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  • Identification and biochemical characterization of unique secretory nucleases of the human enteric pathogen, Entamoeba histolytica. 17766245

    The ancient eukaryotic human pathogen, Entamoeba histolytica, is a nucleo-base auxotroph (i.e. lacks the ability to synthesize purines or pyrimidines de novo) and therefore is totally dependent upon its host for the supply of these essential nutrients. In this study, we identified two unique 28-kDa, dithiothreitol-sensitive nucleases and showed that they are constitutively released/secreted by parasites during axenic culture. Using several different molecular approaches, we identified and characterized the structure of EhNucI and EhNucII, genes that encode ribonuclease T2 family proteins. Homologous episomal expression of epitope-tagged EhNucI and EhNucII chimeric constructs was used to define the functional and biochemical properties of these released/secreted enzymes. Results of coupled immunoprecipitation-enzyme activity analyses demonstrated that these secretory enzymes could hydrolyze a variety of synthetic polynucleotides, as well as the natural nucleic acid substrate RNA. Furthermore, our results demonstrated that sera from acutely infected amebiasis patients recognized and immunoprecipitated these parasite secretory enzymes. Based on these observations, we hypothesize that within its host, these secretory nucleases could function, at a distance away from the parasite, to harness (i.e. hydrolyze/access) host-derived nucleic acids to satisfy the essential purine and pyrimidine requirements of these organisms. Thus, these enzymes might play an important role in facilitating the survival, growth, and development of this important human pathogen.
    Document Type:
    Reference
    Product Catalog Number:
    ECM600
    Product Catalog Name:
    uPA Activity Assay Kit

  • Molecular characterization of c-Abl/c-Src kinase inhibitors targeted against murine tumour progenitor cells that express stem cell markers. 21152443

    The non-receptor tyrosine kinases c-Abl and c-Src are overexpressed in various solid human tumours. Inhibition of their hyperactivity represents a molecular rationale in the combat of cancerous diseases. Here we examined the effects of a new family of pyrazolo [3,4-d] pyrimidines on a panel of 11 different murine lung tumour progenitor cell lines, that express stem cell markers, as well as on the human lung adenocarcinoma cell line A549, the human hepatoma cell line HepG2 and the human colon cancer cell line CaCo2 to obtain insight into the mode of action of these experimental drugs.Treatment with the dual kinase inhibitors blocked c-Abl and c-Src kinase activity efficiently in the nanomolar range, induced apoptosis, reduced cell viability and caused cell cycle arrest predominantly at G0/G1 phase while western blot analysis confirmed repressed protein expression of c-Abl and c-Src as well as the interacting partners p38 mitogen activated protein kinase, heterogenous ribonucleoprotein K, cyclin dependent kinase 1 and further proteins that are crucial for tumour progression. Importantly, a significant repression of the epidermal growth factor receptor was observed while whole genome gene expression analysis evidenced regulation of many cell cycle regulated genes as well integrin and focal adhesion kinase (FAK) signalling to impact cytoskeleton dynamics, migration, invasion and metastasis.Our experiments and recently published in vivo engraftment studies with various tumour cell lines revealed the dual kinase inhibitors to be efficient in their antitumour activity.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Base specificity and idiotypy of anti-DNA autoantibodies reactive with synthetic nucleic acids. 3876384

    Synthetic nucleic acid reactivities and the distribution of idiotypes associated with poly(dA) and poly(dT) specificities were evaluated among both monoclonal and polyclonal anti-DNA antibodies from autoimmune New Zealand mice. Ten monoclonal anti-DNA antibodies (IgG2a or IgG2b), derived from NZB/NZW mice and reactive with natural DNA (duplex and/or heat-denatured), were found to collectively exhibit a diverse binding pattern with six deoxyribohomopolymers. Several monoclonal antibodies displayed reactivity with poly(dT) comparable to that with natural DNA. Serologic studies indicated that polyclonal anti-DNA autoantibodies from NZW/NZW mice and both parental strains also cross-reacted with various homopolymers and bound preferentially with those containing pyrimidines, particularly poly(dT), relative to purines. Detailed binding analyses with two poly(dT)-reactive monoclonal antibodies demonstrated that stable DNA/anti-DNA complexes were formed with synthetic oligomers containing six to 10 nucleotides; binding to such antigens was relatively insensitive to ionic strength and inversely dependent on temperature. Both antibodies exhibited preferential binding (greater than or equal to 10-fold) with poly(dT) relative to poly(dU), suggesting the importance of the C5-methyl group and/or helical conformation in pyrimidine base recognition. Idiotypes on poly(dA)-specific and poly(dT)-specific monoclonal antibodies were found to be reciprocally distinct, localized at or near active site residues, and expressed at low levels (less than 10 to 130 ng/ml) in anti-DNA sera from all three New Zealand strains. These findings suggest that: nucleotide base determinants are significantly involved in DNA/anti-DNA interactions; poly(dT) represents a major cross-reactive synthetic antigen; and idiotype expression among lupus autoantibodies which recognize such determinants may be diverse.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Rhesus cytomegalovirus is similar to human cytomegalovirus in susceptibility to benzimidazole nucleosides. 15215146

