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1.
Molecules ; 26(12)2021 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-34199200

RESUMO

Glycan-targeting antibodies and pseudo-antibodies have been extensively studied for their stoichiometry, avidity, and their interactions with the rapidly modifying glycan shield of influenza A. Broadly neutralizing antiviral agents bind in the same order when they neutralize enveloped viruses regardless of the location of epitopes to the host receptor binding site. Herein, we investigated the binding of cyanovirin-N (CV-N) to surface-expressed glycoproteins such as those of human immunodeficiency virus (HIV) gp120, hemagglutinin (HA), and Ebola (GP)1,2 and compared their binding affinities with the binding response to the trimer-folded gp140 using surface plasmon resonance (SPR). Binding-site knockout variants of an engineered dimeric CV-N molecule (CVN2) revealed a binding affinity that correlated with the number of (high-) affinity binding sites. Binding curves were specific for the interaction with N-linked glycans upon binding with two low-affinity carbohydrate binding sites. This biologically active assembly of a domain-swapped CVN2, or monomeric CV-N, bound to HA with a maximum KD of 2.7 nM. All three envelope spike proteins were recognized at a nanomolar KD, whereas binding to HIV neutralizing 2G12 by targeting HA and Ebola GP1,2 was measured in the µM range and specific for the bivalent binding scheme in SPR. In conclusion, invariant structural protein patterns provide a substrate for affinity maturation in the membrane-anchored HA regions, as well as the glycan shield on the membrane-distal HA top part. They can also induce high-affinity binding in antiviral CV-N to HA at two sites, and CVN2 binding is achieved at low-affinity binding sites.


Assuntos
Proteínas de Bactérias/metabolismo , Ebolavirus/metabolismo , HIV-1/metabolismo , Orthomyxoviridae/metabolismo , Polissacarídeos/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas do Envelope Viral/metabolismo , Proteínas de Bactérias/farmacologia , Sítios de Ligação , Ebolavirus/imunologia , Ebolavirus/isolamento & purificação , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/imunologia , HIV-1/isolamento & purificação , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/metabolismo , Doença pelo Vírus Ebola/virologia , Humanos , Influenza Humana/imunologia , Influenza Humana/metabolismo , Influenza Humana/virologia , Orthomyxoviridae/imunologia , Orthomyxoviridae/isolamento & purificação , Polissacarídeos/imunologia , Ligação Proteica , Proteínas Recombinantes/isolamento & purificação , Proteínas do Envelope Viral/imunologia
2.
Carcinogenesis ; 41(4): 483-489, 2020 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31840161

RESUMO

Intestinal microbiota are considered a sensor for molecular pathways, which orchestrate energy balance, immune responses, and cell regeneration. We previously reported that microbiota restriction promoted higher levels of systemic radiation-induced genotoxicity, proliferative lymphocyte activation, and apoptotic polarization of metabolic pathways. Restricted intestinal microbiota (RM) that harbors increased abundance of Lactobacillus johnsonii (LBJ) has been investigated for bacterial communities that correlated radiation-induced genotoxicity. Indicator phylotypes were more abundant in RM mice and increased in prevalence after whole body irradiation in conventional microbiota (CM) mice, while none of the same ten most abundant phylotypes were different in abundance between CM mice before and after heavy ion irradiation. Muribaculum intestinale was detected highest in female small intestines in RM mice, which were lacking Ureaplasma felinum compared with males, and thus these bacteria could be contributing to the differential amounts of radiation-induced systemic genotoxicity between the CM and RM groups. Helicobacter rodentium and M.intestinale were found in colons in the radiation-resistant CM phenotype. While the expression of interferon-γ was elevated in the small intestine, and lower in blood in CM mice, high-linear energy transfer radiation reduced transforming growth factor-ß with peripheral interleukin (IL)-17 in RM mice, particularly in females. We found that female RM mice showed improved micro-architectural bone structure and anti-inflammatory radiation response compared with CM mice at a delayed phase 6 weeks postexposure to particle radiation. However, microbiota restriction reduced inflammatory markers of tumor necrosis factor in marrow, when IL-17 was reduced by intraperitoneal injection of IL-17 neutralizing antibody.


