Morphometric analysis of the Brasiliorchis picta complex (Orchidaceae)

One of the largest genera of Orchidaceae in the Neotropics with about 450 species, Maxillaria presents several taxonomic uncertainties about its generic circumscription and the delimitation of species groups, mainly due to the large variability of some species. The present study aims at verifying the morphological variation and species delimitation in the Brasiliorchis picta complex, a recent new genus derived from Maxillaria, using morphometric multivariate analysis. A total of 340 specimens belonging to six species (B. chrysantha (Barb. Rodr.) R.B. Singer, S. Koehler & Carnevali, B. gracilis (Lodd.) R.B. Singer, S. Koehler & Carnevali, B. marginata (Lindl.) R.B. Singer, S. Koehler & Carnevali, B. picta (Hook.) R. Singer, S. Koehler & Carnevali, B. porphyrostele (Rchb. f.) R.B. Singer, S. Koehler & Carnevali and B. ubatubana (Hoehne) R.B. Singer, S. Koehler & Carnevali) were analyzed using multivariate methods (PCA, CVA, DA, and Cluster Analysis with UPGMA). B. gracilis shows the largest morphological discontinuity, mainly due to its smaller size. The other species tend to form distinct groups, but intermediate characteristics between pairs of species induce overlaps among the individuals of different species and thus confuse the distinction of each one. Hybridization and geographic distribution can be involved in the differentiation of the species and lineages in this complex. Because the species classified a priori in this work cannot be recognized by the quantitative characters measured here, such other tools as geometric morphometry and molecular data should be employed in future works to clarify species relationships in this complex.

Flavianate, an amino acid precipitant, is a competitive inhibitor of trypsin at pH 3.0

Textile dyes bind to proteins leading to selective co-precipitation of a complex involving one protein molecule and more than one dye molecule of opposite charge in acid solutions, in a process of reversible denaturation that can be utilized for protein fractionation. In order to understand what occurs before the co-precipitation, a kinetic study using bovine ß-trypsin and sodium flavianate was carried out based on reaction progress curve techniques. The experiments were carried out using a-CBZ-L-Lys-p-nitrophenyl ester as substrate which was added to 50 mM sodium citrate buffer, pH 3.0, containing varying concentrations of ß-trypsin and dye. The reaction was recorded spectrophotometrically at 340 nm for 30 min, and the families of curves obtained were analyzed simultaneously by fitting integrated Michaelis-Menten equations. The dye used behaved as a competitive inhibitor of trypsin at pH 3.0, with Ki = 99 µM; kinetic parameters for the substrate hydrolysis were: Km = 32 µM, and kcat = 0.38/min. The competitive character of the inhibition suggests a specific binding of the first dye molecule to His-57, the only positively charged residue at the active site of the enzyme.

Studies on ATP-diphosphohydrolase nucleotide-binding sites by intrinsic fluorescence

Potato apyrase, a soluble ATP-diphosphohydrolase, was purified to homogeneity from several clonal varieties of Solanum tuberosum. Depending on the source of the enzyme, differences in kinetic and physicochemical properties have been described, which cannot be explained by the amino acid residues present in the active site. In order to understand the different kinetic behavior of the Pimpernel (ATPase/ADPase = 10) and Desirée (ATPase/ADPase = 1) isoenzymes, the nucleotide-binding site of these apyrases was explored using the intrinsic fluorescence of tryptophan. The intrinsic fluorescence of the two apyrases was slightly different. The maximum emission wavelengths of the Desirée and Pimpernel enzymes were 336 and 340 nm, respectively, suggesting small differences in the microenvironment of Trp residues. The Pimpernel enzyme emitted more fluorescence than the Desirée apyrase at the same concentration although both enzymes have the same number of Trp residues. The binding of the nonhydrolyzable substrate analogs decreased the fluorescence emission of both apyrases, indicating the presence of conformational changes in the neighborhood of Trp residues. Experiments with quenchers of different polarities, such as acrylamide, Cs+ and I- indicated the existence of differences in the nucleotide-binding site, as further shown by quenching experiments in the presence of nonhydrolyzable substrate analogs. Differences in the nucleotide-binding site may explain, at least in part, the kinetic differences of the Pimpernel and Desirée isoapyrases.

