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R Mercier

Publications and source records attributed to R Mercier.

At least 19 recordsLinked to original sources
PubMed2005

Purification and mass spectroscopic analysis of human CB1 cannabinoid receptor functionally expressed using the baculovirus system.

The cannabinoid receptor 1 (CB1) cannabinoid receptor is an essential component of the cannabinergic system. It has been recognized as a therapeutic target for treating numerous diseases and is currently receiving considerable attention by the pharmaceutical community. Target-based drug design, utilizing three-dimensional information of receptor structure and ligand-binding motifs, requires significant amounts of purified protein. To facilitate the purification of CB1, we have expressed the receptor fused to various epitope tags using the baculovirus expression system. In addition, expression levels and ligand-binding profiles corresponding to the expressed fusion proteins have been compared. C-terminal histidine (His)-tagged CB1 gave a Bmax higher than most other systems previously reported in the literature, and was selected for subsequent metal affinity chromatography purification and mass spectroscopic (MS) analysis. Moreover, cells expressing C-terminal His-tagged CB1 were shown to inhibit forskolin-stimulated cyclic adenosine 3',5'-monophosphate (cAMP) production in a concentration-dependent manner in the presence of CP-55,940, confirming the expressed receptor's functional characteristics. A Western blot analysis of the purified receptor showed several forms of CB1, the most abundant being a 57 kDa monomeric protein. The purified CB1 preparations were subjected to protein digestion followed by MS. Fragments corresponding to >70% of the receptor were identified by this method, confirming the identity and purity of the expressed protein. The work presented here demonstrates that epitope-tagged CB1 can be expressed in sufficient amounts and purified to homogeneity for MS analysis. Moreover, these results will serve as a basis for future experiments aimed at characterizing the ligand-binding domains using covalently reacting receptor probes.

Amino Acid Sequence↗
PubMed2005

A strategy to investigate the plant meiotic proteome.

The analysis of meiosis in higher plants has benefited considerably in recent years from the completion of the genome sequence of the model plant Arabidopsis thaliana and the development of cytological techniques for this species. A combination of forward and reverse genetics has provided important routes toward the identification of meiotic genes in Arabidopsis. Nevertheless identification of certain meiotic genes remains a challenge due to problems such as limited sequence conservation between species, existence of closely related gene families and in some cases functional redundancy between gene family members. Hence there is a requirement to develop new experimental approaches that can be used in conjunction with existing methods to enable a greater range of plant meiotic genes to be identified. As one potential route towards this goal we have initiated a proteomics-based approach. Unfortunately, the small size of Arabidopsis anthers makes an analysis in this species technically very difficult. Therefore we have initially focussed on Brassica oleracea which is closely related to Arabidopsis, but has the advantage of possessing significantly larger anthers. The basic strategy has been to use peptide mass-finger printing and matrix-assisted laser desorption ionization time of flight mass spectrometry to analyse proteins expressed in meiocytes during prophase I of meiosis. Initial experiments based on the analysis of proteins from staged anther tissue proved disappointing due to the low level of detection of proteins associated with meiosis. However, by extruding meiocytes in early prophase I from individual anthers prior to analysis a significant enrichment of meiotic proteins has been achieved. Analysis suggests that at least 18% of the proteins identified by this route have a putative meiotic function and that this figure could be as high as one-third of the total. Approaches to increase the enrichment of proteins involved in meiotic recombination and chromosome synapsis are also described.

Brassica↗
PubMed2004

Anandamide transport is independent of fatty-acid amide hydrolase activity and is blocked by the hydrolysis-resistant inhibitor AM1172.

The endogenous cannabinoid anandamide is removed from the synaptic space by a high-affinity transport system present in neurons and astrocytes, which is inhibited by N-(4-hydroxyphenyl)-arachidonamide (AM404). After internalization, anandamide is hydrolyzed by fatty-acid amide hydrolase (FAAH), an intracellular membrane-bound enzyme that also cleaves AM404. Based on kinetic evidence, it has recently been suggested that anandamide internalization may be mediated by passive diffusion driven by FAAH activity. To test this possibility, in the present study, we have investigated anandamide internalization in wild-type and FAAH-deficient (FAAH(-/-)) mice. Cortical neurons from either mouse strain internalized [(3)H]anandamide through a similar mechanism, i.e., via a rapid temperature-sensitive and saturable process, which was blocked by AM404. Moreover, systemic administration of AM404 to either wild-type or FAAH(-/-) mice enhanced the hypothermic effects of exogenous anandamide, a response that was prevented by the CB(1) cannabinoid antagonist rimonabant (SR141716A). The results indicate that anandamide internalization in mouse brain neurons is independent of FAAH activity. In further support of this conclusion, the compound N-(5Z, 8Z, 11Z, 14Z eicosatetraenyl)-4-hydroxybenzamide (AM1172) blocked [(3)H]anandamide internalization in rodent cortical neurons and human astrocytoma cells without acting as a FAAH substrate or inhibitor. AM1172 may serve as a prototype for novel anandamide transport inhibitors with increased metabolic stability.

