Biomedical subjects

A J Brown

Publications and source records attributed to A J Brown.

At least 19 recordsLinked to original sources

PubMed1999

Characterization of a new hemoprotein in the yeast Saccharomyces cerevisiae.

The Saccharomyces cerevisiae gene YNL234w encodes a 426-amino acid-long protein that shares significant similarities with the globin family. Compared with known globins from unicellular organisms, the Ynl234wp polypeptide is characterized by an unusual structure. In this protein, a central putative heme-binding domain of about 140 amino acids is flanked by two sequences of about 160 and 120 amino acids, respectively, which share no similarity with known polypeptides. Northern analysis indicates that YNL234w transcription is very low in cells grown under normal aerobic conditions but is induced by oxygen-limited growth conditions and by other stress conditions such as glucose repression, heat shock, osmotic stress, and nitrogen starvation. However, the deletion of the gene had no detectable effect on yeast growth. The Ynl234wp polypeptide has been expressed in Escherichia coli, and the hemoprotein nature of the recombinant protein was demonstrated by heme staining after SDS/polyacrylamide gel electrophoresis and spectroscopic analysis. Our data indicate that purified recombinant Ynl234wp possesses a noncovalently bound heme molecule that is predominantly found in a low spin form.

Amino Acid Sequence↗

PubMed1999

Oligomeric structure and regulation of Candida albicans glucosamine-6-phosphate synthase.

Candida albicans glucosamine-6-phosphate (GlcN-6-P) synthase was purified to apparent homogeneity with 52% yield from recombinant yeast YRSC-65 cells efficiently overexpressing the GFA1 gene. The pure enzyme exhibited Km(Gln) = 1.56 mM and Km(Fru-6-P) = 1.41 mM and catalyzed GlcN-6-P formation with kcat = 1150 min-1. The isoelectric point of 4.6 +/- 0.05 was estimated from isoelectric chromatofocusing. Gel filtration, native polyacrylamide gel electrophoresis, subunit cross-linking, and SDS-polyacrylamide gel electrophoresis showed that the native enzyme was a homotetramer of 79.5-kDa subunits, with an apparent molecular mass of 330-340 kDa. Results of chemical modification of the enzyme by group-specific reagents established an essential role of a cysteinyl residue at the glutamine-binding site and histidyl, lysyl, arginyl, and tyrosyl moieties at the Fru-6-P-binding site. GlcN-6-P synthase in crude extract was effectively inhibited by UDP-GlcNAc (IC50 = 0.67 mM). Purification of the enzyme markedly decreased the sensitivity to the inhibitor, but this could be restored by addition of another effector, glucose 6-phosphate. Binding of UDP-GlcNAc to the pure enzyme in the presence of Glc-6-P showed strong negative cooperativity, with nH = 0.54, whereas in the absence of this sugar phosphate no cooperative effect was observed. Pure enzyme was a substrate for cAMP-dependent protein kinase, the action of which led to the substantial increase of GlcN-6-P synthase activity, correlated with an extent of protein phosphorylation. The maximal level of activity was observed for the enzyme molecules containing 1. 21 +/- 0.08 mol of phosphate/mol of GlcN-6-P synthase. Monitoring of GlcN-6-P synthase activity and its sensitivity to UDP-GlcNAc during yeast --> mycelia transformation of C. albicans cells, under in situ conditions, revealed a marked increase of the former and a substantial fall of the latter.

Animals↗

PubMed1999

Associations between amino acids in the evolution of HIV type 1 protease sequences under indinavir therapy.

