Jung-Suk Sung

Removal of Oxidative DNA Damage via FEN1-Dependent Long-Patch Base Excision Repair in Human Cell Mitochondria

ABSTRACT Repair of oxidative DNA damage in mitochondria was thought limited to short-patch base excision repair (SP-BER) replacing a single nucleotide. However, certain oxidative lesions cannot be processed by SP-BER. Here we report that 2-deoxyribonolactone (dL), a major type of oxidized abasic site, inhibits replication by mitochondrial DNA (mtDNA) polymerase γ and interferes with SP-BER by covalently trapping polymerase γ during attempted dL excision. However, repair of dL was detected in human mitochondrial extracts, and we show that this repair is via long-patch BER (LP-BER) dependent on flap endonuclease 1 (FEN1), not previously known to be present in mitochondria. FEN1 was retained in protease-treated mitochondria and detected in mitochondrial nucleoids that contain known mitochondrial replication and transcription proteins. Results of immunofluorescence and subcellular fractionation studies were also consistent with the presence of FEN1 in the mitochondria of intact cells. Immunodepletion experiments showed that the LP-BER activity of mitochondrial extracts was strongly diminished in parallel with the removal of FEN1, although some activity remained, suggesting the presence of an additional flap-removing enzyme. Biological evidence for a FEN1 role in repairing mitochondrial oxidative DNA damage was provided by RNA interference experiments, with the extent of damage greater and the recovery slower in FEN1-depleted cells than in control cells. The mitochondrial LP-BER pathway likely plays important roles in repairing dL lesions and other oxidative lesions and perhaps in normal mtDNA replication.

Roles of base excision repair subpathways in correcting oxidized abasic sites in DNA.

Base excision DNA repair (BER) is fundamentally important in handling diverse lesions produced as a result of the intrinsic instability of DNA or by various endogenous and exogenous reactive species. Defects in the BER process have been associated with cancer susceptibility and neurodegenerative disorders. BER funnels diverse base lesions into a common intermediate, apurinic/apyrimidinic (AP) sites. The repair of AP sites is initiated by the major human AP endonuclease, Ape1, or by AP lyase activities associated with some DNA glycosylases. Subsequent steps follow either of two distinct BER subpathways distinguished by repair DNA synthesis of either a single nucleotide (short‐patch BER) or multiple nucleotides (long‐patch BER). As the major repair mode for regular AP sites, the short‐patch BER pathway removes the incised AP lesion, a 5′‐deoxyribose‐5‐phosphate moiety, and replaces a single nucleotide using DNA polymerase (Polβ). However, short‐patch BER may have difficulty handling some types of lesions, as shown for the C1′‐oxidized abasic residue, 2‐deoxyribonolactone (dL). Recent work indicates that dL is processed efficiently by Ape1, but that short‐patch BER is derailed by the formation of stable covalent crosslinks between Ape1‐incised dL and Polβ. The long‐patch BER subpathway effectively removes dL and thereby prevents the formation of DNA–protein crosslinks. In coping with dL, the cellular choice of BER subpathway may either completely repair the lesion, or complicate the repair process by forming a protein–DNA crosslink.

Long-patch Base Excision DNA Repair of 2-Deoxyribonolactone Prevents the Formation of DNA-Protein Cross-links with DNA Polymerase β

Oxidized abasic sites are a major form of DNA damage induced by free radical attack and deoxyribose oxidation. 2-Deoxyribonolactone (dL) is a C1′-oxidized abasic site implicated in DNA strand breakage, mutagenesis, and formation of covalent DNA-protein cross-links (DPCs) with repair enzymes such as DNA polymerase β (polβ). We show here that mammalian cell-free extracts incubated with Ape1-incised dL substrates under non-repair conditions give rise to DPCs, with a major species dependent on the presence of polβ. DPC formation was much less under repair than non-repair conditions, with extracts of either polβ-proficient or -deficient cells. Partial base excision DNA repair (BER) reconstituted with purified enzymes demonstrated that Flap endonuclease 1 (FEN1) efficiently excises a displaced oligonucleotide containing a 5′-terminal dL residue, as would be produced during long-patch (multinucleotide) BER. Simultaneous monitoring of dL repair and dL-mediated DPC formation demonstrated that removal of the dL residue through the combined action of strand-displacement DNA synthesis by polβ and excision by FEN1 markedly diminished DPC formation with the polymerase. Analysis of the patch size distribution associated with DNA repair synthesis in cell-free extracts showed that the processing of dL residues is associated with the synthesis of ≥2 nucleotides, compared with predominantly single nucleotide replacement for regular abasic sites. Our observations reveal a cellular repair process for dL lesions that avoids formation of DPCs that would threaten the integrity of DNA and perhaps cell viability.

