We report the development of a double-antibody system for radioimmunoassay of CK-B subunit in isoenzymes CK-MB and CK-BB of creatine phosphokinase. With our method, 4.1 ng/ml of isoenzyme CK-BB can be detected. Within-run and between-run coefficients of variation are respectively 5.3% and 19.6% Analytical recovery of added antigen is 98.9 +/- 14.7%. The CK-B concentration in sera from 20 healthy adults was less than 75 ng/ml. In sera from 25 patients with a rise in CK-total activity, but without CK-MB or CK-BB activity tested with the previous reported immunological methods, we found CK-B concentrations in the range 0--86 ng/ml. In contrast, in 23 sera from patients with acute myocardial infarction within the last 48 hours CK-B concentration was in the range 104--225 ng/ml.

Combined somatosensory evoked potential (SEP) and regional brain oxygen saturation (rSO(2)) monitoring and simultaneous measurement of plasma levels of S100Beta and creatine kinase-isozyme BB (CK-BB) were performed to evaluate how reliable these diagnostic modality complexes are in the early prediction of neurological complications after surgery. Between 1999 and 2002, intraoperative SEP and rSO(2) monitoring combined with measurements of S100Beta and CK-BB levels in blood were performed in 82 consecutive patients undergoing cardiovascular operations with cardiopulmonary bypass (CPB). Twelve (14.6%) of these patients were diagnosed as having neurological complications after surgery; seven with transient neurological dysfunction (8.5%), and five with permanent stroke (6.1%). Twenty one of 82 patients in whom rSO(2) was recorded were judged abnormal; however, only nine of the 21 (42.9%) were diagnosed as having brain damage - diagnostic sensitivity and specificity being 75.0% and 82.9%, respectively. All six patients who showed abnormal SEP during surgery had neurological complications, but normal SEP was recorded in six other patients with apparent evidence of neurological complications - diagnostic sensitivity and specificity being 50% and 100%, respectively. There were no significant differences in S100Beta levels between patients with and without brain complications at 1 h and 24 h after CPB, but significant differences were detected in CK-BB levels at 24 h after CPB. In conclusion, simultaneous abnormalities detected in SEP and rSO(2) are highly predictive of cerebral neurocirculatory disturbances, but they are not so sensitive in diagnosing restricted focal cerebral lesions. Additional determinations of blood CK-BB levels might be valuable only to confirm the newly established brain complications.

Prolonged ischemia of skeletal muscle tissue, followed by reperfusion, leads to ischemia/reperfusion injury (IRI), which is a feared local and systemic inflammatory reaction. With respect to the 3Rs, we wanted to determine which parameters for assessment of IRI require a reperfusion time of 24 h and for which 2 h of reperfusion are sufficient. Rats were subjected to 3 h of hind limb ischemia and 2 h or 24 h of reperfusion. Human plasma derived C1 inhibitor was used as a drug to prevent reperfusion injury. For 2 h of reperfusion the rats stayed under anesthesia throughout (severity grade 1), whereas for 24 h they were awake under analgesia during reperfusion (grade 2). The femoral artery was clamped and a tourniquet was placed, under maintenance of venous return. C1 esterase inhibitor was systemically administered 5 min before the induction of ischemia. No differences in local muscle edema formation and depositions of immunoglobulin G and immunoglobulin M were observed between 2 h and 24 h (P>> 0.05), whereas lung edema was only observed after 24 h. Muscle viability was significantly lower after 24 h vs 2 h reperfusion (P < 0.05). Increased plasma creatine kinase (CK)-MM and platelet-derived growth factor (PDGF)-bb could be detected after 2 h, but not after 24 h of reperfusion. By contrast, depositions of C3b/c and fibrin in muscle were only detected after 24 h (P < 0.001). In conclusion, for a first screening of drug candidates to reduce IRI, 2 h reperfusions are sufficient, and these reduce the severity of the animal experiment. Twenty-four-hour reperfusions are only needed for in-depth analysis of the mechanisms of IRI, including lung damage.

