A selective inhibitor of ceramide synthase 1 reveals a novel role in fat metabolism

Specific forms of the lipid ceramide, synthesized by the ceramide synthase enzyme family, are believed to regulate metabolic physiology. Genetic mouse models have established C16 ceramide as a driver of insulin resistance in liver and adipose tissue. C18 ceramide, synthesized by ceramide synthase 1 (CerS1), is abundant in skeletal muscle and suggested to promote insulin resistance in humans. We herein describe the first isoform-specific ceramide synthase inhibitor, P053, which inhibits CerS1 with nanomolar potency. Lipidomic profiling shows that P053 is highly selective for CerS1. Daily P053 administration to mice fed a high-fat diet (HFD) increases fatty acid oxidation in skeletal muscle and impedes increases in muscle triglycerides and adiposity, but does not protect against HFD-induced insulin resistance. Our inhibitor therefore allowed us to define a role for CerS1 as an endogenous inhibitor of mitochondrial fatty acid oxidation in muscle and regulator of whole-body adiposity

Regulation of expression of Na+,K+-ATPase in androgen-dependent and androgen-independent prostate cancer

The β1-subunit of Na+,K+-ATPase was isolated and identified as an androgen down-regulated gene. Expression was observed at high levels in androgen-independent as compared to androgen-dependent (responsive) human prostate cancer cell lines and xenografts when grown in the presence of androgens. Down-regulation of the β1-subunit was initiated at concentrations between 0.01 nM and 0.03 nM of the synthetic androgen R1881 after relatively long incubation times (> 24 h). Using polyclonal antibodies, the concentration of β1-subunit protein, but not of the α1-subunit protein, was markedly reduced in androgen-dependent human prostate cancer cells (LNCaP-FGC) cultured in the presence of androgens. In line with these observations it was found that the protein expression of total Na+,K+-ATPase in the membrane (measured by 3H-ouabain binding) was also markedly decreased. The main function of Na+,K+-ATPase is to maintain sodium and potassium homeostasis in animal cells. The resulting electrochemical gradient is facilitative for transport of several compounds over the cell membrane (for example cisplatin, a chemotherapeutic agent experimentally used in the treatment of hormone-refractory prostate cancer). Here we observed that a ouabain-induced decrease of Na+,K+-ATPase activity in LNCaP-FGC cells results in reduced sensitivity of these cells to cisplatin-treatment. Surprisingly, androgen-induced decrease of Na+,K+-ATPase expression, did not result in significant protection against the chemotherapeutic agent. © 1999 Cancer Research Campaig

Disrupted postnatal lung development in heme oxygenase-1 deficient mice

BACKGROUND: Heme oxygenase (HO) degrades cellular heme to carbon monoxide, iron and biliverdin. The HO-1 isoform is both inducible and cyto-protective during oxidative stress, inflammation and lung injury. However, little is known about its precise role and function in lung development. We hypothesized that HO-1 is required for mouse postnatal lung alveolar development and that vascular expression of HO-1 is essential and protective during postnatal alveolar development. METHODS: Neonatal lung development in wildtype and HO-1 mutant mice was evaluated by histological and molecular methods. Furthermore, these newborn mice were treated with postnatal dexamethasone (Dex) till postnatal 14 days, and evaluated for lung development. RESULTS: Compared to wildtype littermates, HO-1 mutant mice exhibited disrupted lung alveolar structure including simplification, disorganization and reduced secondary crest formation. These defects in alveolar development were more pronounced when these mice were challenged with Dex treatment. Expression levels of both vascular endothelial and alveolar epithelial markers were also further decreased in HO-1 mutants after Dex treatment. CONCLUSIONS: These experiments demonstrate that HO-1 is required in normal lung development and that HO-1 disruption and dexamethasone exposure are additive in the disruption of postnatal lung growth. We speculate that HO-1 is involved in postnatal lung development through modulation of pulmonary vascular development

Expression of CD11c Is Associated with Unconventional Activated T Cell Subsets with High Migratory Potential

Ajudes rebudes: Marie Curie Career Integration Grant; Dexeus Foundation for Women's Health Research; i Contratos Ramón y CajalCD11c is an α integrin classically employed to define myeloid dendritic cells. Although there is little information about CD11c expression on human T cells, mouse models have shown an association of CD11c expression with functionally relevant T cell subsets. In the context of genital tract infection, we have previously observed increased expression of CD11c in circulating T cells from mice and women. Microarray analyses of activated effector T cells expressing CD11c derived from naïve mice demonstrated enrichment for natural killer (NK) associated genes. Here we find that murine CD11c+ T cells analyzed by flow cytometry display markers associated with non-conventional T cell subsets, including γδ T cells and invariant natural killer T (iNKT) cells. However, in women, only γδ T cells and CD8+ T cells were enriched within the CD11c fraction of blood and cervical tissue. These CD11c+ cells were highly activated and had greater interferon (IFN)-γ secretory capacity than CD11c- T cells. Furthermore, circulating CD11c+ T cells were associated with the expression of multiple adhesion molecules in women, suggesting that these cells have high tissue homing potential. These data suggest that CD11c expression distinguishes a population of circulating T cells during bacterial infection with innate capacity and mucosal homing potential

