Preparation of 169Yb Calibration Sources for the Measurement of Electron Anti-Neutrino Mass

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NOD × 129.H2g7 Backcross Delineates 129S1/SvImJ-Derived Genomic Regions Modulating Type 1 Diabetes Development in Mice

OBJECTIVE: Introduction of genes targeted in 129/Sv embryonic stem (ES) cells into NOD mice brings about linked genes that may modulate type 1 diabetes. Our objective was to identify 129S1/SvJ non-MHC regions contributing type 1 diabetes resistance or susceptibility in backcross to NOD/LtJ. RESEARCH DESIGN AND METHODS: After congenic transfer of the NOD H2(g7) haplotype onto 129S1/Sv, 310 females were produced by NOD x (NOD x 129.H2(g7))F1 backcross (N2). A genome scan for quantitative trait locus (QTL) affecting clinical diabetes, age of diabetes onset, and insulitis severity was performed using subphenotype characteristics to improve power and resolution for detection of diabetes susceptibility loci. RESULTS: Thirty-six of 310 (11.6%) N2 females developed type 1 diabetes between 14 and 40 weeks. Significant evidence of linkage for only a single previously reported Idd complex locus (Idd10/17/18, chromosome [Chr] 3) was indicated for clinical diabetes. The quantitative traits of insulitis either alone or combined with age at type 1 diabetes onset were significantly linked to known Idd regions on Chr 1 (Idd5 region), Chr 4 (Idd9 region), Chr 8 (Idd22), Chr 11 (Idd4.3), and proximal Chr 17 (Idd16 region). Significant 129S1/Sv resistance contributions were identified on Chr 1, 15 (two loci), and 19, with suggestive evidence for additional novel 129/Sv resistance QTL on Chr 5 and 17 and susceptibility on Chr 2. CONCLUSIONS: The 129S1/SvJ genome harbors collections of both known and potentially novel non-MHC Idd loci. Investigators targeting 129/Sv genes mapping within chromosomal regions reported herein or elsewhere in the genome need to exclude potential contributions from linked Idd loci by generating a NOD.129 control strain expressing the nontargeted allele

Generation of Stable Pluripotent Stem Cells From NOD Mouse Tail-Tip Fibroblasts

OBJECTIVE: The NOD mouse strain has been widely used to investigate the pathology and genetic susceptibility for type 1 diabetes. Induced pluripotent stem cells (iPSCs) derived from this unique mouse strain would enable new strategies for investigating type 1 diabetes pathogenesis and potential therapeutic targets. The objective of this study was to determine whether somatic fibroblasts from NOD mice could be reprogrammed to become iPSCs, providing an alternative source of stem cells for the production of genetically modified NOD cells and mice. RESEARCH DESIGN AND METHODS: Adult tail-tip fibroblasts from male NOD mice were reprogrammed by retroviral transduction of the coding sequences of three transcription factors, OCT4, SOX2, and KLF4, in combination with a histone deacetylase inhibitor, valproic acid. RESULTS: Eighteen NOD iPSC lines were generated, and three of these cell lines were further characterized. All three cell lines exhibited silencing of the three reprogramming transgenes and reactivation of endogenous pluripotent markers (OCT4, SOX2, NANOG, REX1, and SSEA1). These NOD iPSCs readily differentiated in vitro to form embryoid bodies and in vivo by teratoma formation in immunodeficient mice. Moreover, NOD iPSCs were successfully transfected with a reporter transgene and were capable of contributing to the inner cell mass of C57BL/6 blastocysts, leading to the generation of a chimeric mouse. CONCLUSIONS: Adult tail-tip fibroblasts from NOD mice can be reprogrammed, without constitutive ectopic expression of transcription factors, to produce iPSCs that exhibit classic mouse embryonic stem cell (ESC) features. These NOD iPSCs can be maintained and propagated under normal ESC culture conditions to produce genetically altered cell lines, differentiated cells, and chimeric mice

Sfrp Controls Apicobasal Polarity and Oriented Cell Division in Developing Gut Epithelium

