Myung-Sang Kwon

Increased ferric iron content and iron-induced oxidative stress in the brains of scrapie-infected mice

Scrapie is a transmissible neurodegenerative disease of sheep and goats. The neuropathological changes include vacuolation, astrocytosis, the development of amyloid plaques in some instances, and neuronal loss. The mechanisms involved in neuronal cell death in scrapie are not known. Recently, we reported the presence of oxidative stress in the brains of scrapie-infected animals and suggested that this is the main mechanism that induces neuronal cell loss. It is known that oxidative stress induced by free radicals is associated with iron accumulation; this association led to an examination of the levels of iron (total iron, Fe(2+) and Fe(3+)) in the brains of control and scrapie-infected mice by biochemical methods. In the scrapie-infected group, both the level of total iron and the Fe(3+) level were significantly increased in cerebral cortex, striatum, and brainstem as compared to the values in the control group. A shift in the ratio of Fe(2+)/Fe(3+) was observed in the same regions of infected mice. Additionally, in this scrapie model, we confirmed the presence of oxidative stress, as evidenced by the increase of free malondialdehyde. These results suggest that iron metabolism is changed and that iron-induced oxidative stress partly contributes to neurodegeneration in scrapie infection.

Effect of transition metals (Mn, Cu, Fe) and deoxycholic acid (DA) on the conversion of PrPC to PrPres

The PMCA (protein misfolding cyclic amplification) technique has been shown to drive the amplification of misfolded prion protein by PrPSc seeds during several cycles of incubation‐sonication. Here, we report that cyclic amplification of normal hamster brain homogenates treated with a number of transition metals (manganese [Mn], copper [Cu], and iron [Fe]) leads to conversion of PrPC into protease‐resistant PrPres. The efficiency of PrPres formation and the glycoforms induced by Mn were different from those obtained by Cu and Fe. Previous results have shown higher Mn and lower Cu levels in the affinity‐purified PrPSc from the brain of prion diseases compared with normal hamster brain homogenates. We focused on Mn because we observed higher levels of Mn in whole brain, mitochondria, and scrapie‐associated fibril‐enriched fractions from the brains of animals with prion disease. In the presence of minute quantities of Mn‐induced PrPres template with a large amount of PrPC, PrPres amplification is observed. A metal chelater, EDTA reverses the effect of Mn on PrPres amplification, suggesting that Mn may play a role in the formation of PrPres. It has been proposed that metal‐catalyzed oxidation of PrP leads to the oxidation of amino acids and extensive aggregation of oxidized PrP. Carboxyl acids such as deoxycholic acid (DA) are oxidized molecules produced by 3′ oxidation pathway. In in vitro studies, the potent effect of Mn on PrPres amplification is augmented by DA in a dose‐dependent manner. On the basis of the evidence of the elevated Mn levels in scrapie‐associated fibril (SAF)‐enriched preparations from the brains of animals with prion disease, Mn‐loaded PrP and oxidized molecules such as carboxyl acids may contribute to the formation of the scrapie isoform of PrP in prion diseases.

Hematological and serum biochemical values in cynomolgus monkeys anesthetized with ketamine hydrochloride.

Abstract:  The effects of ketamine anesthesia on both hematological and serum biochemical variables were investigated in 19 male and 15 female cynomolgus monkeys. Blood samples were obtained from the cephalic vein within 30 minutes of an intramuscular injection of ketamine hydrochloride (10 mg/kg). Ketamine anesthesia caused a reduction in leukocyte counts and a significant reduction in lymphocytes percentages. Ketamine anesthesia also increased the serum activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and creatine phosphokinase (CPK), but reduced the serum concentrations of glucose, inorganic phosphate, sodium and potassium. The alterations of hematological and serum biochemical values will be discussed. These alterations should be considered when designing studies for and interpreting data from cynomolgus monkeys.

