Maintained coupling of oxidative phosphorylation to creatine kinase activity in sarcomeric mitochondrial creatine kinase-deficient mice

The importance of mitochondrial creatine kinase (mi-CK) in oxidative muscle was tested by studying the functional properties of in situ mitochondria in saponin-skinned muscle fibres from sarcomeric mi-CK-deficient (mutant) mice. Biochemical analyses showed that the lack of mi-CK in mutant muscle was associated with a decrease in specific activity of MM-CK in mutant ventricle, and increase in mutant soleus (oxidative) muscle. Lactate dehydrogenase activity and isoenzyme analysis showed an increased glycolytic metabolism in mutant soleus. No change was observed in ventricular muscle. In control animals, the apparent K(m) of mitochondrial respiration for ADP in ventricle and soleus (232 +/- 36 and 381 +/- 63 microM, respectively) was significantly reduced in the presence of creatine (52 +/- 8 and 45 +/- 12 microM, respectively). There was no change in the K(m) in oxidative fibres from mutant mice (258 +/- 27 and 399 +/- 66 microM, respectively) compared with control, though surprisingly, it was also significantly decreased in the presence of creatine (144 +/- 8 and 150 +/- 27 microM, respectively) despite the absence of mi-CK. It is proposed that in mutant (and perhaps normal) oxidative tissue, cytosolic MM-CK can relocate to the outer mitochondrial membrane, where it is coupled to oxidative phosphorylation by close proximity to porin, and the adenine nucleotide translocase. Such an effect can preserve the functioning of the CK shuttle and the energetic properties of mi-CK deficient tissue.     

Interactions of mitochondrial ATP synthesis and the creatine kinase equilibrium in skeletal muscle

This study aimed to evaluate the effect of FR128998, (1s,6s)-1-benzyl-10-(3-pyridyl-methyl)-7-thia-10-azaspiro [5,6]-dodecan-11-one 7,7-dioxide hydrochloride, a novel platelet activating factor (PAF) receptor antagonist, on endotoxin lipopolysaccharide-induced disseminated intravascular coagulation in rats. Experimental disseminated intravascular coagulation was induced by an infusion of lipopolysaccharide at 0.25 mg/kg/h for 4 h. Simultaneous infusion of FR128998 (0.25 and 1.0 mg/kg/h) with lipopolysaccharide dose dependently inhibited thrombocytopenia, but not leukopenia. The changes in coagulation parameters of disseminated intravascular coagulation, i.e., prolongation of activated partial thromboplastin time and elevated levels of fibrinogen/fibrin degradation products, were also prevented by the treatment with FR128998. In addition, FR128998 attenuated the increase in serum tumor necrosis factor (TNF) which appeared during the initial stage of disseminated intravascular coagulation. FR128998 (10 microM) also inhibited the TNF production by peripheral blood leukocytes or alveolar macrophages stimulated by lipopolysaccharide in vitro. Furthermore, TNF production induced by PAF itself in vitro was also inhibited in the presence of FR128998. These data indicate that PAF plays a pivotal role in the development of disseminated intravascular coagulation via TNF production.     

Effect of vitamin D3 and 20-hydroxyecdysone on the content of ATP, creatine phosphate, carnosine and Ca2+ in skeletal muscles

The effect of vitamin D3, 20-hydroxyecdysone and extract from Serratula coronata containing 20-hydroxyecdysone on the level of basic metabolites in the skeletal muscles of rats has been studied. It was shown that development of D-hypovitaminosis is accompanied by the decrease in content of ATP, creatine phosphate, carnosine, and by the increase of Ca2+ content. Against the background of D-hypovitaminosis the 20-hydroxyecdysone and the extract from Serratula coronata which contains 20-hydroxyecdysone promote the increase of the amount of these metabolites up to the control of one and normalize Ca2+ content in them.     

