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1.
Microb Pathog ; 173(Pt A): 105858, 2022 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-36349569

RESUMEN

In this study, we investigated the antibacterial mechanism of forsythoside A against the kiwifruit canker pathogen, which provided the theoretical basis for the prevention and control of canker disease and the development of plant-based fungicides. The pathogenic bacteria were isolated from kiwifruit diseased tissues and the specific primers Psa_A1 F2 and Psa_A1 R1 were used for preliminary identification. Four pairs of housekeeping genes, including gapA, gltA, gyrB, and rpoD, were used for polygenic typing identification. The inhibition effect of forsythoside A on Psa was evaluated by the filter paper bacteriostasis method. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Psa were determined by the 96-well plate absorbance and colony counts. The changes in Psa biofilm formation, motility, IAA synthesis, iron utilization, and respiratory chain dehydrogenase activity were determined. The Psa morphology was observed by Scanning electron microscope (SEM) and transmission electron microscope (TEM). The expression of some virulence genes was analyzed by qPCR. The results showed that the pathogen was Pseudomonas syringae pv. actinidiae(Psa). The inhibitory effect of forsythoside A on Psa was positively correlated with its concentration. while the MIC and MBC were 2.0 and 5.0 mg/mL, respectively. The biofilm formation and motility of Psa were not only obviously inhibited, but also the substance and energy metabolism were interfered, while obvious deformity and rupture of the cells were occurred in Psa Bacteria. In addition, The transcription of the Psa pathogenic genes was affected. The infection investigation of kiwifruit leaves indicated that forsythiaside A inhibits Psa pathogenicity and had a protective effect. This study concluded that forsythoside A has a certain control effect on kiwifruit canker, and has the potentiality to be developed as a novel plant fungicide.


Asunto(s)
Actinidia , Pseudomonas syringae , Pseudomonas syringae/genética , Enfermedades de las Plantas/microbiología , Glicósidos/farmacología , Antibacterianos/farmacología
2.
Can J Microbiol ; 65(10): 738-749, 2019 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-31206319

RESUMEN

This study explored the differences among various artificial morel cultivations as well as the factors that influence these differences, including soil bacterial community structure, yield, and mineral element contents of ascocarp and the cultivated soil. High-throughput sequencing results revealed that the dominant bacterial phyla in all the samples, including Proteobacteria, Acidobacteria, Chloroflexi, Bacteroides, and Gemmatimonadetes, were found not only in morel soils (experimental group) but also in wheat soil (control group); the highest richness and diversity in the soil bacteria were observed during the primordial differentiation stage. The M6 group exhibited the highest yield (271.8 g/m2) and had an unexpectedly high proportion of Pseudomonas (25.30%) during the primordial differentiation stage, which was 1.77∼194.62 times more than the proportion of Pseudomonas in other samples. Pseudomonas may influence the growth of morel. The mineral element contents of the different soil groups and the ascocarp were determined by electrothermal digestion and inductively coupled plasma mass spectrometry. The results revealed that morel had high enrichment effects on phosphorus (P, bioconcentration factor = 16.83), potassium (K, 2.18), boron (B, 1.47), zinc (Zn, 1.36), copper (Cu, 1.15), and selenium (Se, 2.27). P levels were the highest followed by Se and K, and the mineral element contents in ascocarp were positively correlated with the soil element contents.


Asunto(s)
Bacterias/aislamiento & purificación , Microbiota , Minerales/análisis , Microbiología del Suelo , Suelo/química
3.
Anim Biotechnol ; 22(4): 223-35, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22132815

