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1.
Plant Cell ; 34(10): 3665-3684, 2022 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-35897146

RESUMO

The endoplasmic reticulum-localized DnaJ family 3B (ERdj3B), is a component of the stromal cell-derived factor 2 (SDF2)-ERdj3B-binding immunoglobulin protein (BiP) chaperone complex, which functions in protein folding, translocation, and quality control. We found that ERdj3B mutations affected integument development in the Ler ecotype but not in the Col-0 ecotype of Arabidopsis (Arabidopsis thaliana). Map-based cloning identified the ERECTA (ER) gene as a natural modifier of ERdj3B. The double mutation of ERdj3B and ER caused a major defect in the inner integument under heat stress. Additional mutation of the ER paralog ERECTA-LIKE 1 (ERL1) or ERL2 to the erdj3b er double mutant exacerbated the defective integument phenotype. The double mutation of ER and SDF2, the other component of the SDF2-ERdj3B-BiP complex, resulted in similar defects in the inner integument. Furthermore, both the protein abundance and plasma membrane partitioning of ER, ERL1, and ERL2 were markedly reduced in erdj3b plants, indicating that the SDF2-ERdj3B-BiP chaperone complex might control the translocation of ERECTA-family proteins from the endoplasmic reticulum to the plasma membrane. Our results suggest that the SDF2-ERdj3B-BiP complex functions in ovule development and the heat stress response in coordination with ERECTA-family receptor kinases.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Resposta ao Choque Térmico , Óvulo Vegetal/metabolismo , Proteínas Serina-Treonina Quinases
2.
PLoS Pathog ; 17(11): e1010120, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34843593

RESUMO

Horizontal gene transfer is widespread in insects bearing intracellular symbionts. Horizontally transferred genes (HTGs) are presumably involved in amino acid synthesis in sternorrhynchan insects. However, their role in insect-symbiont interactions remains largely unknown. We found symbionts Portiera, Hamiltonella and Rickettsia possess most genes involved in lysine synthesis in the whitefly Bemisia tabaci MEAM1 although their genomes are reduced. Hamiltonella maintains a nearly complete lysine synthesis pathway. In contrast, Portiera and Rickettsia require the complementation of whitefly HTGs for lysine synthesis and have lysE, encoding a lysine exporter. Furthermore, each horizontally transferred lysine gene of ten B. tabaci cryptic species shares an evolutionary origin. We demonstrated that Hamiltonella did not alter the titers of Portiera and Rickettsia or lysine gene expression of Portiera, Rickettsia and whiteflies. Hamiltonella also did not impact on lysine levels or protein localization in bacteriocytes harboring Portiera and ovaries infected with Rickettsia. Complementation with whitefly lysine synthesis HTGs rescued E. coli lysine gene knockout mutants. Silencing whitefly lysA in whiteflies harboring Hamiltonella reduced lysine levels, adult fecundity and titers of Portiera and Rickettsia without influencing the expression of Hamiltonella lysA. Furthermore, silencing whitefly lysA in whiteflies lacking Hamiltonella reduced lysine levels, adult fecundity and titers of Portiera and Rickettsia in ovarioles. Therefore, we, for the first time, demonstrated an essential amino acid lysine synthesized through HTGs is important for whitefly reproduction and fitness of both obligate and facultative symbionts, and it illustrates the mutual dependence between whitefly and its two symbionts. Collectively, this study reveals that acquisition of horizontally transferred lysine genes contributes to coadaptation and coevolution between B. tabaci and its symbionts.


