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A Morello

Publications and source records attributed to A Morello.

At least 37 records · Page 2Linked to original sources

Purification and preliminary sequencing of Tc-45, an immunodominant Trypanosoma cruzi antigen: absence of homology with cruzipain, cruzain, and a 46-kilodalton protein.

In spite of being separated by more than 20 million years of evolution, the murine and human immune systems share extensive similarities. Thus, experimental results obtained with the murine model may have predictive value for human Chagas' disease. Challenge of the H-2 congenic mouse stains A.SW (H-2s) and A.CA (H-2f) with Trypanosoma cruzi yields different results. The A.CA animals die approximately 12 days postinfection, while A.SW mice survive indefinitely. A 45-kD protein (Tc45), an antigen differentially recognized by the A.SW strain, is present in cultured epimastigotes and blood trypomastigotes. We describe here its purification from epimastigotes. The presence of Tc45 was monitored and a single band was detected. Since the molecular weights of Tc45, cruzipain, cruzain, and a 46-kD parasite polypeptide are similar, it was important to determine if these molecules are related. A complete lack of homology was observed when the sequence of cruzain, cruzipain, and the 46-kD polypeptide were compared with the preliminary sequence of Tc45.

Amino Acid Sequence↗

Trypanocidal effect of boldine and related alkaloids upon several strains of Trypanosoma cruzi.

The alkaloids boldine, glaucine, predicentrine, apomorphine, coclaurine, norarmepavine and codeine were tested against the epimastigotes of the Tulahuén and LQ strains and the DM 28c clone of Trypanosoma cruzi. The micromolar concentration to inhibit 50% of the culture growth (Tulahuén strain) for apomorphine, glaucine, predicentrine, boldine, norarmepavine, coclaurine and codeine were 29, 90, 85, 110, 310, 580 and > 1000 respectively. Similar values were obtained with the LQ strain and the DM 28c clone. The most active compounds in inhibiting culture growth also inhibited cell respiration, suggesting that these drugs may act by blocking mitochondrial electron transport. The trypanocidal effects of these alkaloids appear to be correlated with their antioxidative activities.

Alkaloids↗

Effects of 2(3)-tert-butyl-4-hydroxyanisole (BHA) on in situ mitochondria of Trypanosoma cruzi.

Results obtained with in situ mitochondria of Trypanosoma cruzi showed that this protozoon had only two energy coupling sites, sites II and III that correspond to higher eukaryote mitochondria. Rotenone did not inhibit the oxygen uptake of the parasite. These results suggest that the NADH-ubiquinone segment of the respiratory chain has no activity. Studies with in situ mitochondria confirmed that BHA, an antioxidant food additive, blocks the mitochondrial electron transport chain at the succinate-cytochrome b segment being the molecular basis of this trypanocidal action.

Adenosine Diphosphate↗

Effects of hydroquinones on intact Trypanosoma cruzi epimastigotes.

1. Hydroquinones inhibited the culture growth of Trypanosoma cruzi epimastigotes at concentrations lower than 1 mM. 2. Hydroquinones inhibited the oxygen consumption on the intact Trypanosoma cruzi cells. I50 values for hydroquinone, terbutylhydroquinone and 2,5-di-t-butylhydroquinone were 24.87 mM, 0.88 mM and 0.26 mM, respectively. t-Butylhydroquinone and 2,5-di-t-butylhydroquinone had a Michaelian type kinetic inhibition; hydroquinone also showed a Michaelian type kinetic inhibition at low concentrations but at higher concentrations it showed a positive cooperativity. 3. These hydroquinones changed the NAD(P) redox-state to a more reduced state and that of cytochrome b to a more oxidized state. The magnitude of the redox-state change was dependent of the hydrophobicity of the derivates. 4. These results suggest that the growth and oxygen uptake inhibition by the hydroquinones is due to a blockage of the mitochondrial electron transport chain before cytochrome b.

