The effect of autografting on the myosin composition in skeletal muscle fibers

The purpose of the study was to investigate the changes in myosin heavy chain (MHC) and myosin light chain (MLC) isoforms following autotransplantation of extensor digitorum longus muscles. Muscles were grafted in "standard" and "nerve-intact" conditions. MHC and MLC isoforms were analyzed by sodium dodecyl sulphate gel electrophoresis. Changes in MHC isoforms 10, 30, and 60 days after grafting were similar in the "standard" and the "nerve-intact" grafts. In contrast to MHC, changes in MLC were different in the 10th day groups, but the same in the 30th day groups. Sixty days after grafting the content of MLC isoforms was the same as the control muscles. These data indicate that transient loss of functional innervation, even for a short time, has permanent effect on the composition of MHC but not MLC isoforms in regenerating skeletal muscle fibers.     

Native myosin from adult rabbit skeletal muscle: isoenzymes and states of aggregation

The globular heads of skeletal muscle myosin have been shown to exist as isoenzymes S1 (A1) and S1 (A2), and there are also isoforms of the heavy chains. Using capillary electrophoresis, we found two dominant isoenzymes of the whole native myosin molecule, in agreement with what has previously been found by various techniques for native and nondenatured myosin from adult rabbits. Findings about possible states of aggregation of myosin and its heads are contradictory. By analytical ultracentrifugation, we confirmed the existence of a tail-tail dimer. By laser light scattering, we found a head-head dimer in the presence of MgATP. Capillary electrophoresis coupled with analytical ultracentrifugation and laser light scattering led us to refine these results. We found tail-tail dimers in a conventional buffer. We found tail-tail and head-head dimers in the presence of 0.5 mM MgATP and pure head-head dimers in the presence of 6 mM MgATP. All the dimers were homodimers. Naming the dominant isoenzymes of myosin a and b, we observed tail-tail dimers with isoenzyme a (TaTa) and with isoenzyme b (TbTb) and also head-head dimers with isoenzyme a (HaHa) and with isoenzyme b (HbHb).     

The regulatory light chains of myosin modulate cross-bridge cycling in skeletal muscle

We investigated the kinetics of Ca2+ activation of skeletal muscle contraction elicited by the photolysis of caged Ca2+. Previously we showed that partial extraction of the 18-kDa regulatory light chains (RLCs) of myosin decreased the rate of force development and was subsequently increased by approximately 20% following reconstitution with RLCs (Potter, J. D., Zhao, J. and Pan, B. S. (1992) FASEB J. 6, A1240). We extend here the RLC-extraction study to the complete removal of the RLCs. The complete removal of RLCs was achieved by a combination of 5,5'-dithiobis-(2-nitrobenzoic acid) and EDTA treatment followed by reduction of oxidized sulfydryl groups by dithiothreitol. Under these conditions the complete extraction of RLCs was accompanied by the extraction of endogenous troponin C, resulting in the loss of isometric tension. Steady state force was restored to 65-75% following troponin C reconstitution and increased to 75-85% as a result of RLC reincorporation into the fibers. The rates of force transients generated by UV-flash photolysis of 1-(2-nitro-4,5-dimethoxyphenyl)-N,N,N',N' -tetrakis[(oxycarbonyl)methyl]-1,2-ethanediamine) or nitrophenyl-EGTA, photoliberating bound Ca2+, decreased 2-fold after RLC extraction and troponin C reconstitution and then increased to the values of intact fibers after RLC reconstitution. These results support our earlier findings that the regulatory light chains of myosin play an important role in the kinetics of cross-bridge cycling.     

Effect of trivalent and pentavalent arsenic on early developmental stages of the chick embryo

The structural element of an eukaryotic chromosome is the so-called chromatin fibre. It is a DNA-protein complex of about 100-200 A thickness and most probably running through from one end of a chromatid to the other. The fine structure of this DNA-protein fibre suggests a core of globular histone subunits around which the DNA-molecule is wound. The single strandedness of chromatids is suggested by the structure of premature condensed chromosomes. The course G-banding seen in metaphase chromosomes is presumably caused by groups of much finer bands seen in decondensed chromosomes. The number of such fine bands in the human genome is estimated to be 10 000-100 000, figures which are in the range of the number of genes in man.     

The role of motor neuron impulse activity in regulation of myosin composition of slow-twitch skeletal muscle

Information technology is growing rapidly in both its capability and capacity to support the delivery of health care. This paper describes the work of the Dental Interspec project, commissioned by the Department of Health. The aim of the project is to provide a framework for the development of information management and technology in dentistry. It is hoped that the work of this project can be built on to eventually produce integrated information systems which would have the potential to improve primary dental care delivery for both patients and dentists.     

