흰쥐 두개관 및 간에서 Purine염기의 대사적 운명

김관식,민병무,김세원,이진 대한구강생물학회 1989 International Journal of Oral Biology Vol.13 No.1

As an attempt to clarify the metabolic fate of purine bases in rat calvaria and liver, the activities of xanthine oxidase, guanine aminohydrolase and HGPRT were assayed. The portions of calvaria containing frontal and parietal bone, and liver were dissected from rats. Specimens were assayed for their activities of xanthine oxidase and guanine aminohydrolase in its cytosol and HGPRT in its mitochondrial fraction. Specific activity of hepatic xanthine oxidase with hypoxanthine or xanthine as its substrate was 4.18 or 7.43mU/mg of protein and guanine aminohydrolase was 11.00mU/mg of protein, respectively. In contrast, calvarial xanthine oxidase and guanine aminohydrolase was 1.49 or 1.88 and 0.98, respectively, which were significantly lower than those of hepatic enzymes. Specific activity of calvarial HGPRT was 21.70 and hepatic HGPRT was 13.65mU/mg of protein when hypoxanthine was employed as its substrate while no measurable activity was detected in calvaria when guanine was employed. Comparing to purine-catabolizing enzymes, activity of HGPRT, salvage-enzyme for purine bases, was relatively higher in both tissues and hypoxanthine was utilized preferentially as substrate than guanine. Besides the production of GMP or IMP by HGPRT, concentration of other purine nucleotides such as GMP from guanine or adenylates form hypoxanthine were also increased in hepatic HGPRT assay but no detectable changes in calvaria. Assuming these increments as the consequences of HGPRT activity, it can be deduced that activity of HGPRT in liver would be greater than that of calvaria. Taken together, these results indicated thet ⅰ) in calvaria, most of purine bases would be reutilized by salvage enzymes rather than completely catabolized ⅱ) both of catabolic pathway to final metabolites and salvage pathway for purines would be active as well as de novo synthesis in liver ⅲ) metabolic fate of purine bases varied from tissue to tissue within same species.

Lidocaine이 흰쥐 해마박편에서 Gamma-Aminobutyric Acid 유리에 미치는 영향에 관한 연구

김형룡,김관식,정동균 대한구강생물학회 1988 International Journal of Oral Biology Vol.12 No.2

Present study was conducted to clarify the role of GABA in lidocaine-induced convulsion employing hippocampal slice. Hippocampal slices(300-400㎛) were prepared from hippocampal tissue blocks using a glass guide while the tissue was continuously moistened with oxygenated (95% O_2-5% CO_2) Krebs-bicarbonate media (KB) at 37℃. After preparation, the slices were equilibrated for 1hr before biochemical testing in KB. Individual equilibrated slices were transferred to tissue holder, incubated for 5min in fresh oxygenated KB, and then exposed for 5min to 50mM K^+ KB and transferred again to fresh KB for 30min rest period. Each slice served as its own control by exposing it a second time to 50mM K^+ KB for 5min after the 30min rest and a second 5min incubation in fresh KB. After the 30min rest and a second 5min incubation in fresh KB, slices were transferred again to fresh KB containing 1, 2, 3, or 4mM lidocaine, incubated for 10min and then exposed to 50mM K^+ KB+lidocaine solution for 5min. The results were as follows : 1. The release of GABA induced by the first and second 5min exposure of 50mM K^+ was 95.9±17.28nmol and 80.6±13.04nmol, respectively. When compared with released amounts of GABA during the corresponding spontaneous periods, these were 4.7 and 4.9-fold increase respectively and revealed that consecutive K^+─exposure increased GABA release with similar fashion. 2. The concentration of lidocaine necessary for 50 percent inhibition(IC_50) of potassium─induced release of GABA was 1.82mM.

