Biomedical subjects

G H Coombs

Publications and source records attributed to G H Coombs.

At least 19 recordsLinked to original sources

PubMed2006

Differences in enzyme activities between two species of Hematodinium, parasitic dinoflagellates of crustaceans.

Parasitic dinoflagellates of the genus Hematodinium infect several commercially important decapod crustaceans. Different species of Hematodinium have different levels of virulence in their respective hosts. Enzyme activities were studied from two species of Hematodinium, one isolated from the Norway lobster (Nephrops norvegicus) and the other from the American blue crab (Callinectes sapidus). We report the identification of differences in secretion of acid phosphatase (AP) and leucine arylamidase from two parasite species. Leucine arylamidase was only contained and secreted by the species infecting the blue crab. Both parasite species contained AP, but only the species infecting the Norway lobster secreted this enzyme. In this species, AP activity was predominantly in the soluble fraction (69.5%). AP activity was localized to cytoplasmic granules and on the membranes surrounding the cell nucleus. In addition to providing information on the cellular metabolism of the parasite, the pattern of activities of these enzymes may also be useful in distinguishing among different species of Hematodinium.

Acid Phosphatase↗

PubMed2006

Molecular detection of Hematodinium spp. in Norway lobster Nephrops norvegicus and other crustaceans.

The Norway lobster Nephrops norvegicus (L.) from the coastal waters of Scotland is seasonally infected by a parasitic dinoflagellate of the genus Hematodinium. Methods used to detect infection include a morphological index (pleopod diagnosis) and several immunoassays. The present study describes the development and application of a set of Hematodinium-specific polymerase chain reaction (PCR) primers and DNA probes based on Hematodinium ribosomal DNA (rDNA). In the PCR assay, a diagnostic band of 380 bp was consistently amplified from total genomic DNA isolated from Hematodinium-infected N. norvegicus. The sensitivity of the assay was 1 ng DNA, which is equivalent to 0.6 parasites. The primer pair also detected Hematodinium DNA in preparations of the amphipod Orchomene nanus, indicating that the amphipod may be infected with the same Hematodinium sp. infecting N. norvegicus. DNA probes detected Hematodinium parasites in heart, hepatopancreas and gill tissues from N. norvegicus, and hepatopancreas and gill tissues from Carcinus maenas, confirming Hematodinium infection in the latter.

Amphipoda↗

PubMed2006

Vaccination with a preparation based on recombinant cysteine peptidases and canine IL-12 does not protect dogs from infection with Leishmania infantum.

Cysteine peptidases (CPs) have been implicated in various processes central to the pathogenicity of Leishmania parasites, and are thought to be key factors in the host-parasite interaction. In order to fully evaluate the potential of the CPs as vaccine candidates, studies in natural host species are required. In the study we report here, recombinant L. infantum CPs CPA and CPB were used to vaccinate dogs. In order to induce an appropriate response against the antigens, recombinant canine IL-12 was added as an adjuvant either by itself or in combination with Quil A. After vaccination, dogs were given an intravenous challenge with promastigotes of L. infantum JPC strain. In both vaccinated groups (CPs with IL-12 or CPs with IL-12 and Quil A) CP-specific antibodies were detected after vaccination, indicating that there was a reaction to the vaccine. However, all dogs were found parasite-positive and all developed some degree of clinical leishmaniosis. The observed lack of efficacy of the candidate vaccines could be due, completely or in part, to a number of factors associated with the vaccine antigen, the adjuvant or host-parasite interactions. When compared to results from other studies, it seems less likely that the molecular conformation of the rCPs or rIL-12 caused this lack of efficacy. More plausible explanations are the dose and timing of the IL-12 application and the potentially different effects IL-12 induces as an adjuvant in either the murine or the canine leishmaniosis model.

Adjuvants, Immunologic↗

PubMed2005

Identification and partial characterisation of metalloproteases secreted by a Mesanophrys-like ciliate parasite of the Norway lobster Nephrops norvegicus.

