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Opposite transitions of chick brain catalytically active cytosolic creatine kinase isoenzymes during development.

Postnatally the rat brain synthesizes catalytic forms of muscle type (MM) and heart type (MB) creatine kinase (CK), besides the supposedly sole type vertebrate brain-specific (BB) CK. We intended to demonstrate that in Rhode Island chicken brain, cytosolic (c) CK isoenzymatic transitions. (for example BB-CK is followed by the appearance of MB-CK and MM-CK during muscle differentiation), can also occur during development and aging. Cytosolic post 125000 x g, mitochondrial CK-free, brain samples were obtained for zone electrophoresis separation and identification of catalytically active cCK isoforms. BB-CK was never found during chicken brain ontogeny. Against the accepted view, an opposite isoenzyme transition pattern from MM through BB-CK was found in the chicken embryonic brain from the very early stages of development up to day 2 post-hatching. At very early stages of chicken brain ontogeny constitutive MM- and MB-CK isoenzymes were present before the advent of creatine. It seems to be that typical and atypical brain MM- and MB-CK could be working as ATPases in the absence of creatine before embryonic stage 28 (day 5.5) and/or such CK isoforms may begin to form part of the slow component b in developing early neurons and later in the nuclei of glial cells to be used by the CK/phosphocreatine (PC) system as the neural tissues mature. The post-hatching transition pattern showed simultaneous expression of more than one CK isoenzyme within the same neural sample as in post-natal rat brain, presumably due to regional differential transphosphorylation requirements. Strain-dependent enzymatic specific activities have been reported in several species. Since equivalent values of brain CK specific activity were obtained previously from the embryonic plateau phase of CK activity during White Leghorn development, and those from Rhode Island brain neurons cultured 11 days, we compared if, in vivo, a similar brain CK specific activity pattern was physiologically equivalent during Rhode Island and White Leghorn chicken ontogeny. We found quantitatively different strain-specific CK specific activity patterns during this period. Rhode Island brain CK activity values were approximately 4.5-fold those of White Leghorn ones. This indicates that production of energy from anaerobic metabolism and transphosphorylation by the CK/PC system to synthesize ATP more efficiently is strain-specific. In Rhode Islands, there was an age-dependent increase of CK specific activity, mostly in older animals (440% above the value found during the embryonic plateau), when the Krebs cycle and glycolysis lose capacity. During adult life and aging, under physiological conditions, the three CK isoenzymes may participate in diverse functions of the different cell compartments of brain glia and neurons with regard to their high and fluctuating energy demands that are not completely covered by anaerobic and aerobic glycolisis.

Animals↗

Studies on the stability of creatine kinase isozymes.

Research on the stabilizing properties of creatine kinase isozymes CK-BB, CK-MB, and CK-MM showed that minor alteration of their sequence and structure influenced their stability significantly. An analysis of the stability of the isozymes in storage after freeze drying indicates that creatine kinase isozymes are all in monomer form because of the loss of subunit interactions. Freeze-drying leads to the oxidization of CK-BB and rearrangement of CK-MB. There are also differences in the unfolding of the isozymes in urea. CK-BB and CK-MB are unfolded in lower urea concentrations than CK-MM. Differences in the thermal unfolding were also examined by differential scanning calorimetry. This paper discusses the potential biological significance of these results.

Animals↗

Production of recombinant human creatine kinase (r-hCK) isozymes by tandem repeat expression of M and B genes and characterization of r-hCK-MB.

BACKGROUND: Serum creatine kinase-MB isoenzyme (CK-MB) is widely used as a marker of myocardial injury. We prepared recombinant human CK (r-hCK) MB isoenzyme and examined its potential for use as a control material for assay of CK-MB in serum. METHODS: cDNAs encoding CK-M and CK-B subunits were inserted into the same plasmid vector, followed by transformation of Escherichia coli. The resulting three types of CK isoenzymes were purified by conventional chromatography. RESULTS: The ratio of MB to MM to BB was 50:40:10 on the basis of CK activity. Highly purified CK-MB with a specific activity of 533 U/mg was produced in a yield of 5.7 mg/g of packed cells. Purified r-hCK-MB had the isoelectric point (pI 5.3) and molecular size (46 kDa for the subunit) of native CK-MB. Its immunoreactivity in an ELISA using antibody against native heart enzyme was similar to that of cardiac CK-MB. The r-hCK-MB retained >90% activity for at least 4 months at 11 degrees C in a delipidated serum matrix in a liquid form at a concentration of 118 U/L. CONCLUSIONS: r-hCK-MB shows key properties of the native cardiac isoenzyme and may be useful as a control and calibrator for serum assays of CK-MB.

