Development of a RP-HPLC method for screening potentially counterfeit anti-diabetic drugs

Pharmaceutical counterfeiting is becoming a serious problem in the world, especially in developing countries including China. Herein an isocratic reversed-phase high performance liquid chromatography (RP-HPLC) method was developed for screening counterfeit medicines and adulterated dietary supplement products. The developed method could be employed to separate and determine simultaneously six anti-diabetic drugs (glipizide, gliclazide, glibenclamide, glimepiride, gliquidone, repaglinide) on an isocratic solvent system using an Alltima C18 column (5 microm, 150 mmx4.6 mm) with an isocratic mobile phase of methanol-phosphate buffer (pH 3.0; 0.01 mol/L) (70:30, v/v), at a flow rate of 1.0 mL/min and at a wavelength of 230 nm. The proposed method was successfully applied to the analysis of medicinal and dietary supplement samples purchased from the local market in China.     

Development and validation of a LC-MS/MS method for the determination of viaminate in human plasma

A sensitive and specific method for determination of viaminate in human plasma by using high-performance liquid chromatography coupled with electrospray tandem mass spectrometry (LC-MS/MS) was developed in this study. The plasma samples were simply deproteinated, extracted, evaporated, and then reconstituted in 200 microl of methanol prior to analysis. Chromatographic separation was carried out on a Shimadzu VP-ODS column (250 mm x 2.0 mm, 5 microm) with a mobile phase of methanol-water (95:5, v/v) at a flow rate of 0.2 ml/min. Quantification was performed in the negative-ion electrospray ionization mode by selected ion monitoring of the product ions at m/z 164 for viaminate and m/z 109 for testosterone propionate which was used as the internal standard. The corresponding parent ions were m/z 446 and m/z 345. A linear calibration curve was observed within the concentration range of 0.10-200 ng/ml. The lowest limit of quantitation (LLOQ) was 0.1 ng/ml. The extraction-efficiency at three concentrations was 100.7, 93.6, and 99.7%. Practical utility of this new LC-MS/MS method was confirmed in pilot pharmacokinetic studies in humans following oral administration.     

Quantitative determination of trans-polydatin, a natural strong anti-oxidative compound, in rat plasma and cellular environment of a human colon adenocarcinoma cell line for pharmacokinetic studies

A simple, accurate, precise, specific and reproducible high-performance liquid chromatography (HPLC) method was developed for determination of trans-polydatin, a natural strong anti-oxidative compound, in rat plasma and cell suspension. The assay procedure involved simple liquid-liquid extraction, the supernatant liquid was added an equal volume of water to avoid solvent effect. The detection of the analyte peak was achieved by monitoring the eluate using a UV detector set at 303 nm. The analysis used a Hypersil ODS2 C18 column (5 microm, 4.6 mm x 250 mm) and methanol/distilled water as the mobile phase (flow rate=1 mL/min). A total analytical run was achieved within 6.0 min and calibration curve was linear over a wide concentration range of 0.25-40 microg/mL for plasma sample and 1.0-500 microM for cell suspension, the coefficients of correlation were 0.9997 and 0.9999 or better, respectively. There was 80.7+/-7.86%, 96.8+/-3.20% and 102.7+/-9.72% recovery from 0.5, 10, and 40 microg/mL plasma samples, respectively. Intra- and inter-batch accuracy and precision were acceptable for the both matrices. The RSD of intra- and inter-day assay variations were all less than 10%. Both analyte and IS were stable in the battery of stability studies, freeze-thaw cycles. The described assay method was applied to pharmacokinetic studies in rats and a human colon adenocarcinoma cell line (Caco-2) successfully. The application of the assay to determine the pharmacokinetic is described.     

Biotechnological production of lactic acid integrated with fishmeal wastewater treatment by <Emphasis Type="Italic">Rhizopus oryzae</Emphasis>

Fishmeal wastewater, a seafood processing waste, was utilized for production of lactic acid and fungal biomass by Rhizopus oryzae AS 3.254 with the addition of sugars. The 30 g/l exogenous glucose in fishmeal wastewater was superior to starch in view of productivities of lactic acid and fungal biomass, and COD reduction. Fishmeal wastewater can be a replacement for peptone which was the most suitable nitrogen source for lactic acid production among the tested organic or inorganic nitrogen sources. Exogenous NaCl (12 g/l) completely inhibited the production of lactic acid and fungal growth. In the medium of COD 5,000 mg/l fishmeal wastewater with the addition of 30 g/l glucose, the maximum productivity of lactic acid was 0.723 g/l h corresponding to productivity of fungal biomass 0.0925 g/l h, COD reduction 84.9% and total nitrogen removal 50.3% at a fermentation time of 30 h.     

小麦秆锈抗性遗传及抗性基因研究进展

目前国际上已发现近80个小麦抗秆锈基因,其中45个抗秆锈基因已被正式定名,58个抗秆锈基因已定位在小麦特定染色体上,其中12个基因被标记。本文对小麦抗秆锈病基因抗源、抗秆锈性遗传、分子标记研究现状及存在问题加以综述,并对抗秆锈分子遗传前景进行展望。     

