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Biomedical subjects

D J Miles

Publications and source records attributed to D J Miles.

11 recordsLinked to original sources
PubMed2001

Effect of macrophages and serum of fish susceptible or resistant to epizootic ulcerative syndrome (EUS) on the EUS pathogen, Aphanomyces invadans.

Epizootic ulcerative syndrome (EUS) is one of the most destructive diseases of fresh and brackish water farmed and wild fish in the Asia-Pacific region. The in vitro germination and growth of the propagules of the EUS pathogen, Aphanomyces invadans (= A. piscicida), were assessed in the presence of the head-kidney macrophages, serum, and serum heated to inactivate complement proteins, of three EUS-susceptible and one resistant fish species. The susceptible species were: striped snakehead (Channa striata), giant gourami (Osphronemus gouramy) and silver barb [Barbodes (= Puntius) gonionotus], and the resistant species was Nile tilapia (Oreochromis niloticus). Fish of all species were acclimatised to either low temperature (20 degrees C +/- 1.6) at which EUS is known to occur, or to high temperature (32 degrees C +/- 5.0) at which EUS does not occur, except for giant gouramis which were only studied at low temperature. The respiratory burst of the macrophages was assessed in the presence of A. invadans or the stimulant phorbol myristate acetate (PMA), and compared to that of controls. Anti-A. invadans antibody concentrations were assessed in all species except silver barbs. All assays were carried out at the same temperature, regardless of the temperature that the fish were kept at. Macrophages of all species other than snakeheads inhibited fungal germination at both temperatures, though only silver barb and gourami macrophages could inhibit germling growth. PMA increased the respiratory burst in nearly all cases. The respiratory burst in the presence of A. invadans was consistently lower than that of controls, though the difference was only significant in the case of snakeheads. The respiratory burst of all macrophage treatments was higher at a low temperature. Except in the case of PMA-stimulated macrophages, regressions between respiratory burst and inhibitory action were only found in susceptible species, suggesting that the respiratory burst is important in those species, but is unable to prevent the proliferation of A. invadans. Serum inhibited fungal germination in all cases other than low temperature tilapia, indicating that the EUS resistance of tilapia is not due to the serum. Inhibition of germling growth by serum only occurred in silver barbs and gourami. Heated serum did not inhibit germination in any case except that of high temperature snakehead, and in fact stimulated germination in the case of tilapia. Heating serum did not affect the growth inhibiting activity of silver barbs and gouramis, but it stimulated growth in some groups. At high temperatures snakeheads had high anti-A. invadans antibody concentrations, which may explain the inhibitory activity of their heated serum. A role for complement and antibodies in defence against A. invadans in susceptible species is suggested.

Animals↗
PubMed1995

Recombinant soluble human tissue factor secreted by Saccharomyces cerevisiae and refolded from Escherichia coli inclusion bodies: glycosylation of mutants, activity and physical characterization.

Tissue factor (TF) is the cell-surface transmembrane receptor that initiates both the extrinsic and intrinsic blood coagulation cascades. The abilities of TF to associate with Factor VIIa and Factor X in a ternary complex and to enable proteolytic activation of Factor X by Factor VIIa reside in the extracellular domain of TF. We describe the expression of the surface domain of TF (truncated TF, tTF) in both Saccharomyces cerevisiae and Escherichia coli and the biochemical and physical characterization of the recombinant proteins. Wild-type tTF and several glycosylation-site mutants were secreted efficiently by S. cerevisiae under the control of the yeast prepro-alpha-signal sequence; the T13A,N137D double mutant was the most homogeneous variant expressed in milligram quantities. Wild-type tTF was expressed in a non-native state in E. coli inclusion bodies as a fusion protein with a poly(His) leader. The fusion protein could be fully renatured and the leader removed by proteolysis with thrombin; the correct molecular mass (24,729 Da) of the purified protein was confirmed by electrospray mass spectrometry. Recombinant tTFs from yeast, E. coli and Chinese hamster ovary cells were identical in their abilities to bind Factor VIIa, to enhance the catalytic activity of Factor VIIa and to enhance the proteolytic activation of Factor X by Factor VIIa. Furthermore, CD, fluorescence emission and NMR spectra of the yeast and E. coli proteins indicated that these proteins are essentially identical structurally.

Amino Acid Sequence↗
PubMed1992

Tissue factor residues 157-167 are required for efficient proteolytic activation of factor X and factor VII.

The cell surface receptor tissue factor (TF) initiates coagulation by supporting the proteolytic activation of factors X and IX as well as VII to active serine proteases. Architectural similarity of TF to the cytokine receptor family suggests a strand-loop-strand structure for TF residues 151-174. Site-directed Ala exchanges in the predicted surface loop demonstrated that residues Tyr157, Lys159, Ser163, Gly164, Lys165, and Lys166 are important for function. Addition of side chain atoms at the Ser162 position decreased function, whereas the Ala exchange was tolerated. The dysfunctional mutants bound VII with high affinity and fully supported the catalysis of small peptidyl substrates by the mutant TF.VIIa complex. Lys159-->Ala substitution was compatible with efficient activation of factor X, whereas the Try157-->Ala exchange and mutations in the carboxyl aspect of the predicted loop resulted in diminished activation of factor X. The specific plasma procoagulant activity of all functionally deficient mutants increased 7- to 200-fold upon the supplementation of VIIa suggesting that TF residues 157-167 also provide important interactions that accelerate the activation of VII to VIIa. These data are consistent with assignment of the TF 157-167 region as contributing to protein substrate recognition and cleavage by the TF.VIIa complex.

