高温影响番茄小孢子发育的细胞学研究

以番茄‘Micro-Tom’为材料,利用形态观察、DAPI染色、石蜡切片等方法对正常情况下番茄小孢子发生过程进行时期划分.通过连续7d的高温胁迫((35±1)℃/(30±1)℃)处理试验,结合细胞学观察,研究高温对番茄花粉小孢子发育的影响.研究表明,高温胁迫不仅导致花粉畸形或败育、花粉数量减少、活力低萌发力差,而且还导致花药绒毡层、药隔组织、药室内壁、花药表皮、环状细胞簇等花药细胞结构的发育异常.结果有助于阐明热胁迫对番茄小孢子发育的影响,并为培育耐高温农作物新品种提供思路.     

Intratumoral heterogeneity determines discordant results of diagnostic tests for human epidermal growth factor receptor (HER) 2 in gastric cancer specimens

Accurate assessment of human epidermal growth factor receptor (HER) 2 is essential for efficient selection of patients who may benefit from therapies targeting this surface receptor (e.g., trastuzumab). Intratumoral heterogeneity of HER2 expression may potentially contribute to inaccurate assessment of HER2 status. To clarify intratumoral heterogeneity of HER2 expression and its potential clinical impact on assessment of HER2 status, we analyzed 148 endoscopic biopsy specimens and 117 excisional tumor specimens collected from 148 patients with primary gastric cancer. Specifically, we assessed HER2 protein overexpression and gene amplification using, respectively, immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). There were 28 IHC-positive cases and 25 FISH-positive cases among these 148 patients. Heterogeneous HER2 protein expression was demonstrated in 23 of 29 (79.3%) IHC-positive cases, while gene expression heterogeneity was found in 11 of 25 (44.0%) FISH-positive cases. Intratumoral heterogeneity was the main reason of discordant results between IHC and FISH or between endoscopic biopsy and excisional tumor specimens. The clinical significance and impact of intratumoral HER2 expression heterogeneity on treatment outcome in gastric cancer require further studies.     

激素引起伊贝母愈伤组织微管蛋白变化的免疫细胞化学研究(简报)

微管蛋白聚合形成微管。微管在维持细胞结构、物质运输、分裂及植物细胞壁的建成等过程中起着重要的作用。70年代后期,在微管生物化学研究取得很大进展的基础上,免疫细胞化学技术与微管研究结合起来,使人们能够从整体水平观察以微管蛋白为主要成份的细胞骨架的动态变化。我们采用免疫酶标技术,对生长在含不同激素培养基上的伊贝母愈伤组织的微管及微管蛋白变化进行了观察和分析,结果表明,激素种类和微管的存在形式是相关的。     

高压蒸汽灭菌器中温度与压力的关系*

应用Clausius-Clapeyron方程、波义耳定律和道尔顿分压定律,分析高压蒸汽灭菌器中混合气体温度和压力的关系。以献数据建立了灭菌器中残留不同量空气时的Antoine方程,导出以压力表读数计算温度的方法。对灭菌时排放空气的必要性给予理论解释。     

尼日利亚菌素高产菌株的选育

通过对致死率和正负突变率的测定,确定了紫外线、亚硝酸、紫外线加吖啶橙3种诱变方式对吸水链霉菌NND-52的29号菌株进行诱变处理的合适剂量,并采用复合诱变得到了一株脱葡萄糖阻遏的高产菌株A19,其尼日利亚菌素的摇瓶产量比出发菌株提高了128％。传代结果表明,在传代的同时结合自然分离,可以保持稳定的产抗特性。     

An improved particle bombardment for the generation of transgenic plants by direct immobilization of relleasable Tn5 transposases onto gold particles

We have developed a modified particle bombardment method for plant transgenesis. An intein-tag and a 6×Cys-tag were successively fused to the N-terminus of a hyperactive Tn5 transposase. The modified transposase was immobilized on bare gold microscopic particles via covalent binding of a 6×Cys-tag sulfydryl groups to the gold surface. The tethered transposase can bind the transposon DNA in vitro to form the transposome in the absence of Mg2? ions. After bombardment of the gold particles carrying the transposomes into the plant cells, the transposomes will be released from the carrier due to the activated self-cleavage function of intein-tag. Our data showed this procedure integrated foreign DNA into the plant genome with an increased transformation frequency as compared to the conventional particle bombardment method. A single copy insertion can also be obtained by decreasing of the assembled transposon DNA amount in relation to plant cell biomass.     

