Estrogen-dependent dynamic profile of eNOS-DNA associations in prostate cancer

In previous work we have documented the nuclear translocation of endothelial NOS (eNOS) and its participation in combinatorial complexes with Estrogen Receptor Beta (ERβ) and Hypoxia Inducible Factors (HIFs) that determine localized chromatin remodeling in response to estrogen (E2) and hypoxia stimuli, resulting in transcriptional regulation of genes associated with adverse prognosis in prostate cancer (PCa). To explore the role of nuclear eNOS in the acquisition of aggressive phenotype in PCa, we performed ChIP-Sequencing on chromatin-associated eNOS from cells from a primary tumor with poor outcome and from metastatic LNCaP cells. We found that: 1. the eNOS-bound regions (peaks) are widely distributed across the genome encompassing multiple transcription factors binding sites, including Estrogen Response Elements. 2. E2 increased the number of peaks, indicating hormone-dependent eNOS re-localization. 3. Peak distribution was similar with/without E2 with ≈ 55% of them in extragenic DNA regions and an intriguing involvement of the 5′ domain of several miRs deregulated in PCa. Numerous potentially novel eNOS-targeted genes have been identified suggesting that eNOS participates in the regulation of large gene sets. The parallel finding of downregulation of a cluster of miRs, including miR-34a, in PCa cells associated with poor outcome led us to unveil a molecular link between eNOS and SIRT1, an epigenetic regulator of aging and tumorigenicity, negatively regulated by miR-34a and in turn activating eNOS. E2 potentiates miR-34a downregulation thus enhancing SIRT1 expression, depicting a novel eNOS/SIRT1 interplay fine-tuned by E2-activated ER signaling, and suggesting that eNOS may play an important role in aggressive PCa

In Silico Evidence for Gluconeogenesis from Fatty Acids in Humans

The question whether fatty acids can be converted into glucose in humans has a long standing tradition in biochemistry, and the expected answer is “No”. Using recent advances in Systems Biology in the form of large-scale metabolic reconstructions, we reassessed this question by performing a global investigation of a genome-scale human metabolic network, which had been reconstructed on the basis of experimental results. By elementary flux pattern analysis, we found numerous pathways on which gluconeogenesis from fatty acids is feasible in humans. On these pathways, four moles of acetyl-CoA are converted into one mole of glucose and two moles of CO2. Analyzing the detected pathways in detail we found that their energetic requirements potentially limit their capacity. This study has many other biochemical implications: effect of starvation, sports physiology, practically carbohydrate-free diets of inuit, as well as survival of hibernating animals and embryos of egg-laying animals. Moreover, the energetic loss associated to the usage of gluconeogenesis from fatty acids can help explain the efficiency of carbohydrate reduced and ketogenic diets such as the Atkins diet

MUC4 activates HER2 signalling and enhances the motility of human ovarian cancer cells

The mucin MUC4 is a high molecular weight transmembrane glycoprotein. It consists of a mucin-type subunit (MUC4α) and a transmembrane growth factor-like subunit (MUC4β). The mucin MUC4 is overexpressed in many epithelial malignancies including ovarian cancer, suggesting a possible role in the pathogenesis of these cancers. In this study, we investigated the functional role of MUC4 in the human ovarian cancer cell line SKOV3. The mucin MUC4 was ectopically expressed by stable transfection, and its expression was examined by western blot and confocal microscopy analyses. The in vitro studies demonstrated an enhanced motility of MUC4-expressing SKOV3 cells compared with the vector-transfected cells. The mucin MUC4 expression was associated with apparent changes in actin organisation, leading to the formation of microspike, lammelopodia and filopodia-like cellular projections. An enhanced protein expression and activation of HER2, a receptor tyrosine kinase, was also seen, although no significant change was observed in HER-2 transcript levels in the MUC4-transfected SKOV3 cells. Reciprocal co-immunoprecipitation revealed an interaction of MUC4 with HER2. Further, the MUC4-overexpressing SKOV3 cells exhibited an increase in the phosphorylation of focal adhesion kinase (FAK), Akt and ERK, downstream effectors of HER2. Taken together, our findings demonstrate that MUC4 plays a role in ovarian cancer cell motility, in part, by altering actin arrangement and potentiating HER2 downstream signalling in these cells

