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1.
Sheng Wu Gong Cheng Xue Bao ; 40(4): 1076-1088, 2024 Apr 25.
Artículo en Chino | MEDLINE | ID: mdl-38658150

RESUMEN

Flavin-containing monooxygenase (FMO) is the key enzyme in the biosynthesis pathway of CSOs with sulfur oxidation. In order to explore the molecular regulatory mechanism of FMO in the synthesis of onion CSOs, based on transcriptome database and phylogenetic analysis, one AcFMO gene that may be involved in alliin synthesis was obtained, the AcFMO had a cDNA of 1 374 bp and encoded 457 amino acids, which was evolutionarily closest to the AsFMO of garlic. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) indicated that AcFMO was the highest in the flowers and the lowest in the leaf sheaths. The results of subcellular localization showed that the AcFMO gene product was widely distributed throughout the cell A yeast expression vector was constructed, and the AcFMO gene was ecotopically overexpressed in yeast to further study the enzyme function in vitro and could catalyze the synthesis of alliin by S-allyl-l-cysteine. In summary, the cloning and functional identification of AcFMO have important reference value for understanding the biosynthesis of CSOs in onions.


Asunto(s)
Clonación Molecular , Cisteína/análogos & derivados , Cebollas , Cebollas/genética , Cebollas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Cisteína/biosíntesis , Cisteína/metabolismo , Oxigenasas/genética , Oxigenasas/metabolismo , Secuencia de Aminoácidos , Filogenia , Disulfuros/metabolismo , Datos de Secuencia Molecular , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
2.
Pestic Biochem Physiol ; 156: 72-79, 2019 May.
Artículo en Inglés | MEDLINE | ID: mdl-31027583

RESUMEN

Metalloenzyme SODs play important roles in insects dealing with environmental stress. Here, we cloned the Cu/ZnSOD (LdCZS) and MnSOD (LdMS) mRNA of Lymantria dispar by rapid amplification of cDNA ends (RACE). Afterwards their expression patterns were detected by quantitative real-time polymerase chain reaction (qPCR) after bioinformatic analysis. We found that both LdCZS and LdMS were widely detected in all gypsy moth larvae and all five tissues that we analyzed, and both of them were up-regulated after larvae were fed with avermectin of sublethal concentration and LC10. The LdCZS expression value are always higher than LdMS after treating with avermectin of sublethal concentrations. In addition, temporal expression profile in avermectin treated larvae showed that LdCZS expressed highest at 2nd hour, and LdMS expressed highest at 6th hour. The cuticulas transcribed LdCZS and LdMS significantly higher than heads, fat bodies, Malpighian tubes, and midguts after spraying avermectin of sublethal concentration. These results suggested that both Cu/ZnSOD and MnSOD are important antioxidant enzymes in L. dispar defensing against pesticide stress, and LdCZS always responded rapider and stronger than LdMS.


Asunto(s)
Ivermectina/análogos & derivados , Larva/metabolismo , Mariposas Nocturnas/metabolismo , Superóxido Dismutasa/metabolismo , Secuencia de Aminoácidos , Animales , Biología Computacional , ADN Complementario/genética , Ivermectina/farmacología , Larva/efectos de los fármacos , Larva/genética , Datos de Secuencia Molecular , Mariposas Nocturnas/efectos de los fármacos , Mariposas Nocturnas/genética , Plaguicidas/farmacología , Reacción en Cadena de la Polimerasa , Superóxido Dismutasa/química , Superóxido Dismutasa/genética
3.
J Agric Food Chem ; 66(48): 12748-12755, 2018 Dec 05.
Artículo en Inglés | MEDLINE | ID: mdl-30441891

RESUMEN

Microalgae starch is receiving increasing attention as a renewable feedstock for biofuel production. Raw microalgae starch from Tetraselmis subcordiformis was proven to be very efficiently hydrolyzed by an α-amylase (AmyP) of glycoside hydrolase subfamily GH13_37 below the temperature of gelatinization (40 °C). The hydrolysis degree reached 74.4 ± 2.2% for 4% raw microalgae starch and 53.2 ± 1.7% for 8% raw microalgae starch after only 2 h. The hydrolysis efficiency was significantly stimulated by calcium ions. The enzyme catalysis of AmyP and its mutants (Q306A and E347A) suggested that calcium ions contributed to the hydrolysis of cyclic structures in raw microalgae starch by a distinctive calcium-binding site Ca2 of AmyP. The study explored raw microalgae starch as a new resource for cold enzymatic hydrolysis and extended our knowledge on the function of calcium in amylolytic enzyme.


