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1.
Br Poult Sci ; 60(3): 202-208, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30968708

RESUMO

1. The slow skeletal muscle troponin I (TNNI1) gene has been found to be specifically expressed in slow muscle fibres and plays an important role in muscle development. The aim of this study was to determine the active control area of duck TNNI1 and identify the potential cis-regulatory elements in the promoter. 2. In this study, the TNNI1 promoter was first cloned by genome walking and the sequences were analysed using bioinformatics software. Firefly luciferase reporter gene vectors, driven by a series of constructs with progressive deletions, were used to identify the core transcriptional regulatory region of the duck TNNI1 gene. The methylation status of the CpG island in the TNNI1 promoter was detected in skeletal muscle on embryonic days 21 and 27, by bisulphite sequencing PCR (BSP). 3. The results showed two CpG islands presented in the promoter region, with one of the CpG islands located in the core transcriptional regulatory region (-2078/-885 bp). The total methylation levels of the 14 CpG sites were not altered between breast and leg muscles on embryonic days 21 and 27. However, four CpG sites (loci of positions 4, 11, 13, and 14) showed dramatically different methylation levels between breast and leg muscles at embryonic days 21 and 27. Analysis showed that multiple CpG sites had a significant correlation between the methylation levels of the CpG sites and mRNA expressions in skeletal muscle. Multiple transcription factor binding sites including Sp1, c-Myc, Oct-1 and NF-kB motifs were identified and might be responsible for transcriptional regulation of the TNNI1 gene. 4. These findings contribute to further understanding of the fundamental mechanism for transcriptional regulation of the TNNI1 gene in ducks.


Assuntos
Proteínas Aviárias/genética , Metilação de DNA , Patos/genética , Regulação da Expressão Gênica , Músculo Esquelético/metabolismo , Troponina I/genética , Animais , Proteínas Aviárias/metabolismo , Sequência de Bases , Ilhas de CpG , Patos/metabolismo , Regiões Promotoras Genéticas , Troponina I/metabolismo
2.
Anim Genet ; 50(3): 279-282, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30974000

RESUMO

Glutaminyl-peptide cyclotransferase-like (QPCTL) is an isoenzyme of glutaminyl-peptide cyclotransferase (QPCT). QPCTL and QPCT catalyze the formation of N-terminal modified pyroglutamate-fractalkine and the chemokine CCL2. The objective of this study was to investigate the association between insertions/deletions in the chicken QPCTL promoter region with growth traits in chickens. We first detected two insertion/deletion variants of QPCTL via whole-genome resequencing analysis of DNA samples from Xichuan chickens. A total of 1896 individuals from 12 breeds were genotyped for 52- and 224-bp insertions/deletions. We found two novel insertions/deletions in the promoter region of the chicken QPCTL gene and studied their association with chicken body weight and carcass traits. Our findings show that QPCTL can be a molecular marker for chicken genetics and breeding programs.


Assuntos
Aminoaciltransferases/genética , Galinhas/genética , Mutação INDEL , Regiões Promotoras Genéticas , Animais , Proteínas Aviárias/genética , Galinhas/classificação
3.
Anim Genet ; 50(3): 287-292, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30994195

RESUMO

Plasma cholinesterase (PCHE) activity is an important auxiliary test in human clinical medicine. It can distinguish liver diseases from non-liver diseases and help detect organophosphorus poisoning. Animal experiments have confirmed that PCHE activity is associated with obesity and hypertension and changes with physiological changes in an animal's body. The objective of this study was to locate the genetic loci responsible for PCHE activity variation in ducks. PCHE activity of Pekin duck × mallard F2 ducks at 3 and 8 weeks of age were analyzed, and genome-wide association studies were conducted. A region of about 1.5 Mb (21.8-23.3 Mb) on duck chromosome 9 was found to be associated with PCHE activity at both 3 and 8 weeks of age. The top SNP, g.22643979C>T in the butyrylcholinesterase (BCHE) gene, was most highly associated with PCHE activity at 3 weeks (-logP = 21.45) and 8 weeks (-logP = 27.60) of age. For the top SNP, the strong associations of CC and CT genotypes with low PCHE activity and the TT genotype with high PCHE activity indicates the dominant inheritance of low PCHE activity. Problems with block inheritance or linkage exist in this region. This study supports that BCHE is a functional gene for determining PCHE levels in ducks and that the genetic variations around this gene can cause phenotypic variations of PCHE activity.


