Mutational analysis of whole mitochondrial DNA in patients with MELAS and MERRF diseases

Choi, Byung-Ok,Hwang, Jung-Hee,Cho, Eun-Min,Jeong, Eun-Hye,Hyun, Young-Se,Jeon, Hyeon-Jeong,Seong, Ki-Min,Cho, Nam-Soo,Chung, Ki-Wha Korean Society for Biochemistry and Molecular Bion 2010 Experimental and molecular medicine Vol.42 No.6

Mitochondrial diseases are clinically and genetically heterogeneous disorders, which make the exact diagnosis and classification difficult. The purpose of this study was to identify pathogenic mtDNA mutations in 61 Korean unrelated families (or isolated patients) with MELAS or MERRF. In particular, the mtDNA sequences were completely determined for 49 patients. From the mutational analysis of mtDNA obtained from blood, 5 confirmed pathogenic mutations were identified in 17 families, and 4 unreported pathogenically suspected mutations were identified in 4 families. The m.3243A>G in the tRNA$^{Leu(UUR)}$ was predominantly observed in 10 MELAS families, and followed by m.8344A>G in the tRNA$^{Lys}$ of 4 MERRF families. Most pathogenic mutations showed heteroplasmy, and the rates were considerably different within the familial members. Patients with a higher rate of mutations showed a tendency of having more severe clinical phenotypes, but not in all cases. This study will be helpful for the molecular diagnosis of mitochondrial diseases, as well as establishment of mtDNA database in Koreans.

Snapshot of degenerative aging of porcine intervertebral disc: a model to unravel the molecular mechanisms

Cho, Hong-Sik,Park, Sang-Hyug,Lee, Sang-Min,Kang, Mi-Ji,Hasty, Karen A.,Kim, Song-Ja Korean Society for Biochemistry and Molecular Bion 2011 Experimental and molecular medicine Vol.43 No.6

Larger animal models, such as porcine, have been validated as appropriate models of the human disc with respect to biomechanics and biochemistry. They are advantageous for research as the models are relatively straightforward to prepare and easily obtainable for research to perform surgical techniques. The intention of this study was to quantitatively analyze gene expression for collagen and proteoglycan components of the extracellular matrix and for collagenase (MMP-1) in porcine discs of varying ages (Newborn; 2-3weeks, Mature; 6-9 month, Older; 2-3 years). In this study, we observed that the cell number and GAG (glycosaminoglycan) formation dramatically decreased with aging. Also, gene expression in the annulus fibrosus (AF) and nucleus pulposus (NP) cells changed with aging. The level of MMP-1 mRNA increased with age and both type I, II collagens decreased with age. The level of aggrecan mRNA was highest in the mature group and decreased significantly with aging. In the mature group, MMP-1 expression was minimal compared to the newborn group. In AF cells, type II collagen was expressed at a high level in the mature group with a higher level of aggrecan, when aged NP showed a decrease in type II collagen. The model of IVD degeneration in the porcine disc shows many changes in gene expression with age that have been previously documented for human and may serve as a model for studying changes in IVD metabolism with age. We concluded that the porcine model is excellent to test hypotheses related to disc degeneration while permitting time-course study in biologically active systems.

Rottlerin enhances IL-$1{\beta}$-induced COX-2 expression through sustained p38 MAPK activation in MDA-MB-231 human breast cancer cells

Park, Eun-Jung,Kwon, Taeg-Kyu Korean Society for Biochemistry and Molecular Bion 2011 Experimental and molecular medicine Vol.43 No.12

Cyclooxygenase-2 (COX-2) is an important enzyme in inflammation. In this study, we investigated the underlying molecular mechanism of the synergistic effect of rottlerin on interleukin$1{\beta}$ (IL-$1{\beta}$)-induced COX-2 expression in MDA-MB-231 human breast cancer cell line. Treatment with rottlerin enhanced IL-$1{\beta}$-induced COX-2 expression at both the protein and mRNA levels. Combined treatment with rottlerin and IL-$1{\beta}$ significantly induced COX-2 expression, at least in part, through the enhancement of COX-2 mRNA stability. In addition, rottlerin and IL-$1{\beta}$ treatment drove sustained activation of p38 Mitogen-activated protein kinase (MAPK), which is involved in induced COX-2 expression. Also, a pharmacological inhibitor of p38 MAPK (SB 203580) and transient transfection with inactive p38 MAPK inhibited rottlerin and IL-$1{\beta}$-induced COX-2 upregulation. However, suppression of protein kinase C ${\delta}$ (PKC ${\delta}$) expression by siRNA or over-expression of dominant-negative PKC ${\delta}$(DN-PKC-${\delta}$) did not abrogate the rottlerin plus IL-$1{\beta}$-induced COX-2 expression. Furthermore, rottlerin also enhanced tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$), phorbol myristate acetate (PMA), and lipopolysaccharide (LPS)-induced COX-2 expression. Taken together, our results suggest that rottlerin causes IL-$1{\beta}$-induced COX-2 upregulation through sustained p38 MAPK activation in MDA-MB-231 human breast cancer cells.

