薄层色谱和高效液相色谱联用测定暗纹东方鲀肌肉中肌肽和谷胱甘肽的含量

通过薄层色谱(TLC)与高效液相色谱(HPLC)联用技术鉴定了暗纹东方鲀(Takifugu obscurus)肌肉中存在肌肽和谷胱甘肽,同时利用高效液相色谱法测定了肌肽和谷胱甘肽(GSH)的含量。薄层层析采用的展开剂为正丁醇:乙酸:水(4∶2∶1),层析板为硅胶板。高效液相色谱利用Kromasil C18反相柱分析,流动相为10%的纯乙腈和90%含有0.05%三氟乙酸的超纯水。结果表明:通过薄层色谱和高效液相色谱鉴定了暗纹东方鲀肌肉中肌肽和谷胱甘肽,其中暗纹东方鲀肌肉中肌肽含量约213μg/g(鲜重),还原性谷胱甘肽含量约211μg/g(鲜重)。本法样品无需衍生,操作简便,适合于暗纹东方鲀肌肉中肌肽和谷胱甘肽的测定。本文为生物体内肌肽和谷胱甘肽的研究提供借鉴意义。     

菊黄东方鲀、暗纹东方鲀及其杂交F1代肌肉营养成分的比较分析

为了了解菊黄东方鲀(Takifugu flavidus)、暗纹东方鲀(T.obscurus)及其杂交F1代的肌肉营养特征,利用生物化学方法,从每类实验样本中取9尾对其肌肉中的粗蛋白、粗脂肪、水分、粗灰分和氨基酸成分进行了测定和分析。结果显示:(1)杂交F1代在生长方面具有明显的杂交优势,与亲本之间存在着显著差异(P0.05),杂交F1代的体重为其亲本的1.48~1.77倍;(2)杂交F1代肌肉水分含量与其母本含量相近,但粗脂肪含量均较亲本少(P0.05),粗蛋白含量则与亲本差异不显著(P0.05);(3)除色氨酸和胱氨酸外,16种氨基酸均在肌肉样本中被检测到,除甲硫氨酸外,其余15种氨基酸间含量均存在着显著性差异(P0.05)。菊黄东方鲀(♀)×暗纹东方鲀(♂)杂交F1代的总氨基酸含量最高,而暗纹东方鲀(♀)×菊黄东方鲀(♂)F1代总氨基酸含量则介于两亲本之间。对其必需氨基酸总量进行分析发现,菊黄东方鲀与其正反杂交F1代之间均存在显著性差异(P0.05),而暗纹东方鲀与其正反杂交F1代之间差异不显著(P0.05);(4)肌肉营养品质评价结果表明,菊黄东方鲀(♀)×暗纹东方鲀(♂)杂交F1代的鲜味氨基酸含量为26.68%,明显高于双亲样本(菊黄东方鲀22.28%、暗纹东方鲀25.20%),而暗纹东方鲀(♀)×菊黄东方鲀(♂)F1代的鲜味氨基酸总量(23.30%)较其父本偏高,但低于其母本。研究结果表明,杂交东方鲀的肌肉营养综合了双亲的优良特性,特别是是菊黄东方鲀(♀)×暗纹东方鲀(♂)杂交F1代,拥有最高的鲜味氨基酸含量,具有推广价值。     

聚乙二醇-4000对暗纹东方鲀肠上皮电生理的影响

以暗纹东方鲀(Takifugu obscurus)为研究对象,采用Ussing chamber技术研究了聚乙二醇-4000(PEG-4000)对肠上皮开路电压、短路电流(Isc)、短路电流变化("Isc)、电导(G)、跨膜电位(TEP)的影响。结果表明:PEG-4000在肠道中6 h残留浓度为1.83 g·L-1,显著高于其在水中浓度(P0.05),残留率达184%; PEG处理组暗纹东方鲀离体肠上皮Isc为69.20μA·cm~(-2),显著低于对照组(P0.05);ΔIsc显著低于对照组(P0.05),仅为对照组的68.57%; PEG处理组暗纹东方鲀离体肠上皮电导为24.97 m S·cm~(-2),显著低于对照组(36.22 m S·cm~(-2))(P0.05); PEG-4000在暗纹东方鲀肠道中极易残留、不易渗透,使得肠道上皮的转运蛋白或者离子通道受阻,从而显著影响肠上皮Isc、"Isc以及电导,降低了离子的主动流动量。本研究结果为鱼类肠道电生理平台的构建提供了技术参数,该平台不仅能用于评估PEG等污染物对鱼类肠道的影响,还能用于鱼类肠道上皮细胞离子转运机制的研究。     

