RESUMO
Microtubule-associated fibers from the basal apparatus of the green flagellate alga Spermatozopsis similis exhibit a complex cross-striation pattern with 28-nm periodicity and consist of 2-nm filaments arranged in several layers. Fibers enriched by mechanical disintegration and high salt extraction (2 M NaCl) of isolated basal apparatuses are soluble in 2 M urea. Dialysis of solubilized fibers against 150 mM KCl yields paracrystals which closely resemble the native fibers in filament arrangement and striation pattern. Paracrystals purified through several cycles of disassembly and reassembly are greatly enriched (greater than 90%) in a single protein of 34 kD (assemblin) as shown by SDS-PAGE. A rabbit polyclonal antibody raised against assemblin labels the striated fibers as shown by indirect immunofluorescence of isolated cytoskeletons or methanol permeabilized cells and immunogold EM. Two-dimensional electrophoresis (isoelectric focusing and SDS-PAGE) resolves assemblin into at least four isoforms (a-d) with pI's of 5.45, 5.55, 5.75, and 5.85. The two more acidic isoforms are phosphoproteins as shown by in vivo 32PO4-labeling and autoradiography. Amino acid analysis of assemblin shows a high content of helix-forming residues (leucine) and a relatively low content of glycine. We conclude that assemblin may be representative of a class of proteins that form fine filaments alongside microtubules.
Assuntos
Clorófitas/ultraestrutura , Proteínas Associadas aos Microtúbulos/ultraestrutura , Microtúbulos/ultraestrutura , Aminoácidos/análise , Clorófitas/fisiologia , Cristalização , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Imunofluorescência , Immunoblotting , Microscopia Imunoeletrônica , Proteínas Associadas aos Microtúbulos/isolamento & purificação , Proteínas Associadas aos Microtúbulos/fisiologia , Microtúbulos/fisiologia , Peso Molecular , FosforilaçãoRESUMO
The flagellar basal apparatus comprises the basal bodies and the attached fibrous structures, which together form the organizing center for the cytoskeleton in many flagellated cells. Basal apparatus were isolated from the naked green flagellate Spermatozopsis similis and shown to be composed of several dozens of different polypeptides including a protein band of 95 kD. Screening of a cDNA library of S. similis with a polyclonal antibody raised against the 95-kD band resulted in a full-length clone coding for a novel protein of 834 amino acids (90.3 kD). Sequence analysis identified nonhelical NH2- and COOH-terminal domains flanking a central domain of approximately 650 residues, which was predicted to form a series of coiled-coils interrupted by short spacer segments. Immunogold labeling using a polyclonal antibody raised against the bacterially expressed 95-kD protein exclusively decorated the striated, wedge-shaped fibers, termed sinister fibers (sf-fibers), attached to the basal bodies of S. similis. Striated fibers with a periodicity of 98 nm were assembled in vitro from the purified protein expressed from the cloned cDNA indicating that the 95-kD protein could be a major component of the sf-fibers. This structure interconnects specific triplets of the basal bodies with the microtubular bundles that emerge from the basal apparatus. The sf-fibers and similar structures, e.g., basal feet or satellites, described in various eukaryotes including vertebrates, may be representative for cytoskeletal elements involved in positioning of basal bodies/centrioles with respect to cytoskeletal microtubules and vice versa.
