40 research outputs found
ํจ์ ํ์ฑ ์ธก์ ๊ณผ ์๋ฌผ ์์ํ๋ฅผ ์ํ ๋ฐ์๊ธฐ๋ฐ ํ์นจ์ ๊ฐ๋ฐ
ํ์๋
ผ๋ฌธ (๋ฐ์ฌ)-- ์์ธ๋ํ๊ต ๋ํ์ ์์ฐ๊ณผํ๋ํ ํํ๋ถ, 2017. 8. ํ์ข
์ธ.The detection of enzyme activity provides many clues to the nature of living systems and the interaction between biomolecules. Enzyme reaction triggers chemical transformation of substrate to regulate its function relating to many biological events. The most general approach for enzyme activity assay is a reaction-based strategy using the chemical transformation of substrates. Numerous probes have been developed based on the enzymatic reaction. However, only a few strategies to control a signal have been developed, and most of them have not been applied practically. Herein, several probes based on two basic strategies are proposed.
The chemical transformation of a probe by enzymatic reaction triggers intramolecular cyclization to form fluorescent N-methylisoindole, which is a basic strategy to detect the enzyme activity.
Sulfatases catalyze the hydrolysis of sulfate esters that are present in a range of biomolecules. We have developed a new activity-based sulfatase probe (probe 1) that generates a fluorescent N-methylisoindole upon hydrolysis by sulfatase. Because of the autoxidation of N-methylisoindole, the sulfatase activity was also tested under reducing conditions containing either glutathione (GSH) or tris(2-carboxyethyl)phosphine (TCEP), which exhibited little change in the kinetic parameters, but a stronger emission than non-reducing conditions. Probe 1 also showed stronger intensity upon treatment with sulfatase under neutral conditions than acidic conditions, but it still has limitations in the selectivity of a specific sulfatase. Nevertheless, the fluorescent signal of the released N-methylisoindole provides a new assay for measuring sulfatase activity that could be applied for high-throughput screening (HTS).
Bacterial arylsulfatases are crucial to biosynthesis in many microorganisms, as they cleave aromatic sulfate esters for use as a sulfur source. They are associated with pathogenesis and are applied in many areas, such as industry and agriculture. The hydrolysis of probe 1 by sulfatases induced fluorescence intensity enhancement and the generation of colored precipitates through the polymerization of N-methylisoindole, which enabled the monitoring of bacterial arylsulfatase activity and the discrimination of periplasmic sulfatases from cytosolic sulfatases through liquid- and solid-phase colony-based assays.
For imaging analysis, the fluorescence of N-methylisoindole, the product of probe 1 from the cleavage upon sulfatase activity emitted a signal at the shorter wavelength. To overcome this limitation, fluorescence resonance energy transfer (FRET), aggregation-induced emission (AIE), and the excimer formation process were introduced. The FRET-based probe showed no fluorescence changes upon the treatment of sulfatase because the distance between N-methylisoindole and naphthalimide, an FRET acceptor, was 6.6 ร
, which was out of range of the Fรถrster distance. The AIE-based probe exhibited decreasing fluorescence and color changes, meaning that N-methylisoindole generation and polymerization did not intensify the AIE process. The excimer formation-based probe showed decreasing excimer emission, but increasing monomer and N-methylisoindole emissions. It also generated colored precipitates. When the probe was applied to bacteria species, the tendency of the emission was different from that in the results of the test in the homogenous condition, but precipitates were still formed. Thus, the excimer formation-based probe was only applicable to staining periplasmic sulfatase-expressing bacteria.
The next strategy to design a probe was based on intramolecular charge transfer (ICT). The basic scaffold was 2-dicyanomethylene-3-cyano-2,5-dihydrofuran (DCDHF) unit conjugated to a modified electron-donating group.
Leucine aminopeptidases (LAPs) are widely distributed in organisms, from bacteria to humans, and play crucial roles in cell maintenance and growth. Thus, assays for LAP are necessary for measuring its activity and inhibitor potency. In this paper, we report a small-molecule probe, DCDHF conjugated to a leucine residue, which exhibits colorimetric and fluorogenic changes according to LAP activity.
Human steroid sulfatase (STS) plays a pivotal role in the regulation of biologically active steroid to bind to the estrogen receptor (ER), which is related to hormone-dependent diseases. DCDHF conjugated with phenyl sulfate ester exhibits pH-dependent emission, which was appropriate for discriminating STS, whose optimal pH is 7other arylsulfatases usually exhibit the maximal activity at pH 5. DCDHF conjugated with phenyl sulfate interposing a self-immolative spacer between them is a ratiometric probe that could detect and monitor sulfatase activity in a complex cellular context.I. Introduction 1
1. Enzyme activity assays 2
2. Bioimaging 9
3. Fluorescent probe for detecting enzyme activity 16
4. Design strategy of fluorescent probe 21
II. Probes inducing signal changes by intramolecular cyclization upon the treatment of enzyme 34
1. Sulfatase activated probe generating N-methyl isoindole in buffer containing reducing agents 35
1.1. Introduction 35
1.2. Results and Discussion 37
1.3. Experiments 43
2. Detection of bacterial sulfatase activity and discrimination of periplasmic sulfatase activity through liquid- and solid-phase colony-based assays 59
2.1. Introduction 59
2.2. Results and Discussion 63
2.3. Experiments 74
3. Efforts for improvement of probe 1 using diverse signal generation strategies 86
3.1. Introduction 86
3.2. Results and Discussion 89
3.3. Experiments 97
III. Probes inducing fluorescence changes by intramolecular charge transfer process upon the treatment of enzyme 116
1. Small-molecule probe using dual signals to monitor leucine aminopeptidase activity 125
1.1. Introduction 117
1.2. Results and Discussion 118
1.3. Experiments 124
2. Selectvie detection of Steroide sulfatase (STS) and ratiometric detection of sulfatase activity in living cells 131
1.1. Introduction 131
1.2. Results and Discussion 134
1.3. Experiments 134
References 155
๊ตญ๋ฌธ ์ด๋ก (Abstract in Korean) 173Docto
Comparison between Fluid Intake and Output Measurement Methods of the Patients Hospitalized in Medical Units
Purpose: The purpose of this study was to compare the fluid intake and output (I&O) measurement methods in order to figure out more effective and easier method for medical patients.
