表达增强型绿色荧光蛋白标记的hBax 和hHGF 双基因的慢病毒 载体构建

目的:构建绿色荧光蛋白标记的hBax和hHGF双基因共表达的重组慢病毒并鉴定。方法:通过重叠PCR技术构建attB1-K-hBAX/T2A/eGFP/P2A/hHGF-attB2基因片段,利用gateway technology构建慢病毒载体质粒pLV.EX2d.null-EF1A>hBAX/T2A/eGFP/P2A/hHGF和阴性对照质粒pLV.EX2d.null-EF1A>eGFP并测序,上述两种质粒分别与辅助质粒共转染293FT细胞包装病毒,荧光显微镜检测病毒滴度。结果:经鉴定慢病毒载体质粒构建正确,荧光显微镜检测hBax和hHGF共表达慢病毒滴度为7.8×107TU/mL,仅表达绿色荧光蛋白的阴性病毒滴度为9×107TU/mL。结论:表达增强型绿色荧光蛋白标记的hBax和hHGF双基因的慢病毒构建成功并获得高滴度的病毒感染液。     

多光谱成像技术在生物医学中的应用进展

多光谱成像(multispectral imaging,MSI)技术在生物医学可视化方面是一种新技术,它结合了两个已建立的光学模块:成像学和光谱学。它的原理是基于液晶可调谐滤光片,从可见光到近红外波长(400-970nm)区域获取多光谱图像。自从MSI系统加上显微镜商品化以来,MSI已经成为一种快速发展的领域,可应用于细胞生物学、临床前药物开发和临床病理学等。国外已有大量关于MSI在生物医学中应用的研究报道,但国内报道少见。本文主要对多光谱成像的基本原理,近三年内该技术在生物医学领域的应用进展作一简要综述。     

扩散张量成像对颈椎病脊髓早期损伤的研究

目的:探讨磁共振(MR)扩散张量成像(DTI)作为定量分析方法,对脊髓型颈椎病(CSM)脊髓早期损伤诊断的应用价值.方法:选择45例经临床及影像诊断为脊髓型颈椎病患者,颈椎常规MRI检查显示脊髓内无异常信号,使用单次激发自旋回波平面(SE-EPI)序列,进行DTI扫描.测量压迫部位脊髓的ADC值及FA值作为病例组,选择病变上或下方两个节段以上未受压正常脊髓作为正常对照组,测量其ADC值及FA值.分析病例组与对照组间ADC及FA值差别,计算ADC值及FA值诊断脊髓损伤的敏感性.结果:所有脊髓型颈椎病患者经DTI检查均可得到ADC图及FA图,经图像后处理,脊髓显示清晰,图像无变形及伪影.3例脊髓型颈椎病患者ADC值降低,42例脊髓型颈椎病患者ADC值增高,平均ADC值为(1.388± 0.149)x 10-3 mm2/s.44名脊髓型颈椎病患者FA值降低,1名脊髓型颈椎病患者FA值增高,平均FA值为0.476±0.085,受压处脊髓平均ADC值升高,平均FA值下降,与正常值比较差别有统计学意义.ADC值诊断的敏感性为93.33％,FA值诊断的敏感性为97.78％.结论:DTI与常规MR比较,能早期而准确地诊断脊髓型颈椎病脊髓早期损伤.     

腱鞘巨细胞瘤的影像学诊断

目的:分析腱鞘巨细胞瘤的影像学表现以提高对该病的认识。方法:回顾性分析10例经手术病理证实的腱鞘巨细胞瘤的X线平片及MRI表现,其中10例行X线平片检查,8例行MRI平扫及增强扫描。结果:X线平片显示局部稍高密度软组织肿块影,邻近骨质未见明显异常或轻度侵蚀破坏。MRI表现为相应部位软组织肿块影,T1WI多呈较低信号,内可见条片状更低信号影,增强后强化明显;T2WI呈高低混杂信号影;病灶与邻近肌腱关系密切,其中一例包绕指屈肌腱蔓状生长;局部骨皮质可受侵。结论:腱鞘巨细胞瘤的影像学表现具有一定的特征性。     

Parasitoids as vectors of facultative bacterial endosymbionts in aphids

Heritable bacterial endosymbionts play an important role in aphid ecology. Sequence-based evidence suggests that facultative symbionts such as Hamiltonella defensa or Regiella insecticola also undergo horizontal transmission. Other than through male-to-female transfer during the sexual generation in autumn, the routes by which this occurs remain largely unknown. Here, we tested if parasitoids or ectoparasitic mites can act as vectors for horizontal transfer of facultative symbionts. Using symbiont-specific primers for diagnostic PCR, we demonstrate for the first time, to our knowledge, that parasitoids can indeed transfer H. defensa and R. insecticola by sequentially stabbing infected and uninfected individuals of their host, Aphis fabae, establishing new, heritable infections. Thus, a natural route of horizontal symbiont transmission is also available during the many clonal generations of the aphid life cycle. No transmissions by ectoparasitic mites were observed, nor did parasitoids that emerged from symbiont-infected aphids transfer any symbionts in our experiments.     

