PubMed Health⌕ Search

Biomedical subjects

G H Coombs

Publications and source records attributed to G H Coombs.

140 records · Page 8Linked to original sources
PubMed1981

Proteinases of Leishmania mexicana amastigotes and promastigotes: analysis by gel electrophoresis.

The proteinases of Leishmania mexicana mexicana amastigotes and promastigotes have been analysed by electrophoresis on polyacrylamide gels containing denatured haemoglobin. Eleven bands of activity were detected indicating multiple proteinases. These were significant quantitative and qualitative differences between the proteinases of the two developmental forms. Four, B-E, were present in both forms but were of much higher activity in the amastigote. There were two major activities in promastigotes, A and D. The other proteinases, F-K, were of lower activity; I and K were not detected in promastigotes. All proteinases were active optimally at pH 4.0. Most of them, including the major proteinases A-E, were thiol proteinases since they were stimulated by 1 mM dithiothreitol and were sensitive to inhibitors such as HgCl2, leupeptin, antipain and iodoacetic acid.

Animals↗
PubMed1981

Respiration of Leishmania mexicana amastigotes and promastigotes.

Promastigotes of Leishmania mexicana mexicana recently derived from amastigotes by transformation in vitro respired at a rate (17 nmol O2/min per 10(8) parasites) 4-5 times higher than that of amastigotes, but when the difference in cell protein content between the two preparations was taken into account the rates were not significantly different (32 nmol O2/min per mg protein). The respiration of both amastigotes and promastigotes was sensitive to cyanide, azide, antimycin A, 2-n-heptyl-4-hydroxyquinoline-N-oxide and high concentrations of amytal, but insensitive to rotenone and salicyl-hydroxamic acid, indicating that the two developmental forms possess a similar cytochrome-containing respiratory chain. D-Glucose and non-esterified fatty acids stimulated promastigote respiration and amastigote transformation to promastigotes in vitro; possibly these substances are important exogenous energy substrates for both forms of the parasites. Amino acids (incuding L-proline) and proteins did not appear to be used as energy substrates. The respiration rate of promastigotes was found to rise significantly upon continued sub-culture in vitro; at the same time cell size and protein content increased.

Amino Acids↗
PubMed1981

The effects of carbon dioxide and oxygen upon the growth and in vitro transformation of Leishmania mexicana mexicana.

Growth of Leishmania mexicana promastigotes is highly dependent upon O2 tension. There was a strong positive correlation between the level of O2, growth rate and maximum parasite density. Promastigotes under low oxygen tension decreased in size, protein content and motility, and deaths occurred. Changes in the carbon dioxide concentration (0.1-5.0%) had little effect on promastigote growth. Transformation in vivo of L. mexicana amastigotes to promastigotes also required oxygen, but a low level (0.4%) was sufficient for a high percentage of the amastigotes to transform. At high O2 concentrations, transformation was a little speedier but the number of parasites transforming was little affected. A greater effect was found with CO2. At 5%, transformation was much more rapid than at 0.1% and also an even greater percentage of amastigotes transformed within 48 h. The results give some indication that amastigotes are adpated for growth at low oxygen tensions encountered in vivo and that high carbon dioxide levels may act as a trigger for transformation of the amastigote to promastigote after it is taken up by the sandfly.

Animals↗
PubMed1981

A quick, simple method for purifying Leishmania mexicana amastigotes in large numbers.

A rapid method for the bulk isolation of purified Leishmania mexicana mexicana amastigotes from parasite-induced lesions in experimentally infected mice is described. The procedure includes purification steps based on differences in net cell charge, lysis susceptibility and buoyant density between parasite and host cells. Yields of up to 2 x 10(10) untransformed amastigotes with minimal contamination with host cells and cell debris can be obtained. At least 90% of the purified amastigotes are viable as judged by light and electron microscopy, the staining of their lysosomes with acridine orange, their ability to transform to promastigotes and their infectivity to macrophages in vivo and in vitro.

Animals↗
PubMed1975

Growth in vitro and metabolism of Plasmodium vinckei chabaudi.

An in vitro system, based on the rocker dilution technic, has been developed that supports intraerythrocytic growth of a rat-adapted strain of Plasmodium vinckei chabaudi from ring to schizont stages; some reinvasion was obtained, although invariably, this was associated with a decrease in parasite numbers. Pertinent features were the very high buffer content of the medium and the low oxygen tension of the gaseous phase. Lactate production, glucose utilization, and 3H-leucine and 3H-adenosine incorporations were investigated for their suitability to monitor parasite growth. Throughout an 18-hr incubation there was a continuous and increasing production of lactate and utilization of glucose, which correlated well with the development of the parasites from ring to schizont stages. During the same period, there was a low but continuous and increasing incorporation of 3H-leucine into parasite protein. However, 3H-adenosine was incorporated only for the 1st hr of incubation, after which time no net incorporation occurred. Parasites grew normally from ring to schizont stages even in the absence of adenosine from the dilution medium.