Differential Gene Expression Profiles of Radioresistant Non–Small-Cell Lung Cancer Cell Lines Established by Fractionated Irradiation: Tumor Protein p53-Inducible Protein 3 Confers Sensitivity to Ionizing Radiation

PURPOSE Despite the widespread use of radiotherapy as a local and regional modality for the treatment of cancer, some non-small-cell lung cancers commonly develop resistance to radiation. We thus sought to clarify the molecular mechanisms underlying resistance to radiation. METHODS AND MATERIALS We established the radioresistant cell line H460R from radiosensitive parental H460 cells. To identify the radioresistance-related genes, we performed microarray analysis and selected several candidate genes. RESULTS Clonogenic and MTT assays showed that H460R was 10-fold more resistant to radiation than H460. Microarray analysis indicated that the expression levels of 1,463 genes were altered more than 1.5-fold in H460R compared with parental H460. To evaluate the putative functional role, we selected one interesting gene tumor protein p53-inducible protein 3 (TP53I3), because that this gene was significantly downregulated in radioresistant H460R cells and that it was predicted to link p53-dependent cell death signaling. Interestingly, messenger ribonucleic acid expression of TP53I3 differed in X-ray-irradiated H460 and H460R cells, and overexpression of TP53I3 significantly affected the cellular radiosensitivity of H460R cells. CONCLUSIONS These results show that H460R may be useful in searching for candidate genes that are responsible for radioresistance and elucidating the molecular mechanism of radioresistance.

Inflammatory response in rat lungs with recurrent exposure to welding fumes: a transcriptomic approach.

As chronic exposure to welding fumes causes pulmonary diseases, such as pneumoconiosis, public concern has increased regarding continued exposure to these hazardous gases in the workplace. In a previous study, the inflammatory response to welding fume exposure was analysed in rat lungs in the case of recurrent exposure and recovery periods. Thus using lung samples, well-annotated by histological observation and biochemical analysis, this study examines the gene expression profiles to identify phenotype-anchored genes corresponding to lung inflammation and the repair phenomenon after recurrent welding fume exposure. Seven genes (Mmp12, Cd5l, LOC50101, LOC69183, Spp1, and Slc26a4) were found to be significantly up-regulated according to the severity of the lung injury. In addition, the transcription and translation of Trem2, which was up-regulated in response to the repair process, were validated using a real-time polymerase chain reaction, Western blotting, and immunohistochemistry. The differentially expressed genes in the exposure and recovery groups were also classified using k-means and hierarchical clustering, plus their toxicological function and canonical pathways were further analysed using Ingenuity Pathways Analysis Software. As a result, this comprehensive and integrative analysis of the transcriptional changes that occur during repeated exposure provides important information on the inflammation and repair processes after welding-fume-induced lung injury.

Blockade of hERG K(+) channel by antimalarial drug, primaquine.

Lengthening of the Q-T interval and proarrhythmia are adverse effects associated with antimalarial agents. Also, lengthening of the Q-T interval is a definite outcome when patients are administered with an overdose of primaquine. Inhibition of potassium current IKr and resultant QT prolongation is suggested as the reason behind drug-induced arrhythmias. The present study investigated the molecular mechanisms of voltage-dependent inhibition of human Ether-a-go-go Related Gene (hERG) delayed rectifier K+ channels expressed in HEK-293 cells by primaquine. Primaquine inhibited hERG current in a concentration-dependent manner with the half-maximal inhibitory concentration (IC50) of 21.5 µM. The voltage-dependent inhibition of hERG current resulted in the activation curve to be shifted to a negative voltage after primaquine exposure in a dose-dependent manner. Blockade of hERG by primaquine was also found to be time-dependent, occurring rather rapidly. Blockade of wild-type hERG channel by primaquine was similar to those of both the S6 residue hERG mutants (F656A and Y652A) and the pore region mutants (T623A). In conclusion, these results indicate that primaquine preferentially inhibits the hERG potassium channel, but blockade of hERG channel by primaquine may not be related to the S6 residue or the pore region, but may be induced through other pathways such as binding other region or effect by drug binding receptor which indicates a need for further exploration.

Toxicoproteomic analysis of phalloidin-induced cholestasis in mouse liver

Phalloidin induces cholestasis by preventing microfilament depolymerization. Phalloidin has been widely used as an agent to induce intrahepatic cholestasis in experimental animals. The objective of this study was to examine the effects of phalloidin on protein expression profiles in mouse liver, so as to identify potential biomarkers of intrahepatic cholestasis. Phalloidin was administered to BALB/c mice at a predetermined dose of 1 mg/kg for 7 days, and phalloidin-induced cholestasis was observed. Hepatic protein expression was investigated via two-dimensional (2D) electrophoresis, and 21 protein spots showing significantly different expression between the treated and control groups were excised from the gels and identified by MALDI-TOF/TOF. The identified proteins were involved in cytoskeletal changes, lipid metabolism, gluconeogenesis, detoxification, and transport mechanisms. Among these proteins, the up-regulation of HSP90-β in phalloidin-treated mice was confirmed by Western blot analysis and then by RT-PCR, indicating that it may serve as a useful biomarker of cholestasis. In summary, these results provide insight into the mechanism involved in phalloidin-induced cytoskeletal change and cholestasis.

