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Biomedical subjects

A Platt

Publications and source records attributed to A Platt.

At least 19 recordsLinked to original sources
PubMed2005

Use of polarised equine endothelial cell cultures and an in vitro thrombosis model for potential characterisation of EHV-1 strain variation.

Equine herpesvirus-1 (EHV-1) is responsible for respiratory disease and abortion in pregnant mares. Some high virulence isolates of EHV-1 also cause neurological disease. The pathogenesis of both abortion and neurological disease relates in part, to thrombus formation occurring in the pregnant uterus and central nervous system. The differences in disease outcome may relate to differing abilities of high and low virulence EHV-1 isolates to cause cell-associated viraemia, infect endothelial cells and cause thrombosis at sites distant from the respiratory tract. This study attempted to identify in vitro assays, which could be used to characterise the interaction between these isolates, equine endothelial cells and clotting factors. No significant difference was found between the growth kinetics of high and low virulence isolates of EHV-1 in polarised endothelial cells. For both isolates, virus was released preferentially from the apical surface of the polarised cells. The functional effects of viral infection on endothelial cells, with reference to virally-induced thrombosis were then investigated. Endothelial cells were grown on microcarrier beads, infected with EHV-1 and assayed for procoagulant activity. No significant difference in clotting time was observed between mock and EHV-1 infected endothelial cells in microcarrier cultures. Thus the degree of thrombosis may reflect a more complex interaction between endothelial cells, circulating leucocytes and other factors in the microenvironment.

Animals↗
PubMed2005

A distinct subset of chemokines dominates the mucosal chemokine response in inflammatory bowel disease.

BACKGROUND: Inflammatory bowel disease (IBD) is characterised by intense mucosal recruitment of activated leukocytes. Chemokines determine inflammatory leukocyte recruitment and retention. AIM: To compare expression of the entire chemokine family within colonic mucosa from IBD patients and uninflamed controls. METHODS: A microarray of cDNAs, representing every member of this superfamily and their cognate receptors, was hybridised with probes derived from colonoscopic biopsies. RESULTS: A distinct subset of chemokines, consisting of CXCLs 1-3 and 8 and CCL20, was upregulated in active colonic IBD, compared with uninflamed areas or tissue from controls. Increased expression of their cognate receptors, CXCR1, CXCR2 and CCR6, was confirmed by quantitative PCR and immunohistochemistry. An identical chemokine response was induced in Caco-2 cells by stimulation with interleukin (IL)-1beta, but not tumour necrosis factor-alpha (TNF-alpha). By contrast, IL-1beta and TNF-alpha were synergistic in an HT29 cell line and primary keratinocytes. CONCLUSIONS: IL-1beta and TNF-alpha appear to be the pivotal mediators of a previously unidentified coordinated epithelial chemokine response that dominates the mucosal chemokine environment in inflamed IBD tissue.

Caco-2 Cells↗
PubMed2003

p73 gene mutations in gastric adenocarcinomas.

BACKGROUND/AIMS: The p73 gene encodes a protein that shares structural and functional homology with the p53 gene product. The highest degree of homology is in the DNA binding domain, which is the region of p53 that is most frequently mutated in cancer. In contrast to p53 there is little evidence that p73 acts as a classic tumour suppressor gene. Because of the similarities between the p53 and p73 genes and the high frequency of mutation of p53, this study was designed to investigate the p73 gene in patients with gastric adenocarcinoma. METHODS: The mutational status of the p73 gene was investigated in a series of 13 cases of gastric adenocarcinoma from the antro-pyloric region and the gastro-oesophageal junction, using the polymerase chain reaction, single strand conformational polymorphism, and direct DNA sequencing. RESULTS: A glutamine to arginine mutation was detected in exon 5 of the p73 gene in a case of adenocarcinoma at the gastro-oesophageal junction. CONCLUSION: Although limited to a small series of cases, these results suggest that p73 may have a potential pathogenetic role in this tumour.

Adenocarcinoma↗
PubMed2000

The insertion of two amino acids into a transcriptional inducer converts it into a galactokinase.

