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2.
Theor Appl Genet ; 137(7): 150, 2024 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-38847846

RESUMO

Grain size is a crucial agronomic trait that determines grain weight and final yield. Although several genes have been reported to regulate grain size in rice (Oryza sativa), the function of Wall-Associated Kinase family genes affecting grain size is still largely unknown. In this study, we identified GRAIN WEIGHT AND NUMBER 1 (GWN1) using map-based cloning. GWN1 encodes the OsWAK74 protein kinase, which is conserved in plants. GWN1 negatively regulates grain length and weight by regulating cell proliferation in spikelet hulls. We also found that GWN1 negatively influenced grain number by influencing secondary branch numbers and finally increased plant grain yield. The GWN1 gene was highly expressed in inflorescences and its encoded protein is located at the cell membrane and cell wall. Moreover, we identified three haplotypes of GWN1 in the germplasm. GWN1hap1 showing longer grain, has not been widely utilized in modern rice varieties. In summary, GWN1 played a very important role in regulating grain length, weight and number, thereby exhibiting application potential in molecular breeding for longer grain and higher yield.


Assuntos
Grão Comestível , Oryza , Proteínas de Plantas , Sementes , Oryza/genética , Oryza/crescimento & desenvolvimento , Oryza/enzimologia , Grão Comestível/genética , Grão Comestível/crescimento & desenvolvimento , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sementes/crescimento & desenvolvimento , Sementes/genética , Fenótipo , Regulação da Expressão Gênica de Plantas , Clonagem Molecular , Mapeamento Cromossômico , Haplótipos , Parede Celular/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Genes de Plantas
3.
Parasites Hosts Dis ; 62(2): 205-216, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38835261

RESUMO

Sigma-class glutathione transferase (GST) proteins with dual GST and prostaglandin synthase (PGS) activities play a crucial role in the establishment of Clonorchis sinensis infection. Herein, we analyzed the structural and enzymatic properties of sigma-class GST (CsGST-σ) proteins to obtain insight into their antioxidant and immunomodulatory functions in comparison with mu-class GST (CsGST-µ) proteins. CsGST-σ proteins conserved characteristic structures, which had been described in mammalian hematopoietic prostaglandin D2 synthases. Recombinant forms of these CsGST-σ and CsGST-µ proteins expressed in Escherichia coli exhibited considerable degrees of GST and PGS activities with substantially different specific activities. All recombinant proteins displayed higher affinities toward prostaglandin H2 (PGS substrate; average Km of 30.7 and 3.0 µm for prostaglandin D2 [PGDS] and E2 synthase [PGES], respectively) than those toward CDNB (GST substrate; average Km of 1,205.1 µm). Furthermore, the catalytic efficiency (Kcat/Km) of the PGDS/PGES activity was higher than that of GST activity (average Kcat/Km of 3.1, 0.7, and 7.0×10-3 s-1µm-1 for PGDS, PGES, and GST, respectively). Our data strongly suggest that the C. sinensis sigma- and mu-class GST proteins are deeply involved in regulating host immune responses by generating PGD2 and PGE2 in addition to their roles in general detoxification.


Assuntos
Clonorchis sinensis , Glutationa Transferase , Oxirredutases Intramoleculares , Glutationa Transferase/metabolismo , Glutationa Transferase/química , Glutationa Transferase/genética , Clonorchis sinensis/enzimologia , Clonorchis sinensis/genética , Animais , Oxirredutases Intramoleculares/metabolismo , Oxirredutases Intramoleculares/química , Oxirredutases Intramoleculares/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Lipocalinas/metabolismo , Lipocalinas/genética , Lipocalinas/química , Lipocalinas/imunologia , Escherichia coli/genética , Prostaglandina H2/metabolismo , Prostaglandina H2/química , Cinética
4.
Parasites Hosts Dis ; 62(2): 226-237, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38835263

