Characterization and expression analysis of TNF-related apoptosis inducing ligand (TRAIL) in grass carp Ctenopharyngodon idella

TRAIL (Apo2 ligand) described as a type II transmembrame protein belonging to the TNF superfamily can induce apoptotic cell death in a variety of cell types. In the present study, a putative cDNA sequence encoding the 299 amino acids of TRAIL (GC-TRAIL) and its genomic organization were identified in grass carp Ctenopharyngodon idella. The predicted GC-TRAIL sequence showed 44 and 41% identities to chicken and human TRAILs, respectively. In a domain search, a tumor necrosis factor homology domain (THD) was identified in the C-terminal portion of TRAILs. The GC-TRAIL gene consists of five exons, with four intervening introns, spaced over approximately 4 kb of genomic sequence. Analysis of GC-TRAlL promoter region revealed the presence of a number of putative transcription factor binding sites, such as Sp1, NF-kappaB, AP-1, GATA, NFAT, HNF, STAT, P53 and IRFI sequences which are important for the expression of other TNF family members. Phylogenetic analysis placed GC-TRAIL and the putative zebrafish (Danio rerio) TRAIL obtained from searching the zebrafish database into one separate cluster near mammalian TRAIL genes, but apart from the reported zebrafish TRAIL-like protein, indicating that the GC-TRAIL is an authentic fish TRAIL. Expression analysis revealed that GC-TRAIL is expressed in many tissues, such as in gills, liver, trunk kidney, head kidney, intestine and spleen. (c) 2005 Elsevier B.V. All rights reserved.

Molecular cloning of TRAF2 binding protein gene and its promoter region from the grass carp Ctenopharyngodon idellus

A tumor necrosis factor receptor-associated factor 2 binding protein (T2BP) gene was isolated from the grass carp (Ctenopharyngodon idellus) by utilizing suppression subtractive hybridization (SSH) and rapid amplification of cDNA ends (RACE). The grass carp T2BP (GT2BP) gene contains an open reading frame of 579 nucleotide(s) (nt), encoding 193 amino acids, with 23 nt 5'-untranslated region and a long 3'-untranslated region of 434 nt including poly (A), 1 AUUUA motif and 4 AUUUUA motifs. No signal peptide has been detected in the predicted GT2BP, but a characteristic forkhead associated domain is present. The GT2BP mRNA shares 83% identity with the zebrafish DNA sequence, and they both have no introns in the genomic DNA. The putative transcription factor binding sites of GT2BP include two C/EBP alpha binding sites, and one c-Jun binding, one AP-1 binding, and one nuclear factor kappa B (NF kappa B) binding sites. Southern blot analysis revealed that the GT2BP was a single-copy gene. Individual difference was observed in GT2BP expression in examined organs of healthy grass carp. However, the expression of GT2BP in all examined organs in a fish with the highest copepod infection level and the significantly higher expression level in spleen and liver in infected fish may indicate its up-regulation with the parasite infection. (c) 2005 Elsevier B.V. All rights reserved.

Effect of p53 on lung carcinoma cells irradiated by carbon ions or X-rays

The study is to investigate the feasibility and advantages of heavy ion beams on radiotherapy. The cellular cycle and apoptosis, cell reproductive death and p53 expression evaluated with flow cytometry, clonogenic survival assays and Western blot analysis were examined in lung carcinoma cells after exposure to 89.63 MeV/u carbon ion and 6 MV X-ray irradiations, respectively. The results showed that the number colonyforming assay of A549 was higher than that of H1299 cells in two radiation groups; A549 cellular cycle was arrested in G(2)/M in 12 It and the percentage of apoptosis ascended at each time point of carbon ion radiation with doses, the expression of p53 upregulated with doses exposed to X-ray or carbon ion. The cell number in G(2)/M of H1299 and apoptosis were increasing at all time points with doses in C-12(6+) ion irradiation group. The results suggested that the effects of carbon ions or X rays irradiation on lung carcinoma cells were different, C-12(6+) ion irradiation could have more effect on upregulating the expression of p53 than X-ray, and the upregulated expression of p53 might produce the cellular cycle G(2)/M arrested, apoptosis increasing; and p53 gene might affect the lung cancer cells radiosensitivity.

