毛木耳漆酶纯化及其部分漆酶特性的研究

对毛木耳AuriculariapolytrichaAP4的粗酶液进行PAGE电泳后发现含有三种漆酶同工酶,并且通过运用NativeSDS-PAGE获得三种漆酶的分子量大小分别约为:LacA(110kD);LacB(84kD);LacC(65kD)。对漆酶粗酶液通过硫酸铵分级沉淀和离子交换柱层析进行纯化,用SDS-PAGE证明获得纯化的单一漆酶LacB。LacB漆酶的反应的最适温度为30℃,最适pH为3.0。此酶氧化ABTS的Km值为6.64×10-mmol/L,金属离子对酶活的影响很大,其中5Ca2+,Mg2+,Zn2+,Na2+,Ag2+对漆酶LacB有明显的激活作用;Co2+,Hg2+,Fe3+,Fe2+,Ba2+等对酶活有明显的抑制作用。LacB和其它真菌漆酶一样具有底物专一性不强的特点,并且LacB对RB亮兰染料有很好的脱色作用。     

以圆币草发酵液为碳源时硫酸盐还原菌处理重金属废水

【目的】探索以圆币草(Hydrocotyle verticillata)发酵液作为碳源时硫酸盐还原菌处理重金属废水的效果,以便于高效去除废水中的重金属离子。【方法】以厌氧污泥为硫酸盐还原菌接种菌群,添加大型水生植物圆币草发酵液,并以乙醇、乳酸钠、葡萄糖、蔗糖和乙酸钠为对照,测定不同碳源下硫酸盐还原效率,分析其对废水中重金属离子(Pb2+,Cd2+,Cu2+,Ni2+)的去除能力。【结果】硫酸盐还原菌能有效利用圆币草发酵液中有机物,在COD/SO42-为1.2、5.0和7.0时硫酸盐最大还原率分别为24.4%、43.6%和60.0%。以发酵液为碳源时硫酸盐还原效率高于葡萄糖、蔗糖和乙酸钠,但低于乙醇和乳酸钠。在添加圆币草发酵液的批次试验反应器中,对低浓度4种重金属离子混合废水具有良好的处理效果,Cd2+、Cu2+、Pb2+和Ni2+的去除率分别为95.2%、98.7%、93.0%和89.6%。当Cd2+、Cu2+、Pb2+和Ni2+浓度为10 mg/L时,以圆币草发酵液为碳源的批次反应器对4种重金属离子仍具有良好的处理效果,去除率均超过90%,且硫酸盐还原菌的活性没有受到抑制。【结论】大型水生植物发酵液作为硫酸盐还原菌的碳源,不仅能有效进行重金属废水的生物深度处理,而且可以实现大型水生植物的资源化。     

发光细菌法快速监测和评价工业废水综合毒性

以汞、镉、铅、砷和六价铬的混合后对发光细菌的综合毒性为参照标准,对工业废水进行快速监测和毒性分级的研究,实验得出上述混合毒物与发光细菌的发光强度的相关关系是y＝40.9131-23.7200lnx,r-0.995。根据工业废水的相对发光强度,建议将工业废水划分为微毒,低毒、中等毒,高毒和剧毒五个毒性级别,并可根据上述线性关系,推算出有毒物质的浓度范围和相当于国家标准的倍数。应用此法对22份工业废水进行了实际测定与评价;还对四个企业工业废水处理前后的毒性进行了测定与毒性比较,结果表明处理后较处理前的毒性的毒性明显降低。     

冬虫夏草菌丝体γ-谷氨酰转肽酶的探测和部分性质研究

本研究首次发现冬虫夏草发酵菌丝体含有较高活力的γ-谷氨酰转肽酶（简称CSGT）,并且通过硫酸铵分级沉淀、疏水层析、凝胶过滤层析、阴离子交换层析和制备电泳的提取纯化程序,将CSGT纯化了2300倍,然后对CSGT的基本酶学性质进行了研究。CSGT的稳定pH范围和温度范围分别为pH8-11和0-20℃, 当pH 9-10 、30℃并且以L-谷氨酸-对-硝基苯胺（简称GpNA）和双甘肽为底物时CSGT的活力达到最大值。几种还原剂均能激活CSGT,说明其活性中心含有巯基。Zn2+, Cu2+, Hg2+ , Mn2+ 等金属离子均强烈抑制CSGT活性,而K+, Ca2+, Mg2+ 和Na+等对CSGT活性没有影响。     

