Search PubMed⌕ Search

Biomedical subjects

R J Simpson

Publications and source records attributed to R J Simpson.

At least 127 records · Page 7Linked to original sources
PubMed1996

Complex assembly of calgranulins A and B, two S100-like calcium-binding proteins from pig granulocytes.

Calgranulin A (CAGA) and calgranulin B (CAGB) are two S100-like calcium-binding proteins that in human, bovine and mouse granulocytes are associated into a heterocomplex. We have previously identified in pig granulocytes the porcine homologue of CAGA and a novel S100-like protein which was named calgranulin C (CAGC). As pig CAGA is not associated with CAGC, we herein investigate its possible association with other proteins. CAGA was purified from pig granulocytes by gel filtration followed by Mono Q chromatography. The purified fractions were analysed by SDS-polyacrylamide gel electrophoresis, isoelectric focusing, mass spectrometry, chemical cross-linking and hydrophobic interaction chromatography. The CAGA-associated protein was further characterized by amino acid sequencing. Two CAGA-containing fractions were isolated. One of them was identified as a CAGA homodimer. The other fraction consists of a heterocomplex containing CAGA and a pI 7.0 calcium-binding protein; this protein has a molecular mass of 15,877.9 +/- 3.8 Da (mean +/- SD) whereas it migrates on 10 and 16% polyacrylamide gels as a 24- and 20-kDa protein, respectively. The pI 7.0 protein was identified by internal amino acid sequencing as the porcine homologue of CAGB. The stoichiometry of the heterocomplex was estimated to be 1:1. Both the CAGA homodimer and CAGA/CAGB were found to be non-covalently associated. Unlike the homodimer, CAGA/CAGB was bound to a Phenyl Superose column in a calcium-dependent manner. Our results suggest that pig granulocytes contain, in addition to CAGC, a CAGA homodimer and a CAGA/CAGB heterodimer. It is proposed that CAGB/CAGB and the CAGA homodimer may play different roles in vivo.

Amino Acid Sequence↗
PubMed1996

Population-based study of heart rate variability and prevalent myocardial infarction. The Atherosclerosis Risk in Communities Study.

Clinically based studies have indicated that patients who have suffered myocardial infarction (MI) exhibit lower heart rate variability (HRV). Such associations have not been reported at the population level. To investigate whether MI is related to lower HRV in the general population, the authors examined a stratified random sample of 2,243 men and women aged 45-64 years, of whom 260 had had MI and 1,983 were free of any coronary heart disease manifestations. All were part of the biracial cohort of the Atherosclerosis Risk in Communities study sampled from four United States communities. Resting, 2-minute supine heart rate data were analyzed using spectral analysis to estimate high-frequency (0.16-0.35 Hz) and low-frequency (0.025-0.15 Hz) spectral powers as conventional indices of HRV. The mean levels of high-frequency powers were 1.02 and 1.23 (beats/min)2 for MI and non-MI groups, respectively (P < .05), and the low-frequency means were 2.46 and 3.11 (beats/min)2 for MI and non-MI groups, respectively (P < .01). Age, race, and sex-adjusted odds ratios (95% confidence interval) of lower high-frequency and low-frequency powers contrasting MI to non-MI individuals were 1.52 (range, 1.09-2.10) and 1.54 (range, 1.12-2.10), respectively. After adjustment for use of beta-blocker medication, the odds ratios (95% confidence interval) were 1.26 (range, 0.88-1.81) and 1.22 (range, 0.87-1.69) for lower high-frequency and low-frequency powers, respectively. The findings from this first population-based study of prevalent MI and HRV provide support for an inverse association between MI and lower HRV. However, adjusting for beta-blocker use eliminates the statistical association. These findings confirm that impaired HRV is found in individuals with prevalent MI, even when drawn as a sample of community-dwelling individuals. This impaired HRV may contribute to the increased risk of subsequent cardiovascular events previously observed in survivors of acute MI. The cross-sectional data also suggest that use of beta-blockers diminishes the odds of lower HRV associated with MI.

Adrenergic beta-Antagonists↗
PubMed1996

The isolation and characterization of a novel collagenolytic serine protease allergen (Der p 9) from the dust mite Dermatophagoides pteronyssinus.

