Fast-to-slow muscle conversion by chronic electrostimulation: effects on mitochondrial respiratory chain function with possible implications for the gracilis neosphincter procedure

The effects of chronic, around the clock, low-frequency electrostimulation on the respiratory chain activity and cytochrome content of freshly isolated mitochondria were evaluated in rabbit skeletal muscle before and after 30 days of continuous or cyclical electrostimulation using a totally implantable system and a training programme now used in humans. The respiratory activity measured in state III increased strongly after electrostimulation. The efficiency of the respiratory chain increased significantly after electrostimulation but the activity of complex [(reduced nicotinamide adenine dinucleotide dehydrogenase) did not increase. The amount of cytochromes a and a3, b562, and c and c1 increased clearly after electrostimulation. The respiratory activity rate of mitochondria obtained after continuous electrostimulation was apparently higher than after cyclical electrostimulation. Chronic uninterrupted low-frequency electrostimulation, using a clinical training programme, induces an increase in mitochondrial respiratory chain activity in purified mitochondria of skeletal muscle. These changes are the basis of induced resistance to fatigue in fast-to-slow muscle conversion by chronic electrostimulation.     

Presence of laminin alpha5 chain and lack of laminin alpha1 chain during human muscle development and in muscular dystrophies

There is currently a great interest in identifying laminin isoforms expressed in developing and regenerating skeletal muscle. Laminin alpha1 has been reported to localize to human fetal muscle and to be induced in muscular dystrophies based on immunohistochemistry with the monoclonal antibody 4C7, suggested to recognize the human laminin alpha1 chain. Nevertheless, there seems to be no expression of laminin alpha1 protein or mRNA in developing or dystrophic mouse skeletal muscle fibers. To address the discrepancy between the results obtained in developing and dystrophic human and mouse muscle we expressed the E3 domain of human laminin alpha1 chain as a recombinant protein and made antibodies specific for human laminin alpha1 chain (anti-hLN-alpha1G4/G5). We also made antibodies to the human laminin alpha5 chain purified from placenta. In the present report we show that hLN-alpha1G4/G5 antibodies react with a 400-kDa laminin alpha1 chain and that 4C7 reacts with a 380-kDa laminin alpha5 chain. Immunohistochemistry with the hLN-alpha1G4/G5 antibody and 4C7 revealed that the two antibodies stained human kidney, developing and dystrophic muscle in distinct patterns. Our data indicate that the previously reported expression patterns in developing, adult, and dystrophic human muscle tissues with 4C7 should be re-interpreted as an expression of laminin alpha5 chain. Our data are also consistent with earlier work in mouse, indicating that laminin alpha1 is largely an epithelial laminin chain not present in developing or dystrophic muscle fibers.     

Primary structure, developmental expression, and immunolocalization of the murine laminin alpha4 chain

The complete primary structure of the mouse laminin alpha4 chain was derived from cDNA clones. The translation product contains a 24-residue signal peptide preceding the mature alpha4 chain of 1,792 residues. Northern analysis on whole mouse embryos revealed that the expression was weak at day 7, but it later increased and peaked at day 15. In adult tissues the strongest expression was observed in lung and cardiac and skeletal muscles. Weak expression was also seen in other adult tissues such as brain, spleen, liver, kidney, and testis. By in situ hybridization of fetal and newborn tissues, expression of the laminin alpha4 chain was mainly localized to mesenchymal cells. Strong expression was seen in the villi and submucosa of the developing intestine, the mesenchymal stroma surrounding the branching lung epithelia, and the external root sheath of vibrissae follicles, as well as in cardiac and skeletal muscle fibers. In the developing kidney, intense but transient expression was associated with the differentiation of epithelial kidney tubules from the nephrogenic mesenchyme. Immunohistologic staining with affinity-purified IgG localized the laminin alpha4 chain primarily to lung septa, heart, and skeletal muscle, capillaries, and perineurium.     

