Vectors for Cu2+-inducible production of glutathione S-transferase-fusion proteins for single-step purification from yeast

We describe six new yeast episomal vectors which encode glutathione S-transferase (GST) affinity tags. These allow for the production of GST-fusion proteins in Saccharomyces cerevisiae under the control of the CUP1 promoter. Affinity chromatography with glutathione-Sepharose permits convenient purification of the fusion protein from a yeast lysate. The presence of a protease cleavage site facilitates subsequent removal of the GST tag. The expression and single-step purification of both GST and a functional GST-metallothionein fusion from yeast are shown as an example of the application of these vectors.     

α-Methylene-β-Lactone Scaffold for Developing Chemical Probes at the Two Ends of the Selectivity Spectrum

The utilities of an α-methylene-β-lactone (MeLac) moiety as a warhead composed of multiple electrophilic sites are reported. We demonstrate that a MeLac-alkyne not only reacts with diverse proteins as a broadly reactive measurement probe, but also recruits reduced endogenous glutathione (GSH) to assemble a selective chemical probe of GSH-β-lactone (GSH-Lac)-alkyne in live cells. Tandem mass spectrometry reveals that MeLac reacts with nucleophilic cysteine, serine, lysine, threonine, and tyrosine residues, through either Michael or acyl addition. A peptide-centric proteomics platform demonstrates that the proteomic selectivity profiles of orlistat and parthenolide, which have distinct reactivities, are measurable by MeLac-alkyne as a high-coverage probe. The GSH-Lac-alkyne selectively probes the glutathione S-transferase P responsible for multidrug resistance. The assembly of the GSH-Lac probe exemplifies a modular and scalable route to develop selective probes with different recognizing moieties.     

哈密瓜脱氢抗坏血酸还原酶基因的鉴定及冷胁迫表达分析

为阐明哈密瓜脱氢抗坏血酸还原酶(dehydroascorbate reductase,DHAR)基因的基本性质及在响应冷胁迫过程中的表达水平,基于生物信息学对2个哈密瓜DHAR基因(CmDHAR)的基本信息、多序列比对、系统进化关系、共线性关系、基因结构域特征、蛋白质三级结构、蛋白质-蛋白质相互作用及冷胁迫下的抗坏血酸...     

Investigation of Bemethyl Biotransformation Pathways by Combination of LC–MS/HRMS and In Silico Methods

Bemethyl is an actoprotector, an antihypoxant, and a moderate psychostimulant. Even though the therapeutic effectiveness of bemethyl is well documented, there is a gap in knowledge regarding its metabolic products and their quantitative and qualitative characteristics. Since 2018, bemethyl is included to the Monitoring Program of the World Anti-Doping Agency, which highlights the challenge of identifying its urinary metabolites. The objective of the study was to investigate the biotransformation pathways of bemethyl using a combination of liquid chromatography-high-resolution mass spectrometry and in silico studies. Metabolites were analyzed in a 24 h rat urine collected after oral administration of bemethyl at a single dose of 330 mg/kg. The urine samples were prepared for analysis by a procedure developed in the present work and analyzed by high performance liquid chromatography–tandem mass spectrometry. For the first time, nine metabolites of bemethyl with six molecular formulas were identified in rat urine. The most abundant metabolite was a benzimidazole–acetylcysteine conjugate; this biotransformation pathway is associated with the detoxification of xenobiotics. The BioTransformer and GLORY computational tools were used to predict bemethyl metabolites in silico. The molecular docking of bemethyl and its derivatives to the binding site of glutathione S-transferase has revealed the mechanism of bemethyl conjugation with glutathione. The findings will help to understand the pharmacokinetics and pharmacodynamics of actoprotectors and to improve antihypoxant and adaptogenic therapy.     

Phytochemistry, traditional uses and pharmacology of Eugenia jambolana Lam. (black plum): A review

Eugenia jambolana Lam. (syn. Syzigium cumini (L.) SKEELS; S. jambolana DC; Family: Myrtaceae), commonly known as black plum or Jamun is a plant native to India. Annually the trees produce oblong or ellipsoid fruits (berries). They are green when raw and purplish black when fully ripe. The ripe fruits are sweetish sour to taste and are used to prepare health drinks, squashes, juices, jellies and wine. Studies have shown that the berries contain carbohydrates, minerals and the pharmacologically active phytochemicals like flavonoids, terpenes, and anthocyanins. Jamun is a plant with known ethnomedicinal uses. Before the discovery of insulin, Jamun was useful in the treatment of diabetes and is an integral part in the various alternative systems of medicine. Scientific studies have shown that the various extracts of Jamun possess a range of pharmacological properties such as antibacterial, antifungal, antiviral, anti-genotoxic, anti-inflammatory, anti-ulcerogenic, cardioprotective, anti-allergic, anticancer, chemopreventive, radioprotective, free radical scavenging, antioxidant, hepatoprotective, anti-diarrheal, hypoglycemic and antidiabetic effects. The present paper reviews these aspects and also addresses the lacunas in the existing knowledge.     

