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R J Simpson

Publications and source records attributed to R J Simpson.

At least 217 records · Page 12Linked to original sources
PubMed1992

The purification of a Rap1 GTPase-activating protein from bovine brain cytosol.

Two GTPase-activating proteins (GAPs) have been detected in extracts from bovine brain: GAP-1, which is specific for the activation of ras GTPases, and GAP-3, which is specific for the activation of the rap1 GTPases. We present a strategy for the purification to homogeneity of a cytosolic form of GAP-3 from bovine brain. The 100,000 x g supernatant from homogenized brains was chromatographed sequentially on DEAE Fast Flow, green H-E4BD Sepharose, Bio-Gel A1.5, hydroxyapatite, and phenyl-Sepharose prior to high resolution separation on Mono Q HR 5/5, phenyl-Superose HR 5/5, Mono Q PC 1.6/5, and Superose 12 PC 3.2/30. This procedure resulted in an approximately 18,000-fold purification, yielding 50 micrograms of GAP-3 from 1.6 kg of tissue. Purified cytosolic GAP-3 migrated as a single band of apparent Mr 55,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, on gel filtration cytosolic GAP-3 chromatographed as a dimer with an apparent Mr 92,000. Purified GAP-3 does not activate ras or rho GTPases and possesses no intrinsic GTPase activity. Amino acid sequence data indicated a proline-rich N terminus. The amino acid sequences of peptides generated by Staphylococcus aureus V8 digestion of reduced and pyridine-ethylated GAP-3 showed no similarity to the predicted primary structure of GAP-1 or any other proteins in the nucleic acid or protein data bases. By comparison with the data of Rubinfeld et al. (Rubinfeld, B., Munemitsu, S., Clark, R., Conroy, L., Watt, K., Crosier, W.J., McCormick, F., and Polakis, P. (1991) Cell 65, 1033-1042), it appears that the membrane-associated (Mr 85,000-95,000) and cytosolic forms of GAP-3 are derived from equivalent, or closely related, genes.

Amino Acid Sequence↗
PubMed1992

Workshop on two-dimensional gel protein databases.

A workshop on two-dimensional gel electrophoresis (2-DE) protein database, organized by the Committee on Data for Science and Technology (CODATA) of the International Council of Scientific Unions Task Group on Biological Macromolecules, was held at the CODATA Secretariat in Paris on March 9, 1992. Eleven scientists from eight different countries represented various aspects of 2-DE analysis--namely, cellular protein database development and protein microsequencing methodologies. The purpose of the workshop was to explore means of integrating the rapidly expanding body of information on 2-DE resolved proteins from different laboratories. A major proposal emanating from the workshop was the establishment of an intermediary or "relational" 2-DE gel protein database. This intermediary database, which would catalogue pertinent information on 2-DE resolved proteins (experimental source, 2-DE loci, biological information, etc.) could be an adjunct to, and accessed through, the existing international protein sequence databanks. It would function as a pointer for researchers to the individual 2-DE protein databases where primary and more specialized 2-DE data would be housed.

Databases, Factual↗
PubMed1992

Effect of hypoxic exposure on iron absorption in heterozygous hypotransferrinaemic mice.

Heterozygous hypotransferrinaemic mice show serum iron, haemoglobin and reticulocyte levels similar to those of normal (+/+) controls but the plasma apotransferrin level is significantly reduced. Male heterozygous hypotransferrinaemic Balb/cJ mice and control (+/+) littermates were exposed to 1-3 days' hypoxia at 0.5 atmospheres. Controls increased their haemoglobin and iron absorption significantly during the first day of exposure; serum transferrin levels were increased and iron absorption had returned to normal levels by the third day or serum transferrin above normal levels and the elevated iron absorption persisted for longer than in controls. Serum iron levels were lower following hypoxic exposure than in controls. Liver iron loading was significantly enhanced by hypoxia in heterozygotes, but not in controls. These observations demonstrate the importance of the reservoir of plasma apotransferrin in the initial phase of enhanced erythropoiesis, and that the ability to enhance plasma levels of iron-bound transferrin is required in order to increase haemoglobin levels under the hypoxic stress.

