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1.
Int J Nanomedicine ; 14: 5257-5270, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31409988

RESUMO

Background: In recent years, green synthesized silver nanoparticles have been increasingly investigated for their anti-cancer potential. In the present study, we aimed at the biosynthesis of silver nanoparticles (AgNPs) using a curcumin derivative, ST06. Although, the individual efficacies of silver nanoparticles or curcumin derivatives have been studied previously, the synergistic cytotoxic effects of curcumin derivative and silver nanoparticles in a single nanoparticulate formulation have not been studied earlier specifically on animal models. This makes this study novel compared to the earlier synthesized curcumin derivative or silver nanoparticles studies. The aim of the study was to synthesize ST06 coated silver nanoparticles (ST06-AgNPs) using ST06 as both reducing and coating agent. Methods: The synthesized nanoparticles AgNPs and ST06-AgNPs were characterised for the particle size distribution, morphology, optical properties and surface charge by using UV-visible spectroscopy, dynamic light scattering (DLS) and transmission electron microscopy (TEM). Elemental composition and structural properties were studied by energy dispersive X-ray spectroscopy (EDX) and X-ray diffraction spectroscopy (XRD). The presence of ST06 as capping agent was demonstrated by Fourier transform infrared spectroscopy (FTIR). Results: The synthesized nanoparticles (ST06-AgNPs) were spherical and had a size distribution in the range of 50-100 nm. UV-Vis spectroscopy displayed a specific silver plasmon peak at 410 nm. The in vitro cytotoxicity effects of ST06 and ST06-AgNPs, as assessed by MTT assay, showed significant growth inhibition of human cervical cancer cell line (HeLa). In addition, studies carried out in EAC tumor-induced mouse model (Ehrlich Ascites carcinoma) using ST06-AgNPs, revealed that treatment of the animals with these nanoparticles resulted in a significant reduction in the tumor growth, compared to the control group animals. Conclusion: In conclusion, green synthesized ST06-AgNPs exhibited superior anti-tumor efficacy than the free ST06 or AgNPs with no acute toxicity under both in vitro and in vivo conditions. The tumor suppression is associated with the intrinsic apoptotic pathway. Together, the results of this study suggest that ST06-AgNPs could be considered as a potential option for the treatment of solid tumors.


Assuntos
Carcinoma de Ehrlich/patologia , Curcumina/farmacologia , Química Verde/métodos , Nanopartículas Metálicas/química , Prata/farmacologia , Neoplasias do Colo do Útero/patologia , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 9/metabolismo , Morte Celular/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Células HeLa , Humanos , Camundongos , Tamanho da Partícula , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espectrometria por Raios X , Espectroscopia de Infravermelho com Transformada de Fourier , Distribuição Tecidual/efeitos dos fármacos , Difração de Raios X
2.
Mol Microbiol ; 100(1): 173-87, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26713845

RESUMO

Twinfilin is an evolutionarily conserved actin-binding protein, which regulates actin-dynamics in eukaryotic cells. Homologs of this protein have been detected in the genome of various protozoan parasites causing diseases in human. However, very little is known about their core functions in these organisms. We show here that a twinfilin homolog in a human pathogen Leishmania, primarily localizes to the nucleolus and, to some extent, also in the basal body region. In the dividing cells, nucleolar twinfilin redistributes to the mitotic spindle and remains there partly associated with the spindle microtubules. We further show that approximately 50% depletion of this protein significantly retards the cell growth due to sluggish progression of S phase of the cell division cycle, owing to the delayed nuclear DNA synthesis. Interestingly, overexpression of this protein results in significantly increased length of the mitotic spindle in the dividing Leishmania cells, whereas, its depletion adversely affects spindle elongation and architecture. Our results indicate that twinfilin controls on one hand, the DNA synthesis and on the other, the mitotic spindle elongation, thus contributing to karyokinesis in Leishmania.


