RESUMO
Biliary obstruction is rarely caused by foreign objects; therefore, the precise diagnosis may be challenging. Even in rare situations, cases of biliary obstruction caused by plant seeds have not been reported previously. To our knowledge, herein, we report the first case of biliary obstruction caused by accumulated plant seeds forming a solid mass with inflammatory cells and bile juice, which were identified as Solanum lycopersicum, Brassica, and Citrus species by DNA analysis and pathological assessment of the specimen after surgical resection for biliary obstruction suggestive of cholangiocarcinoma.
Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Colestase , Humanos , Colangiocarcinoma/diagnóstico , Neoplasias dos Ductos Biliares/cirurgia , Colestase/etiologia , Ductos Biliares Intra-Hepáticos/patologia , Sementes/efeitos adversosRESUMO
Pulmonary arterial hypertension (PAH) is a fatal disease, which is characterized by occlusive pulmonary vascular disease (PVD) in small pulmonary arteries. It remains unknown whether perinatal insults aggravate occlusive PVD later in life. We tested the hypothesis that perinatal hypoxia aggravates PVD and survival in rats. PVD was induced in rats with/without perinatal hypoxia (embryonic day 14 to postnatal day 3) by injecting SU5416 at 7 wk of age and subsequent exposure to hypoxia for 3 wk (SU5416/hypoxia). Hemodynamic and morphological analyses were performed in rats with/without perinatal hypoxia at 7 wk of age (baseline rats, n = 12) and at 15 wk of age in 4 groups of rats: SU5416/hypoxia or control rats with/without perinatal hypoxia (n = 40). Pulmonary artery smooth muscle cells (PASMCs) from the baseline rats with/without perinatal hypoxia were used to assess cell proliferation, inflammation, and genomic DNA methylation profile. Although perinatal hypoxia alone did not affect survival, physiological, or pathological parameters at baseline or at the end of the experimental period in controls, perinatal hypoxia decreased weight gain and survival rate and increased right ventricular systolic pressure, right ventricular hypertrophy, and indices of PVD in SU5416/hypoxia rats. Perinatal hypoxia alone accelerated the proliferation and inflammation of cultured PASMCs from baseline rats, which was associated with DNA methylation. In conclusion, we established the first fatal animal model of PAH with worsening hemodynamics and occlusive PVD elicited by perinatal hypoxia, which was associated with hyperproliferative, proinflammatory, and epigenetic changes in cultured PASMCs. These findings provide insights into the treatment and prevention of occlusive PVD.
Assuntos
Hipertensão Pulmonar , Hipertensão Arterial Pulmonar , Doenças Vasculares , Animais , Proliferação de Células , Modelos Animais de Doenças , Hipertensão Pulmonar Primária Familiar , Hipertensão Pulmonar/patologia , Hipóxia , Indóis , Inflamação/patologia , Artéria Pulmonar/patologia , Pirróis , Ratos , Doenças Vasculares/patologiaRESUMO
AIMS: GJB4 encodes a transmembrane connexin protein (Cx30.3) that is a component of gap junctions. This study investigated whether GJB4 plays an important role in human heart disease and function. METHODS AND RESULTS: We examined a patient and her older brother who both presented with complicated severe hypertrophic cardiomyopathy (HCM) and whose parents are healthy married cousins. The gene exome analysis showed 340 single nucleotide polymorphisms (SNPs) that caused amino acid changes for which the patient was homozygous and both parents were heterozygous. After excluding all known common (>10%) SNP gene mutations, the gene for GJB4 was the only identified gene that is possibly associated with cardiac muscle. The resultant E204A substitution exists in the 4th transmembrane domain. GJB4-E204A impaired the binding with gap junction protein A1 (GJA1) compared with GJB4-WT. The expression of GJB4 was induced in rat disease models of left and right ventricle hypertrophy and mouse disease models of adriamycin-induced cardiomyopathy and myocardial infarction, while it was not detected at all in control. An immunohistochemical study was performed for autopsied human hearts and the explanted heart of the patient. GJB4 was expressed and colocalized with GJA1 in intercalated discs in human diseased hearts, which was extensively enhanced in the explanted heart of the patient. The abnormal expression and localization of GJB4 were observed in beating spheres of patient's induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs). We generated knockout zebrafish of GJB4 by CRISPR/Cas9 and the endodiastolic volume and the ventricular ejection fraction were significantly lower in GJB4-deficient than in wild-type zebrafish at five days post-fertilization. CONCLUSIONS: These results indicate both that GJB4 is defined as a new connexin in diseased hearts, of which mutation can cause a familial form of HCM, and that GJB4 may be a new target for the treatment of cardiac hypertrophy and dysfunction.
