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1.
Eur J Med Chem ; 209: 112550, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-33268144

RESUMO

Current research suggests therapy-induced senescence (TIS) of cancer cells characterized by distinct morphological and biochemical phenotypic changes represent a novel functional target that may enhance the effectiveness of cancer therapy. In order to identify novel small-molecule inducers of cellular senescence and determine the potential to be used for the treatment of melanoma, a new method of high-throughput screening (HTS) and high-contents screening (HCS) based on the detection of morphological changes was designed. This image-based and whole cell-based technology was applied to screen and select a novel class of antiproliferative agents on cancer cells, 4H-chromeno[2,3-d]pyrimidin-4-one derivatives, which induced senescence-like phenotypic changes in human melanoma A375 cells without serious cytotoxicity against normal cells. To evaluate structure-activity relationship (SAR) study of 4H-chromeno[2,3-d]pyrimidin-4-one scaffold starting from hit 3, a focused library containing diversely modified analogues was constructed and which led to the identification of 38, a novel compound to have remarkable anti-melanoma activity in vitro with good metabolic stability.


Assuntos
Antineoplásicos/farmacologia , Benzopiranos/farmacologia , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Melanoma/tratamento farmacológico , Pirimidinas/farmacologia , Animais , Antineoplásicos/química , Benzopiranos/química , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Masculino , Melanoma/patologia , Camundongos Endogâmicos BALB C , Pirimidinas/química
2.
Cell Rep ; 20(13): 3188-3198, 2017 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-28954234

RESUMO

Pathogens have evolved a range of mechanisms to counteract host defenses, notably to survive harsh acidic conditions in phagosomes. In the case of Mycobacterium tuberculosis, it has been shown that regulation of phagosome acidification could be achieved by interfering with the retention of the V-ATPase complexes at the vacuole. Here, we present evidence that M. tuberculosis resorts to yet another strategy to control phagosomal acidification, interfering with host suppressor of cytokine signaling (SOCS) protein functions. More precisely, we show that infection of macrophages with M. tuberculosis leads to granulocyte-macrophage colony-stimulating factor (GM-CSF) secretion, inducing STAT5-mediated expression of cytokine-inducible SH2-containing protein (CISH), which selectively targets the V-ATPase catalytic subunit A for ubiquitination and degradation by the proteasome. Consistently, we show that inhibition of CISH expression leads to reduced replication of M. tuberculosis in macrophages. Our findings further broaden the molecular understanding of mechanisms deployed by bacteria to survive.


Assuntos
Mycobacterium tuberculosis/patogenicidade , Fagossomos/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Animais , Camundongos , Mycobacterium tuberculosis/metabolismo , Transdução de Sinais
3.
J Med Chem ; 57(12): 5293-305, 2014 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-24870926

RESUMO

A critical unmet clinical need to combat the global tuberculosis epidemic is the development of potent agents capable of reducing the time of multi-drug-resistant (MDR) and extensively-drug-resistant (XDR) tuberculosis therapy. In this paper, we report on the optimization of imidazo[1,2-a]pyridine amide (IPA) lead compound 1, which led to the design and synthesis of Q203 (50). We found that the amide linker with IPA core is very important for activity against Mycobacterium tuberculosis H37Rv. Linearity and lipophilicity of the amine part in the IPA series play a critical role in improving in vitro and in vivo efficacy and pharmacokinetic profile. The optimized IPAs 49 and 50 showed not only excellent oral bioavailability (80.2% and 90.7%, respectively) with high exposure of the area under curve (AUC) but also displayed significant colony-forming unit (CFU) reduction (1.52 and 3.13 log10 reduction at 10 mg/kg dosing level, respectively) in mouse lung.


Assuntos
Antituberculosos/química , Imidazóis/química , Piridinas/química , Animais , Antituberculosos/síntese química , Antituberculosos/farmacologia , Farmacorresistência Bacteriana Múltipla , Humanos , Imidazóis/síntese química , Imidazóis/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Camundongos , Microssomos Hepáticos/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Piridinas/síntese química , Piridinas/farmacologia , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico
4.
Nat Med ; 19(9): 1157-60, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23913123

