• Journal of Microbiology and Biotechnology
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• v.30 no.1
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• pp.62-70
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• 2020

Zygosaccharomyces rouxii is an important yeast that is required in the food fermentation process due to its high salt tolerance. In this study, the responses and resistance strategies of Z. rouxii against salt stress were investigated by performing physiological analysis at membrane level. The results showed that under salt stress, cell integrity was destroyed, and the cell wall was ruptured, which was accompanied by intracellular substance spillover. With an increase of salt concentrations, intracellular Na⁺ content increased slightly, whereas intracellular K⁺ content decreased significantly, which caused the increase of the intracellular Na⁺/K⁺ ratio. In addition, in response to salt stress, the activity of Na⁺/K⁺-ATPase increased from 0.54 to 2.14 μmol/mg protein, and the ergosterol content increased to 2.42-fold to maintain membrane stability. Analysis of cell membrane fluidity and fatty acid composition showed that cell membrane fluidity decreased and unsaturated fatty acid proportions increased, leading to a 101.21% rise in the unsaturated/saturated fatty acid ratio. The results presented in this study offer guidance in understanding the salt tolerance mechanism of Z. rouxii, and in developing new strategies to increase the industrial utilization of this species under salt stress.

• Journal of Chest Surgery
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• v.44 no.6
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• pp.406-412
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• 2011

Background: Development of thoracic aortic aneurysms and aortic dissections (TAAD) is attributed to unbearable wall tension superimposed on defective aortic wall integrity and impaired aortic repair mechanisms. Central to this repair mechanisms are well-balanced and adequately functional cellular components of the aortic wall, including endothelial cells, smooth muscle cells (SMCs), inflammatory cells, and adventitial fibroblasts. Adventitial fibroblasts naturally produce aortic extracellular matrix (ECM), and, when aortic wall is injured, they can be transformed into SMCs, which in turn are involved in aortic remodeling. We postulated the hypothesis that adventitial fibroblasts in patients with TAAD may have defects in ECM production and SMC transformation. Materials and Methods: Adventitial fibroblasts were procured from the adventitial layer of fresh aortic tissues of patients with TAAD (Group I) and of multi-organ donors (Group II), and 4-passage cell culture was performed prior to the experiment. To assess ECM production, cells were treated with TNF-${\alpha}$ (50 pM) and the expression of MMP-2/MMP-3 was analyzed using western blot technique. To assess SMC transformation capacity, cells were treated with TGF-${\beta}1$ and expression of SM ${\alpha}$-actin, SM-MHC, Ki-67 and SM calponin was evaluated using western blot technique. Fibroblasts were then treated with TGF-${\beta}1$ (10 pM) for up to 10 days with TGF-${\beta}1$ supplementation every 2 days, and the proportion of transformed SMC in the cell line was measured using immunofluorescence assay for fibroblast surface antigen every 2 days. Results: MMP-3 expression was significantly lower in group I than in group II. TGF-${\beta}1$-stimulated adventitial fibroblasts in group I expressed less SM ${\alpha}$-actin, SM-MHC, and Ki-67 than in group II. SM-calponin expression was not different between the two groups. Presence of fibroblast was observed on immunofluorescence assay after more than 6 days of TGF-${\beta}1$ treatment in group I, while most fibroblasts were transformed to SMC within 4 days in group II. Conclusion: ECM production and SMC transformation are compromised in adventitial fibroblasts from patients with TAAD. This result suggests that functional restoration of adventitial fibroblasts could well be a novel approach for the prevention and treatment of TAAD.

• Journal of Applied Biological Chemistry
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• v.56 no.3
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• pp.165-170
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• 2013

The SCO3388 gene from Streptomyces coelicolor is homologous to tmrB, the tunicamycin resistance gene of Bacillus subtilis. The SCO3388-inactivation strain (SY-tbl-1) was generated by replacing SCO3388 with thiostrepton resistance gene. Spores of S. coelicolor derivatives were prepared on mannitol-soy flour (MS) agar on which SY-tbl-1 displayed no significant defect in growth and development. When plated on R4 agar, spores of SYtbl-1 displayed retardation in growth and sporulation, whereas its mycelium gave rise to normal growth. Thus, SCO3388 is suggested to be involved in the dormant spore germination. Expression of SCO3388 under the ermE1 promoter restored but only partially the ability to sporulate in SY-tbl-1. Neither SY-tbl-1 nor SY-tbl-1/ermE1p-SCO3388 showed a difference in tunicamycin resistance to the wild type whereas, interestingly, the introduction of ermE1p-SCO3388 dramatically enhanced spore survival to heat and detergent treatments, suggesting that SCO3388 might play a role in the maintenance of spore cell wall integrity.

