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1.
Biomed Res Int ; 2017: 9579736, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29457039

RESUMO

The carmine spider mite Tetranychus cinnabarinus is a major pest of crop and vegetable plants worldwide. Previous studies have shown that scopoletin is a promising acaricidal compound against Tetranychus cinnabarinus. However, the acaricidal mechanism of scopoletin remains unclear. In the present study, 12 full-length cDNAs of chitinase (CHIT) genes from Tetranychus cinnabarinus (designated TcCHITs) were cloned and characterized. Although TcCHITs were expressed throughout all life stages, their expression levels were significantly upregulated during the larval and nymphal stages. TcCHITs were downregulated 24 h after treatment with scopoletin and upregulated 24 h after treatment with diflubenzuron (DFB, a chitin synthesis inhibitor). Feeding double-stranded RNA effectively silenced TcCHIT transcription in Tetranychus cinnabarinus, thus increasing its susceptibility to scopoletin but reducing that to DFB. Meanwhile, TcCHIT silencing in larvae and adult resulted in an extremely low molting rate (7.3%) and high mortality rate (53.3%), respectively, compared with those in the control group. CHIT genes are closely related to arthropod survival, molting, and development in Tetranychus cinnabarinus, suggesting that acaricidal mechanisms of scopoletin and DFB may occur by inhibition and activation of CHIT gene expression, respectively. TcCHIT constitutes a possible target of scopoletin and DFB in Tetranychus cinnabarinus.


Assuntos
Acaricidas/farmacologia , Quitinases/antagonistas & inibidores , Escopoletina/farmacologia , Tetranychidae/efeitos dos fármacos , Animais , DNA Complementar/antagonistas & inibidores , Inativação Gênica , Controle de Pragas , Tetranychidae/enzimologia
2.
Inflamm Bowel Dis ; 18(2): 344-8, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21618356

RESUMO

BACKGROUND: Dextran sulfate sodium (DSS) induces experimental colitis and promotes colitis-associated cancer in rodents. Here we document potent inhibition of real-time quantitative polymerase chain reaction (qPCR) using cDNA from DSS-exposed mouse tissues, which complicates gene expression analysis. METHODS: We characterize DSS inhibition of qPCR in-vitro and in a wide array of murine tissues following ingestion of DSS. We examine different approaches to RNA purification prior to cDNA synthesis in order to optimize real-time polymerase chain reaction amplification and gene expression analysis. RESULTS: DSS inhibits qPCR amplification of cDNA between 1 and 10 nM. Orally administered DSS interferes with qPCR amplification of cDNA derived from multiple tissues. Poly-A purification of DSS-exposed RNA allows reliable and cost-effective gene expression analysis in DSS-exposed tissue. CONCLUSIONS: DSS is a potent inhibitor of real-time qPCR amplification and interferes with tissue-specific gene expression analysis in DSS-exposed mice. Poly-A purification of tissue-derived RNA results in reliable and cost-effective gene expression analysis in DSS-exposed mice.


Assuntos
DNA Complementar/antagonistas & inibidores , Sulfato de Dextrana/efeitos adversos , Perfilação da Expressão Gênica , Poli A/isolamento & purificação , RNA Mensageiro/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , DNA Complementar/biossíntese , Sulfato de Dextrana/administração & dosagem , Camundongos , Camundongos Endogâmicos C57BL , RNA/isolamento & purificação
3.
Nucleus ; 1(5): 422-31, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-21326825

RESUMO

Unlike other retroviruses, human immunodeficiency virus type-1 (HIV-1) can infect terminally differentiated cells, due to the ability of its pre-integration complex (PIC) to translocate via the host nuclear pore complex (NPC). The PIC Nuclear import has been suggested to be mediated by the viral integrase protein (IN), via either the importin α or transportin 3 (TNPO3/transportin-SR2) pathways.We show that in virus-infected cells, IN interacts with both importin α and TNPO3, simultaneously or separately, suggesting a multiple use of nuclear import pathways. Disruption of either the IN-importin α or IN-TNPO3 complexes in virus-infected cells by specific cell-permeable-peptides resulted in inhibition of IN and viral cDNA nuclear import. Here we show that peptides which disrupt either one of these complexes block virus infection, indicating involvement of both pathways in efficient viral replication. Formation of IN-importin α and IN-TNPO3 complexes has also been observed in IN-transfected cultured cells. Using specific peptides, we demonstrate that in transfected cells but not in virus infected cells the importin α pathway overrides that of TNPO3. The IN-importin α and IN-TNPO3 complexes were not observed in virus-infected Rev-expressing cells, indicating the Rev protein's ability to disrupt both complexes.Our work suggests that IN nuclear import requires the involvement of both importin α and TNPO3. The ability to inhibit nuclear import of the IN-DNA complex and consequently, virus infection by peptides that interrupt IN's interaction with either importin α or TNPO3 indicates that for efficient infection, nuclear import of IN should be mediated by both nuclear-import receptors.


