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J Léger

Publications and source records attributed to J Léger.

At least 55 records · Page 3Linked to original sources
PubMed1993

Monoclonal antibodies targeted against the C-terminal domain of dystrophin or utrophin.

The structure-function relationships of dystrophin, a protein which is absent or defective in patients with Duchenne or Becker muscular dystrophies, and utrophin can only be compared if specific antibodies are produced. We expressed C-terminal parts of dystrophin and utrophin in expression vectors. Mice were immunized with recombinant proteins and 26 monoclonal antibodies were produced and analyzed. Their respective epitopes were determined using other overlapping recombinant products. We observed antibody specificity towards 400 kDa dystrophin and/or utrophin protein bands, either by Western blot analysis or immunodetection in human skeletal (quadriceps) and smooth (uterus) muscles. These antibodies have been used to compare the relative abundance of both dystrophin and utrophin relative to the structures analyzed.

Animals↗
PubMed1993

Genomic organization and expression of the rat TRPM-2 (clusterin) gene, a gene implicated in apoptosis.

We describe the genomic structure of the rat TRPM-2 gene. This gene is induced de novo during the regression of the prostate and other hormone-dependent tissues after hormone ablation and plays an important role in apoptosis, or active cell death. TRPM-2 is a single copy gene, organized into nine exons, ranging in size from 47 base pairs (exon I) to 412 base pairs (exon IV), spanning a region of 13,750 base pairs. Comparison with sequences registered in the data bases shows that it has extensive homology to SGP-2, a gene that is expressed constitutively in the testis, although the sequences diverge dramatically in the 5'-untranslated region of the mRNA (coded for by exon I in TRPM-2), raising the possibility of alternative exon I usage in SGP-2. Primer extension, S1 nuclease protection, and extensive polymerase chain reaction analysis suggest that the TRPM-2 transcript from the prostate and the SGP-2 transcript from the testis are in fact identical and only contain the exon I sequence identified in the TRPM-2 genomic clone. Analysis of the promoter region of the TRPM-2 gene demonstrates that the putative control region contains several potential regulatory elements that may regulate the complex tissue-specific control of a gene which must be constitutively expressed in some tissues but repressed in others until induced during active cell death.

Amino Acid Sequence↗
PubMed1993

Variability of serum human growth hormone levels in different commercial assays: specificity of growth hormone-releasing hormone stimulation.

Serum human growth hormone (hGH) levels were measured with three different commercial kits, comprising a radioimmunoassay (RIA) (bioMérieux, Marcy-l'Etoile, France, 'bmpoly') and two immunoradiometric assays (IRMA) (bioMérieux, 'bmmono', and CIS bioInternational, Gif-sur-Yvette, France, 'cismono'). Samples were collected after various stimulation tests [arginine-insulin, growth hormone-releasing hormone (GHRH), L-dopa and glucagon-beta-axolol] from children who were undergoing evaluation for short stature. Values obtained with the IRMAs were consistently lower than those obtained with the RIA. Furthermore, the cismono/bmmono and cismono/bmpoly values ratios were always significantly higher when samples were collected during GHRH stimulation than during the other stimulation tests. These data indicate that GHRH could induce a particular form of hGH molecule (in nature or in amount), recognized by the monoclonal antibodies in the cismono kit and that a specific form of hGH may be released by GHRH stimulation.

Child↗
PubMed1993

[Bone demineralization and elevation of serum osteocalcin concentrations in young children with hyperthyroidism].