Asunto(s)
Proteínas Bacterianas/química , Chlorophyta/química , Microalgas/química , Extractos Vegetales/química , Almidón/química , alfa-Amilasas/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biocatálisis , Glicósido Hidrolasas/química , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Hidrólisis , Cinética , Datos de Secuencia Molecular , Familia de Multigenes , Extractos Vegetales/metabolismo , Alineación de Secuencia , Almidón/metabolismo , Especificidad por Sustrato , Temperatura , alfa-Amilasas/genética , alfa-Amilasas/metabolismo
4.
Sci Rep ; 8(1): 13405, 2018 09 07.
Artículo en Inglés | MEDLINE | ID: mdl-30194320

RESUMEN

Thymol, as a dietary monoterpene, is a phenol derivative of cymene, which is the major component of the essential oil of Trachyspermum ammi (L.). It shows multiple biological activities: antifungal, antibacterial, antivirus and anti-inflammatory. T. ammi, commonly known as ajowan, belongs to Apiaceae and is an important medicinal seed spice. To identify the putative genes involved in thymol and other monoterpene biosynthesis, we provided transcriptomes of four inflorescence tissues of two ajowan ecotypes, containing different thymol yield. This study has detected the genes encoding enzymes for the go-between stages of the terpenoid biosynthesis pathways. A large number of unigenes, differentially expressed between four inflorescence tissues of two ajowan ecotypes, was revealed by a transcriptome analysis. Furthermore, differentially expressed unigenes encoding dehydrogenases, transcription factors, and cytochrome P450s, which might be associated with terpenoid diversity in T. ammi, were identified. The sequencing data obtained in this study formed a valuable repository of genetic information for an understanding of the formation of the main constituents of ajowan essential oil and functional analysis of thymol-specific genes. Comparative transcriptome analysis led to the development of new resources for a functional breeding of ajowan.


Asunto(s)
Apiaceae , Regulación de la Expresión Génica de las Plantas/fisiología , Plantas Medicinales , Timol/metabolismo , Transcriptoma/fisiología , Apiaceae/genética , Apiaceae/metabolismo , Vías Biosintéticas/fisiología , Sistema Enzimático del Citocromo P-450/biosíntesis , Sistema Enzimático del Citocromo P-450/genética , Inflorescencia/citología , Inflorescencia/metabolismo , Datos de Secuencia Molecular , Oxidorreductasas/biosíntesis , Oxidorreductasas/genética , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/genética , Plantas Medicinales/genética , Plantas Medicinales/metabolismo , Factores de Transcripción/metabolismo
5.
J Agric Food Chem ; 66(34): 9034-9041, 2018 Aug 29.
Artículo en Inglés | MEDLINE | ID: mdl-30085665

RESUMEN

A novel KG51 gene was isolated from a metagenomic library of Korean black goat rumen and its recombinant protein was characterized as a bifunctional enzyme (cellulase/hemicellulase). In silico sequence and domain analyses revealed that the KG51 gene encodes a novel carbohydrate-active enzyme that possesses a salad-bowl-like shaped glycosyl hydrolase family 5 (GH5) catalytic domain but, at best, 41% sequence identity with other homologous GH5 proteins. Enzymatic profiles (optimum pH values and temperatures, as well as pH and thermal stabilities) of the recombinant KG51 bifunctional enzyme were also determined. On the basis of the substrate specificity data, the KG51 enzyme exhibited relatively strong cellulase (endo-ß-1,4-glucanase [EC 3.2.1.4]) and hemicellulase (mannan endo-ß-1,4-mannosidase [EC 3.2.1.78] and endo-ß-1,4-xylanase [EC 3.2.1.8]) activities, but no exo-ß-1,4-glucanase (EC 3.2.1.74), exo-ß-1,4-glucan cellobiohydrolase (EC 3.2.1.91), and exo-1,4-ß-xylosidase (EC 3.2.1.37) activities. Finally, the potential industrial applicability of the KG51 enzyme was tested in the preparation of prebiotic konjac glucomannan hydrolysates.


Asunto(s)
Celulasa/química , Glicósido Hidrolasas/química , Cabras/genética , Rumen/enzimología , Secuencia de Aminoácidos , Amorphophallus/química , Animales , Celulasa/genética , Celulasa/metabolismo , Estabilidad de Enzimas , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Concentración de Iones de Hidrógeno , Mananos/química , Metagenómica , Datos de Secuencia Molecular , Extractos Vegetales/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rumen/química , Alineación de Secuencia , Especificidad por Sustrato , Temperatura
6.
J Agric Food Chem ; 66(22): 5699-5706, 2018 Jun 06.
Artículo en Inglés | MEDLINE | ID: mdl-29756783

