OBJECTIVE: The individual cascades of the insulin-like growth factor-1 (IGF-1) signaling pathway and the molecular mechanism of aging have not been fully clarified. In the current study, we explored the effect of DNA polymerase delta 1 (POLD1) on the IGF-1 signaling pathway in cell aging. METHODS: First, we analyzed the relationship between IGF-1 and POLD1 expression in aging. To investigate the effect of IGF-1 on POLD1 expression and aging, the 2BS cells were incubated with young-age or old-age human serum, IGF-1 protein, or linsitinib. Next, the effect of IGF-1 on aging was examined in the 2BS cells with increased or decreased POLD1 expression to clarify the molecular mechanism. RESULTS: In this study, we found that IGF-1 expression increased and POLD1 expression decreased with aging in human serum and hippocampal tissues of SAMP8 mice, and a negative relationship between IGF-1 and POLD1 expression was observed. Furthermore, the cells cultured with old-age human serum or IGF-1 showed lower POLD1 expression and more pronounced senescence characteristics, and the effect could be reversed by treatment with linsitinib or overexpression of POLD1, while the effect of linsitinib on cell aging could be reversed with the knockdown of POLD1. CONCLUSION Taken collectively, our findings demonstrate that IGF-1 promotes aging by binding to IGF-1R and inhibiting the expression of POLD1. These findings offer a new target for anti-aging strategies.
Humans ; Animals ; Mice ; Insulin-Like Growth Factor I/pharmacology* ; Cellular Senescence ; Aging ; Hippocampus ; DNA Polymerase III

PURPOSE: Dermatofibrosarcoma protuberans (DFSP) carries a translocation resulting in the collagen type I alpha 1 (COL1A1)-platelet-derived growth factor beta (PDGFB) fusion gene, which is responsible for PDGFB activation. The purpose of this study is to evaluate the clinicopathological, genetic, and therapeutic features of DFSP in Korean patients. MATERIALS AND METHODS: Clinicopathological features of 37 patients with DFSP were reviewed. Multiplex reverse transcriptase-polymerase chain reaction (PCR) was carried out in 16 patients using formalin-fixed, paraffin-embedded tissues and specific primers for COL1A1 and PDGFB. RESULTS: The mean age of 37 patients was 37.4 years old. The most common tumor location was the trunk. All patients were treated primarily with surgery: 34 (91.7%) cases with Mohs micrographic surgery (MMS) and 3 (8.3%) cases with wide local excision. The median follow-up time was 33.7 months. Two patients, one in each treatment group, demonstrated local recurrence during the follow-up period. The COL1A1-PDGFB fusion gene was expressed in 14 (87.5%) cases, demonstrated by reverse transcriptase PCR analysis. No association was found among the different COL1A1-PDGFB fusion transcripts, the various histological subtypes and clinical features. CONCLUSION: Our results support the effectiveness of MMS in treating DFSP. The COL1A1-PDGFB fusion transcript was observed in 87.5% of patients. Therefore, COL1A1-PDGFB is a useful and accurate tool in diagnosing DFSP in Koreans.
Adolescent ; Adult ; Asian Continental Ancestry Group/*genetics ; Collagen Type I/*genetics ; DNA Primers ; Dermatofibrosarcoma/ethnology/*genetics/*pathology/surgery ; Female ; Humans ; Male ; Middle Aged ; Mohs Surgery ; Multiplex Polymerase Chain Reaction ; Neoplasm Recurrence, Local ; Oncogene Proteins, Fusion/*genetics ; Proto-Oncogene Proteins c-sis/*genetics ; Republic of Korea ; Reverse Transcriptase Polymerase Chain Reaction ; Skin Neoplasms/ethnology/*genetics/*pathology/surgery ; Treatment Outcome

