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1.
Genes Dev ; 33(17-18): 1191-1207, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31371435

RESUMO

The vast majority of eukaryotes possess two DNA recombinases: Rad51, which is ubiquitously expressed, and Dmc1, which is meiosis-specific. The evolutionary origins of this two-recombinase system remain poorly understood. Interestingly, Dmc1 can stabilize mismatch-containing base triplets, whereas Rad51 cannot. Here, we demonstrate that this difference can be attributed to three amino acids conserved only within the Dmc1 lineage of the Rad51/RecA family. Chimeric Rad51 mutants harboring Dmc1-specific amino acids gain the ability to stabilize heteroduplex DNA joints with mismatch-containing base triplets, whereas Dmc1 mutants with Rad51-specific amino acids lose this ability. Remarkably, RAD-51 from Caenorhabditis elegans, an organism without Dmc1, has acquired "Dmc1-like" amino acids. Chimeric C. elegans RAD-51 harboring "canonical" Rad51 amino acids gives rise to toxic recombination intermediates, which must be actively dismantled to permit normal meiotic progression. We propose that Dmc1 lineage-specific amino acids involved in the stabilization of heteroduplex DNA joints with mismatch-containing base triplets may contribute to normal meiotic recombination.


Assuntos
Aminoácidos/metabolismo , Rad51 Recombinase/química , Rad51 Recombinase/metabolismo , Recombinases/química , Recombinases/metabolismo , Recombinação Genética/genética , Aminoácidos/genética , Animais , Pareamento Incorreto de Bases , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Sequência Conservada , Mutação , Rad51 Recombinase/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Recombinases/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
2.
J Agric Food Chem ; 67(33): 9277-9285, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31353906

RESUMO

3,3'-Diindolylmethane (DIM), a digestive metabolite originating from cruciferous vegetables, has dietary potential for the treatment of various human intestinal diseases. Although intestinal permeability dysfunction is closely related to the initiation and progression of human intestinal inflammatory diseases (IBDs), the effect of DIM on intestinal permeability is unclear. We evaluated the effect of DIM on the intestinal permeability of human intestinal cell monolayers and the animal model Caenorhabditis elegans, which were treated with IL-1ß and Pseudomonas aeruginosa, respectively, to mimic IBD conditions. DIM substantially restored the intestinal permeability of differentiated Caco-2 cells by enhancing the expression of tight junction proteins (including occludin and ZO-1). Compared to the IL-1ß single treatment (551.0 ± 49.0 Ω·cm2), DIM (10 µM) significantly increased the transepithelial electrical resistance (TEER) of Caco-2 cell monolayers (919.0 ± 66.4 Ω·cm2, p < 0.001). DIM also ameliorated the impaired intestinal permeability and extended the lifespan of C. elegans fed P. aeruginosa. The mean lifespan of DIM-treated worms (10.8 ± 1.3 days) was higher than that of control-treated worms (9.7 ± 1.1 days, p < 0.01). Thus, DIM is a potential nutraceutical candidate for the treatment of leaky gut syndrome by improving intestinal permeability.


Assuntos
Indóis/administração & dosagem , Doenças Inflamatórias Intestinais/tratamento farmacológico , Mucosa Intestinal/efeitos dos fármacos , Animais , Células CACO-2 , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Mucosa Intestinal/metabolismo , Intestinos , Masculino , Ocludina/genética , Ocludina/metabolismo , Permeabilidade/efeitos dos fármacos , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismo
3.
Nat Commun ; 10(1): 3042, 2019 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-31316054

RESUMO

Stress resistance and longevity are positively correlated but emerging evidence indicates that they are physiologically distinct. Identifying factors with distinctive roles in these processes is challenging because pro-longevity genes often enhance stress resistance. We demonstrate that TCER-1, the Caenorhabditis elegans homolog of human transcription elongation and splicing factor, TCERG1, has opposite effects on lifespan and stress resistance. We previously showed that tcer-1 promotes longevity in germline-less C. elegans and reproductive fitness in wild-type animals. Surprisingly, tcer-1 mutants exhibit exceptional resistance against multiple stressors, including infection by human opportunistic pathogens, whereas, TCER-1 overexpression confers immuno-susceptibility. TCER-1 inhibits immunity only during fertile stages of life. Elevating its levels ameliorates the fertility loss caused by infection, suggesting that TCER-1 represses immunity to augment fecundity. TCER-1 acts through repression of PMK-1 as well as PMK-1-independent factors critical for innate immunity. Our data establish key roles for TCER-1 in coordinating immunity, longevity and fertility, and reveal mechanisms that distinguish length of life from functional aspects of aging.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Regulação da Expressão Gênica/fisiologia , Imunidade Inata/genética , Longevidade/genética , Fatores de Alongamento de Peptídeos/metabolismo , Estresse Fisiológico/imunologia , Envelhecimento/genética , Envelhecimento/imunologia , Animais , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/imunologia , Suscetibilidade a Doenças/imunologia , Fertilidade/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Modelos Animais , Mutação , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/imunologia , Estresse Fisiológico/genética
4.
Nat Commun ; 10(1): 2902, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31263173