Effects of an exhaustive eccentric exercise (EE) on the motor control of maximal velocity rhythmic elbow extension/flexion movement (RM) were examined in eight male students. The exhaustive EE consisted of 100 maximal eccentric actions of the elbow flexor muscles. Movement range was 40-170 degrees in EE at an angular velocity of 2rads(-1). A directive scaled RM of 60 degrees with visual feedback was performed in a sitting position, with the right forearm fixed to the lever arm in horizontal plane above protractor. Surface electromyographic activity (EMG) was recorded from the biceps brachii (BB) and triceps brachii (TB) muscles. Maximal isokinetic eccentric and concentric tests and RM test were conducted before, after, 0.5h, 2 days and 7 days after the exercise. Dynamic force production was deteriorated after EE (P<.001), and did not recover fully within 7 days. The delayed recovery phase was characterized by delayed onset of muscle soreness (DOMS) and elevated serum creatine kinase (CK) activity. The RM test revealed a delayed increase of the fatigued BB muscle EMG activity, but the maximal RM velocity could be preserved. The present results emphasize the capacity of the neuromuscular system to compensate for prolonged eccentric-induced contractile failure by optimizing antagonistic muscles coordination in a demanding rhythmic task. The underlying compensatory mechanisms could be related to increased sensitization of small diameter muscle nerve endings.

Previous studies have demonstrated that brain protein synthesis declines after global ischemia and reperfusion. To investigate the role of the translation system in this phenomenon, we examined the ability of partially purified ribosomes, ribosome-bound mRNA and translation cofactors derived from the transiently ischemic cerebral cortex to synthesize protein in vitro. Samples were prepared from canines subjected to 20-min cardiac arrest and after 2 or 8 h of post-resuscitation intensive care. There was no significant decrease in the rate of in vitro protein synthesis as a consequence of either ischemia or reperfusion. Northern hybridization of ribosome-bound RNA revealed a discrete band of mRNA for brain-specific creatine kinase (ck-bb) that was consistent in presence and intensity in all groups. However, mRNA for heat shock 70 protein (hsp-70) was observed only during reperfusion and markedly increased between 2 and 8 h reperfusion. Thus, we conclude that (1) the transcription system is intact during reperfusion and hsp-70 mRNA is made and translocated to the ribosomes during reperfusion, (2) mRNA for ck-bb is not displaced from ribosomes by the appearance of hsp-70 during reperfusion and (3) isolated ribosomes maintain their ability to translate in vitro during the first 8 h of reperfusion after global brain ischemia. Therefore, the early reduction in protein synthesis observed in vivo during post-ischemic brain reperfusion is not due to an intrinsic dysfunction of the ribosomes.

CK-isoenzymes were measured in 31 patients hospitalised for suspected myocardial infarctions who had an increase in serum creatine kinase (CK) above 50 U/l. Of 26 patients with definite evidence of myocardial infarction, MB-isoenzyme--specific for myocardial necrosis--was demonstrated in 24. MB-isoenzyme was no longer detectable in two patients hospitalised 48 hours after the onset of symptoms. In the remaining five patients only MM-isoenzyme was found, the elevated CK activity in three patients having been due to an intramuscular injection, and in two others due to pulmonary embolism. Measurement of CK isoenzymes proved of great diagnostic value in three patients with sudden circulatory arrest of, at first, unknown cause after successful resuscitation. Acute myocardial infarction was proven by the presence of MB-isoenzyme. In one of these patients an additional BB-isoenzyme was seen, possibly due to concomitant cerebral ischaemia. In all other patients (with angina, after cardioversion, or after major surgical operations) only MM-isoenzyme was detected. MB-CK-isoenzyme was found to be a highly specific, as well as sensitive, indicator of myocardial necrosis. This being a rather difficult method, its use is not justified in the routine diagnosis, but in doubtful instances its value can hardly be overestimated.

OBJECTIVE

The analytical and clinical performance of the Evidence Cardiac Panel were evaluated.

METHODS

The Evidence Cardiac Panel, an automated protein biochip microarray system, allows the simultaneous determination of creatine kinase MB (CK-MB), myoglobin (MYO), glycogen phosphorylase BB (GPBB), heart-type fatty acid-binding protein (H-FABP), carbonic anhydrase III (CA III), cardiac troponin I (cTnI). Precision: 3 levels of quality control (QC) and 2 in house pools (P) were assayed. Method comparison: MYO and cTnI concentrations measured on Evidence (E) and on Dimension RxL (D) analyzers were compared. Clinical study: 132 non-consecutive patients admitted to the Emergency Department for chest pain were enrolled.

