光系统II反应中心D1/D2/Cyt b559复合物的光谱特性及其光破坏作用的研究

采用柱层析法从菠菜叶绿体中分离纯化得到高等植物光系统Ⅱ(PSⅡ)反应中心色素蛋白复合体Dl/D2/Cyt b559，并对其性质，特别是光破坏作用的分子机理进行了研究。主要结果如下： 1、PSⅡ反应中心复合物所含的色素比大约为Chla／2 Pheo a=6.0。其四阶导数光谱在红区有两个峰，表明该反应中心至少存在两种结合状态的Chla。 2、Dl/D2/Cyt b559复合物的荧光相对产率及发射光谱的谱带位置与样品的浓度直接相关。只有当样品的浓度达到足够稀的程度(Chla和Pheo a总浓度小于1μg／ml)，才能得到较真实的荧光光谱，其峰位在681nm处。 3、Dl/D2/Cyt b559复合物的CD光谱在红区（Qy带）有一对反向谱带，正蜂为680nm,负峰为660nm，而在β-胡萝卜素的吸收区没有明显的CD信号。当该反应中心复合物受光破坏后，CD信号明显下降，而且当正峰完全消失后，负峰仍然存在，说明负峰不仅包含P680 的信号，也包含其它色素分子的信号，很可能有部分来源于Pheo a。 4、Dl/D2/Cyt b559复合物在488nm处激发的共振拉曼光谱显示四个主要谱带，其峰位分别在1532(ν1)、1165(ν2)、1010(ν3)和970cm-1（ν4）处，表明PSⅡ反应中心结合的B-胡萝卜素分子是全反式构型。Dl/D2/Cyt b559复合物的色素抽提液的拉曼光谱也显示四个主要的拉曼峰，其中ν4谱带的强度急剧下降，说明PSⅡ反应中心内部结合的β-胡萝卜素分子与抽提液中自由的β-胡萝卜素分子的构象不同，而与光合细菌反应中心内部的类胡萝卜素分子的构象相似，其共轭多烯链的平面也处于扭曲状态。 5、光照使PSⅡ反应中心的原初电子供体P680受到破坏，在光照后的暗放置过程中P680分子继续受到破坏，表明在光照过程中很可能有一个相对稳定的反应中间体产生，以至于光照后暗放置过程中Dl/D2/Cyt b559复合物的光谱特性继续发生变化。也就是说，PSⅡ反应中心Dl/D2/Cyt b559复合物的光破坏不是一步反应，而是一个多步反应或多条途径。 6、光照使Dl/D2/Cyt b559复合物中的组氨酸(His)残基受到很大程度的破坏，甲硫氨酸(Met）残基的含量也略有下降，而其它氨基酸的含量基本保持不变。His残基的破坏很可能与光照后暗放置过程中Dl/D2/Cyt b559复合物的光谱特性变化相关。我们认为His残基的光照破坏很可能是Dl/D2/Cyt b559复合物受光照破坏的另一分子机理。 7、人工电子受体癸基质体醌(DPQ)可以与Dl/D2/Cyt b559复合物进行重组。Dl/D2/Cyt b559复合物的荧光衰减分析表明，在DPQ重组之后，两个长寿命荧光组分（24ns和73ns）的寿命减小，而且占整个荧光的分数也下降，表明这两个长寿命荧光衰减组分均来源于电荷重组过程。同时，β-胡萝卜素分子在DPQ重组之后更易于被光照破坏，这个过程可能与β-胡萝卜素分子的生理功能相关。 8、在没有外加人工电子受体的情况下，光照使DDl/D2/Cyt b559 复合物的多肽组成发生一定变化。SDS-PAGE图谱中出现一个约40KDa的新谱带，同时Dl与D2多肽的表观分子量增加，谱带染色强度下降。 9、本文根据以上实验结果，着重对Dl/D2/Cyt b559复合物光破坏的分子机理进行了分析和讨论，并在D1蛋白裂解的两种可能途经中又增加了一个新的可能导致Dl蛋白裂解的途径，即：His残基的光照破坏可以作为Dl/D2/Cyt b559复合物光破坏及Dl蛋白裂解的又一分子机理，这为深入研究PSⅡ反应中心的光破坏提供了新的线索，也为今后研究活体内光抑制现象的分子机制打下了良好的基础

