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R J Simpson

Publications and source records attributed to R J Simpson.

At least 235 records · Page 13Linked to original sources
PubMed1991

Isolation of cDNA encoding a newly identified major allergenic protein of rye-grass pollen: intracellular targeting to the amyloplast.

We have identified a major allergenic protein from rye-grass pollen, tentatively designated Lol pIb of 31kDa and with pI 9.0. A cDNA clone encoding Lol pIb has been isolated, sequenced, and characterized. Lol pIb is located mainly in the starch granules. This is a distinct allergen from Lol pI, which is located in the cytosol. Lol pIb is synthesized in pollen as a pre-allergen with a transit peptide targeting the allergen to amyloplasts. Epitope mapping of the fusion protein localized the IgE binding determinant in the C-terminal domain.

Allergens↗
PubMed1991

Molecular cloning and expression of cDNA encoding the enzyme that controls conversion of high-mannose to hybrid and complex N-glycans: UDP-N-acetylglucosamine: alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I.

UDP-GlcNAc:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT I; EC 2.4.1.101) catalyzes an essential first step in the conversion of high-mannose N-glycans to hybrid and complex N-glycans. Cloning of the gene encoding this enzyme was carried out by mixed oligonucleotide-primed polymerase chain reaction amplification of rabbit liver single-stranded cDNA using sense and antisense 20- to 24-base-pair (bp) primers. A rabbit liver library in phage lambda gt10 yielded a 2.5-kilobase (kb) cDNA with a 447-amino acid coding sequence. None of the nine asparagine residues were in an Asn-Xaa-(Ser or Thr) sequence, indicating that the protein is not N-glycosylated. There is no sequence homology to other previously cloned glycosyltransferases, but GnT I appears to have a domain structure typical of these enzymes--i.e., a short amino-terminal domain, a transmembrane domain, a "neck" region, and a large carboxyl-terminal catalytic domain. RNA was transcribed off the 2.5-kb cDNA, and in vitro translation with rabbit reticulocyte lysate yielded a 52-kDa protein with GnT I activity.

Amino Acid Sequence↗
PubMed1991

House dust mite-derived amylase: allergenicity and physicochemical characterization.

Amylase activity was found in extracts of both Dermatophagoides pteronyssinus whole mite (0.16 U/mg) and spent growth medium (0.01 U/mg) but not in unused growth medium. It was also detected in all extracts of house dust obtained from mattresses (n = 20; geometric mean, 1.95 U/gm) and in 18 extracts of dust obtained from lounge room carpets (n = 20; geometric mean, 0.54 U/gm). Although the origins of amylase in dust are unclear, enzyme activity correlated with mite counts (n = 40; r = 0.35; p less than 0.05) and Der p I concentrations (r = 0.41; p less than 0.01). Mite amylase was purified from spent growth medium by affinity chromatography, gel filtration, and chromatofocusing. It was physicochemically similar to mammalian amylase with regard to molecular weight (60,000), charge heterogeneity (isoelectric point, 5 to 7) and the capacity to bind to an organomercurial affinity matrix. The optimum pH for enzymatic activity was revealed to be 6.4. IgE immunoblot studies demonstrated that the enzyme was allergenic and that its expression was dependent on the integrity of intrachain disulfide bonds. Sera from 25% of mite-allergic children and 46% of mite-allergic adults contained specific IgE to mite amylase. IgE to amylase was associated (p less than 0.01) with increased concentrations of total mite-specific IgE determined with a direct RAST assay.

Adult↗
PubMed1991

Purification and partial amino acid sequence of annexin V from porcine gastric mucosal membranes.

1. Annexin V has been purified from Triton X-100 extracts of porcine gastric mucosal membranes by a combination of chromatography on concanavalin A-Sepharose and DEAE-Sepharose, and preparative gel electrophoresis. 2. No N-terminal amino acid sequence was detected. 3. The sequences of 11 tryptic peptides were determined, amounting to a total of 121 amino acids, or 38% of the molecule. 4. When the peptides were compared with the cDNA-derived sequence of human annexin V, only three substitutions were observed. 5. Human and porcine annexin V are 97% homologous within the sequenced regions.

Amino Acid Sequence↗
PubMed1991

Iron absorption by hypotransferrinaemic mice.