Methods: 71 hospitalized patients participated in the study. In โliquid only (LO)โ method, all amount of water was summed up including any liquid types of food and IV fluids. In โwhole food(WF) intake,โ all liquid and solid food intake and IV fluids were added up.
Results: The average amount of fluid intake was 2105.29 ml for LO method and 2523.54 ml for WF method. The average amount of fluid output was 2148.98 ml. The intra-class correlations (ICC) between the intake and output measures by the two different methods was 0.803 and 0.826, respectively. The correlation between the differences of intake/output and body weight change in two different methods was r=.347 (p=.003), and r=.376 (p=.001), respectively.
Conclusion: The results of this study indicate that both LO and WF method may be useful in monitoring patientsโ fluid balance. Given the comparability of using LO over WF, it is suggested that measuring just liquid only intake as the indicator of patientโs intake is applicable in clinical setting.ope
MVRDV ๊ฑด์ถ์ datascape ๊ณต๊ฐ์กฐ์ง๋ฐฉ๋ฒ ์ฐ๊ตฌ : Villa VPRO, 1997์ Media Galaxy(Eyebeam Institute), 2001๋ฅผ ์ค์ฌ์ผ๋ก
ํ์๋
ผ๋ฌธ(์์ฌ)--์์ธ๋ํ๊ต ๋ํ์ :๊ฑด์ถํ๊ณผ,2003.Maste
๊ธ๋ฃจํ์ฐ ๋ถํดํจ์(glutamate dehydrogenase)์ ๊ตฌ์กฐ, ์๋ก์คํ ๋ฆญ ์กฐ์ , ๋ฐ ์์ฉ๊ธฐ์
Dept. of Medical Technology/๋ฐ์ฌ[์๋ฌธ]
[ํ๊ธ]
๊ธ๋ฃจํ์ฐ ๋ถํดํจ์ (GDH)์ ๊ตฌ์กฐ ๋ฐ ๋ถ์์ ํน์ฑ์ ์์ ๋ณด๊ธฐ ์ํ์ฌ ๋ค์๊ณผ ๊ฐ์ ์ฐ๊ตฌ
๋ค์ ์ํํ์๋ค. ์ฐ์ ์กฐ๋ฏธ๋ฃ๋ก ๋ง์ด ์ด์ฉ๋๊ณ ์์ผ๋ฉฐ Chinese restaurant syndrome์
์ ๋ฐ์ํค๋ ๋ฌผ์ง๋ก ์ ์๋ ค์ ธ ์๋ ๊ธ๋ฃจํ์ฐ๋ํธ๋ฅจ (MSG)์ด ๋์กฐ์ง ๋ด์ GDH์ ๋ฏธ์น๋ ์
ํฅ์ ์กฐ์ฌํ์๋ค. ์ฅ๊ธฐ๊ฐ MSG ๋ณต์ฉ์ ์ํ ๋์ ๋ฏธ์น๋ ์ํฅ์ ์์๋ณด๊ธฐ ์ํด, 1์ฃผ๋ alb
ino ์ฅ์๊ฒ 1๋
๊ฐ ์์์ MSG๋ฅผ ์ฒจ๊ฐํ์ฌ ๋จน์ธ ๊ทธ๋ฃน๊ณผ ๋จน์ด์ง ์์ ๋ ๊ทธ๋ฃน์ผ๋ก ๋๋์ด
์คํํด ๋ณด์๋ค. ์ฅ์ ๋ชธ๋ฌด๊ฒ์ ๋์ ๋ฌด๊ฒ, ์ด DNA์ RNA์ ๊ทธ๋ฆฌ๊ณ ๋จ๋ฐฑ์ง ์์๋ ๋ ๊ทธ๋ฃน
๊ฐ์ ์ ์ํ ์ฐจ์ด๋ฅผ ๋ณด์ด์ง ์์์ผ๋ ๋์ ์ ์ถ์ถ๋ฌผ์์ ๊ธ๋ฃจํ์ฐ ๋๋๋ฅผ ๋น๊ตํ ๊ฒฐ๊ณผ,
MSG๋ฅผ ๋จน์ธ ๊ทธ๋ฃน์์๋ ๋์กฐ๊ตฐ์ ๋นํด 2๋ฐฐ์ ๋ ๋๊ฒ ๋ํ๋ฌ๋ค. ๋ํ, ๊ธ๋ฃจํ์ฐ ๋ถํดํจ์(
GDH)์ mRNA ์์ ๋ ๊ทธ๋ฃน๊ฐ์ ์ ์ํ ์ฐจ์ด๋ฅผ ๋ณด์ด์ง ์์์ผ๋, western blot ๊ฒ์ฌ๋ฅผ ์ค
์ํ ๊ฒฐ๊ณผ ํจ์์ ์์ MSG๋ฅผ ๋จน์ธ ๊ทธ๋ฃน์ด ํ์ ํด ๊ฐ์ํ ๊ฒ์ผ๋ก ๋ณด์, GDH ๋ฐํ์ ์์ด
ํ์ ์ฌ ์กฐ์ ์ ์ํฅ์ ๋ฏธ์น๊ฑฐ๋ GDH์ ๋ถํด์จ์ ์ฆ๊ฐ์ํค๋ ๊ฒ์ผ๋ก ๋ณด์ฌ์ง๋ค. ์ด ๊ฒฐ๊ณผ๋ก,
์ฅ๊ธฐ๊ฐ MSG๋ฅผ ์ญ์ทจํ๊ฒ ๋๋ฉด ์ฅ ๋ ์์ ์กด์ฌํ๋ GDH ํ์ฑ์ ๊ฐ์์ํด์ผ๋ก ๊ธ๋ฃจํ์ฐ์
๋ถํด๊ฐ ์ ํ๋จ์ ์ ์ ์์๋ค.
