研究报告: 组织蛋白酶B介导NLRP3小体在砷致小胶质细胞炎症激活中的作用

目的 探究组织蛋白酶B（CTSB）介导NLRP3小体在砷致小胶质细胞（BV-2）炎症激活中的作用。方法 取处于对数生长期的BV-2细胞，分别暴露于终浓度为0、2、4、8 μmol/L亚砷酸钠（NaAsO₂）溶液培养24 h，检测细胞活性，测定各组细胞内CTSB和细胞焦亡相关蛋白NLRP3、Caspase-1、IL-18、IL-1β的表达水平。流式细胞仪检测胞内溶酶体膜稳定性。基于实验结果，增设CTSB抑制剂组（5 μmol/L CA074-Me +8 μmol/L NaAsO₂、10 μmol/L CA074-Me+8 μmol/L NaAsO₂），检测两组细胞内炎症相关蛋白NLRP3、Caspase-1和IL-1β、IL-18的表达水平。结果 与对照组比较，各染砷组细胞抑制率增高，呈现剂量效应关系，溶酶体膜稳定性下降，差异有统计学意义（P<0.01），胞内CTSB、NLRP3、IL-1β、IL-18、Caspase-1表达增高，差异有统计学意义（P<0.01）；与对照组（8 μmol/L NaAsO₂）比较，抑制剂组BV-2细胞胞内CTSB、NLRP3、IL-1β、IL-18、Caspase-1水平均降低，差异有统计学差异（P<0.01）。结论 NaAsO₂通过诱导小胶质细胞内CTSB水平的上升，介导NLRP3炎症小体激活小胶质细胞，促其释放炎性因子，致神经系统损伤。     

水体硝化体系中砷的解毒机制探讨

硝化是目前废水生物脱氮中应用最为广泛的工艺之一,其功能菌为化能自养型细菌,生长缓慢,对重金属十分敏感。砷是一种剧毒的类金属元素,主要以无机形式的亚砷酸盐[AsO₂^-,As（Ⅲ）]和砷酸盐[AsO₄^3-,As（Ⅴ）]存在,尤以As（Ⅲ）毒性最强。但研究发现,在硝化体系中,高浓度As（Ⅲ）(约400mg/L)未对硝化功能微生物产生明显毒性。深入比较发现,As（Ⅲ）的生物氧化与硝化过程具有一定的关联性。化能自养型As（Ⅲ）氧化菌不仅可在有氧条件下将As（Ⅲ）氧化,还可在缺氧条件下以NO₂^–或NO₃^–为电子受体氧化As（Ⅲ）。而硝化细菌也是典型的化能自养菌,且硝化体系内存在氧气及硝化产物NO₂^–、NO₃^–等电子受体,理论上均可接受电子实现As（Ⅲ）的氧化。本文结合硝化反应特性,综述了As（Ⅲ）在硝化体系下的解毒机制,主要为胞外聚合物的...     

A novel arsenate reductase from the bacterium Thermus thermophilus HB27: Its role in arsenic detoxification

Microorganisms living in arsenic-rich geothermal environments act on arsenic with different biochemical strategies, but the molecular mechanisms responsible for the resistance to the harmful effects of the metalloid have only partially been examined. In this study, we investigated the mechanisms of arsenic resistance in the thermophilic bacterium Thermus thermophilus HB27. This strain, originally isolated from a Japanese hot spring, exhibited tolerance to concentrations of arsenate and arsenite up to 20 mM and 15 mM, respectively; it owns in its genome a putative chromosomal arsenate reductase (TtarsC) gene encoding a protein homologous to the one well characterized from the plasmid pI258 of the Gram + bacterium Staphylococcus aureus. Differently from the majority of microorganisms, TtarsC is part of an operon including genes not related to arsenic resistance; qRT-PCR showed that its expression was four-fold increased when arsenate was added to the growth medium. The gene cloning and expression in Escherichia coli, followed by purification of the recombinant protein, proved that TtArsC was indeed a thioredoxin-coupled arsenate reductase with a k_cat/K_M value of 1.2 × 10⁴ M^− 1 s^− 1. It also exhibited weak phosphatase activity with a k_cat/K_M value of 2.7 × 10^− 4 M^− 1 s^− 1. The catalytic role of the first cysteine (Cys7) was ascertained by site-directed mutagenesis. These results identify TtArsC as an important component in the arsenic resistance in T. thermophilus giving the first structural–functional characterization of a thermophilic arsenate reductase.     

