Discovery of Novel 3,4-Dihydro-2(1H)-Quinolinone Sulfonamide Derivatives as New Tubulin Polymerization Inhibitors with Anti-Cancer Activity.

In this paper, a small series of novel quinoline sulfonamide derivatives was synthesized, and their structure of the target compounds were confirmed by 1H NMR and MS. The screening of the news target compounds' in vitro cytotoxic activities against tumor cell lines by the MTT method was performed. Among them, compound D13 (N-(4-methoxybenzyl)-2-oxo-N-(3,4,5-trimethoxyphenyl)-1,2,3,4-tetrahydroquinoline-6-sulfonamide exhibited the strongest inhibitory effect on the proliferation of HeLa (IC50: 1.34 µM), and this value correlated well with the inhibitory activities of the compound against tubulin polymerization (IC50: 6.74 µM). In summary, a new type of quinoline-sulfonamide derivative with tubulin polymerization inhibitory activity was discovered, and it can be used as a lead compound for further modification.

Effects of Integrated Chinese Traditional Medicine and Conventional Western Medicine on the Quality of Life of Breast Cancer Patients: A Systematic Review and Meta-Analysis.

BACKGROUND: Traditional Chinese medicine has been widely used, in conjunction with conventional Western medicine, in clinical practice around the world to treat breast cancer. The study systematically reviewed and summarized the quality of life of breast cancer patients treated with integrated treatment method vs. conventional Western medicine. METHODS: Eight databases including PubMed, EMBASE, Web of Science, the Cochrane Library, Chinese National Knowledge Infrastructure, China Biology Medicine Disc, Chinese Scientific Journal Database, and Wanfang Data knowledge service platform were searched in this study. The retrieval period was set from January 1, 2005, to December 31, 2020. RESULTS: Twenty-two high-quality publications were included in this study. The total sample size was 1689 patients including 844 in the intervention group receiving traditional Chinese medicine combined with conventional Western medicine and 845 patients in the control group receiving conventional Western medicine only. Compared with the single-used conventional Western medicine treatment, an integrated approach to treat breast cancer can increase quality of life measured by rating scales (SMD = 1.29, 95% CI (1.07, 1.52) and P=0.01) and ranking scales (RR = 1.53, 95% CI (1.39 1.68) and P=0.02) and also decrease adverse reactions measured by rating scales (Z = 10.89, P < 0.05; Group 1: I 2 = 9.0%, P=0.258, SMD = 1.03; and Group 2: I 2 = 31.6%, P=0.199, SMD = 1.56). For further analysis, chemotherapy with epirubicin exhibited higher quality of life than the chemotherapy without epirubicin among breast cancer patients [Z = 19.80, P < 0.05; Group 1: I 2 = 62.4%, P=0.070, SMD = 1.61; and Group 2: I 2 = 9.0%, P=0.359, SMD = 1.04]. Despite the heterogeneity, which was due to a portion of relative low-quality literature or other factors, the results were satisfactory. In terms of secondary results, the patients with lower tumor markers (CEA and CA153) had better efficiency in quality of life with a statistically significant difference (SMD = 1.39, 95% CI: 1.10,1.67) for rating scales. In addition, secondary results related to high incidence of gastrointestinal adverse reactions (RR = 1.33, 95% CI (1.20, 1.48)) and the traditional Chinese medicine syndrome (RR = 1.50, 95% CI (1.28, 1.80))showed lower quality of life in the intervention group than the control group for ranking scales. CONCLUSION: Traditional Chinese medicine, when used in conjunction with the conventional Western medicine, could be an effective way in improving the quality of life and alleviating incidence of associated adverse symptoms such as gastrointestinal adverse reactions, value of tumor markers, and the incidence of traditional Chinese medicine syndrome. Further investigation of larger and methodologically sound trials with longer follow-up periods and appropriate comparison groups is needed.

LINC01087 is Highly Expressed in Breast Cancer and Regulates the Malignant Behavior of Cancer Cells Through miR-335-5p/Rock1.