RESUMEN

CJP-4 is an allergen found in pollen of the Japanese cedar Cryptomeria japonica. The protein is a two-domain family GH19 (class IV) Chitinase consisting of an N-terminal CBM18 domain and a GH19 catalytic domain. Here, we produced recombinant CJP-4 and CBM18-truncated CJP-4 (CJP-4-Cat) proteins. In addition to solving the crystal structure of CJP-4-Cat by X-ray crystallography, we analyzed the ability of both proteins to hydrolyze chitin oligosaccharides, (GlcNAc) n, polysaccharide substrates, glycol chitin, and ß-chitin nanofiber and examined their inhibitory activity toward fungal growth. Truncation of the CBM18 domain did not significantly affect the mode of (GlcNAc) n hydrolysis. However, significant effects were observed when we used the polysaccharide substrates. The activity of CJP-4 toward the soluble substrate, glycol chitin, was lower than that of CJP-4-Cat. In contrast, CJP-4 exhibited higher activity toward ß-chitin nanofiber, an insoluble substrate, than did CJP-4-Cat. Fungal growth was strongly inhibited by CJP-4 but not by CJP-4-Cat. These results indicate that the CBM18 domain assists the hydrolysis of insoluble substrate and the antifungal action of CJP-4-Cat by binding to chitin. CJP-4-Cat was found to have only two loops (loops I and III), as reported for ChiA, an allergenic class IV Chitinase from maize.


Asunto(s)
Quitinasas/química , Cryptomeria/enzimología , Proteínas de Plantas/química , Polen/enzimología , Secuencia de Aminoácidos , Dominio Catalítico , Quitinasas/genética , Quitinasas/metabolismo , Cryptomeria/química , Cryptomeria/genética , Hidrólisis , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Polen/química , Unión Proteica , Homología de Secuencia de Aminoácido , Especificidad por Sustrato
7.
mBio ; 9(3)2018 05 08.
Artículo en Inglés | MEDLINE | ID: mdl-29739899

RESUMEN

The precursors of the diffusible signal factor (DSF) family signals of Xanthomonas campestris pv. campestris are 3-hydroxyacyl-acyl carrier protein (3-hydroxyacyl-ACP) thioesters having acyl chains of 12 to 13 carbon atoms produced by the fatty acid biosynthetic pathway. We report a novel 3-oxoacyl-ACP reductase encoded by the X. campestris pv. campestris XCC0416 gene (fabG2), which is unable to participate in the initial steps of fatty acyl synthesis. This was shown by the failure of FabG2 expression to allow growth at the nonpermissive temperature of an Escherichia colifabG temperature-sensitive strain. However, when transformed into the E. coli strain together with a plasmid bearing the Vibrio harveyi acyl-ACP synthetase gene (aasS), growth proceeded, but only when the medium contained octanoic acid. In vitro assays showed that FabG2 catalyzes the reduction of long-chain (≥C8) 3-oxoacyl-ACPs to 3-hydroxyacyl-ACPs but is only weakly active with shorter-chain (C4, C6) substrates. FabG1, the housekeeping 3-oxoacyl-ACP reductase encoded within the fatty acid synthesis gene cluster, could be deleted in a strain that overexpressed fabG2 but only in octanoic acid-supplemented media. Growth of the X. campestris pv. campestris ΔfabG1 strain overexpressing fabG2 required fabH for growth with octanoic acid, indicating that octanoyl coenzyme A is elongated by X. campestris pv. campestrisfabH Deletion of fabG2 reduced DSF family signal production, whereas overproduction of either FabG1 or FabG2 in the ΔfabG2 strain restored DSF family signal levels.IMPORTANCE Quorum sensing mediated by DSF signaling molecules regulates pathogenesis in several different phytopathogenic bacteria, including Xanthomonas campestris pv. campestris DSF signaling also plays a key role in infection by the human pathogen Burkholderia cepacia The acyl chains of the DSF molecules are diverted and remodeled from a key intermediate of the fatty acid synthesis pathway. We report a Xanthomonas campestris pv. campestris fatty acid synthesis enzyme, FabG2, of novel specificity that seems tailored to provide DSF signaling molecule precursors.


