RESUMO
We investigated the expression of insulin-like growth factor 1 (IGF-1) and IGF-1 type 1 receptor (IGF-1R) in skeletal muscle fiber types in chickens with hepatic fibrosis induced by bile duct ligation (BDL). Eleven hens, approximately 104 weeks old, were randomly assigned to BDL (n = 4) and sham surgery (SHAM; n = 7) groups. In BDL hens, histopathology revealed marked bile duct proliferation and liver fibrosis. The cross-sectional area (CSA) of myofibers from both the pectoralis (PCT) muscles significantly decreased in the BDL group compared with the SHAM group (P < 0.01). In contrast, the CSA of myofibers from the femorotibialis lateralis (FTL) muscle did not decrease in the BDL group. Type I fibers were large, round, and hypertrophic. Elongated type IIA and IIB fibers were also present. For IGF-1 immunostaining, the immunoreaction intensity was higher in the PCT in the BDL group than the SHAM group. Within the BDL group, type I fibers from FTL had a stronger immunoreaction intensity than the type II fibers. For IGF-1R immunostaining, the intensity of the immunoreactions was similar within the PCT in the BDL group compared with the SHAM group. For FTL, type I fibers had stronger reactions to IGF-1R than type II fibers in the BDL group. These results suggest that type I fibers express both IGF-1 and IGF-1R and become hypertrophic in chickens with hepatic fibrosis.
Assuntos
Galinhas , Fator de Crescimento Insulin-Like I , Animais , Feminino , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Fígado/metabolismo , Cirrose Hepática/veterinária , Fibras Musculares Esqueléticas/metabolismo , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismoRESUMO
3D rotational angiography provides remarkable spatial resolution for cerebrovascular disorders; however, it cannot be integrated directly into gamma knife planning due to the discrepancy of DICOM "tag" information, and most physicians still cannot benefit from 3D rotational angiography. Here, we describe a simple and easy technique to enable the integration of 3D rotational angiography.
Assuntos
Angiografia Cerebral/métodos , Imageamento Tridimensional/métodos , Radiocirurgia/métodos , Planejamento da Radioterapia Assistida por Computador/métodos , Feminino , Humanos , MasculinoRESUMO
We compared the effects of polypyrimidine tract-binding protein (PTB) on hepatitis C virus (HCV genotype IIa), encephalomyocarditis virus (EMCV) and poliovirus internal ribosome entry site (IRES) activities in vitro. It bound strongly to EMCV IRES, but weakly to PV and HCV RNAs. PV IRES showed the strongest dependency to PTB and it showed less than one-tenth of IRES activity after the immuno-depletion of PTB from HeLa S10 lysate with pre-coated anti-PTB IgG beads, comparing to the normal IgG beads-treated S10 lysate. EMCV IRES activity was approximately 40% of that of normal control after PTB depletion. Especially, HCV IRES activity was approximately 95%, and most weekly affected by the depletion of PTB. Repletion of PTB to depleted S10 lysate restored activities of PV and EMCV IRESs. The data suggest that PTB plays an important role in picornaviral IRESs, but not in HCV IRES.
Assuntos
Vírus da Encefalomiocardite/genética , Regulação Viral da Expressão Gênica/fisiologia , Hepacivirus/genética , Poliovirus/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/fisiologia , Sítio de Iniciação de Transcrição/fisiologia , Animais , Anticorpos Antivirais/biossíntese , Cobaias , Células HeLa , Humanos , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Biossíntese de Proteínas , RNA Viral/metabolismo , CoelhosRESUMO
Poliovirus receptor (hPVR/CD155) is a cell surface glycoprotein that belongs to the immunoglobulin superfamily but its natural function remains unknown. Two membrane-bound isoforms, hPVRalpha and hPVRdelta, are known to date, and they differ only in the amino acid sequence of their cytoplasmic domains. To gain an insight into the possible function of the cytoplasmic domains, we examined the localization of introduced hPVRalpha and hPVRdelta in polarized epithelial cells deficient of native hPVRs. Basolateral sorting of hPVRalpha was observed in Madine-Darby canine kidney cells expressing mu1B, but not in LLC-PK1 porcine kidney cells deficient in mu1B. Distribution of hPVRdelta, however, occurred both on the apical and basolateral plasma membranes of these two cell lines. Basolateral sorting of hPVRalpha was also seen in LLC-PK1 cells that expressed an intact exogenous mu1B, but not in the cells that expressed a mutant mu1B lacking binding ability to tyrosine-containing signals. These results indicate that mu1B is involved in the distribution of hPVRalpha to the basolateral membrane. Comparative distribution analysis of hPVRalpha using a series of mutants with truncations and substitutions in the cytoplasmic tail demonstrated that determinant for the basolateral sorting resided in the tyrosine-containing motif of the cytoplasmic tail. Furthermore, yeast two hybrid analysis strongly suggested that the tyrosine motif directly interacted with mu1B protein. Thus, basolateral sorting of hPVRalpha appears to involve the interaction with mu1B through a tyrosine motif existing in the cytoplasmic domain.
