Estimate of the intrafamilial correlation for serum creatine kinase and pyruvate kinase in females at risk for Duchenne and Becker muscular dystrophies.

In order to verify the possibility of nonrandom X-inactivation in females heterozygous for Duchenne (DMD) and Becker (BMD) muscular dystrophies, intrafamilial correlations and the heritabilities for serum creatine kinase (CK) and pyruvate kinase (PK) were estimated in a large sample of females belonging to families with affected patients. The results of the present investigation suggest that the apparent intrafamilial correlations for serum CK reported in previous studies in DMD families are not related with the presence of the DMD/BMD gene. Our data do not seem to support the hypothesis of a gene leading to a preferential inactivation of the X-chromosome in females at risk for the dystrophin gene.     

Female transmitters of Duchenne's myopathy: detection by quantitative ultrasonography combined with blood creatine kinase

Duchenne muscular dystrophy (DMD) is a disease inherited in an X-linked recessive manner. Nothing is yet known about the gene defect involved. The problem of DMD carrier detection is very important and now without satisfactory solution. Presently the best method, for this detection, is the determination of the serum creatine-kinase (CK) level. However about 30% of DMD children's mothers have normal CK levels. In this study we associated CK determination and a new echographic method. This physical investigation is based on the muscle attenuation measurements through the evolution of the running spectral moments. The parameter expressed is the slope of the ultrasound attenuation signal (in decibels by centimetre and by megahertz; dB . cm-1 . MHz-1). We measured this parameter in vivo on the vastus medialis. The use of the chemical technique and of the physical one improves largely the detection of obligate carriers of Duchenne muscular dystrophy than when one of these methods is employed alone. One such combination recognizes 17 out of the 19 obligate carriers in our series. The difference of the results obtained by these two unrelated methods cannot be yet interpreted, but can reflect genetic heterogeneity in DMD and the inactivation of the X chromosomes.     

Duchenne muscular dystrophy: Pathogenetic aspects and genetic prevention

Summary Duchenne muscular dystrophy (DMD) is the most common sex linked lethal disease in man (one case in about 4000 male live births). The patients are wheelchair bound around the age of 8–10 years and usually die before the age of 20 years. The mutation rate, estimated by different methods and from different population studies, is in the order of 7×10^-5, which is higher than for any other X-linked genetic disease. Moreover, unlike other X linked diseases such as hemophilia A or Lesh-Nyhan's disease, there seems to be no sex difference for the mutation rates in DMD. Several observations of DMD in girls bearing X-autosomal translocations and linkage studies on two X chromosomal DNA restriction fragment length polymorphisms indicate that the DMD locus is situated on the short arm of the X chromosome, between Xp11 and Xp22. It may be of considerable length, and perthaps consisting of actively coding and non-active intervening DNA sequences. Thus unequal crossing over during meiosis in females could theoretically account for a considerable proportion of new mutations.However, there is no structurally or functionally abnormal protein known that might represent the primary gene product, nor has any pathogenetic mechanism leading to the observed biochemical and histological alterations been elucidated. Among the numerous pathogenetic concepts the hypothesis of a structural or/and functional defect of the muscular plasma membrane is still the most attractive. It would explain both the excess of muscular constituents found in serum of patients and carriers, such as creatine kinase (CK), as well as the excessive calcium uptake by dystrophic muscle fibres, which, prior to necrosis, could lead to hypercontraction, rupture of myofilaments in adjacent sarcomeres and by excessive Ca uptake to mitochondrial damage causing crucial energy loss.The results of studies on structural and functional memthrane abnormalities in cells other than muscle tissue, e.g., erythrocytes, lymphocytes and cultured fibroblasts, indicate that the DMD mutation is probably demonstrable in these tissues. However, most of the findings are still difficult to reproduce or even controversial.DMD is an incurable disease; therefore most effort, in research as well as in practical medicine, is concentrated upon its prevention. Unfortunately the disease cannot yet be diagnosed prenatally. Potential DMD carriers among female relatives of the patients may be identified by pathological heterozygote tests, of which determination of serum CK activity is probably still the most reliable method, allowing the detection of about 70% of adult and probably up to 90% of carriers at school age. Because of the high mutation rate, assessment of individual heterozygote risks in female relatives of isolated DMD cases is of special importance. For the calculations a maximum of genealogical and phenotype information on unaffected male and on heterozygote tests in female relatives is needed to obtain credible risk figures. However, estimating a consultand's risk and passing on this information is only one aspect of genetic counselling in DMD. At least as important is information on the medical, psychological and social impacts of the disease (burden) and the possibility of maintaining a long-term contact between the couples at risk and the team involved in medical, genetic and social problems of the disease. Neonatal CK screening for DMD, although without any therapeutical consequence, could theoretically lead to the prevention of secondary cases, accounting for some 15% of all DMD patients born, but an almost equal prevention rate of such cases would be achieved if CK examinations were limited to all boys with delayed motor development during the first 2 years of life. Finally, it is believed that the two most important preventive problems in DMD, carrier detection and prenatal diagnosis, will ultimately be solved by means of the rapidly advancing DNA technology.This work was dedicated to Professor P.E. Becker in honour of his 75th birthday     

