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J Leger

Publications and source records attributed to J Leger.

At least 19 recordsLinked to original sources
PubMed1992

Proteolytic susceptibility of the central domain in chicken gizzard and skeletal muscle dystrophins.

We investigated proteolytic susceptibility of the central domain in dystrophin molecules from chicken smooth and skeletal muscles. Dystrophin-enriched preparations from both muscles were made as described in Pons et al. (Proc. Natl. Acad. Sci. USA (1990) 87, 7851-7855). These preparations contained other protein components in addition to dystrophin. Three enzymes (Staphylococcus aureus proteinase, chymotrypsin and trypsin) having different proteolytic specificities were used. Time-courses of proteinase degradation were examined by the Western immunoblot technique using a specific polyclonal serum directed against a fragment (residues 1173-1728) of the dystrophin central domain. We observed accumulation of some major proteinase-resistant fragments, in the 110-160 kDa range originating from that central region of the molecule. Cleavage patterns of the smooth and skeletal muscle preparations were quite similar, but molecular weights of the breakdown products differed slightly. Interpretation of the results was based on two predictive structural models of the dystrophin central domain (Koenig and Kunkel (1990) J. Biol. Chem. 265, 4560-4566 and Cross et al. (1990) FEBS Lett. 262, 87-90). Skip residues at the end of repeat 13 (around the 1740th residue of the dystrophin amino acid sequence), as hypothesized in the Cross model, constitute probably the most sensitive site within the dystrophin central domain for any exogenous (or even endogenous) proteinase. Variations observed between dystrophins from skeletal and smooth muscles also suggest that the structures of both dystrophins differ slightly even within the dystrophin central domain. This precise identification of proteinase-resistant dystrophin fragments of variable lengths is a first step towards further physicochemical studies on the very large and rare dystrophin molecule.

Animals↗
PubMed1992

Probing myosin light chain 1 structure with monoclonal antibodies.

Five monoclonal antibodies that react with different regions of myosin light chain 1 from human ventricular myocardial muscle were used to obtain information on interactions between the light chain 1 and heavy chains and generally on the tertiary structure of the light chain 1 within the myosin head. We performed Western blot assays of the five antibodies with myosins from different cardiac and skeletal muscles, with different proteolytic fragments of bovine ventricular myosin light chain 1 (LC1) and to different recombinant fragments of human ventricular LC1 and rat fast skeletal light chain LC1/LC3. The five antibodies were mapped in three different regions of the light chain 1: two antibodies mapped within the first eight amino-terminal residues, two between residues 71 and 74, and one between residues 129 and 134. The apparent dissociation constants of the last three antibodies, determined by antibody-antigen equilibria in solution, were lower than when isolated light chains were used as antigens. It is probable that the corresponding amino acids involved in the antibody epitopes were either involved in interactions between the light and heavy myosin subunits, or somehow hindered by the myosin heavy chain bulk. In contrast, the apparent dissociation constants measured for both other antibodies were higher when myosin, rather than isolated light chains, was used as antigen. Thus LC1 fixation to heavy chains within the myosin molecule induced conformation changes at the amino-terminal end of the light chain 1. No difference in the accessibility of this mobile LC1 segment was detected in the presence of actin. Finally, observed differences in epitope accessibility on the light chain LC1 in myosin, as compared with chymotryptic subfragment 1 (SF1), indicated conformational differences between native myosin and extensively studied SF1 molecules.

Amino Acid Sequence↗
PubMed1992

Properties of chicken cardiac dystrophin.

We investigated the presence of dystrophin by immunoblot and immunofluorescence analyses, negative staining, rotatory shadowing and immunogold electron microscopy in chicken cardiac muscle. Saponin was found to be better than Triton X-100 for providing a new 'dystrophin-enriched' solution for use in biochemical studies of the molecule. By Western blot analysis, only a 400-kDa band was revealed with polyclonal antibodies directed against a central region (residues 1178-1723) of the dystrophin molecule and no cross-reactions with other proteins or degraded products were observed. Specific cleavage of the dystrophin molecule showed that the central rod-shaped domain corresponded to a resistant 'core'. This structure might rigidify the protein. By immunofluorescence, dystrophin was localized at the periphery of cardiac ventricular cells. The molecule was examined by electron microscopy and found to have variable lengths (140-160 nm for the monomeric from and about 260 +/- 10 nm or more for oligomeric forms). These oligomeric structures are considered to be associated molecules which are only partially overlapped lengthwise. The precise distribution of dystrophin within the cardiac muscle was determined by visualisation of gold particles in immuno-electron microscopy. Gold particles were found on the sarcolemma with no evidence of any association with cytoplasmic structures. The present data provide further details on the cardiac dystrophin molecule and suggest that its capacity of self-association may elasticize the dystrophin dimer.

