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R Siman

Publications and source records attributed to R Siman.

64 records · Page 4Linked to original sources
PubMed1985

Distribution of calpain I, an enzyme associated with degenerative activity, in rat brain.

The calcium-activated protease calpain I was localized in rat brain by immunocytochemistry. Calpain I-like immunoreactivity (CLI) was prominent in several structures in which degeneration is an ongoing feature, e.g. spinal motoneurons, olfactory nerve. Also noteworthy was the presence of CLI in regions susceptible to age-related pathologies, e.g. cerebellar Purkinje cells, substantia nigra and subiculum. This distribution suggests that calpain I may be involved with both normal and pathological neuronal degeneration.

Animals↗
PubMed1985

Regional distribution of soluble calcium activated proteinase activity in neonatal and adult rat brain.

Calcium dependent proteolytic activity in the soluble fraction of various rat brain regions was assayed using 14C-radiolabelled denatured casein as a substrate. Two forms of activity, distinguishable by their calcium requirement for half maximal activation (5 and 80 microM), were found; both were blocked by sulfhydryl alkylating agents and thiol proteinase inhibitors. Preincubation at 58 degrees C for 10 min also eliminated the high threshold activity. These characteristics are identical to those reported for calcium activated neutral proteinases ('calpains') found in other tissues. Calpain activity varied markedly across brain regions. The greatest values for the high threshold enzyme were found in pons-medulla followed by cerebellum/mesencephalon and finally the telencephalon. The low threshold enzyme had low levels of activity throughout the brainstem and diencephalon and was barely detectable in telencephalic structures. In contrast, a previously described endogenous inhibitor of calpain, 'calpastatin', was found not to vary in its activity across brain regions. Calpain activity was high in the prenatal brain, but while the hindbrain maintained high levels of activity into adulthood, the activity in the forebrain dropped 80% during the early postnatal period. The differences between forebrain and hindbrain activity levels were evident during the first 5 days of the neonatal period, suggesting that glial cell differentiation is not responsible for the regional variations found in the adult. These results are discussed with regard to the possibility that the turnover of anatomical structures differs between brain regions.

Animals↗
PubMed1984

Brain fodrin: substrate for calpain I, an endogenous calcium-activated protease.

The calcium-activated thiol-protease calpain I, which is present in cytosolic and membrane preparations from rat brain, was tested for its capacity to degrade the neuronal spectrin-like protein fodrin. In the presence of micromolar calcium concentrations purified calpain I degraded both purified fodrin and the fodrin present in hippocampal and cerebellar membranes. Fodrin was identified as a high molecular weight protein present in brain membranes by the following criteria: (i) comigration on NaDodSO4/polyacrylamide gels with purified fodrin, (ii) reactivity with antibodies to purified fodrin, and (iii) a proteolytic map following calpain activation comparable to that found after calpain-mediated degradation of purified fodrin. The fodrin breakdown was selective in that calpain I did not affect at least 15 other membrane-associated polypeptides. Fodrin degradation by the protease was rapid and was accompanied by the appearance of a lower molecular weight breakdown product. Calpain I had a high affinity for fodrin, with a Km for degradation of about 50 nM. Purified calpain I also degraded purified spectrin and the spectrin present in erythrocyte membranes. Calpain I-mediated degradation of spectrin-like proteins could provide a mechanism by which brief increases in intracellular free calcium levels modify the structure of the submembraneous cytoskeleton and the distribution of cell surface receptors and alter cell shape.

Animals↗
PubMed1983

Regulation by calcium ions of glutamate receptor binding in hippocampal slices.

Hippocampal slices were incubated in a Krebs-bicarbonate buffer with various concentrations of calcium and [3H]glutamate receptor binding was measured in crude synaptic membranes derived from these slices. Increasing the calcium concentration from 0 to 2.5 mM resulted in a 2.2-fold increase in the maximal number of the Na-independent [3H]glutamate binding sites without changes in their affinity for [3H]glutamate. This effect was totally blocked by the addition of the protease inhibitor leupeptin (50 microM) to the slice incubation medium. No effect was observed on the Na-dependent [3H]glutamate binding nor on the Na-independent [ 3H ]glutamate binding measured in the presence of a concentration of calcium of 250 microM. Increasing the calcium concentration also resulted in an increased proteolytic activity which was inhibited by about 70% by the addition of leupeptin. Finally, increasing the calcium concentration induced the degradation of high-molecular weight proteins, the microtubule-associated proteins (MAPs) and the 220 000 dalton doublet protein corresponding to fodrin. Both effects were partially prevented by the addition of leupeptin in the slice incubation medium. These results indicate that the same calcium-dependent processes which were previously shown to regulate [ 3H ]glutamate receptor binding to hippocampal membranes occur in the hippocampal slice preparation, and they suggest a mechanism by which fluctuations in calcium levels can activate a calcium-dependent proteinase, the degradation of cytoskeletal-associated proteins and the unmasking of additional glutamate receptors. The participation of such processes in various forms of plasticity is discussed.

Animals↗
PubMed1983

Purification from synaptosomal plasma membranes of calpain I, a thiol protease activated by micromolar calcium concentrations.

Synaptosomal plasma membranes (SPMs) were prepared from whole rat brain and assayed for calcium-stimulated proteolytic activity. Addition of calcium to SPMs caused a dose-dependent increase in trichloroacetic acid-soluble protein. Two peaks of protease activity directed against a casein substrate were detectable when SPMs were incubated with low-ionic-strength buffer and the extract was fractionated on DEAE-cellulose. The enzyme in peak 1 required less than 1/10 the calcium concentration for activation as the peak 2 protease (Kact1 = 35 microM; Kact2 = 500 microM). The specific thiol-protease inhibitors leupeptin and antipain and the alkylator iodoacetate blocked enzyme activity. The low-sensitivity protease was converted to a high-sensitivity enzyme (Kact = 20 microM) by substrate affinity chromatography in the presence of calcium. This protease was purified 550-fold from SPMs. The high- and low-sensitivity membrane-associated calcium-dependent proteases are part of a family of enzymes, the calpains, previously reported in cytosolic fractions of several tissues.

