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B Dunbar

Publications and source records attributed to B Dunbar.

At least 19 recordsLinked to original sources
PubMed1999

Primary structure of the helical domain of porcine collagen X.

The entire primary structure of the collagen X helical region is presented, including identification of the extensive post-ribosomal modifications by amino acid sequencing and mass spectrometry. As in collagen I, a single residue of 3-hydroxyproline was identified, but for collagen X this was located near the N-terminal end of the helix. Lysine residues in collagen X are extensively hydroxylated/glycosylated: at least 11 sites were localized and shown to be fully glycosylated, exclusively as glucosyl-galactosyl derivatives. The lysine-derived crosslinks, dihydroxylysinonorleucine and hydroxylysinonorleucine, were shown to be present in a 3:2 molar ratio primarily within the C-terminal portion of the helix.

Amino Acid Sequence↗
PubMed1999

The covalent attachment of polyamines to proteins in plant mitochondria.

Plant mitochondria from both potato and mung bean incorporated radioactivity into acid insoluble material when incubated with labelled polyamines (spermine, spermidine and putrescine). Extensive washing of mitochondrial precipitates with trichloroacetic acid and the excess of cold polyamine failed to remove bound radioactivity. Addition of nonradioactive polyamine stopped further incorporation of radioactivity but did not release radioactivity already bound. The radioactivity is incorporated into the membrane fraction. The labelling process has all the features of an enzymatic reaction: it is long lasting with distinctive kinetics peculiar to each polyamine, it is temperature dependent and is affected by N-ethylmaleimide. The latter inhibits the incorporation of putrescine but stimulates the incorporation of spermine and spermidine. Treatment of prelabelled mitochondria with pepsin releases bound radioactivity thus indicating protein to be the ligand for the attachment of polyamines. HPLC of mitochondrial hydrolysates revealed that the radioactivity bound to mitochondria is polyamines; traces of acetyl polyamines were also found in some samples. On autoradiograms of SDS/PAGE gels several radioactive bands of proteins were detected. Protein sequencing of labelled spots from a 2D gel gave a sequence which was 60% identical to catalase. We suggest that the attachment of polyamines to mitochondrial proteins occurs cotranslationally possibly via transglutaminases.

Amino Acid Sequence↗
PubMed1998

Structural characterization of viral neutralizing monoclonal antibodies to hepatitis B surface antigen.

In this study we describe the viral neutralizing activity of murine monoclonal antibodies (MAb) specific for hepatitis B surface antigen (HBsAg). This viral neutralizing activity was assessed in vitro by employing Hepatitis Delta Virus (HDV) and human hepatocytes as target cells. To further characterize these viral neutralizing antibodies we generated a panel of anti-idiotypic (anti-Id) reagents and serologically characterized these antibodies for epitope specificity, Id specificity, and Id heterogeneity. Direct binding and competitive inhibition solid phase enzyme immunoassay have demonstrated that two murine MAb specific for HBsAg (anti-HBs), designated A1.2 and A3.1, recognize similar or overlapping epitopes on HBsAg, while monoclonal anti-HBs, designated A2.1 recognizes a unique HBsAg epitope. Further, Id analysis using monoclonal and polyclonal anti-Id reagents have identified both a private and a cross-reactive Id, respectively, on the anti-HBs, A1.2 preparation. The source of the idiotypic cross-reactivity between A1.2 and A3.1 has been identified, using Western blot analysis, to conformational determinants expressed by the heavy (H) and light (L) chains of these monoclonal anti-HBs. Lastly, the intrastrain antibody repertoire induced following HBsAg immunization was found to be relatively restricted in heterogeneity by clonotype analysis using isoelectric focusing and affinity immunoblot analysis. Interspecies variability in the anti-HBs response was observed based on epitope recognition using purified anti-HBs from a variety of species.

Animals↗
PubMed1998

From famine to feast: the role of methylglyoxal production in Escherichia coli.

The enzyme methylglyoxal synthase (MGS) was partially purified from Escherichia coli extracts, and the amino-terminal sequence of candidate proteins was determined, based on the native protein being a tetramer of about 69 kDa. Database analysis identified an open reading frame in the E. coli genome, YccG, corresponding to a protein of 16.9 kDa. When amplified and expressed from a controlled promoter, it yielded extracts that contained high levels of MGS activity. MGS expressed from the trc promoter accumulated to approximately 20% of total cell protein, representing approximately 900-fold enhanced expression. This caused no detriment during growth on glucose, and the level of methylglyoxal (MG) in the medium rose to only 0.08 mM. High-level expression of MGS severely compromised growth on xylose, arabinose and glycerol. A mutant lacking MGS was constructed, and it grew normally on a range of carbon sources and on low-phosphate medium. However, the mutant failed to produce MG during growth on xylose in the presence of cAMP, and growth was inhibited.