Antibodies to Serine Proteases in the Antiphospholipid Syndrome

It is generally accepted that the major autoantigen for antiphospholipid antibodies (aPL) in the antiphospholipid syndrome (APS) is β2-glycoprotein I (β2GPI). However, a recent study has revealed that some aPL bind to certain conformational epitope(s) on β2GPI shared by the homologous enzymatic domains of several serine proteases involved in hemostasis and fibrinolysis. Importantly, some serine protease–reactive aPL correspondingly hinder anticoagulant regulation and resolution of clots. These results extend several early findings of aPL binding to other coagulation factors and provide a new perspective about some aPL in terms of binding specificities and related functional properties in promoting thrombosis. Moreover, a recent immunological and pathological study of a panel of human IgG monoclonal aPL showed that aPL with strong binding to thrombin promote in vivo venous thrombosis and leukocyte adherence, suggesting that aPL reactivity with thrombin may be a good predictor for pathogenic potentials of aPL

The direct effect of Focal Adhesion Kinase (FAK), dominant-negative FAK, FAK-CD and FAK siRNA on gene expression and human MCF-7 breast cancer cell tumorigenesis

<p>Abstract</p> <p>Background</p> <p>Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that plays an important role in survival signaling. FAK has been shown to be overexpressed in breast cancer tumors at early stages of tumorigenesis.</p> <p>Methods</p> <p>To study the direct effect of FAK on breast tumorigenesis, we developed Tet-ON (tetracycline-inducible) system of MCF-7 breast cancer cells stably transfected with FAK or dominant-negative, C-terminal domain of FAK (FAK-CD), and also FAKsiRNA with silenced FAK MCF-7 stable cell line. Increased expression of FAK in isogenic Tet-inducible MCF-7 cells caused increased cell growth, adhesion and soft agar colony formation <it>in vitro</it>, while expression of dominant-negative FAK inhibitor caused inhibition of these cellular processes. To study the role of induced FAK and FAK-CD <it>in vivo</it>, we inoculated these Tet-inducible cells in nude mice to generate tumors in the presence or absence of doxycycline in the drinking water. FAKsiRNA-MCF-7 cells were also injected into nude mice to generate xenograft tumors.</p> <p>Results</p> <p>Induction of FAK resulted in significant increased tumorigenesis, while induced FAK-CD resulted in decreased tumorigenesis. Taq Man Low Density Array assay demonstrated specific induction of FAKmRNA in MCF-7-Tet-ON-FAK cells. DMP1, encoding cyclin D binding myb-like protein 1 was one of the genes specifically affected by Tet-inducible FAK or FAK-CD in breast xenograft tumors. In addition, silencing of FAK in MCF-7 cells with FAK siRNA caused increased cell rounding, decreased cell viability <it>in vitro </it>and inhibited tumorigenesis <it>in vivo</it>. Importantly, Affymetrix microarray gene profiling analysis using Human Genome U133A GeneChips revealed >4300 genes, known to be involved in apoptosis, cell cycle, and adhesion that were significantly down- or up-regulated (p < 0.05) by FAKsiRNA.</p> <p>Conclusion</p> <p>Thus, these data for the first time demonstrate the direct effect of FAK expression and function on MCF-7 breast cancer tumorigenesis <it>in vivo </it>and reveal specific expression of genes affected by silencing of FAK.</p