Altered secondary myogenesis in transgenic animals expressing the neural cell adhesion molecule under the control of a skeletal muscle alpha-actin promoter

The majority of skeletal muscle fibers are generated through the process of secondary myogenesis. Cell adhesion molecules such as NCAM are thought to be intricately involved in the cell-cell interactions between developing secondary and primary myotubes. During secondary myogenesis, the expression of NCAM in skeletal muscle is under strict spatial and temporal control. To investigate the role of NCAM in the regulation of primary-secondary myotube interactions and muscle fusion in vivo, we have examined muscle development in transgenic mice expressing the 125-kD muscle-specific, glycosylphosphatidylinositol-anchored isoform of human NCAM, under the control of a human skeletal muscle alpha-actin promoter that is active from about embryonic day 15 onward. Analysis of developing muscle from transgenic animals revealed a significantly lower number of myofibers encased by basal lamina at postnatal day 1 compared with nontransgenic littermates, although the total number of developing myofibers was similar. An increase in muscle fiber size and decreased numbers of VCAM-1-positive secondary myoblasts at postnatal day 1 was also found, indicating enhanced secondary myoblast fusion in the transgenic animals. There was also a significant decrease in myofiber number but no increase in overall muscle size in adult transgenic animals; other measurements such as the number of nuclei per fiber and the size of individual muscle fibers were significantly increased, again suggesting increased secondary myoblast fusion. Thus the level of NCAM in the sarcolemma is a key regulator of cell-cell interactions occurring during secondary myogenesis in vivo and fulfills the prediction derived from transfection studies in vitro that the 125-kD NCAM isoform can enhance myoblast fusion.     

Alterations in skeletal muscle gene expression in the rat with chronic congestive heart failure

Congestive heart failure is often associated with skeletal muscle abnormalities that contribute to early fatigue and acidosis. Up to the present time, however, the mechanisms responsible for these changes are unclear. Myocardial infarctions were produced by coronary ligation in adult Sprague-Dawley rats. At 20 weeks, 10 control rats, and 15 animals with heart failure [defined by elevated LVEDP (26.1 +/- 3.1 v 2.5 +/- 0.5 mmHg) and RV hypertrophy (300 +/- 21 g v 158 +/- 9 mg)] underwent in vivo measurements of total body, and soleus total protein and myosin heavy chain (MHC) synthesis by [3H]leucine constant infusion. Soleus muscle was also analysed for protein content, and MHC isoenzyme content by SDS-PAGE. Northern blotting also was used to determine levels of the mRNA's encoding type I, IIa, IIb, and IIx MHC, alpha-skeletal actin, COX III, SDH and GAPDH. Soleus muscles in heart failure rats were smaller than controls (112 +/- 6 v 126 +/- 5 mg) and the degree of atrophy was significant when corrected for body mass (0.38 +/- 0.02 v 0.46 +/- 0.02 mg/g. P = 0.007). Although there was no significant difference in plasma leucine flux (an index of whole-body protein synthesis), soleus muscle total and MHC synthesis was reduced in heart failure animals. Whereas the Type I MHC isoenzyme (beta MHC) was the only MHC detected in the soleus of control animals, type II MHC isoenzyme comprised 11.8 +/- 3.1% of the MHC in the heart failure group. Furthermore, steady-state mRNA levels encoding beta MHC were significantly depressed in the heart failure rats, where those encoding Types IIb and IIx MHC were increased. Steady-state mRNA levels of alpha-skeletal actin, cytochrome C oxidase (COX III) and succinate dehydrogenase (SDH) were also significantly depressed. This animal model of chronic heart failure is associated with quantitative and qualitative alterations in skeletal muscle gene expression that are similar to those reported in skeletal muscle of patients with chronic heart failure. The altered phenotype and impaired metabolic capacity may contribute to exercise intolerance in CHF.     

Electron transport chain defects in Alzheimer's disease brain

Previous work suggested a deficiency in the terminal complex of the mitochondrial electron transport chain, cytochrome c oxidase (COX), in platelet mitochondria of Alzheimer's disease (AD) patients. The present study extends this observation to AD brain mitochondria through assay of electron transport chain activities in mitochondria isolated from autopsied brain samples from AD patients (n = 9) and from controls with and without known neurologic disease (n = 8). AD brain mitochondria demonstrated a generalized depression of activity of all electron transport chain complexes. This depression was most marked in COX activity (p < 0.001). Concentrations of cytochromes b, c1, and aa3 were similar in AD and controls. The electron transport chain is defective in AD brain, and the defect centers about COX.     

