Properties of cell surface carbohydrates in sexual reproduction of the Closterium peracerosum–strigosum–littorale complex (Zygnematophyceae,Charophyta)

The Closterium peracerosum–strigosum–littorale complex is a best characterized zygnematophycean green alga with respect to the process of sexual reproduction. Intercellular communication mediated by two sex pheromones has been well documented in this organism, but information concerning direct cell–cell recognition and fusion of cells involved in conjugation processes has not yet been clarified. In this study, we examined the properties of cell surface carbohydrates in vegetative and reproductive cells using a variety of fluorescein isothiocyanate labeled lectins as probes. Among 20 lectins tested, 10 bound to the Closterium cell surface, and eight of these were specific for the cells involved in sexual reproduction. In addition, some of the lectins inhibited the progress of zygote formation. In particular, Lycopersicon esculentum lectin (LEL) and ConcanavalinA (ConA) considerably inhibited zygote formation (23.6% and 0% of zygotes formed, respectively, compared with the control). LEL mainly accumulated on conjugation papillae and on the surface and lumens of empty cell walls remaining after zygote formation. ConA bound to both vegetative and sexually reproductive cells and strongly accumulated on the conjugation papillae of the latter, indicating ConA binding material(s) are non‐specifically present in Closterium cells but some of the material(s) would be essential for zygote formation. These results suggest that different carbohydrates specifically recognized by these lectins are involved in cell recognition and/or fusion during conjugation processes in the C. psl. complex.     

Time-course analysis of nuclear events during conjugation in the marine ciliate Euplotes vannus and comparison with other ciliates (Protozoa,Ciliophora)

Ciliates represent a morphologically and genetically distinct group of single-celled eukaryotes that segregate germline and somatic functions into two types of nuclei and exhibit complex cytogenetic events during the sexual process of conjugation, which is under the control of the so-called “mating type systems”. Studying conjugation in ciliates may provide insight into our understanding of the origins and evolution of sex and fertilization. In the present work, we studied in detail the sexual process of conjugation using the model species Euplotes vannus, and compared these nuclear events with those occurring in other ciliates. Our results indicate that in E. vannus: 1) conjugation requires about 75 hours to complete: the longest step is the development of the new macronucleus (ca. 64h), followed by the nuclear division of meiosis I (5h); the mitotic divisions usually take only 2h; 2) there are three prezygotic divisions (mitosis and meiosis I and II), and two of the eight resulting nuclei become pronuclei; 3) after the exchange and fusion of the pronuclei, two postzygotic divisions occur; two of the four products differentiate into the new micronucleus and macronucleus, respectively, and the parental macronucleus degenerates completely; 4) comparison of the nuclear events during conjugation in different ciliates reveals that there are generally three prezygotic divisions while the number of postzygotic divisions is highly variable. These results can serve as reference to investigate the mating type system operating in this species and to analyze genes involved in the different steps of the sexual process.     

Regeneration of plantlets fromEnteromorpha (Ulvales,Chlorophyta) protoplasts in axenic culture

High yields of protoplasts have been obtained from vegetative thalli of three species ofEnteromorpha by enzymatic degradation of the cell wall. Several commercial and crude enzymes prepared from the digestive system and hepatopancrease of abalone and top-shell were tested at different concentrations and combinations to evaluate the yield. Commercial enzymes in combination with either abalone or top-shell crude enzymes, consistently produced a high yield of protoplasts from all three species. High regeneration rate (85–95%) occurred in the protoplasts cultured at a density greater than 1.72 × 10³ cells cm⁻² at 20 and 25°C. Light intensities tested in the present study did not affect protoplast wall formation and regeneration. Protoplasts, after regenerating the cell wall, followed different types of developmental patterns under identical culture conditions. In one type some cells underwent repeated cell divisions and formed a round and oval shaped hollow thallus with a single layer of cells. In the second type many cells underwent one or two cell divisions (occasionally no division) and soon matured and discharged many motile spores, which on germination grew into normal plantlets. In the third type some cells divided irregularly to form a mass of callus-like cells (exceptE. prolifera). Culture medium supplemented with either mannitol, sorbitol, dextrose, saccharose or NaCl at higher concentrations (> 0.4 M) inhibited cell division and further differentiation in all species. author for correspondence     

