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1.
Mol Med Rep ; 28(2)2023 Aug.
Article in English | MEDLINE | ID: mdl-37350391

ABSTRACT

Polydeoxyribonucleotide (PDRN) is a mixture of deoxyribonucleotides. It serves as an anti­inflammatory and tissue­regenerating agent. The mitogen­activated protein kinase pathway modulates cell growth and collagen accumulation. It also regulates inflammation by suppressing the expression of proinflammatory cytokines. In the present study, it was attempted to elucidate the molecular mechanism of PDRN in skin healing by confirming the effects of PDRN treatment on skin keratinocytes and fibroblasts, and by assessing the levels of collagen and inflammatory cytokines regulated by the extracellular signal­regulated kinase (ERK) pathway. The potential effects of PDRN on skin regeneration were investigated. Fibroblast and keratinocyte proliferation and migration were analyzed using the water­soluble tetrazolium­8 and wound healing assays. The upregulation of collagen synthesis by PDRN­induced ERK activation was analyzed in fibroblasts with or without an ERK inhibitor. Inflammatory cytokine expression levels in keratinocytes were determined using reverse transcription­quantitative polymerase chain reaction. PDRN promoted the proliferation and migration of keratinocytes and fibroblasts. However, PDRN­induced ERK phosphorylation differed between keratinocytes and fibroblasts; PDRN increased ERK phosphorylation and collagen accumulation in fibroblasts, while it inhibited matrix metalloproteinase expression. By contrast, PDRN inhibited ERK phosphorylation in keratinocytes, and it decreased inflammatory cytokine expression levels. PDRN affects skin cell proliferation and migration, and collagen and inflammatory cytokine expression levels via ERK signaling. Overall, PDRN exerts a positive effect on skin regeneration, but the mechanism by which it promotes skin regeneration varies among different skin cell types.


Subject(s)
Polydeoxyribonucleotides , Skin , Humans , Phosphorylation , Polydeoxyribonucleotides/pharmacology , Polydeoxyribonucleotides/metabolism , Skin/metabolism , Keratinocytes/metabolism , Collagen/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Cytokines/metabolism , Fibroblasts/metabolism
2.
Molecules ; 27(15)2022 Aug 02.
Article in English | MEDLINE | ID: mdl-35956878

ABSTRACT

Nicotinamide nucleotide transhydrogenase (NNT) is involved in decreasing melanogenesis through tyrosinase degradation induced by cellular redox changes. Nicotinamide is a component of coenzymes, such as NAD+, NADH, NADP+, and NADPH, and its levels are modulated by NNT. Vitamin C and polydeoxyribonucleotide (PDRN) are also known to decrease skin pigmentation. We evaluated whether a mixture of nicotinamide, vitamin C, and PDRN (NVP-mix) decreased melanogenesis by modulating mitochondrial oxidative stress and NNT expression in UV-B-irradiated animals and in an in vitro model of melanocytes treated with conditioned media (CM) from UV-B-irradiated keratinocytes. The expression of NNT, GSH/GSSG, and NADPH/NADP+ in UV-B-irradiated animal skin was significantly decreased by UV-B radiation but increased by NVP-mix treatment. The expression of NNT, GSH/GSSG, and NADPH/NADP+ ratios decreased in melanocytes after CM treatment, although they increased after NVP-mix administration. In NNT-silenced melanocytes, the GSH/GSSG and NADPH/NADP+ ratios were further decreased by CM compared with normal melanocytes. NVP-mix decreased melanogenesis signals, such as MC1R, MITF, TYRP1, and TYRP2, and decreased melanosome transfer-related signals, such as RAB32 and RAB27A, in UV-B-irradiated animal skin. NVP-mix also decreased MC1R, MITF, TYRP1, TYRP2, RAB32, and RAB27A in melanocytes treated with CM from UV-irradiated keratinocytes. The expression of MC1R and MITF in melanocytes after CM treatment was unchanged by NNT silencing. However, the expression of TYRP1, TYRP2, RAB32, and RAB27A increased in NNT-silenced melanocytes after CM treatment. NVP-mix also decreased tyrosinase activity and melanin content in UV-B-irradiated animal skin and CM-treated melanocytes. In conclusion, NVP-mix decreased mitochondrial oxidative stress by increasing NNT expression and decreased melanogenesis by decreasing MC1R/MITF, tyrosinase, TYRP1, and TYRP2.


