Showing papers by "Cooperative Research Centre published in 1992"

Novel recombinant fusion protein analogues of insulin-like growth factor (IGF)-I indicate the relative importance of IGF-binding protein and receptor binding for enhanced biological potency

Abstract: An efficient expression system in Escherichia coli for several biologically active insulin-like growth factor-I (IGF-I) fusion peptide analogues is described These novel IGF-I fusion protein analogues have properties that make them very useful reagents in the investigation of IGF-I action The analogues comprise an IGF-I sequence and the first 11 amino acids of methionyl porcine growth hormone (pGH) and include [Met1]-pGH(1-11)-Val-Asn-IGF-I, which contains the authentic IGF-I sequence, and two analogues, [Met1]-pGH(1-11)-Val-Asn-[Gly3]-IGF-I and [Met1]-pGH(1-11)-Val-Asn-[Arg3]-IGF-I, where Glu-3 in the human IGF-I sequence has been replaced by Gly or Arg respectively The three peptides are referred to as Long IGF-I, Long [Gly3]-IGF-I or Long [Arg3]-IGF-I depending on the IGF-I sequence present Production of the purified fusion peptides was aided by folding the reduced and denatured fusion peptide sequence under conditions that gave very high yields of biologically active product Introduction of a hydrophobic N-terminal extension peptide appears to facilitate the correct folding of the IGF-I analogues compared with that obtained previously when folding normal-length IGFs The biological activities of the IGF-I fusion peptides were compared with authentic IGF-I and the truncated analogue, des(1-3)IGF-I In L6 rat myoblasts, all the analogues were more potent than authentic IGF-I in their abilities to stimulate protein and DNA synthesis and inhibit protein breakdown In H35 hepatoma cells, where the IGFs act through the insulin receptor, the Long IGF-I analogues maintained a similar potency relative to IGF-I as was observed in the L6 myoblasts The order of biological potency in cell lines secreting IGF-binding proteins (IGFBPs) into the medium was Long [Arg3]-IGF-I-des(1-3)IGF-I greater than Long [Gly3]-IGF-I greater than Long IGF-I greater than IGF-I In chicken embryo fibroblasts, a cell line that does not secrete detectable IGFBPs into the medium, Long [Arg3]-IGF-I, was less potent than IGF-I Investigation of receptor and IGFBP association by these analogues reinforced our previous findings that N-terminal analogues of IGF-I show increased biological potency due to changes in the degree of their IGFBP interactions

Unsaturated diether phospholipids in the Antartic methanogen Methanococcoides burtonii

Abstract: The Antarctic methanogen Methanococcoides burtonii contained only diether phospholipids. These membrane components were analysed by gas chromatography and gas chromatography mass spectrometry. Of particular interest was the occurrence of unsaturated diether lipids in M. burtonii; unsaturated ether lipids accounted for 57% of the diether phospholipids. To our knowledge, unsaturated ether lipids have not been previously reported in a methanogen. The presence of the unsaturated ether lipids in M. burtonii is probably the result of temperature adaptation by the bacterium. It may be possible to use these components as a chemical signature for methanogens in Antarctic and Southern Ocean environments.

Production and characterization of recombinant chicken insulin-like growth factor-I from Escherichia coli.

Abstract: Recombinant chicken insulin-like growth factor-I (cIGF-I) has been produced in Escherichia coli after first modifying a plasmid that coded for a human IGF-I (hIGF-I) fusion protein, in order to introduce codons for the eight amino acid substitutions. The cIGF-I fusion protein, deposited in bacterial inclusion bodies, was dissolved under reducing conditions, desalted, subjected to anion-exchange chromatography to remove proteinases, refolded and partially purified by reverse-phase high-performance liquid chromatography. The fusion protein was cleaved with hydroxylamine after which cIGF-I was purified to homogeneity by three additional chromatographic steps. Recombinant cIGF-I was equipotent with hIGF-I in cell culture bioassays of protein synthesis and breakdown using rat L6 myoblasts and chick embryo fibroblasts. Binding of radiolabelled cIGF-I and hIGF-I was also equivalent in the two cell lines, as was their binding in ligand blots of chicken, sheep and human plasma. The cross-reactivity of cIGF-I in a polyclonal hIGF-I radioimmunoassay was 60% of that observed with hIGF-I. The availability of recombinant cIGF-I will facilitate investigations into the role of IGF-I in chicken growth and development.

Cell wall-less, free-living spirochetes in Antarctica

Abstract: The phylogeny of an Antarctic, cell wall-less, bacterial strain was determined by sequencing PCR amplified 16S rDNA, and comparison of the sequence with other bacterial 16S rRNA sequences available in databanks. Although the strain was phenotypically very similar to members of the genus Anaeroplasma, phylogenetic analyses showed it was a member of the order Spirochaetales. Until now, the order was one of the few bacterial orders in which phylogeny was reflected in a uniform morphology of its members. The viability of wall-less cells in cultures of spirochetes and spirochetal infective material warrants reinvestigation.

A myxoma virus intergenic transient dominant selection vector.

Abstract: The purpose of this study was to construct an intergenic transfer vector which can be used for the generation of recombinant myxoma viruses (MVs) expressing a foreign gene insert. Recombinant MVs expressing the Escherichia coli lacZ gene were constructed in vitro by transfection of MV-infected rabbit cells with a transfer expression vector, and isolated under growth conditions selecting for transient expression of the E. coli gpt gene. The effect of inserting foreign DNA sequences between the viral thymidine kinase gene and open reading frame MF8a upon the transcription of these genes was investigated.