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ABSTRACT: It has been reported that phosphoinositide 3-kinase (PI 3-kinase) and its downstream target, protein kinase B (PKB), play a central role in the signaling of cell survival triggered by neurotrophins (NTs). In this report, we have analyzed the involvement of Ca2+ and calmodulin (CaM) in the activation of the PKB induced by NTs. We have found that reduction of intracellular Ca2+ concentration or functional blockade of CaM abolished NGF-induced activation of PKB in PC12 cells. Similar results were obtained in cultures of chicken spinal cord motoneurons treated with brain-derived neurotrophic factor (BDNF). Moreover, CaM inhibition prevented the cell survival triggered by NGF or BDNF. This effect was counteracted by the transient expression of constitutive active forms of the PKB, indicating that CaM regulates NT-induced cell survival through the activation of the PKB. We have investigated the mechanisms whereby CaM regulates the activation of the PKB, and we have found that CaM was necessary for the proper generation and/or accumulation of the products of the PI 3-kinase in intact cells.
The Journal of Cell Biology 09/2001; 154(3):585-97. · 10.26 Impact Factor
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ABSTRACT: One of the more time-consuming procedures in the study of exogenously expressed proteins in cell lines is the selection of individual transfected clones. In recent years, green fluorescent protein variants with excitation/emission spectra matching the typical flow cytometer configurations have been generated and are in common use. We employed PC12 cells transfected with vectors encoding fluorescent proteins and a fluorescence selection procedure using a fluorescence-activated cell-sorter. In order to select the optimal co-electroporation and sorting conditions, we used the simultaneous detection of two variants of the green fluorescent protein, that possess separable emission peaks when excited at 488 nm. Using these variants and the adequate combination of band-pass filters, we were able to analyze and establish the conditions for identifying and sorting cells transfected with enhanced green fluorescent protein, that simultaneously express another plasmid of interest. Using this procedure, the cells sorted that express both plasmids exceeded 90%. The whole procedure did not alter the physiological responsiveness of the transfected cells to growth factors, and has been successfully applied to the constitutive activation of the mitogen-activated protein kinase pathway, resulting in the spontaneous differentiation of PC12 cells. Also, this procedure has been used with other set of expression vectors encoding proteins that protect PC12 cells from apoptosis caused by different stimuli. The method that we present here provides an easy and fast procedure to obtain a high proportion of positively transfected populations of PC12 cells.
Journal of Neuroscience Methods 08/2000; 100(1-2):63-9. · 1.98 Impact Factor
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ABSTRACT: Nerve growth factor is a member of the neurotrophin family of trophic factors that have been reported to be essential for the survival and development of sympathetic neurons and a subset of sensory neurons. Nerve growth factor exerts its effects mainly by interaction with the specific receptor TrkA, which leads to the activation of several intracellular signaling pathways. Once activated, TrkA also allows for a rapid and moderate increase in intracellular calcium levels, which would contribute to the effects triggered by nerve growth factor in neurons. In this report, we analyzed the relationship of calcium to the activation of the Ras/extracellular signal-regulated kinase pathway in PC12 cells. We observed that calcium and calmodulin are both necessary for the acute activation of extracellular signal-regulated kinases after TrkA stimulation. We analyzed the elements of the pathway that lead to this activation, and we observed that calmodulin antagonists completely block the initial Raf-1 activation without affecting the function of upstream elements, such as Ras, Grb2, Shc, and Trk. We have broadened our study to other stimuli that activate extracellular signal-regulated kinases through tyrosine kinase receptors, and we have observed that calmodulin also modulates the activation of such kinases after epidermal growth factor receptor stimulation in PC12 cells and after TrkB stimulation in cultured chicken embryo motoneurons. Calmodulin seems to regulate the full activation of Raf-1 after Ras activation, since functional Ras is necessary for Raf-1 activation after nerve growth factor stimulation and calmodulin-Sepharose is able to precipitate Raf-1 in a calcium-dependent manner.
