德国小蠊变应原Bla g 2蛋白变复性的荧光光谱研究

包涵体中的重组蛋白经抽提后可以在变性状态下纯化,而纯化后的复性过程是基因工程下游处理的重要环节。通过对变应原Bla g 2蛋白复性前后荧光光谱的比较和分析、变性剂(尿素和SDS)对复性后Bla g 2蛋白的荧光滴定实验、以及复性后Bla g 2在不同pH下的荧光光谱分析,推断出Bla g 2蛋白分子在不同环境下构象的变化及其光谱学特征,初步建立了一种新的检测重组变应原蛋白变复性的光谱实验方法。     

一种新型温度自补偿高灵敏度折射率计

基于高频CO2激光脉冲写入的新型超长周期光纤光栅(ULPFG),提出了一种可实现温度自补偿的新型高灵敏度折射率计。理论与实验表明,新型超长周期光纤光栅不同闪耀阶次谐振峰对外界折射率与温度变化的灵敏度各自不同,特别的是,该光栅存在对外界折变不敏感的谐振峰,测量中除了可以利用该峰实现温度同时测量外,还可以补偿另一个测量折变的谐振峰因温度变化带来的测量误差。该折射率计具有制作简单、成本低、强度好,灵敏度高等优点,当外界折射率在1.43~1.45范围内变化时,其折射率测量灵敏度可达每单位折射率240 nm,在实际工业应用中具有较大的潜在实用价值。     

The Biological Fate of Pharmaceutical Excipient β-Cyclodextrin: Pharmacokinetics,Tissue Distribution,Excretion, and Metabolism of β-Cyclodextrin in Rats

β-cyclodextrin has a unique annular hollow ultrastructure that allows encapsulation of various poorly water-soluble drugs in the resulting cavity, thereby increasing drug stability. As a bioactive molecule, the metabolism of β-cyclodextrin is mainly completed by the flora in the colon, which can interact with API. In this study, understanding the in vivo fate of β-cyclodextrin, a LC-MS/MS method was developed to facilitate simultaneous quantitative analysis of pharmaceutical excipient β-cyclodextrin and API dextromethorphan hydrobromide. The established method had been effectively used to study the pharmacokinetics, tissue distribution, excretion, and metabolism of β-cyclodextrin after oral administration in rats. Results showed that β-cyclodextrin was almost wholly removed from rat plasma within 36 h, and high concentrations of β-cyclodextrin distributed hastily to organs with increased blood flow velocities such as the spleen, liver, and kidney after administration. The excretion of intact β-cyclodextrin to urine and feces was lower than the administration dose. It can be speculated that β-cyclodextrin metabolized to maltodextrin, which was further metabolized, absorbed, and eventually discharged in the form of CO₂ and H₂O. Results proved that β-cyclodextrin, with relative low accumulation in the body, had good safety. The results will assist further study of the design and safety evaluation of adjuvant β-cyclodextrin and promote its clinical development.     

Use of TLC-Densitometric Method for Determination of Valproic Acid in Capsules

Determination of valproic acid in the drug was carried out on the aluminum silica gel 60F₂₅₄ plates and using acetone–water–chloroform–ethanol–ammonia at a volume ratio of 30:1:8:5:11 as the mobile phase, respectively. Two methods of detection of valproic acid were used. The first was a 2% aqueous CuSO₄×5H₂O solution, and the second was a 2′,7′-dichlorofluorescein-aluminum chloride-iron (III) chloride system. The applied TLC-densitometric method is selective, linear, accurate, precise, and robust, regardless of the visualizing reagent used for the determination of valproic acid in Convulex capsules. It has low limits of detection (LOD) and limits of quantification (LOQ), which are equal to 5.8 μg/spot and 17.4 μg/spot using a 2% aqueous CuSO₄×5H₂O solution as visualizing agent and also 0.32 μg/spot and 0.97 μg/spot using a 2′,7′-dichlorofluorescein-aluminum chloride-iron (III) chloride system as visualizing reagent, respectively. The described analytical method can additionally be used to study the identity of valproic acid in a pharmaceutical preparation. The linearity range was found to be 20.00–80.00 μg/spot and 1.00–2.00 μg/spot for valproic acid detected on chromatographic plates using a 2% aqueous CuSO₄×5H₂O solution and the 2′,7′-dichlorofluorescein-aluminum chloride-iron (III) chloride system, respectively. A coefficient of variation that was less than 3% confirms the satisfactory accuracy and precision of the proposed method. The results of the assay of valproic acid equal 96.2% and 97.0% in relation to the label claim that valproic acid fulfill pharmacopoeial requirements. The developed TLC-densitometric method can be suitable for the routine analysis of valproic acid in pharmaceutical formulations. The proposed TLC-densitometry may be an alternative method to the modern high-performance liquid chromatography and square wave voltammetry in the control of above-mentioned substances, and it can be applied when other analytical techniques is not affordable in the laboratory.     

An Improved Approach for Practical Synthesis of 5-Hydroxymethyl-2′-deoxycytidine (5hmdC) Phosphoramidite and Triphosphate

5-Hydroxymethyl-2′-deoxycytidine (5hmdC) phosphoramidite and triphosphate are important building blocks in 5hmdC-containing DNA synthesis for epigenetic studies. However, efficient and practical methods for the synthesis of these compounds are still limited. The current research provides an intensively improved synthetic method that enables the preparation of commercially available cyanoethyl-protected 5hmdC phosphoramidite with an overall yield of 39% on 5 g scale. On the basis of facile and efficient accesses to cyanoethyl protected-5hmdU and 5hmdC intermediates, two efficient synthetic routes for 5hmdC triphosphate were also developed.     

