LC/ESI-MS method applied to characterization of flavonoids glycosides in B. forficata subsp. pruinosa

Bauhinia forficata is used in folk medicine for its hypoglycemiant effect. In the south of Brazil, the subspecies pruinosa is found in a region with the characteristic flora, pampa biome. This species has been consumed by the local population as a tea for diabetes treatment. We studied the chemical composition of hydroethanolic extracts using LC/ESI-MS. The leaf extracts were prepared by percolation with 50% (v/v) ethanol. The chromatographic analyses were performed using a reverse-phase system, gradient elution with acetonitrile:phosphoric acid 0.05%, and ESI-MS in the positive ion mode. The chemical profile of the flavonoids was suggested to involve four quercetin and kaempferol glycosides.

Genetic variability in salt tolerance during germination of Stylosanthes humilis H.B.K. and association between salt tolerance and isozymes

Variation in salt tolerance of six natural populations of Stylosanthes humilis from three ecogeographic regions, Mata (wet tropical climate), Agreste and Sertão (semi-arid tropical climate) of Pernambuco State, Northeast Brazil, was evaluated on germination in 201 mM NaCl. There were significant differences among families of all populations for germination percentage and of five populations (except Tamandaré, from Mata) for germination rate. Populations from semi-arid regions presented high coefficients of genetic variation, those from Agreste being higher than those from Sertão. Populations from Mata showed low coefficients of genetic variation. The coefficients of genotypic determination were high for five populations, except Tamandaré, both for germination percentage ( > or = 0.89) and for germination rate ( > or = 0.79), indicating the possibility of selection for salt tolerance in these populations. An electrophoretic analysis of esterase and peroxidase isozymes was also performed in the six populations, and correlations were estimated between salt tolerance and allelic frequencies. The analysis of salt tolerant and salt sensitive families of populations from Agreste suggested an association of alleles of a peroxidase locus with salt tolerance during germination in the Caruaru population

pH titration of native and unfolded ß-trypsin: evaluation of the D D G0 titration and the carboxyl pK values

The stabilizing free energy of ß-trypsin was determined by hydrogen ion titration. In the pH range from 3.0 to 7.0, the change in free energy difference for the stabilization of the native protein relative to the unfolded one (D D G0 titration) was 9.51 ± 0.06 kcal/mol. An isoelectric point of 10.0 was determined, allowing us to calculate the Tanford and Kirkwood electrostatic factor w. This factor presented a nonlinear behavior and indicated more than one type of titratable carboxyl groups in ß-trypsin. In fact, one class of carboxyl group with a pK = 3.91 ± 0.01 and another one with a pK = 4.63 ± 0.03 were also found by hydrogen ion titration of the protein in the folded state

High dietary calcium decreases blood pressure in normotensive rats

This study evaluates the influence of different concentrations of calcium on blood pressure of normotensive rats. Four groups of Wistar rats (A, B, C and D) had free access to modified isocaloric and isoproteic diets containing 0.2, 0.5, 2 and 4 g% calcium as calcium carbonate for a period of 30 days. Systolic and diastolic arterial blood pressures were monitored in awake rats by the indirect tail cuff method using a Physiograph equipped with transducers and preamplifiers. Body weight and length and food intake were monitored. Under the conditions of the present experiment, the systolic and diastolic arterial blood pressures of group D rats fed a diet containing 4 g% calcium were significantly (P<0.05) lower compared to rats of the other groups.

Molecular identification of Sicilian (dß)º-thalassemia associated with ß-thalassemia and hemoglobin S in Brazil

We describe the clinical and molecular characteristics of two unrelated Brazilian families with an association of the Sicilian form of (dß)º-thalassemia with hemoglobin S and ß-thalassemia. Direct sequencing of the ß-globin gene showed only the hemoglobin S mutation in patient 1 and the ß-thalassemia IVS1-110 in patient 2. The other allele was deleted in both patients and PCR of DNA samples of the breakpoint region of both patients showed a band of approximately 1,150 bp, expected to be observed in the DNA of carriers of Sicilian (dß)º-thalassemia. The nucleotide sequence of this fragment confirmed the Sicilian deletion. There are few reports concerning the Hb S/(dß)º-thalassemia association and patient 2 is the first reported case of Sicilian type of (dß)º-thalassemia in association with ß-thalassemia documented at the molecular level.

