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M Caron

Publications and source records attributed to M Caron.

At least 37 records · Page 2Linked to original sources
PubMed2000

Analysis of galectin 1-mediated cell signaling by combined precipitation and electrophoresis techniques.

Galectin-1 (GAL1) is a beta-galactoside-binding protein that has been implicated in the regulation of viability of lymphoid cells. However, the signaling pathway governed by the binding of GAL1 to the cell membrane is not understood yet. As a first step toward the elucidation of GAL1-initiated signaling events, electrophoresis techniques such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and two-dimensional electrophoresis (2-DE) were used together with precipitation techniques. This allowed us to identify the membrane receptor of GAL1, and to characterize the signal resulting from the binding of GAL1 to this receptor. Our results demonstrate that the tyrosine phosphatase CD45 is the receptor for GAL1, and that the src-type tyrosine kinase Lyn is a target for the effects of GAL1/CD45 interactions in B-cells. Furthermore, these results show the usefulness of combined precipitation and electrophoresis techniques to analyze phosphotyrosine-dependent mechanisms during the study of cell functions.

Blotting, Western↗
PubMed2000

Protein analysis by mass spectrometry and sequence database searching: a proteomic approach to identify human lymphoblastoid cell line proteins.

Lymphoblastoid cell lines correspond to in vitro EBV-immortalized lymphocyte B-cells. These cells display a suitable model for experiments dealing with changes in protein expression occurring upon B-cell differentiation, after drug treatment, or after inhibition of some transcription factors. For all these reasons we have undertaken an effort aimed at developing a hematopoietic cell line protein two-dimensional electrophoresis (2-DE) database, containing B-lymphoblastoid 2-DE maps. In this work, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) peptide mass fingerprinting analysis was adopted for protein identification. The peptide mass fingerprinting identification and the sequence coverage obtained on colloidal Coomassie blue (CBB) stained gel was close to that obtained using zinc-imidazole staining. Everything considered, CBB being more comfortable for subsequent spot manipulations, CBB staining was chosen for identification of a larger number of polypeptides. The results suggest that reticulation of the gel can interfere preventing the uptake of the enzyme during the in-gel digestion step. Consequently, low molecular mass proteins appear more difficult to identify by mass fingerprinting. Finally, the information provided in this study allows the construction of a new annoted reference map of human lymphoblastoid cell proteins. Among the identified proteins 60% were not yet positioned on 2-DE maps in three of the most important well-documented databases. The annoted map will be accessible via Internet on the LBPP server at URL:http:// www-smbh.univ-paris13.fr/lbtp/index.htm.

Acrylic Resins↗
PubMed2000

Regulation of CD45-induced signaling by galectin-1 in Burkitt lymphoma B cells.

It has been well established that Galectin-1 (GAL1), a beta-galactoside-binding protein, regulates the viability of lymphoid cells. However, the signaling pathway governed by the binding of GAL1 to the cell membrane is not understood. As a first step towards the elucidation of GAL1-initiated signaling events leading to a reduced viability of Burkitt lymphoma B cells, we tried to characterize the initial events induced by the binding of GAL1 to its receptor. This characterization was performed in BL36 cells, a Burkitt lymphoma cell line sensitive to GAL1. The results were as follows: (1) when solubilized cell membrane lysates were affinity bound to immobilized GAL1 and eluted by competition, the tyrosine phosphatase glyco-protein CD45 was found in the eluate, highlighting the role of CD45 as a receptor of GAL1; (2) the phosphatase activity of cell membranes diminished after incubation with GAL1; (3) immunoprecipitation experiments demonstrated that the phosphotyrosine kinase Lyn was dysregulated in cells that have been cultured in medium containing 700 nM GAL1, and (4) that the ratio between two isoforms of Lyn was modified during the treatment with GAL1. The regulation of Lyn therefore seems to be a key event in the action of GAL1.

B-Lymphocytes↗
PubMed2000

Insulin-mediated cell proliferation and survival involve inhibition of c-Jun N-terminal kinases through a phosphatidylinositol 3-kinase- and mitogen-activated protein kinase phosphatase-1-dependent pathway.

