A sensitive and continuous fluorometric assay for phospholipase A2 using pyrene-labeled phospholipids in the presence of serum albumin

Phospholipase A2 activity can be determined fluorometrically in the presence of serum albumin using phospholipids labeled at the sn-2-acyl position with 10-pyrenyldecanoic acid. In the water reaction medium 10-pyrene phospholipids form vesicles and the monomer fluorescence of the pyrene is negligible due to pyrene-pyrene interaction. Upon phospholipid hydrolysis 10-pyrenyldecanoic acids are produced and tightly bind to albumin so that a monomer pyrene fluorescence is observed. We obtained an excellent parallelism between hydrolysis determined by a classical extraction method and that followed by direct and continuous spectrofluorometric recording of the monomer emission of pyrene. This assay can measure picomole amounts of phospholipids hydrolyzed per minute so that picogram quantities of phospholipases A2 from pancreas or from venoms can be measured. Phospholipase activity remains proportional to enzyme concentration over three orders of magnitude. The method can be used to quantify the phospholipase A2 activity of crude extracts of low specific activity.     

EGF-induced PGE2 release is synergistically enhanced in retinoic acid treated fetal rat lung cells

Retinoic acid has been shown to induce a 2.5-fold increase in 125I-EGF binding capacity through increased EGF receptor synthesis in a fetal rat lung (FRL) cell line (1). In FRL cells, incubation with either EGF or retinoic acid induces a modest increase in PGE2 secretion (80% or 40%, respectively). However, in the presence of both EGF and retinoic acid, FRL cells exhibit a 6.4-fold increase in PGE2 secretion. Retinoic acid and EGF dose-response curves demonstrate that the effect on PGE2 secretion correlates with the retinoic acid induced increase in EGF receptors. These data suggest a relationship between increased EGF receptor expression and increased EGF responsiveness. Furthermore, these data indicate a potential mechanism by which EGF and retinoic acid may interact in lung physiology.     

Modulation of the mitogenic response of an epidermal growth factor-dependent keratinocyte cell line by dexamethasone, insulin, and transforming growth factor-beta

The stimulation of DNA synthesis by epidermal growth factor (EGF) has been studied for a cell line having properties useful for investigating the mechanism of action of EGF in epithelial cell populations. These studies employ a mouse keratinocyte cell line (MK), isolated by Weissman and Aaronson (1983), which is stringently dependent on exogenous EGF for growth in serum containing medium. The studies reported here characterize the compliment of EGR receptors present on the surface of MK cells and demonstrate the regulatory influence of other hormones on the capacity of EGF to stimulate DNA synthesis. Up-regulated MK cells contain approximately 22,000 EGF receptors per cell, but when the cells are grown in the presence of EGF the receptor number is reduced to about 4,000. It is estimated that only a small number of high-affinity receptors (less than 500) are required for EGF-dependent cell proliferation. In contrast to its action in fibroblastic cells, dexamethasone is a strong inhibitor of EGF-stimulated DNA synthesis of MK cells. Insulin at high concentrations, or insulin-like growth factors I or II (IGF-I, IGF-II) at physiological concentrations, synergistically enhance the EGF response. Interestingly, insulin or IGF-I or II are also able to reverse most of the dexamethasone inhibition of DNA synthesis. Transforming growth factor-beta (TGF-beta) inhibits, in reversible manner, the EGF stimulation of DNA synthesis and this inhibition is not overcome by insulin. TGF-beta receptors have been measured in MK cells and Scatchard analysis indicates approximately 20,000 receptors per cell. None of the modulatory hormones (insulin, dexamethasone, TGF-beta) significantly altered 125I-EGF binding characteristics in MK cells, suggesting a point of action distal to 125I-EGF binding.     

Inhibition of macrophage activation by isoquinolinesulfonamides, phenothiazines, and a napthalenesulfonamide

The influence of isoquinolinesulfonamides (H-7 and H-8), phenothiazines(trifluoperazine and fluphenazine), and a naphthalenesulfonamide (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) on stimulated superoxide anion production and phosphatidyl inositol (PI) cycle activity was investigated in the guinea pig alveolar macrophage. All five drugs were able to inhibit superoxide anion production stimulated by n-formyl-nel-leu-phe (FNLP), leukotriene B4 (LTB4), and phorbol-12,13-dibutyrate (PDB). The order of potency was trifluoperazine greater than or equal to fluphenazine greater than H-7 = W-7 greater than H-8. The dose response curves could be shifted to less efficacy by increasing extracellular calcium. By itself, W-7 markedly stimulated 45Ca+2 efflux, fluphenazine and trifluoperazine slightly stimulated 45Ca+2 efflux, while H-7 and H-8 had no effect on 45Ca+2 efflux from macrophages preloaded with 45Ca+2. Consistent with these results, W-7 markedly stimulated PI cycle activity, fluphenazine and trifluoperazine slightly stimulated PI cycle activity, while H-7 and H-8 had no significant effects on PI cycle activity. In addition, W-7 by itself was able to stimulate a weak and short-lived "burst" of superoxide anion production. In order to evaluate whether a site of action of the inhibitors was at protein kinase C and whether protein kinase C was involved in terminating the normally short-lived FNLP- and LTB4-stimulated macrophage activation, fluphenazine and H-7 were used to evaluate the duration of FNLP- and LTB4-stimulated PI cycle activity, at concentrations of the inhibitors that significantly blocked stimulated superoxide anion production. In all cases, FNLP and LTB4 still stimulated PI cycle activity, which still terminated even though protein kinase C was inhibited. These results suggest that all five drugs block protein kinase C, but H-7 was the most specific in its action at protein kinase C, while the phenothiazines and W-7 have multiple sites of action. In addition, these results suggest that protein kinase C may not function to cause the termination of FNLP- and LTB4-stimulated PI cycle activity and subsequent superoxide anion production.     

