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R J Simpson

Publications and source records attributed to R J Simpson.

At least 397 records · Page 22Linked to original sources
PubMed1978

Additional evidence for re-entry within the bundle branches in man.

A repetitive, non-paced ventricular beat (V3) often occurs during ventricular extrastimulus testing at short coupling intervals when critical retrograde His-Purkinje conduction delay is achieved. Though re-entry within the bundle branches has been proposed as the mechanism for V3, it has been difficult to exclude local re-entry. In the present case, we consistently recorded a right bundle branch potential as well as a bundle of His potential before each V3. The anterograde activation sequence of these potentials provides evidence against local re-entry and supports re-entry involving the bundle branches as the mechanism for V3.

Arrhythmias, Cardiac↗
PubMed1976

Studies on 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe) from Escherichia coli K12. 1. Purification and subunit structure.

1. 3-Deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe) from Escherichia coli K12 has been purified to near homogeneity. The purified enzyme has a specific activity of 67 units/mg which is about 1000 times that found in cell-free extracts of wild-tupe E. coli K12. 2. The minimum molecular weight of the enzyme was estimated by dodecylsuphate-gel electrophoresis to be 33000. Re-estimation of the native molecular weight by gel filtration confirmed the previously determined value of 110000. 3. Amino acid anaktsus abd tryptic fingerprints indicated that the subunits of the enzyme are very similar and possibly identical. 4. The purified enzyme does not contain Co2+.

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed1976

Studies on 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe)from Escherichia coli K12. 2. Kinetic properties.

1. Co2+ is not a cofactor for 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe). 2. The following analogues of phosphoenolpyruvate were tested as inhibitors of 3-deoxy-D-arabinoheptolosonate-7-phosphate synthetase(phe): pyruvate, lactate, glycerate, 2-phosphoglycerate, 2,3-bisphosphoglycerate, 3-methylphosphoenolpyruvate, 3-ethylphosphoenolpyruvate and 3,3-demethylphosphoenolpyruvate. The rusults obtained indicate that the binding of phosphoenolpyruvate to the enzyme requires a phosphoryl group on the C-2 position of the substrate and one free hydrogen atom at the C-3 position. 3. The dead-end inhibition pattern observed with the substrate analogue 2-phosphoglycerate when either phosphoenolpyruvate or erythrose 4-phosphate was the variable substrate is inconsistent with a ping-pong mechanism and indicates that the reaction mechanism for this enzyme must be sequential. The following kinetic constants were determined:Km for phosphoenolpyruvate, 0.08 +/- 0.04 mM; Km for erythrose 4-phosphate, 0.9 +/- 0.3 mM; K is for competitive inhibition by 2-phosphoglycerate with respect to phosphoenolpyruvate, 1.0 +/- 0.1 mM. 4. The enzyme was observed to have a bell-shaped pH PROFILE WITH A PH OPTIMUM OF 7.0. The effects of pH ON V and V/(Km for phosphoenolpyruvate) indicated that an ionizing group of pKa 8.0-8.1 is involved in the catalytic activity of the enzyme. The pKa of this group is unaffected by the binding of phosphoenolpyruvate.

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed1976

Studies on 3-deoxy-d-arabinoheptulosonate-7-phosphate synthetase(phe) from escherichia coli k12. 3. Structural studies.

1. Investigations with structural analogues of phenylalanine indicated an absolute requirement for the aromatic ring and both the alpha-carboxyl and alpha-amino groups of phenylalanine for inhibition of 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe) activity. Replacement of the alpha-H atom with a methyl group does not decrease the inhibition greatly. Varying degrees of inhibition were observed with o, m and p mono-substituted fluoro, chloro and hydroxy phenylalanines. D-Phenylalanine and several metabolites of the aromatic biosynthetic pathways do not inhibit enzymic activity. 2. Circular dichroism studies indicated that the native enzyme possesses approximately 26% alpha-helix. Both circular dichroic and ultraviolet difference spectra indicated that the addition of phenylalanine to the synthetase induces a conformational change involving a small alteration of the secondary structure and large alterations in th interactions of some of the aromatic residues of the enzyme. In particular, a tryptophan residue moves from an extremly hydrophobic environment to one less hydrophobic. 3. Kd for the binding of phenylalanine to the enzyme was determined spectrophotometrically to be 75 muM. 4. Chemical modification studies suggested that a sulphydryl group and possibly a lysine residue may be implicated in the catalytic activity of the enzyme.

