Chiral evaluation of fluvastatin in human plasma by high-performance liquid chromatography electrospray mass spectrometry

The report describes for the first time the enantioselective analysis of fluvastatin in plasma using LC-MS-MS. The enantiomers of fluvastatin (FV) were extracted from plasma with diisopropyl ether at pH 5.0. The enantiomers were separated on a ChiralCel OD-R column with a mobile phase consisting of a mixture of acetonitrile, methanol and water (24:36:40) containing 0.1% formic acid. The protonated ions and their respective product ions were monitored in two functions, 410.6>348.2 for FV enantiomers and 307.1>161.6 for the internal standard (warfarin). Recoveries were higher than 90% and the quantitation limit was 1.5 ng mL(-1) plasma for both enantiomers. The coefficients of variation and the relative errors obtained for the validation of the intra- and interassay precision and accuracy were less than 10%. The method was applied to the investigation of the enantioselective pharmacokinetics of FV administered in a single dose of 40 mg (Lescol, Novartis, S?o Paulo, SP, Brazil) to a patient with primary hypertension and hypercholesterolemia and genotyped as CYP2C9*1/*1. The data showed higher plasma concentrations of the (-)-3S,5R-fluvastatin enantiomer, with an AUC (-)/(+) of 1.84. Oral clearance values (CL/F) were 29.27 and 49.58 L/h, respectively, for the (-)-3S,5R- and (+)-3R,5S-fluvastatin enantiomers.     

Possible biphasic sweating response during short-term heat acclimation protocol for tropical natives

The aim of the present study was to evaluate the sweat loss response during short-term heat acclimation in tropical natives. Six healthy young male subjects, inhabitants of a tropical region, were heat acclimated by means of nine days of one-hour heat-exercise treatments (40+/-0 degrees C and 32+/-1% relative humidity; 50% (.)VO(2peak) on a cycle ergometer). On days 1 to 9 of heat acclimation whole-body sweat loss was calculated by body weight variation corrected for body surface area. On days 1 and 9 rectal temperature (T(re)) and heart rate (HR) were measured continuously, and rating of perceived exertion (RPE) every 4 minutes. Heat acclimation was confirmed by reduced HR (day 1 rest: 77+/-5 b.min(-1); day 9 rest: 68+/-3 b.min(-1); day 1 final exercise: 161+/-15 b.min(-1); day 9 final exercise: 145+/-11 b.min(-1), p<0.05), RPE (13 vs. 11, p<0.05) and T(re) (day 1 rest: 37.2+/-0.2 degrees C; day 9 rest: 37.0+/-0.2 degrees C; day 1 final exercise: 38.2+/-0.2 degrees C; day 9 final exercise: 37.9+/-0.1 degrees C, p<0.05). The main finding was that whole-body sweat loss increased in days 5 and 7 (9.49+/-1.84 and 9.56+/-1.86 g.m(-2).min(-1), respectively) compared to day 1 (8.31+/-1.31 g.m(-2).min(-1), p<0.05) and was not different in day 9 (8.48+/-1.02 g.m(-2).min(-1)) compared to day 1 (p>0.05) of the protocol. These findings are consistent with the heat acclimation induced adaptations and suggest a biphasic sweat response (an increase in the sweat rate in the middle of the protocol followed by return to initial values by the end of it) during short-term heat acclimation in tropical natives.     

Enzymatic properties of two beta-glucosidases from Ceriporiopsis subvermispora produced in biopulping conditions