RESUMO

New therapeutic strategies are needed to combat the tuberculosis pandemic and the spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) forms of the disease, which remain a serious public health challenge worldwide. The most urgent clinical need is to discover potent agents capable of reducing the duration of MDR and XDR tuberculosis therapy with a success rate comparable to that of current therapies for drug-susceptible tuberculosis. The last decade has seen the discovery of new agent classes for the management of tuberculosis, several of which are currently in clinical trials. However, given the high attrition rate of drug candidates during clinical development and the emergence of drug resistance, the discovery of additional clinical candidates is clearly needed. Here, we report on a promising class of imidazopyridine amide (IPA) compounds that block Mycobacterium tuberculosis growth by targeting the respiratory cytochrome bc1 complex. The optimized IPA compound Q203 inhibited the growth of MDR and XDR M. tuberculosis clinical isolates in culture broth medium in the low nanomolar range and was efficacious in a mouse model of tuberculosis at a dose less than 1 mg per kg body weight, which highlights the potency of this compound. In addition, Q203 displays pharmacokinetic and safety profiles compatible with once-daily dosing. Together, our data indicate that Q203 is a promising new clinical candidate for the treatment of tuberculosis.


Assuntos
Trifosfato de Adenosina/biossíntese , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Tuberculose Extensivamente Resistente a Medicamentos/tratamento farmacológico , Imidazóis/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Piperidinas/farmacologia , Piridinas/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Complexo III da Cadeia de Transporte de Elétrons/genética , Imidazóis/farmacocinética , Camundongos , Camundongos Endogâmicos BALB C , Piperidinas/farmacocinética , Piridinas/farmacocinética , Ratos , Ratos Sprague-Dawley
5.
Appl Microbiol Biotechnol ; 97(3): 1213-22, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22825833

RESUMO

Identification of secondary metabolites produced by cryptic gene in bacteria may be difficult, but in the case of nonribosomal peptide (NRP)-type secondary metabolites, this study can be facilitated by bioinformatic analysis of the biosynthetic gene cluster and tandem mass spectrometry analysis. To illustrate this concept, we used mass spectrometry-guided bioinformatic analysis of genomic sequences to identify an NRP-type secondary metabolite from Streptomyces peucetius ATCC 27952. Five putative NRPS biosynthetic gene clusters were identified in the S. peucetius genome by DNA sequence analysis. Of these, the sp970 gene cluster encoded a complete NRPS domain structure, viz., C-A-T-C-A-T-E-C-A-T-C-A-T-C domains. Tandem mass spectrometry revealed that the functional siderophore peptide produced by this cluster had a molecular weight of 644.4 Da. Further analysis demonstrated that the siderophore peptide has a cyclic structure and an amino acid composition of AchfOrn-Arg-hOrn-hfOrn. The discovery of functional cryptic genes by analysis of the secretome, especially of NRP-type secondary metabolites, using mass spectrometry together with genome mining may contribute significantly to the development of pharmaceuticals such as hybrid antibiotics.


Assuntos
Estudos de Associação Genética , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Sideróforos/genética , Sideróforos/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Biologia Computacional , DNA Bacteriano/química , DNA Bacteriano/genética , Genoma Bacteriano , Genômica , Espectrometria de Massas , Peso Molecular , Família Multigênica , Peptídeos Cíclicos/química , Peptídeos Cíclicos/genética , Peptídeos Cíclicos/metabolismo , Análise de Sequência de DNA , Sideróforos/química
6.
PLoS Pathog ; 6(9): e1001100, 2010 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-20844580

RESUMO

The ability of the tubercle bacillus to arrest phagosome maturation is considered one major mechanism that allows its survival within host macrophages. To identify mycobacterial genes involved in this process, we developed a high throughput phenotypic cell-based assay enabling individual sub-cellular analysis of over 11,000 Mycobacterium tuberculosis mutants. This very stringent assay makes use of fluorescent staining for intracellular acidic compartments, and automated confocal microscopy to quantitatively determine the intracellular localization of M. tuberculosis. We characterised the ten mutants that traffic most frequently into acidified compartments early after phagocytosis, suggesting that they had lost their ability to arrest phagosomal maturation. Molecular analysis of these mutants revealed mainly disruptions in genes involved in cell envelope biogenesis (fadD28), the ESX-1 secretion system (espL/Rv3880), molybdopterin biosynthesis (moaC1 and moaD1), as well as in genes from a novel locus, Rv1503c-Rv1506c. Most interestingly, the mutants in Rv1503c and Rv1506c were perturbed in the biosynthesis of acyltrehalose-containing glycolipids. Our results suggest that such glycolipids indeed play a critical role in the early intracellular fate of the tubercle bacillus. The unbiased approach developed here can be easily adapted for functional genomics study of intracellular pathogens, together with focused discovery of new anti-microbials.


