The pyruvate kinase isoenzyme M2 (Tu M2-PK) as a tumour marker for renal cell carcinoma

The M2 isoenzyme of pyruvate kinase (M2-PK) is specially expressed by tumour cells (Tu M2-PK) and has been detected in the peripheral blood of patients with renal cell carcinoma (RCC). We analysed the benefit of using Tu M2-PK as a tumour marker for primary detection of RCC by receiver operating characteristic (ROC) analysis. The area under the curve was 0.674, and the sensitivity, specificity and positive predictive value (PPV) were 44.4%, 87.5% and 88%, respectively, at the ROC optimal cut-off of 28.2 kU/L. We examined 71 patients. Since the marker sensitivity for detection of the early stages T1 and T2 was only 47% it is not suggested to use this marker for primary diagnosis of RCC. Its use as part of the confirmatory preoperative evaluation might be considered in view of its high PPV.     

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

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

Kinetics and localization of brain phosphate activated glutaminase.

The cellular concentration of phosphate, the main activator of phosphate activated glutaminase (PAG) is rather constant in brain and kidney. The enzyme activity, however, is modulated by a variety of compounds affecting the binding of phosphate, such as glutamate, calcium, certain long chain fatty acids, fatty acyl CoA derivatives, members of the tricarboxylic acid cycle and protons (Kvamme et al. [2000] Neurochem. Res. 25:1407-1419). Therefore, the kinetic and allosteric properties of the enzyme are essential for regulating the enzyme activity in situ, especially because the enzymically active pool of PAG is assumed to have an external localization in the inner mitochondrial membrane, being exposed to cytosolic variation in the content of effectors. This has largely been overlooked. A hypothetical model for the allosteric interactions based on the sequential induced fit allosteric model by Koshland et al. ([1966] Biochemistry 5:365-385) is presented. Furthermore, it has been generally accepted that there exist only two isoforms of PAG, the kidney PAG that is similar to brain PAG, and the liver PAG. Therefore, the immunoreactivity of brain cells against kidney PAG antibodies has been considered a measure of PAG protein. Gomez-Fabre et al. ([2000] Biochem. J. 345:365-375) recently found, however, that a PAG mRNA from human breast cancer ZR75 cells is present in human brain and liver, but not in the kidney. We observed only traces of PAG immunoreactivity in cultured astrocytes and cultured neuroblastoma cells, regardless whether antibodies against the C- and N-termini of kidney PAG or antibodies against liver PAG were used, but considerable enzyme activity, demonstrating hitherto unknown isoforms of PAG (Torgner et al. [2001] FEBS Lett. 268(Suppl 1):PS2-031).     

病毒性心肌炎患儿血清肌酸激酶同工酶及心肌肌钙Ⅰ蛋白的动态变化

目的 :通过连续观测急性病毒性心肌炎 ( VMC)患儿血清肌酸激酶同工酶 ( CK- MB)和心肌肌钙蛋白 ( c Tn I)与外周血淋巴细胞等的变化 ,探讨 VMC心肌细胞受损的机制。方法 :对 32例急性期和 1 2例迁延期及慢性期 VMC患儿血清 c Tn I、CK- MB,外周血 T淋巴细胞亚群、B淋巴细胞、自然杀伤细胞 ,肠道病毒 RNA,柯萨奇病毒 ( CBV)特异性抗体 Ig M( CBV- Ig M) ,淋巴细胞增殖活性 ( PI) ,补体 C3,左室射血分数 ( LVEF)进行测定。结果 :1急性期 VMC病程 <2周时 ,血清 c Tn I,CK- MB水平与对照组比较显著升高 (均 P <0 .0 1 ) ,CD3 ,CD4 ,CD8,CD4 /CD8,CD4 9,CD56细胞 ,PI,C3接近正常水平 ,血清 CBV- Ig M、外周血淋巴细胞 CBV- RNA检出阳性率较对照组明显增高 (χ2 值分别为 1 .667和 8.53,P值分别 >0 .0 5和 <0 .0 5) ;2病程 4～ 8周时 ,血清 c T-n I、CK- MB水平再次出现显著增高 ,与对照组比较 ,CD3 、CD4 、CD8、CD56、L VEF均显著降低 (均 P<0 .0 5) ,CD4 /CD8升高 ( P <0 .0 5) ,CBV- Ig M及 CBV- RNA检出率明显下降。结论 :血清 c Tn I、CK- MB是小儿急性 VMC时心肌损伤较敏感和特异的血清标志物 ,病程早期血清水平的升高可能为病毒的直接作用 ,晚期可能与机体免疫功能紊乱引起心肌损伤有关。     

犬实验性肺血栓栓塞症后血清乳酸脱氢酶同工酶3和血浆D-二聚体的动态研究

目的　动态监测犬肺血栓栓塞症 (PTE)后血清乳酸脱氢酶同工酶 3(L DH3)与血浆 D-二聚体的变化 ,探讨两者在急性 PTE诊断中的价值。方法　采用健康杂种犬 18只 ,随机分为三组。栓塞 组采用自体血加凝血酶及人纤维蛋白原制备的自体血栓 ,由股静脉输入建立急性 PTE模型。栓塞 组不加人纤维蛋白原 ,余同栓塞 组。对照组由股静脉输入生理盐水加人纤维蛋白原。三组均于栓塞前后做肺动脉造影及螺旋 CT血管造影 ,栓塞前及栓塞后多时点留取血样测 L DH3及血浆 D-二聚体浓度。结果　肺动脉造影及螺旋 CT血管造影均证实犬 PTE模型制备成功。栓塞 组血浆 D-二聚体于栓塞后 30 m in明显上升 ,2 4 h后明显下降 ,自身前后比较及与栓塞 组、对照组比较 ,差异有显著性 (P<0 .0 5 )。栓塞 ( 、 )组栓塞后 2 h至 4 d L DH3明显升高 ,与栓塞前及对照组比较差异有显著性 (P<0 .0 5 )。结论　血浆 D-二聚体、L DH3在急性 PTE早期有一个相对特征性的动态变化过程 ,联合检测 D-二聚体、L DH3有助于急性 PTE的早期诊断。     

