18 research outputs found
μ¬λ κΈλ£¨νμ° λΆν΄ν¨μμ λμν¨μλ€μ ꡬ쑰μ κΈ°λ₯μ λν λΉκ΅ μ°κ΅¬
Dept. of Biomedical Laboratory Science/λ°μ¬[νκΈ]
μ¬λμ‘°μ§μμ κΈλ£¨νμ° λΆν΄ν¨μ (GDH)λ μ μ μ κΈ°μμ΄ λ€λ₯Έ λ κ°μ§ μ΄μμ λμν¨μ (hGDH1, hGDH2)λ‘ μ‘΄μ¬νλ€. hGDH1μ μ΄μ λΉκ΅μ μμ νκ³ , λͺΈ μ 체μμ λ°νλλ λ°λ©΄μ hGDH2λ μ΄μ μ½νκ³ κ³ νμ‘°μ§μ΄λ μ κ²½μ‘°μ§μμ νΉμ΄μ μΌλ‘ λ°νλλ€. GDH λμν¨μμ ꡬ쑰μ κΈ°λ₯μ λν΄μ μμ보기 μνμ¬, 1557-base-pairμ μ¬λ GDH2λ₯Ό μ μ μλ₯Ό ν©μ±νμ¬ λμ₯κ· μ£Όλ₯Ό μ΄μ©ν΄ νμ±μ μ§λ ν¨μλ‘ λ°ννμλ€. μ΄κ²μ μ΄μ©νμ¬ μ¬λ GDH λμν¨μ κ°μ μ΄μ μμ μ± μ°¨μ΄μ κ΄μ¬νλ μ£Όλ μλ―Έλ
Έμ°μ λμ νκ³ μ νμλ€. 45C (pH 7.0)μμ μ΄μ λν ν¨μμ λΆνμ±μ μλ‘μ€ν
λ¦ μ‘°μ μ κ° μμλ hGDH1 λ³΄λ€ hGDH2μμ λμ± λΉ λ₯΄κ² μ§νλμλ€. νμ§λ§ hGDH1, hGDH2 λͺ¨λ 1 mM ADP μ 3mM L-Leuμ μ‘΄μ¬ μμλ μ΄μ μν ν¨μμ λΆνμ± μλκ° λλ €μ‘λ€. λμ°λ³μ΄λ₯Ό μΌμΌν€λ λ°©λ²μΌλ‘ hGDH2 μ μ μμμμ Ser443μ κ·Έμ λμνλ hGDH1 μ μ μμ Arg443μΌλ‘ μΉνν κ²°κ³Ό hGDH2κ° μ§λκ³ μλ μ΄μ λν λΆμμ μ±μ΄ μ¬λΌμ‘λ€. λ°λ©΄μ κ°λ₯μ±μ΄ μλ€κ³ 보μ¬μ§λ λͺ κ°μ§ λ€λ₯Έ μλ―Έλ
Έμ°μ λν λμ°λ³μ΄λ€μ (L415M, A456G, H470R) μ΄μ μμ μ±μ λν νΉλ³ν λ³νλ₯Ό 보μ¬μ£Όμ§ λͺ»νλ€. λ°λΌμ μ¬λ GDH λμμμμμ Ser443 μμΉκ° μ΄ μμ μ±μ μ€μν μν μ νλ κ³³μμ μκ² λμλ€.
λ€μμΌλ‘ μ¬λ GDH λμν¨μμ κΈλ£¨νμ° κ²°ν©λΆμλ₯Ό μμ보기 μνμ¬ λͺ κ°μ§ κ°λ₯μ±μ΄ μλ λΆμ (K94, G96, K118, K130, or D172)μ λν΄ λμ°λ³μ΄λ°©λ²μ μ¬μ©νμλ€. hGDH1κ³Ό hGDH2μ K94, G96, K118μμμ λμ°μ²΄μμ ν¨μνμ±μ΄ ν¬κ² κ°μνμκ³ κΈλ£¨νμ°μ λν Km κ°μ4-10λ°°μ λ μ¦κ°νμλ€. K130Y λμ°μ²΄μμλ κΈλ£¨νμ°μ λν Km κ°μ΄ 1.6λ°°μ λμ λΆκ³Όν λ°λ©΄ νμ±ν¨μ¨μ μμ°νμ 2~3%λ₯Ό 보μ¬μ£Όμλ€. λ°λΌμ K130 μμΉλ κΈλ£¨νμ° κ²°ν©λ³΄λ€λ ν¨μνμ±μ μμ΄μ μ€μν κ³³μμ μκ² λμλ€. λν D172Y λμ°μ²΄μμ μλ‘μ€ν
λ¦ νμ±μ μΈ ADPμ λν νμ±μ κ°μκ° νμΈλμλ€.
