Accumulation of mitochondrial DNA deletions in myotubes cultured from muscles of patients with mitochondrial myopathies

 Myoblast cultures were established from muscle biopsies of two patients harboring heteroplasmic mitochondrial (mt) DNA deletions. The accumulation kinetics of the deleted mtDNA was followed during myoblast to myotube differentiation. The percent- age of deleted mtDNA was determined by quantitative PCR in myoblasts, myotubes, and muscle biopsies. The deleted form accounted for 65% of the mtDNA present in a muscle biopsy from a patient harboring a 5.6-kb deletion. The percentage of deleted mtDNA was 1.2% in myoblasts and increased progressively after differentiation, up to 12% at 21 days after the commitment time. In a second patient harboring a 2.8-kb deletion, the percentage of deleted mtDNA increased much more slowly: from 0.07% in myoblasts to 0.21% after 22 days of differentiation, as compared with 45% in the muscle biopsy. Thus, a three- and ten-fold increase, respectively, in the fraction of deleted mtDNA occurred during the differentiation of myoblasts to myotubes from the two patients. The faster accumulation of deleted mtDNA in the first patient’s cells was linked to an earlier myoblast to myotube differentiation, suggesting that the level of deleted mtDNA is inversely related to the rate of cell proliferation. Received: 16 April 1996/Accepted: 29 July 1996     

Sensory characteristics of the P afferent neurone of the crab thoracic-coxal muscle receptor organ

Intracellular recordings were made from the P fibre, the smallest of the three afferent neurones innervating the thoracic-coxal muscle receptor organ of the crab (Carcinus maenas). While the two larger afferents are nonspiking, the response of the P fibre to a trapezoidal change in receptor muscle length consists of a single action potential signalling the onset of stretch superimposed on a graded amplitude receptor potential. The P fibre is sensitive to the velocity of the applied stretch, but is insensitive to static joint position, stretch amplitude and the velocity of the release phase. The presence and amplitude of the action potential depends on the initial length of the receptor muscle, the tension caused by efferent activation of the receptor muscle prior to receptor stretch, and on the velocity of stretch. Length constant (1.9 mm) and specific membrane resistance (76 K · cm²) values obtained for the P fibre, together with its small diameter (7 m) suggest that this neurone is less well adapted to conveying passive signals to the thoracic ganglion than are the S and T fibres. It is likely that the P fibre complements the length sensitivity of the S fibre and the tension and velocity sensitivity of the T fibre by signalling the onset of receptor stretch via single action potentials.Abbreviations TCMRO thoracic-coxal muscle receptor organ - TTX tetrodotoxin     

Effects of various environmental conditions on growth and reproduction of the sea hareAplysia oculifera (Adams and Reeve, 1850)

We studied the influences of food type, food quantity, water currents, starvation and light on growth and reproduction of the sea hareaplysia oculifera (Adams and Reeve, 1850) under laboratory conditions. Out of five species of algae served as food,Enteromorpha intestinalis promoted the fastest growth ofA. oculifera, Ulva spp. slower growth,Cladophora sp. allowed maintenance spp. slower growth,Cladophora sp. allowed maintenance of steady body mass, and the brown algaeColpomenia sp. andPadina pavonia were rejected by the sea hares. When sea hares were exposed to four levels of water currents, growth rates decreased as water currents increased. Sea hares fed on 50% ration grew slower than those fed on 100% ration (ad libitum). During 10 days of starvation sea hares lost weight, but when subsequently fed 100% ration they recovered and grew at a rate similar to those fed continuously with 100% ration. Under shade and under natural sunlight sea hares grew at the same rates. Whenever growth rates decreased, sea hares began to spawn at a smaller body size.A. oculifera demonstrated physiological plasticity that adapted them to varied and unpredictable environmental conditions. At different conditions of food availability they applied different tactics of resource allocation between growth and reproduction.     

Culture of rat mesenteric arteriolar smooth muscle cells: effects of platelet-derived growth factor, angiotensin, and nitric oxide on growth

We cultured smooth muscle cells as explants from rat mesenteric arterioles (40–200m in diameter) obtained by injecting a suspension of iron oxide intraarterially and magnetically separating the arterioles after collagenase digestion of adventitial tissue. In third-passaged cells we ascertained smooth muscle purity of >98% by characteristic morphology, contraction responses, and specific immunofluorescence staining. Treatment of growth-arrested (in 0.4% fetal calf serum) cells with platelet-derived growth factor (0.3–7.5 nM) or angiotensin II (0.001–1000 nM) induced ³H-thymidine incorporation and cell proliferation in a dose-dependent manner (P<0.01). S-nitroso-N acetylpencillamine (0.05–0.5 mM), a nitric oxide-generating compound, inhibited 10% fetal calf serum-induced ³H-thymidine incorporation (P<0.05) and cell proliferation (P<0.01). The antimitogenic effect of S-nitroso-N-acetylpencillamine was significantly reduced by hemoglobin and potentiated by superoxide dismutase (P<0.01). In addition to a new technique for culturing mesenteric arteriolar smooth muscle cells, these findings provide evidence that platelet-derived growth factor, angiotensin II, and nitric oxide may be involved in their growth control.     