Amidohydrolases↗
PubMed2004

Purification and mass spectroscopic analysis of human CB2 cannabinoid receptor expressed in the baculovirus system.

The cannabinergic system is present in a variety of organs and tissues that perform a wide range of essential physiologic functions making it an inherently important therapeutic target for drug discovery. In order to augment our knowledge regarding the interactions between cannabinoid receptors (CBs) and their ligands, efficient and effective tools are essential for robust expression and purification of these membrane-bound proteins. In this report, we describe a suitable method for purification of the human cannabinoid receptor 2 (CB2) to a qualitative and quantitative level sufficient for mass spectral analysis. We utilized a baculovirus expression system, incorporating several epitope tags to facilitate purification and to ameliorate the effect the tags have on CB2 expression and function. Expressed protein encoded by a carboxy (C)-terminal His-tagged CB2 construct displayed a B(max) value of 9.3 pmol/mg with a K(D) of 7.30 nM using [3(H)]CP-55(940), a standard cannabinoid radioligand, and was selected for subsequent purification experiments. Western blot analysis of purified membrane protein yielded several forms of CB2, the most abundant being a 41 kDa peptide. A second protein species was observed with an apparent molecular weight of 46 kDa representing a glycosylated form of CB2. In addition, a CB2 homodimer was also identified. The purified receptor was subjected to mass spectroscopic analysis to confirm its identity and purity. Mass spectra corresponding to the intracellular, extracellular and transmembrane domains were obtained. These experiments exemplify the importance of high-level expression systems when developing membrane-bound protein purification strategies. This work will aid in the identification of receptor-ligand binding sites, the characterization of molecular features involved in receptor activation, and the elucidation of the CB2 receptor tertiary structure.

Amino Acid Sequence↗
PubMed2001

SWITCH1 (SWI1): a novel protein required for the establishment of sister chromatid cohesion and for bivalent formation at meiosis.

We have characterized a new gene, SWI1, involved in sister chromatid cohesion during both male and female meiosis in Arabidopsis thaliana. A first allele, swi1.1, was obtained as a T-DNA tagged mutant and was described previously as abnormal exclusively in female meiosis. We have isolated a new allele, swi1.2, which is defective for both male and female meiosis. In swi1.2 male meiosis, the classical steps of prophase were not observed, especially because homologs do not synapse. Chromatid arms and centromeres lost their cohesion in a stepwise manner before metaphase I, and 20 chromatids instead of five bivalents were seen at the metaphase plate, which was followed by an aberrant segregation. In contrast, swi1.2 female meiocytes performed a mitotic-like division instead of meiosis, indicating a distinct role for SWI1 or a different effect of the loss of SWI1 function in both processes. The SWI1 gene was cloned; the putative SWI1 protein did not show strong similarity to any known protein. Plants transformed with a SWI1-GFP fusion indicated that SWI1 protein is present in meiocyte nuclei, before meiosis and at a very early stage of prophase. Thus, SWI1 appears to be a novel protein involved in chromatid cohesion establishment and in chromosome structure during meiosis, but with clear differences between male and female meiosis.

Alleles↗
PubMed1999

Platelet surface p-selectin, platelet-granulocyte heterotypic aggregates, and plasma-soluble p-selectin during plateletpheresis.