RESUMO

Exogenous metabolites from microbial and dietary origins have profound effects on host metabolism. Here, we report that a sub-population of lipid droplets (LDs), which are conserved organelles for fat storage, is defined by metabolite-modulated targeting of the C. elegans seipin ortholog, SEIP-1. Loss of SEIP-1 function reduces the size of a subset of LDs while over-expression of SEIP-1 has the opposite effect. Ultrastructural analysis reveals SEIP-1 enrichment in an endoplasmic reticulum (ER) subdomain, which co-purifies with LDs. Analyses of C. elegans and bacterial genetic mutants indicate a requirement of polyunsaturated fatty acids (PUFAs) and microbial cyclopropane fatty acids (CFAs) for SEIP-1 enrichment, as confirmed by dietary supplementation experiments. In mammalian cells, heterologously expressed SEIP-1 engages nascent lipid droplets and promotes their subsequent expansion in a conserved manner. Our results suggest that microbial and polyunsaturated fatty acids serve unexpected roles in regulating cellular fat storage by promoting LD diversity.


Assuntos
Caenorhabditis elegans/metabolismo , Retículo Endoplasmático/metabolismo , Ácidos Graxos/metabolismo , Gotículas Lipídicas/metabolismo , Animais , Caenorhabditis elegans/química , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Linhagem Celular , Retículo Endoplasmático/química , Retículo Endoplasmático/genética , Subunidades gama da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Humanos , Transporte Proteico
5.
Anticancer Res ; 39(7): 3651-3660, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31262891

RESUMO

BACKGROUND/AIM: Cytochrome P450 epoxygenase is a major enzyme involved in the metabolism of ω-3 polyunsaturated fatty acids (PUFAs) to produce biologically active ω-3 epoxy fatty acids (ω-3 epoxides). In general, all epoxy PUFAs including ω-3 epoxides are quickly metabolized/inactivated by soluble epoxide hydrolase (sEH) to form diol products. The aims of this study were to determine the effect and mechanism of fat-1 transgene, and ω-3 PUFA combined with sEH gene knockout or inhibitor on inhibiting pancreatic cancer and the related mechanisms involved. MATERIALS AND METHODS: PK03-mutant KrasG12D murine pancreatic carcinoma cells were inoculated into mouse models including fat-1, sEH-/- and C57BL/6J mice. The mice were fed with AIN-76A diet with or without ω-3 PUFA supplementation or treated with sEH inhibitor. In addition to tumor growth (tumor size and weight), cell proliferation, mutant Kras-mediated signaling, inflammatory reaction and angiogenesis were analyzed immunohisto-chemically and by western blot assay. ω-3 PUFA metabolism, particularly focusing on ω-3 epoxy fatty acids (ω-3 epoxides), was measured using a liquid chromatography with tandem mass spectrometry (LC-MS/MS) approach. RESULTS: Significant decreases of weight and size of the PK03 pancreatic carcinoma were observed in the fat-1 transgenic mice treated with sEH inhibitor compared to those of C57BL/6J control mice fed with AIN-76A diet (weight: 0.28±0.04 g vs. 0.58±0.06 g; size: 187.0±17.5 mm3 vs. 519.3±60.6 mm3). In a separate experiment, sEH-/- mice fed ω-3 PUFA supplement and C57BL/6J mice treated with sEH inhibitor and fed ω-3 PUFA supplement exhibited a significant reduction in the weight and size of the pancreatic carcinoma compared to C57BL/6J control mice (weight: 0.26±.26 g and 0.39±.39 g vs. 0.69±0.11 g, respectively; size: 274.2±36.2 mm3 and 296.4±99.8 mm3 vs. 612.6±117.8 mm3, respectively). Moreover, compared to the pancreatic tumors in C57BL/6J control mice, the tumors in fat-1 transgenic mice treated with sEH inhibitor showed a significant less inflammatory cell infiltrate (62.6±9.2/HPF (high power field) vs. 8.0±1.2/HPF), tumor cell proliferation (48.5±1.7% vs. 16.5±1.6%), and angiogenesis (micro-vessel density (MVD): 35.0±1.0 vs. 11.1±0.5) immunohistochemically, as well as significantly increased caspase-3 labeled apoptosis (0.44±0.06% vs. 0.69±0.06%, respectively). Using western blot approach, significant inhibition of mutant Kras-activated signals including phosphorylated Serine/threonine kinases (cRAF), Mitogen-activated protein kinase kinase (MEK), and extracellular signal-regulated kinase (ERK) were identified in pancreatic carcinoma of fat-1 transgenic mice treated with sEH inhibitor. Eicosanoic acid metabolic profiling of the serum specimens detected a significant increase of the ratios of epoxides to dihydroxy fatty acid (DiHDPE) for docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), and epoxides/dihydroxy octadecenoic acid (DiHOME) for arachidonic acid (ARA) and linoleic acid (LA), as well as a significant increase of epoxy metabolites of DHA, EPA, ARA and LA in fat-1 transgenic mice treated with a sEH inhibitor. CONCLUSION: ω-3 epoxy products from ω-3 PUFA metabolism play a crucial role in inhibiting pancreatic cancer growth, and use of ω-3 PUFAs combined with sEH inhibition is a strategy with high potential for pancreatic cancer treatment and prevention.