Assuntos
Colinesterases/genética , Patos/genética , Animais , Proteínas Aviárias/sangue , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Butirilcolinesterase/genética , Colinesterases/sangue , Colinesterases/metabolismo , Cruzamentos Genéticos , Patos/sangue , Patos/metabolismo , Feminino , Estudos de Associação Genética , Masculino , Polimorfismo de Nucleotídeo Único
4.
Theriogenology ; 131: 146-152, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30965207

RESUMO

Lead (Pb) is an environmental pollutant and has toxic effect on birds. Selenium (Se) has alleviative effect on Pb poisoning. This study investigated mitigative effect of Se on autophagy in Pb-treated chicken testes. Seven-day-old male chickens were randomly divided into four groups with 45 birds in each group. The birds of the control group were offered drinking water (DW) and commercial diet (CD) (0.49 mg/kg Se). The birds of the Se group were offered DW and CD containing sodium selenite (SeCD) (1.00 mg/kg Se). The birds of the Pb group were offered DW containing lead acetate (PbDW) (350.00 mg/L Pb) and CD. The birds of the Pb/Se group were offered PbDW and SeCD. On the 30th, 60th, and 90th days, respectively; histology, antioxidant indexes (hydrogen peroxide (H2O2), catalase (CAT), total antioxidant capacity (TAOC), reduced glutathione (GSH), and superoxide dismutase (SOD)), mRNA and protein levels of autophagy-related genes (autophagy-related proteins 5, Beclin 1, Dynein, light chain 3 (LC3)-I, LC3-II, and mammalian target of rapamycin (mTOR)) were performed in chicken testes. The results of this study showed that Pb caused histological changes; increased H2O2 content; decreased CAT, TAOC, and SOD activities and GSH content; and increased mRNA and protein levels of the above autophagy-related genes except that mTOR decreased in chicken testes. Se alleviated the above changes. Se alleviated histological damage, oxidative stress, and autophagy in the Pb-treated chicken testes.


Assuntos
Autofagia/efeitos dos fármacos , Galinhas , Poluentes Ambientais/toxicidade , Chumbo/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Selênio/farmacologia , Testículo/efeitos dos fármacos , Animais , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Biomarcadores/metabolismo , Masculino , RNA Mensageiro/metabolismo , Distribuição Aleatória
5.
Res Vet Sci ; 124: 256-262, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30999161

RESUMO

Interferon-induced proteins with tetratricopeptide repeats (IFITs) are a family of proteins strongly induced downstream of type I interferon signaling. The function of IFITs has been investigated extensively in mammals. IFIT5 is the sole protein in this family found in birds and little information is available about the function of avian IFIT5. In this study, duck IFIT5 was cloned from peripheral blood mononuclear cells. Multiple amino acid sequence alignment and phylogenetic analysis showed that duck IFIT5 is highly homologous to chicken IFIT5. Tissue specificity analysis demonstrated that duck IFIT5 was ubiquitously expressed in all examined tissues of five-day-old ducklings, with the highest expression levels in heart, followed by thymus, cerebrum, liver, and lung; kidney expressed the lowest. Quantitative real-time PCR (qRT-PCR) analysis revealed that duck IFIT5 expression rapidly increased both in vitro and in vivo after stimulation with polyinosinic:polycytidylic acid [poly (I:C)] and infection with virulent duck hepatitis A virus type 3 (DHAV-3), respectively. Altogether, these results indicate that the expression of duck IFIT5 is positively correlated with viral load and may play an important role in the immune response to DHAV-3 infection. This study lays a foundation for further research into the innate antiviral immune responses of ducklings.


Assuntos
Patos/genética , Patos/imunologia , Proteínas de Neoplasias/genética , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos/genética , Animais Recém-Nascidos/imunologia , Proteínas Aviárias/química , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Sequência de Bases , Clonagem Molecular , Vírus da Hepatite do Pato/fisiologia , Hepatite Viral Animal/imunologia , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Fases de Leitura Aberta , Filogenia , Infecções por Picornaviridae/imunologia , Infecções por Picornaviridae/veterinária , Poli I-C/farmacologia , Doenças das Aves Domésticas/imunologia , Alinhamento de Sequência
6.
Mol Biotechnol ; 61(6): 400-409, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30945164

RESUMO

Transgenic chickens are of great interest for the production of recombinant proteins in their eggs. However, the use of constitutive strong promoters or the tissue-specific ovalbumin promoter for the generation of the transgenic chickens have different drawbacks that have to be overcome in order to make chicken bioreactor an efficient production system. This prompted us to investigate the use of an alternative tissue-specific promoter, the vitellogenin promoter, which could overcome the difficulties currently found in the generation of chicken bioreactors. In the present work we establish and characterize a DNA construct consisting of a fragment of the 5´-flanking region of the chicken vitellogenin II gene cloned in a reporter vector. This construct is capable of showing the ability of the promoter to drive expression of a reporting gene in a tissue-specific manner and in a way that closely resembles physiologic regulation of vitellogenin, making it an ideal candidate to be used in the future for generation of avian bioreactors. Besides, we validate an in vitro culture system to test the performance of the DNA construct under study that could be used as a practical tool before generating any transgenic chicken. These results are important since they provide the proof of concept for the use of the vitellogenin promoter for future genetic modification of chickens bioreactors with improved characteristics in terms of quality of the recombinant protein produced.


