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PubMed2006

Exploring the role of the active site cysteine in human muscle creatine kinase.

All known guanidino kinases contain a conserved cysteine residue that interacts with the non-nucleophilic eta1-nitrogen of the guanidino substrate. Site-directed mutagenesis studies have shown that this cysteine is important, but not essential for activity. In human muscle creatine kinase (HMCK) this residue, Cys283, forms part of a conserved cysteine-proline-serine (CPS) motif and has a pKa about 3 pH units below that of a regular cysteine residue. Here we employ a computational approach to predict the contribution of residues in this motif to the unusually low cysteine pKa. We calculate that hydrogen bonds to the hydroxyl and to the backbone amide of Ser285 would both contribute approximately 1 pH unit, while the presence of Pro284 in the motif lowers the pKa of Cys283 by a further 1.2 pH units. Using UV difference spectroscopy the pKa of the active site cysteine in WT HMCK and in the P284A, S285A, and C283S/S285C mutants was determined experimentally. The pKa values, although consistently about 0.5 pH unit lower, were in broad agreement with those predicted. The effect of each of these mutations on the pH-rate profile was also examined. The results show conclusively that, contrary to a previous report (Wang et al. (2001) Biochemistry 40, 11698-11705), Cys283 is not responsible for the pKa of 5.4 observed in the WT V/K(creatine) pH profile. Finally we use molecular dynamics simulations to demonstrate that, in order to maintain the linear alignment necessary for associative inline transfer of a phosphoryl group, Cys283 needs to be ionized.

Adenosine Diphosphate↗
PubMed2003

Postnatal suppression of myomesin, muscle creatine kinase and the M-line in rat extraocular muscle.

The M-line and its associated creatine kinase (CK) M-isoform (CK-M) are ubiquitous features of skeletal and cardiac muscle. The M-line maintains myosin myofilaments in register, links the contractile apparatus to the cytoskeleton for external force transfer and localizes CK-based energy storage and transfer to the site of highest ATP demand. We establish here that the muscle group responsible for movements of the eye, extraocular muscle (EOM), is divergent from other striated muscles in lacking both an M-line and its associated CK-M. Although an M-line forms during myogenesis, both in vivo and in vitro, it is actively repressed after birth. Transcripts of the major M-line structural proteins, myomesin 1 and myomesin 2, follow the same pattern of postnatal downregulation, while the embryonic heart-specific EH-myomesin 1 transcript is expressed early and retained in adult eye muscle. By immunocytochemistry, myomesin protein is absent from adult EOM sarcomeres. M-line suppression does not occur in organotypic co-culture with oculomotor motoneurons, suggesting that the mechanism for suppression may lie in muscle group-specific activation or workload patterns experienced only in vivo. The M-line is, however, still lost in dark-reared rats, despite the developmental delay this paradigm produces in the visuomotor system and EOMs. EOM was low in all CK isoform transcripts except for the sarcomeric mitochondrial (Ckmt2) isoform. Total CK enzyme activity of EOM was one-third that of hindlimb muscle. These findings are singularly unique among fast-twitch skeletal muscles. Since EOM exhibits isoform diversity for other sarcomeric proteins, the M-line/CK-M divergence probably represents a key physiological adaptation for the unique energetics and functional demands placed on this muscle group in voluntary and reflexive eye movements.

Adaptation, Physiological↗
PubMed2001

Coagulation and fibrinolysis disorder in muscular dystrophy.

To investigate whether there are any basic abnormalities of coagulation and fibrinolysis in muscular dystrophy, we measured serum levels of the MM isozyme of creatine kinase (CK-MM), fibrin and fibrinogen degradation products (FDP), plasma levels of fibrinogen, antithrombin (AT), and D-dimer in 36 patients with Duchenne muscular dystrophy (DMD), 11 with Becker muscular dystrophy (BMD), 5 with Fukuyama congenital muscular dystrophy (FCMD), 5 with myotonic dystrophy (MyD), and 5 with spinal muscular atrophy (SMA) type 2. FDP levels were elevated in the patients with DMD, BMD, and FCMD (1.0 to 84.9 microg/ml), but not in the patients with MyD and SMA type 2. In DMD, BMD, and FCMD, FDP levels significantly correlated with CK-MM, but not with age, fibrinogen, AT, D-dimer, and type of dystrophy (multiple regression analysis; r(2) = 0.814, P < 0.0001). These findings suggested that enhanced coagulation and fibrinolysis are associated with muscle degeneration in patients with DMD, BMD, and FCMD.

