Arachidonic acid stimulates DNA synthesis in brown preadipocytes through the activation of protein kinase C and MAPK

Arachidonic acid (AA) is a polyunsaturated fatty acid that stimulates the proliferation of many cellular types. We studied the mitogenic potential of AA in rat brown preadipocytes in culture and the signaling pathways involved. AA is a potent mitogen which induces 4-fold DNA synthesis in brown preadipocytes. The AA mitogenic effect increases by NE addition. AA also increases the mitogenic action of different growth factor combinations. Other unsaturated and saturated fatty acids do not stimulate DNA synthesis to the same extent as AA. We analyzed the role of PKC and MEK/MAPK signaling pathways. PKC inhibition by bisindolilmaleimide I (BIS) abolishes AA and phorbol ester stimulation of DNA synthesis and reduces the mitogenic activity of different growth factors in brown preadipocytes. Brown preadipocytes in culture express PKC α, δ, ε and ζ isoforms. Pretreatment with high doses of the phorbol ester PDBu, induces downregulation of PKCs ε and δ and reproduces the effect of BIS indicating that AA-dependent induction of DNA synthesis requires PKC activity. AA also activates MEK/MAPK pathway and the inhibition of MEK activity inhibits AA stimulation of DNA synthesis and brown adipocyte proliferation. Inhibition of PKC δ by rottlerin abolishes AA-dependent stimulation of DNA synthesis and MAPK activation, whereas PKC ε inhibition does not produce any effect. In conclusion, our results identify AA as a potent mitogen for brown adipocytes and demonstrate the involvement of the PDBu-sensitive PKC δ isoform and MEK/MAPK pathway in AA-induced proliferation of brown adipocytes. Increased proliferative activity might increase the thermogenic capacity of brown fat.     

Oxidative activity of human polymorphonuclear leukocytes stimulated by the long-chain phosphatidic acids

It has already been suggested that phosphatidic acids (PAs) play an important role in the regulation of signaling pathways involved in the production of reactive oxygen species (ROS) by human polymorphonuclear leukocytes (PMNs). The present study was performed to elucidate the effects of extracellularly added PA-- 1,2-distearoyl- (DSPA) and 1-stearoyl-2-arachidonoyl-sn-glycero-phosphate (SAPA)--on the ROS production and on the elastase release by human PMNs. ROS production was monitored by luminol-amplified chemiluminescence and the elastase activity was measured in the supernatant of the PA-stimulated human PMNs by colorimetric assay. Obtained effects were compared with those of cells stimulated by either a chemotactic tripeptide, phorbol ester or calcium ionophore. Our results show that long-chain PAs at concentrations higher than 3 x 10(-5) mol/l stimulate the ROS production by human PMNs, whereas they were ineffective in promoting the elastase release. The chemiluminescence pattern of the SAPA-stimulated cells exhibited a biphasic curve, whereas cell stimulation with DSPA resulted in a monophasic chemiluminescence curve. Stimulation of the ROS production by PAs in dependence of the fatty acid composition required the activity of protein kinases.     

Phosphatidic acid may stimulate membrane receptors mediating adenylate cyclase inhibition and phospholipid breakdown in 3T3 fibroblasts

Incubation of 3T3 fibroblasts with phosphatidic acid (PA) from egg lecithin or with thrombin resulted in decreases in cellular cAMP due to inhibition of adenylate cyclase, in rapid increases in inositol 1,4,5-tris-,1,4-bis-, and 1-monophosphates probably due to activation of phospholipase C, and in arachidonic acid release. Synthetic PAs consisting of unsaturated fatty acid diesters were as effective as PA from egg lecithin, whereas PAs with saturated fatty acids were only slightly effective and antagonized the effect of active PAs selectively, despite the fact that both types of PA analogues (sodium salts) were apparently dissolved in the incubation medium. PA-induced decreases in cAMP were not affected by omission of Ca2+ from incubation medium but were abolished by prior exposure of cells to islet-activating protein (pertussis toxin). This islet-activating protein treatment of cells was without effect on PA- or thrombin-induced generation of inositol phosphates. Thus, PA-induced inhibition of adenylate cyclase was (but activation of phospholipase C was not) mediated by an islet-activating protein substrate GTP-binding protein. Homologous desensitization was observed with thrombin-, bradykinin-, and PA-induced decreases in cAMP in 3T3 cells; prior exposure of the cells to any one of these agents abolished or greatly diminished the subsequent response to the same agent but did not affect the responses to others. The effects of PA were cell-specific; it failed to decrease cAMP in rabbit platelets in which labeled PA rapidly increasing in response to thrombin or A23187 was mostly outside the cells. Based on these results, it is proposed that PA interacts with its own specific membrane receptors, thereby triggering multiple effector systems in 3T3 cells.     