RESUMEN

The present study was designed to construct the eukaryotic expression vector for pGH mature peptide (mpGH) and to investigate its transfection mediated by cationic liposome (CLs) in COS-7 cells and mice. The cDNA of mpGH ORF was successfully cloned by reverse transcription-PCR (RT-PCR) using the adult pig pituitary gland RNA. The recombinant eukaryotic expression vector, VmpGH, was constructed by ligating the cDNA fragment to the vector VR1020. The successful construction was confirmed by restriction enzyme digestion, and the expression of mpGH was confirmed by RT-PCR, immunofluorescence analyses (IFA), and ELISA in COS-7 cells. The VmpGH and VR1020 plasmids were entrapped with CLs, and four experimental groups of male Kunming mice were administrated with VmpGH / lipoplex or naked VmpGH plasmids at two dosages (0.5 and 1.0 mg/kg), while the mice injected with VR1020-lipoplex at the dosage of 0.5 mg/kg body weight (BW) were used as control. The BWs of the mice administrated with VmpGH-lipoplex at both dosages were significantly higher than not only those of the control (P < 0.01) but also those of mice injected with naked plasmids (P < 0.01), from 30 to 60 days post-transfection. The transcription of VmpGH was detected by RT-PCR in six tissues, including the liver, kidney, spleen, heart, muscle, and blood, of the mice injected with VmpGH-lipoplex, but not in the same tissues of control mice. Furthermore, the mice injected with VmpGH-lipoplex showed higher plasma GH contents than the control mice (P < 0.05), although their IgG contents did not show much difference. Our study demonstrates that the VmpGH plasmids' transfection mediated by CLs can significantly promote the growth of mice, which may be used to improve the livestock production.


Asunto(s)
Clonación Molecular/métodos , Vectores Genéticos/genética , Hormona del Crecimiento/genética , Liposomas/química , Transfección/métodos , Análisis de Varianza , Animales , Peso Corporal/efectos de los fármacos , Peso Corporal/genética , Células COS , Cationes/química , Chlorocebus aethiops , Escherichia coli , Hormona del Crecimiento/química , Hormona del Crecimiento/metabolismo , Masculino , Ratones , Especificidad de Órganos , Plásmidos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Porcinos
4.
PLoS One ; 13(3): e0194284, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29543859

RESUMEN

Curcuma longa possesses powerful antifungal activity, as demonstrated in many studies. In this study, the antifungal spectrum of Curcuma longa alcohol extract was determined, and the resulting EC50 values (mg/mL) of its extract on eleven fungi, including Fusarium graminearum, Fusarium chlamydosporum, Alternaria alternate, Fusarium tricinctum, Sclerotinia sclerotiorum, Botrytis cinerea, Fusarium culmorum, Rhizopus oryzae, Cladosporium cladosporioides, Fusarium oxysporum and Colletotrichum higginsianum, were 0.1088, 0.1742, 0.1888, 0.2547, 0.3135, 0.3825, 0.4229, 1.2086, 4.5176, 3.8833 and 5.0183, respectively. Among them, F. graminearum was selected to determine the inhibitory effects of the compounds (including curdione, isocurcumenol, curcumenol, curzerene, ß-elemene, curcumin, germacrone and curcumol) derived from Curcuma longa. In addition, the antifungal activities of curdione, curcumenol, curzerene, curcumol and isocurcumenol and the synergies of the complexes of curdione and seven other chemicals were investigated. Differential proteomics of F. graminearum was also compared, and at least 2021 reproducible protein spots were identified. Among these spots, 46 were classified as differentially expressed proteins, and these proteins are involved in energy metabolism, tRNA synthesis and glucose metabolism. Furthermore, several fungal physiological differences were also analysed. The antifungal effect included fungal cell membrane disruption and inhibition of ergosterol synthesis, respiration, succinate dehydrogenase (SDH) and NADH oxidase.


Asunto(s)
Antifúngicos/farmacología , Proteínas Fúngicas/metabolismo , Fusarium/efectos de los fármacos , Extractos Vegetales/farmacología , Proteoma/efectos de los fármacos , Antifúngicos/química , Membrana Celular/efectos de los fármacos , Curcuma/química , Ergosterol/biosíntesis , Fusarium/metabolismo , Complejos Multienzimáticos/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Extractos Vegetales/química , Proteoma/metabolismo , Proteómica , Succinato Deshidrogenasa/metabolismo
5.
PLoS One ; 12(3): e0174618, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28350840

