An immunofluorescence study on lectin binding sites in gametes of Ectocarpus siliculosus (Ectocarpales, Phaeophyceae)

The distribution of binding sites for the lectins WGA, DSA. RCA I, PNA, AAA, MAA. SNA, GNA. and Con A in gametes of both sexes of the brown alga, Ectocarpus siliculosus (Dillw.) Lyngbye, was investigated by fluorescence microscopy. Digoxigenin-conjugated lectins and an FITC-anti-digoxigenin antibody were used as a high sensitivity detection system. Organelles and other distinct cellular domains could be distinguished by their binding specificities. Glycoconjugates associated with one flagellum were found to be associated with the axoneme by lectin binding to isolated flagellar apparatuses. In addition, changes in the distribution of carbohydrate epitopes during the attachment of gametes to the substratum were revealed by differential lectin binding.     

Salt Tolerance of Ectocarpus siliculosus (Dillw.) Lyngb.: Comparison of Gametophytes,Sporophytes and Isolates of Different Geographic Origin

Forty Ectocarpus siliculosus isolates from a wide geographical range, including gametophyte and sporophyte plants, have all been acclimated to the same salinity for several years. Their salinity tolerances in respect of cell viability, photosynthesis and dark respiration were evaluated over the salinity range: 8 to 96 ‰. Significant differences in the physiological tolerances to salt stress compared with viability measurements were evident. Genotypic differences in salt tolerances between groupings of the isolates, and also differences in responses of gametophyte and sporophyte generations were found. However, diploid and haploid sporophyte material had similar tolerances. Triploid and tetraploid sporophytes did not have improved tolerances over those of diploid plants. Culture plants originating from low salinities in the Baltic Sea had broader tolerances than field material collected from Baltic waters of similar salinity.     

REFLECTIVE PROPERTIES OF THE STIGMA IN MALE GAMETES OF ECTOCARPUS SILICULOSUS (PHAEOPHYCEAE) STUDIED BY CONFOCAL LASER SCANNING MICROSCOPY1

The reflection properties of the stigma in male gametes of Ectocarpus siliculosus (Dillw.) Lyngbye were investigated using confocal laser scanning microscopy in the epireflection contrast mode. The complex reflection pattern obtained after optical xy (horizontal) and xz (vertical) sectioning was consistent with stigma ultrastructure as revealed by serial thin sections. The intensity and pattern of the reflection signal varied with the orientation of the cell/stigma to the incident laser light. Maximal reflection occurred only in approximately normal orientation of the stigma to the light source. Focusing of reflected light from an elongated concave depression of the stigma on the region of the flagellar swelling was observed in xy and xz sections of living and fixed gametes. The results indicate the importance of mechanisms (focusing) other than quarter-wave interference reflection in signal amplification by the eyespot of flagellate algae.     

Maullinia ectocarpii gen. et sp. nov. (Plasmodiophorea), an intracellular parasite in Ectocarpus siliculosus (Ectocarpales, Phaeophyceae) and other filamentous brown algae

An obligate intracellular parasite infecting Ectocarpus spp. and other filamentous marine brown algae is described. The pathogen forms an unwalled multinucleate syncytium (plasmodium) within the host cell cytoplasm and causes hypertrophy. Cruciform nuclear divisions occur during early development. Mature plasmodia become transformed into single sporangia, filling the host cell completely, and then cleave into several hundred spores. The spores are motile with two unequal, whiplash-type flagella inserted subapically and also show amoeboid movement. Upon settlement, cysts with chitinous walls are formed. Infection of host cells is accomplished by means of an adhesorium and a stachel apparatus penetrating the host cell wall, and injection of the cyst content into the host cell cytoplasm. The parasite is characterized by features specific for the plasmodiophorids and is described as a new genus and species, Maullinia ectocarpii.     

The metareticulate pollen morphology of Alternanthera Forssk. (Gomphrenoideae,Amaranthaceae) and its taxonomic implications

Alternanthera (Amaranthaceae) is a diverse genus largely restricted to the American Tropics that belongs to the alternantheroid clade containing C₄ and C₃–C₄ intermediate species. This research focuses on the study of pollen characters by studying 13 species, representatives of the two major clades and subclades of Alternanthera. General palynological comparisons were conducted with light microscopy (LM), scanning electron microscopy (SEM) and with confocal laser scanning microscopy (CLSM) for exine ultrastructure. Twenty-five characters were measured and described for Alternanthera and among these, 14 pollen characters were used to discriminate pollen groups using cluster analysis and canonical analysis of principal coordinates (CAP). Pollen form and ornamentation, pores number, spines length, number of ektexinous bodies and nanospines on the ektexinous bodies on pore membranes, arrangement of nanopores and spines on structural elements, and metareticula form were taxonomically important and therefore used to construct the first palynological key to the alternantheroid clade species. Our study indicates that the seemingly subtle morphological variation of pollen is useful for recognising three main pollen types within Alternanthera. The much needed palynological terminology for describing the mesoporium in the metareticulate pollen of Amaranthaceae is provided.     