CONCLUSIONS

The between-day imprecision was CK-MB=6.80-10.08%; MYO=5.36-16.50%; GPBB=6.51-12.12%; H-FABP=6.26-12.63%; CA III=6.98-13.61%; cTnI=6.02-9.80%. Method comparison: E-MYO vs. D-MYO, Bias=-29.22, 95% CI from -40.25 to -18.18; E-cTnI vs. D-cTnI, Bias=-2.75, 95% CI from -4.04 to -1.46. In patients studied (at discharge: AMI, acute myocardial infarction n=42; non-AMI, n=90) H-FABP showed the highest accuracy (ROC analysis, AUC=0.92) and "cTnI+H-FABP" the greatest diagnostic efficacy (89.4%) in AMI diagnosis.

To investigate brain changes in induced deep core hypothermia (18 degrees C) with or without circulatory arrest, four groups of dogs were subjected to cardiopulmonary bypass (CPB) under the following conditions: (1) differential head perfusion with pulsatile flow and simultaneous circulatory arrest to the rest of the body; (2) differential perfusion to the head with a nonpulsatile flow; (3) total circulatory arrest; and (4) continuous hypothermic perfusion. Parameters analyzed were: (1) blood flow distribution; (2) creatine kinase isoenzyme (CK-BB) elevation in the cerebrospinal fluid (CSF) and in the brain venous return; and (3) microscopy of the brain in animals killed at 30 minutes, 24 and 48 hours, 1 and 2 weeks, and 1 month. Although minor brain tissue flow differences were found at 37 degrees C among the groups, flows equalized at 18 degrees C. A significant seven-fold brain flow increase followed the period of circulatory arrest in Group III. Rise of CK-BB levels occurred in brain venous return but not in CSF in all groups. Microscopic cellular damage appeared in all groups with an equal degree of severity, regardless of the method of hypothermia and perfusion implemented.

The antigenic and physical properties of several representative invertebrate phosphagen kinases have been examined in order to further characterize the relationship between taxonomic assignment, quaternary protein structure and evolution of this class of enzymes. Antibodies against dimeric arginine kinase from the sea cucumber cross-reacted with dimeric arginine kinase purified from sea urchin eggs, but failed to react with extracts from any species known to contain monomeric arginine kinase. However, strong immunoreactivity was observed when antibodies against purified dimeric arginine kinase were reacted with pure creatine kinase from the human muscle (CK-MM) and brain (CK-BB) as well as extracts from several species known to contain dimeric creatine kinase. Of particular interest with regard to evolution of the phosphagen kinases, we confirm the presence of creatine kinase activity in the very primitive sponge Tethya aurnatium and detect a reaction with antibodies against dimeric, but not monomeric, arginine kinase. This observation is consistent with recent studies of phosphagen kinase evolution. Substrate utilization was very specific with creatine kinase using only creatine. Arginine kinase catalyzed phosphorylation of arginine but enzymes from several species could also phosphorylate canavanine. No activities were detected with d-arginine. Isoelectric points, evaluated for several pure arginine kinases suggest that generally the monomeric forms are more acidic than the dimeric proteins. Heat inactivation of arginine kinase in several species indicated a wide range of stabilities, which did not appear to be correlated with quaternary structure, but rather distinguished by the organism's environment. On the other hand, homodimeric arginine kinase proteins from species inhabiting disparate environments are sufficiently homologous to form a catalytically active hybrid.

OBJECTIVE

To determine whether biochemical markers can selectively identify those intoxicated patients with presumed minor head injuries who are likely to have CT evidence of intracranial injury.

METHODS

Patients presenting to the ED with simultaneous presumed minor head trauma and ethanol intoxication were prospectively entered into this cross-sectional study. Following phlebotomy, all patients received cranial CT. Associations between the presence of an abnormal CT scan for injury and serum levels of the following biochemical markers were sought: serum catecholamines, creatine kinase-brain band (CK-BB), and serum amylase. Serum levels are reported as mean +/- SEM.