Insulin-dependent suppression of cholesterol $7{\alpha}$-hydroxlase is a possible link between glucose and cholesterol metabolisms

Park, Wook-Ha,KimPak, Young-Mi Korean Society for Biochemistry and Molecular Bion 2011 Experimental and molecular medicine Vol.43 No.10

Cholesterol $7{\alpha}$-hydroxylase (CYP7A1) regulates the balance between cholesterol supply and metabolism by catalyzing the rate-limiting step of bile acid biosynthesis. The transcriptional activity of CYP7A1 is tightly controlled by various nuclear receptors. A forkhead transcription factor O1 (FOXO1) plays a critical role in metabolism, and insulin inactivates FOXO1 through Akt-dependent phosphorylation and nuclear exclusion. We investigated the role of insulin- Akt-FOXO1 signaling pathway in CYP7A1 transcriptional regulation since we found putative insulin-response elements, FOXO1 binding sequences, in both rat and human CYP7A1 promoters. However, ectopic expression of FOXO1 increased the rat CYP7A1-, but mildly reduced human CYP7A1-promoter activities in a dose-dependent manner. Similarly to bile acids, insulin treatment increased small heterodimer partner (SHP) mRNA rapidly and transiently, leading to the suppression of CYP7A1 transcription in both human and rodents. Chromatin immunoprecipitation showed that FOXO1 directly bound to rat CYP1A1 promoter in the absence of insulin. FOXO1 binding to the rat promoter was diminished by insulin treatment as well as by expression of SHP. Our results suggest that the stimulation of insulin- signaling pathway of Akt-FOXO1 and SHP expression may regulate cholesterol/bile acid metabolisms in liver, linking carbohydrate and cholesterol metabolic pathways. A prolonged exposure of insulin in hyperinsulinemic insulin resistance or diabetic status represses CYP7A1 transcription and bile acid biosynthesis through SHP induction and FOXO1 inactivation, leading to impairment of the hepatic cholesterol/bile acid metabolisms.

Preclinical studies for pharmacokinetics and biodistribution of Ad-stTRAIL, an adenovirus delivering secretable trimeric TRAIL for gene therapy

Kim, Chae-Young,Park, Soon-Hye,Jeong, Moon-Sup,Kwon, O-Seo,Doh, Hyoun-Mie,Kang, Su-Hyung,Robbins, Paul D.,Kim, Byong-Moon,Seol, Dai-Wu,Kim, Byung-Gee Korean Society for Biochemistry and Molecular Bion 2011 Experimental and molecular medicine Vol.43 No.10

Malignant glioma is the most frequent type in brain tumors. The prognosis of this tumor has not been significantly improved for the past decades and the average survival of patients is less than one year. Thus, an effective novel therapy is urgently needed. TNF-related apoptosis inducing ligand (TRAIL), known to have tumor cell-specific killing activity, has been investigated as a novel therapeutic for cancers. We have developed Ad-stTRAIL, an adenovirus delivering secretable trimeric TRAIL for gene therapy and demonstrated the potential to treat malignant gliomas. Currently, this Ad-stTRAIL gene therapy is under phase I clinical trial for malignant gliomas. Here, we report preclinical studies for Ad-stTRAIL carried out using rats. We delivered Ad-stTRAIL intracranially and determined its pharmacokinetics and biodistribution. Most Ad-stTRAIL remained in the delivered site and the relatively low number of viral genomes was detected in the opposite site of brain and cerebrospinal fluid. Similarly, only small portion of the viral particles injected was found in the blood plasma and major organs and tissues, probably due to the brain-blood barrier. Multiple administrations did not lead to accumulation of Ad-stTRAIL at the injection site and organs. Repeated delivery of Ad-stTRAIL did not show any serious side effects. Our data indicate that intracranially delivered Ad-stTRAIL is a safe approach, demonstrating the potential as a novel therapy for treating gliomas.