Dimerization of native myosin LC2(RLC)-free subfragment 1 from adult rabbit skeletal muscle

We reinvestigated whether the native myosin LC2-free-subfragment 1 (S1) dimer exists by using viscometry, capillary electrophoresis, and laser light scattering. We found that the intrinsic viscosity of the monomer is [eta]m = 6.7 cm3/g and its translation diffusion coefficient is (c = 0) = 4.43 x 10(-)7 cm2/s. For the dimer, [eta]d = 19.8 cm3/g and (c = 0) = 2.54 x 10(-)7 cm2/s. Using the Svedberg equation and introducing the values of the sedimentation coefficients (5.05 S for the monomer and 6.05 S for the dimer), we find the following molecular weights: Mr,m = 108 000 Da and Mr,d = 213 000 Da, which agree well with previous determinations. Capillary electrophoresis successfully separated S1(A1) and S1(A2), in a monomer buffer, and S1(A1) and S1(A2) and a heterodimer S1(A1)-S1(A2), in a dimer buffer. An interesting feature of the monomer-dimer equilibrium is the presence of temperature transitions, whose positions and widths depend upon the buffer conditions. At low temperatures, a pure dimer was observed, whereas at high temperatures only the monomer was present. The dimerization site on both myosin and S1 is extremely labile.     

Identification of 1,4-dihydropyridine binding domains within the primary structure of the alpha 1 subunit of the skeletal muscle L-type calcium channel

Damage to the spinal cord in course of the treatment of diseases of the infrarenal aorta is a rare but calamitous complication. The reported incidence is about 0.2%. The neurological loss is usually complete flaccid paraplegia with high mortality and rare full or partial recovery. Between 1980 and 1991, 1070 reconstructive procedures of the infrarenal aorta were performed: 821 due to aneurysm (316 elective procedures [mortality 1.6%] and 505 emergency procedures [mortality 24.5%]) and 249 due to aorto-iliac occlusive disease. Damage to the spinal cord occurred in 2 patients (2/1070, 0.19%). One patient had incomplete paraparesis following repair of an unruptured abdominal aortic aneurysm with gradual return of all neurological symptoms to normal. The second patient developed complete paraplegia following repair of a ruptured infrarenal aneurysm. There war no recovery of the symptoms. The patient died from septicaemia 4 months later.     

Protein kinase C-mediated regulation of L-type Ca channels from skeletal muscle requires phosphorylation of the alpha 1 subunit

Dihydropyridine-sensitive, L-type Ca channels are hetero-oligomeric proteins that are modulated in certain cell types by protein kinase C (PKC). In native skeletal muscle membranes, PKC phosphorylates the alpha 1 and beta subunits of these Ca channels and modulates channel activity. However, it is unknown if phosphorylation of both subunits is necessary for PKC-mediated channel regulation. Here we report that stoichiometric phosphorylation of the alpha 1 subunit was required for activation of these Ca channels by PKC, while PKC-mediated phosphorylation of the beta subunit alone did not modify channel activity. Furthermore, reversal of the functional effects of PKC by protein phosphatase-1c was quantitatively correlated with dephosphorylation of the alpha 1 subunit.     

The effect of propranolol upon chick embryo cardiogenesis

The teratogenic effects of propranolol HCl on cardiac development were studied in chick embryos of days 3 and 4 of incubation. Propranolol was injected into the yolk sac at doses ranging from 0.05 to 0.6 mg per egg. All the treated and control embryos were examined on day 7. The LD50 for the embryos treated on the 3rd and 4th day was 0.15 and 0.35 mg per embryo, respectively. Cardiac anomalies such as aortic stenosis ventricular septal defects and common truncus arteriosus were observed. Other malformations included atrial septal defects, thin atrial wall and defects of the pulmonic, aortic and atrioventricular valves. The incidence of cardiac anomalies in the controls was very low. Propranolol was observed to slow the heart rate in the experimental embryos. It is suggested that slowing of heart rate at the early stages of heart development caused aberrant bloodstream flow patterns which probably resulted in the genesis of cardiac anomalies. The results of this study indicate that propranolol has teratogenic effects on chick embryo cardiogenesis.     

The stiffness of skeletal muscle in isometric contraction and rigor: the fraction of myosin heads bound to actin

Step changes in length (between -3 and +5 nm per half-sarcomere) were imposed on isolated muscle fibers at the plateau of an isometric tetanus (tension T0) and on the same fibers in rigor after permeabilization of the sarcolemma, to determine stiffness of the half-sarcomere in the two conditions. To identify the contribution of actin filaments to the total half-sarcomere compliance (C), measurements were made at sarcomere lengths between 2.00 and 2.15 microm, where the number of myosin cross-bridges in the region of overlap between the myosin filament and the actin filament remains constant, and only the length of the nonoverlapped region of the actin filament changes with sarcomere length. At 2.1 microm sarcomere length, C was 3.9 nm T0(-1) in active isometric contraction and 2.6 nm T0(-1) in rigor. The actin filament compliance, estimated from the slope of the relation between C and sarcomere length, was 2.3 nm microm(-1) T0(-1). Recent x-ray diffraction experiments suggest that the myosin filament compliance is 1.3 nm microm(-1) T0(-1). With these values for filament compliance, the difference in half-sarcomere compliance between isometric contraction and rigor indicates that the fraction of myosin cross-bridges attached to actin in isometric contraction is not larger than 0.43, assuming that cross-bridge elasticity is the same in isometric contraction and rigor.     