Assuntos
Cardiomiopatia Hipertrófica Familiar/genética , Conexinas/genética , Infarto do Miocárdio/patologia , Miocárdio/patologia , Adulto , Substituição de Aminoácidos , Angiotensina II/toxicidade , Animais , Animais Geneticamente Modificados , Células COS , Cardiomiopatia Hipertrófica Familiar/diagnóstico , Cardiomiopatia Hipertrófica Familiar/patologia , Cardiomiopatia Hipertrófica Familiar/cirurgia , Criança , Chlorocebus aethiops , Conexina 43/metabolismo , Conexinas/metabolismo , Análise Mutacional de DNA , Modelos Animais de Doenças , Doxorrubicina/toxicidade , Feminino , Junções Comunicantes/patologia , Técnicas de Inativação de Genes , Testes Genéticos , Transplante de Coração , Humanos , Células-Tronco Pluripotentes Induzidas , Masculino , Camundongos , Infarto do Miocárdio/etiologia , Miocárdio/citologia , Miócitos Cardíacos , Linhagem , Cultura Primária de Células , Domínios Proteicos/genética , Ratos , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Self-incompatibility (SI) is a sophisticated system for pollen selectivity to prevent pollination by genetically identical pollen. In Brassica, it is genetically controlled by a single, highly polymorphic S-locus, and the male and female S-determinant factors have been identified as S-locus protein 11 (SP11)/S-locus cysteine-rich protein (SCR) and S-locus receptor kinase (SRK), respectively. However, the overall molecular system and identity of factors in the downstream cascade of the SI reaction remain unclear. Previously, we identified a self-compatible B. rapa mutant line, TSC28, which has a disruption in an unidentified novel factor of the SI signaling cascade. Here, in a genetic analysis of TSC28, using an F2 population from a cross with the reference B. rapa SI line Chiifu-401, the causal gene was mapped to a genetic region of DNA containing markers BrSA64 and ACMP297 in B. rapa chromosome A1. By fine mapping using an F2 population of 1,034 plants, it was narrowed down to a genetic region between DNA markers ACMP297 and BrgMS4028, with physical length approximately 1.01 Mbp. In this genomic region, 113 genes are known to be located and, among these, we identified 55 genes that were expressed in the papilla cells. These are candidates for the gene responsible for the disruption of SI in TSC28. This list of candidate genes will contribute to the discovery of a novel downstream factor in the SP11-SRK signaling cascade in the Brassica SI system.
Assuntos
Brassica rapa/genética , Glicoproteínas/genética , Proteínas de Plantas/genética , Pólen/genética , Polinização/genética , Sequência de Aminoácidos/genética , Brassica rapa/crescimento & desenvolvimento , Flores/genética , Flores/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas/genética , Haplótipos/genética , Proteínas Mutantes/genética , Especificidade de Órgãos/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Pólen/crescimento & desenvolvimento , Alinhamento de Sequência , Análise de Sequência de RNARESUMO
[This corrects the article DOI: 10.1371/journal.pone.0197165.].
RESUMO
Crop rotation and intercropping with Allium plants suppresses Fusarium wilt in various crops. However, the mechanisms underlying this phenomenon have not been fully elucidated. This study was designed to assess the role of microorganisms inhabiting Allium rhizospheres and antifungal compounds produced by Allium roots in Fusarium wilt suppression by Allium cultivation. Suppression of cucumber Fusarium wilt and the pathogen multiplication by Allium (Welsh onion and/or onion)-cultivated soils were eliminated by heat treatment at 60 °C, whereas those by Welsh onion-root extract were lost at 40 °C. The addition of antibacterial antibiotics eliminated the suppressive effect of Welsh onion-cultivated soil on pathogen multiplication, suggesting the contribution of antagonistic gram-negative bacteria to the soil suppressiveness. The Illumina MiSeq sequencing of 16S rRNA gene amplicons revealed that genus Flavobacterium was the predominant group that preferentially accumulated in Allium rhizospheres. Flavobacterium species recovered from the rhizosphere soils of these Allium plants suppressed Fusarium wilt on cucumber seedlings. Furthermore, confocal laser scanning microscopy revealed that Flavobacterium isolates inhibited the multiplication of the pathogen in soil. Taken together, we infer that the accumulation of antagonistic Flavobacterium species plays a key role in Fusarium wilt suppression by Allium cultivation.
