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1.
Mycologia ; 107(1): 157-68, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25232071

RESUMO

A new species of Didymium (Myxomycetes), D. xerophilum, is described, and some details of its life cycle are provided. The new species was collected during studies of arid areas of Argentina and Peru. It can be distinguished by the persistent funnel-shaped invagination of the peridium, the top of which appears as a deep umbilicus in closed sporothecae, and the calcareous hypothallus shared among several sporocarps. This combination of characters, with a circumscissile dehiscence of the sporotheca and a cream stalk packed with rhombic lime crystals, is unknown in other described species. Morphology was examined with scanning electron microscopy and light microscopy, and micrographs of relevant details are included here. Phylogenetic analysis with 18S rDNA sequences of different species of Didymium supports the distinct identity of this new species. Some collections of this myxomycete were made at up to 4600 m, an altitude almost unknown for this group of microorganisms.


Assuntos
Mixomicetos/classificação , Mixomicetos/isolamento & purificação , Altitude , Argentina , Dados de Sequência Molecular , Mixomicetos/genética , Mixomicetos/crescimento & desenvolvimento , Peru , Filogenia , Esporos de Protozoários/classificação , Esporos de Protozoários/enzimologia , Esporos de Protozoários/genética , Esporos de Protozoários/isolamento & purificação
2.
J Biol Chem ; 288(12): 8198-8208, 2013 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-23372162

RESUMO

Ribonucleotide reductases (RNRs) catalyze the only pathway for de novo synthesis of deoxyribonucleotides needed for DNA replication and repair. The vast majority of eukaryotes encodes only a class I RNR, but interestingly some eukaryotes, including the social amoeba Dictyostelium discoideum, encode both a class I and a class II RNR. The amino acid sequence of the D. discoideum class I RNR is similar to other eukaryotic RNRs, whereas that of its class II RNR is most similar to the monomeric class II RNRs found in Lactobacillus spp. and a few other bacteria. Here we report the first study of RNRs in a eukaryotic organism that encodes class I and class II RNRs. Both classes of RNR genes were expressed in D. discoideum cells, although the class I transcripts were more abundant and strongly enriched during mid-development compared with the class II transcript. The quaternary structure, allosteric regulation, and properties of the diiron-oxo/radical cofactor of D. discoideum class I RNR are similar to those of the mammalian RNRs. Inhibition of D. discoideum class I RNR by hydroxyurea resulted in a 90% reduction in spore formation and decreased the germination viability of the surviving spores by 75%. Class II RNR could not compensate for class I inhibition during development, and an excess of vitamin B12 coenzyme, which is essential for class II activity, did not improve spore formation. We suggest that class I is the principal RNR during D. discoideum development and growth and is important for spore formation, possibly by providing dNTPs for mitochondrial replication.


Assuntos
Dictyostelium/enzimologia , Proteínas de Protozoários/metabolismo , Ribonucleotídeo Redutases/metabolismo , Regulação Alostérica , Complexos de Coordenação/química , Cistina Difosfato/química , Dictyostelium/genética , Dictyostelium/fisiologia , Inibidores Enzimáticos/farmacologia , Radicais Livres/química , Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Guanosina Difosfato/química , Ferro/química , Cinética , Filogenia , Proteínas de Protozoários/genética , Ribonucleotídeo Redutases/antagonistas & inibidores , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/genética , Espectrofotometria Ultravioleta , Esporos de Protozoários/enzimologia , Esporos de Protozoários/genética , Tirosina/química
3.
Parasitol Res ; 112(3): 1221-7, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23271570

RESUMO

A key challenge in the successful treatment of Acanthamoeba infections is its ability to transform into a dormant cyst form that is resistant to physiological conditions and pharmacological therapies, resulting in recurrent infections. The carbohydrate linkage analysis of cyst walls of Acanthamoeba castellanii showed variously linked sugar residues, including xylofuranose/xylopyranose, glucopyranose, mannopyranose, and galactopyranose. Here, it is shown that exogenous xylose significantly reduced A. castellanii differentiation in encystation assays (P < 0.05 using paired t test, one-tailed distribution). Using small interfering RNA (siRNA) probes against xylose isomerase and cellulose synthase, as well as specific inhibitors, the findings revealed that xylose isomerase and cellulose synthase activities are crucial in the differentiation of A. castellanii. Inhibition of both enzymes using siRNA against xylose isomerase and cellulose synthase but not scrambled siRNA attenuated A. castellanii metamorphosis, as demonstrated by the arrest of encystation of A. castellanii. Neither inhibitor nor siRNA probes had any effect on the viability and extracellular proteolytic activities of A. castellanii.