Glutathione S-Transferase M3 Is Associated with Glycolysis in Intrinsic Temozolomide-Resistant Glioblastoma Multiforme Cells

Glioblastoma multiforme (GBM) is a malignant primary brain tumor. The 5-year relative survival rate of patients with GBM remains <30% on average despite aggressive treatments, and secondary therapy fails in 90% of patients. In chemotherapeutic failure, detoxification proteins are crucial to the activity of chemotherapy drugs. Usually, glutathione S-transferase (GST) superfamily members act as detoxification enzymes by activating xenobiotic metabolites through conjugation with glutathione in healthy cells. However, some overexpressed GSTs not only increase GST activity but also trigger chemotherapy resistance and tumorigenesis-related signaling transductions. Whether GSTM3 is involved in GBM chemoresistance remains unclear. In the current study, we found that T98G, a GBM cell line with pre-existing temozolomide (TMZ) resistance, has high glycolysis and GSTM3 expression. GSTM3 knockdown in T98G decreased glycolysis ability through lactate dehydrogenase A activity reduction. Moreover, it increased TMZ toxicity and decreased invasion ability. Furthermore, we provide next-generation sequencing–based identification of significantly changed messenger RNAs of T98G cells with GSTM3 knockdown for further research. GSTM3 was downregulated in intrinsic TMZ-resistant T98G with a change in the expression levels of some essential glycolysis-related genes. Thus, GSTM3 was associated with glycolysis in chemotherapeutic resistance in T98G cells. Our findings provide new insight into the GSTM3 mechanism in recurring GBM.     

Purification of glutathione S-transferase from lamb muscle and its effect on lipid peroxidation

Lamb muscle contained 0.4 mg glutathione S-transferase (EC 2.5.1.18) per gram of tissue. On storage at 1°C, 20% of the activity was lost after 3 weeks. Glutathione S-transferase was isolated from lamb muscle by affinity chromatography on S-hexyglutathione Sepharose 6B. One major form of the enzyme was present in the purified preparation, a dimer with subunit molecular weight of 22 900 and isoelectric point of 6.7. This enzyme fits into the Pi class on the basis of substrate specificity. Linoleic acid hydroperoxides and trans, trans-2,4-decadienal, products of linoleate peroxidation, were both substrates (0.11 and 0.24 μmol min^?1 mg^?1 protein respectively). Glutathione S-transferase inhibited copper-stimulated peroxidation of arachidonate in the presence of glutathione. The inhibition was only slightly less efficient in the absence of glutathione. The latter effect probably arises from the presence of enzyme-bound glutathione (4–5 mol mol^?1 enzyme subunit) and also from a small but significant glutathione-independent peroxidase activity. The latter was detected using an HPLC assay which monitors formation of product directly. Reduced glutathione per se showed only a slight inhibition of arachidonate peroxidation.     

Cloning and in vitro and in vivo expression of plant glutathione S-transferase zeta class genes

The cDNAs encoding the zeta class of glutathione S-transferases (GSTs) (GSTZs), which are multifunctional enzymes, were cloned from Arabidopsis thaliana L. and three lines of Brassica napus L. by RT PCR, and named AtGSTZ for A. thaliana ecotype Columbia gll, and BnGSTZ-A, BnGSTZ-B and BnGSTZ-C for B. napus cv. Shan 2A, Shan 213 and Ken C1, respectively. Sequence analysis revealed that the sequences of Shan 2A and Shan 2B were identical, and Ken C1 was different only in 3.8% of the sequence. Comparison of these sequences with published sequences in GeneBank showed that the sequences of the Brassica species were unpublished. The cDNAs were then inserted into two vectors for in vivo expression in Escherichia coli and in vitro expression in a wheat-germ cell-free protein synthesis system. All GSTZs were well expressed both in vivo and in vitro as an enzymatically active form, showing that all of them had both dichloroacetic acid dechlorinating and maleylacetone isomerase activities.     

PolyHIPEs: Recent advances in emulsion-templated porous polymers

Porous polymers with well-defined porosities and high specific surface areas in the form of monoliths, films, and beads are being used in a wide range of applications (reaction supports, separation membranes, tissue engineering scaffolds, controlled release matrices, responsive and smart materials) and are being used as templates for porous ceramics and porous carbons. The surge in the research and development of porous polymer systems is a rather recent phenomenon. PolyHIPEs are porous emulsion-templated polymers synthesized within high internal phase emulsions (HIPEs). HIPEs are highly viscous, paste-like emulsions in which the major, “internal” phase, usually defined as constituting more than 74% of the volume, is dispersed within the continuous, minor, “external” phase. This review focuses upon the recent advances in polyHIPEs involving innovations in polymer chemistry, macromolecular structure, multiphase architecture, surface functionalization, and nanoparticle stabilization. The effects of these innovations upon the natures of the resulting polyHIPE-based materials (including bicontinuous polymers, nanocomposites, hybrids, porous ceramics, and porous carbons) and upon the applications involving polyHIPEs are discussed. The advances in polyHIPEs described in this review are now being used to generate new families of porous materials with novel porous architectures and unique properties.