Animals↗
PubMed1992

Relationship between the cytolysins tenebrosin-C from Actinia tenebrosa and equinatoxin II from Actinia equina.

The chemical, physical and biological properties of the cytolysin tenebrosin-C from Actinia tenebrosa have been compared with those of equinatoxin II from Actinia equina. The two proteins are indistinguishable by reverse-phase and cation-exchange HPLC and capillary zone electrophoresis, and give similar peptide fragments upon cyanogen bromide cleavage (as judged by the chromatographic behaviour, ultraviolet absorption spectra, amino acid composition and N-terminal amino acid sequences of the peptides). Their cardiac stimulatory activities are identical, and their haemolytic activities are similar, with equinatoxin II having slightly greater activity. These data indicate that the two molecules are either identical in all 179 amino acid positions, or differ by no more than one or two residues. These findings are discussed in the context of the taxonomic relationship between the two species of sea anemone.

Amino Acid Sequence↗
PubMed1992

Nutrition education for cardiovascular disease prevention among low income populations--description and pilot evaluation of a physician-based model.

Low income Americans are at greatest risk for coronary heart disease but have least access to health promotion programs for life style modification. Primary care physicians may represent one of the few sources of preventive care available to the poor. However, the majority of physicians feel unprepared to help patients achieve dietary change, and few existing nutrition intervention programs address the special needs of low literacy populations. The Food for Heart Program was developed to facilitate dietary counseling experienced by primary care physicians who care for low literacy patients and to overcome barriers to behavior change faced by patients. The program consists of three components: (1) a validated dietary risk assessment that rapidly identifies atherogenic eating habits and requires no nutritional expertise to administer or interpret, (2) a structured diet treatment program that is culturally specific for a southern patient population and links practical behavior change recommendations with results of the diet assessment, and (3) a system for monitoring and reinforcement that prompts physicians to review progress, reinforce prior messages, and reward positive change. Behavior change theory is used to guide the intervention and readability of the material has been assessed at the 5-6th grade level. An evaluation study of the Food for Heart Program suggests that it has a positive impact on physician counseling and that patients are responding favorably to these efforts.

Cardiovascular Diseases↗
PubMed1992

Application of selective extraction to the study of iron species present in diet and rat gastrointestinal tract contents.

Iron speciation in rodent diet and rat gastrointestinal tract lumen during dietary digestion and absorption was investigated with a novel selective extraction technique. Five Fe fractions were identified, namely exchangeable (soluble in 1 M-magnesium chloride), carbonate-bound (soluble in mild acid), oxide-bound (soluble in hydroxylamine-acetic acid), organic-bound (soluble after treatment with peroxide in nitric acid) and residual. Fe from the pelleted diet was mobilized by rat stomach to the exchangeable fraction, then redistributed to the carbonate- and oxide-bound fractions on passage through the proximal small intestine. In vitro incubation of diet with hydrochloric acid failed to mimic the in vivo effect of the stomach. In vitro neutralization of stomach contents with bicarbonate was found to produce a similar effect on Fe speciation to that seen when diet passed the proximal small intestine in vivo. Comparison of 59Fe speciation in extrinsically labelled diet with endogenous Fe speciation showed that extrinsic labelling does not uniformly label all endogenous species. The experiments suggest that selective extraction may provide a useful approach to the study of Fe species present in diets, in vitro digestions and gastrointestinal contents.

Animals↗
PubMed1992

A leaf phosphorus assay for seedlings of Acacia mangium.

Concentrations of extractable and total phosphorus in leaves, stem, root and nodules of 12-week-old seedlings of two provenances of Acacia mangium Willd. were analyzed to identify the fraction of phosphorus and the plant part most suitable for predicting the phosphorus nutritional status of the seedlings.For both provenances, concentrations of extractable phosphorus were more sensitive to changes in soil phosphorus status and varied less among different plant parts than concentrations of total phosphorus. Concentrations of extractable phosphorus in the youngest fully expanded leaf (Leaf 3 from the apex) and the next two older leaves correlated closely with seedling dry mass and may be used to assess the phosphorus nutritional status of Acacia mangium seedlings.

Journal Article↗
PubMed1992

Recombinant interleukin-6 protects mice against experimental bacterial infection.

