Spironolactone Attenuates Methylglyoxal-induced Cellular Dysfunction in MC3T3-E1 Osteoblastic Cells

Background@#Methylglyoxal (MG) is associated with the pathogenesis of age- and diabetes-related complications. Spironolactone is a competitive antagonist of aldosterone that is widely employed in the treatment of hypertension and heart failure. This study examined the effects of spironolactone on MG-induced cellular dysfunction in MC3T3-E1 osteoblastic cells. @*Methods@#MC3T3-E1 cells were treated with spironolactone in the presence of MG. The mitochondrial function, bone formation activity, oxidative damage, inflammatory cytokines, glyoxalase I activity, and glutathione (GSH) were measured. @*Results@#Pretreatment of MC3T3-E1 osteoblastic cells with spironolactone prevented MG-induced cell death, and improved bone formation activity. Spironolactone reduced MG-induced endoplasmic reticulum stress, production of intracellular reactive oxygen species, mitochondrial superoxides, cardiolipin peroxidation, and inflammatory cytokines. Pretreatment with spironolactone also increased the level of reduced GSH and the activity of glyoxalase I. MG induced mitochondrial dysfunction, but markers of mitochondrial biogenesis such as mitochondrial membrane potential, adenosine triphosphate, proliferator-activated receptor gamma coactivator 1α, and nitric oxide were significantly improved by treatment of spironolactone. @*Conclusion@#Spironolactone could prevent MG-induced cytotoxicity in MC3T3-E1 osteoblastic cells by reduction of oxidative stress. The oxidative stress reduction was explained by spironolactone's inhibition of advanced glycation end-product formation, restoring mitochondrial dysfunction, and anti-inflammatory effect.

Spironolactone Attenuates Methylglyoxal-induced Cellular Dysfunction in MC3T3-E1 Osteoblastic Cells

Background@#Methylglyoxal (MG) is associated with the pathogenesis of age- and diabetes-related complications. Spironolactone is a competitive antagonist of aldosterone that is widely employed in the treatment of hypertension and heart failure. This study examined the effects of spironolactone on MG-induced cellular dysfunction in MC3T3-E1 osteoblastic cells. @*Methods@#MC3T3-E1 cells were treated with spironolactone in the presence of MG. The mitochondrial function, bone formation activity, oxidative damage, inflammatory cytokines, glyoxalase I activity, and glutathione (GSH) were measured. @*Results@#Pretreatment of MC3T3-E1 osteoblastic cells with spironolactone prevented MG-induced cell death, and improved bone formation activity. Spironolactone reduced MG-induced endoplasmic reticulum stress, production of intracellular reactive oxygen species, mitochondrial superoxides, cardiolipin peroxidation, and inflammatory cytokines. Pretreatment with spironolactone also increased the level of reduced GSH and the activity of glyoxalase I. MG induced mitochondrial dysfunction, but markers of mitochondrial biogenesis such as mitochondrial membrane potential, adenosine triphosphate, proliferator-activated receptor gamma coactivator 1α, and nitric oxide were significantly improved by treatment of spironolactone. @*Conclusion@#Spironolactone could prevent MG-induced cytotoxicity in MC3T3-E1 osteoblastic cells by reduction of oxidative stress. The oxidative stress reduction was explained by spironolactone's inhibition of advanced glycation end-product formation, restoring mitochondrial dysfunction, and anti-inflammatory effect.

Characterization of Clostridium difficile Strains Isolated from Patients with C. difficile-associated Disease in Korea

OBJECTIVES: Studies on Clostridium difficile are rare in Korea. We investigated the epidemiological characteristics of C. difficile isolates from patients with C. difficile-associated disease (CDAD) in Korea. METHODS: Multiplex polymerase chain reaction was performed to detect the presence of tcdA and tcdB toxin genes. Antimicrobial susceptibility test was carried out by the disk-dilution method. C. difficile strains were subtyped by automated repetitive-element palindromic PCR (rep-PCR). RESULTS: Among patients with CDAD, 73 (25.8%), 32 (11.3%), 32 (11.3%), and 26 (9.2%) suffered from pneumonia, cancer or neoplasm, diabetes, and colitis, respectively. Of all stool samples, 43 samples (15.2%) were positive for C. difficile strains. We observed two expression patterns of toxin genes: tcdA+/tcdB+ (86% isolates) and tcdA−/tcdB+ (14% isolates), with all isolates expressing tcdB. Furthermore, some isolates were resistant to clindamycin (65%), ampicillin (56%), and cefazolin (40%), but all were susceptible to vancomycin and metronidazole. The tested samples were classified into diverse clusters using automated rep-PCR. CONCLUSION: Our findings revealed the characteristics and antibiotic resistance of C. difficile isolates from patients in Korea. The epidemiological data may provide valuable insight into development of treatment strategies for C. difficile infections in Korea.

