RESUMO
Antibiotic binding sites are located in important domains of essential enzymes and have been extensively studied in the context of resistance mutations; however, their study is limited by positive selection. Using multiplex genome engineering1 to overcome this constraint, we generate and characterize a collection of 760 single-residue mutants encompassing the entire rifampicin binding site of Escherichia coli RNA polymerase (RNAP). By genetically mapping drug-enzyme interactions, we identify an alpha helix where mutations considerably enhance or disrupt rifampicin binding. We find mutations in this region that prolong antibiotic binding, converting rifampicin from a bacteriostatic to bactericidal drug by inducing lethal DNA breaks. The latter are replication dependent, indicating that rifampicin kills by causing detrimental transcription-replication conflicts at promoters. We also identify additional binding site mutations that greatly increase the speed of RNAP.Fast RNAP depletes the cell of nucleotides, alters cell sensitivity to different antibiotics and provides a cold growth advantage. Finally, by mapping natural rpoB sequence diversity, we discover that functional rifampicin binding site mutations that alter RNAP properties or confer drug resistance occur frequently in nature.
Assuntos
Antibacterianos , Sítios de Ligação , RNA Polimerases Dirigidas por DNA , Escherichia coli , Mutação , Rifampina , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/genética , Quebras de DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Farmacorresistência Bacteriana/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Nucleotídeos/deficiência , Nucleotídeos/metabolismo , Regiões Promotoras Genéticas , Rifampina/química , Rifampina/metabolismo , Rifampina/farmacologia , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacosRESUMO
Replication fork maintenance pathways preserve chromosomes, but their faulty application at nonallelic repeats could generate rearrangements causing cancer, genomic disorders and speciation. Potential causal mechanisms are homologous recombination and error-free postreplication repair (EF-PRR). Homologous recombination repairs damage-induced DNA double-strand breaks (DSBs) and single-ended DSBs within replication. To facilitate homologous recombination, the recombinase RAD51 and mediator BRCA2 form a filament on the 3' DNA strand at a break to enable annealing to the complementary sister chromatid while the RecQ helicase, BLM (Bloom syndrome mutated) suppresses crossing over to prevent recombination. Homologous recombination also stabilizes and restarts replication forks without a DSB. EF-PRR bypasses DNA incongruities that impede replication by ubiquitinating PCNA (proliferating cell nuclear antigen) using the RAD6-RAD18 and UBC13-MMS2-RAD5 ubiquitin ligase complexes. Some components are common to both homologous recombination and EF-PRR such as RAD51 and RAD18. Here we delineate two pathways that spontaneously fuse inverted repeats to generate unstable chromosomal rearrangements in wild-type mouse embryonic stem (ES) cells. Gamma-radiation induced a BLM-regulated pathway that selectively fused identical, but not mismatched, repeats. By contrast, ultraviolet light induced a RAD18-dependent pathway that efficiently fused mismatched repeats. Furthermore, TREX2 (a 3'â5' exonuclease) suppressed identical repeat fusion but enhanced mismatched repeat fusion, clearly separating these pathways. TREX2 associated with UBC13 and enhanced PCNA ubiquitination in response to ultraviolet light, consistent with it being a novel member of EF-PRR. RAD18 and TREX2 also suppressed replication fork stalling in response to nucleotide depletion. Interestingly, replication fork stalling induced fusion for identical and mismatched repeats, implicating faulty replication as a causal mechanism for both pathways.
