Properties of creatine kinase-BB from canine and human brain tissues

Creatine kinase (EC 2.7.3.2) BB isoenzyme (CK-BB) was purified to homogeneity from canine and human brain tissues. The purified protein from both sources exhibits Mr of 84,700 daltons. The canine isoenzyme exhibits several properties similar to human isoenzyme with respect to reactive and total thiol groups, UV spectra, isoelectric points and reaction kinetics. While both canine and human CK-BB isoenzymes are unstable compared to other CK isoenzymes, canine CK-BB is even less stable than the human enzyme, losing most of its activity within 20 h at 4 degrees C at pH 5.0. Addition of 2-mercaptoethanol does not prevent rapid loss of the enzyme activity. Increasing the pH to 9.0, however, increases the stability of both CK-BB isoenzymes. Agarose electrophoresis demonstrated the presence of MM as well as BB isoenzyme in various parts of brain tissues. BB was present at an activity of 90.8-93.3 U/mg and MM at 6.7-9.2 U/mg.     

Purification of creatine kinase-BB isoenzymes from human and canine tissues

We describe a procedure for the purification of creatine kinase BB isoenzyme from canine and human brain tissue. The purification involves sequentially 50-70% (NH4)2SO4 fractionation, DEAE-Sephacel, Blue-Sepharose CL-6B, and chromatofocusing chromatography. This method produces a homogeneous protein purification as verified by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing chromatography.     

Diagnostic value of lactate dehydrogenase isoenzymes in cerebrospinal fluid

To evaluate the diagnostic value of lactate dehydrogenase (LD) isoenzymes in cerebrospinal fluid (CSF), 93 consecutive CSF specimens were analyzed. These specimens were from patients of four categories: tumors, infections, hemorrhages, and others. It was found that the isoenzyme patterns overlapped among different categories, but they differed within each category and were thus helpful in differential diagnosis. For instance, metastatic tumors showed prominent LD-5, whereas a primary brain tumor demonstrated an increase in all fractions. Viral encephalitis revealed an increase in the first three isoenzymes and bacterial meningitis, the last two. In acquired immune deficiency syndrome (AIDS) cases, however, LD isoenzyme changes were demonstrated in CSF when only cryptococcal meningitis and not when encephalitis was present. Both subdural and subarachnoid hemorrhages showed elevation of all fractions in our study. Elevation of the first three fractions was usually due to brain tissue damage or hemorrhage, as proven by our isoenzyme study of hemolysate mixed with CSF. The prominence of the last two fractions was related to anaerobic metabolism in the central nervous system or to granulocytic infiltration. In conclusion, LD isoenzyme analysis in CSF is helpful in differential diagnosis of various CNS disorders, although its sensitivity awaits further improvement.     

Rapid purification of creatine kinase MB isoenzyme on preparative polyacrylamide slabs

We describe a procedure for purification of creatine kinase (EC 2.7.3.2) MB isoenzyme (CK-MB) from human cardiac muscle by preparative electrophoresis on polyacrylamide gel. From a 50-g portion of human myocardium we isolated 21 mg of CK-MB, which had a specific creatine kinase activity of 405 kU/g. The CK-MB exhibited an enzyme band on polyacrylamide gel electrophoresis (PAGE) with staining for enzyme activity. The preparation showed single protein bands on sodium dodecyl sulfate--PAGE, pore gradient electrophoresis, and isoelectric focusing electrophoresis, the relative molecular masses of the subunit and of the whole enzyme being 43,000 and 86,000, respectively, and the isoelectric point being 5.0. In addition, the purified CK-MB showed desirable immunological specificity and affinity (K = 2.4 x 10(11) L/mol) when measured by radioimmunoassay.     

