Biomedical subjects

R Zippel

Publications and source records attributed to R Zippel.

At least 37 records · Page 2Linked to original sources

PubMed1999

CDC25(Mm)/Ras-GRF1 regulates both Ras and Rac signaling pathways.

The Ras-GRF1 exchange factor molecule contains in addition to the catalytic domain two pleckstrin homology (PH1 and PH2), one IQ and one Dbl homology (DH) domains. In this study we investigated the role of such additional domains. We found that a Ras-GRF1 mutant lacking PH1 and IQ domains is sufficient to activate c-fos promoter in response to lysophosphatidic acid (LPA). The same mutant did not increase external stimuli-regulated kinase (ERK) activity, suggesting an additional mechanism for the induction of gene transcription. Isolated DH-PH2 module activates c-Jun NH(2)-terminal kinase and the c-fos promoter in response to LPA, providing the basis for an ERK-independent mechanism. These results provide evidence that Ras-GRF1 acts as a bifunctional molecule on both ERK-dependent and independent pathways.

3T3 Cells↗

PubMed1999

Expression of Ras-GRF in the SK-N-BE neuroblastoma accelerates retinoic-acid-induced neuronal differentiation and increases the functional expression of the IRK1 potassium channel.

Ras-GRF, a neuron-specific Ras exchange factor of the central nervous system, was transfected in the SK-N-BE neuroblastoma cell line and stable clones were obtained. When exposed to retinoic acid, these clones showed a remarkable enhancement of Ras-GRF expression with a concomitant high increase in the level of active (GTP-bound) Ras already after 24 h of treatment. In the presence of retinoic acid, the transfected cells stopped growing and acquired a differentiated neuronal-like phenotype more rapidly than the parental ones. Cells expressing Ras-GRF also exhibited a more hyperpolarized membrane potential. Moreover, treatment with retinoic acid led to the appearance of an inward rectifying potassium channel with electrophysiological properties similar to IRK1. This current was present in a large number of cells expressing Ras-GRF, while only a small percentage of parental cells exhibited this current. However, Northern analysis with a murine cDNA probe indicated that IRK1 mRNA was induced by retinoic acid at a similar level in both kinds of cells. Brief treatment with a specific inhibitor of the mitogen-activated protein kinase (MAPK) pathway reduced the number of transfected cells showing IRK1 activity. These findings suggest that activation of the Ras pathway accelerates neuronal differentiation of this cell line. In addition, our results suggest that Ras-GRF and/or Ras-pathway may have a modulatory effect on IRK1 channel activity.

Antineoplastic Agents↗

PubMed1998

Opioid modulation of extracellular signal-regulated protein kinase activity is ras-dependent and involves Gbetagamma subunits.

Although it is well-established that G protein-coupled receptor signaling systems can network with those of tyrosine kinase receptors by several mechanisms, the point(s) of convergence of the two pathways remains largely undelineated, particularly for opioids. Here we demonstrate that opioid agonists modulate the activity of the extracellular signal-regulated protein kinase (ERK) in African green monkey kidney COS-7 cells transiently cotransfected with mu-, delta-, or kappa-opioid receptors and ERK1- or ERK2-containing plasmids. Recombinant proteins in transfected cells were characterized by binding assay or immunoblotting. On treatment with corresponding mu- ([D-Ala2,Me-Phe4,Gly-ol5]enkephalin)-, delta- ([D-Pen2,D-Pen5]enkephalin)-, or kappa- (U69593)-selective opioid agonists, a dose-dependent, rapid stimulation of ERK1 and ERK2 activity was observed. This activation was inhibited by specific antagonists, suggesting the involvement of opioid receptors. Pretreatment of cells with pertussis toxin abolished ERK1 and ERK2 activation by agonists. Cotransfection of cells with dominant negative mutant N17-Ras or with a betagamma scavenger, CD8- beta-adrenergic receptor kinase-C, suppressed opioid stimulation of ERK1 and ERK2. When epidermal growth factor was used to activate ERK1, chronic (>2-h) opioid agonist treatment resulted in attenuation of the stimulation by the growth factor. This inhibition was blocked by the corresponding antagonists and CD8- beta-adrenergic receptor kinase-C cotransfection. These results suggest a mechanism involving Ras and betagamma subunits of Gi/o proteins in opioid agonist activation of ERK1 and ERK2, as well as opioid modulation of epidermal growth factor-induced ERK activity.

Animals↗

PubMed1997

The Ras Guanine nucleotide Exchange Factor CDC25Mm is present at the synaptic junction.

