Biomedical subjects

J J Leger

Publications and source records attributed to J J Leger.

At least 19 recordsLinked to original sources

PubMed2000

Effects of 14-day spaceflight on myosin heavy chain expression in biceps and triceps muscles of the rhesus monkey.

In rats, changes in myosin expression are induced by the chronic elimination of weight-bearing activity, particularly in the postural muscles. This occurs during spaceflight and hindlimb suspension. Myosin heavy chain (MHC) changes affect fast and slow fiber types differently depending on muscle function. An increase in co-expression of different MHC within the same fiber will signal early changes in muscle fibers. In the rat soleus muscles, the spaceflight-induced increase in fast MHC expression appears to be essentially due to the enhanced or de novo synthesis of IID or IIX MHC. In response to microgravity, the expression of slow-type myosin decreases, while that of fast-type increases. There is scarce information concerning the effect of microgravity on rhesus monkeys (Macaca mulatta), especially on their upper limbs. We investigated the expression of MHC using an immunocytochemical approach to determine the nature and magnitude of the changes in biceps and triceps muscles of rhesus monkeys during the Bion 11 14-day mission.

Adaptation, Physiological↗

PubMed1998

Effect of a 14-day spaceflight on dystrophin associated proteins complex in rat soleus muscle.

One of the most obvious effects of hypokinesia and hypodynamia is muscular atrophy. Changes in myosin expression are induced by the chronic elimination of the weight-bearing activity, particularly in the postural muscles, which occur during spaceflight and hindlimb suspension. Other morphological changes, such as the remodeling of myotendinous junction, are also induced by this reduction in mechanical stress. Moreover, the transversal interface between the cytoskeleton and the extracellular matrix of the muscle fiber can also be modified by the functional demand imposed on muscle. Dystrophin and its associated proteins appear to be essential for the stability of this interface and the deletion of one of these proteins results in a pathological phenotype. Changes in their expression appears to be induced by muscle disuse. We have quantified the changes in the expression of dystrophin and associated proteins induced by a 14-day spaceflight.

Animals↗

PubMed1995

The cardiac myosin heavy chain Arg-403-->Gln mutation that causes hypertrophic cardiomyopathy does not affect the actin- or ATP-binding capacities of two size-limited recombinant myosin heavy chain fragments.

Our aim was to investigate the potential functional consequences of myosin heavy chain (MHC) mutations identified in patients with familial hypertrophic cardiomyopathy. We observed the presence of a mutated beta-MHC mRNA in a formalin-fixed paraffin-embedded myocardial tissue of a proband from family A, which Geisterfer-Lowrance et al. [Geisterfer-Lowrance, Kass, Tanigawa, Vosberg, McKenna, Seidman and Seidman (1990) Cell 62, 999-1006] identified as carrying the Arg-403 to Gln mutation. Recombinant DNA methods were then used to obtain size-limited, soluble and undenatured fragments of mutated myosin subfragment 1 focused around the 403 mutation. The present analysis indicated that the 403 mutation did not quantitatively alter the actin- or ATP-binding capacities of two 246-residue or 524-residue-long recombinant MHC fragments containing this mutation. The absence of any apparent impact of the 403 mutation in the recombinant MHC fragments on interactions between actin and ATP is discussed in relation to numerous biochemical and structural reports which demonstrate the crucial role of the central MHC segment, where the 403 mutation occurs, in myosin functions.

Actins↗

PubMed1993

Actin-dystrophin interface.

Dystrophin, an elongated cytoskeletal molecule which is deficient in Duchenne muscular disease, contains an actin-binding domain in its N-terminal portion. We show that this part interacted with actin in the native molecule. By molecular biology techniques, four recombinant proteins were expressed in Escherichia coli using the pMAL vector which allowed us to obtain soluble proteins directly after purification. These constructions were tested for their ability to bind actin under various conditions, and their apparent dissociation constants were determined. The effects of other actin-binding proteins such as caldesmon and tropomyosin were analyzed in comparison to the actin-binding properties of these constructions. These results support the potential concept of a multiple actin-binding contact in the N-terminal region of dystrophin. Differences in the functional domains are discussed relative to similar alpha-actinin-actin-binding sites.