A ciliate parasite, tentatively identified as Mesanophrys sp. of Norway lobsters Nephrops norvegicus, is demonstrated to secrete several proteases into the culture medium (modified Nephrops saline). Analyses using substrate-impregnated sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed 12 activity bands differing greatly in mobility in the gels. The complete inhibition of proteolytic activity by 1,10-phenanthroline indicated that the proteases are of the metallo class. The proteases were active at the physiological temperature (8 degrees C) and haemolymph pH (7.8) of the host. The proteases were selective in the degradation of several host proteins, including the myosin heavy chain, which is a major structural component of lobster muscle. Consequently, these proteases may have important roles in several aspects of the host-parasite interaction including invasion, nutrient uptake by the ciliate, and pathogenesis.

Animals↗

PubMed2005

A parasitic scuticociliate infection in the Norway lobster (Nephrops norvegicus).

A histophagous ciliate infection was discovered in a number of Norway lobsters (Nephrops norvegicus) from the Clyde Sea Area, Scotland. Silver-carbonate staining of cultured ciliates revealed an oral apparatus and additional structural features that are morphologically similar to scuticociliates in the genus Mesanophrys, which are known to parasitize crustaceans. However, ribosomal DNA sequences (ITS1/5.8S/ITS2) of the ciliate were identical to Orchitophyra stellarum, a parasitic scuticociliate of sea stars with a different morphology from Mesanophrys spp. and to the ciliate from N. norvegicus. Associated pathology included degeneration and necrosis of the myocardial heart muscle, and large numbers of ciliates in the gill filaments.

Animals↗

PubMed2003

Functional conservation of a natural cysteine peptidase inhibitor in protozoan and bacterial pathogens.

Cysteine peptidase inhibitor genes (ICP) of the chagasin family have been identified in protozoan (Leishmania mexicana and Trypanosoma brucei) and bacterial (Pseudomonas aeruginosa) pathogens. The encoded proteins have low sequence identities with each other and no significant identity with cystatins or other known cysteine peptidase inhibitors. Recombinant forms of each ICP inhibit protozoan and mammalian clan CA, family C1 cysteine peptidases but do not inhibit the clan CD cysteine peptidase caspase 3, the serine peptidase trypsin or the aspartic peptidases pepsin and thrombin. The functional homology between ICPs implies a common evolutionary origin for these bacterial and protozoal proteins.

Amino Acid Sequence↗

PubMed2003

Biochemical and genetic evidence for a family of heterotrimeric G-proteins in Trichomonas vaginalis.

We have cloned a single copy gene from the human parasite Trichomonas vaginalis that encodes a putative protein of 402 amino acids with approximately 35% sequence identity to known alpha subunits of heterotrimeric G-proteins. It contains the characteristic GTP binding domains G-1 to G-5 with the key residues conserved. The new sequence has an unusual N-terminal extension of approximately 70 residues that cannot be aligned to reference G-proteins and which is characterised by proline-rich repeats. To investigate the expression and cellular localisation of the protein we produced specific antisera against a recombinant fusion protein. The antisera recognised a protein of an apparent molecular mass of 51 kDa in protein extracts from T. vaginalis and immunofluorescent microscopy established that the protein is localised to discrete endomembranes. Using a protocol designed to purify mammalian heterotrimeric G-proteins incorporating a GTPgammaS binding assay, we isolated two proteins from Trichomonas that are recognised by an heterologous GA/1 antisera raised to a peptide of the conserved G-1 domain of G-protein alpha subunits. These two proteins have an apparent molecular mass of 61 and 48 kDa, respectively, larger and smaller than the translation product of the cloned gene. Consistent with these results, the GA/1 antisera did not cross-react with the fusion protein produced from the gene we have cloned. These data suggest T. vaginalis possesses more than one heterotrimeric G-protein alpha subunit. Based on the sequence features of the cloned gene and the biochemical properties of the purified proteins, we suggest that these alpha subunits are likely to be part of classic heterotrimeric G-protein complexes.

