Biomedical subjects

Z Kiss

Publications and source records attributed to Z Kiss.

At least 37 records · Page 2Linked to original sources

PubMed1999

Potentiation of calcium-mediated stimulation of DNA synthesis by ethanol in human and mouse fibroblasts.

Alcohol abuse is a risk factor for cancers of the gastrointestinal tract, and it also can precipitate psoriasis characterized by hyperproliferation of epidermal cells. Because these effects of alcohol may involve stimulation of cell growth, and ethanol (EtOH) was shown to enhance DNA synthesis in mouse fibroblasts and epidermal cells, we conducted a study to determine whether EtOH can also stimulate mitogenesis in human fibroblasts and keratinocytes. In keratinocytes, EtOH had no effects on mitogenesis after shorter (17-hr) treatments, but it partially prevented inhibition of DNA synthesis elicited by longer treatments (3-4 days) with 2 mM calcium (Ca2+), a differentiation-inducing agent. In contrast, treatment of serum-starved zinc-treated (40 microM) human skin fibroblasts with 50-60 mM EtOH for 17 hr resulted in increased DNA synthesis. EtOH-induced DNA synthesis was blocked by 1 mM EGTA, a specific Ca2+ chelator. Despite the presence of 1.8 mM Ca2+ in the cell culture medium, the addition of 1 mM extra Ca2+ (final concentration, 2.8 mM) for 17 hr induced DNA synthesis, presumably mediated by Ca2+ receptors. In eight independent human skin fibroblast lines examined, treatment with EtOH for 46 hr, but not for 17 hr, invariably enhanced the effects of Ca2+ on DNA synthesis, consistent with synergistic stimulation of cell proliferation by EtOH and Ca2+. Neomycin, a Ca2+ receptor agonist, and EtOH also exerted synergistic effects on DNA synthesis after longer (46-hr) treatments. In mouse NIH 3T3 fibroblasts, both EtOH- and Ca2+-enhanced DNA synthesis after 17-hr treatment, but they stimulated cell proliferation only in combination. The results indicate that in human fibroblasts, EtOH can potentiate the longer-term effects of high concentrations of Ca2+ on DNA synthesis whereas, in keratinocytes, EtOH may inhibit Ca2+-induced differentiation.

Animals↗

PubMed1999

Application of an anti-HQIgY antibody for the measurement of IgY concentrations of hen's and quail's serum and yolk.

The development of a sensitive ELISA for the measurement of quail IgY (QIgY) was the main purpose of our study. The suitable antibody (AB) was prepared in rabbits. Both quail IgY (QIgY) and hen IgY (HIgY) were precipitated by this developed AB. For this reason it was marked as anti-hen-quail-IgY (a-HQIgY). The purified AB was conjugated with horseradish peroxidase (aHQIgY-HRP) and a sensitive direct ELISA was developed, based on this labeled AB. The prepared aHQIgY AB which was used in this developed ELISA method was suitable for the measurement of total and specific IgY concentration in domestic hen (Gallus domesticus) and Japanese quail (Coturnix coturnix japonica) either. As a result of our experiments it is very likely that there are identical sequences of IgYs of both species. This part of IgY has good antigen character at the same time. Probably, this phenomenon has occurrence in other Galliform species, too. Further investigations will be carried out in this field.

Animals↗

PubMed1998

Increased synthesis of phosphocholine is required for UV-induced AP-1 activation.

Exposure of mammalian cells to UV irradiation stimulates phosphatidylcholine hydrolysis and activates the transcription factor AP-1. Since phosphocholine (PCho), a phospholipid metabolite, is a potential regulator of mitogenesis and carcinogenesis, we examined the effect of UV exposure on the formation of PCho and the possible mediatory role of PCho in UVB-and UVC-induced activation of AP-1 in mouse JB6 epidermal cells. We found that both UVB and UVC irradiation resulted in increased PCho levels. Hemicholinium-3 (HC-3), an inhibitor of choline kinase, strongly inhibited UV-induced AP-1 activity. By contrast, relatively low levels of PCho (80 microM) or choline (20 microM) nearly doubled UV-induced AP-1 activity, while higher (2-20 mM) concentrations of PCho alone stimulated AP-1 activity 6-8-fold. Importantly, HC-3 inhibited only the stimulatory effect of choline, but not of PCho, on AP-1 activity. Of the mitogen-activated protein (MAP) kinases involved in the regulation of AP-1 activity, UVC stimulated the MAP kinase family ERK-1/ERK-2, JNK as well as p38 kinase activity. These UVC effects were all inhibited by HC-3. With UVB, by contrast, only the activation of ERK-1/ERK-2 was inhibited by HC-3. The data suggest that increased formation of PCho is required for UV-induced activation of AP-1 by an ERK-1/ERK-2-dependent mechanism.

