Human cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis in Santiago del Estero, Argentina: identification of parasites by monoclonal antibodies and isoenzymes

Diagnostic and parasite characterization and identification studies were carried out in human patients with cutaneous leishmaniasis lesions in Santiago del Estero, Northern Province of Argentina. Diagnostic procedures were biopsies of lesions for smears and inoculations in hamster, needle aspirations of material from ulcers for "in vitro" cultures. Immunodiagnostic techniques applied were IFAT-IgG and Montenegro skin test. Primary isolation of eight stocks of leishmanial parasites was achieved from patients with active lesions. All stocks were biologically characterized by their behaviour in hamster, measurements of amastigote and promastigotes and growth "in vitro". Eight stocks were characterized and identified at species level by their reactivity to a cross-panel of sub-genus and specie-specific Monoclonal Antibodies through an Indirect Immunofluorescence technique and a Dot-ELISA. We conclude from the serodeme analysis of Argentina stocks that: stocks MHOM/AR/92/SE-1; SE-2; SE-4; SE-8; SE-8-I; SE-30; SE-34 and SE-36 are Leishmania (Viannia) braziliensis. Three Leishmania stocks (SE-1; SE-2 and SE-30) did not react with one highly specie-specific Monoclonal Antibody (Clone: B-18, Leishmania (Viannia) braziliensis marker) disclosing two serodeme group patterns. Five out of eight soluble extracts of leishmanial promastigotes were electrophoresed on thin-layer starch gels and examined for the enzyme MPI, Mannose Phosphate Isomerase; MDH, Malate Dehydrogenase; 6PGD, 6 Phosphogluconate Dehydrogenase; NH, Nucleoside Hydrolase, 2-deoxyinosinc as substrate; SOD, Superoxide Dismutase; GPI, Glucose Phosphate Isomerase and ES, Esterase. From the isoenzyme studies we concluded that stocks: MHOM/AR/92/SE-1; SE-2; SE-4; SE-8 and SE-8-I are isoenzymatically Leishmania (Viannia) braziliensis. We need to analyze more enzymes before assigning them to a braziliensis zymodeme.

Giardia duodenalis: INTER-STRAIN VARIABILITY OF PROTEINS, ANTIGENS, PROTEASES, ISOENZYMES AND NUCLEIC ACIDS

Giardia duodenalis isolates from asymptomatic or symptomatic patients and from animals present similarities and differences in the protein composition, antigenic profile, pattern of proteases and isoenzymes, as well as in nucleic acids analysis. In the present overview, these differences and similarities are reviewed with emphasis in the host-parasite interplay and possible mechanisms of virulence of the protozoon.

Isoenzymes Detect Variation in Populations of Triatoma brasiliensis (Hemiptera: Reduviidae: Triatominae)

Triatoma brasiliensis is one of the most important vectors of Chagas disease in the semiarid zone of the northeast of Brazil. Intraspecific morphological and behavioural variation has been reported for different populations. Results for four distinct populations using eight isoenzymes are reported here. The literature describes three subspecies: T. brasiliensis brasiliensis Neiva, 1911; T. brasiliensis melanica Neiva & Lent, 1941 and T. brasiliensis macromelasoma Galvão, 1956. These subspecies differ mainly in their cuticle colour pattern and were regarded as synonyms by Lent and Wygodzinsky (1979). In order to evaluate whether the chromatic pattern is a morphological variation of different melanic forms within T. brasiliensis or due to interspecific variation, field collections were performed in localities where these three subspecies have been described: Caicó (Rio Grande do Norte), the type-locality for T. b. brasiliensis; Petrolina (Pernambuco) for T. b. macromelasoma and Espinosa (Minas Gerais) for T. b. melanica. A fourth distinct chromatic pattern was found in Juazeiro (Bahia). A total of nine loci were studied. Values of Nei's genetic distance (D) were calculated. T. b. brasiliensis and T. b. macromelasoma are the closest populations with a D=0.295. T. b. melanica had a D ³ 0.537 when compared to the others, a distance in the range of interspecific variation for other triatomine species

Characterization of Rhodnius neglectus from Two Regions of Brazil Using Isoenzymes, Genitalia Morphology and Morphometry

