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.     

Physical activity patterns in chronic hemodialysis patients: comparison of dialysis and nondialysis days.

OBJECTIVE: To determine physical activity patterns in chronic hemodialysis patients with a specific emphasis on the difference between dialysis and nondialysis days. Design A cross-sectional single-center study. SETTING: Vanderbilt University Outpatient Dialysis Unit. PATIENTS: Twenty current chronic hemodialysis patients: 10 male, 10 female; 15 black, 5 white; mean age, 50.1 +/- 9.9 years; height, 164.5 +/- 10.9 cm; weight, 82.5 +/- 15.4 kg; length on dialysis, 57.3 +/- 45.3 months. METHODS: Minute-by-minute physical activity was assessed over a 7-day period using a triaxial accelerometer, which consists of raw numbers or counts calculated by the 3 axes of the accelerometer (PA counts). PA counts were extrapolated on a daily and hourly basis. Physical functioning tests included: sit-to-stand, 6-minute walk, and 1-repetition maximal leg press exercise. Laboratory values for serum concentrations of albumin, prealbumin, C-reactive protein, and cholesterol were also collected. MAIN OUTCOME MEASURE: PA counts. RESULTS: Total PA counts were significantly lower on dialysis days when compared with nondialysis days (128,279 +/- 74,009 versus 168,744 +/- 95,168, respectively, P = .025). The average PA counts during the 4-hour dialysis time period were significantly lower on dialysis days when compared with nondialysis days (3,086 +/- 3,749 versus 11,070 +/- 7,695, respectively, P = .001). At postdialysis hours 1 and 2, PA counts on dialysis days were significantly higher than on nondialysis days (11,410 +/- 5,340 versus 9,082 +/- 6,646, P = .008, and 14,048 +/- 9,728 versus 8,662 +/- 6,433, P = .016, respectively). By postdialysis hour 4, PA counts on dialysis days had significantly decreased when compared with nondialysis days (6,068 +/- 6,268 versus 10,512 +/- 7,420 PA counts, P = .01, respectively). From postdialysis hours 5 to 20, there was no significant difference in PA counts between dialysis and nondialysis days. CONCLUSION: This study shows that physical activity is lower on dialysis days when compared with nondialysis days, and this decrease is caused by the lack of activity during the 4-hour hemodialysis procedure. New behavior modification strategies involving physical activity, both during hemodialysis and on nondialysis days, must be examined in this patient population.     

Intentional mis-reporting of food consumption and its relationship with body mass index and psychological scores in women

BACKGROUND: The reasons for mis-reporting food consumption warrant investigation. OBJECTIVE: To document intention to mis-report food consumption and its associations with psychological measures in women. DESIGN: A total of 184 female volunteers aged 18-65 years, comprising 50 seeking help in primary care to lose weight with a body mass index (BMI) >/=30 kg m(-2) (obese-clinical group) and 134 nurses (nonclinical groups) (BMI <25 kg m(-2), n = 52; BMI 25-29.9 kg m(-2), n = 45; BMI >/=30 kg m(-2), n = 37) were studied. A questionnaire was administered containing three psychological tests (self-esteem, psychological well-being and Stunkard's three-factor eating questionnaire) and new items to address food intake mis-reporting. RESULTS: Overall, 68% of participants declared an inclination to mis-report (64% nonclinical, 78% clinical). Inclination to under-report was 29, 33 and 51% in the three nonclinical groups; and 46% among the obese clinical patients. Among the same groups, inclination to over-report were 39, 29, 11 and 32%. After adjusting for social deprivation and BMI, women inclined to mis-report had higher hunger (P = 0.008) and disinhibition (P = 0.005) scores than those intending to report accurately. These variables were associated with current dieting, frequency of dieting, self-reported bingeing and dissatisfaction with body weight. CONCLUSIONS: These findings indicate that intentional under-reporting and over-reporting of food consumption are common in women of all BMI categories and are associated with eating behaviour. Current dieting, frequency of dieting in the past, self-reported bingeing and dissatisfaction with body weight seem to mediate this relationship.     

Parvalbumin immunoreactive neurons and fibres in the teleost cerebellum

Summary The distribution of parvalbumin-(PV) immunopositive cell bodies and fibres in the cerebellum of two species of freshwater teleosts (Salmo gairdneri and Barbus meridonalis) was studied using a monoclonal antibody and the avidin-biotin immunoperoxidase technique. A clear laminated pattern of PV immunoreactivity was observed. After PV-immunostaining, Purkinje cells were strongly labelled in their cell bodies, the initial segments of the axons and the dendritic trees. In the molecular layer, only the dendritic branches of the Purkinje cells were PV-positive. In the granule cell layer, extensive axonal plexuses and scattered cell bodies were observed. Most of the immunopositive perikarya were unequivocally identified as displaced Purkinje cells, whereas a reduced number of smaller neurons with unstained dendrites was also found. Eurydendroid cells, the efferent neurons of the teleost cerebellum, were negative; however, they were impinged upon by numerous PV-positive boutons, corresponding to terminals of Purkinje cell axons. Parallel fibres and climbing fibres, as well as stellate cells and granule cells were negative. Basket cells (or deep stellate cells) whose existence in the teleost cerebellum is discussed, were also not observed. The immunoreactivity distribution pattern for PV in the teleost cerebellum differs from previous observations on the localization of this protein in the cerebellum of amniotes.     

Distribution of calbindin D-28K and parvalbumin immunoreactivities in the nucleus olfactorius anterior of the rat

The distributions of calbindin D-28K (CaBP) and parvalbumin (PV) in the rat nucleus olfactorius anterior (NOA) were described using monoclonal antibodies and the avidin-biotin-peroxidase method. The NOA showed a high immunoreactivity for CaBP, with a rostrocaudal increase in the positive neurons and fibres. Pars externa (NOAe) was the only subdivision which showed a low CaBP immunostaining. PV-positive elements were less abundant than those CaBP immunostained. The main difference in the distributions for both proteins was observed in the pars medialis which was practically PV negative. PV- and CaBP-stained neurons showed similar morphologies in the subdivisions where they were present. In NOAe, we observed a characteristic PV- and CaBP-positive neuronal type, with an oriented dendritic pattern. Transition areas were clearly observable in both CaBP- and PV-labelled sections.     

In vitro characterization of peptide-modified p(AAm-co-EG/AAc) IPN-coated titanium implants.

Interpenetrating polymer networks (IPNs) of poly(acrylamide-co-ethylene glycol/acrylic acid) [p(AAm-co-EG/AAc)] functionalized with an -Arg-Gly-Asp- containing peptide derived from rat bone sialoprotein [bsp-RGD(15)] were grafted to titanium implants in an effort to modulate osteoblast behavior in vitro. Surface characterization data were consistent with the presence of an IPN, and ligand density measurements established that the range of peptide density on the modified implants spanned three orders of magnitude (0.01-20 pmol/cm2). In vitro biological characterization of the modified implants employing the primary rat calvarial osteoblast (RCO) model resulted in the identification of a critical ligand density (0.01相似文献