Genome-wide linkage analysis of 1,233 prostate cancer pedigrees from the International Consortium for prostate cancer Genetics using novel sumLINK and sumLOD analyses

BACKGROUND Prostate cancer (PC) is generally believed to have a strong inherited component, but the search for susceptibility genes has been hindered by the effects of genetic heterogeneity. The recently developed sumLINK and sumLOD statistics are powerful tools for linkage analysis in the presence of heterogeneity. METHODS We performed a secondary analysis of 1,233 PC pedigrees from the International Consortium for Prostate Cancer Genetics (ICPCG) using two novel statistics, the sumLINK and sumLOD. For both statistics, dominant and recessive genetic models were considered. False discovery rate (FDR) analysis was conducted to assess the effects of multiple testing. RESULTS Our analysis identified significant linkage evidence at chromosome 22q12, confirming previous findings by the initial conventional analyses of the same ICPCG data. Twelve other regions were identified with genome-wide suggestive evidence for linkage. Seven regions (1q23, 5q11, 5q35, 6p21, 8q12, 11q13, 20p11–q11) are near loci previously identified in the initial ICPCG pooled data analysis or the subset of aggressive PC pedigrees. Three other regions (1p12, 8p23, 19q13) confirm loci reported by others, and two (2p24, 6q27) are novel susceptibility loci. FDR testing indicates that over 70% of these results are likely true positive findings. Statistical recombinant mapping narrowed regions to an average of 9 cM. CONCLUSIONS Our results represent genomic regions with the greatest consistency of positive linkage evidence across a very large collection of high-risk PC pedigrees using new statistical tests that deal powerfully with heterogeneity. These regions are excellent candidates for further study to identify PC predisposition genes. Prostate 70: 735–744, 2010. © 2010 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/71371/1/21106_ftp.pd

Comparative effects of RRR-alpha- and RRR-gamma-tocopherol on proliferation and apoptosis in human colon cancer cell lines

BACKGROUND: Mediterranean societies, with diets rich in vitamin E isoforms, have a lower risk for colon cancer than those of northern Europe and the Americas. Vitamin E rich diets may neutralize free radicals generated by fecal bacteria in the gut and prevent DNA damage, but signal transduction activities can occur independent of the antioxidant function. The term vitamin E represents eight structurally related compounds, each differing in their potency and mechanisms of chemoprevention. The RRR-γ-tocopherol isoform is found primarily in the US diet, while RRR-α-tocopherol is highest in the plasma. METHODS: The effectiveness of RRR-α- and RRR-γ-tocopherol at inhibiting cell growth and inducing apoptosis in colon cancer cell lines with varying molecular characteristics (SW480, HCT-15, HCT-116 and HT-29) and primary colon cells (CCD-112CoN, nontransformed normal phenotype) was studied. Colon cells were treated with and without RRR-α- or RRR-γ-tocopherol using varying tocopherol concentrations and time intervals. Cell proliferation and apoptosis were measured using the trypan blue assay, annexin V staining, DNA laddering and caspase activation. RESULTS: Treatment with RRR-γ-tocopherol resulted in significant cell death for all cancer cell lines tested, while RRR-α-tocopherol did not. Further, RRR-γ-tocopherol treatment showed no cytotoxicity to normal colon cells CCD-112CoN at the highest concentration and time point tested. RRR-γ-tocopherol treatment resulted in cleavage of PARP, caspase 3, 7, and 8, but not caspase 9. Differences in the percentage cell death and apoptosis were observed in different cell lines suggesting that molecular differences in these cell lines may influence the ability of RRR-γ-tocopherol to induce cell death. CONCLUSION: This is the first study to demonstrate that multiple colon cancer cell lines containing varying genetic alterations will under go growth reduction and apoptosis in the presence of RRR-γ-tocopherol without damage to normal colon cells. The amount growth reduction was dependent upon the molecular signatures of the cell lines. Since RRR-γ-tocopherol is effective at inhibition of cell proliferation at both physiological and pharmacological concentrations dietary RRR-γ-tocopherol may be chemopreventive, while pharmacological concentrations of RRR-γ-tocopherol may aid chemotherapy without toxic effects to normal cells demonstrated by most chemotherapeutic agents

