Function of the fully conserved residues Asp99, Tyr52 and Tyr73 in phospholipase A2

In the active centre of pancreatic phospholipase A2 His48 is at hydrogen-bonding distance to Asp99. This Asp-His couple is assumed to act together with a water molecule as a catalytic triad. Asp99 is also linked via an extended hydrogen bonding system to the side chains of Tyr52 and Tyr73. To probe the function of the fully conserved Asp99, Tyr52 and Tyr73 residues in phospholipase A2, the Asp99 residue was replaced by Asn, and each of the two tyrosines was separately replaced by either a Phe or a Gln. The catalytic and binding properties of the Phe52 and Phe73 mutants did not change significantly relative to the wild-type enzyme. This rules out the possibility that either one of the two Tyr residues in the wild-type enzyme can function as an acyl acceptor or proton donor in catalysis. The Gln73 mutant could not be obtained in any significant amounts probably due to incorrect folding. The Gln52 mutant was isolated in low yield. This mutant showed a large decrease in catalytic activity while its substrate binding was nearly unchanged. The results suggest a structural role rather than a catalytic function of Tyr52 and Tyr73. Substitution of asparagine for aspartate hardly affects the binding constants for both monomeric and micellar substrate analogues. Kinetic characterization revealed that the Asn99 mutant has retained no less than 65% of its enzymatic activity on the monomeric substrate rac 1,2-dihexanoyldithio-propyl-3-phosphocholine, probably due to the fact that during hydrolysis of monomeric substrate by phospholipase A2 proton transfer is not the rate-limiting step. The Asp to Asn substitution decreases the catalytic rate on micellar 1,2-dioctanoyl-sn-glycero-3-phosphocholine 25-fold. To explain this remaining activity we suggest that in the mutant the Asn99 orients His48 in the same way as Asp99 orients His48 in native phospholipase A2 and that the lowered activity is caused by a reduced stabilization of the transition state

Evidence for the involvement of tyrosine-69 in the control of stereospecificity of porcine pancreatic phospholipase A2

We have studied the role of Tyr-69 of porcine pancreatic phospholipase A2 in catalysis and substrate binding, using site-directed mutagenesis. A mutant was constructed containing Phe at position 69. Kinetic characterization revealed that the Phe-69 mutant has retained enzymatic activity on monomeric and micellar substrates, and that the mutation has only minor effects on kcat and Km. This shows that Tyr-69 plays no role in the true catalytic events during substrate hydrolysis. In contrast, the mutation has a profound influence on the stereospecificity of the enzyme. Whereas the wild-type phospholipase A2 is only able to catalyse the degradation of sn-3 phospholipids, the Phe-69 mutant hydrolyses both the sn-3 isomers and, at a low (1-2%) rate, the sn-1 isomers. Despite the fact that the stereospecificity of the mutant phospholipase has been altered, Phe-69 phospholipase still requires Ca2+ ions as a cofactor and also retains its specificity for the sn-2 ester bond. Our data suggest that in porcine pancreatic phospholipase A2 the hydroxyl group of Tyr-69 serves to fix and orient the phosphate group of phospholipid monomers by hydrogen bonding. Because no such interaction can occur between the Phe-69 side-chain and the phosphate moiety of the substrate monomer, the mutant enzyme loses part of its stereospecificity but not its positional specificity

Inter-practice variation in diagnosing hypertension and diabetes mellitus: a cross-sectional study in general practice

BACKGROUND: Previous studies of inter-practice variation of the prevalence of hypertension and diabetes mellitus showed wide variations between practices. However, in these studies inter-practice variation was calculated without controlling for clustering of patients within practices and without adjusting for patient and practice characteristics. Therefore, in the present study inter-practice variation of diagnosed hypertension and diabetes mellitus prevalence rates was calculated by 1) using a multi-level design and 2) adjusting for patient and practice characteristics. METHODS: Data were used from the Netherlands Information Network of General Practice (LINH) in 2004. Of all 168.045 registered patients, the presence of hypertension, diabetes mellitus and all available ICPC coded symptoms and diseases related to hypertension and diabetes, were determined. Also, the characteristics of practices were used in the analyses. Multilevel logistic regression analyses were performed. RESULTS: The 95% prevalence range for the practices for the prevalence of diagnosed hypertension and diabetes mellitus was 66.3 to 181.7 per 1000 patients and 22.2 to 65.8 per 1000 patients, respectively, after adjustment for patient and practice characteristics. The presence of hypertension and diabetes was best predicted by patient characteristics. The most important predictors of hypertension were obesity (OR = 3.5), presence of a lipid disorder (OR = 3.0), and diabetes mellitus (OR = 2.6), whereas the presence of diabetes mellitus was particularly predicted by retinopathy (OR = 8.5), lipid disorders (OR = 2.8) and hypertension (OR = 2.7). CONCLUSION: Although not the optimal case-mix could be used in this study, we conclude that even after adjustment for patient (demographic variables and risk factors for hypertension and diabetes mellitus) and practice characteristics (practice size and presence of a practice nurse), there is a wide difference between general practices in the prevalence rates of diagnosed hypertension and diabetes mellitu

Is green space in the living environment associated with people's feelings of social safety?