发光细菌法测定环境中金属毒性的研究进展

在环境污染物的毒性评价和监测中,发光细菌法是一种具有快速、灵敏和廉价等优点的直接生物测试方法.本文简要回顾了发光细菌法的测定原理及其在水环境中的应用;总结了发光细菌法测定水环境中金属毒性的主要影响因素(如pH、有机无机配体和交互作用);重点评述了发光细菌法在土壤样品金属毒性测定方面的应用、不同提取方法的优缺点以及土壤金属毒性与形态之间的关系;提出今后应加强土壤中金属对发光细菌的毒害机理、土壤环境中发光细菌法标准化以及发光细菌法与其他测试方法的关系等研究.快速、廉价、标准化发光细菌法的建立对土壤环境中金属风险评价和监测具有重要的意义.     

重金属诱导拟南芥原生质体DNA 损伤的单细胞凝胶电泳检测

以拟南芥原生质体为实验体系, 研究不同浓度的3种重金属离子对拟南芥原生质体的毒性和DNA损伤的差异。结果表明, 用1-5 mmol.L-1的Zn2+、Cd2+ 和Cu2+分别处理的拟南芥原生质体, 2 小时内活力逐渐下降, 并表现出明显的浓度依赖性;与相同浓度的Cd2+ 和Cu2+ 相比, Zn2+对拟南芥原生质体活力的影响程度较小, 表现出较低的毒性。单细胞凝胶电泳检测发现,用0.1-0.8 mmol .L-1的Zn2+、Cd2+ 和Cu2+ 分别处理拟南芥原生质体30 分钟, 以OTM值表示的原生质体DNA损伤量随重金属离子浓度的增加而递增; 相同浓度(0.5 mmol.L-1)的3种重金属离子相比, Zn2+对原生质体的遗传毒性明显低于Cu2+ 和Cd2+。综合原生质体活力和DNA损伤的单细胞凝胶电泳检测结果, 发现Zn2+对拟南芥原生质体的遗传毒性较低, 而Cd2+ 和Cu2+的遗传毒性较高。本研究建立的拟南芥原生质体实验体系, 结合运用单细胞凝胶电泳技术, 能够快速、灵敏地检测重金属对植物细胞的遗传毒性。     

金属离子对十字花科黑腐病菌不同致病系统表达的影响

十字花科黑腐病菌(Xanthomonas campestris pv.campestris,Xcc)是引起十字花科植物黑腐病的病原细菌,其通过不同的分泌系统将效应物蛋白或细胞毒素转运进真核宿主细胞产生病害,对农业生产造成巨大的经济损失。本研究利用GUS融合报告系统,考察了常见的几种不同金属离子(Zn2+,Mn2+,Ca2+,Fe2+)对Xcc 8004不同致病系统装置基因表达的影响。结果表明,在1滋mol/L~1 mmol/L浓度区间,Zn2+和Mn2+同时对Xcc域型分泌系统(T2SS)和芋型分泌系统(T3SS)有抑制作用;在大于0.01 mmol/L浓度时,Ca2+特异性抑制T3SS;Fe2+同时抑制T2SS、T3SS和郁型分泌系统(T4SS)装置基因的表达。金属离子Zn2+、Mn2+、Ca2+和Fe2+在不同浓度下对Xcc的致病系统均有抑制作用。本研究为分析病原细菌不同分泌系统响应金属离子的模式奠定了基础。     

烟草头孢霉汞还原酶特性的研究

经检测,抗汞真菌烟草头孢霉(Cephalosporium tabacinum)F2菌株中具有汞五原酶活性。该酶是一种胞内酶,需NADH作为电子供体,催化Hg2+还原成为元素汞(Hgo)。该酶促反应还需硫氢基化合物,反应最适温度为30℃,最适pH范围为7．0-8.0。在25—30℃保温40分钟或在pH7．0保温2小时,酶活力均是稳定的。金属离子Ag+,Co2+,Cu2+,Zn2+,Mn2+和Ni2+在浓变为0．2-1．0mmol／L的范围内,对酶活力有不同程度的抑制作用,一定浓度的乙酸苯汞和铁氰化钾对酶活力也有部分抑制作用。     