Assuntos
Evolução Molecular , Transferência Genética Horizontal , Halomonadaceae/fisiologia , Hemípteros/microbiologia , Lisina/metabolismo , Rickettsia/fisiologia , Simbiose , Animais , Hemípteros/genética , Hemípteros/crescimento & desenvolvimento , Lisina/genética
3.
Fish Shellfish Immunol ; 135: 108653, 2023 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-36868540

RESUMO

Echinacea purpurea (Linn.) Moench (EP) is a globally popular herbal medicine, which showed effects on growth promotion, antioxidant and immunomodulatory activities in fish culture world widely. However, there are few studies about the effects on miRNAs by EP in fish. The hybrid snakehead fish (Channa maculate♀ × Channa argus ♂) was new important economic specie of freshwater aquaculture in China with high market value and demand while there were only a few reports about its miRNAs. To overview immune-related miRNAs of the hybrid snakehead fish and to further understand the immune regulating mechanism of EP, we herein constructed and analyzed three small RNA libraries of immune tissues including liver, spleen and head kidney of the fish with or without EP treatment via Illumina high-throughput sequencing technology. Results showed that EP can affect the immune activities of fish by the miRNA-regulated ways. Totally, 67 (47 up and 20 down) miRNAs in liver, 138 (55 up and 83 down) miRNAs in spleen, and 251 (15 up and 236 down) miRNAs in spleen were detected, as well as 30, 60, 139 kinds of immune-related miRNAs belonging to 22, 35 and 66 families of the three tissues respectively. The expressions of 8 immune-related miRNA family members were found in all the three tissues, including miR-10, miR-133, miR-22 and etc. Some miRNAs have been identified involved in the innate and adaptive immune responses, such as the miR-125, miR-138, and miR-181 family. Ten miRNA families with antioxidant target genes were also discovered, including miR-125, miR-1306, and miR-138, etc. Results from Gene Ontology (GO) and KEGG pathway analysis further confirmed there are a majority immune response targets of the miRNAs involved in the EP treatment process. Our study deepened understanding roles of miRNAs in fish immune system and provides new ideas for the study of immune mechanism of EP.


Assuntos
Echinacea , MicroRNAs , Animais , MicroRNAs/genética , MicroRNAs/metabolismo , Antioxidantes , Peixes
4.
Appl Environ Microbiol ; 88(3): e0208921, 2022 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-34818107

RESUMO

Nutritional symbionts are restricted to specialized host cells called bacteriocytes in various insect orders. These symbionts can provide essential nutrients to the host. However, the cellular mechanisms underlying the regulation of these insect-symbiont metabolic associations remain largely unclear. The whitefly Bemisia tabaci MEAM1 hosts "Candidatus Portiera aleyrodidarum" (here, "Ca. Portiera") and "Candidatus Hamiltonella defensa" (here, "Ca. Hamiltonella") bacteria in the same bacteriocyte. In this study, the induction of autophagy by chemical treatment and gene silencing decreased symbiont titers and essential amino acid (EAA) and B vitamin contents. In contrast, the repression of autophagy in bacteriocytes via Atg8 silencing increased symbiont titers, and amino acid and B vitamin contents. Furthermore, dietary supplementation with non-EAAs or B vitamins alleviated autophagy in whitefly bacteriocytes, elevated TOR (target of rapamycin) expression, and increased symbiont titers. TOR silencing restored symbiont titers in whiteflies after dietary supplementation with B vitamins. These data suggest that "Ca. Portiera" and "Ca. Hamiltonella" evade autophagy of the whitefly bacteriocytes by activating the TOR pathway via providing essential nutrients. Taken together, we demonstrate that autophagy plays a critical role in regulating the metabolic interactions between the whitefly and two intracellular symbionts. Therefore, this study reveals that autophagy is an important cellular basis for bacteriocyte evolution and symbiosis persistence in whiteflies. The whitefly symbiosis unravels the interactions between cellular and metabolic functions of bacteriocytes. IMPORTANCE Nutritional symbionts, which are restricted to specialized host cells called bacteriocytes, can provide essential nutrients for many hosts. However, the cellular mechanisms of regulation of animal-symbiont metabolic associations have been largely unexplored. Here, using the whitefly-"Ca. Portiera"/"Ca. Hamiltonella" endosymbiosis, we demonstrate autophagy regulates the symbiont titers and thereby alters the essential amino acid and B vitamin contents. For persistence in the whitefly bacteriocytes, "Ca. Portiera" and "Ca. Hamiltonella" alleviate autophagy by activating the TOR (target of rapamycin) pathway through providing essential nutrients. Therefore, we demonstrate that autophagy plays a critical role in regulating the metabolic interactions between the whitefly and two intracellular symbionts. This study also provides insight into the cellular basis of bacteriocyte evolution and symbiosis persistence in the whitefly. The mechanisms underlying the role of autophagy in whitefly symbiosis could be widespread in many insect nutritional symbioses. These findings provide a new avenue for whitefly control via regulating autophagy in the future.