Asunto(s)
Proteína Transportadora de Acilo/metabolismo , Proteínas Bacterianas/metabolismo , Oxidorreductasas/metabolismo , Xanthomonas campestris/enzimología , Proteína Transportadora de Acilo/química , Proteína Transportadora de Acilo/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Ácidos Grasos/química , Ácidos Grasos/metabolismo , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Oxidorreductasas/química , Oxidorreductasas/genética , Alineación de Secuencia , Transducción de Señal , Xanthomonas campestris/genética , Xanthomonas campestris/crecimiento & desarrollo
8.
Curr Eye Res ; 42(10): 1368-1377, 2017 10.
Artículo en Inglés | MEDLINE | ID: mdl-28628342

RESUMEN

PURPOSE: Crystallin is a major protein present in eye lens. Peptide fragment αA(66-80) derived from αA-crystallin possesses high aggregation propensity and forms amyloid-like structures. αA(66-80) aggregates are known to interact with soluble crystallins and destabilize native structures that subsequently undergo aggregation. Crystallin aggregation in eye lens leads to reduction in lens opacity, the condition generally referred to as a cataract. Thus, αA(66-80) aggregation appears to be an important event during cataract development, and therefore, inhibition of αA(66-80) aggregation may be an attractive strategy to intervene in cataract development. MATERIALS AND METHODS: αA(66-80) peptide derived from αA-crystallin possesses high aggregation potential and has a crucial role in cataract development. In order to inhibit the aggregation of αA(66-80) peptide, epigallocatechin-3-gallate (EGCG), a major active constituent of green tea, was employed. The inhibitory effect was assessed by Congo Red (CR) spectral shift assay, Thioflavin-T binding assay, transmission electron microscopy and fluorescence microscopy. RESULTS: The inhibitory potential of EGCG toward αA-crystallin was clearly observed as in the presence of EGCG, the αA(66-80) aggregation was considerably inhibited and the pre-formed fibrillary aggregates of αA(66-80) were found to be disassembled. CONCLUSION: In the present study, we are able to successfully demonstrate that EGCG efficiently blocks the aggregation of αA(66-80) peptide in a concentration-dependent manner. Furthermore, it is also evident that EGCG is able to disaggregate pre-formed αA(66-80) aggregates. The study suggests that EGCG can be a potential molecule that can prevent the initiation of cataract as well as be helpful in the disease reversal.


Asunto(s)
Antioxidantes/farmacología , Catarata/prevención & control , Catequina/análogos & derivados , Fragmentos de Péptidos/metabolismo , Agregación Patológica de Proteínas/tratamiento farmacológico , Té/química , Cadena A de alfa-Cristalina/metabolismo , Secuencia de Aminoácidos , Amiloide/metabolismo , Catarata/metabolismo , Catequina/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Datos de Secuencia Molecular , Agregación Patológica de Proteínas/metabolismo , Cadena A de alfa-Cristalina/ultraestructura
9.
Arch Virol ; 162(6): 1731-1736, 2017 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-28204895

RESUMEN

Solanum nodiflorum mottle virus (SNMoV) was isolated from a small-flowered nightshade (Solanum nodiflorum) in Queensland, Australia. It has been included in the genus Sobemovirus based on virion morphology and serological relationships. Here, we report the sequence of the complete genome of SNMoV. Sequence analysis confirmed that SNMoV has the characteristic genome organization of sobemoviruses. Phylogenetic analysis showed that it clusters most closely with velvet tobacco mottle virus (VTMoV), another sobemovirus native to Australia. Their genomes show 56.8 % sequence identity.


Asunto(s)
Genoma Viral , Enfermedades de las Plantas/virología , Virus de Plantas/genética , Virus ARN/genética , Australia , Secuencia de Bases , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Filogenia , Virus de Plantas/clasificación , Virus de Plantas/aislamiento & purificación , Virus ARN/clasificación , Virus ARN/aislamiento & purificación , ARN Viral/genética , Solanum/virología
10.
Artículo en Inglés | MEDLINE | ID: mdl-28125060

RESUMEN

A 17ß-estradiol (E2)-degrading bacterium E2S was isolated from the activated sludge in a sewage treatment plant (STP). The morphology, biological characteristics, and 16S ribosomal RNA (rRNA) gene sequence of strain E2S indicated that it belonged to the genus Novosphingobium. The optimal degrading conditions were 30 °C and pH 7.0. The ideal inoculum volume was 5% (v/v), and a 20-mL degradation system was sufficient to support the removal ability of strain E2S. The addition of extra NaCl to the system did not benefit the E2 degradation in batch culture by this strain. Strain E2S exhibited high degradation efficiency with initial substrate concentrations of 10-50 mg·L-1. For example, in mineral salt medium containing 50 mg·L-1 of E2, the degradation efficiency was 63.29% after seven days. In cow manure samples supplemented with 50 mg·L-1 of E2, strain E2S exhibited 66.40% degradation efficiency after seven days. The finding of the E2-degrading strain E2S provided a promising method for removing E2 from livestock manure in order to reduce the potential environmental risks of E2.


