Production of recombinant human creatine kinase (r-hCK) isozymes by tandem repeat expression of M and B genes and characterization of r-hCK-MB. (1/46)

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.  (+info)

Identification of novel markers for monitoring minimal residual disease in acute lymphoblastic leukemia. (2/46)

To identify new markers of minimal residual disease (MRD) in B-lineage acute lymphoblastic leukemia (ALL), gene expression of leukemic cells obtained from 4 patients with newly diagnosed ALL was compared with that of normal CD19(+)CD10(+) B-cell progenitors obtained from 2 healthy donors. By cDNA array analysis, 334 of 4132 genes studied were expressed 1.5- to 5.8-fold higher in leukemic cells relative to both normal samples; 238 of these genes were also overexpressed in the leukemic cell line RS4;11. Nine genes were selected among the 274 overexpressed in at least 2 leukemic samples, and expression of the encoded proteins was measured by flow cytometry. Two proteins (caldesmon and myeloid nuclear differentiation antigen) were only weakly expressed in leukemic cells despite strong hybridization signals in the array. By contrast, 7 proteins (CD58, creatine kinase B, ninjurin1, Ref1, calpastatin, HDJ-2, and annexin VI) were expressed in B-lineage ALL cells at higher levels than in normal CD19(+)CD10(+) B-cell progenitors (P <.05 in all comparisons). CD58 was chosen for further analysis because of its abundant and prevalent overexpression. An anti-CD58 antibody identified residual leukemic cells (0.01% to 1.13%; median, 0.03%) in 9 of 104 bone marrow samples from children with ALL in clinical remission. MRD estimates by CD58 staining correlated well with those of polymerase chain reaction amplification of immunoglobulin genes. These results indicate that studies of gene expression with cDNA arrays can aid the discovery of leukemia markers. (Blood. 2001;97:2115-2120)  (+info)

Comparisons of the effects of tamoxifen, toremifene and raloxifene on enzyme induction and gene expression in the ovariectomised rat uterus. (3/46)

This study compares the actions of oestradiol, tamoxifen, toremifene and raloxifene on enzyme and gene expression in uterine tissues of ovariectomised rats over 72 h. The time-course for the induction of ornithine decarboxylase by the compounds showed a rapid biphasic response, while for creatine kinase brain type (BB) there was a continued increase over 72 h. The efficacy of induction showed that, with both markers, oestradiol gave the highest induction level, followed by tamoxifen or toremifene and then raloxifene. RT-PCR demonstrated that all compounds decreased oestrogen receptor (ER) alpha, ERbeta and ERbeta2 gene expression, 8-24 h after the first dose, suggesting that down-regulation of ER is not the primary cause of the difference in efficacy between these compounds. Using cDNA arrays, expression of 512 genes was examined in the uteri of oestradiol- or tamoxifen-treated rats. Both compounds resulted in the up-regulation of heat-shock protein 27, telomerase-associated protein 1 and secretin. However, most surprising was the marked down-regulation of Wilms' tumour and retinoblastoma genes. We speculate that this may result in a loss of regulation of the transition from the G1 to the S phase in the cell cycle and may make cells more vulnerable to the carcinogenic effects of tamoxifen in this tissue.  (+info)

Effect of weight bearing on recovery from nerve injury in skeletal muscle. (4/46)

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.  (+info)

Expression of brain subtype creatine kinase in the zebrafish embryo. (5/46)

Creatine kinases (CK) play crucial roles in intracellular energy transfer. We have isolated a cDNA from zebrafish embryos, which encodes a CK highly related to the mammalian brain subtype creatine kinase (BCK). The bck mRNA is expressed maternally in the zebrafish embryo and transcripts are distributed uniformly in blastula and gastrula stages. Expression becomes restricted to the prechordal plate and the nervous system during subsequent somitogenesis stages. bck transcripts are abundant in primary neurons in the developing central nervous system of the 1-day-old embryo. While some bck expression persists in the hindbrain, expression vanishes in the spinal cord of the 2-day-old embryo. In summary, the expression pattern of bck is highly dynamic and suggests a role for bck during gastrulation and neuronal differentiation.  (+info)

Postoperative patterns and kinetics of cTnI, cTnT, CK-MB-activity and CK-activity after elective aortic valve replacement. (6/46)

