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M J Carrier

Publications and source records attributed to M J Carrier.

32 records · Page 2Linked to original sources
PubMed1997

Investigation of role for oxidant stress in vascular tolerance development to glyceryl trinitrate in vitro.

1. The role of reactive oxygen species (ROS) during the development of vascular cellular tolerance to glyceryl trinitrate (GTN), was studied in the rat isolated aorta. 2. Nitrate tolerance induced by a 30 min incubation with GTN (30 or 100 microM) in vitro, was not affected by pretreatment with the intracellular superoxide anion scavenger, tiron (10 mM), or the intracellular scavenger of peroxynitrite anion and hydroxyl radical, dimethylsulphoxide (DMSO, 0.2% v v-1). In contrast, pretreatment with the intracellular sulphydryl donor, N-acetyl-L-cysteine (NAC, 1 mM), significantly attenuated GTN-induced tolerance. 3. Pretreatment with a putative inhibitor of oxidant stress-mediated, transcription factor NF-kappa B activation, pyrrolidine dithiocarbamate (PDTC, 50 microM), an inhibitor of gene activation by NF-kappa B, dexamethasone (1 microM) or an inhibitor of protein synthesis, cycloheximide (10 microM), failed to affect tolerance development to GTN. 4. Pretreatment with DMSO (0.2% v v-1) or PDTC (50 microM) depressed non-tolerant vasorelaxation to GTN (1 nM 1 microM) per se. 5. Tiron (10 mM) abolished the reduction of ferricytochrome c by a superoxide anion generating system, assessed photometrically in vitro. In contrast, DMSO (0.2% v v-1), NAC (1 mM) and PDTC (50 microM) were without effect. 6. Our data suggests that neither oxidant stress nor nuclear activation, is important in the development of cellular tolerance to GTN in rat isolated aortic smooth muscle.

Animals↗
PubMed1996

Investigation of endogenous nitric oxide vascular function in the carotid artery of cholesterol-fed rabbits.

1. The function of endogenous nitric oxide (NO) at the level of vascular smooth muscle, was assessed in a popular experimental model of accelerated atherosclerosis, the cholesterol-fed rabbit. 2. Endothelium-dependent vasorelaxation in response to acetylcholine (ACh, 1 microM) was significantly impaired in the carotid artery from rabbits maintained on a 1% (W/W) cholesterol diet for 8-10 weeks. Furthermore, the ability of an inhibitor of nitric oxide synthase (NOS), NG-nitro-L-arginine methyl ester (L-NAME, 1-300 microM), to enhance the contractile reactivity to a submaximal concentration of noradrenaline (NA, 3 microM), was significantly attenuated in hypercholesterolaemia. 3. A significant linear correlation between the maximal contractile effect of L-NAME (300 microM) and maximal vasorelaxation to ACh (1 microM) was determined in the carotid artery from control rabbits. In contrast, no such linear correlation was found in the carotid artery from hypercholesterolaemic rabbits. 4. We conclude that there are lesions both in agonist-stimulated, endogenous NO-dependent vasorelaxation and in the regulation of vasoconstrictor reactivity by endogenous NO in the hypercholesterolaemic rabbit carotid artery. Furthermore, the normal linear relationship between the contractile effect of L-NAME and vasorelaxation to ACh is lost after cholesterol-feeding.

Acetylcholine↗
PubMed1995

pp125FAK tyrosine kinase activity is not required for the assembly of F-actin stress fibres and focal adhesions in cultured mouse aortic smooth muscle cells.

The observed increase in phosphotyrosine content of focal adhesion-associated proteins, in response to integrin engagement, indicates a role for integrin-regulatable tyrosine kinase(s) in cytoskeletal re-organisation. The tyrosine kinase pp125FAK, by virtue of its focal adhesion localisation in fibroblasts, represents a prime candidate to perform this function. We have investigated whether pp125FAK performs a similar function in mouse aortic smooth muscle cells (MASMC). MASMC cultured for 16 hours exhibit F-actin stress fibres and focal adhesions. We have shown that vinculin, pp125FAK and tyrosine-phosphorylated proteins are localised in focal adhesions during this time period. MASMC, under these culture conditions exhibit elevated pp125FAK tyrosine kinase activity, as measured by an increased autophosphorylation potential. We investigated the development of F-actin stress fibres and focal adhesions in MASMC in response to adherence to fibronectin, conditions shown to promote cytoskeletal reorganisation in fibroblasts. Within 30 minutes, MASMC exhibited well-developed F-actin stress fibres and prominent focal adhesions which immunostained intensely for vinculin, pp125FAK and phosphotyrosine. Adherence to fibronectin has been reported to activate pp125FAK tyrosine kinase in fibroblasts, leading to the proposal that pp125FAK plays a critical role in focal adhesion formation. Therefore pp125FAK activation, in response to adherence to fibronectin, was investigated in MASMC. Anti-phosphotyrosine immunoblotting and in vitro kinase assays of MASMC lysates have revealed that, under conditions which promote focal adhesion formation, pp125FAK remains inactive. Since overnight cultures of MASMC exhibited elevated pp125FAK tyrosine kinase activity, we investigated whether these cells deposit their own combination of extracellular matrix (ECM) molecules and/or secrete factors into their conditioned medium which are capable of activating pp125FAK tyrosine kinase. Our results indicate that MASMC-elaborated ECM, but not their conditioned medium, supported pp125FAK tyrosine kinase activation. Furthermore, MASMC exposed to MASMC-ECM displayed a poorly defined F-actin stress fibre network and rudimentary focal adhesions. Thus we have demonstrated the existence of two adhesion-mediated situations in MASMC; one in which fibronectin promotes cytoskeletal reorganisation in the absence of pp125FAK tyrosine kinase activity and the other in which cells adhering to MASMC-ECM display elevated pp125FAK tyrosine kinase activity in association with an impaired ability to promote F-actin stress fibre and focal adhesion formation. These results indicate that in MASMC, pp125FAK tyrosine kinase activity is not involved in F-actin stress fibre assembly and focal adhesion formation.