Linear competitive inhibition of human tissue kallikrein by 4-aminobenzamidine and benzamidine and linear mixed inhibition by 4-nitroaniline and aniline

Hydrolysis of D-valyl-L-leucyl-L-arginine p-nitroanilide (7.5-90.0 µM) by human tissue kallikrein (hK1) (4.58-5.27 nM) at pH 9.0 and 37ºC was studied in the absence and in the presence of increasing concentrations of 4-aminobenzamidine (96-576 µM), benzamidine (1.27-7.62 mM), 4-nitroaniline (16.5-66 µM) and aniline (20-50 mM). The kinetic parameters determined in the absence of inhibitors were: Km = 12.0 ± 0.8 µM and k cat = 48.4 ± 1.0 min-1. The data indicate that the inhibition of hK1 by 4-aminobenzamidine and benzamidine is linear competitive, while the inhibition by 4-nitroaniline and aniline is linear mixed, with the inhibitor being able to bind both to the free enzyme with a dissociation constant Ki yielding an EI complex, and to the ES complex with a dissociation constant Ki', yielding an ESI complex. The calculated Ki values for 4-aminobenzamidine, benzamidine, 4-nitroaniline and aniline were 146 ± 10, 1,098 ± 91, 38.6 ± 5.2 and 37,340 ± 5,400 µM, respectively. The calculated Ki' values for 4-nitroaniline and aniline were 289.3 ± 92.8 and 310,500 ± 38,600 µM, respectively. The fact that Ki'>Ki indicates that 4-nitroaniline and aniline bind to a second binding site in the enzyme with lower affinity than they bind to the active site. The data about the inhibition of hK1 by 4-aminobenzamidine and benzamidine help to explain previous observations that esters, anilides or chloromethyl ketone derivatives of Nalpha-substituted arginine are more sensitive substrates or inhibitors of hK1 than the corresponding lysine compounds.

Nitric oxide synthase blockade and body fluid volumes

The influence of chronic nitric oxide synthase inhibition with N G-nitro-L-arginine methyl ester (L-NAME) on body fluid distribution was studied in male Wistar rats weighing 260-340 g. Extracellular, interstitial and intracellular spaces, as well as plasma volume were measured after a three-week treatment with L-NAME (~70 mg/kg per 24 h in drinking water). An increase in extracellular space (16.1 ± 1.1 vs 13.7 ± 0.6 ml/100 g in control group, N = 12, P<0.01), interstitial space (14.0 ± 0.9 vs 9.7 ± 0.6 ml/100 g in control group, P<0.001) and total water (68.7 ± 3.9 vs 59.0 ± 2.9 ml/100 g, P<0.001) was observed in the L-NAME group (N = 8). Plasma volume was lower in L-NAME-treated rats (2.8 ± 0.2 ml/100 g) than in the control group (3.6 ± 0.1 ml/100 g, P<0.001). Blood volume was also lower in L-NAME-treated rats (5.2 ± 0.3 ml/100 g) than in the control group (7.2 ± 0.3 ml/100 g, P<0.001). The increase in total ratio of kidney wet weight to body weight in the L-NAME group (903 ± 31 vs 773 ± 45 mg/100 g in control group, P<0.01) but not in total kidney water suggests that this experimental hypertension occurs with an increase in renal mass. The fact that the heart weight to body weight ratio and the total heart water remained constant indicates that, despite the presence of high blood pressure, no modification in cardiac mass occurred. These data show that L-NAME-induced hypertension causes alterations in body fluid distribution and in renal mass.

