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Biomedical subjects

A Aitken

Publications and source records attributed to A Aitken.

At least 91 records · Page 5Linked to original sources
PubMed1992

Enteroaggregative Escherichia coli strains secrete a heat-labile toxin antigenically related to E. coli hemolysin.

A protein toxin of approximately 120,000 Da secreted by nonhemolytic enteroaggregative Escherichia coli strains cross-reacted in Western blots (immunoblots) with antibodies raised against the C-terminal region of E. coli hemolysin. Treatment of HEp-2 cells with enteroaggregative E. coli or culture supernatants caused elevation of intracellular calcium and stimulated calcium-dependent protein phosphorylation.

Antigens, Bacterial↗
PubMed1991

Chemical modification of serine at the active site of penicillin acylase from Kluyvera citrophila.

The site of reaction of penicillin acylase from Kluyvera citrophila with the potent inhibitor phenylmethanesulphonyl fluoride was investigated by incubating the inactivated enzyme with thioacetic acid to convert the side chain of the putative active-site serine residue to that of cysteine. The protein product contained one thiol group, which was reactive towards 2,2'-dipyridyl disulphide and iodoacetic acid. Carboxymethylcysteine was identified as the N-terminal residue of the beta-subunit of the carboxy[3H]methylthiol-protein. No significant changes in tertiary structure were detected in the modified penicillin acylase using near-u.v. c.d. spectroscopy. However, the catalytic activity (kcat) with either an anilide or an ester substrate was decreased in the thiol-protein by a factor of more than 10(4). A comparison of sequences of apparently related acylases shows no other extensive regions of conserved sequence containing an invariant serine residue. The side chain of this residue is proposed as a candidate nucleophile in the formation of an acyl-enzyme during catalysis.

Amino Acid Sequence↗
PubMed1991

Purification of a 20 kDa phosphoprotein from epithelial cells and identification as a myosin light chain. Phosphorylation induced by enteropathogenic Escherichia coli and phorbol ester.

Previous studies on the mechanism of enteropathogenic Escherichia coli (EPEC) infection have revealed an increase in the phosphorylation state of a number of proteins in human laryngeal HEp-2 cells. The most prominent was an acidic phosphoprotein(s) of Mr 20-21 kDa. The present study reports: (a) a simple method for purification of phosphorylated 20 kDa protein; (b) identification of the 20 kDa phosphoprotein as myosin light chain; and (c) that the phorbol ester, TPA, also increased the phosphorylation of the 20 kDa myosin light chain. In contrast to the effects of EPEC, TPA stimulation resulted in the dissociation of myosin from the cytoskeleton to the cytosol.

Amino Acids↗
PubMed1991

Removal of N-acetyl groups from blocked peptides with acylpeptide hydrolase. Stabilization of the enzyme and its application to protein sequencing.

Acylpeptide hydrolase, an enzyme that removes the modified residue from N-terminally acetylated peptides, has been purified from ovine liver and developed as a tool in sequencing blocked peptides and proteins. Its instability imposes a major limitation on the use of the mammalian enzyme in protein chemistry. Coupling to Sepharose followed by intramolecular cross-linking with dimethyl-suberimidate increased its thermostability and rendered it more resistant to inactivation by either SDS or N,N-dimethylformamide. The resulting enzyme preparation is reusable and more effective at cleaving longer acetylated peptides. It is therefore useful for unblocking acetylated proteins prior to protein sequence analysis. Intact proteins and many isolated peptides are still too large to be cleaved directly, but in this paper we describe a procedure for overcoming this difficulty. The protein is fragmented and non-acetylated peptides are then absorbed out with isothiocyanato-glass. The N-terminal peptide remains in solution and is unblocked with stabilised acylpeptide hydrolase. No chromatographic separation are required. The N-terminal sequence can then be obtained by automated Edman degradation. This procedure has been successfully demonstrated on a large synthetic peptide.

Amino Acid Sequence↗
PubMed1991

Enzyme-mediated peptide synthesis using acylpeptide hydrolase.

