RESUMO
A successful pregnancy depends on a complex process that establishes fetomaternal tolerance. Seminal plasma is known to induce maternal immune tolerance to paternal alloantigens, but the seminal factors that regulate maternal immunity have yet to be characterized. Here, we show that a soluble form of CD38 (sCD38) released from seminal vesicles to the seminal plasma plays a crucial role in inducing tolerogenic dendritic cells and CD4(+) forkhead box P3(+) (Foxp3(+)) regulatory T cells (Tregs), thereby enhancing maternal immune tolerance and protecting the semiallogeneic fetus from resorption. The abortion rate in BALB/c females mated with C57BL/6 Cd38(-/-) males was high compared with that in females mated with Cd38(+/+) males, and this was associated with a reduced proportion of Tregs within the CD4(+) T-cell pool. Direct intravaginal injection of sCD38 to CBA/J pregnant mice at preimplantation increased Tregs and pregnancy rates in mice under abortive sonic stress from 48 h after mating until euthanasia. Thus, sCD38 released from seminal vesicles to the seminal plasma acts as an immunoregulatory factor to protect semiallogeneic fetuses from maternal immune responses.
Assuntos
ADP-Ribosil Ciclase 1/imunologia , Tolerância Imunológica , Troca Materno-Fetal , Sêmen/imunologia , ADP-Ribosil Ciclase 1/genética , Animais , Células Dendríticas/imunologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , GravidezRESUMO
A Bacillus sp. strain named BRC1 is capable of producing 2,3-butanediol (2,3-BD) using hydrolysates of the Jerusalem artichoke tuber (JAT), a rich source of the fructose polymer inulin. To enhance 2,3-BD production, we undertook an extensive analysis of the Bacillus sp. BRC1 genome, identifying a putative gene (sacC) encoding a fructan hydrolysis enzyme and characterizing the activity of the resulting recombinant protein expressed in and purified from Escherichia coli. Introduction of the sacC gene into Bacillus sp. BRC1 using an expression vector increased enzymatic activity more than twofold. Consistent with this increased enzyme expression, 2,3-BD production from JAT was also increased from 3.98 to 8.10 g L-1. Fed-batch fermentation of the recombinant strain produced a maximal level of 2,3-BD production of 28.6 g L-1, showing a high theoretical yield of 92.3%.
Assuntos
Bacillus/genética , Butileno Glicóis/metabolismo , Glicosídeo Hidrolases/metabolismo , Helianthus/química , Extratos Vegetais/química , Tubérculos/química , Sequência de Aminoácidos , Bacillus/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Técnicas de Cultura Celular por Lotes , Clonagem Molecular , Escherichia coli/genética , Genes Bacterianos , Glicosídeo Hidrolases/genética , Inulina/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Proteínas RecombinantesRESUMO
CpBck1, an ortholog of the cell-wall integrity mitogen-activated protein kinase kinase kinase of Saccharomyces cerevisiae, was cloned and characterized from the chestnut blight fungus Cryphonectria parasitica. The CpBck1-null mutant displayed cell wall integrity-related phenotypic changes such as abnormal cell morphology and wall formation and hypersensitivity to cell wall-disrupting agents. In addition, the mutant showed severely retarded growth without any sign of normal development, such as hyphal differentiation, conidiation, or pigmentation. As the culture proceeded, the mutant colony showed sporadic sectorization. Once sectored, the sectored phenotype of robust mycelial growth without differentiation was stably inherited. Compared with the wild type, both the parental CpBck1-null mutant and the sectored progeny exhibited marked impaired virulence. The present study revealed that a mutation in a signaling pathway component related to cell-wall integrity resulted in sporadic sectorization and these sectored phenotypes were stably inherited, suggesting that this signal transduction pathway is implicated in adaptive genetic changes for sectorization.