RESUMO

Ticks, blood-sucking ectoparasites, spread diseases to humans and animals. Haemaphysalis longicornis is a significant vector for tick-borne diseases in medical and veterinary contexts. Identifying protective antigens in H. longicornis for an anti-tick vaccine is a key tick control strategy. Enolase, a multifunctional protein, significantly converts D-2-phosphoglycerate and phosphoenolpyruvate in glycolysis and gluconeogenesis in cell cytoplasm. This study cloned a complete open reading frame (ORF) of enolase from the H. longicornis tick and characterized its transcriptional and silencing effect. We amplified the full-length cDNA of the enolase gene using rapid amplification of cDNA ends. The complete cDNA, with an ORF of 1,297 nucleotides, encoded a 432-amino acid polypeptide. Enolase of the Jeju strain H. longicornis exhibited the highest sequence similarity with H. flava (98%), followed by Dermacentor silvarum (82%). The enolase motifs identified included N-terminal and C-terminal regions, magnesium binding sites, and several phosphorylation sites. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that enolase mRNA transcripts were expressed across all developmental stages of ticks and organs such as salivary gland and midgut. RT-PCR showed higher transcript levels in syn-ganglia, suggesting that synganglion nerves influence enolase,s role in tick salivary glands. We injected enolase double-stranded RNA into adult unfed female ticks, after which they were subsequently fed with normal unfed males until they spontaneously dropped off. RNA interference significantly (P<0.05) reduced feeding and reproduction, along with abnormalities in eggs (no embryos) and hatching. These findings suggest enolase is a promising target for future tick control strategies.


Assuntos
Sequência de Aminoácidos , Clonagem Molecular , Ixodidae , Fosfopiruvato Hidratase , Animais , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/metabolismo , Ixodidae/genética , Ixodidae/enzimologia , Feminino , Dados de Sequência Molecular , Estágios do Ciclo de Vida/genética , Inativação Gênica , Masculino , Filogenia , Sequência de Bases , DNA Complementar/genética , Haemaphysalis longicornis
5.
Front Cell Infect Microbiol ; 14: 1392015, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38841113

RESUMO

Trehalose-6-phosphate synthase (TPS1) was identified as a virulence factor for Cryptococcus neoformans and a promising therapeutic target. This study reveals previously unknown roles of TPS1 in evasion of host defenses during pulmonary and disseminated phases of infection. In the pulmonary infection model, TPS1-deleted (tps1Δ) Cryptococci are rapidly cleared by mouse lungs whereas TPS1-sufficent WT (H99) and revertant (tps1Δ:TPS1) strains expand in the lungs and disseminate, causing 100% mortality. Rapid pulmonary clearance of tps1Δ mutant is T-cell independent and relies on its susceptibility to lung resident factors and innate immune factors, exemplified by tps1Δ but not H99 inhibition in a coculture with dispersed lung cells and its rapid clearance coinciding with innate leukocyte infiltration. In the disseminated model of infection, which bypasses initial lung-fungus interactions, tps1Δ strain remains highly attenuated. Specifically, tps1Δ mutant is unable to colonize the lungs from the bloodstream or expand in spleens but is capable of crossing into the brain, where it remains controlled even in the absence of T cells. In contrast, strains H99 and tps1Δ:TPS1 rapidly expand in all studied organs, leading to rapid death of the infected mice. Since the rapid pulmonary clearance of tps1Δ mutant resembles a response to acapsular strains, the effect of tps1 deletion on capsule formation in vitro and in vivo was examined. Tps1Δ cryptococci form capsules but with a substantially reduced size. In conclusion, TPS1 is an important virulence factor, allowing C. neoformans evasion of resident pulmonary and innate defense mechanisms, most likely via its role in cryptococcal capsule formation.


Assuntos
Criptococose , Cryptococcus neoformans , Modelos Animais de Doenças , Glucosiltransferases , Pulmão , Fatores de Virulência , Animais , Cryptococcus neoformans/patogenicidade , Cryptococcus neoformans/genética , Cryptococcus neoformans/enzimologia , Cryptococcus neoformans/imunologia , Criptococose/microbiologia , Criptococose/imunologia , Camundongos , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Pulmão/microbiologia , Pulmão/patologia , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Interações Hospedeiro-Patógeno , Encéfalo/microbiologia , Baço/microbiologia , Feminino , Camundongos Endogâmicos C57BL , Imunidade Inata , Evasão da Resposta Imune , Deleção de Genes
6.
Carbohydr Polym ; 340: 122317, 2024 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-38858030

RESUMO

Brown macroalgae synthesize large amounts of fucoidans, sulfated fucose-containing polysaccharides, in the ocean. Fucoidans are of importance for their recently discovered contribution to marine carbon dioxide sequestration and due to their potential applications in biotechnology and biomedicine. However, fucoidans have high intra- and intermolecular diversity that challenges assignment of structure to biological function and the development of applications. Fucoidan-active enzymes may be used to simplify this diversity by producing defined oligosaccharides more applicable for structural refinement, characterization, and structure to function assignment for example via bioassays. In this study, we combined MALDI mass spectrometry with biocatalysis to show that the endo-fucoidanases P5AFcnA and Wv323 can produce defined oligosaccharide structures directly from unrefined macroalgal biomass. P5AFcnA released oligosaccharides from seven commercial fucoidan extracts in addition to unrefined biomass of three macroalgae species indicating a broadly applicable approach reproducible across 10 species. Both MALDI-TOF/TOF and AP-MALDI-Orbitrap systems were used, demonstrating that the approach is not instrument-specific and exploiting their combined high-throughput and high-resolution capabilities. Overall, the combination of MALDI-MS and endo-fucoidanase assays offers high-throughput evaluation of fucoidan samples and also enables extraction of defined oligosaccharides of known structure from unrefined seaweed biomass.


