• Journal of Microbiology and Biotechnology
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• v.1 no.1
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• pp.22-30
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• 1991

In order to secrete the Bacillus subtilis cytidine deaminase (CDase, cytidine/2'-deoxycytidine deaminase) encoded by the B. subtilis cdd gene in E. coli by the aid of signal sequences, the cdd gene was fused in-frame to either amyE or penP signal sequences and the gene expression and CDase localization were examined. For the penP signal sequence::cdd fusion, the cdd gene with 9 amino acids truncated from the 5'-terminus was fused in-frame to the signal sequence, then the $cdd^{+}$ colonies were not occurred from the minimal plate by cdd complementation. The result suggests that 9 amino acids on the $NH_2-terminal$ of CDase have an essential function in the enzyme activity. The hybrid protein obtained by fused gene amyE signal sequence::cdd structural gene gave $cdd^{+}$ phenotype and about half of the total CDase activity was found to be secreted in the periplasm of E. coli transformant JF611/pSO202. The periplasmic CDase activity of JF611 harboring pSO52 containing the intact cdd gene was considerablely lower than that of the cells harboring pSO202 carrying the hybrid cdd gene. This suggests that the CDase was secreted to the periplasm through the cytoplasmic membrane by the aid of the amyE signal sequence in the E. coli transformant.

• 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.

• 한국생물공학회:학술대회논문집
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• 2005.10a
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• pp.608-609
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• 2005

• IMMUNE NETWORK
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• v.2 no.2
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• pp.102-108
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• 2002

Background: This study was aimed to differentiate two forms of CTLA-4 (CD152) in activated peripheral blood lymphocyte and clarify the mechanism how cytoplasmic form of this molecule is targeted to cell surface. Methods: For this purpose we generated 2 different anti-human CD152 peptide antibodies and 5 different N'-terminal deletion mutant CTLA4Ig fusion proteins and carried out a series of Western blot and ELISA analyses. Antipeptide antibodies made in this study were anti-CTLA4pB and anti-CTLA4pN. The former recognized a region on extracellular single V-like domain and the latter recognized N'-terminal sequence of leader domain of human CD152. Results: In Western blot, the former antibody recognized recombinant human CTLA4Ig fusion protein as an antigen. And this recognition was completely blocked by preincubating antipeptide antibody with the peptide used for the antibody generation at the peptide concentration of 200 ug/ml. These antibodies were recognized human CD152 as a cytoplasmic sequestered- and a membrane bound- forms in phytohemagglutinin (PHA)-stimulated peripheral blood lymphocyte (PBL). These two forms of CD152 were further differentiated by using anti-CTLA4pN and anti-CTLA4pB antibodies such that former recognized cytosolic form only while latter recognized both cytoplasmic- and membraneforms of this molecule. Furthermore, in a transfection expression study of 5 different N'-terminal deletion mutant CTLA4Ig, mutated proteins were secreted out from transfected cell surface only when more than 6 amino acids from N'-terminal were deleted. Conclusion: Our results implies that cytosolic form of CTLA-4 has leader sequence while membrane form of this molecule does not. And also suggested is that at least N'-terminal 6 amino acid residues of human CTLA-4 are required for regulation of targeting this molecule from cytosolic- to membrane- area of activated human peripheral blood T lymphocyte.

• Journal of Korean Dental Science
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• v.8 no.2
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• pp.74-81
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• 2015

Purpose: In osteoclast differentiation, actin-rich membrane protrusions play a crucial role in cell adhesion. Odontogenic ameloblast-associated protein (Odam) contributes to cell adhesion by inducing actin rearrangement. Odam-mediated RhoA activity may play a significant role in multinucleation of osteoclasts. However, the precise function of Odam in osteoclast cell adhesion and differentiation remains largely unknown. Here, we identify a critical role for Odam in inducing osteoclast adhesion and differentiation. Materials and Methods: The expression of Odam in osteoclasts was evaluated by immunohistochemistry. Primary mouse bone marrow and RAW264.7 cells were used to test the cell adhesion and actin ring formation induced by Odam. Result: Odam was expressed in osteoclasts around alveolar bone. Odam transfection induced actin filament rearrangement and cell adhesion compared with the control or collagen groups. Overexpression of Odam promoted actin stress fiber remodeling and cell adhesion, resulting in increased osteoclast fusion. Conclusion: These results suggest that Odam expression in primary mouse osteoclasts and RAW264.7 cells promotes their adhesion, resulting in the induction of osteoclast differentiation.

