A screening assay for thyroid hormone signaling disruption based on thyroid hormone-response gene expression analysis in the frog Pelophylax nigromaculatus

Amphibian metamorphosis provides a wonderful model to study the thyroid hormone (TH) signaling disrupting activity of environmental chemicals, with Xenopus laevis as the most commonly used species. This study aimed to establish a rapid and sensitive screening assay based on TH-response gene expression analysis using Pelophylax nigromaculatus, a native frog species distributed widely in East Asia, especially in China. To achieve this, five candidate TH-response genes that were sensitive to T3 induction were chosen as molecular markers, and T3 induction was determined as 0.2 nmol/L T3 exposure for 48 hr. The developed assay can detect the agonistic activity of T3 with a lowest observed effective concentration of 0.001 nmol/L and EC50 at around 0.118–1.229 nmol/L, exhibiting comparable or higher sensitivity than previously reported assays. We further validated the efficiency of the developed assay by detecting the TH signaling disrupting activity of tetrabromobisphenol A (TBBPA), a known TH signaling disruptor. In accordance with previous reports, we found a weak TH agonistic activity for TBBPA in the absence of T3, whereas a TH antagonistic activity was found for TBBPA at higher concentrations in the presence of T3, showing that the P. nigromaculatus assay is effective for detecting TH signaling disrupting activity. Importantly, we observed non-monotonic dose-dependent disrupting activity of TBBPA in the presence of T3, which is difficult to detect with in vitro reporter gene assays. Overall, the developed P. nigromaculatus assay can be used to screen TH signaling disrupting activity of environmental chemicals with high sensitivity.     

Modelling sulphate-enhanced cadmium uptake by Zea mays from nutrient solution under conditions of constant free Cd2+ ion activity

A controlled hydroponic experiment was undertaken to investigate Cd uptake in relation to the activity of Cd species in solution other than the free ion (Cd²⁺) by maintaining a constant Cd²⁺ activity under variable SO₄₂⁻ and Cl⁻ concentrations exposed to maize (Zea mays var. Cameron) plants. The objectives of these experiments were: (1) to distinguish and quantify the different uptake rates of free and inorganic-complexed Cd from nutrient solution, and (2) to model the uptake of Cd by maize with a Biotic Ligand Model (BLM) in a system which facilitates the close examination of root characteristics. Results of the current experiments suggest that, in addition to the free ion, CdSO₄⁰ complexes are important factors in determining Cd uptake in nutrient solution by maize plants. Higher nominal SO₄²⁻ concentrations in solution generally resulted in a greater Cd accumulation by maize plants than predicted by the Cd²⁺ activity. A better integration of the complete dataset for the 3 harvest times (6, 9 and 11 days after treatment) was achieved by including consideration of both the duration of Cd exposure and especially the root surface area to express Cd uptake. Similarly, the fit of the BLM was also improved when taking into account exposure time and expressing uptake in terms of root morphological parameters.     

镧对酸雨胁迫下水稻叶片质膜H+-ATPase活性的影响

为了明晰稀土提高植物抗酸性的内在机制,采用水培法研究了镧(La,15 mg·L~(-1))对酸雨(AR,pH=3.5、2.5)胁迫下水稻叶片质膜H+-ATPase活性的影响.结果显示:与CK相比,pH=3.5 AR组水稻相对生长速率减小,质膜H+-ATPase活性升高,胞内H+增多,CAT活性升高,H_2O_2和MDA含量升高,质膜脂肪酸不饱和度指数(IUFA)降低.La+pH 3.5 AR组水稻各项指标优于pH=3.5 AR组,表明La促使AR下质膜H+-ATPase活性升高,将胞内过多的H+泵出,减缓AR伤害,维持水稻正常生长.另外,La能增强CAT活性,清除过多的H_2O_2,减轻膜脂过氧化程度,使质膜IUFA升高,膜的流动性增强,提高质膜H+-ATPase活性.pH=2.5 AR组水稻相对生长速率、质膜H+-ATPase活性、CAT活性与质膜IUFA均降低,而胞内H+、H_2O_2和MDA含量升高.La+pH 2.5 AR组与pH=2.5 AR组水稻各指标均无明显差异,La对pH=3.5 AR组的缓解效果优于pH=2.5 AR组.综上可知,La对AR下质膜H+-ATPase活性的调节增强了植物耐酸性,这与La能维持AR下较好的质膜环境有关.同时,La的调控效果受AR强度限制.     