Significant diversity exists in amino acid sequences encoding HIV-1 protease in individuals naive for protease inhibitors, which could influence the rate of evolution of resistance. High-level resistance to indinavir requires multiple substitutions among at least 11 amino acid sites, and no single substitution was observed in all of 29 resistant isolates obtained from patients on long-term indinavir monotherapy. We have analyzed the evolution of PR in these sequences. The divergence from the baseline amino acid sequence by week 24 was 4%, increasing more than 7% by week 60. The mean difference between sequences from different patients at baseline was 6% (3-9%), rising to 10% after 40 weeks (3-16%), although at all time points nonsynonymous substitutions were less frequent than synonymous nucleotide changes. Analysis of associations between variants at different amino acid sites using a mutual information statistic revealed four pairs of sites to be significantly associated. In three cases these associations included residue 82. Clusters of baseline and week 24 amino acid sequences identified by maximum parsimony did not correlate significantly with the IC95 to indinavir, although a weak correlation of baseline clusters with phenotype at the week 24 time point was suggested.

Amino Acid Sequence↗

PubMed1999

Over-expression of Candida albicans mitochondrial ribosomal protein S9 (MrpS9p) disturbs mitochondrial function in Saccharomyces cerevisiae.

A Candida albicans mitochondrial ribosomal protein S9 (MRPS9) cDNA was identified in a screen for sequences whose expression induce galactose lethality in Saccharomyces cerevisiae. MRPS9 appears to encode a protein of 346 amino acids with an N-terminal mitochondrial targeting sequence and an internal S9 signature that is conserved amongst eukaryotic mitochondrial and prokaryotic ribosomal protein S9 sequences. Expression of a GAL1-CaMRPS9 fusion in S. cerevisiae caused the slow development of a galactose-negative phenotype upon repeated subculturing, and this correlated with an increased frequency of petite mutant formation. Therefore, over-expression of CaMRPS9 interferes with S. cerevisiae mitochondrial function, which accounts for the inhibition of growth on galactose.

Amino Acid Sequence↗

PubMed1999

Oxysterols and atherosclerosis.

Oxysterols are present in human atherosclerotic plaque and are suggested to play an active role in plaque development. Moreover, the oxysterol:cholesterol ratio in plaque is much higher than in normal tissues or plasma. Oxysterols in plaque are derived both non-enzymically, either from the diet and/or from in vivo oxidation, or (e.g. 27-hydroxycholesterol) are formed enzymically during cholesterol catabolism. While undergoing many of the same reactions as cholesterol, such as being esterified by cells and in plasma, certain oxysterols in some animal and in vitro models exhibit far more potent effects than cholesterol per se. In vitro, oxysterols perturb several aspects of cellular cholesterol homeostasis (including cholesterol biosynthesis, esterification, and efflux), impair vascular reactivity and are cytotoxic and/or induce apoptosis. Injection of relatively large doses of oxysterols into animals causes acute angiotoxicity whereas oxysterol-feeding experiments have yielded contrary results as far as their atherogenicity is concerned. There is no direct evidence yet in humans that oxysterols contribute to atherogenesis. However, oxysterol levels are elevated in human low-density lipoprotein (LDL) subfractions that are considered potentially atherogenic and two recent studies have indicated that raised plasma levels of a specific oxysterol (7beta-hydroxycholesterol) may be associated with an increased risk of atherosclerosis. At the present time there are a number of significant and quite widespread problems with current literature which preclude more than a tentative suggestion that oxysterols have a causal role in atherogenesis. Further studies are necessary to definitively determine the role of oxysterols in atherosclerosis, and considering the wide-ranging tissue levels reported in the literature, special emphasis is needed on their accurate analysis, especially in view of the susceptibility of the parent cholesterol to artifactual oxidation.

Animals↗

PubMed1999

Regulation of vitamin D action.

The control of gene transcription by vitamin D compounds is initiated by binding to the VDR, which enhances the receptor's ability to heterodimerize to RXR, interact with response elements in target genes and attract components of the transcriptional initiation complex. A number of factors are capable of influencing this process, including (i) the rate of uptake and catabolism of the ligand, (ii) the nature of the conformational change induced by a specific ligand, (iii) the cellular content of the VDR, (iv) post-translational modifications of the VDR and (v) the availability of other transcriptional components. Vitamin D analogues may affect these factors differently to 1,25(OH)2D3 to produce unique biological profiles that can be exploited for therapeutic use.

