Vertebrates as a Bactericidal Agent.

In Brazil, although a large number of animals are used in traditional medicine (at least 354 species), information about their biological activities is scarce. In this context, the objective of this study was to evaluate the bactericidal potential of zootherapeutic by-products from animals used in Brazilian traditional medicine and discuss the ecological and cultural consequences of such practices. The species analyzed were: Tupinambis merianae (skin), Iguana iguana (skin and body fat), Crotalus durissus (skin and body fat), Boa constrictor (skin), Euphractus sexcinctus (body fat) and Coendou prehensilis (quills). Experiments were performed with standard clinical strains of Escherichia coli (EC-ATCC10536) and Staphylococcus aureus (SA-ATCC 25923). For the microbiological assay, the zootherapeutics were evaluated using serial microdilutions. The results indicate that none of the samples possess inhibitory activity against standard bacterial strains. The in vitro ineffectiveness of the analyzed products demonstrate a necessity for new pharmacological research that encompass a large number of species of medicinal animals as well as highlight the importance of zootherapy in the context of plans for animal conservation.

Seroepidemiological survey of equine infectious anemia in Rondônia state, Brazil / Levantamento soroepidemiológico de anemia infecciosa equina no Estado de Rondônia, Brasil

Based on the importance of equine infectious anemia (EIA), this study aimed to analyze the spatial distribution and report the occurrence rates of EIA through a seroepidemiological survey and evaluate the actions taken by the Agrosilvopastoral Defence Agency of the State of Rondônia (IDARON) from January 2014 to December 2015. There was a direct relationship between the human development index (HDI) and the number of horses in the different areas of the Rondonia. 7,729 properties were analyzed, in which 19,805 horses were examined, with a mean seropositivity for EIA of 1.15% in 2014 and 1.01% in 2015. Despite the low incidence of seropositive horses for the EIA virus, sanitary control procedures imposed by IDARON with the sacrifice of 95.97% of sick animals in 2014 and 90% in 2015 resulted in a decrease of 28.22% of positive animals between the years 2014 and 2015. Therefore, to achieve an effective reduction in the prevalence of this disease, all positive animals should be isolated and subsequently sacrificed, as they are disseminators of the disease. Many farmers, however, do not understand the real importance of the proliferation of EIA by the presence of carrier animals in the herds.(AU)

Com base na importância da Anemia infecciosa Equina (AIE), a presente investigação teve como objetivo analisar a distribuição espacial e relatar os índices de ocorrência de AIE por meio de levantamento soroepidemiológico e ações realizadas pela Agência de Defesa Agrosilvopastoril do Estado de Rondônia (IDARON) no período de janeiro de 2014 a dezembro de 2015. Observou-se uma relação direta entre o índice de desenvolvimento humano (IDH) e o número de equinos nas diferentes regiões do estado de Rondônia. Foram analisadas 7.729 propriedades, nas quais 19.805 equinos foram examinados, obtendo-se uma soropositividade média de 1,15% em 2014 e 1,01% em 2015 para AIE. Apesar da baixa ocorrência de equinos sorologicamente positivos para o vírus da AIE, os procedimentos de controle sanitário impostos pela IDARON com o sacrifício dos animais enfermos 95,97% em 2014 e 90% em 2015 resultaram em diminuição de 28,22% de animais positivos entre os anos de 2014 e 2015. Portanto, para se conseguir boa redução na prevalência dessa enfermidade, todo o animal positivo deve ser sacrificado, por ser disseminador da doença. Embora inúmeros criadores, não entendam a real importância para a disseminação da AIE nos plantéis pela presença de animais portadores.(AU)