Creatine Kinase↗

[Creatine kinase and its isozymes].

Creatine kinase (CK; EC 2.7.3.2) is found in a variety of striated and smooth muscles, and the brain, and is an important enzyme regulator of high-energy phosphate production and utilization within contractile tissues. Serum CK activity is measured routinely as a sensitive indicator of injuries to the skeletal muscle and myocardium. CK has three isozymes (CK-MM, CK-MB and CK-BB) in cytoplasm and two isozymes (non-sarcomeric and sarcomeric) in mitochondria. CK isozymes provide more specific information about injured tissue because of their tissue distribution. CK-MM is useful in skeletal muscle diseases, such as muscle dystrophy, CK-MB in acute myocardial infarction (AMI), and CK-BB in brain damage and malignant tumor of the gastro-intestinal tract. CK-MB is measured either by enzyme activity or mass concentration and is measured as a marker not only in the diagnosis of AMI but also in suspected AMI and unstable angina. Mitochondrial CK, a useful indicator for pinpointing the severeness of muscle injuries, and CK-linked immunoglobulin are recognized as macro-CK because of their large molecular size.

Angina, Unstable↗

Creatine kinase isoenzyme patterns upon chronic exposure to cigarette smoke: protective effect of Bacoside A.

Cigarette smoking is implicated as a major risk factor in the development of cardiovascular and cerebrovascular diseases. Creatine kinase (CK) and its isoforms (CK-MM, MB, BB) have been advocated as sensitive markers in the assessment of cardiac and cerebral damage. Therefore, in the present study, we report the isoenzyme patterns of CK in rats upon exposure to cigarette smoke and the protective effect of Bacoside A against chronic smoking induced toxicity. Adult male albino rats were exposed to cigarette smoke and simultaneously administered with Bacoside A, the active constituent from the plant Bacopa monniera, for a period of 12 weeks. The activity of CK was assayed in serum, heart and brain, and its isoenzymes in serum were separated electrophoretically. Rats exposed to cigarette smoke showed significant increase in serum CK activity with concomitant decrease in heart and brain. Also cigarette smoke exposure resulted in a marked increase in all the three isoforms in serum. Administration of Bacoside A prevented these alterations induced by cigarette smoking. Cigarette smoking is known to cause free radical mediated lipid peroxidation leading to increased membrane permeability and cellular damage in the heart and brain resulting in the release of CK into the circulation. The protective effect of Bacoside A on the structural and functional integrity of the membrane prevented the leakage of CK from the respective tissues, which could be attributed to its free radical scavenging and anti-lipid peroxidative effect.

Animals↗

Sexual dimorphism in rat cerebrum and cerebellum: different patterns of catalytically active creatine kinase isoenzymes during postnatal development and aging.

During postnatal development, maturation and aging the Wistar rat cerebrum and cerebellum synthesize, in a different sex-dependent manner, catalytically active dimeric cytosolic (c) muscle-type (MM) and heart-type (MB) creatine kinase (CK), besides the supposedly sole type brain-specific (BB) CK. In both sexes, typical and atypical neuromuscular cCK isoenzymes were present during the study for 26 months. As in rat heart, females showed more cerebral cCK variants (41%) in comparison to males. Female rats exhibited about 93% more cerebellar variants of cCK isoenzymes as compared to males. The male cerebellum showed predominantly BB- and MB-CK during the whole study in comparison to the female one that contained all neuromuscular cCK variants. Only female rats showed decreases and increases of cerebral CK specific activity. In contrast to males, coinciding with the weaning period, cerebral female CK activity decreased 45% from 14 to 21 days and increased about 3-fold in female rats and only 1.3-fold in males from 21 to 45 days of age. Contrary to the remarkable 4-fold increase of chicken brain CK specific activity exhibited at old age, the rat did not show another cerebral CK activity increase during senescence in either sex. However, sex differences of CK specific activity appeared in the cerebellum at all ages. From the sex-specific plateau phase at 45-60 days until 2.2 years of age, about a 41% independent increase of cerebellar CK specific activity was observed in both sexes. After puberty, the differential cerebellum-cerebrum values of CK specific activity were higher for female rats than males during youth, adulthood and senescence. The present work shows that in rat cerebrum and cerebellum, production of ATP through anaerobic transphosphorylation by the CK/PC system is sex-and age-specific, especially in the cerebellum, when glycolysis and the Krebs cycle lose capacity. As in rat heart, under physiological conditions at all ages the several cCK isoenzymes do participate in a gender-specific manner, in favor of females, in diverse functions of the different cell compartments of glial and neuronal cells with regard to their high and fluctuating energy demands not completely covered by anaerobic and aerobic glycolysis.

