When quiescent cells ate stimulated to enter the cell cycle, the thymidine kinase(TK) gene is transcriptionally activated at the border of Gl and 5. In this report we show that the human TK promoter contains multiple protein-binding sites. By site-directed mutagenesis, we identified a protein-binding site on the human TK promoter requited for conferring Gl-S-regulated transcription to a heterologous promoter and dissociated it functionally from an adjacent protein-binding domain containing an inverted CCAAT motif requited for high basal level expression. Substitution-mutation of this site results in constitutive expression of the neo reporter gene in serum-stimulated fibroblasts, as well as in cells arrested in mid-Gl by a temperature-sensitive mutation. The regulatory domains for the human TK promoter exhibit interesting symmetrical features, including a set of CCAAT motifs and sites similar to the novel Yi protein-binding site recently discovered in the mouse TK promoter. Thus, components of the hTK complex is important for hTK gene regulation.

대부분의 인슐린의 작용들은 인슐린 수용체를 통하여 이루어진다. 인슐린이 수용체에 결합하면, 수용체 고유의 tyrosine kinase 효소활성의 증가를 유발시키며, 결과적으로 세포내에 존재하는 기질 단백질, IRS-1, 의 tyrosine 잔기의 인산화를 증가시키게 된다. 이후, 여러 형태의 serine / threonine protein kinase 의 연속적인 활성화가 일어난다. 이들에 부가해서, 인슐린의 효자는 세포핵 내에까지 전달되어 유전자 발현의 조절과 같은 세포핵 고유의 활동에도 관여한다. 현재, 세포막에서 시작된 인슐린의 신호들이 세포핵까지 전달되는 정확한 기전에 대해서는 알려진 바 없지만, 최근의 연구에 의하면 MAP Kinase 와 S6 Kinase 그리고 Transcription Factor AP-1의 중요성이 제시되고 있다. 특히 유전자 조절 기전에는 핵단백질인 transcription factor의 인산화 반응이 큰 역할을 한다고 보고되고 있는바, 본 연구에서 AP-1. transcription factor 의 인산화 반응이 인슐린의 신호전달계에 미치는 역할에 대하여 고찰하였다. 요약하면, AP-1 transcription factor의 구성원인 c-Jun, c-Fos 그리고 Fos 관련 단백질들의 인산화가 인슐린에 의해 증가되며, 동시에 그들의. DNA-binding activity 와 유전자 발현의 활성이 증가됨을 밝힘으로써, AP-1 transcription factor의 인산화 반응이 인슐린의 핵 내에서의 작용기전에 중요한 역할을 함이 제시되고 있다. 또한 AP-1 의 인산화 반응에 관여하는 세포핵 protein kinase로서 Casein Kinase II 의 중요성이 밝혀졌다.

세포내에서의 유전자 발현의 다양성은 여러 종류의 외부자극에 의해 항상 조절되어 지고 있다. 유전자 발현의 조절기전과정에서 여러 transcription factor들이 중심적 역할을 하는 것이 알려져 있다. Transcription factor의 활성도는 원핵 생물과 진핵 생물 공히 protein phosphorylation과정을 통하여 조절되어지는 공통의 경로를 거치게 된다. 이러한 protein 인산화과정은 상창에 따른 post-translational modification과정으로서 세포표면에 위치한 각각의 수용체(receptor)들이 신호를 인지하여 그 반응으로서 신속하게 transcription factor의 활성을 조절하기 위한 것이다.

Thymopoietin(TP) was originally isolated from bovine thymic extracts on the basis of its ability to affect neuromuscular transmission when injected into mice (Goldstein, 1974). A 49 amino acid polypeptide was isolated and sequenced (Schlesinger and Goldstein, 1975). It is now evident that this molecule was created by proteolytic cleavage of larger thymopoietin proteins during isolation, and represents the N-terminal sequence of these proteins. Nevertheless, this proteolytic fragment was active in both neurophysiological and immunological experiments, and enabled the identification of an active pentapeptide. (amino acids 32 to 36, Arg-Lys-Asp-Val-Tyr, thymopentin), which. has been studied as an immunomodulatory drug.