细胞分裂素基因（T-cyt）对转基因植物生长发育的调控

高等植物基因表达过程中的信号传导是目前植物分子生物学研究的前沿和热点之一。不少研究者已将脱落酸、乙烯、细胞分裂素及其它植物激素的作用一起归之于植物基因表达的信号传导系统。细胞分裂素作为—类重要的植物激素在植物的生长和发育过程中起重要的调节控制作用。因此研究细胞分裂素的基因与植物发育过程的关系是十分重要的。 为研究细胞分裂素对植物基因表达的调节，本文从转录和翻译水平上测定了黄瓜子叶在外源细胞分裂素诱导下微管蛋白基因表达的活性。发现经BAP处理的黄瓜子叶中α,β-tubulin mRNA迅速积累，微管蛋白的含量迅速增加。这表明外源细胞分裂素在诱导黄瓜子叶膨大的过程中激活了微管蛋白基因的表达。 为探索不同启动子驱动下的细胞分裂素基因转入植物后的表达对转基因植物生长发育的调控，本文将来自根癌农杆菌的细胞分裂素基因(T-cyt)分别置于CaMV 35S启动子，rbc S启动子和T-cyt基因自身启动子的调控下，构建了嵌合表达质粒，分别转化烟草和马铃薯。转基因烟草和马铃薯的PCR检测和Southern杂交鉴定均证实T-cyt基因已分别整合进烟草和马铃薯的核基因组中。标志基因NPTⅡ的酶活性测定表明有外源基因的表达。转基因烟草的Northern分析表明：CaMV 35s启动子驱动的T-cyt基因的mRNA在叶、茎和根中均有表达;rbc S启动子指导的T-cyt基因在叶中表达最强，茎中较弱，在根中几乎没有表达。转细胞分裂素基因的烟草在生长发育上与未转化的对照相比有明显不同。转基因烟草中叶绿素a，b含量明显增加，叶面积减小，叶衰老迟缓。T-cyt基因转化的烟草顶端优势受到抑制，侧芽生长旺盛;与对照相比，其节间短，株高降低，根生长受抑制。 本文还构建了T-cyt基因自身启动子与报告基因GUS编码区的嵌合表达质粒，转化烟草和马铃薯以研究T-cyt启动子在植物中的表达。组织化学定位测定表明，T-cyt启动子在植物的茎，叶中的表达较强，特别是在腋芽的生长点有很高的表达活性，但在根中的表达较弱。诱导性表达试验表明，T-cyt启动子的表达强度受细胞分裂素的诱导，而生长素对T-cyt启动子的表达无明显影响。这提示T-cyt启动子是一个细胞分裂素诱导性表达的启动子。 总之，将T-cyt基因转入植物，作为调节内源细胞分裂素的一种手段，可以对植物的生长发育进行调控。尤其是利用发育阶段特异性和各种器官特异性表达的启动子可以调节T-cyt基因的表达活性，有可能创造出具有经济价值的、具有新遗传特性的植物。

Cyt b-559 链Arg18和Ser24的定点突变及其对PSII功能的影响

Cyt b-559是光系统II反应中心的成分之一，它由亚基和亚基组成的。在Cyt b-559中，血红素辅基与两个亚基中的组氨酸连接成有功能的蛋白，并维持PSII的功能稳定性。前人曾将与血红素相连的His突变，导致Cyt b-559功能和PSII稳定性的丧失。基于此研究，本文采用定点突变技术，将亚基中与His23位置最近的上游氨基酸Arg18分别用Gly和Glu取代，下游氨基酸Ser24用Phe取代，获得了衣藻Cyt b-559的突变体。对突变体的分析，有以下新结果：突变体都能进行光合自养，但无论在异养培养基上还是自养培养基上，和对照相比，其生长速度非常缓慢; PSII的活性分析，表明PSII的放氧活性为野生衣藻细胞的50%～80%， Fv/Fm 的荧光参数为40%～70%;对突变体进行强光（1000μE•m-2•s-1）照射，10min后，其放氧活性都降低为0，而野生型衣藻还保持35%的活性;提取类囊体膜蛋白，进行SDS-PAGE电泳和Western-blotting分析，显示突变体的膜蛋白与对照无显著差异。这些结果说明对围绕血红素环境的固有氨基酸的改变，虽然并没有明显影响类囊体膜蛋白的表达和组成，但是却影响了衣藻细胞的生长和PSII的活性，增加了衣藻细胞对强光的敏感性，降低了衣藻细胞自身的光保护能力。这说明靠近血红素配位环境的氨基酸Arg和Ser，尤其是Arg，对Cyt b-559的功能维持不可缺少，对于维持PSII的活性也很重要。

