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Biomedical subjects

A J Brown

Publications and source records attributed to A J Brown.

At least 19 recordsLinked to original sources
PubMed1992

Metabolism of 22-oxacalcitriol by a vitamin D-inducible pathway in cultured parathyroid cells.

Catabolism of 22-oxacalcitriol (OCT) in parathyroid cells was compared to that of the parent hormone, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Catabolism of both compounds was greatly accelerated by pretreatment of the cells with 1,25-(OH)2D3 or OCT. The rate of degradation of OCT was slightly greater than that of 1,25-(OH)2D3. Excess unlabeled OCT or 1,25-(OH)2D3 inhibited metabolism of both tritiated substrates. Ketoconazole, a cytochrome P450 inhibitor, blocked catabolism of both compounds. The major OCT metabolite appeared to be 1,20-dihydroxy-22,23,24,25,26,27-hexanor-vitamin D3 which was not active in suppressing PTH secretion. We conclude that OCT appears to be metabolized by the same vitamin D-inducible side chain oxidation pathway that catabolizes other vitamin D compounds and that its higher than expected suppression of PTH secretion is not due to slower cellular metabolism.

Animals↗
PubMed1992

Protein folding within the cell is influenced by controlled rates of polypeptide elongation.

Previous studies have proposed that specific translational pauses have evolved to promote protein folding inside the cell by temporally separating the folding of specific regions of some polypeptide chains during their synthesis. Here we show that this is the case for a bifunctional protein in Saccharomyces cerevisiae. The yeast TRP3 gene contains a translational pause comprising ten contiguous non-preferred codons within its second functional domain (indoleglycerol phosphate synthase). Site-directed mutagenesis was used to remove this translational pause by increasing the codon bias of the region without changing the amino acid sequence of the protein (to create the gene TRP3pr: pause replaced). The TRP3pr gene was able to complement a trp3:: URA3 null mutation in yeast. No significant differences in the doubling times of TRP3 or TRP3pr yeast transformants were observed during growth at 25 degrees C, 30 degrees C or 37 degrees C, or in the presence of sublethal concentrations of the analogue, 5-methyltryptophan. However, further analysis of TRP3 and TRP3pr transformants revealed that the removal of the translational pause causes a 1.5-fold decrease in indoleglycerol phosphate synthase activity per TRP3 mRNA. This observation which is statistically significant (P < 0.05) and reproducible, suggests that translational pausing promotes the correct intracellular folding of the TRP3 protein.

Amino Acid Sequence↗
PubMed1992

The compact domain conformation of human Glu-plasminogen in solution.

A complete understanding of the accelerating mechanisms of plasminogen activation and fibrinolysis necessarily requires structural information on the conformational forms of plasminogen. Given the absence of high-resolution structural data on plasminogen the use of lower resolution approaches has been adopted. Two such approaches have previously indicated a compact conformation of Glu-plasminogen (Tranqui, L., Prandini, M., and Chapel, A. (1979) Biol. Cellulaire, 34, 39-42; Bányai, L. and Patthy, L. (1985) Biochim. Biophys. Acta, 832, 224-227) whereas a third has suggested a fairly extended conformation (Mangel, W., Lin, B. and Ramakrishnan, V. (1990) Science, 248, 69-73). Native Glu-plasminogen has been investigated using small-angle X-ray scattering (SAXS) experiments. It is concluded that this molecule in solution is compact (radius of gyration, RG 3.05 +/- 0.02 nm and maximum intramolecular distance, Im 9.1 +/- 0.3 nm) and that the data are consistent with the right-handed spiral structure observed using electron microscopy by Tranqui et al. (1979). A spiral structure of native plasminogen would have important implications for the conformational response of plasminogen to fibrin and concomitant stimulation of plasminogen activation.

Computer Simulation↗
PubMed1992

Convergent and divergent sequence evolution in the surface envelope glycoprotein of human immunodeficiency virus type 1 within a single infected patient.

In an investigation of the evolution of the third hypervariable loop of gp120 (V3), the principal neutralization determinant of human immunodeficiency virus type 1, we have analyzed 89 V3 sequences of plasma viral RNA purified from peripheral blood samples donated over 7 years by an infected hemophiliac. Considerable sequence diversity in the V3 region was found at all time points after seroconversion. Phylogenetic analysis revealed that an important diversification had occurred by 3 years postinfection and that, subsequently, most sequences could be allocated to either one of two major lineages that persisted throughout the remainder of the infection. Rapid changes in frequency of the most common sequences and the observation that the same hexapeptide motif (GPGSAV) at the crown of the V3 loop has evolved convergently provide strong evidence that selective processes determine the evolutionary fate of sequence variants in this region.