Assuntos
Allium/crescimento & desenvolvimento , Cucumis sativus/crescimento & desenvolvimento , Flavobacterium/crescimento & desenvolvimento , Fusarium/crescimento & desenvolvimento , Doenças das Plantas/prevenção & controle , Allium/microbiologia , Produtos Agrícolas , Cucumis sativus/microbiologia , DNA Ribossômico/genética , Flavobacterium/genética , Flavobacterium/isolamento & purificação , Temperatura Alta , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/microbiologia , RNA Ribossômico 16S/genética , Microbiologia do SoloRESUMO
DBP5, also known as DDX19, GLE1 and inositol hexakisphosphate (IP6) function in messenger RNA (mRNA) export at the cytoplasmic surface of the nuclear pore complex in eukaryotic cells. DBP5 is a DEAD-box RNA helicase, and its activity is stimulated by interactions with GLE1 and IP6. In addition, these three factors also have unique role(s). To investigate how these factors influenced the cytoplasmic mRNA expression and cell phenotype change, we performed RNA microarray analysis to detect the effect and function of DBP5, GLE1 and IP6 on the cytoplasmic mRNA expression. The expression of some cytoplasmic mRNA subsets (e.g. cell cycle, DNA replication) was commonly suppressed by the knock-down of DBP5, GLE1 and IPPK (IP6 synthetic enzyme). The GLE1 knock-down selectively reduced the cytoplasmic mRNA expression required for mitotic progression, results in an abnormal spindle phenotype and caused the delay of mitotic process. Meanwhile, G1/S cell cycle arrest was observed in DBP5 and IPPK knock-down cells. Several factors that function in immune response were also down-regulated in DBP5 or IPPK knock-down cells. Thereby, IFNß-1 mRNA transcription evoked by poly(I:C) treatment was suppressed. These results imply that DBP5, GLE1 and IP6 have a conserved and individual function in the cytoplasmic mRNA expression. Variations in phenotype are due to the difference in each function of DBP5, GLE1 and IPPK in intracellular mRNA metabolism.
Assuntos
RNA Helicases DEAD-box/metabolismo , Fase G1 , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ácido Fítico/metabolismo , RNA Mensageiro/metabolismo , Fase S , Transporte Biológico Ativo/genética , Citoplasma/genética , Citoplasma/metabolismo , RNA Helicases DEAD-box/genética , Células HeLa , Humanos , Interferon beta/genética , Interferon beta/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , RNA Mensageiro/genéticaRESUMO
Cassava bacterial blight (CBB) disease caused by Xanthomonas axonopodis pv. manihotis (Xam) is a severe disease in cassava worldwide. In addition to causing significant cassava yield loss, CBB disease has not been extensively studied, especially in terms of CBB resistance genes. The present research demonstrated the molecular mechanisms underlining the defense response during Xam infection in two cassava cultivars exhibiting different degrees of disease resistance, Huay Bong60 (HB60) and Hanatee (HN). Based on gene expression analysis, ten of twelve putative defense-related genes including, leucine-rich repeat receptor-like kinases (LRR-RLKs), resistance (R), WRKY and pathogenesis-related (PR) genes, were differentially expressed between these two cassava cultivars during Xam infection. The up-regulation of defense-related genes observed in HB60 may be the mechanism required for the reduction of disease severity in the resistant cultivar. Interestingly, priming with salicylic acid (SA) or methyl jasmonate (MeJA) for 24 h before Xam inoculation could enhance the defense response in both cassava cultivars. The disease severity was decreased 10% in the resistant cultivar (HB60) and was remarkably reduced 21% in the susceptible cultivar (HN) by SA/MeJA priming. Priming with Xam inoculation modulated cassava4.1_013417, cassava4.1_030866 and cassava4.1_020555 (highest similarity to MeWRKY59, MePR1 and AtPDF2.2, respectively) expression and led to enhanced resistance of the susceptible cultivar in the second infection. The putative cis-regulatory elements were predicted in an upstream region of these three defense-related genes. The different gene expression levels in these genes between the two cultivars were due to the differences in cis-regulatory elements in their promoter regions. Taken together, our study strongly suggested that the induction of defense-related genes correlated with defense resistance against Xam infection, and exogenous application of SA or MeJA could elevate the defense response in both cultivars of cassava. This finding should pave the way for management to reduce yield loss from disease and genetic improvement in cassava.