Comparison of the homeostasis model assessment and quantitative insulin sensitivity check index with data from forearm metabolic studies for the in vivo assessment of insulin sensitivity

The present study was designed to compare the homeostasis model assessment (HOMA) and quantitative insulin sensitivity check index (QUICKI) with data from forearm metabolic studies of healthy individuals and of subjects in various pathological states. Fifty-five healthy individuals and 112 patients in various pathological states, including type 2 diabetes mellitus, essential hypertension and others, were studied after an overnight fast and for 3 h after ingestion of 75 g of glucose, by HOMA, QUICKI and the forearm technique to estimate muscle uptake of glucose combined with indirect calorimetry (oxidative and non-oxidative glucose metabolism). The patients showed increased HOMA (1.88 ± 0.14 vs 1.13 ± 0.10 pmol/l x mmol/l) and insulin/glucose (I/G) index (1.058.9 ± 340.9 vs 518.6 ± 70.7 pmol/l x (mg/100 ml forearm)-1), and decreased QUICKI (0.36 ± 0.004 vs 0.39 ± 0.006 (µU/ml + mg/dl)-1) compared with the healthy individuals. Analysis of the data for the group as a whole (patients and healthy individuals) showed that the estimate of insulin resistance by HOMA was correlated with data obtained in the forearm metabolic studies (glucose uptake: r = -0.16, P = 0.04; non-oxidative glucose metabolism: r = -0.20. P = 0.01, and I/G index: r = 0.17, P = 0.03). The comparison of QUICKI with data of the forearm metabolic studies showed significant correlation between QUICKI and non-oxidative glucose metabolism (r = 0.17, P = 0.03) or I/G index (r = -0.37, P < 0.0001). The HOMA and QUICKI are good estimates of insulin sensitivity as data derived from forearm metabolic studies involving direct measurements of insulin action on muscle glucose metabolism.

IgA response in serum and gut secretion in sensitized mice fed with the dust mite Dermatophagoides pteronyssinus extract

Induced oral tolerance to mucosal-exposed antigens in immunized animals is of particular interest for the development of immunotherapeutic approaches to human allergic diseases. This is a unique feature of mucosal surfaces which represent the main contact interface with the external environment. However, the influence of oral tolerance on specific and natural polyreactive IgA antibodies, the major defense mechanism of the mucosa, is unknown. We have shown that oral administration of an extract of the dust mite Dermatophagoides pteronyssinus (Dp) to primed mice caused down-regulation of IgE responses and an increase in tumor growth factor-ß secretion. In the present study, we observed that primed inbred female A/Sn mice (8 to 10 weeks old) fed by gavage a total weight of 1.0-mg Dp extract on the 6th, 7th and 8th days post-immunization presented normal secretion of IL-4 and IL-10 in gut-associated lymphoid tissue and a decreased production of interferon gamma induced by Dp in the draining lymph nodes (13,340 ± 3,519 vs 29,280 ± 2,971 pg/ml). Mice fed the Dp extract also showed higher levels of serum anti-Dp IgA antibodies and an increase of IgA-secreting cells in mesenteric lymph nodes (N = 10), reflecting an increase in total fecal IgA antibodies (N = 10). The levels of secretory anti-Dp IgA antibodies increased after re-immunization regardless of Dp extract feeding. Oral tolerance did not interfere with serum or secretory IgA antibody reactivity related to self and non-self antigens. These results suggest that induction of oral tolerance to a Dp extract in sensitized mice triggered different regulatory mechanisms which inhibited the IgE response and stimulated systemic and secretory IgA responses, preserving the natural polyreactive IgA antibody production.

Analysis of five streptokinase formulations using the euglobulin lysis test and the plasminogen activation assay

