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PubMed2001

Catabolism of methionine and threonine in vitro by mixed ruminal bacteria and protozoa.

In vitro studies were conducted to examine the metabolism of methionine (Met) and threonine (Thr) using mixed ruminal bacteria (B), mixed ruminal protozoa (P), and a combination of these two (BP). Rumen microorganisms were collected from fistulated goats fed with lucerne cubes (Medicago sativa) and a concentrate mixture twice a day. Microbial suspensions were anaerobically incubated with or without 1 mM each of the substrates at 39 degrees C for 12h. Met, Thr and their related amino compounds in both the supernatants and microbial hydrolyzates of the incubation were analyzed by HPLC. Met was degraded by 58.7, 22.1, and 67.3% as a whole in B, P, and BP suspensions, respectively, during 12h incubation. In the case of Thr, these values were 67.3, 33.4, and 76.2% in B, P, and BP, respectively. Met was catabolized by all of the three microbial suspensions to methionine sulfoxide and 2-aminobutyric acid. Catabolism of Thr by B and BP resulted in the production of glycine and 2-aminobutyric acid, while P produced only 2-aminobutyric acid. From these results, the existence of diverse catabolic routes of Met and Thr in rumen microorganisms was indicated.

Aminobutyrates↗
PubMed2002

Aromatic amino acid biosynthesis and production of related compounds from p-hydroxyphenylpyruvic acid by rumen bacteria, protozoa and their mixture.

Aromatic amino acid biosynthesis and production of related compounds from p-hydroxyphenylpyruvic acid (HPY) by mixed rumen bacteria (B), protozoa (P), and their mixture (BP) in an in vitro system were quantitatively investigated. Microbial suspensions prepared from mature, fistulated goats fed Lucerne ( Medicago sativa) cubes and a concentrate mixture were anaerobically incubated at 39 degrees C for 12 h. Tyrosine (Tyr), phenylalanine (Phe), tryptophan (Trp) and other related compounds in both supernatants and hydrolyzates of all incubations were analyzed by HPLC. Large amounts of Tyr (27.0, 47.0 and 50.8% of disappeared HPY in B, P and BP, respectively) were produced from 1 mM HPY during a 12-h incubation period. The formation of Tyr in P was 1.8 and 1.6 times higher than those in B and BP, respectively. Appreciable amounts of Phe (3-12% of the disappeared HPY) and Trp (2-10% of the disappeared HPY) were also produced from HPY in B, P, and BP. Phe synthesis in B and P was almost similar but Trp synthesis in B was 1.8 times higher than that in P. The biosynthesis of both Phe and Trp from HPY in BP was higher than those in B plus P. A large amount of p-hydroxyphenylacetic acid (about 45% of the disappeared HPY) was produced from HPY in B which was 1.9 times higher than that in P. p-Hydroxybenzoic acid produced from HPY in P was 1.6 times higher than that in B. Considerable amounts of phenylpropionic acid, phenyllactic acid, and phenylpyruvic acid (2-6% of the disappeared HPY) were produced only in B.

Amino Acids, Aromatic↗
PubMed1990

Thymidylate synthase-dihydrofolate reductase in protozoa.

In protozoa, thymidylate synthase (TS) and dihydrofolate reductase (DHFR) exist on the same polypeptide. The DHFR domain is on the amino terminus, TS is on the carboxy terminus, and the domains are separated by a junction peptide of varying size depending on the source. The native protein is a dimer of two such subunits and is 110-140 kDa. Most studies of bifunctional TS-DHFR have been performed with the protein from anti-folate resistant strains of Leishmania major, which show amplification of the TS-DHFR gene and overproduction of the bifunctional protein. The Leishmania TS-DHFR has also been highly expressed in heterologous systems. There is extensive communication between domains, and channeling of the H2folate product of TS to DHFR. Anti-folates commonly used to treat microbial infections are poor inhibitors of L. major DHFR. However, selective inhibitors of L. major vs human DHFR have been found. The TS-DHFR from Plasmodium falciparum has also been cloned and sequenced. Interestingly, pyrimethamine-resistant strains of P. falciparum have a common point mutation in the DHFR coding sequence which causes decreased binding of the folate analog. A detailed knowledge of the structure and function of protozoan TS-DHFRs will soon be available.