Acylpeptide hydrolase is shown to catalyse the specific addition of a single amino acid to the N-terminus of a peptide. The stabilised Sepharose-coupled form of the enzyme is used to couple a carboxy-methylated N-formyl (or N-acetyl) amino acid to a short pre-existing peptide. The yield is improved by optimal timing of the reaction and the presence of moderate concentrations (5%) of N,N-dimethylformamide. Two tripeptides, Ac-Ala-Ala-Ala and fMet-Leu-Phe (f, formyl) were synthesized by this technique (in yields of 2% and 0.064% respectively). The products were characterised by HPLC, amino acid analysis, mass spectroscopy and protein sequencing. The synthetic fMet-Leu-Phe also had biological activity, in that it stimulated superoxide generation by granulocytes. Acylpeptide hydrolase could therefore be a very useful tool for the synthesis and modification of peptides.

Amino Acids↗
PubMed1991

A protein secreted in vivo by Echinococcus granulosus inhibits elastase activity and neutrophil chemotaxis.

A cDNA encoding the carboxy-terminal of the 12-kDa subunit of antigen B of Echinococcus granulosus has been cloned and sequenced. In addition, an amino acid sequence has been generated for the amino-terminal which is tentatively contiguous with the open reading frame of the DNA-derived sequence. Comparison of the inferred sequence of the 12-kDa antigen with other known sequences indicated a limited similarity to alpha-1 antitrypsin. In functional assays, gel-purified native 12-kDa antigen from natural infections inhibited elastase but not trypsin or chymotrypsin, providing further evidence that this antigen is a parasite protease inhibitor. Possibly unrelated to its anti-protease activity but a potentially important function of the 12-kDa antigen was its ability to inhibit recruitment of neutrophils. These functions may be important to the viability of the parasite in the face of the host immune response. In addition, the match between the DNA-derived sequence and the protein sequence was imperfect, with some residues having, according to the amino acid sequencing, two alternatives in approximately equal concentrations, and four DNA-derived residues failing to match with the protein sequence at all. The 12-kDa antigen may be expressed as isoforms from a polymorphic gene and, as far as aware, this observed sequence polymorphism has not, to date, been described for any other flatworm antigen.

Amino Acid Sequence↗
PubMed1991

Elevation of intracellular free calcium levels in HEp-2 cells infected with enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) are a class of diarrheagenic organisms that induce a characteristic attaching and effacing lesion in enterocytes and various cultured cell lines. Infection of cultured HEp-2 cells by EPEC isolates 2036-80 (serotype O119) and E2348-69 (serotype O127) resulted in significant elevation of intracellular free calcium levels, determined quantitatively with the fluorescent calcium indicator dye 2-([2-bis(carboxymethyl)amino-5-methylphenoxy]methyl)-6-methoxy-8- bis(carboxymethyl)aminoquinoline. This effect, which was not observed on infection with non-lesion-forming E. coli strains, was inhibited by dantrolene, a drug that prevents calcium mobilization from intracellular stores. Moreover, activated protein kinase C in infected cells was dissociated from cell membranes by a process that was inhibited by cyclosporin A, suggesting involvement of the calcium-dependent protease calpain. A qualitative method for observing intracellular calcium fluxes by fluorescence microscopy with the recently described fluorescein-based indicator fluo-3 was used to screen a collection of well-characterized E. coli isolates from patients with infantile enteritis. Increased localized calcium-dependent fluo-3 fluorescence was observed only in HEp-2 cells infected with known lesion-forming EPEC strains. We propose that enhancement of intracellular free calcium levels in enterocytes infected with EPEC would result in formation of the characteristic lesion by calcium-dependent activation of actin-depolymerizing proteins, with eventual loss of absorptive capacity.

Calcium↗
PubMed1991

Pyroglutamylglutamylprolineamide is present in rat anterior and posterior pituitary gland.

