Maintenance enteral electrolyte solutions for neonatal calves: sodium acetate and osmolarity effects / Soluções eletrolíticas enterais de manutenção para bezerros neonatos: efeitos do acetato de sódio e da osmolaridade

The use of hypotonic electrolytic solutions in enteral fluid therapy is still understudied in calves. The objective of the present study was to evaluate the effects of maintenance enteral electrolytic solutions with different concentrations of sodium acetate and different osmolarities in calves. For this, 18 Holstein calves, six male and 12 female, 20 days old and weighing around 52kg, were used. The animals were randomly divided into three groups and each group received one of the treatments. The three electrolytic solutions contained the same components in different concentrations, resulting in a hyposmotic, an isosmotic and a hyperosmotic solution. Each animal was maintained in enteral fluid therapy for 12 hours with infusion rate of 15mL kg-1 h-1. Abdominal circumference, body weight, feces consistency, glucose and plasma lactate, pH, pCO2, HCO- 3 and BE were measured at the following times: T0h, T6h, T12h and T24h. The hyposmotic solution did not generate the onset of diarrhea, while the isosmotic and the hyperosmotic did. Regardless of the dose used, acetate did not cause metabolic alkalosis in the evaluated animals. The results suggest that the use of hyposmotic solution in diarrheic calves, dehydrated and without metabolic acidosis, may be clinically important.(AU)

O uso de soluções eletrolíticas hipotônicas na hidratação enteral ainda é pouco estudado em bezerros. O objetivo do presente estudo foi avaliar os efeitos de soluções eletrolíticas enterais de manutenção com diferentes concentrações de acetato de sódio e diferentes osmolaridades em bezerros. Para isso, foram utilizados 18 bezerros, seis machos e 12 fêmeas, holandeses, com 20 dias de nascidos e pesando por volta dos 52kg. Os animais foram divididos aleatoriamente em três grupos e cada grupo recebeu um dos tratamentos. As três soluções eletrolíticas continham os mesmos componentes, mas em diferentes concentrações, resultando em uma solução hiposmótica, uma isosmótica e uma hiperosmótica. Cada animal foi mantido em hidratação enteral durante 12 horas com taxa de infusão de 15mL kg-1h-1. Foram aferidos perímetro abdominal, peso corporal, consistência das fezes, glicose e lactato plasmático, pH, pCO2, HCO- 3 e excesso de base nos seguintes tempos: T0h, T6h, T12h e T24h. A solução hiposmótica não gerou aparecimento de diarreia, enquanto a isosmótica e a hiperosmótica geraram. Independentemente da dose utilizada, o acetato não causou alcalose metabólica nos animais avaliados. Os resultados sugerem que o uso da solução hiposmótica em bezerros diarreicos, desidratados e sem acidose metabólica, pode ser clinicamente importante.(AU)

Edaphic macrofauna in degradation of animal and vegetable residues / Macrofauna edáfica no processo de vermicompostagem de resíduos animais e vegetais

Abstract The objective of this study was to evaluate the ability to degrade organic matter by edaphic macrofauna (worms), carbon/nitrogen (C/N) ratio and hydrogenation potential (pH) during the vermicomposting process in different organic residues. The treatments were constituted by organic residues of animal origin (bovine, ovine and equine manure) and vegetable (herb-checkmate and coffee drag), which were conditioned in plastic pots with a capacity of 10 liters, comprising five treatments in a completely randomized experimental design, with five replications. Were inoculated 150 earthworms of the species Eisenia foetida, into each plot. After 87 days, the evaluation of the multiplication of the earthworms was carried out, through its manual count and its cocoons. At the beginning and at the end of the experiment, the samples were submitted to analysis of humidity at 60 °C, pH, volumetric density, chemical analysis of macronutrients and C/N ratio. There was a dominance of worms and cocoons in the process of vermicomposting in the residues of ovine manure and herb-checkmate. The macronutrients (P, K and Mg) and C/N ratio were higher in the vegetal residues, while for N higher values were found in ovine manure and coffee drag treatments, and for Ca higher value among treatments was observed in the coffee drag treatment at the end and the lowest value at initiation. The results obtained in this study demonstrate the importance of the edaphic macrofauna to the vermicomposting process, since it allows more information about its influence on the continuity of soil organic matter decomposition processes.

