Biomedical subjects

G H Coombs

Publications and source records attributed to G H Coombs.

At least 73 records · Page 4Linked to original sources

PubMed1994

Effects of proteinase inhibitors on the growth and differentiation of Trypanosoma cruzi.

Three proteinase inhibitors, one peptidyl acyloxymethyl ketone (AMK), Z-Phe-Lys-CH2-OCO-(2,4,6-Me3)Ph.HCl, and two diazomethyl ketones (DMKs), Z-Phe-Phe-DMK and Z-Phe-Ala-DMK, have been studied for their effects in vitro on the four developmental stages of Trypanosoma cruzi. The three inhibitors penetrated living parasites and inhibited the major cysteine proteinase, cruzipain. The AMK was the most potent inhibitor of cruzipain itself and at 20 microM caused lysis of epimastigotes and trypomastigotes. When at lower concentrations, however, it had little effect on epimastigote growth but reduced metacyclogenesis. The DMKs had no effect against epimastigotes but inhibited differentiation to metacyclics. All three inhibitors markedly reduced infection of Vero cells by the parasite and the multiplication of the intracellular amastigotes, whereas release of trypomastigotes was almost entirely prevented. The results confirm the importance of cysteine proteinases in the life cycle of T. cruzi, and suggest that the differentiation steps are the most susceptible to cysteine proteinase inhibitors.

Animals↗

PubMed1994

Eimeria tenella contains a pyrophosphate-dependent phosphofructokinase and a pyruvate kinase with unusual allosteric regulators.

Sporozoites and unsporulated oocysts of Eimeria tenella were shown to contain a pyrophosphate-dependent phosphofructokinase (PPi-PFK) but apparently lack an ATP-specific activity. The PPi-PFK resembles those that occur in a number of other protists in being reversible and not subject to metabolic control. In contrast, the ADP-utilising pyruvate kinase, present in two developmental stages of the parasite, exhibited strong positive cooperativity with respect to its substrate, phosphoenolpyruvate, and was shown to be allosterically activated by glucose 6-phosphate, fructose 6-phosphate and AMP. It is suggested that the PPi-PFK represents an adaptation of the parasite towards life in an environment containing only low concentrations of oxygen and that the unusual allosteric regulation of pyruvate kinase evolved to compensate for glycolysis not being controlled at the PPi-PFK step.

Allosteric Regulation↗

PubMed1994

Null mutants for the lmcpa cysteine proteinase gene in Leishmania mexicana.

The parasitic protozoon Leishmania mexicana possesses an abundance of developmentally regulated cathepsin L-like cysteine proteinases expressed at highest levels in amastigotes. We recently characterised lmcpa, a single-copy gene encoding one such proteinase, LmCPa, which differs from other homologues by possessing a 3-amino-acid insertion at the amino terminal of the predicted mature proteinase. To investigate the role of LmCPa in L. mexicana, we used gene-targeting of promastigotes with hygromycin- and phleomycin-resistance markers to generate null mutants by disrupting sequentially both alleles of lmcpa. The promastigote null mutants did not differ significantly from wild-type L. mexicana in growth rate or morphology and could differentiate to metacyclics and the amastigote-like form, both of which could infect the J774G8 macrophage-like cell line. The null mutant amastigote-like form obtained from the J774G8 cells could also establish rump lesions in CBA mice. By these criteria, therefore, LmCPa appears to be non-essential although there is the possibility that LmCPa could be required during development in the sandfly, a stage not analysed here. The apparent redundancy of LmCPa in amastigotes may be due to the presence of other cysteine proteinases and has implications for the choice of candidate targets for rationally designed anti-leishmanial drugs.

Alleles↗

PubMed1994

Identification and molecular cloning of four cysteine proteinase genes from the pathogenic protozoon Trichomonas vaginalis.