We previously reported that long term treatment with insulin led to sustained inhibition of c-Jun N-terminal kinases (JNKs) in CHO cells overexpressing insulin receptors. Here we investigated the signaling molecules involved in insulin inhibition of JNKs, focusing on phosphatidylinositol 3-kinase (PI 3-K) and mitogen-activated protein kinase phosphatase-1 (MKP-1). In addition, we examined the relevance of JNK inhibition for insulin-mediated proliferation and survival. Insulin inhibition of JNKs was mediated by PI 3-K, as it was blocked by wortmannin and LY294002 and required the de novo synthesis of a phosphatase(s), as it was abolished by orthovanadate and actinomycin D. MKP-1 was a good candidate because 1) insulin stimulation of MKP-1 expression correlated with insulin inhibition of JNKs; 2) insulin stimulation of MKP-1 expression, like insulin inhibition of JNKs, was mediated by PI 3-K; and 3) the transient expression of an antisense MKP-1 RNA reduced the insulin inhibitory effect on JNKs. The overexpression of a dominant negative JNK1 mutant increased insulin stimulation of DNA synthesis and mimicked the protective effect of insulin against serum withdrawal-induced apoptosis. The overexpression of wild-type JNK1 or antisense MKP-1 RNA reduced the proliferative and/or antiapoptotic responses to insulin. Altogether, these results demonstrate that insulin inhibits JNKs through a PI 3-K- and MKP-1-dependent pathway and provide evidence for a key role for JNK inhibition in insulin regulation of proliferation and survival.

Animals↗
PubMed1999

Antiinsulin receptor autoantibodies induce insulin receptors to constitutively associate with insulin receptor substrate-1 and -2 and cause severe cell resistance to both insulin and insulin-like growth factor I.

We report here that antiinsulin receptor (anti-IR) autoantibodies (AIRs) from a newly diagnosed patient with type B syndrome of insulin resistance induced cellular resistance not only to insulin but also to insulin-like growth factor I (IGF-I) for the stimulation of phosphatidylinositol 3-kinase and mitogen-activated protein kinase activities and of glycogen and DNA syntheses. The molecular mechanisms of this dual resistance were investigated. Patient AIRs bound the IR at the insulin-binding site and caused insulin resistance at the IR level by inducing a 50% decrease in cell surface IRs and a severe defect in the tyrosine kinase activity of the residual IRs, manifested by a loss of insulin-stimulated IR autophosphorylation and IR substrate-1 (IRS-1)/IRS-2 phosphorylation. In contrast, cell resistance to IGF-I occurred at a step distal to IGF-I receptors (IGF-IRs), as AIRs altered neither IGF-I binding nor IGF-I-induced IGF-IR autophosphorylation, but inhibited the ability of IGF-IRs to mediate tyrosine phosphorylation of IRS-1 and IRS-2 in response to IGF-I. Coimmunoprecipitation assays showed that in AIR-treated cells, IRs, but not IGF-IRs, were constitutively associated with IRS-1 and IRS-2, strongly suggesting that AIR-desensitized IRs impeded IGF-I action by sequestering IRS-1 and IRS-2. Accordingly, AIRs had no effect on the stimulation of mitogen-activated protein kinase activity or DNA synthesis by vanadyl sulfate, FCS, epidermal growth factor, or platelet-derived growth factor, all of which activate signaling pathways independent of IRS-1/IRS-2. Thus, AIRs induced cell resistance to both insulin and IGF-I through a novel mechanism involving a constitutive and stable association of IRS-1 and IRS-2 with the IR.

Aged↗
PubMed1999

Tibiotalar joint arthrodesis for the treatment of severe ankle joint degeneration secondary to rheumatoid arthritis.

The technical aspects of fusion of the rheumatoid ankle do not deviate from those in the post-traumatic or osteoarthritic ankle. Screw fixation can usually be achieved, and rarely is fixation failure a problem in rheumatoid ankle arthrodesis. If fixation is difficult because of deformity or bone quality, external fixation or locking intramedullary nails should be used. The placement of cannulated screws and adequacy of screw fixation has not been a problem (Fig. 13). Screw fixation provides compression and prevents rotation. The surgeon, however, needs to be assured that no screws invade the subtalar joint and that all threads are beyond the arthrodesis site. A washer may be necessary for further stability if this screw is not inserted at too great an angle. The authors have found that troughing out of the cortical surface of the tibia with a power bur aids in screw insertion. Not only does the trough act as a countersink, but it also provides a path for screw insertion and prevents palpable screw irritation. Malalignment is unforgiving. The foot must be placed neutral to dorsiflexion and plantarflexion. Equinus positioning places added stress on the tibia and a back-knee gait occurs. Approximately 5 degrees of valgus is recommended, and varus positioning is unforgiving. Internal and external rotation is determined by the position of the contralateral extremity. Nonunion does not seem to be a problem with rigid internal fixation to any greater degree in patients with RA. Despite this, patients may continue to have pain despite solid fusion, which can be caused by incomplete correction of deformity, painful internal fixation, or adjacent joint pathology. Additionally, patients may experience supramalleolar pain above the fusion site consistent with tibial stress fracture, which is more common if the subtalar or midtarsal joint is rigid or if the patient is obese. A rocker sole shoe with impact-absorbing soles used after brief periods of guarded mobilization in a removable walking cast alleviates this stress on the tibia. Neurovascular insult can be avoided with careful dissection direct to bone, incisions placed in nerve-free zones, and avoidance of plunging deep posteriorly-medially and anteriorly when dissecting and resecting surfaces. Arthrodesis of the tibiotalar joint in the patient with RA should be performed to relieve severe pain caused by advanced arthrosis. Achieving a solid arthrodesis does not seem to be a problem and provides the patient with pain relief; however, marked improvement in patient function and level of activity remains limited by the nature of RA and adjacent joint involvement.