Assuntos
Proteínas Aviárias/genética , Galinhas/genética , Vetores Genéticos/química , Proteínas Recombinantes de Fusão/genética , Vitelogeninas/genética , Região 5'-Flanqueadora , Animais , Animais Geneticamente Modificados , Proteínas Aviárias/metabolismo , Reatores Biológicos , Embrião de Galinha , Galinhas/metabolismo , Clonagem Molecular , Estradiol/farmacologia , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Vetores Genéticos/metabolismo , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Luciferases/genética , Luciferases/metabolismo , Cultura Primária de Células , Regiões Promotoras Genéticas , Receptores Estrogênicos , Proteínas Recombinantes de Fusão/metabolismo , Transfecção/métodos , Vitelogeninas/metabolismo , Zigoto/efeitos dos fármacos , Zigoto/crescimento & desenvolvimento , Zigoto/metabolismo
7.
Int J Mol Sci ; 20(8)2019 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-31013627

RESUMO

Overcoming P-glycoprotein (P-gp) efflux is a strategy to improve the absorption and pharmacokinetics of its substrate drugs. Berberine inhibits P-gp and thereby increases the bioavailability of the P-gp substrate digoxin in rodents. However, the effects of berberine on P-gp in chickens are still unclear. Here, we studied the role of berberine in modulating broilers P-gp expression and function through both in situ and in vitro models. In addition, molecular docking was applied to analyze the interactions of berberine with P-gp as well as with chicken xenobiotic receptor (CXR). The results showed that the mRNA expression levels of chicken P-gp and CXR decreased in the ileum following exposure to berberine. The absorption rate constant of rhodamine 123 increased after berberine treatment, as detected using an in situ single-pass intestinal perfusion model. Efflux ratios of P-gp substrates (tilmicosin, ciprofloxacin, clindamycin, ampicillin, and enrofloxacin) decreased and the apparent permeability coefficients increased after co-incubation with berberine in MDCK-chAbcb1 cell models. Bidirectional assay results showed that berberine could be transported by chicken P-gp with a transport ratio of 4.20, and this was attenuated by verapamil (an inhibitor of P-gp), which resulted in a ratio of 1.13. Molecular docking revealed that berberine could form favorable interactions with the binding pockets of both CXR and P-gp, with docking scores of -7.8 and -9.5 kcal/mol, respectively. These results indicate that berberine is a substrate of chicken P-gp and down-regulates P-gp expression in chicken tissues, thereby increasing the absorption of P-gp substrates. Our findings suggest that berberine increases the bioavailability of other drugs and that drug-drug interactions should be considered when it is co-administered with other P-gp substrates with narrow therapeutic windows.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Berberina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Animais , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Berberina/química , Galinhas , Cães , Células Madin Darby de Rim Canino , Modelos Moleculares , Conformação Proteica , RNA Mensageiro/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Relação Estrutura-Atividade
8.
Gene ; 702: 182-193, 2019 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-30910561

RESUMO

Programmed death-1 (PD-1) has a pivotal role in the attenuation of adaptive immune responses and peripheral tolerance. Here we describe the identification of the Pekin duck programmed death-1 orthologue (duPD-1). The duPD-1 cDNA encodes a 283-amino acid polypeptide that has an amino acid identity of 70%, 32% and 31% with chicken, murine and human PD-1, respectively. The duck PD-1 gene shares five conserved exons with chicken, murine and human PD-1 genes. A cluster of putative regulatory elements within the conserved region B (CR-B) of the basal promotor is conserved. Homology modeling was most compatible with the two ß-sheet IgV domain structure of murine PD-1. Contact residues, shown to be critical for binding of the respective human and murine PD-1 ligands are mostly conserved between avian and mammalian species, whereas residues that define the cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM) are highly conserved across higher vertebrates and frog. Constitutive expression of duPD-1 transcripts was predominantly found in lymphocyte-rich tissues, and mitogen-stimulation of duck peripheral blood mononuclear cells transiently increased duPD-1 mRNA expression. A soluble duPD-1 protein was expressed and shown to engage the identified duck PD-1 ligands. Our observations show considerable evolutionary conservation between mammalian and avian PD-1 orthologues. This work will facilitate further investigation of the role of PD-1 signaling in adaptive immunity in the Pekin duck, a non-mammalian vertebrate and pathogen host with relevance for human and animal health.


