Localization of laminin-5, HD1/plectin, and BP230 in the submandibular glands of developing and adult mice

We studied the distributions of laminin-5 and hemidesmosome components, HD1/plectin and BP230, in the submandibular glands of adult and developing mice. In adult mice, laminin-5 was expressed in the basement membranes of both the myoepithelial cells and excretory ducts. The former expression was predictable because laminin-5 is a ligand for hemidesmosomes, which appear in myoepithelial cells and stratified epithelium. However, the latter expression pattern suggested that the non-stratified epithelium of the excretory duct might also be associated with hemidesmosomes. During fetal development, laminin-5 was found in the basement membrane of developing ducts but not epithelial end buds in which future lobules are formed by epithelial branching. The expression of HD1/plectin but not BP230 was noted in the developing duct at early embryonic stages, indicating the presence of type II hemidesmosomes. Expression of BP230 appeared in the excretory duct epithelium at around the day of birth. At this stage, the typical hemidesmosome was observed in the duct epithelium. Our results suggest that laminin-5 is involved in duct development rather than epithelial branching. The results also suggest that the developing duct epithelium interacts with laminin-5 through the type II hemidesmosome, which later matures into a typical hemidesmosome upon the onset of expression of BP230. Accepted: 12 October 1999     

VILIP-1 Expression In Vivo Results in Decreased Mouse Skin Keratinocyte Proliferation and Tumor Development

VILIP-1, a member of the neuronal Ca²⁺ sensor protein family, is able to act as a tumor suppressor in carcinoma cells by inhibiting cell proliferation and migration. In order to study the role of VILIP-1 in skin carcinogenesis we generated transgenic mice overexpressing VILIP-1 in epidermis under the control of the bovine keratin K5 promoter (K5-VILIP-1). We studied the susceptibility of FVB wild type and VILIP-1 transgenic mice to chemically mediated carcinogenesis. After 30 weeks of treatment with a two-stage carcinogenesis protocol, all animals showed numerous skin tumors. Nevertheless, K5-VILIP-1 mice showed decreased squamous cell carcinoma (SCC) multiplicity of ∼49% (p<0.02) with respect to the corresponding SCC multiplicity observed in wild type (WT) mice. In addition, the relative percentage of low-grade cutaneous SCCs grade I (defined by the differentiation pattern according to the Broders grading scale) increased approximately 50% in the K5-VILIP1 mice when compared with SCCs in WT mice. Similar tendency was observed using a complete carcinogenesis protocol for skin carcinogenesis using benzo(a)pyrene (B(a)P). Further studies of tumors and primary epidermal keratinocyte cultures showed that matrix metalloproteinase 9 (MMP-9) levels and cell proliferation decreased in K5-VILIP-1 mice when compared with their wild counterparts. In addition tissue inhibitor of metalloproteinase 1 (TIMP-1) expression was higher in K5-VILIP-1 keratinocytes. These results show that VILIP-1 overexpression decreases the susceptibility to skin carcinogenesis in experimental mouse cancer models, thus supporting its role as a tumor suppressor gene.     

Malabsorption of Dietary Folate (Pteroylpolyglutamates) in Adult Coeliac Disease and Dermatitis Herpetiformis

Tests of absorption of folic acid (pteroylglutamic acid) and of dietary folates (pteroylpolyglutamates) were performed in 10 patients with untreated adult coeliac disease, five with dermatitis herpetiformis, and three with nutritional folate deficiency. Absorption of pteroylglutamic acid was impaired in eight patients with coeliac disease and in two with dermatitis herpetiformis. Absorption of pteroylpolyglutamates was impaired in all 10 coeliac patients and in four of the five patients with dermatitis herpetiformis. Absorption of both forms of folate was normal in all three patients with nutritional folate deficiency.     

