• Clinical and Experimental Pediatrics
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• v.59 no.6
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• pp.262-270
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• 2016

Purpose: Pulmonary arterial hypertension (PAH) leads to right ventricular failure (RVF) as well as an increase in pulmonary vascular resistance. Our purpose was to study the effect of sildenafil on right ventricular remodeling in a rat model of monocrotaline (MCT)-induced RVF. Methods: The rats were distributed randomly into 3 groups. The control (C) group, the monocrotaline (M) group (MCT 60 mg/kg) and the sildenafil (S) group (MCT 60 mg/kg+ sildenafil 30 mg/kg/day for 28 days). Masson Trichrome staining was used for heart tissues. Western blot analysis and immunohistochemical staining were performed. Results: The mean right ventricular pressure (RVP) was significantly lower in the S group at weeks 1, 2, and 4. The number of intra-acinar arteries and the medial wall thickness of the pulmonary arterioles significantly lessened in the S group at week 4. The collagen content also decreased in heart tissues in the S group at week 4. Protein expression levels of B-cell lymphoma-2 (Bcl-2)-associated X, caspase-3, Bcl-2, interleukin (IL)-6, matrix metalloproteinase (MMP)-2, endothelial nitric oxide synthase (eNOS), endothelin (ET)-1 and ET receptor A (ERA) in lung tissues greatly decreased in the S group at week 4 according to immunohistochemical staining. According to Western blotting, protein expression levels of troponin I, brain natriuretic peptide, caspase-3, Bcl-2, tumor necrosis factor-${\alpha}$, IL-6, MMP-2, eNOS, ET-1, and ERA in heart tissues greatly diminished in the S group at week 4. Conclusion: Sildenafil alleviated right ventricular hypertrophy and mean RVP. These data suggest that sildenafil improves right ventricular function.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.37 no.3
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• pp.161-168
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• 2011

Purpose: The odontogenic keratocysts demonstrated a high recurrence rate and a biologically aggressive nature. This might be due to unknown factors inherent in the epithelium or enzymatic activity in the fibrous wall. Bcl-2 protein is characterized by its ability to inhibit apoptosis. The aim of this study was to evaluate the expression and distribution of bcl-2 in the OKCs, its possible relationship with the tumorous characteristics, such as the aggressive nature and high recurrence rate, and its usefulness to differentiate OKCs from dentigerous cysts. Materials and Methods: Formalin fixed paraffin-embedded tissue sections of 53 OKCs, and 44 dentigerous cyst were immunohistochemically analyzed quantitatively for the immunoreactivity of the bcl-2 protein with i-solution. Results: More Bcl-2 expression was observed in the OKCs (mean34.387%) than dentigerous cyst (mean11.144%) with statistical significance (P<0.001). Seventeen and 15 of the 32 OKCs in this study showed positivity in the basal layer and basal/suprabasal layers, respectively. In dentigerous cyst, 2 of 3 showed positivity in the basal cell layer. Conclusion: Considering that bcl-2 over expression may lead to the increased survival of epithelial cells, this study demonstrated a possible relationship between the aggressive nature of OKC and the intrinsic growth potential of its lining epithelium. Furthermore, the basal/suprabasal distribution of bcl-2 positive cells was observed in some OKCs, which might have a significant impact on the behavior of cysts. The bcl-2 expression of OKCs can be useful for differentiating OKCs from dentigerous cysts.

• Proceedings of the Botanical Society of Korea Conference
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• 1996.07a
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• pp.48-60
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• 1996

We previously identified and characterized a predominantly pollen-expressed gene of Petunia inflata that encodes a receptor-like kinase named PRK1. The extracellular domain of PRK1 contains leucine-rich repeats which have been implicated in protein-protein interactions, and the cytoplasmic domain was found to autophosphorylate on serine and tyrosine. To investigate the function PRK1 in pollen development, we transformed P. inflata plants with a construct containing the promoter of a predominantly pollen-expressed gene of tomato, LAT52, fused to an antisense PRK1 cDNA corresponding to part of the extracellular domain of PRK1, There transgenic plants were found to each produce approximately equal amounts of normal and aborted pollen. Analysis of the inheritance of the transgene inserts in two of the transgenic plants, ASRK-13 and ASRK-20, to their progeny revealed that certain transgene inserts cosegregated with the pollen abortion phenotype. Microscopic examination of the aborted pollen grains showed that their outer wall, the exine, was essentially normal, but that their cytoplasm contained only starch-like granules. Staining of the nuclei of the microspores at different stages of uninucleate stage. However, at subsequent stages half of the microspores completed mitosis and developed into normal binucleate pollen, but the other half initially remained uninucleate, then lost their nucleio. Analysis of the amounts of PRK1 mRNA and the antisense PRK1 transcript suggested that the pollen abortion phenotype most likely resulted from down-regulation of the PRK1 gene by the antisense PRK1 transgene. These results suggest that PRK1 plays an essential role in a signal transduction pathway that mediates post-meiotic development of microspores.