A Marine Euplotes (Ciliophora, Hypotrichida) with Reduced Number of Prezygotic Micronuclear Divisions

In contradistinction to the pattern of 3 prezygotic micronuclear divisions found in 10 species of Euplotes, a marine species resembling Euplotes crassus in structure, has only 2 divisions. This atypical division pattern was observed in all matings involving the 4 available mating types. The critical stages of the nuclear events are demonstrated by using special strains having the micronuclear DNA content and chromosome number only 1/2 the normal values. The employment of such strains facilitates differentiation between the stationary and migratory pronuclei in a given conjugant and determination of the times of conclusion of prezygotic divisions and of the pronuclear exchange.     

Ultrastructural detection of proteins, lipids and carbohydrates in oocytes of Amblyomma triste (Koch, 1844) (Acari; Ixodidae) during the vitellogenesis process

The ovary of the tick Amblyomma triste is classified as panoistic, which is characterized by the presence of oogonia without nurse and follicular cells. The present study has demonstrated that the oocytes in all developmental stages (I-IV) are attached to the ovary through a pedicel, a cellular structure that synthesizes and provides carbohydrate, lipids and proteins supplies for the oocytes during the vitellogenesis process. The lipids are deposited during all oocyte stages; they are freely distributed as observed in stages II, III and IV or they form complexes with other elements. The proteins are also deposited in all stages of the oocytes, however, in lower concentration in the stage IV. There is carbohydrate deposition from oocytes in the stage II as well as in stages III and IV. In addition, the present work has demonstrated that the oocyte yolk of A. triste has a glycolipoprotein nature and the elements are deposited in the following sequence: firstly the lipids and proteins, and finally the carbohydrates.     

Eyespot behavior during the fertilization of gametes in Ulva arasakii Chihara (Ulvophyceae, Chlorophyta)

Behavior of the eyespots during the fertilization of Ulva arasakii Chihara was studied using field emission scanning electron microscopy (FE‐SEM). FE‐SEM enabled the visualization of the eyespot of biflagellate male and female gametes. The smaller male gamete has one protruded smaller (1.3 ± 0.15 μm× 1.0 ± 0.29 μm) eyespot and the larger female gamete has a larger (1.6 ± 0.2 μm× 1.1 ± 0.13 μm) one on a posterior position of the cell. The cell membrane over the eyespot region is relatively smooth compared to other parts of the cell body and exhibits hexagonal arranged lipid globules. Because the size of the cell and the morphology of the eyespot are different between male and female gametes, we could follow the fate of the eyespots during the fertilization. The initial cytoplasmic contact and fusion of the gametes takes place at their anterior end, slightly posterior to the flagellar base. The morphology of the fusing gametes followed two clearly distinguishable patterns. About half the gamete pairs lie side‐by‐side with their longitudinal axes nearly parallel, while the rest are oriented anti‐parallel to each other. In all cases, the larger female gamete fused along the same side as the eyespot, while the smaller male gamete fused along the side away from its eyespot. As fusion proceeds, the gamete pair is transformed into the quadriflagellate planozygote, in which the eyespots are positioned side‐by‐side on the region of cell fusion. These observations indicated that the opposite positioning of the eyespot relative to the cell fusion site in male and female gametes is important for the proper arrangement of the eyespots in the planozygote. The significance of this feature in advanced green algae is briefly discussed.     

<Emphasis Type="Italic">Volvox</Emphasis> (Chlorophyta,Volvocales) as a model organism in developmental biology

Model systems based on two or more related species with different types of development are finding increasing use in current comparative embryology. Green algae of the genus Volvox offer an interesting opportunity to study sex pheromones, morphogenesis as well as the formation of a somatic cell line undergoing terminal differentiation, senescence, and death as well as a line of reproductive cells, which at first grow and then undergo a series of consecutive divisions that give rise to new organisms. However, almost all studies of the recent years were conducted on a single species, Volvox carteri f. nagariensis. The goal of this publication was to advertise the cosmopolitan alga V. aureus as a model species in developmental biology. Published data on V. aureus are briefly reviewed in comparison with the development of V. carteri and outlooks of further studies are specified. In particular, the expediency of collecting new V. aureus strains from nature to study their development in clonal culture is outlined.     