High-efficiency transfer and expression of AdCMV-p53 in human hepatocellular carcinoma cells induced by low-dose carbon-ion radiation

Objective To investigate whether the irradiation with C-beam could enhance adenovirus-mediated transfer and expression of p53 in human hepatocellular carcinoma. Materials and methods HepG2 cells were exposed to C-beam or gamma-ray and then infected with replicationdeficient adenovirus recombinant vectors containing human wild-type p53 or green fluorescent protein, respectively. The transfer efficiency and expression level of the exogenous gene were detected by flow cytometric analysis. Cell survival fraction was detected by clonogenic assay. Results The transfer frequency in C-beam or gamma-irradiated groups increased by 50-83% and 5.7-38.0% compared with the control, respectively (P < 0.05). Compared with C-beam alone, p53 alone, and gamma-ray with p53, the percentages of p53 positive cells for 1 Gy C-beam with p53 increased by 56.0-72.0%, 63.5-82.0%, and 31.3-72.5% on first and third day after the treatments, respectively (P < 0.05). The survival fractions for the 2Gy C-bearn and AdCMV-p53 infection groups decreased to similar to 2%. Conclusion C-beam irradiation could significantly promote AdCMV-green fluorescent protein transfer and expression of p53.

Potentiality of phosphorylation of BRCA1 at Ser 1524 to activate p21 in response to X-ray irradiation

The breast and ovarian cancer susceptibility gene BRCA1 encodes a nuclear phosphoprotein, which functions as a tumor suppressor gene. Many studies suggested that multiple functions of BRCA1 may contribute to its tumor suppressor activity, including roles in cell cycle checkpoints, apoptosis and transcription. It is postulated that phosphorylation of BRCA1 is an important means by which its cellular functions are regulated. In this study, we employed phospho-Ser-specific antibody recognizing Ser-1524 to study BRCA1 phosphorylation under conditions of DNA damage and the effects of phosphorylation on BRCA1 functions. The results showed that 10 Gy X-ray treatment significantly induced phosphorylation of Ser-1524 but not total BRCA1 protein levels. The expression both of p53 and p21 increased after irradiation, but ionizing radiation (IR) -induced activation of p21 was prior to that of p53. The percentages of G0/G1 phase remarkably increased after IR. In addition, no detectable levels of 89 kDa fragment of PARP, a marker of apoptotic cells, were observed. Data implied that IR-induced phosphorylation of BRCA1 at Ser-1524 might activatep21 protein, by which BRCA1 regulated cell cycle, but play no role in apoptosis.