Resumo Este estudo objetivou avaliar a capacidade de degradar a matéria orgânica pela macrofauna edáfica (minhocas), a relação carbono/nitrogênio (C/N) e o potencial hidrogeniônico (pH), durante o processo da vermicompostagem em diferentes resíduos orgânicos. Os tratamentos foram constituídos por resíduos orgânicos de origem animal (esterco bovino, ovino e equino) e vegetal (resíduo de erva-mate e borra-de-café), os quais foram acondicionados em vasos plásticos com capacidade de 10 litros, compondo cinco tratamentos em delineamento experimental inteiramente casualisado, com cinco repetições. Foram inoculadas 150 minhocas da espécie Eisenia foetida, em cada recipiente. Após 87 dias, foi realizada a avaliação da multiplicação das minhocas, através da sua contagem manual e seus casulos. Os resíduos foram submetidos, ao inicio e ao final do experimento, a análises de umidade a 60 °C, pH, densidade volumétrica, análise química de macronutrientes e relação C/N. Houve uma dominância de minhocas e casulos no processo da vermicompostagem nos resíduos de esterco ovino e erva-mate. Observou-se para os macronutrientes (P, K e Mg) e para a relação C/N uma maior quantidade nos resíduos vegetais, enquanto que para N valores maiores foram encontrados nos tratamentos esterco ovino e borra-de-café, e para Ca o maior valor entre os tratamentos foi observado no tratamento com borra de café ao final e o menor valor no inicio. Os resultados obtidos neste estudo demonstram a importância da macrofauna edáfica para o processo da vermicompostagem por possibilitar maiores informações sobre sua influência na continuidade dos processos de decomposição da matéria orgânica.

Edaphic macrofauna in degradation of animal and vegetable residues.

The objective of this study was to evaluate the ability to degrade organic matter by edaphic macrofauna (worms), carbon/nitrogen (C/N) ratio and hydrogenation potential (pH) during the vermicomposting process in different organic residues. The treatments were constituted by organic residues of animal origin (bovine, ovine and equine manure) and vegetable (herb-checkmate and coffee drag), which were conditioned in plastic pots with a capacity of 10 liters, comprising five treatments in a completely randomized experimental design, with five replications. Were inoculated 150 earthworms of the species Eisenia foetida, into each plot. After 87 days, the evaluation of the multiplication of the earthworms was carried out, through its manual count and its cocoons. At the beginning and at the end of the experiment, the samples were submitted to analysis of humidity at 60 °C, pH, volumetric density, chemical analysis of macronutrients and C/N ratio. There was a dominance of worms and cocoons in the process of vermicomposting in the residues of ovine manure and herb-checkmate. The macronutrients (P, K and Mg) and C/N ratio were higher in the vegetal residues, while for N higher values were found in ovine manure and coffee drag treatments, and for Ca higher value among treatments was observed in the coffee drag treatment at the end and the lowest value at initiation. The results obtained in this study demonstrate the importance of the edaphic macrofauna to the vermicomposting process, since it allows more information about its influence on the continuity of soil organic matter decomposition processes.

Passive adaptation to stress in adulthood after short-term social instability stress during adolescence in mice.

This study reports that short-term social instability stress (SIS) in adolescence increases passive-coping in adulthood in male mice. Short-term SIS decreased the latency of immobility and increased the frequency and time of immobility in tail suspension test. These findings support the hypothesis that adolescent stress can induce a passive adaptation to stress in adulthood, even if it is a short period of stress.