Subject(s)
NADP Transhydrogenases , Animals , Ascorbic Acid/metabolism , Ascorbic Acid/pharmacology , Glutathione Disulfide/metabolism , Melanins , Melanocytes/metabolism , Monophenol Monooxygenase/metabolism , NADP/metabolism , NADP Transhydrogenases/metabolism , Niacinamide/metabolism , Niacinamide/pharmacology , Polydeoxyribonucleotides/metabolism , Vitamins/metabolism
3.
Mol Med Rep ; 24(5)2021 11.
Article in English | MEDLINE | ID: mdl-34505634

ABSTRACT

Gastritis refers to inflammation caused by injury to the gastric epithelium, which is usually due to excessive alcohol consumption and prolonged use of nonsteroidal anti­inflammatory drugs. Millions of individuals worldwide suffer from this disease. However, the lack of safe and promising treatments makes it urgent to explore and develop leads from natural resources. Therefore, food as medicine may be the best approach for the treatment of these disorders. The present study described the protective effects of food­polydeoxyribonucleotides (f­PDRNs) in a rat model of gastric mucosal injury induced by HCl­EtOH. Administration of f­PDRN was performed with low­PRF002 (26 mg/kg/day), medium­PRF002 (52 mg/kg/day) and high­PRF002 (78 mg/kg/day) on the day of autopsy. The site of damage to the mucous membrane was also analysed. In addition, an increase in gastric juice pH, total acidity of gastric juice and decrease in gastric juice secretion were confirmed, and gastric juice secretion­related factors corresponding to the administration of f­PDRN were analysed. Administration of f­PDRN reduced the mRNA expression of histamine H2 receptor, muscarinic acetylcholine receptor M3, cholecystokinin 2 receptor and H+/K+ ATPase related to gastric acid secretion and downregulation of histamine, myeloperoxidase and cyclic adenosine monophosphate. In addition, it was histologically confirmed that the loss of epithelial cells and the distortion of the mucosa were recovered in the group in which f­PDRN was administered compared to the model group with gastric mucosa damage. In summary, the present study suggested that f­PDRN has therapeutic potential and may have beneficial effects if taken regularly as a food supplement.


Subject(s)
Gastric Mucosa/metabolism , Gastritis/drug therapy , Polydeoxyribonucleotides/pharmacology , Alcohol Drinking/adverse effects , Animals , Cyclic AMP/metabolism , Disease Models, Animal , Ethanol/adverse effects , Food , Gastric Juice/chemistry , Gastric Juice/drug effects , Gastric Mucosa/injuries , Histamine/metabolism , Male , Polydeoxyribonucleotides/metabolism , Rats , Rats, Sprague-Dawley
4.
Int Immunopharmacol ; 83: 106444, 2020 Jun.
Article in English | MEDLINE | ID: mdl-32234670

ABSTRACT

Acute lung injury (ALI) is characterized by disruption of the alveolar-capillary membrane resulting in pulmonary edema and accumulation of associated proteinaceous alveolar exudate. Initiation of ALI upregulates tumor necrosis factor-α (TNF-α), which activates nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPK) that induce various pro-inflammatory mediators. Polydexyribonucleotide (PDRN) is an adenosine A2A receptor agonist that exerts anti-inflammatory effects by suppressing the production of pro-inflammatory cytokines and apoptosis. We investigated the therapeutic efficiency of PDRN on ALI induced by lipopolysaccharide (LPS) in rats. ALI was induced by intratracheal instillation of LPS (5 mg/kg) in 200 µL saline. The PDRN treatment group received a single intraperitoneal injection of 500 µL saline including PDRN (8 mg/kg) 1 h after ALI induction. To confirm the involvement of the adenosine A2A receptor in PDRN, 8 mg/kg 7-dimethyl-1-propargylxanthine (DMPX) was applied with PDRN treatment. Rats were then sacrificed 12 h after PDRN and DMPX treatments. Intratracheal administration of LPS caused lung tissue damage and significantly increased the lung injury scores and levels of pro-inflammatory cytokines, and apoptotic factors. In addition, MAPK/NF-κB signaling factors were increased by ALI initiation. PDRN treatment potently suppressed expressions of MAPK/NF-κB signaling factors compared to the PDRN + DMPX co-treated group. These alterations led to a reduction of pro-inflammatory cytokines, apoptotic factors, and NF-κB and MAPK signaling, which promoted the recovery of damaged lung tissue. PDRN therapy demonstrated therapeutic effects for LPS-induced ALI compared to the non-treated and DMPX-treated groups. Therefore, PDRN may be used as a therapy for initial treatment of ALI.


