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Biomedical subjects

J J Leger

Publications and source records attributed to J J Leger.

At least 37 records · Page 2Linked to original sources
PubMed1986

Purification and characterization of myosins from human and rabbit skeletal muscles by using specific monoclonal antibodies.

By using immunoaffinity column chromatography slow (I) and fast (IIA, IIB) myosins were isolated from human (vastus lateralis) and rabbit (tibialis anterior, psoas and conoidal bundle) skeletal muscles. The peptide pattern revealed that slow (I) and fast (IIA, IIB) myosin heavy chains are quite distinct, as are those from pure slow (conoidal bundle) and fast (psoas) rabbit skeletal muscles. Unlike Billeter et al. (1981) the authors observed that fast human myosins were always associated with a small amount of slow myosin light chains. The fast myosins (IIA, IIB) from rabbit tibialis anterior muscle did not appear very distinct and contained only fast myosin light chains. These myosins were different from the IIB myosin from the psoas muscle. Ten per cent of the fibres revealed histochemically as fast IIA also reacted with an anti-slow myosin antibody. The classical histochemical techniques appear inadequate to demonstrate the existing differences among fibre types, but the monoclonal antibodies hold promise.

Animals↗
PubMed1986

Myosin detection in human myometrium with a monoclonal antibody.

A monoclonal antibody was prepared from a mouse immunized with human gravid uterine myosin. This monoclonal antibody is specific for the myosin heavy chains of human smooth muscle as determined by radioimmunoassay and immunoblotting experiments. Frozen cryostat sections isolated from different uterine regions were studied by immunofluorescence in order to detect myosin distribution within cells of nongravid, gravid, and pathologic human uteri. Myosin was detectable in the cytoplasm of all uterine muscle cells. No fiber heterogeneity with regard to myosin distribution was detected among or within the different uterine regions, regardless of which physiologic or pathophysiologic situation was studied. A large increase in the cell size was observed during pregnancy. These preliminary observations suggest the value of further production of monoclonal antibodies specific for the different putative molecular variants of uterine myosin and their potential use in identifying individual cells containing different myosin variants.

Antibodies, Monoclonal↗
PubMed1986

Long-term expression of isomyosins and myoendocrine functions in ectopic grafts of atrial tissue.

Tissue fragments of newborn rat atria were transplanted under the dorsal skin or into the bed of the anterior tibial muscle of nude mice. After 5-11 weeks, the grafts, which had reorganized into beating atrium-like structures, were analyzed and compared to ventricular tissue transplanted the same way. As revealed by monoclonal antibodies against alpha- and beta-type myosin heavy chains, atrial grafts retained a typical pattern of myosin expression distinct from that of ventricular grafts. The majority of ectopic atrial myocytes contained specific atrial granules in which cardiodilatin-immunoreactive material has been localized. Specific granules and cardiodilatin immunoreactivity were not found in myocytes of ventricular grafts. We conclude that the long-term maintenance of isomyosin expression and of the myoendocrine function of atrial tissue is largely independent of the anatomical environment.

Animals↗
PubMed1985

Fractionation and characterization of two molecular variants of myosin from adult human atrium.

Monoclonal antibodies (MAb) to myosin heavy chains were prepared from one adult human ventricular myocardium. Several of these MAb reacted by indirect immunofluorescence in a heterogenous way on cryostat transverse sections of fibers from human atrial myocardium, suggesting the presence of different forms of myosin within the human atrium and prompting the further use of the MAb to attempt to fractionate preparations of native atrial myosins. Two molecular variants of human atrial myosins or myosin fragments were thus separated by immunoaffinity chromatography performed with one antiventricular myosin MAb. Seven MAb located at different positions along the myosin heavy chains, as deduced from blotting and immuno-electron microscopy experiments, were used to characterize the structural relationships between the separated human atrial isomyosins and between each of them and the main human ventricular myosin. As deduced from competitive radioimmunoassay measurements, the primary structures of the two atrial myosins differ in at least five antigenic determinants and share at least two of them; similarly located structural differences were observed between one of the atrial myosins and the ventricular myosin. Conversely, the primary structures of the other atrial myosin and of the ventricular myosin differ in at least two antigenic determinants and share at least five of them. Differences in the primary structures of the human cardiac myosins were confirmed by analysis of the peptides produced by limited enzymatic digestion of the heavy chains; a few peptide differences were consistently found. To summarize the two separated forms of atrial myosin have different heavy chains, but they have similar if not identical in vitro ATPase activities and the same light chains. One of the atrial myosins is immunologically close to the ventricular myosin, but they each differ with respect to their heavy chains, light chains, and enzymatic activities.