AIMS: Ceriporiopsis subvermispora produces endoglucanase and beta-glucosidase when cultivated on cellulose or wood, but biodegradation of cellulose during biopulping by C. subvermispora is low even after long periods. To resolve this discrepancy, we grew C. subvermispora on Pinus taeda wood chips and purified the major beta-glucosidases it produced. Kinetic parameters were determined to clear if this fungus produces enzymes capable of yielding assimilable glucose from wood. METHODS AND RESULTS: Ceriporiopsis subvermispora was grown on P. taeda wood chips under solid-state fermentation. After 30 days, the crude extract obtained from enzyme extraction with sodium acetate buffer 50 mmol l(-1), pH 5.4, was filtrated in membranes with a molecular mass exclusion limit of 100 kDa. Enzyme purification was carried out using successively Sephacryl S-300 gel filtration. The retained fraction attained 76% of beta-glucosidase activity with 3.7-fold purification. Two beta-glucosidases were detected with molecular mass of 110 and 53 kDa. We have performed a characterization of the enzymatic properties of the beta-glucosidase of 110 kDa. The optimum pH and temperature were 3.5 and 60 degrees C, respectively. The K(m) and V(max) values were respectively 3.29 mmol l(-1) and 0.113 micromol min(-1) for the hydrolysis of p-nitrophenyl-beta-glucopyranoside (pNPG) and 2.63 mmol l(-1) and 0.103 micromol min(-1), towards cellobiose. beta-Glucosidase activity was strongly increased by Mn(2+) and Fe(3+), while Cu(2+) severely inhibited it. CONCLUSIONS: Ceriporiopsis subvermispora produces small amounts of beta-glucosidase when grown on wood. The gel filtration and polyacrylamide gel electrophoresis data revealed the existence of two beta-glucosidases with 110 and 53 kDa. The 110 kDa beta-glucosidase from C. subvermispora can be efficiently purified in a single step by gel filtration chromatography. The enzyme has an acid pH optimum with similar activity on pNPG and cellobiose and is thus typical beta-glucosidase. SIGNIFICANCE AND IMPACT OF THE STUDY: Ceriporiopsis subvermispora produces beta-glucosidase with limited action during wood decay making able its use for the production of biomechanical and biochemical pulps. The results presented in this paper show the importance of studying the behaviour of beta-glucosidases during biopulping.     

Knowledge and attitudes of dentists regarding ageing and the elderly

doi: 10.1111/j.1741‐2358.2011.00534.x
Knowledge and attitudes of dentists regarding ageing and the elderly Objective: This study was aimed to identify the knowledge and attitudes of dentists regarding ageing and the elderly. Background: As the population ages, there is a greater demand for differentiated care from health professionals. However, the treatment needs of the elderly population are not sufficiently addressed, particularly in developing countries. Materials and methods: An official list of all dentists registered in the city of Belo Horizonte, Brazil, was obtained from the Regional Dentistry Council. A questionnaire was delivered to 276 randomly selected dentists; the survey contained questions designed to characterise the sample and identify dentists’ conduct towards elderly patients. A Likert scale was used to identify knowledge regarding care for the elderly and attitudes towards ageing. Results: Women demonstrated more positive attitudes towards the elderly. Graduates who worked in the public sector demonstrated a greater knowledge of geriatric dentistry (p < 0.05). Attending specific continuing education courses had no influence over the dentists’ willingness to treat the elderly population. Significant associations were not found between attitudes and knowledge regarding the elderly. Conclusion: Dentists who worked in the public sector demonstrated a greater knowledge regarding the elderly. This knowledge did not influence attitudes towards ageing. Women demonstrated a more positive attitude regarding ageing and dental care.     

The population genetics of Quechuas, the largest native South American group: autosomal sequences, SNPs, and microsatellites evidence high level of diversity

Elucidating the pattern of genetic diversity for non-European populations is necessary to make the benefits of human genetics research available to individuals from these groups. In the era of large human genomic initiatives, Native American populations have been neglected, in particular, the Quechua, the largest South Amerindian group settled along the Andes. We characterized the genetic diversity of a Quechua population in a global setting, using autosomal noncoding sequences (nine unlinked loci for a total of 16 kb), 351 unlinked SNPs and 678 microsatellites and tested predictions of the model of the evolution of Native Americans proposed by (Tarazona-Santos et al.: Am J Hum Genet 68 (2001) 1485-1496). European admixture is <5% and African ancestry is barely detectable in the studied population. The largest genetic distances were between African versus Quechua or Melanesian populations, which is concordant with the African origin of modern humans and the fact that South America was the last part of the world to be peopled. The diversity in the Quechua population is comparable with that of Eurasian populations, and the allele frequency spectrum based on resequencing data does not reflect a reduction in the proportion of rare alleles. Thus, the Quechua population is a large reservoir of common and rare genetic variants of South Amerindians. These results are consistent with and complement our evolutionary model of South Amerindians (Tarazona-Santos et al.: Am J Hum Genet 68 (2001) 1485-1496), proposed based on Y-chromosome data, which predicts high genomic diversity due to the high level of gene flow between Andean populations and their long-term effective population size.     