丝颖针茅ScTIP1;1基因的克隆及对非生物胁迫的应答

利用丝颖针茅(Stipa capillacea Keng)一条EST序列并结合cDNA末端快速扩增(RACE)技术,克隆了丝颖针茅的一个液泡膜内在蛋白(tonoplast intrinsic proteins,TIPs)基因ScTIP1;1的全长编码区序列。该基因开放阅读框长度为753 bp,编码250个氨基酸,其蛋白质分子量为25.8 kD,理论等电点为6.16;序列比对及系统进化分析表明,ScTIP1;1和拟南芥(Arabidopsis thaliana)AtTIP1;1蛋白的亲缘关系较近;亚细胞定位结果显示,该蛋白位于液泡膜上;实时荧光qRT-PCR检测表明,盐、干旱及低温胁迫可诱导ScTIP1;1基因的表达,且低温处理后基因的表达量变化最为明显。本研究结果为理解丝颖针茅的生态适应性提供了理论依据。     

蔗茅EfMYB1基因的克隆与表达分析

为了充分利用蔗茅(Erianthus fulvus)野生资源,挖掘其优良的抗性基因,丰富转基因甘蔗育种候选基因库,该研究结合蔗茅转录组数据,以蔗茅99 1无性系为试验材料,利用RT PCR技术克隆蔗茅MYB基因,并对其进行生物信息学分析及胁迫表达分析,以解析蔗茅的耐寒机理,为转基因甘蔗育种奠定理论基础。结果表明：(1)成功克隆得到一个蔗茅MYB基因,命名为EfMYB1基因(登录号ON586646)。(2)生物信息学分析表明,EfMYB1基因全长1 000 bp,ORF为759 bp,编码251个氨基酸;编码蛋白具有一个保守的SANT结构域,无跨膜结构和信号肽,有多个磷酸化位点;二级结构与三级结构主要以α螺旋和无规则卷曲为主;与南荻相似性最高,遗传距离最近。(3)qRT PCR分析结果发现,EfMYB1基因在蔗茅根和叶组织中的相对表达量随低温胁迫时间的持续而逐渐显著上调,并于胁迫72 h时达到最大值,而在茎中的表达则几乎没有变化;茉莉酸甲酯胁迫下,EfMYB1基因的相对表达量呈先升高后降低的趋势,且在处理6 h时达到最高值;脱落酸胁迫下EfMYB1基因的表达水平较0 h时极显著降低。研究认为,EfMYB1基因属于低温胁迫响应基因,可能参与蔗茅低温胁迫下的应答反应。     

山茶CjMYB1基因的克隆及表达分析

该研究通过序列比对分析,以野生红山茶和不同花色品种山茶为材料,采用PCR方法克隆CjMYB1基因,并通过生物信息学和表达分析对其进行初步研究,为深入研究山茶CjMYB1基因在花色形成和花发育过程的调控机理奠定理论基础。结果表明：(1)成功克隆获得山茶CjMYB1基因(GenBank登录号为OL347930),其开放阅读框长为879 bp,编码292个氨基酸,相对分子质量为33.17 kD;CjMYB1基因属于R2R3-MYB转录因子,且与拟南芥MYB基因家族的第7亚组处于同一分支。(2)荧光定量PCR分析发现,山茶CjMYB1基因在野生红山茶花芽中表达量最高,在萼片、花瓣、雄蕊和心皮中都有较高的表达量,推测其在山茶花器官发育中发挥着重要作用;在红色山茶品种中表达量较高,而在粉色、淡黄色、白色山茶品种中表达量较低,说明CjMYB1基因可能在红色山茶品种的花色苷合成途径中起到了关键作用。(3)亚细胞定位实验表明,CjMYB1蛋白定位在细胞核。     

Autoreactive T and B cells from bullous pemphigoid (BP) patients recognize epitopes clustered in distinct regions of BP180 and BP230