RESUMEN

This study described the effects of elements (including Fe, Zn, Mn and their complexes) on the following factors in artificial morel cultivation: the characteristics of mycelia and sclerotia, soil bacterial community structures, yields and contents of microelements. The results indicated that the groups containing Mn significantly promoted mycelia growth rates, and all the experimental groups resulted in higher yields than the control (P<0.01), although their mycelia and sclerotia did not show obvious differences. It was also found that Proteobacteria, Chloroflexi, Bacteroides, Firmicutes, Actinobacteria, Acidobacteria and Nitrospirae were the dominated bacterial phyla. The Zn·Fe group had an unexpectedly high proportion (75.49%) of Proteobacteria during the primordial differentiation stage, while Pseudomonas also occupied a high proportion (5.52%) in this group. These results suggested that different trace elements clearly affected morel yields and soil bacterial community structures, particularly due to the high proportions of Pseudomonas during the primordial differentiation stage.


Asunto(s)
Ascomicetos/crecimiento & desarrollo , Bacterias/crecimiento & desarrollo , Hierro/metabolismo , Manganeso/metabolismo , Microbiología del Suelo , Zinc/metabolismo , Bacterias/clasificación , Bacterias/genética , Biomasa , Ecosistema , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Micelio/crecimiento & desarrollo , ARN Ribosómico 16S/genética , Suelo/química , Especificidad de la Especie , Oligoelementos/metabolismo
6.
Pest Manag Sci ; 62(3): 283-7, 2006 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-16475219

RESUMEN

An acaricidal substance extracted from the external seed coat of Ginkgo biloba L. was identified by UV (ultraviolet), IR (infrared), EI-MS (electron impact ion source mass spectrometry), (1)H NMR (nuclear magnetic resonance) and (13)C NMR as 6-[(Z)-10-heptadecenyl]-2-hydroxybenzoic acid (compound 1). Laboratory bioassay on citrus red mite, Panonychus citri (Mcg), showed that compound 1 possessed the following properties. (i) Powerful contact toxicity with an LC(50) of 5.2 mg litre(-1) after 24 h that was similar to that of pyridaben (LC(50) = 3.4 mg litre(-1)) and significantly superior to that of omethoate (LC(50) = 122 mg litre(-1)). Furthermore, its LC(90) was 13.4 mg litre(-1) after 24 h, which is significantly superior to both pyridaben (LC(90) = 69.6 mg litre(-1)) and omethoate (LC(90) = 453 mg litre(-1)). (ii) Quick-acting acaricidal activity. At identical concentrations, compound 1 was much faster-acting than pyridaben or omethoate. (iii) Compound 1 had strong corrosive action on the cuticle of P. citri but no phytotoxicity to plants.


Asunto(s)
Ginkgo biloba/química , Insecticidas/aislamiento & purificación , Ácaros , Salicilatos/aislamiento & purificación , Animales , Citrus/parasitología , Insecticidas/química , Estructura Molecular , Salicilatos/química
7.
Yi Chuan Xue Bao ; 33(11): 1037-46, 2006 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-17112976

RESUMEN

The chitinase Chi58 is an extracellular chitinase produced by Sanguibacter sp.strain C4. The gene-specific PCR primers were used to detect the presence of the chiA gene in strain C4. A chiA fragment (chiA-F) was amplified from the C4 genomic DNA and was used to blast-search the related sequences from the GenBank database. By alignment and selection of the highly conserved regions of the homologous sequences, two pairs of primers were designed to amplify the open reading frame (ORF) of the chitinase from strain C4 by nested PCR. The results revealed that the Chi58 ORF consisted of 1 692 nucleotides encoding a protein of 563 amino acid residues. The molecular weight of the mature protein was predicted to be 58.544 kDa. The Chi58 ORF was a modular enzyme composed of a signal peptide sequence, a polycystic kidney disease I domain, and a glycosyl hydrolase family 18 domain. The chitinase of C4 exhibited a high level of similarity to the chitinase A of Serratia (88.9%-99.6%) at the amino acid sequence level. The Chi58 gene was cloned into the expression vector pET32a to construct the recombinant plasmid pChi58 and was expressed in E. coli BL-21 (DE3) cells with IPTG induction. The molecular weight of the Trx-Chi58 fusion protein was estimated to be 81.1 kDa by SDS-PAGE.


