Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 7 de 7
Filter
Add more filters










Database
Language
Publication year range
1.
Methods Mol Biol ; 2626: 399-444, 2023.
Article in English | MEDLINE | ID: mdl-36715918

ABSTRACT

Citizen science is a productive approach to include non-scientists in research efforts that impact particular issues or communities. In most cases, scientists at advanced career stages design high-quality, exciting projects that enable citizen contribution, a crowdsourcing process that drives discovery forward and engages communities. The challenges of having citizens design their own research with no or limited training and providing access to laboratory tools, reagents, and supplies have limited citizen science efforts. This leaves the incredible life experiences and immersion of citizens in communities that experience health disparities out of the research equation, thus hampering efforts to address community health needs with a full picture of the challenges that must be addressed. Here, we present a robust and reproducible approach that engages participants from Grade 5 through adult in research focused on defining how diet impacts disease signaling. We leverage the powerful genetics, cell biology, and biochemistry of Drosophila oogenesis to define how nutrients impact phenotypes associated with genetic mutants that are implicated in cancer and diabetes. Participants lead the project design and execution, flipping the top-down hierarchy of the prevailing scientific culture to co-create research projects and infuse the research with cultural and community relevance.


Subject(s)
Drosophila , Public Health , Animals , Research
2.
J Immunol ; 197(6): 2382-9, 2016 09 15.
Article in English | MEDLINE | ID: mdl-27527593

ABSTRACT

Regulation of NF-κB nuclear translocation and stability is central to mounting an effective innate immune response. In this article, we describe a novel molecular mechanism controlling NF-κB-dependent innate immune response. We show that a previously unknown protein, termed as Charon, functions as a regulator of antibacterial and antifungal immune defense in Drosophila Charon is an ankyrin repeat-containing protein that mediates poly(ADP-ribose) polymerase-1 (PARP-1)-dependent transcriptional responses downstream of the innate immune pathway. Our results demonstrate that Charon interacts with the NF-κB ortholog Relish inside perinuclear particles and delivers active Relish to PARP-1-bearing promoters, thus triggering NF-κB/PARP-1-dependent transcription of antimicrobial peptides. Ablating the expression of Charon prevents Relish from targeting promoters of antimicrobial genes and effectively suppresses the innate immune transcriptional response. Taken together, these results implicate Charon as an essential mediator of PARP-1-dependent transcription in the innate immune pathway. Thus, to our knowledge, our results are the first to describe the molecular mechanism regulating translocation of the NF-κB subunit from cytoplasm to chromatin.


Subject(s)
Carrier Proteins/metabolism , Drosophila Proteins/metabolism , Gene Expression Regulation , NF-kappa B/metabolism , Poly (ADP-Ribose) Polymerase-1/immunology , Animals , Carrier Proteins/genetics , DNA-Binding Proteins/genetics , Drosophila , Drosophila Proteins/genetics , Drosophila Proteins/immunology , Immunity, Innate , NF-kappa B/immunology , Promoter Regions, Genetic , Transcription Factors/immunology , Transcription Factors/metabolism , Transcriptional Activation
3.
PLoS Genet ; 8(1): e1002442, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22242017

ABSTRACT

Poly(ADP-ribose) polymerase 1 (PARP1), a nuclear protein, utilizes NAD to synthesize poly(AD-Pribose) (pADPr), resulting in both automodification and the modification of acceptor proteins. Substantial amounts of PARP1 and pADPr (up to 50%) are localized to the nucleolus, a subnuclear organelle known as a region for ribosome biogenesis and maturation. At present, the functional significance of PARP1 protein inside the nucleolus remains unclear. Using PARP1 mutants, we investigated the function of PARP1, pADPr, and PARP1-interacting proteins in the maintenance of nucleolus structure and functions. Our analysis shows that disruption of PARP1 enzymatic activity caused nucleolar disintegration and aberrant localization of nucleolar-specific proteins. Additionally, PARP1 mutants have increased accumulation of rRNA intermediates and a decrease in ribosome levels. Together, our data suggests that PARP1 enzymatic activity is required for targeting nucleolar proteins to the proximity of precursor rRNA; hence, PARP1 controls precursor rRNA processing, post-transcriptional modification, and pre-ribosome assembly. Based on these findings, we propose a model that explains how PARP1 activity impacts nucleolar functions and, consequently, ribosomal biogenesis.