BACKGROUND: Dust mites have been shown to contain a serine protease distinct from the previously reported trypsin and chymotrypsin. The latter enzymes have been shown to be allergens, but the allergenic importance of the former is unknown. OBJECTIVE: This study was performed to isolate and characterize the novel mite serine protease and determine its allergenicity. METHODS: The mite serine protease was isolated from feces-enriched extracts of Dermatophagoides pteronyssinus by ion-exchange chromatography and affinity chromatography, and its physicochemical properties were determined. The allergenicity of the protease was assessed by using the RAST. RESULTS: The protease was enzymatically similar to chymotrypsin and cathepsin G-like enzymes from a variety of sources and was shown to cleave collagen. It had a molecular mass of 23,780 d. N-terminal sequence analysis (18 residues) indicated homology with the mite tryptic allergen, Der p 3, and the chymotryptic allergen, Der p 6. RAST analyses showed that the frequencies of reactivity to the novel allergen and to Der p 1, Der p 2, Der p 3, and Der p 6 were 92%, 97%, 100%, 97%, and 65%, respectively (n = 35). RAST inhibition studies showed some cross-reactivity between the protease and Der p 3 but not Der p 6. CONCLUSIONS: A novel mite serine protease was isolated from D. pteronyssinus and found to be a major allergen. This allergen has been tentatively designated Der p 9.

Allergens↗
PubMed1996

Regulation of pancreatitis-associated protein (HIP/PAP) mRNA levels in mouse pancreas and small intestine.

1. In the present study we have examined the expression of pancreatitis-associated protein mRNA in mouse pancreas and small intestine and determined the effect of a number of factors on the steady-state level of the RNA. 2. The normally low level of pancreatitis-associated protein mRNA in pancreas increased severalfold after 6 h of hypoxia, reaching peak levels (approximately 10-fold greater than normal) after 24 h hypoxia. After 3 days' hypoxia pancreatitis-associated protein mRNA levels were again undetectable. 3. In the pancreas the level of pancreatitis-associated protein mRNA was also increased by alcohol and iron overload, but not by paracetamol. 4. In the small intestine expression of pancreatitis-associated protein mRNA was higher in normal ileum than in duodenum. In the ileum pancreatitis-associated protein mRNA levels were increased 7 to 15-fold after 6 h hypoxia, reaching peak levels by 24 h. Levels declined after 3 days' hypoxia, but remained higher than normal. 5. In the ileum long-term (4 weeks) dietary iron deficiency reduced pancreatitis-associated protein mRNA levels compared with control fed mice, whereas parenteral iron overload increased pancreatitis-associated protein mRNA levels. 6. The data presented suggest regulation of pancreatitis-associated protein gene expression by both oxygen tension and iron status.

Acetaminophen↗
PubMed1996

Molecular characterization of a stigma-specific gene encoding an arabinogalactan-protein (AGP) from Nicotiana alata.

Arabinogalactan-proteins (AGPs) were isolated from the pistils of Nicotiana alata, deglycosylated, and the protein backbones fractionated by reversed-phase HPLC as previously reported. A major fraction, RT35 was isolated and peptide sequences were obtained after protease digestion. A gene-specific degenerate oligonucleotide was designed according to the amino acid sequences and a 380 bp PCR fragment was amplified in vitro from pistil RNA. The PCR fragment was used to screen a pistil cDNA library and a 762 bp cDNA clone (AGPNa3) was isolated and sequenced. The AGPNa3 cDNA encodes a 169 amino acid protein which consists of three domains: an N-terminal secretion signal, a Pro-rich domain and a C-terminal Cys-rich domain. The mature protein has 145 amino acid residues (16.7 kDa) and a predicted pl of 7.5. Northern blot analyses showed that the AGPNa3 gene was only expressed in the pistils of N. alata and of closely related Nicotiana species but not in other plants or suspension-cultured cells. Further Northern blot analysis and in situ hybridization showed that within the pistil, it was primarily expressed in the stigmatic tissues of mature flowers.

Amino Acid Sequence↗
PubMed1996

Benign prostatic hyperplasia in an unselected community-based population: a survey of urinary symptoms, bothersomeness and prostatic enlargement.