Because of reports of high levels of interleukin-6 (IL-6) in patients during infection, we studied the role of IL-6 in experimental infection. Mice infected with the facultative intracellular pathogen Listeria monocytogenes displayed high levels of IL-6 in their sera and tissues, particularly the spleen, 1 to 3 days after infection. At this time, the IL-6 titers correlated with bacterial numbers in individual mice and in groups of mice given graded doses of Listeria organisms. However, the presence of IL-6 in serum declined after 4 days, even when a large initial dose of bacteria meant that bacterial numbers were still increasing at this time. Recombinant mouse IL-6 injected intraperitoneally before infection protected mice in a dose-dependent manner. It was effective when given 4 h before infection but not when administration was delayed for 24 h postinfection. It is therefore believed that IL-6 plays a role in early priming of the immune response to infection. Its exact function in this model is being investigated.

Animals↗
PubMed1992

Characterization of bovine heparin-binding neurotrophic factor (HBNF): assignment of disulfide bonds.

The topology of the disulfides in native heparin-binding neurotrophic factor (HBNF) isolated from bovine brain was studied by proteolytic digestion using trypsin, Asp-N endoproteinase and chymotrypsin and peptide mapping. Disulfide-linked peptides were identified by automated Edman degradation. It has been shown that there are disulfide bonds between Cys15-Cys44, Cys23-Cys53, Cys30-Cys57, Cys67-Cys99 and Cys77-Cys109.

Amino Acid Sequence↗
PubMed1992

Intestinal iron absorption in chronic alcoholics.

Chronic alcohol misusers frequently accumulate significant amounts of excess iron, but the mechanism of this loading is unknown. In vivo whole-body retention studies demonstrated, on average, a two-fold increase in intestinal iron absorption in six male chronic alcoholics. Degrees of iron loading as assessed by serum ferritin or hepatic iron levels did not correlate with alcohol consumption or liver function tests. In vitro studies of iron uptake at varying medium iron concentrations by duodenal mucosa biopsies showed increased iron uptake by tissue from the chronic alcoholics, particularly at the highest medium iron concentration used. Analysis of the uptake data showed similar Michaelis-Menten kinetic constants for uptake by tissue from control subjects and alcoholics. The analysis showed, in addition, a linear component for 59Fe uptake. This component was five-fold greater for the tissue from the chronic alcoholics compared to the controls at the highest medium iron concentration. 57Co-cyanocobalamin was included in the incubation medium as a tissue extracellular fluid marker (ECF). It was found that the apparent distribution volume of the ECF marker, reflecting tissue permeability, was 75% higher for the biopsies from the alcoholics compared to control subjects. These results, together with the previous reports of enhanced in vitro and in vivo intestinal permeability to 51Cr-EDTA in chronic alcoholics, indicate that unregulated increased iron absorption via the non-carrier-mediated paracellular route contributes to the iron overload in chronic alcoholics.

Adult↗
PubMed1992

The group III allergen from the house dust mite Dermatophagoides pteronyssinus is a trypsin-like enzyme.

Faecally enriched extracts of Dermatophagoides pteronyssinus were shown to contain a trypsin-like enzyme which was allergenic. Chromatofocusing studies revealed the presence of nine major isoforms in D. pteronyssinus, with pI in the range 4 to greater than 8, but only two (range 4-5) in D. farinae. Trypsin isolated from D. pteronyssinus by benzamidine-Sepharose 6B affinity chromatography and gelfiltration was found to be a 31-kDa protein which was enzymatically similar to both invertebrate and vertebrate trypsins. The N-terminal sequence obtained (IVGGEXALAGEXPYQISL) was identical to that reported for the mite allergen Der p III and showed homology with crayfish trypsin and Der f III from D. farinae. Mite trypsin underwent autolysis and the N-terminal sequences of two fragments were found to be ALAGEXPYQI and NNQVXGI respectively. Both showed homology with crayfish trypsin, and the former sequence was identical to residues 7-18 of the native enzyme and Der p III. All isoforms of mite trypsin were showed to be allergenic by radioallergosorbent assay and further studies indicated that the trypsin degradation products were also allergenic. The enzyme was compared with other mite allergens and the rank order of allergenic potency was shown to be: whole mite extract greater than Der p I greater than trypsin. However, all sera from a panel of mite allergic individuals showed IgE reactivity to trypsin, comparable to that seen using whole mite extract and Der p I. These data indicate that mite trypsin is a major allergen corresponding to the previously described allergen, Der p III.

