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1.
In myotonic muscular dystrophy, abnormal muscle Na currents underlie myotonic discharges. Since the myotonic muscular dystrophy gene encodes a product, human myotonin protein kinase, with structural similarity to protein kinases, we tested the idea that human myotonin protein kinase modulates skeletal muscle Na channels. Coexpression of human myotonin protein kinase with rat skeletal muscle Na channels in Xenopus oocytes reduced the amplitude of Na currents and accelerated current decay. The effect required the presence of a potential phosphorylation site in the inactivation mechanism of the channel. The mutation responsible for human disease, trinucleotide repeats in the 3' untranslated region, did not prevent the effect. The consequence of an abnormal amount of the kinase would be altered muscle cell excitability, consistent with the clinical finding of myotonia in myotonic dystrophy.  相似文献   

2.
Myotonic dystrophy (DM) is associated with expansion of CTG repeats in the 3'-untranslated region of the myotonin protein kinase (DMPK) gene. The molecular mechanism whereby expansion of the (CUG)n repeats in the 3'-untranslated region of DMPK gene induces DM is unknown. We previously isolated a protein with specific binding to CUG repeat sequences (CUG-BP/hNab50) that possibly plays a role in mRNA processing and/or transport. Here we present evidence that the phosphorylation status and intracellular distribution of the RNA CUG-binding protein, identical to hNab50 protein (CUG-BP/hNab50), are altered in homozygous DM patient and that CUG-BP/hNab50 is a substrate for DMPK both in vivo and in vitro. Data from two biological systems with reduced levels of DMPK, homozygous DM patient and DMPK knockout mice, show that DMPK regulates both phosphorylation and intracellular localization of the CUG-BP/hNab50 protein. Decreased levels of DMPK observed in DM patients and DMPK knockout mice led to the elevation of the hypophosphorylated form of CUG-BP/hNab50. Nuclear concentration of the hypophosphorylated CUG-BP/hNab50 isoform is increased in DMPK knockout mice and in homozygous DM patient. DMPK also interacts with and phosphorylates CUG-BP/hNab50 protein in vitro. DMPK-mediated phosphorylation of CUG-BP/hNab50 results in dramatic reduction of the CUG-BP2, hypophosphorylated isoform, accumulation of which was observed in the nuclei of DMPK knockout mice. These data suggest a feedback mechanism whereby decreased levels of DMPK could alter phosphorylation status of CUG-BP/hNab50, thus facilitating nuclear localization of CUG-BP/hNab50. Our results suggest that DM pathophysiology could be, in part, a result of sequestration of CUG-BP/hNab50 and, in part, of lowered DMPK levels, which, in turn, affect processing and transport of specific subclass of mRNAs.  相似文献   

3.
Thiothrix spp., sulfide-oxidizing filamentous bacteria, were found to be a principal bacterial component of aquatic biofilms causing biofouling in selected municipal water storage tanks, private wells, and drip irrigation systems in Florida. Treatments of up to 200 ppm chlorine in the affected systems could not prevent return of the biofouling problem. The water originated from the upper Floridan aquifer and associated surficial aquifers in central and north Florida. Samples were examined where visible biofilms had a white, filamentous appearance, indicative of Thiothrix spp. The detection of Thiothrix spp. was confirmed by enzyme-liked immunosorbent assay (ELISA), indirect immunofluorescence (IIF), and microbiological procedures. It was estimated through immunocytochemical procedures that Thiothrix spp. comprised 18% of the biofilm in the municipal water storage tanks. These observations confirm that specific biological and chemical interactions may induce physical changes leading to significant biofouling.  相似文献   

4.
Myotonic dystrophy (DM) is an autosomal dominant neuromuscular disorder characterized by a great variability in its clinical manifestations. The mutational basis underlying DM consists of an unstable (CTG)n trinucleotide repeat in the 3' untranslated region of the myotonic dystrophy protein kinase gene (DMPK). Conflicting results on DMPK gene expression in congenitally affected infants (CDM) have been published. Moreover, the prominence of satellite cells seen in muscle of CDM infants supports the notion that the congenital form is associated with an arrest in muscle development and suggests a role for the DMPK gene during differentiation and maturation of muscle. In order to clarify these findings, a comparative study of DMPK and myogenic factor mRNA levels was performed in developing mouse muscle tissues and cultured muscle cells at different developmental stages. Results show that DMPK gene expression is upregulated at a late stage of muscular development. This upregulation does not seem to depend on a given muscle specific bHLH factor.  相似文献   