Assuntos
Resistência à Doença/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Manihot , Fitocromo/farmacologia , Doenças das Plantas/microbiologia , Transcrição Gênica/efeitos dos fármacos , Xanthomonas axonopodis/crescimento & desenvolvimento , Manihot/metabolismo , Manihot/microbiologiaRESUMO
The discovery of biomarkers to predict the potential for lymph node (LN) metastasis in patients with colorectal cancer (CRC) is essential for developing improved strategies for treating CRC. In the present study, they used isobaric tags for relative and absolute quantitation to conduct a proteomic analysis designed to identify novel biomarkers for predicting LN metastasis in patients with CRC. They identified 60 differentially expressed proteins specifically associated with LN metastasis in CRC patients and classified the molecular and functional characteristics of these proteins by bioinformatic approaches. A literature search led them to select heat shock protein 47 (HSP47) as the most suitable candidate biomarker for predicting LN metastasis. Validation analysis by immunohistochemistry showed that HSP47 expression in patients with CRC and the number of HSP47-positive spindle cells in the tumor stroma were significantly higher compared with those in adjacent normal colonic mucosa, and the number of the latter cells increased with tumor progression. Further, the number of HSP47-positive spindle cells in stroma was a more informative marker for identifying LN metastasis than HSP47expression. Multivariate analysis identified spindle cells that expressed elevated levels of HSP47 as an independent predictive biomarker for CRC with LN metastasis. Moreover, these cells served as an independent marker of disease-free and overall survival of patients with CRC. Their data indicate that the number of HSP47-positive spindle cells in the stroma of CRC may serve as a novel predictive biomarker of LN metastasis, early recurrence and poor prognosis.
Assuntos
Adenocarcinoma/química , Neoplasias Colorretais/química , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico HSP47/análise , Metástase Linfática/genética , Proteínas de Neoplasias/análise , Proteômica/métodos , Adenocarcinoma/genética , Adenocarcinoma/mortalidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Colo/química , Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Intervalo Livre de Doença , Feminino , Genes ras , Proteínas de Choque Térmico HSP47/biossíntese , Proteínas de Choque Térmico HSP47/genética , Humanos , Mucosa Intestinal/química , Estimativa de Kaplan-Meier , Masculino , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Modelos de Riscos Proporcionais , Proteínas Proto-Oncogênicas B-raf/genética , Células Estromais/químicaRESUMO
Colorectal cancer (CRC)-associated mortality is primarily caused by lymph node (LN) and distant metastasis, highlighting the need for biomarkers that predict LN metastasis and facilitate better therapeutic strategies. We used an Isobaric Tags for Relative and Absolute Quantification (iTRAQ)-based comparative proteomics approach to identify novel biomarkers for predicting LN metastasis in CRC patients. We analyzed five paired samples of CRC with or without LN metastasis, adjacent normal mucosa, and normal colon mucosa, and differentially expressed proteins were identified and subsequently validated at the protein and/or mRNA levels by immunohistochemistry and qRT-PCR, respectively. We identified 55 proteins specifically associated with LN metastasis, from which we selected ezrin for further analysis and functional assessment. Expression of ezrin at both the protein and mRNA levels was significantly higher in CRC tissues than in adjacent normal colonic mucosa. In univariate analysis, high ezrin expression was significantly associated with tumor progression and poor prognosis, which was consistent with our in vitro findings that ezrin promotes the metastatic capacity of CRC cells by enabling cell invasion and migration. In multivariate analysis, high levels of ezrin protein and mRNA in CRC samples were independent predictors of LN metastasis. Our data thus identify ezrin as a novel protein and mRNA biomarker for predicting LN metastasis in CRC patients.
