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A Brunet

Publications and source records attributed to A Brunet.

At least 55 records · Page 3Linked to original sources
PubMed1997

Elaboration and use of nickel planar macrocyclic complex-based sensors for the direct electrochemical measurement of nitric oxide in biological media.

We describe here the electrochemical detection of nitric oxide, NO, in biological systems by using chemically modified ultramicro carbon electrodes. In the first part of the paper, the different steps involved in the electrochemical preparation and characterization of the nickel-based sensor are described. This is illustrated by the use of nickel(II) tetrasulfonated phthalocyanine complex. The second part of the paper describes two examples of the direct electrochemical measurement of NO production in human blood platelets and endothelial cells from umbilical cord vein.

Biosensing Techniques↗
PubMed1997

[Treatment of advanced kidney cancer using recombinant erythropoietin].

OBJECTIVES: Study of the antitumour effects of erythropoietin on metastatic renal cell carcinoma. METHODS: After giving their informed consent, 20 patients with histologically proven metastatic renal cell carcinoma received subcutaneous recombinant erythropoietin three times a day at a dose of 150 IU/kg when haemoglobin was less than or equal to 12 g/dL or 75 IU/kg when haemoglobin was higher than 12 g/dL. Treatment was continued for a minimum of 8 weeks before reassessment and was continued thereafter, except in the case of progression or excessive toxicity. A staging assessment was performed every 8 weeks and the response was assessed on the basis of WHO criteria. A clinical and laboratory assessment was performed every two months to evaluate toxicity, graded according to the WHO scale. All but one of the patients had received immunotherapy or chemotherapy prior to inclusion in the study. RESULTS: One complete response (> 12 months), one partial response (8 months), two minor responses, 10 cases of stabilisation and 6 cases of progression were observed. 15 patients received treatment at full doses. In 5 patients, the duration of treatment was reduced before the 8 weeks initially defined because of tumour progression in one patient and because of haemoglobin persistently greater than 15 g/dL in 4 other patients. Adverse effects consisted of 1 case of moderate headache, 2 cases of transient bone pain, and 1 case of transient hypertension. CONCLUSION: Erythropoietin exerts a moderate antitumour effect which needs to be confirmed by a phase II trial of first-line treatment in selected patients.

Aged↗
PubMed1997

Mammalian MAP kinase modules: how to transduce specific signals.

MAPK modules are composed of a cascade of three intracellular protein kinases (MKKK, MKK and MAPK) which are activated successively by phosphorylation events. They are used to transduce a variety of information in organisms as diverse as yeasts, worms, flies or mammals. MAPK modules integrate signals coming from membrane receptor activation and, by the ability of MAPK to translocate into the nucleus and phosphorylate nuclear targets such as transcription factors, they relay extracellular signals into a genomic response. Since several MAPK modules transducing different information are expressed in the same cell, in yeast or in mammals, the question arises as to how fidelity is maintained between the distinct MAPK modules of a single cell. Two levels of specificity have been documented: the molecular selectivity of each enzyme for its substrate, which is particularly evident for the MKK-MAPK couple, permits specificity within one particular module; exogenous proteins, such as the yeast Ste5 protein, may serve as 'chaperone' proteins to tether all the members of a module and restrict signal transduction to this module. In mammalian cells, the MAPK modules are not strictly independent and one pathway may interfere with another. It remains to be determined whether this interference is of physiological relevance.

Amino Acid Sequence↗
PubMed1996

Cyclin D1 expression is regulated positively by the p42/p44MAPK and negatively by the p38/HOGMAPK pathway.

