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Gilgenkrantz, H. „Inactivation de l'X : un gène actif spécifique de l'X inactif ?“ médecine/sciences 7, Nr. 4 (1991): 375. http://dx.doi.org/10.4267/10608/4363.
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Fontés, M. „Empreinte génomique parentale et inactivation de l'X : l'antisens a t-il un sens ?“ médecine/sciences 15, Nr. 11 (1999): 1277. http://dx.doi.org/10.4267/10608/1256.
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Byun, Do-Sun, Naseem Ahmed, Shannon Nasser, Joongho Shin, Sheren Al-Obaidi, Sanjay Goel, Georgia A. Corner et al. „Intestinal epithelial-specific PTEN inactivation results in tumor formation“. American Journal of Physiology-Gastrointestinal and Liver Physiology 301, Nr. 5 (November 2011): G856—G864. http://dx.doi.org/10.1152/ajpgi.00178.2011.
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Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a negative regulator of phosphatidylinositol 3-kinase (PI3K) signaling that is frequently inactivated in colorectal cancer through mutation, loss of heterozygosity, or epigenetic mechanisms. The aim of this study was to determine the effect of intestinal-specific PTEN inactivation on intestinal epithelial homeostasis and tumorigenesis. PTEN was deleted specifically in the intestinal epithelium, by crossing PTEN Lox/ Lox mice with villin Cre mice. PTEN was robustly expressed in the intestinal epithelium and maximally in the differentiated cell compartment. Targeted inactivation of PTEN in the intestinal epithelium of PTEN Lox/ Lox/villin Cre mice was confirmed by genotyping, immunohistochemistry, and qPCR. While intestinal-specific PTEN deletion did not have a major effect on cell fate determination or proliferation in the small intestine, it did increase phosphorylated (p) protein kinase B (AKT) expression in the intestinal epithelium, and 19% of animals developed small intestinal adenomas and adenocarcinomas at 12 mo of age. These tumors demonstrated pAKT and nuclear β-catenin staining, indicating simultaneous activation of the PI3K/AKT and Wnt signaling pathways. These findings demonstrate that, while PTEN inactivation alone has a minimal effect on intestinal homeostasis, it can facilitate tumor promotion upon deregulation of β-catenin/TCF signaling, further establishing PTEN as a bona fide tumor suppressor gene in intestinal cancer.
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Staiculescu, Marius Catalin, Jungsil Kim, Robert P. Mecham und Jessica E. Wagenseil. „Mechanical behavior and matrisome gene expression in the aneurysm-prone thoracic aorta of newborn lysyl oxidase knockout mice“. American Journal of Physiology-Heart and Circulatory Physiology 313, Nr. 2 (01.08.2017): H446—H456. http://dx.doi.org/10.1152/ajpheart.00712.2016.
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Mutations in lysyl oxidase (LOX) are associated with thoracic aortic aneurysm and dissection (TAAD). Mice that do not express Lox ( Lox−/−) die soon after birth and have 60% and 40% reductions in elastin- and collagen-specific cross-links, respectively. LOX inactivation could also change the expression of secreted factors, the structural matrix, and matrix-associated proteins that constitute the aortic matrisome. We hypothesized that absence of Lox will change the mechanical behavior of the aortic wall because of reduced elastin and collagen cross-linking and alter the expression levels of matrisome and smooth muscle cell (SMC) genes in a vascular location-specific manner. Using fluorescence microscopy, pressure myography, and gene set enrichment analysis, we visualized the microarchitecture, quantified the mechanical behavior, and examined matrisome and SMC gene expression from ascending aortas (AAs) and descending aortas (DAs) from newborn Lox+/+ and Lox−/− mice. Even though Lox−/− AAs and DAs have fragmented elastic laminae and disorganized SMCs, the unloaded outer diameter and wall thickness were similar to Lox+/+ AAs and DAs. Lox−/− AAs and DAs have altered opening angles, circumferential stresses, and circumferential stretch ratios; however, only Lox−/− AAs have increased pressurized diameters and tangent moduli. Gene set enrichment analysis showed upregulation of the extracellular matrix (ECM) regulator gene set in Lox−/− AAs and DAs as well as differential expression of secreted factors, collagens, ECM-affiliated proteins, ECM glycoproteins, and SMC cell cycle gene sets that depend on the Lox genotype and vascular location. These results provide insights into the local chemomechanical changes induced by Lox inactivation that may be important for TAAD pathogenesis. NEW & NOTEWORTHY Absence of lysyl oxidase ( Lox) causes thoracic aortic aneurysms. The aortic mechanical behavior of Lox−/− mice is consistent with reduced elastin and collagen cross-linking but demonstrates vascular location-specific differences. Lox−/− aortas show upregulation of matrix remodeling genes and location-specific differential expression of other matrix and smooth muscle cell gene sets.
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Appelmelk, Ben J., M. Celeste Martino, Eveline Veenhof, Mario A. Monteiro, Janneke J. Maaskant, Riccardo Negrini, Frank Lindh, Malcolm Perry, Giuseppe Del Giudice und Christina M. J. E. Vandenbroucke-Grauls. „Phase Variation in H Type I and Lewis a Epitopes ofHelicobacter pylori Lipopolysaccharide“. Infection and Immunity 68, Nr. 10 (01.10.2000): 5928–32. http://dx.doi.org/10.1128/iai.68.10.5928-5932.2000.
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ABSTRACT Helicobacter pylori NCTC 11637 lipopolysaccharide (LPS) expresses the human blood group antigens Lewis x (Lex), Ley, and H type I. In this report, we demonstrate that the H type I epitope displays high-frequency phase variation. One variant expressed Lex and Ley and no H type I as determined by serology; this switch was reversible. Insertional mutagenesis in NCTC 11637 of JHP563 (a poly(C) tract containing an open reading frame homologous to glycosyltransferases) yielded a transformant with a serotype similar to the phase variant. Structural analysis of the NCTC 11637 LPS confirmed the loss of the H type I epitope. Sequencing of JHP563 in strains NCTC 11637, an H type I-negative variant, and an H type I-positive switchback variant showed a C14 (gene on), C13 (gene off), and C14 tract, respectively. Inactivation of strain G27, which expresses Lex, Ley, H type I, and Lea, yielded a transformant that expressed Lex and Ley. We conclude that JHP563 encodes a β3-galactosyltransferase involved in the biosynthesis of H type I and Lea and that phase variation in H type I is due to C-tract changes in this gene. A second H type I-negative variant (variant 3a) expressed Lex and Lea and had lost both H type I and Leyexpression. Inactivation of HP093-HP094 resulted in a transformant expressing Lex and lacking Ley and H type I. Structural analysis of a mutant LPS confirmed the serological data. We conclude that the HP093-HP094 α2-fucosyltransferase (α2-FucT) gene product is involved in the biosynthesis of both Ley and Lex. Finally, we inactivated HP0379 in strain 3a. The transformant had lost both Lex and Leaexpression, which demonstrates that the HP0379 gene product is both an α3- and an α4-FucT. Our data provide understanding at the molecular level of how H. pylori is able to diversify in the host, a requirement likely essential for successful colonization and transmission.
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Gao, Xiquan, Marion Brodhagen, Tom Isakeit, Sigal Horowitz Brown, Cornelia Göbel, Javier Betran, Ivo Feussner, Nancy P. Keller und Michael V. Kolomiets. „Inactivation of the Lipoxygenase ZmLOX3 Increases Susceptibility of Maize to Aspergillus spp.“ Molecular Plant-Microbe Interactions® 22, Nr. 2 (Februar 2009): 222–31. http://dx.doi.org/10.1094/mpmi-22-2-0222.
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Plant and fungal lipoxygenases (LOX) catalyze the oxidation of polyunsaturated fatty acids, creating fatty-acid hydroperoxides (oxylipins). Fungal oxylipins are required for normal fungal development and secondary metabolism, and plant host–derived oxylipins interfere with these processes in fungi, presumably by signal mimicry. The maize LOX gene ZmLOX3 has been implicated previously in seed-Aspergillus interactions, so we tested the interactions of a mutant maize line (lox3-4, in which ZmLOX3 is disrupted) with the mycotoxigenic seed-infecting fungi Aspergillus flavus and Aspergillus nidulans. The lox3-4 mutant was more susceptible than wild-type maize to both Aspergillus species. All strains of A. flavus and A. nidulans produced more conidia and aflatoxin (or the precursor sterigmatocystin) on lox3-4 kernels than on wild-type kernels, in vitro and under field conditions. Although oxylipins did not differ detectably between A. flavus–infected kernels of the lox3-4 and wild-type (WT) maize, oxylipin precursors (free fatty acids) and a downstream metabolite (jasmonic acid) accumulated to greater levels in lox3-4 than in WT kernels. The increased resistance of the lox3-4 mutant to other fungal pathogens (Fusarium, Colletotrichum, Cochliobolus, and Exserohilum spp.) is in sharp contrast to results described herein for Aspergillus spp., suggesting that outcomes of LOX-governed host-pathogen interactions are pathogen-specific.
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McGrail, Maura, Fang Liu, Sekhar Kambakam und Zhitao Ming. „Abstract PR-010: Asc1lb progenitor-specific RB conditional inactivation in zebrafish models rare CNS primitive neuroectodermal tumors“. Cancer Research 84, Nr. 5_Supplement_1 (04.03.2024): PR—010—PR—010. http://dx.doi.org/10.1158/1538-7445.brain23-pr-010.
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Abstract We generated a new zebrafish model of human brain cancer by Cre/lox regulated conditional inactivation of the tumor suppressor RB in embryonic neural progenitors. An ascl1b progenitor Cre driver was used to inactivate RB by Cre recombination of an intronic floxed mRFP gene trap cassette, leading to mRFP expression and lineage labeling of RB mutated cells. Beginning at 6 months of age adult fish developed poorly differentiated brain tumors, similar to our previous zebrafish brain tumor model generated by CRISPR targeting of RB in embryos. Tumor transcriptome analysis revealed a proneural transcription factor molecular signature of the olig2+/sox10+ human CNS primitive neuroectodermal tumor subtype. Overexpression of chromatin remodelers suggest epigenetic mechanisms may contribute to tumorigenesis. Our model will allow sorting of RB mutant cells from the earliest stage of mutagenesis in the embryo to induction of cellular transformation in juveniles. With this approach we can profile the epigenetic time course of cellular transformation and reveal new insight into the molecular mechanisms driving PNET oncogenesis. Our approach to model cancer in fish with Cre/lox conditional inactivation can be applied to other cell lineages and brain tumor types. Citation Format: Maura McGrail, Fang Liu, Sekhar Kambakam, Zhitao Ming. Asc1lb progenitor-specific RB conditional inactivation in zebrafish models rare CNS primitive neuroectodermal tumors [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr PR-010.
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Epp, Nikolas, Gerhard Fürstenberger, Karsten Müller, Silvia de Juanes, Michael Leitges, Ingrid Hausser, Florian Thieme, Gerhard Liebisch, Gerd Schmitz und Peter Krieg. „12R-lipoxygenase deficiency disrupts epidermal barrier function“. Journal of Cell Biology 177, Nr. 1 (02.04.2007): 173–82. http://dx.doi.org/10.1083/jcb.200612116.
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12R-lipoxygenase (12R-LOX) and the epidermal LOX-3 (eLOX-3) constitute a novel LOX pathway involved in terminal differentiation in skin. This view is supported by recent studies showing that inactivating mutations in 12R-LOX and eLOX-3 are linked to the development of autosomal recessive congenital ichthyosis. We show that 12R-LOX deficiency in mice results in a severe impairment of skin barrier function. Loss of barrier function occurs without alterations in proliferation and stratified organization of the keratinocytes, but is associated with ultrastructural anomalies in the upper granular layer, suggesting perturbance of the assembly/extrusion of lamellar bodies. Cornified envelopes from skin of 12R-LOX–deficient mice show increased fragility. Lipid analysis demonstrates a disordered composition of ceramides, in particular a decrease of ester-bound ceramide species. Moreover, processing of profilaggrin to monomeric filaggrin is impaired. This study indicates that the 12R-LOX–eLOX-3 pathway plays a key role in the process of epidermal barrier acquisition by affecting lipid metabolism, as well as protein processing.
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Dionellis, Vasilis S., Maxim Norkin, Angeliki Karamichali, Giacomo G. Rossetti, Joerg Huelsken, Paloma Ordonez-Moran und Thanos D. Halazonetis. „Genomic Instability Profiles at the Single Cell Level in Mouse Colorectal Cancers of Defined Genotypes“. Cancers 13, Nr. 6 (12.03.2021): 1267. http://dx.doi.org/10.3390/cancers13061267.
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The genomes of many human CRCs have been sequenced, revealing a large number of genetic alterations. However, the molecular mechanisms underlying the accumulation of these alterations are still being debated. In this study, we examined colorectal tumours that developed in mice with Apclox/lox, LSL-KrasG12D, and Tp53lox/lox targetable alleles. Organoids were derived from single cells and the spectrum of mutations was determined by exome sequencing. The number of single nucleotide substitutions (SNSs) correlated with the age of the tumour, but was unaffected by the number of targeted cancer-driver genes. Thus, tumours that expressed mutant Apc, Kras, and Tp53 alleles had as many SNSs as tumours that expressed only mutant Apc. In contrast, the presence of large-scale (>10 Mb) copy number alterations (CNAs) correlated strongly with Tp53 inactivation. Comparison of the SNSs and CNAs present in organoids derived from the same tumour revealed intratumoural heterogeneity consistent with genomic lesions accumulating at significantly higher rates in tumour cells compared to normal cells. The rate of acquisition of SNSs increased from the early stages of cancer development, whereas large-scale CNAs accumulated later, after Tp53 inactivation. Thus, a significant fraction of the genomic instability present in cancer cells cannot be explained by aging processes occurring in normal cells before oncogenic transformation.