Assuntos
Glicosídeo Hidrolases , Polissacarídeos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Polissacarídeos/química , Glicosídeo Hidrolases/metabolismo , Glicosídeo Hidrolases/química , Hidrólise , Alga Marinha/química , Phaeophyceae/química , Phaeophyceae/enzimologia , Oligossacarídeos/química , Biomassa
7.
Methods Mol Biol ; 2792: 19-27, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38861075

RESUMO

Besides the historical and traditional use of nuclear magnetic resonance (NMR) spectroscopy as a structure elucidation tool for proteins and metabolites, its quantification ability allows the determination of metabolite amounts and therefore enzymatic activity measurements. For this purpose, 1H-NMR with adapted water pulse pre-saturation sequences and calibration curves with commercial standard solutions can be used to quantify the photorespiratory cycle intermediates, 2-phosphoglycolate and glycolate, associated with the phosphoglycolate phosphatase reaction. The intensity of the 1H-NMR signal of glycolate produced by the activity of purified recombinant Arabidopsis thaliana PGLP1 can therefore be used to determine PGLP1 enzymatic activities and kinetic parameters.


Assuntos
Arabidopsis , Glicolatos , Espectroscopia de Ressonância Magnética , Monoéster Fosfórico Hidrolases , Glicolatos/metabolismo , Glicolatos/química , Monoéster Fosfórico Hidrolases/metabolismo , Arabidopsis/metabolismo , Arabidopsis/enzimologia , Espectroscopia de Ressonância Magnética/métodos , Proteínas de Arabidopsis/metabolismo , Ensaios Enzimáticos/métodos , Cinética , Proteínas Recombinantes/metabolismo
8.
Methods Mol Biol ; 2792: 29-39, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38861076

RESUMO

Phosphoglycolate phosphatase (PGLP) dephosphorylates 2-phosphoglycolate to glycolate that can be further metabolized to glyoxylate by glycolate oxidase (GOX) via an oxidative reaction that uses O2 and releases H2O2. The oxidation of o-dianisidine by H2O2 catalyzed by a peroxidase can be followed in real time by an absorbance change at 440 nm. Based on these reactions, a spectrophotometric method for measuring PGLP activity using a coupled reaction with recombinant Arabidopsis thaliana GOX is described. This protocol has been used successfully with either purified PGLP or total soluble proteins extracted from Arabidopsis rosette leaves.


Assuntos
Oxirredutases do Álcool , Arabidopsis , Monoéster Fosfórico Hidrolases , Proteínas Recombinantes , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Arabidopsis/enzimologia , Arabidopsis/genética , Arabidopsis/metabolismo , Oxirredutases do Álcool/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/química , Monoéster Fosfórico Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases/genética , Glicolatos/metabolismo , Ensaios Enzimáticos/métodos , Peróxido de Hidrogênio/metabolismo , Oxirredução , Folhas de Planta/metabolismo , Folhas de Planta/enzimologia , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Espectrofotometria/métodos
9.
Methods Mol Biol ; 2792: 51-75, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38861078

RESUMO

Mitochondrial dihydrolipoamide dehydrogenase (mtLPD1) is a central enzyme in primary carbon metabolism, since its function is required to drive four multienzymes involved in photorespiration, the tricarboxylic acid (TCA) cycle, and the degradation of branched-chain amino acids. However, in illuminated, photosynthesizing tissue a vast amount of mtLPD1 is necessary for glycine decarboxylase (GDC), the key enzyme of photorespiration. In light of the shared role, the functional characterization of mtLPD1 is necessary to understand how the three pathways might interact under different environmental scenarios. This includes the determination of the biochemical properties and all potential regulatory mechanisms, respectively. With regards to the latter, regulation can occur through multiple levels including effector molecules, cofactor availability, or posttranslational modifications (PTM), which in turn decrease or increase the activity of each enzymatic reaction. Gaining a comprehensive overview on all these aspects would ultimately facilitate the interpretation of the metabolic interplay of the pathways within the whole subcellular network or even function as a proof of concept for genetic engineering approaches. Here, we describe the typical workflow how to clone, express, and purify plant mtLPD1 for biochemical characterization and how to analyze potential redox regulatory mechanisms in vitro and in planta.