The Activity of Class I, II, III, and IV Alcohol Dehydrogenase Isoenzymes and Aldehyde Dehydrogenase in Colorectal Cancer

Chronic ethanol consumption is associated with an increased risk for cancer of the colorectum. The highly toxic and carcinogenic compound is acetaldehyde, the product of ethanol metabolism. Ethanol is metabolized to acetaldehyde by alcohol dehydrogenase (ADH) in colorectal mucosa and bacteria. The enzyme responsible for oxidation of acetaldehyde is aldehyde dehydrogenase. The aim of this study was to compare ADH isoenzymes and ALDH activity in colorectal cancer with the activity in normal colonic mucosa. Total ADH activity was measured by a photometric method with p-nitrosodimethylaniline (NDMA) as substrate, and ALDH activity by a fluorometric method with 6-methoxy-2-naphthaldehyde as a substrate. For measurement of the activity of class I and II isoenzymes we employed fluorometric methods, with class-specific fluorogenic substrates. The activity of class III ADH was measured by the photometric method with n-octanol as substrate, and class IV with m-nitrobenzaldehyde as substrate. Samples were taken surgically during routine operations of colorectal carcinomas from 32 patients. The activities of total ADH and, the most important in colon mucosa, class I ADH were significantly higher in cancer than in healthy tissues. The other tested classes of ADH had a tendency to higher-level activity in cancer cells than in healthy mucosa. ALDH activity was not significantly lower in the cancer cells. The activities of all tested enzymes and isoenzymes were not significantly higher in drinkers than in nondrinkers both in colorectal cancer and in normal mucosa. The differences in activities of total ADH and class I isoenzyme between cancer tissues and normal colon mucosa might be a factor for metabolic changes and disturbances in low-mature cancer cells and, additionally, might be a reason for the higher level of acetaldehyde, which can intensify carcinogenesis.     

Genetic structuring and differentiation of Echinococcus multilocularis in Slovakia assessed by sequencing and isoenzyme studies

Summary Nucleotide sequencing of the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene and isoenzyme analysis were used to survey the genetic variability in Echinococcus multilocularis populations from Slovakia. A sample of 12 isolates acquired from 10 different districts from red foxes exhibited identical sequences. Compared with the previously described E. multilocularis variants, one base substitution was consistently observed relative to the M1 variant (detected in China, Alaska, North America, Japan) and three base substitutions were recorded relative to the M2 variant (detected in Germany) in the CO1 fragment. These data, along with the recently gathered data from French isolates, are indicative of a genetically unique population occurring in Central and Western Europe. Electrophoretic examination of enzymes produced by 14 gene loci revealed intraspecific polymorphism only with the glucose-phosphate isomerase (two distinct patterns) and the mannosephosphate isomerase (four genotypes composed of three alleles) enzyme systems. To allow a fast species differentiation of E. multilocularis and E. granulosus (specifically, the G7 genotype occurring in Slovakia), discriminative electrophoretic characters between the species were obtained by isoenzyme analysis. Fixed genetic differences between the species were detected in the glucose-phosphate isomerase, esterase and aldolase systems, and partial differences were detected in four additional systems.     

Geographic and temporal genetic patterns of Aedes aegypti populations in Rio de Janeiro, Brazil

Rio de Janeiro is considered as the most important entry point for dengue viruses in Brazil. Using isoenzyme markers, we investigated the genetic structure of the mosquito vector Aedes aegypti sampled at three-month intervals in 14 districts in Rio de Janeiro from December 2002 to December 2003. We detected high levels of genetic differentiation (i.e. high F(ST) values and significant P values), which tended to persist throughout the year. The species does not take advantage of routes and railways to disperse. Genetic structuring was higher in the rainy season, suggesting low dispersion of Ae. aegypti at this time of year when all dengue epidemics have been reported in the city.     

Expression of mdrl, mrp, topoisomerase IIα/β, and cyclin A in primary or relapsed states of acute lymphoblastic leukaemias

Summary. In a series of 60 ALL samples drawn during different stages of the disease we used a cDNA-PCR approach to analyse the relative mRNA levels of the MDR-associated genes encoding mdrl/P-glycoprotein, mrp, and the topoisomerase II isozymes α and β. Expression analysis of the cyclin A gene was included to examine cellular proliferation activity. The expression of gapdh served as an internal standard. Calculating the mean values we found: (i) a distinctly lower mdrl gene expression in primary ALL and first relapses compared to bone marrow from healthy donors, (ii) no change in mdrl and mrp, but a decreased topoisomerase IIα gene expression in first relapses of ALL compared to the primary leukaemia, and (iii) increased mdrl and mrp levels combined to decreased topoisomerase IIα levels in recurrent relapses of ALL showing significant correlations (mdrl/mrp: r_s=+0.6833, P <0.05: mdrl/topollα: r_s− 0.6727, P < 0.05). The expression of the topoisomerase IIá gene was correlated to that of cyclin A, indicating a link of its expression to cellular proliferation. Our findings suggest that a multifactorial MDR including mrp appears particularly in recurrent relapses of ALL. which often do not respond to chemotherapy. Nonetheless, some individual samples showed gene expression levels very different from the mean values calculated for a particular state of the leukaemia, indicating the need of an individual expression analysis of MDR-associated genes.