Neurotransmitter transporters as molecular targets for addictive drugs

The mdx mouse, an animal model of the Duchenne muscular dystrophy, was used for the investigation of changes in mitochondrial function associated with dystrophin deficiency. Enzymatic analysis of skeletal muscle showed an approximately 50% decrease in the activity of all respiratory chain-linked enzymes in musculus quadriceps of adult mdx mice as compared with controls, while in cardiac muscle no difference was observed. The activities of cytosolic and mitochondrial matrix enzymes were not significantly different from the control values in both cardiac and skeletal muscles. In saponin-permeabilized skeletal muscle fibers of mdx mice the maximal rates of mitochondrial respiration were about two times lower than those of controls. These changes were also demonstrated on the level of isolated mitochondria. Mdx muscle mitochondria had only 60% of maximal respiration activities of control mice skeletal muscle mitochondria and contained only about 60% of hemoproteins of mitochondrial inner membrane. Similar findings were observed in a skeletal muscle biopsy of a Duchenne muscular dystrophy patient. These data strongly suggest that a specific decrease in the amount of all mitochondrial inner membrane enzymes, most probably as result of Ca2+ overload of muscle fibers, is the reason for the bioenergetic deficits in dystrophin-deficient skeletal muscle.     

Contribution of growth, fatness, and activity to weight disturbance after septohypothalamic cuts in adult hamsters.

Investigated the mechanism responsible for weight stability in 80 adult female golden hamsters by (a) transsecting the dorsoventrally oriented nerve pathways between the septal area and hypothalamus (SH cuts) and (b) partitioning the observed increases in the rate of weight gain into 3 contributory components: changes in somatic growth, in body fatness, and in energy expended as voluntary activity on horizontal disks. 60–70% of the weight increase after SH cuts was due to acquisition of lean body mass, and 30–40% consisted of excess body fat. After SH cuts, serum growth hormone and insulin concentrations were increased on Day 14, food intake was increased between Day 2 and Day 42, skeletal lengths were greater on Day 77, and voluntary activity levels were 84% lower on Days 10–45, relative to controls. It is concluded that dorsoventrally oriented nerve pathways in the septal area are involved in the control of growth, maintenance of body fat reserves, and voluntary activity. (51 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

A non-radioisotopic human natural killer cell assay using rhodamine-123 fluorescent dye as labelling probe

A non-radioisotopic method for assessment of human natural killer (NK) cell activity in peripheral blood mononuclear cells (PBMC) was established by labelling K562 erythroleukemia target cells with a fluorescent dye, rhodamine-123 (Rh-123). The labelling and assay conditions were determined for minimizing spontaneous release (SR). In order to investigate whether NK activity assessed by measuring Rh-123 release agrees with the activity determined by a 51Cr release assay, the NK activity of PBMC was measured simultaneously by both assay methods. Statistical analysis demonstrates that NK activities determined by Rh-123 release correlate well with those measured by 51Cr release. The Rh-123 release assay under the conditions determined was found to be applicable to measurement of the enhanced NK activity resulting from pretreatment of effector leukocytes with interferon-alpha. It is concluded that the Rh-123 release assay with use of K-562 labelled target cells is practical for the assessment of human NK activity in laboratories where use of radioisotopes is not permitted or undesirable.     

Differential heparin sensitivity of alpha-dystroglycan binding to laminins expressed in normal and dy/dy mouse skeletal muscle

