Molecular cloning and expression of a novel alternative splice variant of BRDT gene

In this study, a human adult testis cDNA microarray was constructed and hybridized with (33)P-labeled human adult testis, embryo testis and sperm cDNA probes, respectively. A novel alternative splice variant of BRDT gene, named BRDT-NY, presumably involved in testicular function was cloned. It was expressed 3.96-fold more in human adult than embryo testis and also expressed in human spermatozoa. Similarly, RT-PCR revealed a differential expression pattern of this gene in human adult testes and fetal testes. The full length of BRDT-NY was 3438 bp and contained a 2883 bp open reading frame, encoding a 960-amino-acid protein. Sequence analysis showed that it has two bromodomains in N-terminal of the protein. Multiple tissue RT-PCR results showed that BRDT-NY was exclusively expressed in testis. mRNA expression of BRDT-NY gene was deleted in some azoospermic patients' testes. These experiments suggested that BRDT-NY gene may have an important role in the process of spermatogenesis and may be correlated with male infertility.     

Establishment of hybridoma cell lines producing monoclonal antibodies against hepatitis B virus surface antigens (a, d, and r) and development of sensitive ELISA diagnostic test

A new treble-coated enzyme-linked immunoadsorbent assay (ELISA) kit of detecting Hepatitis B virus (HBV) surface antigen subtypes a, d and r (HBsAg-a, -d, -r) was developed by using four established hybridoma cell lines, of which two specifically secrete monoclonal antibodies (MAbs) against HBsAg-a (anti-HBsAg-a), one against -d (anti-HBsAg-d), and one against -r (anti-HBsAg-r). The approach of hybridoma cell lines' establishment were by fusing myeloma cells (SP2/0) with splenocytes from BALB/c mice immunized with a mixture of HBsAg-a, -d, -r. The ascitic MAb productivity of the four cell lines was at the titres of 1:10(6)-1:10(8). A treble-coated ELISA based HBV diagnostic kit was developed for detecting all of the three responding subtypes of HBsAgs. A 96-well ELISA microplate was coated with anti-HBSAg-a, -d, -r at a ratio of 3: 1: 0.5, with a horseradish peroxidase (HRP) conjugated anti-HBsAg-a as the labelled antibody. For clinical application, the new developed diagnostic kit detected HBsAgs of adr, adw, ayr, and ayw at a rate of lower than 0.25, 0.25, 0.5, and 0.5 ng/mL, respectively. Results indicated that this kit was more rapid and sensitive than that other current ELISA-based kits coated with a single MAb (e.g., anti-HBsAg-a).     

The involvement of calcium mobilization in the calcium-activated potassium currents activated by hyposmotic swelling in gastric antral circular myocytes of the guinea-pig

In our study of the effects of hyposmotic swelling on the Ca(2+)-activated potassium currents [I(K(Ca))] and its mechanism, we employed the whole-cell patch clamp technique using the gastric antral circular myocytes of the guinea-pig. Hyposmotic swelling efficiently increased I(K(Ca)), and the extent of changes in I(K(Ca)) was sharply dependent on the osmolarity of the perfusion solutions. When the calcium-free solution (EGTA 10 microM added in calcium-free solution) was superfused, I(K(Ca)) was not increased by the hyposmotic swelling. Gadolinium (Gd(3+)) 100 nM, a blocker of the stretch-activated nonselective cation channel, blocked the activation of I(K(Ca)) induced by hyposmotic swelling, but nicardipine 5 microM (the L-type calcium channel blocker) did not. Heparin 3 mg/ml, a potent inhibitor of inositol triphosphate receptor (InsP(3)R), did not inhibit the response, and caffeine 1 mM (the agonist for calcium-induced calcium release [CICR]) imitated the effect of hyposmotic swelling. Ryanodine (15 microM), markedly inhibited the effect. These results suggest that hyposmotic swelling activates I(K(Ca)), and the activation is associated with CICR, which is triggered by extracellular calcium influx through the stretch-activated channel (SA channel).     

Activated CD8(+) T cells from aged mice exhibit decreased activation-induced cell death

To uncouple the defects of activation and apoptosis of T cells from aged mice, we used anti-CD3 plus IL-2 stimulation to induce an activation response and analyzed the subsequent activation-induced cell death (AICD) response of T cells from 16-month-old mice. The results herein demonstrate that T cells from 16-month-old mice could be activated by anti-CD3-induced activation signals but exhibited distinct phenotypic and functional features compared to young (2-month-old) mice. These include a decrease in AICD, a delayed entry into the cell cycle, and a decreased telomerase activity. The decreased AICD of T cells from 16-month-old mice is associated with a decreased expression of Fas and Fas ligand (FasL), decreased susceptibility to anti-Fas-induced apoptosis, and an increased expansion of a CD8(+) T-cell population. Prior to activation, these T cells exhibit a phenotype that is CD44(hi)CD62L(hi). After stimulation, these T cells produced high levels of the pro-inflammatory cytokine, IFN-gamma, and developed an increased population of IFN-gamma(+)IFN-gamma R(-) T cells. Our results suggest that there is a dysregulation in T-cell homeostasis in aged mice associated with a decrease in AICD of CD8(+) T cells.     