Inheritance pattern of chloroplast DNA is correlated with gamete types based on sex-specific arrangement of the cell fusion site in Caulerpa (Ulvophyceae, Chlorophyta)

Using field emission scanning electron microscopy (FE‐SEM) and fluorescence microscopy, the respective relationships between the arrangement of the gamete cell‐fusion site and the inheritance pattern of chloroplast DNA (cp‐DNA) were studied for Caulerpa brachypus Harvey, C. okamurae Weber‐van Bosse, C. racemosa (Forsskål) J. Agardh var. laete‐virens (Montagne) Weber‐van Bosse, and C. serrulata (Forsskål) J. Agardh var. serrulata f. lata (Weber‐van Bosse) Tseng. The eyespot of the biflagellate gamete was visualized using FE‐SEM. The female gamete, but not the male, has one eyespot on the cell body posterior. In most mating pairs, the female gamete is fused at the anterior left side of the eyespot and the male gamete at a cell surface that is perpendicular to the plane of the flagellar beat when both gametes are mixed. Then, the inheritance pattern of cp‐DNA was observed using fluorescence microscopy after staining with 4′6‐diamidino‐2‐phenylindole. Male and female gametes have one cell nucleus and one chloroplast each. Chloroplasts of the female gamete usually contain 1–11 spherical or rod‐shaped nucleoids. In contrast, nucleoids are not usually detected in the male gamete’s chloroplast. After mixing male and female gametes, the male gamete without nucleoids and female gametes with nucleoids are always associated at the lateral side and become planozygotes. Such a correlation between the arrangement of the cell fusion site and the inheritance pattern of cp‐DNA was found in another member of Caulerpales, Bryopsis maxima Okamura. These results suggest the possibility that the arrangement of the cell fusion site in the gamete is not determined randomly regardless of sex, but is rather correlated with specific mating types. The relation of these results to those for Chlamydomonas is discussed.     

<Emphasis Type="Italic">Siderocelis irregularis</Emphasis> (Chlorophyta,Trebouxiophyceae) in Lake Tanganyika (Africa)

Siderocelis irregularis Hindák, representing a genus Siderocelis (Naumann) Fott that is known from European temperate waters, was identified as a common phytoplankter in Lake Tanganyika. It was found aposymbiotic as well as ingested (possibly endosymbiotic) in lake heterotrophs, mainly Strombidium sp. and Vorticella spp. The morphology and ultrastructure of the species, studied with LM, SEM and TEM, are described with emphasis on the structure of the cell wall and the pyrenoid. Presented at the International Symposium Biology and Taxonomy of Green Algae V, Smolenice, June 26–29, 2007, Slovakia.     

Development of the duct system during exocrine pancreas differentiation in the grass snake Natrix natrix (Lepidosauria,Serpentes)

We analyzed the development of the pancreatic ducts in grass snake Natrix natrix L. embryos with special focus on the three‐dimensional (3D)‐structure of the duct network, ultrastructural differentiation of ducts with attention to cell types and lumen formation. Our results indicated that the system of ducts in the embryonic pancreas of the grass snake can be divided into extralobular, intralobular, and intercalated ducts, similarly as in other vertebrate species. However, the pattern of branching was different from that in other vertebrates, which was related to the specific topography of the snake's internal organs. The process of duct remodeling in Natrix embryos began when the duct walls started to change from multilayered to single‐layered and ended together with tube formation. It began in the dorsal pancreatic bud and proceeded toward the caudal direction. The lumen of pancreatic ducts differentiated by cavitation because a population of centrally located cells was cleared through cell death resembling anoikis. During embryonic development in the pancreatic duct walls of the grass snake four types of cells were present, that is, principal, endocrine, goblet, and basal cells, which is different from other vertebrate species. The principal cells were electron‐dense, contained indented nuclei with abundant heterochromatin, microvilli and cilia, and were connected by interdigitations of lateral membranes and junctional complexes. The endocrine cells were electron‐translucent and some of them included endocrine granules. The goblet cells were filled with large granules with nonhomogeneous, moderately electron‐dense material. The basal cells were small, electron‐dense, and did not reach the duct lumen.     