SEROEPIDEMIOLOGICAL SURVEY OF EQUINE INFECTIOUS ANEMIA IN RONDONIA STATE, BRAZIL

Based on the importance of equine infectious anemia (EIA), this study aimed to analyze the spatial distribution and report the occurrence rates of EIA through a seroepidemiological survey and evaluate the actions taken by the Agrosilvopastoral Defence Agency of the State of Rondônia (IDARON) from January 2014 to December 2015. There was a direct relationship between the human development index (HDI) and the number of horses in the different areas of the Rondonia. 7,729 properties were analyzed, in which 19,805 horses were examined, with a mean seropositivity for EIA of 1.15% in 2014 and 1.01% in 2015. Despite the low incidence of seropositive horses for the EIA virus, sanitary control procedures imposed by IDARON with the sacrifice of 95.97% of sick animals in 2014 and 90% in 2015 resulted in a decrease of 28.22% of positive animals between the years 2014 and 2015. Therefore, to achieve an effective reduction in the prevalence of this disease, all positive animals should be isolated and subsequently sacrificed, as they are disseminators of the disease. Many farmers, however, do not understand the real importance of the proliferation of EIA by the presence of carrier animals in the herds.

Seroepidemiological survey of equine infectious anemia in Rondônia state, Brazil / Levantamento soroepidemiológico de anemia infecciosa equina no Estado de Rondônia, Brasil

Based on the importance of equine infectious anemia (EIA), this study aimed to analyze the spatial distribution and report the occurrence rates of EIA through a seroepidemiological survey and evaluate the actions taken by the Agrosilvopastoral Defence Agency of the State of Rondônia (IDARON) from January 2014 to December 2015. There was a direct relationship between the human development index (HDI) and the number of horses in the different areas of the Rondonia. 7,729 properties were analyzed, in which 19,805 horses were examined, with a mean seropositivity for EIA of 1.15% in 2014 and 1.01% in 2015. Despite the low incidence of seropositive horses for the EIA virus, sanitary control procedures imposed by IDARON with the sacrifice of 95.97% of sick animals in 2014 and 90% in 2015 resulted in a decrease of 28.22% of positive animals between the years 2014 and 2015. Therefore, to achieve an effective reduction in the prevalence of this disease, all positive animals should be isolated and subsequently sacrificed, as they are disseminators of the disease. Many farmers, however, do not understand the real importance of the proliferation of EIA by the presence of carrier animals in the herds.

Com base na importância da Anemia infecciosa Equina (AIE), a presente investigação teve como objetivo analisar a distribuição espacial e relatar os índices de ocorrência de AIE por meio de levantamento soroepidemiológico e ações realizadas pela Agência de Defesa Agrosilvopastoril do Estado de Rondônia (IDARON) no período de janeiro de 2014 a dezembro de 2015. Observou-se uma relação direta entre o índice de desenvolvimento humano (IDH) e o número de equinos nas diferentes regiões do estado de Rondônia. Foram analisadas 7.729 propriedades, nas quais 19.805 equinos foram examinados, obtendo-se uma soropositividade média de 1,15% em 2014 e 1,01% em 2015 para AIE. Apesar da baixa ocorrência de equinos sorologicamente positivos para o vírus da AIE, os procedimentos de controle sanitário impostos pela IDARON com o sacrifício dos animais enfermos 95,97% em 2014 e 90% em 2015 resultaram em diminuição de 28,22% de animais positivos entre os anos de 2014 e 2015. Portanto, para se conseguir boa redução na prevalência dessa enfermidade, todo o animal positivo deve ser sacrificado, por ser disseminador da doença. Embora inúmeros criadores, não entendam a real importância para a disseminação da AIE nos plantéis pela presença de animais portadores.

Overnourishment during lactation induces metabolic and haemodynamic heart impairment during adulthood.