Ascorbic acid가 두개관 세포군의 cAMP농도에 미치는 영향

정동균,김관식,김세원 대한구강생물학회 1989 International Journal of Oral Biology Vol.13 No.1

Ascorbic acid has long been known to influence the bone formation by hydroxylating the proline and lysine residues of collagen. Although there were no direct evidences, this effect might be due to the effect on the function of osteoblasts. This study was undertaken to investigate the effect of ascorbic acid on the cAMP accumulation in the calvarial bone cell populations. Fetal rat calvaria at 19th day of gestation were sequentially digested with enzyme mixture of collagenase, trypsin and EDTA for 10, 10, 10, 20 and 20 minutes. Each calvarial bone cell population was primarily cultured for 6-7 days, and then the effects of ascorbic acid, PTH and their combination on the cAMP accumulation were determined. The results were as follows. 1. Ascorbic acid, at concentration of 100㎍/ml, had no effect on the cAMP accumulation in all bone cell populations. 2. PTH, at concentration of 0.4U/ml, increased cAMP in population I, II, IV and V and decreased cAMP in population III, but the effect of PTH was great in population IV and V. 3. Ascorbic acid plus PTH increased the cAMP in population I, II, IV and V, and showed the synergistic effect in population IV.

Ascorbic acid 가 골조직세포군의 Phosphatase 에 미치는 영향

김상균,김관식,정동균 대한구강생물학회 1987 International Journal of Oral Biology Vol.11 No.2

Several lines of findings suggest that ascorbic acid might influence the function of osteoblasts although no direct evidences were provided. This study was undertaken to investigate the effect of ascorbic acid on bone cells employing 5 fetal rat calvarial cell populations isolated by sequential enzyme digestion. Fetal rat calvaria were treated 5 times consecutively with enzyme mixture of collagenase, trypsin and EDTA for 10, 10, 10, 20 and 20 minutes. Each bone cell population was primarily cultured for 6-7 days and then the effect of ascorbic acid (10 and 100㎍/ml) on the phosphatase level were determined. The observed results were as follows. 1. Population IV and V had characteristics of osteoblast such as high alkaline phosphatase level and low acid phosphatase. 2. Ascorbic acid decreased the acid phosphatase activity of population IV, regardless its concentration while did not affect other cell populations. 3. Alkaline phosphatase activity of population IV was increased significantly by ascorbic acid. 4. Taken together, these results suggest that ascorbic acid may promote the differentiation of osteoblasts and its effect is restricted in osteoblastic population only, not in other type of bone cells.

마우스 골수세포 배양시 Prostaglandin E_2가 파골세포-유사세포 생성에 미치는 영향에 관한 연구

고성희,김관식,정동균 대한구강생물학회 1990 International Journal of Oral Biology Vol.14 No.2

Osteoclasts are primary cells responsible for bone resorption. It has been reported that osteoclasts are formed by fusion of mononuclear precursors derived from hematopoietic progenitor cells. Prostaglandin E_2(PGE_2) is known to be a potent bone resorbing agent, but its mechanisms are not known. This experiment was performed to study the effect of PGE_2 and calcitonin on the generation of osteoclasts from their precursor cells. The bone marrow cells were prepared from 7 to 9 week-old male mice. The femur and tibia were dissected aseptically and the marrow cavity was flushed with 1ml α-minimum essential medium by slow injection. The collected marrow cells were cultured at 1.5∼2.0×10^6 cells/well in 24-well plate for 5, 8 and 11 days. In experimental group, PGE_2(10^-5,10^-6,10^-7,10^-8M) and calcitonin(5, 30, 90ng/ml) were added. After cultures, staining for tartrate-resistant acid phosphatase(TRACP)-marker enzyme of osteoclast-was performed according to the modified method of Burstone. The TRACP-positive multinucleated cells, which have 3 or more nuclei were counted, The observed results were as follows. 1. In control group, TRACP-positive mononuclear cells were present, but no TRACP-positive multinucleated cells appeared. 2. In 8 days of culture, PGE_2 increased significantly the number of osteoclast-like cells in a dose-dependent manner in the mouse marrow cell cultures. 3. In 5, 8 and 11 days of culture with PGE_2(10^-6M), the number of osteoclast-like cells was increased up to 8 day and decreased thereafter. 4. In 8 days of culture, calcitonin decreased significantly the number of osteoclast-like cells induced by PGE_2(10^-6M) in a dose-dependent manner.