Amino Acid Sequence↗

PubMed2002

Detection of the parasitic dinoflagellate Hematodinium in the Norway lobster Nephrops norvegicus by ELISA.

Norway lobsters Nephrops norvegicus from the coastal waters of Scotland are seasonally infected by a parasitic dinoflagellate of the genus Hematodinium. An enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of the parasite in the haemolymph of N. norvegicus. The ELISA is simple to perform with a detection limit of 5 x 10(4) parasites ml(-1) haemolymph. The ELISA is currently being used to study the prevalence and seasonality of Hematodinium infection in N. norvegicus and other crustacean hosts.

Animals↗

PubMed2002

Characterisation of global protein expression by two-dimensional electrophoresis and mass spectrometry: proteomics of Toxoplasma gondii.

The development of tools for the analysis of global gene expression is vital for the optimal exploitation of the data on parasite genomes that are now being generated in abundance. Recent advances in two-dimensional electrophoresis (2-DE), mass spectrometry and bioinformatics have greatly enhanced the possibilities for mapping and characterisation of protein populations. We have employed these developments in a proteomics approach for the analysis of proteins expressed in the tachyzoite stage of Toxoplasma gondii. Over 1000 polypeptides were reproducibly separated by high-resolution 2-DE using the pH ranges 4-7 and 6-11. Further separations using narrow range gels suggest that at least 3000-4000 polypeptides should be resolvable by 2-DE using multiple single pH unit gels. Mass spectrometry was used to characterise a variety of protein spots on the 2-DE gels. Peptide mass fingerprints, acquired by matrix-assisted laser desorption/ionisation-(MALDI) mass spectrometry, enabled unambiguous protein identifications to be made where full gene sequence information was available. However, interpretation of peptide mass fingerprint data using the T. gondii expressed sequence tag (EST) database was less reliable. Peptide fragmentation data, acquired by post-source decay mass spectrometry, proved a more successful strategy for the putative identification of proteins using the T. gondii EST database and protein databases from other organisms. In some instances, several protein spots appeared to be encoded by the same gene, indicating that post-translational modification and/or alternative splicing events may be a common feature of functional gene expression in T. gondii. The data demonstrate that proteomic analyses are now viable for T. gondii and other protozoa for which there are good EST databases, even in the absence of complete genome sequence. Moreover, proteomics is of great value in interpreting and annotating EST databases.

Animals↗

PubMed2002

Recent advances in the search for new anti-coccidial drugs.

Coccidia provide a rich hunting ground for drug-designers, as there are significant biochemical differences between the parasites and their hosts. Recent years have brought the discovery of the plastid and its possible metabolic machinery, characterisation of acidocalcisomes, reports on the apparent absence from some coccidia of a typical mitochondrion, and the discovery of the mannitol cycle and shikimate pathway in the parasites. Moreover, modern technologies such as genomics and proteomics are bringing new insights into the biochemistry of coccidia and highlighting possible drug targets in abundance. A major issue for would-be drug discoverers is to decide upon the targets to prioritise. This review provides an update on recent findings on how coccidia differ biochemically from vertebrates. It includes discoveries within coccidian parasites themselves but also uses findings in Plasmodium to provide an overview of biochemical features that may be characteristics of many apicomplexan parasites and so potential targets for broad-spectrum drugs.

Animals↗

PubMed2001

The stage-regulated expression of Leishmania mexicana CPB cysteine proteases is mediated by an intercistronic sequence element.

