Regulation and Glucocorticoid-Independent Induction of Lipocortin I in Cultured Astrocytes

Stimulation of prostaglandin (PG) release in rat astroglial cultures by various substances, including phorbol esters, melittin, or extracellular ATP, has been reported recently. It is shown here that glucocorticoids (GCs) reduced both basal and stimulated PGD2 release. Hydrocortisone, however, did not inhibit ATP-, calcium ionophore A23187-, or tetradecanoyl phorbol acetate (TPA)-stimulated arachidonic acid release, and only TPA stimulations were affected by dexamethasone. GC-mediated inhibition of PGD2 release thus appeared to exclude regulation at the phospholipase A2 (PLA2) level. Therefore, the effects of GCs on the synthesis of lipocortin I (LC I), a potent, physiological inhibitor of PLA2, were studied in more detail. Dexamethasone was not able to enhance de novo synthesis of LC I in freshly seeded cultures and failed to increase LC I synthesis in 2-3-week-old cultures. It is surprising that LC I was the major LC synthesized in those cultures, and marked amounts accumulated with culture time, reaching plateau levels at approximately day 10. In contrast, LC I was barely detectable in vivo. This tonic inhibition of PLA2 is the most likely explanation for unsuccessful attempts to evoke PG release in astrocyte cultures by various physiological stimuli. GC receptor antagonists (progesterone and RU 38486) given throughout culture time reduced LC I accumulation and simultaneously increased PGD2 release. Nonetheless, a substantial production of LC I persisted in the presence of antagonists. Therefore, LC I induction did not seem to involve GC receptor activation. This was confirmed in serum- and GC-free brain cell aggregate cultures. Here also a marked accumulation of LC I was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Perception of the auxin signal at the plasma membrane of tobacco mesophyll protoplasts

Auxin-induced variations of transmembrane potential difference have been shown to be a useful tool for analyzing hormone sensitivity in tobacco protoplasts. Using this technique, we demonstrated that protoplasts derived from wild-type, an auxin-resistant mutant and Agrobacterium-rhizogenes transformed plants differed widely in the sensitivity of their electrical response to naphthalene acetic acid. We have used different antibodies, raised to auxin binding proteins (ABP) from maize coleoptiles, or to the axr1 gene product (ABP1), to test whether changes in auxin sensitivity can be correlated with the presence of tobacco proteins immunologically related to this ABP. Titrations indicated that 0.4 nM anti-ABP IgG inhibited 50% of the auxin-specific response of wild-type protoplasts, whereas 0.04 nM or 4 nM anti-ABP IgG were necessary to inhibit the response of mutant and transformed protoplasts, respectively, to the same extent. On wild-type protoplasts, blocking part of the immunoreactive sites with anti-ABP antibodies resulted in a decrease in auxin sensitivity of the electrical response (0.4 nM anti-ABP IgG inducing a 10–fold decrease), whereas addition of maize ABP increased this auxin sensitivity (1 pM ABP1 raised the sensitivity more than 1000–fold). The results obtained suggest that the auxin sensitivity detected by our assay system correlates with the amount of tobacco proteins immunologically related to the axr¹ gene product from maize. A hypothesis accounting for the presence of these proteins at the external surface of tobacco protoplasts and for the effects of hetero-logous maize ABP on auxin sensitivity is proposed.     

Activation of phospholipase C is not correlated to the formation of prostaglandins and superoxide in cultured rat liver macrophages.

This study evaluates the role of inositol phosphates as possible mediators of the activation of phospholipase A2 and NADPH oxidase in cultured rat liver macrophages. Inositol phosphate formation was achieved by zymosan, immune complexes, latex particles and calcium ionophore while the release of arachidonic acid and the formation of prostaglandin E2 was also elicited by phorbol ester and NaF, but not by latex particles; generation of superoxide was obtained by zymosan and phorbol ester only. The kinetics of the formation of inositol phosphates revealed that within the first few minutes after zymosan addition inositol trisphosphate was formed, followed by inositol bisphosphate and inositol monophosphate. Pre-treatment of the cells with dexamethasone or removal of extracellular calcium led to an inhibition of the zymosan-induced formation of inositol phosphates and prostaglandin E2 but had no effect on the generation of superoxide; inhibition of the Na+/H+ exchanger by removal of extracellular sodium ions led to a decrease of the zymosan-induced synthesis of prostaglandin E2, but did not affect the formation of inositol phosphates and superoxide. Pre-treatment of the cells with phorbol ester decreased the zymosan-induced synthesis of prostaglandin E2 and superoxide, but even enhanced the zymosan-induced formation of inositol phosphates. These data indicate that in cultured rat liver macrophages the formation of prostaglandins and superoxide cannot be correlated to an activation of phospholipase C.     