Assuntos
Acanthamoeba castellanii/enzimologia , Acanthamoeba castellanii/crescimento & desenvolvimento , Aldose-Cetose Isomerases/antagonistas & inibidores , Glucosiltransferases/antagonistas & inibidores , RNA Interferente Pequeno/metabolismo , Esporos de Protozoários/enzimologia , Esporos de Protozoários/crescimento & desenvolvimento , Aldose-Cetose Isomerases/genética , Inativação Gênica , Glucosiltransferases/genética , RNA Interferente Pequeno/genética
4.
BMC Dev Biol ; 12: 31, 2012 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-23098648

RESUMO

BACKGROUND: Oxygen sensing is a near universal signaling modality that, in eukaryotes ranging from protists such as Dictyostelium and Toxoplasma to humans, involves a cytoplasmic prolyl 4-hydroxylase that utilizes oxygen and α-ketoglutarate as potentially rate-limiting substrates. A divergence between the animal and protist mechanisms is the enzymatic target: the animal transcriptional factor subunit hypoxia inducible factor-α whose hydroxylation results in its poly-ubiquitination and proteasomal degradation, and the protist E3SCF ubiquitin ligase subunit Skp1 whose hydroxylation might control the stability of other proteins. In Dictyostelium, genetic studies show that hydroxylation of Skp1 by PhyA, and subsequent glycosylation of the hydroxyproline, is required for normal oxygen sensing during multicellular development at an air/water interface. Because it has been difficult to detect an effect of hypoxia on Skp1 hydroxylation itself, the role of Skp1 modification was investigated in a submerged model of Dictyostelium development dependent on atmospheric hyperoxia. RESULTS: In static isotropic conditions beneath 70-100% atmospheric oxygen, amoebae formed radially symmetrical cyst-like aggregates consisting of a core of spores and undifferentiated cells surrounded by a cortex of stalk cells. Analysis of mutants showed that cyst formation was inhibited by high Skp1 levels via a hydroxylation-dependent mechanism, and spore differentiation required core glycosylation of Skp1 by a mechanism that could be bypassed by excess Skp1. Failure of spores to differentiate at lower oxygen correlated qualitatively with reduced Skp1 hydroxylation. CONCLUSION: We propose that, in the physiological range, oxygen or downstream metabolic effectors control the timing of developmental progression via activation of newly synthesized Skp1.


Assuntos
Dictyostelium/crescimento & desenvolvimento , Oxigênio/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Protozoários/metabolismo , Proteínas Quinases Associadas a Fase S/metabolismo , Dictyostelium/enzimologia , Dictyostelium/fisiologia , Glicosilação , Hidroxilação , Oxigênio/fisiologia , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Esporos de Protozoários/citologia , Esporos de Protozoários/enzimologia , Esporos de Protozoários/fisiologia
5.
BMC Microbiol ; 12: 284, 2012 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-23190735

RESUMO

BACKGROUND: Regulation of surface antigenic variation in Giardia lamblia is controlled post-transcriptionally by an RNA-interference (RNAi) pathway that includes a Dicer-like bidentate RNase III (gDicer). This enzyme, however, lacks the RNA helicase domain present in Dicer enzymes from higher eukaryotes. The participation of several RNA helicases in practically all organisms in which RNAi was studied suggests that RNA helicases are potentially involved in antigenic variation, as well as during Giardia differentiation into cysts. RESULTS: An extensive in silico analysis of the Giardia genome identified 32 putative Super Family 2 RNA helicases that contain almost all the conserved RNA helicase motifs. Phylogenetic studies and sequence analysis separated them into 22 DEAD-box, 6 DEAH-box and 4 Ski2p-box RNA helicases, some of which are homologs of well-characterized helicases from higher organisms. No Giardia putative helicase was found to have significant homology to the RNA helicase domain of Dicer enzymes. Additionally a series of up- and down-regulated putative RNA helicases were found during encystation and antigenic variation by qPCR experiments. Finally, we were able to recognize 14 additional putative helicases from three different families (RecQ family, Swi2/Snf2 and Rad3 family) that could be considered DNA helicases. CONCLUSIONS: This is the first comprehensive analysis of the Super Family 2 helicases from the human intestinal parasite G. lamblia. The relative and variable expression of particular RNA helicases during both antigenic variation and encystation agrees with the proposed participation of these enzymes during both adaptive processes. The putatives RNA and DNA helicases identified in this early-branching eukaryote provide initial information regarding the biological role of these enzymes in cell adaptation and differentiation.


