Priming Mesenchymal Stem/Stromal Cells with a Combination of a Low Dose of IFN-γ and Bortezomib Results in Potent Suppression of Pathogenic Th17 Immunity Through the IDO1-AHR Axis.

Preconditioning of mesenchymal stem/stromal cells (MSCs) with the inflammatory cytokine IFN-γ enhances not only their immunosuppressive activity but also their expression of HLA and proinflammatory genes. We hypothesized that prevention of the upregulation of inflammatory cytokines and HLA molecules in IFN-γ-primed MSCs would render these cells more immunosuppressive and less immunogenic. In this study, we discovered the following findings supporting this hypothesis: (1) activated human T cells induced the expression of IDO1 in MSCs via IFN-γ secretion and those MSCs in turn inhibited T-cell proliferation in an AHR-dependent fashion; (2) there was no difference in the expression of IDO1 and HLA-DR in MSCs after priming with a low dose (25 IU/mL) versus a high dose (100 IU/mL) of IFN-γ; (3) the transient addition of bortezomib, a proteasome inhibitor, to culture MSCs after IFN-γ priming decreased the expression of HLA-DR, inflammatory cytokine genes and Vcam1 while increasing the expression of IDO1 and the production of L-kynurenine; finally, MSCs primed with a combination of a low dose of IFN-γ and bortezomib were more effective in inhibiting Th17-mediated idiopathic pneumonia syndrome (IPS) and chronic colitis than unprimed MSCs. Our results suggest that bortezomib significantly eliminates the unfavorable effects of IFN-γ priming of MSCs (increased expression of MHC molecules and inflammatory cytokines and cell aggregation genes) and simultaneously increases their immunosuppressive activity by upregulating IDO1. Taken together, our newly established MSC priming method may contribute to MSC-based cell therapy for inflammatory diseases.

Characterization of a Hypovirus-Regulated Septin Cdc11 Ortholog, CpSep1, from the Chestnut Blight Fungus Cryphonectria parasitica.

We identified a protein spot showing downregulation in the presence of Cryphonectria hypovirus 1 and tannic acid supplementation as a septin subunit with the highest homology to the Aspergillus nidulans aspA gene, an ortholog of the Saccharomyces cerevisiae Cdc11 gene. To analyze the functional role of this septin component (CpSep1), we constructed its null mutant and obtained a total of eight CpSep1-null mutants from 137 transformants. All CpSep1-null mutants showed retarded growth, with fewer aerial mycelia and intense pigmentation on plates of potato dextrose agar supplemented with L-methionine and biotin. When the marginal hyphae were examined, hyperbranching was observed in contrast to the wild type. The inhibition of colonial growth was partially recovered when the CpSep1-null mutants were cultured in the presence of the osmostabilizing sorbitol. Conidia production of the CpSep1-null mutants was significantly increased by at least 10-fold more. Interestingly, the conidial morphology of the CpSep1-null mutants changed to circular in contrast to the typical rod-shaped spores of the wild type, indicating a role of septin in the spore morphology of Cryphonectria parasitica. However, no differences in the germination process were observed. Virulence assays using excised chestnut bark, stromal pustule formation on chestnut stems, and apple inoculation indicated that the CpSep1 gene is important in pathogenicity.

Effects of initial moisture content of Korean traditional wheat-based fermentation starter nuruk on microbial abundance and diversity.

The brewing of makgeolli, one of Korea's most popular alcoholic beverages that is gaining popularity globally, is facilitated by nuruk, a traditional Korean cereal starter. The nuruk microbiome greatly influences the fermentation process as well as the nutritional, hygienic, and aromatic qualities of the product. This study is a continuation of our efforts to examine nuruk biodiversity at a depth previously unattainable. In this study, microfloral dynamics in wheat-based nuruk C, composed of traditional ingredients such as barley, green gram, and wheat and fermented under various internal moisture contents of 20% (C20), 26% (C26), and 30% (C30), was evaluated using 454 pyrosequencing during the 30-day fermentation process. Rarefaction analysis and alpha diversity parameters indicated adequate sampling. C20 showed the greatest fungal richness and diversity, C20 and C26 exhibited similar bacterial richness and diversity, while C30 had low fungal and bacterial richness. Fungal taxonomic assignments revealed that the initial moisture content caused selective enrichment of Aspergillus candidus with a decreasing trend during fermentation, whereas Saccharomycetales sp. exhibited increasing relative abundance with increasing moisture content from day 6 of the fermentation process. Depending on initial moisture level, changes in bacterial communities were also observed in the genera Streptomyces, Bacillus, and Staphylococcus, with decreasing trends whereas Saccharopolyspora exhibited a sigmoidal trend with the highest abundance in C26. These findings demonstrate the possible impact of initial moisture content of nuruk on microfloral richness, diversity, and dynamics; this study is thus a step toward our ultimate goal of enhancing the quality of nuruk.

