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1.
Analyst ; 138(21): 6331-6, 2013 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-24003438

RESUMO

Dysfunctional intracellular enzymatic activity is believed to be an underlying cause of a myriad of diseases. We present the first use of surface enhanced Raman scattering (SERS) as a detection technique capable of reporting intracellular activity of a specific enzyme. Careful choice of reagents allowed the preparation of high resolution cellular activity maps highlighting the specific conversion of the commonly used ELISA reagent 5-bromo-4-chloro-3-indolyl ß-D-galactopyranoside (X-Gal), by wild type ß-galactosidase enzymes. Further, through co-addition of X-Gal substrate and inhibitors we were able to demonstrate that intracellular substrate conversion occurred predominantly through an enzymatically specific pathway. The data presented therefore supports the application of SERS probes as sensitive, specific sensors of biochemical activity and demonstrates the use of SERS probes for the first time as beacons capable of high resolution subcellular localisation of native enzymes.


Assuntos
Líquido Intracelular/química , Líquido Intracelular/enzimologia , Análise Espectral Raman/métodos , Animais , Células Cultivadas , Ativação Enzimática/fisiologia , Medições Luminescentes/métodos , Macrófagos/química , Macrófagos/enzimologia , Camundongos , Camundongos Endogâmicos BALB C , beta-Galactosidase/análise , beta-Galactosidase/metabolismo
2.
PLoS One ; 8(6): e67327, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23805309

RESUMO

The protozoan parasite, Trypanosoma brucei, is spread by the tsetse fly and causes trypanosomiasis in humans and animals. Both the life cycle and cell cycle of the parasite are complex. Trypanosomes have eleven cdc2-related kinases (CRKs) and ten cyclins, an unusually large number for a single celled organism. To date, relatively little is known about the function of many of the CRKs and cyclins, and only CRK3 has previously been shown to be cyclin-dependent in vivo. Here we report the identification of a previously uncharacterised CRK:cyclin complex between CRK12 and the putative transcriptional cyclin, CYC9. CRK12:CYC9 interact to form an active protein kinase complex in procyclic and bloodstream T. brucei. Both CRK12 and CYC9 are essential for the proliferation of bloodstream trypanosomes in vitro, and we show that CRK12 is also essential for survival of T. brucei in a mouse model, providing genetic validation of CRK12:CYC9 as a novel drug target for trypanosomiasis. Further, functional characterisation of CRK12 and CYC9 using RNA interference reveals roles for these proteins in endocytosis and cytokinesis, respectively.


Assuntos
Ciclinas , Complexos Multiproteicos , Proteínas Proto-Oncogênicas c-crk , Proteínas de Protozoários , Trypanosoma brucei brucei , Animais , Proliferação de Células/genética , Ciclinas/genética , Ciclinas/metabolismo , Camundongos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas Proto-Oncogênicas c-crk/genética , Proteínas Proto-Oncogênicas c-crk/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Tripanossomíase Africana/genética , Tripanossomíase Africana/metabolismo
3.
Nanomedicine (Lond) ; 8(2): 181-92, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22891867

RESUMO

AIM: The therapeutic potential of epigallocatechin-3-gallate (EGCG), a green tea polyphenol with anticancer properties, is limited by its inability to specifically reach tumors following intravenous administration. The purpose of this study was to determine whether a tumor-targeted vesicular formulation of EGCG would suppress the growth of A431 epidermoid carcinoma and B16-F10 melanoma in vitro and in vivo. MATERIALS & METHODS: Transferrin-bearing vesicles encapsulating EGCG were administered intravenously to mice bearing subcutaneous A431 and B16-F10 tumors. RESULTS: The intravenous administration of EGCG encapsulated in transferrin-bearing vesicles resulted in tumor suppression in 40% of A431 and B16-F10 tumors. Animal survival was improved by more than 20 days compared with controls. CONCLUSION: Encapsulation of EGCG in transferrin-bearing vesicles is a promising therapeutic strategy.