The alpha-dystroglycan binding properties of laminins extracted from fully differentiated skeletal muscle were characterized. We observed that the laminins expressed predominantly in normal adult rat or mouse skeletal muscle bound alpha-dystroglycan in a Ca2+-dependent, ionic strength-sensitive, but heparin-insensitive manner as we had observed previously with purified placental merosin (Pall, E. A., Bolton, K. M., and Ervasti, J. M. 1996 J. Biol. Chem. 271, 3817-3821). Rat skeletal muscle laminins partially purified by heparin-agarose affinity chromatography also bound alpha-dystroglycan without sensitivity to heparin. We also confirm previous studies of dystrophic dy/dy mouse skeletal muscle showing that the alpha2 chain of merosin is reduced markedly and that the laminin alpha1 chain is not up-regulated detectably. However, we further observed a quantitative decrease in the expression of laminin beta/gamma chain immunoreactivity in alpha2 chain-deficient dy/dy skeletal muscle and reduced alpha-dystroglycan binding activity in laminin extracts from dy/dy muscle. Most interestingly, the alpha-dystroglycan binding activity of residual laminins expressed in merosin-deficient dy/dy skeletal muscle was inhibited dramatically (69 +/- 19%) by heparin. These results identify a potentially important biochemical difference between the laminins expressed in normal and dy/dy skeletal muscle which may provide a molecular basis for the inability of other laminin variants to compensate fully for the deficiency of merosin in some forms of muscular dystrophy.     

The lack of NK cytotoxicity associated with fresh HUCB may be due to the presence of soluble HLA in the serum

Human bone marrow transplantation is becoming more common in the treatment of certain forms of cancer despite the scarcity of HLA matched donors. Because human umbilical cord blood (HUCB) has been used as a source for stem cells in bone marrow transplantation, and because NK cells appear to be important in graft versus leukemia response, we investigated the lytic activity of freshly isolated HUCB NK cells (HUCB-NK) against tumor targets and their ability to differentiate into LAK cells following stimulation with various cytokines. Although cytotoxicity mediated by fresh HUCB-NK was low compared to that of adult peripheral blood lymphocyte-derived NK cells (PBL-NK), the ability of HUCB-NK to bind to K562 target cells (TC) was similar to PBL-NK. In addition, the PBL-NK cytotoxicity of postpartum mothers was also low compared to that of normal adult PBL-NK. When we incubated HUCB for 18 hr in either IL-2 or IL-12, we boosted the level of HUCB-NK cytotoxicity to approximately the level observed in PBL-NK and increased the level of perforin, granzyme A, and granzyme B mRNA expression. In addition, when we incubated HUCB in IL-2, IL-4, IL-7, IL-12, TNF-alpha, IFN-alpha, IFN-gamma, or TGF-beta for 5 days, we observed that HUCB was capable of generating LAK cells only when incubated with either IL-2 or IL-12. In contrast, IL-2, IL-7, IL-12, TNF-alpha, and IFN-gamma all generated LAK cells from adult PBL. When we added to the medium low-dose IL-2 and irradiated K562 as feeder cells (mini-LAK), we were unable to generate LAK activity from HUCB-NK, whereas we could generate it with PBL-NK cells under the same conditions. Addition of serum derived from HUCB in a 4-hr 51Cr release assay with PBL-NK as the effector cells (EC) and K562 as the TC resulted in a 42% decrease in PBL-NK-mediated cytotoxicity. Although we detected no TGF-beta in HUCB serum, we did detect high concentrations of soluble class I MHC (sHLA). To our knowledge, sHLA has not previously been shown to inhibit NK cytotoxicity, although the expression of class I HLA on the surface of TC has been shown to inhibit NK cytotoxicity. To study further the effect of sHLA on cell-mediated cytotoxicity, we added various concentrations of sHLA to EC mediating NK, ADCC, and CTL activities. All were inhibited in a dose-dependent manner.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of myb-related genes in the moss, Physcomitrella patens