A Mutant of the Bck1 Homolog from Cryphonectria parasitica Resulted in Sectorization with an Impaired Pathogenicity.

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.

Heterokaryon analysis of a Cdc48-like gene, CpCdc48, from the chestnut blight fungus Cryphonectria parasitica demonstrates it is essential for cell division and growth.

Functional analysis of a cell division cycle 48 (CDC48) ortholog, CpCdc48, from Cryphonectria parasitica was performed via construction of a CpCdc48-null mutant. Genotype analysis revealed that the putative CpCdc48-null mutant was a heterokaryotic transformant containing two different types of nuclei (i.e., one with the wild-type CpCdc48 allele and the other with the CpCdc48-null mutant allele). Although stable mycelial growth of the heterokaryotic transformant was observed on media containing hygromycin B, neither germination nor growth of the resulting spores was observed on selection media, suggesting that the CpCdc48 gene is essential. Microscopic analysis of germinated conidia from the heterokaryon demonstrated that although there were normal germinating spores due to the wild-type conidia, there were many residual conidia that did not germinate. However, with prolonged incubation, non-germinating conidia began to swell into gigantic globose spores. DAPI staining and FACS analysis of the gigantic spores revealed the presence of multiple nuclei. These gigantic conidia did not show any signs of polarized growth and underwent autolysis with further incubation. These findings indicate that the CpCdc48 gene is responsible for delayed cell cycle during spore germination, resulting in some karyokinesis, but not following spore cytokinesis. Thus, CpCdc48 is essential for cell division and polarized growth.

Metagenomic analysis of fungal diversity in Korean traditional wheat-based fermentation starter nuruk.

Nuruk, a traditional natural starter, is extensively used in the brewing of Makgeolli, one of Korea's most popular alcoholic beverages that has been recently gaining global popularity. Thus, the quality of traditional nuruk needs to be enhanced. The nuruk mycobiome greatly influences both fermentation process as well as palatability enhancement. Limitations of culture-dependent identification restrict an accurate analysis of fungal diversity and distribution in nuruks. 454 pyrosequencing of two traditional wheat-based nuruks, prepared at two representative temperature conditions revealed a total of 153 and 53 OTUs for nuruks A and B, respectively, from a total of 33,157 ITS sequences. Phylogenetic assignments indicated that nuruk A mycobiota was dominated by the genera Aspergillus and Mucorales, whereas nuruk B by Rhizomucor. Species-level identification indicated that Mucorales sp., Aspergillus candidus, and Aspergillus cibarius predominated in nuruk A mycoflora whereas Rhizomucor pusillus, Mucorales sp., and Thermoascus crustaceus in nuruk B. The alpha diversity indices suggest nuruk A mycobiota to be more diverse than that of nuruk B at almost all time points of fermentation. Resemblances of patterns of predominant species composition and succession between culture-dependent and -independent phylogenetic analysis creates the potential to reconstruct the nuruk mycobiome in vitro, which allows the establishment of a standard inoculum for scientific comparison.

Microtubule plus end-tracking proteins play critical roles in directional growth of hyphae by regulating the dynamics of cytoplasmic microtubules in Aspergillus nidulans.

Cytoplasmic microtubules (MTs) serve as a rate-limiting factor for hyphal tip growth in the filamentous fungus Aspergillus nidulans. We hypothesized that this function depended on the MT plus end-tracking proteins (+TIPs) including the EB1 family protein EBA that decorated the MT plus ends undergoing polymerization. The ebAΔ mutation reduced colony growth and the mutant hyphae appeared in an undulating pattern instead of exhibiting unidirectional growth in the control. These phenotypes were enhanced by a mutation in another +TIP gene clipA. EBA was required for plus end-tracking of CLIPA, the Kinesin-7 motor KipA, and the XMAP215 homologue AlpA. In addition, cytoplasmic dynein also depended on EBA to track on most polymerizing MT plus ends, but not for its conspicuous appearance at the MT ends near the hyphal apex. The loss of EBA reduced the number of cytoplasmic MTs and prolonged dwelling times for MTs after reaching the hyphal apex. Finally, we found that colonies were formed in the absence of EBA, CLIPA, and NUDA together, suggesting that they were dispensable for fundamental functions of MTs. This study provided a comprehensive delineation of the relationship among different +TIPs and their contributions to MT dynamics and unidirectional hyphal expansion in filamentous fungi.