Assuntos
Di-Hidrolipoamida Desidrogenase , Oxirredução , Di-Hidrolipoamida Desidrogenase/metabolismo , Di-Hidrolipoamida Desidrogenase/genética , Mitocôndrias/metabolismo , Mitocôndrias/genética , Mitocôndrias/enzimologia , Arabidopsis/genética , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Clonagem Molecular/métodos
10.
Methods Mol Biol ; 2792: 3-17, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38861074

RESUMO

Determining enzyme activities involved in photorespiration, either in a crude plant tissue extract or in a preparation of a recombinant enzyme, is time-consuming, especially when large number of samples need to be processed. This chapter presents a phosphoglycolate phosphatase (PGLP) activity assay that is adapted for use in a 96-well microplate format. The microplate format for the assay requires fewer enzymes and reagents and allows rapid and less expensive measurement of PGLP enzyme activity. The small volume of reaction mix in a 96-well microplate format enables the determination of PGLP enzyme activity for screening many plant samples, multiple enzyme activities using the same protein extract, and/or identifying kinetic parameters for a recombinant enzyme. To assist in preparing assay reagents, we also present an R Shiny buffer preparation app for PGLP and other photorespiratory enzyme activities and a Km and Vmax calculation app.


Assuntos
Ensaios Enzimáticos , Monoéster Fosfórico Hidrolases , Extratos Vegetais , Folhas de Planta , Proteínas Recombinantes , Folhas de Planta/química , Folhas de Planta/metabolismo , Folhas de Planta/enzimologia , Monoéster Fosfórico Hidrolases/metabolismo , Cinética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Ensaios Enzimáticos/métodos , Extratos Vegetais/química , Ensaios de Triagem em Larga Escala/métodos
11.
Methods Mol Biol ; 2792: 83-95, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38861080

RESUMO

We describe an assay for measuring the activity of D-glycerate 3-kinase (GLYK) in a 96-well microplate format with the use of a set of coupling enzymes. The assay is appropriate for use with a crude protein extract prepared from leaf tissue and with the recombinant purified enzyme. The 96-well microplate format reduces the needed amounts of reagents and coupling enzymes, making the assay less expensive, high throughput, and suitable for the determination of kinetic parameters Km and Vmax. In addition, we provide a two-step discontinuous assay modified from past work, making it possible to measure the activity of GLYK at temperatures higher than 45 °C.


Assuntos
Ensaios Enzimáticos , Extratos Vegetais , Folhas de Planta , Proteínas Recombinantes , Folhas de Planta/química , Folhas de Planta/enzimologia , Proteínas Recombinantes/metabolismo , Cinética , Ensaios Enzimáticos/métodos , Extratos Vegetais/química , Ensaios de Triagem em Larga Escala/métodos
12.
Planta ; 260(1): 26, 2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38861179

RESUMO

MAIN CONCLUSION: CaTPS2 and CaTPS3 were significantly expressed in flowers of Curcuma alismatifolia 'Shadow' and demonstrated bifunctional enzyme activity, CaTPS2 generated linalool and nerolidol as products, and CaTPS3 catalyzed ß-myrcene and ß-farnesene formation. This study presents the discovery and functional characterization of floral terpene synthase (TPS) genes in Curcuma alismatifolia 'Shadow', a cultivar renowned for its unique fragrance. Addressing the gap in understanding the genetic basis of floral scent in this species, we identified eight TPS genes through comprehensive transcriptome sequencing. Among these, CaTPS2 and CaTPS3 were significantly expressed in floral tissues and demonstrated bifunctional enzyme activity corresponding to the major volatile compounds detected in 'Shadow'. Functional analyses, including in vitro assays complemented with rigorous controls and alternative identification methods, elucidated the roles of these TPS genes in terpenoid biosynthesis. In vitro studies were conducted via heterologous expression in E. coli, followed by purification of the recombinant protein using affinity chromatography, enzyme assays were performed with GPP/FPP as the substrate, and volatile products were inserted into the GC-MS for analysis. Partially purified recombinant protein of CaTPS2 catalyzed GPP and FPP to produce linalool and nerolidol, respectively, while partially purified recombinant protein of CaTPS3 generated ß-myrcene and ß-farnesene with GPP and FPP as substrates, respectively. Real-time quantitative PCR further validated the expression patterns of these genes, correlating with terpenoid accumulation in different plant tissues. Our findings illuminate the molecular mechanisms underpinning floral fragrance in C. alismatifolia and provide a foundation for future genetic enhancements of floral scent in ornamental plants. This study, therefore, contributes to the broader understanding of terpenoid biosynthesis in plant fragrances, paving the way for biotechnological applications in horticulture plant breeding.


