Recombinant protein of heptad-repeat HR212, a stable fusion inhibitor with potent anti-HIV action in vitro

HR212, a recombinant protein expressed in Escherichia coli, has been previously reported to inhibit HIV-1 membrane fusion at low nanomolar level. Here we report that HR212 is effective in blocking laboratory strain HIV-1IIIB entry and replication with EC50 values of 3.92±0.62 and 6.59±1.74 nM, respectively, and inhibiting infection by clinic isolate HIV-1KM018 with EC50 values of 44.44±10.20 nM, as well as suppressing HIV-1- induced cytopathic effect with an EC50 value of 3.04±1.20 nM. It also inhibited HIV-2ROD and HIV-2CBL-20 entry and replication in the μM range. Notably, HR212 was highly effective against T20-resistant strains with EC50 values ranging from 5.09 to 7.75 nM. Unlike T20, HR212 showed stability sufficient to inhibit syncytia formation in a time-of-addition assay, and was insensitive to proteinase K digestion. These results suggest that HR212 has great potential to be further developed as novel HIV-1 fusion inhibitor for treatment of HIV/ AIDS patients, particularly for those infected by T20-resistant variants.

Photoluminescence of ZnS clusters in zeolite-Y

Two obvious emissions are observed from the ZnS clusters encapsulated in zeolite-Y. The emission around 355 nm is sharp and weak, locating at the onset of the absorption edge. The band around 535 nm is broad, strong and Stokes-shifted. Both the two emissions shift to blue and their intensities firstly increase then decrease as the loading of ZnS in zeolite-Y or clusters size decreases. Through investigation, the former is attributed to the excitonic fluorescence, and the latter to the trapped luminescence from surface states. The cluster size-dependence of the luminescence may be explained qualitatively by considering both the carrier recombination and the nonradiative recombination rates. Four peaks appearing in the excitation spectra are assigned to the transitions of 1S-1S, 1S-1P, 1S-1D and surface state, respectively. The excitation spectra of the clusters do not coincide with their absorption spectra. The states splitted by quantum-size confinement are detected in the excitation spectra, but could not be differentiated in the optical absorption spectra due to inhomogeneous broadening. The size-dependence of the excitation spectra is similar to that of the absorption spectra. Both the excitation spectra of excitonic and of trapped emissions are similar, but change in relative intensity and shift in position are observed.

Anti-HER-2-SEC2融合免疫毒素的构建和活性研究

以PCR技术从金黄色葡萄球菌基因组DNA中首次克隆编码成熟SECZ蛋白的全基因sec2。该基因共717bp，编码239个氨基酸，Genbank Accession number：AY450554。构建了SEC2的表达载体pET-28a-sec2，并在大肠杆菌BL21（DE3）中高效表达可溶性rSEC2蛋白。经亲和层析纯化，其纯度在95％以上，平均回收量为每升培养物40mg。纯化的rSEcZ保持了与野生型相当的生物学活性。以限制性核酸内切酶连接技术分别将两个抗人表皮生长因子受体HER-2单链抗体基因通过DNA Linker与sec2融合，构建融合基因b-l-sec2和ml小sec2，并以两种方式表达纯化。以pET-32a表达载体在E，coliAD494（DE3）中以氨基端融合大肠杆菌硫氧还蛋白（TrxA）形式高效表达融合蛋白TRX-B-L-SEC2和TRX-ML-L-SEC2，经亲和层析纯化，并以肠激酶切割得到成熟融合免疫毒素B-L-SEC2和ML-L-SEC2，其纯度在95％以上，平均回收量为每升培养物smg；以构建的新型表达载体pASK-75-EX在E.coliBL21（ED3）中以不溶性包涵体形式表达融合免疫毒素蛋白，经变性、纯化和复性后得到具有生物学活性的融合免疫毒素，其纯度在95％以上，平均回收量为每升培养物30mg。以两种方式制备的融合免疫毒素都保持了SECZ蛋白的免疫原性，都能有效刺激人外周血单个核细胞的增殖，并且都显示出在体外与HER-2过表达的乳腺癌细胞SK-Br-3特异性结合能力，具有显著的靶向性抑瘤作用。用PcR方法扩增了编码TrxA蛋白的基因trxA并克隆至表达载体pET-28a启动子上游，构建了一种在单质粒中利用两个相同的启动子游离共表达硫氧还蛋白与目的蛋白的表达载体。利用该载体可使TrxA与外源蛋白在大肠杆菌BL21（DE3）中以非融合形式高效共表达。共表达的TrxA可明显促进外源蛋白单链抗体ML3.9（scFv-ML）、3一轻基苯甲酸-6-单加氧酶（3HBA）的可溶性表达；并明显减少肠毒素C2（SEC2）、结核杆菌螺旋酶A亚基（GYRA）的包涵体表达。

Total-internal-reflection fluorescence microscopy with W-shaped axicon mirrors

A scheme based on a W-shaped axicon mirror device for total-internal-reflection fluorescence microscopy (TIRFM) is presented. This approach combines the advantages of higher efficiency compared with traditional TIRFM, adjustable illumination area, and simple switching between wide-field and TIRF imaging modes. TIRF images obtained with this approach are free of shadow artifacts and of interference fringes. Example micrographs of fluorescently labeled polystyrene beads, of Convallaria majalis tissue, and of Propidium-iodide-labeled Chinese hamster ovary cells are shown, and the capabilities of the scheme are discussed. (C) 2010 Optical Society of America

Fluorescence Hydrogen Peroxide Probe Based on a Microstructured Polymer Optical Fiber Modified with a Titanium Dioxide Film

A highly sensitive microstructured polymer optical fiber (MPOF) probe for hydrogen peroxide was made by forming a rhodamine 6G-doped titanium dioxide film on the side walls of array holes in an MPOF. It was found that hydrogen peroxide only has a response to the MPOF probe in a certain concentration of potassium iodide in sulfuric acid solution. The calibration graph of fluorescence intensity versus hydrogen peroxide concentration is linear in the range of 1.6 x 10(-7) mol/L to 9.6 x 10(-5) mol/L. The method, with high sensitivity and a wide linear range, has been applied to the determination of trace amounts of hydrogen peroxide in a few real samples, such as rain water and contact lens disinfectant, with satisfactory results.