Polyphyllin D Shows Anticancer Effect through a Selective Inhibition of Src Homology Region 2-Containing Protein Tyrosine Phosphatase-2 (SHP2)

Natural products have continued to offer tremendous opportunities for drug development, as they have long been used in traditional medicinal systems. SHP2 has served as an anticancer target. To identify novel SHP2 inhibitors with potential anticancer activity, we screened a library containing 658 natural products. Polyphyllin D was found to selectively inhibit SHP2 over SHP1, whereas two other identified compounds (echinocystic acid and oleanolic acid) demonstrated dual SHP1 and SHP2 inhibition. In a cell-based assay, polyphyllin D exhibited cytotoxicity in Jurkat cells, an acute lymphoma leukemia cell line, whereas the other two compounds were ineffective. Polyphyllin D also decreased the level of phosphorylated extracellular signal-regulated kinase (p-ERK), a proliferation marker in Jurkat cells. Furthermore, knockdown of protein tyrosine phosphatase (PTP)N6 (SHP1) or PTPN11 (SHP2) decreased p-ERK levels. However, concurrent knockdown of PTPN6 and PTPN11 in Jurkat cells recovered p-ERK levels. These results demonstrated that polyphyllin D has potential anticancer activity, which can be attributed to its selective inhibition of SHP2 over SHP1.     

A Quadruple‐Action Platinum(IV) Prodrug with Anticancer Activity Against KRAS Mutated Cancer Cell Lines

We developed a novel Pt^IV prodrug that simultaneously releases four different bioactive moieties inside the cancer cell. Its cytotoxicity against monolayer cultures (2D) and spheroid (3D) cancer cells is significantly better than cisplatin. It is 200–450‐fold more potent than cisplatin against KRAS mutated pancreatic and colon cancers and is 40‐fold more selective towards KRAS mutated cells compared to non‐cancerous. This is important since RAS proteins play a role in regulating cell differentiation, proliferation, and survival and KRAS is mutated in 90 % of pancreatic adenocarcinomas, 45 % of colorectal cancers, and 35 % of lung adenocarcinomas. The selectivity index, determined by dividing the IC₅₀ value in non‐cancerous cells by that of a cancerous cell line, is two‐fold better than cisplatin, attesting to preferential cytotoxicity towards neoplastic cells.     

Surface plasmon resonance detection of oligonucleotide sequences of the rpoB genes of Mycobacterium tuberculosis

Oligonucleotide sequences related to the normal and mutated rpoB genes of Mycobacterium tuberculosis are detected using a surface plasmon resonance (SPR) biosensor system. A bioselective element was prepared by immobilizing the thiol-modified oligonucleotides of the selected sequence (the capture probe P2) that contains the mutated TCG → TTG codon 531 (evoking drug resistance) of the rpoB gene of M. tuberculosis on a gold sensor surface. Specific hybridization between immobilized probe P2 and complementary target T2 gave the highest sensor response, single-base mismatched oligonucleotide TN (corresponding to the normal gene sequence) produced somewhat smaller response and no response was observed at injection of noncomplementary oligonucleotide TC. The P2-T2 hybridization efficiency is calculated ca. 30% (5 × 10¹² molecules cm⁻²), and the lowest detection limit of T2 was 10 nM. An extended T2E oligonucleotide sequence consisting of T2 sequence and additional 24 nucleotides was shown to cause more pronounced sensor response (at least 5 nM T2E was easily detected). Injection into the sensor cell of the oligonucleotides complementary to the free additional part of T2E after P2-T2E hybridization gave a significant additional SPR response, thus showing that the sandwich hybridization format further improves the sensor sensitivity and decreases the lowest detection limit. The experimental results on surface hybridization between the studied oligonucleotides were in good agreement with thermodynamic parameters of the hybridization calculated for solution conditions. The described approach could be proposed as a basis for creating a biosensor for real-time and label-free diagnostics of drug resistant tuberculosis.     