Allergens↗
PubMed1992

Binding of anti-human-interleukin-6 monoclonal antibodies to synthetic peptides of human interleukin-6 studied using surface plasmon resonance.

Biosensor technology employing surface plasmon resonance (SPR) detection provides a highly-sensitive (sub ng), non-extrinsic labelling approach for monitoring protein interactions in real-time. We have used this approach to map the binding sites on human interleukin-6 (hIL-6) for a series of anti-hIL-6 monoclonal antibodies (mAbs). Epitopes were localised by monitoring the ability of ten synthetic peptides, spanning the sequence of hIL-6, to inhibit the binding of anti-hIL-6 mAbs to immobilised hIL-6. Peptide P8 (Pro139-Gln153) inhibited binding of anti-IL-6-mAbs 1, 2 and 7. To increase the sensitivity of detection of antibody-synthetic peptide interactions, a procedure was developed for immobilising the synthetic peptides directly to the sensor surface of the SPR instrument. From this study, association equilibrium constants of 2.1 x 10(6)M-1 and 3.6 x 10(4)M-1 were calculated for the mAb7-immobilised P8 and mAb7-free P8 interactions, respectively.

Antibodies, Monoclonal↗
PubMed1991

Identification of a functional domain of human granulocyte colony-stimulating factor using neutralizing monoclonal antibodies.

Human granulocyte colony-stimulating factor (G-CSF) is a hemopoietic growth factor that is being used successfully to treat various forms of neutropenia. To define functionally important regions of G-CSF, we have prepared 37 monoclonal anti-G-CSF antibodies and mapped the regions of G-CSF recognized by different antibody groups. Antibodies recognizing similar epitopes were identified by competition assays, neutralization assays, conformation dependence and cross-reactivity with canine G-CSF. Seven of eight neutralizing antibodies fell into two related epitope groups and were conformation-dependent. The eighth was unrelated and conformation-independent. Peptides of G-CSF were generated by chemical or enzymatic digestion and tested for antibody reactivity. One of the neutralizing antibodies (LMM351) recognized a small, disulfide-bonded peptide from the V8 protease digest (residues 34-46). A synthetic peptide (residues 20-58) was recognized by all the neutralizing antibodies, implicating this disulfide-bonded loop in receptor binding. The epitopes recognized by nonneutralizing antibodies were found throughout G-CSF. Thus, regions of G-CSF that are not involved in receptor binding have also been defined. A CNBr peptide (residues 1-121) had greatly reduced biological activity, indicating that the COOH terminus is required for receptor binding. We predict that residues 20-46 and the COOH terminus bind to the G-CSF receptor.

Amino Acid Sequence↗
PubMed1991

In vitro studies of intestinal drug absorption. Determination of partition and distribution coefficients with brush border membrane vesicles.

Brush border membrane vesicles (BBMV) were isolated from rat small intestine and characterized in terms of relative enrichment of specific organelle marker enzymes (20-fold enrichment; 20% yield), contamination by other subcellular organelles (less than 1%) and functional integrity (Na(+)-dependent glucose uptake). Using these vesicles, techniques were developed for the determination of partition and distribution coefficients for the model solutes, nitrobenzene, toluene and benzoic acid. No gender, age or regional variation along the small intestine in partition coefficient (log P) values was detected. There was no temperature (10-40 degrees) or pH (4.5-8.0) dependence in partition coefficients of nitrobenzene and toluene. Fair agreement was obtained for log P and log D values for these two solutes determined with BBMV and those reported with octanol and propylene glycol dipelargonate. Selective removal of proteins, both ecto-brush border and micro-villus core proteins, did not alter the partition coefficients of the three model solutes. In contrast, depletion of the BBMV of non-esterified fatty acids significantly decreased the partition coefficients. Liposomes prepared from BBMV lipid extracts were also used for partition coefficient determinations and gave similar values to intact BBMV; addition of increasing amounts of cholesterol to the lipid extract caused small increases in the partition coefficients of the model solutes in the liposomes. It was concluded that the partition coefficients of the BBMV were related to the lipid and not to the protein composition of the vesicles. The method offers a rapid and reliable means of measuring the partition coefficient of non-protein bound drugs and nutrients in isolated intestinal BBMV and should assist in the subsequent modelling and prediction of intestinal absorption in vivo.

