Phorbol myristate acetate stimulates formation of phosphatidyl inositol 4-phosphate and phosphatidyl inositol 4,5-bisphosphate in human platelets

There are conflicting data in the literature as to whether or not the Ca2+ activation of phospholipase A2 is mediated by the calcium binding protein calmodulin. In the present study the membrane-bound phospholipase A2 enzymes in rat and human platelets were shown to be absolutely Ca2+ dependent but were not stimulated by the addition of calmodulin. A partially purified phospholipase A2 from rat platelet membrane, which contained little endogenous calmodulin, also was not stimulated by calmodulin addition. Both isolated and membrane-bound phospholipase A2 were inhibited by the non-specific calmodulin antagonist trifluoperazine but the inhibition was not overcome by adding calmodulin. There was thus no evidence from these studies that phospholipase A2 is calmodulin regulated.     

Carbon monoxide dehydrogenase and acetate thiokinase in Methanothrix soehngenii

Abstract In Methanothrix soehngenii acetate is first activated by an acetate thiokinase rather than a phosphotransacetylase. The specific activity of the acetate thiokinase was 5.29 μmol acetate activated min⁻¹ mg⁻¹ protein with a half maximum rate at 0.74 mM acetate and at 0.047 mM CoA. In cell-free extracts a CO-dehydrogenase activity was measured of 3.02 μmol min⁻¹ mg⁻¹ protein with a half maximum rate at 0.44 mM CO and at 0.18 mM methylviologen. NADP and NAD could not replace methylviologen. F₄₂₀ showed only low activity as electron acceptor.     

Effect of Acetate on Esterase C Activity During the Growth Cycle of Paramecium*

SYNOPSIS Enhanced esterase C activity could be demonstrated by starch gel electrophoresis in various stocks of Paramecium spp. (P. primaurelia stocks 90 and 540, P. biaurelia stock 93, P. tetraurelia stock 29. P. pentaurelia stock 87, P. octaurelia stocks 31 and 300, and P. multimicronucleatum species 3, stock 8 MO) grown in Adaptation Medium. This esterase, however, was barely detectable when they were cultivated in Axenic Medium. Addition of trypticase to Adaptation Medium resulted in reduction of esterase C in the ciliates. This effect is ascribable to Na acetate present in trypticase. Since esterase C increased with the decrease in acetate concentration (as estimated by gas-liquid chromatography) during growth of Paramecium, acetate appears to be utilized by the cells. Sensitivity of esterase C to acetate occurs in all 6 species of Paramecium examined. Different stocks within a species may have different levels of sensitivity; in one case this is genetically determined. The results emphasize the importance of controlling and manipulating growth conditions for the assessment of inter- and intraspecies variations in the isozymes of Paramecium.     

Proliferation of lamellar whorls in arcuate neurons of the hypothalamus of male rats treated with estradiol benzoate or cyproterone acetate

Summary The arcuate nucleus of the hypothalamus (AH) of male rats which had been treated either with estradiol benzoate (E²B) or cyproterone acetate (CPA) was examined ultrastructurally for the presence of whorls of endoplasmic reticulum. The incidence of whorl containing neurons (WCN) was 2–4 times higher in the AH of animals treated for 2–3 weeks with E²B or for 2 weeks with CPA than in the AH of oil treated controls. CPA is a powerful anti-androgen while E²B acts both peripherally and centrally to limit testosterone production. These findings, together with previous evidence that whorls proliferate in AH of male rats deprived of androgen by morphine treatment or castration, suggest that steroid feedback (androgen alone or both androgen and estrogen) plays an important role in AH whorl proliferation. The possibility that WCN may be LH-RH containing neurons is suggested by the close correspondence between the number and location of WCN within AH as determined in this study and the distribution of LH-RH containing cells reported by others.The authors are indebted to Schering AG for supplying cyproterone acetate for this study. This work was supported by grants DA-00259, NS-09156 and MH-14677 from U.S.P.H.S.Research Scientist Development Award MH-38894Research Scientist Development Award MH-70180     