Assuntos
Proteínas Aviárias/química , Proteínas Aviárias/genética , Receptor de Morte Celular Programada 1/química , Receptor de Morte Celular Programada 1/genética , Animais , Proteínas Aviárias/classificação , Mapeamento Cromossômico , Clonagem Molecular , Patos , Expressão Gênica , Ligantes , Modelos Moleculares , Filogenia , Receptor de Morte Celular Programada 1/classificação , Receptor de Morte Celular Programada 1/metabolismo , Domínios Proteicos , Estrutura Secundária de Proteína , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Análise de Sequência de Proteína , Distribuição Tecidual
9.
Gen Comp Endocrinol ; 276: 69-76, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30851298

RESUMO

The function of oocyte-derived growth differentiation factor 9 (GDF9) in ovarian follicles has thus far been poorly defined in avian species compared with the defined function in mammals. Our aim here is to investigate the effects of GDF9 on steroidogenesis and on chicken ovarian granulosa cell (GC) mitosis. Primary GCs from both prehierarchical (6-8 mm in diameter, phGCs) and preovulatory follicles (F1-F5, poGCs) were cultured in the presence or absence of the GDF9 protein. The progesterone (P4) levels in the culture medium were then measured by radioimmunoassay (RIA), and the expression levels of steroidogenesis genes were detected by quantitative PCR. We found that GDF9 alone showed no significant effect on the P4 levels by regulating the expression of steroidogenesis genes, such as STAR, CYP11A1 and HSD3B. Further experiments indicated that GDF9 promoted follicle-stimulating hormone (FSH)-induced P4 production and STAR expression. GDF9 also rescued the FSH-induced decrease of FSH receptor (FSHR) expression but had no effect on the forskolin-induced P4, STAR and forskolin-inhibited FSHR expression levels, suggesting that GDF9 might achieve its regulatory role of P4 by enhancing FSHR and STAR expression. In addition, GDF9 also promoted GC cell cycle progression, regulated the gene transcription of related genes, potentiated DNA replication and inhibited apoptosis. Interestingly, these effects differed between the phGCs and the poGCs. To our knowledge, this is the first report that illustrates the function of GDF9 on chicken GCs and the effects on ovarian steroidogenesis. Our findings highlight the regulation of central oocytes on the surrounding granulosa cells and emphasize the interaction between paracrine signals and endocrine hormones on ovarian progesterone production; these findings contribute to the understanding of the development of avian ovarian follicles.


Assuntos
Galinhas/metabolismo , Hormônio Foliculoestimulante/farmacologia , Células da Granulosa/metabolismo , Fator 9 de Diferenciação de Crescimento/farmacologia , Progesterona/biossíntese , Animais , Apoptose/efeitos dos fármacos , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Colforsina/farmacologia , DNA/biossíntese , Replicação do DNA/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Células da Granulosa/efeitos dos fármacos , Humanos , Ovulação/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Radioimunoensaio
10.
Gene ; 701: 72-81, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30898701

RESUMO

Avian leukosis virus subgroup J (ALV-J) is an oncogenic retrovirus that causes severe economic losses to the poultry industry worldwide. Circular RNAs (circRNAs) are a class of non-coding RNAs that has been described in various biological systems and pathogenic processes. However, the immune mechanisms in response to circRNAs remain unknown. In this study, high-throughput transcriptome sequencing was used to detect circRNAs present in chicken macrophage (HD11) and chick embryo fibroblast (CEF) cells infected with ALV-J. We identified 7684 circRNAs from diverse genomic locations in CEF and HD11 after ALV-J infection, these RNAs showed complex expression patterns that differed based on the cells type and infection time. In total, 302 differentially expressed (DE) circRNAs and 164 DE circRNAs were identified in CEF and HD11 after ALV-J infection, respectively. CircRNA7419-associated with KDM4C- and circRNA6679 and circRNA6680-associated with TNFAIP6- were involved in the immune response upon ALV-J infection in CEF. Host genes were analyzed through further bioinformatics analysis. The result confirmed that a large number of DE circRNAs corresponded to several immune-associated or tumor-associated terms and pathways, such as Mucin type O-Glycan biosynthesis, MAPK signaling pathway, B cell receptor signaling, and Wnt signaling pathway in CEF, as well as Jak-STAT signaling pathway, apoptosis, and MAPK signaling pathway in HD11. CircRNAs related to the B cell receptor signaling pathway in CEF, and the Jak-STAT signaling pathway in HD11, were selected for circRNA-miRNA interaction network analyses. Our study indicates that circRNAs expression was altered by ALV-J infection in both CEF and HD11, and may play a key role in the progression of ALV-J infection.