Streptokinase, a 47-kDa protein isolated and secreted by most group A, C and G ß-hemolytic streptococci, interacts with and activates human protein plasminogen to form an active complex capable of converting other plasminogen molecules to plasmin. Our objective was to compare five streptokinase formulations commercially available in Brazil in terms of their activity in the in vitro tests of euglobulin clot formation and of the hydrolysis of the plasmin-specific substrate S-2251™. Euglobulin lysis time was determined using a 96-well microtiter plate. Initially, human thrombin (10 IU/ml) and streptokinase were placed in individual wells, clot formation was initiated by the addition of plasma euglobulin, and turbidity was measured at 340 nm every 30 s. In the second assay, plasminogen activation was measured using the plasmin-specific substrate S-2251™. Streptase™ was used as the reference formulation because it presented the strongest fibrinolytic activity in the euglobulin lysis test. The Unitinase™ and Solustrep™ formulations were the weakest, showing about 50% activity compared to the reference formulation. All streptokinases tested activated plasminogen but significant differences were observed. In terms of total S-2251™ activity per vial, Streptase™ (75.7 ± 5.0 units) and Streptonase™ (94.7 ± 4.6 units) had the highest activity, while Unitinase™ (31.0 ± 2.4 units) and Strek™ (32.9 ± 3.3 units) had the weakest activity. Solustrep™ (53.3 ± 2.7 units) presented intermediate activity. The variations among the different formulations for both euglobulin lysis test and chromogenic substrate hydrolysis correlated with the SDS-PAGE densitometric results for the amount of 47-kDa protein. These data show that the commercially available clinical streptokinase formulations vary significantly in their in vitro activity. Whether these differences have clinical implications needs to be investigated.

Incidence, risk factors and prognostic factors of acute renal failure in patients admitted to an intensive care unit

The objective of the present study was to assess the incidence, risk factors and outcome of patients who develop acute renal failure (ARF) in intensive care units. In this prospective observational study, 221 patients with a 48-h minimum stay, 18-year-old minimum age and absence of overt acute or chronic renal failure were included. Exclusion criteria were organ donors and renal transplantation patients. ARF was defined as a creatinine level above 1.5 mg/dL. Statistics were performed using Pearsons' chi2 test, Student t-test, and Wilcoxon test. Multivariate analysis was run using all variables with P < 0.1 in the univariate analysis. ARF developed in 19.0% of the patients, with 76.19% resulting in death. Main risk factors (univariate analysis) were: higher intra-operative hydration and bleeding, higher death risk by APACHE II score, logist organ dysfunction system on the first day, mechanical ventilation, shock due to systemic inflammatory response syndrome (SIRS)/sepsis, noradrenaline use, and plasma creatinine and urea levels on admission. Heart rate on admission (OR = 1.023 (1.002-1.044)), male gender (OR = 4.275 (1.340-13642)), shock due to SIRS/sepsis (OR = 8.590 (2.710-27.229)), higher intra-operative hydration (OR = 1.002 (1.000-1004)), and plasma urea on admission (OR = 1.012 (0.980-1044)) remained significant (multivariate analysis). The mortality risk factors (univariate analysis) were shock due to SIRS/sepsis, mechanical ventilation, blood stream infection, potassium and bicarbonate levels. Only potassium levels remained significant (P = 0.037). In conclusion, ARF has a high incidence, morbidity and mortality when it occurs in intensive care unit. There is a very close association with hemodynamic status and multiple organ dysfunction.

Genotyping hepatitis C virus from hemodialysis patients in Central Brazil by line probe assay and sequence analysis

The present study examined the distribution of hepatitis C virus (HCV) genotypes and subtypes in a hemodialysis population in Goiás State, Central Brazil, and evaluated the efficiency of two genotyping methods: line probe assay (LiPA) based on the 5' noncoding region and nucleotide sequencing of the nonstructural 5B (NS5B) region of the genome. A total of 1095 sera were tested for HCV RNA by RT-nested PCR of the 5' noncoding region. The LiPA assay was able to genotype all 131 HCV RNA-positive samples. Genotypes 1 (92.4%) and 3 (7.6%) were found. Subtype 1a (65.7%) was the most prevalent, followed by subtypes 1b (26.7%) and 3a (7.6%). Direct nucleotide sequencing of 340 bp from the NS5B region was performed in 106 samples. The phylogenetic tree showed that 98 sequences (92.4%) were classified as genotype 1, subtypes 1a (72.6%) and 1b (19.8%), and 8 sequences (7.6%) as subtype 3a. The two genotyping methods gave concordant results within HCV genotypes and subtypes in 100 and 96.2% of cases, respectively. Only four samples presented discrepant results, with LiPA not distinguishing subtypes 1a and 1b. Therefore, HCV genotype 1 (subtype 1a) is predominant in hemodialysis patients in Central Brazil. By using sequence analysis of the NS5B region as a reference standard method for HCV genotyping, we found that LiPA was efficient at the genotype level, although some discrepant results were observed at the subtype level (sensitivity of 96.1% for subtype 1a and 95.2% for subtype 1b). Thus, analysis of the NS5B region permitted better discrimination between HCV subtypes, as required in epidemiological investigations.

