RESUMO
Cytochrome P450s (P450s) comprise a gene superfamily encoding enzymes that are involved in diverse plant metabolic pathways that produce primary and secondary metabolites such as phenylpropanoids, terpenoids, nitrogen-containing compounds, and plant hormones. They comprise one of the most diverse gene families in plant evolution. Although there are many studies that aim to characterize P450s in plants, there is no report on the characterization of this superfamily in Coffea arabica, where they might be related to plant tolerance to biotic and abiotic stresses, as well as aroma-related compounds. In this study, we report the characterization and annotation of 87 putative P450s from C. arabica obtained from the Brazilian Coffee Genome Project and describe their transcriptional pattern in different tissues and coffee organs. To validate our approach, we measured the transcriptional profile of the CaCYP81D8_1 gene by quantitative polymerase chain reaction in leaves, flowers, and fruits. This study is the first effort to present and analyze the P450 superfamily in C. arabica, which may assist in understanding the chemical diversity of coffee secondary metabolites.
Assuntos
Coffea/genética , Sistema Enzimático do Citocromo P-450/genética , Perfilação da Expressão Gênica , Família Multigênica , Proteínas de Plantas/genética , Coffea/enzimologia , Sistema Enzimático do Citocromo P-450/classificação , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Isoenzimas/classificação , Isoenzimas/genética , Filogenia , Proteínas de Plantas/classificação , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Three ω-3 fatty acid desaturase genes (CtFAD3, CtFAD7, and CtFAD8) were isolated from safflower (Carthamus tinctorius L.). Transcript analysis showed that the highest transcript levels were detected for CtFAD3 and the low transcript levels were detected for CtFAD7 and CtFAD8 in flowers. This result indicates that CtFAD3 enzyme activity is important for fatty acid desaturation in flowers. The low transcript level of CtFAD3 in developing seeds was consistent with the recorded high level of linoleic acid (18:2) and lack of linolenic acid (18:3) in safflower seed oil. At low temperatures, the induced transcription levels of ω-3 fatty acid desaturase genes in the stems and petioles were consistent with increased polyunsaturated fatty acids (PUFAs). In the roots, ω-3 fatty acid desaturase noticeably increased at low temperatures, whereas PUFA levels decreased. Interestingly, C18:3(Δ9,12,15) alcohol was specifically found in safflower roots, and showed a significant increase, indicating a flux in the acid to alcohol ratio of this compound in safflower roots.
Assuntos
Carthamus tinctorius/genética , Ácidos Graxos Dessaturases/genética , Proteínas de Plantas/genética , Temperatura , Sequência de Aminoácidos , Carthamus tinctorius/enzimologia , Carthamus tinctorius/crescimento & desenvolvimento , DNA Complementar/química , DNA Complementar/genética , Ácidos Graxos Dessaturases/classificação , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos/metabolismo , Ácidos Graxos Insaturados/metabolismo , Flores/enzimologia , Flores/genética , Flores/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Isoenzimas/classificação , Isoenzimas/genética , Isoenzimas/metabolismo , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/metabolismo , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Caules de Planta/enzimologia , Caules de Planta/genética , Caules de Planta/crescimento & desenvolvimento , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sementes/enzimologia , Sementes/genética , Sementes/crescimento & desenvolvimento , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
The aim of this study was to characterize the sarcoplasmic-endoplasmic reticulum Ca-ATPase (SERCA) isoforms in rabbit masticatory muscles compared with those in fast-twitch muscle. It was hypothesized that combined expression of the SERCA isoforms in fast- and slow-twitch muscles accounts for lower Ca-ATPase activity. SERCA was isolated by differential centrifugation, the isoforms were determined by ELISA, and the activity of each isoform was measured using a colorimetric method. Activity was tested for significance by anova, and the distribution of isoforms was assessed using the chi-square test (P < 0.05) and correlated to SERCA activity using Spearman's rank correlation. SERCA1 was predominant (90.5%) in fast-twitch muscle, whereas a mixture of SERCA isoforms was found in masticatory muscles: 62-78% was SERCA2, 20-37% was SERCA1, and the SERCA3 content was negligible. Depressor muscles showed a significantly higher content (77.8%) of SERCA2, and elevator muscles showed a higher content (35.4%) of SERCA1. Elevator muscles showed higher expression of SERCA2a (58%), and depressor muscles showed higher expression of SERCA2b (20%). The SERCA1 content was mainly SERCA1a and significantly higher for elevator muscles (33%), whereas depressor muscles showed a higher content of SERCA1b (4%). The SERCA1 content of fast-twitch muscle was mainly SERCA1a (88.5%). It is concluded that the mixture of different SERCA isoforms, along with a substantial content of SERCA2b, in masticatory muscles would support lower Ca-ATPase activity and calcium transport.