Asunto(s)
Biodegradación Ambiental , Estradiol/metabolismo , Estrógenos/metabolismo , Estiércol/microbiología , ARN Ribosómico 16S/genética , Sphingobacterium/genética , Sphingobacterium/metabolismo , Animales , Bovinos , China , Femenino , Datos de Secuencia Molecular , Filogenia
11.
Eur Surg Res ; 58(1-2): 51-68, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-27838689

RESUMEN

BACKGROUND/PURPOSE: Liver transplantation is the treatment of choice in patients with end-stage liver disease. During liver transplantation, ischemia-reperfusion injury (IRI) occurs, which is an inevitable consequence of the transplantation process. To reduce the extent of cellular injury, one of the proteins that have been extensively investigated is heme oxygenase 1 (HO-1), which plays an important role in protecting the organs against IRI. The aim of this study was to introduce an active and functional HO-1 protein conjugated to a cell-penetrating peptide (CPP) in vitro and ex vivo into liver cells in hypothermic and anoxic conditions and to assert its cytoprotective effects. METHODS: We generated an enzymatically active soluble (s)HO-1-CPP recombinant protein. The ability of the sHO-1-CPP protein to penetrate McA-RH7777, Clone 9, and Hep G2 cells, primary hepatocytes, and Kupffer and human umbilical vein endothelial cells in vitro, as well as its ability to penetrate a whole liver ex vivo under hypothermic and anoxic conditions, was assessed. An in vitro hypoxia-reoxygenation (HR) model was used to determine the cytoprotective effect of the sHO-1-CPP protein. RESULTS: We showed that our recombinant protein sHO-1-CPP can cross cell membranes into rodent and human liver cells in vitro, and the results were further validated ex vivo, where rodent livers were perfused with an organ preservation solution supplemented with sHO-1-CPP under anoxic and hypothermic conditions. Immunohistochemistry revealed an intracellular localization of sHO-1-CPP in zones 1-3 of the perfused livers. The CPP did not exert any significant toxicity on the cells. Treating cells with sHO-1-CPP showed significant cytoprotection in the in vitro HR model. CONCLUSIONS: Our findings show that the recombinant protein sHO-1-CPP can be successfully delivered to cells of a whole organ in an ex vivo hypothermic and anoxic perfusion model and that it provides cytoprotection to hepatocytes in an in vitro HR model. These results hold great potential for future repair and protection of donor organs. Future experiments are planned to confirm these data in in vivo models of IRI.


Asunto(s)
Péptidos de Penetración Celular , Citoprotección , Hemo-Oxigenasa 1/administración & dosificación , Hígado/citología , Daño por Reperfusión/prevención & control , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Isquemia Fría , Sistemas de Liberación de Medicamentos , Evaluación Preclínica de Medicamentos , Células Hep G2 , Humanos , Técnicas In Vitro , Trasplante de Hígado , Datos de Secuencia Molecular , Perfusión , Ratas Wistar , Proteínas Recombinantes
12.
Arch Virol ; 162(3): 885-889, 2017 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-27858290

RESUMEN

The complete bipartite genome (RNA1 and RNA2) of a new nepovirus infecting potato was obtained using small RNA sequencing and assembly complemented by Sanger sequencing. Each RNA encodes a single polyprotein, flanked by 5' and 3' untranslate regions (UTR) and followed by a poly (A) tail. The putative polyproteins encoded by RNA1 and RNA2 had sets of motifs which are characteristic of viruses in the genus Nepovirus. Sequence comparisons using the Pro-Pol region and the coat protein, including phylogenetic analysis of these regions, showed closest relationships with nepoviruses. The data obtained support the taxonomical status of this new virus (putative named Potato virus B, PVB) as a member of the genus Nepovirus, subgroup B.


Asunto(s)
Variación Genética , Nepovirus/genética , Nepovirus/aislamiento & purificación , Enfermedades de las Plantas/virología , Solanum tuberosum/virología , Secuencia de Bases , Genoma Viral , Datos de Secuencia Molecular , Nepovirus/clasificación , Perú , Filogenia , ARN Viral/genética , Análisis de Secuencia de ADN , Proteínas Virales/genética
13.
Virus Genes ; 53(2): 323-327, 2017 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-28004232

RESUMEN

Beet curly top Iran virus (BCTIV) is a distinct geminivirus which has been reported from sugar-beet-growing farms in Iran. In this study, the role of the splicing in expression of complementary-sense genes of BCTIV was studied. Total RNA was extracted from BCTIV-infected tissue, and the predicted intron position of complementary-sense mRNA transcripts was amplified by RT-PCR followed by cloning of the amplicons. Sequence confirmed that both spliced and unspliced mRNAs are synthesized by the same transcription unit. Sequence comparison showed that a 155-nt segment (intron) corresponding to nucleotides 1890-2044 of the viral genome has been removed from the latter transcript and therefore fusion of the C1:C2 genes resulted creation of a continuous reading frame for potential production of intact replication initiator protein (Rep). BCTIV intron comprises of most consensus splicing signals required for splicing in eukaryotes and several plant viruses including mastre- and capulaviruses.