Aging↗

Creatine phosphokinase in ectopic pregnancy revisited: significant diagnostic value of its MB and MM isoenzyme fractions.

OBJECTIVE: Although evidence on elevated creatine phosphokinase (CPK) as a tool for the diagnosis of ectopic pregnancy (EP) is rather conflictive, no previous study on CPK isoenzyme levels and their relative ratio was found in the literature. STUDY DESIGN: Forty women with EP, 20 with intrauterine (IU) abortive gestation, and 20 regular pregnant women (controls) were studied. Serum total CPK, CPK-MB, and CPK-MM levels were measured at the time of presentation and 24 hours after surgery. Relative operating characteristic (ROC) curves were plotted. RESULTS: Women with EP had significantly higher CPK concentrations and a significantly decreased CPK-MB relative ratio (CPK-MB%), compared with both women with IU abortive pregnancy and controls. The ROC curves demonstrated a significant discriminatory ability of both increased total CPK and decreased CPK-MB% ratio for the diagnosis of EP. CONCLUSION: Determination of CPK isoenzyme fractions can significantly enhance the diagnostic value of total maternal CPK in the prediction of ectopic pregnancy.

Abortion, Spontaneous↗

Over-expression, purification and characterization of the oligomerization dynamics of an invertebrate mitochondrial creatine kinase.

Mitochondrial creatine kinase (MtCK) plays a central role in energy homeostasis within cells that display high and variable rates of ATP turnover. Vertebrate MtCKs exist primarily as octamers but readily dissociate into constituent dimers under a variety of circumstances. MtCK is an ancient protein that is also found in invertebrates including sponges, the most primitive of all multi-cellular animals. We have cloned, expressed, and purified one of these invertebrate MtCKs from a marine polychaete worm, Chaetopterus variopedatus (CVMtCK). Size exclusion chromatography and dynamic light scattering (DLS) were used to characterize oligomeric state in comparison with that of octameric chicken sarcomeric isoform (SarMtCK). At protein concentrations >1 mg/ml, CVMtCK was predominantly octameric (>90%). When diluted to 0.1 mg/ml, CVMtCK dissociated into dimers much more rapidly than SarMtCK when observed under identical conditions. The rate of dissociation for both MtCKs increased as temperature rose from 2 to 28 degrees C, and in CVMtCK, fell at higher incubation temperatures. The fraction of octameric CVMtCK at equilibrium increased with temperature and then fell. Temperature transition studies showed that octamers and dimers were rapidly interconvertible on a similar time scale. Importantly, when CVMtCK was converted to the transition state analog complex (TSAC), both size exclusion chromatography and DLS showed that there was minimal dissociation of octamers into dimers while SarMtCK octamers were highly unstable as the TSAC. These results clearly show distinct differences in octamer stability between CVMtCK and SarMtCK, which could impact function under physiological circumstances. Furthermore, the large yield of recombinant protein and high stability of CVMtCK in the TSAC suggest that this protein might be a good target for crystallization efforts.

Adenosine Diphosphate↗

Functional properties and [Ca(2+)](i) metabolism of creatine kinase--KO mice myocardium.

One major function of the creatine kinase system is to maintain energy demand of myofibrillar contraction processes. Loss of the CK-system led to adaptations in skeletal muscle. To analyze the impact on myocardial function contractile parameters and intracellular calcium metabolism of transgenic mice lacking mitochondrial CK (ScCKmit(-/-)) alone or both mitochondrial and cytoplasmic ScCK (CK(-/-)) were investigated compared to wild type at various workload conditions using isolated intact muscle fibers. Force development at baseline conditions, force-frequency relationship (60-600/min), and rapid frequency switch (60-600/min) were unaltered in myocardium of transgenic mice compared to wild type. Intracellular calcium metabolism revealed unchanged amplitude of the intracellular calcium transients (ICT), refilling of the sarcoplasmic reticulum (calcium reuptake, post-rest behavior) in the ScCKmit(-/-) and CK(-/-) mice. The results demonstrate the effectiveness of myocardial energy-recruiting compensatory mechanisms at baseline as well as under stress conditions in CK depleted myocardium of transgenic mice.

Adaptation, Physiological↗

Cardiac troponins and creatine kinase content of striated muscle in common laboratory animals.

