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151.
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153.
Castilleja A Ward NE O'Brian CA Swearingen B Swan E Gillogly MA Murray JL Kudelka AP Gershenson DM Ioannides CG 《Molecular and cellular biochemistry》2001,217(1-2):21-33
We investigated the ubiquitination and degradation of a tumor antigen, the HER-2/neu (HER-2) protooncogene product which is overexpressed in epithelial cancers. HER-2 degradation was investigated in the ovarian tumor line, SKOV3.A2, that constitutively overexpressed long-life HER-2. We used as agonist geldanamycin (GA), which initiated downmodulation of HER-2 from the cell surface. HER-2 was polyubiquitinated and degraded faster in the presence than in the absence of GA. GA did not decrease HLA-A2 expression. Presentation of the immunodominant cytotoxic T lymphocyte (CTL) epitope, E75 (369–377) from SKOV.A2 was inhibited by proteasome inhibitors, such as LLnL but was enhanced by cysteine protease inhibitors such as E64, indicating that both the proteasome and cysteine proteases are involved in epitope formation but have different effects. Enhanced tumor recognition was not an immediate or early effect of GA treatment, but was evident after 20 h of GA treatment. In contrast, 20 h GA treatment did not increase tumor sensitivity to LAK cell lysis. Twenty hour GA-treated SKOV3.A2 cells expressed an unstable HER-2 protein synthesized in the presence of GA, of faster electrophoretic mobility than control HER-2. This suggested that the newly synthesized HER-2 in the presence of GA was the main source of epitopes recognized by CTL. Twenty hour GA-treated SKOV3.A2 cells were better inducers of CTL activity directed to a number of HER-2 CTL epitopes, in peripheral blood mononuclear cells compared with control untreated SKOV3.A2 cells. Thus, induction of HER-2 protein instability enhanced the sensitivity of tumor for CTL lysis. Increased HER-2 CTL epitopes presentation may have implications for overcoming the poor immuno-genicity of human tumors, and design of epitope precursors for cancer vaccination. 相似文献
154.
PCR-Denaturing Gradient Gel Electrophoresis Profiling of Inter- and Intraspecies 18S rRNA Gene Sequence Heterogeneity Is an Accurate and Sensitive Method To Assess Species Diversity of Arbuscular Mycorrhizal Fungi of the Genus Gigaspora 总被引:1,自引:0,他引:1 下载免费PDF全文
Francisco A. de Souza George A. Kowalchuk Paula Leeflang Johannes A. van Veen Eric Smit 《Applied microbiology》2004,70(3):1413-1424
Despite the importance of arbuscular mycorrhizal fungi in the majority of terrestrial ecosystems, their ecology, genetics, and evolution are poorly understood, partly due to difficulties associated with detecting and identifying species. We explored the inter- and intraspecies variations of the 18S rRNA genes of the genus Gigaspora to assess the use of this marker for the discrimination of Gigaspora isolates and of Gigasporaceae populations from environmental samples. Screening of 48 Gigaspora isolates by PCR-denaturing gradient gel electrophoresis (DGGE) revealed that the V3-V4 region of the 18S rRNA gene contained insufficient variation to discriminate between different Gigaspora species. In contrast, the patterns of 18S ribosomal DNA (rDNA) heterogeneity within the V9 region of this marker could be used for reliable identification of all recognized species within this genus. PCR-DGGE patterns provided insight into some putative misidentifications and could be used to differentiate geographic isolates of G. albida, G. gigantea, and G. margarita but not G. rosea. Two major clusters were apparent based upon PCR-DGGE ribotype patterns, one containing G. albida, G. candida, G. ramisporophora, and G. rosea and the other containing G. decipiens and G. margarita. Dissection of the DGGE patterns by cloning, DGGE screening, and sequencing confirmed these groupings and revealed that some ribotypes were shared across species boundaries. Of the 48 isolates examined, only two displayed any spore-to-spore variation, and these exceptions may be indicative of coisolation of more than one species or subspecies within these cultures. Two Brazilian agricultural soils were also analyzed with a Gigasporaceae-specific nested PCR approach, revealing a dominance of G. margarita within this family. 相似文献
155.