Adolescent↗
PubMed2004

A survey on exposure level, health status, and biomarkers in workers exposed to 1-bromopropane.

BACKGROUND: The aim of this study was to evaluate the health effects of exposure mainly to 1-bromopropane, which is an alternative to ozone-depleting solvents, and to establish biomarkers for assessing 1-bromopropane exposure. METHODS: Twenty-four female and 13 male workers of a 1-bromopropane-factory were interviewed, and their urine and blood samples were collected. Measured parameters included 1-bromopropane levels in the factory, as well as individual exposure levels, urinary 1-bromopropane levels, enzymatic activity and M subunit's concentration of serum creatine kinase (CK). RESULTS: Frequent symptoms reported by workers exposed to 1-bromopropane were nose, throat, and eyes irritation or malaise and/or headache. Urinary 1-bromopropane levels correlated significantly with individual exposure levels, but enzymatic activity or CK-M subunit did not. CONCLUSIONS: The symptoms suggested irritation of the mucous membrane and possible adverse effects on the central nervous system. There were no severe chronic symptoms suggestive of neurological damage in workers exposed to less than 170 ppm. Urinary 1-bromopropane level may be a good indicator of exposure. Am. J. Ind. Med. 45:63-75, 2004.

Adult↗
PubMed2006

S100B stimulates myoblast proliferation and inhibits myoblast differentiation by independently stimulating ERK1/2 and inhibiting p38 MAPK.

The Ca2+-modulated protein of the EF-hand type, S100B, was shown to inhibit rat L6 myoblast differentiation and myotube formation by interacting with a high affinity with an unidentified receptor (Sorci et al., 2003). We show here that S100B independently inhibits the MKK6-p38 MAPK pathway and stimulates the Ras-MEK-ERK1/2 pathway. The inhibitory effect of S100B on p38 MAPK translates into a defective induction of the muscle-specific transcription factor myogenin and the antiproliferative factor p21(WAF1), while S100B's stimulatory effect on ERK1/2 results in stimulation of myoblast proliferation via cyclin D1 induction and Rb phosphorylation and protection against apoptosis via activation of NF-kappaB transcriptional activity. Also, the S100B's effects that are mediated by the Ras-MEK-ERK1/2 pathway that is, stimulation of proliferation and protection against apoptosis, depend on reactive oxygen species production, being inhibited by antioxidants, while the S100B inhibitory effect on the MKK6-p38 MAPK pathway is not. We propose that S100B might participate in the regulation of myoblast differentiation by stimulating myoblast proliferation, protecting myoblasts against apoptosis, and modulating myotube formation.

Animals↗
PubMed2006

Comparison of promoter region constructs for in vivo intramuscular expression.

BACKGROUND: High transgene expression is generally expected after gene transfer. However, different level, kinetics and localization of expression might be needed for relevant therapeutic applications. Former studies have compared various promoter regions driving gene expression leading to conflicting results. In the present work, two promoter families have been compared using the efficient in vivo intramuscular electrotransfer technique. METHODS: Three promoter regions were constructed by associating the strong ubiquitous cytomegalovirus (CMV) enhancer-promoter to its homologous intron A or to a heterologous intron, or to a hybrid intron. Promoter regions derived from the muscle creatine kinase (MCK) promoter were also studied. The expression of the same transgene (SeAP or neurotrophin-3) under control of these different promoters was compared after plasmid electrotransfer in mouse tibialis-cranialis skeletal muscle. RESULTS: Heterologous intron association to the CMV promoter did not modify gene expression kinetics nor increase gene expression level. Usefulness of intron A or hybrid intron association to the CMV promoter depended on the gene. The various MCK promoters drove efficient gene expression but lower than that obtained with the CMV promoter. Furthermore, peak value was reached earlier with MCK promoter regions (14 days). CONCLUSION: For applications of gene transfer restricted to skeletal muscle, the MCK promoter or a MCK promoter variant would be a promising alternative to the CMV promoter. Indeed, it has been demonstrated that the use of MCK promoter limits humoral and cell-mediated immune responses. Furthermore, the MCK promoter decreases the initial expression peak that may be detrimental, drives a sustained gene expression, and improves gene transfer safety.