Genotyping of two single nucleotide polymorphisms in 5,10-methylenetetrahydrofolate reductase by multiplex polymerase chain reaction and capillary electrophoresis

Two single nucleotide polymorphisms (SNPs) of 5,10-methylenetetrahydrofolate reductase (MTHFR) gene, A1298C and C677T, were widely considered to be related with various neoplasia disorders. We established a simple and effective capillary electrophoresis (CE) method for detection of two SNPs in MTHFR gene simultaneously. DNA samples were amplified by multiplex PCR with universal fluorescence-labeled primer and analyzed by single-strand conformation polymorphism (SSCP)-CE method. The CE method was performed using 1.5% hydroxyethyl cellulose in 1× TBE buffer containing 1 M urea. The PCR products after SSCP procedure were electrokinetically injected at −10 kV, 30 s. Separation voltage was −6 kV and the temperature was set at 20 °C. The optimal SSCP-CE method was applied to detect two polymorphisms in MTHFR gene of acute lymphoblastic leukemia (ALL) and attention-deficit/hyperactivity disorder (ADHD) patients. Genotyping results were evaluated in terms of relationships between outcomes for ADHD patients after ALL chemotherapy and ALL disease. The SSCP-CE method and multiplex PCR with universal fluorescence primer were used as the fast technique for screening two SNPs in MTHFR gene, A1298C and C677T. The genotyping data were coincident with DNA sequencing. This SSCP-CE method was found feasible for detecting mutation of MTHFR gene in populations.     

Trehalose-6-Phosphate as a Potential Lead Candidate for the Development of Tps1 Inhibitors: Insights from the Trehalose Biosynthesis Pathway in Diverse Yeast Species

In some pathogens, trehalose biosynthesis is induced in response to stress as a protection mechanism. This pathway is an attractive target for antimicrobials as neither the enzymes, Tps1, and Tps2, nor is trehalose present in humans. Accumulation of T6P in Candida albicans, achieved by deletion of TPS2, resulted in strong reduction of fungal virulence. In this work, the effect of T6P on Tps1 activity was evaluated. Saccharomyces cerevisiae, C. albicans, and Candida tropicalis were used as experimental models. As expected, a heat stress induced both trehalose accumulation and increased Tps1 activity. However, the addition of 125 μM T6P to extracts obtained from stressed cells totally abolished or reduced in 50 and 60 % the induction of Tps1 activity in S. cerevisiae, C. tropicalis, and C. albicans, respectively. According to our results, T6P is an uncompetitive inhibitor of S. cerevisiae Tps1. This kind of inhibitor is able to decrease the rate of reaction to zero at increased concentrations. Based on the similarities found in sequence and function between Tps1 of S. cerevisiae and some pathogens and on the inhibitory effect of T6P on Tps1 activity observed in vitro, novel drugs can be developed for the treatment of infectious diseases caused by organisms whose infectivity and survival on the host depend on trehalose.     

Evolution of cluster models in quantum chemical analysis of spectroscopic characteristics of surface structures on oxides at the IOC RAS

The author’s review integrates and analyzes the results of quantum chemical studies on a common topic carried out at the Institute of Organic Chemistry of the RAS (IOC RAS). The cluster approaches to quantum chemical analysis of adsorption and heterogeneous catalysis processes on various oxides proposed and developed at the IOC RAS are characterized. The approaches comprise the construction of covalent and also neutral and partially charged ionic cluster models of surface active sites and their complexes with molecules and free radicals. Examples of chemically acceptable cluster models specially designed for the review and calculated by density functional theory (DFT) with the B3LYP functional and the 6-311G(d) basis set clearly demonstrate the efficiency of using such models to retrieve structure-chemical information from radiospectroscopic, ultraviolet, and infrared spectra of simple molecules and free radicals adsorbed on oxide systems that are widely used in practice.     

