• Molecular & Cellular Toxicology
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• 제4권1호
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• pp.11-15
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• 2008

The expression of neuronal nitric oxide synthase (nNOS) is regulated by various spliced first exons (exon 1a-1i), sharing differentially common exon 2 in diverse human tissues. The highly complex structure and regulation of human nNOS gene gave limitations of information for the precise mechanism of nNOS regulation. In the present study, we report that the repeats of polymorphic dinucleotides $(GT)^nA(TG)^n$ repeats located in just upstream to the exon 1f in human nNOS gene play suppressive role in transcription, as shown in the characteristics of Z-DNA motif in other genes. In neuronal and trophoblast cells transfected transiently with luciferase construct without dinucleotide repeats at the 5'-flanking region of exon 1f in nNOS gene, the luciferase activity was increased markedly. However, the presence of the dinucleotide repeats dramatically suppressed the luciferase activity to the basal level, and which was dependent on the length of $(GT)^n$ and $(TG)^n$ repeats. More importantly, we found the polymorphisms in the length of dinucleotide repeats in human. Furthermore, we show for the first time here that there is a significant association of the lengths of polymorphic dinucleotide $(GT)^n$ and $(TG)^n$ repeats with the risk of schizophrenia.

• Molecular & Cellular Toxicology
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• 제4권1호
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• pp.66-71
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• 2008

Long term exposure of Jurkat cells to 2 ATA pressure resulted in the inhibition of cell growth. Under a 2 ATA pressure, the morphological changes in the cells were visualized by electron microscopy. The cells exhibited significant inhibitory responses after three passages. However, short-term exposure study was carried out, 2 ATA pressure may have beneficial effects. The Jurkat cells were exposed to $H_2O_2$ (25 and $50{\mu}M$) in order to induce DNA damage, and then incubated under at either normal pressure or 2 ATA for 1 or 2 hours in order to recover the DNA damage. The extent of DNA damage was determined via Comet assay. More recovery from DNA damage was observed at 2 ATA than at normal pressure. The activity of the DNA repair enzymes, DNA polymerase-$\beta$, was also evaluated at both normal pressure and 2 ATA. The activity of DNA polymerase-$\beta$ was observed to have increased significantly at the 2 ATA than at normal pressure. In conclusion, the effects of hyperbaric pressure from 1 ATA to 2 ATA on biochemical systems can be either beneficial or harmful. Long term exposure to hyperbaric pressure clearly inhibited cell proliferation and caused genotoxic effects, but short-term exposure to hyperbaric pressure proved to be beneficial in terms of bolstering the DNA repair system. The results of the present study have clinical therapeutic application, and might prove to be an useful tool in the study of genotoxicity in the future.

• Molecular & Cellular Toxicology
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• 제4권1호
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• pp.85-91
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• 2008

These days there is a constant possibility of exposure to UV radiation which can cause abnormal production of melanin and result in skin disease such as hyperpigmentation and melanoma. Many materials were investigated for skin whitening and protection against UV radiation. In this study, we assessed the melanogenesis inhibitory activities of Korean Red Ginseng (KRG, Ginseng Radix Rubra) and Ginkgo (EGb 761 Ginkgo Biloba) in an attempt to develop a new skin whitening agent derived from natural products. B16F10 melanoma cells were treated for 48 hr with KRG and EGb 761. The inhibitory effect on melanogenesis was measured and related cytokines and proteins expression were also investigated by RT-PCR and Western blotting. In addition, we also assessed the effects of these substances on the skin of C57BL/6 mice. Cell growth, melanin content and tyrosinase activity were inhibited effectively in B16F10 melanoma cells treated with KRG and EGb 761. Moreover, tyrosinase mRNA expression was inhibited clearly and melanogenesis related proteins (MRPs) containing tyrosinase, TRP1 and TRP2 were also reduced by KRG and EGb761, while cytokines such as IL-$1{\beta}$ and IL-6 were induced. In the case of UV irradiated mice, we observed induction of cytokine mRNA levels and reduction of MRPs mRNA expression. In addition, a decrease in pigmentation from treatment with KRG and EGb 761 on the skin of mice was observed. These results indicate that KRG and EGb 761 inhibit melanogenesis in B16F10 cells and have display protective activities against UVB. Therefore, we suggest that KRG and EGb 761 are good candidates to be used as whitening agents and UVB protectors for the skin.

