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Biomedical subjects

C M Newman

Publications and source records attributed to C M Newman.

33 records · Page 2Linked to original sources
PubMed1993

Post-translational processing of Schizosaccharomyces pombe YPT5 protein. In vitro and in vivo analysis of processing mutants.

SpYPT5p is a member of the rab/YPT small GTP-binding protein family, which is believed to be involved in the regulation of intracellular trafficking. The protein sequence terminates with a CXC motif, and in our previous report (Newman, C. M. H., Giannakouros, T., Hancock, J. F., Fawell, E. H., Armstrong, J., and Magee, A. I. (1992) J. Biol. Chem. 267, 11329-11336) we have shown that SpYPT5p is prenylated both in vivo and in vitro, where geranylgeranylation was confirmed, and carboxyl-methylated. In order to dissect the role of prenylation of each cysteine, we have generated C-terminal mutants where either one or both cysteine(s) were replaced by serine and expressed them in vitro in reticulocyte lysates and in vivo in transfected COS cells. Our results suggest that both cysteines of the CXC motif are prenylated but that the rate of prenylation of the two cysteines is different. The upstream cysteine was found to be preferentially prenylated in reticulocyte lysates unless cytosol from COS cells was added. A separate activity could therefore be required for prenylation of the second cysteine, or the presence of an additional factor is needed to allow accumulation of doubly prenylated SpYPT5p. However, the modification of the upstream cysteine is not a prerequisite for the prenylation of the other. Furthermore, gene replacement in Schizosaccharomyces pombe revealed that each cysteine of the CXC motif can individually support function. Carboxyl methylation occurred only on protein which had been prenylated on the C-terminal cysteine and was required for efficient membrane binding in vitro.

Base Sequence↗
PubMed1993

Processing of the small GTP-binding protein SpYPT1p in Schizosaccharomyces pombe and in mammalian COS cells.

SpYPT1p belongs to a family of ras-like GTP-binding proteins which is believed to be involved in the regulation of intracellular vesicular trafficking. We have analyzed the processing of this protein in its natural environment, the fission yeast Schizosaccharomyces pombe, and when expressed in transfected mammalian COS cells. In COS cells SpYPT1p exists in two forms: a cytosolic 24 kDa protein which represents the unprocessed precursor form and a prenylated 23.5 kDa protein which is equally distributed between membranes and cytosol. In contrast, in S.pombe we have been unable to detect any prenylation of the protein, despite the presence of a potential C-terminal CysCys prenylation site. In addition a 23.5 kDa form was localized exclusively in the cytosol and a third membrane-bound 23 kDa form was also detected. Pulse-chase experiments revealed that in S.pombe SpYPT1p is co-translationally or immediately after translation converted to the 23.5 kDa form which is then rapidly processed to the 23 kDa membrane bound form. We have been unable to detect any significant soluble pool of the protein.

Animals↗
PubMed1993

A novel N-terminal motif for palmitoylation of G-protein alpha subunits.

We have examined the post-translational processing of G alpha subunits expressed endogenously in rat PC12 and NG108-15 rat/mouse hybrid cells, and after transfection of cDNA expression constructs into COS cells. Thioester-linked palmitoylation of alpha o, alpha s, alpha q/alpha 11 and alpha 12 has been detected by metabolic labelling with [3H]palmitate and immunoprecipitation. Palmitoylation of alpha o occurs post-translationally in cells treated with protein-synthesis inhibitors, suggesting possible dynamic acylation. Palmitoylation of the C-terminal CAAX motif has been excluded. Site-directed mutagenesis of alpha o has been used to implicate the site of modification as a cysteine residue next to the N-terminal myristoylated glycine, in a novel protein-lipid modification motif Met-Gly-Cys. The non-palmitoylated alpha o mutant is still myristoylated but shows reduced membrane binding, suggesting that reversible palmitoylation may regulate G alpha localization and function.