Assuntos
DNA Viral/antagonistas & inibidores , Integrase de HIV/metabolismo , HIV-1/fisiologia , Poro Nuclear/metabolismo , alfa Carioferinas/metabolismo , beta Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Antivirais/síntese química , Antivirais/farmacologia , Linhagem Celular Tumoral , DNA Complementar/antagonistas & inibidores , DNA Complementar/biossíntese , DNA Viral/biossíntese , Integrase de HIV/genética , Interações Hospedeiro-Patógeno , Humanos , Dados de Sequência Molecular , Poro Nuclear/genética , Peptídeos/síntese química , Peptídeos/farmacologia , Ligação Proteica , Transfecção , Replicação Viral/efeitos dos fármacos , alfa Carioferinas/genética , beta Carioferinas/genética , Produtos do Gene rev do Vírus da Imunodeficiência Humana/genética , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismo
4.
Mol Immunol ; 44(13): 3297-304, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17416419

RESUMO

We determined the interferon-gamma (IFN-gamma) cDNA sequence from three porcine breeds, Duroc, Landrance/Duroc hybrid, and Landrance breeds. Five single nucleotide polymorphisms (SNPs) of porcine IFN-gamma (PoIFN-gamma) were identified, respectively, at positions 269 (A/G), 376 (C/T), 426 (T/C), and 465 (T/C) of the coding sequence in Landrance/Duroc hybrid, and at position 251 (A/G) in Landrance breed. Among them, A269G and A251G polymorphisms resulted in Q67R and K61R replacements in the mature protein. PoIFN-gamma cDNAs of Duroc breed (PoIFN-gamma-W) and Landrance/Duroc hybrid (PoIFN-gamma-M), which, respectively, encoded Q67 and R67, were introduced into a prokaryotic expression vector pET32 to express recombinant PoIFN-gamma-W (rPoIFN-gamma-W) and rPoIFN-gamma-M protein variants in Escherichia coli. The identity of both protein variants was further confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We then compared bioactivities of these two recombinant proteins. Although both recombinant protein variants exhibited comparable activities in antiproliferation of PK-15 cells and in nitric oxide (NO) induction of porcine peripheral monocytes, antiviral activity of rPoIFN-gamma-W protein was significantly higher (P<0.001) than that of rPoIFN-gamma-M protein in a plaque inhibition assay using pseudorabies virus (PRV). IC50 values of rPoIFN-gamma-W and rPoIFN-gamma-M protein in anti-PRV assay were determined as 5.3+/-1.3 and 9.3+/-4.3nM, respectively. In conclusion, we have identified five novel SNPs in PoIFN-gamma cDNA, including two missense polymorphisms that result in Q67R and K61R replacements. Our results further demonstrate that Q67R can markedly reduce antiviral activity of the PoIFN-gamma protein. This is the first report that shows the functional SNP in the coding region of IFN-gamma. In the future, it is imperative to determine whether Q67R replacement in IFN-gamma may have disease association.