PURPOSE: Long non-coding RNA is involved in the genesis and development of various tumors, and it has been found through database screening that LINC01087 is highly expressed in breast cancer (BC), but mechanisms of LINC01087 in BC are still under investigation. Therefore, this study aimed to study relevant mechanisms of LINC01087 in BC to provide potential therapeutic targets for the disease in clinic practice. PATIENTS AND METHODS: The qRT-PCR assay was applied to determine the LINC01087 expression in BC, and the cell counting kit-8 (CCK8) assay, transwell assay, and flow cytometry were used to analyze the proliferation, apoptosis, and invasion of breast cancer cells (BCCs), respectively. The Western blot assay was used to determine the ROCK1 expression, and the luciferase reporter gene assay, RNA-binding protein immunoprecipitation (RIP), and RNA pull-down assays were applied to study the interaction between LINC01087 and miR-335-5p. Moreover, tumor xenotransplantation was conducted in nude mice to explore the effects of LINC01087 on BCCs. RESULTS: The qRT-PCR assay revealed that the LINC01087 expression in BC tissues was higher than that in corresponding tumor-adjacent tissues, and survival analysis revealed an unfavorable prognosis of patients with high expression of LINC01087. Down-regulation of LINC01087 could slow down the proliferation, invasion, and migration of BCCs and accelerate apoptosis of them in vitro. Luciferase reporter gene assay results revealed that LINC01087 enhanced the expression of ROCK1 by regulating miR-335-5p, and LINC01087 could be adopted as a miR-335-5p sponge to inhibit ROCK1 expression. CONCLUSION: LINC01087 is overexpressed in cases with BC, and patients with high expression of it suffer a poor survival. Furthermore, LINC01087 can act as a miR-335-5p sponge to affect the expression of ROCK1 and affect the invasion and migration of BCCs.

Therapeutic Effect of Mongolian Medicine RuXian-I on Hyperplasia of Mammary Gland Induced by Estrogen/Progesterone through CRYAB-Promoted Apoptosis.

The traditional Mongolian medicine (TMM) RuXian-I is an empirical formula specifically used for treating the hyperplasia of mammary gland (HMG) in clinic based on the principles of traditional Mongolian medicine, but the treatment mechanism is not completely clear. In this paper, we elaborated the mechanism of RuXian-I in the treatment of HMG induced by estrogen and progestogen from its toxicity and activity. Firstly, RuXian-I exhibited no toxic effect on HMG rats through no changes of body weight and food intake measurement and no pathologic changes of the organs (heart, liver, spleen, lung, and kidney) detected. Secondly, RuXian-I could decrease the increased nipple height and diameter and remarkably relieve the pathologic changes of HMG rats and also alleviate serum sex hormone levels (estradiol (E2), luteinizing hormone (LH), progesterone (P), and testosterone (T)) of HMG rats. Finally, RuXian-I could obviously inhibit the upregulation level of antiapoptotic protein CRYAB of HMG rats and promote mammary gland cell apoptosis of HMG rats via increases of promoting apoptosis protein caspases-3, 8, and 9 and Bax and tumor suppressor protein p53, decreases of antiapoptosis protein Bcl-2, and release of cytochrome c. These results suggested that RuXian-I has protective and therapeutic effects on HMG rats induced by estrogen and progestogen possibly via promoting apoptotic pathway regulated by CRYAB and is a promising agent for treating HMG.

Synthesis, in vitro and in vivo biological evaluation of novel lappaconitine derivatives as potential anti-inflammatory agents.