The parasitic protozoon Trichomonas vaginalis produces multiple forms of cysteine proteinase (CP). The molecular basis for this has now been examined by cloning DNA fragments encoding CPs. Using generic degenerate oligonucleotide primers based on two well-conserved regions within the central region of all eukaryotic CPs, several polymerase chain reaction fragments were isolated from T. vaginalis genomic DNA and shown to encode different CPs. One fragment with a well-represented sequence was used as a general probe to screen a T. vaginalis cDNA library at moderate stringency and five different cDNA clones were isolated. Preliminary sequencing showed that they encoded similar but distinct CPs. In the process of confirming the 5' end of one of these cDNA clones using RACE-PCR (rapid amplification of cDNA 5' ends-polymerase chain reaction), an additional sequence encoding a different CP was identified. The corresponding clone (TvCP3) and the three longest clones from the library screen (TvCP1, TvCP2 and TvCP4) were characterized further. TvCP1 and TvCP2 were full-length and TvCP3 and TvCP4 were apparently slightly less than full-length. Comparison of the predicted amino acid sequences of the four clones showed that TvCP1 and TvCP4 are related (72% identity). TvCP2 is closer to TvCP1 (60%) and TvCP4 (65%) than is TvCP3, which has 53%, 59% and 56% identity to TvCP1, TvCP2 and TvCP4, respectively. Comparison with the sequences of other known CPs indicated that the T. vaginalis gene products all belong to the cathepsin L/cathepsin H/papain branch of the papain superfamily. The TvCP1, TvCP2 and TvCP4 sequences are related (38-45% identity) to those of CP2 of Dictyostelium discoideum, human cathepsin L, three CPs from lobster and CPs from black gram, oilseed rape and rice (oryzains alpha and beta). TvCP3 shows less identity to the other eukaryotic CPs but is most similar to D. discoideum CP2 (38%). The four predicted amino acid sequences share some features distinct from the majority of CPs, which suggests they might have had a common evolutionary origin. The most striking feature of sequences TvCP1, TvCP2 and TvCP3 is the apparent lack of a pre-sequence (signal sequence) for TvCP1 and very short pre-sequences for TvCP2 and TvCP3. Southern analysis indicated that the organization of the genes corresponding to the TvCP cDNAs differs. The TvCP1, TvCP2 and TvCP3 genes are single-copy, whereas the TvCP4 gene appeared to be multiple-copy.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1994

Multiple high activity cysteine proteases of Leishmania mexicana are encoded by the Imcpb gene array.

The interrelationship of the multiple cysteine proteases (CPs) found characteristically at high activity in Leishmania mexicana amastigotes has been investigated. The mature forms of the five enzymes of groups B and C, which have subtly different substrate preferences, are the same size. Enzymically deglycosylated group A CP proteins also have the same molecular mass. Proteases of all three groups are specifically recognized by antisera raised against the group B or group C CPs. In addition, CPs of groups A, B and C have highly similar N-terminal amino acid sequences. The consensus sequence matches that predicted from the sequenced Imcpb gene, which occurs in a tandem array of over ten similar genes. Thus, the results are consistent with the groups A, B and C CPs being products of different Imcpb genes within the array, the different genes encoding CPs with identical N-termini, but with limited amino acid substitutions within the mature enzyme accounting for the different properties of the CPs. Evidence is also presented to indicate membrane-association of proteolytically active but less processed forms of Imcpb products.

Amino Acid Sequence↗

PubMed1993

Purification of cysteine proteinases from trichomonads using bacitracin-Sepharose.

Bacitracin affinity chromatography has been used to purify proteinases of the parasitic protozoon Tritrichomonas foetus. It proved superior to other affinity chromatography methods we have tested for the purification of trichomonad proteinases and should prove a useful procedure for purifying cysteine proteinases from these parasites and other parasitic protozoa. The main cysteine proteinases of T. foetus were purified over 100-fold to be free from the majority of other cell proteins. About 90 micrograms of protein containing 1.56-fold more proteinase activity than was detectable in the original cell lysate was obtained from 10(9) cells (7.2 mg protein). SDS-PAGE revealed that the eluate contained two main Coomassie blue-staining bands. N-terminal amino acid sequence analysis of these proteins confirmed that one of them was a cysteine proteinase with unusual features. Cysteine proteinases were also purified from cell lysates of Trichomonas vaginalis and a N-terminal sequence determined. This is the first amino acid sequence information that has been obtained for trichomonad cysteine proteinases. The method was also used to purify proteinases from the medium of T. foetus cultures. Some selectivity in binding of the proteinases to the affinity column was found.

Amino Acid Sequence↗

PubMed1993

Cathepsin B-like cysteine proteases of Leishmania mexicana.

