IMMUNOLOCALIZATION OF CENTER IN THE FLAGELLAR APPARATUS OF MALE GAMETES OF ECTOCARPUS SILICULOSUS (PHAEOPHYCEAE) AND OTHER BROWN ALGAL MOTILE CELLS1

Centrin or a centrin homologue was localized using immunofluorescence in the flagellar basal body region in zoids of five brown algal species: Ectocarpus siliculosus (Dillw.) Lyngb., Scytosiphon lomentaria (Lyngb.) Link, Laminaria digitata (Huds.) Lamour., Sphacelaria rigidula (Kütz.) Prud'homme van Reine, and Fucus serratus L. The antigen is restricted to short rods extending along the basal body(ies) and towards the nucleus, which always remains firmly linked to the flagellar apparatus in isolated cytoskeletons. To identify these antigenic sites, pre- and postembedding immunogold electron microscopy was applied to male gametes of E. siliculosus. At least three different structures associated with the basal bodies were antigenic: a fibrous structure connecting the proximal end of the posterior basal body to the nucleus (nucleus-basal body connector), a striated band that links the two basal bodies to each other and is located in the angel formed by them, and amorphous material interconnecting the basal bodies in their most proximal regions. In addiction, specific labeling occurs along the external surface and within the lumen of both basal bodies and in the flagellar transitional region. The possible function of these centrin-containing structures is discussed.     

CROSSING EXPERIMENTS,LIPID COMPOSITION,AND THE SPECIES CONCEPT IN ECTOCARPUS SILICULOSUS AND E. FASCICULATUS (PHAEOPHYCEAE,ECTOCARPALES)1

Sporophytes of Ectocarpus siliculosus (Dillwyn) Lyngbye and E. fasciculatus Harvey were collected in the vicinity of Roscoff Brittany, France. Gametophytes derived from meiospores were used for intra- and interspecific crosses. Intraspecific gamete combinations gave viable zygotes, which developed into fertile sporophytes. Interspecific crosses were unsuccessful. Gamete fusions did not occur between female gametes of E. fasciculatus and male gametes of E. siliculosus. Hybrid zygotes were formed in the reciprocal combination but died soon after germination. We conclude that the two species of Ectocarpus at Roscoff represent distinct taxonomic entities, which are separated by pre- and postzygotic compatibility barriers. These biological findings are confirmed by the differential occurrence of the chemotaxonomic marker betaine-lipid diacetylglycerylhydroxymethyltrimethyl-β-alanine, which is present in our cultures of E. fasciculatus but absent in E. siliculosus.     

应用激光扫描共聚焦显微镜FRAP技术研究兔早期胚胎发育中细胞间隙连接介导通讯

本研究应用激光扫描共聚焦显微镜的光漂白恢复技术(FRAP)分析兔早期胚胎卵裂球之间通过间隙连接介导的细胞通讯(GJIC)。研究结果发现,用强激光分别将4-细胞期胚胎、异裂胚胎和8-细胞期胚胎的一个卵裂球荧光光漂白后,经过15分钟的荧光恢复,4-细胞期胚胎的光漂白恢复率为17.8％,异裂胚胎的光漂白恢复率为23.7％,二者之间没有明显的差异;8-细胞期胚胎的光漂白恢复率为78.2％,与前二者之间存在明显的差异。推测兔早期胚胎卵裂球细胞间隙连接建立的时间在8-细胞阶段,胚胎卵裂球间隙连接通讯可能是兔胚胎正常发育的重要条件。     

