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.

Eradication of Helicobacter pylori infection in patients with duodenal ulcer and non-ulcer dyspepsia and analysis of one-year reinfection rates

Helicobacter pylori (HP) infection is endemic worldwide. The proposed treatment is expensive and there are few reports regarding reinfection rates in Brazil. The aim of this study was to compare the eradication rates obtained with two therapeutic options and to evaluate reinfection one year after treatment. This was a prospective randomized trial with 55 patients. Thirty-nine patients had active duodenal ulcer (DU) and 16 non-ulcer dyspepsia (NUD), and all tested positive for HP. Diagnosis was based on at least two positive tests: ultrarapid urease test, histology and/or culture. Patients were randomized to two groups: group OMC treated with 40 mg omeprazole (once a day), 500 mg metronidazole and 250 mg clarithromycin (twice daily) for 7 days, or group NA treated with 300 mg nizatidine (once a day) and 1000 mg amoxicillin (twice daily) for 14 days. Those patients in whom HP was eradicated were followed up for one year to evaluate reinfection. Twenty-five patients were randomized for OMC and 30 for NA. HP eradication occurred in 20/25 patients (80%) treated with OMC and 13/30 (43%) treated with NA (P = 0.01). After reallocation because of initial treatment failure, the overall eradication rate was 44/51 patients (86%). After an average follow-up of one year, we evaluated 34 patients (23 with DU and 11 with NUD). Reinfection occurred in 3/34 patients (7.6%). We conclude that OMC is effective for HP eradication, and that NA should not be used. Reinfection occurs in 7.6% of the patients in the first year after eradication.

Serum leptin concentration during puberty in healthy nonobese adolescents

Data obtained during the past five years have indicated that there are important age- and gender-based differences in the regulation and action of leptin in humans. To study the physiological changes of leptin during puberty in both sexes, and its relationship with body composition and sexual maturation, we measured leptin concentrations in 175 healthy adolescents (80 girls, 95 boys, 10-18 years of age), representing all pubertal stages. We excluded individuals with a body mass index (BMI) below the 5thor above the 95th percentile relative to age. Serum concentrations of leptin were determined by a monoclonal antibody-based immunofluorimetric assay, developed in our laboratory. Body composition was determined by dual-energy X-ray absorptiometry. Pubertal stage was assigned by physical examination, according to Tanner criteria for breast development in females and genital development in males. Leptin concentration in girls (N = 80) presented a positive linear correlation with age (r = 0.35, P = 0.0012), BMI (r = 0.65, P < 0.0001) and %fat mass (r = 0.76, P < 0.0001). In boys (N = 95) there was a positive correlation with BMI (r = 0.49, P < 0.0001) and %fat mass (r = 0.85, P < 0.0001), but a significant negative linear correlation with Tanner stage (r = -0.45, P < 0.0001) and age (r = -0.40, P < 0.0001). The regression equation revealed that %fat mass and BMI are the best parameters to be used to estimate leptin levels in both sexes. Thus, the normal reference ranges for circulating leptin during adolescence should be constructed according to BMI or %fat mass to assure a correct evaluation.

Aurapten, a coumarin with growth inhibition against Leishmania major promastigotes

Several natural compounds have been identified for the treatment of leishmaniasis. Among them are some alkaloids, chalcones, lactones, tetralones, and saponins. The new compound reported here, 7-geranyloxycoumarin, called aurapten, belongs to the chemical class of the coumarins and has a molecular weight of 298.37. The compund was extracted from the Rutaceae species Esenbeckia febrifuga and was purified from a hexane extract starting from 407.7 g of dried leaves and followed by four silica gel chromatographic fractionation steps using different solvents as the mobile phase. The resulting compound (47 mg) of shows significant growth inhibition with an LD50 of 30 µM against the tropical parasite Leishmania major, which causes severe clinical manifestations in humans and is endemic in the tropical and subtropical regions. In the present study, we investigated the atomic structure of aurapten in order to determine the existence of common structural motifs that might be related to other coumarins and potentially to other identified inhibitors of Leishmania growth and viability. This compound has a comparable inhibitory activity of other isolated molecules. The aurapten is a planar molecule constituted of an aromatic system with electron delocalization. A hydrophobic side chain consisting of ten carbon atoms with two double bonds and negative density has been identified and may be relevant for further compound synthesis.