Leslie Redpath J Bengtsson U DeSimone J Lao X Wang X Stanbridge EJ 《Radiation research》2003,159(1):57-71
We have studied the impact of TP53 status on the extent and nature of chromosome damage seen in human skin fibroblasts after gamma irradiation beyond the G1-phase checkpoint but prior to the G2-phase checkpoint. Mitotic cells were examined in the absence and presence of treatment with nocodazole and the yield of aberrations was scored as a function of time postirradiation. The results revealed substantially greater damage in the absence of nocodazole, indicating that damage was being masked in its presence. While metaphase aberrations were seen exclusively in the presence of nocodazole, anaphase aberrations were seen principally in its absence. Furthermore, these were mostly of an unseparated, or "sticky", type that showed separation of the chromatids in the centromeric region, indicating normal degradation of cohesin, with retention of adhesion further out on the chromatid arms. Using postirradiation BrdU labeling and the absence of nocodazole, we were able to identify mitotic figures up to the third postirradiation mitosis. Analysis of the data revealed that in cells wild-type for TP53 the aberrant anaphases were lost after the first postirradiation mitosis, although they were still found in gradually decreasing amounts into the second and third postirradiation mitoses in E6-expressing cells. The data indicate that the formation of these sticky anaphases is independent of TP53 status, an observation that is consistent with the TP53 independence of transient G2-phase arrest. However, the consequences of the formation of these lesions appear to be very different. In the case of cells wild-type for TP53 this is chronic G1-phase arrest, while in E6 cells it is anaphase catastrophe. 相似文献
156.
Eric S. J. Harris Shugeng Cao Sean D. Schoville Chengming Dong Wenquan Wang Zaiyou Jian Zhongzhen Zhao David M. Eisenberg Jon Clardy 《PloS one》2012,7(11)
Oridonin is a diterpenoid with anti-cancer activity that occurs in the Chinese medicinal plant Isodon rubescens and some related species. While the bioactivity of oridonin has been well studied, the extent of natural variation in the production of this compound is poorly known. This study characterizes natural variation in oridonin production in order to guide selection of populations of Isodon with highest oridonin yield. Different populations of I. rubescens and related species were collected in China, and their offspring were grown in a greenhouse. Samples were examined for oridonin content, genotyped using 11 microsatellites, and representatives were sequenced for three phylogenetic markers (ITS, rps16, trnL-trnF). Oridonin production was mapped on a molecular phylogeny of the genus Isodon using samples from each population as well as previously published Genbank sequences. Oridonin has been reported in 12 out of 74 species of Isodon examined for diterpenoids, and the phylogeny indicates that oridonin production has arisen at least three times in the genus. Oridonin production was surprisingly consistent between wild-collected parents and greenhouse-grown offspring, despite evidence of gene flow between oridonin-producing and non-producing populations of Isodon. Additionally, microsatellite genetic distance between individuals was significantly correlated with chemical distance in both parents and offspring. Neither heritability nor correlation with genetic distance were significant when the comparison was restricted to only populations of I. rubescens, but this result should be corroborated using additional samples. Based on these results, future screening of Isodon populations for oridonin yield should initially prioritize a broad survey of all species known to produce oridonin, rather than focusing on multiple populations of one species, such as I. rubescens. Of the samples examined here, I. rubescens or I. japonicus from Henan province would provide the best source of oridonin. 相似文献
157.
Disentangling environmental and host sources of fungal endophyte communities in an experimental beachgrass study 下载免费PDF全文
Disentangling the ecological factors that contribute to the assembly of the microbial symbiont communities within eukaryotic hosts is an ongoing challenge. Broadly speaking, symbiont propagules arrive either from external sources in the environment or from internal sources within the same host individual. To understand the relative importance of these propagule sources to symbiont community assembly, we characterized symbiotic fungal endophyte communities within the roots of three species of beachgrass in a field experiment. We manipulated two aspects of the external environment, successional habitat and physical disturbance. To determine the role of internal sources of propagules for endophyte community assembly, we used beachgrass individuals with different pre‐existing endophyte communities. Endophyte species richness and community composition were characterized using culture‐based and next‐generation sequencing approaches. Our results showed that external propagule sources associated with successional habitat, but not disturbance, were particularly important for colonization of most endophytic taxa. In contrast, internal propagule sources played a minor role for most endophytic taxa but were important for colonization by the dominant taxon Microdochium bolleyi. Our findings highlight the power of manipulative field experiments to link symbiont community assembly to its underlying ecological processes, and to ultimately improve predictions of symbiont community assembly across environments. 相似文献
158.
