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21.
Jeffrey D. Carter Estrella G. Gonzales Xi Huang Adam N. Smith Ian Mitchelle S. de Vera Peter W. D’Amore James R. Rocca Maureen M. Goodenow Ben M. Dunn Gail E. Fanucci 《FEBS letters》2014
Conformational sampling of pre- and post-therapy subtype B HIV-1 protease sequences derived from a pediatric subject infected via maternal transmission with HIV-1 were characterized by double electron–electron resonance spectroscopy. The conformational ensemble of the PRE construct resembles native-like inhibitor bound states. In contrast, the POST construct, which contains accumulated drug-pressure selected mutations, has a predominantly semi-open conformational ensemble, with increased populations of open-like states. The single point mutant L63P, which is contained in PRE and POST, has decreased dynamics, particularly in the flap region, and also displays a closed-like conformation of inhibitor-bound states. These findings support our hypothesis that secondary mutations accumulate in HIV-1 protease to shift conformational sampling to stabilize open-like conformations, while maintaining the predominant semi-open conformation for activity. 相似文献
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The observed lengthening of the C period in the presence of a defective ribonucleoside diphosphate reductase has been assumed to be due solely to the low deoxyribonucleotide supply in the nrdA101 mutant strain. We show here that the nrdA101 mutation induces DNA double-strand breaks at the permissive temperature in a recB-deficient background, suggesting an increase in the number of stalled replication forks that could account for the slowing of replication fork progression observed in the nrdA101 strain in a Rec(+) context. These DNA double-strand breaks require the presence of the Holliday junction resolvase RuvABC, indicating that they have been generated from stalled replication forks that were processed by the specific reaction named "replication fork reversal." Viability results supported the occurrence of this process, as specific lethality was observed in the nrdA101 recB double mutant and was suppressed by the additional inactivation of ruvABC. None of these effects seem to be due to the limitation of the deoxyribonucleotide supply in the nrdA101 strain even at the permissive temperature, as we found the same level of DNA double-strand breaks in the nrdA(+) strain growing under limited (2-microg/ml) or under optimal (5-microg/ml) thymidine concentrations. We propose that the presence of an altered NDP reductase, as a component of the replication machinery, impairs the progression of the replication fork, contributing to the lengthening of the C period in the nrdA101 mutant at the permissive temperature. 相似文献
24.
Riola J Guarino E Guzmán EC Jiménez-Sánchez A 《Cellular & molecular biology letters》2007,12(1):70-81
NDP reductase activity can be inhibited either by treatment with hydroxyurea or by incubation of an nrdA
ts mutant strain at the non-permissive temperature. Both methods inhibit replication, but experiments on these two types of
inhibition yielded very different results. The chemical treatment immediately inhibited DNA synthesis but did not affect the
cell and nucleoid appearance, while the incubation of an nrdA101 mutant strain at the non-permissive temperature inhibited DNA synthesis after more than 50 min, and resulted in aberrant
chromosome segregation, long filaments, and a high frequency of anucleate cells. These phenotypes are not induced by SOS.
In view of these results, we suggest there is an indirect relationship between NDP reductase and the chromosome segregation
machinery through the maintenance of the proposed replication hyperstructure. 相似文献
25.
Molina L Duque E Gómez MJ Krell T Lacal J García-Puente A García V Matilla MA Ramos JL Segura A 《Environmental microbiology》2011,13(8):2315-2327
Pseudomonas putida DOT-T1E has the capacity to grow in the presence of high concentrations of toluene. This ability is mainly conferred by an efflux pump encoded in a self-transmissible 133 kb plasmid named pGRT1. Sequence analysis of the pGRT1 plasmid revealed several key features. Most of the genes related to the plasmid maintenance functions show similarity with those encoded on pBVIE04 from Burkholderia vietnamensis G4, and knock-out mutants in several of these genes confirmed their roles. Two additional plasmid DNA fragments were incorporated into the plasmid backbone by recombination and/or transposition; in these DNA regions, apart from multiple recombinases and transposases, several stress-related and environmentally relevant functions are encoded. We report that plasmid pGRT1 not only confers the cells with tolerance to toluene but also resistance to ultraviolet light. We show here the implication of a new protein in solvent tolerance which controls the level of expression of the TtgGHI efflux pump, as well as the implication of a protein with homology to the universal stress protein in solvent tolerance and ultraviolet light resistance. Furthermore, this plasmid encodes functions that allow the cells to chemotactically respond to toluene and participate in iron scavenging. 相似文献
26.