The tandemly arranged CPB genes of Leishmania mexicana are polycistronically transcribed and encode cysteine proteases that are differentially stage-specific; CPB1 and CPB2 are expressed predominantly in metacyclics, whereas CPB3-CPB18 are expressed mainly in amastigotes. The mechanisms responsible for this differential expression have been studied via gene analysis and re-integration of individual CPB genes, and variants thereof, into a CPB-deficient parasite mutant. Comparison of the nucleotide sequences of the repeat units of CPB1 and CPB2 with CPB2.8 (typical of CPB3-CPB18) revealed two major regions of divergence as follows: one of 258 base pairs (bp) corresponding to the C-terminal extension of CPB2.8; another, designated InS, of 120 bp, with insertions totaling 57 bp, localized to the intercistronic region downstream of CPB1 and CPB2. Cell lines expressing CPB2.8 or CPB2 with the 3'-untranslated region and intercistronic sequence of CPB2.8 showed up-regulation in amastigotes. Conversely, metacyclic-specific expression occurred with CPB2 or CPB2.8 with the 3'-untranslated region and intercistronic sequence of CPB2. Moreover, the InS down-regulated expression in amastigotes of a reporter gene integrated into the CPB locus. It is proposed that the InS mediates metacyclic-specific stage-regulated expression of CPB by affecting the maturation of polycistronic pre-mRNA. This is the first well defined cis-regulatory element implicated in post-transcriptional stage-specific gene expression in Leishmania.

3' Untranslated Regions↗

PubMed2001

Development and application of an immunoassay diagnostic technique for studying Hematodinium infections in Nephrops norvegicus populations.

Patent Hematodinium infections of the Norway lobster Nephrops norvegicus can be detected with a morphological method (pleopod diagnosis), but this fails to identify low-level haemolymph (sub-patent) and any tissue-based (latent) infections. The current study describes the development and application of an immunoassay for the detection of antigens of the parasite Hematodinium in the Norway lobster N. norvegicus. Infected tissue and haemolymph samples were detected as multiple-band reactions to a polyclonal antibody (anti-Hematodinium). The sensitivity limit of the method was 204 parasites mm(-3), approximately 10 times more sensitive than the pleopod diagnosis method. Use of the immunoassay on tissue samples taken from catches in the Clyde Sea area, Scotland, UK, showed that the pleopod method considerably under-diagnosed infection prevalence in the early part of the season, though this under-diagnosis decreased as infected lobsters in the field progressed from latent and sub-patent to patent infections. However, the immunoassay failed to detect any infected lobsters during the summer months, suggesting that infection may not be carried over from one season to the next. The data presented suggest that this immunoassay allows for the accurate estimation of Hematodinium infection prevalence in the field and should be employed, where possible, for the routine monitoring of infection prevalence in N. norvegicus.

Animals↗

PubMed2001

Identification of peptides inhibitory to recombinant cysteine proteinase, CPB, of Leishmania mexicana.

We have identified peptides that are relatively resistant to hydrolysis by a recombinant cysteine proteinase, CPB2.8DeltaCTE, of Leishmania mexicana, and yet exhibit inhibition constant (K(i)) values in the nanomolar range. Common to these peptides is a basic-hydrophobic-hydrophobic motif in the P3-P1 sites, which is also present in the pro-region of the enzyme. A nine-amino acid stretch, FAARYLNGA, which has good homology to the pro-region of mammalian cathepsin L was identified as the part of the pro-region most likely to interact with the active site of the parasite enzyme. This peptide is not hydrolyzed by CPB2.8DeltaCTE and inhibited it with a K(i) of 4 microM. Extension of this sequence at both the N- and C-termini and the introduction of ortho-aminobenzoic acid at the N-terminal site reduced the K(i) value to 30 nM. The best substrate for CPB2.8DeltaCTE was also well hydrolyzed by cathepsin L, however the best inhibitor of the parasite enzyme inhibit poorly cathepsin L, with K(i) value two order of magnitude higher than against the parasite enzyme. These promising data provide insights into the peculiar specificity of the parasite enzyme and will aid the design of antiparasitic drugs directed against the leishmanial enzyme.

Amino Acid Sequence↗

PubMed2001

Substrate specificity of recombinant cysteine proteinase, CPB, of Leishmania mexicana.