Assuntos
Variação Antigênica , DNA Helicases/metabolismo , Giardia lamblia/enzimologia , Giardia lamblia/crescimento & desenvolvimento , RNA Helicases/metabolismo , Esporos de Protozoários/crescimento & desenvolvimento , Biologia Computacional , DNA Helicases/genética , Genoma de Protozoário/genética , Giardia lamblia/genética , RNA Helicases/genética , Esporos de Protozoários/enzimologia
6.
Parasitol Res ; 109(2): 417-23, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21286750

RESUMO

Entamoeba histolytica forms chitin-walled cysts during encystation process, where formation of the cyst wall needs not only chitin synthase but also chitinase. During excystation, quadruplet amoebae emerge from the chitin-walled cysts by dissolving the wall, so that chitinase may be necessary for excystation process as well. There is, however, no report on chitinase expression during excystation. In this study, we used Entamoeba invadens, a reptilian amoeba, as a model for encystation and excystation of E. histolytica, and studied chitinase mRNA expression in those processes. Although expression of three E. invadens chitinases designated EiChit1, EiChit2, and EiChit3 during encystation has been reported, we identified another enzyme named as EiChit4 in the E. invadens genome database. Therefore, we investigated the primary structure and mRNA expression of these four chitinases of Ei in the excystation as well as the encystation by real-time reverse transcription polymerase chain reaction (RT-PCR). Like EiChit1, EiChit4 had an 8 × Cys chitin-binding domain (CBD) and a hydrophilic spacer between the CBD and catalytic domain, and was also closer to EiChit1 than EiChit2 and EiChit3 in the phylogenetic tree. During encystation, the expression of all four chitinases increased in the early phase; the increase in EiChit1 and EiChit4 was much higher than in EiChit2 and EiChit3. Then, the expression of all four chitinases sharply decreased in the later phase. In cysts, EiChit1 was most abundantly expressed and EiChit4 was at a lower level, while the expressions of EiChit2 and EiChit3 were virtually absent. Following the induction of excystation, mRNA levels of EiChit1 and EiChit4 in cysts 5 h after induction were significantly lower than those in cysts before induction, while those of EiChit2 and EiChit3 were remarkably higher than before induction. The mRNAs of only EiChit2 and EiChit3 remarkably increased when the excystation was induced in the presence of cytochalasin D. These data demonstrate different structures and expressions of four chitinases in the differentiation of E. invadens.


Assuntos
Quitinases/biossíntese , Quitinases/genética , Entamoeba/enzimologia , Entamoeba/crescimento & desenvolvimento , RNA Mensageiro/biossíntese , Esporos de Protozoários/enzimologia , Esporos de Protozoários/crescimento & desenvolvimento , Sequência de Aminoácidos , Análise por Conglomerados , DNA de Protozoário/química , DNA de Protozoário/genética , Entamoeba/genética , Entamoeba/metabolismo , Regulação da Expressão Gênica , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Esporos de Protozoários/genética , Esporos de Protozoários/metabolismo
7.
Sci Rep ; 8(1): 6643, 2018 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-29704004

RESUMO

Sporulation in Dictyostelium fruiting bodies evolved from amoebozoan encystation with both being induced by cAMP acting on PKA, but with downstream components still being unknown. Using tagged mutagenesis to find missing pathway components, we identified a sporeless mutant defective in a nuclear protein, SpaA. Expression of prespore genes was strongly reduced in spaA- cells, while expression of many spore stage genes was absent. Chromatin immunoprecipitation (ChIP) of a SpaA-YFP gene fusion showed that (pre)spore gene promoters bind directly to SpaA, identifying SpaA as a transcriptional regulator. SpaA dependent spore gene expression required PKA in vivo and was stimulated in vitro by the membrane-permeant PKA agonist 8Br-cAMP. The PKA agonist also promoted SpaA binding to (pre)spore promoters, placing SpaA downstream of PKA. Sequencing of SpaA-YFP ChIPed DNA fragments revealed that SpaA binds at least 117 (pre)spore promoters, including those of other transcription factors that activate some spore genes. These factors are not in turn required for spaA expression, identifying SpaA as the major trancriptional inducer of sporulation.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dictyostelium/crescimento & desenvolvimento , Esporos de Protozoários/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo , Imunoprecipitação da Cromatina , Análise Mutacional de DNA , DNA de Protozoário/metabolismo , Dictyostelium/enzimologia , Dictyostelium/genética , Dictyostelium/metabolismo , Deleção de Genes , Perfilação da Expressão Gênica , Regiões Promotoras Genéticas , Ligação Proteica , Esporos de Protozoários/enzimologia , Esporos de Protozoários/genética , Esporos de Protozoários/metabolismo , Fatores de Transcrição/genética
8.
Biochim Biophys Acta ; 1641(1): 55-64, 2003 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-12788229