Assuntos
Catequina/análogos & derivados , Chá/química , Administração Intravenosa , Animais , Carcinoma de Células Escamosas/tratamento farmacológico , Catequina/administração & dosagem , Catequina/uso terapêutico , Linhagem Celular Tumoral , Feminino , Humanos , Melanoma/tratamento farmacológico , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão , Ensaios Antitumorais Modelo de Xenoenxerto
4.
PLoS One ; 7(9): e44617, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22957089

RESUMO

Signalling through post-translational modification (PTM) of proteins is a process central to cell homeostasis, development and responses to external stimuli. The best characterised PTM is protein phosphorylation which is reversibly catalysed at specific residues through the action of protein kinases (addition) and phosphatases (removal). Here, we report characterisation of an orphan protein phosphatase that possesses a domain architecture previously only described in Plantae. Through gene disruption and the production of active site mutants, the enzymatically active Protein Phosphatase containing Kelch-Like domains (PPKL, PBANKA_132950) is shown to play an essential role in the development of an infectious ookinete. PPKL is produced in schizonts and female gametocytes, is maternally inherited where its absence leads to the development of a malformed, immotile, non-infectious ookinete with an extended apical protrusion. The distribution of PPKL includes focussed localization at the ookinete apical tip implying a link between its activity and the correct deployment of the apical complex and microtubule cytoskeleton. Unlike wild type parasites, ppkl(-) ookinetes do not have a pronounced apical distribution of their micronemes yet secretion of microneme cargo is unaffected in the mutant implying that release of microneme cargo is either highly efficient at the malformed apical prominence or secretion may also occur from other points of the parasite, possibly the pellicular pores.


Assuntos
Monoéster Fosfórico Hidrolases/química , Plasmodium berghei/enzimologia , Plasmodium berghei/patogenicidade , Plasmodium falciparum/enzimologia , Plasmodium falciparum/patogenicidade , Animais , Catálise , Domínio Catalítico , Movimento Celular , Citoesqueleto/metabolismo , Feminino , Regulação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Masculino , Microtúbulos/metabolismo , Fenótipo , Fosforilação , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Proteínas de Protozoários/fisiologia
5.
Cell Microbiol ; 14(8): 1271-86, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22486816

RESUMO

Leishmania ISPs are ecotin-like natural peptide inhibitors of trypsin-family serine peptidases, enzymes that are absent from the Leishmania genome. This led to the proposal that ISPs inhibit host serine peptidases and we have recently shown that ISP2 inhibits neutrophil elastase, thereby enhancing parasite survival in murine macrophages. In this study we show that ISP1 has less serine peptidase inhibitory activity than ISP2, and in promastigotes both are generally located in the cytosol and along the flagellum. However, in haptomonad promastigotes there is a prominent accumulation of ISP1 and ISP2 in the hemidesmosome and for ISP2 on the cell surface. An L. major mutant deficient in all three ISP genes (Δisp1/2/3) was generated and compared with Δisp2/3 mutants to elucidate the physiological role of ISP1. In in vitro cultures, the Δisp1/2/3 mutant contained more haptomonad, nectomonad and leptomonad promastigotes with elongated flagella and reduced motility compared with Δisp2/3 populations, moreover it was characterized by very high levels of release of exosome-like vesicles from the flagellar pocket. These data suggest that ISP1 has a primary role in flagellar homeostasis, disruption of which affects differentiation and flagellar pocket dynamics.