The primary S(1) subsite specificity of a recombinant cysteine proteinase, CPB2.8 Delta CTE, of Leishmania mexicana was investigated in a systematic way using a series of peptides derived from Abz-KLRFSKQ-EDDnp in which Arg was substituted by all natural amino acids (where Abz is ortho-amino-benzoyl and EDDnp is N-[2,4-dinitrophenyl]-ethylenediamine). The peptides from this series with charged side chain amino acids, Cys, Cys(SBzl), and Thr(OBzl) were well hydrolysed. All other substitutions resulted in peptides that were resistant or hydrolysed very slowly and inhibited the enzyme with K(i) values in the range of 9--400 nM. Looking for natural substrates for CPB2.8, we observed that the recombinant enzyme failed to release kinin from human kininogen, an activity earlier observed with cruzipain from Trypanosoma cruzi (Del Nery et al., J. Biol. Chem. 272 (1997) 25713.). This lack of activity seems to be a result of the resistance to hydrolysis of the sequence at the N-terminal site of bradykinin in the human kininogen. The preferences for the S(3), S(2) and S(1)'-S(3)' for some amino acids were also examined using substrates derived from Abz-KLRFSKQ-EDDnp with variations at Lys, Leu, Phe, Ser and Lys, using the amino acids Ala, Phe, Leu, His or Pro. Peptides with Phe at P(1)' presented the highest affinity to the leishmanial enzyme. For comparison, some of the obtained peptides were also assayed with recombinant human cathepsin L and cruzain. The best substrates for CPB2.8 Delta CTE were also well hydrolysed by cathepsin L, however, the best inhibitors of the parasite enzyme have low affinity to cathepsin L. These promising data provide leads for the design of anti-parasitic drugs directed against the leishmanial enzyme.

Amino Acid Sequence↗

PubMed2001

Analysis of the S(2) subsite specificities of the recombinant cysteine proteinases CPB of Leishmania mexicana, and cruzain of Trypanosoma cruzi, using fluorescent substrates containing non-natural basic amino acids.

We have explored the specificity of the S(2) subsite of recombinant cysteine proteinases from Leishmania mexicana (CPB2.8 Delta CTE) and from Trypanosoma cruzi (cruzain) employing a series of fluorogenic substrates based on the peptide Bz-F-R-MCA, in which Bz is the benzoyl group and the Phe residue has been substituted for by Arg, His and non-natural basic amino acids that combine a basic group with an aromatic or hydrophobic group at the side chain: 4-aminomethyl-phenylalanine (Amf), 4-guanidine phenylalanine (Gnf), 4-aminomethyl-N-isopropyl-phenylalanine (Iaf), 3-pyridyl-alanine (Pya), 4-piperidinyl-alanine (Ppa), 4-aminomethyl-cyclohexyl-alanine (Ama), and 4-aminocyclohexyl-alanine (Aca). Bz-F-R-MCA was hydrolyzed well by CPB2.8 Delta CTE and cruzain, but all the substitutions of Phe resulted in less susceptible substrates for the two enzymes. CPB2.8 Delta CTE has a restricted specificity to hydrophobic side chains as with cathepsin L. However, the peptides with the residues Amf and Ama presented higher affinity to CPB2.8 Delta CTE, and the latter was an inhibitor of the enzyme. Although, cruzain accepts basic as well as hydrophobic residues at the S(2) subsite, it is more restrictive than cathepsin B and no inhibitor was found amongst the examined peptides.

Amino Acids, Basic↗

PubMed2001

Targeting polyamines of parasitic protozoa in chemotherapy.

All parasitic protozoa contain polyamines and in recent years they, and their associated enzymes, have attracted attention as drug targets because they might reveal novel antiparasite therapies. How justified is this approach to drug discovery? In this review, Sylke Müller, Graham Coombs and Rolf Walter summarize the current status of research into drugs that exploit polyamine metabolism of trypanosomatid and malaria parasites, and propose priorities for research into such drugs. This review was inspired by an Expert Meeting entitled 'Polyamine Metabolism of Parasitic Protozoa as a Drug Target'.