• Biodesign
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• v.5 no.4
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• pp.136-140
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• 2017

Lam6 is a member of sterol-specific ${\underline{l}ipid$ transfer proteins ${\underline{a}}nchored$ at ${\underline{m}ebrane$ contact sites (LAMs). Lam6 localizes to the ER-mitochondria contact sites by its PH-like domain and the C-terminal transmembrane helix. Here, we purified and crystallized the Lam6 PH-like domain from Saccharomyces cerevisiae. To aid crystallization of the Lam6 PH-like domain, T4 lysozyme was fused to the N-terminus of the Lam6 PH-like domain with a short dipeptide linker, GlySer. The fusion protein was crystallized under the condition of 0.1 M HEPES-HCl pH 7.0, 10% (w/v) PEG 8000, and 0.1 M $Na_3$ Citrate at 293K. X-ray diffraction data of the crystals were collected to $2.4{\AA}$ resolution using synchrotron radiation. The crystals belong to the orthorhombic space group $P2_12_12_1$ with unit cell parameters $a=59.5{\AA}$, $b=60.1{\AA}$, and $c=105.6{\AA}$. The asymmetric unit contains one T4L-Lam6 molecule with a solvent content of 58.7%. The initial attempt to solve the structure by molecular replacement using the T4 lysozyme structure was successful.

• Development and Reproduction
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• v.7 no.2
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• pp.81-88
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• 2003

Initiation and activation of sperm motility are prerequisite processes for the contact and fusion of male and female gametes at fertilization. The phenomena are under the regulation of CAMP and $Ca^{2+}$ in vertebrates and invertebrates. Mammalian sperm requires $Ca^{2+}$and cyclic AMP for the activation of sperm motility. Cell signaling for the initiation and activation of sperm motility has been well studied in the ascidians, Ciona intestinalis and C. savignyi and salmonid fishes. In Ciona, whose cell signaling for activation of sperm motility has been established, the sperm-activating and -attracting factor released from unfertilized egg requires extracellular $Ca^{2+}$ for activating sperm motility and eliciting chemotactic behavior of the activated sperm toward the egg. On the other hand, the cyclic AMP-dependent phosphorylation of protein is essential for the initiation of sperm motility in salmonid fishes. A decrease in the environmental Ti concentration surrounding the spawned sperm causes a li efflux and $Ca^{2+}$ influx through the specific $K^{+}$ channel and dihydropyridine-sensitive L-/T- type $Ca^{2+}$ channel, respectively, thereby leading to the membrane hyperpolarization and $Ca^{2+}$ influx. The membrane hyperpolarization synthesizes cyclic AMP, which triggers the luther Process of cell signaling, i.e., cyclic AMP-dependent protein phosphorylation, to initiate sperm motility in salmond fishes.almond fishes.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.81.2-82
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• 2003