泡桐丛枝植原体tRNA异戊烯基焦磷酸转移酶基因克隆、原核表达及功能分析

【目的】为了鉴定植原体tRNA异戊烯基焦磷酸转移酶基因(tRNA-ipt)的表达及蛋白功能,探索植原体致病机理。【方法】对泡桐丛枝、桑萎缩、长春花绿变及苦楝丛枝植原体tRNA-ipt基因完整序列进行PCR扩增和生物信息学分析。对泡桐丛枝植原体tRNA-ipt基因进行原核表达并制备抗体。利用Western blot和FITC间接免疫荧光显微镜检测其在植原体中的表达。使用分光光度计分析该基因对大肠杆菌生长的影响,用ELISA测定转化菌株细胞分裂素含量。【结果】首次发现泡桐丛枝、桑萎缩、长春花绿变及苦楝丛枝植原体中完整tRNA-ipt基因,大小为876 bp,编码291个氨基酸,且N端均含有ATP/GTP结合位点保守序列(GPTASGKT)。4种植原体tRNA-IPT之间的氨基酸序列相似率为99.1%-99.5%,与同组植原体同源性在95.4%-99.3%,与其他组植原体同源性低于70%。SDS-PAGE结果显示tRNA-IPT蛋白在大肠杆菌中得到表达。首次获得泡桐丛枝植原体tRNA-IPT抗体并检测到该蛋白在泡桐发病组织中的特异表达。经过对转化菌株生长曲线及玉米素含量的测定,发现该基因能促进大肠杆菌后期生长和玉米素核苷的积累。【结论】4种植原体tRNA-ipt基因编码相同特性的功能蛋白,泡桐丛枝植原体tRNA-IPT蛋白能够在植原体中表达,根据该基因对异源菌株生长速率和激素合成的影响推断该蛋白可能参与植原体的细胞分裂素合成,在致病过程中起到重要作用。     

Real-time detection of Escherichia coli O157:H7 sequences using a circulating-flow system of quartz crystal microbalance

A DNA piezoelectric biosensing method for real-time detection of Escherichia coli O157:H7 in a circulating-flow system was developed in this study. Specific probes [a 30-mer oligonucleotide with or without additional 12 deoxythymidine 5′-monophosphate (12-dT)] for the detection of E. coli O157:H7 gene eaeA, synthetic oligonucleotide targets (30 and 104 mer) and PCR-amplified DNA fragments from the E. coli O157:H7 eaeA gene (104 bp), were used to evaluate the efficiency of the probe immobilization and hybridization with target DNA in the circulating-flow quartz crystal microbalance (QCM) device. It was found that thiol modification on the 5′-end of the probes was essential for probe immobilization on the gold surface of the QCM device. The addition of 12-dT to the probes as a spacer, significantly enhanced (P < 0.05) the hybridization efficiency (H%). The results indicate that the spacer enhanced the H% by 1.4- and 2-fold when the probes were hybridized with 30- and 104-mer targets, respectively. The spacer reduced steric interference of the support on the hybridization behavior of immobilized oligonucleotides, especially when the probes hybridized with relatively long oligonucleotide targets. The QCM system was also applied in the detection of PCR-amplified DNA from real samples of E. coli O157:H7. The resultant H% of the PCR-amplified double-strand DNA was comparable to that of the synthetic target T-104AS, a single-strand DNA. The piezoelectric biosensing system has potential for further applications. This approach lays the groundwork for incorporating the method into an integrated system for rapid PCR-based DNA analysis.     

An isoform of GTPase regulator DOCK4 localizes to the stereocilia in the inner ear and binds to harmonin (USH1C)

The driving forces for the regulation of cell morphology are the Rho family GTPases that coordinate the assembly of the actin cytoskeleton. This dynamic feature is a result of tight coupling between the cytoskeleton and signal transduction and is facilitated by actin-binding proteins (ABPs). Mutations in the actin bundling and PDZ domain-containing protein harmonin are the causes of Usher syndrome type 1C (USH1C), a syndrome of congenital deafness and progressive blindness, as well as certain forms of non-syndromic deafness. Here, we have used the yeast two-hybrid assay to isolate molecular partners of harmonin and identified DOCK4, an unconventional guanine exchange factor for the Rho family of guanosine triphosphatases (Rho GEF GTPases), as a protein interacting with harmonin. Detailed molecular analysis revealed that a novel DOCK4 isoform (DOCK4-Ex49) is expressed in the brain, eye and inner ear tissues. We have further provided evidence that the DOCK4-Ex49 binds to nucleotide free Rac as effectively as DOCK2 and DOCK4 and it is a potent Rac activator. By immunostaining using a peptide antibody specific to DOCK4-Ex49, we showed its localization in the inner ear within the hair bundles along the stereocilia (SC). Together, our data indicate a possible Rac-DOCK4-ABP harmonin-activated signaling pathway in regulating actin cytoskeleton organization in stereocilia.     

人GDNF基因在昆虫细胞中的高效表达

应用昆虫杆状病毒表达系统在昆虫细胞Ｔｎ－５Ｂ１－４中高效表达了人胶质细胞源性神经营养因子（ＧＤＮＦ）,ＰＡＧＥ分析表达量占细胞可溶性蛋白质的３０％左右,表达产物经亲和层析纯化后纯度达８０％以上,活性研究表明,昆虫细胞表达的ＧＤＮＦ蛋白能显著促进多巴胺能神经元的存活,此研究为进一步研究ＧＤＮＦ结构与功能打下了良好的基础。