• Journal of Microbiology and Biotechnology
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• v.30 no.8
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• pp.1142-1148
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• 2020

Mitochondria play a vital role in iron uptake and metabolism in pathogenic fungi, and also influence virulence and drug tolerance. However, the regulation of iron transport within the mitochondria of Cryptococcus neoformans, a causative agent of fungal meningoencephalitis in immunocompromised individuals, remains largely uncharacterized. In this study, we identified and functionally characterized Mrs3/4, a homolog of the Saccharomyces cerevisiae mitochondrial iron transporter, in C. neoformans var. grubii. A strain expressing an Mrs3/4-GFP fusion protein was generated, and the mitochondrial localization of the fusion protein was confirmed. Moreover, a mutant lacking the MRS3/4 gene was constructed; this mutant displayed significantly reduced mitochondrial iron and cellular heme accumulation. In addition, impaired mitochondrial iron-sulfur cluster metabolism and altered expression of genes required for iron uptake at the plasma membrane were observed in the mrs3/4 mutant, suggesting that Mrs3/4 is involved in iron import and metabolism in the mitochondria of C. neoformans. Using a murine model of cryptococcosis, we demonstrated that an mrs3/4 mutant is defective in survival and virulence. Taken together, our study suggests that Mrs3/4 is responsible for iron import in mitochondria and reveals a link between mitochondrial iron metabolism and the virulence of C. neoformans.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.16
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• pp.6899-6904
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• 2014

NGAL (neutrophil gelatinase-associated lipocalin) is a novel cancer-related protein involves multiple functions in many cancers and other diseases. We previously overexpressed NGAL to analyze its role in esophageal squamous cell carcinoma (ESCC). In this study, a protein-protein interaction (PPI) was constructed and the shortest paths from NGAL to transcription factors in the network were analyzed. We found 28 shortest paths from NGAL to RELA, most of them obeying the principle of extracellular to cytoplasm, then nucleus. These shortest paths were also prioritized according to their normalized intensity from the microarray by the order of interaction cascades. A systems approach was developed in this study by linking differentially expressed genes with publicly available PPI data, Gene Ontology and subcellular localizaton for the integrated analyses. These shortest paths from NGAL to DEG transcription factors or other transcription factors in the PPI network provide important clues for future experimental identification of new pathways.

• Microbiology and Biotechnology Letters
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• v.20 no.5
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• pp.511-518
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• 1992

We subcloned a 58 KD chitinase gene of Serratia marcescens into Escherichia coli and investigated the expression and secretion of the chitinase. Chitinase was produced in E. coli by using its own promoter but the levels of enzyme were very low, less than 5 mU/m$\ell$. However, by the combined action of the chitinase and lac promoters, the chitinase activity increased up to about 80 mU/m$\ell$. The most of the chitinase produced in E. coli was localized in periplasm and the small amounts were observed in cytosol and culture medium. Intracellular chitinase activities increased in proportion to the growth of E. coli up to the early stationary phase but rapidly decreased thereafter, which was assumed to be degradation of the chitinase by E. coli proteolytic enzymes.

• The korean journal of orthodontics
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• v.44 no.6
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• pp.320-329
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• 2014

Objective: To investigate the involvement of ephrinB2 in periodontal tissue remodeling in compression areas during orthodontic tooth movement and the effects of compressive force on EphB4 and ephrinB2 expression in osteoblasts and osteoclasts. Methods: A rat model of experimental tooth movement was established to examine the histological changes and the localization of ephrinB2 in compressed periodontal tissues during experimental tooth movement. RAW264.7 cells and ST2 cells, used as precursor cells of osteoclasts and osteoblasts, respectively, were subjected to compressive force in vitro. The gene expression of EphB4 and ephrinB2, as well as bone-associated factors including Runx2, Sp7, NFATc1, and calcitonin receptor, were examined by quantitative real-time polymerase chain reaction (PCR). Results: Histological examination of the compression areas of alveolar bone from experimental rats showed that osteoclastogenic activities were promoted while osteogenic activities were inhibited. Immunohistochemistry revealed that ephrinB2 was strongly expressed in osteoclasts in these areas. Quantitative real-time PCR showed that mRNA levels of NFATc1, calcitonin receptor, and ephrinB2 were increased significantly in compressed RAW264.7 cells, and the expression of ephrinB2, EphB4, Sp7, and Runx2 was decreased significantly in compressed ST2 cells. Conclusions: Our results indicate that compressive force can regulate EphB4 and ephrinB2 expression in osteoblasts and osteoclasts, which might contribute to alveolar bone resorption in compression areas during orthodontic tooth movement.

