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M S Shearman

Publications and source records attributed to M S Shearman.

At least 37 records · Page 2Linked to original sources
PubMed1996

Cytotoxicity of prion protein peptide (PrP106-126) differs in mechanism from the cytotoxic activity of the Alzheimer's disease amyloid peptide, A beta 25-35.

The abnormal form of the prion protein (PrPSc), a synthetic prion protein peptide fragment (PrP106-126) and fragments of the Alzheimer's protein precursor, APP, have been shown to be cytotoxic in vitro. We have used synchronous, clonal cell models originally developed to study the toxicity of the Alzheimer's disease amyloid peptide, A beta 25-35, to investigate the actions of PrP peptides. We found that the cytotoxicity of the PrP106-126 depends on its state of aggregation and the cellular expression of PrPc, and is independent of a loss of MTT reduction activity in the absence of cell death associated with the cellular effects of A beta 25-35. These factors may play a role in the lesion specificity of different pathological phenotypes of prion-protein related diseases.

Amino Acid Sequence↗
PubMed1996

Cellular MTT reduction distinguishes the mechanism of action of beta-amyloid from that of tachykinin receptor peptides.

Inhibition of the reduction of the redox dye 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide by rat phaeochromocytoma PC12 cells is a specific, early response to nanomolar concentrations of the beta-amyloid peptide fragment beta (25-35), and appears to be a reliable indicator of the mechanism of beta-amyloid toxicity. Neither selective tachykinin receptor agonists, nor tachykinin receptor peptide and non-peptide antagonists elicited such a response. Furthermore, tachykinin receptor peptides did not block the effects of beta-amyloid in PC12 cells, or in two other beta-amyloid-sensitive cell lines. These experimental model systems allow the mechanism of action of beta-amyloid to be distinguished from that of tachykinin receptor peptides, and prove that the neurotoxic action of beta-amyloid, as measured by the inhibition of cellular 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide reduction is not mediated by an interaction with tachykinin receptors.

Amyloid beta-Peptides↗
PubMed1996

Cyclic AMP-dependent protein kinase but not protein kinase C regulates the cardiac Ca2+ channel through phosphorylation of its alpha 1 subunit.

The voltage-dependent L-type Ca2+ channel in the heart is regulated by cAMP-dependent protein kinase (PKA) and possibly by protein kinase C (PKC). We have investigated the channel modulation through phosphorylation by these protein kinases, using liposomes into which Ca2+ channels from bovine heart were reconstituted. Phosphorylation of the proteoliposomes with PKA increased the dihydropyridine-sensitive Ca2+ efflux from them by about 70%. PKA rapidly phosphorylated membrane proteins of 210 and 170 kDa. A dihydropyridine-class Ca2+ channel blocker, [3H]azidopine, specifically photo-labeled a protein of 210 kDa, suggesting that the 210-kDa phosphoprotein might be the alpha 1 subunit of the Ca2+ channel. In contrast, phosphorylation of the proteoliposomes with PKC failed to modulate the Ca2+ efflux. Although PKC catalyzed the phosphorylation of membrane proteins of 150, 130, 95, 67, and 62 kDa, the 210- and 170-kDa proteins were not phosphorylated by this kinase. These results suggest that phosphorylation of the 210-kDa protein in the cardiac sarcolemma by PKA may be responsible for modulation of the channel function, whereas modulation of the channel by PKC, if it occurs, must be the result of an indirect mechanism, e.g. phosphorylation of a cytoplasmic protein or an associated channel polypeptide, that cannot function in the reconstituted system.

Animals↗
PubMed1995

Sulphated compounds attenuate beta-amyloid toxicity by inhibiting its association with cells.

Agents that interfere with the toxic effects of beta-amyloid protein may be therapeutically useful against Alzheimer's disease. We reported recently that several sulphated glycosaminoglycans and sulphonated dyes attenuate the toxic effects of beta-amyloid fragments beta 25-35 and beta 1-40 in two clonal cell lines. We now demonstrate that this protective effect is due to interference with beta-amyloid cell association rather than effects on beta-amyloid structure. Using an enzyme-linked immunoabsorbance assay to detect cell-associated beta 1-40, we found in a range of compounds a strong correlation between inhibition of HeLa cell association of beta 1-40 and attenuation of cellular toxicity as measured by inhibition of 3-[4,5-dimethylthia-zol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction. In contrast, effects on peptide structure, as measured by Congo red binding, were generally inconsistent with the attenuating effects of the compounds on cellular toxicity. These results suggest that by binding beta-amyloid these agents prevent its interaction with cells.