Sites of selective cAMP-dependent phosphorylation of the L-type calcium channel alpha 1 subunit from intact rabbit skeletal muscle myotubes

The principal (alpha 1) subunit of purified skeletal muscle dihydropyridine-sensitive (L-type) calcium channels is present in full-length (212 kDa) and COOH-terminal truncated (190 kDa) forms, which are both phosphorylated by cAMP-dependent protein kinase (cA-PK) in vitro. Immunoprecipitation of the calcium channel from rabbit muscle myotubes in primary cell culture followed by phosphorylation with cA-PK, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and two-dimensional phosphopeptide mapping revealed comparable phosphorylation of three COOH-terminal phosphopeptides found in the purified full-length alpha 1 subunit. Stimulation of muscle myotubes with a permeant cAMP analogue, 8-(4-chlorophenylthio) adenosine 3',5'-cyclic monophosphate, prior to immunoprecipitation of alpha 1 results in a 60-80% reduction of cA-PK catalyzed "back" phosphorylation of each of these sites in vitro in calcium channels purified from the cells, indicating that these sites are phosphorylated in vivo in response to increased intracellular cAMP. Serine 687, the most rapidly phosphorylated site in the truncated 190-kDa alpha 1 subunit, was observed as a minor phosphopeptide whose level of phosphorylation was not significantly affected by stimulation of endogenous cA-PK in the myotubes. The COOH-terminal sites, designated tryptic phosphopeptides 4, 5, and 6, were identified as serine 1757 (phosphopeptides 4 and 6) and 1854 (phosphopeptide 5) by a combination of protease cleavage, phosphorylation of synthetic peptides and fusion proteins, specific immunoprecipitation, and phosphopeptide mapping. Phosphorylation of serines 1757 and 1854 in the COOH-terminal region of the 212-kDa alpha 1 subunit in intact skeletal muscle cells may play a pivotal role in the regulation of calcium channel function by cA-PK.     

The subunit structure of alpha-acetohydroxyacid isomeroreductase from Salmonella typhimurium

Alpha-Acetohydroxyacid isomeroreductase from Salmonella typhimurium has a native molecular weight of 220,000. The constituent polypeptide chains exhibit anomalous but unimodal electrophoretic migration on sodium dodecyl sulfate-urea polyacrylamide gels. The subunit molecular weight, determined by sedimentation equilibrium in 6 M guanidine hydrochloride, is 57,000. The apparent tetrameric nature of the native enzyme was confirmed by determining the types of oligomers formed upon cross-linking with dimethylsebacimidate. Analysis of tryptic peptides suggests that the polypeptide chains have an identical amino acid sequence. Carbohydrate analysis, ultraviolet absorption spectrum, and atomic absorption spectrum are consistent with the lack of cobalamine and cobalt. The Michaelis constants are as follows: alpha-acetolactate, 2.9 x 10-4 M; alpha-aceto-alpha-hydroxybutyrate, 7.8 x 10-4 M; NADPH, 1.5 x 10-5 M; Mg2+, 7.7 x 10-4 M. The catalytic constants (molecules of substrate catalyzed per min per molecules of enzyme) for alpha-acetolactate and alpha-aceto-alpha-hydroxybutyrate are 1,100 and 4,700, respectively. Comparative tryptic peptide analysis and immunological analysis show that alpha-acetohydroxyacid isomero-reductase and biosynthetic L-threonine deaminase bear no structural relationship and therefore rule out a "shared structure" hypothesis for the putative involvement of L-threonine deaminase in the synthesis of alpha-acetohydroxyacid isomeroreductase.     

Altered expression of myosin heavy chain in human skeletal muscle in chronic heart failure

To explore further alterations in skeletal muscle in chronic heart failure (CHF), we examined myosin heavy chain (MHC) isoforms from biopsies of the vastus lateralis in nine male patients with class II-III (CHF) (left ventricular ejection fraction (LVEF) 26 +/- 11%, peak oxygen consumption (peak VO2) 12.6 +/- 2 mL.kg-1.min-1) and nine age-matched sedentary normal males (NL). The relative content of MHC isoforms I, IIa, and IIx was determined by gel electrophoresis as follows: The normal sedentary group (NL) had a higher percent of MHC type I when compared with the patients (NL 48.4 +/- 7% vs CHF patients 24 +/- 21.6%, P < 0.05, no difference between MCH IIa (NL 45.1 +/- 10.5% vs CHF 56.0 +/- 12.5%), and CHF patients had a higher relative content of MHC type IIx than did the normal group (NL 6.5 +/- 9.6% vs CHF 20.0 +/- 12.9%, P < 0.05. Three of nine patients had no detectable MHC type I. In patients relative expression of MHC type I (%) was related to peak VO2 (r = 0.70, P < 0.05). Our results indicate that major alterations in MHC isoform expression are present in skeletal muscle in CHF. These alterations parallel previously reported changes in fiber typing that may affect contractile function i skeletal muscle and possibly exercise performance. The absence of MHC type I in some CHF patients suggests that skeletal muscle changes in this disorder are not solely a result of deconditioning, buy may reflect a specific skeletal muscle myopathy in this disorder.