Detection and identification of myoglobin in serum by immunoblotting. Effect of exercise on patients with Duchenne muscular dystrophy

The diurnal changes and the effect of pre- and postexercise on serum myoglobin (Mb) levels and creatine kinase (CK) activity were investigated and compared in 11 male patients with Duchenne muscular dystrophy (DMD) and 11 normal male controls. The Mb levels and the CK activity in patients with DMD were significantly higher (p less than 0.001) than in the controls under both resting and exercise conditions. No correlation was found between serum Mb levels and CK activity in patients with DMD. The results indicate that Mb is also a useful parameter in both the RIA and the immunoblotting technique for diagnosing muscle-fiber degeneration and screening DMD.     

Selectivity of 4,5,6,7-tetrabromobenzimidazole as an ATP-competitive potent inhibitor of protein kinase CK2 from various sources

Like the previously reported 4,5,6,7-tetrabromobenzotriazole (TBBt), the structurally related 4,5,6,7-tetrabromobenzimidazole (TBBz) is a selective ATP-competitive inhibitor of protein kinase CK2 from such divergent sources as yeast, rat liver, Neurospora crassa and Candida tropicalis, with K(i) values in the range 0.5-1 microM. It is virtually inactive vs. PKA, PKC, and a very weak inhibitor of protein kinase CK1. The corresponding tetrachlorobenzimidazole (TCBz) is a much weaker inhibitor of CK2, like tetrachlorobenzotriazole (TCBt) relative to TBBt. Bearing in mind the similarity of the van der Waals radii of Br (1.95 A) and CH(3) (2.0 A), the corresponding much less hydrophobic 4,5,6,7-tetramethylbenzotriazole (TMeBt) was prepared and found to be a very weak inhibitor of CK2, as well as of CK1. An unexpected, and significant, difference between TBBt and TBBz are their inhibitory activities vs. the yeast protein kinase PK60S, which phosphorylates, both in vitro and in intact yeast cells, three of the five pp13 kDa ribosomal surface acidic proteins in yeast cells. TBBt was previously noted to be a more effective inhibitor of PK60S than of yeast CK2; by contrast, TBBz is a relatively feeble inhibitor of PK60S, hence more selective than TBBt vs. CK2 in yeast cells. TMeBt was virtually inactive vs PK60S. Like TBBt, TBBz is an additional lead compound for development of more potent inhibitors of CK2.     

Phosphorylation of cytokeratin 17 by herpes simplex virus type 2 US3 protein kinase

We previously reported the establishment of an HEp2 cell line which expresses the US3 protein kinase (PK) of herpes simplex virus type 2 (HSV-2) upon induction with IPTG. Here we report that expression, phosphorylation and ubiquitination of cytokeratin 17 (CK17) are enhanced in US3-expressing HEp2 cells. In vitro kinase and co-immunoprecipitation assays provided evidence that US3 PK directly phosphorylates CK17. Expression of US3 PK caused a significant decrease in filamentous staining of CK17, suggesting that phosphorylation of CK17 by US3 PK causes a disruption of intermediate filaments. Our observations suggest a role for US3 in the regulation of CKs and intermediate filaments in cells. Moreover, we found that infection of a keratinocyte-derived cell line, A431, with a US3-deficient virus, results in cytopathic effects that are morphologically distinct from those induced by wild-type and revertant viruses, suggesting that US3 PK may be important for interaction between HSV-2 and peripheral epithelial cells.     