Epithelial tubular morphogenesis leading to alteration of organ shape has important physiological consequences. However, little is known regarding the mechanisms that govern epithelial tube morphogenesis. Here, we show that inactivation of Sfrp1 and Sfrp2 leads to reduction in fore-stomach length in mouse embryos, which is enhanced in the presence of the Sfrp5 mutation. In the mono-cell layer of fore-stomach epithelium, cell division is normally oriented along the cephalocaudal axis; in contrast, orientation diverges in the Sfrps-deficient fore-stomach. Cell growth and apoptosis are not affected in the Sfrps-deficient fore-stomach epithelium. Similarly, cell division orientation in fore-stomach epithelium diverges as a result of inactivation of either Stbm/Vangl2, an Fz/PCP component, or Wnt5a. These observations indicate that the oriented cell division, which is controlled by the Fz/PCP pathway, is one of essential components in fore-stomach morphogenesis. Additionally, the small intestine epithelium of Sfrps compound mutants fails to maintain proper apicobasal polarity; the defect was also observed in Wnt5a-inactivated small intestine. In relation to these findings, Sfrp1 physically interacts with Wnt5a and inhibits Wnt5a signaling. We propose that Sfrp regulation of Wnt5a signaling controls oriented cell division and apicobasal polarity in the epithelium of developing gut

Wnt expression is not correlated with β-catenin dysregulation in Dupuytren's Disease

BACKGROUND: Dupuytren's contracture or disease (DD) is a fibro-proliferative disease of the hand that results in finger flexion contractures. Increased cellular β-catenin levels have been identified as characteristic of this disease. As Wnts are the most widely recognized upstream regulators of cellular β-catenin accumulation, we have examined Wnt gene expression in surgical specimens and in DD-derived primary cell cultures grown in two-dimensional monolayer culture or in three-dimensional FPCL collagen lattice cultures. RESULTS: The Wnt expression profile of patient-matched DD and unaffected control palmar fascia tissue was determined by a variety of complimentary methods; Affymetrix Microarray analysis, specific Wnt and degenerative primer-based Reverse Transcriptase (RT)-PCR, and Real Time PCR. Microarray analysis identified 13 Wnts associated with DD and control tissues. Degenerate Wnt RT-PCR analysis identified Wnts 10b and 11, and to a lesser extent 5a and 9a, as the major Wnt family members expressed in our patient samples. Competitive RT-PCR analysis identified significant differences between the levels of expression of Wnts 9a, 10b and 11 in tissue samples and in primary cell cultures grown as monolayer or in FPCL, where the mRNA levels in tissue > FPCL cultures > monolayer cultures. Real Time PCR data confirmed the down-regulation of Wnt 11 mRNA in DD while Wnt 10b, the most frequently isolated Wnt in DD and control palmar fascia, displayed widely variable expression between the methods of analysis. CONCLUSION: These data indicate that changes in Wnt expression per se are unlikely to be the cause of the observed dysregulation of β-catenin expression in DD

Cadherin–catenin expression in primary colorectal cancer: a survival analysis

Both cell adhesion and cell signalling events are mediated by components of the cadherin-catenin complex. Loss of expression of the components of this complex have been shown to correlate with invasive behaviour in many tumour types although their exact role in colorectal cancer remains unclear. Immunohistochemical analysis of the expression of components of the cadherin-catenin complex in colorectal cancers from 60 patients was undertaken. Loss of memberanous expression of E-cadherin, alpha-catenin and beta-catenin was demonstrated in 52%, 85% and 40% of tumours respectively. Focal nuclear expression of beta-catenin ( 75% of tumour cells per section) was seen in 11 (18%) tumours. Loss of membranous alpha-catenin expression significantly correlated with tumour de-differentiation (P = 0.009). There was a trend towards an association between advanced tumour stage and loss of membranous expression of alpha-catenin or beta-catenin, although these associations were not statistically significant. Univariate analysis revealed that advanced Dukes' stage, tumour de-differentiation, loss of membranous beta-catenin expression, cytoplasmic beta-catenin expression and widespread nuclear expression of beta-catenin all correlated with short survival following apparently curative resection of the primary tumour. However, only Dukes' stage (P = 0.002), tumour grade (P = 0.02) and widespread nuclear expression of beta-catenin (P = 0.002) were independent predictors of short survival. Disturbed growth signalling events in colorectal tumours are thought to result in nuclear accumulation of beta-catenin. Consequently, tumours with widespread nuclear expression of beta-catenin are likely to have severely abnormal growth characteristics, and which therefore might be predictive of short survival in these patients

