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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.

Biotransformation of lignocellulose by microbial fermentation is usually preceded by thermo-chemical pretreatments followed by enzymatic hydrolysis of cellulose. Derivatives formed during the pretreatment of the lignocellulosic biomass inhibit enzymatic hydrolysis as well as microbial fermentation. Pretreated lignocellulose hydrolysate contains many derivatives of either furanic or phenolic inhibitory derivatives. In the present study, laccase was used to detoxify three different types of lignocellulosic derivatives that are highly toxic to microbial fermentation due to their low hydrophilic nature, namely furfural, acetosyringone, and coniferyl aldehyde. A minimal inhibitory concentration (MIC) test was carried out with Saccharomyces cerevisiae . The MIC of furfural, acetosyringone, and coniferyl aldehyde was 12 mM, 24 mM, and 1.5 mM, respectively. Laccase was immobilized on to cellulose nanofiber produced by Gluconacetobacter xylinus . Immobilized laccase showed a better pH and thermal stability than free laccase. Reuse of immobilized laccase retains 85% of its enzyme activity after 16 recycles. Immobilized laccase completely degraded the three lignocellulose inhibitory derivatives after 36 h of incubation at 40 °C. Finally, the degradation was confirmed by ultraviolet visible spectroscopy (UV–VIS spectrum), high performance liquid chromatography and liquid chromatography mass spectrometry. Interestingly, it was found that the effect of enzymatic degradation depends on the structural variation of the lignocellulosic derivatives as laccase alone detoxified the furfural and coniferyl aldehyde, whereas a redox mediator HOBt was needed for the detoxification of ketone based lignin derivative acetosyringone.

A putative laccase was cloned from cDNA of Fusarium oxysporum HUIB02, which resulted in a highly specific amplicon of 1.8 kb. Nucleotide sequence consisting of 1875 nucleotides, encoding for a polypeptide of 623 amino acids and shared 99% similarity to other F. oxysporum putative laccase gene. Hence, the gene was named as FoLacc5. FoLacc5 was expressed in P. pastoris and qualified the expression as extracellular protein. SDS-PAGE and western blot analysis indicated rFoLacc5 has a molecular weight above 100 kDa in glycosylated form. Optimal culture conditions enhanced the enzyme production level to 21,966 U/L. The optimal culture conditions was YPM medium, initial pH of 6.5, shaking speed of 210 rpm, inoculation temperature of 30 °C, and Cu2+ concentration of 0.08 mM. rFoLacc5 exhibited maximal activity at pH of 3.5 and temperature of 40 °C. Fe3+ and Cu2+ showed different effect on rFoLacc5 which Fe3+ ion completely inhibited, whereas Cu2+ ion increased enzyme activity. rFoLacc5 removed various synthetic dyes including brommothymol blue, methylene blue, methyl orange, remazol brilliant blue R, aniline blue, evans blue, indigo carmine, and orange II. This is the first study conducted on characterization of recombinant laccase rFoLacc5 and its application on synthetic dyes removal. rFoLacc5 shows great potential for biotechnology application on dyes removal.

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.

Phleichrome, a pigment produced by the phytopathogenic fungus Cladosporium phlei, is a fungal perylenequinone whose photodynamic activity has been studied intensively. To determine the biological function of phleichrome and to engineer a strain with enhanced production of phleichrome, we identified the gene responsible for the synthesis of phleichrome. Structural comparison of phleichrome with other fungal perylenequinones suggested that phleichrome is synthesized via polyketide pathway. We recently identified four different polyketide synthase (PKS) genes encompassing three major clades of fungal PKSs that differ with respect to reducing conditions for the polyketide product. Based on in silico analysis of cloned genes, we hypothesized that the non-reducing PKS gene, Cppks1, is involved in phleichrome biosynthesis. Increased accumulation of Cppks1 transcript was observed in response to supplementation with the application of synthetic inducer cyclo-(l-Pro-l-Phe). In addition, heterologous expression of the Cppks1 gene in Cryphonectria parasitica resulted in the production of phleichrome. These results provide convincing evidence that the Cppks1 gene is responsible for the biosynthesis of phleichrome.

