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D Coates

Publications and source records attributed to D Coates.

At least 19 recordsLinked to original sources
PubMed2003

Molecular detection of anthrax spores on animal fibres.

AIMS: To develop a rapid, specific and sensitive diagnostic test for the detection of the spores of Bacillus anthracis on commercial samples of animal fibres (e.g. wool and cashmere). METHODS AND RESULTS: Extraction of DNA from spores using a mechanical disruption method based on bead beating was evaluated but subsequently abandoned as it compromised the sensitivity of the overall protocol. A multiplex PCR and two nested amplification reactions designed for B. anthracis were developed during this study. CONCLUSIONS: A simple selective incubation step in combination with multiplex PCR was found to be more effective than generic DNA extraction coupled to a sensitive nested amplification reaction. SIGNIFICANCE AND IMPACT OF THE STUDY: The rapid diagnostic test could be applied to the analysis of commercial fibre samples for the detection of anthrax as required by health and safety legislation resulting in considerable savings in time and expense.

Animals↗
PubMed2002

Improved detection of Sugarcane yellow leaf virus using a real-time fluorescent (TaqMan) RT-PCR assay.

Yellow leaf syndrome (YLS) of sugarcane has been associated with Sugarcane yellow leaf virus (ScYLV) and has been reported from most sugarcane growing countries around the world. As sugarcane is vegetatively propagated, it is important to use effective and sensitive detection methods to screen new propagating material. Virus detection in symptomatic tissue is currently achieved using enzyme linked immunosorbent assay (ELISA), tissue blot immunoassay (TBIA) or a conventional RT-PCR based assay. This paper reports the development of an improved assay based on multiplex real-time fluorescent RT-PCR. The new assay is 100-fold more sensitive than conventional RT-PCR, and incorporates a novel 'RNA specific' internal positive control (based around the intron of the caffeic acid 3-o-methyltransferase gene) to guard against false negative results. The paper also describes the comparison of eight RNA extraction methods for sugarcane tissue giving a number of alternatives for different laboratory situations. The sensitivity of this assay has allowed the detection of ScYLV in many samples that were thought to be healthy following conventional testing (RT-PCR, ELISA or TBIA). The detection of ScYLV using this TaqMan assay can be applied to the production of ScYLV-free plants and prevents its spread through the propagation material.

Luteovirus↗
PubMed2002

Genetic diversity within and divergence between rare and geographically widespread taxa of the Acacia acuminata Benth. (Mimosaceae) complex.

The Acacia acuminata complex is a group of woody shrubs and small trees comprised of three formally described taxa (A. oldfieldii, A. acuminata ssp. acuminata and ssp. burkittii) and two informal taxa ("small seed" and "narrow phyllode") with contrasting geographical distributions within the south-west of Western Australia. In addition, a series of variant populations of possible hybrid origin exist. Population genetic structure was investigated in 25 populations representing the taxa and variants using 16 polymorphic allozyme loci. All taxa and variants exhibited relatively high levels of genetic variation compared with other woody angiosperms with similar geographic distributions. Levels of genetic diversity in the widely distributed ssp. burkittii and "narrow phyllode" taxon were considerably higher (H(e), 0.311 and 0.319, respectively) than expected for widespread woody shrubs. In contrast, the rare and highly restricted A. oldfieldii exhibited significantly lower levels of genetic diversity (H(e), 0.173) compared with the other taxa in the complex, but higher than other rare woody shrubs. Although morphologically close, associations based on genetic distance showed A. oldfieldii was highly divergent from the other taxa (D = 0.85) while including variant populations confused systematic alliances. The unusual placement of some of these populations and high degree of population differentiation (G(ST), 12.7%) supports the suggestion that these may represent a series of hybridisation events between the various taxa.

Acacia↗
PubMed2001

The neprilysin (NEP) family of zinc metalloendopeptidases: genomics and function.

Neprilysin (NEP), a thermolysin-like zinc metalloendopeptidase, plays an important role in turning off peptide signalling events at the cell surface. It is involved in the metabolism of a number of regulatory peptides of the mammalian nervous, cardiovascular, inflammatory and immune systems. Examples include enkephalins, tachykinins, natriuretic and chemotactic peptides. NEP is an integral plasma membrane ectopeptidase of the M13 family of zinc peptidases. Other related mammalian NEP-like enzymes include the endothelin-converting enzymes (ECE-1 and ECE-2), KELL and PEX. A number of novel mammalian homologues of NEP have also recently been described. NEP family members are potential therapeutic targets, for example in cardiovascular and inflammatory disorders, and potent and selective inhibitors such as phosphoramidon have contributed to understanding enzyme function. Inhibitor design should be facilitated by the recent three-dimensional structural solution of the NEP-phosphoramidon complex. For several of the family members, however, a well-defined physiological function or substrate is lacking. Knowledge of the complete genomes of Caenorhabditis elegans and Drosophila melanogaster allows the full complement of NEP-like activities to be analysed in a single organism. These model organisms also provide convenient systems for examining cell-specific expression, developmental and functional roles of this peptidase family, and reveal the power of functional genomics.