Assuntos
Complexo 1 de Proteínas Adaptadoras , Subunidades mu do Complexo de Proteínas Adaptadoras , Proteínas de Transporte/metabolismo , Polaridade Celular/fisiologia , Células Epiteliais/metabolismo , Proteínas de Membrana/metabolismo , Sinais Direcionadores de Proteínas/fisiologia , Receptores Virais/metabolismo , Proteínas Adaptadoras de Transporte Vesicular , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Linhagem Celular , Cães , Humanos , Imuno-Histoquímica , Proteínas de Membrana/química , Dados de Sequência Molecular , Isoformas de Proteínas , Subunidades Proteicas , Transporte Proteico , Receptores Virais/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
Yap1p, a crucial transcription factor in the oxidative stress response of Saccharomyces cerevisiae, is transported in and out of the nucleus under nonstress conditions. The nuclear export step is specifically inhibited by H(2)O(2) or the thiol oxidant diamide, resulting in Yap1p nuclear accumulation and induction of transcription of its target genes. Here we provide evidence for sensing of H(2)O(2) and diamide mediated by disulfide bond formation in the C-terminal cysteine-rich region (c-CRD), which contains 3 conserved cysteines and the nuclear export signal (NES). The H(2)O(2) or diamide-induced oxidation of the c-CRD in vivo correlates with induced Yap1p nuclear localization. Both were initiated within 1 min of application of oxidative stress, before the intracellular redox status of thioredoxin and glutathione was affected. The cysteine residues in the middle region of Yap1p (n-CRD) are required for prolonged nuclear localization of Yap1p in response to H(2)O(2) and are thus also required for maximum transcriptional activity. Using mass spectrometry analysis, the H(2)O(2)-induced oxidation of the c-CRD in vitro was detected as an intramolecular disulfide linkage between the first (Cys(598)) and second (Cys(620)) cysteine residues; this linkage could be reduced by thioredoxin. In contrast, diamide induced each pair of disulfide linkage in the c-CRD, but in this case the cysteine residues in the n-CRD appeared to be dispensable for the response. Our data provide evidence for molecular mechanisms of redox signal sensing through the thiol-disulfide redox cycle coupled with the thioredoxin system in the Yap1p NES.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/química , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Glutationa/metabolismo , Oxirredução , Transdução de Sinais , Tiorredoxinas/metabolismo , Fatores de Transcrição/químicaRESUMO
The genome of hepatitis C virus (HCV) is a single-stranded RNA of positive polarity that has a poly(U/C) tract followed by a highly conserved 98-nt sequence, termed the X region, in the 3' untranslated region (UTR). To investigate the effect of the 3'UTR on the HCV translation that depends on the internal ribosomal entry site (IRES), we prepared a deletion HCV RNA, MA delta, that lacked the RNA region from nt 1286 to 8785. A series of MA delta RNAs that differ in the primary structure of their 3'UTR, were generated and examined for their translation efficiencies in reticulocyte lysates. Deletion of the poly(U/C) tract and/or stem-loop structure (SL) 3 region of 3'X resulted in enhancement of the translation efficiency. Translation of MA delta RNA was inhibited by the addition of recombinant polypyrimidine tract-binding protein (PTB). A similar inhibition by PTB, however, was observed when an RNA lacking the poly(U/C) tract or SL3 region was used. The inhibitory effect by PTB was not obvious for MA delta (1041) RNA composed of nt 1 to 1041 but MA delta (8928) RNA composed of nt 1 to 1285 and nt 8786 to 8928. These results suggest that the observed down-regulation of HCV translation by the 3'UTR is mediated by some host factor(s) other than PTB, and that a PTB site for inhibition resides in the coding sequence of nt 1042 to 8928 of MA delta RNA.