Assuntos
Biomarcadores/metabolismo , Osso e Ossos/anatomia & histologia , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Microbioma Gastrointestinal/fisiologia , Animais , Osso e Ossos/microbiologia , Osso e Ossos/efeitos da radiação , Feminino , Microbioma Gastrointestinal/efeitos da radiação , Interleucina-17/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Filogenia
3.
Carcinogenesis ; 39(10): 1207-1215, 2018 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-30060078

RESUMO

Since its initial sales in the 1970s, the herbicide glyphosate attained widespread use in modern agriculture, becoming the most commercially successful and widely used herbicide of all time as of 2016. Despite a primary mechanism that targets a pathway absent from animal cells and regulatory studies showing safety margins orders of magnitude better than many other, more directly toxic herbicides, the safety status of glyphosate has recently been brought into question by a slow accumulation of studies suggesting more subtle health risks, especially when considered in combination with the surfactants it is usually applied with. Current, official views of respected international regulatory and health bodies remain divided on glyphosate's status as a human carcinogen, but the 2015 International Agency for Research on Cancer decision to reclassify the compound as Category 2A (probably carcinogenic to humans) marked a sea change in the scientific community's consensus view. The goal of this review is to consider the state of science regarding glyphosate's potential as a human carcinogen and genotoxin, with particular focus on studies suggesting mechanisms that would go largely undetected in traditional toxicology studies, such as microbiome disruption and endocrine mimicry at very low concentrations.


Assuntos
Exposição Ambiental/efeitos adversos , Glicina/análogos & derivados , Herbicidas/efeitos adversos , Neoplasias/induzido quimicamente , Medição de Risco/métodos , Animais , Carcinógenos/análise , Glicina/efeitos adversos , Humanos , Microbiota , Mutagênicos/efeitos adversos , Mutagênicos/análise , Glifosato
4.
Arch Toxicol ; 92(11): 3441, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30267108

RESUMO

The original article can be found online.

5.
Mutagenesis ; 31(5): 491-509, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27209205

RESUMO

Potential ionising radiation exposure scenarios are varied, but all bring risks beyond the simple issues of short-term survival. Whether accidentally exposed to a single, whole-body dose in an act of terrorism or purposefully exposed to fractionated doses as part of a therapeutic regimen, radiation exposure carries the consequence of elevated cancer risk. The long-term impact of both intentional and unintentional exposure could potentially be mitigated by treatments specifically developed to limit the mutations and precancerous replication that ensue in the wake of irradiation The development of such agents would undoubtedly require a substantial degree of in vitro testing, but in order to accurately recapitulate the complex process of radiation-induced carcinogenesis, well-understood animal models are necessary. Inbred strains of the laboratory mouse, Mus musculus, present the most logical choice due to the high number of molecular and physiological similarities they share with humans. Their small size, high rate of breeding and fully sequenced genome further increase its value for use in cancer research. This chapter will review relevant m. musculus inbred and F1 hybrid animals of radiation-induced myeloid leukemia, thymic lymphoma, breast and lung cancers. Method of cancer induction and associated molecular pathologies will also be described for each model.


Assuntos
Modelos Animais de Doenças , Camundongos , Neoplasias Induzidas por Radiação , Animais , Feminino , Masculino , Neoplasias Induzidas por Radiação/genética
6.
Arch Toxicol ; 90(9): 2109-2130, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27311821

RESUMO

Chronic inflammation is strongly associated with approximately one-fifth of all human cancers. Arising from combinations of factors such as environmental exposures, diet, inherited gene polymorphisms, infections, or from dysfunctions of the immune response, chronic inflammation begins as an attempt of the body to remove injurious stimuli; however, over time, this results in continuous tissue destruction and promotion and maintenance of carcinogenesis. Here, we focus on intestinal inflammation and its associated cancers, a group of diseases on the rise and affecting millions of people worldwide. Intestinal inflammation can be widely grouped into inflammatory bowel diseases (ulcerative colitis and Crohn's disease) and celiac disease. Long-standing intestinal inflammation is associated with colorectal cancer and small-bowel adenocarcinoma, as well as extraintestinal manifestations, including lymphomas and autoimmune diseases. This article highlights potential mechanisms of pathogenesis in inflammatory bowel diseases and celiac disease, as well as those involved in the progression to associated cancers, most of which have been identified from studies utilizing mouse models of intestinal inflammation. Mouse models of intestinal inflammation can be widely grouped into chemically induced models; genetic models, which make up the bulk of the studied models; adoptive transfer models; and spontaneous models. Studies in these models have lead to the understanding that persistent antigen exposure in the intestinal lumen, in combination with loss of epithelial barrier function, and dysfunction and dysregulation of the innate and adaptive immune responses lead to chronic intestinal inflammation. Transcriptional changes in this environment leading to cell survival, hyperplasia, promotion of angiogenesis, persistent DNA damage, or insufficient repair of DNA damage due to an excess of proinflammatory mediators are then thought to lead to sustained malignant transformation. With regard to extraintestinal manifestations such as lymphoma, however, more suitable models are required to further investigate the complex and heterogeneous mechanisms that may be at play.