Subject(s)
Acute Lung Injury/genetics , Adenosine A2 Receptor Agonists/metabolism , Polydeoxyribonucleotides/metabolism , Acute Lung Injury/immunology , Animals , Cytokines/metabolism , Disease Models, Animal , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , Inflammation Mediators/metabolism , Lipopolysaccharides , Male , NF-kappa B , Polydeoxyribonucleotides/genetics , Rats , Rats, Sprague-Dawley , Signal Transduction
5.
J Biol Chem ; 292(32): 13187-13196, 2017 08 11.
Article in English | MEDLINE | ID: mdl-28592487

ABSTRACT

To find their DNA target sites in complex solution environments containing excess heterogeneous DNA, sequence-specific DNA-binding proteins execute various translocation mechanisms known collectively as facilitated diffusion. For proteins harboring a single DNA contact surface, long-range translocation occurs by jumping between widely spaced DNA segments. We have configured biosensor-based surface plasmon resonance to directly measure the affinity and kinetics of this intersegmental jumping by the ETS-family transcription factor ETS variant 6 (ETV6). To isolate intersegmental target binding in a functionally defined manner, we pre-equilibrated ETV6 with excess salmon sperm DNA, a heterogeneous polymer, before exposing the nonspecifically bound protein to immobilized oligomeric DNA harboring a high-affinity ETV6 site. In this way, the mechanism of ETV6-target association could be toggled electrostatically through varying NaCl concentration in the bulk solution. Direct measurements of association and dissociation kinetics of the site-specific complex indicated that 1) freely diffusive binding by ETV6 proceeds through a nonspecific-like intermediate, 2) intersegmental jumping is rate-limited by dissociation from the nonspecific polymer, and 3) dissociation of the specific complex is independent of the history of complex formation. These results show that target searches by proteins with an ETS domain, such as ETV6, whose single DNA-binding domain cannot contact both source and destination sites simultaneously, are nonetheless strongly modulated by intersegmental jumping in heterogeneous site environments. Our findings establish biosensors as a general technique for directly and specifically measuring target site search by DNA-binding proteins via intersegmental translocation.


Subject(s)
DNA/metabolism , Models, Molecular , Proto-Oncogene Proteins c-ets/metabolism , Repressor Proteins/metabolism , Animals , Binding Sites , Biosensing Techniques , DNA/chemistry , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/metabolism , ETS Motif , Facilitated Diffusion , Humans , Kinetics , Nucleotide Motifs , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Polydeoxyribonucleotides/chemistry , Polydeoxyribonucleotides/metabolism , Protein Conformation , Protein Interaction Domains and Motifs , Proto-Oncogene Proteins c-ets/chemistry , Proto-Oncogene Proteins c-ets/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Repressor Proteins/chemistry , Repressor Proteins/genetics , Response Elements , Salmon , Static Electricity , Surface Plasmon Resonance , Thermodynamics , ETS Translocation Variant 6 Protein
6.
J Cell Physiol ; 232(9): 2299-2307, 2017 Sep.
Article in English | MEDLINE | ID: mdl-27791262