Assuntos
Glicolipídeos/metabolismo , Lipopolissacarídeos/metabolismo , Macrófagos/microbiologia , Mycobacterium tuberculosis/metabolismo , Fagossomos/fisiologia , Tuberculose/metabolismo , Tuberculose/patologia , Animais , Feminino , Macrófagos/citologia , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mutação/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Fagocitose , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tuberculose/microbiologia
7.
Am J Vet Res ; 71(2): 169-75, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20113224

RESUMO

OBJECTIVE: To determine whether triamcinolone acetonide diffuses from the distal interphalangeal joint (DIPJ) to the navicular bursa, diffusion is direct or systemic, and addition of sodium hyaluronan has an effect on diffusion in horses. ANIMALS: 11 adult horses without forelimb lameness. PROCEDURES: 1 randomly chosen forelimb DIPJ of each horse received an injection of 10 mg of triamcinolone acetonide plus 20 mg of sodium hyaluronan (group 1), and the contralateral forelimb DIPJ received an injection of 10 mg of triamcinolone acetonide plus 2 mL of lactated Ringer's solution (group 2). Synovial fluid samples were taken from both forelimb navicular bursae and 1 hind limb navicular bursa (systemic control group) at 6 hours. Triamcinolone acetonide concentrations in synovial fluid were quantified by use of high-performance liquid chromatography plus tandem mass spectrometry. Data were logarithmically transformed, and contrast analysis was performed on the 3 groups. RESULTS: Triamcinolone acetonide was detected in navicular bursal samples in all groups. Groups 1 and 2 had significantly greater concentrations of triamcinolone acetonide than the systemic control group. There was no significant difference between groups 1 and 2. CONCLUSIONS AND CLINICAL RELEVANCE: Triamcinolone acetonide diffused directly from the DIPJ into the navicular bursa in clinically normal horses, and diffusion was not affected by addition of hyaluronan. Injection into the DIPJ with triamcinolone acetonide or a triamcinolone acetonide-hyaluronan combination can potentially be used for treatment of navicular syndrome, but further studies are needed to determine whether triamcinolone acetonide diffuses similarly in horses with navicular syndrome.


Assuntos
Injeções Intra-Articulares/veterinária , Triancinolona Acetonida/administração & dosagem , Triancinolona Acetonida/farmacologia , Animais , Feminino , , Ácido Hialurônico/administração & dosagem , Ácido Hialurônico/farmacologia , Masculino
8.
Int J Gynecol Cancer ; 19(6): 1068-73, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19820370

RESUMO

INTRODUCTION: The purpose of this study was to evaluate the efficacy of conservative treatment with progestin and pregnancy outcomes in women with early-stage endometrial cancer. METHODS: We retrospectively analyzed the medical records of 35 patients with endometrial adenocarcinoma, who were treated with progestin from January 1996 to December 2006. Women with early-stage grade 1 endometrioid endometrial adenocarcinoma, who wanted to receive conservative treatment or preserve fertility, were included. All women were treated with medroxyprogesterone acetate or megestrol acetate, with regular dilation and curettage performed. Complete remission (CR) was defined as no evidence of endometrial adenocarcinoma or hyperplasia. Partial remission was diagnosed when the patient developed endometrial hyperplasia, and persistent disease was defined as residual endometrial adenocarcinoma by pathologic confirmation. RESULTS: The median age was 31 years (range, 21-43 years), and the median follow-up period was 39 months (range, 5-108 months). Complete remission was achieved in 22 patients (62.9%), partial remission was achieved in 1 patient (2.9%), and 12 patients (34.3%) had persistent disease. The median time to CR was 9 months (range, 2-12 months). Of the 22 patients with CR, 9 (40.9%) had recurrent disease, and the median time to recurrence was 12 months (range, 8-48 months). Ten (83.3%) of the 12 patients with CR who tried to conceive were successful, and 8 of the 10 pregnancies resulted in live births. There were no congenital anomalies in babies associated with progestin treatment. CONCLUSIONS: Conservative treatment with progestin can be considered a good therapeutic option in patients with well-differentiated early-stage endometrioid endometrial adenocarcinoma who wish to preserve their uteri or become pregnant.