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Liu, Lei, Huichun Tong und Xiuzhu Dong. „Function of the Pyruvate Oxidase-Lactate Oxidase Cascade in Interspecies Competition between Streptococcus oligofermentans and Streptococcus mutans“. Applied and Environmental Microbiology 78, Nr. 7 (27.01.2012): 2120–27. http://dx.doi.org/10.1128/aem.07539-11.
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ABSTRACTComplex interspecies interactions occur constantly between oral commensals and the opportunistic pathogenStreptococcus mutansin dental plaque. Previously, we showed that oral commensalStreptococcus oligofermentanspossesses multiple enzymes for H2O2production, especially lactate oxidase (Lox), allowing it to out-competeS. mutans. In this study, through extensive biochemical and genetic studies, we identified a pyruvate oxidase (pox) gene inS. oligofermentans. Apoxdeletion mutant completely lost Pox activity, while ectopically expressedpoxrestored activity. Pox was determined to produce most of the H2O2in the earlier growth phase and log phase, while Lox mainly contributed to H2O2production in stationary phase. Bothpoxandloxwere expressed throughout the growth phase, while expression of theloxgene increased by about 2.5-fold when cells entered stationary phase. Since lactate accumulation occurred to a large degree in stationary phase, the differential Pox- and Lox-generated H2O2can be attributed to differential gene expression and substrate availability. Interestingly, inactivation ofpoxcauses a dramatic reduction in H2O2production from lactate, suggesting a synergistic action of the two oxidases in converting lactate into H2O2. In anin vitrotwo-species biofilm experiment, thepoxmutant ofS. oligofermentansfailed to inhibitS. mutanseven thoughloxwas active. In summary,S. oligofermentansdevelops a Pox-Lox synergy strategy to maximize its H2O2formation so as to win the interspecies competition.
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Heaps, Cristine L., Janet L. Parker, Michael Sturek und Douglas K. Bowles. „Altered calcium sensitivity contributes to enhanced contractility of collateral-dependent coronary arteries“. Journal of Applied Physiology 97, Nr. 1 (Juli 2004): 310–16. http://dx.doi.org/10.1152/japplphysiol.01400.2003.
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Coronary arteries distal to chronic occlusion exhibit enhanced vasoconstriction and impaired relaxation compared with nonoccluded arteries. In this study, we tested the hypotheses that an increase in peak Ca2+ channel current density and/or increased Ca2+ sensitivity contributes to altered contractility in collateral-dependent coronary arteries. Ameroid occluders were surgically placed around the proximal left circumflex coronary artery (LCX) of female miniature swine. Segments of epicardial arteries (∼1 mm luminal diameter) were isolated from the LCX and nonoccluded left anterior descending (LAD) arteries 24 wk after Ameroid placement. Contractile responses to depolarization (10–100 mM KCl) were significantly enhanced in LCX compared with size-matched LAD arterial rings [concentration of KCl causing 50% of the maximal contractile response (EC50); LAD = 41.7 ± 2.3, LCX = 34.3 ± 2.7 mM]. However, peak Ca2+ channel current was not altered in isolated smooth muscle cells from LCX compared with LAD (−5.29 ± 0.42 vs. −5.68 ± 0.55 pA/pF, respectively). Furthermore, whereas half-maximal activation of Ca2+ channel current occurred at nearly the same membrane potential in LAD and LCX, half-maximal inactivation was shifted to a more positive membrane potential in LCX cells. Simultaneous measures of contractile tension and intracellular free Ca2+ (fura 2) levels in arterial rings revealed that significantly more tension was produced per unit change in fura 2 ratio in LCX compared with LAD in response to KCl but not during receptor-agonist stimulation with endothelin-1. Taken together, our data indicate that coronary arteries distal to chronic occlusion display increased Ca2+ sensitivity in response to high KCl-induced depolarization, independent of changes in whole cell peak Ca2+ channel current. Unaltered Ca2+ sensitivity in endothelin-stimulated arteries suggests more than one mechanism regulating Ca2+ sensitization in coronary smooth muscle.
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Kasi, Pashtoon Murtaza, Jessica Kim Lee, Hanna Tukachinsky, Lincoln W. Pasquina, Pierre Vanden Borre, Brennan James Decker, Dean C. Pavlick et al. „Liquid-biopsy detection of FGFR2 and other actionable rearrangements in GI malignancies.“ Journal of Clinical Oncology 41, Nr. 16_suppl (01.06.2023): 4085. http://dx.doi.org/10.1200/jco.2023.41.16_suppl.4085.
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4085 Background: Actionable rearrangements (RE) represent an emerging therapeutic target for GI malignancies. However, there remains uncertainty whether liquid biopsy (LBx) assays testing circulating tumor DNA (ctDNA) can detect RE with the same fidelity as tissue biopsy. Here we report the performance of ctDNA-based comprehensive genomic profiling (CGP) for the detection of RE leveraging an FDA-approved >300-gene platform, with a focus on data from patients with GI malignancies. Methods: An institutional research database of clinical CGP results for tissue biopsy (TBx, FoundationOneCDx) and LBx (FoundationOneLiquid CDx) from patients with cancers of the GI tract was reviewed. Sensitivity for FGFR2 RE was studied in patients with cholangiocarcinoma (CCA) or carcinoma of unknown primary (CUP) and both TBx and LBx CGP available. Results: Across 7870 GI LBx, 1094 predicted pathogenic RE were detected in 826 cases (10%) including 283 oncogenic kinase RE, 686 inactivating RE, and 125 other gain-of-function RE. FGFR2 was the most frequently rearranged gene, enriched in CCA (4.4%, 37/833) and stomach (3.0%, 11/368). EGFR RE (38) occurred across 6 cancer types and included 8 fusions, 15 c-terminal truncations, 4 intragenic deletions/inversions, and 3 kinase domain duplications. BRAF RE (31) were detected among in colorectal (22) and pancreas (9) cases. LBx detected 44 exon 3 skipping (protein stabilizing) RE in CTNNB1 (beta-catenin) across all samples. Other frequent activating RE were detected in MYC (36), FGFR3, RET, ROS1 (21 each), and ALK (13). Potentially targetable inactivating RE were detected in BRCA1 (28), ARID1A (24), STK11 (22), PTEN (17), TSC2 (14), and BRCA2 (8). Focusing on CCA, FGFR2 RE were more prevalent in cases with elevated (≥1%) ctDNA tumor fraction (TF, 26/342, 7.6%), matching the prevalence in TBx (7.8%, 505/6,492) with a similar spectrum of fusion partners seen for both TBx and LBx (30% BICC1, 70% rare for both). In 13 CCA/CUP patients positive for FGFR2 RE by TBx, LBx detected 12 (92%) including 8 different fusion partners; the false negative case had TF <1%. In 11 CCA LBx samples with FGFR2 resistance mutations detected, LBx CGP successfully detected a driver FGFR2 RE in 10 and driver C382R mutation in 1. Conclusions: Rearrangements are represented across GI malignancies, including many that result in known oncogenic drivers that may be targetable with available therapies. We observe reliable detection of FGFR2 fusions in ctDNA, in contrast to some prior publications. ctDNA represents a pragmatic analyte for detection of rearrangements and other actionable alterations, offering timely result return when tissue is inadequate or unavailable. [Table: see text]
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Raab, Sabine, Heike Beck, Andreas Gaumann, Ali Yüce, Hans-Peter Gerber, Karl Plate, Hans-Peter Hammes, Napoleone Ferrara und Georg Breier. „Impaired brain angiogenesis and neuronal apoptosis induced by conditional homozygous inactivation of vascular endothelial growth factor“. Thrombosis and Haemostasis 91, Nr. 03 (2004): 595–605. http://dx.doi.org/10.1160/th03-09-0582.
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SummaryVascular endothelial growth factor (VEGF) is essential for the differentiation of the primitive embryonic vascular system and has been implicated in the vascularization of organs. Recently, VEGF has also been proposed to play a role in neural development, neuroprotection, and adult neurogenesis. Here we have investigated the function of VEGF in the developing brain by cre-lox technology. We show that VEGF produced by the embryonic neuroectoderm is required for the vascularization and the development of the brain. Both the invasion and the directed growth of capillaries were severely impaired in the fore-, midand hindbrain of VEGFlox/lox/nestin-cre mouse embryos homozygous for a VEGF mutation in the neural tube. These observations demonstrate thatVEGF, via local secretion by neural progenitors, induces brain angiogenesis and guides the growth of capillaries toward the ventricular zone. VEGF deficiency led to developmental retardation and progressive destruction of neural tissue in all brain regions.The defect was most pronounced in telencephalic structures, such as the hippocampus, and caused microcephaly.Taken together, the findings establish the critical importance of neuroectoderm-derived VEGF in the morphogenesis of the brain. VEGF acts as a key regulator of brain angiogenesis and provides instructive cues for the correct spatial organization of the vasculature.
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Amić, Ana, Jasmina M. Dimitrić Marković, Zoran Marković, Dejan Milenković, Žiko Milanović, Marko Antonijević, Denisa Mastiľák Cagardová und Jaime Rodríguez-Guerra Pedregal. „Theoretical Study of Radical Inactivation, LOX Inhibition, and Iron Chelation: The Role of Ferulic Acid in Skin Protection against UVA Induced Oxidative Stress“. Antioxidants 10, Nr. 8 (18.08.2021): 1303. http://dx.doi.org/10.3390/antiox10081303.
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Ferulic acid (FA) is used in skin formulations for protection against the damaging actions of the reactive oxygen species (ROS) produced by UVA radiation. Possible underlying protective mechanisms are not fully elucidated. By considering the kinetics of proton-coupled electron transfer (PCET) and radical-radical coupling (RRC) mechanisms, it appears that direct scavenging could be operative, providing that a high local concentration of FA is present at the place of •OH generation. The resulting FA phenoxyl radical, after the scavenging of a second •OH and keto-enol tautomerization of the intermediate, produces 5-hydroxyferulic acid (5OHFA). Inhibition of the lipoxygenase (LOX) enzyme, one of the enzymes that catalyse free radical production, by FA and 5OHFA were analysed. Results of molecular docking calculations indicate favourable binding interactions of FA and 5OHFA with the LOX active site. The exergonicity of chelation reactions of the catalytic Fe2+ ion with FA and 5OHFA indicate the potency of these chelators to prevent the formation of •OH radicals via Fenton-like reactions. The inhibition of the prooxidant LOX enzyme could be more relevant mechanism of skin protection against UVA induced oxidative stress than iron chelation and assumed direct scavenging of ROS.
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Teder, Tarvi, Stefanie König, Rajkumar Singh, Bengt Samuelsson, Oliver Werz, Ulrike Garscha und Jesper Z. Haeggström. „Modulation of the 5-Lipoxygenase Pathway by Chalcogen-Containing Inhibitors of Leukotriene A4 Hydrolase“. International Journal of Molecular Sciences 24, Nr. 8 (19.04.2023): 7539. http://dx.doi.org/10.3390/ijms24087539.
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The 5-lipoxygenase (5-LOX) pathway gives rise to bioactive inflammatory lipid mediators, such as leukotrienes (LTs). 5-LOX carries out the oxygenation of arachidonic acid to the 5-hydroperoxy derivative and then to the leukotriene A4 epoxide which is converted to a chemotactic leukotriene B4 (LTB4) by leukotriene A4 hydrolase (LTA4H). In addition, LTA4H possesses aminopeptidase activity to cleave the N-terminal proline of a pro-inflammatory tripeptide, prolyl-glycyl-proline (PGP). Based on the structural characteristics of LTA4H, it is possible to selectively inhibit the epoxide hydrolase activity while sparing the inactivating, peptidolytic, cleavage of PGP. In the current study, chalcogen-containing compounds, 4-(4-benzylphenyl) thiazol-2-amine (ARM1) and its selenazole (TTSe) and oxazole (TTO) derivatives were characterized regarding their inhibitory and binding properties. All three compounds selectively inhibit the epoxide hydrolase activity of LTA4H at low micromolar concentrations, while sparing the aminopeptidase activity. These inhibitors also block the 5-LOX activity in leukocytes and have distinct inhibition constants with recombinant 5-LOX. Furthermore, high-resolution structures of LTA4H with inhibitors were determined and potential binding sites to 5-LOX were proposed. In conclusion, we present chalcogen-containing inhibitors which differentially target essential steps in the biosynthetic route for LTB4 and can potentially be used as modulators of inflammatory response by the 5-LOX pathway.
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Ortiz, Maria Eugenia, Marta Inés Bühler und Liliana Isabel Zelarayán. „Involvement of PLA2, COX and LOX in Rhinella arenarum oocyte maturation“. Zygote 22, Nr. 4 (27.02.2013): 440–45. http://dx.doi.org/10.1017/s096719941200069x.
