Raúl N. Ondarza

Brindley's gland exocrine products of Triatoma infestans.

olatile exocrine products of the metathoracic Brindleys 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 esters were also found to be emitted into the air, apparently for defence. No volatile products were detected in the metasternal glands.

Enzyme Regulation by Biological Disulfides

More than a dozen enzymes have been found to be activated or inhibitedin vitro by disulfide-exchange between the protein and small-molecule disulfides. Accordingly, thiol/disulfide ratio changesin 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.

Characterization of a nucleotide-peptide from rat liver

A nucleotide-peptide has been isolated from trichloroacetic acid and HClO4 extracts obtained from rat liver. After purification by paper chromatography and paper ionophoresis, structural and quantitative features of this nucleotide-peptide were determined. 1. 1. It is an adenine ribonucleotide-peptide containing stoichiometric amounts of cysteine, glutamic acid, glycine, taurine and β-alanine. Glutamic acid is the amino terminal amino acid of the peptide. 2. 2. The ratio adenine/ribose/total phosphorus is of 1:1:3. Two of the phosphates have been located, one is in the 3′ position and the other in the 5t of the ribose of the nucleotide. 3. 3. Performic acid oxidation splits the compound into two components, one containing the taurine and β-alanine, the other cysteic acid, glycine and glutamic acid.

On the active site of the NADPH-dependent CoA-SS-Glutathione reductase from yeast and rat liver

We have described a new NADPHdependent CoASSG*-reductase from rat liver and yeast [ 1,2] . Since this enzyme catalyzes a reaction similar to those of lipoamide dehydrogenase (EC 1.6.4.3), GSSG*reductase (EC 1.6.4.2) and thioredoxin reductase, which are well-known pyridine nucleotide-dependent flavoproteins [3-51, we decided to see if the new re ductase contains an active site with the particular characteristics of this group of enzymes. Our studies with arsenite and 5-nitrofuran derivatives, utilised as inhibitors, indicate that the CoASSG reductase contains a disulfide bridge in its active site and suggest the presence of FAD as prosthetic group.

Characterization of a NADPH-dependent coencyme A-SS-glutathione reductase from yeast

Abstract A new NADPH-dependent CoA-SS-glutathione reductase from yeast cells, as from commercially purified yeast GSSG reductase, has been established. According to the data obtained from polyacrylamide column gel electrophoresis and isoelectric-focusing technique, the yeast GSSG reductas (EC 1.6.4.2) can be separated from the new enzymic activity. This enzyme has the following characteristics: (1) pH optimum = 5.5; (2) K m = 20·10 −5 M; (3) mol. wt. = 108·103; (4) produces GSH and CoASH from CoASSG as substrate in the presence of NADPH and does not require GSH; (5) catalyzes an irreversible reaction; (6) does not reduce CoASSCys, GSSCys, or CysSSCys; (7) is partially inhibited at 25 mM phosphate ion concentration.

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 [N1,N8‐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 γ‐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.

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 μM after 24 h of culture under aerobic conditions, caused a substantial decrease in the number of E. histolytica HK9 trophozoites, from approx. 15×106 to 5·37×106 cells, and at 100 μM to 0·8×106 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.

CoAS-Sglutathione and GSSG reductases from rat liver: Two disulfide oxidoreductase activities in one protein entity

Abstract The characteristics of the NADPH-dependent CoAS-Sglutathione reductase from rat liver have been studied. In contrast to the enzyme obtained from yeast during the purification procedure using different types of column chromatography and isoelectric-focusing technique, the new reducing enzyme in rat liver was found to purify together with GSSG reductase. The data indicate that in liver both enzymatic activities belong to the same protein entity, since they cannot be separated by any of the above regular physical methods. The CoAS-Sglutathione and GSSG reducing activities have a molecular weight of 42 500; a pI of 6.85 and do not reduce cystine, cystamine, pantethine or insulin. On the other hand, an NADPH-dependent non-specific disulfide reductase also present in rat liver extracts can be easily separated and distinguished from the GSSG and CoAS-Sglutathione-reducing enzymes by ion-exchange column chromatography and isoelectric-focusing.

Thiol compounds from a free-living pathogenic opportunistic amoeba, Acanthamoeba polyphaga

New bimane‐reacting compounds from perchloric acid extracts have been detected by HPLC from Acanthamoeba polyphaga. The main compounds detected are cysteine, glutathione and other novel thiol compounds. All of these compounds must be thiols, since they disappear or decrease substantially when treated by N‐ethylmaleimide prior to acetonitrile/bimane derivatization. Cysteine and glutathione increase in quantity when dithiothreitol reduction is applied to the fresh extract. This means that they are likely to be present in their oxidized and reduced form and indicates the possible presence of a corresponding thiol/disulphide enzymic system. There are other compounds that have a different behaviour, since although they can react with bimane, they do not disappear if treated previously by N‐ethylmaleimide. This shows that they are not thiols but can react with bimane. The main thiol compounds found to be present, in both the parasite and the host lymphocyte cells, were cysteine and glutathione. We were unable to detect ovothiol A in Acanthamoeba but instead we found another thiol compound that could be structurally related to trypanothione. The new thiol compounds unique to this parasite and not present in lymphocytes will permit the study of disulphide‐reducing enzymes as potential drug targets.

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, N1,N8‐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 Mg2+ 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.