p-Chloromercuribenzoic acidλ μ¬λ GDHμ νμ±μ κ°μμν¨λ€. [14C]κ° νμ§λ peptideλ₯Ό νμΈν κ²°κ³Ό PCMBμ μμ©νλ κ³³μ C323μ΄μλ€. hGDH λμν¨μ μμ°μ²΄μλ λ€λ₯΄κ² Arg, Gly, Leu, Met λ Tyrλ‘ μΉνμν¨ C323 λμ°μ²΄λ€μ [14C]p-chloromercuribenzoic acid μ κ²°ν©νμ§ μμλ€. μ΄ λμ°μ²΄λ€μ ν¨μ νμ± ν¨μ¨μ μμ°μ²΄μ 11%~14%μ΄μκ³ μ΄λ¬ν μ¬μ€μ λ κ°μ§μ μ¬λ GDH λμν¨μμμ λ€λ₯΄μ§ μμλ€. μλ‘μ€ν
λ¦ μν₯μ μΈ ADPμ GTPλ [14C]p-chloromercuribenzoic acidκ³Όμ κ²°ν©μ μ΄λ ν μν₯λ λΌμΉμ§ μμλ€. μ§κΈκΉμ§μ κ²°κ³Όλ C323 λΆμκ° μ¬λ GDH λμν¨μμ ν¨μνμ±μ κ΄μ¬νλ€λ κ²μ 보μ¬μ£Όκ³ μλ€.
μ루미λμ µMμ λλ λ²μμμ pseudo-first-order reactionμ μν΄ GDHλ₯Ό λΆνμ±μν¨λ€. μ΄λ¬ν λΆνμ±μ pHμ μμ‘΄νκ³ μ°μ± μνμμ μ¦κ°νλ€. λΆνμ±λ GDHλ ν¨μ λ¨μ λ¨λ체 λΉ 2λͺ°μ μ루미λκ³Ό κ²°ν©νλ€. μ¬λ¬ ν‘μ°©μ λ€ μ€μμ ꡬμ°μ°κ³Ό μΌνλΆμκ° μ루미λκ³Ό ν¨μμ 볡ν©μ²΄μμ μ루미λμ λΆλ¦¬μν¨λ€. κ·Έ λΆλ¦¬μμλ 5.3 µMμ΄λ€. ADP, NAD+λ GTP κ²°ν©λΆμμμμ μ€νμ κ·Έ λΆμκ° GDHμ λν μ루미λ λΉνμ±κ³Ό κ΄λ ¨μ΄ μλ€λ μ¬μ€μ 보μ¬μ€λ€. μνΈκ΄ μ΄μμ± μΈ‘μ μ€νμμ GDHμ λν μ루미λμ κ²°ν©μ ν¨μμ Ξ±-λμ ꡬ쑰 μ Ξ²-λ³νꡬ쑰λ₯Ό κ°μμν€κ³ λλ€μ½μΌκ΅¬μ‘°λ₯Ό μ¦κ°μν¨λ€λ μ¬μ€μ νμΈνμλ€. λ°λΌμ μ루미λμ μν GDHμ λΆνμ±νλ μ루미λ κ²°ν©μ μν ꡬ쑰μ μΈ λ³νμ κΈ°μΈν κ²μΌλ‘ μκ°ν μ μκ³ μ΄λ¬ν μ¬μ€μ κΈλ£¨νμ° λμ¬μ κ΄μ¬νλ ν¨μμμ μ루미λμ μν μν₯μ΄ μ루미λμ μν μ κ²½λ
μ±μ μμΈ μ€ νλμΌ κ²μ΄λΌλ κ°λ₯μ±μ μ μν΄ μ€λ€.