ABSTRACT

Polydeoxyribonucleotides (PDRNs) are low molecular weight DNA molecules of natural origin that stimulate cell migration and growth, extracellular matrix (ECM) protein production, and reduce inflammation. Most preclinical and clinical studies on tissue regeneration with PDRNs focused on skin, and only few are about musculoskeletal tissues. Starting from an overview on skin regeneration studies, through the analysis of in vitro, in vivo, and clinical studies (1990-2016), the present review aimed at defining the effects of PDRN and their mechanisms of action in the regeneration of musculoskeletal tissues. This would also help future researches in this area. A total of 29 studies were found by PubMed and www.webofknowledge.com searches: 20 were on skin (six in vitro, six in vivo, one vitro/vivo, seven clinical studies), while the other nine regarded bone (one in vitro, two in vivo, one clinical studies), cartilage (one in vitro, one vitro/vivo, two clinical studies), or tendon (one clinical study) tissues regeneration. PDRNs improved cell growth, tissue repair, ECM proteins, physical activity, and reduced pain and inflammation, through the activation of adenosine A2A receptor. PDRNs are currently used for bone, cartilage, and tendon diseases, with a great variability regarding the PDRN dosage to be used in clinical practice, while the dosage for skin regeneration is well established. PDRNs are usually administered from a minimum of three to a maximum of five times and they act trough the activation of A2A receptor. Further studies are advisable to confirm the effectiveness of PDRNs and to standardize the PDRN dose. J. Cell. Physiol. 232: 2299-2307, 2017. © 2016 Wiley Periodicals, Inc.


Subject(s)
Musculoskeletal System/metabolism , Polydeoxyribonucleotides/metabolism , Receptors, Adenosine A2/metabolism , Regeneration , Skin/metabolism , Animals , Bone Remodeling , Cell Movement , Cell Proliferation , Extracellular Matrix/metabolism , Humans , Musculoskeletal System/pathology , Musculoskeletal System/physiopathology , Signal Transduction , Skin/pathology , Skin/physiopathology , Wound Healing
7.
Chem Biol Drug Des ; 88(1): 122-8, 2016 07.
Article in English | MEDLINE | ID: mdl-26867976

ABSTRACT

In 80-90% tumor cells, telomerase becomes active and stabilizes the length of telomeres. The formation and stabilization of G-quadruplexes formed from human telomeric sequences have been proved able to inhibit the activity of telomerase, thus human telomeric G-quadruplex structure has become a potential target for the development of cancer therapy. Hence, structure of G-quadruplex formed in K(+) solution has been an attractive hotspot for further studies. However, the exact structure of human telomeric G-quadruplex in K(+) is extremely controversial, this study provides information for the understanding of different G-quadruplexes. Here, we report that 22nt and 24nt human telomeric sequences form unimolecular hybrid-type mixed parallel/antiparallel G-quadruplex in K(+) solution elucidated utilizing Circular Dichroism, Differential Scanning Calorimetry, and gel electrophoresis. Moreover, individual configuration of these two sequences was speculated in this study. The detailed structure information of the G-quadruplex formed under physiologically relevant condition is necessary for structure-based rational drug design.


Subject(s)
G-Quadruplexes , Models, Molecular , Telomere Shortening , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Calorimetry, Differential Scanning , Circular Dichroism , Drug Design , Humans , Molecular Weight , Native Polyacrylamide Gel Electrophoresis , Polydeoxyribonucleotides/chemistry , Polydeoxyribonucleotides/metabolism , Potassium/chemistry , Sodium/chemistry , Solubility , Telomerase/chemistry , Telomerase/metabolism
8.
J Biol Chem ; 291(3): 1235-42, 2016 Jan 15.
Article in English | MEDLINE | ID: mdl-26511320

ABSTRACT

Transcription factor-DNA interactions are central to gene regulation. Many transcription factors regulate multiple target genes and can bind sequences that do not conform strictly to the consensus. To understand the structural mechanism utilized by the transcription regulators to bind diverse target sequences, we have employed the repressor AraR from Bacillus subtilis as a model system. AraR is known to bind to eight different operator sites in the bacterial genome. Although there are differences in the sequences of four of these operators, ORE1, ORX1, ORA1, and ORR3, the AraR-DNA binding domain (AraR-DBD) as well as full-length AraR unexpectedly binds to each of these sequences with similar affinities as measured by fluorescence anisotropy experiments. We have determined crystal structures of AraR-DBD in complex with two different natural operators ORE1 and ORX1 up to 2.07 and 1.97 Å resolution, respectively. These structures were compared with the previously reported structures of AraR-DBD bound to two other natural operators (ORA1 and ORR3). Interactions of two molecules of AraR-DBD with the symmetric operator, ORE1, are identical, but their interaction with the non-symmetric operator ORX1 results in breakdown of the symmetry in protein-DNA interactions. The novel interactions observed are accompanied by local conformational change in the DNA. ChIP-sequencing (ChIP-Seq) data on other transcription factors has shown that they can bind to diverse targets, and hence the plasticity exhibited by AraR may be a general phenomenon. The ability of transcription factors to form alternate interactions may be important for employment in new functions and evolution of novel regulatory circuits.