Subject(s)
Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Poly Adenosine Diphosphate Ribose/metabolism , Poly(ADP-ribose) Polymerases/genetics , Poly(ADP-ribose) Polymerases/metabolism , RNA, Ribosomal/genetics , Ribosomes/metabolism , Animals , Animals, Genetically Modified , Cell Nucleolus/enzymology , Cell Nucleolus/ultrastructure , Gene Expression Regulation , In Situ Hybridization, Fluorescence , Mutation , Nuclear Proteins/metabolism , Poly (ADP-Ribose) Polymerase-1 , RNA Interference , RNA Precursors/genetics , RNA Precursors/metabolism , RNA, Ribosomal/metabolism , Ribosomes/genetics , Ribosomes/ultrastructure
4.
Chem Res Toxicol ; 23(7): 1151-62, 2010 Jul 19.
Article in English | MEDLINE | ID: mdl-20536192

ABSTRACT

The mitomycin derivative 10-decarbamoyl mitomycin C (DMC) more rapidly activates a p53-independent cell death pathway than mitomycin C (MC). We recently documented that an increased proportion of mitosene1-beta-adduct formation occurs in human cells treated with DMC in comparison to those treated with MC. Here, we compare the cellular and molecular response of human cancer cells treated with MC and DMC. We find the increase in mitosene 1-beta-adduct formation correlates with a condensed nuclear morphology and increased cytotoxicity in human cancer cells with or without p53. DMC caused more DNA damage than MC in the nuclear and mitochondrial genomes. Checkpoint 1 protein (Chk1) was depleted following DMC, and the depletion of Chk1 by DMC was achieved through the ubiquitin proteasome pathway since chemical inhibition of the proteasome protected against Chk1 depletion. Gene silencing of Chk1 by siRNA increased the cytotoxicity of MC. DMC treatment caused a decrease in the level of total ubiquitinated proteins without increasing proteasome activity, suggesting that DMC mediated DNA adducts facilitate signal transduction to a pathway targeting cellular proteins for proteolysis. Thus, the mitosene-1-beta stereoisomeric DNA adducts produced by the DMC signal for a p53-independent mode of cell death correlated with reduced nuclear size, persistent DNA damage, increased ubiquitin proteolysis and reduced Chk1 protein.


Subject(s)
DNA Adducts/chemistry , Mitomycins/chemistry , Proteasome Endopeptidase Complex/metabolism , Protein Kinases/metabolism , Tumor Suppressor Protein p53/metabolism , Apoptosis , Cell Line, Tumor , Checkpoint Kinase 1 , DNA Damage , Gene Silencing , Humans , Mitomycin/toxicity , Mitomycins/toxicity , RNA Interference , RNA, Small Interfering/metabolism , Tumor Suppressor Protein p53/genetics
5.
Chem Res Toxicol ; 21(12): 2370-8, 2008 Dec.
Article in English | MEDLINE | ID: mdl-19053323

ABSTRACT

The antitumor antibiotic and cancer chemotherapeutic agent mitomycin C (MC) alkylates and crosslinks DNA, forming six major MC-deoxyguanosine adducts of known structures in vitro and in vivo. Two of these adducts are derived from 2,7-diaminomitosene (2,7-DAM), a nontoxic reductive metabolite of MC formed in cells in situ. Several methods have been used for the analysis of MC-DNA adducts in the past; however, a need exists for a safer, more comprehensive and direct assay of the six-adduct complex. Development of an assay, based on mass spectrometry, is described. DNA from EMT6 mouse mammary tumor cells, Fanconi Anemia-A fibroblasts, normal human fibroblasts, and MCF-7 human breast cancer cells was isolated after MC or 10-decarbamoyl mitomycin C (DMC) treatment of the cells, digested to nucleosides, and submitted to liquid chromatography electrospray-tandem mass spectrometry. Two fragments of each parent ion were monitored ("multiple reaction monitoring"). Identification and quantitative analysis were based on a standard mixture of six adducts, the preparation of which is described here in detail. The lower limit of detection of adducts is estimated as 0.25 pmol. Three initial applications of this method are reported as follows: (i) differential kinetics of adduct repair in EMT6 cells, (ii) analysis of adducts in MC- or DMC-treated Fanconi Anemia cells, and (iii) comparison of the adducts generated by treatment of MCF-7 breast cancer cells with MC and DMC. Notable results are the following: Repair removal of the DNA interstrand cross-link and of the two adducts of 2,7-DAM is relatively slow; both MC and DMC generate DNA interstrand cross-links in human fibroblasts, Fanconi Anemia-A fibroblasts, and MCF-7 cells as well as EMT6 cells; and DMC shows a stereochemical preference of linkage to the guanine-2-amino group opposite from that of MC.


Subject(s)
Cross-Linking Reagents/pharmacology , DNA Adducts/biosynthesis , Mitomycin/pharmacology , Mitomycins/pharmacology , Spectrometry, Mass, Electrospray Ionization/methods , Tandem Mass Spectrometry/methods , Animals , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Cell Line, Tumor , Chromatography, High Pressure Liquid , Cross-Linking Reagents/chemistry , Cross-Linking Reagents/metabolism , DNA Adducts/chemistry , DNA Damage , DNA Repair , DNA, Neoplasm/chemistry , DNA, Neoplasm/drug effects , Fibroblasts/drug effects , Fibroblasts/metabolism , Humans , Mammary Neoplasms, Animal/drug therapy , Mammary Neoplasms, Animal/genetics , Mammary Neoplasms, Animal/metabolism , Mice , Mitomycin/chemistry , Mitomycin/metabolism , Mitomycins/chemistry , Mitomycins/metabolism
6.
ACS Chem Biol ; 2(6): 399-407, 2007 Jun 15.
Article in English | MEDLINE | ID: mdl-17530733