Comparative study of codon substitution patterns in foot-and-mouth disease virus (serotype O)

Ahn, In-Sung,Bae, Se-Eun,Son, Hyeon-Seok Korean Society for Biochemistry and Molecular Bion 2011 Experimental and molecular medicine Vol.43 No.10

We compared genetic variations in the VP1 gene of foot-and-mouth disease viruses (FMDVs) isolated since 2000 from various region of the world. We analyzed relative synonymous codon usage (RSCU) and phylogenetic relationship between geographical regions, and calculated the genetic substitution patterns between Korean isolate and those from other countries. We calculated the ratios of synonymously substituted codons (SSC) to all observed substitutions and developed a new analytical parameter, EMC (the ratio of exact matching codons within each synonymous substitution group) to investigate more detailed substitution patterns within each synonymous codon group. We observed that FMDVs showed distinct RSCU patterns according to phylogenetic relationships in the same serotype (serotype O). Moreover, while the SSC and EMC values of FMDVs decreased according to phylogenetic distance, G + C composition at the third codon position was strictly conserved. Although there was little variation among the SSC values of 18 amino acids, more dynamic differences were observed in EMC values. The EMC values of 4- and 6-fold degenerate amino acids showed significantly lower values while most 2-fold degenerate amino acids showed no significant difference. Our findings suggest that different EMC patterns among the 18 amino acids might be an important factor in determining the direction of evolution in FMDV.

$In$ $vitro$ migration capacity of human adipose tissue-derived mesenchymal stem cells reflects their expression of receptors for chemokines and growth factors

Baek, Sun-Jin,Kang, Sung-Keun,Ra, Jeong-Chan Korean Society for Biochemistry and Molecular Bion 2011 Experimental and molecular medicine Vol.43 No.10

The homing properties of adipose tissue-derived mesenchymal stem cells (AdMSCs) have stimulated intravenous applications for their use in stem cell therapy. However, the soluble factors and corresponding cellular receptors responsible for inducing chemotaxis of AdMSCs have not yet been reported. In the present study, the migration capacity of human AdMSCs (hAdMSCs) toward various cytokines or growth factors (GFs) and the expression of their receptors were determined. In a conventional migration assay, PDGF-AB, TGF-${\beta}1$, and TNF-${\alpha}$ showed the most effective chemoattractant activity. When AdMSCs were pre-incubated with various chemokines or GF, and then allowed to migrate toward medium containing 10% FBS, those preincubated with TNF-${\alpha}$ showed the highest migratory activity. Next, hAdMSCs were either pre-incubated or not with TNF-${\alpha}$, and allowed to migrate in response to various GFs or chemokines. Prestimulation with TNF-${\alpha}$ increased the migration activity of hAdMSCs compared to unstimulated hAdMSCs. When analyzed by FACS and RT-PCR methods, hAdMSCs were found to express C-C chemokine receptor type 1 (CCR1), CCR7, C-X-C chemokine receptor type 4 (CXCR4), CXCR5, CXCR6, EGF receptor, fibroblast growth factor receptor 1, TGF-${\beta}$ receptor 2, TNF receptor superfamily member 1A, PDGF receptor A and PDGF receptor B at both the protein and the mRNA levels. These results indicate that the migration capacity of hAdMSCs is controlled by various GFs and chemokines. Prior $in$ $vitro$ modulation of the homing capacity of hAdMSCs could stimulate their movement into injured sites $in$ $vivo$ when administered intravenously, thereby improving their therapeutic potential.

Simvastatin inhibits osteoclast differentiation by scavenging reactive oxygen species

Moon, Ho-Jin,Kim, Sung-Eun,Yun, Young-Pil,Hwang, Yu-Shik,Bang, Jae-Beum,Park, Jae-Hong,Kwon, Il-Keun Korean Society for Biochemistry and Molecular Bion 2011 Experimental and molecular medicine Vol.43 No.11

Osteoclasts, together with osteoblasts, control the amount of bone tissue and regulate bone remodeling. Osteoclast differentiation is an important factor related to the pathogenesis of bone-loss related diseases. Reactive oxygen species (ROS) acts as a signal mediator in osteoclast differentiation. Simvastatin, which inhibits 3-hydroxy-3-methylglutaryl coenzyme A, is a hypolipidemic drug which is known to affect bone metabolism and suppresses osteoclastogenesis induced by receptor activator of nuclear factor-${\kappa}B$ ligand (RANKL). In this study, we analyzed whether simvastatin can inhibit RANKL-induced osteoclastogenesis through suppression of the subsequently formed ROS and investigated whether simvastatin can inhibit $H_2O_2$-induced signaling pathways in osteoclast differentiation. We found that simvastatin decreased expression of tartrate-resistant acid phosphatase (TRAP), a genetic marker of osteoclast differentiation, and inhibited intracellular ROS generation in RAW 264.7 cell lines. ROS generation activated $NF-{\kappa}B$, protein kinases B (AKT), mitogen-activated protein kinases signaling pathways such as c-JUN N-terminal kinases, p38 MAP kinases as well as extracellular signal- regulated kinase. Simvastatin was found to suppress these $H_2O_2$-induced signaling pathways in osteoclastogenesis. Together, these results indicate that simvastatin acts as an osteoclastogenesis inhibitor through suppression of ROS-mediated signaling pathways. This indicates that simvastatin has potential usefulness for osteoporosis and pathological bone resorption.