Subject(s)
Bacillus subtilis/physiology , Bacterial Proteins/metabolism , DNA, Bacterial/metabolism , Heat-Shock Proteins/metabolism , Models, Biological , Operator Regions, Genetic , Repressor Proteins/metabolism , AT Rich Sequence , Anisotropy , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Binding Sites , Consensus Sequence , DNA, Bacterial/chemistry , Fluorescence Polarization , Heat-Shock Proteins/chemistry , Heat-Shock Proteins/genetics , Kinetics , Nucleic Acid Conformation , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Polydeoxyribonucleotides/chemistry , Polydeoxyribonucleotides/metabolism , Protein Conformation , Protein Interaction Domains and Motifs , Repressor Proteins/chemistry , Repressor Proteins/genetics , Response Elements , Sequence Alignment
9.
Food Chem Toxicol ; 84: 89-98, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26277627

ABSTRACT

This study aimed to investigate the degradation of three transgenic Bacillus thuringiensis (Bt) genes (Cry1Ab, Cry1Ac, and Cry1Ab/Ac) and the corresponding encoded Bt proteins in KMD1, KF6, and TT51-1 rice powder, respectively, following autoclaving, cooking, baking, or microwaving. Exogenous Bt genes were more stable than the endogenous sucrose phosphate synthase (SPS) gene, and short DNA fragments were detected more frequently than long DNA fragments in both the Bt and SPS genes. Autoclaving, cooking (boiling in water, 30 min), and baking (200 °C, 30 min) induced the most severe Bt protein degradation effects, and Cry1Ab protein was more stable than Cry1Ac and Cry1Ab/Ac protein, which was further confirmed by baking samples at 180 °C for different periods of time. Microwaving induced mild degradation of the Bt and SPS genes, and Bt proteins, whereas baking (180 °C, 15 min), cooking and autoclaving led to further degradation, and baking (200 °C, 30 min) induced the most severe degradation. The findings of the study indicated that degradation of the Bt genes and proteins somewhat correlated with the treatment intensity. Polymerase chain reaction, enzyme-linked immunosorbent assay, and lateral flow tests were used to detect the corresponding transgenic components. Strategies for detecting transgenic ingredients in highly processed foods are discussed.


Subject(s)
Bacillus thuringiensis/chemistry , Bacterial Proteins/analysis , DNA, Bacterial/analysis , Food Inspection/methods , Oryza/chemistry , Plants, Genetically Modified/chemistry , Seeds/chemistry , Bacillus thuringiensis/genetics , Bacillus thuringiensis/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , China , Cooking , DNA Cleavage/radiation effects , DNA, Bacterial/metabolism , Feasibility Studies , Food, Genetically Modified/radiation effects , Hot Temperature , Microwaves , Oligodeoxyribonucleotides/analysis , Oligodeoxyribonucleotides/metabolism , Oryza/genetics , Peptide Fragments/analysis , Peptide Fragments/metabolism , Plants, Genetically Modified/genetics , Polydeoxyribonucleotides/analysis , Polydeoxyribonucleotides/metabolism , Protein Stability , Proteolysis/radiation effects , Seeds/genetics
10.
Bioorg Med Chem ; 23(15): 4769-4776, 2015 Aug 01.
Article in English | MEDLINE | ID: mdl-26081762

