GADD45α mediates arsenite‐induced cell apoptotic effect in human hepatoma cells via JNKs/AP‐1‐dependent pathway

Arsenite (As(III)), an effective chemotherapeutic agent for the acute promyelocytic leukemia (APL) and multiple myeloma (MM), might be also a promise for the therapy of other cancers, including the solid tumors. However, the molecular bases of arsenite‐induced cytotoxicity in the tumor cells have not been fully defined. In this study, we have disclosed that arsenite effectively induces the apoptotic response in the HepG2 human hepatoma cells by triggering GADD45α induction and the subsequent activation of JNKs/AP‐1 cell death pathway. However, signaling events relating to GADD45α/JNKs/AP‐1 pathway activation have not been observed in HL7702 human diploid hepatic cells under the same arsenite exposure condition. Our results thus have illustrated the selective pro‐apoptotic role of arsenite in the hepatoma cells by activating GADD45α‐dependent cell death pathway whereas with little effect on the normal hepatic cells. The approaches to up‐regulate GADD45α levels might be helpful in improving the chemotherapeutic action of arsenite on certain solid tumors including hepatoma. J. Cell. Biochem. 109: 1264–1273, 2010. © 2010 Wiley‐Liss, Inc.     

Synthesis,evaluation and CoMFA/CoMSIA study of nitrofuranyl methyl N-heterocycles as novel antitubercular agents

A series of novel nitrofuranyl methyl N-heterocycles based on the structure of IIIM-MCD-211 were designed and synthesized. Compounds 6d, 8b and 12a show excellent activity against MTB H37Rv strain (MIC: 0.031–0.062?μg/mL) roughly comparable to INH and IIIM-MCD-211. In addition, a three-dimensional quantitative structure-activity relationship (3D-QSAR) study was performed on the above mentioned chemical series employing comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) techniques. The developed CoMFA and CoMSIA models display high external predictability (r²_pred of 0.954 and 0.935, respectively) and good statistical robustness. More importantly, the newly designed compounds 16a and 16b (MIC: <0.016?μg/mL) based on the two models, as expected, were found to be more active than 12a and IIIM-MCD-21. Design and synthesis of more potent nitrofuranyl methyl N-heterocycles as anti-TB agents are currently in progress.     

毕赤酵母发酵生产颗粒性乙肝表面抗原的条件优化

乙型肝炎病毒的流行对人们的生命健康造成了极大的威胁, 而有效准确的诊断和预防性疫苗是阻止其流行的主要手段, 乙肝表面抗原是诊断试剂和疫苗的主要成分。本试验在构建稳定表达HBsAg的毕赤酵母菌株后, 对其发酵条件进行了研究。采用摇瓶分批培养方法, 探讨了不同培养基、溶解氧、诱导物甲醇的浓度以及pH值等因素对菌体生长与重组蛋白表达的影响。在10 L发酵罐上采用分批补料培养的方法研究了进行扩大培养生产重组HBsAg。结果表明, FBS无机盐合成培养基是理想的工业发酵培养基, 溶解氧对菌体的生长与表达有显著的影响, 甲醇诱导最佳终浓度为1% (V/V), 发酵的最适pH值为5.4~6.0。发酵罐放大培养后, ELISA和 SDS-PAGE分析表明重组HBsAg获得了高效表达, 最终菌体生物量达到310 OD600, 表达量达到27 mg/L。电子显微镜观察表达重组乙肝抗原可以自组装为22 nm类病毒颗粒, 为HBV的新一代早期血清学诊断和疫苗的大规模生产提供了一定的参考。     

