The objective of this review is to provide an overview of the use of biochemical markers for the detection of Central Nervous System (CNS) complications after cardiac surgery and extracorporeal circulation (ECC). A computerized literature search in MEDLINE from 1966 onward was the basis for the references. The literature covering the following biochemical markers is reviewed: adenylkinase, creatine phosphokinase isoenzyme BB (CK-BB), lactate, neuron-specific enolase (NSE), S-100 protein, myelin basic protein, lactate dehydrogenase, aspartate aminotransferase, glutathione, vasointestinal neuropeptide, and 7B2-specific neuropeptide. For clinical purposes, it is necessary to have a biochemical marker that can be measured in blood. Lactate, although a primary marker of anaerobic metabolism, and CK-BB values, calculated from the arterio-internal jugular venous difference, appear to correlate with periods of ischemia during ECC. S-100 protein levels have been shown to correlate with duration of ECC, and when combined with NSE values, could be used to identify patients with CNS dysfunction after cardiac surgery. The use of NSE may be limited by its presence in erythrocytes and platelets because the high levels that can result from hemolysis can render it less specific. Although recently introduced, S-100 protein may have the potential to be a valuable marker for CNS dysfunction after ECC.

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.

Creatinine kinase BB (CK-BB) is elevated in many tumours including those of the breast. We have recently described a new, highly sensitive and specific method for measuring CK-BB, based on monoclonal antibodies and time-resolved fluorometry. Using this method, we quantitated CK-BB in 172 breast tumour cytosols and examined the associations between CK-BB and other clinicopathological variables and patient survival. High CK-BB levels were seen more frequently in tumours from patients who were younger (age < 50 years), patients who qualified for chemotherapy and patients with oestrogen receptor-positive tumours. No association was seen between CK-BB and tumour stage, grade, size, histological type or the progesterone receptor. In univariate analysis, the risk of relapse or death was higher in the group with tumours containing high CK-BB levels but the difference did not reach statistical significance. In multivariate analysis, the risk of death was statistically significantly higher in the high-CK-BB group. Analysis of subsets of patients revealed that patients with oestrogen receptor-negative cancer have higher risk of death if their tumours contain high levels of CK-BB. Our data suggest that, in general, CK-BB is associated with more aggressive tumours but its value as a prognostic indicator is limited. CK-BB content of breast tumours may be more useful as an aid in selecting therapy directed at inhibiting this enzyme activity and thus depriving tumour cells of their energy source.

BACKGROUND

Creatine kinase (CK) exists as three cytosolic isoenzymes, CK-MM, CK-MB, and CK-BB, and one mitochondrial isoenzyme. Animal and human observations suggest that the CK-MB content of myocardium is dynamic and may increase in response to ischemia, but the response of the myocardial CK system to chronic coronary artery occlusion is not well-defined.

RESULTS

We measured serial changes in myocardial total CK, percent CK-MB, and percent CK-BB before and 3 weeks after coronary artery occlusion in 17 pentobarbital-anesthetized dogs. Tissue biopsies were obtained from the left anterior descending (LAD) coronary artery myocardium, the right coronary artery (RCA) myocardium, and the circumflex coronary artery myocardium at baseline and 3 weeks after LAD occlusion (n = 6), RCA occlusion (n = 5), and no coronary artery occlusion (n = 6). Tissue samples were assayed for total CK, percent CK-MB, and percent CK-BB. Samples were also examined by electron microscopy for evidence of ischemic myopathy. Total myocardial CK activity did not change over 3 weeks. Percent CK-MB increased significantly in the tissue supplied by the occluded artery (4.1-fold in dogs with LAD occlusion and 6.7-fold in dogs with RCA occlusion). Percent CK-BB did not change. Dogs with LAD occlusion had ultrastructural evidence of myopathic fibers interspersed with normal fibers in the LAD myocardium. Dogs with RCA occlusion had no ultrastructural evidence of myopathic fibers in the RCA myocardium.

CONCLUSIONS

Chronic coronary artery occlusion causes a pronounced change in the canine myocardial CK system that is limited to the tissue supplied by the occluded coronary artery. These biochemical alterations do not correlate with any cellular ultrastructural changes. Myocardial CK-MB content is dynamic, varies geographically within the heart, and increases rapidly after coronary artery occlusion.