Assuntos
Halomonadaceae , Hemípteros , Complexo Vitamínico B , Animais , Autofagia , Halomonadaceae/genética , Hemípteros/microbiologia , Simbiose/genética , Complexo Vitamínico B/metabolismo
5.
Macromol Rapid Commun ; 43(7): e2100891, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-34939252

RESUMO

Regenerated silk fibers typically fall short of silkworm cocoon fibers in mechanical properties due to reduced fiber crystal structure and alignment. One approach to address this has been to employ inorganic materials as reinforcing agents. The present study avoids the need for synthetic additives, demonstrating the first use of exfoliated silk nanofibers to control silk solution crystallization, resulting in all-silk pseudocomposite fibers with remarkable mechanical properties. Incorporating only 0.06 wt% silk nanofibers led to a ≈44% increase in tensile strength (over 600 MPa) and ≈33% increase in toughness (over 200 kJ kg-1 ) compared with fibers without silk nanofibers. These remarkable properties can be attributed to nanofiber crystal seeding in conjunction with fiber draw. The crystallinity nearly doubled from ≈17% for fiber spun from pure silk solution to ≈30% for the silk nanofiber reinforced sample. The latter fiber also shows a high degree of crystal orientation with a Herman's orientation factor of 0.93, a value which approaches that of natural degummed B. mori silk cocoon fiber (0.96). This study provides a strong foundation to guide the development of simple, eco-friendly methods to spin regenerated silk with excellent properties and a hierarchical structure that mimics natural silk.


Assuntos
Bombyx , Fibroínas , Nanofibras , Animais , Bombyx/química , Fibroínas/química , Nanofibras/química , Seda/química , Resistência à Tração
6.
J Am Chem Soc ; 143(1): 85-91, 2021 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-33373216

RESUMO

A palladium-catalyzed asymmetric Markovnikov hydroaminocarbonylation of alkenes with anilines has been developed for the atom-economical synthesis of 2-substituted propanamides bearing an α-stereocenter. A novel phosphoramidite ligand L16 was discovered which exhibited very high reactivity and selectivity in the reaction. This asymmetric Markovnikov hydroaminocarbonylation employs readily available starting materials and tolerates a wide range of functional groups, thus providing a facile and straightforward method for the regio- and enantioselective synthesis of 2-substituted propanamides under ambient conditions. Mechanistic studies revealed that the reaction proceeds through a palladium hydride pathway.

7.
J Am Chem Soc ; 143(19): 7298-7305, 2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-33970621

RESUMO

Hydroaminocarbonylation of alkenes is one of the most promising yet challenging methods for the synthesis of amides. Herein, we reported the development of a novel and effective Pd-catalyzed Markovnikov hydroaminocarbonylation of 1,1-disubstituted or 1,1,2-trisubstituted alkenes with aniline hydrochloride salts to afford amides bearing an α quaternary carbon. The reaction makes use of readily available starting materials, tolerates a wide range of functional groups, and provides a facile and straightforward approach to a diverse array of amides bearing an α quaternary carbon. Mechanistic investigations suggested that the reaction proceeded through a palladium hydride pathway. The hydropalladation and CO insertion are reversible, and the aminolysis is probably the rate-limiting step.