Assuntos
Clorófitas/metabolismo , Proteínas do Citoesqueleto/metabolismo , Sequência de Aminoácidos , Formação de Anticorpos , Sequência de Bases , Clonagem Molecular , Citoplasma/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/isolamento & purificação , DNA Complementar , Microtúbulos/metabolismo , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de DNARESUMO
We report the isolation of striated flagellar roots from the Prasinophycean green alga Tetraselmis striata using sedimentation in gradients of sucrose and flotation on gradients of colloidal silica. PAGE in the presence of 0.1% SDS demonstrates that striated flagellar roots are composed of a number of polypeptides, the most predominant one being a protein of 20,000 Mr. The 20,000 Mr protein band represents approximately 63% of the Coomassie Brilliant Blue staining of gels of isolated flagellar roots. Two-dimensional gel electrophoresis (isoelectric focusing and SDS PAGE) resolves the major 20,000 Mr flagellar root protein into two components of nearly identical Mr, but of differing isoelectric points (i.e., pl's of 4.9 and 4.8), which we have designated 20,000-Mr-alpha and 20,000-Mr-beta, respectively. Densitometric scans of two-dimensional gels of cell extracts indicate that the 20,000-Mr-alpha and -beta polypeptides vary, in their stoichiometry, between 2:1 and 1:1. This variability appears to be related to the state of contraction or extension of the striated flagellar roots at the time of cell lysis. Incubation of cells with 32PO4 followed by analysis of cell extracts by two-dimensional gel electrophoresis and autoradiography reveals that the more acidic 20,000-Mr-beta component is phosphorylated and the 20,000-Mr-alpha component contains no detectable label. These results suggest that the 20,000-Mr-alpha component is converted to the more acidic 20,000-Mr-beta form by phosphorylation. Both the 20,000-Mr-alpha and -beta flagellar root components exhibit a calcium-induced reduction in relative electrophoretic mobilities in two-dimensional alkaline urea gels. Antiserum raised in rabbits against the 20,000-Mr protein binds to both the 20,000-Mr-alpha and 20,000-Mr-beta forms of the flagellar root protein when analyzed by electrophoretic immunoblot techniques. Indirect immunofluorescence on vegetative or interphase cells demonstrate that the antibodies bind to two cyclindrical organelles located in the anterior region of the cell. Immunocytochemical investigations at ultrastructural resolution using this antiserum and a colloidal gold-conjugated antirabbit-IgG reveals immunospecific labeling of striated flagellar roots and their extensions. We conclude that striated flagellar roots are simple ion-sensitive contractile organelles composed predominantly of a 20,000 Mr calcium-binding phosphoprotein, and that this protein is largely responsible for the motile behavior of these organelles.
Assuntos
Proteínas de Ligação ao Cálcio/isolamento & purificação , Cálcio/farmacologia , Clorófitas/ultraestrutura , Flagelos/ultraestrutura , Organoides/ultraestrutura , Fosfoproteínas/isolamento & purificação , Proteínas de Plantas , Fracionamento Celular , Centrifugação com Gradiente de Concentração , Eletroforese em Gel de Poliacrilamida , Flagelos/efeitos dos fármacos , Imunofluorescência , Microscopia Eletrônica , Peso Molecular , Organoides/efeitos dos fármacosRESUMO
A rapid, Ca2+-dependent change in the angle between basal bodies (up to 180 degrees) is associated with light-induced reversal of swimming direction (the "photophobic" response) in a number of flagellated green algae. In isolated, detergent-extracted, reactivated flagellar apparatus complexes of Spermatozopsis similis, axonemal beat form conversion to the symmetrical/undulating flagellar pattern and basal body reorientation (from the antiparallel to the parallel configuration) are simultaneously induced at greater than or equal to 10(-7) M Ca2+. Basal body reorientation, however, is independent of flagellar beating since it is induced at greater than or equal to 10(-7) M Ca2+ when flagellar beating is inhibited (i.e., in the presence of 1 microM orthovanadate in reactivation solutions; in the absence of ATP or dithiothreitol in isolation and reactivation solutions), or when axonemes are mechanically removed from flagellar apparatuses. Although frequent axonemal beat form reversals were induced by varying the Ca2+ concentration, antiparallel basal body configuration could not be restored in isolated flagellar apparatuses. Observations of the photophobic response in vivo indicate that even though the flagella resume the asymmetric, breaststroke beat form 1-2 s after photostimulation, antiparallel basal body configuration is not restored until a few minutes later. Using an antibody generated against the 20-kD Ca2+-modulated contractile protein of striated flagellar roots of Tetraselmis striata (Salisbury, J. L., A. Baron, B. Surek, and M. Melkonian, 1984, J. Cell Biol., 99:962-970), we have found the distal connecting fiber of Spermatozopsis similis to be immunoreactive by indirect immunofluorescence and immunogold electron microscopy. Electrophoretic and immunoblot analysis indicates that the antigen of S. similis flagellar apparatuses consists, like the Tetraselmis protein, of two acidic isoforms of 20 kD. We conclude that the distal basal body connecting fiber is a contractile organelle and reorients basal bodies during the photophobic response in certain flagellated green algae.