Actins↗

PubMed1993

Dystrophin and dystrophin-related protein expression in Torpedo marmorata electric organ.

The presence of different dystrophin-related protein forms was investigated in electric organ as compared to cardiac, white or red skeletal muscles from Torpedo marmorata. Two strategies were followed. First, we used specific C-terminal dystrophin and dystrophin-related protein monoclonal antibodies which we characterized in the present study. 400 kDa protein bands were detected in the tissues mentioned above with both specific types of antibodies. Second, we produced monoclonal antibodies raised against a dystrophin-enriched preparation from T. marmorata electric organ. Western blot and immunofluorescence analyses showed the tissue specificity of T. marmorata antibodies and allowed us to classify them as types I, II and III. Vessel walls and neuromuscular junctions were labeled with T. marmorata type II and III antibodies in human muscles (skeletal and smooth). Both approaches demonstrated that the T. marmorata electric organ contained different proteins related with dystrophin: a dystrophin form, a dystrophin-related protein form and a dystrophin-related protein isoform, homologous to the dystrophin-related protein present in muscle vessel walls and at the neuromuscular junctions of human tissues. The presence of dystrophin and dystrophin-related protein is finally discussed relative to their functions and organ specificities.

Animals↗

PubMed1993

Probing functional regions in cardiac isomyosins with monoclonal antibodies.

Seven Mabs prepared against subfragment 1 (S1) of either bovine cardiac beta-specific or rabbit fast skeletal muscle myosin were used to identify functional regions in cardiac isomyosin heavy chains. This approach was designed to improve the understanding of structure-function relationships within the myosin molecule and between alpha and beta myosin heavy chains (MHCs). We used bacterial expression of human beta myosin fragments and determined that the seven antibodies were localized within four different MHC subdomains: amino acid residues 33-37 (one beta-specific antibody), 67-84 (one alpha/beta-specific antibody), 85-106 (four alpha/beta-specific antibodies) and 215-248 (one alpha/beta-specific antibody). All epitopes were accessible on myosin and actomyosin with the same affinities. Therefore, none of these MHC epitopes were located on the interfaces between the myosin head and actin. Three antibodies reacting at three out of the four investigated epitopes enhanced acto-S1 ATPase activities but not myosin, S1, or actomyosin activities. One antibody, which was strictly beta-specific and bound to five amino acid residues near the most N-terminal MHC end, substantially inhibited all myosin or S1 ATPase activities measured with or without actin. The epitope of this antibody coincides with one difference cluster observed between both cardiac MHC isoforms [McNally et al (1989) J. Mol. Biol. 210, 665-671], suggesting that this small variable MHC area could be one of the structural bases to explain observed functional differences in cardiac alpha and beta myosin isoforms.

Amino Acid Sequence↗

PubMed1993

Mdx transgenic mouse: restoration of recombinant dystrophin to the dystrophic muscle.

We report the restoration of the 430-kD dystrophin in mdx, the mouse model of Duchenne muscular dystrophy, by expression of a single-copy recombinant dystrophin transgene. Muscle-specific expression was achieved using a creatine kinase promoter influenced by two enhancers. Immunostaining with anti-Xp21-coded dystrophin monoclonal antibodies showed that the recombinant dystrophin was localized to the muscle fiber membrane. However, there was variability in the level of dystrophin expression in various animals with aging, between fast and slow muscles, and within different regions of the same muscle. Curiously, recombinant dystrophin was relatively absent in the diaphragm muscle of these mdx transgenic animals. Our studies indicate that there is a direct correlation between the level of muscle fibers expressing recombinant dystrophin and the level of muscle fibers with peripheral nuclei, indicating an improvement in muscle pathology. These studies indicate that the regional expression of recombinant dystrophin in dystrophic muscle leads to regional restoration of normal muscle morphology.