鼻咽癌细胞对HA的粘附作用及微丝的共聚焦显微镜观察

本文研究了人鼻咽癌上皮细胞系ＣＮＥ－２Ｚ对透明质酸（ＨＡ）的粘附作用及其粘附后微丝的改变,结果表明,鼻咽癌细胞粘附的ＯＤ值随ＨＡ浓度的升高而升高,３００μｇ／ｍｌ、１２５０μｇ／ｍｌ组与对照组比较有极显著性意义,Ｐ＜０．０１;癌细胞粘附的ＯＤ值随培养时间延长而升高,２４０ｍｉｎ组及１２０ｍｉｎ组与６０ｍｉｎ组比较有极显著性意义,Ｐ＜０．０１。ＬＳＭ观察ＨＡ浓度为３００μｇ／ｍｌ时培养４ｈ和２４ｈ的癌细胞微丝的分布明显不同,培养４ｈ的癌细胞呈圆形,微丝均匀地分布于胞浆内;培养２４ｈ的癌细胞呈不规则形,微丝主要分布于与ＨＡ的粘附面上。上述结果提示ＨＡ可促进鼻咽癌细胞粘附,癌细胞与ＨＡ结合后可诱导ＭＦ重组     

INHERITANCE AND MEIOTIC ELIMINATION OF A VIRUS GENOME IN THE HOST ECTOCARPUS SZLZCULOSUS (PHAEOPHYCEAE)1

The marine brown alga Ectocarpus siliculosus (Dillwyn) Lyngbye is frequently infected by a latent DNA virus that multiplies in modified sporangia and gametangia of the host. We describe a polymerase chain reaction (PCR) procedure for the amplification and detection of viral DNA in Ectocarpus. PCR analysis of parents and progeny plants confirmed that virus DNA passes through meiosis like a Mendelian trait. An infected sporophyte produced equal numbers of gametophytes with and without the viral genome. Thus, meiosis in sexual populations of the host acts as a mechanism for the creation of virus-free progeny.     

THE EFFECT OF LABELING INTENSITY,ESTIMATED BY REAL-TIME CONFOCAL LASER SCANNING MICROSCOPY,ON FLOW CYTOMETRIC APPEARANCE AND IDENTIFICATION OF IMMUNOCHEMICALLY LABELED MARINE DINOFLAGELLATES1

Two different fluorescein isothiocyanate (FITC) conjugates were used to analyze the effect of labeling intensity on the flow cytometric appearance of marine dinoflagellates labeled with antibodies that specifically recognized the outer cell wall. Location of the labeling was revealed by epifluorescence and real-time confocal laser scanning microscopy using an anti-rabbit IgG/FITC-conjugated secondary antiserum. Flow cytometric measurements showed that cells of Prorocentrum species labeled this way could not always be distinguished from unlabeled cells. The labeling intensity increased several times when a biotinylated anti-rabbit IgG secondary antiserum was used in combination with a streptavidin/FITC conjugate. Flow cytometry indicated that the labeling intensity had increased 50%, which resulted in an improved separation of clusters of labeled and unlabeled cells.     

共聚焦显微技术研究ABA诱导蚕豆气孔保卫细胞H_2O_2的产生(简报)

逆境下,植物细胞内ABA含量急剧增加,同时植物也可通过一些酶代谢反应积累活性氧,如H_2O_2,O_2~-。ABA作为逆境信号对气孔运动的显著调节作用已被诸多实验所证实,但关于其对气孔运动调节的细节还知之甚少。H_2O_2作为氧化信号分子在植物抗病信号转导中已得到广泛研究,但H_2O_2是否介导保卫细胞的气孔运动还缺乏直接的证据。我们已初步发现H_2O_2可参与外源ABA诱     

共聚焦镜观察凋亡巨噬细胞内pH的变化

用透射电镜观察巨噬细胞的形态学改变,结果显示,地塞米松处理８小时后,大部分巨噬细胞发生凋亡特征变化：胞突缩短、减少,胞膜完整。胞体皱缩,胞质密度增加,其中出现大量空泡。胞核染色质边聚、浓缩。另外用激光扫描共聚焦显微镜（ＡＣＡＳ５７０）和ｐＨ荧光探针ＳＮＡＲＦ┐１／ＡＭ实时检测地塞米松处理巨噬细胞胞浆ｐＨ的动态变化。加入地塞米松,多数巨噬细胞胞浆马上发生快速和短期的碱化。随后,胞浆ｐＨ缓慢降低,胞浆酸化。结果表明,胞浆酸化是细胞凋亡发展的必然过程,胞浆碱化则很可能与细胞凋亡的发生相关,也可能与细胞种类、细胞功能状态相关     