碳离子束联合p53腺病毒重组体转染对肿瘤细胞生物学效应研究

肿瘤是严重威胁人类生命健康的常见病、多发病，不仅病因复杂、发生发展异常迅速，而且到目前为止，发病机理不完全清楚，尚无适应范围广和有特异疗效的治疗方法。因此，肿瘤治疗方法的探索依然是医学、生物学及其相关学科研究的热点。肿瘤的重离子治疗和基因治疗是近年来发展起来的新的肿瘤治疗方法。但它们同样或多或少存在一些不足。在肿瘤治疗方法的探索中，将两种或两种以上理化特性或生物学作用原理不尽相同的现有治疗方法有机结合，充分利用各自优势，取长补短，使治疗效果叠加，对肿瘤发挥协同或相加抑制作用。本研究将重离子辐射与p53腺病毒重组体(AdCMV-p53)转染有机结合，探讨了重离子辐射联合p53基因转导对肿瘤细胞的生物学作用及其可能机理。在低剂量γ辐射联合AdCMV-p53/GFP转染HT-29和PC-3细胞研究基础上，我们用不同剂量的AdCMV-p53/GFP转染经0.5 Gy、1.0 Gy、2.0 Gy 12C6+束/γ射线预辐射处理的人非小细胞肺癌(H1299细胞系，nullp53)，人肝癌细胞(HepG2细胞系，wtp53)和人宫颈癌细胞(Hela细胞系，wtp53，wtp53低水平表达)。用流式细胞分析法检测肿瘤细胞绿色荧光蛋白(GFP)、p53蛋白表达水平和细胞周期。DAPI染色后用荧光显微镜检测细胞凋亡。用RT-PCR检测外源性p53转录。用Western Blot检测外源性p53、MDM2和p21蛋白表达。用克隆形成法测定肿瘤细胞存活。通过与γ辐射联合腺病毒重组体转染组比较，观察了12C6+ 辐射联合腺病毒重组体转染对肿瘤细胞外源性p53蛋白表达、细胞周期阻滞、细胞凋亡和细胞增殖的影响。结果显示，12C6+ 辐射对AdCMV-GFP转染H1299、HepG2和Hela细胞的诱导作用明显强于γ辐射(p<0.05)。与γ辐射诱导AdCMV-GFP转染组相比，0.5 Gy 12C6+束辐射联合20 MOI AdCMV-p53转染组H1299细胞GFP阳性率增加约50% (其GFP阳性率提高到约90%)。0.5 Gy、1.0 Gy 12C6+辐射联合40 MOI AdCMV-p53转染组HepG2细胞GFP阳性率增加约44%(其阳性率分别达56.6%和76.4%)。0.5 Gy、1.0 Gy 12C6+ 束辐射联合40 MOI AdCMV-p53转染组Hela细胞GFP阳性率分别增加37.8%和50%(其阳性率分别达43.4%和59.8%)。12C6+ 辐射对AdCMV-p53转染H1299、HepG2和Hela细胞外源性p53蛋白表达的增强作用明显强于γ辐射(p<0.05)。12C6+ 辐射联合AdCMV-p53转染组各种细胞p53阳性率明显高于其它处理组同种细胞p53阳性率(p<0.05)。转染后第5天，γ辐射联合AdCMV-p53转染组3种细胞p53阳性率均降至对照水平。转染后第13天，12C6+ 辐射联合AdCMV-p53转染组3种细胞p53阳性率仍高达6－44%。12C6+ 辐射联合AdCMV-p53转染H1299细胞G0/G1、G2/M期细胞所占比例明显高于其它处理组G0/G1、G2/M期细胞所占比例(p<0.05)。与γ辐射联合AdCMV-p53转染组相比，12C6+ 辐射联合AdCMV-p53转染组G0/G1期细胞增加6－36%，G2/M期细胞增加了13－86%。12C6+ 辐射联合AdCMV-p53转染HepG2细胞G0/G1期细胞所占比例明显高于其它处理组G0/G1期细胞所占比例(p<0.05)；转染后第5天，1.0、2.0 Gy 12C6+ 辐射联合AdCMV-p53转染组G2/M期细胞所占比例明显高于γ辐射联合AdCMV-p53转染组G2/M期细胞所占比例(p<0.05)。各12C6+束辐射联合AdCMV-p53转染Hela细胞G0/G1和G2/M期细胞所占比例均明显高于单纯12C6+ 辐射组和γ射线辐射联合AdCMV-p53转染组G0/G1和G2/M期细胞所占比例(p<0.05)。各12C6+ 辐射联合AdCMV-p53转染H1299、HepG2和Hela细胞凋亡率明显高于等剂量12C6+ 单纯辐射和等剂量γ辐射联合AdCMV-p53转染组细胞凋亡率(p<0.05)。与等剂量单纯12C6+辐射和等剂量γ辐射联合AdCMV-p53转染组相比，12C6+ 辐射联合AdCMV-p53转染H1299细胞凋亡率分别增加8.0－66.0%和9.3－63.5%；12C6+束辐射联合AdCMV-p53转染HepG2细胞凋亡率分别增加0.8－32.7%和4.5－27.1%； 12C6+束辐射联合AdCMV-p53转染Hela细胞凋亡率分别增加4.8－30.7%和3.1－22.7%。低剂量12C6+ 辐射联合AdCMV-p53转染细胞存活率明显低于其它处理组同种细胞存活率(p<0.05)。结果提示，低剂量碳离子辐射对腺病毒重组体转染肿瘤细胞和靶细胞内外源p53蛋白表达的促进作用明显强于低剂量γ辐射。碳离子辐射联合AdCMV-p53转染通过促进外源性p53转导、靶细胞外源性p53蛋白表达、细胞周期阻滞和细胞凋亡等增强对肿瘤细胞的抑制。碳离子辐射联合AdCMV-p53转染对肿瘤细胞生物学作用与肿瘤细胞内在p53基因状态有关。总之，我们的研究表明，低剂量碳离子辐射联合AdCMV-p53转染，可通过促进腺病毒重组体对肿瘤细胞的转染、增强靶细胞外源性p53蛋白稳定表达及其由此而诱发的细胞周期阻滞与细胞凋亡等有效抑制肿瘤细胞。在临床上，碳离子辐射联合AdCMV-p53转染有望在提高肿瘤治疗效果的基础上，进一步降低碳离子辐射与AdCMV-p53转染的各自临床用量，减少碳离子辐射的毒副作用，降低AdCMV-p53转染的潜在生物危险性

Applications of homemade kit in mutation detection of genes

Several methods of mutation detection, such as single-strand conformation polymorphism (SSCP), tandem SSCP/heteroduplex analysis and SNaPshot analysis were developed using homemade kit on ABI 310 genetic analyzer, and were successfully applied to mutation detection of 31 colorectal tumor samples. The sieving capability of homemade kit and commercial kit were compared, results demonstrate that homemade kit has higher resolution and shorter analysis time. In clinical tumor samples, 26% K-ras (exon 1) and 24% p53 (exons 7-8) were found to have mutations, and all mutations were single point variations. A majority of mutations occurred in one gene, only 1 tumor contained alterations in the two genes, which indicates that development of colorectal cancer lies on alternate pathways, and may correlate with different gene mutations.

