Defense reactions of mosquitoes to filarial worms: potential mechanism for avoidance of the response by Brugia pahangi microfilariae

The melanization response of Aedes trivittatus and the black-eyed Liverpool (LVP) and Rocke-feller (RKF) strains of Aedes aegypti against intrathoracically inoculated Brugia pahangi microfilariae (mff) that previously had penetrated LVP, RKF, or A. trivittatus midguts in vitro was assessed at 1, 3, and 5 days postinoculation (PI). LVP and RKF midgut-derived mff almost totally avoided the melanization response and developed normally in LVP strain A. aegypti, and although over 90% of these mff died by 5 days PI in RKF mosquitoes, the majority of these were not melanized. A. aegypti midgut-derived mff also were able to avoid the response of A. trivittatus in 33–43% of the cases. Penetration through A. trivittatus midguts, however, did not significantly affect the ability of mff to avoid the melanization response in any of the mosquitoes examined. Allogeneic and xenogeneic tissue inplants were accepted by all three mosquito species examined. Data presented support the hypothesis that mff avoid immune recognition in compatible mosquitoes by coating themselves with midgut material(s) during penetration of the midgut in their migration to the hemocoel.     

Isolation of silencer-containing sequences causing a tissue-specific position effect on alcohol dehydrogenase expression in Drosophila melanogaster

A transient expression assay has been used to investigate the cause of a tissuespecific position effect on Adh expression from a transgene insertion in Drosophila. A 15.4-kb genomic clone containing the 3.2-kb Adh insert along with flanking regions of genomic DNA is expressed in this assay in a tissue-specific pattern resembling the abnormal expression pattern of the position effect. The 3.2-kb Adh insert is expressed normally without the flanking sequences. A silencer element is located upstream of the Adh gene within a 2-kb fragment that acts in both orientations and at a distance of at least 6.5 kb from the larval Adh promoter to suppress ADH expression in a nontissue specific fashion. The DNA sequence of the 2-kb fragment indicates that it is a noncoding region. A 17-bp sequence is repeated within this region and may be associated with the silencer activity, since subclones from the 2-kb fragment, each containing one of the repeated regions, both retain full silencer activity. This silencer fails to suppress expression from an α₁-tubulin promoter-LacZ fusion construct or an hsp70 promoter-Ach fusion construct. In addition to the silencer, another element is located downstream of the Adh gene that produces a higher level of anterior than posterior midgut expression. These results suggest that the 5′ silencer and the 3′ element act together to create the tissue specific pcsition effect characteristic of the GC-1 line. © 1994 Wiley-Liss, Inc.     

Neuron-restrictive silencer factor functions to suppress Sp1-mediated transactivation of human secretin receptor gene

In the present study, a functional neuron restrictive silencer element (NRSE) was initially identified in the 5′ flanking region (− 83 to − 67, relative to ATG) of human secretin receptor (hSCTR) gene by promoter assays coupled with scanning mutation analyses. The interaction of neuron restrictive silencer factor (NRSF) with this motif was later indicated via gel mobility shift and ChIP assays. The silencing activity of NRSF was confirmed by over-expression and also by shRNA knock-down of endogenous NRSF. These studies showed an inverse relationship between the expression levels of NRSF and hSCTR in the cells. As hSCTR gene was previously shown to be controlled by two GC-boxes which are regulated by the ratio of Sp1 to Sp3, in the present study, the functional interactions of NRSF and Sp proteins to regulate hSCTR gene was investigated. By co-immunoprecipitation assays, we found that NRSF could be co-precipitated with Sp1 as well as Sp3 in PANC-1 cells. Interestingly, co-expressions of these factors showed that NRSF could suppress Sp1-mediated, but not Sp3-mediated, transactivation of hSCTR. Taken together, we propose here that the down-regulatory effects of NRSF on hSCTR gene expression are mediated via its suppression on Sp1-mediated transactivation.     