To increase the folding efficiency of proinsulin, we have designed a series of mini-proinsulins having the central C-peptide region replaced with sequences forming reverse turns. These proteins were produced as fusion proteins in E. coli in the form of inclusion bodies. After isolation process of the sulfonated mini-proinsulins, the subsequent refolding experiments indicate that the mini-proinsulins, with non-native penta-peptide sequences inserted between two of the enzyme processing sites, show substantially increased folding yields compared with the proinsulin. The correct disulfide connections were verified by fingerprint analysis using Glu-C endoproteinase. These novel mini-proinsulins could be used for the study of folding mechanism of proinsulin.

Protein kinase C (PK-C) represents a central element transducing signals generated by a broad range of pathways which produce the lipid second messenger sn-1,2-diacylglycerol (DAG) directly or indirectly. Many dominant oncogenes have proven to function, at least in part, through this pathway Likewise, this pathway is involved in expression of other aspects of the transformed phenotype, such as tumor invasion or multidrug resistance. As expected from its broad role in cell signaling, PK-C Is also important in a range of other physiological and pathophysiological processes, including inflammation, differentiation, and nerve function.

후천성 면역결핍증(AIDS)이 전 세계적으로 문제가 된 질병으로 인식된 이래 AIDS의 치료를 위한 새로운 치료제의 개발이 전 세계적으로 중요한 목표가 되어 왔다. 수많은 개발된 물질 중에서 lymphocytes와 macrophage에서의 면역결핍성 바이러스(HIV)에 의한 급성간염을 억제하거나 제거하는데 있어 뉴크레오사이드 유도체들이 아주 중요한 역할을 해오고 있다. 지금까지 개발된 새로운 뉴크레오사이드 유도체중에서 현재 4종류의 뉴크레오사이드 유도체가 FDA에 꼭해 공인이 되어 AIDS환자의 치료에 사용되고 있다.

reverse transcription은 AIDS를 일으킨다고 알려진 바이러스인 HIV-1의 번식에는 필수적이나 인체 세포에는 필수적이 아니기에 이 단계를 표적으로 하는 AIDS 치료제가 우선적으로 개발되었다. 그 단계에 필요한 효소가 바이러스에 의해 만들어진 RT이며 이 효소의 작용을 저해하는 nucleoside 유도체들인 AZT, DDC, DDI 들이 현재 AIDS 환자의 치료에 사용되고 있다. 이들 nucleoside 유도체들은 세포안으로 들어가 triphosphate 형태로 변화된 후 dNTP와 상경적으로 경쟁하며 합성 중인 바이러스의 DNA에 들어가 DNA의 합성을 정지시켜 바이러스의 증식을 억제한다. 그러나, 이들 nucleoside 유도체들은 치료용량에서 심한 독성을 나타낼 뿐만 아니라 장기 투여시 내성을 나타내는 바이러스가 생겨나 AIDS의 치료를 불가능하게 하고 있다.

표본수를 결정하는 방법에는 크게 sequential design과 fixed sample size design이 있다. Fixed sample size design은 연구를 시행하기 전에 표본수를 합리적으로 결정하고 정해진 표본내에서 연구를 진행하는 방법이며, sequential design은 연구를 진행하면서 결과의 차이가 있는가 또는 없는가에 대해 미리 정해진 한계영역을 기준으로 계속적으로 연구대상을 추출하여 연구를 진행하는 방법이다. 여기서는 많이 사용되는 fixed sample size design에 대해서만 생각하기로 한다.

