High-resolution structural profile of hylaseptin-4: Aggregation, membrane topology and pH dependence of overall membrane binding process.

Hylaseptin-4 (HSP-4, GIGDILKNLAKAAGKAALHAVGESL-NH2) is an antimicrobial peptide originally isolated from Hypsiboas punctatus tree frog. The peptide has been chemically synthetized for structural investigations by CD and NMR spectroscopies. CD experiments reveal the high helical content of HSP-4 in biomimetic media. Interestingly, the aggregation process seems to occur at high peptide concentrations either in aqueous solution or in presence of biomimetic membranes, indicating an increase in the propensity of the peptide for adopting a helical conformation. High-resolution NMR structures determined in presence of DPC-d38 micelles show a highly ordered α-helix from amino acid residues I2 to S24 and a smooth bend near G14. A large separation between hydrophobic and hydrophilic residues occurs up to the A16 residue, from which a shift in the amphipathicity is noticed. Oriented solid-state NMR spectroscopy show a roughly parallel orientation of the helical structure along the POPC lipid bilayer surface, with an insertion of the hydrophobic N-terminus into the bilayer core. Moreover, a noticeable pH dependence of the aggregation process in both aqueous and in biomimetic membrane environments is attributed to a single histidine residue (H19). The protonation degree of the imidazole side-chain might help in modulating the peptide-peptide or peptide-lipid interactions. Finally, molecular dynamics simulations confirm the orientation and preferential helical conformation and in addition, show that HSP-4 tends to self-aggregate in order to stabilize its active conformation in aqueous or phospholipid bilayer environments.

Herd-level factors associated with piglet weight at weaning, kilograms of piglets weaned per sow per year and sow feed conversion.

To understand the production factors that affect conclusive parameters of sow herd performance can improve the use of the resources and profitability of farm. The objective of this study was to identify associations and quantify the effects of a set of factors related to piglet weight at weaning (PWW), kilograms of piglets weaned per sow per year (kgPWSY) and sow feed conversion (SFC). Data from 150 farms were collected, for a total study population of 135 168 sows, including gilt replacement, breeding (mating), gestation and farrowing/lactation phases. A questionnaire focusing on reproductive performance, management, facilities, feeding, health and biosafety was administered. Multiple linear regression models were used to assess associations among factors with each of the three dependent variables. Increased duration of lactation was positively associated with PWW, kgPWSY and SFC. The increase in the number of live born pigs per litter was positively associated with kgPWSY and with SFC. Farms with higher PWW had farrowing room humidifiers, did not surgically castrate male piglets and used quaternary ammonia compounds for farrowing room disinfection. Farms with higher kgPWSY used lined ceilings in farrowing rooms and winter feeds with higher CP percentages in gestation; they also had more farrowings per sow per year. Sow feed conversion was worse in farms with partly slatted floors during gestation, in farms feeding lactating sows six times a day or ad libitum and farms with a higher sow-handler ratio. This study indicates that farms can increase PWW and kgPWSY and improve the SFC by changing one or more management, biosafety and feeding practices or facilities as well as by focusing on improving several performance parameters, particularly increasing the duration of lactation and the number of live born pigs per litter.

MSX1 gene polymorphisms in non-syndromic cleft lip and/or palate.

OBJECTIVE: The aim of this study was to investigate the contribution of 6 polymorphic variants of the MSX1 gene in non-syndromic cleft lip and/or palate (NSCL/P). METHODS: Three hundred and fifty-eight individuals (158 NSCL/P cases and 200 controls) were genotyped by TaqMan allelic discrimination using predesigned SNP assays. Statistical analyses were conducted using the software spss 15.0 and the r statistical suite. Haplotype block structure and haplotype frequencies were determined using the Haploview. A P-value of 0.05 and confidence interval of 95% were used for all of statistical tests. RESULTS: The patients with non-syndromic cleft lip and/or palate were characterized by similar distribution of MSX1 genotypes and allele in comparison to subjects without oral clefts (P > 0.05). Two haplotype blocks were constructed with polymorphisms of MSX1 gene and haplotypes formed showed a similar frequency in patients with and without oral clefts. CONCLUSIONS: The present study provides no evidence that MSX1 polymorphisms (rs3775261, rs1042484, rs12532, rs6446693, rs4464513 and rs1907998) play a major role in NSCL/P.

Influence of excipients and technological process on anti-inflammatory activity of quercetin and Achyrocline satureioides (Lam.) D.C. extracts by oral route.