Animals↗

A chemiluminescent immunoassay for urinary thromboxane B2.

Because of the vasoactive properties of thromboxane A2 and other related prostaglandins, much research has been conducted on drugs which alter their levels. Urinary levels of thromboxane B2 and 2,3-dinor thromboxane B2 (major urinary metabolite of thromboxane B2) are used as an indication of thromboxane production in-vivo. In order to accurately measure urinary TXB2 levels of subjects on investigative drugs which lower TXA2 and subsequently TXB2, a simple and sensitive analytical tool becomes necessary. We have thus developed a non-radioisotopic (chemiluminescent) assay for urinary TXB2. Sensitivity has been demonstrated to 5 pg/ml. The method correlates well with gas chromatography/mass spectrometry (the accepted reference method) even without column chromatographic purification prior to the conduct of the chemiluminescent assay (r = 0.96). In addition, we have demonstrated feasibility for a chemiluminescent assay to measure urinary 2,3-dinor TXB2.

Acridines↗

An immunogenetically defined and immunodominant Trypanosoma cruzi antigen.

Two strains of mice, A. SW (H-2s) and A.CA (H-2f), were immunized with live trypomastigotes or epimastigotes of the Tulahuen strain of Trypanosoma cruzi or with their sonicates. By immunowestern blotting, sera from A.SW mice, but not from A.CA, recognized, in an immunodominant fashion, a 45 kDal polypeptide (Tc45) present in both epimastigotes and trypomastigotes. Since A.SW and A.CA strains are congenic for the major histocompatibility H-2 complex, recognition of Tc45 seems to be controlled by this genetic region or by gene(s) located in its immediate vicinity. Subcellular fractionation revealed that Tc45 is mainly present at the cytoplasmic compartment.

Animals↗

Trypanosoma cruzi: a possible control of transfusion-induced Chagas' disease by phenolic antioxidants.

The following phenolic antioxidant food additives were evaluated against Trypanosoma cruzi epimastigotes: BHT, BHA, gallic acid and its methyl, propyl, octyl, and lauryl esters, 2,4-di-tert-butyl-6-(4-methoxybenzyl)-phenol, 4,4'-isopropilidenediphenol, and protocatechuic acid and its ethyl ester. The inhibition of the respiration; the changes in motility, shape, and lysis of the parasites; and the human blood hemolysis caused by these chemicals were studied. Human blood samples experimentally contaminated with 2000 or 150,000 trypomastigotes per milliliter were freed of parasites after treatment for 24 hr at 4 degrees C with 5 or 10 mM BHT (2,6-di-tert-butyl-4-hydroxytoluene), respectively. Consequently, BHT and other phenolic compounds deserve further study to determine their role in preventing the transmission of Chagas' disease by blood transfusion.

Animals↗

Role of glutathione in the susceptibility of Trypanosoma cruzi to drugs.

1. Glutathione (G-SH) concentration, gamma-glutamyltranspeptidase and glutathione S-transferase activities were studied in several strains of T. cruzi epimastigotes. GSH varied from 1.04 mM for the LQ strain to 0.61 mM for the Tulahuen strain. 2. Cultures of the LQ strain presented more resistance to drugs than those of the Tulahuen. It was necessary a concentration of nifurtimox 4 times higher and one of benznidazole 10 times higher in order to inhibit approximately to 50% the growth of LQ strain cultures when compared with the Tulahuen strain. 3. Buthionine sulfoximine decreased the concentration of glutathione to about 50% in the LQ and Tulahuen strains and potentiated the toxicity of nifurtimox and benznidazole in T. cruzi epimastigote cultures. These results suggest that glutathione is an important factor in the resistance of T. cruzi to nifurtimox and benznidazole.

Animals↗

Effects of t-butyl-4-hydroxyanisole and other phenolic antioxidants on tumoral cells and Trypanosoma parasites.