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed1976

Complete amino acid analysis of proteins from a single hydrolysate.

An analytical procedure which affords the precise amino acid composition of a protein or a peptide from a single hydrolysate is described. This method utilizes 4 N methanesulfonic acid containing 0.2% 3-(2-aminoethyl)indole, rather then 6N HCl as a catalyst for hydrolysis. The hydrolysis is carried out in vacuo (20 mu) at 115 degrees for 22 to 72 hours. Half-cystine is determined as S-sulfocysteine by treating the hydrolysate with dithiothreitol followed by an excess of tetrathionate. The values of all amino acids, including tryptophan and half-cystine, were close to the expected theoretical values for the proteins examined. The method has the advantage that the neutralized hydrolysate can be applied directly to an ion exchange column. Further, the method is capable of distinguishing between free sulfhydryl groups as S-carbosymethylcysteine and disulfides as S-sulfocysteine. A limitation of the procedure is that tryptophan remains sensitive to the presence of carbohydrate in the sample.

Amino Acids↗
PubMed1971

Purification and properties of 3-deoxy-d-arabinoheptulosonic acid-7-phosphate synthetase (phe) from a lambda aroG+ transductant of Escherichia coli.

By incorporating aroG, the structural gene for 3-deoxy-d-arabinoheptulosonic acid-7-phosphate (DAHP) synthetase (phe), into the genome of a heat-inducible susR60 mutant of phage lambda, it has been possible to increase the intracellular levels of DAHP synthetase (phe) in a lysogenized strain of Escherichia coli some 15-fold over levels found in the wild-type strain. By using this strain, the enzyme has been purified approximately 2,000-fold compared with wild type, and various kinetic parameters of the purified enzyme have been studied. In contrast to previous reports, the inhibition by phenylalanine was found to exhibit sigmoidal kinetics, suggestive of cooperative interactions between phenylalanine binding sites. Stimulation of enzyme activity by Co(2+) was minimal (14%).

Aldehyde-Lyases↗
PubMedDate not supplied

Identification and characterization of recombinant murine interleukin-6 with a C-terminal pentapeptide extension using capillary reversed phase HPLC-MS and edman degradation.

We have identified a preparation of recombinant murine interleukin-6 (mIL-6) that, in addition to the anticipated product, also contained approximately equal amounts of mIL-6 with a C-terminal pentapeptide extension. The extension mutant was generated by readthrough of the stopcodon, and termination at a second in-frame stopcodon 12 base pairs 3' in the expression vector. Aliquots of the preparation were subjected to proteolytic digestion with Asp-N and Lys-C-endopeptidase. The resultant peptides were separated by reversed-phase capillary HPLC, and analysed using a combination of mass spectrometry and N-terminal sequence analysis. These data revealed a C-terminal pentapeptide (Gln-Gly-Ser-Val-Asp) extension, with the authentic stopcodon being translated as glutamine. The extension mutant was isolated by reversed-phase HPLC and shown to have similar mitogenic activity to mIL-6 on murine hybridoma 7TD1 cells.

Animals↗
PubMedDate not supplied

Proteome analysis of polyacrylamide gel-separated proteins visualized by reversible negative staining using imidazole-zinc salts.

Identification and characterization of proteins isolated from natural sources by polyacrylamide gel electrophoresis has become a routine technique. However, efficient sample proteolysis and subsequent peptide extraction is still problematic. Here, we present an improved protocol for the rapid detection of polyacrylamide gel-separated proteins, in situ protein modification, proteolytic digestion and peptide extraction for subsequent protein identification and characterization by capillary high-performance liquid chromatography/tandem mass spectrometry. This simple technique employs the rapid imidazole-zinc reverse stain, in-gel S-pyridylethylation and proteolytic digestion of microcrushed polyacrylamide gel pieces with proteases. This technique obviates the need for buffer exchange or gel lyophilisation due to all of the sample manipulation steps being carried out at near neutral pH and thus lends itself readily to automation.