Induction of Heat Shock Proteins and Antioxidant Enzymes in 2,3,7,8-TCDD-Induced Hepatotoxicity in Rats

2,3,7,8-Tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) is an environmental toxicant with a polyhalogenated aromatic hydrocarbon structure and is one of the most toxic man-made chemicals. Exposure to 2,3,7,8-TCDD induces reproductive toxicity, immunotoxicity, and hepatotoxicity. In this study, we evaluated how 2,3,7,8-TCDD-induced hepatotoxicity affect the expression of heat shock proteins and antioxidant enzymes using the real-time polymerase chain reaction (PCR) in rat. 2,3,7,8-TCDD increased heat shock protein (Hsp27, alpha-B-crystallin, Mortalin, Hsp105, and Hsp90s) and antioxidant enzymes (SOD-3, GST and catalase) expression after a 1 day exposure in livers of rats, whereas heat shock protein (alpha-B-crystallin, Hsp90, and GRP78) and antioxidant enzymes (SOD-1, SOD-3, catalase, GST, and GPXs) expression decreased on day 2 and then slowly recovered back to control levels on day 8. These results suggest that heat shock proteins and antioxidant enzymes were induced as protective mechanisms against 2,3,7,8-TCDD induced hepatotoxicity, and that prolonged exposure depressed their levels, which recovered to control levels due to reduced 2,3,7,8-TCDD induced hepatotoxicity.

Novel Mutation in PRKAR1A in Carney Complex

A case of Carney complex in a Korean patient is presented. The patient had the characteristics of Carney complex including skin lesions, positive family history, and multiple myxomas including a superficial angiomyxoma in the perianal area. An extensive genetic analysis revealed a novel mutation in the protein kinase A type I-a regulatory subunit (PRKAR1A) gene, but not in the phosphodiesterase type 11A (PDE11A) gene. This is the first case wherein extensive genetic studies were performed in a patient with Carney complex in Korea.

Expression of Growth Factors and Cytokines in Patients withDegenerated Disc Disease and Herniated Nucleus Pulposus

OBJECTIVE: To investigate the expression of cytokines and growth factors in disc specimens obtained from small numbers of patients with herniated nucleus pulposus (HNP) and degenerated disc disease (DDD). METHOD: A total of ten human intervertebral disc samples consisted of five painful degenerative discs and five herniated intervertebal discs were obtained from surgery. MRI and Western blot analyses on these disc samples were performed to determine the levels of disc degeneration and the expression levels of cytokines and growth factors. RESULTS: The levels of IL-6 were significantly greater in the DDD patients than in the HNP patients, but no statistical differences were observed in the expression of IL-1beta, IL-8 and TNF-alpha between the HNP and DDD patients. In addition, the expression of TGF beta was significantly higher in the DDD patients than in the HNP patients. CONCLUSION: The higher levels of cytokine and growth factor expression in the DDD than in the HNP show why discogenic patients usually have more severe back pain than patients with herniated discs.

Analysis of HPV-other Samples by Performing HPV DNA Sequencing

BACKGROUND: HPV-other samples are designated as being positive on HPV-PCR, but negative when using specific HPV hybridization probes. We wanted to determine the types on the HPV-other samples by performing sequencing, and to know the pathologic status of the uterine cervix according to the HPV type detected on sequencing. METHODS: For HPV genotying, we used the commercially available HPV DNA Chip test, which contains 15 types of high-risk HPV and 9 types of low-risk HPV. The HPV DNA sequencing was performed for the HPV-other samples of 209 patients who subsequently underwent cervical biopsy. RESULTS: For 204 of the 209 samples, the HPV types detected by sequencing were absent types at used HPV DNA chip. For the remaining 5 samples, sequencing was impossible due to mixed peaks. HPV-81 (19.6%), HPV-61 (18.6%), HPV-62 (16.7%) and HPV-84 (13.9%) were frequently detected. For the HPV-81, -62, -71, and -72 samples, most of the samples displayed normal or LSIL. However, HPV-84 and -61 were more associated with HSIL or worse, as compared to the other types. Conclusion: HPV-81, -61, -62 and -84 were frequently found on sequencing analysis of the HPV-other samples. The pathologic status was diverse, according to the HPV type detected on sequencing.

