• BMB Reports
• /
• 제32권2호
• /
• pp.196-202
• /
• 1999

The existence of the Ras-independent signal transduction pathway of insulin leading to DNA synthesis was investigated in Rat-1 fibroblasts overexpressing human insulin receptor (HIRc-B) using the single-cell microinjection technique. Microinjection of a dominant-negative mutant $Ras^{N17}$ protein into quiescent HIRc-B cells inhibited the DNA synthesis stimulated by insulin. Microinjection of oncogenic H-$Ras^{V12}$ protein ($H-Ras^{V12}$) (0.1 mg/ml) induced DNA synthesis by 35%, whereas that of control-injected IgG was induced by 20%. When the marginal amount of oncogenic H-$Ras^{V12}$ protein was coinjected with a dominant-negative mutant of the H-Ras protein ($Ras^{N17}$), DNA synthesis was 35% and 74% in the absence and presence of insulin, respectively. This full recovery of DNA synthesis by insulin suggests the existence of the Ras-independent pathway. The same recovery was observed in the cells coinjected with either H-$Ras^{V12}$ plus H-$Ras^{N17}$ plus SH2 domain of the p85 subunit of PI3-kinase ($p85^{SH2-N}$) or H-$Ras^{V12}$ plus H-$Ras^{N17}$ plus interfering anti-Shc antibody. When co-injected with a dominant-negative H-$Ras^{N17}$, the DNA synthesis induced by the Ras-independent pathway was blocked. These results indicate that the Ras-independent pathway of insulin leading to DNA synthesis exists, bypassing the p85 of PI3-kinase and Shc protein, and requires Rac1 protein.

• Biomolecules & Therapeutics
• /
• 제6권4호
• /
• pp.345-350
• /
• 1998

The adverse effects of ethanol on cell proliferation have been described for a variety of tissues and cells. In the present study, we investigated whether chronic ethanol intoxication impairs the cell proliferation and DNA synthesis induced by oncogenic $H-ras^{V12}$ - and $v-K-ras^{V12}$-transformed cells. Ethanol treatment inhibited the cell proliferation and the DNA synthesis of control parental fibroblasts in a time- and dose-dependent manner. In contrast, ethanol did not suppress the proliferation of either oncogenic $H-ras^{V12}$ - or $v-K-ras^{V12}$ -transformed fibroblasts. Microinjection of oncogenic $H-Ras^{V12}$ protein induces DNA synthesis and ethanol treatment did not interfere with the DNA synthesis. The antiproliferative toxicity of ethanol was rescued by antioxidants, such as N-acetylcysteine and 4-methlpyrazole. These results indicate that the antiproliferative action site of ethanol toxicity lies upstream or is independent of Ras and ethanol exerts its toxicity through a free radical formation.

• The Korean Journal of Physiology and Pharmacology
• /
• 제24권3호
• /
• pp.267-276
• /
• 2020

T In the present study, we investigated the effect of oncogenic H-Ras on rat mdr1b expression in NIH3T3 cells. The constitutive expression of H-Ras^V12 was found to downregulate the mdr1b promoter activity and mdr1b mRNA expression. The doxorubicin-induced mdr1b promoter activity of the H-Ras^V12 expressing NIH3T3 cells was markedly lower than that of control NIH3T3 cells. Additionally, there is a positive correlation between the level of H-Ras^V12 expression and a sensitivity to doxorubicin toxicity. To examine the detailed mechanism of H-Ras^V12-mediated down-regulation of mdr1b expression, antioxidant N-acetylcysteine (NAC) and NADPH oxidase inhibitor diphenylene iodonium (DPI) were used. Pretreating cells with either NAC or DPI significantly enhanced the oncogenic H-Ras-mediated down-regulation of mdr1b expression and markedly prevented doxorubicin-induced cell death. Moreover, NAC and DPI treatment led to a decrease in ERK activity, and the ERK inhibitors PD98059 or U0126 enhanced the mdr1b-Luc activity of H-Ras^V12-NIH3T3 and reduced doxorubicin-induced apoptosis. These data suggest that Ras^V12 expression could downregulate mdr1b expression through intracellular reactive oxygen species (ROS) production, and ERK activation induced by ROS, is at least in part, contributed to the downregulation of mdr1b expression.

