厚壳贻贝whirlin类贝壳基质蛋白的重组表达与功能分析

贝壳历来是生物工程和材料学研究的重要对象。贝壳中的贝壳基质蛋白质在贝壳的形成与发育过程中具有重要的调控作用。Whirlin类蛋白质(Whirlin-like protein,WLP)是一种从厚壳贻贝(Mytilus coruscus)中鉴定的新型贝壳基质蛋白质。序列分析结果显示,该蛋白质含有PDZ(postsynaptic density/Discs large/Zonula occludens)结构域,而该结构域对贝壳生物矿化的影响目前尚无报道。为深入了解WLP在贝壳形成中对碳酸钙晶体的影响,在序列分析基础上,采用密码子优化结合原核重组表达,获得其重组表达产物后,开展了重组WLP对碳酸钙晶体形貌及晶型的影响研究,结晶速度抑制以及碳酸钙晶体结合分析。分析结果表明,重组WLP能诱导文石型碳酸钙晶体的形貌和方解石型碳酸钙晶体的晶型发生改变;同时重组WLP对碳酸钙晶体具有结合作用,且能抑制碳酸钙晶体的结晶速度。上述结果表明,WLP对贝壳的形成及发育具有重要影响,并可能在贝壳肌棱柱层的形成中发挥了重要作用。     

Immunological Properties of Recombinant Mycobacterium bovis Bacillus Calmette-Guérin Strain Expressing Fusion Protein IL-2-ESAT-6

The live vaccine Mycobacterium bovis bacillus Calmette-Guérin (BCG) provides variableefficacy against adult pulmonary tuberculosis (TB).Recombinant BCG,expressing either immunodominantantigens or Th1 cytokines,is a promising strategy for developing a new TB vaccine.However,not much isknown about whether the introduction of cytokine and specific antigen genes concurrently into the BCGstrain could improve the immunogenicity of BCG.In this study,a recombinant BCG strain (rBCG) expressingthe fusion protein human interleukin (IL)-2 and ESAT-6 (early secreted antigenic target-6 kDa) antigen ofMycobacterium tuberculosis was constructed.Six weeks after BALB/c mice (H-2~d) were immunizedwith 10~6 colony forming units (CFUs) BCG or rBCG,splenocyte proliferation was determined with MTT[3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide] assay,IL-4 and interferon (IFN)-γproducedby splenocytes were tested by enzyme linked immunosorbent assay (ELISA,) and the cytotoxicity ofsplenocytes from immunized mice to P815 cells (H-2~d) expressing ESAT-6 protein was measured usingCytoTox 96 Non-Radioactive Cytotoxicity Assay.Compared with native BCG-vaccinated mice,rBCG inducedstronger Th1 responses that were confirmed by high lymphoproliferative responses and IFN-γproductionto culture filtrate protein (CFP) or ESAT-6 protein.Moreover,rBCG induced significant enhanced CTLresponses against P815-ESAT-6 cells.Results from rBCG-immunized mice demonstrated that introducingthe il-2 and esat-6 genes into BCG could enhance Th1 type immune responses to ESAT-6.Further investigationis needed by introducing other Th1 cytokines and antigens into BCG to optimize the protective efficacyagainst TB.     

耐药细菌蛋白质的分泌——新抗攻击的靶点

几十年来,由于抗生素的大量使用,导致了细菌对很多药物的抗药性.为了克服细菌的抗药性问题,需要用新的思路去发掘新的抗生素,包括发掘细菌细胞中存在的抗生素作用的新靶点.蛋白质的分泌过程对于细菌是生死攸关的,它可能成为新药物的适合靶点.     

Preparation of capacitor’s electrode from sunflower seed shell

Series of nanoporous carbons are prepared from sunflower seed shell (SSS) by two different strategies and used as electrode material for electrochemical double-layer capacitor (EDLC). The surface area and pore-structure of the nanoporous carbons are characterized intensively using N₂ adsorption technique. The results show that the pore-structure of the carbons is closely related to activation temperature and dosage of KOH. Electrochemical measurements show that the carbons made by impregnation-activation process have better capacitive behavior and higher capacitance retention ratio at high drain current than the carbons made by carbonization-activation process, which is due to that there are abundant macroscopic pores and less interior micropore surface in the texture of the former. More importantly, the capacitive performances of these carbons are much better than ordered mesoporous carbons and commercial wood-based active carbon, thus highlighting the success of preparing high performance electrode material for EDLC from SSS.     

Regeneration of Stereocilia of Hair Cells by Forced Atoh1 Expression in the Adult Mammalian Cochlea

The hallmark of mechanosensory hair cells is the stereocilia, where mechanical stimuli are converted into electrical signals. These delicate stereocilia are susceptible to acoustic trauma and ototoxic drugs. While hair cells in lower vertebrates and the mammalian vestibular system can spontaneously regenerate lost stereocilia, mammalian cochlear hair cells no longer retain this capability. We explored the possibility of regenerating stereocilia in the noise-deafened guinea pig cochlea by cochlear inoculation of a viral vector carrying Atoh1, a gene critical for hair cell differentiation. Exposure to simulated gunfire resulted in a 60–70 dB hearing loss and extensive damage and loss of stereocilia bundles of both inner and outer hair cells along the entire cochlear length. However, most injured hair cells remained in the organ of Corti for up to 10 days after the trauma. A viral vector carrying an EGFP-labeled Atoh1 gene was inoculated into the cochlea through the round window on the seventh day after noise exposure. Auditory brainstem response measured one month after inoculation showed that hearing thresholds were substantially improved. Scanning electron microscopy revealed that the damaged/lost stereocilia bundles were repaired or regenerated after Atoh1 treatment, suggesting that Atoh1 was able to induce repair/regeneration of the damaged or lost stereocilia. Therefore, our studies revealed a new role of Atoh1 as a gene critical for promoting repair/regeneration of stereocilia and maintaining injured hair cells in the adult mammal cochlea. Atoh1-based gene therapy, therefore, has the potential to treat noise-induced hearing loss if the treatment is carried out before hair cells die.     