Animals↗
PubMed2001

Functional expression and characterization of the cytoplasmic aminopeptidase P of Caenorhabditis elegans.

Aminopeptidase P (AP-P; X-Pro aminopeptidase; EC 3.4.11.9) cleaves the N-terminal X-Pro bond of peptides and occurs in mammals as both cytosolic and plasma membrane forms, encoded by separate genes. In mammals, the plasma membrane AP-P can function as a kininase, but little is known about the physiological role of the cytosolic enzyme. The C. elegans genome contains a single gene encoding AP-P (W03G9.4), analysis of which predicts regions displaying high levels of amino-acid sequence homology between the predicted gene product and mammalian cytoplasmic AP-P, with the absolute conservation of key catalytic residues. The sequence of an EST (yk91g4), comprising the open reading frame of W03G9.4, confirmed the predicted genomic structure of the gene and the prediction that W03G9.4 codes for a nonsecreted protein with a molecular mass of 68 kDa. Nematodes transformed with a promoter reporter construct, W03G9.4:GFP, showed high levels of fluorescence in the intestine of larvae and adult hermaphrodites, indicating that the intestine is a major site of W03G9.4 expression. yk91g4 tagged with a hexahistidine and DLYDDDDK peptide epitope was expressed in Escherichia coli to yield, after affinity purification, a recombinant protein with a molecular mass of 71 kDa. The recombinant W03G9.4 removed the N-terminal amino acid from bradykinin (RPPGFSPFR), a Caenorhabditis elegans neuropeptide (KPSFVRFamide) and Lem Trp 1 (APSGFLGVRamide), but did not display activity towards angiotensin I (NRVYIHPFHL), des-Arg bradykinin and AF1 (KNEFIRFamide). The activity towards bradykinin was inhibited by EDTA and 1, 10 phenanthroline, as expected for a metalloenzyme, and also by apstatin (IC50, 1 microM), a selective inhibitor of mammalian AP-P. A Km of 45 microM and an optimum pH of 7-8 was observed with bradykinin as the substrate. The activity of the nematode AP-P, like its mammalian counterparts, was strongly influenced by metal ions, with Co2+, Mn2+ and Zn2+ all inhibiting the hydrolysis of bradykinin. We conclude that W03G9.4 codes for a cytoplasmic AP-P with very similar enzymatic properties to those of mammalian AP-P, and we suggest that the enzyme has a physiological role in the intracellular hydrolysis of proline-containing peptides absorbed from the lumen of the intestine.

Amino Acid Sequence↗
PubMed2001

An evaluation of the use of chlorine dioxide (Tristel One-Shot) in an automated washer/disinfector (Medivator) fitted with a chlorine dioxide generator for decontamination of flexible endoscopes.

Microbiological tests were carried out to evaluate a new chlorine dioxide sterilant: Tristel OneShot. Preliminary in vitro suspension tests showed that solutions containing around 140 ppm chlorine dioxide achieved a reduction factor exceeding 10(6) of Staphylococcus aureus in 1min and of Bacillus subtilis spores in 2.5 min in the presence of 3g/L bovine albumin. Subsequent tests evaluated the effectiveness of Tristel One-Shot in a Medivator washer/disinfector fitted with a Tristel Generator for processing flexible endoscopes. Each test run involved three stages. In the first, the instrument and air-water channels of a gastroscope were inoculated with a suspension of Pseudomonas aeruginosa (10(8)cfu/ml) in 10% sodium glutamate and serum (0, 5 or 10%) and then drained, partially dried, and saline flushed through for total viable counts (TVCs). In the second stage, the channels were re-inoculated with test organisms; detergent was flushed through the channels which were then brushed; and saline was flushed through for TVCs. In the third stage, the channels were re-inoculated; detergent was flushed through the channels which were then brushed; the endoscope was processed in the Medivator; and saline was flushed through for TVCs. Carrying out all three stages enabled determination of (1) the contribution played by manual cleaning of channels prior to processing in the Medivator, and (2) the combined effect of manual cleaning followed by processing. Two series of test runs were done. In the first, the Tristel Generator was set to generate 230ppm chlorine dioxide, and in the second 150ppm. In the first, cleaning followed by processing in the Medivator consistently achieved a >/= 10(6)-fold reduction of test organisms, and in the second a >/= 10(5)-fold reduction. Pre-cleaning of channels was very important-when done the initial concentration of serum in the inoculum (0-10%) had no affect on the results obtained after processing.

