Structure and post-translational modifications of the web silk protein spidroin-1 from Nephila spiders

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Leishmania replication protein A-1 binds in vivo single-stranded telomeric DNA

Replication protein A (RPA) is a highly conserved heterotrimeric single-stranded DNA-binding protein involved in different events of DNA metabolism. In yeast, subunits 1 (RPA-1) and 2 (RPA-2) work also as telomerase recruiters and, in humans, the complex unfolds G-quartet structures formed by the 3' G-rich telomeric strand. In most eukaryotes, RPA-1 and RPA-2 bind DNA using multiple OB fold domains. In trypanosomatids, including Leishmania, RPA-1 has a canonical OB fold and a truncated RFA-1 structural domain. In Leishmania amazonensis, RPA-1 alone can form a complex in vitro with the telomeric G-rich strand. In this work, we show that LaRPA-1 is a nuclear protein that associates in vivo with Leishmania telomeres. We mapped the boundaries of the OB fold DNA-binding domain using deletion mutants. Since Leishmania and other trypanosomatids lack homologues of known telomere end binding proteins, our results raise questions about the function of RPA-1 in parasite telomeres. (C) 2007 Elsevier B.V. All rights reserved.

Cell localization of the anti-inflammatory protein annexin 1 during experimental inflammatory response

The localization of the glucocorticoid-inducible protein annexin 1 (ANX-1) in leukocytes during the process of experimental inflammation has been studied using immunocytochemistry. ANX-1 immunoreactivity was detected in extravasated neutrophils and eosinophils as well as in resident tissue mast cells. Following injection of carrageenin, the mesenteric tissue was highly inflamed with large presence of leukocytes (predominantly neutrophils with a small percentage of eosinophils) adherent to post-capillary venules and extravasated in the perivascular tissue. ANX-1 immunoreactivity was detected in the cytosol of neutrophils and eosinophils mainly associated with granules and/or vesicles. A good degree of localization in the endosomes was observed in the neutrophils, In both cell types, some ANX-1 immunoreactivity in the nucleus and in the plasma membrane was also detected. Resident mast cells were also activated. Mast cells were positive for ANX-1, without apparent changes in protein content in relation to their activation status. Degranulated mast cells still presented ANX-1 associated with the granule matrix. In conclusion, this study demonstrated the presence of ANX-1 in leukocytes that play a central role in the host inflammatory response. These are the extravasating polymorphonuclear cells, or the resident mast cells. These data provide morphological support to the notion that endogenous and exogenous ANX-1 are able to modulate the reactivity of these cell types, and more generally, of the experimental inflammatory reaction.

Immunohistochemical detection of metalloproteinase-9 (MMP-9), anti-oxidant like 1 protein (AOP-1) and synaptosomal-associated protein (SNAP-25) in the cerebella of dogs naturally infected with spontaneous canine distemper

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Melatonin decreases breast cancer metastasis by modulating Rho-associated kinase protein-1 expression

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Time-dependent expression of annexin 1 in a model of chronic granulomatous inflammation

Objective and design: To determine the expression pattern and distribution of the glucocorticoid-inducible protein annexin 1 (ANXA1) in a murine model of chronic granulomatous inflammation.Materials or subjects: TO Mouse.Treatment: Chronic granulomatous inflammation was induced by injecting into dorsal sub-cutaneous air-pouches in mice, a mixture of croton oil and Freund's complete adjuvant (CO/FCA).Methods: Western and northern analysis, corticosterone assay, and immunohistochemistry. Statistical analysis was performed using ANOVA followed by Tukey's pair-wise comparisons or Dunnett's multiple comparisons.Results: ANXA1 protein levels changed significantly throughout the 4-week time course, with an initial peak at day 7 and a later elevation at 28 days. ANXA1 mRNA levels peaked at days 1 and 3, with a significant decline at day 7 followed by an upward trend to day 28. Plasma corticosterone measurements taken throughout the time course revealed an increase from 14 days onward, suggesting that corticosterone does not influence ANXA1 expression during the initial stages of the model. Immunogold staining revealed that ANXA1 expression in the inflamed tissue was mainly in extravasated neutrophils, with intact protein (37 kDa) being predominantly observed on the cell membrane.Conclusions: the pattern of ANXA1 expression indicates that infiltrated neutrophils are responsible for the majority of ANXA1 present both at early and later stages of this model of granulomatous inflammation.

