Epigallocatechin‐3‐Gallate Inhibits Ethanol‐Induced Apoptosis Through Neurod1 Regulating CHOP Expression in Pancreatic β‐Cells

Epiga‐llocatechin‐3‐gallate (EGCG) is one kind of polyphenol abundant extracted from green tea which has a potent antidiabetic activity. However, the molecular mechanisms mediating the protection procession of EGCG are still unclear. The aim of this study was to investigate the protective effect of EGCG on pancreatic β‐cells exposed to ethanol and the possible underlying mechanisms. To observe the effect of EGCG, we assessed apoptosis in βTC‐6 and INS‐1 cells, which were in complete medium containing 60 mM ethanol, or coincubation with different concentration of EGCG. We also evaluated the roles of Neurod1 in CHOP expression and ethanol‐mediated damage through plasmid overexpression. Treatment with EGCG decreased CHOP expression and apoptosis, whereas its treatment increased Neurod1 expression in ethanol‐treated βTC‐6 and INS‐1 cells. Overexpression of Neurod1 caused the decrease of CHOP expression and apoptosis in ethanol‐treated cells. Furthermore, Neurod1 inhibited CHOP expression by deacetylation of Histone H4 at the CHOP gene promoter. In addition, EGCG partially restores the activity of Neurod1 binding to CHOP promoter in ethanol‐treated cells. In conclusion, EGCG protected β‐cell against ethanol‐induced β‐cell apoptosis by Neurod1 regulating CHOP expression. Anat Rec, 299:573–582, 2016. © 2016 Wiley Periodicals, Inc.     

High frequency recombination betweenloxP sites in human chromosomes mediated by an adenovirus vector expressing Cre recombinase

An adenovirus vector (AdCre1) expressing Cre recombinase has been used to induce recombination betweenloxP sites in human chromosomes. G418 resistant cells with oneloxP site, generated by transfection with a plasmid containingloxP between the SV40 promoter and the G418 resistance (neo) gene, were infected with AdCre1 and transfected with a plasmid containingloxP adjacent to a promoterless hisD gene. This resulted in integration of hisD downstream of the SV40 promoter with gain of histidinol and loss of G418 resistance. Since AdCre1 is non-replicating and Cre expression transient, histidinol resistant cells containing the hisD gene flanked byloxP sites were stable. Reinfection of these cells with AdCre1 induced excision of hisD in over 90% of infected cells. This high efficiency of site-specific recombination suggests that AdCre1 may be exploited for temporal and tissue-specific regulation of gene expression and for chromosome engineering in vitro and in animals.     

双重复合糖基转移酶的ABO基因启动子CpG岛甲基化

背景：在ABO血型抗原的研究中,绝大多数样本是相同的ABO基因表达出正常的相同的ABH抗原。但有一定数量的样本表现出具有相同分子遗传背景但表达出来的抗原强度却随着家系\个体不同而有所差异,说明了ABO血型中复杂的表达调控机制。分析一类罕见的双重复合型标本的ABO血型血清学与基因背景情况,深入表观遗传学的研究,有利于部分揭示ABO基因表达机制。 目的：探讨双重复合型ABO糖基转移酶表达相关的ABO基因启动子CpG岛甲基化水平与ABH抗原表达的关系。 方法：6例经血型血清学定为CisAB或B(A)型的标本,进行ABO基因编码区全长序列和启动子序列的测定,采用重亚硫酸盐处理法检测ABO基因启动子CpG岛甲基化程度。 结果与结论：6例双重复合AB型的标本中,在B101等位基因基础上存在nt803C > G突变的2个CisAB05/B(A)06等位基因,在ABO启动子CpG岛区域两者在nt-33(30%)、nt+27(50%)、nt+49(50%)具有甲基化差异;在A101等位基因序列的基础上存在nt803C > G突变的2个CisAB01等位基因,在ABO启动子CpG岛区域两者在nt-26(10%)位置有甲基化差异;在B101等位基因基础上存在nt640A > G突变的2个B(A)04等位基因ABO启动子CpG岛,在nt-33(10%)、nt+16(50%)、nt+57(60%)、nt+59(60%)、nt+68(60%)和nt+74(60%)位有甲基化差异。全部的6例标本ABO基因启动子区域DNA序列无任何突变异常。结果提示在相同的ABO遗传基因背景下,ABO基因启动子CpG岛区域某些位点甲基化可能影响ABH抗原在红细胞膜表面的表达。     