Synthesis and DNA binding behavior of a naphthalene diimide derivative carrying two dicobalt hexacarbonyl complexes as an infrared DNA probe

Naphthalene diimide having two dicobalt hexacarbonyl complexes at its substituent termini 1 was designed and synthesized as an infrared probe for double stranded DNA. Spectrophotometric and viscometric DNA binding studies of 1 and naphthalene diimide having two acetylene moieties 2 as its precursor were carried out to prove their threading intercalation binding modes. Fourier Transform Infrared Reflection-Absorption Spectroscopy (FT-IR RAS) measurements were achieved for DNA-immobilized on a gold surface before and after hybridization with complementary DNA strand. Distinguished absorption peaks assigned to the dicobalt hexacarbonyl complexes were obtained in the case of the formation of double stranded DNA after treatment with 1. This result revealed that 1 can be used as an infrared probe for monitoring double stranded DNA.     

Development of a pharmacophore for cruzain using oxadiazoles as virtual molecular probes: quantitative structure–activity relationship studies

Chagas’s is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. According to the World Health Organization, 7 million people are infected worldwide leading to 7000 deaths per year. Drugs available, nifurtimox and benzimidazole, are limited due to low efficacy and high toxicity. As a validated target, cruzain represents a major front in drug discovery attempts for Chagas disease. Herein, we describe the development of 2D QSAR (\(r_{{{\text{pred}}}}^{2}\)?=?0.81) and a 3D-QSAR-based pharmacophore (\(r_{{{\text{pred}}}}^{2}\)?=?0.82) from a series of non-covalent cruzain inhibitors represented mostly by oxadiazoles (lead compound, IC₅₀?=?200 nM). Both models allowed us to map key intermolecular interactions in S1′, S2 and S3 cruzain sub-sites (including halogen bond and C?H/π). To probe the predictive capacity of obtained models, inhibitors available in the literature from different classes displaying a range of scaffolds were evaluate achieving mean absolute deviation of 0.33 and 0.51 for 2D and 3D models, respectively. CoMFA revealed an unexplored region where addition of bulky substituents to produce new compounds in the series could be beneficial to improve biological activity.     

Experimental detection of proteolytic activity in a signal peptide peptidase of Arabidopsis thaliana

Background

The ubiquitin ligase COP1, COnstitutively Photomorphogenic 1, functions in many biological responses in mammalian cells, but its downstream pathway remains unclear.

Results

Here, we identified FIP200, a key regulator of mammalian autophagy, as a novel COP1-interacting protein by yeast two-hybrid screening. The interaction was confirmed by a GST-pulldown assay. Split-GFP analysis revealed that interaction between COP1 and FIP200 predominantly occurred in the cytoplasm and was enhanced in cells treated with UV irradiation. Different forms of FIP200 protein were expressed in cultured mammalian cells, and ectopic expression of COP1 reduced one of such forms.

Conclusions

These data suggest that COP1 modulates FIP200-associated activities, which may contribute to a variety of cellular functions that COP1 is involved in.     

A Comprehensive Review on <Emphasis Type="SmallCaps">l</Emphasis>-Asparaginase and Its Applications

l-asparaginase (LA) catalyzes the degradation of asparagine, an essential amino acid for leukemic cells, into ammonia and aspartate. Owing to its ability to inhibit protein biosynthesis in lymphoblasts, LA is used to treat acute lymphoblastic leukemia (ALL). Different isozymes of this enzyme have been isolated from a wide range of organisms, including plants and terrestrial and marine microorganisms. Pieces of information about the three-dimensional structure of l-asparaginase from Escherichia coli and Erwinia sp. have identified residues that are essential for catalytic activity. This review catalogues the major sources of l-asparaginase, the methods of its production through the solid state (SSF) and submerged (SmF) fermentation, purification, and characterization as well as its biological roles. In the same breath, this article explores both the past and present applications of this important enzyme and discusses its future prospects.     