Maternal protein restriction affects gene expression and enzyme activity of intestinal disaccharidases in adult rat offspring

This study investigated the consequences of intrauterine protein restriction on the gastrointestinal tract and particularly on the gene expression and activity of intestinal disaccharidases in the adult offspring. Wistar rat dams were fed isocaloric diets containing 6% protein (restricted, n = 8) or 17% protein (control, n = 8) throughout gestation. Male offspring (n = 5-8 in each group) were evaluated at 3 or 16 weeks of age. Maternal protein restriction during pregnancy produced offspring with growth restriction from birth (5.7 ± 0.1 vs 6.3 ± 0.1 g; mean ± SE) to weaning (42.4 ± 1.3 vs 49.1 ± 1.6 g), although at 16 weeks of age their body weight was similar to control (421.7 ± 8.9 and 428.5 ± 8.5 g). Maternal protein restriction also increased lactase activity in the proximal (0.23 ± 0.02 vs 0.15 ± 0.02), medial (0.30 ± 0.06 vs 0.14 ± 0.01) and distal (0.43 ± 0.07 vs 0.07 ± 0.02 U·g-1·min-1) small intestine, and mRNA lactase abundance in the proximal intestine (7.96 ± 1.11 vs 2.38 ± 0.47 relative units) of 3-week-old offspring rats. In addition, maternal protein restriction increased sucrase activity (1.20 ± 0.02 vs 0.91 ± 0.02 U·g-1·min-1) and sucrase mRNA abundance (4.48 ± 0.51 vs 1.95 ± 0.17 relative units) in the duodenum of 16-week-old rats. In conclusion, the present study shows for the first time that intrauterine protein restriction affects gene expression of intestinal enzymes in offspring.

Energy and protein requirements for maintenance and growth of Boer crossbred kids

Meat production by goats has become an important livestock enterprise in several parts of the world. Nonetheless, energy and protein requirements of meat goats have not been defined thoroughly. The objective of this study was to determine the energy and protein requirements for maintenance and growth of 34 3/4 Boer x 1/4 Saanen crossbred, intact male kids (20.5 +/- 0.24 kg of initial BW). The baseline group was 7 randomly selected kids, averaging 21.2 +/- 0.36 kg of BW. An intermediate group consisted of 6 randomly selected kids, fed for ad libitum intake, that were slaughtered when they reached an average BW of 28.2 +/- 0.39 kg. The remaining kids (n = 21) were allocated randomly on d 0 to 3 levels of DMI (treatments were ad libitum or restricted to 70 or 40% of the ad libitum intake) within 7 slaughter groups. A slaughter group contained 1 kid from each treatment, and kids were slaughtered when the ad libitum treatment kid reached 35 kg of BW. Individual body components (head plus feet, hide, internal organs plus blood, and carcass) were weighed, ground, mixed, and subsampled for chemical analyses. Initial body composition was determined using equations developed from the composition of the baseline kids. The calculated daily maintenance requirement for NE was 77.3 +/- 1.05 kcal/kg(0.75) of empty BW (EBW) or 67.4 +/- 1.04 kcal/kg(0.75) of shrunk BW. The daily ME requirement for maintenance (118.1 kcal/g(0.75) of EBW or 103.0 kcal/kg(0.75) of shrunk BW) was calculated by iteration, assuming that the heat produced was equal to the ME intake at maintenance. The partial efficiency of use of ME for NE below maintenance was 0.65. A value of 2.44 +/- 0.4 g of net protein/kg(0.75) of EBW for daily maintenance was determined. Net energy requirements for growth ranged from 2.55 to 3.0 Mcal/kg of EBW gain at 20 and 35 kg of BW, and net protein requirements for growth ranged from 178.8 to 185.2 g/kg of EBW gain. These results suggest that NE and net protein requirements for growing meat goats exceed the requirements previously published for dairy goats. Moreover, results from this study suggest that the N requirement for maintenance for growing goats is greater than the established recommendations.

Utilization of fish protein hydrolysate in prepared diets for pacu, Piaractus mesopotamicus, larvae

Growth and survival rates of pacu, Piaractus mesopotamicus, larvae fed prepared diets containing different animal protein sources were evaluated. Four diets with the same level of crude protein (CP) (36%) and calories (4.02 kcal gross energy/g of diet) were fed to the larvae. Diets were formulated to contain one of four protein sources: (1) fish meal (FM), (2) tilapia residue silage (TS), (3) protein hydrolysate from tilapia residue (HT), and (4) eviscerated tilapia residue (HET). Larvae were fed Artemia nauplii for six days, prior to the start of the study, and the prepared diet was supplied from day 7 until the study concluded. Variance analysis showed no significant differences (P > 0.05) for survival rates and larval final lengths among treatments. However, final average weights were significantly different (P < 0.05 for larvae fed FM and HT. Average survival rates were relatively high and ranged from 68.1% to 73.9%. After the live food was replaced by prepared diets, no larval growth was observed for any treatment. Fish protein hydrolysate (HT and HET) and fish silage showed potential to be used as ingredients in the diet of pacu larvae. However, hydrolysate inclusion levels, processing methods to minimize nutrient lixiviation, and the best moment to replace live food with an inert diet (weaning) need further investigation. © 2003 by The Haworth Press, Inc. All rights reserved.