用氧吸收速率(OUR)表征活性污泥硝化活性的研究

研究了用氧吸收速率（ＯｘｙｇｅｎＵｐｔａｋｅＲａｔｅ，简称ＯＵＲ）表征活性污泥硝化活性的方法．利用生物抑制剂丙烯基硫脲（ａｌｙｌｔｈｉｏｕｒｅａ，简称ＡＴＵ）和氯酸钠（ＮａＣｌＯ３）可以选择性地抑制亚硝酸细菌和硝酸细菌的活性．通过测量不同时间的ＯＵＲ，可以分别计算出污泥的亚硝化活性和硝化活性．结果表明：该方法的测量结果与实际反应器中的硝化反应情况相当一致．该方法简单易行，快速方便，适用于硝化系统中硝化活性的测量．     

磁性吸附剂CeO2/MZFS去除水中盐酸四环素

利用废碱性电池制备得到尖晶石结构的锰锌铁氧体Mn_0.6Zn_0.4Fe₂O₄（MZF）,利用原位聚合制备得到硅包覆的磁性纳米复合物MZF@SiO₂（MZFS）,采用浸渍法制备得到集磁分离、吸附与类Fenton催化活性于一体的CeO₂/MZFS磁性吸附剂,并对其形貌和磁性能等进行了表征.结果表明,CeO₂/MZFS对水中盐酸四环素（TH）表现出良好的吸附性能,160 min吸附过程可达平衡,TH的去除率达到95%.吸附过程可用准二级动力学方程描述,化学吸附为速控步骤;Freundlich等温吸附模型能较好地描述该吸附过程,热力学计算结果表明,CeO₂/MZFS对TH的吸附是以物理吸附为主的自发吸热过程;利用红外光谱初步揭示了吸附机制.吸附TH后的CeO₂/MZFS可利用其非均相类Fenton催化活性以H₂O₂作为再生剂进行原位再生,6次循环使用后,CeO₂/MZFS对TH仍能保持较高的平衡吸附量.研究结果可为抗生素废水的治理提供理论指导和技术支持.     

Direct calorimetry of free-moving eels with manipulated thyroid status

In birds and mammals, the thyroid gland secretes the iodothyronine hormones of which tetraiodothyronine (T4) is less active than triiodothyronine (T3). The action of T3 and T4 is calorigenic and is involved in the control of metabolic rate. Across all vertebrates, thyroid hormones also play a major role in differentiation, development and growth. Although the fish thyroidal system has been researched extensively, its role in thermogenesis is unclear. In this study, we measured overall heat production to an accuracy of 0.1 mW by direct calorimetry in a free-moving European eel (Anguilla anguilla L.) with different thyroid status. Hyperthyroidism was induced by injection of T3 and T4, and hypothyroidism was induced with phenylthiourea. The results show for the first time at the organismal level, using direct calorimetry, that neither overall heat production nor overall oxygen consumption in eels is affected by hyperthyroidism. Therefore, we conclude that the thermogenic metabolism-stimulating effect of thyroid hormones (TH) is not present with a cold-blooded fish species like the European eel. This supports the concept that TH does not stimulate thermogenesis in poikilothermic species.     

Low intensity ultrasound stimulates biological activity of aerobic activated sludge

This work aims to explore a procedure to improve biological wastewater treatment efficiency using low intensity ultrasound. The aerobic activated sludge from a municipal wastewater treatment plant was used as the experimental material. Oxygen uptake rate (OUR) of the activated sludge (AS) was determined to indicate the changes of AS activity stimulated by ultrasound at 35 kHz for 0–40 min with ultrasonic intensities of 0–1.2 W/cm². The highest OUR was observed at the ultrasonic intensity of 0.3 W/cm² and an irradiation period of 10 min; more than 15% increase was achieved immediately after sonication. More significantly, the AS activity stimulated by ultrasound could last 24 h after sonication, and the AS activity achieved its peak value within 8 h after sonication, or nearly 100% higher than the initial level after sonication. Therefore, to improve the wastewater treatment efficiency of bioreactors, ultrasound with an intensity of 0.3 W/cm² could be employed to irradiate a part of the AS in the bioreactor for 10 min every 8 h. Translated from Environmental Science, 2005, 26(4): 124–128 [译自: 环境科学]     

