Nelia M. Gerez de Burgos

Arginine kinase of the flagellate protozoa Trypanosoma cruzi: Regulation of its expression and catalytic activity

In epimastigotes of Trypanosoma cruzi, the etiological agent of Chagas’ disease, arginine kinase activity increased continuously during the exponential phase of growth. A correlation between growth rate, enzyme‐specific activity and enzyme protein was observed. Arginine kinase‐specific activity, expressed as a function of enzyme protein, remains roughly constant up to 18 days of culture. In the whole range of the culture time mRNA levels showed minor changes indicating that the enzyme activity is post‐transcriptionally regulated. Arginine kinase could be proposed as a modulator of energetic reserves under starvation stress condition.

Inhibition by gossypol of oxidoreductases from Trypanosoma cruzi

The effects of gossypol, a polyphenolic compound isolated from the cotton plant upon six oxidoreductases from cultured epimastigotes of Typanosoma cruzi were studied. Gossypol was a powerful inhibitor of the alpha-hydroxyacid and malate dehydrogenases, NAD-linked enzymes, and of glutamate dehydrogenase, malic enzyme and glucose-6-phosphate dehydrogenase, NADP-dependent enzymes. The drug did not have an effect on succinate dehydrogenase, a flavoprotein. The Ki values with respect to substrate were 0.73, 0.3 and 3.5 microM for alpha-hydroxyacid, malate and glutamate dehydrogenases, respectively, and 1.1, 0.19 and 7.8 microM with respect to the coenzyme. Inhibition was noncompetitive with respect to substrate and uncompetitive in relation to the coenzyme.

The sexual cycle of male bats: Changes of testicular lactate dehydrogenase isoenzymes

Abstract 1. The sexual activity of bats of the speciesTadarida brasiliensis takes place once a year, at the end of winter. 2. Histological sections of testis from these bats showed spermatogenic activity only during the winter months. 3. Starch gel electrophoretic patterns of testis extracts revealed the apperance of additional lactate dehydrogenase isoenzymes when spermatogenic activity was initiated. 4. The cyclic synthesis of peculiar isoenzymatic forms affords a very useful index to study the factors regulating gene activity.

Properties of α-hydroxyacid dehydrogenase isozymes from Trypanosoma cruzi

Whole cell extracts of culture epimastigotes of Trypanosoma cruzi (Tulahuen strain) have α-hydroxyacid dehydrogenase activity which catalyzes the NAD-linked reaction α-ketoacid ⇋ α-hydroxyacid, with a variety of substrates. Two molecular forms of the enzyme have been separated by means of gel electrophoresis. These isozymes were partially purified by DEAE-cellulose chromatography and ammonium sulfate precipitation. Molecular weights were estimated and some catalytic properties were determined with purified isozymes. The faster migrating fraction (isozyme I) has a molecular weight of 85 500 and showed significant activity against linear 3–5 carbon chain substrates. The lowest Km value was obtained for pyruvate. Isozyme II (MW 60 500) utilizes linear and branched chain substrates with 4–6 carbon atoms. Its highest activity and lowest Km value were recorded with α-keto-isocaproate as substrate.

Testicular Lactate Dehydrogenase Isozyme: Cyclic Appearance in Bats

In male bats Tadarida brasiliensis spermatogenesis occurs only during winter and spring. Two additional lactate dehydrogenase isozymes appear in testes containing mature spermatozoa. This type of isozymic change implies cyclic activation of genes and affords valuable markers for study of the factors involved.

Trypanosoma cruzi: cruzipain and membrane-bound cysteine proteinase isoform(s) interacts with human α2-macroglobulin and pregnancy zone protein

Plasmatic levels of pregnancy zone protein (PZP) increase in children with acute Chagas disease. PZP, as well as alpha2-macroglobulin (alpha2-M), are able to interact with Trypanosoma cruzi proteinases. The interaction of alpha2-M and PZP with cruzipain, the major cysteine proteinase of T. cruzi, was investigated. Several molecular changes on both alpha-M inhibitors under reaction with cruzipain were found. PAGE analysis showed: (i) formation of complexes of intermediate mobility and tetramerization of native alpha2-M and PZP, respectively; (ii) limited proteolysis of bait region in alpha2-M and PZP, and (iii) covalent binding of cruzipain to PZP and alpha2-M. Conformational and structural changes experimented by alpha-Ms correlate with modifications of the enzyme electrophoretic mobility and activity. Cruzipain-alpha-M complexes were also detected by gelatin SDS-PAGE and immunoblotting using polyclonal anti-cruzipain antibodies. Concomitantly, alpha2-M and PZP impaired the activity of cruzipain towards Bz-Pro-Phe-Arg-pNA substrate. In addition, alpha-Ms were able to form covalent complexes with membrane isoforms of cysteine proteinases cross-reacting with cruzipain. The present study suggests that both human alpha-macroglobulin inhibitors could prevent or minimize harmful action of cruzipain on hosts molecules and hypothetically regulate parasite functions controlled by cruzipain.