๋ค์์ผ๋ก, GDH์ ๊ตฌ์กฐ์ , ๊ธฐ๋ฅ์ ์กฐ์ ํํ๋ฅผ ์ฌ๋ ์๊ฒ ์ฐ๊ตฌํ๊ธฐ ์ํด 1557-base-pair์
์ธ๊ฐ GDH ์ ์ ์๋ฅผ ํฉ์ฑํ์ฌ ๋์ฅ๊ท ์ฃผ๋ฅผ ์ด์ฉํด ํ์ฑ์ ์ง๋ ํจ์๋ก ๋ฐํ์์ผฐ๋ค. ์ด๋ ๊ฒ
๋ฐํ๋ ํจ์๋ ์ฌ๋๊ณผ ์์ ์กฐ์ง์์ ์ถ์ถํ ํจ์์ ์ฌ๋ฌ ๊ฐ์ง ์ํํ์ ์ธก๋ฉด์์ ์ ์ฌ
ํ ์์์ ๋ณด์๋ค. ์๋ฆฌ์ง์ ๋์ฐ๋ณ์ด๋ฐฉ๋ฒ์ ์ด์ฉํด ์ด ํจ์์ Lys130 ์์น๋ฅผ ๋ค๋ฅธ ์๋ฏธ
๋
ธ์ฐ์ผ๋ก ๋ฐ๊พผ ๋์ฐ์ฒด๋ฅผ ์ด์ฉํด ์คํํด ๋ณธ ๊ฒฐ๊ณผ, Lys130 ์์น๋ GDH์ ๊ธฐ์ง์ด๋ ๋ค๋ฅธ ์กฐ
ํจ์๊ฐ ๊ฒฐํฉํ๋ ๊ณณ์ด ์๋ ์ด๋งค์์ฉ์ ์ผ์ผํค๋๋ฐ ๊ด์ฌํ๋ ์ค์ํ ๊ณณ์์ ์๊ฒ ๋์๋ค.
๋ค์์ผ๋ก, ์ธ๊ฐ GDH์ ์๋ก์คํ
๋ฆญ ํ์ฑ์ ์ธ ADP์ ์กฐํจ์์ธ NAD^+ ๊ฒฐํฉ๋ถ์๋ฅผ ๊ท๋ช
ํ๊ธฐ
์ํด, ๊ฐ๊ฐ ์ด์ ํด๋น๋๋ Tyr187๊ณผ Glu279๋ถ์์ ๋ํด ๋์ฐ๋ณ์ด๋ฅผ ์ผ์ผ์ผ ์๋ฏธ๋
ธ์ฐ์ ํฌ
๊ธฐ์ ์์์ฑ, ๊ณ๊ฐ์ง์ ์ด์จํ ์ ๋์ ๋ฐ๋ผ ๋ค์ํ ๋์ฐ์ฒด๋ค์ ๋ง๋ค์ด ์คํํด ๋ณด์๋ค. GD
H์ ์์ฐํ์ ADP์ ๋ฐ์์ํค๋ฉด 3๋ฐฐ์ ๋ ํ์ฑ๋๊ฐ ์ฆ๊ฐ๋ ๋ฐ๋ฉด ADP ๊ฒฐํฉ๋ถ์์ ๊ฐ ๋์ฐ์ฒด
ํจ์๋ค(Tyr187 ๋์ฐ์ฒด)์ ํ์ฑ์ ์ ์ํ ์ฐจ๋ฅผ ๋ณด์ด์ง ์์๋ค. ์ ์ง์ํ์ ์ญํ์น์๋ฅผ ๊ตฌ
ํ ๊ฒฐ๊ณผ Tyr187 ๋์ฐ์ฒด ํจ์๋ค์, 2-oxoglutarate์ NADH์ ๋ํ K_m ๊ฐ์๋ ์ ์ํ ์ฐจ์ด
๋ฅผ ๋ณด์ด์ง ์์์ผ๋ V_(max) ๊ฐ์๋ ์์ฐํ์ ๋นํด 4๋ฐฐ ์ ๋ ๊ฐ์๋ ๊ฒฐ๊ณผ๋ฅผ ์ป์๋ค. Glu2
79 ๋์ฐ์ฒด ํจ์๋ค์ NAD^+ ์ ๊ธ๋ฃจํ์ฐ์ ๋ํด V_(max) ๊ฐ์ 22 ~ 28๋ฐฐ ์ ๋ ๊ฐ์ํ ๋ฐ๋ฉด
K_m ๊ฐ์ NAD^+์ ๋ํด์๋ง 20 ~ 25๋ฐฐ ์ ๋ ๊ฐ์ํ์๋ค. ADP์ NAD^+ ๊ฒฐํฉ๋ถ์๋ฅผ ๊ท๋ช
ํ
๊ธฐ ์ํด [ฮฑ-^(32)P]8-azidoadenosine 5โฒ-diphosphate (8NโADP)์ [^(32)P]nicotinamid
e 2-azidoadenosine dinucleotide (2NโNAD^+)๋ฅผ ์ด์ฉํ์ฌ ๊ดํก์ฐฉํ์ง๋ฅผ ์ค์ํ ๊ฒฐ๊ณผ GDH
์ ์์ฐํ์ ๋ํ [ฮฑ-^(32)P]8NโADP์ [^(32)P]2NโNAD^+์ ๊ดํก์ฐฉ ํฌํ๋์ ๋ํ K_d
๊ฐ์ 25 ฮผM ๊ณผ 55 ฮผM๋ก ๋ํ๋ฌ๋ค. ์ด๋, ์์ฐํ์ ๊ฒฝ์ฐ์๋ ADP์ NAD^+๋ฅผ ์ฒจ๊ฐํ๋ฉด 8N
โADP์ 2NโNAD^+์ ๊ดํก์ฐฉ์ด ํ์ ํ ๊ฐ์๋์์ผ๋, Tyr187์ Glu279 ๋์ฐ์ฒด๋ค์ ๊ฐ๊ฐ 8
NโADP ๋ฐ 2NโNAD^+์ ๋ฐ์ํ์ง ์์๋ค. ์ฆ, ์ธ๊ฐ GDH ์ ์ ์์ ๋์ฐ๋ณ์ด์ ๊ดํก์ฐฉํ์ง
๋ฒ์ ํตํด GDH์ Tyr187๊ณผ Glu279 ์์น๋ ๊ฐ๊ฐ ADP์ NAD^+์ ์ผ๊ธฐ๊ฐ ๊ฒฐํฉํ๋ ๋ถ์์์
์ฆ๋ช
ํ ์ ์์๋ค.