Assuntos
Divisão do Núcleo Celular , Replicação do DNA , Leishmania/genética , Leishmania/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas de Protozoários/metabolismo , Fuso Acromático/metabolismo , Ciclo Celular/genética , Divisão Celular , Nucléolo Celular/metabolismo , Expressão Gênica , Ligação Proteica , Transporte Proteico
3.
Cytoskeleton (Hoboken) ; 72(12): 621-32, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26662514

RESUMO

Coronin proteins bind with actin filaments and participate in regulation of actin-dependent processes. These proteins contain a coiled-coil domain at their C-terminus, which is responsible for their dimeric or trimeric forms. However, the functional significance of these oligomeric configurations in organizing the actin cytoskeleton is obscure. Here, we report that the Leishmania coronin exists in a higher oligomeric form through its coiled-coil domain, the truncation of which ablates the ability of Leishmania coronin to assist actin-filament formation. F-actin co-sedimentation assay using purified proteins shows that the coiled-coil domain does not interact with actin-filaments and its absence does not abrogate actin-coronin interaction. Furthermore, it was shown that unlike other coronins, Leishmania coronin interacts with actin-filaments through its unique region. These results provided important insights into the role of coronin oligomerization in modulating actin-network.


Assuntos
4-Butirolactona/análogos & derivados , Citoesqueleto de Actina/metabolismo , 4-Butirolactona/metabolismo , Leishmania , Ligação Proteica
4.
Mol Microbiol ; 91(3): 562-78, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24354789

RESUMO

Actin-related proteins are ubiquitous actin-like proteins that show high similarity with actin in terms of their amino acid sequence and three-dimensional structure. However, in lower eukaryotes, such as trypanosomatids, their functions have not yet been explored. Here, we show that a novel actin-related protein (ORF LmjF.13.0950) is localized mainly in the Leishmania mitochondrion. We further reveal that depletion of the intracellular levels of this protein leads to an appreciable decrease in the mitochondrial membrane potential as well as in the ATP production, which appears to be accompanied with impairment in the flagellum assembly and motility. Additionally, we report that the mutants so generated fail to survive inside the mouse peritoneal macrophages. These abnormalities are, however, reversed by the episomal gene complementation. Our results, for the first time indicate that apart from their classical roles in the cytoplasm and nucleus, actin-related proteins may also regulate the mitochondrial function, and in case of Leishmania donovani they may also serve as the essential factor for their survival in the host cells.


Assuntos
Actinas/metabolismo , Flagelos/fisiologia , Leishmania donovani/imunologia , Leishmania donovani/fisiologia , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Proteínas de Protozoários/metabolismo , Actinas/genética , Trifosfato de Adenosina/biossíntese , Animais , Sobrevivência Celular , Células Cultivadas , Deleção de Genes , Teste de Complementação Genética , Leishmania donovani/genética , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/parasitologia , Camundongos , Proteínas de Protozoários/genética
5.
Eukaryot Cell ; 11(6): 752-60, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22492507

RESUMO

Leishmania, like other eukaryotes, contains large amounts of actin and a number of actin-related and actin binding proteins. Our earlier studies have shown that deletion of the gene corresponding to Leishmania actin-depolymerizing protein (ADF/cofilin) adversely affects flagellum assembly, intracellular trafficking, and cell division. To further analyze this, we have now created ADF/cofilin site-specific point mutants and then examined (i) the actin-depolymerizing, G-actin binding, and actin-bound nucleotide exchange activities of the mutant proteins and (ii) the effect of overexpression of these proteins in wild-type cells. Here we show that S4D mutant protein failed to depolymerize F-actin but weakly bound G-actin and inhibited the exchange of G-actin-bound nucleotide. We further observed that overexpression of this protein impaired flagellum assembly and consequently cell motility by severely impairing the assembly of the paraflagellar rod, without significantly affecting vesicular trafficking or cell growth. Taken together, these results indicate that dynamic actin is essentially required in assembly of the eukaryotic flagellum.


Assuntos
Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Cofilina 1/metabolismo , Flagelos/metabolismo , Leishmania donovani/metabolismo , Mutação/genética , Proteínas de Protozoários/genética , Substituição de Aminoácidos/genética , Animais , Ácido Aspártico/metabolismo , Eletroforese em Gel de Poliacrilamida , Flagelos/ultraestrutura , Espaço Intracelular/metabolismo , Leishmania donovani/ultraestrutura , Nucleotídeos/metabolismo , Fenótipo , Polimerização , Ligação Proteica , Transporte Proteico , Proteínas de Protozoários/metabolismo , Coelhos , Serina/metabolismo , Vesículas Transportadoras/metabolismo
6.
J Cell Sci ; 123(Pt 11): 1894-901, 2010 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-20460437

RESUMO

ADF/cofilin is an actin-dynamics-regulating protein that is required for several actin-based cellular processes such as cell motility and cytokinesis. A homologue of this protein has recently been identified in the protozoan parasite Leishmania, which has been shown to be essentially required in flagellum assembly and cell motility. However, the role of this protein in cytokinesis remains largely unknown. We show here that deletion of the gene encoding ADF/cofilin in these organisms results in several aberrations in the process of cell division. These aberrations include delay in basal body and kinetoplast separation, cleavage furrow progression and flagellar pocket division. In addition to these changes, the intracellular trafficking and actin dynamics are also adversely affected. All these abnormalities are, however, reversed by episomal complementation. Together, these results indicate that actin dynamics regulates early events in Leishmania cell division.


