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S A Hughes

Publications and source records attributed to S A Hughes.

15 recordsLinked to original sources
PubMed1995

Characterization of the leader papain-like proteinase of MHV-A59: identification of a new in vitro cleavage site.

Sequence analysis of the mouse hepatitis virus, strain A59 (MHV-A59) genome predicts the presence of two papain-like proteinases encoded within the first open reading frame (ORF 1a) of the replicase gene. The more 5' of these domains, the leader papain-like proteinase, is responsible for the cleavage of the amino terminal protein, p28. The core of this proteinase domain was defined to between amino acids 1084 and 1316 from the beginning of ORF 1a. Through the use of deletion analysis coupled with in vitro expression, we studied the role of the coding region between p28 and the leader papain-like proteinase on the cleavage of p28 itself. Expression of a series of deletion mutants showed processing of p28, albeit at lower levels. Reduced p28 production resulting from a 0.4-kb deletion positioned between p28 and the proteinase domain suggests an involvement of this region in catalytic processing. Some mutants displayed cleavage patterns indicative of a second cleavage site. Interestingly, this new cleavage site identified in vitro maps to a position similar to the expected cleavage site of a p65 polypeptide detected in MHV-A59-infected cells. Mutagenesis of the catalytic His1272 residue demonstrates that both cleavages observed are mediated by the leader papain-like proteinase encoded in ORF 1a.

Amino Acid Sequence↗
PubMed1995

Identification and characterization of a 65-kDa protein processed from the gene 1 polyprotein of the murine coronavirus MHV-A59.

A 65-kDa protein has been detected in mouse hepatitis virus A59 (MHV-A59)-infected DBT cells using polyclonal antibodies directed against polypeptides encoded by the 5' 1.8 kb of gene 1. The presence of this 65-kDa protein (p65) was previously predicted from immunoprecipitation studies of gene 1 expression in MHV-A59-infected DBT cells with other antisera (1). p65 was rapidly labeled in virus-infected cells at late times of infection; however, its cleavage from the polyprotein was significantly delayed compared to the amino-terminal gene 1 polyprotein cleavage product, p28. Similar to p28, p65 was cleaved from the growing polyprotein without detectable intermediate precursors. Kinetic analysis of p65 with specific antibodies indicates that p65 is immediately adjacent to p28 in the gene 1 polyprotein. The proteolytic activity responsible for the carboxy-terminal cleavage of p65, as well as the function of the p65 protein, remains to be determined.

Animals↗
PubMed1995

Oral terbutaline in the outpatient management of preterm labor.

OBJECTIVE: Our purpose was to prove our hypothesis that once preterm uterine contractions and/or labor is controlled with intravenous tocolysis, oral terbutaline, as a maintenance drug, does not prolong pregnancy. STUDY DESIGN: Before discharge, 184 patients between 24 and 35 completed weeks' gestation were prospectively randomized to continued bed rest either with or without oral terbutaline. Assignment was made with stratification into four groups: group 1, those patients with a Bishop score > or = 5 with oral terbutaline (n = 50); group 2, those with a Bishop score > or = 5 without oral terbutaline (n = 53); group 3, those with a Bishop score < 5 with oral terbutaline (n = 41); group 4, those with a Bishop score < 5 without oral terbutaline (n = 40). Oral terbutaline was discontinued at 37 completed weeks. RESULTS: No statistically significant differences were found in the number of readmissions, the number of unscheduled hospital visits, and the neonatal outcomes among the four groups. The gestational age at delivery and percent of deliveries at > or = 37 weeks were not significantly different when group 1 was compared with group 2 and group 3 was compared with group 4. CONCLUSION: Oral terbutaline maintenance does not improve pregnancy outcome in patients who have had initial successful intravenous tocolysis.

Administration, Oral↗
PubMed1995

Identification of the murine coronavirus p28 cleavage site.