PURPOSE: Dermatofibrosarcoma protuberans (DFSP) carries a translocation resulting in the collagen type I alpha 1 (COL1A1)-platelet-derived growth factor beta (PDGFB) fusion gene, which is responsible for PDGFB activation. The purpose of this study is to evaluate the clinicopathological, genetic, and therapeutic features of DFSP in Korean patients. MATERIALS AND METHODS: Clinicopathological features of 37 patients with DFSP were reviewed. Multiplex reverse transcriptase-polymerase chain reaction (PCR) was carried out in 16 patients using formalin-fixed, paraffin-embedded tissues and specific primers for COL1A1 and PDGFB. RESULTS: The mean age of 37 patients was 37.4 years old. The most common tumor location was the trunk. All patients were treated primarily with surgery: 34 (91.7%) cases with Mohs micrographic surgery (MMS) and 3 (8.3%) cases with wide local excision. The median follow-up time was 33.7 months. Two patients, one in each treatment group, demonstrated local recurrence during the follow-up period. The COL1A1-PDGFB fusion gene was expressed in 14 (87.5%) cases, demonstrated by reverse transcriptase PCR analysis. No association was found among the different COL1A1-PDGFB fusion transcripts, the various histological subtypes and clinical features. CONCLUSION: Our results support the effectiveness of MMS in treating DFSP. The COL1A1-PDGFB fusion transcript was observed in 87.5% of patients. Therefore, COL1A1-PDGFB is a useful and accurate tool in diagnosing DFSP in Koreans.
Adolescent ; Adult ; Asian Continental Ancestry Group/*genetics ; Collagen Type I/*genetics ; DNA Primers ; Dermatofibrosarcoma/ethnology/*genetics/*pathology/surgery ; Female ; Humans ; Male ; Middle Aged ; Mohs Surgery ; Multiplex Polymerase Chain Reaction ; Neoplasm Recurrence, Local ; Oncogene Proteins, Fusion/*genetics ; Proto-Oncogene Proteins c-sis/*genetics ; Republic of Korea ; Reverse Transcriptase Polymerase Chain Reaction ; Skin Neoplasms/ethnology/*genetics/*pathology/surgery ; Treatment Outcome

BACKGROUND: Human rhinoviruses are the major cause of asthma exacerbation in both children and adults. Recently, impaired antiviral interferon (IFN) response in asthmatics has been indicated as a primary reason of the susceptibility to respiratory virus, but the mechanism of defective IFN production is little understood to date. The expression of IFN regulatory factor 7 (IRF7), a transcriptional factor for virus-induced type I IFN production is known to be regulated epigenetically by DNA methylation. OBJECTIVE: We aimed to investigate the expression of IFN-α, IFN-β, and IRF7 in response to rhinovirus infection in the adult asthmatics and evaluate DNA methylation status of IRF7 gene promotor. METHODS: Twenty symptomatic adult asthmatics and 10 healthy subjects were enrolled and peripheral blood was collected from each subject. Peripheral blood mononuclear cells (PBMCs) were isolated, cultured, and ex vivo stimulated with rhinovirus-16. The mRNA expressions of IFN-α, IFN-β, and IRF7 were analyzed using real time quantitative polymerase chain reaction. Genomic DNA was isolated from untreated PBMCs and the methylation status of IRF7 gene promotor was investigated using bisulfite pyrosequencing. RESULTS: The mean age of asthmatics was 45.4 ± 15.7 years and 40% was male, which were not different with those of control group. Asthmatics showed significantly decreased mRNA expressions (relative expression to beta-actin) of IFN-α and IFN-β compared with normal control. The mRNA expression of IRF7 in the asthmatics was also significantly lower than those in the normal control. No significant difference of DNA methylation was observed between asthmatics and controls in all analyzed positions of IRF7 promotor CpG loci. CONCLUSION: The mRNA expression of type I IFN in response to rhinovirus was impaired in the PBMCs of adult asthmatics. The mRNA expression of IRF7, transcriptional factor inducing type I IFN was also reduced, but not caused by altered DNA methylation in the IRF7 gene promotor.
Adult ; Asthma ; Child ; DNA Methylation ; DNA ; Healthy Volunteers ; Humans ; Interferon Type I ; Interferons ; Male ; Methylation ; Polymerase Chain Reaction ; Rhinovirus ; RNA, Messenger

Antiporters ; genetics ; Base Sequence ; Female ; Glycogen Storage Disease Type I ; diagnosis ; genetics ; Humans ; Infant ; Monosaccharide Transport Proteins ; genetics ; Mutation ; Polymerase Chain Reaction ; Sequence Analysis, DNA

OBJECTIVEPrevious studies have demonstrated that two homozygous missense MYO1E mutations are associated with childhood autosomal recessive focal segmental glomerulosclerosis in steroid-resistant nephrotic syndrome (SRNS) families from Italy and Turkey. Non-disease-causing heterozygous MYO1E variants were also found in other SRNS patient cohorts. However, the role of MYO1E mutations in Chinese sporadic SRNS has not been established.