8.
Biomacromolecules ; 22(2): 788-799, 2021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33337131

RESUMO

Low-molecular weight (LMW) silk was utilized as a LMW silk plasticizer for regenerated silk, generating weak physical crosslinks between high-molecular weight (HMW) silk chains in the amorphous regions of a mixed solution of HMW/LMW silk. The plasticization effect of LMW silk was investigated using mechanical testing, Raman spectroscopy, and wide-angle X-ray scattering (WAXS). Small amounts (10%) of LMW silk resulted in a 19.4% enhancement in fiber extensibility and 37.8% increase in toughness. The addition of the LMW silk facilitated the movement of HMW silk chains during drawing, resulting in an increase in molecular chain orientation when compared with silk spun from 100% HMW silk solution. The best regenerated silk fibers produced in this work had an orientation factor of 0.94 and crystallinity of 47.82%, close to the values of natural degummedBombyx mori silk fiber. The approach and mechanism elucidated here can facilitate artificial silk systems with enhanced properties.


Assuntos
Bombyx , Fibroínas , Animais , Peso Molecular , Seda , Análise Espectral Raman
9.
Int J Mol Sci ; 22(24)2021 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-34948043

RESUMO

MYB transcription factors of plants play important roles in flavonoid synthesis, aroma regulation, floral organ morphogenesis, and responses to biotic and abiotic stresses. Cymbidium ensifolium is a perennial herbaceous plant belonging to Orchidaceae, with special flower colors and high ornamental value. In this study, a total of 136 CeMYB transcription factors were identified from the genome of C. ensifolium, including 27 1R-MYBs, 102 R2R3-MYBs, 2 3R-MYBs, 2 4R-MYBs, and 3 atypical MYBs. Through phylogenetic analysis in combination with MYB in Arabidopsis thaliana, 20 clusters were obtained, indicating that these CeMYBs may have a variety of biological functions. The 136 CeMYBs were distributed on 18 chromosomes, and the conserved domain analysis showed that they harbored typical amino acid sequence repeats. The motif prediction revealed that multiple conserved elements were mostly located in the N-terminal of CeMYBs, suggesting their functions to be relatively conserved. CeMYBs harbored introns ranging from 0 to 13 and contained a large number of stress- and hormone-responsive cis-acting elements in the promoter regions. The subcellular localization prediction demonstrated that most of CeMYBs were positioned in the nucleus. The analysis of the CeMYBs expression based on transcriptome data showed that CeMYB52, and CeMYB104 of the S6 subfamily may be the key genes leading to flower color variation. The results lay a foundation for the study of MYB transcription factors of C. ensifolium and provide valuable information for further investigations of the potential function of MYB genes in the process of anthocyanin biosynthesis.


Assuntos
Mapeamento Cromossômico/métodos , Perfilação da Expressão Gênica/métodos , Genes myb , Orchidaceae/fisiologia , Sequenciamento Completo do Genoma/métodos , Sequência de Aminoácidos , Núcleo Celular/metabolismo , Cor , Sequência Conservada , Evolução Molecular , Flores/genética , Flores/fisiologia , Regulação da Expressão Gênica de Plantas , Sequenciamento de Nucleotídeos em Larga Escala , Família Multigênica , Orchidaceae/genética , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Análise de Sequência de RNA
10.
Angew Chem Int Ed Engl ; 60(43): 23117-23122, 2021 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-34240535

RESUMO

Asymmetric hydroxycarbonylation is one of the most fundamental yet challenging methods for the synthesis of carboxylic acids. Herein, we reported the development of a palladium-catalyzed highly enantioselective Markovnikov hydroxycarbonylation of vinyl arenes with CO and water. A monodentate phosphoramidite ligand L6 plays vital role in the reaction. The reaction tolerates a range of functional groups, and provides a facile and atom-economical approach to an array of 2-arylpropanoic acids including several commonly used non-steroidal anti-inflammatory drugs. The catalytic system has also enabled an asymmetric Markovnikov hydroalkoxycarbonylation of vinyl arenes with alcohols to afford 2-arylpropanates. Mechanistic investigations suggested that the hydropalladation is irreversible and is the regio- and enantiodetermining step, while hydrolysis/alcoholysis is probably the rate-limiting step.

