Preparation of Tyr-C-peptide from genetically altered human insulin precursor

C-peptide radioimmunoassay (C-peptide RIA) is widely used in determination of pancreatic B-cell secretion activity.¹²⁵I labeled TyrC-peptide is indispensable in C-peptide RIA kit. Herein we discuss a way of obtaining recombinant Tyr-C-peptide. Arg32Tyr human proinsulin mutant (R32Y-proinsulin) gene was constructed by site-directed mutagenesis and overexpressed inEscherichia coli. Purified R32Y-proinsulin was converted to insulin and Tyr-C-peptide by trypsin and carboxypeptidase B codigestion. Tyr-C-peptide was isolated through reverse-phase HPLC (RP-HPLC) and identified by C-peptide RIA and amino acid analysis.     

高效液相色谱法测定血浆中表阿霉素浓度

报道了高效液相色谱法测定血浆中表阿霉素浓度的实验方法。采用μＢｏｎｄａｐａｋＣ＿（１８）色谱柱,正磷酸、甲醇、乙腈、异丙醇为流动相。荧光检测器检测,激发波长为４５０ｎｍ,发射波长为５３０ｎｍ。血浆经离心后,取血清,用甲醇沉淀蛋白。方法的平均相对回收率为９９．４％±４．６％,日内和日间平均ＲＳＤ小于５％和６％。最低检测限为３ｎｇ。本方法快速、准确,灵敏度高。     

高效液相色谱法检测人肝组织中黄曲霉毒素

以人肝癌组织为研究材料,丙酮、氯仿为主要抽提溶剂,建立了用高效液相色谱技术检测肝组织中黄曲霉毒素Ｂ＿１的方法。采用μ－Ｐｏｒａｓｉｌ色谱往,氯仿、环己烷、乙腈、异丙醇为流动相,荧光检测器检测,激发波长３６５ｎｍ,发射波长４３５ｎｍ。方法的平均回收率为９６．８５％,变异系数为４．５２％,最低检测限为０．０５ｎｇ。１０例肝癌组织中有７例检出５。４９～７３．７０ｎｇ／ｇ不等的黄曲霉毒素Ｂ＿１。方法快速、准确、灵敏度高。     

对甲氧基苯磺酰氯柱前衍生测定尿中游离羟脯氨酸与脯氨酸

以对甲氧基苯磺酰氯为衍生试剂,建立了分离测定人尿中游离羟脯氨酸和脯氨酸的高效液相色谱方法。考察了衍生温度、衍生缓冲液pH值、衍生时间、衍生剂用量对衍生反应的影响以及流动相组成、流动相缓冲液浓度、pH值和柱温对分离的影响。在优化条件下,羟脯氨酸和脯氨酸分别在5～100μmol/L和5～250μmol/L范围内呈良好线性,相关系数分别为0.999 4、0.999 5,检出限(S/N=3)分别为0.50 nmol/L和0.20 nmol/L,回收率(n=5)分别为95%～99%和96%～102%,相对标准偏差分别为2.1%～4.3%和2.0%～4.8%。该方法可用于人尿中游离羟脯氨酸和脯氨酸的定性定量分析。     

Adenovirus adenine nucleotide translocator-2 shRNA effectively induces apoptosis and enhances chemosensitivity by the down-regulation of ABCG2 in breast cancer stem-like cells

Cancer stem cells (CSCs) are resistant to chemo- and radio-therapy, and can survive to regenerate new tumors. This is an important reason why various anti- cancer therapies often fail to completely control tumors, although they kill and eliminate the bulk of cancer cells. In this study, we determined whether or not adenine nucleotide translocator-2 (ANT2) suppression could also be effective in inducing cell death of breast cancer stem-like cells. A sub-population (SP; CD44+/ CD24-) of breast cancer cells has been reported to have stem/progenitor cell properties. We utilized the adeno- ANT2 shRNA virus to inhibit ANT2 expression and then observed the treatment effect in a SP of breast cancer cell line. In this study, MCF7, MDA-MB-231 cells, and breast epithelial cells (MCF10A) mesenchymally-transdifferentiated through E-cadherin knockdown were used. ANT2 expression was high in both stem-like cells and non-stem-like cells of MCF7 and MDA-MB-231 cells, and was induced and up-regulated by mesenchymal transdifferentiation in MCF10A cells (MCF10A(EMT)). Knockdown of ANT2 by adeno-shRNA virus efficiently induced apoptotic cell death in the stem-like cells of MCF7 and MDA-MB-231 cells, and MCF10A(EMT). Stem-like cells of MCF7 and MDA-MB-231, and MCF10A(EMT) cells exhibited increased drug (doxorubicin) resistance, and expressed a multi-drug resistant related molecule, ABCG2, at a high level. Adeno-ANT2 shRNA virus markedly sensitized the stem-like cells of MCF7 and MDA-MB-231, and the MCF10A(EMT) cells to doxorubicin, which was accompanied by down-regulation of ABCG2. Our results suggest that ANT2 suppression by adeno-shRNA virus is an effective strategy to induce cell death and increase the chemosensitivity of stem-like cells in breast cancer.     