๋ง์ง๋ง์ผ๋ก, GDH ๊ฒฐํ์ผ๋ก ์ ๊ฒฝํดํ์ฑ ์งํ์ด ์๊ธด ํ์์๊ฒ ๋ถ์กฑํ GDH๋ฅผ ๋จ๋ฐฑ์ง ์ฐจ์
์์ ๋ณด์ถฉํ๊ธฐ์ํ ์๋ก์ด ๋ฐฉ๋ฒ์ ์๋ํ์๋ค. ์ด๋ฅผ ์ํด์ ์ธ๊ฐ GDH ์ ์ ์์ ์ธ๊ฐ ๋ฉด์ญ
๊ฒฐํ ๋ฐ์ด๋ฌ์ค์ ์กด์ฌํ๋ TAT ๋จ๋ฐฑ์ง์ ์ ์ด ๋ถ์๋ฅผ ์ตํฉ ์์ผ TAT-GDH ์ตํฉ ๋จ๋ฐฑ์ง์
๋ฐํ์์ผฐ๋ค. ์ด๋ ๊ฒ ์ป์ด์ง TAT-GDH ์ตํฉ ๋จ๋ฐฑ์ง์ ๋ณ์ฑ ์์ผ ์ ๊ฒฝ์ธํฌ ๋ฐฐ์์ก์ ๋ถ์ฃผํ
๊ฒฐ๊ณผ ์ ๊ฒฝ์ธํฌ ๋ด๋ก ์ ์นจํฌํ์๊ณ , ์ผ๋จ ์ธํฌ ๋ด๋ก ๋ค์ด๊ฐ๊ฒ ๋๋ฉด ๋ณ์ฑ๋ ๋จ๋ฐฑ์ง์ด ๋ค์
์ ํํ๋ฅผ ๊ฐ์ถ๊ฒ ๋์ด ์ ์์ ์ธ ํจ์ ํ์ฑ์ ๊ฐ์ง๊ฒ ๋์๋ค. ์ฆ, TAT ์๊ธฐ๋ฅผ GDH์ ์ฒจ
๋ถํ์ฌ๋ GDH ํ์ฑ์๋ ๋ณ ์ํฅ์ ๋ฏธ์น์ง ์์ผ๋ฉฐ, ์ธํฌ ๋ด ๋
์ฑ๋ ๋ํ๋ด์ง ์์์ ์๊ฒ
๋์๋ค. ์ด๋ฌํ ๊ฒฐ๊ณผ๋ GDH ๊ฒฐํ์ผ๋ก ์ธํด ์ผ๊ธฐ๋ ์ฌ๋ฌ ์งํ์ ์๊ณ ์๋ ํ์์๊ฒ GDH๋ฅผ
์ง์ ์ ๋ฌํ ์ ์๋ ์๋ก์ด ๋ฐฉ๋ฒ์ ์ ์ํด ์ฃผ๊ณ ์๋ค๊ณ ์ฌ๋ฃ๋๋ค.
--------------------
ํต์ฌ์ด: ๊ธ๋ฃจํ์ฐ ๋ถํดํจ์, ๊ธ๋ฃจํ์ฐ๋ํธ๋ฅจ, ๋์ฐ๋ณ์ด, ADP ๊ฒฐํฉ๋ถ์, NAD^+ ๊ฒฐํฉ๋ถ์,
์ด๋งค์์ฉ๋ถ์, ๊ดํก์ฐฉํ์ง๋ฒ, TAT ์ตํฉ ๋จ๋ฐฑ์ง, ๋จ๋ฐฑ์ง ์น๋ฃ, ํดํ์ฑ ์ ๊ฒฝ์งํ, ์ ์ ์
๊ณตํ, ๋จ๋ฐฑ์ง ๊ณตํ
Structure, Allosteric Regulation, and Reaction Mechanism of Glutamate Dehydrogenase
Glutamate in the form of its sodium salt (MSG; monosodium glutamate) is a widely
used food additive and reported to be a cause of the Chinese restaurant syndrome.
To investigate a long-term effect of MSG ingestion on the brain, one-week-old
albino rats were kept for 1 year in equal groups with or without MSG in their
drinking water. The concentrations of the glutamate in the brain crude extracts of
the MSG treated group were 2-fold higher than those of the control group. There
were no significant change in body weight, brain weight, and contents of protein,
total RNA and DNA in the two groups of rats. The concentration of the enzyme on the
western blot analysis was significantly decreased in the MSG treated group, whereas
the level of GDH mRNA remained unchanged, suggesting a post-transcriptional control
of the expression of GDH or an increased rate of degradation of enzyme protein.
These results indicate that the prolonged MSG feeding reduces the activity of GDH
and subsequently decreases the catabolism of glutamate in rat brain.
To gain a deeper insight into the structural and regulatory basis of GDH, a
1557-base-pair gene that encodes human GDH has been synthesized and expressed in
Escherichia coli as a soluble protein. The recombinant enzymes were
indistinguishable in its biochemical properties from those isolated from human and
bovine tissues. The results from the site-directed mutagenesis of Lys130 site
indicate that Lys130 plays an important role in the catalysis of GDH and that
Lys130 is an essential residue required for the catalysis of GDH but not for the
substrate or coenzyme binding.
