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1.
Oncogene ; 27(37): 5011-8, 2008 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-18469860

RESUMO

The co-chaperone protein, BAG3, which belongs to the BAG protein family, has an established antiapoptotic function in different tumor cell lines. Here we demonstrated that treatment of the human neuroblastoma cell line, SK-N-MC, with fibroblast growth factor-2 (FGF-2) results in induction of BAG3 expression. Induction of BAG3 protein by FGF-2 occurs at the transcriptional level; it requires the extracellular regulated kinase1/2 pathway and is dependent on the activity of Egr-1 upon the BAG3 promoter. Targeted suppression of BAG3 by small-interfering RNA results in dysregulation of cell-cycle progression most notably at S and G(2) phases, which corroborates the decreased level of cyclin B1 expression. These observations suggest a new role for BAG3 in regulation of the cell cycle.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteína 1 de Resposta de Crescimento Precoce/fisiologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Neuroblastoma/genética , Regulação para Cima/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose , Sequência de Bases , Sítios de Ligação , Ciclo Celular/efeitos dos fármacos , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neuroblastoma/patologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Células Tumorais Cultivadas
2.
Oncogene ; 27(1): 32-43, 2008 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-17653096

RESUMO

The human immunodeficiency virus type 1 (HIV-1) viral protein R (vpr) gene is an evolutionarily conserved gene among the primate lentiviruses. Several functions are attributed to Vpr including the ability to cause cell death, cell cycle arrest, apoptosis and DNA damage. The Vpr domain responsible for DNA damage as well as the mechanism(s) through which Vpr induces this damage is unknown. Using site-directed mutagenesis, we identified the helical domain II within Vpr (aa 37-50) as the region responsible for causing DNA damage. Interestingly, Vpr Delta(37-50) failed to cause cell cycle arrest or apoptosis, to induce Ku70 or Ku80 and to suppress tumor growth, but maintained its capability to activate the HIV-1 LTR, to localize to the nucleus and to promote nonhomologous end-joining. In addition, our cytogenetic data indicated that helical domain II induced chromosomal aberrations, which mimicked those induced by cisplatin, an anticancer agent. This novel molecular mimicry function of Vpr might lead to its potential therapeutic use as a tumor suppressor.


Assuntos
Antineoplásicos Alquilantes/toxicidade , Cisplatino/toxicidade , Dano ao DNA/efeitos dos fármacos , HIV-1/genética , Mimetismo Molecular/genética , Proteínas Supressoras de Tumor/genética , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/genética , Sequência de Aminoácidos , Animais , Fármacos Anti-HIV/toxicidade , Linhagem Celular Tumoral , Dano ao DNA/genética , Feminino , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C3H , Mimetismo Molecular/efeitos dos fármacos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/genética , Proteínas Supressoras de Tumor/fisiologia , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/fisiologia
3.
J Neurosci Methods ; 85(1): 13-20, 1998 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-9874136

RESUMO

We tested the hypothesis that other polymers of lysine would be better substrates for culture of CNS neurons than polylysine itself. In a serum-free medium optimized for survival of hippocampal neurons grown on substrates of poly-D-lysine, 13% more neurons survived on substrates to which a sequential copolymer of lysine and alanine (LAS) was applied (P = 0.006). The effect was specific for the sequential polymer, in contrast to the random copolymer of lysine and alanine. This suggests that average cationic charge density is not as important as the spacing of these charges. More dramatically, immunostaining for the axon-associated microtubule-associated protein, tau, indicated a 2-fold higher rate of fiber growth on LAS. The somatodendritic cytoskeletal component MAP2 also appeared to be increased in cells cultured on LAS. This suggests that cytoskeletal differentiation in general and axon formation in particular are stimulated by the LAS substrate. Scanning electron microscopy supported this conclusion. By circular dichroism, the conformation of LAS in phosphate-buffered saline appeared to be a random coil, indistinguishable from poly-D-lysine. These results indicate that LAS is a superior substrate to polylysine for growth of CNS neurons. LAS may be useful for regeneration of damaged circuits in the CNS as well as a substrate for connections to a neuroprosthesis.