AIM: In this study, the effects of postnatal overfeeding on heart energy homoeostasis and cardiac haemodynamics in adult male Swiss mice were examined. METHODS AND RESULTS: During the suckling period, the mice were divided into four groups of control or overfed pups in combination with baseline or ischaemia/reperfusion treatments (control group baseline, CGBL; overfed group baseline, OGBL; control group ischaemia/reperfusion, CGIR; and overfed group ischaemia/reperfusion, OGIR). End diastolic pressure (EDP), heart contraction speed (Max dP/dt), relaxation speed (Min dP/dt), isovolumetric relaxation time (Tau) and frequency by beats per minute (BPM) were measured. During baseline and ischaemia/reperfusion, key proteins such as AKT1, AKT2, AKT3, pAKT, adenosine monophosphate-activated protein kinase (AMPK), pAMPK, insulin receptor beta (IRß), protein tyrosine phosphatase 1B (PTP1B), insulin receptor substrate 1 (IRS1), fatty acid binding protein (FABP), CD36, phosphoinositide 3-kinase (PI3K) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) were studied. The expression of atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), carnitine palmitoyltransferase 1 (CPT1) and uncoupling protein 3 (UCP3) was studied as a marker of cardiac hypertrophy and energetic metabolism. Cardiac fibrosis was analyzed by quantifying collagen deposition, which is increased in the OGBL and OGIR groups compared with the control groups. CONCLUSIONS: The OGBL group showed reduced EDP compared with the CGBL group and high Max dP/dt compared with the OGBL group. Ischaemia/reperfusion increased EDP and Min dP/dt in the intragroup comparison. By contrast, Tau and frequency were not significantly different among groups. The OGIR mice showed significant alterations in heart metabolism proteins, including AKT2, pAKT/AKT1, pAKT/AKT2, AMPK, pAMPK/AMPK, PTP1B, IRS1, FABP and CD36. Furthermore, alterations in ANP, BNP, CPT1 and UCP3 messenger RNA (mRNA) expression indicated hypertrophy and reduction in their efficiency, such that exclusive overnutrition in childhood induces a long-term effect on haemodynamics, metabolism and heart remodelling.

Progenitor cells and TNF-alpha involvement during morphological changes in pancreatic islets of obese mice.

Overnutrition during pregnancy and lactation lend increasing support to the development of obesity and several chronic diseases in adulthood such as type 2 diabetes mellitus, which leads to beta-cell dysfunction and insulin resistance. In this work, we aimed to study the effects of early life overnutrition on the development of obesity, analyzing the morphological changes, expression of TNF-α, and also the stem cell marker CD133 in the pancreatic islets of young and adult mice. Overnutrition during lactation phase was used as an experimental model to induce obesity. The animals were analyzed at 28 and 150 days of age, when pancreata were collected for histological, ultrastructural and western blotting analysis. The results showed that islet hypertrophy is established in obese groups at day 28 and remained until adulthood. CD133+ cells were observed as small cells within pancreatic islets in both control and obese young mice. However, at day 150, these cells were observed only in the islet peripheries and near ducts of the obese group. Furthermore, TNF-α expression in pancreatic islets was increased in both young and adult obese groups when compared to control groups. This work shows interesting data about CD133 receptor and TNF-α roles in the pancreas during obesity development.

Increased expression of keratinase and other peptidases by Candida parapsilosis mutants.

Keratinases are enzymes of great importance involved in pathogenic processes of some fungi. They also have a widespread ecological role since they are responsible for the degradation and recycling of keratin. On the one hand, studying them furthers our knowledge of pathogenicity mechanisms, which has important implications for human health, and on the other hand, understanding their ecological role in keratin recycling has biotechnological potential. Here, a wild-type keratinolytic Candida parapsilosis strain isolated from a poultry farm was treated with ethyl methanesulfonate in order to generate mutants with increased keratinase activity. Mutants were then cultured on media with keratin extracted from chicken feathers as the sole source of nitrogen and carbon. Approximately 500 mutants were screened and compared with the described keratinolytic wild type. Three strains, H36, I7 and J5, showed enhanced keratinase activity. The wild-type strain produced 80 U/mL of keratinolytic activity, strain H36 produced 110 U/mL, strain I7, 130 U/mL, and strain J5, 140 U/mL. A 70% increase in enzyme activity was recorded for strain J5. Enzymatic activity was evaluated by zymograms with proteic substrates. A peptidase migrating at 100 kDa was detected with keratin, bovine serum albumin and casein. In addition, a peptidase with a molecular mass of 50 kDa was observed with casein in the wild-type strain and in mutants H36 and J5. Gelatinase activity was detected at 60 kDa. A single band of 35 kDa was found in wild-type C. parapsilosis and in mutants with hemoglobin substrate.

