外源基因导入部分脱壁的大麦细胞及其转基因植株的再生

采用ＰＥＧ法、电击法以及ＰＥＧ＋电击法将带有ｇｕｓ和ｎｐｔⅡ基因的ｐＢＩ１２１质粒导入部分脱壁的大麦细胞，通过Ｓｏｕｔｈｅｒｎ杂交、ＧＵＳ和ＮＰＴⅡ蛋白活性检测，证实了外源报告基因已在大麦基因组中整合并在转化细胞中获得表达。     

GHRP—scu—PA—32K突变体的构建,表达及纯化产物的部分性 …

在ｓｃｕ－ＰＡ３２Ｋ的ｃＤＮＡ分子基础上经定点突变,在Ｎ端紧接Ｌｅｕ＾１以前引入编码ＧＨＲＰ四肽的寡核苷酸序列（ＧＧＴＣＡＴＡＧＧＣＣＴ）,构建了ＧＨＲＰ－ｓｃｕ－ＰＡ－３２Ｋ的突变体ｃＤＮＡ。将它克隆到表达载体ｐＣＭ－β－ｄｈｆｒ共转染ＣＨＯ／ＤＨＦＲ＾－细胞。筛选到的稳定表达株在无 清培养基的表达量为５８０ＩＲ／（１０＾６细胞．２４ｈ）。经锌离子螯合亲和柱纯化的产物,ＳＤＳ－ＰＡＧＥ显示为一蛋     

血小板衍化生长因子及其受体在人胰腺癌局部浸润转移中作用的实验研究

观察胰腺组织及其癌变标本中血小板衍化生长因子（ＰＤＧＦ）及其受体（ＰＤＧＦＲ）基因表达的变化关系,以探讨它们在人胰腺癌局部侵袭和转移过程中的作用。用诺真法（Ｎｏｒｔｈｅｒｎ－ｂｌｏｔ）和原位杂交（ｉｎｓｉｔｕｈｙｂｒｉｄｉｚａｔｉｏｎ,ＩＳＨ）方法,采用ｃＤＮＡ探针（ＰＤＧＦＡ、Ｂ,ＰＤＧＦＲα、β）对两株胰腺癌细胞（Ｐａｔｕ８９８８、Ｐａｔｕ８９０２）、１５例胰腺癌手术切除标本和８例正常胰腺组织标本进行杂交检测。胰腺癌组织中,ＰＤＧＦＡ、ＰＤＧＦＢ在ＲＮＡ水平的表达与正常胰腺组织相近;而ＰＤＧＦＲα、ＰＤＧＦＲβ尤其是ＰＤＧＦＲα在ＲＮＡ水平的表达,较正常胰腺组织相对提高。ＰａＴｕ细胞株中,有ＰＤＧＦＡ及其ＰＤＧＦＲβ的表达。ＩＳＨ结果显示：ＰＤＧＦＡ、ＰＤＧＦＢ阳性反应深浅不一,胰腺癌与正常胰腺组织无明显差异。胰腺癌细胞中ＰＤＧＦ和ＰＤＧＦＲ均有表达,而ＰＤＧＦＲβ则比较多地定位于结缔组织中的纤维细胞和内皮细胞。ＰＤＧＦ、ＰＤＧＦＲ可能通过对胰腺癌细胞外基质蛋白降解代谢的调控,共同影响着肿瘤的侵袭和转移过程     

应用重组Etp28抗原免疫预防球虫病的研究

将构建好的可表达ＧＳＴ融合蛋白的重组病毒ＡｃＭＮＰＶ－ＯＣＣ－Ｅｔｐ２８感染Ｓｆ９细胞,７２ｈ后取感染了病毒的细胞裂解物上清液进行ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｍ－ｂｌｏｔ分析,结果显示５３ｋＤａ的融合蛋白在昆虫Ｓｆ９细胞中得到了高效表达,表达水平为占细胞总蛋白的２１．３％;将ＧＳＴ－６ｘＨｉｓ－Ｅｔｐ２８注射维菌鸡进行免疫保护试验,结果表明免疫组比对照组在平均增重和盲肠粘膜层卵囊计数方面有一定程度     

大鼠谷胱甘肽S—转移酶P1基因增强因子及其反式作用因子的研究

探讨大鼠谷胱甘肽Ｓ－转移酶Ｐ１基因上游增强子元件及作用于其上特异性结合蛋白与该基因在癌细胞中高表达的关系,用荧光素酶的报告系统在ＨｅＬａ及ＣＢＲＨ７９１９细胞中增强子元件Ｉ（ＧＰＥ Ｉ）及增强子元件Ⅱ（ＧＰＥⅡ）的活性,并将ＧＰＥⅡ的增强子活性部位定位于其上游８４ｂｐ的ＧＰＥⅡ－１区域内。分析对比不同细胞中结合于ＧＰＥⅠ核心序列（ｃＧＰＥⅠ）、ＧＰＥⅡ－１上特异性核合蛋白,发现ＨｅＬａＣＢＲＨ７９     