暗纹东方鲀线粒体基因组核苷酸全序列测定与分析

以暗纹东方鲀(Takifugu fasciatus)肝的线粒体DNA为模板,参照红鳍东方鲀(T.rubripes)等近源鱼类的线粒体基因组DNA序列,设计合成14对特异引物,进行PCR扩增并测序,首次获得了暗纹东方鲀线粒体基因组全序列。结果表明,暗纹东方鲀线粒体基因组序列全长16 444 bp(GenBank登录号为GQ409967),A+T含量为55.8%,其mtDNA结构与其他脊椎动物相似,由22个tRNA基因、2个rRNA基因、13个蛋白质编码基因和1段819 bp非编码的控制区(D-loop)所组成。蛋白质基因除COⅠ和ND6的起始密码子为GTG、CCT以外,均为典型的起始密码子ATG。ND1、ATPase8、COⅢ、ND4L、ND5、Cyt b使用典型的终止密码子TAA,其他的使用不完全终止密码子。除ND6和tRNAGln、tRNAAla、tRNAAsn、tRNACys、tRNATyr、tRNASer、tRNAGlu、tRNAPro在L-链上编码之外,其余基因均在H-链编码。基因排列顺序与已测定的鲀类一致,这显示了鲀类线粒体基因排列顺序上的保守性。tRNA基因核苷酸长度为64～73nt,预测了22个tRNA基因的二级结构,均呈较为典型的三叶草状。基于19种鲀类mtDNA全序列构建的进化树表明,暗纹东方鲀与红鳍东方鲀、中华东方鲀(T.chinensis)聚成一个姊妹群。结果还支持东方鲀属鱼类为一单系类群。     

脊椎动物性别决定和分化的分子机制研究进展

哺乳类性别决定是多种转录因子和生长因子相继表达和相互调控的结果。SRY的表达启动雄性通路并诱导下游雄性特异基因SOX9、AMH等的表达。FOXL2在雌性未分化性腺表达,WNT-4和DAX1也在雌性性别决定或分化时期表达,表明雌性通路也是受特定基因调控的,而并非“默认通路”。鸟类的性别也是由遗传基因决定的,EFT1(雌性)和DMRT1(雄性)可能是性别决定候选基因。爬行类为温度性别决定的典型,温度可能通过调节雌激素水平和控制性别特异遗传基因表达决定性别。大部分两栖类性别受环境因素影响,但发现DMRT1和DAX1可能与其精巢发育有关。鱼类性别决定和分化方式差异很大,多种因素(遗传基因、环境因素、类固醇激素等)参与了这一过程。从青Q鳉Y染色体定位克隆的DMY,被认为是第一个非哺乳类脊椎动物雄性性别决定基因。所有这些表明脊椎动物性别决定和分化机制是多样化的。     

暗纹东方鲀耐寒相关基因CIRBP、HMGB1和AFP-Ⅳ的分子特征和对低温胁迫的响应

为了探索暗纹东方鲀(Takifugu obscurus)对低温环境的响应机制,克隆了暗纹东方鲀耐寒相关基因CIRBP、HMGB1和AFP-Ⅳ的cDNA序列,并进行了基因的分子特征和功能分析。组织分布检测显示CIRBP和HMGB1在下丘脑、肝脏和肌肉中具有高表达,而AFP-Ⅳ则主要在肝脏中表达。在受到低温胁迫后, 3种基因在肝脏和下丘脑中的表达呈现不同的变化趋势,其中CIRBP基因在肝脏中于48h表达量显著增加,在下丘脑中于12h和48h有上调表达; HMGB1基因在肝脏中呈现逐渐上升的趋势,于48h达到最大值,而在下丘脑中呈现先上升后下降的趋势,处理后2h达到最大, 2—8h下降,于8h下降至最低,随后恢复至初始水平;肝脏中的AFP-Ⅳ在0—24h无显著变化,在48h上升至最大值。进一步通过大肠杆菌原核表达系统研究了AFP-Ⅳ的抗冻功能,发现AFP-Ⅳ融合蛋白在–80℃下具有抗冻活性,并且抗冻活性随着浓度的增加而提高。研究结果表明3种基因都参与了暗纹东方鲀对低温胁迫的应答过程,为深入探索暗纹东方鲀的耐低温机制奠定了基础。     

