Restin, belonging to the melanoma-associated antigen superfamily, was firstly cloned from the differentiated HL-60 cells when induced by all-trans retinoic acid (ATRA) in our lab. Our previous results showed that restin might be correlated to cell cycle arrest. Due to the importance of p53 in the regulation of cell growth and the relationship between p53 and ATRA, we tried to test the relationship between p53 and restin. Firstly, transfection results showed that p53 was able to upregulate the expression of restin at the transcriptional level when p53 was transfected into eukaryotic cells. Secondly, the bioinformatics analysis revealed that the upstream sequence (about 2 kb) from the first ATG of the ORF of restin gene contained a p53 binding site. In order to confirm that p53 was involved in the transcriptional regulation of restin, we cloned the upstream sequence of restin and constructed the promoter luciferase reporter system. From the luciferase activity, we demonstrated that the promoter of restin gene could be induced by ATRA. Then, another two luciferase reporter plasmids driven by the reporter of restin with no (RPΔp53-luc) or mutant (mRP-luc) p53 binding site were constructed to see the regulation of restin by p53. Results showed that the transcriptional upregulation of restin gene was not due to the putative p53 binding site on the upstream of restin gene. We proposed that p53 upregulated restin transcription through an indirect way rather than direct interaction with the cis-activating element of the restin promoter.

Human Apr3 was first cloned from HL-60 cells treated by ATRA. In this study, we further demonstrated that Apr3 could be obviously upregulated by ATRA in many other ATRA sensitive cells, suggesting a common role of Apr3 in ATRA effects. Indirect immunofluorescence assay indicates that Apr3 is a membrane protein, while its truncated form without the predicted transmembrane and intracellular domain, was likely a secreted one. Furthermore, FACS analysis showed that Apr3 overexpression could cause an obvious G1/S phase arrest which might be induced by dramatic reduction of cyclin D1 expression. Strikingly, the truncated Apr3 antagonized the negative role of Apr3 on cell cycle and cyclin D1. Taken together, our data suggest that Apr3 should play an important role in ATRA signal pathway and the predicted transmembrane and/or the intracellular domain mediates Apr3 membrane localization and is vital for the negative regulation on cell cycle and cyclin D1.

OBJECTIVE:To study the biological properties of human dental pulp cells (HDPC) by cloning and analysis of genes differentially expressed in HDPC in comparison with human gingival fibroblasts (HGF).METHODS:HDPC and HGF were cultured and identified by immunocytochemistry. HPDC and HGF subtractive cDNA library was established by PCR-based modified subtractive hybridization, genes differentially expressed by HPDC were cloned, sequenced and compared to find homogeneous sequence in GenBank by BLAST.RESULTS:Cloning and sequencing analysis indicate 12 genes differentially expressed were obtained, in which two were unknown genes. Among the 10 known genes, 4 were related to signal transduction, 2 were related to trans-membrane transportation (both cell membrane and nuclear membrane), and 2 were related to RNA splicing mechanisms.CONCLUSION:The biological properties of HPDC are determined by the differential expression of some genes and the growth and differentiation of HPDC are associated to the dynamic protein synthesis and secretion activities of the cell.

Little is known about the origin and genetic diversity of swamp buffaloes in China. To obtain more knowledge on genetics of the water buffalo in China, the complete mitochondrial D-loop sequences of 30 samples from 6 native types were investigated. The results revealed 12 mitochondrial haplotypes with 50 polymorphic sites. Among these polymorphic sites, there were 49 transitions and 1 transversion. The average nucleotide diversity and haplotype diversity estimated from mtDNA D-loop region in 6 Chinese water buffalo types were 0.00684 and 0.798, respectively, showing rather abundant mitochondrial genetic diversity. The Neighbor-Joining (NJ) tree of mtDNA of Chinese water buffaloes was constructed according to the 12 haplotypes. The NJ tree indicated two lineages being designated lineage A and lineage B, in which lineage A was predominant, and lineage B was at low frequency. The new lineage B was first discovered and defined in 6 Chinese water buffalo types. These results showed that two different maternal lineages were involved in the origin of domestic swamp buffaloes in China and the lineage B was probably an introgression from Southeast Asian buffaloes.

AIM: To clone the promoter and construct its luciferase report vector of our newly found gene restin and to analyze its response to the stimulation with all-trans-retinoic acid (ATRA). METHODS: Approximate 2000 bp up from the ATG of gene restin was bioinformatics analyzed with computer. The fragment was generated by polymerase chain reaction and cloned the upstream of luciferase report gene into plasmid pG5-luc, replacing the five GAL-4 binding sites and adenovirus promoter to generate the plasmid Rp-luc. Rp-luc was transfected into HeLa cells and luciferase activities were measured with or without the treatment of ATRA at indicated time. RESULTS: We successfully cloned and identified the promoter of the gene restin and constructed its luciferase report plasmid. Obvious response to ATRA was found when transfected into HeLa cells and a high expression of luciferase was detected when induced with ATRA. CONCLUSION: The luciferase report system of restin gene promoter we have constructed can be applied for further study of the function and transcriptional regulation of restin.

