RESUMO
Cardiosporidium cionae (Apicomplexa), from the ascidian Ciona intestinalis L., is redescribed with novel ultrastructural, phylogenetic and prevalence data. Ultrastructural analysis of specimens of C. intestinalis collected from the Gulf of Naples showed sporonts and plasmodia of C. cionae within the host pericardial body. Several merogonic stages and free merozoites were found in the pericardial body, together with sexual stages. All stages showed typical apicomplexan cell organelles, i.e. apicoplasts, rhoptries and subpellicular microtubules. Merogonic stages of C. cionae were also produced inside haemocytes. A fragment of the rSSU gene of C. cionae was amplified by PCR using DNA extracted from the pericardial bodies. The amplified product showed closest affinity with other apicomplexan representatives and a 66bp unique insertion, specific for C. cionae, at position 1644. Neighbour-joining phylogenetic analysis placed C. cionae in a clade with other piroplasm genera, including Cytauxzoon, Babesia and Theileria spp. The parasite was found in different populations of C. intestinalis with highest prevalence in October-November. Ultrastructural and DNA data showed that the organism, described in 1907 from the same host but not illustrated in detail, is a member of a novel marine apicomplexan radiation of tunicate parasites.
Assuntos
Hemócitos/parasitologia , Piroplasmida/classificação , Urocordados/parasitologia , Animais , Sequência de Bases , DNA de Protozoário/análise , DNA de Protozoário/isolamento & purificação , Hemócitos/ultraestrutura , Interações Hospedeiro-Parasita , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Filogenia , Piroplasmida/genética , Piroplasmida/crescimento & desenvolvimento , Piroplasmida/ultraestrutura , Reação em Cadeia da Polimerase , Prevalência , Alinhamento de Sequência , Urocordados/ultraestruturaRESUMO
Mouse erythroleukemia (Mel) cells have a cell cycle-dependent high sensitivity to chemical and physical mutagens. This report shows that a 5 h exposure to 0.1 or 0.01 microg/ml metavanadate during the initial period of erythroid differentiation induction was sufficient to permanently damage the ability of treated Mel cells and their progeny to undergo erythroid differentiation, without affecting cell viability and proliferation. Conversely, a 5 h pulse of metavanadate at 1 or 10 microg/ml inhibited both differentiation and cell proliferation. The cell cycle-dependent period of mutagenesis was essential for fixation of damage in the cell genome and the progeny of the cells treated with 0.1 or 0.01 microg/ml metavanadate stably inherited an impaired capacity to differentiate. The efficiency of the DNA repair synthesis machinery during the specific period of exposure of Mel cells seemed directly involved in damage fixation. In fact, the mutagenic effects of a 0.1 microg/ml metavanadate pulse was further increased in the presence of 1 mM hydroxyurea, an inhibitor of DNA repair synthesis. In contrast, 5 microg/ml vanillin, an antimutagenic agent that stimulates repair, completely restored the capacity of progeny of cells treated with 0.1 microg/ml metavanadate to complete differentiation. Determination of [(3)H]deoxythymidine in acid-insoluble DNA indicated that incorporation was stimulated by metavanadate alone and was further increased by metavanadate plus vanillin; conversely, incorporation of thymidine was reduced in the presence of hydroxyurea. The capacity of metavanadate to permanently damage Mel cell erythroid differentiation appeared to depend on the cell cycle-related efficiency of the DNA repair systems, activated to correct the induced alteration, rather than on a specific concentration.
Assuntos
Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Leucemia Eritroblástica Aguda/patologia , Vanadatos/toxicidade , Animais , Reparo do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Vírus da Leucemia Murina de Friend , Leucemia Eritroblástica Aguda/genética , Leucemia Eritroblástica Aguda/metabolismo , Camundongos , Células Tumorais CultivadasRESUMO
The "Henze solution", derived originally from the aqueous extraction of pelleted whole blood from the ascidian Phallusia mammillata, was examined using spectral studies. The aqueous extraction of fractionated blood cells including compartment cells, signet ring cells, and morula cells obtained using cell separation techniques were also examined. It was found that this Henze solution, and the Henze precipitate itself derived from this solution, emanated solely from the morula cells. Furthermore, it was found that this solution is formed independently of the vanadium metal ions otherwise associated with the vanadocytes. Observation of the Henze precipitate by light microscopy shows that this material partially forms crystallites or microglasses.