Quercetin inhibits the invasion and mobility of murine melanoma B16-BL6 cells through inducing apoptosis via decreasing Bcl-2 expression

Quercetin has been known to have anti-tumor and anti-oxidation activities. In the present study, we have investigated its in vitro anti-metastatic activity. Quercetin inhibited the invasion and mobility of murine melanoma B16-BL6 cells in a dose-dependent manner but did not affect their adhesion to either laminin, fibronectin, or type VI collagen. Moreover, quercetin significantly inhibited the proliferation of B16-BL6 cells only in the case of time incubation longer than 48 h. Quercetin dose-dependently decreased the cell rates in S and G2–M phases of cell cycle. The effect of quercetin to cause a remarkable apoptosis of B16-BL6 cells was also demonstrated by flow cytometric assay as well as DNA fragmentation with a typical 180-bp ladder band in agarose electrophoresis and a quantitative analysis. Furthermore, quercetin markedly inhibited the expression of anti-apoptotic protein Bcl-2 but hardly influenced Bcl-X_L. These results suggest that the inhibition of quercetin on invasiveness and migration of B16-BL6 cells are closely associated with the arrest of cell cycle as well as the induction of apoptosis by decreasing the Bcl-2 expression. This revised version was published online in July 2006 with corrections to the Cover Date.     

HLA-DPA1 promoter haplotypes are differently distributed in southern Chinese ethnic groups

DPA1 gene is one of the human leukocyte antigen (HLA) class II genes and its promoter is highly polymorphic. From comparative studies among five southern Chinese populations, Jing, Li, Bai, Lahu, and Meizhou Han, we describe their single-nucleotide polymorphism (SNP)/haplotype frequency data of HLA-DPA1 gene promoter in this study. Within the 760-bp promoter region, we have identified 21 SNPs and nine possible haplotypes. Pair-wise comparisons show similar frequencies distribution of the HLA-DPA1 promoter haplotypes among Jing, Li, and Bai, whereas all pair-wise comparisons involved with Lahu or Meizhou Han and other ethnic groups show remarkable difference. The differences in frequencies of HLA-DPA1 promoter alleles may reveal different ethnic origins and demographic histories of the five populations. Our study may help distinguishing each of these populations by sequence variations of HLA-DPA1 promoter, which may be served as functional molecular markers for clinical and immunological studies involving the DPA1 locus.     

Cloning and characterization of a novel gene which encodes a protein interacting with the mitosis-associated kinase-like protein NTKL

NTKL is an evolutionarily conserved kinase-like protein. The cell-cycle-dependent centrosomal localization of NTKL suggested that it was involved in centrosome-related cellular function. The mouse NTKL protein is highly homologous with human NTKL. A novel mouse protein was identified as an NTKL-binding protein (NTKL-BP1) by yeast two-hybrid screening, and the full-length cDNA was amplified based on the result of a sequence data analysis cloning strategy. The full-length cDNA sequence of the NTKL-BP1 gene consists of 2,537 bp, which encode 368 amino acids. A database search revealed that homologues of NTKL-BP1 exist in different organisms, including Arabidopsis thaliana, Drosophila melanogaster, Plasmodium falciparum, Geobacter metallireducens, Anopheles gambiae and human. It suggests that NTKL-BP1 is an evolutionarily conserved protein. The expression of NTKL-BP1 was observed in multiple normal mouse tissues. The interaction of the two proteins was confirmed by co-immunoprecipitation. Moreover, immunofluorescent staining indicated that NTKL and NTKL-BP1 were all localized in the cytoplasm.     

免疫组织化学-激光显微切割-聚合酶链反应技术在胃癌石蜡切片中检测p53突变的应用

目的 探索在石蜡切片中应用免疫组织化学-激光显微切割-聚合酶链反应(PCR)技术,检测进展期胃癌p53蛋白表达阴性或阳性的p53基因外显子5～8的突变情况。方法 对41例进展期胃癌标本进行p53蛋白的免疫组织化学标记,再用激光显微切割(LMD)技术切取p53表达一致的癌组织及远离癌灶的正常胃黏膜腺体。经消化后直接进行p53基因外显子5～8的PCR扩增、单链构象多态性分析及直接测序。结果 41例进展期胃癌标本均扩增出特异的目的条带。其中有11例p53蛋白表达阳性,15例p53基因检测到突变。蛋白表达阳性者中有8例突变(8／11);表达阴性者中有7例突变(7／30,23．3％)。p53蛋白表达和p53基因突变具显著相关(P=0．004)。结论 免疫组织化学-LMD-PCR技术应用于石蜡切片可获得满意的结果,此技术可将细胞特定基因的结构状态和表达水平很好地结合起来检测分析。