Assuntos
Ascomicetos/genética , Eleocharis/microbiologia , MAP Quinase Quinase Quinases/genética , Doenças das Plantas/microbiologia , Transdução de Sinais , Ascomicetos/patogenicidade , Ascomicetos/ultraestrutura , Parede Celular/metabolismo , Eleocharis/imunologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Hifas , MAP Quinase Quinase Quinases/metabolismo , Fenótipo , Casca de Planta/imunologia , Casca de Planta/microbiologia , Doenças das Plantas/imunologia , Análise de Sequência de DNA , Deleção de Sequência , VirulênciaRESUMO
A small heat-shock protein gene, CpHsp24, of Cryphonectria parasitica was selected based on its expression pattern, which showed that it was tannic acid inducible and that its induction was severely hampered by a hypovirus. The predicted protein sequence of CpHsp24 consisted of a hallmark α-crystalline domain flanked by a variable N-terminal and a short C-terminal region. Disruption of CpHsp24 resulted in a slow growth rate under standard growth conditions. The CpHsp24-null mutant showed enhanced sensitivity to heat shock, which was consistent with Northern and Western analyses displaying the heat-shock induction of the CpHsp24 gene and protein, respectively. Virulence tests on the excised bark revealed a severe decrease in the necrotic area of the CpHsp24-null mutant. When the hypovirus was transferred, virus-containing CpHsp24-null progeny displayed severely retarded growth patterns with hypovirulent characteristics of reduced pigmentation and sporulation. Because the tannic-acid-inducible and hypoviral-suppressible expression and the severely impaired virulence are also characteristics of the laccase3 gene (lac3), lac3 expression in the CpHsp24-null mutant was also examined. The resulting lac3 induction was severely affected in the CpHsp24-null mutant, suggesting that CpHsp24 is important for lac3 induction and that CpHsp24 may act as a molecular chaperone for the lac3 protein.
Assuntos
Ascomicetos/genética , Cyperaceae/microbiologia , Regulação Fúngica da Expressão Gênica , Proteínas de Choque Térmico Pequenas/metabolismo , Doenças das Plantas/microbiologia , Taninos/farmacologia , Ascomicetos/efeitos dos fármacos , Ascomicetos/patogenicidade , Ascomicetos/fisiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Teste de Complementação Genética , Proteínas de Choque Térmico Pequenas/genética , Temperatura Alta , Lacase/genética , Lacase/metabolismo , Fenótipo , Casca de Planta/microbiologia , Estrutura Terciária de Proteína , Vírus de RNA/fisiologia , Deleção de Sequência , Estresse Fisiológico , Árvores , VirulênciaRESUMO
A synthetic consensus gene was designed based on residues of the amino acid sequences of dengue envelope domain III (scEDIII) from all four serotypes, and codon optimization for expression was conducted using baker's yeast, Saccharomyces cerevisiae. The synthetic gene was cloned into a yeast episomal expression vector, pYEGPD-TER, which was designed to direct cloned gene expression using the glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter, a functional signal peptide of the amylase 1A protein from rice, and the GAL7 terminator. PCR and back-transformation into Escherichia coli confirmed the presence of the scEDIII gene-containing plasmid in the transformants. Northern blot analysis showed the presence of the scEDIII-specific transcript. Western blot analysis indicated that expressed scEDIII, with mobility similar to purified EDIII from E. coli, was successfully secreted into the culture media. Quantitative ELISA revealed that the recombinant scEDIII comprised approximately 0.1-0.6% of cell-free extract. In addition, 0.1-0.6 mg of scEDIII protein per liter of culture filtrate was detected on day 1 and peaked on day 3 after cultivation. The secreted scEDIII protein can be purified to ≥90% purity with 85% recovery using a simple ion-exchange FPLC followed by molecular weight cut-off. Upon administration of the purified protein to mice, mouse sera contained antibodies that were specific to all four serotypes of dengue virus. Moreover, a balanced immune response against all four serotypes was observed, suggesting that it may be possible to develop an effective tetravalent dengue vaccine using S. cerevisiae.