Identification of the Third Binding Site of Arsenic in Human Arsenic (III) Methyltransferase

Arsenic (III) methyltransferase (AS3MT) catalyzes the process of arsenic methylation. Each arsenite (iAs³⁺) binds to three cysteine residues, methylarsenite (MMA³⁺) binds to two, and dimethylarsenite (DMA³⁺) binds to one. However, only two As-binding sites (Cys156 and Cys206) have been confirmed on human AS3MT (hAS3MT). The third As-binding site is still undefined. Residue Cys72 in Cyanidioschyzon merolae arsenite S-adenosylmethyltransferase (CmArsM) may be the third As-binding site. The corresponding residue in hAS3MT is Cys61. Functions of Cys32, Cys61, and Cys85 in hAS3MT are unclear though Cys32, Cys61, and Cys85 in rat AS3MT have no effect on the enzyme activity. This is why the functions of Cys32, Cys61, and Cys85 in hAS3MT merit investigation. Here, three mutants were designed, C32S, C61S, and C85S. Their catalytic activities and conformations were determined, and the catalytic capacities of C156S and C206S were studied. Unlike C85S, mutants C32S and C61S were completely inactive in the methylation of iAs³⁺ and active in the methylation of MMA³⁺. The catalytic activity of C85S was also less pronounced than that of WT-hAS3MT. All these findings suggest that Cys32 and Cys61 markedly influence the catalytic activity of hAS3MT. Cys32 and Cys61 are necessary to the first step of methylation but not to the second. Cys156 and Cys206 are required for both the first and second steps of methylation. The S^C32 is located far from arsenic in the WT-hAS3MT-SAM-As model. The distances between S^C61 and arsenic in WT-hAS3MT-As and WT-hAS3MT-SAM-As models are 7.5 Å and 4.1 Å, respectively. This indicates that SAM-binding to hAS3MT shortens the distance between S^C61 and arsenic and promotes As-binding to hAS3MT. This is consistent with the fact that SAM is the first substrate to bind to hAS3MT and iAs is the second. Model of WT-hAS3MT-SAM-As and the experimental results indicate that Cys61 is the third As-binding site.     

接种丛枝菌根真菌对云南石漠化土壤呼吸的影响

为探明"丛枝菌根（Arbuscular mycorrhiza，AM）真菌-植物-土壤"耦合作用对石漠化土壤呼吸季节动态的影响，采用LI-6400-09土壤呼吸室和便携式光合作用测量系统，对圆柏（Sabina chinensis）接种摩西斗管囊霉（Funneliformis mosseae，FM）、根内根孢囊霉（Rhizophagus intraradices，RI）2种AM菌种处理下土壤呼吸季节动态进行野外连续定位观测，并探究AM真菌接种处理下石漠化土壤呼吸速率与植物生长、土壤理化性质之间的关系。结果表明：（1）相较于对照，接种AM真菌对石漠化生境土壤呼吸季节动态产生了显著影响（P<0.01）。AM真菌处理具有较高的土壤呼吸季节变化幅度，即根内根孢囊霉处理土壤呼吸速率（1.55-9.10 μmol m^-2 s^-1）显著高于摩西斗管囊霉（1.62-8.29 μmol m^-2 s^-1）和对照（1.23-4.46 μmol m^-2 s^-1）；（2） AM真菌接种处理下土壤温湿度变化对土壤呼吸的影响显著大于对照，即土壤温度与水分对土壤呼吸的平均解释量大小顺序为：RI （44.84%；52.35%）>FM （17.18%；41.65%）>CK （2.66%；16.55%）；（3）2种菌种处理下土壤呼吸速率均与土壤有机质、硝态氮、全氮、速效钾、树高、胸径及根系生物量呈显著或极显著正相关（P<0.01或0.05），而与pH呈极显著负相关（P<0.01），但对照处理土壤呼吸速率除与pH呈显著负相关（P<0.05）外，与其它土壤理化指标相关性不显著；（4）土壤温度和水分、硝态氮、铵态氮、有机质、易氧化有机碳、速效钾、全氮及全磷对土壤呼吸变化的贡献最大，而胸径、树高、有效磷、微生物生物量、根系生物量及pH的影响次之。因此，"AM真菌-寄主植物-土壤"相互作用对石漠化生境土壤呼吸季节动态的影响，主要取决于不同AM真菌接种处理对土壤微气候（如含水量）、碳素（有机质、易氧化有机碳）、无机氮库（铵态氮、硝态氮）、根系生物量及磷钾养分可利用性的调控。