The transcriptional induction of the GAL genes of Saccharomyces cerevisiae occurs when galactose and ATP interact with Gal3p. This protein-small molecule complex associates with Gal80p to relieve its inhibitory effect on the transcriptional activator Gal4p. Gal3p shares a high degree of sequence homology to galactokinase, Gal1p, but does not itself possess galactokinase activity. By constructing chimeric proteins in which regions of the GAL1 gene are inserted into the GAL3 coding sequence, we have been able to impart galactokinase activity upon Gal3p as judged in vivo and in vitro. Remarkably, the insertion of just two amino acids from Gal1p into the corresponding region of Gal3p confers galactokinase activity onto the resultant protein. The chimeric protein, termed Gal3p+SA, retains its ability to efficiently induce the GAL genes. Kinetic analysis of Gal3p+SA reveals that the K(m) for galactose is similar to that of Gal1p, but the K(m) for ATP is increased. The chimeric enzyme was found to have a decreased turnover number in comparison to Gal1p. These results are discussed in terms of both the mechanism of galactokinase function and that of transcriptional induction.

Amino Acid Sequence↗
PubMed2000

Anabolic steroid accelerated multicompartment syndrome following trauma.

The case is reported of a 23 year old male body builder who was involved in a road traffic accident after taking anabolic steroids. The resulting trauma caused a severe life threatening acute multicompartment syndrome resulting in the need for urgent multiple fasciotomies.

Accidents, Traffic↗
PubMed1998

The yeast galactose genetic switch is mediated by the formation of a Gal4p-Gal80p-Gal3p complex.

Saccharomyces cerevisiae responds to galactose as the sole source of carbon by activating the GAL genes encoding the enzymes of the Leloir pathway. Here, we show in vitro that the switch from repressed to activated gene expression involves the interplay of three proteins [an activator (Gal4p), a repressor (Gal80p) and an inducer (Gal3p)] and two small molecules (galactose and ATP). We also show that the galactose- and ATP-dependent interaction between Gal3p and Gal80p occurs without disruption of the Gal80p-Gal4p interaction. Thus, Gal3p-mediated activation of transcription occurs via the formation of a tripartite protein complex.

Adenosine Triphosphate↗
PubMed1997

Signaling activation and repression of RNA polymerase II transcription in yeast.

Activators of RNA polymerase II transcription possess distinct and separable DNA-binding and transcriptional activation domains. They are thought to function by binding to specific sites on DNA and interacting with proteins (transcription factors) binding near to the transcriptional start site of a gene. The ability of these proteins to activate transcription is a highly regulated process, with activation only occurring under specific conditions to ensure proper timing and levels of target gene expression. Such regulation modulates the ability of transcription factors either to bind DNA or to interact with the transcriptional machinery. Here we discuss recent advances in our understanding of these mechanisms of transcriptional regulation in yeast.

DNA, Fungal↗
PubMed1996

Nitrogen metabolite signalling involves the C-terminus and the GATA domain of the Aspergillus transcription factor AREA and the 3' untranslated region of its mRNA.

AREA is a GATA transcription factor which mediates nitrogen metabolite repression in Aspergillus nidulans in response to intracellular glutamine levels. We have identified and localized three elements important to modulation of AREA function: a region of 13 residues within the DNA-binding GATA domain which forms a putative extended loop structure, the 12 C-terminal residues, and sequences within a 218 nucleotide region of the 3' UTR. The 12 C-terminal residues are also required for transcriptional activation at a subset of loci under areA control. Specific deletions within the 3' UTR and the C-terminus cause similar levels of derepression and the mutations are additive, implicating two principal signal transduction pathways. The contribution of the 3' UTR to AREA modulation is effected at the level of transcript stability such that the areA mRNA is at least five times more stable under nitrogen-derepressing conditions than it is under repressing growth conditions.

Amino Acid Sequence↗
PubMed1996

Mutational analysis of the C-terminal region of AREA, the transcription factor mediating nitrogen metabolite repression in Aspergillus nidulans.