RESUMO

Changes in the levels of reactive oxygen species (ROS) have been associated previously with cell differentiation and development in several systems. Thus, there is interest in studying the developmental regulation of antioxidant enzymes, whose activities may modulate ROS levels and subsequent oxidant-mediated signal transduction events in specific tissues. Our recent identification in Dictyostelium discoideum of the prespore-specific catalase B (CatB) enzyme suggested (a) that the CatB enzyme functions to provide protection to the mature spores, and (b) that the CatB enzyme may have a regulatory role in cell differentiation and morphogenesis. We have now confirmed both these hypotheses. We specifically disrupted the catB gene by homologous recombination. The resulting catB null strain displays a 4-h delay in development at the time of normal catB gene expression, followed by slow and asynchronous development of fruiting bodies, taking 10 h longer than the isogenic parent strain. The expression of both prestalk- and prespore-specific genes was altered in the mutant both temporally and quantitatively, and the resultant mutant spores had increased sensitivity to H(2)O(2). This study supports the idea that CatB functions in the development of D. discoideum by regulating the level of ROS, and adds to the growing body of evidence for regulatory roles for ROS.


Assuntos
Catalase/metabolismo , Dictyostelium/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Esporos de Protozoários/enzimologia , Animais , Catalase/genética , Diferenciação Celular , Sobrevivência Celular , Dictyostelium/citologia , Temperatura Alta , Peróxido de Hidrogênio/farmacologia , Morfogênese , Mutação , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Recombinação Genética , Esporos de Protozoários/efeitos dos fármacos
9.
FEMS Microbiol Lett ; 247(1): 81-90, 2005 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-15927751

RESUMO

Microsporidia are fungal-like unicellular eukaryotes which develop as obligate intracellular parasites. They differentiate into resistant spores that are protected by a thick cell wall composed of glycoproteins and chitin. Despite an extensive description of the fibrillar structure of this wall, very little is known about its protein components and deposit mechanisms. In this study on the human pathogen Encephalitozoon cuniculi, we identify by mass spectrometry the target of polyclonal antibodies previously raised against a 33-kDa protein located at the outer face of the parasite plasma membrane. This 254-amino acid protein is encoded by the ECU11_0510 open reading frame and presents two isoforms of 33 and 55 kDa. Sequence analysis supports an assignment to the polysaccharide deacetylase family with a suspected chitin deacetylase activity (EcCDA). As demonstrated by TEM studies, EcCDA is present at the plasma membrane of the early stages of E. cuniculi life-cycle. At the sporoblast stage, the enzyme accumulates especially in paramural bodies which are convolutions of the plasma membrane opened to the wall. The identification of an EcCDA homologue in the insect parasite Antonospora locustae (ex Nosema locustae) suggests a widespread distribution of this enzyme among Microsporidia. This characterization of a new microsporidian surface protein creates new perspectives to understand spore wall formation and spore resistance.


Assuntos
Amidoidrolases/fisiologia , Parede Celular/fisiologia , Encephalitozoon cuniculi/fisiologia , Amidoidrolases/genética , Amidoidrolases/isolamento & purificação , Sequência de Aminoácidos , Animais , Parede Celular/enzimologia , Encephalitozoon cuniculi/enzimologia , Encephalitozoon cuniculi/genética , Imuno-Histoquímica , Isoenzimas/genética , Isoenzimas/isolamento & purificação , Isoenzimas/fisiologia , Microscopia Eletrônica de Transmissão , Peso Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência , Esporos de Protozoários/enzimologia
10.
Parazitologiia ; 37(4): 333-42, 2003.
Artigo em Russo | MEDLINE | ID: mdl-14515511

RESUMO

Some differences in trehalose catabolism were found for terrestrial and aquatic microsporidian species (Undeen, Van der Meer, 1999). In microsporidia species from aquatic hosts, the spore extrusion causes the intrasporal trehalose hydrolysis by trehalase that is followed by the drastic rise of reducing sugars (glucose) concentration. On the contrary, in tested terrestrial microsporidian species, total and reducing sugars remain unchanged through the germination. In this study we demonstrate by means of the enzymatic and paper chromatography methods, that in spores of microsporidia Nosema grylli, infecting fat bodies of crickets Gryllus bimaculatus, neither an increase of glucose concentration nor a reduction in intrasporal trehalose content takes place during the spore discharge. In this respect N. grylli is close to other terrestrial species. However, we have revealed in N. grylli spores activity of alpha,alpha-trehalase (EC 3.2.1.28) with acid pH-optimum like it was found by other authors in spores of aquatic microsporidia N. algerae. This result differs from the neutral pH-optimum (7.0) of trehalse of other terrestrial microsporidia N. apis. Concentration of trehalose in N. grylli spores reduces during long-term storage. All attempts to detect an activity of trehalose phosphorylase (synthase) (K phi 2.4.1.64), other potential key enzyme for trehalose catabolism in N. grylli spores have failed. The absence of changes of the sugar content in terrestrial microsporidian spores during the extrusion indicates, that the main physiological role of trehalose hydrolysis by trehalase in these species is catabolism of energy reserves for providing the long-term survival in the environment.