Assuntos
Carcinoma Endometrioide/tratamento farmacológico , Carcinoma Endometrioide/reabilitação , Neoplasias do Endométrio/tratamento farmacológico , Neoplasias do Endométrio/reabilitação , Resultado da Gravidez , Progestinas/uso terapêutico , Adulto , Algoritmos , Antineoplásicos Hormonais/efeitos adversos , Antineoplásicos Hormonais/uso terapêutico , Feminino , Fertilidade/efeitos dos fármacos , Seguimentos , Procedimentos Cirúrgicos em Ginecologia/efeitos adversos , Humanos , Infertilidade Feminina/prevenção & controle , Acetato de Medroxiprogesterona/efeitos adversos , Acetato de Medroxiprogesterona/uso terapêutico , Acetato de Megestrol/efeitos adversos , Acetato de Megestrol/uso terapêutico , Gravidez , Progestinas/efeitos adversos , Indução de Remissão/métodos , Estudos Retrospectivos , Resultado do Tratamento , Adulto Jovem
9.
Planta ; 227(5): 1047-61, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18097685

RESUMO

The seed protein IAP1 from bean (PvIAP1; Phaseolus vulgaris L.) that is modified by the phytohormone indole-3-acetic acid (IAA) was heterologously expressed in the two reference plant species Arabidopsis thaliana and Medicago truncatula. For the transformation of Medicago we devised a novel protocol using seedling infiltration. When PvIAP1 was overexpressed under the control of the constitutive 35SCaMV promoter in Arabidopsis, the plants showed signs of earlier bolting and enhanced branching. Expression of a fusion protein of PvIAP1 with both a green fluorescence protein (GFP) as reporter and 6x histidine (His) tag under the control of the native bean IAP1 promoter resulted in the accumulation of the protein in both plant species exclusively in seeds as shown by immunoblotting and by fluorescence microscopy. During seed development, PvIAP1 was first expressed in the vascular bundle of Arabidopsis, whereas in later stages GFP fluorescence was visible essentially in all tissues of the seed. Fluorescence decreased rapidly after imbibition in the seeds for both Arabidopsis and Medicago, although the fluorescence persisted longer in Arabidopsis. GFP fluorescence was distributed evenly between an organelle fraction, the microsomal membrane fraction, and the cytosol. This was also confirmed by immunoblot analysis. Clusters of higher GFP fluorescence were observed by confocal microscopy. Although PvIAP1 protein accumulated in seeds of both Arabidopsis and Medicago, neither species post-translationally modified the protein with an indoleacyl moiety as shown by quantitative GC-MS analysis after alkaline hydrolysis. These results indicate an apparent specificity for IAA attachment in different plant species.


Assuntos
Arabidopsis/metabolismo , Ácidos Indolacéticos/metabolismo , Medicago truncatula/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Cromatografia Gasosa-Espectrometria de Massas , Regulação da Expressão Gênica de Plantas , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Immunoblotting , Medicago truncatula/genética , Medicago truncatula/crescimento & desenvolvimento , Microscopia de Fluorescência , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sementes/genética , Sementes/crescimento & desenvolvimento , Sementes/metabolismo
10.
Planta ; 224(5): 1015-22, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16683161

RESUMO

Achenes and receptacle tissue of Fragaria vesca, L. cultivar Yellow Wonder were shown to contain conjugated indole-3-acetic acid (IAA) that was not soluble in organic solvents and yielded IAA after strong alkaline hydrolysis, suggestive of IAA attached to plant proteins. This solvent insoluble conjugated IAA accounted for between 0.4 and 4 ng of IAA per gram fresh weight of tissue in both achenes and receptacles. To investigate this strawberry conjugate class further, a polyclonal antibody was produced to IAA-glycine attached to BSA that detected neutral indole acid esters, monocarboxylic-amino acid IAA conjugates and IAA proteins. Using immunoblotting, both achenes and receptacles of strawberry were shown to have primarily an immuno-detectable band at 76 kDa. Two-dimensional polyacrylamide gel electrophoresis yielded a wide band that was analyzed by LC-MS/MS analysis following in-gel trypsin digestion. Peptides derived from the immuno-detectable band were tentatively identified by peptide fragment analysis as being from either a chaperonin related to the hsp60 class of proteins or, alternatively, an ATP synthase. This is one of the first reports of an IAA modified protein in fruit tissue.


