The Canadian Institutes of Health Research response to antimicrobial resistance.

Antimicrobial resistance (AMR) has been a research priority for the Canadian Institutes of Health Research (CIHR), Institute of Infection and Immunity (III) since its inception, and a number of strategic research initiatives have been launched to address this global health problem by promoting and supporting research related to mechanisms and processes that impact the emergence and spread of resistance among individuals and within the environment. Here we will present research initiatives on AMR led by CIHR-III, which include national programs as well as international partnerships with the United Kingdom and the European Union, in addition to interesting outcomes of these initiatives.

Altered expression of the poly(ADP-ribosyl)ation enzymes in uveal melanoma and regulation of PARG gene expression by the transcription factor ERM.

Purpose: Poly(ADP-ribosyl)ation is a reversible post-translational modification that requires the contribution of the enzymes poly(ADP-ribose) polymerase-1 (PARP-1) and poly(ADP-ribose) glycohydrolase (PARG). Our study explores expression and activity of PARP-1 and PARG in uveal melanoma cell lines with varying tumorigenic properties.

Methods: Gene profiling on microarrays was conducted using RNA prepared from the uveal melanoma cell lines T97, T98, T108, and T115.

Poly(ADP-ribose) metabolism analysis in the nematode Caenorhabditis elegans.

Poly(ADP-ribose) polymerases (PARPs) are a well-conserved family of enzymes found in many species. These enzymes catalyze poly(ADP-ribosyl)ation, a modification of proteins implicated in a variety of nuclear processes, such as DNA damage signaling and repair, cell death and survival, and transcription. Poly(ADP-ribose) glycohydrolase (PARG) is responsible for the specific hydrolysis of poly(ADP-ribose) (PAR), the product of poly(ADP-ribosyl)ation, and its action is required for the modified proteins to regain their original function in the cell.

Photoactivated multivitamin preparation induces poly(ADP-ribosyl)ation, a DNA damage response in mammalian cells.

Multivitamin preparation (MVP) is part of total parenteral nutrition given to premature infants. Photoactivated MVP carries an important load in peroxides, but their cellular effects have not yet been determined. We hypothesized that these peroxides may elicit a DNA-damage response.

Activation of the abundant nuclear factor poly(ADP-ribose) polymerase-1 by Helicobacter pylori.

Modification of eukaryotic proteins is a powerful strategy used by pathogenic bacteria to modulate host cells during infection. Previously, we demonstrated that Helicobacter pylori modify an unidentified protein within mammalian cell lysates in a manner consistent with the action of a bacterial ADP-ribosylating toxin. Here, we identified the modified eukaryotic factor as the abundant nuclear factor poly(ADP-ribose) polymerase-1 (PARP-1), which is important in the pathologies of several disease states typically associated with chronic H.

The DNA damage-inducible C. elegans tankyrase is a nuclear protein closely linked to chromosomes.

Tankyrases are protein members of the poly(ADP-ribose) polymerase family bearing several ankyrin domain and a WGR domain. They have functional role in telomere maintenance, are found at centrosome, and are associated with vesicular secretion. This diversity in localization and function makes it difficult to identify a unified role for tankyrases.

Regulation of poly(ADP-ribose) polymerase-1 (PARP-1) gene expression through the post-translational modification of Sp1: a nuclear target protein of PARP-1.

Background: Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that plays critical functions in many biological processes, including DNA repair and gene transcription. The main function of PARP-1 is to catalyze the transfer of ADP-ribose units from nicotinamide adenine dinucleotide (NAD+) to a large array of acceptor proteins, which comprises histones, transcription factors, as well as PARP-1 itself. We have previously demonstrated that transcription of the PARP-1 gene essentially rely on the opposite regulatory actions of two distinct transcription factors, Sp1 and NFI.

Caenorhabditis elegans LET-767 is able to metabolize androgens and estrogens and likely shares common ancestor with human types 3 and 12 17beta-hydroxysteroid dehydrogenases.

Mutations that inactivate LET-767 are shown to affect growth, reproduction, and development in Caenorhabditis elegans. Sequence analysis indicates that LET-767 shares the highest homology with human types 3 and 12 17beta-hydroxysteroid dehydrogenases (17beta-HSD3 and 12). Using LET-767 transiently transfected into human embryonic kidney-293 cells, we have found that the enzyme catalyzes the transformation of both 4-androstenedione into testosterone and estrone into estradiol, similar to that of mouse 17beta-HSD12 but different from human and primate enzymes that catalyze the transformation of estrone into estradiol.

Altered DNA damage response in Caenorhabditis elegans with impaired poly(ADP-ribose) glycohydrolases genes expression.

Poly(ADP-ribosyl)ation is one of the first cellular responses induced by DNA damage. Poly(ADP-ribose) is rapidly synthesized by nick-sensor poly(ADP-ribose) polymerases, which facilitate DNA repair enzymes to process DNA damage. ADP-ribose polymers are rapidly catabolized into free ADP-ribose units by poly(ADP-ribose) glycohydrolase (PARG).

The Caenorhabditis elegans FancD2 ortholog is required for survival following DNA damage.

Fanconi anemia (FA) is an autosomal recessive disease characterized by bone-marrow failure, congenital abnormalities, and cancer susceptibility. There are 11 FA complementation groups in human where 8 genes have been identified. We found that FancD2 is conserved in evolution and present in the genome of the nematode Caenorhabditis elegans.

Ionizing radiations in Caenorhabditis elegans induce poly(ADP-ribosyl)ation, a conserved DNA-damage response essential for survival.

