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191.
We have examined the removal of thymine residues from T-G mismatches in DNA by the thymine-DNA mismatch glycosylase from Methanobacterium thermoautrophicum (Mig-Mth), within the context of the base excision repair (BER) pathway, to investigate why this glycosylase has such low activity in vitro. Using single-turnover kinetics and steady-state kinetics, we calculated the catalytic and product dissociation rate constants for Mig-Mth, and determined that Mig-Mth is inhibited by product apyrimidinic (AP) sites in DNA. Electrophoretic mobility shift assays (EMSA) provide evidence that the specificity of product binding is dependent upon the base opposite the AP site. The binding of Mig-Mth to DNA containing the non-cleavable substrate analogue difluorotoluene (F) was also analyzed to determine the effect of the opposite base on Mig-Mth binding specificity for substrate-like duplex DNA. The results of these experiments support the idea that opposite strand interactions play roles in determining substrate specificity. Endonuclease IV, which cleaves AP sites in the next step of the BER pathway, was used to analyze the effect of product removal on the overall rate of thymine hydrolysis by Mig-Mth. Our results support the hypothesis that endonuclease IV increases the apparent activity of Mig-Mth significantly under steady-state conditions by preventing reassociation of enzyme to product. 相似文献
192.
Adsorption of DNA to mica mediated by divalent counterions: a theoretical and experimental study 下载免费PDF全文
Pastré D Piétrement O Fusil S Landousy F Jeusset J David MO Hamon L Le Cam E Zozime A 《Biophysical journal》2003,85(4):2507-2518
The adsorption of DNA molecules onto a flat mica surface is a necessary step to perform atomic force microscopy studies of DNA conformation and observe DNA-protein interactions in physiological environment. However, the phenomenon that pulls DNA molecules onto the surface is still not understood. This is a crucial issue because the DNA/surface interactions could affect the DNA biological functions. In this paper we develop a model that can explain the mechanism of the DNA adsorption onto mica. This model suggests that DNA attraction is due to the sharing of the DNA and mica counterions. The correlations between divalent counterions on both the negatively charged DNA and the mica surface can generate a net attraction force whereas the correlations between monovalent counterions are ineffective in the DNA attraction. DNA binding is then dependent on the fractional surface densities of the divalent and monovalent cations, which can compete for the mica surface and DNA neutralizations. In addition, the attraction can be enhanced when the mica has been pretreated by transition metal cations (Ni(2+), Zn(2+)). Mica pretreatment simultaneously enhances the DNA attraction and reduces the repulsive contribution due to the electrical double-layer force. We also perform end-to-end distance measurement of DNA chains to study the binding strength. The DNA binding strength appears to be constant for a fixed fractional surface density of the divalent cations at low ionic strength (I < 0.1 M) as predicted by the model. However, at higher ionic strength, the binding is weakened by the screening effect of the ions. Then, some equations were derived to describe the binding of a polyelectrolyte onto a charged surface. The electrostatic attraction due to the sharing of counterions is particularly effective if the polyelectrolyte and the surface have nearly the same surface charge density. This characteristic of the attraction force can explain the success of mica for performing single DNA molecule observation by AFM. In addition, we explain how a reversible binding of the DNA molecules can be obtained with a pretreated mica surface. 相似文献
193.