Assuntos
Adenocarcinoma/terapia , Proteínas de Caenorhabditis elegans/genética , Suplementos Nutricionais , Compostos de Epóxi/farmacologia , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Ômega-3/farmacologia , Neoplasias Pancreáticas/terapia , Adenocarcinoma/patologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias Pancreáticas/patologia
6.
Genes Dev ; 33(13-14): 857-870, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31147388

RESUMO

Piwi proteins are important for germ cell development in most animals. These proteins are guided to specific targets by small guide RNAs, referred to as piRNAs or 21U RNAs in Caenorhabditis elegans In this organism, even though genetic screens have uncovered 21U RNA biogenesis factors, little is known about how these factors interact or what they do. Based on the previously identified 21U biogenesis factor PID-1 (piRNA-induced silencing-defective 1), we here define a novel protein complex, PETISCO (PID-3, ERH-2, TOFU-6, and IFE-3 small RNA complex), that is required for 21U RNA biogenesis. PETISCO contains both potential 5' cap and 5' phosphate RNA-binding domains and interacts with capped 21U precursor RNA. We resolved the architecture of PETISCO and revealed a second function for PETISCO in embryonic development. This essential function of PETISCO is mediated not by PID-1 but by the novel protein TOST-1 (twenty-one U pathway antagonist). In contrast, TOST-1 is not essential for 21U RNA biogenesis. Both PID-1 and TOST-1 interact directly with ERH-2 using a conserved sequence motif. Finally, our data suggest a role for TOST-1:PETISCO in SL1 homeostasis in the early embryo. Our work describes a key complex for 21U RNA processing in C. elegans and strengthens the view that 21U RNA biogenesis is built on an snRNA-related pathway.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Embrião não Mamífero/fisiologia , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , RNA Nucleolar Pequeno/biossíntese , Animais , RNA Nuclear Pequeno/metabolismo
7.
Environ Pollut ; 251: 871-878, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31234252

RESUMO

Di(2-ethylhexyl)phthalate (DEHP) is an ubiquitous and emerging contaminant that is widely present in food, agricultural crop, and the environment, posing a potential risk to human health. This study utilized the nematode Caenorhabditis elegans to decipher the toxic effects of early life exposure to DEHP on aging and its underlying mechanisms. The results showed that exposure to DEHP at 0.1 and 1.5 mg/L inhibited locomotive behaviors. In addition, DEHP exposure significantly shortened the mean lifespan of the worms and further adversely affected pharyngeal pumping rate and defecation cycle in aged worms. Moreover, DEHP exposure also further enhanced accumulation of age-related biomarkers including lipofuscin, lipid peroxidation, and intracellular reactive oxygen species in aged worms. In addition, exposure to DEHP significantly suppressed gene expression of hsp-16.1, hsp-16.49, and hsp-70 in aged worms. Further evidences showed that mutation of genes involved in insulin/IGF-1-like signaling (IIS) pathway (daf-2, age-1, pdk-1, akt-1, akt-2, and daf-16) restored lipid peroxidation accumulation upon DEHP exposure in aged worms, whereas skn-1 mutation resulted in enhanced lipid peroxidation accumulation. Therefore, IIS and SKN-1 may serve as an important molecular basis for DEHP-induced age-related declines in C. elegans. Since IIS and SKN-1 are highly conserved among species, the age-related declines caused by DEHP exposure may not be exclusive in C. elegans, leading to adverse human health consequences due to widespread and persistent DEHP contamination in the environment.