11.
Anal Chem ; 92(9): 6207-6212, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32250596

RESUMO

Rapid and sensitive detection of metabolites and chemical residues in human tears is highly beneficial for understanding eye health. In this study, Schirmer paper was used for noninvasive microsampling of human tears, and then paper spray mass spectrometry (PSMS) was performed for direct analysis of human tears. Schirmer PSMS was successfully used for rapid diagnosis of dry-eye syndrome by detecting the volume and metabolites of human tears. Drugs of abuse, therapeutic drugs, and pharmacodynamics in human tears were also investigated by Schirmer PSMS. Furthermore, specific markers of environmental exposures in the air to human eyes, including volatile organic compounds, aerosol, and smoke, were unambiguously sampled and detected in human tears using Schirmer PSMS. Excellent analytical performances were achieved, including single-use, low-sample consumption (1.0 µL), rapid analysis (the whole analytical procedure completed within 3 min), high sensitivity (absolute limit of detection less than or equal to 0.5 pg, signal-to-noise ratio greater than or equal to 3), good reproducibility (relative standard deviation less than 10%, n = 3), and accurate quantitation (average deviation less than 3%, n = 3). Overall, our results showed that Schirmer PSMS is a highly effective method for direct tear analysis and is expected to be a convenient tool for human tear analysis in significant clinical applications.


Assuntos
Espectrometria de Massas/métodos , Papel , Lágrimas/química , Antibacterianos/análise , Síndromes do Olho Seco/diagnóstico , Humanos , Limite de Detecção , Metaboloma , Soluções Oftálmicas/química , Sistemas Automatizados de Assistência Junto ao Leito , Análise de Componente Principal , Reprodutibilidade dos Testes , Razão Sinal-Ruído , Manejo de Espécimes/métodos , Compostos Orgânicos Voláteis/análise
12.
Phytochem Anal ; 31(6): 739-746, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32304155

RESUMO

INTRODUCTION: Vinca alkaloids are important sources for producing anticancer drugs from Catharanthus roseus. The phosphorus of soil is one of crucial factors for planting C. roseus. OBJECTIVES: We aim to develop an in vivo sampling technique coupled with direct mass spectrometry with wooden tip for investigating distributions and changes of alkaloids in flowers, leaves, stems, veins and roots of living C. roseus under low-phosphorus stress. MATERIALS AND METHODS: Living C. roseus were prepared under low-phosphorus stress (n = 10) and control conditions (n = 10). Wooden-tip electrospray ionisation mass spectrometry and conventional liquid chromatography-mass spectrometry were applied to analyse living C. roseus and extracts of C. roseus, respectively. RESULTS: Distributions and changes of serpentine, vindoline, catharanthine, and anhydrovinblastine in living C. roseus under low-phosphorus stress and control conditions were successfully obtained. CONCLUSION: Compared to control soil conditions, low-phosphorus soil was found to induce C. roseus to generate more serpentine but less catharanthine and vindoline in leaves, veins, stems and roots, and to generate more anhydrovinblastine in flowers, leaves, stems and roots. Overall, our results showed a simple, rapid, and effective method for in vivo sampling and direct analysis of living plants.


Assuntos
Catharanthus , Cromatografia Líquida de Alta Pressão , Fósforo , Folhas de Planta , Espectrometria de Massas por Ionização por Electrospray
13.
Rapid Commun Mass Spectrom ; 33(24): 1877-1883, 2019 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-31429145

RESUMO

RATIONALE: Herbal dietary supplements (HDSs) adulterated with undeclared synthetic drugs can lead to serious health problems METHODS: A fast-switching positive/negative high-voltage (+/- HV) was developed to apply on electrospray ionization mass spectrometry (ESI-MS) with porous tips for rapid screening of five antirheumatic drugs in antirheumatic HDSs. The fast-switching (switch-time: 100 ms) negative and positive ions were alternately generated to perform full-MS and tandem-MS analysis, providing an effective method for rapid detection of analytes in whichever mode of detection was most suitable (negative or positive ion mode). The use of different tips and solvents was also optimized in this work. RESULTS: The limits of detection of the five antirheumatic drugs were found to be less than 0.1 ng/g (S/N > 3). The reproducibility of the five drugs was measured to be 10.0-23.3% (n = 5). A single sample analysis could be completed within 1 min. Rapid screening of a total of 28 real HDS samples collected from the market was examined by the fast-switching HV substrate-tip ESI-MS method, and the screening result was further validated by conventional liquid chromatography/mass spectrometry. CONCLUSIONS: Overall, our results demonstrated that fast-switching HV substrate-tip ESI-MS is a rapid, reliable, and effective method for simultaneous screening of various analytes in complex samples.