A new TRH-like peptide pyroglutamylglutamylprolineamide (pGlu-Glu-ProNH2) has recently been purified and characterized from both the rabbit prostate complex and human semen. In this study, TRH-immunoreactive peptides were extracted from anterior pituitary, posterior pituitary and hypothalamus and subjected to gel exclusion chromatography. For each tissue, TRH was resolved from pGlu-Glu-ProNH2 by anion-exchange chromatography at pH 7.6. In the anterior pituitary, 63% of the TRH immunoreactivity was chromatographically identical to pGlu-Glu-ProNH2 whereas in the posterior pituitary the new peptide represented less than 5% of the total TRH immunoreactivity. Only trace levels of pGlu-Glu-ProNH2 were observed in hypothalamus, suggesting that the acidic TRH-related peptide found in the anterior pituitary may not be of hypothalamic origin. The new TRH-like peptide was purified from whole pituitaries by gel exclusion and ion-exchange chromatography, followed by high power liguid chromatography and was shown to have chromatographic properties identical to pGlu-Glu-ProNH2. Amino acid analysis of the purified peptide revealed glutamic acid and proline residues in the ratio Glx:2 Pro:1, which is the expected composition of pGlu-Glu-ProNH2 after acid hydrolysis.

Animals↗
PubMed1990

Muscarinic acetylcholine receptors. Peptide sequencing identifies residues involved in antagonist binding and disulfide bond formation.

Muscarinic acetylcholine receptors (mAChR) were purified from rat brain and labeled either with the site-directed affinity label [3H]propylbenzilylcholine mustard (PrBCM) or with the sulfhydryl-specific label [3H]N-ethylmaleimide (NEM), using a protocol designed to give selective incorporation of the label into disulfide-bonded cysteines. m1 mAChRs were purified from CHO-K1 cells stably expressing the cloned receptor sequence and labeled with [3H]PrBCM. The labeled receptors were cleaved with the lysine-specific protease Lys-C and, after fractionation of the products, subcleaved with cyanogen bromide. Two major CNBr cleavage products were found with a molecular mass of approximately 3.9 and approximately 2.4 kDa, labeled either by [3H]PrBCM or [3H]NEM. The results obtained from CNBr cleavage of purified forebrain receptors were consistent with those obtained from the purified cloned m1 mAChR. Edman degradation was applied to the CNBr peptides. The results were compatible with the attachment of the [3H]PrBCM label to a conserved aspartic acid residue in transmembrane helix 3 of the mAChR (corresponding to Asp-105, m1 sequence) and of [3H]NEM to a conserved cysteine residue (corresponding to Cys-98, m1 sequence). These results support the hypothesis that the cysteine residue participates in a disulfide bond on the extracellular surface of the mAChRs and related G-protein-coupled receptors, while the aspartic acid residue is involved in binding the positively charged headgroup of muscarinic antagonists.

Amino Acid Sequence↗
PubMed1990

Protein kinase C inhibitor proteins. Purification from sheep brain and sequence similarity to lipocortins and 14-3-3 protein.

Potent inhibitors of protein kinase C have been isolated from sheep brain by DEAE-cellulose, phenyl-Sepharose CL-4B and Mono Q anion-exchange chromatography. Analysis by one- and two-dimensional SDS/polyacrylamide gel electrophoresis showed the purified preparation to contain three bands ranging over 29-33 kDa in molecular mass, each consisting of several charge isomers with similar pI values (5.4-5.7). Peptide mapping, amino acid analysis and sequencing suggested that the proteins are related, with the possibility that some species are distinct gene products. The concentration of inhibitor proteins required for half-maximal inhibition of protein kinase C activity is 1.7 microM. Inhibitory activity could not be affected by increasing the substrate, cofactor or ATP concentration in the standard protein kinase C assay, but was abolished by heat treatment. The inhibitor preparation did not affect the binding of phorbol dibutyrate to protein kinase C and could inhibit phosphorylation over a wide range of calcium concentrations. Inhibitory activity could be removed by immunoprecipitation of the purified inhibitor proteins with polyclonal antibodies raised against synthetic peptides, the sequences corresponding to those of peptide fragments obtained from protein digests. Amino acid sequence analysis of the inhibitors confirms they are novel proteins although similarities exist with a neuronal specific protein termed 14-3-3 and the carboxy terminus of the calcium-lipid binding series (endonexin/calpactin/lipocortin).

