RESUMO
Solavetivone (1), cytotoxic to OVCAR-3 cells with an IC(50) value of 0.1 mM, has been isolated from Solanum indicum. In addition, a novel solafuranone (2) and three known compounds, scopoletin, N-(p-trans-coumaroyl)tyramine, and N-trans-feruloyltyramine, were isolated for the first time from this plant. The structures of the above compounds were established by means of spectroscopic and X-ray analyses.
Assuntos
Antineoplásicos Fitogênicos/isolamento & purificação , Furanos/isolamento & purificação , Solanaceae/química , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Cromatografia , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Furanos/química , Humanos , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética , Medicina Tradicional Chinesa , Conformação Molecular , Estrutura Molecular , Neoplasias Ovarianas , Raízes de Plantas/química , Plantas Medicinais/química , Espectrofotometria Infravermelho , Espectroscopia de Infravermelho com Transformada de Fourier , Estereoisomerismo , Relação Estrutura-Atividade , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Hepatitis delta virus (HDV) encodes two isoforms of delta antigens (HDAgs). The small form of HDAg is required for HDV RNA replication, while the large form of HDAg inhibits the viral replication and is required for virion assembly. In this study, we found that the expression of B23, a nucleolar phosphoprotein involved in disparate functions including nuclear transport, cellular proliferation, and ribosome biogenesis, is up-regulated by these two HDAgs. Using in vivo and in vitro experimental approaches, we have demonstrated that both isoforms of HDAg can interact with B23 and their interaction domains were identified as the NH(2)-terminal fragment of each molecule encompassing the nuclear localization signal but not the coiled-coil region of HDAg. Sucrose gradient centrifugation analysis indicated that the majority of small HDAg, but a lesser amount of the large HDAg, co-sedimented with B23 and nucleolin in the large nuclear complex. Transient transfection experiments also indicated that introducing exogenous full-length B23, but not a mutated B23 defective in HDAg binding, enhanced HDV RNA replication. All together, our results reveal that HDAg has two distinct effects on nucleolar B23, up-regulation of its gene expression and the complex formation, which in turn regulates HDV RNA replication. Therefore, this work demonstrates the important role of nucleolar protein in regulating the HDV RNA replication through the complex formation with the key positive regulator being small HDAg.
Assuntos
Antígenos de Hepatite/metabolismo , Vírus Delta da Hepatite/genética , Proteínas Nucleares/metabolismo , RNA Viral/fisiologia , Replicação Viral , Vírus Delta da Hepatite/imunologia , Antígenos da Hepatite delta , Humanos , Nucleofosmina , Conformação Proteica , RNA Mensageiro/metabolismo , RNA Viral/biossíntese , Células Tumorais Cultivadas , Regulação para CimaRESUMO
Phage AR1 is similar to phage T4 in several essential genes but differs in host range. AR1 infects various isolates of Escherichia coli O157:H7 but does not infect K-12 strains that are commonly infected by T4. We report here the determinants that confer this infection specificity. In T-even phages, gp37 and gp38 are components of the tail fiber that are critical for phage-host interaction. The counterparts in AR1 may be similarly important and, therefore, were characterized. The AR1 gp37 has a sequence that differs totally from those of T2 and T4, except for a short stretch at the N terminus. The gp38 sequence, however, has some conservation between AR1 and T2 but not between AR1 and T4. The sequences that are most closely related to the AR1 gp37 and gp38 are those of phage Ac3 in the T2 family. To identify the AR1-specific receptor, E. coli O157:H7 was mutated by Tn10 insertion and selected for an AR1-resistant phenotype. A mutant so obtained has an insertion occurring at ompC that encodes an outer membrane porin. To confirm the role of OmpC in the AR1 infection, homologous replacement was used to create an ompC disruption mutant (RM). When RM was complemented with OmpC originated from an O157:H7 strain, but not from K-12, its AR1 susceptibility was fully restored. Our results suggest that the host specificity of AR1 is mediated at least in part through the OmpC molecule.