We have previously shown that the persistent activation of p42/p44(MAPK) is required to pass the G1 restriction point in fibroblasts (Pagès, G., Lenormand, P., L'Allemain, G., Chambard, J. C., Meloche, S., and Pouysségur, J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8319-8323) and postulated that MAPKs control the activation of G1 cyclin-dependent complexes. We examined the mitogen-dependent induction of cyclin D1 expression, one of the earliest cell cycle-related events to occur during the G0/G1 to S-phase transition, as a potential target of MAPK regulation. Effects exerted either by the p42/p44(MAPK) or the p38/HOGMAPK cascade on the regulation of cyclin D1 promoter activity or cyclin D1 expression were compared in CCL39 cells, using a co-transfection procedure. We found that inhibition of the p42/p44(MAPK) signaling by expression of dominant-negative forms of either mitogen-activated protein kinase kinase 1 (MKK1) or p44(MAPK), or by expression of the MAP kinase phosphatase, MKP-1, strongly inhibited expression of a reporter gene driven by the human cyclin D1 promoter as well as the endogenous cyclin D1 protein. Conversely, activation of this signaling pathway by expression of a constitutively active MKK1 mutant dramatically increased cyclin D1 promoter activity and cyclin D1 protein expression, in a growth factor-independent manner. Moreover, the use of a CCL39-derived cell line that stably expresses an inducible chimera of the estrogen receptor fused to a constitutively active Raf-1 mutant (DeltaRaf-1:ER) revealed that in absence of growth factors, activation of the Raf > MKK1 > p42/p44MAPK cascade is sufficient to fully induce cyclin D1. In marked contrast, the p38(MAPK) cascade showed an opposite effect on the regulation of cyclin D1 expression. In cells co-expressing high levels of the p38(MAPK) kinase (MKK3) together with the p38(MAPK), a significant inhibition of mitogen-induced cyclin D1 expression was observed. Furthermore, inhibition of p38(MAPK) activity with the specific inhibitor, SB203580, enhanced cyclin D1 transcription and protein level. Altogether, these results support the notion that MAPK cascades drive specific cell cycle responses to extracellular stimuli, at least in part, through the modulation of cyclin D1 expression and associated cdk activities.

Animals↗
PubMed1996

Endothelin-3, Ca2+ mobilization and cyclic GMP content in human platelets.

As previously described for endothelin-3, platelet exposure to cyclic GMP-elevating agents such as sodium nitroprusside and M&B-22948 (2-o-propoxyphenyl-8-azapurin-6-one), a cGMP phosphodiesterase inhibitor, lowered Ca2+ mobilization in response to thrombin. Interestingly, when cGMP phosphodiesterases were blocked, endothelin-3 produced a dose-dependent cGMP accumulation (P < 0.001). Since endothelin-3 has been proposed to decrease the activity of Ca2+ accumulating pumps, we examined whether this latter effect could be mediated by a rise in cGMP content. Cyclic GMP decreased in a dose-dependent manner the initial rate and plateau value of the ATP-dependent 45Ca2+ uptake in platelet membrane vesicles (P = 0.006 for each). Furthermore, combined treatment with endothelin-3 and M&B-22948 or a moderate concentration of Na(+)-nitroprusside further reduced the thrombin-evoked Ca2+ discharge (P = 0.004 and 0.01, respectively), suggesting that endothelin-3 pre-exposure had reduced the amount of mobilizable Ca2+. We propose that the depletion of platelet Ca2+ stores and the reduction of Ca2+ release evoked by endothelin-3 could be due, at least in part, to the elevation of cGMP content and to a decrease in Ca2+ accumulating pump activity.

Analysis of Variance↗
PubMed1996

Identification of MAP kinase domains by redirecting stress signals into growth factor responses.

Mitogen-activated protein kinase (MAPK) cascades, termed MAPK modules, channel extracellular signals into specific cellular responses. Chimeric molecules were constructed between p38 and p44 MAPKs, which transduce stress and growth factor signals, respectively. A discrete region of 40 residues located in the amono-terminal p38MAPK lobe directed the specificity of response to extracellular signals, whereas the p44MAPK chimera, expressed in vivo, redirected stress signals into early mitogenic responses, demonstrating the functional independence of these domains.

Amino Acid Sequence↗
PubMed1996

Low levels of chimerism in rabbit fetuses produced from preimplantation embryos microinjected with fetal gonadal cells.