OBJECTIVES: To determine the prevalence of (anatomical) benign prostatic hyperplasia/enlargement (BPH/BPE) in an unselected population and to assess the relationships between BPE, urinary flow rates (UFRs) and symptoms in men with BPH. SUBJECTS AND METHODS: All men aged 40-79 years and living in three ex-mining villages in Scotland were invited to undergo a measurement of UFR, transrectal ultrasonography (TRUS) and to complete a symptom score and a previously validated lifestyle questionnaire. RESULTS: Of 597 eligible men, 367 (61%) completed the urinary symptom questionnaire and 310 (52%) underwent TRUS and measurement of UFR. The response rate in the age group 50-69 was > 60%. Age-specific prevalence rates for BPE (prostate size > 20 g) per 1000 men were: 40-49 years, 615; 50-59 years, 776; 60-69 years, 892; and 70-79 years, 889, giving an overall rate for all ages of 765. There was no significant relationship between prostate size and symptoms, nor between size and peak UFR, nor any clear association between size and interference with activities of daily living. However, men with a UFR of > 20 mL/s were very unlikely to have prostates > 40 g. Relationships were found between some urinary symptoms and UFR. Age, weight and the level of prostate-specific antigen (PSA) were the only independent variables associated with prostate size. CONCLUSION: Compared to published studies, BPE was substantially more prevalent than assumed previously. This study further reinforces the need for the subtle and detailed evaluation of patients who have a syndrome of BPH in assigning them to intervention, be it medical or surgical, or non-intervention/watchful waiting.

Adult↗
PubMed1996

Dietary iron levels and hypoxia independently affect iron absorption in mice.

The effect of dietary iron levels on the hypoxic response of iron absorption was investigated by feeding mice diets of different iron composition and determining iron absorption. Eight groups of mice were fed a purified diet consisting of casein, corn oil and sucrose with vitamins and iron-free mineral supplements. Groups A-D were fed a nonpurified diet until 6 wk old and then switched to purified diet for 4 d. Groups E-H were fed purified diet for 4 wk from weaning. Groups C, D, G and H were exposed to hypoxia (53.3 kPa) for the last 3 d of the study. Groups A, C, E and G received the low iron purified diet (<1 mg iron per kg); the other groups received the same diet supplemented with 62 mg/kg iron as FeCl3 x 6H2O. Hypoxic exposure raised iron absorption (P < 0.001) by a similar proportion (2.5- to 5.0-fold) in all mice but did not affect duodenal nonheme iron levels. Low iron diet feeding raised iron absorption but reduced duodenal and liver nonheme iron levels. In a second experiment, mice fed nonpurified diet were divided into four groups and exposed to normal atmosphere or hypoxia (53.3 kPa) with or without dosing with 100 microg iron by stomach tube 1 h before determining iron absorption. There was no effect of iron dosing on iron absorption at either pressure. The data suggest that the control of iron absorption by tissue oxygen acts through a mechanism independent of the control exerted by dietary or mucosal iron levels.

Animals↗
PubMed1996

Ventricular fibrillation induction using nonsynchronized low energy external shock during rapid ventricular pacing: method of induction when fibrillation mode of ICD fails.

Third-generation implantable cardioverter defibrillators (ICD) are frequently implanted with nonthoracotomy systems and provide noninvasive methods for electrical stimulation and ventricular fibrillation induction. These modalities facilitate postoperative testing of the ICD. Rapid right ventricular burst pacing via the defibrillator is commonly used for initiation of ventricular tachyarrhythmias. However, with the available third-generation devices, ventricular fibrillation (VF) induction may be impossible in up to 19% of the patients. In these cases, transvenous placement of a right ventricular catheter has been required to generate VF and appropriately evaluate the device. We report a new technique of noninvasive induction of VF using a low energy external nonsynchronized shock delivered during ICD fibrillation induction pacing. In three patients, after all efforts to induce VF by the Ventritex Cadence V-100 had failed, a 20 J nonsynchronized shock was delivered during rapid RV pacing. This resulted in VF on the first attempt in all patients. This noninvasive technique of VF initiation may provide a useful clinical approach to ICD testing that eliminates the costs and risks of an invasive procedure.