从线粒体Cyt b基因探讨红瘰疣螈物种地位的有效性

疣螈属的红瘰疣螈(Tylototriton shanjing)和棕黑疣螈(T.verrucosus)的物种界限一直不清楚.测定了来自中国西南地区14个地点的T.shanjing和T.verrucosus共40只标本的线粒体DNA Cyt b基因(753 bp).结果表明:(1)用邻接法、最大简约法和贝叶斯法等3种系统发育分析法分别重建棕黑疣螈种组系统发育树的拓扑结构不支持T.shanjing是单系群;(2)T.shanjing与T.verrucsus的mtDNA Cyt b序列差异平均值仅为1.2%.未达到种级水平.因此,全部T.shanjing样品都属于同一个物种,即T.verrucosus,不支持T.shanjing的物种地位,T.shanjing为T.verrucosus的同物异名,并建议恢复T.verrucosus的中文名红瘰疣螈.根据基于40个样品Cvt b基因序列的系统发育树和遗传变异以及地理分布,这些红瘰疣螈(T.verrucosus)样品聚为3支,即中国西南地区的红瘰疣螈可分为片马、滇中滇西和滇东南3个地理居群.

黄河裸裂尻鱼群体遗传结构和Cyt b序列变异

测定了来自黄河上游和柴达木盆地托索湖的裸裂尻鱼共16个个体的Cytb基因全序列(1141bp),探讨了种群结构和遗传多样性。用MEGA2.1软件分析了碱基组成和序列变异;以青海湖裸鲤、花斑裸鲤和极边扁咽齿鱼为外类群,用PAUP*4.0b10程序构建了单倍型NJ树;用Arlequin Ver.2000程序计算了群体间遗传变异值(Fst)和Nm值以及群体分化概率值。结果显示,来自柴达木水系托索湖的裸裂尻鱼没有形成单系群,Fst=0.204(P<0.05),Nm=1.95。初步判断,黄河和柴达木水系托索湖的裸裂

The direct electrochemistry behavior of Cyt c on the modified glassy carbon electrode by SBA-15 with a high-redox potential

It is discovered that SBA-15 (santa barbara amorphous) can provide the favorable microenvironments and optimal direct electron-transfer tunnels (DETT) of immobilizing cytochrome c (Cyt c) by the preferred orientation on it. A high-redox potential (254 mV vs. Ag/AgCl) was obtained on glassy carbon (GC) electrode modified by immobilizing Cyt c on rod-like SBA-15. With ultraviolet-visible (UV-vis), circular dichroism (CD), FTIR and cyclic voltammetry, it was demonstrated that immobilization made Cyt c exhibits stable and ideal electrochemical characteristics while the biological activity of immobilized Cyt c is retained as usual.

Phylogeographical, ecological and biological patterns shown by nuclear (ssrRNA and gGAPDH) and mitochondrial (Cyt b) genes of trypanosomes of the subgenus Schizotrypanum parasitic in Brazilian bats

The genetic diversity and phylogeographical patterns of Trypanosoma species that infect Brazilian bats were evaluated by examining 1043 bats from 63 species of seven families captured in Amazonia, the Pantanal, Cerrado and the Atlantic Forest biomes of Brazil. The prevalence of trypanosonne-infected bats, as estimated by haemoculture, was 12.9%, resulting in 77 Cultures of isolates, most morphologically identified as Trypanosoma cf. cruzi, classified by barcoding using partial sequences from ssrRNA gene into the subgenus Schizotrypanum and identified as T. cruzi (15), T cruzi marinkellei (37) or T. cf. dionisii (25). Phylogenetic analyses using nuclear ssrRNA, glycosomal glyceraldehyde 3-phosphate dehydrogenase (gGAPDH) and mitochondrial cytochrome b (Cyt b) gene sequences generated three clades, which clustered together forming the subgenus Schizotrypanum. In addition to vector association, bat trypanosomes were related by the evolutionary history, ecology and phylogeography of the bats. Tryponosoma cf. dionisii trypanosomes (32.4%) infected 12 species from four bat families captured in all biomes, from North to South Brazil, and clustered with T. dionisii from Europe despite being separated by some genetic distance. Trypanosoma cruzi marinkellei (49.3%) was restricted to phyllostomid bats from Amazonia to the Pantanal (North to Central). Trypanosoma cruzi (18.2%) was found mainly in vespertilionid and phyllostomid bats from the Pantanal/Cerrado and the Atlantic Forest (Central to Southeast), with a few isolates from Amazonia. (C) 2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Relações filogenéticas das famílias Coreidae e Pentatomidae (Heteroptera: Pentatomomorpha) baseadas nas sequências de genes mitocondriais (Cyt b, COI e 16S), nucleares (28S e 18S) e informações morfológicas

