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.     

Pollination Ecology and Endemic Trends in Pedicularis bracteosa var. atrosanguinea Pennell & Thompson (Scrophulariaceae) in North America

Abstract Pedicularis bracteosa var. atrosanguinea occurs locally in association with P. racemosa or P. groenlandica in the Olympic Mountains in Washington. Other plant species, e.g., Polygonum bistortoides, Lupinus argenteus var. parviflorus and Valeriana sitchensis compete for space and bumblebee pollinators within the study area. Pollinator sharing, resulting from such competition, may increase the frequency of unvisited flowers of P. bracteosa var. atrosanguinea. P. bracteosa var. atrosanguinea , with blood purple nectariferous flowers, is presumed to lack intense reflections of blue spectral components from its corollas in attracting bumblebees compared to those of P. racemosa and P. groenlandica . Six species of bumblebees ( Bombus ) and cuckoo bees ( Psithyrus ) pollinate P. bracteosa var. atrosanguinea . Of these, Bombus mixtus and B. occidentalis occur in higher frequencies and are the major pollinators of P. bracteosa var. atrosanguinea . Queen and larger worker bumblebees pollinate nototribically as they probe for nectar, while smaller worker bumblebees pollinate sternotribically while scraping pollen from anthers deeply hidden in the dome-shaped galea. Corbicular pollen loads of bumblebees collected in the study area contain Pedicularis pollen alone/in combination of Polygonum, Valeriana, Lupinus, Erigeron and Bidens , or exclusively of Polygonum or Valeriana. P. bracteosa var. atrosanguinea does not suffer seriously from deficient pollination but seedlings resulting from pollinated flowers may be subjected to natural selection pressure exerted by colonial plant species for space. P. bracteosa var. atrosanguinea does not propagate asexually but resumes aerial growth seasonally from the self-same underground root stocks. If seedlings are under continuous selection pressure for lack of space, P. bracteosa var. atrosanguinea is presumed to regenerate primarily by perennial root stocks. This behavior may favor endemism in P. bracteosa var. atrosanguinea .     

Transformed 3T3 cells have reduced levels and altered subcellular distribution of the major PKC substrate protein marcks

The MARCKS (myristylated alanine-rich C-kinase substrate) protein is an abundant calmodulin-binding protein that is a major and specific endogenous substrate of protein kinase C (PKC). Stimulation of cells with phorbol esters or other activators of PKC has been shown previously to result in rapid phosphorylation of MARCKS proteins and redistribution of these myristylated C-kinase substrates from membrane to cytosol. Here we show that NIH3T3 murine fibroblasts transformed by p21-HA-C-RAS or pp60-V-SRC oncoproteins have markedly reduced levels of p68-MARCKS and that most of the remaining MARCKS protein is found in the cytosol. 3T3 cells containing a nontransforming oncoprotein p26-BCL2, in contrast, exhibited normal levels and distribution of p68-MARCKS. When taken together with recent evidence that MARCKS proteins are involved in regulating organization of the membrane cytoskeleton, our findings suggest that oncoprotein-mediated alterations in MARCKS protein levels and subcellular distribution may contribute to the development or maintenance of the transformed phenotpe.     

Reductive cleavage and regeneration of the disulfide bonds inStreptomyces subtilisin inhibitor (SSI) as studied by the carbonyl13C NMR resonances of cysteinyl residues

Summary Four enhanced carbonyl carbon resonances were observed whenStreptomyces subtilisin inhibitor (SSI) was labeled by incorporating specifically labeled [1-¹³C]Cys. The¹³C signals were assigned by the¹⁵N,¹³C double-labeling method along with site-specific mutagenesis. Changes in the spectrum of the labeled protein ([C]SSI) were induced by reducing the disulfide bonds with various amounts of dithiothreitol (DTT). The results indicate that, in the absence of denaturant, the Cys⁷¹-Cys¹⁰¹ disulfide bond of each SSI subunit can be reduced selectively. This disulfide bond, which is in the vicinity of the reactive site scissile bond Met⁷³-Val⁷⁴, is more accessible to solvent than the other disulfide bond. Cys³⁵-Cys⁵⁰, which is embedded in the interior of SSI. This half-reduced SSI had 65% of the inhibitory activity of native SSI and maintained a conformation similar to that of the fully oxidized SSI. Reoxidation of the half reduced-folded SSI by air regenerates fully active SSI which is indistinguishable with intact SSI by NMR. In the presence of 3 M guanidine hydrochloride (GuHCl), however, both disulfide bonds of each SSI subunit were readily reduced by DTT. The fully reduced-unfolded SSI spontaneously refolded into a native-like structure (fully reduced-folded state), as evidenced by the Cys carbonyl carbon chemical shifts, upon removing GuHCl and DTT from the reaction mixture. The time course of disulfide bond regeneration from this state by air oxidation was monitored by following the NMR spectral changes and the results indicated that the disulfide bond between Cys⁷¹ and Cys¹⁰¹ regenerates at a much faster rate than that between Cys³⁵ and Cys⁵⁰.Nomenclature of the various states of SSI that are observed in the present study Fully oxidized-folded native or intact (without GuHCl or DTT) - half reduced-folded (Cys⁷¹-Cys¹⁰¹ reduced; DTT without GuHCl) - inversely half reduced-folded (Cys³⁵-Cys⁵⁰ reduced; a reoxidation intermediate from fully reduced-folded state) - fully reduced-unfolded (reduced by DTT in the presence of GuHCl) - fully reduced-folded (an intermediate state obtained by removing DTT and GuHCl from the fully reduced-unfolded SSI reaction mixture)     

The effect of vitamin E analogues and long hydrocarbon chain compounds on calcium-induced muscle damage. A novel role for α-tocopherol?