发光细菌JMU07的分离鉴定及其在生物毒性测定的初步研究

从乌贼表皮通过分离纯化得到一株发光细菌JMU07。该菌的菌落形态呈典型细菌菌落特征;显微镜下观察其为球杆状菌,革兰氏染色阴性。用荧光分光光度计测定其发光波长在420-650 nm之间,最大发光波长为477 nm。16S rDNA法测序,构建系统进化树,初步鉴定发光细菌JMU07为鳆发光杆菌(Photobacterium leiog-nathi)。生长发光曲线测定表明,发光细菌JMU07发光强度最高出现在对数中后期,相比明亮发光杆菌,JMU07具有发光强度高,持续发光时间长的特点。根据国标GB/T15441-1995研究HgCl2浓度与发光细菌JMU07发光强度抑制率的关系得到:HgCl2浓度与发光细菌JMU07发光强度抑制呈良好线性关系;JMU07 EC50为0.11 mg/L,略低于明亮发光杆菌的0.14 mg/L,表明JMU07对HgCl2的毒性更敏感。因此新分离得到的发光细菌JMU07有希望用于环境检测、食品卫生与安全等领域综合毒性的快速检测。     

重金属对芝麻种子萌发及幼苗生长的影响

采用培养皿滤纸发芽法研究镉(Cd2+)、镍(Ni2+)、铬(Cr6+)、汞(Hg2+)、铅(Pb2+)等5种重金属离子对芝麻Sesamum indicum种子萌发及幼苗生长的影响。结果表明,重金属离子胁迫对芝麻种子露白萌发影响较小,对胚根生长影响最大;随着重金属离子浓度增加,芝麻幼苗芽长、根长和鲜重减小;在Cd2+、Ni2+和Cr6+浓度20 mg·L-1,Hg2+浓度50 mg·L-1,Pb2+浓度100 mg·L-1时,芝麻种子萌发后胚根基本不生长,难以成苗,这些浓度可视为对应重金属离子对芝麻种子萌发毒害的致死浓度。综合比较,5种重金属离子对芝麻种子萌发及芽期幼苗生长的毒害程度依次为Ni2+ > Cr6+ > Cd2+ > Hg2+ > Pb2+。     

Improved high-performance liquid chromatographic method for the pharmacokinetic studies of a novel iron chelator, CP502, in rats

An improved reverse-phase high-performance liquid chromatographic method (RP-HPLC) for the determination of a novel iron chelator CP502 (1,6-dimethyl-3-hydroxy-4-(1H)-pyridinone-2-carboxy-(N-methyl)-amide hydrochloride) in rat plasma, urine and feces was developed and validated. The separation was performed on a polymeric column using a mobile phase composed of 1mM ethylenediaminetetra-acetic acid disodium salt (EDTA), acetonitrile, methanol and methylene chloride. Separation of CP502 from plasma, urine or feces endogenous compounds was achieved by gradient elution. Retention times of CP502 and its major metabolite (glucuronide) were about 13 and 4 min, respectively. The method was validated in terms of limit of detection (LOD), limit of quantification (LOQ), selectivity (endogenous from plasma, urine or feces), linearity, extraction recovery, robustness (column selection, mobile phase composition, detection mode, internal standard (IS) selection, analyte stability), day-to-day reproducibility and system suitability (repeatability, peak symmetry and resolution). The method is applicable to bioavailability and pharmacokinetic studies of CP502 in rats.     

A Simple Method of Obtaining Ultrathin-Sections from a Semithin-Section

The Epon-812 semithin-section, which had been observed under light microscope, was stuck to a trimmed resin block with 502 instant glue and was ultrasectioned for electron microscopy. This method, which is simple and tepid, gives a good result and may solve some problems happened in ultrasectioning some special material which needs to be exactly located.     

Calibration of the Minolta SPAD-502 leaf chlorophyll meter

Use of leaf meters to provide an instantaneous assessment of leaf chlorophyll has become common, but calibration of meter output into direct units of leaf chlorophyll concentration has been difficult and an understanding of the relationship between these two parameters has remained elusive. We examined the correlation of soybean (Glycine max) and maize (Zea mays L.) leaf chlorophyll concentration, as measured by organic extraction and spectrophotometric analysis, with output (M) of the Minolta SPAD-502 leaf chlorophyll meter. The relationship is non-linear and can be described by the equation chlorophyll (mol m^–2)=10(^M0.265), r ²=0.94. Use of such an exponential equation is theoretically justified and forces a more appropriate fit to a limited data set than polynomial equations. The exact relationship will vary from meter to meter, but will be similar and can be readily determined by empirical methods. The ability to rapidly determine leaf chlorophyll concentrations by use of the calibration method reported herein should be useful in studies on photosynthesis and crop physiology.Abbreviations Chl- chlorophyll - M- SPAD-502 meter value     

