• International Journal of Industrial Entomology and Biomaterials
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• v.17 no.2
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• pp.223-228
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• 2008

A novel beetle antimicrobial protein from stimulated Copris tripartitus and the corresponding gene were isolated in parallel through differential display-PCR and expression in Escherichia coli. To find cDNA clones responsible for bacteria resistance, the suppression subtractive hybridization and GeneFishing differentially expressed genes system were employed in the dung beetle, Copris tripartitus immunized with lipopolysaccaride. One cDNA clone from eight subtracted clones was selected through dot blot analysis and confirmed by northern blot analysis. The 516-bp, selected cDNA clone was determined by 5' and 3' rapid amplication of cDNA ends and cloned into the GST fusion expression vector pGEX-4T-1 for expression of the protein. The expressed protein was predicted 14.7 kDa and inhibited the growth of gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa. These results implied that the expressed protein is related to immune defense mechanism against microorganism.

• Korean Journal of Radiology
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• v.22 no.7
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• pp.1021-1033
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• 2021

Objective: To assess the expression of vascular normalization genes in different molecular subtypes of breast cancer and to determine whether molecular subtypes with a higher vascular normalization gene expression can be identified using dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) and intravoxel incoherent motion (IVIM) diffusion-weighted imaging (DWI). Materials and Methods: This prospective study evaluated 306 female (mean age ± standard deviation, 50 ± 10 years), recruited between January 2014 and August 2017, who had de novo breast cancer larger than 1 cm in diameter (308 tumors). DCE MRI followed by IVIM DWI studies using 11 different b-values (0 to 1200 s/mm²) were performed on a 1.5T MRI system. The Tofts model and segmented biexponential IVIM analysis were used. For each tumor, the molecular subtype (according to six [I-VI] subtypes and PAM50 subtypes), expression profile of genes for vascular normalization, pericytes, and normal vascular signatures were determined using freshly frozen tissue. Statistical associations between imaging parameters and molecular subtypes were examined using logistic regression or linear regression with a significance level of p = 0.05. Results: Breast cancer subtypes III and VI and PAM50 subtypes luminal A and normal-like exhibited a higher expression of genes for vascular normalization, pericyte markers, and normal vessel function signature (p < 0.001 for all) compared to other subtypes. Subtypes III and VI and PAM50 subtypes luminal A and normal-like, versus the remaining subtypes, showed significant associations with K^trans, k_ep, v_p, and IAUGC_BN90 on DEC MRI, with relatively smaller values in the former. The subtype grouping was significantly associated with D, with relatively less restricted diffusion in subtypes III and VI and PAM50 subtypes luminal A and normal-like. Conclusion: DCE MRI and IVIM parameters may identify molecular subtypes of breast cancers with a different vascular normalization gene expression.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.2
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• pp.155-162
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• 2020

Equine follicle stimulating hormone receptor (eFSHR) has a large extracellular domain and an intracellular domain containing approximately 10 phosphorylation sites within the G protein-coupled receptor. This study was conducted to analyze the function of phosphorylation sties at the eFSHR C-terminal region. We constructed a mutant of eFSHR, in which the C-terminal cytoplasmic tail was truncated at residue 641 (eFSHR-t641). This removed 10 potential phosphorylation sites from the C-terminal region of the intracellular loop. The eFSHR-wild type (eFSHR-wt) and eFSHR-t641 cDNAs were subcloned into the pCMV-ARMS1-PK2 expression vector. These plasmids were transfected into PathHunter CHO-K1 Parental cells expressing β-arrestin 2 enzyme acceptor fusion protein and analyzed for agonist-induced cAMP response. The cAMP response in cells expressing eFSHR-t641 was lower than the response in cells expressing eFSHR-wt. EC₅₀ values of eFSHR-wt and eFSHR-t641 were 1079 ng/mL and 1834 ng/mL, respectively. eFSHR-t641 was approximately 0.58-fold compared with that of eFSHR-wt. The maximal response in eFSHR-wt and eFSHR-t641 was 24.7 nM and 16.7 nM, respectively. The Rmax value of phosphorylation sites in eFSHR-t641 was also decreased to approximately 68.4% of that in eFSHR-wt. The collective data implicate that the phosphorylation sites in the eFSHR C-terminal region have a pivotal role in signal transduction in PathHunter CHO-K1 cells, and indicate that β-arrestin is involved in coupling the activated receptors to the internalization system.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.8
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• pp.4031-4036
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• 2012