Iron absorption rates by homozygous and wild-type mice from a hypotransferrinaemic mouse colony were examined with in vivo tied-off duodenal segments and in vitro incubated duodenal fragments. Enhanced initial rates of mucosal uptake and carcass transfer by homozygotes, compared to wild-types, were observed. The changes in vivo and in in vitro uptake kinetics resemble changes seen in iron deficient or hypoxic mice, suggesting that the liver iron loading shown by homozygotes is due to a failure of the normal mechanism for regulation of iron absorption. In vivo mucosal uptake and carcass transfer of radioiron showed an inverse correlation with liver non-haem iron content in homozygous hypotransferrinaemic mice, suggesting that some degree of control of absorption, albeit at markedly reduced sensitivity, can operate in these mice. No correlation between haemoglobin level and iron absorption was observed in homozygous hypotransferrinaemic mice, suggesting that this regulator of iron absorption does not function in these mice. The precise pathogenic mechanism of the enhanced iron absorption in hypotransferrinaemia remains to be determined. Mucosal transferrin levels were found to parallel serum transferrin levels in homozygotes, heterozygotes and wild-type mice. This supports previous suggestions that mucosal transferrin is derived from plasma transferrin and that the enhancement of iron absorption, by physiological mechanisms, does not require the presence of mucosal transferrin.

Absorption↗
PubMed1991

Absorption of iron from iron succinyl-protein complexes by mouse small intestine.

The absorption of iron from iron succinyl-protein complexes was investigated in mice. 59Fe-labelled succinyl-casein and -albumin complexes, [59Fe]ferritin and 59FeSO4, at doses of 20 or 200 micrograms of iron, were administered orally to normal mice or mice with absorption enhanced by chronic hypoxia. 59Fe from iron succinyl-protein was well absorbed in normal mice (greater than 10% of dose) and showed enhanced absorption in hypoxic mice (greater than 40% of dose). Intestinal uptake was predominantly by the duodenum for all compounds studied. In-vivo absorption of 59Fe from an iron succinyl-protein complex was studied using tied-off segments of mouse duodenum, jejunum or ileum of normal or hypoxic mice. Incubation for up to 15 min in duodenum or 60 min in ileum showed very little absorption of 59Fe. No enhancement of absorption was seen in hypoxic mice. It was concluded that absorption of the intact iron succinyl-protein complex cannot explain absorption seen after oral dosing.

Animals↗
PubMed1991

Alpha B crystallin accumulation is a specific response to Ha-ras and v-mos oncogene expression in mouse NIH 3T3 fibroblasts.

The conditional expression of the v-mos and Ha-ras(EJ) oncogenes in NIH 3T3 cells leads to the accumulation of a 23-kDa protein (p23) (R. Klemenz, S. Hoffmann, R. Jaggi, and A.-K. Werenskiold, Oncogene 4:799-803, 1989). We purified p23 to homogeneity and determined part of the amino acid sequence. The obtained sequence is identical with that of the eye lens protein alpha B crystallin. Northern (RNA) blot and Western immunoblot experiments were performed to demonstrate that alpha B crystallin mRNA and protein do indeed accumulate as a consequence of v-mos and Ha-ras oncogene expression. Comparison of cDNA clones obtained from the mRNA of eye lenses and of oncogene-expressing fibroblasts revealed identity between them. The major transcription initiation site of the alpha B crystallin gene in our experimental system was shown by primer extension experiments to be identical with the one used in eye epithelial cells. In addition, we identified a second minor initiation site 49 nucleotides further upstream. Serum growth factors did not stimulate alpha B crystallin expression in growth-arrested cells.

Amino Acid Sequence↗
PubMed1991

Allergenicity and physicochemical characterization of house dust mite derived amylase.

The enzyme amylase was shown to be present in extracts prepared from both house dust and spent growth medium used in the culture of the mite Dermatophagoides pteronyssinus. In dust, it was shown to correlate with both mite counts and concentrations of the faecally derived mite allergen, Der p I. Mite amylase was isolated from the culture medium and shown to be a single chain protein with a molecular weight of 56,000. The enzyme contained free sulphydryl groups and had the N-terminal sequence, KYXNPHFIGXRSVITXLME. It was found to be an allergen using sera from adults (46% positive) and children (25%) who were mite allergic. The expression of allergenicity was dependent on the integrity of intra-chain disulphide bonds.

Allergens↗
PubMed1991

A brief dietary assessment to guide cholesterol reduction in low-income individuals: design and validation.