Mouse hepatitis virus strain A59 encodes a papain-like cysteine proteinase (PLP-1) that, during translation of ORF1a, cleaves p28 from the amino terminus of the growing polypeptide chain. In order to determine the amino acid sequences surrounding the p28 cleavage site, the first 4.6 kb of murine hepatitis virus strain A59 ORF1a was expressed in a cell-free transcription-translation system. Amino-terminal radiosequencing of the resulting downstream cleavage product demonstrated that cleavage occurs between Gly-247 and Val-248. Site-directed mutagenesis of amino acids surrounding the p28 cleavage site revealed that substitutions of Arg-246 (P2) and Gly-247 (P1) nearly eliminated cleavage of p28. Single-amino-acid substitutions of other residues between P7 and P2' were generally permissive for cleavage, although a few changes did greatly reduce proteolysis. The relationship between the p28 cleavage site and other viral and cellular papain proteinase cleavage sites is discussed.

Amino Acid Sequence↗
PubMed1994

Coronavirus polyprotein processing.

MHV gene 1 contains two ORFs in different reading frames. Translation proceeds through ORF 1a into ORF 1b via a translational frame-shift. ORF 1a potentially encodes three protease activities, two papain-like activities and one poliovirus 3C-like activity. Of the three predicted activities, only the more amino terminal papain-like domain has been demonstrated to have protease activity. ORF 1a polypeptides have been detected in infected cells by the use of antibodies. The order of polypeptides encoded from the 5' end of the ORF is p28, p65, p290. p290 is processed into p240 and p50. Processing of ORF1a polypeptides differs during cell free translation of genome RNA and in infected cells, suggesting that different proteases may be active under different conditions. Two RNA negative mutants of MHV-A59 express greatly reduced amounts of p28 and p65 at the non-permissive temperature. These mutants may have defects in one or more viral protease activities. ORF 1b, highly conserved between MHV and IBV, potentially contains polymerase, helicase and zinc finger domains. None of these activities have yet been demonstrated. ORF 1b polypeptides have yet been detected in infected cells.

Animals↗
PubMed1994

Left ventricular rupture associated with mitral valve replacement.

Rupture of the left ventricle during the immediate postoperative period is a serious, but uncommon complication of mitral valve replacement. This review article outlines the pathological findings, possible causative mechanisms and current management of this cardiac surgical catastrophe.

Aged↗
PubMed1993

A newly identified MHV-A59 ORF1a polypeptide p65 is temperature sensitive in two RNA negative mutants.

Polypeptide products of MHV-A59 gene 1 have been identified in infected DBT cells and in the products of in vitro translations of genome RNA. In this paper we report the identification in infected cell lysates of a 65-kDa polypeptide (p65) encoded in ORF 1a. Studies on the kinetics of appearance and processing of p65 show that p65 is detectable after p28 but before the appearance of p290, p240 and p50. No homologue of the p65 polypeptide identified in infected cell lysates was immunoprecipitated from in vitro translations of genomic RNA, providing further evidence that in vitro processing of polypeptides encoded in ORF 1a of gene 1 differs from that which occurs late in infection of DBT cells. Although the function of p65 is unknown, two MHV-A59 ts mutants isolated and characterized by Baric et al. (3,4) do not produce detectable levels of p65 at the non-permissive temperature indicating that p65 may play an important role in the virus life cycle.

Animals↗
PubMed1993

Gastrin and gastrin-related responses to infection with Ostertagia ostertagi in the calf.

The effects of a single challenge with 60,000 infective Ostertagia ostertagi larvae on blood and gastrointestinal mucosal gastrin concentrations, gastrin-producing G-cell numbers in the pyloric mucosa and growth of different parts of the gut were investigated in 16, two-and-a-half-month-old calves. Infected calves exhibited a rise in abomasal pH which was accompanied by a 145 per cent increase in wet weight of the fundic mucosa (P < 0.05) and a significant rise in blood total gastrin concentrations (P < 0.01). Circulating little gastrin (G-17) was unaffected. Pyloric mucosal total gastrin concentrations remained unaltered in the infected calves until day 28 when levels fell to 36.9 per cent of control group values (P < 0.01). Pyloric mucosal G-cell numbers declined during the experiment in the infected group. It is suggested that release of previously stored tissue gastrin and not a change in G-cell numbers contributes to the hypergastrinaemia associated with ostertagia infection in the calf.