Basement membrane laminin is a multidomain glycoprotein that interacts with itself, heparin and cells. The distal long arm plays major cell and heparin interactive roles. The long arm consists of three subunits (A, B1, B2) joined in a coiled-coil rod attached to a terminal A chain globule (G). The globule is in turn subdivided into five subdomains (Gl-5). In order to analyze the functions of this region, recombinant G domains (rG, rAiG, rG5, rGΔ2980-3028) were expressed in Sf9 insect cells using a baculovirus expression vector. A hybrid molecule (B-rAiG), consisting of recombinant A chain(rAiG) and the authentic B chains (E8-B)was assembled in vitro. The intercalation of rAiG into E8-B chains suppressed a heparin binding activity identified in subdomain Gl-2. By the peptide napping and ligand blotting, the relative affinity of each subeomain to heparin was assigned as Gl> G2= G4> G5> G3, such that G1 bound strongly and G3 not at all. The active heparin binding site of G domain in intact laminin appears to be located in G4 and proximal G5. Cell binding was examined using fibrosarcoma Cells. Cells adhered to E8, B-rAiG, rAiG and rG, did not bind on denatured substrates, poorly bound to the mixture of E8-B and rG. Anti-${\alpha}$6 and anti-${\beta}$1 integrin subunit separately blocked cell adhesion on E8 and B-rAiG, but not on rAiG. Heparin inhibited cell adhesion on rAiG, partially on B-rAiG, and not on E8. In conclusion, 1) There are active and cryptic cell and heparin binding activities in G domain. 2) Triple-helix assembly inactivates cell and heparin binding activities and restores u6131 dependent cell binding activities.

The purpose of the present study was to address whether the in vivo noradrenergic neural activities in the locus coeruleus are involved in the regulation of blood pressure. Two groups of the animals were prepared, 1) SHR and 2) age-matched normotensive control, WKY. At the age of 6 and 16 weeks, blood pressure and the releases of NE from the locus coeruleus in SHR and KWY were measured by in vivo microdialysis at three different conditions: 1) normal, 2) elevated state of blood pressure by systemic injected phenylephrine and 3) increased state of neural activity by perfused phenylephrine into the locus coeruleus. The basal release of NE of SHR were significantly higher than that of WKY, Phenylephrine treatment caused elevation of blood pressure in both SHR and WKY in dose-dependent manner. Following phenylephrine injection, the releases of NE from the locus coeruleus of SHR were significantly decreased, whereas the significant change of NE in WKY was observed in the highest dose of phenylephrine. Phenylephrine perfusion into the locus coeruleus through microdialysis probe caused pressor responses and the pressor response in SHR was greater compared with that in WKY. The results from the present study suggests that the noradrenergic nervous system in the locus coeruleus may contribute as one of the development and maintenance factors for hypertension in SHR.

In neurons, nitric oxide(NO) is produced by neuronal nitric oxide synthase following stimulation of N-methyl-D-aspartate(NMDA) receptors and the subsequent influx of Ca$\^$2+/. NO, induced in this manner, reportedly plays critical roles in neuronal plasticity, including neurite outgrowth, synaptic transmission, and long-term potentiation(LTP) (1-7). However, excessive activation of NMDA receptors has also been shown to be associated with various neurological disorders, including focal ischemia, epilepsy, trauma, neuropathic pain and chronic neurodegenerative maladies, such as Parkinson's disease, Hungtington's disease and amyotrophic lateral sclerosis(8). The paradox that nitric oxide(NO) has both neuroprotective and neurodestructive effects may be explained, at least in part, by the finding that NO effects on neurons are dependent on the redox state. This claim may be supported by the recent finding that tissue concentrations of cysteine approach 700 ${\mu}$M in settings of cerebral ischemia (9), levels of thiol that is expected to influence both the redox state of the system and the NO group itself(10).

Resent advances in computer technology have introduced a sophisticated capability for computing the biological fate of toxicants in a biological system. This methodology, which has drastically altered risk assessment skill in toxicology, is designed using all the mechanistic information, and all claim better accuracy with extrapolating capability Iron animal to people than conventional pharmacokinetic methods. Biologically based mathematical models in which the specific mechanistic steps governing tissue disposition(pharmacokinetics) and toxic action (pharmacodynamics) of chemicals are constructed in quantitative terms by a set of equations loading to prediction of the outcome of specific toxicological experiments by computer simulation. pharmacokinetic and pharmacodynamic models are useful in risk assessment because their mechanistic biological basis permits the high-to-low dose, route to route and interspecies extrapolation of the tissue disposition and toxic action of chemicals.

