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1.
Clin J Gastroenterol ; 15(6): 1158-1163, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36198986

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 adversos
2.
Am J Physiol Lung Cell Mol Physiol ; 323(2): L178-L192, 2022 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-35762603

RESUMO

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/patologia
3.
PLoS One ; 15(10): e0240129, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33048975

RESUMO

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/metabolismo
5.
Sci Rep ; 9(1): 1715, 2019 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-30737419

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 Solo
6.
PLoS One ; 13(5): e0197165, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29746542

RESUMO

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/genética
7.
Oncotarget ; 8(63): 106935-106947, 2017 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-29291001

RESUMO

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.

8.
Int J Cancer ; 140(6): 1425-1435, 2017 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-27925182

RESUMO

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/química
9.
Pediatr Blood Cancer ; 63(2): 221-7, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26469522

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 Tandem
10.
DNA Res ; 20(6): 593-603, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23903436

RESUMO

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 DNA
11.
Plant Cell Physiol ; 54(6): 848-58, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23628996

RESUMO

By using high-resolution two-dimensional PAGE followed by phosphoprotein-specific staining and peptide mass fingerprint analysis along with other assays, we found that α-tubulin is phosphorylated in response to hyperosmotic stress in rice and Arabidopsis. The onset of the phosphorylation response was as early as 2 min after hyperosmotic stress treatment, and a major proportion of α-tubulin was phosphorylated after 60 min in root tissues. However, the phosphorylated form of α-tubulin was readily dephosphorylated upon stress removal. The phosphorylation site was identified as Thr349 by comprehensive mutagenesis of serine/threonine residues in a rice α-tubulin isoform followed by evaluation in cultured cell protoplasts. This residue is located at the surface for the interaction with ß-tubulin in polymerized α-ß tubulin dimers and has been proposed to be directly involved in this interaction. Thus, α-tubulin phosphorylation was considered to occur on free tubulin dimers in response to hyperosmotic stress. The incorporation of green fluorescent protein (GFP)-α-tubulin into cortical microtubules was completely inhibited in transgenic Arabidopsis when Thr349 was substituted with glutamate or aspartate. Using transgenic Arabidopsis plants expressing GFP-α-tubulin, we found that hyperosmotic stress causes extensive cortical microtubule depolymerization. Microtubule-destabilizing treatments such as propyzamide or oryzalin and temperature stresses resulted in α-tubulin phosphorylation, whereas hyperosmotic stress-induced α-tubulin phosphorylation was partially inhibited by taxol, which stabilizes microtubules. These results and the three-dimensional location of the phosphorylation site suggested that microtubules are depolymerized in response to hyperosmotic stress via α-tubulin phosphorylation. Together, the results of the present study reveal a novel mechanism that globally regulates the microtubule polymerization.


Assuntos
Arabidopsis/metabolismo , Oryza/metabolismo , Pressão Osmótica , Proteínas de Plantas/metabolismo , Estresse Fisiológico , Tubulina (Proteína)/metabolismo , Arabidopsis/efeitos dos fármacos , Células Cultivadas , Microtúbulos/metabolismo , Oryza/citologia , Fosfoproteínas/metabolismo , Fosforilação , Fosfotreonina/metabolismo , Polimerização , Multimerização Proteica , Proteômica , Protoplastos/metabolismo , Plântula/metabolismo , Temperatura
12.
Planta ; 237(5): 1187-98, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23328898

RESUMO

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/microbiologia
13.
Plant J ; 44(6): 939-49, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16359387

RESUMO

The plant hormone abscisic acid (ABA) induces gene expression via the ABA-response element (ABRE) present in the promoters of ABA-regulated genes. A group of bZIP proteins have been identified as ABRE-binding factors (ABFs) that activate transcription through this cis element. A rice ABF, TRAB1, has been shown to be activated via ABA-dependent phosphorylation. While a large number of signalling factors have been identified that are involved in stomatal regulation by ABA, relatively less is known about the ABA-signalling pathway that leads to gene expression. We have shown recently that three members of the rice SnRK2 protein kinase family, SAPK8, SAPK9 and SAPK10, are activated by ABA signal as well as by hyperosmotic stress. Here we show that transient overexpression in cultured cell protoplasts of these ABA-activated SnRK2 protein kinases leads to the activation of an ABRE-regulated promoter, suggesting that these kinases are involved in the gene-regulation pathway of ABA signalling. We further show several lines of evidence that these ABA-activated SnRK2 protein kinases directly phosphorylate TRAB1 in response to ABA. Kinetic analysis of SAPK10 activation and TRAB1 phosphorylation indicated that the latter immediately followed the former. TRAB1 was found to be phosphorylated not only in response to ABA, but also in response to hyperosmotic stress, which was interpreted as the consequence of phosphorylation of TRAB1 by hyperosmotically activated SAPKs. Physical interaction between TRAB1 and SAPK10 in vivo was demonstrated by a co-immunoprecipitation experiment. Finally, TRAB1 was phosphorylated in vitro by the ABA-activated SnRK2 protein kinases at Ser102, which is phosphorylated in vivo in response to ABA and is critical for the activation function.