Assuntos
Vírus da Leucose Aviária , Leucose Aviária , Galinhas , Sistema de Sinalização das MAP Quinases , Doenças das Aves Domésticas , RNA , Via de Sinalização Wnt , Animais , Leucose Aviária/genética , Leucose Aviária/metabolismo , Leucose Aviária/patologia , Vírus da Leucose Aviária/genética , Vírus da Leucose Aviária/metabolismo , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Linhagem Celular , Embrião de Galinha , Galinhas/genética , Galinhas/metabolismo , Galinhas/virologia , Doenças das Aves Domésticas/genética , Doenças das Aves Domésticas/metabolismo , Doenças das Aves Domésticas/patologia , Doenças das Aves Domésticas/virologia , RNA/genética , RNA/metabolismo
11.
Gene ; 701: 82-88, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30902784

RESUMO

The goose is one of the most important waterfowl, having lowing laying rate. Previous studies have shown the SNPs in the introns of MAGI-1 (Record-106975) and ACSF2 (Record-106582) significantly associated with egg production in geese. However, the mechanism of those SNPs influencing egg production remains unclear. In this study, the three goose breeds (Yangzhou geese, Zhedong white geese, and Carlos geese) with obviously different egg production were selected, and the allele frequency distribution and functions of those SNPs were investigated. The results suggested that the allele frequency distribution of ACSF2 was significantly different among the three goose breeds (χc2 = 92.377, Pc = 2.29 × 10-22), with the C allele appearing at frequencies of 0.29 in the Yangzhou geese and 0.94 in the Carlos geese. In contrast, the allele frequencies of MAGI-1 were not significantly different among the different goose breeds. Quantitative Reverse Transcription PCR (qRT-PCR) showed that the expression of MAGI-1 with the AG genotype individuals was significantly higher than those of the AA and GG genotype. For ACSF2, the CC genotype had significantly higher expression than both the AC genotype and the AA genotype. The luciferase reporter analysis revealed that the site-directed mutation ACSF2 (A>C) significantly drove the expression activity. Further analysis suggested that the mutation altered the binding site of the transcription factor BARHL2. Binding of BARHL2 to the ACSF2 intron was confirmed by electrophoretic mobility shift assay (EMSA) analysis. Thus, our findings revealed the A>C mutation of ACSF2 (Record-106582) could promote the expression by regulating the binding of BARHL2, resulting in differences in egg performance, which provided molecular insights into the effect of the polymorphism in ACSF2 on egg performance in geese.


Assuntos
Proteínas Aviárias/genética , Cruzamento , Moléculas de Adesão Celular Neuronais/genética , Coenzima A Ligases/genética , Gansos/genética , Íntrons , Polimorfismo Genético , Animais , Proteínas Aviárias/biossíntese , Moléculas de Adesão Celular Neuronais/biossíntese , Coenzima A Ligases/biossíntese , Gansos/metabolismo
12.
Anim Genet ; 50(3): 283-286, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30883845

RESUMO

The agouti signaling protein gene (ASIP) is a widely studied pigmentation gene that plays an important role in melanin synthesis. To determine the variety of ASIP expression in the Muchuan Black-Bone chicken, we examined genetic variation in the ASIP promoter region. A single nucleotide polymorphism (c.-1826A>T) was found to be associated with the skin color (dorsal and subalar) of black-bone chicken. Individuals with TT and AT genotypes had higher ASIP mRNA levels in the skin than did those with the AA genotype (P < 0.01). In addition, individuals with the TT genotype had higher ASIP mRNA levels than did those with the AT genotype (P < 0.05). Expression of melanogenesis-related genes (melanocortin 1 receptor and tyrosinase genes) was higher in the skin of chickens with the TT and AT genotypes than in those with the AA genotype (P < 0.01). A luciferase assay showed that promoter activity was higher in chickens with the TT genotype than in those with the AA genotype. Putative transcription factor prediction suggested that the c.-1826A>T mutation might shift the promoter binding affinity with differential transcription factors. In summary, we identified a novel mutation in the ASIP gene promoter that may affect chicken skin color by altering ASIP transcriptional activity.