Creatine supplementation in health and disease. Effects of chronic creatine ingestion in vivo: down-regulation of the expression of creatine transporter isoforms in skeletal muscle

Interest in creatine (Cr) as a nutritional supplement and ergogenic aid for athletes has surged over recent years. After cellular uptake, Cr is phosphorylated to phosphocreatine (PCr) by the creatine kinase (CK) reaction using ATP. At subcellular sites with high energy requirements, e.g. at the myofibrillar apparatus during muscle contraction, CK catalyzes the transphosphorylation of PCr to ADP to regenerate ATP, thus preventing a depletion of ATP levels. PCr is thus available as an immediate energy source, serving not only as an energy buffer but also as an energy transport vehicle. Ingestion of creatine increases intramuscular Cr, as well as PCr concentrations, and leads to exercise enhancement, especially in sprint performance. Additional benefits of Cr supplementation have also been noticed for high-intensity long-endurance tasks, e.g. shortening of recovery periods after physical exercise. The present article summarizes recent findings on the influence of Cr supplementation on energy metabolism, and introduces the Cr transporter protein (CreaT), responsible for uptake of Cr into cells, as one of the key-players for the multi-faceted regulation of cellular Cr homeostasis. Furthermore, it is suggested that patients with disturbances in Cr metabolism or with different neuro-muscular diseases may benefit from Cr supplementation as an adjuvant therapy to relieve or delay the onset of symptoms. Although it is still unclear how Cr biosynthesis and transport are regulated in health and disease, so far there are no reports of harmful side effects of Cr loading in humans. However, in this study, we report that chronic Cr supplementation in rats down-regulates in vivo the expression of the CreaT. In addition, we describe the presence of CreaT isoforms most likely generated by alternative splicing.     

Expression of brain-type creatine kinase and ubiquitous mitochondrial creatine kinase in the fetal rat brain: evidence for a nuclear energy shuttle

To test the hypothesis that embryonic brain cells utilize a creatine phosphate energy shuttle, we examined the pattern of creatine kinase (CK) isoform expression and localization in the fetal rat brain. Moderate levels of CK activity are present at embryonic day 14 (7 U/mg protein) and decrease slightly until 3 days postpartum followed by a rapid, fourfold up-regulation to adult levels by 1 month (18 U/mg protein). In parallel with changes in enzyme activity, there is a biphasic and coordinate pattern of expression of brain-type CK (BCK) and ubiquitous mitochondrial CK (uMtCK) determined by nondenaturing electrophoresis and immunoblot analysis. The localization of CK isoforms was examined by immunocytochemistry, and, during the fetal period, BCK and uMtCK immunoreactivity was detected throughout the central and peripheral nervous system, especially in neuroepithelial regions of the cerebral vesicles and spinal cord. In large cells within the olfactory neuroepithelium and ventral spinal cord, differential compartmentation of CK isoforms was evident, with BCK localized primarily in cell nuclei, whereas uMtCK immunoreactivity was present in the cell body (but not within nuclei). In olfactory bulb neuroepithelium, both isoforms were expressed in the middle zone of the germinal layer associated with DNA synthesis. In embryonic skeletal and cardiac muscle, which also express BCK, the same compartmentation of BCK was seen, with BCK localized primarily in the cell nucleus of cardiac and skeletal myoblasts. These results demonstrate a coordinate pattern of expression and compartmentation of BCK and uMtCK isoforms in the fetal brain that, in some cells, provides the anatomic basis for a nuclear energy shuttle.     

Post-traumatic reconnection of the cervical spinal cord with skeletal striated muscles. Study in adult rats and marmosets

In an attempt at repairing the injured spinal cord of adult mammals (rat, dog and marmoset) and its damaged muscular connections, we are currently using: 1) peripheral nerve autografts (PNG), containing Schwann cells, to trigger and direct axonal regrowth from host and/or transplanted motoneurons towards denervated muscular targets; 2) foetal spinal cord transplants to replace lost neurons. In adult rats and marmosets, a PNG bridge was used to joint the injured cervical spinal cord to a denervated skeletal muscle (longissimus atlantis [rat] or biceps brachii [rat and marmoset]). The spinal lesion was obtained by the implantation procedure of the PNG. After a post-operative delay ranging from 2 to 22 months, the animals were checked electrophysiologically for functional muscular reconnection and processed for a morphological study including retrograde axonal tracing (HRP, Fast Blue, True Blue), histochemistry (AChE, ATPase), immunocytochemistry (ChAT) and EM. It was thus demonstrated that host motoneurons of the cervical enlargement could extend axons all the way through the PNG bridge as: a) in anaesthetized animals, contraction of the reconnected muscle could be obtained by electrical stimulation of the grafted nerve; b) the retrograde axonal tracing studies indicated that a great number of host cervical neurons extended axons into the PNG bridge up to the muscle; c) many of them were assumed to be motoneurons (double labelling with True Blue and an antibody against ChAT); and even alpha-motoneurons (type C axosomatic synapses in HRP labelled neurons seen in EM in the rat); d) numerous ectopic endplates were seen around the intramuscular tip of the PNG. In larger (cavitation) spinal lesions (rat), foetal motoneurons contained in E14 spinal cord transplants could similarly grow axons through PNG bridges up to the reconnected muscle. Taking all these data into account, it can be concluded that neural transplants are interesting tools for evaluating both the plasticity and the repair capacities of the mammalian spinal cord and of its muscular connections.     