Previous studies have indicated that both cytodifferentiation of Clara cells and the onset of pulmonary cytochrome P450 activity are postnatal events. However, the relationship between these two events during lung development remains poorly understood. To determine how these events interrelate, we examined rabbit Clara cells during postnatal differentiation, with the following goals in mind: 1) to identify the patterns of intracellular expression of cytochrome P450 monooxygenase isozymes 2B and 4B and cytochrome P450 reductase, 2) to describe the biogenesis of the organelles with which these isozymes are associated, namely smooth and rough endoplasmic reticulum, and 3) to compare the patterns of expression with cytochrome P450 activity in the whole lung over the same period. Lungs of rabbits ranging in age from 24 days gestational age (DGA) to 25 weeks postnatally were studied. Ultrastructural morphometry showed that smooth endoplasmic reticulum averaged < 5% of the Clara cell volume in late gestational (24-30 DGA) and neonatal rabbits [0-7 days postnatally (DPN)], grew to 20-30% of the cell volume in 14-21-DPN animals, and approximated adult levels (> 40%) in 28-DPN rabbits. In contrast, rough endoplasmic reticulum decreased from > 10% of the cell volume at 27 DGA to < 5% in adults. All postnatal animals showed considerable heterogeneity in the abundance of smooth endoplasmic reticulum among individual cells. Immunohistochemistry revealed that cytochrome P450 reductase appeared in Clara cells earlier (28 DGA) than did either isozyme 2B or 4B (1 DPN). Each antigen was detected first in the apical borders of the cells, then throughout the cytoplasm in a few cells by 7 DPN, and finally in adult abundance by 28 DPN. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting showed that cytochrome P450 protein concentrations increased postnatally. Cytochrome P450 heme protein was not detected spectrophotometrically in the lungs of animals younger than 3 DPN but increased to approximately 70% of adult levels by 28 DPN. Likewise, cytochrome P450 activity (measured as ethoxy- and pentoxyresorufin O-dealkylation) was not detected in animals younger than 2 DPN but increased to approximately 75% of adult levels by 28 DPN.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparative electronmicroscopical investigations on the influences of altered gravity on cytochrome oxidase in the inner ear of fish: a spaceflight study

The regional metabolic activity in the otolithic sensory epithelia of the inner ear of a cichlid fish (Oreochromis mossambicus) was investigated on light- and electronmicroscopical level using the cytochemical method for detection of cytochrome oxidase activity. In adult animals a characteristic distribution of mitochondria with high enzyme activity was found in sensory and non-sensory cells of otolithic sensory epithelia, which was correlated with regions with a high energy demand. These findings were the basis for studies on the influence of long-term altered gravity conditions in developing larvae: hypogravity (10(-4) g in spaceflight), normal gravity (1 g in a centrifuge in space and 1 g on earth) and hypergravity (3 g in a laboratory centrifuge). Cytochrome oxidase activity was quantified in different parts of the sensory hair cell synapse in the vestibular sensory epithelia utricle and saccule: apical and basal cytoplasm, postsynaptic area of the afferent synapse and presynaptic region of the efferent synapse. Our results show that the energy metabolism of utricle, but not of saccule is decreased after microgravity exposure during the 2nd German Spacelab Mission D-2. However, a general effect of the spaceflight is detectable in both sensory epithelia. Long-term exposure to increased acceleration (3 g) had no effects on cytochrome oxidase activity in inner ear sensory epithelia.     

Plasmatocyte spreading peptide is encoded by an mRNA differentially expressed in tissues of the moth Pseudoplusia includens

We investigated in rats the effect of 4 wk of hypodynamia on the respiration of mitochondria isolated from four distinct muscles [soleus, extensor digitorum longus, tibial anterior, and gastrocnemius (Gas)] and from subsarcolemmal (SS) and intermyofibrillar (IMF) regions of mixed hindlimb muscles that mainly contained the four cited muscles. With pyruvate plus malate as respiratory substrate, 4 wk of hindlimb suspension produced an 18% decrease in state 3 respiration for IMF mitochondria compared with those in the control group (P < 0.05). The SS mitochondria state 3 were not significantly changed. Concerning the four single muscles, the mitochondrial respiration was significantly decreased in the Gas muscle, which showed a 59% decrease in state 3 with pyruvate + malate (P < 0.05). The other muscles presented no significant decrease in respiratory rate in comparison with the control group. With succinate + rotenone, there was no significant difference in the respiratory rate compared with the respective control group, whatever the mitochondrial origin (SS, or IMF, or from single muscle). We conclude that 4 wk of hindlimb suspension alters the respiration of IMF mitochondria in hindlimb skeletal muscles and seems to act negatively on complex I of the electron-transport chain or prior sites. The muscle mitochondria most affected are those isolated from the Gas muscle.     