Assuntos
Leishmania major/fisiologia , Inibidores de Proteases/metabolismo , Proteínas Secretadas Inibidoras de Proteinases/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Células Cultivadas , Flagelos/metabolismo , Flagelos/ultraestrutura , Técnicas de Inativação de Genes , Interações Hospedeiro-Parasita , Leishmania major/genética , Leishmania major/metabolismo , Leishmania major/ultraestrutura , Macrófagos Peritoneais/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Inibidores de Proteases/química , Transporte Proteico , Proteínas Secretadas Inibidoras de Proteinases/química , Proteínas Secretadas Inibidoras de Proteinases/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Vesículas Secretórias/metabolismo , Vesículas Secretórias/ultraestrutura , Serina Proteases/química
6.
Mol Microbiol ; 84(1): 77-92, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22329999

RESUMO

AIR9 is a cytoskeleton-associated protein in Arabidopsis thaliana with roles in cytokinesis and cross wall maturation, and reported homologues in land plants and excavate protists, including trypanosomatids. We show that the Trypanosoma brucei AIR9-like protein, TbAIR9, is also cytoskeleton-associated and colocalizes with the subpellicular microtubules. We find it to be expressed in all life cycle stages and show that it is essential for normal proliferation of trypanosomes in vitro. Depletion of TbAIR9 from procyclic trypanosomes resulted in increased cell length due to increased microtubule extension at the cell posterior. Additionally, the nucleus was re-positioned to a location posterior to the kinetoplast, leading to defects in cytokinesis and the generation of aberrant progeny. In contrast, in bloodstream trypanosomes, depletion of TbAIR9 had little effect on nucleus positioning, but resulted in aberrant cleavage furrow placement and the generation of non-equivalent daughter cells following cytokinesis. Our data provide insight into the control of nucleus positioning in this important pathogen and emphasize differences in the cytoskeleton and cell cycle control between two life cycle stages of the T. brucei parasite.


Assuntos
Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/citologia , Animais , Pontos de Checagem do Ciclo Celular , Linhagem Celular , Citocinese , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Protozoários/genética , Interferência de RNA , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/ultraestrutura , Moscas Tsé-Tsé
7.
Pharm Res ; 29(3): 782-94, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21971828

RESUMO

PURPOSE: To investigate the use of nano self-assemblies formed by polyallylamine (PAA) modified with 5 or 10% mole fluorenylmethoxy carbonyl (Fmoc(5)/(10)), dimethylamino-1-naphthalenesulfonyl (Dansyl(5)/(10)) and 5% mole cholesteryl group (Ch(5)) for oral hydrophobic drug delivery. METHODS: Propofol, griseofulvin and prednisolone were loaded into amphiphilic PAAs. Particle size and morphology of drug-loaded self-assemblies were determined using photon correlation spectroscopy and transmission electron microscopy. Solubilising capacity, in vitro drug release and formulation stability were analysed by HPLC, and in vitro biocompatibility studies (haemolysis and cytotoxicity) were carried out on bovine erythrocytes and Caco-2 cells, respectively. Dansyl(10) and Ch(5) griseofulvin formulations were administered intra-gastrically to rats, and drug plasma levels were analysed by HPLC. RESULTS: Drug-encapsulated self-assemblies typically have hydrodynamic size of 300-400 nm. Dansyl(10) exhibited universal drug solubiliser property and had significantly improved prednisolone, griseofulvin and propofol solubility by 145, 557 and 224-fold, respectively. Fmoc polymers resulted in modest drug solubility improvement. These polymers were non-haemolytic, did not enhance cytotoxicity compared to unmodified PAA, and demonstrated significant increase in griseofulvin plasma concentration compared to griseofulvin in water after oral administration. CONCLUSIONS: Ch(5) and Dansyl(10) showed promising potential as nano-carriers for oral hydrophobic drug delivery.


Assuntos
Anti-Inflamatórios/administração & dosagem , Portadores de Fármacos/química , Griseofulvina/administração & dosagem , Hipnóticos e Sedativos/administração & dosagem , Poliaminas/química , Prednisolona/administração & dosagem , Propofol/administração & dosagem , Administração Oral , Animais , Anti-Inflamatórios/farmacocinética , Células CACO-2 , Bovinos , Sobrevivência Celular , Colestenos/química , Colestenos/toxicidade , Compostos de Dansil/química , Compostos de Dansil/toxicidade , Portadores de Fármacos/toxicidade , Fluorenos/química , Fluorenos/toxicidade , Griseofulvina/farmacocinética , Hemólise/efeitos dos fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Hipnóticos e Sedativos/farmacocinética , Masculino , Poliaminas/toxicidade , Prednisolona/farmacocinética , Propofol/farmacocinética , Ratos , Ratos Sprague-Dawley
8.
J Biol Chem ; 286(46): 39914-25, 2011 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-21949125