Assuntos
Técnicas de Cultura de Células/métodos , Hipocampo/citologia , Neurônios/citologia , Peptídeos/farmacologia , Polímeros/farmacologia , Alanina/farmacologia , Animais , Dicroísmo Circular , Imunofluorescência , Hipocampo/ultraestrutura , Lisina/farmacologia , Microscopia Eletrônica , Neurônios/ultraestrutura , Polilisina/farmacologia , Ratos , Ratos Sprague-Dawley
4.
J Biomed Mater Res ; 34(3): 381-92, 1997 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-9086408

RESUMO

The lysosomal degradability of poly(alpha-amino acids) based on poly(L-glutamic acid) and its derivatives/copolymers was evaluated to gain insight into the subcellular fate of the macromolecules as water soluble polymeric drug carriers. The results indicate that both the incorporation of hydrophobic comonomers and modification of the carboxylic groups of glutamic acid side chains with hydroxyalkylamine increase the lysosomal degradability of the copolymers. Decreased lysosomal degradability of L-glutamic acid copolymers containing tripeptides terminated in p-nitroanilide (drug model) in the side chains confirmed that drug conjugation alters the degradation pattern of the polymeric carriers. The percentages of the enzymatic release of p-nitroaniline from its polymeric complex with time is relatively independent of the contents of the tripeptidyl p-nitroanilides attached to the polymeric conjugates. Determination of the degradation products by electrospray mass spectroscopy showed that no fragments less than 10(3) D were generated by lysosomal enzymes, whereas the main degradation products by papain and chymotrypsin were tripeptides and tetrapeptides. The conclusions derived from these data strongly suggest that these macromolecules, if used as lysosomotropic drug carriers, may accumulate in the lysosomes and limit their usefulness in some applications.


Assuntos
Materiais Biocompatíveis/metabolismo , Lisossomos/metabolismo , Peptídeos/metabolismo , Animais , Materiais Biocompatíveis/química , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Técnicas In Vitro , Espectrometria de Massas , Teste de Materiais , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Peptídeos/química , Ácido Poliglutâmico/análogos & derivados , Ácido Poliglutâmico/química , Ácido Poliglutâmico/metabolismo , Ratos
5.
Virology ; 199(2): 484-90, 1994 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-8122378

RESUMO

A detailed knowledge of the pathogenesis of infections caused by thymidine-kinase (TK)-deficient herpes simplex virus type 1 (HSV-1) strains is important because such mutants can arise during treatment of HSV infections with acyclovir--especially in immunocompromised patients--and also because TK-negative mutants may become useful for the therapy of intracranial tumors. In this work, we studied the pathogenesis of a genetically engineered TK-negative HSV-1 strain dlsptk, in SCID mice (mice with severe combined immunodeficiency) after corneal infection. We found that dlsptk established a persistent infection that kills SCID mice within 80.2 +/- 21.3 days. The cause of death seemed to be related to uncontrolled viral replication in the superficial and deep facial tissues of the animals. Viremia probably did not occur, as judged by the inability to detect infectious virus and viral gene expression in various internal organs. However, the virus did reach the nervous system, most probably by axonal transport from the primary site of the infection. Virus-specific DNA reached low but detectable levels in the trigeminal ganglia and the brainstems by 7 days p.i. and remained at low levels for up to 50 days p.i. as determined by spot blot analysis. By in situ hybridization and immunostaining we determined that, in some of the neurons of the trigeminal ganglia infected by the virus, viral latency was established. However, our results suggested that in other infected neurons viral replication occurred and virus spread to surrounding nonneuronal cells and to the central nervous system. This work provides a new model in which the pathogenesis of infections caused by TK-deficient HSV strains in immunocompromised hosts can be effectively studied and which may also help to identify the potential side effects of the therapy of intracranial tumors with TK-negative HSV strains.


Assuntos
Herpes Simples/microbiologia , Herpesvirus Humano 1/patogenicidade , Neurônios/microbiologia , Timidina Quinase/fisiologia , Replicação Viral/fisiologia , Animais , Modelos Animais de Doenças , Herpesvirus Humano 1/enzimologia , Herpesvirus Humano 1/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID
6.
Virology ; 192(2): 618-30, 1993 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-8380666

RESUMO

A Herpes simplex virus type I (HSV-I) strain 17 mutant deleted between the NotI and HpaI restriction sites of the latency associated transcript (LAT) region has been constructed. The mutant, therefore, contains a deletion of the putative LAT promoter and is called 17N/H. The 17N/H isolate established latent infections in mice nearly as efficiently as its wildtype parent. However, like other LAT null mutants, 17N/H reactivates from explanted ganglia with much slower kinetics than its LAT competent parent. In tissue culture, although 17N/H produces as much virus per cell as its strain 17 parent, it produces small plaques. The small plaque phenotype appears to be due to the inability of the virus to be released from the infected cell into the medium, following low but not high multiplicities of infection (m.o.i.). The mutant was also shown to produce an aberrant LAT homologous transcript of 1.1 kb as well as overproduce an approximately 29,000-Da HSV-specific polypeptide, which is barely detectable in wildtype infected cells. Rescuants of the 17N/H defect were constructed using a 10-kb restriction fragment containing viral sequences spanning the deletion, make large plaques, and have reactivation patterns and infected cell gene product profiles indistinguishable from the 17 parent. This shows that the phenotypes observed in 17N/H are reversed when the deletion, or at most sequences within 5 kb of each side of the deletion, is corrected. The possibilities that the defect in viral egress from infected cell, the small LAT homologous transcript, and the accumulation of the 29,000 Da polypeptide are related to the delayed reactivation kinetics are discussed.