Oligonucleotide Sequence Motifs as Nucleosome Positioning Signals

To gain a better understanding of the sequence patterns that characterize positioned nucleosomes, we first performed an analysis of the periodicities of the 256 tetranucleotides in a yeast genome-wide library of nucleosomal DNA sequences that was prepared by in vitro reconstitution. The approach entailed the identification and analysis of 24 unique tetranucleotides that were defined by 8 consensus sequences. These consensus sequences were shown to be responsible for most if not all of the tetranucleotide and dinucleotide periodicities displayed by the entire library, demonstrating that the periodicities of dinucleotides that characterize the yeast genome are, in actuality, due primarily to the 8 consensus sequences. A novel combination of experimental and bioinformatic approaches was then used to show that these tetranucleotides are important for preferred formation of nucleosomes at specific sites along DNA in vitro. These results were then compared to tetranucleotide patterns in genome-wide in vivo libraries from yeast and C. elegans in order to assess the contributions of DNA sequence in the control of nucleosome residency in the cell. These comparisons revealed striking similarities in the tetranucleotide occurrence profiles that are likely to be involved in nucleosome positioning in both in vitro and in vivo libraries, suggesting that DNA sequence is an important factor in the control of nucleosome placement in vivo. However, the strengths of the tetranucleotide periodicities were 3–4 fold higher in the in vitro as compared to the in vivo libraries, which implies that DNA sequence plays less of a role in dictating nucleosome positions in vivo. The results of this study have important implications for models of sequence-dependent positioning since they suggest that a defined subset of tetranucleotides is involved in preferred nucleosome occupancy and that these tetranucleotides are the major source of the dinucleotide periodicities that are characteristic of positioned nucleosomes

Contributions of Histone H3 Nucleosome Core Surface Mutations to Chromatin Structures, Silencing and DNA Repair

Histone H3 mutations in residues that cluster in a discrete region on the nucleosome surface around lysine 79 of H3 affect H3-K79 methylation, impair transcriptional silencing in subtelomeric chromatin, and reveal distinct contributions of histone H3 to various DNA-damage response and repair pathways. These residues might act by recruitment of silencing and DNA-damage response factors. Alternatively, their location on the nucleosome surface suggests a possible involvement in nucleosome positioning, stability and nucleosome interactions. Here, we show that the yeast H3 mutants hht2-T80A, hht2-K79E, hht2-L70S, and hht2-E73D show normal nucleosome positioning and stability in minichromosomes. However, loss of silencing in a subtelomeric URA3 gene correlates with a shift of the promoter nucleosome, while nucleosome positions and stability in the coding region are maintained. Moreover, the H3 mutants show normal repair of UV lesions by photolyase and nucleotide excision repair in minichromosomes and slightly enhanced repair in the subtelomeric region. Thus, these results support a role of those residues in the recruitment of silencing proteins and argue against a general role in nucleosome organization

Genetic Diversity among Enterococcus faecalis

Enterococcus faecalis, a ubiquitous member of mammalian gastrointestinal flora, is a leading cause of nosocomial infections and a growing public health concern. The enterococci responsible for these infections are often resistant to multiple antibiotics and have become notorious for their ability to acquire and disseminate antibiotic resistances. In the current study, we examined genetic relationships among 106 strains of E. faecalis isolated over the past 100 years, including strains identified for their diversity and used historically for serotyping, strains that have been adapted for laboratory use, and isolates from previously described E. faecalis infection outbreaks. This collection also includes isolates first characterized as having novel plasmids, virulence traits, antibiotic resistances, and pathogenicity island (PAI) components. We evaluated variation in factors contributing to pathogenicity, including toxin production, antibiotic resistance, polymorphism in the capsule (cps) operon, pathogenicity island (PAI) gene content, and other accessory factors. This information was correlated with multi-locus sequence typing (MLST) data, which was used to define genetic lineages. Our findings show that virulence and antibiotic resistance traits can be found within many diverse lineages of E. faecalis. However, lineages have emerged that have caused infection outbreaks globally, in which several new antibiotic resistances have entered the species, and in which virulence traits have converged. Comparing genomic hybridization profiles, using a microarray, of strains identified by MLST as spanning the diversity of the species, allowed us to identify the core E. faecalis genome as consisting of an estimated 2057 unique genes