Assuntos
Regiões 3' não Traduzidas , Hepacivirus/genética , Biossíntese de Proteínas , RNA Viral/genética , Animais , Regulação para Baixo , Iniciação Traducional da Cadeia Peptídica , Proteína de Ligação a Regiões Ricas em Polipirimidinas , Polirribonucleotídeos/metabolismo , Estabilidade de RNA , RNA Líder para Processamento/genética , RNA Viral/biossíntese , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Deleção de SequênciaRESUMO
Plasmid pSV2MT-I encoding mouse metallothionein-I (MT-I) designed to be expressed under the control of an SV40 promoter was introduced into human HeLa S3 cells. Several transformants (HeLa/MTH) carrying multi-copies of mouse MT-I cDNA in their genomes were isolated. These transformants produced 4 to 20-fold larger amounts of MT than their parent cells. The MT levels in HeLa/MTH were well correlated with the extent of resistance to cadmium, but not with that to cis-platinum (cis-DDP) in vitro. To study the role of MT in resistance to cis-DDP in vivo, nude mice were inoculated subcutaneously with two independent HeLa/MTH clones. MT levels in these tumors were about 3-fold higher than those in the parental cells. The growth of tumors derived from either HeLa/MTH clone was not inhibited in the presence of 15 micromol/kg of cis-DDP, which completely inhibited the growth of tumors derived from the parental HeLa cells. These data strongly suggest that the elevated level of MT confers resistance to cis-DDP in vivo but not in vitro. Thus, the results of this study indicate that in vitro determinations of the influence of MT on cis-DDP resistance may underestimate its importance in in vivo situations.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Metalotioneína/biossíntese , Animais , Antioxidantes/metabolismo , Northern Blotting , Southern Blotting , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Glutationa/metabolismo , Células HeLa , Humanos , Camundongos , Camundongos Nus , Plasmídeos , Fatores de Tempo , TransfecçãoRESUMO
We have isolated a novel actin filament-binding protein, named afadin, localized at cadherin-based cell-cell adherens junctions (AJs) in various tissues and cell lines. Afadin has one PDZ domain, three proline-rich regions, and one actin filament-binding domain. We found here that afadin directly interacted with a family of the immunoglobulin superfamily, which was isolated originally as the poliovirus receptor-related protein (PRR) family consisting of PRR1 and -2, and has been identified recently to be the alphaherpes virus receptor. PRR has a COOH-terminal consensus motif to which the PDZ domain of afadin binds. PRR and afadin were colocalized at cadherin-based cell-cell AJs in various tissues and cell lines. In E-cadherin-expressing EL cells, PRR was recruited to cadherin-based cell-cell AJs through interaction with afadin. PRR showed Ca2+-independent cell-cell adhesion activity. These results indicate that PRR is a cell-cell adhesion molecule of the immunoglobulin superfamily which is recruited to cadherin-based cell-cell AJs through interaction with afadin. We rename PRR as nectin (taken from the Latin word "necto" meaning "to connect").