Assuntos
Monoterpenos Acíclicos , Alquil e Aril Transferases , Curcuma , Flores , Sesquiterpenos , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Flores/genética , Flores/enzimologia , Flores/metabolismo , Sesquiterpenos/metabolismo , Monoterpenos Acíclicos/metabolismo , Curcuma/genética , Curcuma/enzimologia , Curcuma/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas , Terpenos/metabolismo , Compostos Orgânicos Voláteis/metabolismo , Filogenia , Odorantes
13.
J Agric Food Chem ; 72(23): 12935-12945, 2024 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-38822796

RESUMO

Blister beetles of Epicauta impressicornis have attracted attention because they contain a large amount of cantharidin (CTD). To date, however, the synthesis and transfer of CTD in adults of E. impressicornis are largely unknown. Here, we showed that the larvae E. impressicornis are capable of synthesizing CTD and they consume CTD during pupation. Before sexual maturity, both male and female adults synthesized a small amount of CTD, while after sexual maturity, males produced larger amounts of CTD, but females did not. The newly synthesized CTD in males first appeared in the hemolymph and then accumulated in the reproductive system. During the mating, the males transferred CTD to the reproductive system of females. In addition, a farnesyl pyrophosphate synthase (FPPS) gene was identified in male E. impressicornis. RNA-seq analysis, quantitative RT-PCR, and RNA interference analyses were conducted to investigate expression patterns and the functional roles of E. impressicornis FPPS (EiFPPS). Our results indicate that EiFPPS is highly expressed in the fat body of males. Moreover, the knock-down of EiFPPS led to a significant decrease in CTD synthesis. The current study indicates that EiFPPS is expressed in the fat body to regulate CTD synthesis in male E. impressicornis blister beetles.


Assuntos
Cantaridina , Besouros , Corpo Adiposo , Geraniltranstransferase , Proteínas de Insetos , Animais , Besouros/genética , Besouros/metabolismo , Besouros/enzimologia , Cantaridina/metabolismo , Masculino , Corpo Adiposo/metabolismo , Corpo Adiposo/enzimologia , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Geraniltranstransferase/genética , Geraniltranstransferase/metabolismo , Feminino , Larva/crescimento & desenvolvimento , Larva/genética , Larva/metabolismo
14.
Biochem J ; 481(12): 779-791, 2024 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-38829839

RESUMO

ent-Kaurene is a biosynthetic intermediate diterpene of phytohormone gibberellins, and is biosynthesized from geranylgeranyl diphosphate via ent-copalyl diphosphate (ent-CDP). The successive cyclization is catalyzed by two distinct diterpene synthases, ent-CDP synthase (ent-CPS) and ent-kaurene synthase (KS). Homologs of these diterpene synthase genes have been reported to be involved in the biosynthesis of specialized-metabolic diterpenoids for defense in several plant species, including rice (Oryza sativa). These diterpene synthases consist of three domains, αßγ domains. Active sites of ent-CPS exist at the interface of ß and γ domain, while those of KS are located within the α domain. We herein carried out domain-deletion experiments using several KSs and KS like enzymes (KSLs) to obtain insights into the roles of domains other than active-site domains. As previously reported in taxadiene synthase, deletion of γ or ßγ domains drastically decreased activities of specialized-metabolic OsKSL5, OsKSL8, OsKSL7 and OsKSL10 in O. sativa. However, unexpectedly, only α domains of several gibberellin-biosynthetic KSs, including OsKS1 in O. sativa, AtKS in Arabidopsis thaliana, TaKS in wheat (Triticum aestivum) and BdKS1 in Brachypodium distachyon, retained their original functions. Additionally, the specialized-metabolic OsKSL4, which is closely related to OsKS1, also functioned without its ßγ domains. Domain-swapping experiments showed that replacing ßγ domains in OsKSL7 with those from other KS/KSLs retained the OsKSL7 activity. Moreover, deletion of ßγ domains of bifunctional PpCPS/KS in moss (Physcomitrella patens) drastically impaired its KS-related activity. Thus, we demonstrate that monofunctional gibberellin-biosynthetic KSs are the unique diterpene synthases that retain their functions without ßγ domains.