Assuntos
Nosema/metabolismo , Trealose/metabolismo , Animais , Cromatografia em Papel , Corpo Adiposo/parasitologia , Gryllidae/parasitologia , Concentração de Íons de Hidrogênio , Esporos de Protozoários/enzimologia , Esporos de Protozoários/metabolismo , Fatores de Tempo , Trealose/análise
11.
Cell Signal ; 26(2): 453-9, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24184654

RESUMO

Amoebas survive environmental stress by differentiating into encapsulated cysts. As cysts, pathogenic amoebas resist antibiotics, which particularly counteracts treatment of vision-destroying Acanthamoeba keratitis. Limited genetic tractability of amoeba pathogens has left their encystation mechanisms unexplored. The social amoeba Dictyostelium discoideum forms spores in multicellular fruiting bodies to survive starvation, while other dictyostelids, such as Polysphondylium pallidum can additionally encyst as single cells. Sporulation is induced by cAMP acting on PKA, with the cAMP phosphodiesterase RegA critically regulating cAMP levels. We show here that RegA is deeply conserved in social and pathogenic amoebas and that deletion of the RegA gene in P. pallidum causes precocious encystation and prevents cyst germination. We heterologously expressed and characterized Acanthamoeba RegA and performed a compound screen to identify RegA inhibitors. Two effective inhibitors increased cAMP levels and triggered Acanthamoeba encystation. Our results show that RegA critically regulates Amoebozoan encystation and that components of the cAMP signalling pathway could be effective targets for therapeutic intervention with encystation.


Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Amoeba/enzimologia , AMP Cíclico/metabolismo , Proteínas de Protozoários/metabolismo , 3',5'-AMP Cíclico Fosfodiesterases/genética , Acanthamoeba/enzimologia , Acanthamoeba/fisiologia , Amoeba/fisiologia , Sequência de Bases , Dictyostelium/enzimologia , Dictyostelium/fisiologia , Dados de Sequência Molecular , Proteínas de Protozoários/classificação , Proteínas de Protozoários/genética , Esporos de Protozoários/enzimologia , Esporos de Protozoários/metabolismo
12.
Cell Signal ; 26(2): 409-18, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24511612

RESUMO

Cyclic AMP (cAMP) is an important intracellular signaling molecule for many G protein-mediated signaling pathways but the specificity of cAMP signaling in cells with multiple signaling pathways is not well-understood. In Dictyostelium, at least two different G protein signaling pathways, mediated by the Gα2 and Gα4 subunits, are involved with cAMP accumulation, spore production, and chemotaxis and the stimulation of these pathways results in the activation of ERK2, a mitogen-activated protein kinase that can down regulate the cAMP-specific phosphodiesterase RegA. The regA gene was disrupted in gα2(−) and gα4(−) cells to determine if the absence of this phosphodiesterase rescues the development of these G protein mutants as it does for erk2(−) mutants. There gA(−) mutation had no major effects on developmental morphology but enriched the distribution of the Gα mutant cells to the prespore/prestalk border in chimeric aggregates. The loss of RegA function had no effect on Gα4- mediated folate chemotaxis. However, the regA gene disruption in gα4(−) cells, but not in gα2(−) cells, resulted in a substantial rescue and acceleration of spore production. This rescue in sporulation required cell autonomous signaling because the precocious sporulation could not be induced through intercellular signaling in chimeric aggregates. However, intercellular signals from regA(−) strains increased the expression of the prestalk gene ecmB and accelerated the vacuolization of stalk cells. Intercellular signaling from the gα4(−)regA(−) strain did not induce ecmA gene expression indicating cell-type specificity in the promotion of prestalk cell development. regA gene disruption in a Gα4(HC) (Gα4 overexpression) strain did not result in precocious sporulation or stalk cell development indicating that elevated Gα4 subunit expression can mask regA(−) associated phenotypes even when provided with wild-type intercellular signaling. These findings indicate that the Gα2 and Gα4-mediated pathways provide different contributions to the development of spores and stalk cells and that the absence of RegA function can bypass some but not all defects in G protein regulated spore development.


Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , AMP Cíclico/metabolismo , Dictyostelium/enzimologia , Dictyostelium/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Proteínas de Protozoários/metabolismo , 3',5'-AMP Cíclico Fosfodiesterases/genética , Dictyostelium/crescimento & desenvolvimento , Dictyostelium/metabolismo , Regulação para Baixo/efeitos dos fármacos , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Ácido Fólico/farmacologia , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/deficiência , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/genética , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/deficiência , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Mutação , Proteínas de Protozoários/genética , Transdução de Sinais , Esporos de Protozoários/enzimologia , Esporos de Protozoários/genética , Complexo Vitamínico B/farmacologia
13.
Cell Rep ; 3(3): 622-9, 2013 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-23434509

RESUMO

Signaling pathways controlled by reversible protein phosphorylation (catalyzed by kinases and phosphatases) in the malaria parasite Plasmodium are of great interest, for both increased understanding of parasite biology and identification of novel drug targets. Here, we report a functional analysis in Plasmodium of an ancient bacterial Shewanella-like protein phosphatase (SHLP1) found only in bacteria, fungi, protists, and plants. SHLP1 is abundant in asexual blood stages and expressed at all stages of the parasite life cycle. shlp1 deletion results in a reduction in ookinete (zygote) development, microneme formation, and complete ablation of oocyst formation, thereby blocking parasite transmission. This defect is carried by the female gamete and can be rescued by direct injection of mutant ookinetes into the mosquito hemocoel, where oocysts develop. This study emphasizes the varied functions of SHLP1 in Plasmodium ookinete biology and suggests that it could be a novel drug target for blocking parasite transmission.


Assuntos
Fosfoproteínas Fosfatases/metabolismo , Plasmodium berghei/enzimologia , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Deleção de Genes , Células Germinativas/enzimologia , Dados de Sequência Molecular , Fosfoproteínas Fosfatases/genética , Plasmodium berghei/crescimento & desenvolvimento , Plasmodium berghei/patogenicidade , Proteínas de Protozoários/genética , Esporos de Protozoários/enzimologia , Esporos de Protozoários/crescimento & desenvolvimento , Virulência/genética , Zigoto/enzimologia
14.
PLoS One ; 8(3): e59365, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23555023

RESUMO

In Dictyostelium discoideum, AprA and CfaD are secreted proteins that inhibit cell proliferation. We found that the proliferation of cells lacking CnrN, a phosphatase and tensin homolog (PTEN)-like phosphatase, is not inhibited by exogenous AprA and is increased by exogenous CfaD. The expression of CnrN in cnrN cells partially rescues these altered sensitivities, suggesting that CnrN is necessary for the ability of AprA and CfaD to inhibit proliferation. Cells lacking CnrN accumulate normal levels of AprA and CfaD. Like cells lacking AprA and CfaD, cnrN cells proliferate faster and reach a higher maximum cell density than wild type cells, tend to be multinucleate, accumulate normal levels of mass and protein per nucleus, and form less viable spores. When cnrN cells expressing myc-tagged CnrN are stimulated with a mixture of rAprA and rCfaD, levels of membrane-associated myc-CnrN increase. AprA also causes chemorepulsion of Dictyostelium cells, and CnrN is required for this process. Combined, these results suggest that CnrN functions in a signal transduction pathway downstream of AprA and CfaD mediating some, but not all, of the effects of AprA and CfaD.


Assuntos
Núcleo Celular/genética , Quimiotaxia/genética , Dictyostelium/genética , PTEN Fosfo-Hidrolase/genética , Proteínas de Protozoários/genética , Esporos de Protozoários/genética , Contagem de Células , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/enzimologia , Proliferação de Células/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Dictyostelium/efeitos dos fármacos , Dictyostelium/enzimologia , Regulação da Expressão Gênica , PTEN Fosfo-Hidrolase/deficiência , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Esporos de Protozoários/efeitos dos fármacos , Esporos de Protozoários/enzimologia
15.
Acta Trop ; 123(1): 39-46, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22498829

RESUMO

Giardia intestinalis is considered an early-branching eukaryote and is therefore a valuable model for studying primordial cellular processes. This work reports the characterization of the ubiquitin-activating enzyme (E1) during growth and different stages of trophozoite differentiation into cysts. We found that in Giardia E1 expression (both at mRNA and protein levels) is regulated during encystation. The enzyme is proteolytically processed mainly into two fragments of 68kDa (N-terminal) and 47kDa (C-terminal). This phenomenon has not been described for any other E1. In trophozoites, this enzyme localized at spots within the cytoplasm as detected by using polyclonal antibodies against either E1 N- or C-terminal fragments. This pattern changed during encystation into a diffuse localization throughout the cytoplasm of encysting cells. E1 localizes in mature cysts at cytoplasmic spots and in the cyst wall. Our antisense silencing experiments suggested that E1 is an essential gene for parasite viability. On the other hand, E1 over-expression greatly increased the encystation rate, indicating a relationship between E1 and Giardia differentiation.


