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T Combes

Publications and source records attributed to T Combes.

At least 19 recordsLinked to original sources
PubMed1999

Cloning and characterization of PRAX-1. A new protein that specifically interacts with the peripheral benzodiazepine receptor.

Using a cytoplasmic domain of the peripheral benzodiazepine receptor (PBR) as a bait in the yeast two-hybrid system, we have isolated a cDNA encoding a new protein that specifically interacts with PBR. We named it PRAX-1, for peripheral benzodiazepine receptor-associated protein 1. PRAX-1 is a 1857-amino acid protein, the sequence of which was structurally unrelated to any known proteins. The gene encoding PRAX-1 is located in the q22-q23 region of the long arm of the human chromosome 17. The PRAX-1 mRNA is 7.5 kilobase pairs, predominantly expressed in the central nervous system, pituitary gland, and thymus. At the protein level, we found the PRAX-1 as a single 220-250-kDa protein in the brain and in many different human cell lines tested using specific antibody raised against PRAX-1. Parallel analysis of the PRAX-1 mRNA and protein expression performed in mouse and rat gave similar results. Immunocytochemistry analysis carried out to define the distribution of the PRAX-1 protein in the rat brain showed that PRAX-1 was prevalent in the mesolimbic system, specially abundant in the CA1 subfield of the hippocampus. Exhibiting several domains involved in protein-protein interaction (three proline-rich domains, three leucine-zipper motifs, and an Src homology region 3-like domain), the PRAX-1 may be looked upon as a new adaptator protein. We show that both the Src homology region 3-like domain and a proline-rich domain in PRAX-1 are required for the interaction with PBR. PRAX-1 is a cytoplasmic protein that also partially colocalizes with PBR in the mitochondria, as determined by confocal microscopy and Western blotting. Altogether our observations support a model of interaction implicating PBR and this newly described protein, PRAX-1. As being the first cytoplasmic protein associated with PBR, PRAX-1 is a new tool that opens new fields for exploring PBR biological roles.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Colocalization of sterol isomerase and sigma(1) receptor at endoplasmic reticulum and nuclear envelope level.

SR31747A is a sigma ligand previously described as having original immunosuppressive properties. Two SR31747A targets were recently identified and termed sigma(1) or SR-BP-1 (SR31747A-binding protein-1) and hSI (human sterol isomerase). In order to characterize these proteins further, we examined their expression and localization at the subcellular level. Based on the amino acid sequence deduced from the cloned hSI, anti-hSI polyclonal antibody was raised against the N-terminal fragment of the protein. Using this antibody, we performed Western-blot experiments to demonstrate the presence of hSI in various B and T cell lines, and hSI expression was quantified in these cell lines by flow cytometry and estimated at 15 000-30 000 sites per cell. Subcellular localization studies by both confocal and electron microscopy, performed on THP1 cells with anti-hSI antibody and with the previously described anti-(SR-BP-1) monoclonal antibody, demonstrated that: (a) hSI was colocalized with SR-BP-1; (b) hSI and SR-BP-1 were associated with the endoplasmic reticulum and with the outer and inner membranes of the nuclear envelope; (c) both proteins were delocalized during the cell cycle at the mitosis step when the nuclear membranes disappeared. Taken together our results suggest that both SR31747A-binding proteins not only play a role in sterol metabolism but indirectly affect lipoprotein functions.

Antibodies, Monoclonal↗
PubMed1999

Gi protein modulation induced by a selective inverse agonist for the peripheral cannabinoid receptor CB2: implication for intracellular signalization cross-regulation.

The peripheral cannabinoid receptor (CB2) is a G protein-coupled receptor that is both positively and negatively coupled to the mitogen-activated protein kinase (MAPK) and cAMP pathways, respectively, through a Bordetella pertussis toxin-sensitive G protein. CB2 receptor-transfected Chinese hamster ovary cells exhibit high constitutive activity blocked by the CB2-selective ligand, SR 144528, working as an inverse agonist. We showed here that in addition to the inhibition of autoactivated CB2 in this model, we found that SR 144528 inhibited the MAPK activation induced by Gi-dependent receptors such as receptor-tyrosine kinase (insulin, insulin-like growth factor 1) or G protein-coupled receptors (lysophosphatidic acid), but not by Gi-independent receptors such as the fibroblast growth factor receptor. We showed that this SR 144528 inhibitory effect on Gi-dependent receptors was mediated by a direct Gi protein inhibition through CB2 receptors. Indeed, we found that through binding to the CB2 receptors, SR 144528 blocked the direct activation of the Gi protein by mastoparan analog in Chinese hamster ovary CB2 cell membranes. Furthermore, we described that sustained treatment with SR 144528 induced an up-regulation of the cellular Gi protein level as shown in Western blotting as well as in confocal microscopic experiments. This up-regulation occurred with a concomitant loss of SR 144528 ability to inhibit the insulin or lysophosphatidic acid-induced MAPK activation. This inverse agonist-induced modulation of the Gi strongly suggests that the modulated protein is functionally associated with the complex SR 144528/CB2 receptors, and that the Gi level may account for the heterologous desensitization phenomena.

