Seung-Moon Park

Zinc is a key factor in controlling alternation of two types of L31 protein in the Bacillus subtilis ribosome

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 fraction 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 between two types of L31 protein in the ribosome of B. subtilis.

Characterization of HOG1 homologue, CpMK1, from Cryphonectria parasitica and evidence for hypovirus-mediated perturbation of its phosphorylation in response to hypertonic stress

We examined the biological function of cpmk1, which encodes a MAPK of Cryphonectria parasitica, and its regulation by mycovirus. Sequence comparisons revealed that cpmk1 had highest homology with osm1, a hog1‐homologue from Magnaporthe grisea. A growth defect was observed in the cpmk1‐null mutant under hyperosmotic conditions, indicating that cpmk1 functionally belongs to a hog1 subfamily. Immunoblot analyses indicated that the CpMK1 pathway was affected specifically in hyperosmotic conditions by the hypovirus CHV1‐EP713. Moreover, the virus‐infected hypovirulent UEP1 strain also exhibited severe osmosensitivity compared to the virus‐free isogenic strain EP155/2, thus providing additional evidence for viral regulation of cpmk1 in response to a hypertonic stress. Besides osmosensitivity, disruption of cpmk1 resulted in several, but not all, hypovirulence‐associated changes, such as reduced pigmentation, conidiation, laccase production and cryparin expression. However, the cpmk1‐null mutant exhibited an increased accumulation of pheromone gene transcripts. Virulence assays of the cpmk1‐null mutant revealed reduced canker area, but not as severe as that of UEP1. These results suggest that mycoviruses modulate the MAPK and thereby provoke the aberrant expression of target genes, some of which are likely to be implicated in viral symptom development.

Inhibition of VEGF blocks TGF-β1 production through a PI3K/Akt signalling pathway

Vascular endothelial growth factor (VEGF) is a mediator of airway inflammation and remodelling in asthma. Transforming growth factor (TGF)-β1 plays pivotal roles in diverse biological processes, including tissue remodelling and repair in a number of chronic lung diseases. However, there are few studies elucidating the interactions between VEGF and TGF-β1 in allergic airway disease. A murine model of allergic airway disease was used to define the mechanism by which VEGF induces subepithelial fibrosis and to investigate a potential relationship between VEGF and TGF-β1 and the mechanisms by which VEGF signalling regulates TGF-β1 expression in allergic airway disease. The ovalbumin (OVA)-inhaled murine model revealed the following typical pathophysiological features of allergic airway disease in the lungs: increased numbers of inflammatory cells of the airways, airway hyperresponsiveness, increased peribronchial fibrosis, and increased levels of VEGF and TGF-β1. Administration of VEGF inhibitors reduced the pathophysiological signs of allergic airway disease and decreased the increased TGF-β1 levels and peribronchial fibrosis, including phosphoinositide 3-kinase (PI3K) activity after OVA inhalation. In addition, the increased TGF-β1 levels and collagen deposition after OVA inhalation were decreased by administration of PI3K inhibitors. These results suggest that inhibition of vascular endothelial growth factor attenuates peribronchial fibrosis, at least when mediated by regulation of transforming growth factor-β1 expression through phosphoinositide 3-kinase/Akt pathway in a murine model of allergic airway disease.

Phosphoinositide 3-kinase δ inhibitor suppresses interleukin-17 expression in a murine asthma model

Phosphoinositide 3-kinases (PI3Ks) contribute to the pathogenesis of asthma by regulating the activation of inflammatory mediators, inflammatory cell recruitment and immune cell function. Recent findings have indicated that PI3Ks also regulate the expression of interleukin (IL)-17, which has been recognised as an important cytokine involved in airway inflammation. In the present study, we investigated a role of PI3K&dgr; in the regulation of IL-17 expression in allergic airway disease using a murine model of asthma. After ovalbumin inhalation, administration of a selective p110&dgr; inhibitor, IC87114, significantly attenuated airway infiltration of total cells, lymphocytes, neutrophils and eosinophils, as well as airway hyperresponsiveness, and attenuated the increase in IL-17 protein and mRNA expression. Moreover, IC87114 reduced levels of IL-4, -5 and -13, expression of keratinocyte chemoattractant protein and mRNA, and nuclear factor (NF)-&kgr;B activity. In addition, a NF-&kgr;B inhibitor, BAY 11-7085 substantially reduced the increase in IL-17 protein levels. Our results also showed that inhibition of IL-17 activity with an anti-IL-17 antibody remarkably reduced airway inflammation and hyperresponsiveness. These findings suggest that inhibition of the p110&dgr; signalling pathway suppresses IL-17 expression through regulation of NF-&kgr;B activity and, thus, has therapeutic potential in asthma.