The presence of high level soluble herpes virus entry mediator in sera of gastric cancer patients

The development of gastric cancer (GC) is closely related to chronic inflammation caused by Helicobacter pylori infection, and herpes virus entry mediator (HVEM) is a receptor expressed on the surface of leukocytes that mediates potent inflammatory responses in animal models. However, the role of HVEM in human GC has not been studied. Previously, we showed that the interaction of HVEM on human leukocytes with its ligand LIGHT induces intracellular calcium mobilization, which results in inflammatory responses including induction of proinflammatory cytokine production and anti-bacterial activities. In this study, we report that leukocytes from GC patients express lower levels of membrane HVEM (mHVEM) and have lower LIGHT-induced bactericidal activities than those from healthy controls (HC). In contrast, levels of soluble HVEM (sHVEM) in the sera of GC patients were significantly higher than in those of HC. We found that monocyte membrane-bound HVEM is released into the medium when cells are activated by proinflammatory cytokines such as TNF-α and IL-8, which are elevated in the sera of GC patients. mHVEM level dropped in parallel with the release of sHVEM, and release was completely blocked by the metalloprotease inhibitor, GM6001. We also found that the low level of mHVEM on GC patient leukocytes was correlated with low LIGHT-induced bactericidal activities against H. pylori and S. aureus and production of reactive oxygen species. Our results indicate that mHVEM on leukocytes and sHVEM in sera may contribute to the development and/or progression of GC.     

CHROMATOGRAPHIC SEPERATION AND PURIFICATION OF HUMAN CHORIONIC GONADOTROPIN

ＣＨＲＯＭＡＴＯＧＲＡＰＨＩＣＳＥＰＥＲＡＴＩＯＮＡＮＤＰＵＲＩＦＩＣＡＴＩＯＮＯＦＨＵＭＡＮＣＨＯＲＩＯＮＩＣＧＯＮＡＤＯＴＲＯＰＩＮＷａｎｇＨａｉｆｅｎｇ；ＸｕＹｕｔａｉ；ＲｅｎＲｏｕｇｌｉ；ＴａｎｇＬｅｉ；ＷａｎｇＨａｉｒｅｎ（Ｄｅｐａｒｔｍｅ...     

双柱双泵高效液相色谱－电化学法测定尿中香草扁桃酸

采用双柱、双泵和双阀，通过住切换技术对尿中的香草扁桃酸（ＶＭＡ）进行直接进样分离，并用电化学检测器检测。样品先进入柱Ⅰ，初步分离后，包含ＶＭＡ的组分被切换入柱Ⅱ进一步分离。结果显示，此法操作简单、快速，回收率高，重复性好。     

A liquid chromatography-tandem mass spectrometry assay for the determination of nemonoxacin (TG-873870), a novel nonfluorinated quinolone, in human plasma and urine and its application to a single-dose pharmacokinetic study in healthy Chinese volunteers

Nemonoxacin (TG‐873870) is a novel C‐8‐methoxy nonfluorinated quinolone with higher activity than ciprofloxacin, levofloxacin and moxifloxacin against Gram‐positive pathogens including methicillin‐susceptible or methicillin‐resistant Staphylococcus aureus and Streptococcus pneumoniae with various resistant phenotypes. A rapid, sensitive and selective liquid chromatography–tandem mass spectrometric (LC‐MS/MS) method was developed and validated to determine the concentration of nemonoxacin in human plasma and urine. Protein precipitation and liquid–liquid extraction were employed for plasma and urine sample preparations, respectively, and extract was then injected into the system. Separation was performed on a C₁₈ reversed‐phase column using acetonitrile–0.1% formic acid as a mobile phase. Both analyte and internal standard (gatifloxacin) were determined using electrospray ionization and the MS data acquisition via the selected reaction monitoring in positive‐ion mode. The lower limit of quantification was 5 ng/mL and the calibration curves were linear in the concentration range of 5–1000 ng/mL. The accuracy, precision, selectivity, linearity, recovery, matrix effect and stability were validated for TG‐873870 in human plasma and urine. The method was successfully applied to a pharmacokinetic study enrolling 12 healthy Chinese volunteers administered nemonoxacin malate capsules. Copyright © 2012 John Wiley & Sons, Ltd.