2-Hydroxyestradiol Overcomes Mesenchymal Stem Cells-Mediated Platinum Chemoresistance in Ovarian Cancer Cells in an ERK-Independent Fashion

Ovarian cancer (OC) is the second most common type of gynecological malignancy. Platinum (Pt)-based chemotherapy is the standard of care for OC, but toxicity and acquired chemoresistance has proven challenging. Recently, we reported that sensitivity to platinum was significantly reduced in a co-culture of OC cells with MSC. To discover compounds capable of restoring platinum sensitivity, we screened a number of candidates and monitored ability to induce PARP cleavage. Moreover, we monitored platinum uptake and expression of ABC transporters in OC cells. Our results showed that 2-hydroxyestradiol (2HE2), a metabolite of estradiol, and dasatinib, an Abl/Src kinase inhibitor, were significantly effective in overcoming MSC-mediated platinum drug resistance. Dasatinib activity was dependent on ERK1/2 activation, whereas 2HE2 was independent of the activation of ERK1/2. MSC-mediated platinum drug resistance was accompanied by reduced intracellular platinum concentrations in OC cells. Moreover, MSC co-cultured with OC cells resulted in downregulation of the expression of cellular transporters required for platinum uptake and efflux. Exposure to 2HE2 and other modulators resulted in an increase in intracellular platinum concentrations. Thus, 2HE2 and dasatinib might act as sensitizers to restore platinum drug sensitivity to OC cells and thus to limit TME-mediated chemoresistance in OC.     

Supercritical CO2 Plant Extracts Show Antifungal Activities against Crop-Borne Fungi

Fungal infections of cultivated food crops result in extensive losses of crops at the global level, while resistance to antifungal agents continues to grow. Supercritical fluid extraction using CO₂ (SFE-CO₂) has gained attention as an environmentally well-accepted extraction method, as CO₂ is a non-toxic, inert and available solvent, and the extracts obtained are, chemically, of greater or different complexities compared to those of conventional extracts. The SFE-CO₂ extracts of Achillea millefolium, Calendula officinalis, Chamomilla recutita, Helichrysum arenarium, Humulus lupulus, Taraxacum officinale, Juniperus communis, Hypericum perforatum, Nepeta cataria, Crataegus sp. and Sambucus nigra were studied in terms of their compositions and antifungal activities against the wheat- and buckwheat-borne fungi Alternaria alternata, Epicoccum nigrum, Botrytis cinerea, Fusarium oxysporum and Fusarium poae. The C. recutita and H. arenarium extracts were the most efficacious, and these inhibited the growth of most of the fungi by 80% to 100%. Among the fungal species, B. cinerea was the most susceptible to the treatments with the SFE-CO₂ extracts, while Fusarium spp. were the least. This study shows that some of these SFE-CO₂ extracts have promising potential for use as antifungal agents for selected crop-borne fungi.     

Important Requirements for the Selection of Internal Standards during the Development of Desorption/Ionization Assays for Drug Quantification in Biological Matrices—A Practical Example

Desorption/ionization mass spectrometry (DI-MS) approaches allow for the rapid quantification of drugs in biological matrices using assays that can be validated according to regulatory guidelines. However, specific adaptations must be applied to create reliable quantification methods, depending on the approach and instrumentation used. In the present article, we demonstrate the importance of the molecular weight, the fragmentation pattern, and the purity of the internal standard for the development of matrix-assisted laser desorption/ionization (MALDI)-ion mobility (IM)-tandem MS and MS/MS methods. We present preliminary results of method development for the quantification of selinexor in microdialysis fluids with a stable isotopically labeled internal standard. In addition, we discuss the selection of internal standards for MALDI-MS assays using different instrumentations.     

Structural Diversity of Silver Fluorinated β-Diketonates: Effect of the Terminal Substituent and Solvent

In order to demonstrate the role of the fluorination and some solvents in the structural organization of the Ag(I) coordination polymers with β-diketonate ligands (R¹C(O)C_αHC(O)R²)⁻ we synthesized a series of the compounds containing tfac- (R₁ = CH₃, R² = CF₃) and pfpac- (R₁ = CH₃, R² = C₂F₅) anions. Solvent-free [Ag(L)]_∞ (L = tfac 1, pfpac 2) compounds and the corresponding acetonitrile and toluene adducts have been characterized by elemental analysis and/or NMR, IR and single-crystal XRD. This series includes five new coordination polymers. Compound 1 is a 3D coordination framework based on Ag–O_{chelate/bridge}, Ag–C_α bonds, and argentophilic interactions. An increase in the fluorinated group leads to a chain coordination polymer 2 of an unusual structural organization. These chains can be represented as a “DNA-type”, where two intertwined helices based on Ag–O_chelate and Ag–C_α bonds are connected through Ag–O_bridge ones. Two structural types of chain coordination polymers, [Ag(tfac)(CH₃CN)] and [Ag₂(L)₂(solvent)], have been revealed for the adducts. The latter structural type differs significantly from the previously studied toluene and acetonitrile adducts of fluorinated Ag(I) β-diketonates of the same stoichiometry. Thermal analysis in helium showed that both 1 and 2 decompose to metallic silver with the compound of pfpac-ligand being slightly more stable.