Assuntos
Instabilidade Cromossômica/genética , Cromossomos de Mamíferos/genética , Reparo do DNA/genética , Replicação do DNA/genética , Recombinação Homóloga/genética , Sequências Repetidas Invertidas/genética , Animais , Sequência de Bases , Quebra Cromossômica , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Exodesoxirribonucleases/metabolismo , Hidroxiureia/farmacologia , Camundongos , Nucleotídeos/deficiência , Nucleotídeos/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Rad51 Recombinase/metabolismo , RecQ Helicases/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação/efeitos da radiação , Raios UltravioletaRESUMO
Here we employ a set of RNA Polymerase II (Pol II) activity mutants to determine the consequences of increased or decreased Pol II catalysis on gene expression in Saccharomyces cerevisiae. We find that alteration of Pol II catalytic rate, either fast or slow, leads to decreased Pol II occupancy and apparent reduction in elongation rate in vivo. However, we also find that determination of elongation rate in vivo by chromatin immunoprecipitation can be confounded by the kinetics and conditions of transcriptional shutoff in the assay. We identify promoter and template-specific effects on severity of gene expression defects for both fast and slow Pol II mutants. We show that mRNA half-lives for a reporter gene are increased in both fast and slow Pol II mutant strains and the magnitude of half-life changes correlate both with mutants' growth and reporter expression defects. Finally, we tested a model that altered Pol II activity sensitizes cells to nucleotide depletion. In contrast to model predictions, mutated Pol II retains normal sensitivity to altered nucleotide levels. Our experiments establish a framework for understanding the diversity of transcription defects derived from altered Pol II activity mutants, essential for their use as probes of transcription mechanisms.
Assuntos
RNA Polimerase II/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Biocatálise , Imunoprecipitação da Cromatina , Regulação Fúngica da Expressão Gênica , Meia-Vida , Mutação , Nucleotídeos/deficiência , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/enzimologia , Elongação da Transcrição Genética , Sítio de Iniciação de TranscriçãoRESUMO
UNLABELLED: To date, the majority of work on RNA virus replication fidelity has focused on the viral RNA polymerase, while the potential role of other viral replicase proteins in this process is poorly understood. Previous studies used resistance to broad-spectrum RNA mutagens, such as ribavirin, to identify polymerases with increased fidelity that avoid misincorporation of such base analogues. We identified a novel variant in the alphavirus viral helicase/protease, nonstructural protein 2 (nsP2) that operates in concert with the viral polymerase nsP4 to further alter replication complex fidelity, a functional linkage that was conserved among the alphavirus genus. Purified chikungunya virus nsP2 presented delayed helicase activity of the high-fidelity enzyme, and yet purified replication complexes manifested stronger RNA polymerization kinetics. Because mutagenic nucleoside analogs such as ribavirin also affect intracellular nucleotide pools, we addressed the link between nucleotide depletion and replication fidelity by using purine and pyrimidine biosynthesis inhibitors. High-fidelity viruses were more resistant to these conditions, and viral growth could be rescued by the addition of exogenous nucleosides, suggesting that mutagenesis by base analogues requires nucleotide pool depletion. This study describes a novel function for nsP2, highlighting the role of other components of the replication complex in regulating viral replication fidelity, and suggests that viruses can alter their replication complex fidelity to overcome intracellular nucleotide-depleting conditions. IMPORTANCE: Previous studies using the RNA mutagen ribavirin to select for drug-resistant variants have highlighted the essential role of the viral RNA-dependent RNA polymerase in regulating replication fidelity. However, the role of other viral replicase components in replication fidelity has not been studied in detail. We identified here an RNA mutagen-resistant variant of the nsP2 helicase/protease that conferred increased fidelity and yet could not operate in the same manner as high-fidelity polymerases. We show that the alphavirus helicase is a key component of the fidelity-regulating machinery. Our data show that the RNA mutagenic activity of compounds such as ribavirin is coupled to and potentiated by nucleotide depletion and that RNA viruses can fine-tune their replication fidelity when faced with an intracellular environment depleted of nucleotides.