Assuntos
Proteína Agouti Sinalizadora/genética , Proteínas Aviárias/genética , Galinhas/genética , Pigmentação , Polimorfismo de Nucleotídeo Único , Animais , Feminino
13.
Proc Natl Acad Sci U S A ; 116(14): 6766-6774, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30877242

RESUMO

Focal adhesion kinase (FAK) is a key signaling molecule regulating cell adhesion, migration, and survival. FAK localizes into focal adhesion complexes formed at the cytoplasmic side of cell attachment to the ECM and is activated after force generation via actomyosin fibers attached to this complex. The mechanism of translating mechanical force into a biochemical signal is not understood, and it is not clear whether FAK is activated directly by force or downstream to the force signal. We use experimental and computational single-molecule force spectroscopy to probe the mechanical properties of FAK and examine whether force can trigger activation by inducing conformational changes in FAK. By comparison with an open and active mutant of FAK, we are able to assign mechanoactivation to an initial rupture event in the low-force range. This activation event occurs before FAK unfolding at forces within the native range in focal adhesions. We are also able to assign all subsequent peaks in the force landscape to partial unfolding of FAK modules. We show that binding of ATP stabilizes the kinase domain, thereby altering the unfolding hierarchy. Using all-atom molecular dynamics simulations, we identify intermediates along the unfolding pathway, which provide buffering to allow extension of FAK in focal adhesions without compromising functionality. Our findings strongly support that forces in focal adhesions applied to FAK via known interactions can induce conformational changes, which in turn, trigger focal adhesion signaling.


Assuntos
Trifosfato de Adenosina/química , Proteínas Aviárias/química , Proteína-Tirosina Quinases de Adesão Focal/química , Simulação de Dinâmica Molecular , Desdobramento de Proteína , Trifosfato de Adenosina/metabolismo , Animais , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Galinhas , Ativação Enzimática , Proteína-Tirosina Quinases de Adesão Focal/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Adesões Focais/enzimologia , Adesões Focais/genética , Mecanotransdução Celular/genética , Domínios Proteicos , Relação Estrutura-Atividade
14.
Mol Genet Genomics ; 294(3): 679-692, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30834967

RESUMO

Cathartidae is a small family of large-bodied carrion-feeding birds, of which the turkey vulture (Cathartes aura, Cathartidae) is the most widespread distributed. To investigate the chemoreception system, detoxification system, and immune system in the turkey vulture, we compared its genome to 14 other avian genomes. Comparative genomics demonstrated the expansion in the chemoreception system, especially the olfactory receptors, while the genes in the detoxification system of the turkey vulture did not show apparent expansion. We identified five positively selected genes associated with the immune system in the turkey vulture, which was likely to strengthen the immune defense against pathogenic invasion. Functional enrichment analysis indicated that many positively selected genes were involved in the regulation of immune system processes, implying important reorganization of the immune system in the turkey vulture. The turkey vulture-specific missense mutations were found in one positively selected gene (BCL6), and all the missense mutations were classified as deleterious by PolyPhen-2, possibly contributing to immune adaptation to the carrion feeding. Furthermore, we identified four turkey vulture-specific missense mutations in three ß-defensin genes of the turkey vulture, which was an indispensable part in the innate immunity (a natural barrier against invasive microbes including bacteria, fungi, and viruses). Our genomic analyses in the turkey vulture provided insights into the genetic signatures of the adaptation to the carrion feeding.


Assuntos
Proteínas Aviárias/genética , Aves/genética , Genoma/genética , Genômica/métodos , Sequência de Aminoácidos , Animais , Bactérias/patogenicidade , Aves/classificação , Aves/microbiologia , Comportamento Alimentar , Fungos/patogenicidade , Sistema Imunitário/metabolismo , Sistema Imunitário/microbiologia , Filogenia , Homologia de Sequência de Aminoácidos , Virulência , Vírus/patogenicidade , beta-Defensinas/genética
15.
Biomed Environ Sci ; 32(2): 107-120, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30862342

RESUMO

OBJECTIVE: Our aim was to explore whether heat stress protein (HSP) 9 preferentially expresses under heat stress and affects the expression of other heat stress proteins as well as to explore the effect of HSPB9 overexpression and knockdown on apoptosis in DF-1. METHODS: We used gene cloning to construct an overexpression vector of the target gene, and synthesized the target gene interference fragment to transfect the chicken fibroblast cell line. Gene and protein expression, as well as apoptosis, were detected by RT-qPCR, Western blot, and flow cytometry. RESULTS: Chicken DF-1 cells showed an early state of apoptosis in the early stages of HSPB9 overexpression. In the later stages, as HSPB9 expression increased, the cells showed inhibition of apoptosis. When the cells were under heat stress, HSPB9 expression was much higher and earlier than the expression of HSPB1 and HSPA2. In addition, high expression of HSPB9 had a negative effect on HSPB1 and HSPA2 expression. This negative feedback decreased the percentage of early stages of apoptotic cells and promoted cell survival. CONCLUSION: HSPB9 expression, although rapid, is detrimental to cell survival early during its overexpression. In heat stress, HSPB9 overexpression, while inhibiting the expression of HSPA2 and HSPB1, is beneficial to cell survival.


