Batroxase, a new metalloproteinase from B. atrox snake venom with strong fibrinolytic activity

The structures and functional activities of metalloproteinases from snake venoms have been widely studied because of the importance of these molecules in envenomation. Batroxase, which is a metalloproteinase isolated from Bothrops atrox (Para) snake venom, was obtained by gel filtration and anion exchange chromatography. The enzyme is a single protein chain composed of 202 amino acid residues with a molecular mass of 22.9 kDa, as determined by mass spectrometry analysis, showing an isoelectric point of 7.5. The primary sequence analysis indicates that the proteinase contains a zinc ligand motif (HELGHNLGISH) and a sequence C164I165M166 motif that is associated with a "Met-turn" structure. The protein lacks N-glycosylation sites and contains seven half cystine residues, six of which are conserved as pairs to form disulfide bridges. The three-dimensional structure of Batroxase was modeled based on the crystal structure of BmooMP alpha-I from Bothrops moojeni. The model revealed that the zinc binding site has a high structural similarity to the binding site of other metalloproteinases. Batroxase presented weak hemorrhagic activity, with a MHD of 10 mu g, and was able to hydrolyze extracellular matrix components, such as type IV collagen and fibronectin. The toxin cleaves both a and beta-chains of the fibrinogen molecule, and it can be inhibited by EDTA. EGTA and beta-mercaptoethanol. Batroxase was able to dissolve fibrin clots independently of plasminogen activation. These results demonstrate that Batroxase is a zinc-dependent hemorrhagic metalloproteinase with fibrin(ogen)olytic and thrombolytic activity. Published by Elsevier Ltd.

Arthrospira platensis biomass with high protein content cultivated in continuous process using urea as nitrogen source

Aims: Arthrospira platensis has been studied for single-cell protein production because of its biomass composition and its ability of growing in alternative media. This work evaluated the effects of different dilution rates (D) and urea concentrations (N0) on A.similar to platensis continuous culture, in terms of growth, kinetic parameters, biomass composition and nitrogen removal. Methods and results: Arthrospira platensis was continuously cultivated in a glass-made vertical column photobioreactor agitated with Rushton turbines. There were used different dilution rates (0.040.44 day-1) and urea concentrations (0.5 and 5 mmol l-1). With N0 = 5 mmol l-1, the maximum steady-state biomass concentration was1415 mg l-1, achieved with D = 0.04 day-1, but the highest protein content (71.9%) was obtained by applying D = 0.12 day-1, attaining a protein productivity of 106.41 mg l-1 day-1. Nitrogen removal reached 99% on steady-state conditions. Conclusions: The best results were achieved by applying N0 = 5 mmol l-1; however, urea led to inhibitory conditions at D = 0.16 day-1, inducing the system wash-out. The agitation afforded satisfactory mixture and did not harm the trichomes structure. Significance and Impact of the Study: These results can enhance the basis for the continuous removal of nitrogenous wastewater pollutants using cyanobacteria, with an easily assembled photobioreactor.

B Lymphocyte-Induced Maturation Protein-1 Contributes to Intestinal Mucosa Homeostasis by Limiting the Number of IL-17-Producing CD4(+) T Cells

The transcription factor B lymphocyte induced maturation protein-1 (Blimp-1) plays important roles in embryonic development and immunity. Blimp-1 is required for the differentiation of plasma cells, and mice with T cell specific deletion of Blimp-1 (Blimp-1CKO mice) develop a fatal inflammatory response in the colon. Previous work demonstrated that lack of Blimp-1 in CD4(+) and CD8(+) T cells leads to intrinsic functional defects, but little is known about the functional role of Blimp-1 in regulating differentiation of Th cells in vivo and their contribution to the chronic intestinal inflammation observed in the Blimp1CKO mice. In this study, we show that Blimp-1 is required to restrain the production of the inflammatory cytokine IL-17 by Th cells in vivo. Blimp-1CKO mice have greater numbers of IL-17 producing TCR beta(+)CD4(+)cells in lymphoid organs and in the intestinal mucosa. The increase in IL-17 producing cells was not restored to normal levels in wild-type and Blimp-1CKO mixed bone marrow chimeric mice, suggesting an intrinsic role for Blimp-1 in constraining the production of IL-17 in vivo. The observation that Blimp-1 deficient CD4(+) T cells are more prone to differentiate into IL-17(+)/IFN-gamma(+) cells and cause severe colitis when transferred to Rag1-deficient mice provides further evidence that Blimp-1 represses IL-17 production. Analysis of Blimp-1 expression at the single cell level during Th differentiation reveals that Blimp-1 expression is induced in Th1 and Th2 but repressed by TGF-beta in Th17 cells. Collectively, the results described here establish a new role for Blimp-1 in regulating IL-17 production in vivo. The Journal of Immunology, 2012,189: 5682-5693.

