Synthesis and anticancer and lipophilic properties of 10-dialkylaminobutynyl derivatives of 1,8- and 2,7-diazaphenothiazines

New derivatives of two isomeric types of azaphenothiazines, 1,8- and 2,7-diazaphenothiazine, containing the triple bond substituents and additionally tertiary cyclic and acyclic amine groups, were synthesized and tested for their anticancer activity. The compounds exhibited differential inhibitory activities. Better results were obtained when the acetylenic group was transformed via the Mannich reaction to the dialkylaminobutynyl groups. The most active was 2,7-diazaphenothiazine with the N-methylpiperazine-2-butynyl substituent against the human ductal breast epithelial tumor cell line T47D, more potent than cisplatin. The 2,7-diazaphenothiazine system turned out to be more active than isomeric 1,8-diaza one. For the most active compound, the expression of TP53, CDKN1A, BCL-2 and BAX genes was detected by the RT-QPCR method. The gene expression ratio BACL-2/BAX suggests the mitochondrial apoptosis in T47D cells. The synthesis makes possible to obtain many new bioactive phenothiazines with the dialkylaminoalkynyl substituents inserting various tertiary cyclic and acyclic amine moieties to the substituents.     

Isolation and biological activities of 3-hydroxy-4(1H)-pyridone

3-Hydroxy-4(4H)-pyridone (3,4-DHP), a degraded product of mimosine [β-[N-(3-hydroxy-4-oxypyridyl)]-α-aminopropionic acid], is known to cause goiters, loss of hair, and infertility in animals, but limits of 3,4-DHP on separation and purification have prevented efforts on investigating other toxicity and biological properties of 3,4-DHP. By this study, a novel and simple isolation of 3,4-DHP was developed either from Leucaena leaves using an ion-exchanged resin or mimosine degraded in high temperature (110°C, 6?h). The inhibition of mimosine on the growth of barnyardgrass was approximately fourfold higher (IC₅₀?=?0.04?mg?g^?1) than that of 3,4-DHP (IC₅₀?=?0.15?mg?g^?1). In general, the antifungal activity of mimosine is much stronger than that of 3,4-DHP, but it differs depending on the kind of fungi. The 1,1-diphyenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of 3,4-DHP, in contrast with the growth inhibitory activity, is about fourfold stronger [EC₅₀?=?2.4?mg?g^?1 gallic acid equivalent (GAE)] than that of mimosine [EC₅₀?=?10.3?mg?g^?1 GAE]. This study is the first to report on the herbicidal, antifungal, and antioxidant activities of 3,4-DHP.     

Establishment of an effective TLC bioautographic method for the detection of Mycobacterium tuberculosis H37Ra phosphoglucose isomerase inhibition by phosphoenolpyruvate

A bioautographic assay based on thin layer chromatography was developed for phosphoenolpyruvate (PEP) detecting as a known but rarely studied inhibitor of phosphoglucose isomerase (PGI). The protocol with NADP⁺/NBT/PMS (β-nicotinamide adenine dinucleotide phosphate/nitrotetrazolium blue chloride/phenazine methosulfate) staining was capable of detecting Mycobacterium tuberculosis H₃₇Ra PGI inhibition using PEP. According to this method, visibly brighter spots (zones) against purple background are observed in the area of inhibition of the above-mentioned enzyme activity. The detection limit for PEP as an inhibitor of Mycobacterium tuberculosis H₃₇Ra PGI was 226?μg per spot/zone. Noteworthy is that we are the first authors to have successfully used a bioautographic assay to detect Mycobacterium tuberculosis H₃₇Ra PGI inhibition by PEP.     

Design and synthesis of antimicrobial,anticoagulant, and anticholinesterase hybrid molecules from 4-methylumbelliferone

We designed and synthesized new series of diverse triazoles, isoxazoles, isoxazolines, and aziridines linked 4-methylumbelliferone 1 using intermolecular 1,3-dipolar cycloaddition reactions. Structures of these compounds were established on the basis of ¹H NMR, ¹³C NMR, and ESI-HRMS. All prepared compounds were evaluated for their antimicrobial, anticoagulant, and anticholinesterase activities. Interestingly, among the tested molecules, some of the analogs displayed better activities than the parent 4-methylumbelliferone 1 such as 6a and 6d for their antifungal properties. Moreover, compounds 4, 5, 6, and 7 showed the importance of the added fragments to 4-methylumbelliferone 1 via the linker methylene to have good activity.     