Amino Acid Sequence↗
PubMed1992

22-oxacalcitriol suppresses 25-hydroxycholecalciferol-1 alpha-hydroxylase in rat kidney.

22-oxacalcitriol can decrease the serum level of 1,25-dihydroxycholecalciferol by increasing its metabolic clearance rate and decreasing its production rate. To determine whether 22-oxacalcitriol suppressed the renal 25-hydroxycholecalciferol-1 alpha- hydroxylase we treated rats with 200 ng of 22-oxacalcitriol daily for 1 week. Enzyme activity was measured in vitro by measurement of production of 1,25-dihydroxycholecalciferol by renal slices incubated with 25-hydroxycholecalciferol. 22-oxacalcitriol significantly decreased the 25-hydroxycholecalciferol-1 alpha-hydroxylase activity from 797 +/- 208 pg of 1,25-dihydroxycholecalciferol/g of kidney/h in the control group to 257 +/- 150 pg of 1,25-dihydroxycholecalciferol/g of kidney/h (p less than 0.05). This is the first demonstration of suppression of the renal 25-hydroxycholecalciferol-1 alpha-hydroxylase by the vitamin D3 analog, 22-oxacalcitriol.

Animals↗
PubMed1992

Suppression of PTH and decreased action on bone are partially responsible for the low calcemic activity of 22-oxacalcitriol relative to 1,25-(OH)2D3.

We previously showed that OCT, an analog of 1,25-(OH)2D3 with little calcemic activity, can decrease PTH mRNA levels in normal rats and inhibit PTH secretion in cultured bovine parathyroid cells with the same potency as 1,25-(OH)2D3 and that in normal rats fed a normal calcium diet, administration of OCT (500 ng) for 5 days did not increase plasma Ca. Thus, to determine if PTH suppression by OCT contributes to its lack of calcemic activity and to further characterize the effects of OCT on Ca metabolism, we performed several studies in parathyroidectomized (PTX) rats. PTX rats, maintained on a normal diet (0.9% Ca), received daily injections of vehicle, 1,25-(OH)2D3 (200 ng/day), or OCT (200 ng/day) for 6 days. Plasma Ca was measured daily. Plasma Ca in control rats stayed between 6.60 and 7.40 mg/dl, whereas Ca increased to 12.9 +/- 0.42 mg/dl in 1,25-(OH)2D3-treated rats and to 9.53 +/- 0.35 mg/dl in OCT-treated rats after 6 days. With a Ca-deficient diet, control rats maintained a plasma Ca between 4.25 and 4.60 mg/dl, but Ca increased to 13.7 +/- 0.24 mg/dl with 1,25-(OH)2D3 and to 7.29 +/- 0.17 mg/dl with OCT. Since the elevation in Ca by OCT was similar with both diets, OCT appears to act primarily on bone. PTX rats were infused with PTH (1.84 micrograms/kg/day) via an Alzet pump to achieve normal plasma Ca and then treated daily with either vehicle or OCT (200 ng/day). After 6 days, OCT increased serum Ca to 10.7 +/- 0.21 mg/dl over a control value of 8.58 +/- 0.29 mg/dl.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Positive regulation of the LPD1 gene of Saccharomyces cerevisiae by the HAP2/HAP3/HAP4 activation system.

The LPD1 gene of Saccharomyces cerevisiae, encoding lipoamide dehydrogenase (LPDH), is subject to catabolite repression. The promoter of this gene contains a number of motifs for DNA-binding transcriptional activators, including three which show strong sequence homology to the core HAP2/HAP3/HAP4 binding motif. Here we report that transcription of LPD1 requires HAP2, HAP3 and HAP4 for release from glucose repression. In the wild-type strain, specific activity of LPDH was increased 12-fold by growth on lactate, 10-fold on glycerol and four- to five-fold on galactose or raffinose, compared to growth on glucose. In hap2, hap3 and hap4 null mutants, the specific activities of LPDH in cultures grown on galactose and raffinose showed only slight induction above the basal level on glucose medium. Similar results were obtained upon assaying for beta-galactosidase production in wild-type, or hap2, hap3 or hap4 mutant strains carrying a single copy of the LPD1 promoter fused in frame to the lacZ gene of Escherichia coli and integrated at the URA3 locus. Transcript analysis in wild-type and hap2 mutants confirmed that the HAP2 protein regulates LPD1 expression at the level of transcription in the same way as it does for the CYC1 gene. Site-directed mutagenesis of the putative HAP2/HAP3/HAP4 binding site at -204 relative to the ATG start codon showed that this element was required for full derepression of the LPD1 gene on non-fermentable substrates.