Bullous pemphigoid (BP) is a well-characterized model of autoantibody-mediated autoimmunity, which presumably depends on autoreactive Th cells that promote the activation of autoreactive B cells. The two major autoantigens of BP are BP180 and BP230, two components of dermoepidermal adhesion complexes. Both, autoreactive Th cell responses and autoantibody profiles were characterized in 35 patients with acute onset BP using BP180 and BP230 proteins. Our findings indicate the following: 1) autoreactive Th cells recognized epitopes within the NH2-terminal (77.1%), COOH-terminal (65.7%), and central portion (57.1%) of the BP180 ectodomain; 2) IgG autoantibodies were found to exhibit similar or identical reactivity against the NH2-terminal (82.8%), COOH-terminal (77.1%), and central portion (37.1%) of the BP180 ectodomain; 3) T and B cell reactivity with the NH2-terminal portion of the BP180 ectodomain was associated with extensive BP, whereas the central portion was more frequently recognized in limited BP; 4) only 7 of 16 (43.7%) and 6 of 16 (37.5%) BP patients showed a Th cellular response against the COOH- and NH2-terminal regions of BP230, respectively, whereas 5) IgG reactivity against the COOH- and NH2-termini of BP230 was detected in 5 of 16 (31.3%) and 6 of 16 (37.5%) patients, respectively. These results demonstrate that Th and B cell reactivities against BP180, are, in contrast to BP230 reactivity, almost constantly detectable in BP patients, and differential epitope recognition of BP180 seems to be associated with distinct clinical severity. These observations support the concept that BP180, but not BP230, is the primary autoantigen of BP critical for disease development.     

Lef-1基因在棕色和白色羊驼皮肤差异表达

本实验旨在研究Lef-1在不同毛色羊驼皮肤中的表达和定位,探索其与毛色间的关系.采用实时荧光定量PCR、Western blotting以及免疫组织化学方法,研究白色和棕色羊驼皮肤中的mRNA、蛋白表达水平和定位.实时荧光定量PCR结果显示,Lef-1在棕色羊驼皮肤组织相对基因表达量是3.3727±0.1989,在白色羊驼皮肤组织是1.0003±0.0227; Western blotting结果显示,在羊驼皮肤组织总蛋白中存在分子量约44 KD与兔抗Lef-1多克隆抗体发生免疫阳性反应的蛋白条带,棕色羊驼平均蛋白表达量显著高于白色羊驼;免疫组织化学结果显示,在棕色羊驼皮肤组织中多表达在毛球部、表皮也有分布,在白色羊驼皮肤组织中多表达在表皮,在棕色和白色羊驼皮肤组织中外根鞘都有较强表达.结果显示Lef-1在棕色和白色羊驼皮肤的定位和含量存在差异,提示Lef-1可能影响了羊驼被毛颜色的形成.     

Decreased Expression Of apM1 in Omental and Subcutaneous Adipose Tissue of Humans With Type 2 Diabetes

We have screened a subtracted cDNA library in order to identify differentially expressed genes in omental adipose tissue of human patients with Type 2 diabetes. One clone (#1738) showed a marked reduction in omental adipose tissue from patients with Type 2 diabetes. Sequencing and BLAST analysis revealed clone #1738 was the adipocyte-specific secreted protein gene apM1 (synonyms ACRP30, AdipoQ, GBP28). Consistent with the murine orthologue, apM1 mRNA was expressed in cultured human adipocytes and not in preadipocytes. Using RT-PCR we confirmed that apM1 mRNA levels were significantly reduced in omental adipose tissue of obese patients with Type 2 diabetes compared with lean and obese normoglycemic subjects. Although less pronounced, apM1 mRNA levels were reduced in subcutaneous adipose tissue of Type 2 diabetic patients. Whereas the biological function of apM1 is presently unknown, the tissue specific expression, structural similarities to TNFα and the dysregulated expression observed in obese Type 2 diabetic patients suggest that this factor may play a role in the pathogenesis of insulin resistance and Type 2 diabetes.     

Decreased Expression of Beclin 1 Correlates Closely with Bcl-xL Expression and Poor Prognosis of Ovarian Carcinoma

Background

It has been suggested that autophagy-related Beclin 1 plays a critical role in the regulation of tumor development and/or progression, but its prognostic significance and relationship with Bcl-xL expression in ovarian carcinoma are unclear.