λ§μ§λ§μΌλ‘ GDHμ λν siRNAλ₯Ό λ§λ€ μ μλ 벑ν°λ₯Ό μ¬μ©ν¨μΌλ‘μ¨ νν¨μ¨μ¨ λ³ κ°μ μ κ²½ν΄νμ± μ§νμμ GDHμ κΈ°λ₯μ μ΄ν΄λ³΄κ³ μ νμλ€. siRNAλ 무μ²μΆλλ¬Όμμμ RNA κ°μ λμμ RNA λΆν΄λ₯Ό μΌκΈ°μν€λ μ€κ°μ²΄λ‘μ μμ©μ νλ 19-21 λ΄ν΄λ μ΄ν°λμ μ΄μ€λμ RNAμ΄λ€. phGDH-siRNA3 κ³Ό phGDH-siRNA4μ co-transfectionμ GDH λ°νμ μ΅μ μν€κ³ mRNA μμ κ°μμν¨λ€λ κ²μ νμΈνμλ€. λν TUNEL κ²μ¬λ₯Ό ν΅ν΄μ μ΄λ¬ν GDHμ μ μ μ μλ©Έμ΄ μ κ²½μΈν¬μμ μΈν¬μ¬λ₯Ό μΌμΌν¨λ€λ μ¬μ€κ³Ό caspase 3 λ°νμ μ¦κ°κ° μ λλλ€λ μ μ νμΈνμλ€. λ°λΌμ μ κ²½μΈν¬μμ μ¬λ GDHμ μ΅μ λ μΈν¬μ¬μ κ΄λ ¨μ΄ μλ€λ μ¬μ€μ μ¦λͺ
νμμΌλ©° μ΄λ μ κ²½μΈν¬ λ΄μμ GDHκ° μ€μν μμ©μ νλ€λ κ²μ μ μν΄μ£Όκ³ μλ€.
[μλ¬Έ]Molecular biological studies confirmed that two human glutamate dehydrogenase isozymes (hGDH1 & hGDH2) of distinct genetic origin are expressed in human tissues. hGDH1 is heat stable and expressed widely, whereas hGDH2 is heat-labile and specific for neural and testicular tissues. To gain a insight into the structural and regulatory basis of GDH isozymes, a 1557-base-pair gene that encodes human GDH2 has been synthesized and expressed in E. coli as a soluble protein. At 45oC (pH 7.0), heat inactivation processed faster for hGDH2 (half-life = 45 min) than for hGDH1 (half-life = 310 min) in the absence of allosteric regulators. Both hGDH1 and hGDH2, however, showed much slower heat inactivation processes in the presence of 1 mM ADP or 3 mM L-Leu. In hGDH isozymes, the 443 site is Arg in hGDH1 and Ser in hGDH2, respectively. Substitution of Ser into Arg at 443 site by cassette mutagenesis abolished the heat-lability of hGDH2 with a similar half-life of hGDH1. These results suggest that the Ser 443 residue plays an important role in the different thermal stability of hGDH isozymes.
The cassette mutagenesis at several putative positions (K94, G96, K118, K130, or D172) was performed to examine the residues involved in the glutamate-binding of the hGDH isozymes (hGDH1 and hGDH2). There was dramatic reduction in the catalytic efficiency in mutant proteins at K94, G96, K118, or K130 site, but not at D172 site. The Km values for glutamate were 4~10-fold greater for the mutants at K94, G96, or K118 site than for the wild-type hGDH1 and hGDH2. The decreased catalytic efficiency of the K130 mutant mainly results from the reduced kcat value, suggesting a possibility that the K130Y residue may be involved in the catalysis rather than in the glutamate-binding. The reduction of ADP activation in D172Y mutant was more profoundly observed in hGDH2 than in hGDH1. It is suggested that K94, G96, and K118 residues play an important role, although at different degrees, in the binding of glutamate to hGDH isozymes.
Reaction of hGDH1 and hGDH2 with p-chloromercuribenzoic acid resulted in a loss of enzyme activity. A reactive cysteine residue was identified as C323 in the overall sequence of [14C]-labeled peptide. In contrast to the wild-type hGDH isozymes, no incorporation of [14C]p-chloromercuribenzoic acid was observed with the C323 mutants containing Arg, Gly, Leu, Met, or Tyr at position 323 using synthetic hGDH genes. The enzyme efficiency (kcat
Km) of the mutants showed only 11%~14% of the wild type hGDH isozymes, suggesting that the decreased efficiencies of the mutants mainly result from the decrease in kcat values. There were no differences between the two hGDH isozymes in sensitivities to the mutagenesis at C323 site. It is suggested that C323 plays an important role for the catalysis of hGDH isozymes.