Animals↗

PubMed1998

Molecular analysis of CaMnt1p, a mannosyl transferase important for adhesion and virulence of Candida albicans.

There is an immediate need for identification of new antifungal targets in opportunistic pathogenic fungi like Candida albicans. In the past, efforts have focused on synthesis of chitin and glucan, which confer mechanical strength and rigidity upon the cell wall. This paper describes the molecular analysis of CaMNT1, a gene involved in synthesis of mannoproteins, the third major class of macromolecule found in the cell wall. CaMNT1 encodes an alpha-1, 2-mannosyl transferase, which adds the second mannose residue in a tri-mannose oligosaccharide structure which represents O-linked mannan in C. albicans. The deduced amino acid sequence suggests that CaMnt1p is a type II membrane protein residing in a medial Golgi compartment. The absence of CaMnt1p reduced the ability of C. albicans cells to adhere to each other, to human buccal epithelial cells, and to rat vaginal epithelial cells. Both heterozygous and homozygous Camnt1 null mutants of C. albicans showed strong attenuation of virulence in guinea pig and mouse models of systemic candidosis, which, in guinea pigs, could be attributed to a decreased ability to reach and/or adhere internal organs. Therefore, correct CaMnt1p-mediated O-linked mannosylation of proteins is critical for adhesion and virulence of C. albicans.

Amino Acid Sequence↗

PubMed1998

Apolipoprotein J (clusterin) induces cholesterol export from macrophage-foam cells: a potential anti-atherogenic function?

Apolipoprotein J (apo J) is a secreted glycoprotein of which the exact function remains a matter for speculation. Apo J has been implicated in such diverse processes as sperm maturation, regulation of complement activation, programmed cell death, tissue remodelling and lipid transport. In this study a possible role for apo J in lipid transport was explored. Mouse peritoneal macrophages were incubated with acetylated low-density lipoprotein (AcLDL) to produce foam cells containing cholesterol and cholesteryl esters. Incubation of the foam cells with physiological concentrations of purified apo J led to a dose-dependent export of cholesterol. The appearance of cholesterol in the medium was associated predominantly with a decline in intracellular cholesteryl esters rather than intracellular free cholesterol. The kinetics of cholesterol release to apo J were similar to apo A-I, an established promoter of cholesterol efflux. Apo J was also shown to induce phospholipid efflux from cells, whereas the cholesterol exported to the medium was associated with the apo J. Studies using foam cells from apo E-null mice showed that the cholesterol exported to the medium was independent of apo E production by the cells. These results present the first evidence that apo J can promote cholesterol efflux from foam cells and indicates that it might have a function in cellular cholesterol homoeostasis in both normal and pathological situations.

Animals↗

PubMed1998

Molecular epidemiological analysis of HIV in sexual networks in Uganda.

OBJECTIVE: To investigate the suitability of HIV sequence analysis, based on the p17 region of the gag gene, to characterize the sexual networks in and around a trading town in south-west Uganda. METHODS: Blood samples were obtained from 54 HIV-seropositive members of three distinct sexual networks and phylogenetic analysis carried out on proviral DNA sequences obtained from the p17 region of gag from 53 individuals. RESULTS: Despite documented evidence of very little sexual mixing between residents of the trading town, fishing village and surrounding rural area, there was no evidence of clustering of sequences associated with place of residence. More strikingly, known sexual partners failed to show significantly related sequences, and the two pairs of sequences that did show significant similarity came from individuals who had no known social or sexual contact. CONCLUSIONS: Sequence analyses such as those described here have proved effective in confirming or identifying epidemiological links not only following single transmission events but also within risk groups. However, the results from Uganda contrast markedly with those from Europe and the United States. The length of time that the community has been infected, the number of occasions when the virus has been introduced and the high degree of partner change may contribute to the lack of supportive evidence for sociological studies of sexual networks in Uganda.