Animals↗
PubMed1992

Insulin-like growth-factor-binding protein gene expression and protein production by human tumour cell lines.

The secretion of insulin-like growth-factor-binding proteins (IGFBPs) and expression of the genes encoding IGFBP-1, IGFBP-2 and IGFBP-3 have been studied in a panel of cell lines derived from breast carcinomas, Wilms' tumour, neuroblastoma, retinoblastoma, colon carcinoma, liver adenocarcinoma, Burkitt's lymphoma and a non-small-cell lung carcinoma. All cell lines, with the exception of the Burkitt's lymphoma cell line, secreted IGFBPs, as detected by affinity labelling. A 34-kDa BP was present in the conditioned media of all IGFBP-secreting cell lines, whereas BPs ranging from 18 kDa to 53 kDa were variably secreted. All IGFBP-secreting cell lines expressed the IGFBP-2 gene as determined by Northern blot analysis. The Wilms' tumour, the neuroblastoma and the retinoblastoma cell line expressed the IGFBP-2 gene only. All other cell lines, with the exception of the Burkitt's lymphoma, expressed the IGFBP-2 gene and, in addition, either the IGFBP-1 gene and/or the IGFBP-3 gene. IGFBP-1 gene expression could be detected by reverse transcriptase polymerase chain reaction only. IGFBP-3 gene expression was detected by Northern blot analysis, but transcripts were less abundant than IGFBP-2 mRNAs. These findings indicate that the expression of multiple BP genes and the secretion of BPs may be a common property of tumour cells.

Affinity Labels↗
PubMed1992

Cloning and expression of a human ATP-citrate lyase cDNA.

A full-length cDNA clone of 4.3 kb encoding the human ATP-citrate lyase enzyme has been isolated by screening a human cDNA library with the recently isolated rat ATP-citrate lyase cDNA clone [Elshourbagy et al. (1990) J. Biol. Chem. 265, 1430]. Nucleic-acid sequence data indicate that the cDNA contains the complete coding region for the enzyme, which is 1105 amino acids in length with a calculated molecular mass of 121,419 Da. Comparison of the human and rat ATP-citrate lyase cDNA sequences reveals 96.3% amino acid identity throughout the entire sequence. Further sequence analysis identified the His765 catalytic phosphorylation site, the ATP-binding site, as well as the CoA binding site. The human ATP-citrate lyase cDNA clone was subcloned into a mammalian expression vector for expression in African green monkey kidney cells (COS) and Chinese hamster ovary cells (CHO) cells. Transfected COS cells expressed detectable levels of an enzymatically active recombinant ATP-citrate lyase enzyme. Stable, amplified expression of ATP-citrate lyase in CHO cells as achieved by using coamplification with dihydrofolate reductase. Resistant cells expressed high levels of enzymatically active ATP-citrate lyase (3 pg/cell/d). Site-specific mutagenesis of His765----Ala diminishes the catalytic activity of the expressed ATP-citrate lyase protein. Since catalysis of ATP-citrate lyase is postulated to involve the formation of phosphohistidine, these results are consistent with the pattern of earlier observations of the significance of the histidine residue in catalysis of the human ATP-citrate lyase.

ATP Citrate (pro-S)-Lyase↗
PubMed1992

Intracellular processing of the N-terminal ORF 1a proteins of the coronavirus MHV-A59 requires multiple proteolytic events.

