参考文献

X-linked dystonia-parkinsonism (XDP) is a hereditary neurodegenerative disorder involving a progressive loss of striatal medium spiny neurons. The mechanisms underlying neurodegeneration are not known,in part because there have been few cellular models available for studying the disease. The XDP haplotype consists of multiple sequence variations in a region of the X chromosome containingTAF1,a large gene with at least 38 exons,and a multiple transcript system (MTS) composed of five unconventional exons. A previous study identified an XDP-specific insertion of a SINE-VNTR-Alu (SVA)-type retrotransposon in intron 32 ofTAF1,as well as a neural-specific TAF1 isoform,N-TAF1,which showed decreased expression in post-mortem XDP brain compared with control tissue. Here,we generated XDP patient and control fibroblasts and induced pluripotent stem cells (iPSCs) in order to further probe cellular defects associated with this disease. As initial validation of the model,we compared expression ofTAF1and MTS transcripts in XDP versus control fibroblasts and iPSC-derived neural stem cells (NSCs). Compared with control cells,XDP fibroblasts exhibited decreased expression ofTAF1transcript fragments derived from exons 32-36,a region spanning the SVA insertion site. N-TAF1,which incorporates an alternative exon (exon 34'),was not expressed in fibroblasts,but was detectable in iPSC-differentiated NSCs at levels that were ∼threefold lower in XDP cells than in controls. These results support the previous findings that N-TAF1 expression is impaired in XDP,but additionally indicate that this aberrant transcription might occur in neural cells at relatively early stages of development that precede neurodegeneration. View Publication

In view of recent reports documenting pervasive translation outside of canonical protein-coding sequences,we wished to determine the proportion of major histocompatibility complex (MHC) class I-associated peptides (MAPs) derived from non-canonical reading frames. Here we perform proteogenomic analyses of MAPs eluted from human B cells using high-throughput mass spectrometry to probe the six-frame translation of the B-cell transcriptome. We report that ∼ 10% of MAPs originate from allegedly noncoding genomic sequences or exonic out-of-frame translation. The biogenesis and properties of these 'cryptic MAPs' differ from those of conventional MAPs. Cryptic MAPs come from very short proteins with atypical C termini,and are coded by transcripts bearing long 3'UTRs enriched in destabilizing elements. Relative to conventional MAPs,cryptic MAPs display different MHC class I-binding preferences and harbour more genomic polymorphisms,some of which are immunogenic. Cryptic MAPs increase the complexity of the MAP repertoire and enhance the scope of CD8 T-cell immunosurveillance. View Publication

Studies of repertoires of mouse monoclonal CD4(+) T cells have revealed several mechanisms of self-tolerance; however,which mechanisms operate in normal repertoires is unclear. Here we studied polyclonal CD4(+) T cells specific for green fluorescent protein expressed in various organs,which allowed us to determine the effects of specific expression patterns on the same epitope-specific T cells. Peptides presented uniformly by thymic antigen-presenting cells were tolerated by clonal deletion,whereas peptides excluded from the thymus were ignored. Peptides with limited thymic expression induced partial clonal deletion and impaired effector T cell potential but enhanced regulatory T cell potential. These mechanisms were also active for T cell populations specific for endogenously expressed self antigens. Thus,the immunotolerance of polyclonal CD4(+) T cells was maintained by distinct mechanisms,according to self-peptide expression patterns. View Publication