Molecular characterization of the Aspergillus nidulans fbxA encoding an F-box protein involved in xylanase induction

The filamentous fungus Aspergillus nidulans has been used as a fungal model system to study the regulation of xylanase production. These genes are activated at transcriptional level by the master regulator the transcriptional factor XInR and repressed by carbon catabolite repression (CCR) mediated by the wide-domain repressor CreA. Here, we screened a collection of 42 A. nidulans F-box deletion mutants grown either in xylose or xylan as the single carbon source in the presence of the glucose analog 2-deoxy-D-glucose, aiming to identify mutants that have deregulated xylanase induction. We were able to recognize a null mutant in a gene (fbxA) that has decreased xylanase activity and reduced xInA and xInD mRNA accumulation. The Delta fbxA mutant interacts genetically with creAd-30, creB15, and creC27 mutants. FbxA is a novel protein containing a functional F-box domain that binds to Skp1 from the SCF-type ligase. Blastp analysis suggested that FbxA is a protein exclusive from fungi, without any apparent homologs in higher eukaryotes. Our work emphasizes the importance of the ubiquitination in the A. nidulans xylanase induction and CCR. The identification of FbxA provides another layer of complexity to xylanase induction and CCR phenomena in filamentous fungi. (C) 2011 Elsevier Inc. All rights reserved.

Haplotypes of susceptibility to chronic periodontitis in the Interleukin 8 gene do not influence protein level in the gingival crevicular fluid

Objective: Previously, we identified that the ATC/TTC haplotype formed by polymorphisms in the Interleukin-(IL)8 gene conferred susceptibility to chronic periodontitis (CP). The aim of the study was to investigate whether the IL8 haplotype ATC/TTC was associated with the volume of gingival crevicular fluid (GCF), the concentration of interleukin IL-8 in the GCF, as well as periodontal conditions in patients with CP in comparison to controls without CP. Methods: Seventy-nine individuals (CP: n = 41, controls: n = 38) were grouped according to the presence (susceptible for CP) or absence (not susceptible for CP) of the IL8 ATC/TTC haplotype. After periodontal clinical evaluation, they were subdivided by the presence or absence of CP. GCF was collected from each patient and the IL-8 levels were determined by ELISA. The GCF volume of each subject was measured by means of a calibrated electronic device. Comparisons of means between carriers and non-carriers of the ATC/TTC haplotype were evaluated using the Mann-Whitney test. Linear regression and stepwise linear regression analysis were used to analyse the association of the GCF volume with potential covariates and their contribution for the phenotype. Results: We did not find significant differences of both periodontal conditions and IL-8 concentration in the GCF of patients with the presence or absence of the IL8 ATC/TTC haplotype. However, the GCF volume was significantly higher amongst the patients affected by CP that are absent for the IL8 ATC/TTC haplotype. In addition, linear regression analysis showed a statistically significant association between GCF volume and CP, IL8 haplotype ATC/TTC and IL-8 concentration. Conclusions: The IL8 haplotype of susceptibility to CP was neither associated with IL-8 cytokine levels nor with clinical periodontal parameters. Also, CP, IL8 haplotype and IL-8 concentration showed a positive association with the GCF volume levels in the studied patients. (c) 2012 Published by Elsevier Ltd.