Enhanced detection of human immunodeficiency virus type 1-specific T-cell responses to highly variable regions by using peptides based on autologous virus sequences 下载免费PDF全文
Altfeld M Addo MM Shankarappa R Lee PK Allen TM Yu XG Rathod A Harlow J O'Sullivan K Johnston MN Goulder PJ Mullins JI Rosenberg ES Brander C Korber B Walker BD 《Journal of virology》2003,77(13):7330-7340
The antigenic diversity of human immunodeficiency virus type 1 (HIV-1) represents a significant challenge for vaccine design as well as the comprehensive assessment of HIV-1-specific immune responses in infected persons. In this study we assessed the impact of antigen variability on the characterization of HIV-1-specific T-cell responses by using an HIV-1 database to determine the sequence variability at each position in all expressed HIV-1 proteins and a comprehensive data set of CD8 T-cell responses to a reference strain of HIV-1 in infected persons. Gamma interferon Elispot analysis of HIV-1 clade B-specific T-cell responses to 504 overlapping peptides spanning the entire expressed HIV-1 genome derived from 57 infected subjects demonstrated that the average amino acid variability within a peptide (entropy) was inversely correlated to the measured frequency at which the peptide was recognized (P = 6 x 10(-7)). Subsequent studies in six persons to assess T-cell responses against p24 Gag, Tat, and Vpr peptides based on autologous virus sequences demonstrated that 29% (12 of 42) of targeted peptides were only detected with peptides representing the autologous virus strain compared to the HIV-1 clade B consensus sequence. The use of autologous peptides also allowed the detection of significantly stronger HIV-1-specific T-cell responses in the more variable regulatory and accessory HIV-1 proteins Tat and Vpr (P = 0.007). Taken together, these data indicate that accurate assessment of T-cell responses directed against the more variable regulatory and accessory HIV-1 proteins requires reagents based on autologous virus sequences. They also demonstrate that CD8 T-cell responses to the variable HIV-1 proteins are more common than previously reported. 相似文献
194.
While morphologic and biochemical aspects of degenerative joint disease (osteoarthritis [OA]) have been elucidated by numerous studies, the molecular mechanisms underlying the progressive loss of articular cartilage during OA development remain largely unknown. The main focus of the present study was to gain more insight into molecular changes during the very early stages of mechanically induced cartilage degeneration and to relate molecular alterations to histological changes at distinct localizations of the joint. Studies on human articular cartilage are hampered by the difficulty of obtaining normal tissue and early-stage OA tissue, and they allow no progressive follow-up. An experimental OA model in dogs with a slow natural history of OA (Pond-Nuki model) was therefore chosen. Anterior cruciate ligament transection (ACLT) was performed on 24 skeletally mature dogs to induce joint instability resulting in OA. Samples were taken from different joint areas after 6, 12, 24 and 48 weeks, and gene expression levels of common cartilage molecules were quantified in relation to the histological grading (modified Mankin score) of adjacent tissue. Histological changes reflected early progressive degenerative OA. Soon after ACLT, chondrocytes responded to the altered mechanical conditions by significant and stable elevation of collagen type II, collagen type I and YKL40 expression, which persisted throughout the study. In contrast to the mild to moderate histological alterations, these molecular changes were not progressive and were independent of the joint localization (tibia, femur, lateral, medial) and the extent of matrix degeneration. MMP13 remained unaltered until 24 weeks, and aggrecan and tenascinC remained unaltered until 48 weeks after ACLT. These findings indicate that elevated collagen type II, collagen type I and YKL40 mRNA expression levels are early and sensitive measures of ACLT-induced joint instability independent of a certain grade of morphological cartilage degeneration. A second phase of molecular changes in OA may begin around 48 weeks after ACLT with altered expression of further genes, such as MMP13, aggrecan and tenascin. Molecular changes observed in the present study suggest that dog cartilage responds to degenerative conditions by regulating the same genes in a similar direction as that observed for chondrocytes in late human OA. 相似文献
195.
Zheng Guo Tianwen Zhang Xia Li Qi Wang Jianzhen Xu Hui Yu Jing Zhu Haiyun Wang Chenguang Wang Eric J Topol Qing Wang Shaoqi Rao 《BMC bioinformatics》2005,6(1):1-12
Background
Despite the continuous production of genome sequence for a number of organisms, reliable, comprehensive, and cost effective gene prediction remains problematic. This is particularly true for genomes for which there is not a large collection of known gene sequences, such as the recently published chicken genome. We used the chicken sequence to test comparative and homology-based gene-finding methods followed by experimental validation as an effective genome annotation method.Results
We performed experimental evaluation by RT-PCR of three different computational gene finders, Ensembl, SGP2 and TWINSCAN, applied to the chicken genome. A Venn diagram was computed and each component of it was evaluated. The results showed that de novo comparative methods can identify up to about 700 chicken genes with no previous evidence of expression, and can correctly extend about 40% of homology-based predictions at the 5' end.Conclusions
De novo comparative gene prediction followed by experimental verification is effective at enhancing the annotation of the newly sequenced genomes provided by standard homology-based methods. 相似文献196.