Assuntos
Vacinas contra Dengue/genética , Vírus da Dengue/genética , Epitopos/genética , Saccharomyces cerevisiae/genética , Vacinas Sintéticas/genética , Proteínas do Envelope Viral/genética , Animais , Formação de Anticorpos , Antígenos Virais/química , Antígenos Virais/genética , Antígenos Virais/imunologia , Antígenos Virais/isolamento & purificação , Sequência de Bases , Sequência Consenso , Vacinas contra Dengue/química , Vacinas contra Dengue/imunologia , Vacinas contra Dengue/isolamento & purificação , Vírus da Dengue/química , Vírus da Dengue/imunologia , Epitopos/química , Epitopos/imunologia , Epitopos/isolamento & purificação , Feminino , Vetores Genéticos/genética , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Terciária de Proteína , Transformação Genética , Vacinas Sintéticas/química , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificação , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/isolamento & purificaçãoRESUMO
Putative arabinanase (PcARA) was cloned from cDNA of Phanerochaete chrysosporium. The gene sequencing indicated that PcARA consisted of 939 nucleotides that encodes for 312 amino acid arabinanase-polypeptide chain, including a signal peptide of 19 amino acids. Three-dimensional homology indicated that this enzyme is a five-bladed ß-propeller, belonging to glycosidase family 43 and its secondary structure is consisted of 24 ß-sheets. The PcARA-cDNA was expressed in Pichia pastoris using pPICZαC. SDS-PAGE of purified arabinanase showed a single band of 33 kDa that is very close to theoretical molecular mass of 33.9 kDa calculated by its amino acid content. Recombinant arabinanase (rPcARA) exhibited maximum activity at pH and temperature of 5.0 and 60 °C, respectively. End-product analysis of debranched arabinan hydrolysis by thin-layer chromatography indicated that rPcARA acted as endo-type. The synergistic action of rPcARA with recombinant xylanase resulted in 72 and 9.3 % release of total soluble sugar of arabinoxylan and NaOH-pretreated barley straw, respectively.
Assuntos
Clonagem Molecular , Endo-1,4-beta-Xilanases , Proteínas Fúngicas , Temperatura Alta , Phanerochaete , Polissacarídeos/química , Endo-1,4-beta-Xilanases/biossíntese , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/genética , Estabilidade Enzimática , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Phanerochaete/enzimologia , Phanerochaete/genética , Pichia/enzimologia , Pichia/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
To lower the cost of biomass harvesting, the growth of natural biofilm is considered to be an optimal alternative to microalgae aggregation. This study investigated algal mats that naturally agglomerate into a lump and float on water surfaces. Halomicronema sp., a filamentous cyanobacterium with high cell aggregation and adhesion to substrates, and Chlamydomonas sp., which grows rapidly and produces high extracellular polymeric substances (EPS) in certain environments, are the main microalgae that make up selected mats through next-generation sequencing analysis. These two species play a major role in the formation of solid mats, and showed a symbiotic relationship as the medium and nutritional source, particularly owing to the large amount of EPS formed by the reaction between EPS and calcium ions through zeta potential and Fourier-transform infrared spectroscopy analysis. This led to the formation of an ecological biomimetic algal mat (BAM) that mimics the natural algal mat system, and this is a way to reduce costs in the biomass production process as there is no separate treatment process for harvesting.
RESUMO
The present study reported the improvement of biological treatment for the removal of recalcitrant dyes including aniline blue, reactive black 5, orange II, and crystal violet in contaminated water. The biodegradation efficiency of Fusarium oxysporum was significantly enhanced by the addition of mediators and by adjusting the biomass density and nutrient composition. A supplementation of 1% glucose in culture medium improved the biodegradation efficiency of aniline blue, reactive black 5, orange II, and crystal violet by 2.24, 1.51, 4.46, and 2.1 folds, respectively. Meanwhile, the addition of mediators to culture medium significantly increased the percentages of total removal for aniline blue, reactive black 5, orange II, and crystal violet, reaching 86.07%, 68.29%, 76.35%, and 95.3%, respectively. Interestingly, the fungal culture supplemented with 1% remazol brilliant blue R boosted the biodegradation up to 97.06%, 89.86%, 91.38%, and 86.67% for aniline blue, reactive black 5, orange II, and crystal violet, respectively. Under optimal culture conditions, the fungal culture could degrade these synthetic dyes concentration up to 104 mg/L. The present study demonstrated that different recalcitrant dye types can be efficiently degraded using microorganism such as F. oxysporum.