• The Journal of Korean Society of Virology
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• v.28 no.1
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• pp.53-62
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• 1998

Human papillomavirus (HPV) 16, E7 proteins derived from the prototype (Bac73) and natural variant (Bac101) E7 open reading frame were produced in Sf9 insect cells. The variant E7 gene occurred naturally by substitution mutation at the position of 88 nucleotide, resulting serine instead of asparagine. Using E7 specific monoclonal antibody (VD6), both E7 proteins were identified in recombinant baculovirus infected SF9 cells. Radiolabelling and immunoprecipitation analysis revealed that both E7 proteins were phosphoproteins. Immunostaining result showed that E7 proteins were mainly localized in the cytoplasm. Nuclear form of E7 proteins was also detected after a sequential fractionation procedure for removing chromatin structure. Considering that the VD6 recognition site in E7 protein is located within 10 amino acid at the N-terminus, this region appears to be blocked by the nuclear component. Western blot analysis revealed that nuclear form was more abundant than cytoplasmic E7 proteins. Time course immunostaining showed that the primary location of E7 protein was the nucleus and exported to the cytoplasm as proteins were accumulated. These events occurred similarly in both Bac73 and Bac101 infected Sf9 cells, suggesting that these two proteins may have similar biological functions.

• Parasites, Hosts and Diseases
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• v.44 no.3
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• pp.187-196
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• 2006

The mammalian trematode Paragonimus westermani is a typical digenetic parasite, which can cause paragonimiasis in humans. Host tissues and blood cells are important sources of nutrients for development, growth and reproduction of P. westermani. In this study, a cDNA clone encoding a 47 kDa hemoglobinase of P. westermani was characterized by sequencing analysis, and its localization was investigated immunohistochemically. The phylogenetic tree prepared based on the hemoglobinase gene showed high homology with hemoglobinases of Fasciola hepatica and Schistosoma spp. Moreover, recombinant P. westermani hemoglobinase degradaded human hemoglobin at acidic pH (from 3.0 to 5.5) and its activity was almost completely inhibited by E-64, a cysteine proteinase inhibitor. Immunohistochemical studies showed that P. westermani hemoglobinase was localized in the epithelium of the adult worm intestine implying that the protein has a specific function. These observations suggest that hemoglobinase may act as a digestive enzyme for acquisition of nutrients from host hemoglobin. Further investigations may provide insights into hemoglobin catabolism in P. westermani.

• Journal of Plant Biotechnology
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• v.36 no.1
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• pp.68-74
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• 2009

We have previously demonstrated that overexpression and characterization of Brassica rapa type-l metallothionein gene (BrMT1) in Arabidopsis which showed enhanced resistance to cadmium and ROS. Here, we present the consistent study of our previous report about BrMTs. BrMT3 expressing DTY167 cells showed resistance to Zn and Pb as well as Cd. Thus, we have developed the BrMT3 overexpression Arabidopsis to enhance capacity for metal stresses. Successful expression and localization were achieved using the rubisco transit peptides of RbcS-BrMT3-GFP protein, which was confirmed by western blot analysis with the GFP antibody and green fluorescence signal from the chloroplast. BrMT3 overexpression Arabidopsis plants exhibited a higher resistance to cadmium compared to control plants. This result indicates that BrMT3 would be applicable to the development of plants with enhanced resistance against heavy metal stresses.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.4
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• pp.1531-1537
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• 2014