Asunto(s)
Geminiviridae/genética , Filogenia , Empalme del ARN/genética , Proteínas Virales/genética , Beta vulgaris/virología , Geminiviridae/patogenicidad , Genoma Viral , Irán , Datos de Secuencia Molecular , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/virología , Virión/genética
14.
BMC Complement Altern Med ; 16(1): 519, 2016 Dec 16.
Artículo en Inglés | MEDLINE | ID: mdl-27986088

RESUMEN

BACKGROUND: Different strains of influenza virus are affecting a large number of people worldwide. Many synthetic antiviral medicines are available for influenza virus in the market. But still there is a need for the development of universal drugs against these strains of influenza virus. METHODS: For this purpose conserved residues within the influenza virus nucleoprotein have been retrieved. The drugs, previously known to have antiviral properties, were screened to identify the best candidate universal drug against Influenza virus strains. Compounds from leaf extracts of neem, were also screened to identify the natural drugs without side effects. RESULT: Molecular docking identified three potential compounds (Nimbaflavone, Rutin, and Hyperoside) having perfect binding with reported conserved residues (ASP302, SER50) of influenza virus nucleoprotein that is involved in the binding of drugs. Further analysis showed Hyperoside as a universal drug against various influenza strains. Some chemical drugs were also evaluated through screening against nucleoprotein. The results showed six drugs (OMS, CBX, LGH, Naproxen, BMS-883559, and BMS-885838) which were interacting with same conserved residues (ASP302, TYR52, SER50, GLY288, SER376, and ARG99) as were found in the case of neem phytochemicals. Hyperoside from neem leaf extract along with drugs LGH, Naproxen, BMS-885838, and BMS-883559 showed best interactions with conserved residues of nucleoprotein. CONCLUSION: The compound Hyperoside from neem leaf extract along with drugs LGH, Naproxen, BMS-885838, and BMS-883559 showed best interactions with conserved residues of nucleoprotein. So these compounds have been identified for their potential against influenza strains to be utilized as a universal drug.


Asunto(s)
Antivirales/farmacología , Azadirachta/química , Virus de la Influenza A/efectos de los fármacos , Gripe Humana/virología , Extractos Vegetales/farmacología , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas del Núcleo Viral/antagonistas & inhibidores , Secuencia de Aminoácidos , Antivirales/química , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Humanos , Virus de la Influenza A/química , Virus de la Influenza A/genética , Virus de la Influenza A/metabolismo , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Proteínas de la Nucleocápside , Extractos Vegetales/química , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Alineación de Secuencia , Proteínas del Núcleo Viral/química , Proteínas del Núcleo Viral/genética , Proteínas del Núcleo Viral/metabolismo
15.
Chin J Nat Med ; 14(11): 801-812, 2016 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-27914524

RESUMEN

Phenolic compounds, metabolites of the phenylpropanoid pathway, play an important role in the growth and environmental adaptation of many plants. Phenylalanine ammonia-lyase (PAL) is the first key enzyme of the phenylpropanoid pathway. The present study was designed to investigate whether there is a multi-gene family in I. Indigotic and, if so, to characterize their properties. We conducted a comprehensive survey on the transcription profiling database by using tBLASTn analysis. Several bioinformatics methods were employed to perform the prediction of composition and physicochemical characters. The expression levels of IiPAL genes in various tissues of I. indigotica with stress treatment were examined by quantitative real-time PCR. Protoplast transient transformation was used to observe the locations of IiPALs. IiPALs were functionally characterized by expression with pET-32a vector in Escherichia colis strain BL21 (DE3). Integration of transcripts and metabolite accumulations was used to reveal the relation between IiPALs and target compounds. An new gene (IiPAL2) was identified and both IiPALs had the conserved enzymatic active site Ala-Ser-Gly and were classified as members of dicotyledon. IiPAL1 and IiPAL2 were expressed in roots, stems, leaves, and flowers, with the highest expression levels of IiPAL1 and IiPAL2 being observed in stems and roots, respectively. The two genes responded to the exogenous elicitor in different manners. Subcellular localization experiment showed that both IiPALs were localized in the cytosol. The recombinant proteins were shown to catalyze the conversion of L-Phe to trans-cinnamic acid. Correlation analysis indicated that IiPAL1 was more close to the biosynthesis of secondary metabolites than IiPAL2. In conclusion, the present study provides a basis for the elucidation of the role of IiPALs genes in the biosynthesis of phenolic compounds, which will help further metabolic engineering to improve the accumulation of bioactive components in I. indigotica.