Amyloid beta-Peptides↗
PubMed1995

Sulfonated dyes attenuate the toxic effects of beta-amyloid in a structure-specific fashion.

We recently reported that several sulfate-containing glycosaminoglycans, a class of compounds associated with the beta-amyloid plaques of Alzheimer's disease, attenuate the toxic effects of beta-amyloid fragments beta 25-35 and beta 1-40. The amyloid-binding sulfonated dye Congo Red was shown to have a similar effect. Using two clonal cell lines, we now demonstrate that several sulfonated dyes attenuate beta-amyloid toxicity and that the protective effect appears specific for compounds whose sulfonate groups can interact with the beta-pleated structure of aggregated amyloid. These results suggest that by binding beta-amyloid these compounds may prevent toxic interactions of the peptide with cells.

Amyloid beta-Peptides↗
PubMed1995

Formation of signal transfer complexes between stem cell and platelet-derived growth factor receptors and SH2 domain proteins in vitro.

Cellular growth and differentiation signals are generated and defined by the interaction of specific phosphotyrosine residues of activated receptor tyrosine kinases (RTKs) and src homology-2 (SH2) domain-containing intracellular signal transducers. This appears to involve for both the p145c-kit and beta platelet-derived growth factor receptor (PDGF-R) cytoplasmic domains the formation of multiprotein signal transfer complexes, which include combinations of noncatalytic and enzymatically active subunits of phosphatidylinositol 3'-kinase (PI3'-K), phospholipase C-gamma (PLC gamma), and guanosine trisphosphatase activating protein (GAP). In vitro association experiments indicate that PLC gamma and PI3'-K bind the beta PDGF-R simultaneously, while these two SH2 proteins compete for association to p145c-kit binding sites, with p85/PI3'-K exhibiting higher affinity. Interestingly, GAP and p85/PI3'-K binding to distinct p145c-kit phosphotyrosines is cooperative, enhancing formation of a heterotetrameric signaling complex, which may include different combinations of p85 alpha and p85 beta with p110, p112, and p116 by interaction with the same tyrosine 721 docking site. The diversity of molecular interactions observed for PDGF-R and p145c-kit suggests a new mode of signal definition and modulation.

Amino Acid Sequence↗
PubMed1995

Sulfated glycosaminoglycans and dyes attenuate the neurotoxic effects of beta-amyloid in rat PC12 cells.

Glycosaminoglycan (GAG)-containing proteoglycans are associated with the neuritic plaques and cerebrovascular beta-amyloid deposits of Alzheimer's disease as well as with the amyloid deposits of prion and other disorders. GAGs and other sulfate-containing compounds have previously been shown to bind beta-amyloid peptide in vitro, suggesting possible effects of beta-amyloid deposition and/or toxicity in vivo. Using reduction of the redox dye 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to measure beta-amyloid neurotoxicity in rat pheochromocytoma PC12 cells, several polysulfated GAGs and synthetic sulfate-containing compounds were found to attenuate the neurotoxic effects of beta-amyloid fragments beta 25-35 and beta 1-40. These results suggest that by binding beta-amyloid these compounds may prevent toxic interactions of the peptide with cells.

Amyloid beta-Peptides↗
PubMed1995

The intracellular component of cellular 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) reduction is specifically inhibited by beta-amyloid peptides.