Animals↗

PubMed1998

Phorbol ester stimulation of phosphatidylcholine synthesis requires expression of both protein kinase C-alpha and phospholipase D.

The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) stimulates both the synthesis and phospholipase D (PLD)-mediated hydrolysis of phosphatidylcholine (PtdCho). Here, attached and suspended NIH 3T3 fibroblasts as well as variants of the MCF-7 human breast carcinoma cell line expressing PKC-alpha and a PtdCho-specific PLD activity at widely different levels were used to determine the possible role of PKC-alpha, PtdCho hydrolysis, and choline uptake in the mediation of PMA effect on PtdCho synthesis. In wild-type MCF-7 cells, which express both PKC-alpha and PLD activities at very low levels, PMA had little effects on the uptake or incorporation [14C]choline into PtdCho. In multidrug resistant MCF-7/MDR1 cells, which highly express PKC-alpha but lack the PtdCho-specific PLD activity, 100-nM PMA had relatively small stimulatory effects on the uptake of [14C]choline (approximately 1.5-fold) and [14C]PtdCho synthesis (1.5- to 2-fold). In NIH 3T3 fibroblasts and MCF-7/PKC-alpha cells, both expressing PKC-alpha and PLD activities at high levels, 10-100-nM PMA enhanced [14C]choline uptake only slightly (1.7- to 2.2-fold), while it had much greater (approximately 4-9-fold) stimulatory effects on PtdCho synthesis. PMA significantly enhanced the formation of phosphatidic acid (PtdOH) in MCF-7/PKC-alpha cells (2.8-fold increase), but not in MCF-7/MDR1 cells (1.4-fold increase), while in both cell lines it had only small (1.3-1.5-fold) stimulatory effects on 1,2-diacylglycerol (1, 2-DAG) formation. In suspended NIH 3T3 cells, 200-300-mM ethanol blocked the stimulatory effect of PMA on PtdOH formation without affecting PtdCho synthesis indicating that neither PtdOH nor 1,2-DAG derived from it is a mediator of PMA effect on PtdCho synthesis. In attached NIH 3T3 cells, dimethylbenz[a]anthracene enhanced phosphocholine formation and, thus, choline uptake without increasing PtdCho synthesis or modifying the effect of PMA. While the results indicate that the stimulatory effect of PMA on PtdCho synthesis requires the expression of both PKC-alpha and a PtdCho-specific PLD, they do not support a role for 1,2-DAG, PtdOH or choline in the mediation of PMA effect.

3T3 Cells↗

PubMed1998

Inhibitors of calcineurin block expression of cyclins A and E induced by fibroblast growth factor in Swiss 3T3 fibroblasts.