In this study, we reported a laccase producing fungus from hard wood samples. The isolation produced laccase as extracellular enzymes under liquid culture using rice straw and wood chips as carbon sources. The enzyme accumulated highest activity after 12 days and 6 days cultivation on rice straw and wood chips, reaching 186 U/mL and 2.5 U/mL, respectively. Fe2＋ inhibited activity whereas Co2＋, Cu 2 + , and Mg 2 + significantly enhanced its activity. The optimal pH and temperature were 5.0 and 45°C, respectively. Molecular identification indicated that the isolation belonged in Fusarium genus and named as Fusairum oxysporum HUIB02. On the other hand, F. oxysporum HUIB02 showed excellent synthetic dyes decolouration for bromothymol blue, methyl orange, remazol brilliant blue R, indigo carmine with degree approximately 100%. The strain also exhibited great decolouration on malachite green and evans blue. These synthetic dyes enhanced laccase accumulation up to 12.6-fold in compared with culture in rice straw medium. SDS-PAGE zymogram analysis indicated F. oxysporum HUIB02 secreted two main laccases with molecular weight of 110 kDa (Lacc110) and 42 kDa (Lacc42). Laccase gene expression was affected by synthetic dyes. The Lacc110 expression level was suppressed whereas the Lacc42 expression level was induced. The results remarked F. oxysporum HUIB02 is a good source in laccase production and dye decolouration as well as synthetic dyes are directly affected on gene expression pathway in organism.

Results to date suggest that microalgal Thraustochytrids family strains can be used to produce high-functional omega-3 rich oil (~ 30-70% of dry cell weight) and carotenoid-based antioxidant pigments simultaneously with value-added bioactive potential. In the present study, we describe the isolation and characterization of a new Thraustochytrid Schizochytrium sp. from the west coastal area of Korea. This newly isolated Thraustochytrid, identified as Schizochytrium sp. through 18S rRNA analysis and named SH104, simultaneously produces high levels of DHA and carotenoid-based antioxidant pigments. An improved Schizochytrium mutant, named SHG104, was obtained from the original host strain by γ-irradiation-induced mutagenesis. Under combined temperature-shift cultivation conditions employing white-light LEDs (light-emitting diodes), Schizochytrium sp. SHG104 yielded 10.8 g L-1 of biomass comprising 45.8% total lipids (32.1% DHA) and 4.6 mg L-1 of astaxanthin. In addition to DHA, the main fatty acids produced by Schizochytrium sp. SHG104 were palmitic acid and a trace of other long-chain fatty acids. The carotenoid profile of SH104 and SHG104 was β-carotene, astaxanthin, canthaxanthin, pheonicoxanthin and echinenone, which analyzed by HPLC and LC/APCI-MS. Furthermore, genomic analysis of Schizochytrium and Aurantiochytrium microalgae confirmed that the presence of carotenogenesis pathway enzymes and genes including geranylgeranyl diphosphate, phytoene synthase, lycopene cyclase, and cytochrome P450 hydroxylase that necessary for the production of antioxidants via a complete biosynthetic KEGG synthesis pathway. This newly isolated Schizochytrium microalga potentially have wide application as a source of antioxidants for astaxanthin-containing pigments, commercial omega-3 lipids and feed additives, such as nutritional supplements for aquaculture.

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.

Recombinant acetyl xylan esterase (rAXE) from Aspergillus ficcum, which mediated the production of peracetic acid (PAA), was covalently immobilized with magnetic Fe3O4–chitosan nanoparticles (Fe3O4–CSN) using glutaraldehyde. Fe3O4–CSN were prepared by co-precipitation of Fe2+ and Fe3+ ions in ammonia solution followed by coating of chitosan with sodium tripolyphosphate (TPP), and were characterized by FESEM, SEM, FTIR and XRD. The immobilization rAXE on Fe3O4–CSN was optimized using response surface methodology (RSM) by examining immobilization cross-linking time, enzyme concentration, and glutaraldehyde (GA) concentration. Based on the experimental values the predicted variables for the maximum immobilization of rAXE in terms of specific activity (0.042 U) were found to be 13.65 μg of rAXE protein and 0.265% of GA concentrations, where the optimum cross-linking time was 11.33 h. The immobilized rAXE onto Fe3O4–CSN nanoparticles shows better stability at thermal and pH ranges than soluble free rAXE. The immobilized rAXE was stable for around 90% of activity in the aqueous phase, whereas it retains only 60% of its activity in the semi-aqueous phase after 10 cycles of reuse.