CDC25Mm, a mouse Ras-Guanine nucleotide Exchange Factor, is specifically expressed as a product of 140 kDa (p140) in the postnatal and adult brain. Immunohistochemical analysis indicates that it is present throughout the brain particularly concentrated in discrete punctate structures. Subcellular fractionation of the mouse brain shows that p140 is present in synaptosomes but not in highly purified synaptic vesicles. Moreover, isolated postsynaptic densities (PSDs) are largely enriched in CDC25Mm. This protein can be phosphorylated by calcium/calmodulin kinase II, the most abundant protein in PSDs. Altogether these results suggest that CDC25Mm is present at synaptic junctions and that it may be involved in synaptic signal transduction leading to Ras activation.

Animals↗

PubMed1997

Ras-GRF, the activator of Ras, is expressed preferentially in mature neurons of the central nervous system.

In rodents, the Ras-specific guanine-nucleotide exchange factor (Ras-GRF) is expressed in different areas of the brain and, at a reduced level, also in the spinal cord. No expression of the 140 kDa Ras-GRF was detected in dorsal root ganglia and all other tissues tested. Analysis of primary cultures derived from brain reveals that this exchange factor is only present in neurons of the central nervous system. In primary hippocampal cultures, the expression of Ras-GRF increases in parallel with the onset of a neuronal network and in the whole brain it increases sharply after birth.

Animals↗

PubMed1996

The brain specific Ras exchange factor CDC25 Mm: modulation of its activity through Gi-protein-mediated signals.

CDC25Mm is a mouse guanine nucleotide exchange factor specific for Ras, exclusively expressed in the brain. We used a reporter gene containing a Ras-responsive fos-promoter in order to gain information on the role played by this exchange factor in signal transduction. Transient expression of CDC25Mm in CHO cells activates Ras. Moreover serum, but not insulin, can upregulate the response mediated by CDC25Mm and this modulation requires that the CDC25Mm maintains its N-terminal region. NIH3T3 fibroblasts, stably overexpressing this exchange factor, show a partially transformed phenotype, suggesting that the Ras-dependent pathway is constitutively active. In these cells serum and lysophosphatidic acid (LPA) stimulate Ras activity above the basal level while PDGF does not. Both serum and LPA-induced Ras activations in CDC25Mm overexpressing cells can be completely inhibited by pertussis toxin. Moreover, these responses are strongly reduced by coexpression of a truncated version of CDC25Mm lacking the C-terminal catalytic portion. This construct behaves in a dominant negative manner suggesting that it may compete with CDC25Mm by sequestering in an unproductive way signalling components activated by these factors. The data presented indicate that CDC25Mm does not participate in connecting tyrosine kinase receptors with Ras, while it could mediate Ras activation induced by pertussis toxin sensitive Gi-coupled receptors.

3T3 Cells↗

PubMed1996

Predictive factors for acute renal failure in thoracic and thoracoabdominal aortic aneurysm surgery.

PURPOSE: The purpose of this study was to analyze the factors associated with acute renal failure in total descending thoracic and thoracoabdominal aortic aneurysm surgery. METHODS: A total of 234 patients underwent thoracoabdominal aortic aneurysm or total descending thoracic aneurysm repair between January 1991 and January 1994. Eighty-five women and 149 men were evaluated. The median age was 67 years (range 8 to 88 years). Seventy-seven patients had type I thoracoabdominal aortic aneurysm, 99 had type II, 51 had type III or IV, and 7 had total descending thoracic aneurysm. Factors such as age, sex, aneurysm type, and visceral and distal aortic perfusion were examined with univariate fourfold table and multivariate logistic regression analysis. RESULTS: Acute renal failure, defined as an increase in serum creatinine by 1 mg/dl per day for two consecutive days after surgery, occurred in 41 (17.5%) of 234 patients. Thirty-six (15%) of 234 patients required dialysis. Twenty (49%) of 41 patients with acute renal failure died. Of the 21 survivors with renal failure, renal failure resolved in 18 (86%) within 30 days of surgery. The univariate odds ratio of death, given acute renal failure, was 6.7 (95% confidence interval [CI] 3.2 to 14.2, p < 0.0001). No significant association was found between the probability of acute renal failure and age, sex, hypertension, right renal artery reattachment, or renal bypass. Factors associated with increased risk of acute renal failure in multivariate analysis were visceral perfusion (odds ratio [OR] = 3.6 95%, CI 1.2 to 11.0, p < 0.02), left renal artery reattachment (OR = 4.4 95%, CI 1.6 to 11.9, p < 0.004), preoperative creatinine > or = 2.8 mg/dl (OR = 10.3, 95% CI 12.0 to 411.8, p < 0.0001), and simple clamp technique (OR = 3.4 95%, CI 1.07 to 10.76, p < 0.04). Direct univariate correlation was seen between preoperative creatinine and acute renal failure (OR = 3.2 per mg/dl increase, 95% CI 2.7 to 10.1, p < 0.0001). CONCLUSION: Postoperative acute renal failure after thoracoabdominal and total descending thoracic aortic aneurysm surgery is associated with preoperative creatinine level, visceral perfusion, left renal artery reattachment, and simple cross-clamp technique.

