Rescue of defective G protein–coupled receptor function in vivo by intermolecular cooperation

G protein–coupled receptors (GPCRs) are ubiquitous mediators of signaling of hormones, neurotransmitters, and sensing. The old dogma is that a one ligand/one receptor complex constitutes the functional unit of GPCR signaling. However, there is mounting evidence that some GPCRs form dimers or oligomers during their biosynthesis, activation, inactivation, and/or internalization. This evidence has been obtained exclusively from cell culture experiments, and proof for the physiological significance of GPCR di/oligomerization in vivo is still missing. Using the mouse luteinizing hormone receptor (LHR) as a model GPCR, we demonstrate that transgenic mice coexpressing binding-deficient and signaling-deficient forms of LHR can reestablish normal LH actions through intermolecular functional complementation of the mutant receptors in the absence of functional wild-type receptors. These results provide compelling in vivo evidence for the physiological relevance of intermolecular cooperation in GPCR signaling.     

Antiviral Activity of Ribosome-Inactivating Proteins

Ribosome-inactivating proteins (RIPs) are rRNA N-glycosylases from plants (EC 3.2.2.22) that inactivate ribosomes thus inhibiting protein synthesis. The antiviral properties of RIPs have been investigated for more than four decades. However, interest in these proteins is rising due to the emergence of infectious diseases caused by new viruses and the difficulty in treating viral infections. On the other hand, there is a growing need to control crop diseases without resorting to the use of phytosanitary products which are very harmful to the environment and in this respect, RIPs have been shown as a promising tool that can be used to obtain transgenic plants resistant to viruses. The way in which RIPs exert their antiviral effect continues to be the subject of intense research and several mechanisms of action have been proposed. The purpose of this review is to examine the research studies that deal with this matter, placing special emphasis on the most recent findings.     

Microarray analysis of genes differentially expressed in melatonin‐rich transgenic rice expressing a sheep serotonin N‐acetyltransferase

Transgenic rice plants overexpressing a sheep serotonin N‐acetyltransferase led to an enhanced production of melatonin with various physiological effects, including seminal root elongation and resistance against cold and oxidative stress, which raises the possibility that melatonin may alter gene expression profiles in the transgenic rice. Therefore, we performed a microarray analysis to investigate the regulatory role of melatonin using the melatonin‐rich transgenic rice. We identified 260 and 204 genes that were up‐ or downregulated in the melatonin‐rich transgenic rice when compared with the wild type. Of these, 20 upregulated genes were identified in the seedlings of melatonin‐rich rice at more than twice the levels in the wild type (P < 0.05), while 23 downregulated genes were also detected. The representative upregulated genes included caleosin, a Ca²⁺‐binding oil‐body surface protein involved in the degradation of lipids stored in oil bodies and various signaling proteins such as a cyclin F‐box protein and leucine‐rich repeat protein. In contrast, jasmonate‐induced protein, senescence‐associated protein, and polygalacturonase were included in the downregulated gene group. These results suggest that melatonin has an important role in modulating a wide range of gene expression, reflecting its pleiotropic physiological roles in plant growth and development.     

转基因马铃薯中人白介素-12的生物学活性

目的 检测转基因马铃薯体系中表达的人白介素-12(human interleukin 12,hIL-12)的肿瘤杀伤活性.方法 通过ELISA法检测转基因马铃薯中hIL-12刺激外周血单个核细胞(PBMC)产生IFN-γ的量以反映生物学活性,并观察hIL-12单克隆抗体对此生物活性的阻断效应;MTT法检测经转基因马铃薯中hIL-12作用的PBMC对NK敏感肿瘤细胞K562的杀伤作用,肿瘤细胞凋亡以Annexin V/PI双染后激光共聚焦显微镜观察.结果 转基因马铃薯叶和块茎中表达的hIL-12与重组人白介素-12(rhIL-12)标准品在25～400 pg/ml间均能诱生IFN-γ,其中转基因马铃薯叶和块茎中hIL-12刺激PBMC产生IFN-γ的量分别为(163.34±22.17)～(12 107.10±3 996.51)pg/ml和(224.20±25.76)～(11 196.30±4 067.48)pg/ml,与野生对照组均有统计学差异(P<0.01),而hIL-12单克隆抗体能完全阻断此诱生IFN-γ效应.同时200 pg/ml转基因马铃薯叶、块茎中提取的hIL-12对人红白血病细胞K562的杀伤率分别为(28.4±8.7)%、(32.3±6.9)%,而与野生对照组(10.3±4.9)%的杀伤率相比,有统计学差异(P<0.05).激光共聚焦显微镜观察到经转基因马铃薯表达hIL-12作用的K562细胞出现早、中、晚各期的凋亡特征.结论 转基因马铃薯表达的hIL-12具有诱导PBMC产生IFN-γ及杀伤肿瘤细胞K562的生物学活性.     

