• Title/Summary/Keyword: subunits$(\alpha'-,\

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COOH-Terminal Animo Acids of Tethered-Buman Glycoprotein Bormone $\alpha$-Subunit Play an Important Role for Secretion

  • Min, K.S;Yoon, J.K.
    • Korean Journal of Animal Reproduction
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    • v.26 no.4
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    • pp.395-399
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    • 2002
  • Human chorionic gonadotropin (hCG) is a member of the glycoprotein hormone family which includes FSH. hCG TSH. These hormone family is characterized by a heterodimeric structure composed a common $\alpha$-subunit noncovalently linked to a hormone specific $\beta$-subunit. To determine u and $\beta$ -subunits can be synthesized as a single polypeptide chain (tethered-hCG) and also display biological activity, the tethered-hCC and -FSH molecule by fusing the carboxyl terminus of the hCG $\beta$-subunit to the amino terminus of the $\alpha$-subunit was constructed. To determine the importance of $\alpha$ COOH -terminal amino acid, we also deleted the $\alpha$ COOH-terminal amino acids. The expressing vectors were transfected into CHO-K 1 cells. The tethered-wthCG and -wtFSH was efficiently secreted. The $\alpha$ Δ83hCG and $\alpha$ Δ 83FSH mutants had no secretion. These results are the first conclusive evidence that COOH-terminal amino acids are very important for secretion in human glycoprotein hormone $\alpha$-subunit. These results demonstrated that the $\alpha$ Δ83hCG and $\alpha$ Δ 83FSH mutants could be play a pivotal role in the secretion of tethered-molecule.

Binding Symmetry of External Divalent Cations to Cyclic Nucleotide-gated IonChannel Reveled by Channel Tandem Dimers

  • Kwon, Ryuk-Jun;Park, Chul-Seung
    • Proceedings of the Korean Biophysical Society Conference
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    • 2001.06a
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    • pp.37-37
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    • 2001
  • Cyclic nucleotide-gated (CNG) channels are composed of homo or hetero tetramer of ${\alpha}$ and ${\beta}$ subunits. The a subunits of these channels have a conserved glutamate residue within the pore-forming region. This residue determines the selectivity as well as the affinity for the extracellular divalent cations. Using the high affinity mutant (E363D) of bovine retinal CNG channel in which the Glu was replaced to Asp at position 363, we constructed tandem dimers and investigated the binding symmetry of divalent cation to the site.(omitted)

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The Effects of Daesiho-tang Extracts on Hypoxic E18 Cortical Neuroblast (대시호탕이 배양신경세포를 이용한 저산소증에 미치는 영향)

  • 김지형;정승현;신길조;문일수;이원철
    • The Journal of Korean Medicine
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    • v.22 no.1
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    • pp.63-77
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    • 2001
  • Objectives : In acute stage of CVA, many patients experience Bugibultong as a symptom. Daesiho-tang is one of the most useful prescriptions for constipated stroke patients. This study investigated the effects of Daesiho-tang extracts on hypoxic E18 cortical neuroblast. Methods : To evaluate the effect of Daesiho-tang extracts on enuronal death caused by hypocia the neuronal viability and protein expressions of NMDA receptor subunits, ${\alpha}CaMK$ II PDE2 PSD95 densin-180, $eEF-l{\alpha}$ and GFAP were investigated. Results : We guessed that Daesiho-tang extracts worked to protect against cell damage through effecting on the protein expression of PSD(post synatpic density), especially increase of ${\alpha}CaMK$ II, $eEF-l{\alpha}$ PDE2, PSD95 and densin-180 Conclusions : According to the above results, it is supposed that Daesiho-tang is clinically applicable to stroke patients in the acute stage.

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Determination of Monoclonal Antibodies Capable of Recognizing the Native Protein Using Surface Plasmon Resonance

  • Kim, Deok-Ryong
    • BMB Reports
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    • v.34 no.5
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    • pp.452-456
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    • 2001
  • Surface plasmon resonance has been used for a biospecific interaction analysis between two macromolecules in real time. Determination of an antibody that is capable of specifically interacting with the native form of antigen is very useful for many biological and medical applications. Twenty monoclonal antibodies against the $\alpha$ subunit of E. coli DNA polymerase III were screened for specifically recognizing the native form of protein using surface plasmon resonance. Only four monoclonal antibodies among them specifically recognized the native $\alpha$ protein, although all of the antibodies were able to specifically interact with the denatured $\alpha$ subunit. These antibodies failed to interfere with the interaction between the $\tau$ and $\alpha$ subunits that were required for dimerization of the two polymerases at the DNA replication fork. This real-time analysis using surface plasmon resonance provides an easy method to screen antibodies that are capable of binding to the native form of the antigen molecule and determine the biological interaction between the two molecules.