Lappaconitine (LA), a natural compound with a novel C18-diterpenoid alkaloid skeleton, displayed extensive biological profile. Recent research on LA is focused mainly on its anti-tumor and analgesic effects, and therefore we aimed to investigate its anti-inflammatory potential. A series of novel LA derivatives with various substituents on the 20-N position was designed and synthesized. In the initial screening of LA derivatives against NO production, all the target compounds, except compound E2, exhibited excellent inhibitory ability relative to that of LA. Particularly, compound A4 exhibited the most potent inhibition with IC50 of 12.91 µmol/L. The elementary structure-activity relationships (SARs) of NO inhibitory activity indicated that replacement of the benzene ring with an electron donating group could improve the anti-inflammatory efficacy. Furthermore, compound A4 shows an anti-inflammatory mechanism by inhibiting NO, PGE2, and TNF-α generation via the suppression of NF-κB and MAPK signaling pathways. Notably, compound A4 could exert a significant therapeutic effect on LPS-induced acute lung injury (ALI) in vivo. Based on the above research, we further investigated the preliminary pharmacokinetic property of A4 in rats. Therefore, compound A4 could be a promising candidate for the development of anti-inflammatory agents in the future.

Discovery and evaluation of novel synthetic 5-alkyl-4-oxo-4,5-dihydro-[1,2,4]triazolo[4,3-a]quinoxaline-1-carbox-amide derivatives as anti-inflammatory agents.

To develop novel anti-inflammatory agents, a series of 5-alkyl-4-oxo-4,5-dihydro-[1, 2, 4]triazolo[4,3-a]quinoxaline-1-carboxamide derivatives were designed, synthesised, and evaluated for anti-inflammatory effects using RAW264.7 cells. Structures of the synthesised compounds were determined using 1H NMR, 13 C NMR, and HRMS. All the compounds were screened for anti-inflammatory activity based on their inhibitory effects against LPS-induced NO release. Among them, 5-(3,4,5-trimethoxybenzyl)-4-oxo-4,5-dihydro-[1, 2, 4]triazolo[4,3-a]quinoxaline-1-carboxamide (6p) showed the highest anti-inflammatory activity and inhibited NO release more potently than the lead compound D1. Further studies revealed that compound 6p reduced the levels of NO, TNF-α, and IL-6, and that its anti-inflammatory activity involves the inhibition of COX-2 and iNOS and downregulation of the mitogen-activated protein kinases (MAPK) signal pathway. Notably, compound 6p displayed more prominent anti-inflammatory activity than D1 and the positive control ibuprofen in the in vivo acute inflammatory model. Overall, these findings indicate that compound 6p is a therapeutic candidate for the treatment of inflammation.

Mongolian Medicine RuXian-I Treatment of Estrogen-Induced Mammary Gland Hyperplasia in Rats Related to TCTP Regulating Apoptosis.

Mongolian medicine RuXian-I is composed of 30 Mongolian herbs, which is a traditional Mongolian recipe for clinical treatment of breast "Qi Su Bu Ri Le Du Sen" disease (hyperplasia of mammary glands, HMG). Based on the previous study, this dissertation further explores the therapeutic mechanism of RuXian-I on estrogen-induced HMG in rats. RuXian-I had no effect on the body weight and food intake of HMG rats and had no toxic effects on the five organs (heart, lung, spleen, and kidney). RuXian-I reduced the diameter and height of nipple, organ index, and pathological changes and alleviated the sex hormone levels oh HMG; RuXian-I reduced the upregulation of TCTP, Mcl-1, and Bcl-xL in breast tissue of mammary gland hyperplasia and increased the downregulation of p53, Bax, caspase-9, and caspase-3 protein. RuXian-I has an effective therapeutic activity on HMG rats, and its possible therapeutic mechanism is closely related to antiapoptosis protein TCTP-regulated apoptosis.

Synthesis and biological evaluation of ursolic acid derivatives bearing triazole moieties as potential anti-Toxoplasma gondii agents.