Erythrocytes may contain a ouabain-insensitive K+-ATPase which plays a role in internal K+ balance

Erythrocytes are useful in evaluating K+ transport pathways involved in internal K+ balance. Several forms of H+,K+-ATPase have been described in nephron segments active in K+ transport. Furthermore, the activity of a ouabain-insensitive isoform of H+,K+-ATPase expressed in collecting duct cells may be modulated by acid-base status. Various assays were performed to determine if a ouabain-insensitive K+-ATPase is present in rat erythrocytes and, if so, whether it plays a role in internal K+ balance. Kinetic studies demonstrated that maximal stimulation of enzyme activity was achieved with 2.5 mM K+ at pH 7.4. Subsequent experiments were performed on erythrocyte membranes collected from animals submitted to varying degrees of K+ homeostasis: control rats, K+-depleted rats, K+-loaded rats, and rats rendered hyperkalemic due to acute renal failure. As observed in the collecting duct cell studies, there was a significant decrease in the activity of ouabain-insensitive K+-ATPase in the erythrocytes of both K+-loaded and metabolically alkalotic K+-depleted rats. However, this enzyme activity in erythrocyte membranes of rats with metabolic acidosis-related hyperkalemia was similar to that of control animals. This finding may be interpreted as resulting from two potentially modulating factors: the stimulating effect that metabolic acidosis has on K+-ATPase and the counteracting effect that hyperkalemia and uremia have on metabolic acidosis. In summary, we present evidence of a ouabain-insensitive K+-ATPase in erythrocytes, whose activity is modulated by acid-base status and K+ levels.

Techniques used to identify the Brazilian variant of HIV-1 subtype B

The purpose of the present study was to compare the sensitivity and specificity of V3 enzyme immunoassay (solid phase EIA and EIA inhibition) and restriction fragment length polymorphism (RFLP) with the DNA sequencing "gold standard" to identify the Brazilian HIV-1 variants of subtype B and B"-GWGR. Peripheral blood mononuclear cells were collected from 61 HIV-1-infected individuals attending a clinic in São Paulo. Proviral DNA was amplified and sequentially cleaved with the Fok I restriction enzyme. Plasma samples were submitted to a V3-loop biotinylated synthetic peptide EIA. Direct partial DNA sequencing of the env gene was performed on all samples. Based on EIA results, the sensitivity for detecting B-GPGR was 70%, compared to 64% for the Brazilian variant B"-GWGR while, the specificity of B-GPGR detection was 85%, compared to 88% for GWGR. The assessment of RFLP revealed 68% sensitivity and 94% specificity for the B-GPGR strain compared to 84 and 90% for the B"-GWGR variant. Moreover, direct DNA sequencing was able to detect different base sequences corresponding to amino acid sequences at the tip of the V3 loop in 22 patients. These results show a similar performance of V3 serology and RLFP in identifying the Brazilian variant GWGR. However, V3 peptide serology may give indeterminate results. Therefore, we suggest that V3 serology be used instead of DNA sequencing where resources are limited. Samples giving indeterminate results by V3 peptide serology should be analyzed by direct DNA sequencing to distinguish between B-GPGR and the Brazilian variant B"-GWGR.

There is no difference in hepatic fibrosis rates of patients infected with hepatitis C virus and those co-infected with HIV

Some studies have suggested that human immunodeficiency virus (HIV) infection modifies the natural history of hepatitis C virus (HCV) infection, accelerating the progression of fibrosis and the development of cirrhosis. Our objective was to evaluate the fibrosis progression rate (FPR) in HCV/HIV-co-infected patients, and to identify factors that may influence it. HCV-mono-infected and HCV/HIV-co-infected patients with a known date of HCV infection (transfusion or injection drug use) and a liver biopsy were included. The FPR was defined as the ratio between the fibrosis stage (Metavir score) and the estimated length of infection in years and the result was reported as fibrosis units per year. The factors studied were gender, age at infection, consumption of alcohol, aminotransferase levels, histological activity grade, HCV genotype and viral load, CD4 cell count, HIV viral load, and the use of antiretroviral therapy. Sixty-five HCV-infected (group 1) and 53 HCV/HIV-co-infected (group 2) patients were evaluated over a period of 19 months. The mean FPR of groups 1 and 2 was 0.086 ± 0.074 and 0.109 ± 0.098 fibrosis units per year, respectively (P = 0.276). There was a correlation between length of HCV infection and stage of fibrosis in both groups. The age at infection, the aspartate aminotransferase level (r = 0.36) and the inflammatory activity grade were correlated with the FPR (P < 0.001). No difference in FPR was found between HCV-mono-infected and HCV/HIV-co-infected patients.