To identify ADP (allosteric activator) and NAD^+ (coenzyme) binding sites within
human GDH, a series of cassette mutations at Tyr187 and Glu279 positions were
constructed for ADP and NAD^+ binding, respectively. The wild type GDH was
activated up to 3-fold by ADP, whereas no significant activation by ADP was
observed with the Tyr187 mutant GDH regardless of their size, hydrophobicity, and
ionization of the side chains. Studies of the steady-state velocity of Tyr187
mutant enzymes revealed essentially unchanged apparent K_m values for
2-oxoglutarate and NADH, but an approximately 4-fold decrease in the respective
apparent V_(max) values. The Glu279 mutant proteins showed a 22 ~ 28-fold decrease
in the respective apparent V_(max) values and 20 ~ 25-fold increase in K_m values
for NAD^+ with essentially unchanged K_m values for glutamate. The identification
of the ADP and NAD^+ binding sites was further performed using photoaffinity
labeling with [ฮฑ-^(32)P]8-azidoadenosine 5โฒ-diphosphate (8NโADP) and
[^(32)P]nicotinamide 2-azidoadenosine dinucleotide (2NโNAD^+). Saturation of
photoinsertion with [ฮฑ-^(32)P]8NโADP and [^(32)P]2NโNAD^+ occurred apparent K_d
values near 25 ฮผM and 55 ฮผM, respectively, for wild type GDH. The photoinsertions
of 8NโADP and 2NโNAD^+ were significantly decreased by ADP and NAD^+,
respectively. Unlike wild type GDH, none of the mutant enzymes at Tyr187 or Glu279
was able to interact with 8NโADP and 2NโNAD^+, respectively. The results with
cassette mutagenesis and photoaffinity labeling indicate that the Tyr187 and Glu279
are required for efficient base-binding of ADP and NAD^+ to GDH, respectively.
In an effort to replenish the GDH activity in the patients with the GDH-deficient
neurodegenerative disorders, human GDH was transduced into PC12 cells by fusing
with a gene fragment encoding the protein transduction domain of human
immunodeficiency virus TAT protein to produce genetic in-frame TAT-GDH fusion
protein. The TAT-GDH protein can enter PC12 cells efficiently when added
exogenously in culture media. Once inside the cells, the transduced denatured
TAT-GDH protein showed a full activity of GDH indicating that the TAT-GDH fusion
protein was correctly refolded after delivery into cells and the activities of GDH
in the TAT-GDH fusion protein was not affected by the addition of the TAT sequence.
TAT-GDH showed no cytotoxicity as determined by the ability to inhibit protein
synthesis. These results may suggest new possibilities for direct delivery of GDH
into the patients with the GDH-deficient disorders.ope
ํ์ฒ ์ง์ญ์ด์ ์์ด๋ก ์ ์ฐ๊ตฌ
ํ์๋
ผ๋ฌธ(์์ฌ)--์์ธ๋ํ๊ต ๋ํ์ :๊ตญ์ด๊ตญ๋ฌธํ๊ณผ ๊ตญ์ดํ์ ๊ณต,2001.Maste
์๋ก์ด ํ๊ด ๋จ๋ฐฑ์ง ํ์นจ : ๋ถ์ ์ผ๊ฐ์ฒด
Thesis(master`s)--์์ธ๋ํ๊ต ๋ํ์ :ํํ๋ถ ์ ๊ธฐํํ์ ๊ณต,2005.Maste
้ๅ็ฃ ํฅ๋๋ฌด ๋ น๋ณ๊ท (Gymnosporangium)์ ๅฝขๆ ็ ็นๅพต, ไบบๅทฅๆฅ็จฎ, ๅๅญ็็ฉๅญธ์ ๏ง็จํ ๅๅฎ, ๅฅไธป็น็ฐๆง ๋ฐ ็ณป็ตฑๅ้ก์ ๊ดํ ็ก็ฉถ
Thesis (master`s)--์์ธ๋ํ๊ต ๋ํ์ :์ฐ๋ฆผ์์ํ๊ณผ,2003.Maste
Photoaffinity labeling์ ์ด์ฉํ ๋์กฐ์ง glutamate dehydrogenase isoproteins์ ADP์ NAD+ ๊ฒฐํฉ๋ถ์ ๋ถ์
The Graduate School Yonsei University/์์ฌ[ํ๊ธ]
๋ณธ ์ฐ๊ตฌ์์๋ ์ ๋์กฐ์ง์ผ๋ก๋ถํฐ ๋ถ๋ฆฌ ์ ์ ํ 2๊ฐ์ง glutamate dehydngenase isoproteins (GDH โ
๋ฐ GDH โ
ก)์ ADP ๊ฒฐํฉ๋ถ์์ ์๋ฏธ๋
ธ์ฐ ์์ด์ ๊ท๋ช
ํ๊ธฐ ์ํด [ฮฑ**32P] 8N-azidoadenosine 5-diphosphate (8N^^3 ADP)๋ก photolabeling ์ ํ์๋ค. Photolysis ๋ฅผ ์ํค์ง ์์์ ๋, 8N^^3 ADP์ ์ ์ฌํ๊ฒ GDHโ
๊ณผ GDHโ
ก ์ ํ์ฑ์ ์ฆ๊ฐ์์ผฐ๋ค. Trypsin์ ์ฒ๋ฆฌํ ํ, photolabel์ ํฌํจํ๊ณ ์๋ peptide๋ฅผ immobilized aluminum affinity chromatography์ reversed-phase HPLC๋ฅผ ์ด์ฉํ์ฌ ๋ถ๋ฆฌํ์์ผ๋ฉฐ, GDHโ
๊ณผ GDHโ
ก์ ๊ฒฐ๊ณผ๋ ๊ฐ๊ฐ E-M-S-W-I-A-D-T-Y-A-S-T-I-G-H-Y-D๊ณผ E-M-S-W-I-A-D-T-Y-A-S-T-I-G-H-Y-D-I-N ์ผ๋ก ๋์๋ค. ํํธ 1mM ADP์ ์กด์ฌํ์์ photolysis๋ฅผ ์ํฌ ๊ฒฝ์ฐ, peptide์ photolabeling์ด 90%์ ๋ ๊ฐ์ํ์์ผ๋ ๋ค๋ฅธ nucleotides๋ฅผ ์ฌ์ฉํ ๊ฒฝ์ฐ์๋ ADP์ฒ๋ผ ํจ๊ณผ์ ์ผ๋ก photoinsertion ์ ์์ ๊ฐ์์ํค์ง ๋ชปํ์๋ค. ์ด๋ฌํ ๊ฒฐ๊ณผ๋ก ๋ฏธ๋ฃจ์ด ADP ๊ฒฐํฉ๋ถ์์ ๋ํ photoprobedml ํน์ด์ฑ์ ์
์ฆํ๊ฒ ๋์๊ณ , ๋ณธ ์คํ์์ ์ฌ์ฉํ photoprobe์ธ photoprobe์ธ [ฮฑ**32 P] 8N^^3 ADP ๋ก ํ์ธํ peptide๊ฐ ๋์กฐ์ง GDH isoproteinsdml ADP ๊ฒฐํฉ domain์ ์์นํจ์ ์ ์ ์์ด๋ค. ๋ํ [(32)**P] nicotinamide 2-azidoadenosine diphosphate (2N^^3 NAD**+) ์ photoaffinity probe๋ก ์ฌ์ฉํ์ฌ ์ญ์ ์ ๋์กฐ์ง์์ ๋ถ๋ฆฌํ GDHโ