Ursolic acid (UA), a plant-derived compound, has many properties beneficial to health. In the present study, we synthesised three series of novel UA derivatives and evaluated their anti-Toxoplasma gondii activity both in vitro and in vivo. Most derivatives exhibited an improved anti-T. gondii activity in vitro when compared with UA (parent compound), whereas compound 3d exhibited the most potent anti-T. gondii activity in vivo. Spiramycin served as the positive control. Additionally, determination of biochemical parameters, including the liver and spleen indexes, indicated compound 3d to effectively reduce hepatotoxicity and significantly enhance anti-oxidative effects, as compared with UA. Furthermore, our molecular docking study indicated compound 3d to possess a strong binding affinity for T. gondii calcium-dependent protein kinase 1 (TgCDPK1). Based on these findings, we conclude that compound 3d, a derivative of UA, could act as a potential inhibitor of TgCDPK1.

Comprehensive Circular RNA Profiling Reveals the Regulatory Role of the CircRNA-0067835/miR-155 Pathway in Temporal Lobe Epilepsy.

BACKGROUND/AIMS: Temporal lobe epilepsy (TLE) is the most common form of adult localization-related epilepsy that is accompanied by progressive etiopathology and high incidences of drug resistance. Circular RNAs (circRNAs) play important roles in fine-tuning gene expression, however, the expression profile and clinical significance of circRNAs in TLE remains unknown. METHODS: Circular RNA microarray was conducted to identify TLE-related circRNAs. CCK8 assays and flow cytometric assays were conducted to clarify the role of circRNA in TLE in vitro. Bioinformatics analysis and in vitro experiments were conducted to clarify the mechanism of circRNA-mediated gene regulation in TLE cell. RESULTS: 586 differentially expressed circRNAs were identified between TLE and the control tissues. The expression of circRNA-0067835 was significantly down-regulated in tissues and plasma from TLE patients. Lower circRNA-0067835 correlated to increased seizure frequency, HS, and higher Engel's score. Overexpression of circRNA-0067835 observably decreased SH-SY5Y cell proliferation by causing G1 arrest and promoting apoptosis. Bioinformatics online programs predicted that circRNA-0067835 acted as miR-155 sponge to regulate FOXO3a expression, which was validated using luciferase reporter assay. CONCLUSION: Our experiments showed that circRNA-0067835 regulated refractory epilepsy progression by acting as a sponge of miR-155 to promote FOXO3a expression, indicating that circRNA-0067835 may serve as a potential therapeutic target for patients with TLE.

Synthesis and characterisation of (Z)-styrylbenzene derivatives as potential selective anticancer agents.

To identify anticancer agents with high potency and low toxicity, a series of (Z)-styrylbenzene derivatives were synthesised and evaluated for anticancer activities using a panel of nine cancer cell lines and two noncancerous cell lines. Most derivatives exhibited significant anti-proliferative activities against five cancer cell lines, including MGC-803 and BEL-7402. (Z)-3-(p-Tolyl)-2-(3,4,5-trimethoxyphenyl)acrylonitrile (6h) showed a strong inhibitory effect on MGC-803 cells (IC50 < 0.01 µM) and exhibited stronger anti-proliferative activity than taxol (IC50 < 0.06 ± 0.01 µM). The IC50 value of 6h in L-02 cells was 10,000-fold higher than in MGC-803 cells. Compound 6h inhibited proliferation of BEL-7402 cells by arresting at the G2/M phase through up-regulation of cyclin B1 expression, down-regulation of cyclin A and D1 expression, and induction of apoptosis. In addition, 6h inhibited the migration of BEL-7402 cells and the formation of cell colonies.

MiR-153 regulates expression of hypoxia-inducible factor-1α in refractory epilepsy.

Mesial temporal lobe epilepsy (mTLE), the most common type of temporal lobe epilepsy (TLE), is particularly relevant due to its high frequency of therapeutic resistance of anti-epileptic therapies. MicroRNAs (miRNAs) have been shown to be dysregulated in epilepsy and neurodegenerative diseases, and we hypothesized that miRNAs could be involved in the pathogenesis of MTLE. The present study aimed to explore the expression and functions of miRNA-153 in mTLE. The expression levels of miRNA-153 in refractory TLE patients were evaluated. The bioinformatics analysis showed that the potential target genes of miR-153 were involved in biological processes, molecular functions, and cellular components. miRNA-153 is significantly dysregulated in temporal cortex and plasma of mTLE patients. We identify HIF-1α as a direct target of miRNA-153, and luciferase reporter assays demonstrated that miR-153 could regulate the HIF-1αexpression via 3'-UTR pairing. These data suggest that miR-153 might represent a useful biomarker and treatment target for patients with mTLE.