Assuntos
Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Antígenos de Protozoários/metabolismo , DNA de Cinetoplasto/genética , Leishmania/fisiologia , Fatores de Despolimerização de Actina/genética , Fatores de Despolimerização de Actina/imunologia , Actinas/imunologia , Antígenos de Protozoários/imunologia , Células Cultivadas , Citocinese/genética , Citoesqueleto/metabolismo , Imunofluorescência , Microscopia Eletrônica , Organismos Geneticamente Modificados , Multimerização Proteica/genética , Transporte Proteico/genética , Deleção de Sequência/genética
7.
J Cell Sci ; 123(Pt 12): 2035-44, 2010 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-20501700

RESUMO

Actin-based myosin motors have a pivotal role in intracellular trafficking in eukaryotic cells. The parasitic protozoan organism Leishmania expresses a novel class of myosin, myosin XXI (Myo21), which is preferentially localized at the proximal region of the flagellum. However, its function in this organism remains largely unknown. Here, we show that Myo21 interacts with actin, and its expression is dependent of the growth stage. We further reveal that depletion of Myo21 levels results in impairment of the flagellar assembly and intracellular trafficking. These defects are, however, reversed by episomal complementation. Additionally, it is shown that deletion of the Myo21 gene leads to generation of ploidy, suggesting an essential role of Myo21 in survival of Leishmania cells. Together, these results indicate that actin-dependent trafficking activity of Myo21 is essentially required during assembly of the Leishmania flagellum.


Assuntos
Flagelos/metabolismo , Leishmania/metabolismo , Miosinas/metabolismo , Proteínas de Protozoários/metabolismo , Actinas/genética , Actinas/metabolismo , Flagelos/genética , Leishmania/genética , Miosinas/genética , Ligação Proteica , Transporte Proteico , Proteínas de Protozoários/genética
8.
Nucleic Acids Res ; 38(10): 3308-17, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20147461

RESUMO

Leishmania actin (LdACT) is an unconventional form of eukaryotic actin in that it markedly differs from other actins in terms of its filament forming as well as toxin and DNase-1-binding properties. Besides being present in the cytoplasm, cortical regions, flagellum and nucleus, it is also present in the kinetoplast where it appears to associate with the kinetoplast DNA (kDNA). However, nothing is known about its role in this organelle. Here, we show that LdACT is indeed associated with the kDNA disc in Leishmania kinetoplast, and under in vitro conditions, it specifically binds DNA primarily through electrostatic interactions involving its unique DNase-1-binding region and the DNA major groove. We further reveal that this protein exhibits DNA-nicking activity which requires its polymeric state as well as ATP hydrolysis and through this activity it converts catenated kDNA minicircles into open form. In addition, we show that LdACT specifically binds bacterial type II topoisomerase and inhibits its decatenation activity. Together, these results strongly indicate that LdACT could play a critical role in kDNA remodeling.


Assuntos
Actinas/metabolismo , DNA Topoisomerases Tipo II/metabolismo , DNA de Cinetoplasto/metabolismo , Leishmania/metabolismo , Actinas/química , Animais , Linhagem Celular , Cromatina , DNA Catenado/metabolismo , Desoxirribonuclease I/metabolismo , Escherichia coli/enzimologia , Leishmania/genética
9.
Mol Biochem Parasitol ; 170(1): 41-4, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19925831

RESUMO

Compartmentalization of glycolytic enzymes in glycosomes is vital in trypanosomatid parasites. Retention of these enzymes in the cytosol induces sugar toxicity and accumulation of intermediate metabolites, notably the hexokinase product glucose-6-phosphate. However, the role of hexokinase in sugar mediated toxicity remains unexplored. We have generated Leishmania donovani transfectants expressing a catalytically active cytosolic mutant of hexokinase. In the presence of glucose, these transfectants exhibited toxicity during log and stationary phases of growth. These results suggest that targeting of hexokinase to the glycosome is required to prevent uncontrolled and cytotoxic glucose phosphorylation in L. donovani parasites.