人Ⅰ型丙氨酸氨基转移酶的可溶性表达及抗血清反应模式

目的克隆表达重组人Ⅰ型丙氨酸氨基转移酶(ALT1)蛋白及制备抗血清，探讨建立ALT1特异性检测方法思路。方法逆转录聚合酶链反应(RT-PCR)扩增人ALT1 cDNA，插入pGEX-2T，构建原核表达载体pGEX-2T—ALT1，并转人大肠杆菌进行表达。SDS-PAGE，活力测定和活性染色鉴定表达产物。表达产物免疫小鼠所得抗血清对天然ALT1，重组ALT1及重组ALT2进行Western blot分析。结果重组质粒测序和酶切结果显示ALT1基因已经成功克隆到pGEX-2T载体。SDS-PAGE和活性染色表明所表达蛋白具有天然活性。抗ALT1血清除识别重组ALT1外，还可识别天然蛋白和重组ALT2。结论重组人ALT1得到高效可溶性表达。用目前方法制备的抗血清特异性不高，如建立ALT1的免疫学检测方法需要采用特异性识别ALT1的抗体，以避免交叉反应引起的假性升高。     

Susceptibility of Bacteroides ureolyticus to antimicrobial agents and identification of a tetracycline resistance determinant related to tetM

The in-vitro activity of ten antimicrobial agents was evaluated for 28 clinical isolates of Bacteroides ureolyticus, an obligate anaerobe associated with non-gonococcal urethritis. The isolates were characterized by plasmid DNA profile and PAGE protein pattern. All isolates were inhibited at concentrations equal to or lower than the recommended breakpoint concentration for ampicillin (16 mg/l), metronidazole (16 mg/l) and erythromycin (4 mg/l). Twenty-seven isolates were inhibited by less than or equal to 2 mg/l of ciprofloxacin, pefloxacin and ofloxacin. Four isolates were tetracycline-resistant requiring 2-64 mg/l of tetracycline, minocycline or doxycycline for inhibition. In two tetracycline-resistant isolates tetM was demonstrated by dot-blot and Southern hybridizations. These two isolates did not contain a plasmid and had a PAGE protein pattern type III. These data confirm the spread of the tetM determinant in various bacteria of the genital tract.     

Early elevation of S-100B protein in blood after cardiac surgery is not a predictor of ischemic cerebral injury

BACKGROUND: We hypothesized that early changes in S-100B levels after cardiac surgery are nonspecific and mostly reflect damage to tissues outside the brain rather than ischemic brain damage. METHODS: We measured serum levels of S-100B at several times perioperatively in 21 patients undergoing cardiac surgery. In addition, we measured levels of neuron specific enolase (NSE), glial fibrillary acidic protein (GFAP), creatine kinase (CK), the cardiac isoenzyme of CK (CK-MB), and myoglobin (MB) in these patients. RESULTS: Early increases in serum S-100B concentration were significantly (p<0.01) correlated with increases in markers of tissue injury outside the brain: S-100B/CK: r(2)=0.69; S-100B/CK-MB: r(2)=0.64; S-100B/myoglobin: r(2)=0.60; S-100B/NSE: r(2)=0.51; CK/NSE: r(2)=0.60; CK-MB/NSE: r(2)=0.59; and myoglobin/NSE: r(2)=0.54. CONCLUSIONS: Our findings indicate that increases in S-100B in the early phase after cardiac surgery are not due to release of S-100B from brain alone but also from tissue outside the brain.     

Comparison of enzymatic characteristics of creatine kinase BB from human healthy and tumor stomach tissues

Creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2, CK) BB isoenzyme from stomach tumor tissue was partially purified and its characteristics were compared with those from healthy tissue. Molecular mass of tumor CK-BB was estimated to be 82 000 by polyacrylamide gel electrophoresis. Tumor CK-BB was separated into 2 main subbands around pH 4.5 and 11, minor subbands around pH 5-7.5 by agarose isoelectric focusing. The isoenzyme reacted with anti-human brain CK-BB antibodies and formed a hybrid, CK-MB, with CK-MM prepared from healthy human skeletal muscle. The above physicochemical and immunological characteristics of tumor CK-BB were the same as those of normal CK-BB from normal stomach tissue. Optimum pH of tumor CK-BB was more acidic than that of normal CK-BB. Affinity for creatine phosphate and heat sensitivity of tumor CK-BB were slightly lower than those of normal CK-BB. Tumor CK-BB was more stable after iodoacetamide and urea treatments.     