Assuntos
Antirreumáticos/análise , Contaminação de Medicamentos , Medicamentos de Ervas Chinesas/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Suplementos Nutricionais/análise , Contaminação de Medicamentos/estatística & dados numéricos , Sensibilidade e Especificidade
14.
Yi Chuan ; 40(8): 620-631, 2018 Aug 16.
Artigo em Chinês | MEDLINE | ID: mdl-30117418

RESUMO

Single-cell whole genome amplification (WGA) is a new technology, which can amplify small amounts of DNA from single cell and obtain the high coverage whole genome DNA template for revealing cell heterogeneity. Single cell WGA methods mainly include primer extension preamplification PCR (PEP-PCR), degenerate oligonucleotide primed PCR (DOP-PCR), multiple displacement amplification (MDA), and multiple annealing and looping-based amplification cycles (MALBAC). In this review, we describe the principles and applications of different single cell genome wide amplification, and we evaluate and compare their amplification efficiency, including the coverage of genome, homogeneity, reproducibility, and detection power of single-nucleotide variants (SNV) and copy number variants (CNV). The results show that MALBAC have the highest amplification homogeneity, the lowest allelic gene knockdown rate, the best reproducibility, and the best detection effect on CNV and SNV. We also describe the applications of MALBAC in human single sperm meiosis, aneuploidy analysis, and human oocyte genome research.


Assuntos
Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Reprodução Assistida , Análise de Célula Única/métodos , Animais , DNA/genética , Genômica , Humanos , Reprodução
15.
Qual Life Res ; 26(4): 1007-1014, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27696109

RESUMO

PURPOSE: This study aimed to test the effects of horticulture therapy on activities of daily living, happiness, meaning of life, and interpersonal intimacy of nursing home older adults in southern Taiwan. METHODS: A quasi-experimental study was applied. Eighty-five older adults aged 65 or older who lived in nursing homes in southern Taiwan were recruited conveniently. All participants completed the study: experimental group (n = 41) and control group (n = 44). The experimental group received horticulture therapy for 1 h once a week for 8 weeks, while the control group continued their routine daily activities. The following questionnaires were administered before and after the intervention period: (1) Barthel Index (BI), (2) Chinese Happiness Inventory short version (CHI), (3) Meaning of Life Scale (MLS), and (4) Interpersonal Intimacy Scale (IIS). RESULTS: The BI, CHI, MLS, and IIS scores significantly improved in the experimental group (p < .05). After 8 weeks of horticulture therapy, the BI, CHI, and IIS scores of experimental group participants were significantly better than the scores of control group participants (p < .05); however, the MLS scores of two groups showed no significant differences (p = .738). CONCLUSIONS: Horticulture therapy improved activities of daily living, happiness, and interpersonal intimacy of older adults in nursing homes. We recommend that nursing homes recruit and train personnel to lead horticultural therapy and to incorporate the therapy as routine daily activities in the facilities.


Assuntos
Atividades Cotidianas , Idoso Fragilizado/psicologia , Horticultura Terapêutica , Casas de Saúde , Qualidade de Vida , Idoso , Idoso de 80 Anos ou mais , Feminino , Serviços de Saúde para Idosos , Humanos , Masculino , Inquéritos e Questionários , Taiwan
16.
J Biol Chem ; 290(33): 20556-64, 2015 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-26149688

