Characterization of a phospholipase Dalpha cDNA from tomato fruit.

Phospholipase D (PLD) initiates phospholipid (PL) catabolism in plant cells and is also involved in signal transduction and retailoring of membrane PL. Total PL declines and phosphatidic acid increases in pericarp tissue during tomato fruit ripening, suggesting that increased PLD activity alters membrane structure. To assess the role of PLD in tomato ripening, we have begun a molecular genetic approach. Using a castor bean PLDalpha cDNA as a probe, a PLDalpha cDNA (LEPLD2) was isolated from our tomato fruit library. It has an open reading frame of 2421 nucleotides, encoding a polypeptide of 807 amino acids with a molecular mass of 92 kDa. The deduced LEPLD2 PLDalpha shares >75% sequence identity with PLDalphas from castor bean, tobacco and tomato. LEPLD2 transcript, detected by RNA gel-blot analysis, was very low in roots, low in stems, moderate in leaves, high in flowers, and increased in fruit during development and ripening. Expression of LEPLD2 in Escherichia coli yielded active enzyme, and a FLAG-PLDalpha fusion protein produced by transformed E. coli migrated close to the calculated 94 kDa on SDS/PAGE.

Evidence for prostanoid biosynthesis as a biochemical feature of certain subclasses of non-small cell carcinomas of the lung as determined in established cell lines derived from human lung tumors.

Detectable levels (greater than or equal to 0.2 pmol/10(6) cells) of one or more prostanoid species resultant to calcium ionophore A23187-induced biosynthesis from endogenous arachidonic acid were distributed in 28 cell lines derived from different histological classes of lung tumors as follows: large cell undifferentiated carcinoma (3 of 3 cell lines); adenosquamous carcinoma (1 of 2 cell lines); squamous cell carcinoma (0 of 2 cell lines); adenocarcinoma (9 of 10 cell lines); bronchioloalveolar cell carcinoma (2 of 2 cell lines); and small cell carcinoma (1 of 9 cell lines). Using the mean levels of 9 alpha,11 beta-prostaglandin F2, prostaglandin F2 alpha, prostaglandin D2, prostaglandin E2, thromboxane B2 and 6-keto-prostaglandin F1 alpha as an index of prostaglandin H (PGH) synthase activity, the distribution in cell lines representative of the different histological classes of human lung tumors exhibiting PGH synthase activity exceeding mean values greater than or equal to 2 pmol/10(6) cells was as follows: large cell undifferentiated carcinoma (3 of 3 cell lines), adenosquamous carcinoma (1 of 2 cell lines), adenocarcinoma (8 of 10) cell lines), bronchioloalveolar cell carcinoma (2 of 2 cell lines) and small cell carcinoma (0 of 9 cell lines). Three different prostanoid species accumulated to mean levels greater than or equal to 2 pmol/10(6) cells. Prostaglandin E2 levels exceeded 2 pmol/10(6) cells in 14 of the 16 cell lines in which this prostanoid accumulated to detectable levels. Cumulative levels of prostaglandin F2 alpha exceeded 2 pmol/10(6) cells in 9 of the 15 cell lines in which prostaglandin F2 alpha reached detectable levels. Detectable levels of thromboxane B2 were observed in five cell lines with thromboxane B2 accumulation exceeding 2 pmol/10(6) cells in two of the five cell lines. 9 alpha,11 beta-prostaglandin F2 and 6-keto-prostaglandin F1 alpha accumulated to detectable levels in the culture medium of one cell line, while prostaglandin D2 accumulation to detectable levels was observed in two cell lines. Stimulation of cultured human lung tumor cells exhibiting PGH synthase activity greater than or equal to 2 pmol/10(6) cells in the presence of 10(-5) M exogenous arachidonic acid resulted in a 2- to 4-fold increase in the accumulation of individual prostanoids, while the inclusion of a 10(-5) M exogenous concentration of arachidonic acid failed to stimulate detectable prostanoid production in human lung tumor cells in which PGH synthase activity was not previously expressed.(ABSTRACT TRUNCATED AT 400 WORDS)

Vasopressin-, angiotensin II-, and alpha 1-adrenergic-induced inhibition of Ca2+ transport by rat liver plasma membrane vesicles.

A rapid method for isolating highly purified rat liver plasma membrane vesicles using isotonic medium and Percoll self-forming gradient centrifugation is described. The vesicles were characterized by enzyme markers and electron microscopy. The method also yielded a fraction rich in nuclei. The vesicles transported Ca2+ in an ATP-dependent manner and this was enhanced by oxalate. The Vmax for Ca2+ uptake was 0.65 +/- 0.08 nmol/mg X min, which was approximately 18-fold higher than for other liver plasma membrane preparations, and the Km for Ca2+ was 5.2 +/- 0.4 nM. Calcium uptake was inhibited by 40-50% in vesicles isolated from rat livers perfused for 3 min with 10(-7)M vasopressin. The half-maximally effective concentration of vasopressin was 5 X 10(-10)M which correlates with that for raising cytosolic Ca2+ and phosphorylase a. Inhibition was not significant in vesicles from livers perfused with vasopressin for only 1 min, indicating that inhibition of the Ca2+ pump may not be involved in the rise in cytosolic Ca2+ observed at 1-2 s with this hormone. Epinephrine (10(-5)M) and angiotensin II (10(-7)M) inhibited Ca2+ uptake by 31 +/- 10 and 26 +/- 5%, respectively, at 3 min. Glucagon (10(-7)M) had no effect. It is proposed that the inhibitory action of the Ca2+-dependent hormones on the plasma membrane Ca2+ pump plays an important role in the actions of these hormones by prolonging the elevation in cytosolic Ca2+.