Background: The negative signaling provided by interactions of the co-inhibitory molecule, programmed death-1 (PD-1), and its ligands, B7-H1 (PD-L1) and B7-DC (PD-L2), is a critical mechanism contributing to tumor evasion; blockade of this pathway has been proven to enhance cytotoxic activity and mediate antitumor therapy. Here we evaluated the anti-tumor efficacy of AAV-mediated delivery of the extracellular domain of murine PD-1 (sPD-1) to a tumor site. Material and Methods: An rAAV vector was constructed in which the expression of sPD-1, a known negative regulator of TCR signals, is driven by human cytomegalovirus immediate early promoter (CMV-P), using a triple plasmid transfection system. Tumor-bearing mice were then treated with the AAV/sPD1 construct and expression of sPD-1 in tumor tissues was determined by semi quantitative RT-PCR, and tumor weights and cytotoxic activity of splenocytes were measured. Results: Analysis of tumor homogenates revealed sPD-1 mRNA to be significantly overexpressed in rAAV/sPD-1 treated mice as compared with control levels. Its use for local gene therapy at the inoculation site of H22 hepatoma cells could inhibit tumor growth, also enhancing lysis of tumor cells by lymphocytes stimulated specifically with an antigen. In addition, PD-1 was also found expressed on the surfaces of activated CD8+ T cells. Conclusion: This study confirmed that expression of the soluble extracellular domain of PD-1 molecule could reduce tumor microenvironment inhibitory effects on T cells and enhance cytotoxicity. This suggests that it might be a potential target for development of therapies to augment T-cell responses in patients with malignancies.

• Korean Journal of Microbiology
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• v.45 no.4
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• pp.419-424
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• 2009

To provide the basis of biosensor based on the lux genes from bioluminescent bacteria of Photobacterium leiognathi and Vibrio harveyi, we test the expression of lux genes in several strains of Escherichia coli. The expression of the recombinant plasmid of PlXba.pT7-3, containing all lux genes requiring for light emission without adding substrate, in E. coli 43R was so strong to see the blue-green light in single colony as well as in the alginate immobilized cell. In addition, the light intensity was decreased by adding heavy metal ion such as cadmium and zinc ions. These result raise the possibility that a biosensor can be developed using the lux genes system.

• Genes and Genomics
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• v.40 no.11
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• pp.1225-1235
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• 2018

Hypoxia seriously affects the innate immune system of fish. However, the roles of suppressor of cytokine signaling (SOCS), pivotal anti-inflammatory genes, in response to hypoxia/reoxygenation remain largely unexplored. The primary objective of this study was to elucidate the function of SOCS genes under acute hypoxia and reoxygenation in pufferfish (Takifugu fasciatus). In the present study, SOCS1, 2 and 3 were identified in T. fasciatus referred to as TfSOCS1, 2 and 3. Then, qRT-PCR and western blot analysis were employed to assess their expressions at both the mRNA and protein levels. Tissue distribution demonstrated that the three SOCS genes were predominantly distributed in gill, brain and liver. Under hypoxia challenge ($1.63{\pm}0.2mg/L$ DO for 2, 4, 6 and 8 h), the expressions of TfSOCS1 and 3 in brain and liver at the mRNA and protein levels were significantly decreased, while their expressions showed an opposite trend in gill. Different from the expressions of TfSOCS1 and 3, the expression of TfSOCS2 was inhibited in gill, along with its increased expression in brain and liver. After normoxic recovery ($7.0{\pm}0.3mg/L$ of DO for 4 and 12 h), most of TfSOCS genes were significantly altered at R4 (reoxygenation for 4 h) and returned to the normal level at R12 (reoxygenation for 12 h). SOCS genes played vital roles in response to hypoxia/reoxygenation challenge. Our findings greatly strengthened the relation between innate immune and hypoxia stress in T. fasciatus.

• Journal of Microbiology and Biotechnology
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• v.25 no.7
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• pp.1007-1014
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• 2015