Bone mineralization and serum osteocalcin level were evaluated in 15 children with Grave's disease. Two groups were constituted according to the presence (group I: n = 9) or absence (group II: n = 6) of a severe bone demineralization. A spontaneous fracture and a collapsed vertebra were found in one group I patient. Patients in group I were younger than in group II (8.3 +/- 4.9 vs 11.5 +/- 4.3 yrs). One patient in group II and six in group I were prepubertal with advanced bone age and increased growth velocity. Osteocalcin measurement (Oc) was performed in 10 patients (group I: n = 6; group II: n = 4) at the time of biological hyperthyroidism. The six patients with bone demineralization had elevated Oc levels. In group II, two patients had normal Oc levels and two had elevated Oc levels. In treated patients with good control of hyperthyroidism, all group II patients except one, had normal serum Oc levels and bone mineralization remain normal (n = 5) after 0.6 to 4.6 yrs of follow-up. In group I patients, although height velocity was normal, elevated (n = 4) or slightly elevated (n = 1) serum Oc levels and severe bone demineralization (n = 7 cases) persisted after 0.5 to 3 yrs of good control of the hyperthyroidism. Although the method used for measuring bone mineralization is potentially less precise than bone densitometry and not all the patients had serum osteocalcin measurements at the same time of the illness, our results emphasize that skeletal demineralization may be particularly marked in young children with Grave's disease and should be carefully evaluated.

Absorptiometry, Photon↗
PubMed1993

[Psychiatric sequelae of diencephalic tumors in children. 3 case reports].

Three cases of behavioral disorders in children previously treated for a diencephalic tumor are reported. In such cases, classical psychiatric evaluation is difficult because reference data on childhood psycho-organic syndromes are lacking. Another complicating feature is that multiple factors contribute to the impairment of social interactions, including maladaptive reactions to disease-related stress, sensory deficits, and cognitive disorders. A structured evaluation should be performed in order to assess the child's behavior, the psychological and educational environment, and impairment of neuropsychological and neurophysiological function.

Adaptation, Psychological↗
PubMed1992

A homologue of dystrophin is expressed at the blood vessel membrane of DMD and BMD patients: immunological evidence.

Muscles from Becker muscular dystrophy (BMD) and Duchenne muscular dystrophy (DMD) patients were analysed using monoclonal and polyclonal antibodies raised against different regions of the dystrophin molecule. On blot, two of the antibodies detected a protein of Mr 400K in muscle extracts from all patients, including a BMD patient with a deletion which spanned more than 40% of the central rod domain of the Xp21 encoded dystrophin. Immunocytochemical labelling of tissue sections from the same patients showed that the same two antibodies labelled a protein at the surface membrane of smooth muscle fibers in blood vessels of both BMD and DMD muscles. Thus we have demonstrated a 400K blood vessel-associated protein, which is immunologically homologous with dystrophin, for at least two epitopes from the carboxy terminal and the central rod domains must be encoded by another gene than the dystrophin gene.

Adult↗
PubMed1992

Patterns of dystrophin expression in developing, adult and regenerating tail skeletal muscle of Amphibian urodeles.

The patterns of expression of dystrophin were investigated by indirect immunofluorescence and by immunoblotting in developing, adult and regenerating tail skeletal muscle of newts Pleurodeles waltl and Notophthalmus viridescens. In this study, a monoclonal antibody H-5A3 directed against the C-terminal region (residues 3357-3660) and a polyclonal antibody raised to the central domain (residues 1173-1738) of the chicken skeletal muscle dystrophin were used. Western blot analysis showed that these antibodies recognized a 400 kDa band of dystrophin (and may be of dystrophin-related protein) in the adult muscle tissues and in newt tail regenerates. During skeletal muscle differentiation or epimorphic regeneration (blastema), anti-dystrophin immunoreactivity gradually accumulated over the periphery of the myofibers. Dystrophin and laminin were first and concomitantly observed at the ends of the newly formed myotubes where they were anchored on connective tissue septa or bone processes by dystrophin-rich myotendinous structures. It is noteworthy that neuromuscular junctions, which most probably also contain dystrophin, are established in urodeles near the ends of the myofibers as shown by histochemical localization of AChE activity or fluorescent bungarotoxin detection of AChRs. In the stump transition zone close to the tail amputation level where tissue regeneration of injured muscle fibers took place, dystrophin staining located on the cytoplasmic surface of myofibers progressively disappeared during the dedifferentiation process which seemed to occur during muscle regeneration as suggested by electron microscopy. Furthermore, double labeling experiments using anti-dystrophin and anti-laminin antibodies showed a good correlation between the remodeling processes of the muscle fiber basal lamina and the loss of dystrophin along the sarcolemma of damaged and presumably dedifferentiating muscle cells.