Re-evaluation of thyroid hormone signaling antagonism of tetrabromobisphenol A for validating the T3-induced Xenopus metamorphosis assay

We developed the T3-induced Xenopus metamorphosis assay, which is supposed to be able to sensitively detect thyroid hormone(TH) signaling disruption of chemicals. The present study aimed to validate the T3-induced Xenopus metamorphosis assay by re-evaluating the TH signaling antagonism of tetrabromobisphenol A(TBBPA), a known TH signaling disruptor. According to the assay we developed, Xenopus tadpoles at stage 52 were exposed to 10–500 nmol/L TBBPA in the presence of 1 nmol/L T3. After 96 hr of exposure, TBBPA in the range of 10–500 nmol/L was found to significantly inhibit T3-induced morphological changes of Xenopus tadpoles in a concentration-dependent manner in term of body weight and four morphological endpoints including head area(HA), mouth width(MW), unilateral brain width/brain length(ULBW/BL), and hind-limb length/snout-vent length(HLL/SVL).The results show that these endpoints we developed are sensitive for characterizing the antagonistic effects of TBBPA on T3-induced metamorphosis. Following a 24-hr exposure,we found that TBBPA antagonized expression of T3-induced TH-response genes in the tail,which is consistent with previous findings in the intestine. We propose that the tail can be used as an alternative tissue to the intestine for examining molecular endpoints for evaluating TH signaling disruption. In conclusion, our results demonstrate that the T3-induced Xenopus metamorphosis assay we developed is an ideal in vivo assay for detecting TH signaling disruption.     

循环流廊道湿地中氮归趋过程模拟研究

针对新型高效脱氮循环流廊道(CFC)湿地,构建了涵盖6种氮形态、3类介质、10种代谢途径的N循环模型,探索了湿地内N迁移转化模式.结果表明,沸石吸附(53.3%)、植物吸收NH+4-N(27.6%)、NO-3-N反硝化(10.2%)、植物吸收NO-3-N(2.9%)和NO-2-N短程反硝化(1.5%)对TN去除贡献依次降低.NH+4-N去除机制存在季节差异,其中1月NH+4-N主要通过沸石吸附去除(84.5%);4~6月通过植物吸收去除(76.4%~85.3%);7月通过沸石吸附(36.1%)、亚硝化(45.8%)及植物吸收(21.4%)共同去除.此外,定期收获植物、按期再生沸石及种植水生植物可分别提升TN去除率1.7%~7.7%、43.1%~72.2%和19.8%~36.2%.综之,CFC湿地去除途径多样性保障了TN的长期高效去除.     

In vitro fluorescence displacement investigation of thyroxine transport disruption by bisphenol A

Bisphenol A (BPA) is a chemical with high production volume and wide applications in many industries. Although BPA is known as an endocrine disruptor, its toxic mechanisms have not been fully characterized. Due to its structural similarity to thyroid hormones thyroxine (T4) and triiodothyronine (T3), one possible mechanism of BPA toxicity is disruption of hormone transport by competitive binding with the transport proteins. In this study, the binding interactions of BPA, T4, and T3 with three thyroid hormone transport proteins, human serum albumin (HSA), transthyretin (TTR), and thyroxine-binding globulin (TBG) were investigated by fluorescence measurement. Using two site-specific fluorescence probes dansylamide and dansyl-L-proline, the binding constants of BPA with HSA at drug site I and site II were determined as 2.90 104 and 3.14 104 L/mol, respectively. By monitoring the intrinsic fluorescence of tryptophan, a binding constant of 4.70 103 L/mol was obtained. Similarly, by employing 8-anilino-1-naphthalenesulfonic acid as fluorescence probe, the binding a nity of BPA with TTR and TBG was measured to be 3.10 105 and 5.90 105 L/mol, respectively. In general, BPA showed lower binding a nity with the proteins than T3 did, and even lower a nity than T4. Using these binding constants, the amount of BPA which would bind to the transport proteins in human plasma was estimated. These results suggest that the concentrations of BPA commonly found in human plasma are probably not high enough to interfere with T4 transport.     

