RESUMO
Faithful chromosome segregation is required for both mitotic and meiotic cell divisions and is regulated by multiple mechanisms including the anaphase-promoting complex/cyclosome (APC/C), which is the largest known E3 ubiquitin-ligase complex and has been implicated in regulating chromosome segregation in both mitosis and meiosis in animals. However, the role of the APC/C during plant meiosis remains largely unknown. Here, we show that Arabidopsis APC8 is required for male meiosis. We used a combination of genetic analyses, cytology and immunolocalisation to define the function of AtAPC8 in male meiosis. Meiocytes from apc8-1 plants exhibit several meiotic defects including improper alignment of bivalents at metaphase I, unequal chromosome segregation during anaphase II, and subsequent formation of polyads. Immunolocalisation using an antitubulin antibody showed that APC8 is required for normal spindle morphology. We also observed mitotic defects in apc8-1, including abnormal sister chromatid segregation and microtubule morphology. Our results demonstrate that Arabidopsis APC/C is required for meiotic chromosome segregation and that APC/C-mediated regulation of meiotic chromosome segregation is a conserved mechanism among eukaryotes.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Subunidade Apc8 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Meiose , Subunidade Apc8 do Ciclossomo-Complexo Promotor de Anáfase/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos , Cromossomos de Plantas/genética , Sequência Conservada , Variação Genética , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Mitose , Modelos Biológicos , Fenótipo , Mutação Puntual/genética , Fuso Acromático/metabolismo , CoesinasRESUMO
Plant cytochrome P450 enzymes play vital roles in the biosynthesis of plant secondary metabolites, including phenylpropanoids and phytoalexins. Isoflavone-2'-hydroxylase (AmI2'H) from Astragalus membranaceus Bge. Var. mongolicus (Bge.) Hsiao is a membrane protein and an eukaryotic cytochrome P450 enzyme involved in isoflavonoid biosynthesis. We cloned the AmI2'H gene by employing RACE methods and modified the gene sequence to facilitate protein expression and increase protein solubility. Two vectors, pET-28a(+) and pCW ori(+), were used to express AmI2'H in Escherichia coli. The expression efficiency and purity of target protein were analyzed and demonstrated that pET-28a(+) vector containing the AmI2'H gene could produce larger amounts of target proteins with higher purity than pCWori(+). The purified proteins were identified as AmI2'H by LC-ESI-MS/MS analysis and their proper folding was assessed by CO difference spectrum.
Assuntos
Astragalus propinquus/enzimologia , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/isolamento & purificação , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Sequência de Aminoácidos , Astragalus propinquus/química , Astragalus propinquus/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Dados de Sequência Molecular , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Alinhamento de SequênciaRESUMO
A chalcone reductase (CHR) gene was isolated from Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao (A. mongholicus). The full-length cDNA of A. mongholicus CHR, designated as Amchr (GenBank accession No. HM357239), was 1196 bp long. It had a 957 bp open reading frame encoding a 318-amino acid protein of 35 kDa, a 67 bp 5' non-coding region and a 172 bp 3'-untranslated region. The putative AmCHR protein showed striking similarity to CHR from other leguminous species. Two-dimensional structure modeling showed that AmCHR consisted of 45.28% α-helix, 10.38% extended strand and 44.34% random coil. Prediction showed that three-dimensional AmCHR was a global protein containing an aldo-ket-red domain, with a putative Asp-Tyr-Lys-His catalytic tetrad in the center. The AmCHR gene was 1251 bp long, consisting of three exons and two introns. Intron I was 125 bp and intron II was 169 bp long. Southern blot analysis indicated that Amchr belonged to a small multigene family. Under natural conditions, Amchr was expressed differentially in the root, stem and leaf tissues of A. mongholicus, with a preferential expression in the root. The recombinant AmCHR protein was successfully expressed in Escherichia coli strain BL21 with pET42a vector. The result showed that the expressed AmCHR protein had molecular weight of about 35 kDa, which matched the size of the predicted protein by bioinformatic analysis. This study opened avenues towards understanding of the function of AmCHR protein and the role of the Amchr gene in the calycosin-7-O-ß-D: -glucoside branch pathway in A. mongholicus.
