Adiponectin, HOMA-Adiponectin, HOMA-IR in Children and Adolescents: Ouro Preto Study.

OBJECTIVES: To examine the association and predictive capacity of adiponectin levels, HOMA-AD and HOMA-IR indexes with metabolic risk markers in children and adolescents. METHODS: A cross-sectional study was conducted with 691 children and adolescents (7-14 y), of both sexes. Demographic (sex, age), anthropometric (weight, height, body mass index, waist circumference, body fat), biochemical [total cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL), triglycerides, fasting glycemia, insulin and adiponectin] and clinical parameters (arterial blood pressure) were analyzed. RESULTS: In multiple linear regression models, metabolic risk were analyzed in relation to adiponectin levels, HOMA-AD and HOMA-IR. ROC curve analysis was used to define the cut-off for metabolic syndrome for each method studied. Adiponectin level was inversely correlated with weight (r = -0.12; p = 0.01), waist circumference (WC) (r = -0.12; p = 0.01), and triglycerides (r = -0.11; p = 0.02); it was directly correlated with HDL (r = 0.10; p = 0.03) only in the adolescents. In the final linear regression model, after adjustment, only triglycerides (p = 0.03) and HDL (p = 0.04) remained significant. However, HOMA-AD and HOMA-IR were associated with metabolic risk and were the most suitable methods for metabolic syndrome screening in both age groups. For children, independent variables explained 16.0% and 14.5% of HOMA-AD and HOMA-IR, respectively. For adolescents, R2 was higher in HOMA-AD and HOMA-IR models (R2adjusted = 31.9% and R2adjusted = 29.6%, respectively). CONCLUSIONS: HOMA-AD and HOMA-IR are better explained by metabolic markers than adiponectin levels.

High levels of chemerin associated with variants in the NOS3 and APOB genes in rural populations of Ouro Preto, Minas Gerais, Brazil.

Chemerin is an adipokine that has been associated with components of metabolic syndrome. It has been described to affect adipocyte metabolism and inflammatory responses in adipose tissue, as well as the systemic metabolism of lipids and glucose. Few epidemiological studies have evaluated classical and genetics cardiovascular risk factors (CVRFs) in the mixed adult rural population in Brazil. Therefore, the present study explored possible associations between CVRFs and chemerin. This cross-sectional study included 508 adults from the rural localities of Lavras Novas, Chapada, and Santo Antônio do Salto in Ouro Preto, Minas Gerais, Southeast Brazil. Demographic, behavioral, clinical, biochemical, anthropometric variables, and 12 single nucleotide polymorphisms (SNPs) linked with metabolic syndrome phenotypes were evaluated for associations with chemerin level. There was a significant association of high triglyceride levels [odds ratio (OR)=1.91, 95%CI: 1.23-2.98], insulin resistance (OR=1.82, 95%CI: 1.03-3.22), age (OR=1.64, 95%CI: 1.08-2.49), and sex (OR=1.99, 95%CI: 1.35-2.95) with high levels of chemerin. High chemerin levels were significantly associated with the genetic polymorphisms rs693 in the APOB gene (OR=1.50, 95%CI: 1.03-2.19) and rs1799983 in the NOS3 gene (OR=1.46, 95%CI: 1.01-2.12) for the AA and GT+TT genotypes, respectively. In the concomitant presence of genotypes AA of rs693 and GT+TT of rs1799983, the chance of presenting high levels of chemerin showed a 2.21-fold increase (95%CI: 1.25-3.88) compared to the reference genotype. The development of classical CVRFs in this population may be influenced by chemerin and by two risk genotypes characteristic of variants in well-studied genes for hypertension and dyslipidemia.