Soil Profile Arsenic Concentration Distributions in Missouri Soils Having Cambic and Argillic Soil Horizons

Understanding of the pedogenic pathways associated with arsenic (As) transformations in soil is important to understanding arsenic soil chemistry and discriminating between natural background and anthropogenic arsenic (As). Twenty-one soil series, some with multiple pedons, were assessed to determine if the As distributions in soil profiles exhibit discrete maxima that correspond to the presence of agrillic horizons. The majority of pedons exhibiting argillic horizon expression show a Fe-oxyhydroxide and As maxima corresponding precisely with the argillic horizon. Pearson correlation coefficients verify the close correspondence of Fe and As. Soil profiles having cambic horizons, and lacking argillic horizons, may also show As and Fe accumulations at soil depths. Some coarse-textured, well-drained to moderately well-drained Entisols and Inceptisols have Fe-oxyhydroxide accumulation in their cambic horizons, promoting As accumulation. Conversely, silty-textured and poorly drained to somewhat poorly drained Entisols and Inceptisols have C and Cg horizons that show somewhat uniform Fe and As concentrations throughout their soil profiles. Analysis of selected pedons having well-drained to moderately well-drained soil profiles demonstrates that clay fraction Fe and As concentrations are closely correlated and that the As and Fe concentrations are greater than those from the corresponding whole soil. The somewhat poorly drained Crowley pedon exhibited cohesive masses of Fe and Mn accumulation (sand separate) that had greater arsenic concentrations than those of the clay and silt separates. These pedogenic nodules with enhanced arsenic concentrations reveal alternative pathways involving arsenic transformation.     

Multiple cysteine residues are necessary for sorting and transport activity of the arsenite permease Acr3p from Saccharomyces cerevisiae

The yeast transporter Acr3p is a low affinity As(III)/H⁺ and Sb(III)/H⁺ antiporter located in the plasma membrane. It has been shown for bacterial Acr3 proteins that just a single cysteine residue, which is located in the middle of the fourth transmembrane region and conserved in all members of the Acr3 family, is essential for As(III) transport activity. Here, we report a systematic mutational analysis of all nine cysteine residues present in the Saccharomyces cerevisiae Acr3p. We found that mutagenesis of highly conserved Cys151 resulted in a complete loss of metalloid transport function. In addition, lack of Cys90 and Cys169, which are conserved in eukaryotic members of Acr3 family, impaired Acr3p trafficking to the plasma membrane and greatly reduced As(III) efflux, respectively. Mutagenesis of five other cysteines in Acr3p resulted in moderate reduction of As(III) transport capacities and sorting perturbations. Our data suggest that interaction of As(III) with multiple thiol groups in the yeast Acr3p may facilitate As(III) translocation across the plasma membrane.     

Optimization of arsenic phytoremediation using Eichhornia crassipes

This study aimed to evaluate the pH, phosphate, and nitrate in the process of arsenic absorption by Eichhornia crassipes (water hyacinth), using the surface response methodology, in order to optimize the process. The plants were exposed to a concentration of arsenic of 0.5 mg L^?1 (NaAsO₂) over a period of 10 days. The results indicated optimal levels for the absorption of arsenic by E. crassipes at pH equal to 7.5, absence of phosphate, and minimum nitrate level of 0.0887 mmol L^?1. For the tested concentration, E. crassipes was able to accumulate 498.4 mg kg^?1 of As (dry base) in its plant tissue and to reduce 83% of the initial concentration present in the aqueous medium where it was cultivated. The concentration of phosphorus in solution linearly increased the phosphorus content in the plants and negatively influenced the absorption of arsenic. The concentration of 0.5 mg L^?1 of As did not significantly affect the relative growth rate (RGR) and the tolerance index (TI). 94% of As (III) initially solubilized in water was converted by the end of the experiment period into As (V). The water hyacinth was important in the phytoremediation of arsenic when cultivated under optimal conditions for its removal.