Plants have evolved along with pathogens, and they have developed sophisticated defense systems against specific microorganisms to survive. G-protons are considered one of the upstream signaling components working as a key for the defense signal transduction pathway. For activation and inactivation of G-protein, GTP-biding proteins are involved. GTP -binding proteins are found in all organisms. Small GTP-binding proteins, having masses of 21 to 30kD, belong to a superfamily, often named the Ras supefamily because the founding members are encoded by human Ras genes initially discovered as cellular homologs of the viral ras oncogene. Members of this supefamily share several common structural features, including several guanine nucleotide binding domains and an effector binding domain. However, exhibiting a remarkable diversity in both structure and function. They are important molecular switches that cycle between the GDP-bound inactive form into the GTP-bound active form through GDP/GTP replacement. In addition, most GTP-binding proteins cycle between membrane-bound and cytosolic forms. such as the RAC family are cytosolic signal transduction proteins that often are involved in processing of extracellular stimuli. Plant RAC proteins are implicated in regulation of plant cell architecture secondary wall formation, meristem signaling, and defense against pathogens. But their molecular mechanisms and functions are not well known. We isolated a RacB homolog from rice to study its role of defense against pathogens. We introduced the constitutively active and the dominant negative forms of the GTP-hinging protein OsRacB into the wild type rice. The dominant negative foms are using two forms (full-sequence and specific RNA interference with RacB). Employing southern, and protein analysis, we examine to different things between the wild type and the transformed plant. And analyzing biolistic bombardment of onion epidermal cell with GFP-RacB fusion protein revealed association with the nucle.

• Korean Journal of Microbiology
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• v.38 no.4
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• pp.218-218
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• 2002

Using two drugs, tunicamycin and brefeldin A, which affect protein processing, we investigated the intracellular processing mechanism of secreted aspartyl proteinase 1 (SAPl) of Candide albicans. Three intracellular forms of SAPI were detected by immunoblotting using menoclonal antibody (MAb) CAPl. Their molecular weights were approximately 40, 41 and 45 kDa, respectively. The 41 kDa protein is a glycoprotein and may be the same as the extracellular form judging by its molecular mass. The 40 kDa protein was the unglycosylated form and its molecular mass coincided with deglycosylated SAPl and the 45 kDa protein was also the unglycosylated form. Neither the 40 and 45 kDa proteins were detected in the culture supernatant of C. albicans. These suggested that the 40 and 45 kDa proteins might be intracellular precursor forms of SAPI. These results show that SAPI is translated as a 45 kDa precusor form in the endoplasmic reticulum and the 45 kDa precursor farm undergoes proteolytic cleavage after translocation into the Golgi apparatus, generating the 40 kDa precursor form. This 40 kDa precursor is converted into a 41 kDa mature form through glycosylation in the Golgi apparatus. The mature form of the 41 kDa protein is sorted into secretary vesicles and finally released into the extracellular space through membrane fusion. When the glycan region of SAPl was digested with N-glycosidase F, both stability and activity of the enzyme decreased. These results indicate that the glycan attached to the enzyme may, at least in parti be related to enzyme stability and activity.

• Journal of Microbiology
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• v.38 no.4
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• pp.218-223
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• 2000

Using two drugs, tunicamycin and brefeldin A, which affect protein processing, we investigated the intracellular processing mechanism of secreted aspartyl proteinase 1 (SAPl) of Candide albicans. Three intracellular forms of SAPI were detected by immunoblotting using menoclonal antibody (MAb) CAPl. Their molecular weights were approximately 40, 41 and 45 kDa, respectively. The 41 kDa protein is a glycoprotein and may be the same as the extracellular form judging by its molecular mass. The 40 kDa protein was the unglycosylated form and its molecular mass coincided with deglycosylated SAPl and the 45 kDa protein was also the unglycosylated form. Neither the 40 and 45 kDa proteins were detected in the culture supernatant of C. albicans. These suggested that the 40 and 45 kDa proteins might be intracellular precursor forms of SAPI. These results show that SAPI is translated as a 45 kDa precusor form in the endoplasmic reticulum and the 45 kDa precursor farm undergoes proteolytic cleavage after translocation into the Golgi apparatus, generating the 40 kDa precursor form. This 40 kDa precursor is converted into a 41 kDa mature form through glycosylation in the Golgi apparatus. The mature form of the 41 kDa protein is sorted into secretary vesicles and finally released into the extracellular space through membrane fusion. When the glycan region of SAPl was digested with N-glycosidase F, both stability and activity of the enzyme decreased. These results indicate that the glycan attached to the enzyme may, at least in parti be related to enzyme stability and activity.