RESUMO
BACKGROUND: Malignant potential in unfavorable neuroblastoma (NB) is dependent on an undifferentiated status. The aim of this study was to identify a novel biomarker associated with the undifferentiated status of NB in vitro and to evaluate its prognostic implication. PROCEDURE: Shotgun proteomic analysis was performed in undifferentiated and all trans-retinoic acid induced differentiated NB cells in vitro. An identified protein was verified by multiple reaction monitoring (MRM) and evaluated by Western blot analysis. Immunohistochemistry (IHC) was used to examine the expression of the identified protein in 33 primary NB tissues. RESULTS: Twelve proteins, including ATP-dependent RNA helicase (DDX39A), were only detected in undifferentiated NB cells. A peptide of DDX39A was detected at a significantly higher level in undifferentiated IMR-32 (P = 0.002) and LA-N-1 (P < 0.001) cells by MRM. Western blot analysis revealed that DDX39A expression was significantly higher in undifferentiated IMR-32 (P = 0.02) and LA-N-1 (P = 0.025) cells. IHC demonstrated that DDX39A was highly expressed in the primary tumor tissues of patients with poor prognosis, and univariate and multivariate survival analyses showed that DDX39A expression could be an independent unfavorable prognostic factor (P = 0.027). CONCLUSIONS: DDX39A is a potential biomarker for unfavorable NB using a proteomic approach. Evaluation of DDX39A protein expression in NB tumor tissues may provide complementary prognostic information for further subclassification of these tumors.
Assuntos
Biomarcadores Tumorais/análise , RNA Helicases DEAD-box/biossíntese , Neuroblastoma/patologia , Proteômica/métodos , Western Blotting , Diferenciação Celular , Criança , Pré-Escolar , RNA Helicases DEAD-box/análise , Feminino , Humanos , Imuno-Histoquímica , Lactente , Recém-Nascido , Estimativa de Kaplan-Meier , Masculino , Neuroblastoma/mortalidade , Prognóstico , Espectrometria de Massas em TandemRESUMO
Naturally occurring autoantibodies have natural physiologic functions related to normal cell processes. However, the repertoire of naturally occurring autoantibodies against neuronal antigens in CSF is unclear. The purpose of this study was to identify naturally occurring autoantibodies against neuronal antigens in CSF from patients with various neurologic diseases by proteomics-based analysis. The CSF samples were collected from 77 patients with various neurologic disorders. The antigen source for 2-dimensional immunoblotting was the SH-SY5Y human neuroblastoma cell line. There were 8 spots recognized in CSF from more than one-fourth of the 77 patients including all patient groups and these spots were recognized in intravenous immunoglobulin preparations. These antigen spots were identified as heat shock 105-kDa/110-kDa protein 1, isoform CRA_b, 78-kDa glucose-regulated protein, heat shock cognate 71-kDa protein, tubulin beta chain, vimentin (2 spots), and 60-kDa heat shock protein, mitochondrial; we could not identify the protein name corresponding to 1 of the 8 spots. In summary, there were 6 proteins identified that were main target antigens that reacted with naturally occurring autoantibodies in CSF from patients with varied neurologic disorders; the functions of autoantibodies against the identified antigens are unknown and may be clarified with further studies. BIOLOGICAL SIGNIFICANCE: Naturally occurring autoantibodies may have important functions in tissue homeostasis. In this study, we identified 6 common target antigens that reacted with autoantibodies in cerebrospinal fluid (CSF) from patients, independent of disease type. These findings may clarify the importance of naturally occurring autoantibodies in CSF and the use of these antibodies potentially may be a novel therapy for various neurologic disorders.
Assuntos
Antígenos/química , Antígenos/imunologia , Autoanticorpos/química , Autoanticorpos/imunologia , Líquido Cefalorraquidiano/imunologia , Doenças do Sistema Nervoso/líquido cefalorraquidiano , Líquido Cefalorraquidiano/química , Feminino , Humanos , Immunoblotting/métodos , MasculinoRESUMO
Stage-specific transcription is a fundamental biological process in the life cycle of the Plasmodium parasite. Proteins containing the AP2 DNA-binding domain are responsible for stage-specific transcriptional regulation and belong to the only known family of transcription factors in Plasmodium parasites. Comprehensive identification of their target genes will advance our understanding of the molecular basis of stage-specific transcriptional regulation and stage-specific parasite development. AP2-O is an AP2 family transcription factor that is expressed in the mosquito midgut-invading stage, called the ookinete, and is essential for normal morphogenesis of this stage. In this study, we identified the genome-wide target genes of AP2-O by chromatin immunoprecipitation-sequencing and elucidate how this AP2 family transcription factor contributes to the formation of this motile stage. The analysis revealed that AP2-O binds specifically to the upstream genomic regions of more than 500 genes, suggesting that approximately 10% of the parasite genome is directly regulated by AP2-O. These genes are involved in distinct biological processes such as morphogenesis, locomotion, midgut penetration, protection against mosquito immunity and preparation for subsequent oocyst development. This direct and global regulation by AP2-O provides a model for gene regulation in Plasmodium parasites and may explain how these parasites manage to control their complex life cycle using a small number of sequence-specific AP2 transcription factors.