14-3-3 Proteins↗
PubMed1990

Platelet protein phosphorylation and protein kinase C activation by phorbol esters with different biological activity and a novel synergistic response with Ca2+ ionophore.

Phorbol esters with different biological activities have been tested for their ability to induce the phosphorylation of human platelet proteins. We have shown that only the potent platelet aggregatory phorbol esters were able to stimulate the phosphorylation of proteins of 76, 68, 47, 30 and 20 kDa in intact platelets. The ability of these esters to stimulate phosphorylation of the 47-kDa protein ('p47') correlated with their ability to cause platelet aggregation. When a non-platelet aggregatory deoxyphorbol (12-deoxyphorbol 13-phenylacetate 20-acetate) was combined with a subthreshold dose of the Ca2+ ionophore, A23187, a large increase in phosphorylation of p47 and a fourfold decrease in Ka was observed. This was in contrast to a barely detectable stimulation of phosphorylation at micromolar levels of this phorbol ester in the absence of the ionophore. This synergism was not evident for the potent platelet aggregatory derivatives. The Ka for DOPPA with a mixture of total platelet protein kinase C was 530 nM in the absence of calcium decreasing to 120 nM in the presence of calcium. In the presence of calcium, 12-deoxyphorbol 13-phenylacetate 20-acetate was shown to stimulate preferentially one of the isoforms of protein kinase C.

Blood Platelets↗
PubMed1990

Protein phosphorylation by protein kinase C in HEp-2 cells infected with enteropathogenic Escherichia coli.

Infection of HEp-2 monolayers with enteropathogenic Escherichia coli 2036-80 (O119) stimulated phosphorylation of several target cell proteins, the most prominent of which had apparent molecular weights of 21,000 and 29,000. Proteins of the same size were phosphorylated in response to known activators of the calcium-phospholipid-dependent protein kinase C. Screening of clinical isolates of various O serogroups revealed that all strains able to form the characteristic attaching and effacing lesion of enteropathogenic E. coli showed elevated phosphorylation of 21,000- and 29,000-dalton protein species.

Bacterial Adhesion↗
PubMed1989

The distribution, purification, and pharmacological action of an amphibian neuromedin U.

The distribution, primary structure, and relative biological activity of neuromedin U has been determined from the frog Rana temporaria. Following sequential column chromatography of a gastrointestinal extract, the peptide was sufficiently pure to enable characterization by micro-sequence analysis. The entire sequence was found to be an icosapentapeptide which displays marked sequence similarity to both porcine and rat neuromedin U. The sequence of the biologically active, COOH-terminal region is almost completely conserved across all species. Synthetic, COOH-terminally amidated amphibian neuromedin U, like the porcine and rat peptides, stimulates rat uterine contraction in vitro thereby fulfilling the criterion upon which the nomenclature of this peptide family is based. In addition, the peptide demonstrates parallel pressor effects when infused systemically into rats. The high degree of amino acid sequence conservation is indicative of strong evolutionary pressure acting to retain the presence of this possibly physiologically important peptide across the vertebrate subphylum.

Amino Acid Sequence↗
PubMed1989

Sequence analysis of the cloned streptococcal cell surface antigen I/II.

The gene spa P (formerly designated as spa P1) encoding the Mr 185,000 surface antigen (I/II) of Streptococcus mutans, serotype c (NG5), has been sequenced. The gene (4683 bp) encodes a protein of 1561 amino acid residues including putative signal peptide (residues 1-38) and transmembrane (residues 1537-1556) sequences. The N-terminal region (60-550) has alanine-rich repeats and is predicted to be alpha-helical. However, the C-terminal region (800-1540) is proline-rich and favours an extended structure. Except for a short central variable region the sequences appear to be highly conserved for S. mutans serotype c. N-Terminal sequencing of separated antigen I and antigen II polypeptides suggests that the former represents the N-terminal and the latter the C-terminal portions of the intact antigen.

Amino Acid Sequence↗