Identification and expression of TRAF6 (TNF receptor-associated factor 6) gene in Zhikong Scallop Chlamys farreri

Tumor necrosis factor receptor-associated factor 6 (TRAF6), a key signaling adaptor molecule common to the TNFR superfamily and IL-IR/TLR family, is important not only for a diverse array of physiological processes functions of the TNFR superfamily, but also is involved in adaptive immunity and innate immunity. In this report, the first bivalve TRAF6 (named as CfTRAF6) gene is identified and characterized from Zhikong scallop Chlamys farreri. The full-length cDNA of CfTRAF6 is of 2510 bp, consisting of a 5'-terminal untranslated region (UTR) of 337 bp, a 3'-terminal UTR of 208 bp with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame (ORF) encoding a polypeptide of 655 amino acids. The predicted amino acid sequence of CfTRAF6 comprises characteristic motifs of the TRAF proteins, including a Zinc finger of RING-type, two Zinc fingers of TRAF-type, a coiled-coil region, and a MATH (the meprin and TRAF homology) domain. The overall amino acid sequence identity between CfTRAF6 and other TRAF6s is 28-68%. Phylogenetic analyses of CfTRAF6 sequence with TRAF sequences from other organisms indicate that CfTRAF6 is a true TRAF6 orthologue. The mRNA expression of CfTRAF6 in various tissues is measured by Real-time RT-PCR. The mRNA transcripts are constitutively expressed in tissues of haemocyte, muscle, mantle, heart, gonad and gill, but the highest expression is observed in the gonad. The temporal expressions of CfTRAF6 mRNA in the mixed primary cultured haemocytes are recorded after treatment with 20 mu g mL(-1) and 0.5 mu g mL(-1) peptido-glycan (PGN). The expression level of CfTRAF mRNA is down-regulated from 1.5 h to 3 h after the treatment with 0.5 mu g mL(-1) PGN, and then recovers to the original level. While the expression of CfTRAF6 is obviously decreased after treatment with 20 mu g mL(-1) PGN, and reach the lowest point (only about 1/9 times to control) at 3 h. The result Suggests that CfTRAF6 can be greatly regulated by PGN and it may be involved in signal transduction and immune response of scallop. (C) 2008 Published by Elsevier Ltd.

Cloning and expression of tumor necrosis factor (TNF alpha) cDNA from red seabream Pagrus major

A fragment of TNFalpha cDNA sequence from red seabream was cloned by homology cloning approach with two degenerated primers which were designed based on the conserved regions of other animals' TNF sequences. The sequence was elongated by 3' and 5' RACE to get the full length CDS sequence. This sequence contained 1264 nucleotides that included a 5' UTR of 85 bp, a 3' UTR of 514 bp and an open reading frame (ORF) of 666 bp which could encode 222 amino acids propeptide. In 3' UTR, there were several mRNA instability motifs and three endotoxin-responsive sequences, but the sequence lacked the polyadenylation signal. The deduced peptide had a clear transmembrane domain, a TNFalpha family signature and a TNF2 family profile. The cell attachment sequence and the glycosaminoglycan attachment sites were also found in the sequence. The red seabream TNF sequence shared relatively high similarity with both mammalian TNFalpha and TNFbeta by multiple sequence alignments. Phylogenetic analysis showed that the piscine TNFalpha were located independently in a different branch compared with mammalian TNFalpha and TNFbeta. Based on the primary and secondary structure analysis and gene expression study, we could concluded that the red seabream TNF should be a TNFalpha, not TNFbeta. RT-PCR was used to study TNFa transcript expression. 24 h after the red seabream was challenged by Vibrio anguillarum, the RS TNFalpha transcript expression were detected in blood, brain, gill, heart, head kidney, kidney, Ever, muscle and spleen. Results showed that TNFalpha mRNA was constitutively expressed in parts of the tissues both in stimulated and unstimulated fish and the expression could be enhanced after the pathogen infection.