Assuntos
Ácido Abscísico/farmacologia , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Regulação da Expressão Gênica de Plantas , Oryza/enzimologia , Oryza/genética , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Fatores de Transcrição de Zíper de Leucina Básica/química , Fatores de Transcrição de Zíper de Leucina Básica/genética , Ativação Enzimática , Cinética , Dados de Sequência Molecular , Pressão Osmótica , Fosforilação , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas Recombinantes/metabolismo , Elementos de Resposta , Serina/metabolismo , Transdução de Sinais
14.
Plant Cell ; 16(5): 1163-77, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15084714

RESUMO

To date, a large number of sequences of protein kinases that belong to the sucrose nonfermenting1-related protein kinase2 (SnRK2) family are found in databases. However, only limited numbers of the family members have been characterized and implicated in abscisic acid (ABA) and hyperosmotic stress signaling. We identified 10 SnRK2 protein kinases encoded by the rice (Oryza sativa) genome. Each of the 10 members was expressed in cultured cell protoplasts, and its regulation was analyzed. Here, we demonstrate that all family members are activated by hyperosmotic stress and that three of them are also activated by ABA. Surprisingly, there were no members that were activated only by ABA. The activation was found to be regulated via phosphorylation. In addition to the functional distinction with respect to ABA regulation, dependence of activation on the hyperosmotic strength was different among the members. We show that the relatively diverged C-terminal domain is mainly responsible for this functional distinction, although the kinase domain also contributes to these differences. The results indicated that the SnRK2 protein kinase family has evolved specifically for hyperosmotic stress signaling and that individual members have acquired distinct regulatory properties, including ABA responsiveness by modifying the C-terminal domain.


Assuntos
Ácido Abscísico/farmacologia , Oryza/enzimologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Ativação Enzimática , Genoma de Planta , Dados de Sequência Molecular , Oryza/efeitos dos fármacos , Oryza/genética , Concentração Osmolar , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
15.
Plant Cell ; 16(1): 33-44, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14688295

RESUMO

GAMYB was first isolated as a positive transcriptional regulator of gibberellin (GA)-dependent alpha-amylase expression in barley aleurone cells, and its molecular and biochemical properties have been well characterized. However, the role of GAMYB elsewhere in the plant is not well understood. To investigate the molecular function of GAMYB outside of the aleurone cells, we isolated loss-of-function mutants from a panel of rice mutants produced by the insertion of a retrotransposon, Tos17. Through PCR screening using primers for rice GAMYB (OsGAMYB) and Tos17, we isolated three independent mutant alleles that contained Tos17 inserted in the exon region. No alpha-amylase expression in the endosperm was induced in these mutants in response to GA treatment, indicating that the Tos17 insertion had knocked out OsGAMYB function. We found no significant defects in the growth and development of the mutants at the vegetative stage. After the phase transition to the reproductive stage, however, shortened internodes and defects in floral organ development, especially a defect in pollen development, were observed. On the other hand, no difference was detected in flowering time. High-level OsGAMYB expression was detected in the aleurone cells, inflorescence shoot apical region, stamen primordia, and tapetum cells of the anther, but only low-level expression occurred in organs at the vegetative stage or in the elongating stem. These results demonstrate that, in addition to its role in the induction of alpha-amylase in aleurone, OsGAMYB also is important for floral organ development and essential for pollen development.


Assuntos
Flores/enzimologia , Oryza/enzimologia , Proteínas de Plantas/genética , Sementes/enzimologia , Fatores de Transcrição/genética , alfa-Amilases/genética , Elementos de DNA Transponíveis/genética , Elementos de DNA Transponíveis/fisiologia , Indução Enzimática , Flores/genética , Flores/crescimento & desenvolvimento , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Giberelinas/farmacologia , Mutagênese Insercional/métodos , Mutação , Oryza/genética , Oryza/crescimento & desenvolvimento , Fenótipo , Proteínas de Plantas/metabolismo , Reprodução/genética , Reprodução/fisiologia , Sementes/genética , Sementes/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo , alfa-Amilases/metabolismo
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