Asynchronous expression of the alcohol and supernatant malate dehydrogenase loci during Barbus hybrid development (Cypriniformes, Teleostei)

The tissue specificity and ontogeny of supernatant malate dehydrogenase (s-MDH) and alcohol dehydrogenase (ADH) are reported for the tiger barb (Barbus tetrazona), the rosy barb (Barbus conchonius) and their reciprocal hybrids. The tissue distribution of s-MDH and ADH isozymes in both species is consistent with spatial profiles reported for other teleosts. The expression of alleles of paternal origin at the s-Mdh-B and Adh loci are delayed in reciprocal hybrids as compared to their expression intraspecifically; suggestive of a low degree of affinity between maternally derived regulatory factors and paternal regulative elements controlling structural gene activation.     

Comparison of the spatial and temporal expression of supernatant malate dehydrogenase in Barbus hybrids (Cypriniformes, Teleostei)

The tissue specificity and ontogeny of supernatant malate dehydrogenase (s-MDH) are reported for the tiger barb, cherry barb, and their reciprocal hybrids. The tissue distribution of s-MDH isozymes in Barbus is consistent with the patterns reported in other teleosts. The expression of the Mdh-B locus is correlated with the initial muscle contractions of the developing embryos. It is suggested that the state of muscle cell differentiation may be the stimulus necessary for the expression of this locus in Barbus. Expression of maternal and paternal alleles at the B locus are synchronously delayed in reciprocal hybrids, as compared to their expression intraspecifically.     

Ontogenetic patterns of enzyme locus expression in Barbus hybrids (Cypriniformes, Teleostei)

The tissue specific patterns and ontogeny of sorbitol dehydrogenase (SDH, EC 1.1.1.14). lactate dehydrogenase (LDH, EC 1.1.1.27) and isocitrate dehydrogenase (IDH, EC 1.1.1.42) are reported for Barbus tetrazona (tiger barb), B. conchonius (rosy barb), B. nigrofasciatus (black ruby barb), B. titteya (cherry barb), B. sachsi (gold barb), and in interspecific hybrids where B. tetrazona is the maternal parent. The spatial and temporal expression of SDH, LDH and IDH isozymes in Barbus is consistent with those reported for other teleosts. As the genetic distance between the parentals used in forming the hybrid increases, allelic expression proceeds from synchronous to asynchronous, with an increasing delay in embryonic gene expression. These observations are consistent with the hypothesis that parental sensor genes differ in their response to maternally controlled regulatory signals; indicative of species specific effector/activator RNA molecule concentrations and sensor/receptor gene induction thresholds.     

Heterogeneous cellular expression of creatine kinase isoenzyme during normal rat heart development

The degree to which developmentally related alterations in cardiac creatine kinase (CK) activity reflect modification of CK isoenzyme gene expression remains uncertain. The present studies addressed this question by assessing multiple aspects of CK in rat heart during the perinatal to adult transition. In addition to whole tissue, isolated and purified muscle and nonmuscle cells were studied, as well as myofibrillar, mitochondrial, and cytosolic subcellular fractions. Whole homogenate CK enzyme specific activity nearly doubled during the weanling to adult developmental period. Muscle cell CK activity increased by a similar magnitude. Nonmuscle cell activity decreased. In the adult heart, both myofibrillar and mitochondrial CK activities were augmented versus the weanling heart. The cytoplasmic fraction activity held constant during development. Electrophoretic isoenzyme analyses of both weanling and adult cardiac muscle cells indicated the presence of mitochondrial CK and MM-CK isoforms. Weanling heart nonmuscle cells contained mitochondrial, MM, MB, and BB isoforms; however, BB isoform was not detected in the adult heart nonmuscle cells. Arrhenius plots provided information regarding heart muscle and nonmuscle cell alterations during development. CK activation energies were also determined for whole tissue, muscle/nonmuscle cells, myofibrils, mitochondria, and cytosol. Results demonstrate that heterogeneous muscle/nonmuscle cellular composition and differential myofibrillar/mitochondrial subcellular composition account for normal, developmentally related changes in heart CK enzyme activity. CK isoenzyme gene expression changes were not detected in cardiac muscle cells, and transition of CK-B to CK-M gene expression is limited to nonmuscle cells during normal, weanling to adult development in the rat heart.     