Adenosine Triphosphatases↗
PubMed1985

Levels of ventricular myosin fragments in human sera after myocardial infarction, determined with monoclonal antibodies to myosin heavy chains.

Serum levels of ventricular myosin heavy chains were quantitated in patients with acute myocardial infarction using a competitive radioimmunoassay involving monoclonal antibodies to the b-type myosin heavy chains of a human ventricle. Among the seven antibodies selected for their higher affinity for ventricular myosin heavy chains, only four antibodies detected significant and variable myosin amounts in the serum samples of nineteen patients with acute myocardial infarction; the same antibodies occasionally detected, if at all, low myosin amounts in the sera of patients with no clinical sign of myocardial infarction, and no myosin in the serum of the healthy control subjects. The peak levels of myosin release were observed 4.6 +/- 0.5 days (n = 13, P less than 0.01) after myocardial infarction and correlated rather well with the measured creatine kinase peak levels (the correlation coefficients were between 0.75 and 0.81, P less than 0.01, depending on the monoclonal antibody used for myosin determination). The time courses of myosin release varied according to the complexity of the heart attack observed. It was concluded that the titration of serum myosin was probably of little clinical value for therapeutic intervention during the acute phase of myocardial infarction; it could, however be an effective tool for retroactive detection of an infarct and for late estimation of infarct size.

Adult↗
PubMed1984

Fiber types and myosin types in human atrial and ventricular myocardium. An anatomical description.

Hybridomas were prepared from mice immunized with myosin from the enlarged left ventricle of a 53-year-old female with an obstructive cardiomyopathy. The specificity of 15 monoclonal antibodies to myosin heavy chains was assessed by the reactivity of muscle extracts and of chymotryptic myosin fragments of different sizes with these antibodies, as determined by the immune replicate technique; some of the monoclonal antibodies cross-reacted only with the ventricular V3-type myosin from hypothyroid rats, whereas the other antibodies cross-reacted both with the latter and with the ventricular V1-type myosins from normal young rats. Immunological heterogeneity of the fibers from human atrial muscles and from human ventricular muscles was detected by some of the antimyosin antibodies by means of indirect immunofluorescence. Histochemical fiber heterogeneity was also detected by adenosine triphosphatase staining of the same tissues. Because of the close correspondence observed between the immunological and histochemical responses of atrial fibers, it has been postulated that at least two distinct types of myosin exist in the human atrium, each myosin form being histochemically related to either alpha- or beta-like ventricular myosin heavy chains. In contrast, there was no direct correspondence between the two experimental approaches in human ventricles, and it is postulated that at least three distinct types of myosin exist within the human ventricles, one V1-type myosin, presumably corresponding to the very rare fibers with an alkaline-stable adenosine triphosphatase activity, and two other V3-type myosins corresponding to immunologically different fibers, each having an alkaline-labile adenosine triphosphatase activity. Monoclonal antibodies that can distinguish among the different myosin variants were further used to provide the basis for an anatomical description of fiber types and myosin types within the human atrial and ventricular myocardium in the whole hearts of two young boys who died sudden violent deaths. Small zones of myosin variation were seen to be scattered, but probably not randomly distributed, within large areas of myocardium in which the cellular distribution of myosin was constant; the large areas had one myosin distribution specific for each cardiac cavity. No clear-cut conclusions can yet be made concerning the physiological role of the regional variations observed in the distribution of the different molecular forms of myosin.

Adenosine Triphosphatases↗
PubMed1983

Myosin light chains in normal and pathological human skeletal muscles.