RESULTS

Nine of the 107 patients (8.4%; 95% CI 3.9-15.4%) had evidence of intracranial injury on CT. Mean serum CK-BB (16.1 +/- 3.7 vs 13.2 +/- 9.6 ng/mL), serum norepinephrine (913 +/- 117 vs 1,089 +/- 76 pg/mL), and serum amylase (64.9 +/- 14.8 vs 84 +/- 4.7 U/L) levels were not significantly different in patients with and without CT evidence of intracranial injury, respectively. Mean serum epinephrine (298 +/- 54 vs 167 +/- 18 pg/mL; p = 0.03) and serum dopamine (218 +/- 50 vs 130 +/- 9 pg/mL; p = 0.014) levels were significantly elevated in the group with intracranial injury on CT. A threshold level of serum dopamine>> or = 140 pg/mL yields a sensitivity of 89% (95% CI 52-100%) and a specificity of 80% (95% CI 70-87%) for CT-evident injury. A threshold level of serum epinephrine>> or = 218 pg/mL yields a sensitivity of 89% (95% CI 52-100%) and a specificity of 80% (95% CI 70-87%) for CT-evident injury.

CONCLUSIONS

Elevated serum epinephrine and dopamine levels are associated with intracranial CT-evident injury for ethanol-intoxicated patients with presumed minor head injuries. The potential use of these biochemical markers to guide a more selective approach to cranial CT scanning warrants further evaluation.

In the present study, 78 patients with a blunt thorax trauma were examined. Fifty-four percent of the patients had been injured in traffic. Creatinine kinase (CK) and its isoenzymes (MM, MB and BB), ASAT, ALAT, ECG and thorax X-ray were taken. In 24% of the patients examined, the activity of serum MB isoenzyme was greater than or equal to 6% of the total activity of CK, and in 40% no MB activity was observed. Pathological ECG changes were detected in 89% in the MB greater than or equal to 6% group and in 32% in the MB = 0% group. ECG changes cannot be regarded as specific indicators of cardiac contusion. Arrhythmias, conduction defects, ST segment and T wave changes, and a pathologically long QTc interval were present significantly more often (P less than 0.01) in the MB greater than or equal to 6% group than in the MB = 0% group. Roentgenologically, pulmonary contusion, cardiac dilatation and venous congestion-pulmonary edema were diagnosed more frequently in the MB greater than or equal to 6% group (P less than 0.01) than in the MB = 0% group. Eleven of the patients died. A forensic autopsy was performed on these patients, and a macroscopic heart injury was detected in five of them. Serum MB activity had been found in all these five.

Creatine kinase a key enzyme in cellular energy homeostasis of vertebrates offers the promise of engineering plants with enhanced stress tolerance. In order to provide plants with such an energy buffering system, tobacco was transformed with a cDNA, encoding the cytosolic brain-type isoform of chicken creatine kinase (BB-CK), the expression of which was under the control of the cauliflower mosaic virus 35S (CaMV 35S) promoter. Transgenic tobacco plants were selected and suspension cultures generated. Both transgenic plants and suspension cultures were shown to stably express enzymatically active BB-CK in vitro and in vivo, and in most cases for three successive generations (T0-T2). Exogenously supplied creatine was shown to enter the plant cells and resulted in only a slight reduction in root growth at concentrations up to 10 mM. Furthermore, the BB-CK expressing tobacco plants and cell suspension cultures were able to convert creatine into phosphocreatine.