Expression of TNF-alpha gene in Anabaena sp PCC 7120 and purification of its product by affinity chromatography

The effects of N (NaNO3) and C (NaAc) source in medium on the expression of tumor necrosis factor-alpha (TNF-alpha) gene in transgenic Anabaena sp. PCC 7120 were compared. The data showed that N source stabilized the expression of foreign protein and C source altered the synthesis of cell walls. Comparing several methods for breaking the cells, supersonic was able to extract TNF-alpha better than others. For purification of TNF-alpha, transgenic Anabaena cells were broken, the extracts were precipitated with ammonia sulfate, and the impure TNF-alpha was eluted from DEAE ion exchange chromatography. Electrophoresis (PAGE-SDS) showed a single band at 17 kD position.

The expression of AmphiTCTP, a TCTP orthologous gene in amphioxus related to the development of notochord and somites

The translationally controlled tumor protein (TCTP) is highly conserved and has been widely found in eukaryotic organisms. Here, we report the phylogenetic analysis and developmental expression of AmphiTCTP, a TCTP homologous gene in cephalochordate amphioxus. Phylogenetic analysis indicates that the putative protein of AmphiTCTP is close to its vertebrate orthologs. The mRNA of AmphiTCTP is found in fertilized eggs, early cleavage embryo and most of the early developmental stages by in situ hybridization and RT-PCR, but its expression is not detectable from late cleavage stage to mid-gastrula. The expression of AmphiTCTP in zygotes and early cleavage stages shows that AmphiTCTP may be a maternal gene. From the early neurula stage onward, AmphiTCTP transcript is localized in the presumptive notochord, presomitic mesoderm, and nascent somites. However, its expression is gradually down-regulated after the notochord and somites have been formed. The expression pattern of AmphiTCTP thus coincides with the differentiation of the notochord and somites, this suggests that AmphiTCTP may not be a housekeeping gene and may play an important role in mesoderm development. (c) 2007 Elsevier Inc. All rights reserved.

Molecular coordinated regulation of gene expression during ovarian development in the penaeid shrimp

To understand the molecular events of ovarian development in penaeid shrimp, RNA arbitrarily primed polymerase chain reaction (RAP-PCR) was used to identify differentially expressed genes during ovarian maturation in Metapenaeus ensis. From a screening of 700 clones in a cDNA library of the shrimp ovary by the products of RAP-PCR of different maturation stages, 91 fragments with differentially expressed pattern as revealed by dot-blot hybridization were isolated and sequenced. Forty-two of these fragments show significant sequence similarity to known gene products and the differentially expressed pattern of 10 putative genes were further characterized via Northern hybridization. Putative glyceraldehyde-3-phosphate dehydrogenase and arginine kinase are related to provision of energy for active cellular function in oocyte development. Translationally controlled tumor protein, actin, and keratin are related to the organization of cytoskeleton to accomplish growth and development of oocytes. High mobility group protein DSP1, heat shock protein 70, and nucleoside diphosphate kinase may act as repressors before the onset of ovarian maturation. Peptidyl-prolyl cis-trans isomerase and glutathione peroxidase are related to the stabilization of proteins and oocytes. This study provides new insights on the molecular events in the ovarian development in the shrimp.

Highly sensitive determination of the methylated p16 gene in cancer patients by microchip electrophoresis

The p16 tumor suppressor gene is inactivated by promoter region hypermethylation in many types of tumor. Recent studies showed that aberrant methylation of the p16 gene is an early event in many tumors, especially in lung cancer, and may constitute a new biomarker for early detection and monitoring of prevention trials. We detected tumor-associated aberrant hypermethylation of the p16 gene in plasma and tissue DNA from 153 specimens using a modified semi-nested methylation-specific PCR (MSP) combining plastic microchip electrophoresis or slab gel electrophoresis, respectively. Specimens were from 79 lung cancer patients, 15 abdominal tumor patients, 30 positive controls and 30 negative controls. The results showed that the positive rate obtained by microchip electrophoresis was more than 26.6% higher and the same speciticity was kept when compared with slab gel electrophoresis. The microchip electrophoresis can rapidly and accurately analyze the PCR products of methylated DNA and obviously improve the positive rate of diagnosis of cancer patients when compared with gel electrophoresis. This method with the high assay sensitivity might be used for detection of methylation of p16 gene and even to facilitate early diagnosis of cancer patients. (C) 2004 Elsevier B.V. All rights reserved.