High levels of chemerin associated with variants in the NOS3 and APOB genes in rural populations of Ouro Preto, Minas Gerais, Brazil

Chemerin is an adipokine that has been associated with components of metabolic syndrome. It has been described to affect adipocyte metabolism and inflammatory responses in adipose tissue, as well as the systemic metabolism of lipids and glucose. Few epidemiological studies have evaluated classical and genetics cardiovascular risk factors (CVRFs) in the mixed adult rural population in Brazil. Therefore, the present study explored possible associations between CVRFs and chemerin. This cross-sectional study included 508 adults from the rural localities of Lavras Novas, Chapada, and Santo Antônio do Salto in Ouro Preto, Minas Gerais, Southeast Brazil. Demographic, behavioral, clinical, biochemical, anthropometric variables, and 12 single nucleotide polymorphisms (SNPs) linked with metabolic syndrome phenotypes were evaluated for associations with chemerin level. There was a significant association of high triglyceride levels [odds ratio (OR)=1.91, 95%CI: 1.23−2.98], insulin resistance (OR=1.82, 95%CI: 1.03−3.22), age (OR=1.64, 95%CI: 1.08−2.49), and sex (OR=1.99, 95%CI: 1.35−2.95) with high levels of chemerin. High chemerin levels were significantly associated with the genetic polymorphisms rs693 in the APOB gene (OR=1.50, 95%CI: 1.03−2.19) and rs1799983 in the NOS3 gene (OR=1.46, 95%CI: 1.01−2.12) for the AA and GT+TT genotypes, respectively. In the concomitant presence of genotypes AA of rs693 and GT+TT of rs1799983, the chance of presenting high levels of chemerin showed a 2.21-fold increase (95%CI: 1.25−3.88) compared to the reference genotype. The development of classical CVRFs in this population may be influenced by chemerin and by two risk genotypes characteristic of variants in well-studied genes for hypertension and dyslipidemia.

IGF2, LEPR, POMC, PPARG, and PPARGC1 gene variants are associated with obesity-related risk phenotypes in Brazilian children and adolescents

Association studies of genetic variants and obesity and/or obesity-related risk factors have yielded contradictory results. The aim of the present study was to determine the possible association of five single-nucleotide polymorphisms (SNPs) located in the IGF2, LEPR, POMC, PPARG, and PPARGC1 genes with obesity or obesity-related risk phenotypes. This case-control study assessed overweight (n=192) and normal-weight (n=211) children and adolescents. The SNPs were analyzed using minisequencing assays, and variables and genotype distributions between the groups were compared using one-way analysis of variance and Pearson's chi-square or Fisher's exact tests. Logistic regression analysis adjusted for age and gender was used to calculate the odds ratios (ORs) for selected phenotype risks in each group. No difference in SNP distribution was observed between groups. In children, POMC rs28932472(C) was associated with lower diastolic blood pressure (P=0.001), higher low-density lipoprotein (LDL) cholesterol (P=0.014), and higher risk in overweight children of altered total cholesterol (OR=7.35, P=0.006). In adolescents, IGF2 rs680(A) was associated with higher glucose (P=0.012) and higher risk in overweight adolescents for altered insulin (OR=10.08, P=0.005) and homeostasis model of insulin resistance (HOMA-IR) (OR=6.34, P=0.010). PPARG rs1801282(G) conferred a higher risk of altered insulin (OR=12.31, P=0.003), and HOMA-IR (OR=7.47, P=0.005) in overweight adolescents. PARGC1 rs8192678(A) was associated with higher triacylglycerols (P=0.005), and LEPR rs1137101(A) was marginally associated with higher LDL cholesterol (P=0.017). LEPR rs1137101(A) conferred higher risk for altered insulin, and HOMA-IR in overweight adolescents. The associations observed in this population suggested increased risk for cardiovascular diseases and/or type 2 diabetes later in life for individuals carrying these alleles.

IGF2, LEPR, POMC, PPARG, and PPARGC1 gene variants are associated with obesity-related risk phenotypes in Brazilian children and adolescents.