Fuciños P Atanes E López-López O Esperanza Cerdán M Isabel González-Siso M Pastrana L Luisa Rúa M 《Protein expression and purification》2011,78(2):120-130
Two N-terminally truncated variants of the esterase E34Tt from Thermus thermophilus HB27 (YP_004875.1) were expressed in Kluyveromyces lactis. Production and biochemical properties of both recombinant proteins were investigated. The esterase activity was greatly increased compared to the wild-type strain. In particular, the extracellular production of the ΔN16 variant (KLEST-3S) was 50-fold higher than that obtained with T. thermophilus HB27. Response surface methodology was applied to describe the pH and temperature dependence of both activity and stability. When compared with the wild type esterase, the optimal temperature of reaction decreased 35 and 15 °C for ΔN16 and ΔN26, respectively. KLEST-3S showed a maximum of activity at pH 7.5 and 47.5 °C, and maximal stability at pH 8.1 and 65 °C. KLEST-5A (ΔN26) did not show an absolute maximum of activity. However, best results were obtained at 40 °C and pH 8.5. KLEST-5A showed also a lower stability. In the presence of a surfactant, both proteins showed lower stability at 85 °C (t(?)< 5 min) than the wild-type enzyme (t(?)=135 min). However, in the absence of detergent, the stability of KLEST-3S was higher (t(?)=230 min, at 85 °C) than that of the mutant KLEST-5A (12 min) or the wild type enzyme (19 min). Minor differences were observed in the substrate specificity. Our results suggest that the N-terminal segment is critical for maintaining the hyperthermophilic function and stability. 相似文献
27.
Rosario Linacero Julia Rueda Estrella Esquivel Alberto Bellido Angel Domingo Ana M. V��zquez 《In vitro cellular & developmental biology. Plant》2011,47(5):618-628
In vitro regenerated plants of rye, Secale cereale L., Ailés and Merced cultivars, were studied to verify if genetic and/or epigenetic changes were promoted by in vitro conditions. Inter-simple sequence repeat (ISSR) fingerprints on HpaII/MspI-digested and uncut DNA were generated. DNA digested with methylation-sensitive isoschizomers revealed epigenetic modifications, while modification of ISSR patterns obtained with undigested DNA indicated genetic changes. With this technique, it was possible to study both genetic and/or epigenetic changes within the same DNA sequences. The frequency of plants with at least one variation was high: 73% and 30% of rye plants showed at least one genetic change, and 50% and 73% carried at least one methylation change, in the Ailés and Merced cultivars, respectively. Further analyses revealed that a considerable number of variable markers showed both types of modifications, indicative of both genetic and epigenetic changes. Moreover, genetic variation was related to the presence of the CCGG target in the analyzed bands. These results indicate the possible existence of a common mechanism connecting both types of variation. 相似文献
28.