Animals↗
PubMed1999

SR 144528, an antagonist for the peripheral cannabinoid receptor that behaves as an inverse agonist.

In the present report, we investigated in detail the effects of SR 144528, a selective antagonist of the peripheral cannabinoid receptor (CB2), on two well-characterized functions mediated by CB2: the induction of the early response gene krox24 and the inhibition of adenylyl cyclase. We generated Chinese hamster ovary cells doubly transfected with human CB2 and a luciferase reporter gene linked to either the murine krox24 regulatory sequence or multiple cAMP responsive elements. Our results show that (1) SR 144528 antagonizes the effect of receptor agonists-it inhibits the krox24 reporter activity and prevents the inhibition of forskolin-induced cAMP reporter activity mediated by CP 55,940; (2) CB2 is autoactivated-CB2 mediates signaling in the absence of ligand, and this basal activity is reduced by pretreating the cells with pertussis toxin; (3) SR 144528 is an inverse agonist-it reproduces the effects of pertussis toxin; and (4) inhibition of precoupled CB2 by a long-term pretreatment of cells with SR 144528 potentiates krox24 response to cannabinoid receptor agonists and restores activation of adenylyl cyclase. Taken together, these data provide evidences for the inverse agonist property of SR 144528 and the constitutive activation of CB2 in Chinese hamster ovary-expressing cells.

Adenylyl Cyclase Inhibitors↗
PubMed1998

Neurotensin type 1 receptor-mediated activation of krox24, c-fos and Elk-1: preventing effect of the neurotensin antagonists SR 48692 and SR 142948.

Stimulation of neurotensin (NT) type 1 receptors (NT1-R) in transfected CHO cells is followed by the activation of mitogen-activated protein kinases and the expression of the early response gene krox24. By making point mutations and internal deletions in the krox24 promoter, we show that proximal serum responsive elements (SRE) are involved in transcriptional activation by NT. In addition, we show that the related early response gene c-fos and the Ets protein Elk-1 are also induced by NT. The involvement of NT1-R in NT-mediated activation of krox24, c-fos and Elk-1 was demonstrated by the preventing effect of the specific antagonists SR 48692 and SR 142948. Finally, we show that the activation of krox24 and Elk-1 on the one hand, and that of c-fos on the other hand, result from independent transduction pathways since the former are pertussis toxin-sensitive whereas the latter is insensitive to pertussis toxin.

Adamantane↗
PubMed1996

Development of a monoclonal antibody to immuno-cytochemical analysis of the cellular localization of the peripheral benzodiazepine receptor.

Based on the amino acid sequence deduced from the cloned human peripheral benzodiazepine receptor (PBR) gene, monoclonal antibody (Mab 8D7) was produced against the C-terminal fragment of the receptor. Immunoblot experiments, performed against purified PBR, indicated that the antipeptide antibody recognized, under denaturing conditions, the corresponding amino acid sequence of the PBR. When mitochondrial membranes from PBR transfected yeast or from THP1 and U937 cells were used on immunoblot analysis, a high level of immunoreactivity was observed at 18 kDa, the PBR molecular mass deduced from cDNA, establishing the specificity of the antibody for the receptor. Moreover, binding experiments realized with intact mitochondria demonstrated that the immunogenic sequence was accessible to the antibody indicating that the C-terminal fragment of the PBR faces the cytosol. Using this Mab we developed a technique which allowed precise quantification of PBR density per cell. Furthermore, cellular localization studies by flow cytometric analysis and confocal microscopy on cell lines displaying different levels of PBR showed that Mab 8D7 was entirely colocalized with an antimitochondria Mab.

Amino Acid Sequence↗
PubMed1995

[Changes in urethral pressure after intravenous injection of moxisylyte hydrochloride in urethral instability in women. Preliminary results].