The flavonoids quercetin, 3-O-methylquercetin and luteolin play an important role in the anti-inflammatory activity of Achyrocline satureioides ethanol extracts when administered intraperitoneally. The present work describes the oral anti-inflammatory effect of quercetin and A. satureioides extracts and the role played by the solvent concentration, adjuvant and drying processes of freeze-drying (FD) or spray-drying (SD) on the effect. The best anti-edema effect was observed with 250 mg/kg body wt of the freeze-dried powder (FDP), prepared with 40% (v/v) ethanol (FDP40). In contrast, 250 mg/kg body wt of FDP80, prepared with ethanol 80% (ES80), did not significantly inhibit the carrageenan-induced rat paw edema. However, when ES80 was freeze-dried in the presence of polysorbate 80 (FDP80-P80) or spray-dried in the presence of colloidal silicon dioxide (CSD) and P80 (SDP80), both dried extracts became more active. Quercetin suspension in saline did not inhibit paw edema, but the mixture of quercetin with polysorbate 80 was effective in edema inhibition by the oral route. Aqueous extract (ESAQ), freeze-dried (FDPAQ, FDPAQ-P80) or spray-dried (SDPAQ) did not exhibit the edema-inhibition effect. Taken together, the results point to the following order of efficacy (at 4 h, for example): FDP40 > indomethacin > SDP40 > SDP80 = FDP80-80 > Quercetin-P80. Additionally, the FDP40, SDP40 (prepared from 40% v/v ethanol added of CSD) and SDP80 reduced the total leukocyte and polymorphonuclear cell migration in the pleural cavity.

Antioxidant, a pro-oxidant and cytotoxic effects of Achyrocline satureioides extracts.

In this study we compared the antioxidant properties of five different extracts of different composition obtained from Achyrocline satureioides' inflorescences (Compositae), a widely used Brazilian folk medicinal herb. All of the extracts presented significant antioxidant potential identified by TRAP assay, which increased in the presence of human plasma. Characterization of the content of flavonoids in each extract showed that the FDP80 (ethanol 80%) and FFr (enriched flavonoid fraction) extracts contained a higher content of flavonoids. Cytotoxicity of the extracts as determined in Sertoli cell culture showed that FDP80 and FFr were highly toxic at most concentrations tested. The extracts induced a significant increase in lipid peroxidation levels in Sertoli cells. These results suggest that medicinal herb extracts that contain higher flavonoid concentrations and shows higher antioxidant protection in vitro might not always produce the greatest benefit.

LC determination of flavonoids: separation of quercetin, luteolin and 3-O-methylquercetin in Achyrocline satureioides preparations.

The pharmacological activities of the flavonoids show the interest in quantifying these constituents in phytopharmaceutical preparations, as well as in the validation of the analytical methodologies. LC methods have been reported to quantify isolated flavonoids or these compounds in complex biological matrices, such as herbal raw materials and extractive preparations. This work was designed, therefore, to develop an LC system to separate quercetin, luteolin and 3-O-methylquercetin and to quantify them in extractive solutions from Achyrocline satureioides. The main validation parameters of the method are also determined. The method showed linearity for quercetin and luteolin in the range 1-10 microg/ml. The aqueous and ethanol 80% extractive solutions showed linear response in the range 2.5-20 microl/ml and ethanol 40% extractive solution in the range 2.5-10 microl/ml. Precision and accuracy were determined for ethanol 80% extractive solution, in the concentration of 10 microl/ml. The LC method showed an excellent performance in separating the flavonoids quercetin, luteolin and 3-O-methylquercetin in A. satureioides extracts, since the presence of interference has been previously evaluated.

The adjuvants aerosil 200 and Gelita-Sol-P influence on the technological characteristics of spray-dried powders from Passiflora edulis var. flavicarpa.

Passiflora edulis (passionflower) is a plant widely used in the Brazilian popular medicine and phytopharmaceutical industry for its sedative activity. This work refers to the development of spray-dried powders (SDPs) from the 40% ethanolic extractive solution of P. edulis aerial parts. The SDPs were prepared with a Büchi 190 Mini-spray dryer using as drying adjuvants Aerosil 200 alone (SDP1), an Aerosil 200: Gelita-Sol-P (1:1) mixture (SDP2) and an Aerosil 200:Gelita-Sol-P (1:3) mixture (SDP3). All the powders were obtained using 40 parts adjuvant and 60 parts extract dried residue. The comparison criteria applied were particle size distribution, hygroscopicity at 95%, 60%, and 35% relative humidity (RH), as well as the flavonoid process recovery. The particle size distributions were analyzed by way of (a) normal distribution parameters, (b) the RRSB grid and (c) considering diameter values in terms of an equivalent sphere. All the powders presented nonnormal distribution, and the RRSB analysis appeared to be, therefore, the analysis method of choice. The total flavonoid recovery was around 80%, and it was practically not affected by the SDP1, SDP2, and SDP3 compositions. At the 60% and 90% RH atmospheres, the SDP3 and SDP2 moisture uptakes were higher than that of the SDP1. All the formulations were, on the contrary, stable at 35% RH, showing a slight moisture loss tendency. The results showed in sum that the SDP prepared using Aerosil 200 as the drying adjuvant alone presented the best technological characteristics of all.