RESUMO

Metacaspases are caspase family cysteine peptidases found in plants, fungi, and protozoa but not mammals. Trypanosoma brucei is unusual in having five metacaspases (MCA1-MCA5), of which MCA1 and MCA4 have active site substitutions, making them possible non-enzymatic homologues. Here we demonstrate that recombinant MCA4 lacks detectable peptidase activity despite maintaining a functional peptidase structure. MCA4 is expressed primarily in the bloodstream form of the parasite and associates with the flagellar membrane via dual myristoylation/palmitoylation. Loss of function phenotyping revealed critical roles for MCA4; rapid depletion by RNAi caused lethal disruption to the parasite's cell cycle, yet the generation of MCA4 null mutant parasites (Δmca4) was possible. Δmca4 had normal growth in axenic culture but markedly reduced virulence in mice. Further analysis revealed that MCA4 is released from the parasite and is specifically processed by MCA3, the only metacaspase that is both palmitoylated and enzymatically active. Accordingly, we have identified that the multiple metacaspases in T. brucei form a membrane-associated proteolytic cascade to generate a pseudopeptidase virulence factor.


Assuntos
Caspases/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/enzimologia , Fatores de Virulência/metabolismo , Animais , Caspases/genética , Flagelos/genética , Flagelos/metabolismo , Lipoilação/fisiologia , Camundongos , Proteínas de Protozoários/genética , Fatores de Virulência/genética
9.
Biomaterials ; 32(25): 5889-99, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21596431

RESUMO

The possibility of using non-viral gene delivery systems for the treatment of cancer is currently limited by their lower transfection efficacy compared to viral systems. On the basis that amino acids such as arginine, lysine and leucine were involved in enhancing DNA transportation into cells, we hypothesized that the grafting of these amino acids to the highly promising generation 3 diaminobutyric polypropylenimine (DAB) dendrimer would improve its transfection efficacy in cancer cells. In this work we demonstrated that the conjugation of arginine, lysine and leucine to the dendrimer led to an enhanced anti-proliferative activity of the polyplexes, by up to 47-fold for DAB-Lys in T98G cancer cells compared to the unmodified polyplex in vitro. In vivo, the intravenous administration of amino acid-bearing DAB polyplexes resulted in a significantly improved tumor gene expression, with the highest gene expression level observed after treatment with DAB-Lys polyplex. Arginine, lysine and leucine-bearing generation 3 polypropylenimine polymers are therefore highly promising gene delivery systems for gene transfection in tumors.


Assuntos
Arginina/administração & dosagem , Expressão Gênica , Leucina/administração & dosagem , Lisina/administração & dosagem , Neoplasias/patologia , Polipropilenos/química , Animais , Feminino , Humanos , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Endogâmicos BALB C
10.
J Control Release ; 154(1): 20-6, 2011 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-21539872

RESUMO

The therapeutic potential of tocotrienol, a vitamin E extract with anti-cancer properties, is hampered by its failure to specifically reach tumors after intravenous administration. In this work, we demonstrated that novel transferrin-bearing, tocopheryl-based multilamellar vesicles entrapping tocotrienol significantly improved tocotrienol uptake by cancer cells overexpressing transferrin receptors. This led to a dramatically improved therapeutic efficacy in vitro, ranging from 17-fold to 72-fold improvement depending on the cell lines, compared to the free drug. In vivo, the intravenous administration of this novel tocotrienol formulation led to complete tumor eradication for 40% of B16-F10 murine melanoma tumors and 20% of A431 human epidermoid carcinoma tumors. Animal survival was improved by more than 20 days compared to controls, for the two tumor models tested. These therapeutic effects, together with the lack of toxicity, potentially make transferrin-bearing vesicles entrapping tocotrienol a highly promising therapeutic system as part as an anti-cancer therapeutic strategy.