Animal models are important for the investigation of human heart pathology, novel treatments, and medical or surgical interventions for disease. Serum markers of myocardial damage may also be important tools within this field of research. In order to assess the cardiac specificity of widely utilised serum markers, we measured the cardiac troponins and creatine kinase (CK) isoenzymes in cardiac and skeletal muscle samples taken from dog, monkey, pig and rat. These samples were also analysed by immunoblotting for cardiac troponin I (cTnI) and cardiac troponin T (cTnT). The content of cTnI and cTnT in skeletal muscle was below 0.6% of that found in heart for all animal species studied. This low immunoreactivity in skeletal muscle was confirmed by immunoblot analysis. The content of CK was higher in skeletal muscle than in heart muscle for all species. The CK-MB/total CK ratio was lower in skeletal muscle than in cardiac muscle for all species. The differences in CK-MB content of skeletal muscle and heart muscle were much less pronounced than the tissue differences in the amounts of the cardiac troponins. The cardiac troponins are potentially useful serum markers of myocardial damage, with high specificity for myocardial muscle in these common laboratory animals. Creatine kinase-MB is much less cardiac-specific.

Animals↗

Myositis in children with meningococcal disease: a role for tumour necrosis factor-alpha and interleukin-8?

OBJECTIVES: Myalgia is under-recognized in meningococcal disease (MCD). In septic shock, myositis is thought to be mediated by pro-inflammatory cytokines such as tumour necrosis factor-alpha (TNF-alpha), interleukin-8 (IL-8) and interleukin-6 (IL-6) but this has never previously been studied in MCD. We aimed to demonstrate whether muscle damage mediated via TNF-alpha and other pro-inflammatory cytokines occurs in MCD, as estimated by creatine kinase skeletal muscle isoenzyme (CK-MM) and cardiac isoenzyme (CK-MB) concentrations. METHODS: A total of 68 children, median age 2.7 years, with a diagnosis of MCD were prospectively studied. Severity of disease was measured using the Glasgow Meningococcal Septicaemia Prognostic Score (GMSPS). Severe disease was defined as a GMSPS of > or =8. TNF-alpha, IL-8, IL-6 and IL-1Ra concentrations were determined on samples taken on admission. RESULTS: CK-MM correlated significantly with TNF-alpha, IL-8 and GMSPS. There was no significant correlation between CK-MB and TNF-alpha or IL-6, but CK-MB correlated with GMSPS and IL-8. Fifty-six percent of children with MCD had evidence of muscle damage as manifested by elevated CK-MM. CONCLUSIONS: TNF-alpha and IL-8 may be potential mediators in the pathophysiology of skeletal muscle damage in MCD.

Adolescent↗

In vitro measurement of enzymatic markers as a tool to detect mouse cardiomyocytes injury.

Primary cultures of cardiomyocytes represent a useful model for analyzing cardiac cell biology as well as pathogenesis of several cardiovascular disorders. Our aim was to standardize protocols for determining the damage of cardiac cells cultured in vitro by measuring the creatine kinase and its cardiac isotype and lactate dehydrogenase activities in the supernatants of mice cardiomyocytes submitted to different protocols of cell lysis. Our data showed that due to its higher specificity, the cardiac isotype creatine kinase was the most sensitive as compared to the others studied enzymatic markers, and can be used to monitor and evaluate cardiac damage in in vitro assays.

Animals↗

Electrophoresis of creatine kinase isoforms: a highly sensitive fluorescence scanning method.

OBJECTIVE: To develop an agarose electrophoretic method for creatine kinase (CK) isoforms, using highly sensitive fluorescence scanning. METHODS: A discontinuous buffer system was used. Electrophoresis on agarose gel was performed under constant current and low voltage. CK isoforms were separated within 30 minutes and detected by fluorescence scanning. RESULTS: There were no significant differences when the activities of CK-MM were between 853.0 U/L and 14.0 U/L and those of CK-MB between 152.0 U/L and 2.4 U/L. The detection limits of stain method for CK-MM and CK-MB isoforms were 36.0 U/L and 12.3 U/L, respectively; while those of fluorescence method were 12.0 U/L and 2.1 U/L. The experimental results showed good precision for CK-MM isoforms, as well for CK-MB isoforms and isoenzymes. CONCLUSION: An agarose electrophoretic method has been developed to measure CK isoenzymes and isoforms clinically. This method is rapid, simple, sensitive, highly reproducible and inexpensive. It is suitable for general laboratories.