Assuntos
Doença Celíaca/complicações , Transformação Celular Neoplásica , Colite Ulcerativa/complicações , Doença de Crohn/complicações , Neoplasias Intestinais/etiologia , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Doença Celíaca/genética , Doença Celíaca/metabolismo , Doença Celíaca/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Colite Ulcerativa/genética , Colite Ulcerativa/metabolismo , Colite Ulcerativa/patologia , Doença de Crohn/genética , Doença de Crohn/metabolismo , Doença de Crohn/patologia , Dano ao DNA , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Humanos , Mediadores da Inflamação/metabolismo , Neoplasias Intestinais/genética , Neoplasias Intestinais/metabolismo , Neoplasias Intestinais/patologia , Camundongos , Fatores de Risco , Transdução de Sinais , Microambiente Tumoral
7.
Hum Genomics ; 8: 13, 2014 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-25062865

RESUMO

The use of radiation therapy is a cornerstone of modern cancer treatment. The number of patients that undergo radiation as a part of their therapy regimen is only increasing every year, but this does not come without cost. As this number increases, so too does the incidence of secondary, radiation-induced neoplasias, creating a need for therapeutic agents targeted specifically towards incidence reduction and treatment of these cancers. Development and efficacy testing of these agents requires not only extensive in vitro testing but also a set of reliable animal models to accurately recreate the complex situations of radiation-induced carcinogenesis. As radiation-induced leukemic progression often involves genomic changes such as rearrangements, deletions, and changes in methylation, the laboratory mouse Mus musculus, with its fully sequenced genome, is a powerful tool in cancer research. This fact, combined with the molecular and physiological similarities it shares with man and its small size and high rate of breeding in captivity, makes it the most relevant model to use in radiation-induced leukemia research. In this work, we review relevant M. musculus inbred and F1 hybrid animal models, as well as methods of induction of radiation-induced myeloid leukemia. Associated molecular pathologies are also included.


Assuntos
Leucemia Mieloide/genética , Lesões Experimentais por Radiação/genética , Animais , Carcinogênese/genética , Carcinogênese/efeitos da radiação , Humanos , Leucemia Mieloide/patologia , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Lesões Experimentais por Radiação/patologia , Tolerância a Radiação
8.
Mutagenesis ; 30(6): 841-9, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26122113

RESUMO

Non-homologous end joining (NHEJ) directly joins two broken DNA ends without sequence homology. A distinct pathway called microhomology-mediated end joining (MMEJ) relies on a few base pairs of homology between the recombined DNA. The majority of DNA double-strand breaks caused by endogenous oxygen species or ionizing radiation contain damaged bases that hinder direct religation. End processing is required to remove mismatched nucleotides and fill in gaps during end joining of incompatible ends. POL3 in Saccharomyces cerevisiae encodes polymerase δ that is required for DNA replication and other DNA repair processes. Our previous results have shown that POL3 is involved in gap filling at 3' overhangs in POL4-independent NHEJ. Here, we studied the epistatic interaction between POL3, RAD50, XRS2 and POL4 in NHEJ using a plasmid-based endjoining assay in yeast. We demonstrated that either rad50 or xrs2 mutation is epistatic for end joining of compatible ends in the rad50 pol3-t or xrs2 pol3-t double mutants. However, the pol3-t and rad50 or pol3-t and xrs2 mutants caused an additive decrease in the end-joining efficiency of incompatible ends, suggesting that POL3 and RAD50 or POL3 and XRS2 exhibit independent functions in NHEJ. In the rad50 pol4 mutant, end joining of incompatible ends was not detected. In the rad50 or xrs2 mutants, NHEJ events did not contain any microhomology at the rejoined junctions. The pol3-t mutation restored MMEJ in the rad50 or xrs2 mutant backgrounds. Moreover, we demonstrated that NHEJ of incompatible ends required RAD50 and POL4 more than POL3. In conclusion, POL3 and POL4 have differential functions in NHEJ, independent of the RAD50-mediated repair pathway.