The protein kinase 60S is a free catalytic CK2alpha' subunit and forms an inactive complex with superoxide dismutase SOD1

The 60S ribosomes from Saccharomyces cerevisiae contain a set of acidic P-proteins playing an important role in the ribosome function. Reversible phosphorylation of those proteins is a mechanism regulating translational activity of ribosomes. The key role in regulation of this process is played by specific, second messenger-independent protein kinases. The PK60S kinase was one of the enzymes phosphorylating P-proteins. The enzyme has been purified from yeast and characterised. Pure enzyme has properties similar to those reported for casein kinase type 2. Peptide mass fingerprinting (PMF) has identified the PK60S as a catalytic alpha(') subunit of casein kinase type 2 (CK2alpha(')). Protein kinase activity is inhibited by SOD1 and by highly specific CK2 inhibitor-4,5,6,7-tetrabromo-benzotriazole (TBBt). The possible mechanism of regulation of CK2alpha(') activity in stress conditions, by superoxide dismutase in regulation of 80S-ribosome activity, is discussed.     

Creatine kinase increases the solubility and enzymatic activity of pyruvate kinase by means of diazymatic coupling

Five separate methods, paper chromatography, ethanol solubility, pH dependence, enzymatic activity, and NMR saturation transfer, were used to study the coupling of pyruvate kinase (PK) and creatine kinase (CK). Each method demonstrated specific coupling of the two proteins. The coupling with CK showed that PK had increased ethanol solubility, enzymatic activity, pH-dependent aqueous solubility, and ability to receive ATP directly from CK even at very high (1.2 M) ionic strength at equilibrium conditions. The robustness of the coupling between two enzymes with highin vivo concentrations indicates the necessity for considering kinetic schemes utilizing high enzyme concentrations, as opposed to the dilute properties of Michaelis — Menten kinetics. The direct transfer of ATP from PK to CK constitutes a net transfer of the glycolytically produced high-energy phosphate onto creatine, maintaining a high ADP and low ATP concentration in the vicinity of the glycolytic complex, conditions suited to maintaining a high glycolytic flux. The demonstration of the physical and functional coupling of two soluble enzymes, termed diazymes, suggests that glycolysis may be regulated by diazymatic coupling and that other cellular processes could be regulated similarly.     

Nucleotide promiscuity of 3-phosphoglycerate kinase is in focus: implications for the design of better anti-HIV analogues

The wide specificity of 3-phosphoglycerate kinase (PGK) towards its nucleotide substrate is a property that allows contribution of this enzyme to the effective phosphorylation (i.e. activation) of nucleotide-based pro-drugs against HIV. Here, the structural basis of the nucleotide-PGK interaction is characterised in comparison to other kinases, namely pyruvate kinase (PK) and creatine kinase (CK), by enzyme kinetic analysis and structural modelling (docking) studies. The results provided evidence for favouring the purine vs. pyrimidine base containing nucleotides for PGK rather than for PK or CK. This is due to the exceptional ability of PGK in forming the hydrophobic contacts of the nucleotide rings that assures the appropriate positioning of the connected phosphate-chain for catalysis. As for the D-/L-configurations of the nucleotides, the L-forms (both purine and pyrimidine) are well accepted by PGK rather than either by PK or CK. Here again the dominance of the hydrophobic interactions of the L-form of pyrimidines with PGK is underlined in comparison with those of PK or CK. Furthermore, for the l-forms, the absence of the ribose OH-groups with PGK is better tolerated for the purine than for the pyrimidine containing compounds. On the other hand, the positioning of the phosphate-chain is an even more important term for PGK in the case of both purines and pyrimidines with an L-configuration, as deduced from the present kinetic studies with various nucleotide-site mutants of PGK. These characteristics of the kinase-nucleotide interactions can provide a guideline for designing new drugs.     

The theory of diazymes and functional coupling of pyruvate kinase and creatine kinase

The physical-chemical principles governing the interactions of enzymes having common metabolic products are presented. Methods for comparing the dissociation rates of the metabolic product and the rates of enzyme-enzyme interaction are given. Using muscle pyruvate kinase (PK) and creatine kinase (CK) as an example, it is shown that the probability of forming an enzyme-product-enzyme complex is much greater than the rate of ATP dissociation from either enzyme. Experimental evidence using 31P-NMR demonstrates that in the presence of both pyruvate kinase and creatine kinase, there is exchange of phosphate between phosphocreatine and phosphoenolpyruvate without a change in the intermediate, ATP. This confirms the formation of a PK.ATP.CK complex in an aqueous solution without enzyme attachment to a substructure. Enzymes capable of forming these mobile clusters are defined as diazymes, and the criteria for their formation are given. The metabolic implications of diazymes are discussed.     