Abnormal lactate dehydrogenase isoenzyme in serum and tumor tissue of a patient with neuroblastoma

Serum and tumor tissue of a patient with neuroblastoma contained an abnormal isoenzyme of lactate dehydrogenase (LDH; EC 1.1.1.27), which, on agarose gel electrophoresis, migrated between LDH-2 and LDH-3 with a mobility the same as that of the extra LDH isoenzyme found in normal human erythrocytes. On surgical removal of the tumor, the high total LDH activity (775 U/L) in the serum of the patient rapidly decreased to normal (70-220 U/L), and the abnormal LDH isoenzyme was no longer detected. The total LDH activity of the abnormal LDH isoenzyme per gram of hemoglobin in the tumor tissue was 26 times that of erythrocytes, suggesting that the abnormal isoenzyme originated mainly from the tumor cells themselves rather than the erythrocytes contained in the tumor tissue. This first report on the appearance of the abnormal LDH isoenzyme in a patient with neuroblastoma suggests that this abnormal LDH isoenzyme may have some significance as a marker enzyme for neurogenic tumors.     

Isolation of creatine kinase BB isoenzyme with high specific activity and adequate purity for radioimmunoassay from human placenta on preparative polyacrylamide gel electrophoresis

This report describes the procedures for isolation of creatine kinase BB isoenzyme (CK-BB) from human placenta on preparative polyacrylamide gel electrophoresis. 2.5 mg of CK-BB was purified from a 100-g portion of the human placenta, which had a mean specific activity of 957 kU/g and a mean yield of 16%. The placenta CK-BB exhibited single protein bands on several electrophoretic techniques. In addition, both of the placenta and brain CK-BB preparations were individually iodinated and the identical immunological properties of both the CK-BB preparations were confirmed in radioimmunoassay.     

Brain specific creatine kinase isoenzyme behavior in rat serum after bile acid, sodium oleate and albumin injection.

In this investigation we have shown that severe liver disease with cerebral involvement can be followed by the presence of BB creatine kinase isoenzyme in serum. Groups of rats were injected with a bile acid mixture, oleic acid, albumin and normal saline respectively. Bile acids or/and oleic acid induced BB isoenzyme to leak from central nervous tissue and this can be measured in serum. Albumin binding prevented this leakage. The experiments support our hypothesis, based on biochemical findings in human liver failure, that detergent and surface activity properties of the above mentioned components are able to produce this specific brain isoenzyme leakage.     

PAGE4 is a cytoplasmic protein that is expressed in normal prostate and in prostate cancers

PAGE4 is an X chromosome-linked cancer-testis antigen that was identified by expressed sequence tags database mining and a functional genomic approach. PAGE4 is preferentially expressed in normal male and female reproductive tissues and also in a variety of cancers including prostate. In the present study, we have used in situ hybridization to show that PAGE4 mRNA is expressed only in the epithelial cells of normal and prostate-cancer specimens. Analysis of the protein product encoded by the PAGE4 mRNA reveals that it encodes a Mr 16,000 protein and is detected in tissue extracts from both normal prostate and prostate cancer. Cell fractionation analysis of PAGE4 protein indicates that PAGE4 is localized in the cytoplasm of the cell. Furthermore, cDNA microarray analysis indicates that the expression of lipoprotein lipase, a gene frequently deleted in prostate cancer, is down-regulated in a cell line that expresses PAGE4.     