Assuntos
Giardia lamblia/enzimologia , Giardia lamblia/crescimento & desenvolvimento , Enzimas Ativadoras de Ubiquitina/metabolismo , Animais , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Viabilidade Microbiana , Proteólise , Esporos de Protozoários/enzimologia , Esporos de Protozoários/crescimento & desenvolvimento , Trofozoítos/enzimologia , Trofozoítos/crescimento & desenvolvimento
16.
Mol Biochem Parasitol ; 184(2): 118-21, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22575601

RESUMO

Genes for mannitol-metabolizing enzymes, mannitol phosphate dehydrogenase (MPDH) and mannitol dehydrogenase (MDH), have been recently identified in the genome of Acanthamoeba castellanii and their potential role in stress tolerance was proposed. Using qRT-PCR, comparison has been made of mRNA levels of the enzymes for mannitol metabolism at various time intervals during the stress defence reactions of encystation and pseudocyst formation. Gradual decrease of both enzymes during encystation and slight increases at the beginning of pseudocyst formation were observed. Detailed analysis of mRNA sequences of the two genes revealed similarities with various alcohol dehydrogenases rather than mannitol dehydrogenases. Our results indicate there is probably no protective role for mannitol in Acanthamoeba as no mannitol was detected using HILIC ESI MS, in any Acanthamoeba life cycle stage. Possible misinterpretation of previously published sequences as encoding enzymes of the mannitol metabolic pathway is discussed.


Assuntos
Acanthamoeba castellanii/enzimologia , Manitol Desidrogenases/metabolismo , Manitol/metabolismo , Proteínas de Protozoários/metabolismo , Acanthamoeba castellanii/metabolismo , Acanthamoeba castellanii/fisiologia , Sequência de Aminoácidos , Metabolismo dos Carboidratos , Sequência Conservada , Regulação Enzimológica da Expressão Gênica , Manitol Desidrogenases/genética , Anotação de Sequência Molecular , Dados de Sequência Molecular , Proteínas de Protozoários/genética , Reação em Cadeia da Polimerase em Tempo Real , Esporos de Protozoários/enzimologia , Estresse Fisiológico , Transcrição Gênica
17.
FEMS Microbiol Lett ; 319(1): 82-7, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21438914

RESUMO

The genome of Dictyostelium contains two novel hybrid-type polyketide synthases (PKSs) known as 'Steely'; the Steely enzyme is formed by the fusion of type I and type III PKSs. One of these enzymes, SteelyB, is known to be responsible for the production of the stalk cell-inducing factor DIF-1 in vivo. On the other hand, the product(s) and expression pattern of SteelyA are not clearly understood, because there are two different reports associated with the in vitro products of SteelyA and its expression pattern. To solve this problem, we first examined the expression pattern using two different primer sets and found that it was quite similar to that shown in the dictyExpress database. stlA expression peaked at approximately 3 h and declined, but showed a small peak around the end of development. Next, we examined the in vivo product of SteelyA using a stlA null mutant and found that the mutant lacked 4-methyl-5-pentylbenzene-1,3-diol (MPBD). This null mutant showed aberrant, glassy sori, and most of the cells in the sori remained amoeba-like without a cell wall. This defect was restored by adding 200 nM of MPBD to the agar. These results indicate that SteelyA produces MPBD in vivo and induces spore maturation.


Assuntos
Dictyostelium/enzimologia , Policetídeo Sintases/metabolismo , Proteínas de Protozoários/metabolismo , Resorcinóis/metabolismo , Esporos de Protozoários/crescimento & desenvolvimento , Dictyostelium/genética , Dictyostelium/crescimento & desenvolvimento , Dictyostelium/metabolismo , Policetídeo Sintases/genética , Proteínas de Protozoários/genética , Esporos de Protozoários/enzimologia , Esporos de Protozoários/genética , Esporos de Protozoários/metabolismo
18.
Protein J ; 29(4): 283-9, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20496101

RESUMO

Metalloproteinases (MMP) belong to the family of cation dependent endopeptidases that degrade matrices at physiological pH and to cleave extracellular matrix proteins. They play an important role in diverse physiological and pathological processes; not only there diverse types of MMP differ in structure and functionally, but also their enzymatic activity is regulated at multiple levels. Trying to shed some light over the processes that govern the pathology of African Trypanosomiasis, the aim of the present study was to examine the proteolytic activity of the crude trypanosome protein extract obtained from the bloodstream forms of Trypanosoma brucei brucei parasites. We hereby report the partial biochemical characterization of a neutral Trypanosoma brucei-metalloproteinase that displays marked proteolytic activities on gelatin and casein, with a molecular mass of approximately 40 kDa, whose activity is strongly dependent of pH and temperature. Furthermore, we show that this activity can be inhibited by classical MMP inhibitors such as EDTA, EGTA, phenantroline, and also by tetracycline and derivatives. This study has a relevant role in the search for new therapeutical targets, for the use of metalloproteinases inhibitors as treatment strategies, or as enhancement to trypanocidal drugs used in the treatment of the disease.