Association studies of genetic variants and obesity and/or obesity-related risk factors have yielded contradictory results. The aim of the present study was to determine the possible association of five single-nucleotide polymorphisms (SNPs) located in the IGF2, LEPR, POMC, PPARG, and PPARGC1 genes with obesity or obesity-related risk phenotypes. This case-control study assessed overweight (n=192) and normal-weight (n=211) children and adolescents. The SNPs were analyzed using minisequencing assays, and variables and genotype distributions between the groups were compared using one-way analysis of variance and Pearson's chi-square or Fisher's exact tests. Logistic regression analysis adjusted for age and gender was used to calculate the odds ratios (ORs) for selected phenotype risks in each group. No difference in SNP distribution was observed between groups. In children, POMC rs28932472(C) was associated with lower diastolic blood pressure (P=0.001), higher low-density lipoprotein (LDL) cholesterol (P=0.014), and higher risk in overweight children of altered total cholesterol (OR=7.35, P=0.006). In adolescents, IGF2 rs680(A) was associated with higher glucose (P=0.012) and higher risk in overweight adolescents for altered insulin (OR=10.08, P=0.005) and homeostasis model of insulin resistance (HOMA-IR) (OR=6.34, P=0.010). PPARG rs1801282(G) conferred a higher risk of altered insulin (OR=12.31, P=0.003), and HOMA-IR (OR=7.47, P=0.005) in overweight adolescents. PARGC1 rs8192678(A) was associated with higher triacylglycerols (P=0.005), and LEPR rs1137101(A) was marginally associated with higher LDL cholesterol (P=0.017). LEPR rs1137101(A) conferred higher risk for altered insulin, and HOMA-IR in overweight adolescents. The associations observed in this population suggested increased risk for cardiovascular diseases and/or type 2 diabetes later in life for individuals carrying these alleles.

Genetic composition of a Brazilian population: the footprint of the Gold Cycle.

Ancestry-informative markers (AIMs) are powerful tools for inferring the genetic composition of admixed populations. In this study, we determined the genetic ancestry of the Ouro Preto (Brazil) population and evaluated the association between ancestry and self-reported skin color. The genetic ancestry of 189 children and adolescents was estimated by genotyping 15 AIMs. The estimate of population admixture was determined using the Bayesian Markov Chain Monte Carlo (MCMC) method implemented in two different programs (STRUCTURE and ADMIXMAP). Volunteers self-reported their skin colors. The European ancestry contribution ranged from 0.503 to 0.539, the African contribution ranged from 0.333 to 0.425, and the Amerindian component ranged from 0.04 to 0.164. The relative contributions of African (P < 0.016) and European (P < 0.011) ancestry differed significantly among skin color groups, except between black and dark-brown groups. The population of Ouro Preto has a higher contribution of African ancestry compared to the mean for the southeast region of Brazil. Therefore, extrapolating the African ancestry contribution for southeastern Brazil to the Ouro Preto population would underestimate the actual value for this city. We also showed that self-reported skin color could be appropriate for describing the genetic structure of this particular population.

Anthropometric methods for obesity screening in schoolchildren: the Ouro Preto Study.

BACKGROUND AND AIMS: Childhood obesity is increasing dramatically in last decades. To evaluate the usefulness of body mass index (BMI), skinfold thickness (ST), waist circumference (WC), and foot-to-foot bioelectrical impedance (BIA-FF) for screening for obesity in mixed-race population, using the tetrapolar bioelectrical impedance (BIA-T) technique as reference method. METHODS AND RESULTS: A cross-sectional-based population study was performed in the city of Ouro Preto, Brazil, in 2006. Schoolchildren aged 6-15 years (n = 788) was randomly selected according to age and sex stratified by the proportion of students in each schools of the city. Nonparametric receiver operating characteristic (ROC) analysis was used to define the sensitivity and specificity for each method studied using the tetrapolar method as reference. The BMI and the BIA-FF were the most suitable for adiposity screening in pre-pubertal and pubertal stages because they present a better balance between sensitivity and specificity, and smaller misclassification. For post-pubertal boys, the BF-ST-D was the best body fat assessment method. CONCLUSION: The results suggest that BIA-FF and BMI are choice methods for obesity screening in mixed population and that the method choice for body fat screening must be done according to sexual maturity of boys and girls. The present study demonstrates the need to perform studies in wider mixed-race population to determine anthropometric parameters and to examine the predictive ability of methods and cut-offs here elucidated in the development of obesity.

Persistence of PCR-positive tissue in benznidazole-treated mice with negative blood parasitological and serological tests in dual infections with Trypanosoma cruzi stocks from different genotypes.