In the previous work, acetyl xylan esterase (AXE) of Aspergillus ficcum was successfully produced in Pichia pastoris as host. In this study, the recombinant AXE was immobilized on graphite oxide and used for the production of peracetic acid. Immobilization efficiency was enhanced by modifying graphite oxide via surface functionalization. The conditions for enzyme immobilization were also investigated and the optimal conditions were determined as 4℃ of temperature, 24 h of reaction time and pH 7. The activity of immobilized AXE was found to be 62.53 U/g-support. With the immobilized AXE, about 134 mM of peracetic acid was produced under 37℃ of temperature and 30 min of reaction time. Enzyme activity remained at > 50% of the original after 10 production cycles.

LEDs light offer several advantages over the conventional lamps, thereby being considered as the optimal light sources for microalgal cultivation. In this study, various light-emitting diodes (LEDs) especially red and blue color with different light wavelengths were employed to explore the effects of light source on phototrophic cultivation of Chlorella vulgaris. Blue light illumination led to significantly increased cell size, whereas red light resulted in small-sized cell with active divisions. Based on the discovery of the effect of light wavelengths on microalgal biology, we then applied appropriate wavelength at different growth stages; blue light was illuminated first and then shifted to red light. By doing so, biomass and lipid productivity of C. vulgaris could be significantly increased, compared to that in the control. These results will shed light on a novel approach using LED light for microalgal biotechnology.

Laccases are widely distributed in plants, bacteria, and fungi, and are used for a wide range of applications, including bioremediation. In the present work laccase was immobilized on an electrospun zein polyurethane nanofiber via crosslinking with glutaraldehyde. FESEM and FTIR analysis clearly demonstrates the binding efficiency of laccase on the nanofiber. The relative activity of immobilized laccase was 85% that of free laccase. Immobilized laccase had a better pH and thermal stability than free laccase. The immobilized laccase completely degraded chloroxuron up to 25 reuse cycles in the presence of 1 mM HoBt. Paddy seeds soaked with solution containing chloroxuron treated with immobilized laccase showed a germination percentage closer to the distilled water control; whereas no damage or fatality of paddy seedlings were noticed in treated chloroxuron solution, which demonstrates the ability of immobilized laccase to detoxify the phenyl urea herbicide chloroxuron.

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.

dsRNA was found in malformed cultures of Lentinula edodes strain FMRI0339, one of the three most popular sawdust cultivated commercial strains of shiitake, and was also found in healthy-looking fruiting bodies and actively growing mycelia. Cloning of the partial genome of the dsRNA revealed the presence of the RdRp sequence of a novel L. edodes mycovirus (LeV), and sequence comparison of the cloned amplicon showed identical sequences sequence to known RNA-dependent RNA polymerase genes of LeV found in strain HKA. The meiotic stability of dsRNA was examined by measuring the ratio of the presence of dsRNA among sexual monokaryotic progeny. More than 40% of the monokaryotic progeny still contained the dsRNA, indicating the persistence of dsRNA during sexual reproduction. Comparing the mycelia growth of monokaryotic progeny suggested that there appeared to be a tendency toward a lower frequency of virus incidence in actively growing progeny.

A bifunctional xylosidase/arabinofuranosidase gene (PcXyl) was cloned from the cDNA library of Phanerochaete chrysosporium and further expressed in Pichia pastoris. Enzymatic assay indicated that P. pastoris produced rPcXyl at a level of 26,141 U l⁻¹. The xylosidase and arabinofuranosidase activities of rPcXyl were maximized, respectively, at pHs of 5.0 and 5.5 and temperatures of 45°C and 50°C. SDS-PAGE revealed a single band of purified rPcXyl of 83 kDa. Cu²⁺ and Zn²⁺ completely inhibited the enzyme activity of rPcXyl. The enzyme activity of rPcXyl was increased 151%, 126% and 123%, respectively, in the presence of glucose, xylose and arabinose at concentrations of 5 mM. rPcXyl hydrolyzed xylobiose to xylose and xylotriose to xylose and xylobiose, indicating rPcXyl acts as an exo-type enzyme. Additionally, rPcXyl enhanced xylose release from xylan substrates in synergy with rPcXynC.