Animals↗

PubMed1993

Developmental changes in the myosin composition of guinea pig ventricular muscle. Relation to thyroid state and mechanical properties.

In a variety of mammalian species, thyroid hormone regulates the contractile properties of the heart as well as the expression of the alpha and beta heavy chains of myosin. We have previously shown that the plasma levels of thyroid hormone reach a peak immediately after birth in guinea pigs and decline with maturation. We therefore studied age-related changes in the expression of the myosin heavy chains in the guinea pig ventricle in relation to the ventricular mechanical properties and the levels of thyroid hormone. The composition of the myosin heavy chains was characterized by gel electrophoresis and immunoblotting. Anti-beta-chain antibody stained equally myosins from newborns (0-5 days) and adults (75-90 days), while anti-alpha-chain positively decorated only the myosins of euthyroid newborns or of hyperthyroid adults, but not myosins of embryos, hypothyroid newborns or hypothyroid adults. Myosin of euthyroid adults was faintly stained by anti-alpha-chain. The alterations in the composition of myosin corresponded with the "thyroid state" of these groups. The plasma levels of total T3 were 24.3 +/- 2.7, 9.04 +/- 1.2 and 139.0 +/- 9.3 ng/dl (mean +/- SEM) in the euthyroid, hypothyroid and hyperthyroid adults, respectively. In euthyroid and hypothyroid newborns, the plasma levels of T3 were 56.5 +/- 11.9 and 26.5 +/- 9.8 ng/dl, respectively. Within each age group the thyroid state corresponded with maximal twitch tension (Tmax), rates of development of tension and relaxation, time to peak tension and rate of activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

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

Expression of various NCAM isoforms in human embryonic muscles: correlation with myosin heavy chain phenotypes.

Neural cell adhesion molecules (NCAM) are known to play a pivotal role in regulating cell-cell interactions in various tissues. The diversity of NCAM is made by alternative splicing of a single gene and by post-translational modifications. The spatio-temporal expression of the various isoforms is developmentally regulated and may modulate cell interactions. We investigated the expression of NCAM isoforms, in particular polysialylated and phosphatidylinositol-anchored isoforms, in developing psoas and quadriceps human muscle from 15 weeks of gestation to term. In parallel, we examined the expression of the myosin heavy chain phenotype (another developmentally regulated system) to determine whether polysialylated-NCAM molecules (the so-called embryonic NCAM) and developmental myosin heavy chains are coexpressed. Our results showed an expression of polysialylated-NCAM and phosphatidylinositol-anchored isoforms during the early stages of myotube maturation. The expression of polysialylated-NCAM on developing myotube was always associated with the expression of developmental myosin heavy chains. However, the loss of polysialylated-NCAM from maturing myotubes was not correlated with the disappearance of the developmental myosin heavy chains, but rather with the appearance of an adult myosin heavy chain phenotype. The relationship between polysialylated-NCAM and myosin heavy chain phenotype was similar in psoas and in quadriceps muscles. We observed that maturation of quadriceps muscle takes place earlier than psoas. Biochemical analysis showed that phosphatidylinositol-anchored molecules were never polysialylated; this indicates different roles of these isoforms in muscle development.

Aging↗

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

Metabolic and morphologic properties of single muscle fibers in the rat after spaceflight, Cosmos 1887.

