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  • RhoA-mediated Ca2+ sensitization in erectile function. 12060659

    A Rho-kinase inhibitor increases corpus cavernosum (CC) pressure in an in vivo rat model (Chitaley, K., Wingard, C. J., Webb, R. C., Branam, H., Stopper, V. S., Lewis, R. W., and Mills, T. M. (2001) Nat. Med. 7, 119-122) suggesting that Rho-mediated Ca(2+) sensitization of CC smooth muscle maintains the flaccid (contracted) state. We directly demonstrate Ca(2+) sensitization of permeabilized rabbit and human CC and identify a highly expressed molecular component of this pathway. Ca(2+) sensitization of force induced by endothelin or GTPgammaS was significantly greater in CC than in rabbit ileum smooth muscle and was accompanied by a 17-fold higher RhoA content. Pull-down assays with the RhoA binding domain of mDia showed the high RhoA content of CC to be available for activation by GTPgammaS. Ca(2+) sensitization induced by endothelin, phenylephrine, or GTPgammaS was completely relaxed by the Rho kinase inhibitor Y-27632. Human and rabbit CC both express the phosphatase inhibitor CPI-17, the myosin phosphatase regulatory (MYPT-1) and catalytic (PP1delta) subunits, and two isoforms of Rho kinase. We suggest that high expression of RhoA contributes, through RhoA-mediated Ca(2+) sensitization, to the flaccid state of CC that can be reversed by a water-soluble, orally active Rho kinase inhibitor suitable for therapy of erectile dysfunction.
    Document Type:
    Reference
    Product Catalog Number:
    05-773
    Product Catalog Name:
    Anti-phospho-MYPT1 (Thr850) Antibody, clone SA19, rabbit monoclonal

  • Discovery of drug mode of action and drug repositioning from transcriptional responses. 20679242

    A bottleneck in drug discovery is the identification of the molecular targets of a compound (mode of action, MoA) and of its off-target effects. Previous approaches to elucidate drug MoA include analysis of chemical structures, transcriptional responses following treatment, and text mining. Methods based on transcriptional responses require the least amount of information and can be quickly applied to new compounds. Available methods are inefficient and are not able to support network pharmacology. We developed an automatic and robust approach that exploits similarity in gene expression profiles following drug treatment, across multiple cell lines and dosages, to predict similarities in drug effect and MoA. We constructed a "drug network" of 1,302 nodes (drugs) and 41,047 edges (indicating similarities between pair of drugs). We applied network theory, partitioning drugs into groups of densely interconnected nodes (i.e., communities). These communities are significantly enriched for compounds with similar MoA, or acting on the same pathway, and can be used to identify the compound-targeted biological pathways. New compounds can be integrated into the network to predict their therapeutic and off-target effects. Using this network, we correctly predicted the MoA for nine anticancer compounds, and we were able to discover an unreported effect for a well-known drug. We verified an unexpected similarity between cyclin-dependent kinase 2 inhibitors and Topoisomerase inhibitors. We discovered that Fasudil (a Rho-kinase inhibitor) might be "repositioned" as an enhancer of cellular autophagy, potentially applicable to several neurodegenerative disorders. Our approach was implemented in a tool (Mode of Action by NeTwoRk Analysis, MANTRA, http://mantra.tigem.it).
    Document Type:
    Reference
    Product Catalog Number:
    5001

  • Remediation of waters contaminated with ionic herbicides by sorption on polymerin. 17904611

    This study investigated the sorption of paraquat and 2,4-D on polymerin, the humic acid-like fraction of olive mill wastewater. Effects of pH, contact time, initial concentration and sorbent dosage on the sorption of both herbicides were studied. The sorption mechanism of paraquat on polymerin was consistent with the ion exchange of this herbicide with Ca, Mg and K natively occurring in the sorbent; in contrast, 2,4-D was bound to polymerin by hydrogen bonding. Simulated wastewaters contaminated with paraquat were purified after three sorption cycles on polymerin renewed at each cycle, at a solid/liquid ratio of 0.5, whereas those containing 2,4-D showed a maximal residue removal of 44% after two sorption cycles at the same ratio. The possible application of this model to other water-soluble herbicides, as well as the possible exploitation of polymerin as a bio-filter for the decontamination of pollution point sources is briefly discussed.
    Document Type:
    Reference
    Product Catalog Number:
    5500