Molecular and Cellular Biology 04/2000; 20(6):1931-46. · 5.53 Impact Factor
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ABSTRACT: Evidence suggests that membrane depolarization is able to promote neuronal survival through a sustained, although moderate, increase in the intracellular calcium. We have used the PC12 cell line to study the possible intracellular pathways that can be activated by calcium influx. Previously, we observed that membrane depolarization-induced calcium influx was able to activate the extracellular-regulated kinase (ERK)/mitogen-activated protein kinase pathway and most of this activation was calmodulin-dependent. We demonstrated that a part of the ERK activation is due to the phosphorylation of the epidermal growth factor receptor. Here, we show that both the epidermal growth factor receptor phosphorylation and the Shc-Grb2-Ras activation are not calmodulin-modulated. Moreover, dominant negative mutant Ha-ras (Asn-17) prevents the activation on ERKs by membrane depolarization, suggesting that Ras and calmodulin are both necessaries to activate ERKs by membrane depolarization. We failed to observe any significant induction and/or modulation of the A-Raf, B-Raf or c-Raf-1 kinase activities, thus suggesting the existence of a MEK kinase different from the classical Raf kinases that directly or indirectly can be modulated by Ca2+/calmodulin.
Journal of Biological Chemistry 02/1999; 274(1):75-85. · 4.77 Impact Factor
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ABSTRACT: In the absence of neurotrophic factors, chronic depolarization of plasma membrane has been shown to maintain several populations of primary neurons in culture. We report that in the PC12 cell line, depolarization causes Ca2+ influx through voltage-gated Ca2+ channels, which is able to stimulate extracellular-regulated kinase (ERK) activity. We studied which mediators were responsible for ERK activation resulting from increased levels of Ca2+ in the cytoplasm and found that calmodulin was involved in this process. The addition of W13, a calmodulin inhibitor, to the culture medium, prevented ERK activation when PC12 cells were depolarized. In addition, we show that high K+ treatment did not induce Trk A phosphorylation, thus excluding the possibility of Ca2+ operating through this receptor to activate the ERK signal transduction pathway. Moreover, although high K+ treatment is able to phosphorylate the epidermal growth factor receptor (EGFR) and thus to activate the ERK signal transduction pathway, we demonstrate that W13 did not alter the state of EGFR phosphorylation in conditions that almost completely blocked ERK activation. These data suggest that calmodulin mediates ERK activation induced by increases in intracellular Ca2+ concentration in PC12 cells by a mechanism that seems to be independent of Trk A and EGFR activation.
Journal of Neurochemistry 07/1998; 70(6):2554-64. · 4.06 Impact Factor
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ABSTRACT: High-affinity iron uptake in Saccharomyces cerevisiae involves the extracytoplasmic reduction of ferric ions by FRE1 and FRE2 reductases. Ferrous ions are then transported across the plasma membrane through the FET3 oxidase-FTR1 permease complex. Expression of the high-affinity iron uptake genes is induced upon iron deprivation. We demonstrate that AFT1 is differentially involved in such regulation. Aft1 protein is required for maintaining detectable non-induced level of FET3 expression and for induction of FRE2 in iron starvation conditions. On the contrary, FRE1 mRNA induction is normal in the absence of Aft1, although the existence of AFT1 point mutations causing constitutive expression of FRE1 (Yamaguchi-Iwai et al., EMBO J. 14: 1231-1239, 1995) indicates that Aft1 may also participate in FRE1 expression in a dispensable way. The alterations in the basal levels of expression of the high-affinity iron uptake genes may explain why the AFT1 mutant is unable to grow on respirable carbon sources. Overexpression of AFT1 leads to growth arrest of the G1 stage of the cell cycle. Aft1 is a transcriptional activator that would be part of the different transcriptional complexes interacting with the promoter of the high-affinity iron uptake genes. Aft1 displays phosphorylation modifications depending on the growth stage of the cells, and it might link induction of genes for iron uptake to other metabolically dominant requirement for cell growth.