A novel inhibitory interaction between dimethylsulfoniopropionate (DMSP) and the denitrification pathway

Dimethylsulfoniopropionate (DMSP) is an abundant organic sulfur compound in marine algae and denitrification influences nitrogen availability to primary producers, the key regulators of coastal eutrophication. In this study, we tested the effect of DMSP on the nitrous oxide (N₂O) reduction step of denitrification in sediments and biofilms from the Douro and Ave estuaries (NW Portugal) and in pure cultures of a denitrifying bacterium, Ruegeria pomeroyi. N₂O accumulation rates were monitored in sediment slurries and bacterial cell suspensions amended with DMSP concentrations ranging from 0 to 5 mM. In these treatments N₂O accumulation rates increased linearly with DMSP concentration (R ² from 0.89 to 0.99, p < 0.001), suggesting an inhibitory effect of DMSP on the nitrous oxide reductase activity. The addition of DMSP to sediments and bacterial culture resulted in accumulation of dimethylsulfide (DMS) as well as N₂O. However, no direct inhibition on N₂O reductase activity by DMS was observed. Natural concentrations of DMSP in the different estuarine sites were found to be linearly correlated to natural N₂O effluxes (R ² = 0.64, p < 0.001), suggesting that DMSP may negatively affect N₂O reductase in situ. This newly identified interaction between DMSP and N₂O emissions may have a significant ecological role as the inhibition of the nitrous oxide reduction enhances nitrogen loss via N₂O. Since N₂O is a powerful greenhouse gas, the results from our study may be important for evaluating climate change scenarios.     

Steady-state level of insulin-like growth factor-I (IGF-I) receptor mRNA and the effect of IGF-I on the in vitro culture of caprine preantral follicles

The objectives were to quantify insulin-like growth factor receptor-1 (IGFR-1) mRNA in preantral follicles on Days 0 and 18 of in vitro culture in the presence or absence of FSH, and to evaluate the effects of IGF-I on the rate of normal follicles, antral cavity formation, and in vitro growth and maturation of caprine oocytes on Days 0, 6, 12, and 18 of culture. The expression of IGFR-1 was analyzed using real-time RT-PCR before and after follicle culture. Preantral follicles were isolated from the cortex of caprine ovaries and individually cultured for 18 d in the presence or absence of bovine IGF-I (50 or 100 ng/mL). At the end of the culture period, the oocytes were submitted to IVM. The expression of IGFR-1 mRNA in preantral follicles cultured in vitro only approached being significantly higher in follicles supplemented with FSH when compared to follicles immediately after recovery (P < 0.06) and cultured without FSH (P < 0.1). There was a higher (P < 0.05) percentage of normal follicles on Days 6, 12, and 18 of culture in IGF-I 50 (97, 92, 67%, respectively) and IGF-I 100 (100, 90, 80%) groups versus the control (90, 64, 36%). In addition, the rate of antrum formation at 6 and 12 d of culture was higher (P < 0.05) in IGF-I groups (IGF-I 50: 72 and 90% and IGF-I 100: 69 and 85%) than the control group (41 and 59%). After 18 d of culture, the percentages of grown oocytes acceptable for IVM were higher (P < 0.05) in follicles cultured in the presence of IGF-I (82 vs 49%). Furthermore, follicles cultured in the presence of IGF-I 50 and IGF-I 100 had higher (P < 0.05) meiotic resumption rates (63 and 66%, respectively) than the control group (11%). In conclusion, treatment with FSH tended to increase IGFR-1 mRNA expression during the in vitro culture of preantral follicles and the addition of IGF-I to the culture medium clearly improved the in vitro development of caprine preantral follicles.     