Analysis of Base Excision DNA Repair of the Oxidative Lesion 2‐Deoxyribonolactone and the Formation of DNA–Protein Cross‐Links

DNA base lesions arising from oxidation or alkylation are processed primarily by the base excision repair pathway (BER). The damaged bases are excised by DNA N-glycosylases, which generate apurinic/apyrimidinic (AP) sites; AP sites produced by hydrolytic decay of DNA or the spontaneous loss of damaged bases are also processed by BER. Free radicals produce various types of abasic lesions as oxidative damage. This chapter focuses on the analysis of DNA repair and other reactions that occur with the lesion 2-deoxyribonolactone (dL), which has received much attention recently. DNA substrates with site-specific dL lesions are generated by photolysis of a synthetic precursor residue; both small oligonucleotide and plasmid-based substrates can be produced. The dL residue is readily incised by AP endonucleases such as the mammalian Ape1 protein, which would bring the lesion into BER. However, the second enzyme of the canonical BER pathway, DNA polymerase beta, instead of excising Ape1-incised dL, forms a stable DNA-protein cross-link with the lesion. Such cross-links are analyzed by polyacrylamide gel electrophoresis. Incubation of Ape1-incised dL substrates with mammalian cell-free extracts shows that other proteins can also form such cross-links, although DNA polymerase beta appears to be the major species. This chapter presents methods for analyzing the extent of DNA repair synthesis (repair patch size) associated with dL in whole cell extracts. These analyses show that dL is processed nearly exclusively by the long patch BER pathway, which results in the repair synthesis of two or more nucleotides.

Bioinert membranes prepared from amphiphilic poly(vinyl chloride)-g-poly(oxyethylene methacrylate) graft copolymers

Poly(vinyl chloride) (PVC) membrane was hydrophilically modified by grafting with poly(oxyethylene methacrylate) (POEM) using atom transfer radical polymerization (ATRP). The successful grafting of PVC main chain by POEM was characterized by Fourier transform infrared spectroscopy (FT-IR). The molecular weight and hydrophilicity of membranes increased with the amount of POEM grafting, as characterized by gel permeation chromatography (GPC) and contact angle measurement, respectively. Transmission electron microscope (TEM) and small angle X-ray scattering (SAXS) analysis revealed the microphase-separated structure of PVC-g-POEM and the domain spacing increased from 59.3 to 86.1 nm with increasing grafting degree. Scanning electron microscopy (SEM) was used for the direct visualization of the mouse embryonic fibroblast (MEF) cell and bacteria adhesion on the membrane surface. Protein adsorption and eukaryotic and prokaryotic cell adhesion tests showed that the bioinert properties of membranes were significantly increased with POEM content.

Cytotoxicity and antibacterial assessment of gallic acid capped gold nanoparticles.

Cytotoxicity of nanoparticles needs to be examined for their biocompatibility and suitability in biomedical applications. Gallic acid method was used for the high concentration synthesis (1mM) of gold nanoparticles (AuNPs) having narrow size-distribution at ambient temperature (25°C). Gallic acid capped AuNPs were characterized by different techniques such as, UV-vis spectroscopy, TEM, EDAX, SAED, and XPS. In-vitro stability of AuNPs with biomolecules, such as glucose and bovine serum albumin (BSA) was explored at different concertation ranges. The result reveals that AuNPs are biocompatible with normal cell line mouse embryonic fibroblast (MEF) cells up to 100ppm and cell viability can be retained more than 50% even after increasing the dose from 200 to 400ppm. Antibacterial application of AuNPs revealed that both particles and surface chemistry seems to be safer for both gram positive and gram negative bacterial cultures.

Modified Panax ginseng Extract Inhibits uPAR-Mediated α5β1-Integrin Signaling by Modulating Caveolin-1 to Induce Early Apoptosis in Lung Cancer Cells

Urokinase receptor (uPAR) is enhanced in many human cancer cells and is frequently an indicator of poor prognosis. Activation of [Formula: see text]1-integrin requires caveolin-1 and is regulated by uPAR. However, the underlying molecular mechanism responsible for the interaction between uPAR and [Formula: see text]1-integrin remains obscure. We found that modified regular Panax ginseng extract (MRGX) had a negative modulating effect on the uPAR/[Formula: see text]1-integrin interaction, disrupted the uPAR/integrin interaction by modulating caveoline-1, and caused early apoptosis in cancer cells. Additionally, we found that siRNA-mediated caveoline-1 downregulation inhibited uPAR-mediated [Formula: see text]1-integrin signaling, whereas caveoline-1 up-regulation stimulated the signaling, which suppressed p53 expression, thereby indicating negative crosstalk exists between the integrin [Formula: see text]1 and the p53 pathways. Thus, these findings identify a novel mechanism whereby the inhibition of [Formula: see text]1 integrin and the activation of p53 modulate the expression of the anti-apoptotic proteins that are crucially involved in inducing apoptosis in A549 lung cancer cells. Furthermore, MRGX causes changes in the expressions of members of the Bcl-2 family (Bax and Bcl-2) in a pro-apoptotic manner. In addition, MGRX-mediated inhibition of [Formula: see text]1 integrin attenuates ERK phosphorylation (p-ERK), which up-regulates caspase-8 and Bax. Therefore, ERK may affect mitochondria through a negative regulation of caspase-8 and Bax. Taken together, these findings reveal that MRGX is involved in uPAR-[Formula: see text]1-integrin signaling by modulating caveolin-1 signaling to induce early apoptosis in A549 lung-cancer cells and strongly indicate that MRGX might be useful as a herbal medicine and may lead to the development of new herbal medicine that would suppress the growth of lung-cancer cells.

