Design and Characterization of a New pVII Combinatorial Phage Display Peptide Library for Protease Substrate Mining Using Factor VII Activating Protease (FSAP) as Model

We describe a novel, easy and efficient combinatorial phage display peptide substrate-mining method to map the substrate specificity of proteases. The peptide library is displayed on the pVII capsid of the M13 bacteriophage, which renders pIII necessary for infectivity and efficient retrieval, in an unmodified state. As capture module, the 3XFLAG was chosen due to its very high binding efficiency to anti-FLAG mAbs and its independency of any post-translational modification. This library was tested with Factor-VII activating protease (WT-FSAP) and its single-nucleotide polymorphism variant Marburg-I (MI)-FSAP. The WT-FSAP results confirmed the previously reported Arg/Lys centered FSAP cleavage site consensus as dominant, as well as reinforcing MI-FSAP as a loss-of-function mutant. Surprisingly, rare substrate clones devoid of basic amino acids were also identified. Indeed one of these peptides was cleaved as free peptide, thus suggesting a broader range of WT-FSAP substrates than previously anticipated.     

Occupational Factors and 5-Year Weight Change Among Men in a Danish National Cohort.

This study explored whether factors related to the work environment could predict changes in body mass index (BMI) and whether the effect of psychosocial factors was dependent on baseline BMI. The sample consisted of 1,980 male employees from the Danish National Work Environment Cohort Study. Changes in BMI between 1995 and 2000 were analyzed, by multiple regression, as a function of background variables and a series of occupational variables obtained in 1995. Age, baseline BMI, job insecurity, and psychological demands predicted changes in BMI. Job insecurity and high or low psychological demands increased the likelihood of weight gain among obese employees, whereas they increased the likelihood of weight loss among employees with a low BMI. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Nonlinear protein binding and enzyme heterogeneity: effects on hepatic drug removal

The kinetics of substrate removal by the liver and the resulting nonlinear changes in unbound fraction along the flow path at varying input drug concentrations were examined by a model simulation study. Specifically, we varied the binding association constant, KA, and the Michaelis-Menten constants (Km and Vmax) to examine the steady state drug removal (expressed as hepatic extraction ratio E) and changes in drug binding for (i) unienzyme systems and (ii) simple, parallel metabolic pathways; zonal metabolic heterogeneity was also added as a variable. At low KA, E declined with increasing input drug concentration, due primarily to saturation of enzymes; only small differences in binding were present across the liver. At high KA, a parabolic profile for E with concentration was observed; changes in unbound fraction between the inlet and the outlet of the liver followed in parallel fashion. Protein binding was the rate-determining step at low input drug concentrations, whereas enzyme saturation was the rate-controlling factor at high input drug concentration. Heterogeneous enzymic distribution modulated changes in unbound fraction within the liver and at the outlet. Despite marked changes in unbound fraction occurring within the liver for different enzymic distributions, the overall transhepatic differences were relatively small. We then investigated the logarithmic average unbound concentration and the length averaged concentration as estimates of substrate concentration in liver in the presence of nonlinear drug binding. Fitting of simulated data, with and without assigned random error (10%), to the Michaelis-Menten equation was performed; fitting was repeated for simulated data obtained with presence of a specific inhibitor of the high-affinity, anteriorly distributed pathway. Results were similar for both concentration terms: accurate estimates were obtained for anterior, high affinity pathways; an overestimation of parameters was observed for the lower affinity posteriorly distributed pathways. Improved estimations were found for posteriorly distributed pathways upon inhibition with specific inhibitors; with added random error, however, the improvement was much decreased. We applied the method for fitting of several sets of metabolic data obtained from rat liver perfusion studies performed with salicylamide (SAM) (i) without and (ii) with the presence of 2,6-dichloro-4-nitrophenol (DCNP), a SAM sulfation inhibitor. The fitted results showed that SAM sulfation was a high-affinity high-capacity pathway; SAM glucuronidation was of lower affinity but comparable capacity as the sulfation pathway, whereas SAM hydroxylation was of lower affinity and lower capacity.     

Removal of nitrates from groundwater through the use of artificial wetlands

Intensive fertilizer use has led to nitrate contamination of groundwater in many areas. This article examines the possibility of removing nitrates through the construction of artificial wetlands. A pilot study conducted in Denmark has found that the method is effective and economical, at least for small waterworks, though further treatment is necessary before an acceptable microbiological water quality is attained.     

Semiparametric Estimation in Time‐Series Regression with Long‐Range Dependence

Abstract. We consider semiparametric estimation in time‐series regression in the presence of long‐range dependence in both the errors and the stochastic regressors. A central limit theorem is established for a class of semiparametric frequency domain‐weighted least squares estimates, which includes both narrow‐band ordinary least squares and narrow‐band generalized least squares as special cases. The estimates are semiparametric in the sense that focus is on the neighbourhood of the origin, and only periodogram ordinates in a degenerating band around the origin are used. This setting differs from earlier studies on time‐series regression with long‐range dependence, where a fully parametric approach has been employed. The generalized least squares estimate is infeasible when the degree of long‐range dependence is unknown and must be estimated in an initial step. In that case, we show that a feasible estimate which has the same asymptotic properties as the infeasible estimate, exists. By Monte Carlo simulation, we evaluate the finite‐sample performance of the generalized least squares estimate and the feasible estimate.     