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SummaryIn Rhinella arenarum, progesterone is the physiological nuclear maturation inducer that interacts with the oocyte surface and starts a cascade of events that leads to germinal vesicle breakdown (GVBD). Polyunsaturated fatty acids and their metabolites produced through cyclooxygenase (COX) and lipoxygenase (LOX) pathways play an important role in reproductive processes. In amphibians, to date, the role of arachidonic acid (AA) metabolites in progesterone (P4)-induced oocyte maturation has not been clarified. In this work we studied the participation of three enzymes involved in AA metabolism – phospholipase A2 (PLA2), COX and LOX in Rhinella arenarum oocyte maturation. PLA2 activation induced maturation in Rhinella arenarum oocytes in a dose-dependent manner. Oocytes when treated with 0.08 μM melittin showed the highest response (78 ± 6% GVBD). In follicles, PLA2 activation did not significantly induce maturation at the assayed doses (12 ± 3% GVBD). PLA2 inhibition with quinacrine prevented melittin-induced GVBD in a dose-dependent manner, however PLA2 inactivation did not affect P4-induced maturation. This finding suggests that PLA2 is not the only phospholipase involved in P4-induced maturation in this species. P4-induced oocyte maturation was inhibited by the COX inhibitors indomethacin and rofecoxib (65 ± 3% and 63 ± 3% GVBD, respectively), although COX activity was never blocked by their addition. Follicles showed a similar response following the addition of these inhibitors. Participation of LOX metabolites in maturation seems to be correlated with seasonal variation in ovarian response to P4. During the February to June period (low P4 response), LOX inhibition by nordihydroguaiaretic acid or lysine clonixinate increased maturation by up to 70%. In contrast, during the July to January period (high P4 response), LOX inhibition had no effect on hormone-induced maturation.
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Appelmelk, Ben J., Steve L. Martin, Mario A. Monteiro, Chris A. Clayton, Andrew A. McColm, Pengyuan Zheng, Theo Verboom et al. „Phase Variation in Helicobacter pyloriLipopolysaccharide due to Changes in the Lengths of Poly(C) Tracts in α3-Fucosyltransferase Genes“. Infection and Immunity 67, Nr. 10 (01.10.1999): 5361–66. http://dx.doi.org/10.1128/iai.67.10.5361-5366.1999.
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ABSTRACT The lipopolysaccharide (LPS) of Helicobacter pyloriexpresses the Lewis x (Lex) and/or Ley antigen. We have shown previously that H. pylori LPS displays phase variation whereby an Lex-positive strain yields variants with different LPS serotypes, for example, Lex plus Ley or nonfucosylated polylactosamine. H. pylori has two α3-fucosyltransferase genes that both contain poly(C) tracts. We now demonstrate that these tracts can shorten or lengthen randomly, which results in reversible frameshifting and inactivation of the gene products. We provide genetic and serological evidence that this mechanism causes H. pylori LPS phase variation and demonstrate that the on or off status of α3-fucosyltransferase genes determines the LPS serotypes of phase variants and clinical isolates. The role of the α3-fucosyltransferase gene products in determining the LPS serotype was confirmed by structural-chemical analysis of α3-fucosyltransferase knockout mutants. The data also show that the two α3-fucosyltransferase genes code for enzymes with different fine specificities, and we propose the names futA and futB to designate the orthologs of the H. pylori 26695 α3-fucosyltransferase genes HP0379 and HP0651, respectively. The data also show that the α3-fucosylation in H. pylori precedes α3-fucosyltransferase, an order of events opposite to that which prevails in mammals. Finally, the data provide an understanding at the molecular level of the mechanisms underlying LPS diversity in H. pylori, which may play an important role in adaptation to the host.
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Taşoyan, İzlem Cansu, und Elif Turabi Yolacaner. „Physical Properties of Some Soy Powders and Functional and Sensory Properties of Milk Chocolates Prepared with These Powders“. Turkish Journal of Agriculture - Food Science and Technology 11, Nr. 2 (28.02.2023): 246–57. http://dx.doi.org/10.24925/turjaf.v11i2.246-257.5444.
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Soybean is a nutritious crop commonly used for food enrichment due to its rich nutritional content and valuable functional characteristics. Soy products and also soymilk are potential ingredients for substitution of milk powder in food products with respect to providing high nutritious quality, lowering production cost and being an alternative for vegan and vegetarian diets. In this study, soymilk powder and soy protein isolate were added to milk chocolate to obtain a functional food product. Some chemical, physical and functional properties of powder ingredients were determined. Soymilk powder was found to have 44.43 % protein, 18.14 % fat and 6.06 % ash content. According to the chemical analysis, inactivation of 99.1 % for LOX-1, 100 % for LOX-3 and 98.5 % for trypsin inhibitors was achieved by heat treatment of 98 °C for 20 minutes. Functionality of chocolates was evaluated in terms of total phenolic content and total antioxidant capacity. The results were significantly higher than the literature data. Considering all results in terms of functionality, it can be stated that soymilk powder and soy protein isolate can be added to milk chocolate in order to obtain a functional food product.
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Terpstra, Leonieke, Josée Prud'homme, Alice Arabian, Shu Takeda, Gérard Karsenty, Shoukat Dedhar und René St-Arnaud. „Reduced chondrocyte proliferation and chondrodysplasia in mice lacking the integrin-linked kinase in chondrocytes“. Journal of Cell Biology 162, Nr. 1 (30.06.2003): 139–48. http://dx.doi.org/10.1083/jcb.200302066.
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Chondrocyte proliferation and differentiation requires their attachment to the collagen type II–rich matrix of developing bone. This interaction is mediated by integrins and their cytoplasmic effectors, such as the integrin-linked kinase (ILK). To elucidate the molecular mechanisms whereby integrins control these processes, we have specifically inactivated the ILK gene in growth plate chondrocytes using the Cre-lox methodology. Mice carrying an ILK allele flanked by loxP sites (ILK-fl) were crossed to transgenic mice expressing the Cre recombinase under the control of the collagen type II promoter. Inactivation of both copies of the ILK-fl allele lead to a chondrodysplasia characterized by a disorganized growth plate and to dwarfism. Expression of chondrocyte differentiation markers such as collagen type II, collagen type X, Indian hedgehog and the PTH-PTHrP receptor was normal in ILK-deficient growth plates. In contrast, chondrocyte proliferation, assessed by BrdU or proliferating cell nuclear antigen labeling, was markedly reduced in the mutant growth plates. Cell-based assays showed that integrin-mediated adhesion of primary cultures of chondrocytes from mutant animals to collagen type II was impaired. ILK inactivation in chondrocytes resulted in reduced cyclin D1 expression, and this most likely explains the defect in chondrocyte proliferation observed when ILK is inactivated in growth plate cells.
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Leitner, Gerda C., Gerhard Hagn, Laura Niederstaetter, Andrea Bileck, Kerstin Plessl-Walder, Michaela Horvath, Vera Kolovratova et al. „INTERCEPT Pathogen Reduction in Platelet Concentrates, in Contrast to Gamma Irradiation, Induces the Formation of trans-Arachidonic Acids and Affects Eicosanoid Release during Storage“. Biomolecules 12, Nr. 9 (07.09.2022): 1258. http://dx.doi.org/10.3390/biom12091258.
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Pathogen inactivation techniques for blood products have been implemented to optimize clinically safe blood components supply. The INTERCEPT system uses amotosalen together with ultraviolet light wavelength A (UVA) irradiation. Irradiation-induced inactivation of nucleic acids may actually be accompanied by modifications of chemically reactive polyunsaturated fatty acids known to be important mediators of platelet functions. Thus, here, we investigated eicosanoids and the related fatty acids released upon treatment and during storage of platelet concentrates for 7 days, complemented by the analysis of functional and metabolic consequences of these treatments. Metabolic and functional issues like glucose consumption, lactate formation, platelet aggregation, and clot firmness hardly differed between the two treatment groups. In contrast to gamma irradiation, here, we demonstrated that INTERCEPT treatment immediately caused new formation of trans-arachidonic acid isoforms, while 11-hydroxyeicosatetraenoic acid (11-HETE) and 15-HETE were increased and two hydroperoxyoctadecadienoic acid (HpODE) isoforms decreased. During further storage, these alterations remained stable, while the release of 12-lipoxygenase (12-LOX) products such as 12-HETE and 12-hydroxyeicosapentaenoic acid (12-HEPE) was further attenuated. In vitro synthesis of trans-arachidonic acid isoforms suggested that thiol radicals formed by UVA treatment may be responsible for the INTERCEPT-specific effects observed in platelet concentrates. It is reasonable to assume that UVA-induced molecules may have specific biological effects which need to be further investigated.
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Poulsen, Søren Brandt, Jeppe Praetorius, Helle H. Damkier, Lance Miller, Raoul D. Nelson, Edith Hummler und Birgitte Mønster Christensen. „Reducing αENaC expression in the kidney connecting tubule induces pseudohypoaldosteronism type 1 symptoms during K+ loading“. American Journal of Physiology-Renal Physiology 310, Nr. 4 (15.02.2016): F300—F310. http://dx.doi.org/10.1152/ajprenal.00258.2015.
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Genetic inactivation of the epithelial Na+ channel α-subunit (αENaC) in the renal collecting duct (CD) does not interfere with Na+ and K+ homeostasis in mice. However, inactivation in the CD and a part of the connecting tubule (CNT) induces autosomal recessive pseudohypoaldosteronism type 1 (PHA-1) symptoms in subjects already on a standard diet. In the present study, we further examined the importance of αENaC in the CNT. Knockout mice with αENaC deleted primarily in a part of the CNT (CNT-KO) were generated using Scnn1alox/lox mice and Atp6v1b1:: Cre mice. With a standard diet, plasma Na+ concentration ([Na+]) and [K+], and urine Na+ and K+ output were unaffected. Seven days of Na+ restriction (0.01% Na+) led to a higher urine Na+ output only on days 3–5, and after 7 days plasma [Na+] and [K+] were unaffected. In contrast, the CNT-KO mice were highly susceptible to a 2-day 5% K+ diet and showed lower food intake and relative body weight, lower plasma [Na+], higher fractional excretion (FE) of Na+, higher plasma [K+], and lower FE of K+. The higher FE of Na+ coincided with lower abundance and phosphorylation of the Na+-Cl− cotransporter. In conclusion, reducing ENaC expression in the CNT induces clear PHA-1 symptoms during high dietary K+ loading.
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Canalis, Ernesto, Stefano Zanotti, Wesley G. Beamer, Aris N. Economides und Anna Smerdel-Ramoya. „Connective Tissue Growth Factor Is Required for Skeletal Development and Postnatal Skeletal Homeostasis in Male Mice“. Endocrinology 151, Nr. 8 (09.06.2010): 3490–501. http://dx.doi.org/10.1210/en.2010-0145.
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Connective tissue growth factor (CTGF), a member of the cysteine-rich 61 (Cyr 61), CTGF, nephroblastoma overexpressed (NOV) (CCN) family of proteins, is synthesized by osteoblasts, and its overexpression inhibits osteoblastogenesis and causes osteopenia. The global inactivation of Ctgf leads to defective endochondral bone formation and perinatal lethality; therefore, the consequences of Ctgf inactivation on the postnatal skeleton are not known. To study the function of CTGF, we generated Ctgf+/LacZ heterozygous null mice and tissue-specific null Ctgf mice by mating Ctgf conditional mice, where Ctgf is flanked by lox sequences with mice expressing the Cre recombinase under the control of the paired-related homeobox gene 1 (Prx1) enhancer (Prx1-Cre) or the osteocalcin promoter (Oc-Cre). Ctgf+/LacZ heterozygous mice exhibited transient osteopenia at 1 month of age secondary to decreased trabecular number. A similar osteopenic phenotype was observed in 1-month-old Ctgf conditional null male mice generated with Prx1-Cre, suggesting that the decreased trabecular number was secondary to impaired endochondral bone formation. In contrast, when the conditional deletion of Ctgf was achieved by Oc-Cre, an osteopenic phenotype was observed only in 6-month-old male mice. Osteoblast and osteoclast number, bone formation, and eroded surface were not affected in Ctgf heterozygous or conditional null mice. In conclusion, CTGF is necessary for normal skeletal development but to a lesser extent for postnatal skeletal homeostasis.
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Klochendler-Yeivin, Agnes, Eli Picarsky und Moshe Yaniv. „Increased DNA Damage Sensitivity and Apoptosis in Cells Lacking the Snf5/Ini1 Subunit of the SWI/SNF Chromatin Remodeling Complex“. Molecular and Cellular Biology 26, Nr. 7 (01.04.2006): 2661–74. http://dx.doi.org/10.1128/mcb.26.7.2661-2674.2006.
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ABSTRACT The gene encoding the SNF5/Ini1 core subunit of the SWI/SNF chromatin remodeling complex is a tumor suppressor in humans and mice, with an essential role in early embryonic development. To investigate further the function of this gene, we have generated a Cre/lox-conditional mouse line. We demonstrate that Snf5 deletion in primary fibroblasts impairs cell proliferation and survival without the expected derepression of most retinoblastoma protein-controlled, E2F-responsive genes. Furthermore, Snf5-deficient cells are hypersensitive to genotoxic stress, display increased aberrant mitotic features, and accumulate phosphorylated p53, leading to elevated expression of a specific subset of p53 target genes, suggesting a role for Snf5 in the DNA damage response. p53 inactivation does not rescue the proliferation defect caused by Snf5 deficiency but reduces apoptosis and strongly accelerates tumor formation in Snf5-heterozygous mice.
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Salahudeen, Ameen Abdulla, Xingnan Li, Michael Cantrell und Calvin Jay Kuo. „Gastrointestinal organoid cultures for functional evaluation of oncogenic loci.“ Journal of Clinical Oncology 33, Nr. 3_suppl (20.01.2015): 85. http://dx.doi.org/10.1200/jco.2015.33.3_suppl.85.