Animals↗
PubMed1992

In vitro digestion of dystrophin by calcium-dependent proteases, calpains I and II.

Dystrophin is a cytoskeletal protein which is thought to play an important role in membrane physiology since its absence (due to gene deficiency) leads to the symptoms of Duchenne muscular dystrophy (DMD). Some disruption in the regulation of intracellular free Ca2+ levels could lead to DMD-like symptoms. In this study, calpains, which are very active calcium-dependent proteases, were examined for their capacity to hydrolyse dystrophin in vitro. The results show that calpains are able to split dystrophin and produce breakdown products of different sizes (the degree of cleavage being dependent on the incubation time with proteases). The time-course of protease degradation was examined by Western immunoblot using three polyclonal sera which were characterized as being specific to the central (residues 1173-1728) and two distal parts of the molecule ie specific to the N-terminal (residues 43-760) or the C-terminal (residues 3357-3660) extremities of the dystrophin molecule. The cleavage patterns of dystrophin showed an accumulation of some major protease-resistant fragments of high relative molecular mass (250-370 kDa). These observations demonstrate that calpains digest dystrophin very rapidly when the calcium concentration is compatible with their activation. For instance, it is clear that calpains first give rise to large dystrophin products in which the C-terminal region is lacking. These observations suggest that dystrophin antibodies specific to the central domain of the molecule should be used to detect dystrophin for diagnostic purposes and before any conclusion as to the presence or absence of dystrophin can be deduced from results obtained using immunoanalyses of muscle biopsies.

Animals↗
PubMed1992

Direct double monoclonal immunoradiometric assay of urinary human growth hormone.

Several reports indicate that urinary growth hormone (GH) excretion might reflect central release of the hormone, and that measurement of urinary GH shows promise in the investigation of physiological and pathological GH secretion. We have developed and evaluated a direct immunoradiometric assay (IRMA) in which two monoclonal antibodies are used to measure GH in the urine of children. The detection limit is approximately 0.018 pmol/L for a sample volume of 2 mL. Within- and between-run variations (CVs) were 5.6% and 14.2%, respectively. Analytical recovery and dilution experiments showed the specificity of the method for GH. In normal-stature prepubertal children ages 3-12 years, 24-h urinary GH excretion was 0.189 (SD 0.100) pmol and correlated well with the amount of GH in the first morning miction, which showed wide day-to-day variations. Like others, we found a strong correlation between GH concentrations in serum and urine during stimulation tests with GH-releasing hormone (somatoliberin) and (or) during physiological nocturnal secretion, confirming that urinary GH measurement may be of help in investigating patients (particularly young children) with diseases in which GH secretion is impaired.

Antibodies, Monoclonal↗
PubMed1991

Ultrastructural localization of dystrophin in chicken smooth muscle.

We investigated the presence of dystrophin in gizzard smooth muscle by immunofluorescence assay, immunoblot detection and an immunogold electron microscopy technique. Western blot analyses, using antibodies raised against sequences 1173-1728 and 3357-3660 of the dystrophin molecule, revealed the presence of a major intact 400 kDa protein band and an immunofluorescence localization restricted to the periphery of the smooth muscle cells. We were able to precisely determine the dystrophin distribution along the plasmalemma whereas caldesmon molecules were present in the cytoplasm. The most commonly observed distance between two neighbouring dystrophin molecules suggested a self-associating arrangement. We discuss these findings in relation to the function of dystrophin in the smooth muscle cell structure.

Animals↗
PubMed1991

Expression of myosin heavy chain isoforms in Duchenne muscular dystrophy patients and carriers.

The expression of MHC isoforms in the skeletal muscles of nine patients with Duchenne muscular dystrophy (DMD) (from 2.5 to 15 yr of age) and three DMD carriers was studied using different specific anti-MHC MAbs. We also analyzed muscle fiber size and fiber reactivity with acridine orange and/or with a surface antigen marker. One-quarter of all fibers of DMD patients, or less with age, were of normal size and contained only adult slow MHC. Half of the muscle fibers contained adult and developmental MHCs. Only half of these fibers were representative of an active regenerative process. MHC co-expression also altered the proportion of normal fast or slow fibers. Adult fast MHCs were expressed as unique MHC only in small and very small fibers in the oldest DMD patients. In DMD carrier muscles, the greatest alterations in MHC expression were observed in patients with the most reduced dystrophin expression. However, MHC changes in dystrophin-positive fibers were similar to those observed in dystrophin-free fibers. In conclusion, disruptions or delays in the switching of all genes coding for adult fast and slow MHC and developmental MHC coincided with dystrophin deletion and with perturbations in its expression.

Adolescent↗
PubMed1990

Activity of cefotiam in combination with beta-lactam antibiotics on enterobacterial hospital strains.