Assuntos
Cálcio/farmacologia , Clorófitas/ultraestrutura , Proteínas Contráteis/fisiologia , Flagelos/ultraestrutura , Organoides/ultraestrutura , Clorófitas/fisiologia , Flagelos/fisiologia , Imunofluorescência , Luz , Microscopia Eletrônica , Organoides/fisiologiaRESUMO
The microtubule associated system I fibers of the basal apparatus of the flagellate green alga Spermatozopsis similis are noncontractile and display a 28-nm periodicity. Paracrystals with similar periodicities are formed in vitro by SF-assemblin, which is the major protein component of system I fibers. We have determined the amino acid sequence of SF-assemblin and show that it contains two structural domains. The NH2-terminal 31 residues form a nonhelical domain rich in proline. The rod domain of 253 residues is alpha-helical and seems to form a segmented coiled coil with a 29-residue repeat pattern based on four heptads followed by a skip residue. The distinct cluster of acidic residues at the COOH-terminal end of the motifs (periodicity about 4 nm) may be related to tubulin binding of SF-assemblin and/or its self assembly. A similar structure has been predicted from cDNA cloning of beta-giardin, a protein of the complex microtubular apparatus of the sucking disc in the protozoan flagellate Giardia lamblia. Although the rod domains of SF-assemblin and beta-giardin share only 20% sequence identity, they have exactly the same length and display 42% sequence similarity. These results predict that system I fibers and related microtubule associated structures arise from molecules able to form a special segmented coiled coil which can pack into 2-nm filaments. Such molecules seem subject to a strong evolutionary drift in sequence but not in sequence principles and length. This conservation of molecular architecture may have important implications for microtubule binding.
Assuntos
Clorófitas/química , Endopeptidases/química , Microtúbulos/química , Serina Endopeptidases , Sequência de Aminoácidos , Clorófitas/ultraestrutura , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Homologia de Sequência de AminoácidosRESUMO
The content of lipid peroxidation products and the metabolism of gangliosides and galactosylceramides in brain tissue were studied in the presence of ischemia and when an agent containing nicotinic acid and cysteine is administered. The above agent was found to have an antioxidative effect that was evident in the lower levels of lipid peroxides and in the normalized qualitative and quantitative content of galactosylceramides and gangliosides.
Assuntos
Antioxidantes/farmacologia , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/metabolismo , Cisteína/farmacologia , Glicolipídeos/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Niacina/farmacologia , Animais , Química Encefálica/efeitos dos fármacos , Masculino , Ratos , Complexo Vitamínico B/farmacologiaRESUMO
Quantitative shifts in the content of interleukine-1, -2 and -6 of the myocardium of mice under the conditions of acoustic stress and delta-sleep inducing peptide action are studied. It has been shown that injection of delta-sleep inducing peptide has no effect on the level of interleukine-1 and -2 in the myocardial tissue, whereas the quantity of interleukine-6 increased. The level of interleukine-1- and -6 in the myocardium were increased and no significant changes were observed in the level of interleukine-2 under the noise action. Interleukine-1- and -2 were not detected in the hypophysis of experimental animals of all groups (intact, under acoustic stress, under delta-sleep inducing peptide application). The level of interleukine-6 in the hypothalamus decreased under conditions of acoustic stress, whereas administration of delta-sleep inducing peptide has no effect on its level. The obtained data are considered in the context of immune modulating properties of delta-sleep inducing peptide.