Localization of Tubulin and a Centrin-Homologue in Vegetative Cells and Developing Gametangia of Ectocarpus siliculosus (Dillw.) Lyngb. (Phaeophyceae,Ectocarpales)

The distribution of tubulin and centrin in vegetative cells and during gametogenesis of Ectocarpus siliculosus was studied by immunofluorescence. In interphase cells bundles of microtubules are focused on the centriolar region near the nuclear surface. Some of the bundles ensheath the nucleus while others traverse the cytoplasm in various directions, sometimes reaching the cell cortex. Evaluation of serial optical sections by confocal laser scanning microscopy (CLSM) revealed that the perinuclear and “cytoplasmic” microtubule bundles presumably constitute a single complex. In interphase cells centrin is localized as a single bright spot in the centriolar region. In dividing cells duplication and separation of the microtubular complex and the centrin spot takes place. In post-mitotic cells with two nuclei, the centrioles are located at opposite cell poles, short microtubule bundles emanate from them and partially encompass the nucleus. During gametogenesis a gradual transformation of the vegetative cytoskeleton to the gametic flagellar apparatus occurs.     

激光共聚焦显微镜研究大白鼠肝癌FSK7902细胞系的实体瘤中角蛋白和嗜铬颗粒素A的关系

应用免疫组织化学ＡＢＣ法和免疫荧光组织化学方法结合激光共聚焦显微镜观察了嗜铬颗粒素Ａ和角蛋白在大白鼠肝癌ＦＳＫ７９０２细胞系形成的实体瘤癌细胞中的表达,结果表明在ＦＳＫ７９０２实体瘤内,大多数癌细胞呈角蛋白和ＣｇＡ免疫反应阳性,反应产物位于核周胞质或核旁的某些区域。     

PERIPHYTON COLONIZATION OF ROCK SURFACES IN A BOREAL FOREST STREAM STUDIED BY SCANNING ELECTRON MICROSCOPY AND TRACK AUTORADIOGRAPHY1

Periphyton colonization of natural rock surfaces and granite tiles was followed experimentally in the Matamek River, an acidic (pH 5.5) sixth order boreal stream in northeast Quebec, Canada. Accumulation of chlorophyll a and freshweight algal biomass was logarithmic over a 25 day colonization period. The major colonizers were Tabellaria flocculosa (Roth) Kütz., T. fenestrata (Lyngb.) Kütz., Mougeotia sp., and Eunotia pectinalis (Kütz) Rabh., and its varieties. The microcolonization sequence on granite tiles, followed over 27 days with scanning electron microscopy, showed an initial accumulation of algal cells on the upstream and downstream margins and in depressions, followed by a gradual filling-in of the flat surfaces. It is hypothesized that the observed slow rate of colonization was due to the high surface tension of the granite substratum and the absence of preconditioning by an organic film. It is further hypothesized that the increase in cellular carbon fixation rates of T. flocculosa measured over a 23 day period using nuclear track autoradiography parallels the development of an algal-detrital microcosm on the granite tile, and is evidence for the establishment of localized tight nutrient spiralling. It is suggested that the periphyton community may be regarded as a nutrient recycling system at a microenvironmental level which may be especially important in oligotrophic systems.     

ASYMMETRY OF EYESPOT AND MATING STRUCTURE POSITIONS IN ULVA COMPRESSA (ULVALES,CHLOROPHYTA) REVEALED BY A NEW FIELD EMISSION SCANNING ELECTRON MICROSCOPY METHOD1

