阿嗪米特原料药中潜在基因毒性杂质氯乙酰氯和氯乙酸的含量测定

采用化学衍生化-高效液相色谱法分别测定阿嗪米特原料药中潜在基因毒性杂质氯乙酰氯和氯乙酸。以2-硝基苯肼为衍生化试剂,经衍生化后以Thermo syncronis C₁₈(250 mm×4.6 mm,5μm)为色谱柱,以0.1%磷酸水-乙腈为流动相进行梯度洗脱;柱温40℃,检测波长226 nm;流速1 mL/min。空白溶剂、衍生化试剂及阿嗪米特均不干扰待测物质出峰;氯乙酰氯和氯乙酸的检测限分别为7.5 ng/mL和15 ng/mL,均在30～300 ng/mL的范围内与色谱峰面积呈现良好的线性关系;加样回收率在87.37%～109.75%范围内。本研究所建立的两种方法专属性好、精密度好、灵敏度高、操作简便,可用于阿嗪米特原料药中潜在基因毒性杂质氯乙酰氯及氯乙酸的痕量测定。     

使用适应性设计的随机对照试验报告规范解读

如何提高适应性设计随机对照试验报告质量成为目前的关注热点。使用适应性设计的随机对照试验研究报告规范（adaptive designs CONSORT Extension,ACE）在CONSORT 2010声明基础上进行了针对适应性设计随机对照试验的拓展,为规范适应性设计随机对照试验报告提供了指导。本文将重点对ACE拓展...     

Fluorimetric determination of chloroxine using manual and flow-injection methods

A reliable and highly sensitive method is described for the determination of chloroxine in pharmaceutical preparations. It involves the formation of a complex between chloroxine and aluminum(III) in a micellar medium. The complex is a very fluorescent species, and there is a linear relationship between chloroxine concentration and fluorescence intensity over the range {ce:inline-formula}2.0 × 10 ⁻⁸ −5.1 × 10 ⁻⁵mol1 ⁻¹{/ce:inline-formula}. The limit of detection is {ce:inline-formula}5 × 10 ⁻⁹mol1 ⁻¹{/ce:inline-formula}. The method can be easily adapted to a flow system using a three-channel manifold, the peak height being proportional to the chloroxine concentration over the range {ce:inline-formula}5.6 × 10 ⁻⁷ − 5.6 × 10 ⁻⁵mol1 ⁻¹{/ce:inline-formula}. Manual and flow-injection procedures permit the determination of chloroxine in the presence of chlorquinaldol, and have been successfully applied to the determination of chloroxine in pharmaceutical preparations.     

Absence of the canalicular isoform of the MRP gene-encoded conjugate export pump from the hepatocytes in Dubin-Johnson syndrome

The Dubin-Johnson syndrome is characterized by an inherited defect in the secretion of amphiphilic anionic conjugates from hepatocytes into the bile. We have recently identified the membrane protein mediating the adenosine triphosphate (ATP)-dependent transport of glutathione and glucuronate conjugates as a multidrug-resistance protein (MRP) and localized it to the canalicular as well as to the lateral hepatocyte plasma membrane. In the present study we show the selective absence of the canalicular isoform of MRP (cMRP) from the hepatocytes in a patient with Dubin-Johnson syndrome by double-label immunofluorescence and confocal laser scanning microscopy using antibodies directed against MRP and dipeptidyl-peptidase IV (DPPIV). Another isoform of MRP was detected, however, in the lateral hepatocyte membrane of the patient. Moreover, MRP was present on immunoblots of erythrocyte membranes from Dubin-Johnson syndrome and normal humans. These findings are analogous to our recent observations on the localization of the rat homolog of MRP and its canalicular isoform, cMRP, in normal and transport- deficient GY/TR- Wistar rat liver. The elucidation of the selective absence of an isoform of MRP and from the canalicular membrane domain in conjunction with the defined substrate specificity of the MRP and cMRP gene-encoded conjugate export pumps contributes to the molecular definition of the transport defect in Dubin-Johnson syndrome. (Hepatology 1996 May;23(5):1061-6)     