Randomized phase II trial of nimotuzumab plus irinotecan versus irinotecan alone as second-line therapy for patients with advanced gastric cancer.

BACKGROUND: This multicenter, randomized phase II trial was conducted to compare the efficacy and safety of nimotuzumab plus irinotecan (N-IRI) versus irinotecan alone (IRI) in patients with advanced gastric cancer (AGC) showing disease progression after previous 5-fluorouracil-based therapy. METHODS: Irinotecan-naive patients (n = 82) received N-IRI (nimotuzumab 400 mg weekly plus irinotecan 150 mg/m(2) biweekly) or IRI (irinotecan 150 mg/m(2) biweekly) until disease progression. The primary endpoint was progression-free survival (PFS), and the secondary endpoints were overall survival (OS), response rate (RR), safety, tolerability, and the correlation between efficacy and tumor epidermal growth factor receptor (EGFR) expression. RESULTS: Of 83 patients, 40 and 43 patients were randomly assigned to the N-IRI and IRI groups, respectively. In the N-IRI/IRI treatment group, median PFS was 73.0/85.0 days (P = 0.5668), and median OS and RR at 18 months were 250.5/232.0 days (P = 0.9778) and 18.4/10.3 %, respectively. Median PFS and OS in the EGFR 2+/3+ subgroups were 118.5/59.0 and 358.5/229.5 days, respectively. The RR was 33.3/0.0 % in the N-IRI/IRI treatment group. The incidence of grade 3 or higher adverse events was 77.5/64.3 %. No adverse events of grade 3 or higher skin rash or grade 3 or higher infusion-related reaction were reported. CONCLUSIONS: There was no superiority of N-IRI over IRI alone in terms of PFS in 5-fluorouracil-refractory AGC patients. However, N-IRI showed potential improvement in the EGFR 2+/3+ subgroup based on improved RR, PFS, and OS.

TIMP-1 modulates chemotaxis of human neural stem cells through CD63 and integrin signalling.

We recently reported that hNSCs (human neural stem cells) have the interesting characteristic of migration towards an intracranial glioma. However, the molecules and mechanisms responsible for tumour tropism are unclear. In the present study, we used microarray and proteomics analyses to identify a novel chemoattractant molecule, TIMP-1 (tissue inhibitor of metalloproteinase-1), secreted from human brain tumour tissues. We demonstrate that TIMP-1 significantly enhances hNSC adhesion and migration in a cell culture system. These effects were critically dependent on CD63, as shRNA-mediated ablation of CD63 expression attenuated the response. TIMP-1 significantly increased the number of FAs (focal adhesions) and cytoskeletal reorganization for cell migration in hNSCs, whereas knockdown of CD63 resulted in decreased hNSC spreading, FAs and migration, even after TIMP-1 treatment. In addition, TIMP-1 binding to CD63 activated ß1 integrin-mediated signalling through Akt and FAK phosphorylation, leading to pattern changes in distribution of vinculin and F-actin (filamentous actin). Furthermore, inactivation of ß1 integrin by use of a blocking antibody or inhibition of PI3K (phosphoinositide 3-kinase) signalling impaired the migration of hNSCs towards TIMP-1. Collectively, our results underline TIMP-1 as a novel and effective key regulator of CD63 and ß1 integrin-mediated signalling, which regulates hNSC adhesion and migration.

Functional analysis of a tannic-acid-inducible and hypoviral-regulated small heat-shock protein Hsp24 from the chestnut blight fungus Cryphonectria parasitica.

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.

Expression and purification of an immunogenic dengue virus epitope using a synthetic consensus sequence of envelope domain III and Saccharomyces cerevisiae.

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.

Agarolytic Pathway in the Newly Isolated Aquimarina sp. Bacterial Strain ERC-38 and Characterization of a Putative ß-agarase.