模拟酸雨对水稻叶片质膜H+-ATPase活性与胞内Ca2+浓度的影响

在水培条件下研究了模拟酸雨(pH=2.5～5.5)对水稻叶片胞内Ca2+浓度和质膜H+-ATPase活性的影响.结果表明:与对照组(CK)相比,酸雨处理5 d(胁迫期)后,pH=5.5和5.0处理组的水稻叶片胞内H+浓度、质膜H+-ATPase活性、胞内Ca2+浓度、质膜Ca2+-ATPase活性无显著变化;pH=4.0和3.5处理组各指标显著升高,且H+-ATPase活性随Ca2+浓度升高而上升;pH=3.0和2.5处理组各指标显著降低,此时胞内Ca2+缺失,对H+-ATPase活性的调节作用受到限制.经正常条件培养5 d(恢复期)后,pH=4.0和3.5处理组各指标均恢复至CK的处理水平,表明H+-ATPase活性受到Ca2+调控已恢复到正常;pH=3.0和2.5处理组的Ca2+浓度高于CK及胁迫期,H+-ATPase活性低于CK但高于胁迫期,表明H+-ATPase活性受Ca2+调控得到部分恢复.因此,酸雨胁迫下胞内Ca2+对质膜H+-ATPase活性有一定调节作用,且受酸雨强度的制约.     

Phosphatase activity and culture conditions of the yeast Candida mycoderma sp. and analysis of organic phosphorus hydrolysis ability

Orthophosphate is an essential but limiting macronutrient for plant growth. About 67% cropland in China lacks sufficient phosphorus, especially that with red soil. Extensive soil phosphorus reserves exist in the form of organic phosphorus, which is unavailable for root uptake unless hydrolyzed by secretory acid phosphatases. Thus, many microorganisms with the ability to produce phosphatase have been exploited. In this work, the activity of an extracellular acid phosphatase and yeast biomass from Candida mycoderma was measured under different culture conditions, such as pH, temperature, and carbon source. A maximal phosphatase activity of 8.47 × 10^5±0.11× 10^5 U/g was achieved by C. Mycoderma in 36 hr under the optimal conditions. The extracellular acid phosphatase has high activity over a wide pH tolerance range from 2.5 to S.0 （optimum pH 3.5）. The effects of different phosphorus compounds on the acid phosphatase production were also studied. The presence of phytin, lecithin or calcium phosphate reduced the phosphatase activity and biomass yield significantly. In addition, the pH of the culture medium was reduced significantly by lecithin. The efficiency of the strain in releasing orthophosphate from organic phosphorus was studied in red soil （used in planting trees） and rice soil （originating as red soil）. The available phosphorus content was increased by 230% after inoculating 20 days in rice soil and decreased by 50% after inoculating 10 days in red soil. This work indicates that the yeast strain C. mycoderma has potential application for enhancing phosphorus utilization in plants that grow in rice soil.     

杭州湾潮滩湿地土壤碱性磷酸酶活性分布及其与磷形态的关系

以杭州湾潮滩作为研究区域,对土壤中碱性磷酸酶活性、磷形态、有机磷细菌数量及理化性质的时空分布特征进行分析,揭示碱性磷酸酶活性与磷形态分布及转化的关系. 结果表明,杭州湾潮滩湿地土壤中碱性磷酸酶活性具有显著的时空差异,总体表现为7月最高（38. 59~104.74 mg·kg^-1·h^-1）,2月最低（8. 21~40.61 mg·kg^-1·h^-1）,且各月之间有显著性差异（p<0.05）;不同植被型土壤碱性磷酸酶活性之间差异总体表现为芦苇（39.72~104.63 mg·kg^-1·h^-1）>互花米草（32.18~73.23 mg·kg^-1·h^-1）≈海三棱藨草（9.68~83.48 mg·kg^-1·h^-1）>光滩（8.21~49.50 mg·kg^-1·h^-1）,其中光滩与其他类型土壤具有显著性差异（p<0.05）. 无机磷形态中的可交换态磷（Exch-P）、铁/铝结合态磷（Fe/Al-P）及钙结合态磷（Ca-P）的含量均表现为：5月>7月>10月> 2月,5月与2月之间具有显著性差异（p<0.05）. 有机磷形态中的活性有机磷（L-P）的含量动态变化为：7月>5月>10月>2月;中等活性有机磷（ML-P）的含量表现为：2月> 5月>10月>7月;非活性有机磷（NL-P）的含量没有明显的动态变化. 相关性分析结果显示,碱性磷酸酶活性与有机磷细菌数量呈显著的正相关关系（r=0.58,p<0.01）,有机磷细菌是影响碱性磷酸酶活性的主要因素之一.土壤碱性磷酸酶活性与无机磷组分中的（Fe/Al）-P（r=0.39,p<0.05）、 有机磷组分中的L-P （r=0.37,p<0.05）、NL-P（r=0.31,p<0.05）呈正相关,潮滩湿地土壤中的NL-P是生物有效磷的潜在磷源.     