Chronic overload due to an experimental abdominal aortic stenosis in rats causes hypertrophy of ventricles and a parallel increase of the MB and BB isoforms of creatine kinase (CK) in cardiac tissue. The CK isoenzyme profile was determined using a new two-step method combining anion exchange chromatography and electrophoresis. In overloaded ventricles a shift was observed from the MM isoform which decreased (from 407 +/- 10 mumol X min-1 X g-1 ww in sham-operated to 370 +/- 0.13, in the overloaded group, p less than 0.05) towards the MB and BB forms whose activity was enhanced (from 56 +/- 4 and 6.5 +/- 0.7 to 77 +/- 6 and 10.1 +/- 1.2, respectively, p less than 0.02). These modifications were more pronounced (318 +/- 15, 83 +/- 15 and 15.1 +/- 2.5, for the MM, MB and BB forms respectively, p less than 0.01) in rats having a very marked hypertrophy and whose ventricular/body weight ratio (expressed in mg of ventricles per g) was above 3. Moreover this ratio correlates both with the amount of the MB form (r = 0.32, p less than 0.05) and with the percentage of the B monomer (r = 0.51, p less than 0.01). This shift, like that previously described for lactate dehydrogenase and myosin, favoured the fetal form and this supports the hypothesis that overloaded myocytes improve their efficiency by expressing some of the isoforms normally present in the fetus.

After occlusion of the right common carotid artery in the gerbil, we monitored the progression of ischemic damage and postischemic damage and the repair process in the brain immunohistochemically by using tubulin, creatine kinase BB-isoenzyme (CK-BB), and neuron-specific enolase as the neuronal markers and astroprotein, glial fibrillary acidic protein, and CK-BB as the astrocytic markers. The earliest ischemic lesion was detected in the hippocampus and the cerebral cortex after ischemia for 5 minutes as loss of the reaction in the neuropil, nerve cell bodies, and dendrites. The reaction disappeared more promptly in the dendrites than in the nerve cell bodies. The reaction for tubulin was the most sensitive for detection of the neuronal ischemic damage. After an ischemic period of 30 minutes and subsequent reestablishment of cerebral circulation, the immunohistochemical lesions affecting the neuronal structure expanded during the first 3 hours and then slowly afterward for up to 12 hours. Reactive astrocytes were already identified 24 hours after reperfusion. The current investigation demonstrated that early ischemic damage can be clearly visualized by use of the immunohistochemical technique soon after the onset of cerebral ischemia but that considerable heterogeneity exists not only in different anatomic regions but also within the neuronal structure. This technique has potential for further investigation of cerebral ischemia or other pathophysiologic conditions when used in combination with other morphologic, physiologic, or biochemical techniques.

OBJECTIVE

Cationic lipid/DNA complexes have been proposed as a method of in vivo gene delivery via intravenous or intramuscular injection. A concern with using these polycationic molecules is whether they are associated with tissue toxicity at the injection site. Therefore, the objective of these studies was to investigate the myotoxic potential of selected non-viral gene delivery macromolecules (e.g., cationic lipids and polymers) with and without plasmid DNA (pDNA) in vitro.

METHODS

Myotoxicity was assessed by the cumulative release of creatine kinase (CK) over 90 minutes from the isolated rodent extensor digitorum longus muscle into a carbogenated balanced salt solution (BBS, pH 7.4, 37 degrees C) following a 15 microL injection of the test formulation. Phenytoin (Dilantin) and normal saline served as positive and negative controls, respectively.

RESULTS

The myotoxicity of plasmid DNA (pDNA, approximately 5000bp, 1 mg/ml) was not statistically different from normal saline. However, the myotoxicity of Dilantin was 16-times higher than either normal saline or pDNA (p < 0.05). Cationic liposomes were found to be less myotoxic than polylysine and PAMAM dendrimers. Polylysine's myotoxicity was found to be dependent upon concentration and molecular weight. The myotoxicity of formulations of cationic liposomes(s), lower molecular weight polylysine (25,000) and higher concentration of PAMAM dendrimers with pDNA were found to be statistically less significant than those formulations without pDNA.

CONCLUSIONS

The cationic liposomes were less myotoxic compared to the dendrimers and polylysine. Myotoxicity was dependent upon the type of cationic lipid macromolecule, concentration, molecular weight and the presence of pDNA. A possible explanation for this reduced tissue damage in cationic lipids complexed with pDNA is that the formation of complex reduces the overall positive charge of the injectable system resulting in less damage.