Assuntos
Reparo do DNA por Junção de Extremidades , DNA Polimerase III/metabolismo , DNA Polimerase beta/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , DNA Polimerase III/genética , DNA Polimerase beta/genética , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Mutação
9.
Mutat Res ; 749(1-2): 58-65, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23748015

RESUMO

Cigarette smoke causes direct oxidative DNA damage as well as indirect damage through inflammation. Epidemiological studies show a strong relationship between secondhand smoke and cancer; however, the mechanisms of secondhand smoke-induced cancer are not well understood. Animal models with either (i) deficient oxidative DNA damage repair, or (ii) a decreased capacity to combat oxidative stress may help determine the pathways important in mitigating damage caused by smoke. In this study, we used mice lacking Ogg1 and Myh, both of which are involved in base excision repair by removing oxidatively damaged DNA bases. Gclm-deficient mice, which have decreased levels of glutathione (GSH), were used to look at the role of smoke-induced oxidative damage. Ex vivo experiments show significantly elevated levels of DNA single-strand breaks and chromosomal aberrations in peripheral blood lymphocytes from Ogg1(-/-)Myh(-/-) double knockout mice compared to wild type (WT) mice after 24h of exposure to cigarette smoke extract (CSE). The average γH2AX foci per cell was significantly elevated 3h after exposure to CSE in cells from Ogg1(-/-)Myh(-/-) double knockout mice compared to wildtype mice. In vivo we found that all mice had increased markers of DNA damage after exposure to side-stream tobacco smoke (SSTS). Ogg1(-/-)Myh(-/-) and Gclm(-/-) mice had altered levels of peripheral blood glutathione after SSTS exposure whereas wild type mice did not. This may be due to differential regulation of glutathione synthesis in the lung. We also found that Ogg1(-/-)Myh(-/-) mice had a decreased lifespan after oral gavage with benzo[a]pyrene compared to wildtype mice and sham-exposed Ogg1(-/-)Myh(-/-) mice. Our results are important in investigating the roles of oxidative stress and oxidative DNA damage repair in cigarette smoke-induced cancers and characterizing the role of genetic polymorphisms in smoke-related disease susceptibility.


Assuntos
Células Sanguíneas/efeitos dos fármacos , Distúrbios no Reparo do DNA/genética , Glutationa/deficiência , Estresse Oxidativo/genética , Poluição por Fumaça de Tabaco/efeitos adversos , Animais , Células Sanguíneas/metabolismo , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , DNA Glicosilases/genética , Distúrbios no Reparo do DNA/sangue , Distúrbios no Reparo do DNA/patologia , Feminino , Glutamato-Cisteína Ligase/genética , Glutationa/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estresse Oxidativo/efeitos dos fármacos
10.
Mutagenesis ; 27(1): 77-86, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21980144

RESUMO

Chronic inflammatory diseases are characterised by systemically elevated levels of tumour necrosis factor (TNF)-α, a proinflammatory cytokine with pleiotropic downstream effects. We have previously demonstrated increased genotoxicity in peripheral leukocytes and various tissues in models of intestinal inflammation. In the present study, we asked whether TNF-α is sufficient to induce DNA damage systemically, as observed in intestinal inflammation, and whether tumour necrosis factor receptor (TNFR) signalling would be necessary for the resultant genotoxicity. In the wild-type mice, 500 ng per mouse of TNF-α was sufficient to induce DNA damage to multiple cell types and organs 1-h post-administration. Primary splenic T cells manifested TNF-α-induced DNA damage in the absence of other cell types. Furthermore, TNFR1(-/-)TNFR2(-/-) mice demonstrated decreased systemic DNA damage in a model of intestinal inflammation and after TNF-α injection versus wild-type mice, indicating the necessity of TNFR signalling. Nuclear factor (NF)-κB inhibitors were also able to decrease damage induced by TNF-α injection in wild-type mice. When TNF-α administration was combined with interleukin (IL)-1ß, another proinflammatory cytokine, DNA damage persisted for up to 24 h. When combined with IL-10, an anti-inflammatory cytokine, decreased genotoxicity was observed in vivo and in vitro. TNF-α/TNFR-mediated signalling is therefore sufficient and plays a large role in mediating DNA damage to various cell types, subject to modulation by other cytokines and their mediators.


Assuntos
Dano ao DNA , Inflamação/patologia , Receptores do Fator de Necrose Tumoral/genética , Fator de Necrose Tumoral alfa/administração & dosagem , Animais , Sobrevivência Celular , Colite/induzido quimicamente , Ensaio Cometa , Modelos Animais de Doenças , Combinação de Medicamentos , Inflamação/genética , Interleucina-10/administração & dosagem , Interleucina-1beta/administração & dosagem , Interleucina-1beta/toxicidade , Leucócitos/metabolismo , Camundongos , Camundongos Knockout , Transdução de Sinais
11.
Radiat Res ; 197(2): 184-192, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-35130347