In Swiss 3T3 fibroblasts, growth factor-stimulated progression from G1 to S phase involves activation of the Ca2+/calmodulin-dependent serine/threonine-specific protein phosphatase 2B (calcineurin). Here we report that both cobalt and the calcium chelator EGTA, inhibitors of calcium uptake, as well as cyclosporin A and FK-506, specific inhibitors of calcineurin function, abolished fibroblast growth factor (FGF)-induced expression of cyclins A and E, but not cyclin D1. At 0.1 microM concentration cyclosporin A completely blocked FGF-induced expression of cyclins E and A and it inhibited FGF-stimulated DNA synthesis by 40%; full inhibition of DNA synthesis required 10 microM cyclosporin A. PD 98059, an inhibitor of mitogen-activated protein (MAP) kinase kinase, and hemicholinium-3, an inhibitor of FGF-induced MAP kinase activity, did not inhibit the stimulatory effect of FGF on the expression of cyclin E. On the other hand, the inhibitory effect of 0.1 microM cyclosporin A on FGF-stimulated DNA synthesis was additive with that of hemicholinium-3, suggesting that the two inhibitors acted by different mechanisms. The inhibitors of calcineurin and calcium uptake also completely blocked the stimulatory effects of lysophosphatidic acid on the expression of cyclins E and A, but not cyclin D1. The results suggest that FGF- or lysophosphatidic acid-induced transcription of cyclin A and cyclin E genes is mediated by calcineurin involving a MAP kinase-independent mechanism and that increased expression of cyclins A and E is required for the maximal stimulatory effects of these mitogens on DNA synthesis.

3T3 Cells↗

PubMed1998

The choline kinase inhibitor hemicholinium-3 can inhibit mitogen-induced DNA synthesis independent of its effect on phosphocholine formation.

In NIH 3T3 cells, phosphocholine (PCho) stimulates mitogenesis in synergism with insulin, ATP, and sphingosine-1-phosphate (S1P) via an extracellular target. Intracellular PCho also has been suggested to mediate the mitogenic effects of fibroblast growth factor (FGF) and several other growth factors based, in part, on the observed inhibition of growth factor-induced mitogenesis by the choline kinase inhibitor hemicholinium-3 (HC-3). Here we examined the specificity of HC-3 effects on mitogenesis in serum-starved NIH 3T3 and Swiss 3T3 cells. In both cell lines, FGF greatly enhanced DNA synthesis in a medium containing 28 microM choline, and it also stimulated the formation of -14C-PCho from both 50 microM and 5 mM [14C]choline. HC-3 (2 mM) inhibited basal or FGF-induced formation of [14C]PCho and [14C]phosphatidylcholine as well as the uptake of -14C-choline only at the 50 microM, but not the 5 mM, concentration of [14C]choline. In addition, HC-3 (1 mM) from three different sources (95-99.9% purity) inhibited FGF-stimulated DNA synthesis by 53-58% which was not reversed by 5 mM choline. The choline analogue dimethylethanolamine (1 mM) also inhibited FGF-stimulated formation of [14C]PCho from 50 microM -14C-choline, but it had no effect on FGF-induced DNA synthesis. Of the other growth regulators examined, synergistic stimulation of DNA synthesis by extracellular PCho and S1P or PCho and ATP via choline kinase-independent mechanisms was inhibited by 2 mM HC-3. However, HC-3 failed to inhibit the synergistic mitogenic effects of PCho and insulin or S1P and insulin. The results suggest that FGF-induced mitogenesis does not require PCho formation and that HC-3 can inhibit DNA synthesis independent of its inhibitory effects on choline metabolism.

3T3 Cells↗

PubMed1998

Phosphorylation of ethanolamine, methylethanolamine, and dimethylethanolamine by overexpressed ethanolamine kinase in NIH 3T3 cells decreases the co-mitogenic effects of ethanolamines and promotes cell survival.