Aluminum inactivated hGDH isozymes by a pseudo-first-order reaction at µM concentration. Double reciprocal plot gave a straight line with a kinact of 2.7 min-1 and indicated the presence of a binding step prior to inactivation. The inactivation was strictly pH-dependent and a marked increase in the sensitivity to aluminum was observed as the pH decreased. When preincubated with enzyme, several chelators such as N-(2-hydroxyethyl) ethylenediaminetriacetic acid, ethylenediaminetriacetic acid, citrate, or NaF efficiently protected the enzyme against the aluminum inactivation. In a related experiment, only citrate and NaF released the aluminum from the completely inactivated aluminum-enzyme complex and fully recovered the enzyme activity. The dissociation constant for the aluminum-enzyme complex was calculated to be 5.3 µM. Circular dichroism studies showed that the binding of aluminum to the enzyme induced a decrease in Ξ±-helix and Ξ²-sheet and an increase in random coil. Therefore, it is suggested that inactivation of hGDH by aluminum is due to the conformational change induced by aluminum binding. These results suggest a possibility that aluminum-induced alterations in enzymes of the glutamate system may be one of the causes of aluminum-induced neurotoxicity.
GDH function may be of importance for neurodegenerative disease such as Parkinson''s disease. In this study, several vector-based siRNA of hGDH was constructed and directly expressed intracellularly from a plasmid DNA. Using immunoblotting method, it was confirmed that hGDH expression were knockdown by most of phGDH-siRNAs in human neuroblastoma cells. RT-PCR analysis showed that mRNA level also decreased like as hGDH protein level. TUNEL assay after 48 h co-transfection revealed that inhibition of hGDH expression induced apoptotic condition and increased caspase-3 activity in human neuroblastoma cells. It is shown that inhibition of hGDH expression in neuronal cell are related with apoptosis.restrictio
(A)Study on rhetorical expression of public information design : focus on information design case for Seoul public transportation
νμλ
Όλ¬Έ(μμ¬)--μμΈλνκ΅ λνμ :λμμΈνλΆ μκ°λμμΈμ 곡,2005.Maste
Apoptosis and signal transduction by chobalt chloride in C6 glioma cells
μμλ³λ¦¬νκ³Ό/μμ¬λλΉν(brain ischemia)μ hypoxiaλ₯Ό μΌκΈ°νλλ°, μ΄κ²μ λμμκ³Ό μ€μΆμ κ²½κ³ κ²½μμ
μΌμΌν€λ μ£Όμν λ³λ¦¬νμ μμμ΄λ€. λ³Έ μ°κ΅¬μμλ normoxiaμμ hypoxia μνλ₯Ό μ λν
λ κ²μΌλ‘ μλ €μ§ cobalt chlorideλ‘ μΈν¬λ°°μλ C6 glioma μΈν¬μμ hypoxiaλ₯Ό μ λνμ¬
κ·Έμ λ°λ₯Έ apoptosisμμ MAP kinase κ° κ΄μ¬ν¨μ μμλ³΄κ³ μ νμλ€.