Long-lasting monoaminergic and behavioral dysfunctions in a mice model of socio-environmental stress during adolescence.

Adolescence is one of the critical periods of development and has great importance to health for an individual as an adult. Stressors or traumatic events during this period are associated with several psychiatric disorders as related to anxiety or depression and cognitive impairments, but whether negative experiences continue to hinder individuals as they age is not as well understood. We determined how stress during adolescence affects behavior and neurochemistry in adulthood. Using an unpredictable paradigm (2 stressors per day for 10days) in Balb/c mice, behavioral, hormonal, and neurochemical changes were identified 20days after the cessation of treatment. Adolescent stress increased motor activity, emotional arousal and vigilance, together with a reduction in anxiety, and also affected recognition memory. Furthermore, decreased serotonergic activity on hippocampus, hypothalamus and cortex, decreased noradrenergic activity on hippocampus and hypothalamus, and increased the turnover of dopamine in cortex. These data suggest behavioral phenotypes associated with emotional arousal, but not depression, emerge after cessation of stress and remain in adulthood. Social-environmental stress can induce marked and long-lasting changes in HPA resulting from monoaminergic neurotransmission, mainly 5-HT activity.

Transenamel and transdentinal penetration of hydrogen peroxide applied to cracked or microabrasioned enamel.

The present study evaluated transenamel and transdentinal penetration of hydrogen peroxide during tooth whitening recognized in altered enamel by the presence of cracks or microabrasion. We used 72 experimental units (n=20) obtained from bovine incisors: GI-sound enamel; GII-teeth showing visible enamel cracks (4 mm to 5.7 mm in length); and GIII-microabrasioned enamel. The 12 remaining specimens were used to analyze the enamel surface morphology using scanning electron microscopy. The specimens were cylindrical and 5.7 mm in diameter and 3.5 mm thick. A product based on 35% hydrogen peroxide was used for bleaching, following the manufacturer's recommendations for use. To quantify the H2O2 penetration, the specimens were placed in artificial pulp chambers containing an acetate buffer solution. After bleaching, the solution was collected and adequately proportioned with leucocrystal violet, peroxidase enzyme, and deionized water. The resulting solution was evaluated using ultraviolet visible reflectance spectrophotometer equipment. The data were analyzed by analysis of variance (ANOVA) and Fisher's PLSD at a significance level of 0.05, and significant differences in the penetration of peroxide in different substrate conditions were observed (p<0.0001). The penetration of hydrogen peroxide was more intense in cracked teeth. The group in which the enamel was microabraded showed intermediate values when compared to the control group. Microabrasion and the presence of cracks in the enamel make this substrate more susceptible to penetration of hydrogen peroxide during in-office whitening.

Cysteine peptidase inhibitors in trypanosomatid parasites.

Parasitic diseases caused by pathogenic protozoa remain a challenge for public health. Despite efforts to control transmission, to improve early diagnosis and to optimize patient care, millions of infected people, mainly in poor areas of the globe, develop debilitating pathologies that are often fatal. For most of those disorders, the current treatments are greatly unsatisfactory and the continuous search for alternative chemotherapies remains at the center of research. Over the last decades, cysteine peptidases of protozoa feature as highly promising drug targets and their validation in laboratory models of disease or experimental infections instigated growing efforts in medicinal chemistry, aiming at the development of compounds with therapeutical potential. More recently, it was uncovered that protozoa also express new families of endogenous proteinaceous peptidase inhibitors that act as potential virulence factors. In this review, we will cover the main findings that contributed to the validation of cysteine peptidases from Trypanosoma cruzi, Trypanosoma brucei and Leishmania as drug targets and the current knowledge of their biological roles in those organisms. We give an overview of the development of small molecule cysteine peptidase inhibitors with anti-parasite activity and describe the current background on natural peptidase inhibitors in trypanosomatids.

Migration of transjugular intrahepatic portosystemic shunt to the right atrium: complications in the intraoperative period of liver transplantation.