Assuntos
Expressão Gênica , Glucose/toxicidade , Hexoquinase/metabolismo , Leishmania donovani/enzimologia , Proteínas de Protozoários/metabolismo , Citosol/química , Citosol/metabolismo , Hexoquinase/química , Hexoquinase/genética , Leishmania donovani/genética , Leishmania donovani/metabolismo , Receptor 2 de Sinal de Orientação para Peroxissomos , Ligação Proteica , Transporte Proteico , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo
10.
J Cell Sci ; 122(Pt 10): 1691-9, 2009 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-19401334

RESUMO

In general, coronins play an important role in actin-based processes, and are expressed in a variety of eukaryotic cells, including Leishmania. Here, we show that Leishmania coronin preferentially distributes to the distal tip during cytokinesis, and interacts with microtubules through a microtubule-based motor, kinesin K39. We further show that reduction in coronin levels by 40-50% in heterozygous coronin mutants results in generation of bipolar cells (25-30%), specifically in the log phase, owing to unregulated growth of the corset microtubules. Further analysis of bipolar cells revealed that the main cause of generation of bipolar cell morphology is the intrusion of the persistently growing corset microtubules into the other daughter cell corset from the opposite direction. This defect in cytokinesis, however, disappears upon episomal gene complementation. Additionally, our attempts to prepare homozygous mutants were unsuccessful, as only the aneuploid cells survive the selection process. These results indicate that coronin regulates microtubule remodeling during Leishmania cytokinesis and is essentially required for survival of these parasites in culture.


Assuntos
4-Butirolactona/análogos & derivados , Citocinese , Leishmania donovani/metabolismo , Microtúbulos/metabolismo , Proteínas de Protozoários/metabolismo , 4-Butirolactona/deficiência , 4-Butirolactona/genética , 4-Butirolactona/metabolismo , Aneuploidia , Animais , Antígenos de Protozoários/metabolismo , Citocinese/efeitos dos fármacos , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/genética , Leishmania donovani/crescimento & desenvolvimento , Leishmania donovani/ultraestrutura , Macrolídeos/farmacologia , Microtúbulos/efeitos dos fármacos , Microtúbulos/ultraestrutura , Mutação , Proteínas de Protozoários/genética , Fatores de Tempo , Moduladores de Tubulina/farmacologia
11.
Mol Biochem Parasitol ; 164(2): 105-10, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19121339

RESUMO

Leishmania major genome analysis revealed the presence of putative genes corresponding to two myosins, which have been designated to class IB and a novel class, class XXI, specifically present in kinetoplastids. To characterize these myosin homologs in Leishmania, we have cloned and over-expressed the full-length myosin XXI gene and variable region of myosin IB gene in bacteria, purified the corresponding proteins, and then used the affinity purified anti-sera to analyze the expression and intracellular distribution of these proteins. Whereas myosin XXI was expressed in both the promastigote and amastigote stages, no expression of myosin IB could be detected in any of the two stages of these parasites. Further, myosin XXI expression was more predominant in the promastigote stage where it was preferentially localized in the proximal region of the flagellum. The observed flagellar localization was not dependent on the myosin head region or actin but was exclusively determined by the myosin tail region, as judged by over-expressing GFP conjugates of full-length myosin XXI, its head domain and its tail domain separately in Leishmania. Furthermore, immunofluorescence and immuno-gold electron microscopy analyses revealed that this protein was partly associated with paraflagellar rod proteins but not with tubulins in the flagellar axoneme. Our results, for the first time, report the expression and detailed analysis of cellular localization of a novel class of myosin, myosin XXI in trypanosomatids.


Assuntos
Flagelos/química , Leishmania major/química , Miosinas/análise , Miosinas/genética , Animais , Fusão Gênica Artificial , DNA de Protozoário/química , DNA de Protozoário/genética , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de DNA
12.
Mol Microbiol ; 70(4): 837-52, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18793337

RESUMO

ADF/cofilins are ubiquitous actin dynamics-regulating proteins that have been mainly implicated in actin-based cell motility. Trypanosomatids, e.g. Leishmania and Trypanosoma, which mediate their motility through flagellum, also contain a putative ADF/cofilin homologue, but its role in flagellar motility remains largely unexplored. We have investigated the role of this protein in assembly and motility of the Leishmania flagellum after knocking out the ADF/cofilin gene by targeted gene replacement. The resultant mutants were completely immotile, short and stumpy, and had reduced flagellar length and severely impaired beat. In addition, the assembly of the paraflagellar rod was lost, vesicle-like structures were seen throughout the length of the flagellum and the state and distribution of actin were altered. However, episomal complementation of the gene restored normal morphology and flagellar function. These results for the first time indicate that the actin dynamics-regulating protein ADF/cofilin plays a critical role in assembly and motility of the eukaryotic flagellum.