λ¨Όμ cobalt chlorideλ₯Ό C6 glioma μΈν¬μ μ²λ¦¬νμ¬ μΈν¬ λ
μ±λ₯μ λ³Έ κ²°κ³Ό, IC_(50)μ΄
400 ΞΌMλ‘ λνλ¬λ€. λ cobalt chloride μ²λ¦¬μ hypoxia-inducible factor 1(HIF-1)μ
λ°νμ μ¦κ°λ‘ cobalt chloride μ²λ¦¬λ‘ hypoxiaκ° μ λλ¨μ μ¦λͺ
νμλ€. Cobalt chloride
λ₯Ό 24μκ° μ²λ¦¬μ 300 ΞΌM μ΄μμ λλμμ DNA ladderκ° νμ±λ¨μ 1.8% agarose μ κΈ°μ
λμ ν΅ν΄ νμΈνμκ³ , Hoechst 33258 μΌμμ ν΅ν΄ ν΅μ λΆμ μ νμΈνμμΌλ©°, μ μνλ―Έ
κ²½μ ν΅ν΄ apoptosisλ‘ μ§νλλ μΈν¬μ νννμ μΈ λ³νλ₯Ό κ΄μ°°νμλ€. λν caspase-3
μ νμ± μ¦κ°λ₯Ό ν΅ν΄ apoptosis mediatorμ μμ©μ νμΈνμλ€. λ€μμΌλ‘ cobalt chlorid
eμ μν΄ μ λλλ apoptosisμ μ νΈ μ λ¬ κΈ°μ μ λν΄ μμ보μλ€. 400 ΞΌM cobalt chlo
rideλ₯Ό μ²λ¦¬μ μΈν¬μ§μμμ ERK 1/2μ νμ±νκ° 1μκ°λΆν° μμνμ¬ 6μκ°μμ κ°μ₯ κ°
νκ² λνλ¨μ μ μ μμκ³ , μ΄λ κ² νμ±νλ ERK 1/2λ μΈν¬μ§μμ νμ±νκ° λ μκ°(1
μκ°)μ ν΅μΌλ‘ μ΄λνμ¬ 3μκ°λΆν° κ°μνμλ€. ν΅λ΄ μ΄λν λ°νλλ transcription fa
ctorλ NFΞΊBμ μ¦κ°λ₯Ό immunoblottingμ ν΅ν΄ νμΈνμλ€. λν MEK1μ inhibitorμΈ PD9
8059μ μ μ²λ¦¬μ cobalt chlorideλ‘ μ λλλ DNA λΆμ λ₯μ μ΅μ μ μ μνλ―Έκ²½μΌλ‘ λ³Έ μΈ
ν¬μμ νννμ λ³νμ κ°μλ₯Ό νμΈν¨μΌλ‘μ apoptosisμ MAPKμ κ΄λ ¨μ±μ λ·λ°μΉ¨νμλ€
.
μμ μ€ν κ²°κ³Όλ₯Ό ν΅ν΄ C6 glioma μΈν¬μμ cobalt chlorideκ° μΈν¬ μ±μ₯ μ΅μ ν¨κ³Όμ a
poptosisλ₯Ό μ λνλ©° κ·Έμ μ νΈ μ λ¬μ MAP kinase family μ€μμλ ERK 1/2 pathwayλ₯Ό
ν΅νλ©° transcription factorμΈ NFΞΊBλ₯Ό νμ±μν΄μ μ μ μμλ€.
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ν΅μ¬λλ λ§ : Cobalt chloride, μΈν¬ λ
μ±λ₯, Apoptosis, ERK 1/2, NFΞΊB.
Apoptosis and Signal Trasduction by Cobalt Chloride in C6 Glioma cells
Brain ischemia brings about hypoxic insults. Hypoxia is one of the major
pathological factors inducing neuronal injury and cenrtral nervous system
infaction. In this study, I investigated the involvement of MAP kinase in
hypoxia-induced apoptosis using cobalt chloride in C6 glioma cells. The cobalt
chloride was used for the induction of hypoxia and its IC_(50) was 400 ΞΌM. I
demonstrated DNA fragmentation after incubation with concentrations more than 300
ΞΌM cobalt chloride for 24 h. I also evidenced nuclear cleavage with Hoechst33258
stianing and morphological changes of the cells undergoing apoptosis with electron
microscopy. Furthermore I confirmed activation of caspase-3, one of the mediator of
apoptosis. Next I examined the signal pathway of cobalt chloride-induced apoptosis
in C6 cells. The activation of extracellular signal-regulated protein kinase 1/2
(ERK 1/2) started to increase at 1h and more activated at 6h after treatment of 400
ΞΌM cobalt chloride. At the same time, the activated ERK 1/2 translocated into the
nucleus and activated transcriptional factor, NFΞΊB not c-Jun. In addition,
pretreatment of PD98059 inhibited cobalt chloride-induced DNA fragmentation and
apoptotic cell morphology. These results suggest that cobalt chloride is able to
induce the apoptotic activity in C6 glioma cells and its apoptotic mechanism may be
associated with signal transduction via MAP kinase (ERK 1/2).ope