Gametes of the marine green alga Ulva compressa L. are biflagellate and pear shaped, with one eyespot at the posterior end of the cell. The species is at an early evolutionary stage between isogamy and anisogamy. In the past, zygote formation of green algae was categorized solely by the relative sizes of gametes produced by two mating types (+ and ?). Recently, however, locations of cell fusion sites and/or mating structures of gametes have been observed to differ between mating types in several green algae (asymmetry of cell fusion site and/or mating structure positions). To use this asymmetry for determining gamete mating type, we explored a new method, field emission scanning electron microscopy (FE‐SEM), for visualizing the mating structure of U. compressa. When gametes were subjected to drying stress in the process of a conventional critical‐point‐drying method, a round structure was observed on the cell surfaces. In the mating type MGEC‐1 (mt⁺), this structure was located on the same side of the cell as the eyespot, whereas it was on the side opposite the eyespot in the mating type MGEC‐2 (mt^?). The gametes fuse at the round structures. TEM showed an alignment of vesicles inside the cytoplasm directly below the round structures, which are indeed the mating structures. Serial sectioning and three‐dimensional construction of TEM micrographs confirmed the association of the mating structure with flagellar roots. The mating structure was associated with 1d root in the MGEC‐1 gamete but with 2d root in the MGEC‐2 gamete.     

去卵巢致骨质疏松症大鼠腺垂体FS细胞中Cx43、S-100蛋白表达--激光扫描共聚焦显微镜观察

检测间隙连接蛋白Cx43、神经组织蛋白S-100在去卵巢致骨质疏松症(OVX-OP)大鼠腺垂体滤泡星形细胞(FS细胞)中的表达.实验采用10月龄未孕产SD雌性大鼠40只,随机均分为卵巢切除组(OVX组,n=20)和假性手术对照组(Sham组,n=20).于术后6周末用双能X线骨吸收测量法(DEXA)测量大鼠全身及腰椎4-6(L4-6)骨密度(BMD).取两组大鼠垂体,制成连续切片.应用FITC标记的IgG探针,对腺垂体组织中Cx43和S-100进行间接免疫荧光染色,并用激光扫描共聚焦显微镜(LSCM)定位和定量分析腺垂体FS细胞中Cx43、S-100的表达.结果发现,术后6周末OVX组大鼠全身及L4-6BMD均明显低于Sham组值(P<0.01,P<0.01).Cx43阳性荧光反应主要定位于相邻的FS细胞的胞浆中和/或胞膜上.OVX组Cx43阳性表达荧光强度和表达阳性率均显著低于Sham组(P<0.01).S-100蛋白表达定位于FS细胞的胞浆中,两组间S-100阳性表达荧光强度和表达阳性率无显著差异(P>0.05).本研究提示,SD大鼠OVX术后6周出现骨质疏松变化;OVX大鼠腺垂体FS细胞数量无明显变化、而Cx43表达显著下降,后者的变化可能与大鼠OVX-OP发生相关.     

CELL FORM AND SURFACE PATTERNS IN CHROOMONAS and CRYPTOMONAS CELLS (CRYPTOPHYTA) AS REVEALED BY SCANNING ELECTRON MICROSCOPY1

Fifteen freshwater cryptomonad species were freeze-dried and examined with the scanning electron microscope. Surveys of cell surfaces revealed four general cell types. Chroomonas type cells lack a furrow but possess a shallow vestibular depression where the flagella are inserted. The presence of a gullet could not be detected. Cryptomonas spp. displayed three morphological types, all lacking gullets. The first type of Cryptomonas has a simple, shallow furrow with ridges that apparently can close to form a raphe but an oval opening or stoma remains at the posterior end and an opening from the vestibulum is formed at the anterior end. The second Cryptomonas type consists of a complex furrow with furrow ridges and folds that extend almost two-thirds of the cell length. A sloma is present in the central region of the closed furrow. The folds apparently can separate thereby exposing the underlying furrow. The third type of Cryptomonas possesses a simple, non-closing furrow. At the anterior end there is a vestibular ligule which extends from the dorsalleft side of the cell and covers the region of the vestibulum where the contractile vacuole discharges.     