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Influence of historical landscapes, drainage evolution and ecological traits on patterns of genetic diversity in Southeast Asian freshwater snakehead fishes

Snakehead fishes in the family Channidae are obligate freshwater fishes represented by two extant genera, the African Parachannna and the Asian Channa. These species prefer still or slow flowing water bodies, where they are top predators that exercise high levels of parental care, have the ability to breathe air, can tolerate poor water quality, and interestingly, can aestivate or traverse terrestrial habitat in response to seasonal changes in freshwater habitat availability. These attributes suggest that snakehead fishes may possess high dispersal potential, irrespective of the terrestrial barriers that would otherwise constrain the distribution of most freshwater fishes. A number of biogeographical hypotheses have been developed to account for the modern distributions of snakehead fishes across two continents, including ancient vicariance during Gondwanan break-up, or recent colonisation tracking the formation of suitable climatic conditions. Taxonomic uncertainty also surrounds some members of the Channa genus, as geographical distributions for some taxa across southern and Southeast (SE) Asia are very large, and in one case is highly disjunct. The current study adopted a molecular genetics approach to gain an understanding of the evolution of this group of fishes, and in particular how the phylogeography of two Asian species may have been influenced by contemporary versus historical levels of dispersal and vicariance. First, a molecular phylogeny was constructed based on multiple DNA loci and calibrated with fossil evidence to provide a dated chronology of divergence events among extant species, and also within species with widespread geographical distributions. The data provide strong evidence that trans-continental distribution of the Channidae arose as a result of dispersal out of Asia and into Africa in the mid–Eocene. Among Asian Channa, deep divergence among lineages indicates that the Oligocene-Miocene boundary was a time of significant species radiation, potentially associated with historical changes in climate and drainage geomorphology. Mid-Miocene divergence among lineages suggests that a taxonomic revision is warranted for two taxa. Deep intra-specific divergence (~8Mya) was also detected between C. striata lineages that occur sympatrically in the Mekong River Basin. The study then examined the phylogeography and population structure of two major taxa, Channa striata (the chevron snakehead) and the C. micropeltes (the giant snakehead), across SE Asia. Species specific microsatellite loci were developed and used in addition to a mitochondrial DNA marker (Cyt b) to screen neutral genetic variation within and among wild populations. C. striata individuals were sampled across SE Asia (n=988), with the major focus being the Mekong Basin, which is the largest drainage basin in the region. The distributions of two divergent lineages were identified and admixture analysis showed that where they co-occur they are interbreeding, indicating that after long periods of evolution in isolation, divergence has not resulted in reproductive isolation. One lineage is predominantly confined to upland areas of northern Lao PDR to the north of the Khorat Plateau, while the other, which is more closely related to individuals from southern India, has a widespread distribution across mainland SE Asian and Sumatra. The phylogeographical pattern recovered is associated with past river networks, and high diversity and divergence among all populations sampled reveal that contemporary dispersal is very low for this taxon, even where populations occur in contiguous freshwater habitats. C. micropeltes (n=280) were also sampled from across the Mekong River Basin, focusing on the lower basin where it constitutes an important wild fishery resource. In comparison with C. striata, allelic diversity and genetic divergence among populations were extremely low, suggesting very recent colonisation of the greater Mekong region. Populations were significantly structured into at least three discrete populations in the lower Mekong. Results of this study have implications for establishing effective conservation plans for managing both species, that represent economically important wild fishery resources for the region. For C. micropeltes, it is likely that a single fisheries stock in the Tonle Sap Great Lake is being exploited by multiple fisheries operations, and future management initiatives for this species in this region will need to account for this. For C. striata, conservation of natural levels of genetic variation will require management initiatives designed to promote population persistence at very localised spatial scales, as the high level of population structuring uncovered for this species indicates that significant unique diversity is present at this fine spatial scale.