Assuntos
Metaloproteases/química , Trypanosoma brucei brucei/enzimologia , Animais , Cromatografia por Troca Iônica , Precursores Enzimáticos/metabolismo , Concentração de Íons de Hidrogênio , Metaloproteases/isolamento & purificação , Metaloproteases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Esporos de Protozoários/enzimologia , Temperatura
19.
Clinics ; 70(3): 157-161, 03/2015. tab
Artigo em Inglês | LILACS | ID: lil-747105

RESUMO

OBJECTIVE: To determine whether Tai Chi Chuan or ballroom dancing promotes better performance with respect to postural balance, gait, and postural transfer among elderly people. METHODS: We evaluated 76 elderly individuals who were divided into two groups: the Tai Chi Chuan Group and the Dance Group. The subjects were tested using the NeuroCom Balance Master¯ force platform system with the following protocols: static balance tests (the Modified Clinical Tests of Sensory Interaction on Balance and Unilateral Stance) and dynamic balance tests (the Walk Across Test and Sit-to-stand Transfer Test). RESULTS: In the Modified Clinical Test of Sensory Interaction on Balance, the Tai Chi Chuan Group presented a lower sway velocity on a firm surface with open and closed eyes, as well as on a foam surface with closed eyes. In the Modified Clinical Test of Sensory Interaction on Unilateral Stance, the Tai Chi Chuan Group presented a lower sway velocity with open eyes, whereas the Dance Group presented a lower sway velocity with closed eyes. In the Walk Across Test, the Tai Chi Chuan Group presented faster walking speeds than those of the Dance Group. In the Sit-to-stand Transfer Test, the Tai Chi Chuan Group presented shorter transfer times from the sitting to the standing position, with less sway in the final standing position. CONCLUSION: The elderly individuals who practiced Tai Chi Chuan had better bilateral balance with eyes open on both types of surfaces compared with the Dance Group. The Dance Group had better unilateral postural balance with eyes closed. The Tai Chi Chuan Group had faster walking speeds, shorter transfer times, and better postural balance in the final standing position during the Sit-to-stand Test. .


Assuntos
/metabolismo , AMP Cíclico/metabolismo , Dictyostelium/enzimologia , Dictyostelium/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Proteínas de Protozoários/metabolismo , /genética , Dictyostelium/crescimento & desenvolvimento , Dictyostelium/metabolismo , Regulação para Baixo/efeitos dos fármacos , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Ácido Fólico/farmacologia , /deficiência , /genética , /metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/deficiência , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Mutação , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas de Protozoários/genética , Transdução de Sinais , Esporos de Protozoários/enzimologia , Esporos de Protozoários/genética , Complexo Vitamínico B/farmacologia
20.
J Basic Microbiol ; 44(6): 459-70, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15558817

RESUMO

UDPgalactose:polysaccharide galactosyl-transferase is the enzyme that is specifically localized in prespore cells of Dictyostelium discoideum and its activity sharply changes in response to differentiation and dedifferentiation. To clarify the nature of this enzyme, we first developed an improved assay method for the enzyme, and by using this method, we partially purified the enzyme through DEAE-sepharose, phenyl-sepharose and ATP-sepharose chromatography. The apparent molecular mass of the enzyme was ca. 200 KDa (by non-denaturing polyacrylamide gel gradient analysis) and the isoelectric point was around pH 7. The enzyme exhibited a hitherto undescribed property, that is the reaction proceeds faster at 0 degrees C than at 21 degrees C, with a smaller K(m) value and an unchanged V(max) value. This low-temperature resistant property of the enzyme is consistent with the previous observation (Maeda 1984, J. Cell Sci. 69, 159-165) that prespore differentiation is favored at low temperatures. The reaction appears to proceed in a double displacement manner. ATP reversibly inhibited the enzyme with a K(i) value of 2 mM, suggesting the possibility that ATP regulates its activity in vivo.


Assuntos
Dictyostelium/enzimologia , Galactosiltransferases/isolamento & purificação , Galactosiltransferases/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática , Galactosiltransferases/química , Ponto Isoelétrico , Peso Molecular , Esporos de Protozoários/enzimologia , Temperatura
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