The potential pluripotency of rabbit fetal germ cells has been investigated by using them to make chimeric embryos. Gonial cells, isolated from enzyme-dispersed male and female transgenic fetal rabbit gonads of 18-22 days gestation, were microinjected in groups of about 10 into 640 nontransgenic rabbit embryos between the two-cell and expanded blastocyst stages. Injections were made with primary isolations of gonial cells, within 48 hr of their collection. The injected embryos were transferred, with or without non-injected control embryos, into 49 recipient rabbits. Tissues from 159 resulting fetuses, implantation sites, and a few liveborn young were examined by PCR analysis for the two transgenes used (alpha-1 antitrypsin or luciferase). The overall pregnancy rate (about 80%) was not affected by the stage of development of the embryo injected, nor by co-transfer of control embryos. The survival rate of injected embryos (18% overall, 23.6% in pregnant recipients) was almost identical to that of 243 control embryos. Chimerism was detectable in tissues produced from 4 of 159 (2.5%) of the injected embryos, all four of which had been injected at the 8- to 16-cell stage. This low rate of success indicates that, although passage of rabbit gonial cells is not an absolute requirement for pluripotency, further investigation should pay particular attention to improving culture conditions with a view to deriving EG cell lines.

Animals↗
PubMed1996

MMPI profiles of acute and chronic PTSD in a civilian sample.

In a treatment setting, a group of 165 subjects presenting with either acute or chronic posttraumatic stress disorder (PTSD) were compared to 72 subjects presenting with panic disorder only in order to determine whether the MMPI PTSD assessment strategy developed with Vietnam veterans could be validly used with civilians. Results indicated that the MMPI profile, codetype, diagnostic decision rule, and PK scale developed with samples of Vietnam veterans did not apply well to civilians, especially those presenting with acute PTSD. It is thus recommended that specific assessment strategies be developed for these populations.

Acute Disease↗
PubMed1996

Social desirability in the assessment of trauma.

Since admitting distress is often considered undesirable, the role of social desirability as a moderator variable in the assessment of traumatic events scores, on the PTSD Interview, and diagnosis was investigated. A sample of 342 city bus drivers were grouped by their lifetime exposure to trauma (nonexposed, stressful but nontraumatic event, traumatic event). Analyses replicate and extend findings from the literature on life events: social desirability plays a marginal role as a moderator variable in the assessment of trauma.

Bias↗
PubMed1996

[Recurrent exposure to traumatic events: inoculation or growing vulnerability?].

Two opposed hypotheses have been proposed regarding the impact of a traumatic event in regards to future adversity: gradual inoculation or vulnerability. A thorough examination of research supporting these hypotheses indicate they are not as antagonistic as they appear. It seems possible to integrate the two into a singular theory in so far as circumstances favoring one and the other become better known. A few research strategies are proposed.

Habituation, Psychophysiologic↗
PubMed1996

[Prevalence of post-traumatic stress disorder in bus drivers].

Many recent reports indicate that public transport employees constitute a group of workers at high risk of going through a traumatic event. Yet, very few scientific data on the impact of these events on this population are available. The authors present results of an epidemiological study conducted among Montreal bus drivers. A little more than a third of bus drivers have reported a traumatic event at work. Among the whole of traumatic events occurring during the course of their work, serious threats and assault constitute the majority of these events. Traumatic events are more frequent at work than in their private lives. The prevalence of PTSD for those exposed to a traumatic event at work is of 10.7% and of 21.3% for those bus drivers exposed to traumatic events in their private lives. Nine out of 100 drivers exposed to a traumatic event at work suffered a PTSD in the month preceding the study. When the authors consider the events occurring in the private lives, almost 15% of those exposed were victims of PTSD. When the two levels of expositions are reunited, 12% of those exposed presented a PTSD in the month preceding the research.

Adult↗
PubMed1995

The mouse p44 mitogen-activated protein kinase (extracellular signal-regulated kinase 1) gene. Genomic organization and structure of the 5'-flanking regulatory region.