Concordant segregation of human brain-type creatine kinase, CK(BB), expression with nucleoside phosphorylase (NP) expression and the presence of chromosome 14 was observed in 53 independently derived cell hybrids between CK(BB)-positive human cells and CK(BB)-negative rodent cells. Further analysis of 26 subclones of two CK(BB)-positive human-mouse cell hybrids confirmed the positive correlation of CK(BB) expression with NP expression. The results suggest that the gene coding for human CK(BB) can be assigned to chromosome 14.

An increase in the biosynthetic rate of the brain-type isozyme of creatine kinase (CKBB, first described in the uterus as the "estrogen-induced protein") was found in the ovary, vagina and estrogen receptor-rich regions of the brain (preoptic area, anterior hypothalamus and median eminence), one hour after injection of 5 micrograms of estradiol-17 beta into 25-28 day-old rats. The increase in synthetic rate in the ovary, detected by 35S methionine incorporation, peaked at 1h, but still remained higher than in control ovaries at 6 h and was reflected in a longer-term increase in ovarian CK specific activity after 4 daily injections. Both ovary and vagina, similarly to brain, contained exclusively the BB isozyme of CK. These findings suggest that the entire female reproductive system can respond to estrogen by a rapid induction of CKBB.

Isoenzymes of creatine kinase (CK, EC 2.7.3.2) in guinea-pig smooth, cardiac and skeletal muscles as well as in brain were analyzed by cellulose acetate electrophoresis and FPLC gel permeation chromatography. In crude tissue extracts of smooth muscles brain type BB-CK and the hybrid form MB-CK were detected, but in enriched mitochondrial fractions from different guinea-pig smooth muscles, mitochondrial type Mi-CK was unambiguously identified. Smooth muscle Mi-CK displayed the same electrophoretic mobility as Mi-CK from brain, which migrates slower than cardiac Mi-CK. Identical to parallel experiments with Mi-CK from cardiac muscle and brain, smooth muscle Mi-CK could be resolved into dimeric and octameric species, the latter being remarkably stable. In contrast to guinea-pig smooth muscles, Mi-CK was not detected in chicken gizzard tissue extracts nor in enriched mitochondrial fractions thereof. The presence of Mi-CK, predominantly in octameric form, in guinea-pig smooth muscles, but not in chicken gizzard, may represent a clue for the different physiological properties of these muscles and may provide the molecular basis for the dependence of the PCr production on oxidative metabolism observed in the guinea-pig taenia caeci.

Amyloid beta-peptide (Abeta), the main constituent of senile plaques in Alzheimer's disease (AD) brain, is hypothesized to be a key factor in the neurodegeneration seen in AD. Recently it has been shown that the neurotoxicity of Abeta occurs in conjunction with free-radical oxidative stress associated with the peptide. In the present study, we investigated the temporal relations among the formation of Abeta-associated free radicals, the oxidative damage to, and the activation of antioxidant defense mechanisms in rat embryonic hippocampal neuronal culture subjected to toxic Abeta(25-35). Temporal electron paramagnetic resonance (EPR) spectroscopy results show that synthetic Abeta(25-35) forms free radicals rapidly after solubilization with a high signal intensity at initial time points. At those time points, neuronal toxicity and oxidative stress gradually increase as assessed by reduction of 3-[4,5-dimethylthiazol-2-yl)-2,5-diphenyl] tetrazolium bromide, trypan blue exclusion, formation of reactive oxygen species, and detection of protein carbonyl levels. The latter occurs before neurotoxicity. When the EPR signal intensity of Abeta solution decreases at later time points, neuronal toxicity levels off and remains the same until the end of the experiment. The oxidative-sensitive enzyme creatine kinase (CK) (brain isoform) (CK-BB) content increases at initial points of the Abeta treatment in correlation with the EPR signal to keep the CK activity constant, presumably to overcome the Abeta-induced oxidative insult. CK-BB content returns to normal levels by the end of the experiment. CK activity normalized to CK content implies the presence of inactivated CK molecules during the treatment. Both Mn SOD and Cu/Zn superoxide dismutase (SOD) mRNA levels show robust increases initially, which later return to control level with decreasing oxidative insult. These results are consistent with the notion that Abeta(25-35) promotes a rapid free-radical oxidative stress to neurons, which respond by modulating various oxidative stress-handling genes.

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.