Chlorine Compounds↗
PubMed2000

Functional conservation of the active sites of human and Drosophila angiotensin I-converting enzyme.

Human somatic angiotensin I-converting enzyme (sACE) has two active sites present in two homologous protein domains, resulting from a tandem gene duplication. It has been proposed that the N- and C-terminal active sites can have specific in vivo roles. In Drosophila melanogaster, Ance and Acercode for two ACE-like single-domain proteins, also predicted to have distinct physiological roles. We have investigated the relationship of Ance and Acer to the N- and C-domains of human sACE by genomic sequence analysis and by using domain-selective inhibitors, including RXP 407, a selective inhibitor of the human N-domain. These phosphinic peptides were potent inhibitors of Acer, but not of Ance. We conclude that the active sites of the N-domain and of Acer share structural features that permit the binding of the unusual RXP407 inhibitor and the hydrolysis of a broader range of peptide structures. In comparison, Ance, like the human C-domain of ACE, displays greater inhibitor selectivity. From the analysis of the published sequence of the Adh region of Drosophila chromosome 2, which carries Ance, Acer, and four additional ACE-like genes, we also suggest that this functional conservation is reflected in an ancestral gene structure identifiable in both protostome and deuterostome lineages and that the duplication seen in vertebrate genomes predates the divergence of these lineages. The conservation of ACE enzymes with distinct active sites in the evolution of both vertebrate and invertebrate species provides further evidence that these two kinds of active sites have different physiological functions.

Acetylation↗
PubMed2000

Expression and functional characterization of a Drosophila neuropeptide precursor with homology to mammalian preprotachykinin A.

Peptides structurally related to mammalian tachykinins have recently been isolated from the brain and intestine of several insect species, where they are believed to function as both neuromodulators and hormones. Further evidence for the signaling role of insect tachykinin-related peptides was provided by the cloning and characterization of cDNAs for two tachykinin receptors from Drosophila melanogaster. However, no endogenous ligand has been isolated for the Drosophila tachykinin receptors to date. Analysis of the Drosophila genome allowed us to identify a putative tachykinin-related peptide prohormone (prepro-DTK) gene. A 1.5-kilobase pair cDNA amplified from a Drosophila head cDNA library contained an 870-base pair open reading frame, which encodes five novel Drosophila tachykinin-related peptides (called DTK peptides) with conserved C-terminal FXGXR-amide motifs common to other insect tachykinin-related peptides. The tachykinin-related peptide prohormone gene (Dtk) is both expressed and post-translationally processed in larval and adult midgut endocrine cells and in the central nervous system, with midgut expression starting at stage 17 of embryogenesis. The predicted Drosophila tachykinin peptides have potent stimulatory effects on the contractions of insect gut. These data provide additional evidence for the conservation of both the structure and function of the tachykinin peptides in the brain and gut during the course of evolution.

Animals↗
PubMed2000

Comparative genomics of the eukaryotes.

A comparative analysis of the genomes of Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae-and the proteins they are predicted to encode-was undertaken in the context of cellular, developmental, and evolutionary processes. The nonredundant protein sets of flies and worms are similar in size and are only twice that of yeast, but different gene families are expanded in each genome, and the multidomain proteins and signaling pathways of the fly and worm are far more complex than those of yeast. The fly has orthologs to 177 of the 289 human disease genes examined and provides the foundation for rapid analysis of some of the basic processes involved in human disease.

Animals↗
PubMed2000

Conserved roles for peptidases in the processing of invertebrate neuropeptides.