Laminarionema elsbetiae gen. et sp. nov. (Ectocarpales, Phaeophyceae), a new endophyte in Laminaria sporophytes

Laminarionema elsbetiae gen. et sp, nov, (Ectocarpaceae, Ectocarpales. Phaeophyceae), a new endophyte of Laminaria japonica Areschoug (Laminariales, Phaeophyceae), is described from Muroran. Hokkaido, Japan. Laminarionema elsbetiae grows in the host tissues forming networks in the epidermal and subcortical layers as well as penetrating into the cortical and medullary layers. Only phaeophycean hairs emerge from the surface of the host tissue. No reproductive cells were found in field material. However, under host-free culture the species formed three morphologically different reproductive structures. Macrosporangia containing a single large motile spore were formed under long and short day conditions below 20°C, transformed from vegetative cells, conical to elongated in shape, 50–75 μm in length and ca. 10 μm in diameter. Microsporangia were linear to lanceolate, sometimes branched, formed under long and short day conditions below 15°C. Unilocular sporangia were more or less irregular in shape, formed under short day conditions of 5–15°C, 60–75 μm in length and 40–45 μm in diameter. Sexual fusion between macro- and microspores was not seen. In mixed cultures of L. elsbetiae with young sporophytes of L Japonica Areschoug as well as Saccorhiza dermatodea (de la Pylaie) J. Agardh, L elsbetiae infected both hosts, grew in the same manner as in natural hosts, and formed macrosporangia between host epidermal cells.     

Body musculature of Beauchampiella eudactylota (Gosse, 1886) (Rotifera: Euchlanidae) with additional new data on its trophi and overall morphology

This study presents the results of confocal laser scanning microscopy and fluorescence‐labelled phalloidin used to visualize the system of body musculature in Beauchampiella eudactylota. Moreover, the poorly known trophi of B. eudactylota are described based on scanning electron microscopy. In total, four paired longitudinal muscles (musculi longitudinales I–IV) and three circular muscles (musculi circulares I–III) were identified. Among these are the musculus longitudinalis ventralis, the musculus longitudinalis dorsalis and the musculus circumpedalis as documented in previous studies for other rotifer species. Compared to other species, B. eudactylota is characterized by the low number of lateral longitudinal muscles and the absence of some longitudinal muscles (musculi longitudinales capitum) and circular muscles (corona sphincter, musculus pars coronalis). Moreover, scanning electron microscopic data on the trophi of B. eudactylota reveal a number of striking similarities to the trophi in some species of Epiphanidae. This suggests that either (1) these similarities represent plesiomorphic characters present both in Epiphanidae and B. eudactylota or (2) they are synapomorphic features of B. eudactylota and some species of Epiphanidae, which would question the monophyly of Euchlanidae.     

Surface colonization of lodgepole pine (Pinus contorta var. latifolia [Dougl. Engelm.]) roots by Pseudomonas fluorescens and Paenibacillus polymyxa under gnotobiotic conditions

Indirect immunofluorescence techniques and confocal scanning laser microscopy were used to identify rhizobacterial strains on the root surfaces of pine seedlings, which were grown from seeds under gnotobiotic conditions. Conifer plant growth promoting rhizobacterial strains Paenibacillus polymyxa L6 and Pw-2, and the forest soil isolate Pseudomonas fluorescens M20, were inoculated onto surface-disinfested pine seeds, singly, or in dual combinations: strains L6 + M20, or strains Pw-2 + M20. Segments containing particular root microsites (root tip, root hair zone, or areas of lateral root emergence) were sampled randomly from roots 7 or 13 weeks after inoculation, and the colonization of roots by each bacterium was observed. Root segments were also sampled from individual roots at six different points along the length of the root, and the qualitative colonization of younger areas, closer to the root tip, contrasted with that of older areas, closer to the root base. The ability of strain M20 to colonize root areas adjacent to sites of lateral root emergence improves in the presence of either P. polymyxa strain, while the ability of the P. polymyxa strains to colonize these areas was not affected. More rhizobacteria were also generally observed on younger root tissues than on areas closer to the root base.     

Transfer of a marine DNA virus fromEctocarpus toFeldmannia (Ectocarpales,Phaeophyceae): aberrant symptoms and restitution of the host

Summary The marine brown algaEctocarpus siliculosus is invaded by a polyhedric virus, whose genome consists of circular, double-stranded DNA. In laboratory experiments this virus can infect a different host species,Feldmannia simplex. InfectedFeldmannia plants show severe somatic malformations. However, no functional virus particles are formed. SuchFeldmannia plants recover to resume a normal, symptom-free appearance. This result raises the possibility of intergeneric gene transfer in the natural habitat.     