On the role of cell surface associated,mucin-like glycoproteins in the pennate diatom Craspedostauros australis (Bacillariophyceae)

Diatoms are single-celled microalgae with silica-based cell walls (frustules) that are abundantly present in aquatic habitats, and form the basis of the food chain in many ecosystems. Many benthic diatoms have the remarkable ability to glide on all natural or man-made underwater surfaces using a carbohydrate- and protein-based adhesive to generate traction. Previously, three glycoproteins, termed FACs (F rustule A ssociated C omponents), have been identified from the common fouling diatom Craspedostauros australis and were implicated in surface adhesion through inhibition studies with a glycan-specific antibody. The polypeptide sequences of FACs remained unknown, and it was unresolved whether the FAC glycoproteins are indeed involved in adhesion, or whether this is achieved by different components sharing the same glycan epitope with FACs. Here we have determined the polypeptide sequences of FACs using peptide mapping by LC–MS/MS. Unexpectedly, FACs share the same polypeptide backbone (termed CaFAP1), which has a domain structure of alternating Cys-rich and Pro-Thr/Ser-rich regions reminiscent of the gel-forming mucins. By developing a genetic transformation system for C. australis, we were able to directly investigate the function of CaFAP1-based glycoproteins in vivo. GFP-tagging of CaFAP1 revealed that it constitutes a coat around all parts of the frustule and is not an integral component of the adhesive. CaFAP1-GFP producing transformants exhibited the same properties as wild type cells regarding surface adhesion and motility speed. Our results demonstrate that FAC glycoproteins are not involved in adhesion and motility, but might rather act as a lubricant to prevent fouling of the diatom surface.     

Late type of daughter cell wall synthesis in one of the Chlorellaceae, <Emphasis Type="Italic">Parachlorella kessleri</Emphasis> (Chlorophyta,Trebouxiophyceae)

Autosporulation is a common mode of propagation for unicellular algae. Autospore-forming species of Chlorellaceae, Chlorella vulgaris Beijerinck, C. sorokiniana Shihira et Krauss, C. lobophora Andreyeva, and Parachlorella kessleri (Fott et Nováková) Krienitz et al. have glucosamine as the main constituent of their rigid cell wall. Recent phylogenetic analyses have showed that the Chlorellaceae divided into two sister groups: the Chlorella-clade and the Parachlorella-clade. We compared the cell wall structure and synthesis of the daughter cell wall in the four species by electron microscopy using rapid freezing and freeze substitution methods. The cell wall of C. vulgaris, C. sorokiniana, and C. lobophora consisted of an electron-dense thin layer with an average thickness of 17–20, 22, and 19 nm, respectively. In these three species, daughter cell wall synthesis occurred on the outer surface of the plasma membrane in the early cell-growth phase. The cell wall of P. kessleri, however, was electron-transparent and 54–59 nm in thickness. Ruthenium red staining of P. kessleri indicated that ruthenium-red-specific polysaccharides accumulated over the outer surface of the plasma membrane. Immunoelectron microscopic observation with an anti--1, 3-glucan antibody and staining with wheat germ agglutinin (WGA) indicated that the cell wall contained -1, 3-glucan and WGA specific N-acetyl--D-glucosamine. In P. kessleri, daughter cell wall synthesis began after successive protoplast division. The daughter cell wall synthesis during autosporulation in the four species of Chlorellaceae can be classified into two types—the early and the late types.     