• Animal cells and systems
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• v.15 no.1
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• pp.29-36
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• 2011

Insect lysozymes are basic, cationic proteins synthesized in fat body and hemocytes in response to bacterial infections and depolymerize the bacterial cell wall. The c-type lysozyme of the insect Spodoptera litura (SLLyz) is a single polypeptide chain of 121 residues with four disulfide bridges and 17 rare codons and is approximately 15 kDa. The full-length SLLyz cDNA is 1039 bp long with a poly(A) tail, and contains an open reading frame of 426 bp long (including the termination codon), flanked by a 54 bp long 5' UTR and a 559 bp long 3' UTR. As a host for the production of high-level recombinant proteins, E. coli is used most commonly because of its low cost and short generation time. However, the soluble expression of heterologous proteins in E. coli is not trivial, especially for disulfide-bonded proteins. In order to prevent inclusion body formation, GST was selected as a fusion partner to enhance the solubility of recombinant protein, and fused to the amplified products encoding mature SLLyz. The expression vector pGEX-4T-1/rSLLyz was then transformed into E. coli BL21(DE3)pLysS for soluble expression of rSLLyz, and the soluble fusion protein was purified successfully. Inhibition zone assay demonstrated that rSLLyz showed antibacterial activity against B. megaterium. These results demonstrate that the GST fusion expression system in E. coli described in this study is efficient and inexpensive in producing a disulfide-bonded rSLLyz in soluble, active form, and suggest that the insect lysozyme is an interesting system for future structural and functional studies.

• 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.

• The Plant Pathology Journal
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• v.21 no.4
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• pp.301-309
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• 2005

Trichoderma species/isolates exhibited varied degree of agglutination on sclerotial (Sc) and hyphal (Hy) surface of Macrophomina phaseolina. The agglutination efficiencies on Sc and Hy ranged from $11\;to\;57\%$. Isolates of T. harzianum (Th) and T. viride (Tv) showed greater agglutination on Sc ($23-57\%$) and Hy ($16-47\%$). Different enzymes (trypsin, pepsin, proteinase k, a-chymotrypsin, lyticase and glucosidase) and inhibitors (tunicamycin, cycloheximide, brefeldin A, sodium azide, dithiothreitol and SDS) reduced the agglutination potential of conidia of Th-23/98 and Tv-25/98; however, the extent of response varied greatly in different treatments. Different fractions of Th-23/98 and Tv-25/98 exhibited haemagglutinating reaction with human blood group A, B, AB and O. Haemagglutinating activity was inhibited by different sugars and glycoproteins tested. Crude haemagglutinating protein from outer cell wall protein fraction of Th-23/98 and Tv-25/98 were eluted on Sephadex G-100 column. Initially Th-23/98 and Tv-25/98 exhibited two peaks showing no agglutination activity; however, lectin activity was detected in the third peak. Similar to crude lectin, the purified lectin also exhibited haemagglutinating activity with different erythrocyte source. SDS-PAGE analysis of partially purified lectin revealed single band with an estimated molecular mass of 55 and 52 kDa in Th-23/98 and Tv-25/98, respectively. Trypsin, chymotrypsin and b-1,3-glucanase totally inhibited lectin activity. Similarly, various pH also affected the haemagglutinating activity of Th-23/98 and Tv-25/98. From the present observations, it can be concluded that the recognition/attachment of mycoparasite (T. harzianum and T. viride) to the host surface (M. phaseolina) may be most likely due to lectin-carbohydrate interaction.

• 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.