  • Functional stability of a mixed microbial consortium producing PHA from waste carbon sources. 18478444

    Polyhydroxyalkanoates (PHAs) represent an environmentally effective alternative to synthetic thermoplastics; however, current production practices are not sustainable. In this study, PHA production was accomplished in sequencing batch bioreactors utilizing real wastewaters and mixed microbial consortia from municipal activated sludge as inoculum. Polymer production reached 85, 53, and 10% of the cell dry weight from methanol-enriched pulp and paper mill foul condensate, fermented municipal primary solids, and biodiesel wastewater, respectively. Using denaturing gradient gel electrophoresis of 16S-rDNA from polymerase chain reaction-amplified DNA extracts, distinctly different communities were observed between and within wastewaters following enrichment. Most importantly, functional stability was maintained despite differing and contrasting microbial populations.
    Document Type:
    Reference
    Product Catalog Number:
    07-909
    Product Catalog Name:
    Anti-phospho-Src (Tyr418) Antibody

  • Morphological and neoplastic transformation of C3H/10T1/2 Cl 8 mouse embryo cells by insoluble carcinogenic nickel compounds. 2548861

    We studied induction of cytotoxicity and morphological transformation in C3H/10T1/2 Cl 8 (10T1/2) mouse embryo fibroblasts by soluble and insoluble carcinogenic nickel compounds. Soluble nickel sulfate and nickel chloride caused dose-dependent cytotoxicity in the concentration range from 0.5 microM to 100 microM after 48 hr treatments, but neither compound induced morphological transformation even at concentrations causing up to 94% cytotoxicity. Insoluble nickel subsulfide, nickel monosulfide, and nickel oxide caused dose-dependent cytotoxicity and a low, dose-dependent frequency of morphological transformation in the concentration ranges from 0.5 to 40 microM, 5 to 50 microM, and 50 to 400 microM, respectively, after 48 hr exposure of cells to these compounds. Foci were predominantly of type II morphology; type III foci were rare. The insoluble nickel compounds studied caused no induction of base substitution mutations to ouabain resistance in 10T1/2 cells over concentration ranges that induced morphological transformation. Nickel subsulfide and nickel monosulfide were taken into cells by phagocytosis, since particles were visible in intracytoplasmic vacuoles. Numerous nickel oxide particles were found associated with cells, but true phagocytic uptake was difficult to detect since no vacuoles were observed. We twice cloned type II and type III foci induced by insoluble nickel compounds, established independent cell lines, and characterized their phenotypes. Four of seven of these cell lines had three- to fourfold increased saturation densities compared to 10T1/2 cells, formed type II and type III foci in reconstruction assays, and grew in soft agarose. One cell line induced by nickel oxide formed tumors in nude mice. These data indicate that insoluble carcinogenic nickel compounds induced type II foci in 10T1/2 cells, some of which were tumorigenic, and that the 10T1/2 cell system is suitable for studying mechanisms of nickel compound-induced morphological transformation in mammalian cells.
    Document Type:
    Reference
    Product Catalog Number:
    5100
    Product Catalog Name:

  • Implication of DNA demethylation and bivalent histone modification for selective gene regulation in mouse primordial germ cells. 23029374