Acrylic Resins↗
PubMedDate not supplied

Two-dimensional electrophoretic analysis of proteins expressed by normal and cancerous human crypts: application of mass spectrometry to peptide-mass fingerprinting.

Protein patterns of normal human colonic crypts, isolated from different regions of the large intestine, and several colorectal cancer cell lines were compared using two-dimensional electrophoresis gels (2-DE). As detected by intrinsic radiolabeling and Coomassie Brilliant Blue staining, the protein patterns for normal crypts isolated from the ascending, and descending, regions of the colon and the rectum, were almost (> 95%) identical. While 75-80% of the protein spots from normal crypts and the colorectal cancer cell line (LIM 1863), a cell line that grows as organoids and differentiates spontaneously into crypt-like structures in vitro, can be matched, the relative expression levels of a large number of proteins differ. At least two protein spots (undetectable in the protein pattern from normal cells), proteins a (M(r) approximately 18,000, pI 6.7-6.9) and b (M(r) approximately 24,000, pI 5.9-6.0), were detected in the 2-DE gel protein pattern in the three cell lines LIM 1863, LIM 1215 and LIM 1899. The identity of these proteins is not yet known and further studies are required before they can be considered as potential colon tumor markers. Approximately 60% of the cellular proteins from LIM 1215 cells, a colon carcinoma cell line that exhibits many properties associated with columnar cells, can be matched with LIM 1863 cells. The results presented here represent an initial phase in our efforts to develop a comprehensive protein database for normal human colon cells and several colorectal cancer cell lines. While our initial protein identification relied on microsequencing methodologies, we are presently evaluating peptide-mass fingerprinting, utilizing capillary reversed-phase high-performance liquid chromatography (RP-HPLC) and electrospray mass spectrometry, as a means for rapid identification of proteins at subpicomole levels. Using this approach, protein #3 (M(r) approximately 66,000, pI 6.2) was identified as heat shock protein 60 from as few as seven tryptic peptide masses when they were screened against the molecular weight search (MOWSE) peptide-mass database.

Amino Acid Sequence↗
PubMedDate not supplied

Two-dimensional electrophoretic analysis of human breast carcinoma proteins: mapping of proteins that bind to the SH3 domain of mixed lineage kinase MLK2.

MLK2, a member of the mixed lineage kinase (MLK) family of protein kinases, first reported by Dorow et al. (Eur. J. Biochem. 1993, 213, 701-710), comprises several distinct structural domains including an src homology-3 (SH3) domain, a kinase catalytic domain, a unique domain containing two leucine zipper motifs, a polybasic sequence, and a cdc42/rac interactive binding motif. Each of these domains has been shown in other systems to be associated with a specific type of protein interaction in the regulation of cellular signal transduction. To study the role of MLK2 in recruiting specific substrates, we constructed a recombinant cDNA encoding the N-terminal 100 amino acids of MLK2 (MLK2N), including the SH3 domain (residues 23-77), fused to glutathione S-transferase. This fusion protein was expressed in Escherichia coli, purified using gluthathione-Sepharose affinity chromatography and employed in an affinity approach to isolate MLK2-SH3 domain binding proteins from lysates of 35S-labelled MDA-MB231 human breast tumour cells. Electrophoretic analysis of bound proteins revealed that two low-abundance proteins with a molecular weights (Mr) of approximately 31,500 and approximately 34,000, bound consistently to the MLK2N protein. To establish accurately the Mt / isoelectric point (pI) loci of these MLK2-SH3 domain binding proteins, a number of abundant proteins in a two-dimensional electrophoresis (2-DE) master gel were identified to serve as triangulation marker points. Proteins were identified by (i) direct Edman degradation following electroblotting onto polyvinylidene difluoride (PVDF) membranes, (ii) Edman degradation of peptides generated by in-gel proteolysis and fractionation by rapid (approximately 12 min) microbore column (2.1 mm ID) reversed-phase high performance liquid chromatography (HPLC), (iii) mass spectrometric methods including peptide-mass fingerprinting and electrospray (ESI)-mass spectrometry (MS)-MS utilizing capillary (0.2-0.3 mm ID) column chromatography, or (iv) immunoblot analysis. Using this information, a preliminary 2-DE protein database for the human breast carcinoma cell line MDA-MB231, comprising 21 identified proteins, has been constructed and can be accessed via the World Wide Web (URL address: http:(/)/ www.ludwig.edu.au/www/jpsl/jpslhome.htm l).

