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1.
Nucleic Acids Res ; 36(21): 6739-51, 2008 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-18948292

RESUMEN

Drug-resistant variants of HIV-1 reverse transcriptase (RT) are also known to be resistant to anti-RT RNA aptamers. In order to be able to develop diagnostics and therapies that can focus on otherwise drug-resistant viruses, we have isolated two aptamers against a well-known, drug-resistant HIV-1 RT, Mutant 3 (M3) from the multidrug-resistant HIV-1 RT panel. One aptamer, M302, bound M3 but showed no significant affinity for wild-type (WT) HIV-1 RT, while another aptamer, 12.01, bound to both M3 and WT HIV-1 RTs. In contrast to all previously selected anti-RT aptamers, neither of these aptamers showed observable inhibition of either polymerase or RNase H activities. Aptamers M302 and 12.01 competed with one another for binding to M3, but they did not compete with a pseudoknot aptamer for binding to the template/primer cleft of WT HIV-1 RT. These results represent the surprising identification of an additional RNA-binding epitope on the surface of HIV-1 RT. M3 and WT HIV-1 RTs could be distinguished using an aptamer-based microarray. By probing protein conformation as a correlate to drug resistance we introduce an additional and useful measure for determining HIV-1 drug resistance.


Asunto(s)
Aptámeros de Nucleótidos/química , Farmacorresistencia Viral/genética , Transcriptasa Inversa del VIH/análisis , VIH-1/efectos de los fármacos , Fármacos Anti-VIH/uso terapéutico , Secuencia de Bases , Transcriptasa Inversa del VIH/química , Transcriptasa Inversa del VIH/genética , VIH-1/genética , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , Análisis por Matrices de Proteínas
2.
Mol Cell Proteomics ; 2(1): 19-28, 2003 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-12601079

RESUMEN

Insect resistance to the Cry toxins of Bacillus thuringiensis (Bt) has been examined previously using a number of traditional biochemical and molecular techniques. In this study, we utilized a proteomic approach involving two-dimensional differential gel electrophoresis, mass spectrometry, and function-based activity profiling to examine changes in the gut proteins from the larvae of an Indianmeal moth (IMM, Plodia interpunctella) colony exhibiting resistance to Bt. We found a number of changes in the levels of certain specific midgut proteins that indicate increased glutathione utilization, elevation in oxidative metabolism, and differential maintenance of energy balance within the midgut epithelial cells of the Bt-resistant IMM larva. Additionally, the electrophoretic migration pattern of a low molecular mass acidic protein, which apparently is an ortholog of F(1)F(0)-ATPase, was considerably altered in the Bt-resistant insect indicating that variations in amino acid content or modifications of certain proteins also are important components of the resistance phenomenon in the IMM. Furthermore, there was a dramatic decrease in the level of chymotrypsin-like proteinase in the midgut of the Bt-resistant larva, signifying that reduction of chymotrypsin activity, and subsequently decreased activation of Cry toxin in the insect midgut, is an important factor in the resistant state of the IMM. The proteomic analysis of larval gut proteins utilized in this study provides a useful approach for consolidating protein changes and physiological events associated with insect resistance to Bt. Our results support the hypothesis that physiological adaptation of insects and resistance to Bt is multifaceted, including protein modification and changes in the synthesis of specific larval gut proteins. We believe that increased oxidative metabolism may be an adaptive response of insects that undergo survival challenge and that it could mediate detoxification as well as higher rates of generalized and localized mutations that enhance their resistance and provide survival advantage.


Asunto(s)
Bacillus thuringiensis/metabolismo , Larva/microbiología , Proteoma , Animales , Quimotripsina/farmacología , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Glutatión/metabolismo , Insectos , Espectrometría de Masas , Mariposas Nocturnas , Oxígeno/metabolismo , Control Biológico de Vectores
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