Methodology/Principal Findings

In the present study, the methods of Western blotting and immunohistochemistry (IHC) were utilized to investigate the expression status of Beclin 1 and Bcl-xL in fresh ovarian tissues and paraffin-embedded epithelial ovarian tumor tissues. Decreased expression of Beclin 1 was examined by IHC in 8.3% of normal ovaries, in 15.4% of cystadenomas, in 20.0% of borderline tumors, and in 55.6% of ovarian carcinomas, respectively. In ovarian carcinomas, decreased expression of Beclin 1 was correlated closely with ascending histological grade, later pT/pN/pM status and/or advanced clinical stage (P<0.05). In univariate survival analysis, a highly significant association between low-expressed Beclin 1 and shortened patient survival was evaluated in ovarian carcinoma patients (P<0.01), and Beclin 1 expression was an independent prognostic factor as evidenced by multivariate analysis (P = 0.013). In addition, decreased expression of Beclin 1 was inversely correlated with altered expression of Bcl-xL in ovarian carcinoma cohort, and combined analysis further showed that the low Beclin 1/high Bcl-xL group had the lowest survival rate.

Conclusions/Significance

Our findings suggest that Beclin 1 expression, as examined by IHC, could be served as an additional tool in identifying ovarian carcinoma patients at risk of tumor progression, and predicting patient survival in ovarian carcinomas with increased expression of Bcl-xL.     

ZNF230/荧光蛋白融合基因表达载体的构建及其在Cos细胞中的表达与定位

为了用绿色荧光蛋白标记观察人类无精症相关基因ZNF230在Cos7细胞中的蛋白质表达及定位,用PCR方法扩增得到突变的人和小鼠mt-ZNF230和mt-znf230基因,使其3′端的终止密码TGA突变为TGG,并装入T-载体,双酶切后通过定向克隆将其与真核表达载体pEGFP-N1的绿色荧光蛋白（green fluorescence protein,GFP）基因融合,构建了ZNF230—荧光蛋白融合基因表达载体。然后经真核表达质粒－脂质体介导,导入Cos7细胞系。荧光显微镜观察显示：在空白载体pEGFP-N1转染的Cos细胞中荧光布满整个细胞,而在转染阳性载体pEGFP-ZNF230的Cos细胞中荧光主要聚集在细胞核中。表明转染的Cos细胞系能高效表达人ZNF230和小鼠znf230蛋白,ZNF基因表达的蛋白定位于细胞核内。Abstract: To use green fluorescent protein as a marker to studythe localization of the fusion protein, the mutant full length cDNAs of human ZNF230 and mouse znf230 with their stop codon TGA changed to TGG were obtained by PCR amplification., and then cloned into pGEM-Teasy vector. After the double enzyme cutting, the mutated human and mouse ZNF230(znf230) were inserted into mammalian expression plasmid pEGFP-N1.Thus we constructed the plasmid with fusion gene of ZNF230 and green fluorescent protein(GFP).Then the Cos cell was transfected with the fused gene by liposome. Fluorescence microscopy showed that green fluorescence protein expressed over the whole cell when transfected with vector pEGFP-N1.While after the transfection with pEGFP-ZNF230, the fluorescence located mainly on the nuclei of the cells. We demonstrated that the transfected Cos cell line can express human ZNF230 and mouse znf230 with high efficiency.When transfected with the constructed recombinant pEGFP-ZNF230 vector, the ZNF230 protein localizes mainly on the nucleus.     

ZNF230/荧光蛋白融合基因表达载体的构建及其在Cos细胞中的表达与定位

为了用绿色荧光蛋白标记观察人类无精症相关基因ZNF230在Cos7细胞中的蛋白质表达及定位,用PCR方法扩增得到突变的人和小鼠mt ZNF230和mt znf230基因,使其3′端的终止密码TGA突变为TGG,并装入T 载体,双酶切后通过定向克隆将其与真核表达载体pEGFP N1的绿色荧光蛋白(greenfluorescenceprotein,GFP)基因融合,构建了ZNF230—荧光蛋白融合基因表达载体。然后经真核表达质粒-脂质体介导,导入Cos7细胞系。荧光显微镜观察显示:在空白载体pEGFP N1转染的Cos细胞中荧光布满整个细胞,而在转染阳性载体pEGFP ZNF230和pEGFP znf230的Cos细胞中荧光主要聚集在细胞核中。表明转染的Cos细胞系能高效表达人ZNF230和小鼠znf230蛋白,ZNF230基因表达的蛋白定位于细胞核内。     