Influence of renal denervation on blood pressure, sodium and water excretion in acute total obstructive apnea in rats

Obstructive apnea (OA) can exert significant effects on renal sympathetic nerve activity (RSNA) and hemodynamic parameters. The present study focuses on the modulatory actions of RSNA on OA-induced sodium and water retention. The experiments were performed in renal-denervated rats (D; N = 9), which were compared to sham (S; N = 9) rats. Mean arterial pressure (MAP) and heart rate (HR) were assessed via an intrafemoral catheter. A catheter was inserted into the bladder for urinary measurements. OA episodes were induced via occlusion of the catheter inserted into the trachea. After an equilibration period, OA was induced for 20 s every 2 min and the changes in urine, MAP, HR and RSNA were recorded. Renal denervation did not alter resting MAP (S: 113 ± 4 vs D: 115 ± 4 mmHg) or HR (S: 340 ± 12 vs D: 368 ± 11 bpm). An OA episode resulted in decreased HR and MAP in both groups, but D rats showed exacerbated hypotension and attenuated bradycardia (S: -12 ± 1 mmHg and -16 ± 2 bpm vs D: -16 ± 1 mmHg and 9 ± 2 bpm; P < 0.01). The basal urinary parameters did not change during or after OA in S rats. However, D rats showed significant increases both during and after OA. Renal sympathetic nerve activity in S rats increased (34 ± 9%) during apnea episodes. These results indicate that renal denervation induces elevations of sodium content and urine volume and alters bradycardia and hypotension patterns during total OA in unconscious rats.

Effect of exercise training on Ca2+ release units of left ventricular myocytes of spontaneously hypertensive rats

In cardiomyocytes, calcium (Ca2+) release units comprise clusters of intracellular Ca2+ release channels located on the sarcoplasmic reticulum, and hypertension is well established as a cause of defects in calcium release unit function. Our objective was to determine whether endurance exercise training could attenuate the deleterious effects of hypertension on calcium release unit components and Ca2+ sparks in left ventricular myocytes of spontaneously hypertensive rats. Male Wistar and spontaneously hypertensive rats (4 months of age) were divided into 4 groups: normotensive (NC) and hypertensive control (HC), and normotensive (NT) and hypertensive trained (HT) animals (7 rats per group). NC and HC rats were submitted to a low-intensity treadmill running protocol (5 days/week, 1 h/day, 0% grade, and 50-60% of maximal running speed) for 8 weeks. Gene expression of the ryanodine receptor type 2 (RyR2) and FK506 binding protein (FKBP12.6) increased (270%) and decreased (88%), respectively, in HC compared to NC rats. Endurance exercise training reversed these changes by reducing RyR2 (230%) and normalizing FKBP12.6 gene expression (112%). Hypertension also increased the frequency of Ca2+ sparks (HC=7.61±0.26 vs NC=4.79±0.19 per 100 µm/s) and decreased its amplitude (HC=0.260±0.08 vs NC=0.324±0.10 ΔF/F0), full width at half-maximum amplitude (HC=1.05±0.08 vs NC=1.26±0.01 µm), total duration (HC=11.51±0.12 vs NC=14.97±0.24 ms), time to peak (HC=4.84±0.06 vs NC=6.31±0.14 ms), and time constant of decay (HC=8.68±0.12 vs NC=10.21±0.22 ms). These changes were partially reversed in HT rats (frequency of Ca2+ sparks=6.26±0.19 µm/s, amplitude=0.282±0.10 ΔF/F0, full width at half-maximum amplitude=1.14±0.01 µm, total duration=13.34±0.17 ms, time to peak=5.43±0.08 ms, and time constant of decay=9.43±0.15 ms). Endurance exercise training attenuated the deleterious effects of hypertension on calcium release units of left ventricular myocytes.