Restin, isolated from tumor differentiation cell induced by ATRA, is the novel member of MACE （melanoma antigen encoding gene） family. To obtain some clues of its biological function, this work is going to investigate the restin expression level in variety of tumors and cells. Using the code region of restin labeled with #alpha#-~(32)P-dCTP as a probe, the mRNA expression of restin was examined in multiple tissue northern（MTN） blot membrane and multiple tissue express （MTE）array by Northern blot and dot blot hybridization, respectively. The mRNA expression of restin was examined by in situ hybridization with restin probe labeled with dig in 11 tumor tissues. Northern blot showed that all 12 normal tissues displayed hybrid bands with same location, approximately 1.8 kb. These results suggested that the restin mRNA could not present some splicing variants. Dot blot showed that the restin high expressed in all normal tissues, but less or not expressed in tumor cell lines. In 11 tumor tissues, 8 malignant tumors displayed no expression, but 3 benign tumors displayed lower expression. These results suggested that human restin was rarely expressed in cancer ceil lines, tumor tissues and but ubiquitously expressed in many normal tissues. These results were quite different from the expression of other MAGEs, such as MAGE-A, B, and C. Considering the inducible expression of restin in tumor tissues by ATRA, we deduced that the restin was possibly to be a regulator of cell proliferation and differentiation.

In this paper, we have analyzed the nuclear DNA of Alectoris magna samples, collected from the Longzhong Loess Plateau. We used allelic variation at eight microsatellite markers to describe the genetic structure of A. magna populations. The primary goal of this study was to examine the population genetic structure and determine the extent of population differentiation among populations of A. magna . The average value of H E (0.455) was smaller than H O (0.477), and there was a heterozygote deficit at the MCW135 locus in the Lanzhou population and the Beidao population. The AB063167 locus in each population (except that of Jingyuan) was not in equilibrium ( P < 0.05). The Lixian edge population had a lower proportion of genetic diversity than the central geographic populations; the Haiyuan and Jingyuan populations had higher genetic diversity than the central geographic populations. Analysis of population structure revealed clear differentiation among the eight populations of A. magna , suggesting strong isolation of these populations and correspondingly low levels of migration or gene flow. The A. magna populations of Longzhong Loess Plateau are separated into eastern and western populations by a clustering chart. Genetic data indeed suggest that patterns of speciation and population diversification of A. magna in the Longzhong Plateau have been affected by the stability of the climate, natural selection, and human intervention.

AIM:To obtain recombinant human defensin alpha(HDalpha) and detect its biological activity, so as to facilitate further research.METHODS:The HDalpha gene fragment with hydroxylamine cleavage site was synthesized, and then cloned into pBV220-IL-4 vector to construct pBV220-IL-4-HDalpha. The constructed vector which was confirmed to be correct by sequencing was transformed into E.coli DH5alpha and the IL-4-HDalpha fusion protein was expressed under temperature induction. After fusion protein was cleaved to remove IL-4 by hydroxylamine, purification and renaturation was performed. HDalpha's characteristics were identified by SDS-PAGE and bioactivity detection.RESULTS:After temperature induction, the expressed fusion protein which accounted for about 20% of total bacterial protein existed mainly in the form of inclusion body. After cleaving by hydroxylamine, the purity of obtained HDalpha was about 99.8%. Bacteriostatic test and clone forming test showed that recombinant HDalpha could obviously inhibit the growth of bacteria.CONCLUSION:The recombinant expression plasmid for HDalpha gene has been constructed successfully and obtained engineering bacteria can stably express target protein. Furthermore, techniques of purification and renaturation was set out, which lays the foundation for further functional study and application of HDalpha.

Activating transcription factor 3 (ATF3), a member of the activating transcription factor/cAMP responsive element binding protein (ATF/CREB) family of transcription factors, is induced by many physiological stresses. To investigate the activity of ATF/CREB in cells with physiological stresses, we developed a practical reporter vector, the plasmid pATF/CRE-luc, bearing activating transcription factor/cAMP responsive element (ATF/CRE) binding sites. This plasmid was constructed by inserting three repeats of the ATF/CRE binding element into the plasmid pG5luc, replacing the GAL-4 binding sites. The plasmids pACT/ATF3 and pATF/CRE-luc were transfected into HeLa and NIH3T3 cells, respectively, and the results showed that the expression of luciferase was increased in a dose-dependent manner on plasmid pACT/ATF3. The data suggested that the plasmid pATF/CRE-luc could be used as a sensitive and convenient reporter system of ATF3 activity.

