Distribution and induction of cytochrome P450 1A1 in the rainbow trout brain

Cytochrome P450 (CYP) 1A1 participates in the activation as well as detoxification of environmental pollutants such as aromatic hydrocarbons. This CYP form is also efficiently induced by aromatic hydrocarbons. The presence of CYP 1A1 in the brain might thus be of physiological and toxicological importance. In the present investigation on rainbow trout, the distribution of 7-ethoxyresorufin-O-deethylase (EROD) activity, a cytochrome CYP 1A1 catalyzed reaction, was measured in whole tissue homogenates from brain parts. In control fish, a relatively high activity was found in the rainbow trout olfactory bulb compared to the other brain parts. Although an EROD induction (3 to 7-fold) by β-naphthoflavone (BNF) was recorded in all brain parts from the rainbow trout, the highest induced activity was measured in the olfactory bulbs. To ascertain the distribution of EROD activity in cells, whole brain tissue was subfractionated by differential centrifugation. The fractionation scheme separated mitochondria (P2 fraction) and microsomes (P3 fraction) as determined by marker enzymes and electron microscopy. In control rainbow trout, a low EROD activity could be measured in the P2 fraction. BNF induced the EROD activity in both P2 and P3 fractions. Western blotting showed the induction by BNF of a protein band in the P2 and P3 fractions with a molecular mass around 58,000 when highly specific anti-cod CYP 1A1 antibodies were used. ELISA measurements confirmed the induction of CYP 1A1 protein in the rainbow trout brain subcellular fractions.     

Mitochondrial NAD(P)-malic enzyme from herring skeletal muscle

Mitochondrial NAD(P)-dependent malic enzyme [EC 1.1.1.39, L-malate: NAD⁺ oxidoreductase (decarboxylating)] was purified from herring skeletal muscle to a specific activity of 8.2 mol/min/mg. The purification procedure involved chromatography on DEAE-cellulose, Red Agarose and a Sephacryl S-300 with a final recovery of 38% of enzyme activity. This enzyme catalyzes the oxidative decarboxylation of malate in the presence of either NAD or NADP in the presence of Mn²⁺. Some kinetic characteristics of this enzyme were determined. The pH optimum of activity is 7.0. ATP was shown to be a competitive inhibitor with malate. The inhibition by ATP displayed hyperbolic competitive kinetics with a K_i (ATP) of 0.28 mM in the presence of NAD and 0.75 mM in the presence of NADP. Fumarate reversed ATP inhibition.In vivo, regulation of NAD(P)-dependent malic enzyme might respond to changing levels of mitochondrial ATP and fumarate with the enzyme undergoing kinetic activation by an increase in the concentration of mitochondrial fumarate which could reverse enzyme inhibition by ATP.     

Effect of salinity on survival, growth, oxygen consumption and ammonia-N excretion of juvenile whiteleg shrimp, Litopenaeus vannamei

In this study, we tested the lower salinity tolerance of juvenile shrimps (Litopenaeus vannamei) at a relatively low temperature (20 °C). In the first of two laboratory experiments, we first abruptly transferred shrimps (6.91 ± 0.05 g wet weight, mean ± SE) from the rearing salinity (35 000 mg L^?1) to salinities of 5000, 15 000, 25 000, 35 000 (control) and 40 000 mg L^?1 at 20 °C. The survival of L. vannamei juvenile was not affected by salinities from 15 000 to 40 000 mg L^?1 during the 96‐h exposure periods. Shrimps exposed to 5000 mg L^?1 were significantly affected by salinity, with a survival of 12.5% after 96 h. The 24‐, 48‐ and 96‐h lethal salinity for 50% (LS₅₀) were 7020, 8510 and 9540 mg L^?1 respectively. In the second experiment, shrimps (5.47 ± 0.09 g wet weight, mean ± SE) were acclimatized to the different salinity levels (5000, 15 000, 25 000, 35 000 and 40 000 mg L^?1) and then maintained for 30 days at 20 °C. Results showed that the survival was significantly lower at 5000 mg L^?1 than at other salinity levels, but the final wet weight under 5000 mg L^?1 treatment was significantly higher than those under other treatments (P<0.05). Feed intake (FI) of shrimp under 5000 mg L^?1 was significantly lower than those of shrimp under 150 00–40 000 mg L^?1; food conversion efficiency (FCE), however, showed a contrasting change (P<0.05). Furthermore, salinity significantly influenced the oxygen consumption rates, ammonia‐N excretion rates and the O/N ratio of test shrimps (P<0.05). The results obtained in our work provide evidence that L. vannamei juveniles have limited capacity to tolerate salinities <10 000 mg L^?1 at a relatively low temperature (20 °C). Results also show that L. vannamei juvenile can recover from the abrupt salinity change between 15 000 and 40 000 mg L^?1 within 24 h.     