Alkaline Phosphatase↗
PubMed2006

CAV3 gene mutation analysis in patients with idiopathic hyper-CK-emia.

As caveolin-3 deficiencies may explain persistent hyper-CK-emia, we performed CAV3 gene mutation analysis and immunohistochemistry for caveolin-3 in 31 patients with idiopathic hyper-CK-emia. In 2 of 29 patients who donated blood, variants in the CAV3 gene were detected. Although immunohistochemical analysis strongly suggested that caveolin-3 was properly localized in the muscle tissue of the two affected patients, it may not function normally and could thus explain their persistent hyper-CK-emia. Our findings contribute to the clarification of unexplained persistent hyper-CK-emia, but further research is needed before CAV3 gene mutation analysis becomes part of the routine evaluation of these patients.

Adult↗
PubMed2005

Proteomic analysis of organ-specific post-translational lysine-acetylation and -methylation in mice by use of anti-acetyllysine and -methyllysine mouse monoclonal antibodies.

Post-translational lysine-acetylation and -methylation are two major PTMs of lysine residues in proteins. Recently, we established pan-reactive anti-acetyllysine mouse mAbs, which can bind to Nepsilon-acetylated lysine residues in various contexts of amino acid sequences. In the present study, we established pan-reactive anti-methyllysine mouse mAbs comparable to the anti-acetyllysine ones. By using these anti-acetyllysine and -methyllysine antibodies, we found that the pattern of lysine-acetylated and -methylated proteins in mouse organs showed extreme variation from organ to organ. We selected brain and skeletal muscle as model cases to be further analyzed by 2-DE followed by Western blotting. In brain, alpha-tubulin at its basal level was found to be extremely acetylated; and alpha-enolase was shown to be a newly recognized possibly acetylated protein. NF-L protein, Hsc70, alpha-tubulin fragments, beta-actin, and brain-type creatine kinase were identified as putative lysine-methylated proteins in mouse brain. In skeletal muscle, lysine-methylation of alpha-actin and both lysine-acetylation and -methylation of muscle-type creatine kinase were found as novel putative lysine-modified proteins. The approach presented here might be useful to find novel disease markers and/or drug target molecules that would not be noticed by use of the traditional proteomic approach only.

Acetylation↗
PubMed2002

The short MCK1350 promoter/enhancer allows for sufficient dystrophin expression in skeletal muscles of mdx mice.

First-generation adenovirus vectors (AdV) have been used successfully to transfer a human dystrophin minigene to skeletal muscle of mdx mice. In most studies, strong viral promoters such as the cytomegalovirus promoter/enhancer (CMV) were used to drive dystrophin expression. More recently, a short version of the muscle creatine kinase promoter (MCK1350) has been shown to provide muscle-specific reporter gene expression after AdV-mediated gene delivery. Therefore, we generated a recombinant AdV where dystrophin expression is controlled by MCK1350 (AdVMCKdys). AdVMCKdys was injected by the intramuscular route into anterior tibialis muscle of mdx mice shortly after birth. Dystrophin expression was assessed at 20, 30, and 60 days after AdV-injection. At 20 days, muscles of AdVMCKdys-injected mdx mice showed a high number of dystrophin-positive fibers (mean: 365). At 60 days, the number of dystrophin-positive fibers was not only maintained, but increased significantly (mean: 600). In conclusion, MCK1350 allows for sustained dystrophin expression after AdV-mediated gene transfer to skeletal muscle of newborn mdx mice. In contrast to previous studies, where strong viral promoters were used, dystrophin expression driven by MCK1350 peaks at later time points. This may have implications for the future use of muscle-specific promoters for gene therapy of Duchenne muscular dystrophy.