restin 基因是Zhu等人从全反式维甲酸(all-trans retinoic acid, ATRA)诱导肿瘤细胞分化时克隆得到的一种黑色素瘤抗原相关基因. 前期研究表明, 该基因与细胞周期阻滞有关. 由于p53在细胞增殖调控中占据重要地位, 并且与维甲酸具有诱导关系, 因此本研究试图揭示维甲酸诱导 restin 基因表达是否与p53有关. 将p53转染真核细胞, 研究 restin 与p53的表达关系. 结果表明p53能诱导 restin 基因转录增加. 进一步分析发现, restin 基因5¢端上游约2 kb基因组序列中存在p53蛋白结合位点. 扩增该序列构建荧光报告系统, 检测到该报告系统可以接受维甲酸的诱导调控. 在此基础上, 研究p53对缺失p53结合位点的截短体报告质粒和p53结合位点突变后的报告质粒的作用, 结果显示p53调控restin基因的表达与该基因上游序列区中的p53结合位点无关, 推测p53对 restin 基因的表达调控可能还存在其他分子的相互作用.

The oncoprotein c-Jun is a component of the activator protein-1 transcription factor complex, which is involved in cellular proliferation, transformation, and death. The stabilization of c-Jun is critically important for its function. The phosphorylation of c-Jun by c-Jun NH(2)-terminal kinase 1 and extracellular signal-regulated protein kinases reduces c-Jun ubiquitination resulting in increased stabilization of c-Jun. In this report, we showed that COOH-terminal Src kinase (CSK) binds with and phosphorylates c-Jun at Y26 and Y170. Phosphorylation of c-Jun by CSK, in opposition to c-Jun NH(2)-terminal kinase 1 and extracellular signal-regulated protein kinases, promoted c-Jun degradation and reduced stability. By promoting c-Jun degradation, CSK helps to maintain a low steady-state level of c-Jun, thereby inhibiting activator protein-1 activity and cell transformation caused by c-Jun. These results indicated that this function of CSK controls cell proliferation under normal growth conditions and may have implications for CSK loss of function in carcinogenesis.

AIM:To clone a new human gene, restin, from retinoic acid-treated promyelocytic cell line HL-60, express the protein in E.coli and prepare the antisera against the protein.METHODS:The restin gene was amplified from retinoic acid-treated promyelocytic cell line HL-60 by RT-PCR and cloned into a prokaryotic expression vector. The recombinant restin was induced to express in E. coli by temperature. After preliminary purification by SDS-PAGE, the restin protein was used to immunize rabbits to obtain antisera. The titers and specificity of the rabbit anti-restin antisera were tested by Western blot and immunofluorescence analysis.RESULTS:Recombinant restin with M(r) being about 26,000 was highly expressed in E. coli. The titers of the anti-sera to restin ranged from 1:100 to 1:800. Immunofluorescence analysis showed that restin distributed mainly in the nuclei of COS-7 cells.CONCLUSION:We successfully prepared the antisera against restin, which are useful for further investigation of biological functions of restin.

目的:为进一步研究在肝癌上过表达的癌相关基因Gankyrin的功能,进行GST-Gankyrin融合蛋白表达载体的构建、原核表达、及抗体制备.方法:采用逆转录PCR(RT-PCR)法从人肝癌细胞系hepG2中扩增Gankyrin Cdna编码区,并将其重组于谷胱甘肽硫转移酶(GST)融合蛋白表达质粒Pgex-4T2中,经酶切、序列鉴定分析后,用该重组质粒转化大肠杆菌DH-5α,经IPTG诱导获得表达,并将Gankyrin蛋白免疫家兔,经过Western blot检测抗Gankyrin抗体的产生.结果:成功构建了Gankyrin原核表达载体,并在大肠杆菌DH-5α中获得表达,Western blot检测证实获得了抗Gankyrin抗体.结论:成功表达了Gankyrin蛋白并制备了其特异性抗体.

防御素(Defensin)是一类阳离子大环形多肽,在人体多种器官和组织广泛存在,起着重要的屏障防御作用,对G+/-菌、真菌及病毒都有杀灭作用.临床观察表明,测定血浆及各种体液中防御素的改变有助于某些炎症性疾患的诊断,如脓毒症、细菌感染及早产等.此外,提纯或重组防御素作为"超级抗生素"来取代传统的抗生素已成为药学界的研究热点.本研究拟在大肠杆菌中表达人防御素-α,建立稳定表达的工程菌和纯化、复性技术途径,获得活性蛋白,为进一步研究提供材料.