Assuntos
Antineoplásicos/administração & dosagem , Carcinoma de Células Escamosas/tratamento farmacológico , Sistemas de Liberação de Medicamentos/métodos , Melanoma Experimental/tratamento farmacológico , Tocotrienóis/administração & dosagem , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Transporte Biológico , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Composição de Medicamentos , Feminino , Humanos , Injeções Intravenosas , Melanoma Experimental/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão , Receptores da Transferrina/biossíntese , Receptores da Transferrina/metabolismo , Propriedades de Superfície , Análise de Sobrevida , Tocotrienóis/química , Tocotrienóis/uso terapêutico , Transferrina/química , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Nanomedicine ; 7(5): 615-23, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21333755

RESUMO

The potential of gene therapy to treat cancer is currently limited by the low expression of therapeutic genes in the tumors. Because amino acids are known to have excellent properties in cell penetration and gene expression regulation, we investigated if the conjugation of arginine (Arg), lysine (Lys) and leucine (Leu) onto the surface of the gene delivery system polyethylenimine (PEI) could lead to an improved gene expression in tumors. The intravenous administration of Arg-, Lys- and Leu-bearing PEI polyplexes led to a significant increase of gene expression in the tumor, with a ß-galactosidase expression amount at least threefold higher than that obtained after treatment with unmodified PEI polyplex. The three amino acid-bearing PEI polyplexes led to similar levels of gene expression in the tumor. The treatments were well tolerated by the mice. Arg-, Lys- and Leu-bearing PEI polyplexes are therefore highly promising gene delivery systems for cancer therapy.


Assuntos
Arginina/administração & dosagem , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Terapia Genética/métodos , Leucina/administração & dosagem , Lisina/administração & dosagem , Neoplasias/terapia , Polietilenoimina/química , Animais , Arginina/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Expressão Gênica/efeitos dos fármacos , Técnicas de Transferência de Genes , Humanos , Leucina/química , Lisina/química , Camundongos , Camundongos Endogâmicos BALB C , beta-Galactosidase/efeitos dos fármacos , beta-Galactosidase/genética
13.
J Pharm Sci ; 99(11): 4642-57, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20845462

RESUMO

Cationic polyamines such as the poly(propylenimine) dendrimers (DAB16) are anti-tumour agents (Dufes et al., 2005, Cancer Res 65:8079-8084). Their mechanism of action is poorly understood, but the lack of in vivo toxicity suggests cancer specificity. To explore this polyamine pharmacophore we cross-linked the aza-cyclic compound, hexacyclen, with 1,4-dibromobutane or 1,8-dibromooctane to yield the polyamines [poly(butylhexacyclen)--CL4] or [poly(octylhexacyclen)--CL8] respectively, both free of primary amines. We characterised the compounds and their respective nanoparticles and examined their interaction with the putative targets of the cationic polyamines: the cell membrane and DNA. Like DAB 16, CL4 and CL8 bind plasmid DNA and all three compounds interrupted the cell cycle of A431 epidermoid carcinoma cells in the S-phase. Additionally all three compounds disrupted erythrocyte membranes, with CL8 and DAB 16 being more active, in this respect, than CL4. CL4 (IC(50) =775.1 µg mL(-1)) and CL8 (IC(50) =8.4 µg mL(-1)), in a similar manner to DAB 16, were anti-proliferative against A431 cells; although unlike DAB 16, CL4 and CL8 were not tumouricidal against A431 xenografts in mice, indicating that primary amines may play an important role in the in vivo activity of DAB 16.