DNA, Viral↗

PubMed1998

mRNA translation in yeast during entry into stationary phase.

The expression of some Saccharomyces cerevisiae genes is induced as cells enter stationary phase. Their mRNAs are translated during a period in the growth cycle when the translational apparatus is relatively inert, thereby raising the possibility that these mRNAs compete effectively for a limiting pool of translation factors. To test this idea, the translation of mRNAs carrying different 5'-leaders was compared during exponential growth and after entry into stationary phase upon glucose starvation. Closely related sets of lacZ mRNAs, carrying 5'-leaders from the PYK1, PGK1, RpL3, Rp29, HSP12, HSP26 or THI4 mRNAs, were studied. These mRNAs displayed differing translational efficiencies during exponential growth, but their relative translatabilities were not significantly affected by entry into stationary phase, indicating that they compete just as effectively under these conditions. Polysome analysis revealed that the wild-type PYK1, ACT1 and HSP26 mRNAs are all translated efficiently during stationary phase, when the translational apparatus is relatively inert. Also, significant levels of the translation initiation factors eIF-2alpha, eIF-4E and eIF-4A were maintained during the growth cycle. These data are consistent with the idea that, while translational activity decreases dramatically during entry into stationary phase, yeast cells maintain excess translational capacity under these conditions.

Artificial Gene Fusion↗

PubMed1998

Direct observation of lipid hydroperoxides in phospholipid vesicles by electrospray mass spectrometry.

Positive ion electrospray ionization mass spectrometry was used to obtain a lipid profile of vesicles prepared from egg yolk lethicin and enriched with arachidonylstearoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine. The vesicles were oxidized by treatment with tert-butylhydroperoxide and iron (II) sulfate, and the formation of hydroperoxides of the polyunsaturated lipid arachidonylstearoyl phosphatidylcholine was observed. The native lipid signal at 832 a.m.u. decreased and new signals appeared at 864, 896, and 928 a.m.u., corresponding to the addition of one (+32), two (+64), and three (+96) molecules of dioxygen. The dihydroperoxide was found to be the most favourable peroxide product, but it appeared that a degradation of the hydroperoxides was occurring concomitant with their formation, and only their net formation was observed. The rate of depletion of the polyunsaturated lipid and the rate of accumulation of the hydroperoxides was found to increase with the Fe2+ concentration between 10 microM and 2 mM, and was also dependent on the tert-butylhydroperoxide concentration. This is the first report of analysis of lipid hydroperoxides by electrospray mass spectrometry, showing that technique offers a sensitive, direct, and informative approach to the study of oxidative damage to biological membranes.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1998

Maternal antibody to hepatitis B core antigen detected in dried neonatal blood spot samples.

Despite Department of Health recommendations, universal antenatal testing for hepatitis B virus (HBV) is not performed throughout Scotland. We describe the evaluation of an assay to document past or present infection with HBV, by identifying maternal antibody in routine Guthrie dried neonatal blood spot samples taken when infants are 7 days old. A modified haemagglutination assay to detect antibody to hepatitis B core antigen (CORECELL, Green Cross) was validated and found to be 79% sensitive (44/56) and 100% (105/105) specific when used with dried blood spot samples made from panels of serum of known reactivity. Ninety-three percent (13/14) of HBV carriers were CORECELL positive. Sixty-six (0.5%) of 14044 routine Guthrie samples taken from babies born in Scotland from June August 1992 were CORECELL positive indicating past or present maternal infection with HBV. A cross-sectional survey would document the maternity hospitals where universal antenatal hepatitis B screening should be urgently established.

Adult↗

PubMed1998

Mechanism of vitamin D action and its regulation.