Increased expression of keratinase and other peptidases by Candida parapsilosis mutants

Keratinases are enzymes of great importance involved in pathogenic processes of some fungi. They also have a widespread ecological role since they are responsible for the degradation and recycling of keratin. On the one hand, studying them furthers our knowledge of pathogenicity mechanisms, which has important implications for human health, and on the other hand, understanding their ecological role in keratin recycling has biotechnological potential. Here, a wild-type keratinolytic Candida parapsilosis strain isolated from a poultry farm was treated with ethyl methanesulfonate in order to generate mutants with increased keratinase activity. Mutants were then cultured on media with keratin extracted from chicken feathers as the sole source of nitrogen and carbon. Approximately 500 mutants were screened and compared with the described keratinolytic wild type. Three strains, H36, I7 and J5, showed enhanced keratinase activity. The wild-type strain produced 80 U/mL of keratinolytic activity, strain H36 produced 110 U/mL, strain I7, 130 U/mL, and strain J5, 140 U/mL. A 70 percent increase in enzyme activity was recorded for strain J5. Enzymatic activity was evaluated by zymograms with proteic substrates. A peptidase migrating at 100 kDa was detected with keratin, bovine serum albumin and casein. In addition, a peptidase with a molecular mass of 50 kDa was observed with casein in the wild-type strain and in mutants H36 and J5. Gelatinase activity was detected at 60 kDa. A single band of 35 kDa was found in wild-type C. parapsilosis and in mutants with hemoglobin substrate.

Left ventricular hypertrophy induced by overnutrition early in life.

BACKGROUND AND AIMS: Human overnutrition has caused a rise in the prevalence of obesity in recent years. In addition to the deleterious effects of obesity during childhood, the long-term effects in adulthood have also been described. For instance, cardiovascular diseases, particularly left ventricular hypertrophy, and type 2 diabetes are among the diseases associated with a history of obesity. However, it remains uncertain during which period of life this association is established. Several authors have suggested that the early period of life is critical for the emergence of cardiac disorders. METHODS AND RESULTS: In this study, we examined the impact of overnutrition on the heart morphology and stereology of obese animals using an experimental model to induce an overweight phenotype. In these animals, overfeeding during lactation was able to induce a significant increase in body weight and visceral fat starting at the 10th day of life, and this increased weight persisted until 21 days of age. Impairments in triglyceride levels and cholesterol were also observed in these animals. Moreover, an increased heart weight/tibia length ratio was observed, indicating an enlarged heart. The overfed animals also had left ventricular hypertrophy with an increased area of cardiomyocytes and a decreased vessel density. CONCLUSIONS: Our findings showed that overnutrition during early life induced obesity and cardiac hypertrophy in the pups, perhaps due to a decrease in the intramyocardial vessel.

Crystal structure of Dioclea rostrata lectin: insights into understanding the pH-dependent dimer-tetramer equilibrium and the structural basis for carbohydrate recognition in Diocleinae lectins.

The legume lectins from the subtribe Diocleinae, often referred to as concanavalin A-like lectins, are a typical example of highly similar proteins that show distinct biological activities. The pH-dependent oligomerization that some of these lectins undergo and the relative position of amino acids within the carbohydrate-binding site are factors that have been reported to contribute to these differences in the activities of Diocleinae lectins. In the present work, we determined the amino acid sequence and the crystal structure of the lectin of Dioclea rostrata seeds (DRL), with the aim of investigating the structural bases of the different behavior displayed by this lectin in comparison to other Diocleinae lectins and determining the reason for the distinct pH-dependent dimer-tetramer equilibrium. In addition, we discovered a novel multimeric arrangement for this lectin.

Temporal variation of chemical composition and relaxant action of the essential oil of Ocimum gratissimum L. (Labiatae) on guinea-pig ileum.