Assuntos
Caderinas/metabolismo , Moléculas de Adesão Celular/genética , Junções Intercelulares/metabolismo , Proteínas dos Microfilamentos/metabolismo , Receptores do Fator de Necrose Tumoral , Receptores Virais , Processamento Alternativo/genética , Sequência de Aminoácidos , Animais , Células COS/química , Células COS/metabolismo , Cálcio/metabolismo , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/metabolismo , Agregação Celular/fisiologia , Células Epiteliais/química , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Junções Intercelulares/química , Junções Intercelulares/ultraestrutura , Cinesinas , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Proteínas dos Microfilamentos/química , Microscopia Eletrônica , Miocárdio/química , Miocárdio/citologia , Miocárdio/metabolismo , Miosinas , Nectinas , Estrutura Terciária de Proteína , Coelhos , Membro 14 de Receptores do Fator de Necrose Tumoral , Vinculina/metabolismoRESUMO
We recently reported that rat luteinized ovary tissue and primary cultures of rat ovarian granulosa cells reveal a remarkably tight functional correlation between expressed selective uptake of lipoprotein cholesteryl esters and the expression of an HDL receptor protein, scavenger receptor, class B, type I (SR-BI). In the current study, we examine these same processes in C57 mouse granulosa cells and report a different correlation. Unlike the rat cells, non-hormone stimulated mouse granulosa cells are able to effectively carry out their selective pathway functions and secrete HDL-derived progestins despite low levels of SR-BI and barely detectable levels of SR-BII (an isoform of SR-BI). Once stimulated with trophic hormones or Bt2cAMP, small (30-40%) increases are observed in selective pathway functions, but major (approximately 20-fold) increases are seen in SR-BI and SR-BII expression: thus, relatively little is gained in selective cholesteryl ester uptake by mouse granulosa cells even though SR-BI and SR-BII levels are greatly increased. The importance of the HDL receptor proteins to the selective pathway remains clear, however, since a significant portion of the selective process in both basal and stimulated granulosa cells is inhibitable by the use of blocking antibody. Another surface protein, caveolin, previously reported to co-localize with SR-BI in mouse cells shows no change in expression during periods when SR-BI/BII levels are undergoing major shifts.
Assuntos
Antígenos CD36/metabolismo , Proteínas de Transporte , Caveolinas , Células da Granulosa/metabolismo , Proteínas de Ligação a RNA , Receptores Imunológicos , Receptores de Lipoproteínas/metabolismo , Sequência de Aminoácidos , Animais , Bucladesina/farmacologia , Antígenos CD36/genética , Caveolina 1 , Ésteres do Colesterol/metabolismo , Feminino , Células da Granulosa/efeitos dos fármacos , Técnicas In Vitro , Lipoproteínas HDL/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Progesterona/biossíntese , Ratos , Receptores de Lipoproteínas/genética , Receptores Depuradores , Receptores Depuradores Classe B , Especificidade da Espécie , Distribuição TecidualRESUMO
The La autoantigen (also known as SS-B), a cellular RNA binding protein, may shuttle between the nucleus and cytoplasm, but it is mainly located in the nucleus. La protein is redistributed to the cytoplasm after poliovirus infection. An in vitro translation study demonstrated that La protein stimulated the internal initiation of poliovirus translation. In the present study, a part of the La protein was shown to be cleaved in poliovirus-infected HeLa cells, and this cleavage appeared to be mediated by poliovirus-specific protease 3C (3Cpro). Truncated La protein (dl-La) was produced in vitro from recombinant La protein by cleavage with purified 3Cpro at only one Gln358-Gly359 peptide bond in the 408-amino-acid (aa) sequence of La protein. The dl-La expressed in L cells was detected in the cytoplasm. However, green fluorescence protein linked to the C-terminal 50-aa sequence of La protein was localized in the nucleus, suggesting that this C-terminal region contributes to the steady-state nuclear localization of the intact La protein in uninfected cells. The dl-La retained the enhancing activity of translation initiation driven by poliovirus RNA in rabbit reticulocyte lysates. These results suggest that La protein is cleaved by 3Cpro in the course of poliovirus infection and that the dl-La is redistributed to the cytoplasm. dl-La, as well as La protein, may play a role in stimulating the internal initiation of poliovirus translation in the cytoplasm.