Assuntos
Envelhecimento/efeitos dos fármacos , Caenorhabditis elegans/efeitos dos fármacos , Dietilexilftalato/toxicidade , Poluentes Ambientais/toxicidade , Fator de Crescimento Insulin-Like I/metabolismo , Longevidade/efeitos dos fármacos , Plastificantes/toxicidade , Animais , Biomarcadores/metabolismo , Proteínas de Caenorhabditis elegans/biossíntese , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Choque Térmico/biossíntese , Insulina/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Lipofuscina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/genética
8.
Cell Mol Life Sci ; 76(20): 4131-4144, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31053883

RESUMO

ABCB6 belongs to the family of ATP-binding cassette (ABC) transporters, which transport various molecules across extra- and intra-cellular membranes, bearing significant impact on human disease and pharmacology. Although mutations in the ABCB6 gene have been linked to a variety of pathophysiological conditions ranging from transfusion incompatibility to pigmentation defects, its precise cellular localization and function is not understood. In particular, the intracellular localization of ABCB6 has been a matter of debate, with conflicting reports suggesting mitochondrial or endolysosomal expression. ABCB6 shows significant sequence identity to HMT-1 (heavy metal tolerance factor 1) proteins, whose evolutionarily conserved role is to confer tolerance to heavy metals through the intracellular sequestration of metal complexes. Here, we show that the cadmium-sensitive phenotype of Schizosaccharomyces pombe and Caenorhabditis elegans strains defective for HMT-1 is rescued by the human ABCB6 protein. Overexpression of ABCB6 conferred tolerance to cadmium and As(III) (As2O3), but not to As(V) (Na2HAsO4), Sb(V), Hg(II), or Zn(II). Inactivating mutations of ABCB6 abolished vacuolar sequestration of cadmium, effectively suppressing the cadmium tolerance phenotype. Modulation of ABCB6 expression levels in human glioblastoma cells resulted in a concomitant change in cadmium sensitivity. Our findings reveal ABCB6 as a functional homologue of the HMT-1 proteins, linking endolysosomal ABCB6 to the highly conserved mechanism of intracellular cadmium detoxification.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Cádmio/toxicidade , Proteínas de Caenorhabditis elegans/genética , Inativação Metabólica/genética , Poluentes Químicos da Água/toxicidade , Transportadores de Cassetes de Ligação de ATP/deficiência , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Antimônio/toxicidade , Arseniatos/toxicidade , Trióxido de Arsênio/toxicidade , Cádmio/metabolismo , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Linhagem Celular Tumoral , Sequência Conservada , Expressão Gênica , Teste de Complementação Genética , Células HeLa , Humanos , Mercúrio/toxicidade , Mutação , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo , Poluentes Químicos da Água/metabolismo , Zinco/toxicidade
9.
Parasit Vectors ; 12(1): 187, 2019 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-31036054

RESUMO

BACKGROUND: Signalling pathways have been extensively investigated in the free-living nematode Caenorhabditis elegans, but very little is known about these pathways in parasitic nematodes. Here, we constructed a model for the dauer-associated signalling pathways in an economically highly significant parasitic worm, Haemonchus contortus. METHODS: Guided by data and information available for C. elegans, we used extensive genomic and transcriptomic datasets to infer gene homologues in the dauer-associated pathways, explore developmental transcriptomic, proteomic and phosphoproteomic profiles in H. contortus and study selected molecular structures. RESULTS: The canonical cyclic guanosine monophosphate (cGMP), transforming growth factor-ß (TGF-ß), insulin-like growth factor 1 (IGF-1) and steroid hormone signalling pathways of H. contortus were inferred to represent a total of 61 gene homologues. Compared with C. elegans, H. contortus has a reduced set of genes encoding insulin-like peptides, implying evolutionary and biological divergences between the parasitic and free-living nematodes. Similar transcription profiles were found for all gene homologues between the infective stage of H. contortus and dauer stage of C. elegans. High transcriptional levels for genes encoding G protein-coupled receptors (GPCRs), TGF-ß, insulin-like ligands (e.g. ins-1, ins-17 and ins-18) and transcriptional factors (e.g. daf-16) in the infective L3 stage of H. contortus were suggestive of critical functional roles in this stage. Conspicuous protein expression patterns and extensive phosphorylation of some components of these pathways suggested marked post-translational modifications also in the L3 stage. The high structural similarity in the DAF-12 ligand binding domain among nematodes indicated functional conservation in steroid (i.e. dafachronic acid) signalling linked to worm development. CONCLUSIONS: Taken together, this pathway model provides a basis to explore hypotheses regarding biological processes and regulatory mechanisms (via particular microRNAs, phosphorylation events and/or lipids) associated with the development of H. contortus and related nematodes as well as parasite-host cross talk, which could aid the discovery of new therapeutic targets.