Assuntos
Músculos da Mastigação/enzimologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/análise , Animais , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Isoenzimas/análise , Isoenzimas/classificação , Masculino , Músculo Masseter/enzimologia , Fibras Musculares de Contração Rápida/enzimologia , Fibras Musculares de Contração Lenta/enzimologia , Músculos do Pescoço/enzimologia , Músculos Pterigoides/enzimologia , Coelhos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/classificação , Músculo Temporal/enzimologiaRESUMO
Class III peroxidases are present as large multigene families in all land plants. This large number of genes together with the diversity of processes catalyzed by peroxidases suggests possible functional specialization of each isoform. However, assigning a precise role for each individual peroxidase gene has continued to be a major bottleneck. Here we investigated the enzyme activity and translational profile of class III peroxidases during stem development of sugarcane as a first step in the estimation of physiological functions of individual isoenzymes. Internodes at three different developmental stages (young, developing and mature) were divided into pith (inner tissue) and rind (outer tissue) fractions. The rind of mature internodes presented the highest enzymatic activity and thus could be considered the ideal tissue for the discovery of peroxidase gene function. In addition, activity staining of 2DE gels revealed different isoperoxidase profiles and protein expression regulation among different tissue fractions. In-gel tryptic digestion of excised spots followed by peptide sequencing by LC-MS/MS positively matched uncharacterized peroxidases in the sugarcane database SUCEST. Multiple spots matching the same peroxidase gene were found, which reflects the generation of more than one isoform from a particular gene by post-translational modifications. The identified sugarcane peroxidases appear to be monocot-specific sequences with no clear ortholog in dicot model plant Arabidopsis thaliana.
Assuntos
Peroxidases/metabolismo , Proteínas de Plantas/metabolismo , Caules de Planta/metabolismo , Proteômica/métodos , Saccharum/metabolismo , Eletroforese em Gel Bidimensional , Isoenzimas/classificação , Isoenzimas/genética , Isoenzimas/metabolismo , Espectrometria de Massas/métodos , Peroxidases/classificação , Peroxidases/genética , Filogenia , Proteínas de Plantas/genética , Caules de Planta/genética , Caules de Planta/crescimento & desenvolvimento , Biossíntese de Proteínas , Proteoma/genética , Proteoma/metabolismo , Saccharum/genética , Saccharum/crescimento & desenvolvimento , Fatores de TempoRESUMO
At the present, no secreted phospholipase A2 (sPLA2) from soybean (Glycine max) was investigated in detail. In this work we identified five sequences of putative secreted sPLA2 from soybean after a BLAST search in G. max database. Sequence analysis showed a conserved PA2c domain bearing the Ca²âº binding loop and the active site motif. All the five mature proteins contain 12 cysteine residues, which are commonly conserved in plant sPLA2s. We propose a phylogenetic tree based on sequence alignment of reported plant sPLA2s including the novel enzymes from G. max. According to PLA2 superfamily, two of G. max sPLA2s are grouped as XIA and the rest of sequences as XIB, on the basis of differences found in their molecular weights and deviating sequences especially in the N- and C-terminal regions of the isoenzymes. Furthermore, we report the cloning, expression and purification of one of the putative isoenzyme denoted as GmsPLA2-XIA-1. We demonstrate that this mature sPLA2 of 114 residues had PLA2 activity on Triton:phospholipid mixed micelles and determine the kinetic parameters for this system. We generate a model based on the known crystal structure of sPLA2 from rice (isoform II), giving first insights into the three-dimensional structure of folded GmsPLA2-XIA-1. Besides describing the spatial arrangement of highly conserved pair HIS-49/ASP-50 and the Ca⺲ loop domains, we propose the putative amino acids involved in the interfacial recognition surface. Additionally, molecular dynamics simulations indicate that calcium ion, besides its key function in the catalytic cycle, plays an important role in the overall stability of GmsPLA2-XIA-1 structure.