Prevalence and Genotype Distribution of Cervical Human Papillomavirus DNA in Korean Women: A Multicenter Study

Background: DNA prevalence and type distribution of human papillomavirus (HPV) varies geographically. We investigated HPV prevalence and type distribution in Korean women using the MyHPV DNA chip testing. Methods: A total of 2,368 women from five regions of the country underwent Pap smear examination and MyHPV chip testing. Results: Overall HPV positivity was 15.8% and 78.4% in women with normal and abnormal cytology, respectively. High-risk HPV infection was strongly correlated with cytological atypia. In women with abnormal cytology, the five most common HPV types were 16, 58, 18, 52, and 56/53, and HPV16 was significantly the most common type in most geographical regions. After HPV16, HPV58, and 52 were the next most frequently detected types. Women with normal cytology, in contrast, showed heterogeneity in HPV type distribution. High-grade intraepithelial lesions infected with HPV16, 18, 31 or 45 are more likely to progress to carcinoma. Conclusions: The HPV chip test can provide useful data regarding HPV positivity and type. The most common HPV type in Korean women with abnormal cytology is HPV16, with HPV58 and 52 being frequently present. Our data may have important implications for vaccination programs and the development of cervical screening.

Overexpression of Metastatic Tumor Antigen in Osteosarcoma: Comparison between Conventional High-Grade and Central Low-Grade Osteosarcoma / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: The metastatic tumor antigen (MTA) gene is a recently identified metastasis-associated gene which has implications in the signal transduction or regulation of gene expression. However, the expression of MTA in osteosarcoma and its potential relationship with metastasis have not been examined, forming the basis of this study. MATERIALS AND METHODS: We compared the expression levels of the MTA1 protein between 32 cases of high- grade osteosarcomas and 21 cases of low-grade osteosarcomas by immunohistochemistry. In addition, the mRNA expression levels of MTA1, 2, 3 in these osteosarcoma cell lines and control fibroblasts were evaluated by real-time quantitative PCR. RESULTS: MTA1 immunoreactivity was present in 81.25% of high-grade osteosarcoma specimens. Its expression was predominantly localized to the nucleus or cytoplasm of osteosarcoma cells. Thirteen (86.6%) of 15 patients who died of osteosarcomas displayed strong MTA1 expression. Both primary bone and pulmonary metastatic lesions exhibited MTA1 expression. All low- grade osteosarcomas were negative for MTA1 except for focal weak reactivity in two cases. The tested high-grade osteosarcoma cell lines showed marked amplification of MTA1 and MTA2 mRNA compared to control cells. CONCLUSION: These results suggest that MTA might be involved in the progression of high-grade osteosarcoma, particularly in hematogenous metastasis of osteosarcoma.

Characterization of Cyclic AMP Response Element (CRE) in the Promoter of the Rat Thyrotropin Releasing Hormone (TRH) Gene / 대한내분비학회지

We investigated whether the two variant elements of CRE(TGcCGTCA[5'CRE], TGACcTCA[3'CRE]) in the 5'flanking region of the rat TRH gene, which are different from the CRE consensus sequence(5'-TGACGTCA-3') by one base pair, are responsive to cAMP, and whether the one base pair difference is responsible for the degree of cAMP responsiveness of the gene. When CA 77 cells were stimulated with forskolin and isobutylmethylxanthine for 4 hours, the level of TRH mRNA was increased by only two fold. The transient gene expression study using serial 5'deletion of the TRH gene in PC12 cells showed that the region between-113 and-77, which includes 5'CRE, was crucial for the cAMP resonsiveness. When the plasmid, which contains the 30 bp oligonucleotide including either 5'CRE or 3'CRE ligated to the enhancerless RSV promoter, was transfected into PC12 cells, it did not significantly affect not only the basal transcription but cAMP responsiveness. The 65 bp oligonucleotide including both 5'CRE and 3'CRE, however, increased both of the basal transcription and cAMP-stimulated transcription by 2-3 fold. When the sequence of 5'CRE was converted to that of the CRE consensus by replacing one base pair, the cAMP responsiveness was increased by two fold although the basal transcription was not increased. The one base pair mutant of 3'CRE increased both of the basal and cAMP-stimulated transcription by 3-4 fold. These results suggest that there are the two variant CREs in rat TRH gene, which are relatively weak CRE compared to the CREs of other neuropeptide genes and cooperative for the activation of both the basal and cAMP-stimulated transcription. The one base pair difference of the variant CREs from the CRE consensus sequence is responsible for the weak responsiveness to cAMP.