Structural effects and lipid membrane interactions of the pH-responsive GALA peptide with fatty acid acylation

GALA is a pH-responsive, membrane-perturbing peptide designed to fold from a random coil at physiological pH to an amphipathic α-helix under mildly acidic conditions. Because of its pH-activated function, GALA has been sought-after as a component of intracellular drug delivery systems that could actively propel endosomal escape. In this study, we conjugated GALA with lauryl and palmitoyl fatty acid tails as model hydrophobic moieties and examined the physicochemical characteristics and activities of the resulting peptide amphiphiles (PAs). The fatty acid variants of GALA exhibited distinctly different membrane perturbing mechanisms at pH 7.5 and 5.5. At physiological pH, the PAs ruptured liposomes through a surfactant-like mechanism. At pH 5.5, lauryl-GALA was shown to form transmembrane pores with a higher potency as compared to its unmodified peptide counterpart; however, after prolonged exposure it also caused liposome lysis. The lytic activity of fatty acid-conjugated GALA did not impair cell viability. Lauryl-GALA was tolerated well by SJSA-1 osteocarcinoma cells and enhanced cell internalization of the PA was observed. Our findings are discussed with the overarching goal of developing efficient therapeutic delivery systems.     

Polyunsaturated fatty acids inhibit mouse hepatic glucocorticoid receptor activation in vitro

The effect of saturated fatty acids (SFAs) stearic and palmitic acids and polyunsaturated fatty acids (PUFAs) oleic, linoleic and arachidonic acids was studied on in vitro heat activation of mouse hepatic glucocorticoid receptor (GR) complex, as assessed by binding to DNA-cellulose and purified nuclei. Significant dose-dependent inhibition of heat activation of hormone-receptor complex by the PUFAs was observed. Linoleic and arachidonic acids were found to be more potent (caused approximately 70% inhibition maximally at 160 microM) inhibitors of GR heat activation, compared to oleic acid (approximately 38% inhibition at 40 microM). However, stearic and palmitic acids were unable to modulate GR heat activation, suggesting that the unsaturated moieties in PUFAs are possibly the important determinants of receptor activation. Thus, our study shows an inhibitory effect of PUFAs on in vitro hepatic GR activation.     

Accumulation of C20 and C22 unsaturated fatty acids in triacylglycerols from developing seeds of Limnanthes alba

In Limnanthes alba seeds, an accumulation of phosphatidic acid (PA) occurred during seed development until the day 15 after pollination (DAP). Between the 15 and the 20 DAP, a rapid synthesis of triacylglycerols (TG) took place with a simultaneous decrease of PA. During the same period, very long-chain fatty acids (VLCFA), viz. 20: 1 Δ⁵, 22:1 Δ¹³ and 22:2Δ^5,13 were synthesized and rapidly channelled into TG. These results suggest the involvement of glycerol-3-phosphate in TG biosynthesis. Moreover, TG synthesis appears to be a time-compartmentalized event; the first accumulation of PA having short-chain acyl moieties then, from the 15 DAP, synthesis of VLCFA which are esterified in TG originating from the PA accumulated during the first period.     