We have discovered a ubiquitin (Ub)-specific cysteine protease encoded within the N-terminal approximately 500 residues of the UL36 gene product, the largest (3164 aa) tegument protein of herpes simplex virus 1 (HSV-1). Enzymatic activity of this fragment, UL36USP, is detectable only after cleavage of UL36USP from full-length UL36 and occurs late during viral replication. UL36USP bears no homology to known deubiquitinating enzymes (DUBs) or Ub binding proteins. Sequence alignment of the large tegument proteins across the family Herpesviridae indicates conservation of key catalytic residues amongst these viruses. Recombinant UL36USP exhibits hydrolytic activity toward Ub-AMC and ubiquitinated branched peptides in vitro. In addition, recombinant UL36USP can cleave polyUb chains and appears to be specific for Lys48 linkages. Mutation of the active site cysteine residue (Cys65) to alanine abolishes this enzymatic activity. The lack of homology between UL36USP and eukaryotic DUBs makes this new family of herpesvirus ubiquitin-specific proteases attractive targets for selective inhibition. 相似文献
197.
Fernando WA Leininger E Simkin J Li N Malcom CA Sathyamoorthi S Han M Muneoka K 《Developmental biology》2011,350(2):409-310
Amputation of the distal region of the terminal phalanx of mice causes an initial wound healing response followed by blastema formation and the regeneration of the digit tip. Thus far, most regeneration studies have focused in embryonic or neonatal models and few studies have examined adult digit regeneration. Here we report on studies that include morphological, immunohistological, and volumetric analyses of adult digit regeneration stages. The regenerated digit is grossly similar to the original, but is not a perfect replacement. Re-differentiation of the digit tip occurs by intramembranous ossification forming a trabecular bone network that replaces the amputated cortical bone. The digit blastema is comprised of proliferating cells that express vimentin, a general mesenchymal marker, and by comparison to mature tissues, contains fewer endothelial cells indicative of reduced vascularity. The majority of blastemal cells expressing the stem cell marker SCA-1, also co-express the endothelial marker CD31, suggesting the presence of endothelial progenitor cells. Epidermal closure during wound healing is very slow and is characterized by a failure of the wound epidermis to close across amputated bone. Instead, the wound healing phase is associated with an osteoclast response that degrades the stump bone allowing the wound epidermis to undercut the distal bone resulting in a novel re-amputation response. Thus, the regeneration process initiates from a level that is proximal to the original plane of amputation. 相似文献
198.
199.
Muro I Berry DL Huh JR Chen CH Huang H Yoo SJ Guo M Baehrecke EH Hay BA 《Development (Cambridge, England)》2006,133(17):3305-3315
Caspase family proteases play important roles in the regulation of apoptotic cell death. Initiator caspases are activated in response to death stimuli, and they transduce and amplify these signals by cleaving and thereby activating effector caspases. In Drosophila, the initiator caspase Nc (previously Dronc) cleaves and activates two short-prodomain caspases, Dcp-1 and Ice (previously Drice), suggesting these as candidate effectors of Nc killing activity. dcp-1-null mutants are healthy and possess few defects in normally occurring cell death. To explore roles for Ice in cell death, we generated and characterized an Ice null mutant. Animals lacking Ice show a number of defects in cell death, including those that occur during embryonic development, as well as during formation of adult eyes, arista and wings. Ice mutants exhibit subtle defects in the destruction of larval tissues, and do not prevent destruction of salivary glands during metamorphosis. Cells from Ice animals are also markedly resistant to several stresses, including X-irradiation and inhibition of protein synthesis. Mutations in Ice also suppress cell death that is induced by expression of Rpr, Wrinkled (previously Hid) and Grim. These observations demonstrate that Ice plays an important non-redundant role as a cell death effector. Finally, we demonstrate that Ice participates in, but is not absolutely required for, the non-apoptotic process of spermatid differentiation. 相似文献
200.