The medicinal plant Ocimum gratissimum L. (Labiatae) is widely encountered in the Northeast of Brasil where it is used to treat digestive problems. Its leaves have an essential oil (EOOG) content whose chemical composition varies according to the time of plant collection. We have compared the effects of the EOOG, collected at 08:00 a.m. (EOOG8) and at 12:00 a.m. (EOOG12), on the relaxation of guinea-pig isolated ileum. Both EOOG8 and EOOG12 (30-300 microg/ml) reversibly relaxed the spontaneous tonus of the guinea-pig ileum in a concentration-dependent manner, with similar IC50 values (49.3 and 23.8 microg/ml, respectively). The magnitude of the decrease in resting tonus was similar to that of the recognised smooth muscle relaxant papaverine. EOOG8 and EOOG12 relaxed 60 mM KCl-precontracted preparations similarly (38.33 +/- 9.91 microg/ml and 35.53 +/- 6.70), whereas a significantly more potent relaxant effect of EOOG12 compared to EOOG8 was observed when tissues were contracted using 10 microM acetylcholine (IC50 values of 69.55 +/- 4.93 and 128.16 +/- 15.70 microg/ml, respectively; p < 0.05). The principal constituents of the essential oil, eugenol and cineole, also relaxed KCl-precontracted preparations, although they were less potent than EOOG, suggesting that they alone were not responsible for EOOG-induced relaxations. Our results show that the essential oil extracted from the leaves of O. gratissimum L., collected at different time periods, exerts significant relaxant effects on isolated guinea-pig ileum which may underlie the therapeutic action of the plant.

Inhibitory effects of the essential oil of Mentha pulegium on the isolated rat myometrium.

The effects of the essential oil of Mentha pulegium L. (EOMP), a plant commonly known as "pennyroyal" or "poejo" that is used in folk medicine as an abortifaceant, were assessed on the isolated rat myometrium. Myometrial strips were stimulated with 10 nM oxytocin or 10 microM PGF (2alpha). EOMP (10 - 300 microg/mL) concentration-dependently and reversibly inhibited the amplitude of oscillatory contractions, being approximately 3-fold more active against contractions stimulated by oxytocin than those by PGF (2alpha) (IC (50) values of 45.7 +/- 5.6 microg/mL and 160.9 +/- 5.9 microg/mL , respectively), although the maximal inhibitory effect occurred at the same concentration (300 microg/mL ) in both cases. This action was shared by pulegone (30 - 300 microM), the principal component of the essential oil (IC (50) values of 21.8 +/- 2.1 microg/mL and 12.7 +/- 4.6 microg/mL , respectively). Nifedipine (30 nM - 30 microM) also abolished agonist-stimulated contractions, and was approximately twice and 12 times as potent as EOMP in inhibiting oxytocin- and prostaglandin F (2alpha) (PGF (2alpha))-stimulated contractions, respectively. In conclusion, our results show that the essential oil of the abortifaceant plant Mentha pulegium exerts an inhibitory effect on the contractile activity of the isolated rat myometrium. This oil shares a common effect with the voltage-dependent calcium channel (VDCC) blocker nifedipine, although ostensibly acting via a different mechanism. It thus appears that EOMP and pulegone do not exert direct toxic effects on the myometrium per se that would cause abortion, and other possibilities such as systemic metabolism of plant constituents may rather underlie the abusive use of Mentha pulegium in popular medicine.

Impairment of inflammatory response in adult rats submitted to maternal undernutrition during early lactation: role of insulin and glucocorticoid.

OBJECTIVE: Early nutritional environment may program permanent metabolic alterations, predisposing to later diseases. We have investigated the interference of maternal malnutrition during lactation with the development of acute inflammation in adult rats. MATERIALS AND METHODS: Adult rats, offspring of dams fed with either protein-free diet (UN group) or 22% protein diet (C group) during the first 10 days of lactation, were submitted to pleurisy with carrageenan (500 microg/cavity). Pleural edema, neutrophil migration and ICAM expression, were evaluated 4 h after and correlated with alterations in plasma insulin and corticosterone. Leukocyte-endothelium interactions were evaluated by intravital microscopy 1 h after inflammation. RESULTS: Compared to controls, UN rats showed a decrease in pleural edema formation (50%), neutrophil migration (50%), endothelial ICAM-1 expression on pulmonary tissue, and impairment in leukocyte adhesion (50%) and migration (80%) through endothelium. Circulating insulin was lower (42%) and corticosterone was higher (34%) in UN, compared to controls. Pre-treatment of UN with insulin (5 IU/day) or RU486 (20 mg/kg/day), inhibitor of glucocorticoid receptor, restored leukocyte functions and ICAM-1 expression. CONCLUSION: Postnatal maternal malnutrition, programming for permanent alterations in insulin and glucocorticoid secretion in progeny, that were unable to properly mount an inflammatory response, probably predisposes to chronic diseases in adult life.