Assuntos
Antineoplásicos/farmacologia , Compostos Aza/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Poliaminas/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Compostos Aza/química , Compostos Aza/uso terapêutico , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , DNA/metabolismo , Feminino , Humanos , Camundongos , Camundongos Nus , Poliaminas/química , Poliaminas/uso terapêutico
14.
J Control Release ; 147(2): 289-97, 2010 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-20705108

RESUMO

The cationic peptide, salmon calcitonin (sCT) was complexed with the cationic amphiphilic polyelectrolyte, poly(allyl)amine, grafted with palmitoyl and quaternary ammonium moieties at pH 5.0 and 7.4 to yield particulates (sCT-QPa). The complexes were approximately 200 nm in diameter, had zeta potentials ranging from +20 to +50 mV, and had narrow polydispersity indices (PDIs). Differential scanning calorimetry revealed the presence of an interaction between sCT and QPa in the complexes. Electron microscopy confirmed the zeta-size data and revealed a vesicular bilayer structure with an aqueous core. Tyrosine- and Nile red fluorescence indicated that the complexes retained gross physical stability for up to 7 days, but that the pH 5.0 complexes were more stable. The complexes were more resistant to peptidases, serum and liver homogenates compared to free sCT. In vitro bioactivity was measured by cAMP production in T47D cells and the complexes had EC50 values in the nM range. While free sCT was unable to generate cAMP following storage for 7 days, the complexes retained approximately 33% activity. When the complexes were injected intravenously to rats, free and complexed sCT (pH 5.0 and 7.4) but not QPa reduced serum calcium over 120 min. Free and complexed sCT but not QPa also reduced serum calcium over 240 min following intra-jejunal administration. In conclusion, sCT-QPa nanocomplexes that have been synthesised are stable, bioactive and resistant to a range of peptidases. These enhanced features suggest that they may have the potential for improved efficacy when formulated for injected and oral delivery.


Assuntos
Conservadores da Densidade Óssea/administração & dosagem , Calcitonina/administração & dosagem , Portadores de Fármacos/química , Nanopartículas/química , Poliaminas/química , Compostos de Amônio Quaternário/química , Tensoativos/química , Animais , Conservadores da Densidade Óssea/farmacologia , Calcitonina/farmacologia , Cálcio/sangue , Varredura Diferencial de Calorimetria , Composição de Medicamentos , Estabilidade de Medicamentos , Masculino , Microscopia Eletrônica de Transmissão , Tamanho da Partícula , Ratos , Ratos Wistar , Propriedades de Superfície
15.
Microbiology (Reading) ; 156(Pt 10): 3079-3084, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20616103

RESUMO

Human small and large intestinal tissue was used to study the interaction of Campylobacter jejuni with its target tissue. The strain used for the study was 81-176 (+pVir). Tissue was processed for scanning and transmission electron microscopy, and by immunohistochemistry for light microscopy. Organisms adhered to the apical surface of ileal tissues at all time points in large numbers, in areas where mucus was present and in distinct groups. Microcolony formation was evident at 1-2 h, with bacteria adhering to mucus on the tissue surface and to each other by flagellar interaction. At later time points (3-4 h), biofilm formation on ileal tissue was evident. Flagellar mutants did not form microcolonies or biofilms in tissue. Few organisms were observed in colonic tissue, with organisms present but not as abundant as in the ileal tissue. This study shows that C. jejuni 81-176 can form microcolonies and biofilms on human intestinal tissue and that this may be an essential step in its ability to cause diarrhoea in man.


Assuntos
Biofilmes/crescimento & desenvolvimento , Campylobacter jejuni/crescimento & desenvolvimento , Intestino Grosso/microbiologia , Intestino Delgado/microbiologia , Aderência Bacteriana , Humanos , Técnicas In Vitro , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão
16.
J Control Release ; 143(2): 215-21, 2010 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-19944722