Assuntos
Glycine max/enzimologia , Glycine max/genética , Fosfolipases A2/genética , Proteínas de Plantas/genética , Sequência de Aminoácidos , Sequência de Bases , Biocatálise , Cálcio/química , Cálcio/metabolismo , Clonagem Molecular , Simulação por Computador , Eletroforese em Gel de Poliacrilamida , Eritrócitos/metabolismo , Hemólise , Humanos , Isoenzimas/classificação , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Modelos Moleculares , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Fosfolipases A2/química , Fosfolipases A2/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Espectrometria de FluorescênciaRESUMO
La Leishmaniasis es una enfermedad endémica en la provincia de Salta, Argentina, con cientos de casos anuales. La identificación de la especie de Leishmania es necesaria debido a diferencias de presentación clínica y de respuesta al tratamiento entre especies. La técnica PS-PCR ha sido validada con el método de isoenzimas para la identificación de especies de Leishmania en Argentina. OBJETIVOS: optimizar la técnica PS-PCR para identificar la especie de parásitos aislados por cultivo y en muestras clínicas de pacientes de Salta y correlacionarla con sus características clínicas y su respuesta al tratamiento. RESULTADOS: La PS-PCR permitió identificar la especie de Leishmania en las 24 muestras analizadas: 9 aislados de cultivo y 15 muestras clínicas de raspado de lesiones (100% de especificidad). Las especies halladas fueron:L(V) braziliensis (19/24; p< 0,0001), L(L) amazonensis (4/24) yL(V) guyanensis (1/24). La primera estuvo relacionada con todos los casos severos (8 con lesiones mucocutáneas y 1 con enfermedad diseminada), y con todas las fallas al tratamiento con Glucantime(4) observadas. En los casos de L(L) amazonensis, no se observó enfermedad grave ni resistencia al tratamiento. El caso deL(V) guyanensis no era autóctono de la zona. CONCLUSIONES: Este estudio demuestra que la técnica PS-PCR es apropiada para el diagnóstico específico de la leishmaniasis. El diagnóstico deL(V) braziliensis obliga a considerar tratamientos alternativos para los casos severos de la enfermedad
Leishmaniasis is endemic in the province of Salta, Argentina, with hundreds of cases annually. Identifying the species of Leishmania is necessary due to differences in clinical presentation and treatment response between species. PS-PCR technique has been validated by the method of isozymesto identify Leishmania species in Argentina. OBJECTIVES:To optimize the PS-PCR technique to identify the species of parasites isolated by culture and in clinical specimens from patients of Salta and its correlation with clinical characteristics and response to treatment. RESULTS: The PS-PCR allowed us to identify the species of Leishmania in 24 samples analyzed: 9 isolated from clinical specimens and 15 culture swabs taken from lesions (100% specificity). The species found were: L(V)braziliensis (19/24, P <0.0001), L(L) amazonensis (4/24) andL(V) guyanensis (1/24). The first one was related to all severe cases (8 with mucocutaneous lesions and 1 with disseminated disease), and to all failures to treatment with Glucantime (4)observed. In the cases of L(L) amazonensis, there were noserious disease or resistance to treatment. The case of L(V)guyanensis was not indigenous to the area. CONCLUTIONS:This study demonstrates that PS-PCR technique is suitable for specific diagnosis of leishmaniasis. The diagnosis of L(V)braziliensis under takes to consider alternative treatments for severe cases of the disease
Assuntos
Humanos , Citocromos b , Isoenzimas/classificação , Leishmania/parasitologia , Reação em Cadeia da Polimerase/métodosRESUMO
The possible regulation of amino acid remobilization via the phloem in wheat (Triticum aestivum L.) by the primary enzyme in nitrogen (N) assimilation and re-assimilation, glutamine synthetase (GS, E.C. 6.3.1.2) was studied using two conditions known to alter N phloem transport, N deficiency and cytokinins. The plants were grown for 15 days in controlled conditions with optimum N supply and then N was depleted from and/or 6-benzylaminopurine was added to the nutrient solution. Both treatments generated an induction of GS1, monitored at the level of gene expression, protein accumulation and enzyme activity, and a decrease in the exudation of amino acids to the phloem, obtained with EDTA technique, which correlated negatively. GS inhibition by metionine sulfoximide (MSX) produced an increase of amino acids exudation and the inhibitor successfully reversed the effect of N deficiency and cytokinin addition over phloem exudation. Our results point to an important physiological role for GS1 in the modulation of amino acids export levels in wheat plants.