Antinociceptive properties of the essential oil of Ocimum gratissimum L. (Labiatae) in mice.

We have investigated the antinociceptive effects of the essential oil of Ocimum gratissimum L. (Labiatae) (EOOG) in two classical models of pain in male Swiss mice (25-35 g), the writhing test and the formalin test. At doses of 30, 100 and 300 mg/kg (po), EOOG produced a dose-dependent inhibition (from 58.3 4.4 to 40.7 6.3, 36.4 3.6 and 24.6 3.6, respectively; N = 8-10, P<0.05) of acetic acid-induced writhing, causing up to a ~60% inhibition at the highest dose used, comparable to that obtained with indomethacin (10 mg/kg, po). At the same doses, EOOG predominantly inhibited the late (inflammatory) phase of the formalin-induced pain response (from 59.3 8.3 to 40.4 4.8, 23.2 2.8 and 25.3 5.5, respectively; N = 6, P<0.05), with a maximal reduction of ~60% of the control, although a significant reduction of the initial (neurogenic) phase was also observed at 300 mg/kg (from 62.5 6.07 to 37 5.9; P<0.05). On the basis of these data, we conclude that EOOG possesses interesting antinociceptive properties in the writhing and formalin tests. Due to the relatively low toxicity of EOOG, further detailed examination is strongly indicated for a better characterization of its pharmacological properties and its potential therapeutic value.

Antinociceptive properties of the essential oil of Ocimum gratissimum L. (Labiatae) in mice

We have investigated the antinociceptive effects of the essential oil of Ocimum gratissimum L. (Labiatae) (EOOG) in two classical models of pain in male Swiss mice (25-35 g), the writhing test and the formalin test. At doses of 30, 100 and 300 mg/kg (po), EOOG produced a dose-dependent inhibition (from 58.3 ± 4.4 to 40.7 ± 6.3, 36.4 ± 3.6 and 24.6 ± 3.6, respectively; N = 8-10, P<0.05) of acetic acid-induced writhing, causing up to a 60 percent inhibition at the highest dose used, comparable to that obtained with indomethacin (10 mg/kg, po). At the same doses, EOOG predominantly inhibited the late (inflammatory) phase of the formalin-induced pain response (from 59.3 ± 8.3 to 40.4 ± 4.8, 23.2 ± 2.8 and 25.3 ± 5.5, respectively; N = 6, P<0.05), with a maximal reduction of 60 percent of the control, although a significant reduction of the initial (neurogenic) phase was also observed at 300 mg/kg (from 62.5 ± 6.07 to 37 ± 5.9; P<0.05). On the basis of these data, we conclude that EOOG possesses interesting antinociceptive properties in the writhing and formalin tests. Due to the relatively low toxicity of EOOG, further detailed examination is strongly indicated for a better characterization of its pharmacological properties and its potential therapeutic value

A phase III trial of efficacy of the FML-vaccine against canine kala-azar in an endemic area of Brazil (São Gonçalo do Amaranto, RN).

Protection against canine kala-azar was investigated in naturally exposed dogs of an endemic area, vaccinated with the fucose mannose ligand (FML)-vaccine of Leishmania donovani. A total of 97% of vaccinees were seropositive to FML and 100% showed intradermal reaction to L. donovani lysate, 7 months after vaccination. The absorbency values and size of intradermal reaction were both significantly higher in vaccinees than in controls (ANOVA, P<0.0001). After 2 years, 92% (chi(2)=6.996; P<0.0025) protection was achieved: only 8% of vaccinees showed mild signs of kala-azar with no deaths while 33% of controls developed clinical or fatal disease. The FML-vaccine induced a significant, long-lasting and strong protective effect against canine kala-azar in the field.