Yeast 07/1997; 13(7):621-37. · 1.89 Impact Factor
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ABSTRACT: In order to characterize new yeast genes regulating cell proliferation, a number of overexpression-sensitive clones have been isolated from a Saccharomyces cerevisiae cDNA library in a multicopy vector under the control of the GAL1 promoter, on the basis of growth arrest phenotype under galactose-induction conditions. Thirteen of the independent clones isolated in this way correspond to previously known genes (predominantly coding for morphogenesis-related proteins or for multifunctional transcriptional factors), while the remaining 11 independent clones represent new genes with unknown functions. The more stringent conditions employed in this screening compared with previous ones that also employed a dominant genetics approach to isolate overexpression-sensitive genes has allowed us to extend the number of yeast genes that exhibit this phenotype. The effect of overexpression of MCM1 (whose product participates in the regulation of a number of apparently unrelated cellular functions) has been studied in more detail. Galactose-induced overexpression of MCM1 leads to rapid growth arrest at the G1 or S cell cycle stages, with many morphologically-abnormal cells. Several of the other clones also exhibit a G1 arrest terminal phenotype when overexpressed.
Yeast 02/1995; 11(1):25-32. · 1.89 Impact Factor
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ABSTRACT: The hormonal regulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression was studied in the rat hepatoma cell line FAO-1. Both 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase activities were detected in FAO-1 cells, at 68% of the levels found in rat liver. Northern blot analysis showed that FAO-1 cells, like rat liver, contained a predominant species of bifunctional enzyme mRNA, which is 2.2 kb in size. A sensitive RNAase protection assay revealed the presence in FAO-1 cells of an additional mRNA species, which is generated when transcription is initiated from the skeletal muscle promoter of the rat liver/skeletal muscle gene. The liver/skeletal muscle mRNA ratio in FAO-1 cells was 10:1, which is similar to that observed in rat liver. In contrast, in another rat hepatoma cell line, FTO-2B, only the skeletal muscle mRNA was detected. Insulin and dexamethasone induced the liver bifunctional enzyme mRNA in FAO-1 cells by 2-4-fold and 10-20-fold respectively in a concentration- and time-dependent manner, and their effects were antagonized by cyclic AMP. Transcription of the gene in FAO-1 cells, measured by nuclear run-on assays, was also enhanced by dexamethasone and insulin. It is concluded that the FAO-1 cell line is similar to liver with respect to both the preferential use of the liver promoter of the gene and its regulation by hormones, and is therefore an excellent model for the study of the hepatic expression of this gene.
Biochemical Journal 08/1993; 293 ( Pt 1):173-9. · 4.90 Impact Factor
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ABSTRACT: At least two genes encode isoenzymes of rat 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Alternative splicing of one of these genes generates a skeletal muscle-specific transcript from an upstream promoter and a liver-specific transcript from a downstream promoter. A potent glucocorticoid response element was identified in the first intron of the gene, i.e. between liver exon I and exon II. The element is approximately 3.5 kilobase pairs (kb) downstream of the liver isoenzyme transcription start site and 13 kb upstream of exon II of the gene and confers dexamethasone-sensitive expression of chloramphenicol acetyltransferase (CAT) activity from a heterologous thymidine kinase promoter and from both homologous 5'-flanking regions of the gene. This glucocorticoid response element also exhibits androgen- but not estrogen-sensitive expression of CAT activity in HeLa cells cotransfected with the appropriate receptor expression vector. DNase footprint and sequence analysis revealed that the element is comprised minimally of two adjacent 15-mer glucocorticoid receptor dimer binding sites situated in opposite orientations. Glucocortcoid regulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression in liver and skeletal muscle is mediated by a single complex glucocorticoid response element located in the first intron of the skeletal muscle/liver gene.