Age Factors↗
PubMed1991

Imaging of myocardial infarction in dogs and humans using monoclonal antibodies specific for human myosin heavy chains.

The use of three different monoclonal antibodies specific for human ventricular myosin heavy chains in the visualization of the location and extent of necrosis in dogs with experimental acute myocardial infarction and in humans is described. Using a classic immunohistochemical method or ex vivo analysis of heart slices in dogs with acute myocardial infarction subjected to intravenous injection of unlabeled antimyosin antibodies or antimyosin antibodies labeled with indium-111, it was observed that all antibody fragments specifically reached the targeted necrotic zone less than 2 h after antibody injection and remained bound for up to 24 h. In a limited but significant number of cases (5 of the 12 humans and 11 of 43 dogs), it was possible to image the necrotic zone in vivo as early as 2 to 4 h after antibody injection. In other cases, individual blood clearance variations retarded or even prevented in vivo necrosis detection. Higher antimyosin fixation values were obtained in the necrotic zones in dogs with a rapid blood clearance relative to that of the other dogs. It is concluded that antimyosin antibodies always reached necrotic areas within 2 h. If blood clearance was rapid, in vivo imaging of the necrotic area was possible 2 to 6 h after necrosis, even in humans. In some cases, however, uncontrolled individual variations in the timing required for sufficient blood clearance hampered this rapid in vivo detection of myocardial necrosis.

Aged↗
PubMed1991

Growth factors acting via tyrosine kinase receptors induce HSP90 alpha gene expression.

Hsp90 is a heat-shock protein constitutively expressed in most cells. Besides regulation by thermal stress, the expression of hsp90 is also positively regulated by developmental and mitogenic stimuli. The effect of serum and insulin on protein and hsp90 alpha-mRNA levels has been studied in the chicken hepatoma cell line DU249. The culture of cells in serum-free medium resulted in a decrease of hsp90 alpha-mRNA level. A transient increase was observed at 6-9 h after serum restimulation. The expression of hsp90 gene was also increased by insulin alone in a dose-dependent manner and was maximum between 6 and 9 h treatment. The insulin induced increase of hsp90 alpha-mRNA was suppressed by cycloheximide (10 micrograms/ml) but not by an inhibitor of DNA synthesis, demonstrating that this induction requires protein neosynthesis. In serum starved cells, other growth factors (IGF1, EGF and bFGF) showed a positive effect on hsp90 alpha-mRNA level which took place before DNA synthesis with the same time-course as that of insulin. With PDGF, the induction of hsp90 alpha-mRNA occurred earlier. The time interval between the maximum of hsp90 alpha-mRNA induction and that of DNA synthesis was the same for all growth factors studied. From these results, we conclude that growth factors acting via tyrosine kinase receptors up-regulate hsp90 alpha-mRNA level in a DNA synthesis independent manner, possibly in late G1.

Blood↗
PubMed1991

Immunoradiometric assay of myosin heavy chain fragments in plasma for investigation of myocardial infarction.

Estimation of the extent and location of infarct is important for the prognosis and hence therapeutic strategy in patients with acute myocardial infarction (AMI). Because cardiac myosin is the major structural protein of the myocardium, and may thus reflect the extent of injured tissue, we established a new sensitive immunoradiometric assay, using a pair of monoclonal antibodies (Mabs) that specifically bind the myosin heavy chain fragments liberated from the myocyte into plasma after a heart attack. A first Mab is linked to a magnetic solid phase. A second Mab, radiolabeled with 125I, is used to detect myosin trapped on the solid phase by the first Mab during a 3-h incubation. This assay can detect 10 micro-units of myosin per liter and is highly reproducible.

Antibodies, Monoclonal↗
PubMed1990

Isolated dystrophin molecules as seen by electron microscopy.