Stimulation of calcium uptake in rat calvaria by prostacyclin

Treatment of newborn rat calvaria discs with a variety of unsaturated fatty acids led to a 50% enhancement of calcium uptake. Arachidonic acid was effective at lower concentrations than cis-vaccenic or oleic acid, while trans-vaccenic acid and saturated fatty acids did not enhance calcium uptake. Cyclooxygenase inhibitors indomethacin and acetylsalicylic acid abolished the enhancement of calcium uptake seen in response to cis-vaccenic acid and inhibited calcium uptake by otherwise untreated bones. Prostacyclin was found to produce up to 2 fold stimulation of calcium uptake with an EC50 of approximately 0.1 microM. No statistically significant stimulation of calcium uptake was seen in response to PGE2 or PGE1 alpha up to 25 microM, while slight stimulation was produced by 6-keto PGE1 alpha but only at concentrations of 10 microM. Prostacyclin production by calvaria was demonstrated and was stimulated over 50% by cis-vaccenic acid. These results suggest that not only is enhanced prostacyclin production responsible for elevation of calcium uptake in response to unsaturated fatty acids, but also that prostacyclin may be an important regulator of bone calcium homeostasis.     

The plasminogen activator family from the salivary gland of the vampire bat Desmodus rotundus: cloning and expression

Complementary DNAs coding for four Desmodus rotundus salivary plasminogen activators (DSPAs) were isolated and characterized. The predicted amino acid sequences display structural features also found in tissue-type plasminogen activator. The largest forms (DSPA alpha 1 and -alpha 2) contain a signal peptide, a finger (F), an epidermal growth factor (EGF), a kringle, and a serine protease domain, whereas DSPA beta and -gamma lack the F and F-EGF domains, respectively. Additional differences between the four forms suggest that distinct genes code for the members of the DSPA family. Transfection of DSPA-encoding cDNAs, placed under the control of the simian virus 40 late promoter, into COS-1 cells resulted in the secretion of highly fibrin-dependent PAs.     

Regulation of fatty acid biosynthesis as a possible mechanism for the mitoinhibitory effect of fumonisin B1 in primary rat hepatocytes

The mitoinhibitory effect of fumonisin B1 (FB1) on the mitogenic response of epidermal growth factor (EGF) was investigated in primary hepatocyte cultures with respect to the alterations in the omega6 fatty acid metabolic pathway. Fatty acid analyses of hepatocytes showed that EGF treatment resulted in a significant decrease in the relative levels of 20:4omega6 (arachidonic acid) and an increase in 18:2omega6 (linoleic acid). Supplementation of the hepatocyte cultures with 20:4omega6 in the absence of EGF resulted in an increase in the total omega6 and omega6/omega3 fatty acid ratio. Addition of 20:5omega3 (eicosapentaenoic acid) resulted in an increase of the relative levels of the long chain omega3 fatty acids at the expense of the omega6 fatty acids. When 20:4omega6 and 20:5omega3 was added in the presence of EGF, the mitogenic response of EGF was increased and decreased respectively. When compared to the fatty acid profiles in the absence of EGF, the decreased mitogenic response coincided with a decrease of total omega6 fatty acids and total polyunsaturated fatty acids (PUFA). In addition, the saturated and mono-unsaturated fatty acids increased and the polyunsaturated/saturated (P/S) fatty acid ratio decreased which implied a more rigid membrane structure. Addition of prostaglandin E2 (PGE2) and prostaglandin E1 (PGE1) stimulated and inhibited the mitogenic response respectively. Ibuprofen, a known cyclooxygenase inhibitor, and FB1 inhibited the EGF-induced mitogenic response in a dose-dependent manner. The mitoinhibitory effect of FB1 on the EGF response was counteracted by the addition of PGE2. FB1 also disrupts the omega6 fatty acid metabolic pathway in primary hepatocytes, resulting in the accumulation of C18:2omega6 in phospatidylcholine and triacylglicerol. The disruption of the omega6 fatty acid metabolic pathway and/or prostaglandin synthesis is likely to be an important event in the mitoinhibitory effect of FB1 on growth factor responses.     