Rapid on-site evaluation of fine needle aspiration specimens by cytology scientists : a review of 3032 specimens

Objectives To determine: (1) the accuracy of cytology scientists at assessing specimen adequacy by rapid on-site evaluation (ROSE) at fine needle aspiration (FNA) cytology collections; and (2) whether thyroid FNA with ROSE has lower inadequacy rates than non-attended FNAs. Methods The ROSE of adequacy for 3032 specimens from 17 anatomical sites collected over a 20-month period was compared with the final report assessment of adequacy. ROSE was performed by 19 cytology scientists. The report profile for 1545 thyroid nodules with ROSE was compared with that for 1536 consecutive non-ROSE thyroid FNAs reported by the same cytopathologists during the study period. Results ROSE was adequate in 75% (2276/3032), inadequate in 12% (366/3032) and in 13% (390/3032) no opinion was rendered. Of the 2276 cases assessed as adequate by ROSE, 2268 (99.6%) were finally reported as adequate for assessment; eight specimens had adequacy downgraded on the final report. Fifty eight per cent of cases with a ROSE assessment of inadequate were reported as adequate (212/366), whereas 93% (363/390) with no opinion rendered were reported as adequate. The overall final report adequacy rate for the 3032 specimens was 94% (2843/3032). Confirmation of a ROSE of adequacy at reporting was uniformly high amongst the 19 scientists, ranging from 98% to 100%. The inadequacy rate for thyroid FNAs with ROSE (6%) was significantly (P < 0.0001) lower than for non-ROSE thyroid FNAs (17%). A significantly (P = 0.02) higher proportion of adequate ROSE thyroid specimens was reported with abnormalities, compared with non-ROSE thyroid collections. Conclusions Cytology scientists are highly accurate at determining specimen adequacy at ROSE for a wide range of body sites. ROSE of thyroid FNAs can significantly reduce inadequate reports.

Potential-dependent surface-enhanced resonance raman spectroscopy at nanostructured TiO2 : A case study on cytochrome b5

Abstract: Nanostructured titanium dioxide (TiO2) electrodes, prepared by anodization of titanium, are employed to probe the electron-transfer process of cytochrome b5 (cyt b5) by surface-enhanced resonance Raman (SERR) spectroscopy. Concomitant with the increased nanoscopic surface roughness of TiO2, achieved by raising the anodization voltage from 10 to 20 V, the enhancement factor increases from 2.4 to 8.6, which is rationalized by calculations of the electric field enhancement. Cyt b 5 is immobilized on TiO2 under preservation of its native structure but it displays a non-ideal redox behavior due to the limited conductivity of the electrode material. The electron-transfer efficiency which depends on the crystalline phase of TiO2 has to be improved by appropriate doping for applications in bioelectrochemistry. Nanostructured TiO2 electrodes are employed to probe the electron-transfer process of cytochrome b5 by surface-enhanced resonance Raman spectroscopy. Concomitant with the increased nanoscopic surface roughness of TiO2, the enhancement factor increases, which can be attributed to the electric field enhancement. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Tunable electric field enhancement and redox chemistry on TiO2 island films via covalent attachment to Ag or Au nanostructures

Abstract Ag-TiO2 and Au-TiO2 hybrid electrodes were designed by covalent attachment of TiO2 nanoparticles to Ag or Au electrodes via an organic linker. The optical and electronic properties of these systems were investigated using the cytochrome b5 (Cyt b5) domain of sulfite oxidase, exclusively attached to the TiO2 surface, as a Raman marker and model redox enzyme. Very strong SERR signals of Cyt b 5 were obtained for Ag-supported systems due to plasmonic field enhancement of Ag. Time-resolved surface-enhanced resonance Raman spectroscopic measurements yielded a remarkably fast electron transfer kinetic (k = 60 s -1) of Cyt b5 to Ag. A much lower Raman intensity was observed for Au-supported systems with undefined and slow redox behavior. We explain this phenomenon on the basis of the different potential of zero charge of the two metals that largely influence the electronic properties of the TiO2 island film. © 2013 American Chemical Society.

Omega-Hydroxylation of acyclic monoterpene alcohols by rat lung microsomes

Rat lung microsomes were shown to �-hydroxylate acyclic monoterpene alcohols in the presence of NADPH and O2. NADH could neither support hydroxylation efficiently nor did it show synergistic effect. The hydroxylase activity was greater in microsomes prepared from β-naphthoflavone (BNF)-treated rats than from phenobarbital (PB)-treated or control microsomal preparations. Hydroxylation was specific to the C-8 position in geraniol and has a pH optimum of 7.8. The inhibition of the hydroxylase activity by SKF-525A, CO, N-ethylmaleimide, ellipticine, α-naphthoflavone, cyt. Image and p-CMB indicated the involvement of the cyt. P-450 system. However, NaN3 stimulated the hydroxylase activity to a significant level. Rat kidney microsomes were also capable of �-hydroxylating geraniol although the activity was lower than that observed with lungs.