Carbon-Oxygen Lyases↗
PubMed1998

It takes a village and time... a collaborative case study.

Today's approach to health care presents a challenge to those providers who work with patients with multifaceted complex issues that require the time and the efforts of several clinicians. These patients do not easily fit into a short-term, predetermined mold of care. This is an ongoing issue that warrants further investigation. This article presents a collaborative case study that chronicles the case of a patient who had multiple problems, such as homelessness, depression, substance abuse, anger, and serious emotional and physical scars contributing to a damaged self-image. In this case, three clinicians in three programs collaboratively assisted the patient in his complicated trek toward an improved quality of life.

Alcoholism↗
PubMed1997

Photoreceptor rim protein: partial sequences of cDNA show a high degree of similarity to ABC transporters.

PURPOSE: To isolate and sequence cDNA for bovine rim protein, a large membrane-bound glycoprotein found in photoreceptor outer segments. METHODS: Bovine rim protein was N-terminally sequenced (22 residues) and fragments were prepared by partial proteolysis. Two internal sequences of 21 and 18 amino acid residues were obtained from 35 kDa and 32 kDa fragments, respectively. Sense and anti-sense oligonucleotide primers were constructed, based on the peptide sequences derived from the 35 kDa and 32 kDa fragments, respectively, and the polymerase chain reaction (PCR) was used to amplify a 150 bp sequence from bovine retinal cDNA. RESULTS: The amplified sequence coded for the remainder of the peptide sequence determined from the 35 kDa fragment, which was not present in the primer, confirming that it was derived from the rim protein. The 150 bp sequence was translated to give a 50 amino acid peptide. Part of this peptide was compared with Dna sequence databases using the TFastA program, which found 94.6% identity with an EST derived from human retina and 86.1% identity to the mouse abc1 transporter. CONCLUSIONS: It is proposed that rim protein is a member of the ATP transporter family of proteins. It may be involved in transport of molecules involved in visual transduction across the photoreceptor disk membrane.

ATP-Binding Cassette Transporters↗
PubMed1996

Evidence for a novel class of microbial 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase in Streptomyces coelicolor A3(2), Streptomyces rimosus and Neurospora crassa.

The tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthases from Streptomyces coelicolor A3(2), Streptomyces rimosus and Neurospora crassa have been purified to homogeneity. All three enzymes have a subunit Mr of 54,000. The S. coelicolor DAHP synthase was physically and kinetically characterized and the N-terminal amino acid sequence was obtained. The N-terminal amino acid sequence could not be obtained for the enzymes from S. rimosus and N. crassa, their N-termini apparently being blocked. However, following proteolytic digestion, internal amino acid sequences were obtained from both enzymes. A comparison with the known DAHP synthase sequences indicated that these DAHP synthases are unrelated to other microbial DAHP synthase sequences but are similar to plant DAHP synthases. Up until now, two distinct classes of DAHP synthase have been described, one comprising exclusively enzymes from plants, the other restricted to enzymes from micro-organisms. These studies indicate that the class containing the plant DAHP synthases also contains enzymes from a microbial eukaryote and from several bacteria.

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed1994

The characterisation of the shikimate pathway enzyme dehydroquinase from Pisum sativum.

Peptides accounting for 157 residues of the bifunctional shikimate pathway enzyme, dehydroquinase/shikimate dehydrogenase, of Pisum sativum were sequenced. Three of the peptides were homologous to regions in Escherichia coli dehydroquinase and two to E. coli shikimate dehydrogenase. The pea dehydroquinase activity was inhibited by treatment with dehydroquinate plus sodium borohydride, establishing it as a type I dehydroquinase. Synthetic oligonucleotides designed from the amino acid sequence were used as PCR primers to amplify fragments of P. sativum cDNA. DNA sequence analysis showed that these amplified products were derived from dehydroquinase/shikimate dehydrogenase cDNA. The complete amino acid sequence of the dehydroquinase domain has been defined; it is homologous to all other type I dehydroquinases and is N-terminal.

Amino Acid Sequence↗
PubMed1994

Evidence for a new cytochrome P450 form induced by 3-methylcholanthrene in rats.

