• Archives of Pharmacal Research
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• 제16권4호
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• pp.271-275
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• 1993

In order to prepare a novel human insulin analogue suhbstituted with homoserine at B$^{30}$ / position, (B$^{30}$ /-homoserine) human insulin, a synthetic gene was designed by linking directly a gene for B chain with that for A chain. This gene was constructed by enzymatic joining of 10 different synthetic oligonucleotides, and then inserted at the polylinker region of pUC19 plasmid. To achieve a high level of gene expression, the gene fusion technique region of pUC19 plasmid. To achieve a high level of gene expression, the gene fusion technique was employed using amino terminal regions of lacZ gene up to Clal or hpal, and either of them has been located under tac promoter. The chemical induction of these fused genes by isopropyl-.betha.-D-thiogalactopyranoside (IPTG) gave a satisfactory level of expression in Escherichia coli harboring the ocnstructed plasmids. It was observed that the fused gene product as a single chain insulin precusor was produced more than 30% of total cell protein of E. coli as a form of inclusion body.

• BMB Reports
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• 제39권4호
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• pp.457-463
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• 2006

Insulin resistance is commonly observed in patients prior to the development of type 2 diabetes and may predict the onset of the disease. We tested the hypothesis that impairment in insulin stimulated glucose-disposal in insulin resistant patients would be reflected in the gene expression profile of skeletal muscle. We performed gene expression profiling on skeletal muscle of insulin resistant and insulin sensitive subjects using microarrays. Microarray analysis of 19,000 genes in skeletal muscle did not display a significant difference between insulin resistant and insulin sensitive muscle. This was confirmed with real-time PCR. Our results suggest that insulin resistance is not reflected by changes in the gene expression profile in skeletal muscle.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 2001년도 추계학술대회 및 정기총회
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• pp.100-100
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• 2001

Hypoxia is a pathophysiological condition that occurs during injury, ischemia, and stroke. Hypoxic stress induces the expression of genes associated with increased energy flux, including the glucose transporters Glutl and Glut3, several glycolytic enzymes, nitric oxide synthase, erythropoietin and vascular endothelial growth factor. Induction of these genes is mediated by a common basic helix-loop-helix PAS transcription complex, the hypoxia-inducible factor-l${\alpha}$ (HIF-1${\alpha}$)/ aryl hydrocarbon receptor nuclear translocator (ARNT). Insulin plays a central role in regulating metabolic pathways associated with energy storage and utilization. It triggers the conversion of glucose into glycogen and triglycerides and inhibits gluconeogenesis. Insulin also induced hypoxia-induced genes. However the underlying mechanism is unestablished. Here, we study the possibility that transcription factor HIF-1${\alpha}$ is involved in insulin-induced gene expression. We investigate the mechanism that regulates hypoxia-inducible gene expression In response to insulin We demonstrate that insulin increases the transcription of hypoxia- inducible gene. Insulin-induced transcription is not detected in Arnt defective cell lines. Under hypoxic condition, HIF- l${\alpha}$ stabilizes but does not under insulin treatment. Insulin-induced gene expression is inhibited by presence of PI-3 kinase inhibitor and Akt dominant negative mutant, whereas hypoxia-induced gene expression is not. ROS inhibitor differently affects insulin-induced gene expressions and hypoxia-induced gene expressions. Our results demonstrate that insulin also regulates hypoxia-inducible gene expression and this process is dependent on Arnt. However we suggest HIF-l${\alpha}$ is not involved insulin-induced gene expression and insulin- and hypoxia- induces same target genes via different signaling pathway.