This article reports the case of a patient who underwent transjugular intrahepatic portosystemic shunt, which migrated to the right atrium. During liver transplantation, the extracardiac portion was sectioned and the portion adherent inside the atrium was managed expectantly.

Ten polymorphic microsatellite loci for Allobates femoralis, an Amazonian dendrobatoid frog.

Within the anuran amphibians, dendrobatoids (poison-arrow frogs) are renowned for their parental care, but the lack of highly variable DNA markers so far precluded precise and comprehensive measurements of their genetic mating system. Here we present 10 polymorphic microsatellites for Allobates femoralis, a widespread dendrobatoid from the Amazon basin. In 24 field-collected individuals, we found between seven and 15 alleles per locus, without deviations from Hardy-Weinberg equilibrium or linkage disequilibrium. The loci will be employed to quantify reproductive success in view of male territoriality and female mate choice, as well as to determine the fine-scale genetic structure of local populations.

Down-regulation of the cardiac sarcoplasmic reticulum ryanodine channel in severely food-restricted rats.

We have shown that myocardial dysfunction induced by food restriction is related to calcium handling. Although cardiac function is depressed in food-restricted animals, there is limited information about the molecular mechanisms that lead to this abnormality. The present study evaluated the effects of food restriction on calcium cycling, focusing on sarcoplasmic Ca2+-ATPase (SERCA2), phospholamban (PLB), and ryanodine channel (RYR2) mRNA expressions in rat myocardium. Male Wistar-Kyoto rats, 60 days old, were submitted to ad libitum feeding (control rats) or 50% diet restriction for 90 days. The levels of left ventricle SERCA2, PLB, and RYR2 were measured using semi-quantitative RT-PCR. Body and ventricular weights were reduced in 50% food-restricted animals. RYR2 mRNA was significantly decreased in the left ventricle of the food-restricted group (control = 5.92 +/- 0.48 vs food-restricted group = 4.84 +/- 0.33, P < 0.01). The levels of SERCA2 and PLB mRNA were similar between groups (control = 8.38 +/- 0.44 vs food-restricted group = 7.96 +/- 0.45, and control = 1.52 +/- 0.06 vs food-restricted group = 1.53 +/- 0.10, respectively). Down-regulation of RYR2 mRNA expressions suggests that chronic food restriction promotes abnormalities in sarcoplasmic reticulum Ca2+ release.

Down-regulation of the cardiac sarcoplasmic reticulum ryanodine channel in severely food-restricted rats

We have shown that myocardial dysfunction induced by food restriction is related to calcium handling. Although cardiac function is depressed in food-restricted animals, there is limited information about the molecular mechanisms that lead to this abnormality. The present study evaluated the effects of food restriction on calcium cycling, focusing on sarcoplasmic Ca2+-ATPase (SERCA2), phospholamban (PLB), and ryanodine channel (RYR2) mRNA expressions in rat myocardium. Male Wistar-Kyoto rats, 60 days old, were submitted to ad libitum feeding (control rats) or 50 percent diet restriction for 90 days. The levels of left ventricle SERCA2, PLB, and RYR2 were measured using semi-quantitative RT-PCR. Body and ventricular weights were reduced in 50 percent food-restricted animals. RYR2 mRNA was significantly decreased in the left ventricle of the food-restricted group (control = 5.92 ± 0.48 vs food-restricted group = 4.84 ± 0.33, P < 0.01). The levels of SERCA2 and PLB mRNA were similar between groups (control = 8.38 ± 0.44 vs food-restricted group = 7.96 ± 0.45, and control = 1.52 ± 0.06 vs food-restricted group = 1.53 ± 0.10, respectively). Down-regulation of RYR2 mRNA expressions suggests that chronic food restriction promotes abnormalities in sarcoplasmic reticulum Ca2+ release.