Assuntos
Peptídeo Indutor do Sono Delta/farmacocinética , Insuficiência Cardíaca/imunologia , Interleucina-1/imunologia , Interleucina-2/imunologia , Interleucina-6/imunologia , Miocárdio/imunologia , Miocárdio/metabolismo , Ruído/efeitos adversos , Fases do Sono/fisiologia , Animais , Peptídeo Indutor do Sono Delta/administração & dosagem , Camundongos , Hipófise/imunologia , Hipófise/metabolismoRESUMO
Essentials Hemorrhagic risk of antiplatelet drugs is generally thought to be lower than anticoagulants. We systematically reviewed trials comparing antiplatelet and anticoagulant drugs in older patients. Overall, the risk of major bleeding was similar with antiplatelet and with anticoagulant drugs. In elderly patients, risks and benefits of antiplatelet drugs should be carefully weighted. SUMMARY: Background The hemorrhagic risk of antiplatelet drugs in older patients could be higher than is usually assumed. Objective To compare the bleeding risk of antiplatelet drugs and oral anticoagulants in elderly patients. Methods We carried out a systematic review and meta-analysis. We searched PubMed, EMBASE and the Cochrane Library up to January 2016 for randomized and non-randomized controlled trials (RCTs) and parallel cohorts comparing antiplatelet drugs and oral anticoagulants in patients aged 65 years or older. Two independent authors assessed studies for inclusion. The pooled relative risk (RR) of major bleeding was estimated using a random model. Results Seven RCTs (4550 patients) and four cohort studies (38 649 patients) met the inclusion criteria. The risk of major bleeding when on aspirin or clopidogrel was equal to that when on warfarin in RCTs (RR, 1.01; 95% confidence interval (95% CI), 0.69-1.48; moderate-quality evidence), lower than when on warfarin in non-randomized cohort studies (RR, 0.87; 95% CI, 0.77-0.99; low-quality evidence) and not different when all studies were combined (RR, 0.86; 95% CI, 0.73-1.01). Bleeding of any severity (RR, 0.70; 95% CI, 0.57-0.86) and intracranial bleeding (RR, 0.46; 95% CI, 0.30-0.73) were less frequent with antiplatelet drugs than with warfarin. All-cause mortality was similar (RR, 0.99). Subgroup analysis suggested that major bleeding might be higher with warfarin than with aspirin in patients over 80 years old. Conclusion Elderly patients treated with aspirin or clopidogrel suffer less any-severity bleeding but have a risk of major bleeding similar to that of oral anticoagulants, with the exception of intracranial bleeding.
Assuntos
Anticoagulantes/efeitos adversos , Fibrilação Atrial/tratamento farmacológico , Inibidores da Agregação Plaquetária/efeitos adversos , Administração Oral , Idoso , Idoso de 80 Anos ou mais , Aspirina/efeitos adversos , Clopidogrel , Estudos de Coortes , Feminino , Hemorragia , Humanos , Hemorragias Intracranianas , Masculino , Ensaios Clínicos Controlados Aleatórios como Assunto , Risco , Acidente Vascular Cerebral/etiologia , Ticlopidina/efeitos adversos , Ticlopidina/análogos & derivados , Resultado do Tratamento , Vitamina K/antagonistas & inibidores , Varfarina/efeitos adversosRESUMO
In eukaryotes, protein deacetylation is carried out by two well-conserved histone deacetylase (HDAC) families: RPD3/HDA1 and SIR2. Intriguingly, model plants such as Arabidopsis express an additional plant-specific HDAC family, termed type-2 HDACs (HD2s). Transcriptomic analyses from more than 1300 green plants generated by the 1000 plants (1KP) consortium showed that HD2s appeared early in green plant evolution, the first members being detected in several streptophyte green alga. The HD2 family has expanded via several rounds of successive duplication; members are expressed in all major green plant clades. Interestingly, angiosperm species express new HD2 genes devoid of a zinc-finger domain, one of the main structural features of HD2s. These variants may have been associated with the origin and/or the biology of the ovule/seed.