Marine microbes, particularly Bacteroidetes, are a rich source of enzymes that can degrade diverse marine polysaccharides. Aquimarina sp. ERC-38, which belongs to the Bacteroidetes phylum, was isolated from seawater in South Korea. It showed agar-degrading activity and required an additional carbon source for growth on marine broth 2216. Here, the genome of the strain was sequenced to understand its agar degradation mechanism, and 3615 protein-coding sequences were predicted, which were assigned putative functions according to their annotated functional feature categories. In silico genome analysis revealed that the ERC-38 strain has several carrageenan-degrading enzymes but could not degrade carrageenan because it lacked genes encoding κ-carrageenanase and S1_19A type sulfatase. Moreover, the strain possesses multiple genes predicted to encode enzymes involved in agarose degradation, which are located in a polysaccharide utilization locus. Among the enzymes, Aq1840, which is closest to ZgAgaC within the glycoside hydrolase 16 family, was characterized using a recombinant enzyme expressed in Escherichia coli BL21 (DE3) cells. An enzyme assay revealed that recombinant Aq1840 mainly converts agarose to NA4. Moreover, recombinant Aq1840 could weakly hydrolyze A5 into A3 and NA2. These results showed that Aq1840 is involved in at least the initial agar degradation step prior to the metabolic pathway that uses agarose as a carbon source for growth of the strain. Thus, this enzyme can be applied to development and manufacturing industry for prebiotic and antioxidant food additive. Furthermore, our genome sequence analysis revealed that the strain is a potential resource for research on marine polysaccharide degradation mechanisms and carbon cycling.

Comparative proteomic analysis of chestnut blight fungus, Cryphonectria parasitica, under tannic-acid-inducing and hypovirus-regulating conditions.

Chestnut blight fungus, Cryphonectria parasitica , and its hypovirus present a useful model system for investigating the mechanisms of hypoviral infection. To identify gene products associated with fungal pathogenicity and hypoviral regulation, we attempted a proteomic analysis of the virus-free EP155/2 strain and its isogenic virus-infected UEP1 strain in response to tannic acid (TA), which is abundant in the bark of chestnut trees. In this study, pretreatment of mycelia grown on TA-supplemented media was developed for proteomic analysis. Approximately 704 proteins from the mycelia of the EP155/2 strain were reproducibly present in 3 independent extractions. Among these, 111 and 79 spots were found to be responsive to hypovirus infection and TA supplementation, respectively. The TA-grown UEP1 strain yielded 28 spots showing an expression pattern different from that of untreated UEP1. Thirty protein spots showing considerable differences in spot density were selected for further analysis. Hybrid tandem LC-MS/MS spectrometry of the 30 selected protein spots revealed that 29 were identified while 1 was unidentified. Among the identified 29 proteins, 15 were metabolic enzymes; 5 were stress-related, of which 4 were heat-shock proteins and 1 was glutathione S-transferase; 5 were signaling and cellular process-related proteins; 2 were structural proteins; and 2 matched proteins of hypothetical genes.

Genomic analysis of the basal lineage fungus Rhizopus oryzae reveals a whole-genome duplication.

Rhizopus oryzae is the primary cause of mucormycosis, an emerging, life-threatening infection characterized by rapid angioinvasive growth with an overall mortality rate that exceeds 50%. As a representative of the paraphyletic basal group of the fungal kingdom called "zygomycetes," R. oryzae is also used as a model to study fungal evolution. Here we report the genome sequence of R. oryzae strain 99-880, isolated from a fatal case of mucormycosis. The highly repetitive 45.3 Mb genome assembly contains abundant transposable elements (TEs), comprising approximately 20% of the genome. We predicted 13,895 protein-coding genes not overlapping TEs, many of which are paralogous gene pairs. The order and genomic arrangement of the duplicated gene pairs and their common phylogenetic origin provide evidence for an ancestral whole-genome duplication (WGD) event. The WGD resulted in the duplication of nearly all subunits of the protein complexes associated with respiratory electron transport chains, the V-ATPase, and the ubiquitin-proteasome systems. The WGD, together with recent gene duplications, resulted in the expansion of multiple gene families related to cell growth and signal transduction, as well as secreted aspartic protease and subtilase protein families, which are known fungal virulence factors. The duplication of the ergosterol biosynthetic pathway, especially the major azole target, lanosterol 14alpha-demethylase (ERG11), could contribute to the variable responses of R. oryzae to different azole drugs, including voriconazole and posaconazole. Expanded families of cell-wall synthesis enzymes, essential for fungal cell integrity but absent in mammalian hosts, reveal potential targets for novel and R. oryzae-specific diagnostic and therapeutic treatments.

Preparation of self-assembled zinc oxide nanoparticles multilayer films under ultrasonic irradiation.