METHODPeripheral blood samples were collected for genetic analysis from 54 children with sporadic SRNS in Chinese Han ethnic group and a normal control group of 59 healthy adult volunteers. None of the patients carried mutations in NPHS2 or WT1. Genomic DNA was extracted from peripheral blood leukocytes. Twenty-eight exons and exon-intron boundaries of the MYO1E gene were amplified by polymerase chain reaction. Mutational analysis was performed by direct DNA sequencing and restriction endonuclease digestion.

RESULTFifty-one variants in the MYO1E gene were identified in 54 children with sporadic SRNS. Among them, 10 MYO1E mutations of IVS1-11T>C, IVS2-86T>A, 279T>C (D93D), IVS6-181G>A, 718C>T (L240F), 1678A>G (T560A), IVS16-35A>G, IVS18+48T>A, IVS19+38G>A and IVS25+13C>T were detected in 11 patients, whereas they were absent in the 59 normal Chinese controls. Forty-one variants in MYO1E were identified and all of them were published in single nucleotide polymorphism database from national center for biotechnology information. Furthermore, all the 10 MYO1E mutations were in heterozygous states.

CONCLUSIONMYO1E mutations are not a major cause of Chinese children with sporadic SRNS in the study.

Adolescent ; Case-Control Studies ; Child ; Child, Preschool ; China ; ethnology ; DNA Mutational Analysis ; Ethnic Groups ; genetics ; Exons ; Female ; Humans ; Infant ; Male ; Mutation ; genetics ; Myosin Type I ; genetics ; Nephrotic Syndrome ; congenital ; ethnology ; genetics ; Polymerase Chain Reaction ; Polymorphism, Genetic

In addition to single-nucleotide polymorphisms (SNP), copy number variation (CNV) is a major component of human genetic diversity. Among many whole-genome analysis platforms, SNP arrays have been commonly used for genomewide CNV discovery. Recently, a number of CNV defining algorithms from SNP genotyping data have been developed; however, due to the fundamental limitation of SNP genotyping data for the measurement of signal intensity, there are still concerns regarding the possibility of false discovery or low sensitivity for detecting CNVs. In this study, we aimed to verify the effect of combining multiple CNV calling algorithms and set up the most reliable pipeline for CNV calling with Affymetrix Genomewide SNP 5.0 data. For this purpose, we selected the 3 most commonly used algorithms for CNV segmentation from SNP genotyping data, PennCNV, QuantiSNP; and BirdSuite. After defining the CNV loci using the 3 different algorithms, we assessed how many of them overlapped with each other, and we also validated the CNVs by genomic quantitative PCR. Through this analysis, we proposed that for reliable CNV-based genomewide association study using SNP array data, CNV calls must be performed with at least 3 different algorithms and that the CNVs consistently called from more than 2 algorithms must be used for association analysis, because they are more reliable than the CNVs called from a single algorithm. Our result will be helpful to set up the CNV analysis protocols for Affymetrix Genomewide SNP 5.0 genotyping data.
Coat Protein Complex I ; DNA Copy Number Variations ; Genetic Variation ; Humans ; Polymerase Chain Reaction

PURPOSE: The specific aim of this study is to unravel a DNA copy number alterations, and to search for novel genes that are associated with the development of Korean gastric cancer. MATERIALS AND METHODS: We investigated a DNA copy number changes in 23 gastric adenocarcinomas by array-comparative genomic hybridization and quantitative real-time polymerase chain reaction analyses. Besides, the expression of UQCRFS1, which shows amplification in array-CGH, was examined in 186 gastric cancer tissues by an immunohistochemistry, and in 9 gastric cancer cell lines, as well as 24 gastric cancer tissues by immunoblotting. RESULTS: We found common gains at 48 different loci, and a common loss at 19 different loci. Amplification of UQCRFS1 gene at 19q12 was found in 5 (21.7%) of the 23 gastric cancers in an array-comparative genomic hybridization and DNA copy number were increased in 5 (20.0%) out of the 25 gastric cancer in quantitative real-time polymerase chain reaction. In immunohistochemistry, the overexpression of the protein was detected in 105 (56.5%) out of the 186 gastric cancer tissues. Statistically, there was no significant relationship between the overexpression of UQCRFS1 and clinicopathologic parameters (P>0.05). In parallel, the overexpression of UQCRFS1 protein was confirmed in 6 (66.7%) of the 9 gastric cancer cell lines, and 12 (50.0%) of the 24 gastric cancer tissues by immunoblotting. CONCLUSIONS: These results suggest that the overexpression of UQCRFS1 gene may contribute to the development and/or progression of gastric cancer, and further supported that mitochondrial change may serve as a potential cancer biomarker.
Adenocarcinoma ; Cell Line ; Coat Protein Complex I ; DNA ; DNA Copy Number Variations ; Immunohistochemistry ; Nucleic Acid Hybridization ; Real-Time Polymerase Chain Reaction ; Stomach Neoplasms