A cDNA encoding for manganese peroxidase isozyme H4 (MnPH4), isolated from Phanerochaete chrysosporium, was expressed in Pichia pastoris, under the control of alcohol oxidase I promoter. The recombinant MnPH4 was efficiently secreted onto media supplemented with hemin at a maximum concentration of 500 U/L, after which purified rMnPH4 was used to decolorize the triarylmethane dye malachite green (MG). Response surface methodology (RSM) was employed to optimize three different operational parameters for the decolorization of MG. RSM showed that the optimized variables of enzyme (0.662 U), MnSO4 (448 μM), and hydrogen peroxide (159 μM) decolorized 100 mg/L of MG completely at 3 h. Additionally, UV-VIS spectra, high-performance liquid chromatography, gas chromatography-mass spectrometry, and liquid chromatography-electrospray ionization/mass spectrometry analysis confirmed the degradation of MG by the formation of main metabolites 4-dimethylamino-benzophenone hydrate, N, N-dimethylaniline (N,N-dimethyl-benzenamine), and methylbenzaldehyde. Interestingly, it was found that rMnPH4 mediates hydroxyl radical attack on the central carbon of MG. Finally, rMnPH4 degraded MG resulted in the complete removal of its toxicity, which was checked under in vitro conditions.

A wild-type bacteria producing an antimicrobial substance was isolated from Korean traditional fermented soybean paste, and identified as Bacillus subtilis. The antimicrobial substance purified by TLC, tentatively named UV254-B, displayed a specific antimicrobial activity against pathogenic Bacillus cereus and Listeria monocytogenes, without inhibiting the growth of soybean-fermenting Bacillus species. The antimicrobial substance was susceptible to proteinase K and lipase but resistant to esterase. Antimicrobial activity was observed over a wide range of pH from 3 to 11, with the maximum activity at pH 9, and thermal stability up to 80°C. The minimum inhibitory concentration of the antimicrobial substance was found to be 64 μg/mL for B. cereus and 128 μg/mL for L. monocytogenes. This antimicrobial substance has a putative molecular weight either at 1,133.6 or 1,700.5, which differs from that of other antimicrobial substances described for B. subtilis such as iturin, surfactin, fengycin, and subtilisin.

In previous studies, the biological characteristics of the fungus Cladosporium phlei and its genetic manipulation by transformation were assessed to improve production of the fungal pigment, phleichrome, which is a fungal perylenequinone that plays an important role in the production of a photodynamic therapeutic agent. However, the low production of this metabolite by the wild-type strain has limited its application. Thus, we attempted to clone and characterize the genes that encode polyketide synthases (PKS), which are responsible for the synthesis of fungal pigments such as perylenequinones including phleichrome, elsinochrome and cercosporin. Thus, we performed genomic DNA PCR using 11 different combinations of degenerate primers targeting conserved domains including β-ketoacyl synthase and acyltransferase domains. Sequence comparison of the PCR amplicons revealed a high homology to known PKSs, and four different PKS genes showing a high similarity to three representative types of PKS genes were amplified. To obtain full-length PKS genes, an ordered gene library of a phleichrome-producing C. phlei strain (ATCC 36193) was constructed in a fosmid vector and 4800 clones were analyzed using a simple pyramidal arrangement system. This hierarchical clustering method combines the efficiency of PCR with enhanced specificity. Among the three representative types of PKSs, two reducing, one partially reducing, and one non-reducing PKS were identified. These genes were subsequently cloned, sequenced, and characterized. Biological characterization of these genes to determine their roles in phleichrome production is underway, with the ultimate aim of engineering this pathway to overproduce the desired substance.

cDNA of the glx1 gene encoding glyoxal oxidase (GLX) from Phanerochaete chrysosporium was isolated and expressed in Pichia pastoris. The recombinant GLX (rGLX) produces H(2)O(2) over 7.0 nmol/min/mL using methyl glyoxal as a substrate. Use of rGLX as a generator of H(2)O(2) improved the coupled reaction with recombinant manganese peroxidase resulting in decolorization of malachite green up to 150 µM within 90 min.

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.

A coexpression strategy in Saccharomyces cerevisiae using episomal and integrative vectors for the Escherichia coli heat-labile enterotoxin B subunit (LTB) and a fusion protein of an ApxIIA toxin epitope produced by Actinobacillus pleuropneumoniae coupled to LTB, respectively, was adapted for the hetero-oligomerization of LTB and the LTB fusion construct. Enzyme-linked immunosorbent assay (ELISA) with GM1 ganglioside indicated that the LTB fusion construct, along with LTB, was oligomerized to make the functional heteropentameric form, which can bind to receptors on the mucosal epithelium. The antigen-specific antibody titer of mice orally administered antigen was increased when using recombinant yeast coexpressing the pentameric form instead of recombinant yeast expressing either the LTB fusion form or antigen alone. Better protection against challenge infection with A. pleuropneumoniae was also observed for coexpression in recombinant yeast compared with others. The present study clearly indicated that the coexpression strategy enabled the LTB fusion construct to participate in the pentameric formation, resulting in an improved induction of systemic and mucosal immune responses.