Adenosine↗
PubMedDate not supplied

Characterisation of three groups of cysteine proteinases in the amastigotes of Leishmania mexicana mexicana.

The multiple cysteine proteinases characteristic of the amastigote forms of Leishmania mexicana mexicana have been shown to be of three types. The groups of enzymes are distinguished by their substrate specificities and physical properties and have been purified free from other proteinases and most other proteins. One group (A) comprises at least four enzymes that bind to Con A. The cysteine proteinases comprising the other two groups (B and C) were separated by ion exchange chromatography. These last two groups of enzymes show different specificities towards a range of peptidyl aminomethylcoumarins. Notably, the two group C proteinases are more active towards compounds with a basic amino acid in the P1 position, whereas the three enzymes of group B are as active towards substrates with tyrosine in this position. All the cysteine proteinases show preference for compounds with bulky amino acids at positions P2 and P3 and all are equally susceptible to a range of inhibitors characteristically active against cysteine proteinases. Other proteinases present in amastigotes of L. m. mexicana were shown to differ significantly from the cysteine proteinases with respect to their substrate preferences and susceptibility to inhibitors.

Animals↗
PubMedDate not supplied

Comparison of the phosphofructokinase and pyruvate kinase activities of Cryptosporidium parvum, Eimeria tenella and Toxoplasma gondii.

Oocysts of Cryptosporidium parvum were shown to contain a pyrophosphate-dependent phosphofructokinase (PPi-PFK) similar to those previously described for Eimeria tenella and Toxoplasma gondii. PPi-PFK of C. parvum displayed simple hyperbolic kinetics with respect to its substrate fructose 6-phosphate and was not affected by fructose 2,6-bisphosphate, the major allosteric activator of most ATP-PFKs. Inorganic pyrophosphatase was not detectable in any of the three parasites. T. gondii tachyzoites and C. parvum cysts both contained a pyruvate kinase (PK) specific for ADP rather than PPi/AMP. The PK of T. gondii was similar to that of E. tenella in that it displayed strong positive cooperativity with respect to its substrate phosphoenolpyruvate and was heterotropically activated by glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-bisphosphate. PK of C. parvum showed no evidence of allosteric properties. The results suggest that the three coccidia are similar in depending heavily on anaerobic energy production via glycolysis but that the mechanisms for regulating glycolysis are not common to all species.

Anaerobiosis↗
PubMedDate not supplied

Combinatorial library of peptide isosters based on Diels-Alder reactions: identification of novel inhibitors against a recombinant cysteine protease from Leishmania mexicana.

A combinatorial split-and-mix library of peptide isosters based on a Diels-Alder reaction was synthesized as a "one-bead-two-compounds" library and encoded by ladder synthesis for facile analysis by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. In the "one-bead-two-compounds" library approach, each bead contains a library member as a putative protease inhibitor along with a fluorescence-quenched substrate for the protease. When the library was screened with CPB2.8 DeltaCTE, a recombinant cysteine protease from L. mexicana, several beads containing compounds with inhibitory activity could be selected from the library and analyzed by MALDI-TOF MS for structure elucidation. Two types of inhibitors were revealed. One novel class of inhibitors had the bicyclic Diels-Alder product isosteric element incorporated internally in a peptide, while the other type was an N-terminal alpha,beta-unsaturated ketone Michael acceptor used as starting material for the Diels-Alder reaction. Selected hit sequences and constructed consensus sequences based on the observed frequencies of amino acids in different subsites were resynthesized and assayed in solution for inhibitor activity and were shown to have IC(50) values in the high nanomolar to low micromolar range.

Animals↗
PubMedDate not supplied

Expression of cysteine proteinases by metacyclic promastigotes of Leishmania mexicana.

The expression of cysteine proteinases by metacyclic promastigotes of Leishmania mexicana was investigated using gelatin polyacrylamide gel electrophoresis. Two prominent bands were detected which distinguished metacyclics from multiplicative promastigotes, lacking detectable cysteine proteinase activity, and amastigotes, with a distinct banding pattern composed of multiple enzymes. A correlation between relative activity of the metacyclic-specific bands and the prevalence of metacyclics was found both during the growth cycle in vitro as metacyclogenesis occurred, and by comparison of stationary phase populations from consecutive subpassages in vitro. Irreversible inhibition of the metacyclic activities using N-benzyloxycarbonyl-phenylalanyl-alanyl diazomethane did not inhibit metacyclic to amastigote transformation in vitro. These activities provide a useful biochemical marker for the metacyclic promastigotes of L. mexicana.

Animals↗