Among the triatomines considered as secondary in the epidemiology of Chagas disease, Rhodnius neglectus is frequently captured in artificial ecotopes, especially peridomiciliary ones, rarely producing colonies indoors. Nevertheless, the presence of breeding colonies in houses was unquestionably demonstrated in some areas of the State of Goiás, Brazil. Previous isoenzyme comparisons of this species with morphologically close triatomines, such as R. prolixus, R. robustus or R. nasutus, did not produce definitive conclusions because of doubt about the geographical origin of the R. neglectus. We present here, for the first time, the isoenzyme profile of topotypes of R. neglectus. In addition, wild caught specimens from the type locality, Uberaba (Minas Gerais, Brazil), were compared to wild caught specimens from Jaraguá (Goiás, Brazil), where R. neglectus is more frequently reported invading houses. We used isoenzyme, morphology and morphometry analysis. Neither morphological nor enzymatic differences were found between areas, but metric, size-related divergence was evidenced between them.

Selective distribution of lactate dehydrogenase isoenzymes in neurons and astrocytes of human brain.

In vertebrates, the interconversion of lactate and pyruvate is catalyzed by the enzyme lactate dehydrogenase. Two distinct subunits combine to form the five tetrameric isoenzymes of lactate dehydrogenase. The LDH-5 subunit (muscle type) has higher maximal velocity (Vmax) and is present in glycolytic tissues, favoring the formation of lactate from pyruvate. The LDH-1 subunit (heart type) is inhibited by pyruvate and therefore preferentially drives the reaction toward the production of pyruvate. There is mounting evidence indicating that during activation the brain resorts to the transient glycolytic processing of glucose. Indeed, transient lactate formation during physiological stimulation has been shown by 1H-magnetic resonance spectroscopy. However, since whole-brain arteriovenous studies under basal conditions indicate a virtually complete oxidation of glucose, the vast proportion of the lactate transiently formed during activation is likely to be oxidized. These in vivo data suggest that lactate may be formed in certain cells and oxidized in others. We therefore set out to determine whether the two isoforms of lactate dehydrogenase are localized to selective cell types in the human brain. We report here the production and characterization of two rat antisera, specific for the LDH-5 and LDH-1 subunits of lactate dehydrogenase, respectively. Immunohistochemical, immunodot, and western-blot analyses show that these antisera specifically recognize their homologous antigens. Immunohistochemistry on 10 control cases demonstrated a differential cellular distribution between both subunits in the hippocampus and occipital cortex: neurons are exclusively stained with the anti-LDH1 subunit while astrocytes are stained by both antibodies. These observations support the notion of a regulated lactate flux between astrocytes and neurons.

Characterization of rat heart alkaline phosphatase isoenzymes and modulation of activity

Alkaline phosphatase (ALP) is important in calcification and its expression seems to be associated with the inflammatory process. We investigated the in vitro acute effects of compounds used for the prevention or treatment of cardiovascular diseases on total ALP activity from male Wistar rat heart homogenate. ALP activity was determined by quantifying, at 410 nm, the p-nitrophenol released from p-nitrophenylphosphate (substrate in Tris buffer, pH 10.4). Using specific inhibitors of ALP activity and the reverse transcription-polymerase chain reaction, we showed that the rat heart had high ALP activity (31.73 ± 3.43 nmol p-nitrophenol·mg protein-1·min-1): mainly tissue-nonspecific ALP but also tissue-specific intestinal ALP type II. Both ALP isoenzymes presented myocardial localization (striated pattern) by immunofluorescence. ALP was inhibited a) strongly by 0.5 mM levamisole, 2 mM theophylline and 2 mM aspirin (91, 77 and 84%, respectively) and b) less strongly by 2 mM L-phenylalanine, 100 mL polyphenol-rich beverages and 0.5 mM progesterone (24, 21 to 29 and 11%, respectively). β-estradiol and caffeine (0.5 and 2 mM) had no effect; 0.5 mM simvastatin and 2 mM atenolol activated ALP (32 and 36%, respectively). Propranolol (2 mM) tended to activate ALP activity and corticosterone activated (18%) and inhibited (13%) (0.5 and 2 mM, respectively). We report, for the first time, that the rat heart expresses intestinal ALP type II and has high total ALP activity. ALP activity was inhibited by compounds used in the prevention of cardiovascular pathology. ALP manipulation in vivo may constitute an additional target for intervention in cardiovascular diseases.