组织因子抑制剂高通量筛选模型的建立及DT-13抗肿瘤作用的研究（英文）

目的：建立新型的细胞水平组织因子抑制剂高通量药物筛选模型, 对本中心库存天然化合物进行筛选, 得到对TF有较好抑制作用的天然化合物, 用于进一步评估抗肿瘤转移药物。方法：利用发色底物法, 建立细胞水平TF抑制剂高通量筛选模型, 用该细胞株进行抗肿瘤转移药物筛选。结果：选用人乳腺癌细胞MDA-MB-435成功建立了快速稳定、灵敏度高、操作简便的抗肿瘤转移药物筛选模型。筛选1260种化合物, 经过初筛、复筛和体内评价发现短葶山麦冬皂苷-C（DT-13）对TF的抑制作用比较明显, 并具有一定的抗肿瘤转移作用。结论：通过建立的TF抑制剂筛选模型, 对库存天然产物进行筛选, 得到具有抗肿瘤转移的天然产物DT-13。     

Antigenicity and immunogenicity of HIV-1 gp140 with different combinations of glycan mutation and V1/V2 region or V3 crown deletion

The carbohydrate moieties on HIV-1 envelope glycoprotein (Env) act as shields to mask conserved neutralizing epitopes, while the hyperimmunogenic variable regions are immunodominant in inducing non-neutralizing antibodies, representing the major challenge for using Env as a vaccine candidate to induce broadly neutralizing antibodies (bNAbs). In this study, we designed a series of HIV-1 gp140 constructs with the removal of N276/N463 glycans, deletion of the V1/V2 region and the V3 crown, alone or in combination. We first demonstrated that all the constructs had a comparable level of expression and were mainly expressed as trimers. Following purification of gp140s from mammalian cells, we measured their binding to bNAbs and non-NAbs in vitro and capability in inducing bNAbs in vivo. Antibody binding assay showed that removal of N276/N463 glycans together with the deletion of V1/V2 region enhanced the binding of gp140s to CD4-binding site-targeting bNAbs VRC01 and 3BNC117, and CD4-induced epitopes-targeting non-NAbs A32, 17b and F425 A1g8, whereas further deletion of V3 crown in the gp140 mutants demonstrated slightly compromised binding capability to these Abs. Immunogenicity study showed that the above mutations did not lead to the induction of a higher Env-specific IgG response via either DNA-DNA or DNA-protein prime-boost strategies in mice, while neutralization assay did not show an apparent difference between wild type and mutated gp140s. Taken together, our results indicate that removal of glycans at N276/N463 and deletion of the V1/V2 region can expose the CD4-binding site and CD4-induced epitopes, but such exposure alone appears incapable of enhancing the induction of bNAbs in mice, informing that additional modification or/and immunization strategies are needed. In addition, the strategies which we established for producing gp140 proteins and for analyzing the antigenicity and immunogenicity of gp140 provide useful means for further vaccine design and assessment.     

Genotypic and phenotypic characterization of West Nile virus NS5 methyltransferase mutants

Although West Nile virus (WNV) causes annual cases of neurological disease and deaths in humans, a vaccine has not been licensed for human use. Several WNV genes have been targeted for mutagenesis in attempts to generate live attenuated vaccine candidates, including the non-structural protein NS5. Specifically, mutation of WNV NS5-K61A or NS5-E218A in the catalytic tetrad of the methyltransferase decreases enzyme activity of the NS5 protein and correspondingly attenuates the virus in mice. In this report, NS5-K61A, NS5-E218A, and a double mutant encoding both mutations (NS5-K61A/E218A) were compared both in vitro and in vivo. Each single mutant was strongly attenuated in highly susceptible outbred mice, whereas the double mutant unexpectedly was not attenuated. Sequencing analysis demonstrated that the double mutant was capable of reversion at both residues NS5-61 and NS5-218, whereas the genotype of the single mutants did not show evidence of reversion. Overall, either NS5-K61A or NS5-E218A methyltransferase mutations could be potential mutations to include in a candidate live WNV vaccine; however, multiple mutations in the catalytic tetrad of the methyltransferase are not tolerated.