Achievement of complete mycological cure by topical antifungal agent NND-502 in guinea pig model of tinea pedis

We examined the therapeutic effect of a 1% cream preparation of NND-502, a novel topical antifungal agent, in a guinea pig tinea pedis model produced by infecting the plantar skin of guinea pigs with Trichophyton mentagrophytes. Animals developing tinea pedis were divided into two groups: an untreated control group and a treated group. In the latter group, after confirming infection had been established, the infected animals were topically treated with the NND-502 cream once daily for one week. The animals were reared in a clean environment free from exposure to exogenous dermatophytes. At one week (5 weeks post-infection), 6 weeks (10 weeks post-infection) and 16 weeks (20 weeks post-infection) after completion of the treatment, plantar skin samples were taken from a certain number of both groups of animals. The results demonstrated that all of the animals in the untreated control group and none of those in the treated group were culture-positive in this animal model of tinea pedis. The topical treatment with NND-502 achieved a mycological cure. Thus NND-502 can be considered a promising candidate as a new anti-dermatophytic agent for topical use.     

Hsa_circ_0003596 enhances the development of cell renal clear cell carcinoma through the miR-502-5p/IGF1/PI3K/AKT axis

Accumulating research findings have shown that circular RNAs (circRNAs) play an indispensable role in tumorigenesis and tumor progression. The current study aimed to explore the role and modulatory mechanism of hsa_circ_0003596 in clear cell renal cell carcinoma (ccRCC). Quantitative real-time polymerase chain reaction was adopted to detect the expression of hsa_circ_0003596 in ccRCC tissue and cell lines. 5-Ethynyl-2′-deoxyuridine, cell counting kit 8 and the colony formation assay were utilized to assess the proliferation potential of the ccRCC cells. Transwell along with wound healing assays were adopted to quantify infiltration coupled with the migration potential of the cells. The current research study found that the circRNA hsa_circ_0003596 was overexpressed in ccRCC tissue and cell lines. Further, result showed that hsa_circ_0003596 was associated with distant metastasis of renal cancer. Notably, the knockdown of hsa_circ_0003596 can lower the proliferation, infiltration and migration potential of ccRCC cells. The results of in vivo experiments found that the reduction of hsa_circ_0003596 significantly hampered the growth of tumors in mice. In addition, it was evident that hsa_circ_0003596 acts as a “molecular sponge” for miR-502-5p to upregulate the expression of the microRNA-502-5p (miR-502-5p) target insulin-like growth factor 1 (IGF1R). Furthermore, it was found that the phosphatidylinositol 3-kinase (PI3K)/AKT signaling was the downstream cascade of hsa_circ_0003596/miR-502-5p/IGF1R cascade, which is partly responsible for the cancer-promoting effect. Overall, the results of the present study showed that hsa_circ_0003596 facilitated the proliferation, infiltration and migration of ccRCC through the miR-502-5p/IGF1R/PI3K/AKT axis. Therefore, it was evident that hsa_circ_0003596 can serve as a possible biomarker and therapeutic target against ccRCC.     

Comparison of Chlorophyll Meter Readings with Leaf Chlorophyll Concentration in Amaranthus vlitus: Correlation with Physiological Processes1

The portable chlorophyll meter (SPAD-502) has been successfully used for a rapid and direct estimation of total chlorophyll content (TCHL) in the leaves of some crops. In this work, SPAD-502 meter readings and TCHL concentration were compared for the leaves of Amaranthus vlitus L., a common weed. SPAD readings were linearly and positively correlated to TCHL concentration in the leaves. A linear correlation was also shown between SPAD-502 readings and some physiological parameters of the leaves, such as photosynthesis, transpiration, and stomatal conductance.     