Previous studies have demonstrated that supplemental α-tocopherol inhibited calcium-induced cytosolic enzyme efflux from normal rat skeletal muscles incubated in vitro and suggested that the protective action was mediated by the phytyl chain of α-tocopherol [1]. In order to investigate this further a number of hydrocarbon chain analogues of tocopherol (7.8-dimethyl tocol, 5,7-dimethyl tocol, tocol, α-tocotrienol, α-tocopherol [10], vitamin K1, vitamin K1 [10], vitamin K1 diacetate, vitamin K2 [20], phytyl ubiquinone and retinol) were tested for any ability to inhibit calcium ionophore, A23187, induced creatine kinase (CK) enzyme efflux. Some compounds were found to be very effective inhibitors and comparison of their structures and ability and to inhibit TBARS production in muscle homogenates revealed that the effects did not appear related to antioxidant capacity or chromanol methyl groups, but rather the length and structure of the hydrocarbon chain was the important mediator of the effects seen.     

Restoration of vigor and rooting competence in stem tissues of mature citrus by repeated grafting of their shoot apices onto freshly germinated seedlings in vitro

Summary Repeated grafting of 0.2-cm shoot tips from fruiting-age trees ofCitrus reticulata Blanco ‘Ponkan’ mandarin andC. sinensis Osbeck ‘Liu Tseng’ sweet orange onto freshly germinated ‘Troyer’ citrange [Poncirus trifoliata (L.) Raf. X.C. sinensis Osbeck] seedlings in vitro resulted in progressive restoration of rooting competence and vigor of regenerated roots and shoots. The restored traits were retained through the course of the investigation and suggested a phase reversal phenomenon.     

Effects of diphenyl ether herbicides on porphyrin accumulation by cultured hepatocytes

Several diphenyl ether herbicides, such as acifluorfen methyl, have been previously shown to cause large accumulations of the heme and chlorophyll precursor, protoporphyrin, in plants. Lightinduced herbicidal damage is mediated by the photoactive porphyrin. Here we investigate whether diphenyl ether herbicides can affect porphyrin synthesis in rat and chick hepatocytes. In rat hepatocyte cultures, protoporphyrin, as well as coproporphyrin, accumulated after treatment with acifluorfen or acifluorfen methyl. Combination of acifluorfen methyl with an esterase inhibitor to prevent the conversion of acifluorfen methyl to acifluorfen resulted in a greater accumulation of porphyrins than caused by acifluorfen methyl or acifluorfen alone. In vitro enzyme studies of hepatic mitochondria isolated from rat and chick embryos demonstrated that protopor-phyrinogen oxidase, the penultimate enzyme of heme biosynthesis, was inhibited by low concentrations of acifluorfen, nitrofen, or acifluorfen methyl with the latter being the most potent inhibitor. These findings indicate that diphenyl ether treatment can cause protoporphyrin accumulation in rat hepatocyte cultures and suggest that this accumulation was associated with the inhibition of protoporphyrinogen oxidase. In cultured chick embryo hepatocytes, treatment with acifluorfen methyl plus an esterase inhibitor caused massive accumulation of uroporphyrin rather than protoporphyrin or coproporphyrin. Specific isozymes of cytochrome P450 were also induced in chick embryo hepatocytes. These effects were not observed in the absence of an esterase inhibitor. These results suggest that diphenyl ether herbicides can cause uroporphyrin accumulation similar to that induced by other cytochrome P450-inducing chemicals such as polyhalogenated aromatic hydrocarbons in the chick hepatocyte system.     

Influence of single and concurrent clofibrate and phenobarbital administration on cytochrome P450-dependent mixed function oxidase activities and peroxisome proliferation in male rat liver

The influence of both single and concurrent administration of phenobarbital and clofibrate on hepatomegaly, cytochrome P450-depen-dent mixed function oxidase activities, and peroxisome proliferation in male rat liver have been studied. Both xenobiotics separately increase the liver :body weight ratio and their combined administration results in greater hepatomegaly than either compound alone. Both compounds induce NADPH-cytochrome c(P450) reductase activity and laurate ω- and ω-1-hydroxylase activities, but only phenobarbital induces pentoxyresorufin-O-de-alkylase. None of the drug treatments induced microsomal cytochrome b5. Phenobarbital did not cause peroxisome proliferation and inhibited the corresponding clofibrate-dependent proliferation. Taken collectively, our studies have demonstrated that concomitant treatment with phenobarbital and clofibrate are largely permissive with respect to the hepatic mixed function oxidase system but have opposing effects on the phenomenon of peroxisome proliferation in the same tissue.     

Variation in tissue element concentrations in Quercus ilex L. over a range of different soils

In order to study the variability in nutrient concentrations in four tissues of Q. ilex in relation to soil properties, we selected fifteen stands in both Quercus ilex forests and Q. ilex-Pinus halepensis mixed forests. These stands had developed on soils derived from eight different parent materials. Three soil groups were differentiated according to their chemical properties: calcareous soils, siliceous soils, and volcanic soils. Across sites, nutrient concentrations were generally less variable in current-year tissues than in older tissues. Nitrogen and potassium showed the lowest variability among sites, their concentrations in current-year leaves ranging from 1.17% to 1.39% for N and from 0.53% to 0.68% for K. There were few statistically significant correlations between tissue element concentrations, the most frequent being the antagonistic relationship between calcium and magnesium. Nitrogen concentration in current-year leaves was negatively correlated with soil chemical fertility (nitrogen, phosphorus and potassium). This may reflect a nutritional imbalance between nitrogen and other nutrients, some of which may be more limiting than nitrogen to Q. ilex growth in Catalonia forests. Negative correlations were also found between plant magnesium and soil calcium, and positive correlations between plant calcium and soil calcium.