Dopamine neuron glutamate cotransmission: frequency-dependent modulation in the mesoventromedial projection

Mesoventromedial dopamine neurons projecting from the medial ventral tegmental area to the ventromedial shell of the nucleus accumbens play a role in attributing incentive salience to environmental stimuli that predict important events, and appear to be particularly sensitive to the effects of psychostimulant drugs. Despite the observation that these dopamine neurons make up almost the entire complement of neurons in the projection, stimulating their cell bodies evokes a fast glutamatergic response in accumbens neurons. This is apparently due to dopamine neuron glutamate cotransmission, suggested by the extensive coexpression of vesicular glutamate transporter 2 (VGLUT2) in the neurons. To examine the interplay between the dopamine and glutamate signals, we used acute quasi-horizontal brain slices made from DAT-YFP mice in which the intact mesoventromedial projection can be visualized. Under current clamp, when dopamine neurons were stimulated repeatedly, dopamine neuron glutamate transmission showed dopamine-mediated facilitation, solely at higher, burst-firing frequencies. Facilitation was diminished under voltage clamp and flipped to inhibition by intracellular Cs⁺ or GDPβS, indicating that it was mediated postsynaptically. Postsynaptic facilitation was D1 mediated, required activation of NMDA receptors and closure of voltage gated K⁺-channels. When postsynaptic facilitation was blocked, D2-mediated presynaptic inhibition became apparent. These counterbalanced pre- and postsynaptic actions determine the frequency dependence of dopamine modulation; at lower firing frequencies dopamine modulation is not apparent, while at burst firing frequency postsynaptic facilitation dominates and dopamine becomes facilitatory. Dopamine neuron glutamate cotransmission may play an important role in encoding the incentive salience value of conditioned stimuli that activate goal-directed behaviors, and may be an important subtract for enduring drug-seeking behaviors.     

mdr1启动子调控CD∷UPP基因对紫杉醇耐药卵巢癌细胞的杀伤作用

目的:探讨腺病毒介导的mdr1启动子调控胞嘧啶脱氨酶∷尿嘧啶磷酸核糖转移酶(CD∷UPP)融合基因联合5-氟胞嘧啶(5-FC)对紫杉醇耐药卵巢癌细胞的特异性杀伤作用。方法:扩增、纯化含有mdr1-CD∷UPP基因的重组腺病毒,转染人卵巢癌紫杉醇耐药细胞株A2780/Taxol和亲本细胞株A2780,RT-PCR检测mdr1和CD∷UPP基因的表达水平;之后加入5-FC,MTT法检测细胞抑制情况及旁观者效应,并观察腺病毒转染后裸鼠移植瘤的生长情况。结果:mdr1和CD∷UPP基因在A2780/Taxol细胞中可稳定表达,转染后A2780/Taxol组的细胞生长明显低于A2780组;转基因的A2780/Taxol细胞联合5-FC后可通过旁观者效应杀伤周围未转基因的耐药细胞;耐药组移植瘤生长明显受到抑制,肿瘤体积为(569.10±187.93)mm3,对照组肿瘤体积为(2111.98±230.82)mm3,差异有统计学意义(P<0.01)。结论:mdr1启动子可调控CD∷UPP基因特异性表达并特异性杀伤紫杉醇耐药卵巢癌细胞。     