RESUMO

Microbiota can both negatively and positively impact radiation-induced bone loss. Our prior research showed that compared to mice with conventional gut microbiota (CM), mice with restricted gut microbiota (RM) reduced inflammatory tumor necrosis factor (TNF) in bone marrow, interleukin (IL)-17 in blood, and chemokine (C-C motif) ligand 20 (CCL20) in bone marrow under anti-IL-17 treatment. We showed that Muribaculum intestinale was more abundant in intestinal epithelial cells (IECs) from the small intestine of female RM mice and positively associated with augmented skeletal bone structure. Female C57BL/6J pun RM mice, which were injected with anti-IL-17 antibody one day before exposure to 1.5 Gy 28Si ions of 850 MeV/u, showed high trabecular numbers in tibiae at 6 weeks postirradiation. Irradiated CM mice were investigated for lower interferon-γ and IL-17 levels in the small intestine than RM mice. IL-17 blockage resulted in bacterial indicator phylotypes being different between both microbiota groups before and after irradiation. Analysis of the fecal bacteria were performed in relation to bone quality and body weight, showing reduced tibia cortical thickness in irradiated CM mice (-15%) vs. irradiated RM mice (-9.2%). Correlation analyses identified relationships among trabecular bone parameters (TRI-BV/TV, Tb.N, Tb.Th, Tb.Sp) and Bacteroides massiliensis, Muribaculum sp. and Prevotella denticola. Turicibacter sp. was found directly correlated with trabecular separation in anti-IL-17 treated mice, whereas an unidentified Bacteroidetes correlated with trabecular thickness in anti-IL-17 neutralized and radiation-exposed mice. We demonstrated radiation-induced osteolytic damage to correlate with bacterial indicator phylotypes of the intestinal microbiota composition, and these relationships were determined from the previously discovered dose-dependent particle radiation effects on cell proliferation in bone tissue. New translational approaches were designed to investigate dynamic changes of gut microbiota in correlation with conditions of treatment and disease as well as mechanisms of systemic side-effects in radiotherapy.


Assuntos
Microbioma Gastrointestinal
12.
Int J Cancer ; 129(8): 1815-25, 2011 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-21520038

RESUMO

Chronic intestinal inflammation leads to increased risk of colorectal and small intestinal cancers and is also associated with extraintestinal manifestations such as lymphomas, other solid cancers and autoimmune disorders. We have previously found that acute and chronic intestinal inflammation causes DNA damage to circulating peripheral leukocytes, manifesting a systemic effect in genetically and chemically induced models of intestinal inflammation. Our study addresses the scope of tissue targets and genotoxic damage induced by inflammation-associated genotoxicity. Using several experimental models of intestinal inflammation, we analyzed various types of DNA damage in leukocyte subpopulations of the blood, spleen, mesenteric and peripheral lymph nodes and in intestinal epithelial cells, hepatocytes and the brain. Genotoxicity in the form of DNA single- and double-stranded breaks accompanied by oxidative base damage was found in leukocyte subpopulations of the blood, diverse lymphoid organs, intestinal epithelial cells and hepatocytes. The brain did not demonstrate significant levels of DNA double-stranded breaks as measured by γ-H2AX immunostaining. CD4(+) and CD8(+) T-cells were most sensitive to DNA damage versus other cell types in the peripheral blood. In vivo measurements and in vitro modeling suggested that genotoxicity was induced by increased levels of systemically circulating proinflammatory cytokines. Moreover, genotoxicity involved increased damage rather than reduced repair, as it is not associated with decreased expression of the DNA double-strand break recognition and repair protein, ataxia telangiectasia mutated. These findings suggest that levels of intestinal inflammation contribute to the remote tissue burden of genotoxicity, with potential effects on nonintestinal diseases and cancer.


Assuntos
Colite/genética , Dano ao DNA , Animais , Encéfalo/citologia , Colite/imunologia , Citocinas/sangue , Hepatócitos/citologia , Inflamação/genética , Doenças Inflamatórias Intestinais/genética , Mucosa Intestinal/citologia , Leucócitos/citologia , Sistema Linfático/citologia , Camundongos
13.
Mol Genet Genomics ; 285(6): 471-84, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21512733

