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1.
Biochem Biophys Res Commun ; 354(4): 949-54, 2007 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-17276393

RESUMO

Propagation of the scrapie isoform of the prion protein (PrP(Sc)) depends on the expression of endogenous cellular prion (PrP(C)). During oral infection, PrP(Sc) propagates, by conversion of the PrP(C) to PrP(Sc), from the gastrointestinal tract to the nervous system. Intestinal epithelium could serve as the primary site for PrP(C) conversion. To investigate PrP(C) sorting in epithelia cells, we have generated both a green fluorescent protein (EGFP) or hemagglutinin (HA) tagged human PrP(C) (hPrP(C)). Combined molecular, biochemical, and single living polarized cell imaging characterizations suggest that hPrP(C) is selectively targeted to the apical side of Madin-Darby canine kidney (MDCKII) and of intestinal epithelia (Caco2) cells.


Assuntos
Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Proteínas PrPC/metabolismo , Animais , Células CHO , Células CACO-2/metabolismo , Polaridade Celular , Cricetinae , Cricetulus , Cães , Humanos , Proteínas PrPC/biossíntese , Transporte Proteico
2.
J Cell Biol ; 150(2): 321-34, 2000 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-10908575

RESUMO

Programmed cell death or apoptosis leads to the activation of the caspase-activated DNase (CAD), which degrades chromosomal DNA into nucleosomal fragments. Biochemical studies revealed that CAD forms an inactive heterodimer with the inhibitor of caspase-activated DNase (ICAD), or its alternatively spliced variant, ICAD-S, in the cytoplasm. It was initially proposed that proteolytic cleavage of ICAD by activated caspases causes the dissociation of the ICAD/CAD heterodimer and the translocation of active CAD into the nucleus in apoptotic cells. Here, we show that endogenous and heterologously expressed ICAD and CAD reside predominantly in the nucleus in nonapoptotic cells. Deletional mutagenesis and GFP fusion proteins identified a bipartite nuclear localization signal (NLS) in ICAD and verified the function of the NLS in CAD. The two NLSs have an additive effect on the nuclear targeting of the CAD-ICAD complex, whereas ICAD-S, lacking its NLS, appears to have a modulatory role in the nuclear localization of CAD. Staurosporine-induced apoptosis evoked the proteolysis and disappearance of endogenous and exogenous ICAD from the nuclei of HeLa cells, as monitored by immunoblotting and immunofluorescence microscopy. Similar phenomenon was observed in the caspase-3-deficient MCF7 cells upon expressing procaspase-3 transiently. We conclude that a complex mechanism, involving the recognition of the NLSs of both ICAD and CAD, accounts for the constitutive accumulation of CAD/ICAD in the nucleus, where caspase-3-dependent regulation of CAD activity takes place.


Assuntos
Apoptose/fisiologia , Núcleo Celular/metabolismo , Fragmentação do DNA/fisiologia , Desoxirribonucleases/metabolismo , Proteínas/metabolismo , Proteínas Reguladoras de Apoptose , Caspase 3 , Caspases/metabolismo , Compartimento Celular/fisiologia , Dimerização , Transdução de Sinais/fisiologia , Células Tumorais Cultivadas
3.
J Biol Chem ; 275(3): 1625-9, 2000 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-10636854