Degradation of serum amyloid A protein by surface-associated enzymes of human blood monocytes

Peripheral blood monocytes incubated in a serum-free medium degraded serum amyloid A (SAA) protein along three pathways. Of 20 normal subjects, 8 degraded SAA completely with no detectable intermediates. Eight subjects transiently produced an amyloid A (AA)-like intermediate which comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) with tissue AA protein and reacted with antisera to AA, whereas four subjects yielded a persistent AA-like intermediate on PAGE. This group also failed to degrade tissue AA protein. Cells from 10 patients with amyloidosis fell into the second group. The responsible enzymes appear to be serine proteases because they are inhibited by disopropyl fluorophosphate. They were not affected by epsilon-amino caproic acid, L-1-tosylamide-2-phenylethyl chloromethyl ketone, or N-alpha-p-tosyl-L-lysine chlormethyl ketone. It appears possible that the enzymes are associated with the outer membrane of the cell because only a small fraction of the activity is secreted into the medium and because enzyme activity remains after fixation of the cells with glutaraldehyde which completely stops phagocytosis. Perhaps differences in patterns of proteolysis may play a role in the predisposition to amyloidosis.     

Acute exposure of the neonatal rat to tributyltin results in decreases in biochemical indicators of synaptogenesis and myelinogenesis

Assays of neuron-localized (neurotypic) and glia-localized (gliotypic) proteins were used to detect and characterize the toxic effects of tributyltin (TBT) on the developing rat central nervous system. Four proteins associated with specific aspects of neuronal and glial development were evaluated: 1) p38, a synaptic vesicle-associated protein; 2) neurofilament 200, an intermediate filament protein of the neuronal cytoskeleton; 3) myelin basic protein, an oligodendroglia and myelin-sheath associated protein; and 4) glial fibrillary acidic protein, an intermediate filament protein of astrocytes. On postnatal days 13, 22 and 60, the amount of each protein in homogenates of cerebellum, forebrain and hippocampus was determined by radioimmunoassay. A single administration of TBT (2, 3 or 4 mg/kg i.p.) on postnatal day 5 caused dose- and region-dependent decreases in brain weight with the cerebellum being most affected. These decrements were not associated with light microscopic evidence of altered brain development (on postnatal day 61) but were accompanied by large dose- and region-dependent decreases in p38 and myelin basic protein. Decrements in both the per tissue (total) and per milligram of tissue protein (concentration) values for these proteins were observed in cerebellum and forebrain; hippocampus was largely unaffected. TBT-induced reductions in p38 and myelin basic protein were seen at dosages that did not affect brain, thymus or body weight. At dosages of TBT that did not affect body weight, reductions in brain weight, p 38 and myelin basic protein did not persist into adulthood. The data indicate that exposure to TBT on postnatal day 5 is toxic to the developing nervous system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lactate dehydrogenase and alkaline phosphatase isoenzymes and protein-bound sialic acid in regenerating rat liver

Lactate dehydrogenase (LDH) and alkaline phosphatase (AP) isoenzyme patterns and protein-bound sialic acid content were compared between normal, regenerating rat liver 10 days after partial hepatectomy and fetal rat liver. For this purpose, liver from ten adult rats and two pools of ten fetal livers each were examined. Isoenzymes were separated by electrophoresis on cellulose acetate and their percent distribution calculated after quantitation by densitometry of the bands. LDH-5 and LDH-4 combined represented in all the tissues examined 90%-94% of the total activity. LDH-5/LDH-4 ratios were nearly equivalent in the normal and regenerated liver (7.14, 6.41), but substantially lower in fetal liver (2.50). Two bands of AP were visualized in electropherograms. AP-1/AP-2 ratio was lower in regenerated liver (1.57) as compared to normal liver (2.27) and still lower in fetal liver (1.06). Protein-bound sialic acid was, on protein basis, slightly but not significantly higher in regenerated liver (1.71 microgram/mg protein) than in normal liver (1.43), and significantly higher in fetal liver (1.87). The relatively small differences in isoenzyme patterns and in protein-bound sialic acid between regenerated and normal liver as compared to those between fetal and normal tissue add support to the view that the cells in regenerated liver are not of embryonic origin.     