Operant serial feature-positive conditional discrimination with composite features and different topography responses

In a serial feature-positive conditional discrimination procedure the properties of a target stimulus A are defined by the presence or not of a feature stimulus X preceding it. In the present experiment, composite features preceded targets associated with two different topography operant responses (right and left bar pressing); matching and non-matching-to-sample arrangements were also used. Five water-deprived Wistar rats were trained in 6 different trials: X-R®Ar and X-L®Al, in which X and A were same modality visual stimuli and the reinforcement was contingent to pressing either the right (r) or left (l) bar that had the light on during the feature (matching-to-sample); Y-R®Bl and Y-L®Br, in which Y and B were same modality auditory stimuli and the reinforcement was contingent to pressing the bar that had the light off during the feature (non-matching-to-sample); A- and B- alone. After 100 training sessions, the animals were submitted to transfer tests with the targets used plus a new one (auditory click). Average percentages of stimuli with a response were measured. Acquisition occurred completely only for Y-L®Br+; however, complex associations were established along training. Transfer was not complete during the tests since concurrent effects of extinction and response generalization also occurred. Results suggest the use of both simple conditioning and configurational strategies, favoring the most recent theories of conditional discrimination learning. The implications of the use of complex arrangements for discussing these theories are considered.

Calculation of viscoelastic properties of edible films: application of three models

The viscoelastic properties of edible films can provide information at the structural level of the biopolymers used. The objective of this work was to test three simple models of linear viscoelastic theory (Maxwell, Generalized Maxwell with two units in parallel, and Burgers) using the results of stress relaxation tests in edible films of myofibrillar proteins of Nile Tilapia. The films were elaborated according to a casting technique and pre-conditioned at 58% relative humidity and 22ºC for 4 days. The testing sample (15mm x 118mm) was submitted to tests of stress relaxation in an equipment of physical measurements, TA.XT2i. The deformation, imposed to the sample, was 1%, guaranteeing the permanency in the domain of the linear viscoelasticity. The models were fitted to experimental data (stress x time) by nonlinear regression. The Generalized Maxwell model with two units in parallel and the Burgers model represented the relaxation curves of stress satisfactorily. The viscoelastic properties varied in a way that they were less dependent on the thickness of the films.

Phase transitions in biodegradable films based on blends of gelatin and poly (vinyl alcohol)

The aim of this work was to study the effect of the hydrolysis degree (HD) and the concentration (C PVA) of two types of poly (vinyl alcohol) (PVA) and the effect of the type and the concentration of plasticizers on the phase properties of biodegradable films based on blends of gelatin and PVA, using a response-surface methodology. The films were made by casting and the studied properties were their glass (Tg) and melting (Tm) transition temperatures, which were determined by diferential scanning calorimetry (DSC). For the data obtained on the first scan, the fitting of the linear model was statistically significant and predictive only for the second melting temperature. In this case, the most important effect on the second Tm of the first scan was due to the HD of the PVA. In relation to the second scan, the linear model could be fit to Tg data with only two statistically significant parameters. Both the PVA and plasticizer concentrations had an important effect on Tg. Concerning the second Tm of the second scan, the linear model was fit to data with two statistically significant parameters, namely the HD and the plasticizer concentration. But, the most important effect was provoked by the HD of the PVA.

Physiological potential of rice seeds treated with rhizobacteria or the insecticide thiamethoxam

Development of new technologies, aiming at increasing productivity in different crops, involves constant research on the effectiveness and application of these techniques in seed treatment. In this study, it was aimed at evaluating physiological potential of rice seeds treated with plant growth-promoting rhizobacteria (PGPR) (strains DFs185, DFs223, DFs306, DFs416), or with two dosages of the insecticide thiamethoxam. The variables assessed were: germination (G); first count of germination (FCG); cold test (CT); length of seedlings aerial parts (LAP), root system (LRS), and total length (TL); emergence speed index (ESI); emergence (E), at 14 days; and dry phytomass (DP). Treatments have had a positive effect on percentage of G, FCG and E. The strain DFs185 has promoted increase in percentage emergence, for five of the six lots assessed. The variables: LAP; LRS; TL; ESI; and DP have undergone low or none influence of treatments; and there has been no toxic effect of rhizobacteria or insecticide thiamethoxam. In the cold test, a negative effect of treatments has been detected. Seed treatment with rhizobacteria, as well as with thiamethoxam, improve quality of low quality rice seeds. The strain DFs185 is promising for treating rice seeds, once it stimulates seed germination and emergence.