Genetic studies of Psammolestes tertius (Hemiptera : Reduviidae : Triatominae) using male genital morphology, morphometry, isoenzymes, and random amplified polymorphic DNA

Two Brazilian populations of Psammolestes tertius (Ceara and Minas Gerais) collected from thornbird nests (Furnariidae) were compared by male genital morphology, morphametry, isoenzymes, and random amplified polymorphic DNA (RAPD). Merle genitalia showed no difference between the populations. In contrast, morphometry, isoenzyme, and RAPD clearly distinguished the two populations. Possible mechanisms of dispersal and the origin of Psammolestes are discussed.

Molecular weight estimation of esterase isoenzymes in closely related Drosophila Species (Diptera: Drosophilidae) in non-denaturing polyacrylamide gel electrophoresis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Invited review Giardia duodenalis: Inter-strain variability of proteins, antigens, proteases, isoenzymes and nucleic acids

Giardia duodenalis isolates from asymptomatic or symptomatic patients and from animals present similarities and differences in the protein composition, antigenic profile, pattern of proteases and isoenzymes, as well as in nucleic acids analysis. In the present overview, these differences and similarities are reviewed with emphasis in the host-parasite interplay and possible mechanisms of virulence of the protozoon.

CYP-450 isoenzymes catalyze the generation of hazardous aromatic amines after reaction with the azo dye Sudan III

This work describes the mutagenic response of Sudan III, an adulterant food dye, using Salmonella typhimurium assay and the generation of hazardous aromatic amines after different oxidation methods of this azo dye. For that, we used metabolic activation by S9, catalytic oxidation by ironporphyrin and electrochemistry oxidation in order to simulate endogenous oxidation conditions. The oxidation reactions promoted discoloration from 65% to 95% of Sudan III at 1×10-4molL-1 and generation of 7.6×10-7molL-1 to 0.31×10-4molL-1 of aniline, o-anisidine, 2-methoxi-5-methylaniline, 4-aminobiphenyl, 4,4'-oxydianiline; 4,4'-diaminodiphenylmethane and 2,6-dimethylaniline. The results were confirmed by LC-MS-MS experiments. We also correlate the mutagenic effects of Sudan III using S. typhimurium with the strain TA1535 in the presence of exogenous metabolic activation (S9) with the metabolization products of this compound. Our findings clearly indicate that aromatic amines are formed due to oxidative reactions that can be promoted by hepatic cells, after the ingestion of Sudan III. Considering that, the use of azo compounds as food dyestuffs should be carefully controlled. © 2013 Elsevier Ltd.

12-Oxophytodienoate-10,11-Reductase: Occurrence of Two Isoenzymes of Different Specificity against Stereoisomers of 12-Oxophytodienoic Acid1

The reduction of 12-oxophytodienoic acid (OPDA) to 3-oxo-2(2′[Z]-pentenyl)-cyclopentane-1-octanoic acid is catalyzed by 12-oxophytodienoate-10,11-reductase (OPR). Analysis of the isomer preference of OPR has indicated that the activity is composed of two isoenzymes exhibiting different stereoselectivities. The two isoforms of OPR have been separated, using protein extracts of Rock Harlequin (Corydalis sempervirens) as the starting material. OPRI, the enzyme reported earlier from the same species and corresponding to the cloned OPR from Arabidopsis, utilized 9R,13R-OPDA >> 9S,13R-OPDA but not the 13S-configured isomers, whereas the new activity, OPRII, effectively reduced all four OPDA isomers, including the natural 9S,13S-OPDA (cis-[+]-OPDA). OPRII activity is characterized in detail. The enzyme's enzymatic, biochemical, and immunological properties prove that it is a close relative of OPRI. The roles of OPRI and OPRII in octadecanoid biology are discussed.

Caractérisation de l'activité phénotypique des Isoenzymes du cytochrome P450 chez des patients avec maladie du foie

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Caractérisation de l'activité phénotypique des Isoenzymes du cytochrome P450 chez des patients avec maladie du foie

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.