Plectasin, the first defensin extracted from a fungus (the saprophytic ascomycete Pseudoplectania nigrella), is attractive as a prospective antimicrobial agent. The purpose of this study was to establish a bacterium-based production system and evaluate the antimicrobial activity of the resulting plectasin. A gene encoding plectasin, with the codon preference of Escherichia coli, was optimized based on its amino acid sequence, synthesized using genesplicing with overlap extension PCR, and inserted into the expression vector pGEX-4T-1. The fusion protein was expressed in the soluble fraction of E. coli and purified using glutathione Stransferase affinity chromatography. Plectasin was cleaved from the fusion protein with thrombin and purified by ultrafiltration. The purified plectasin showed strong, concentrationdependent antimicrobial activity against gram-positive bacteria, including antibiotic-resistant bacteria, especially penicillin-resistant Enterococcus faecium. This antimicrobial activity was equal to chemically synthesized plectasin and was maintained over a wide range of pH and temperatures. This soluble recombinant expression system in E. coli is effective for producing plectasin at a relatively lower cost, and higher purity and efficiency than prior systems, and might provide a foundation for developing a large-scale production system. Overall, plectasin shows potential as a novel, high-performance, and safe antibiotic for the treatment of refractory diseases caused by drug-resistant bacterial strains.

• The Journal of Korean Society of Virology
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• v.26 no.1
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• pp.77-90
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• 1996

Nucleocapsid protein (NP)which exists in the particle of hantavirus and surrounds the viral RNA genome is one of the major structural proteins and plays role of antigen to elicit the antibody detected predorminantly right after infection of the virus in the patients of hemorragic fever with renal syndrome (HFRS)or experimental animals. NP is important target antigen in serological diagnostic system of HFRS utilizing whole antigens from the native virus particle, such as IFA, ELISA and Western blotting. Therefore, the preparation of this protein in the level of higher quantity and purity is desirasble for developed dianosis of the disease. The purpose of this study is the cloning of NP gene which exists in the S genome segment of Maaji (MAA) virus and expression of the gene to obtain qualified, genetically engineered NP to be utilized as an immunodiagnostic antigen. First of all, for the purpose of amplifing the MAA-NP gene by PCR, the specific primers were built from the known nucleotide sequence of Hantaan viral NP gene. The viral cDNA of the NP gene was synthesized by using the primers and RNase $H^-$ AMV reverse transcriptase. Thereafter, using this cDNA as a template, the NP gene was amplified specifically by Taq DNA polymrerase. The pT7blue (R)T-overhang vector systems were used for cloning of the amplified NP gene. The expression system was consisted of BL21 (DE3)pLysS and pET16b as a host and a plasmid repectively. Into Ndel site of pET16b, NP gene was ligated with cohesive end for the expression. Insertion of NP gene in the plasmid was confirmed by PCR and mini prep methods. For expression, IPTG was used and the expressed protein was characterized by Western blotting. The MAA-NP was expressed as the form of inclusion body (insoluble fraction)and the protein purified by affinity and metal chealating columns reacted specifically with the sera from patients of HFRS as to be tested by ELISA and Western blotting.

• Journal of Microbiology
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• v.45 no.2
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• pp.175-178
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• 2007

An endonuclease from Xanthomonas oryzae pathovar oryzae KACC 10331, XorII, was recombinantly produced in Escherichia coli using a T7 system. XorII was purified using a combination of ion exchange and immobilized metal affinity chromatography (IMAC). An optimized washing protocol was carried out on an IMAC in order to obtain a high purity product. The final amount of purified XorII was approximately 2.5 mg/L of LB medium. The purified recombinant XorII was functional and showed the same cleavage pattern as PvuI. The enzyme activity tested the highest at $25^{\circ}C$ in 50 mM NaCl, 10 mM Tris-HCl, 10 mM $MgCl_{2}$, and 1 mM dithiothreitol at a pH of 7.9.

• Toxicological Research
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• v.28 no.4
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• pp.255-262
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• 2012

Inflammation is the immune system's response to infection and injury-related disorders, and is related to pro-inflammatory factors (NO, $PGE_2$, cytokines, etc.) produced by inflammatory cells. Atopic dermatitis (AD) is a representative inflammatory skin disease that is characterized by increasing serum levels of inflammatory chemokines, including macrophage-derived chemokine (MDC). Carpinus tschonoskii is a member of the genus Carpinus. We investigated the anti-inflammatory activity of C. tschonoskii by studying the effects of various solvent fractions prepared from its leaves on inflammatory mediators in HaCaT and RAW264.7 cells. We found that the chloroform fraction of C. tschonoskii inhibited MDC at both the protein and mRNA levels in HaCaT cells, acting via the inhibition of STAT1 in the IFN-${\gamma}$ signaling pathway. In addition, the chloroform fraction significantly suppressed the expression of inflammatory factors induced by lipopolysaccharide stimulation, except COX-2 and TNF-${\alpha}$. These results suggest that the chloroform fraction of C. tschonoskii leaves may include a component with potential anti-inflammatory activity.