Ankle Joint↗
PubMed1998

Glycoaffinity chromatography and biological recognition.

The potential of bioaffinity chromatography as a tool for study of biological recognition mechanisms is gaining increasing recognition. Biochromatographic methods allow the separation of proteins according to both the structure of their polypeptidic chain and their post-translational modifications. Among the various post-translational modifications which proteins undergo, glycosylation has conducted to the development of original methods (glycotechnologies). This review discusses the applications of glycotechnologies in bioaffinity chromatography, and particularly the use of biochromatography to elucidate mechanisms involved in glycobiology.

Binding Sites↗
PubMed1998

Affinity purification and characterization of recombinant human galectin-1.

Galectin-1, a polypeptidic factor that can have major effects on cell growth and apoptosis, was overexpressed in E. coli. This protein was purified to homogeneity by affinity chromatography on lactose coupled to divinylsulfone-activated agarose. The recombinant galectin-1 (rGAL1) was compared with the homologous protein purified from human brain tissue using two-dimensional electrophoresis on immobilized pH gradient (IPG-DALT). rGAL1 had a major isoelectric point of 5.4 (major pI of tissular galectin-1, 5.1) and its subunit molecular mass was 14500. Addition of rGAL1 to Jurkat T-lymphoblastoid cells induced cell death in a concentration-dependent manner.

Brain Chemistry↗
PubMed1998

Insulin differentially regulates SAPKs/JNKs and ERKs in CHO cells overexpressing human insulin receptors.

In the present study, we compared the ability of insulin to regulate SAPKs/JNKs and ERKs in CHO cells overexpressing human insulin receptors. We show that acute insulin treatment induced a time-dependent increase both in SAPK/JNK and ERK activity but with distinct kinetics. PI-3-kinase inhibition by wortmannin completely blocked insulin activation of SAPKs/JNKs, whereas it partially decreased ERK activation. Prolonged exposure to insulin caused a marked inhibition of SAPK/JNK activity while it induced a sustained activation of ERKs. Insulin inhibition of SAPKs/JNKs was partly due to decreased tyrosine phosphorylation of JNK2. These data indicate that insulin differentially regulates SAPKs/JNKs and ERKs. Moreover, they provide the first evidence that insulin exerts opposite effects on SAPK/JNK activity according to the time of cell treatment.

Androstadienes↗
PubMed1998

Realistic expectations for pedal bypass grafts in patients with end-stage renal disease.

PURPOSE: Limb-threatening ischemia in patients with end-stage renal disease (ESRD) represents a challenging clinical problem. Multiple series have shown the inferior limb salvage rate for femoropopliteal or femorotibial bypass grafts in this group. This outcome study is restricted to those patients with ESRD who require pedal bypass grafts for attempted limb salvage. METHODS: Between December 1, 1990, and December 31, 1997, 34 patients with ESRD underwent pedal bypass grafting on 41 limbs. This review explores the patient and bypass graft outcomes and their relationships to typical risk factors. RESULTS: The average age in the study was 64 years (range, 39 to 85 years). Twenty patients (59%) were men, 31 (91%) had diabetes, 32 (94%) were hypertensive, and 28 (82%) had coronary artery disease, but only 10 patients (29%) were smokers. All the patients were undergoing dialysis except 2 patients with functioning renal transplants. All bypass grafting procedures were performed for limb salvage. The follow-up periods ranged from 1 to 84 months (average, 13.5 months). With life-table analysis, the cumulative assisted primary patency rate was 62% at 1 year and 62% at 2 years. The limb salvage rate was 56% and 50% at 1 and 2 years, respectively. All the patients who were seen with heel gangrene had early limb loss or died. Seven of the 16 amputations (44%) were performed despite patent bypass grafts. Ten of the 16 amputations (63%) occurred within 3 months of the surgery. The survival rate was 64% at 1 year and 52% at 2 years. After the bypass graft procedure, the mean ankle brachial index and the toe pressure rose from 0.48 to 1.05 and 18 to 86, respectively. CONCLUSION: Modest success can be expected with pedal bypass grafts in patients with ESRD, with most failures occurring in the first 3 months. Limb salvage rates lag behind graft patency rates because of progressive necrosis despite a hemodynamically functioning bypass graft. Heel gangrene is a strong predictor for a negative outcome. Lastly, overall patient survival rates are poor but comparable with the rates of other patients with ESRD.