Calcium-regulated fusion of yolk granules is important for yolk degradation during early embryogenesis of Rhodnius prolixus Stahl.

This study examined the process of membrane fusion of yolk granules (YGs) during early embryogenesis of Rhodnius prolixus. We show that eggs collected at days 0 and 3 after oviposition contain different populations of YGs, for example day-3 eggs are enriched in large YGs (LYGs). Day-3 eggs also contain the highest free [Ca(2+)] during early embryogenesis of this insect. In vitro incubations of day-0 YGs with [Ca(2+)] similar to those found in day-3 eggs resulted in the formation of LYGs, as observed in vivo. Fractionation of LYGs and small YGs (SYGs) and their subsequent incubation with the fluorescent membrane marker PKH67 showed a calcium-dependent transference of fluorescence from SYGs to LYGs, possibly as the result of membrane fusion. Acid phosphatase and H(+)-PPase activities were remarkably increased in day-3 LYGs and in calcium-treated day-0 LYGs. Both fractions were found to contain vitellins as major components, and incubation of YGs with calcium induced yolk proteolysis in vitro. Altogether, our results suggest that calcium-induced membrane fusion events take part in yolk degradation, leading to the assembly of the yolk mobilization machinery.

Relato de 7 casos de pacientes portadores de Hepatite B em acompanhamento no Ambulatório de Hepatologia do Hospital do Servidor Público Municipal / Report of 7 cases of patients with hepatitis B Hepatology Outpatient Clinic of the Hospital do Servidor Publico Municipal

Vários estudos têm demonstrado a preocupação em relação ao HBV devido a sua evolução para formas crônicas e o aparecimento de hepatocarcinoma. Hoje temos um novo arsenal de medicamentos para diminuir a carga viral dos portadores HBV quando em atividade replicativa, sendo a carga viral extremamente importante, num futuro próximo os genótipos também orientarão para o prognóstico (AU)

Relato de 7 casos de pacientes portadores de Hepatite B em acompanhamento no Ambulatório de Hepatologia do Hospital do Servidor Público Municipal

Vários estudos têm demonstrado a preocupação em relação ao HBV devido a sua evolução para formas crônicas e o aparecimento de hepatocarcinoma. Hoje temos um novo arsenal de medicamentos para diminuir a carga viral dos portadores HBV quando em atividade replicativa, sendo a carga viral extremamente importante, num futuro próximo os genótipos também orientarão para o prognóstico

Prolactin and cortisol levels in women with endometriosis.

Endometriosis is a progressive estrogen-dependent disease affecting women during their reproductive years. The objective of the present study was to investigate whether endometriosis is associated with stress parameters. We determined cortisol and prolactin levels in serum, peritoneal and follicular fluid from infertile women with endometriosis and fertile women without the disease. The extent of the disease was staged according to the revised American Fertility Society classification (1997). Serum and peritoneal fluid were collected from 49 women aged 19 to 39 years undergoing laparoscopy. Eighteen women had stage I-II endometriosis and 10 had stage III-IV. Controls were 21 women undergoing laparoscopy for tubal sterilization. Follicular fluid was obtained from 39 women aged 25-39 years undergoing in vitro fertilization (21 infertile women with endometriosis and 18 infertile women without endometriosis). Serum prolactin levels were significantly higher in infertile women with stage III-IV endometriosis (28.9 +/- 2.1 ng/mL) than in healthy controls (13.2 +/- 2.1 ng/mL). Serum cortisol levels were significantly higher in infertile women with stage III-IV endometriosis (20.1 +/- 1.3 ng/mL) than in controls (10.5 +/- 1.4 ng/mL). Cortisol and prolactin levels in follicular fluid and peritoneal fluid did not differ significantly between groups. The high levels of cortisol and prolactin in the serum from women with endometriosis might contribute to the subfertility frequently associated with the disease. Moreover, since higher levels of cortisol and prolactin are often associated with stress, it is probable that stress might contribute to the development of endometriosis and its progression to advanced stages of the disease.