RESUMO

Genes in the RAD52 epistasis group are involved in repairing DNA double-stranded breaks via homologous recombination. We have previously shown that RAD50 is involved in mitotic nonhomologous integration but not in homologous integration. However, the role of Rad50 in nonhomologous integration has not previously been examined. In the current work, we report that the rad50∆ mutation caused a tenfold decrease in the frequency of nonhomologous integration with the majority of nonhomologous integrants showing an unstable Ura(+) phenotype. Sequencing analysis of the integration target sites showed that integration events of both ends of the integrating vector in the rad50∆ mutant occurred at different chromosomal locations, resulting in large deletions or translocations on the genomic insertion sites. Interestingly, 47% of events in the rad50∆ mutant were integrated into repetitive sequences including rDNA locus, telomeres and Ty elements and 27% of events were integrated into non-repetitive sequences as compared to 11% of events integrated into rDNA and 70% into non-repetitive sequences in the wild-type cells. These results showed that deletion of RAD50 significantly changes the distribution of different classes of integration events, suggesting that Rad50 is required for nonhomologous integration at non-repetitive sequences more so than at repetitive ones. Furthermore, Southern analysis indicated that half of the events contained deletions at one or at both ends of the integrating DNA fragment, suggesting that Rad50 might have a role in protecting free ends of double-strand breaks. In contrast to the rad50∆ mutant, the rad50S mutant (separation of function allele) slightly increases the frequency of nonhomologous integration but the distribution of integration events is similar to that of wild-type cells with the majority of events integrated into a chromosomal locus. Our results suggest that deletion of RAD50 may block the major pathway of nonhomologous integration into a non-repetitive chromosomal locus and Rad50 may be involved in tethering two ends of the integrating DNA into close proximity that facilitates nonhomologous integration of both ends into a single chromosomal locus.


Assuntos
Proteínas de Ligação a DNA/genética , Mutação , Recombinação Genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sequência de Bases , Cromossomos , Sequências Repetitivas de Ácido Nucleico , Deleção de Sequência , Translocação Genética
14.
Drug Chem Toxicol ; 34(4): 433-9, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21740348

RESUMO

Aminoglycoside antibiotics have been in use since 1944 with the discovery of streptomycin. The aim of this study was to derive a new, highly resistant multicopy neo(R) transgenic mouse strain, named TgN3Ems, by random insertion of the plasmid, pPGKneobpA, and compare the level of drug resistance of wild-type and transgenic mice in vivo and corresponding primary mouse embryonic fibroblasts (MEFs) in vitro to a model neomycin analog, G418. The expression neoR in transgenic animals caused a 5-fold increase in the approximate lethal dose of G418, compared to wild type. No adverse pathological changes were found for the transgenic mice treated with G418, as they all died within minutes after injection. In contrast, the G418 treatment of wild-type mice resulted in a marked liver and kidney toxicity detected microscopically and via increases of serum biomarkers for liver and kidney damage. In addition, there was a mild bone marrow and lymphoid depletion. In in vitro studies, the transgenic MEFs survived 20-fold higher G418 levels, compared to the wild-type MEF cells. Therefore, TgN3Ems transgenic mice could be used as a source of G418-resistant feeder cells for gene targeting. Since the expression of drug-resistance genes in transgenic animals confers resistance to toxicity, the TgN3Ems mice might serve as a tool applicable in drug design.


Assuntos
Resistência a Medicamentos/genética , Células Alimentadoras/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Marcação de Genes , Gentamicinas/toxicidade , Canamicina Quinase/genética , Animais , Southern Blotting , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Células Alimentadoras/citologia , Células Alimentadoras/enzimologia , Fibroblastos/citologia , Fibroblastos/enzimologia , Gentamicinas/farmacologia , Dose Letal Mediana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Plasmídeos , Regiões Promotoras Genéticas , Testes de Toxicidade Aguda , Transgenes
15.
Mol Genet Genomics ; 284(4): 231-42, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20677012

RESUMO

The integrase of the Saccharomyces cerevisiae retrotransposon Ty1 integrates Ty1 cDNA into genomic DNA likely via a transesterification reaction. Little is known about the mechanisms ensuring that integrase does not integrate non-Ty DNA fragments. In an effort to elucidate the conditions under which Ty1 integrase accepts non-Ty DNA as substrate, PCR fragments encompassing a selectable marker gene were transformed into yeast strains overexpressing Ty1 integrase. These fragments do not exhibit similarity to Ty1 cDNA except for the presence of the conserved terminal dinucleotide 5'-TG-CA-3'. The frequency of fragment insertion events increased upon integrase overexpression. Characterization of insertion events by genomic sequencing revealed that most insertion events exhibited clear hallmarks of integrase-mediated reactions, such as 5 bp target site duplication and target site preferences. Alteration of the terminal dinucleotide abolished the suitability of the PCR fragments to serve as substrates. We hypothesize that substrate specificity under normal conditions is mainly due to compartmentalization of integrase and Ty cDNA, which meet in virus-like particles. In contrast, recombinant integrase, which is not confined to virus-like particles, is able to accept non-Ty DNA, provided that it terminates in the proper dinucleotide sequence.


