Induction of net mass lipid transfer reactions in plasma by wine consumption with dinner.

Moderate alcohol consumption is associated with a reduced risk of coronary heart disease. Alcohol may exert protection through its effects on the metabolism of plasma lipoproteins. In the present study we investigated the effects of moderate wine consumption with an evening dinner on lipoprotein composition and parameters of reverse cholesterol transport (plasma lipid transfer reactions and cholesterol esterification) in eight healthy middle-aged men. Wine consumption, if compared with mineral water, resulted in increased postprandial plasma levels of triglyceride-(TG)-rich lipoproteins (P < 0.005 or < 0.002 at two different time points) and in increased net mass transfer of cholesterylesters (CE) from high-density lipoprotein (HDL) to apolipoprotein B-containing lipoproteins during in vitro incubation of plasma (P < 0.001). Net mass transfer of TG (in the opposite direction) was also significantly increased by wine (P = 0.014). The concentrations of total plasma cholesterol, HDL-cholesterol and apolipoproteins A-I, A-II and B did not change postprandially and were not affected significantly by wine, but the CE TG-1 in HDL was affected postprandially and decreased by wine consumption. It is concluded that moderate wine consumption with evening dinner induces transfer reactions of CE and TG between HDL and TG-rich lipoproteins. Due to the fact that wine raises plasma TG, it also causes changes in plasma cholesterol metabolism and lipoprotein composition, without major effects on total plasma cholesterol concentration.

Moderate alcohol consumption and platelet aggregation in healthy middle-aged men.

Changes in platelet aggregation have often been proposed as an explanation for the protective effect of moderate alcohol consumption on the development of coronary heart disease, observed in epidemiological studies. To test the tenability of this assumption, the acute effect of moderate alcohol consumption on platelet aggregation was studied in eight healthy middle-aged men in a controlled study. The intake of alcohol consisted of two glasses of red wine at dinner and two glasses of distilled liquor (Hollands gin), combined with a snack, before bedtime. No acute effects of moderate alcohol consumption on platelet aggregation were observed.

Effects of a moderate dose of alcohol on blood lipids and lipoproteins postprandially and in the fasting state.

Effects of a moderate dose of alcohol on blood lipids and lipoproteins were studied in volunteers of two age groups (20-30 and 45-55 years), each consisting of eight healthy men. The alcohol (30 g in red port and wine) was consumed during a standard dinner. Two blood samples were drawn: one in the postprandial phase, and one the next morning after fasting overnight. In the postprandial phase, one hour after intake, alcohol increased high-density lipoprotein cholesterol (HDL-C) by 11.5%, triglycerides (TG) by 15.3% and apolipoprotein A2 (Apo-A2) by 7.3% (P = 0.002, P = 0.044 and P = 0.024, respectively). The increase in HDL-C appeared to be mainly attributed to the HDL2-C subfraction which increased by 15.3% (P = 0.066). Furthermore, the increases in HDL-C, HDL2-C and TG were more pronounced in the middle-aged men then in the young men. After fasting overnight the effects of alcohol had disappeared.

Isolation and characterization of different activated forms of factor VIII, the human antihemophilic A factor.

Factor VIII was purified 1200-fold from commercial concentrates (Centre National de Transfusion Sanguine) by immunoaffinity chromatography using an anti-(80-kDa light chain) monoclonal antibody. The different molecular forms isolated were subsequently separated and analyzed using Fast Protein Liquid Chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis. The different factor-VIII forms obtained, consisted of 80-kDa light chains, each being associated with one more-or-less fragmented heavy chain ranging over 90-210 kDa. The specific activity of these different forms was 7000 U/mg. At different stages of activation of factor VIII by thrombin, various forms were separated and identified. Activated complexes were found to result from the association of the 70-kDa light chain (generated from the 80-kDa light chain) with heavy chains ranging over 90-210 kDa. Two different thrombin activation steps were characterized. The first step corresponding to the cleavage of the 80-kDa light chain led to a sixfold increase in the procoagulant activity, and yielded a stable activated intermediate form. Compared with normal factor VIII, the ratio of von Willebrand activity to factor-VIII activity, measured in the activated fractions, decreased, indicating that von Willebrand factor dissociates from factor VIII after proteolysis of the light chain by thrombin. In the second step, the 90-kDa heavy chain was cleaved into two polypeptides of 45 kDa and 50 kDa, which were associated with the 70-kDa proteolyzed light chain, generating the final activated complex (45-50-70 kDa). The new intermediate forms we described imply a new scheme for the multistep activation process of factor VIII.