Nitrate reductase in needles,roots and trunk wood of spruce trees [Picea abies (L.) Karst.]

Summary Nitrate reductase (EC 1.6.6.2) activity (NRA), as measured by an in vivo assay, is present in needle leaves and mycorrhizal fine root tips of adult Norway spruce [Picea abies (L.) Karst.] in at least equal amounts on a fresh weight basis, in both adult and 5-year-old trees. NRA could also be demonstrated in trunk wood of deroted trees after fertilization with 5 mM , exhibiting a longitudinal profile in the trunk. Inducibility in needles can more efficiently be achieved by NO₂ (100 g·m^-3) than by 5 mM nitrate, which is effective only in root-amputated trees. A remarkably high level of needle-NRA in unfertilized trees, which are characterized by a very low level of nitrate in the xylem sap, suggests that NRA in spruce needles may in part be constitutive. Organic-N is a major nitrogen source for the needles even in root-amputated trees, indicating pronounced exchange processes between ray parenchyma and trunk xylem, which in turn are modified by the nitrogen source fed to the trunk stump. Intact trees exhibit a very similar amino acid composition of the xylem sap, regardless of whether or has been fed. The amino acid pattern of the needles is not thrown out of balance by flooding with and , which occurs in fertilized derooted trees. This indicates a distinct potential for homoeostasis of nitrogen entrance-metabolism (i.e. NRA and glutamine synthetase activity) in the needles. In the ectomycorrhiza/fine root-system (EMC), marked differences in NRA were observed depending on root-tip diameter and along the longitudinal profile of the fine roots. EMC-nitrate reductase is strongly enhanced by . Needle-NRA exhibits a circannual rhythm. An early summer maximum is followed by a December minimum. This activity pattern matches well the transitory increase of soluble nitrogen in spring and the total protein maximum in winter. In an indirect way assimilatory NRA may well contribute to nitrogen overfertilization (by consumption of NO_X) as one possible cause of the contemporary decline of spruce populations.     

Ganglioside biosynthesis in rat liver: different distribution of ganglioside synthases in hepatocytes, Kupffer cells, and sinusoidal endothelial cells

The activities of five glycolipid-glycosyltransferases, GL2, GM3, GM2, GM1, and GD1a synthase, were determined in a cell-free system with homogenate protein of total rat liver, isolated hepatocytes, Kupffer cells, and sinusoidal endothelial cells. In rat liver parenchymal and nonparenchymal cells ganglioside synthases were distributed differently. Compared to hepatocytes, Kupffer cells expressed a nearly sevenfold greater activity of GM3 synthase, but only 14% of GM2, 19% of GM1, and 67% of GD1a synthase activity. Sinusoidal endothelial cells expressed a pattern of enzyme activities quite similar to that of Kupffer cells with the exception of higher GM2 synthase activity. Activity of GL2 synthase was distributed unifromly in parenchymal and nonparenchymal cells of rat liver, but differed by sex. It was 1 to 2 orders of magnitude below that of all the other ganglioside synthases investigated. The results indicate GL2 synthase regulates the total hepatic ganglioside content, and hepatocytes but not nonparenchymal liver cells have high enzymatic capacities to form a-series gangliosides more complex than GM3.     

Bioelectric responses at fertilization: Separation of the events associated with insemination from those due to the cortical reaction in sea urchin,Lytechinus variegatus

The bioelectric responses at fertilization of the sea urchin Lytechinus variegatus are a complex series of membrane potential and resistance changes that occur concomitant with gamete fusion, ionic fluxes, and the cortical granule discharge. This work attempts to separate the electrical effects of sperm-egg interactions from those of the cortical reactions. Two approaches were taken to discern the electrical events associated with insemination, distinct from cortical granule discharge: (1) fertilization of eggs treated with 3% urethane, 10 mM procaine, or 10 mM nicotine, to prevent the cortical reaction and (2) refertilization of fertilized eggs (denuded with 1 mM aminotriazole containing 1 mg/ml soybean trypsin inhibitor). Cortical granule discharge in the absence of sperm incorporation was investigated by artificial activation with 5 μM A23187 or by fertilization in the presence of 10 μM cytochalasin D, which prevents incorporation. These results are consistent with a model in which the sperm-egg interaction triggers both a rapid (50–400 msec), but minor (?10 mV), electrical transient that leads to an action potential and then both the Na⁺-dependent fast block to polyspermy and the late block resulting from the secretion of the cortical granules.     