Effect of insulin-like growth factor-I on some quality traits and fertility of cryopreserved ovine semen

The objective was to evaluate the effects of insulin-like growth factor-I (IGF-I) on the quality and fertility of frozen/thawed ovine semen. Five rams (five ejaculates/ram) were used for evaluation of semen parameters. Before cryopreservation, ejaculates were divided into four aliquots and extended with Tris alone or supplemented with human IGF-I (50, 100, or 250 ng/mL). Semen was evaluated immediately after thawing (T0), after 1 h (T1) and 2 h (T2) post-incubation at 37 °C. The percentage of live cells (fluorescence analysis-calcein and ethidium), acrosome integrity (NAR) and motility were analyzed, and hypo-osmotic swelling tests (HOST) were used to evaluate membrane resistance. In addition, AI was performed using 121 ewes to compare the optimal concentration of IGF-I vs. Tris alone on pregnancy rates after laparoscopic insemination. Pregnancy diagnosis was performed by transrectal ultrasonography. After 1 and 2 h post-incubation, in every group, percentage motile sperm, NAR and HOST decreased compared to semen at T0. Motility was higher (P < 0.05) in the IGF-I 100 and IGF-I 250 groups when compared to the IGF-I 50 and Tris groups (76.2 and 74.4% vs. 66.2 and 64.4 percent, respectively) at T0, after 1 h (67 and 63.6% vs. 56.2 and 54.7%) and 2 h post-incubation (58.2 and 55.8% vs. 48 and 47.2%). Furthermore, viability was higher (P < 0.05) in the insulin-like growth factor-I (IGF-I) 100 and IGF-I 250 groups than in the IGF-I 50 and Tris groups (88.7 and 88.3% vs. 76.6 and 77.6%, respectively) at T0. There was no difference (P > 0.05) in NAR or hypo-osmotic swelling tests (HOST) among groups. There were no differences (P > 0.05) in fertility between the IGF-I 100 and Tris groups. In conclusion, IGF-I improved subjective sperm motility and structural integrity of the plasma membrane without a significant effect on 45-day pregnancy rates after laparoscopic insemination of ewes with frozen-thawed semen.     

Taurine supplementation prevents morpho-physiological alterations in high-fat diet mice pancreatic β-cells

Taurine (Tau) is involved in beta (β)-cell function and insulin action regulation. Here, we verified the possible preventive effect of Tau in high-fat diet (HFD)-induced obesity and glucose intolerance and in the disruption of pancreatic β-cell morpho-physiology. Weaning Swiss mice were distributed into four groups: mice fed on HFD diet (36 % of saturated fat, HFD group); HTAU, mice fed on HFD diet and supplemented with 5 % Tau; control (CTL); and CTAU. After 19 weeks of diet and Tau treatments, glucose tolerance, insulin sensitivity and islet morpho-physiology were evaluated. HFD mice presented higher body weight and fat depots, and were hyperglycemic, hyperinsulinemic, glucose intolerant and insulin resistant. Their pancreatic islets secreted high levels of insulin in the presence of increasing glucose concentrations and 30 mM K⁺. Tau supplementation improved glucose tolerance and insulin sensitivity with a higher ratio of Akt phosphorylated (pAkt) related to Akt total protein content (pAkt/Akt) following insulin administration in the liver without altering body weight and fat deposition in HTAU mice. Isolated islets from HTAU mice released insulin similarly to CTL islets. HFD intake induced islet hypertrophy, increased β-cell/islet area and islet and β-cell mass content in the pancreas. Tau prevented islet and β-cell/islet area, and islet and β-cell mass alterations induced by HFD. The total insulin content in HFD islets was higher than that of CTL islets, and was not altered in HTAU islets. In conclusion, for the first time, we showed that Tau enhances liver Akt activation and prevents β-cell compensatory morpho-functional adaptations induced by HFD.