Invertebrates use a wide range of peptides as transmitters and hormones to regulate complex behaviour, physiology and development. These animals, especially those that are amenable to genetic study and are the subject of genome-sequencing projects, provide powerful model systems for understanding the functions of peptidases in controlling the bioactivity of peptides. Neprilysin, a zinc metallopeptidase and a key enzyme in the metabolism of mammalian peptides, is also implicated in the inactivation of peptides at synapses and of circulating peptide hormones in insects and nematodes. A family of neprilysin-like genes are present in the genomes of both Drosophila melanogaster and Caenorhabditis elegans; in C. elegans it seems that individual family members have evolved to take on different physiological functions, because they are expressed in a tissue-specific manner. Angiotensin I-converting enzymes (peptidyl dipeptidase A, angiotensin-converting enzyme) are another group of zinc metallopeptidases found in some invertebrates that lack angiotensin peptides. In D. melanogaster there are two functional angiotensin-converting enzymes that are essential for normal development. One of these (Acer) is expressed in the embryonic heart, whereas the second enzyme (Ance) is expressed in several tissues at different stages of the life cycle. The accumulation of Ance within secretory vesicles of some peptide-synthesizing cells suggests a role for the enzyme in the intracellular processing of insect peptides. Ance is very efficient at cleaving pairs of basic residues from the C-terminus of partly processed peptides, suggesting a novel role for the enzyme in prohormone processing. Invertebrates will continue to provide insights into the evolutionarily conserved functions of known peptidases and of those additional family members that are expected to be identified in the future from genome-sequencing projects.

Animals↗
PubMed2000

Exploring the Caenorhabditis elegans and Drosophila melanogaster genomes to understand neuropeptide and peptidase function.

Comparison of peptidase gene families in the newly released Drosophila melanogaster and Caenorhabditis elegans genomes highlights important differences in peptidase distributions with relevance to the evolution of both form and function in these two organisms and can help to identify the most appropriate model when using comparative studies relevant to the human condition.

Animals↗
PubMed1999

A mosquito (Anopheles stephensi) angiotensin I-converting enzyme (ACE) is induced by a blood meal and accumulates in the developing ovary.

Angiotensin I-converting enzyme (ACE) has a key role in regulating levels of several circulating peptides in mammals and has a vital role in male fertility. ACE has recently been found in insects, where its role is unclear. A mutant allele of the ACE gene (Ance) of Drosophila melanogaster is embryonic lethal, indicating an important role for the enzyme in development. We now report the presence of ACE in female Anopheles stephensi mosquitoes and that the enzyme is induced by a blood meal. ACE accumulates in developing ovaries and passes into the mosquito eggs, where it may play a role in the metabolism of peptides during embryogenesis. The ovarian ACE has an Mr of 70 kDa and is inhibited by captopril and lisinopril with IC50 values of 0.1 microM and 0.6 microM, respectively.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1999

The construction of a cDNA expression library for the sheep scab mite Psoroptes ovis.

The need for alternative control strategies for sheep scab is critical. One approach is to develop vaccines based on 'concealed' antigens derived from Psoroptes ovis. This strategy requires the identification and characterisation of potential target antigens, which has been hampered by the problem of limited biological material for isolation of protein antigens. To aid the discovery of P. ovis antigens and to provide a resource for generating recombinant protein, we constructed a P. ovis cDNA expression library, using total RNA isolated from 250 mg of mixed-stage P. ovis and the Clontech SMART cDNA synthesis kit. The presence of P. ovis-specific sequences was confirmed using PCR amplification and sequencing of actin. The sequences of cDNA inserts from six random clones included one with high homology to the Dermatophagoides pteronyssinus (house dust mite) antigen p Dp15. This is a glutathione S-transferase known to be an important house dust mite antigen. We conclude that this library will be a useful tool for the identification of potential target antigens for the immunological control of P. ovis and to further our understanding of the pathology of sheep scab.

Amino Acid Sequence↗
PubMed1999

Divergence in the chloroplast genome and nuclear rDNA of the rare western australian plant lambertia orbifolia gardner (Proteaceae)

The population genetic structure of the Australian plant Lambertia orbifolia was investigated for chloroplast DNA (cpDNA) and rDNA based on restriction fragment length polymorphism. Variation was assessed in 14-20 individuals from six populations with probes covering the majority of the chloroplast genome and the whole rRNA gene unit. For cpDNA, eight mutations were detected which were distributed over five haplotypes. Nucleotide diversity in the species was high and the majority of this diversity was distributed between populations with diversity within populations restricted to a single population. There was significant differentiation between the two regions in the species distribution with the Narrikup region being distinguished by a single haplotype that was characterized by six unique mutations. Variation in rDNA was detected with three gene length variants present in most individuals. However, the Narrikup region was characterized by homogenization of the gene unit to a single length variant in all individuals. The divergence of the Narrikup region suggests that the disjunction in the species distribution has been present for a long time and the two regions represent separate evolutionary lineages.