Plastic remodeling of the synaptic apparatus of skeletal muscles under physiological and pathological conditions

The synaptic apparatus of skeletal muscles undergoes continuous remodelling, by adapting to the functioning level of the peripheral neuromuscular apparatus under normal conditions, compensating and restoring its impaired function in pathology. This reorganization has specific features depending on the factors that influence it, such as increased function, decreased or no function, neuronal and nerve damage, synaptic toxin intoxication, autoantibody action, disturbances of trophic influences, etc. Damage to any link of the neuromuscular apparatus due to the above factors is likely to be an essential condition for remodelling.     

Glycogen content in the liver and skeletal muscles in certain types of experimental nanism

The presence of various types of birthmarks was determined in 1,058 newborn infants under 72 hours of age. Of these, 79.5% were white, 6.2% were black, 11.2% were ladinos, and 2.6% were Asiatic. Mongol spots were present in 9.6% of the white babies, 95.5% of the black babies, 81% of the Asiatic babies, and 70.1% of ladino infants. Pigmented lesions were present in 42 (4%) of the infants. Biopsies obtained in 34 (3.2%) revealed that only one-third (11) of these were melanocytic nevi. Salmon patches were present in 40.3% of the infants, recognizable early strawberry marks in 2.6%, and port-wine strains in 0.3%. In addition to birthmarks, it was determined that 30.3% of the 508 babies examined at one of the two hospitals had toxic erythema of the newborn.     

Dextran strongly increases the Michaelis constants of oxidative phosphorylation and of mitochondrial creatine kinase in heart mitochondria

A minimal model of glycogen metabolism in muscle tissue is analyzed in accordance with metabolic control analysis. The model contains two branch points. Rather than contributing to complexity of the analysis, this branching allows expression of the control coefficients in a simplified form. Glucose 6-phosphate is the metabolite at the first branch point, and the analysis is simplified further by the fact that glucose 6-phosphate is the substrate for enzymes which catalyze near-equilibrium reactions. Control of the concentration of glucose 6-phosphate is of interest because of its pivotal location in the metabolic system, but also because it interacts with an allosteric site on glycogen synthase to stimulate glycogen synthase activity. It is shown that the control which the transporter and enzymes involved in glycogen synthesis exert on glycolytic flux is proportional to the control which these components exert on glucose 6-phosphate concentration. Thus, glycolysis plays a major role in control of glucose 6-phosphate concentration. It is concluded that control of glycogen synthesis is not a rigid parameter of any component of this metabolic system. Rather the distribution of control is flexible and shifts from one portion of the system to another in response to shifts in the physiological state. An important element in determining the distribution of control of glycogen synthesis is the change in the sensitivity of the allosteric site of glycogen synthase to glucose 6-phosphate which is brought about by conversion of glycogen synthase to the dephosphorylated, glucose 6-phosphate-independent, state.     

Features of the organization of the cytochrome systems and oxygen regimen in skeletal muscles of marine fishes

The organization of cytochrome systems in pelagic (Mugil so-iuy) and benthic (Platichthys flesus luscus) species of sea fishes were investigated. The total cytochrome content was 3.6-3.7 times higher (p < 0.001) in muscles of active M. so-iuy and made 67.5 +/- 5.3 nM g-1 for red and 5.91 +/- 0.58 nM g-1 for white muscle tissue. Cytochrome stechiometry had non-compensation character in benthic P. flesus luscus. Cytochrome ratio-b/aa3, was less than one and was equivalent 0.80-0.83 for both muscle groups. On the contrary it was higher than one--1.47-1.66 (p < 0.001) in M. so-iuy. Differences in cytochrome organization were connected with oxygen state of muscle tissue of these fishes. Mean oxygen tension in P. flesus luscus muscles was lower for 20.1-22.5% (p < 0.001). A number of anoxic and hypoxic zones (less than 6 mm Hg) in muscle tissue of this fish made 70 and 4-6% for white and red muscles accordingly. This value was 1.7-2.5 times lower in M. so-iuy muscles.     