Assuntos
Apoptose/genética , Proteínas Aviárias/genética , Proteínas de Choque Térmico/genética , Resposta ao Choque Térmico/genética , Animais , Linhagem Celular , Galinhas
16.
Environ Sci Pollut Res Int ; 26(11): 10529-10536, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30767103

RESUMO

Ammonia (NH3) is a highly irritant, alkaline gas. Atmospheric emission of NH3 was recognized as an environmental challenge. As a global issue, the NH3 emission survey with spatially detailed information demonstrated that the sources of atmospheric NH3 include agriculture (livestock wastes, fertilizers) and some industrial activities. As an environmental pollution, excessive NH3 exposure can induce many bird dysfunction. Neutrophils respond to multiple invading pathogens through different mechanisms. In order to investigate the effect of NH3 exposure on broilers' neutrophil, 1-day-old broilers were treated with/without NH3 for 28 days. We extracted neutrophils from peripheral blood of chicken with/without NH3 exposure and subsequently stimulated with PMA. Changes of cytokines and inflammatory bodies, heat shock proteins (HSPs), and glucose metabolism of neutrophil were examined in both cases. We not only explored that the index associated with inflammation changed due to NH3 exposure but also observed the status of neutrophils which was treated with PMA stimulation. After NH3 exposure, IL-1ß and IL-6 were significantly increased on broilers neutrophil. Inflammatory-related factors (NLRP3, ASC, and caspase-1) were significantly elevated. The mRNA expression of HSP70 and HSP90 was increased significantly. All glucose metabolism indicators were reduced. In summary, we concluded that NH3 enhanced inflammation and disrupted glucose metabolism, and increased the expression of HSPs and inflammatory factors. In addition, the sensitivity of neutrophils to exogenous stimuli was diminished. This information can not only be used to evaluate the damage of NH3-spiked neutrophils to chickens, but also provide clues for human health pathophysiology caused by excess NH3, providing valuable information for NH3 risk management.


Assuntos
Amônia/toxicidade , Proteínas Aviárias/genética , Citocinas/metabolismo , Glucose/metabolismo , Proteínas de Choque Térmico/genética , Inflamação/veterinária , Neutrófilos/efeitos dos fármacos , Doenças das Aves Domésticas/genética , Animais , Proteínas Aviárias/metabolismo , Galinhas , Citocinas/genética , Feminino , Fertilizantes/análise , Fertilizantes/toxicidade , Transtornos do Metabolismo de Glucose/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Inflamação/etiologia , Inflamação/genética , Inflamação/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Masculino , Neutrófilos/metabolismo , Doenças das Aves Domésticas/etiologia , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/metabolismo
17.
MAbs ; 11(3): 532-545, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30735467

RESUMO

In antibody discovery, in-depth analysis of an antibody library and high-throughput retrieval of clones in the library are crucial to identifying and exploiting rare clones with different properties. However, existing methods have technical limitations, such as low process throughput from the laborious cloning process and waste of the phenotypic screening capacity from unnecessary repetitive tests on the dominant clones. To overcome the limitations, we developed a new high-throughput platform for the identification and retrieval of clones in the library, TrueRepertoire™. This new platform provides highly accurate sequences of the clones with linkage information between heavy and light chains of the antibody fragment. Additionally, the physical DNA of clones can be retrieved in high throughput based on the sequence information. We validated the high accuracy of the sequences and demonstrated that there is no platform-specific bias. Moreover, the applicability of TrueRepertoire™ was demonstrated by a phage-displayed single-chain variable fragment library targeting human hepatocyte growth factor protein.


Assuntos
Proteínas Aviárias , Técnicas de Visualização da Superfície Celular/métodos , Anticorpos de Cadeia Única , Animais , Proteínas Aviárias/biossíntese , Proteínas Aviárias/química , Proteínas Aviárias/genética , Bacteriófagos/genética , Galinhas , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética
18.
Dev Comp Immunol ; 95: 89-95, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30753854

RESUMO

The chicken yolk sac (YS) plays an important role in nutrient absorption and immune function for the developing embryo. The avian ß-defensins (AvBD) are cationic peptides that are important members of the innate immune system. The objective of this study was to profile AvBD mRNA expression patterns and distribution of cells expressing AvBD mRNA in the chicken YS. Expression of AvBD1, 2, 7, and 10 mRNA was low at embryonic day 7 (e7), increased to e9 through e13 and then declined to e19. Using in situ hybridization, AvBD10 mRNA was found to be expressed in endodermal epithelial cells, while AvBD1, 2, and 7 mRNA were expressed in heterophils. The developmental expression pattern and distribution of AvBD mRNA in the YS reveals the importance of these genes to protection of the developing chick embryo.


