干扰SET8调节血管平滑肌细胞增殖、凋亡促进血管钙化

背景与目的血管平滑肌细胞(VSMC)凋亡在许多血管钙化疾病的病理过程中起关键作用。近期研究表明,赖氨酸甲基转移酶SET8参与调节细胞增殖、凋亡过程。文章旨在探寻SET8是否通过调节VSMC增殖、凋亡而影响血管钙化。方法体外培养原代大鼠VSMC,将VSMC随机分为正常对照组、空质粒组和SET8-shRNA组。以脂质体Lipofectamine~(TM)2000为载体,转染VSMC。采用茜素红染色法和钙含量测定法判断细胞钙化程度;采用MTT法检测三组细胞的增殖能力;实时荧光定量PCR、Western blot法检测三组细胞中增殖基因Survivin和凋亡基因Caspase-3的表达水平,分析干扰SET8对VSMC增殖、凋亡及其相关基因和蛋白表达的影响。结果 (1)干扰SET8可成功抑制VSMC中SET8蛋白的表达(P0.05)。(2)转染VSMC后,与正常对照组和空质粒组比较,SET8-shRNA组钙含量明显升高(P0.05)。(3)MTT结果显示,在第12 h、24 h、36 h、48 h,SET8-shRNA组VSMC的增殖能力较正常对照组和空质粒组明显降低(P0.05)。(4)实时荧光定量PCR和Western blot结果显示SET8-shRNA组Survivin的mRNA和蛋白表达较正常对照组和空质粒组明显下降,而Caspase-3的mRNA和蛋白表达则相反(P0.05)。结论干扰SET8能够增加促凋亡基因表达,抑制增殖基因表达,SET8有可能参与调节大鼠血管钙化。     

Identification and characterization of doublesex in Bemisia tabaci

Bemisia tabaci (Gennadius) is an important agricultural pest with a worldwide distribution. Although B. tabaci is known to have a unique haplodiploid reproductive strategy, its sex determination mechanism is largely unknown. In this study, we cloned the full‐length sequence of B. tabaci doublesex (Btdsx) and found that Btdsx has 28 splicing isoforms. We found two new splicing isoforms of transformer 2 (Bttra2), which encode two proteins. We also confirmed that both genes lack sex‐specific splicing isoforms. Real‐time quantitative PCR analysis showed that the expression of Btdsx and Bttra2 is higher in males than in females. RNA interference of Bttra2 affected the expression of Btdsx and vice versa. Furthermore, silencing of Bttra2 or Btdsx caused malformation of the male genitalia (anal style). It did not affect the female phenotype, but reduced the expression of vitellogenin gene in females. These results indicate that Btdsx is associated with sex determination in B. tabaci and that Btdsx and Bttra2 affect each other and are important for male genitalia formation. In addition to increasing our understanding of the roles of dsx and tra2 in the sex determination of B. tabaci, the results will be useful for studies of sex determination in other haplodiploid species.     

SH-SY5Y神经细胞中α7神经型尼古丁受体基因沉默对CaM、CaMKⅡ蛋白水平的影响

目的构建稳定的α7 n AChR沉默的神经母细胞瘤细胞(SH-SY5Y)细胞,研究α7神经型尼古丁受体(n AChR)基因沉默对钙调蛋白(Ca M)、钙调素依赖性蛋白激酶Ⅱ(Ca MKⅡ)水平的影响,了解α7 n AChR神经保护作用及其与阿尔茨海默病(AD)发病机制的关系。方法将α7 n AChR shRNA重组质转染到SH-SY5Y,用含嘌呤霉素的培养液筛选,挑选阳性克隆后采用实时荧光定量PCR和蛋白质印迹方法(Western-blot)检测细胞中α7n AChR mRNA及蛋白表达水平的变化;Western-blot方法测定Ca M、Ca MKⅡ蛋白表达水平。结果获得稳定转染α7 n AChR shRNA重组质粒的细胞克隆株,与对照组相比,α7 n AChR mRNA及蛋白表达量分别减少了95%和80%。Ca M、Ca MKⅡ蛋白表达量分别减少了48.5%和35%。结论成功构建了α7 n AChR mRNA沉默的SH-SY5Y细胞细胞株,α7 n AChR沉默降低了Ca M、Ca MKⅡ的蛋白水平,可能影响信号通路转导,这可能与阿尔茨海默病(AD)的发病有一定的关系。     

Susceptibility to interference and intrusion errors in consequence of the dominant hemisphere’s hippocampal infarct: A case report

We present a case of a man with an ischemic lesion of the left hippocampus. Detailed neuropsychological assessment revealed susceptibility to retroactive interference and a tendency to make intrusion errors in addition to mild deficits in the verbal memory processes. Although retroactive interference and intrusion errors are normally considered to be the manifestations of frontal lobe dysfunctions, the idea of susceptibility to interference has recently begun to emerge in the literature, as an explanation of medial temporal lobe amnesia. Our data support this new theory, suggesting that one role of the hippocampus is to decrease the interference during the learning processes.     