In vitro cell culture model systems for investigating the biochemical mechanisms involved in the neurodegenerative actions of beta-amyloid peptide (beta-AP) have been established. Using rat pheochromocytoma PC12 or human epitheloid HeLa cell lines, submicromolar concentrations of the beta-AP fragments beta 1-40, beta 1-39, and beta 25-35, but not beta 1-28, were found to inhibit the reduction of the redox dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). In both cell lines, the beta-AP-sensitive component represented approximately 70% of total cellular MTT reduction. When the reduction of a series of structurally related dyes was compared with that of MTT, the reduction of 3 alpha-naphthyl-2-phenyl-5-(4-nitrophenyl)-2H-tetrazolium chloride (NTV) was also found to be sensitive to beta 25-35, but that of seven other redox dyes was not. A property common to MTT and NTV is that they are both readily taken up into PC12 and HeLa cells and do not require an artificial electron coupling agent to be reduced. Microscopic analysis of MTT-formazan product formation in PC12 and HeLa cells following beta 25-35 treatment revealed that it was the intracellular component of the reduction of this dye that was abolished. These results support the hypothesis that the cellular reduction of MTT represents a specific indicator of the initial events underlying the mechanism of beta-AP toxicity.

Amyloid beta-Peptides↗
PubMed1994

Inhibition of PC12 cell redox activity is a specific, early indicator of the mechanism of beta-amyloid-mediated cell death.

An in vitro tissue culture cell model system for investigating the biochemical mechanisms involved in the neurodegenerative actions of beta-amyloid has been established. Using rat pheochromocytoma PC12 cells, it was found that an early, specific response of cells to the beta-amyloid protein or the beta-amyloid fragment 25-35 was a potent inhibition of cellular redox activity, as measured by 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) reduction. This inhibitory response was rapid and occurred at nanomolar concentrations of peptide, concentrations at which no equivalent decreases in cell proliferation or cell survival were observed. The inhibition of PC12 cell MTT reduction was initially reversible upon removal of the peptide; if sustained for several days, however, by repeated peptide application, it became associated with a dramatic reduction in cell survival. Inhibition of MTT reduction may, therefore, be an early indicator of the mechanism of beta-amyloid-mediated cell death.

Amino Acid Sequence↗
PubMed1993

Phosphatidylinositol 3'-kinase associates with p145c-kit as part of a cell type characteristic multimeric signalling complex.

p145c-kit is expressed in cell lineages of diverse origin and appears to regulate distinct cell type characteristic functions. Independent mutations at the murine Dominant White Spotting (W) locus result in the alteration of p145c-kit tyrosine kinase activity and signalling potential, which differentially affects melanocyte migration, germ cell regeneration and hematopoietic cell differentiation. Molecules that may be involved in mediation and definition of p145c-kit signalling pathways were investigated in cell lines of hematopoietic, melanogenic and central nervous system origin. High-affinity association of endogenous cellular proteins with activated p145c-kit was limited to a characteristic set of molecules that correlated with the presence of phosphatidylinositol (PtdIns) 3'-kinase activity. The observed association pattern of proteins was cell type characteristic, and all of the proteins were displaced from the receptor by competition with excess receptor binding subunit of PtdIns 3'-kinase. Our data indicate that PtdIns 3'-kinase associates with p145c-kit as part of a multimeric signalling complex, and suggest that the cell type characteristic composition of this complex influences the signalling potential of p145c-kit in the diverse cell types in which it is expressed and thereby defines its cell type-specific functions.

Amino Acid Sequence↗
PubMed1993

Haemopoietic stem cell development to neutrophils is associated with subcellular redistribution and differential expression of protein kinase C subspecies.

Multipotential FDCP-Mix A4 (A4) cells can be induced either to self-renew or to differentiate and develop into mature neutrophils in liquid culture, depending on the haemopoietic growth factors with which they are cultured. When cultured in low concentrations of interleukin 3 (IL-3, 1 unit/ml)) plus Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) and Granulocyte-CSF (G-CSF), A4 cells proliferate with accompanying development to form cells which resemble mature, postmitotic neutrophils. The presence of high concentrations of IL-3 (100 units/ml) blocks the development of A4 cells even in the presence of GM-CSF plus G-CSF. A4 cell development to neutrophils is accompanied by major changes in the expression of protein kinase C (PKC) subspecies in these cells. The predominant subspecies present in multipotent A4 cells, as judged by direct chromatographic analysis, was the type III enzyme (alpha) subspecies, whereas in mature A4 cell neutrophils, the type II (beta I + beta II) enzymes were predominant. Phorbol esters added to immature A4 cells resulted in a proliferative response, but when added to postmitotic A4 cells resembling neutrophils they elicited a large increase in reactive oxygen intermediate production. This suggests that the type III (alpha) subspecies may mediate proliferative responses in stem cells, whilst the type II (beta I + beta II) enzymes are more important for the mature cell functions of postmitotic neutrophils. In cultures containing IL-3 (100 units/ml) both the type III, and also the type II subspecies were predominantly membrane-associated for prolonged periods (> 24 hours).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Differential effects of W mutations on p145c-kit tyrosine kinase activity and substrate interaction.