Role of nuclear factor kappa B and reactive oxygen species in the tumor necrosis factor-a-induced epithelial-mesenchymal transition of MCF-7 cells

The microenvironment of the tumor plays an important role in facilitating cancer progression and activating dormant cancer cells. Most tumors are infiltrated with inflammatory cells which secrete cytokines such as tumor necrosis factor-a (TNF-a). To evaluate the role of TNF-a in the development of cancer we studied its effects on cell migration with a migration assay. The migrating cell number in TNF-a -treated group is about 2-fold of that of the control group. Accordingly, the expression of E-cadherin was decreased and the expression of vimentin was increased upon TNF-a treatment. These results showed that TNF-a can promote epithelial-mesenchymal transition (EMT) of MCF-7 cells. Further, we found that the expression of Snail, an important transcription factor in EMT, was increased in this process, which is inhibited by the nuclear factor kappa B (NFkB) inhibitor aspirin while not affected by the reactive oxygen species (ROS) scavenger N-acetyl cysteine. Consistently, specific inhibition of NFkB by the mutant IkBa also blocked the TNF-a-induced upregulation of Snail promoter activity. Thus, the activation of NFkB, which causes an increase in the expression of the transcription factor Snail is essential in the TNF-a-induced EMT. ROS caused by TNF-a seemed to play a minor role in the TNF-a-induced EMT of MCF-7 cells, though ROS per se can promote EMT. These findings suggest that different mechanisms might be responsible for TNF-a - and ROS-induced EMT, indicating the need for different strategies for the prevention of tumor metastasis induced by different stimuli.

How frequently should a patient taking amiodarone be screened for thyroid dysfunction?

Amiodarone-induced thyroid dysfunction (AITD) is a common complication of amiodarone therapy and its prevalence varies according to iodine intake, subclinical thyroid disorders and the definition of AITD. There is no consensus about the frequency of screening for this condition. We evaluated 121 patients on chronic regular intake of amiodarone (mean intake = 248.5 ± 89 mg; duration of treatment = 5.3 ± 3.9 years, range = 0.57-17 years) and with stable baseline cardiac condition. Those with no AITD were followed up for a median period of 3.2 years (range: 0.6-6.7) and the incidence rate of AITD, defined by clinical and laboratorial findings as proposed by international guidelines, was obtained (62.8 per 1000 patients/year). We applied the Cox proportional hazard model to adjust for potential confounding factors and used sensitivity analysis to identify the best screening time for follow-up. We detected thyroid dysfunction in 59 (48.7%) of the 121 patients, amiodarone-induced hypothyroidism in 50 (41.3%) and hyperthyroidism in 9 (7.5%). Compared with patients without AITD, there was no difference regarding dosage or duration of therapy, heart rhythm disorder or baseline cardiac condition. During the follow-up of the 62 patients without AITD at baseline evaluation, 11 developed AITD (interquartile range, IR: 62.8 (95%CI: 31.3-112.3) cases per 1000 patients/year), 9 of them with hypothyroidism - IR: 11.4 (95%CI: 1.38-41.2), and 2 hyperthyroidism - IR: 51.3 (95%CI: 23.4-97.5). Age, gender, dose, and duration of treatment were not significant after adjustment. During the first 6 months of follow-up the incidence rate for AITD was 39.3 (9.2-61.9) cases per 1000 patients/year. These data show that AITD is quite common, and support the need for screening at 6-month intervals, unless clinical follow-up dictates otherwise or further information regarding the prognosis of untreated subclinical AITD is available.