Ethanolamine (Etn), as well as its N-methyl (MeEtn) and N,N-dimethyl (Me2Etn) analogues, were recently shown to potentiate the stimulatory effect of insulin on DNA synthesis in serum-starved NIH 3T3 fibroblasts. In the present work we assessed the impact of the co-mitogenic effects of Etn and its methyl analogues on cell proliferation and cell survival, and examined whether the cell growth regulatory effects of these ethanolamines involve an Etn-kinase-mediated phosphorylation step. For this purpose, NIH 3T3 sublines highly overexpressing Drosophila Etn kinase and an appropriate vector control line were utilized and the effects of Etn, MeEtn, Me2Etn, methylamine (MeNH2), and dimethylamine (Me2NH) were studied. 31P-NMR analysis of the water-soluble cell metabolites revealed that both MeEtn and Me2Etn, but not choline, are excellent substrates for the expressed Etn kinase. The methylated ethanolamines (MeEtn and Me2Etn) and methylamines (MeNH2, Me2NH) were used as Etn models that can or cannot be phosphorylated, respectively. In serum-starved vector control cells, both MeNH2 (1 mM) and Me2NH (1 mM) were more effective than Etn in enhancing insulin-induced DNA synthesis, and both were almost as effective as MeEtn and Me2Etn. However, in the Etn kinase overexpressor cells the potentiating effects of Etn, MeEtn and Me2Etn, but not those of MeNH2 and Me2NH, were significantly reduced. Moreover, in the overexpressor cells, lower concentrations of Etn (50-200 microM) inhibited the combined mitogenic effects of Me2NH (1 mM) and insulin. These data are consistent with a mechanism in which the phosphorylated and non-phosphorylated ethanolamines are negative and positive regulators of insulin-induced mitogenesis, respectively. After incubating the cells for 13 days in serum-free medium in 96-well microplates, there was a steady decrease in cell numbers in both cell lines. However, between 6-13 days, 0.1-1 mM MeEtn and, particularly, Me2Etn provided significant protection against cell death in the Etn kinase overexpressor cells. In vector control cells, only Me2Etn in combination with insulin had similar effects on cell survival. The data suggest that phosphorylated ethanolamines may function as promoters of cell survival.

3T3 Cells↗

PubMed1998

Stimulation of DNA synthesis in untransformed cells by the antiviral and antitumoral compound tricyclodecan-9-yl-xanthogenate (D609).

The antiviral and antitumor xanthate compound tricyclodecan-9-yl-xanthogenate (D609) is best known for its inhibitory effect on phosphatidylcholine-specific phospholipase C activity. Now we report that in NIH 3T3 cells, but not in several transformed cell types tested, D609 stimulated DNA synthesis when phosphocholine (PCho), insulin, or ATP was also present. Maximal co-mitogenic effects of D609 were observed at 5 microg/mL, a concentration 4-6 times lower than that required to inhibit phospholipase C activity. The synergistic mitogenic effects of D609 and PCho, but not of D609 and insulin, were associated with activation of p42 and, to a lesser extent, p44 mitogen-activated protein (MAP) kinases. The results raise the possibility that the mitogenic activity of D609 in untransformed cells may contribute to its antiviral and antitumor effects.

3T3 Cells↗

PubMed1998

Ethanol potentiates the stimulatory effects of insulin and phosphocholine on mitogenesis by a zinc-dependent and rapamycin-sensitive mechanism in fibroblasts and JB6 cells.

In most cellular systems ethanol inhibits growth factor-induced cell growth. Here we examined the effects of ethanol on DNA synthesis and cell proliferation induced by insulin and phosphocholine (PCho) in NIH3T3 fibroblasts, Swiss 3T3 fibroblasts and mouse epidermal JB6 cells. In serum-starved low (12-18) passage NIH3T3 fibroblasts, 60 mM ethanol enhanced the mitogenic effect of insulin in the absence or presence of 25 microM zinc about 2- or 12-fold, respectively. In contrast, in serum-starved high (30-47) passage NIH3T3 cells 60 mM ethanol had large (20-40-fold) potentiating effects on insulin-induced DNA synthesis even in the absence of zinc. Furthermore, ethanol also enhanced the effects of PCho on DNA synthesis in both the absence and presence of insulin. The potentiating effects of ethanol on insulin- and PCho-induced DNA synthesis were associated with 1.2-1.3-fold stimulation of cell proliferation. Rapamycin, an inhibitor of p70 S6 kinase action, strongly inhibited the potentiating effects of ethanol on insulin- and PCho-induced mitogenesis. Unexpectedly, ethanol inhibited synergistic activation of p42/p44 mitogen-activated protein kinases by insulin and PCho. In both Swiss 3T3 and JB6 cells, ethanol potentiated insulin-induced DNA synthesis only in the presence of zinc. In these cells, ethanol also increased the effects of PCho on both DNA synthesis and cell proliferation in the co-presence of either insulin or ATP. The results indicate that in various cell lines physiologically relevant concentrations of ethanol can increase the ability of insulin and PCho to induce DNA synthesis and, to smaller extents, cell proliferation. In low passage NIH3T3 cells as well as in Swiss 3T3 and JB6 cells potentiation of insulin-induced DNA synthesis by ethanol requires the presence of zinc.