ABSTRACT

10-Decarbamoyl-mitomycin C (DMC), a mitomycin C (MC) derivative, generates an array of DNA monoadducts and interstrand cross-links stereoisomeric to those that are generated by MC. DMC was previously shown in our laboratory to exceed the cytotoxicity of MC in a human leukemia cell line that lacks a functional p53 pathway (K562). However, the molecular signal transduction pathway activated by DMCDNA adducts has not been investigated. In this study, we have compared molecular targets associated with signaling pathways activated by DMC and MC in several human cancer cell lines. In cell lines lacking wild-type p53, DMC was reproducibly more cytotoxic than MC, but it generated barely detectable signal transduction markers associated with apoptotic death. Strikingly, DMCs increased cytotoxicity was not associated with an increase in DNA double-strand breaks but was associated with early poly(ADP-ribose) polymerase (PARP) activation and Chk1 kinase depletion. Alkylating agents can induce increased PARP activity associated with programmed necrosis, and the biological activity of DMC in p53-null cell lines fits this paradigm. In cell lines with a functional p53 pathway, both MC and DMC induced apoptosis. In the presence of p53, both MC and DMC activate procaspases; however, the spectrum of procaspases involved differs for the two drugs, as does induction of p73. These studies suggest that in the absence of p53, signaling to molecular targets in cell death can shift in response to different DNA adduct structures to induce non-apoptotic cell death.


Subject(s)
DNA Adducts/physiology , Mitomycins/pharmacology , Signal Transduction/physiology , Tumor Suppressor Protein p53/physiology , Animals , Cell Death/drug effects , Cell Death/physiology , Cell Line, Tumor , DNA Adducts/genetics , Humans , Mice , Signal Transduction/drug effects
7.
Photodermatol Photoimmunol Photomed ; 19(6): 295-302, 2003 Dec.
Article in English | MEDLINE | ID: mdl-14617104

ABSTRACT

PURPOSE: Bicyclic monoterpene (BMT) diols are small-molecule compounds that mimic ultraviolet radiation (UVR) by inducing melanogenesis. The objective of this study was to compare the effects of 2,2-dimethyl-3-propanyldiol-norbornane (AGI-1140), a novel BMT diol, and ultraviolet B (UVB) on additional cellular responses. METHODS: S91 mouse melanoma cells were treated with a range of concentrations of AGI-1140, and examined for induction of melanogenesis and nitric oxide (NO). The effect of AGI-1140 on dendrite outgrowth from human melanocytes was examined by quantitative microscopy. The effect of AGI-1140 and UVB on phosphorylation of p53 serine 15 in human keratinocytes was examined by Western blotting, while the release of tumor necrosis factor-alpha (TNF-alpha) and prostaglandin E2 (PGE2) was determined by enzyme-linked immunosorbent assay. The effects of AGI-1140 and UVB on cell cycle arrest of human melanocytes, keratinocytes, fibroblasts, and endothelial cells were compared using fluorescence-activated cell sorting. RESULTS: Similar to UVB, AGI-1140 induced both melanogenesis and NO in melanoma cells. AGI-1140 also induced dendrite outgrowth from melanocytes, indicative of differentiation. However, whereas UVB induced G2 cell cycle arrest with phosphorylation of p53 at serine 15, AGI-1140 induced G1 cell cycle arrest without this phosphorylation. Additionally, unlike UVB, AGI-1140 did not increase the secretion of TNF-alpha or PGE2, mediators of UVB-induced immunosuppressive and inflammatory responses in the skin that may contribute to carcinogenesis. CONCLUSION: This study shows that melanogenesis can be induced by AGI-1140 without many of the deleterious effects associated with UVB.


Subject(s)
Monoterpenes/pharmacology , Norbornanes/pharmacology , Skin Pigmentation/drug effects , Skin Pigmentation/radiation effects , Ultraviolet Rays , Animals , Blotting, Western , Cell Cycle/drug effects , Cell Cycle/radiation effects , Cell Line, Tumor/drug effects , Cell Line, Tumor/metabolism , Cell Line, Tumor/radiation effects , Dendrites/drug effects , Dendrites/metabolism , Dendrites/radiation effects , Dinoprostone/biosynthesis , Dose-Response Relationship, Drug , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Endothelial Cells/radiation effects , Enzyme-Linked Immunosorbent Assay , Fibroblasts/drug effects , Fibroblasts/metabolism , Fibroblasts/radiation effects , Keratinocytes/drug effects , Keratinocytes/metabolism , Keratinocytes/radiation effects , Melanocytes/drug effects , Melanocytes/metabolism , Melanocytes/radiation effects , Melanoma, Experimental/pathology , Mice , Monoterpenes/administration & dosage , Nitric Oxide/biosynthesis , Norbornanes/administration & dosage , Tumor Necrosis Factor-alpha/biosynthesis
SELECTION OF CITATIONS
SEARCH DETAIL
...