SUMMARY The gene CpSte11 of Cryphonectria parasitica, which encodes a yeast Ste11 homologue, was cloned and characterized. Gene replacement analysis revealed a high frequency of CpSte11 null mutants. When compared with the wild-type parent strain, CpSte11 null mutants showed no difference in terms of growth rate or pigmentation. However, CpSte11 null mutants showed a marked decrease in both the number and size of stromal pustules on chestnut twigs. The virulence test showed that, in comparison with those of the wild-type and virus-infected hypovirulent strains, CpSte11 null mutants produced necrotic areas of intermediate size. Disruption of the CpSte11 gene also resulted in defects in female fertility. Down-regulation of transcripts for the mating pheromone precursor gene, Mf2/2, and mating response transcription factors, such as cpst12 and pro1, was observed in CpSte11 null mutants. The down-regulation of Mf2/2, cpst12 and pro1 was also observed in the mutant phenotype of Cpmk2, a mating response Fus3-like mitogen-activated protein kinase (MAPK) gene, but not in the mutant of Cpmk1, a high-osmolarity glycerol Hog1-like MAPK gene. These results indicate that the cloned CpSte11 gene is functionally involved in the mating response pathway and acts through downstream targets, including Cpmk2, cpst12, pro1 and Mf2/2. However, the characteristics of the CpSte11 null mutant were fully phenocopied only in the cpst12 null mutant, but not in other studied null mutants of components of the putative mating response pathway.

UV-mutagenesis was performed to obtain mutant strains that demonstrate altered production of phleichrome, a secondary metabolite of Cladosporium phlei. Among fifty mutants selected, based on the increased area and intensity of the purple pigment surrounding the colonies, the strain M0035 showed the highest production of phleichrome, more than seven fold over wild type. Plate cultures of the M0035 strain resulted in a total of 592 mg phleichrome consisting of 146 mg and 446 mg from the mycelia and agar media, respectively. The M0035 strain displayed a growth rate and a mycelial mass comparable to the parental strain but had significantly reduced asexual sporulation.

The cDNA of endo-1,4-β-xylanaseA, isolated from Phaenerocheate chrysosporium was expressed in Pichia pastoris. Using either the intrinsic leader peptide of XynA or the α-factor signal peptide of Saccharomyces cerevisiae, xylanaseA is efficiently secreted into the medium at maximum concentrations of 1,946 U/L and 2,496 U/L, respectively.

The cDNA of endo-1,4-β-xylanaseC, isolated from Phanerochaete chrysosporium, was expressed in Pichia pastoris, under the control of the alcohol oxidase I promoter. Using either the intrinsic leader peptide of xylanaseC or the α-factor signal peptide of Saccharomyces cerevisiae, xylanaseC is efficiently secreted into the medium, at a maximum concentration of 2500 U·l(-1).

Objectives The seed of Phaseolus calcaratus Roxburgh (PHCR) has traditionally been used as a herbal medicine, considered to have anti-inflammatory potential. Here we examined the ability of PHCR seed extract to inhibit inflammatory responses of macrophages to bacterial toxin and the mechanism involved. Methods In the present study, we prepared four fractions from an ethanol extract of PHCR seed and investigated their effects on the production of nitric oxide and cytokines, and the expression of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. Key findings The fractions inhibited LPS-induced nitric oxide production and cyclooxygenase-2 (COX-2) expression in the cells. The ethyl acetate fraction at 100 µg/ml almost completely suppressed NO production, iNOS and COX-2 expression, and TNF-α and IL-6 secretion in cells stimulated with LPS. The fraction also inhibited phosphorylation of extracellular signal-regulated kinase (ERK) and p38 in LPS-stimulated cells with the attendant suppression of IκBα nuclear translocation and nuclear factor (NF)-κB activation. Furthermore, PHCR seed extracts contained a large number of phenolic compounds having antioxidant potentials against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and hydroxyl radicals. We identified catechin-7-O-β-d-glucopyranoside as one of the active compounds responsible for the biological activity of PHCR seed extract. Conclusions These results suggest for the first time that ethanol extracts from PHCR seed have anti-inflammatory potential on LPS-stimulated macrophages through the down-regulation of ERK/p38- and NF-κB-mediated signalling pathways.