5.
Myotonic dystrophy (DM) is caused by an expansion of a CTG repeat sequence in the 3' noncoding region of a protein kinase gene (DMPK) at 19q13.3. We used in situ hybridization to analyse the replication timing of the genomic region containing DMPK in fibroblasts and myoblasts from controls and myotonic dystrophy patients. In this method the relative proportion of singlet to doublet hybridization signals is used to infer the relative time of replication of specific loci or regions. Our results show that in cells from normal individuals approximately 65% of signals appear as doublets, indicating early replication. In DM patients with a number of CTG repeats ranging from about 600-1800 we observed a significant increase of singlet-doublets compared to the background level. These results suggest the existence of replication alternations and/or structural differences between the normal and mutant alleles induced by the presence of the DM mutation.  相似文献   

6.
Myotonic dystrophy (DM), the most prevalent muscular disorder in adults, is caused by (CTG)n-repeat expansion in a gene encoding a protein kinase (DM protein kinase; DMPK) and involves changes in cytoarchitecture and ion homeostasis. To obtain clues to the normal biological role of DMPK in cellular ion homeostasis, we have compared the resting [Ca2+]i, the amplitude and shape of depolarization-induced Ca2+ transients, and the content of ATP-driven ion pumps in cultured skeletal muscle cells of wild-type and DMPK[-/-] knockout mice. In vitro-differentiated DMPK[-/-] myotubes exhibit a higher resting [Ca2+]i than do wild-type myotubes because of an altered open probability of voltage-dependent l-type Ca2+ and Na+ channels. The mutant myotubes exhibit smaller and slower Ca2+ responses upon triggering by acetylcholine or high external K+. In addition, we observed that these Ca2+ transients partially result from an influx of extracellular Ca2+ through the l-type Ca2+ channel. Neither the content nor the activity of Na+/K+ ATPase and sarcoplasmic reticulum Ca2+-ATPase are affected by DMPK absence. In conclusion, our data suggest that DMPK is involved in modulating the initial events of excitation-contraction coupling in skeletal muscle.  相似文献   

7.
The merosin M-chain (or laminin-alpha 2) is one of three subunits of laminin-2 which is highly expressed in striated muscle and peripheral nerve. Complete lack of laminin-alpha 2 expression in skeletal muscle is the hallmark of one form of congenital muscular dystrophy which is characterized by dysmyelination of the central nervous system (CNS), links to chromosome 6q2 and is common among Caucasians. Laminin-alpha 2 expression was also found to be significantly reduced in Fukuyama congenital muscular dystrophy which links to chromosome 9q3. We report consistently preserved laminin-2 expression, including laminin-alpha 2, as detected by immunofluorescence in skeletal muscle from five patients with Walker-Warburg syndrome which is characterized by congenital muscular dystrophy and, in addition, type II lissencephaly or pachygyria, defective CNS myelination, and ocular dysgenesis. These findings show that in spite of partial phenotypic overlap between Fukuyama CMD and Walker-Warburg syndrome the two disorders are nosologically separate disease entities. They also exclude that Walker-Warburg syndrome is allelic to the common form of congenital muscular dystrophy with laminin-alpha 2 deficiency.  相似文献   