3T3 Cells↗

PubMed1998

Clinical application of the nitroglycerin lingual spray.

The effect of sublingually administered nitrate spray was investigated with noninvasive methods. During 3 months, 82 patients were entered into the study: 40 with angina pectoris, 15 with acute myocardial infarction, 18 with hypertensive crisis, and 9 with left ventricular failure or acute pulmonary edema. The hemodynamic effects of two jets of nitroglycerin spray (0.8 mg Nitrolingual spray; Pohl-Boskamp, Hohenlocksted, Germany) was measured on heart rate, blood pressure, and flow velocity at baseline and 1, 5, and 10 minutes after drug administration. Flow velocities were measured through the left ventricular outflow tract and the mitral valve (early diastolic wave and atrial wave) with bedside Doppler echocardiography. The time to improvement and occurrence of adverse events was analyzed. Heart rate was constant after the therapy (75 +/- 8, 75 +/- 10, 75 +/- 10, and 75 +/- 9 beats per min; not significant), and systolic blood pressure decreased significantly 1 minute after administration and remained decreased throughout the examination (135 +/- 27, 124 +/- 21, 125 +/- 19, and 124 +/- 22 mm Hg, respectively; p < 0.001). The diastolic blood pressure was also significantly decreased (82 +/- 17, 79 +/- 14, 78 +/- 12, 78 +/- 14 mm Hg; p < 0.001). A significant increase in flow velocities in the left ventricular outflow tract was detected (90 +/- 8, 101 +/- 10, and 114 +/- 13 cm/s; p < 0.001) concomitantly with a significant increase in the early diastolic flow (46 +/- 4, 65 +/- 6, and 76 +/- 8 cm/s; p < 0.001) and the atrial wave (101 +/- 9, 110 +/- 10, and 118 +/- 9 cm/s; p < 0.001). This increase of flow velocity was less pronounced through the mitral valve than through the left ventricular outflow tract.

Administration, Sublingual↗

PubMed1997

Extracellular sphingosine 1-phosphate stimulates formation of ethanolamine from phosphatidylethanolamine: modulation of sphingosine 1-phosphate-induced mitogenesis by ethanolamine.

In this work, we determined the effects of sphingosine 1-phosphate (S1P) on phospholipase D (PLD)-mediated hydrolysis of phosphatidylethanolamine (PtdEtn), and evaluated the effects of the water-soluble product ethanolamine on S1P-induced DNA synthesis in NIH 3T3 cells. In [14C]ethanolamine-labelled cells, S1P (0.5-5 microM) stimulated PLD-mediated hydrolysis of PtdEtn 1.5-2.1-fold. Down-regulation of protein kinase C by chronic (24 h) treatment of cells with 300 nM PMA, or pretreatments (10 min) with the cell-permeant calcium chelator 1,2-bis-(O-aminophenoxy)-ethane-N,N, N',N'-tetra-acetic acid tetra-acetoxymethyl ester led to the inhibition of S1P-induced PtdEtn hydrolysis. S1P alone was a weak inducer of DNA synthesis, but its effects were enhanced by phosphocholine (PCho), insulin, ATP or PMA. Ethanolamine (5-100 microM) did not modify the mitogenic effect of S1P alone, whereas at 50-100 microM concentrations it actually enhanced the mitogenic effect of PCho via a mitogen-activated protein (MAP) kinase-independent mechanism. In contrast, 5-20 microM concentrations of ethanolamine, which correspond to normal blood ethanolamine levels in humans, strongly inhibited DNA synthesis induced by S1P plus PCho via a MAP kinase-dependent mechanism; importantly, less or no inhibition was observed with 50-100 microM concentrations of ethanolamine. At 5-50 microM concentrations, ethanolamine also inhibited the synergistic mitogenic effects of both S1P plus insulin (22-27% inhibition) and PCho plus ATP (45-73% inhibition) but not those of S1P plus PMA or S1P plus ATP. The results indicate that S1P stimulates PLD-mediated hydrolysis of PtdEtn by a mechanism that may involve a regulatory protein kinase C isoform. Increased formation of ethanolamine by PLD-mediated PtdEtn hydrolysis or by other means may be required for maximal stimulation of DNA synthesis by S1P in the presence of insulin, and particularly PCho.