Assuntos
DNA Fúngico/genética , Integrases/genética , Retroelementos/genética , Saccharomyces cerevisiae/genética , Sequência Conservada , Primers do DNA , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Plasmídeos/genética , Reação em Cadeia da Polimerase , RNA Fúngico/genética , DNA Polimerase Dirigida por RNA/genética , Mapeamento por Restrição , Ribonuclease H/genética , Saccharomyces cerevisiae/enzimologia , Sequências Repetidas Terminais/genética
16.
Mutat Res ; 705(1): 40-59, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20298806

RESUMO

Chronic inflammation is strongly associated with approximately 1/5th of all human cancers. Arising from combinations of factors such as environmental exposures, diet, inherited gene polymorphisms, infections, or from dysfunctions of the immune response, chronic inflammation begins as an attempt of the body to remove injurious stimuli; however, over time, this results in continuous tissue destruction and promotion and maintenance of carcinogenesis. Here we focus on intestinal inflammation and its associated cancers, a group of diseases on the rise and affecting millions of people worldwide. Intestinal inflammation can be widely grouped into inflammatory bowel diseases (ulcerative colitis and Crohn's disease) and celiac disease. Long-standing intestinal inflammation is associated with colorectal cancer and small-bowel adenocarcinoma, as well as extraintestinal manifestations, including lymphomas and autoimmune diseases. This article highlights potential mechanisms of pathogenesis in inflammatory bowel diseases and celiac disease, as well as those involved in the progression to associated cancers, most of which have been identified from studies utilizing mouse models of intestinal inflammation. Mouse models of intestinal inflammation can be widely grouped into chemically induced models; genetic models, which make up the bulk of the studied models; adoptive transfer models; and spontaneous models. Studies in these models have lead to the understanding that persistent antigen exposure in the intestinal lumen, in combination with loss of epithelial barrier function, and dysfunction and dysregulation of the innate and adaptive immune responses lead to chronic intestinal inflammation. Transcriptional changes in this environment leading to cell survival, hyperplasia, promotion of angiogenesis, persistent DNA damage, or insufficient repair of DNA damage due to an excess of proinflammatory mediators are then thought to lead to sustained malignant transformation. With regards to extraintestinal manifestations such as lymphoma, however, more suitable models are required to further investigate the complex and heterogeneous mechanisms that may be at play.


Assuntos
Doença Celíaca/imunologia , Neoplasias Colorretais/etiologia , Inflamação/etiologia , Doenças Inflamatórias Intestinais/imunologia , Animais , Doença Celíaca/complicações , Modelos Animais de Doenças , Humanos , Doenças Inflamatórias Intestinais/complicações , Camundongos
17.
RSC Adv ; 10(19): 11079-11087, 2020 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-35495330

RESUMO

Cyanovirin-N (CV-N) has been shown to reveal broad neutralizing activity against human immunodeficiency virus (HIV) and to specifically bind Manα(1→2)Manα units exposed on various glycoproteins of enveloped viruses, such as influenza hemagglutinin (HA) and Ebola glycoprotein. Chemically synthesized dimannosylated HA peptides bound domain-swapped and dimeric CV-N with either four disulfide-bonds (Cys-Cys), or three Cys-Cys bonds and an intact fold of the high-affinity binding site at an equilibrium dissociation constant K D of 10 µM. Cys-Cys mutagenesis with ion-pairing amino-acids glutamic acid and arginine was calculated by in silico structure-based protein design and allowed for recognizing dimannose and dimannosylated peptide binding to low-affinity binding sites (K D ≈ 11 µM for one C58-C73 bond, and binding to dimannosylated peptide). In comparison, binding to HA was achieved based on one ion-pairing C58E-C73R substitution at K D = 275 nM, and K D = 5 µM for two C58E-C73R substitutions. We were utilizing a triazole bioisostere linkage to form the respective mannosylated-derivative on the HA peptide sequence of residues glutamine, glycine, and glutamic acid. Thus, mono- and dimannosylated peptides with N-terminal cysteine facilitated site-specific interactions with HA peptides, mimicking a naturally found N-linked glycosylation site on the HA head domain.

