奥利亚罗非鱼DMO蛋白结构和功能预测的研究

目的:对奥利亚罗非鱼DNO蛋白的结构和功能进行预测研究.方法:利用生物信息学的方法对DMO基因推导的氨基酸序列进行结构特征和功能域预测分析,探讨了DMO的信号肽、亲/疏水性、跨膜拓扑结构、卷曲螺旋结构、基序、功能域及高级结构.结果:DMO在第1～17,39～113,153～301.372～410区段为高亲水性区域,属亲水性基团,具有两个螺旋卷曲区域,97～112,155～168氨基酸区域,没有信号肽,含有两个跨膜结构域,发生跨膜运动;DMO包含两个保守的DM-domain和DNA锌指结构等功能结构域;DNO含有1个亮氨酸拉链结构(21～42),7个蛋白激酶C磷酸化位点(49～51,184～186,191～193,235～237,270～272,294～296,333～335),5个酪蛋白激酶Ⅱ磷酸化位点(189～192,225～228,227～230,231～234,233～236).1个cAMP-andcGMP-依赖蛋白激酶磷酸化位点(296～299)10个肉豆蔻酰化位点(59～64,121～126,125～130,129～134,135～140,301～306,304～309,307～312,310～315,350～355);DMO蛋白含有27.38%的α-螺旋,8.8%的β-转角和53.79%的无规卷曲;DMO的高级结构中含有两个α-螺旋区域.结论:DMO蛋白与性别调控有关,并在细胞信号传导中发挥作用,且其生物活性可能接受信号途径中多种信号的调控,这为进一步研究奥利亚罗非鱼DMO基因编码蛋白的功能,明确DMO基因与性别调控的关系奠定了基础.     

Crystal Structure of Dicamba Monooxygenase: A Rieske Nonheme Oxygenase that Catalyzes Oxidative Demethylation

Dicamba (3,6-dichloro-2-methoxybenzoic acid) is a widely used herbicide that is efficiently degraded by soil microbes. These microbes use a novel Rieske nonheme oxygenase, dicamba monooxygenase (DMO), to catalyze the oxidative demethylation of dicamba to 3,6-dichlorosalicylic acid (DCSA) and formaldehyde. We have determined the crystal structures of DMO in the free state, bound to its substrate dicamba, and bound to the product DCSA at 2.10-1.75 Å resolution. The structures show that the DMO active site uses a combination of extensive hydrogen bonding and steric interactions to correctly orient chlorinated, ortho-substituted benzoic-acid-like substrates for catalysis. Unlike other Rieske aromatic oxygenases, DMO oxygenates the exocyclic methyl group, rather than the aromatic ring, of its substrate. This first crystal structure of a Rieske demethylase shows that the Rieske oxygenase structural scaffold can be co-opted to perform varied types of reactions on xenobiotic substrates.     

Accumulation of weak bases in relation to intralysosomal pH in cultured human skin fibroblasts

The volume of the lysosomal compartment in cultured human skin fibroblasts was estimated from the distribution between the cells and the medium of tracer amounts of labelled methylamine and chloroquine, which accumulate in the lysosomes, 2,2-dimethyloxazolidine-2,4-dione, which accumulates in the soluble cytoplasmic compartment relative to the lysosomes, and sucrose, which is excluded by the cells. In a foetal fibroblast line, the fractional volume of the lysosomal compartment was $\text{0.044 ± 0.007}$ ($\text{n}\text{= 8}$). In fibroblasts from a patient with the I-cell disease, the fractional volume was 0.15.The fractional volume of the lysosomal compartment was used to calculate the intralysosomal pH from the accumulation of the weak bases in the cells. The mean value obtained was $\text{5.29 ± 0.04}$ ($\text{n}\text{= 8}$).In fibroblasts incubated with various concentrations of chloroquine, the fractional volume of the lysosomal compartment and the accumulation of chloroquine in the cells were used to calculate the concentration of chloroquine in the lysosomes. The intralysosomal concentration increased from 3 to 114 mM as the extracellular concentration increased from 1 to 100 μM. Concomitantly, the intralysosomal pH increased from 5.3 in the absence of chloroquine to 5.9 in the presence of 100 μM chloroquine. A similar increase in intralysosomal pH could be calculated in fibroblasts incubated with different concentrations of ammonia.     