The influence of beta subunit structure on the stability of Na+/K(+)-ATPase complexes and interaction with K+

Heterologous expression of the beta subunit of H+/K(+)-ATPase (HK beta) with alpha subunits of Na+/K(+)-ATPase (NK alpha) in yeast leads to the formation of ouabain binding complexes, indicating assembly of the two subunits into active ion pumps (Eakle, K. A., Kim, K. S., Kabalin, M. A., and Farley, R. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 2834-2838). Complexes of NK alpha and HK beta are less sensitive to inhibition of ouabain binding by K+, suggesting that HK beta lowers the affinity of K+ binding sites. This effect is particularly pronounced when HK beta is combined with the alpha 3 isoform of NK alpha. In this case, titration with K+ yields a biphasic curve, suggesting that there are two nonequivalent sites for K+ binding. Attempts at purifying complexes formed with either alpha 1 + HK beta or alpha 3 + HK beta using SDS extraction of microsomal membranes resulted in the loss of ouabain binding. Controls show that alpha 1 + beta 1 and alpha 3 + beta 1 complexes still retain ouabain binding after SDS extraction under the same conditions. This suggests that the HK beta subunit forms a less stable complex with NK alpha subunits. We have created chimeric beta subunits comprised of the amino-terminal cytoplasmic and transmembrane regions of HK beta combined with the carboxyl-terminal extracellular region of Na+/K(+)-ATPase beta 1 (HN beta 1) and the complementary chimera with amino-terminal cytoplasmic and transmembrane regions of beta 1 combined with the carboxyl-terminal extracellular region of HK beta (NH beta 1). When NH beta 1 is combined with either alpha 1 or alpha 3, the complexes show profiles of K+ inhibition of ouabain binding that are very similar to HK beta combined with either alpha 1 or alpha 3. The data suggest that the extracellular region of HK beta is primarily responsible for the effect on apparent K+ affinity. When the HN beta 1 subunit is expressed with the alpha 3 subunit, less than 5% of the amount of ouabain binding complexes are formed compared with HN beta 1 + alpha 1. This observation suggests that the HN beta 1 subunit either assembles poorly or forms an unstable complex with alpha 3. After SDS extraction, complexes of alpha 1 + NH beta 1 and alpha 3 + NH beta 1 retain ouabain binding, while alpha 1 + HN beta 1 complexes are sensitive to SDS extraction.(ABSTRACT TRUNCATED AT 400 WORDS)     

The subunit structure of myosin from skeletal muscle of the early chick embryo

The subunit composition and immunological properties of two types of myosins, the 3S and 6S myosin components, from skeletal muscle of early chick embryos were studied by SDS-acrylamide gel electrophoresis and immunodiffusion techniques. It was shown that the 6S myosin in the early embryonic stage was composed of two heavy chains and three kinds of light chains, as is well-known in the complete myosin molecule, having the same molecular weights and the same antigenicities as corresponding subunits of the myosin from adult chicken skeletal muscle. The heavy chain of 6S myosin was also reactive with the antibody against the heavy chain of cardiac myosin. The embryonic 3S myosin was shown to be composed of a heavy chain which was roughly the same in molecular weight but not the same in antigenicity as those of adult or embryonic 6S myosin. No light chains were detected either electrophoretically or immunologically in the 3S myosin component.     

The Saccharomyces cerevisiae succinate-ubiquinone reductase contains a stoichiometric amount of cytochrome b562

The Saccharomyces cerevisiae succinate-ubiquinone reductase or succinate dehydrogenase (SDH) is a tetramer of non-equivalent subunits encoded by the SDH1, SDH2, SDH3, and SDH4 genes. In most organisms, SDH contains one or two endogenous b-type hemes. However, it is widely believed that the yeast SDH does not contain heme. In this report, we demonstrate the presence of a stoichiometric amount of cytochrome b562 in the yeast SDH. The cytochrome is detected as a peak present in fumarate-oxidized, dithionite-reduced mitochondria. The peak is centered at 562 nm and is present at a heme:covalent FAD molar ratio of 0.92+/-0.11. The cytochrome is not detectable in mitochondria isolated from SDH3 and SDH4 deletion strains. These observations strongly support our conclusion that cytochrome b562 is a component of the yeast SDH.