OBJECTIVES: To assess different methodologies to better define an early post-therapeutic cure criterion after benznidazole treatment in BALB/c mice following mixed infection with dual Trypanosoma cruzi genotypes. METHODS: According to the classical cure criteria, animals were classified as treated not cured (TNC = 76.4%), treated cured (TC = 12.5%) and dissociated (DIS = 11.1%) using parasitological [fresh blood examination (FBE), blood culture (BC) and blood PCR] and serological methods [conventional serology (CS-ELISA) and non-conventional serology (NCS-FC-ALTA)]. Tissues were also evaluated by PCR. RESULTS: FBE was able to detect patent parasitaemia in only 18.1% of TNC and therapeutic failure was detected in 79.1% and 97.2% of TNC by BC and blood PCR, respectively. CS-ELISA should not be used before 3 months after treatment since it may lead to false-negative results. At 3 months after treatment with benznidazole, NCS-FC-ALTA was more efficient for categorizing the groups of treated mice. In the TNC group, although a decreased frequency of PCR-positive tissue was observed in several host tissues, increased positivity was also observed, despite the T. cruzi genotype combination. All TC animals presented at least two positive tissue-PCR results. CONCLUSIONS: Our results confirm that NSC-FC-ALTA and blood PCR are the most suitable methods to early detect therapeutic failure in acute murine T. cruzi infection. Additionally, our data show that BC positivity is highly dependent upon the T. cruzi genotype combination. Moreover, our findings demonstrated that PCR tests performed on tissues from animals considered cured after benznidazole treatment still detected T. cruzi DNA, most probably indicating residual infection.

Impact of dual infections on chemotherapeutic efficacy in BALB/c mice infected with major genotypes of Trypanosoma cruzi.

The aim of this work was to investigate the impact of dual infections with stocks of Trypanosoma cruzi major genotypes on benznidazole (BZ) treatment efficacy. For this purpose, T. cruzi stocks representative of the genetic T. cruzi lineages, displaying different susceptibilities to BZ, belonging to the major T. cruzi genotypes broadly dispersed in North and South America and important in Chagas' disease epidemiology were used. Therapeutic efficacy was observed in 27.8% of the animals treated. Following BZ susceptibility classification, significant differences were observed in dual infections on the major genotype level, demonstrating that combinations of genotypes 19+39 and genotypes 19+32 led to a shift in the expected BZ susceptibility profile toward the resistance pattern. Analysis on the T. cruzi stock level demonstrated that 9 out of 24 dual infections shifted the expected BZ susceptibility profile compared with the respective single infections, including shifts toward lower and higher BZ susceptibilities. Microsatellite identification was able to identify a mixture of T. cruzi stocks in 7.7% of the T. cruzi isolates from infected and untreated mice (6.9%) and infected and treated but not cured mice (9.0%), revealing in some mixtures of BZ-susceptible and -resistant stocks that the T. cruzi stock identified after BZ treatment was previously susceptible in single infections. Considering the clonal structure and evolution of T. cruzi, an unexpected result was the identification of parasite subpopulations with distinct microsatellite alleles in relation to the original stocks observed in 12.2% of the isolates. Taken together, the data suggest that mixed infections, already verified in nature, may have an important impact on the efficacy of chemotherapy.

Apolipoprotein E polymorphism in Brazilian dyslipidemic individuals: Ouro Preto study.

The influence of apolipoprotein E alleles and genotypes on plasma lipid levels was determined in 185 individuals of mixed ethnicity living in Ouro Preto, Brazil. DNA was obtained from blood samples and the genotypes were determined by an RFLP-PCR procedure. The *3 allele was the most frequent (72%), followed by *4 (20%) and *2 (8%); *4 frequency was higher and *2 frequency was lower in the dyslipidemic group than in the normal control group. The *2 carriers presented lower LDL and total cholesterol levels compared to the *3 and *4 carriers. All six expected genotypes were observed in the individuals genotyped: E2/2 (2.1%), E4/4 (2.7%), E2/4 (3.7%), E2/3 (8.0%), E3/3 (53.3%), E3/4 (29.9%); no difference in genotype frequencies was found between the normal and dyslipidemic groups. Compared with *2, the presence of *3 increases more than two times the risk for dyslipidemia (OR = 2.31; P = 0.025; 95% CI = 1.06-5.06) and the presence of *4 increases it three times (OR = 3.31; P = 0.006; 95% CI = 1.36-8.04). The only significant effect of genotype was an increased risk for dyslipidemia in the *4 genotype carriers (E3/4 + E4/4) compared with the *2 genotype carriers (E2/2 + E2/3) with OR = 3.69 (95% CI = 1.25-10.88). The present study indicates that in the Ouro Preto admixed population the presence of APOE *2 can confer a protective effect, whereas the presence of APOE *4 implies an enhanced risk for dyslipidemia.