Adult↗
PubMed1996

A duodenal mucosal abnormality in the reduction of Fe(III) in patients with genetic haemochromatosis.

BACKGROUND: Previous in vitro studies have shown that the uptake of Fe(III) by freshly isolated duodenal mucosal biopsy specimens is increased in patients with genetic haemochromatosis. Moreover, in the mouse it has recently been found that reduction of Fe(III) to Fe(II) is a prerequisite for iron uptake by the proximal intestine. AIMS/METHODS: This study used the in vitro technique to investigate the rates of reduction and uptake of 59Fe(III) by duodenal mucosal biopsy specimens obtained at endoscopy from treated and untreated patients with genetic haemochromatosis. RESULTS: The rate of reduction of iron in the medium was proportional to the incubation time and was not caused by the release of reducing factors from the tissue fragments. Ferrozine, a specific Fe(II) chelator and ferricyanide, a non-permeable oxidising agent, inhibited uptake of 59Fe showing that reduction of Fe(III) precedes uptake. The rates (all values given as pmol/mg/min) of reduction (152 (49) v 92 (23)) and uptake (8.3 (4.0) v 3.6 (1.3), mean (SD)), were significantly increased in biopsy specimens from the untreated group (n = 6) compared with those from 10 control subjects (p < 0.04). Furthermore, the reduction and uptake rates were still increased in five patients in whom iron stores were normal after venesection treatment. CONCLUSIONS: These results show that there is a persistent abnormality in the reduction and uptake of iron by the intestine in genetic haemochromatosis.

Analysis of Variance↗
PubMed1996

Expression of genes involved in iron metabolism in mouse intestine.

Intestinal nonheme iron levels and mRNA levels of genes implicated in iron metabolism were measured in mice with altered iron metabolism [chronic (4 wk) and acute (4 days) dietary iron deficiency; iron overload and hypoxia] to investigate their role in the process and regulation of intestinal iron absorption. Mucosal nonheme iron levels were decreased by both chronic and acute iron deficiency and increased by iron overload but were not affected by hypoxia. There was evidence of a gradient of mucosal nonheme iron along the small intestine (duodenum, jejunum > ileum). There were also regional differences in H-ferritin (duodenum > ileum) and transferrin receptor (ileum > duodenum) mRNA levels. Iron overload produced a decrease in transferrin receptor (TfR) mRNA in the duodenum, with ferritin mRNA levels unaffected in both the duodenum and ileum. Chronic iron deficiency induced a twofold increase in TfR mRNA levels in both the duodenum and ileum, whereas H- and L-ferritin mRNA levels did not change significantly. The ratio of H- to L-ferritin mRNA decreased significantly during exposure to hypoxia; however, individual ferritin and TfR mRNA levels were not significantly altered. Calreticulin (mobilferrin), cysteine-rich intestinal protein, and H(+)-adenosinetriphosphatase mRNA levels were virtually unchanged in all models. A comparison with previously published data on changes in iron absorption leads us to conclude that 1) iron absorption can be altered independently of effects on transcripts of genes for iron-related proteins, and 2) it is not essential for iron absorption to be coordinated with regulation of mucosal iron metabolism.

Animals↗
PubMed1995

Relationship between duodenal cytosolic aconitase activity and iron status in the mouse.

Cytosolic aconitase activity was assayed in duodenal mucosa from mice subjected to a variety of manipulations known to modulate duodenal iron status and duodenal iron absorption. No changes in cytosolic aconitase activity were observed 1 h after oral FeSO4 dosing or intramuscular desferrioxamine treatment. Three days of hypoxic exposure and two weeks treatment with intramuscular iron dextran also had no effect on cytosolic aconitase. Three weeks growth on an iron deficient diet significantly reduced cytosolic aconitase activity. In no situation was there any evidence for significant amounts of inactive aconitase which could be activated in vitro with FeSO4/cysteine. These data suggest that duodenal cytosolic aconitase is not sensitive to acute changes in mucosal iron levels and is generally much less sensitive to body iron status than is duodenal iron absorption. There is evidence that chronic iron depletion reduces cytosolic aconitase to a relatively small degree but generally activity is maintained, consistent with an important metabolic role for the enzyme.