Step initiation interferes with working memory in nondisabled patients with the earliest multiple sclerosis–A dual-task study

Balance and cognition are affected by multiple sclerosis (MS). Cognitive-motor interference (CMI) is important for balance impairment in MS, however little is known about CMI at the earliest stages of the disease. Step initiation (SI) with anticipatory postural adjustments (APAs) has been linked to postural instability and falls in subjects with MS, therefore we aimed to assess CMI between SI and the two storage systems of working memory in patients with clinically isolated syndrome (presented as optic neuritis-ON) suggestive of MS. Twenty patients with normal/near normal visual acuity and 20 age-, weight-, height-, sex- and education-matched control subjects were included. APAs were studied using center of pressure measures in three conditions: SI alone, SI + Brooks’ spatial- and SI + 2-back verbal working memory task. Decrements (% change) in performance on cognitive tasks and in APA parameters were calculated. CMI was assessed combining the two decrements scores. Performance on both cognitive tasks was more affected by dual-tasking in patients compared to healthy subjects. In both groups APA parameters were not influenced by dual-tasking. CMI was higher in patients compared to healthy subjects. Our results suggest that the disease affects CMI in its earliest stages. Since both cognitive tasks were similarly affected by dual-tasking in patients and controls central executive seems to play the major role in CMI between SI and working memory. Patients prioritizing motor over cognitive task for balance maintenance suggests reduced divided attention capacity as a cause of increased CMI in the earliest MS.     

The P450 enzyme Shade mediates the hydroxylation of ecdysone to 20‐hydroxyecdysone in the Colorado potato beetle,Leptinotarsa decemlineata

Ecdysone 20‐monooxygenase (E20MO), a cytochrome P450 monooxygenase (CYP314A1), catalyses the conversion of ecdysone (E) to 20‐hydroxyecdysone (20E). We report here the cloning and characterization of the Halloween gene Shade (Shd) encoding E20MO in the Colorado potato beetle, Leptinotarsa decemlineata. LdSHD has five conserved motifs typical of insect P450s, ie the Helix‐C, Helix‐I, Helix‐K, PxxFxPE/DRF (PERF) and heme‐binding motifs. LdShd was expressed in developing eggs, the first to fourth instars, wandering larvae, pupae and adults, with statistically significant fluctuations. Its mRNA was ubiquitously distributed in the head, thorax and abdomen. The recombinant LdSHD protein expressed in Spodoptera frugiperda 9 (Sf9) cells catalysed the conversion of E to 20E. Dietary introduction of double‐stranded RNA (dsRNA) of LdShd into the second instar larvae successfully knocked down the LdShd expression level, decreased the mRNA level of the ecdysone receptor (LdEcR) gene, caused larval lethality, delayed development and affected pupation. Moreover, ingestion of LdShd‐dsRNA by the fourth instars also down‐regulated LdShd and LdEcR expression, reduced the 20E titre, and negatively influenced pupation. Introduction of 20E and a nonsteroidal ecdysteroid agonist halofenozide into the LdShd‐dsRNA‐ingested second instars, and of halofenozide into the LdShd‐dsRNA‐ingested fourth instars almost completely relieved the negative effects on larval performance. Thus, LdSHD functions to regulate metamorphotic processes by converting E to 20E in a coleopteran insect species Le. decemlineata.     

Parkinson’s disease and the Stroop color word test: processing speed and interference algorithms

Objective: Processing speed alters the traditional Stroop calculations of interference. Consequently, alternative algorithms for calculating Stroop interference have been introduced to control for processing speed, and have done so in a multiple sclerosis sample. This study examined how these processing speed correction algorithms change interference scores for individuals with idiopathic Parkinson’s disease (PD, n = 58) and non-PD peers (n = 68). Method: Linear regressions controlling for demographics predicted group (PD vs. non-PD) differences for Jensen’s, Golden’s, relative, ratio, and residualized interference scores. To examine convergent and divergent validity, interference scores were correlated with standardized measures of processing speed and executive function. Results: PD–non-PD differences were found for Jensen’s interference score, but not Golden’s score, or the relative, ratio, and residualized interference scores. Jensen’s score correlated significantly with standardized processing speed but not executive function measures. Relative, ratio, and residualized scores correlated with executive function but not processing speed measures. Golden’s score did not correlate with any other standardized measures. Conclusions: The relative, ratio, and residualized scores were comparable for measuring Stroop interference in processing speed-impaired populations. Overall, the ratio interference score may be the most useful calculation method to control for processing speed in this population.