Amino Acid Sequence↗
PubMedDate not supplied

A two-dimensional gel database of human colon carcinoma proteins.

The master two-dimensional gel database of human colon carcinoma cells currently lists cellular proteins from normal crypts and the colorectal cancer cell lines LIM 1863, LIM 1215 and LIM 1899 (Ward et al., Electrophoresis 1990, 11, 883-891; Ji et al., Electrophoresis 1994, 15, 391-405). Updated two-dimensional electrophoretic (2-DE) maps of cellular proteins from LIM 1215 cells, acquired under both nonreducing and reducing conditions, are presented. Fifteen cellular proteins are identified in the reducing 2-DE gel map, and seven in the nonreducing gel map, along with a tabular listing of their M(r)/pI loci and mode of identification. We also include our mass spectrometric based procedures for identifying 2-DE resolved proteins. This procedure relies on a combination of capillary column (0.10-0.32 mm internal diameter) reversed-phase HPLC peptide mapping of in-gel digested proteins, peptide mass fingerprinting, sequence analysis by either collision-induced dissociation or post-source-decay fragmentation, and protein identification using available database search algorithms. These data, and descriptions of the micro-techniques employed in this laboratory for identifying 2-DE resolved proteins can be accessed via the internet URL: http:(/)/www.ludwig.edu.au.

Amino Acid Sequence↗
PubMedDate not supplied

Electrophoretic analysis of the novel antigen for the gastrointestinal-specific monoclonal antibody, A33.

The murine monoclonal antibody A33 (mAbA33) recognises a human cell membrane-associated antigen selectively expressed in epithelial cells of the lower gastrointestinal tract and > 90% of colonic cancers, but is not detected in a wide range of other normal tissues by immunohistochemical analysis. In phase I/II clinical triasl, mAbA33 has been shown to target advanced colon cancers and the humanized version is currently being evaluated in therapy studies. Although the mAbA33 has been well characterised by immunohistochemical and clinical studies, until recently, the target antigen has remained poorly defined. This was largely attributable to the antigenic determinant recognised by mAbA33 being dependent on the native spatial conformation of the A33 antigen which impeded its identification by conventional two-dimensional electrophoresis (2-DE) and immunoblot analysis. We have developed an immunoblot method, based on nonreducing/non-urea precast 2-DE gels, that has facilitated the purification of the detergent (0.3% Triton X-100) solubilised A33 antigen from the human colon cancer cell lines LIM1215 and SW1222. Under these 2-DE conditions, the A33 antigen electrophoreses with an apparent M(r) approximately 41000 and pI 5.0-6.0. Attempts to isolate the A33 antigen from 2-DE gels for direct structural analysis were unsuccessful, due to its co-electrophoresis with actin and cytokeratin proteins. However, using Western blot and biosensor detection the A33 antigen has been purified chromatographically and N-terminal sequence analysis was possible. Using polyclonal antibodies raised against a synthetic peptide corresponding to the N-terminal region of the A33 antigen we have used Western blot analysis to localise the molecule in our master 2-DE protein database for normal human colon crypts and several colon carcinoma cell lines (URL address: http:(/)/www.ludwig.edu.au). Under reducing 2-DE conditions, the A33 antigen electrophoresis as 6 differentially charged isoforms (pI 4.6-4.8) with a single molecular weight species at M(r) approximately 55000.