• Mycobiology
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• v.37 no.3
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• pp.161-170
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• 2009

Cryptococcus neoformans is a basidiomycete human fungal pathogen that causes meningoencephalitis in both immunocompromised and immunocompetent individuals. The ability to sense and respond to diverse extracellular signals is essential for the pathogen to infect and cause disease in the host. Four major stress-activated signaling (SAS) pathways have been characterized in C. neoformans, including the HOG (high osmolarity glycerol response), PKC/Mpk1 MAPK (mitogen-activated protein kinase), calcium-dependent calcineurin, and RAS signaling pathways. The HOG pathway in C. neoformans not only controls responses to diverse environmental stresses, including osmotic shock, UV irradiation, oxidative stress, heavy metal stress, antifungal drugs, toxic metabolites, and high temperature, but also regulates ergosterol biosynthesis. The PKC(protein kinase C)/Mpk1 pathway in C. neoformans is involved in a variety of stress responses, including osmotic, oxidative, and nitrosative stresses and breaches of cell wall integrity. The $Ca^{2+}$/calmodulin- and Ras-signaling pathways also play critical roles in adaptation to certain environmental stresses, such as high temperature and sexual differentiation. Perturbation of the SAS pathways not only impairs the ability of C. neoformans to resist a variety of environmental stresses during host infection, but also affects production of virulence factors, such as capsule and melanin. A drug(s) capable of targeting signaling components of the SAS pathway will be effective for treatment of cryptococcosis.

• Journal of Microbiology and Biotechnology
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• v.18 no.5
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• pp.845-851
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• 2008

TolC is an outer membrane porin protein and an essential component of drug efflux and type-I secretion systems in Gram-negative bacteria. TolC comprises a periplasmic $\alpha$-helical barrel domain and a membrane-embedded $\beta$-barrel domain. TdeA, a functional and structural homolog of TolC, is required for toxin and drug export in the pathogenic oral bacterium Actinobacillus actinomycetemcomitans. Here, we report the expression of the periplasmic domain of TdeA as a soluble protein by substitution of the membrane-embedded domain with short linkers, which enabled us to purify the protein in the absence of detergent. We confirmed the structural integrity of the TdeA periplasmic domain by size-exclusion chromatography, circular dichroism spectroscopy, and electron microscopy, which together showed that the periplasmic domain of the TolC protein family fold correctly on its own. We further demonstrated that the periplasmic domain of TdeA interacts with peptidoglycans of the bacterial cell wall, which supports the idea that completely folded TolC family proteins traverse the peptidoglycan layer to interact with inner membrane transporters.

• Journal of Life Science
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• v.12 no.6
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• pp.803-811
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• 2002

The present study was carried out for research and development of natural antimicrobial from extract of Stachys sieboldii MIQ against food borne bacteria. The hexane extract of Stachys sieboldii MIQ at 250$\mu\textrm{g}$/$m\ell$ per disc showed 15 ~ 20 mm inhibition zone against gram positive and gram negative barteria. Minimum inhibitory concentration (MIC) of hexane extract was 250${\mu}g$/$m\ell$against Bacillus cereus, 250~500${\mu}g$/$m\ell$against Listeria monocytogenes, 500${\mu}g$/$m\ell$ against Staphylococcus aureus, Psedomonas aeruginosa. Observation by transmission electron microscope, showed that disruption of the cell wall assumed to be due to the bactericidal activity. In addition, the membrane integrity of the sensitive cells was disrupted by exposure to Stachys sieboldii MIQ extract on the D-$\beta$-galatosidase activity as substrate of O-nitrophenol-$\beta$-D-galacto-pyranoside. The hexane extract of Stachys sieboldii MIQ was very stable on the pH and thermal stability.

• Journal of Ginseng Research
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• v.44 no.1
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• pp.161-167
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• 2020

Background: The ascomycete fungi Cylindrocarpon destructans (Cd) and Fusarium solani (Fs) cause ginseng root rot and significantly reduce the quality and yield of ginseng. Cd produces the secondary metabolite radicicol, which targets the molecular chaperone Hsp90. Fs is resistant to radicicol, whereas other fungal genera associated with ginseng disease are sensitive to it. Radicicol resistance mechanisms have not yet been elucidated. Methods: Transcriptome analyses of Fs and Cd mycelia treated with or without radicicol were conducted using RNA-seq. All of the differentially expressed genes (DEGs) were functionally annotated using the Fusarium graminearum transcript database. In addition, deletions of two transporter genes identified by RNA-seq were created to confirm their contributions to radicicol resistance. Results: Treatment with radicicol resulted in upregulation of chitin synthase and cell wall integrity genes in Fs and upregulation of nicotinamide adenine dinucleotide dehydrogenase and sugar transporter genes in Cd. Genes encoding an ATP-binding cassette transporter, an aflatoxin efflux pump, ammonium permease 1 (mep1), and nitrilase were differentially expressed in both Fs and Cd. Among these four genes, only the ABC transporter was upregulated in both Fs and Cd. The aflatoxin efflux pump and mep1 were upregulated in Cd, but downregulated in Fs, whereas nitrilase was downregulated in both Fs and Cd. Conclusion: The transcriptome analyses suggested radicicol resistance pathways, and deletions of the transporter genes indicated that they contribute to radicicol resistance.