Sludge reed bed facilities: operation and problems.

Short operating periods and problems with dewatering efficiency, vegetation, mineralisation and odour are primarily caused by incorrect construction of the filter, poor capillary connections, an inadequate number of basins, insufficient basin area and overloading during commissioning and during subsequent operation. Dimensioning should be based on the sludge quality including the dewatering qualities and not solely on calculations of the sludge production. Loading after planting depends on the development level of the vegetation. The loading program should ensure that reed establishment is not impeded and should prevent the sludge residue from growing so quickly horizontally and vertically that the reeds cannot colonise the sludge residue. Overloading results in an anaerobic sludge residue with ensuing methane production. Typically, a sludge reed bed facility with a loading period of maximum 5 days, must be built with 10 basins to permit a rest phase of about 40 days. Facilities with 8 basins, where it is possible to load 1 basin for 7 days, will be able to have a 7-week rest phase before the first basin is loaded again. Facilities with for example 8-13 basins and loading period of between 4 and 10 days are able to achieve rest periods of up to 2 to 2(1/2) months, which results in the optimal evapotranspiration and minerilization. The conditions and thus the possibilities vary depending upon the type of sludge.     

Preparation of glucose oxidase immobilized in different carriers using radiation polymerization

Glucose oxidase (EC 1.1.3.4) was immobilized on different polymeric materials using different immobilization techniques (entrapping by γ‐irradiation, and covalent binding using epichlorohydrin). Studies were carried out to increase the thermal stability of glucose oxidase (GOD) for different applications. The activity and stability of the resulting biopolymers have been compared with those of free GOD. The effect of different polyvinyl alcohol/polyacrylamide (PVA/PAAm) compositions of the copolymer carrier on the enzymatic activity of the immobilized GOD was studied. The maximum enzymatic activity was obtained with the composition ratio of PVA/PAAm of 60:40. The behaviour of the free and immobilized enzyme was analysed as a function of pH. A broadening in the pH profile (5.5–8) was observed for immobilized preparations. The activity and stability of the resulting biopolymers produced by immobilization of GOD onto different carriers have been compared, in both aqueous and organic media, with those of the free GOD. The enzyme's tolerance toward both heat and organic solvent was enhanced by immobilization onto polymers. The addition of different concentrations of organic solvents (10–50%, v/v) to the enzyme at higher temperature (60 °C) was found to stabilize the enzyme molecule. The strongest stabilizing effect on the enzymatic activity was achieved at a concentration of 10%. Copyright © 2005 Society of Chemical Industry     

Long-term effect of lisinopril and atenolol on kidney function in hypertensive NIDDM subjects with diabetic nephropathy

The aim of our study was to evaluate whether inhibition of ACE (lisinopril 10-20 mg/day) can reduce the rate of decline in kidney function more than reducing blood pressure with conventional antihypertensive treatment (atenolol 50-100 mg/day), usually in combination with a diuretic. We performed a prospective, randomized, parallel study for 42 months, double blind for the first 12 months and single blind thereafter. Forty-three (21 lisinopril and 22 atenolol) hypertensive NIDDM patients with diabetic nephropathy were enrolled. Data from 36 patients (17 lisinopril and 19 atenolol, 60 +/- 7 years of age, 27 men) who completed at least 12 months of the study period are presented. At baseline, the two groups were comparable: glomerular filtration rate (51Cr-EDTA plasma clearance) was 75 +/- 6 and 74 +/- 8 ml x min(-1) x 1.73 m(-2), mean 24-h ambulatory blood pressure (A&D TM2420) was 110 +/- 3 and 114 +/- 2 mmHg, and 24-h urinary albumin excretion rate was 961 (range 331-5,727) and 1,578 (476-5,806) mg/24 h in the lisinopril and atenolol groups, respectively. The mean follow-up time was similar, 37 and 35 months in the lisinopril and atenolol groups, respectively. Mean ambulatory blood pressure was equally reduced in the two groups, 12 +/- 2 and 10 +/- 2 mmHg in the lisinopril and atenolol groups, respectively. Glomerular filtration rate declined in a biphasic manner with a faster initial (0 to 6 months) change of 1.25 +/- 0.49 and 0.81 +/- 0.29 ml x min(-1) x month(-1) followed by a slower sustained decline (6 to 42 months) of 0.59 +/- 0.10 and 0.54 +/- 0.13 ml x min(-1) x month(-1) in the lisinopril and atenolol groups, respectively. No significant differences were observed in either initial or sustained decline in glomerular filtration rate between the two groups. Urinary albumin excretion was reduced (% reduction of baseline) more in the lisinopril than in the atenolol group, at 55 (95% CI 29-72) and 15% (-13 to 34), respectively (P = 0.01). In conclusion, the relentless decline in kidney function characteristically found in hypertensive NIDDM patients with diabetic nephropathy can be reduced equally effectively by two antihypertensive treatments, the beta-blocker atenolol and the ACE inhibitor lisinopril.