Conversion of Glucose to 2-Keto-l-Gulonate, an Intermediate in l-Ascorbate Synthesis, by a Recombinant Strain of Erwinia citreus.

A gene for 2,5-diketo-d-gluconate (25DKG) reductase, which encodes an enzyme composed of 277 amino acid residues catalyzing the reduction of 25DKG to 2-keto-l-gulonate (2KLG), was cloned from Corynebacterium sp. strain SHS752001 and expressed in Erwinia citreus SHS2003, a strain which oxidizes glucose to 25DKG. The recombinant microorganism converted glucose to 2KLG, a compound which can be readily converted to l-ascorbate (vitamin C). Improvements in the yield of 2KLG were obtained by changing fermentation conditions, using the p(l) promoter of bacteriophage lambda to express the reductase, and selecting a mutant of E. citreus which could use neither 25DKG nor 2KLG as a sole carbon source for growth. When a culture of the recombinant strain was fed with glucose to a total of 40 g/liter, 49.4% of the glucose was converted to 2KLG during a 72-h fermentation.

Characterization of four monoclonal antibodies to factor VIII coagulant protein and their use in immunopurification of factor VIII.

Four monoclonal anti-VIII:C antibodies were obtained from the fusion of the splenocytes of one Balb/C mouse with a specific activity ranging from 2.3 to 45,000 U/mg when purified from ascitic fluid. Only one antibody was able to inhibit completely Factor VIII:C in normal plasma. The four antibodies could bind Factor VIII:CAg in plasma and commercial concentrate both in liquid and solid phase, and were suitable for immunopurification of Factor VIII:C. Three antibodies competed with polyclonal anti-VIII:CAg Fab' in a liquid phase IRMA, and all of them were able to displace their own binding to Factor VIII:CAg. Competition studies between monoclonal antibodies for the binding to Factor VIII:CAg were performed and showed the recognition of different epitopes and various functional impact. These studies indicate that at least one antibody, with the lowest anti-VIII:C titer clearly recognizes a different epitope of VIII:C than those recognized by the others. Affinity constants ranged from 10(9) to 10(10) l/mole.

Replication and gene functions of the bacteriocinogenic plasmid CloDF13.

The replication and genetic constitution of plasmid CloDF13 was studied using mutants of CloDF13 obtained by NTG mutagenesis, insertion of the ampicillin transposon Tn901, or deletion of particular CloDF13 DNA regions. Analysis of the polypeptides encoded by these mutant plasmids enabled us to locate six genes on the CloDF13 physical map. These genes cover about 60% of the coding capacity of CloDF13. A large part of the CloDF13 genome (about 30%) is involved in the conjugal transfer of this plasmid. This transfer region codes for at least two polypeptides, polypeptide B (61,000 daltons) and C (24,000 daltons). Those CloDF13 DNA regions responsible for the synthesis of the cloacin protein and immunity protein were also mapped on the plasmid genome. In addition we were able, using a copy mutant of CloDF13, CloDF13-cop3, to locate those DNA sequences involved in interaction with male-specific RNA phages and bacteriophage P1. For replication of CloDF13, two regions are essential. One region, from 43% to 64%, affects the stability of CloDF13-cop3 plasmids. In the case of the CloDF13-cop3 mutant, deletion of this region results in the generation of multimeric plasmid molecules accompanied by an impaired segregation of plasmid DNA molecules to daughter cells. The second region, from about 1.8% to 11.5%, contains an origin of replication as well as well as DNA sequences involved in the control of CloDF13 replication. The replication of CloDF13 starts at about 3% on the CloDF13 physical map and proceeds unidirectionally counter-clockwise.