Assuntos
Haemonchus/genética , Haemonchus/metabolismo , Transdução de Sinais , Animais , Proteínas de Caenorhabditis elegans/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Genômica , Redes e Vias Metabólicas , Modelos Moleculares , Proteômica , Fatores de Transcrição/genética , Transcriptoma
10.
PLoS Genet ; 15(4): e1008122, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31034475

RESUMO

Early exposure to some mild stresses can slow down the aging process and extend lifespan, raising the question of how early life stress might impact the somatic health of aged animals. Here, we reveal that early life heat experience triggers the establishment of epigenetic memory in soma, which promotes long-lasting stress responses and longevity in C. elegans. Unlike lethal heat shock, mild heat activates a unique transcriptional program mimicking pathogen defense responses, characterized by the enhanced expression of innate immune and detoxification genes. Surprisingly, the expression of defense response genes persists long after heat exposure, conferring enhanced stress resistance even in aged animals. Further studies identify the histone acetyltransferase CBP-1 and the chromatin remodeling SWI/SNF complex as epigenetic modulators of the long-lasting defense responses. Histone acetylation is elevated by heat stress and maintained into agedness thereafter. Accordingly, histone acetylation levels were increased on the promoters of defense genes. Moreover, disruption of epigenetic memory abrogates the longevity response to early hormetic heat stress, indicating that long-lasting defense responses are crucial for the survival of aged animals. Together, our findings provide mechanistic insights into how temperature stress experienced in early life provides animals with lifetime health benefits.


Assuntos
Resposta ao Choque Térmico , Histonas/metabolismo , Longevidade , Acetilação , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Epigênese Genética , Temperatura Alta , Imunidade Inata , Desentoxicação Metabólica Fase I , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Regiões Promotoras Genéticas
12.
J Agric Food Chem ; 67(17): 4774-4781, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30963762

RESUMO

Targeted analysis of Coffea arabica and Coffea canephora green coffees (total sample size n = 57) confirmed 2- O-ß-d-glucopyranosyl-carboxyatractyligenin (6) as the quantitatively dominating carboxyatractyligenin derivative. Its abundance in Arabicas (2425 ± 549 nmol/g, n = 48) exceeded that in Robustas (34 ± 12 nmol/g, n = 9) roughly by a factor of 70. Coffee processing involving heat (e.g., steam treatment and decaffeination) reduced concentrations of 6 and increased those of the decarboxylated derivative. The bioavailability of compound 6 in Caenorhabditis elegans was demonstrated by ultraperformance liquid chromatography-tandem mass spectrometry analysis of extracts prepared from nematode cultures incubated in a liquid medium containing 6. A toxicity assay performed to assess the impact of 6 in vivo showed a 20-fold higher median lethal dose (LD50 = 11.7 ± 1.2 mM) concentration compared to that of the known phytotoxic adenine-nucleotide transporters inhibitor carboxyatractyloside (2, LD50 = 0.61 ± 0.05 mM), whereas 1 mM 6 and 0.1 mM 2 were sufficient to decrease the survival of wild type C. elegans, already 10-20-fold lower doses reduced reproduction. Because the insulin/insulin-like growth factors signaling cascade (IIS) is a key regulator of life span and stress resistance, the impact of compound 6 on the survival of long-living daf-2 C. elegans was tested. As the susceptibility of these nematodes to 6 was as high as that in wild type, an impact on central metabolic processes independent of IIS was suggested. Analysis of the in vivo adenosine triphosphate (ATP) content of adult C. elegans revealed no changes after 1 and 24 h, but a 50% reduction after treatment with 1 mM 6 during the entire postembryonic development. These data speak for a developmental-stage-dependent modulation of the ATP pool by 6.