A fluorescence depolarization study of the orientational distribution of crossbridges in muscle fibres

A fluorescence depolarization study of the orientational distribution of crossbridges in dye-labelled muscle fibres is presented. The characterization of this distribution is important since the rotation of crossbridges is a key element in the theory of muscle contraction. In this study we exploited the advantages of angle-resolved experiments to characterize the principal features of the orientational distribution of the crossbridges in the muscle fibre. The directions of the transition dipole moments in the frame of the dye and the orientation and motion of the dye relative to the crossbridge determined previously were explicitly incorporated into the analysis of the experimental data. This afforded the unequivocal determination of all the second and fourth rank order parameters. Moreover, this additional information provided discrimination between different models for the orientational behaviour of the crossbridges. Our results indicate that no change of orientation takes place upon a transition from rigor to relaxation. The experiments, however, do no rule out a conformational change of the myosin S 1 during the transition. Correspondence to: Y. K. Levine     

Y-intercept of the maximal work-duration relationship and anaerobic capacity in cyclists

The degree to which the y-intercept (Y-int) of the linear regression of maximal work output on exercise duration represented anaerobic capacity was determined in ten well-trained male cyclists [peak oxygen uptake ( = 69.8 (SD 4.2) ml · kg ^–1 · min ^–1). Each cyclist performed three exhausting cycle sessions on separate occasions; the mean exercise durations were 312, 243 and 141 s for the low (approximately 104% , medium (approximately 108% and high (approximately 113% intensities respectively, and Y-int (kilojoules; joules per kilogram was derived from the regression of work output on exercise duration. The muscle anaerobic adenosine 5-triphosphate (ATP) yield (ATP) and anaerobic capacity (AC) were estimated from changes in metabolites in the vastus lateralis muscle and blood lactate concentration during the high intensity cycling session. The activities of glycogen phosphorylase, phosphofructokinase and citrate synthase, as well as muscle buffer value (in vitro ) were also determined. The Y-int (kilojoules) was positively correlated (P0.05) with AC (r=0.73), ATP (r=0.70) and in vitro (r=0.71); similar correlations (P0.05) were observed for Y-int (joules per kilogram). The Y-int was not correlated (P>0.05) with any enzyme activity. When the Y-int was transformed into oxygen equivalents [litres of oxygen equivalent (1 O₂ Eq)] it was, on average, 0.92 1 O₂ Eq lower than AC (P0.05); however, an alternative method of establishing the work-duration regression yielded a mean Y-int which was only 0.19 1 O₂ Eq less than AC (P0.05). These findings support the validity of Y-int as a work estimate of anaerobic capacity in well-trained cyclists.     

Endurance training alters alanine and glutamine release from muscle during contractions

The release of alanine and glutamine from perfused muscle of trained and control animals was investigated. Release rates did not differ between trained and control muscle at rest. During contractions in trained muscle, alanine release was higher than at rest, while glutamine release was transiently increased. Phenylalanine release did not differ between trained and control muscle, implying that protein degradation was not increased in trained muscle. The muscle cellular adaptations to training include a selective modification of amino acid output, which could potentially influence gluconeogenic flux and alter muscle ammonia levels during contractions.     

Physiology, thermoregulation and bipedalism

It has long been recognized that the bipedal posture reduces the surface area of the body exposed to the sun. In recent years, a theory has been developed by Wheeler that bipedalism evolved in the ancestor of the Hominidae in order to help relieve thermal stress on the animals in open equatorial environments. Bipedalism was said to afford a distinct adaptive advantage over quadrupedalism by permitting hominids to remain active in the open throughout the day. The heat load of the hypothetical hominid comprises the external environment as modelled by Wheeler and the animal's internal environment (i.e., the internal heat generated by its metabolic and locomotor activities, and its evaporative and respirative cooling capacities). When these factors are integrated in the calculation of the animal's thermal budget, the putative advantage of the bipedal over the quadrupedal posture is considerably reduced. The simulations conducted in this study suggest that the increased time afforded to early hominids in the open by bipedalism was relatively short and, therefore, of little or no adaptive significance. These results suggest that thermoregulatory considerations cannot be implicated as a first cause in the evolution of bipedalism in the hominid ancestor.     

Flight metabolism in the African fruit beetle,Pachnoda sinuata

Metabolite concentrations in flight muscles and in abdomen of beetles (Pachnoda sinuata) were measured after various periods of tethered flight and subsequent rest. Three distinct phases of energy metabolism are found in active flight muscles: (1) during the first minutes of flight proline is used as main substrate and concomitantly alanine accumulated as an end product; (2) the second phase is characterized by a large-scale degradation of glycogen; (3) after about 8 min of flight the metabolite levels stabilize, while flight performance appears unchanged. After the termination of flight the preflight proline concentration (70 mol·g^-1 fw) is re-established in less than 60 min, whereas restoration of resting levels of other metabolites requires longer. The pattern of maximal enzyme activities and the respiratory rates of mitochondria with different substrates confirm the significance of proline and carbohydrates as the main fuels of working flight muscles.Abbreviations CS citrate synthetase - Cytox cytochrome c oxidase - EDTA ethylenediaminetetra-acetate - fw fresh weight - GluDH glutamate dehydrogenase - GPT alanine aminotransferase - HOAD hydroxyacyl-coenzyme A dehydrogenase - HPLC high pressure liquid chromatography - ME malic enzyme - PCA perchloric acid - RQ repiratory quotient - TRA triethanolamine