We have compared the cell and tissue selective estrogenic and antiestrogenic activities of tamoxifen, raloxifene, ICI 164,384 and a permanently ionized derivative of tamoxifen--tamoxifen methiodide (TMI). This non-steroidal antiestrogen has limited ability to cross the blood brain barrier and is therefore less likely to cause the central nervous system disturbances caused by tamoxifen. We have used the stimulation of the specific activity of the "estrogen induced protein", creatine kinase BB, as a response marker in bone, cartilage, uterine and adipose cells and in rat skeletal tissues, uterus and mesometrial adipose tissue. In vitro, TMI, tamoxifen and raloxifene mimicked the agonistic action of 17beta-estradiol in ROS 17/2.8 rat osteogenic osteosarcoma, female calvaria, and SaOS2 human osteoblast cells. In Ishikawa endometrial cancer cells, tamoxifen showed reduced agonistic effects and raloxifene showed no stimulation. However, as antagonists, tamoxifen and raloxifene were equally effective in Ishikawa or SaOS2 cells. In immature rats, all four of the antiestrogens inhibited estrogen action in diaphysis, epiphysis, uterus and mesometrial adipose tissue; when administered alone, tamoxifen stimulated creatine kinase (CK) specific activity in all these tissues. Raloxifene and TMI, however, stimulated only the skeletal tissues and had no stimulatory effect in the uterus or mesometrial fat, and the pure antiestrogen ICI 164,384 showed no stimulatory effect in any of the tissues. The simultaneous injection of estrogen, plus an antiestrogen which acted as an agonist, resulted in lower CK activity than after injection of either agent alone. These differential effects, in vivo and in vitro, may point the way to a wider therapeutic choice of an appropriate antiestrogen which, although antagonizing E2 action in mammary cancer, can still protect against osteoporosis and cardiovascular disease and not stimulate the uterus with its attendant undesirable changes, or interfere with the beneficial action of E2 in the brain.

OBJECTIVE

It is still unclear whether cerebral perfusion is affected during off-pump coronary bypass grafting (OPCABG). We investigated the predictive value of the neurobiochemical markers of brain damage and cerebral perfusion in relation to early neuropsychological outcome after OPCABG.

METHODS

We performed OPCABG in ten patients (mean age, 63.4 +/- 5.5 years). A 5.5 F oximetric catheter was placed in the jugular bulb to continuously measure jugular oxygen saturation (SjO(2)) during OPCABG. We also examined the activity of daily living (ADL) index and performed the Mini-Mental State Examination (MMSE) to assess neuropsychological state preoperatively and 7 days postoperatively. Venous serum levels of neuron-specific enolase (NSE) and brain-specific creatine kinase (CK-BB) were measured preoperatively and 24 h after skin closure.

RESULTS

The mean arterial blood pressure and the SjO(2) during anastomosis of the left circumflex coronary artery (Cx) were significantly lower than that of the left anterior descending coronary artery (LAD) (P < 0.001). None of the patients died. There was no transient or permanent neurologic deficit. Cognitive decline was evident in two patients with a low SjO(2) and a high postoperative NSE level. The postoperative CK-BB value was normal in all patients.

CONCLUSIONS

Monitoring intraoperative continuous cerebral oxygen desaturation and postoperative NSE levels could be useful for predicting early neuropsychological outcome after OPCABG.

Searching for alternatives to the imprecise spectrophotometric tests for low-concentration creatine kinase (EC 2.7.3.2) isoenzyme MB (CK-MB), we investigated the analytical performance of two potentially superior approaches--a bioluminescent immunoinhibition assay (I, LKB-Wallac) and an ELISA (enzyme-labeled immunosorbent assay) technique (II, Hybritech)--in comparison with an electrophoretic method (III, Beckman). Only I showed good between-day precision (CV 8.3%) at the upper reference limit, allowing reproducible assay of CK-B subunit activity down to at least 3 U/L. In conditions where CK isoenzyme assays remained unaffected by CK-MM concentrations, test results were proportional to the amount of CK-MB in the sample up to at least 50 U/L for I, 120 micrograms/L for II, and 100 U/L for III (r greater than 0.998 by linear regression analysis). For CK-MB-positive samples, the data by I correlated more closely with values by III (n = 24; r = 0.994) than did results by II (n = 15; r = 0.909), but both methods were equally effective in discriminating between samples with or without electrophoretically supranormal CK-MB activity (93% sensitivity). II was entirely CK-MB specific, whereas CK-B activity by I was consistently (18/18) increased in CK-MB-negative samples containing CK-BB (n = 6; r = 0.996) or macro CK, types 1 or 2 (n = 12; r = 0.930). I is highly sensitive for screening for increased non-MM CK activity, the nature of which should be subsequently clarified by electrophoresis.