Assuntos
Atractilosídeo/análogos & derivados , Caenorhabditis elegans/efeitos dos fármacos , Coffea/química , Preparações de Plantas/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Atractilosídeo/farmacocinética , Atractilosídeo/farmacologia , Disponibilidade Biológica , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Coffea/toxicidade , Café/química , Feminino , Insulina/genética , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Dose Letal Mediana , Masculino
13.
Gene ; 706: 19-31, 2019 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-31028869

RESUMO

The fem-1 gene in Caenorhabditis elegans is involved in sex differentiation; it is specifically required for all aspects of male development. In this study, the full-length cDNA of the fem-1 (Pvfem-1) gene was isolated from the Pacific whiteleg shrimp Penaeus vannamei. The Pvfem-1 transcript is 3778 nt long and encodes a putative protein (PvFEM-1) of 638 amino acids that presented eight ankyrin repeats. The translated protein showed a significant (P < 0.05) structural similitude by superposition with C. elegans FEM-1 protein. Pvfem-1 expression was evaluated by qPCR and in situ hybridization (ISH) during embryogenesis, larval development, and gonads of both genders in subadult and adult life stages. Pvfem-1 was found expressed in brain, intestine, hepatopancreas, and in the gonads of both genders in subadults and adults when quantified by RT-qPCR. A significant finding was the discovery of a natural antisense transcript (NAT) of Pvfem-1 by ISH. It was present in the oocyte nucleus of subadult female shrimp gonads but was not seen within oocytes from adult females, although it was detected in follicular cells, suggesting a possible post-transcriptional regulation of Pvfem-1 in female gonad. Conversely, in males, no NAT was observed, and Pvfem-1 was found expressed in spermatogonia of both, subadult and adult shrimps indicating a function in male sexual differentiation and gametes generation. This study represents the first step for future functional analysis that is expected to contribute to clarifying the role of Pvfem-1 in sex differentiation and determination.


Assuntos
Elementos Antissenso (Genética)/fisiologia , Penaeidae/genética , Processos de Determinação Sexual/genética , Sequência de Aminoácidos , Animais , Elementos Antissenso (Genética)/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , DNA Complementar/metabolismo , Feminino , Regulação da Expressão Gênica/genética , Gônadas/metabolismo , Hibridização In Situ , Masculino , Ovário/metabolismo , Filogenia , RNA Mensageiro/genética , Alinhamento de Sequência , Análise de Sequência de DNA/métodos , Fatores Sexuais
14.
Genes Cells ; 24(5): 377-389, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30929290

RESUMO

In Caenorhabditis elegans, germline cells remain transcriptionally silenced during embryogenesis. The transcriptional silencing is achieved by two different mechanisms: One is the inhibition of RNA polymerase II in P2-P4 cells at the establishment stage, and another is chromatin-based silencing in two primordial germ cells (PGCs) at the maintenance stage; however, the molecular mechanism underlying chromatin-based silencing is less understood. We investigated the role of the chromodomain protein MRG-1, which is an essential maternal factor for germline development, in transcriptional silencing in PGCs. PGCs lacking maternal MRG-1 showed increased levels of two histone modifications (H3K4me2 and H4K16ac), which are epigenetic markers for active transcription, and precocious activation of germline promoters. Loss of MES-4, a H3K36 methyltransferase, also caused similar derepression of the germline genes in PGCs, suggesting that both MRG-1 and MES-4 function in chromatin-based silencing in PGCs. In addition, the mrg-1 null mutant showed abnormal chromosome structures and a decrease in homologous recombinase RAD-51 foci in PGCs, but the mes-4 null mutant did not show such phenotypes. Taken together, we propose that MRG-1 has two distinct functions: chromatin-based transcriptional silencing and preserving genomic integrity at the maintenance stage of PGCs.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Cromatina/genética , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Células Germinativas/metabolismo , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Cromatina/metabolismo , Instabilidade Genômica , Células Germinativas/citologia , Código das Histonas , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo
15.
Nat Methods ; 16(5): 437-445, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30988468