ABSTRACT

Previously, we reported our investigations of the interaction between a cyclic naphthalene diimide derivative (cNDI 1) and double stranded DNA (dsDNA) (Bioorg. Med. Chem.2014, 22, 2593). Here, we report the synthesis of the novel cNDI 2, which has shorter linker chains than cNDI 1. We performed comparative investigations of the interactions of both cNDI 1 and cNDI 2 with different types of dsDNA, including analysis of their thermodynamics and kinetics. Interactions between the cNDIs and calf thymus DNA (CT-DNA), poly[d(A-T)]2, or poly[d(G-C)]2 were explored by physicochemical and biochemical methods, including UV-Vis spectroscopy, circular dichroism (CD) spectroscopy, stopped-flow kinetics, and a topoisomerase I assay. Upon addition of cNDIs to CT-DNA, the existence of an induced CD signal at approximately the wavelength of the naphthalene diimide chromophore and unwinding of the DNA duplex, as detected by the topoisomerase I assay, revealed that cNDIs bound to the DNA duplex. As indicated by the steric constraint in the formation of the complex, bis-threading intercalation was the more favorable binding mode. UV-Vis spectroscopic titration of the cNDIs with DNA duplexes showed affinities on the order of 10(5)-10(6)M(-1), with a stoichiometry of one cNDI molecule per four DNA base pairs. Thermodynamic parameters (ΔG, ΔH, and ΔS) based on the van't Hoff equation indicated that exothermic and entropy-dependent hydrophobic interactions played a major role in the reaction. Stopped-flow association and dissociation analysis showed that cNDI interactions with poly[d(G-C)]2 were more stable and had a slower dissociation rate than their interactions with poly[d(A-T)]2 and CT-DNA. Measurement of ionic strength indicated that electrostatic attraction is also an important component of the interaction between cNDIs and CT-DNA. Because of its longer linker chain, cNDI 1 showed higher binding selectivity, a more entropically favorable interaction, and much slower dissociation from dsDNA than cNDI 2.


Subject(s)
DNA/chemistry , Imides/chemistry , Naphthalenes/chemistry , Animals , Cattle , Circular Dichroism , DNA/metabolism , DNA Topoisomerases, Type I/metabolism , Imides/metabolism , Kinetics , Naphthalenes/metabolism , Polydeoxyribonucleotides/chemistry , Polydeoxyribonucleotides/metabolism , Spectrophotometry, Ultraviolet , Thermodynamics
11.
J Biol Chem ; 289(38): 26574-26583, 2014 Sep 19.
Article in English | MEDLINE | ID: mdl-25107903

ABSTRACT

Ultraviolet (UV) photoproducts are removed from genomic DNA by dual incisions in humans in the form of 24- to 32-nucleotide-long oligomers (canonical 30-mers) by the nucleotide excision repair system. How the small, excised, damage-containing DNA oligonucleotides (sedDNAs) are processed in cells following the dual incision event is not known. Here, we demonstrate that sedDNAs are localized to the nucleus in two biochemically distinct forms, which include chromatin-associated, transcription factor II H-bound complexes and more readily solubilized, RPA-bound complexes. Because the nuclear mobility and repair functions of transcription factor II H and RPA are influenced by post-incision gap-filling events, we examined how DNA repair synthesis and DNA ligation affect sedDNA processing. We found that although these gap filling activities are not essential for the dual incision/sedDNA generation event per se, the inhibition of DNA repair synthesis and ligation is associated with a decrease in UV photoproduct removal rate and an accumulation of RPA-sedDNA complexes in the cell. These findings indicate that sedDNA processing and association with repair proteins following the dual incisions may be tightly coordinated with gap filling during nucleotide excision repair in vivo.


Subject(s)
DNA Repair , Polydeoxyribonucleotides/metabolism , Cell Cycle Checkpoints , Cell Line, Tumor , Cell Nucleus/metabolism , DNA/biosynthesis , DNA Cleavage , Humans , Replication Protein A/metabolism , Transcription Factor TFIIH/metabolism
12.
J Phys Chem B ; 118(5): 1288-95, 2014 Feb 06.
Article in English | MEDLINE | ID: mdl-24417409

ABSTRACT

Doxorubicin (DOX) is an important anthracycline antibiotic whose intricate features of binding to DNAs, not yet fully understood, have been the object of intense debate. The dimerization equilibrium has been studied at pH = 7.0, I = 2.5 mM, and T = 25 °C. A thermodynamic and kinetic study of the binding of doxorubicin to DNA, carried out by circular dichroism, viscometry, differential scanning calorimetry, fluorescence, isothermal titration calorimetry, and T-jump relaxation measurements, has enabled us to characterize for the first time two different types of calf thymus DNA (ctDNA)/DOX complexes: PD1 for C(DOX)/C(DNA) < 0.3, and PD2 for higher drug content. The nature of the PD1 complex is described better in light of the affinity of DOX with the synthetic copolymers [poly(dA-dT)]2 and [poly(dG-dC)]2. The formation of PD1 has been categorized kinetically as a two-step mechanism in which the fast step is the groove binding in the AT region, and the slow step is the intercalation into the GC region. This bifunctional nature provides a plausible explanation for the high PD1 constant obtained (K1 = 2.3 × 10(8) M(-1)). Moreover, the formation of an external aggregate complex ctDNA/DOX (PD2) at the expense of PD1, with K2 = 9.3 × 10(5) M(-1), has been evinced.