• Advances in nano research
• /
• 제12권6호
• /
• pp.605-616
• /
• 2022

We prepared two samples of ultrafine ferrihydrite (FH) nanoparticle ensembles of quite a different origin. First is the biosynthesized sample (as a product of the vital activity of bacteria Klebsiella oxytoca (hereinafter marked as FH-bact) with a natural organic coating and negligible magnetic interparticle interactions. And the second one is the chemically synthesized ferrihydrite (hereinafter FH-chem) without any coating and high level of the interparticle interactions. The interparticle magnetic interactions have been tuned by modifying the nanoparticle surface in both samples. The coating of the FH-bact sample has been partially removed by annealing at 150℃ for 24 h (hereinafter FH-annealed). The FH-chem sample, vice versa, has been coated (1.0 g) with biocompatible polysaccharide (arabinogalactan) in an ultrasonic bath for 10 min (hereinafter FH-coated). The changes in the surface properties of nanoparticles have been controlled by XPS. According to the electron microscopy data, the modification of the nanoparticle surface does not drastically change the particle shape and size. A change in the average nanoparticle size in sample FH-annealed to 3.3 nm relative to the value in the other samples (2.6 nm) has only been observed. The estimated particle coating thickness is about 0.2-0.3 nm for samples FH-bact and FH-coated and 0.1 nm for sample FH-annealed. Mössbauer and magnetization measurements are definitely shown that the drastic change in the blocking temperature is caused by the interparticle interactions. The experimental temperature dependences of the hyperfine field hf>(T) for samples FH-bact and FH-coated have not revealed the effect of interparticle interactions. Otherwise, the interparticle interaction energy E_int estimated from the hf>(T) for samples FH-chem and FH-annealed has been found to be 121k_B and 259k_B, respectively.

• Genomics & Informatics
• /
• 제4권4호
• /
• pp.161-166
• /
• 2006

The establishment of DNA microarray technology has enabled high-throughput analysis and molecular profiling of various types of cancers. By using the gene expression data from microarray analysis we are able to investigate diagnostic applications at the molecular level. The most important step in the application of microarray technology to cancer diagnostics is the selection of specific markers from gene expression profiles. In order to select markers of Immortalization and transformation we used c-myc and $H-ras^{V12}$ oncogene-transfected NIH3T3 cells as our model system. We have identified 8751 differentially expressed genes in the immortalization/transformation model by multivariate permutation F-test (95% confidence, FDR<0.01). Using the support vector machine algorithm, we selected 13 discriminative genes which could be used to predict immortalization and transformation with perfect accuracy. We assayed $H-ras^{V12}$-transfected 'transformed' cells to validate our immortalization/transformation dassification system. The selected molecular markers generated valuable additional information for tumor diagnosis, prognosis and therapy development.

• 대한자기공명의과학회:학술대회논문집
• /
• 대한자기공명의과학회 2007년도 학술대회
• /
• pp.45-45
• /
• 2007

• 생명과학회지
• /
• 제22권4호
• /
• pp.538-544
• /
• 2012

에탄올은 사람에 대한 발암물질로 잘 알려져 있다. 또한 여러 조직이나 세포에서의 에탄올에 의한 세포증식억제효과도 잘 알려져 있다. 본 연구에서는 여러 암세포에서 에탄올에 의한 세포증식억제 효과를 조사하였는데 특히 발암원성 $ras$로 형질전환되거나 미세주입된 세포에서의 영향을 조사하였다. 에탄올은 여러 정상세포들의 증식을 억제하였다. 반면에 여러 암세포나 발암원성 Ras에 의한 세포증식은 억제하지 못 하였다. 또한 발암원성 단백질의 세포내 미세주사에 의한 DNA합성 유도도 에탄올에 의해 억제 되지 않았다. 이러한 에탄올의 세포증식억제 효과는 $N$-acetylcysteine이나 4-methylpyrazole과 같은 항산화제에 의해 제거되었다. 이러한 실험 결과는 에탄올에 의한 세포증식억제 효과는 Ras단백질의 upstream에 있거나 또는 Ras와 독립적으로 작용하며, 활성산소 형성과 밀접한 관계가 있다는 것을 알려준다.

• Journal of Animal Science and Technology
• /
• 제59권9호
• /
• pp.20.1-20.9
• /
• 2017

Background: Ribonuclease (RNase) is one of the few toxic proteins that are present constantly in snake venoms of all types. However, to date this RNase is still poorly studied in comparison not only with other toxic proteins of snake venom, but also with the enzymes of RNase group. The objective of this paper was to investigate some properties of RNase from venom of Vietnam cobra Naja atra. Methods: Kinetic methods and gel filtration chromatography were used to investigate RNase from venom of Vietnam cobra. Results: RNase from venom of Vietnam cobra Naja atra has some characteristic properties. This RNase is a thermostable enzyme and has high conformational stability. This is the only acidic enzyme of the RNase A superfamily exhibiting a high catalytic activity in the pH range of 1-4, with $pH_{opt}=2.58{\pm}0.35$. Its activity is considerably reduced with increasing ionic strength of reaction mixture. Venom proteins are separated by gel filtration into four peaks with ribonucleolytic activity, which is abnormally distributed among the isoforms: only a small part of the RNase activity is present in fractions of proteins with molecular weights of 12-15 kDa and more than 30 kDa, but most of the enzyme activity is detected in fractions of polypeptides, having molecular weights of less than 9 kDa, that is unexpected. Conclusions: RNase from the venom of Vietnam cobra is a unique member of RNase A superfamily according to its acidic optimum pH ($pH_{opt}=2.58{\pm}0.35$) and extremely low molecular weights of its major isoforms (approximately 8.95 kDa for RNase III and 5.93 kDa for RNase IV).