Zinc oxide nanoparticles were synthesized and self-assembled on the reactive surface of a glass slide functionalized with (3-mercaptopropyl)-trimethoxysilane under ultrasonic irradiation. The structure, morphology, and optical property of the zinc oxide nanoparticles were investigated by TEM, XRD, and UV-vis spectroscopy. The functionalized glass slide was soaked in an aqueous solution which dispersed zinc oxide nanoparticles under ultrasonic irradiation. Zinc oxide multilayer films grew up to several layers (up to 5 layers) depending on the immersion time. The self-assembled zinc oxide nanoparticles multilayer films were characterized using UV-vis spectroscopy and SEM. Ultrasonic irradiation was an efficient method to make multilayer films on the functionalized glass slide with zinc oxide nanoparticles.

Synthesis of gold nanoparticles using pluronic F127NF under microwave irradiation and catalytic effects.

This study examined the synthesis of gold nanoparticles with pluronic F127NF and KAuCl4 in water under non-classical conditions. The gold nanoparticle products were well dispersed in water and characterized by ultraviolet-visible spectroscopy and transmission electron microscopy. The reaction time for the synthesis was investigated by monitoring the change in color and the peak of the UV-vis spectra under microwave conditions. The gold nanoparticles were used as a catalyst for the reduction of 4-nitrophenol to 4-aminophenol with NaBH4. The resulting product was confirmed by UV-vis spectroscopy and liquid chromatography-mass spectroscopy.

Therapeutic Effect of IL1ß Priming Tonsil Derived-Mesenchymal Stem Cells in Osteoporosis.

BACKGROUND: Stem cell therapies can be a new therapeutic strategy that may rebalance anabolic and anti-resorptive effects in osteoporosis patients. Tonsil-derived mesenchymal stem cells (TMSCs) can be an alternative therapeutic source for chronic degenerative diseases including osteoporosis. MSCs acquire immune regulatory function under the inflammatory cytokines. Since interleukin (IL) 1ß is known to be one of inflammatory cytokines involved in osteoporosis progression, treatment of IL1ß with TMSCs may enhance immunomodulatory function and therapeutic effects of TMSCs in osteoporosis. METHODS: For IL1ß priming, TMSCs were cultured in the presence of the medium containing IL1ß for 1 day. Characteristics of IL1ß priming TMSCs such as multipotent differentiation properties, anti-inflammatory potential, and suppression of osteoclast differentiation were assessed in vitro. For in vivo efficacy study, IL1ß priming TMSCs were intravenously infused twice with ovariectomized (OVX) osteoporosis mouse model, and blood serum and bone parameters from micro computed tomography images were analyzed. RESULTS: IL1ß priming TMSCs had an enhanced osteogenic differentiation and secreted factors that regulate both osteoclastogenesis and osteoblastogenesis. IL1ß priming TMSCs also suppressed proliferation of peripheral blood mononuclear cells (PBMCs) and decreased expression of Receptor activator of nuclear factor kappa-Β ligand (RANKL) in PHA-stimulated PBMCs. Furthermore, osteoclast specific genes such as Nuclear factor of activated T cells c1 (NFATc1) were effectively down regulated when co-cultured with IL1ß priming TMSCs in RANKL induced osteoclasts. In OVX mice, IL1ß priming TMSCs induced low level of serum RANKL/osteoprotegerin (OPG) ratio on the first day of the last administration. Four weeks after the last administration, bone mineral density and serum Gla-osteocalcin were increased in IL1ß priming TMSC-treated OVX mice. Furthermore, bone formation and bone resorption markers that had been decreased in OVX mice with low calcium diet were recovered by infusion of IL1ß priming TMSCs. CONCLUSION: IL1ß priming can endow constant therapeutic efficacy with TMSCs, which may contribute to improve bone density and maintain bone homeostasis in postmenopausal osteoporosis. Therefore, IL1ß priming TMSCs can be a new therapeutic option for treating postmenopausal osteoporosis.

Heterologous expression of a tannic acid-inducible laccase3 of Cryphonectria parasitica in Saccharomyces cerevisiae.

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.

Nucleotide sequences of four segments of chrysovirus in Korean Cryphonectria nitschkei BS122 strain.

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.

Surface-displayed expression of a neutralizing epitope of ApxIIA exotoxin in Saccharomyces cerevisiae and oral administration of it for protective immune responses against challenge by Actinobacillus pleuropneumoniae.

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.