RESUMO

The possibility of using genes as medicines to treat cancer is limited by the lack of safe and efficacious delivery systems able to deliver therapeutic genes selectively to tumors by intravenous administration. We investigate if the conjugation of the polypropylenimine dendrimer to transferrin, whose receptors are overexpressed on numerous cancers, could result in a selective gene delivery to tumors after intravenous administration, leading to an increased therapeutic efficacy. The objectives of this study are to evaluate the targeting and therapeutic efficacies of a novel transferrin-bearing polypropylenimine dendrimer. The intravenous administration of transferrin-bearing polypropylenimine polyplex resulted in gene expression mainly in the tumors. Consequently, the intravenous administration of the delivery system complexed to a therapeutic DNA led to a rapid and sustained tumor regression over one month, with long-term survival of 100% of the animals (90% complete response, 10% partial response).The treatment was well tolerated by the animals, with no apparent signs of toxicity. Transferrin-bearing polypropylenimine may thus be a promising gene delivery system for cancer therapy.


Assuntos
Carcinoma de Células Escamosas/terapia , DNA/administração & dosagem , Dendrímeros/química , Técnicas de Transferência de Genes , Polipropilenos/química , Transferrina/química , Fator de Necrose Tumoral alfa/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células , DNA/genética , Feminino , Expressão Gênica , Terapia Genética/métodos , Glioblastoma/terapia , Camundongos , Camundongos Endogâmicos BALB C , Tamanho da Partícula , Plasmídeos/administração & dosagem , Plasmídeos/genética , Transfecção , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Langmuir ; 26(4): 2301-16, 2010 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-19860445

RESUMO

The shape of dendrimer amphiphiles has an unexpected effect on their self-assembly. A series of diaminobutane poly(propylenimine) generation 3 dendrimer (DAB-dendr-(NH(2))(16)) amphiphiles has been synthesized, bearing an average of five (PD5), three (PD3) and one (PD1) palmitoyl group(s) per dendrimer molecule. Additionally DAB-dendr-(NH(2))(16) was derivatized with a layer of poly(ethylene glycol) (PEG, degree of polymerization = 12) groups and conjugated to an average of 1 palmitoyl group at the PEG end (PPD1). A final amphiphile resulted from the conjugation of DAB-dendr-(NH(2))(16) with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-succinimidylpropionate (DSPE-PEG(3400)-SPA), i.e.: DPD5 (with 4 DSPE-PEG arms). The critical micellar concentration in aqueous media followed the trend: DPD5 < PD5 = PD3 < PD1 < PPD1 and amphiphiles eventually formed 10-20 nm monomolecular or multimolecular micelles and/or 200 nm spheres or tubules. Aggregation was entropy driven, as expected, for DPD5, PD5 and PD1 and enthalpy driven with the most hydrophilic compound PPD1, but was unexpectedly enthalpy driven for PD3. PD3 aggregates formed low capacity hydrophobic domains with a limited capacity for encapsulation of cyclosporine A; encapsulation levels (mole drug per mole polymer) were 0.099, 0.014, 0.099, and 0.735 for PD1, PD3, PD5, and DPD5 and, respectively. We conclude that star shaped amphiphiles such as PD3 are sterically hindered from self-assembling into high capacity hydrophobic domains in aqueous media. Amphiphile-membrane interactions were promoted by hydrophobic groups, but diminished by PEG moieties. DPD5 is the most suitable amphiphile for biomedical applications.


Assuntos
Polipropilenos/química , Tensoativos/química , Dendrímeros/química , Micelas , Estrutura Molecular , Tamanho da Partícula , Polipropilenos/síntese química , Propriedades de Superfície , Tensoativos/síntese química , Termodinâmica
18.
J Control Release ; 140(2): 95-9, 2009 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-19709637