Creatine Kinase↗

Loss of myocardial CK-MB into the circulation following 3.5 hours of swimming in a rat model.

The purpose of this study was to document alterations of creatine kinase-B (CK-B) in the left and right ventricles of rats and CK-MB release into the circulation following a single bout of stressful prolonged intense exercise. Male Sprague-Dawley rats, with 8% bodyweight attached to each tail, were forced to swim 3.5 hours and were then sacrificed immediately (0 h PS), 3 hours (3 h PS), 24 hours (24 h PS), and 48 hours (48 h PS) post swimming, respectively. Sedentary (control) rats were sacrificed at rest. Serum CK-MB mass increased 2.1 times (8.9 microg/L; p < 0.01 vs. controls of 4.3 microg/L) and 1.4 times (6.0 microg/L; P < 0.01 vs. controls) at 0 h PS, and 3 h PS, respectively, and returned to baseline at 24 h PS. Western blot analysis indicated that CK-B of the right ventricle decreased 14% (p < 0.05), 20% (p < 0.01), and 12% (p < 0.05) at 3h PS, 24h PS and 48h PS, respectively. The CK-B of the left ventricles decreased 34% (p < 0.05) at 0 h PS, returned to baseline at 3 h PS, and was increased 39% (P < 0.01) at 48 h PS. Our findings demonstrate that a single bout of stressful, prolonged, intense exercise resulted in CK-B subunit loss from the myocardium, resulting in increased serum CK-MB concentrations, an indication of myocardial injury.

Animals↗

Safety of intramyocardial injection of autologous bone marrow cells to treat myocardial ischemia in pigs.

OBJECTIVE: The purpose of this study is to determine the potential adverse consequences of intracardiac injections of bone marrow mononuclear cells (BMCs) to facilitate the revascularization of ischemic myocardium. BACKGROUND: Bone marrow mononuclear cells are used to treat heart failure, though there are few studies that evaluated the safety of BMC transplantation for chronic myocardial ischemia. METHODS: The pigs received coronary ameroid constrictors to induce chronic myocardial ischemia and left ventricular dysfunction. At 4 weeks, autologous BMCs were injected intramyocardially by Boston Scientific Stiletto catheter with low-dose (10(7) cells) or high-dose BMC (10(8)). Control animals received saline. Blood samples were collected for hematological and chemical indices, including cardiac enzyme levels at regular time intervals postinfarction. At 7 weeks, animals underwent electrophysiological study to evaluate the arrhythmic potential of transplanted BMC, followed by necropsy and histopathology. RESULTS: No mortalities were associated with intramyocardial delivery of BMC or saline. At Day 0, the total creatine phosphokinase (CPK) was in the normal range in all groups. All groups had significant elevations in CPK after ameroid placement, with no significant differences between groups. At 7 weeks, CPK in all groups had returned to pretreatment levels. Electrophysiological assessment revealed that one control animal had an inducible arrhythmia. No arrhythmias were induced in low- or high-dose BMC-treated pigs. There were no histopathological changes associated with BMC injection. CONCLUSION: This study showed, in a clinically relevant large-animal model, that catheter-based intramyocardial injection of autologous BMC into ischemic myocardium is safe.

Animals↗

Isoenzyme-directed selection and characterization of anti-creatine kinase single chain Fv antibodies from a human phage display library.

Epitopes differing among isoenzymes of creatine kinase (CK) are apparently limited in number and poorly immunogenic in vivo. Especially for the BB-CK isoenzyme, very few monoclonal antibodies (mAb) are available. Here, we use in vitro selection with a synthetic human phage display antibody library and develop isoenzyme competition and peptide panning strategies to obtain human single chain Fv (scFv) antibodies against specific CK isoenzymes. We isolated and characterized seven scFv clones that recognize native as well as denatured cytosolic BB-CK in ELISA, immunoblot, immunofluorescence histochemistry and surface plasmon resonance (SPR) spectroscopy. To a variable but minor degree, they also react with cytosolic MM-CK, but not with mitochondrial CK isoenzymes. Epitope mapping revealed that the scFv antibodies recognize different BB-CK epitopes, including the N-terminus and the isoenzyme-specific box, a highly conserved sequence of unknown function for which no mAb were available so far. With a K(D) of 3.5-9.6 x 10(-7) M, the isolated scFv compare favorably with mouse mAb and may overcome certain of their limitations. Our results demonstrate the advantages of in vitro antibody selection for the generation of isoenzyme-specific antibodies.

Amino Acid Sequence↗