Investigation of differentially expressed proteins in rat gastrocnemius muscle during denervation–reinnervation

To have a better insight into the molecular events involved in denervation-induced atrophy and reinnervation-induced regeneration of skeletal muscles, it is important to investigate the changes in expression levels of a great multitude of muscle proteins during the process of denervation–reinnervation. In this study, we employed an experimental model of rat sciatic nerve crush to examine the differentially expressed proteins in the rat gastrocnemius muscle at different time points (0, 1, 2, 3, 4 weeks) after sciatic␣nerve crush by using two-dimensional gel electrophoresis (2-DE) followed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS), collectively referred to as the modern proteomic analysis. The results showed that 16 proteins in the rat gastrocnemius muscle exhibited two distinct types of change pattern in their relative abundance: (1) The relative expression levels of 11 proteins (including alpha actin, myosin heavy chain, etc.)were decreased either within 1 or 2 weeks post-sciatic nerve injury, followed by restoration during the ensuing days until 4 weeks. (2) The other 5 proteins (including alpha enolase, beta enolase, signal peptide peptidase-like 3, etc.) displayed an up-regulation in their relative expression levels within 1 week following sciatic nerve injury, and a subsequent gradual decrease in their relative expression levels until 4 weeks. Moreover, the significance of the changes in expression levels of the 16 proteins during denervation–reinnervation has been selectively discussed.     

PYDDT,a novel phase 2 enzymes inducer,activates Keap1–Nrf2 pathway via depleting the cellular level of glutathione

Keap1–Nrf2 pathway has emerged as a regulator for the endogenous antioxidant response, which is critical in defending cells against carcinogenesis. Herein, we demonstrated that depleting the cellular level of glutathione (GSH) by a novel electrophilic agent 2-(pro-1-ynyl)-5-(5,6-dihydroxypenta-1,3-diynyl) thiophene (PYDDT) could activate Keap1–Nrf2 pathway. In above process, it was found that Keap1 was modified by S-glutathionylation, an important post-translational modification of protein cysteines with critical roles in oxidative stress and signal transduction. We concluded from our findings that conjugation with intracellular GSH by PYDDT might lead to Keap1 S-glutathionylation and was a key event involved in its Nrf2 inducing activity.     

Isolation and analysis of bioactive diterpenoids in Salvia species (Salvia chionantha and Salvia kronenburgiii) by micellar electrokinetic capillary chromatography

In the present work, we isolated two bioactive diterpenoids, horminone and 7-O-acetylhorminone and developed a micellar electrokinetic chromatography (MEKC) method for the simultaneous quantitative analysis of them in Turkish Salvia species. The optimal separation electrolyte was 50 mmol/L SDS and 25% methanol at pH 11.5. The limits of detection (S/N = 3) were 3.269 and 4.518 μg/mL for horminone and 7-O-acetylhorminone, respectively. The method has been applied successfully to analyze these two components in Salvia chionantha and Salvia kronenburgii acetone extracts.     

Fast detection of Candida albicans and/or bacteria in blood plasma by “sample-self-focusing” using capillary electrophoresis-laser-induced fluorescence

Detection of microbial contamination in blood plasma is critical and necessary in different medical and research fields. Most of the current standard procedures for the detection of bacteria and fungi can be time-consuming, for example, direct inoculation methods of microbial cultures in respective growth media can take a few days to several weeks. A fast analysis method with high sensitivity output such as CE-laser-induced florescence becomes an attractive alternative. Previously, a spacer-injection method with the use of zwitterionic surfactant (SB3-10) as a blocking agent to negate the cells’ mobility, induce aggregation and single microbial peak formation in a buffer solution was reported. Here, a fast, simple direct method for microbial detection in blood plasma without using the spacer and blocking agent is reported. To compensate for the natural electrophoretic heterogeneity of microbes, a CTAB additive was used to sweep all microbial cells towards the plasma peak where a single sharp microbial peak is formed and detected. With the use of BacLight™ Green bacterial stain, the microbial peak, generally, can be detected within 10 min in front of the plasma peak using capillary electrophoresis coupled with laser-induced florescence detection. The LOD of microbes detectable were 5 cells per injection. This technique provides a great advantage over traditional, time-consuming microbial inoculation methods.     

Chiral separation by a monofunctionalized cyclodextrin derivative: From selector to permethyl-β-cyclodextrin bonded stationary phase

Preparation of (6-monoureido-6-monodeoxy) permethylated β-cyclodextrin bonded chiral stationary phase from permethylated 6-monoamino-6-monodeoxy-β-cyclodextrin is described. The optimized chiral stationary phase was evaluated by using HPLC separation of racemates of coumarin derivatives. Column characterization was performed by solid-state ¹³C, ¹⁵N, ²⁹Si NMR using cross-polarization at the magic angle spinning. The development process was supported by CE experiments where the complex formation between cyclodextrins and warfarin was investigated. The results demonstrate good enantio-discrimination for coumarin derivatives.     

