs of these proteins in cultured skeletal muscle (Xenopus laevis). The Xenopus homolog is an Mr 48,000 protein and, like dystrophin, is a sarcolemmal protein. Both proteins localized precisely to talin-positive sites, hence with each other, on the substrate-apposed sarcolemma. Therefore, the first sites of appearance of dystrophin on cultured muscle cells are focal adhesions, i.e. specific sites of cytoskeleton/extracellular matrix interaction. These data also add to evidence that dystrophin and the 58 kDa act together.

Animals↗
PubMed1985

Effects of tunicamycin on the expression of beta-adrenergic receptors in human astrocytoma cells during growth and recovery from agonist-induced down-regulation.

Tunicamycin, which inhibits formation of asparagine-linked glycoproteins, caused a concentration-dependent blockade of beta-adrenergic receptor (beta-AR) accumulation in 1321N1 human astrocytoma cells during growth in culture. A concentration of tunicamycin (0.1 microgram/ml) that inhibited receptor accumulation and [3H]mannose or [3H]glucosamine incorporation into glycoproteins by 90% had only a small effect (10%) on [3H]leucine incorporation into protein, and reduced the rate of cell growth. Incubation in drug-free medium subsequent to treatment of 1321N1 cells with tunicamycin for 48 hr resulted in recovery of beta-AR to control levels within an additional 48 hr. Exposure of cultures to isoproterenol (0.1 microM, 12 hr) caused an 80-90% loss of beta-AR in both pre- and postconfluent cultures; beta-AR recovered to control levels upon removal of isoproterenol. Although both tunicamycin and the protein synthesis inhibitor cycloheximide blocked beta-AR accumulation during growth of 1321N1 cells, neither agent inhibited the appearance of beta-AR during recovery from the down-regulated state in preconfluent cultures. However, cycloheximide, but not tunicamycin, blocked recovery of beta-AR after isoproterenol-induced loss of receptors in postconfluent cultures. In a previous report (Mol. Pharmacol. 26:424-429, 1984), we provided direct evidence that recovery of beta-AR from down-regulation in postconfluent cultures requires de novo synthesis of receptor protein. Thus, the results with tunicamycin are consistent with the idea that recovery of beta-AR in postconfluent cultures requires the synthesis of new beta-AR molecules, but as aglycoproteins that exhibit radioligand-binding characteristics similar to those of native glycoprotein beta-AR.

Astrocytoma↗
PubMed1984

Effects of drug-induced changes in brain monoamines on aggression and motor behavior in mice.

Mice maintained on a basal casein diet supplemented with 4% L-tyrosine potentiated L-DOPA effects on aggression. At low doses (12.5-25 mg/kg) L-DOPA increased aggression whereas at high doses (50-100 mg/kg) it decreased aggression. 5-HTP (50-200 mg/kg) produced a dose-dependent decrease in aggression and motor activity which was antagonized by pretreatment with dietary L-tyrosine (4%) or L-DOPA (50 mg/kg). L-DOPA induced reductions in motor activity were, in turn, antagonized by 5-HTP. Increases in motor activity following d-amphetamine (3 mg/kg) were sharply reduced by 5-HTP (50-100 mg/kg), but 5-HTP potentiated reductions in aggression following d-amphetamine. The concentration in brain of tyrosine, DOPA, dopamine (DA), noradrenaline (NA), DOPAC, HVA, tryptophan, serotonin (5-HT), and 5-HIAA were obtained following drug and diet treatments. The changes observed, particularly in DA and 5-HT metabolites, provide further evidence for an inhibitory role of brain 5-HT systems in the mediation of the behavioral effects of d-amphetamine and the catecholamine precursors, L-tyrosine and L-DOPA.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1984

Footshock treatment activates catecholamine synthesis in slices of mouse brain regions.

Synthesis of catecholamines was measured in slices of frontal cortex, hypothalamus, striatum, hippocampus and brainstem by the accumulation of [3H]norepinephrine (NE) and [3H]dopamine (DA) following incubation with [3H]tyrosine. Following acute footshock (60 shocks, 0.3 mA 30 min), consistent increases in [3H]DA accumulation were seen in frontal cortex slices, but no significant effect was seen in striatal slices. The accumulation of [3H]NE was not altered consistently in frontal cortex, hypothalamus, hippocampus or brainstem. Brain slices from mice adrenalectomized 24-48 h before footshock showed similar results. However, in hypophysectomized mice, footshock did not increase the [3H]DA accumulation in slices of frontal cortex. Administration of dexamethasone before footshock prevented the footshock-induced increase in frontal cortex [3H]DA accumulation, but footshock then significantly increased [3H]NE accumulation in the hypothalamus and brainstem. Chronic footshock (5 days) had little effect on frontal cortex [3H]catecholamine accumulation but produced a consistent elevation of [3H]NE accumulation in slices from the hypothalamus. In confirmation that the slice data reflected in vivo metabolism, both dihydroxyphenylacetic acid and homovanillic acid were significantly elevated in the frontal cortex but not the striatum of mice receiving acute footshock. Since previous studies have shown that ACTH administered intracerebroventricularly also accelerated [3H]DA accumulation in frontal cortex slices, these results are consistent with the involvement of ACTH in the effects of footshock on frontal cortex DA. The effects of chronic footshock are consistent with the activation of hypothalamic tyrosine hydroxylase by corticosterone.

Animals↗
PubMed1982

ACTH 1-24 and lysine vasopressin selectively activate dopamine synthesis in frontal cortex.