Assuntos
Bacteriófagos/genética , Escherichia coli/genética , Genes Virais , Porinas/genética , Proteínas do Envelope Viral/genética , Bacteriófagos/química , Bacteriófagos/metabolismo , Clonagem Molecular , Elementos de DNA Transponíveis , Escherichia coli/química , Escherichia coli/virologia , Dados de Sequência Molecular , Mutagênese Insercional , Porinas/metabolismo , Transposases/genética , Proteínas do Envelope Viral/metabolismoRESUMO
Determination of hepatitis D virus (HDV) genotypes is epidemiologically and clinically important. Phylogenic analysis based on sequencing analysis of multiple HDV strains isolated from sera of patients is not convenient for mass screening in routine laboratories. This study was designed to develop genotype-specific antibodies against hepatitis delta antigen (HDAg) and to apply these antibodies for immunohistochemical differentiation of HDV genotypes in formalin-fixed, paraffin-embedded liver biopsies of patients. Divergence in the carboxyl-terminal 19 amino acids of the large HDAg between genotypes I and II is more than 70%. Peptides covering these residues were conjugated to keyhole limpet hemocyanin and were used for immunization. The generated antibodies were confirmed for their specificity by binding to type-specific HDAgs expressed in DNA-transfected Huh-7 hepatoma cells. Liver biopsies from 6 patients who had dominant genotype I HDV and 33 patients who had dominant genotype II HDV in sera were stained with these antibodies. The accuracy for these antibodies was 94.9%, and the agreement between dominant HDV genotypes in serum and dominant hepatic HDV genotypes based on HDAg staining was nearly perfect (kappa = 0.83). In summary, the carboxyl-terminal 19 amino acids of the large HDAg can be used as immunogens to generate genotype-specific antibodies. These antibodies were proven to be useful in immunohistochemical differentiation of HDV genotypes in liver biopsies.
Assuntos
Vírus Delta da Hepatite/genética , Sequência de Aminoácidos/genética , Anticorpos Antivirais/análise , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos , Biópsia , Reações Cruzadas , Genótipo , Antígenos de Hepatite/genética , Antígenos de Hepatite/metabolismo , Vírus Delta da Hepatite/imunologia , Antígenos da Hepatite delta , Humanos , Imuno-Histoquímica , Fígado/imunologia , Fígado/patologia , Fígado/virologia , Dados de Sequência Molecular , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
The HIV pol sequentially encodes protease (PR), reverse transcriptase (RT), and integrase (IN) from the 5'-3' direction. We explored the significance of this gene arrangement. All six possible gene dispositions were examined. In two situations where PR was removed from the leading place and no two genes were in their original location, viral polyprotein processing was abolished. Processing of the polyprotein did not occur when IN was translocated to the front of PR-RT. However, in the following two arrangements, the polyprotein was processed but only at specific sites. First, PR remained in the leading position while the locations of RT and IN were exchanged; viral polyprotein was processed at a site between the upstream transframe peptide (TF) and PR. Second, PR was placed after RT-IN and located at the distal end of Pol. Processing occurred only at the created junction between TF and RT. These results indicated that cleavage after TF occurred autocatalytically but did not proceed to a second site, which needed an extraneous PR for trans-action. Therefore, arranging Pol in the order of PR-RT-IN warrants the streamline processing of the polyprotein once the autocleavage is initiated.
Assuntos
Produtos do Gene pol/metabolismo , Genes pol , HIV-1/genética , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Viral da Expressão Gênica , Produtos do Gene pol/análise , Produtos do Gene pol/química , Integrase de HIV/química , Integrase de HIV/genética , Integrase de HIV/metabolismo , Protease de HIV/química , Protease de HIV/genética , Protease de HIV/metabolismo , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , HIV-1/enzimologia , Peso Molecular , Fases de Leitura Aberta/genética , Testes de Precipitina , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
To study the association between anti-Gag and anti-Nef antibody reactivities and their correlations with disease progression, 174 HIV-1/AIDS patients were followed up for 1 year after they received triple therapy. The antibody reactivities were analyzed using a Western blot test with recombinant Gag and Nef proteins. The results showed that decreasing levels of anti-Gag or anti-Nef antibody correlate with disease progression defined by HIV-1 viral loads or T4 cell counts. After receiving triple treatment for 1 year, 8 of 38 (21.1%) Nef antibody-negative patients became positive, while only 9 of 125 (7.2%) Nef antibody-positive persons lost the antibody reactivity (p < 0.01). Therefore, HIV-1 Nef may serve as a clinical marker of disease progression.
Assuntos
Produtos do Gene nef/imunologia , Anticorpos Anti-HIV/sangue , Infecções por HIV/imunologia , HIV-1/fisiologia , Carga Viral , Western Blotting , Contagem de Linfócito CD4 , Progressão da Doença , Produtos do Gene gag/imunologia , HIV-1/isolamento & purificação , Humanos , RNA Viral/sangue , Proteínas Recombinantes de Fusão/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
Hepatitis delta virus (HDV) is a subviral pathogen that requires pre-existing or concurrent infection with hepatitis B virus (HBV). HDV expresses two forms of a single protein, the delta antigen (HDAg), which are identical except for an additional 19 residues at the C terminus of the large form. Within this C-terminal extension a cysteine residue is isoprenylated; this isoprenylation is critical for interaction with HBV envelope proteins to enable virus assembly and release into the medium. Therefore, large HDAg must be recruited to an extracellular compartment. However, immuno-staining with HDAg-specific antibodies has localized the large antigen mainly to the nucleus and supports the notion that large HDAg suppresses virus replication in the nucleus. Since isoprenylation would increase the hydrophobicity of the protein and may favour transport towards specific membranes, the question remains whether the large HDAg detected in the nucleus carries an isoprenyl group. To address this issue, antibodies against the farnesyl modification were generated to allow direct visualization of the antigen by immunofluorescence microscopy. The anti-farnesyl antibodies specifically stained large HDAg expressed in Huh-7 cells, and the signal was largely restricted to the nucleus; the staining pattern could be superimposed on those of cells stained for large HDAg. The large HDAg translocated into the nucleus was therefore isoprenylated. In addition, antibodies specific for the farnesyl modification should be applicable to the study of other similarly isoprenylated proteins.