Assuntos
Proteínas Aviárias/genética , Desenvolvimento Embrionário/imunologia , Regulação da Expressão Gênica no Desenvolvimento/imunologia , Saco Vitelino/imunologia , beta-Defensinas/genética , Animais , Proteínas Aviárias/imunologia , Embrião de Galinha , Galinhas , Endoderma/citologia , Endoderma/imunologia , Endoderma/metabolismo , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , RNA Mensageiro/metabolismo , Saco Vitelino/crescimento & desenvolvimento , Saco Vitelino/metabolismo , beta-Defensinas/imunologia
19.
Prep Biochem Biotechnol ; 49(2): 192-201, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30734625

RESUMO

In this paper, we report a soluble expression based on Escherichia coli and two-step purification of a novel thioredoxin-tagged chicken interferon-α fusion protein (Trx-rChIFN-α) by using pET32a(+) expression system. The mature ChIFN-α gene was amplified by Reverse transcriptase-polymerase chain reaction (RT-PCR) and subcloned into pET-32a (+) vector prior to transformation into Rosetta (DE3) competent cells. After IPTG induction, the recombinant fusion protein was expressed efficiently in the soluble fraction. The protein purification was performed by nickel affinity chromatography and DEAE anion exchange chromatography. The purified product has a purity of 95% with a yield of 47.3 mg/L of culture. The specific activity of the fusion protein reaches to 2.0 × 107 IU/mg as determined in the CEF/VSV titration system. After excision of the Trx tag by enterokinase, the remaining solo protein was confirmed as rChIFN-α protein by SDS-PAGE, N-terminal sequencing and mass spectrometry. The effects of this Trx-rChIFN-α fusion protein against H9N2 influenza virus infection were also evaluated in ovo. The results showed that the Trx-rChIFN-α protein could significantly reduce the hemagglutination titer of H9N2 virus, and the H9N2 viruses HA gene copy numbers. These findings will enable us to produce large amount and bio-active rChIFN-α protein for future applications.


Assuntos
Antivirais/farmacologia , Proteínas Aviárias/farmacologia , Galinhas/genética , Vírus da Influenza A Subtipo H9N2/efeitos dos fármacos , Influenza Aviária/tratamento farmacológico , Interferon-alfa/farmacologia , Animais , Antivirais/química , Antivirais/isolamento & purificação , Antivirais/metabolismo , Proteínas Aviárias/química , Proteínas Aviárias/genética , Proteínas Aviárias/isolamento & purificação , Escherichia coli/genética , Interferon-alfa/química , Interferon-alfa/genética , Interferon-alfa/isolamento & purificação , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia , Tiorredoxinas/química , Tiorredoxinas/genética , Tiorredoxinas/isolamento & purificação , Tiorredoxinas/farmacologia
20.
Sci Total Environ ; 663: 380-386, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30716628

RESUMO

Hydrogen sulfide (H2S) is a toxic gas and one of the air pollutants of great concern. High-concentrated H2S can induce energy metabolism disturbance and apoptosis. However, the mechanism of H2S-induced liver injuries is unknown. Lipopolysaccharide (LPS), the main component of endotoxin, can cause fulminant hepatitis. Here, we evaluated the effects of H2S combined with LPS on the energy metabolism and apoptosis pathway in the liver using a one-day-old chicken as a model. Our results showed that the expression levels of energy metabolism-related genes (AMP-activated protein kinase (AMPK), Hypoxia-inducible factor-1 (HIF-1), aconitase 2 (ACO2), hexokinase1 (HK1), hexokinase 2 (HK2), lactate dehydrogenase A (LDHA), lactate dehydrogenase B (LDHB), phosphofructokinase (PFK), pyruvate kinase (PK) and succinate dehydrogenase B (SDHB)) tended to decrease, that the status of apoptosis increased, and that the expression levels of apoptosis-related genes (caspase3, BCL2, and bax) increased in H2S group, suggesting that H2S exposure disturbed the energy metabolism in the liver and induced hepatocyte apoptosis through the mitochondrial pathway. In addition, H2S combined with the LPS aggravated the level of energy metabolism disorders and apoptosis, indicating that H2S inhalation-induced energy metabolism disturbance is involved in LPS-mediated hepatocyte apoptosis through the mitochondrial pathway.


Assuntos
Apoptose/efeitos dos fármacos , Galinhas/metabolismo , Metabolismo Energético/efeitos dos fármacos , Sulfeto de Hidrogênio/efeitos adversos , Animais , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Galinhas/genética , Hepatócitos/efeitos dos fármacos , Hepatócitos/fisiologia , Exposição por Inalação/análise , Lipopolissacarídeos/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
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