Cytokeratin 14 expression in rat liver cells in culture and localization in vivo

Rat liver epithelial cells (LECs) are non-parenchymal proliferating cells that readily emerge in primary culture and can be established as cell lines, but their in vivo cell(s) of origin is unclear. We reported recently some evidence indicating that the LEC line, T51B, contains two cytokeratins (CKs) equivalent to human CK8 and CK14 respectively. T51B cells also contain vimentin assembled as a network of intermediate filaments distinct from that of the CKs. In the present study, we examined the expression of CK14 gene in various LEC preparations and a Triton-resistant rat skin cytoskeletal fraction, and then assessed its usefulness as an LEC specific marker in the liver. Northern and Western blot analyses with cDNAs and antibodies for CK8, CK14, CK18 and vimentin confirmed that rat hepatocytes express CK8 and CK18 genes only, whereas T51B cells express CK8, CK14 and vimentin genes in the absence of CK18. CK14 was also present in LECs derived as primary from embryonic-day 12 rat liver and secondary cultures from 4-day-old rat liver. Primary cultures of oval cells isolated from 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) treated rat liver (an enriched source of biliary epithelial cells) contained CK14 mRNAs which were slightly shorter than those in LECs. The analyses of CK5 (the usual partner of CK14) gene expression using specific cDNA and antibody clearly demonstrated its absence in LECs. In situ double immunolocalization analyses by laser scanning confocal microscopy showed that CK14 was not present in hepatocytes (HES6+ cells) and was expressed in some biliary epithelial (BDS7+ cells). CK14-positive cells were also found in the Glisson's capsule. However, CK14-positive cells of the portal region were vimentin negative, whereas those of the Glisson's capsule were vimentin positive. Our results suggest that CK14 gene expression is part of the differentiation program of two types of LECs and that this differential CK14 gene expression can be used as a new means to type LECs in culture and in vivo.     

Molecular analysis of the QM gene from <Emphasis Type="Italic">Penaeus monodon</Emphasis> and its expression on the different ovarian stages of development

In present study, a QM gene was obtained from the ovary and neurosecretory organ in eyestalk cDNA library of black tiger prawn (Penaeus monodon). The full-length black tiger prawn QM (PmQM) cDNA contained a 5′-UTR of 41 bp, an ORF of 663 bp encoding a polypeptide of 220 amino acids with molecular weight 25.5 kDa, and a 3′-UTR of 54 bp. Homology analysis of the deduced amino acid sequence of the PmQM with other known QM sequences by MatGAT software revealed that the PmQM was high homology with other invertebrates. A conserved signature sequence of the QM family was found in the PmQM deduced amino acid sequence. Analysis of the tissue expression pattern of the PmQM gene showed that the PmQM mRNA was expressed in all tissues tested, with highest levels in ovary. Furthermore, the PmQM expression was found to be different in three important ovarian stages of development. The results indicated PmQM might play an important role in ovarian development.     

细胞角蛋白基因13在喉鳞状细胞癌中缺失和表达的研究

为了探讨细胞角蛋白基因13（Cytokeratin13,CK13)在喉癌发生中的作用,在CK13基因内部及附近选择5个微卫星引物进行杂合性丢失（loss of heterozygosity,LOH)分析,于DNA水平间接检测72例喉鳞状细胞癌患者中该基因的缺失,应用Northern Blot检测16例喉鳞癌患者的配对肿瘤及癌旁正常组织中CK13基因的表达差异;同时应用CK13蛋白单克隆抗体对不同分化程度的喉鳞癌组织进行免疫组化染色。结果表明：5个STR位点均存在LOH,其中D17S1964E、D17S2092、D17S791、D17S1665及D17S808位点的LOH频率分别为18．03％、28．13％、27．42％、39．68％和34．85％,其中D17S1665位点的LOH频率最高,至少一个位点出现LOH的病例高达77．78％（56／72）,杂合性丢失与临床分期、淋巴结转移无显著相关,但与肿瘤分化程度高低相关（P＜0．05）。Northern blot结果表明：16例喉鳞癌患者C13基因在正常组织中的表达比肿瘤组织显著增强,免疫组化结果也显示CK13蛋白在正常组织或高分化肿瘤中的表达明显高于低分化者,且存在显著差异（P＜0．01）。证实CK13基因可能在喉鳞状细胞癌的发生中具有重要作用,可能是一个新的抑癌基因。     

大鼠前列腺上皮细胞角蛋白8mRNA表达调节的定位研究

本文应用反义RNA探针原位杂交法,研究雄激素对大鼠腹侧前列腺(VP)上皮细胞角蛋白(CK)8 mRNA表达的影响。发现1.在任何VP组织切片中,CK 8探针专一、大量定位于VP腺上皮细胞中,CK 8 mRNA是前列腺上皮细胞特异而灵敏的标志。2.去睾大鼠VP CK 8 mRNA染色增强,提示CK 8mRNA有过度表达,注射雄激素又可抑制其过度表达。3.与已知受雄激素抑制性基因不同,即使大鼠VP完全萎缩之后达2个月之久,其存留腺上皮细胞CK 8 mRNA表达仍持续增高。4.前列腺发育早期,迅速增殖的幼稚腺上皮细胞高度表达CK 8 mRNA,以后随着体内雄激素水平升高,VP上皮CK 8 mRNA表达下降,分布转移。以上结果进一步支持前列腺CK 8基因是新的一类受雄激素抑制性基因的推测,同时表明前列腺CK 8基因的表达与前列腺干细胞的增殖分化有密切联系,CK 8 mRNA高度表达是前列腺干细胞一个重要特征。     