Assuntos
Histona Desacetilases/metabolismo , Proteínas de Plantas/metabolismo , Viridiplantae/metabolismo , Regulação da Expressão Gênica de Plantas , Histona Desacetilases/genética , Proteínas de Plantas/genética , Viridiplantae/genéticaRESUMO
Several fiber systems are associated with the flagella and basal bodies of eukaryotic cells. Apart from the contractile and Ca(2+)-sensitive system-II fibers, these include the noncontractile system-I fibers that run parallel to flagellar root microtubules. Using electron microscopy and image reconstruction, we have investigated the structure of the system-I fibers of the flagellate green alga Spermatozopsis similis. The fibers were observed in three different states: (1) in situ, (2) after isolation of the intact fibers, (3) after disassembly and reconstitution of fibers in vitro from their 34 kDa subunit protein. The fibers are highly ordered; they show a constant repeat of 28 nm, they are polar, and they contain several transverse and longitudinal striations. A model is discussed showing the system-I fiber to be built from rod-like molecules with a staggered arrangement and identical polarities.
Assuntos
Clorófitas/ultraestrutura , Flagelos/ultraestrutura , Clorófitas/química , Flagelos/química , Microscopia Eletrônica , Microtúbulos/química , Microtúbulos/ultraestrutura , Difração de Raios XRESUMO
Glycoproteins associated with one type of flagellar scale (p-scale) isolated from the flagellate green alga Tetraselmis striata (Prasinophyceae) were shown to bind the mannose-specific lectin GNA (Galanthus nivalis agglutinin). Enzymatic deglycosylation of the glycoproteins with N-glycosidase F led to an electrophoretic mobility shift to lower molecular masses in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and abolished GNA-binding strongly indicating that most of the scale-associated glycoproteins contain asparagine-linked oligosaccharide side chains presumably of the high mannose type. To evaluate the significance of N-linked glycoproteins for scale structure and integrity, p-scales were digested with various proteases or extracted with 8 M urea and their ultrastructure and protein composition determined. The results show that while scale-associated N-linked glycoproteins do not determine the overall structure of the scale subunits (which consist of complex polysaccharides), they are apparently involved in mediating linkages between scale subunits; we have tentatively identified one glycoprotein of Mr 280,000 which may link outer scale subunits to one another. In addition, some scale-associated N-linked glycoproteins may provide connections between the layer of p-scales and the underlying flagellar membrane.
Assuntos
Clorófitas/química , Flagelos/química , Glicoproteínas/química , Clorófitas/ultraestrutura , Flagelos/ultraestrutura , Galanthus , Glicoproteínas/efeitos dos fármacos , Glicosídeo Hidrolases/farmacologia , Lectinas/metabolismo , Microscopia Eletrônica , Oligossacarídeos/química , Lectinas de Plantas , Serina Endopeptidases/farmacologia , Solubilidade , Subtilisinas/farmacologia , Ureia/farmacologiaRESUMO
The reflection properties of different types of eyespots in three unicellular, flagellated green algae (Tetraselmis chui, Chlamydomonas eugametos, Hafniomonas reticulata) were investigated using confocal laser scanning microscopy in the epireflection mode. The eyespots differed with respect to the number of eyespot lipid globule layers and surface appearance (concave/convex). A strong reflection signal was observed in all eyespots, and a detailed quantitative analysis by optical xy (horizontal) and xz (vertical) sectioning was performed. By applying both sectioning capabilities, multi- and single/double-layered eyespots as well as concave and convex eyespot surfaces could be distinguished using living, immobilized cells. Focusing of the reflected light was only observed in eyespots with concave surfaces. In xz series of multi-layered eyespots at reduced laser intensities (0.01%), the intensity profiles of the reflection revealed a series of alternating maxima and minima with increasing reflection intensities toward the cell surface. At very low laser intensities (0.001%), multi-layered eyespots exhibited about twice the reflection intensity at the presumptive photoreceptor site compared to single/double-layered eyespots. Our results provide the first experimental evidence to support the proposal that multi-layered eyespots act as interference reflectors in photoaxis of green algae.