Evidence is presented for a new 3-methylcholanthrene (3MC)-induced form of cytochrome P450, P450MCX, in rat liver microsomes. P450MCX was co-purified with CYP1A1 from 3MC-treated male Sprague-Dawley rats but was resolved by gel electrophoresis. The M(r) of P450MCX (56,700) was intermediate between CYP1A1 (57,000) and CYP1A2 (54,800). Monoclonal antibodies showed that P450MCX was immunorelated to both CYP1A1 and CYP1A2 but not to CYP2B1, CYP2C6 or CYP3A1. Immunoreactive P450MCX was not detectable in liver microsomes from untreated rats but was highly induced by 3MC and Aroclor 1254, although not induced by isosafrole. The N-terminal amino acid sequence of P450MCX, obtained from an electroblotted sample resolved on SDS-PAGE, did not match any known cytochrome P450 or other protein. P450MCX may be a new member of the CYP1 family.

Amino Acid Sequence↗
PubMed1994

The amino acid sequence of the small monomeric phosphoglycerate mutase from the fission yeast Schizosaccharomyces pombe.

The amino acid sequence of the monomeric 2,3-bisphosphoglycerate (BPG)-dependent phosphoglycerate mutase (PGAM) from the fission yeast Schizosaccharomyces pombe has been determined. Amino acid sequencing of proteolytic fragments of the enzyme showed the S. pombe mutase to be similar in sequence to the tetrameric enzyme of baker's yeast (Saccharomyces cerevisiae). An S. pombe cDNA library was screened using a PCR fragment generated from two oligonucleotides complementary to sequences encoding the regions at the two active-site histidine residues. The 0.63 kb cDNA encoded an open reading frame of 210 amino acids. This sequence agreed completely with sequences of peptides derived from the purified protein. The amino acid sequence of S. pombe PGAM is 43% identical with that of S. cerevisiae PGAM and shows an equally high degree of identity with BPG-dependent PGAMs from other sources. However, the sequence of the S. pombe enzyme differs from other BPG-dependent enzymes in three important ways: (i) it does not contain the alanine- and lysine-rich sequence of amino acids at the C-terminus which have been proposed to constitute a flexible tail involved in catalysis; (ii) the sequence spanning residues 122-146 (S. cerevisiae PGAM numbering) is not present in the S. pombe PGAM sequence; in the S. cerevisiae PGAM crystal structure this stretch of sequence has been shown to occur as an extended loop, part of which is involved in inter-subunit interactions; (iii) the amino acid sequence in the region of a second S. cerevisiae inter-subunit contact (residues 74-78) shows radical mutations in the S. pombe enzyme.

Amino Acid Sequence↗
PubMed1993

Complete amino acid sequences of five dimeric and four monomeric forms of metallothionein from the edible mussel Mytilus edulis.

Cadmium-induced metallothioneins from the common sea mussel, Mytilus edulis, were shown to comprise of two groups of isoforms having apparent molecular masses of 10 kDa and 20 kDa. The 10-kDa group was resolved by anion-exchange chromatography into four fractions while the 20-kDa group was resolved into three fractions using this method. After metal removal and S-methylation of the cysteine residues using methyl-p-nitrobenzenesulphonate the complete amino acid sequences were determined. Five isoforms of the 20-kDa group were shown to possess monomeric units consisting of 71 amino acids. These proteins were distinct from the four 72-amino-acid proteins of the 10-kDa group. The FASTA algorithm has been used to compare the degree of similarity between the mussel metallothionein MT-10-IV isoform and other metallothioneins. The mussel MT-10-IV isoform exhibited substantial similarity to other molluscan metallothioneins. Moreover, the mussel metallothionein exhibited more similarity to vertebrate metallothioneins than to those of non-molluscan invertebrates, thus suggesting that the mussel metallothioneins are class I metallothioneins.

Amino Acid Sequence↗
PubMed1993

Purification and characterization of fatty acid-binding proteins from brown adipose tissue of the rat.

Fatty acid-binding proteins (FABPs) have been identified and purified from interscapular brown adipose tissue of the rat. The proteins were characterized and their properties compared with the FABP present in white adipose tissue. FABP was purified to electrophoretic homogeneity from brown adipose tissue by a procedure involving precipitation with 70% ammonium sulphate, followed sequentially by ion-exchange chromatography and gel filtration chromatography. The purified fraction migrated as a single band on SDS-PAGE with an apparent molecular mass of 14,200. Scatchard analysis of [14C]oleate-binding to purified FABP gave a Kd value of approx. 0.80 +/- 0.02 microM and a maximal binding of 0.65 +/- 0.03 mol per mol of protein; these values were similar to that found with the FABP purified from white fat. The FABP concentration in brown adipose tissue was almost twice that of FABP in white adipose tissue. Fatty acid analysis of FABP from brown adipose tissue revealed that the intrinsic arachidonic acid content was proportionately higher than that present in FABP of white adipose tissue. Isoelectric focusing of delipidated FABP indicated that it existed with two charge isoforms (pI 6.85 and 7.35). The purified FABP additionally emerged in two peaks (FABP-I and FABP-II) from a reverse phase HPLC column. Amino acid analysis showed that Gly, Thr, and Ser residues in FABP-I were almost twice as high as in FABP-II. The N-terminals of both FABP-I and -II were not blocked. These components have been partially sequenced and showed a sequence homology only between 25-31 residues from the N-terminal. Further studies are required to elucidate the precise function of the two different isoforms of FABP in brown adipose tissue.