Bacteria of the Bacillus species have been reported as an important microorganism in fermented soybean products. In the present study, thirty Bacillus isolates were screened from Meju, a Korean soybean fermentation starter. The comparative analysis of 16S rDNA sequences, 16S-23S internal transcribed spacer sequences, phenotypic, and biochemical characterizations revealed three phylogenetically distinct groups namely Bacillus atrophaeus,Bacillus polyfermenticus and Bacillus subtilis. The isolates were assayed for poly-γ-glutamate production and fibrinolytic activity. Among the isolates, B. polyfermenticus exhibited maximum poly-γ-glutamate production and fibrinolytic activity. Moreover, the soybean products fermented by B. polyfermenticus have increased the time taken for coagulation and hemorrhage in mice. The results of the present study clearly indicate the functional role of B. polyfermenticus in fermented soybean products.

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 0.026∼0.048% 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.

Genetic diversity of the chrysovirus within the four fungal strains was analyzed by comparing the full-length sequences of cloned chrysoviral genes encoding the RNA-dependent RNA polymerase (RdRp) and capsid protein (CP). Because the morphological characteristics of four chrysovirus-infected Cryphonectria spp. strains were different, strain identification was conducted via sequence comparison of the internal transcribed spacers (ITSs) of the fungal rRNA gene. Phylogenic analysis of the ITS regions revealed that the four strains were closely clustered with the reference strain of Cryphonectria nitschkei, while they were more distantly related to other common Cryphonectria species, indicating that they were likely C. nitschkei. Sequence comparison among chrysoviruses from Korean C. nitschkei strains revealed that similarities of the RdRp and CP genes ranged from 98% to 100% and from 95% to 100%, respectively, at the protein level. Their corresponding nucleotide sequences showed 97% to 100% and 84% to 100% identities, respectively. Compared to RdRp, the CP gene had more divergence, suggesting the presence of genes possessing different evolutionary rates within the chrysovirus genome. Sequence comparisons with other known chrysoviruses showed that the four Korean chrysoviruses were clustered together at the next lineage level. Discovering why two strains (bsl31 and bsl32) containing identical ITS sequences and chrysoviruses display different phenotypes should prove interesting.

To facilitate the genetic manipulation of Cladosporium phlei, a causal agent of leaf spot disease in timothy (Phleum pretense), protoplast-mediated transformation of C. phlei has been developed and the resulting transformants were characterized in this study. Hygromycin B resistance was applied as a dominant selection marker due to the sensitivity of C. phlei to this antibiotic. The transformation efficiency ranged from approximately 20-100 transformants per experiment. Southern blot analysis of stable transformants revealed that transformation occurred by way of stable integration of the vector DNA into the fungal chromosome. PCR analysis and plasmid rescuing of randomly selected transformants suggested that integration of tandem repeat copies of vector DNA was common. In addition, multiple integrations of the transforming vector at different chromosomal sites were also observed. The establishment of a transformation method for C. phlei facilitates strain improvement of this fungus and can be applied as an initial step in the molecular analysis of pigment production in this fungus.

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 endoplasmci 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 inmmunosorbent 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 aligomers 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.

A new laccase gene (lac3) from the chestnut blight fungus Cryphonectria parasitica was induced by the presence of tannic acid, which is abundant in the bark of chestnut trees and is assumed to be one of the major barriers against pathogen infection. However, other commonly known laccase inducers, including ferulic acid, 2,5-xylidine, catechol, and pH, did not induce lac3 transcription. Moreover, the hypovirus modulated the induction of lac3 transcription, abolishing the transcriptional induction of the lac3 gene by tannic acid. A functional analysis of lac3 using a lac3-null mutant indicated that fungal growth and other morphological characteristics, including pigmentation and sporulation, were not affected. However, a virulence assay indicated that the loss of function of a tannic acid–inducible and hypoviral-regulated laccase resulted in reduced virulence without detectable changes in the morphological features. The constitutive expression of lac3 resulted in no significant differences in the necrotic lesions from those caused by the wild type, but its expression in the presence of the hypovirus led to larger lesions than those caused by the hypovirulent strain. These results suggest that the lac3 gene product may not be the only determinant of fungal virulence in chestnut trees but is an important factor.