Assuntos
Vírus Chikungunya/fisiologia , Cisteína Endopeptidases/metabolismo , RNA Helicases/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Replicação Viral/fisiologia , Animais , Antivirais/farmacologia , Sequência de Bases , Linhagem Celular , Vírus Chikungunya/genética , Chlorocebus aethiops , Cricetinae , Cisteína Endopeptidases/genética , Replicação do DNA/efeitos dos fármacos , Células HeLa , Humanos , Mutação/genética , Nucleotídeos/deficiência , Purinas/biossíntese , Pirimidinas/biossíntese , RNA Helicases/genética , RNA Viral/biossíntese , RNA Polimerase Dependente de RNA/genética , Ribavirina/farmacologia , Análise de Sequência de RNA , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Células Vero , Replicação Viral/genéticaRESUMO
The minichromosome maintenance (MCM) helicase, composed of subunits Mcm2-7, is essential for the initiation and elongation phases of DNA replication. Even when DNA synthesis is blocked, MCM continues DNA unwinding to some extent for activation of the replication checkpoint and then stops. However, the mechanism of regulation of MCM-helicase activity remains unknown. Here, we show that truncation of the Mcm4 C-terminal domain (CTD) in fission yeast results in hypersensitivity to replication block caused by dNTP depletion. The truncation mcm4-c84 does not affect the activation of the replication checkpoint pathway but delays its attenuation during recovery from replication block. Two dimensional gel electrophoresis showed that mcm4-c84 delays the disappearance of replication intermediates, indicating that the Mcm4 CTD is required for efficient recovery of stalled replication forks. Remarkably, chromatin immunoprecipitation revealed that mcm4-c84 brings about an increase rather than a decrease in the association of the single-stranded DNA-binding protein RPA to stalled forks, and MCM and the accessory complex GINS are unaffected. These results suggest that the Mcm4 CTD is required to suspend MCM-helicase activity after the formation of single-stranded DNA sufficient for checkpoint activation.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , DNA Helicases/metabolismo , Replicação do DNA , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Ciclo Celular , Componente 4 do Complexo de Manutenção de Minicromossomo , Mutação/genética , Nucleotídeos/deficiência , Estrutura Terciária de Proteína , Schizosaccharomyces/citologia , Schizosaccharomyces/metabolismo , Relação Estrutura-AtividadeRESUMO
Melanoma is the deadliest form of skin cancer. While clinical developments have significantly improved patient prognosis, effective treatment is often obstructed by limited response rates, intrinsic or acquired resistance to therapy, and adverse events. Melanoma initiation and progression are associated with transcriptional reprogramming of melanocytes to a cell state that resembles the lineage from which the cells are specified during development, that is the neural crest. Convergence to a neural crest cell (NCC)-like state revealed the therapeutic potential of targeting developmental pathways for the treatment of melanoma. Neural crest cells have a unique sensitivity to metabolic dysregulation, especially nucleotide depletion. Mutations in the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH) particularly affect neural crest-derived tissues and cause Miller syndrome, a genetic disorder characterized by craniofacial malformations in patients. The developmental susceptibility of the neural crest to nucleotide deficiency is conserved in melanoma and provides a metabolic vulnerability that can be exploited for therapeutic purposes. We review the current knowledge on nucleotide stress responses in neural crest and melanoma and discuss how the recent scientific advances that have improved our understanding of transcriptional regulation during nucleotide depletion can impact melanoma treatment.
Assuntos
Linhagem da Célula , Melanócitos/metabolismo , Melanoma/metabolismo , Crista Neural/metabolismo , Nucleotídeos/metabolismo , Neoplasias Cutâneas/metabolismo , Estresse Fisiológico , Animais , Antimetabólitos Antineoplásicos/uso terapêutico , Regulação Neoplásica da Expressão Gênica , Humanos , Melanócitos/efeitos dos fármacos , Melanócitos/patologia , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Crista Neural/efeitos dos fármacos , Crista Neural/patologia , Nucleotídeos/deficiência , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Transcrição GênicaRESUMO
We examined whether active fruit naringin can reduce the risk of BPA-mediated neurotoxicity in L-NAME induced hypertensive rats and whether the modulation could be linked to improvement of brain NO signaling. Male albino rats were randomly distributed into eight (n = 7) groups. Group I was control animals, Group II was orally-treated with L-NAME, Group III was orally treated with 100 mg/kg BPA, Group IV was orally-treated with L-NAME +100 mg/kg BPA. Group V was orally-administered with L-NAME +80 mg/kg NAR. Group VI was orally-administered with 100 mg/kg BPA +80 mg/kg NAR. Group VII was orally-administered with L-NAME+100 mg/kg BPA +80 mg/kg NAR. Lastly, group VIII was orally-treated with 80 mg/kg NAR. The treatment lasted for 14 days. Sub-acute exposure to L-NAME and BPA induced hypertension and mediated-neuroinflammation at CA-2 and CA-4 of hippocampus cells. It was evident by increase in PDE-51 and enzymes of ATP hydrolysis (ATPase, ADPase and AMPase) with corresponding upsurge in cholinergic (AChE and BuChE), dopaminergic (MAO-A) and adenosinergic (ADA) enzymes as well as movement disorder. The hypertensive-mediated neurotoxicity was related to alteration of NO signaling and higher release of pro-inflammatory cytokines (TNF-α and IL-1ß), apoptotic proteins (P53 and caspace-9) and facilitated entry of T-lymphocytes (CD43+) into CNS through blood brain barrier potentiated by antigen presenting cells. Hence, these features of BPA-mediated neurotoxicity in L-NAME induced hypertensive rats were prohibited by co-administration of NAR through production of neuro-inflammatory mediators, stabilizing neurotransmitter enzymes, normalizing NO signaling and improving brain histology.