Assuntos
DNA Complementar/genética , Variação Genética , Herpesvirus Suídeo 1/imunologia , Interferon gama/antagonistas & inibidores , Interferon gama/genética , Mutação de Sentido Incorreto/genética , Polimorfismo Genético , Sequência de Aminoácidos , Animais , Antivirais/antagonistas & inibidores , Antivirais/metabolismo , Sequência de Bases , Células Cultivadas , DNA Complementar/antagonistas & inibidores , Herpesvirus Suídeo 1/crescimento & desenvolvimento , Humanos , Interferon gama/fisiologia , Dados de Sequência Molecular , Suínos , Ensaio de Placa Viral , Inativação de Vírus
5.
Int J Urol ; 13(9): 1221-5, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16984557

RESUMO

AIM: In this study, we examined whether human Hsp27 is involved in the radiation sensitization induced by gamma radiation in cultured human prostate cancer cells. METHODS: We transfected DU145 cells with full length Hsp27 antisense cDNA to obtain viable cell lines which expressed reduced Hsp27. Selected individual clones were subjected to western blot analyses to confirm reduced expression of Hsp27. RESULTS: Hsp27 belongs to a family of abundant and ubiquitous stress proteins, the small heat shock proteins. It has been shown that Hsp27 can inhibit apoptosis both in a caspase-dependent and independent manner. Colony assays showed that the cells engineered to express reduced Hsp27 levels had a significantly increased sensitivity to gamma radiation compared with control cells that were transfected with the vector alone. However, there was also a significant difference in viability of cells with reduced Hsp27 levels and control cells 72 h after gamma-irradiation. CONCLUSIONS: Our results suggest the possible application of antisense Hsp27 cDNA or other methods to reduce Hsp27 expression as a radiation sensitizer in radiation oncology.


Assuntos
Apoptose/efeitos da radiação , Proteínas de Choque Térmico/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias da Próstata/radioterapia , Tolerância a Radiação/genética , Western Blotting , Ensaio de Unidades Formadoras de Colônias , DNA Antissenso , DNA Complementar/antagonistas & inibidores , DNA Complementar/genética , Regulação para Baixo , Raios gama , Proteínas de Choque Térmico HSP27 , Proteínas de Choque Térmico/genética , Humanos , Masculino , Chaperonas Moleculares , Proteínas de Neoplasias/genética , Plasmídeos , Neoplasias da Próstata/metabolismo , Transfecção , Células Tumorais Cultivadas
6.
Insect Biochem Mol Biol ; 33(10): 1025-33, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-14505696

RESUMO

An inhibitor of Aspergillus oryzae fungal protease was purified to homogeneity from the hemolymph of fifth instar larvae of Antheraea mylitta by ammonium sulfate precipitation, anion exchange and gel filtration (FPLC) chromatography, and termed as AmFPI-1. The extent of purification was checked by two-dimensional gel electrophoresis, and the molecular weight of purified inhibitor was determined by SDS-PAGE as 10.4 kDa. Fifteen N-terminal amino acid sequences of this protein were determined, and degenerate oligonucleotides were synthesized on the basis of these sequences. A cDNA library of A. mylitta integument was constructed, and protease inhibitor cDNA was partially amplified by PCR using degenerate oligonucleotides and CDS primers. A full-length inhibitor cDNA clone obtained by screening the library with PCR amplified DNA as probe was sequenced. The cDNA consists of 543 nucleotides with an ORF of 315 bp and encodes a protein of 105 amino acids. The sequence exhibits similarity to several Bombyx mori ESTs, and in particular to N-terminal amino acid sequence of an inducible serine protease inhibitor (ISPI-1) from Galleria mellonella indicating its relatedness to ISPI-1 of G. mellonella. The presence of this protease inhibitor in the hemolymph may play an important role as a natural defense system against invading microorganisms.


Assuntos
Proteínas de Insetos/genética , Proteínas de Insetos/isolamento & purificação , Mariposas/química , Inibidores de Proteases/isolamento & purificação , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Precipitação Química , Cromatografia em Gel , Cromatografia por Troca Iônica/métodos , Clonagem Molecular , DNA Complementar/antagonistas & inibidores , DNA Complementar/genética , Endopeptidases/efeitos dos fármacos , Endopeptidases/metabolismo , Biblioteca Gênica , Hemolinfa/química , Proteínas de Insetos/metabolismo , Proteínas de Insetos/farmacologia , Larva/química , Larva/crescimento & desenvolvimento , Dados de Sequência Molecular , Inibidores de Proteases/metabolismo , Inibidores de Proteases/farmacologia , Homologia de Sequência de Aminoácidos
7.
Biochemistry ; 37(7): 1880-9, 1998 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-9485313