Assuntos
Alquil e Aril Transferases , Giberelinas , Oryza , Proteínas de Plantas , Giberelinas/metabolismo , Alquil e Aril Transferases/metabolismo , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/química , Oryza/enzimologia , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/química , Domínio Catalítico , Diterpenos do Tipo Caurano/metabolismo , Diterpenos do Tipo Caurano/química , Arabidopsis/genética , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Diterpenos/metabolismo , Diterpenos/química , Domínios Proteicos , Catálise
15.
J Agric Food Chem ; 72(23): 13297-13307, 2024 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-38830127

RESUMO

2-(2-Phenylethyl)chromones (PECs) are the primary constituents responsible for the promising pharmacological activities and unique fragrance of agarwood. However, the O-methyltransferases (OMTs) involved in the formation of diverse methylated PECs have not been reported. In this study, we identified one Mg2+-dependent caffeoyl-CoA-OMT subfamily enzyme (AsOMT1) and three caffeic acid-OMT subfamily enzymes (AsOMT2-4) from NaCl-treated Aquilaria sinensis calli. AsOMT1 not only converts caffeoyl-CoA to feruloyl-CoA but also performs nonregioselective methylation at either the 6-OH or 7-OH position of 6,7-dihydroxy-PEC. On the other hand, AsOMT2-4 preferentially utilizes PECs as substrates to produce structurally diverse methylated PECs. Additionally, AsOMT2-4 also accepts nonPEC-type substrates such as caffeic acid and apigenin to generate methylated products. Protein structure prediction and site-directed mutagenesis revealed that residues of L313 and I318 in AsOMT3, as well as S292 and F313 in AsOMT4 determine the distinct regioselectivity of these two OMTs toward apigenin. These findings provide important biochemical evidence of the remarkable structural diversity of PECs in agarwood.


Assuntos
Metiltransferases , Proteínas de Plantas , Thymelaeaceae , Metiltransferases/genética , Metiltransferases/química , Metiltransferases/metabolismo , Thymelaeaceae/enzimologia , Thymelaeaceae/química , Thymelaeaceae/genética , Proteínas de Plantas/genética , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Madeira/química , Especificidade por Substrato , Ácidos Cafeicos/química , Ácidos Cafeicos/metabolismo , Metilação , Flavonoides
16.
BMC Vet Res ; 20(1): 242, 2024 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-38831422

RESUMO

BACKGROUND: ATPase activity and the antioxidant function of intestinal tissue can reflect intestinal cell metabolic activity and oxidative damage, which might be related to intestinal function. However, the specific influence of intestinal ATPase activity and antioxidant function on growth performance, feed conversion efficiency, and the intestinal microbiota in sheep remains unclear. RESULTS: This study analyzed the correlation between ATPase activity and antioxidant function in the jejunum of 92 Hu sheep and their growth performance and feed conversion efficiency. Additionally, individuals with the highest (H group) and lowest (L group) jejunum MDA content and Na+ K+-ATPase activity were further screened, and the effects of jejunum ATPase activity and MDA content on the morphology and microbial community of sheep intestines were analyzed. There was a significant correlation between jejunum ATPase and SOD activity and the initial weight of Hu sheep (P < 0.01). The H-MDA group exhibited significantly higher average daily gain (ADG) from 0 to 80 days old and higher body weight (BW) after 80 days. ATPase and SOD activities, and MDA levels correlated significantly and positively with heart weight. The jejunum crypt depth and circular muscle thickness in the H-ATP group were significantly higher than in the L-ATP group, and the villus length, crypt depth, and longitudinal muscle thickness in the H-MDA group were significantly higher than in the L-MDA group (P < 0.01). High ATPase activity and MDA content significantly reduced the jejunum microbial diversity, as indicated by the Chao1 index and observed species, and affected the relative abundance of specific taxa. Among species, the relative abundance of Olsenella umbonata was significantly higher in the H-MDA group than in the L-MDA group (P < 0.05), while Methanobrevibacter ruminantium abundance was significantly lower than in the L-MDA group (P < 0.05). In vitro culture experiments confirmed that MDA promoted the proliferation of Olsenella umbonata. Thus, ATPase and SOD activities in the jejunum tissues of Hu sheep are predominantly influenced by congenital factors, and lambs with higher birth weights exhibit lower Na+ K+-ATPase, Ca2+ Mg2+-ATPase, and SOD activities. CONCLUSIONS: The ATPase activity and antioxidant performance of intestinal tissue are closely related to growth performance, heart development, and intestinal tissue morphology. High ATPase activity and MDA content reduced the microbial diversity of intestinal tissue and affect the relative abundance of specific taxa, representing a potential interaction between the host and its intestinal microbiota.