Quantitation of gibberellins and the metabolism of [3H]gibberellin A1 during somatic embryogenesis in carrot and anise cell cultures

In a carrot (Daucus carota L.) cell line lacking the ability to undergo somatic embryogenasis, and in carrot and anise (Pimpinella anisum L.) cell lines in which embryogenesis could be regulated by presence or absence of 2,4-dichlorophen-oxyacetic acid (2,4-D), in the medium (+2,4-D=no embryogenesis,-2,4-D=embryo differentiation and development), the levels of endogenous gibberellin(s) (GA) were determined by the dwarfrice bioassay, and the metabolism of [³H]GA₁ was followed. Embryos harvested after 14 d of subculture in-2,4-D had low levels (0.2–0.3 g g^-1 dry weight) of polar GA (e.g. GA₁-like), but much (3–22 times) higher levels of less-polar GA (GA_4/7-like); GA₁, GA₄ and GA₇ are native to these cultures. Conversely, the undifferentiated cells in a non-embryogenic strain, and proembryos of an embryogenic strain (+2,4-D) showed very high levels of polar GA (2.9–4.4 g g^-1), and somewhat reduced levels of less-polar GA. Cultures of anise undergoing somatic embryo development (-2,4-D) metabolized [³H]GA₁ very quickly, whereas proembryo cultures of anise (+2,4-D) metabolized [³H]GA₁ slowly. The major metabolites of [³H]GA₁ in anise were tentatively identified as GA₈-glucoside (24%), GA₈ (15%), GA₁-glucoside (8%) and the ¹⁽¹⁰⁾GA₁-counterpart (2%). Thus, high levels of a GA₁-like substance and a reduced ability to metabolize GA₁ are correlated with the absence of embryo development, while lowered levels of GA₁-like substance and a rapid metabolism of GA₁ into GA₈ and GA-conjugates are correlated with continued embryo development. Exogenous application of GA₃ is known to reduce somatic embryogenesis in carrot cultures; GA₄ was found to have the same effect in anise cultures. Thus, a role (albeit negative) in somatic embryogenesis for a polar, biologically active GA is implied.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA gibberellin(s) or gibberellin-like substances - GC-RC gas chromatography-radiochromatogram counting - HPLC high-presare liquid chromatography - Rt retention time - TLC thinlaver chromatography     

Isolation of an Hfr donor of Pseudomonas aeruginosa PAO by insertion of the plasmid RP1 into the tryptophan synthase gene

Summary A derivative of the IncP-1 plasmid RP1, temperature-sensitive for maintenance, was inserted into the Pseudomonas aeruginosa chromosome by selection for a plasmid marker (carbenicillin resistance) at nonppermissive temperature. In one strain, PAO 1000, the plasmid was stably integrated in the trpA, B gene cluster mapped at 27 min, as shown by the following evidence. (i) Trp⁺ transductants lost all plasmid markers. (ii) Cleared lysates of PAO 1000 showed no plasmid band typical of the autonomous RP1 in agarose gel electrophoresis. (iii) No transfer of carbenicillin resistance by PAO 1000 was detectable. (iv) PAO 1000 mobilised the chromosome from an origin at, or very near, the plasmid insertion site with high frequency (recovery of proximal markers 10^–3 per donor). Matings on the plate with and without interruption of conjugation showed that chromosome transfer was unidirectional. (v) Recombinants from PAO 1000-mediated crosses did not inherit plasmid markers or the trpA, B mutation. A derivative of PAO 1000 was obtained which had lost the Hfr property and all plasmid markers except carbenicillin resistance. This strain (PAO 1001), when carrying the autonomous RP1 plasmid, was capable of unidirectional chromosome mobilisation like PAO 1000, but with 50-fold lower efficiency. We propose that integration of the temperature-sensitive RP1 plasmid in PAO 1000 occurred via transposition of Tnl, the element specifying carbenicillin resistance.