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A J Collier

Publications and source records attributed to A J Collier.

9 recordsLinked to original sources
PubMed1999

Alterations to both the primary and predicted secondary structure of stem-loop IIIc of the hepatitis C virus 1b 5' untranslated region (5'UTR) lead to mutants severely defective in translation which cannot be complemented in trans by the wild-type 5'UTR sequence.

Cap-independent translation of the hepatitis C virus (HCV) genomic RNA is mediated by an internal ribosome entry site (IRES) within the 5' untranslated region (5'UTR) of the virus RNA. To investigate the effects of alterations to the primary sequence of the 5'UTR on IRES activity, a series of HCV genotype 1b (HCV-1b) variant IRES elements was generated and cloned into a bicistronic reporter construct. Changes from the prototypic HCV-1b 5'UTR sequence were identified at various locations throughout the 5'UTR. The translation efficiencies of these IRES elements were examined by an in vivo transient expression assay in transfected BHK-21 cells and were found to range from 0.4 to 95.8% of the activity of the prototype HCV-1b IRES. Further mutational analysis of the three single-point mutants most severely defective in activity, whose mutations were all located in or near stem-loop IIIc, demonstrated that both the primary sequence and the maintenance of base pairing within this stem structure were critical for HCV IRES function. Complementation studies indicated that defective mutants containing either point mutations or major deletions within the IRES elements could not be complemented in trans by a wild-type IRES.

5' Untranslated Regions↗
PubMed1998

Translation efficiencies of the 5' untranslated region from representatives of the six major genotypes of hepatitis C virus using a novel bicistronic reporter assay system.

The 5' untranslated region (5'UTR) of hepatitis C virus (HCV) contains an internal ribosome entry site (IRES) which directs translation of the viral open reading frame (ORF). The 5'UTR is highly conserved between virus isolates in both primary sequence and predicted secondary structure. We cloned and sequenced the 5' regions (nt 18 of the 5'UTR to nt 15 of the core coding sequence) of HCV isolates representing the six major genotypes and subcloned these into a bicistronic, dual luciferase reporter construct. The relative expression of the two luciferases, one directed by the HCV IRES and the other by cap-dependent ribosome scanning, was used to compare the activities of the different IRES elements in transfected cells. The 5'UTR from a genotype 2b isolate was the most efficient at directing translation in all four cell lines tested: BHK-21, HeLa-T4+, HuH7 and HepG2. In HepG2 cells the 2b 5'UTR was three times as efficient as the type 6a 5'UTR, which was generally the least active IRES tested. These data suggest that HCV isolates are not able to translate their ORF with equal efficiency, and provide a starting point from which further sequence-function studies can be undertaken.

5' Untranslated Regions↗
PubMed1996

TGF-beta isoforms fail to modulate inositol phosphates and cAMP in normal and tumour-derived human oral keratinocytes.

This study examined inositol phosphate and cAMP regulation by TGF-beta 1, -beta 2 and -beta 3 in normal and tumour-derived human oral keratinocytes. Previous findings indicated that the cell lines expressed TGF-beta cell surface receptors and had a range of response to exogenous TGF-beta 1, -beta 2 and -beta 3 from being refractory to the ligand to marked inhibition. Basal levels of inositol phosphates broadly reflected the differentiation status of the cells as demonstrated by involucrin expression, but did not correlate with responsiveness to TGF-beta 1, as measured previously by thymidine incorporation. Treatment of cells with bradykinin or serum caused up-regulation of inositol phosphate levels; by contrast, TGF-beta 1, -beta 2 and -beta 3 failed to modulate inositol phosphates. In two tumour-derived cell lines, the TGF-beta isoforms had no effect on cAMP levels, despite a significant increase in cAMP using a potent agonist of adenylate cyclase (forskolin). Furthermore, the cAMP analogue, dibutyryl cAMP, failed to mimic the inhibitory or refractory responses of TGF-beta in these cells lines. The results demonstrate that in normal and tumour-derived human oral keratinocytes, TGF-beta signal transduction is not mediated by inositol phosphates or cAMP.

Bradykinin↗
PubMed1996

Dysregulation of autocrine TGF-beta isoform production and ligand responses in human tumour-derived and Ha-ras-transfected keratinocytes and fibroblasts.