Effect of ultraviolet B (302 nm) irradiation on viability, metabolic and detoxification functions of goat hepatocytes – in vitro study

The object of the present study was to investigate the effect(s) of UV-B irradiation on the functional integrity, metabolic and detoxifying capacity of the isolated goat hepatocytes. Isolated goat hepatocytes were subjected to UV-B irradiation invitro for 0, 250, 500, 1250, 2500 and 7500 Joules/m² which correspond to the irradiation time of 0, 1, 2, 5, 10 and 30 min. Cells were then analysed for Viability (Trypan blue exclusion test [TBE], 3-[4,5-dimethylthiozol-2yl]-2,5-diphenyltetrazolium bromide [MTT] assay, Membrane integrity (Lactate dehydrogenase [LDH] leakage, Lipid peroxidation) Detoxification (Ureagenesis, Cytochrome P450 activity [CYP450, Diazepam metabolism] and Glutathione-S-Transferase [GST] activity. The results show that there was no difference in functional, metabolic as well as detoxifying parameters of the hepatocytes when irradiated from 0–1250 Joules/m², whereas a significant alteration was appreciable in the parameters such as LDH leakage, lipid peroxidation, and CYP450 activity when irradiated beyond 1250 Joules/m². Our present findings suggest that the biologically compatible and feasible dose of UV-B irradiation for xenotransplantation appears to be 1250 Joules/m².     

Intronic CA-repeat and CA-rich elements: a new class of regulators of mammalian alternative splicing

We have recently identified an intronic polymorphic CA-repeat region in the human endothelial nitric oxide synthase (eNOS) gene as an important determinant of the splicing efficiency, requiring specific binding of hnRNP L. Here, we analyzed the position requirements of this CA-repeat element, which revealed its potential role in alternative splicing. In addition, we defined the RNA binding specificity of hnRNP L by SELEX: not only regular CA repeats are recognized with high affinity but also certain CA-rich clusters. Therefore, we have systematically searched the human genome databases for CA-repeat and CA-rich elements associated with alternative 5' splice sites (5'ss), followed by minigene transfection assays. Surprisingly, in several specific human genes that we tested, intronic CA RNA elements could function either as splicing enhancers or silencers, depending on their proximity to the alternative 5'ss. HnRNP L was detected specifically bound to these diverse CA elements. These data demonstrated that intronic CA sequences constitute novel and widespread regulatory elements of alternative splicing.     

NRSF慢病毒干涉载体的构建及功能初步检测

为研究神经元限制性沉默因子(NRSF)负调控神经元及胰岛细胞中神经特异性基因的表达,拟通过RNAi的方法降低NRSF表达以进一步观察其调控的下游基因的表达情况.构建含人胰岛素启动子-荧光素酶(HIP-LUC)的pcDNA3.1报告载体.通过RNAi序列设计软件进行NRSF干涉片段及脱靶对照片段的设计,然后构建慢病毒干涉载体.包装产生慢病毒干涉毒液,用其感染HeLa细胞获得稳定干涉NRSF的细胞株,利用RT-PCR、实时定量PCR、蛋白质免疫印迹、免疫荧光染色等方法检测NRSF的干涉效果及其下游基因的表达情况,观察干涉NRSF后胰岛素启动子报告载体荧光素酶活性的变化.构建具有NRSF干涉效果的慢病毒干涉载体成功,并获得了稳定干涉NRSF及脱靶对照的HeLa细胞株,RT-PCR及实时定量PCR检测结果表明,NRSF干涉片段的干涉效率为56%(n=6,P<0.01),蛋白质免疫印迹、免疫荧光染色方法检测证实干涉后NRSF蛋白表达水平明显降低.RT-PCR实验表明干涉NRSF后下游基因开始表达,荧光素酶活性分析表明,干涉NRSF后胰岛素启动子活性增强了2.4倍(n=3,P<0.01).上述结果表明,成功构建NRSF的慢病毒干涉载体并获得稳定干涉NRSF的HeLa细胞株,干涉NRSF后,其下游基因特别是胰岛素基因开始表达.这一研究工作有助于我们进一步了解NRSF在胰岛细胞发育分化中的调控作用.