hLMO3基因真核表达载体的构建及蛋白的表达和定位

目的 构建hLMO3真核表达载体并证实融合蛋白在细胞内的表达及定位.方法 以人胎脑文库cDNA为模板,PCR扩增hLMO3基因cDNA全长,亚克隆至pEGFP表达载体中.将构建的重组质粒进行酶切测序鉴定,并转染到人上皮细胞HEK293细胞中,提取细胞蛋白进行Western blot检测.利用激光扫描共聚焦显微镜观察pE...     

Di-lysine motif-like sequences formed by deleting the C-terminal domain of aquaporin-4 prevent its trafficking to the plasma membrane

Aquaporin-4 is a transmembrane water channel protein, the C-terminal domain of which is facing the cytosol. In the process of investigating the role of the C-terminal domain of aquaporin-4 with regard to intracellular trafficking, we observed that a derivative of aquaporin-4, in which the C-terminal 53 amino acids had been removed (Δ271-323), was localized to intracellular compartments, including the endoplasmic reticulum, but was not expressed on the plasma membranes. This was determined by immunofluorescence staining and labeling of the cells with monoclonal antibody specifically recognizing the extracellular domain of aquaporin-4, followed by confocal microscopy and flow cytometry. Deletion of additional amino acids in the C-terminal domain of aquaporin-4 led to its redistribution to the plasma membrane. This suggests that the effect of the 53-amino acid deletion on the subcellular localization of aquaporin-4 could be attributed to the formation of a signal at the C terminus that retained aquaporin-4 in intracellular compartments, rather than the loss of a signal required for plasma membrane targeting. Substitution of the lysine at position 268 with alanine could rescue the Δ271-323-associated retention in the cytosol, suggesting that the C-terminal sequence of the mutant served as a signal similar to a di-lysine motif.     

小鼠神经生长因子的细胞内免疫组织化学阳性反应定位

用免疫组织化学方法对小鼠神经组织和非神经组织中神经生长因子的分布进行了定位观察。实验结果表明 :(1)神经生长因子免疫反应主要局限于神经组织 ,尤其是神经元 ;非神经组织除颌下腺的颗粒曲管外基本上都呈阴性反应 ;(2 )在神经元 ,神经生长因子定位于细胞核与细胞质但以胞核为主 ,少量位于胞质靠近细胞膜的部位 ;(3 )雄性小鼠颌下腺颗粒曲管细胞的NGF主要定位于分泌颗粒的界膜区。神经生长因子在胞内定位的精确性和分布的多样性 ,为进一步研究其作用机制和信号传导提供了重要线索     

不同分子亚型乳腺癌细胞系中RUNX3蛋白表达及定位研究

目的:检测人类Runt相关转录因子3(RUNX3)在不同分子亚型乳腺癌细胞系中的表达及其亚细胞定位情况,为进一步揭示RUNX3的失活机制和发现新的治疗靶点提供理论依据。方法:在5种乳腺癌细胞(MCF-7、T47D、SKBR-3、MDA-MB-231和BT-549)及正常乳腺上皮细胞(MCF-10A)中,通过Western blot和免疫荧光实验检测RUNX3的蛋白表达和亚细胞定位情况;采用来普霉素B(Leptomycin B)抑制RUNX3的出核,利用CCK-8法检测细胞活力的改变,EdU染色检测细胞增殖情况,Western blot和免疫荧光实验检测RUNX3的蛋白表达和亚细胞定位的改变。结果:与MCF-10A细胞相比,5种乳腺癌细胞系中RUNX3的核定位减少、胞浆定位增多。经Leptomycin B处理后,CCK-8实验结果显示5种乳腺癌细胞的活力明显减弱,EdU染色显示5种乳腺癌细胞增殖能力明显降低,Western blot和免疫荧光实验显示5种乳腺癌细胞胞浆中的RUNX3蛋白表达量明显降低、胞核中的RUNX3蛋白表达量明显增多(P0.05)。结论:不同分子亚型乳腺癌细胞中均存在RUNX3的胞浆转位失活现象,针对性地逆转RUNX3的出核过程可以明显降低肿瘤细胞的活力和增殖能力,可能成为乳腺癌潜在的治疗靶点。     

Regulation of glutamatergic and GABAergic neurotransmission in the chick nucleus laminaris: role of N-type calcium channels