8.
Physical exercise can cause marked alterations in the structure and function of human skeletal muscle. However, little is known about the specific signaling molecules and pathways that enable exercise to modulate cellular processes in skeletal muscle. The mitogen-activated protein kinase (MAPK) cascade is a major signaling system by which cells transduce extracellular signals into intracellular responses. We tested the hypothesis that a single bout of exercise activates the MAPK signaling pathway. Needle biopsies of vastus lateralis muscle were taken from nine subjects at rest and after 60 min of cycle ergometer exercise. In all subjects, exercise increased MAPK phosphorylation, and the activity of its downstream substrate, the p90 ribosomal S6 kinase 2. Furthermore, exercise increased the activities of the upstream regulators of MAPK, MAP kinase kinase, and Raf-1. When two additional subjects were studied using a one-legged exercise protocol, MAPK phosphorylation and p90 ribosomal S6 kinase 2, MAP kinase kinase 1, and Raf-1 activities were increased only in the exercising leg. These studies demonstrate that exercise activates the MAPK cascade in human skeletal muscle and that this stimulation is primarily a local, tissue-specific phenomenon, rather than a systemic response to exercise. These findings suggest that the MAPK pathway may modulate cellular processes that occur in skeletal muscle in response to exercise.  相似文献   

9.
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11.
Muscle cells are frequently subjected to severe conditions caused by heat, oxidative, and mechanical stresses. The small heat shock proteins (sHSPs) such as alphaB-crystallin and HSP27, which are highly expressed in muscle cells, have been suggested to play roles in maintaining myofibrillar integrity against such stresses. Here, we identified a novel member of the sHSP family that associates specifically with myotonic dystrophy protein kinase (DMPK). This DMPK-binding protein, MKBP, shows a unique nature compared with other known sHSPs: (a) In muscle cytosol, MKBP exists as an oligomeric complex separate from the complex formed by alphaB-crystallin and HSP27. (b) The expression of MKBP is not induced by heat shock, although it shows the characteristic early response of redistribution to the insoluble fraction like other sHSPs. Immunohistochemical analysis of skeletal muscle cells shows that MKBP localizes to the cross sections of individual myofibrils at the Z-membrane as well as the neuromuscular junction, where DMPK has been suggested to be concentrated. In vitro, MKBP enhances the kinase activity of DMPK and protects it from heat-induced inactivation. These results suggest that MKBP constitutes a novel stress-responsive system independent of other known sHSPs in muscle cells and that DMPK may be involved in this system by being activated by MKBP. Importantly, since the amount of MKBP protein, but not that of other sHSP family member proteins, is selectively upregulated in skeletal muscle from DM patients, an interaction between DMPK and MKBP may be involved in the pathogenesis of DM.  相似文献   

12.
13.
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.  相似文献   

14.
Two recently described proteins in the mitochondrial uncoupling protein (UCP) family, UCP-2 and UCP-3, have been linked to phenotypes of obesity and NIDDM. We determined the mRNA levels of UCP-2 and UCP-3 in skeletal muscle of NIDDM patients and of healthy control subjects. No difference in the mRNA levels or in the protein expression of UCP-2 was observed between the two groups. In contrast, mRNA levels of UCP-3 were significantly reduced in skeletal muscle of NIDDM patients compared with control subjects. In the NIDDM patients, a positive correlation between UCP-3 expression and whole-body insulin-mediated glucose utilization rate was also noted. These results suggest that UCP-3 regulation may be altered in states of insulin resistance.  相似文献   

15.
Like all other muscular dystrophies, Duchenne muscular dystrophy is characterized by the coexistence of degenerative lesions of the muscle fibers and of regenerative changes. The present study has been carried out in order to precise the degree of regeneration at different stages of the disease, by analyzing the expression of several markers of cell proliferation and of muscular differentiation. In the two affected foetuses of our series, the m. quadriceps is histologically normal, except for the absent expression of immunoreactive dystrophin. The quadriceps from the eight children of our series (20 months-16 years) all present clear dystrophic changes. Muscle regeneration is characterized by activation of the satellite cells, by their multiplication followed by their fusion giving birth to regenerative fibers. By studying the expression of muscular markers (vimentin, desmin, isoforms of the myosin heavy chains), it has been possible to define more precisely the degree of maturation and of differentiation of these regenerative fibers. Our results suggest that an abortive regeneration of the muscle fibers in Duchenne muscular dystrophy can explain, at least partly, the progressive evolution of this disease.  相似文献   