RESUMO

The diffusion of DNA in cytoplasm is thought to be an important determinant of the efficacy of gene delivery and antisense therapy. We have measured the translational diffusion of fluorescein-labeled double-stranded DNA fragments (in base pairs (bp): 21, 100, 250, 500, 1000, 2000, 3000, 6000) after microinjection into cytoplasm and nucleus of HeLa cells. Diffusion was measured by spot photobleaching using a focused argon laser spot (488 nm). In aqueous solutions, diffusion coefficients of the DNA fragments in water (D(w)) decreased from 53 x 10(-8) to 0.81 x 10(-8) cm(2)/s for sizes of 21-6000 bp; D(w) was related empirically to DNA size: D(w) = 4.9 x 10(-6) cm(2)/s.[bp size](-0.72). DNA diffusion coefficients in cytoplasm (D(cyto)) were lower than D(w) and depended strongly on DNA size. D(cyto)/D(w) decreased from 0.19 for a 100-bp DNA fragment to 0.06 for a 250-bp DNA fragment and was <0.01 for >2000 bp. Diffusion of microinjected fluorescein isothiocyanate (FITC) dextrans was faster than that of comparably sized DNA fragments of 250 bp and greater. In nucleus, all DNA fragments were nearly immobile, whereas FITC dextrans of molecular size up to 580 kDa were fully mobile. These results suggest that the highly restricted diffusion of DNA fragments in nucleoplasm results from extensive binding to immobile obstacles and that the decreased lateral mobility of DNAs >250 bp in cytoplasm is because of molecular crowding. The diffusion of DNA in cytoplasm may thus be an important rate-limiting barrier in gene delivery utilizing non-viral vectors.


Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , DNA/metabolismo , Biossíntese de Proteínas , Meios de Contraste/farmacologia , Dextranos/metabolismo , Fluoresceína/farmacologia , Células HeLa , Humanos , Microinjeções , Microscopia Confocal , Microscopia de Fluorescência
4.
Gene Ther ; 6(4): 482-97, 1999 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10476208

RESUMO

Inefficient nuclear delivery of plasmid DNA is thought to be one of the daunting hurdles to gene transfer, utilizing a nonviral delivery system such as polycation-DNA complex. Following its internalization by endocytosis, plasmid DNA has to be released into the cytosol before its nuclear entry can occur. However, the stability of plasmid DNA in the cytoplasm, that may play a determinant role in the transfection efficiency, is not known. The turnover of plasmid DNA, delivered by microinjection into the cytosol, was determined by fluorescence in situ hybridization (FISH) and quantitative single-cell fluorescence video-image analysis. Both single- and double-stranded circular plasmid DNA disappeared with an apparent half-life of 50-90 min from the cytoplasm of HeLa and COS cells, while the amount of co-injected dextran (MW 70,000) remained unaltered. We propose that cytosolic nuclease(s) are responsible for the rapid-degradation of plasmid DNA, since (1) elimination of plasmid DNA cannot be attributed to cell division or to the activity of apoptotic and lysosomal nucleases; (2) disposal of microinjected plasmid DNA was inhibited in cytosol-depleted cells or following the encapsulation of DNA in phospholipid vesicles; (3) generation and subsequent elimination of free 3'-OH ends could be detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay (TUNEL), reflecting the fragmentation of the injected DNA; and finally (4) isolated cytosol, obtained by selective permeabilization of the plasma membrane, exhibits divalent cation-dependent, thermolabile nuclease activity, determined by Southern blotting and 32P-release from end-labeled DNA. Collectively, these findings suggest that the metabolic instability of plasmid DNA, caused by cytosolic nuclease, may constitute a previously unrecognized impediment for DNA translocation into the nucleus and a possible target to enhance the efficiency of gene delivery.


Assuntos
Citosol/metabolismo , Técnicas de Transferência de Genes , Plasmídeos/metabolismo , Transfecção , Southern Blotting , Cálcio/metabolismo , Desoxirribonucleases/metabolismo , Digitonina/metabolismo , Células HeLa , Humanos , Hibridização in Situ Fluorescente , Microinjeções , Microscopia de Fluorescência
5.
J Biol Chem ; 274(31): 21873-7, 1999 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-10419506