Acylation↗
PubMed1992

Post-translational processing of Schizosaccharomyces pombe YPT proteins.

ras proteins are post-translationally processed at their carboxyl-terminal CAAX motif by a triplet of modifications: prenylation of C with farnesyl, proteolytic trimming of AAX, and carboxyl-methylation. These modifications co-operate with palmitoylation of nearby sites or a polybasic region to target plasma membrane localization. The related YPT/rab proteins in contrast are localized to compartments of the endo-membrane system and may be involved in directing membrane traffic. These proteins end in XCC or CXC motifs. We have analyzed the processing of members of this subfamily form the fission yeast Schizosaccharomyces pombe. We find using in vitro translation in reticulocyte lysates that YPT1, -3, and -5 are prenylated with geranylgeranyl and that they incorporate label from [3H]mevalonic acid when expressed in transfected COS cells in vivo. Furthermore, prenylation was necessary for membrane binding in vivo. The CXC protein YPT5, but neither of the two XCC proteins YPT1 and YPT3, was carboxyl-methylated in S. pombe and in COS cells in vivo. However, YPT5 was not carboxyl-methylated in vitro in lysates which were able to methylate ras protein. YPT3 was detectably palmitoylated when expressed in COS cells, though at a much lower level than ras.

Amino Acid Sequence↗
PubMed1992

Inhibition in the rat of nitric oxide synthesis in vivo does not attenuate the hypotensive action of acetylcholine, ATP or bradykinin.

The hypotensive action of acetylcholine in vivo may be dependent on the release of the novel vasorelaxant, endothelium-derived relaxing factor (EDRF)/nitric oxide (NO), by the vascular endothelium. However, using two different inhibitors of NO synthesis, NG-monomethyl-L-arginine (L-NMMA, 100 mg/kg) and NG-nitro-L-arginine methyl ester (L-NAME, 100 mg/kg), we have been unable to attenuate the hypotensive action of intravenous (i.v.) boluses of acetylcholine in anesthetized rats. L-NMMA also failed to alter the hypotensive effect of i.v. bradykinin and adenosine triphosphate (ATP). NO generation by a column of cultured endothelial cells was, however, completely abolished by L-NMMA. The hypotensive effect of acetylcholine was not affected by glibenclamide at a dose which blocks the effect of i.v. cromakalim, a drug which opens ATP-sensitive K+ channels. The rapid hypotensive response to i.v. bolus acetylcholine, ATP and bradykinin remains unexplained.

Acetylcholine↗
PubMed1990

Response of angiographically normal and atherosclerotic left anterior descending coronary arteries to acetylcholine.

Acetylcholine-induced constriction of human coronary arteries in vivo is commonly attributed to endothelial dysfunction. To examine the effects of 2 other important determinants of vascular responses--namely, agonist concentration and the segment of circulation under study--the diameters of proximal, middle and distal segments of the left anterior descending artery (LAD) and coronary sinus oxygen saturation were measured in 10 patients with angiographically normal coronary arteries (group 1) and in 7 patients with coronary atherosclerosis (group 2) after intracoronary acetylcholine was infused at concentrations from 10(-7)M to between 10(-4)M and 10(-2)M. In group 1, acetylcholine caused minor (less than or equal to 6%) but progressive dilatation of the LAD up to 10(-4)M, but constriction, particularly of the distal segments and tertiary branches, occurred at higher concentrations. Over the same concentration range, coronary sinus oxygen saturation rose progressively from a basal level of 36 +/- 3% to a maximum of 72 +/- 3% in the absence of changes in heart rate and blood pressure, suggesting marked progressive dilatation of resistance vessels. Concentrations greater than or equal to 10(-3)M caused intense constriction of distal epicardial vessels and, in some cases, anginal pain and objective signs of ischemia. Conversely, in group 2, acetylcholine (infused only up to 10(-4)M for ethical reasons) failed to cause significant changes in LAD diameter. These data suggest that the local acetylcholine concentration and coronary vascular segment under study may determine the observed response to at least an equivalent extent as does the presence or absence of coronary atherosclerosis, raising the question of whether a constrictor response to intracoronary acetylcholine reliably indicates the presence of coronary atherosclerosis.