OBJECTIVE: To study the action of an alpha blocker, Moxisylyte hydrochloride, during an intravenous test on the course of urethral pressure in women with urethral instability associated with urethral hypertonia. METHODS: The population consisted of 20 women with a mean age of 38 years, presenting with a clinical disorder of micturition (urinary incontinence: 15 cases, urgency: 17 cases, frequency, 17 cases) present for an average of 4 years and associated with resting urethral pressure variations ranging from 22 to 88 cm H2O (mean: 44.8 cm H2O) and static urethral pressures ranging 72 to 150 cm H2O (mean: 102.5 cm H2O). An urodynamic assessment was performed before and after intravenous injection of Moxisylyte hydrochloride at the dose of 0.5 mg/kg. RESULTS: Moxisylyte hydrochloride induced a significant reduction of urethral pressure variations, ranging from 8 to 42 cm H2O (mean: 21.9 cm H2O) and static urethral pressures, ranging from 47 to 102 cm H2O (mean: 68.8 cm H2O). Treatment was well tolerated in every case. CONCLUSION: These preliminary results need to be completed by a randomized placebo-controlled study to confirm a statistically significant effect of Moxisylyte hydrochloride on urethral pressure stability in women presenting with urethral instability.

Adult↗
PubMed1992

[The value of a clinical test for the evaluation of bladder-sphincter function in the management of urinary disorders of the elderly].

The authors report the value of a clinical test for the evaluation of vesico-sphincteric function in the demonstration of urinary disorders in the elderly. This simple, inexpensive test which can be performed at the patient's bedside, was performed in 175 elderly patients with a mean age of 81.2 years, mostly suffering from urinary incontinence (66.2%) and mostly (80%) derived from intermediate and long-term geriatric units. The mechanisms most often detected was detrusor instability (41% of patients). Cystomanometric data, obtained in 80 patients, were analysed and compared retrospectively with the data of the test: they demonstrated a good correlation in terms of evaluation of detrusor stability. As part of the overall management, this test constitutes a real diagnostic tool available to nursing staff, particularly in geriatric institutions not equipped with more specialised investigations.

Aged↗
PubMed1991

[Value of a clinical test for the assessment of bladder and sphincter function in the management of urinary disorders in the aged].

The authors report about the merits of a clinical test for the evaluation of bladder and sphincter function in the analysis of urinary disorders in the aged. This simple and inexpensive bedside test has been carried out in 175 subjects whose mean age was 81.2 years, and the majority of whom (66.2%) presented with urinary incontinence; 80% of the patients came from longterm-care geriatric departments. The most frequent mechanism is bladder instability (41% of all patients). The cystomanometric data obtained in 80 subjects were analysed and retrospectively compared to the test data: they showed good correlation for the study of vesical stability. From the viewpoint of general health care, this test is a real diagnostic tool for the practitioners, in particular in geriatric departments without further specialised exploration commodities.

Age Factors↗
PubMed1986

[Beta-lactamase induction in Pseudomonas aeruginosa by cefpiramide and 3 other antipyocyanic cephalosporins].

Cefpiramide (SR 95445) (CPM) is a new cephalosporin with activity against Pseudomonas and a good bioavailability following parenteral administration. This drug is a first rather than second choice treatment in Pseudomonas infections. For this reason, investigation into cefpiramide's capacity to induce beta-lactamase production is especially interesting. A heavy inoculum of P. aeruginosa NCTC 8203, a strain that produces and inducible cephalosporinase (pI = 8.7) was incubated for 4 hours with CPM, cefsulodin (CFS), cefoperazone (CPZ) and ceftazidime (CTZ) in various concentrations. After collection and sonic treatment of the bacteria, the beta-lactamase activity was assayed using an acidimetric method and expressed as units of enzyme activity per mg proteins in the cell-free extract. The smallest increase in beta-lactamase production was recorded with CPM. The strongest inductor was CTZ. CFS and CPZ had an intermediate effect.

Cefoperazone↗
PubMed1986

[Bactericidal activity of cefpiramide on P. aeruginosa using an in vitro pharmacokinetic simulation model].

Cefpiramide (CPM) is a new cephalosporin with good activity against Pseudomonas. Sustained high serum concentrations are observed. We studied CPM killing kinetics using an in vitro model that simulates the pharmacokinetic profile observed in humans following a single intramuscular injection. The strain tested was Pseudomonas aeruginosa NCTC 8203. CPM was compared to cefoperazone (CPZ), cefsulodin (CFS) and ceftazidime (CTZ). These drugs differed only for the time-interval to bacterial regrowth that was in the following ascending order: CFS, CPZ, CTZ and CPM. This finding corroborates the fact that cefpiramide's pharmacokinetic properties allow wider space intervals between doses than for other drugs.

Cefoperazone↗