Assuntos
Fragaria/metabolismo , Frutas/metabolismo , Ácidos Indolacéticos/metabolismo , Proteínas de Plantas/metabolismo , Immunoblotting
11.
Bioorg Med Chem ; 13(9): 3229-40, 2005 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-15809158

RESUMO

Indole-3-acetic acid is an indispensable hormone (auxin) in plants and an important metabolite in humans, animals, and microorganisms. Here we introduce its 5- and 6-(2-aminoethyl)-derivatives for use in the design of novel research tools, such as immobilized and carrier-linked forms of indole-3-acetic acid and its conjugates with biochemical tags or biocompatible molecular probes. The aliphatic nitrogens of 5- and 6-(2-aminoethyl)indole were acetylated and the products were converted to the corresponding 3-(N,N-dimethylamino)methyl derivatives (gramines). These were reacted with cyanide. Saponification of the resulting acetonitriles was accompanied by N-deprotection to yield 5- and 6-(2-aminoethyl)indole-3-acetic acids. The latter were chemically stable and could be linked, via their amino groups, and without prior protection of their carboxyl moieties, to bovine serum albumin and to biotin, including appropriate spacer modules. One of the protein conjugates was used to elicit the formation of monoclonal antibodies, which were evaluated using the biotin conjugates in an enzyme-linked immunosorbent assay employing streptavidin-coupled alkaline phosphatase, and thus shown to recognize predominantly the indole-3-acetic acid moiety.


Assuntos
Ácidos Indolacéticos/química , Ácidos Indolacéticos/síntese química , Animais , Anticorpos Monoclonais/química , Biotina/química , Biotinilação , Bovinos , Linhagem Celular Tumoral , Cristalografia por Raios X , Imunoensaio , Camundongos , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Soroalbumina Bovina/química
12.
Plant Physiol ; 129(3): 1045-53, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12114559

RESUMO

Gibberellins (GAs) in developing seeds of morning glory (Pharbitis nil) were quantified and localized by immunostaining. The starch grains began to be digested after the GA contents had increased and reached a plateau. Immunohistochemical staining with the antigibberellin A(1)-methyl ester-antiserum, which has high affinity to biologically active GAs, showed that GA(1) and/or GA(3) were localized around starch grains in the integument of developing young seeds, suggesting the participation of GA-inducible alpha-amylase in this digestion. We isolated an alpha-amylase cDNA (PnAmy1) that was expressed in the immature seeds, and using an antibody raised against recombinant protein, it was shown that PnAmy1 was expressed in the immature seeds. GA responsiveness of PnAmy1 was shown by treating the young fruits 9 d after anthesis with GA(3). RNA-blot and immunoblot analyses showed that PnAmy1 emerged soon after the rapid increase of GA(1/3). An immunohistochemical analysis of PnAmy1 showed that it, like the seed GA(1/3), was also localized around starch grains in the integument of developing young seeds. The localization of GA(1/3) in the integument coincident with the expression of PnAmy1 suggests that both function as part of a process to release sugars for translocation or for the further development of the seeds.


Assuntos
Convolvulaceae/enzimologia , Giberelinas/metabolismo , Sementes/enzimologia , alfa-Amilases/genética , Northern Blotting , Clonagem Molecular , Convolvulaceae/genética , Convolvulaceae/crescimento & desenvolvimento , DNA Complementar/química , DNA Complementar/genética , Indução Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Giberelinas/farmacologia , Imuno-Histoquímica , Dados de Sequência Molecular , Proteínas de Plantas/metabolismo , RNA de Plantas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sementes/genética , Sementes/crescimento & desenvolvimento , Análise de Sequência de DNA , Amido/metabolismo , alfa-Amilases/metabolismo
13.
Proc Natl Acad Sci U S A ; 99(3): 1718-23, 2002 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-11830675

RESUMO

We show that the expression of an indole-3-acetic acid (IAA)-modified protein from bean seed, IAP1, is correlated to the developmental period of rapid growth during seed development. Moreover, this protein undergoes rapid degradation during germination. The gene for IAP1, the most abundant protein covalently modified by IAA (iap1, GenBank accession no. ) was isolated and cloned from bush bean (Phaseolus vulgaris) seeds. The 957-bp sequence encodes a 35-kDa polypeptide. IAA-modified proteins represent a distinct class of conjugated phytohormones and appear in bean to be the major form of auxin in seeds. IAA proteins also are found at other stages of development in bean plants. Our immunological and analytical data suggest that auxin modification of a small class of proteins may be a feature common to many plants.


Assuntos
Ácidos Indolacéticos/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/genética , Sequência de Aminoácidos , Conjugação Genética , Primers do DNA , Fabaceae/genética , Biblioteca Gênica , Immunoblotting , Dados de Sequência Molecular , Pisum sativum/genética , Proteínas de Plantas/química , Proteínas Recombinantes/metabolismo , Sementes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Zea mays/genética
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