Acute Kidney Injury↗

PubMed1995

kappa-Opioid receptor-transfected cell lines: modulation of adenylyl cyclase activity following acute and chronic opioid treatments.

The opioid receptors mu, delta and kappa have recently been cloned. Here we show that kappa-agonists inhibit adenylyl cyclase activity in Chinese hamster ovary cells stably transfected with rat kappa-opioid receptor cDNA. Chronic exposure of the cells to kappa-agonists did not lead to significant desensitization of the capacity of the agonists to inhibit adenylyl cyclase. On the other hand, withdrawal of the agonist following the chronic treatment led to the phenomenon of supersensitivity ('overshoot') of adenylyl cyclase activity. Both the inhibition of adenylyl cyclase activity by the acute opioid treatment and the chronic agonist-induced supersensitivity are pertussis toxin sensitive, demonstrating involvement of Gi/Go proteins in both processes.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1994

Expression of two different products of CDC25Mm, a mammalian Ras activator, during development of mouse brain.

The CDC25Mm gene codes for Ras-guanine nucleotide exchange factors. Four different full-length cDNA clones derived from the same gene and coding for proteins of different sizes that have in common the last 661 amino acids have been isolated from mouse brain. In order to investigate the expression of the products of this gene in different tissues we have prepared two polyclonal antibodies directed toward two different regions of the protein comprised in the last C-terminal 472 amino acids. While in most of the tested tissues we have been unable to definitely identify CDC25Mm products, in NIH3T3 fibroblasts we have found a poorly expressed 120-kDa protein. In the mouse brain we have identified two proteins of 140 and 58 kDa. While the former is expressed in the adult mouse, the latter is present in the embryo and persists for few days after birth. This finding suggests that differential expression of various forms of CDC25Mm may be involved in brain development.

3T3 Cells↗

PubMed1992

Cloning by functional complementation of a mouse cDNA encoding a homologue of CDC25, a Saccharomyces cerevisiae RAS activator.

In the yeast Saccharomyces cerevisiae genetic and biochemical evidence indicates that the product of the CDC25 gene activates the RAS/adenylyl cyclase/protein kinase A pathway by acting as a guanine nucleotide protein. Here we report the isolation of a mouse brain cDNA homologous to CDC25. The mouse cDNA, called CDC25Mm, complements specifically point mutations and deletion/disruptions of the CDC25 gene. In addition, it restores the cAMP levels and CDC25-dependent glucose-induced cAMP signalling in a yeast strain bearing a disruption of the CDC25 gene. The CDC25Mm-encoded protein is 34% identical with the catalytic carboxy terminal part of the CDC25 protein and shares significant homology with other proteins belonging to the same family. The protein encoded by CDC25Mm, prepared as a glutathione S-transferase fusion in Escherichia coli cells, activates adenylyl cyclase in yeast membranes in a RAS2-dependent manner. Northern blot analysis of mouse brain poly(A)+ RNA reveals two major transcripts of approximately 1700 and 5200 nucleotides. Transcripts were found also in mouse heart and at a lower level in liver and spleen.

Adenylyl Cyclases↗

PubMed1992

Isolation of multiple mouse cDNAs with coding homology to Saccharomyces cerevisiae CDC25: identification of a region related to Bcr, Vav, Dbl and CDC24.