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85 Background: Novel in vitro methods surpassing limitations of current gastrointestinal cancer models such as gastric and esophagus cancer are required to functionally validate putative oncogenic loci discovered by genome sequencing efforts. The in vitro culture of primary, non-transformed tissues as three-dimensional organoids that accurately recapitulate organ structure and physiology has diverse applications including cancer biology. Methods: Mouse wild type, or p53flox/flox in tandem with lox-stop-lox KRASG12D upper digestive tract tissue containing epithelial and mesenchymal components were cultured in an air-liquid-interface and subjected to adenovirus expressing either immunoglobulin Fc (control) or GFP tagged Cre recombinase. Results: 3-dimensional organoids were generated with histological adherence to normal tissue architecture including that seen in esophagus and were able to be maintained in long term culture. Organoids exposed to GFP tagged Cre adenovirus demonstrated green fluorescence not seen in organoids exposed to control virus. Conditional allele organoids that were exposed to Cre adenovirus demonstrated increased rate of growth compared to controls. Histology of these rapidly growing organoids demonstrated cellular features consistent with dysplasia. Conclusions: 3-dimensional organoids can be generated from upper digestive tract tissues, can undergo adenoviral mediated transfection to achieve oncogenic gene expression or inactivation resulting in dysplastic morphology. 3-dimensional organoids are therefore an attractive model to study or identify candidate oncogenic loci identified by recent genomic sequencing studies.
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Balsitis, Scott J., Julien Sage, Stefan Duensing, Karl Münger, Tyler Jacks und Paul F. Lambert. „Recapitulation of the Effects of the Human Papillomavirus Type 16 E7 Oncogene on Mouse Epithelium by Somatic Rb Deletion and Detection of pRb-Independent Effects of E7 In Vivo“. Molecular and Cellular Biology 23, Nr. 24 (15.12.2003): 9094–103. http://dx.doi.org/10.1128/mcb.23.24.9094-9103.2003.
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ABSTRACT Although the human papillomavirus (HPV) E7 oncogene is known to contribute to the development of human cervical cancer, the mechanisms of its carcinogenesis are poorly understood. The first identified and most recognized function of E7 is its binding to and inactivation of the retinoblastoma tumor suppressor (pRb), but at least 18 other biological activities have also been reported for E7. Thus, it remains unclear which of these many activities contribute to the oncogenic potential of E7. We used a Cre-lox system to abolish pRb expression in the epidermis of transgenic mice and compared the outcome with the effects of E7 expression in the same tissue at early ages. Mice lacking pRb in epidermis showed epithelial hyperplasia, aberrant DNA synthesis, and improper differentiation. In addition, Rb-deleted epidermis (i.e., epidermis composed of cells with Rb deleted) exhibited centrosomal abnormalities and failed to arrest the cell cycle in response to ionizing radiation. Transgenic mice expressing E7 in skin display the same range of phenotypes. In sum, few differences were detected between Rb-deleted epidermis and E7-expressing epidermis in young mice. However, when both E7 was expressed and Rb was deleted in the same tissue, increased hyperplasia and dysplasia were observed. These findings indicate that inactivation of the Rb pathway can largely account for E7's phenotypes at an early age, but that pRb-independent activities of E7 are detectable in vivo.
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Kot, Alexander, Zhendong A. Zhong, Hongliang Zhang, Yu-An Evan Lay, Nancy E. Lane und Wei Yao. „Sex dimorphic regulation of osteoprogenitor progesterone in bone stromal cells“. Journal of Molecular Endocrinology 59, Nr. 4 (November 2017): 351–63. http://dx.doi.org/10.1530/jme-17-0076.
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Increasing peak bone mass is a promising strategy to prevent osteoporosis. A mouse model of global progesterone receptor (PR) ablation showed increased bone mass through a sex-dependent mechanism. Cre-Lox recombination was used to generate a mouse model of osteoprogenitor-specific PR inactivation, which recapitulated the high bone mass phenotype seen in the PR global knockout mouse mode. In this work, we employed RNA sequencing analysis to evaluate sex-independent and sex-dependent differences in gene transcription of osteoprogenitors of wild-type and PR conditional knockout mice. PR deletion caused marked sex hormone-dependent changes in gene transcription in male mice as compared to wild-type controls. These transcriptional differences revealed dysregulation in pathways involving immunomodulation, osteoclasts, bone anabolism, extracellular matrix interaction and matrix interaction. These results identified many potential mechanisms that may explain our observed high bone mass phenotype with sex differences when PR was selectively deleted in the MSCs.
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Müller, Kathrin, Dagmar Führer, Jens Mittag, Nora Klöting, Matthias Blüher, Roy E. Weiss, Marie-Christine Many, Kurt Werner Schmid und Knut Krohn. „TSH Compensates Thyroid-Specific IGF-I Receptor Knockout and Causes Papillary Thyroid Hyperplasia“. Molecular Endocrinology 25, Nr. 11 (01.11.2011): 1867–79. http://dx.doi.org/10.1210/me.2011-0065.
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Abstract Although TSH stimulates all aspects of thyroid physiology IGF-I signaling through a tyrosine kinase-containing transmembrane receptor exhibits a permissive impact on TSH action. To better understand the importance of the IGF-I receptor in the thyroid in vivo, we inactivated the Igf1r with a Tg promoter-driven Cre-lox system in mice. We studied male and female mice with thyroidal wild-type, Igf1r+/−, and Igf1r−/− genotypes. Targeted Igf1r inactivation did transiently reduce thyroid hormone levels and significantly increased TSH levels in both heterozygous and homozygous mice without affecting thyroid weight. Histological analysis of thyroid tissue with Igf1r inactivation revealed hyperplasia and heterogeneous follicle structure. From 4 months of age, we detected papillary thyroid architecture in heterozygous and homozygous mice. We also noted increased body weight of male mice with a homozygous thyroidal null mutation in the Igf1r locus, compared with wild-type mice, respectively. A decrease of mRNA and protein for thyroid peroxidase and increased mRNA and protein for IGF-II receptor but no significant mRNA changes for the insulin receptor, the TSH receptor, and the sodium-iodide-symporter in both Igf1r+/− and Igf1r−/− mice were detected. Our results suggest that the strong increase of TSH benefits papillary thyroid hyperplasia and completely compensates the loss of IGF-I receptor signaling at the level of thyroid hormones without significant increase in thyroid weight. This could indicate that the IGF-I receptor signaling is less essential for thyroid hormone synthesis but maintains homeostasis and normal thyroid morphogenesis.
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Saeki, Kiyoshi, Wanglong Qiu, Richard Friedman, Carrie Shawber, Jan Kitajewski, Jianhua Hu und Gloria H. Su. „Abstract PO-073: Inactivation of Notch4 attenuated pancreatic tumorigenesis in mice“. Cancer Research 81, Nr. 22_Supplement (15.11.2021): PO—073—PO—073. http://dx.doi.org/10.1158/1538-7445.panca21-po-073.
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Abstract Expression of the Notch family of receptors are often upregulated in pancreatic ductal adenocarcinoma (PDAC), however, the functional impacts of the Notch signaling network on pancreatic tumorigenesis remain unresolved. In this study, we focused on Notch4, which had not been investigated in PDAC. Leveraging the conventional Notch4 deficient mouse line and previously established genetically engineered mouse models (GEMM) for PDAC, we generated KC (LSL-KrasG12D;p48-Cre), N4−/−KC (Notch4−/−;LSL-KrasG12D;p48-Cre), PKC (p16flox/flox;LSL-KrasG12D;p48-Cre), and N4−/−PKC GEMMs (Notch4−/−; p16flox/lox;LSL-KrasG12D;p48-Cre). We performed caerulein treatment in both KC and N4−/−KC mice, and compared the development of acinar to ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN) between them. The ADM/PanIN lesions were significantly smaller in the N4−/−KC than in the KC GEMM (p=0.01), suggesting that Notch4 deficiency attenuated early pancreatic tumorigenesis. This in vivo result was confirmed by in vitro ADM induction of the explant cultures of mouse pancreatic acinar cells. The number of ADM structures in the N4−/−KC acinar cultures was significantly lower than the KC acinar cultures (p<0.001). To evaluate the role of Notch4 in the later stage of pancreatic tumorigenesis, we compared the histological progression and overall survival between the PKC and N4−/−PKC mice. We found that N4−/−PKC mice had better prognosis (p=0.012) and less tumor burden (PanIN: p=0.018 (2 months), PDAC: p=0.039 (5 months)) compared to the PKC GEMM. RNA-Seq analysis of pancreatic tumor cell lines derived from the PKC and N4−/−PKC GEMMs revealed 408 genes were differentially expressed (FDR<0.05) and the genes related to the NGF processing as novel downstream effectors of the Notch4 signaling pathway(p<0.001). Our study is a novel biological investigation that demonstrated that Notch4 signaling possesses tumor promoting function in pancreatic tumorigenesis. Our study revealed a novel association between the NGF processing pathway and Notch4 signaling in PDAC. Citation Format: Kiyoshi Saeki, Wanglong Qiu, Richard Friedman, Carrie Shawber, Jan Kitajewski, Jianhua Hu, Gloria H. Su. Inactivation of Notch4 attenuated pancreatic tumorigenesis in mice [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-073.
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Highlander, Sarah K., Natalie D. Fedorova, David M. Dusek, Roger Panciera, Laura E. Alvarez und Carol Rinehart. „Inactivation of Pasteurella(Mannheimia) haemolytica Leukotoxin Causes Partial Attenuation of Virulence in a Calf Challenge Model“. Infection and Immunity 68, Nr. 7 (01.07.2000): 3916–22. http://dx.doi.org/10.1128/iai.68.7.3916-3922.2000.
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ABSTRACT The leukotoxin of Pasteurella (Mannheimia)haemolytica is believed to play a significant role in pathogenesis, causing cell lysis and apoptosis that lead to the lung pathology characteristic of bovine shipping fever. Using a system for Cre-lox recombination, a nonpolar mutation within thelktC transacylase gene of the leukotoxin operon was created. The lktC locus was insertionally inactivated using a loxP-aph3-loxP cassette, and then the aph3marker was excised from the chromosome by Cre recombinase expressed from a P. haemolytica plasmid. The resultinglktC strain (SH2099) secretes inactive leukotoxin and carries no known antibiotic resistance genes. Strain SH2099 was tested for virulence in a calf challenge model. We inoculated 3 × 108 or 3 × 109 CFU of wild-type or mutant bacteria into the lungs of healthy, colostrum-deprived calves via transthoracic injection. Animals were observed for clinical signs and for nasal colonization for 4 days, after which they were euthanized and necropsied. The lower inoculum (3 × 108 CFU) caused significantly fewer deaths and allowed lung pathology to be scored and compared, while the 3 × 109 CFU dose of either the wild-type or mutant was lethal to ≥50% of the calves. The estimated 50% lethal dose of SH2099 was four times higher than that of the wild-type strain. Lung lesion scores were reduced twofold in animals inoculated with the mutant, while clinical scores were nearly equivalent for both strains. The wild-type and mutant strains were equally capable of colonizing the upper respiratory tracts of the calves. In this study, the P. haemolytica lktC mutant was shown to be less virulent than the parent strain.
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Haque, Shabirul, Jianhua Li, Ario de Marco, Tomo-o. Ishikawa, Harvey Herschman, Ruben Spretz, Sandra Noriega, Luis Nunez und Patricia Mongini. „Cre-based deletion of COX-2 within B cells in vitro: steps toward specific nanoparticle-driven COX-2 inactivation in vivo. (P4044)“. Journal of Immunology 190, Nr. 1_Supplement (01.05.2013): 44.7. http://dx.doi.org/10.4049/jimmunol.190.supp.44.7.
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Abstract Several lines of evidence indicate that COX-2 and downstream prostaglandin (PG) positively influence germinal center development and Ig heavy chain class switching. Although activated B cells upregulate COX-2 and downstream effector molecules, it is unclear whether autocrine production of PG is a major driver of B cell autoimmunity. One approach to investigate this is through Cre-mediated, B cell lineage-specific COX-2flox recombination in mice prone to autoimmunity. Because Cre insertion into B lineage genes, CD19 and mb-1, compromises expression of molecules needed for BCR-triggered autoimmunity, we are seeking to achieve B cell-specific Cre-Lox recombination through an alternative route: B cell-specific NP bearing Cre as a payload. We here report that purified recombinant Cre protein rapidly achieves specific in vitro recombination within PCR amplicons of COX-2 with inserted pLox sites. Furthermore, one hour exposure of viable splenocytes from NOD.B10 COX-2flox mice to Cre protein at 37oC results in ~ 90% recombination. Cells treated with active Cre and subsequently stimulated with BCR-L, IL-4 and BAFF, display significantly less COX-2 protein upon reculture than cells similarly treated with heat-inactivated Cre. We additionally report the preparation of CD22-specific nanoparticles composed of PEG and PLA. In future studies, similar NP will be employed to target Cre specifically to B cells in Sjogren’s Syndrome-susceptible NOD.B10 COX-2flox mice.
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Masłowska, Katarzyna H., Luisa Laureti und Vincent Pagès. „iDamage: a method to integrate modified DNA into the yeast genome“. Nucleic Acids Research 47, Nr. 20 (16.08.2019): e124-e124. http://dx.doi.org/10.1093/nar/gkz723.