We analysed 676 isolates from 33 Korean Cryphonectria parasitica subpopulations in Korea for dsRNA incidence and diversity. dsRNA was detected in 84 isolates. Although the dsRNA banding patterns varied in several minor bands, infected isolates could be categorized into two groups. The most common banding pattern occurred in 77 isolates and contained a 12.7-kb band indicative of Cryphonectria hypovirus 1 (CHV1), and several accompanying minor bands with sizes ranging from 0.9–5 kb. Northern blot analysis revealed that all 12.7-kb fragments in the dsRNA-containing isolates hybridized to probes corresponding to open reading frames (ORFs) A and B from the reference CHV1 strain (GenBank accession no. M57938 ). In addition, the sequence of a 1.4-kb cDNA fragment from a representative isolate of the most common group showed 99 % sequence similarity to ORF A of CHV1. However, the other group of seven isolates had distinctive bands of 3.5 and 3.3 kb, but not the 12.7-kb band. Sequence comparison showed that cloned fragments of these dsRNAs were similar to those of the coat protein and RNA-dependent RNA polymerase genes of chrysovirus, which indicates the occurrence of chrysovirus in the Korean population. Fungal strain identity was assessed via RFLP analysis of the ITS regions. Among the 84 tested isolates, six had different ITS-RFLP patterns (RFLP-II) from that (RFLP-I) of C. parasitica, and are believed to be C. nitschkei, a sympatric species reported on chestnut trees in Japan. The chrysovirus and CHV1 were detected in strains showing both RFLP patterns. However, the chrysovirus was more frequent in the RFLP-II group.

We have analysed changes in the composition of ribosomal proteins during cell growth in Bacillus subtilis. Ribosome fractions were prepared from B. subtilis cells at different phases of growth and were separated by radical-free and highly reducing (RFHR) two-dimensional polyacrylamide gel electrophoresis. We identified 50 ribosomal proteins, Including two paralogues of L31 protein (RpmE and YtiA). Although the ribosome fraction extracted from exponentially growing cells contained RpmE protein, this protein disappeared during the stationary phase. In contrast, YtiA was detected In the ribosome fractlon extracted after the end of exponential growth. Expression of the ytiA gene encoding YtiA was found to be negatively controlled by Zur, a zinc-specific transcriptional repressor that controls zinc transport operons. Analysis by inductively coupled plasma mass spectrometry (ICP-MS) Indicated that RpmE contains one zinc ion per molecule of protein. in addition, mutagenesis of the rpmE gene encoding RpmE revealed that Cys-36 and Cys-39, located within a CxxC motif, are required not only for binding zinc but also for the accumulation of RpmE in the cell. Taken together, these results indicate that zinc plays an essential role in the alternation Accepted 10 December, 2003. *For correspondence. E-mail kawamura@rikkyo.ne.jp; Tel. and Fax (+81) 33 985 2401. T he first two authors contributed equally to this work. between two types of L31 protein in the ribosome of B. subtilis.

To eliminate porcine reproductive and respiratory syndrome virus (PRRSV) from a supplying boar stud, samples of serum and semen from 118 boars were assessed three times a month by an indirect fluorescent antibody (IFA) test to detect antibodies, and by a nested reverse transcriptase-PCR (nRT-PCR) to detect the genome of PRRSV. The boars detected as persistently infected carriers were culled. A PRRSV-negative population of boars was established after three months and no semen positive for the virus was detected for six months. Subsequently, a three-step plan was introduced to eliminate PRRSV from the seedstock breeding farm during three parity cycles on the farm over 15 months, each step taking five months. In step 1, umbilical cords taken from piglets at birth and serum samples taken from their dams at the start of weaning were subjected to ifa and nRT-pcr analysis. The sows with PRRSV detected in serum by nRT-pcr were regarded as carrier sows and culled. The rates of detection of PRRSV were reduced from 5 percent to 2.5 percent in the sera of the sows, and from 14.8 percent to 1.8 percent in the umbilical cords of the piglets. In step 2, the sows that had farrowed the piglets with PRRSV detected by nRT-PCR in their cords were considered to have transmitted the infection and removed. During step 2, the virus detection rates in umbilical cords by nRT-pcr were reduced, but not completely eliminated. In step 3, 10-week-old nursery pigs with antibodies to PRRSV in their serum by ifa and elisa were culled. The three steps established the PRRSV-negative state of the multisite farm containing the breeding and nursery farm, and the PRRSV-negative state of both the multisite farm and the supplying boar stud was evaluated by monthly monitoring over at least one parity cycle of the farm for five months.