RESUMO

Ribonucleotidyl transferases (rNTases) add untemplated ribonucleotides to diverse RNAs. We have developed TRAID-seq, a screening strategy in Saccharomyces cerevisiae to identify sequences added to a reporter RNA at single-nucleotide resolution by overexpressed candidate enzymes from different organisms. The rNTase activities of 22 previously unexplored enzymes were determined. In addition to poly(A)- and poly(U)-adding enzymes, we identified a cytidine-adding enzyme that is likely to be part of a two-enzyme system that adds CCA to tRNAs in a eukaryote; a nucleotidyl transferase that adds nucleotides to RNA without apparent nucleotide preference; and a poly(UG) polymerase, Caenorhabditis elegans MUT-2, that adds alternating uridine and guanosine nucleotides to form poly(UG) tails. MUT-2 is known to be required for certain forms of RNA silencing, and mutants of the enzyme that result in defective silencing did not add poly(UG) tails in our assay. We propose that MUT-2 poly(UG) polymerase activity is required to promote genome integrity and RNA silencing.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Nucleotidiltransferases/genética , Interferência de RNA , RNA Nucleotidiltransferases/genética , Saccharomyces cerevisiae/genética , Animais , Caenorhabditis elegans/enzimologia , Mutação , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genética
16.
Nat Commun ; 10(1): 1791, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30996251

RESUMO

Apoptotic death of cells damaged by genotoxic stress requires regulatory input from surrounding tissues. The C. elegans scaffold protein KRI-1, ortholog of mammalian KRIT1/CCM1, permits DNA damage-induced apoptosis of cells in the germline by an unknown cell non-autonomous mechanism. We reveal that KRI-1 exists in a complex with CCM-2 in the intestine to negatively regulate the ERK-5/MAPK pathway. This allows the KLF-3 transcription factor to facilitate expression of the SLC39 zinc transporter gene zipt-2.3, which functions to sequester zinc in the intestine. Ablation of KRI-1 results in reduced zinc sequestration in the intestine, inhibition of IR-induced MPK-1/ERK1 activation, and apoptosis in the germline. Zinc localization is also perturbed in the vasculature of krit1-/- zebrafish, and SLC39 zinc transporters are mis-expressed in Cerebral Cavernous Malformations (CCM) patient tissues. This study provides new insights into the regulation of apoptosis by cross-tissue communication, and suggests a link between zinc localization and CCM disease.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Zinco/metabolismo , Animais , Animais Geneticamente Modificados , Apoptose/efeitos da radiação , Proteínas Reguladoras de Apoptose/genética , Encéfalo/patologia , Encéfalo/cirurgia , Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/efeitos da radiação , Proteínas de Caenorhabditis elegans/genética , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Hemangioma Cavernoso do Sistema Nervoso Central/cirurgia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteína KRIT1/genética , Proteína KRIT1/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Mutagênese , Mutação , Fosforilação/fisiologia , Alinhamento de Sequência , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
17.
Cell Physiol Biochem ; 52(5): 970-983, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30977983

RESUMO

BACKGROUND/AIMS: Regulation of mRNA translation is central to protein homeostasis and is optimized for speed and accuracy. Spontaneous recoding events occur virtually at any codon but at very low frequency and are commonly assumed to increase as the cell ages. METHODS: Here, we leveraged the polyglutamine(polyQ)-frameshifting model of huntingtin exon 1 with CAG repeat length in the pathological range (Htt51Q), which undergoes enhanced non-programmed translational -1 frameshifting. RESULTS: In body muscle cells of Caenorhabditis elegans, -1 frameshifting occured at the onset of expression of the zero-frame product, correlated with mRNA level of the non-frameshifted expression and formed aggregates correlated with reduced motility in C. elegans. Spontaneous frameshifting was modulated by IFG-1, the homologue of the nutrient-responsive eukaryotic initiation factor 4G (eIF4G), under normal growth conditions and NSUN-5, a conserved ribosomal RNA methyltransferase, under osmotic stress. CONCLUSION: Our results suggest that frameshifting and aggregation occur at even early stages of development and, because of their intrinsic stability, may persist and accelerate the onset of age-related proteinopathies.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Mutação da Fase de Leitura , Proteína Huntingtina , Doença de Huntington , Expansão das Repetições de Trinucleotídeos , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Modelos Animais de Doenças , Éxons , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/genética , Doença de Huntington/metabolismo , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismo
18.
J Agric Food Chem ; 67(33): 9178-9186, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-30835107