Clinical tumor specimens and cultures of small cell lung cancer (SCLC) produce 10- to 100-fold higher quantities of the BB isoenzyme of creatine kinase (CK-BB) (EC 2.7.3.2) than did other types of lung cancer. Serum CK-BB levels were evaluated in 105 newly diagnosed, previously untreated patients with SCLC. All patients were thoroughly staged, including 42 patients with limited-stage and 63 patients with extensive-stage disease. Serum CK-BB was elevated (greater than 10 ng/ml) in 27 patients (26%) (range, 11 to 522 ng/ml; median, 40 ng/ml). Only 1 of 42 patients with limited disease had an elevated serum CK-BB, while 26 of 63 (41%) of patients with extensive disease did. When patients were subgrouped according to the number of metastatic sites detected in pretreatment staging, a significant association between the presence of an elevated serum CK-BB and the number of metastatic sites was observed (p less than 0.005). No association between the presence of metastatic disease in a specific site and an elevated serum CK-BB could be detected. After adjusting for the number of metastatic sites, survival among patients with a normal pretreatment CK-BB was significantly better than in patients with an elevated CK-BB (p = 0.014). Sequential serum CK-BB determinations in 33 patients revealed an excellent correlation between clinical response to therapy and serum CK-BB levels. Continuous SCLC cell lines established from 13 patients in this study all expressed high levels of CK-BB. These data suggest that serum CK-BB determinations may be of value in estimating the extent of tumor dissemination, assigning prognosis, and monitoring response to therapy in patients with SCLC.

Creatine kinase isoenzyme BB (CK-BB) is found in high concentrations in the brain. Normally concentrations in blood are undetectable or low. Blood concentrations of CK-BB were measured in 16 boxers before and after a fight and in 16 track cyclists before and after a race. Blood CK-BB rose to significantly higher concentrations in the boxers than in the cyclists. The number of blows received to the head was estimated in half the boxers and correlated significantly with the rise in CK-BB. This increase in blood CK-BB may indicate disruption of the blood-brain barrier. This may be one of the mechanisms accounting for brain damage in boxers.

The activity and role of creatine kinase (CK) associated with contractile proteins of smooth muscle have been investigated using skinned guinea-pig taenia coli fibers. Total CK activity was 163 +/- 22 IU/g (ww) and agarose electrophoresis showed BB, MB, and MM isoforms (BB-CK being the predominant isoenzyme). After skinning for 1 h with Triton X-100, BB-CK was specifically associated with the myofibrils, representing 22% of the preskinned CK activity. When relaxed fibers were exposed to pCa 9 in the presence of 250 microM ADP, 0 ATP and 12 mM PCr, tension was not significantly different from resting tension, but changing to pCa 4.5 caused the fibers to generate 59.1 +/- 5.2 percent of maximal tension. When a high-tension rigor state was achieved (250 microM ADP, 0 ATP, 0 PCr, and pCa 9), the addition of 12 mM PCr effected significant relaxation. These observations implicate an endogenous form of BB-CK, associated with the myofilaments and capable of producing enough ATP for submaximal tension generation and significant relaxation from rigor conditions. It was also shown that ADP is bound to the myofibrils and available for rephosphorylation by BB-CK. These results suggest co-localization of ATPase, MLCK and CK on the contractile proteins of the taenia coli. This enzymic association may play a role in the compartmentation of adenine nucleotides in smooth muscle.

Creatine kinase (EC 2.7.3.2) B-subunit activity in serum may be routinely measured as residual activity after specific immunoinhibition of the M-subunit. We assessed the inhibition kinetics, specificity, completeness of inhibition, and inhibitory capacity of three different anti-M preparations, with use of isolated human BB, MM, and MB isoenzymes. The Scandinavian-recommended reaction system was used. We suggest a set of tentative quality requirements for anti-M for use in diagnosing acute myocardial infarction. The need to measure and subtract sample residual adenylate kinase activity was demosntrated. We describe a routine photometric method for determining B-subunit activity in serum. With the Scandinavian CK method the upper reference value for total creatine kinase in serum was found to be 150 U/L for women, 270 U/L for men. By bioluminescence, we found the upper reference value for B-subunit activity to be 6 U/L for both sexes. We discuss three different modes for applying B-subunit determinations to the diagnosis of acute myocardial infarction.