Immunoreactivities for epidermal growth factor (EGF) and for EGF receptors in rats with gastric ulcers

Summary The present study was aimed at assessing whether epidermal growth factor (EGF) and its receptors are present in the gastric mucosa during the healing of gastric ulcers. Immunohistochemical, immunochemical and functional studies were performed in rats after induction of ulcers in the oxyntic mucosa. Controls, which included non-operated and sham-operated animals, displayed only rare cells in the bottom of the oxyntic glands showing EGF-like immunoreactivity. Within one day after ulcer induction, a markedly increased number of chief cells in undamaged mucosa showed intense staining. Concomitantly, there was an increased immunoreactivity for EGF receptors in the mucous neck cells. Maximal immunostaining for both compounds was observed at 3 days after ulcer induction; augmented staining was still demonstrable after 3 weeks. RIA revealed significantly increased EGF concentration in the oxyntic mucosa three days after ulcer induction, and at this stage stimulated gastric acid secretion, measured in a parallel group of chronic fistula rats, indicated significant inhibition. The transient increases in EGF-like and EGF receptor immunoreactivities may stimulate gland cell proliferation. The local release of EGF-like substances may also serve to reduce gastric acidity and thereby promote ulcer healing.     

Life Cycle and Morphology of Bryopsidella ostreobiformis spec. nov. (Bryopsidaceae,Chlorophyta) from the Mediterranean,under Culture Conditions,with Comments on the Phylogeny of the Bryopsis/Derbesia Complex

Bryopsidella ostreobiformis sp. nov. is presented as the second species of the genus. The latter is amended to accomodate this new species. The derbesioid sporophyte is capable of producing three- to eight-rayed stellate propagules. The dioecious gametophyte is a heterotrichous thallus with branched, prostrate filaments and unbranched, erect ones, and does not resemble Bryopsis. The life cycle is distinctly diplohaplontic, with a diploid sporophyte and a haploid gametophyte. B. ostreobiformis is postulated as the starting point of a hypothetical evolutionary sequence of life histories in the Derbesia/Bryopsis complex.     

Behavior of flagella and flagellar root systems in the planozygotes and settled zygotes of the green alga Bryopsis maxima Okamura (Ulvophyceae,Chlorophyta) with reference to spatial arrangement of eyespot and cell fusion site

Behaviors of male and female gametes, planozygotes and their microtubular cytoskeletons of a marine green alga Bryopsis maxima Okamura were studied using field emission scanning electron microscopy, high‐speed video microscopy, and anti‐tubulin immunofluorescence microscopy. After fusion of the biflagellate male and female gametes, two sets of basal bodies lay side by side in the planozygote. Four long female microtubular roots extended from the basal bodies to the cell posterior. Four short male roots extended to nearly half the distance to the posterior end. Two flagella, one each from the male and female gametes, become a pair. Specifically, the no. 2 flagellum of the female gamete and one male flagellum point to the right side of the eyespot of the female gamete, which is located at the cell posterior and which is associated with 2s and 2d roots of the female gamete. This spatial relationship of the flagella, microtubular roots, and the eyespot in the planozygote is retained until settlement. During forward swimming, the planozygote swings the flagella backward and moves by flagellar beating. The male and female flagella in the pair usually beat synchronously. The cell withdraws the flagella and becomes round when the planozygote settles to the substratum 20 min after mixing. The axoneme and microtubular roots depolymerize, except for the proximal part and the basal bodies. Subsequently, distinct arrays of cortical microtubules develop in zygotes until 30 min after mixing. These results are discussed with respect to the functional significance of the spatial relationships of flagellar apparatus‐eyespot‐cell fusion sites in the mating gametes and planozygote of green algae.     