Thirty-nine human skeletal muscle biopsies from 24 individuals were classified as normal, neuropathic, or myopathic muscle according to classical clinical observations and histopathological properties of the muscles. The content in myosin light chains (LC) of each muscle sample was analyzed by means of a new technique of polyacrylamide gel electrophoresis that gives an improved discrimination, involving isoelectrofocusing of the muscle homogenate for the first dimension and successive migration in a urea-containing gel for the second dimension. Four different LC patterns have been observed in the normal muscles; these four patterns and three different ones have been observed in the pathological muscles. No apparent correlation exists between the myosin LC content and the histochemical fiber typing. It is concluded that the myosin LC are apparently not a useful marker to detect the normality or the pathology of human muscle.

Amyotrophic Lateral Sclerosis↗
PubMed1983

Structural differences between atrial and ventricular myosins from normal human hearts.

Comparisons were made between myosins isolated from the right and left ventricles and the atria of normal human hearts. Parameters examined included electrophoretic mobilities of native molecules, K+ and Ca2+ dependent enzymatic activities, light chain composition, peptide patterns from partial proteolytic digests of entire heavy chains or rods, and maps of complete digests of specific 21 and 25 kilodalton heavy chain fragments. Human ventricular and atrial myosins differ in all parameters except in the charge of molecules. Structural differences between cardiac myosins derived from the two sources were apparent in both the head and tail portions of the heavy chains. With respect to the above parameters no differences were found between myosins from left and right human ventricles.

Adenosine Triphosphatases↗
PubMed1983

Characterization and comparison of the contractile proteins from human gravid and non-gravid myometrium.

Electrophoretic systems were used to quantify and characterize the main proteins involved in myometrium contraction, i.e., myosin, actin, and tropomyosin. Skeletin, a cytoskeletal component was also investigated. No difference in their respective concentrations could be found in 13 uteri representing various menstrual cycle stages, postmenopausal conditions, and different gestational ages. The great variability of their tissular level observed in each of the uteri does, however, reflect the heterogeneity of the muscular component of the uterine wall. Comparison of the different forms of actin in gravid and non-gravid uteri showed an increase of the gamma-form in gravid uteri. However, myosins purified from gravid and non-gravid uteri were shown to have identical ATPase activity and peptide patterns following limited chymotryptic proteolysis.

Actins↗
PubMed1982

Comparative sequence of myosin light chains from normal and hypertrophied human hearts.

Myosin light chains from normal and hypertrophied human hearts were partially sequenced in order to see whether structural modifications of these light subunits could provide a molecular basis for the changes observed in heart properties and in myosin enzymatic activity. Normal light chains were prepared form hearts taken at autopsy, weighing 350 g or less and apparently devoid of myocardial disease. "Hypertrophied cardiac myosin light chains" were prepared from two greatly hypertrophied hearts, weighing 600 and750 g. No amino acid substitutions, deletions, or additions were observed in the light chains from hypertrophied hearts. The third light chain previously reported in human cardiac myosin and related to hypertrophy was found to be a proteolytic product of LC2. The comparison between human and beef cardiac myosin light chains indicated that the sequences of these subunits of the myosin molecule are highly conserved.

Amino Acid Sequence↗
PubMed1981

Evidence for new forms of cardiac myosin heavy chains in mechanical heart overloading and in ageing.

Heavy chains of myosin rods and subfragment 1 were isolated from normal hearts and from mechanically overloaded hearts of young and older rats. These myosin heavy-chain fragments were cleaved by cyanogen bromide or partially proteolysed by pronase and by chymotrypsin after denaturation with sodium dodecyl sulfate. The peptides, analyzed by electrophoresis on a one-dimensional polyacrylamide slab gel, varied depending on the origin of the cardiac myosin heavy chains. Some bands present in the peptide patterns of the normal heart of young rats were missing from the pattern of greatly hypertrophied hearts and vice versa. We conclude that mechanical overloading of the heart stimulates the synthesis of cardiac myosin 'isozyme' with a heavy-chain primary structure which is different from that observed in the normal heart of young rat. The patterns from myosin heavy-chain peptides from the hearts of older rats were different from those for peptides from young rat hearts; these results also indicate the presence of a new myosin heavy chain specific to ageing. No difference was detected between the peptide patterns of heavy chains isolated from hypertrophied hearts of young and older rats, and those isolated from normal hearts of older rats.

Aging↗
PubMed1980

Absence of phosphates from the myosin heavy chains of striated muscles.

