Effects of the oxidant potassium permanganate on the expression of gill metallothionein mRNA and its relationship to sublethal whole animal endpoints in channel catfish.

Potassium permanganate is an oxidant heavily used in fish culture. The effects of this compound were examined utilizing molecular (Metallothionein) and whole animal endpoints following an 8-week exposure to nominal concentrations of 0.5 (daily) and 1.0 and 2.0 mg/L (on alternate days) of potassium permanganate (PM). In order to measure MT, a complementary DNA clone of metallothionein (MT) was cloned and sequenced from the liver of channel catfish treated with a single injection of cadmium chloride (10 mg/kg). The cDNA was obtained by reverse transcriptase polymerase chain reaction (RT-PCR), using 3' rapid amplification of cDNA ends (RACE) technique. No significant correlation was observed with gill MT expression or sublethal endpoints indicative of toxicity (weight, length, condition index [CI], or liver somatic index [LSI). MT mRNA expression in gill was significantly reduced only after 8 weeks in the 2.0 mg/L treatment. Decreases in CI were observed in males at all time points after 4 weeks, at the 2.0 mg/L treatment concentration, with a NOEC of 1 mg/L. Reductions in LSI that were not dose dependent were also observed in both males and females throughout the 8-week study and no consistent reduction in weight gain or length was observed. These data demonstrate that minimal changes in sublethal effects occur in fish following 0.5-2.0 mg/L PM treatment after 4 weeks, but recovery from adverse effects is observed by 8 weeks, suggesting that acute (typically less than 1 week) treatment of channel catfish with PM would not significantly affect fish health.

Cloning and expression analysis of murine phospholipase D1.

Activation of phosphatidylcholine-specific phospholipase D(PLD) occurs as part of the complex signal-transduction cascade initiated by agonist stimulation of tyrosine kinase and G-protein-coupled receptors. A variety of mammalian PLD activities have been described, and cDNAs for two PLDs recently reported (human PLD1 and murine PLD2). We describe here the cloning and chromosomal localization of murine PLD1. Northern-blot hybridization and RNase protection analyses were used to examine the expression of murine PLD1 and PLD2 ina variety of cell lines and tissues. PLD1 and PLD2 were expressed in all RNA samples examined, although the absolute expression of each isoform varied, as well as the ratio of PLD1 to PLD2. Moreover, in situ hybridization of adult brain and murine embryo sections revealed high levels of expression of individual PLDs in some cell types and no detectable expression in others. Thus the two PLDs probably carry out distinct roles in restricted subsets of cells rather than ubiquitous roles in all cells.

RalA interacts directly with the Arf-responsive, PIP2-dependent phospholipase D1.

RalA GTPase associates with a phospholipase D (PLD) that is activated in v-Src- and v-Ras-transformed cells. Two mammalian PLDs were recently cloned: PLD1, which is activated by Arf family GTPases and dependent upon phosphatidylinositol-4,5-bisphosphate (PIP2), and PLD2, which is also dependent upon PIP2, but not stimulated by Arf. Another PLD has been described that is stimulated by oleate. Evidence is provided that the RalA-assiciated PLD is PLD1. First, the PLD precipitated by RalA from murine fibroblasts was stimulated by Arf, dependent upon PIP2, and inhibited by oleate. Second, immobilized RalA precipitated PLD1 from sf9 insect cells overexpressing PLD1. Third, a series of RalA mutants precipitated PLD activity from both PLD1-expressing insect cells and murine fibroblasts with the same efficiency. And finally, immobilized RalA precipitated PLD1 from a purified PLD1 preparation. These data argue that RalA associates directly with the Arf-responsive, PIP2-dependent PLD1.

Phospholipase D2, a distinct phospholipase D isoform with novel regulatory properties that provokes cytoskeletal reorganization.

BACKGROUND: Activation of phospholipase D (PLD) is an important but poorly understood component of receptor-mediated signal transduction responses and regulated secretion. We recently reported the cloning of the human gene encoding PLD1; this enzyme has low basal activity and is activated by protein kinase C and the small GTP-binding proteins, ADP-ribosylation factor (ARF), Rho, Rac and Cdc42. Biochemical and cell biological studies suggest, however, that additional and distinct PLD activities exist in cells, so a search was carried out for novel mammalian genes related to PLD1. RESULTS: We have cloned the gene for a second PLD family member and characterized the protein product, which appears to be regulated differently from PLD1: PLD2 is constitutively active and may be modulated in vivo by inhibition. Unexpectedly, PLD2 localizes primarily to the plasma membrane, in contrast to PLD1 which localizes solely to peri-nuclear regions (the endoplasmic reticulum, Golgi apparatus and late endosomes), where PLD activity has been shown to promote ARF-mediated coated-vesicle formation. PLD2 provokes cortical reorganization and undergoes redistribution in serum-stimulated cells, suggesting that it may have a role in signal-induced cytoskeletal regulation and/or endocytosis. CONCLUSIONS: PLD2 is a newly identified mammalian PLD isoform with novel regulatory properties. Our findings suggest that regulated secretion and morphological reorganization, the two most frequently proposed biological roles for PLD, are likely to be effected separately by PLD1 and PLD2.