Epoxyeicosatrienoic acid: the signal transduction

Cytochrome P450 monoxygenase converts arachionic acid to four epoxyeicosatrienoic acid regiosomes: 5, 6-EET (epoxyeicosatrienoic acid); 8, 9-EET; 11, 12-EET and 14, 15-EET. Recent studies show that EETs are involved in signal transduction. EETs open Ca²⁺-sensit ive K⁺ channel and inhibit Na⁺ channel, Ca²⁺-sensitive Cl^- channel and so on. What is more, EETs have been demonstrated to activate PP60^c-src and initiate a tyrosine kinase cascade that mediates mitogenic effects.     

Synergistic and antagonistic effects of atrazine on the toxicity of organophosphorodithioate and organophosphorothioate insecticides to Chironomus tentans (Diptera: Chironomidae)

The acute toxicities of two organophosphorodithioate (dimethoate and disulfoton) and two organophosphorothioate (omethoate and demeton-S-methyl) insecticides were evaluated individually and in binary combination with the herbicide atrazine using fourth-instar larvae of the aquatic midge, Chironomus tentans. Atrazine alone up to 1000 μg/L did not show significant toxicity to the midges in a 48-h bioassay. However, atrazine concentrations as low as 1 μg/L in combination with dimethoate at EC₂₅ (concentration to affect 25% of tested midges), 100 μg/L in combination with disulfoton (EC₂₅), and 10 μg/L in combination with demeton-S-methyl (EC₂₅) significantly enhanced the toxicity of each organophosphate insecticide. In contrast, atrazine concentrations of 10 μg/L and above in combination with omethoate (EC₂₅) significantly decreased the toxicity of the insecticide. Biochemical analysis indicated that increased toxicity of dimethoate, disulfoton, and demeton-S-methyl in binary combination with atrazine correlated to the increased inhibition of acetylcholinesterase. Furthermore, cytochrome P450-dependent O-deethylation activity in the midges exposed to atrazine at 1000 μg/L was 1.5-fold higher than that in the control midges. Thus, atrazine appeared to induce cytochrome P450 monooxygenases in the midges. Elevated cytochrome P450 monooxygenase activity may increase the toxicities of dimethoate, disulfoton, and demeton-S-methyl by enhancing the oxidative activation of dimethoate into omethoate, and disulfoton and demeton-S-methyl into their sulfoxide analogs with increased anticholinesterase activity. In contrast, atrazine reduced the toxicity of omethoate possibly by enhancing the oxidative metabolic detoxification since omethoate does not require oxidative activation.     

Familial Congenital Methemoglobinemia in Pomeranian Dogs Caused by a Missense Variant in the NADH‐Cytochrome B5 Reductase Gene

Background

In veterinary medicine, congenital methemoglobinemia associated with nicotinamide adenine dinucleotide (NADH)‐cytochrome b5 reductase (b5R) deficiency is rare. It has been reported in several breeds of dogs, but little information is available about its etiology.

Objectives

To analyze the NADH‐cytochrome b5 reductase gene, CYB5R3, in a Pomeranian dog family with methemoglobinemia suspected to be caused by congenital b5R deficiency.

Animals

Three Pomeranian dogs from a family with methemoglobinemia were analyzed. Five healthy beagles and 5 nonrelated Pomeranian dogs without methemoglobinemia were used as controls.

Methods

Methemoglobin concentration, b5R activity, and reduced glutathione (GSH) concentration were measured, and a turbidity index was used to evaluate Heinz body formation. The CYB5R3 genes of the affected dog and healthy dogs were analyzed by direct sequencing.

Results

Methemoglobin concentrations in erythrocytes of the affected dogs were remarkably higher than those of the control dogs. The b5R activity of the affected dogs was notably lower than that of the control dogs. DNA sequencing indicated that this Pomeranian family carried a CYB5R3 gene missense variant (ATC→CTC at codon 194) that resulted in the replacement of isoleucine (Ile) by leucine (Leu).

Conclusions and Clinical Importance

This dog family had familial congenital methemoglobinemia caused by b5R deficiency, which resulted from a nonsynonymous variant in the CYB5R3 gene. This variation (c.580A>C) led to an amino acid substitution (p.Ile194Leu), and Ile194 was located in the proximal region of the NADH‐binding motif. Our data suggested that this variant in the canine CYB5R3 gene would affect function of the b5R in erythrocytes.     

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.     

Molecular characterization of Ascaridia galli from Bangladesh and development of a PCR method for distinguishing A. galli from Heterakis spp.

We analyzed the nuclear ribosomal internal transcribed spacer (ITS) 1 and ITS2 sequences for Bangladesh isolates of Ascaridia galli, and we determined that the sequences were unreliable as molecular markers for distinguishing A. galli from other Ascaridia species, because the sequences showed high identity with that of A. columbae. However, the ITS1 sequences were available for designing PCR primers distinguishable between Ascaridia galli and Heterakis spp. Bangladesh isolates of A. galli constituted a monophyletic clade along with other geographical isolates in the cytochrome c oxidase subunit I (COI) phylogenetic tree, however, we could not clarify the phylogenetic relationships between A. galli and other Ascaridia spp., because their available sequences in GenBank were very few. The developed PCR method using DNA from A. galli and Heterakis spp. eggs would enable differential diagnosis of the individual infections in the future.     