Intracellular compartmentation of cardiac fibres from rainbow trout and Atlantic cod--a general design of heart cells

In mammalian cardiomyocytes, mitochondria and adjacent ATPases with participation of creatine kinase (CK) constitute functional compartments with an exchange of ADP and ATP delimited from cytosolic bulk solution. The question arises if this extends to ectothermic vertebrates: their low body temperature and thinner cardiomyocytes with a lower density of membrane structures may reduce the need and structural basis for compartmentation. In saponin-skinned cardiac fibres from rainbow trout and Atlantic cod, we investigated mitochondrial respiration induced by endogenous ADP generated by ATPases and its competition for this ADP with pyruvate kinase (PK) in excess. At low Ca(2+) activity (pCa = 7.0), PK lowered ATP-induced respiration by 40% in trout and 26% in cod. At high Ca(2+) activity (pCa = 5.41), PK had no effect. Additionally, ADP release from the fibres was almost zero but increased drastically upon inhibition of respiration with 1 mM Na-azide. This suggests that fibres are compartmented. PK abolished creatine-stimulated respiration in trout suggesting a less tight coupling of CK to respiration than in mammals. In conclusion, intracellular compartmentation seems to be a general feature of vertebrate cardiomyocytes, whereas the role of CK is unclear, but it seems to be less important for energy transport in species with lower metabolism.     

Carrier detection and genetic counselling in Duchenne muscular dystrophy: a follow-up study.

Assay of serum creatine kinase activity is useful in the detection of carriers of the X-linked gene for Duchenne muscular dystrophy (DMD). For genetic counselling this assay has been used in conjunction with pedigree analysis to improve estimates of the risk that a female relative of a DMD patient is a carrier. To measure the impact of the program, follow-up information was obtained from women who had received genetic counselling for DMD. Their responses showed that the risk of producing an affected son had been a major factor in their attitude toward family planning, and their reproductive performance correlated inversely with their genetic risk. The decision by the majority of proven carriers to prevent the birth of further male offspring was reflected in a recent decline in the frequency of a known family history of DMD among newly ascertained cases.     

Intracellular compartmentation of cardiac fibres from rainbow trout and Atlantic cod—a general design of heart cells

In mammalian cardiomyocytes, mitochondria and adjacent ATPases with participation of creatine kinase (CK) constitute functional compartments with an exchange of ADP and ATP delimited from cytosolic bulk solution. The question arises if this extends to ectothermic vertebrates: their low body temperature and thinner cardiomyocytes with a lower density of membrane structures may reduce the need and structural basis for compartmentation. In saponin-skinned cardiac fibres from rainbow trout and Atlantic cod, we investigated mitochondrial respiration induced by endogenous ADP generated by ATPases and its competition for this ADP with pyruvate kinase (PK) in excess. At low Ca²⁺ activity (pCa = 7.0), PK lowered ATP-induced respiration by 40% in trout and 26% in cod. At high Ca²⁺ activity (pCa = 5.41), PK had no effect. Additionally, ADP release from the fibres was almost zero but increased drastically upon inhibition of respiration with 1 mM Na-azide. This suggests that fibres are compartmented. PK abolished creatine-stimulated respiration in trout suggesting a less tight coupling of CK to respiration than in mammals. In conclusion, intracellular compartmentation seems to be a general feature of vertebrate cardiomyocytes, whereas the role of CK is unclear, but it seems to be less important for energy transport in species with lower metabolism.     

Creatine kinase in regulation of heart function and metabolism. I. Further evidence for compartmentation of adenine nucleotides in cardiac myofibrillar and sarcolemmal coupled ATPase-creatine kinase systems

In isolated and purified cardiac myofibrillar and sarcolemmal preparations, the route of movement of ADP produced in the Mg2+-ATPase reactions was studied by investigating the efficiency of competition between the endogenous creatine kinase and exogenous pyruvate kinase reactions. In the homogeneous control system composed of hexokinase and glucose as ATPase, soluble creatine kinase rapidly rephosphorylated ADP produced in the presence of 1 mM ATP, but the addition of pyruvate kinase in an increasing amount inhibited the reaction of creatine release from phosphocreatine and symmetrically increased the rate of pyruvate production from phosphoenol pyruvate. At a pyruvate-kinase/creatine-kinase activity ratio (PK/CK) of 50, all ADP was used by the pyruvate kinase. In myofibrillar and sarcolemmal preparations containing particulate creatine kinase, the creatine kinase reaction was much less efficiently suppressed by pyruvate kinase, and at PK/CK = 50 half-maximal release of creatine was still observed. The rate of immediate myofibrillar MgADP rephosphorylation in the endogenous creatine-kinase reaction was observed to be governed by the concentration of phosphocreatine in accordance with the kinetics of this enzyme. The physiological significance of these findings is discussed.     