Assuntos
Flavanonas/farmacologia , Hipocampo/efeitos dos fármacos , Hipertensão/complicações , Mediadores da Inflamação/metabolismo , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Síndromes Neurotóxicas/prevenção & controle , Nucleotídeos/deficiência , Estresse Oxidativo/efeitos dos fármacos , Animais , Anti-Inflamatórios/farmacologia , Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Compostos Benzidrílicos , Modelos Animais de Doenças , Hipocampo/metabolismo , Hipocampo/patologia , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Leucossialina/metabolismo , Locomoção/efeitos dos fármacos , Masculino , Neurônios/imunologia , Neurônios/metabolismo , Neurônios/patologia , Síndromes Neurotóxicas/imunologia , Síndromes Neurotóxicas/metabolismo , Síndromes Neurotóxicas/patologia , Óxido Nítrico/metabolismo , Fenóis , Ratos Wistar , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Incidents that slow or stall replication fork progression, collectively known as replication stress, represent a major source of spontaneous genomic instability. Here, we determine the requirement for global protein biosynthesis on DNA replication and associated downstream signaling. We study this response side by side with dNTP deprivation; one of the most commonly used means to investigate replication arrest and replicative stress. Our in vitro interrogations reveal that inhibition of translation by cycloheximide (CHX) rapidly impairs replication fork progression without decoupling helicase and polymerase activities or inducing DNA damage. In line with this, protein deprivation stress does not activate checkpoint signaling. In contrast to the direct link between insufficient dNTP pools and genome instability, our findings suggest that replication forks remain stable during short-term protein deficiency. We find that replication forks initially endure fluctuations in protein supply in order to efficiently resume DNA synthesis upon reversal of the induced protein deprivation stress. These results reveal distinct cellular responses to replication arrest induced by deprivation of either nucleotides or proteins.
Assuntos
Replicação do DNA , Nucleotídeos/deficiência , Biossíntese de Proteínas , Linhagem Celular Tumoral , Cicloeximida/farmacologia , DNA/metabolismo , Dano ao DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/metabolismo , Histonas/metabolismo , Humanos , Nucleotídeos/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Amplification of genes involved in signal transduction and cell cycle control occurs in a significant fraction of human cancers. Loss of p53 function has been proposed to enable cells with gene amplification to arise spontaneously during growth in vitro. However, this conclusion derives from studies employing the UMP synthesis inhibitor N-phosphonacetyl-L-aspartate (PALA), which, in addition to selecting for cells containing extra copies of the CAD locus, enables p53-deficient cells to enter S phase and acquire the DNA breaks that initiate the amplification process. Thus, it has not been possible to determine if gene amplification occurs spontaneously or results from the inductive effects of the selective agent. The studies reported here assess whether p53 deficiency leads to spontaneous genetic instability by comparing cell cycle responses and amplification frequencies of the human fibrosarcoma cell line HT1080 when treated with PALA or with methotrexate, an antifolate that, under the conditions used, should not generate DNA breaks. p53-deficient HT1080 cells generated PALA-resistant variants containing amplified CAD genes at a frequency of >10(-5). By contrast, methotrexate selection did not result in resistant cells at a detectable frequency (<10(-9)). However, growth of HT1080 cells under conditions that induced DNA breakage prior to selection generated methotrexate-resistant clones containing amplified dihydrofolate reductase sequences at a high frequency. These data demonstrate that, under standard growth conditions, p53 loss is not sufficient to enable cells to produce the DNA breaks that initiate amplification. We propose that p53-deficient cells must proceed through S phase under conditions that induce DNA breakage for genetic instability to occur.