Assuntos
Clorófitas/ultraestrutura , Microscopia Eletrônica , Microscopia Eletrônica de VarreduraRESUMO
The planar distribution of 3-beta-hydroxysterols in the plasmalemma of the green flagellate Chlamydomonas reinhardii has been studied with the freeze-fracture technique using the polyene antibiotic filipin and the saponin tomatin as cytochemical markers. Filipin-sterol complexes were predominantly observed as 25 to 30 nm protuberances on the E-face of the plasmalemma with corresponding invaginations on the P-face. Generally filipin-sterol and tomatin-sterol complexes were randomly distributed, but were virtually absent from sites of intramembrane particle arrays (IMP-arrays, i.e. the flagellar necklace, the flagellar bracelet including strut arrays, the eyespot membrane). The results suggest that IMP-arrays of Chlamydomonas are deficient in 3-beta-hydroxysterols and may therefore be regarded as special lipid arrays. IMP-arrays might require an altered lipid environment for proper function. Some functional aspects of IMP-arrays in Chlamydomonas are discussed.
Assuntos
Membrana Celular/ultraestrutura , Chlamydomonas/ultraestrutura , Filipina/metabolismo , Técnica de Fratura por Congelamento , Microscopia Eletrônica , Esteróis , Tomatina/metabolismoRESUMO
Highly purified Golgi membranes were isolated from the scaly green flagellate Scherffelia dubia using osmotic shock for controlled cell rupture, differential centrifugations and a discontinuous sucrose density gradient centrifugation. Three Golgi membrane fractions (based on the distribution of IDPase activity in the gradient) at densities 1.14 g/ml, 1.17 g/ml and 1.20 g/ml were obtained. The specific IDPase activity in these fractions was enriched about 78-fold compared to the crude cell homogenate. The Golgi membrane fractions were further characterized by electron microscopy, SDS-PAGE and lectin blotting. The low density fraction (1.14 g/ml) contained two distinct vesicle populations and scale precursors associated with the outer surface of the larger-size vesicles. The medium density fraction (1.17 g/ml) contained in addition to the larger vesicles, multilamellate vesicles and semicircular cisternae. Finally, in the high density fraction (1.20 g/ml) in addition to small and large vesicles, a tubular membrane reticulum was observed. The three Golgi membrane fractions revealed the same complex overall polypeptide composition when analyzed by SDS-PAGE, but gradual quantitative differences in the polypeptide profile between fractions were observed. The lectins GNA, DSA, and AAA bound to several glycoproteins in all Golgi membrane fractions. Deglycosylation with N-glycosidase F showed that all carbohydrate structures recognized by GNA and DSA, and one recognized by AAA were of the N-glycosidic type indicating the presence of both "high mannose" and "processed" N-glycans in the Golgi apparatus of S. dubia.
Assuntos
Clorófitas/ultraestrutura , Complexo de Golgi/ultraestrutura , Membranas Intracelulares/ultraestrutura , Centrifugação com Gradiente de Concentração , Eletroforese em Gel de Poliacrilamida , Flagelos , Lectinas/metabolismo , Microscopia Eletrônica , Microssomos/ultraestrutura , Pressão Osmótica , Frações Subcelulares/químicaRESUMO
We report on the isolation and purification of structurally intact eyespot apparatuses from the naked, biflagellate green alga Spermatozopsis similis. Two eyespot-enriched fractions, separated by sucrose gradient centrifugation, retained the typical reflective properties of eyespots in situ as demonstrated by reflection confocal laser scanning microscopy. Ultrastructurally, both fractions contained eyespot plates consisting of a single layer of lipid globules. Structurally intact eyespot apparatuses, including patches of plasma membrane and chloroplast envelope overlying the eyespot plate and a single thylakoid subtending the eyespot plate, were particularly enriched in one of the two fractions (fraction 2a). Measurement of several marker enzymes and chlorophyll content (less than 0.001% of total) established the absence of most other cell organelles from the eyespot fractions. The absorption spectra of the two fractions were dominated by carotenoids with an additional shoulder at 540 nm. Following extraction with organic solvents and sodium dodecyl sulfate polyacrylamide gel electrophoresis, several proteins were found to be considerably enriched in the two fractions. In addition to several proteins in the high Mr range, at least 4 polypeptides of 35, 29, 23, and 20 kDa are selectively enriched in fraction 2a with the 29 and 20 kDa proteins being the most prominent. The presence of glycoproteins in fraction 2a was demonstrated by binding of the mannose-specific lectin Galanthus nivalis agglutinin to several high molecular weight polypeptides. In addition, a hydrophobic component with abnormal electrophoretic mobility that reacts strongly with periodic acid-Schiff and thymol/sulfuric acid was prominent in both fractions. Mass isolation and purification of the intact phototactic apparatus of a flagellate green alga now greatly facilitates the biochemical and molecular characterization of the signal transduction chain involved in green algal phototaxis.