Adenoviruses, Human↗
PubMed2001

Regulation of the levels of small heat-shock proteins during differentiation of C2C12 cells.

Levels of the small heat-shock proteins (sHSPs) HSP27 and alphaB-crystallin during differentiation of mouse C2C12 cells were determined using specific immunoassays. Increases of these proteins were about 3-fold and 10-fold, respectively. Under the same conditions, however, the level of HSP70 in C2C12 cells barely increased, indicating selective accumulation of HSP27 and alphaB-crystallin with differentiation. While expression of mRNA for alphaB-crystallin was also markedly increased and that for HSP27 was but to a lesser extent, mRNA for HSP70 could barely be detected during differentiation. Activation of the heat-shock factor was not observed, in contrast to the case with heat-stressed undifferentiated cells. Various inhibitors of protein kinases affected the differentiation and the associated increase of sHSPs. Rapamycin, an inhibitor of p70 S6 kinase, completely inhibited the differentiation and suppressed the accumulation of HSP27 and alphaB-crystallin. SB203580, an inhibitor of p38 MAP kinase, also inhibited differentiation, but the accumulation of alphaB-crystallin was rather enhanced. PD98059, an inhibitor of MAP kinase kinase, significantly increased expression of a differentiation marker for muscle cells, creatine kinase M isozyme, as well as accumulation of alphaB-crystallin. These results suggest that accumulation of sHSPs during differentiation of C2C12 cells is regulated in a complex manner.

Animals↗
PubMed2006

Neuromuscular recovery after a strength training session in elderly people.

Ageing is associated with an increased susceptibility to muscle damage but little is known on how this affects muscle recovery after exercise. Hence, this study is aiming at investigating the effects of a heavy-resistance training session of neuromuscular recovery of the calf muscles of a group of elderly men aged >65. Maximal isometric and isokinetic torque, muscle voluntary activation (VA) capacity, surface electromyographic activity (EMG), peak-to-peak amplitude of action potentials associated with twitch responses of plantar flexors were evaluated before and 5 min (post1), 24 h (post2) and 48 h (post3) after 10 sets of 10 repetitions of a calf raise exercise performed at an intensity of 70% of the individual, one repetition maximum. Blood samples were taken before and 1, 48, 96 and 144 h after the training session and assayed for serum creatine kinase (CK), lactate dehydrogenase (LDH) and myoglobin (Mb). Peak torque during eccentric and concentric (120 degrees s-1) contractions and twitch parameters were significantly reduced at post1, and recovered completely at post2. No significant changes were found in integrated EMG, M-wave amplitudes and VA throughout the entire test period. CK and LDH concentrations reached peak values 48 h after the exercise session and returned to the pre-exercise values 96 h after the training session. Serum Mb level increased by 73.2% 1 h after exercise and recovered at 48 h. The reduction in peak torque following a strength training session in an elderly population could be explained mainly by fatigue of peripheral origin. After 24 h the elders recovered completely their capacities of strength production, despite muscle damage being still evident 48 h after the strength training session.

Aged↗
PubMed2006

Excitability of the soleus reflex arc during intensive stretch-shortening cycle exercise in two power-trained athlete groups.