Characteristics of androgen binding in rat adrenal tissue using [3H]methyltrienolone (R1881) as tracer

A high level of binding of [3H]methyltrienolone (R1881 = 17beta-hydroxy-17alpha-methyl-estra-4, 9, 11-trien-3-one) was found in cytosol prepared from adrenals of castrated male rats. Binding of [3H]R1881 was of high affinity (DK = 6.2 nM) and highly specific for androgens. The [3H]R1881 complex migrates at 7-9S on sucrose gradients in low ionic strength buffer and at 4-5S in buffer containing 0.4M KC1. All binding studies have been performed in parallel with rat ventral prostate and adrenal cytosol. The present data suggest the presence of an androgen binding component in rat adrenal tissue.     

Mitogens for murine embryo cell lines

The growth-promoting activities of fetal bovine serum, cortisol, phorbol myristate acetate, prostaglandin F2α, insulin, epidermal growth factor, and fibroblast growth factor were evaluated on four murine embryo cell lines (Swiss 3T3, Balb 3T3, M2, and C3H10T1/2). Each cell had an unique response spectrum to this collection of reported mitogens. Phorbol myristate acetate and prostaglandin F2α were active only on selected cell lines; cortisol was inactive on all four lines. Serum, epidermal growth factor, and fibroblast growth factor were able to stimulate cell division in all four lines, albeit to varying degrees for the different target cells.     

Thermoluminescence study of the in vivo effects of bicarbonate depletion and acetate/formate presence in the two algae Chlamydobotrys stellata and Chlamydomonas reinhardtii

We investigated the influence of CO₂/HCO₃ ^–-depletion and of the presence of acetate and formate on the in vivo photosynthetic electron transport in the two green algae Chlamydobotrys stellata and Chlamydomonas reinhardtii by means of thermoluminescence technique and mathematical glow curve analysis. The main effects of the removal of CO₂ from the algal cultures was: (1) A shift of the glow curve peak position to lower temperatures resulting from a decrease of the B band and an increase of the Q band. (2) Treatment of CO₂-deficient Chl. stellata with DCMU yielded two thermoluminescence bands in the Q band region peaking at around +12°C and +5°C; in case of Chl. reinhardtii DCMU treatment induced only one band with an emission maximum at +5°C. The presence of acetate or formate in CO₂-depleted algal cultures lowered the intensities of all of the individual TL bands but that of a HT band (TL+37). The effects of CO₂-depletion and of the presence of anions were fully reversible.Abbreviations DCMU 3-(3,4)-dichlorophenyl-1,1-dimethylurea - HT band high temperature TL band - P₆₈₀ reaction center chlorophyll of PS II - Q_A and Q_B primary and secondary quinone acceptors of PS II, respectively - PS II Photosystem II - S_2/3 redox states of the oxygen evolving complex of PS II - TL thermoluminescence     

Vertical profiles of CH4 concentrations,dissolved substrates and processes involved in CH4 production in a flooded Italian rice field

Vertical profiles were measured in soil cores taken from flooded rice fields in the Po valley during July and August 1990. Methane concentrations generally increased with depth and reached maximum values of 150–500 μM in 5–13 cm depth. However, the shape of the profiles was very different when studying different soil cores. The CH₄ content of gas bubbles showed a similar variability which apparently was due to spatial rather than temporal inhomogeneities. Similar inhomogeneities were observed in the vertical profiles of acetate, propionate, lactate, and formate which showed maximum values of 1500, 66, 135, and 153, μM, respectively. However, maxima and minima of the vertical profiles of the different substates usually coincided in one particular soil core. Large inhomogeneities in the vertical profiles were also observed for the rates of total CH₄ production, however, the percentage contribution of H₂/CO₂ to CH₄ production was relatively homogeneous at 24 ± 7% (SD). Similarly, the H₂ content of gas bubbles was relatively constant at 93.3 ± 9.6 ppmv when randomly sampled in the rice field at different times of the day. A small contribution (6%) of H₂/CO₂ to acetate production was also observed. Vertical profiles of the respiratory index (RI) for [2-¹⁴C] acetate showed that acetate was predominantly degraded by methanogenesis in 5–11 cm depth, but by respiration in the surface soil (3 cm depth) and in soil layers below 13–16 cm depth which coincided with a transition of the colour (grey to reddish) and the physical characteristics (porosity, density) of the soil. The observations indicate that the microbial community which degrades organic matter to CH₄ is in itself relatively homogenous, but operates at highly variable rates within the soil structure. Author for correspondence     

Regulation of the transmodulated epidermal growth factor receptor by cholera toxin and the protein phosphatase inhibitor okadaic acid.