Assuntos
Movimento Celular , Clorófitas/ultraestrutura , Organelas/fisiologia , Células Fotorreceptoras/fisiologia , Western Blotting , Clorófitas/fisiologia , Eletroforese em Gel de Poliacrilamida , Galanthus , Luz , Microscopia Eletrônica de Varredura/métodosRESUMO
Isolation of intact photoreceptive organelles (eyespot apparatuses) involved in blue-light mediated photoresponses in a flagellate green alga (Spermatozopsis similis) allowed for the first time the identification of both 11-cis- and all-trans-retinal in a plant cell. Both isomers were identified by HPLC analysis in conjunction with UV spectra. Additionally, reconstitution of a distinct absorption band, centered around 540 nm, was achieved by addition of exogenous 9-cis-retinal to bleached, isolated eyespot apparatuses.
Assuntos
Proteínas de Transporte/química , Clorófitas/química , Organelas/química , Células Fotorreceptoras/química , Retinaldeído/química , Clorófitas/fisiologia , Flagelos , Organelas/fisiologia , Células Fotorreceptoras/fisiologia , Fotossíntese/fisiologia , Retinaldeído/fisiologiaRESUMO
The cortical microtubular cytoskeleton of the colorless, phagotrophic dinoflagellate Oxyrrhis marina has been investigated by immunofluorescence and transmission electron microscopy. It consists of two systems, an anterior system comprising microtubular bands (of 2-4 microtubules each) which extend from a focal point at the cell apex to about three-quarters of the cell length where they either become transversely oriented (on the ventral right surface of the cell) or abut transversely oriented microtubules (on the dorsal and ventral left cell surface); and a posterior system in which microtubular bands extend from a focal point near the basal apparatus posteriorly around the antapex of the cell to become transversely oriented in the region where they meet the abutting anterior microtubular bands. The peripheral cytoskeleton of Oxyrrhis contains no continuous pole-to-pole microtubules and is thus basically similar to that of other dinoflagellates. Upon phagotrophic feeding the peripheral microtubular cytoskeleton undergoes reversible rearrangements. The non-permanent cytostome is located at the right ventral surface of the cell between the ventral ridge microtubules (vrm) and the groove of the longitudinal flagellum. During phagocytosis the anteriorly focused microtubular bands of the peripheral cytoskeleton near the right ventral surface of the cell are 'lifted' or 'pushed' towards the vrm to enable uptake of food organisms of diverse size and shape. Within minutes after phagocytosis the microtubular bands are relocated to their former position. We conclude that the organization of a peripheral microtubular cytoskeleton from two opposite focal points provided the dinoflagellates with the flexibility needed to evolve the multitude of phagocytotic mechanisms that characterize this group of protists today.