In Aspergillus nidulans the positive-acting, wide domain regulatory gene areA mediates nitrogen metabolite repression. Previous analysis demonstrated that the C-terminal 153 residues of the areA product (AREA) are inessential for at least partial expression of most genes subject to regulation by areA. Paradoxically, areAr2, a -1 frameshift replacing the wild-type 122 C-terminal residues with a mutant peptide of 117 amino acids, leads to general loss of function. To determine the basis for the areAr2 mutant phenotype, and as a means of delineating functional domains within the C-terminal region of AREA, we have selected and characterised areAr2 revertants. Deletion analysis, utilising direct gene replacement, extended this analysis. A mutant areA product truncated immediately after the last residue of the highly conserved GATA (DNA-binding) domain retains partial function. The areAr2 product retains some function with respect to the expression of uaZ (encoding urate oxidase) and the mutant allele is partially dominant with respect to nitrate reductase levels. Consistent with the areAr2 product having a debilitating biological activity, we have demonstrated that a polypeptide containing both the wild-type DNA-binding domain and the mutant C-terminus of AREA2 is able to bind DNA in vitro but no longer shows specificity for GATA sequences.

Amino Acid Sequence↗
PubMed1995

The 4-hydroxy-2-oxovalerate aldolase and acetaldehyde dehydrogenase (acylating) encoded by the nahM and nahO genes of the naphthalene catabolic plasmid pWW60-22 provide further evidence of conservation of meta-cleavage pathway gene sequences.

We report the complete nucleotide sequence and over-expression of the nahOM genes for the acetaldehyde dehydrogenase (acylating) and the 4-hydroxy-2-oxovalerate aldolase from the meta pathway operon of the naphthalene catabolic plasmid pWW60-22 from Pseudomonas sp. NCIMB9816. Additional partial sequence analysis of adjacent DNA shows the gene order within the operon to be nahNLOMK, identical to the order found for the isofunctional genes in the meta pathway operons in the toluene/xylene pathway of TOL plasmid pWW0 and the phenol/methylphenol pathway of pVI150. The deduced amino acid sequences of NahO and NahM were significantly homologous to the equivalent enzymes encoded by other Pseudomonas meta pathways, although both were the most divergent in each comparison. The nahOM genes were inserted downstream of the T7 promoter in the expression vector pET3a and similar constructs were also made of the isofunctional regions from pVI150 (dmpFG) and TOL plasmid pDK1 (xyIQK). High expression of all three gene pairs was detected by enzyme assays and by SDS-PAGE.

Aldehyde Oxidoreductases↗
PubMed1994

Nitrogen regulation in fungi.

Nitrogen regulation has been extensively studied in fungi revealing a complex array of interacting regulatory genes. The general characterisation of the systems in Aspergillus nidulans and Neurospora crassa shall be briefly described, but much of this paper will concentrate specifically on the recent molecular characterisation of areA, the principle regulatory gene from A. nidulans which mediates nitrogen metabolite repression. Three areas shall be explored in detail, firstly the DNA binding domain, which has been characterised extensively by both molecular and genetic analysis. Secondly we shall report recent analysis which has revealed the presence of related DNA binding activities in A. nidulans. Finally we shall discuss the mechanism by which the nitrogen state of the cell is monitored by the areA product, in particular localisation of the domain within the areA product which mediates the regulatory response within the protein.

Amino Acid Sequence↗
PubMed1993

C-terminal truncation of the transcriptional activator encoded by areA in Aspergillus nidulans results in both loss-of-function and gain-of-function phenotypes.

Mutations truncating as many as 143 C-terminal residues from the transcriptional activator encoded by the areA gene, mediating nitrogen metabolite repression in Aspergillus nidulans, do not significantly reduce the ability of the areA product to activate expression of most genes under areA control. Such mutations can even have a gain-of-function, derepressed phenotype, consistent with a critical role for this region in modulating the activity of the areA protein. However, expression of a few genes under areA control is substantially impaired by such C-terminal truncations, indicating that regions of an activator protein can play differing roles in the control of different structural genes. This underlines the advantages of being able to monitor effects of areA mutations on expression of large numbers of structural genes. Additionally, it is shown that truncation of as many as 153 C-terminal residues, virtually all amino acids C-terminal to the DNA-binding region, is compatible with retention of some areA function.

Amino Acid Sequence↗