• Molecular & Cellular Toxicology
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• 제2권4호
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• pp.257-261
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• 2006

Signal transducer and activator of transcription 4 (STAT4) is one of the important mediators in generating inflammation and immune responses. To address the role of Stat4 in carrageenan induced acute inflammation, we performed paw edema measurement and 7.4 k mouse cDNA microarray analysis in carrageenan induced acute inflammation in Stat4 knockout (-/-) mice. Male BALB/c (n=8) and Stat4 -/- (n=5) were used and paw edema was induced with injection of $30\;{\mu}L$ of 1% carrageenan into plantar surface of right hind paw. Next, we isolated the mRNA in mouse whole brain and analyzed cDNA microarray profiles for the changes of the brain expression in Stat4 -/- mice. Interestingly, the increase in paw volume of Stat4 -/- mice was reduced by about 30% as compared to that of wild type. The cDNA microarray analysis revealed the altered expressions of several cytokines (Tnf, Il6, and Il4) and pain-associated proteins (Ptgs2, Gabra6, and Gabbr1) in Stat4 -/- mice. Our results suggest that Stat4 may be related to the inhibitory responses on carrageenan induced acute inflammation.

• Molecular & Cellular Toxicology
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• 제2권4호
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• pp.273-278
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• 2006

95 squamous cell lung carcinoma samples (normal tissue: 40 samples, tumor: 55 samples) were analyzed with 8 K cDNA microarray. 1-way ANOVA test was employed to select differentially expressed genes in tumor with FDR<0.01. Among the selected 1,655 genes, final 212 genes were chosen according to the expression fold change and used for following analysis. The expression of up-regulated 64 genes was verified with Reverse Transcription PCR and 10 genes were identified as candidates for SCC markers. In our opinion, those candidates can be exploited as diagnostic or therapeutic purposes. Gene Ontology (GO) based analysis was performed using those 212 genes, and following categories were revealed as significant biological processes: Immune response (GO: 0006955), antigen processing (GO: 0030333), inflammatory response (GO: 0006954), Cell adhesion (GO: 0007155), and Epidermis differentiation (GO: 0008544). Gene set enrichment analysis (GSEA) also carried out on overall gene expression profile with 522 functional gene sets. Glycolysis, cell cycle, K-ras and amino acid biosynthesis related gene sets were most distinguished. These results are consistent with the known characteristics of SCC and may be interconnected to rapid cell proliferation. However, the unexpected results from ERK activation in squamous cell carcinoma gripped our attention, and further studies are under progress.

• Molecular & Cellular Toxicology
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• 제2권2호
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• pp.114-119
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• 2006

Benzo[a]pyrene is a representative ecotoxicant in marine environment and a model compound of polycyclic aromatic hydrocarbons, which has an ability to bioaccumulate in aquatic organisms. This study aimed to identify molecular biomarkers suitable for assessing environmental pollution using a microarray technique. We examined the effects of benzo[a]pyrene on gene expressions in the rockfish, Sebastes schlegeli. We constructed the subtractive cDNA library with hepatic RNA from benzo[a]pyrene-exposed and non-exposed control fish. From the library 10,000 candidate clones were selected randomly and cDNA microarray was constructed. We determined benzo[a]pyrene-responsive genes using a high-density microarray. Statistical analysis showed that approximately 400 genes are significantly induced or reduced by benzo[a]pyrene treatment ($2\;{\mu}m$). Especially gene expression changes of 4 candidate clones among the up- or down-regulated genes were investigated in 6, 12 and 24 hr BaP-exposed fish groups. Many methods have been developed to monitor marine environmental status, which depend on quantifying the levels of the toxic components in polluted seawater or on ecological accessing, such as species diversity or richness. However, those methods could not provide information on physiological or genetic changes induced by such environmental stresses. Comparing with the conventional methods, these data will propose that benzo[a]pyrene-responsive genes can be useful for biological risk assessment of polycyclic aromatic hydrocarbons on marine organism at molecular level.

• Molecular & Cellular Toxicology
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• 제2권2호
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• pp.97-105
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• 2006

Several features of the implant surface, such as roughness, topography, and composition play a relevant role in implant integration with bone. This study was conducted in order to determine the effects of ceramic-coatings on Ti surfaces on the biological responses of a human osteoblast-like cell line (MG63). MG63 cells were cultured on Zr (Zrconium-coated surface), Nb (Niobium-coated surface), and control (Uncoated Titanium) Ti. The morphology of these cells was assessed by SEM. The cDNAs prepared from the total RNAs of the MG63 were hybridized into a human cDNA microarray (1,152 elements). The appearances of the surfaces observed by SEM were different on each of the three dental substrate types. MG63 cells cultured on Zr, Nb and control exhibited cell-matrix interactions. In the expression of several genes were up-, and down-regulated on the different surfaces. The attachment and expression of key osteogenic regulatory genes were enhanced by the surface morphology of the dental materials used.