The accumulation of [3H] catecholamines from [3H] tyrosine in frontal cortical, septal, striatal and hippocampal slices was examined following intracerebroventricular (i.c.v.) injections of ACTH 1-24, lysine vasopressin (LVP) and saline. Both ACTH 1-24 and LVP (1 microgram) selectively increased the accumulation of [3H] dopamine (DA) in frontal cortical slices, but did not affect that of [3H] norepinephrine (NE). LVP but not ACTH 1-24 also inhibited the accumulation of [3H] DA in striatal slices. ACTH 1-24 did not alter the accumulation of [3H] NE in hippocampal slices, nor did LVP alter the accumulation of either catecholamine (CA) in septal slices. In vitro incubations with ACTH analogs of LVP failed to alter the rate of accumulation of [3H] CAs in striatal, substantia nigral and frontal cortical slices, except for an inhibitory effect at high doses. This effect is believed to be an artifact of precursor dilution caused by release of tyrosine following degradation of the peptides. Neither peptide modified the increased [3H] CA accumulation stimulated by 26 mM K+, nor did ACTH 1-24 modify the inhibition of [3H] CA accumulation caused by 3 X 10 -6 M haloperidol or 3 X 10 -7 M apomorphine. Selective activation of the mesocortical DA system has also been reported ot occur in response to footshock, suggesting the possibility that endogenous ACTH and/or LVP might mediate the stress-induced activation of mesocortical DA synthesis. Alternatively, i.c.v. injections of these peptides may themselves be stressful and thus indirectly elicit the response.

Adrenocorticotropic Hormone↗
PubMed1982

Neurotensin induces catalepsy in mice.

Intracerebroventricular (i.c.v.) injection of neurotensin (NT) induced catalepsy in mice at doses greater than or equal to 0.02 microgram. The cataleptic effect progressively increased, reaching a maximum at approx. 2 hr after injection. In contrast, the hypothermic effect of neurotensin reached a maximum 1 hr after the injection, and was declining at 2 hr. Not all mice that showed hypothermia also showed catalepsy, and some mice showed catalepsy without hypothermia. Catalepsy induced by intracerebroventricular injection of neurotensin was not significantly correlated with the hypothermia. Furthermore, oxotremorine induced hypothermia without catalepsy. Thus, several lines of evidence indicate that the catalepsy induced by neurotensin is not the consequence of the neurotensin induced hypothermia. Thyrotropin releasing hormone (TRH), injected either intracerebroventricularly with neurotensin, or intraperitoneally before neurotensin abolished the hypothermia but only diminished the catalepsy scores. The cataleptic effect of neurotensin is consistent with its other neuroleptic-like activities.

Animals↗
PubMed1982

Effect of fusaric acid on aggression, motor activity, and brain monoamines in mice.

The effects of fusaric acid (FA), a dopamine-beta-hydroxylase (D beta H) inhibitor, were determined on aggression, motor activity, and brain monoamines at doses of 3.2 to 60 mg/kg following administration of dietary supplements of L-tyrosine or balanced protein to male albino mice. Compared to saline injected control animals, both aggression and motor activity were reduced by the highest doses of FA. Somewhat more reduction in aggression was observed in animals administered dietary supplements of casein compared to those given the tyrosine supplement. Treatment with FA at doses of 30 to 60 mg/kg decreased brain norepinephrine and dopamine, and decreased brain tyrosine in animals fed the tyrosine supplement. In contrast, FA increased 5-hydroxytryptamine, and caused marked increases in 5-hydroxyindoleacetic acid at the highest doses. The data suggest that the neurochemical effects of FA may not be the same in rats and mice.

Aggression↗
PubMed1981

Benzodiazepines decrease grooming in response to novelty but not ACTH or beta-endorphin.

Excessive grooming in response to intracerebroventricular (ICV) ACTH1-24 was assayed following various doses of diazepam, chlordiazepoxide and flurazepam. Grooming scores were only affected by doses of the benzodiazepines higher than those that depressed locomotor activity. Similarly, diazepam did not affect excessive grooming induced by ICV beta-endorphin, nor did chronic chlordiazepoxide affect ACTH-induced grooming. By contrast similar doses of the benzodiazepines decreased the increased grooming score observed when mice were observed in a novel as opposed to the home cage. This result is consistent with the hypothesis that novel cage-induced grooming is caused by an increase in the ventricular content of ACTH or beta-endorphin, and that the benzodiazepines decrease or prevent this increase. It is not consistent with hypotheses of a functional antagonism between ACTH and benzodiazepines, at least insofar as the mechanisms involved in the production of grooming are concerned.

Adrenocorticotropic Hormone↗
PubMed1979

Differential tolerance to dietary amino acid-induced changes in aggressive behavior and locomotor activity in mice.

Male albino mice were maintained on a semisynthetic 12% casein protein diet for 2 weeks, then switched to diets modified by the addition of a 4% L-amino acid supplement (L-tyrosine, L-phenylalanine, and L-tryptophan) or 4% casein (control). Territorial=induced aggressive behavior increased following 1 week on the amino acid supplements, especially after tyrosine, but an apparent tolerance developed to these effects after 5 weeks on the amino acid supplements. Locomotor activity also increased following 1 week on the supplements, most notably after phenylalanine alone or in combination with tyrosine, and these effects tended to persist after 5 weeks on the supplements. Endogenous whole brain levels of dopamine, norepinephrine, serotonin, 5-hydroxyindoleacetic acid, tyrosine, phenylalanine, and tryptophan showed no tolerance to increased concentrations of brain catecholamines and indoleamines over the 5-week period, and no clear relation between the concentrations of these monoamines and the behavioral changes.

Aggression↗