Assuntos
Dano ao DNA , Fibrossarcoma/patologia , Amplificação de Genes , Tetra-Hidrofolato Desidrogenase/genética , Proteína Supressora de Tumor p53/deficiência , Ácido Aspártico/análogos & derivados , Ácido Aspártico/farmacologia , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Resistência a Medicamentos , Humanos , Metotrexato/farmacologia , Modelos Genéticos , Nucleotídeos/deficiência , Ácido Fosfonoacéticos/análogos & derivados , Ácido Fosfonoacéticos/farmacologia , Seleção Genética , Neoplasias Cutâneas/patologia , Células Tumorais CultivadasRESUMO
We propose that the lethality of commonly used anticancer drugs, e.g., methotrexate and cis-platinum are due, at least in part, to an increase of the E2F-1-mediated apoptotic cascade. The drugs directly or indirectly decrease deoxynucleoside triphosphates. The E2F family acts to provide control of S phase by transcribing genes required for deoxynucleoside triphosphate and DNA synthesis. Thus, a mechanism for control of E2F-1 is essential, a signal safeguarding against aberrant or uncontrolled cell proliferation. We have proposed a feedback control by NTPs that down-regulates E2F-1. Here, we provide evidence in support of this hypothesis.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/metabolismo , Cisplatino/farmacologia , Neoplasias do Colo/tratamento farmacológico , Proteínas de Ligação a DNA/metabolismo , Metotrexato/farmacologia , Nucleotídeos/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Fatores de Transcrição/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Replicação do DNA/efeitos dos fármacos , DNA de Neoplasias/antagonistas & inibidores , DNA de Neoplasias/biossíntese , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Humanos , Masculino , Nucleotídeos/deficiência , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologiaRESUMO
Infestation by the biotrophic pathogen Gymnosporangium asiaticum can be devastating for plant of the family Rosaceae. However, the phytopathology of this process has not been thoroughly elucidated. Using a metabolomics approach, we discovered the intrinsic activities that induce disease symptoms after fungal invasion in terms of microbe-induced metabolic responses. Through metabolic pathway enrichment and mapping, we found that the host altered its metabolite levels, resulting in accumulation of tetrose and pentose sugar alcohols, in response to this fungus. We then used a multiple linear regression model to evaluate the effect of the interaction between this abnormal accumulation of sugar alcohol and the group variable (control/parasitism). The results revealed that this accumulation resulted in deficiency in the supply of specific sugars, which led to a lack of amino sugar and nucleotide sugar metabolism. Halting this metabolism could hamper pivotal functions in the plant host, including cell wall synthesis and lesion repair. In conclusion, our findings indicate that altered metabolic responses that occur during fungal parasitism can cause deficiency in substrates in pivotal pathways and thereby trigger pathological symptoms.