In this study, it was tested whether lead intoxication could change thiamine content and the thiamine related biochemical factor such as activity of transketolase in the brain, and whether the changes of the myelin composition :s well as the seizure threshold induced by lead intoxication in rats be related to these changes of thiamine status and thiamine related biochemical factors. In addition, it was also tested whether administration of excessive thiamine can reverse the toxic manifestation of lead in lead intoxicated animals. Five groups of Wistar rats were prepared: 1)Control group, 2)lead treated group, 3)thiamine treated group, 4)lead plus thiamine treated group and 5)thiamine deficiency group. Each group of animals was divided into three subgroups based on ages: 3, 7 and 10 weeks of age subgroups. Lead concentration, thiamine content, the activity of transketolase and myelin composition in brain areas and threshold of electric shock seizure were tested in each group. Lead concentrations in all brain regions of lead treated group were higher than those of control group, and those of lead plus thiamine treated group were significantly lower than those of lead treated group. Thiamine contents in the brain regions of lead treated group were significantly lower than those of control group, and those of lead plus thiamine treated group were recovered back to those of control group. Activities of transketolase of lead treated group were significantly lower than those of control group, while those of lead plus thiamine treated group were recovered back to those of control group. The cases of which was observed with the concomitant changes of thiamine content and transketolase activity in myelin content or constituent of all the brain regions tested were total amount of myelin protein in the cerebellum of 3 week old rats, and phospholipid in the cerebellum of 3 week old rats and the telencephalon of 16 week old rats. Thresholds of the electroshock seizure of lead-treated group and thiamine-deficient group in 3, 7 week old rats were significantly lower than those of control group, while those of the lead plus thiamine-treated group were similar to those of control group. Changes of the electroshock seizure threshold induced by lead intoxication were observed in 3 week and 7 week old animals with the concomitant decrement of thiamine content in all the brain regions tested. These observations were reversed by the supplementation with thiamine to those animals. However, the changes of seizure threshold induced by lead intoxication corelated with the changes of thiamine contents as well as. transketolase due to lead intoxication. The changes of myelin phospholipid as one of myelin composition and those of myelin Protein content only in the cerebellum of 3 week old rats correlated with the changes of the seizure threshold as well as thiamine content due to lead intoxication. The results from the present study may indicate that neurotoxicity of lead in rats may be mediated at least in part through the changes of thiamine status. Such changes of thiamine status may induce the changes of myelin composition such as myelin phospholipid and those of myelin protein content especially in the cerebellum of 3 week old rats which may eventually affect the threshold of seizure.

To investigate the mechanism of the regulation of cytochrome P450IAl, the 5'-flanking region of a trout cytochrome P4501Al was cloned into the CAT basic expression vector at HindⅢ site. This trout Cytochrome P450IAl upstream DNA containing CAT construct was transfected into Hepa-1 cells .3MC treatment to hepa I cells transfected with trout P450IAl-CAT construct increased CAT protein and mRNA by 2.81 fold when it was compared with that of control. This increase CAT protein and mRNA was decreased by concomitantly treated flavonoids and aminopyrine. The level of CAT protein was 29.2-58.0% of 3MC stimulated CAT protein.

라니티딘은 최근 위궤양 및 십이지장 궤양의 치료에 통상적으로 많이 처방되는 히스타민 H$_2$ receptor antagonist로 작용하는 약물이다. 이 약물의 Pharma-cokinetics에 대해서는 동물 및 사람에 있어서 이미 많은 연구가 되어 있다(1-4). 수용성 약물인 라니티딘은 정상인에 있어서 경구투여 후 흡수가 신속하나 불완전한 것으로 알려져 있다(4). 경구투여 후 개개인에 따른 처고 혈중농도(Cmax)가 상당히 큰 차이를 보이며, 생체내 이용률(Bioavailability)은 평균 50% 이나 최저 27%에서 최고 88%에 이르기까지 넓은 범위를 보이고 있다. 더욱이 공복 시 경구투여 하거나(5-8), jejunum에 직접 bolus 투여후(9) 혹은 심지어는 정맥주사후의 경우(10)에도 소위 'double-peak phenomenon'이라고 불리 우는 최고 혈중농도에 있어서 bimodal pattern을 나타낸다. 이처럼 highly variable한 약물들은 생물학적 동등성(Bioequivalence) 측면에서 제제를 평가할 때 상당히 중요하고도 어려운 과제이므로, 현재 세계적인 issue가 되고 있다.