RESUMO

PAX3 is a transcription factor critical to gene regulation in mammalian development. Mutations in PAX3 are associated with Waardenburg syndrome (WS), but the mechanism of how mutant PAX3 proteins cause WS remains unclear. Here, we found that PAX3 loads on mitotic chromosomes using its homeodomain. PAX3 WS mutants with mutations in homeodomain lose the ability to bind mitotic chromosomes. Moreover, loading of PAX3 on mitotic chromosomes requires arginine methylation, which is regulated by methyltransferase PRMT5 and demethylase JMJD6. Mutant PAX3 proteins that lose mitotic chromosome localization block cell proliferation and normal development of zebrafish. These results reveal the molecular mechanism of PAX3s loading on mitotic chromosomes and the importance of this localization pattern in normal development. Our findings suggest that PAX3 WS mutants interfere with the normal functions of PAX3 in a dominant negative manner, which is important to the understanding of the pathogenesis of Waardenburg syndrome.


Assuntos
Arginina/metabolismo , Cromossomos Humanos , Mitose/genética , Fatores de Transcrição Box Pareados/genética , Síndrome de Waardenburg/genética , Animais , Células HEK293 , Humanos , Larva/metabolismo , Metilação , Fator de Transcrição PAX3 , Proteína-Arginina N-Metiltransferases/metabolismo , Peixe-Zebra/crescimento & desenvolvimento
17.
J Biol Chem ; 289(44): 30785-30799, 2014 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-25225287

RESUMO

Pigment epithelium-derived factor (PEDF), a potent antiangiogenesis agent, has recently attracted attention for targeting tumor cells in several types of tumors. However, less is known about the apoptosis-inducing effect of PEDF on human lung cancer cells and the underlying molecular events. Here we report that PEDF has a growth-suppressive and proapoptotic effect on lung cancer xenografts. Accordingly, in vitro, PEDF apparently induced apoptosis in A549 and Calu-3 cells, predominantly via the Fas-L/Fas death signaling pathway. Interestingly, A549 and Calu-3 cells are insensitive to the Fas-L/Fas apoptosis pathway because of the low level of cell surface Fas. Our results revealed that, in addition to the enhancement of Fas-L expression, PEDF increased the sensitivity of A549 and Calu-3 cells to Fas-L-mediated apoptosis by triggering the translocation of Fas protein to the plasma membrane in a p53- and FAP-1-dependent manner. Similarly, the up-regulation of Fas-L by PEDF was also mediated by p53. Furthermore, peroxisome proliferator-activated receptor γ was determined to be the upstream regulator of p53. Together, these findings uncover a novel mechanism of tumor cell apoptosis induced by PEDF and provide a potential therapeutic strategy for tumors that are insensitive to Fas-L/Fas-dependent apoptosis because of a low level of cell surface Fas.


Assuntos
Antineoplásicos/farmacologia , Apoptose , Proteínas do Olho/farmacologia , Proteína Ligante Fas/genética , Fatores de Crescimento Neural/farmacologia , Serpinas/farmacologia , Proteína Supressora de Tumor p53/fisiologia , Receptor fas/metabolismo , Animais , Antineoplásicos/uso terapêutico , Caspase 8/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Proteínas do Olho/fisiologia , Proteínas do Olho/uso terapêutico , Proteína Ligante Fas/metabolismo , Humanos , Neoplasias Pulmonares , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Neovascularização Patológica/prevenção & controle , Fatores de Crescimento Neural/fisiologia , Fatores de Crescimento Neural/uso terapêutico , PPAR gama/metabolismo , Transporte Proteico , Proteína Tirosina Fosfatase não Receptora Tipo 13/metabolismo , Serpinas/fisiologia , Serpinas/uso terapêutico , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
18.
J Biol Chem ; 289(47): 32628-38, 2014 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-25296756