FLP recombinase-mediated site-specific recombination in silkworm, Bombyx mori

A comprehensive understanding of gene function and the production of site-specific genetically modified mutants are two major goals of genetic engineering in the post-genomic era. Although site-specific recombination systems have been powerful tools for genome manipulation of many organisms, they have not yet been established for use in the manipulation of the silkworm Bombyx mori genome. In this study, we achieved site-specific excision of a target gene at predefined chromosomal sites in the silkworm using a FLP/FRT site-specific recombination system. We first constructed two stable transgenic target silkworm strains that both contain a single copy of the transgene construct comprising a target gene expression cassette flanked by FRT sites. Using pre-blastoderm microinjection of a FLP recombinase helper expression vector, 32 G3 site-specific recombinant transgenic individuals were isolated from five of 143 broods. The average frequency of FLP recombinase-mediated site-specific excision in the two target strains genome was approximately 3.5%. This study shows that it is feasible to achieve site-specific recombination in silkworms using the FLP/FRT system. We conclude that the FLP/FRT system is a useful tool for genome manipulation in the silkworm. Furthermore, this is the first reported use of the FLP/FRT system for the genetic manipulation of a lepidopteran genome and thus provides a useful reference for the establishment of genome manipulation technologies in other lepidopteran species.     

New mechanism of nerve injury in Alzheimer’s disease: β‐amyloid‐induced neuronal pyroptosis

The present study was designed to investigate the role of β‐amyloid (Aβ_1‐42) in inducing neuronal pyroptosis and its mechanism. Mice cortical neurons (MCNs) were used in this study, LPS + Nigericin was used to induce pyroptosis in MCNs (positive control group), and Aβ_1‐42 was used to interfere with MCNs. In addition, propidium iodide (PI) staining was used to examine cell permeability, lactate dehydrogenase (LDH) release assay was employed to detect cytotoxicity, immunofluorescence (IF) staining was used to investigate the expression level of the key protein GSDMD, Western blot was performed to detect the expression levels of key proteins, and enzyme‐linked immunosorbent assay (ELISA) was utilized to determine the expression levels of inflammatory factors in culture medium, including IL‐1β, IL‐18 and TNF‐α. Small interfering RNA (siRNA) was used to silence the mRNA expression of caspase‐1 and GSDMD, and Aβ_1‐42 was used to induce pyroptosis, followed by investigation of the role of caspase‐1‐mediated GSDMD cleavage in pyroptosis. In addition, necrosulfonamide (NSA), an inhibitor of GSDMD oligomerization, was used for pre‐treatment, and Aβ_1‐42 was subsequently used to observe the pyroptosis in MCNs. Finally, AAV9‐siRNA‐caspase‐1 was injected into the tail vein of APP/PS1 double transgenic mice (Alzheimer's disease mice) for caspase‐1 mRNA inhibition, followed by observation of behavioural changes in mice and measurement of the expression of inflammatory factors and pyroptosis‐related protein. As results, Aβ_1‐42 could induce pyroptosis in MCNs, increase cell permeability and enhance LDH release, which were similar to the LPS + Nigericin‐induced pyroptosis. Meanwhile, the expression levels of cellular GSDMD and p30‐GSDMD were up‐regulated, the levels of NLRP3 inflammasome and GSDMD‐cleaved protein caspase‐1 were up‐regulated, and the levels of inflammatory factors in the medium were also up‐regulated. siRNA intervention in caspase‐1 or GSDMD inhibited Aβ_1‐42‐induced pyroptosis, and NSA pre‐treatment also caused the similar inhibitory effects. The behavioural ability of Alzheimer's disease (AD) mice was relieved after the injection of AAV9‐siRNA‐caspase‐1, and the expression of pyroptosis‐related protein in the cortex and hippocampus was down‐regulated. In conclusion, Aβ_1‐42 could induce pyroptosis by GSDMD protein, and NLRP3‐caspase‐1 signalling was an important signal to mediate GSDMD cleavage, which plays an important role in Aβ_1‐42‐induced pyroptosis in neurons. Therefore, GSDMD is expected to be a novel therapeutic target for AD.     