Assuntos
Amino Açúcares/deficiência , Basidiomycota/patogenicidade , Interações Hospedeiro-Patógeno , Metaboloma , Nucleotídeos/deficiência , Rosaceae/metabolismo , Basidiomycota/fisiologia , Cromatografia Líquida de Alta Pressão , Frutas/metabolismo , Frutas/microbiologia , Cromatografia Gasosa-Espectrometria de Massas , Redes e Vias Metabólicas/fisiologia , Doenças das Plantas/microbiologia , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Rosaceae/microbiologia , Álcoois Açúcares/metabolismoRESUMO
It is well known that inappropriate nutrient intake accounts for the maintenance of the immunological equilibrium, in humans and animals. Vitamins, elements, lipids, proteins and nucleic acids play an important role in the regulation of cellular and humoral immune responses since single or multiple deficits of these food components have been shown to cause immune abnormalities. For instance, in the course of protein-calorie malnutrition bacterial and/or viral infections represent the major cause of death. Ageing is characterized by a decline of many immune functions, and this process is called immunosenescence. Here, we report novel findings on the inability of superantigens to activate old CD8+, natural killer and B cells, as an expression of cell amnesia. In the elderly, this lack of activation could lead to lethal effects in the case of severe staphylococcal infections. Quite interestingly, recent findings outlined some similarities between human immune deficiency virus (HIV)-1 infection and ageing in terms of immune changes. The model of HIV-infection may be useful for the interpretation of ageing mechanisms and possible therapeutical interventions. Finally, the role of nutrition in different pathological conditions and the use of medical foods for correcting of immune deficits will be described.
Assuntos
Deficiências Nutricionais/imunologia , Imunidade , Fenômenos Fisiológicos da Nutrição , Envelhecimento/imunologia , Deficiência de Vitaminas/imunologia , Humanos , Lipídeos/deficiência , Lipídeos/imunologia , Nucleotídeos/deficiência , Nucleotídeos/imunologia , Desnutrição Proteico-Calórica/imunologia , Oligoelementos/deficiência , Oligoelementos/imunologiaRESUMO
We describe the changes of several brush-border enzymatic activities in different subpopulations of epithelial cells, separated sequentially from the villus tip-to-crypt axis of the small intestine, induced by deprivation of dietary nucleotides for different periods of time in adult rats. Deprivation of dietary nucleotides lead to a decrease in the content and specific activity of alkaline phosphatase, leucine-aminopeptidase, maltase, sucrase and lactase in the villus tip, but had little effect on the crypt zone. The effect of the nucleotide deprivation on the enzymatic activity progressively increased towards the tip of the villus. Since these enzymes are maturation markers of the intestinal cells, these results support the idea that dietary nucleotides affect the maturation status of small-intestine epithelium.
Assuntos
Intestino Delgado/fisiologia , Nucleotídeos/deficiência , Animais , Dieta , Células Epiteliais , Epitélio/enzimologia , Epitélio/fisiologia , Intestino Delgado/citologia , Intestino Delgado/enzimologia , Nucleotídeos/fisiologia , Ratos , Ratos WistarRESUMO
BACKGROUND: We evaluated the effects of nucleosides (NS) and nucleotides (NT) on the rejection of rat allogeneic small intestinal transplants. METHODS: A 2-cm segment of jejunum from fetal Fischer rats (RT-1(lvl)) was transplanted at day 19 of gestation into the abdominal wall of 7-week-old Lewis rats (RT-1(l)) by a nonvascular technique. Two weeks before transplantation, recipient rats were separated into an NS-NT-free group and an NS-NT-supplemented group. At 2 days after transplantation, histologic study of the grafts was performed with hematoxylin-eosin staining and interleukin-2 (IL-2) production estimated in recipient blood using an ELISA method. The morphologic findings were graded in a blind fashion on a scale of 0 to 4, with 0 indicating an intact intestinal structure. RESULTS: Mean plasma IL-2 levels of the NS-NT-free group were significantly lower than those of the NS-NT-supplemented group. The mean rejection score of the NS-NT-free group was also significantly lower than that of the NS-NT-supplemented group. CONCLUSIONS: Administration of an NS-NT-free diet reduces acute rejection in rat small intestinal transplantations.