The antioxidant food additives 2(3)-tert-butyl-4-hydroxyanisole (BHA), 2,6-di(tert-butyl)-p-cresol (BHT) and the methyl and propyl esters of gallic acid inhibited Trypanosoma cruzi culture growth and oxygen consumption. The I50 values for growth and oxygen uptake with BHA were 0.284 and 0.400 and for BHT 0.083 and 0.235 mM, respectively. Moreover, BHA inhibited the respiration of several tumor cells, as well as of the procyclic and bloodstream trypomastigote forms of T. brucei brucei, with I50 in the range 0.29-0.52 mM. Inhibition of the parasites' oxygen uptake by BHA was not of the pure Michaelis-Menten type, but may be of a mixed form. It is postulated that these compounds are inhibitors because they resemble ubiquinone.

Animals↗

The carboxylesterases of Trypanosoma cruzi epimastigotes.

Carboxylesterase activity in Trypanosoma cruzi was found mainly in the microsomal (40%) and the cytosolic fraction (26%). The Vmax for p-nitrophenyl acetate was 28.50 and 17.60 nmol per min and mg of protein for the microsomal and the cytosolic fractions, respectively. The Km was 0.78 mM for the microsomal activity and 0.55 mM for the cytosolic activity. The inhibition rate constant with N-ethylmaleimide were 38.10 M-1 min-1 and 2.56 M-1 min-1 for the cytosolic and the microsomal enzymes, respectively. The rate constants with Paraoxon were 8,360 M-1 X min-1 and 32,600 M-1 X min-1. Polyacrylamide gel electrophoresis under nondenaturing conditions showed three bands of microsomal activity with M.W. of 63, 136 and 153 kDA. Similarly, three bands of cytosolic activity with M.W. of 126, 294 and 479 kDA were identified.

Animals↗

The gamma-glutamyltranspeptidase of Trypanosoma cruzi.

Trypanosoma cruzi epimastigotes show gamma-glutamyltranspeptidase activity which has characteristics significantly different than the mammalian enzyme. The protozoan enzyme is localized in the cytosolic fraction, it has a Km of 1.6 mM and a Vmax of 17.4 nmol/min/mg protein with L-gamma-glutamyl-p-nitroanilide as gamma-glutamyl donor, and an optimun pH range from 7.5 to 8.0. The best amino acid acceptors were L-histidine, L-asparagine, L-aspartate, L-glutamate and L-proline, but L-glutamine was a very poor acceptor. The enzyme was very sensitive to inhibition by 6-diazo-5-oxo-L-norleucine (k2 = 4.0 X 10(5)/M per min) and O-diazo-acetyl-L-serine (k2 = 1.1 X 10(4)/M per min). Phenobarbital (k2 = 8.38/M per min) and L-serine borate (Ki = 34 mM) were poor inhibitors. The activity of the enzyme was not correlated with the logarithmic phase of growth of the parasites and steadily decreases with the age of the cultures.

Chagas Disease↗

t-Butyl-4-hydroxyanisole, a novel respiratory chain inhibitor. Effects on Trypanosoma cruzi epimastigotes.

t-Butyl-4-hydroxyanisole, an antioxidant food additive, inhibited the growth of Trypanosoma cruzi by almost 100% at 0.5 mM concentration. This compound inhibited 70% of oxygen consumption of epimastigotes. The redox level of NAD(P) was shifted to a more reduced state and inversely the redox level of cytochrome b changed to a more oxidized state. This hydroxyanisole thus is a new electron transport chain inhibitor. This compound and related ones, or the respiratory chain of T. cruzi, may be important in the design of antichagasic drugs.

Animals↗

Phosphatase activity in Trypanosoma cruzi. Phosphate removal from ATP, phosphorylated proteins and other phosphate compounds.