Assuntos
Corantes , Águas Residuárias , Corantes/química , Violeta Genciana , Biodegradação Ambiental , Têxteis , Lacase/metabolismoRESUMO
Numerous studies have shown that hydrogen peroxide (H(2)O(2)) inhibits proliferation and osteoblastic differentiation in bone-like cells. Human periodontal ligament fibroblasts (PLF) are capable of differentiating into osteoblasts and are exposed to oxidative stress during periodontal inflammation. However, the cellular responses of PLF to H(2)O(2) have not been identified. In this study, we examined how H(2)O(2) affects the viability and proliferation of PLF by exposing the cells to glucose oxidase (GO) or direct addition of H(2)O(2). We also explored the effects of GO on the osteoblastic differentiation of PLF and the mechanisms involved. The viability and proliferation in PLF were increased with the addition of 10 mU/ml GO but not by volumes greater than 15 mU/ml or by H(2)O(2) itself. GO-stimulated DNA synthesis was correlated with the increase in cyclin E protein levels in the cells. Osteoblastic differentiation of PLF was also augmented by combined treatment with GO, as evidenced by the increases in alkaline phosphatase activity, mineralization, collagen synthesis, and osteocalcin content in the cells. The inductions of runt-related transcription factor 2 and osterix mRNA and proteins were further increased in PLF incubated in combination with GO compared to those in untreated cells. These results demonstrate that the continuous presence of H(2)O(2) stimulates the proliferation of PLF and augments their potential to differentiate into osteoblasts through the up-regulation of bone-specific transcription factors. Collectively, we suggest that H(2)O(2) may elicit the functions of PLF in maintaining the dimensions of the periodontal ligament and in mediating a balanced metabolism in alveolar bone.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Osteoblastos/efeitos dos fármacos , Ligamento Periodontal/efeitos dos fármacos , Adulto , Sequência de Bases , Western Blotting , Colágeno/metabolismo , Meios de Cultura , Primers do DNA , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Masculino , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Ligamento Periodontal/citologia , Ligamento Periodontal/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The antigen I/II (AgI/II) protein is a major surface protein that mediates the attachment of Streptococcus mutans (S. mutans) to the saliva-coated pellicle. Numerous studies have investigated not only the mechanisms by which AgI/II signaling is transduced within cells, but have also attempted to use AgI/II-specific antibodies to treat dental caries and host immune responses. However, little information is available about the effects of AgI/II on basic cellular events in bone cells. In this study, we examined the effects of the His-tagged recombinant N-terminal half of the AgI/II protein (rAgI/II-N) generated from S. mutans GS-5 on the viability, proliferation, and cell cycle progression of primary calvarial osteoblasts. We also investigated the mechanisms involved in the rAgI/II-N-mediated survival of serum-starved osteoblasts. We found that rAgI/II treatment attenuated the serum deprivation-induced decrease in cell viability and proliferation of osteoblasts. rAgI/II-N also prevented the loss of mitochondrial membrane potential (MMP), alterations in levels of two key mitochondrial Bcl-2 family proteins, and the accumulation of numerous cells into the sub-G(1) phase that were observed in serum-starved osteoblasts. Pharmacological inhibitors of phosphoinositide 3-kinase (PI3K), but not of extracellular signal-regulated kinase or Ras, blocked the rAgI/II-N-mediated protection against serum deprivation-induced cell death. Additional experiments revealed that the integrin α5ß1-mediated PI3K pathway is required for rAgI/II-N-mediated Akt phosphorylation in osteoblasts. Collectively, these results suggest that rAgI/II-N induces survival signals in serum-starved osteoblasts through integrin-induced PI3K/Akt signaling pathways.