๊ณผ GDHโ
ก์ NAD**+ ๊ฒฐํฉ๋ถ์์ ๋ํ์ฌ๋ ์กฐ์ฌํ์๋ค. photolysis๋ฅผ ์ํค์ง ์์์ ๋, 2N^^3 NAD**+๋ NAD**+์ ์ ์ฌํ๊ฒ GDH isoproteinsdml ๊ธฐ์ง๋ก์ ์์ฉํ์๋ค. Probedml photoinsertion ์ GTP๋ Glutarate ์ํด์๋ ์ฆ๊ฐํ๊ณ NAD**+๋ ADP์ ์ํด์๋ ๊ฐ์ํ์๋ค. Trypsin ์ฒ๋ฆฌ ํ immobilized boronate affinity chromatography์ reversed-phase HPLC ๋ก photolabel-containing peptides๋ฅผ ๋ถ๋ฆฌํ์๋ค. ๊ทธ ๊ฒฐ๊ณผ๋ GDH isoproteins ๋ ๋ค ๋์ผํ๊ฒ C-I-A-V-G-X-S-D-G-S-I-W-N-P-D-G-I-D-P-K ๋ก ๋์๋ค. ์ด๊ฒ์ ์๋ฏธ๋
ธ์ฐ ์์ด์ด ์ ๋ฐํ์ง ์ ๊ฐ์กฐ์ง GDH์ Cys**270 ์์ Lys**289 ์ ์์น์ ์ผ์นํจ์ ์๊ฒ ๋์๋ค. ์ฌ๊ธฐ์, 'X'๋ ๋จ๋ฐฑ์ง sequencing ๊ณผ์ ์์ ๋ฏธํ์ธ ์๋ฏธ๋
ธ์ฐ์ผ๋ก ๋ถ์๋ ๊ฒ์ธ๋ฐ ๊ธฐ์กด์ ์๋ ค์ง GDH ์ ์๋ฏธ๋
ธ์ฐ ์์ด๊ณผ ๋น๊ตํด ๋ณผ ๋, photolabeled glutamate ์์ ์ ์ ์์๋ค. NAD**+ ์กด์ฌํ์์ photolysis๋ฅผ ์ํจ ๊ฒฝ์ฐ, peptide์ photolabeling ์ด ๊ฐ์ํ๋ ๊ฒ์ผ๋ก ๋ณด์ photoprobe์ NAD**+ ๊ฒฐํฉ๋ถ์๋ด์ ํน์ด์ฑ์ ์
์ฆํ ์ ์์๊ณ , ์ฌ์ฉํ photoprobe ๊ฐ ๋์กฐ์ง GDH isoproteins์ NAD**+ ๊ฒฐํฉ domain์ ์์นํจ์ ์ ์ ์์๋ค.
[์๋ฌธ]
Photoffinity labeling with [ฮฑ**32P]8-azidhosphate (8N^^3 ADP) was used to identify the ADP binding site with two types of glutamate dehyrogenase isoproteins (GDH โ
and GDH โ
ก) isolated from bovine brain, 8N^^3 ADP, without photolysis, mimicked the activator properties of ADP on GDH โ
and GDH โ
ก sctivities, although maximal activity with 8N^^3 ADP was about 75% of maximal ADP - stimulated activity. Saturation of photoinsertion with [ฮฑ**32 P] 8N^^3 ADP occurred at around 40-50ฮผm photoprobe with apparent K^^4 values near 25 ฮผm and 40ฮผm for GDHโ
and GDHโ
ก,
respectively. Photoinsertion of [ฮฑ**32 P] 8N^^3 ADP was decreased best by ADP in comparison to other nucleotides. With the combination of immobilized alumminum affinity chromatograph and reversed-phase high performance liquid chromatography (HPLC), photolabel-containing peptides generated by tryptic digestion were isolated This identified a portion of the adenine ring binding domain of GDH isoproteins as region containing the sequence, E-M-S-W-I-A-D-T-Y-A-S-T-I-G-H-D and E-M-S-W-I-A-D-T-Y-A-S-T-I-G-H-Y-D-I-N for GDHโ
and GDHโ
ก, respectively. Photolabeling of the peptide was prevented over 90% by the presence of 1 mM ADP. These results demonstrate selectivity of the photoprobe for ADP binding site and suggest that the peptide identified using the photoprobe islocaed in the ADP binding domain of the brain GDH ispproteins. Another photoaffinity probe, [(32)**P] nicotinamide 2 - azidoadenosine dinucleoteins (2N^^3 NAD**+), was also used to identify the NAD**+ binding site within ghnamate dehydrogenase isoproteins(GDHโ
and GDHโ
ก) isolated from bvine brain. In the absence of photolysis, 2N^^3 NAD**+ was a substrate for the GDH isoproteins. When the enzymes were covalently modified by photolysis in the persence of saturating amounts of photoprobe, about 50% inhibition of the GDH activites was oberved. Photoinsertion of probe was increased by GTP or glutarate and decreased by NAD**+ or ADP. With the combination of
immobilized boronate affinity chromatography and reversed-phase HPLC, photolabel-containing peptides generated with trypsin were isolated This identified a portion of the adenine ring binding domain of GDH isoproteins as the region containing the sequence, C-I-A-V-G-X-S-D-G-S-I-W-N-P-D-G-I-D-P-K for both GDH isoproteins, corresponding to Cys**270 through Lys**289 of the amino acid sequence of well known bovine liver GDH. The symbol X indicate a position for which no phenylthiohydantoin-amino acid could be assigned. The missing residue, however, can be designed as a photolabeled glutamate since the sequences inchuding the glutamate residue in question have a complete identity with those of NAD**+ during photolysis. These results demonstrate selectivity of the photoprobe for the NAD**+ binding site and suggest that the peptide identified using the photoprobe is located is located in the NAD**+ binding domain of the brain GDH isoproteins. Both amino acid sequencing and compositional analysis identified Glu**275 as the site of photoinsertionrestrictio
Development of a nursing intervention protocol based on nursing diagnosis of critical medical-surgical patients.