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Abstract In order to explore the mechanisms employed by living cells to deal with DNA alterations, we have developed a method by which we insert a modified DNA into a specific site of the yeast genome. This is achieved by the site-specific integration of a modified plasmid at a chosen locus of the genome of Saccharomyces cerevisiae, through the use of the Cre/lox recombination system. In the present work, we have used our method to insert a single UV lesion into the yeast genome, and studied how the balance between error-free and error-prone lesion bypass is regulated. We show that the inhibition of homologous recombination, either directly (by the inactivation of Rad51 recombinase) or through its control by preventing the polyubiquitination of PCNA (ubc13 mutant), leads to a strong increase in the use of Trans Lesion Synthesis (TLS). Such regulatory aspects of DNA damage tolerance could not have been observed with previous strategies using plasmid or randomly distributed DNA lesions, which shows the advantage of our new method. The very robust and precise integration of any modified DNA at any chosen locus of the yeast genome that we describe here is a powerful tool that will enable the exploration of many biological processes related to replication and repair of modified DNA.
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Abravaya, Klara, Claudia Esping, Robert Hoenle, Jacek Gorzowski, Robert Perry, Paul Kroeger, John Robinson und Richard Flanders. „Performance of a Multiplex Qualitative PCR LCx Assay for Detection of Human Immunodeficiency Virus Type 1 (HIV-1) Group M Subtypes, Group O, and HIV-2“. Journal of Clinical Microbiology 38, Nr. 2 (2000): 716–23. http://dx.doi.org/10.1128/jcm.38.2.716-723.2000.
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Early detection of human immunodeficiency virus (HIV) in blood and blood products can be achieved by a sensitive nucleic acid amplification-based assay. We report on the performance of a PCR-based qualitative assay that detects both HIV type 1 (HIV-1) and HIV-2 with a sensitivity of 20 to 50 copies/ml. The assay has a specificity of 99.6% and an inhibition rate of 1.7%. One milliliter of sample is processed with a manifold system and Qiagen columns, and one-third of the extracted sample is used for PCR amplification. An internal control sequence, which is processed and amplified with each sample, monitors for amplification inhibition. Samples are reverse transcribed and are then amplified by reverse transcription-coupled PCR, after which HIV-1- and HIV-2-specific probes are hybridized to the amplified products. Following hybridization, samples are detected in the LCx instrument by microparticle enzyme immunoassay techniques. The detection system has an automated inactivation step that controls for PCR contamination. The HIV-1/2 qualitative RNA assay detects HIV-1 group M subtypes A, B, C, D, E, F, and G and group O. Testing of several HIV-1 seroconversion panels has demonstrated that the HIV-1/2 qualitative RNA assay detects HIV infection on the average of 6 days before p24 antigen can be detected and 11 days before antibodies can be detected.
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Prevost, Gaëtan, Arnaud Arabo, Long Jian, Eddy Quelennec, Dorthe Cartier, Sahar Hassan, Anthony Falluel-Morel et al. „The PACAP-Regulated Gene Selenoprotein T Is Abundantly Expressed in Mouse and Human β-Cells and Its Targeted Inactivation Impairs Glucose Tolerance“. Endocrinology 154, Nr. 10 (01.10.2013): 3796–806. http://dx.doi.org/10.1210/en.2013-1167.
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Selenoproteins are involved in the regulation of redox status, which affects several cellular processes, including cell survival and homeostasis. Considerable interest has arisen recently concerning the role of selenoproteins in the regulation of glucose metabolism. Here, we found that selenoprotein T (SelT), a new thioredoxin-like protein of the endoplasmic reticulum, is present at high levels in human and mouse pancreas as revealed by immunofluorescence and quantitative PCR. Confocal immunohistochemistry studies revealed that SelT is mostly confined to insulin- and somatostatin-producing cells in mouse and human islets. To elucidate the role of SelT in β-cells, we generated, using a Cre-Lox strategy, a conditional pancreatic β-cell SelT-knockout C57BL/6J mice (SelT-insKO) in which SelT gene disruption is under the control of the rat insulin promoter Cre gene. Glucose administration revealed that male SelT-insKO mice display impaired glucose tolerance. Although insulin sensitivity was not modified in the mutant mice, the ratio of glucose to insulin was significantly higher in the SelT-insKO mice compared with wild-type littermates, pointing to a deficit in insulin production/secretion in mutant mice. In addition, morphometric analysis showed that islets from SelT-insKO mice were smaller and that their number was significantly increased compared with islets from their wild-type littermates. Finally, we found that SelT is up-regulated by pituitary adenylate cyclase-activating polypeptide (PACAP) in β-pancreatic cells and that SelT could act by facilitating a feed-forward mechanism to potentiate insulin secretion induced by the neuropeptide. Our findings are the first to show that the PACAP-regulated SelT is localized in pancreatic β- and δ-cells and is involved in the control of glucose homeostasis.
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Dzhamieiev, V. Y. „Modern concepts of auxin’s action. 1. History of discovery, metabolism, transport“. Vìsnik Harkìvsʹkogo nacìonalʹnogo agrarnogo unìversitetu. Serìâ Bìologiâ 2020, Nr. 3 (30.10.2020): 98–123. http://dx.doi.org/10.35550/vbio2020.03.098.
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Auxin (indolyl-3-acetic acid, IAA) is one of the key classical phytohormones with a very wide range of physiological effects. The first part of the scientific lecture describes the main stages of discovery of the hormone. The main pathways of auxin synthesis in plant tissues, which is carried out in two different ways: tryptophan-dependent and tryptophan-independent, are considered in detail. At the same time, multiple pathways of the auxin formation from tryptophan have been found in plant tissues. Among them, the mechanisms that occur with the formation of such intermediate metabolites as indole-3-acetaldoxime, indole-3-pyruvate and indole-3-acetamide are considered. The indole-3-pyruvate pathway is currently considered the main mechanism of hormone synthesis. Experimental evidence has also been obtained for the functioning of the tryptophan-independent pathway of auxin synthesis, the key enzyme of which is cytoplasmic indole synthase. It is assumed that the precursor of auxin in the tryptophan-independent pathway may be some intermediate metabolite between anthranilic acid and tryptophan. The article also describes the routes of auxin inactivation through the formation of conjugated forms and oxidation. A brief characterization of IAA dioxygenases, belonging to the 2-oxoglutarate-Fe (II)-oxygenases family, which are currently considered the main catalytic systems for auxin oxidation, is presented. The mechanisms and significance of polar and lateral transport of auxin are discussed. The characteristics of transmembrane auxin transporters belonging to the families PIN/PIL, ABCB/PGP and AUX/LAX are given.
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Won, Minho, Hyunju Ro und Igor B. Dawid. „Lnx2 ubiquitin ligase is essential for exocrine cell differentiation in the early zebrafish pancreas“. Proceedings of the National Academy of Sciences 112, Nr. 40 (21.09.2015): 12426–31. http://dx.doi.org/10.1073/pnas.1517033112.
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The gene encoding the E3 ubiquitin ligase Ligand of Numb protein-X (Lnx)2a is expressed in the ventral-anterior pancreatic bud of zebrafish embryos in addition to its expression in the brain. Knockdown of Lnx2a by using an exon 2/intron 2 splice morpholino resulted in specific inhibition of the differentiation of ventral bud derived exocrine cell types, with little effect on endocrine cell types. A frame shifting null mutation in lnx2a did not mimic this phenotype, but a mutation that removed the exon 2 splice donor site did. We found that Lnx2b functions in a redundant manner with its paralog Lnx2a. Inhibition of lnx2a exon 2/3 splicing causes exon 2 skipping and leads to the production of an N-truncated protein that acts as an interfering molecule. Thus, the phenotype characterized by inhibition of exocrine cell differentiation requires inactivation of both Lnx2a and Lnx2b. Human LNX1 is known to destabilize Numb, and we show that inhibition of Numb expression rescues the Lnx2a/b-deficient phenotype. Further, Lnx2a/b inhibition leads to a reduction in the number of Notch active cells in the pancreas. We suggest that Lnx2a/b function to fine tune the regulation of Notch through Numb in the differentiation of cell types in the early zebrafish pancreas. Further, the complex relationships among genotype, phenotype, and morpholino effect in this case may be instructive in the ongoing consideration of morpholino use.
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Beare, Paul A., Charles L. Larson, Stacey D. Gilk und Robert A. Heinzen. „Two Systems for Targeted Gene Deletion in Coxiella burnetii“. Applied and Environmental Microbiology 78, Nr. 13 (20.04.2012): 4580–89. http://dx.doi.org/10.1128/aem.00881-12.
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ABSTRACTCoxiella burnetiiis a ubiquitous zoonotic bacterial pathogen and the cause of human acute Q fever, a disabling influenza-like illness.C. burnetii's former obligate intracellular nature significantly impeded the genetic characterization of putative virulence factors. However, recent host cell-free (axenic) growth of the organism has enabled development of shuttle vector, transposon, and inducible gene expression technologies, with targeted gene inactivation remaining an important challenge. In the present study, we describe two methods for generating targeted gene deletions inC. burnetiithat exploit pUC/ColE1ori-based suicide plasmids encodingsacBfor positive selection of mutants. As proof of concept,C. burnetiidotA anddotB, encoding structural components of the type IVB secretion system (T4BSS), were selected for deletion. The first method exploited Cre-lox-mediated recombination. Two suicide plasmids carrying different antibiotic resistance markers and aloxPsite were integrated into 5′ and 3′ flanking regions ofdotA. Transformation of this strain with a third suicide plasmid encoding Cre recombinase resulted in the deletion ofdotAunder sucrose counterselection. The second method utilized a loop-in/loop-out strategy to deletedotAanddotB.A single suicide plasmid was first integrated into 5′ or 3′ target gene flanking regions. Resolution of the plasmid cointegrant by a second crossover event under sucrose counterselection resulted in gene deletion that was confirmed by PCR and Southern blot. ΔdotAand ΔdotBmutants failed to secrete T4BSS substrates and to productively infect host cells. The repertoire ofC. burnetiigenetic tools now allows ready fulfillment of molecular Koch's postulates for suspected virulence genes.
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Cardoso, Marcos S., Tânia M. Silva, Mariana Resende, Rui Appelberg und Margarida Borges. „Lack of the Transcription Factor Hypoxia-Inducible Factor 1α (HIF-1α) in Macrophages Accelerates the Necrosis of Mycobacterium avium-Induced Granulomas“. Infection and Immunity 83, Nr. 9 (22.06.2015): 3534–44. http://dx.doi.org/10.1128/iai.00144-15.
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The establishment of mycobacterial infection is characterized by the formation of granulomas, which are well-organized aggregates of immune cells, namely, infected macrophages. The granuloma's main function is to constrain and prevent dissemination of the mycobacteria while focusing the immune response to a limited area. In some cases these lesions can grow progressively into large granulomas which can undergo central necrosis, thereby leading to their caseation. Macrophages are the most abundant cells present in the granuloma and are known to adapt under hypoxic conditions in order to avoid cell death. Our laboratory has developed a granuloma necrosis model that mimics the human pathology ofMycobacterium tuberculosis, using C57BL/6 mice infected intravenously with a low dose of a highly virulent strain ofMycobacterium avium. In this work, a mouse strain deleted of the hypoxia inducible factor 1α (HIF-1α) under the Cre-lox system regulated by the lysozyme M gene promoter was used to determine the relevance of HIF-1α in the caseation of granulomas. The genetic ablation of HIF-1α in the myeloid lineage causes the earlier emergence of granuloma necrosis and clearly induces an impairment of the resistance againstM. aviuminfection coincident with the emergence of necrosis. The data provide evidence that granulomas become hypoxic before undergoing necrosis through the analysis of vascularization and quantification of HIF-1α in a necrotizing mouse model. Our results show that interfering with macrophage adaptation to hypoxia, such as through HIF-1α inactivation, accelerates granuloma necrosis.
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Danziger, Natalie, Elise C. Kohn, Julia C. F. Quintanilha, Gerald Li, Julia A. Elvin und Douglas I. Lin. „Gynecologic-cancer analysis of ARID1A alterations detected in tissue and liquid biopsies.“ Journal of Clinical Oncology 41, Nr. 16_suppl (01.06.2023): 5593. http://dx.doi.org/10.1200/jco.2023.41.16_suppl.5593.
