Biomedical subjects
Publications and source records attributed to T J Harris.
Faecal samples were collected from 209 randomly selected sheep farms in southern England, and the nematode eggs extracted were tested by an egg hatch assay and by a larval development test for their resistance to benzimidazoles. Benzimidazole resistance was found on 35 per cent of farms tested in East Sussex, 44 per cent in Oxfordshire and 61 per cent in West Sussex.
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Disulphide bonds in human recombinant tissue inhibitor of metalloproteinases (TIMP) were assigned by resolving proteolytic digests of TIMP on reverse-phase h.p.l.c. and sequencing those peaks judged to contain disulphide bonds by virtue of a change in retention time on reduction. This procedure allowed the direct assignment of Cys-145-Cys-166 and the isolation of two other peptides containing two disulphide bonds each. Further peptide cleavage in conjunction with fast-atom-bombardment m.s. analysis permitted the assignments Cys-1-Cys-70, Cys-3-Cys-99, Cys-13-Cys-124 and Cys-127-Cys-174 from these peptides. The sixth bond Cys-132-Cys-137 was assigned by inference, as the native protein has no detectable free thiol groups.
Five thousand oocysts of each of two species of coccidia, Eimeria crandallis and E ovinoidalis or 30,000 infective larvae of Nematodirus battus, given as single infections to three- to five-week-old lambs, caused only transient diarrhoea and had no effect on growth. Lambs infected first with coccidia and two weeks later with N battus suffered severe diarrhoea, weight loss and some deaths. Simultaneous administration of the coccidia and the nematodes increased the clinical severity of the syndrome and increased the numbers of nematode eggs produced.
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As a consequence of gene cloning and DNA sequencing several gene families are emerging in the field of cell-cell recognition. These include immunoglobulins, integrins, certain extracellular glycoproteins and a family of functionally unrelated proteins which include factor B. We report here the cloning and sequencing of a gene from Plasmodium falciparum, coding for a protein we call thrombospondin related anonymous protein (TRAP), which shares certain sequence motifs common to other well-characterized proteins. The most significant homology is based around the sequence Trp-Ser-Pro-Cys-Ser-Val-Thr-Cys-Gly (WSPCSVTCG), present in three copies in region I of thrombospondin (TSP), six copies in properdin (P) and one copy in all the circumsporozoite (CS) proteins sequenced so far. TRAP also shares with certain extracellular glycoproteins, including TSP, the cell-recognition signal Arg-Gly-Asp (RGD), which has been shown to be crucial in the interaction of several extracellular glycoproteins with members of the integrin superfamily. Unlike the CS protein, TRAP is expressed during the erythrocytic stage of the parasite life cycle.
The complete amino acid sequence of the human complement system regulatory protein, factor H, has been derived from sequencing three overlapping cDNA clones. The sequence consists of 1213 amino acids arranged in 20 homologous units, each about 60 amino acids long, and an 18-residue leader sequence. The 60-amino-acid-long repetitive units are homologous with those found in a large number of other complement and non-complement proteins. Two basic C-terminal residues, deduced from the cDNA sequence, are absent from factor H isolated from outdated plasma. A tyrosine/histidine polymorphism was observed within the seventh homologous repeat unit of factor H. This is likely to represent a difference between the two major allelic variants of factor H. The nature of the cDNA clones indicates that there is likely to be an alternative splicing mechanism, resulting in the formation of at least two species of factor H mRNA.
Superficial skin cancer is the most common malignancy in man, and radiotherapy has played an important curative role since the early part of the 20th century. We present an overview of the changing pattern of care for patients with superficial skin cancer at the Queensland Radium Institute (QRI), Brisbane--the skin cancer "capital" of the world. Although some 90,000 clinically-diagnosed skin cancers have been treated by radiotherapy at the QRI during the period 1944-1985, we document a dramatic decline in radiotherapy usage for superficial skin cancers over the past 10-15 years. We identify and discuss the major reasons for this changing pattern of care: (a) policy changes initiated by radiation oncologists because of unsightly radiation scars caused by the Queensland climate, (b) improvements in the availability and technical aspects of surgery and dermatology, and (c) surgical preference.