The cultural characteristics of the fungusCladosporium phlei were assessed in order to develop an improved method for the production of the fungal pigment, phleichrome, which is an intermediate in the production of a photodynamic therapeutic agent. The growth ofC. phlei, as measured by the hyphal growth rate and increase in biomass, varies significantly depending on the culture media utilized (V8 juice-based medium proved optimal for both growth rate and biomass increase). How-ever, even on a V8 juice plate, the growth ofC. phlei occurred slowly and in a limited fashion, in that the colony covered only 75% of the agar surface after more than 4 weeks of cultivation at 20°C. Supplementations of glucose, fructose, galactose, and sucrose increased both hyphal expansion and mass production, whereas supplementations of other carbon sources, including glycerol and sorbitol, exerted no detectable effects. The effect of inorganic nitrogen supplementation was negligible, whereas organic nitrogen evidenced significant effects, with enhanced growth with malt extract and growth inhibition with yeast extract and tryptone. Sporulation was enhanced under conditions of continuous light, and a minimum of 103 spores per mL of liquid media was found to be necessary for the optimal mass increase. A simple extraction procedure was established in order to isolate the deep red pigment which was subsequently identified as phleichrome via NMR analysis. WhenC. phlei was cultured on V8 medium containing 5% glucose and 2% malt extract, the quantity of mycelial mass was estimated as 20.6 g (dry weight) per liter of culture. The expected phleichrome yields from the mycelia and culture filtrates were estimated to be 43 and 2 mg/L, repectively.

Notes on Three Species of the Genus Euphoriomyces (Laboulbeniales) from KoreaTaxonomic Studies on Myelochroa from KoreaMolecular Analysis of Korean Anzia opuntiella (Lichenized Ascomycota) Based on...

Vascular endothelial cells release proteinases that degrade the extracellular matrix (ECM), thus enabling cell migration during angiogenesis and vasculogenesis. Sildenafil citrate stimulates the nitric oxide-cyclic guanosine monophosphate pathway through inhibition of phosphodiesterase type V (PDE5). In this report, we examined the mechanisms underlying sildenafil citrate-induced cell migration using cultured mouse aortic endothelial cells (MAECs). Sildenafil citrate induced migration and proteinase secretion by murine endothelial cells. Sildenafil citrate induced the secretion of matrix metalloproteinase-2 (MMP-2) and MMP-9, which is inhibited by NF-κB inhibitors. Sildenafil citrate also induced the secretion of plasmin, which is inhibited by Pl 3′-kinase inhibitors. It is suggested that sildenafil citrate-induced migrating activity in endothelial cells may be accomplished by increased secretion of proteinases.

Actinobacillus pleuropneumoniae is an important pig pathogen, which is responsible for swine pleuropneumonia, a highly contagious respiratory infection. To develop subunit vaccines forA. pleuropneumoniae infection, the Apx toxin genes,apxI andapxII, which are thought to be important for protective immunity, were expressed inSaccharomyces cerevisiae, and the induction of immune responses in mice was examined. TheapxI andapxII genes were placed under the control of a yeast hybridADH2-GPD promoter (AG), consisting of alcohol dehydrogenase II (ADH2) and theGPD promoter. Western blot analysis confirmed that both toxins were successfully expressed in the yeast. The ApxIA and ApxIIA-specific IgG antibody response assays showed dose dependent increases in the antigen-specific IgG antibody titers. The challenge test revealed that ninety percent of the mice immunized with ApxIIA or a mixture of ApxIA and ApxIIA, and sixty percent of mice immunized with ApxIA survived, while none of those in the control groups survived longer than 36 h. These results suggest that vaccination of the yeast expressing the ApxI and ApxII antigens is effective for the induction of protective immune responses againstA. pleuropneumoniae infections in mice.

We have studied noise properties and magnetocardiographic performance of single-layer second-order YBCO SQUID gradiometers made on STO substrates. A three-loop pickup coil is directly coupled to a step-edge junction SQUID with the coupling polarity of the center loop opposite to that of two side loops. Optimized device parameters for balancing were obtained from 8.8 mm /spl times/ 3.0 mm devices on 10 mm /spl times/ 10 mm substrates and scaled up to the larger devices for use in cardiomagnetic measurements. A 17.6 mm /spl times/ 6 mm gradiometer with a 5.8 mm baseline showed an unshielded gradient noise of 0.84 pT/cm/sup 2//Hz/sup 1/2/ at 1 Hz, which corresponds to an equivalent field noise of 280 fT/Hz/sup 1/2/. A large balancing factor of 10/sup 3/ allowed us to record magnetocardiogram of a human subject.