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Expression of Low Voltage-Activated $Ca^{2+}$ Channels in Xenopus Oocytes

  • Lee, Jung-Ha;Han, Dong-Pyo
    • Journal of Microbiology and Biotechnology
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    • v.11 no.4
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    • pp.614-618
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    • 2001
  • Low-threshold T-type $Ca^{2+}$ channels are distinctive voltage-operated gates for external $Ca^{2+}$ entry around a resting membrane potential due to their low voltage activation. These phenomena have already been extensively studied due to their relevance in diverse physiological functions. Recently, three T-type $Ca^{2+}$ channel ${\alpha}$$_1$subunits were cloned and their biophysical properties were characterized after expression in mammalian expression systems. In this study, ${\alpha_IG} and {\alpha_IH}$ low-threshold $Ca^{2+}$ channels were expressed and characterized in Xenopus oocytes after adding 5' and 3'untranslated portions of a Xenopus ${\beta}$ globin to improve their expression levels. The added portions dramatically enhanced the expression levels of the ${\alpha_IG} and {\alpha_IH}$ T-type channels. When currents were recorded in 10 mM $Ba^{2+}$ as the charge carrier, the activation thresholds were about -60 mV, peak currents appeared at -20 mV, and the reversal potentials were between +40 and +45. The activation time constants were very similar to each other, while the inactivation time constants of the ${\alpha_IG}$ currents were smaller than those of ${\alpha_IH}$. Taken together, the electrophysiological properties of the ${\alpha_IG} and {\alpha_IH}$ channels expressed in Xenopus oocytes were similar to the previously reported characteristics of low-threshold $Ca^{2+}$ channel currents.

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The Alpha Subunit of Go Interacts with Brain Specific High Mobility Group Box Containing Protein

  • Park, Jung-Sik;Ghil, Sung-Ho
    • Biomedical Science Letters
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    • v.12 no.4
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    • pp.405-411
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    • 2006
  • Heterotrimeric GTP binding proteins (G proteins) mediate signal transduction generated by neurotransmitter and hormones. Among G-proteins, Go is classified as a member of the Go/Gi family and the most abundant heterotrimeric G protein in brain. Most of the mechanistic analyses on the activation of Go indicated its action to be mediated by the $G{\beta}{\gamma}$ dimer because downstream effectors for its ${\alpha}$ subunit have not been clearly defined. To determine the downstream effectors of alpha subunits of Go ($Go{\alpha}$), we used yeast two-hybrid system to screen $Go{\alpha}$ interacting partners in cDNA library from the human brain. A brain specific high mobility group box containing protein (BHX), A possible transcription factor, was identified as a $Go{\alpha}$ interacting protein. We confirmed interaction between $Go{\alpha}$ and BHX employing in vitro affinity binding assay. Moreover, active form of $Go{\alpha}$ preferentially interacts with BHX than inactive farm. Our findings indicate that $Go{\alpha}$ could modulate gene expression via interaction with BHX during neuronal or brain development.

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Tumor Cell Clone Expressing the Membrane-bound Form of IL-12p35 Subunit Stimulates Antitumor Immune Responses Dominated by $CD8^+$ T Cells

  • Lim, Hoyong;Do, Seon Ah;Park, Sang Min;Kim, Young Sang
    • IMMUNE NETWORK
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    • v.13 no.2
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    • pp.63-69
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    • 2013
  • IL-12 is a secretory heterodimeric cytokine composed of p35 and p40 subunits. IL-12 p35 and p40 subunits are sometimes produced as monomers or homodimers. IL-12 is also produced as a membrane-bound form in some cases. In this study, we hypothesized that the membrane-bound form of IL-12 subunits may function as a costimulatory signal for selective activation of TAA-specific CTL through direct priming without involving antigen presenting cells and helper T cells. MethA fibrosarcoma cells were transfected with expression vectors of membrane-bound form of IL-12p35 (mbIL-12p35) or IL-12p40 subunit (mbIL-12p40) and were selected under G418-containing medium. The tumor cell clones were analyzed for the expression of mbIL-12p35 or p40 subunit and for their stimulatory effects on macrophages. The responsible T-cell subpopulation for antitumor activity of mbIL-12p35 expressing tumor clone was also analyzed in T cell subset-depleted mice. Expression of transfected membranebound form of IL-12 subunits was stable during more than 3 months of in vitro culture, and the chimeric molecules were not released into culture supernatants. Neither the mbIL-12p35-expressing tumor clones nor mbIL-12p40-expressing tumor clones activated macrophages to secrete TNF-${\alpha}$. Growth of mbIL-12p35-expressing tumor clones was more accelerated in the $CD8^+$ T cell-depleted mice than in $CD4^+$ T cell-depleted or normal mice. These results suggest that $CD8^+$ T cells could be responsible for the rejection of mbIL-12p35-expressing tumor clone, which may bypass activation of antigen presenting cells and $CD4^+$ helper T cells.