Assuntos
Butadienos/imunologia , Farneseno Álcool/imunologia , Hemiterpenos , Antígenos de Hepatite/análise , Vírus Delta da Hepatite/imunologia , Pentanos , Sequência de Aminoácidos , Animais , Anticorpos Anti-Hepatite/imunologia , Antígenos da Hepatite delta , Humanos , Dados de Sequência Molecular , Prenilação de Proteína , Coelhos , Células Tumorais CultivadasRESUMO
Bioassay-directed fractionation of an EtOH extract of Curcuma zedoaria led to isolation of an active curcuminoid, which was identified as demethoxycurcumin (2) by comparison of its 1H and 13C NMR spectra with literature data and by direct comparison with synthetic material. Curcumin (1) and bisdemethoxycurcumin (3) were also obtained. Curcuminoids (1-3) were synthesized and demonstrated to be cytotoxic against human ovarian cancer OVCAR-3 cells. The observed CD50 values of 1, 2, and 3 were 4.4, 3.8, and 3.1 microg/mL, respectively. Three additional novel compounds, 3, 7-dimethylindan-5-carboxylic acid (4), curcolonol (5), and guaidiol (6), were also isolated from the EtOH extract. The structures and relative stereochemistry of 4-6 were determined by spectroscopic methods and X-ray crystallographic analysis.
Assuntos
Antineoplásicos Fitogênicos/isolamento & purificação , Curcumina/farmacologia , Plantas Medicinais/química , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , China , Cromatografia Líquida de Alta Pressão , Cristalografia por Raios X , Curcumina/análogos & derivados , Curcumina/isolamento & purificação , Ensaios de Seleção de Medicamentos Antitumorais , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Espectrofotometria Ultravioleta , Células Tumorais CultivadasRESUMO
The N-terminal region of the hepatitis-C virus (HCV) core protein is rich in basic residues, while the C-terminal end of the protein comprises of a stretch of hydrophobic amino acids. Between these two extremes is an amphipathic region with two predicted alpha-helical segments. This region embodies Leu or hydrophobic residues in positions of heptad repeats and is possibly capable of self-association. To investigate this possibility, the core sequence was divided into two fragments and expressed separately as recombinant proteins. Recombinant proteins with the N-terminal fragment remained as monomers even at high concentrations in SDS/PAGE. Recombinant protein with the C-terminal fragment appeared largely monomeric on denaturing gels but some oligomers were also detected. Furthermore, proline mutations in either one of the predicted alpha helices adversely affected the observed oligomerization. The self-association capacity of the core protein C-terminal region was further supported by results from a yeast two-hybrid system. To affirm our conclusion, a peptide covering the heptad repeats and the predicted alpha helices was synthesized. Data from mass spectrometry and gel-filtration chromatography concluded that this peptide readily self-associated into the homodimer. Therefore, our results suggest that the oligomerization motifs of the HCV core protein may not be limited to the previously suggested N-terminal region.