Microtubule organization in the apical cell of Sphacelaria (Phaeophyceae) and its related protoplast

The growth of the filamentous brown alga Sphacelaria depends on a large, strongly polarized, apical cell. The protoplast derived from this cell can be distinguished in a heterogeneous suspension by cytological markers, so it is possible to study development of the cytoskeleton during protoplast isolation and the first steps of regeneration. In the initial cell, microtubules show an asymmetric distribution along the axis; they are mainly located at the distal part around the physodes. After protoplast isolation, this polarity initially seems to be maintained; subsequently, the microtubules radiate from the two centrioles and spread out to the plasmalemma. This experimental model is suitable for investigating the development of the polarity of the initial cell, and the sequence of the first morphogenetic events leading to protoplast regeneration.     

用免疫荧光标记对人组织中肥大细胞亚型的分选与形态观察

方法:利用中性蛋白酶成分、特征性酶抗体的免疫荧光染色和流式细胞仪确定分选肥大细胞亚型,以激光扫描共聚焦显微镜显示肥大细胞内分泌颗粒。结果:三种免疫表型被确定:肥大细胞的类胰蛋白酶阳性(MCT)、类糜蛋白酶阴性;类糜蛋白酶阳性(MCC)、类胰蛋白酶阴性和类胰蛋白酶阳性、类糜蛋白酶阳性(MCTC)。肥大细胞内分泌颗粒分散或聚集存在,分泌颗粒突起分泌或以分散的方式释放。分泌颗粒大范围释放后,肥大细胞的形态结构发生了改变。结论:利用肥大细胞的特征性酶抗体、免疫荧光标记和流式细胞仪可将人组织中的肥大细胞分选纯化为三种亚型;以共聚焦显微镜显示肥大细胞含有丰富的分泌颗粒,它说明肥大细胞具备了为人体I型变态反应提供快速反应的物质基础。     

Cadmium adsorption by non-living biomass of the semi-macroscopic brown alga,Ectocarpus siliculosus,grown in axenic mass culture and localisation of the adsorbed Cd by transmission electron microscopy

Non-living, freeze-dried material of the brown algaEctocarpus siliculosus (Phaeophyceae) demonstrated high equilibrium uptake of Cd from aqueous solutions (Fehrmann & Pohl, 1993). The alga was grown in 250-L photobioreactors under various growth conditions (light, salinity and nutrient concentrations) in order to obtain larger quantities of biomass and to improve its Cd adsorption capacity. To derive further knowledge on the biosorbant phenomenon different adsorption parameters such as pH for the sorption process and kinetics of Cd adsorption were tested. The maximum adsorption capacity of the freeze-dried biomass exceeded 41 mg Cd per g biomass. After repeated addition of low Cd concentrations the maximum adsorption capacity was lower (31.4 mg Cd per g biomass). In comparison with other adsorbing materials (activated carbon, silica gel, siliceous earth)E. siliculosus showed significantly higher adsorption capacity. Desorption of deposited Cd with 0.1 M HCl resulted in no changes of the adsorption capacity through five subsequent adsorption-/desorption-cycles. Hence, driedE. siliculosus appears to be an efficient material for the elimination of Cd from industrial waste water.Transmission electron microscopic investigations showed an electron dense area in the outer surface layers of the cell wall after Cd adsorption indicating the most likely location of Cd fixation.Author for correspondence     

Differentiation of Cells in the Hypophysis and in Pancreatic Islets

Deparaffinized, 3-5μ, sections are brought to water, oxidized 3.5 min in an equal-parts mixture of 0.3% H₂SO₄ and 0.3% KMnO₄, and decolorized with 4% K₂S₂O₅. Nuclei are stained with Gomori's (1939) chromium-hematoxylin, and cell granules with Cason's (1950) mixture. The eosinophilic cells of the hypophysis and the alpha cells of pancreatic islets (of Langerhans) stain carmine red; basophilic and beta cells stain dark blue. Heidenhain's _susa is the most suitable fixative for hypophysis, Bouin's fluid for pancreas; but a satisfactory result is obtainable after formalin-sublimate or plain formalin. Besides studying the ratio of the cell types in the hypophysis or in pancreatic islets, it is possible to estimate the granule content of the cells. The method works on human autopsy material provided fixation of hypophysis occurs within 24 hr, and. pancreas, 12 hr post mortem, and it is suitable also for quite fresh organs.     