RESUMO

Defective cAMP-stimulated chloride conductance of the plasma membrane of epithelial cell is the hallmark of cystic fibrosis (CF) and results from mutations in the cystic fibrosis transmembrane conductance regulator, CFTR. In the majority of CF patients, mutations in the CFTR lead to its misfolding and premature degradation at the endoplasmic reticulum (ER). Other mutations impair the cAMP-dependent activation or the ion conductance of CFTR chloride channel. In the present work we identify a novel mechanism leading to reduced expression of CFTR at the cell surface, caused by C-terminal truncations. The phenotype of C-terminally truncated CFTR, representing naturally occurring premature termination and frameshift mutations, were examined in transient and stable heterologous expression systems. Whereas the biosynthesis, processing, and macroscopic chloride channel function of truncated CFTRs are essentially normal, the degradation rate of the mature, complex-glycosylated form is 5- to 6-fold faster than the wild type CFTR. These experiments suggest that the C terminus has a central role in maintaining the metabolic stability of the complex-glycosylated CFTR following its exit from the ER and provide a plausible explanation for the severe phenotype of CF patients harboring C-terminal truncations.


Assuntos
Códon de Terminação/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Fibrose Cística/genética , Mutação da Fase de Leitura , Deleção de Sequência , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Células COS , Linhagem Celular , Membrana Celular/fisiologia , Cricetinae , Regulador de Condutância Transmembrana em Fibrose Cística/biossíntese , Primers do DNA , Bases de Dados Factuais , Heterozigoto , Humanos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Reação em Cadeia da Polimerase , Dobramento de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Transfecção
6.
J Neurochem ; 67(3): 988-95, 1996 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-8752104

RESUMO

The presence of P-glycoprotein in the cell plasma membrane limits the penetration of many cytotoxic substances into cells that express the gene product. There is considerable evidence also to indicate that P-glycoprotein is expressed as part of the normal blood-brain barrier in the luminal membranes of the cerebral capillary endothelial cells, where it presumably performs a protective function for the brain. This report describes the functional expression of P-glycoprotein in an immortalised cell line, RBE4, derived from rat cerebral capillary endothelial cells. The expression of P-glycoprotein is demonstrated by western immunoblotting and by immunogold and fluorescent staining with monoclonal antibodies. The cellular accumulation of [3H]colchicine and [3H]-vinblastine is investigated and shown to be enhanced by the presence of azidothymidine, chlorpromazine, verapamil, cyclosporin A, and PSC 833 ([3'-keto-Bmt1]-[Val2]-cyclosporin) at 50 or 100 microM concentration. It is concluded that the RBE4 cell line is a valuable tool for investigating the mechanisms of P-glycoprotein activity both in the blood-brain barrier and in multidrug resistance in general.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/fisiologia , Encéfalo/citologia , Animais , Transporte Biológico/fisiologia , Linhagem Celular , Linhagem Celular Transformada/efeitos dos fármacos , Linhagem Celular Transformada/metabolismo , Colchicina/farmacocinética , Endotélio/metabolismo , Humanos , Immunoblotting , Imuno-Histoquímica , Células KB/efeitos dos fármacos , Células KB/metabolismo , Ratos , Células Tumorais Cultivadas/metabolismo , Vimblastina/farmacocinética
7.
Ann Biol Clin (Paris) ; 54(1): 31-6, 1996.
Artigo em Inglês | MEDLINE | ID: mdl-8731793

RESUMO

The blood-brain barrier is formed by the cerebral capillary endothelial cells, joined together by tight junctions. These cells express the general endothelial cell markers as well as specific markers found on endothelial cells forming physiological barriers such as gamma-glutamyltranspeptidase, the glucose transporter Glut1 and the neutral amino-acid transporter. Using the monoclonal antibodies C219 and MRK16, we have revealed by Western blot and immuno-histochemistry the expression of the multidrug resistance P-glycoprotein on isolated rat cerebral cortex capillaries. On the other hand, P-glycoprotein was not detectable in brain cortex homogenates. P-glycoprotein thus appears to be a blood-brain barrier endothelium-specific marker which could regulate brain penetration of xenobiotics and thus participate in the neuroprotection of the brain.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/efeitos dos fármacos , Barreira Hematoencefálica/efeitos dos fármacos , Resistência a Múltiplos Medicamentos , Animais , Western Blotting , Córtex Cerebral/química , Humanos , Imuno-Histoquímica , Ratos , Ratos Sprague-Dawley
8.
Cell Biol Toxicol ; 11(5): 283-93, 1995 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-8608409