    Primordial germ cells (PGCs) sequentially induce specific genes required for their development. We focused on epigenetic changes that regulate PGC-specific gene expression. mil-1, Blimp1, and Stella are preferentially expressed in PGCs, and their expression is upregulated during PGC differentiation. Here, we first determined DNA methylation status of mil-1, Blimp1, and Stella regulatory regions in epiblast and in PGCs, and found that they were hypomethylated in differentiating PGCs after E9.0, in which those genes were highly expressed. We used siRNA to inhibit a maintenance DNA methyltransferase, Dnmt1, in embryonic stem (ES) cells and found that the flanking regions of all three genes became hypomethylated and that expression of each gene increased 1.5- to 3-fold. In addition, we also found 1.5- to 5-fold increase of the PGC genes in the PGCLCs (PGC-like cells) induced form ES cells by knockdown of Dnmt1. We also obtained evidence showing that methylation of the regulatory region of mil-1 resulted in 2.5-fold decrease in expression in a reporter assay. Together, these results suggested that DNA demethylation does not play a major role on initial activation of the PGC genes in the nascent PGCs but contributed to enhancement of their expression in PGCs after E9.0. However, we also found that repression of representative somatic genes, Hoxa1 and Hoxb1, and a tissue-specific gene, Gfap, in PGCs was not dependent on DNA methylation; their flanking regions were hypomethylated, but their expression was not observed in PGCs at E13.5. Their promoter regions showed the bivalent histone modification in PGCs, that may be involved in repression of their expression. Our results indicated that epigenetic status of PGC genes and of somatic genes in PGCs were distinct, and suggested contribution of epigenetic mechanisms in regulation of the expression of a specific gene set in PGCs.
    Document Type:
    Reference
    Product Catalog Number:
    07-449
    Product Catalog Name:
    Anti-trimethyl-Histone H3 (Lys27) Antibody

  • Alterations in AP-1 and AP-1 regulatory genes during HPV-induced carcinogenesis. 18219112

    BACKGROUND: Previous studies demonstrated a functional involvement of the AP-1 transcription factor in HPV-induced cervical carcinogenesis. Here, we aimed to obtain further insight in expression alterations of AP-1 family members during HPV-mediated transformation and their relationship to potential regulatory (Notch1, Net) and target (CADM1) genes. METHODS: mRNA expression levels of c-Jun, JunB, junD, c-Fos, FosB, Fra-1, Fra-2, Notch1, Net and CADM1 were determined by quantitative RT-PCR in primary keratinocytes (n=5), early (n=4) and late (n=4) passages of non-tumorigenic HPV-immortalized keratinocytes and in tumorigenic cervical cancer cell lines (n=7). In a subset of cell lines protein expression and AP-1 complex composition was determined. RESULTS: Starting in immortal stages c-Fos, Fra-2 and JunB expression became up regulated towards tumorigenicity, whereas Fra-1, c-Jun, Notch1, Net and CADM1 became down regulated. The onset of deregulated expression varied amongst the AP-1 members and was not directly related to altered Notch1, Net or CADM1 expression. Nevertheless, a shift in AP-1 complex composition from Fra-1/c-Jun to c-Fos/c-Jun heterodimers was only observed in tumorigenic cells. CONCLUSION: HPV-mediated transformation is associated with altered AP-1, Notch1, Net and CADM1 transcription. Whereas the onset of deregulated expression of various AP-1 family members became already manifest during the immortal state, a shift in AP-1 complex composition appeared a rather late event associated with tumorigenicity.
    Document Type:
    Reference
    Product Catalog Number:
    06-341

  • Changes in the ratio of free NEDD8 to ubiquitin triggers NEDDylation by ubiquitin enzymes. 22004789