Higher infiltration by Th17 cells compared with regulatory T cells is associated with severe acute T-cell-mediated graft rejection

Chung, Byung-Ha,Oh, Hye-Jwa,Piao, Shang Guo,Sun, In-O,Kang, Seok-Hui,Choi, Sun-Ryoung,Park, Hoon-Suk,Choi, Bum-Soon,Choi, Yeong-Jin,Park, Cheol-Whee,Kim, Yong-Soo,Cho, Mi-La,Yang, Chul-Woo Korean Society for Biochemistry and Molecular Bion 2011 Experimental and molecular medicine Vol.43 No.11

The aim of this study was to evaluate whether the Th17 and Treg cell infiltration into allograft tissue is associated with the severity of allograft dysfunction and tissue injury in acute T cell-mediated rejection (ATCMR). Seventy-one allograft tissues with biopsy-proven ATCMR were included. The biopsy specimens were immunostained for FOXP3 and IL-17. The allograft function was assessed at biopsy by measuring serum creatinine (Scr) concentration, and by applying the modified diet in renal disease (MDRD) formula, which provides the estimated glomerular filtration rate (eGFR). The severity of allograft tissue injury was assessed by calculating tissue injury scores using the Banff classification. The average numbers of infiltrating Treg and Th17 cells were $11.6{\pm}12.2cells/mm^2$ and $5.6{\pm}8.0cells/mm^2$, respectively. The average Treg/Th17 ratio was $5.6{\pm}8.2$. The Treg/Th17 ratio was significantly associated with allograft function (Scr and MDRD eGFR) and with the severity of interstitial injury and tubular injury ($P$ < 0.05, all parameters). In separate analyses of the number of infiltrating Treg and Th17 cells, Th17 cell infiltration was significantly associated with allograft function and the severity of tissue injury. By contrast, Treg cell infiltration was not significantly associated with allograft dysfunction or the severity of tissue injury. The results of this study show that higher infiltration of Th17 cell compared with Treg cell is significantly associated with the severity of allograft dysfunction and tissue injury.

Down-regulation of survivin suppresses uro-plasminogen activator through transcription factor JunB

Lee, Kyung-Hee,Choi, Eun-Young,Koh, Sung-Ae,Kim, Min-Kyoung,Kim, Kyeong-Ok,Lee, Si-Hyung,Jang, Byung-Ik,Kim, Se-Won,Kim, Sang-Woon,Song, Sun-Kyo,Choi, Joon-Hyuk,Kim, Jae-Ryong Korean Society for Biochemistry and Molecular Bion 2011 Experimental and molecular medicine Vol.43 No.9

Survivin, a member of the inhibitors of apoptosis protein family, is expressed during development and in various human cancers. However, the clinical relevance of survivin in cancer is still a matter of debate. Genes induced by hepatocyte growth factor (HGF) were screened using cDNA microarray technology in the stomach cancer cell lines, NUGC3 and MKN28. The levels of JunB, survivin, and uro-plasminogen activator (uPA) were up-regulated in cells treated with HGF in a dose-dependent manner. HGF-induced up regulation of JunB, survivin, and uPA was inhibited by pre-treatment with a MEK inhibitor (PD 98059). HGF-induced up-regulation of uPA was repressed by survivin knockdown. HGF enhanced the binding activity of JunB to the survivin promoter in control cells, but not in the JunB-shRNA cells. Transfection with survivin- shRNA resulted in a decrement of cell proliferation, as determined with MTT assays. In an $in$ $vitro$ invasion assay, significantly fewer cells transfected with survivin shRNA than control cells were able to invade across a Matrigel membrane barrier. In conclusion, survivin appeared to play an important role in the up-regulation of uPA induced by HGF $via$ JunB and might contribute to HGF-mediated tumor invasion and metastasis, which may serve as a promising target for gastric cancer therapy.