人LIGHT胞外区基因的克隆及融合蛋白的表达

目的：克隆人LIGHT胞外区基因（hsLIGHT），构建其原核表达质粒，并诱导其在大肠杆菌中表达融合蛋白。方法：采用RT-PCR方法从HL60细胞中扩增hsLIGHT，将其克隆人原核表达质粒pGEX-4T-2，构建其重组表达质粒pGEX-4T-2／hsLIGHT，以不同浓度IPTG诱导表达，于不同时间经SDS-PAGE和Western blot分析、鉴定。结果：经RT-PCR扩增获得的hsLIGHT序列与Genebank报道的LIGHT基因胞外区序列完全一致；SDS-PAGE和Westernblot分析证实重组质粒可表达出相对分子量为47000的蛋白，与GST-hsLIGHT融合蛋白分子量一致。结论：成功完成了人LIGHT胞外区基因的克隆及其原核表达质粒的构建，在大肠杆菌E-coli BL21中经IPTG诱导表达了融合蛋白GST-hsLIGHT，为进一步探讨LIGHT的抗肿瘤生物学活性、探索肿瘤免疫治疗新方法奠定了基础。     

Biolistic nuclear transformation of Saccharomyces cerevisiae and other fungi

Summary The yeast Saccharomyces cerevisiae has been engineered to synthesize and secrete desulfato-hirudin (hirudin), a thrombin inhibitor from the leech Hirudo medicinalis. The synthetic gene coding for hirudin was expressed constitutively under the control of four size-variants of the yeast glyceraldehyde-3-phosphate dehydrogenase promoter (GAP) and cloned into a 2 based multicopy yeast vector. The constitutive action of the four promoter variants was confirmed by demonstrating that the expression and secretion of hirudin is growth-related. The different efficiencies of the promoter variants not only affected hirudin expression but also led to changes in several cellular parameters, such as cell growth, average plasmid copy number and plasmid stability. The observed changes show that yeast cells establish a specific equilibrium for each promoter variant. We conclude, that the adjustment of cellular parameters in response to the expression levels of a heterologous protein is regulated by two counteracting selective forces: (1) the need for complementation of the auxotrophic host marker by the plasmid-encoded selection gene which, in the case of dLEU2, requires several plasmid copies; and (2) a selective advantage of cells with a lower copy number enabling them to escape the burden of heterologous protein production.     

Differentiation of human hematopoietic cells increases expression of a gene transferred by a retroviral vector

To study expression of a retroviral vector in human hematopoietic lineages, two established human hematopoietic cell lines (HL60 and K562) and a human adherent stromal cell line (KM101) were infected with the vector pZIP-SV(X). Expression of the transferred neomycin resistance gene (neor) of pZIP-SV(X) was evaluated as the ability of the cells to form colonies (greater than 50 cells) in an agar assay in the presence of the neomycin analogue, G418. After infection, all three cell lines produced colonies resistant to G418. The level of neor mRNA in separate colonies was analyzed by Northern blot analysis. The neor gene transferred by the vector pZIP-SV(X) was expressed in both human hematopoietic and stromal cell lines. In addition, primary adherent human stromal cells infected with pZIP-SV(X) grew in the presence of G418. To determine if differentiation of hematopoietic cells affects expression of the retroviral vector, HL60 cells infected with pZIP-SV(X) were induced to differentiate, and the level of neor mRNA measured. The amount of neor mRNA increased when HL60 cells were induced to differentiate along the granulocytic pathway. Conversely, when HL60 cells were induced toward monocytoid differentiation (TPA), the level of neor mRNA did not significantly increase. We conclude that the neor gene transferred by a retroviral vector, pZIP-SV(X), is functionally expressed. In addition, expression of the transferred neor gene is regulated during myeloid differentiation of HL60 cells.     