Design and synthesis of novel oridonin analogues as potent anticancer agents.

To identify anticancer agents with higher potency and lower toxicity, a series of oridonin derivatives with substituted benzene moieties at the C17 position were designed, synthesised, and evaluated for their antiproliferative properties. Most of the derivatives exhibited antiproliferative effects against AGS, MGC803, Bel7402, HCT116, A549, and HeLa cells. Compound 2p (IC50 = 1.05 µM) exhibited the most potent antiproliferative activity against HCT116 cells; it was more potent than oridonin (IC50 = 6.84 µM) and 5-fluorouracil (5-FU) (IC50 = 24.80 µM). The IC50 value of 2p in L02 cells was 6.5-fold higher than that in HCT116 cells. Overall, it exhibited better selective antiproliferative activity and specificity than oridonin and 5-FU. Furthermore, compound 2p arrested HCT116 cells at the G2 phase of the cell cycle and increased the percentage of apoptotic cells to a greater extent than oridonin.

Design, synthesis, anti-inflammatory activity, and molecular docking studies of perimidine derivatives containing triazole.

We report here the design, synthesis, and anti-inflammatory activities of a series of perimidine derivatives containing triazole (5a-s). The chemical structures of the synthesized compounds have been assigned on the basis of IR, 1H NMR, 13C NMR, and HRMS spectral analyses. The anti-inflammatory properties of the synthesized perimidine derivatives were evaluated in a lipopolysaccharide (LPS)-stimulated inflammation model. Among the tested compounds, compound 7-(3-methylbenzyl)-7H-[1,2,4]triazolo[4,3-a]perimidine (hereafter referred to as 5h) and compound 7-(2-fluorobenzyl)-7H-[1,2,4]triazolo[4,3-a]perimidine (hereafter referred to as 5n) caused a reduction in the levels of the pro-inflammatory cytokines-tumor necrosis factor (TNF)-α and interleukin (IL)-6-in RAW264.7 cells. The anti-inflammatory potential of compounds 5h and 5n was also evaluated in vivo in a xylene-induced ear inflammation model. Compound 5n showed the most potent anti-inflammatory activity with an inhibition of 49.26% at a dose of 50mg/kg. This activity is more potent than that of the reference drug ibuprofen (28.13%), and slightly less than that of indometacin (49.36%). To further elucidate the mechanisms underlying these inhibitory effects, LPS-induced nuclear factor-κB (NF-κB) activation and mitogen-activated protein kinase (MAPK) phosphorylation were studied. The results of western blotting showed that the extract obtained from compound 5n inhibited NF-κB (p65) activation and MAPK (extracellular signal-regulated kinase (ERK) and p38) phosphorylation in a dose-dependent manner. Moreover, the results of a docking study of compound 5n into the COX-2 binding site revealed that its mechanism was possibly similar to that of naproxen, a COX-2 inhibitor. The effect of compound 5n on COX-2 antibody was showed it could significantly inhibit COX-2 activity.

Sugemule-3 Protects against Isoprenaline-induced Cardiotoxicity In vitro.