Assuntos
Fatores de Despolimerização de Actina/metabolismo , Destrina/metabolismo , Flagelos/ultraestrutura , Leishmania donovani/genética , Proteínas de Protozoários/metabolismo , Fatores de Despolimerização de Actina/genética , Actinas/genética , Actinas/metabolismo , Animais , Movimento Celular , Clonagem Molecular , DNA de Protozoário/genética , Destrina/genética , Deleção de Genes , Técnicas de Inativação de Genes , Células HeLa , Humanos , Leishmania donovani/metabolismo , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Mutação , Proteínas de Protozoários/genética , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
13.
J Biol Chem ; 283(33): 22760-73, 2008 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-18539603

RESUMO

Leishmania actin was cloned, overexpressed in baculovirus-insect cell system, and purified to homogeneity. The purified protein polymerized optimally in the presence of Mg2+ and ATP, but differed from conventional actins in its following properties: (i) it did not polymerize in the presence of Mg2+ alone, (ii) it polymerized in a restricted range of pH 7.0-8.5, (iii) its critical concentration for polymerization was found to be 3-4-fold lower than of muscle actin, (iv) it predominantly formed bundles rather than single filaments at pH 8.0, (v) it displayed considerably higher ATPase activity during polymerization, (vi) it did not inhibit DNase-I activity, and (vii) it did not bind the F-actin-binding toxin phalloidin or the actin polymerization disrupting agent Latrunculin B. Computational and molecular modeling studies revealed that the observed unconventional behavior of Leishmania actin is related to the diverged amino acid stretches in its sequence, which may lead to changes in the overall charge distribution on its solvent-exposed surface, ATP binding cleft, Mg2+ binding sites, and the hydrophobic loop that is involved in monomer-monomer interactions. Phylogenetically, it is related to ciliate actins, but to the best of our knowledge, no other actin with such unconventional properties has been reported to date. It is therefore suggested that actin in Leishmania may serve as a novel target for design of new antileishmanial drugs.


Assuntos
Actinas/química , Actinas/metabolismo , Leishmania donovani/química , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Actinas/ultraestrutura , Trifosfato de Adenosina/metabolismo , Animais , Sequência de Bases , Simulação por Computador , Concentração de Íons de Hidrogênio , Cinética , Leishmania donovani/fisiologia , Magnésio/metabolismo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteínas de Protozoários/ultraestrutura , Spodoptera/parasitologia
15.
Mol Biochem Parasitol ; 143(2): 152-64, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16024104

RESUMO

The presence of actin in Leishmania has recently been demonstrated, but the functional form of this protein (filamentous actin) has not yet been identified. We report here that the putative coronin homologue identified in the Leishmania genome is invariably associated with the filament-like structures of actin in Leishmania promastigotes. The occurrence of filamentous structures is significantly increased upon overexpression of Leishmania coronin as its GFP fusion product in Leishmania cells. However, expression of Leishmania actin or coronin alone in mammalian cells does not result in formation of any filament-like structures of Leishmania actin or association of Leishmania coronin with mammalian filamentous actin, but coexpression of both the proteins in these cells leads to formation of filamentous structures containing Leishmania actin and coronin. The high specificity of Leishmania coronin for Leishmania actin could be attributed to its unique structure as it differs from other coronins not only in the unique region but also in the actin-binding site and leucine zipper motif. These results taken together indicate that Leishmania contains a novel form of coronin which colocalizes with actin in filament-like structures in these cells.