Cardiac markers in the early hours of acute myocardial infarction: clinical performance of creatine kinase, creatine kinase MB isoenzyme (activity and mass concentration), creatine kinase MM and MB subform ratios, myoglobin and cardiac troponin T

We compared early markers of acute myocardial infarction (AMI) in the first 6 h from the onset of symptoms in 133 non-traumatized patients arriving at the emergency department with chest pain suggestive of AMI. Clinical performance parameters were calculated on the basis of 45 patients with AMI and 88 patients with a non-AMI diagnosis. At admission and in the first 0-3 h after the onset of chest pain the creatine kinase-MB (CK-MB) subform ratio was the most sensitive test at a comparable specificity level of 0.95. In the time interval of 3-5 h, myoglobin, the CK-MB mass concentration and the CK-MB subform ratio were associated with the greatest areas under receiver operating characteristic (ROC) curves, but differences between these tests were small and non-significant. At 6 h from the onset of pain, differences in clinical performance between the same three tests were even smaller whether or not samples drawn after the start of thrombolytic treatment were included in the test comparison. For confirmation of AMI at 6 h after onset of pain, CK-MB (activity and mass concentration) demonstrated the highest positive likelihood ratio, and for exclusion of AMI at 6 h the CK-MB subform ratio was associated with the highest negative likelihood ratio. However, differences between the CK-MB subform ratio, CK-MB mass concentration and myoglobin were not significant as estimated by the substantial overlap between the confidence intervals of the likelihood ratios and the ROC areas at 6 h. Cardiac troponin T (cTnT) demonstrated an ROC area equal to the CK-MB isoform ratio and myoglobin at 6 h. However, the likelihood ratio for ruling out AMI was lower, mostly due to the elevated cTnT in unstable coronary disease not defined as AMI. We conclude that the CK-MB subform ratio, CK-MB mass concentration and myoglobin do not demonstrate any significant differences in clinical performance for ruling in or ruling out acute myocardial infarction at 6 h after the onset of chest pain.     

Cholinergic sites in skeletal muscle. II. Interaction of an agonist and two antagonists with the acetylcholine site

The equilibrium interactions of alpha-bungarotoxin, d-tubocurarine, and carbamylcholine with junctional and extrajunctional skeletal muscle acetylcholine receptors were examined. d-Tubocurarine is a competitive inhibitor of the bindings of alpha-bungarotoxin to the acetylcholine receptor. No substantive difference was observed in the association of d-tubocurarine with the junctional and extrajunctional receptors. In contrast, the carbamylcholine inhibition of toxin binding is not competitive. The data indicate that either the single set of alpha-bungarotoxin and d-tubocurarine bindings sites contains two subsets of carbamylcholine sites or that the carbamylcholine binds in a cooperative manner to a single set of sites. In addition, the affinity of carbamylcholine for extrajunctional receptors may be higher than the affinity for junctional receptors.     

Muscle power and shoulder joint function after removal of the latissimus dorsi muscle

Understanding the functional dynamics of the oral cavity helps researchers design methods to predict clinical outcomes. Most information about the oral cavities of companion animals must be drawn from animal studies designed to understand human oral disease and from human studies. Based upon functional criteria for any animal or experimental research study, supragingival plaque and calculus can be assessed within four to six weeks. Although longer studies may be more appealing, such study designs may compromise the oral soft tissue health of the animals and are complicated by oral cavity dynamics. Thus, they are less sensitive and predictive of longer term clinical outcomes. Additional research is necessary to relate tooth surface accumulation phenomena with oral soft tissue health.     