RESUMO

Human plasminogen kringle 5 (K5) is known to display its potent anti-angiogenesis effect through inducing endothelial cell (EC) apoptosis, and the voltage-dependent anion channel 1 (VDAC1) has been identified as a receptor of K5. However, the exact role and underlying mechanisms of VDAC1 in K5-induced EC apoptosis remain elusive. In the current study, we showed that K5 increased the protein level of VDAC1, which initiated the mitochondrial apoptosis pathway of ECs. Our findings also showed that K5 inhibited the ubiquitin-dependent degradation of VDAC1 by promoting the phosphorylation of VDAC1, possibly at Ser-12 and Thr-107. The phosphorylated VDAC1 was attenuated by the AKT agonist, glycogen synthase kinase (GSK) 3ß inhibitor, and siRNA, suggesting that K5 increased VDAC1 phosphorylation via the AKT-GSK3ß pathway. Furthermore, K5 promoted cell surface translocation of VDAC1, and binding between K5 and VDAC1 was observed on the plasma membrane. HKI protein blocked the impact of K5 on the AKT-GSK3ß pathway by competitively inhibiting the interaction of K5 and cell surface VDAC1. Moreover, K5-induced EC apoptosis was suppressed by VDAC1 antibody. These data show for the first time that K5-induced EC apoptosis is mediated by the positive feedback loop of "VDAC1-AKT-GSK3ß-VDAC1," which may provide new perspectives on the mechanisms of K5-induced apoptosis.


Assuntos
Apoptose/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Fragmentos de Peptídeos/farmacologia , Plasminogênio/farmacologia , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Apoptose/genética , Western Blotting , Caspases/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Retroalimentação Fisiológica/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Células HEK293 , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fragmentos de Peptídeos/genética , Fosforilação/efeitos dos fármacos , Plasminogênio/genética , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Ubiquitina/metabolismo , Canal de Ânion 1 Dependente de Voltagem/genética
19.
Biochim Biophys Acta ; 1839(7): 579-91, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24852358

RESUMO

Aberrant expression levels of transcriptional regulators result in alterations in transcriptional control. STAF65γ is a structural subunit of the GCN5 transcriptional co-activator complex. Reports showed that STAF65γ is highly expressed in several human cancer cells, but the consequences of this aberrant expression pattern remain elusive. Here, we show that the STAF65γ protein is highly expressed in lung adenocarcinoma patients and high levels of STAF65γ correlate with poor prognosis. High levels of STAF65γ cause repression of the c-Myc oncogene through physical association with transcription factor YY1 and co-repressors HDACs. Physical interactions between STAF65γ and class IIa HDACs facilitate nuclear enrichment and regulate the assembly of HDAC complexes. Moreover, SUMOylation of STAF65γ is necessary for maintaining the co-repressor complex containing YY1 and class IIa HDACs at the promoter. Our findings reveal a distinct role of STAF65γ in nuclear import, transcriptional repression, and cell cycle regulation at high levels of expression, which is associated with poor clinical outcomes of lung adenocarcinoma.


Assuntos
Adenocarcinoma/genética , Histona Desacetilases/genética , Neoplasias Pulmonares/genética , Regiões Promotoras Genéticas , Transativadores/genética , Transcrição Gênica , Transporte Ativo do Núcleo Celular/genética , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Adulto , Idoso , Ciclo Celular/genética , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteínas Repressoras/genética , Sumoilação , Fator de Transcrição YY1/genética
20.
Guang Pu Xue Yu Guang Pu Fen Xi ; 35(9): 2544-7, 2015 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-26669164

RESUMO

As a common spectral characterization technique, Raman spectroscopy is widely used and has a specified calibration procedure. Based on laser confocal micro-Raman spectrometer, in this paper, we briefly introduced the principle, configuration and main components of Raman spectrometer. In addition, the calibration procedures were also presented, with an emphasis on the calibration of spectrometer (spectrograph) and that of excitation laser wavelength. On the basis of conventional calibration method, a novel and more accurate method was proposed to obtain the actual excitation wavelength, that is, calibration at the point of Raman shift Δν=0. Using this novel calibration method of excitation wavelength, Raman frequency shift values of sulfur were measured, and compared with the standard values from American Society Testing and Materials (ASTM). As a result, the measured values after calibration were consistent with those ASTM values, which indicated that the calibration method is accurate. Thus, a more reasonable calibration procedure of the laser confocal micro-Raman spectrometer was provided.

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