Assuntos
Deleção de Genes , Genoma Viral , Simplexvirus/fisiologia , Transcrição Gênica/genética , Ativação Viral/genética , Sequência de Bases , Linhagem Celular , DNA Viral/genética , DNA Viral/isolamento & purificação , Humanos , Cinética , Dados de Sequência Molecular , Mutagênese , Oligodesoxirribonucleotídeos , RNA Viral/genética , RNA Viral/isolamento & purificação , Mapeamento por Restrição , Simplexvirus/genética , Fatores de Tempo , Transfecção , Ensaio de Placa Viral
7.
J Virol ; 65(8): 4142-52, 1991 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-1649322

RESUMO

In a mouse model for herpes simplex virus type 1 (HSV-1) latency in which the virus was inoculated via the eye after corneal scarification, HSV-1 replicated in corneal epithelial cells and infected the nerve cell endings. HSV-1 reached the trigeminal ganglia by fast axonal transport between 2 and 10 days postinfection (p.i.) and established a latent infection in neuronal cells or replicated and spread to nonneuronal cells. By using in situ hybridization, we showed that cellular transcription factors are stimulated by HSV-1 infection in trigeminal ganglia. This stimulation is biphasic, peaking at 1 and 3 to 4 days p.i. The first peak involves c-jun and oct-1 expression in neurons, and the second involves c-jun, c-fos, and oct-1 expression in neurons and nonneuronal cells. Corneal scarification, alone or followed by infection with UV-inactivated HSV-1, induced monophasic c-jun and oct-1 expression in some neurons of the trigeminal ganglia, with a peak at 1 day p.i. Corneal infection without prior scarification induced c-jun, c-fos, and oct-1 expression in some neuronal and nonneuronal cells of the trigeminal ganglia 2 to 9 days p.i. Explanation of ganglia from latently infected animals resulted in reactivation of the latent virus. Independently of the presence of latent HSV-1 in explanted ganglia, expression of c-fos, c-jun, and oct-1 was induced first in nonneuronal cells, peaking 6 to 10 h postexplantation, and then in neuronal cells, with a peak at 24 h after explantation when expression of viral replicative genes was first detectable. Since ocular HSV-1 infection, corneal scarification, and explantation of trigeminal ganglia all resulted in induction of expression of cellular transcription factors in ganglia, these factors may play a critical role in the permissiveness of cells for HSV-1 replication during acute infection, latency, and reactivation.


Assuntos
Ceratite Dendrítica/microbiologia , Simplexvirus/fisiologia , Fatores de Transcrição/biossíntese , Gânglio Trigeminal/microbiologia , Animais , Córnea/microbiologia , Sondas de DNA , DNA Viral/análise , Proteínas de Ligação a DNA/biossíntese , Modelos Animais de Doenças , Feminino , Fator C1 de Célula Hospedeira , Camundongos , Camundongos Endogâmicos BALB C , Hibridização de Ácido Nucleico , Fator 1 de Transcrição de Octâmero , Técnicas de Cultura de Órgãos , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas c-fos , Proteínas Proto-Oncogênicas c-jun , Simplexvirus/genética , Transcrição Gênica , Replicação Viral
9.
Int J Pept Protein Res ; 12(2): 57-68, 1978 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-711371

RESUMO

The acid catalyzed O leads to C migration of the benzyl group in the side chain of tyrosine could be reduced by applying HBr in a mixture of phenol and p-cresol instead of BHr in trifluoroacetic acid for acidolytic deprotection. This side reaction occurs also during the removal of Boc groups. The loss of O-benzyl protection and the formation of 3-benzyltyrosine residues could be suppressed by the application of a 7:3 mixture of trifluoroacetic acid and acetic acid. The acid- and base-catalyzed ring closure of beta-benzylaspartyl residues to aminosuccinyl derivatives was also studied. In this case HBr in trifluoroacetic acid was found to be relatively harmless. Deprotection with HBr in a mixture of trifluoroacetic acid and p-cresol can be applied for peptides that contain both beta-benzylaspartyl and O-benzyltyrosyl residues. An attempt to reduce the rate of the base-catalyzed side reaction by application of hindered tertiary amines was abandoned because the tertiary amines which were effective in this respect let to significant reduction of the rate of the desired reaction, the aminolysis of active esters, as well. A satisfactory solution for the problem was found in the selective catalysis of the active ester reaction with 1-hydroxybenzotriazole or 4-dimethyl-aminopyridine. These catalysts do not enhance the rate of ring closure and in their presence essentially pure beta-benzylaspartyl peptides can be produced in good yield.


Assuntos
Ácido Aspártico , Peptídeos/síntese química , Tirosina , Compostos de Benzil , Fenômenos Químicos , Química , Métodos
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