BJ-48, a novel thrombin-like enzyme from the Bothrops jararacussu venom with high selectivity for Arg over Lys in P1: Role of N-glycosylation in thermostability and active site accessibility.

BJ-48, a serine protease from the venom of Bothrops jararacussu, was purified to homogeneity using affinity chromatography on p-aminobenzamidine-agarose followed by HPLC gel filtration. BJ-48 presented 52kDa by SDS-PAGE analysis and 48,036Da by electron spray mass spectrometry. The enzyme was shown to be highly glycosylated with 42% of N-linked carbohydrates composed of Fuc(1):GalN(4):GlcN(5):Gal(1):Man(2) and a high content of sialic acid residues (8-12%). BJ-48 had optimal esterase activity at pH 7.5 and displayed maximum catalytic rate at 50 degrees C. Its hydrolytic activity was strongly inhibited by aprotinin and dithiothreitol while N-tosyl-l-phenylalanine chloromethyl ketone, 6-aminocaproic acid, E-64 and soybean trypsin inhibitor (SBTI) were ineffective. The kinetics of BJ-48 with chromogenic substrates revealed an unprecedented selectivity (10(4)-fold) for Arg over Lys in P1. BJ-48 proved to be a thrombin-like enzyme (TLE) with a specific fibrinogen-clotting activity of 73.4NIH units/mg. The TLE rapidly digested human fibrinogen Bbeta chain, but the Aalpha chain was cleaved specifically to release fibrinopeptide A with k(cat)/K(m)=2.1muM(-1)s(-1). The TLE showed no activity toward other thrombin substrates like protein C, protease-activated receptor-1 or inhibitors such as hirudin and antithrombin. A non-denaturing procedure using PNGase F and neuraminidase followed by hydrophobic interaction chromatography was employed to obtain active BJ-48 forms with variable carbohydrate content. Compared to the native enzyme, total or partially deglycosylated BJ-48 forms presented up to 2-fold reduction in their specific activities upon heating at 55/65 degrees C or treatment with SBTI. These results point out a role for BJ-48 glycosylation in thermostability and controlling the access of some canonical protein inhibitors to the active site.     

Well-being therapy of generalized anxiety disorder

BACKGROUND: There is increasing awareness that the goal of treatment in generalized anxiety disorder (GAD) should not simply be a response, but restoration of normal function. The aim of this study was to apply a novel psychotherapeutic approach for increasing the level of remission in GAD. METHODS: Twenty patients with DSM-IV GAD devoid of comorbid conditions were randomly assigned to 8 sessions of cognitive behavioral therapy (CBT) or the sequential administration of 4 sessions of CBT followed by other 4 sessions of well-being therapy (WBT). Assessment methods included the Anxiety and Depression Scales of Paykel's Clinical Interview for Depression, Ryff's Psychological Well-being Scales and Kellner's Symptom Questionnaire. A one-year follow-up was undertaken. RESULTS: Significant advantages of the CBT-WBT sequential combination over CBT only were observed with both observer and self-rated methods after treatment. Such gains were maintained at follow-up. CONCLUSIONS: These preliminary results suggest the feasibility and clinical advantages of adding WBT to the treatment of GAD. They lend support to a sequential use of treatment components for achieving a more sustained recovery.     

Antibacterial activity and inhibition against Staphylococcus aureus NorA efflux pump by ferulic acid and its esterified derivatives

Objective: To evaluate the inhibitory activity of ferulic acid and four of its esterified derivatives (methyl, ethyl, propyl, and butyl) against resistance mech...     

Gastroprotective effect of a flavone from Lonchocarpus araripensis Benth. (Leguminosae) and the possible mechanism

The gastroprotective effect of DDF (3,6-dimethoxy-6', 6'-dimethyl-[2', 3' : 7,8]-chromeneflavone) from Lonchocarpus araripensis Benth. (Leguminosae) on gastric damage induced by absolute ethanol (96%, 0.2 mL/mouse) and indometacin (30 mg kg(-1), p.o.) in mice was investigated. Intraperitoneally administered DDF at dose levels of 50, 100 and 200 mg kg(-1) markedly reduced the gastric lesions in the ethanol model by 62, 72 and 96%, and in the indometacin model by 34, 70 and 75%, respectively, as compared with misoprostol (50 microg kg(-1), p.o.), the reference compound that caused lesion suppression by 67% in ethanol model and by 72% against indometacin-induced ulceration. The ED50 of DDF in reducing gastric lesions induced by ethanol and indometacin (dose of the DDF that reduced the gastric lesion area by 50% in relation to the control value) was 50.87 and 61.56 mg kg(-1), respectively. Mechanistic studies were carried out at 100 mg kg(-1) DDF using the ethanol model. Compared with N-acetylcysteine (750 mg kg(-1), p.o.), a donor of sulfhydryls, DDF only partially replenished the ethanol-induced depletion of gastric mucosal NP-SH. Pretreatment with TRPV1 antagonist capsazepine (5 mg kg(-1), i.p.) or the non-selective cyclooxygenase inhibitor indometacin (10 mg kg(-1), p.o.) effectively blocked the gastroprotective effect of DDF (100 mg kg(-1)) against ethanol damage. Furthermore, the effect of DDF was significantly reduced in mice pretreated with L-NAME, or glibenclamide, the respective inhibitors of nitric oxide synthase and K+ ATP channel activation. These data provide evidence to show that DDF affords gastroprotection against gastric damage induced by ethanol and indometacin by different and complementary mechanisms, which include involvement of endogenous prostaglandins, nitric oxide release, the activation of TRPV1 receptor or K+ ATP channels, besides a sparing effect on NP-SH reserve.     

Epidemiology and Control of Child Toxocariasis in the Western Brazilian Amazon – A Population-Based Study

Toxocara spp. infection and the seroconversion rate in the Amazon have been poorly investigated. This study analyzed individual and household-level risk factors for the presence of IgG antibodies to Toxocara spp. in urban Amazonian children over a period of 7 years and evaluated the seroconversion rates over a 1-year follow-up. In children < 59 months of age, the overall prevalence rate was 28.08% in 2003 and 23.35% in 2010. The 2010–2011 seroconversion rates were 13.90% for children 6–59 months of age and 12.30% for children 84–143 months of age. Multilevel logistic regression analysis identified child age, previous wheezing, and current infection with hookworm as significant associated factors for Toxocara spp. seropositivity in 2003. In 2010, age, previous helminthiasis, and having a dog were associated with seropositivity, whereas having piped water inside the household was a protective factor. Control programs mainly need to target at-risk children, water quality control, and animal deworming strategies.