3T3 Cells↗

PubMed1997

Ethanolamine, but not phosphoethanolamine, potentiates the effects of insulin, phosphocholine, and ATP on DNA synthesis in NIH 3T3 cells--role of mitogen-activated protein-kinase-dependent and protein-kinase-independent mechanisms.

NIH 3T3 fibroblasts express a phospholipase D activity hydrolyzing phosphatidylethanolamine (PtdEtn) which produces ethanolamine (Etn) in response to a variety of growth regulating agents. The main objective of this work was to evaluate the effects of Etn on mitogenesis and to determine whether these effects require its metabolism to phosphoethanolamine (PEtn) or PtdEtn. To increase conversion of Etn to PEtn, an Etn-specific kinase derived from Drosophila was highly expressed in NIH 3T3 cells. Overexpression of this Etn kinase resulted in large (10-12.5-fold) increases in PEtn formation, but only in modest (1.2-1.7-fold) increases in PtdEtn synthesis. In both vector control and Etn kinase overexpressor cells, Etn had biphasic effects on insulin-induced DNA synthesis with maximal (approximately 2-fold) potentiating effects being observed at 0.5-1 mM concentrations, followed by an inhibitory phase at higher Etn concentrations. In the Etn kinase overexpressor lines, the inhibitory phase was elicited by lower Etn concentrations and it was partially blocked by 5 mM choline due to decreased formation of PEtn. In both vector control and Etn kinase overexpressor cells, phosphocholine (PCho) and insulin synergistically stimulated DNA synthesis; their effects were further enhanced by physiologically relevant (5-60 microM) concentrations of Etn by a mechanism independent of mitogen-activated protein (MAP) kinase. Concentrations of Etn >50 microM also enhanced the effects of both PCho and the synergistic effects of PCho plus ATP; however, in the latter case 20 microM Etn was inhibitory. The magnitude of both the potentiating and inhibitory effects of Etn on PCho-induced as well as PCho + ATP-induced DNA synthesis were similar in the vector control and Etn kinase overexpressor cells; they were associated with stimulation and inhibition, respectively, of p42 MAP kinase activity. The results indicate that in NIH 3T3 cells Etn exerts significant effects on DNA synthesis which, except inhibition of insulin-induced DNA synthesis by higher concentrations of Etn, do not correlate with the metabolism of Etn to PEtn or PtdEtn.

3T3 Cells↗

PubMed1997

Expression of protein kinase C-beta promotes the stimulatory effect of phorbol ester on phosphatidylethanolamine synthesis.

Stimulation of phosphatidylethanolamine (PtdEtn) synthesis by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) has reportedly been found only in hepatocytes expressing the alpha-, betaII-, epsilon-, and zeta-PKC isozymes. In contrast, stimulation of phosphatidylcholine synthesis by PKC activators, known to be mediated by PKC-alpha, is widespread in mammalian cells. In this work, various cell lines exhibiting characteristic differences in their PKC systems were used to determine the role of specific PKC isozymes in the mediation of PMA effect on PtdEtn synthesis. In NIH 3T3 fibroblasts, which express high levels of PKC-alpha but none of the beta (betaI or betaII) isoforms, PMA did not stimulate PtEtn synthesis. In contrast, in Rat-6 fibroblasts overexpressing PKC-betaI, 10-100 nM PMA considerably (1.7- to 2.6-fold) enhanced PtdEtn synthesis. In wild-type or multidrug resistant MCF-7 human breast carcinoma cells, which express PKC-alpha and PKC-betaII (to varying extents) but not PKC-betaI, PMA had only small or no effects on PtdEtn synthesis. In contrast, in MCF-7 cells overexpressing PKC-alpha, and as a consequence also expressing the betaI- and betaII-PKC isoforms, PMA effectively stimulated the synthesis of PtdEtn. Finally, in HL60 human leukemia cells, which contains PKC-betaII as the major PKC isoform, PMA again stimulated PtdEtn synthesis. The results establish that while stimulation of PtdEtn synthesis by PMA occurs only in selected cell lines, this phenomenon is not restricted to hepatocytes. Furthermore, the data indicate that expression of either PKC-betaI or PKC-betaII, but not PKC-alpha, correlates with the effect of PMA on PtdEtn synthesis. Overall, these observations strongly suggest that regulation of PtdEtn and PtdCho synthesis by PMA involves separate PKC isozymes, i.e., PKC-beta and PKC-alpha, respectively.