Unscheduled DNA synthesis in spermatogenic cells of mice treated in vivo with the indirect alkylating agents cyclophosphamide and mitomen

Cyclophosphamide (CPA) and mitomen (DMO) are chemical mutagens that require metabolic activation to produce their biological effect. We have used an in vivo UDS assay in various meiotic and postmeiotic germ-cell stages of male mice to study DNA repair after treatment with these chemicals. EMS, a compound requiring no metabolic activation, was also used for comparative purposes.CPA and DMO induced UDS in meiotic through early-to-midspermatid stages, but no UDS was detected in late spermatids and mature sperm. While EMS produced a maximum UDS response in the germ cells immediately after treatment, CPA and DMO did not produce a maximum response until ～0.5 to 1 h after injection. This delay is attributed to the time required for CPA and DMO to be enzymatically vonverted active alkylating metabolites.Unlike the results found with EMS, mutation frequencies (dominant lethals, translocations, specific-locus mutations) following CPA treatment are not noticeably reduced in germ-cell stages in which UDS occurred. In the case of DMO, mutations are induced only in mature spermatozoa, and these germ-cell stages represent only a fraction of those in which no UDS is detected. The results with CPA and DMO thus still leave unclear the relationship between DNA repair and the differential spermatogenic response of mice to genetic damage.     

奥利亚罗非鱼DMO和DMT蛋白二级结构和B细胞抗原表位的预测

以DMO和DMT氨基酸序列为基础,采用Garnier-Robson法、Chou-Fasman法和Karplus-Schulz法预测蛋白质的二级结构;按Kyte-Doolittle法、Emini法和Jameson-Wolf法预测DMO和DMT蛋白的B细胞抗原表位。预测结果表明：在DMO蛋白N-端第80～112,144～147,193～194,251～255,260～269区段和279～283区段,DMT蛋白N－端61～86,98～105,140～146,239～241区段和第269～273区段,可能是α-螺旋中心;DMO蛋白N-端第59～61,69～70,148～150区段和383～390区段,DMT蛋白的N-端第125～129,207～213,255～264区段和第281～284区段,可能是β-折叠中心;在DMO蛋白分子N-端40～41, 44～45,50～51,128～129,189～192,204～207,216～222,226～233,244～246,298～299区段和第323～326区段和DMT蛋白分子N-端第12～13,26～27,43～44,58～60,93～95,115～120,136～139区段和第149～151区段具有较柔软的结构,这些区段有可能进行一定幅度的摆动或折叠而形成较复杂的三级结构。DMO蛋白N-端第1～5,41～51,65～67,86～89,98～110,154～170,183～203,205～248,258～264,284～291,293～298,270～375,389～392,402～410区域和DMT蛋白N-端第1～9,17～28,77～84,114～123,131～139,157～184,196～207区域可能是B细胞表位优势区域。以蛋白质的二级结构预测作为辅助手段,用抗原指数,亲水性参数和可及性参数预测DMO和DMT蛋白的B细胞表位,为DMO和DMT蛋白单克隆抗体的制备提供了线索,为系统研究奥利亚罗非鱼DMO和DMT基因的性别调控机理研究提供参考。     

An estimation of the light-induced electrochemical potential difference of protons across the membrane of Halobacterium halobium

The light-dependent uptake of triphenylmethylphosphonium (TPMP⁺) and of 5,5-dimethyloxazolidine-2,4-dione (DMO) by starved purple cells of Halobacterium halobium was investigated. DMO uptake was used to calculate the pH difference (ΔpH) across the membrane, and TPMP⁺ was used as an index of the electrical potential difference, Δψ.Under most conditions, both in the light and in the dark, the cells are more alkaline than the medium. In the light at pH 6.6, ΔpH amounts to 0.6–0.8 pH unit. Its value can be increased to 1.5–2.0 by either incubating the cells with TPMP⁺ (10^?3 M) or at low external pH (5.5). — ΔpH can be lowered by uncoupler or by nigericin. The TPMP⁺ uptake by the cells indicates a large Δψ across the membrane, negative inside. It was estimated that in the light, at pH 6.6, Δψ might reach a value of about 100 mV and that consequently the electrical equivalent of the proton electrochemical potential difference, , amounts under these conditions to about 140 mV.The effects of different ionophores on the light-driven proton extrusion by the cells were in agreement with the effects of these compounds on — ΔpH.