RESUMO

The P-glycoprotein mdr is expressed not only in tumoral cells, but also in nontransformed cells, including the specialized endothelial cells of brain capillaries which build up the blood-brain barrier. Since all previously identified blood-brain barrier markers are rapidly lost when cerebral capillary endothelial cells are maintained in primary culture, we have investigated whether P-glycoprotein (P-gp) would follow the same rule, in order to address the influence of the cerebral environment on the specific P-gp expression in the brain endothelium. As compared to freshly isolated purified cerebral capillaries, P-glycoprotein was detected by immunochemistry at a high level in 5-7 day primary cultures. In our culture conditions, P-glycoprotein was immunodetected at a lower molecular weight than that found in freshly isolated capillaries. Enzymatic deglycosylation led to the same 130 kDa protein for both fresh and cultured samples, suggesting that P-gp post-translational modifications were altered in primary cultures. However, studies on the uptake and efflux of the P-gp substrate [3H]vinblastine, and on the effect of various mdr reversing agents on the uptake and efflux, clearly indicated that the efflux pump function of the P-glycoprotein was maintained in primary cultures of bovine cerebral capillary endothelial cells. P-Glycoprotein may thus represent the first blood-brain barrier marker which is maintained in cerebral endothelial cells cultured in the absence of factors originating from the brain parenchyma.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Barreira Hematoencefálica/fisiologia , Endotélio Vascular/química , Endotélio Vascular/fisiologia , Vimblastina/farmacocinética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/análise , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Animais , Biomarcadores , Capilares/citologia , Bovinos , Células Cultivadas/química , Células Cultivadas/fisiologia , Córtex Cerebral/irrigação sanguínea , Endotélio Vascular/enzimologia , Imuno-Histoquímica , Trítio/farmacocinética , gama-Glutamiltransferase/metabolismo
9.
Exp Cell Res ; 220(1): 161-70, 1995 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-7664832

RESUMO

An immortalized brain capillary endothelial cell line displaying blood-brain barrier characteristics may represent a useful tool for studying blood-brain barrier endothelial cell differentiation and for the in vitro prediction of drug brain penetration. In the present study, we have established a rat cerebral capillary endothelial cell line (CR3) by genomic introduction of the immortalizing SV40 large T gene under the control of the human vimentin promoter. The CR3 cell line displayed endothelial morphological and biochemical characteristics for up to 30 passages. However, the CR3 cell line did not spontaneously express the specific blood-brain barrier markers gamma-glutamyl transpeptidase and mdr P-glycoprotein. However, when the cells were treated with the cell differentiating agent all-trans-retinoic acid, the blood-brain barrier markers were induced. Retinoic acid-treated CR3 cells may thus represent a useful tool for biological and pharmacological research related to the blood-brain barrier.


Assuntos
Barreira Hematoencefálica/fisiologia , Encéfalo/fisiologia , Linhagem Celular/fisiologia , Endotélio Vascular/fisiologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/análise , Animais , Biomarcadores , Encéfalo/citologia , Capilares/citologia , Capilares/fisiologia , Diferenciação Celular , Linhagem Celular/citologia , Transformação Celular Viral , Técnicas de Cultura/métodos , Endotélio Vascular/citologia , Imunofluorescência , Cariotipagem , Lipoproteínas LDL/metabolismo , Ratos , Vimentina/isolamento & purificação , Vimblastina/metabolismo , gama-Glutamiltransferase/análise , Fator de von Willebrand/isolamento & purificação
10.
Biochem Pharmacol ; 48(7): 1528-30, 1994 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-7945455