The inhibitory effect of polyunsaturated fatty acids on human CYP enzymes

The inhibitory effect of saturated fatty acids (SFAs): palmitic acid (PA), stearic acid (SA) and polyunsaturated fatty acids (PUFAs): linoleic acid (LA), linolenic acid (LN), arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on six human drug-metabolizing enzymes (CYP1A2, 2C9, 2C19, 2D6, 2E1 and 3A4) was studied. Supersomes from baculovirus-expressing single isoforms were used as the enzyme source. Phenacetin O-deethylation (CYP1A2), diclofenac 4-hydroxylation (CYP2C9), mephenytoin 4-hydroxylation (CYP2C19), dextromethorphan O-demethylation (CYP2D6), chlorzoxazone 6-hydroxylation (CYP2E1) and midazolam 1-hydroxylation (CYP3A4) were used as the probes. Results show that all the five examined PUFAs competitively inhibited CYP2C9- and CYP2C19-catalyzed metabolic reactions, with Ki values ranging from 1.7 to 4.7 microM and 2.3 to 7.4 microM, respectively. Among these, AA, EPA and DHA tended to have greater inhibitory potencies (lower IC(50) and Ki values) than LA and LN. In addition, these five PUFAs also competitively inhibited the metabolic reactions catalyzed by CYP1A2, 2E1 and 3A4 to a lesser extent (Ki values>10 microM). On the other hand, palmitic and stearic acids, the saturated fatty acids, had no inhibitory effect on the activities of six human CYP isozymes at concentrations up to 200 microM. Incubation of PUFAs with CYP2C9 or CYP2C19 in the presence of NADPH resulted in the decrease of PUFA concentrations in the incubation mixtures. These results indicate that the PUFAs are potent inhibitors as well as the substrates of CYP2C9 and CYP2C19.     

Inhibition of brain prostaglandin D synthetase and prostaglandin D2 dehydrogenase by some saturated and unsaturated fatty acids

The activities of rat brain prostaglandin D synthetase and swine brain prostaglandin D2 dehydrogenase were inhibited by some saturated and unsaturated fatty acids. Myristic acid was most potent among saturated straight-chain fatty acids so far tested. The IC50 values of this acid were 80 microM for prostaglandin D synthetase and 7 microM for prostaglandin D2 dehydrogenase, respectively. Little inhibition was found with methyl myristate and myristyl alcohol. The IC50 values of these derivatives were more than 200 microM for both enzymes, suggesting that the free carboxyl group was essential for the inhibition. The effects of cis double bond structure of fatty acids on the inhibition potency were examined by the use of the carbon 18 and 20 fatty acids. The inhibition potencies for both enzymes increased with the number of cis double bonds; the IC50 values of stearic, oleic, linoleic and linolenic acid were, respectively, more than 200, 60, 30 and 30 microM for prostaglandin D synthetase, and 20, 10, 8.5 and 7 microM for prostaglandin D2 dehydrogenase. Arachidonic acid also inhibited the activities of both enzymes with respective IC50 values of 40 microM for prostaglandin D synthetase and 3.9 microM for prostaglandin D2 dehydrogenase, while arachidic acid showed little inhibition. The kinetic studies with myristic acid and arachidonic acid demonstrated that the inhibition by these fatty acids was competitive and reversible for both enzymes. Myristic acid and other fatty acids also inhibited the activities of several enzymes in prostaglandin metabolism, although to a lesser extent. The IC50 values of myristic acid for prostaglandin E isomerase, thromboxane synthetase and NAD-linked prostaglandin dehydrogenase (type I) were 200, 700 and 100 microM, respectively. However, this fatty acid showed little inhibition on fatty acid cyclooxygenase (20% at 800 microM), glutathione-requiring prostaglandin D synthetase from rat spleen (20% at 800 microM), and NADP-linked prostaglandin dehydrogenase (type II) (no inhibition at 200 microM).     