Genome-Wide Transcriptional Response During the Development of Bleomycin-Induced Pulmonary Fibrosis in Sprague-Dawley Rats

Pulmonary fibrosis is a common consequence of many lung diseases and a leading cause of morbidity and mortality. The molecular mechanisms underlying the development of pulmonary fibrosis remain poorly understood. One model used successfully to study pulmonary fibrosis over the past few decades is the bleomycin-induced pulmonary fibrosis model. We aimed to identify the genes associated with fibrogenesis using an Affymetrix GeneChip system in a bleomycin-induced rat model for pulmonary fibrosis. To confirm fibrosis development, several analyses were performed, including cellular evaluations using bronchoalveolar lavage fluid, measurement of lactate dehydrogenase activity, and histopathological examinations. Common aspects of pulmonary fibrosis such as prolonged inflammation, immune cell infiltration, emergence of fibroblasts, and deposition of extracellular matrix and connective tissue elements were observed. Global gene expression analysis revealed significantly altered expression of genes (≥ 1.5-fold, p < 0.05.) in a time-dependent manner during the development of pulmonary fibrosis. Our results are consistent with previous results of well-documented gene expression. Interestingly, the expression of triggering receptor expressed on myeloid cells 2 (Trem2) , secreted phosphoprotein 1 (Spp1) , and several proteases such as Tpsab1, Mcpt1, and Cma1 was considerably induced in the lung after bleomycin treatment, despite little evidence that they are involved in pulmonary fibrogenesis. These data will aid in our understanding of fibrogenic mechanisms and contribute to the identification of candidate biomarkers of fibrotic disease development.

The stoichiometric relationship between KCNH-2 and KCNE-2 in IKr channel formation ☆

KCNH-2 and KCNE-2 may encode the channel-forming alpha- and regulatory beta-subunits, respectively, of I(Kr) channels, which are involved in inherited or acquired long QT syndrome. However, in contrast to other multimeric channels, the stoichiometry of KCNH-2 and KCNE-2, which should be reasonably maintained if they are to be accepted as the components of a multi-molecular complex, has not been established, yet. In this study, we found that the protein expression of KCNE-2 was adequate to support the formation of a complex with KCNH-2; however, the level of transcription was not. This finding, together with previous data from electrophysiological and molecular biological studies, supports that KCNH-2 and KCNE-2 are molecular components of I(Kr) channels.

Analysis of Gene Expression in 4,4'-Methylenedianiline-induced Acute Hepatotoxicity

4,4′-Methylenedianiline (MDA) is an aromatic amine that is widely used in the industrial synthetic process. Genotoxic MDA forms DNA adducts in the liver and is known to induce liver damage in human and rats. To elucidate the molecular mechanisms associated with MDA-induced hepatotoxicity, we have identified genes differentially expressed by microarray approach. BALB/c male mice were treated once daily with MDA (20 mg/kg) up to 7 days via intraperitoneal injection (i.p.) and hepatic damages were revealed by histopathological observation and elevation of serum marker enzymes such as AST, ALT, ALP, cholesterol, DBIL, and TBIL. Microarray analysis showed that 952 genes were differentially expressed in the liver of MDA-treated mice and their biological functions and canonical pathways were further analyzed using Ingenuity Pathways Analysis (IPA). Toxicological functional analysis showed that genes related to hepatotoxicity such hyperplasia/hyperproliferation (Timpl), necrosis/cell death (Cd14, Mt1f, Timpl, and Pmaipl), hemorrhaging (Mt1f), cholestasis (Akr1c3, Hpx, and Slc10a2), and inflammation (Cd14 and Hpx) were differentially expressed in MDA-treated group. This gene expression profiling should be useful for elucidating the genetic events associated with aromatic amine-induced hepatotoxicity and for discovering the potential biomarkers for hepatotoxicity.