重组腺相关病毒介导的HSV-tk/GCV体系对兔晶状体上皮细胞的抑制作用

目的 探讨2型重组腺相关病毒(rAAV2)载体介导的单纯疱疹病毒胸苷激酶基因/丙氧鸟苷体系(HSV-tk/GCV)对体外培养兔晶状体上皮细胞(N/N1003A)的抑制作用,以及晶状体上皮细胞死亡的机制.方法 实验研究.rAAV2载体介导增强型绿色荧光蛋白基因(EGFP)转染体外培养N/N1003A细胞,倒置荧光显微镜观察细胞中EGFP的表达,流式细胞仪检测病毒的转染效率.重组病毒rAAV2/HSV-tk转染体外培养的N/N1003A细胞为实验组,以没有转染重组病毒的细胞作为对照组,MTT法检测HSV-tk/GCV体系对细胞作用的浓度依赖性、时间依赖性和旁观者效应;相差显微镜、透射电镜、Hoechst33258染色观察细胞凋亡、坏死改变,流式细胞仪检测细胞凋亡率和细胞周期的变化.不同浓度GCV对两组细胞的作用结果采用两因素析因设计的方差分析;两组细胞周期和凋亡的比较采用两样本t检验.结果 rAAV2载体能介导EGFP基因稳定高效转染N/N1003A细胞.GCV对两组细胞的杀伤作用有剂量-效应依赖关系(F=13 076.239,P<0.001);实验组N/N1003A-tk细胞的存活率低于对照组,差异有统计学意义(F=53 947.119,P<0.001).实验组GCV的IC50为2 mg/L,而对照组IC50为524 mg/L.GCV的杀伤效应随时间延长而增强,且存在明显的旁观者效应.N/N1003A-tk细胞在GCV作用下出现明显的细胞凋亡和坏死,细胞的凋亡率7.18%±2.04%,与对照组(3.50%±0.56%)比较差异有统计学意义(t=3.83,P<0.01);S期细胞比例明显增加,G0/G1期细胞比例明显减少,与对照组比较差异有统计学意义(S期细胞比例:t=3.55,P<0.01;G0/G1期细胞比例:t=4.29,P<0.01).结论 GCV可有效杀伤重组腺相关病毒rAAV2/HSV-tk转染的兔晶状体上皮细胞,并具有较强的旁观者效应.     

转sck基因水稻中CpTI蛋白的细胞内定位

目的转sck基因水稻中CpTI蛋白细胞内定位。方法运用免疫组织化学方法,应用免疫电镜技术,观察在转sck基因水稻根部细胞、叶片细胞中CpTI蛋白的定位情况。结果在转sck基因水稻根尖部细胞的内质网中和内质网附近有胶体金颗粒大量存在,细胞的质体、叶绿体、细胞质、细胞核中也散在一些胶体金颗粒,在对照水稻的根部内质网和其他部位未发现胶体金颗粒存在。水稻叶片细胞由于有叶泡的存在,内质网不明显,但是细胞核、细胞质和质体内也发现胶体金颗粒,在非转基因水稻的叶片中未发现胶体金颗粒。结论转sck基因水稻中的CpTI蛋白在内质网中大量存在,在细胞的质体、叶绿体、细胞质、细胞核中也存在少量CpTI蛋白。     

核化线粒体组学与转基因人分析法在人类进化研究中的应用1例

5年前,作者提出转基因人分析法,即以人类染色体DNA与线粒体DNA的重组状况作为研究对象,并将这种研究线粒体DNA与染色体DNA之间相互作用的科学称之为核化线粒体组学.分析当时可利用的尼安特人的线粒体DNA高变区数百个碱基,认为不能排除尼安特人与现代人在基因水平上可能存在亲缘关系.最近,细胞杂志发表了尼安特人的线粒体DNA完全序列,通过利用核化线粒体组学方法 ,使5年前的推论得到了确认,即尼安特人与现代人在基因水平上存在亲缘关系.这一应用实例对分子考古学具有一定的参考价值.