草鱼肝微粒体的提取及CYP酶活性的测定

CYP酶代谢是药物生物转化的主要途径,其数量和活性大小直接影响药物在体内的活化与代谢。我们对草鱼肝微粒体CYP酶含量及其活性进行了初步研究,以差速离心法提取草鱼肝微粒体,以CO还原差示光谱法测得CYP酶及细胞色素b5含量分别为0.619±0.102 nmol/mg、0.264±0.042 nmol/mg。以7-乙氧异吩噁唑酮-O-脱乙基反应、苯胺-4-羟化反应、氨基比林-N-脱甲基反应作为CYP1A、CYP2E、CYP3A的探针反应,测得EROD酶活为0.043±0.004 nmol/mg/min,ANH酶活为0.028±0.002 nmol/mg/min,AMND酶活为0.207±0.035 nmol/mg/m in。结果表明草鱼肝微粒体中CYP酶发育完好,并且具有参与药物代谢的3种主要亚型活性,其含量与活性大小与其它实验动物相差较大。本实验的方法与结果为草鱼CYP酶的系统研究提供可靠手段,最终为指导水产合理用药提供理论依据。     

Identification and characterization of the enzymes responsible for the metabolism of the non‐steroidal anti‐inflammatory drugs,flunixin meglumine and phenylbutazone,in horses

The in vivo metabolism and pharmacokinetics of flunixin meglumine and phenylbutazone have been extensively characterized; however, there are no published reports describing the in vitro metabolism, specifically the enzymes responsible for the biotransformation of these compounds in horses. Due to their widespread use and, therefore, increased potential for drug–drug interactions and widespread differences in drug disposition, this study aims to build on the limited current knowledge regarding P450‐mediated metabolism in horses. Drugs were incubated with equine liver microsomes and a panel of recombinant equine P450s. Incubation of phenylbutazone in microsomes generated oxyphenbutazone and gamma‐hydroxy phenylbutazone. Microsomal incubations with flunixin meglumine generated 5‐OH flunixin, with a kinetic profile suggestive of substrate inhibition. In recombinant P450 assays, equine CYP3A97 was the only enzyme capable of generating oxyphenbutazone while several members of the equine CYP3A family and CYP1A1 were capable of catalyzing the biotransformation of flunixin to 5‐OH flunixin. Flunixin meglumine metabolism by CYP1A1 and CYP3A93 showed a profile characteristic of biphasic kinetics, suggesting two substrate binding sites. The current study identifies specific enzymes responsible for the metabolism of two NSAIDs in horses and provides the basis for future study of drug–drug interactions and identification of reasons for varying pharmacokinetics between horses.     