BACKGROUND: Sugemule-3 (SD) is a traditional Chinese medicine with protective effect of myocardium. However, the underlying mechanisms of the effect had not been elucidated. MATERIALS AND METHODS: In the present study, the serum of SD was prepared. A model of ß-adrenergic agonist isoprenaline (ISO)-induced H9c2 cardiomyocytes injury was established in vitro. The changes in cell viability were examined to determine the available concentration of ISO and serum of SD. ELISA, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay, and flow cytometry were used to detect the effect of serum of SD on oxidative stress and apoptosis. The expression levels of the mitochondria-dependent apoptotic pathway and mitogen-activated protein kinase signalling-related proteins were analyzed. RESULTS: Incubation with different dose of ISO (0.015, 0.01, 0.005, and 0.0025 mol/L) for 24 h caused dose-dependent loss of cell viability and 0.01 mol/L of ISO approximately reduced the cell viability to 50%. Pretreatment with 50 µ mol/L serum of SD effectively decreased the levels of ISO-induced cell toxicity. Serum of SD relived ISO-induced oxidative stress and apoptosis in H9c2 cardiomyocytes. A further mechanism study indicated that serum of SD inhibited the mitochondria-dependent apoptotic pathways and regulated the expression levels of Bcl-2 family. ISO activated ERK and P38, whereas serum of SD inhibited their activation. CONCLUSION: Serum of SD inhibits the ISO-induced activation of the mitochondria-dependent apoptotic pathway, oxidative stress, and ERK, P38 inactivation. Serum of SD is used for the treatment of ISO-induced cardiomyopathy. SUMMARY: The serum of SD pretreatment significantly ameliorated ISO-induced H9c2 cardiomyocytes injuries.The protective effect related with apoptosis and oxidative stressInhibition of MAPK pathway was involed in serum of SD induced cardioprotection.The serum of SD is used for the treatment of ISO-induced cardiomyopathy. Abbreviations used: ELISA: Enzyme-linked Immunosorbent Assay; TUNEL: TdT-mediated dUTP nick end labeling; MTT: 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, DMSO: dimethyl sulfoxide; MDA: Malondialdehyde; SOD: Superoxide Dismutase; GSH-Px: Glutathione peroxidase.

Synthesis and anti-inflammatory activity evaluation of a novel series of 6-phenoxy-[1,2,4]triazolo[3,4-a]phthalazine-3-carboxamide derivatives.

The transcription factor nuclear factor-κB (NF-κB) controls many physiological processes including inflammation, immunity, and apoptosis. In this study, a novel series of 6-phenoxy-[1,2,4]triazolo[3,4-a]phthalazine-3-carboxamide derivatives were synthesized as potent anti-inflammatory agents, which acted on tumor necrosis factor (TNF-α) as inhibitors of NF-κB activation. We showed that compounds 6h (6-(2,4-dichlorophenoxy)-[1,2,4]triazolo[3,4-a]phthalazine-3-carboxamide) and 6i (6-(3-tolyloxy)-[1,2,4]triazolo[3,4-a]phthalazine-3-carboxamide) showed more prominent anti-inflammatory activity than other compounds, with similar activities as the reference drug dihydrotanshinone; compound 6i showed the lowest cellular toxicity among the tested compounds. In vivo evaluation of the anti-inflammatory activity showed that compound 6i exhibited excellent anti-inflammatory activity with 58.19% inhibition at 50mg/kg intraperitoneal (i.p.), with equal efficacy as the positive control indomethacin (100mg/kg i.p.; 59.21% inhibition).

Current Research on Antiepileptic Compounds.

Epilepsy affects about 1% of the world's population. Due to the fact all antiepileptic drugs (AEDs) have some undesirable side effects and about 30% of epileptic patients are not seizure-free with the existing AEDs, there is still an urgent need for the development of more effective and safer AEDs. Based on our research work on antiepileptic compounds and other references in recent years, this review covers the reported work on antiepileptic compounds which are classified according to their structures. This review summarized 244 significant anticonvulsant compounds which are classified by functional groups according to the animal model data, although there are some limitations in the data. This review highlights the properties of new compounds endowed with promising antiepileptic properties, which may be proven to be more effective and selective, and possibly free of unwanted side effects. The reviewed compounds represent an interesting possibility to overcome refractory seizures and to reduce the percentage of patients with a poor response to drug therapy.

Tetrazolium compounds: synthesis and applications in medicine.