Assuntos
Dieta , Terapia de Imunossupressão/métodos , Intestino Delgado/transplante , Nucleosídeos/deficiência , Nucleotídeos/deficiência , Transplante Isogênico/imunologia , Animais , Intestino Delgado/patologia , Modelos Animais , Ratos , Ratos Endogâmicos Lew , Transplante Isogênico/patologiaRESUMO
Nucleotide deficiency causes replication stress (RS) and DNA damage in dividing cells. How nucleotide metabolism is regulated in vivo to prevent these deleterious effects remains unknown. In this study, we investigate a functional link between nucleotide deficiency, RS, and the nucleoside salvage pathway (NSP) enzymes deoxycytidine kinase (dCK) and thymidine kinase (TK1). We show that inactivation of dCK in mice depletes deoxycytidine triphosphate (dCTP) pools and induces RS, early S-phase arrest, and DNA damage in erythroid, B lymphoid, and T lymphoid lineages. TK1(-/-) erythroid and B lymphoid lineages also experience nucleotide deficiency but, unlike their dCK(-/-) counterparts, they still sustain DNA replication. Intriguingly, dCTP pool depletion, RS, and hematopoietic defects induced by dCK inactivation are almost completely reversed in a newly generated dCK/TK1 double-knockout (DKO) mouse model. Using NSP-deficient DKO hematopoietic cells, we identify a previously unrecognized biological activity of endogenous thymidine as a strong inducer of RS in vivo through TK1-mediated dCTP pool depletion. We propose a model that explains how TK1 and dCK "tune" dCTP pools to both trigger and resolve RS in vivo. This new model may be exploited therapeutically to induce synthetic sickness/lethality in hematological malignancies, and possibly in other cancers.
Assuntos
Replicação do DNA/fisiologia , Hematopoese/fisiologia , Redes e Vias Metabólicas/fisiologia , Modelos Biológicos , Nucleosídeos/metabolismo , Nucleotídeos/deficiência , Estresse Fisiológico/fisiologia , Animais , Western Blotting , Bromodesoxiuridina , Desoxicitidina Quinase/genética , Desoxicitidina Quinase/metabolismo , Nucleotídeos de Desoxicitosina/metabolismo , Citometria de Fluxo , Imunofenotipagem , Camundongos , Camundongos Knockout , Nucleotídeos/metabolismo , Timidina Quinase/genética , Timidina Quinase/metabolismoRESUMO
In the intrinsic death pathway, cytochrome C (CC) released from mitochondria to the cytosol triggers Apaf-1 apoptosome formation and subsequent caspase activation. This process can be recapitulated using recombinant Apaf-1 and CC in the presence of nucleotides ATP or dATP [(d)ATP] or using fresh cytosol and CC without the need of exogenous nucleotides. Surprisingly, we found that stored cytosols failed to support CC-initiated caspase activation. Storage of cytosols at different temperatures led to the loss of all (deoxy)nucleotides including (d)ATP. Addition of (d)ATP to such stored cytosols partially restored CC-initiated caspase activation. Nevertheless, CC could not induce complete caspase-9/3 activation in stored cytosols, even with the addition of (d)ATP, despite robust Apaf-1 oligomerization. The Apaf-1 apoptosome, which functions as a proteolytic-based molecular timer appeared to be defective as auto-processing of recruited procaspase-9 was inhibited. Far Western analysis revealed that procaspase-9 directly interacted with Apaf-1 and this interaction was reduced in the presence of physiological levels of ATP. Co-incubation of recombinant Apaf-1 and procaspase-9 prior to CC and ATP addition inhibited CC-induced caspase activity. These findings suggest that in the absence of nucleotide such as ATP, direct association of procaspase-9 with Apaf-1 leads to defective molecular timer, and thus, inhibits apoptosome-mediated caspase activation. Altogether, our results provide novel insight on nucleotide regulation of apoptosome.