Apolipoprotein E polymorphism in Brazilian dyslipidemic individuals: Ouro Preto study

The influence of apolipoprotein E alleles and genotypes on plasma lipid levels was determined in 185 individuals of mixed ethnicity living in Ouro Preto, Brazil. DNA was obtained from blood samples and the genotypes were determined by an RFLP-PCR procedure. The *3 allele was the most frequent (72 percent), followed by *4 (20 percent) and *2 (8 percent); *4 frequency was higher and *2 frequency was lower in the dyslipidemic group than in the normal control group. The *2 carriers presented lower LDL and total cholesterol levels compared to the *3 and *4 carriers. All six expected genotypes were observed in the individuals genotyped: E2/2 (2.1 percent), E4/4 (2.7 percent), E2/4 (3.7 percent), E2/3 (8.0 percent), E3/3 (53.3 percent), E3/4 (29.9 percent); no difference in genotype frequencies was found between the normal and dyslipidemic groups. Compared with *2, the presence of *3 increases more than two times the risk for dyslipidemia (OR = 2.31; P = 0.025; 95 percent CI = 1.06-5.06) and the presence of *4 increases it three times (OR = 3.31; P = 0.006; 95 percent CI = 1.36-8.04). The only significant effect of genotype was an increased risk for dyslipidemia in the *4 genotype carriers (E3/4 + E4/4) compared with the *2 genotype carriers (E2/2 + E2/3) with OR = 3.69 (95 percent CI = 1.25-10.88). The present study indicates that in the Ouro Preto admixed population the presence of APOE *2 can confer a protective effect, whereas the presence of APOE *4 implies an enhanced risk for dyslipidemia.

Trypanosoma cruzi: Impact of dual-clone infections on parasite biological properties in BALB/c mice.

Herein, we have analyzed major biological properties following dual-clone Trypanosoma cruzi infections in BALB/c mice. Eight T. cruzi clonal stocks, two of each principal genotype, including genotype 19 and 20 (T. cruzi I), hybrid genotype 39 (T. cruzi) and 32 (T. cruzi II) were combined into 24 different dual-clone infections. Special attention was given to characterize biological parameters assayed including: prepatent period, patent period, maximum of parasitemia, day of maximum parasitemia, area under the parasitemia curve, infectivity, mortality, and hemoculture positivity. Our findings clearly demonstrated that features resultant of dual-clone infections of T. cruzi clonal stocks did not display either the characteristics of the corresponding monoclonal infections or the theoretical mixture based on the respective monoclonal infections. Significant changes in the expected values were observed in 4.2-79.2% of the mixtures considering the eight biological parameters studied. A lower frequency of significant differences was found for mixtures composed by phylogenetically distant clonal stocks. Altogether, our data support our hypothesis that mixed T. cruzi infections have a great impact on the biological properties of the parasite in the host and re-emphasizes the importance of considering the possible occurrence of natural mixed infections in humans and their consequences on the biological aspects of ongoing Chagas' disease.

Influence of the long-term Trypanosoma cruzi infection in vertebrate host on the genetic and biological diversity of the parasite.

The influence of the long-term Trypanosoma cruzi infection in vertebrate host on the biological and genetic properties of the parasite was evaluated. Four T. cruzi isolates obtained from different chronic chagasic dogs infected with Berenice-78 T. cruzi strain during 2 and 7 years were comparatively analyzed. The long-term T. cruzi infection has led to alterations in parasitemia, virulence and pathogenicity of Be-78 strain for mice. These biological parameters varied from low to high in realation to the parental strain. Randomly amplified polymorphic DNA and isoenzyme profiles detected two distinct genetic groups of parasites. The first group included the parental strain and two T. cruzi isolates, and the second group the two other isolates. Interestingly, the isolates of the second group showed a reversibility of the genetic profile to the parental strain after 25 passages in mice. No correlation between the genetic groups and biological properties of the isolates was observed. Our findings confirmed the population heterogeneity of the Be-78 strain, and showed how differently it responds to the long-term infection in the same vertebrate hosts.