Assuntos
Antígenos de Bactérias/fisiologia , Sobrevivência Celular/imunologia , Osteoblastos/microbiologia , Osteoblastos/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Streptococcus mutans/imunologia , Animais , Antígenos de Bactérias/administração & dosagem , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/imunologia , Ciclo Celular , Proliferação de Células , Células Cultivadas , Meios de Cultura Livres de Soro , Interações Hospedeiro-Patógeno/imunologia , Camundongos , Mitocôndrias/metabolismo , Modelos Biológicos , Osteoblastos/imunologia , Osteoblastos/metabolismo , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Transdução de Sinais , Streptococcus mutans/patogenicidade , Estresse FisiológicoRESUMO
Since aflatoxin B(1) (AFB(1))-mediated hepatic damage is related to the production of AFB(1)-8,9-epoxide and reactive oxygen species, bioactive compounds having antioxidant potentials are suggested to be capable of reducing AFB(1)-induced toxicity. We previously purified a mixture of flavonoids that we named RCMF (Rhus verniciflua Stokes chloroform-methanol fraction), from a traditional Korean food additive and herbal medicine. RCMF exhibited various biological effects, including antioxidant and antitumor activities. In this study, we examined whether RCMF protects against AFB(1)-induced liver injury using in vitro and in vivo systems. Pretreatment of HepG2 cells with RCMF significantly reduced AFB(1)-stimulated production of ROS and malondialdehyde (MDA) to the control levels. RCMF also prevented the reduction in HepG2 cell viability caused by AFB(1). Oral administration of RCMF to mice significantly suppressed an AFB(1)-induced increase in serum levels of alanine aminotransferase, alkaline phosphatase and lactate dehydrogenase. It also prevented MDA formation and blocked decreases in glutathione levels and superoxide dismutase activities in the livers of AFB(1)-treated mice. In addition, RCMF supplementation prevented an AFB(1) -induced decrease in serum titers of IgA and IgG1. Collectively, these results suggest that RCMF attenuates AFB(1)-mediated damage to the liver, and that this effect is at least partially related to the restoration of antioxidant defense systems and an increase in AFB(1)-GSH conjugate formation.
Assuntos
Aflatoxina B1/toxicidade , Antioxidantes/uso terapêutico , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Flavonoides/uso terapêutico , Extratos Vegetais/uso terapêutico , Rhus/química , Aflatoxina B1/antagonistas & inibidores , Animais , Sobrevivência Celular/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/imunologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Flavonoides/análise , Glutationa/metabolismo , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Isotipos de Imunoglobulinas/sangue , Masculino , Malondialdeído/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Extratos Vegetais/química , Distribuição Aleatória , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismoRESUMO
Lactobacillus acidophilus is a gram-positive, microaerophilic, and acidophilic bacterial species. L. acidophilus strains in the gastrointestinal tracts of humans and other animals have been profiled, but strains found in the canine gut have not been studied yet. Our study helps in understanding the genetic features of the L. acidophilus C5 strain found in the canine gut, determining its adaptive features evolved to survive in the canine gut environment, and in elucidating its probiotic functions. To examine the canine L. acidophilus C5 genome, we isolated the C5 strain from a Korean dog and sequenced it using PacBio SMRT sequencing technology. A comparative genomic approach was used to assess genetic relationships between C5 and six other strains and study the distinguishing features related to different hosts. We found that most genes in the C5 strain were related to carbohydrate transport and metabolism. The pan-genome of seven L. acidophilus strains contained 2,254 gene families, and the core genome contained 1,726 gene families. The phylogenetic tree of the core genes in the canine L. acidophilus C5 strain was very close to that of two strains (DSM20079 and NCFM) from humans. We identified 30 evolutionarily accelerated genes in the L. acidophilus C5 strain in the ratio of non-synonymous to synonymous substitutions (dN/dS) analysis. Five of these thirty genes were associated with carbohydrate transport and metabolism. This study provides insights into genetic features and adaptations of the L. acidophilus C5 strain to survive the canine intestinal environment. It also suggests that the evolution of the L. acidophilus genome is closely related to the host's evolutionary adaptation process.