Journal of Biological Chemistry 09/1992; 267(22):15673-80. · 4.77 Impact Factor
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ABSTRACT: The hormonal control of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression was studied in the rat hepatoma cells, FTO-2B. In contrast to another hepatoma cell line (HTC), the enzyme in FTO-2B cells displays both kinase and bisphosphatase activities. As in rat liver, the mRNA in FTO-2B cells is 2.2-kilobases in length. However, the 5' region of the mRNA differs from the mRNA in the liver in that it contains sequences unique to 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase mRNA from skeletal muscle. These results suggest that the mRNA in FTO-2B cells may represent an additional alternative splicing product of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene. Exposure of FTO-2B cells to media containing either insulin (10(-7) M) or dexamethasone (10(-6) M) induced about a 10-fold increase in the level of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase mRNA within 6-10 h of hormone treatment. The concentrations of insulin or dexamethasone giving half-maximal stimulation were 10(-9) M and 2 x 10(-8) M, respectively, and dibutyryl cyclic AMP (5 x 10(-7) M) completely prevented the increase in enzyme mRNA induced by these hormones. Exposure of cells to glucose-free medium abolished the insulin-mediated enhancement in 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase mRNA, but not that induced by dexamethasone. No alteration in the degradation rate of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase mRNA was noted when cells were treated with insulin. Run-on transcription assays with isolated nuclei showed an increase in the relative transcription rate of the gene in cells treated with either insulin or dexamethasone. The time course of transcription activation preceded the increase in the level of the mRNA, indicating that the main mechanism for the induction of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase expression by insulin and dexamethasone is mediated by stimulation of gene transcription.
Journal of Biological Chemistry 02/1991; 266(3):1557-63. · 4.77 Impact Factor
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ABSTRACT: Adenosine can be metabolized by chicken erythrocytes as a carbon source maintaining ATP levels. In addition, it stimulates glucose uptake and lactate production. Adenosine effects do not involve binding to membrane receptors. They are dependent on the provision of a carbon source to glycolysis and correlate with the increase of fructose 2,6-bisphosphate levels that activating phosphofructokinase would increase the glycolytic flux.
FEBS Letters 12/1989; 258(1):143-6. · 3.54 Impact Factor
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ABSTRACT: Chicken erythrocytes contain fructose 2,6-P2 at a concentration (0.6 nmol/10(9) cells) lower than that of glucose 1,6-P2 (5.4 nmol/10(9) cells) and similar to that of 2,3-bis phosphoglycerate (1.2 nmol/10(9) cells). In chick embryo erythrocytes the content of both bisphosphorylated hexoses is much lower. They begin to increase at hatching and reach chicken values in a few days. Fructose 2,6-P2 at microM concentration activates phosphofructokinase from chicken erythrocytes and releases the inhibition produced by ATP, 2,3-bisphosphoglycerate and inositol hexaphosphate. Glucose 1,6-P2 has similar effects but at much greater concentration. Rabbit reticulocytes contain fructose 2,6-P2 at a concentration (39 pmol/10(9) cells) much lower than that of glucose 1,6-P2 (74 nmol/10(9) cells).
Biomedica biochimica acta 02/1987; 46(2-3):S258-62.
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ABSTRACT: The metabolization curves for both ethanol and acetaldehyde after an acute intragastric or intravenous administration to the mother, have been studied. Metabolization of ethanol followed a very similar pattern in both the pregnant and their control virgin rats, whereas the levels of acetaldehyde derived from the metabolism of the administered ethanol were significantly higher in the pregnant animals, this fact implying that, in late gestation, there is a decrease in the mother's capacity for acetaldehyde metabolism. At the foetal side of the placenta, 150 min after the administration, the concentration of ethanol was similar to that found in the mother's circulation, thus proving a fluid transit of this metabolite through the placenta. The concentration of acetaldehyde in the foetus was relatively high, after the intragastric administration of the ethanol dose; we conclude that at certain ethanol concentrations, acetaldehyde can cross the rat placenta.
Archives internationales de physiologie et de biochimie 01/1985; 92(5):339-44.
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ABSTRACT: Cell cycle control in Saccharomyces cerevisiae is exerted mainly at the START point in the G1 phase of the cycle. At this point, yeast cells check physiological signals and external parameters such as nutrient availability or the presence of sexual pheromones, before proceeding to next stages of the cycle. G1 cyclins regulate START by activating Cdc28, the p34 protein kinase, involving physical association between the cyclin and the kinase catalytic subunit. As a consequence of the accumulation and subsequent degradation of G1 cyclins, a peak of p34 protein kinase activity is reached before START, which would regulate the pass through this point by phosphorylating specific substrates. Here we review the molecular aspects of START and the mechanisms, both positive and negative, that regulate this cell cycle point. We also describe a new gene that links nutrient availability to G1 cyclin expression and the possible connections with the Ras-cAMP pathway.
Microbiología (Madrid, Spain) 10(1-2):27-36.