Chrysin-piperazine conjugates as antioxidant and anticancer agents.

Synthesis of 7-(4-bromobutoxy)-5-hydroxy-2-phenyl-4H-chromen-4-one intermediate treating chrysin with 1,4-dibromobutane facilitated combination of chrysin with a wide range of piperazine moieties which were equipped via reacting the corresponding amines with bis(2-chloroethyl)amine hydrochloride in diethylene glycol monomethyl ether solvent. Free radical scavenging potential of prepared products was analyzed in vitro adopting DPPH and ABTS bioassay in addition to the evaluation of in vitro anticancer efficacies against cervical cancer cell lines (HeLa and CaSki) and an ovarian cancer cell line SK-OV-3 using SRB assay. Bearable toxicity of 7a-w was examined employing Madin-Darby canine kidney (MDCK) cell line. In addition, cytotoxic nature of the presented compounds was inspected utilizing Human bone marrow derived mesenchymal stem cells (hBM-MSCs). Overall, 7a-w indicated remarkable antioxidant power in scavenging DPPH(·) and ABTS(·+), particularly analogs 7f, 7j, 7k, 7l, 7n, 7q, 7v, 7w have shown promising free radical scavenging activity. Analogs 7j and 7o are identified to be highly active candidates against HeLa and CaSki cell lines, whereas 7h and 7l along with 7j proved to be very sensitive towards ovarian cancer cell line SKOV-3. None of the newly prepared scaffolds showed cytotoxic nature toward hBM-MSCs cells. From the structure-activity point of view, nature and position of the electron withdrawing and electron donating functional groups on the piperazine core may contribute to the anticipated antioxidant and anticancer action. Different spectroscopic techniques (FT-IR, (1)H NMR, (13)C NMR, Mass) and elemental analysis (CHN) were utilized to confirm the desired structure of final compounds.

Anti-adipogenic effects of sesamol on human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) from adult bone marrow are able to differentiate into adipocytes, osteoblasts, chondrocytes and neuronal cells. Adipocytes in bone marrow are primarily responsible for the maintenance of bone structure by maintaining cell number balance with other stromal cells. However, the number of adipocytes in the bone marrow increases with age, leading to an imbalance of the bone marrow microenvironment, which results in a disruption of bone structure. In addition, the excessive number of adipocytes in bone marrow can cause diseases, such as osteoporosis or anemia. In this study, we investigated the effect of sesamol, a major natural phenolic compound of sesame oil, on the adipogenic differentiation of hMSCs. Numerous studies have reported the anti-oxidant property of sesamol, but its effect on cell differentiation has not yet been shown. We first found that sesamol treatment during adipogenic differentiation of hMSCs reduced intracellular lipid accumulation, which was unrelated to lipolysis. Interestingly, sesamol diminished the expression of genes responsible for adipogenesis, but increased the expression of osteogenic genes. In addition, sesamol decreased the expression of genes necessary for adipocyte maturation without affecting the expression of hMSC-specific genes. Studies concerning intracellular signaling in hMSCs showed that the extracellular signal-regulated kinase 1/2 (ERK1/2) was decreased by sesamol, which was similar with the effect of an ERK1/2 inhibitor. Overall, this study demonstrates that sesamol can attenuate the adipogenic differentiation of hMSCs without affecting its characteristics through the inhibition of ERK1/2 pathway. Herein, this study reports for the first time the effect of sesamol on hMSC differentiation and suggests the possibility of using sesamol as a therapeutic agent to treat intraosseous disruption triggered by the excessive adipogenesis of hMSCs.

Recent trends on the stent research for blood arteries by bibliometric analysis

The research trends on stent for blood arteries are reviewed by bibliometric analysis using 7,790 journal articles published from 1986 to 2013 of the Web of Science database. The bibliometric indicators are applied to analyze the journal article data, which are simple number of publications for selecting key players, citation indicators for measuring qualitative research performance, collaboration indicators for figuring out the degree of international collaboration and keyword mapping for identifying the research trends. The studies of stent for blood arteries are investigated on the basis of the analysis by countries, institutions and topic changing. The leading countries and institutions published many high-quality journal articles with strong international collaboration. In this report, the current status and future of research trends are clearly revealed from the periodic topic changing analysis. The keywords such as ‘drug eluting stent’, ‘stent coated with new polymers’ and ‘drug delivery systems’ have come into the recent stent-related research, which means lots of efforts are under way to overcome the present limitations of the research.