๊ฐํธํ๊ณผ/์์ฌ[ํ๊ธ]
21์ธ๊ธฐ ๊ฐํธ๋ ๋
์์ ์ธ ์ค๋ฌด๋ฅผ ํตํ ์ ๋ฌธ์ง ๊ฐํธ๋ฅผ ์๊ตฌํ๋ค. ๊ฐํธ์ง๋จ๊ณผ ๊ฐํธ์ค์ฌ๋ฅผ ์ฐ๊ณํ ๊ฐํธ์ค์ฌ ํ๋กํ ์ฝ์ ํ์ค์ ์ธ ๊ฐํธํ๋์ ์ ์ํ๊ณ ๊ฐํธ๋ฌธ์ ์ ๋ฐ๊ฒฌ์์ ์ง๋จ, ์ํํ๋ ๋ชจ๋ ํ๋์ ๋
์์ ์ผ๋ก ์ํํ๊ธฐ ์ํ ๋๊ตฌ๋ก ์ฌ์ฉ๋ ์ ์๋ค. ๋ฐ๋ผ์ ๋ณธ ์ฐ๊ตฌ์ ๋ชฉ์ ์ ๋ด์ธ๊ณผ ์คํ์์๊ฒ ํ์ํ ๊ฐํธ์ง๋จ์ ํ์
ํ๊ณ ์ด์ ๋ฐ๋ฅธ ๊ฐํธ์ค์ฌ ๋ฐ ํ๋์ ๊ท๋ช
ํจ์ผ๋ก์, ๋ด์ธ๊ณผ ์คํ์ ๊ฐํธ์ํ์ ์ง์นจ์ด ๋๋ ๊ฐํธ์ค์ฌ ํ๋กํ ์ฝ์ ๊ฐ๋ฐํ๋ ๊ฒ์ด๋ค.
์ฐ๊ตฌ๋ฐฉ๋ฒ์ ์์ธ ์์ฌ Y๋ํ ๋ถ์ ๋ณ์ ์คํ์์ค 5๊ณณ์ ๊ทผ๋ฌด ์ค์ธ ๊ฐํธ์ฌ ์ค ์คํ์์ค 3๋
์ด์์ ๊ฐํธ๊ฒฝ๋ ฅ์ ๊ฐ์ง 55๋ช
์ ๊ฐํธ์ฌ๋ฅผ ๋์์ผ๋ก, North American Nursing Diagnosis Association(NANDA)์ ๊ฐํธ์ง๋จ์ ๊ธฐ๋ณธ์ผ๋ก ํ ์ค๋ฌธ์ง๋ฅผ ํตํด ์ฑ์ธ ๋ด์ธ๊ณผ ์คํ์์๊ฒ ํ์ํ ๊ฐํธ์ง๋จ์ ๋ด์ฉํ๋น๋ ๊ฒ์ฆ์ ๊ฑฐ์ณ ์ ์ ํ์๋ค.
์ ์ ๋ ๊ฐํธ์ง๋จ์ ๋ฐ๋ผ Nursing Intervention Classification(NIC)์์ ์ ์ํ ๊ฐํธ์ค์ฌ๋ฅผ ๊ธฐ์ด๋ก, ์ ๋ฌธ๊ฐ ์ง๋จ์ด ๋ชจ๋ ํฉ์ํ ๊ฐํธ์ค์ฌ์ ์ฐ์ ์ค์ฌ๋ฅผ ๊ฐํธ์ค์ฌ๋ก ์ ์ ํ์๋ค.
์ ๋ฌธ๊ฐ ์ง๋จ์ 55๋ช
์ ๊ฐํธ์ฌ ์ค ์คํ์ ๊ฐํธ๊ฒฝ๋ ฅ 5๋
์ด์ ๋ ๊ฐํธ์ฌ์ ๊ฐํธ๋ํ ๊ต์ 3์ธ์ผ๋ก ๊ตฌ์ฑํ์๋ค.
์ ์ ๋ ๊ฐํธ์ค์ฌ์ ๋ํ ๊ฐํธํ๋๋ชฉ๋ก์ NIC์์ ์ ์ํ ๊ฐํธํ๋ ๋ชฉ๋ก์ ํ๊ธ์์ ์์
์ ๊ฑฐ์ณ ์๋น ๊ฐํธ์ค์ฌ ํ๋กํ ์ฝ๋ก ์์ฑํ๊ณ ์ด์ ๋ํ ์ ๋ฌธ๊ฐ ์ง๋จ์ ๋ด์ฉํ๋น๋ ๊ฒ์ฆ๋จ๊ณ๋ฅผ ๊ฑฐ์น ํ ์ต์ข
๋ด์ธ๊ณผ ์คํ์ ๊ฐํธ์ค์ฌ ํ๋กํ ์ฝ์ ๊ฐ๋ฐํ์๋ค.
๋ณธ ์ฐ๊ตฌ์ ๊ฒฐ๊ณผ๋ ๋ค์๊ณผ ๊ฐ๋ค.