• Mycobiology
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• v.45 no.4
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• pp.362-369
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• 2017

We assessed the regulation of cryparin, a class II hydrophobin, using three representative mitogen-activated protein kinase (MAPK) pathways in Cryphonectria parasitica. Mutation of the CpSlt2 gene, an ortholog of yeast SLT2 in the cell wall integrity (CWI) pathway, resulted in a dramatic decrease in cryparin production. Similarly, a mutant of the CpBck1 gene, a MAP kinase kinase kinase gene in the CWI pathway, showed decreased cryparin production. Additionally, mutation of the cpmk1 gene, an ortholog of yeast HOG1, showed decreased cryparin production. However, mutation of the cpmk2 gene, an ortholog of yeast Kss1/Fus3, showed increased cryparin production. The easy-wet phenotype and accumulation of the cryparin transcript in corresponding mutants were consistent with the cryparin production results. In silico analysis of the promoter region of the cryparin gene revealed the presence of binding motifs related to downstream transcription factors of CWI, HOG1, and pheromone responsive pathways including MADS-box- and Ste12-binding domains. Real-time reverse transcriptase PCR analyses indicated that both CpRlm1, an ortholog of yeast RLM1 in the CWI pathway, and cpst12, an ortholog of yeast STE12 in the mating pathway, showed significantly reduced transcription levels in the mutant strains showing lower cryparin production in C. prasitica. However, the transcription of CpMcm1, an ortholog of yeast MCM1, did not correlate with that of the mutant strains showing downregulation of cryparin. These results indicate that three representative MAPK pathways played a role in regulating cryparin production. However, regulation varied depending on the MAPK pathways: the CWI and HOG1 pathways were stimulatory, whereas the pheromone-responsive MAPK was repressive.

• Molecules and Cells
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• v.42 no.4
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• pp.363-375
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• 2019

Fungal sectorization is a complex trait that is still not fully understood. The unique phenotypic changes in sporadic sectorization in mutants of CpBck1, a mitogen-activated protein kinase kinase kinase (MAPKKK) gene, and CpSlt2, a mitogen-activated protein kinase (MAPK) gene, in the cell wall integrity pathway of the chestnut blight fungus Cryphonectria parasitica have been previously studied. Although several environmental and physiological factors cause this sectoring phenotype, genetic variants can also impact this complex morphogenesis. Therefore, RNA sequencing analysis was employed to identify candidate genes associated with sectorization traits and understand the genetic mechanism of this phenotype. Transcriptomic analysis of CpBck1 and CpSlt2 mutants and their sectored progeny strains revealed a number of differentially expressed genes (DEGs) related to various cellular processes. Approximately 70% of DEGs were common between the wild-type and each of CpBck1 and CpSlt2 mutants, indicating that CpBck1 and CpSlt2 are components of the same MAPK pathway, but each component governs specific sets of genes. Functional description of the DEGs between the parental mutants and their sectored progenies revealed several key pathways, including the biosynthesis of secondary metabolites, translation, amino acid metabolism, and carbohydrate metabolism; among these, pathways for secondary metabolism and translation appeared to be the most common pathway. The results of this comparative study provide a better understanding of the genetic regulation of sector formation and suggest that complex several regulatory pathways result in interplays between secondary metabolites and morphogenesis.

• International Journal of Highway Engineering
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• v.3 no.4 s.10
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• pp.127-136
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• 2001

To increase the structural integrity of concrete box culvert good compaction by the dynamic compaction roller with bi9 capacity is as effective as good backfill materials. It is needed for effective compaction that a compaction roller closes to concrete structure with high frequency. However structural distress of the culvert could be occur due to the excessive earth pressure by great dynamic compaction load. To investigate the characteristics of Induced stress by compaction, a box culvert was constructed with changing cushion materials and compaction methods. Two types of cushion material such as tire rubber chip and EPS(Expanded Polystyrene) were used as cushion panels and they are set on the culverts before backfill construction. Laboratory test result of cushion material says that the value of dynamic elastic modulus of rubber is lesser than that of EPS. On the other hand, material damping of rubber material is greater than that of EPS. In most case, dynamic compaction rollers with 10.5 ton weights were used and vibration frequency was applied 30Hz for the great compaction energy. This paper presents the main results on the characteristics of dynamic earth pressures during compaction. The amounts of induced dynamic pressures$(\Delta\sigma\;h)$ by compaction are affected with construction condition such as compaction frequency, depth of pressure cell, distance between roller and the wall of culvert and roller direction. Based on the measured values dynamic lateral pressure on the culverts, it could be said that orthogonal direction of roller to the length of culvert is more effective to compaction efficiency than parallel direction.