Base Sequence↗
PubMed1992

A 3' transcriptional enhancer within the coding sequence of a yeast gene encoding the common subunit of two multi-enzyme complexes.

A well-defined set of isogenic yeast strains has been constructed whereby each strain contains a different LPD::lacZ gene fusion integrated at the ura3 locus. These LPD::lacZ fusions differ in the amount of the LPD1 gene (encoding lipoamide dehydrogenase) that is fused to the lacZ reporter. Comparison of the beta-galactosidase activities of each strain during growth on glucose or ethanol revealed that some part of the LPD1 coding region between +13 and +700 is involved in activating gene expression in a carbon source-dependent manner. This activation occurs at the mRNA level, and is not mediated by changes in mRNA stability. Therefore, the LPD1 gene appears to contain a transcriptional enhancer that lies 3' to the transcriptional start site, and which responds to carbon source.

Base Sequence↗
PubMed1992

Binding and metabolism of 1,25-dihydroxyvitamin D3 in cultured bovine parathyroid cells.

Several laboratories, including ours, have reported that receptors for 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] are decreased in parathyroid glands of uremic animals and patients. To elucidate the factors involved in receptor regulation in this tissue, we have characterized the receptor in primary cultures of bovine parathyroid cells. Extracts from these cells contain a single binding component that binds 1,25-(OH)2D3 with a Kd of 58 pM and sediments in sucrose density gradients at 3.4S, indicating the continued expression of the vitamin D receptor in these cells. Labeling of the intact parathyroid cells with tritiated 1,25-(OH)2D3 was maximal by 2 h, and binding affinity by this method was estimated to be 22 pM. Longer incubation of the cells with tritiated 1,25-(OH)2D3 resulted in a loss of specific binding to 10% maximal by 12 h. The decrease in binding correlated temporally with degradation of 1,25-(OH)2D3 in the medium. This metabolic activity was absent in vitamin D-deficient cells and was first detectable 3-4 h after the addition of 1,25-(OH)2D3, indicating that 1,25-(OH)2D3 induces its own metabolism in parathyroid cells. Replenishment of the cultures after 12 h with fresh tritiated 1,25-(OH)2D3 restored maximal binding, demonstrating that the loss of binding was not due to down-regulation of receptor. Inclusion of the cytochrome P450 inhibitor ketoconazole did not alter maximal binding at 2 h, but blocked both the metabolism of 1,25-(OH)2D3 and the decrease in binding after 3 h. In contrast to other cell types, such as osteosarcoma cells, no homologous up-regulation was seen in cultured parathyroid cells even after 12 h in the presence of 0.5 nM 1,25-(OH)2D3. Furthermore, receptor levels in preparations from cells treated for 20 h with unlabeled 1,25-(OH)2D3 at concentrations of 0.1, 1.0, and 10 nM were not different from controls. Thus, it appears that the vitamin D receptors in parathyroid cell cultures are not up-regulated by their ligand.

Animals↗
PubMed1991

A comparison of the reactivity and stability of wild type and His388----Gln mutant phosphoglycerate kinase from yeast.

A variety of physico-chemical techniques have been used to probe the possible interactions between the characteristic structural domains of yeast phosphoglycerate kinase by comparison of the wild-type enzyme with the specific H388Q mutant in which a potential interaction between His388 and Glu190 in the crucial interdomain region is disrupted. Enzyme kinetic studies indicate that, despite being structurally remote from the active site, this mutation has significant effects on both the Vmax and Km values for various substrates. The single cysteine residue in the N domain of the protein is markedly more reactive in the mutant, and this enhanced accessibility is moderated by binding of substrates and various anions. Differences are also observed in the near-ultraviolet CD spectra of these proteins. The chemical and thermal stability of the mutant enzyme is reduced, as indicated from guanidinium chloride and differential-scanning calorimetry denaturation studies. Moreover, interdomain interactions seem to be altered in the mutant, resulting in the appearance of independent thermal transition for the two domains, in contrast to the single cooperative transition observed for the wild-type enzyme. The conformational and/or dynamic effects of the mutation on the H388Q enzyme are therefore various and not solely localised in the hinge region.