Fluorescence-topographic NSOM directly visualizes peak-valley polarities of GM1/GM3 rafts in cell membrane fluctuations

Simultaneous fluorescence-topographic nanoscale imaging of cell-surface molecules in the context of membrane ultra-structures has not been reported. Here, near-field scanning optical microscopy (NSOM)-based direct fluorescence-topographic imaging indicated that GM3 rafts/nanodomains (190.0 +/- 49.8 nm ranging 84.5-365.0 nm) were localized predominantly on the peaks of microvillus-like protrusions in the apical membrane of GM3 + Madin-Darby canine kidney cells, whereas GM1 rafts/nanodomains (159.5 +/- 63.8 nm ranging 42-360 nm) were distributed mainly on the slops of protrusions or the valleys between protrusions in the plasma membranes of GM1 + MDCK cells. The data demonstrated that gangliosides polarized not only in a well-known apical-basolateral manner but also in the more microscopic peak-valley manner, implicating unique distribution of GM1 or GM3 in cell-surface fluctuations on the apical membrane of polarized cells. The peak-valley polarities of gangliosides also implicated their different functions relevant to lipid rafts, microvilli, or cellular processes. Importantly, our study demonstrated for the first time that the NSOM-based direct fluorescence-topographic imaging is unique and powerful for elucidating nanoscale distribution of specific cell-surface molecules in membrane fluctuations.     

Divalent metal ion complexes of S100B in the absence and presence of pentamidine

As part of an effort to inhibit S100B, structures of pentamidine (Pnt) bound to Ca²⁺-loaded and Zn²⁺,Ca²⁺-loaded S100B were determined by X-ray crystallography at 2.15 Å (R_free = 0.266) and 1.85 Å (R_free = 0.243) resolution, respectively. These data were compared to X-ray structures solved in the absence of Pnt, including Ca²⁺-loaded S100B and Zn²⁺,Ca²⁺-loaded S100B determined here (1.88 Å; R_free = 0.267). In the presence and absence of Zn²⁺, electron density corresponding to two Pnt molecules per S100B subunit was mapped for both drug-bound structures. One Pnt binding site (site 1) was adjacent to a p53 peptide binding site on S100B (± Zn²⁺), and the second Pnt molecule was mapped to the dimer interface (site 2; ± Zn²⁺) and in a pocket near residues that define the Zn²⁺ binding site on S100B. In addition, a conformational change in S100B was observed upon the addition of Zn²⁺ to Ca²⁺-S100B, which changed the conformation and orientation of Pnt bound to sites 1 and 2 of Pnt-Zn²⁺,Ca²⁺-S100B when compared to Pnt-Ca²⁺-S100B. That Pnt can adapt to this Zn²⁺-dependent conformational change was unexpected and provides a new mode for S100B inhibition by this drug. These data will be useful for developing novel inhibitors of both Ca²⁺- and Ca²⁺,Zn²⁺-bound S100B.     

Functional intracellular antibody fragments do not require invariant intra-domain disulfide bonds

Intracellular antibody fragments that interfere with molecular interactions inside cells are valuable in investigation of interactomes and in therapeutics, but their application demands that they function in the reducing cellular milieu. We show here a 2.7-Å crystal structure of intracellular antibody folds based on scaffolds developed from intracellular antibody capture technology, and we reveal that there is no structural or functional difference with or without the intra-domain disulfide bond of the variable domain of heavy chain or the variable domain of light chain. The data indicate that, in the reducing in vivo environment, the absence of the intra-domain disulfide bond is not an impediment to correction of antibody folding or to interaction with antigen. Thus, the structural constraints for in-cell function are intrinsic to variable single-domain framework sequences, providing a generic scaffold for isolation of functional intracellular antibody single domains.     

In vitro high affinity alpha-synuclein binding sites for the amyloid imaging agent PIB are not matched by binding to Lewy bodies in postmortem human brain

Amyloid containing deposits are a defining neuropathological feature of a wide range of dementias and movement disorders. The positron emission tomography tracer PIB (Pittsburgh Compound-B, 2-[4'-(methylamino)phenyl]-6-hydroxybenzothiazole) was developed to target senile plaques, an amyloid containing pathological hallmark of Alzheimer's disease, formed from the amyloid-beta peptide. Despite the fact that PIB was developed from the pan-amyloid staining dye thioflavin T, no detailed characterisation of its interaction with other amyloid structures has been reported. In this study, we demonstrate the presence of a high affinity binding site (K(d) approximately 4 nM) for benzothiazole derivatives, including [3H]-PIB, on alpha-synuclein (AS) filaments generated in vitro, and further characterise this binding site through the use of radioligand displacement assays employing 4-N-methylamino-4'-hydroxystilbene (SB13) (K(i) = 87 nM) and 2-(1-{6-[(2-fluoroethyl(methyl)amino]-2-naphthyl}ethylidene)malononitrile (FDDNP) (K(i) = 210 nM). Despite the presence of a high-affinity binding site on AS filaments, no discernible interaction of [3H]-PIB was detected with amygdala sections from Parkinson's disease cases containing frequent AS-immunoreactive Lewy bodies and related neurities. These findings suggest that the density and/or accessibility of AS binding sites in vivo are significantly less than those associated with amyloid-beta peptide lesions. Lewy bodies pathology is therefore unlikely to contribute significantly to the retention of PIB in positron emission tomography imaging studies.