In Saccharomyces cerevisiae, the product of the CDC25 gene is an essential Ras activator that appears to function by stimulating guanine nucleotide exchange on Ras. Using the ability of a mouse cDNA expression library to complement yeast cells lacking functional CDC25, Martegani et al. have identified a 1.7 kb partial cDNA from a gene, designated CDC25Mm, with homology to CDC25. We have now screened a mouse brain cDNA library to identify full-length clones of CDC25Mm. This cloning has led to the isolation of six distinct full-length cDNAs, each of which appear to be derived from the CDC25Mm gene, since their 3' 2 kb appear to be identical and to encode the same 661 C-terminal amino acids. Three cDNAs are predicted to encode protein products of 666 or 667 amino acids. The other three cDNAs encode products that are 836, 1120 and 1260 amino acids, respectively. A 241 amino acid region near the N-terminus of the two largest products was found to have homology to a domain shared by Bcr, Vav, Dbl and CDC24. Polyclonal antibodies raised to a peptide encoded by all the cDNAs have identified at least two protein products in NIH3T3 fibroblasts. Their apparent molecular weights are 75 and 95 kDa, which correspond closely to those predicted to be encoded, respectively, by the two shorter classes of cDNAs. In NIH3T3, the 95 kDa form is much more abundant than the 75 kDa form, while PC-12 pheochromocytoma cells contain relatively high levels of the 75 kDa form. We conclude that CDC25Mm is a complex gene whose protein products are regulated in a tissue-specific manner.

3T3 Cells↗

PubMed1992

[Pressure behavior in muscle compartments of the foot in defined stress, metatarsal and ankle joint fractures].

In addition to the importance of compartment syndrome in the lower leg, a growing interest is being shown in the muscle compartments of the foot region. The standard pressure rates in the muscle compartments of the foot comprise up to 4 kPa for men and up to 2.5 kPa for women. During graded exercise (n = 9) on the treadmill the pressure increased in line with the strain and declined again rapidly when the exercise stopped without showing any significant differences in pressure between the muscle compartments studied. In metatarsal and ankle joint fractures post-traumatic pressures were found which were as high as or higher than after 20 minutes on the treadmill and would make the development of a compartment syndrome possible. This points to the necessity of measuring the pressure in the muscle compartments of the patient's foot when there is a clinical suspicion of a compartment syndrome.

Adolescent↗

PubMed1991

Characterization of the tyrosine phosphorylation of calpactin I (annexin II) induced by platelet-derived growth factor.

Stimulation in vivo of Swiss 3T3 fibroblasts with platelet-derived growth factor (PDGF) in the presence of orthovanadate induces the tyrosine phosphorylation of a 39 kDa protein, identified as the phosphorylated slow-migrating form of calpactin I (annexin II) heavy chain, p36. In fact, in PDGF-stimulated cells, anti-(calpactin I) antibodies recognize a doublet of bands, p36 and p39, and the latter disappears upon treatment with phosphatase. In many regards phosphorylation of p39 differs from the rapid and transient phosphorylation of the PDGF receptor and of other substrates: (a) it has slower kinetics but is then stable for longer periods of time; (b) it occurs at 37 degrees C but not at 4 degrees C; and (c) whereas most of the tyrosine-phosphorylated proteins are associated with membrane-enriched preparations, membrane association of p39 only occurs in the presence of Ca2+. Moreover, calpactin I leaks out of permeabilized cells at 0.1 microM free Ca2+, whereas it remains associated with the cells at concentrations of Ca2+ greater than or equal to 1 microM. PDGF does not stimulate phosphoinositide turnover (and thus Ca2+ mobilization) at 4 degrees C; thus it can be suggested that the Ca(2+)-dependent translocation of the protein to membrane/cytoskeletal structures is a necessary condition for its phosphorylation. In addition, calpactin I may not be a direct substrate for the PDGF receptor kinase, but rather the substrate of another tyrosine kinase activated by the receptor.

Animals↗

PubMed1991

Bombesin stimulates a high affinity GTPase activity in membranes of Swiss 3T3 fibroblasts.

Peptides of the bombesin family are mitogenic for Swiss 3T3 fibroblasts and in these cells stimulate the turnover of polyphosphoinositides. Recent studies have suggested that G protein(s) may be involved in the signal transduction pathway triggered by bombesin. In this study we have found and characterized a high affinity GTPase activity stimulated by bombesin in membranes of Swiss 3T3 fibroblasts. Our results support the involvement of a G protein in the response of Swiss 3T3 cells to bombesin.

Animals↗

PubMed1990

Kinetics of tyrosine phosphorylation and internalization of human EGF receptors overexpressed in NIH 3T3 fibroblasts.