Animals↗

PubMed2001

Aspartic proteases of Plasmodium falciparum and other parasitic protozoa as drug targets.

All parasitic protozoa contain multiple proteases, some of which are attracting attention as drug targets. Aspartic proteases are already the targets of some clinically useful drugs (e.g. chemotherapy of HIV infection) and a variety of factors make these enzymes appealing to those seeking novel antiparasite therapies. This review provides a critical analysis of the current knowledge on Plasmodium aspartic proteases termed plasmepsins, proposes a definitive nomenclature for this group of enzymes, and compares these enzymes with aspartic proteases of humans and other parasitic protozoa. The present status of attempts to obtain specific inhibitors of the parasite enzymes that will be useful as drugs is outlined and suggestions for future research priorities are proposed.

Amino Acid Sequence↗

PubMed2001

S1 subsite specificity of a recombinant cysteine proteinase, CPB, of Leishmania mexicana compared with cruzain, human cathepsin L and papain using substrates containing non-natural basic amino acids.

We have explored the substrate specificity of a recombinant cysteine proteinase of Leishmania mexicana (CPB2.8 Delta CTE) in order to obtain data that will enable us to design specific inhibitors of the enzyme. Previously we have shown that the enzyme has high activity towards substrates with a basic group at the P1 position [Hilaire, P.M.S., Alves, L.C., Sanderson, S.J., Mottram, J.C., Juliano, M.A., Juliano, L., Coombs, G.H. & Meldal M. (2000) Chem. Biochem. 1, 115--122], but we have also observed high affinity for peptides with hydrophobic residues at this position. In order to have substrates containing both features, we synthesized one series of internally quenched fluorogenic peptides derived from the sequence ortho-amino-benzoyl-FRSRQ-N-[2,4-dinitrophenyl]-ethylenediamine, and substituted the Arg at the P1 position with the following non-natural basic amino acids: 4-aminomethyl-phenylalanine (Amf), 4-guanidine-phenylalanine (Gnf), 4-aminomethyl-N-isopropyl-phenylalanine (Iaf), 3-pyridyl-alanine (Pya), 4-piperidinyl-alanine (Ppa), 4-aminomethyl-cyclohexyl-alanine (Ama), and 4-aminocyclohexyl-alanine (Aca). For comparison, the series derived from ortho-amino-benzoyl-FRSRQ-N-[2,4-dinitrophenyl]-ethylenediamine was also assayed with cruzain (the major cysteine proteinase of Trypanosoma cruzi), human cathepsin L and papain. The peptides ortho-amino-benzoyl-FAmfSRQ-N-[2,4-dinitrophenyl]-ethylenediamine (k(cat)/K(m) = 12,000 mM(-1) x s(-1)) and ortho-amino-benzoyl-FIafSRQ-N-[2,4-dinitrophenyl]-ethylenediamine (k(cat)/K(m) = 27,000 mM(-1) x s(-1)) were the best substrates for CPB2.8 Delta CTE. In contrast, ortho-amino-benzoyl-FAmaSRQ-N-[2,4-dinitrophenyl]-ethylenediamine and ortho-amino-benzoyl-FAcaSRQ-N-[2,4-dinitrophenyl]-ethylenediamine were very resistant and inhibited this enzyme with K(i) values of 23 nM and 30 nM, respectively. Cruzain hydrolyzed quite well the substrates in this series with Amf, Ppa and Aca, whereas the peptide with Ama was resistant and inhibited cruzain with a K(i) of 40 nM. Human cathepsin L presented an activity on these peptides very similar to that of CPB2.8 Delta CTE and papain hydrolyzed all the peptides with high efficiency. In conclusion, we have demonstrated that CPB2.8 Delta CTE has more restricted specificity at the S1 subsite and it seems possible to design efficient inhibitors with amino acids such as Ama or Aca at the P(1) position.

Animals↗