食蚊鱼CYP19b基因克隆及环境激素暴露对其表达的影响

为探讨17-雌二醇(E2)、17-甲基睾酮(MT)和雄烯二酮(AED)暴露对食蚊鱼脑组织CYP19b表达的影响,首次克隆和分析了食蚊鱼(Gambusia affinis) CYP19b cDNA的全系列。采用静水暴露方法, 分别设置2、10、50和100 ng/L E2、MT和AED各4个实验组, 并设置对照组和平行组, 定量测定了1、3、5和7d脑组织中的CYP19b mRNA表达水平的变化。成功克隆了食蚊鱼CYP19b基因全长, 获得基因片段总长1670 bp, ORF(OpenReading Frame)是从52 bp到1554 bp, 共编码501个氨基酸。通过与其他相关鱼类CYP19b基因编码的氨基酸序列相似性作比较, 食蚊鱼CYP19b基因氨基酸序列的同源性很高。结果显示, 短时间的暴露, E2较低浓度组(2和10 ng/L)对食蚊鱼作用不明显, 但E2相对较高浓度组(50和100 ng/L)能显著地上调食蚊鱼CYP19b基因的表达(P0.01)。暴露早期MT对CYP19b表达无明显影响, 随着暴露时间的持续延长, 除了MT较高浓度组(50 ng/L)外, 其他各浓度组MT对CYP19b的表达有显著的抑制作用(P0.01), 并呈一定的剂量相关关系。AED作为雄激素类似物与MT的作用效果相似, 相对较高浓度(50和100 ng/L)的AED抑制CYP19b的表达(P0.01)。研究结果初步表明, 3种典型环境激素对食蚊鱼CYP19b基因表达的影响十分显著, 但不同的暴露浓度及不同的取样时间点可能表现为促进或抑制CYP19b基因的表达。     

怒江东方墨头鱼的年龄结构与生长特性

2006年10月至2008年10月于怒江采集东方墨头鱼(Garra orientalis)136尾,体长54～167mm,体重3.0～114.6g。研究了其年龄、生长等生物学特性,通过耳石轮纹估算东方墨头鱼的年龄,显示怒江东方墨头鱼由10个年龄组组成。耳石半径与体长关系为:L=0.061R1.131(n=125,R2=0.907),体长与体重关系为:W=0.1×10-4L3.160。von Bertalanffy生长参数由退算体长估算:雄性L∞=224.79mm,k=0.094/年,t0=0.51年;雌性L∞=228.91mm,k=0.091/年,t0=0.639年;总体L∞=227.78mm,k=0.091/年,t0=0.588年。体重生长拐点雄性12.15龄、雌性13.75龄。     

饲料中添加壳聚糖和益生菌对暗纹东方纯幼鱼生长及部分消化酶活性的影响

在基础饲料中分别添加不同浓度的益生菌(0.1%、0.2%、0.4%)、壳聚糖(0.2%、0.5%、1.0%)、壳聚糖与益生菌混合物、甘露聚糖与益生菌混合物,在室内水泥池(5.0m×2.0m×1.0m)中喂养暗纹东方鲀幼鱼(3.15±0.05g),用基础饲料投喂暗纹东方鲀作为对照,每组三个重复,60d后测定鱼体的生长、肌肉生化成分、肝胰脏、肠道蛋白酶和淀粉酶活性和白肌RNA/DNA。结果显示,对照组相对增重率为476.4%,饲料中添加0.2%的壳聚糖、0.1%的益生菌对暗纹东方鲀的促生长作用最明显,相对增重率显著提高。壳聚糖与益生菌混合物、甘露聚糖与益生菌混合物对生长无显著影响。对照组肌肉蛋白质和脂肪含量分别为82.94%、11.21%,壳聚糖、益生菌与甘露聚糖在饲料中的添加,能够显著降低肌肉脂肪含量,显著提高肌肉蛋白质含量,也能在一定程度上降低肝体比。0.2%壳聚糖和0.1%益生菌能使肠道淀粉酶活性显著增强(P<0.05),白肌RNA/DNA显著增大(P<0.05),说明肠道淀粉酶活性增强和蛋白质合成增加是壳聚糖和益生菌促进暗纹东方鲀生长的重要原因。     

雌激素对中华鳖性腺分化及DMRT1、SOX9基因表达的影响

中华鳖（Pelodiscus sinensis）性别决定的方式一直存在较大的争议,分子机制更是不清楚。在大部分脊椎动物中,雌激素在性别决定和性腺分化中扮演重要的调控作用。实验通过对性别分化前胚胎进行雌二醇（E2）和芳香化酶抑制剂（AI）处理,研究雌激素在中华鳖性腺分化中的作用及机理。实验结果显示,与对照组（雌性比例49%）相比,E2处理组中雌性中华鳖仔鳖比例显著增加,高达92.3%;而在AI处理组中,雌性比例显著下调至13.1%。HE染色分析表明,ZZ（雄性）和ZW（雌性）胚胎分别经过E2和AI处理后,ZZ和ZW性腺结构呈现明显的雌性化和雄性化特征。同时,通过RT-PCR和免疫荧光染色发现,E2能显著降低雄性性别关键因子DMRT1和SOX9 mRNA和蛋白表达水平;AI则表现相反的调节作用。综上所述,雌激素通过抑制雄性性别关键因子DMRT1和SOX9的表达来抑制雄性分化,促进雌性分化,揭示雌激素在中华鳖雌性性别分化中起着重要的调控作用。       