By using checkerboard titrations the effect of cefotiam combined with different beta-lactam antibiotics on fifty strains of Enterobacteriaceae moderately susceptible (minimal inhibiting concentration greater than or equal to 8 mg/l) or resistant (minimal inhibiting concentration greater than or equal to 64 mg/l) to cefotiam was evaluated. The following compounds were tested: cefamandole, cefazolin, cefmenoxime, cefotaxime, cefotiam, ceftazidime, cefuroxime, mecillinam and piperacillin. The synergistic effect varied markedly. The combination cefotiam-mecillinam showed the highest rate of synergistic activity. Antagonism was found in 1% of the combinations.

Anti-Bacterial Agents↗
PubMed1990

Screening for congenital hypothyroidism in France. Misdiagnosed cases: collaborative study of screening centres in France.

Misdiagnosed cases of congenital-hypothyroidism (CH) during the first 9 years of the French screening program were analysed. A total of 50 cases were missed (3% of total diagnosed) which represents a severe failure of the system. Failures were caused by technical errors of sample collection or TSH assay (n = 27) or due to normal TSH (n = 22) or T4 (n = 1) concentrations in the newborn blood specimens. We conclude that screening methods should be improved and that physicians should remain alert to clinical signs of hypothyroidism.

Congenital Hypothyroidism↗
PubMed1990

Thyroid hormones influences sex steroid binding protein levels in infancy: study in congenital hypothyroidism.

In order to test the hypothesis of a role of thyroid hormones on the circulating levels of sex steroid binding protein (SHBG) in children, SHBG levels were determined in 15 infants with congenital hypothyroidism at diagnosis and during the first 18 months of T4 therapy and in a separate group of 13 children with congenital hypothyroidism (7.1 +/- 0.5 yr, mean +/- SD), treated before 1 month of age, both during adequate L-T4 therapy and 4 weeks after withdrawing therapy. SHBG levels were significantly lower in hypothyroid infants than in controls (48.2 +/- 6.5 vs. 77.8 +/- 7.9 nmol/L; P less than 0.01), and significantly lower in infants with athyreosis compared to those with ectopic or eutopic glands (P less than 0.05). In patients with low values at diagnosis, SHBG increased rapidly and remained normal during LT4 therapy. After 1.5-18 months of treatment, a positive correlation (P less than 0.01) was found between SHBG, FT4, and FT3 levels. In older hypothyroid children, 4 weeks after withdrawal of therapy a significant (P less than 0.05) decrease in SHBG concentrations was observed. Analysis of these results as well as previous reports in adults, indicates that thyroid hormones influence SHBG concentrations in infants and children. This study also indicates that thyroid hormones may play a role in the physiological postnatal increase of SHBG.

Child↗
PubMed1989

Congenital hypothyroidism: decreased growth velocity in the first weeks of life.

The growth velocity of 46 infants with congenital hypothyroidism, classified into three groups: athyreosis (n = 12), ectopic (n = 25) and eutopic gland (n = 9), was analyzed. Height, expressed as the standard deviation score (SDS), was measured at birth, at diagnosis and 2, 4, and 8 weeks after initiation of therapy. Bone age was retarded at birth and at diagnosis, epiphyseal surfaces of the knee were correlated with serum T4 values. By contrast, height (mean +/- SD) at birth was normal (0.3 +/- 0.8 SDS) with identical distribution among the three subgroups. Growth velocity was decreased during the first weeks of life: height at diagnosis was -0.1 +/- 0.6 SDS, a value significantly different from size at birth (p less than 0.02). After therapy was started, a continuous decline in growth velocity was observed, reaching -0.2 +/- 0.7 and -0.2 +/- 0.6 SDS, respectively, at 2 and 4 weeks of therapy (p less than 0.01 vs. size at birth). Growth retardation was correlated with the degree of thyroid deficiency. Mean height after 2 weeks of therapy was at -0.6, -0.1, 0.1 SDS in patients with athyreosis, ectopic and eutopic gland, respectively. Catch-up growth occurred thereafter, and the mean height reached 0.3 SDS at 12 weeks for the entire group. A correlation between the decrease in growth velocity and T4 levels at diagnosis was found (p less than 0.05). The normal size at birth and the decreased growth rate observed at diagnosis seem to indicate that growth becomes thyroid hormone dependent immediately after birth.

Body Height↗
PubMed1989

Anti-myosin heavy chain monoclonal antibodies reveal two IIB (fast) fiber subtypes.