Contribution of phospholipase D in endothelin-1-mediated extracellular signal-regulated kinase activation and proliferation in rat uterine leiomyoma cells

Endothelin (ET)-1 is a mitogenic factor in numerous cell types, including rat myometrial cells. In the present study, we investigated the potential role of ET-1 in the proliferation of tumoral uterine smooth muscle cells (ELT-3 cells). We found that ET-1 exerted a more potent mitogenic effect in ELT-3 cells than in normal myometrial cells, as indicated by the increase in [3H]thymidine incorporation, cell number, and bromodeoxyuridine incorporation. The ET-1 was more efficient than platelet-derived growth factor and epidermal growth factor to stimulate proliferation. The ET-1-mediated cell proliferation was inhibited in the presence of U0126, a specific inhibitor of (mitogen-activated protein kinase ERK kinase), indicating that extracellular signal-regulated kinase (ERK) activation is involved. Additionally, ET-1 induced the activation of phospholipase (PL) D, leading to the synthesis of phosphatidic acid (PA). The ET-1-induced activation of PLD was twofold higher in ELT-3 cells compared to that in normal cells. The two cell types expressed mRNA for PLD1a and PLD2, whereas PLD1b was expressed only in ELT-3 cells. The exposure of cells to butan-1-ol reduced ET-1-mediated production of PA by PLD and partially inhibited ERK activation and DNA synthesis. Addition of exogenous PLD or PA in the medium reproduced the effect of ET-1 on ERK activation and cell proliferation. Collectively, these data indicate that ET-1 is a potent mitogenic factor in ELT-3 cells via a signaling pathway involving a PLD-dependent activation of ERK. This highlights the potential role of ET-1 in the development of uterine leiomyoma, and it reinforces the role of PLD in tumor growth.     

A transient rise in cytosolic calcium follows stimulation of quiescent cells with growth factors and is inhibitable with phorbol myristate acetate

We have used aequorin as an indicator for the intracellular free calcium ion concentration [( Ca++]i) of Swiss 3T3 fibroblasts. Estimated [Ca++]i of serum-deprived, subconfluent fibroblasts was 89 (+/-20) nM, almost twofold higher than that of subconfluent cells growing in serum, whose [Ca++]i was 50 (+/-19) nM. Serum, partially purified platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF) stimulated DNA synthesis by the serum-deprived cells, whereas epidermal growth factor (EGF) did not. Serum immediately and transiently elevated the [Ca++]i of serum-deprived cells, which reached a maximal value of 5.3 microM at 18 s poststimulation but returned to near prestimulatory levels within 3 min. Moreover, no further changes in [Ca++]i were observed during 12 subsequent h of continuous recording. PDGF produced a peak rise in [Ca++]i to approximately 1.4 microM at 115 s after stimulation, and FGF to approximately 1.2 microM at 135 s after stimulation. EGF caused no change in [Ca++]i. The primary source of calcium for these transients was intracellular, since the magnitude of the serum-induced rise in [Ca++]i was reduced by only 30% in the absence of exogenous calcium. Phorbol 12-myristate 13-acetate (PMA) had no effect on resting [Ca++]i. When, however, quiescent cells were treated for 30 min with 100 nM PMA, serum-induced rises in [Ca++]i were reduced by sevenfold. PMA did not inhibit growth factor-induced DNA synthesis and was by itself partially mitogenic. We suggest that if calcium is involved as a cytoplasmic signal for mitogenic activation of quiescent fibroblasts, its action is early, transient, and can be partially substituted for by PMA. Activated protein kinase C may regulate growth factor-induced increases in [Ca++]i.     

Frequent gain and loss of pyrrolizidine alkaloids in the evolution of Senecio section Jacobaea (Asteraceae)

Pyrrolizidine alkaloids (PAs) of the macrocyclic senecionine type are secondary metabolites characteristic for most species of the genus Senecio (Asteraceae). These PAs are deterrent and toxic to most vertebrates and insects and provide plants with a chemical defense against herbivores. We studied the PA composition of 24 out of 26 species of Senecio section Jacobaea using GC-MS. The PA profiles of eight of these species have not been studied before and additional PAs were identified for most other species that were included in previous studies. With one exception (senecivernine) all 26 PAs identified in sect. Jacobaea can be regarded as derivatives of the biosynthetic backbone structure senecionine. Based on the PA profiles of the species of sect. Jacobaea and the results of previous tracer studies, we constructed two hypothetical biosynthetic scenarios of senecionine diversification. Both scenarios contain two major reactions: the conversion of the necine base moiety retronecine into the otonecine moiety and site-specific epoxidations within the necic acid moiety. Further reactions are site-specific hydroxylations, sometimes followed by O-acetylations, site-specific dehydrogenations, E, Z-isomerizations, and epoxide hydrolysis and chlorolysis. The GC-MS data and both biosynthetic scenarios were subsequently used to study the evolution of PA formation in sect. Jacobaea by reconstructing the evolutionary history of qualitative PA variation in this section. This was achieved by optimizing additive presence/absence data of PAs and types of enzymatic conversions on a maximum parsimony cladogram of section Jacobaea inferred from DNA sequence and morphological data. Besides showing large intra- and interspecific variation, PA distribution appears to be largely incidental within the whole clade. These results together with the finding that all but one of the PAs identified in sect. Jacobaea are also present in species of other sections of Senecio indicate that differences in PA profiles in Senecio can not be explained by the gain and loss of PA specific genes, but rather by a transient switch-off and switch-on of the expression of genes encoding PA pathway-specific enzymes.     