Mitogen-activated protein kinase (MAPK) or extracellular signal-regulated kinase are ubiquitous kinases conserved from fungi to mammals. Their activity is regulated by phosphorylation on both threonine and tyrosine, and they play a crucial role in the regulation of proliferation and differentiation. We report here the cloning of the murine p44 MAP kinase (extracellular signal-regulated kinase 1) gene, the determination of its intron/exon boundaries, and the characterization of its promoter. The gene spans approximately eight kilobases (kb) and can be divided into nine exons and eight introns, each coding region exon containing from one to three of the highly conserved protein kinase domains. Primer extension analysis reveals the existence of two major start sites of transcription located at -183 and -186 base pairs (bp) as well as four discrete start sites for transcription located at -178, -192, -273, and -292 bp of the initiation of translation. However, the start site region lacks TATA-like sequences but does contain initiator-like sequences proximal to the major start sites obtained by primer extension. 1 kb of the promoter region has been sequenced. It contains three putative TATA boxes far upstream of the main start sites region, one AP-1 box, one AP-2 box, one Malt box, one GAGA box, one half serum-responsive element, and putative binding sites for Sp1 (five), GC-rich binding factor (five), CTF-NF1 (one), Myb (one), p53 (two), Ets-1 (one), NF-IL6 (two), MyoD (two), Zeste (one), and hepatocyte nuclear factor-5 (one). To determine the sites critical for the function of the p44 MAPK promoter, we constructed a series of chimeric genes containing variable regions of the 5'-flanking sequence of p44 MAPK gene and the coding region for luciferase. Activity of the promoter, measured by its capacity to direct expression of a luciferase reporter gene, is strong, being comparable with the activity of the Rous sarcoma virus promoter. Progressive deletions of the approximately 1 kb (-1200/-78) promoter region allowed us to define a minimal region of 186 bp (-284/-78) that has maximal promoter activity. Within this context, deletion of the AP-2 binding site reduces by 30-40% the activity of the promoter. Further deletion of this minimal promoter that removes the major start sites (-167/-78) surprisingly preserves promoter activity. This result implicates a major role of this region that contains the Sp1 sites.(ABSTRACT TRUNCATED AT 400 WORDS)

Alternative Splicing↗
PubMed1995

Direct measurement of nitric oxide production in platelets: relationship with cytosolic Ca2+ concentration.

NO production in platelets has been followed by electrochemical detection. It was undetectable in unstimulated platelets and in thrombin or ADP-stimulated platelets, but dose-dependently stimulated by collagen. A production of 5 10(-19) mol/platelet was reached with 9 micrograms collagen. In collagen-stimulated platelets, preincubation with 1 mM L-Arg, D-Arg or L-NMMA increased by 77%, left unchanged or decreased by 63% NO production, respectively. NO production did not parallel cytosolic Ca2+ changes, although it decreased in low Ca2+ medium or when Ca2+ transients were attenuated by intracellular Ca2+ buffer. These results confirm that human platelets can generate NO. They demonstrate that cytosolic [Ca2+], although participating in the regulation of its synthesis, is not the messenger for NO synthase activation. Platelet NO production could become functionally important when collagen fibrils of the sub-endothelium are accessible.

Adenosine Diphosphate↗
PubMed1995

B-Raf protein isoforms interact with and phosphorylate Mek-1 on serine residues 218 and 222.

The B-raf/c-Rmil proto-oncogene belongs to the raf/mil family of serine/threonine protein kinases. It encodes multiple protein isoforms resulting from alternative splicing of two exons located upstream of the kinase domain. Recent studies suggested that B-Raf could be the intermediate molecule between Ras and Mek-1 (MAP Kinase Kinase) in signalling pathways specific of neural cells. However, there has been no evidence for a direct interaction between B-Raf and Mek-1. We report here that different B-Raf isoforms can be co-immunoprecipitated with anti-Mek-1 antisera in COS-1 cells and that the kinase activity of B-Raf is not required for its interaction with Mek-1. We also show that all B-Raf isoforms tested phosphorylate Mek-1 in a time-dependent manner, whereas kinase defective mutants fail to do so. Finally, we demonstrate that the constitutively activated S218D, S222D and S218D/S222D mutants of Mek-1 interact similarly with B-Raf. However, only the S218D and S222D mutants, and not the S218D/S222D double mutant, can be phosphorylated by B-Raf isoforms. Therefore, serine residues 218 and 222, previously shown to regulate Mek-1 activity, appear to be the major phosphorylation sites by B-Raf in vitro.