Assuntos
Regulação da Expressão Gênica , Genoma de Protozoário , Malária/genética , Plasmodium berghei/genética , Proteínas de Protozoários/genética , Fator de Transcrição AP-2/genética , Sequência de Aminoácidos , Animais , Imunoprecipitação da Cromatina , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Estágios do Ciclo de Vida , Malária/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Oocistos/crescimento & desenvolvimento , Oocistos/metabolismo , Oocistos/parasitologia , Plasmodium berghei/crescimento & desenvolvimento , Plasmodium berghei/isolamento & purificação , Proteínas de Protozoários/metabolismo , RNA de Protozoário , Homologia de Sequência de Aminoácidos , Fator de Transcrição AP-2/metabolismoRESUMO
With the aim of understanding relationship between genetic and phenotypic variations in cultivated tomato, single nucleotide polymorphism (SNP) markers covering the whole genome of cultivated tomato were developed and genome-wide association studies (GWAS) were performed. The whole genomes of six tomato lines were sequenced with the ABI-5500xl SOLiD sequencer. Sequence reads covering ~13.7× of the genome for each line were obtained, and mapped onto tomato reference genomes (SL2.40) to detect ~1.5 million SNP candidates. Of the identified SNPs, 1.5% were considered to confer gene functions. In the subsequent Illumina GoldenGate assay for 1536 SNPs, 1293 SNPs were successfully genotyped, and 1248 showed polymorphisms among 663 tomato accessions. The whole-genome linkage disequilibrium (LD) analysis detected highly biased LD decays between euchromatic (58 kb) and heterochromatic regions (13.8 Mb). Subsequent GWAS identified SNPs that were significantly associated with agronomical traits, with SNP loci located near genes that were previously reported as candidates for these traits. This study demonstrates that attractive loci can be identified by performing GWAS with a large number of SNPs obtained from re-sequencing analysis.
Assuntos
Genoma de Planta , Polimorfismo de Nucleotídeo Único , Solanum lycopersicum/genética , Produtos Agrícolas/genética , Loci Gênicos , Marcadores Genéticos/fisiologia , Estudo de Associação Genômica Ampla , Genótipo , Desequilíbrio de Ligação , Análise de Sequência de DNARESUMO
Induced penetration resistance is triggered by failed penetration attempts of nonpathogenic fungi. The resistance mechanism is an important nonhost reaction in plants that can block the invasion of filamentous pathogens such as fungi and oomycetes. However, it remains unclear whether the mechanical stimuli accompanying fungal penetration play a role in induced penetration resistance, whereas the perforation of the cell wall may provide significant stimuli to plant cells. Here, we used microneedles or biolistic bombardment to mimic fungal penetration pegs and a micromanipulation transfer technique of the bio-probe, a germling of Blumeria graminis hordei, to the wounded cells to demonstrate that microwounds derived from fungal penetration attempts may trigger induced penetration resistance in plant cells. When preinoculated with the nonpathogenic fungi Erysiphe pisi and Colletotrichum orbiculare, which were unable to penetrate a barley cell, the penetration of a bio-probe that was transferred by micromanipulation onto the same cell was completely blocked. Fungal penetration was essential to the triggering of induced penetration resistance because a penetration-peg-defective mutant of C. orbiculare completely lacked the ability to trigger resistance. The artificial microwounds significantly, but not completely, blocked the penetration of the bio-probe. Treatment with the actin polymerization inhibitor cytochalasin A or expression of the actin depolymerizing protein HvPro1 caused complete ablation of the induced penetration resistance triggered by either failed fungal penetration or artificial microwounds. These results strongly suggest that microwounding may trigger actin-dependent induced penetration resistance. Manipulation of induced penetration resistance may be a promising target to improve basic disease resistance in plants.