The 600 X g particulate fraction, obtained from the homogenates of human heart muscle, contained large quantities of an atypical creatine kinase (CK-Z). Creatine kinase Z migrated cathodically relative to CK-MM on agarose gel electrophoresis, and was not inhibited by antibodies directed against human CK-MM and CK-BB. Creatine kinase Z had an apparent Km for Mg-ADP and creatine phosphate of 0.04 mmol/l and 1.3 mmol/l, respectively. This enzyme existed in two molecular forms; one form of molecular weight 33 000-38 000 in the presence of a buffer containing Tris-HCl (0.05 mol/l), EDTA (0.001 mol/l), and 2-mercaptoethanol (0.010 mol/l), pH 8.0; and another form having a molecular weight of 62 000-68 000 in the presence of a buffer containing sodium phosphate (0.020 mol/l) and EDTA (0.001 mol/l), pH 8.0. Creatine kinase Z had biochemical properties which were different from those of the other soluble creatine kinase isoenzymes (MM, MB and BB), but similar to those reported for mitochondrial creatine kinases isolated from other animal tissues.

Duchenne muscular dystrophy (DMD) is a progressive, lethal X-linked neuromuscular disorder with an average worldwide incidence of 1:5000. Blood spot creatine kinase (CK) enzyme assays previously used in newborn screening programs for DMD are nonspecific because measured CK enzyme activity is attributable to 3 isoenzyme forms of CK (CK-MM, CK-MB, and CK-BB) and it is the CK-MM isoform that is found predominantly in skeletal muscle. CK-MM is increased in boys with DMD owing to muscle damage. We describe a sensitive and specific automated immunoassay for CK-MM to screen for DMD in blood spots.

The prototype assay was developed on the PerkinElmer GSP® analyzer to enable high-throughput screening. CK-MM was assayed using a solid phase, 2-site immunofluorometric system. Purified human CK-MM was used to create calibrators and controls.

The limit of blank (LOB), detection (LOD), and quantification (LOQ) values were <1, 3, and 8 ng/mL, respectively. The analytical measurement range was 4-8840 ng/mL. Interassay (n = 40) imprecision was <7% across the analytical range. Cross-reactivity was <5% for CK-MB and 0% for CK-BB. The mean recovery of CK-MM was 101% (range 87%-111%). Blood spots from newborn infants (n = 277) had a mean CK-MM concentration of 155 ng/mL and a 99th centile of 563 ng/mL. The mean blood spot CK-MM concentration from 10 cases of DMD was 5458 ng/mL (range 1217-9917 ng/mL).

CK-MM can be reliably quantified in blood spots. The development of this CK-MM assay on a commercial immunoassay analyzer would enable standardized and high-throughput newborn blood spot screening of DMD.

The effects of electroconvulsive therapy (ECT) on serum levels of the acute-phase reactant C-reactive protein (CRP) and intracellular enzymes such as alkaline phosphatase (ALP), lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatine kinase (CK), have received little attention. If brain cells are damaged, CK-BB, LDH and AST levels are expected to show (minor) elevations. We measured serum levels of prolactin, AST, ALT, LDH, ALP, CK and CRP before and 5 min, 30 min, 4 h, 1 day, 2 days, and 3 days after ECT in 15 consecutive patients (eight women and seven men; mean 53.9 years old, range 3082) who did not receive ECT in the preceding 2 weeks. Prolactin levels increased (P = 0.001), but none of the other mean concentrations significantly increased over time. All concentrations remained within the normal range in every patient, except for five samples with elevated CK levels (range 333-675 IU/l). CK-MB and CK-BB fractions, however, remained low, indicating that skeletal muscle was the source of the CK elevation. Serum levels of markers of brain cell leakage and inflammation remained low following one ECT session, suggesting that ECT does not cause direct brain cell leakage, nor an inflammatory response.