Use of solid-phase microextraction coupled to gas chromatography–mass spectrometry for determination of urinary volatile organic compounds in autistic children compared with healthy controls

Autism spectrum disorders (ASDs) are a group of neurodevelopmental disorders which have a severe life-long effect on behavior and social functioning, and which are associated with metabolic abnormalities. Their diagnosis is on the basis of behavioral and developmental signs usually detected before three years of age, and there is no reliable biological marker. The objective of this study was to establish the volatile urinary metabolomic profiles of 24 autistic children and 21 healthy children (control group) to investigate volatile organic compounds (VOCs) as potential biomarkers for ASDs. Solid-phase microextraction (SPME) using DVB/CAR/PDMS sorbent coupled with gas chromatography–mass spectrometry was used to obtain the metabolomic information patterns. Urine samples were analyzed under both acid and alkaline pH, to profile a range of urinary components with different physicochemical properties. Multivariate statistics techniques were applied to bioanalytical data to visualize clusters of cases and to detect the VOCs able to differentiate autistic patients from healthy children. In particular, orthogonal projections to latent structures discriminant analysis (OPLS-DA) achieved very good separation between autistic and control groups under both acidic and alkaline pH, identifying discriminating metabolites. Among these, 3-methyl-cyclopentanone, 3-methyl-butanal, 2-methyl-butanal, and hexane under acid conditions, and 2-methyl-pyrazine, 2,3-dimethyl-pyrazine, and isoxazolo under alkaline pH had statistically higher levels in urine samples from autistic children than from the control group. Further investigation with a higher number of patients should be performed to outline the metabolic origins of these variables, define a possible association with ASDs, and verify the usefulness of these variables for early-stage diagnosis.

Chemometrics-Assisted Optimization of Beta-/Gamma-Tocol Separation on a C<Subscript>30</Subscript> Stationary Phase in Reversed-Phase LC

This paper presents a chemometrics-assisted optimization study to improve the separation of tocopherol (-T) and tocotrienol (-TT) homologues on a C₃₀ stationary phase in reversed-phase HPLC. The HPLC settings were optimized using a central composite design and the response surface methodology. Flow rate, column temperature, and mobile phase composition were chosen as independent variables. Peak resolution (R_s), analysis time (t_R), and peak symmetries of the tocopherol isomers were chosen as response variables. Optimum performance in terms of R_s was obtained at a flow rate of 0.31 mL min^?1, a temperature of 8.70 °C, and % B content (methyl tert-butyl ether: methanol: water, 80:18:2, v/v/v) in the mobile phase of 38.12%. The analysis of variance and regression analysis gave adjusted R² values of 0.9841 for R_s, 0.9850 for t_R-(α-T), 0.9853 for t_R-(β-T), and 0.9204 for the peak symmetry of β-T. This confirms the good agreement of experimental data with predicted values. The close eluting peaks of β-/γ-tocol could be baseline separated at the optimized conditions at a minimized analysis time. Empirical second-order polynomial models were derived that gave statistically high significances (P?<?0.0001). Hence, the models can be successfully employed to predict the optimum separation conditions of co-eluting peaks of β-/γ-tocols. The optimized method was successfully applied to determine the individual tocol homologues in various cold pressed edible oils. Total contents ranged from 15 to almost 2600 mg tocol kg^?1 oil.     

Synthesis and evaluation of N-(2-[18F]fluoro-4-nitrobenzoyl)glucosamine: a preliminary report

d-glucosamine at concentration of certain range could kill tumor cells without influencing normal cells. There are also some reports on the antitumor activity of d-glucosamine and its derivatives in murine models. It was therefore postulated that d-glucosamine might have the potential to invade tumor cells. We designed and radiosynthesized a glucosamine derivative, N-(2-[¹⁸F]fluoro-4-nitrobenzoyl)glucosamine ([¹⁸F]FNBG([¹⁸F]7)). Evaluations in vitro and in vivo were performed on tumor bearing mice. Excitingly, the radiochemical purity of [¹⁸F]FNBG([¹⁸F]7) was 99%, and besides the best radiochemical yield was up to 35%. The best T/Bl (Tumor/Blood) and T/M (Tumor/Muscle) ratios of [¹⁸F]FNBG([¹⁸F]7) were 4.40 and 4.84. Although [¹⁸F]FNBG([¹⁸F]7) deserved further studies, the results revealed it might become a potential PET imaging agent for detecting tumors.     