Amino Acid Sequence↗
PubMed1992

Cofactor residues lysine 165 and 166 are critical for protein substrate recognition by the tissue factor-factor VIIa protease complex.

High affinity binding of factor VIIa (VIIa) to its cellular receptor tissue factor (TF), as well as association of factor X with phospholipid are required for optimal assembly of the extrinsic activation complex. In addition to the interactions of substrate with phospholipid and enzyme, we here provide evidence that cofactor residues Lys-165 and Lys-166 specifically contribute to the recognition of macromolecular substrate. Ala for Lys replacement in TFA165A166 was compatible with high affinity binding of VIIa when analyzed on cell surfaces as well as in the absence of phospholipid. Dissociation of TFA165A166.VIIa did not occur with a faster rate compared to TF.VIIa, further supporting unaltered VIIa binding function of TFA165A166. Cleavage of chromogenic peptidyl substrate by TFA165A166.VIIa complexes was not diminished, demonstrating that TFA165A166 supported enhancement of catalytic function of the VIIa protease domain. In contrast, factor X activation was reduced in the presence and absence of phospholipid. Further, TFA165A166 effectively competed with wild-type TF in the cleavage of factor X at limited VIIa concentrations. Selective reduction in macromolecular substrate hydrolysis combined with normal VIIa binding by TFA165A166 indicates that the cofactor TF does contribute, either directly or indirectly via specific interactions with VIIa, to factor X recognition.

Amino Acid Sequence↗
PubMed1992

The third Trp-Lys-Ser (WKS) tripeptide motif in tissue factor is associated with a function site.

The tripeptide sequence Trp-Lys-Ser (WKS) is repeated three times in the extracellular ligand binding domain of human Tissue Factor (TF). Using site-directed mutagenesis, we replaced each of the WKS motifs in human TF by Arg-Lys-Gly (RKG), the least conserved replacement for the motif found in murine TF. This substitution in the first repeat W14KS, as well as a Trp14----Arg substitution, resulted in a structurally altered protein, whereas a conservative hydrophobic Trp14----Phe substitution resulted in a functionally normal protein. This suggests that Trp14 may contribute to a hydrophobic core rather than involvement of this motif in function. Replacement of the W45KS and W158KS motifs was associated with no detectable structural alterations; however, function was diminished with the RKG replacement of the third repeat. Mutant proteins with Lys159----Ala and Tyr157----Ala substitutions exhibited loss of function, whereas Tyr156----Ala and Ser160----Ala substitutions flanking the YWK sequence resulted in functional proteins. These data demonstrate that the W158KS motif in human TF is associated with a functional site and identify Lys159 in this motif as a functionally important residue.

Amino Acid Sequence↗
PubMed1988

Construction and characterization of a haploid strain of Saccharomyces cerevisiae that completely lacks all genomic CYH2 sequences.

A diploid strain of the yeast Saccharomyces cerevisiae has been constructed that has one copy of the ribosomal protein gene CYH2 completely deleted and replaced with the TRP1 gene using the method of Rothstein (1983). There are only small differences in growth rate and no detectable difference in steady state level of CYH2 mRNA between the diploid that is heterozygous for the CYH2 deletion and the parent diploid with two normal copies of this gene. This suggests that the diploid must partially compensate for the loss of one CYH2 gene. Tetrad dissection shows that haploid spores lacking the CYH2 gene cannot germinate. The lethality of this deletion can be rescued by a CYH2 cDNA on a low copy vector. Haploids which lack the genomic copy of the CYH2 gene, but contain a plasmid copy of the CYH2 cDNA are able to grow normally. These CYH2 deleted yeast haploids should be useful to analyze mutationally altered CYH2 genes and genes homologous to CYH2 from other organisms without interference from a genomic copy.

Fungal Proteins↗
PubMed1985

Microplate solid-phase radioimmunoassay for rat prolactin.

A rat prolactin solid-phase radioimmunoassay has been developed that uses 96-well microtiter plates with removable wells to which the antibody is firmly adsorbed, resulting in a solid-phase antibody. Antigen as either reference or unknown competes with radioactivity labeled antigen for binding sites on the solid-phase antibody. After immunoreaction, free antigen is removed by washing the wells with phosphosaline solution. The solid-phase antibody-antigen complex is counted for quantitation with data reduction methods currently used in routine radioimmunoassay procedures. This microplate solid-phase radioimmunoassay has several advantages over conventional methods without sacrificing specificity, sensitivity, or accuracy. This method is rapid, compact, economical, easily automated, and could be readily established in other laboratories.

Animals↗