Assuntos
Adenosina Trifosfatases , Antioxidantes , Microbioma Gastrointestinal , Jejuno , Animais , Jejuno/microbiologia , Jejuno/enzimologia , Antioxidantes/metabolismo , Microbioma Gastrointestinal/fisiologia , Adenosina Trifosfatases/metabolismo , Ovinos , Masculino , Malondialdeído/metabolismo , Superóxido Dismutase/metabolismo
17.
J Vet Sci ; 25(3): e48, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38834516

RESUMO

IMPORTANCE: Early diagnosis of canine pancreatitis is challenging due to non-specific clinical signs. Currently, abdominal ultrasonography and measurement of canine pancreatic lipase (cPL) have been employed for the diagnosis of pancreatitis. OBJECTIVE: Many qualitative and quantitative commercial cPL tests have been developed and used in veterinary clinics. This study aimed to compare three different methodologies SNAP cPL, Spec cPL, and Vcheck cPL tests to assess the concordance of these assays. METHODS: Fifty serum samples were collected from 36 dogs with or without pancreatitis and subjected to SNAP cPL, Spec cPL, and Vcheck cPL tests. Agreement and correlation coefficients were calculated between the test results, and correlations were determined during the management of the patients. RESULTS: The results of the three cPL assays were strongly correlated in 47/50 serum samples (94%). Cohen's kappa analysis between the Spec cPL and Vcheck cPL showed near perfect agreement (κ = 0.960, p < 0.001), SNAP cPL and Vcheck cPL (κ = 0.920, p < 0.001), and Spec cPL and SNAP cPL (κ = 0.880, p < 0.001). The correlation coefficients (r) between data from Spec cPL and Vcheck cPL tests was calculated by Spearman's correlation test (r = 0.958, p < 0.001). Furthermore, the patterns of change in serum cPL concentrations determined using Spec cPL and Vcheck cPL were significantly consistent during the monitoring period in 11 patients. CONCLUSIONS AND RELEVANCE: Our data illustrated that Spec cPL and Vcheck cPL tests are compatible for clinical use in the diagnosis and monitoring of canine pancreatitis.


Assuntos
Doenças do Cão , Lipase , Pancreatite , Animais , Cães , Lipase/sangue , Pancreatite/veterinária , Pancreatite/diagnóstico , Pancreatite/sangue , Doenças do Cão/diagnóstico , Doenças do Cão/sangue , Masculino , Feminino , Pâncreas/enzimologia
18.
J Enzyme Inhib Med Chem ; 39(1): 2346523, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38847581

RESUMO

Toxoplasmosis, induced by the intracellular parasite Toxoplasma gondii, holds considerable implications for global health. While treatment options primarily focusing on folate pathway enzymes have notable limitations, current research endeavours concentrate on pinpointing specific metabolic pathways vital for parasite survival. Carbonic anhydrases (CAs, EC 4.2.1.1) have emerged as potential drug targets due to their role in fundamental reactions critical for various protozoan metabolic processes. Within T. gondii, the Carbonic Anhydrase-Related Protein (TgCA_RP) plays a pivotal role in rhoptry biogenesis. Notably, α-CA (TcCA) from another protozoan, Trypanosoma cruzi, exhibited considerable susceptibility to classical CA inhibitors (CAIs) such as anions, sulphonamides, thiols, and hydroxamates. Here, the recombinant DNA technology was employed to synthesise and clone the identified gene in the T. gondii genome, which encodes an α-CA protein (Tg_CA), with the purpose of heterologously overexpressing its corresponding protein. Tg_CA kinetic constants were determined, and its inhibition patterns explored with inorganic metal-complexing compounds, which are relevant for rational compound design. The significance of this study lies in the potential development of innovative therapeutic strategies that disrupt the vital metabolic pathways crucial for T. gondii survival and virulence. This research may lead to the development of targeted treatments, offering new approaches to manage toxoplasmosis.


Assuntos
Inibidores da Anidrase Carbônica , Anidrases Carbônicas , Clonagem Molecular , Toxoplasma , Toxoplasma/enzimologia , Anidrases Carbônicas/metabolismo , Anidrases Carbônicas/genética , Cinética , Inibidores da Anidrase Carbônica/farmacologia , Inibidores da Anidrase Carbônica/química , Inibidores da Anidrase Carbônica/síntese química , Relação Estrutura-Atividade , Relação Dose-Resposta a Droga , Estrutura Molecular , Ânions/química , Ânions/farmacologia , Ânions/metabolismo
19.
Curr Microbiol ; 81(7): 217, 2024 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-38852107