Aconitate Hydratase↗
PubMed1995

Complete nucleotide sequence, expression, and chromosomal localisation of human mixed-lineage kinase 2.

Protein kinases play pivotal roles in the control of many cellular processes. In a search for protein kinases expressed in human epithelial tumour cells, we discovered two members of a novel protein kinase family [Dorow, D. S., Devereux, L., Dietzsch, E. & de Kretser, T. A. (1993) Eur. J. Biochem. 213, 701-710]. Due to the unique mixture of structural domains within their amino acid sequences, we named the family mixed-lineage kinases (MLK). We initially isolated clones encoding partial cDNAs of MLK1 and 2 from a human colonic cDNA library. The MLK2 cDNA was subsequently used to screen a human brain cDNA library and we have now cloned and sequenced a 3454-bp cDNA encoding the full-length MLK2 protein. The predicted MLK2 polypeptide has 954 amino acids and contains a src homology 3 (SH3) domain, a kinase catalytic domain, a double leucine zipper and basic domain, and a large C-terminal domain. The 22-amino-acid N-terminal region has four glutamic acid residues immediately following the initiator methionine. Beginning at amino acid 23, the 55-amino-acid SH3 domain contains a 5-amino-acid insert in a position corresponding to inserts of 6 and 15 residues in the SH3 domains of n-src and the phosphatidylinositol 3'-kinase. Adjacent to the SH3 domain is a kinase catalytic domain with conserved motifs associated with both serine/threonine and tyrosine specificity. Beginning nine residues C-terminal to the catalytic domain, there are two leucine/isoleucine zippers separated by a 13-amino-acid spacer sequence and followed by a stretch of basic residues. The polybasic sequence contains a motif that is similar to nuclear localisation signals from several proteins. The C-terminal domain is composed of 491 amino acids of which 17% are serine or threonine and 16% are proline. This domain also has a biased ratio of basic to acidic amino acids with a calculated pI of 9.38. When used as a probe to examine mRNA expression in human tissues, a MLK2 cDNA hybridised to a species of 3.8 kb that was expressed at highest levels in RNA from brain and skeletal muscle. The 3454-bp cDNA was also used for fluorescence in situ hybridisation to localise the MLK2 gene to human chromosome 19 q13.2.

Amino Acid Sequence↗
PubMed1995

Age, race, and sex differences in autonomic cardiac function measured by spectral analysis of heart rate variability--the ARIC study. Atherosclerosis Risk in Communities.

To investigate the distribution of heart rate variability (HRV) spectral power in an unselected sample of the population, and to ascertain the population correlates of HRV, we examined 1,984 healthy persons, aged 45 to 64 years, randomly selected from the Atherosclerosis Risk in Communities (ARIC) study cohort. Resting, supine, 2-minute, beat-to-beat heart rate data were collected between 7 A.M. and 12 noon. The race- and sex-adjusted geometric means of low-frequency component (LF, 0.025 to 0.15 Hz) were 4.00 and 3.13 (beats/min)2; of high-frequency component (HF, 0.16 to 0.35 Hz), 1.65 and 1.21 (beats/min)2; and of the HF/LF ratio, 0.41 and 0.39, for 45-to-54 and 55-to-64 years age groups, respectively (test of mean difference by age, p < 0.01, p < 0.01, and p = 0.11 for LF, HF, and HF/LF ratio, respectively). Comparing black with white examinees, the age- and sex-adjusted geometric means of LF were 3.06 and 3.70 (beats/min)2; of HF, 1.66 and 1.36 (beats/min)2; of HF/LF, 0.54 and 0.37, respectively (test of mean difference by race, p < 0.01, p < 0.01, and p < 0.01). The age- and race-adjusted geometric means of LF for women and men were 3.12 and 4.10 (beats/min)2; of HF, 1.46 and 1.38 (beats/min)2; and of HF/LF, 0.47 and 0.34, respectively (test of mean difference, p < 0.01, p = 0.34, and p < 0.01). We conclude that HRV spectral indexes are associated with age, race, and sex. With increasing age, the parasympathetic and sympathetic spectral power components decrease.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗
PubMed1995

Equilibrium denaturation of recombinant murine interleukin-6: effect of pH, denaturants, and salt on formation of folding intermediates.