Simultaneous visualization of the actin plate and new cell partition membrane during cytokinesis in the brown alga Sphacelaria rigidula (Sphacelariales,Phaeophyceae)

In many brown algae, cytokinesis is accomplished through the centrifugal expansion of the membrane structure formed by the fusion of Golgi vesicles and flat cisternae. In contrast, it has been reported that cytokinesis in Sphacelaria rigidula progresses centripetally by adding Golgi vesicles and flat cisternae to cleaving furrows of the plasma membrane. The reason why this cytokinetic pattern was observed only in Sphacelaria species is unknown. In either cytokinesis pattern, a plate-like actin structure (the actin plate) coincides with the cytokinetic plane between the daughter nuclei. However, it is unclear how the actin plate is related to cytokinesis progression. In this study, we re-examined cytokinesis in the apical cells of S. rigidula using transmission electron microscopy. Double staining of the actin plate and the developing membrane was followed by fluorescence microscopy analysis to determine the relationship between these two formations. The results showed that cytokinesis in S. rigidula, as in many brown algae, was completed by centrifugal growth of the new cell partition membrane. A furrow of the plasma membrane was observed at the beginning of cytokinesis; however, further invagination did not occur. The actin plate arose at the center of the cytokinetic plane before membrane fusion and extended parallel to the expansion of the new cell partition membrane. When cytokinesis was slow due to insufficient Golgi vesicle supply to the cytokinetic plane in the cells under brefeldin A treatment, the extension of the actin plate was also suspended. In this study, the spatiotemporal relationship between the occurrence and expansion of the actin plate and the new cell partition membrane was revealed. These observations indicate that the actin plate might promote membrane fusion or lead to the growth of a new cell partition membrane.     

Ultrastructural variations in Microcystis aeruginosa (Chroococcales,Cyanophyta) during a surface bloom induced by high incident light irradiance

ABSTRACT

We evaluated the ultrastructural variations occurring during a surface bloom in Microcystis aeruginosa Kützing PCC 7005, by comparing cultures exposed to high incident light irradiance (90 µmol photons m^-2 s^-1) and to low irradiance (9 µmol photons m^-2 s^-1). At 12h, and 15 and 30 days in culture, the cells were fixed in glutaraldehyde and subjected to cytochemical assays. Exposure to high irradiance induced structural changes in the cell wall, and differences in the photosynthetic apparatus and granule reserves. After 15 days of high irradiance, the following features were observed: a reduced number of thylakoids and changes in their arrangement; high accumulation of glycogen, poly-beta-hydroxybutyrate, lipid bodies and cyanophycin; conspicuous structural changes in the cell wall. At day 30 of high irradiance, the viable cells had few thylakoids, arranged parallel to each other and perpendicular to the plasma membrane, and abundant glycogen. Nitrogen, calcium and phosphorus, as detected by electron spectroscopic imaging, were present in different sites of cells grown at low and high irradiance. The results of this study, together with previous data on variation in Fe-superoxide dismutase (Canini et al., 2001a), suggest that defense mechanisms against high and prolonged irradiance only operate for a short time.     

Down-regulation of Bcl2 and Survivin,and up-regulation of Bax involved in copper (II) phenylthiosemicarbazone complex-induced apoptosis in leukemia stem-like KG1a cells

Previous studies suggested that phenylthiosemicarbazones are considered as a new apoptosis-inducing agent. In this study, anti-proliferative and apoptotic effects of the copper (II) phenylthiosemicarbazone complex (Cu-PTSC) were investigated in human acute myeloid leukemia KG1a cell line. The KG1a cells were treated with various concentrations (20−140 μM) of the Cu-PTSC, and cell viability was determined by MTT assay. The IC₅₀ value of 80 ± 2.5 μM was selected for further evaluations. Apoptosis, as the antitumor strategy in the cells, was investigated morphologically by acridine orange/ethidium bromide (AO/EtBr) double staining, and surface expression assay of phosphatidylserine by Annexin V/PI technique was studied via flow cytometry. Results indicated that the cells undergo morphologic changes with chromatin condensation and G₀/G₁ cell cycle arrest after treatment with Cu-PTSC. The presence of phosphatidylserine on the outer surface of the cell membrane confirmed the apoptosis occurrence in the KG1a cells. Real-time PCR and western blot analyses revealed that the incubation of KG1a cells with Cu-PTSC down-regulated the expression of Bcl-2 (anti-apoptotic protein) and Survivin (as an IAP protein) while induced the expression of Bax (pro-apoptotic protein). Based on present data, it seems that Cu-PTSC may provide a novel therapeutic approach for the treatment of acute myeloid leukemia.