Adipose Tissue, Brown↗
PubMed1993

The purification and properties of ferredoxin-NADP(+)-oxidoreductase from roots of Pisum sativum L.

A ferredoxin-NADP(+)-oxidoreductase (FNR) was purified to homogeneity from pea root plastids to a specific activity of 200 nkat.mg protein-1, following acetone precipitation and ferredoxin affinity chromatography. The molecular weight of the enzyme was estimated to be 36,000 and 33,800 by SDS-polyacrylamide gel electrophoresis and molecular exclusion chromatography, respectively. The absorption spectrum of the enzyme suggests it contains flavin as a prosthetic group. The enzyme requires NADPH and did not use NADH as an electron donor. The Km values for NADPH and ferredoxin were calculated to be 28 and 5 microM, respectively. The enzyme exhibited optimal activity at pH 8.0. Although resembling the leaf enzyme in most properties, amino terminal sequencing demonstrates clear differences between the leaf and root proteins and suggests closer homology of the pea root enzyme with the enzyme from spinach roots. A polyclonal antibody against the pea root plastid enzyme was raised by the immunization of rabbits. Judging by immunodiffusion only partial identity was observed between the root plastid and chloroplast FNR. The root plastid FNR enzyme activity was precipitated with increasing concentrations of the antibody, in contrast to the chloroplast enzyme which was not inhibited. The potential usefulness of these antibodies is discussed.

Amino Acid Sequence↗
PubMed1993

The ABA-1 allergen of the nematode Ascaris suum: epitope stability, mass spectrometry, and N-terminal sequence comparison with its homologue in Toxocara canis.

ABA-1 is a major allergen of nematode parasites of the genus Ascaris which includes the large roundworms of humans and pigs, A. lumbricoides and A. suum, respectively. The allergen was purified from A. suum by immunoaffinity chromatography for immunochemical examination. The IgE antibody repertoire is under MHC control in infected rodents and the IgE-binding epitopes were robust to treatment with heat or periodate, and electroblotting on nitrocellulose. This implies that the IgE epitopes comprise primary peptide sequence or an unusually stable secondary or tertiary structure. The molecular mass of ABA-1 is controversial, but mass spectrometry analysis indicated that there were five components of similar size, with the major species being 14,643.2 +/- 1.4 D. Finally, N-terminal sequence analysis of ABA-1 and TBA-1 (the homologue in the canine nematode infective to humans, Toxocara canis) revealed a high degree of similarity, and we have previous evidence that ABA-1 homologues are widespread amongst ascaridid parasites of humans. ABA-1 and its homologues might, therefore, be important to the immunopathology of many infections with nematode parasites, upon which the genetic constitution of the hosts will also have a bearing.

Allergens↗
PubMed1992

Sequences directing dihydrolipoamide dehydrogenase (E3) binding are located on the 2-oxoglutarate dehydrogenase (E1) component of the mammalian 2-oxoglutarate dehydrogenase multienzyme complex.

Sequences located in the N-terminal region of the high M(r) 2-oxoglutarate dehydrogenase (E1) enzyme of the mammalian 2-oxoglutarate dehydrogenase multienzyme complex (OGDC) exhibit significant similarity with corresponding sequences from the lipoyl domains of the dihydrolipoamide acetyltransferase (E2) and protein X components of eukaryotic pyruvate dehydrogenase complexes (PDCs). Two additional features of this region of E1 resemble lipoyl domains: (i) it is readily released by trypsin, generating a small N-terminal peptide with an apparent M(r) value of 10,000 and a large stable 100,000 M(r) fragment (E1') and (ii) it is highly immunogenic, inducing the bulk of the antibody response to intact E1. This 'lipoyl-like' domain lacks a functional lipoamide group. Selective but extensive degradation of E1 with proteinase Arg C or specific conversion of E1 to E1' with trypsin both cause loss of overall OGDC function although the E1' fragment retains full catalytic activity. Removal of this small N-terminal peptide promotes the dissociation of dihydrolipoamide dehydrogenase (E3) from the E2 core assembly and also affects the stability of E1 interaction. Thus, structural roles which are mediated by a specific gene product, protein X in PDC and possibly also the E2 subunit, are performed by similar structural elements located on the E1 enzyme of the OGDC.

Amino Acid Sequence↗