A study of functionally active amino acids involved in the interaction of HLA-A2 or HLA-A3 molecules with cytolytic T lymphocytes

A large series of HLA-A2/HLA-A3 recombinant genes were generated by using the in vivo recombination technique. These genes have each been modified in the last two-thirds of the third exon such that one or several HLA-A2-specific substitutions have been made in the HLA-A3 gene and vice versa. The recombinant genes were transfected into the murine cell line P815 and the transfectants were used as targets for a series of 20 human CTL lines or clones specific for HLA-A2 or HLA-A3, or restricted by HLA-A2 and specific for influenza A. Several patterns of anti-HLA-A2, anti-HLA-A3, and HLA-A2-restricted anti-influenza CTL activity were observed and when uncloned cell lines were studied, a progressive selection of some clones with a similar pattern of activity was regularly found. From the comparison of these different patterns the following conclusions can be drawn: 1) In most but not all cases both domains of the class I molecule were essential for CTL recognition, but residue 152 was critically important for the majority of CTL tested; 2) amino acids 114/116 were also critical in most cases, and their position close to amino acid 152 in the tertiary structure of the molecule may have some functional significance; and 3) amino acid 161, although highly conserved, plays an unexpected but very important role in CTL function.     

Linkage relationships of seven enzyme and two pigmentation loci in the snail Biomphalaria glabrata

Crossing experiments with inbred stocks of the snail (Biomphalaria glabrata) demonstrated that variants at two loci determining pigmentation and seven enzyme-determining loci exhibited normal Mendelian segregation ratios in F2 progeny. Among 39 pairwise comparisons for joint segregation, there was evidence of genetic linkage between a locus controlling mantle pigmentation (S) and 6-phosphogluconate dehydrogenase (Pgd) and confirmation of a previously described linkage between esterase-2 (Est-2) and catalase (Cat). Recombination fractions were estimated to be 17 +/- 4 for S-Pgd and 33 +/- 5 for Est-2-Cat. The remaining five loci--Acon-1, Pgm-1, Lap-1, Lap-2, and Pgd--assorted independently. This brings to 17 the number of loci examined for segregation and assortment in this medically important species. As Biomphalaria has a chromosome number n = 18, markers should soon be available for most or all of the linkage groups.     

BCNU-induced protection from hyperbaric hyperoxia: role of glutathione metabolism

To explore the role of the glutathione oxidation-reduction cycle in altering the sensitivity of rats to the effects of hyperbaric hyperoxia, we administered N,N-bis(2-chloroethyl)-N-nitrosourea (BCNU) to decrease tissue glutathione reductase activity. We then exposed these animals and their matched vehicle-treated controls to 100% O2 at 4 ATA. Animals that received BCNU and were immediately exposed to hyperbaric O2 showed enhanced toxicity by seizing earlier in the exposure than controls. Animals that received BCNU 18 h before the hyperbaric O2 exposure were paradoxically protected from the effects of the exposure with a prolongation of their time to initial seizure and a marked increase in their survival time during the exposure. Tissue glutathione concentrations were also measured in the various groups and the hyperbaric O2 exposure produced marked decreases in hepatic glutathione levels in all control animals. In animals treated with BCNU 18 h before exposure, hepatic glutathione concentrations also decreased, but the concentrations had significantly increased during the 18-h waiting period, allowing these animals to maintain hepatic levels in the normal range even during their hyperbaric exposures. We conclude that treatment of rats with BCNU 18 h before exposure to hyperbaric hyperoxia results in enhanced protection of the animals during the exposure.     

Glyphosate tolerant flax plants from Agrobacterium mediated gene transfer

Agrobacterium tumefaciens carrying a disarmed Ti-plasmid vector containing a chimeric NPT-II gene and a glyphosate resistance plant-derived 5-enolpyruvylshikimate-3-phosphate synthase gene was used to transform flax hypocotyl tissues. Transformed shoots could be regenerated from the inoculated tissue and were proven to be transgenic by the combination of leaf callus assays, nopaline assays and progeny tests. Co-segregation was observed in the progeny for kanamycin and glyphosate resistance.     

The cytogenetic and hepatotoxic effects of dioxin on mouse liver cells

The cytogenetic and hepatotoxic effects of 2,3,7,8-tetrachlorodibenzo p-dioxin (TCDD) on mouse liver cells were investigated. Male C57BL/6J strain mice, which have TCDD receptors, were given single intraperitoneal injections of 25, 37.5, 75 and 150 g of TCDD/kg body weight or corn oil carrier alone. Two-thirds hepatectomies were carried out at 1 or 7 days after injection and chromosomal aberrations and mitotic indexes of the regenerating hepatocytes were scored 54 hr after hepatectomy. Liver sections from additional intact mice were studied for TCDD-hepatotoxicity at 1, 7 and 30 days after injection. The three high doses of TCDD caused hepatotoxicity with necrosis of liver cells and focal architectural collapse by 30 days after injection. No evidence was obtained of an increase in the frequency of chromosomal structural aberrations at doses that allowed sufficient mitotic activity for cytogenetic evaluation. We conclude that TCDD is not a clastogen for mouse hepatocytes, although high doses cause marked hepatocellular necrosis.Abbreviations CSD chromosome deletion - META metacentric chromosome - TCDD 2,3,7,8-tetrachlorobenzo-p-dioxin