Runt-related transcription factor 3 (RUNX3) is a tumor suppressor gene whose reduced expression may play an important role in the development and progression of esophageal squamous cell cancer (ESCC). The aim of this study was to investigate the clinical relevance of RUNX3 in ESCC patients and effects of overexpression on biological behaviour of Eca109 cells in vitro and in vivo. Immunohistochemistry was performed to detect the clinical relevance of RUNX3 and lymph node metastasis in 80 ESCC tissues and 40 non-cancerous tissues using the SP method. RT-PCR and Western blotting were applied to assess the RUNX3 level and verify the Eca109 cell line with stable overexpression. Localization of RUNX3 proteins was performed by cell immunofluorescence. CCK-8 and Scrape motility assays were used to determine proliferation and migration and the TUNEL assay to analyze cell apoptosis. Invasive potential was assessed in cell transwell invasion experiments. In nude mice, tumorigenesis in vivo was determined. Results showed decreased expression of RUNX3 in esophageal tissue to be significantly related to lymph node metastasis (LNM) (P<0.01). In addition, construction of a recombinant lentiviral vector and transfection into the human ESCC cell line Eca109 demonstrated that overexpression could inhibit cell proliferation, migration and invasion, and induce apoptosis. The in vivo experiments in mice showed tumorigenicity and invasiveness to be significantly reduced. Taken together, our studies indicate that underexpression of RUNX3 in human ESCC tissue is significantly correlated with progression. Restoration of RUNX3 expression significantly inhibits ESCC cells proliferation, migration, invasion and tumorigenesis.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.2
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• pp.272-278
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• 2010

In this study, we cloned, sequenced and characterized porcine y+L Amino Acid Transporter-1 (y+LAT1). By screening a translated EST database with the protein sequence of the human $y^{+}$LAT1 and by using rapid amplification of cDNA ends (RACE), the full-length cDNA encoding porcine $y^{+}$LAT1 was isolated from porcine intestine RNA. It was 2,111 bp long, encoding a 511 amino acid trans-membrane glycoprotein composed of 12 transmembrane domains. The predicted amino acid sequence was found to be 91%, 90%, 87% and 87% identical to those of cattle, human, mouse and rat $y^{+}$LAT1 respectively. Real-time RT-PCR results indicated that the small intestine had the highest $y^{+}$LAT1 mRNA abundance and the lung had the lowest $y^{+}$LAT1 mRNA abundance. Baby hamster kidney (BHK) cells transfected with green fluorescent protein (GFP) tagged porcine $y^{+}$LAT1 cDNA indicated that the cellular localization of the gene product in BHK was on the plasma membrane.

• Biomedical Science Letters
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• v.7 no.4
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• pp.167-172
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• 2001

Nebulin is an approximately 700 kDa filamentous protein in vertebrate skeletal muscle. It binds to the Z line and also binds side-by-side to the entire thin actin filament in a sarcomere. The correlation of nebulin size with thin filament length have led to the suggestion that nebulin acts as a molecular ruler for the length of thin filaments. The C-terminal part of human nebulin is anchored in the sarcomeric Z-disk and contains an SH3 domain. SH3 domains have been identified in an ever-increasing number of proteins important for a wide range of cellular processes, from signal transduction to cytoskeleton assembly and membrane localization. However, the exact physiological role of SH3 domains remains, in many cases, unclear. To explore the role of nebulin SH3 in the cytoskeletal rearrangement that accompanies myoblast differentiation, we transfected sense and antisense nebulin SH3 domain fused to enhanced green fluorescent protein in myoblast. Cells expressing nebulin SH3 fragment showed decrease of cell-cell adhesion, and cells transfected with antisense nebulin SH3 gene showed a rounded cell morphology and loss of cell-matrix adhesion. No alteration in cell shape and differentiation were observed in control cells expressing enhanced green fluorescent protein. Perturbation of nebulin altered the cell shape and disrupted cell adhesion in myoblast, demonstrating that nebulin can affect cytoskeleton rearrangement.