A group of Leishmania mexicana cysteine proteases that differ from those previously found in this protozoon are described. The enzymes characteristically have a preference for peptidyl substrates with a phenylalanyl-valyl-arginyl moiety, do not hydrolyse gelatin in substrate-sodium dodecyl sulphate polyacrylamide gels, are stimulated by thiol-reducing agents and are sensitive to inhibitors specific for cysteine proteases. They have unusual solubility properties that indicate that the enzymes are amphiphilic proteins. Two of the cysteine proteases have been purified from L. mexicana amastigotes and shown to have molecular masses of 31 and 33 kDa. Their N-terminal amino acid sequences are very similar and show high homology to the mammalian cysteine protease, cathepsin B.

Amino Acid Sequence↗

PubMed1992

Characterization of a multi-copy gene for a major stage-specific cysteine proteinase of Leishmania mexicana.

lmcpb, a gene from Leishmania mexicana that encodes a major cysteine proteinase in the parasite, has been cloned and sequenced. LmCPb is related more to cysteine proteinases from Trypanosoma brucei and Trypanosoma cruzi than to a previously characterized cysteine proteinase, LmCPa, of L. mexicana. It contains a long C-terminal extension characteristic of similar enzymes of T. brucei and T. cruzi. The gene is multi-copy and tandemly arranged. lmcpb RNA levels are developmentally regulated with steady state levels being high in amastigotes, low in metacyclic promastigotes and undetectable in multiplicative promastigotes. This variation correlates with and may account for the stage-specific expression of LmCPb enzyme activity.

Amino Acid Sequence↗

PubMed1992

Cystatin-like cysteine proteinase inhibitors of parasitic protozoa.

A new method has been developed for detecting cystatins and other cysteine proteinase inhibitors. The method, which involves protein separation by SDS-PAGE followed by a cysteine proteinase overlay step, is more sensitive than previously reported techniques: as little as 1 ng of recombinant human cystatin C can be detected and cysteine proteinase inhibitors could also be detected in complex protein mixtures such as bovine foetal serum. The method has been used to show, for the first time, cysteine proteinase inhibitors in lysates of a range of parasitic protozoa (Trypanosoma brucei, Leishmania mexicana mexicana, Toxoplasma gondii and Tritrichomonas foetus) and to confirm that one occurs in the free-living ciliate Tetrahymena pyriformis. Cystatin-like inhibitory activity was also demonstrated in boiled lysates of L. mexicana mexicana using conventional assays methods.

Animals↗

PubMed1992

Stage-specific proteinases of Leishmania mexicana mexicana promastigotes.

Four distinct bands of cysteine proteinase activity were detected when stationary-phase populations of Leishmania mexicana mexicana were subjected to gelatin-SDS-PAGE. The highest mobility band contained at least three isoforms separable by mono Q anion exchange chromatography. These high mobility activities were distinct from all the major amastigote enzymes. Stationary-phase promastigote populations also contained two acid-activable precursor forms of the promastigote-specific band. It is suggested that these promastigote-specific activities occur in the infective metacyclic stage of the parasite and may have a role in parasite survival upon inoculation into a mammal.

Animals↗

PubMed1992

Hydrolysis of L-leucine methyl ester by Leishmania mexicana mexicana amastigote cysteine proteinases.

The main cysteine proteinases of the amastigote form of Leishmania mexicana mexicana were partially purified by gel filtration and ion exchange chromatography. The latter procedure resulted in the separation of some individual cysteine proteinases, as demonstrated by gelatin-sodium dodecyl sulphatepolyacrylamide gel electrophoresis. Fractions containing the partially purified proteinases rapidly hydrolysed L-leucine methyl ester to leucine. The activity towards this compound co-eluted with and resembled the parasite's cysteine proteinase activity. The results suggest that amastigotes of L.m.mexicana are susceptible to L-leucine methyl ester because this compound is rapidly hydrolysed by cysteine proteinases that occur in abundance in the megasomes of this stage.

Animals↗

PubMed1992

Axenic cultivation and characterization of Leishmania mexicana amastigote-like forms.

A new method is described which has made possible the long-term axenic cultivation of Leishmania mexicana amastigote-like forms in Schneider's Drosophila medium supplemented with 20% (v/v) foetal calf serum. Unlike previous methods, it utilizes direct culture of parasites obtained from the lesions of infected animals rather than adaptation of promastigotes in vitro. Ultrastructural (possession of megasomes), biochemical (cysteine proteinase activity and gelatin SDS-PAGE banding pattern) and infectivity (in vivo) data are presented which show the close similarity of the cultured forms to lesion amastigotes. The axenically cultured forms grew optimally at a temperature of 32-33 degrees C, providing further evidence for their amastigote nature. It was found that adjustment of the pH of the growth medium to 5.4 was required in order to retain the amastigote morphology of the cultured parasites. This supports the notion that leishmanial amastigotes are acidophiles.