Adult↗
PubMed1998

Production and characterization of a monoclonal antibody able to discriminate galectin-1 from galectin-2 and galectin-3.

Antisera raised against galectin-1 exhibit crossreactivities with other galectins or related molecules. In order to overcome this problem, a monoclonal antibody to human brain galectin-1 was obtained by selecting clones without reactivity toward galectin-3. This mAb specifically bound galectin-1 of various animal origins but neither galectin-2 nor galectin-3. Western-blotting analysis of soluble human brain extracts after 2D gel electrophoresis revealed only the two most acidic isoforms of galectin-1. The ability of this mAb to bind galectin-1/asialofetuin complexes indicates that its epitope is not localized in the carbohydrate recognition domain of galectin-1. This particularity induces with efficiency its monospecificity.

Amino Acid Sequence↗
PubMed1998

Increase in extracellular glutamate caused by reduced cerebral perfusion pressure and seizures after human traumatic brain injury: a microdialysis study.

OBJECT: To determine the extent and duration of change in extracellular glutamate levels after human traumatic brain injury (TBI), 17 severely brain injured adults underwent implantation of a cerebral microdialysis probe and systematic sampling was conducted for 1 to 9 days postinjury. METHODS: A total of 772 hourly microdialysis samples were obtained in 17 patients (median Glasgow Coma Scale score 5+/-2.5, mean age 39.4+/-20.4 years). The mean (+/-standard deviation) glutamate levels in the dialysate were evaluated for 9 days, during which the mean peak concentration reached 25.4+/-13.7 microM on postinjury Day 3. In each patient transient elevations in glutamate were seen each day. However, these elevations were most commonly seen on Day 3. In all patients there was a mean of 4.5+/-2.5 transient elevations in glutamate lasting a mean duration of 4.4+/-4.9 hours. These increases were seen in conjunction with seizure activity. However, in many seizure-free patients the increase in extracellular glutamate occurred when cerebral perfusion pressure was less than 70 mm Hg (p < 0.001). Given the potential injury-induced uncoupling of cerebral blood flow and metabolism after TBI, these increases in extracellular glutamate may reflect a degree of enhanced cellular crisis, which in severe head injury in humans appears to last up to 9 days. CONCLUSIONS: Extracellular neurochemical measurements of excitatory amino acids may provide a marker for secondary insults that can compound human TBI.

Adult↗
PubMed1997

Anti-galectin-1 autoantibodies in serum of patients with neurological diseases.

The presence of autoantibodies to human brain galectin-1 was investigated in serum from patients with multiple sclerosis, patients with or without evidence of other neurological disorders, and healthy controls, using an ELISA on purified brain galectin-1. Levels of autoantibodies to galectin-1 were significantly higher in patients than in healthy controls. Comparison of levels of anti-galectin-1 and anti-idiotypic antibodies mimicking human brain galectin-1 (L-IgG) showed that the highest levels of autoantibodies were present in patients with low levels of L-IgG. This finding can be explained by hypothesizing that the concentration of autoantibodies to galectin-1 is possibly associated with impairment of the regulation of the immune system.

Adult↗
PubMed1997

Stimulation of the angiotensin II type I receptor on bovine adrenal glomerulosa cells activates a temperature-sensitive internalization-recycling pathway.

Angiotensin II (Ang II) is an important regulator of aldosterone production by bovine adrenal glomerulosa cells. On these cells Ang II interacts with the AT1 receptor that is coupled to a G protein controlling the activity of phospholipase C. A primary culture of bovine adrenal glomerulosa cells was used to study the internalization-recycling mechanism of the AT1 receptor after stimulation with Ang II. When cells were pretreated with 10 nM Ang II for 30 min at 37 degrees C and binding studies were performed at 12 degrees C we observed a 48% loss in [125I]Ang II binding. Scatchard analysis revealed that this loss in binding translated into a decreased affinity of the AT1 receptor without any loss in the total amount of binding sites. Under the same conditions an important internalization of [125I]Ang II was invariably observed. These observations suggest that a mechanism was at work to recycle the internalized receptors to the cell surface during the binding studies. Following internalization we indeed observed an externalization of [125I]Ang II. This phenomenon relatively rapid at 37 degrees C was much slower at 12 degrees C and completely inhibited at 4 degrees C. When cells were pretreated with 10 nM Ang II for 30 min at 37 degrees C binding assays at 4 degrees C no longer revealed a loss of binding affinity but rather a 54% reduction in the total amount of binding sites. The maximal binding capacity could be recovered during incubations at 12 degrees C. These results reveal the existence of a dynamic recycling process for the AT1 receptor. In accordance with this interpretation the phenomenon was blocked by monensin, a known inhibitor of receptor recycling. These studies suggest that the stimulation of the AT1 receptor sets in motion an internalization-recycling process that seems to be a fundamental aspect of the AT1 receptor transduction mechanism.