Most of the biological actions of 1,25(OH)2D3, the hormonal form of vitamin D, are mediated by the vitamin D receptor (VDR), a member of the steroid/thyroid receptor superfamily. The VDR functions as a ligand-induced transcription factor regulating the rate of expression of genes involved not only in the control of calcium homeostasis and bone remodeling, but also in immunomodulation, the control of hormone secretion, Inhibition of cell growth, and induction of cell differentiation. This section discusses the molecular mechanisms involved in the transcriptional control of gene expression by 1,25(OH)2D3.

Animals↗

PubMed1998

Vitamin D analogues.

The plethora of actions attributed to 1,25(OH)2D3 throughout the body have suggested potential therapeutic applications for the treatment of hyperproliferative diseases, immune dysfunction, endocrine disorders, and metabolic bone disease. However, the potent calcemic activity of the natural vitamin D hormone has precluded its use in most cases. New vitamin D analogues are under development that display greater specificity, in most cases, by retaining the therapeutic properties of 1,25(OH)2D3, but with lower calcemic activity. Two analogues have been approved for use in patients: calcipotriol (Dovonex from Leo Pharmaceuticals, Copenhagen, Denmark) for the treatment of psoriasis; and 19-nor-1,25(OH)2D2 (Zemplar from Abbott Laboratories, Abbott Park, IL) for secondary hyperparathyroidism. Many others analogues are currently being tested in preclinical and clinical trials for the treatment of various types of cancer and osteoporosis, and for immunosuppression. The selectivity of the analogues can be attributed to the combined interactions with four proteins: the vitamin D receptor (VDR), the serum vitamin D binding protein (DBP), the vitamin D-24-hydroxylase and to a newly described membrane receptor. Low DBP affinity has been shown to be responsible for the reduced calcemic actions of calcipotriol and 22-oxacalcitriol (OCT), which is being tested for secondary hyperparathyroidism. However, the low calcemic activity of other analogues, including 19-nor-1,25(OH)2D2, involves other, as yet undefined, mechanisms. Understanding of the molecular basis for the selectivity of the vitamin D analogues will allow the design of more effective and safer vitamin D compounds for the treatment of a wide range of clinical disorders.

Humans↗

PubMed1998

Mapping of a yeast G protein betagamma signaling interaction.

The mating pathway of Saccharomyces cerevisiae is widely used as a model system for G protein-coupled receptor-mediated signal transduction. Following receptor activation by the binding of mating pheromones, G protein betagamma subunits transmit the signal to a MAP kinase cascade, which involves interaction of Gbeta (Ste4p) with the MAP kinase scaffold protein Ste5p. Here, we identify residues in Ste4p required for the interaction with Ste5p. These residues define a new signaling interface close to the Ste20p binding site within the Gbetagamma coiled-coil. Ste4p mutants defective in the Ste5p interaction interact efficiently with Gpa1p (Galpha) and Ste18p (Ggamma) but cannot function in signal transduction because cells expressing these mutants are sterile. Ste4 L65S is temperature-sensitive for its interaction with Ste5p, and also for signaling. We have identified a Ste5p mutant (L196A) that displays a synthetic interaction defect with Ste4 L65S, providing strong evidence that Ste4p and Ste5p interact directly in vivo through an interface that involves hydrophobic residues. The correlation between disruption of the Ste4p-Ste5p interaction and sterility confirms the importance of this interaction in signal transduction. Identification of the Gbetagamma coiled-coil in Ste5p binding may set a precedent for Gbetagamma-effector interactions in more complex organisms.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Analysis of human immunodeficiency virus type 1 env and gag sequence variants derived from a mother and two vertically infected children provides evidence for the transmission of multiple sequence variants.