Asunto(s)
Actinomycetales/enzimología , Actinomycetales/genética , Quitinasas/genética , Genes Bacterianos , Actinomycetales/clasificación , Secuencia de Aminoácidos , Secuencia de Bases , Quitinasas/biosíntesis , Quitinasas/química , Quitinasas/metabolismo , Clonación Molecular , Escherichia coli/genética , Expresión Génica , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Solubilidad
8.
World J Gastroenterol ; 15(7): 836-44, 2009 Feb 21.
Artículo en Inglés | MEDLINE | ID: mdl-19230044

RESUMEN

AIM: To investigate the effect of detachment of esophageal cancer cells from extracellular matrix on the localization of death receptor 5 (DR5) and apoptosis. METHODS: Anchorage-dependent EC9706 cells of esophageal squamous cell carcinoma were pretreated or not treated with brefeldin A. Detached cells were harvested by ethylenediaminetetraacetic acid digestion. Expression and localization of DR5 in these cells were determined by immunocytochemical and immunofluorescence assays, as well as flow cytometry analysis. Apoptosis of EC9706 cells was detected by flow cytometry after stained with fluorescein isothiocyanate-labeled annexin V/propidium iodide. Activation of caspase 8 was detected by Western blot analysis. RESULTS: Immunocytochemical assay indicated that DR5 was predominantly perinuclear in adherent cells but was mainly localized in cell membrane in detached cells. In addition, immunofluorescence assay also confirmed the above-mentioned results, and further demonstrated that DR5 was present in the form of coarse granules in detached cells, but in the form of fine granules in adherent cells. Cytometry analysis revealed higher levels of DR5 expression on the surfaces of brefeldin-A-untreated cells than on the surfaces of brefeldin-A-treated cells, but brefeldin A treatment did not affect the total DR5 expression levels. Moreover, nocodazole did not influence the extracelluar DR5 expression levels in EC9706 cells. Apoptosis assay revealed that detached cells were more sensitive to DR5 antibody-induced apoptosis than adherent cells. Western blotting showed that caspase 8 was activated in temporarily detached cells 4 h earlier than in adherent cells. CONCLUSION: Progress from adhesion to detachment of EC9706 cells causes DR5 relocalization, and promotes cytoplasmic translocation of DR5 to cell surfaces via a Golgi-dependent pathway. Moreover, it might also result in DR5 aggregation to render apoptosis of detached cells.


Asunto(s)
Carcinoma de Células Escamosas/patología , Neoplasias Esofágicas/patología , Matriz Extracelular/patología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Apoptosis , Brefeldino A/uso terapéutico , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/enzimología , Carcinoma de Células Escamosas/metabolismo , Caspasa 8/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Activación Enzimática , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias Esofágicas/enzimología , Neoplasias Esofágicas/metabolismo , Matriz Extracelular/metabolismo , Citometría de Flujo , Humanos , Inmunohistoquímica , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética
9.
Mol Cell Endocrinol ; 309(1-2): 32-8, 2009 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-19422880

RESUMEN

The red-spotted grouper, Epinephelus akaara, is a protogynous hermaphroditic fish that shows the characteristic of natural sex change. In this study, 2-year-old female groupers were successfully reversed to functional males by oral administration of 17alpha-methyltestosterone (MT) for 42 days. The protein inhibitor of the neuronal nitric oxide synthase (PIN) gene was cloned from sex-reversed male gonads using modern suppression subtractive hybridization (SSH), cDNA synthesis and rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The full-length cDNA of PIN is 499bp containing a 270bp open reading frame (ORF) that encodes 89 amino acids. Virtual Northern blotting and reverse transcription-PCR (RT-PCR) analysis revealed that PIN was specifically transcribed in sex-reversed male gonads. Tissue-specific expression analysis showed that PIN gene was expressed in the brain, heart, liver, spleen, and kidney but not in the muscle tissue. Analyses of the expression pattern by RT-PCR and Western blotting indicated that transcription and the level of expression of PIN in the gonads increased gradually during the transformation from female to male. The results showed that PIN is strongly expressed in the sex-reversed male gonad but scarcely in the female gonad, and that its expression is upregulated as the change of sex proceeds. Taken together, these findings demonstrate that PIN is associated with the MT-induced sex transition of the red-spotted grouper, but the precise role of the gene in this process remains to be further investigated.