1. ๋ด์ธ๊ณผ ์คํ์์๊ฒ ํ์ํ ๊ฐํธ์ง๋จ์ CVI(Index of Content Validation) .90์ด์์ ์ ์๋ฅผ ๋ฐ์ ๊ฐ์ผ์ํ์ฑ, ๊ธฐ๋ ๊ฐ๋ฐฉ ์ ์ง๋ถ๋ฅ, ๊ธฐ๋ ํก์ธ ์ํ์ฑ, ๋นํจ์จ์ ํธํก ์์, ํต์ฆ, ์กฐ์ง๊ด๋ฅ๋ณํ, ๊ฐ์ค๊ตํ ์ฅ์ , ์ฒด์ก ๋ถ์กฑ, ํผ๋ถ ์์ ์ํ์ฑ, ํธํก๊ธฐ๋ฅ ๋ถ์กฑ ๋ฑ 10๊ฐ๊ฐ ์ ์ ๋์๋ค.
2. 10๊ฐ์ ๊ฐํธ์ง๋จ์ ๋ฐ๋ฅธ ๊ฐํธ์ค์ฌ๋ NIC์์ ์ ์ํ ๊ฐํธ์ค์ฌ ์ค ์ ๋ฌธ๊ฐ๊ฐ ๋ชจ๋ ํฉ์ํ 32๊ฐ์ ๊ฐํธ์ค์ฌ์ 45๊ฐ์ ์ฐ์ ์ค์ฌ๋ฅผ ํฉํด ์ด 67๊ฐ์ ๊ฐํธ์ค์ฌ๋ฅผ ์ ์ ํ์๋ค.
3. ๊ฐํธํ๋์ ์๋น๊ฐํธ์ค์ฌ ํ๋กํ ์ฝ๋ก ์ ๋ฌธ๊ฐ์ง๋จ์ ๋ด์ฉ ํ๋น๋ ๊ฒ์ฆ์ ์ค์ํ ๊ฒฐ๊ณผ CVI๋ฅผ ํตํด .83์ด์์ ์ ์๋ฅผ ๋ฐ์ 979๊ฐ์ ๊ฐํธํ๋์ผ๋ก ์ ์ ํ์๋ค. .83์ด์์ ๊ฐํธํ๋์ด ์๋ ํผ๋ถ์์ ์ํ์ฑ์ ์ง๋จ์ ๋ํ ์ด๋์ฆ์ง์ด๋ผ๋ ์ค์ฌ๋ ํ๋กํ ์ฝ์์ ์ญ์ ํ์๋ค.
4. ๊ทธ ๊ฒฐ๊ณผ, 10๊ฐ์ ๊ฐํธ์ง๋จ, 66๊ฐ์ ๊ฐํธ์ค์ฌ, 979๊ฐ์ ๊ฐํธํ๋์ผ๋ก ๊ตฌ์ฑ๋ ์ต์ข
๋ด์ธ๊ณผ ์คํ์ ๊ฐํธ์ค์ฌ ํ๋กํ ์ฝ์ ๊ฐ๋ฐํ์๋ค.
์ด๋ฌํ ๋ด์ธ๊ณผ ์คํ์ ๊ฐํธ์ค์ฌ ํ๋กํ ์ฝ์ ๊ฐํธ์ฌ์ ์์ฌ๊ฒฐ์ ์ ๋์ ์ผ๊ด์ฑ์๊ณ ๊ณผํ์ ์ธ ๊ฐํธ๋ฅผ ์ ๊ณตํ ์ ์์ผ๋ฉฐ, ์ง์์ ์ธ ๊ฐ๋ฐ์ ํตํด ๊ฐํธ์ง๋จ๊ณผ ์ค์ฌ์ ์ ์ฐํ ๋ฐ ์ ๋ฌธ์ง์ผ๋ก์ ์์์ ๋์ด๋ ๋ฐ ๊ธฐ์ฌํ ์ ์๋ค๊ณ ๋ณธ๋ค.
[์๋ฌธ]
Professional nurses in the 21st century use research to promote independent practice and to link nursing diagnoses and nursing interventions. In this study a nursing intervention protocol for critical medical-surgical patients which can be used for instruction in nursing activities was to developed. Understanding of priority nursing diagnoses and identification of related nursing interventions and activities is an important link for nursing.
The participants were 55 nurses with more than 3 years experience in one of five ICUs in Y medical center in Seoul. Through a survey based on the nursing diagnoses of the North American Nursing Diagnosis Association, content validity of the
nursing diagnoses for critical medical-surgical patients was verified.
For these nursing diagnoses and according to the nursing interventions suggested by Nursing Intervention Classification(NIC), priority nursing interventions were
established and agreed upon by a team of experts. The experts were nurses with careers in ICU of 5 years or more and 3 professors of nursing.
Following translation and updating of the NIC nursing activity list, a nursing activity list for the established nursing interventions was developed as the preliminary nursing intervention protocol. After verification for content validity
by the expert group, a final nursing intervention protocol for critical medical-surgical patients was developed.
A summary of the result of this study is presented below.
1. Nursing diagnoses for these patients were established as those which had a CVI (Index of Content Validation) of .90 or higher. Ten diagnoses met the criteria, Risk for infection, Ineffective airway clearance, Risk for aspiration, Ineffective
breathing pattern, Pain, Altered tissue perfusion, Impaired gas exchange, Fluid volume deficit, Risk for impaired skin integrity, and Inability to sustain spontaneous ventilation,
2. From the nursing interventions suggested by NIC for the 10 diagnoses 32 were selected by all of the experts. As well as these interventions, 45 priority interventions were selected for a total of 67 interventions.
3. Using the preliminary nursing intervention protocol from the expert group, nursing activities were selected through examination of content validity. A total of 979 activities having a CVI of .83 or higher were selected. The intervention, "Exercise promotion" for the nursing diagnosis "Risk for impaired skin integrity" was deleted as none of the nursing activities met the CVI criteria.
4. The final nursing intervention protocol developed for critical medical-surgical patients consisted of 10 nursing diagnoses, 66 nursing interventions, and 979 nursing activities.
Such nursing intervention protocols can help nurses make decisions and provide constant/scientific care. Through continuous development, they will also provide a computerized system of nursing diagnoses and interventions and improve the personal capability of nurses to be professional.ope