Journal Article↗
PubMed1999

Hydrolysis by somatic angiotensin-I converting enzyme of basic dipeptides from a cholecystokinin/gastrin and a LH-RH peptide extended at the C-terminus with gly-Arg/Lys-arg, but not from diarginyl insulin.

Endoproteolytic cleavage of protein prohormones often generates intermediates extended at the C-terminus by Arg-Arg or Lys-Arg, the removal of which by a carboxypeptidase (CPE) is normally an important step in the maturation of many peptide hormones. Recent studies in mice that lack CP activity indicate the existence of alternative tissue or plasma enzymes capable of removing C-terminal basic residues from prohormone intermediates. Using inhibitors of angiotensin I-converting enzyme (ACE) and CP, we show that both these enzymes in mouse serum can remove the basic amino acids from the C-terminus of CCK5-GRR and LH-RH-GKR, but only CP is responsible for converting diarginyl insulin to insulin. ACE activity removes C-terminal dipeptides to generate the Gly-extended peptides, whereas CP hydrolysis gives rise to CCK5-GR and LH-RH-GK, both of which are susceptible to the dipeptidyl carboxypeptidase activity of ACE. Somatic ACE has two similar protein domains (the N-domain and the C-domain), each with an active site that can display different substrate specificities. CCK5-GRR is a high-affinity substrate for both the N-domain and C-domain active sites of human sACE (Km of 9.4 microm and 9.0 microm, respectively) with the N-domain showing greater efficiency (kcat : Km ratio of 2.6 in favour of the N-domain). We conclude that somatic forms of ACE should be considered as alternatives to CPs for the removal of basic residues from some Arg/Lys-extended peptides.

Animals↗
PubMed1999

Insect angiotensin-converting enzyme. A processing enzyme with broad substrate specificity and a role in reproduction.

Insect angiotensin-converting enzyme (ACE) is a peptidyl dipeptidase that removes dipeptides and dipeptideamides from the C-terminus of a broad range of in vitro oligopeptide substrates. In mammals, ACE has important roles in blood homeostasis and a recently recognized, but as yet undefined, role in the fertility of male mice. High levels of ACE are found in the male reproductive tissues of several insect species, and emerging data indicates an important role for the enzyme in insect reproduction. In this paper we review some of the recent findings about insect ACE, and we speculate as to the physiological role of this enzyme in insect reproduction.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1998

The Drosophila melanogaster-related angiotensin-I-converting enzymes Acer and Ance--distinct enzymic characteristics and alternative expression during pupal development.

Drosophila melanogaster express two distinct angiotensin-I-converting enzymes (ACEs) called Ance and Acer, which display a high level of primary structure similarity. We have expressed Acer in the yeast Pichia pastoris and purified the recombinant enzyme with a view to developing biochemical tools to distinguish between Acer and Ance. Purified Acer and Ance expressed in yeast were used to raise anti-Acer Ig and anti-Ance Ig that specifically cross-reacted with the respective enzyme on immunoblotting, but did not act as specific inhibitors. Acer cleaves the C-terminal dipeptides from benzoylglycyl-histidyl-leucine and [Leu5]enkephalin, and Acer and Ance are both able to act as endopeptidases, releasing the C-terminal dipeptideamide from [Leu5]enkephalinamide. However, Acer hydrolyses this substrate at a slightly faster rate than [Leu5]enkephalin, whereas Ance hydrolyses the peptide with a free C-terminus with a kcat 15-fold higher than [Leu5]enkephalinamide. In addition, Acer did not cleave angiotensin I. In contrast, Ance hydrolysed 25% of this substrate at an 8-fold lower enzyme concentration. Furthermore, Acer did not hydrolyse the synthetic substrates Phe-Ser-Pro-Arg-Leu-Gly-Arg-Arg and Phe-Ser-Pro-Arg-Leu-Gly-Lys-Arg, two partially processed putative locustamyotropin precursors, under conditions where Ance produced 82% substrate hydrolysis. Acer was inhibited by captopril, trandolaprilat and enalaprilat, with apparent Ki values in the nanomolar range, whereas lisinopril and fosinoprilat were less potent. We show that the two Drosophila ACEs are alternatively expressed in stages P1 (white puparium)-P15 (eclosion) of pupal development; Ance is expressed predominantly during stages P4-P7, whereas the ACE activity expressed during stages P9-P12 is mainly due to Acer suggesting different roles for the two enzymes during pupal development.

Amino Acid Sequence↗