当归注射液对兔主动脉平滑肌细胞中增殖细胞核抗原表达的影响

目的：观察当归注射液对平滑肌细胞（ Ｓ Ｍ Ｃ） 中增殖细胞核抗原（ Ｐ Ｃ Ｎ Ａ） 表达的影响。方法：用免疫组织化学 Ｌ Ｓ Ａ Ｂ 法检测培养的兔主动脉 Ｓ Ｍ Ｃ 中 Ｐ Ｃ Ｎ Ａ 的表达,同时测定培养液中超氧化物歧化酶（ Ｓ Ｏ Ｄ） 、脂质过氧化物（ Ｌ Ｐ Ｏ） 、前列环素（ Ｐ Ｇ Ｉ２） 及环磷酸腺苷（ｃ Ａ Ｍ Ｐ） 的含量。结果：当归注射液能增加 Ｓ Ｏ Ｄ 活性,降低 Ｌ Ｐ Ｏ 和升高 Ｐ Ｇ Ｉ２ 、ｃ Ａ Ｍ Ｐ 水平,抑制 Ｓ Ｍ Ｃ 中 Ｐ Ｃ Ｎ Ａ 的表达（ Ｐ＜ ００５ ～００１） 。结论：当归有抑制 Ｓ Ｍ Ｃ 增殖的作用。     

不同S180细胞株蛋白质特性的电泳及免疫组化法比较研究

用ＳＤＳ聚丙烯酰胺凝胶电泳（ＳＤＳ－ＰＡＧＥ）和免疫组化染色方法，比较研究４个不同单位保种的小鼠肉瘤（Ｓ１８０）细胞蛋白质的表达。电泳结果显示，北京市肿瘤研究所仲种的Ｓ１８０蛋白质条带数最多，武汉大学保种中心的Ｓ１８０最小。免疫组化染色法测定四个单位Ｓ１８０细胞的Ｒａｆ－１、Ｔｒｋ、Ｇａｉ、Ｇａｏ和ＮＦＫｂＰ６５蛋白表达，Ｇａｉ、ＴｒｋＡ和Ｒａｆ－１阳性率均低于０．２％；Ｇａｏ阳性率均低于４．２％     

ANLL中转录因子GATA—2基因点突变

了解转录因子ＧＡＴＡ－２基因在ＡＮＬＬ中的表达和突变情况,方法：采用ＲＴ－ＰＣＲ检测８５例ＡＮＬＬ病人外周血单个核细胞中ＧＡＴＡ－２基因的表达情况,ＰＣＲ产物进一步经单链构象多态性（ＳＳＣＰ）分析以了解基因突变情况。结果：绝大多数ＡＮＬＬ都表达了ＧＡＴＡ－２基因（８９．４％）,ＳＳＣＰ分析发现一例Ｍ２型的ＰＣＲ产物出现异常迁移带,核苷序列分析显示在ＧＡＴＡ－２基因第８９２位的核苷酸出现点突变,即第     

大麦黄矮病毒（BYDV）外壳蛋白基因在小麦原生质体中的…

实验采用ＰＥＧ介导法转化小麦，用ＧＵＳ基因作为标志，用荧光法测定Ａｃｔｌ－ＧＵＳ，Ｅｍｕ－ＧＵＳ，３５Ｓ－ＧＵＳ三种质粒转化小麦细胞后的瞬间表达强度，以比较Ａｃｔｌ，Ｅｍｕ，３５Ｓ三种启动子在小表中的表达强度，结果表明Ａｃｔｌ和Ｅｍｕ的强度大致相等，均比３５Ｓ强，采用Ｅｍｕ为启动子带ＢＹＤＶＣＰ基因的质粒和经改造过的Ａｃｔｌ为启动子带有抗潮霉素选择记基的质粒转化小麦“济南１７７”的原生质体，转化６     