BACKGROUND: Plateletpheresis components have been shown to contain p-selectin-positive platelets after collection and storage. P-selectin mediates binding of activated platelets to granulocytes and monocytes. This study was undertaken to assess platelet activation, granulocyte activation, platelet-granulocyte heterotypic aggregate formation, and the plasma-soluble p-selectin level during plateletpheresis performed on a particular instrument (MCS+, Haemonetics). STUDY DESIGN AND METHODS: Flow cytometry was used to assay platelet surface p-selectin, granulocyte iC3b receptor, and platelet-granulocyte aggregates in the platelet component, residual blood in the disposable polycarbonate bowl of the MCS+, and in the donor blood with and without the addition of in vitro agonists before, during, and after plateletpheresis. The plasma-soluble p-selectin levels in the platelet component, disposable bowl, and donor venous blood were measured by an enzyme-linked immunosorbent assay. RESULTS: Levels of p-selectin-positive platelets, activated granulocytes, and platelet-granulocyte aggregates were greater in the disposable bowl than in the preapheresis donor blood. Levels of p-selectin-positive platelets, activated granulocytes, and platelet-granulocyte aggregates in the postapheresis donor blood were similar to those in the preapheresis donor blood. The platelet components contained no activated granulocytes or detectable platelet-granulocyte heterotypic aggregates, and only about 10-percent activated platelets. The plasma-soluble p-selectin level in the platelet component was significantly greater than that in the preapheresis donor blood, the residual blood in the disposable bowl, or the postapheresis donor blood. CONCLUSIONS: Measurements of platelet surface p-selectin, platelet-granulocyte heterotypic aggregates, and plasma-soluble p-selectin can be used to detect platelet activation during plateletpheresis.

Antibodies, Heterophile↗
PubMed1997

TPDC-FuHu chemotherapy for the treatment of recurrent metastatic brain tumors.

PURPOSE: To evaluate a combination of thioguanine, procarbazine, dibromodulcitol, CCNU (CCNU), fluorouracil, and hydroxyurea (TPDC-FuHu), designed to improve the efficacy of CCNU, in the treatment of recurrent metastatic brain tumors. PATIENTS AND METHODS: One hundred fifteen patients with progressive or recurrent metastatic brain tumors that failed to respond to surgery and/or radiation therapy were enrolled onto a multicenter prospective study between 1989 and 1995. Patients received TPDC-FuHu in a repeated cycle every 6 weeks until recurrence or until they completed six courses. RESULTS: Ninety-seven patients were assessable at the end of the study. Forty-eight had lung cancer (39 non-small-cell [NSCLC] and nine small-cell [SCLC]), 28 had breast cancer, nine had melanoma, and 12 had adenocarcinoma of different origins (three colon, two kidney, one bladder, one stomach, and five of unknown origin). The response and stable disease (SD) rate (overall response rate) was 52%, 66%, 60%, and 22% in patients with NSCLC, SCLC, breast cancer, and melanoma, respectively. Median time to progression (MTP) was 12, 26, 12, and 6 weeks, respectively, for the four groups. Side effects were mild to moderate in the majority of patients. Severe myelosuppression (grade 4) occurred in only 11% of the patients. CONCLUSION: TPDC-FuHu chemotherapy is an active treatment against recurrent brain metastases from breast cancer and SCLC, and to a lesser extent from NSCLC. This regimen is well tolerated and has acceptable toxicity.

Adenocarcinoma↗
PubMed1989

[Retroperitoneal perforation of duodenal ulcer responsible for abscess].

The authors report a case of retroperitoneal perforation of a duodenal ulcer. This is a rare condition which is exceptionally responsible for abscess formation. If often has a poor prognosis. In the present case, the abscess was infrahepatic. The diagnosis of the origin of the abscess was established by radiological opacification via percutaneous puncture. Surgical drainage and irrigation performed via an extraperitoneal approach centered over the cavity, combined with triple-agent antibiotic therapy (penicillin, gentamicin, metronidazole) ensured collapse followed by sterilisation of the abscess cavity. The ulcer was cured by medical treatment. This case differs from the published case in terms of the diagnostic procedures classically used (upper gastrointestinal endoscopy and small bowel series), the infrahepatic site of the abscess and the extraperitoneal surgical treatment which avoided dissemination to the peritoneal cavity.

Abscess↗
PubMed1986

[Injuries of the thoracic duct during neck surgery].

Although rarely published, operative injuries to thoracic duct in neck are by no means exceptional, and can occur during all types of surgery to lower cervical and supraclavicular regions. A case is reported and used as a basis for an analysis of diagnostic means and therapeutic possibilities of injuries detected during operation or those developing manifestations at a later stage. In the case reported, long-term medical treatment resulted in arrest of lymphorrhea within 37 days.