Angiotensin II↗
PubMed1997

Purification and characterization of a human brain galectin-1 ligand.

Our previous studies have characterized an endogenous lectin from human brain identified as galectin-1. A soluble ligand of galectin-1 was purified from human brain by affinity chromatography and preparative electrophoresis. The purified ligand (termed HBGp82, for human brain galectin-1-binding polypeptide of 82,000 daltons) has an apparent molecular mass of 82 kDa and is glycosylated by N-linked biantennary complex structures. HBGp82 was partially characterized by microsequencing of peptide fragments. Similar peptides were found in a heat shock of protein of 90,000 daltons, hsp90. However, comparison of apparent molecular weights and matrix-assisted laser desorption mass spectrometry clearly showed that HBGp82 differs to some degree from hsp90.

Amino Acid Sequence↗
PubMed1997

Externalization and binding of galectin-1 on cell surface of K562 cells upon erythroid differentiation.

Galectin 1 (GAL1) is a beta-galactoside-binding lectin involved in cell cycle progression. GAL1 overexpression is associated with neoplastic transformation and loss of differentiation. The gene encoding for human GAL1 resides on chromosome 22(q12; q13), and its expression is developmentally regulated. Although devoid of signal peptide GAL1 can be externalized from cells by a mechanism independent of the normal secretory process. We report here on a study of the effects of erythroid differentiation of the human leukemia cell line K562 on GAL1 protein expression. In undifferentiated K562 cells, GAL1 was expressed into the cytosol. However, the amount of GAL1 was surprisingly weaker in K562 cells than in other leukemia cell lines such as TF-1 or KG1a. Treatment of K562 cells with erythropoietin (EPO) or with aphidicolin (APH), an inhibitor for DNA polymerase alpha, induced an erythroid phenotype and led to the externalization of cytosolic GAL1 which was then bound to ligands on cell surface in a galactoside-inhibitable fashion. Our results demonstrate that acquisition of an erythroid phenotype is associated with an externalization of GAL1. The autocrine binding of GAL1 to cell surface ligands of non adherent cells such as K562 suggest that GAL1 is implicated rather in signal transduction than in cell-cell or cell-matrix interaction. Moreover, the reciprocal translocation involving chromosomes 9 and 22 t(9;22) present in K562 cells might explain the weak expression of GAL1 in K562 leukemia cells.

Aphidicolin↗
PubMed1997

Major circadian variations of glucose homeostasis in a patient with Rabson-Mendenhall syndrome and primary insulin resistance due to a mutation (Cys284-->Tyr) in the insulin receptor alpha-subunit.

We have performed clinical, in vitro biochemical, and genetic studies of a patient with severe insulin resistance, considerable growth restriction, and Rabson-Mendenhall syndrome (patient RM-3). The blood IGF-I level was undetectable in this patient, although the GH level was moderately decreased. During the postprandial period, glycemia, ketonuria, and plasma glucagon were very elevated despite high doses of exogenous insulin (glucose levels up to 30 mmol/L). In the postabsorptive state, blood glucose was normalized with small amounts of insulin; ketonuria, and glucagon levels were reduced but remained supranormal. Erythrocytes and cultured skin fibroblasts from the patient displayed a decrease in cell surface insulin receptors (IRs). The ability of physiologic concentrations of insulin to stimulate metabolic processes was altered in patient fibroblasts. Analysis of the IR gene by denaturing gradient gel electrophoresis and direct sequencing showed a homozygous missense mutation in exon 3, replacing Cys284 by Tyr in the alpha-subunit. In conclusion, marked primary insulin resistance was evidenced in patient cells as a result of a structural alteration in the IR alpha-subunit. The in vitro studies could not account alone for the in vivo metabolic alterations because glucose homeostasis varied considerably during the day in the patient.

Amino Acid Sequence↗