Studies on the antioxidant properties of some phytoestrogens

Isoflavones genistein and daidzein are nonsteroidal phytoestrogens occurring mainly in soybean foods. These phytoestrogens possess estrogenic properties and show a variety of health benefits as anti‐inflammatory agents. However, the mechanism of their action has not been identified in detail. The aim of this study is to characterize the antioxidant powers of genistein, daidzein and daidzein metabolite–equol through their activities to scavenge superoxide anion radical (O^?₂^?), hydroxyl radical (HO^?), 2,2–diphenyl–1‐picrylhydrazyl radical (DPPH^?) and hydrogen peroxide (H₂O₂) using chemiluminescence and spectrophotometry techniques. Potassium superoxide in dimethyl sulphoxide (DMSO) and 18‐crown‐6 ether were used as a source of O^?₂^?. Hydroxyl radicals were produced using the Fenton reaction. In free radical assays, genistein had the IC₅₀ values (an amount of antioxidant concentration required to decrease the initial radical concentration by 50%) 0.391 ± 0.012 mM for O^?₂^?, 0.621 ± 0.028 mM for HO^? and 1.89 ± 0.16 mM for DPPH^?. The IC₅₀ values for daidzein for these free radicals were 1.924 ± 0.011 mM, 0.702 ± 0.012 mM and 2.81 ± 0.03 mM, respectively. Equol was the most active the free radical scavenger with IC₅₀ = 0.451 ± 0.018 mM for HO^? and IC₅₀ = 1.36 ± 0.11 mM for DPPH^?. All tested compounds exerted a significant effect on the H₂O₂: IC₅₀ = 18.1 ± 1.1 μM for genistein, IC₅₀ = 2.1 ± 0.5 μM for daidzein, and IC₅₀ = 1.06 ± 0.2 μM for equol. These findings show that genistein, daidzein and equol are effective free radical scavengers and possess high antioxidant power in vitro. Copyright © 2016 John Wiley & Sons, Ltd.     

大鼠室旁核注射orexin-A对体重的影响

目的:探讨大鼠室旁核(PVN)注射orexin-A对体重的影响。方法:大鼠室旁核(PVN)微量注射orexin-A,用大脑置管埋管、组织化学染色等方法探讨PVN注射orexin-A对其体重的影响。结果:与安慰剂组大鼠相比,PVN注射orexin-A组大鼠体重明显减轻(P0.05),而orexin-A组和安慰剂组摄食量无明显差异(P0.05)。注射结束后6天,orexin-A处理大鼠的体重仍显著低于注射前(P0.05),而安慰剂组大鼠则比注射前显著增重(P0.05)。药物注射可显著降低机体脂肪,但并不特异存在于注射orexin-A或安慰剂的大鼠身上。Orexin-A组和安慰剂组大鼠的肌肉量和脂肪量均显著降低(P0.05),但注射orexin-A的大鼠降低更明显。与安慰剂组相比,orexin-A处理后的摄食转化率显著降低(P0.05)。结论:大鼠室旁核(PVN)注射orexin-A可通过增加活动量产生负能量平衡,引起体重减轻。     

Characterization of two monoclonal antibodies, 38F10 and 44D11,against the major envelope fusion protein of Helicoverpa armigera nucleopolyhedrovirus

The envelope fusion protein F of baculoviruses is a class I viral fusion protein which play a significant role during virus entry into insect cells. F is initially synthesized as a precursor(F_0) and then cleaved into a disulfide-linked F_1 and F_2 subunits during the process of protein maturation and secretion. To facilitate further investigation into the structure and function of F protein during virus infection, monoclonal antibodies(mAbs) against the F_2 subunit of Helicoverpa armigera nucleopolyhedrovirus(HearNPV)(Ha F) were generated. Two kinds of mAbs were obtained according to their different recognition epitopes: one kind of mAbs, as represented by 38F10,recognizes amino acid(aa) 85 to 123 of F_2 and the other kind, represented by 44D11, recognizes aa148 to 173 of F_2. Western blot and immunofluorescence assay confirmed that both of the mAbs recognized the F protein expressed in HearNPV infected cells, however, only 44D11 could neutralize HearNPV infection. The results further showed that 44D11 may not interact with a receptor binding epitope, rather it was demonstrated to inhibit syncytium formation in cells expressing the Ha F protein. The results imply that the monoclonal antibody 44D11 recognizes a region within HaF_2 that may be involved in the F-mediated membrane fusion process.     