Creatine kinase BB isoenzyme (CK-BB) was detected in abnormal amounts in serum samples from 11 of 46 patients with Stage D carcinoma of the prostate by electrophoresis. Thirteen of 46 Stage D patients had elevated acid phosphatase values and 10 of these 13 had elevated CK-BB. CK-BB elevations were less frequent in earlier stages of prostatic cancer; Stage C: 0 of 35, Stage B: 1 of 26, Stage A: 0 of 3 and none in a group of 35 with BPH, prostatitis and bladder cancer. Results of CK-BB by a specific radioimmunoassay correlated well with those obtained by electrophoresis in most cases. Several patients were followed over time and data on CK-BB is presented for this interval. The origin of the CK-BB is still unclear. The BB isoenzyme predominates in prostatic tissue and CK-BB is the fetal form of the enzyme in human muscle and myocardium. The increase in serum CK-BB may be related to increased release of the isoenzyme, either from the prostate itself or from a metastatic lesion, or may represent a release of the fetal form of the enzyme from dedifferentiated tumor tissue.

We examined the effect of weight bearing (WB) on muscle recovery after nerve injury. Rats were housed in individual cages for 2 wk under WB or hindlimb suspension (HS) after being subjected to sciatic nerve compression for 1 wk. Sham operated on rats served as controls (sham group). We used 31P- and 19F-nuclear magnetic resonance spectroscopy combined with histochemical, physiological, and biochemical techniques to assess the outcome in the three groups. Creatine kinase-BB (CK-BB) mRNA levels expression, CK activity, and type I fiber density in the WB group were elevated compared with those in the HS group. In addition, sciatic functional index, tetanic tension, energy state, and local circulation dynamics of the WB group were greater than those of the HS group. These results suggested that WB plays an important role in muscle regeneration, inhibits the reduction of CK activity, and facilitates the activation of neural recovery, energy state, and local circulation dynamics.

Two adherent sublines, H69V and H69VZ, have been isolated from the classic SCLC cell line NCI-H69. Significant morphological differences were observed between the parental and the derivative cell lines. While NCI-H69 grew as densely packed free floating cellular aggregates the derivative lines grew as a monolayer of epithelioid cells. The growth rates of both the derivative lines were faster than the parental line with doubling times closer to non-SCLC cell lines in the derivative lines. Both H69V and H69VZ either express very low levels or do not express neuroendocrine cell markers including L-dopa-decarboxylase (DDC), creatine kinase-BB isoenzyme (CK-BB), bombesin-like immunoreactivity (BLI), neuron specific enolase (NSE), and neurosecretory type dense core granules (DGCs), compared to the parental cell line. All the lines stained positive for epithelial markers such as CAM5.2. LDH isoenzyme and chromosome analyses confirmed the human origin of all the cell lines. Therefore, it appears that cell line NCI-H69 contains stem cell subpopulation capable of generating cells of both small and non-small cell like phenotypes.

The importance of creatine kinase (E.C. 2.7.3.2) in endocrine tissues has been generally overlooked. Using a specific radiometric assay, we have demonstrated the existence of CK in the Brockmann body (principal islet) of the Coho salmon. We have purified this protein from insular tissue and concurrently purified CK from brain and muscle of the salmon. Purification characteristics, immunological cross-reactivity, and N-terminal sequence analysis have demonstrated that the predominant cytosolic CK from the Brockmann body is indistinguishable from the BB (brain) isoenzyme. Immunocytochemical studies indicated that the enzyme resides in the endocrine parenchyma. Phosphocreatine may serve as a reservoir of energy in the islet and augment its capacity to secrete hormones. The induction of CK-BB in the islet by other hormones could influence the secretion of insular hormones. Interorgan flux of the substrate creatine may be an undescribed mechanism of physiological regulation.

We describe a sensitive, specific radioimmunoassay for the BB isoenzyme of creatine kinase (CK-BB) in serum. A sequential saturation assay was used to achieve sufficient sensitivity to detect the isoenzyme in 100-microliter serum samples of all healthy persons and patients tested. Bound and free antigen were separated by a second antibody system. Large excesses of purified isoenzyme MM did not react in the assay. Cross reactivity of two preparations of CK-MB was only 1 to 7+. The 95th percentile of serum CK-BB in 208 healthy adults was 6.2 microgram/liter. Within-assay and between-assay precision ranged from 5.5 to 11.9% and 9.7 to 13.6%, respectively.