We have established a novel cell line,designated as TF-1,from a patient with erythroleukemia,which showed complete growth dependency on granulocyte-macrophage colony-stimulating factor (GM-CSF) or on interleukin-3 (IL-3) and carried a homogeneous chromosomal abnormality (54X). Erythropoietin (EPO) also sustained the short-term growth of TF-1,but did not induce erythroid differentiation. These three hematopoietic growth factors acted on TF-1 synergistically. Transforming growth factor-beta and interferons inhibited the factor-dependent growth of TF-1 cells in a dose-dependent fashion,and monocyte-colony stimulating factor and interkeukin-1 enhanced the GM-CSF-dependent growth of TF-1. Ultrastructural studies revealed some very immature features in this cell line. Although TF-1 cells do not express glycophorin A or carbonyl anhydrase I,the morphological and cytochemical features,and the constitutive expression of globin genes,indicate the commitment of TF-1 to erythroid lineage. When induced to differentiate,TF-1 entered two different pathways. Specifically,hemin and delta-aminolevulinic acid induced hemoglobin synthesis,whereas TPA induced dramatic differentiation of TF-1 into macrophage-like cells. In summary,TF-1 is a cell line of immature erythroid origin that requires GM-CSF,IL-3,or EPO for its growth and that has the ability to undergo differentiation into either more mature erythroid cells or into macrophage-like cells. TF-1 is a useful tool for analyzing the human receptors for IL-3,GM-CSF,and EPO or the signal transduction of these hemopoietic growth factors. View Publication

Mutations in RB and PTEN are linked to castration resistance and poor prognosis in prostate cancer. Identification of genes that are regulated by these tumor suppressors in a context that recapitulates cancer progression may be beneficial for discovering novel therapeutic targets. Although various genetically engineered mice thus far provided tumor models with various pathological stages,they are not ideal for detecting dynamic changes in gene transcription. Additionally,it is difficult to achieve an effect specific to tumor progression via gain of functions of these genes. In this study,we developed an in vitro model to help identify RB- and PTEN-loss signatures during the malignant progression of prostate cancers. Trp53(-/-) ; Rb(f/f),Trp53(-/-) ; Pten(f/f),and Trp53(-/-) ; Rb(f/f) ; Pten(f/f) prostate epithelial cells were infected with AD-LacZ or AD-Cre. We found that deletion of Rb,Pten or both stimulated prostasphere formation and tumor development in immune-compromised mice. The GO analysis of genes affected by the deletion of Rb or Pten in Trp53(-/-) prostate epithelial cells identified a number of genes encoding cytokines,chemokines and extracellular matrix remodeling factors,but only few genes related to cell cycle progression. Two genes (Il-6 and Lox) were further analyzed. Blockade of Il-6 signaling and depletion of Lox significantly attenuated prostasphere formation in 3D culture,and in the case of IL-6,strongly suppressed tumor growth in vivo. These findings suggest that our in vitro model may be instrumental in identifying novel therapeutic targets of prostate cancer progression,and further underscore IL-6 and LOX as promising therapeutic targets. textcopyright 2015 Wiley Periodicals,Inc. View Publication

The epigenetics of early commitment to embryonal cardiomyocyte is poorly understood. In this work,we compared the effect of thyroid hormone and that of anacardic acid,a naturally occurring histone acetylase inhibitor,or both in combination,on mouse embryonic stem cells (mES) differentiating into embryonal cardiomyocyte by embryoid bodies (EBs) formation. Although the results indicated that anacardic acid (AA) and thyroid hormone were both efficient in promoting cardiomyocyte differentiation,we noticed that a transient exposure of mES to AA alone was sufficient to enlarge the beating areas of EBs compared to those of untreated controls. This effect was associated with changes in the chromatin structure at the promoters of specific cardiomyogenic genes. Among them,a rapid induction of the transcription factor Castor 1 (CASZ1),important for cardiomyocytes differentiation and maturation during embryonic development,was observed in the presence of AA. In contrast,thyroid hormone (T 3) was more effective in stimulating spontaneous firing,thus suggesting a role in the production of a population of cardiomyocyte with pacemaker properties. In conclusion,AA and thyroid hormone both enhanced cardiomyocyte formation along in apparently distinct pathways. View Publication

An 85- to 95 kDa class of lymphocyte surface molecules,defined in man by antibodies of the Hermes series,is involved in lymphocyte binding to high endothelial venules and is likely of central importance in the process of lymphocyte homing. In this report,we have examined the relationship between these Hermes-defined homing-receptors" and two other 80 to 95 kDa lymphocyte surface molecules that have been extensively studied--CD44 [In(Lu)-related p80] defined by mAb A1G3 and A3D8� View Publication

