Molecular rescue of tumour-specific T cell receptor idiotype from T cell lymphomas

The T cell receptor (TCR) idiotype on T cell lymphomas can serve as a vaccine target. To clone the relevant genes, 5' rapid amplification of cDNA ends (RACE) was performed on 13 T cell lymphomas and nine control samples. Two polymerase chain reactions (PCR) were performed for each TCR chain (alpha and beta) and the proportion of the clonal TCR sequence over the total number of TCR sequences was calculated. For alpha, the average proportions were 0.43 vs. 0.05. For beta these were 0.44 and 0.04. The TCR was identified in 10 of 13 lymphoma samples.     

Evaluation of serum IgE in peach-allergic patients with systemic reaction by using recombinant Pru p 7 (gibberellin-regulated protein)

Background

Lipid transfer protein (LTP) is a major fruit allergen. It has, however, recently been revealed that the systemic reaction in peach-allergic patients is related not only to LTP (Pru p 3) but also to gibberellin-regulated protein (Pru p 7). We investigated recombinant Pru p 7 (rPru p 7) for its potential use in worldwide standardization for the diagnosis of peach allergy.

应用酵母双杂交技术筛选与pGBKT7-CT813编码产物相互作用蛋白的研究

目的以pGBKT7-CT813作为诱饵质粒与HeLa细胞酵母GAL4AD融合cDNA文库进行酵母双杂交试验,以进一步探讨沙眼衣原体包涵体膜蛋白的生物学功能。方法根据STD基因库提供信息设计引物,用PCR法获取目的基因片段CT813,经酶切处理的CT813和pGBKT7质粒,在T4连接酶作用下连接,连接产物转入感受态细胞BL-21,培养后进行菌落PCR验证,对阳性质粒进行测序分析。质粒转化入酵母菌株Y187和AH109中,检测其有无自激活及毒性作用。阳性重组酵母菌株AH109与cDNA文库菌株Y187进行配合,待三叶草（或米奇）形状合子形成后涂布于腺嘌呤、组氨酸、亮氨酸、色氨酸缺陷型培养基和铺有X-Gal的SD/-Ade/-His/-Leu/-Trp平板上初筛,再经过2次筛选,收集阳性菌落。将阳性菌液点种在滤纸上,在液氮中反复冻融2次,然后浸泡在Z缓冲液-β巯基乙醇-X-Gal混合液中室温温育8h。对筛选出的显色阳性菌液进行PCR验证。选取22个PCR阳性菌液提取酵母质粒,将22个质粒再转入感受态细胞E.coli,BL-21,对阳性菌落提取质粒,进行回交试验,对验证阳性的菌落对应的质粒进行测序。结果成功构建了pGBKT7-CT813诱饵质粒,该质粒的表达产物对AH109和Y187均无自激活和毒性作用。将回交试验筛选的阳性菌落对应的质粒进行测序,对测序结果做BLAST检索分析,确定筛选出与pGBKT7-CT813特异性相互作用的基因可能编码的蛋白是：半乳糖凝集素-1（LGALS1）、环腺苷酸应答原件结合蛋白3（CREB3）、核糖体核糖体蛋白L10a（RPL10a）和微管蛋白37E-16（RP1-37E16）。结论筛选出的4种蛋白可能与沙眼衣原体的致病机制相关,但仍需要进一步验证。pGBKT7-CT813编码产物与多种蛋白有相互作用,为进一步研究其生物学功能打下了基础。     

广州管圆线虫Ⅳ期幼虫cDNA文库的筛选及EST序列分析

目的 筛选和鉴定广州管圆线虫未知基因序列,丰富该虫的基因序列数据,为寻找诊断或药物靶点分子提供实验依据.方法 构建广州管圆线虫Ⅳ期幼虫cDNA噬菌体文库,随机挑选单个噬菌斑,提取噬菌体DNA进行PCR,扩增其插入的cDNA片段并对PCR产物进行序列测定;采用生物信息学方法对表达序列标签(expressed sequence tags,EST)序列进行分析.结果 获得了51条广州管圆线虫基因的EST序列,并提交至GenBank,这些基因包括半乳糖凝集素10,半乳糖凝集素140,表皮蛋白,CRE-RPS-24蛋白等.序列分析结果提示,表皮蛋白和CRE-RPS-24蛋白可能为具有诊断价值的抗原分子,值得进一步研究.结论 获得51条广州管圆线虫基因序列,为进一步研究广州管圆线虫致病机制及候选抗原分子提供了新的序列信息.     