Plant-Parasitic Nematodes of South Viet Nam

Between 1974 and 1978, 2,842 identifications of plant-parasitic nematodes were made from more than 1,700 soil and plant samples collected in eight provinces of South Viet Nam. Species in nine genera—Helicotylenchus, Criconemoides, Meloidogyne, Pratylenchus, Tylenchorhynchus, Hoplolaimus, Hirschmanniella, Xiphinema, and Rotylenchulus—comprised 96.1% of the identifications; the remaining 3.9% were species of 11 genera. Fourteen genera were associated with rice which was grown on about 2,500,000 ha in 1970. Of these, Ditylenchus, Hirschmanniella, and Meloidogyne were most important. Ditylenchus angustus caused severe damage to about 50,000 ha of flooded rice in the Mekong Delta in 1976. Hirschmanniella spp. were found in all samples examined from flooded rice fields. Meloidogyne spp. were common in rice seedbeds, upland rice, and rice not kept flooded continuously. Meloidogyne and Pratylenchus spp. were found in roots of 22 of the 32 crop plants sampled. Little or no attempt was made in South Viet Nam to control nematodes.     

Study of types of some species of "Filaria" (Nematoda) parasites of small mammals described by Von Linstow and Molin

Parasitic nematodes from the Berlin (ZMB) and Vienna (NMW) Museum collections referred to the genus Filaria Mueller, 1787 by von Linstow or Molin were studied. Three samples were in good condition and the specimens redescribed. Litomosa hepatica (von Linstow, 1897) n. comb., sample ZMB Vermes Entozoa 3368, from the megachiropteran Pteropus neohibernicus, Bismarck Archipelago, resembles L. maki Tibayrenc, Bain & Ramanchandran, 1979, from Pteropus vampyrus, in Malaysia, but the buccal capsule differs. Both species display particular morphological characters which differ from species of Litomosa parasitic in microchiropterans. The remaining material originates from Brazil. The spicule morphology of Litomosoides circularis (von Linstow, 1899) Chandler, 1931, sample ZMB Vermes Entozoa 1059 from Hesperomys spec. (= Holochilus brasiliensis), Porto Alegre, confirms that it belongs to the sigmodontis group; the microfilaria presents characters of the genus Litomosoides, e.g. body attenuated at both extremities and salient cephalic hook. Taxonomic discussions by others confirm that species of Litomosoides belonging to the sigmodontis group and described subsequently are distinct from L. circularis. Litomosoides serpicula (Molin, 1858) Guerrero, Martin, Gardner & Bain, 2002, is redescribed, sample NMW 6323 from the bat Phyllostoma spiculatum (= Sturnira lilium), Ypanema. It is very close to L. brasiliensis Almeida, 1936, type host Moytis sp., but distinguished by a single ring in the buccal capsule, rather than two, supporting previous conclusions that the taxon L. brasiliensis, as generally regarded, may represent a complex of species. Samples NMW 6322 and NMW 6324, from other bats and also identified by Molin (1858) as Filaria serpicula, contain unidentifiable fragments of Litomosoides incertae sedis. Filaria hyalina von Linstow, 1890, sample ZMB Vermes Entozoa Q 3905 from Sorer vulgaris (= Sorex araneus), is incertae sedis because it contains two unidentifiable posterior parts of male, which might be an acuarid, Stammerinema sp. Filaria vesperuginis von Linstow, 1885, sample ZMB Vermes Entozoa Q 3929, from the bat Vesperugo serotinus (= Eptesicus serotinus), contains encysted nematode larvae and is a nomen dubium.     

Microbial transformation of zaluzanin-D

Microbial transformation of zaluzanin-D using different fungi gave 11,13-dihydrozaluzanin-C, zaluzanin-C, 4,16,11,13 - tetrahydro zaluzanin-C, estafiatone, dihydroestafiatol and dihydroestafiatone.     

