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We have previously identified a potential TGF-beta activation element (TAE) in the rat collagen alpha1(I) promoter at -1624 upstream of the transcriptional start site [Ritzenthaler et al., 1991, 1993]. To determine the importance of the TAE in vivo, we produced transgenic mice carrying 3.6 kb of the rat collagen alpha1(I) promoter linked to the reporter gene chloramphenicol acetyl transferase with and without site-directed mutations that eliminate DNA-protein binding at the TAE site. Tissue-specific expression of the reporter gene in transgenic mice with the mutated collagen promoter was similar to that of transgenic mice with the normal promoter in two genetic backgrounds as judged by in situ hybridization, reporter assays, and immunochemistry. Endotracheal instillation of bleomycin induces lung fibrosis, mediated in part by TGF-beta. Earlier studies indicated that expression of wild-type collagen-reporter gene was upregulated in transgenic mice lungs in response to endotracheal instillation of bleomycin. A similar level of reporter gene upregulation was observed in transgenic mice carrying the mutation in the TAE. Two lines of transgenic mice carrying the mutated promoter construct displayed unexpected neurological abnormalities. In the FVB genetic background, there was a higher than normal incidence of mortality, spontaneous seizures, and an inability to nurture offspring. Histological evidence demonstrated clear abnormalities, including disorderly arrangement of neurons in the hippocampus and significant laminar cortical necrosis in the cerebrum in animals after seizures. In the C57Bl/6 background, there was a high incidence of severe communicating hydrocephalus, early runting, and increased mortality similar to that in transgenic animals with astroglial overexpression of TGF-beta. These animals provide an interesting model system to investigate molecular mechanisms responsible for seizures and hydrocephalus.

The hepatic uptake, transport, and secretion into bile of unesterified cholesterol cannot be directly quantitated because of extensive exchange and equilibration between different pools of unesterified cholesterol. Plant sterols are structurally similar to cholesterol but because of poor intestinal absorption are ordinarily not present in the liver. To quantitate hepatic sterol uptake and transport in the absence of exchange with endogenous sterols, isolated rat livers were perfused with the plant sterol, sitostanol, incorporated in phosphatidylcholine liposomes. Appreciable amounts of sitostanol were taken up by the liver and uptake was independent of the presence of bile salt. In contrast, like unesterified cholesterol, the secretion of sitostanol in bile required bile salt. Sitostanol was detected in bile within 5 min after a perfusion was begun and reached a plateau by about 20 min. The rate of appearance of sitostanol in bile was precisely the same as unesterified cholesterol when both sterols were perfused together. Furthermore, the output of sitostanol in bile was directly proportional to the output of cholesterol. At the peak of biliary sitostanol secretion, the amount of sitostanol relative to unesterified cholesterol was much greater in bile (40-50% of sterols) than in the whole liver (11% of sterols). Selective biliary secretion of sitostanol was associated with much greater concentrations of sitostanol in canalicular membranes than in the interior membranes of the hepatocyte and in newly secreted high density lipoproteins compared to newly secreted very low density lipoproteins. These results indicate that sitostanol parallels the secretion from and distribution of unesterified cholesterol in the liver and suggest that sitostanol can be used as a physiologic analog of unesterified cholesterol to trace the transport of sterols through the liver.

Human platelets secreted phospholipase A 2 in a dose- and time-dependent manner when challenged with thrombin, 12- O -tetradecanoyl-phorbol-13-acetate (TPA), or collagen. Enzyme release was maximal at concentrations of 0.1 units/ml of thrombin, 100 nM TPA, or 2 μg/ml of collagen; and complete by 2 min in platelets treated with thrombin or TPA. Cells challenged with collagen required up to 5 min for maximal secretion. Besides dose and time functions, phospholipase A 2 secretion was also dependent on platelet concentration and the levels of bovine serum albumin in the incubation medium. The secreted enzyme was soluble and exhibited substrate and Ca 2+ requirements similar to a detergent-solubilized, partially purified phospholipase A 2 from whole platelets [Kramer et al., Biochim. Biophys. Acta (1988) 959, 269–279]. The pH optimum of the secreted enzyme, however, was 1–2 units lower than the pH optimum of the phospholipase A 2 from whole cells. Secreted phospholipase A 2 hydrolyzed phosphatidylethanolamine at 5–12 times the rate of phosphatidylcholine when the substrates were present in pure form. These apparent differences in activity were greatly diminished, though, when 1:1 molar mixtures of the two substrates were employed. Because phospholipase A 2 catalyzes a key reaction during the formation of bioactive arachidonate metabolites, the secretion of this enzyme from platelets may be important in the regulation of thrombosis.

Membranes isolated from porcine aortic endothelial cells (PAEC) contain a CoA-independent transacylase enzyme (CoA-IT). CoA-IT, an integral membrane protein, transfers an acyl moiety to added [3H]alkylhydroxyglycerophosphocholine (LPAF) to generate [3H]alkylacylglycerophosphocholine (alkylacyl-GPC). This enzyme exhibits an apparent Km of 0.7 microM and a Vmax of 0.8 nmol/min per mg for the transfer of an acyl group to added [3H]LPAF. The addition of the nonionic detergent Triton X-100 (TX-100) (0.5 mg/ml), the sulfhydryl reagents N-ethylmaleimide (NEM) (200 microM) or thimerosal (200 microM), or pre-incubating the membranes at 95 degrees C for 10 min all decreased LPAF: CoA-IT activity by more than 95%. The inhibitory action of NEM or thimerosal suggests that sulfhydryl group(s) are involved in or are close to the catalytic site of LPAF: CoA-IT.

THE aggregation of washed human platelets in vitro by adenosine diphosphate (ADP) requires calcium and is stimulated by the addition of platelet-free plasma1. The identity of the plasma activity has been the subject of recent conflicting reports. Hellem and Owren2 have suggested that the plasma factor is that entity missing in von Willebrand's disease; but others3,4 have found that ADP-induced platelet aggregation proceeds normally in plasma from patients with established von Willebrand's disease. McLean et al.5 and Cross4 have indicated that fibrinogen may be the plasma co-factor, and Caen6 has recently suggested that both fibrinogen and the anti-von Willebrand factor participate in ADP-induced platelet aggregation.

Using a sonicated dispersion of radiolabeled 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine as substrate, we found that phospholipase A2 activity of human platelets was enhanced 2.4-fold by albumin (1 mg/ml). The enzyme was recovered predominantly in the cytosolic fraction of platelets with less than a third of its activity being associated with the membrane fraction. In the presence of 24 mM n-octyl-beta-D-glucopyranoside (octylglucoside) phospholipase A2 was effectively (more than 90%) extracted from platelet lysates without solubilization of platelet membranes. Ion exchange chromatography of the soluble enzyme yielded a phospholipase A2 of unchanged total activity and great stability. This phospholipase A2 was active only in the presence of divalent cations (Ca2+ greater than Sr2+ greater than Mg2+ = 0), required albumin for optimal activity and exhibited exclusive positional specificity for the acyl ester bond at the 2-position of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine. Indomethacin (500 microM), mepacrine (500 microM) and N-ethylmaleimide (4 mM) inhibited the phospholipase A2 by 69, 62 and 19%, respectively. The results are discussed in the light of previous findings on human platelet phospholipase A2.