Amino Acid Sequence↗
PubMed1991

Fish and fish oil intake: effect on haematological variables related to cardiovascular disease.

To investigate the effects of practical amounts of n-3 highly-unsaturated fatty acids (HUFA) on a number of parameters involved in haemostasis, 12 healthy men were fed three diets in a 3 x 3 cross-over design. The diets, fed as the evening meal for 6-week periods, were: Control diet (essentially fish-free), Fish diet (200 g/d of lean Australian fish flesh) and the same fish-based diet but supplemented with 5 g/d fish oil (Fish + Oil). The diets supplied about 0, 0.6, and 2.0 g n-3 HUFA/d, respectively. Relative to the Control diet, the number of circulating leukocytes was significantly reduced after both the Fish and Fish + Oil diets (by 13% and 15%, respectively). This reduction occurred in the number of neutrophils, although this did not reach significance on the Fish diet alone. Platelet count fell on the Fish + Oil diet (by 6%) but not on the Fish diet alone. There was an apparent enhancement of fibrinolytic potential after both fish-containing diets, which tended to be accentuated with the fish oil supplementation. The mechanisms underlying some if not all of these observations may be eicosanoid-mediated as indicated by a diminution in the platelet arachidonic acid (20:4n-6) to eicosapentaenoic acid (20:5n-3) ratios after the Fish + Oil (13.7 +/- 1.8) and Fish (34.4 +/- 8.9) diets relative to the Control diet (66.1 +/- 15.6). These data suggest that a practical amount of lean fish can improve certain haematological parameters implicated in the etiology of cardiovascular disease.

Animals↗
PubMed1991

Prevalence of maternal HIV infection in Scotland based on unlinked anonymous testing of newborn babies.

Dried blood spot samples from newborn babies have been successfully tested for HIV-1 antibody by the particle agglutination method to assess the prevalence of infection in the mothers. In January, 1990, unlinked anonymous testing of Guthrie cards for HIV antibody was begun in Scotland. 99.6% of Scottish births were tested. 9 mothers spontaneously refused to allow testing of their baby's blood. Samples were coded by district postcodes. Eluates of 65,773 dried blood spots were initially tested for HIV-1 antibody with the Fujirebio technique. Of the 31 positive samples 19 were confirmed to be positive by enzyme-linked radioimmunoassay and western blot (seroprevalence 0.29 per 1000). All these samples came from large metropolitan areas on the east coast. Prevalences were 2.5 per 1000 for Edinburgh city, 1.4 per 1000 for Dundee, and 0.7 per 1000 for Aberdeen. We identified as HIV-positive all babies known to be so in named testing programmes. HIV testing of Guthrie cards can be used to monitor HIV status in mothers who have just given birth. The use of district postcode data in sample identification will allow accurate targetting of prevention strategies and early detection of spread of infection by geographic area.

Agglutination Tests↗
PubMed1991

The complete sequence of a 7.5 kb region of chromosome III from Saccharomyces cerevisiae that lies between CRY1 and MAT.

We report the sequence of a 7.5 kb region lying between the CRY1 and MAT loci of chromosome III from Saccharomyces cerevisiae. This region lies in the overlap between two major contigs used for the generation of the complete nucleotide sequence of this chromosome. Comparison of this sequence with those reported previously for this overlap [Thierry et al. (1990) Yeast 6, 521; Jia et al. (1991) Yeast 7, 413] reveals 38 nucleotide differences, 45% of which generate changes in the amino acid sequences of the four genes in this region (YCR591, YCR592, YCR521 and YCR522). These differences appear to reflect true sequence polymorphisms between the two yeast strains used to generate the clones used in the sequencing project. Three of the four genes in this region display weak homologies to proteins in the PIR database. Some properties of YCR521 are analogous to those of ribosomal protein genes. However, the functions of all four genes remain obscure.

Acetyltransferases↗
PubMed1991

Erythrocyte eicosapentaenoic acid versus docosahexaenoic acid as a marker for fish and fish oil consumption.