Animals↗

PubMed1992

A developmentally regulated cysteine proteinase gene of Leishmania mexicana.

We have isolated a gene encoding a previously unreported class of trypanosomatid cysteine proteinase (CP) from the protozoan parasite Leishmania mexicana. The single-copy gene (lmcpa) [corrected]. has several unusual features that distinguish it from CP genes cloned from the related species Trypanosoma brucei and Trypanosoma cruzi. These include a shorter C-terminal extension of only 10 amino acids and a three-amino-acid insertion, GlyValMet, close to the predicted N-terminus of the mature protein. Northern blot analysis showed that the gene is expressed in all life-cycle stages but at higher levels in the amastigote stage in the mammal and in stationary phase promastigote cultures which contain the infective metacyclic form of the parasite. A precursor protein of 38 kDa was detected in amastigotes and stationary phase promastigotes with antisera specific to the LmCPa pro-region, but was barely detectable in early log-phase promastigotes. Anti-central domain antisera recognized the 38 kDa precursor and 24 and 27 kDa proteins. The major CPs of L. mexicana amastigotes, previously designated types A, B and C, were not detected with the antisera, suggesting that the gene codes for a previously uncharacterized CP in L. mexicana. The 24 kDa protein detected by the antiserum has no activity towards gelatin but apparently hydrolyses the peptide substrate BzPheValArgAMC. The relative levels of the 24 and 27 kDa proteins vary between the different life-cycle stages. The results indicate that expression of this CP is regulated at both the RNA and protein level.

Amino Acid Sequence↗

PubMed1991

Purification and characterization of methionine gamma-lyase from Trichomonas vaginalis.

Methionine gamma-lyase (EC 4.4.1.11) was purified to homogeneity from the anaerobic protozoan parasite Trichomonas vaginalis by a series of f.p.l.c. procedures. The enzyme catalyses alpha gamma- and alpha beta-elimination reactions of a number of derivatives of methionine and cysteine. It also catalyses gamma-replacement reactions of the thiomethyl group of methionine, homocysteine and ethionine to yield the corresponding S-substituted homocysteine derivative. The enzyme is pyridoxal 5'-phosphate-dependent, has a native molecular mass of approx. 160 kDa and consists of four apparently identical subunits of molecular mass 43-45 kDa. The absorption spectrum of the enzyme is typical of those obtained for other pyridoxal 5'-phosphate-dependent enzymes, and the holoenzyme can be resolved to the apoenzyme by incubation with hydroxylamine and reconstituted by addition of the cofactor. The enzyme activity is significantly affected by carbonyl and thiol reagents, is competitively inhibited by a number of substrate analogues and is completely inactivated by the suicide inhibitor DL-propargylglycine. The T. vaginalis enzyme is similar, in terms of activity and properties, to the enzymes found in a number of species of bacteria that metabolize methionine under anaerobic conditions. It is suggested that methionine catabolism may be of particular importance to the survival of T. vaginalis under microaerophilic conditions in its host.

Animals↗

PubMed1991

Conservation of cation-transporting ATPase genes in Leishmania.

DNA fragments isolated from Leishmania donovani ATPase genes were used to analyze the organization and expression of cation transporting ATPase genes in L. donovani, Leishmania tropica, Leishmania mexicana, Leishmania braziliensis, Trypanosoma brucei and Trypanosoma cruzi. The ATPase loci in all Leishmania species contained a tandem pair of ATPase genes arranged in head-to-tail orientation and separated by approximately 2 kb. No restriction site polymorphisms were detected in the internal portions of the Leishmania ATPase genes which contain domains conserved between the L. donovani and other eukaryotic plasma membrane ATPases. The ATPase locus of each of the four Leishmania species was mapped to a single small chromosome of approximately 750 kb. The ATPase locus of L. mexicana was differentially expressed. Promastigotes in exponential growth contained abundant transcripts from the upstream ATPase gene, while transcripts from the downstream gene were relatively scarce. Transcripts from the downstream ATPase gene increased in abundance in promastigotes allowed to reach the stationary phase of growth and were most abundant in amastigotes. The two trypanosome species were found to contain DNA fragments that hybridized strongly to the Leishmania ATPase gene.