Animals↗
PubMed1993

Phospholipid biosynthesis in protozoa.

Because of the diverse nature of the organisms which are all classed as 'protozoa' (and because of the lack of detailed information on phospholipid metabolism about most of them), it will probably never be possible to generalize phospholipid metabolism to the degree that it has been possible to characterize a mammalian metabolism. Nonetheless, patterns have begun to emerge (i.e. the similarities among the ciliates Entodinium, Paramecium and Tetrahymena) and will not doubt be expanded upon in the future.

Animals↗
PubMed1984

Thiol groups on the surface of anaerobic parasitic protozoa.

Evidence is presented that Giardia lamblia and Entamoeba histolytica, phylogenetically unrelated aerotolerant anaerobes, have crucial thiol groups on or easily accessible to their external surface. Both parasites were killed by three structurally unrelated thiol-blocking reagents which penetrate intact cells poorly or not at all. The parasites were protected from p-chloromercuribenzenesulfonic acid (10-100 microM) by cysteine or by reduced glutathione. Killing was arrested with identical kinetics by addition of either cysteine (which quickly penetrates the cells) or bovine serum albumin (which does not penetrate intact cells) at various times after p-chloromercuribenzenesulfonic acid, indicating that the reactive site may be on the outer surface of the cell. Proteins lacking cysteine did not protect. Sensitivity of three other protozoa to p-chloromercuribenzenesulfonic acid was also tested. Trichomonas vaginalis (anaerobic) was at least as sensitive as E. histolytica and G. lamblia, while Crithidia fasciculata and Paramecium tetraurelia (both aerobic) were less sensitive. Thiol groups on the G. lamblia surface were demonstrated directly by fluorescence-activated cell sorter analysis of trophozoites which had been modified with a thiol-specific hapten, N-iodoacetyl-N'-(5-sulfonic-1-naphthyl)ethylenediamine and reacted with fluorescent antibody to this hapten.

Anaerobiosis↗
PubMed1989

Presence of a fructose-2,6-bisphosphate-insensitive pyrophosphate: fructose-6-phosphate phosphotransferase in the anaerobic protozoa Tritrichomonas foetus, Trichomonas vaginalis and Isotricha prostoma.

Extracts of the anaerobic protozoa Tritrichomonas foetus, Trichomonas vaginalis and Isotricha prostoma contained a high activity (0.5-1 mumol min-1 (mg protein)-1) of pyrophosphate:fructose-6-phosphate phosphotransferase (PPi-PFK), but no detectable ATP: fructose-6-phosphate phosphotransferase. PPi-PFK from I. prostoma was purified close to homogeneity by adsorption on phospho-Ultrogel and elution with fructose-1,6-bisphosphate, and subsequent anion-exchange chromatography. The enzyme had an Mr of 95,000 as determined by gel filtration and consisted of subunits of Mr 48,000. PPi-PFK from I. prostoma and from T. foetus displayed hyperbolic kinetics with respect to their substrates and were not affected by fructose-2,6-bisphosphate. In sharp contrast with what has been found in other eukaryotes, no evidence could be found for the presence of fructose-2,6-bisphosphate in the two trichomonads, in I. prostoma and in Entamoeba histolytica.

Animals↗
PubMed1987

Immune response of fish to parasitic protozoa.

Epizootic outbreaks of fish diseases are increasingly common as a result of intensive aquaculture, fish farming and sea ranching. Very few drugs are available for treatment or prophylaxis against fish diseases, and development of such compounds is inhibited by different national regulations governing the use of chemicals in fish for human or animal consumption. Alternative approaches are urgently needed. But although the taxonomy and biology of fish parasites have been extensively studied, relatively little is known about protective immunity in fish and the effects of parasites on the piscine immune system. In this article, Patrick Woo discusses the immune responses of fish to parasitic protozoa, showing that vaccination is a viable control strategy, and stressing the need for a coordinated global research programme on fish diseases.

Journal Article↗
PubMed1989

The surface membrane 3'-nucleotidase/nuclease of trypanosomatid protozoa.