Photocatalytic activity of CdS nanocrystals stabilized by a polymer shell and promoted by cobalt and nickel complexes in the reaction of hydrogen evolution

Nanocrystals (NCs) of CdS with oleate surface (NC-1) and octadecyl thiolate surface (NC-2), stabilized by a polycation shell, were doped with nickel bis(2-aminobenzenethiolate) (1), cobalt(III) chlorobis(dimethylglyoximato)(2-mercaptopyridine) (2), and also with 1,2-ethanedithiol and didodecylsulfide clathrochelates of cobalt(II) (3 and 4). The influence of doping on the photocatalytic activity in the hydrogen evolution reaction was investigated. Complex 1 appeared to be the most effective cocatalyst for H₂ evolution with the reaction rate increased by the factor of 8—11. Accomodating the complex in a polymer shell yields the best result. The rate of H₂ evolution increases monotonically with increasing concentration of this complex until the concentration achieves the ratio of one complex molecule per single NC. It is shown that the chemical composition of the surface has a significant influence on their photocatalytic activity in the hydrogen evolution reaction. The activity of NC-2 is 200 times that of NC-1. The replacement of oleate groups of the latter with sulfide increases the activity of these photocatalysts by a factor of 2000.     

细胞代谢组学用于羽扇豆醇干预人乳腺癌细胞MCF-7的机理探究

应用基于气相色谱-质谱联用（GC-MS）的代谢组学方法结合细胞周期实验,研究羽扇豆醇体外抑制人乳腺癌细胞MCF-7增殖的作用机理。代谢组学的研究结果表明：通过正交偏最小方差判别分析（OPLS-DA）可以很好地区分羽扇豆醇作用的MCF-7细胞代谢谱与对照组细胞代谢谱,模型参数为：R²Ycum=0.988,Q²Ycum=0.964。VIP（variable importance in the projection）值大于1的差异代谢物进一步用t检验进行单位分析,选择t<0.05（VIP>1）的代谢物作为羽扇豆醇作用组的生物标志物,得到琥珀酸、磷酸、亮氨酸、异亮氨酸等11种代谢差异物。结合羽扇豆醇将细胞周期抑制在G1期这一现象,推测羽扇豆醇可能是主要抑制了三羧酸循环中的琥珀酰辅酶A的生成和底物磷酸化生成ATP的反应来抑制MCF-7细胞的增殖。本实验从代谢组学角度为乳腺癌抗肿瘤机制提供新的线索。     

Effect of mutated transporters associated with antigen-processing 2 on characteristic major histocompatibility complex binding peptides: analysis using electrospray ionization tandem mass spectrometry

A novel allele of transporters associated with the antigen-processing (TAP) 2 gene, TAP2*Bky2 (Val(577)), is significantly increased in Japanese patients with Sj?gren's syndrome (SS), and has a strong association with SS-A/Ro autoantibody production in SS and autoantibody including anti-SS-A/Ro and anti-U1 RNP antibody in systemic lupus erythematosus (SLE). To determine the influence of this natural mutated TAP on peptides loaded onto MHC class I, we analyzed the repertoire of peptides loaded onto MHC class I on transfectants with TAP1 and TAP2 or mutated TAP2 by electrospray ionization tandem mass spectrometry (ESI-MS/MS). After comparison of the peptide profiles we identified three peptides from only mutated TAP transfectants. Moreover, one of these peptides is derived from snRNP A, which is a target for anti-U1 RNP antibody. To our knowledge this is the first report to show that the natural mutation of TAP2 changes the peptide profile loaded onto MHC class I molecules.