Animals↗

PubMed1997

Bombesin and zinc enhance the synergistic mitogenic effects of insulin and phosphocholine by a MAP kinase-dependent mechanism in Swiss 3T3 cells.

Simultaneous treatment of serum-starved (24 h) Swiss 3T3 cells with insulin (500 nM) and phosphocholine (PCho) (0.25-1 mM) resulted in synergistic stimulation of DNA synthesis via a mitogen activated protein (MAP) kinase-independent rapamycin-sensitive mechanism. Co-treatment of cells with bombesin (10 nM) or zinc (25 microM) enhanced the combined mitogenic effects of insulin and PCho 2-3-fold; however, in the presence of bombesin or zinc the combined effects of insulin and PCho were not inhibited by rapamycin. The potentiating effects of bombesin and zinc on insulin plus PCho-induced DNA synthesis were accompanied by large stimulation of p42 MAP kinase activity. The results indicate that in Swiss 3T3 cell cultures, synergistic stimulation of DNA synthesis by extracellular insulin and PCho via a p42 MAP kinase-dependent mechanism requires the presence of other growth regulatory agents, such as bombesin or zinc.

3T3 Cells↗

PubMed1997

Alkyl lysophospholipids inhibit phorbol ester-stimulated phospholipase D activity and DNA synthesis in fibroblasts.

The antineoplastic alkyl lysophospholipids (ALP) 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) and 1-S-hexadecylthio-2-methoxymethyl-2-deoxy-rac-glycero-3-phosphocho line (BM41.440) were found to alter phospholipase D (PLD)-mediated phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) hydrolysis in NIH 3T3 fibroblasts. After a shorter (50 min) treatment, 2.5-7.5 microg/ml concentrations of ALP stimulated PtdCho, but not PtdEtn, hydrolysis 2-4-fold. At the same time, 7.5-25 microg/ml concentrations of ALP significantly inhibited the larger (5.8-6.5-fold) stimulatory effects of phorbol 12-myristate 13-acetate (PMA) on both PtdCho and PtdEtn hydrolysis. When a brief (30 min) exposure of cells to 1-2.5 microg/ml concentrations of BM 41.440 was followed by incubation of washed cells for 3-16 h prior to the assay of PLD activity or DNA synthesis, the treated cells exhibited no increased PtdCho hydrolysis, while their responses to the stimulatory PMA effects on both PLD activity and DNA synthesis were strongly reduced. The results suggest that the PLD and protein kinase C systems may be important cellular targets of ALP actions.

3T3 Cells↗

PubMed1997

Phosphocholine and sphingosine-1-phosphate synergistically stimulate DNA synthesis by a MAP kinase-dependent mechanism.

We have previously shown that in NIH 3T3 fibroblasts phosphocholine (PCho) potentiates sphingosine-1-phosphate (S1P)-induced mitogenesis. Here we report that PCho and S1P also synergistically stimulate DNA synthesis in mouse Swiss 3T3 fibroblasts and in mouse JB6 epidermal cells. The combined actions of PCho and S1P on DNA synthesis were associated with synergistic activation of the p42/p44 mitogen-activated protein (MAP) kinases. Ethanolamine (50-100 microM) further enhanced the synergistic effects of PCho and SIP on DNA synthesis but not on MAP kinase activity. The results indicate that the synergistic mitogenic effects of PCho and S1P (i) are not restricted to NIH 3T3 fibroblasts, (ii) are predominantly mediated by the MAP kinase-dependent signal transduction pathway, and (iii) are enhanced by ethanolamine via a MAP kinase-independent mechanism.