The plasma membrane of protozoan parasites has been the subject of intense research interest because it serves as the interface between the parasite and its host] This structure functions in the acquisition of nutrients, in the response to environmental signals, in the secretion and excretion of metabolites, in the recognition of host surfaces and in the avoidance of host-destructive factors including the components of the immune response. Recent technological advances, such as the use of monoclonal antibodies and recombinant DNA methodologies, have led to the identification of specific plasma membrane components and their coding; sequences, often in the absence of information regarding the physiological role of the component. In this article Michael Gottlieb considers the functional activity of o plasma membrane enzyme which has been identified in a number of protozoa belonging to the family Trypanosomatidae, and considers its adaptive significance and possible evolution in the different genera of this family.

Journal Article↗
PubMed1991

Viruses of parasitic protozoa.

Recently, specific viruses have been identified among the parasitic protozoa Trichomonas vaginalis, Giardia lamblia, Leishmania braziliensis, the Eimeria spp and the Babesia spp. These viruses share many features: they are all RNA viruses and most, if not all, doublestranded (ds) RNA viruses with nonsegmented genomes ranging between 5 and 7 kilobases (kb); they are spherical or icosahedral with an average diameter of 30-40 nm. The giardiavirus is one of the best characterized and can infect virus free G. lamblia trophozoites in its freed, pure form. The replicative intermediate of the giardiavirus genome has been isolated from infected cells, and can be introduced into G. lamblia by electroporation to produce giardiavirus, thus raising the possibility of its being used as a specific genetic transfection vector for the parasite.

Journal Article↗
PubMed1994

Coenzyme Q homologs in parasitic protozoa as targets for chemotherapeutic attack.

The central role of coenzyme Q (ubiquinone) in cellular energy metabolism is well established. Recent work has implicated this molecule in a wide range of other cellular functions, including roles in growth control, plasma membrane oxidase and as a cellular antioxidant. In this review, Jayne Ellis presents an overview of the current knowledge of this important cellular component in species of parasitic protozoa, discusses current therapies using its analogs and proposes its potential roles in these organisms.

Journal Article↗
PubMed1986

Application of chemi- and bioluminescence in studies of immunological reactions against protozoa.

The opsonization and lysis of different protozoa by antibodies and/or complement was followed using luminol-dependent chemiluminescence and bioluminescence. The addition of immune serum to variable antigen type populations of Trypanosoma evansi led to the specific opsonization of trypanosomes resulting in an intense metabolic activation and chemiluminescence response of phagocytic cells. In comparison to those of uninfected control mice, the phagocytosis of coccidia merozoites by spleen cells from mice infected with Eimeria falciformis was enhanced during the acute stage of a primary infection. Opsonizing activity was demonstrated in phosphate-buffered saline extracts of gut contents of mice infected for 10 days. The incubation of E. falciformis merozoites together with guinea-pig complement resulted in slow lysis of the cells. The addition of mouse serum collected greater than 6 days after an infection led to an accelerated lysis of the merozoites, indicating the appearance of complement-fixing antibodies in the serum. Heat-inactivated immune serum alone had no lysing activity on merozoites. In the presence of complement, bovine lymphoblastoid cells infected with Theileria annulata were lysed by anti-lymphoblastoid cell serum raised in mice but not by serum from cattle which had developed immunity to Theileria annulata.

Animals↗
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↗
PubMed1989

Actin, tubulin and H4 histone genes in three species of hypotrichous ciliated protozoa.

In hypotrichous ciliated protozoa, genes are transcribed in the macronucleus where the genome consists of 'gene-sized' linear DNA molecules. We have isolated clones of actin, tubulin and H4 histone macronuclear genes from Oxytricha nova, Stylonychia lemnae and Euplotes crassus in an effort to determine if they possess molecules of similar size for a given coding function, and also to determine the size range of non-coding DNA present on these molecules. Our results indicate that while the length of their non-coding DNA can vary slightly, both between different hypotrichs and within the gene family of a single organism, actin and tubulin macronuclear molecules are similarly sized. The sizes observed for these molecules support the hypothesis that each macronuclear molecule encodes a single gene. However, the H4 histone macronuclear molecules show a much wider size range and generally are much longer than necessary to encode the H4 histone. We therefore sequenced a 1700-bp H4 histone macronuclear molecule from O. nova to determine if it might possibly encode additional gene products. Sequence data reveals the presence of nine open reading frames (ORFs) greater than 100 bp in length; however, Northern hybridization analysis of the products of this DNA molecule reveals only a single transcript.

Actins↗
PubMedDate not supplied

[Ciliated protozoa and thanatology].