Influence of picrotoxin and strychnine on the spectral sensitivity of the turtle ERG b- and d-waves: II. Light adaptation

Clinical and laboratory features of acute meningococcal meningitis according to age were studied in 255 patients. Whereas males accounted for three out of five patients aged 0-4 years, females accounted for three out of four patients older than 50 years of age. All patients had clinical signs of nuchal rigidity and fever. Patients older than 30 years of age had less frequent petechiae (62%) than younger patients (81%). Furthermore, elderly patients above 50 years of age were prone to an obtunded mental state and a prolonged disease course with fever. Without relation to age, 2/3 had purulent meningitis and 2/3 had marked peripheral leucocytosis (> 15 x 10(9) cells/l); 90% of patients had at least one of these findings. The cellular inflammatory response in peripheral blood indicated a bacterial aetiology in > 95% of the cases. More than 80% of children and adults had abnormal CSF biochemical findings, but the level of protein and the glucose ratio (CSF/serum) were positively and negatively correlated to increasing age of the patient, respectively: thus, in children these biochemical markers may be unreliable in the differentiation between a bacterial and non-bacterial aetiology. Thrombocytopenia (< 100.000 x 10(9)/I) was not associated with age, though the lowest platelet count was found in elderly patients. The case fatality rate was 7.5%, but neither age, sex nor sign of septicaemia was associated with fatality. Thrombocytopenia, a lowered coagulation index (< 0.5, factors II, VII, X), a moderate anaemia (haemoglobin < 11 g/dl), an obtunded mental state and a history of convulsions were poor prognostic factors; only anaemia was independently correlated to fatality so this should be considered as an important prognostic marker in the acute phase of meningococcal meningitis.     

The preprotein translocase of the mitochondrial inner membrane: function and evolution

Growing mitochondria acquire most of their proteins by the uptake of mitochondrial preproteins from the cytosol. To mediate this protein import, both mitochondrial membranes contain independent protein transport systems: the Tom machinery in the outer membrane and the Tim machinery in the inner membrane. Transport of proteins across the inner membrane and sorting to the different inner mitochondrial compartments is mediated by several protein complexes which have been identified in the past years. A complex containing the integral membrane proteins Tim17 and Tim23 constitutes the import channel for preproteins containing amino-terminal hydrophilic presequences. This complex is associated with Tim44 which serves as an adaptor protein for the binding of mtHsp70 to the membrane. mtHsp70, a 70 kDa heat shock protein of the mitochondrial matrix, drives the ATP-dependent import reaction of the processed preprotein after cleavage of the presequence. Preproteins containing internal targeting information are imported by a separate import machinery, which consists of the intermembrane-space proteins Tim9, Tim10, and Tim12, and the inner membrane proteins Tim22 and Tim54. The proteins Tim17, Tim22, and Tim23 have in common a similar topology in the membrane and a homologous amino acid sequence. Moreover, they show a sequence similarity to OEP16, a channel-forming amino acid transporter in the outer envelope of chloroplasts, and to LivH, a component of a prokaryotic amino acid permease, defining a new PRAT-family of preprotein and amino acid transporters.     

Reduced sarcolemmal dystrophin distribution and upregulation of utrophin in the cardiac and skeletal muscles of CHF-146 dystrophic hamsters

Abnormalities in the dystrophic gene product, dystrophin, have been implicated in initiating the primary membrane defect and excessive intracellular calcium accumulation (EICA), which play fundamental pathogenic roles in hereditary muscular dystrophy (HMD). Two other cytoskeletal proteins, spectrin and utrophin, bear remarkable structural and functional homologies to dystrophin. CHF-146 strain dystrophic hamsters (DH), like patients with Duchenne muscular dystrophy (DMD), die prematurely from cardiopulmonary insufficiency, focal myonecrosis, and progressive degeneration of the cardiac and skeletal muscles with EICA. Although DH present a suitable model for HMD, there are controversies concerning their dystrophin and utrophin status. Using immunocytochemistry and Western blotting, we studied dystrophin, spectrin and utrophin anomalies in the cardiac and skeletal muscles of 6-mo-old male DH. Age- and sex-matched CHF-148 strain albino normal hamsters (NH) served as controls. Sarcolemmal dystrophin staining was much weaker and interruptive in the DH. The densitometric analysis of the immunoblots revealed that dystrophin is reduced in DH by 83% in cardiac muscle (p < 0.0001), and by 50% in skeletal muscle (p < 0.0001). We conclude that sarcolemmal dystrophin distribution is markedly reduced and discontinuous in the cardiac and skeletal muscles of DH, with simultaneous upregulation of utrophin and a varied degree of spectrin labelling. This observation suggests that reduced sarcolemmal dystrophin is associated with membrane hyperpermeability, which leads to progressive muscle degeneration via EICA and segmental necrosis in DH. As in DMD, utrophin appears to play an important compensatory role in hamster dystrophinopathy.