Effects of specific treatment on parasitological and histopathological parameters in mice infected with different Trypanosoma cruzi clonal genotypes.

The goal of this study was to verify the effect of specific treatment on parasitological and histopathological parameters in mice experimentally infected with different Trypanosoma cruzi clonal genotypes. Twenty cloned stocks were selected, representative of the whole phylogenetic diversity of the protozoan and belonging to the clonal genotypes 19 and 20 (T. cruzi I) and 39 and 32 (T. cruzi II). The stocks were inoculated in 40 BALB/c mice divided into four groups: (i) treated with benznidazole, (ii) treated with itraconazole and (iii and iv) untreated control groups (NT) for each drug, respectively. Seven parameters related to parasitaemia curves and histopathological lesions were analysed. Four during the acute phase (AP) and three during both the AP and chronic phase (CP) of infection. Statistical comparison between benznidazole-treated and NT groups for the biological parameters showed significant differences for all genotypes. Benznidazole treatment led to lower patent period, maximum of parasitaemia, day of maximum parasitaemia and area under the parasitaemia curve for all genotypes analysed. Percentage of positive haemoculture during AP and CP was lower for genotypes 19 and 32. Tissue parasitism (TP) and inflammatory process (IP) during AP were lower for genotypes 19 and 32, respectively. In general, itraconazole treatment induced a smaller reduction in these same parameters between treated and NT animals in relation to benznidazole treatment. Our results indicate that phylogenetic divergence among T. cruzi clonal genotypes must be taken in account in chemotherapy and studies dealing with all aspects of the parasite and the disease.

Impact of Trypanosoma cruzi clonal evolution on its biological properties in mice.

Twenty Trypanosoma cruzi stocks attributed to the 19, 20, 39, and 32 clonal genotypes were comparatively studied in BALB/c mice during the acute and chronic phases of the infection to test the working hypothesis that T. cruzi clonal structure has a major impact on its biological properties. Fourteen parameters were assayed: (1) infectivity; (2) prepatent period; (3) patent period; (4) maximum of parasitemia; (5) day of maximum of parasitemia; (6) parasitemia; (7) mortality, (8) percentage of positive hemoculture, (9) tissue parasitism; (10) inflammatory process during the acute phase of the infection; (11) mortality, (12) percentage of positive hemoculture; (13) tissue parasitism; and (14) inflammatory process during the chronic phase of the infection. Statistical comparison showed that the results are overall consistent with the working hypothesis that biological differences are proportional to the evolutionary divergence among the genotypes. Thus, closely related genotypes (19 vs 20 and 32 vs 39) show in general fewer differences than distantly related groups (19 or 20 vs 32 or 39) except for the comparison between 19 and 32. The working hypothesis is even more strongly supported by the result of the nonparametric Mantel test, which showed a highly significant correlation (P = 2.3 x 10(-3)) between biological differences and genetic distances among all pairs of stocks. These data taken together emphasize that it is crucial to take into account the phylogenetic diversity of T. cruzi natural clones in all applied studies dealing with diagnosis, drug and vaccine design, epidemiological surveys, and clinical diversity of Chagas' disease. Index Descriptors and Abbreviations: Trypanosoma cruzi; phylogenetic distance; biological properties; clonal theory; multilocus enzyme electrophoresis (MLEE); randomly amplified polymorphic DNA (RAPD); acute phase (AP); chronic phase (CP); days after inoculation (d.a.i.); liver infusion tryptose (LIT); gastrointestinal tract (GIT); genitourinary tract (GUT); percentage of infectivity (%INF); percentage of mortality during the acute phase (%MORT AP); percentage of mortality during the chronic phase (%MORT CP); prepatent period (PPP); patent period (PP); maximum of parasitemia (MP); day of maximum of parasitemia (DMP); parasitemia (PAR); percentage of positive hemoculture during the acute phase (% + HC AP); percentage of positive hemoculture during the chronic acute phase (% + HC CP); tissue parasitism (TP); inflammatory process (IP); tissue parasitism during the acute phase (TP AP); tissue parasitism during chronic phase (TP CP); inflammatory process during acute phase (IP AP); inflammatory process chronic phase (IP CP); Mann-Whitney test (MW); Kruskal-Wallis (KW); Kolmogorow-Smirnov test (KS).