Regulation of Classical Cadherin Membrane Expression and F-Actin Assembly by Alpha-Catenins, during Xenopus Embryogenesis

Alpha (α)-E-catenin is a component of the cadherin complex, and has long been thought to provide a link between cell surface cadherins and the actin skeleton. More recently, it has also been implicated in mechano-sensing, and in the control of tissue size. Here we use the early Xenopus embryos to explore functional differences between two α-catenin family members, α-E- and α-N-catenin, and their interactions with the different classical cadherins that appear as tissues of the embryo become segregated from each other. We show that they play both cadherin-specific and context-specific roles in the emerging tissues of the embryo. α-E-catenin interacts with both C- and E-cadherin. It is specifically required for junctional localization of C-cadherin, but not of E-cadherin or N-cadherin at the neurula stage. α-N-cadherin interacts only with, and is specifically required for junctional localization of, N-cadherin. In addition, α -E-catenin is essential for normal tissue size control in the non-neural ectoderm, but not in the neural ectoderm or the blastula. We also show context specificity in cadherin/ α-catenin interactions. E-cadherin requires α-E-catenin for junctional localization in some tissues, but not in others, during early development. These specific functional cadherin/alpha-catenin interactions may explain the basis of cadherin specificity of actin assembly and morphogenetic movements seen previously in the neural and non-neural ectoderm

Genomic structure and alternative splicing of murine R2B receptor protein tyrosine phosphatases (PTPκ, μ, ρ and PCP-2)

BACKGROUND: Four genes designated as PTPRK (PTPκ), PTPRL/U (PCP-2), PTPRM (PTPμ) and PTPRT (PTPρ) code for a subfamily (type R2B) of receptor protein tyrosine phosphatases (RPTPs) uniquely characterized by the presence of an N-terminal MAM domain. These transmembrane molecules have been implicated in homophilic cell adhesion. In the human, the PTPRK gene is located on chromosome 6, PTPRL/U on 1, PTPRM on 18 and PTPRT on 20. In the mouse, the four genes ptprk, ptprl, ptprm and ptprt are located in syntenic regions of chromosomes 10, 4, 17 and 2, respectively. RESULTS: The genomic organization of murine R2B RPTP genes is described. The four genes varied greatly in size ranging from ~64 kb to ~1 Mb, primarily due to proportional differences in intron lengths. Although there were also minor variations in exon length, the number of exons and the phases of exon/intron junctions were highly conserved. In situ hybridization with digoxigenin-labeled cRNA probes was used to localize each of the four R2B transcripts to specific cell types within the murine central nervous system. Phylogenetic analysis of complete sequences indicated that PTPρ and PTPμ were most closely related, followed by PTPκ. The most distant family member was PCP-2. Alignment of RPTP polypeptide sequences predicted putative alternatively spliced exons. PCR experiments revealed that five of these exons were alternatively spliced, and that each of the four phosphatases incorporated them differently. The greatest variability in genomic organization and the majority of alternatively spliced exons were observed in the juxtamembrane domain, a region critical for the regulation of signal transduction. CONCLUSIONS: Comparison of the four R2B RPTP genes revealed virtually identical principles of genomic organization, despite great disparities in gene size due to variations in intron length. Although subtle differences in exon length were also observed, it is likely that functional differences among these genes arise from the specific combinations of exons generated by alternative splicing