Binding of epidermal growth factor (EGF) to cells rapidly induces tyrosine phosphorylation of its receptor which is followed by its internalization and dephosphorylation. The kinetics of these processes differs widely in time from minutes to hours according to cell types. In this paper we analyzed EGF receptor phosphorylation and down-regulation in NIH 3T3 cells transfected with the recombinant hEGF-R cDNA which express 4 X 10(5) receptors/cell. In the presence of EGF receptor phosphorylation reached a maximum after 1 min and was then maintained for about 1 h, while during this time the number of EGF-binding sites was reduced to 40% of the initial number. Detailed analysis of the fate of a population of receptors previously activated and autophosphorylated at 4 degrees C, after warming to 37 degrees C in the absence of the ligand, showed that internalization of the cell surface-associated EGF and dephosphorylation of the receptor were rapid (t1/2 15 min) and followed a similar kinetics. Our data indicate that at any given time only a fraction of the total cell surface receptors is phosphorylated on tyrosine and that dephosphorylation occurs at the cell surface or very rapidly after internalization. In addition the data also suggest that a certain recycling of previously internalized receptors may occur in these cells during EGF treatment.

Calcium↗

PubMed1990

Cytosolic calcium and membrane conductance in response to platelet-derived growth factor and bradykinin stimulation in single human fibroblasts.

Bradykinin (BK) and platelet-derived growth factor (PDGF) act as mitogens and stimulate phosphatidylinositol (PI) turnover in human fibroblasts. By coupling whole-cell electrophysiological measurements with cytosolic Ca2+ determinations using fura-2 microfluorimetry, we have studied the changes in cytosolic calcium and in membrane conductance in single cells following stimulation with BK or PDGF. Both agonists produce variable patterns of response which include: single transient, sustained pulsations, damped oscillations, no response. In all cases, there is a very good temporal correlation between increases in intracellular Ca2+ and membrane current. The cytosolic calcium elevation appears to be insensitive to membrane potential changes, indicating that Ca2+ is released from an intracellular source. The Ca2(+)-activated current is not blocked by 1 microM apamin or by 0.5 mM (+)-tubocurarine; it is instead strongly reduced by 5 mM tetraethylammonium (TEA). We can conclude that BK and PDGF induce very similar early responses in human fibroblasts, and that the variable pattern of response does not depend on the particular mitogen used. The membrane currents are due to a kind of Ca2(+)-activated K+ channels which, according to their voltage-dependence and specific blockers, belong to the "maxi K+" class.

Bradykinin↗

PubMed1989

Effect of the different dimeric forms of the platelet-derived growth factor on cellular responses in mouse Swiss 3T3 fibroblasts.

PDGF consists of two polypeptide chains, A and B, and all three possible dimers have been isolated from different sources. Human PDGF, essentially AB, porcine PDGF (BB) and recombinant PDGF-AA were tested on Swiss 3T3 fibroblasts for their ability to stimulate mitogenesis, phosphoinositide turnover and tyrosine phosphorylation of the PDGF receptor. When used in saturating amounts, the three isoforms were equally active in inducing mitogenesis. However, PDGF-AA was less active than AB and BB to induce the phosphorylation of the receptor and the turnover of phosphoinositides (30% and 50%, respectively). These findings suggest that, in Swiss 3T3 fibroblasts, PDGF receptors of the alpha-type are present in a slightly lower amount than beta-type. In addition, the two types of receptor appear to have similar physiological functions.

Animals↗

PubMed1989

Kinetics and regulation of tyrosine phosphorylation of the platelet derived growth factor receptor.

Platelet derived growth factor (PDGF) interaction with the cells induces rapid tyrosine phosphorylation of the PDGF receptor in a dose dependent manner. At 37 degrees C phosphorylation of the receptor is followed by its dephosphorylation and internalization. It is observed that the higher the ligand concentration, the more transient is the response, and the observed kinetics are explained by a simple kinetic model. At 4 degrees C the phosphorylated form of the receptor is more stable; however, if PDGF is dissociated from the cell surface-associated ligand-receptor complexes, the receptors are rapidly dephosphorylated, indicating that phosphatases specific for phosphotyrosine groups are very active within the cells. In fact, addition of orthovanadate stabilizes the phosphorylated form of the receptor and helps in recognizing possible physiological substrates of the PDGF receptor kinase. The expression of PDGF receptors on the cell surface has been investigated under different growth conditions: a positive correlation exists between the amount of PDGF receptors and the duplication times of exponentially growing cultures. Moreover, during exponential growth the PDGF receptors are scarcely expressed, and their number increases reaching a maximal value when the population enters the stationary phase.

Animals↗