RESUMO
Little is known about the effects of mechanical forces on osteoclastogenesis by bone marrow macrophages (BMMs) in the absence of mechanosensitive cells, including osteoblasts and fibroblasts. In this study, we examined the effects of mechanical force on osteoclastogenesis by applying centrifugal force to BMMs using a horizontal microplate rotor. Our findings, as measured by an in vitro model system, show that tumor necrosis factor (TNF)-α is capable of inducing osteoclast differentiation from BMMs and bone resorption in the presence of macrophage-colony stimulating factor (M-CSF) and is further facilitated by receptor activator of nuclear factor-kappaB (NF-κB) ligand (RANKL). Application of force to BMMs accelerated TNF-α-induced osteoclastogenesis; this was inhibited either by anti-TNF-α or anti-TNF-α receptor but not by OPG. TNF-α also increased c-Fms expression at both mRNA and protein levels in BMMs. An anti-c-Fms antibody completely inhibited osteoclast differentiation and bone resorption induced by TNF-α but partially blocked osteoclastogenesis stimulated in combination with RANKL. These results suggest that TNF-α (in the presence of M-CSF) is capable of inducing osteoclastogenesis from BMMs, and that osteoclastogenesis is significantly stimulated by force application through the activation of c-Fms-mediated signaling. Overall, the present study reveals the facilitating effect of mechanical force on osteoclastic differentiation from BMMs without the addition of mechanosensitive cells.
Assuntos
Força Compressiva/fisiologia , Macrófagos/fisiologia , Osteoclastos/citologia , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Animais , Células da Medula Óssea , Diferenciação Celular , Células Cultivadas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/análise , Receptor de Fator Estimulador de Colônias de Macrófagos/análise , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Transdução de Sinais , Fator de Necrose Tumoral alfaRESUMO
BACKGROUND: A tannic acid-inducible and mycoviral-regulated laccase3 (lac3) from the chestnut blight fungus Cryphonectria parasitica has recently been identified, but further characterization was hampered because of the precipitation of protein products by tannic acid supplementation. The present study investigated the heterologous expression of the functional laccase3 using a yeast Saccharomyces cerevisiae. RESULTS: Laccase activity in the culture broth of transformants measured using a laccase-specific substrate suggested that the lac3 gene was successfully expressed and the corresponding protein product secreted into the culture media. In addition, activity staining and Western blot analysis of a native gel revealed that the enzyme activity co-existed with the protein product specific to anti-laccase3 antibody, confirming that the cloned lac3 gene is responsible for the laccase activity. When transformants were grown on plates containing tannic acid-supplemented media, brown coloration was observed around transformed cells, indicating the oxidation of tannic acid. However, the enzymatic activity was measurable only in the selective ura- media and was negligible in nonselective nutrient-rich culture conditions. This was in part because of the increased plasmid instability in the nonselective media. Moreover, the protein product of lac3 appears to be sensitive to the cultured nonselective nutrient-rich broth, because a rapid decline in enzymatic activity was observed when the cultured broth of ura- media was mixed with that of nonselective nutrient-rich broth. In addition, constitutive expression of the lac3 gene resulted in a reduced cell number of the lac3 transformants compared to that of vector-only transformed control. However, the presence of recombinant vector without lac3 induction did not affect the growth of transformants. CONCLUSIONS: The results suggest that expression of the lac3 gene has an inhibitory effect on the growth of transformed S. cerevisiae and that the controlled expression of lac3 is appropriate for the possible application of recombinant yeast to the treatment of phenolic compounds.
Assuntos
Ascomicetos/enzimologia , Lacase/biossíntese , Saccharomyces cerevisiae/metabolismo , Taninos/química , Ascomicetos/genética , Regulação Fúngica da Expressão Gênica , Lacase/genética , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Transformação GenéticaRESUMO
The near full-length genome consisting of four segments of dsRNA from a chrysovirus infecting Korean Cryphonectria nitschkei BS122 strain (CnV1-BS122) was sequenced. The open reading frames of segments 1, 2, 3, and 4 were 2,889, 2,721, 2,475, and 2,232 nucleotides (nt) in length, respectively. Sequence analysis and homology searches of the amino acid sequences deduced from the ORFs of each segment revealed that segments 1, 2, 3, and 4 encoded RNA-dependent RNA polymerase, capsid protein, a putative cysteine protease, and replication-associated protein, respectively. The entire 5' ends of segments 1, 2, and 4 were 82, 242, and 698 nt in length, respectively; the sequence of the 5' end of segment 3 was not determined because of difficulty in amplification. The entire 3' end of segment 3 was 77 nt in length. Partial amplification of the 3' ends of segments 1, 2, and 4 yielded amplimers of 7, 17, and 30 nt, respectively.