激光共聚焦显微镜观察小鼠早期胚胎中PKB/Akt对微丝聚合的影响

在本实验中我们用优化的免疫荧光化学法结合激光共聚焦显微技术,观察了微丝在小鼠卵细胞不同期的分布情况及PKB/Akt对小鼠卵母细胞和早期胚胎的微丝聚合的影响。结果显示,在小鼠卵母细胞及早期胚胎中均有微丝的表达,且主要集中在纺锤体处的质膜处、极体及分裂沟处。注射激活型PKB/Akt mRNA能够增强微丝的聚集。相反,注射激酶失活型的PKB/AktmRNA减弱了微丝的聚合。因而我们认为PKB/Akt可以影响小鼠卵细胞和早期胚胎的微丝聚集。     

钙结合蛋白CIB1在293T细胞中的表达及亚细胞定位研究

分析和比对钙结合蛋白CIB1在人类、大鼠、小鼠中氨基酸序列差异,并研究CIB1蛋白在293T细胞中的表达及亚细胞定位情况。通过Western Blot分析发现293T细胞本身几乎检测不到CIB1的表达。进一步采用CIB1外源性质粒转染,通过免疫荧光分析实验,在激光共聚焦显微镜下观察转染后不同时间293T细胞中CIB1的表达情况及亚细胞定位变化。实验结果表明,随着转染时间的增加,CIB1在293T细胞中的表达有逐渐增强的趋势,并且发生从细胞核向细胞质的转位现象。该实验结果对了解CIB1亚细胞定位变化及功能研究具有重要参考价值。     

In Situ Microspatial Imaging Using Two-Photon and Confocal Laser Scanning Microscopy of Bacteria and Extracellular Polymeric Secretions (EPS) Within Marine Stromatolites

The combination of a hydrophilic embedding resin, Nanoplast, with fluorescent probes, and subsequent imaging using two-photon and confocal laser scanning microscopy (2P-LSM and CLSM) has allowed in imaging of the in situ microspatial arrangements of microbial cells and their extracellular polymeric secretion (EPS) within marine stromatolites. Optical sectioning by 2P-LSM and CLSM allowed imaging of endolithic cyanobacteria cells, Solentia sp., seen within carbonate sand grains. 2P-LSM allowed very clear imaging with a high resolution of bacteria using DAPI, which normally require UV excitation and reduced photo-bleaching of fluorescent probes.     

Life histories of Colpomenia sinuosa and Hydroclathrus clathratus (Scytosiphonaceae, Phaeophyceae) in culture

Life-history studies in culture were carried out on Colpomenia sinuosa (Mertens ex Roth) Derbès et Solier and Hydroclathrus clathratus (C. Agardh) Howe (Scytosiphonales, Phaeophyceae) from Japan. These species showed a heteromorphic life-history pattern with an alternation between erect thalli bearing plurilocular zoidangia and prostrate thalli bearing ectocarpoid plurilocular and unilocular zoidangia. Plurizoids released from erect and prostrate thalli developed into prostrate thalli. Unizoids, however, developed into erect thalli. Prostrate thalli produced plurilocular zoidangia in long-day conditions and unilocular zoidangia in short-day conditions at 10-20°C. Prostrate thalli of C. sinuosa formed ascocysts. Germlings of both species did not grow at 5°C.     

Development,morphological characteristics and viability of temporary cysts of Pyrodinium bahamense var. compressum (Dinophyceae) in vitro

Pellicle or temporary cysts of Pyrodinium bahamense var. compressum (Böhm) Steidinger, Tester & F.J.R. Taylor and their role in bloom dynamics have not yet been adequately characterized and understood. We investigated the role of temperature- and nutrient-mediated stress as factors that could induce pellicle formation in batch cultures. Cellular features and their implications for temporary cyst viability were examined using confocal laser scanning microscopy (CLSM). Our data suggest that temperature change is one of the key factors influencing pellicle formation, preserving viability at low temperature (i.e. 13°C). Hypnocysts (resting cysts) were not observed. During pellicle formation, motile cells generally undergo ecdysis, extrusion of cytoplasmic materials and bacteria, compaction of the nucleus and non-motility. The outermost covering of the temporary cysts shows red autofluorescence and it contains lower concentrations of chlorophyll (chl) a and no detectable chl c. The nuclear region is surrounded by transitional red bodies and other unidentified cellular structures. Temporary cysts can immediately revert back to the motile state upon exposure to optimum conditions. This is accompanied by the expansion of the nuclear region, regeneration of the chloroplasts and enlargement of the cell. Developmental changes during reversal of temporary cysts to motile forms were also observed to cause breaks in the cell covering that could serve as sites for bacterial entry. Though observed in vitro, such behaviour may also be occurring in nature especially as a response to drastic short-lived environmental changes. This is the first detailed report on the characteristics of temporary cysts of P. bahamense var. compressum.