Regulation of bone morphogenetic proteins in early embryonic development

Bone morphogenetic proteins (BMPs), a large subgroup of the TGF- family of secreted growth factors, control fundamental events in early embryonic development, organogenesis and adult tissue homeostasis. The plethora of dose-dependent cellular processes regulated by BMP signalling demand a tight regulation of BMP activity. Over the last decade, a number of proteins have been identified that bind BMPs in the extracellular space and regulate the interaction of BMPs with their cognate receptors, including the secreted BMP antagonist Chordin. In the early vertebrate embryo, the localized secretion of BMP antagonists from the dorsal blastopore lip establishes a functional BMP signalling gradient that is required for the determination of the dorsoventral – or back to belly – body axis. In particular, inhibition of BMP activity is essential for the formation of neural tissue in the development of vertebrate and invertebrate embryos. Here we review recent studies that have provided new insight into the regulation of BMP signalling in the extracellular space. In particular, we discuss the recently identified Twisted gastrulation protein that modulates, in concert with metalloproteinases of the Tolloid family, the interaction of Chordin with BMP and a family of proteins that share structural similarities with Chordin in the respective BMP binding domains. In addition, genetic and functional studies in zebrafish and frog provide compelling evidence that the secreted protein Sizzled functionally interacts with the Chd–BMP pathway, despite being expressed ventrally in the early gastrula-stage embryo. These intriguing discoveries may have important implications, not only for our current concept of early embryonic patterning, but also for the regulation of BMP activity at later developmental stages and tissue homeostasis in the adult.     

金鱼(Carassius auratus L.)对水中游离态铅的吸收积累及鳃分泌粘液的自身保护作用

研究了金鱼在铅暴露实验中的铅吸收过程及鱼鳃分泌物的自身保护作用，水相Ｐｂ＾２＋的鳃吸收机理以及分泌粘液对铅的络作用。结果表明，金鱼因铅暴露导致粘液分泌量增加。结合到鳃上的铅与水相Ｐｂ＾２＋活度关系可用Ｌａｎｇｍｕｉｒ吸附等温式表述。     

Modeling of acute cadmium toxicity in solution to barley root elongation using biotic ligand model theory

Protons（H⁺）as well as different major and trace elements may inhibit cadmium（Cd）uptake in aquatic organisms and thus alleviate Cd toxicity.However,little is known about such interactions in soil organisms.In this study,the independent effects of the cations calcium(Ca²⁺),magnesium(Mg²⁺),potassium（K⁺）,H+and zinc(Zn²⁺)on Cd toxicity were investigated with 5-day long barley root elongation tests in nutrient solutions.The tested concentrations of selected cations and trace metal ions were based on the ranges that occur naturally in soil pore water.The toxicity of Cd decreased with increasing activity of Ca²⁺,Mg²⁺,H⁺and Zn²⁺,but not K⁺.Accordingly,conditional binding constants were obtained for the binding of Cd²⁺,Ca²⁺,Mg²⁺,H⁺,and Zn²⁺ with the binding ligand:log K_CdBL5.19,logK_CaBL2.87,logK_MgBL2.98,logK_HBL5.13 and logK_ZnBL5.42,respectively.Furthermore,it was calculated that on average 29% of the biotic ligand sites needed to be occupied by Cd to induce a 50% decrease in root elongation.Using the estimated constants,a biotic ligand model was successfully developed to predict the Cd toxicity to barley root elongation as a function of solution characteristics.The feasibility and accuracy of its application for predicting Cd toxicity in soils were discussed.     