Asunto(s)
Proteínas de Peces/genética , Perfilación de la Expresión Génica , Gónadas/metabolismo , Organismos Hermafroditas , Perciformes/genética , Procesos de Determinación del Sexo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , ADN Complementario/genética , Dieta , Proteínas de Peces/química , Proteínas de Peces/metabolismo , Regulación de la Expresión Génica , Biblioteca de Genes , Inmunohistoquímica , Masculino , Datos de Secuencia Molecular , Filogenia , Homología de Secuencia de Aminoácido , Testosterona/sangre , Transcripción Genética
10.
Curr Microbiol ; 52(1): 45-9, 2006 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-16391997

RESUMEN

One or more proteinaceous factors with insecticidal activities in the locust pathogen Serratia marcescens HR-3 culture filtrates were found to cause the death of grassland locusts. A novel insecticidal protein was purified to homogeneity. It was a monomer of 61 kDa. The purified protein showed a strong insecticidal effect with a median lethal dosage of 12.1 microg locust(-1) and contained a high level of protease activity (101 U ml(-1)). Insecticidal activity was significantly decreased when the protein was pretreated with ethylene diamine tetraacetic acid and 1-10-phenanthroline, and it was restored when the treated protein was incubated with Zn(2+). The N-terminal amino acid sequence of insecticidal protein showed sequence similarity with metalloprotease from S. marcescens SM6 and Serratia spp. E15. Our results suggested that the factor primarily responsible for insecticidal activity toward locusts was a zinc-dependent 61-kDa metalloprotease.


Asunto(s)
Proteínas Bacterianas/aislamiento & purificación , Insecticidas/aislamiento & purificación , Péptido Hidrolasas/aislamiento & purificación , Serratia marcescens/química , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/farmacología , Quelantes/farmacología , Coenzimas/farmacología , Ácido Edético/farmacología , Saltamontes/efectos de los fármacos , Insecticidas/farmacología , Datos de Secuencia Molecular , Peso Molecular , Péptido Hidrolasas/metabolismo , Fenantrolinas/farmacología , Homología de Secuencia de Aminoácido , Zinc/farmacología
11.
Gen Comp Endocrinol ; 146(3): 296-303, 2006 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-16442533

RESUMEN

The cDNA (GenBank, AY251538) encoding bullfrog growth hormone (fGH) was cloned by RT-PCR from the total RNA of pituitary glands. Its sequence encoded a putative polypeptide of 215 amino acids, including a signal peptide of 25 amino acids with no change to those of other previous reported bullfrog GHs. The fGH precursor shares 98.1, 96.3, and 95.3% homologies to those of other bullfrog GHs (AAB24792, AAB19428, CAA31038) in amino-acid sequence and its nucleotide sequences of the coding region shares 99.1 and 98.5% homologies to those of previous bullfrog GH genes (S52027 and X12520). The fGH cDNA was also efficiently expressed in Escherichia coli carrying a plasmid pGfGH in which the cDNA was under the control of GST promoter of pGEX1-lambdaT. The expressed fusion protein GST-fGH is comprised about 29.3% of the total cellular protein in such bacteria. The purified GST-fGH cannot only showed a obvious dose-response curve when it reacted with the hepatic membrane receptor proteins from bullfrog, but also significantly increased the body weight and length of bullfrog after twice injection and such effects lasted two or three weeks after the last injection with purified GST-fGH.


Asunto(s)
Hormona del Crecimiento/genética , Rana catesbeiana , Secuencia de Aminoácidos , Animales , Clonación Molecular , Glutatión Transferasa/genética , Hormona del Crecimiento/farmacología , Datos de Secuencia Molecular , Rana catesbeiana/crecimiento & desarrollo , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia
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