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.     

Effects of probucol on renal function in rats with bilateral ureteral obstruction

To ascertain the potential role of reactive oxygen metabolites in the pathophysiology of obstructive uropathy, we examined the effect of probucol, an antioxidant agent, on renal function in normal rats and rats with unilateral release of bilateral ureteral obstruction (BUO) of 24 hours duration. Rats were fed either a standard diet or a standard diet containing one percent probucol for two weeks prior to study. Probucol lowered serum cholesterol in both normal and BUO rats. Probucol did not significantly affect renal function in normal rats. BUO rats given probucol had greater inulin and PAH clearances at three to five hours and three days following release of BUO than rats with BUO not given probucol. Kidneys from obstructed rats had higher levels of malondialdehyde, an index of lipid peroxidation, a greater number of leukocytes in the cortex, decreased levels of reduced glutathione and increased levels of oxidized glutathione. Renal cortex from obstructed rats treated with probucol had significantly higher levels of reduced glutathione than kidneys of obstructed rats not given probucol. A decrease in cholesterol, using another lipid-lowering agent, lovastatin, did not modify renal function in rats with BUO. The data can be interpreted to indicate a role for reactive oxygen species in the pathophysiology of obstructive nephropathy. The improved renal function seen in probucol-treated rats with BUO may be due to an effect of this agent in affecting accumulation of reactive oxygen metabolites and/or decreasing the number of leukocytes infiltrating the renal cortex.     

Exercise training ameliorates progressive renal disease in rats with subtotal nephrectomy

To determine the effect of chronic exercise training on renal function in animals with moderate renal insufficiency, rats with 75% renal ablation were either exercise trained by swimming for two months or remained sedentary. Glomerular filtration rate was significantly higher in trained (1.89 +/- 0.07 ml/min) than in sedentary rats (1.52 +/- 0.11 ml/min). No change was observed in renal blood flow or the degree of hypertension. Proteinuria was reduced in trained (13.6 +/- 4.9 mg/24 hr) compared to sedentary animals (33.5 +/- 9.2 mg/24 hr). The degree of glomerulosclerosis was much less prominent in trained animals. Plasma, low-density lipoprotein cholesterol-levels and total triglycerides were reduced in trained compared to sedentary rats. This study suggests that chronic exercise training ameliorates the progression of renal disease and improves plasma lipids in rats with moderate renal insufficiency. The mechanism for this improvement in renal function appears to be independent of the influence of systemic blood pressure.     

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.     

Gated CT imaging using a free-breathing respiration signal from flow-volume spirometry

Respiration-induced tumor motion is known to cause artifacts on free-breathing spiral CT images used in treatment planning. This leads to inaccurate delineation of target volumes on planning CT images. Flow-volume spirometry has been used previously for breath-holds during CT scans and radiation treatments using the active breathing control (ABC) system. We have developed a prototype by extending the flow-volume spirometer device to obtain gated CT scans using a PQ 5000 single-slice CT scanner. To test our prototype, we designed motion phantoms to compare image quality obtained with and without gated CT scan acquisition. Spiral and axial (nongated and gated) CT scans were obtained of phantoms with motion periods of 3-5 s and amplitudes of 0.5-2 cm. Errors observed in the volume estimate of these structures were as much as 30% with moving phantoms during CT simulation. Application of motion-gated CT with active breathing control reduced these errors to within 5%. Motion-gated CT was then implemented in patients and the results are presented for two clinical cases: lung and abdomen. In each case, gated scans were acquired at end-inhalation, end-exhalation in addition to a conventional free-breathing (nongated) scan. The gated CT scans revealed reduced artifacts compared with the conventional free-breathing scan. Differences of up to 20% in the volume of the structures were observed between gated and free-breathing scans. A comparison of the overlap of structures between the gated and free-breathing scans revealed misalignment of the structures. These results demonstrate the ability of flow-volume spirometry to reduce errors in target volumes via gating during CT imaging.     

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.     

Revisiting serum thyroglobulin in the follow-up of patients with differentiated thyroid carcinoma

This study analyzed serum thyroglobulin (Tg) during hypothyroidism in 207 patients with differentiated thyroid carcinoma treated with total thyroidectomy and radioiodine ablation and undetectable anti-Tg antibodies. Disease staging was defined by clinical examination, stimulated Tg, pre- and post-ablative radioiodine scanning, and other imaging methods (X-Ray, US, CT and MIBI-scan). The average interval from initial therapy was 2.3 years. 153 patients (74%) had no evident disease, 34 (16.4%) presented neck/mediastinal disease, and 20 (9.6%) had distant metastases (Mt). The best cut-off for Tg was 1 ng/ml, showing 100% sensitivity for distant Mt and 88.2% for local recurrence or lymph node Mt, and 88.8% specificity for any Mt and 74.8% for distant Mt. In patients with Tg <1 ng/ml, 2.8% showed cervical lymph nodes Mt. Cervical or mediastinal disease were 26% of cases with Tg between 1 and 5 ng/ml. Tg from 5 to 10 ng/ml was associated to distant Mt in 14.2% of the cases and others showed lymph nodes Mt. In patients with Tg >10 ng/ml, 51.3% presented distant Mt. We suggest the need for neck US even in cases with Tg <1 ng/ml. In addition, patients with Tg levels <5 ng/ml should be investigated by neck US and mediastinal CT only, and empirical therapy should be limited to patients with a minimum Tg level >5 ng/ml.