RESUMO

Genotoxic lesions frequently arrest DNA synthesis and, as a consequence, result in the accumulation of incompletely replicated chromosomal segments containing long single-stranded regions of parental DNA. Here, we exploited complementary strand synthesis in Xenopus laevis egg or HeLa cell lysates to test how the eukaryotic replication machinery responds to such damaged single-stranded intermediates. Although both cell lysates promoted efficient conversion of M13 or phi X174 single-stranded templates to double-stranded products, their replication activity was inhibited by DNA damage arising from ultraviolet (UV) radiation or exposure to the alkylating agent N-methyl-N-nitrosourea (MNU). When M13 single-stranded DNA containing UV-or MNU-induced lesions was coincubated with single-stranded substrates containing no lesions, we observed suppression of DNA synthesis on both damaged and undamaged templates. In contrast, complementary strand synthesis remained unaffected in coincubation reactions containing damaged DNA in the double-stranded form. Effective inhibition of complementary strand synthesis on undamaged templates required the presence of at least stoichiometric amounts of UV-or MNU-treated single-stranded DNA, indicating that a DNA polymerase or accessory protein is excluded from DNA synthesis by immobilization at or near the lesion sites. In support of this competitive mode of inhibition, we found that inactivation of DNA synthesis by coincubation with damaged single-stranded DNA was relieved by the addition of an exogenous DNA polymerase that catalyzes processive strand elongation. In summary, this study reveals sequestration of critical components of the eukaryotic replication machinery in a DNA damage-dependent and single-strand-specific manner, thereby providing a potential mechanism to sense arrested replication intermediates during an early recognition step of S phase checkpoint responses.


Assuntos
Dano ao DNA , DNA Complementar/antagonistas & inibidores , DNA Complementar/biossíntese , Óvulo/metabolismo , Animais , Sistema Livre de Células/enzimologia , Sistema Livre de Células/metabolismo , Sistema Livre de Células/efeitos da radiação , Citosol/metabolismo , Citosol/efeitos da radiação , DNA Complementar/efeitos dos fármacos , DNA Complementar/efeitos da radiação , DNA Viral/biossíntese , DNA Viral/efeitos da radiação , DNA Polimerase Dirigida por DNA/farmacologia , Células HeLa , Humanos , Metilnitrosoureia/farmacologia , Óvulo/efeitos dos fármacos , Óvulo/efeitos da radiação , Moldes Genéticos , Raios Ultravioleta , Xenopus laevis
8.
J Biochem ; 119(6): 1070-5, 1996 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-8827439

RESUMO

To investigate the dominant-negative inhibition of Moloney murine leukemia virus (Mo-MuLV) proliferation by polymerization-defective mutants of reverse transcriptase (RT), we constructed several plasmids harboring the Mo-MuLV RT gene in which the YVDD sequence, one of the conserved sequences in RNA-dependent polymerases, was altered, and then transformed mouse NIH3T3 cells and Escherichia coli with the mutant plasmids. Mouse NIH3T3 cells expressing these mutant RT genes were highly resistant to Mo-MuLV proliferation. The mutant RT expressed in E. coli exhibited no polymerization activity, but it retained its binding activity to the template RNA and inhibited in vitro poly(dG) synthesis occurring with the wild-type RT. These results suggest that the competition for binding of the two types of enzymes to the template is responsible for the resistance to Mo-MuLV proliferation and that the YVDD sequence of Mo-MuLV may be a good target for dominant-negative inhibition of retroviral proliferation.


Assuntos
Vírus da Leucemia Murina de Moloney/enzimologia , DNA Polimerase Dirigida por RNA/metabolismo , Células 3T3/metabolismo , Células 3T3/virologia , Sequência de Aminoácidos , Animais , Sequência Conservada , DNA Complementar/antagonistas & inibidores , DNA Complementar/biossíntese , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Virais/genética , Camundongos , Vírus da Leucemia Murina de Moloney/genética , Vírus da Leucemia Murina de Moloney/fisiologia , Mutação , Plasmídeos/genética , Poli A/metabolismo , Poli G/antagonistas & inibidores , Poli G/biossíntese , Ligação Proteica , DNA Polimerase Dirigida por RNA/genética , Transfecção , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral
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