Identification of novel proteins associated with both alpha-synuclein and DJ-1

The molecular mechanisms leading to neurodegeneration in Parkinson disease (PD) remain elusive, although many lines of evidence have indicated that alpha-synuclein and DJ-1, two critical proteins in PD pathogenesis, interact with each other functionally. The investigation on whether alpha-synuclein directly interacts with DJ-1 has been controversial. In the current study, we analyzed proteins associated with alpha-synuclein and/or DJ-1 with a robust proteomics technique called stable isotope labeling by amino acids in cell culture (SILAC) in dopaminergic MES cells exposed to rotenone versus controls. We identified 324 and 306 proteins in the alpha-synuclein- and DJ-1-associated protein complexes, respectively. Among alpha-synuclein-associated proteins, 141 proteins displayed significant changes in the relative abundance (increase or decrease) after rotenone treatment; among DJ-1-associated proteins, 119 proteins displayed significant changes in the relative abundance after rotenone treatment. Although no direct interaction was observed between alpha-synuclein and DJ-1, whether analyzed by affinity purification followed by mass spectrometry or subsequent direct co-immunoprecipitation, 144 proteins were seen in association with both alpha-synuclein and DJ-1. Of those, 114 proteins displayed significant changes in the relative abundance in the complexes associated with alpha-synuclein, DJ-1, or both after rotenone treatment. A subset of these proteins (mortalin, nucleolin, grp94, calnexin, and clathrin) was further validated for their association with both alpha-synuclein and DJ-1 using confocal microscopy, Western blot, and/or immunoprecipitation. Thus, we not only confirmed that there was no direct interaction between alpha-synuclein and DJ-1 but also, for the first time, report these five novel proteins to be associating with both alpha-synuclein and DJ-1. Further characterization of these docking proteins will likely shed more light on the mechanisms by which DJ-1 modulates the function of alpha-synuclein, and vice versa, in the setting of PD.     

Metabolic flux estimation--a self-adaptive evolutionary algorithm with singular value decomposition

Metabolic flux analysis is important for metabolic system regulation and intracellular pathway identification. A popular approach for intracellular flux estimation involves using ¹³C tracer experiments to label states that can be measured by nuclear magnetic resonance spectrometry or gas chromatography mass spectrometry. However, the bilinear balance equations derived from ¹³C tracer experiments and the noisy measurements require a nonlinear optimization approach to obtain the optimal solution. In this paper, the flux quantification problem is formulated as an error-minimization problem with equality and inequality constraints through the ¹³C balance and stoichiometric equations. The stoichiometric constraints are transformed to a null space by singular value decomposition. Self-adaptive evolutionary algorithms are then introduced for flux quantification. The performance of the evolutionary algorithm is compared with ordinary least squares estimation by the simulation of the central pentose phosphate pathway. The proposed algorithm is also applied to the central metabolism of Corynebacterium glutamicum under lysine-producing conditions. A comparison between the results from the proposed algorithm and data from the literature is given. The complexity of a metabolic system with bidirectional reactions is also investigated by analyzing the fluctuations in the flux estimates when available measurements are varied.     

SIRT2 regulates adipocyte differentiation through FoxO1 acetylation/deacetylation

The family of mammalian Sirtuin proteins comprises seven members homologous to yeast Sir2. Here we show that SIRT2, a cytoplasmic sirtuin, is the most abundant sirtuin in adipocytes. Sirt2 expression is downregulated during preadipocyte differentiation in 3T3-L1 cells. Overexpression of SIRT2 inhibits differentiation, whereas reducing SIRT2 expression promotes adipogenesis. Both effects are accompanied by corresponding changes in the expression of PPARgamma, C/EBPalpha, and genes marking terminal adipocyte differentiation, including Glut4, aP2, and fatty acid synthase. The mechanism underlying the effects of reduced SIRT2 in 3T3-L1 adipocytes includes increased acetylation of FOXO1, with direct interaction between SIRT2 and FOXO1. This interaction enhances insulin-stimulated phosphorylation of FOXO1, which in turn regulates FOXO1 nuclear and cytosolic localization. Thus, Sirt2 acts as an important regulator of adipocyte differentiation through modulation of FOXO1 acetylation/phosphorylation and activity and may play a role in controlling adipose tissue mass and function.     

PCR survey of hox genes in the goldfish Carassius auratus auratus

A tetraploidization event took place in the cyprinid lineage leading to goldfishes about 15 million years ago. A PCR survey for Hox genes in the goldfish Carassius auratus auratus (Actinopterygii: Cyprinidae) was performed to assess the consequences of this genome duplication. Not surprisingly, the genomic organization of the Hox gene clusters of goldfish is similar to that of the closely related zebrafish (Danio rerio). However, the goldfish exhibits a much larger number of recent pseudogenes, which are characterized by indels. These findings are consistent with the hypothesis that dosage effects cause selection pressure to rapidly silence crucial developmental regulators after a tetraploidization event.     

慢病毒介导的RNA干涉对乳腺癌SKBR3细胞HER2受体的下调及生长抑制

大约30％的乳腺癌中有表皮生长因子受体家族蛋白HER2的过表达,此类癌症的预后差,恶性程度高。RNA干涉（RNAi）是最近发展起来能特异性抑制哺乳动物细胞中基因表达的新技术。本文在以往获得的能够产生良好基因沉默效应的小干涉RNA（siRNA）的基础上,构建了U6和H1双启动子siRNA表达载体,并转染HER2高表达乳腺癌SKBR3细胞定量测定了其HER2下调效应。随后,siRNA表达盒经LR重组反应被克隆入慢病毒载体中,在成功包装成病毒后,感染SKBR3并经荧光定量PCR、蛋白印迹杂交和流式细胞仪一系列实验证明慢病毒介导的RNAi确实能有效地下调肿瘤抗原HER2的表达。细胞长期增殖实验表明经慢病毒处理后细胞生长得到抑制。我们的研究为进一步阐明HER2与癌症恶化的关系以及发展新的基因治疗药物提供了工具和可能。