백서 두개관 세포군의 골형성능에 관한 연구

안중진,김관식,정동균 대한구강생물학회 1989 International Journal of Oral Biology Vol.13 No.2

To study the osteogenic capacity of rat calvarial cell populations, 5 bone cell populations were prepared from fetal rat calvaria by sequential enzyme digestion. After primary culture for 6-7 days each bone cell population was collected and resuspended at 0.5-1×10^6 cells/35mm culture dish and allowed to attach overnight. Cells were grown in α-MEM plus 15% FBS with or without 50㎍/ml ascorbic acid, 10mM β-glycerophosphate or 10^-7M dexamethasone for 23 days. After long-term culture, the cell layer was fixed with neutral buffered formalin and stained with von Kossa stain. The von Kossa positive nodules were counted. The observed results were as follows. 1. Nodule formation was begun after 7-10 days of culture and was increased with culture periods. 2. Mineralized bone nodules, positive for von Kossa stain, were formed only in population IV and V when cultured in media supplemented with 50㎍/ml ascorbic acid and 10mM β-glycerophosphate. 3. Mineralized nodule was not formed in all bone cell populations when cultured in media supplemented with ascorbic acid or β-glycerophosphate only. 4. Dexamethasone (10^-7M) increased significantly the formation of bone nodule in population IV and V. These results indicate that enzymatically released calvarial cells can form mineralized bone nodules in vitro, and along with biochemical data suggest that population IV and V express the osteoblastic phenotype.

Propranolol이 흰쥐 해마박편에서 Gamma-aminobutyric acid 및 Glutamic acid유리에 미치는 영향에 관한 연구

김경범,김관식,정동균 대한구강생물학회 1990 International Journal of Oral Biology Vol.14 No.1

Present study was performed to clarify the effect of propranolol on the release of amino acid neurotransmitters employing hippocampal slices. Hippocampal slices (300-400㎛ thick) were prepared by the method of Spencer et al.(1976) and pre-equilibrated in Krebs-bicarbonate medium (KBM, pH 7.4) for 1 hr at 37℃. Pre-equilibrated slices were incubated in fresh KBM and then veratrine (25μM)-containing KBM for 10 min period. Basal and veratrine-induced release of GABA and glutamic acid were determined from recovered medium by HPLC. After 30 min resting period, slices were reincubated in propranolol-containing KBM and propranolol plus veratrine-containing medium consecutively for 10 min period each to investigate the effect of propranolol on basal or veratrine-induced amino acid release from hippocampal slices. The observed results were as follows : 1. The release of GABA induced by the first and second 10min-exposure of 25μM veratrine was 246.9±16.82 nmol and 209.9±20.84 nmol, respectively. When compared with released amounts of GABA during the corresponding spontaneous periods, these were 7.3 and 7.3-fold increase respectively. 2. the release of glutamic acid induced by the first and second 10min-exposure of 25μM veratrine was 426.3±63.16 nmol and 377±61.83 nmol, respectively. When compared with released amounts of glutamic acid during the corresponding spontaneous periods, these were 9.9 and 10.2-fold increase respectively. 3. Propranolol (2-8 mM) inhibited veratrine-stimulated GABA release in dose-dependent manner. 4. Propranolol (2-6 mM) enhanced veratrine-stimulated and spontaneous release of glutamic acid in dose-dependent fashion.

Norepinephrine이 흰쥐 해마박편에서 아미노산 신경전달물질 유리에 미치는 영향에 관한 연구

김성룡,김관식,정동균 대한구강생물학회 1990 International Journal of Oral Biology Vol.14 No.1

Present study was performed to clarify the effect of norepinephrine on the release of amino acid neurotransmitters employing hippocampal slices. Hippocampal slices (300-400㎛ thick) were prepared by the method of Spencer et al.(1976) and pre-equilibrated in Krebs-bicarbonate medium (KBM, pH 7.4) for 1 hr at 37℃. Pre-equilibrated slices were incubated in fresh KBM and then veratrine (25μM)-containing KBM for 10 min period. Basal and veratrine-induced release of GABA and glutamic acid were determined from recovered medium by HPLC. After 30 min resting period, slices were reincubated in norepinephrine-containing KBM and test agent plus veratrine-containing medium consecutively for 10 min period each to investigate the effect of norepinephrine on basal or veratrine-induced amino acid release from hippocampal slices. The observed results were as follows: 1. The release of GABA induced by the first and second 10min-exposure of 25μM veratrine was 246.9±16.82 nmol and 209.9±20.84 nmol, respectively. When compared with released amounts of GABA during the corresponding spontaneous periods, these were 7.3 and 7.3-fold increase respectively. 2. The release of glutamic acid induced by the first and second 10min-exposure of 25μM veratrine was 426.3±63.16 nmol and 377±61.83 nmol, respectively. When compared with released amounts of glutamic acid during the corresponding spontaneous periods, these were 9.9 and 10.2-fold increase respectively. 3. Norepinephrine (1-100μM) increased veratrine-stimulated and spontaneous release of GABA in dose-dependent manner. 4. Norepinephrine (1-100μM) inhibited veratrine-stimulated glutamic acid release in dose-dependent fashion.

