pEGFP/hVEGF121融合蛋白真核表达载体的构建及在骨髓间质干细胞中的表达

目的：构建携带人血管内皮细胞生长因子121及绿色荧光蛋白报告基因的融合蛋白真核表达质粒并检测其在骨髓间质干细胞（MSC）中的表达。 方法： 采用PCR技术，以pCD/hVEGF121质粒为模板扩增VEGF121基因全长，采用PCR产物的粘端克隆法，将VEGF121定向克隆入pEGFP-C1的多克隆位点，构建pEGFP/hVEGF121重组质粒，酶切、PCR及序列分析鉴定，脂质体介导转染体外培养的MSC，荧光显微镜及免疫细胞化学染色检测EGFP/VEGF融合蛋白的表达。 结果： PCR、酶切及测序证实目的基因VEGF121正确连接至pEGFP-C1的多克隆位点，pEGFP/hVEGF121重组质粒转染MSC后，荧光显微镜及免疫细胞化学检测EGFP/VEGF蛋白在MSC中存在表达。 结论： 成功构建了携带人VEGF121及EGFP报告基因的融合蛋白真核表达质粒，并在MSC中获得表达，为进一步研究VEGF基因治疗缺血性心血管疾病及MSC的分化奠定了实验基础。     

A PCR–restriction fragment length polymorphism assay to genotype human metapneumovirus

Human metapneumovirus (hMPV) genotypes A and B show epidemiological and probably clinical differences. This report describes a fast and simple PCR–restriction fragment length polymorphism (PCR-RFLP) assay, involving digestion of the fusion protein gene with Tsp 509I, that allows lineages A1, A2, B1 and B2 to be distinguished. The assay should help in elucidating the epidemiology of hMPV, and possibly in predicting the severity of clinical infection.     

磷酸钙人工骨(CPC)在颈前路椎间融合的临床应用

目的探讨磷酸钙人工骨(CPC)在颈椎前路椎间融合手术中的应用效果。方法2001年4月至2003年10月颈前路手术中应用磷酸钙人工骨栓椎间融合结合钛钢板固定治疗颈椎病17例,颈椎间盘突出症5例,颈椎外伤脱位2例,共24例35个节段。采用JOA评分评价神经功能,X线片判定融合效果。结果随访18±6.5个月,术后无感染,无过敏或毒性反应。JOA评分由术前9.28±2.15分增加到14.65±2.18分(P<0.001)。术后X线片未见CPC骨栓塌陷或移位,钛板和螺钉无松动及折断。术后16.5±6.8个月均获得椎间融合。结论颈椎前路椎间融合手术应用磷酸钙人工骨替代自体骨,经济、安全、简便、效果可靠。     

An interaction site of the envelope proteins of Semliki Forest virus that is preserved after proteolytic activation

Semliki Forest virus (SFV) membrane fusion is mediated by the viral E1 protein at acidic pH and regulated by the dimeric interaction of E1 with the E2 membrane protein. During low pH-triggered fusion, the E2/E1 heterodimer dissociates, freeing E1 to drive membrane fusion. E2 is synthesized as a precursor, p62, which is processed to mature E2 by the cellular protease furin. Both the dissociation of the p62/E1 dimer and the fusion reaction of p62 virus have a more acidic pH threshold than that of the mature E2 virus. We have previously isolated SFV mutations that allow virus growth in furin-deficient cells. Here we have used such pci mutations to compare the interactions of the p62/E1 and E2/E1 dimers. Our data suggest that there is an important p62/E1 dimer interaction site identified by an E2 R250G mutation and that this interaction is maintained after processing to the mature E2 protein.     

PD-L2剪切异构体与EGFP融合蛋白的表达及其亚细胞定位研究

目的　探讨PD L2的剪切异构体在哺乳动物细胞的表达和亚细胞定位。方法　以RT PCR方法克隆人PD L2基因的常规剪切体和新型剪切异构体的cDNA ,构建其与EGFP融合的表达载体 ,分别转染K5 6 2细胞进行表达 ,经抗PD L2抗体染色后以流式细胞仪和激光共聚焦显微镜分析融合蛋白的表达及其亚细胞定位。结果　从活化的人白细胞中克隆到常规剪切体PD L2Ⅰ和新型剪切异构体PD L2Ⅱ的cDNA ,后者删除了编码胞外区Ig C结构域的外显子 3。进而构建了PD L2Ⅰ EGFP和PD L2Ⅱ EGFP融合蛋白表达载体 ,分别转染K5 6 2细胞。流式细胞仪分析显示 ,转染前者的细胞中既可检测到EGFP的表达又可在细胞膜上检测到PD L2表达 ;而转染后者的细胞中只能检测到EGFP的表达 ,在其细胞膜上不能检测到PD L2表达。共聚焦显微镜观察显示 ,PD L2Ⅰ EGFP主要分布于细胞膜表面 ,PD L2Ⅱ EGFP则主要分布于细胞内。结论　常规剪切体PD L2Ⅰ能正确定位于细胞膜表面 ,而新型剪切异构体PD L2Ⅱ则位于细胞内 ,不能运输至细胞膜 ,提示不同PD L2剪切异构体可能与其功能的调变有关     