Dystrophin, the protein product of the Duchenne muscular dystrophy locus [Hoffman, E. P., Brown, R. H., Jr., & Kunkel, L. M. (1987) Cell 51, 919-928], is expressed in striated and smooth muscles as well as in non-muscle tissues. Examination of its primary structure has revealed that the molecule is composed of four domains, three of which share many features with the membrane cytoskeletal proteins spectrin and actinin. Dystrophin has thus been predicted to adopt a rod shape [Koenig, M., Monaco, A. P. & Kunkel, L. M. (1988) Cell 53, 219-228]. In the present study, we describe its isolation from the chicken gizzard smooth muscle and present electron microscopic images of the molecule. Polyclonal antibodies were first prepared from a dystrophin fragment derived from the chicken skeletal muscle gene (residues 1173-1728). A dystrophin-enriched membrane preparation from chicken gizzard muscle was then purified by passing it through an affinity chromatography column made with the anti-dystrophin antibodies. Electron microscopy of isolated and rotatory-shadowed dystrophin molecules revealed that the lengths measured for the dystrophin monomers (175 +/- 15 nm) are compatible with a structural arrangement of the repeat sequence segments in triple-barrel alpha-helices connected by short-turn regions, as was earlier postulated for the repeat domains of spectrin and actinin. Electron microscopic images indicate that in addition the dystrophin molecules could present the same capacity of self-association in oligomeric structures as these cytoskeletal proteins and may thus be a part of a complex molecular meshwork essential to muscle cell function.

Animals↗
PubMed1990

Ductal heterogeneity of cytokeratins, gene expression, and cell death in the rat ventral prostate.

The rat ventral prostate is a complex gland composed of numerous ducts. The epithelial cells that line the lumen of the ducts are surrounded by stromal cells. The epithelial cells display a characteristic morphology that is dependent on their anatomical location within the ducts; the cells that line the lumen in the region of the ducts close to the urethra (the proximal region) are cuboidal, while those in the distal regions of the ducts are tall columnar cells. We have examined the regional expression of two genes that are expressed in the prostate: prostate steroid-binding protein (PSBP; a marker for androgen-dependent protein synthesis) and TRPM-2 (a marker for programmed cell death). We have demonstrated that the expression of PSBP, in the presence of androgens, and TRPM-2, in the absence of androgens, is restricted to the luminal epithelial cells in the distal regions of the prostatic ducts. Neither of the genes is expressed in the proximal regions of the ducts. In view of the probable effects of the epithelial-stromal interactions in the gland we have also characterized the cytokeratin composition of the epithelial cells lining the prostatic ducts. We have established that the basal epithelial cells of the prostate are primarily localized in the proximal region of the ducts. We propose that these cells may attenuate the influence of the stromal cells on the luminal epithelium and exert a negative influence on the cytodifferentiation of the secretory epithelial cells. The results also suggest that PSBP, which has been considered to be an androgen-dependent gene may, in fact, be a sequence that is constitutively expressed in the luminal cells that die in the absence of androgens. This has significant implications on the mechanism of androgen action in the rat ventral prostate.

Androgen-Binding Protein↗
PubMed1990

[Presence of dystrophine-like protein at the neuromuscular junction in Duchenne muscular dystrophy and in "mdx" mutant mice].

We have studied by indirect immunofluorescence, using three different polyclonal antidystrophin antibodies raised against fusion proteins, the neuromuscular junctions (NMJs) in muscle biopsies from Duchenne muscular dystrophy (DMD) patients, from human controls and mutant "mdx" mice and normal mice. In controls the periphery of all muscle fibres was strongly labelled by the three dystrophin antibodies and there was a high concentration of labelling at the NMJs (where it was co-localized with acetylcholine receptor labelled by the alpha-bungarotoxin). In DMD and in "mdx" mice the NMJs were equally labelled whereas there was an absence of reaction at the periphery of all (DMD) or most ("mdx" mice) muscle fibers. These findings suggest that a dystrophin-like protein, which was identified by the antibodies we have used, is present at the NMJs in the Duchenne's myopathy and "mdx" mice.

Animals↗
PubMed1990

Study of megakaryocytic progenitors (CFU-MK) in human long-term bone marrow cultures (LTBMC): adjuvant effect of plasma from aplastic patients.