Assuntos
Extratos de Tecidos/isolamento & purificação , Urocordados/citologia , Animais , Células Sanguíneas/química , Células Sanguíneas/citologia , Soluções , Urocordados/químicaRESUMO
Proteolytic activity was studied in the fractionated blood cells of the vanadium accumulating ascidian P. mammillata by separating the cells before measuring their activity. Cells were separated to avoid vanadocyte breakdown and subsequent vanadium diffusion into the assay medium. Our study revealed activity in the morula cell extract that was obtained by sonication and Centricon concentration. After removing part of the extract for enzyme activity assay the remainder was kept at 0 degrees C; it was later found that much of the protein in this latter fraction formed a sediment whereas the protease remained in solution. The serine-protease substrate specificity of the enzyme was measured and the results are discussed in relation to serine protease involvement in immune defense.
Assuntos
Endopeptidases/sangue , Urocordados/metabolismo , Vanádio/metabolismo , Animais , Células Sanguíneas/citologia , Células Sanguíneas/enzimologia , Fracionamento Celular , Separação CelularRESUMO
A study was made on the effect of vanadium, in both the tetravalent state in vanadyl sulphate and in the pentavalent state in sodium meta-vanadate, and ortho-vanadate, on the proteolysis of azocasein by two serine proteases, trypsin and subtilisin and two cysteine proteases bromelain and papain. Also the proteolysis of bovine azoalbumin by serine proteases was considered. An inhibitory effect was present in all cases, except meta-vanadate with subtilisin. The oxidation level of vanadium by itself did not determine the inhibition kinetics, which also depended on the type and composition of the vanadium containing molecule and on the enzyme assayed. The pattern of inhibition was similar for proteases belonging to the same class. The highest inhibition was obtained with meta-vanadate on papain and with vanadyl sulphate on bromelain.
Assuntos
Inibidores de Cisteína Proteinase/farmacologia , Inibidores de Serina Proteinase/farmacologia , Vanádio/farmacologia , Albuminas/metabolismo , Animais , Bromelaínas/antagonistas & inibidores , Bromelaínas/metabolismo , Caseínas/metabolismo , Bovinos , Técnicas In Vitro , Cinética , Oxirredução , Papaína/antagonistas & inibidores , Papaína/metabolismo , Subtilisinas/antagonistas & inibidores , Subtilisinas/metabolismo , Tripsina/metabolismo , Vanadatos/farmacologia , Vanádio/química , Compostos de Vanádio/farmacologiaRESUMO
When the blood cells of ascidians Phallusia mammillata are stained with the ligand 2,2'-bipyridine, those cells which contain vanadium(III), in an easily sequestered form, take up the stain producing in situ, a purple complex. This material extracted displays spectral characteristics consistent with the formation of an oxo-bridge vanadium(III) bipyridine dimer. The staining is localized in the signet ring cell, a bivacuolated cell, a cell type with numerous darkly staining compartments, and also by the vacuolated amoebocyte. The possible ramifications of these observation are discussed in relation to the delineation of the signet ring cell lineage.
Assuntos
Histocitoquímica , Urocordados/citologia , Vanádio/sangue , 2,2'-Dipiridil , Animais , Separação Celular , Centrifugação com Gradiente de Concentração , Corantes , Hemócitos/química , Urocordados/metabolismoRESUMO
We have purified a 34 kDa hatching enzyme from the water in which the embryos of the sea-squirt Ciona intestinalis hatch. This enzyme was obtained in homogeneous form as judged from SDS-PAGE and HPLC gel filtration. The enzyme possesses proteolytic activity and is able to digest the chorion of the egg of C. intestinalis. It is a metalloproteinase and contains one atom of Zn per molecule. The optimum pH is 8.5. The enzyme shows hydrolytic activity towards the -CO-NH- bonds, which are hydrolyzed by the members of the serine proteinase family. It has a trypsin-like activity in that it cuts the bond of Arg and Lys at P1 position of the scissile bond -P1-P1', but it differs from trypsin insofar as it hydrolyzes the peptide bond on either side of Arg and Lys. The purified enzyme is inhibited by the common metal-chelators and by the classical trypsin proteinase inhibitors. The apparent K(m) values at 37 degrees C and pH 8.5 toward tosyl-Gly-Pro-Arg-NHNap, tosyl-Gly-Pro-Lys-NHNap and Bz-Arg-Gly-Arg-NHNap were 0.125, 0.5 and 2.5 mM, respectively. The results obtained in this study suggest that the hatching enzyme from C. intestinalis exhibits both trypsin-like activity and metalloproteinase activity.