Assuntos
Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Astragalus propinquus/enzimologia , Sequência de Bases , Southern Blotting , Clonagem Molecular , Biologia Computacional , DNA Complementar/genética , Perfilação da Expressão Gênica , Dados de Sequência Molecular , Estrutura Molecular , Análise de Sequência de DNARESUMO
Isoflavonoids are a group of phenolic secondary metabolites found almost exclusively in leguminous plants. Formononetin, calycosin and calycosin-7-O-ß-d-glucoside (CG) are isoflavonoid products in the CG pathway. Accumulation of the three isoflavonoids plus daidzein and expression of six genes of enzymes involved in the CG pathway were studied in Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao with ultraviolet (UV) irradiation. Our results showed that (1) main isoflavonoids in roots, stems and leaves were CG, daidzein and calycosin, respectively; they accumulated significantly under the induction of UV irradiation during 8 days but their content declined later; (2) expression of six genes of enzymes involved in the CG pathway was inhibited slightly at early stage but the expression was increased greatly afterward; (3) chalcone synthase, chalcone reductase and chalcone isomerase were expressed to their individual maximum level within shorter hours than were cinnamate 4-hydroxylase, isoflavone synthase (IFS) and isoflavone 3'-hydroxylase and (4) more calycosin but less daidzein accumulated in leaves. IFS was highly expressed in leaves, which might lead to high accumulation of the common precursor of daidzein and 2,7-dihydroxy-4'-O-methoxy-isoflavanone, the latter of which would be converted to formononetin, calycosin and CG via a series of reactions. Little daidzein accumulated in leaves, which suggested that rather than be converted to daidzein, the 2,7,4'-trihydroxyisoflavanone was probably more easily caught by 2-hydroxyisoflavanone 4'-O-methyltransferase and hence provided more precursors for formononetin. The findings were discussed in terms of the influence of UV irradiation in the accumulation of isoflavonoids.
Assuntos
Astragalus propinquus/enzimologia , Astragalus propinquus/genética , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Glucosídeos/metabolismo , Isoflavonas/metabolismo , Transcrição Gênica/efeitos da radiação , Raios Ultravioleta , Astragalus propinquus/efeitos da radiação , Vias Biossintéticas/efeitos da radiação , Cromatografia Líquida de Alta Pressão , Genes de Plantas/genética , Redes e Vias Metabólicas/genética , Redes e Vias Metabólicas/efeitos da radiação , Folhas de Planta/genética , Folhas de Planta/efeitos da radiação , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancer types worldwide, with the lowest 5-year survival rate among all kinds of cancers. Histopathology image analysis is considered a gold standard for PDAC detection and diagnosis. However, the manual diagnosis used in current clinical practice is a tedious and time-consuming task and diagnosis concordance can be low. With the development of digital imaging and machine learning, several scholars have proposed PDAC analysis approaches based on feature extraction methods that rely on field knowledge. However, feature-based classification methods are applicable only to a specific problem and lack versatility, so that the deep-learning method is becoming a vital alternative to feature extraction. This paper proposes the first deep convolutional neural network architecture for classifying and segmenting pancreatic histopathological images on a relatively large WSI dataset. Our automatic patch-level approach achieved 95.3% classification accuracy and the WSI-level approach achieved 100%. Additionally, we visualized the classification and segmentation outcomes of histopathological images to determine which areas of an image are more important for PDAC identification. Experimental results demonstrate that our proposed model can effectively diagnose PDAC using histopathological images, which illustrates the potential of this practical application.
RESUMO
A new thermostable ß-glucosidase gene (bgl) from Chaetomium thermophilum CT2 was cloned, sequenced and expressed. The full-length DNA of bgl was 3,101 bp and included three introns. The full-length cDNA contained an open reading frame of 2,604-bp nucleotides, encoding 867 amino acids with a potential secretion signal. The C. thermophilum CT2 ß-glucosidase gene was functionally expressed in Pichia pastoris. The purified recombinant ß-glucosidase was a 119-kDa glycoprotein with an optimum catalytic activity at pH 5.0 and 60°C. The enzyme was stable at 50°C, and retained 67.7% activity after being kept at 60°C for 1 h; the half-time of the enzyme at 65°C was approximately 55 min, and even retained 29.7% activity after incubation at 70°C for 10 min.