In several explosive types of sport events the leg extensor muscles are subjected to very high impact loads. Thus, extreme requirements exist for the neuromuscular system to develop sufficient muscle stiffness in the lower extremities in order to tolerate these high impact loads. Therefore, it would be challenging to measure reflex modulation during high impact activities, and with different athlete populations. In the present experiment, H-reflex and short latency reflex (M1) sensitivity was measured during drop jump exercises among high jumpers and sprinters. The changes in both reflex peak-to-peak amplitudes showed a significant (P < 0.05) reduction towards the end of the exercise for the sprinters. In addition, the same subject group showed a remarkable increase in serum creatine kinase (CK) activity 2 h after the jumps. Similar changes could not be observed for the high jumpers. These results clearly indicate different neural adaptation strategies for the two athlete groups. Reduction in H-reflex sensitivity and an increase in CK-activity in sprinters were taken as evidence for presynaptic inhibition, probably induced by substances related to muscle damage. Since high jump training includes more high impact loading, it was assumed that it could lead to some structural adaptation and, thus, prevents exercise induced reflex modification to a certain extent.

Adult↗
PubMed2006

Responses of old men to repeated bouts of eccentric exercise of the elbow flexors in comparison with young men.

A bout of eccentric exercise confers protection against subsequent bouts of the same exercise. This study investigated whether the protective effect would be produced similarly between old and young adults. Eight old men (70.5 +/- 4.1 years) and ten young men (20.4 +/- 2.0 years) performed two bouts of eccentric exercise of the elbow flexors (six sets of five eccentric actions) separated by 4 weeks. Changes in maximal isometric strength, range of motion (ROM), upper arm circumference (CIR), plasma creatine kinase (CK) activity, myoglobin (Mb) concentration, and muscle soreness (SOR) before, immediately after, and 1, 24, 48, 72 and 96 h following exercise were compared between bouts, and between groups by a two-way repeated measures ANOVA. Changes in the measures following the first bout were significantly (P < 0.05) smaller for the old than the young group. The young group showed significantly (P < 0.05) smaller changes in all measures following the second bout than the first bout; however, the old group had the protective effect only for ROM, Mb, and SOR. The magnitude of the effect observed for ROM and Mb concentration in the old group was significantly (P < 0.05) smaller compared with that of the young group. These results suggest that the protective effect conferred by the first bout was less for the old than the young group. This may be due to the less muscle damage after the first bout in the old subjects, but it is also possible that the protective effect of old adults does not last as long as that of young adults.

Adolescent↗
PubMed2006

Attenuation of muscle damage by preconditioning with muscle hyperthermia 1-day prior to eccentric exercise.

This study investigated the hypothesis that muscle damage would be attenuated in muscles subjected to passive hyperthermia 1 day prior to exercise. Fifteen male students performed 24 maximal eccentric actions of the elbow flexors with one arm; the opposite arm performed the same exercise 2-4 weeks later. The elbow flexors of one arm received a microwave diathermy treatment that increased muscle temperature to over 40 degrees C, 16-20 h prior to the exercise. The contralateral arm acted as an untreated control. Maximal voluntary isometric contraction strength (MVC), range of motion (ROM), upper arm circumference, muscle soreness, plasma creatine kinase activity and myoglobin concentration were measured 1 day prior to exercise, immediately before and after exercise, and daily for 4 days following exercise. Changes in the criterion measures were compared between conditions (treatment vs. control) using a two-way repeated measures ANOVA with a significance level of P < 0.05. All measures changed significantly following exercise, but the treatment arm showed a significantly faster recovery of MVC, a smaller change in ROM, and less muscle soreness compared with the control arm. However, the protective effect conferred by the diathermy treatment was significantly less effective compared with that seen in the second bout performed 4-6 weeks after the initial bout by a subgroup of the subjects (n = 11) using the control arm. These results suggest that passive hyperthermia treatment 1 day prior to eccentric exercise-induced muscle damage has a prophylactic effect, but the effect is not as strong as the repeated bout effect.

Adult↗
PubMed2007

Monitoring for overreaching in rugby league players.