Low-income Americans are at greatest risk for coronary heart disease. Dietary assessment methods are needed that can efficiently and effectively guide diet counseling to reduce serum cholesterol in this population. The Dietary Risk Assessment is a brief food frequency questionnaire designed to guide an intervention program for cholesterol reduction. It can easily be administered and scored in 10 to 15 minutes by persons who are not trained in nutrition. The assessment is culturally specific for a low-income southern population, identifies positive as well as problematic dietary behaviors, is easily interpreted, and measures potential barriers to dietary change. The assessment was validated against 3 days of dietary recall data in a sample of 42 low-income individuals recruited from the waiting room of an ambulatory care clinic. A Keys score, which measures the serum-cholesterol-raising potential of the diet, was calculated for each patient from their recall data. The Keys and Dietary Risk Assessment scores were significantly correlated (r = .60, P less than .001). We conclude that the Dietary Risk Assessment can rank individuals by level of dietary atherogenic risk adequately to guide a dietary treatment program for low-income patients, an underserved population with a high prevalence of diet-induced elevations in serum cholesterol.

Cholesterol↗
PubMed1990

Strategies for internal amino acid sequence analysis of proteins separated by polyacrylamide gel electrophoresis.

An evaluation has been made of various strategies for obtaining internal amino acid sequence data from electrophoretically separated proteins. Electroblotting, in situ proteolysis and extraction, and direct electroelution are compared. Electroblotting of protein or peptides from gels resulted in poor yields (typically, 1-7%). However, higher yields (3-67%) were achieved by in situ enzymatic cleavage followed by acid extraction of the peptides from the gel. Peptides extracted from the gel were separated by reversed-phase high-performance liquid chromatography (RP-HPLC), on short, small-bore columns (100 x 2.1 mm I.D.), to enable recovery of peptides in small volumes (ca. 50 microliters) suitable for microsequence analysis. Capillary zone electrophoresis under acidic conditions (pH 2.5) was used to assess peptide purity before sequence analysis. Cysteine residues were identified in unmodified proteins or peptides by a characteristic phenylthiohydantoin (PTH)-amino acid derivative during sequence analysis. This derivative does not co-chromatograph with any known PTH-amino acid. Direct electrophoretic elution of protein from gels yielded between 45-50% of applied protein. Proteins recovered from gels by electrophoretic elution required further purification by inverse-gradient RP-HPLC [R. J. Simpson, R. L. Moritz, E. C. Nice and B. Grego, Eur. J. Biochem., 165 (1987) 21] to remove sodium dodecylsulphate and acrylamide-related contaminants for sequence analysis.

Amino Acid Sequence↗
PubMed1990

Location of the dihydroorotase domain within trifunctional hamster dihydroorotate synthetase.

Mammalian dihydroorotase (DHOase, EC 3.5.2.3) is part of a trifunctional protein, dihydroorotate synthetase which catalyzes the first three reactions of de novo pyrimidine biosynthesis. We have subcloned a portion of the cDNA from the plasmid pCAD142 and obtained a nucleotide sequence which extends 2.1 kb in the 5' direction from the sequence encoding the aspartate transcarbamoylase (ATCase) domain at the 3'-end of the cDNA. The DHOase and ATCase domains have been purified from an elastase digest of the trifunctional protein and subjected to amino acid (aa) sequencing from their N termini. The sequence of the N-terminal 24 aa of the DHOase domain has been obtained and aligned with the cDNA sequence. The C-terminal residues of the DHOase domain have been identified as Leu followed by Val which, when taken with partial sequences of the CNBr fragments of this domain, defines the coding sequence of the active, globular DHOase domain released by proteolysis. Prediction of protein secondary structure from the deduced aa sequence showed that the DHOase domain (Mr 37,751) is separated from the C-terminal ATCase domain (Mr 34,323) by a bridging sequence (Mr 12,532) consisting of multiple beta-turns.

Amino Acid Sequence↗
PubMed1990

Complete amino acid sequence of tenebrosin-C, a cardiac stimulatory and haemolytic protein from the sea anemone Actinia tenebrosa.