Physiological Functions of the COPI Complex in Higher Plants

  • Ahn, Hee-Kyung;Kang, Yong Won;Lim, Hye Min;Hwang, Inhwan;Pai, Hyun-Sook
    • Molecules and Cells
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    • v.38 no.10
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    • pp.866-875
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    • 2015
  • COPI vesicles are essential to the retrograde transport of proteins in the early secretory pathway. The COPI coatomer complex consists of seven subunits, termed ${\alpha}-$, ${\beta}-$, ${\beta}^{\prime}-$, ${\gamma}-$, ${\delta}-$, ${\varepsilon}-$, and ${\zeta}$-COP, in yeast and mammals. Plant genomes have homologs of these subunits, but the essentiality of their cellular functions has hampered the functional characterization of the subunit genes in plants. Here we have employed virus-induced gene silencing (VIGS) and dexamethasone (DEX)-inducible RNAi of the COPI subunit genes to study the in vivo functions of the COPI coatomer complex in plants. The ${\beta}^{\prime}-$, ${\gamma}-$, and ${\delta}$-COP subunits localized to the Golgi as GFP-fusion proteins and interacted with each other in the Golgi. Silencing of ${\beta}^{\prime}-$, ${\gamma}-$, and ${\delta}$-COP by VIGS resulted in growth arrest and acute plant death in Nicotiana benthamiana, with the affected leaf cells exhibiting morphological markers of programmed cell death. Depletion of the COPI subunits resulted in disruption of the Golgi structure and accumulation of autolysosome-like structures in earlier stages of gene silencing. In tobacco BY-2 cells, DEX-inducible RNAi of ${\beta}^{\prime}$-COP caused aberrant cell plate formation during cytokinesis. Collectively, these results suggest that COPI vesicles are essential to plant growth and survival by maintaining the Golgi apparatus and modulating cell plate formation.

Purification and Characterization of Quercitrin-Hydrolyzing ${\alpha}$-L-Rhamnosidase from Fusobacterium K-60, a Human Intestinal Bacterium

  • PARK SUN-YOUNG;KIM JOO-HYUN;KIM DONG-HYUN
    • Journal of Microbiology and Biotechnology
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    • v.15 no.3
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    • pp.519-524
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    • 2005
  • An ${\alpha}$-L-rhamnosidase (EC 3.2.1.40.), which transforms quercitrin to quercetin, was purified from Fusobacterium K-60, a human intestinal anaerobic bacterium. The specific activity of the purified ${\alpha}$-L-rhamnosidase was 2.89 mol/min/mg protein. ${\alpha}$-L-Rhamnosidase, whose molecular size was 170 kDa by gel filtration, was composed of four subunits ($M_r$ 41,000 Da) with pI and optimal pH values of 5.2 and 5.5-7.0, respectively. The apparent $K_m$ and $V_{max}$ values for p-nitrophenyl-${\alpha}$-L-rhamnopyranoside and quercitrin were determined to be 0.057 mM and 3.4 mol/min/mg, and 0.077 mM and 5.0 mol/min/mg, respectively. This enzyme was strongly inhibited by $Cu^{2+},\;Mn^{2+}$, L-rhamnose, and p-chlormercuriphenylsulfonic acid. These findings suggest that the biochemical properties and substrate specificity of the purified enzyme are different from those of the previously purified ${\alpha}$-L-rhamnosidase. This is the first reported purification of quercitrin-hydrolyzing ${\alpha}$-L-rhamnosidase from intestinal bacteria.

Mangiferin inhibits tumor necrosis factor-α-induced matrix metalloproteinase-9 expression and cellular invasion by suppressing nuclear factor-κB activity

  • Dilshara, Matharage Gayani;Kang, Chang-Hee;Choi, Yung Hyun;Kim, Gi-Young
    • BMB Reports
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    • v.48 no.10
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    • pp.559-564
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    • 2015
  • We investigated the effects of mangiferin on the expression and activity of metalloproteinase (MMP)-9 and the invasion of tumor necrosis factor (TNF)-$\alpha$-stimulated human LNCaP prostate carcinoma cells. Reverse-transcription polymerase chain reaction (RT-PCR) and western blot analysis showed that mangiferin significantly reversed TNF-$\alpha$-induced mRNA and protein expression of MMP-9 expression. Zymography data confirmed that stimulation of cells with TNF-$\alpha$ significantly increased MMP-9 activity. However, mangiferin substantially reduced the TNF-$\alpha$-induced activity of MMP-9. Additionally, a matrigel invasion assay showed that mangiferin significantly reduced TNF-$\alpha$-induced invasion of LNCaP cells. Compared to untreated controls, TNF-$\alpha$-stimulated LNCaP cells showed a significant increase in nuclear factor-${\kappa}B$ (NF-${\kappa}B$) luciferase activity. However, mangiferin treatment markedly decreased TNF-$\alpha$-induced NF-${\kappa}B$ luciferase activity. Furthermore, mangiferin suppressed nuclear translocation of the NF-${\kappa}B$ subunits p65 and p50. Collectively, our results indicate that mangiferin is a potential anti-invasive agent that acts by suppressing NF-${\kappa}B$-mediated MMP-9 expression.