RESUMO

Chicoric acid is a dicaffeoyl ester with many bioactivities, including antioxidation, antidiabetes, and anti-inflammation. A previous study reported that chicoric acid extended the lifespan in Caenorhabditis elegans; however, the mechanism behind the effect of chicoric acid on the extended lifespan remains unknown. Consistent with the previous report, chicoric acid (25 and 50 µM) extended the maximum lifespan compared to the control (17.5 ± 3.3 and 15.6 ± 5%, respectively; p < 0.001 for both). The declines of the pumping rate and locomotive activity, two indicators of aging, were delayed by chicoric acid. Moreover, chicoric acid enhanced resistance to oxidative stress in C. elegans. It was further determined that the extended lifespan by chicoric acid was in part via aak-2 [a homologue of adenosine monophosphate (AMP)-activated protein kinase] and skn-1 (a homologue of nuclear factor erythroid 2-related factor 2). The current findings suggest that chicoric acid has the potential to be used as an anti-aging bioactive compound.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/crescimento & desenvolvimento , Ácidos Cafeicos/farmacologia , Proteínas de Ligação a DNA/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Succinatos/farmacologia , Fatores de Transcrição/metabolismo , Animais , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ligação a DNA/genética , Feminino , Longevidade/efeitos dos fármacos , Masculino , Estresse Oxidativo/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Fatores de Transcrição/genética
19.
PLoS Genet ; 15(3): e1008004, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30921322

RESUMO

Germ cell immortality, or transgenerational maintenance of the germ line, could be promoted by mechanisms that could occur in either mitotic or meiotic germ cells. Here we report for the first time that the GSP-2 PP1/Glc7 phosphatase promotes germ cell immortality. Small RNA-induced genome silencing is known to promote germ cell immortality, and we identified a separation-of-function allele of C. elegans gsp-2 that is compromised for germ cell immortality and is also defective for small RNA-induced genome silencing and meiotic but not mitotic chromosome segregation. Previous work has shown that GSP-2 is recruited to meiotic chromosomes by LAB-1, which also promoted germ cell immortality. At the generation of sterility, gsp-2 and lab-1 mutant adults displayed germline degeneration, univalents, histone methylation and histone phosphorylation defects in oocytes, phenotypes that mirror those observed in sterile small RNA-mediated genome silencing mutants. Our data suggest that a meiosis-specific function of GSP-2 ties small RNA-mediated silencing of the epigenome to germ cell immortality. We also show that transgenerational epigenomic silencing at hemizygous genetic elements requires the GSP-2 phosphatase, suggesting a functional link to small RNAs. Given that LAB-1 localizes to the interface between homologous chromosomes during pachytene, we hypothesize that small localized discontinuities at this interface could promote genomic silencing in a manner that depends on small RNAs and the GSP-2 phosphatase.


Assuntos
Células Germinativas/metabolismo , Proteína Fosfatase 1/fisiologia , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos , Genoma , Células Germinativas/fisiologia , Meiose/fisiologia , Prófase Meiótica I/fisiologia , Metilação , Monoéster Fosfórico Hidrolases , Proteína Fosfatase 1/metabolismo , Interferência de RNA/fisiologia , RNA Interferente Pequeno
20.
PLoS Genet ; 15(3): e1007706, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30849079

RESUMO

In the natural environment, animals often encounter multiple sensory cues that are simultaneously present. The nervous system integrates the relevant sensory information to generate behavioral responses that have adaptive values. However, the neuronal basis and the modulators that regulate integrated behavioral response to multiple sensory cues are not well defined. Here, we address this question using a behavioral decision in C. elegans when the animal is presented with an attractive food source together with a repulsive odorant. We identify specific sensory neurons, interneurons and neuromodulators that orchestrate the decision-making process, suggesting that various states and contexts may modulate the multisensory integration. Among these modulators, we characterize a new function of a conserved TGF-ß pathway that regulates the integrated decision by inhibiting the signaling from a set of central neurons. Interestingly, we find that a common set of modulators, including the TGF-ß pathway, regulate the integrated response to the pairing of different foods and repellents. Together, our results provide mechanistic insights into the modulatory signals regulating multisensory integration.


Assuntos
Caenorhabditis elegans/fisiologia , Animais , Animais Geneticamente Modificados , Comportamento Animal/fisiologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/fisiologia , Alimentos , Genes de Helmintos , Interneurônios/fisiologia , Cetonas , Mutação , Neuropeptídeos/genética , Neuropeptídeos/fisiologia , Neurotransmissores/fisiologia , Odorantes , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/fisiologia , Células Receptoras Sensoriais/fisiologia , Transdução de Sinais , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/fisiologia
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