Animals↗
PubMed1991

The major colonic cell mitogen extractable from colonic mucosa is an N terminally extended form of basic fibroblast growth factor.

Colonic growth factors (CGFs) were extracted from porcine intestinal epithelium and mucosa. Under acidic conditions, very little mitogenic activity (as assayed using murine 3T3 fibroblasts and a human colonic cell line) was extractable. However, by extracting at neutral or slightly alkaline pH, significant mitogenic activity for both the murine fibroblasts and human colonic carcinoma cell line could be detected. CGFs are present throughout the intestine and cecum. The epithelial mucosa of the distal colorectal region appeared to contain mitogens which were more potent for the colonic cells than the 3T3 fibroblasts. Purification of CGFs from the colonic mucosa required removal of associated mucin by pH precipitation prior to chromatographic fractionation. It was then possible to develop a complete purification (390,000-fold) scheme for the major CGF, an 18-kDa protein which bound to heparin-Sepharose. N-terminal sequence analysis yielded a single sequence (Q)SPGGAMAAGSITTLPALP, i.e. an N-terminally extended form of basic fibroblast growth factor. Apart from the substitution of Gly in bovine basic fibroblast growth factor by a Ser in porcine CGF, the proteins are identical. A similar extraction procedure using purified human colonic crypt epithelial cells yielded a mitogen for the human colonic cell line with similar chromatographic properties.

Amino Acid Sequence↗
PubMed1991

Iron binding to, and release from, the basolateral membrane of mouse duodenal enterocytes.

The basolateral membrane of mouse duodenal enterocytes can be selectively labelled in vitro with 59Fe by incubating intact enterocytes with 59Fe(III)-nitrilotriacetate at 0-4 degrees C. It has been proposed that this labelling represents binding to a site important in the transfer of intracellular Fe to the portal plasma (Snape, S., Simpson, R.J. and Peters, T.J. (1990) Cell Biochem. Funct. 8, 107-115). Studies presented here show binding to intact enterocytes in vitro was complete within 1 h and was proportional to enterocyte protein concentration. Binding to enterocytes isolated from both normal and chronically hypoxic mice showed a hyperbolic dependence on medium Fe(III) concentration, consistent with a single class of binding sites. Neither apparent binding constant nor maximal binding were increased by hypoxic exposure of mice, suggesting that the increased in vivo labelling of this site in hypoxia is not due to an increase in affinity or capacity of this site for iron. Release of iron from intact enterocytes, labelled at 0-4 degrees C, was measured at 37 degrees C and 0-4 degrees C. Release of 59Fe was extensive and more rapid at 37 degrees C with highest release to mouse serum. Iron released to serum was found to be bound to transferrin. Prior dialysis of serum against buffer led to complete failure of enterocytes to release iron. Reconstituting serum by adding back the dialysate restores release to levels seen in fresh serum, suggesting that low molecular weight serum components, notably bicarbonate, mediate iron transfer from the basolateral membrane to serum transferrin. The properties of the basolateral membrane iron binding site described here are consistent with a role in the iron transfer process.

Animals↗
PubMed1991

Cloning and sequencing of Lol pI, the major allergenic protein of rye-grass pollen.

We have isolated a full length cDNA clone encoding the major glycoprotein allergen Lol pI. The clone was selected using a combination of immunological screening of a cDNA expression library and PCR amplification of Lol pI-specific transcripts. Lol pI expressed in bacteria as a fusion protein shows recognition by specific IgE antibodies present in sera of grass pollen-allergic subjects. Northern analysis has shown that the Lol pI transcripts are expressed only in pollen of rye-grass. Molecular cloning of Lol pI provides a molecular genetic approach to study the structure-function relationship of allergens.

Allergens↗