Assuntos
Citoesqueleto de Actina/química , Actinas/análise , Leishmania/química , Proteínas dos Microfilamentos/análise , Citoesqueleto de Actina/genética , Actinas/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Cricetinae , DNA de Protozoário/química , DNA de Protozoário/genética , Expressão Gênica , Leishmania/metabolismo , Leishmania/ultraestrutura , Leishmania donovani/química , Leishmania donovani/metabolismo , Leishmania donovani/ultraestrutura , Leishmania major/química , Leishmania major/metabolismo , Leishmania major/ultraestrutura , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Ligação Proteica , Proteínas de Protozoários/análise , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos
16.
Mol Biochem Parasitol ; 134(1): 105-14, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-14747148

RESUMO

To study the occurrence and subcellular distribution of actin in trypanosomatid parasites, we have cloned and overexpressed Leishmania donovani actin gene in bacteria, purified the protein, and employed the affinity purified rabbit polyclonal anti-recombinant actin antibodies as a probe to study the organisation and subcellular distribution of actin in Leishmania cells. The Leishmania actin did not cross react with antimammalian actin antibodies but was readily recognized by the anti-Leishmania actin antibodies in both the promastigote and amastigote forms of the parasite. About 10(6) copies per cell of this protein (M(r) 42.05 kDa) were present in the Leishmania promastigote. Unlike other eukaryotic actins, the oligomeric forms of Leishmania actin were not stained by phalloidin nor were dissociated by actin filament-disrupting agents, like Latrunculin B and Cytochalasin D. Analysis of the primary structure of this protein revealed that these unusual characteristics may be related to the presence of highly diverged amino acids in the DNase I-binding loop (amino acids 40-50) and the hydrophobic plug (amino acids 262-272) regions of Leishmania actin. The subcellular distribution of actin was studied in the Leishmania promastigotes by employing immunoelectron and immunofluorescence microscopies. This protein was present not only in the flagella, flagellar pocket, nucleus and the kinetoplast but it was also localized on the nuclear, vacuolar and cytoplasmic face of the plasma membranes. Further, the plasma membrane-associated actin was colocalised with subpellicular microtubules, while most of the actin present in the kinetoplast colocalised with the k-DNA network. These results clearly indicate that Leishmania contains a novel form of actin which may structurally and functionally differ from other eukaryotic actins. The functional significance of these observations is discussed.


Assuntos
Actinas/química , Actinas/metabolismo , Leishmania/genética , Leishmania/metabolismo , Microtúbulos/metabolismo , Actinas/genética , Sequência de Aminoácidos , Animais , Compostos Bicíclicos Heterocíclicos com Pontes , Clonagem Molecular , Citocalasina D , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Leishmania/ultraestrutura , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Organelas/química , Faloidina , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Análise de Sequência de DNA , Coloração e Rotulagem , Tiazóis , Tiazolidinas
17.
FEBS Lett ; 515(1-3): 184-8, 2002 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-11943218

RESUMO

Hydrogel nanoparticles of cross-linked polyvinylpyrrolidone (PVP-NP) (35-50 nm in diameter) containing fluoresceinated dextran (FITC-Dx) were encapsulated in reconstituted Sendai viral envelopes containing only the fusion (F) protein (F-virosomes(1)). Incubation of these loaded F-virosomes with human hepatoblastoma cells (HepG2) in culture resulted in membrane-fusion-mediated delivery of NPs to the cell cytoplasm, as inferred from the ability of cells to internalize FITC-Dx loaded PVP-NP (PVP(f)-NP) in the presence of azide (an inhibitor of the endocytotic process). Introduction of PVP(f)-NP into the HepG2 cells was assured by selective accumulation of FITC fluorescence in the cytosolic compartment. The structural integrity of the internalized PVP(f)-NP was also confirmed by fluorescence microscopy and ultracentrifugation analysis. The potential usefulness of PVP-NP-mediated cytosolic release of water soluble drugs both in vitro and in vivo has been established for the first time.


Assuntos
Citosol/metabolismo , Dextranos/administração & dosagem , Fluoresceína-5-Isotiocianato/administração & dosagem , Hidrogéis/administração & dosagem , Vírus Sendai , Proteínas Virais de Fusão/administração & dosagem , Células 3T3 , Animais , Células CHO , Cricetinae , Preparações de Ação Retardada/administração & dosagem , Preparações de Ação Retardada/metabolismo , Dextranos/metabolismo , Relação Dose-Resposta a Droga , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos/métodos , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Hepatoblastoma/tratamento farmacológico , Hepatoblastoma/metabolismo , Humanos , Hidrogéis/química , Hidrogéis/farmacocinética , Fusão de Membrana/efeitos dos fármacos , Camundongos , Microscopia de Fluorescência , Tamanho da Partícula , Povidona/administração & dosagem , Povidona/química , Povidona/farmacocinética , Vírus Sendai/química , Células Tumorais Cultivadas , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/farmacocinética
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