Disentangling the effects of insomnia and night work on cardiovascular diseases: a study in nursing professionals

Cardiovascular diseases (CVDs) are known to be associated with poor sleep quality in general populations, but they have not been consistently associated with specific work schedules. Studies of CVD generally do not simultaneously consider sleep and work schedules, but that approach could help to disentangle their effects. We investigated the association between insomnia and a self-reported physician diagnosis of CVD in day and night workers, considering all sleep episodes during nocturnal and diurnal sleep. A cross-sectional study was conducted in 1307 female nursing professionals from 3 public hospitals, using baseline data from the “Health and Work in Nursing - a Cohort Study.” Participants were divided into two groups: i) day workers with no previous experience in night shifts (n=281) and whose data on insomnia were related to nocturnal sleep and ii) those who worked exclusively at night (n=340) and had data on both nocturnal and diurnal sleep episodes, as they often sleep at daytime. Multiple logistic regression analysis was performed. Among day workers, insomnia complaints increased the odds of CVD 2.79-fold (95% CI=1.01-6.71) compared with workers who had no complaints. Among night workers, reports of insomnia during both nocturnal and diurnal sleep increased the odds of reported CVD 3.07-fold (95% CI=1.30-7.24). Workers with insomnia had similar probabilities of reporting CVD regardless of their work schedule, suggesting a relationship to insomnia and not to night work per se. The results also highlighted the importance of including evaluation of all sleep episodes (diurnal plus nocturnal sleep) for night workers.

OCORRÊNCIA DE Escherichia coli O157: H7 EM PRODUTOS CÁRNEOS E SENSIBILIDADE DOS MÉTODOS DE DETECÇÃO

Foi verificada a ocorrência de Escherichia coli O157:H7 em 340 amostras de produtos cárneos e ambiente industrial, provenientes de frigoríficos do Sul e Sudeste do Brasil, no período de abril/98 a abril/99. A presença de E.coli O157:H7 não foi detectada em nenhuma das amostras analisadas e os resultados da avaliação da sensibilidade dos métodos de detecção evidenciaram que tanto o método cultural quanto o imunoensaio da Neogem foram capazes de detectar a presença de E.coli O157:H7 em cultura pura em concentrações iniciais de menos de 0,5Log UFC/mL do caldo de enriquecimento.

Simulação da dinâmica operacional de uma linha industrial de abate de suínos

Este trabalho teve como objetivo implementar um modelo computacional para simular a dinâmica operacional de uma linha industrial de abate de suínos. O sistema real modelado pertence à empresa Frigorífico Frimesa, sediada no município de Medianeira (PR). O modelo implementado é tipo dinâmico, discreto e estocástico. Este simula 34 operações unitárias e foi estruturado com o uso da linguagem de simulação EXTEND TM. Para validação do modelo foram coletados dados relativos a cinco dias de operação, em que foram abatidos 1.346, 1.630, 1.360, 1.585 e 1.550 suínos, respectivamente. Como parâmetros de comparação entre os dados obtidos a partir do sistema e gerados pelo modelo foram selecionadas as seguintes variáveis: (i) tempo de duração da operação; (ii) tempo de deslocamento da insensibilização até a depiladeira; (iii) tempo deslocamento da insensibilização até a câmara fria; (iv) número de carcaças re-inspecionadas; e (v) número final de carcaças. Na validação do modelo, foi constatado que, para a variável tempo de duração da operação por meio do teste Tukey a 1% de significância, não foram detectadas diferenças estatísticas entre os valores obtidos do sistema real e os gerados pelo modelo. Considerando-se esta e outras análises, foi concluído que o modelo aplica-se à finalidade para a qual foi implementado. Sendo assim, foram realizadas duas análises de sensibilidade. Na primeira, ao alterar o número de suínos a abater de 1 mil para 2 mil, foi determinado que os tempos de duração da operação variariam de 6,20 a 10,10 h, respectivamente. Para a segunda, ao abater 1.340 suínos e alterar a velocidade das nórias de 300 para 600 animais por hora, o tempo de duração da operação passou de 8,10 para 7,40 h, respectivamente.