Prolactin and cortisol levels in women with endometriosis

Endometriosis is a progressive estrogen-dependent disease affecting women during their reproductive years. The objective of the present study was to investigate whether endometriosis is associated with stress parameters. We determined cortisol and prolactin levels in serum, peritoneal and follicular fluid from infertile women with endometriosis and fertile women without the disease. The extent of the disease was staged according to the revised American Fertility Society classification (1997). Serum and peritoneal fluid were collected from 49 women aged 19 to 39 years undergoing laparoscopy. Eighteen women had stage I-II endometriosis and 10 had stage III-IV. Controls were 21 women undergoing laparoscopy for tubal sterilization. Follicular fluid was obtained from 39 women aged 25-39 years undergoing in vitro fertilization (21 infertile women with endometriosis and 18 infertile women without endometriosis). Serum prolactin levels were significantly higher in infertile women with stage III-IV endometriosis (28.9 ± 2.1 ng/mL) than in healthy controls (13.2 ± 2.1 ng/mL). Serum cortisol levels were significantly higher in infertile women with stage III-IV endometriosis (20.1 ± 1.3 ng/mL) than in controls (10.5 ± 1.4 ng/mL). Cortisol and prolactin levels in follicular fluid and peritoneal fluid did not differ significantly between groups. The high levels of cortisol and prolactin in the serum from women with endometriosis might contribute to the subfertility frequently associated with the disease. Moreover, since higher levels of cortisol and prolactin are often associated with stress, it is probable that stress might contribute to the development of endometriosis and its progression to advanced stages of the disease.

Randomized crossover trial of intravenous 5-FU versus oral UFT both modulated by leucovorin: a one-centre experience.

Although intravenous (IV) 5-fluorouracil (5-FU) and uracil/futraful (UFT) have comparable antitumour efficacy in the treatment of metastatic colorectal cancer (MCC), we wanted to assess which of these two regimens would be preferred by our patients. We randomized 20 previously untreated patients with MCC at our centre to receive oral UFT or bolus IV 5-FU both associated with leucovorin. After the first cycle patients were crossed over to the other arm. Before the third cycle we left patients to choose one of the regimens to continue their treatment until disease progression. Two patients chose 5-FU and 18 chose UFT (P < 0.001). Fewer side effects (50%) and convenience of home treatment (40%) were the main reasons for their choice for the oral regimen. UFT induced less mucositis (P = 0. 02) and diarrhoea (P = 0. 01). We conclude that convenience and lower toxicity may explain the observed preference for oral UFT.

Comparison of the specificity, stability and individual rate constants with respective activation parameters for the peptidase activity of cruzipain and its recombinant form, cruzain, from Trypanosoma cruzi.

The Trypanosoma cruzi cysteine protease cruzipain contains a 130-amino-acid C-terminal extension, in addition to the catalytic domain. Natural cruzipain is a complex of isoforms, because of the simultaneous expression of several genes, and the presence of either high mannose-type, hybrid monoantennary-type or complex biantenary-type oligosacharide chains at Asn255 of the C-terminal extension. Cruzipain and its recombinant form without this extension (cruzain) were studied comparatively in this work. S2 to S2' subsite specificities of these enzymes were examined using four series of substrates derived from the internally quenched fluorescent peptide Abz-KLRFSKQ-EDDnp (Abz, ortho-aminobenzoic acid; EDDnp, N-(2,4-dinitrophenyl)-ethylenediamine). Large differences in the kinetic parameters were not observed between the enzymes; however, Km values were consistently lower for the hydrolysis of most of the substrates by cruzain. No difference in the pH-activity profile between the two enzymes was found, but in 1 m NaCl cruzipain presented a kcat value significantly higher than that of cruzain. The activation energy of denaturation for the enzymes did not differ significantly; however, a negative entropy value was observed for cruzipain denaturation whereas the value for cruzain was positive. We determined the individual rate constants (k1, substrate diffusion; k-1, substrate dissociation; k2, acylation; k3, deacylation) and the respective activation energies and entropies for hydrolysis of Abz-KLRFSKQ-EDDnp determining the temperature dependence of the Michaelis-Menten parameters kcat/Km and kcat as previously described [Ayala, Y.M. & Di Cera, E. (2000) Protein Sci. 9, 1589-1593]. Differences between the two enzymes were clearly detected in the activation energies E1 and E-1, which are significantly higher for cruzipain. The corresponding DeltaS1 and DeltaS-1 were positive and significantly higher for cruzipain than for cruzain. These results indicate the presence of a larger energy barrier for cruzipain relating to substrate diffusion and dissociation, which could be related to the C-terminal extension and/or glycosylation state of cruzipain.