Assuntos
Ascomicetos/virologia , Sequência de Bases , Genoma Viral , Vírus de RNA/genética , Coreia (Geográfico) , Dados de Sequência Molecular , Fases de Leitura Aberta , Vírus de RNA/isolamento & purificação , Análise de Sequência de DNA , Proteínas Virais/genéticaRESUMO
A neutralizing epitope fragment of ApxIIA toxin (ApxIIA#5) of the Korean Actinobacillus pleuropneumoniae serotype 2 strain was expressed and immobilized on the cell surface of Saccharomyces cerevisiae for efficient vaccine development. Expression of ApxIIA#5 was confirmed by Western blot analysis using cell-wall proteins, and the surface display of ApxIIA#5 was further visualized under confocal microscopy. Quantitative ELISA revealed that the recombinant ApxIIA#5 directed to the cell surface consisted of approximately 16% cell-wall proteins, estimated to be 35 mg of ApxIIA#5 protein per liter of cultured cells. An immunoassay revealed that antigen-specific antibodies against ApxIIA#5 were present in the sera of mice fed recombinant ApxIIA#5-expressing yeast, but not in mice fed the wild-type nor the vector-only transformant. Moreover, the mice fed the recombinant epitope-expressing yeast were protected from injection of a lethal dose of A. pleuropneumoniae.
Assuntos
Actinobacillus pleuropneumoniae/imunologia , Proteínas de Bactérias/imunologia , Epitopos/genética , Epitopos/imunologia , Proteínas Hemolisinas/imunologia , Saccharomyces cerevisiae/genética , Administração Oral , Animais , Especificidade de Anticorpos , Western Blotting , Ensaio de Imunoadsorção Enzimática , Epitopos/administração & dosagem , Feminino , Expressão Gênica , Camundongos , Microscopia , Plasmídeos/genética , Transformação Genética , VacinaçãoRESUMO
The enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase cleaves the ethylene precursor ACC into alpha-ketobutyrate and ammonia. The decreased level of ethylene allows the plant to be more resistant to a wide environmental stress including plant pathogens. In the present study, we characterized the ACC deaminase activity of a Pseudomonas entomophila strain PS-PJH isolated from the red pepper rhizosphere region of red pepper grown at Jinan, Korea. The isolate produced 23.8 +/- 0.4 micromol of alpha-ketobutyrate/mg of protein/h during ACC deamination under in vitro conditions. Polymerase chain reaction for acdS gene showed that the isolated P. entomophila strain PS-PJH carry sequences similar to the known acdS genes. Results of the multiple sequence alignment revealed >99% identity (nucleotide and amino acid) with acdS gene of Pseudomonas putida strains AM15 and UW4. The isolated bacteria promoted 43.3 and 34.1% of growth in Raphanus sativus and Lactuca sativa plants, respectively. Based on the 16S-23S internal transcribed spacer region sequences, the isolate was identified as P. entomophila. To the best of our knowledge this is the first study to report the acdS gene in P. entomophila.
Assuntos
Carbono-Carbono Liases/genética , Carbono-Carbono Liases/metabolismo , Pseudomonas/enzimologia , Pseudomonas/isolamento & purificação , Microbiologia do Solo , Amônia/metabolismo , Butiratos/metabolismo , Capsicum/microbiologia , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Coreia (Geográfico) , Dados de Sequência Molecular , Filogenia , Raízes de Plantas/microbiologia , Reação em Cadeia da Polimerase , Pseudomonas/genética , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
Cryparin is an abundant cell-wall-associated hydrophobin of Cryphonectria parasitica. Although cryparin is encoded as a single copy gene, it is the most abundant protein produced by this fungus when grown in liquid culture. Studies to characterize the transcriptional regulatory element(s) found that the fragment between nt -188 and the start codon was the minimal but sufficient promoter element for expression of the cryparin gene. To explore the possibility of using this small fragment as a minimal core promoter, three different chimeric reporter genes were constructed using a bacterial hygromycin B resistance gene (hph), an inducible laccase of C. parasitica, and glucose oxidase of Aspergillus niger to examine the promoter properties of the fragment. When using C. parasitica as an expression host, the 188-bp fragment functioned as a promoter for the expression of all three reporter genes. Moreover, a high level of expression was further confirmed by measuring the relative amount of transcripts of hph and an internal control gene, glyceraldehyde-3-phosphate dehydrogenase, using quantitative real-time polymerase chain reaction. The 188-bp fragment also showed promoter activity in other fungi, Gibberella zeae, A. niger, and Aspergillus nidulans, which suggests that this fragment can be applied as a strong core promoter for heterologous gene expression in various fungi.