• Journal of Microbiology and Biotechnology
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• v.31 no.5
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• pp.756-763
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• 2021

Agarose is a linear polysaccharide composed of ᴅ-galactose and 3,6-anhydro-ʟ-galactose (AHG). It is a major component of the red algal cell wall and is gaining attention as an abundant marine biomass. However, the inability to ferment AHG is considered an obstacle in the large-scale use of agarose and could be addressed by understanding AHG catabolism in agarolytic microorganisms. Since AHG catabolism was uniquely confirmed in Vibrio sp. EJY3, a gram-negative marine bacterial species, we investigated AHG metabolism in Streptomyces coelicolor A3(2), an agarolytic gram-positive soil bacterium. Based on genomic data, the SCO3486 protein (492 amino acids) and the SCO3480 protein (361 amino acids) of S. coelicolor A3(2) showed identity with H2IFE7.1 (40% identity) encoding AHG dehydrogenase and H2IFX0.1 (42% identity) encoding 3,6-anhydro-ʟ-galactonate cycloisomerase, respectively, which are involved in the initial catabolism of AHG in Vibrio sp. EJY3. Thin layer chromatography and mass spectrometry of the bioconversion products catalyzed by recombinant SCO3486 and SCO3480 proteins, revealed that SCO3486 is an AHG dehydrogenase that oxidizes AHG to 3,6-anhydro-ʟ-galactonate, and SCO3480 is a 3,6-anhydro-ʟ-galactonate cycloisomerase that converts 3,6-anhydro-ʟ-galactonate to 2-keto-3-deoxygalactonate. SCO3486 showed maximum activity at pH 6.0 at 50℃, increased activity in the presence of iron ions, and activity against various aldehyde substrates, which is quite distinct from AHG-specific H2IFE7.1 in Vibrio sp. EJY3. Therefore, the catabolic pathway of AHG seems to be similar in most agar-degrading microorganisms, but the enzymes involved appear to be very diverse.

• The Plant Pathology Journal
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• v.37 no.2
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• pp.173-181
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• 2021

The genus Streptomyces demonstrates enormous promise in promoting plant growth and protecting plants against various pathogens. Single and consortium treatments of two selected Streptomyces strains (Streptomyces shenzhenensis TKSC3 and Streptomyces sp. SS8) were evaluated for their growth-promoting potential on rice, and biocontrol efficiency through induced systemic resistance (ISR) mediation against Xanthomonas oryzae pv. oryzicola (Xoc), the causal agent of rice bacterial leaf streak (BLS) disease. Seed bacterization by Streptomyces strains improved seed germination and vigor, relative to the untreated seed. Under greenhouse conditions, seed bacterization with consortium treatment TKSC3 + SS8 increased seed germination, root length, and dry weight by 20%, 23%, and 33%, respectively. Single and consortium Streptomyces treatments also successfully suppressed Xoc infection. The result was consistent with defense-related enzyme quantification wherein single and consortium Streptomyces treatments increased peroxidase (POX), polyphenol oxidase, phenylalanine ammonia-lyase, and β,1-3 glucanase (GLU) accumulation compared to untreated plant. Within all Streptomyces treatments, consortium treatment TKSC3 + SS8 showed the highest disease suppression efficiency (81.02%) and the lowest area under the disease progress curve value (95.79), making it the best to control BLS disease. Consortium treatment TKSC3 + SS8 induced the highest POX and GLU enzyme activities at 114.32 µmol/min/mg protein and 260.32 abs/min/mg protein, respectively, with both enzymes responsible for plant cell wall reinforcement and resistant interaction. Our results revealed that in addition to promoting plant growth, these Streptomyces strains also mediated ISR in rice plants, thereby, ensuring protection from BLS disease.

• Applied Biological Chemistry
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• v.50 no.2
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• pp.95-100
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• 2007

The cellulase gene of Bacillus licheniformis K11 which has plant growth-promoting activity by auxin and antagonistic ability by siderophore was cloned in pUC18 using PCR employing heterologous primers. The 1.6kb PCR fragment contained the full sequence of the cellulase gene, denoted celW which has been reported to encode a 499 amino acid protein. Similarity search in protein data base revealed that the cellulase from B. licheniformis K11 was more than 97% identical in amino acid sequence to those of various Bacillus spp. The cellulase protein from B. licheniformis K11, overproduced in E. coli DH5${\alpha}$ by the lac promoter on the vector, had apparent molecular weight of 55 kDa upon CMC-SDS-PAGE analysis. The protein not only had enzymatic activity toward carboxymethyl-cellulose (CMC), but also was able to degrade insoluble cellulose, such as Avicel and filter paper (Whatman$^{\circledR}$ No. 1). In addition, the cellulase could degrade a fungal cell wall of Phytophthora capsici. Consequently B. licheniformis K11 was able to suppress the peperblight causing P. capsici by its cellulase. Biochemical analysis showed that the enzyme had a maximum activity at 60$^{\circ}C$ and pH 6.0. Also, the enzyme activity was activated by Co$^{2+}$ of Mn$^{2+}$ but inhibited by Fe$^{3+}$ or Hg$^{2+}$. Moreover, enzyme activity was not inhibited by SDS or sodium azide.