Restin, a member of melanoma-associated antigen superfamily gene, was first cloned from differentiated leukemia cell induced by all trans-retinoic acid, and was able to inhibit cell proliferation, but the molecular mechanism was not clear. Since Restin was localized in cell nucleus, and its homolog member, Necdin (neuronal growth suppressor factor), could interact with transcription factors p53 and E2F1, we proposed that Restin might also function as Necdin through interacting with some transcription factors. In this study, transcription factors p53, AP1, ATFs and E2Fs were cloned and used in the mammalian two-hybrid system to identify their interaction with Restin. The results showed that only ATF3 had a strong interaction with Restin. It is interesting to know that ATF3 was an important transcription factor for G1 cell cycle initiation in physiological stress response. It was possible that the inhibition of cell proliferation by Restin might be related with the inhibition of ATF3 activity.

[Objective] To determine the expression of Gankyrin gene in hepatocellular carcinoma cell lines and to screen target cells for the study of RNA interference targeting at Gankyrin in hepatocellular carcinomas. [Methods] The expression of Gankyrin in HepG2, SMMC-7721 and HHCC cell lines was detected using RT-PCR and Western blotting analysis. [Results] The expression of Gankyrin mRNA and protein could be detected in all three hepatocellular carcinoma cell lines. [Conclusions] Gankyrin gene expressed in HepG2,SMMC-7721, HHCC cell lines, all three cell lines could be used in the study of suppressing the expression of Gankyrin using RNAi in HCCs.

目的:探索通过RNA干涉技术抑制gankyrin的表达对肝癌细胞系HepG2增殖及细胞周期的影响.方法:建立稳定表达靶向gankyrin的shRNA的HepG2细胞系.通过Western blotting验证基因表达的抑制效果.采用细胞计数及MTT法检测细胞的增殖能力.流式细胞仪分析细胞周期.结果:抑制gankyrin基因的表达显著抑制了HepG2细胞的生长,与转染阴性对照shRNA质粒细胞相比,细胞计数示细胞数目明显减低(d6:24.4×103±5.2×103vs123.3×103±2.8×103,P＜0.05),MTT分析显示细胞增殖率明显下降(144 h:7.53±0.50vs16.30±0.38,P＜0.05),流式细胞分析示细胞周期阻滞在G1期(G1:71.63±3.60%vs52.57±2.82%,P＜0.05).结论:在HepG2细胞中抑制gankyrin的表达可有效抑制细胞生长并导致细胞周期的阻滞.利用RNA干涉技术抑制gankyrin的表达可能会成为一种有效的肝癌基因治疗手段.

Restin是从维甲酸诱导分化的白血病细胞中克隆的一种黑色素瘤相关抗原家族基因, 对细胞增殖具有抑制作用. 由于该基因定位于细胞核, 且其同源基因 Necdin (神经细胞生长抑制因子)与转录因子p53和E2F1具有强烈的相互作用, 推测Restin也可能作用于某种转录因子. 为此, 克隆了转录因子p53, AP1, ATF和E2F, 研究Restin与这些转录因子的相互作用. 结果发现Restin与ATF3表现出强烈的相互作用. ATF3是细胞应激反应中非常重要的转录因子, 推测Restin对细胞增殖的抑制作用可能与ATF3活性的抑制有关.

In order to investigate the varied expression of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) in hypertrophied ventricle induced by pressure overload, the abundance of PPAR-gamma in ventricles of spontaneously hypertensive rats (SHR) were detected. Using age matched Wistar-Kyoto rats (WKY) as control, the body mass (BM), left ventricle (LV) wet mass (WM), interventricular septum (IS) WM and right ventricle (RV) WM from 4-week-old and 16-week-old SHR were detected respectively. Then the protein expression in ventricles of WKY and SHR was detected by immunohistochemistral techniques and Western blot, and the mRNA level was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The ratio of LVWM, ISWM and RVWM to BM in 4-week-old SHR were similar to that in 4-week-old WKY (P>0.05), and the protein and mRNA level of PPAR-gamma in ventricles of 4-week-old SHR were also similar to that of age matched WKY (P>0.05). In 16-week-old SHR, the ratio of LVWM and ISWM to BM were higher compared with age matched WKY (P<0.01), whereas there was no significant difference in RV (P>0.05). The protein and mRNA level in LV and IS of 16-week-old SHR were great lower than that of 16-week-old WKY (P<0.01), while in RV there was no significant difference of protein (P>0.05), and a little decrease of mRNA (P<0.05). The findings suggested that the expression level of PPAR-gamma in hypertrophied ventricles induced by pressure overload ( LV and IS in 16-week-old SHR) was significantly decreased, and the decreased expression of PPAR-gamma in ventricles might contribute to ventricular remodeling in hypertension.