Assuntos
Proteínas Fúngicas/genética , Fungos/genética , Expressão Gênica , Proteínas de Membrana/genética , Regiões Promotoras Genéticas , Sequência de Bases , Fungos/metabolismo , Genes Reporter , Dados de Sequência Molecular , Deleção de SequênciaRESUMO
Although the Escherichia coli heat-labile enterotoxin B subunit (LTB) has already been expressed in several different systems, including prokaryotic and eukaryotic organisms, studies regarding the synthesis of LTB into oligomeric structures of pentameric size in the budding yeast Saccharomyces cerevisiae have been limited. Therefore, this study used a functional signal peptide of the amylase 1A protein from rice to direct the yeast-expressed LTB towards the endoplasmic reticulum to oligomerize with the expected pentameric size. The expression and assembly of the recombinant LTB were confirmed in both the cell-free extract and culture media of the recombinant strain using a Western blot analysis. The binding of the LTB pentamers to intestinal epithelial cell membrane glycolipid receptors was further verified using a GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA). On the basis of the GM1-ELISA results, pentameric LTB proteins comprised approximately 0.5-2.0% of the total soluble proteins, and the maximum quantity of secreted LTB was estimated to be 3 mg/l after a 3-day cultivation period. Consequently, the synthesis of LTB monomers and their assembly into biologically active oligomers in a recombinant S. cerevisiae strain demonstrated the feasibility of using a GRAS microorganism-based adjuvant, as well as the development of carriers against mucosal disease.
Assuntos
Escherichia coli Enterotoxigênica/genética , Enterotoxinas , Saccharomyces cerevisiae/metabolismo , Amilases/genética , Northern Blotting , Western Blotting , Enterotoxinas/biossíntese , Enterotoxinas/química , Enterotoxinas/genética , Ensaio de Imunoadsorção Enzimática , Humanos , Oryza/genética , Ligação Proteica , Sinais Direcionadores de Proteínas , Subunidades Proteicas/biossíntese , Subunidades Proteicas/química , Subunidades Proteicas/genética , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genéticaRESUMO
Plant-based vaccines have been produced in transgenic plants including tobacco, potatoes, corn, and rice. However, these plants are not suitable for administration without cooking. To overcome this obstacle, a fusion gene encoding the synthetic enterotoxigenic Escherichia coli heat-labile enterotoxin B subunit genetically fused with a synthetic neutralizing epitope of porcine epidemic diarrhea virus (sLTB-sCOE) was introduced into lettuce cells (Lactuca sativa) by Agrobacterium-mediated transformation methods. The integration and expression of the sLTB-sCOE fusion gene was confirmed in transgenic lettuce by genomic DNA PCR amplification and Northern blot analysis, respectively. Synthesis and assembly of the LTB-COE fusion protein into oligomeric structures with pentamer size were observed in transgenic plant extracts by Western blot analysis with anti-LTB or anti-COE antibodies. The binding of plantproduced LTB-COE to intestinal epithelial cell membrane glycolipid receptors was confirmed by GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA). Based on the ELISA results, LTB-COE fusion protein made up about 0.026â¼0.048% of the total soluble protein in the transgenic lettuce leaf tissues. The synthesis and assembly of LTB-COE monomers into biologically active oligomers in transgenic lettuce leaf tissues demonstrates the feasibility of using uncooked edible plant-based vaccines for mucosal immunization.