The equilibrium denaturation of an Escherichia coli-derived recombinant murine interleukin-6 (mIL-6) was studied using fluorescence and circular dichroism spectroscopy. The urea-induced unfolding of mIL-6 at pH 4.0 can be described by a two-state unfolding mechanism based on the superimposibility of the CD and fluorescence unfolding transitions. Assuming a two-state mechanism and a linear dependence of the free energy of unfolding on denaturant concentration, a value of 6.9-9.0 kcal/mol was calculated for the free energy of unfolding in the absence of denaturant [delta GU(H2O)]. However, when GuHCl was used as a denaturant at pH 4.0, a biphasic unfolding transition was observed. This unfolding transition has a distinct midpoint occurring at 2.5 M GuHCl, which is indicative of the formation of stable folding intermediates. Similar intermediate folded species were also observed at pH 7.4 when either urea or GuHCl were used as denaturants. The intermediate folded states of mIL-6 exhibited a tendency to aggregate, as judged by the concentration dependence of their fluorescence characteristics. The fluorescence emission maximum of mIL-6 at pH 7.4 in the presence of 1.5 M GuHCl, for example, was blue-shifted from 343 nm at a protein concentration of 50 micrograms/mL to 336 nm at 500 micrograms/mL. Intermediate formation at pH 4.0, using 10 mM sodium acetate buffer and urea as the denaturant, was facilitated by the addition of 0.4 and 0.8 M salt, where the salt was either NaCl or GuHCl.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Molecular cloning and expression of cDNA encoding the rat UDP-N-acetylglucosamine:alpha-6-D-mannoside beta-1,2-N-acetylglucosaminyltransferase II.

UDP-N-acetyl-D-glucosamine:alpha-6-D-mannoside beta-1,2-N-acetylglucosaminyltransferase II (EC 2.4.1.143) (GnT II) is a Golgi resident enzyme that catalyzes an essential step in the biosynthetic pathway leading from high mannose to complex N-linked oligosaccharides. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the enzyme purified from rat liver revealed a polypeptide of 42 kDa. Amino acid sequences were obtained from the N terminus and a tryptic peptide. Overlapping cDNA clones coding for the full-length rat GnT II were obtained. The complete nucleotide sequence revealed a 1326-base pair open reading frame that codes for a polypeptide of 442 amino acids, including a presumptive N-terminal membrane-anchoring domain. The region of cDNA coding for the C-terminal 389 amino acids of rat GnT II was linked in frame to a cDNA segment encoding the cleavable signal sequence of the human interleukin-2 receptor and transiently expressed in COS-7 cells. A 77-fold enhancement of GnT II activity over a control carrying the GnT II cDNA out-of-frame was detected in the culture medium at 72 h after transfection. 1H-NMR spectroscopy confirmed that the oligosaccharide synthesized in vitro by the recombinant enzyme was the product of GnT II activity. These data verify the identity of the cloned GnT II cDNA and demonstrate that the C-terminal region of the protein includes the catalytic domain.

Amino Acid Sequence↗
PubMed1995

NMR studies of the solution properties of recombinant murine interleukin-6.

The effects of solvent, pH and temperature on the 1H-NMR spectra of recombinant murine interleukin-6 (IL-6) are described. Assignments made from two-dimensional homonuclear spectra are presented for resonances of the fifteen aromatic amino-acid side chains. A time-dependent loss of intensity was observed for all resonances in the spectrum of IL-6, probably as a result of aggregation. This aggregation is markedly temperature-dependent. The pKa values of the four histidine residues in murine IL-6 has been measured; one has a value of 5.5, approx. one pH unit less than the value exhibited by the other three. Analysis of the NOESY spectra has allowed a preliminary characterisation of the nature of interactions among the aromatic side chains within the protein fold. 1H and 15N resonances of residues Thr-4 to Val-21 are assigned from three-dimensional 1H-15N correlated spectroscopy, and evidence is presented for these residues comprising a mobile N-terminal tail with little ordered structure. An N-terminal mutant lacking the first 22 residues of the murine IL-6 sequence and known to possess full biological activity was also examined and shown to have essentially retained the tertiary fold of the native molecule.

Amino Acid Sequence↗