Animals↗
PubMedDate not supplied

Mutant and native human beta-amyloid precursor proteins in transgenic mouse brain.

Human beta-amyloid precursor protein (beta APP) has been targeted to transgenic neurons using synapsin I promoter-based chimeric transgenes. Native human beta APP was introduced as well as beta APP containing mutations genetically linked to familial Alzheimer's disease (AD) and to hereditary cerebral hemorrhage with amyloidosis-Dutch type. In mouse brain, human beta APP RNA was up to 60% as abundant as total endogenous beta APP RNA. Human beta APP gene expression was most abundant in the CA subfields of the hippocampus and in the piriform cortex. Correct processing of human beta APP at the beta-secretase cleavage site was demonstrated in transgenic mouse brains. Despite a 40% increase in total beta APP immunoreactivity in lines expressing mutant human beta APP, no evidence of amyloid deposition was found in brains of mice up to 14 months in age. Higher levels of mutant human beta APP, increased age, or other factors may be necessary to elicit beta-amyloid-related neuropathologies in the rodent brain.

Amyloid beta-Protein Precursor↗
PubMedDate not supplied

Diffuse plaques contain C-terminal A beta 42 and not A beta 40: evidence from cats and dogs.

Recent reports have suggested that beta-amyloid (A beta) species of variable length C-termini are differentially deposited within early and late-stage plaques and the cerebrovasculature. Specifically, longer C-terminal length A beta 42/3 fragments (i.e., A beta forms extending to residues 42 and/or 43) are thought to be predominant within diffuse plaques while both A beta 42/3 and A beta 40 (A beta forms terminating at residue 40) are present within a subset of neuritic plaques and cerebrovascular deposits. We sought to clarify the issue of differential A beta deposition using aged canines, a partial animal model of Alzheimer's disease that exhibits extensive diffuse plaques and frequent vascular amyloid, but does not contain neuritic plaques or neurofibrillary tangles. We examined the brains of 20 aged canines, 3 aged felines, and 17 humans for the presence of A beta immunoreactive plaques, using antibodies to A beta 1(-17), A beta 17(-24), A beta 1(-28), A beta 40, and A beta 42. We report that plaques within the canine and feline brain are immunopositive for A beta 42 but not A beta 40. This is the first observation of nascent AD pathology in the aged feline brain. Canine plaques also contained epitopes within A beta 1(-17), A beta 17(-24), and A beta 1(-28). In all species examined, vascular deposits were immunopositive for both A beta 40 and A beta 42. In the human brain, diffuse plaques were preferentially A beta 42 immunopositive, while neuritic plaques and vascular deposits were both A beta 40 and A beta 42 immunopositive. However, not all neuritic plaques contain A beta 40 epitopes.

Aging↗
PubMedDate not supplied

Regulation of glutamate receptor binding by the cytoskeletal protein fodrin.

The erythrocyte cytoskeleton, which consists primarily of a meshwork of spectrin and actin, controls cell shape and the disposition of proteins within the membrane. Proteins similar to spectrin have recently been found in diverse cells and tissues, and it is possible that they mediate the capping of cell-surface receptors, although this has not been demonstrated directly. In neurones, the spectrin-like protein fodrin lines the cortical cytoplasm and may link actin filaments to the membrane. Fodrin has been hypothesized to regulate the number of receptor binding sites on neuronal membranes for the putative neurotransmitter L-glutamate. Micromolar calcium concentrations activate the thiol protease calpain I, induce fodrin degradation and more than double the density of glutamate binding sites; these effects are all blocked by thiol protease inhibitors. We have now used specific antibodies to examine further the role of fodrin proteolysis in regulating glutamate receptors. We report that fodrin antibodies block the fodrin degradation and increase in glutamate binding normally induced by calcium, and so provide direct evidence for control of membrane receptors by a non-erythroid spectrin.

Animals↗
PubMedDate not supplied

Proteasome inhibitor and lymphocyte function: partial inhibition of cell-mediated cytotoxicity and implication that the lymphocyte proteasome may contain multiple chymotryptic domains.

The multicatalytic proteinase complex or proteasome possesses at least 4 distinct proteolytic activities. We have previously reported that the chymotrypsin-like activity of the rat natural killer cell proteasome may play a role in natural killer (NK) cell-mediated cytotoxicity or IL-2 activated NK (A-NK) cell-mediated cytotoxicity. Using a series of novel, Cephalon, Inc, synthetic proteasome inhibitors (CEP-1508, CEP-1612 and CEP-3117) which have been reported to be specific for the chymotrypsin-like activity of the proteasome, we have further investigated the possible role of the proteasome, with emphasis on the chymotryptic activity components, in cell-mediated cytotoxicity. We now report that these compounds can inhibit the rat NK proteasome in a dose dependent manner. Nevertheless, there is only a 50% inhibition of A-NK cell-mediated cytotoxicity. These results confirm and extend our previous results that the proteasome contributes, at least in part, to cell-mediated cytotoxicity. However, as anticipated, since multiple molecular pathways contribute to cell-mediated cytotoxicity, the proteasome contributes only partially to NK cell-mediated cytolytic reactivity. The exact role of the proteasome in NK cell-mediated killing, and whether single or multiple chymotryptic domains function directly or indirectly, remains to be fully determined.

Animals↗