The complete amino acid sequence of the cardiac stimulatory and haemolytic protein tenebrosin-C, from the Australian sea anemone Actinia tenebrosa, has been determined by Edman degradation of the intact molecule and fragments produced by treatment of the polypeptide chain with cyanogen bromide and enzymatic cleavage with endoproteinase Asp-N, thermolysin and trypsin. The molecule is a single-chain polypeptide consisting of 179 amino acid residues with a calculated molecular mass of 19,797 Da. Tenebrosin-C shows a high degree of amino acid sequence similarity (63%) with Stoichactis helianthus cytolysin III [Blumenthal, K. M. and Kem, W. R. (1983) J. Biol. Chem. 258, 5574-5581] and is identical to a partial sequence (90 residues) reported for equinatoxin, a cardiostimulatory and haemolytic protein isolated from the European sea anemone Actinia equina [Ferlan, I. and Jackson, K. (1983) Toxicon Suppl. 3, 141-144]. No amino acid sequence similarity was detected between tenebrosin-C and other protein sequences stored in available databases. The predicted secondary structure of tenebrosin-C suggests that it is a compact, highly structured molecule.

Amino Acid Sequence↗
PubMed1990

Internal amino acid sequencing of proteins by in situ cyanogen bromide cleavage in polyacrylamide gels.

A new method was developed for generating peptide fragments for amino acid sequence analysis from polyacrylamide-gel separated proteins. This method involves in situ CNBr treatment of proteins in the polyacrylamide gel after their separation by electrophoresis. Pure CNBr peptides were recovered either by solvent extraction followed by microbore column reversed-phase HPLC or, alternatively, by a second electrophoretic separation step (SDS-PAGE) followed by electrotransfer of the peptides onto polyvinylidene difluoride (PVDF) membranes. These approaches yielded sequence data at subnanomole levels for a wide range of CNBr fragments recovered from gel-separated proteins.

Amino Acid Sequence↗
PubMed1990

Subcellular localization of recently-absorbed iron in mouse duodenal enterocytes: identification of a basolateral membrane iron-binding site.

The subcellular distribution of newly absorbed iron in isolated mouse duodenal enterocytes was investigated by analytical subcellular fractionation using sucrose density gradient centifugation. Two major peaks of mucosal 59Fe activity were observed: one soluble and one particulate (density 1.18-1.20 g ml-1). The latter was increased following prior exposure of animals to chronic hypoxia. The particulate 59Fe was localized to the basolateral membranes using the marker enzyme Na+, K+ activated, Mg2+ dependent, ATPase and by washing intact enterocytes with the selective plasma membrane perturbant digitonin. The basolateral membrane can be selectively labelled by in vitro incubation of intact enterocytes at 0 degrees C with 59Fe(III)-nitrilotriacetate complex, confirming the presence of a 59Fe binding site on this membrane. No significant difference in in vitro iron binding to this site was observed between normal and chronically hypoxic animals. Iron binding to the basolateral membrane was significantly higher in disrupted, compared to intact enterocytes, indicating that this site is present on both sides of the basolateral membrane. It is therefore suggested that the increased labelling of this site in hypoxia, in vivo, is a consequence of an increase in a mucosal Fe pool which is available for binding to a membrane receptor.

Adenosine Triphosphatases↗
PubMed1990

Development of a database of amino acid sequences for human colon carcinoma proteins separated by two-dimensional polyacrylamide gel electrophoresis.

The tandem use of preparative two-dimensional polyacrylamide gel electrophoresis (2-DE) and electroblotting onto polyvinylidene difluoride membranes has been employed to rapidly isolate a number of proteins from a crude cell extract of a human colon carcinoma cell line (LIM 1863). The immobilized proteins were located by staining with Coomassie Brilliant Blue R-250, and selected protein spots were excised and subjected to Edman degradation. Our results demonstrate that overall sequence yields in the 3-20 pmol range can be achieved on protein spots from four identical 2-DE gels; approximately 150-200 micrograms of total protein was applied to a single 2-DE gel. An approximate two-fold increase in sensitivity of phenylthiohydantoin-amino acid detection (subpicomole range) was achieved by fitting our commercial sequencers with a simple sample transfer device which permitted the analysis of the total phenylthiohydantoin-amino acid derivative. N-Terminal amino acid sequence data was obtained for thirteen electroblotted proteins. All of these sequences positively matched those of proteins of known structure listed in the available protein sequence databases. Approximately 40% of the electroblotted proteins did not yield N-terminal sequence information, presumably because they had blocked N-termini (either naturally or artifactually). Internal amino acid sequence information was obtained from three proteins isolated by preparative 2-DE. This was achieved by in situ digestion of the proteins in the gel matrix with Staphylococcus aureus V8 protease, electrophoresis of the generated peptides in a one-dimensional gel, electrotransfer of the peptides to a polyvinylidene difluoride membrane and microsequence analysis of the electroblotted peptides.

Amino Acid Sequence↗