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Escherichia coli(M15)을 이용한 serotonin N-acetyltransferase 유전자의 발현
김용만,정미영,윤경식,진병관,백행운,조용호,백형환 慶熙大學校 1997 論文集 Vol.26 No.-
AA-NAT cDNA was obtained by RT-PCR technique from total RNA of rat sacrified at night(02:00am). pCRNAT was cloned using the pCRII vector with insertion of AA-NAT cDNA(about 1.4 kb) at EcoRI site. For the expression of the gene, pQECNAT was subcloned, in which the coding region of AA-NAT was inserted into expression vector pQE30 at BamHI and HindIII sites. According to the experimental results, Escherichia coli strain M15, transformed by the expession vector pQECNAT, was selected as a host to express AA-NAT gene with the induction of isopropyl β-D-thiogalactoside(IPTG). Optimal condition for the expression of AA-NAT gene was achieve from the experimental results, showing that the expression of the protein was inducible much 19.800 ± 2,200 dpm per ml of culture volume in 4 hours at the concentration of 2 mM IPTG. Partial purification through the affinity column(Ni-NTA agarose) binding to the continuous 6 histidine residues of protein resulted in 5 times more increase in the specific activity of AA-NAT than that of the homogenate of bacterial pellet. These experimental results will provide basic data in further study for the enzymatic kinetics and antibody production of AA-NAT.
망막아세포종 세포주(Y-79)의 Arylalkylamine N-acetyltransferase 활성에 미치는 Dopamine의 영향
김석민,정미영,윤경식,진병관,백행운,백형환,조용호 慶熙大學校 1997 論文集 Vol.26 No.-
The arylalkylamine N-acetyltransferase(AA-NAT, EC 2.3.1.87) in vertebral pineal gland and retina is rate-limiting enzyme in the pathway of biosynthesis of melatonin. AA-NAT activities are remarkably increased during night with 10-150 folds. Neurohormone melatonin is thought to play roles in a broad range of physiological functions in human, primarily through effects on the biological clock in the suprachiasmatic nucleus of the hypothalamus, This rhythm reflects large changes in the activities of AA-NAT. So the important regulatory role of AA-NAT has made it central interest in research on melatonin biochemistry and neurochemical signal transduction. The neurotransmitter that regulates the AA-NAT activities is norepinephrine, which acts through a cyclic AMP mechanism. The effect of dopamine on AA-NAT in human retinoblastoma Y-79 cells was studied. the obtained results are as follows; AA-NAT activities in Y-79 cells were increased by cyclic AMP, and dopamine inhibited the AA-NAT activities stimulated by forskolin. The pattern of inhibition of dopamine on the AA-NAT activities in Y-79 cells was time and concentration dependent. The results suggest that dopamine participates in the regulation of the AA-NAT activities through the inhibitory mechanism in the human pineal and retinal tissues.
Interleukin 13 on Microglia is Neurotoxic in Lipopolysaccharide-injected Striatum in vivo
홍아름,장재근,정용철,원소윤,진병관 한국뇌신경과학회 2022 Experimental Neurobiology Vol.31 No.1
To explore the potential function of interleukin-13 (IL-13), lipopolysaccharide (LPS) or PBS as a control was unilaterally microinjected into striatum of rat brain. Seven days after LPS injection, there was a significant loss of neurons and microglial activation in the striatum, visualized by immunohistochemical staining against neuronal nuclei (NeuN) and the OX-42 (complement receptor type 3, CR3), respectively. In parallel, IL-13 immunoreactivity was increased as early as 3 days and sustained up to 7 days post LPS injection, compared to PBS-injected control and detected exclusively within microglia. Moreover, GFAP immunostaining and blood brain barrier (BBB) permeability evaluation showed the loss of astro- cytes and disruption of BBB, respectively. By contrast, treatment with IL-13 neutralizing antibody (IL-13NA) protects NeuN+ neurons against LPS- induced neurotoxicity in vivo . Accompanying neuroprotection, IL-13NA reduced loss of GFAP+ astrocytes and damage of BBB in LPS-injected striatum. Intriguingly, treatment with IL-13NA produced neurotrophic factors (NTFs) on survived astrocytes in LPS-injected rat striatum. Taken together, the present study suggests that LPS induces expression of IL-13 on microglia, which contributes to neurodegeneration via damage on as- trocytes and BBB disruption in the striatum in vivo .