Assuntos
Autoantígenos/metabolismo , Cisteína Endopeptidases/fisiologia , Ribonucleoproteínas/metabolismo , Proteínas Virais , Proteases Virais 3C , Animais , Citoplasma/metabolismo , Células HeLa , Humanos , Camundongos , Peso Molecular , Biossíntese de Proteínas , Coelhos , Antígeno SS-BRESUMO
BACKGROUND: The yAP-1 transcription factor is crucial for the oxidative stress response of the budding yeast Saccharomyces cerevisiae; its activity is induced in response to oxidative stress, and as a consequence the expression of a number of target genes is enhanced. We have shown previously that yAP-1 is mainly found in the cytoplasm, but that upon the imposition of oxidative stress it localizes to the nucleus. In this study, we addressed the mechanism through which yAP-1 nuclear localization is regulated. RESULTS: Here we show that yAP-1 localization is mediated by active export from the nucleus, resulting from the activity of Crm1 (XpoI), a conserved protein that functions as an export receptor which recognizes the nuclear export signal (NES). When Crm1 expression was repressed, yAP-1 was localized in the nucleus and induced the expression of a yAP-1 dependent target gene. Our results also suggest that the cysteine rich domain (CRD), at the C-terminus of yAP-1, functions as an export recognition sequence. yAP-1 and Crm1 interact in vivo and this interaction is reduced in response to oxidative stress. CONCLUSIONS: These results suggest a novel regulatory mechanism of nucleocytoplasmic transport which is dependent upon a redox sensitive nuclear export pathway.
Assuntos
Proteínas de Bactérias , Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Proteínas Fúngicas , Carioferinas , Estresse Oxidativo , Receptores Citoplasmáticos e Nucleares , Saccharomyces cerevisiae/metabolismo , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica , Transporte Biológico Ativo , Compartimento Celular , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Reporter , Modelos Biológicos , Ligação Proteica , Saccharomyces cerevisiae/genética , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe , Proteína Exportina 1RESUMO
This study addresses the question of whether the level of expression of SR-BI (an HDL receptor) is linked to the expression of selective lipoprotein-cholesteryl ester delivery in a steroidogenic cell model. Rat ovarian granulosa cells are physiologically normal cells which show no selective uptake of HDL-cholesteryl esters and no progestin production until luteinized by trophic hormones or adenylate cyclase stimulators, after which expression of the selective cholesterol pathway and production of steroid hormone is dramatically up-regulated. The current study demonstrates that at every cell stage studied, the protein content and level of expression of SR-BI mRNA are linked to changes that occur in HDL-cholesteryl ester uptake; i.e., SR-BI is not present in basal (non-luteinized) cells, develops slowly (from 6-9 h) after hormone treatment, increases robustly from 9-48 h after stimulation, and remains high after incubation with HDL. In contrast, another structural protein, caveolin, did not follow this pattern; caveolin expression showed an inverse relationship to selective cholesteryl ester uptake, and was most prominent in basal cells and least prominent in luteinized, HDL-incubated cells. Morphologically, SR-BI appears to be associated with cell surface sites showing high levels of cholesteryl ester uptake (after luteinization and/or incubation with HDL labeled with fluorescent cholesteryl esters), and at the electron microscope level, SR-BI is most clearly associated with microvillar regions on the cell surface which also bind HDL-labeled with colloidal gold. Thus, induction of the SR-BI receptor system and induction of the HDL-selective cholesterol uptake pathway in rat granulosa cells appear to be linked morphologically, biochemically, and functionally.
Assuntos
Antígenos CD36/biossíntese , Proteínas de Transporte , Caveolinas , Ésteres do Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Proteínas de Ligação a RNA , Receptores Imunológicos , Receptores de Lipoproteínas/biossíntese , Esteroides/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , Transporte Biológico Ativo/efeitos dos fármacos , Bucladesina/farmacologia , Antígenos CD36/genética , Caveolina 1 , Membrana Celular/metabolismo , Primers do DNA/genética , Feminino , Expressão Gênica/efeitos dos fármacos , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Células da Granulosa/ultraestrutura , Técnicas In Vitro , Células Lúteas/efeitos dos fármacos , Células Lúteas/metabolismo , Células Lúteas/ultraestrutura , Proteínas de Membrana/metabolismo , Microscopia Eletrônica , Modelos Biológicos , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Depuradores , Receptores Depuradores Classe BRESUMO
Polypyrimidine-tract-binding protein (PTB) is involved in pre-mRNA splicing and internal-ribosomal-entry-site-dependent translation. The biochemical properties of various segments of PTB were analysed in order to understand the molecular basis of the PTB functions. The protein exists in oligomeric as well as monomeric form. The central part of PTB (amino acids 169-293) plays a major role in the oligomerization. PTB contains several RNA-binding motifs. Among them, the C-terminal part of PTB (amino acids 329-530) exhibited the strongest RNA-binding activity. The N-terminal part of PTB is responsible for the enhancement of RNA binding by HeLa cell cytoplasmic factor(s).