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5593 Background: The tumor suppressor gene ARID1A is an emerging target for new cancer treatment strategies including ATR inhibition. This study aimed to describe the landscape of ARID1A alterations ( ARID1Amut) in gynecologic malignancies. Methods: Patients (pts) with a diagnosis of ovarian (OC) or uterine cancer (UC) of different histologies and comprehensive genomic profiling (CGP) by Foundation Medicine Inc. were included in this study. CGP of solid tissue biopsies (Tbx; FoundationOneCDx) included evaluation of genomic loss of heterozygosity (gLOH; gLOH-high as ≥16% as validated in OC), microsatellite instability (MSI), and tumor mutational burden (TMB; high as ≥10 mutations/Megabase). CGP of peripheral whole-blood liquid biopsies (Lbx; FoundationOneLiquidCDx) included evaluation of MSI and tumor fraction (TF), a measure of the relative quantity of circulating tumor DNA (ctDNA). TF was calculated by measures of aneuploidy and allele frequency and binned as TF < 1%, TF 1 to < 10%, or TF ≥10%. Results: Tbx Cohort: 5,778/30,212 (19.1%) cases were ARID1Amut. Pts had a median age of 63 (range 21-89) years. ARID1Amut were observed across many subtypes and most frequently in endometrial endometrioid (n = 3,052, 57.7%) and ovarian clear cell (n = 982, 57.6%) but rarely in serous (OC, n = 12,258, 2.8%; UC, n = 2,682, 8.9%). Pts frequently harbored more than one ARID1Amut (2,360/5,778, 40.8%). Of the 8,484 observed ARID1Amut, 97.6% were short variants (SV), 0.5% were deletions, and 1.9% were inactivating rearrangements. SV were primarily frameshifts (68.5%) and nonsense mutations (27.6%). The most frequent alterations observed were frameshifts at the D1860 codon. SV were predicted to be homozygous in 11.9%, heterozygous in 65.3%, or unknown zygosity in 22.8%. Overall, 16.6% of ARID1Amut cases with SV had at least one homozygous alteration. 6.6% of pts with homozygous ARID1Amut were MSI-high (MSI-H), while 39.4% of pts with only heterozygous or unknown zygosity ARID1Amut were MSI-H (p < 0.0001). Overall, 1,905 (33.0%) of ARID1Amut cases were MSI-H, and 2,183 (37.8%) were TMB high. For ARID1Amut cases with evaluable gLOH (n = 4745), the median gLOH was 2.7%, and 5.9% pts were gLOH-high. The most frequently altered co-occurring genes were PTEN (62.2%), PIK3CA (54.2%), and TP53 (27.6%). 8.7% of ARID1Amut also harbored a BRCA alteration. Lbx Cohort: 59/967 (6.1%) cases were ARID1Amut. 17 (28.8%) were MSI-H. Frequency of ARID1Amut increased as TF increased, with a detected frequency of 1.3%, 6.7%, and 14.0% among Lbx with TF < 1%, TF 1 to < 10%, or TF ≥10% respectively. Conclusions: ARID1Amut are observed across a variety of Gynecologial cancer subtypes but are enriched in clear cell and endometrioid histologies and detected in both tissue and liquid biopsies. ARID1Amut were not associated with elevated gLOH but were often MSI-H and TMB ≥10mut/Mb. Clinical trials targeting ARID1A may wish to consider the context of co-occuring biomarkers.
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Rusch, A., und R. A. Eatock. „A delayed rectifier conductance in type I hair cells of the mouse utricle“. Journal of Neurophysiology 76, Nr. 2 (01.08.1996): 995–1004. http://dx.doi.org/10.1152/jn.1996.76.2.995.
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1. Membrane currents of hair cells in acutely excised or cultured mouse utricles were recorded with the whole cell voltage-clamp method at temperatures between 23 and 36 degrees C. 2. Type I and II hair cells both had delayed rectifier conductances that activated positive to -55 mV. 3. Type I, but not type II, hair cells had an additional delayed rectifier conductance (gK,L) with an activation range that was unusually negative and variable. At 23-25 degrees C, V(1/2) values ranged from -88 to -62 mV in 57 cells. 4. gK,L was very large. At 23-25 degrees C, the average maximum chord conductance was 75 +/- 65 nS (mean +/- SD, n = 57; measured at -54 mV), or approximately 21 nS/pF of cell capacitance. 5. gK,L was highly selective for K+ over Na+ (permeability ratio PNa+/PK+:0.006), but unlike other delayed rectifiers, gK,L was significantly permeable to Cs+ (PCs+/PK+:0.31). gK,L was independent of extracellular Ca2+. 6. At -64 mV, Ba2+ and 4-aminopyridine blocked gK,L with apparent dissociation constants of 2.0 mM and 43 microM, respectively. Extracellular Cs+ (5 mM) blocked gK,L by 50% at -124 mV. Apamin (100 nM) and dendrotoxin (10 nM) has no effect. 7. The kinetic data of gK,L are consistent with a sequential gating model with at least two closed states and one open state. The slow activation kinetics (principal time constants at 23-25 degrees C:600-200 ms) had a thermal Q10 of 2.1. Inactivation (Q10:2.7) was partial at all temperatures. Deactivation followed a double-exponential time course and had a Q10 of 2.0. 8. At 23-25 degrees C, gK,L was appreciably activated at the mean resting potential of type I hair cells (-77 +/- 3.1 mV, n = 62), so that input conductances were often more than an order of magnitude larger than those of type II cells. If these conditions hold in vivo, type I cells would produce unusually small receptor potentials. Warming the cells to 36 degrees C produced parallel shifts in gK,L's activation range (0.8 +/- 0.3 mV/degrees C, n = 8), and in the resting potential (0.6 +/- 0.3 mV/degrees C, n = 4). Thus the high input conductances were not an artifact of unphysiological temperatures but remained high near body temperature. It remains possible that in vivo gK,L's activation range is less negative and input conductances are lower; the large variance in the voltage range of activation suggests that it may be subject to modulation.
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Matsuoka, Kazuhiko, Latifa Bakiri, Lena I. Wolff, Markus Linder, Amanda Mikels-Vigdal, Ana Patiño-García, Fernando Lecanda, Christine Hartmann, Maria Sibilia und Erwin F. Wagner. „Wnt signaling and Loxl2 promote aggressive osteosarcoma“. Cell Research 30, Nr. 10 (20.07.2020): 885–901. http://dx.doi.org/10.1038/s41422-020-0370-1.
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Abstract Osteosarcoma (OS) is the most frequent primary malignant bone tumor in urgent need of better therapies. Using genetically modified mouse models (GEMMs), we demonstrate that Wnt signaling promotes c-Fos-induced OS formation via the actions of the collagen-modifying enzyme Loxl2. c-Fos/AP-1 directly regulates the expression of the Wnt ligands Wnt7b and Wnt9a in OS cells through promoter binding, and Wnt7b and Wnt9a in turn promote Loxl2 expression in murine and human OS cells through the transcription factors Zeb1 and Zeb2. Concordantly, inhibition of Wnt ligand secretion by inactivating the Wnt-less (Wls) gene in osteoblasts in c-Fos GEMMs either early or in a therapeutic setting reduces Loxl2 expression and progression of OS. Wls-deficient osteosarcomas proliferate less, are less mineralized and are enriched in fibroblastic cells surrounded by collagen fibers. Importantly, Loxl2 inhibition using either the pan-Lox inhibitor BAPN or a specific inducible shRNA reduces OS cell proliferation in vitro and decreases tumor growth and lung colonization in murine and human orthotopic OS transplantation models. Finally, OS development is delayed in c-Fos GEMMs treated with BAPN or with specific Loxl2 blocking antibodies. Congruently, a strong correlation between c-FOS, LOXL2 and WNT7B/WNT9A expression is observed in human OS samples, and c-FOS/LOXL2 co-expression correlates with OS aggressiveness and decreased patient survival. Therefore, therapeutic targeting of Wnt and/or Loxl2 should be considered to potentiate the inadequate current treatments for pediatric, recurrent, and metastatic OS.
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Zaiss, Anne-Kathrin, Sodany Son und Lung-Ji Chang. „RNA 3′ Readthrough of Oncoretrovirus and Lentivirus: Implications for Vector Safety and Efficacy“. Journal of Virology 76, Nr. 14 (15.07.2002): 7209–19. http://dx.doi.org/10.1128/jvi.76.14.7209-7219.2002.
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ABSTRACT The expression of reporter genes driven by the same human elongation factor 1α (EF1α) promoter in murine leukemia virus (MLV)- and human immunodeficiency virus type 1 (HIV-1)-based vectors was studied in either transfected or virally transduced cells. The HIV-1 vectors consistently expressed 3 to 10 times higher activity than the MLV vectors at both the RNA and protein levels. The difference was not attributable to transcriptional interference, alternative enhancer/silencer, or differential EF1α intron splicing. Based on nuclear run-on assays, both vectors exhibited similar EF1α transcriptional activity. The reduced RNA levels of MLV vectors could not be explained by the decrease in RNA half-lives. Southern analysis of proviral DNA indicated that both HIV-1 and MLV vectors efficiently propagated the EF1α intron in the transduced cells. To decipher the discrepancy in transgene expression between MLV and HIV-1 vectors, the role of RNA 3′-end processing was examined using a sensitive Cre/lox reporter assay. The results showed that MLV vectors, but not HIV-1 vectors, displayed high frequencies of readthrough of the 3′ polyadenylation signal. Interestingly, the polyadenylation signal of a self-inactivating (SIN) HIV-1 vector was as leaky as that of the MLV vectors, suggesting a potential risk of oncogene activation by the lentiviral SIN vectors. Together, our results suggest that an efficient polyadenylation signal would improve both the efficacy and the safety of these vectors.
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Bojkovic, Jade, Daryl L. Richie, David A. Six, Christopher M. Rath, William S. Sawyer, Qijun Hu und Charles R. Dean. „Characterization of an Acinetobacter baumanniilptDDeletion Strain: Permeability Defects and Response to Inhibition of Lipopolysaccharide and Fatty Acid Biosynthesis“. Journal of Bacteriology 198, Nr. 4 (14.12.2015): 731–41. http://dx.doi.org/10.1128/jb.00639-15.
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ABSTRACTLipid A on the Gram-negative outer membrane (OM) is synthesized in the cytoplasm by the Lpx pathway and translocated to the OM by the Lpt pathway. SomeAcinetobacter baumanniistrains can tolerate the complete loss of lipopolysaccharide (LPS) resulting from the inactivation of early LPS pathway genes such aslpxC. Here, we characterized a mutant deleted forlptD, which encodes an OM protein that mediates the final translocation of fully synthesized LPS to the OM. Cells lackinglptDhad a growth defect comparable to that of anlpxCdeletion mutant under the growth conditions tested but were more sensitive to hydrophobic antibiotics, revealing a more significant impact on cell permeability from impaired LPS translocation than from the loss of LPS synthesis. Consistent with this, ATP leakage andN-phenyl-1-naphthylamine (NPN) fluorescence assays indicated a more severe impact oflptDdeletion than oflpxCdeletion on inner and outer membrane permeability, respectively. Targeted liquid chromatography-mass spectrometry (LCMS) analysis of LPS intermediates from UDP-3-O-R-3-hydroxylauroyl-N-acetyl-α-d-glucosamine through lipid IVAshowed that the loss of LptD caused an accumulation of lipid IVA. This suggested that pathway intermediate accumulation or mislocalization caused by the blockage of later LPS pathway steps impacts envelope integrity. Supporting this notion, chemical inhibition of lipid A precursor enzymes, including LpxC and FabB/F, in thelptDdeletion strain partially rescued growth and permeability defects.IMPORTANCENew antibiotics to treat Gram-negative bacterial infections are urgently needed. Inhibition of LPS biosynthesis is attractive because this would impact viability and cell permeability. Therefore, a better understanding of this pathway is important, especially in strains such asA. baumanniiATCC 19606, where LPS biosynthesis is not essentialin vitro. We show that ATCC 19606 also survives the loss of the final translocation of LPS into the OM (lptDdeletion). Intriguingly, this impaired cell envelope integrity more than the loss of LPS biosynthesis (lpxCdeletion), presumably due to the accumulation of toxic intermediates. Supporting this, chemical inhibition of LPS biosynthesis partially reversed this permeability defect. This extends our understanding of the LPS machinery and provides insights into potential interrelationships of the target steps along this important pathway.
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Wei Li, Wei Yang und Can Yang. „Knockdown of Bcl-3 alleviates psoriasis and dyslipidemia comorbidity by regulating Akt pathway“. Allergologia et Immunopathologia 50, Nr. 6 (01.11.2022): 115–21. http://dx.doi.org/10.15586/aei.v50i6.683.
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Background: Psoriasis is considered as an inflammatory skin disease accompanied by dyslipid-emia comorbidity. B-cell leukemia-3 (Bcl-3) belongs to IκB (inhibitor of nuclear factor kappa B [NF-κB]) family, and regulates inflammatory response through associating with NF-κB. The role of Bcl-3 in psoriasis was investigated in this study. Methods: Apolipoprotein E (ApoE)-deficient mice were treated with imiquimod to induce psoriasis and dyslipidemia. Mice were injected intradermally in the back with lentiviral particles encoding Bcl-3 small hairpin RNA (shRNA). Hematoxylin and eosin were used to detect pathological characteristics. The blood lipid levels were determined by automatic biochemical analyzer, and inflammation was assessed by enzyme-linked-immunosorbent serologic assay and real-time quantitative reverse transcription polymerase chain reaction. Results: Bcl-3 was elevated in imiquimod-induced ApoE-deficient mice. Injection with lentiviral particles encoding Bcl-3 shRNA reduced Psoriasis area and severity index (PASI) score in ApoE-deficient psoriatic mice. Knockdown of Bcl-3 also ameliorated imiquimod-induced psoriasiform skin lesions in ApoE-deficient mice. Moreover, loss of Bcl-3 enhanced expression of loricrin, an epidermal barrier protein, reduced expression of proliferating cell nuclear antigen (PCNA) and lectin-like oxidized LDL (oxLDL) receptor-1 (LOX-1) in imiquimod-induced ApoE-deficient mice. The enhanced levels of blood lipid in ApoE-deficient mice were attenuated by silencing of Bcl-3 with increase of high-density lipoprotein, and reduction of total cholesterol, triglycerides, and low-density lipoprotein cholesterol. Knockdown of Bcl-3 attenuated imiquimod-induced decrease of transforming growth factor beta (TGF-β), and increase of Interleukin (IL)-17A, IL-23, IL-6, and tumor necrosis factor-α (TNF-α) in ApoE-deficient mice. Protein expression of phospho-Akt (p-Akt) and p-GSK3β in ApoE-deficient psoriatic mice was decreased by silencing of Bcl-3. Conclusion: Loss of Bcl-3 exerted anti-inflammatory effect on psoriasis and dyslipidemia comorbidity through inactivation of Akt/GSK3β pathway.