3T3 Cells↗

PubMed1997

[The ursodeoxycholic acid-p-aminobenzoic acid test in the diagnosis of small bowel bacterial overgrowth syndrome].

UNLABELLED: Contaminated small bowel syndrome is frequently associated with meteorism due to excessive gas formation, and diarrhoea as a result of bacterial fermentative processes, including splitting of carbohydrates or deconjugating and dehydroxylating bile salts. In addition to gas production, bacteria capable of metabolizing bile salts have been shown to release p-aminobenzoic acid (PABA) from and Ursodeoxycholic-acid-PABA substrate. Our aim was to determine the possible complementary role of the UDCA-PABA test in the diagnosis of bacterial overgrowth. PATIENTS AND METHODS: The H2 breath and UDCA-PABA tests were performed simultaneously on 46 patients with suspected contaminated small bowel syndrome, and on 7 healthy subjects. The H2 breath test was performed by oral loading of 25 g lactose and/or 10 g lactulose. The UDCA-PABA test was carried out by determining urinary excretion of PABA after oral loading with 250 mg UDCA-PABA conjugate. The diagnosis of bacterial overgrowth was established, when either H2 breath, or UDCA-PABA test proved to be pathological. RESULTS: Based upon the pathologic values of either the H2 breath test, or the UDCA-PABA test, 25 out of 46 patients proved to have contaminated small bowel syndrome. In 10 out of 25 patients only pathologic urinary PABA excretion (12.772 +/- 1.707 vs 4.1 +/- 0.58), indicated bacterial overgrowth, and in 9 out of the same group only positive H2 breath test (early rise of > 20 ppm of H2) indicated the same, while in 6 cases both tests proved to be pathological. In 7 CSBS patients the urinary excretion of PABA significantly decreased following a 10 day Tinidazole treatment (5.48 +/- 1.286 vs 13.068 +/- 2.068). CONCLUSION: The UDCA-PABA test proved to be a valuable complementary method to detect bacterial overgrowth, when H2 production failed to reveal bacterial overgrowth.

4-Aminobenzoic Acid↗

PubMed1997

Promotion-resistant JB6 mouse epidermal cells exhibit defects in phosphatidylethanolamine synthesis and phorbol ester-induced phosphatidylcholine hydrolysis.

The tumour-promotion-sensitive (P+) and -resistant (P-) variants of mouse JB6 epidermis-derived cells have often been used to study the requirements for the tumour-promoting effect of PMA. As part of an effort to identify the defect(s) in JB6 P- cells that might prevent the promoting effect of PMA, stimulation of phospholipase D (PLD)-mediated hydrolysis of phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) by PMA as well as the rate of phospholipid synthesis were compared in three P+ variants, two P- variants and a transformed variant of the JB6 cell line. PMA (5-100 nM) had significantly less stimulatory effect on PtdCho hydrolysis in P- cells than in P+ or transformed JB6 cells. The effects of PMA on PtdEtn hydrolysis in the P+ and P- cell lines were similar, whereas in transformed cells PMA had slightly less effect. Each JB6 cell line was found to contain similar amounts of PtdCho. In contrast, P- cells contained significantly less PtdEtn and a correspondingly higher level of ethanolamine phosphate compared with P+ and transformed cells. P- cells also secreted ethanolamine phosphate into the medium; this process was greatly enhanced by PMA. In the two P- variants the synthesis of PtdEtn from [14C]ethanolamine was reduced to various extents, whereas the rate of PtdCho synthesis was comparable in each JB6 cell line. The synthesis of PtdCho, but not PtdEtn, was greatly stimulated by PMA in both the P+ and P- clones. The results indicate that decreased synthesis/level of PtdEtn and suboptimal functioning of a PtdCho-specific PLD are common characteristics of the P- JB6 cells examined so far. The observed alterations in phospholipid metabolism may play a role in the resistance of P- cells to the tumour-promoting action of PMA.

Animals↗