Assuntos
Proteínas do Core Viral/química , Sequência de Aminoácidos , Sequência de Bases , Biopolímeros , Primers do DNA , Hepacivirus/química , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Proteínas do Core Viral/genéticaRESUMO
Bioassay-directed fractionation of an ethanolic extract of Selaginella moellendorffii has led to the isolation of a known biflavone, ginkgetin (1). A dose-dependent inhibition was observed with 1 on the growth of OVCAR-3 (human ovarian adenocarcinoma) cells with 50% inhibition occurring at 1.8 micrograms/mL. Nonbioactive fractions yielded four additional known biflavones, amentoflavone 7,4',7",4"'-tetramethyl ether, kayaflavone, podocarpusflavone A, and amentoflavone.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Biflavonoides , Flavonoides/farmacologia , Plantas Medicinais/química , Animais , Chlorocebus aethiops , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Células Tumorais Cultivadas , Células VeroRESUMO
An enzyme immunoassay was developed for monitoring protease reactions of human immunodeficiency virus (HIV). The protease and its substrate, the gag precursor, were generated separately in Escherichia coli. The HIV-1 protease was generated with a glutathione-S-transferase expression system and the gag substrate, named Pin17/24, was prepared with a PinPoint expression system. Pin17/24 consists of an N-terminal peptide, which is biotinylated in E. coli, fused with a C-terminal peptide that contains a protease cleavage site flanked by p17 and p24 segments. Through its biotin in the N-terminal region, Pin17/24 bound to ELISA plates coated with avidin, whereas through its C-terminal region, the same molecule of Pin17/24 could be recognized by an anti-p24 monoclonal antibody. When the protease was added to Pin17/24, the p24 fragment was released from the biotinylated fusion protein and could no longer be retained on the avidin plates, and as a result, binding of the anti-p24 monoclonal antibody decreased. The binding was specific and the reaction was inhibited by a known HIV protease inhibitor. Due to the specific interactions between avidin and biotin, monoclonal antibody and antigen, and the HIV protease and the gag substrate, crude preparations of these reagents can be used readily in the assay. The simplicity and feasibility of this method should be useful for simultaneous monitoring of many enzyme reactions, particularly for screening possible HIV protease inhibitors.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Produtos do Gene gag/metabolismo , Protease de HIV/análise , HIV-1/enzimologia , Animais , Biotina , Escherichia coli , Estudos de Viabilidade , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por SubstratoRESUMO
The putative E1 of hepatitis C virus (HCV) was expressed in Escherichia coli using a glutathione-S-transferase (GST) fusion protein system. The full length E1 protein is difficult to express. A series of E1 DNA fragments was generated and used for expression vector construction. Fusion proteins containing the E1 C-terminal region could not be expressed. When this region was truncated, the fusion proteins were synthesized to high levels. The possibility of this C-terminal region hampering the production of fusion protein was further explored. A construct with this segment directly fused to the C-terminus of GST indeed generated no detectable recombinant protein. According to the predicted structure of E1, this region may have membrane-associating properties. The expression results suggest a general approach to facilitate the production of viral membrane proteins in prokaryotes. Furthermore, these recombinant E1 proteins generated as antigens were used for Western blotting with sera from HCV-infected individuals. It was found that E1 is antigenic during HCV natural infection.
Assuntos
Hepacivirus/genética , Proteínas do Envelope Viral/genética , Virologia/métodos , Antígenos Virais/genética , Sequência de Bases , DNA Viral/genética , Escherichia coli/genética , Expressão Gênica , Vetores Genéticos , Hepacivirus/imunologia , Anticorpos Anti-Hepatite/sangue , Hepatite C/imunologia , Anticorpos Anti-Hepatite C , Antígenos da Hepatite C , Dados de Sequência Molecular , Plasmídeos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas do Envelope Viral/imunologiaRESUMO
All animal retroviruses whose nucleotide sequences have been determined contain two or three closely spaced cysteine residues in the extracellular domain of the env-encoded transmembrane protein. Using human immunodeficiency virus type 1 gp41 as a working model, the functional significance of these highly conserved cysteines was investigated. We report here that substituting the two conserved cysteine residues in this domain of gp41 with glycine residues resulted in the loss of viral infectivity, which could be attributed to severe impairment in the processing of gp160 precursor to gp120.
Assuntos
Cisteína , Genes env , Proteína gp41 do Envelope de HIV/genética , HIV-1/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Linhagem Celular , DNA Viral/genética , Produtos do Gene env/análise , Produtos do Gene env/genética , Proteína gp120 do Envelope de HIV/análise , Proteína gp120 do Envelope de HIV/genética , Proteína gp160 do Envelope de HIV , Transcriptase Reversa do HIV , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Precursores de Proteínas/análise , Precursores de Proteínas/genética , Provírus/genética , DNA Polimerase Dirigida por RNA/análise , TransfecçãoAssuntos
Anticorpos Monoclonais/química , Complexo Antígeno-Anticorpo , Antígenos/química , Epitopos/química , Sequência de Aminoácidos , Animais , Antígenos/imunologia , Escherichia coli , Imunoglobulina G/química , Indicadores e Reagentes , Camundongos , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/imunologia , Resinas Vegetais , Ribossomos/ultraestruturaRESUMO
Human immunodeficiency virus (HIV) vaccines targeted at blocking HIV-CD4 interactions are expected to be less affected by the sequence heterogeneity of HIV than those targeted at variable regions of the envelope outercoat glycoprotein, gp120. All potential CD4 binding sites identified thus far in HIV are localized in the C-terminal region of gp120. In this study we demonstrate that the N-terminal region of gp120 also contains conserved residues critical for binding to CD4 and that gp120-CD4 interactions can be blocked by an antiserum with binding specificity to an N-terminal region of gp120. These results suggest that not all potential CD4 binding sites are present in the C-terminal region of gp120 and that an alternative HIV vaccine development strategy may have to include the N-terminal gp120 region as a component to raise effective CD4-blocking antibodies.