The adaptation of a slow (soleus, Sol) and a fast (medial gastrocnemius, MG) skeletal muscle to spaceflight was studied in five young male rats. The flight period was 12.5 days and the rats were killed approximately 48 h after returning to 1 g. Five other rats that were housed in cages similar to those used by the flight rats were maintained at 1 g for the same period of time to serve as ground-based controls. Fibers were classified as dark or light staining for myosin adenosine triphosphatase (ATPase). On the average, the fibers in the Sol of the flight rats atrophied twice as much as those in the MG. Further, the fibers located in the deep (close to the bone and having the highest percentage of light ATPase and high oxidative fibers in the muscle cross section) region of the MG atrophied more than the fibers located in the superficial (away from the bone and having the lowest percentage of light ATPase and high oxidative fibers in the muscle cross-section) region of the muscle. Based on quantitative histochemical assays of single muscle fibers, succinate dehydrogenase (SDH) activity per unit volume was unchanged in fibers of the Sol and MG. However, in the Sol, but not the MG, the total amount of SDH activity in a 10-microns-thick section of a fiber decreased significantly in response to spaceflight. Based on population distributions, it appears that the alpha-glycerophosphate dehydrogenase (GPD) activities were elevated in the dark ATPase fibers in the Sol, whereas the light fibers in the Sol and both fiber types in the MG did not appear to change. The ratio of GPD to SDH activities increased in the dark (but not light) fibers of the Sol and was unaffected in the MG. Immunohistochemical analyses indicate that approximately 40% of the fibers in the Sol of flight rats expressed a fast myosin heavy chain compared with 22% in control rats. Further, 31% of the fibers in the Sol of flight rats expressed both fast and slow myosin heavy chains compared with 8% in control rats. Immunohistochemical changes in the MG were minimal. These data suggest that the magnitude and direction of enzymatic activity and cell volume changes are dependent on the muscle, the region of the muscle, and the type of myosin expressed in the fibers. Further, the ability of fibers to maintain normal or even elevated activities per unit volume of some metabolic enzymes is remarkable considering the marked and rapid decrease in fiber volume.

Adenosine Triphosphatases↗

PubMed1989

Cardiac beta myosin heavy chain diversity in normal and chronically hypertensive baboons.

We have identified two distinct beta-myosin heavy chains (MHCs) present in baboon myocardium by electrophoresis in gradient pore gels and by Western blots with anti-MHC MAb. The two beta-MHCs have molecular masses of 210 and 200 kD and share several antigenic determinants including an epitope recognized by a beta-MHC-specific MAb. A fivefold increase in the level of the 200-kD beta-MHC was observed in the hypertrophied left ventricles of baboons with chronic (5.3 +/- 0.7 yr) renal hypertension. A 60% increase (P less than 0.01) in BP and a 100% increase (P less than 0.001) in left ventricular mass to body weight ratio occurred in hypertensive baboons compared with normotensive animals. The Ca2+-activated myosin ATPase activity in hypertrophied left ventricles was decreased by 35% (P less than 0.05) compared with controls. Normal levels of the 200-kD MHC were detected in the right ventricles and intraventricular septa of the hypertensive animals. These data suggest that cardiac MHCs of primates may exist in alternative molecular forms that are indistinguishable by nondenaturing gel electrophoresis and that increased concentration of a second beta-MHC is associated with ventricular hypertrophy (r = 0.55). The functional significance and mechanisms that control the concentration of beta-MHC subspecies remain to be determined.

Adenosine Triphosphatases↗

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↗

PubMed1988

Ventricular myosin of the shrew Crocidura russula, correlation with contractile properties.

The present study demonstrates that in the shrew ventricular muscle the speed of tension development and relaxation, as well as twitch duration, are much shorter than in the guinea-pig. It also shows that ventricular myosin of the shrew has a high Ca2+-activated ATPase activity and that it is composed of alpha-type heavy chains. Namely, the native molecule is a V1 variety of myosin. These findings advance our knowledge on an as yet uncharacterized mammalian heart and further demonstrate the correlation between mechanical properties and myosin type in heart muscle.

Animals↗

PubMed1987

Expression of myosin light chains during fetal development of human skeletal muscle.