Single-cell expression analysis of BMP15 and GDF9 in mature oocytes and BMPR2 in cumulus cells of women with polycystic ovary syndrome undergoing controlled ovarian hyperstimulation

To detect expression of bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) in oocytes, and their receptor type 2 receptor for BMPs (BMPR2) in cumulus cells in women with polycystic ovary syndrome (PCOS) undergoing in vitro fertilization (IVF), and determine if BMPR2, BMP15, and GDF9 expression correlate with hyperandrogenism in FF of PCOS patients. Prospective case-control study. Eighteen MII-oocytes and their respective cumulus cells were obtained from 18 patients with PCOS, and 48 MII-oocytes and cumulus cells (CCs) from 35 controls, both subjected to controlled ovarian hyperstimulation (COH), and follicular fluid (FF) was collected from small (10-14 mm) and large (> 18 mm) follicles. RNeasy Micro Kit (Qiagen(A (R))) was used for RNA extraction and gene expression was quantified in each oocyte individually and in microdissected cumulus cells from cumulus-oocyte complexes retrieved from preovulatory follicles using qRT-PCR. Chemiluminescence and RIA assays were used for hormone assays. BMP15 and GDF9 expression per oocyte was higher among women with PCOS than the control group. A positive correlation was found between BMPR2 transcripts and hyperandrogenism in FF of PCOS patients. Progesterone values in FF were lower in the PCOS group. We inferred that BMP15 and GDF9 transcript levels increase in mature PCOS oocytes after COH, and might inhibit the progesterone secretion by follicular cells in PCOS follicles, preventing premature luteinization in cumulus cells. BMPR2 expression in PCOS cumulus cells might be regulated by androgens.

DNA repair gene excision repair cross complementing-group 1 (ERCC1) in head and neck squamous cell carcinoma: analysis of methylation and polymorphism (G19007A), protein expression and association with epidemiological and clinicopathological factors

Aims: To evaluate the associations of excision repair cross complementing-group 1 (ERCC1) (DNA repair protein) (G19007A) polymorphism, methylation and immunohistochemical expression with epidemiological and clinicopathological factors and with overall survival in head and neck squamous cell carcinoma (HNSCC) patients. Methods and results: The study group comprised 84 patients with HNSCC who underwent surgery and adjuvant radiotherapy without chemotherapy. Bivariate and multivariate analyses were used. The allele A genotype variant was observed in 79.8% of the samples, GG in 20.2%, GA in 28.6% and AA in 51.2%. Individuals aged more than 45 years had a higher prevalence of the allelic A variant and a high (83.3%) immunohistochemical expression of ERCC1 protein [odds ratio (OR) = 4.86, 95% confidence interval (CI): 1.2-19.7, P = 0.027], which was also high in patients with advanced stage (OR= 5.04, 95% CI: 1.07-23.7, P = 0.041). Methylated status was found in 51.2% of the samples, and was higher in patients who did not present distant metastasis (OR = 6.67, 95% CI: 1.40-33.33, P = 0.019) and in patients with advanced stage (OR = 5.04, 95% CI: 1.07-23.7, P = 0.041). At 2 and 5 years, overall survival was 55% and 36%, respectively (median = 30 months). Conclusion: Our findings may reflect a high rate of DNA repair due to frequent tissue injury during the lifetime of these individuals, and also more advanced disease presentation in this population with worse prognosis.

Genetic relatedness of Plasmodium falciparum isolates and the origin of allelic diversity at the merozoite surface protein-1 (MSP-1) locus in Brazil and Vietnam

Abstract Background Despite the extensive polymorphism at the merozoite surface protein-1 (MSP-1) locus of Plasmodium falciparum, that encodes a major repetitive malaria vaccine candidate antigen, identical and nearly identical alleles frequently occur in sympatric parasites. Here we used microsatellite haplotyping to estimate the genetic distance between isolates carrying identical and nearly identical MSP-1 alleles. Methods We analyzed 28 isolates from hypoendemic areas in north-western Brazil, collected between 1985 and 1998, and 23 isolates obtained in mesoendemic southern Vietnam in 1996. MSP-1 alleles were characterized by combining PCR typing with allele-specific primers and partial DNA sequencing. The following single-copy microsatellite markers were typed : Polyα, TA42 (only for Brazilian samples), TA81, TA1, TA87, TA109 (only for Brazilian samples), 2490, ARAII, PfG377, PfPK2, and TA60. Results The low pair-wise average genetic distance between microsatellite haplotypes of isolates sharing identical MSP-1 alleles indicates that epidemic propagation of discrete parasite clones originated most identical MSP-1 alleles in parasite populations from Brazil and Vietnam. At least one epidemic clone propagating in Brazil remained relatively unchanged over more than one decade. Moreover, we found no evidence that rearrangements of MSP-1 repeats, putatively created by mitotic recombination events, generated new alleles within clonal lineages of parasites in either country. Conclusion Identical MSP-1 alleles originated from co-ancestry in both populations, whereas nearly identical MSP-1 alleles have probably appeared independently in unrelated parasite lineages.