短期培养下抑制剂烯丙基硫脲对土壤硝化作用及微生物的影响

硝化抑制剂烯丙基硫脲（ATU）对土壤硝化作用及温室效应的影响及机理尚不清楚。本研究采集典型旱地土壤,进行21天室内微宇宙培养,探究了氮肥与不同剂量ATU（分别为氮素用量的1%, 5%, 10%, 15%和20%）配施对土壤硝化作用及N2O和CO2排放通量的影响,并通过实时荧光定量PCR和高通量测序16S rRNA基因技术监测硝化微生物群落变化,同时与传统硝化抑制剂双氰胺（DCD）进行了保氮减排效果的对比。结果表明,与未施加氮肥的对照相比（CK）,单施氮肥（N）显著提高了土壤硝化强度并促进了N2O排放。DCD能显著抑制硝态氮和N2O的积累,抑制效率分别为68.6%和93.3%。而低浓度ATU对土壤硝化作用无影响,仅在高浓度具有抑制效应,且抑制效率最高仅为14.7%。所有ATU处理N2O排放量均显著降低,降幅为60.3～68.2%,仍远高于DCD处理。处理间N2O和CO2的综合温室效应强弱顺序为N>ATU+N>DCD+N≈CK,且不同ATU施用量处理之间差异不显著。相关分析发现氨氧化细菌（AOB）,而不是氨氧化古菌（AOA）和全程氨氧化细菌（Comammox）,与土壤硝态氮积累和N2O排放显著正相关,与土壤pH显著负相关。高通量测序结果表明Nitrosovibrio tenuis类型AOB对氮肥诱导的硝化过程起主导作用。除此之外,ATU和DCD还能显著提高Cupriavidus,并降低Patulibacter、Aeromicrobium、Actinomycetospora、Defluviicoccus和Acidipila等微生物属在群落中的相对丰度。该研究为深化土壤碳氮循环理论,合理使用硝化抑制剂以及减缓温室气体排放提供科学依据。     

家蚕细胞色素P450基因CYP9A22的克隆及在中肠和脂肪体的诱导转录

细胞色素P450对昆虫的生长发育、环境适应性以及抗药性具有重要作用。为了探讨氯氰菊酯对家蚕(Bombyxmori)细胞色素P450基因转录的诱导作用,采用RT-PCR技术和cDNA末端快速扩增(RACE)策略,从家蚕5龄幼虫克隆到细胞色素P450基因CYP9A22(GenBank登录号:EF535804)。CYP9A22基因的cDNA编码区长1 596 bp,编码531个氨基酸,推定的蛋白质分子质量为61 kD,等电点为7.71。将该基因的cDNA和家蚕基因组序列比对,有9个内含子;与已知的CYP9家族的棉铃虫(Helicoverpa armigera)CYP9A14基因的相应氨基酸同源性达到62.3%。用半定量RT-PCR方法分析氯氰菊酯诱导家蚕CYP9A22表达水平,结果表明:CYP9A22的mRNA转录表达具有组织特异性,在中肠的表达量高于脂肪体;氯氰菊酯诱导家蚕24 h,在其脂肪体的表达量是未诱导家蚕脂肪体的3.1倍,初步推测CYP9A22的过量表达可能与抗药性有一定关系。     

氮磷钾肥施用量对稻米直链淀粉含量和淀粉粘滞特性的影响

通过盆栽和大田试验,研究了N、P、K肥对稻米直链淀粉含量和淀粉粘滞特性的影响。结果表明,N肥施用量增加,直链淀粉含量、最高粘度和崩解值下降,消碱值和回复值上升。增施P肥对直链淀粉含量影响不明显,对最高粘度、崩解值、消碱值和回复值影响效用,供试品种间变化趋势存在差异。K肥施用量增加,各品种均表现出直链淀粉含量、最高粘度、崩解值上升趋势,消碱值和回复值呈下降趋势。对NPK肥,不同品种反应程度不同,品种与N、P、K之间,N、P、K三者之间都存在互作效用。减少N肥用量,适量增施K肥有利于稻米食味品质的提高。     

茶树叶片荧光性绿斑病的初步研究

采用田间调查、病原生物培养、病理解剖和电子探针多种方法,对茶树叶片荧光性绿斑病的症状、发生和田间分布特点、病因进行了初步研究。结果表明:该病害典型症状表现为叶片下表皮局部凸起、呈绿色、且能发射绿色荧光;其凸起增厚是由于叶片海绵细胞病变、体积增大、细胞之间相互挤压所致;病害的发生与茶树生长势和营养供应水平密切相关;田间分布具分散性和不均衡性,无明显的发病中心;病理解剖和微生物培养没有发现病原生物,但可见细胞膜结构的破坏、细胞质中多泡体的形成以及多种异常细胞,而且病叶海绵细胞中含有较多的草酸钙晶体。综合以上结果可以初步判定:该病害属于生理性病害。