Allele-specific targeting of hsa-miR-657 to human IGF2R creates a potential mechanism underlying the association of ACAA-insertion/deletion polymorphism with type 2 diabetes

The biological mechanism of a recent discovered association of type 2 diabetes with the ACAA-insertion/deletion polymorphism at the 3′UTR of the IGF2R gene has remained unclear. A very recently emerging novel polymorphic control layer by microRNAs (miRNAs) makes it possible to elucidate this issue. In this study, a prediction by web tools MicroInspector and miRanda demonstrated that DNA sequence polymorphism (DSPs) ACAA-insertion/deletion in IGF2R 3′UTR is located within the hsa-miR-657 and hsa-miR-453 binding sites. And luciferase reporter assay revealed that hsa-miR-657 acts directly at the 3′UTR of the IGF2R. Furthermore, ACAA-deletion exerted a further repression compared with ACAA-insertion, indicating that hsa-miR-657 regulates IGF2R gene expression in a polymorphic control manner. Importantly, we also demonstrated that hsa-miR-657 can translationally regulate the IGF2R expression levels in Hep G2 cells. Thus, our findings testify the possibility that the ACAA-insertion/deletion polymorphism may result in the change of IGF2R expression levels at least in part by hsa-miR-657-mediated regulation, contributing to the elucidation for the pathogenesis of type 2 diabetes and raise the possibility that miRNAs or in combination with functional DNA sequence polymorphism may be valuable in the treatment of human type 2 diabetes.     

鲤鳃表面结构扫描电镜研究

鲤的鳃丝表面显示了由三边形一六边形不等的表皮细胞,呈现出各种图案式的结构。     

Low molecular weight polyethylenimines linked by beta-cyclodextrin for gene transfer into the nervous system

BACKGROUND: Polyethylenimines (PEIs) with high molecular weights are effective nonviral gene delivery vectors. However, the in vivo use of these PEIs can be hampered by their cellular toxicity. In the present study we developed and tested a new PEI polymer synthesized by linking less toxic, low molecular weight (MW) PEIs with a commonly used, biocompatible drug carrier, beta-cyclodextrin (CyD). METHODS AND RESULTS: The terminal CyD hydroxyl groups were activated by 1,1'-carbonyldiimidazole. Each activated CyD then linked two branched PEI molecules with MW of 600 Da to form a CyD-containing polymer with MW of 61 kDa, in which CyD served as a part of the backbone. The PEI-CyD polymer developed was soluble in water and biodegradable. In cell viability assays with sensitive neurons, the polymer performed similarly to low-MW PEIs and displayed much lower cellular cytotoxicity compared to PEI 25 kDa. The gene delivery efficiency of the polymer was comparable to, and at higher polymer/DNA ratios even higher than, that offered by PEI 25 kDa in neural cells. Attractively, intrathecal injection of plasmid DNA complexed by the polymer into the rat spinal cord provided levels of gene expression close to that offered by PEI 25 kDa. CONCLUSIONS: The polymer reported in the current study displayed improved biocompatibility over non-degradable PEI 25 kDa and mediated gene transfection in cultured neurons and in the central nervous system effectively. The new polymer would be worth exploring further as an in vivo delivery system of therapeutic genetic materials for gene therapy of neurological disorders.     

磷缓解水鳖镉毒害的生理研究

研究了Cd对水鳖生理生化过程的影响及Pi对Cd胁迫的缓解作用。结果显示,Cd能抑制水鳖的代谢和生长,使叶绿素含量与光合速率下降,呼吸速率与可溶性糖含量增加,POD活性增强;当加入0.6mmol/L Pi后,能相对减轻Cd造成的毒害,表现出Pi对Cd的拮抗和缓解作用。     

白腐菌对染料脱色和降解作用的研究进展

白腐菌应用于废水处理始于二十世纪八十年代。本文对印染废水的处理方法、白腐菌及其对污染物的降解机理作了简要概述 ,着重介绍了白腐菌对染料脱色和降解作用的研究进展。白腐菌对染料的脱色解降作用机理有部分尚待进一步研究 ;同时 ,白腐菌的吸附作用亦不容忽视。