Overexpression of chromatin assembly factor‐1 p60, poly(ADP‐ribose) polymerase 1 and nestin predicts metastasizing behaviour of oral cancer

Mascolo M, Ilardi G, Romano M F, Celetti A, Siano M, Romano S, Luise C, Merolla F, Rocco A, Vecchione M L, De Rosa G & Staibano S
(2012) Histopathology
Overexpression of chromatin assembly factor‐1 p60, poly(ADP‐ribose) polymerase 1 and nestin predicts metastasizing behaviour of oral cancer Aims: The natural history of oral squamous cell carcinomas (OSCCs) is variable and difficult to predict. This study aimed to assess the value of the expression of poly(ADP‐ribose) polymerase 1 (PARP‐1), chromatin assembly factor‐1 (CAF‐1)/p60 and the stem cell markers CD133, CD166, CD44, CD44v6 and nestin as markers of outcome and progression‐free survival in OSCC patients. Methods: Clinical data were collected from 66 patients (41 male and 25 female, aged 29–92 years) who underwent surgery for OSCC of the tongue, floor, lips, and palate. During follow‐up (range: 12–131 months), 14 patients experienced relapse/metastasis and/or death. The study was performed by immunohistochemistry on paraffin‐embedded tumour tissues, western blot analysis of tumour protein lysates and human cell lines, and RNA silencing assays. In addition, the human papillomavirus (HPV) status of primary tumours was evaluated by immunohistochemistry and viral subtyping. Univariate and multivariate analyses were performed to determine the correlation between these parameters and the clinical and pathological variables of the study population. Results and conclusions: We found that a PARP‐1^high/CAF‐1 p60^high/nestin^high phenotype characterized the OSCCs with the worst prognosis (all HPV‐negative). This may be of benefit in clinical management, since radio‐enhancing anti‐PARP‐1 and/or anti‐CAF‐1/p60 agents may allow radioresistance to be bypassed in the nestin‐overexpressing, metastasizing OSCC cells.     

Parp‐1 deficiency in ES cells promotes invasive and metastatic lesions accompanying induction of trophoblast giant cells during tumorigenesis in uterine environment

Embryonic stem (ES) cells deficient in poly(ADP‐ribose) polymerase‐1 (Parp‐1) develop into teratocarcinomas with the appearance of trophoblast giant cells (TGCs) when injected subcutaneously into nude mice. Because the uterus is one of the original organs in which germ cell tumors develop with induction of trophoblast lineage, here we investigated whether Parp‐1 deficiency in ES cells affects teratocarcinoma formation processes by grafting ES cells into the horns of uteri. Teratocarcinomas developed from both wild‐type (Parp‐1^+/+) and Parp‐1^?/? ES cells. The weights of the tumors derived from Parp‐1^?/? ES cells were lower than those of the tumors derived from Parp‐1^+/+ ES cells (P < 0.05). The Parp‐1^?/? tumors showed the appearance of TGCs. Notably, organ metastasis to the lung and liver was observed for the Parp‐1^?/? tumors, but not for the Parp‐1^+/+ tumors (P < 0.05). Invasions were more frequently observed with the Parp‐1^?/? tumors compared with the Parp‐1^+/+ tumors (P < 0.05). Since TGCs are known to have invasive properties, the appearance of TGCs may have supported the metastatic process. The present findings suggest that loss of Parp‐1 during teratocarcinoma formation might augment invasive and metastatic properties of the tumors in the uterine environment.     

Transcriptional regulatory regions of gap43 needed in developing and regenerating retinal ganglion cells

Mammals and fish differ in their ability to express axon growth‐associated genes in response to CNS injury, which contributes to the differences in their ability for CNS regeneration. Previously we demonstrated that for the axon growth‐associated gene, gap43, regions of the rat promoter that are sufficient to promote reporter gene expression in the developing zebrafish nervous system are not sufficient to promote expression in regenerating retinal ganglion cells in zebrafish. Recently, we identified a 3.6‐kb gap43 promoter fragment from the pufferfish, Takifugu rubripes (fugu), that can promote reporter gene expression during both development and regeneration. Using promoter deletion analysis, we have found regions of the 3.6‐kb fugu gap43 promoter that are necessary for expression in regenerating, but not developing, retinal ganglion cells. Within the 3.6‐kb promoter, we have identified elements that are highly conserved among fish, as well as elements conserved among fish, mammals, and birds. Developmental Dynamics 239:482–495, 2010. © 2009 Wiley‐Liss, Inc.