• Molecular & Cellular Toxicology
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• 제2권2호
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• pp.81-88
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• 2006

Sphingolipid metabolites regulate many aspects of cell proliferation, differentiation, and apoptosis. In the present study, we have assessed the effects of the novel phytosphingosine derivative, N-acetylphytospingosine (NAPS), on the depigmentation of murine B16F10 melanoma cells, and have also attempted to identify the possible signaling pathway involved, in comparison with $C_{2}-ceramide$. NAPS and $C_{2}-ceramide$ both inhibited the growth of the B16F10 cells in a dose-dependent manner. Melanin content and tyrosinase activity were significantly reduced in response to treatment with NAPS and $C_{2}-ceramide$ at concentrations in a range between $1-5\;{\mu}M$. However, the levels of tyrosinase mRNA, as well as the levels of tyrosinase related protein-1 (TRP-1) and tyrosinase related protein-2 (TRP-2) genes and the level of tyrosinase protein remained unaffected by treatment with either NAPS or $C_{2}-ceramide$. We also attempted to determine the signaling pathway exploited by NAPS and $C_{2}-ceramide$. Interestingly, the phosphorylation of Akt/PKB at serine 473 by NAPS was reduced at the 5 minute mark, whereas $C_{2}-ceramide$ induced the phosphorylation of Akt/PKB at serine 473. Finally, Akt/PKB activity in the NAPS-treated cells was elevated in comparison with the untreated cells. LY294002, a specific PI3-K inhibitor which is located upstream of Akt/PKB, inhibited the phosphorylation of Akt/PKB, but induced an increase in melanin synthesis. These results suggest that the activation of Akt/PKB at serine 473 is related with the suppression of melanin production in the B16F10 mouse melanoma cells. Therefore, the mechanisms exploited by NAPS and $C_{2}-ceramide$ responsible for the depigmentation of B16F10 cells were concluded to involve the inhibition of melanosomal tyrosinase activity.

• Molecular & Cellular Toxicology
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• 제2권2호
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• pp.75-80
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• 2006

Biomarkers identify various stages and interactions on the pathway from exposure to disease. The three categories of biomarkers are those measuring susceptibility, exposure and effect. Susceptibility biomarkers are identifiable genetic variations affecting absorption, metabolism or response to environmental agents. Biomarkers of exposure indicate the amount of a foreign compound that is absorbed into the body. Biological measurements performed on human tissues are vastly expanding the capabilities of classical epidemiology, which has relied primarily on estimates of human exposure derived form chemical levels in the air, water, and other exposure routes. Biomarkers of exposure indicate the amount of a foreign compound that is absorbed into the body. Biological measurements performed on human tissues are vastly expanding the capabilities of classical epidemiology, which has relied primarily on estimates of human exposure derived form chemical levels in the air, water, and other exposure routes. The biomarker response is typical of chemical pollution by specific classes of compound, such as (i) heavy metals (mercury, cadmium, lead, zinc), responsible for the induction of metallothionein synthesis, and (ii) organochlorinated pollutants (PCBs, dioxins, DDT congeners) and polycyclic aromatic hydrocarbons (PAHs), which induce the mixed function oxygenase (MFO) involved in their bio transformations and elimination. Currently genomic researches are developed in human cDNA clone subarrays oriented toward the expression of genes involved in responses to xenobiotic metabolizing enzymes, cell cycle components, oncogenes, tumor suppressor genes, DNA repair genes, estrogen-responsive genes, oxidative stress genes, and genes known to be involved in apoptotic cell death. Several research laboratories in Korea for kicking off these Environmental Genomics were summarized.

• Molecular & Cellular Toxicology
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• 제2권4호
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• pp.266-272
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• 2006

Carpal tunnel syndrome (CTS) is one of the most common disorders by under pressure of the median nerve at the wrist in these days. However, pathological mechanism of CTS is unknown. We carried out this study to identify the changes of gene expression and to evaluate possible mechanism in CTS. 120 CTS patients and 30 control patients were included in this study. Patients with a history of diabetes, hypertension, thyroid diseases, and arthritis were excluded. CTS patients were divided to three experimental groups-Mild, Moderate, and Severe group-according to elecrodiagnosis. Radioactive cDNA microarrays (Nylon membrane including 1,152 genes) were used to examine the difference of gene expression profile in CTS. We identified up-regulated genes by more than 2.0 value of z-ratio, and down-regulated genes by less than-2.0 value of z-ratio. 20 genes such as the ITGAL, ITGAM, PECAM1, VIL2, TGFBR2, RAB7, RNF5 and NFKB1 were up-regulated, and 28 genes such as PRG5, CASP8, CDH1, IGFBP5, CBX3, HREV107, PIN, and WINT2 were down-regulated. These genes were related with TGF beta signaling pathway, NF-Kb signaling pathway, antiapoptotic pathway and T cell receptor signaling pathway. However, there were no differences in gene expression profiles according to severities of symptoms. We suggest that CTS could be related with proinflammatory mechanism and antiapoptotic mechanism.