Low-density lipoprotein preparation by combined diafiltration and ultracentrifugation

A method for isolating low-density lipoprotein by combining diafiltration and ultracentrifugation is described. Diafiltration separates plasma components by use of an ultrafiltration membrane that excludes particles of molecular weight greater than 300,000. The retentate is concentrated three- to fourfold by ultrafiltration, allowing large-scale preparation of low-density lipoprotein. Low-density lipoprotein prepared in this manner is similar in physical, chemical, and biologic properties to low-density lipoprotein isolated by sequential density ultracentrifugation alone. When low-density lipoprotein, prepared by either method, was added to human umbilical vein endothelial cell cultures, no cytotoxicity was observed. The techniques described reduce the demand on multiple rotors and ultracentrifuges for large-scale preparation of low-density lipoprotein suitable and often needed for tissue culture studies.     

Urinary profiles of volatile and acid metabolites in germfree and conventional rats

Qualitative and quantitative differences in the urinary excretion of volatile and acidic metabolites in germfree and conventional rats were examined by capillary gas chromatography and gas chromatography/mass spectrometry. A number of carbonyl compounds, including several short-chain aliphatic ketones and acetophenone, were higher in the conventional urines, while many heterocyclic compounds (furan derivatives, benzothiazole and others) were lower. Both qualitative and quantitative differences were observed in the urinary excretion of acidic metabolites. Three meta-hydroxy phenolic acids appeared only in the conventional rat urines, while levels of many other aromatic and aliphatic acids were also higher.     

Activation of the acetyl-coenzyme A:lysoplatelet-activating factor acetyltransferase regulates platelet-activating factor synthesis in human endothelial cells.

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) is a phospholipid with many physiological actions. It is synthesized by endothelial cells and a variety of others in response to stimulation with receptor-mediated agonists. In endothelial cells it remains associated with the surface of the cell and serves as a signal for adhesive interactions with leukocytes. Thus, its synthesis must be precisely regulated. In previous work we have shown that PAF synthesis is regulated at the initiating step, a phospholipase A2. Here we demonstrate that the subsequent step of PAF synthesis, the acetyl-CoA:lyso-PAF acetyltransferase, is rapidly activated when cells are exposed to thrombin or other agonists. We found that the activity increased from basal values (5 nmol/mg/min) to approximately 3-fold higher within 1 min following the addition of agonists. The enzyme activity returned to basal levels within 10 min. The pattern of activation and inactivation suggested covalent modification of the enzyme. This was supported in experiments in which we showed that homogenates had stable enhanced activity and that there was no evidence for an activator or inhibitor. Pretreatment of the cells with vanadate, an inhibitor of protein phosphatases, markedly prolonged the activation state. In subsequent studies we pretreated intact cells with vanadate to block inactivation of the enzyme and then measured the accumulation of PAF in response to thrombin. We found that it was markedly augmented and prolonged. From this we conclude that the synthesis of PAF in intact cells is regulated by the activity of the acetyltransferase. We characterized requirements for activation of acetyltransferase and found that it was not dependent on the influx of intracellular calcium but that calcium entry did influence the length of time for which the enzyme was activated. The acetyltransferase in endothelial cells was shown to be a specific enzyme that did not catalyze the transfer of long chain acyl groups from acyl-CoA to lysophospholipids and demonstrated modest specificity for the acceptor lysophospholipids. These results suggest that activation of the acetyltransferase is a crucial determinant of the amount of PAF synthesized in activated endothelial cells.     

Sexual health education interventions for young people: a methodological review.

OBJECTIVES--To locate reports of sexual health education interventions for young people, assess the methodological quality of evaluations, identify the subgroup with a methodologically sound design, and assess the evidence with respect to the effectiveness of different approaches to promoting young people''s sexual health. DESIGN--Survey of reports in English by means of electronic databases and hand searches for relevant studies conducted in the developed world since 1982. Papers were reviewed for eight methodological qualities. The evidence on effectiveness generated by studies meeting four core criteria was assessed. Judgments on effectiveness by reviewers and authors were compared. PAPERS--270 papers reporting sexual health interventions. MAIN OUTCOME MEASURE--The methodological quality of evaluations. RESULTS--73 reports of evaluations of sexual health interventions examining the effectiveness of these interventions in changing knowledge, attitudes, or behavioural outcomes were identified, of which 65 were separate outcome evaluations. Of these studies, 45 (69%) lacked random control groups, 44 (68%) failed to present preintervention and 38 (59%) postintervention data, and 26 (40%) omitted to discuss the relevance of loss of data caused by drop outs. Only 12 (18%) of the 65 outcome evaluations were judged to be methodologically sound. Academic reviewers were more likely than authors to judge studies as unclear because of design faults. Only two of the sound evaluations recorded interventions which were effective in showing an impact on young people''s sexual behaviour. CONCLUSIONS--The design of evaluations in sexual health intervention needs to be improved so that reliable evidence of the effectiveness of different approaches to promoting young people''s sexual health may be generated.