大麦黄矮病毒（BYDV）外壳蛋白基因在小麦原生质体中的稳定转化

实验采用ＰＥＧ介导法转化小麦，用ＧＵＳ基因作为标志，用荧光法测定Ａｃｔｌ－ＧＵＳ、Ｅｍｕ－ＧＵＳ、３５Ｓ－ＧＵＳ三种质粒转化小麦细胞后的瞬间表达强度，以比较Ａｃｔｌ、Ｅｍｕ、３５Ｓ三种启动子在小表中的表达强度．结果表明，Ａｃｔｌ和Ｅｍｕ的强度大致相等，均比３５Ｓ强．采用Ｅｍｕ为启动子带ＢＹＤＶＣＰ基因的质粒和经改造过的Ａｃｔ１为启动子带有抗潮霉素选择标记基因的质粒共转化小麦“济南１７７”的原生质体．转化６ｄ后，用潮霉素进行筛选，最后得到两块抗潮霉素的愈伤组织，转化后４个月，经ＰＣＲ检测，证明ＣＰ基因已整合进一块愈伤组织的细胞基因组中．     

肽泡的形态特征及其成膜蛋白的特性

小麦(Triticum aestivum)贮存蛋白经过纯化获得其中35kD一种蛋白质。该蛋白质在一定浓度的有机溶剂系统中经过超声波处理或搅拌,可以制成囊泡,并能包裹分子量从0.4～150kD的不同化合物(染料及抗体)。这种肽泡具有良好的稳定性,经低温冷冻干燥后,可成鳞片状,加水后复溶仍然保持肽泡的稳定性。这种特性称为DRV(Dehydration Rehydration Vesicles)性质。该蛋白质经修饰制定肽泡能包裹水溶性化合物,如水溶性染料,或不经修饰可包裹脂溶性化合物。这种蛋白质是由16种疏水性氨基酸残基组成,其N-末端为苯丙氨酸。     

甘薯珠心细胞的超微结构研究

本文叙述了甘薯珠心细胞在衰退前的发育过程中的超微结构变化.一般珠心表皮细胞和离胚囊较远的珠心细胞以及胚囊发育初期靠近胚囊的细胞有较大的核质比和清晰的核被膜;细胞质中线粒体、核糖体、高尔基体和内质网等细胞器十分丰富,功能也非常活跃;细胞中有较多的小液泡;细胞壁上具有较多的胞间连丝.随着胚囊的扩展和珠心细胞的发育,在离胚囊稍近的珠心细胞中,出现平行内质网槽库和同心内质网环,同心内质网包围的细胞质被消化后形成小液泡.通过新液泡的形成,原有液泡的扩大与合并,珠心细胞形成大的中央液泡,此时,珠心细胞由具有分生组织细胞特征转化为呈现薄壁组织细胞的典型特征,以后,胚囊的进一步扩展将导致珠心细胞的衰亡.     

Involvement of heterotrimeric G protein in signal transduction of extracellular calmodulin in regulatingrbcS expression

The role of heterotrimeric G protein in signal transduction pathway of extracellular calmodulin in regulating rbcS expression was examined in suspension-cultured cells of transgenic tobacco. Pharmalogical experiments indicated that G protein agonist cholera toxin enhanced rbcS expression and heterotrimeric G protein antagonist pertussis toxin inhibited rbcS expression in transgenic tobacco cells. Pertussis toxin also inhibited the enhancement effect caused by exogenous purified calmodulin on rbcS expression, whereas cholera toxin completely reversed the inhibitory effects caused by anti-calmodulin serum on rbcS expression. The right side-out vesicles from tobacco cell membrane were purified, which contained all of substrates for fluometric assay of GTPase activity. Exogenous purified calmodulin, when adding directly to the medium of plasma membrane vesicles, significantly activated GTPase activity in the right side-out plasma membrane vesicles, and this increase in GTPase activity was completely inhibited both by heterotrimeric G proteins antagonist pertussis toxin and nonhy-drolyzable GTP analogs GMP-PCP. These results provided the evidence that heterotrimeric G proteins may be involved in signal transduction pathways of extracellular calmodulin to regulate rbcS gene expression.     

Compartmental specificity of cellular membrane fusion encoded in SNARE proteins

Membrane-enveloped vesicles travel among the compartments of the cytoplasm of eukaryotic cells, delivering their specific cargo to programmed locations by membrane fusion. The pairing of vesicle v-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) with target membrane t-SNAREs has a central role in intracellular membrane fusion. We have tested all of the potential v-SNAREs encoded in the yeast genome for their capacity to trigger fusion by partnering with t-SNAREs that mark the Golgi, the vacuole and the plasma membrane. Here we find that, to a marked degree, the pattern of membrane flow in the cell is encoded and recapitulated by its isolated SNARE proteins, as predicted by the SNARE hypothesis.     