Assuntos
Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/genética , Reagentes de Ligações Cruzadas , Células HeLa , Humanos , Dados de Sequência Molecular , Mutação , Proteína de Ligação a Regiões Ricas em Polipirimidinas , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas/genética , Análise de Sequência , Relação Estrutura-AtividadeRESUMO
CD26 is a leukocyte-activation antigen that is expressed on T lymphocytes and macrophages and possesses dipeptidyl peptidase IV (DPPIV) activity, whose natural substrates have not been identified yet. CXC chemokines, stromal cell-derived factor 1alpha (SDF-1alpha) and 1beta (SDF-1beta), sharing the receptor CXCR-4, are highly efficacious chemoattractants for resting lymphocytes and CD34(+) progenitor cells, and they efficiently block the CXCR-4-mediated entry into cells of T cell line tropic strains of HIV type 1 (HIV-1). Here we show that both the chemotactic and antiviral activities of these chemokines are abrogated by DPPIV-mediated specific removal of the N-terminal dipeptide, not only when the chemokines are produced in transformed mouse L cell line to express human CD26 but also when they were exposed to a human T cell line (H9) physiologically expressing CD26. Mutagenesis of SDF-1alpha confirmed the critical requirement of the N-terminal dipeptide for its chemotactic and antiviral activities. These data suggest that CD26-mediated cleavage of SDF-1alpha and SDF-1beta likely occurs in human bodies and promotes HIV-1 replication and disease progression. They may also explain why memory function of CD4(+) cells is preferentially lost in HIV-1 infection. Furthermore, CD26 would modulate various other biological processes in which SDF-1alpha and SDF-1beta are involved.
Assuntos
Quimiocinas CXC/imunologia , Citocinas/imunologia , Dipeptidil Peptidase 4/imunologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , Linfócitos T/imunologia , Linfócitos T/virologia , Animais , Linhagem Celular , Quimiocina CXCL12 , Quimiocinas CXC/metabolismo , Quimiotaxia/imunologia , Citocinas/metabolismo , Dipeptidil Peptidase 4/metabolismo , Humanos , Camundongos , Transdução de Sinais/imunologia , Células Estromais/imunologiaRESUMO
Polypeptides of amino acids 1 to 241 (PVR241) and 1 to 330 (PVR330) of the human poliovirus receptor (hPVR) were produced in a baculovirus expression system. PVR241 contained extracellular domains 1 and 2 of hPVR, and PVR330 contained extracellular domains 1, 2, and 3. These peptides were purified by immunoaffinity column chromatography with an anti-hPVR monoclonal antibody (MAb). After the purification, PVR241 and PVR330 appeared to retain their native conformation as judged by reactivity with an anti-PVR MAb that recognized domain 1 of hPVR in a conformation-dependent manner. The virulent Mahoney strain of poliovirus type 1 was mixed with the purified PVRs in various concentrations. An average of at least 43 PVR330 molecules were able to bind to one virion particle under the conditions used. The equilibrium dissociation constant between the PVR330 molecule and the PVR binding site (canyon) on the virion was determined to be 4.50 +/- (0.86) x 10(-8) M at 4 degrees C. Higher rates of conformational change of the virus (160S) to 135S and 80S particles were observed as the concentration of PVR330 was increased. In this in vitro system, the ratio of the amount of the 135S particle to that of the 80S particle seemed to be always constant. After the disappearance of the 160S particle, the amount of the 80S particle was not increased by further incubation at 37 degrees C. These results suggested that the 80S particle was not derived from the 135S particle under the conditions used in this study.