We have compared the levels of creatine kinase (CK) activity and the distribution of CK isoenzymes determined by agarose gel electrophoresis in normal colon, liver and lung tissues, and in colon, liver and lung adenocarcinomas, lung squamous cell carcinomas and lung carcinoids. Colon and lung adenocarcinomas, and squamous cell carcinomas presented lower CK activity than the normal tissues and no differences were found between hepatocarcinoma and normal liver tissue. In contrast, lung carcinoids had higher CK activity than normal lung tissue. Type BB-CK was the predominant isoenzyme in normal lung, colon and liver tissues. Type MM isoenzyme was detected in normal lung and type MB-CK was found in normal colon. In most lung tumours the CK isoenzyme electrophoretic pattern did not change. However, no type BB-CK was detected in some hepatocarcinomas, type MM-CK decreased in lung carcinoids and type MB isoenzyme was not observed in colon adenocarcinomas. It is concluded that in most tumours there is a decrease in the expression of type B- and type M-CK subunits, whereas in lung carcinoid the expression of type B-CK activity increases. Thus, the increase in type BB-CK observed in the serum of patients with lung and colon adenocarcinomas is probably due mainly to enhanced enzyme release as a result of tumour cell necrosis.

We compared results for measurements of creatine kinase isoenzyme MB (CK-MB) by immunoinhibition vs immunoprecipitation, using sera from 53 normal healthy individuals, 55 patients with increased CK-MB associated with acute myocardial infarction, and 42 patients whose blood exhibited one or more abnormal forms of CK by electrophoresis. These last 42 patients, selected from a group of 91 cases exhibiting abnormal forms as detected in a screening of 5000 hospitalized and clinic patients, include: (a) CK-BB bound to IgG (macro CK type 1), (b) a polymeric complex of mitochondrial CK (macro CK type 2), (c) abnormally high activity of free CK-BB isoenzyme, and (d) persistent increases of CK-MB from patients without myocardial infarction. These abnormal forms occur in less than 2% of all patients and are exceedingly rare in patients with acute myocardial infarction. Therefore, the vast majority of CK-MB analyses can be performed rapidly and efficiently by immunoinhibition, which has analytical sensitivity, is associated with high clinical sensitivity, and is easily automated for a low cost per test. In contrast, immunoprecipitation is a more specific analytical measurement of CK-MB but is less efficient and more costly.

We assessed the relationship between phosphocreatine (PCr) and creatine (Cr) content and creatine kinase (CK) activity in skeletal muscle of mice. The PCr and total Cr (tCr) concentrations, as well as CK activity, in hindlimb muscles of mice, with or without the cytosolic and mitochondrial isoforms of muscle creatine kinase (wild-type or CK--/-- mice), were determined by in vivo magnetic resonance (MR) spectroscopy and by biochemical means during postnatal growth and adulthood. In wild-type muscle the [tCr], PCr/ATP ratio and CK activity increased rapidly in the first 4-7 weeks. Remarkably, CK--/-- mice showed a similar increase in the PCr/ATP ratio during the first month in the presence of only minor brain-type BB-CK activity. Uptake of Cr in muscle was seemingly unrelated to CK activity as tCr increased in the same way in the muscles of both mouse types. At older ages the PCr/ATP ratio decreased in CK--/-- muscles, in contrast to wild-type where it still slowly increased, whereas [tCr] was similar for muscle of both mouse types. Using a new in vivo MR approach with application of [4-13C]Cr, a lower PCr/tCr ratio was also observed in CK--/-- muscle. From these data it follows that in vivo global ATP levels at rest are similar in the presence or absence of CK. Although Cr could still be converted to PCr in mature CK--/-- muscle, the immediate availability of PCr decreased, and PCr became partly inconvertible at older ages. Apparently, catalysis of the CK reaction by BB-CK, although significant in muscles of newborn mice, gradually declines to very low levels in adulthood. Part or all of this BB-CK may arise from satellite cells fusing with myotubes, a process that is most active during the first months of life. Finally, our observation that the MR and chemical assessment of muscle [tCr] and PCr/tCr ratio were similar for all mice does not support the existence of a significant MR-invisible or immobile pool of Cr, with a role for CK in this phenomenon.

Concentrations of several proteins that are characteristic of the nervous system were time-sequentially analyzed by radio- and enzyme-immunoassay in the cerebrospinal fluid (CSF) of patients with Creutzfeldt-Jakob disease (CJD). We found abnormally high levels of several proteins, such as neuron-specific enolase (NSE), S-100b protein, brain-type isozyme of creatine kinase (CK-BB) and alpha subunit of GTP binding protein G0 (G0 alpha) in the early stage of the disease. Generally, these protein levels were far higher in CJD patients than in normal controls and other neurological patients in the early stage before the typical clinical manifestations were evident. These levels increased to maxima when the disease activity was most prominent and returned to normal or mildly elevated levels in the terminal stage. The results imply that these protein levels can serve as biochemical markers for the presence of an active destructive process in CJD brain and provide us with a useful indicator for early diagnosis of CJD.