T. cruzi epimastigotes have a lysosomal acid phosphatase (pH 4.0) and acid and alkaline phosphatases (pH 5.5 and 8.0) localized in the cytosolic fraction. The levels of the lysosomal acid phosphatase increase with the age of the cultures, but the cytosolic phosphatases decline after the logarithmic phase of growth. The lysosomal phosphatase preferentially hydrolyses low mol. wt phosphate esters; whereas, the cytosolic alkaline phosphatases primarily act on phosphorylated proteins, and both the cytosolic acid and alkaline phosphatases on uridine nucleotide derivatives. The parasite also contains a microsomal glucose 6-phosphatase, and ATPases (Mg2+ and Ca2+-activated) derived from plasma membranes and mitochondria.

Acid Phosphatase↗

Primate model for the study of hepatic metabolism of lipoprotein cholesterol.

We describe a primate model for the study of net cholesterol flux by splanchnic, portal, and hepatic regions concomitant with that of biliary lipid secretion in conscious healthy fed or fasting baboons with exteriorized but physiologically intact enterohepatic circulations. In five fasting baboons studied, we found evidence for low-density lipoprotein (LDL) cholesterol uptake by the liver of 1.0 mg/min, P less than 0.05. Conversely, splanchnic production of LDL cholesterol was found in the fed state (n = 5), amounting to 0.8 mg/min, P less than 0.05. Uptake or secretion of high-density lipoprotein (HDL) cholesterol by either the liver or the intestine could not be detected by transgradient analysis in our animals in either fed or fasting states. Very low-density lipoprotein secretion of 0.4 mg/min by the liver was also found in the fed state. Isotopic biliary cholesterol derived from labeled plasma HDL or LDL cholesterol reflected hepatic LDL cholesterol production in the fed state but LDL cholesterol uptake in the fasting state. Biliary cholesterol secretion amounted to 8.5% of net hepatic lipoprotein cholesterol flux in the fed state and 10% in the fasting state. We conclude that uptake of LDL cholesterol by the liver is appreciable and contributes to biliary sterol secretion in the fasting baboon. Uptake or secretion of HDL cholesterol was not detected in any metabolic beds by transgradient analysis, but in tracer studies appeared to be a major source of lipoprotein cholesterol transferred to bile in the fed state.

Animals↗

Acid and alkaline phosphatase activity in Trypanosoma cruzi epimastigotes.

Phosphatase activity in intact Trypanosoma cruzi epimastigotes has been demonstrated. After subcellular fractionation three activities were characterized: (a) a membrane-bound microsomal acid activity with an optimum pH of 4.0 and a Km of 1.2 mM, strongly inhibited by tartrate and fluoride; (b) a soluble cytosolic acid activity with an optimum pH of 5.5 and a Km of 0.95 mM, strongly inhibited by p-hydroxymercuribenzoate, EDTA and copper ions and activated by cyanide, manganese and magnesium ions; and (c) a soluble cytosolic alkaline activity with an optimum pH of 8.0 and a Km of 3.8 mM, inhibited by p-hydroxymercuribenzoate, fluoride, EDTA, and copper, calcium and zinc ions. This activity was increased by magnesium and manganese ions.

Acid Phosphatase↗

Inhibitory effect of acetylcholine on muscular tonus of the small intestine of a lizard.

The present report concerns the effects of cholinergic agonists on the isometric tension of "in vitro" preparations of the esophagus and distal part of the small intestine of the lizard Liolaemus gravenhorsti. Acetylcholine (Ach) and carbachol elicited a dose-related increase of isometric tension in the esophagus, whereas the small intestine was slowly relaxed by these drugs. Eserine induced a synergistic action on the cholinergic responses of both organs, and atropine completely antagonized the respective effects. The esophagus and the intestine showed different thresholds for the cholinergic evoked responses, the former being about 30 times more sensitive than the intestine. Assessment of cholinesterase activity revealed that Ach is hydrolyzed at a significant lesser speed in the intestine than in the esophagus. The results do not provide information about the nature of the chemical mediator causing the inhibitory effect observed in the intestine. The eventual role of an adrenergic mechanism mediated by muscarinic receptors is under study in our laboratory.

Acetylcholine↗