Kinetic characterization of rat tissue kallikrein using NÓ-substituted arginine 4-nitroanilides and NÓ-benzoyl-L-arginine ethyl ester as substrates

Hydrolysis of seven N(alpha-substituted L-arginine 4-nitroanilides: henzoyl-arginine p-nitroanilide (Bz-Arg-Nan), tosyl-arginine p-nitroanilide (Tos-Arg-Nan), acetyl-leucyl-arginine p-nitroanilide (Ac-Leu-Arg-Nan), acetyl-phenylalanyl-arginine p-nitroanilide (Ac-Phe-Arg-Nan), benzoyl-phenylalanyl-arginine p-nitroanilide (Bz-Phe-Arg-Nan), tosyl-phenylalanyl-arginine p-nitroanilide (Tos-Phe-Arg-Nan), and D-valyl-leucyl-arginine p-nitroanilide (D-Val-Leu-Arg-Nan), and the N(alpha-substituted L-arginine ester: benzoyl-arginine ethyl ester (Bz-Arg-OEt), by rat tissue kallikrein was studied throughout a wide range of substrate concentrations. The enzyme showed a bimodal behavior with all the substrates tested except Tos-Arg-Nan. At low substrate concentrations (10 to 170 muM for p-nitroanilides and 50 to 190 muM for Bz-Arg-OEt) the hydrolysis followed Michaelis-Menten kinetics, but at higher substrate concentrations (150 to 700 muM for p-nitroanilides and 200 to 1800 muM for Bz-Arg-OEt) a deviation from Michaelis-Menten kinetics was observed with a significant decrease in hydrolysis rates. At high concentrations of the p-nitroanilide substrates, partial enzyme inhibition was observed, whereas complete enzyme inhibition was observed with Bz-Arg-OEt at high concentration. The kinetic parameters reported here were calculated from data for substrate concentrations range where the enzyme followed Michaelis-Menten behavior. D-Val-Leu-Arg-Nan (Km = 24 ñ 2 muM; Vmax 10.42 ñ 0.28 muM/min) was the best substrate tested, followed by Ac-Phe-Arg-Nan (Km = 13 ñ 2 muM; Vmax = 3.21 ñ 0.11 muM/min), while Tos-Arg-Nan (Km = 29 ñ 2 muM; Vmax, = 0. 10 ñ 0.002 muM/min) was the worst of the tested substrates for rat tissue kallikrein. For the hydrolysis of Bz-Arg-OEt (Km = 125 ñ 15 muM; Vmax = 121.3 ñ 7.6 muM/min), the kinetic parameters using a substrate inhibition model can reasonably account for the observed enzyme behavior, with a Ksi value about 13.6 times larger than the estimated Km value.

Utilizaçäo do eluato de sangue dessecado em papel de filtro no diagnóstico sorológico da toxoplasmose caprina / Use of dried blood sample for sorological diagnosis of toxoplasmosis in goats

Foi realizada pesquisa de anticorpos anti-Toxoplasma gondii em 183 amostras de sangue dessecado em papel de filtro utilizando as reaçöes de imunofluorescência indireta ELISA e dot-ELISA, tomando como referência os resultados obtidos nos soros. A análise dos resultados demonstrou que papéis com sangue dessecado podem ser conservados por um período mínimo de 45 dias à temperatura ambiente e por seis meses a 4§C, desde que mantidos livres de umidade pela utilizaçäo de agentes dessecantes como a sílica-gel. A reprodutibilidade das reaçöes, avaliada por meio da curva dos títulos de anticorpos no decorrer do tempo após a coleta do sangue em papéis de filtro, demonstrou uma concordância de 97 a 100 por cento entre os resultados obtidos nos soros e eluatos. Os títulos de anticorpos permaneceram estáveis durante o período observado. Os resultados obtidos com eluato de sangue dessecado foram semelhantes na RIFI, ELISA e dot-ELISA, indicando que qualquer uma das três reaçöes pode ser utilizada em eluatos de sangue dessecado para o diagnóstico da toxoplasmose caprina