Assuntos
Aminoácidos/metabolismo , Glutamato-Amônia Ligase/genética , Floema/metabolismo , Proteínas de Plantas/genética , Triticum/genética , Compostos de Benzil , Transporte Biológico/efeitos dos fármacos , Eletroforese em Gel de Poliacrilamida , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Glutamato-Amônia Ligase/classificação , Glutamato-Amônia Ligase/metabolismo , Isoenzimas/classificação , Isoenzimas/genética , Isoenzimas/metabolismo , Cinetina/farmacologia , Metionina Sulfoximina/farmacologia , Nitratos/farmacologia , Filogenia , Proteínas de Plantas/metabolismo , Purinas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Triticum/enzimologia , Triticum/metabolismoRESUMO
Two hundred and sixty-one accessions of the genus Capsicum were obtained from the Colombian Amazonian germplasm bank at Amazonian Institute of Scientific Research (Sinchi) and were evaluated with five polymorphic enzymatic systems, including esterase (EST), peroxidase (PRX), 6-phosphogluconatedehydrogenase (6-PGDH), aspartate amino transferase (GOT), and malic enzyme (ME). Using a cluster analysis (UPGMA) the genetic variability of these accessions were characterized. Grouping of the species C. baccatum and C. pubescens were observed, while the species C. annuum, C. chinense and C. frutescens did not group independently, a result that has been previously reported in isoenzyme analyses of this genus. Several accessions were deemed of particular interest for future ecological and evolutive studies.
Doscientas sesenta y una accesiones del género Capsicum del banco de germoplasma del Instituto Amazónico de Investigaciones Científicas (Sinchi) se evaluaron a través de cinco sistemas enzimáticos polimórficos: esterasa (EST), peroxidasa (PRX), 6-fosfogluconato deshidrogenasa (6-PGDH), aspartato amino transferasa(GOT) y enzima málica (ME). Se utilizó un análisis de agrupamiento (Upgma) con el fin de determinar la variabilidad genética. Se observó un agrupamiento de las especies C. baccatum y C. pubescens, mientras que las especies C. annuum, C. chinense y C. frutescens no mostraron un agrupamiento independiente, lo cual ya ha sido reportado en estudios por isoenzimas para el género. Varias accesiones mostraron característicasparticulares para estudios ecológicos y evolutivos.
Assuntos
Capsicum/classificação , Capsicum/crescimento & desenvolvimento , Capsicum/enzimologia , Capsicum/microbiologia , Reativadores Enzimáticos , Isoenzimas/análise , Isoenzimas/classificação , Isoenzimas/ultraestruturaRESUMO
The isoforms of cAMP-dependent protein kinase (PKA) show distinct biochemical properties and subcellular localization, suggesting different physiological functions, and conferring the fine-tuning between the activation of cAMP-PKA cascade and the cellular response. The critical role of PKA in memory and synaptic plasticity has been extensively demonstrated both in vertebrates and invertebrates, but the role of PKA isoforms is a matter of debate. Here we present experimental data showing differential PKA activation profiles after two different experiences: an instance of associative contextual learning (context-signal learning) and a single exposure to a novel context, both in the learning and memory model of the crab Chasmagnathus. Differences were found in the temporal course of activation and in the involvement of PKA isoforms. We found increased PKA activity immediately and 6 h after context-signal training correlating with the critical periods during which pharmacological inhibition of PKA disrupts memory formation. In contrast, PKA activity increased immediately but not 6 h after single exposure to a novel context. The amounts of PKA I and PKA II holoenzymes were analyzed to determine changes in holoenzyme levels and/or differential activation induced by both experiences. Results indicate that context-induced PKA activation is at least in part due to PKA II, and that PKA activation 6 h after context-signal learning coincides with an increase in the total level of PKA I. Considering the higher sensitivity of PKA I to cAMP, its increment can account for the PKA activation found 6 h after training and is proposed as a novel mechanism providing the prolonged PKA activation during memory consolidation.
Assuntos
Aprendizagem por Associação/fisiologia , Braquiúros/enzimologia , Encéfalo/enzimologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Memória/fisiologia , Transdução de Sinais/fisiologia , Animais , Proteínas Quinases Dependentes de AMP Cíclico/classificação , Ativação Enzimática , Isoenzimas/classificação , Isoenzimas/metabolismo , MasculinoRESUMO
The non-enzymatic deamidation of asparaginyl residues is a major source of spontaneous damage of several proteins under physiological conditions. In many cases, deamidation and isoaspartyl formation alters the biological activity or stability of the native polypeptide. Rates of deamidation of particular residues depend on many factors including protein structure and solvent exposure. Here, we investigated the spontaneous deamidation of the two NADP-glutamate dehydrogenase isoenzymes from Saccharomyces cerevisiae, which have different kinetic properties and are differentially expressed in this yeast. Our results show that Asn54, present in Gdh3p but missing in the GDH1-encoded homologue, is readily deamidated in vitro under alkaline conditions. Relative to the native enzyme, deamidated Gdh3p shows reduced protein stability. The different deamidation rates of the two isoenzymes could explain to some extent, the relative in vivo instability of the allosteric Gdh3p enzyme, compared to that of Gdh1p. It is thus possible that spontaneous asparaginyl modification could play a role in the metabolic regulation of ammonium assimilation and glutamate biosynthesis.