Actins↗
PubMed1992

Expression of human IL-1 beta in Salmonella typhimurium. A model system for the delivery of recombinant therapeutic proteins in vivo.

The feasibility of using Salmonella typhimurium aroA mutant (SL3261) to deliver protein therapeutic agents was investigated in a murine model system. We have constructed an Escherichia coli expression plasmid designed to express the human protein IL-1 beta. This plasmid expresses IL-1 beta to high levels (greater than 30% total cell protein) in E. coli. In Salmonella the IL-1 beta is expressed constitutively to about 10% total cell protein, as verified by Western blotting analysis using polyclonal rabbit anti-IL-1 beta antibody. The protein is produced in a soluble and biologically active form. BALB/c mice administered orally or i.v. with S. typhimurium aroA mutants carrying the plasmid produced highly significant antibody responses against human IL-1 beta as determined by a solid-phase RIA. Furthermore, mice injected with the construct were significantly protected against lethal gamma-irradiation (850 rad). This study therefore demonstrates that the vaccine strain of Salmonella mutants can also be used effectively to deliver therapeutic proteins in vivo.

Animals↗
PubMed1991

Amino acid misincorporation during high-level expression of mouse epidermal growth factor in Escherichia coli.

To determine whether the high-level expression of foreign proteins in Escherichia coli can lead to frequent translational errors, we analyzed amino acid misincorporation in mouse epidermal growth factor (mEGF) produced as a TrpE fusion protein. The mEGF DNA does not encode phenylalanine and determining the phenylalanine content of the purified protein will measure missense errors. Using this approach, we found an error frequency of about 1 in 40 for codons differing by a single base from those for phenylalanine. This is at least ten times higher than the error rate found for normal E. coli protein synthesis and may be due to limiting supply of charged tRNAs and GTP, brought about by the high-level production of the heterologous protein. The unexpectedly high error rate has implications for the clinical use of E. coli-derived therapeutic proteins.

Amino Acids↗
PubMed1990

Identification of mRNA for PDGF B-chain in human megakaryocytes isolated using a novel immunomagnetic separation method.

A rapid and simple method has been developed for the separation of pure populations of intact human megakaryocytes from whole bone marrow. Megakaryocytes were specifically recognized using monoclonal antibodies coupled to magnetizable articles. Cells labelled with the magnetizable probe were separated from unlabelled cells by introduction of a magnetic field. The technique yields megakaryocyte suspensions with a purity of greater than 98%. Electron microscope examination showed that the ultrastructure of the isolated megakaryocytes was well preserved. Using this method of cell purification, we have investigated expression of the gene for platelet-derived growth factor (PDGF). RNA was isolated from cells harvested from either ribs or posterior iliac crest. The RNA was spotted onto nitrocellulose, and then hybridized using a c-sis riboprobe specific for PDGF B chain mRNA. We demonstrate that mRNA for PDGF B-chain is identifiable in samples of 50,000 cells. We conclude that PDGF is synthesized by the megakaryocyte.

Antibodies, Monoclonal↗
PubMed1984

An effect of codon context on the mistranslation of UGU codons in vitro.

Effects of codon context on nonsense codon suppression may act either through release factor recognition of termination codons or aminoacyl-tRNA selection by the ribosome. The latter hypothesis has been studied by comparing misreading by Escherichia coli UGA suppressor tryptophan tRNA of UGU (cysteine) codons in two synthetic polymers, poly(U-G) and poly( U5 , G), which differ in sequence around the UGU codons. In vitro translation of these polymers in a cell-free system from E. coli yielded selection errors of 4 X 10(-3) and 1.75 X 10(-2) for UGU codons in poly(U-G) and poly( U5 , G), respectively. This difference suggests that codon context may significantly affect misincorporation of amino acids into protein.

Codon↗
PubMedDate not supplied

Endothelial cell dysfunction and the pathogenesis of diabetic macroangiopathy.

Type 2 diabetes mellitus (DM) represents a high risk condition for the development of atherosclerotic and thromboembolic macroangiopathy, which make major contributions to diabetic mortality and morbidity. While many cardiovascular risk factors are common to both atherosclerosis and Type 2 DM, the enhanced risk of diabetic macroangiopathy may be attributable to additional pro-atherogenic mediators associated with insulin resistance syndrome. Given the central pathogenic role of endotheliopathy in atherosclerosis, it is likely that this vascular monolayer is the ultimate target of injury in response to such mediators. Furthermore, a pro-oxidative, dysfunctional endothelium may actively contribute to the pro-atherogenic environment through an inappropriate regulation of vascular tone, permeability, coagulation, fibrinolysis, cell adhesion and proliferation. Such dysfunction may mediate hypertension, dyslipidaemia and altered haemostasis, in addition to aggravating in vivo insulin resistance.

Arteriosclerosis↗
PubMedDate not supplied

Endothelin-1 synthesis reduced by red wine.

Statistical evidence of reduced coronary heart disease in areas of high wine consumption has led to the widespread belief that wine affords a protective effect. Although moderate drinking of any alcohol helps to reduce the incidence of coronary heart disease, there is no clear evidence that red wine confers an additional benefit. Here we show that red wines strongly inhibit the synthesis of endothelin-1, a vasoactive peptide that is crucial in the development of coronary atherosclerosis. Our findings indicate that components specific to red wine may help to prevent coronary heart disease.

Animals↗