Adenosine Triphosphatases↗

PubMed1990

The specificity of trichomonad cysteine proteinases analysed using fluorogenic substrates and specific inhibitors.

The multiple cysteine proteinases of Trichomonas vaginalis and Tritrichomonas foetus, both those retained intracellularly and those released, were separated using gelatin-SDS-PAGE, and their activity towards a range of 15 fluorogenic peptidyl aminomethylcoumarins determined together with their relative sensitivity to inhibitors. Three types of enzyme were apparent in T. vaginalis: (i) an 86-kDa enzyme active only on Z-Arg-Arg-NHMec; (ii) a 54-kDa proteinase which was most active on Z-Phe-Arg-NHMec but also able to hydrolyse N-t-Boc-Val-Leu-Lys-NHMec, Suc-Ala-Phe-Lys-NHMec, H-Pro-Phe-Arg-NHMec and Z-Arg-Arg-NHMec; and (iii) a group of six enzymes which preferentially hydrolysed substrates with bulky residues at the P2 and P3 positions. N-t-Boc-Val-Leu-Lys-NHMec and H-Leu-Val-Tyr-NHMec were the best substrates for the latter group. The 86-kDa proteinase was inactivated by E-64, but only at high concentrations, and was relatively insensitive to the peptidyl diazomethanes. The other proteinases were inhibited by low concentrations of E-64 and by Z-Phe-Ala-CHN2, and to a lesser extent by Z-Phe-Phe-CHN2. Differences between the proteinases of T. foetus were also demonstrated. All of them were active on Z-Arg-Arg-NHMec, but their activity towards other substrates varied. Three predominantly extracellular proteinases (25, 27 and 34 kDa), hydrolysed Z-Arg-Arg-NHMec specifically. Other proteinases (apparent Mr of 20,000 and 32,000) hydrolysed a number of other substrates, with the 32-kDa enzyme having greater activity towards N-t-Boc-Val-Leu-Lys-NHMec and H-Leu-Val-Tyr-NHMec than towards Z-Arg-Arg-NHMec. At a high concentration (270 microM), E-64 inhibited all of the T. foetus enzymes, but lower concentrations were less effective, with the 18-kDa proteinase being particularly insensitive. Z-Phe-Ala-CHN2 and Z-Phe-Phe-CHN2 were relatively poor inhibitors. The results demonstrate that the proteinases of both species are a heterogeneous group with respect to specificity, and have highlighted significant differences between the enzymes of T. vaginalis and T. foetus. The information on the specificities will be useful for assessing the features required in proteinase inhibitors if they are to be of potential value as antitrichomonal agents.

Animals↗

PubMed1990

Changes in hepatic xenobiotic-metabolising enzymes in mouse liver following infection with Leishmania donovani.

Infection of mice with Leishmania donovani resulted in decreased activities of several liver enzymes involved in the metabolism of xenobiotics. Microsomal membranes from infected livers contained reduced amounts of cytochromes P450 and b5 and NADPH-cytochrome P450 reductase. Several cytochrome P450 isoenzymes (P450-PB1, P450-PB3, P450-PCN and P450-UT1) and P450-mediated reactions (aminopyrine demethylase, aniline hydroxylase, benzphentamine demethylase and ethoxycoumarin deethylase) were affected similarly. The metabolism of two carcinogens (nitrosodimethylamine and 7,12-dimethylbenz[a]anthracene) by liver microsomal membrane preparations was also reduced. Leishmania infection caused an increase of cytosolic epoxide hydrolase and microsomal epoxide hydrolase and NADH-cytochrome b5 reductase were unaffected. The results suggest that Leishmania-infected animals are likely to have altered responses to exogenous toxins compared to uninfected animals.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1990

Cysteine proteinases of parasitic protozoa.

Proteinases are involved with many processes in living organisms. In recent years, there has been increasing interest in elucidating the functions the enzymes perform in parasites. These studies have revealed that one class of proteinases, the cysteine proteinases, predominates in many parasitic protozoa. In this article Mick North, Jeremy Mottram and Graham Coombs review what is known about the cysteine proteinases of parasitic protozoa and discuss the approaches being pursued in attempts to design antiparasite drugs based on inhibitors or substrates of these enzymes.

Journal Article↗