Genetics of immunological tolerance: I. Bidirectional selective breeding of mice for oral tolerance.

The polygenic nature of oral tolerance regulation was elucidated by the method of bidirectional selective breeding of mouse strains for tolerance susceptibility (TS) and resistance (TR) starting from a genetically heterogeneous population achieved by the equilibrated intercrossing of eight inbred mouse strains (A/J, DBA/2J, P/J, SWR/J, SJL/J, CBA/J, BALB/cJ and C57BL/6J). Seven days after intragastric administration of 5 mg OVA or BSA, mice were intraperitoneally immunized with 100 microg of the corresponding antigen. The individual antibody titres were measured by haemagglutination. The phenotypes at the highest and lowest extremes were selected for assortative mating, avoiding consanguinity. The second litter of each mating couple was intraperitoneally immunized only to evaluate the immunocompetence of the corresponding generation and to ascertain the non-selection of non-responder mice. A normal distribution of agglutinin titres ranging from 4 to 14 log2 was observed in the F0 population. In the F12 generation, TR and TS strains showed highly significant differences for agglutinin titres (TR=15.06+/-1.80 and TS=8.35+/-2.44), and IgG responses by ELISA. Up to the F12 generation, the mean realized heritability was 0.14+/-0.02. The response to the selection was 0.43 log2 and the selection differential 3.10 log2/generation. A provisional estimation indicated that oral tolerance may be influenced by eight or nine independent loci.

[Prevalence and indirect costs of headache in a Brazilian Company]. / Prevalência e custos indiretos das cefaléias em uma empresa Brasileira.

Employees from a Brazilian oil company research centre (n = 993) were interviewed on the occurrence of headache during a 30 days period. Headache prevalence was 49.8%, with a mean frequency of 4.3 +/- 7.0 attacks per month, lasting 12.2 +/- 21.4 hours each. According to the International Headache Society diagnostic criteria, migraine (5.5%), episodic tension-type headache (26.4%), chronic tension-type headache (1.7%) and headaches not fulfilling the criteria for such disorders (16.2%) were observed. Women suffered comparatively more headache and specifically migraine than men. The pain interfered with work productivity in 10% of the subjects, corresponding to 538.75 hours off. According to an indirect costs estimation for each headache, the company may loose up to US$125.98 per employee annually. Since among headaches migraine has the highest indirect cost, migraine prevention and treatment is particularly important at the working environment. Migraine frequency may be prevented to a large extent, resulting on positive effects in both the quality of life and productivity. The cost-benefit ratio clearly favours therapeutic and preventive programs against chronic headaches.

Allelic associations of two polymorphic microsatellites in intron 40 of the human von Willebrand factor gene.

At intron 40 of the von Willebrand factor (vWF) gene, two GATA-repeat polymorphic sites exist that are physically separated by 212 bp. At the first site (vWF1 locus), seven segregating repeat alleles were observed in a Brazilian Caucasian population, and at the second (vWF2 locus) there were eight alleles, detected through PCR amplifications of this DNA region. Haplotype analysis of individuals revealed 36 different haplotypes in a sample of 338 chromosomes examined. Allele frequencies between generations and gender at each locus were not significantly different, and the genotype frequencies were consistent with their Hardy-Weinberg expectations. Linkage disequilibrium between loci is highly significant with positive allele size association; that is, large alleles at the loci tend to occur together, and so do the small alleles. Variability at each locus appeared to have arisen in a stepwise fashion, suggesting replication slippage as a possible mechanism of production of new alleles. However, we observed an increased number of haplotypes, in contrast with the predictions of a stepwise production of variation in the entire region, suggesting some form of "cooperative" changes between loci that could be due to either gene conversion, or a common control mechanism of production of new variation at these repeat polymorphism sites. The high degree of polymorphism (gene diversity values of 72% and 78% at vWF1 and vWF2, respectively, and of 93% at the haplotype level) makes these markers informative for paternity testing, genetic counseling, and individual-identification purposes.