皮质骨圈在椎弓根钉固定系统中支撑作用的生物力学评价

目的：了解脊椎椎弓根钉固定系统在人体皮质骨圈（allograft fusion cage,AFC)植入椎间隙前后对脊柱稳定性的影响。方法：在8具新鲜成年猪离体腰椎标上，以L2－3，L3－R，L4－5节段为实验对象，测试各节段在正常状态下（正常组）、椎间盘切除并Steffee钢板固定（内固定组）、椎间盘切除AFC植入Steffee钢板固定（AFC组）等三种种状态下的轴向压缩刚度。结果：（1）内固定组节段轴向压缩刚度为正常组的14．0％；（2）AFC组节段的轴向压缩刚度明显增加，达到了正常椎间的轴向压缩刚度；（3）在相同轴向压缩载荷作用下，AFC给椎弓根钉受力移位较内固定组明显减小。结论：脊椎椎弓根钉固定系统在AFC椎间植入后，对脊椎稳定性较无AFC植入显著增强，其受力移位明显减小，即可显著减少临床断钉。钢板折弯的机会。     

Segregation and transmission of mitochondrial markers in fusion products of the asporogenous yeast Torulopsis glabrata

Summary Using a protoplast fusion technique we have been able to locate to the mitochondrial genome of the asporogenous yeast Torulopsis glabrata mutations conferring resistance to oligomycin, antimycin and diuron. When two strains differing in the size of their mtDNAs were fused the mitochondrial markers from the parent with the larger mtDNA (71–91) were transmitted predominantly among the fusion products. Both genetical and physical evidence support the occurrence of recombination in T. glabrata mitochondrial genome. Segregation of the mitochondrial genome appears to take place before the separation of the first bud from the fusion product.     

Anatomic and physiologic development of the photoreceptor of the kitten

Summary The postnatal development of the photoreceptor of the cat was studied using physiological and anatomical methods. The late receptor potential (LRP) was recorded in vitro and the threshold and maximum amplitude determined. The same specimens used in the electrophysiological studies were then prepared for microscopy, and rod cell outer and inner segment length and diameter, photoreceptor density, and inter-receptor distance were determined. A small LRP was first recorded at 9–10 days, but only at very high stimulus intensities. Thereafter, there was a rapid decrease in the threshold and an increase in the amplitude of the LRP. The threshold reached adult values at 17–18 days, while the amplitude of the LRP was adultlike at 23–26 days. Of the anatomical parameters examined, inter-receptor spacing and rod cell diameters seem to be most clearly associated, respectively, with the attainment of adult LRP threshold and amplitude. Outer segment length was adult-like at 35–43 days of age and thus postdated physiological maturity of the photoreceptor. These observations suggest that the surface area of the rod cell outer segment tips is more critical in the development of the adult LRP than is the number of discs in the outer segment. In addition, changes over time in the mean diameter and length of rod cell inner segments follows the pattern of ontogenetic changes in LRP amplitude. These findings imply a close relationship during ontogeny between the metabolic functions of the inner segment and phototransduction at the outer segment disc.This study was supported by the Florida Lions Eye Bank (GST) and by a grant, 2-RO1-EY00376-08, from the National Eye Institute, National Institutes of Health, Bethesda, Maryland (DIH)Andrew Labbie and Joseph Muroff were participants in the Community Laboratory Research Programm sponsored by the Dade County Public School System, Miami, Florida     

The mitochondrial plasmid of the true slime mold <Emphasis Type="Italic">Physarum polycephalum</Emphasis> bypasses uniparental inheritance by promoting mitochondrial fusion

Mitochondrial DNA (mtDNA) is inherited maternally in most eukaryotes. Linear mitochondrial plasmids in higher plants and fungi are also transmitted from the maternal parent to the progeny. However, mF, which is a mitochondrial linear plasmid of Physarum polycephalum, evades uniparental mitochondrial inheritance. We examined 36 myxamoebal strains of Physarum and isolated three novel mF⁺ strains (JE8, TU111, NG111) that harbored free mF plasmids. These strains were mated with the mF^– strain KM88. Of the three mF^– × mF⁺ crosses, only KM88 × JE8 displayed complete uniparental inheritance. However, in KM88 × TU111 and KM88 × NG111, the mtDNA of KM88 and mF of TU111 and NG111 were inherited by the plasmodia and showed recombination. For example, although the mtDNA of TU111 was eliminated, the mF of TU111 persisted and became inserted into the mtDNA of KM88, such that recombinant mtDNA represented 80% of the total mtDNA. The parental mitochondria fused to yield giant mitochondria with two or more mitochondrial nucleoids. The mF appears to exchange mitochondria from the recipient (paternal) to the donor (maternal) by promoting mitochondrial fusion.The first two authors have equally contributed to this work