Charpentier TH Wilder PT Liriano MA Varney KM Pozharski E MacKerell AD Coop A Toth EA Weber DJ 《Journal of molecular biology》2008,382(1):56-73
As part of an effort to inhibit S100B, structures of pentamidine (Pnt) bound to Ca2+-loaded and Zn2+,Ca2+-loaded S100B were determined by X-ray crystallography at 2.15 Å (Rfree = 0.266) and 1.85 Å (Rfree = 0.243) resolution, respectively. These data were compared to X-ray structures solved in the absence of Pnt, including Ca2+-loaded S100B and Zn2+,Ca2+-loaded S100B determined here (1.88 Å; Rfree = 0.267). In the presence and absence of Zn2+, electron density corresponding to two Pnt molecules per S100B subunit was mapped for both drug-bound structures. One Pnt binding site (site 1) was adjacent to a p53 peptide binding site on S100B (± Zn2+), and the second Pnt molecule was mapped to the dimer interface (site 2; ± Zn2+) and in a pocket near residues that define the Zn2+ binding site on S100B. In addition, a conformational change in S100B was observed upon the addition of Zn2+ to Ca2+-S100B, which changed the conformation and orientation of Pnt bound to sites 1 and 2 of Pnt-Zn2+,Ca2+-S100B when compared to Pnt-Ca2+-S100B. That Pnt can adapt to this Zn2+-dependent conformational change was unexpected and provides a new mode for S100B inhibition by this drug. These data will be useful for developing novel inhibitors of both Ca2+- and Ca2+,Zn2+-bound S100B. 相似文献
159.
Rutherford NJ Zhang YJ Baker M Gass JM Finch NA Xu YF Stewart H Kelley BJ Kuntz K Crook RJ Sreedharan J Vance C Sorenson E Lippa C Bigio EH Geschwind DH Knopman DS Mitsumoto H Petersen RC Cashman NR Hutton M Shaw CE Boylan KB Boeve B Graff-Radford NR Wszolek ZK Caselli RJ Dickson DW Mackenzie IR Petrucelli L Rademakers R 《PLoS genetics》2008,4(9):e1000193
The TAR DNA-binding protein 43 (TDP-43) has been identified as the major disease protein in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin inclusions (FTLD-U), defining a novel class of neurodegenerative conditions: the TDP-43 proteinopathies. The first pathogenic mutations in the gene encoding TDP-43 (TARDBP) were recently reported in familial and sporadic ALS patients, supporting a direct role for TDP-43 in neurodegeneration. In this study, we report the identification and functional analyses of two novel and one known mutation in TARDBP that we identified as a result of extensive mutation analyses in a cohort of 296 patients with variable neurodegenerative diseases associated with TDP-43 histopathology. Three different heterozygous missense mutations in exon 6 of TARDBP (p.M337V, p.N345K, and p.I383V) were identified in the analysis of 92 familial ALS patients (3.3%), while no mutations were detected in 24 patients with sporadic ALS or 180 patients with other TDP-43-positive neurodegenerative diseases. The presence of p.M337V, p.N345K, and p.I383V was excluded in 825 controls and 652 additional sporadic ALS patients. All three mutations affect highly conserved amino acid residues in the C-terminal part of TDP-43 known to be involved in protein-protein interactions. Biochemical analysis of TDP-43 in ALS patient cell lines revealed a substantial increase in caspase cleaved fragments, including the approximately 25 kDa fragment, compared to control cell lines. Our findings support TARDBP mutations as a cause of ALS. Based on the specific C-terminal location of the mutations and the accumulation of a smaller C-terminal fragment, we speculate that TARDBP mutations may cause a toxic gain of function through novel protein interactions or intracellular accumulation of TDP-43 fragments leading to apoptosis. 相似文献
160.
Topoisomerase IIIalpha is required for normal proliferation and telomere stability in alternative lengthening of telomeres 下载免费PDF全文
Temime-Smaali N Guittat L Wenner T Bayart E Douarre C Gomez D Giraud-Panis MJ Londono-Vallejo A Gilson E Amor-Guéret M Riou JF 《The EMBO journal》2008,27(10):1513-1524
Topoisomerase (Topo) IIIalpha associates with BLM helicase, which is proposed to be important in the alternative lengthening of telomeres (ALT) pathway that allows telomere recombination in the absence of telomerase. Here, we show that human Topo IIIalpha colocalizes with telomeric proteins at ALT-associated promyelocytic bodies from ALT cells. In these cells, Topo IIIalpha immunoprecipitated with telomere binding protein (TRF) 2 and BLM and was shown to be associated with telomeric DNA by chromatin immunoprecipitation, suggesting that these proteins form a complex at telomere sequences. Topo IIIalpha depletion by small interfering RNA reduced ALT cell survival, but did not affect telomerase-positive cell lines. Moreover, repression of Topo IIIalpha expression in ALT cells reduced the levels of TRF2 and BLM proteins, provoked a strong increase in the formation of anaphase bridges, induced the degradation of the G-overhang signal, and resulted in the appearance of DNA damage at telomeres. In contrast, telomere maintenance and TRF2 levels were unaffected in telomerase-positive cells. We conclude that Topo IIIalpha is an important telomere-associated factor, essential for telomere maintenance and chromosome stability in ALT cells, and speculate on its potential mechanistic function. 相似文献