Halomonas aquamarina盐敏感质膜蛋白组的研究

革兰氏阴性细菌外膜蛋白对细菌外部环境因素的调节功能已受到高度重视,但有关细菌质膜的作用报道极少.为研究质膜蛋白在抗盐机制中所起的作用,采用蛋白质组学技术比较深海中度嗜盐菌Halomonasaquamarina在生理(0.56mol/LNaCl)、较高(1mol/LNaCl)和高(2mol/LNaCl)浓度下质膜蛋白差异表达的结果.结果表明,高盐浓度下出现2个新蛋白,质谱分析证明其为ABC转运蛋白.这些发现对从细胞膜整体角度了解细菌的抗盐机理具有重要意义.     

Messenger RNA targeting of rice seed storage proteins to specific ER subdomains

Rice seeds, a rich reserve of starch and protein, are a major food source in many countries. Unlike the seeds of other plants, which typically accumulate one major type of storage protein, rice seeds use two major classes, prolamines and globulin-like glutelins. Both storage proteins are synthesized on the endoplasmic reticulum (ER) and translocated to the ER lumen, but are then sorted into separate intracellular compartments. Prolamines are retained in the ER lumen as protein bodies whereas glutelins are transported and stored in protein storage vacuoles. Mechanisms responsible for the retention of prolamines within the ER lumen and their assembly into intracisternal inclusion granules are unknown, but the involvement of RNA localization has been suggested. Here we show that the storage protein RNAs are localized to distinct ER membranes and that prolamine RNAs are targeted to the prolamine protein bodies by a mechanism based on RNA signal(s), a process that also requires a translation initiation codon. Our results indicate that the ER may be composed of subdomains that specialize in the synthesis of proteins directed to different compartments of the plant endomembrane system.     

Localization of calmodulin and calmodulin-like protein and their functions in biomineralization in P. fucata

Calmodulin （CAM） and calmodulin-like protein （CaLP） are two proteins involved in biomineralization. Their localizations in Pinctada fucata mantle epithelia were studied by Western blot （WB） analysis of the nuclear/cytosol fraction of primary cultured P. fucata mantle cells and immunogold electron microscopy. The results showed a completely different distribution of these two proteins at the subcellular level. CaM was distributed throughout both the nucleus and cytoplasm of the mantle epithelium but CaLP was distributed only in the cytoplasm. The functions of these two proteins in biomineralization were investigated by shell regeneration. During this pro- cess, the expressions of CaM and CaLP were greatly enhanced in different organelles of the mantle epithelium. Overexpression of these two proteins and a mutant of calmodulin-like protein （M-CaLP） that lacks an extra C-terminal tail in MC3T3-E1 promoted the mRNA expression of osteopontin, a biomineralization marker for osteoblasts. All of the results indicated that CaM and CaLP have completely different distributions in the mantle epithelium and affect the biomineralization process at different levels. The extra C-terminal tail of CaLP is important for its functions in biomineralization in P. fucata.     

锯缘青蟹Y器结构与卵巢发育的研究

锯缘青蟹的Y器位于前鳃腔、眼柄的后外侧，其显著特点是细胞紧密排列，细胞核几乎占据了整个腺细胞，异染色质极其丰富，细胞突起多，细胞器少。线粒体具管状嵴，细胞内常见不同大小的空泡，卵巢成熟之前，Y器具有巨大脂肪球；腺细胞胞质少，空泡数量较多，以小泡为主，线粒体基质电子密度高。卵巢将成熟时，腺细胞胞质体积有所增大，多泡小体、髓样小体和大空泡增多，线粒体基质电子密度低，内嵴膨大或消失，表明腺细胞分泌作用逐渐减弱。锯缘青蟹Y器超微结构的变化，为其蜕皮类固醇参与卵巢发育的生理作用提供了形态学依据。     

Bovine papillomavirus E5 oncoprotein binds to the 16K component of vacuolar H(+)-ATPases.

The major transforming protein of bovine papillomavirus type 1, E5, is mainly associated with endomembranes, specifically binding to a cellular protein of relative molecular mass 16,000 (16K). At the same time as transformation, E5 causes the phosphorylation of tyrosine residues in epidermal and platelet-derived growth factor receptors. We show here that the 16K protein associated with E5 is the 16K component of vacuolar ATPases. This protein is known to be an integral membrane protein in endosomes, bovine chromaffin granules, synaptic vesicles, fungal and plant vacuoles and clathrin-coated vesicles, as well as a component of gap-junction-like membrane complexes. Because proton pumps are critical for the function of cellular compartments that process growth-factor receptors, the interaction of E5 with the 16K protein could explain the pleiomorphic features of cells transformed by E5.