RESUMO
Complete nuclear-encoded SSU rRNA sequences have been obtained from three taxa of streptophyte green algae (Klebsormidium nitens, Nitella capillaris, Chaetosphaeridium globosum) and two strains of the scaly green flagellate Mesostigma viride. Phylogenetic analyses of 70 taxa of Viridiplantae (Chlorophyta and Streptophyta) and 57 taxa of streptophyte green algae and embryophyte plants using distance, parsimony and likelihood methods revealed a novel monophyletic lineage among the Streptophyta comprising the genera Mesostigma and Chaetosphaeridium. This lineage is described here as the Mesostigmatophyceae classis nova. Our analyses demonstrate that (1) scaly green flagellates (prasinophytes) are polyphyletic, (2) a scaly green flagellate is a member of the Streptophyta and forms a clade with the oogamous, filamentous Chaetosphaeridium to the exclusion of all other known streptophyte green algae, (3) a previously published SSU rRNA sequence of Chaetosphaeridium (AF113506) is chimeric and contains part of a fungal SSU rRNA, and (4) the phylogenetic relationships between the Mesostigmatophyceae and other streptophyte green algae remain unresolved by SSU rRNA sequence comparisons.
Assuntos
Clorófitas/classificação , DNA de Plantas/genética , DNA Ribossômico/genética , Clorófitas/genética , Clorófitas/ultraestrutura , Classificação , Evolução Molecular , Funções Verossimilhança , Filogenia , Especificidade da Espécie , Terminologia como AssuntoRESUMO
Partial sequencing of cDNA libraries to generate expressed sequence tags (ESTs) is an effective means of gene discovery, generation of molecular markers and characterization of transcription patterns. We have constructed an EST-database of the scaly green flagellate Scherffelia dubia (Chlorophyta) containing 361 sequences. cDNAs were obtained from interphase cells and from cells regenerating flagella. Analysis of the ESTs identified 138 EST-groups with significant similarity to known sequences. 134 EST-groups showed no significant similarity to any sequences in the databases. Most of the ESTs with similarity to known proteins are associated with typical interphase cell functions of a photosynthetic plant cell: assimilation of nutrients and biosynthesis of proteins. Others are related to the activation of the secretory pathway or the biogenesis of scales (e.g. kdo-synthase). Comparison of S. dubia ESTs with the genome of Arabidopsis thaliana and the EST database of Chlamydomonas reinhardtii revealed that S. dubia ESTs with similarity to known proteins were more similar to sequences in C. reinhardtii than to those of A. thaliana. Additionally, ESTs for guanylyl cyclase and cGMP phosphodiesterase are present in the two flagellates, but so far these gene products have not been found in embryophytes.
Assuntos
Clorófitas/genética , Eucariotos/genética , Etiquetas de Sequências Expressas , Genes de Protozoários , Regiões 3' não Traduzidas , Animais , Arabidopsis/genética , Chlamydomonas/genética , Bases de Dados Factuais , Biblioteca Gênica , Genoma de PlantaRESUMO
Highly purified flagella of the green alga Tetraselmis striata (Chlorophyta) were extracted by Triton X-114 phase partitioning. SDS-PAGE analysis revealed that most proteins were present in the aqueous phase, only two prominent flagellar membrane proteins (fmp) of apparent molecular weight 145 and 57 kDa (fmp145 and fmp57) were enriched in the detergent phase. Fmp145 was purified by gel permeation chromatography. Glycosidase treatment in combination with lectin blot analysis showed that fmp145 is a glycoprotein containing 3-5 N-glycans of the high mannose and/or hybrid type. A polyclonal antibody (anti-fmp136) was raised against the deglycosylated form of fmp145 and used to localize fmp145 by immunofluorescence and immunoelectron microscopy. Immunogold labeling showed fmp145 to be present between the scale layers and the flagellar membrane. During flagellar regeneration fmp145 is incorporated evenly and rapidly into the newly developing flagella. Anti-fmp136 specifically cross-reacted with flagella of only a subgroup of Tetraselmis strains characterized by a specific flagellar hair type (type II according to Marin et al. 1993) and thus could be a useful immunomarker for the identification of Tetraselmis strains by fluorescence microscopy.