This work aimed to determine whether the heavy chains of myosin from different striated muscle were phosphorylated. Myosin and its heavy chains were prepared from cardiac and skeletal muscles of rats injected in vivo with radioactive phosphates. The results for radioactive phosphate localization indicate the absence of phosphate from pure heavy chains and from any of their purified fragments, whatever the striated muscle used. In addition, phosphates are present in the myosin phosphorylated light chain and in a contaminating protein closely associated to the myosin heavy chain.

Animals↗
PubMed1978

The separation of the myosin light chains on agarose beads.

The myosin light chains from rabbit skeletal muscle and bovine and human hearts are separated according to their molecular weights by filtration on agarose beads. The purity of the isolated components and the yield of such columns are compared to the results obtained by other techniques of separation.

Animals↗
PubMed1976

A comparative study of reactive--SH groups of cardiac and skeletal muscle myosins.

Cardiac and skeletal muscle myosins have been treated by N-ethylmaleimide in presence or absence of Mg-ADP. The variations of Ca2+ and K+-ATPase activities and the incorporation of N-[14C]ethylmaleimide into the whole myosin molecule and into its separated subunits (heavy and light chains) have been measured with N-ethylmaleimide treatment for different lengths of time. The results reported here show the following: 1. The Ca2+-ATPase activity of cardiac myosin is activated by N-ethylmaleimide treatment to a lesser extent than that of skeletal myosin. 2. The K+-ATPase activity of both myosins is inhibited in the same quantitative way. 3. The cardiac light chain L1 contains one highly reactive thiol group which is absent from the skeletal light chains. 4. The labelling of three SH-groups localized in the heavy subunits of both myosins induced the same degree of inactivation. 5. The difference observed between the degree of inhibition of the Ca2+-ATPase activity for the two types of myosin with longer treatments appears to be due to differences in the reactivity of the fourth--SH group labelled on the heavy chains.

Adenosine Triphosphatases↗
PubMed1975

Human cardiac myosin ATPase and light subunits. A comparative study.

Myosin was extracted from normal human hearts (autopsy material) and compared to that of pig heart and rabbit white skeletal muscle. Myosin light subunits were isolated by a preparative urea gel electrophoresis. These subunits were shown by urea and sodium dodecylsulfate gel electrophoresis to be only slightly affected by the time lapse between death and the beginning of myosin extraction. This was also true for myosin ATPases. The Ca-2+-activated ATPases of pig and human heart myosins have the same apparent Km and V, whereas white skeletal muscle myosin ATPase has the same Km with a higher V. Human myosin light subunits, when compared to those of pig heart possess: (i) different molecular weights: 27 999 and 18 000 datlons for pig heart, and 25 000 and 19 000 daltons for human heart. (ii) for both the light chains, different ultraviolet spectra and a higher helical content for the subunit molecular weight 25 000. (iii) a different composition for several amino acids (Tyr, Pro, Lys). A third light subunit (molecular weight 15 000) was occasionally seen in human as well as pig heart myosin. It concentration varied inversely with that of the subunit molecular weight 27 000-25 000, and so was probably a degradation product of the heaviest subunit.

Adenosine Triphosphatases↗
PubMed1975

Cardiac myosin: preparation, ATPase in chronic heart hypertrophy.

A low myofibrillar ATPase seems to be established definitely in several experimental models of chronic heart hypertrophy as well as in humans, but the biochemical pathogenesis of this defect is still unclear. Three different preparations of myosin were studied. Their purity was estimated by measuring MgATPase or by polyacrylamide gel electrophoresis. The first preparation was highly contaminated by actin and tropomyosin; the second was rather pure, and the third (chromatography on DEAE-Sephadex) was pure but slightly denaturated. Heart myosin CaATPase (ionic strength 0.6 or 0.06) was decreased in chronic aortic insufficiency in the rabbits (CAI) when all three preparations were tested. Two (molecular weight 18,000 and 26,000), sometimes three light subunits were found in heart myosin. Their charge and molecular weight are normal in CAI. The third subunit (molecular weight 15,500) was found in control as well as in CAI. Search for an inhibitor was unsuccessful since the two myosin ATPases are additive. The nucleoprotein peak separated from myosin during chromatography was identical in control and CAI. Therefore, myosin seems to be abnormal in CAI.

Adenosine Triphosphatases↗