Clinical adaptation of a high-performance liquid chromatographic method for the assay of pyridoxal 5′-phosphate in human plasma

Vitamin B6, measured as pyridoxal 5′-phosphate (PLP), is a co-enzyme in the transsulfuration pathway of homocysteine metabolism. Since depletion of PLP has been suggested as an independent risk factor for coronary artery disease, PLP is frequently measured to guide patient care. By a change and utilization of an Aquasil C18 column and the addition of an acetonitrile clean-up gradient to the potassium phosphate, with sodium perchlorate and bisulfite buffer between samples we report the modification of a previously described method for analysis of PLP. The result is a more practical, efficient, reliable and robust method for daily clinical use. We also determined and report that it is critical to protect freshly prepared standard PLP samples from light exposure during assay preparation.     

Enhancing the fungicidal activity of amphotericin B via vacuole disruption by benzyl isothiocyanate,a cruciferous plant constituent

Amphotericin B (AmB), a typical polyene macrolide antifungal agent, is widely used to treat systemic mycoses. In the present study, we show that the fungicidal activity of AmB was enhanced by benzyl isothiocyanate (BITC), a cruciferous plant-derived compound, in the budding yeast, Saccharomyces cerevisiae. In addition to forming a molecular complex with ergosterol present in fungal cell membranes to form K⁺-permeable ion channels, AmB has been recognized to mediate vacuolar membrane disruption resulting in lethal effects. BITC showed no effect on AmB-induced plasma membrane permeability; however, it amplified AmB-induced vacuolar membrane disruption in S. cerevisiae. Furthermore, the BITC-enhanced fungicidal effects of AmB significantly decreased cell viability due to the disruption of vacuoles in the pathogenic fungus Candida albicans. The application of the combinatorial antifungal effect of AmB and BITC may aid in dose reduction of AmB in clinical antifungal therapy and consequently decrease side effects in patients. These results also have significant implications for the development of vacuole-targeting chemotherapy against fungal infections.     

Cripto-1 localizes to dynamic and shed filopodia associated with cellular migration in glioblastoma cells

Cripto-1 is a protein participating in tissue orientation during embryogenesis but has also been implicated in a wide variety of cancers, such as colon, lung and breast cancer. Cripto-1 plays a role in the regulation of different pathways, including TGF-β/Smad and Wnt/β-catenin, which are highly associated with cell migration both during embryonal development and cancer progression. Little is known about the detailed subcellular localization of cripto-1 and how it participates in the directional movement of cells. In this study, the subcellular localization of cripto-1 in glioblastoma cells was investigated in vitro with high-resolution microscopy techniques. Cripto-1 was found to be localized to dynamic and shed filopodia and transported between cells through tunneling nanotubes. Our results connect the refined subcellular localization of cripto-1 to its functions in cellular orientation and migration.     

The large bat Helitron DNA transposase forms a compact monomeric assembly that buries and protects its covalently bound 5′-transposon end

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Use of a radioimmunoassay technique to measure the titer of anti-cow-zona antibodies in marmoset monkeys

A double antibody radioimmunoassay technique is described for the measurement of anti-zona antibodies in the peripheral plasma of marmosets actively immunized against intact cow zonae pellucidae. The method has been shown to be a reliable, repeatable indicator of antibody titer, enabling direct comparison of the response obtained between marmosets. This is the first report of a procedure describing the active immunization of a primate against zona antigens, and the first time a radioimmunoassay technique has been used to monitor profiles of anti-cow zona antibody production following active immunization. The method should prove to be a useful tool in the evaluation of zona antigens as agents for immunocontraception.     

环氧树脂固定化的Bacillus sp.DL-2胞外蛋白酶在拆分(±)-乙酸苏合香酯中的应用

环氧基是一个非常活跃的基团,它能与酶、蛋白质和核酸等生物分子发生反应形成共价键,有利于生物分子的固定化。经共价结合法固定化的酶其稳定性及重复使用性可得到显著提高。用环氧树脂ES-103B为载体采用共价结合法对海洋细菌Bacillus sp. DL-2的胞外蛋白酶进行固定化,经过单因素实验优化条件得出最优固定化条件为:p H 8. 0的胞外蛋白酶溶液,25 g/L的ES-103B,45℃下反应8h。采用此最优条件下的固定化酶拆分(±)-乙酸苏合香酯制备出了e. e. p=97. 5%的(R)-1-苯乙醇(产率为45. 0%)和e. e. s=99. 2%的(S)-乙酸苏合香酯(产率为83. 9%)。该固定化酶拆分(±)-乙酸苏合香酯在重复使用8次后制备出的(R)-1-苯乙醇的e. e. p仍大于90%,且固定化胞外蛋白酶在4℃下具有较好的储存稳定性。     

恶性疟原虫抗原表位的亚单位疫苗与DNA疫苗的联合免疫

构建了含有恶性疟原虫抗原基因 ( AWTE)的真核表达质粒 p CMV- AWTE,以及能在大肠杆菌中得到分泌性表达的原核表达质粒 p MC0 5 ,表达的蛋白 AWTE保持了疟原虫抗原的抗原性。将 p CMV- AWTE以及 AWTE两者混合各 1 0μg鼻腔免疫小鼠 ,一次后诱导机体产生了较高水平的体液免疫及细胞免疫     

Generation of a purely clonal defective interfering influenza virus

Defective interfering (DI) influenza viruses carry a large deletion in a gene segment that interferes with the replication of infectious virus; thus, such viruses have potential for antiviral therapy. However, because DI viruses cannot replicate autonomously without the aid of an infectious helper virus, clonal DI virus stocks that are not contaminated with helper virus have not yet been generated. To overcome this problem, we used reverse genetics to generate a clonal DI virus with a PB2 DI gene, amplified the clonal DI virus using a cell line stably expressing the PB2 protein, and confirmed its ability to interfere with infectious virus replication in vitro. Thus, our approach is suitable for obtaining purely clonal DI viruses, will contribute to the understanding of DI virus interference mechanisms and can be used to develop DI virus‐based antivirals.     

Kinetochore life histories reveal an Aurora-B-dependent error correction mechanism in anaphase

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