Protein turnover, amino acid requirements and recommendations for athletes and active populations

Skeletal muscle is the major deposit of protein molecules. As for any cell or tissue, total muscle protein reflects a dynamic turnover between net protein synthesis and degradation. Noninvasive and invasive techniques have been applied to determine amino acid catabolism and muscle protein building at rest, during exercise and during the recovery period after a single experiment or training sessions. Stable isotopic tracers (13C-lysine, 15N-glycine, ²H5-phenylalanine) and arteriovenous differences have been used in studies of skeletal muscle and collagen tissues under resting and exercise conditions. There are different fractional synthesis rates in skeletal muscle and tendon tissues, but there is no major difference between collagen and myofibrillar protein synthesis. Strenuous exercise provokes increased proteolysis and decreased protein synthesis, the opposite occurring during the recovery period. Individuals who exercise respond differently when resistance and endurance types of contractions are compared. Endurance exercise induces a greater oxidative capacity (enzymes) compared to resistance exercise, which induces fiber hypertrophy (myofibrils). Nitrogen balance (difference between protein intake and protein degradation) for athletes is usually balanced when the intake of protein reaches 1.2 g·kg-1·day-1 compared to 0.8 g·kg-1·day-1 in resting individuals. Muscular activities promote a cascade of signals leading to the stimulation of eukaryotic initiation of myofibrillar protein synthesis. As suggested in several publications, a bolus of 15-20 g protein (from skimmed milk or whey proteins) and carbohydrate (± 30 g maltodextrine) drinks is needed immediately after stopping exercise to stimulate muscle protein and tendon collagen turnover within 1 h.

Expression and accumulation of the two-domain odorant-binding protein AaegOBP45 in the ovaries of blood-fed Aedes aegypti

BACKGROUND: Aedes aegypti mosquitoes are the main vectors of dengue viruses. Despite global efforts to reduce the prevalence of dengue using integrated vector management strategies, innovative alternatives are necessary to help prevent virus transmission. Detailed characterizations of Ae. aegypti genes and their products provide information about the biology of mosquitoes and may serve as foundations for the design of new vector control methods. FINDINGS: We studied the Ae. aegypti gene, AAEL010714, that encodes a two-domain odorant-binding protein, AaegOBP45. The predicted gene structure and sequence were validated, although single nucleotide polymorphisms were observed. Transcriptional and translational products accumulate in the ovaries of blood fed females and are not detected or are at low abundance in other tissues. CONCLUSIONS: We validated the Ae. aegypti AAEL010714 gene sequence and characterized the expression profile of a two-domain OBP expressed in ovaries. We propose that AaegOBP45 function as a component of the mosquito eggshell.

Interaction of leptospira elongation factor tu with plasminogen and complement factor h: a metabolic leptospiral protein with moonlighting activities

The elongation factor Tu (EF-Tu), an abundant bacterial protein involved in protein synthesis, has been shown to display moonlighting activities. Known to perform more than one function at different times or in different places, it is found in several subcellular locations in a single organism, and may serve as a virulence factor in a range of important human pathogens. Here we demonstrate that Leptospira EF-Tu is surface-exposed and performs additional roles as a cell-surface receptor for host plasma proteins. It binds plasminogen in a dose-dependent manner, and lysine residues are critical for this interaction. Bound plasminogen is converted to active plasmin, which, in turn, is able to cleave the natural substrates C3b and fibrinogen. Leptospira EF-Tu also acquires the complement regulator Factor H (FH). FH bound to immobilized EF-Tu displays cofactor activity, mediating C3b degradation by Factor I (FI). In this manner, EF-Tu may contribute to leptospiral tissue invasion and complement inactivation. To our knowledge, this is the first description of a leptospiral protein exhibiting moonlighting activities