    Ubiquitin and UBL (ubiquitin-like) modifiers are small proteins that covalently modify other proteins to alter their properties or behaviours. Ubiquitin modification (ubiquitylation) targets many substrates, often leading to their proteasomal degradation. NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8) is the UBL most closely related to ubiquitin, and its best-studied role is the activation of CRLs (cullin-RING ubiquitin ligases) by its conjugation to a conserved C-terminal lysine residue on cullin proteins. The attachment of UBLs requires three UBL-specific enzymes, termed E1, E2 and E3, which are usually well insulated from parallel UBL pathways. In the present study, we report a new mode of NEDD8 conjugation (NEDDylation) whereby the UBL NEDD8 is linked to proteins by ubiquitin enzymes in vivo. We found that this atypical NEDDylation is independent of classical NEDD8 enzymes, conserved from yeast to mammals, and triggered by an increase in the NEDD8 to ubiquitin ratio. In cells, NEDD8 overexpression leads to this type of NEDDylation by increasing the concentration of NEDD8, whereas proteasome inhibition has the same effect by depleting free ubiquitin. We show that bortezomib, a proteasome inhibitor used in cancer therapy, triggers atypical NEDDylation in tissue culture, which suggests that a similar process may occur in patients receiving this treatment.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1510

  • Distinct functions of BMP4 during different stages of mouse ES cell neural commitment. 20504958

    Bone morphogenetic protein (BMP) signaling plays a crucial role in maintaining the pluripotency of mouse embryonic stem cells (ESCs) and has negative effects on ESC neural differentiation. However, it remains unclear when and how BMP signaling executes those different functions during neural commitment. Here, we show that a BMP4-sensitive window exists during ESC neural differentiation. Cells at this specific period correspond to the egg cylinder stage epiblast and can be maintained as ESC-derived epiblast stem cells (ESD-EpiSCs), which have the same characteristics as EpiSCs derived from mouse embryos. We propose that ESC neural differentiation occurs in two stages: first from ESCs to ESD-EpiSCs and then from ESD-EpiSCs to neural precursor cells (NPCs). We further show that BMP4 inhibits the conversion of ESCs into ESD-EpiSCs during the first stage, and suppresses ESD-EpiSC neural commitment and promotes non-neural lineage differentiation during the second stage. Mechanistic studies show that BMP4 inhibits FGF/ERK activity at the first stage but not at the second stage; and IDs, as important downstream genes of BMP signaling, partially substitute for BMP4 functions at both stages. We conclude that BMP signaling has distinct functions during different stages of ESC neural commitment.
    Document Type:
    Reference
    Product Catalog Number:
    EZRADP-62K

  • Identification of a small-molecule inhibitor of the PICK1 PDZ domain that inhibits hippocampal LTP and LTD. 20018661

    Proteins containing PSD-95/Discs-large/ZO-1 homology (PDZ) domains play key roles in the assembly and regulation of cellular signaling pathways and represent putative targets for new pharmacotherapeutics. Here we describe the first small-molecule inhibitor (FSC231) of the PDZ domain in protein interacting with C kinase 1 (PICK1) identified by a screening of ~44,000 compounds in a fluorescent polarization assay. The inhibitor bound the PICK1 PDZ domain with an affinity similar to that observed for endogenous peptide ligands (K(i)~10.1 muM). Mutational analysis, together with computational docking of the compound in simulations starting from the PDZ domain structure, identified the binding mode of FSC231. The specificity of FSC231 for the PICK1 PDZ domain was supported by the lack of binding to PDZ domains of postsynaptic density protein 95 (PSD-95) and glutamate receptor interacting protein 1 (GRIP1). Pretreatment of cultured hippocampal neurons with FSC231 inhibited coimmunopreciptation of the AMPA receptor GluR2 subunit with PICK1. In agreement with inhibiting the role of PICK1 in GluR2 trafficking, FSC231 accelerated recycling of pHluorin-tagged GluR2 in hippocampal neurons after internalization in response to NMDA receptor activation. FSC231 blocked the expression of both long-term depression and long-term potentiation in hippocampal CA1 neurons from acute slices, consistent with inhibition of the bidirectional function of PICK1 in synaptic plasticity. Given the proposed role of the PICK1/AMPA receptor interaction in neuropathic pain, excitotoxicity, and cocaine addiction, FSC231 might serve as a lead in the future development of new therapeutics against these conditions.
    Document Type:
    Reference
    Product Catalog Number:
    MAB397
    Product Catalog Name:
    Anti-Glutamate Receptor 2 Antibody, extracellular, clone 6C4