The aim of this study was to identify indicators of non-functional overreaching (NFOR) in team sport athletes undertaking intensive training loads. Eighteen semi-professional rugby league players were randomly assigned into two pair matched groups. One group completed 6 weeks of normal training (NT) whilst the other group was deliberately overreached through intensified training (IT). Both groups then completed the same 7-day stepwise training load reduction taper. Multistage fitness test (MSFT) performance, VO2 (max), peak aerobic running velocity (V (max)), maximal heart rate, vertical jump, 10-s cycle sprint performance and body mass were measured pre- and post-training period and following the taper. Hormonal, haematological and immunological parameters were also measured pre-training and following weeks 2, 4 and 6 of training and post-taper. MANOVA for repeated measures with contrast analysis indicated that MSFT performance and VO2 (max) were significantly reduced in the IT group over time and condition, indicating that a state of overreaching was attained. However, the only biochemical measure that was significantly different between the IT and NT group was the glutamine to glutamate (Gln/Glu) ratio even though testosterone, testosterone to cortisol (T/C) ratio, plasma glutamate, and CK activity were significantly changed after training in both groups. Positive endurance and power performance changes were observed post-taper in the IT group confirming NFOR. These changes were associated with increases in the T/C ratio and the Gln/Glu ratio and decreases in plasma glutamate and CK activity. These results indicate that although there was no single reliable biochemical marker of NFOR in these athletes, the Gln/Glu ratio and MSFT test may be useful measures for monitoring responses to IT in team sport athletes.

Adult↗
PubMed2005

Functional coupling between nucleoside diphosphate kinase of the outer mitochondrial compartment and oxidative phosphorylation.

In rat liver mitochondria all nucleoside diphosphate kinase of the outer compartment is associated with the outer surface of the outer membrane (Lipskaya, T. Yu., and Plakida, K. N. (2003) Biochemistry (Moscow), 68, 1136-1144). In the present study, three systems operating as ADP donors for oxidative phosphorylation have been investigated. The outer membrane bound nucleoside diphosphate kinase was the first system tested. Two others employed yeast hexokinase and yeast nucleoside diphosphate kinase. The two enzymes exhibited the same activity but could not bind to mitochondrial membranes. In all three systems, muscle creatine phosphokinase was the external agent competing with the oxidative phosphorylation system for ADP. Determination of mitochondrial respiration rate in the presence of increasing quantities of creatine phosphokinase revealed that at large excess of creatine phosphokinase activity over other kinase activities (of the three systems tested) and oxidative phosphorylation the creatine phosphokinase reaction reached a quasi-equilibrium state. Under these conditions equilibrium concentrations of all creatine phosphokinase substrates were determined and K(eq)app of this reaction was calculated for the system with yeast hexokinase. In samples containing active mitochondrial nucleoside diphosphate kinase the concentrations of ATP, creatine, and phosphocreatine were determined and the quasi-equilibrium concentration of ADP was calculated using the K(eq)app value. At balance of quasi-equilibrium concentrations of ADP and ATP/ADP ratio the mitochondrial respiration rate in the system containing nucleoside diphosphate kinase was 21% of the respiration rate assayed in the absence of creatine phosphokinase; in the system containing yeast hexokinase this parameter was only 7% of the respiration rate assayed in the absence of creatine phosphokinase. Substitution of mitochondrial nucleoside diphosphate kinase with yeast nucleoside diphosphate kinase abolished this difference. It is concluded that oxidative phosphorylation is accompanied by appearance of functional coupling between mitochondrial nucleoside diphosphate kinase and the oxidative phosphorylation system. Possible mechanisms of this coupling are discussed.

Adenosine Diphosphate↗
PubMed1985

Phorbol ester inhibits myoblast fusion and activates beta-adrenergic receptor coupled adenylate cyclase.

Primary cultures of myoblasts, derived from embryonic chick pectoral muscle, were treated with phorbol ester (TPA) for 8-96 h. TPA treatment blocked the fusion of myoblasts along with the expression of the MM form of creatine kinase. Interestingly, TPA treatment markedly increased the activity of beta-adrenergic receptor coupled adenylate cyclase (AC) activity. The study suggests that TPA treatment augments the functional interaction between a coupling Ns protein and catalytic unit of AC. The likely significance of these results is briefly presented.

Adenylyl Cyclases↗