Addition of tumor promoting phorbol esters, such as phorbol 12-myristate 13-acetate (PMA), to many cell lines results in a decrease of 125I-epidermal growth factor (EGF) binding and increased serine/threonine phosphorylation of the EGF receptor in a process termed transmodulation. It is, however, unclear whether or not receptor phosphorylation is causally related to the inhibition of high affinity EGF binding. We have investigated the significance of phosphorylation/dephosphorylation events in the mechanism of PMA-induced transmodulation using the adenylate cyclase activator cholera toxin and the serine/threonine protein phosphatase inhibitor okadaic acid. In Rat-1 fibroblasts treated at 37 degrees C, PMA induced a rapid decrease in EGF binding which persisted for 3 hours. In contrast, cells exposed to PMA in the presence of cholera toxin exhibited a marked recovery of binding within 60 minutes. The PMA-stimulated decrease in binding correlated with a rapid increase in the phosphorylation state of the EGF receptor. While phosphorylation of the receptor was sustained at an elevated level for at least three hours in cells receiving PMA alone, EGF receptor phosphorylation decreased between 1 and 3 hours in cells treated with PMA and cholera toxin. Furthermore, the cholera toxin-stimulated return of EGF binding was inhibited by treatment with the phosphatase inhibitor okadaic acid. These results suggest that a cholera toxin-activated phosphatase can increase binding capacity of the transmodulated EGF receptor in Rat-1 cells. Cholera toxin treatment elicited a qualitatively similar response in cells transmodulated by platelet-derived growth factor (PDGF). Okadaic acid antagonized the natural return of binding observed in cells stimulated with PDGF alone, indicating that a dephosphorylation event may be required for the recovery of normal EGF binding after receptor transmodulation.     

Oxysterol activation of arachidonic acid release and protaglindin E2 biosynthesis in NRK 49F cells is partially dependent on protein kinase activity

We previously demonstrated that the oxysterol potentiation of arachidonic acid release and prostaglandin biosynthesis induced by foetal calf serum activation of normal rat kidney (NRK) cells (fibroblastic clone 49F) was not related to a direct effect of oxysterols on cell free Ca²⁺ level. Since both Ca²⁺ variations and protein C are involved in arachidonic acid release in some models, we looked for a possible modulation by protein C in the oxysterol effect on arachidonic acid release. We show that when the phorbol ester 12-O-tetradecanoyl-phorbol-13acetate (TPA), a protein kinase C activator, was added to the culture medium, the oxyterol effect on arachidonic acid release and prostaglandin synthesis clearly increased. Moreover, the effect of TPA was dose-dependent and TPA EC₅₀ (4 × 10⁻⁹ M) was unchanged in the presence of the oxysterol. Preincubation of cells with TPA for 24 h prevented the arachidonic acid release induced by TPA alone, whereas the oxysterol effect was decreased but not abolished. In the absence of serum, TPA and ionomycin added together induced the same noticeable (arachidonic acid) release and PGE₂ synthesis as serum alone. Nevertheless, the potentiating effect of cholest-5-ene-3β,25-diol was much higher when serum itself was used to activate NRK cells than it was in the present serum-mimicking experimental conditions. Thus, the presence of growth factors is probably required to obtain a full oxysterol effect. We conclude that the oxysterol effect was synergistic with, but not fully dependent on, protein kinase C and Ca²⁺ ion fluxes, therefore oxysterols could affed earlier events triggered by serum growth factor binding to their cell membrane receptors.