Multilocus phylogenetic analysis of the genus Aeromonas

A broad multilocus phylogenetic analysis (MLPA) of the representative diversity of a genus offers the opportunity to incorporate concatenated inter-species phylogenies into bacterial systematics. Recent analyses based on single housekeeping genes have provided coherent phylogenies of Aeromonas. However, to date, a multi-gene phylogenetic analysis has never been tackled. In the present study, the intra- and inter-species phylogenetic relationships of 115 strains representing all Aeromonas species described to date were investigated by MLPA. The study included the independent analysis of seven single gene fragments (gyrB, rpoD, recA, dnaJ, gyrA, dnaX, and atpD), and the tree resulting from the concatenated 4705 bp sequence. The phylogenies obtained were consistent with each other, and clustering agreed with the Aeromonas taxonomy recognized to date. The highest clustering robustness was found for the concatenated tree (i.e. all Aeromonas species split into 100% bootstrap clusters). Both possible chronometric distortions and poor resolution encountered when using single-gene analysis were buffered in the concatenated MLPA tree. However, reliable phylogenetic species delineation required an MLPA including several “bona fide” strains representing all described species.     

Fate map of the distal portion of Drosophila proboscis as inferred from the expression and mutations of basic patterning genes

The late-third-instar labial disc is comprised of two disc-proper cell layers, one representing mainly the ventral half of the anterior compartment (L-layer) and the other, the dorsal half of the anterior compartment and most, if not all, of the posterior compartment (M-layer). In the L-layer, Distal-less represses homothorax whereas no Distal-less-dependent homothorax repression occurs in the M-layer where Distal-less is coexpressed with homothorax. In wild-type labial discs, clawless, one of the two homeobox genes expressed in distal cells receiving maximum (Decapentaplegic+Wingless) signaling activity in leg and antennal discs, is specifically repressed by proboscipedia. A fate map, inferred from data on basic patterning gene expression in larval and pupal stages and mutant phenotypes, indicates the inner surface of the labial palpus, which includes the pseudotracheal region, to be a derivative of the distal portion of the M-layer expressing wingless, patched, Distal-less and homothorax. The outer surface of the labial palpus with more than 30 taste bristles derives from an L-layer area consisting of dorsal portions of the anterior and posterior compartments, each expressing Distal-less. Our analysis also indicates that, in adults and pupae, the anterior-posterior boundary, dividing roughly equally the outer surface of the distiproboscis, runs along the outer circumference of the inner surface of distiproboscis.     

Spatial and temporal regulation of the homeotic selector gene Antennapedia is required for the establishment of leg identity in Drosophila

Antennapedia is one of the homeotic selector genes required for specification of segment identity in Drosophila. Dominant mutations that ectopically express Antennapedia cause transformation of antenna to leg. Loss-of-function mutations cause partial transformation of leg to antenna. Here we examine the role of Antennapedia in the establishment of leg identity in light of recent advances in our understanding of antennal development. In Antennapedia mutant clones in the leg disc, Homothorax and Distal-less are coexpressed and act via spineless to transform proximal femur to antenna. Antennapedia is negatively regulated during leg development by Distal-less, spineless, and dachshund and this reduced Antennapedia expression is needed for the proper development of distal leg elements. These findings suggest that the temporal and spatial regulation of the homeotic selector gene Antennapedia in the leg disc is necessary for normal leg development in Drosophila.     

Molecular phylogeny of Phoma and allied anamorph genera: Towards a reclassification of the Phoma complex

The present generic concept of Phoma is broadly defined, with nine sections being recognised based on morphological characters. Teleomorph states of Phoma have been described in the genera Didymella, Leptosphaeria, Pleospora and Mycosphaerella, indicating that Phoma anamorphs represent a polyphyletic group. In an attempt to delineate generic boundaries, representative strains of the various Phoma sections and allied coelomycetous genera were included for study. Sequence data of the 18S nrDNA (SSU) and the 28S nrDNA (LSU) regions of 18 Phoma strains included were compared with those of representative strains of 39 allied anamorph genera, including Ascochyta, Coniothyrium, Deuterophoma, Microsphaeropsis, Pleurophoma, Pyrenochaeta, and 11 teleomorph genera. The type species of the Phoma sections Phoma, Phyllostictoides, Sclerophomella, Macrospora and Peyronellaea grouped in a subclade in the Pleosporales with the type species of Ascochyta and Microsphaeropsis. The new family Didymellaceae is proposed to accommodate these Phoma sections and related anamorph genera. The present study demonstrated that Phoma radicina, the type species of Phoma sect. Paraphoma and Phoma heteromorphospora, the type species of Phoma sect. Heterospora can be assigned to the Phaeosphaeriaceae and Leptosphaeriaceae respectively.