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- Nilsson, IngMarie, et al.
(författare)
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Photocross-linking of nascent chains to the STT3 subunit of the oligosaccharyltransferase complex
- 2003
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Ingår i: Journal of Cell Biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 161:4, s. 715-725
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Tidskriftsartikel (refereegranskat)abstract
- In eukaryotic cells, polypeptides are N glycosylated after passing through the membrane of the ER into the ER lumen. This modification is effected cotranslationally by the multimeric oligosaccharyltransferase (OST) enzyme. Here, we report the first cross-linking of an OST subunit to a nascent chain that is undergoing translocation through, or integration into, the ER membrane. A photoreactive probe was incorporated into a nascent chain using a modified Lys-tRNA and was positioned in a cryptic glycosylation site (-Q-K-T- instead of -N-K-T-) in the nascent chain. When translocation intermediates with nascent chains of increasing length were irradiated, nascent chain photocross-linking to translocon components, Sec61alpha and TRAM, was replaced by efficient photocross-linking solely to a protein identified by immunoprecipitation as the STT3 subunit of the OST No cross-linking was observed in the absence of a cryptic sequence or in the presence of a competitive peptide substrate of the OST. As no significant nascent chain photocross-linking to other OST subunits was detected in these fully assembled translocation and integration intermediates, our results strongly indicate that the nascent chain portion of the OST active site is located in STT3.
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- Shao, X-W, et al.
(författare)
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Application of a colorimetric chain-termination assay for characterization of reverse transcriptase from 3-azido-2,3-deoxythymidine-resistant HIV isolates
- 2002
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Ingår i: Biotechnology and applied biochemistry. - 0885-4513 .- 1470-8744. ; 35, s. 155-164
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Tidskriftsartikel (refereegranskat)abstract
- wo different enzyme assays, both based on the interaction of native reverse transcriptase(IRT) and 3'-azido-2',3'-deoxythymidine triphosphate (AZT-TP), were used to characterize the enzymesfrom 18 HIV-I isolates with decreased sensitivity to AZT in cell culture. The first assay, which measures the balance between incorporation and excision of AZT monophosphate in the presence of dNTP substrate (in terms of IC50), gave an approx. 9-fold variation in sensitivity to AZT-TP. There was a correlation between the IC50 values and the sensitivity of the corresponding virus to AZT in cell culture (r = 0.60, P < 0.01). The second assay, which was designed specifically for measurement of chain termination in the absence of dNTP substrate (as the concentration of AZT-TP giving 50% residual primer function, or CT50), revealed a more than 600-fold difference between the different isolate RTs. For the majority ofenzymes there was a strict correlation between the results from the two assays; however, four isolatesexhibited significantly higher CT50/IC50 ratios than the other isolates. These differences were not related to sensitivity of the corresponding viruses to AZT but to the occurrence of certain mutations in their pol gene. The four deviating isolates contained either a minimum of four AZT-specific substitutions, including Thr-215 --> Tyr (isolates 134 and 143), or some of the known specific substitutions combined with Thr-39 --> Ala (isolates 80 and 157). The Thr-39 Ala substitution has previously been recorded in connection with AZT/Foscarnet combination therapy.
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- Shao, Xing-Wu, et al.
(författare)
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Use of HIV-1 reverse transcriptase recovered from human plasma for phenotypic drug susceptibility testing
- 2003
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Ingår i: AIDS. ; 17, s. 1463-1471
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Tidskriftsartikel (refereegranskat)abstract
- Objective: To demonstrate the use of HIV-1 reverse transcriplase (RT) recovered directly fromplasma for phenotypic drug susceptibility testing. Methods: Plasma from HIV-1 infected individuals with and without drug resistance-associated mutations were selected for the study. The blind coded plasmas were treated to inactivate cellular enzymes. The virions were immobilized on a gel and washed to remove antiretroviral drugs and RT activity blocking antibodies. The immobilized virions were lysed; the viral RTeluted and quantified, all according to the ExaVir Load procedure. The drug sensitivity profiles of each RT were determined using serially diluted drugs and modified Cavidi HS Lenti RT kits. Results: The phenotypic drug sensitivity profiles of the RT and the patterns of drug resistance mutations were highly concordant. Plasma RT from virions devoid of mutations associated with drug resistance had average 50% inhibitory concentrations (IC50) of 1.5 +/- 0.93 muM for nevirapine, 0.21 +/- 0.099 muM forefavirenz, 7.1 +/- 3.2 muM for delavirdine, 0.42 +/- 0.15 muM for azidothymidine triphosphate and 0.059 +/- 0.018 muM for didehydrothymidine triphosphate. The increase in IC50 value for RT with drugresistance associated substitutions was from 3- to more than 65-fold for non-nucleoside inhibitors and between 2- and 30-fold for thymidine analogue drugs. Conclusion: RT derived from virions recovered from the plasma of HIV infected individuals can be used foranalysis of phenotypic drug susceptibility. The methods presented provide rapid alternatives for analysingphenotypic drug susceptibility especially when the therapy is based on non-nucleoside RT inhibitors and thymidine-analogue drugs.
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- Zhang, Zong Xian, et al.
(författare)
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On study of influences of loading rate on fractal dimensions of fracture surfaces in Gabbro
- 2001
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Ingår i: Rock Mechanics and Rock Engineering. - : Springer Science and Business Media LLC. - 0723-2632 .- 1434-453X. ; 34:3, s. 235-242
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Tidskriftsartikel (refereegranskat)abstract
- Studies of the effects of loading rates on rock fracture were performed for decades. However, the previous work on static or dynamic rock fracture was mainly limited to a macro-experimental study. The present investigation measures the fractal dimensions of the fracture surfaces of the gabbro specimens fractured at various loading rates covering static and dynamic loading, and explores the relationship between the fractal dimensions and the fracture toughness of the rock.
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