Assuntos
Actinas/metabolismo , Colletotrichum/patogenicidade , Hordeum/microbiologia , Doenças das Plantas/microbiologiaRESUMO
The crystal structure of Acidithiobacillus thiooxidans isocitrate dehydrogenase complexed with NAD+ and citrate has been solved to a resolution of 1.9 A. The protein fold of this NAD+-dependent enzyme shares a high similarity with those of NADP+-dependent bacterial ICDHs. The NAD+ and the citrate are clearly identified in the active site cleft with a well-defined electron density. Asp-357 is the direct cofactor-specificity determinant that interacts with 2'-OH and 3'-OH of the adenosine ribose. The adenosine ribose takes a C2'-endo puckering conformation as previously reported for an NAD+-specific isopropylmalate dehydrogenase. The nicotinamide moiety of NAD+ has the amide NH2 group oriented in cis conformation with respect to the C4 carbon of the nicotinamide ring, slanted toward the bound citrate molecule with a dihedral angle of -21 degrees . The semi-empirical molecular orbital calculation suggests that the pro-R hydrogen atom at C4 of NADH would bear the largest negative charge when the amide NH2 group is in such conformation, suggesting that the amide group has a catalytically significant role in stabilizing the transition state as NADH is being formed during the hydride transfer catalysis.
Assuntos
Hidrogênio/química , Isocitrato Desidrogenase/química , NAD/química , Teoria Quântica , Sequência de Aminoácidos , Cristalografia por Raios X , Ligação de Hidrogênio , Isocitrato Desidrogenase/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Catalase plays a key role in protecting cells against toxic reactive oxygen species. Here we report on the cloning, purification and characterization of a catalase (KatA, DR1998) from the extremely radioresistant bacterium Deinococcus radiodurans. The size of purified D. radiodurans KatA monomer was 65 kDa while gel filtration revealed that the size of the enzyme was 240 kDa, suggesting that KatA formed a homotetramer in solution. Purified KatA displayed a final specific activity of 68,800 U/mg of protein. The catalase activity of KatA was inhibited by sodium azide, sodium cyanide and 3-amino-1,2,4-triazole. The absorption spectrum of KatA exhibited a Soret band at 408 nm. The position of the spectral peak remained unchanged following reduction of KatA with dithionite. No peroxidase activity was found for KatA. These results demonstrate that D. radiodurans KatA is a typical monofunctional heme-containing catalase. The stability of KatA with respect to H2O2 stress was superior to that of commercially available Aspergillus niger and bovine liver catalases. The relative abundance of KatA in cells in addition to the H2O2 resistance property may play a role in the survival strategy of D. radiodurans against oxidative damage.
Assuntos
Catalase/química , Deinococcus/enzimologia , Sequência de Aminoácidos , Cromatografia em Gel , Clonagem Molecular , DNA Bacteriano/química , Escherichia coli/metabolismo , Peróxido de Hidrogênio/química , Dados de Sequência Molecular , Plasmídeos/metabolismo , Proteínas/química , Homologia de Sequência de AminoácidosRESUMO
The actin cytoskeleton is a key player in defense responses during early stages of infection by fungal pathogens. To investigate molecular mechanisms of actin-related defense responses, a cultured tobacco ( Nicotiana tabacum L.) BY-2 cell system was devised. When conidia were directly deposited on BY-2 cells, neither a pathogen, Erysiphe cichoracearum, nor a non-pathogen, Erysiphe pisi, was able to form appressoria or haustoria on BY-2 cells. On the other hand, conidia of the powdery mildews formed appressoria on BY-2 cells if they were covered with a thin hydrophobic membrane of Formvar. Percentages of appressoria formation of the powdery mildews on the Formvar-covered BY-2 cells were mostly the same as those on leaf epidermal cells. The pathogen successfully penetrated through the membrane into BY-2 cells and formed haustoria, whereas penetration attempts of the non-pathogen were completely rejected by the BY-2 cells similar to attempts on leaf epidermal cells. On the other hand, when BY-2 cells were treated with actin cytoskeleton-depolymerizing agents, cytochalasins, the non-pathogen became able to penetrate and form haustoria in BY-2 cells. Simultaneously, cytochalasin inhibited callose deposition at penetration sites of the non-pathogen. These results demonstrated that the actin cytoskeleton plays an important role in defense mechanisms against fungal penetration, even in the dedifferentiated cultured cells. The newly devised Formvar-covered cultured cell system will be a useful tool for molecular dissection of signal perception and defense mechanisms of plant cells during the early stage of fungal attack.