BACKGROUND: Ventilator-associated pneumonia (VAP) requires prompt and appropriate treatment. Since methicillin-resistant Staphylococcus aureus (MRSA) is a frequent pathogen in VAP, rapid identification of it, is pivotal. Our aim was to evaluate the utility of quantitative polymerase chain reaction (qPCR) as a useful method for etiologic diagnoses of MRSA pneumonia. METHODS: We performed qPCR for mecA, S. aureus-specific femA-SA, and S. epidermidis-specific femA-SE genes from bronchoalveolar lavage or bronchial washing samples obtained from clinically-suspected VAP. Molecular identification of MRSA was based on the presence of the mecA and femA-SA gene, with the absence of the femA-SE gene. To compensate for the experimental and clinical conditions, we spiked an internal control in the course of DNA extraction. We estimated number of colony-forming units per mL (CFU/mL) of MRSA samples through a standard curve of a serially-diluted reference MRSA strain. We compared the threshold cycle (Ct) value with the microbiologic results of MRSA. RESULTS: We obtained the mecA gene standard curve, which showed the detection limit of the mecA gene to be 100 fg, which corresponds to a copy number of 30. We chose cut-off Ct values of 27.94 (equivalent to 1x10(4) CFU/mL) and 21.78 (equivalent to 1x10(5) CFU/mL). The sensitivity and specificity of our assay were 88.9% and 88.9% respectively, when compared with quantitative cultures. CONCLUSION: Our results were valuable for diagnosing and identifying pathogens involved in VAP. We believe our modified qPCR is an appropriate tool for the rapid diagnosis of clinical pathogens regarding patients in the intensive care unit.
Adenosine ; Bronchoalveolar Lavage ; Coat Protein Complex I ; DNA ; Humans ; Critical Care ; Intensive Care Units ; Limit of Detection ; Methicillin Resistance ; Methicillin-Resistant Staphylococcus aureus ; Pneumonia ; Pneumonia, Ventilator-Associated ; Polymerase Chain Reaction ; Real-Time Polymerase Chain Reaction ; Sprains and Strains ; Stem Cells

OBJECTIVE: Tributyltin (TBT), an endocrine disrupting chemical, has been reported to decrease ovarian function by causing apoptosis in the ovary, but the mechanism is not fully understood. Therefore, we examined whether TBT increases the expression of adipogenesis-related genes in the ovary and the increased expression of these genes is associated with apoptosis induction. METHODS: Three-week-old Sprague-Dawley rats were orally administered TBT (1 or 10 mg/kg body weight) or sesame oil as a control for 7 days. The ovaries were obtained and weighed on day 8, and then they were fixed for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) or frozen for RNA extraction. Using the total RNA of the ovaries, adipogenesis- and apoptosis-related genes were analyzed by real-time polymerase chain reaction (PCR). RESULTS: The ovarian weight was significantly decreased in rats administered 10 mg/kg TBT compared to that in control rats. As determined by the TUNEL assay, the number of apoptotic follicles in ovary was significantly increased in rats administered 10 mg/kg TBT. The real-time PCR results showed that the expression of adipogenesis-related genes such as PPARgamma, aP2, CD36, and PEPCK was increased after TBT administration. In addition, apoptosis-related genes such as TNFalpha and TNFR1 were expressed more in the TBT-administered rats compared with the control rats. CONCLUSION: The present study demonstrates that TBT induces the expression of adipogenesis- and apoptosis-related genes in the ovary leading to apoptosis in the ovarian follicles. These results suggest that the increased expression of adipogenesis-related genes in the ovary by TBT exposure might induce apoptosis resulting in a loss of ovarian function.
Adipogenesis ; Animals ; Apoptosis ; DNA Nucleotidylexotransferase ; Female ; In Situ Nick-End Labeling ; Ovarian Follicle ; Ovary ; PPAR gamma ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Receptors, Tumor Necrosis Factor, Type I ; RNA ; Sesame Oil ; Trialkyltin Compounds ; Tumor Necrosis Factor-alpha