RESUMO

The expression of the multidrug transporter P-glycoprotein has been studied in two human intestinal epithelial cell lines. No functional expression of P-glycoprotein was found in the differentiated HT29-18-C1 cell line. The expression of P-glycoprotein in the Caco-2 cell line was very high, as judged by immunoblotting and by active efflux of vinblastine. The polarized transport of vinblastine in the basolateral to apical direction was temperature and energy dependent, and was reduced by P-glycoprotein inhibitors such as verapamil, chlorpromazine and reserpine. This adds further evidence that the polarized transport of vinblastine across Caco-2 monolayers is mediated by P-glycoprotein. The anticancer drug docetaxel (Taxotere) was transported in a polarized manner: basolateral to apical permeability was 20-fold higher than in the reverse direction. This polarized transport was inhibited by verapamil, chlorpromazine and reserpine, thus demonstrating that docetaxel is a substrate of P-glycoprotein. The implications of these results for the pharmacokinetics and toxicity of taxoids are discussed.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/fisiologia , Antineoplásicos Fitogênicos/metabolismo , Mucosa Intestinal/metabolismo , Paclitaxel/análogos & derivados , Taxoides , Vimblastina/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Transporte Biológico Ativo , Linhagem Celular , Docetaxel , Epitélio/metabolismo , Humanos , Immunoblotting , Paclitaxel/metabolismo
11.
Eur J Neurosci ; 5(5): 449-53, 1993 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-8261121

RESUMO

Axonal transport of the vesicular monoamine transporter was assayed in the rat brain by in vivo binding of the specific ligand [3H]reserpine. Because of the marked localization of reserpine binding sites in dopaminergic cell bodies and nerve terminals, the dopaminergic nigrostriatal pathway was chosen for the study of the axonal transport of the monoamine carrier present in the membrane of synaptic vesicles. When labelled reserpine was injected into the substantia nigra, a delayed accumulation of radioactivity in the ipsilateral striatum was observed approximately 4 h after the injection. Similarly, injection into the right striatum was followed by a substantial accumulation of radioactivity in the ipsilateral substantia nigra, which was prevented by peripheral injection of unlabelled reserpine or tetrabenazine. This process was rapid and dependent on microtubules. In senescent rats, the amount of retrogradely transported [3H]reserpine was significantly reduced. These results demonstrate that labelled reserpine may be used to monitor in vivo fast axonal transport in central neurons.


Assuntos
Axônios/metabolismo , Dopamina/metabolismo , Reserpina , Vesículas Sinápticas/metabolismo , Envelhecimento/metabolismo , Animais , Sítios de Ligação , Transporte Biológico , Colchicina/farmacologia , Corpo Estriado/metabolismo , Masculino , Microtúbulos/efeitos dos fármacos , Microtúbulos/fisiologia , Coelhos , Ratos , Ratos Sprague-Dawley , Substância Negra/metabolismo , Trítio
12.
Neurosci Lett ; 124(1): 111-4, 1991 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-1713313

RESUMO

It is now clearly established that fast anterograde axonal transport can be altered during ageing, both in the central and the peripheral nervous systems, but no information is yet available concerning the modifications of fast retrograde axonal transport during senescence. In the present paper, we report the changes occurring in the retrograde axonal transport of neurotensin in dopaminergic neurons of old rats. When iodinated neurotensin was injected into the striatum, a diminution of approximately 50% in the amount of the labelling measured in the ipsilateral substantia nigra was observed in senescent rats by comparison with young adult rats. Nevertheless, the rate of neurotensin transport was not modified. Our results clearly indicate that less neurotensin is transported from the nerve terminals towards the cell bodies in senescent rats which may have possible consequences for dopaminergic neurons.


Assuntos
Transporte Axonal , Corpo Estriado/metabolismo , Dopamina/fisiologia , Neurotensina/farmacocinética , Substância Negra/metabolismo , Animais , Hidroxidopaminas , Masculino , Microtúbulos/fisiologia , Neurônios/metabolismo , Oxidopamina , Ratos , Ratos Endogâmicos , Receptores de Neurotensina , Receptores de Neurotransmissores/metabolismo
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