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The molecular cloning of a cDNA coding for human gastric lipase and its expression in yeast is described. A lipase present in human gastric aspirates was purified and its N-terminal amino-acid sequence was determined. This was found to be homologous with the N-terminal sequence of rat lingual lipase. A cDNA library was constructed from mRNA isolated from human stomach tissue and probed with cloned rat lingual lipase DNA. One clone, pGL17, consisting of approximately 1450 base-pairs, contained the entire coding sequence for a human gastric lipase. The amino-acid sequence from the isolated protein and the DNA sequence obtained from the cloned gene indicated that human gastric lipase consists of a 379 amino acid polypeptide with an unglycosylated Mr of 43,162. Human gastric lipase and rat lingual lipase amino-acid sequences were closely homologous but were unrelated to porcine pancreatic lipase apart from a 6 amino-acid sequence around the essential Ser-152 of porcine pancreatic lipase. A yeast expression plasmid containing the phosphoglycerate kinase promoter and terminator sequences together with the human gastric lipase gene was constructed. Yeast transformed with this vector synthesised the lipolytically active enzyme.
A cDNA clone of the mRNA coding for the human complement system control protein Factor I has been isolated. The predicted amino acid sequence obtained from the DNA sequence demonstrates a protein consisting of a heavy chain (Mr 35,400) linked to a light chain (Mr 27,600), both of which contain three sites for N-linked glycosylation. The light chain has clear homology with other serine proteinases, most notably in the region of the catalytically active and structurally important amino acids and shares some of the features characteristic of the plasminogen activators. The heavy chain has a clear 'mosaic' nature typical of the plasma serine proteinases; in particular it contains class A and class B LDL (low-density lipoprotein) receptor repeats with conserved cysteine residues similar to those found in other complement proteins.
Peptide sequencing of the complement system regulatory protein, factor H, permitted the synthesis of a mixed sequence oligonucleotide probe. Human liver cDNA libraries were screened and factor H-specific clones selected. No full-length clone was obtained, but the largest available clone, R2a, was found to encode the C-terminal 657 amino acids of factor H. The derived amino acid sequence consists of 10 contiguous internally homologous segments, each about 60 amino acids long. Sequences homologous to these are found in several other complement and non-complement proteins. Such sequences are likely to represent a particular type of tertiary structure subunit.
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A comparison of the cDNA-derived amino acid sequences of human stromelysin and collagenase with the N-terminal sequences of purified enzymes reveals that these metalloproteinases are highly conserved and that they are secreted as proenzymes. A putative zinc-binding site was identified by its homology with the zinc-chelating sequence of thermolysin. These sequences permitted the identification of: transin, a protein induced in rat fibroblasts either exposed to growth factors or transformed by oncogenic viruses, as the rat homologue of stromelysin, and XHF1, a protein induced in human fibroblasts after treatment with tumourigenic agents, as collagenase.
The nucleotide sequence for a glutamine synthetase (GS) mRNA from gene-amplified Chinese hamster (CHO) cells was determined from recombinant cDNA clones obtained from both pBR322 and lambda gt10 libraries and by primer extension. The sequence obtained contains about 1400 bp corresponding to a minor species of mRNA terminated by a poly A sequence. The mRNA contains 146 nucleotides of 5'-noncoding region, 1119 bp of coding sequence, and 108 bp of 3'-noncoding sequence with a 32 bp poly(A) tail. The polyadenylation site used shows little homology with efficient polyadenylation sites, but has considerable complementarity with U4 RNA. The predicted amino acid sequence, starting from an initiation codon with the preferred sequence surrounding it, indicates that Chinese hamster GS has high homology with published bovine brain GS peptides and enabled an ordering of these peptides. There is homology between the mammalian GS enzymes and glutamine synthetases obtained from plants and cyanobacteria but no obvious homology between the CHO cell GS sequence and that of other ATP hydrolysing enzymes.
cDNA clones of the mRNA coding for tissue-type plasminogen activator (t-PA) have been obtained and their nucleotide sequences compared to those reported previously. A gene coding for t-PA has been reconstructed and inserted into vectors for expression in prokaryotic cells. Relatively high levels of t-PA accumulated in inclusion bodies in Escherichia coli containing an optimized expression plasmid, but only a small proportion of the insoluble protein was recovered as active enzyme using a variety of solubilization procedures.