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R N Ondarza

Publications and source records attributed to R N Ondarza.

At least 19 recordsLinked to original sources

Inhibitory and lytic effects of phenothiazine derivatives and related tricyclic neuroleptic compounds, on Entamoeba histolytica HK9 and HM1 trophozoites.

It has been shown previously that tricyclic neuroleptics like clomipramine and chlorpromazine have lethal effects on Leishmania donovani and L. major, and other studies indicate that the phenothiazine inhibitors of trypanothione reductase are potential anti-trypanosomal and anti-leishmanial drugs. With this in mind and our original observation on the presence of trypanothione in Entamoeba histolytica HK9, we examined the possible inhibitory effects of various phenothiazine and tricyclic derivatives on this human parasite. We found that drugs like clomipramine (KD002), the most potent in vitro inhibitor of trypanothione reductase among 30 tricyclic compounds tested, at 25 microM after 24 h of culture under aerobic conditions, caused a substantial decrease in the number of E. histolytica HK9 trophozoites, from approx. 15 x 10(6) to 5.37 x 10(6) cells, and at 100 microM to 0.8 x 10(6) cells. A substantial inhibitory effect on cell proliferation could also be demonstrated with metronidazol (used clinically against amoebiasis). Under similar experimental conditions other tricyclic and phenothiazine derivatives (OFKs), designed originally to inhibit the trypanothione reductase of trypanosomatides, had an inhibitory effect of 16 to 95%. For comparison, similar results were obtained using clomipramine and a phenothiazine derivative (OFK006) with Trypanosoma cruzi and Crithidia luciliae, except that with the latter the inhibitory effect of clomipramine was less dramatic. Experiments comparing two E. histolytica strains showed that normal cell proliferation under anaerobiosis was higher in strain HK9 than in HM1, which is highly virulent, but that metronidazol and clomipramine were less effective against HM1. Two other drugs tested, diphenydramine (KD005) and a phenothiazine derivative (OFK008), also had significant but lower inhibitory effects on both strains. The inhibitory activity on cell proliferation and the lytic effects on this human parasite by the tricyclic compounds clomipramine, chlorpromazine and others, as well as by the phenothiazine derivatives, indicate that they can be considered potential anti-amoebic agents.

Aerobiosis↗

Detection by HPLC of a trypanothione synthetase activity in vitro from Entamoeba histolytica.

We have previously demonstrated the presence of glutathione-spermidine (Gsp) and trypanothione [T(SH)(2)] from Entamoeba histolytica trophozoites, on the basis of results obtained with acid extracts purified by Florisil and DEAE-cellulose, derivatized with the fluorescent reagent monobromobimane and separated by HPLC. Gsp was originally found in Escherichia coli and later in trypanosomatids such as Trypanosoma cruzi, T. brucei, T. congolense and the insect trypanosomatid Crithidia fasciculata, along with the novel compound T(SH)(2), N(1), N(8)-bis(glutathionyl)-spermidine. Here we demonstrate the presence of a T(SH)(2) synthetase activity in partly purified extracts from Entamoeba histolytica HK9, incubated at two pH values (6.5 and 7.5) with reduced glutathione (GSH), spermidine and ATP, in the presence of Mg(2+) at different time intervals. The thiol products were detected by HPLC in picomole amounts and compared with commercial Gsp and T(SH)(2) standards. We have used also an extract of Crithidia luciliae as a reference, to compare our results with C. fasciculata, in which the presence of this enzyme has previously been demonstrated and was later purified and separated into two synthetase activities from the same source: one for Gsp and the other for T(SH)(2). The presence of a T(SH)(2) synthetase activity in Entamoeba histolytica means that this protozoan has a similar metabolism to that of the trypanosomatids and opens the possibility of establishing a rational drug design against this human parasite.

Amide Synthases↗

Entamoeba histolytica: a eukaryote with trypanothione metabolism instead of glutathione metabolism.

Entamoeba histolytica is a human pathogen that lacks the capacity to synthesize glutathione but can incorporate it, from the growth media or presumably from the human host, to form trypanothione [N(1), N(8)-bis(glutathionyl)-spermidine conjugate]. This novel thiol compound has previously been found in trypanosomatids, as has its precursor glutathionyl-spermidine, which was originally detected in Escherichia coli. Previously we showed the presence of these two thiol compounds in extracts from cultures of Entamoeba histolytica HK9. Here we report that when Entamoeba histolytica HK9 is grown in a culture medium that lacks glutathione (treated with the enzyme gamma-glutamyl transpeptidase), trypanothione is not formed, although the trophozoites can continue dividing for at least 60 h but at 25% lower cell density. The finding of a trypanothione metabolism in Entamoeba histolytica raises many questions: one concerns the possibility of a phylogenetic relationship, in this respect, with trypanosomatids such as Trypanosoma cruzi, T. brucei and Crithidia fasciculata; another concerns its role in cell metabolism; a third concerns it possible use as a target for a rational drug design strategy against this parasite.