Effect of cooling rate on human and murine hemopoietic precursor cell recovery

The effect of cooling rate on recovery of human and murine hemopoietic precursor cells was studied. In the presence of 10% Me₂SO, a cooling rate of 7 °C/min from ?4 to ?30 °C was optimal for recovery of both human and murine precursor cells which give rise to colonies in diffusion chambers implanted in mice (CFU-DG). Cooling of human marrow at a rate between 3 and 7 °C/min resulted in the best CFU-C recovery, although no good correlation between the cooling rate and murine CFU-C recovery was demonstrated. These data suggest that recovery of the primitive hemopoietic precursor cells can be improved by changing the standard cryopreservation programs used presently. However, improved recovery of CFU-DG does not necessarily translate into faster reconstitution of hemopoiesis. No significant difference was observed in overall recovery of bone marrow cellularity in lethally irradiated mice following injection of untreated marrow and marrow cooled at a rate of 1 and 7 °C/min.     

叶绿素计SPAD-502在林业上应用

叶绿素是植物光合作用的色素,传统方法测定叶绿素一般采用分光光度法.本研究采用便携式叶绿素计SPAD-502测定落叶松人工林下4个主要阔叶树种绿色度（SPAD值）的季节变化,并与分光光度法测定的叶绿素含量进行相关性分析.结果表明,SPAD值与叶绿素含量具有显著的相关性,SPAD值能较好地反映树木叶绿素含量的变化.因此,使用叶绿素计测定树木的叶绿素含量是完全可行的,在一定条件下可代替叶绿素含量的直接测定.由于叶绿素计SPAD-502携带方便、测定简便、迅速,且不损坏叶片,应在林业研究中积极推广使用.     

Study on Omentin-1 and miR-502-3p in osteoporotic fracture

Objectives:To explore the expression and correlation of Omentin-1 and miR-502-3p in serum of patients with osteoporotic fracture (OPF).Methods:Sixty OPF patients diagnosed and treated in our hospital from June 2018 to December 2019 were included in group A. Fifty-six osteoporosis patients without fractures were included in group B. Omentin-1 and miR-502-3p levels were detected by enzyme-linked immunosorbent assay (ELISA) and real-time quantitative PCR (qRT-PCR). Their predictive value for diagnostic efficiency was assessed by ROC curve. Spearman’s rank correlation test was used for correlation analysis. The risk factors related to the prognosis of OPF were analyzed by Logistic univariate and multivariate analysis.Results:The expression of Omentin-1 and miR-502-3p in group A was markedly lower than in group B (P<0.001). Spearman correlation analysis showed that in OPF, there was a negative correlation between serum Omentin-1 and TNF-α (r=0.8579, P<0.001), a negative correlation between serum miR-502-3p and TNF-α (r= 0.8653, P<0.001), and a positive correlation between serum Omentin-1 and miR-502-3p (r= 0.8764, P<0.001).Conclusions:Omentin-1 and miR-502-3p were down-regulated in serum of patients with OPF, both of which could be used as potential biomarkers for the diagnosis and disease evaluation of OPF.     

An important role of a pyrethroid-sensing residue F1519 in the action of the N-alkylamide insecticide BTG 502 on the cockroach sodium channel

Deltamethrin, a pyrethroid insecticide, and BTG 502, an alkylamide insecticide, target voltage-gated sodium channels. Deltamethrin binds to a unique receptor site and causes prolonged opening of sodium channels by inhibiting deactivation and inactivation. Previous ²²Na⁺ influx and receptor binding assays using mouse brain synaptoneurosomes showed that BTG 502 antagonized the binding and action of batrachotoxin (BTX), a site 2 sodium channel neurotoxin. However, the effect of BTG 502 has not been examined directly on sodium channels expressed in Xenopus oocytes. In this study, we examined the effect of BTG 502 on wild-type and mutant cockroach sodium channels expressed in Xenopus oocytes. Toxin competition experiments confirmed that BTG 502 antagonizes the action of BTX and possibly shares a common receptor site with BTX. However, unlike BTX which causes persistent activation of sodium channels, BTG 502 reduces the amplitude of peak sodium current. A previous study showed that BTG 502 was more toxic to pyrethroid-resistant house flies possessing a super-kdr (knockdown resistance) mechanism than to pyrethroid-susceptible house flies. However, we found that the cockroach sodium channels carrying the equivalent super-kdr mutations (M918T and L1014F) were not more sensitive to BTG 502 than the wild-type channel. Instead, a kdr mutation, F1519I, which reduces pyrethroid binding, abolished the action of BTG 502. These results provide evidence the actions of alkylamide and pyrethroid insecticides require a common sodium channel residue.