Systemic administration of antisense oligonucleotides simultaneously targeting CK2?? and ???? subunits reduces orthotopic xenograft prostate tumors in mice

CK2 is a highly conserved, ubiquitous, signal responsive protein serine/threonine kinase. CK2 promotes cell proliferation and suppresses apoptosis, and increased CK2 expression is observed in all cancers examined. We previously reported that direct injection of antisense (AS) CK2α phosphorothioate oligonucleotides (PTO) into xenograft prostate tumors in mice significantly reduced tumor size. Downregulation of CK2α in tumor cells in vivo appeared to result in overexpression of CK2α' protein. This suggested that in cancer cells downregulation of CK2α might be compensated by CK2α' in vivo, prompting us to design a bispecific (bs) AS PTO (bs-AS-CK2) targeting both catalytic subunits. bs-AS-CK2 reduced CK2α and α' protein expression, decreased cell proliferation, and induced apoptosis in cultured cells. Biodistribution studies of administered bs-AS-CK2 oligonucleotide demonstrated its presence in orthotopic prostate xenograft tumors. High dose injections of bs-AS-CK2 resulted in no damage to normal liver or prostate, but induced extensive cell death in tumor tissue. Intraperitoneal treatment with bs-AS-CK2 PTO decreased orthotopic tumor size and downregulated both CK2 mRNA and protein expression. Tumor reduction was accomplished using remarkably low doses and was improved by dividing the dose using a multi-day schedule. Decreased expression of the key signaling pathway proteins NF-κB p65 and AKT was also observed. We propose that the molecular downregulation of CK2 through bispecific targeting of the two catalytic subunits may be uniquely useful for therapeutic elimination of tumors.     

草河车治疗不同阶段胃癌病变的实验动物研究

为检测用草河车治疗胃癌前病变(precancerous lesions of gastric cancer,PLGC)不同阶段模型大鼠过程中MUC1和CK18基因(细胞角蛋白基因)的表达变化,在48只大鼠采用关木通乙醇提取物灌胃制造PLGC模型的过程中,从第13周开始,每2周随机抽取6只大鼠,3只直接解剖作为模型组,3只用草河车灌胃治疗2周后解剖作为治疗组,另取10只正常大鼠用生理盐水灌胃作为空白对照组,观察各组病变程度,提取血液和胃黏膜组织后用Real-time PCR技术分别检测胃癌标志基因MUC1和CK18在治疗前后的表达变化。结果显示,治疗前,随着大鼠胃癌病变逐步加重,血浆中CK18表达水平逐渐升高,MUC1则在胃黏膜组织逐渐降低表达,用草河车提取液灌胃后,治疗组中各级病变的MUC1表达相对于模型组均有不同程度的上调,且前期比后期上调更明显;而CK18的表达均有不同程度的下调,且后期比前期下调更明显。这些结果说明,MUC1和CK18在胃癌发生和发展过程中起重要作用,而草河车的某些成分可能通过调控它们的表达来缓解病变。     

金鱼酯酶同工酶的研究——Ⅰ.鲫鱼和红虎头金鱼各组织器官酯酶同工酶的比较

本文采用pH不连续系统聚丙烯酰胺垂直平板电泳方法,对鲫鱼和红虎头金鱼的眼、肝脏、肾脏、肌肉、卵巢(或精巢)、脑和心脏等7种新鲜组织的酯酶(EST)同工酶进行了比较研究。其结果是:鲫鱼(Carassius auratus)和红虎头金鱼(Carassius auratus var.)不仅具有丰富的EST同工酶和组织特异性,而且在它们的各个相应组织之间也显示出差异。表明酯酶同工酶在金鱼种内是有差异的。这对于探讨金鱼种内各品种之间的亲缘关系将是一个很有用的手段。     

Evolution of isozyme loci and their differential tissue expression

Summary The phylogeny of the creatine kinase (CK, EC 2.7.3.2) isozyme loci and their differential tissue expressions were determined for representatives of 65 families of vertebrates, with emphasis on the fishes. The transition from the single creatine kinase locus, characteristic of certain echinoderms, to the two creatine kinase loci which are orthologous to those present in all vertebrates, occurred early in the chordate line. The majority of pre-teleostean fishes possesses only these two CK loci (A and C). These loci are relatively generalized in their tissue expressions which are variable among species of primitive fishes. The third and fourth creatine kinase loci (B and D) arose separately in the ancestors of the bony fishes and appear to be the result of regional genome duplications. Concomitant with the increase in the number of isozyme loci has been an increase in the specificity of their tissue expression. In the advanced teleost fishes the four CK loci are differentially expressed in a characteristic manner. The A₂ isozyme predominates in skeletal muscle, the B₂ isozyme in eye and brain, the C₂ isozyme in stomach muscle, and the D₂ isozyme is found exclusively in testis. We propose a phylogeny of the creatine kinase genes in the lower chordates based on the time of appearance of new CK loci, the sequence in which the loci achieve a tissue restricted expression, and the immunochemical relatedness of the orthologous and paralogous gene products.