PASSAGE OF A MARINE BROWN ALGAL DNA VIRUS FROM ECTOCARPUS FASCICULATUS (ECTOCARPALES,PHAEOPHYCEAE) TO MYRIOTRICHIA CLAVAEFORMIS (DICTYOSIPHONALES,PHAEOPHYCEAE): INFECTION SYMPTOMS AND RECOVERY1

A dsDNA virus (EfasV-1) isolated from Ectocarpus fasciculatus Harvey infected Myriotrichia clavaeformis Harvey, a species belonging to a different brown algal order. The virus did not complete its infection cycle in the foreign host but caused infertility due to malformed reproductive structures. After some time in culture, the host's reproductive capacity was sometimes restored with concomitant loss of at least part of the viral genome. This incidence of interordinal virus transfer is discussed in relation to possibilities for virus-mediated horizontal gene transfer in brown algae.     

鳋科（寄生桡足类）胸板的扫描电镜比较研究

本文利用扫描电镜观察了我国鳋科的5个属的种类。首次发现在它们的胸部腹面游泳足连接板之下均有一板状结构,其形状与栉齿的着生情况在不同属之间存在着明显的差异,同属不同种之间也不尽相同,这是一个过去未曾被注意过的结构,权且称之为胸板。它是一个独立的结构,不与连接板相连。胸板上的栉齿从形态-功能-适应三位一体分析,它与附着寄主有夭,以免被水流冲走,与无栉齿的相比,应是一个进步性状。     

IMMUNOFLUORESCENCE MICROSCOPY OF FERTILIZATION AND PARTHENOGENESIS IN LAMINARIA ANGUSTATA (PHAEOPHYTA)1

Normal fertilization and parthenogenesis of unfertilized eggs were observed in Laminaria angustata Kjellman by indirect immunofluorescence microscopy using a tubulin antibody. Sperm aster formation did not occur at plasmogamy. The centrosome of the egg gradually disappeared. Shortly after karyogamy, one centrosome reappeared near the zygote nucleus. During mitosis, the centrosome replicated and the daughter centrosomes migrated to opposite poles. The mitotic spindle was formed by microtubules that elongated from both poles. After the first cell division, each of the daughter cells received one centrosome that persisted throughout the development of the sporophyte. During parthenogenetic development, abnormal mono-, tri-, and multi-polar spindles were formed. These abnormal spindles caused abnormal nuclear and cytoplasmic division. Thus, cells were produced with 1) no nuclei, 2) multiple nuclei, 3) irregular numbers of chromosomes, and/or 4) no centrosomes. This is one of the reasons for the abortion and abnormal morphogenesis during parthenogenesis. Ultrastructural observations showed that, although cells of some parthogenetic sporophytes have centrioles, cells of almost all abnormally shaped parthenogenetic sporophytes lack centrioles. These results suggest that centrioles are required for normal centrosomal functions in Laminaria. Although centrioles are inherited paternally, some centrosomal material appears to be present or produced de novo in unfertilized eggs.     

SCANNING ELECTRON MICROSCOPY OF CORALLINA AND HALIPTILON (CORALLINACEAE,RHODOPHYTA): SURFACE FEATURES AND THEIR TAXONOMIC IMPLICATIONS1

Scanning electron microscopy of intergenicula in members of the subfamily Corallinoideae reveals two distinctive surface morphologies: a Corallina-type (C-type) with round to irregular cell outlines and round trichocyte bases, and a Jania-type (J-type) with elongate, polygonal cell outlines and elongate trichocyte bases with excentric pores. The surface results from the calcified lateral walls of the epithallial cells projecting up from around collapsed protoplasts. Since J-type surfaces and trichocytes only occur in unequivocal members of the tribe Janieae—especially the genera Jania and Haliptilon, the presence of J-type surfaces in questionable members of Corallina reveals that they in fact belong to Haliptilon. Thus the two surface types clarify previously difficult taxonomic distinctions between Haliptilon and Corallina and allow identification to genus from purely vegetative material. Seventeen new combinations in Haliptilon are proposed. These results have considerable biogeographic implications with tropical species found to belong to Haliptilon, and Corallina sensu stricto being recognized primarily as a temperate and cold water genus.