Effect of vitamin D therapy in addition to amitriptyline on migraine attacks in pediatric patients

The purpose of this study was to investigate the effect of supplementary vitamin D therapy in addition to amitriptyline on the frequency of migraine attacks in pediatric migraine patients. Fifty-three children 8-16 years of age and diagnosed with migraine following the International Headache Society 2005 definition, which includes childhood criteria, were enrolled. Patients were classified into four groups on the basis of their 25-hydroxyvitamin D [25(OH)D] levels. Group 1 had normal 25(OH)D levels and received amitriptyline therapy alone; group 2 had normal 25(OH)D levels and received vitamin D supplementation (400 IU/day) plus amitriptyline; group 3 had mildly deficient 25(OH)D levels and received amitriptyline plus vitamin D (800 IU/day); and group 4 had severely deficient 25(OH)D levels and was given amitriptyline plus vitamin D (5000 IU/day). All groups were monitored for 6 months, and the number of migraine attacks before and during treatment was determined. Calcium, phosphorus alkaline phosphatase, parathormone, and 25(OH)D levels were also determined before and during treatment. Results were compared between the groups. Data obtained from the groups were analyzed using one-way analysis of variance. The number of pretreatment attacks in groups 1 to 4 was 7±0.12, 6.8±0.2, 7.3±0.4, and 7.2±0.3 for 6 months, respectively (all P>0.05). The number of attacks during treatment was 3±0.25, 1.76±0.37 (P<0.05), 2.14±0.29 (P<0.05), and 1.15±0.15 (P<0.05), respectively. No statistically significant differences in calcium, phosphorus, alkaline phosphatase, or parathormone levels were observed (P>0.05). Vitamin D given in addition to anti-migraine treatment reduced the number of migraine attacks.

Detection of enterotoxins produced by B. cereus through PCR analysis of ground and roasted coffee samples in Rio de Janeiro, Brazil

Coffee is one of the most appreciated drinks in the world. Coffee ground is obtained from the fruit of a small plant that belongs to the genus Coffea. Coffea arabica and Coffea canephora robusta are the two most commercially important species. They are more commonly known as arabica and robusta, respectively. Two-thirds of Coffea arabica plants are grown in South and Central America, and Eastern Africa - the place of origin for this coffee species. Contamination by microorganisms has been a major matter affecting coffee quality in Brazil, mainly due to the harvesting method adopted. Brazilian harvests are based on fruits collected from the ground mixed with those that fall on collection cloths. As the Bacillus cereus bacterium frequently uses the soil as its environmental reservoir, it is easily capable of becoming a contaminant. This study aimed to evaluate the contamination and potential of B. cereus enterotoxin genes encoding the HBL and NHE complexes, which were observed in strains of ground and roasted coffee samples sold in Rio de Janeiro. The PCR (Polymerase Chain Reaction) results revealed high potential of enterotoxin production in the samples. The method described by Speck (1984) was used for the isolation of contaminants. The investigation of the potential production of enterotoxins through isolates of the microorganism was performed using the B. cereus enterotoxin Reverse Passive Latex Agglutination test-kit (BCET-RPLA, Oxoid), according to the manufacturer's instructions. The potential of enterotoxin production was investigated using polymerase chain reaction (PCR) methods for hblA, hblD and hblC genes (encoding hemolysin HBL) and for nheA, nheB and nheC genes (encoding non-hemolytic enterotoxin - NHE). Of all the 17 strains, 100% were positive for at least 1 enterotoxin gene; 52.9% (9/17) were positive for the 3 genes encoding the HBL complex; 35.3% (6/17) were positive for the three NHE encoding genes; and 29.4% (5/17) were positive for all enterotoxic genes.

Inactivation of E. coli and B. subtilis spores in ozonized cassava starch

In the present study, the efficacy of ozone inactivation of B. subtilis spores and E. coli in cassava starch was evaluated. Cassava starch with 18 and 30% moisture content was processed with ozone at concentrations of 40-118 ppm and exposure times of 15-120 minutes. The processing at 113 ppm/120 minutes (maximum exposure level to ozone evaluated) at 18% of moisture content did not cause significant reduction of B. subtilis spores and caused the reduction of only 2 decimal of E. coli. On the other hand, when the ozonation process was carried out for 120 minutes at 30% of moisture content, 3.6 decimal reduction of B. subtilis was achieved at 40 ppm of ozone and total B. subtilis load reduction (>5 log cycles) was observed at 118 ppm of ozone. Similarly, total E. coli load reduction (>7 log cycles) was achieved at 40 ppm of ozone exposure for 60 minutes. Therefore, the results indicate that the ozone efficacy against microorganisms in cassava starch was mainly dependent on the sample moisture content and to ozone concentration and exposure time. Moreover, it was observed that ozone is a promising technology to reduce microbial counts in dried food.