Adult↗
PubMed1985

[Mediastinoscopy in the evaluation of the operability of bronchial cancer].

Mediastinoscopy is an invasive technique used to explore the anterior and upper mediastinum. The axial mediastinum and its lymph nodes are explored through the cervical approach, and the pulmonary vessels and anterior mediastinal lymph nodes through the parasternal approach. Bronchial carcinomas which extend to the mediastinum, either via the lymphatic system or by contiguity, are of extremely severe prognosis. The advent of mediastinoscopy has considerably reduced the number of exploratory thoracotomies and therefore the operative mortality rate, while increasing the resectability rate to 95%. It has also narrowed the indications for surgery without decreasing the number of curative resections. In the hands of experienced operators, the technique has a low morbidity rate. CT examination is not as accurate as mediastinoscopy to evaluate the mediastinal extension of bronchial carcinomas, but it can help to lay down the indications for the invasive technique, thereby limiting its use to selected cases.

Aged↗
PubMed1984

Prophylactic use of cefotaxime in colonic and rectal surgery.

Two hundred and seventeen patients, undergoing abdominal colonic and rectal surgery, received after randomization, the following regimen: group A (74 patients): cefotaxime 1 g intravenous at the induction of anaesthesia, the beginning of the resection, 4 and 8 h later; group B (72 patients): cefotaxime in the same regimen associated with ornidazole or metronidazole 0.5 g intravenous at the induction of anaesthesia and 0.5 g intravenous with the last injection of cefotaxime; group C (71 patients):cefotaxime following the same regimen as groups A and B and metronidazole orally 0.5 tds 3 days before surgery. All wounds were assessed daily, until discharge from hospital. Severe sepsis included: septicaemia, peritonitis, intra-abdominal abscess and extra-abdominal infections with death. Non-severe sepsis included all others. All the patients having a history of allergy to beta-lactam antibiotics and those with pre-operative infection were excluded. Mean age of the population was: 64.5 years. Seventy-seven patients had rectal cancer and 82 patients cancer of the colon; Twenty-five patients had inflammatory bowel disease, and in 33 others disease such as polyposis was present. Risk factors of post-operative infection were present in 115 cases (A, 36 patients; B, 37 patients; C, 42 patients). All three groups were very well matched for age, sex, type of intervention and diagnosis. Non-infectious complications appeared in 56 patients. Sepsis developed in 76 patients (A, 27 patients; B, 27 patients; C, 22 patients, no significant difference). Severe sepsis occurred in 14 patients (A, 6 patients; B, 4 patients; C, 4 patients, no significant difference) and in 62 patients non severe sepsis (A, 21 patients; B, 23 patients; C, 18 patients, no significant difference). Post-operative peritonitis was not seen. This study suggests that cefotaxime alone 4 g peri-operatively is useful in prophylaxis during rectal and colonic surgery.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1984

[Cystic abdomino-mediastinal lymphangioma. Apropos of a case. Review of the literature].

A further case of cystic lymphangioma is reported, characteristic features being the multiple abdominal and thoracic localizations of the lesions. Apart form splenic sites, it is rare to detect lesions in the mesogastric and mediastinal regions, particularly as propagation of abdominal lymphangioma towards the mediastinum is an exceptional occurrence. A literature review provides data on the various regions in which cysts have been reported in the body, and the diagnostic problems they raise.

Abdominal Neoplasms↗
PubMed1983

[Explosion of intestinal gas during surgery].

Two cases of colonic gas explosion during surgery are reported. The treatment of the lesions required a partial colectomy in one case and a total colectomy in the other case. The different factors involved in such accidents are discussed. Three factors are necessary to trigger off an explosion of intestinal gases: the presence of combustible gases (hydrogen, methane), the presence of combustive gases (oxygen, nitrous oxide) and an initiating heat source (endoscopic or surgical electrocautery). The mannitol used for bowel cleansing undergoes partial colonic bacterial fermentation increasing the intraluminal concentration of hydrogen. During anaesthesia the oxygen-nitrous oxide mixture increases the intestinal concentration of these two major combustive gases. Electrocautery provides the spark triggering the explosion. The use of mannitol for colonic preparation should be questioned; the use of electrocautery to open the colon is advised against.

Aged↗