Neurons in the chicken nucleus laminaris (NL), the third order auditory nucleus involved in azimuth sound localization, receive bilaterally segregated (ipsilateral vs contralateral) glutamatergic excitation from the cochlear nucleus magnocellularis and GABAergic inhibition from the ipsilateral superior olivary nucleus (SON). Here, I investigate the voltage-gated calcium channels (VGCCs) that trigger the excitatory and the inhibitory transmission in the NL. Whole-cell recordings were performed in acute brainstem slices. The excitatory transmission was predominantly mediated by N-type VGCCs, as the specific N-type blocker ω-Conotoxin-GVIA (ω-CTx-GVIA, 1–2.5 μM) inhibited excitatory postsynaptic currents (EPSCs) by ∼90%. Blockers for P/Q- and L-type VGCCs produced no inhibition, and blockade of R-type VGCCs produced a small inhibition. In individual cells, the effect of each VGCC blocker on the EPSC elicited by activation of the ipsilateral input was the same as that on the EPSC elicited by activation of the contralateral input, and the two EPSCs had similar kinetics, suggesting physiological symmetry between the two glutamatergic inputs to single NL neurons. The inhibitory transmission in NL neurons was almost exclusively mediated by N-type VGCCs, as ω-CTx-GVIA (1 μM) produced a ∼90% reduction of inhibitory postsynaptic currents, whereas blockers for other VGCCs produced no inhibition. In conclusion, N-type VGCCs play a dominant role in triggering both the excitatory and the inhibitory transmission in the NL, and the presynaptic VGCCs that mediate the two bilaterally segregated glutamatergic inputs to individual NL neurons are identical. These features may play a role in optimizing coincidence detection in NL neurons.     

The risk of urinary incontinence after partial urethral resection in patients with anterior vulvar cancer

Objective

The number of women with vulvar carcinoma located in the anterior fourchette in immediate proximity to the urethral opening has increased. A retrospective analysis was performed in order to evaluate the risk of urinary incontinence after tumor-resection, standard inguinal lymphadenectomy and additional partial urethral resection.

Study design

Between 2002 and 2007, 19 women with vulvar carcinomas located close to the urethral opening and consequently treated by additional partial urethral resection of up to 1.5 cm, were evaluated for urinary loss postoperatively by standard incontinence questionnaire. All patients complaining about some kind of urinary loss underwent urodynamic measurement. Results were compared with 21 controls (women with anterior vulvar cancer treated without urethral resection).

Results

Five of 19 women (26%) of the study group complained about urinary disturbances and received urodynamic evaluation. Ninety-five percent of the patients (18/19 women) were continent by urodynamic criteria; in one woman the measurement was unreliable. One patient in the control group (1/21 women) complained of an increase of urge symptoms that had been present preoperatively.

Conclusions

Twenty-six percent of our patients after partial urethral resection reported incontinence symptoms, though this was not always confirmed by urodynamics. We conclude that the risk of urinary stress incontinence after partial urethral resection in anterior vulvar carcinoma is acceptable.     

Nuclear import strategies of high-risk HPV18 L2 minor capsid protein

We have investigated the nuclear import strategies of high-risk HPV18 L2 minor capsid protein. HPV18 L2 interacts with Kap α₂ adapter, and Kap β₂ and Kap β₃ nuclear import receptors. Moreover, binding of RanGTP to either Kap β₂ or Kap β₃ inhibits their interaction with L2, suggesting that these Kap β/L2 complexes are import competent. Mapping studies show that HPV18 L2 contains two NLSs: in the N-terminus (nNLS) and in the C-terminus (cNLS), both of which can independently mediate nuclear import. Both nNLS and cNLS form a complex with Kap α₂β₁ heterodimer and mediate nuclear import via a classical pathway. The nNLS is also essential for the interaction of HPV18 L2 with Kap β₂ and Kap β₃. Interestingly, both nNLS and cNLS interact with the viral DNA and this DNA binding occurs without nucleotide sequence specificity. Together, the data suggest that HPV18 L2 can interact via its NLSs with several Kaps and the viral DNA and may enter the nucleus via multiple import pathways mediated by Kap α₂β₁ heterodimers, Kap β₂ and Kap β₃.     

肿瘤抑制因子PTEN亚细胞定位与缺血-缺氧性脑损伤

第10号染色体缺失的磷酸酶和张力蛋白同源等位基因(PTEN)具有脂质和蛋白双重磷酸酶活性。脑组织缺血-缺氧后PTEN发生活化并向线粒体或胞核内转移,而这种在神经元内的不同亚细胞定位可能最终决定细胞命运。因此,阐明PTEN在脑缺血-缺氧后的亚细胞定位机制,将为各种急慢性神经退行性变疾病的靶向治疗和药物研发提供新思路。