• 대한한방내과학회지
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• 제31권1호
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• pp.25-39
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• 2010

Background : At high glucose levels in $\beta$-cells, cell viability and insulin secretion are decreased by glucotoxicity. Sopyung-tang(SPT) had an effect on blood glucose level decrease and antioxidant enzyme activities in streptozotocin-induced diabetic rats. Objectives : This study performed a series of experiment to verify the effects of SPT extract on the cell viability, antioxidant enzyme activities, insulin secretion and insulin mRNA expression at hyperglycemic states of RIN-m5F. Methods : After treatment at various concentrations of SPT added to the RIN-m5F cells, cell viability by MTT assay, free radical-scavenging activity, SOD activity and insulin secretion were measured. Additionally, insulin-related gene expression was measured using real-time RT-PCR. Results : Compared to the control group, SPT extract showed considerable effects on RIN-m5F cell viability, DPPH radical-scavenging activity, superoxide dismutase (SOD) activity, insulin secretion and insulin-related gene expression. Conclusions : This study showed that SPT extract has an effect on $\beta$-cell cell viability, insulin secretion and insulin-related gene expression. Thus, SPT extract may be used for treatment of diabetes and its complications. Further mechanism studies of SPT seem to be necessary on the glucotoxicity and oxidative stress.

• Biotechnology and Bioprocess Engineering:BBE
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• 제1권1호
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• pp.9-12
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• 1996

In order to produce the single chain precursor of a novel human insulin analogue, (B30-Homoserine) insulin, the fermentative behaviors of Escherichia coli JM103 were studied, which harbors pKBA plasmid carrying a hybrid gene in which the gene for a single chain precursor was fused with lacZ gene under tac promoter. The maximal induction of gene expression was achieved when more than 0.05 mM of isopropyl-$\beta$-D-thiogalactopyranoside(IPTG) was supplemented to fermentation medium after 4 h cultivation of E. coli, and followed by longer than 2-h fermentation. The hybrid protein of the single chain insulin precursor was isolated from cytoplasmic inclusion bodies by dissolving in 8M urea solution, and purified through DEAE-Sephacel and Sephadex G-200 column chromatographies with a recovery of 35%. The finally purified hybrid protein showed a single band on sodium dodecyl sulfate-polyacrylamide gel.

• 약학회지
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• 제37권5호
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• pp.504-510
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• 1993

Human insulin gene is consisted of the polymorphic region with the repeating units, the regulatory sequence, the structural gene including the intervening sequence, and 3'-flanking region. The polymerase chain reaction, which amplifies the target DNA between two specific primers, has been performed for the amplification of human insulin gene and simple one-step cloning of it into Escherichia coli. Out of 1727 nuceotides compared, only 4 sites were variable: 5'-regulatory region(G2101$\rightarrow$AGG); IVS I(T2401$\rightarrow$A); Exon II(C2411 deletion); IVS II(A2740 dejection). The variations at the G2101 and T2401 were the same as those found in one American allele. The other two variations were observed only in the specific Korean allele. And, the enzyme digestion patterns among normal, insulin dependent diabetes mellitus, and non-insulin dependent diabetes mellitus were the same. On the other hand, PCR method showed the possibility of the quickaccess for the polymorphic region in terms of the restriction fragment length of polymorphism.

• Journal of Nutrition and Health
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• 제35권2호
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• pp.207-212
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• 2002

Acetyl-CoA carboxylase (ACC) is the enzyme that controls no devo fatty acid biogynthesis, and this enzyme catalyzes the carboxylation pathway of acetyl-CoA to malonyl-CoA. Acetyl-CoA carboxylase gene expression was regulated by nutritional and hormonal status. The present study was performed to identify the regulation mechanism of ACC gene promoter I. The fragments of ACC promoter I -1.2-kb region wert recombined to pGL3-Basic vector with luciferase as a reporter gene. The primary hepatocytes from the rat were used to investigate the hormonal regulation of ACC promoter I activity. ACC PI (-1.2)/Luc plasmid was trtransferred into primary hepatocytes using lipofectin. Activity of luciferase was increased two-fold by 10-9M, three-fold by 10-8M, 10-6M, 3.5-fold by 10-6M, and 4.5-fold by 10-7M insulin treatment, respectively. In the presence of dexamethasone (1 $\mu$M), the effects of insulin increased about 1.5-fold, showing the additional effects of dexamethasone. Moreover, the activity of luciferase increased with insulin+dexamethasone, insulin+T3, dexamethasone+T3, and dexamethasone+insulin+T3 treatment approximately 6-, 4-, 6.5-, and 10-fold, respectively. Therefore it can be postulated that 1) these hormones coordinately regulate acetyl-CoA caroxylase gene expression via regulation of promoter activity, 2) the -1.2-kb region of ACC promoter I may have the response element sequences for insulin, dexamethasone, and T3.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XII)
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• pp.34-38
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• 2003