Wnt/β-Catenin Signaling Induces the Aging of Mesenchymal Stem Cells through the DNA Damage Response and the p53/p21 Pathway

Recent studies have demonstrated the importance of cellular extrinsic factors in the aging of adult stem cells. However, the effects of an aged cell–extrinsic environment on mesenchymal stem cell (MSC) aging and the factors involved remain unclear. In the current study, we examine the effects of old rat serum (ORS) on the aging of MSCs, and explore the effects and mechanisms of Wnt/β-catenin signaling on MSC aging induced by ORS treatment. Senescence-associated changes in the cells are examined with SA-β-galactosidase staining and ROS staining. The proliferation ability is detected by MTT assay. The surviving and apoptotic cells are determined using AO/EB staining. The results suggest that ORS promotes MSC senescence and reduces the proliferation and survival of cells. The immunofluorescence staining shows that the expression of β-catenin increases in MSCs of old rats. To identify the effects of Wnt/β-catenin signaling on MSC aging induced with ORS, the expression of β-catenin, GSK-3β, and c-myc are detected. The results show that the Wnt/β-catenin signaling in the cells is activated after ORS treatment. Then we examine the aging, proliferation, and survival of MSCs after modulating Wnt/β-catenin signaling. The results indicate that the senescence and dysfunction of MSCs in the medium containing ORS is reversed by the Wnt/β-catenin signaling inhibitor DKK1 or by β-catenin siRNA. Moreover, the expression of γ-H2A.X, a molecular marker of DNA damage response, p16INK4a, p53, and p21 is increased in senescent MSCs induced with ORS, and is also reversed by DKK1 or by β-catenin siRNA. In summary, our study indicates the Wnt/β-catenin signaling may play a critical role in MSC aging induced by the serum of aged animals and suggests that the DNA damage response and p53/p21 pathway may be the main mediators of MSC aging induced by excessive activation of Wnt/β-catenin signaling

Inhibition of Monkeypox virus replication by RNA interference

The Orthopoxvirus genus of Poxviridae family is comprised of several human pathogens, including cowpox (CPXV), Vaccinia (VACV), monkeypox (MPV) and Variola (VARV) viruses. Species of this virus genus cause human diseases with various severities and outcome ranging from mild conditions to death in fulminating cases. Currently, vaccination is the only protective measure against infection with these viruses and no licensed antiviral drug therapy is available. In this study, we investigated the potential of RNA interference pathway (RNAi) as a therapeutic approach for orthopox virus infections using MPV as a model. Based on genome-wide expression studies and bioinformatic analysis, we selected 12 viral genes and targeted them by small interference RNA (siRNA). Forty-eight siRNA constructs were developed and evaluated in vitro for their ability to inhibit viral replication. Two genes, each targeted with four different siRNA constructs in one pool, were limiting to viral replication. Seven siRNA constructs from these two pools, targeting either an essential gene for viral replication (A6R) or an important gene in viral entry (E8L), inhibited viral replication in cell culture by 65-95% with no apparent cytotoxicity. Further analysis with wild-type and recombinant MPV expressing green fluorescence protein demonstrated that one of these constructs, siA6-a, was the most potent and inhibited viral replication for up to 7 days at a concentration of 10 nM. These results emphasis the essential role of A6R gene in viral replication, and demonstrate the potential of RNAi as a therapeutic approach for developing oligonucleotide-based drug therapy for MPV and other orthopox viruses

Mega-evolutionary dynamics of the adaptive radiation of birds

The origin and expansion of biological diversity is regulated by both developmental trajectories and limits on available ecological niches. As lineages diversify, an early and often rapid phase of species and trait proliferation gives way to evolutionary slow- downs as new species pack into ever more densely occupied regions of ecological niche space. Small clades such as Darwin’s finches demonstrate that natural selection is the driving force of adaptive radiations, but how microevolutionary processes scale up to shape the expansion of phenotypic diversity over much longer evolutionary timescales is unclear. Here we address this problem on a global scale by analysing a crowd-sourced dataset of three-dimensional scanned bill morphology from more than 2,000 species. We find that bill diversity expanded early in extant avian evolutionary history, before transitioning to a phase dominated by packing of morphological space. However, this early phenotypic diversification is decoupled from temporal variation in evolutionary rate: rates of bill evolution vary among lineages but are comparatively stable through time. We find that rare, but major, discontinuities in phenotype emerge from rapid increases in rate along single branches, sometimes leading to depauperate clades with unusual bill morphologies. Despite these jumps between groups, the major axes of within-group bill-shape evolution are remarkably consistent across birds. We reveal that macroevolutionary processes underlying global-scale adaptive radiations support Darwinian and Simpsonian ideas of microevolution within adaptive zones and accelerated evolution between distinct adaptive peaks