RESUMO

The application of enzymes in agricultural fields has been little explored. One potential application of fungal lytic enzymes (chitinases, lipases, and proteases) is as an additive to current biopesticides to increase their efficacy and reduce the time of mortality. For this, a screening of lytic overproducer fungi under submerged fermentation with a chemical-defined medium was performed. Then, the enzymatic crude extract (ECE) was concentrated and partially characterized. This characterization consisted of measuring the enzymatic activity (lipase, protease and, chitinase) and determining the enzyme stability after storage at temperatures of - 80, - 20 and, 4 °C. And lastly, the application of these concentrated enzymatic crude extracts (C-ECE) as an enhancer of spores-based fungal biopesticide was proven. Beauveria were not as good producers of lytic enzymes as the strains from Trichoderma and Metarhizium. The isolate M. robertsii Mt015 was selected for the co-production of chitinases and proteases; and the isolate T. harzianum Th180 for co-production of chitinases, lipases, and proteases. The C-ECE of Mt015 had a protease activity of 18.6 ± 1.1 U ml-1, chitinase activity of 0.28 ± 0.01 U ml-1, and no lipase activity. Meanwhile, the C-ECE of Th180 reached a chitinase activity of 0.75 U ml-1, lipase activity of 0.32 U ml-1, and protease activity of 0.24 U ml-1. Finally, an enhancing effect of the enzymatic extracts of M. robertsii (66.7%) and T. harzianum (43.5%) on the efficacy of B. bassiana Bv064 against Diatraea saccharalis larvae was observed. This work demonstrates the non-species-specific enhancing effect of enzymatic extracts on the insecticidal activity of conidial-based biopesticides, which constitutes a contribution to the improvement of biological control agents' performance.


Assuntos
Quitinases , Fermentação , Peptídeo Hidrolases , Quitinases/metabolismo , Peptídeo Hidrolases/metabolismo , Animais , Lipase/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Agentes de Controle Biológico/farmacologia , Agentes de Controle Biológico/metabolismo , Fungos/metabolismo , Controle Biológico de Vetores/métodos , Beauveria/enzimologia , Beauveria/metabolismo , Estabilidade Enzimática
20.
Respir Res ; 25(1): 230, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38824593

RESUMO

BACKGROUND: Airway epithelium is an important component of airway structure and the initiator of airway remodeling in asthma. The changes of extracellular matrix (ECM), such as collagen deposition and structural disturbance, are typical pathological features of airway remodeling. Thus, identifying key mediators that derived from airway epithelium and capable of modulating ECM may provide valuable insights for targeted therapy of asthma. METHODS: The datasets from Gene Expression Omnibus database were analyzed to screen differentially expressed genes in airway epithelium of asthma. We collected bronchoscopic biopsies and serum samples from asthmatic and healthy subjects to assess lysyl oxidase like 2 (LOXL2) expression. RNA sequencing and various experiments were performed to determine the influences of LOXL2 knockdown in ovalbumin (OVA)-induced mouse models. The roles and mechanisms of LOXL2 in bronchial epithelial cells were explored using LOXL2 small interfering RNA, overexpression plasmid and AKT inhibitor. RESULTS: Both bioinformatics analysis and further experiments revealed that LOXL2 is highly expressed in airway epithelium of asthmatics. In vivo, LOXL2 knockdown significantly inhibited OVA-induced ECM deposition and epithelial-mesenchymal transition (EMT) in mice. In vitro, the transfection experiments on 16HBE cells demonstrated that LOXL2 overexpression increases the expression of N-cadherin and fibronectin and reduces the expression of E-cadherin. Conversely, after silencing LOXL2, the expression of E-cadherin is up-regulated. In addition, the remodeling and EMT process that induced by transforming growth factor-ß1 could be enhanced and weakened after LOXL2 overexpression and silencing in 16HBE cells. Combining the RNA sequencing of mouse lung tissues and experiments in vitro, LOXL2 was involved in the regulation of AKT signaling pathway. Moreover, the treatment with AKT inhibitor in vitro partially alleviated the consequences associated with LOXL2 overexpression. CONCLUSIONS: Taken together, the results demonstrated that epithelial LOXL2 plays a role in asthmatic airway remodeling partly via the AKT signaling pathway and highlighted the potential of LOXL2 as a therapeutic target for airway remodeling in asthma.


Assuntos
Remodelação das Vias Aéreas , Aminoácido Oxirredutases , Asma , Ovalbumina , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Animais , Aminoácido Oxirredutases/metabolismo , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/biossíntese , Ovalbumina/toxicidade , Remodelação das Vias Aéreas/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Camundongos , Humanos , Asma/patologia , Asma/metabolismo , Asma/enzimologia , Asma/genética , Transdução de Sinais/fisiologia , Feminino , Camundongos Endogâmicos BALB C , Masculino , Transição Epitelial-Mesenquimal/fisiologia
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