利用shRNA真核表达载体干涉雌性鸡胚性腺中芳香化酶基因(CYP19A1)的表达

本研究通过把构建的短发夹结构RNA(shRNA)真核表达载体导入鸡胚,检测鸡胚性腺分化期质粒载体在鸡胚体内代谢情况及雌性鸡胚性腺中芳香化酶基因(CYP19A1)mRNA表达效率,进而探讨利用该方法在鸡胚体内进行特定基因干涉的可行性.实验针对CYP19A1基因构建了4条特异性表达载体,一条非特异性表达载体.每个实验组选取45个新鲜种蛋作为实验材料进行胚盘下腔注射,并设立空白对照组.鸡胚发育12d时,检测各处理组鸡胚肝脏组织中质粒存在情况,并取其左侧性腺组织进行目标基因的荧光定量分析.研究结果表明,导入组在12日胚龄时所有鸡胚基因组中均可检测到绿色荧光蛋白基因(EGFP);荧光定量结果显示,导入特异性表达载体cyp-580、cyp-1083和cyp-1295后,对应雌性鸡胚性腺CYP19A1 mRNA表达效率显著低于空白对照组,干涉效率分别为:73％、52％和85％;特异性表达载体cyp-1403组CYP19A1mRNA表达效率与空白对照组相比有所降低但无显著性差异.本实验为诱导鸡胚性反转提供了新方法并建立了鸡胚发育期特定基因体内干涉新平台.     

蚯蚓（Eiseniafetida）细胞色素P450及抗氧化酶系对环境浓度苯并（a）芘的响应

以赤子爱胜蚓（Eiseniafetida）为供试生物,草甸棕壤为供试土壤,以蚯蚓微粒体细胞色素P450含量、抗氧化酶系以及谷胱甘肽转移酶活性为指标,进行了土壤中苯并（a）芘[B（a）P]暴露与酶活性的量-效关系研究。结果表明,在接近沈抚灌区实地污染状况B（a）P（0．1～2．0mg·kg-1）暴露下,第1、3、7以及第14d取样时,P450和SOD有较好的响应：SOD在第1、3d显著升高,而在第7、14d时降低;P450总体表现为低浓度B（a）P诱导下降低,高浓度下升高的趋势。CAT、POD以及GST的敏感性相对较差。比照其他4个指标,蚯蚓P450的敏感性更为优异,具有较好的应用前景。指标敏感性总体表现为：P450〉SOD〉CAT／POD〉GST。     

Short-term population dynamics of ammonia oxidizing bacteria in an agricultural soil

Ammonia oxidizing bacteria (AOB) control the rate limiting step of nitrification, the conversion of ammonia (NH₄⁺) to nitrite (NO₂⁻). The AOB therefore have an important role to play in regulating soil nitrogen cycling. Tillage aerates the soil, stimulating rapid changes in soil N cycling and microbial communities. Here we report results of a study of the short term responses of AOB and net nitrification to simulated tillage and NH₄⁺ addition to soil. The intensively farmed vegetable soils of the Salinas Valley, California, provide the context for this study. These soils are cultivated frequently, receive large N fertilizer inputs and there are regional concerns about groundwater N concentrations. An understanding of N dynamics in these systems is therefore important. AOB population sizes were quantified using a real-time PCR approach. In a 15 day experiment AOB populations, increased rapidly following tillage and NH₄⁺ addition and persisted after the depletion of soil NH₄⁺. AOB population sizes increased to a similar degree, over a 1.5-day period, irrespective of the amount of NH₄⁺ supplied. These data suggest selection of an AOB community in this intensively farmed and C-limited soil, that rapidly uses NH₄⁺ that becomes available. These data also suggest that mineralization may play an especially important role in regulating AOB populations where NH₄⁺ pool sizes are very low. Methodological considerations in the study of soil AOB communities are also discussed.     

土壤粒度和磁化率特征对绰墩古水稻土成因的指示

本文研究了绰墩农业遗址P-02原状土剖面的粒度分布,定量分析了土壤黏粒的矿物组成,测定了土壤/沉积物及其各粒级组分(黏粒、粉砂粒、细砂粒和粗砂粒)的磁化率。结果表明：在0 ~ 30 cm和134 ~ 154 cm两层段,黏粒含量从下向上有降低的趋势,这可能预示着长期的水耕作业造成黏粒从表层向亚表层移动;在148 ~ 200 cm层段,土壤磁化率从下向上有降低的趋势,可能说明该处存在长期淹水的状况。总之,黏粒含量和质量磁化率数据说明长江下游地区的先民在马家浜文化时期已开始采用“水溽”方式进行水稻的栽培。