Human long-term bone marrow cultures (LTBMC) provide a very interesting tool for studying the events that are involved in stem cell commitment. At the present time, megakaryocyte (MK) progenitor cells have never been demonstrated in this system. In an effort to detect this cell lineage, we modified the culture medium by substituting fetal calf serum (FCS) and horse serum (HS) mix with human plasma obtained from treated aplastic leukemic patients. This plasma was harvested between days 15 and 21 following induction chemotherapy or conditioning regimen for autograft or allogeneic bone marrow transplantation. Using LTBMC, 17 normal marrows were cultivated for 11 weeks in Iscove's modified Dulbecco's medium containing either 20% human aplastic plasma or control FCS/HS mixture. In this plasma medium we observed the development of an adherent layer morphologically comparable to that observed with standard medium. We demonstrated presence of MK cells at all stages of maturation for 10 weeks and MK colony-forming cells (CFU-MK) for 11 weeks in the culture supernatants. An increased production of nonadherent cells and granulocyte-macrophage progenitors (CFU-GM) was also observed. LTBMC in aplastic plasma medium provide a new method for studying megakaryocytopoiesis, especially in human hematological diseases.

Adolescent↗
PubMed1989

Myosin: a highly sensitive indicator of myocardial necrosis after cardiac operations.

Plasma levels of ventricular myosin fragments, determined with monoclonal antibodies to myosin heavy chains, were studied in 27 patients after cardiac operations (17 aorta-coronary bypass grafts and 10 valve replacements) to assess their possible role as a marker of perioperative myocardial necrosis. Five patients had perioperative myocardial necrosis after aorta-coronary bypass grafts as indicated by changes in the electrocardiogram and elevated levels of the MB isoenzyme of creatine kinase. Six more patients were also studied after thoracic operations performed by the same sternotomy approach. After cardiac operations, myosin levels increased from postoperative day 3 and reached peak values on day 7. Peak myosin values in patients with perioperative myocardial necrosis after aorta-coronary bypass grafting were significantly higher than in patients after an identical operation but without perioperative myocardial infarction (3793 +/- 592 versus 369 +/- 47 ng/ml; p less than 0.001). These results suggest that plasma myosin is a sensitive marker of myocardial necrosis. Furthermore, peak plasma levels of ventricular myosin after coronary bypass grafting without myocardial infarction (mean value 369 +/- 47 ng/ml) were not significantly different from peak levels after thoracic operations (mean value 253 +/- 52 ng/ml), whereas they were significantly higher after valve replacement (mean value 794 +/- 149 ng/ml; p less than 0.01). These results indicate that a certain degree of myocardial necrosis occurs during value replacement that is undetectable by the usual diagnostic criteria for perioperative myocardial infarction. We conclude that the plasma level of ventricular myosin fragments is a more specific and accurate marker of perioperative myocardial necrosis than changes in the electrocardiogram or elevated creatine kinase MB levels. Therefore the detection of myosin fragments, which appear in the serum on the third day after cardiac operations, may be useful for precise comparisons of different techniques of myocardial protection.

Biomarkers↗
PubMed1988

Histochemistry of ventricular heavy-chain myosins in cardiomyopathic Syrian hamsters treated with D-600.

Monoclonal antibodies (MAb) have been used to study the distribution of ventricular heavy-chain (HC) myosins in cardiomyopathic UM-X7.1 Syrian hamsters. The Ab were identified as alpha and beta anti-HC myosins because of their ability to cross-react with ventricular V1 and V3 myosins, respectively. Cryostat frozen sections from the midventricle region of normal and myopathic hearts were processed for demonstration of these isomyosins by indirect immunofluorescence. In myopathic hearts, there was a shift of predominant alpha myosin toward the beta isoform with the time course of the hamster cardiomyopathy. A D-600 treatment while preventing cardiac necrotic lesions had little or no effect on the beta isomyosin conversion. It is inferred that the isomyosin shift during the progression of the hamster cardiomyopathy is unrelated to the necrotizing process and merely reflects the hypokynetism of the cardiomyocytes.

Animals↗