Assuntos
Proteínas de Membrana , Poliovirus/metabolismo , Receptores Virais/metabolismo , Animais , Sítios de Ligação , Bombyx , Linhagem Celular , Centrifugação com Gradiente de Concentração , Fracionamento Químico , Células HeLa , Humanos , Cinética , Poliovirus/ultraestrutura , Receptores Virais/genética , Receptores Virais/isolamento & purificação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Vírion/metabolismo , Vírion/ultraestruturaRESUMO
Adeno-associated virus (AAV) vectors have a limited capacity for packaging DNA. To insert both a therapeutic gene and a selectable marker gene in the same AAV vector efficiently, we developed a novel dicistronic AAV vector containing a 230 base pairs (bp) internal ribosome entry site (IRES) element derived from hepatitis C virus (HCV) genome and a 420 bp blasticidin S-resistance gene (bsr) as a small selectable marker in the second cistron. The 650 bp HCV IRES-bsr construct was placed downstream of the 3' end of the luciferase gene (Luc) under the control of the human cytomegalovirus (CMV) promoter. This dicistronic gene conferred blasticidin S-resistance to 293 cells besides luciferase activity, when examined not only by transfection but also by transduction using AAV vectors. The dicistronic AAV vector harbouring HCV IRES-bsr is capable of expressing a therapeutic gene of up to 3.6 kilobases (kb) (including promoter/enhancer elements) as well as a selectable marker gene. If a selectable marker gene is not necessary, this vector is able to incorporate two different kinds of therapeutic genes more easily than that containing EMCV IRES. The dicistronic AAV vector described here is useful for expressing many kinds of cDNA besides a selectable marker.
Assuntos
Dependovirus/genética , Genes Virais , Vetores Genéticos , Genoma Viral , Hepacivirus/genética , Proteínas Estruturais Virais/genética , Animais , Sequência de Bases , Linhagem Celular , Citomegalovirus/genética , Primers do DNA , Resistência Microbiana a Medicamentos/genética , Marcadores Genéticos , Humanos , Luciferases/biossíntese , Plasmídeos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Biossíntese de Proteínas , Coelhos , Proteínas Recombinantes de Fusão/biossíntese , Reticulócitos/metabolismo , TransfecçãoRESUMO
Poliovirus receptor (PVR) is a cell surface glycoprotein that belongs to the immunoglobulin superfamily. Although MPH was initially reported as the mouse homolog of human PVR, recent data strongly suggest that MPH is the mouse homolog of human PRR2, a PVR-related gene 2 product, and not that of human PVR. Thus MPH is renamed mPRR2 in this study. Physiological functions of the PVR-related gene products have not been elucidated, although PVR has been well characterized as the poliovirus receptor. In this study, a possible function of mPRR2 (MPH), which is not a functional receptor for poliovirus, was investigated. Mouse L cells expressing mPRR2 were prepared. Those mouse cells showed a higher activity of cell aggregation than the parental mouse L cells. Enhancement of cell aggregation was also observed for insect Sf9 cells infected with recombinant baculovirus carrying mPRR2 cDNA. On the other hand, L cells expressing human PVR or monkey PVR (AGM alpha1 or AGM alpha2) did not show increased cell aggregation. The cell aggregation activity of L cells expressing mPRR2 was inhibited by the addition of anti-mPRR2 monoclonal antibodies or a soluble mPRR2 molecule produced by the baculovirus expression system. An immunofluorescence study revealed that mPRR2 protein was localized to the cell-cell contact sites between cells expressing mPRR2. A similar localization of mPRR2 was observed for intrinsic mPRR2 molecules of the mouse neuroblastoma cell line NS20Y. The contact site-specific localization of mPRR2 was not observed on the border between mPRR2-expressing and nonexpressing HeLa cells. Furthermore, mPRR2 proteins directly bound to each other in vitro. mPRR2 was detected on various types of cultured cells of mouse origin and in various mouse tissues. These results suggest that mPRR2 is an intercellular adhesion molecule with a homophilic binding manner.
