Adenoviral vectors expressing siRNAs for discovery and validation of gene function

RNA interference is a powerful tool for studying gene function and for drug target discovery in diverse organisms and cell types. In mammalian systems, small interfering RNAs (siRNAs), or DNA plasmids expressing these siRNAs, have been used to down-modulate gene expression. However, inefficient transfection protocols, in particular, for primary cell types, have hampered the use of these tools in disease-relevant cellular assays. To be able to use this technology for genome-wide function screening, a more robust transduction protocol, resulting in a longer duration of the knock-down effect, is required. Here, we describe the validation of adenoviral vectors that express hairpin RNAs that are further processed to siRNAs. Infection of cell lines, or primary human cells, with these viruses leads to an efficient, sequence-specific, and prolonged reduction of the corresponding target mRNA, resulting in a reduction of the encoded protein level in the cell. For knock-down of one of the targets, GalphaS, we have measured inhibition of ligand-dependent, G-protein-coupled signaling. It is expected that this technology will prove to be of great value in target validation and target discovery efforts.     

A new variant of type II von Willebrand disease with aberrant multimeric structure of plasma but not platelet von Willebrand factor (type IIF)

A patient with a lifelong bleeding disorder was diagnosed as having Type II von Willebrand disease. The larger multimers of von Willebrand factor were absent from her plasma but present in platelets. A high- resolution electrophoretic technique was used to study the complex structure of individual von Willebrand factor multimers. In normal plasma, each multimer could be resolved into five bands: a more intense central one and four less intense, two moving faster and two slower than the central band. In normal platelets, each multimer could also be resolved into five bands. The central one had a mobility similar to that in plasma, whereas the four satellite bands had a mobility that differed from that of the corresponding plasma bands. In the patient, platelet von Willebrand factor antigen content and ristocetin cofactor activity were normal, and von Willebrand factor showed the same structure of individual multimers as seen in normal platelets. On the other hand, plasma von Willebrand factor antigen and ristocetin cofactor activity were decreased, and the structure of individual von Willebrand factor multimers was different from that of normal plasma and similar to that seen in normal and patient's platelets. After infusion of 1-deamino-8-D-arginine vasopressin, the largest von Willebrand factor multimers, as well as new satellite bands with a mobility similar to those in normal plasma, appeared in the patient plasma, and the levels of von Willebrand factor antigen and ristocetin cofactor activity became normal. Yet no relevant change in the prolonged bleeding time was observed. This new variant of von Willebrand disease, therefore, is characterized by the presence of a dysfunctional von Willebrand factor molecule that exhibits unique structural abnormalities in plasma but appears to be normal in platelets. The designation of Type IIF is proposed for this type of von Willebrand disease in accordance with the terminology that has been previously used.     

自体髂骨植骨带锁钢板内固定材料置入并一期前后路减压治疗多节段脊髓型颈椎病12例:临床资料回顾分析

目的:严重的多节段脊髓型颈椎病单纯前路或后路手术都有其局限性。观察一期前后路联合手术并自体髂骨植骨及带锁钢板内固定材料置入在治疗多节段脊髓型颈椎病中的应用价值。方法:选择2004-11/2006-12本院12例多节段脊髓型颈椎病患者,均采用一期前后路减压、自体髂骨植骨融合、带锁钢板内固定联合手术。其中男9例,女性3例,年龄49～75岁;3节段受累9例,4节段受累3例(突出节段分布:C3～66例,C4～73例,C3～73例)。全部病例进行临床随访,患者均对本试验知情同意。采用mJOA评分标准对患者神经功能改善情况进行评定;术前颈椎侧位片测量,以D值(C4椎体后下缘到齿突后缘与C7椎体后下缘连线的垂直距离)评价颈椎(C2～7)弧度;根据颈椎伸屈动态侧位片C2和C7椎体后缘切线相交所成的夹角之和评价颈椎(C2～7)活动范围。主要以电话随访和问卷填写的方式,分别从神经功能改善情况、颈椎弧度、活动范围及术后并发症等进行随访观察。结果:①12例患者全部得到随访,术后随访时间6～28个月,平均(16±6)个月。②所有植骨均获得骨性愈合;疗效结果中优4例(33.3%);良6例(50%);无效2例(16.7%);颈椎D值术前(3.9±1.4)mm,术后即刻(8.5±1.7)mm,随访时(8.1±2.5)mm。术前与术后差异有显著性(P<0.01),术后与随访时差异无显著性(P=0.251);颈椎活动范围术前(36.3±4.0)°,随访时(10.6±2.7)°,与术前相比差异具有显著性(P<0.01)。③术后C5神经根麻痹1例,为感觉及运动混合型,8个月随访时,感觉功能恢复,肩关节外展肌力从术后Ⅱ级恢复至Ⅳ级;1例术后6个月出现"S"畸形而再次压迫脊髓,神经功能改善停滞,目前处于随访中。结论:一期前后路手术并自体髂骨植骨及带锁钢板内固定材料置入减压充分、彻底,而且前路手术能重建颈椎稳定性,恢复颈椎生理前凸和椎间高度,并且后路减压术又能预防相邻颈椎退变引起的脊髓继发的压迫。     