Investigation of the ability of several naturally occurring and synthetic polyanions to bind to and potentiate the biological activity of acidic fibroblast growth factor

The ability of several animal, plant, and bacterial derived polyanions (PAs) as well as synthetic PAs to compete with heparin for the binding of acidic fibroblast growth factor (aFGF) was correlated with their ability to potentiate the mitogenic and neurotrophic actions of this factor. Dextran sulphate, K-carrageenan, pentosan sulphate, polyanethole sulfonate, heparin, and fucoidin competed for the heparin binding site on aFGF at relatively low concentrations (≤50 μg/ml). λ-carrageenan, ι-carrageenan, and polyvinyl sulphate exhibited lower affinity for aFGF, whereas hyaluronic acid, dermatan sulphate, chondroitin-6-sulphate, chondroitin-4-sulphate, and uncharged dextran displayed very low or no demonstrable affinity. Potentiation of the mitogenic action of aFGF for Balb/c 3T3 fibroblasts tended to be in general agreement with the aFGF binding affinity of the PAs. However, polyanethole sulfonate, the carrageenans, polyvinyl sulphate, fucoidin, and pentosan sulphate exerted a mitogenic action on the 3T3 cells that was independent of, and in addition to, the ability of these GAGs to potentiate the action of aFGF. The ability to potentiate the neurotrophic action of aFGF for E8 chick ciliary neurons was a general property of those PA with low or no activity in the mitogen assay. Thus hyaluronic acid, dermatan sulphate, chondroitin-4-sulphate, chondroitin-6-sulphate, and even uncharged dextran all potentiated aFGF induced neuronal survival. The differential effects of these PA in potentiating the biological activities of aFGF are discussed in relation to their ability to compete for the heparin-binding site of aFGF. © 1993 Wiley-Liss, Inc.     

Role of polyamines in regulating silymarin production in Silybum marianum (L.) Gaertn (Asteraceae) cell cultures under conditions of calcium deficiency

As part of our efforts to identify the possible role of polyamines (PAs) in silymarin (Sm) production, the effects of calcium deprivation on cell growth and on endogenous PAs levels and Sm production by milk thistle (Silybum marianum (L.) Gaertn) grown in cell cultures were examined. Young cultured cells of the H2 line of S. marianum were transferred to a medium without calcium and with ethylene glycol-bis-(β-aminoethyl) ether-N,N,N′,N′-tetraacetic acid present to chelate any free calcium in order to analyze the effects of this medium on the levels of PAs and Sm produced by the cells. During the 17 days of exposure to this calcium-free medium most of the cell populations were in the G0/G1 phase (from day 7 to day 14 of culture) while PA levels underwent a progressive decline up to day 17, after which they were no longer detectable. We observed that putrescine (Put) accumulation was always lower than that observed under normal conditions. The lack of calcium in the MS medium advances the onset of the stationary phase, whose beginning is marked by an increase in the Put/spermidine (Spd) index, raising the production of Sm; the suspensions were productive for a longer time and hence produced more of the substance. Our results indicate that under stress conditions the production of Sm in young-cell suspensions of S. marianum is not associated with high levels of PAs in the medium – contrary to what one would expect – allowing us to conclude that growth inhibition appears to be the factor responsible for the maximum Sm accumulation while PAs are not directly involved in the Sm synthesis pathway by milk thistle grown in culture.     