Several polypeptide products of MHV-A59 ORF 1a were characterized in MHV-A59 infected DBT cells, using antisera directed against fusion proteins encoded in the first 6.5 kb of ORF1a. These included the previously identified N-terminal ORF 1a product, p28, as well as 290-, 240-, and 50-kDa polypeptides. P28 was always detected as a discrete band without larger precursors, suggesting rapid cleavage of p28 immediately after its synthesis. Once p28 was cleaved there was little degradation of the protein over a 2-hr period. The intracellular cleavage of p28 was not inhibited by the protease inhibitor leupeptin, in contrast to results obtained during in vitro translation of genome RNA (Denison and Perlman, 1986). These data suggest that different protease activities may be responsible for the cleavage of p28 in vitro and in vivo. The 290-kDa protein was an intermediate cleavage product derived from a precursor of greater than 400 kDa. The 290-kDa product was subsequently cleaved into secondary products of 50 and 240 kDa. The intracellular cleavage of the 290-kDa polypeptide was inhibited by leupeptin at concentrations which did not inhibit the early cleavage of p28 or the cleavage of the 290-kDa product from its larger polyprotein precursor. In the presence of zinc chloride, a product of greater than 320 kDa was detected, which appears to incorporate p28 at its amino terminus. This suggests that at least two protease activities may be necessary for processing of ORF1a proteins, one of which cleaves p28 and is sensitive to zinc chloride but resistant to leupeptin, and the other which cleaves the 290-kDa precursor and is sensitive to both inhibitors. Both the 290- and 240-kDa proteins should contain sequences predicted to encode two papain-like protease activities.

Leupeptins↗
PubMed1992

Neuropathology of the chronic epileptic syndrome induced by intrahippocampal tetanus toxin in rat: preservation of pyramidal cells and incidence of dark cells.

A few nanograms of tetanus toxin injected into a rat hippocampus causes a chronic epileptic syndrome characterized by brief seizures that recur intermittently for about 6 weeks. Cognitive and other behavioural impairments persist after the seizures and other epileptic electrographic activity have remitted, and may be permanent. Our previous studies suggested that the behavioural changes following seizure remission were an indication of functional impairment associated with decreased neuronal excitability rather than with neuronal loss. The conclusion that neurons were preserved relied on qualitative histological observations and, indirectly, on electrophysiological measurements of the amplitudes of antidromic population spikes. Recently, gross histopathology has been described in a quantitative histological study of rats 7-10 days after they had received rather higher doses of intrahippocampal tetanus toxin. Here we report a quantitative histological study of hippocampi from rats which had gained remission from seizures induced by low doses of tetanus toxin. Adult Sprague Dawley rats received unilateral injections of 3-4 ng (about 6-8 mouse LD50) tetanus toxin, or vehicle, into the dorsal hippocampus. The first experiment confirmed that postsynaptic evoked responses recorded from pyramidal cells were depressed 10-19 weeks after injection. Unexpectedly, there also was a decrease of 20% in the antidromic response from CA3a contralateral to the injection. However, cell counts in these hippocampi revealed no change in pyramidal cell numbers. The second experiment used rats from two breeding colonies, prepared for histology 7 weeks after injection. Hippocampal pyramidal cell numbers were within the normal range in all but three of the 24 rats that had received tetanus toxin. These three had lesions of the CA1 pyramidal layer contralateral to the injection. The lesions were of the order of 2 mm in diameter, and were associated with glial proliferation. When these three cases were excluded, there remained a small increase in glial density in CA1 of the toxin-injected rats. In addition, toxin-injected rats from one of the colonies were susceptible to a pathology known as acidophylic or dark cell change. These occurred in 11 of 18 toxin-injected rats from this colony, in all divisions of the pyramidal layer, in both the injected and the contralateral hippocampus (where parallel studies revealed independent secondary epileptic foci). We conclude that loss of pyramidal neurons is not necessary for the persistent behavioural changes in this model.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMedDate not supplied

Physeal injuries of the cervical spine.

Four cases of cervical spine growth plate ("ring apophysis") injury are described. Two nonfatal injuries involved anterior displacement of a portion of the inferior physis and epiphyseal ossification center in the lower cervical spine. Neither injury was associated with neurological damage. Follow-up roentgenograms demonstrated healing, but with a spur-like prominence in one patient. A third patient sustained a fatal separation through the inferior end plate of C2. The fourth patient sustained a complete separation of C6 from C7. These injuries are correlated with pathologic specimens and normal developmental anatomy.

Adolescent↗