Tetrazoles represent a class of five-membered heterocyclic compounds with polynitrogen electron-rich planar structural features. This special structure makes tetrazole derivatives useful drugs, explosives, and other functional materials with a wide range of applications in many fields of medicine, agriculture, material science, etc. Based on our research works on azoles and other references in recent years, this review covers reported work on the synthesis and biological activities of tetrazole derivatives.

Analysis on the interaction domain of VirG and apyrase by pull-down assay.

VirG is outer membrane protein of Shigella and affects the spread of Shigella. Recently it has been reported that apyrase influences the location of VirG, although the underlying mechanism remains poorly understood. The site of interaction between apyrase and VirG is the focus of our research. First we constructed recombinant plasmid pHIS-phoN2 and pS-(v1-1102, v53-758, v759-1102, v53-319, v320-507, v507-758) by denaturation-renaturation, the phoN2:kan mutant of Shigella flexneri 5a M90T by a modified version of the lambda red recombination protocol originally described by Datsenko and Wanner and the complemented strain M90TΔphoN2/pET24a(PhisphoN2). Second, the recombinant plasmid pHIS-phoN2 and the pS-(v1-1102, v53-758, v759-1102, v53-319, v320-507, v507-758) were transformed into E. coli BL21 (DE3) and induced to express the fusion proteins. Third, the fusion proteins were purified and the interaction of VirG and apyrase was identified by pull-down. Fourth, VirG was divided and the interaction site of apyrase and VirG was determined. Finally, how apyrase affects the function of VirG was analyzed by immunofluorescence. Accordingly, the results provided the data supporting the fact that apyrase combines with the α-domain of VirG to influence the function of VirG.

Recombinant expressed vector pET32a (+) S constructed by ligation independent cloning.

The aim of this work was to develop a new method for constructing vectors, named ligation-independent cloning (LIC) method. We constructed the S label expression vector and recombinant pET32a (+) S-phoN2 by LIC. The recombinant proteins were expressed in E. coli at a high level, and then the specificity of the recombinant proteins was identified by western blot. The target band was detected by S monoclonal antibody and Apyrase polyclonal antibodies but not Trx monoclonal antibody and HIS monoclonal antibody. Finally, we obtained protein Apyrase in E. coli (BL21), with a protein-only expression S tag. Collectively, our results demonstrated that LIC is effective for the construction of new vectors and recombinant plasmids. Free from the limitations of restriction enzyme sites and with a higher positive rate, LIC processes should find broad applications in molecular biology research.

Synthesis and Anticonvulsant Evaluation of some New 6-(Substituted-phenyl)thiazolo[3,2-b][1,2,4]triazole Derivatives in Mice.

Epilepsy is the most frequent nearological affiction and afflicts 1% about of the world's population. Currently there is an urgent need for the development of novel anticonvulsants with higher levels of potency and lower levels of toxicity. In this paper, a series of new 6-(substituted-phenyl)thiazolo[3,2-b][1,2,4]triazole derivatives were synthesized and tested for their anticonvulsant activities using the maximal electroshock (MES) and subcutaneous pentylenetetrazole (PTZ) screens, which are the most widely employed seizure models for early identification of candidate anticonvulsants. Their neurotoxicity was determined applying the rotarod test. In these compounds, 6-(4-fluorophenyl)thiazolo[3,2-b][1,2,4]triazole (3c) showed selective protection against the MES seizures with an ED50 value of 49.1 mg/Kg and a TD50 value of 94.1 mg/Kg, which provided compound 3c a protective index (PI = TD50/ED50) of 1.9 in the MES test. 6-(4-Propoxyphenyl)thiazolo[3,2-b][1,2,4]triazole (5b) was found to be active in both models, i.e. MES test and PTZ test. In the PTZ screen, compound 5b gave an ED50 of 63.4 mg/Kg and a TD50 of 105.6 mg/Kg, resulting in a PI value of 1.7 which is higher than carbamazepine.