Castagno LN Estrella MJ Sannazzaro AI Grassano AE Ruiz OA 《Journal of applied microbiology》2011,110(5):1151-1165
Aims: To isolate and characterize phosphate‐solubilizing strains from a constrained environment such as the Salado River Basin and to assess their phosphate‐solubilizing mechanisms, to further selection of the most promising strains to inoculate and improve the implantation and persistence of Lotus tenuis in the most important area devoted to meat‐cow production in Argentina. Methods and Results: Fifty isolates were obtained and through BOX‐PCR analysis, 17 non‐redundant strains were identified. Subsequently, they were found to be related to Pantoea, Erwinia, Pseudomonas, Rhizobium and Enterobacter genera, via 16S rRNA gene sequence analysis. This was in agreement with the clusters obtained by antibiotic resistance analysis. All isolates were tested for their phosphate‐solubilizing activity and selected strains were inoculated onto L. tenuis plants. The most efficient isolate, was identified as Pantoea eucalypti, a novel species in terms of plant growth‐promoting rhizobacteria. Conclusions: The isolates obtained in this study showed a significant in vitro plant‐growth promoting activity onto Lotus tenuis and the best of them solubilizes phosphate mainly via induction of the metabolism through secretion and oxidation of gluconic acid. Singnificance and Impact of the Study: The use of these bacteria as bioinoculants, alone or in combination with nitrogen‐fixing micro‐organisms, could be a sustainable practice to facilitate the nutrient supply to Lotus tenuis plants and preventing negative side‐effects such as eutrophication. 相似文献
29.
Lasry-Levy E Hietaharju A Pai V Ganapati R Rice AS Haanpää M Lockwood DN 《PLoS neglected tropical diseases》2011,5(3):e981
Background
Neuropathic pain has been little studied in leprosy. We assessed the prevalence and clinical characteristics of neuropathic pain and the validity of the Douleur Neuropathique 4 questionnaire as a screening tool for neuropathic pain in patients with treated leprosy. The association of neuropathic pain with psychological morbidity was also evaluated.Methodology/Principal Findings
Adult patients who had completed multi-drug therapy for leprosy were recruited from several Bombay Leprosy Project clinics. Clinical neurological examination, assessment of leprosy affected skin and nerves and pain evaluation were performed for all patients. Patients completed the Douleur Neuropathique 4 and the 12-item General Health Questionnaire to identify neuropathic pain and psychological morbidity.Conclusions/Significance
One hundred and one patients were recruited, and 22 (21.8%) had neuropathic pain. The main sensory symptoms were numbness (86.4%), tingling (68.2%), hypoesthesia to touch (81.2%) and pinprick (72.7%). Neuropathic pain was associated with nerve enlargement and tenderness, painful skin lesions and with psychological morbidity. The Douleur Neuropathique 4 had a sensitivity of 100% and specificity of 92% in diagnosing neuropathic pain. The Douleur Neuropathique 4 is a simple tool for the screening of neuropathic pain in leprosy patients. Psychological morbidity was detected in 15% of the patients and 41% of the patients with neuropathic pain had psychological morbidity. 相似文献30.
Perfahl H Byrne HM Chen T Estrella V Alarcón T Lapin A Gatenby RA Gillies RJ Lloyd MC Maini PK Reuss M Owen MR 《PloS one》2011,6(4):e14790
We investigate a three-dimensional multiscale model of vascular tumour growth, which couples blood flow, angiogenesis, vascular remodelling, nutrient/growth factor transport, movement of, and interactions between, normal and tumour cells, and nutrient-dependent cell cycle dynamics within each cell. In particular, we determine how the domain size, aspect ratio and initial vascular network influence the tumour's growth dynamics and its long-time composition. We establish whether it is possible to extrapolate simulation results obtained for small domains to larger ones, by constructing a large simulation domain from a number of identical subdomains, each subsystem initially comprising two parallel parent vessels, with associated cells and diffusible substances. We find that the subsystem is not representative of the full domain and conclude that, for this initial vessel geometry, interactions between adjacent subsystems contribute to the overall growth dynamics. We then show that extrapolation of results from a small subdomain to a larger domain can only be made if the subdomain is sufficiently large and is initialised with a sufficiently complex vascular network. Motivated by these results, we perform simulations to investigate the tumour's response to therapy and show that the probability of tumour elimination in a larger domain can be extrapolated from simulation results on a smaller domain. Finally, we demonstrate how our model may be combined with experimental data, to predict the spatio-temporal evolution of a vascular tumour. 相似文献