The expression of myosin light chains (MLCs) during the development of human skeletal muscle was investigated by using two different two-dimensional electrophoretic techniques. In both electrophoretic systems the predominant light chain 1 (LC1) expressed during the whole fetal period was found to co-migrate with the adult fast LC1 (LC1F). The main LC2 expressed during the whole fetal period was found to be different from the main fast LC2 (LC2F) and slow LC2 (LC2S) usually present in adult muscle, but co-migrated with a minor component often present in adult muscle. This fetal LC2 was phosphorylatable, and the phosphorylated form co-migrated with the main component of LC2F expressed in the adult. The adult fast LC3 appeared as early as week 20 of gestation, whereas the adult slow light chains (LC1S and LC2S) appeared only during the late fetal period. A minor component of LC1, previously described in humans as an 'embryonic LC' (LCemb.) [Strohman, Micou-Eastwood, Glass & Matsuda (1983) Science 221, 955-957], was only expressed in the early fetal period and was found to co-migrate with atrial LC1 (ALC1). We discuss the expression of these specific developmental forms of MLCs co-existing with immature myosin heavy chains during fetal life.

Adult↗

PubMed1987

Distribution of alpha- and beta-myosin heavy chains in the ventricular fibers of the postnatal developing rat.

Four monoclonal antibodies, two raised against alpha-myosin heavy chain (MHC) and two against beta-MHC, have been used to investigate in situ the fiber distribution of alpha- and beta-MHC in rat cardiac ventricles during postnatal development. Eighteen ventricles from 2-day-old to 1-year-old rats were analyzed. Three fiber populations were determined according to their immunofluorescent labeling: one with only alpha-MHC, one only beta-MHC, and one with mixed alpha- and beta-MHC. Large variations in the proportions of these three fiber populations according to age indicate that: (1) alpha-MHC are expressed in all fibers until the second month; they then disappear in a small endocardial fiber population and in a few apparently conductive fibers around the vessels. (2) beta-MHC are also first expressed in all fibers and then disappear gradually from epicardium to endocardium between the second and fourth weeks, except in the conductive fibers; they reappear during the second month sequentially from endocardium to epicardium; and they are then expressed in almost all fibers, except in a small epicardial fiber population, proportionally larger in the right ventricle than in the left. Immunological characterization of MHC isolated from a 22-day-old-rat ventricle, using anti-beta immunoaffinity chromatography, suggests that MHC of conductive fibers are probably at least partially in an alpha beta heterodimeric form.

Aging↗

PubMed1987

Development changes in the human cardiac isomyosin distribution: an immunohistochemical study using monoclonal antibodies.

With monoclonal antibodies (Mab) specific for myosin heavy chain (MHC) isozymes, we have investigated the isomyosin content of atrial, ventricular and conductive fibers of 19 human fetuses (ranging from 14-36 weeks of gestation) and 3 newborns (2 days-2 weeks). In addition, the conduction system of 2 human adult hearts was studied. The fetal atrium is composed mostly of alpha-MHC during the first 23 weeks of gestation. beta-MHC is already expressed as traces at 14 weeks of gestation, and its expression increases progressively until birth, resulting in a great augmentation in beta-MHC. During this course, beta-MHC always predominates in certain areas (the crista terminalis and the interatrial septum) but not in other areas (the auricles). Preceding birth, the fetal ventricle is composed mostly of beta-MHC. From 14 weeks of gestation to birth, alpha-MHC is expressed in very rare fibers. Then, after birth, a large number of fibers simultaneously synthesize alpha-MHC. The AV node and His bundle system were labelled with anti-alpha and anti-beta Mab in fetal, newborn, and adult hearts with a double gradient of distribution: spatial (a higher proportion of alpha-containing fibers in the AV node than in the distal portion of the bundle of branches) and temporal (a higher proportion of alpha-containing fibers at a given point in fetal development than in the adult heart). One of the twenty-five hearts studied had an isomyosin distribution pattern not accorded to its age. Interestingly, it was clinically diagnosed as having idiopathic hypertrophic cardiomyopathy.

Antibodies, Monoclonal↗