(+/-)-3,4-Methylenedioxymethamphetamine administration to rats does not decrease levels of the serotonin transporter protein or alter its distribution between endosomes and the plasma membrane

We showed that the serotonin (5-HT) neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) reduces brain tissue 5-HT, decreases expression of 5-HT transporter (SERT) protein, and increases expression of glial fibrillary acidic protein (GFAP). In contrast, doses of (+/-)-3,4-methylenedioxymethamphetamine (MDMA) that decrease brain tissue 5-HT fail to alter expression of SERT or GFAP. Using a new and highly sensitive anti-SERT antibody, we determined whether MDMA alters the subcellular distribution of SERT protein by measuring SERT expression in endosomes and plasma membranes 2 weeks after MDMA administration. Rat brain tissues (caudate, cortex, and hippocampus) were collected 3 days and 2 weeks after MDMA (7.5 mg/kg i.p., every 2 h x 3 doses) or 5,7-DHT (150 microg/rat i.c.v.) administration. Representative results from cortex are as follows. At both 3 days and 2 weeks postinjection, MDMA decreased tissue 5-HT (65%) and had no effect on GFAP expression. MDMA increased heat shock protein 32 (HSP32; a marker for microglial activation) expression (30%) at 3 days, but not 2 weeks. MDMA did not alter SERT expression at either time point and did not alter SERT levels in either endosomes or plasma membranes (2 weeks). 5,7-DHT decreased tissue 5-HT (80%), increased HSP32 expression at both time points (about 50%), and increased GFAP expression at 2 weeks (40%). 5,7-DHT decreased SERT expression (33%) at 2 weeks, but not at 3 days. These findings indicate that a dosing regimen of MDMA that depletes brain 5-HT does not alter SERT protein expression or the distribution of SERT between endosomes and the plasma membrane and does not produce detectable evidence for neurotoxicity.     

DEAE-cellulose chromatography of creatine kinase isoenzymes--effect of pH and serum.

DEAE-cellulose chromatography (pH 7.0) of human heart extracts revealed the presence of three creatine kinase isoenzymes. The CK3 (skeletal muscle) isoenzyme was not retained on the column under these conditions. The CK2 (heart) and CK1 (brain) isoenzymes eluted at a conductivity of 5.5 +/- 0.6 m omega-1 and 11.4 +/- 1.2m omega-1, respectively. When DEAE-cellulose chromatography was performed at pH 8.0, CK2 eluted at a slightly higher conductivity, 6.5 m omega-1, whereas CK1 eluted as before 12.0 m omega-1. DEAE-cellulose chromatography of CK2 and CK1 isoenzymes in the presence of serum protein, and serum albumin had no significant effect on the elution of CK2 at pH 7.0 and 7.4, and on the elution of CK1 at pH 7.0 and 8.0 However, serum and serum albumin decreased the affinity of CK2 for DEAE-celluose at pH 8.0, and caused this isoenzyme to elute at a conductivity of 3.0-3.5 m omega-1. The decreased affinity of CK2 for DEAE-cellulose was not due to aggregation of CK2 with albumin or some other serum protein, but was related to the amount of albumin applied to the column.     