Animals↗
PubMed1995

[MAP kinase module: role in the control of cell proliferation].

A kinase cascade highly conserved throughout evolution, Raf/MAP kinase kinase kinase (MAPKKK)-->MAP kinase kinase (MAPKK)-->MAP kinase (MAPK)-->ribosomal S6 kinase (p90 RSK), is thought to play a crucial role in signal transduction from the membrane to the nucleus. In mammalian cells, this cascade is connected both to tyrosine kinase receptors and G protein-coupled receptors. Although the mode of activation at the receptor level differs, all mitogens activate the ubiquitously expressed isoforms of MAPK, p42 and p44. We have cloned, epitope tagged and expressed in fibroblasts, the Hamster MAPKK and p44 MAPK in order to analyze their time-course of activation, their subcellular localization, their regulatory phosphorylation sites and their role in cell cycle entry. We have demonstrated that MAPK activation was rapid, biphasic and persistent. The sustained phase of activation is only obtained with potent mitogenic agents, correlating with their ability to elicit cell cycle entry. Activation of MAPKK is also rapid and persistent but does not distinguish between mitogenic and non mitogenic factors, indicating that a distinction occurs at the MAPK level, probably by the action of specific phosphatases such as MAPK phosphatase MKP-1. Both isoforms of MAPK are translocated into the nucleus upon growth factor addition whereas the upstream activators (MAPKKK, Raf and MAPKK) remain cytoplasmic. MAPK translocation, together with the ability of MAPK to phosphorylate transcription factors, indicates that MAPK might constitute a relay between cytoplasmic and nuclear events. Finally we show that interfering with the MAP kinase cascade, by expressing either MAPK antisense, a MAPK dominant negative mutant or the MAPK specific phosphatase, MKP-1, suppresses the growth factor induced G0 to G1 transition. In addition, permanently activated versions of MAPKK reduce growth factor requirement, allow autonomous cell growth and induce tumor formation in nude mice. We therefore conclude that MAP kinase activation is both necessary and sufficient to trigger cell cycle entry.

Animals↗
PubMed1994

Constitutive mutant and putative regulatory serine phosphorylation site of mammalian MAP kinase kinase (MEK1).

In response to various external stimuli, MAP kinases are activated by phosphorylation on tyrosine and threonine by MAP kinase kinase (MAPKK), a dual specificity kinase. This kinase is in turn activated via Raf-1 and MAPKK kinase (MAPKKK). To determine regulatory phosphorylation sites of MAPKK, we isolated a Chinese hamster cDNA, that we epitope-tagged and expressed in fibroblasts. This hamster MAPKK (MEK1 isoform) can reactivate recombinant p44mapk when immunoprecipitated from growth factor-stimulated cells or when incubated with an active form of MAPKKK. Mutations at either of two residues that are conserved among kinases, D208N or S222A, abolished MAPKK activity. However, only S222A/MAPKK showed a reduction in phosphorylation in response to active MAPKKK and exerted a dominant negative effect on the serum-stimulated endogenous MAPKK. Finally, replacing Ser222 with Asp, a negatively charged residue, restored MAPKK activity independently of the upstream kinase. These results strongly suggest that Ser222 represents one key MAPKKK-dependent phosphorylation site switching on and off the activity of MAPKK, an event crucial for growth control.

Amino Acid Sequence↗
PubMed1994

Growth factor-stimulated MAP kinase induces rapid retrophosphorylation and inhibition of MAP kinase kinase (MEK1).

The MAP kinase module (Raf/MAPKKK-MAPKK-MAPK) has been shown to be sequentially activated after mitogenic stimulation. Here we demonstrate, by site directed mutagenesis, that MAPK is able to retrophosphorylate its own activator, MAPKK, on two threonine residues Thr-292 and Thr-386 in vitro, and that these sites are also phosphorylated in vivo. A comparison of the kinetics of serum-mediated activation of a wild-type MAPKK and of a mutant unable to undergo phosphorylation by MAPK suggests that this retrophosphorylation may be involved in a negative feedback control of the cascade in vivo.

Animals↗