The c-kit gene, mapped to the dominant white spotting (W) locus of the mouse (Chabot, B., Stephenson, D. A., Chapman, V. M., Besmer, P., and Bernstein, A. (1988) Nature 335, 88-89; Geissler, E. N., Ryan, M. A., and Housman, D. E. (1988) Cell 55, 185-192), encodes a receptor tyrosine kinase, p145c-kit. Germline mutations at the W locus lead to loss of function alterations in p145c-kit, and result in mice with developmental defects of varying severity in the melanocytic, hematopoietic stem cell, and primordial germ cell lineages. To investigate in more detail the effect of W mutations on p145c-kit signaling, three mutations, W42, Wv, and W41, that confer severe, intermediate, and mild phenotypic characteristics, respectively, were introduced into the human p145c-kit tyrosine kinase domain. These mutations attenuated the intrinsic tyrosine kinase activity of the receptor to different degrees. In addition, they had differential effects on the interaction of the p145c-kit substrates, phospholipase C gamma, GTPase-activating protein, and the receptor-binding subunit of phosphatidylinositol 3'-kinase, p85. Notably, the Wv mutation, while retaining significant receptor tyrosine kinase activity, was unable to bind phospholipase C gamma and GTPase-activating protein, but could still associate with p85. These results suggest that the location of W mutations may be an important determinant of the specificity of substrate association and phosphorylation, and may explain, at least in part, the cell type-specific defects associated with certain W alleles.

Amino Acid Sequence↗
PubMed1991

Protein kinase C subspecies in adult rat hippocampal synaptosomes. Activation by diacylglycerol and arachidonic acid.

Synaptosomes isolated from the adult rat hippocampus contain the alpha- and beta-subspecies of protein kinase C (PKC), but not the gamma-subspecies which is abundantly expressed in the pyramidal cells in this brain region. Although the gamma-subspecies is known to respond significantly to free arachidonic acid, it is found that both the alpha- and beta-subspecies are also activated dramatically by arachidonic acid in synergistic action with diacylglycerol. Oleic, linoleic, and linolenic acids are all active. It is possible that unsaturated fatty acids may take part in the activation of alpha- and beta-subspecies of PKC which are present in the presynaptic nerve endings terminating at the hippocampal pyramidal cells.

Animals↗
PubMed1991

Synaptosomal protein kinase C subspecies: A. Dynamic changes in the hippocampus and cerebellar cortex concomitant with synaptogenesis.

The expression of protein kinase C (PKC) subspecies in synaptosomes prepared from a number of adult brain regions was compared. Cerebral cortical and thalamic/striatal synaptosomes were found to express three peaks of enzyme activity upon hydroxyapatite chromatography, corresponding to the type I(gamma), type II(beta), and type III(alpha) subspecies. Synaptosomes prepared from either the hippocampus or the cerebellar cortex, however, contained only two major peaks, corresponding to the alpha- and beta-subspecies, with barely detectable levels of the gamma-subspecies, even though these tissue areas were enriched in the latter enzyme. When the ontogenic pattern of hippocampal synaptosomal PKC subspecies was examined, it was found that at postnatal day 7, significant quantities of the gamma-subspecies were present and that this subspecies reached its peak levels at around postnatal day 14, before steadily declining to its adult level. Similar changes were observed also for the gamma-subspecies in cerebellar cortex synaptosomes. The dynamic changes in the synaptosomal PKC subspecies take place at a critical period in the development of the rat brain, concomitant with an active period of synaptogenesis, suggesting that it may play a role in synaptogenesis.

Aging↗