Assuntos
Apoptossomas/metabolismo , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Caspase 9/metabolismo , Nucleotídeos/deficiência , Trifosfato de Adenosina/deficiência , Caspases/metabolismo , Citocromos c/metabolismo , Citosol , Fibroblastos , Humanos , Ligação ProteicaAssuntos
Antígenos de Superfície/análise , Biomarcadores/análise , Proteínas Alimentares/farmacologia , Linfócitos/imunologia , Nucleotídeos/farmacologia , Deficiência de Proteína/imunologia , Animais , Anticorpos Monoclonais , Feminino , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Linfonodos/transplante , Transfusão de Linfócitos , Linfócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Nucleotídeos/deficiência , Valores de Referência , Transplante Homólogo , Transplante IsogênicoRESUMO
The aim of the present study was to investigate the influence of dietary nucleotides on liver morphology. Adult rats were fed for 21 d on a nucleotide-containing diet or the same diet free of nucleotides. Liver sections were examined by light and transmission electron microscopy, as well as for nucleic acid and protein contents. Morphometric analysis was performed for different variables. Deprivation of dietary nucleotides resulted in a reduction in hepatocyte nuclear and nucleolar areas as well as in nuclear chromatin condensation. In addition, the rough endoplasmic reticulum was reduced, as were ribosome association and abundance, whereas fat accumulated. These findings portray dietary nucleotides as required nutrients for the liver under normal physiological conditions and suggest that an inadequate supply of nucleotides for a certain period of time has transient negative effects on liver ultrastructure and function.
Assuntos
Fígado/citologia , Nucleotídeos/deficiência , Animais , Nucléolo Celular/ultraestrutura , Núcleo Celular/ultraestrutura , Cromatina/genética , DNA/análise , Retículo Endoplasmático/ultraestrutura , Fígado/química , Fígado/ultraestrutura , Masculino , Microscopia Eletrônica , Conformação de Ácido Nucleico , Nucleotídeos/administração & dosagem , Proteínas/análise , RNA/análise , Ratos , Ratos Wistar , Ribossomos/ultraestruturaRESUMO
BACKGROUND & AIMS: Dietary nucleotides are reported to influence the growth and functioning of the liver and small intestine. The aim of this study was to examine the mechanism by which nucleotides exert their effects in these tissues by assessing protein synthesis activity and related parameters in the presence or absence of dietary nucleotides. METHODS: Rats were fed a purified diet with or without nucleotides for 10 days. Fractional protein synthesis rate, RNA and DNA concentrations, polysome size distribution, and number of ribosomes were assessed. RESULTS: Fractional protein synthesis rates of the liver and small intestine were lower in the nucleotide-deprived group than in the control group. In the liver, RNA concentration was also lower in the nucleotide-deprived group, but values in the small intestine were similar in the two groups. In the liver, deprivation of nucleotides resulted in a reduction in the number of ribosomes and in polysome breakdown. Protein and DNA concentrations did not vary in the liver; however, the concentration of DNA was lower in the small intestine of the nucleotide-deprived group than in the control group. CONCLUSIONS: Dietary nucleotides can modulate protein synthesis in the liver and small intestine as a result of tissue-specific nucleic acid changes.
Assuntos
Intestino Delgado/metabolismo , Fígado/metabolismo , Nucleotídeos/administração & dosagem , Nucleotídeos/deficiência , Biossíntese de Proteínas , Animais , DNA/metabolismo , Dieta , Fígado/ultraestrutura , Masculino , Polirribossomos/ultraestrutura , RNA/metabolismo , Ratos , Ratos Wistar , Valores de Referência , Ribossomos/ultraestruturaRESUMO
Having shown rapid trafficking of aminoglycosides to the Golgi complex in cell culture, we focused on the injurious interaction that occurs when gentamicin administration is preceded by renal ischemia. Using Texas red-labeled gentamicin as a tracer, we determined that 15 min of cellular nucleotide depletion did not significantly increase subsequent uptake. However, cells previously depleted of nucleotides accumulated significantly more Texas red-labeled gentamicin within a dispersed Golgi complex. Using Ricinus communis and Lens culinaris lectins, which label specific compartments of the Golgi complex (trans-Golgi network/trans and medial/cis compartments, respectively), we determined that the medial/cis compartment dispersed after 15 min of nucleotide depletion but the trans-Golgi network/trans compartment remained unaffected. An increase in the number of cells exhibiting disrupted medial/cis-Golgi morphology after repletion in physiological media containing gentamicin was also seen. In summary, the increase in nephrotoxicity seen when ischemia precedes aminoglycoside uptake may be part of a complex mechanism initially involving increased Golgi accumulation and prolonged Golgi dispersion. The Golgi complex must then endure the effects of gentamicin accumulated in larger quantities in an aberrant physiological state.