DFNB74, a novel autosomal recessive nonsyndromic hearing impairment locus on chromosome 12q14.2-q15

Autosomal recessive nonsyndromic hearing impairment (ARNSHI) segregating in three unrelated, large consanguineous Pakistani families (PKDF528, PKDF859 and PKDF326) is linked to markers on chromosome 12q14.2-q15. This novel locus is designated DFNB74 . Maximum two-point limit of detection (LOD) scores of 5.6, 5.7 and 2.6 were estimated for markers D 12 S 313, D 12 S 83 and D 12 S 75 at θ = 0 for recessive deafness segregating in these three families. Haplotype analyses identified a critical linkage interval of 5.35 cM (5.36 Mb) defined by D 12 S 329 at 74.58 cM and D 12 S 313 at 79.93 cM. DFNB74 is the second ARNSHI locus mapped to chromosome 12, but the physical intervals do not overlap with one another. A locus contributing to the early onset, rapidly progressing hearing loss of A/J mice ( ahl4 , age-related hearing loss 4) was reported to map to chromosome 10 in a region of conserved synteny to DFNB74 , suggesting that ahl4 and DFNB74 may be due to mutations of the same gene in these two species.     

Immunolocalization of Le(y) oligosaccharide in endometrium during menstrual cycle and effect of early luteal phase mifepristone administration on its expression in implantation stage endometrium of the rhesus monkey

Changes in carbohydrate expression on endometrial and blastocyst cell surfaces may play a critical role in the process of implantation. Le(y) is an oligosaccharide antigen which has been shown to be involved in blastocyst attachment in the mouse. In the present study, immunohistochemical distribution of Le(y) in endometrium during proliferative and secretory phases of normal menstrual cycles in the rhesus monkey was examined. Endometrial samples were collected on cycle days 7 (n=4), 13 (n= 4), 16 (n=4), 20 (n=4) and 25 (n=3). There was a gradual increase of Le(y) in luminal surface from proliferative to periovulatory (P < 0.001), and from periovulatory to postovulatory (P < 0.05), phases. Le(y) then remained constant in the midsecretory phase and decreased (P < 0.01) during the premenstrual phase. Le(y) score in glands did not change between the phases, except in midsecretory phase when it was higher than that in other phases (P < 0.05). The stromal compartment showed no statistically significant changes. The profiles of endometrial Le(y) on day 6 after ovulation in mated fecund cycles with or without early luteal phase mifepristone treatment were also examined. Females were allowed to cohabit with males during days 8-16 of their ovulatory cycles and were injected s.c. with vehicle (n=7) only, or with a single dose of mifepristone (2 mg/kg body weight; n=8) on day 2 after ovulation. Significant decreases in the area and optical absorption of immunoprecipitate were observed in the epithelial compartment (P < 0.01) following mifepristone treatment. There was no change in the stromal compartment either in area or in optical absorption of immunoprecipitate for Le(y) with or without mifepristone treatment. The expression of Le(y) in the endometrial epithelial compartment appears to be influenced by progesterone and may be associated with endometrial receptivity prior to implantation in the rhesus monkey.