Abstract. The authors investigate whether the percentage of green space in people's living environ- ment affects their feelings of social safety positively or negatively. More specifically they investigate the extent to which this relationship varies between urban and rural areas, between groups in the community that can be identified as more or less vulnerable, and the extent to which different types of green space exert different influences. The study includes 83736 Dutch citizens who were interviewed about their feelings of social safety. The percentage of green space in the living environment of each respondent was calculated, and data analysed by use of a three-level latent variable model, controlled for individual and environmental background characteristics. The analyses suggest that more green space in people's living environment is associated with enhanced feelings of social safetyöexcept in very strongly urban areas, where enclosed green spaces are associated with reduced feelings of social safety. Contrary to the common image of green space as a dangerous hiding place for criminal activity which causes feelings of insecurity, the results suggest that green space generally enhances feelings of social safety. The results also suggest, however, that green space in the most urban areas is a matter of concern with respect to social safety.

Trust, openness and continuity of care influence acceptance of antibiotics for children with respiratory tract infections: a four country qualitative study

Background. Clinician–parent interaction and health system influences on parental acceptance of prescribing decisions for children with respiratory tract infections (RTIs) may be important determinants of antibiotic use. Objective. To achieve a deeper understanding of parents’ acceptance, or otherwise, of clinicians’ antibiotic prescribing decisions for children with RTIs. Methods. Qualitative interviews with parents of child patients who had recently consulted in primary care with a RTI in four European countries, with a five-stage analytic framework approach (familiarization, developing a thematic framework from interview questions and emerging themes, indexing, charting and interpretation). Results. Fifty of 63 parents accepted clinicians’ management decisions, irrespective of antibiotic prescription. There were no notable differences between networks. Parents ascribed their acceptance to a trusting and open clinician–patient relationship, enhanced through continuity of care, in which parents felt able to express their views. There was a lack of congruence about antibiotics between parents and clinicians in 13 instances, mostly when parents disagreed about clinicians’ decision to prescribe (10 accounts) rather than objecting to withholding antibiotics (three accounts). All but one parent adhered to the prescribing decision, although some modified how the antibiotic was administered. Conclusions. Parents from contrasting countries indicated that continuity of care, open communication in consultations and clinician–patient trust was important in acceptance of management of RTI in their children and in motivating adherence. Interventions to promote appropriate antibiotic use in children should consider a focus on eliciting parents’ perspectives and promoting and building on continuity of care within a trusting clinician–patient relationship

Extended lymph node dissection for gastric cancer from a European perspective

Surgical oncolog

Minor surgery in general practice and effects on referrals to hospital care: Observational study

<p>Abstract</p> <p>Background</p> <p>Strengthening primary care is the focus of many countries, as national healthcare systems with a strong primary care sector tend to have lower healthcare costs. However, it is unknown to what extent general practitioners (GPs) that perform more services generate fewer hospital referrals. The objective of this study was to examine the association between the number of surgical interventions and hospital referrals.</p> <p>Methods</p> <p>Data were derived from electronic medical records of 48 practices that participated in the Netherlands Information Network of General Practice (LINH) in 2006-2007. For each care-episode of benign neoplasm skin/nevus, sebaceous cyst or laceration/cut it was determined whether the patient was referred to a medical specialist and/or minor surgery was performed. Multilevel multinomial regression analyses were used to determine the relation between minor surgery and hospital referrals on the level of the GP-practice.</p> <p>Results</p> <p>Referral rates differed between diagnoses, with 1.0% of referrals for a laceration/cut, 8.2% for a sebaceous cyst and 10.2% for benign neoplasm skin/nevus. The GP practices performed minor surgery for a laceration/cut in 8.9% (SD:14.6) of the care-episodes, for a benign neoplasm skin/nevus in 27.4% (SD:14.4) of cases and for a sebaceous cyst in 26.4% (SD:13.8). GP practices that performed more minor surgery interventions had a lower referral rate for patients with a laceration/cut (-0.38; 95%CI:-0.60- -0.11) and those with a sebaceous cyst (-0.42; 95%CI:-0.63- -0.16), but not for people with benign neoplasm skin/nevus (-0.26; 95%CI:-0.51-0.03). However, the absolute difference in referral rate appeared to be relevant only for sebaceous cysts.</p> <p>Conclusions</p> <p>The effects of minor surgery vary between diagnoses. Minor surgery in general practice appears to be a substitute for specialist medical care only in relation to sebaceous cysts. Measures to stimulate minor surgery for sebaceous cysts may induce substitution.</p