Assuntos
Ciona intestinalis/enzimologia , Metaloendopeptidases/isolamento & purificação , Animais , Ciona intestinalis/embriologia , Embrião não Mamífero/enzimologia , Concentração de Íons de Hidrogênio , Cinética , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/metabolismo , Especificidade por Substrato , TemperaturaRESUMO
A novel polypeptide containing the unusual posttranslationally modified amino acids L-3,4,5-trihydroxyphenylalanine (TOPA) and L-6-bromotryptophan (6-BrW) has been isolated from the morula cells of the vanadium-accumulating ascidian, Phallusia mammillata. The polypeptide, designated Morulin Pm, has a molecular weight of 3825 +/- 0.6 and has a simple amino acid composition consisting mainly of TOPA and 6-BrW as well as Ser, Leu, Phe, and Ala. To our knowledge, this is the first reported example of multiple sites of brominated tryptophan in a polypeptide of this size. Edman degradation revealed the N-terminal sequence to be BrW-Leu-Phe-BrW before sequencing was blocked. While the N-terminal tripeptide could be isolated from chymotrypsin digests of Morulin Pm, the rest of the polypeptide resisted further cleavage by the proteases, a feature common among this class of peptides. However, unlike other ascidian blood cell peptides examined to date, microheterogeneity was minimal. For the first time a detailed NMR investigation could be undertaken on a member of this class of polypeptides. In addition to signals assignable to the constituent amino acids by extensive 2D experiments, resonances were present both in the 13C and 1H spectra not typical of a simple linear peptide. Two proton resonances were identified with a cross peak in the correlation spectrum strongly indicative of a C-terminal decarboxy-delta 2,3-unsaturated TOPA residue as observed in certain tunichromes and clionamide. Chemical degradation experiments were undertaken in an effort to produce identifiable fragments to which these signals could be assigned, including full and partial acid hydrolysis and tryptophan-targeted BNPS-skatole treatment. However, the nature of the modification remains unknown. Possible structures for the modification, which may represent the source of the difficulties encountered in the structural elucidation of this and related peptides, are assessed. Conjecture is made as to the biological relevance of Morulin Pm, based on its localization and chemical characteristics.
Assuntos
Células Sanguíneas/química , Di-Hidroxifenilalanina/análogos & derivados , Peptídeos/química , Triptofano/análogos & derivados , Urocordados/química , Aminoácidos/análise , Animais , Cromatografia Líquida de Alta Pressão , Quimotripsina/metabolismo , Di-Hidroxifenilalanina/análise , Eletroforese em Gel de Poliacrilamida , Cromatografia Gasosa-Espectrometria de Massas , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Estrutura Molecular , Peso Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Peptídeos/isolamento & purificação , Processamento de Proteína Pós-Traducional , Análise de Sequência , Espectrofotometria , Triptofano/análise , Urocordados/citologiaRESUMO
Cryofixed blood morula cells of Phallusia mammillata (Cuvier), which are considered to be vanadium-accumulating cells, were examined by X-ray microanalysis using STEM (scanning transmission electron microscopy) and SEM (scanning electron microscopy). It is thought that cryopreparation preserves the native distribution of diffusible elements such as sodium, chlorine, and potassium, and prevents the displacement of vanadium, all of which may occur during conventional preparation. The results show that morula cell globules contain a large amount of sulphur and chlorine, and some sodium, magnesium, bromium and potassium, but very little or no vanadium.
Assuntos
Urocordados/análise , Vanádio/análise , Animais , Microanálise por Sonda Eletrônica , Congelamento , Mórula/análise , Urocordados/citologia , Urocordados/embriologiaRESUMO
X-ray histospectrographic analysis at the scanning and transmission electron microscope (STEM) are made on the blood cells of Phallusia mamillata Cuvier and Ciona intestinalis, to study the 'direct' intracellular sites of accumulation of vanadium. The results show a clear accumulation of vanadium on the membrane and in the granules of vacuoles of amebocytes, signet ring cell, compartment cell and traces of metal in the 'vanadophores' of vanadocytes.
Assuntos
Células Sanguíneas/análise , Urocordados/metabolismo , Vanádio/análise , Animais , Microscopia Eletrônica , Especificidade da EspécieRESUMO
An ultrastructural study of the nucleolus of embryos of Paracentrotus lividus was carried out after treatment with Actinomycin D. It was shown that the fibrillar component of the nucleolus persists in the embryos treated with Actinomycin D in the mesenchyme blastula stage and fixed 24 and 48 hr after fertilization. The results are discussed in relation to the synthesis of RNA.