Soft Agar Assay를 이용한 배양골조직과 골세포의 Transforming Growth Factor-β유리에 관한 연구

백정화,김관식,정동균 대한구강생물학회 1991 International Journal of Oral Biology Vol.15 No.2

To study the effect of PTH, osteotropic hormone, on the TGF-β activity in conditioned medium prepared from bone explants, fetal rat ulnae and radii were removed at 19-day of gestation and organ cultured for 24 hours. Then media were changed with fresh BGJb media for control group or PTH-supplemented media (50 or 200 ng/㎖) for experimental group respectively and conditioned media were prepared by 2-day culture of explants. To study the cellular orgin of matrix-associatied TGF-β and the effect of PTH on the TGF-β activity in conditioned media prepared from bone cells, five bone cell populations were prepared from fetal rat calvaria by sequential enzyme digestion. After primary culture, each bone cell population was collected and resuspended as Ⅰ-Ⅱ, Ⅲ and Ⅳ-Ⅴ groups. After 24 hours, media were changed with fresh minimum essential medium(MEM) or 600 ng/㎖ PTH-supplemented media and conditioned media were prepared by 2-day culture. For activation of latent TGF-β activity in bone cell-conditioned media, media were acidified and then neutralized. TGF-β activities of conditioned media were measured by anchorage-independent growth of NRK fibroblasts using modified Todaro soft agar assay method(1987) and the number of colonies ≥ 50 ㎛ was counted. The observed results were as follows. 1. TGF-β, without 2 ng/㎖ EGF, did not induce colony formation in soft agar suspensions. 2. With 2 ng/㎖ EGF, TGF-β induced NRK cells to form large colonies in soft agar suspensions and colony numbers increased proportionally to TGF-β concentrations from 50 pg/㎖ to 1600 pg/㎖. 3. When ulnae and radii were incubated with 50 ng/㎖ PTH, TGF-β activity in conditioned media was not significantly different from control conditioned media. In contrast, TGF-β activity in conditioned media prepared from culture with supplementation of 200 ng/㎖ PTH, was significantly increased(p < 0.01). 4. TGF-β activity was detectable in all bone cell populations and the amount was not significantly different between cell populations. 5. When bone cell populations were incubated with 600 ng/㎖ PTH. TGF-β activity in conditioned media from each population was not significantly different from control conditioned media. These results suggest that all cell populations isolated from calvaria synthesize TGF-β and PTH has no apparent effects on the TGF-β production of bone cell populations. In addition, increased TGF-β activity in bone explant conditioned media by 200 ng/㎖ PTH appears to be due to the enhanced release of TGF-β from bone matrix secondary to bone resorptive process.

부갑상선 호르몬이 조골세포군의 교원분해효소 유리에 미치는 영향에 관한 연구

이인석,김관식,정동균 대한구강생물학회 1990 International Journal of Oral Biology Vol.14 No.1

Bone resorption is accomplished by the removal of both mineral and the organic matrix. The cellular origin of bone collagenase that is responsible for the removal of unmineralized organic matrix has remained equivocal. This study was performed to investigate the effect of PTH on the release of collagenase and cellular origin of bone collagenase in bone cell populations. Calvaria from fetal rat at 19-day of gestation, were sequentially digested by the enzyme solution consisted of collagenase, trypsin and EDTA for 10 min (PopulationI), 10 min (II), 10 min (III), 20 min (IV) and 20 min (V). Each bone cell population was primarily cultured for 6-7 days and splited to 5.0×10^5 cells/35mm culture dish and then incubated for 6 days with MEM with or without PTH (1.0U/ml). Collected medium was incubated with type I collagen for 6h at 37℃ and then used for hydroxyproline determination by Woessner's method. The results were as follows. 1. There were no significant differences of collagenase activity among 5 bone cell populations. 2. PTH (1.0U/ml) had no effect on the collagenase activity in the population I, II, III and V. 3. PTH (1.0U/ml) significantly increased collagenase activity in population IV.