Poly(ADP-ribosyl)ation is one of the first responses to DNA damage in mammals. Although it is involved in base excision repair, its exact role has not been ascertained yet. Poly(ADP-ribose) polymerase-1 (PARP-1) and PARP-2 mediate most of the poly(ADP-ribosyl)ation response in mammals and are well conserved in evolution.

Regulation of the poly(ADP-ribose) polymerase-1 gene expression by the transcription factors Sp1 and Sp3 is under the influence of cell density in primary cultured cells.

PARP-1 [poly(ADP-ribose) polymerase-1) is a nuclear enzyme that is involved in several cellular functions, including DNA repair, DNA transcription, carcinogenesis and apoptosis. The activity directed by the PARP-1 gene promoter is mainly dictated through its recognition by the transcription factors Sp1 and Sp3 (where Sp is specificity protein). In the present study, we investigated whether (i) both PARP-1 expression and PARP-1 enzymatic activity are under the influence of cell density in primary cultured cells, and (ii) whether its pattern of expression is co-ordinated with that of Sp1/Sp3 at varying cell densities and upon cell passages.

The C. elegans gene pme-5: molecular cloning and role in the DNA-damage response of a tankyrase orthologue.

Tankyrases are recently identified proteins characterized by ankyrin repeats and a poly(ADP-ribose) polymerase (PARP) signature motif. In vertebrates, tankyrases mediate protein-protein interactions via the ankyrin domain. Many partners have been identified that could function in telomere maintenance, signal transduction in vesicular transport, and cell death.

Single amino acid substitution enhances bacterial expression of PARP-4D214A.

Poly(ADP-ribose) polymerase-1 (PARP-1) is the canonical member of the PARP family of enzymes and modulates many crucial nuclear functions. PARP-1 is involved in apoptosis and is the substrate of caspase-3, a protease that cleaves PARP-1 at the conserved sequence 211DEVD214. To generate a caspase-3-uncleavable PARP-1, we introduced an amino acid substitution D214-->A214 at the site of cleavage.

The genes pme-1 and pme-2 encode two poly(ADP-ribose) polymerases in Caenorhabditis elegans.

Poly(ADP-ribose) polymerases (PARPs) are an expanding, well-conserved family of enzymes found in many metazoan species, including plants. The enzyme catalyses poly(ADP-ribosyl)ation, a post-translational modification that is important in DNA repair and programmed cell death. In the present study, we report the finding of an endogenous source of poly(ADP-ribosyl)ation in total extracts of the nematode Caenorhabditis elegans.

Poly(ADP-ribosyl)ation reactions in the regulation of nuclear functions.

Poly(ADP-ribosyl)ation is a post-translational modification of proteins. During this process, molecules of ADP-ribose are added successively on to acceptor proteins to form branched polymers. This modification is transient but very extensive in vivo, as polymer chains can reach more than 200 units on protein acceptors.

6-Aminonicotinamide sensitizes human tumor cell lines to cisplatin.

The nicotinamide analogue 6-aminonicotinamide (6AN) is presently undergoing evaluation as a potential modulator of the action of various antineoplastic treatments. Most previous studies of this agent have focused on a three-drug regimen of chemical modulators that includes 6AN. In the present study, the effect of single-agent 6AN on the efficacy of selected antineoplastic drugs was assessed in vitro.

Interaction between the C. elegans cell-death regulators CED-9 and CED-4.

Programmed cell death (apoptosis) is an evolutionarily conserved process used by multicellular organisms to eliminate cells that are not needed or are potentially detrimental to the organism. Members of the Bcl-2 family of mammalian proteins are intimately involved in the regulation of apoptosis, but, their precise mechanism of action remains unresolved. In Caenorhabditis elegans, the Bcl-2 homologue CED-9 prevents cell death by antagonizing the death-promoting activities of CED-3, a member of the Caspase family of death proteases, and of CED-4, a protein with no known mammalian homologue.

Characterization of anti-peptide antibodies directed towards the automodification domain and apoptotic fragment of poly (ADP-ribose) polymerase.

Poly(ADP-ribose) polymerase (PARP; EC 2.4.2.

Alteration of the nucleolar localization of poly(ADP-ribose) polymerase upon treatment with transcription inhibitors.

The effect of RNA, DNA, and protein synthesis inhibitors on the subnuclear localization of poly(ADP-ribose) polymerase (PARP) was examined. Indirect immunofluorescence indicated that PARP was distributed throughout the nuclei but concentrated in nucleoli of MDBK, HeLa, and CHO cells. Treatment with the DNA synthesis inhibitor cytosine arabinoside or the protein synthesis inhibitor cycloheximide did not change the distribution of PARP.

Association of poly(ADP-ribose) polymerase with nuclear subfractions catalyzed with sodium tetrathionate and hydrogene peroxide crosslinks.

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme which catalyzes the transfer of ADP-ribose units from NAD+ to a variety of nuclear proteins under the stimulation of DNA strand break. To examine its role in DNA repair, we have been studying the interaction of PARP with other nuclear proteins using disulfide cross-linking, initiated by sodium tetrathionate (NaTT). Chinese Hamster Ovary (CHO) cells were extracted sequentially with Nonidet P40 (detergent), nucleases (DNase+RNase), and high salt (1.

Complete inhibition of poly(ADP-ribose) polymerase activity prevents the recovery of C3H10T1/2 cells from oxidative stress.

Activation of the poly(ADP-ribose) polymerase after oxidative damage is implicated in different responses of the cells, for example, cell recovery after sublethal damage or cell death after lethal damage. However, the extent and mechanism of involvement of the enzyme in these two processes appear to be different. Inhibitors of this polymerase, such as benzamides, which do not completely inhibit PARP have been shown to protect the cells from killing by massive oxidant damage, could neither reduce the cellular recovery after mild oxidant damage nor completely inhibit DNA repair in vitro.