Serum tartrate-resistant acid phosphatase isoforms in rheumatoid arthritis

Objectives: Our objective was to evaluate the significance and source of serum tartrate-resistant acid phosphatase (TRACP) in patients with rheumatoid arthritis (RA). Methods: Thirty-five RA, 32 osteoarthritis (OA) and 16 control subjects were studied. Serum TRACP-5b activity and total TRACP protein were determined by immunoassay. TRACP isoforms were analyzed by non-denaturing polyacrylamide gel electrophoresis (PAGE). Serum bone alkaline phosphatase (BAP), cross-linked N-terminal telopeptides (NTx), and C-terminal telopeptides (ICTP) of type I collagen were estimated as markers of bone turnover. C-reactive protein (CRP) was measured as a marker of chronic inflammation. Macrophages and dendritic cells (DC) were developed from peripheral blood monocytes. Cell lysates and culture supernatants were analyzed for TRACP isoforms by immunoassay and PAGE. Results: In RA, mean TRACP-5b activity was normal, but median total TRACP protein was increased twofold (p<0.001). In OA, TRACP-5b activity and protein were normal. In RA, TRACP-5b activity correlated weakly with ICTP (r=0.56) while TRACP protein levels correlated weakly with NTx (r=0.43). Additionally, TRACP protein, but not TRACP-5b activity correlated significantly with CRP (r=0.42). Macrophage and DC lysates contained TRACP-5b, while tissue culture supernatants contained TRACP-5a. Conclusions: Increased total TRACP protein in RA sera was probably due to TRACP-5a and not derived from osteoclasts. Rather, it could be a secreted product of inflammatory macrophages and DC.     

Regulated delivery of glial cell line-derived neurotrophic factor into rat striatum, using a tetracycline-dependent lentiviral vector

In this study, a tetracycline-regulated lentiviral vector system, based on the tetracycline-dependent transactivator rtTA2(S)-M2, was developed for controlled expression of glial cell line-derived neurotrophic factor (GDNF) in the rat brain. Expression of the marker gene green fluorescent protein (GFP) and GDNF was tightly regulated in a dose-dependent manner in neural cell lines in vitro. Injection of high-titer lentiviral vectors into the rat striatum resulted in a 7-fold induction of GDNF tissue levels (1060 pg/mg tissue), when doxycycline (a tetracycline analog) was added to the drinking water. However, low levels of GDNF (150 pg/mg tissue) were also detected in animals that did not receive doxycycline, indicating a significant background leakage from the vector system in vivo. The level of basal expression was markedly reduced when a 10-fold lower dose of the tetracycline-regulated GDNF vector was injected into the striatum (3-11 pg/mg tissue), and doxycycline-induced GDNF tissue levels obtained in these animals were about 190 pg/mg tissue. Doxycycline-induced expression of GDNF resulted in a significant downregulation of the tyrosine hydroxylase (TH) protein in the intact striatum. Removal of doxycycline from the drinking water rapidly (within 3 days) turned off transgenic GDNF mRNA expression and GDNF protein levels in the tissue were completely reduced by 2 weeks, demonstrating the dynamics of the system in vivo. Accordingly, TH protein expression returned to normal by 2-8 weeks after removal of doxycycline, indicating that GDNF-induced downregulation of TH is a reversible event.     

SPECIFICITY IN THE EFFECTS ON BRAIN METABOLISM OF TWO DIFFERING NEUROTROPIC VIRUSES

1. Brain tissue infected with the virus of Western equine encephalomyelitis shows specific differences in metabolism from brain tissue infected with the virus of poliomyelitis. 2. With added glucose concentration of 121 mg. per cent, oxygen utilization of poliomyelitic brain is significantly below normal and that of encephalitic brain is not. 3. With added glucose concentration of 217 mg. per cent, oxygen utilization of encephalitic brain is significantly below normal and that of poliomyelitic brain is not. 4. With lactate-glucose as the substrate, oxygen utilization of encephalitic brain is significantly below normal and that of poliomyelitic brain is not. 5. With pyruvate-glucose as the substrate, neither encephalitic brain nor poliomyelitic brain differs significantly from normal in oxygen consumption. 6. With succinate-glucose as the substrate, oxygen utilization of poliomyelitic brain is significantly above that of the normal control in the 1st hour. 7. With added glucose concentration of 37.5 mg. per cent, anaerobic metabolism of encephalitic brain is significantly below the normal whereas that of poliomyelitic brain is not. 8. With added glucose concentration of 229.5 mg. per cent, anaerobic metabolism of both encephalitic brain and poliomyelitic brain is significantly below the normal.