For the production of $B^{30}-homoserine$ insulin analog as a novel anti-diabetic drug, the fermentative study was attempted for the maximal gene expression of HTS-fused $B^{30}-homoserine$ insulin precursor in the recombinant Escherichia coli cells. In a batch fermentation, the maximal production of insulin precursor as much as 38.95 mg/L-h, which occupied more than 12.8% of total cell protein. was achieved when the gene expression was induced by 0.5 mM IPTG at the middle logarithmic growth phase. The HTS-fused $B^{30}-homoserine$ insulin precursor was recovered from a batch culture through the processes of cell harvest, collection of insoluble fraction after sonication and purification by nickel affinity column chromatography. The isolated insulin precursor was 14 mg/L with a recovery yield of 35.9% of expressed gene product. The insulin A and B chain mixture was recovered after the insulin precursor was subjected to CNBr cleavage and purified by nickel affinity column chromatography. The isolated insulin chains were then sulfitolyzed with sodium thiosulfat and sodium tetrathionate, and reconstituted to insulin analog with ${\beta}-mercaptoethanol$, followed by purification with CM-Sepharose C-25 column chromatography.

• Animal cells and systems
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• 제5권4호
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• pp.341-346
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• 2001

Hypertension is a complex disease with strong genetic influences. Essential hypertension has been shown to be associated with insulin resistance. To clarify the genetic basis of insulin resistance in Hypertension, case-control association studies were performed to examine candidate genes for insulin resistance in hypertension. Polymorphisms investigated were the BstO I polymorphism of the $\beta$3-adrenergic receptor (ADRB3) gene, the Xba I Polymorphism of the glycogen synthase (GSY) gene, the Dde I polymorphism of the protein phosphatase 1 G subuit (PP1G) gene, the BstE II polymorphism of the glucagon receptor (GCG-R) gene, the Pst 1 polymorphism of the insulin (INS) gene and the Acc I polymorphism of the glucokinase (GCK) gene. No significant differences were observed in the distribution of alleles and genotypes of the ADRB3, GSY PP1G, GCG-R, INS, and GCK genes between hypertensive and normotensive groups. Although the frequencies in each of these polymorphisms were not significantly different between essential hypertensive and normotensive individuals, our results may provide additional information for linkage analysis and associative studies of disorders in carbohydrate metabolism or in cardiovascular disease.

• 생명과학회지
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• 제20권5호
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• pp.670-675
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• 2010

Autoimmune regulator gene (Aire)는 흉선에서 발현되며 promiscuous genes으로 알려진 흉선에서 자가항원 발현을 조절한다. Aire 와 promiscuous genes은 흉선에서 T세포 tolerance와 자가면역에 관여한다. 말초 조직 즉 림프절에서 Aire의 역할을 알아보고자 림프절 구성 세포중 하나인 fibroblastic reticular cell (FRC)을 분리 확립하였다. 마우스 림프절로부터 분리된 FRC에서 Aire의 발현을 확인하였고 또한 promiscuous antigen인 insulin의 발현도 확인하였다. Aire 과발현 플라스미드로 형질전환 후 배양 FRC에서 Insulin의 발현이 증가하였다. 이것은 Aire가 FRC에서 promiscuous gene의 발현을 조절한다는 것을 보여주며 peripheral selection과 연관되어 있을 수 있다는 것을 제시한다.