18.
DNA Repair (Amst) ; 7(9): 1531-41, 2008 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-18606574

RESUMO

Nonhomologous end joining connects DNA ends in the absence of extended sequence homology and requires removal of mismatched DNA ends and gap-filling synthesis prior to a religation step. Pol4 within the Pol X family is the only polymerase known to be involved in end processing during nonhomologous end joining in yeast. The Saccharomyces cerevisiae POL3/CDC2 gene encodes polymerase delta that is involved in DNA replication and other DNA repair processes. Here, we show that POL3 is involved in nonhomologous end joining using a plasmid-based end-joining assay in yeast, in which the pol3-t mutation caused a 1.9- to 3.2-fold decrease in the end-joining efficiency of partially compatible 5' or 3' ends, or incompatible ends, similar to the pol4 mutant. The pol3-t pol4 double mutation showed a synergistic decrease in the efficiency of NHEJ with partially compatible 5' ends or incompatible ends. Sequence analysis of the rejoined junctions recovered from the wild-type cells and mutants indicated that POL3 is required for gap filling at 3' overhangs, but not 5' overhangs during POL4-independent nonhomologous end joining. We also show that either Pol3 or Pol4 is required for simple religation of compatible or blunt ends. These results suggest that Pol3 has a generalized function in end joining in addition to its role in gap filling at 3' overhangs to enhance the overall efficiency of nonhomologous end joining. Moreover, the decreased end-joining efficiency seen in the pol3-t mutant was not due to S-phase arrest associated with the mutant. Taken together, our genetic evidence supports a novel role of Pol3 in nonhomologous end joining that facilitates gap filling at 3' overhangs in the absence of Pol4 to maintain genomic integrity.


Assuntos
DNA Polimerase III/fisiologia , Reparo do DNA , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Sequência de Bases , Instabilidade Cromossômica , Dano ao DNA , DNA Polimerase Dirigida por DNA/fisiologia , Mutação , Fase S , Proteínas de Schizosaccharomyces pombe/fisiologia
19.
Radiat Res ; 172(2): 141-51, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19630519

RESUMO

Abstract Illegitimate recombination can repair DNA double-strand breaks in one of two ways, either without sequence homology or by using a few base pairs of homology at the junctions. The second process is known as microhomology-mediated recombination. Previous studies showed that ionizing radiation and restriction enzymes increase the frequency of microhomology-mediated recombination in trans during rejoining of unirradiated plasmids or during integration of plasmids into the genome. Here we show that radiation-induced microhomology-mediated recombination is reduced by deletion of RAD52, RAD1 and RAD10 but is not affected by deletion of RAD51 and RAD2. The rad52 mutant did not change the frequency of radiation-induced microhomology-mediated recombination but rather reduced the length of microhomology required to undergo repair during radiation-induced recombination. The rad1 and rad10 mutants exhibited a smaller increase in the frequency of radiation-induced microhomology-mediated recombination, and the radiation-induced integration junctions from these mutants did not show more than 4 bp of microhomology. These results suggest that Rad52 facilitates annealing of short homologous sequences during integration and that Rad1/Rad10 endonuclease mediates removal of the displaced 3' single-stranded DNA ends after base-pairing of microhomology sequences, when more than 4 bp of microhomology are used. Taken together, these results suggest that radiation-induced microhomology-mediated recombination is under the same genetic control as the single-strand annealing apparatus that requires the RAD52, RAD1 and RAD10 genes.


Assuntos
Enzimas Reparadoras do DNA/genética , Reparo do DNA/genética , DNA Bacteriano/genética , Endonucleases/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Recombinação Genética/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , DNA Bacteriano/efeitos da radiação , Mutação/genética , Recombinação Genética/efeitos da radiação , Saccharomyces cerevisiae/efeitos da radiação , Homologia de Sequência , Endonucleases Específicas para DNA e RNA de Cadeia Simples/genética
20.
Radiat Res ; 171(4): 454-63, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19397446

RESUMO

DNA double-strand breaks repaired through nonhomologous end joining require no extended sequence homology as a template for the repair. A subset of end-joining events, termed microhomology-mediated end joining, occur between a few base pairs of homology, and such pathways have been implicated in different human cancers and genetic diseases. Here we investigated the effect of exposure of yeast and mammalian cells to ionizing radiation on the frequency and mechanism of rejoining of transfected unirradiated linear plasmid DNA. Cells were exposed to gamma radiation prior to plasmid transfection; subsequently the rejoined plasmids were recovered and the junction sequences were analyzed. In irradiated yeast cells, 68% of recovered plasmids contained microhomologies, compared to only 30% from unirradiated cells. Among them 57% of events used>or=4 bp of microhomology compared to only 11% from unirradiated cells. In irradiated mammalian cells, 54% of plasmids used>or=4 bp of microhomology compared to none from unirradiated cells. We conclude that exposure of yeast and mammalian cells to radiation prior to plasmid transfection enhances the frequency of microhomology-mediated end-joining events in trans. If such events occur within genomic locations, they may be involved in the generation of large deletions and other chromosomal aberrations that occur in cancer cells.


Assuntos
Radiação Ionizante , Recombinação Genética , Saccharomyces cerevisiae/efeitos da radiação , Animais , Sequência de Bases , Células CHO , Aberrações Cromossômicas , Cricetinae , Cricetulus , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Modelos Genéticos , Dados de Sequência Molecular , Plasmídeos/metabolismo , Homologia de Sequência do Ácido Nucleico
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