RESUMO

The therapeutic potential of tocotrienol, an extract of vitamin E with anti-cancer properties, is hampered by its failure to specifically reach tumors after intravenous administration, without secondary effects on normal tissues. We hypothesize that the encapsulation of tocotrienol-rich fraction (TRF) within vesicles bearing transferrin, whose receptors are overexpressed on many cancer cells, could result in a selective delivery to tumors after intravenous administration. The objectives of this study are therefore to prepare and characterize transferrin-targeted vesicles encapsulating TRF, and to evaluate their therapeutic efficacy in vitro and in vivo. The entrapment of TRF in transferrin-bearing vesicles led to a 3-fold higher TRF uptake and more than 100-fold improved cytotoxicity in A431 (epidermoid carcinoma), T98G (glioblastoma) and A2780 (ovarian carcinoma) cell lines compared to TRF solution. The intravenous administration of TRF encapsulated in transferrin-bearing vesicles led to tumor regression and improvement of animal survival in a murine xenograft model, contrary to that observed with controls. The treatment was well tolerated by the animals. This work corresponds to the first preparation of a tumor-targeted delivery system able to encapsulate tocotrienol. Our findings show that TRF encapsulated in transferrin-bearing vesicles is a highly promising therapeutic system, leading to tumor regression after intravenous administration without visible toxicity.


Assuntos
Antineoplásicos/administração & dosagem , Portadores de Fármacos , Neoplasias Pulmonares/tratamento farmacológico , Tocotrienóis/administração & dosagem , Transferrina/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/toxicidade , Transporte Biológico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Química Farmacêutica , Relação Dose-Resposta a Droga , Composição de Medicamentos , Feminino , Concentração Inibidora 50 , Injeções Intravenosas , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Tamanho da Partícula , Receptores da Transferrina/metabolismo , Solventes/química , Fatores de Tempo , Tocotrienóis/química , Tocotrienóis/metabolismo , Tocotrienóis/toxicidade , Transferrina/química , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Analyst ; 134(1): 170-5, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19082189

RESUMO

Bone marrow-derived immune cells (macrophages) treated with gold and silver nanoparticles before fixation and dye staining have been analysed by multiple wavelength line scanning surface enhanced resonance Raman scattering (SERRS) mapping. The method yields high selectivity and sensitivity within short analysis times, identifying nanoparticle aggregates in secondary lysosomes. Using routine cell stains, the output from fluorescence, Raman and SERRS is quantified at four wavelengths of excitation, demonstrating the potential at longer biologically compatible wavelengths of using nanoparticles with cell stains for superior cell mapping.


Assuntos
Macrófagos/ultraestrutura , Corantes , Ouro , Nanopartículas , Sensibilidade e Especificidade , Prata , Análise Espectral Raman/métodos
20.
Langmuir ; 24(18): 9997-10004, 2008 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-18690736

RESUMO

Self-assembly is fundamental to the biological function of cells and the fabrication of nanomaterials. However, the origin of the shape of various self-assemblies, such as the shape of cells, is not altogether clear. Polymeric, oligomeric, or low molecular weight amphiphiles are a rich source of nanomaterials, and controlling their self-assembly is the route to tailored nanosystems with specific functionalities. Here, we provide direct evidence that a particular molecular architecture, polymeric branching, leads to a rare form of self-assembly, the planar nanodisc. Cholesterol containing self-assemblies formed from amphiphilic linear or branched cetyl poly(ethylenimine) (Mn approximately 1000 Da) or amphiphilic cetyl poly(propylenimine) dendrimer derivatives (Mn approximately 2000 Da) show that branching, by reducing the hydrophilic headgroup area, alters the shape of the self-assemblies transforming closed 60 nm spherical bilayer vesicles to rare 50 nm x 10 nm planar bilayer discs. Increasing the hydrophilic headgroup area, by the inclusion of methoxy poly(ethylene glycol) moieties into the amphiphilic headgroup, transforms the planar discs to 100 nm spherical bilayer vesicles. This study provides insight into the key role played by molecular shape on molecular self-organization into rare nanodiscs.


Assuntos
Dendrímeros/química , Nanotecnologia/métodos , Polímeros/química , Colesterol/química , Relação Dose-Resposta a Droga , Bicamadas Lipídicas/química , Microscopia Eletrônica de Transmissão , Modelos Químicos , Conformação Molecular , Nanopartículas/química , Nanoestruturas/química , Polietilenoglicóis , Polipropilenos/química , Propriedades de Superfície , Termodinâmica
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