Indirect immunofluorescence analysis of different rat skeletal muscles using anti-myosin heavy chain (MHC) monoclonal antibodies (MAb) revealed the presence of two immunologically distinct kinds of fibers within the IIB fibers, histochemically identified by myosin ATPase staining. Some IIB fibers (designated here as IIB1) were unreactive with one anti-fast MHC MAb, whereas they did react with another anti-fast MHC MAb; other IIB fibers (designated here as IIB2) reacted with both anti-fast MAbs. Neither of the two IIB fiber subtypes was significantly reactive with a neonatal MHC MAb. The number of each IIB fiber subtype was age-dependent, at least in the plantaris muscle. IIB1 fibers were observed only in the superficial portion of the plantaris and gastrocnemius muscle. The ratio of IIB1:IIB2 fibers was about the same throughout the extensor digitorum longus and extraocular muscles. Therefore, the two kinds of IIB fibers here observed have a different myosin heavy chain content. On the basis of their specific immunoreactivities, we suggest that IIB1 fibers contain the previously described MHCB. IIB2 fibers contain either a unique new MHC isoform or a mixture of at least two MHC, possibly composed of the MHCB and either the previously described MHCA or a new MHC isoform.

Adenosine Triphosphatases↗
PubMed1989

No evidence for a defect in growth hormone binding to liver membranes in thalassemia major.

To test the hypothesis of a defect in GH-receptor interaction, which could explain the growth failure of thalassemic children, the binding of [125I]human (h) GH to membrane fractions prepared from liver biopsies was studied. Small amounts of liver were obtained from 6 girls and 11 boys with homozygous beta-thalassemia, aged 3-15 yr, all prepubertal, at the time of splenectomy. Specific binding of [125I]hGH ranged from 0.37-5.11% of the added radioactivity/100 micrograms liver membrane protein, with variations in both receptor number and binding affinity. This 14-fold variation in hGH binding to liver membranes of thalassemic children was comparable to that in membrane fractions of livers obtained from normal donors at the time of liver transplant. The binding of insulin to liver membranes from the thalassemic patients ranged from 9.8-17.9% of the added radioactivity/100 micrograms membrane protein and from 2.8-15.0%/100 micrograms membrane protein in the normal donors. Insulin and GH binding to liver membranes did not vary in a consistent way. A 3-fold difference was found in 5'-nucleotidase activity of the membrane fractions. Histological hepatic modifications were assessed with respect to siderosis and fibrosis. No correlation was found between these parameters and GH binding. These results suggest that possible membrane alterations are not the only reason for the variations in hGH binding. All patients had retarded growth, and all but 2 had low plasma insulin-like growth factor I levels. No relationship was found between the level of GH binding to liver membranes and the growth failure. Thus, a defect in GH binding to liver membranes is probably not the cause of the growth retardation of thalassemic children.

Adolescent↗
PubMed1989

Normal growth hormone (GH) response to GH-releasing hormone in children with thalassemia major before puberty: a possible age-related effect.

The plasma GH response to a single iv bolus dose of 2 micrograms/kg BW synthetic GHRH-(1-44)NH2 was evaluated in 13 prepubertal children with thalassemia major (mean age, 7.6 +/- 0.8 yr) with growth retardation and in 15 prepubertal children with nonendocrine short stature. All of the patients showed a significant increase in plasma GH concentration, with a mean peak of 31.4 +/- 4.5 micrograms/L at 15 min (P less than 0.001 vs. basal values; range, 18.4-65 micrograms/L) after GHRH, which was not different from that of the control group of idiopathic short stature children (40.1 +/- 3.4 micrograms/L; range, 21-65.4 micrograms/L). All but 1 of the thalassemic patients had a normal GH response to the arginine-insulin stimulation test. The mean plasma insulin-like growth factor-I level was low (0.12 +/- 0.05 U x 10(3)/L; range, less than 0.02-0.61 U x 10(3)/L). Analysis of these results as well as previously reported data indicating that older thalassemic patients have an impaired GH response indicates that there may be an age-related pituitary and/or hypothalamic dysfunction in thalassemic children. This study also confirms that the insulin-like growth factor-I decrease occurs before any alteration in GH secretion. These changes might play a role in the early growth retardation that occurs in these patients.

Age Factors↗
PubMed1989

[Prophylaxis of vitamin D deficiency in hypothyroidism in the newborn infant].

This study deals with the relationship between the occurrence of hypercalcemia and the administration of prophylactic doses of vitamin D in children with hypothyroidism, before and during L-thyroxine (LT4) treatment. The goal of the study was to determine the dosage of vitamin D necessary to prevent rickets without inducing hypercalcemia. There was a 23% prevalence of hypercalcemia at the time of the diagnosis of hypothyroidism by screening whereas it was 21% in the children who were not given vitamin D during the first 3 months of LT4 treatment. This figure was significantly higher in those who were given vitamin D during the first 3 months of treatment and reached 70%. However, one of the 19 children not given vitamin D presented with biological signs evoking vitamin D deficiency. In conclusion, in hypothyroid infants, vitamin D should be administered carefully during the first 6 months of treatment and restricted to children at risk for developing vitamin D deficiency.

Calcifediol↗
PubMed1988

[Fasting].

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Fasting↗