Two-dimensional electrophoretic analysis of crude microtubule preparations from the rat brain revealed the presence of three polypeptides in positions corresponding to those of the isovariants of purified rat brain creatine kinase (CK-BB). By the use of [gamma-32P]ATP, the two more acidic forms of these polypeptides were shown to be phosphorylated. Their identity as phosphorylated forms of CK-BB was established by using various peptide mapping techniques. Thus CK-BB is a phosphoprotein and its isoelectric variation may be attributed to phosphorylation.

Total creatine kinase (CK) and CK MB activities were determined in gastrocnemius muscle and serum obtained from 14 female marathon runners. The level of CK MB in muscle increased significantly (p less than 0.05) after chronic exercise training from 5.3% to 10.5% of the total CK activity, but not after acute exercise (post-marathon 8.9%). No significant differences in total CK activities were detected. However, the total CK activity in the muscles were significantly (p less than 0.05) less than those previously reported from the muscle of men runners (1800 U/g, 3000 U/g respectively). No significant correlation existed between fiber type and muscle CK MB activity. Additionally, trace amounts of mitochondrial CK and CK BB were present in muscle homogenates. A significant correlation was observed in the increase in mean serum total CK (597 UL-1) and CK MB (23 UL-1) activities 24 h after the race (r = 0.97, p less than 0.05). These results suggest that gastrocnemius muscle in women adapts to training with increased CK MB activities and imply that skeletal muscle is the major source of elevated serum CK MB activities in women marathon runners.

In higher eukaryotes three different types of creatine kinases (CK) are expressed: the muscle-specific M-CK, the ubiquitous cytoplasmic B-CKs, and the mitochondrial Mi-CKs. They fulfill multiple tasks in cells with an intensive energy metabolism. Isolated chicken B-CK can be resolved by two-dimensional gel electrophoresis into a major acidic Ba-CK and a major basic Bb-CK protein species which are very likely produced from the unique chicken B-CK gene (Wirz, T., Hossle, J. P., Soldati, T., and Perriard, J.-C. (1989) Experientia (Basel) 45, 32 (abstr.]. However, close inspection of the gels indicates the presence of additional B-CK species. This additional heterogeneity is generated by two distinct post-transcriptional processes. Post-translational phosphorylation was shown to contribute to heterogeneity of both Ba- and Bb-CK isoproteins and appears to modulate their enzymatic activity (A. F. Q. Quest, H. M. Eppenberger, and T. Wallimann, manuscript in preparation). Alternative ribosomal initiation of Bb-CK synthesis at multiple sites was shown to occur in cell free systems as well as in vivo, resulting in proteins differing in the length of their amino termini. Using site-directed mutagenesis to "switch off" each of the first four methionine codons of a full length Bb-CK cDNA, we were able to correlate each protein product with one distinct translational start site. An additional protein species appears to be produced by initiation at a noncanonical start codon. We propose that a leucine codon may be used as a translational start site. Evidence is presented to support the role of these amino-terminal truncated subunits in the regulation of the enzyme.

OBJECTIVE

Compound K (CK), an intestinal metabolite of ginsenosides, has pharmacological properties such as anti-angiogenesis, anti-inflammation, anti-platelet and anti-cancer activities. In the present study, we investigated the inhibitory effect of CK on vascular smooth muscle cell (VSMC) proliferation and migration in vitro and neointima formation in a rat carotid artery injury model.

RESULTS

CK significantly inhibited both the proliferation and migration of PDGF-BB-stimulated VSMCs in a concentration-dependent manner. In accordance with these findings, CK blocked the PDGF-BB-induced progression of synchronized cells through the G0/G1 phase of the cell cycle. CK also decreased the expressions of cell cycle-related proteins, including cyclin-dependent kinase (CDK) 2, cyclin E, CDK4, cyclin D1, and proliferative cell nuclear antigen (PCNA) in response to PDGF. However, CK did not affect early signal transduction through PDGF-Rβ, Akt, ERK1/2 and PLC-γ1 phosphorylation. CK attenuated PDGF-BB-induced VSMC migration by inhibiting MMP-2 and MMP-9 expression. Furthermore, the CK-treated groups showed a significant reduction in neointima formation vs. the control group. Immunohistochemical staining demonstrated decreased expression of PCNA in the neointima of the CK-treated group.

CONCLUSIONS

Our findings demonstrated that CK was capable of suppressing the abnormal VSMC proliferation and migration. It suggested that CK can be a therapeutic agent to control pathologic cardiovascular conditions such as restenosis and atherosclerosis.