Epigenetic and physiological effects of gibberellin inhibitors and chemical pruners on the floral transition of azalea

The ability to control the timing of flowering is a key strategy in planning the production of ornamental species such as azaleas; however, it requires a thorough understanding of floral transition. DNA methylation is involved in controlling the functional state of chromatin and gene expression during floral induction pathways in response to environmental and developmental signals. Plant hormone signalling is also known to regulate suites of morphogenic processes in plants and its role in flowering-time control is starting to emerge as a key controlling step. This work investigates if the gibberellin (GA) inhibitors and chemical pinching applied in improvement of azalea flowering alter the dynamics of DNA methylation or the levels of polyamines (PAs), GAs and cytokinins (CKs) during floral transition, and whether these changes could be related to the effects observed on flowering ability. DNA methylation during floral transition and endogenous content of PAs, GAs and CKs were analysed after the application of GA synthesis inhibitors (daminozide, paclobutrazol and chlormequat chloride) and a chemical pruner (fatty acids). The application of GA biosynthesis inhibitors caused alterations in levels of PAs, GAs and CKs and in global DNA methylation levels during floral transition; also, these changes in plant growth regulators and DNA methylation were correlated with flower development. DNA methylation, PA, GA and CK levels can be used as predictive markers of plant floral capacity in azalea.     

Polyunsaturated fatty acids stimulate phosphatidylcholine synthesis in PC12 cells

The synthesis of phospholipids in mammalian cells is regulated by the availability of three critical precursor pools: those of choline, cytidine triphosphate and diacylglycerol. Diacylglycerols containing polyunsaturated fatty acids (PUFAs) apparently are preferentially utilized for phosphatide synthesis. PUFAs are known to play an important role in the development and function of mammalian brains. We therefore studied the effects of unsaturated, monounsaturated and polyunsaturated fatty acids on the overall rates of phospholipid biosynthesis in PC12 rat pheochromocytoma cells. Docosahexaenoic acid (DHA, 22:6n-3), eicosapentaenoic acid (EPA, 20:5n-3) and arachidonic acid (AA, 20:4n-6) all significantly stimulated the incorporation of (14)C-choline into total cellular phospholipids. In contrast, monounsaturated oleic acid (OA) and the saturated palmitic (PA) and stearic (SA) acids did not have this effect. The action of DHA was concentration-dependent between 5 and 50 microM; it became statistically significant by 3 h after DHA treatment and then increased over the ensuing 3 h. DHA was preferentially incorporated into phosphatidylethanolamine (PE) and phosphatidylserine (PS), while AA predominated in phosphatidylcholine (PC).     

Suppressive effect of endogenous regucalcin on deoxyribonuclic acid synthesis in the nuclei of rat renal cortex

The effect of regucalcin, a regulatory protein of Ca2+ signaling, on deoxyribonucleic acid (DNA) synthesis activity in the nuclei isolated from rat renal cortex was investigated. The addition of calcium chloride (10-100 microM) in the reaction mixture containing the nuclei caused a significant decrease in DNA synthesis activity. Nuclear DNA synthesis activity was significantly raised in the presence of EGTA (1 mM), a chelator of Ca2+, indicating that nuclear Ca2+ has an inhibitory effect. Regucalcin (0.1-0.5 microM) added in the reaction mixture in the presence of either EGTA (1 mM) or calcium chloride (50 microM) had a significant inhibitory effect on nuclear DNA synthesis activity. The presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) in the reaction mixture caused a significant increase in DNA synthesis activity. This increase was completely abolished by the addition of regucalcin (0.5 microM). The effect of anti-regucalcin monoclonal antibody in increasing DNA synthesis was enhanced in the presence of EGTA. Additionally, an inhibitory effect of calcium chloride (10 or 50 microM) was enhanced in the presence of anti-regucalcin monoclonal antibody (25 ng/ml). The present study demonstrates that endogenous regucalcin has a suppressive effect on DNA synthesis in the nuclei of rat renal cortex.