Three-year follow-up of Interleukin 6 and C-reactive protein in chronic obstructive pulmonary disease

Background: Past studies have shown that mean values of Interleukin-6 (IL-6) and C-reactive protein (CRP) do not change significantly in COPD patients over a one-year period. However, longer period follow-up studies are still lacking. Thus, the aim of this study is to evaluate plasma CRP and IL-6 concentration over three years in COPD patients and to test the association between these inflammatory mediators and disease outcome markers. Methods: A cohort of 77 outpatients with stable COPD was evaluated at baseline, and 53 (mean FEV1, 56% predicted) were included in the prospective study. We evaluated Interleukin-6 (IL-6), C-reactive protein (CRP), six-minute walking distance (6MWD), and body mass index (BMI) at baseline and after three years. Plasma concentration of IL-6 was measured by high sensitivity ELISA, and CRP was obtained by high sensitivity particle-enhanced immunonephelometry. Results: IL-6 increased significantly after 3 years compared to baseline measurements [0.8 (0.5-1.3) vs 2.4 (1.3-4.4) pg/ml; p < 0.001] and was associated with worse 6MWD performance. In the Cox regression, increased IL-6 at baseline was associated with mortality [Hazard Ratio (95% CI) = 2.68 (0.13, 1.84); p = 0.02]. CRP mean values did not change [5 (1.6-7.9) vs 4.7 (1.7-10) pg/L; p = 0.84], although eleven patients (21%) presented with changes >3 mg/L in CRP after 3 years. Conclusions: The systemic inflammatory process, evaluated by IL-6, seems to be persistent, progressive and associated with mortality and worse physical performance in COPD patients. Trial registration: No.:NCT00605540. © 2013 Ferrari et al; licensee BioMed Central Ltd.

Methylation pattern of THBS1, GATA-4, and HIC1 in pediatric and adult patients infected with helicobacter pylori

Background: Helicobacter pylori infection is usually acquired in childhood and persists into adulthood if untreated. The bacterium induces a chronic inflammatory response, which is associated with epigenetic alterations in oncogenes, tumor-suppressor genes, cell-cycle regulators, and cell-adhesion molecules. Aim: The aim of this study was to analyze the effect of H. pylori infection on the methylation status of Thrombospondin-1 (THBS1), Hypermethylated in cancer 1 (HIC1) and Gata binding protein-4 (GATA-4) in gastric biopsy samples from children and adults infected or uninfected with the bacterium and in samples obtained from gastric cancer patients. Methods: The methylation pattern was analyzed with methylation-specific PCR. Results: Our results showed that H. pylori infection was associated with methylation of the promoter regions of the THBS1 and GATA-4 genes in pediatric and adult samples (p < 0.01). HIC1 showed the lowest level of methylation, which was not an early event during gastric carcinogenesis. Conclusions: The results from this study indicate that methylation of THBS1 and GATA-4 occurs in the early stages of chronic gastritis and gastric cancer in association with H. pylori infection; however, in gastric cancer samples, other mechanisms cooperate with the down-regulation of these genes. Methylation of HIC1 may not be the principal mechanism implicated in its down-regulation in gastric cancer samples. © 2013 Springer Science+Business Media New York.

Functional and Topological Studies with Trp-Containing Analogs of the Peptide StII(1-30) Derived From the N-Terminus of the Pore Forming Toxin Sticholysin II: Contribution to Understand its Orientation in Membrane

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Modelo experimental de conjuntivite alérgica: efeito do tratamento farmacológico com a proteína anti-inflamatória galectina - 1

Pós-graduação em Genética - IBILCE

Efeitos de absorventes de umidade e de aditivos químicos e microbianos sobre o valor nutritivo, o perfil fermentativo e as perdas em silagens de capim-marandu

The objective of this trial was to evaluate the nutritional value, fermentation profile and dry matter losses of Palisadegrass silages ensiled with either dried citrus pulp, soybean hulls, chemical or microbial additives. The trial was carried out in a completely randomized experimental design and in a factorial arrangement (3 x 5), with three dry matter levels (wet forage or forage ensiled with pelleted citrus pulp or pelleted soybean hulls) and five additives (without or with the presence of bacterial inoculants or the addition of: sodium benzoate, formic acid in the concentration of 62% or 44%), totalizing 15 treatments and 60 experimental silos. The variables analyzed were: nutritional value, losses due to gases and effluents, and dry matter recovery. The use of dried citrus pulp or soybean hulls at the ensiling time increased the dry matter content (29.4 and 28,9%) and decreased the effluent production (4.1 and 3.8 kg/t of fresh matter), also providing fermentable substrate to microorganisms, resulting in increased fermentation coefficient and digestibility of silages. The use of formic acid resulted in silages with higher digestibility and increased water-soluble carbohydrates and crude protein content. This additive was also effective in reducing the losses due to gases and, as a result, increased the total dry matter recovery. The treatment containing homolactic bacteria showed similar trend of increasing the digestibility and reducing the losses due to gases. The use of sodium benzoate was less effective in altering the fermentation pattern of tropical silages. The nutritional value and total dry matter losses of silages ensiled without additives can be considered satisfactory. However, wet forage ensiled with dried citrus pulp and, mainly, with soybeans hulls showed the best results. Treatments containing formic acid had a beneficial effect on the fermentation profile of tropical grass silages.