Different ciliated protozoa are observed by the immersion of dead bodies in soft water. In death from drowning a ciliated Tetrahymena kind was discovered whose size and hemotactism facilitate in a priviliged way the penetration in the internal medium through pulmonary channels. Diagnosis in blood was easy by putting this organism in culture. This method had an astonishing power of reproduction. This search fortunately completes the series about diatoms by means of corroboration on the diagnosis of drowning.

Adolescent↗
PubMed1987

Radioligand binding characterization of beta-adrenergic receptors in the protozoa Trypanosoma cruzi.

A direct radioligand binding technique utilizing the beta-adrenergic antagonist [3H]dihydroalprenolol was employed in the identification and characterization of Trypanosoma cruzi beta-adrenergic receptors. [3H]DHA binding was saturable (Bmax = 1.5 pmol/10(6) cells) with an apparent equilibrium dissociation constant (Kd) of 127 nM. Binding of [3H]DHA was displaced by propranolol in a concentration-dependent manner. The relative potency order of adrenergic ligands in displacing [3H]DHA binding was: propranolol greater than or equal to alprenolol greater than epinephrine. 5-Hydroxytryptamine, phentolamine and catechol had no effect. The experimental results support the suggestion that beta-adrenergic receptors are present in the pathogenic protozoa Trypanosoma cruzi.

Animals↗
PubMedDate not supplied

The intracellular multiplication of Legionella pneumophila in protozoa from hospital plumbing systems.

Between October 1987 and March 1989, we tested 144 water samples obtained from the plumbing and cooling tower systems of 5 Paris hospitals for the presence of legionellae and amoebae. Of the samples tested for Legionella, 67 out of 144 (46.5%) were positive, and 82 out of 116 tested for amoebae (70.7%) were positive. The ability of protozoa to support the multiplication of legionella was shown by incubating samples at 35.5 degrees C for 7-15 days. Prior to determining the presence of legionellae and amoebae, 51 of the 144 samples were incubated. After incubation, 22 out of 25 (88%) samples which were positive for the presence of both Legionella and amoebae showed multiplication of Legionella. In 3 out of the 25 (12%) samples containing Legionella and amoebae, Legionella failed to multiply. Six out of the 51 (11.8%) samples which were negative in direct culture for Legionella but positive for amoebae, became positive after incubation. Legionella did not multiply in samples negative for amoebae, nor was there proliferation in samples after filtration through a 1.2-microns membrane followed by incubation for the same period and temperature. Strains of Legionella pneumophila serogroup 1 and serogroup 6 (SG1 and SG6), including 3 patient isolates and 2 environmental isolates, were cocultured with 2 strains of amoebae and Tetrahymena pyriformis. Plate counts, Gimenez staining and electron microscopy demonstrated that intracellular legionellae proliferation occurred.

Air Conditioning↗
PubMed1994

Ancient origin of RNA editing in kinetoplastid protozoa.

A rooted phylogenetic tree of the kinetoplastid protozoa has been constructed that, together with a comparative analysis of editing of several genes, leads to the surprising conclusion that extensive or pan-editing with multiple overlapping guide RNAs is more ancient than 5'-editing. The mechanism of editing is still uncertain, but multiple ribonucleoprotein complexes have been identified that contain components of the enzymatic machinery.

Animals↗
PubMed2005

Ultrastructural alterations in ciliated protozoa under heavy metal exposure.

Transmission electron microscopy was used to study the ultrastructural changes induced by exposure to Cd or Zn in three species of ciliated protozoa: Colpoda steinii, Cyrtolophosis elongata and Drepanomonas revoluta. The main cytoplasmic alterations were partial mitochondrial degeneration, cytoplasmic vacuolisation, accumulation of membranous debris and autophagosome formation. At the nuclear level we detected nucleolar fusion in the macronucleus, and micronuclear membrane modifications. We compared these modifications with those coinciding with ciliate encystment (a differentiation process induced by environmental nutritional stress) and with changes in eukaryotic cells treated with staurosporine, a potent protein kinase inhibitor considered to be an apoptosis inducer. Exposure to heavy metals also coincided with the appearance of electron-dense accumulations in the cytoplasm, which might be related to metallothionein-mediated detoxification. The results are compared with previously reported data from ciliates and microalgae treated with heavy metals.

Animals↗