Asunto(s)
Clonación Molecular , Isatis/enzimología , Fenilanina Amoníaco-Liasa/genética , Proteínas de Plantas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Regulación de la Expresión Génica de las Plantas , Isatis/genética , Datos de Secuencia Molecular , Familia de Multigenes , Fenilanina Amoníaco-Liasa/química , Fenilanina Amoníaco-Liasa/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Alineación de Secuencia
16.
Biomed Res Int ; 2016: 5986519, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27703977

RESUMEN

The shell of the pearl oyster (Pinctada fucata) mainly comprises aragonite whereas that of the Pacific oyster (Crassostrea gigas) is mainly calcite, thereby suggesting the different mechanisms of shell formation between above two mollusks. Calmodulin (CaM) is an important gene for regulating the uptake, transport, and secretion of calcium during the process of shell formation in pearl oyster. It is interesting to characterize the CaM in oysters, which could facilitate the understanding of the different shell formation mechanisms among mollusks. We cloned the full-length cDNA of Pacific oyster CaM (cgCaM) and found that the cgCaM ORF encoded a peptide of 113 amino acids containing three EF-hand calcium-binding domains, its expression level was highest in the mantle, hinting that the cgCaM gene is probably involved in shell formation of Pacific oyster, and the common ancestor of Gastropoda and Bivalvia may possess at least three CaM genes. We also found that the numbers of some EF hand family members in highly calcified species were higher than those in lowly calcified species and the numbers of these motifs in oyster genome were the highest among the mollusk species with whole genome sequence, further hinting the correlation between CaM and biomineralization.


Asunto(s)
Calmodulina/química , Calmodulina/genética , Clonación Molecular/métodos , Crassostrea/genética , Crassostrea/metabolismo , ADN Complementario/genética , Secuencia de Aminoácidos , Animales , Calmodulina/metabolismo , Crassostrea/clasificación , Datos de Secuencia Molecular , Especificidad de la Especie
17.
Chin J Nat Med ; 14(8): 607-14, 2016 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-27608950

RESUMEN

It is hypothesized that protease inhibitors play an essential role in survival of venomous animals through protecting peptide/protein toxins from degradation by proteases in their prey or predators. However, the biological function of protease inhibitors in scorpion venoms remains unknown. In the present study, a trypsin inhibitor was purified and characterized from the venom of scorpion Mesobuthus eupeus, which enhanced the biological activities of crude venom components in mice when injected in combination with crude venom. This protease inhibitor, named MeKTT-1, belonged to Kunitz-type toxins subfamily. Native MeKTT-1 selectively inhibited trypsin with a Kivalue of 130 nmol·L(-1). Furthermore, MeKTT-1 was shown to be a thermo-stable peptide. In animal behavioral tests, MeKTT-1 prolonged the pain behavior induced by scorpion crude venom, suggesting that protease inhibitors in scorpion venom inhibited proteases and protect the functionally important peptide/protein toxins from degradation, consequently keeping them active longer. In conclusion, this was the first experimental evidence about the natural existence of serine protease inhibitor in the venom of scorpion Mesobuthus eupeus, which preserved the activity of venom components, suggests that scorpions may use protease inhibitors for survival.


Asunto(s)
Inhibidores de Proteasas/química , Venenos de Escorpión/química , Escorpiones/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Femenino , Cinética , Masculino , Ratones , Datos de Secuencia Molecular , Inhibidores de Proteasas/toxicidad , Venenos de Escorpión/genética , Venenos de Escorpión/toxicidad , Escorpiones/genética , Tripsina/química
18.
Chin J Nat Med ; 14(9): 677-682, 2016 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-27667513

RESUMEN

The present study was designed to identify immunomodulatory components from the leech salivary gland of Haemadipsa sylvestris. The Sephadex G-50, Resource(TM) S column chromatography and reverse-phase high performance liquid chromatography (RP-HPLC) were used to isolate and purify the salivary gland extracts (SGE). Structural analysis of isolated compounds was based on Edman degradation and matrix assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS). The cDNA encoding the precursor of the compound was cloned from the cDNA library of the salivary gland of H. sylvestris. The levels of inflammatory mediators, including tumor necrosis factor-α (TNF-α), interferon γ (IFN-γ), interleukin-6 (IL-6), and monocyte chemotactic protein-1 (MCP-1) were assayed using an enzyme-linked immunosorbent assay (ELISA). The effects on cell proliferation and cell viability were observed using MTT assay. A novel neuropeptide Y (Neuropeptide Y-HS) from the leech salivary gland of H. sylvestris was purified and characterized. It was composed of 36 amino acid residues and the amino acid sequence was determined to be FLEPPERPAVFTSVEQMKSYIKALNDYYLLLGRPRF-NH2, containing an amidated C-terminus. It showed significant inhibitory effects on the production of inflammatory cytokines including TNF-α, IFN-γ, IL-6, and MCP-1. Neuropeptide Y was identified from leeches for the first time. The presence of neuropeptide Y-HS in leech salivary gland may help get blood meal from hosts and inhibit inflammation.