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Tothova, Zuzana, Ramya Kollipara, Ronald A. DePinho und D. Gary Gilliland. „The Role of Forkhead Transcription Factors FoxO1, FoxO3 and FoxO4 in Hematopoiesis and Leukemogenesis.“ Blood 106, Nr. 11 (16.11.2005): 1350. http://dx.doi.org/10.1182/blood.v106.11.1350.1350.
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Abstract FoxO is a family of forkhead transcription factors that negatively regulate proliferation and survival signals in hematopoietic cells. We and others have previously shown that inhibition of the three members of this family (FoxO1, FoxO3 and FoxO4) by leukemogenic tyrosine kinase fusion genes results in enhanced proliferative and survival signaling in leukemic cells. For example, the transforming activities of the lymphoma associated NPM-ALK (nucleophosmin-anaplastic lymphoma kinase) fusion, BCR-ABL, or FLT3-ITD, are mediated in part by inactivation of FoxO through phosphorylation and ubiquitin mediated degradation by constitutively active Akt (Gu TL, et al. Blood 2004), with subsequent induction of proliferative and survival signals. Furthermore, inhibition of FoxO is required for efficient transformation of hematopoietic cells. However, the roles of FoxO in adult hematopoiesis are unknown. We have initiated studies to examine the role of FoxO in the context of normal hematopoiesis and leukemogenesis using a triple conditional knockout mouse for each of the FoxO1, FoxO3 and FoxO4 alleles. The FoxO alleles are flanked by lox-P sites and conditional excision is mediated by Cre expression under the control of the interferon inducible Mx1 promoter. Based on the normal function of FoxO family members to repress proliferative and survival signals, we hypothesized that the deletion of FoxO subfamily members would lead to an enhanced proliferation and survival in the hematopoietic compartment, and might contribute to the development of a myeloproliferative and/or lymphoproliferative phenotype in vivo. Triple homozygous conditional FoxO knockout mice were generated in an Mx1-Cre background to allow for excision of the FoxO alleles in the hematopoietic stem cell compartment after treatment with pIpC. Complete excision of each of the three alleles in the hematopoietic compartment was confirmed. However, in contrast with our working hypothesis, we observed that loss of function of FoxO family members was associated with a relatively subtle hematopoietic phenotype with 12 months of follow-up. The phenotype includes a non-fatal mild myeloproliferative phenotype that is progressive over time and characterized by modest splenomegaly, extramedullary hematopoiesis and increased mature myeloid populations in bone marrow and spleen. In addition, there are subtle alterations in both B and T lymphoid cell populations, including a decrease in both immature and mature B cells in the spleen and bone marrow; and abnormalities of CD4+CD8+ double positive and CD4+ and CD8+ T cells in the thymus. Examination of stem and progenitor populations also revealed subtle differences in the HSC and CLP progenitor populations at 4 weeks post pIpC. Thus, these data indicate that complete loss of FoxO function in the adult hematopoietic compartment results in a relatively subtle hematopoietic phenotype. They further demonstrate that although inhibition of FoxO family members is required for efficient transformation of hematopoietic cells by leukemogenic fusion tyrosine kinases, loss of FoxO function alone is not sufficient to induce a leukemic phenotype.
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Shin, Dong-Myung, Rui Liu, Wu Wan, Janina Ratajczak, Mariusz Z. Ratajczak und Magdalena Kucia. „Polycomb Group Protein-Mediated Bivalent Domains Regulate Pluripotency of Oct4+ Very Small Embryonic-Like (VSEL) Stem Cells In Adult Bone Marrow“. Blood 116, Nr. 21 (19.11.2010): 4788. http://dx.doi.org/10.1182/blood.v116.21.4788.4788.
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Abstract Abstract 4788 Recently, we identified a population of very small embryonic-like (VSEL) stem cells (SCs) in adult bone marrow (BM; Leukemia 2006;20:857). These Oct4+CXCR4+SSEA-1+Sca-1+CD45-Lin- VSELs are capable of differentiation in vitro into cells of all three germ lineages and are able to form spheres (VSEL-derived spheres[VSEL-DS]) composed of primitive stem cells in co-cultures with C2C12 myoblasts. Cells isolated from VSEL-DS are also able to differentiate into all three germ layers (cardiomyocytes, neural cells, and insulin-producing cells). Our previous epigenetic study proved that VSELs exhibit open chromatin structures in the promoters of pluripotent stem cell markers, such as Oct4 and Nanog (Leukemia 2009;23:2042). However, it still remains unclear whether the pluripotency of VSELs could be orchestrated by a mechanism similar to that seen in embryonic stem cells (ESCs). To address this issue, we evaluated whether adult BM-derived VSELs, in a similar way as ESCs, display bivalent-domain–marked histone modifications in the promoter region of homeodomain-containing developmental master transcription factors (TFs), such as Dlx-, Irx-, Lhx-, Pou-, Pax-, and Six-family proteins. To overcome the problem that VSELs sorted by FACS are heterogeneous, we constructed a cDNA library from a small number of cells (~25) by employing the protocols used for single-cell analysis (Nat Protoc 2007;2:739). Analysis of this cDNA library indicated that Oct4+ VSELs highly express several epigenetic regulators, especially polycomb repressive complex 2 (PRC2) proteins, including Ezh2, Suz12, and Eed. It is known that PRC2, due to its histone methyltransferase activity on lysine27 of histone3 (H3K27), is responsible for transcriptionally repressive trimethylation of H3K27 (HKK27me3) in promoters for several homeobox domain-containing developmental TFs, such as Sox21, Nkx2.2, Dlx1, Zfpm2, Irx2, Lbx1h, Hlxb9, and Pax5. Moreover, we observed that most of these promoters are “bivalent” and marked with the transcriptionally active histone code that includes trimethylated H3K4 together with H3K27me3, similarly to ESCs. Such bivalent epigenetic marks prevent the premature expression of these TFs in freshly isolated quiescent VSELs. Furthermore, we noticed that during formation of VSEL-DS, Ezh2 expression was decreased and most of the bivalent domains in VSELs were transformed into unmodified histone marks, resulting in the expression of some bivalent-targeted genes, including Hlxb9, HoxA3, and Evx1. Moreover, most of the pluripotent SC markers were also reduced during VSEL-DS progression, which was paralleled by inactivation of the Oct4 promoter. In conclusion, our results suggest that the pluripotency of adult tissue-derived VSELs is modulated, in a similar manner as in ESCs, by transient repression of developmental master TFs through bivalent epigenetic marks. Although PRC2 proteins stimulate cellular proliferation and are highly expressed in various tumors, unique epigenetic reprogramming of genomic imprinting maintains VSEL quiescence under steady-state conditions and also protects these cells from teratoma formation (Leukemia 2009;23:2042). Thus, we suggest that attenuation of this protective mechanism in VSELs, for example during chronic tissue injury and ageing, may lead to tumor formation. Disclosures: No relevant conflicts of interest to declare.
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Duran-Ortiz, Silvana, Edward O. List, Stephen Bell, Reetobrata Basu, Jonathan Young, Todd McHugh, Samuel Casey Mathes et al. „Growth Hormone Receptor Gene Disruption in Mature-Adult Mice Improves Glucose Metabolism and Lifespan in Females“. Journal of the Endocrine Society 5, Supplement_1 (01.05.2021): A445—A446. http://dx.doi.org/10.1210/jendso/bvab048.910.
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Abstract Growth hormone (GH) serves an important role in early and adult life. Reduction of GH action has been shown to increase life span in many species of animals. In fact, mice bearing a congenital disruption of GH receptor (GHR) gene (GHRKO) hold the record for the longest-lived laboratory mice. In addition to extended life span, these mice show improved health with lower rates of cancer, increased insulin sensitivity, and resistance to age-associated cognitive decline. Furthermore, humans with decreased GH action due to inactivating mutations in the GHR (Laron Syndrome patients) are resistant to cancer and diabetes. Even though the beneficial effects of congenital Ghr gene disruption are well studied, the consequences of postnatal disruption of GH action were unknown. Previously our laboratory generated a mouse line with disrupted GH action at 1.5 months of age (1.5mGHRKO mice). Results showed that these mice had improved insulin sensitivity and increased maximal lifespan only in females, yet growth retardation was still present. To consider decreased GH action as a possible therapeutic to extend healthy lifespan, it was imperative to elucidate the effects of disrupting Ghr gene at a mature-adult age, well after the developmental and growth period of the mice. To this end, we hypothesized that removal of GH action in adult life would convey some of the same health and life span benefits seen in the GHRKO mice without the reduced body length. To test this hypothesis, we used the cre-lox system to generate mice with a disrupted Ghr gene at a mature-adult age (6 months), referred as 6mGHRKO mice. We then performed a phenotypic and lifespan characterization, and tested for molecular mechanisms known to be associated with extended longevity, namely oxidative stress resistance and mTOR modulation. We found that similar to GHRKO and 1.5mGHRKO mice, disruption of GHR at 6 months of age resulted in mice with increased adipose tissue mass, decreased lean mass, high circulating GH, but decreased insulin growth factor-1 levels compared to control mice. Furthermore, the 6mGHRKO mice displayed significantly improved insulin sensitivity in males, with no changes in glucose tolerance. Also, serum levels of inflammatory markers and liver triglycerides were unchanged in these mice. Experiments to evaluate the status of oxidative damage and mTOR activation in liver, skeletal muscle and subcutaneous adipose tissue of male and female 6mGHRKO mice showed a tissue-specificity and sexual dimorphism in these results. Importantly, male and female 6mGHRKO mice showed no change in body length, but mean, median and maximal lifespan were significantly extended in females. In conclusion, disruption of GH action well past sexual maturation produces beneficial effects on insulin sensitivity and aging in mice. Acknowledgements: This work is supported by the State of Ohio’s Eminent Scholar Program, by NIH grant AG059779 and by the AMVETS.
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Shehata, Medhat, Susanne Schnabl, Elena Ponath, Stefanie Tauber, Dita Demirtas, Martin Hilgarth, Martin Bilban et al. „Lumiliximab Triggers Apoptosis Mechanisms in CLL Cells through the Inhibition of PI3-K/Akt Pathway.“ Blood 114, Nr. 22 (20.11.2009): 2371. http://dx.doi.org/10.1182/blood.v114.22.2371.2371.
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Abstract Abstract 2371 Poster Board II-348 Chronic lymphocytic leukemia (CLL) is a clonal expansion of B cells which is characterized by a defect in apoptosis and is associated with the co-expression of CD19, CD5 and CD23. Lumiliximab is a monoclonal antibody against CD23 which has been shown to exert a promising therapeutic effect in CLL in vivo and to induce apoptosis in CD23+ lymphoma cells in vitro. The aim of this study was to investigate the direct effect of lumiliximab on cell viability and to explore its molecular mechanism of action in primary CLL cells. PBMC from twenty CLL patients were used in this study. Nine patients had previous therapy and 11 were untreated. Nine patients had unmutated and 11 had mutated IgVH genes. The mean percentage of CD19+/CD5+/CD23+ cells was 80% (range 53-98%). PBMC were exposed to various concentrations of lumiliximab (1-50 μg/mL) for various durations (1-15 days). The long term incubation with lumiliximab was performed in a microenvironment co-culture model using primary human stromal cells which prevent spontaneous apoptosis of CLL cells. Cell viability was assessed by flow cytometric analysis using annexin V/PI staining and by MTT assays. The results showed that single exposure to lumiliximab had a minimal effect on cell viability (< 1 fold increase in apoptosis rate compared to untreated cells and to the isotype control antibody). Cross-linking with goat anti-human IgG was more effective in inducing cell death. Interestingly, repeated exposure to lumiliximab without cross-linking had a significant pro-apoptotic effect selectively in the CD19+/CD5+ cells. Western blotting analysis demonstrated a significant biological response to the single and repeated exposure to lumiliximab in spite of the moderate pro-apoptotic effect. Lumiliximab induced a significant decrease in Bcl-2, Mcl-1 and Hsp70 protein expression. In addition, it resulted in a significant decrease in the phosphorylation of Akt1 at serine residue 473 and dephosphorylation (activation) of the tumor suppressor PTEN at serine residue 380 suggesting the involvement of the PI3-K/Akt/PTEN cascade in priming CLL cells to undergo apoptosis by lumiliximab. Pre-incubation of CLL cells with lumiliximab enhanced the pro-apoptotic effect of PI3-K inhibitor LY292004 and the CK2 inhibitor apigenin. Consistent with previous observations, pre-exposure of CLL cells to lumiliximab augmented the cytotoxic effect of Fludarabine. The in vitro response to lumiliximab did not appear to be influenced by IgVH mutation status, cytogenetics or by previous therapy. To gain further insight into the downstream targets of lumiliximab in CLL, microarray analysis and pathway exploration was performed. The data revealed that lumiliximab regulates several sets of genes which are involved in chemokine signaling, cytokine/cytokine receptor interaction, oxidative stress, PI3-K and integrin signaling, complement cascade and Toll-like receptor signaling. Single exposure to lumiliximab led to down-regulation of several genes including LY9, GPR183, RGS1, HSPA6, and INPP5F and up-regulation of FN1, FAIM3 and LOX. Repeated exposure to Lumiliximab led to a significant down-regulation of TXNIP, CTSB, SODS, IFI44, IRF7, TNFSF13B, MS4A7, CCL4, CCL7, CCL8, cathepsin B, MARKS, ADAMS and FCER1G and up-regulation of SPP1, C10orf10, ANGPTL6, RBBP4 and GSTA4. In conclusion, these data demonstrate that repeated exposure to Lumiliximab is effective in priming CLL cells to undergo apoptosis through inactivation of the PI3-K/Akt pathway and render the cells more sensitive to cytotoxic compounds. The data also provide further evidence of a promising therapeutic role for lumiliximab in CLL and a rationale for lumiliximab-based drug combinations to improve treatment of this incurable disease. Disclosures: Shehata: Biogen Idec: Research Funding. Hughes:Biogen Idec: Employment. Maclaren:Biogen Idec: Employment. Jaeger:Biogen Idec: Research Funding.