铁皮石斛钙网蛋白基因DoCRT1的分子克隆与表达研究

该研究采用RACE克隆铁皮石斛钙网蛋白(calreticulin,CRT)基因DoCRT1,进行生物信息学分析,并借助定量PCR检测基因表达模式,为揭示该基因在铁皮石斛生长发育及逆境生理中的分子作用奠定基础。结果表明:(1)DoCRT1基因(GenBank登录号KT957551)cDNA全长1 672bp,ORF长1 275bp,编码一条由424个氨基酸组成的多肽,分子量49.05kD,等电点4.42,具有CRT蛋白保守的钙网蛋白/钙联接蛋白P结构域(205~320)和类伴刀豆球蛋白凝集素/葡聚糖酶结构域A(20~222)以及多个基序;蛋白N端含有一个信号肽(1~23)和一个跨膜区域(8~24),预测结果显示主要定位于细胞液泡、胞外,与多种植物CRT蛋白一致性很高(80%~87%)。(2)DoCRT1蛋白与OsCRT1/2、ZmCRT1亲缘关系近,聚在CRT进化树的CRT1/2分支。(3)qRT-PCR检测结果显示,DoCRT1基因转录本在石斛根中表达量较高,为叶中的2.23倍,且茎与叶中表达量无显著差异。     

CCHCR1 Is Up-Regulated in Skin Cancer and Associated with EGFR Expression

Despite chronic inflammation, psoriatic lesions hardly ever progress to skin cancer. Aberrant function of the CCHCR1 gene (Coiled-Coil α-Helical Rod protein 1, HCR) within the PSORS1 locus may contribute to the onset of psoriasis. As CCHCR1 is expressed in certain cancers and regulates keratinocyte (KC) proliferation in a transgenic mouse model, we studied its relation to proliferation in cutaneous squamous cell cancer (SCC) cell lines by expression arrays and quantitative RT-PCR and in skin tumors by immunohistochemistry. CCHCR1 protein was detected in the pushing border of SCC and lining basal cell carcinoma islands. Different from psoriasis, Ki67 had a similar expression pattern as CCHCR1. The most intense CCHCR1 staining occurred in areas positive for epidermal growth factor receptor (EGFR). Expression of CCHCR1 mRNA was upregulated 30–80% in SCC lines when compared to normal KCs and correlated positively with Ki67 expression. The most aggressive and invasive tumor cell lines (RT3, FaDu) expressed CCHCR1 mRNA less than non-tumorigenic HaCaT cells. Moreover, the tumor promoters okadaic acid and menadione downregulated CCHCR1 mRNA. We conclude that both in psoriasis and the early stages of KC transformation, CCHCR1 may function as a negative regulator of proliferation, but beyond a certain point in oncogenesis cannot control this phenomenon any longer.     

表没食子儿茶素-3-没食子酸酯抑制TNF-a和IL-1β在小鼠皮肤烫伤修复期间的表达

肿瘤坏死因子-a(tumor necrosis factor-a,TNF-a)和白细胞介素-1β(interleukin, IL-1β)在创伤修复中起着至关重要的作用.本研究利用小鼠皮肤深II度烫伤模型,采用逆转录聚合酶链反应(RT-PCR)和酶联免疫吸附试验(ELISA)检测烫伤部位组织Tnf-a mRNA和Il-1β mRNA的表达水平以及TNF-a和IL-1β的含量,以探讨表没食子儿茶素-3-没食子酸酯(EGCG)对小鼠皮肤烫伤修复期间TNF-a和IL-1β表达的影响.结果显示,用0.2 mg/g EGCG膏剂涂敷烫伤皮肤,处理12 h可致组织Tnf-a mRNA表达水平和TNF-a含量下降,处理24 h可致组织Il-1β mRNA表达水平和IL-1β含量下降.上述结果提示0.2 mg/g EGCG处理能抑制烫伤组织TNF-a和IL-1β的表达,减弱创伤组织的炎症反应,有助于创伤组织的修复.