Asunto(s)
Sanguijuelas/química , Neuropéptido Y/administración & dosificación , Neuropéptido Y/química , Secuencia de Aminoácidos , Animales , Factores Inmunológicos/administración & dosificación , Factores Inmunológicos/química , Factores Inmunológicos/genética , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Interferón gamma/inmunología , Interleucina-6/inmunología , Espectrometría de Masas , Ratones , Datos de Secuencia Molecular , Neuropéptido Y/genética , Mapeo Peptídico , Glándulas Salivales/química , Factor de Necrosis Tumoral alfa/inmunología
19.
Biomed Res Int ; 2016: 3981478, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27631006

RESUMEN

Conotoxins are a kind of neurotoxin which can specifically interact with potassium, sodium type, and calcium channels. They have become potential drug candidates to treat diseases such as chronic pain, epilepsy, and cardiovascular diseases. Thus, correctly identifying the types of ion channel-targeted conotoxins will provide important clue to understand their function and find potential drugs. Based on this consideration, we developed a new computational method to rapidly and accurately predict the types of ion-targeted conotoxins. Three kinds of new properties of residues were proposed to use in pseudo amino acid composition to formulate conotoxins samples. The support vector machine was utilized as classifier. A feature selection technique based on F-score was used to optimize features. Jackknife cross-validated results showed that the overall accuracy of 94.6% was achieved, which is higher than other published results, demonstrating that the proposed method is superior to published methods. Hence the current method may play a complementary role to other existing methods for recognizing the types of ion-target conotoxins.


Asunto(s)
Conotoxinas/química , Canales Iónicos/química , Simulación del Acoplamiento Molecular/métodos , Mapeo de Interacción de Proteínas/métodos , Análisis de Secuencia de Proteína/métodos , Algoritmos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión , Aprendizaje Automático , Datos de Secuencia Molecular , Reconocimiento de Normas Patrones Automatizadas/métodos , Unión Proteica
20.
Cell Stress Chaperones ; 21(6): 1077-1088, 2016 11.
Artículo en Inglés | MEDLINE | ID: mdl-27581971

RESUMEN

Upon diapause termination and exposure to favorable environmental conditions, cysts of the crustacean Artemia franciscana reinitiate development, a process dependent on the resumption of metabolic activity and the maintenance of protein homeostasis. The objective of the work described herein was to characterize molecular chaperones during post-diapause growth of A. franciscana. An Hsp40 complementary DNA (cDNA) termed ArHsp40 was cloned and shown to encode a protein with an amino-terminal J-domain containing a conserved histidine, proline, and aspartic acid (HPD) motif. Following the J-domain was a Gly/Phe (G/F) rich domain, a zinc-binding domain which contained a modified CXXCXGXG motif, and the carboxyl-terminal substrate binding region, all characteristics of type I Hsp40. Multiple alignment and protein modeling showed that ArHsp40 is comparable to Hsp40s from other eukaryotes and likely to be functionally similar. qRT-PCR revealed that during post-diapause development, ArHsp40 messenger RNA (mRNA) varied slightly until the E2/E3 stage and decreased significantly upon hatching. The immunoprobing of Western blots demonstrated that ArHsp40 was also relatively constant until E2/E3 and then declined dramatically. The drop in ArHsp40 when metabolism and protein synthesis were increasing was unexpected and demonstrated developmental regulation. The reduction in ArHsp40 at such an active life history stage indicates, as one possibility, that A. franciscana possesses additional Hsp40s, one or more of which replaces ArHsp40 as development progresses. Increased synthesis upon heat shock established that in addition to being developmentally regulated, ArHsp40 is stress inducible and, because it is found in mature cysts, ArHsp40 has the potential to contribute to stress tolerance during diapause.


Asunto(s)
Artemia/metabolismo , Proteínas de Artrópodos/metabolismo , Proteínas del Choque Térmico HSP40/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Artemia/crecimiento & desarrollo , Proteínas de Artrópodos/genética , Clonación Molecular , Diapausa , Proteínas del Choque Térmico HSP40/genética , Respuesta al Choque Térmico , Larva/metabolismo , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Alineación de Secuencia , Temperatura
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