What is the status of gene therapy for primary immunodeficiency?

The efforts to find satisfactory treatments for seriously ill patients with primary immunodeficiency have resulted in the development of important new therapeutic procedures with benefits reaching far beyond the relatively small number of patients affected with these rare disorders. Allogeneic bone marrow transplantation, immunoglobulin and enzyme replacement treatments and more recently gene therapy have all been introduced into clinical medicine as treatments for one or more of the primary immunodeficiency diseases. Beginning in 1990, gene-corrected T cells were first used to treat ADA deficiency SCID. With this demonstration that the gene-transfer procedure could be safely used to introduce functional transgenes into patient cells, clinical trials for a broad range of inherited disorders and cancer were started in the mid 90s. Of all these early clinical experiments, those addressing primary immunodeficiency have also been the most successful. Both ADA and X-SCID have now been cured using gene insertion into autologous bone marrow stem cells. In addition some patients with chronic granulomatous disease (CGD) have shown an unexpectedly high level of functionally corrected granulocytes in their blood following infusion of autologous gene-corrected bone marrow. There remain however a great many significant challenges to be overcome before gene therapy becomes the treatment of choice for these and other disorders. The use of genes as medicines is the most complex therapeutic system ever attempted and it may rake several more decades of work before its real potential as a treatment for both inherited and sporadic disorders if finally realized. Presented at the First Robert A Good Society Symposium, St. Petersburg, FL 2006.     

Characterization of angiotensin II-receptor subtypes in podocytes

Glomerular podocytes play a key role in maintaining the integrity of the glomerular filtration barrier. This function may be regulated by angiotensin II (Ang II) through activation of cell-surface receptors. Although studies suggest that podocytes express receptors for Ang II, the Ang II binding site has not been characterized with radioligand binding techniques. We therefore used iodine 125-labeled Ang II to monitor Ang II-receptor density during differentiation of a mouse podocyte cell line. Scatchard analyses of equilibrium binding data revealed a single class of high-affinity binding sites (dissociation constant approximately 3 nmol/L) in both differentiated and nondifferentiated cells. During differentiation, the density of Ang II-receptor sites increased roughly 15-fold in differentiated podocytes (maximal density of specific binding sites 881 fmol/mg protein) compared with that in nondifferentiated cells (52 fmol/mg protein; P<.005). Glomerular podocytes expressed messenger RNA for AT1A, AT1B, and AT2 receptor subtypes, and competitive binding studies found that differentiated podocytes expressed mostly AT1 receptors (approximately 75%) with lesser amounts of AT2 (approximately 25%). Up-regulation of Ang II-receptor number was associated with increased Ang II-receptor responsiveness, as evidenced by enhanced Ang II-stimulated inositol phosphate (IP) generation and incorporation of tritiated thymidine. Both [3H]thymidine incorporation and IP generation were mediated by AT1-receptor activation. These data suggest that glomerular podocytes express a high-affinity binding site for Ang II with pharmacologic characteristics of both AT1 and AT2 receptors. This receptor site is up-regulated during podocyte differentiation, and receptor activation induces both IP generation and DNA synthesis by AT1-dependent mechanisms. We speculate that activation of podocyte Ang II receptors contributes to glomerular damage in disease states.     

Differential expression and cellular localization of activin and inhibin mRNA in the rainbow trout ovary and testis

An inhibin cDNA from rainbow trout consisted of 1305 bp, which coded for 352 amino acid residues. The deduced amino acid sequence of mature inhibin was 50 to 60% identical to mammalian sequences. Distribution of inhibin alpha and activin beta A and beta B in different ovarian and testis compartments was studied in rainbow trout by in situ hybridization with complementary RNA probes. In testis tissue, inhibin alpha and activin beta A and beta B were expressed only in the testicular interstitia between the seminal lobules, where Sertoli cells and Leydig cells are distributed. The localizations and intensities of the reactions were constant throughout the maturation of the testis. Within ovarian tissue, the theca cell layers of follicles showed strong reactions of Dig-labeled antisense mRNA probes hybridizing against inhibin alpha and activin beta A and beta B in all samples over the same sampling period. In regressing oocytes, a positive reaction was observed in the granular cell layer of the follicles.