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Visconte, Valeria, Regis Peffault de Latour, Rodrigo T. Calado, Marie Desierto, Jichun Chen und Neal S. Young. „Reduced Hematopoietic Stem Cell Function in a Mouse Model with Conditional Pig-a Gene Deletion.“ Blood 114, Nr. 22 (20.11.2009): 2428. http://dx.doi.org/10.1182/blood.v114.22.2428.2428.
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Abstract Abstract 2428 Poster Board II-405 Hematopoietic stem cells (HSCs) are capable of self-renewal as well as differentiation to produce progenitor cells of all hematopoietic lineages in order to sustain life long blood production. In normal inbred mice, HSCs survive multiple rounds of transplantation and extend function to over 100 months, 3-4 times the lifespan of a normal mouse (Harrison DE, Mech Ageing Dev, 1973). We assessed HSC functions in a murine model of paroxysmal nocturnal hemoglobinuria (PNH). PNH originates with a somatic mutation in a HSC of the X-linked phosphatydylinositol glycan class A (PIG-A) gene. We produced a mouse model with conditional Pig-a deletion (Pig-a-/-) by cross-breeding mice carrying germline insertion of two lox sites flanking exon 6 of Pig-a gene with mice carrying the transgene Cre-recombinase driven by the human c-fes promoter. The resultant B6 Fes-cre-Pig-aflox (Pig-a-/-) mice showed Pig-a gene inactivation specifically in hematopoietic cells. Mice survived without obvious abnormalities and did not show any sign of clinical PNH or bone marrow (BM) failure. In preliminary experiments, we confirmed that BM cells from Pig-a-/- donors had normal HSC functional ability to compete with BM cells from congenic B6-CD45.1 competitors in a competitive repopulation assay, as previously shown by another PNH mouse model with conditional Pig-a gene deletion (Keller P et al, J Exp Med, 2001). We then tested whether Pig-a deletion and the resultant GPI deficiency might affect HSC function long term after serial transplantation. Toward this end, we incubated BM cells from Pig-a-/- donors with aerolysin to lyse GPI-normal cells (GPI+) and then transplanted the residual GPI-deficient (GPI-) cells into lethally-irradiated normal C57BL/6 recipients. Within two months, GPI- BM cells engrafted efficiently in all recipients, and 95-100% of recipient T, B and granulocytes were GPI- cells by flow cytometry. After 11 months, we serially-transplanted the GPI- BM cells into lethally-irradiated secondary recipients. Phenotype analysis at one and three months after secondary transplantation also showed relative stable GPI- cell engraftment in T cells (29 ± 9% and 69 ± 29%), B cells (79 ± 6% and 72 ± 15%) and granulocytes (74 ± 11% and 70 ± 10%). After 8 months in the secondary recipients, we extracted BM cells from three secondary recipients with >70% GPI- cells in T, B, and granulocytes. Flow cytometry analysis showed that their BM cellularity was normal, but marrows contained three to five fold lower proportion of cells staining for the Kit+Sca1+Lin- (KSL) markers. We pooled BM cells from these three secondary recipients and transplanted BM cells into lethally-irradiated third-round recipients. Three out of five recipients did not survive transplantation and died between one and two weeks after irradiation/transplantation. The remaining two recipients showed <5% GPI- cells at two months and <2% GPI- cells at seven months following transplantation, suggesting that functional ability of GPI- BM cells had been diminished or drastically reduced. In order to test the competitive repopulating capacity, we also used pooled BM cells from secondary recipients in a competitive repopulation assay, congenic B6-CD45.1 BM cells serving as competitors. There was essentially no engraftment from GPI- donor cells in all recipients, when measured at two and seven months following transplantation, indicating that, after 19 months and two rounds of transplantation, GPI- BM cells were unable to compete with control BM cells for engraftment. In conclusion, Pig-a deletion and lack of GPI-anchored proteins may cause reduced HSC function long-term. Thus, the expansion of the mutant HSC clone seen in PNH patients is unlikely related to any intrinsic self-renewal or proliferative advantage of the HSC clone carrying Pig-a mutation. Disclosures: No relevant conflicts of interest to declare.
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Visconte, Valeria, Nalini Raghavachari, Keyvan Keyvanfar, Delong Liu, Marie Desierto, Jichun Chen und Neal S. Young. „Clonally-Restricted T-Lymphocytes in PigA Mutant Mice.“ Blood 112, Nr. 11 (16.11.2008): 2040. http://dx.doi.org/10.1182/blood.v112.11.2040.2040.
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Abstract Somatic mutation in the X-linked phosphatydylinositol glycan class A (PIG-A) gene causes glycosyl phosphatidylinositol (GPI) anchor deficiency in hematopoietic stem and progenitor cells, in humans, a requirement for the development of the disease paroxysmal nocturnal hemoglobinuria (PNH). While progress has been made in understanding PNH and especially in treatment of intravascular hemolysis secondary to cell surface deficiency of CD59, why PIG-A mutant stem cells expand in the setting of immune-mediated bone marrow failure remains obscure. We produced a conditional PigA knock-out animal model (PigA−/−) by cross-breeding mice carrying germline insertion of two lox sites flanking exon 6 of PigA gene with mice carrying the transgene Cre-recombinase driven by the human c-fes promoter. The resultant B6 Fes-cre PigAflox (PigA−/−) mice had PigA gene inactivation specifically in hematopoietic cells. We observed that GPI-deficient (GPI−) bone marrow (BM) and spleen cells from PigA−/− mice contained much larger proportions of lymphocytes, especially CD8+ T cells, in comparison to GPI+ cells. The expansion of GPI−CD8+ T cells was not associated with any obvious hematological phenotype, and blood and BM cell counts were relatively normal in PigA−/− mice. In comparison to GPI+ cells analyzed by microarray, GPI− BM cells showed up-regulation in expression of genes important for immune function responses. Pathway analysis revealed that differentially-expressed genes were clustered in several groups related to immunological function, such as lymphocyte markers (CD8b1, CD8a, CD3e, CD3d, CD7, CD2, CD5, CD6, CD28, CD96, CD27), proteins related to T cell activation (Lck, Zap70, Fyn, Zeta, Lat, Traf1, Tcf7, Ctla2a/Ctla2b), TCR components (Tcr-beta-V13, Tcr-beta-V8.2, Tcr-alpha, Tcr-beta- J, Tcr-gamma), chemokines and C-C motifs (Ccl5, CXCR6, Ccr7), and molecules of the killer lectin-like receptor subfamily (Klrc1, Klrc2, Klra7, Klra8). We transplanted into lethally-irradiated recipients BM cells from PigA−/− mice (pre-incubated with aerolysin to lyse GPI+ cells) or BM cells from normal PigA+/+ donors. By microarray, transplanted GPI− cells retained the phenotype of untransplanted GPI− cells, with a much increased CD8+ T cell proportion and up-regulated immune function gene expression in comparison to transplanted normal BM cells. The enlarged GPI−CD8+ T cell pool had a significantly lower proportion of CD11a+ cells than did GPI+CD8+ T cells, suggesting that GPI−CD8+ T cells were generally less active. There was no difference in the proportion of CD44− naive T cells between GPI−CD8+ and GPI+CD8+ T cells; GPI−CD8+ T cells were not NK cells as they lacked surface NK1.1 staining. The percentage of CD4+CD25+FoxP3+ regulatory T cells in GPI− cells was only 10% of that in GPI+ cells in peripheral blood in both untransplanted and transplanted animals, indicating that the expanded T cell population in the GPI− cell fraction contained few cells with immunosuppressive property. We further investigated T cell clonality by usage of T cell receptor beta variable region (Vbeta); approximately 5-6 Vbeta subfamilies were over represented in the GPI− CD8+ T cells. In particular, Vbeta 5.1/5.2 was prominent in GPI−CD8+ T cells, constituting 22-23 ± 5% GPI− T cells from untransplanted and transplanted animals; a significant increase in comparison to 8-9.1 ± 0.5% Vbeta 5.1/5.2 clonal representation in GPI+CD8+ T cells. Our results are consistent with an antigen-driven T cell response in the GPI− lymphocyte population, independent of pancytopenia. Functionally, GPI−CD8 T cells showed no response to lectin stimulation as measured by gamma interferon production, but they were capable of effecting target cell apoptosis when co-incubated with minor-H antigen mismatched BM cells in vitro. Our data agrees with observations in humans, in which an immune process driven by a restricted set of (unknown) antigens appears active in the pathogenesis of PNH (Gargiulo et al., Blood 2007). We conclude that deletion of PigA gene in hematopoietic cells, independent of frank hematopoietic failure, leads to enrichment of lymphocytes, especially CD8 T cells, in the GPI− cell fraction that have an inactive and naive phenotype. These expanded, clonally-restricted, T cells may provide an initial pool of immune effectors, which in the proper immune activated environment, contribute to bone marrow failure in PNH.
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Kotini, Andriana, Ibrahim Boussaad und Eirini P. Papapetrou. „Chromosome 7q Hemizygosity Recapitulates MDS-Related Cellular Phenotypes In Genetically Engineered Human Pluripotent Stem Cells“. Blood 122, Nr. 21 (15.11.2013): 862. http://dx.doi.org/10.1182/blood.v122.21.862.862.
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Abstract Loss of the entire or part of one copy of chromosome 7 [del(7/7q)] is a recurrent cytogenetic abnormality in MDS. Its strong association with previous exposure to alkylating agents, consistently poor response to therapy and discrete gene expression profile strongly suggest that del(7/7q)-MDS is a distinct disease in the MDS spectrum whose pathogenesis is intimately linked to loss of chr7q genetic material. Understanding the role of chr7q loss in cell biology can provide key insights into the pathogenesis of MDS and leukemogenesis. Narrowing down the responsible region on chr7q presents a challenge that has proved intractable with existing approaches. Chr7q deletions physically mapped in large cohorts of patients are typically very large and dispersed along most of the length of chr7q. Modeling in the mouse is problematic for reasons of synteny. Deletion of the syntenic region of one commonly deleted region (7q22 CDR) failed to demonstrate a phenotype. Several lines of evidence point to haploinsufficiency of chr7q genetic material – rather than a 2-hit model – as the underlying mechanism in del(7q)-MDS. No inactivating mutations of candidate 7q genes have been detected by resequencing the remaining allele and haploinsufficiency of coding and miRNA genes has been strongly linked to the pathogenesis of del(5q)-MDS. Haploinsufficiency can only be assessed through functional studies (and not genomic technologies), but these are currently hindered by the lack of a clearly described del(7q)-associated phenotype. With recent advances in human pluripotent stem cell (hPSC) research and genetic engineering technologies, reverse human genetics in an isogenic setting by disruption of genomic elements into their cognate genomic and cellular context - hitherto unthinkable for the human genome – are now a realistic prospect. To determine the impact of hemizygous chr7q loss on the cellular phenotype, we have generated isogenic del(7q)- and normal hPSCs by engineering deletions spanning variable overlapping regions encompassing the entire length of chr7q using adeno-associated virus (AAV)-mediated gene targeting combined with Cre-lox technology. Specifically, we targeted two inverted loxP sites together with a positive (puro) and a negative (HSVtk) selection marker in a near-telomeric region of chromosome 7q (7q36.3) into the H1 hESC line, as well as a karyotypically normal iPSC line (line 2-12) derived from BMMCs of a patient with del(7q)-MDS. Following transient expression of Cre recombinase and ganciclovir selection, clones were screened by qPCR probing different regions along the length of chr7. Four H1-derived and four 2-12-derived clones were selected following screening of 24 and 34 clones, respectively, and after excluding clones with additional chromosomal abnormalities by karyotyping and the exact extent of their chr7q deletions was mapped by aCGH. We focused our phenotypic characterization on two cellular phenotypes that we recently reported in del(7q)-iPSCs derived from MDS BMMCs: cell proliferation and in vitro hematopoietic differentiation potential. 7 of the 8 clones harboring deletions spanning variable lengths along the entire chr7q had a lower (by ½ log) proliferation rate than their corresponding isogenic parental lines. All these clones also exhibited a markedly reduced differentiation potential along all hematopoietic lineages and almost absent clonogenic capacity in methylcellulose. These cellular phenotypes are highly similar to those we find in our del(7q)-MDS-iPSCs. Notably, one of the eight clones (2-12.Cre-44), harboring a smaller deletion spanning 7q11.21-7q.31.1 retained comparable proliferation and differentiation capacity to that of normal isogenic and non-isogenic hPSCs. In conclusion, our results demonstrate that hemizygous loss of chr7q material recapitulates the cellular phenotypes of impaired proliferation and hematopoietic differentiation that we find in del(7q)-MDS-iPSCs, supporting a haploinsufficiency pathogenesis of del(7q)-MDS. Correlation of the phenotypes with the boundaries of chr7q deletions in our collection of hESC and iPSC clones points to a region spanning 7q31.1-7q36.1 as the critical region in del(7q)-MDS. Further studies in additional clones harboring smaller chr7q deletions will further narrow down the responsible region and guide prioritization of candidate genes. Disclosures: No relevant conflicts of interest to declare.