Identification, characterization and localization of chagasin, a tight-binding cysteine protease inhibitor in Trypanosoma cruzi.

Lysosomal cysteine proteases from mammalian cells and plants are regulated by endogenous tight-binding inhibitors from the cystatin superfamily. The presence of cystatin-like inhibitors in lower eukaryotes such as protozoan parasites has not yet been demonstrated, although these cells express large quantities of cysteine proteases and may also count on endogenous inhibitors to regulate cellular proteolysis. Trypanosoma cruzi, the causative agent of Chagas' heart disease, is a relevant model to explore this possibility because these intracellular parasites rely on their major lysosomal cysteine protease (cruzipain) to invade and multiply in mammalian host cells. Here we report the isolation, biochemical characterization, developmental stage distribution and subcellular localization of chagasin, an endogenous cysteine protease inhibitor in T. cruzi. We used high temperature induced denaturation to isolate a heat-stable cruzipain-binding protein (apparent molecular mass, 12 kDa) from epimastigote lysates. This protein was subsequently characterized as a tight-binding and reversible inhibitor of papain-like cysteine proteases. Immunoblotting indicated that the expression of chagasin is developmentally regulated and inversely correlated with that of cruzipain. Gold-labeled antibodies localized chagasin to the flagellar pocket and cytoplasmic vesicles of trypomastigotes and to the cell surface of amastigotes. Binding assays performed by probing living parasites with fluorescein (FITC)-cruzipain or FITC-chagasin revealed the presence of both inhibitor and protease at the cell surface of amastigotes. The intersection of chagasin and cruzipain trafficking pathways may represent a checkpoint for downstream regulation of proteolysis in trypanosomatid protozoa.

Cysteine protease isoforms from Trypanosoma cruzi, cruzipain 2 and cruzain, present different substrate preference and susceptibility to inhibitors.

Cysteine-proteinases from parasitic protozoa have been recently characterized as factors of virulence and pathogenicity in several human and veterinary diseases. In Chagas' disease, the chronic infection caused by Trypanosoma cruzi, structure-functional studies on cysteine proteases were thus far limited to the parasite's major isoform, a cathepsin L-like lysosomal protease designated as cruzipain, cruzain or GP57/51. Encoded by a large gene family, cruzipain is efficiently targeted by synthetic inhibitors, which prevent parasite intracellular growth and differentiation. We have previously demonstrated that the multicopy cruzipain gene family includes polymorphic sequences, which could encode functionally different isoforms. We report here a comparative kinetic study between cruzain, the archetype of the cruzipain family, and an isoform, termed cruzipain 2, which is expressed preferentially by the mammalian stages of T. cruzi. Heterologous expression of the catalytic domain of cruzipain 2 in Saccharomyces cerevisae yielded an enzyme that differs markedly from cruzain with respect to pH stability, substrate specificity and sensitivity to inhibition by natural and synthetic inhibitors of cysteine proteases. We suggest that the structural-functional diversification imparted by genetic polymorphism of cruzipain genes may have contributed to T. cruzi adaptation to vertebrate hosts.