Animals↗

Brindley's gland exocrine products of Triatoma infestans.

Volatile exocrine products of the metathoracic Brindley's glands in Triatoma infestans (Hemiptera: Reduviidae) were obtained by dissection and by sampling the air passed over agitated live adults. Isobutyric acid was the main component in the glands, together with isobutyl, isoamyl and amyl alcohols, 2-phenylethanol and other carboxylic acids and esters. Isobutyric acid, isobutyl, isoamyl and amyl alcohols and ester were also found to be emitted into the air, apparently for defence. No volatile products were detected in the metasternal glands.

Animals↗

Enzyme regulation by biological disulfides.

More than a dozen enzymes have been found to be activated or inhibited in vitro by disulfide-exchange between the protein and small-molecule disulfides. Accordingly, thiol/disulfide ratio changes in vivo may be of great importance in the regulation of cellular metabolism. An awareness of this regulatory mechanism in both host cells and parasites, coupled with information on the presence or absence of key enzymes, may lead to rational drug design against certain diseases involving thiol intermediates, including trypanosomiasis.

Animals↗

Sylvatic focus of Chagas' disease in Oaxaca, Mexico.

During three field trips to the community of 'La Humedad', municipality of Santiago Jamiltepec, Oaxaca (a State in the southern part of the Mexican Republic), live specimens of Triatoma mazzottii Usinger (Hemiptera: Reduviidae) were found infected with Trypanosoma cruzi. The insects were captured in only one of the dwellings inspected in the locality, and in four small caves where bats and other mammals live. These findings suggest the need for further studies on the existence of a sylvatic cycle of Chagas' disease in Mexico, which has not yet been established.

Animals↗

Purification, properties, and oligomeric structure of glutathione reductase from the cyanobacterium Spirulina maxima.

Glutathione reductase [NAD(P)H:GSSG oxidoreductase EC 1.6.4.2] from cyanobacterium Spirulina maxima was purified 1300-fold to homogeneity by a simple three-step procedure involving ammonium sulfate fractionation, ion exchange chromatography on DEAE-cellulose, and affinity chromatography on 2',5'-ADP-Sepharose 4B. Optimum pH was 7.0 and enzymatic activity was notably increased when the phosphate ion concentration was increased. The enzyme gave an absorption spectrum that was typical for a flavoprotein in that it had three peaks with maximal absorbance at 271, 370, and 460 nm and a E1%271 of 23.3 Km values were 120 +/- 12 microM and 3.5 +/- 0.9 microM for GSSG and NADPH, respectively. Mixed disulfide of CoA and GSH was also reduced by the enzyme under assay conditions, but the enzyme had a very low affinity (Km 3.3 mM) for this substrate. The enzyme was specific for NADPH. The isoelectric point of the native enzyme at 4 degrees C was 4.35 and the amino acid composition was very similar to that previously reported from other sources. The molecular weight of a subunit under denaturing conditions was 47,000 +/- 1200. Analyses of pure enzyme by a variety of techniques for molecular weight determination revealed that, at pH 7.0, the enzyme existed predominantly as a tetrameric species in equilibrium with a minor dimer fraction. Dissociation into dimers was achieved at alkaline pH (9.5) or in 6 M urea. However, the equilibrium at neutral pH was not altered by NADPH or by disulfide reducing reagents. The Mr and S20,w of the oligomeric enzyme were estimated to be 177,000 +/- 14,000 and 8.49 +/- 0.5; for the dimer, 99,800 +/- 7000 and 5.96 +/- 0.4, respectively. Low concentrations of urea increased the enzymatic activity, but this increase was not due to changes in the proportions of both forms.

Amino Acids↗

Glutathione reductase in evolution.

The disulfide reducing activities of GSSG-and CoASSG-reductases were measured on partially purified extracts from a variety of prokaryotes and eukaryotes. Glutathione-reductase was found in varying amounts in all eukaryotes and prokaryotes, used in this study, with the exception of the two strict anaerobes Clostridium tartarivorum and Desulfovibrio vulgaris, and the two primitive Archaebacteria Methanosarcina barkeri and Halobacterium halobium. CoASSG-reductase was found in some eukaryotes and prokaryotes, but showed no clear pattern of distribution other than its absence whenever GSSG-reductase was not present. The absence of GSSG-reductase activity in organisms lacking GSH, confirms that glutathione metabolism is not universal and suggests that this enzyme might be useful as a marker in classifying organisms. The data suggest that glutathione-reductase occurs as a result of the change from a reducing to a oxidizing atmosphere in the primitive Earth.

Aerobiosis↗