16.
Previous studies have demonstrated that oxygen consumption and fat oxidation remain elevated in the postexercise period. The purpose of this study was to determine whether malonyl-CoA, an inhibitor of fatty acid oxidation, remains depressed in muscle after exercise. Rats were sprinted for 5 min (40 m/min, 5% grade) or run for 30 min (21 m/min, 15% grade). Red quadriceps malonyl-CoA returned to resting values by 90 min postexercise in the sprinting rats and remained significantly lower at least 90 min postexercise in the 30-min exercise group. AMP-activated protein kinase activity remained significantly elevated (P < 0.05) for 10 min after exercise in both groups. The most rapid rate of glycogen repletion was in the first 30 min postexercise. The respiratory exchange ratio decreased from a nonexercise value of 0.87 +/- 0.01 to an average 0.82 +/- 0.01 during the 90-min period after 30 min of exercise. Thus muscle malonyl-CoA remains depressed and fat oxidation is elevated for relatively prolonged periods after a single bout of exercise. This may allow fat oxidation to contribute more to muscle energy requirements, thus leaving more glucose for replenishment of muscle glycogen.  相似文献   

17.
We studied the effect of mitochondrial extracts from skeletal muscle of patients with Duchenne's muscular dystrophy (DMD) on calmitine from the skeletal muscle of normal mice and control subjects. Our results clearly show the existence of an abnormal proteolytic activity of mitochondria from patients with DMD on calmitine from the normal mouse. This proteolytic activity was not found on calmitine from the control subject. Overall, our observations suggest that calmitine concentration in the muscle of the control subject remains elevated because of the presence of a calmitine-specific protease and an inhibitor of this protease which regulates and/or suppresses the activity of the enzyme according to the requirements of the muscle cell. Conversely, the calmitine deficiency observed in the muscle of patients with DMD would be due to the absence of this inhibitor. This would account for the continual activity of the enzyme in degrading calmitine as soon as it is synthesized. The identification of this inhibitor is currently being investigated in our laboratory.  相似文献   

18.
The serum lipoprotein(a) [Lp(a)] level is a known risk factor for arteriosclerotic coronary artery disease. However, its association with restenosis after percutaneous transluminal coronary angioplasty (PTCA) is controversial. We hypothesized that the Lp(a) level is a significant risk factor for restenosis after angioplasty through a pathophysiological mechanism leading to excess thrombin generation or inhibition of fibrinolysis. We designed a prospective study of the relation of Lp(a) to outcome after PTCA, in which we measured selected laboratory variables at entry and collected clinical, procedural, lesion-related, and outcome data pertaining to restenosis. Restenosis was defined as >50% stenosis of the target lesion by angiography or as ischemia in the target vessel distribution by radionuclide-perfusion scan. Before the patients underwent PTCA, blood was obtained by venipuncture for measurement of Lp(a), total cholesterol, thrombin-antithrombin (TAT) complex, alpha2-antiplasmin-plasmin (APP) complex, and plasminogen activator inhibitor-1 (PAI-1). Evaluable outcome data were obtained on 162 subjects, who form the basis of this report. Restenosis occurred in 61 subjects (38%). The Lp(a) level was not correlated significantly with TAT, APP, PAI-1, or the TAT-APP ratio. Levels of TAT, APP, and PAI-1 were not statistically different in the patients with versus those without restenosis. The median ratio of TAT to APP was 2-fold higher in the restenosis group, and this difference approached statistical significance (P=0.07). Univariate analysis was performed for the association of clinical, lesion-related, and procedural risk factors with restenosis. Lp(a) levels did not differ significantly in the restenosis versus no-restenosis group, whether assessed categorically (>25 mg/dL versus <25 mg/dL) or as a continuous variable by Mann-Whitney U test. The number of lesions dilated and the lack of family history of premature heart disease were significantly associated with restenosis (P=0.002 and P=0.008, respectively). A history of diabetes mellitus was of borderline significance (P=0.055). By multiple logistic regression analysis, the number of lesions dilated was the only variable significantly associated with restenosis (P=0.03). We conclude that the number of lesions dilated during PTCA is a significant risk factor for restenosis, whereas the serum Lp(a) level was not a significant risk factor for restenosis in our patient population. The TAT to APP ratio merits further study as a possible risk factor for restenosis.  相似文献   

19.
20.
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).  相似文献   

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