OBJECTIVES: The aim of this prospective study was to evaluate postoperative kinetics of four different biochemical ischaemic markers after elective aortic valve replacement (AVR). Additionally, pre-, peri- and postoperative data were analysed in order to identify factors with possible impact on the postoperative release of the selected enzymes. DESIGN: Forty patients (14 males, 26 females, aged 70 +/- 11 years; EF = 54 +/- 18% [mean +/- SD]) undergoing elective AVR were prospectively included in this study. For all patients, serum concentrations of cTnI, cTnT, and serum activities of CK-MB and CK were measured preoperatively as well as 0, 6, 12, 24, 48 and 120 hours after removal of the aortic cross-clamp. Clinical data were assessed in all patients and correlated with postoperative enzyme patterns. RESULTS: There were no major complications. Preoperatively, all patients showed enzyme values in the normal range whereas the four ischaemic markers reached higher values postoperatively. cTnI reached its maximum values 24 hours (XMed = 2.35 micrograms/L, 95%-CI [2.0, 3.3]) and cTnT 48 hours after the operation (XMed = 0.239 microgram/L, 95%-CI [0.174, 0.283]). Typical biphasic release kinetics could be demonstrated for cTnT. There was a high linear correlation between cTnI and cTnT at all sampling times. In contrast, a high linear correlation between cTnI, cTnT, and CK-MB-activity was only found 48 hours after aortic unclamping. cTnI nearly was in normal range 120 h postoperatively (XMed = 0.5 microgram/L, 95%-CI [0.2, 0.6]), whereas cTnT still remained pathologically elevated (XMed = 0.223 microgram/L, 95%-CI [0.137, 0.299]). No linear correlation was found between maximum values of the ischaemic markers postoperatively and age, gender, body surface area, ejection fraction, LV-hypertrophy, operating time, ECC time, time of cardiac arrest, lowest body temperature, perfusion pressure, cardioplegia volume, reperfusion time, postoperative septiformic circulatory instability, or ventilation time. CONCLUSIONS: All four ischaemic markers showed individual peak characteristics and kinetics after uncomplicated AVR. In contrast to previous findings, aortic cross-clamping time had no detectable impact on postoperative peak patterns of any ischaemic marker.  (+info)

Two specific markers for neural differentiation of embryonal carcinoma cells. (7/46)

Two multipotential embryonal carcinoma (EC) cell lines, 1003 and 1009, can be induced to form preferentially neural derivatives in vitro. Synthesis of specific proteins during neural differentiation was followed by two-dimensional gel electrophoresis. The comparison of protein patterns obtained with neural and non-neural derivatives of these EC cell lines indicates that two changes are specific for the neural pathway: (i) the appearance of a new beta-tubulin isoform and (ii) the accumulation of the brain isozyme of creatine phosphokinase already present in small amounts in EC stem cells. These changes were found to take place early in the course of differentiation and to occur even when neurite outgrowth was prevented.  (+info)

Expression of creatine kinase isoenzyme genes during postnatal development of rat brain cerebellum: evidence for transcriptional regulation. (8/46)

Transcription and accumulation of brain-type creatine kinase (CKB) mRNA and its protein was examined during postnatal development of rat brain cerebellum, the brain region containing highest CKB mRNA in the adult. CKB protein was extremely low at day 1, increased about 10-fold until week 4 and remained constant until week 10. This time course was paralleled by cerebellar CKB mRNA, which was also extremely low at day 1 and increased 5-fold during the first 3 weeks and then remained constant. High levels of CKB protein were also detected in cultured primary cerebellar granular neurons. Nuclear run-on assays directly showed that CKB mRNA accumulation during postnatal cerebellar development was due to increased transcription. When compared with cerebrum and whole brain, cerebellar CKB mRNA accumulation during postnatal development was temporally delayed. Analysis of myocyte enhancer factor (MEF)-2 and Sp1, factors known to initiate or sustain CKB transcription in tissues other than brain, revealed that MEF-2 in cerebellum was low at week 1 but increased 3.5-fold by week 7, while Sp1 remained unchanged. The increase in CKB protein during cerebellar postnatal development was coincident with that of the ubiquitous mitochondrial CK protein and mRNA, indicating that a functional phosphocreatine energy shuttle probably exists for efficient ATP regeneration in the cerebellum. This should be beneficial for the many energy-demanding requirements during cerebellar development, as indicated by the observed temporal co-expression of CKB with myelin basic protein, which is involved in axon myelination by oligodendrocytes.  (+info)

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