多重连接探针扩增方法在假肥大性肌营养不良产前基因诊断中的应用

假肥大性肌营养不良(Duchenne/Becker muscular dystrophy, DMD/BMD)是一种由于DMD基因突变导致的X连锁隐性致死性遗传病。目前没有有效的治疗方法。为建立一种既可以对携带者进行检测又可以进行产前基因诊断的方法, 文章联合应用多重连接探针扩增技术(Multiplex ligation-dependent probe amplification, MLPA)和短串联重复序列(Short tandem repeats , STR)为遗传标记连锁分析的方法对26例有高风险再生育患儿的假肥大性肌营养不良家系的孕妇通过羊水穿刺进行产前基因诊断。26例进行产前基因诊断的羊水标本中有7例诊断为男性患儿, 4例诊断为女性携带者。MLPA可以作为筛查DMD基因缺失和重复突变的首选方法。联合应用MLPA和STR连锁分析, 可以提高假肥大性肌营养不良的产前基因诊断率。     

Thiazole- and selenazole-comprising high-affinity inhibitors possess bright microsecond-scale photoluminescence in complex with protein kinase CK2

A previously disclosed protein kinase (PK) CK2-selective inhibitor 4-(2-amino-1,3-thiazol-5-yl)benzoic acid (ATB) and its selenium-containing counterpart (ASB) revealed remarkable room temperature phosphorescence when bound to the ATP pocket of the protein kinase CK2. Conjugation of these fragments with a mimic of CK2 substrate peptide resulted in bisubstrate inhibitors with increased affinity towards the kinase. Attachment of the fluorescent acceptor dye 5-TAMRA to the conjugates led to significant enhancement of intensity of long-lifetime (microsecond-scale) photoluminescence of both sulfur- and selenium-containing compounds. The developed photoluminescent probes make possible selective determination of the concentration of CK2 in cell lysates and characterization of CK2 inhibitors by means of time-gated measurement of photoluminescence.     

Mutations in the C-terminal domain of topoisomerase II affect meiotic function and interaction with the casein kinase 2β subunit

Topoisomerase II is a major target of the protein kinase casein kinase 2 (PK CK2) in vivo. All major phosphorylation acceptor sites in the yeast enzyme are found in the C-terminal 350aa. The acceptor sites are generally clustered such that there is more than one modified Ser or Thr within a short peptide. Mutagenesis of the predicted acceptor sites have confirmed that five of the eight predicted sites are targeted in vitro and in vivo by PK CK2. Mutation to nonphosphorylatable, neutral residues provokes at most a 10% increase in mitotic doubling time. Truncation of the enzyme leaves the enzyme catalytically active, but slightly lengthens the doubling time during mitotic growth and impedes progress through meiosis. Since this could reflect the loss of interaction with an important ligand, we have examined whether the C-terminal domain of the yeast enzyme mediates interaction with the regulatory subunit of PK CK2, which was previously reported to bind topoisomerase II. We find that point mutation of the phospho-acceptor sites does not abrogate the interaction with a small region of PK CK2 , while truncation at aal276 or aal236 does. The site of interaction within PK CK2 does not coincide with the highly negatively charged spermine binding site.     

Curcumin alleviates dystrophic muscle pathology in mdx mice

Abnormal activation of nuclear factor kappa B (NF-kappaB) probably plays an important role in the pathogenesis of Duchenne's muscular dystrophy (DMD). In this report, we evaluated the efficacy of curcumin, a potent NF-kappaB inhibitor, in mdx mice, a mouse model of DMD. We found that it improved sarcolemmic integrity and enhanced muscle strength after intraperitoneal (i.p.) injection. Histological analysis revealed that the structural defects of myofibrils were reduced, and biochemical analysis showed that creatine kinase (CK) activity was decreased. We also found that levels of tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1beta) and inducible nitric oxide synthase (iNOS) in the mdx mice were decreased by curcumin administration. EMSA analysis showed that NF-kappaB activity was also inhibited. We thus conclude that curcumin is effective in the therapy of muscular dystrophy in mdx mice, and that the mechanism may involve inhibition of NF-kappaB activity. Since curcumin is a non-toxic compound derived from plants, we propose that it may be useful for DMD therapy.