Variations of Landfill Leachate's Properties in Conjunction with the Treatment Process

A study was done on the variations of water quality parameters, in conjunction with the processing steps, installed for the treatment of leachate from a sanitary landfill. The leachate was primarily subjected to biological treatment, composed of anaerobic digestion, aerobic treatment, and rotating biological contact in series. The effluent from the rotating biological contact process was further treated by combination of flocculation-sedimentation and adsorption processes. Finally, it was polished by the reverse osmosis process before discharge. The quality of raw leachate, of influents to and of effluents from the reverse osmosis process was assessed and compared. To determine size - dependent behavior of organic materials, analyses were also made for the fractionated samples through ultrafiltration. The overall treatment efficiency was about 98 % for removal of organic materials. Most of the organic materials were in the low molecular weight range. In particular, about 95% of the biological oxygen demand was found to be exerted by the fraction of organic material of which cut - off molecular weight was less than 500. The analysis of molecular weight distribution confirmed that the organic materials resistant to the removal by the treatment process were humic substances, of which cut off molecular weight was greater than 500. The reverse osmosis process showed a high efficiency for removal of high molecular weight constituents. However, the final effluent showed an unexpectedly high oxygen demand.

Characterization of a fungal protein kinase from Cryphonectria parasitica and its transcriptional upregulation by hypovirus

The chestnut blight fungus Cryphonectria parasitica and its hypovirus comprise useful model system to study the mechanisms of hypoviral infection. We used degenerate primers based on fungal protein kinases to isolate a gene, cppk1, which encodes a novel Ser/Thr protein kinase of C. parasitica. The gene showed highest homology to ptk1, a Ser/Thr protein kinase from Trichoderma reesei. The encoded protein had a predicted mass of 70.5 kDa and a pI of 7.45. Northern blot analyses revealed that the cppk1 transcript was expressed from the beginning of culture, with a slight increase by 5 days of culture. However, its expression was specifically affected by the presence of virus, and it was transcriptionally upregulated in the fungal strain infected with the hypovirus. A kinase assay using Escherichia coli‐derived CpPK1 revealed CpPK1‐specific phosphorylated proteins with estimated masses of 50 kDa and 44 kDa. In addition, the phosphorylation of both proteins was higher in a cell‐free extract from the hypovirulent strain. The increased expression of cppk1 by the introduction of an additional copy results in a subset of viral symptoms of reduced pigmentation and conidiation in a virus‐free isolate. cppk1 overexpression also causes the downregulation of mating factor genes Mf2/1 and Mf2/2, resulting in female sterility. The present study suggests that the hypovirus disturbs fungal signalling by transcriptional upregulation of cppk1, which results in reduced pigmentation and conidiation and female sterility.

Expression of Heteropolymeric Ferritin Improves Iron Storage in Saccharomyces cerevisiae

ABSTRACT Saccharomyces cerevisiae was engineered to express different amount of heavy (H)- and light (L)-chain subunits of human ferritin by using a low-copy integrative vector (YIp) and a high-copy episomal vector (YEp). In addition to pep4::HIS3 allele, the expression host strain was bred to have the selection markers leu2− and ura3− for YIplac128 and YEp352, respectively. The heterologous expression of phytase was used to determine the expression capability of the host strain. Expression in the new host strain (2805-a7) was as high as that in the parental strain (2805), which expresses high levels of several foreign genes. Following transformation, Northern and Western blot analyses demonstrated the expression of H- and L-chain genes. The recombinant yeast was more iron tolerant, in that transformed cells formed colonies on plates containing more than 25 mM ferric citrate, whereas none of the recipient strain cells did. Prussian blue staining indicated that the expressed isoferritins were assembled in vivo into a complex that bound iron. The expressed subunits showed a clear preference for the formation of heteropolymers over homopolymers. The molar ratio of H to L chains was estimated to be 1:6.8. The gel-purified heteropolymer took up iron faster than the L homopolymer, and it took up more iron than the H homopolymer did. The iron concentrations in transformants expressing the heteropolymer, L homopolymer, and H homopolymer were 1,004, 760, and 500 μg per g (dry weight) of recombinant yeast cells, respectively. The results indicate that heterologously expressed H and L subunits coassemble into a heteropolymer in vivo and that the iron-carrying capacity of yeast is further enhanced by the expression of heteropolymeric isoferritin.

Structure, heterologous expression, and properties of rice (Oryza sativa L.) family 19 chitinases.

We identified four new family 19 chitinases in Oryza sativa L. cv. Nipponbare: one class I (OsChia1d), two class II (OsChia2a and OsChia2b), and one class IV (OsChia4a). OsChia2a resembled (about 60% identity) the catalytic domains of class I chitinases, but OsChia2b was almost identical (95% identity) to that of the class IV enzyme. OsChia1c, OsChia1cΔCBD (a deletion of OsChia1c lacking a chitin-binding domain, CBD), and OsChia2b were separately expressed and purified in Pichia pastoris. OsChia1c inhibited fungal growth significantly more than OsChia1cΔCBD or OsChia2b. The activities of these enzymes on chitin polymers were similar, but they acted differently on N-acetylchitooligosaccharides, (GlcNAC)n. OsChia1c slowly hydrolyzed (GlcNAC)6 and very poorly hydrolyzed (GlcNAC)4 and (GlcNAC)5. In contrast, OsChia2b efficiently hydrolyzed these oligosaccharides. The high antifungal activity and low hydrolytic activity of the class I enzyme towards (GlcNAC)n imply that it participates in the generation of N-acetylchitooligosaccharide elicitors from the cell walls of infecting fungi.

A Tannic Acid-Inducible and Hypoviral-Regulated Laccase3 Contributes to the Virulence of the Chestnut Blight Fungus Cryphonectria parasitica

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.

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.