Acetylcholine↗
PubMed1990

Stability of a novel dithranol ointment formulation, containing ascorbyl palmitate as an anti-oxidant.

An emulsifying ointment-based dithranol formulation is described which was, with the addition of 0.1% w/w ascorbyl palmitate, stable for 52 weeks at room temperature. This is in contrast to the more conventionally used dithranol in Lassar's paste, which is stable for only 4 weeks at room temperature. An emulsifying ointment-based paste was additionally more convenient to use since it was easier to apply and remove.

Anthralin↗
PubMed1990

Rapid tolerance to the hypotensive effects of glyceryl trinitrate in the rat: prevention by N-acetyl-L- but not N-acetyl-D-cysteine.

1. A new model of tolerance to the hypotensive effect of organic nitrates has been developed in the rat. 2. The fall in mean arterial pressure (MAP) in response to bolus doses of sodium nitroprusside (NP) (4 micrograms kg-1) and glyceryl trinitrate (GTN) (10 micrograms kg-1) was recorded both before and after a 60 min infusion of either 0.9% saline, NP (20 micrograms kg-1 min-1) or GTN (40 micrograms kg-1 min-1). 3. The hypotensive effects of NP or GTN were unchanged following saline infusion, but were reduced in both cases by approximately 40% following the infusion of NP. 4. Infusion of GTN for 60 min virtually abolished the hypotensive effect of a GTN bolus (i.e. nitrate tolerance), whilst the effect of a NP bolus was reduced only to a similar extent (30%) as after an infusion of NP. This latter effect is attributed to a degree of non-specific cross-tolerance between GTN and NP. 5. Co-treatment of a group of rats with N-acetyl-L-cysteine (L-NAC) prevented the development of nitrate tolerance, confirming the role of thiols in this phenomenon, whereas N-acetyl-D-cysteine (D-NAC) did not. 6. The stereospecificity in the effect of NAC in preventing this specific tolerance to GTN suggests that the interaction between GTN and NAC and/or cysteine involves an enzyme-dependent step. 7. NAC was unable to prevent the non-specific cross-tolerance to NP which followed infusion of GTN, suggesting that the mechanism does not directly involve NAC and/or cysteine.

Acetylcysteine↗
PubMed1989

Enalapril for severe heart failure in infancy.

Eight infants aged between 4 days and 12 weeks with severe heart failure that was refractory to optimal conventional treatment with diuretics were treated with enalapril. The starting dose was 0.1 mg/kg/day, increasing according to response to 0.12-0.43 mg/kg/day. One infant with severe myocarditis did not tolerate enalapril because of hypotension and later died of intractable heart failure. Six of the remaining patients had congenital systemic to pulmonary shunts and one had a simple aortic coarctation. Two weeks after starting enalapril the clinical features of heart failure had improved in all the infants, the mean (SEM) plasma sodium concentration had increased from 129 (2.4) to 136 (1.1) mmol/l and plasma urea concentration had fallen from 7.0 (0.85) to 2.9 (0.85) mmol/l. These data suggest that enalapril is a potentially useful treatment for severe heart failure in infancy.

Dose-Response Relationship, Drug↗
PubMed1988

The importance of bradykinin and histamine in the skin response to antigen.

On three separate occasions 12 atopic subjects were injected intradermally with two doses of antigen and one of saline as control. Pretreatment with terfenadine 60 mg orally significantly inhibited the flare response to both the lower dose of antigen and to saline (P less than 0.05). Ingestion of enalapril 5 mg orally 3 h before increased the flare response to both doses of antigen. Neither enalapril nor terfenadine affected the weal response when compared with placebo. Both endogenous histamine and bradykinin appear to be released during the intradermal flare response but are not important in the weal reaction to antigen.

Adult↗