The fatty acid composition of erythrocyte membranes was investigated in 21 healthy men after 6 wk of varying intakes of eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3). In one experiment, 12 subjects were fed three diets in a 3 x 3 crossover design: an essentially fish-free control diet, a fish diet (0.15 g EPA/d, 0.41 g DHA/d) and the same fish-based diet supplemented with 5 g/d fish oil (Fish + Oil: 0.99 g EPA/d, 0.99 g DHA/d). A 6 wk wash-out period was allowed between each diet. In another experiment, 11 subjects were supplemented with 5 g/d fish oil alone for 6 wk (0.84 g EPA/d, 0.48 g DHA/d). After fish or fish oil feeding, the percent proportion of EPA and DHA in the erythrocyte membranes rose at the expense of linoleic and arachidonic acids. After 6 wk on the fish-based diets, EPA incorporation approached saturation, with the incremental increases being proportional to the amounts supplied by the diets. In contrast, parallel increases were observed for erythrocyte DHA even though the Fish + Oil diet was supplying twice as much DHA as the fish alone diet. These observations imply different metabolic rates for EPA and DHA and their importance is discussed in terms of the value of erythrocyte EPA versus DHA as markers for fish and fish oil consumption.

Adolescent↗
PubMed1991

Persistent changes in the fatty acid composition of erythrocyte membranes after moderate intake of n-3 polyunsaturated fatty acids: study design implications.

To examine the incorporation of n-3 polyunsaturated fatty acids (PUFAs) into erythrocyte membranes during and after moderate n-3 PUFA intake, 12 healthy men were fed three diets for 6-wk periods in a 3 x 3 crossover design, supplying different amounts of eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3): a control diet, a fish diet (0.15 g EPA/d, 0.41 g DHA/d), and a fish + oil diet (5 g fish oil/d; 0.99 g EPA/d and 0.99 g DHA/d). A 6-wk washout period was allowed between diets. Between 6 and 12 wk after the fish + oil diet, erythrocyte EPA and DHA were still declining and it was only after 18 wk that erythrocyte EPA had returned to baseline whereas DHA had not. Investigators examining variables that are influenced by altered membrane fatty acid composition should be aware of these prolonged effects when designing studies. Protracted washout periods (greater than 18 wk) make the classic crossover design prohibitive and a parallel design becomes essential.

Adult↗
PubMed1991

Detection, quantification and sequencing of HIV-1 from the plasma of seropositive individuals and from factor VIII concentrates.

A highly sensitive and reliable RNA polymerase chain reaction method has been developed which has been used to detect, quantify and sequence cell-free HIV RNA directly from the plasma of seropositive individuals. Plasma from 10 out of 12 haemophiliacs tested was found to contain detectable levels of HIV-1 RNA [log mean value: 1.2 x 10(3) copies for Centers for Disease Control (CDC) group II patients, 5.5 x 10(3) copies for CDC group IV patients]. The presence of cell-free circulating virus in both symptomatic and asymptomatic individuals suggests that viral replication continues throughout the course of infection. The same procedure has been used to detect and sequence HIV-1 RNA in two batches of unheated commercial factor VIII concentrate distributed in 1981 and 1983. The sequences obtained revealed a closer relationship to North American than to African strains of HIV-1.

Amino Acid Sequence↗
PubMed1991

Discontinuous sequence change of human immunodeficiency virus (HIV) type 1 env sequences in plasma viral and lymphocyte-associated proviral populations in vivo: implications for models of HIV pathogenesis.

Sequence change in different hypervariable regions of the external membrane glycoprotein (gp120) of human immunodeficiency virus type 1 (HIV-1) was studied. Viral RNA associated with cell-free virus particles circulating in plasma and proviral DNA present in HIV-infected peripheral blood mononuclear cells (PBMCs) were extracted from blood samples of two currently asymptomatic hemophiliac patients over a 5-year period. HIV sequences were amplified by polymerase chain reaction to allow analysis in the V3, V4, and V5 hypervariable regions of gp120. Rapid sequence change, consisting of regular replacements by a succession of distinct viral populations, was found in both plasma virus and PBMC provirus populations. Significant differences between the frequencies of sequence variants in DNA and RNA populations within the same sample were observed, indicating that at any one time point, the predominant plasma virus variants were antigenically distinct from viruses encoded by HIV DNA sequences in PBMCs. How these findings contribute to current models of HIV pathogenesis is discussed.

Acquired Immunodeficiency Syndrome↗