[Educational program in universal precaution measures: a methodological approach]. / Programa educativo em medidas de precaução universais: uma metodologia de abordagem.

An updating program on measures of universal precautions (M.U.P.) was developed at the Center of Whole Care of Woman's Health (Centro de Atenção Integral à Saúde da Mulher-CAISM). These measures and the procedures in the case of work accident were published in a booklet. First, servants should be aware of the matter of stress and its influence on the quality of life. Then, updating was carried through encouraging the reflection on the consequences of the non-adoption of M.U.P. The answers to 286 pre-tests and 242 post-tests were analyzed and the results showed a significantly higher index of correct answers (p < 0.01), mainly regarding the appropriate use of glove.

The FML (Fucose Mannose Ligand) of Leishmania donovani. a new tool in diagnosis, prognosis, transfusional control and vaccination against human kala-azar.

The Fucose-Mannose Ligand (FML) of Leishmania donovani is a complex glycoproteic fraction. Its potential use as a tool for diagnosis of human visceral leishmaniasis was tested with human sera from Natal, Rio Grande do Norte, Brazil. The FML-ELISA test, showed 100% sensitivity and 96% specificity, identifying patients with overt kala-azar (p < 0.001, when compared to normal sera), and subjects with subclinical infection. More than 20% apparently healthy subjects with positive reaction to FML developed overt kala-azar during the following 10 months. In the screening of human blood donnors, a prevalence of 5% of sororeactive subjects was detected, attaining 17% in a single day. The GP36 glycoprotein of FHL is specifically reconized by human kala-azar sera. The immunoprotective effect of FML on experimental L. donovani infection was tested in swiss albino mice. The protection scheemes included three weekly doses of FML, supplemented or not with saponin by the subcutaneous or intraperitoneal routes and challenge with 2 x 10(7) amastigotes of Leishmania donovani. An enhancement of 80.0% in antibody response (p < 0.001) and reduction of 85.5% parasite liver burden (p < 0.001) was detected in animals immunized with FML saponin, unrespectively of the immunization route.

Kinetic mechanism of the inhibition of human urinary kallikrein by basic pancreatic trypsin inhibitor.

Hydrolysis of D-valyl-L-leucyl-L-arginine p-nitroanilide (D-Val-Leu-Arg-Nan) at five different concentrations (10-20 microM) by human urinary kallikrein was studied in the absence and in the presence of increasing concentrations of basic pancreatic trypsin inhibitor (BPTI) (1.35-9.15 nM). The data indicate that the inhibition of human urinary kallikrein by BPTI is not a simple competitive inhibition as reported by others, but that it is a competitive inhibition of the parabolic type, with two inhibitor molecules binding to one enzyme molecule, with the formation of a ternary enzymatic complex. Statistical analysis of the experimental data supports the kinetic model proposed. The calculated values of the constants Ki and Kii were 16.20 nM and 1.10 nM, respectively. It is noteworthy that the Kii < Ki, i.e., the second BPTI molecule binds to the enzyme with a larger affinity suggesting that this second binding site was probably created or positively modulated as a consequence of the binding of the first BPTI molecule.

Kinetic mechanism of the inhibition of human urinary kallikrein by basic pancreatic trypsin inhibitor

Hydrolysis of D-valvyl-L-leucyl-L-arginine p-nitroanilide (D-Val-Leu-Arg-Nan) at five different concentration (10-20µM) by human urinary kallikrein was studied in the absence and in the presence of increasing concentrations of basic pancreatic trypsin inhibitor (BPTI) (1.35-9.15nM). The data indicate that the inhbition of human urinary kallikrein by BPTI is not a simple competitive inhibition as reported by others, but that it is a competitive inhibition of the parabolic type, with two inhibitor molecules binding to one enzyme molecule, with the formetion of a ternary enzymatic complex. Statistical analysis of the experimental data supports the kinetic model proposed. The calculated values of the constants Ki and Kii were 16.20 nM and 1.10 nM, repectively. It is noteworthy that the Kii < Ki, i.e., the second BPTI molecule binds to the enzyme with a larger affinity suggesting that this second binding site was probably created or positively modulated as a consequence of the binding of the first BPTI molecule