This study examined the autocrine production of TGF-beta 1, -beta 2 and -beta 3 in culture supernatants from tumour-derived (H series, n = 7; BICR series, n = 5), Ha-ras-transfected (n = 4) and normal (n = 2) human keratinocytes using a sandwich enzyme-linked immunosorbent assay (ELISA). Detection limits were 39.0 pg ml-1 for TGF-beta 1, 78.0 pg ml-1 for TGF-beta 2 and 1.9 ng ml-1 for TGF-beta 3. Tumour-derived oral keratinocytes predominantly produced less TGF-beta 1 than normal oral epithelial cells; the expression of endogenous TGF-beta 2 was variable. In keratinocytes containing mutant Ha-ras, TGF-beta 1 production was enhanced and TGF-beta 2 was undetectable. TGF-beta 3 mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) but the protein was not detected in conditioned media, most probably because of the low detection limits of the ELISA for this isoform. Neutralisation experiments indicated that the latent TGF-beta peptide was secreted in keratinocyte conditioned medium. Seven tumour-derived keratinocyte cell lines (H series) and fibroblasts separated from normal (n = 1) and tumour-derived (n = 2) keratinocyte cultures were examined for their response to exogenous TGF-beta 1, -beta 2 and -beta 3. Six of seven tumour-derived keratinocyte cell lines were inhibited by TGF-beta 1 and TGF-beta 2 (-beta 1 > -beta 2); one cell line was refractory to both TGF-beta 1 and TGF-beta 2. Keratinocytes were inhibited (4 of 7), stimulated (1 of 7) or failed to respond (2 of 7) to TGF-beta 3, TGF-beta 1, -beta 2 and -beta 3 stimulated both normal and tumour-associated fibroblasts, but the tumour-associated fibroblasts showed less response to the ligands than their normal counterparts following prolonged treatment with each isoform. The results demonstrate variable autocrine production of TGF-beta isoforms by malignant keratinocytes, with loss of TGF-beta 1 generally associated with the tumour-derived phenotype and modification of endogenous isoform production dependent on the genetic background of the tumour cells. Further, the variable response of the tumour-derived keratinocytes and contiguous fibroblasts to the TGF-beta isoforms suggests that dysregulation of TGF-beta autocrine and paracrine networks are common characteristics of squamous epithelial malignancy.

Cell Line↗
PubMed1994

The binding of hepatitis A virus to cell surfaces shows evidence of positive co-operativity.

Radio-iodinated hepatitis A virus binds to cultured mammalian cells in a saturable manner, with about 1.4 x 10(3) sites/cell and a S0.5 of about 1.4 x 10(-11) M for FRhK-4 cells. This binding to FRhK-4 cells shows evidence of positive co-operativity, with a Hill coefficient of 2.1 (+/- 0.1). This implies that the cellular receptor for the virus may have multiple binding sites and that the affinity of HAV for its receptor is increased if one of the binding sites is occupied by virus. Binding is completely blocked by two neutralising monoclonal antibodies, which also inhibit viral haemagglutination. A non-neutralising monoclonal antibody partially inhibits binding to FRhK-4 cells, but has no effect on haemagglutination.

Animals↗
PubMed1984

Hypocalcaemia associated with high dose rates of zinc oxide to lactating dairy cows.

Clinical hypocalcaemia was associated with single doses of three oi more days; supply of zinc oxide to dairy cows for facial eczema control. Experimental hand dosing of a single dose of zinc oxide (40 and 120 mg Zn/kg b wt) to Jersey cows caused a significant drop in serum calcium concentration at 24 h followed by a rise to concentrations higher than controls at subsequent samplings over four days. At the highest zinc dose rate, 78% of serum calcium values were equal to or lower than the lowest control value at 24 h post dosing. Serum magnesium concentrations had a reciprocal relationship to the calcium levels at all samplings. Serum zinc concentrations were most elevated over the 48 h post dosing, returning to normal levels at the fifth day. Following the highest zinc dose milk yields were reduced by approximately 1.5 kg milk/cow/day for 48 h. Similar results were obtained when cows grazed pasture on which large amounts of zinc oxide had been sprayed.

Journal Article↗