In order to investigate the transmission of human immunodeficiency virus type 1 (HIV-1) from mother-to-child we have examined serial plasma RNA samples obtained from a mother over an eight year period spanning four pregnancies. Child 1 and 2 (born January 1987 and June 1990) were uninfected whilst child 3 and 4 (born July 1992 and February 1994) were HIV positive. Genetic variation was examined within the viral population of the mother and her two infected children for both the V3 loop and flanking regions of the env gene and the p17 region of the gag gene. In one child (child 4) a highly homogeneous virus population was observed within both env and gag in contrast to the more heterogeneous virus population observed within the mother. Viral sequences of child 4 clustered within a single branch within the reconstructed phylogenetic tree. This is consistent with the transmission of a single maternal variant to the child in this case, which may indicate a selective process. By contrast, child 3 showed substantial genetic heterogeneity even within the first samples obtained shortly after birth. Sequences of child 3 clustered in two distinct groups within the phylogenetic tree and were separated by sequences of the mother. These results are not consistent with the selective transmission of a single maternal variant to the child in this case and we therefore propose that the infection within child 3 is the result of the transmission of multiple sequence variants to the child. All transmitted sequence variants were predicted to be of the macrophage-tropic, nonsyncytium-inducing (NSI) phenotype.

Amino Acid Sequence↗

PubMed1998

The TATA-binding protein (TBP) from the human fungal pathogen Candida albicans can complement defects in human and yeast TBPs.

Candida albicans is the major fungal pathogen in humans, yet little is known about transcriptional regulation in this organism. Therefore, we have isolated, characterized, and expressed the C. albicans TATA-binding protein (TBP) gene (TBP1), because this general transcription initiation factor plays a key role in the activation and regulation of eukaryotic promoters. Southern and Northern blot analyses suggest that a single C. albicans TBP1 locus is expressed at similar levels in the yeast and hyphal forms of this fungus. The TBP1 open reading frame is 716 bp long and encodes a functional TBP of 27 kDa. C. albicans TBP is capable of binding specifically to a TATA box in vitro, substituting for the human TBP to activate basal transcription in vitro, and suppressing the lethal delta spt15 mutation in Saccharomyces cerevisiae. The predicted amino acid sequences of TBPs from C. albicans and other organisms reveal a striking pattern of C-terminal conservation and N-terminal variability: the C-terminal DNA-binding domain displays at least 80% amino acid sequence identity to TBPs from fungi, flies, nematodes, slime molds, plants, and humans. Sequence differences between human and fungal TPBs in the DNA-binding domain may represent potential targets for antifungal therapy.

Amino Acid Sequence↗

PubMed1998

Induction of the vitamin D 24-hydroxylase (CYP24) by 1,25-dihydroxyvitamin D3 is regulated by parathyroid hormone in UMR106 osteoblastic cells.

The expression of the vitamin D 24-hydroxylase is highly regulated in target tissues for 1,25-dihydroxyvitamin D3 (1,25(OH)2D), where it may modulate the action of 1,25(OH)2D. In UMR106 osteoblastic cells, 1,25(OH)2D and PTH synergistically induce 24-hydroxylase expression. The purpose of these studies was to characterize the interaction between 1,25(OH)2D and PTH with regard to the messenger RNA (mRNA) levels of the cytochrome P450 component of the 24-hydroxylase (CYP24). PTH alone had no effect on CYP24 mRNA levels, and 1,25(OH)2D alone produced only a modest increase. However, 1,25(OH)2D and PTH together synergistically increased CYP24 mRNA levels 3-fold compared with 1,25(OH)2D alone. PTH also increased the sensitivity of UMR cells to 1,25(OH)2D from 10(-8) to 10(-10) M. PTH worked through the cAMP signaling pathway as evidenced by the lack of effect of PTH (3-34) and by the full activity of 8-bromo-cAMP. PTH in the presence of 1,25(OH)2D increased CYP24 gene transcription as shown by nuclear run-on studies and by activation of a CYP24 promoter-reporter construct after transfection. PTH also increased vitamin D receptor number in UMR cells, but this occurred at times later than the increase in transcription. These studies demonstrate that PTH in the presence of 1,25(OH)2D works through the cAMP-dependent signaling pathway to increase transcription of the CYP24 gene, to increase CYP24 protein levels, and to increase 24-hydroxylase activity.

Animals↗