Assuntos
Adesão Celular/fisiologia , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana , Animais , Cálcio , Cátions Bivalentes , Moléculas de Adesão Celular , Linhagem Celular , Membrana Celular/química , Mapeamento de Epitopos , Haplorrinos , Células HeLa , Humanos , Células L , Magnésio , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Nectinas , Especificidade de Órgãos , Poliovirus , Receptores Virais/análise , Proteínas Recombinantes de Fusão , Spodoptera , TransfecçãoRESUMO
We previously identified a highly conserved 98-nucleotide (nt) sequence, the 3'X, as the extreme 3'-terminal structure of the hepatitis C virus (HCV) genome (T. Tanaka, N. Kato, M.-J. Cho, and K. Shimotohno, Biochem. Biophys. Res. Commun. 215:744-749, 1995). Since the 3' end of positive-strand viral RNA is the initiation site of RNA replication, the 3'X should contribute to HCV negative-strand RNA synthesis. Cellular factors may also be involved in this replication mechanism, since several cellular proteins have been shown to interact with the 3'-end regions of other viral genomes. In this study, we found that both 38- and 57-kDa proteins in the human hepatocyte line PH5CH bound specifically to the 3'-end structure of HCV positive-strand RNA by a UV-induced cross-linking assay. The 57-kDa protein (p57), which had higher affinities to RNA probes, recognized a 26-nt sequence including the 5'-terminal 19 nt of the 3'X and 7 flanking nt, designated the transitional region. This sequence contains pyrimidine-rich motifs and shows similarity to the consensus binding sequence of the polypyrimidine tract-binding protein (PTB), which has been implicated in alternative pre-mRNA splicing and cap-independent translation. We found that this 3'X-binding p57 is identical to PTB. The 3'X-binding p57 was immunoprecipitated by anti-PTB antibody, and recombinant PTB bound to the 3'X RNA. In addition, p57 bound solely to the 3'-end region of positive-strand RNA, not to this region of negative-strand RNA. We suggest that 3'X-PTB interaction is involved in the specific initiation of HCV genome replication.
Assuntos
DNA Viral/metabolismo , Proteínas de Ligação a DNA/metabolismo , Hepacivirus/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequência de Bases , Linhagem Celular , Mapeamento Cromossômico , Genoma Viral , Humanos , Dados de Sequência Molecular , Proteína de Ligação a Regiões Ricas em PolipirimidinasRESUMO
Heterodimerization among the basic-leucine zipper (bZIP) proteins or among the basic-helix-loop-helix-leucine zipper (bHLHZip) proteins confers a multitude of combinational activities to these transcription factors. To further examine the function of the bHLHZip protein, USF, we screened for cellular proteins which could directly interact with USF using the yeast two-hybrid system. A bZip protein, Fra1, was found to efficiently interact with USF. USF specifically interacts with Fra1 but not with other closely related family members, c-Fos, Fra2, FosB, or with c-Jun. Both the bHLHZip and the N-terminal regions of Fra1 are required for efficient interaction with USF. In vivo association between USF and Fra1 has been demonstrated by co-immunoprecipitation. Expression of exogenous USF led to a decrease in AP1-dependent transcription in F9 cells. Co-expression of exogenous Fra1 restored the AP1 activity in a dose-dependent manner. These data show that USF and Fra1 physically and functionally interact demonstrating that cross-talk occurs between factors of distantly related transcription families.
Assuntos
Proteínas de Ligação a DNA , Proteínas Proto-Oncogênicas c-fos/metabolismo , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/metabolismo , Animais , Dimerização , Genes Reporter , Sequências Hélice-Alça-Hélice , Humanos , Zíper de Leucina , Camundongos , Ligação Proteica , Coelhos , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Transcrição Gênica , Fatores Estimuladores Upstream , beta-Galactosidase/biossínteseRESUMO
The YAP1 gene of Saccharomyces cerevisiae encodes a bZIP-containing transcription factor that is essential for the normal response of cells to oxidative stress. Under stress conditions, the activity of yAP-1 is increased, leading to the induced expression of a number of target genes encoding protective enzymes or molecules. We have examined the mechanism of this activation. Upon imposition of oxidative stress, a small increase in the DNA-binding capacity of yAP-1 occurs. However, the major change is at the level of nuclear localization; upon induction the yAP-1 protein relocalizes from the cytoplasm to the nucleus. This regulated localization is mediated by a cysteine-rich domain (CRD) at the C-terminus, its removal resulting in constitutive nuclear localization and high level activity. Furthermore, the CRD of yAP-1 is sufficient to impose regulated nuclear localization of the GAL4 DNA-binding domain. Amino acid substitutions indicated that three conserved cysteine residues in the CRD are essential for the regulation. We suggest therefore, that these cysteine residues are important in sensing the redox state of the cell and hence regulating yAP-1 activity.