3T3 Cells↗
PubMedDate not supplied

Molecular and functional roles of duodenal cytochrome B (Dcytb) in iron metabolism.

Dcytb has been identified as the mammalian transplasma ferric reductase that catalyzes the reduction of ferric to ferrous iron in the process of iron absorption. Its mRNA and protein levels are up-regulated by several independent stimulators of iron absorption. Furthermore, its cDNA encodes putative binding sites for heme and ascorbic acid. Using Northern and Western blots, RT-PCR and confocal microscopy, we studied the expression and localisation of Dcytb in cell lines and tissues of CD1 mice. Dcytb expression and function were modulated by iron. Dcytb and DMT1, both predominantly localised in the apical region of the duodenum were up-regulated in iron deficiency. Dcytb, the iron regulated ferric reductase may also utilize cytoplasmic ascorbate as electron donor for transmembrane reduction of iron. Dcytb expression was found in other tissues apart from the duodenum and its regulation and functions at these other sites are of interest in iron metabolism.

Ascorbic Acid↗
PubMedDate not supplied

Style self-incompatibility gene products of Nicotiana alata are ribonucleases.

Self-incompatibility in flowering plants is often controlled by a single nuclear gene (the S-gene) having several alleles. This gene prevents fertilization by self-pollen or by pollen bearing either of the two S-alleles expressed in the style. Sequence analysis shows that three alleles of the S gene of Nicotiana alata encode style glycoproteins with regions of defined homology. Two of the homologous regions also show precise homology with ribonucleases T2 (ref. 4) and Rh (ref. 5). We report here that glycoproteins corresponding to the S1, S2, S3, S6 and S7 alleles isolated from style extracts of N. alata are ribonucleases. These style S-gene-encoded glycoproteins account for most of the ribonuclease activity recovered from style extracts. The ribonuclease specific activity of style extracts of the self-incompatible species N. alata is 100-1,000-fold higher than that of the related self-compatible species N. tabacum. These observations implicate ribonuclease activity in the mechanism of gametophytic self-incompatibility.

Alleles↗
PubMedDate not supplied

Treatment of ras-induced cancers by the F-actin-bundling drug MKT-077.

A rhodacyanine dye called MKT-077 has shown a highly selective toxicity toward several distinct human malignant cell lines, including bladder carcinoma EJ, and has been subjected to clinical trials for cancer therapy. In the pancreatic carcinoma cell line CRL-1420, but not in normal African green monkey kidney cell line CV-1, it is selectively accumulated in mitochondria. However, both the specific oncogenes responsible for its selective toxicity toward cancer cells, and its target proteins in these cancer cells, still remain to be determined. This study was conducted using normal and ras-transformed NIH 3T3 fibroblasts to determine whether oncogenic ras mutants such as v-Ha-ras are responsible for the selective toxicity of MKT-077 and also to identify its targets, using its derivative called "compound 1" as a specific ligand. We have found that v-Ha-ras is responsible for the selective toxicity of MKT-077 in both in vitro and in vivo. Furthermore, we have identified and affinity purified at least two distinct proteins of 45 kD (p45) and 75 kD (p75), which bind MKT-077 in v-Ha-ras-transformed cells but not in parental normal cells. Microsequencing analysis has revealed that the p45 is a mixture of beta- and gamma-actin, whereas the p75 is HSC70, a constitutive member of the Hsp70 heat shock adenosine triphosphatase family, which inactivates the tumor suppressor p53. MKT-077 binds actin directly, bundles actin filaments by cross-linking, and blocks membrane ruffling. Like a few F-actin-bundling proteins such as HS1, alpha-actinin, and vinculin as well as F-actin cappers such as tensin and chaetoglobosin K (CK), the F-actin-bundling drug MKT-077 suppresses ras transformation by blocking membrane ruffling. These findings suggest that other selective F-actin-bundling/capping compounds are also potentially useful for the chemotherapy of ras-associated cancers.

3T3 Cells↗