Micellar electrokinetic chromatography method development for determination of impurities in Ritonavir

Ritonavir is a synthetic peptidomimetic human immunodeficiency virus (HIV) protease inhibitor employed in the treatment of AIDS since 1996. Synthetic precursors are potential impurities in the final product. In the present work a micellar electrokinetic chromatography (MEKC) method for the separation of Ritonavir from three available synthetic precursors was developed. The optimized separation is performed in a background electrolyte composed of sodium tetraborate (pH 9.6; 15 mM) containing sodium dodecylsulfate (30 mM) and acetonitrile (18%, v/v). Mass spectrometry was used to confirm the identity of the tested substances. Good repeatability was observed for migration time (RSD about 0.4%) and peak area (RSD about 0.8%). The limits of detection (LOD) obtained allow the determination of two of the impurities at levels as low as 0.005% m/m, and one at a level of 0.3% m/m.     

Determination of voriconazole in human serum and plasma by micellar electrokinetic chromatography

The micellar electrokinetic capillary chromatography (MEKC) separation and analysis of voriconazole and UK 115794 (internal standard) were examined and an assay for determination of voriconazole in human plasma and serum was developed. The MEKC medium comprises a 2:15 (v/v) mixture of methanol and a pH 9.3 buffer composed of 5 mM Na₂B₄O₇, 7 mM Na₂HPO₄ and 54 mM SDS. Sample preparation is based upon liquid/liquid extraction with ethylacetate and dichloromethane (75%/25%) at physiological pH. Using this approach with 250 μl serum or plasma and reconstitution of the dried extract into 100 μl of a buffer composed of 0.5 mM Na₂B₄O₇ and 0.7 mM Na₂HPO₄ (pH 9.3), the detection and quantitation limits were determined to be 0.1 and 0.2 μg/ml, respectively, a sensitivity that is suitable for therapeutic drug monitoring of voriconazole (provisional therapeutic range: 1–6 μg/ml) in human plasma and serum samples. The method was validated and compared to an HPLC method, showing excellent agreement between the two for a set of 91 samples that stemmed from patients being treated with voriconazole. The MEKC assay is also demonstrated to be suitable to explore pharmacokinetic data of voriconazole.     

变性梯度凝胶电泳分析川芎可培养内生细菌种群的多样性

目的 了解川芎可培养内生细菌的种群多样性信息.方法 实验采用富集培养法培养内生细菌,从菌液中直接提取总DNA,并扩增细菌16S rDNA V3片段,应用DGGE分析16S rDNA V3片段的多态性.结果 DGGE图谱显示:不同川芎样品的16S RDNA V3片段产物出现的带型及数量有一定差异,不同样品的强度高条带数量及其在泳道中的迁移率各不相同,体现优势种群的种类与数量的区别.结论 川芎根茎的可培养内生细菌具有较丰富的种群多样性,地域因素(产地)可能是影响土壤细菌群落结构的重要因素,其次是品种.     

商陆皂苷甲作用于子宫内膜异位症大鼠腹腔巨噬细胞蛋白质组的差异分析

目的研究商陆皂苷甲作用于子宫内膜异位症大鼠腹腔巨噬细胞蛋白质组的差异,鉴定差异表达的蛋白质。方法采用自体移植法建立子宫内膜异位症大鼠模型。造模成功后取腹腔巨噬细胞,商陆皂苷甲作用24 h后,采用双向凝胶电泳技术分离巨噬细胞总蛋白,并获得药物组、模型组和空白对照组蛋白质组图谱,PDQuest软件分析确认三组细胞的差异蛋白质,最后运用串联飞行时间质谱（MALDI-TOF-TOF MS）鉴定差异蛋白点。结果 M2型丙酮酸激酶、磷酸酰肌醇转移蛋白和热休克蛋白8在子宫内膜异位症大鼠腹腔巨噬细胞中高表达,而磷酸甘油醛脱氢酶表达减少。商陆皂苷甲能抑制造模引起的M2型丙酮酸激酶等四种蛋白的表达变化。结论利用蛋白质组学技术,研究商陆皂苷甲作用于子宫内膜异位症大鼠腹腔巨噬细胞后蛋白表达差异,为探索子宫内膜异位症的发病机制和商陆皂苷甲的作用机制提供新的线索和依据。     

Silk-elastinlike protein polymers for matrix-mediated cancer gene therapy

Silk-elastinlike protein polymers (SELPs) are recombinant polymers designed from silk fibroin and mammalian elastin amino acid repeats. These are versatile materials that have been examined as controlled release systems for intratumoral gene delivery. SELP hydrogels comprise monodisperse and tunable polymers that have the capability to control and localize the release and expression of plasmid DNA and viruses. This article reviews recent developments in the synthesis and characterization of SELP hydrogels and their use for matrix-mediated gene delivery.