    Rhesus and human cytomegalovirus (RhCMV and HCMV, respectively) exhibit comparable inhibition by benzimidazole nucleosides, including 2,5,6-trichloro-(1-beta-d-ribofuranosyl)benzimidazole (TCRB), and pyrrolo[2,3-d]pyrimidines. The two HCMV protein targets of TCRB, UL89 and UL56, are highly conserved with their RhCMV homologues. These data indicate that infection of rhesus macaques with RhCMV represents a useful model to test novel anti-HCMV drugs.
    Document Type:
    Reference
    Product Catalog Number:
    MAB810
    Product Catalog Name:
    Anti-Cytomegalovirus Antibody, clone 8B1.2 (ASR)

  • Expression of P2Y receptors in the rat anterior pituitary. 21567129

    In this study, the distribution patterns of P2Y(1), P2Y(2) P2Y(4), P2Y(6), P2Y(12), and P2Y(13) receptors in the anterior pituitary cells of rat were studied with double-labeling immunofluorescence and Western blot. The results showed that P2Y receptors were widely expressed in the anterior pituitary. P2Y(1) and P2Y(4) receptors were found to be expressed in the majority of gonadotrophs and thyrotrophs, P2Y(2) receptors were expressed in a small subpopulation of lactotrophs and almost all the folliculo-stellate cells, that were also stained with S100 protein immunoreactivity. P2Y(6) receptors were expressed in macrophages. P2Y(13) receptors were expressed in a small subpopulation of cells in the rat anterior pituitary, the identity of which needs to be clarified. P2Y(1) and P2Y(4) receptors are co-expressed in some gonadotrophs and thyrotrophs. Corticotrophs and somatotrophs were found not to express P2Y receptors in this study. FSH and TSH were shown to coexist in the same endocrine cells in rat anterior pituitary. The present data suggests that purines and/or pyrimidines could be involved in regulating the functions of gonadotrophs and thyrotrophs via P2Y(1) and P2Y(4) receptors, some lactotrophs via P2Y(2) receptors, and folliculo-stellate cells via P2Y(2) receptors in the rat anterior pituitary.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • A thymidylate synthase ternary complex-specific antibody, FTS, permits functional monitoring of fluoropyrimidines dosing. 22824673

    5-Fluorouracil (5FU) and similar fluoropyrimidines induce covalent modification of thymidylate synthase (TS) and inhibit its activity. They are often used to treat solid cancers, but drug resistance and toxicity are drawbacks. Therefore, there is an unmet need for a functional assay to quantify fluorouracil activity in tissues, so as to individually tailor dosing. It is cumbersome to separately quantify unmodified and 5FU-modified TS using currently available commercial anti-TS antibodies because they recognize both forms. We report here the first monoclonal antibody (FTS) specific to 5FU-modified TS. By immunoblot assay, the FTS antibody specifically recognizes modified TS in a dose-dependent manner in 5FU-treated cells, in cancer xenograft tissues of 5FU-treated mice, and in the murine tissues. In the same assay, the antibody is nonreactive with unmodified TS in untreated or treated cells and tissues. Speculatively, a high-throughput assay could be enabled by pairing anti-TS antibodies of two specificities, one recognizing only modified TS and another recognizing both forms, to structurally quantify the TS-inhibiting effect of fluorouracil at a cellular or tissue level without requiring prior protein separation. Such a development might aid preclinical analytic studies or make practical the individual tailoring of dosing.
    Document Type:
    Reference
    Product Catalog Number:
    ABN402
    Product Catalog Name:
    Anti-Spartin Antibody

  • P53 represses pyrimidine catabolic gene dihydropyrimidine dehydrogenase (DPYD) expression in response to thymidylate synthase (TS) targeting. 28851987

    Nucleotide metabolism in cancer cells can influence malignant behavior and intrinsic resistance to therapy. Here we describe p53-dependent control of the rate-limiting enzyme in the pyrimidine catabolic pathway, dihydropyrimidine dehydrogenase (DPYD) and its effect on pharmacokinetics of and response to 5-fluorouracil (5-FU). Using in silico/chromatin-immunoprecipitation (ChIP) analysis we identify a conserved p53 DNA-binding site (p53BS) downstream of the DPYD gene with increased p53 occupancy following 5-FU treatment of cells. Consequently, decrease in Histone H3K9AC and increase in H3K27me3 marks at the DPYD promoter are observed concomitantly with reduced expression of DPYD mRNA and protein in a p53-dependent manner. Mechanistic studies reveal inhibition of DPYD expression by p53 is augmented following thymidylate synthase (TS) inhibition and DPYD repression by p53 is dependent on DNA-dependent protein kinase (DNA-PK) and Ataxia telangiectasia mutated (ATM) signaling. In-vivo, liver specific Tp53 loss increases the conversion of 5-FU to 5-FUH2 in plasma and elicits a diminished 5-FU therapeutic response in a syngeneic colorectal tumor model consistent with increased DPYD-activity. Our data suggest that p53 plays an important role in controlling pyrimidine catabolism through repression of DPYD expression, following metabolic stress imposed by nucleotide imbalance. These findings have implications for the toxicity and efficacy of the cancer therapeutic 5-FU.
    Document Type:
    Reference
    Product Catalog Number:
    17-10086
    Product Catalog Name:
    EZ-Magna ChIP™ A/G Chromatin Immunoprecipitation Kit