Presence of a Fatty Acid-Binding Protein and Lipid Stores in Flight Muscles of Dipetalogaster maximus (Hemiptera: Reduviidae)

Abstract A fatty acid-binding protein (FABP) from the cytosolic fraction of the triatomine Dipetalogaster maximus (Uhler) flight muscles was purified by a procedure based on gel filtration, reverse-phase high performance liquid chromatography, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein has an apparent molecular mass of 14 kDa, and its N-terminus is unblocked. Its N-terminal sequence was obtained by submitting an SDS-PAGE band blotted onto a polyvinylidene difluoride membrane to Edman degradation. The sequence obtained indicates that this FABP belongs to the heart type. This is the first time that a fatty acid-binding protein has been reported for a triatomine. The presence of said FABP, abundant mitochondria, and lipid stores in the flight muscles of D. maximus suggests that beta oxidation of fatty acids is used by the triatomine thoracic muscle as an energy source, and could be related to its dispersal capacity.

Gene discovery in Triatoma infestans

BackgroundTriatoma infestans is the most relevant vector of Chagas disease in the southern cone of South America. Since its genome has not yet been studied, sequencing of Expressed Sequence Tags (ESTs) is one of the most powerful tools for efficiently identifying large numbers of expressed genes in this insect vector.ResultsIn this work, we generated 826 ESTs, resulting in an increase of 47% in the number of ESTs available for T. infestans. These ESTs were assembled in 471 unique sequences, 151 of which represent 136 new genes for the Reduviidae family.ConclusionsAmong the putative new genes for the Reduviidae family, we identified and described an interesting subset of genes involved in development and reproduction, which constitute potential targets for insecticide development.

GM1 ganglioside enhances the rewarding properties of cocaine in rats

GM1 pretreatment enhanced the rewarding properties of cocaine as assessed in the conditioned place preference paradigm. This effect was shown by the lower dosage of cocaine necessary to induce conditioning compared with rats receiving cocaine alone, as well as by the fewer number of sessions necessary to induce place preference. GM1 pretreatment did not modify the plasma level of cocaine, but it induced a significant increase in the brain cocaine level compared with animals receiving cocaine alone. In order to evaluate the possibility that GM1 pretreatment may alter the pharmacokinetic parameters of cocaine, the brain and plasma esterase activities, the plasma bound/free cocaine ratio and the brain blood barrier permeability to i.v. Evans Blue administration were assessed. None of these parameters was modified by the GM1 administration. In addition, GM1 (100microM) did not alter the dopamine transporter inhibition induced by cocaine (10(-7)-10(-5)M), as determined by the uptake of [(3-)H]-dopamine in the microsacs of nucleus accumbens. In conclusion, GM1 pretreatment, which did not have any effect per se, increased the rewarding effect of cocaine, a phenomenon correlated with a significant increase in the brain cocaine levels. The different pharmacokinetic parameters evaluated, as well as the inhibitory effect of cocaine on the dopamine transporter, were not modified by GM1, but it modifies the brain cocaine disposition. Thus, the mechanisms by which GM1 enhanced the rewarding effects of cocaine merit further study.

Effects of Temperature and pH on Hexokinase from the Flight Muscles of Dipetalogaster maximus (Hemiptera: Reduviidae)

Abstract Effects of temperature and pH on the catalytic properties of hexokinase (HK, EC 2.7.1.1) from the flight muscles of Dipetalogaster maximus (Uhler) were studied. The enzyme showed a hyperbolic behavior with its two substrates (glucose and ATP). There was no inhibition by glucose. Apparent Km and Vmax increased as pH increased from 7.0 to 8.5. Catalytic efficiency was lowest at pH 7.0. Km, Vmax, and catalytic efficiency were higher at 37°C than at 30 and 20°C. There was marked inhibition by ATP, which was dependent on pH and temperature. Km values for ATP were reduced and catalytic efficiency increased as pH increased. Lowest Vmax was observed at pH 7.0. At this pH there was 87.3% inhibition by ATP, whereas it was only 5.7% at pH 8.5 (at 30°C). Km, Vmax, and catalytic efficiency were higher at 37°C than at 30 and 20°C. The strong inhibition by ATP detected at 20°C (pH 7.6) almost disappeared at 37°C. Therefore, temperature could regulate hexokinase activity by modulating the inhibition produced by ATP. Glucose utilization and ATP production would be promoted when temperature rises from 30 to 37°C. Because insect thoracic muscles increase their temperature over 30°C during flight, this phenomenon elucidates a mechanism enhancing energy supply for muscle activity.