A sustained-release microcarrier effectively prolongs and enhances the antibacterial activity of lysozyme

Bacterial infections have become a great threat to public health in recent years. A primary lysozyme is a natural antimicrobial protein; however, its widespread application is limited by its instability. Here, we present a poly (N-isopropylacrylamide) hydrogel inverse opal particle (PHIOP) as a microcarrier of lysozyme to prolong and enhance the efficiency against bacteria. This PHIOP-based lysozyme (PHIOP-Lys) formulation is temperature-responsive and exhibits long-term sustained release of lysozyme for up to 16 days. It shows a potent antibacterial effect toward both Escherichia coli and Staphylococcus aureus, which is even higher than that of free lysozyme in solution at the same concentration. PHIOPs-Lys were demonstrated to effectively inhibit bacterial infections and enhance wound healing in a full-thickness skin wound rat model. This study provides a novel pathway for prolonging the enzymatic activity and antibacterial effects of lysozyme.     

四溴双酚-A和五溴酚对红鲫甲状腺激素和脱碘酶的影响

将红鲫(Carassius auratus)分别暴露于0.5mg·L-1四滇双酚-A(TBBPA)和0.1 mg·L-1五澳酚(PBP)中28 d,采用放射性免疫分析法和化学发光酶联免疫分析法检测红鲫的血清甲状腺激素水平和肝脏脱碘酶活性.结果显示,TBBPA能使红鲫血清TT4和TT3水平降低,rT3水平、肝脏脱碘酶ID2和ID3活性升高.PBP对红鲫甲状腺系统的干扰要小于TBBPA,它对红鲫血清TT3水平和ID2活性几乎没有影响.上述结果表明,TBBPA和PBP能够干扰红鲫体内甲状腺激素的稳态,加速甲状腺激素在肝脏内的转化和代谢;这可能是通过抑制甲状腺激素与运载蛋白的结合来实现的.     

Molecular characterization and developmental expression patterns of thyroid hormone receptors (TRs) and their responsiveness to TR agonist and antagonist in Rana nigromaculata

Considering some advantages of Rana nigromaculata as an experimental species, we propose that this species, like Xenopus laevis, could be used to assay thyroid hormone（TH） signaling disrupting actions. To validate the utilizability of R. nigromaculata, we investigated the responsiveness of R. nigromaculata to a TH receptor（TR） agonist（T3） and antagonist（amiodarone） by analyzing expression, based on characterizing TR cDNA and developmental expression patterns. With high levels of identity with the corresponding genes in X. laevis, both TRα and TRβ in R. nigromaculata exhibited roughly similar developmental expression patterns to those of X. laevis, in spite of some species-specific differences. Both TRα and TRβ expression had greater changes in the liver and intestine than in the tail and brain during metamorphosis. T3 exposure for 2 days induced more dramatic increases of TRβ expression in stage 27 than in stage34 tadpoles but not in stage 42 tadpoles, showing that the responsiveness of R. nigromaculata to TH decreased with development and disappeared at the onset of metamorphic climax.Corresponding to greater changes of TRβ expression in the liver and intestine than in the tail and brain during metamorphosis, the liver and intestine had higher responsiveness to exogenous T3 than the tail and brain. Amiodarone inhibited T3-induced TRβ expression. Our results show that R. nigromaculata can be used as a model species for assaying TH signaling disrupting actions by analyzing TRβ expression, and intestine tissues at stage 27 are ideal test materials due to high responsiveness and easy accessibility.     

高选择性基因工程菌E.coli SE5000生物富集水体中的镍离子

利用基因工程菌E．coli SE5000对水体中的镍离子进行富集研究．菌体细胞对Ni^2 的富集速率很快,富集过程满足Langmuir等温线模型．经基因改造的基因工程菌不仅最大镍富集容量与原始宿主菌相比增加了4倍多,而且对pH值的变化呈现出更强的适应性．对离子强度及其它共存重金属离子的影响的实验结果表明：Na^ ,Ca^2 ,Cd^2 ,Pb^2 的影响较小,但Mg^2 ,Hg^2 ,Cr^3 和Cu^2 所引起的负面效应较大．金属螯合剂EDTA的存在对基因工程菌的富集行为影响很大．