The bioorthogonal keto group has attracted interest for the site-specific chemical conjugation of recombinant proteins under mild conditions,e.g. with aminooxy-functionalised fluorescent probes,radiometal chelates,toxins or polymers. However,the cotranslational incorporation of the corresponding non-canonical amino acid p-acetyl-L-phenylalanine (Apa) into proteins expressed in Escherichia coli by means of amber suppression using a previously described system with a mutated tRNA and an engineered tyrosyl-tRNA synthetase from Methanococcus jannaschii shows limited efficiency and considerable promiscuity towards endogenous amino acids. Employing a one-plasmid system that encodes all three components required for selection,i.e. the modified aminoacyl-tRNA synthetase (aaRS),the cognate amber suppressor tRNA and the enhanced green fluorescent protein equipped with an amber stop codon and serving as reporter,we have generated an Apa-specific aaRS&tRNA pair with considerably improved efficiency (17-fold increased expression) and also fidelity (6-fold). To this end,both the aaRS and the tRNA were subjected to doped random mutagenesis and selection in altogether four evolutionary cycles using fluorescence-activated bacterial cell sorting as well as automated screening of microcultures. The resulting aaRS&tRNA pair was applied to the functionalisation of an Anticalin with specificity towards oncofetal fibronectin by introducing a keto group at a permissible site for subsequent conjugation with a fluorescent dye,thus allowing visualisation of this tumour target under the microscope. View Publication

There is little information on the function of epididymal basal cells. These cells secrete prostaglandins,can metabolize radical oxygen species,and have apical projections that are components of the blood-epididymis barrier. The objective of this study was to develop a reproducible protocol to isolate rat epididymal basal cells and to characterize their function by gene expression profiling. Integrin-alpha6 was used to isolate a highly purified population of basal cells. Microarray analysis indicated that expression levels of 552 genes were enriched in basal cells relative to other cell types. Among these genes,45 were expressed at levels of 5-fold or greater. These highly expressed genes coded for proteins implicated in cell adhesion,cytoskeletal function,ion transport,cellular signaling,and epidermal function,and included proteases and antiproteases,signal transduction,and transcription factors. Several highly expressed genes have been reported in adult stem cells,suggesting that basal cells may represent an epididymal stem cell population. A basal cell culture was established that showed that these basal cells can differentiate in vitro from keratin (KRT) 5-positive cells to cells that express KRT8 and connexin 26,a marker of columnar cells. These data provide novel information on epididymal basal cell gene expression and suggest that these cells can act as adult stem cells. View Publication

Amphipterygium adstringens is a plant traditionally used to treat gingivitis,gastric ulcer and even gastric cancer but the mechanism involved in the regulation of the immune response is not elucidated yet. The 6-pentadecylsalicylic acid (6SA) is the main anacardic acid found in A. adstringens. In order to evaluate the immune-modulatory abilities of 6SA,we used mouse splenocytes and determined the phosphorylation of the transcription factor NF-κB and MAP kinases ERK1/2,JNK and p38 in helper and cytotoxic T cells,natural killer (NK) cells and F4/80(+) macrophages. Treatment with 6SA was not cytotoxic as measured by both trypan blue exclusion and tetrazolium salts (MTT) tests. Additionally,6SA did not alter the proportion of helper and cytotoxic T lymphocytes,NK cells or macrophages. Moreover,6SA treatment significantly increased the phosphorylation of ERK1/2,JNK,P38 and NF-κB mainly in macrophages. In this cells (peritoneal macrophages),treatment with 6SA increased the secretion of nitric oxide (NO),interleukin (IL)-6 and tumour necrosis factor (TNF)-α and decreased the secretion of IL-4 and IL-10 depending on MAPK and NF-κB phosphorylation. In addition,6SA increased the migration and phagocytic activity of macrophages also depending on the phosphorylation of different kinases. These data suggest that 6SA induces the classical activation pathway in macrophages via the phosphorylation of MAP kinases and NF-κB thus activating the adaptive immune system. View Publication