红缘天牛成虫对杏树挥发物的生理和行为反应

为了研究红缘天牛成虫对寄主植物杏树衰弱树及木段挥发物的生理及行为反应,本文用6种挥发物,分别以不同浓度的单一组份对红缘天牛进行了电生理和行为测试;又在这6种单组份化合物对天牛行为选择表现为引诱活性的浓度范围内选择EAG值最高的浓度,以4-6种化合物等体积制成7种组合配方进行EAG和行为测试。结果表明:单一组份R-柠檬烯、反-2-己烯醛、丁酸丁酯、S-柠檬烯、异戊醇和3-蒈烯等6种单一化合物在预设的8个浓度梯度范围内,都能引起红缘天牛产生一定的生理反应,但行为选择实验的结果没有统计学意义。7种组合配方中,R4对红缘天牛成虫引诱活性最强(P0.01),引诱率达到76.67%,R1次之(P0.05),R4与R1的区别在于增加了丁酸丁酯;即丁酸丁酯在R4配方中具有明显的增效作用。     

Characterization of conserved arginine residues on Cdt1 that affect licensing activity and interaction with Geminin or Mcm complex

All organisms ensure once and only once replication during S phase through a process called replication licensing. Cdt1 is a key component and crucial loading factor of Mcm complex, which is a central component for the eukaryotic replicative helicase. In higher eukaryotes, timely inhibition of Cdt1 by Geminin is essential to prevent rereplication. Here, we address the mechanism of DNA licensing using purified Cdt1, Mcm and Geminin proteins in combination with replication in Xenopus egg extracts. We mutagenized the 223th arginine of mouse Cdt1 (mCdt1) to cysteine or serine (R-S or R-C, respectively) and 342nd and 346th arginines constituting an arginine finger-like structure to alanine (RR-AA). The RR-AA mutant of Cdt1 could not only rescue the DNA replication activity in Cdt1-depleted extracts but also its specific activity for DNA replication and licensing was significantly increased compared to the wild-type protein. In contrast, the R223 mutants were partially defective in rescue of DNA replication and licensing. Biochemical analyses of these mutant Cdt1 proteins indicated that the RR-AA mutation disabled its functional interaction with Geminin, while R223 mutations resulted in ablation in interaction with the Mcm2～7 complex. Intriguingly, the R223 mutants are more susceptible to the phosphorylation-induced inactivation or chromatin dissociation. Our results show that conserved arginine residues play critical roles in interaction with Geminin and Mcm that are crucial for proper conformation of the complexes and its licensing activity.     

EXPRESSION AND EFFECTS OF MUTANT Bombyx mori ACETYLCHOLINESTRASE1 IN BmN CELLS

The main mechanism of toxicity of organophosphate (OP) and carbamate (CB) insecticides is their irreversible binding and inhibition of acetylcholinestrase (AChE), encoded by ace1 (acetylcholinestrase gene 1), leading to eventual death of insects. Mutations in AChE may significantly reduce insects susceptibility to these pesticides. Bombyx mori is an important beneficial insect, and no OP‐ or CB‐resistant strains have been generated. In this study, wild‐type ace1 (wace1) and mutant ace1 (mace1) were introduced into BmN cells, confirmed by screening and identification. The expression of wace1 and mace1 in the cells was confirmed by Western blot and their expression levels were about 21‐fold higher than the endogenous ace1 level. The activities of AChE in wace1 and mace1 transgenic cells were 10.6 and 20.2% higher compared to control cells, respectively. mace1 transgenic cells had higher remaining activity than wace1 transgenic cells under the treatment of physostigmine (a reversible cholinesterase inhibitor) and phoxim (an OP acaricide). The results showed that ace1 transgene can significantly improve ace1 expression, and ace1 mutation at a specific site can reduce the sensitivity to AChE inhibitors. Our study provides a new direction for the exploration of the relationship between AChE mutations and drug resistance.