Subject(s)
DNA Adducts/chemistry , DNA/chemistry , Doxorubicin/chemistry , Animals , Cattle , DNA/metabolism , Hydrogen-Ion Concentration , Kinetics , Poly dA-dT/chemistry , Poly dA-dT/metabolism , Polydeoxyribonucleotides/chemistry , Polydeoxyribonucleotides/metabolism , Thermodynamics , Transition Temperature , Viscosity
13.
J Photochem Photobiol B ; 127: 78-87, 2013 Oct 05.
Article in English | MEDLINE | ID: mdl-23968995

ABSTRACT

The interaction of mitoxantrone with alternating Poly(dG-dC).Poly(dG-dC) and Poly(dA-dT).Poly(dA-dT) duplex has been studied by absorption, fluorescence and Circular Dichroism (CD) spectroscopy at Drug to Phosphate base pair ratios D/P=20.0-0.04. Binding to GC polymer occurs in two distinct modes: partial stacking characterized by red shifts of 18-23nm at D/P=0.2-0.8 and external binding at D/P=1.0-20.0 whereas that to AT polymer occurs externally in the entire range of D/P. The binding constant and number of binding sites is 3.7×10(5)M(-1), 0.3 and 1.3× 10(4)M(-1), 1.5 in GC and AT polymers, respectively at low D/P ratios. CD binding isotherms show breakpoints at D/P=0.1, 0.5 and 0.25, 0.5 in GC and AT polymers, respectively. The intrinsic CD bands indicate that the distortions in GC polymer are significantly higher than that in AT polymer. Docking studies show partial insertion of mitoxantrone rings between to GC base pairs in alternating GC polymer. Side chains of mitoxantrone interact specifically with base pairs and DNA backbone. The studies are relevant to the understanding of suppression or inhibition of DNA cleavage on formation of ternary complex with topoisomerase-II enzyme and hence the anti cancer action.


Subject(s)
Antineoplastic Agents/metabolism , Mitoxantrone/metabolism , Poly dA-dT/metabolism , Polydeoxyribonucleotides/metabolism , Spectrum Analysis , Absorption , Molecular Docking Simulation , Nucleic Acid Conformation , Poly dA-dT/chemistry , Polydeoxyribonucleotides/chemistry
14.
Mol Biol Rep ; 40(9): 5439-50, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23666107

ABSTRACT

Isoquinoline alkaloids and their analogs represent an important class of molecules for their broad range of clinical and pharmacological utility. These compounds are of current interest owing to their low toxicity and excellent chemo preventive properties. These alkaloids can play important role in stabilising the nucleic acid triple helices. The present study has focused on the interaction of five 9-O-(ω-amino) alkyl ether berberine analogs with the DNA triplex poly(dT)·poly(dA)*poly(dT) and the parent duplex poly(dA)·poly(dT) studied using various biophysical techniques. Scatchard analysis of the spectral data indicated that the analogs bind both to the duplex and triplex in a non-cooperative manner in contrast to the cooperative binding of berberine to the DNA triplex. Strong intercalative binding to the DNA triplex structure was revealed from ferrocyanide quenching, fluorescence polarization and viscosity results. Thermal melting studies demonstrated higher stabilization of the Hoogsteen base paired third strand of the DNA triplex compared to the Watson-Crick strand. Circular dichroism studies suggested a stronger perturbation of the DNA triplex conformation by the alkaloid analogs compared to the duplex. The binding was entropy-driven in each case and the entropy contribution to free energy increased as the length of the alkyl side chain increased. The analogs exhibited stronger binding affinity to the triple helical structure compared to the parent double helical structure.


Subject(s)
Berberine Alkaloids/metabolism , DNA/chemistry , Polydeoxyribonucleotides/metabolism , Berberine Alkaloids/chemistry , Biophysics , Calorimetry , Circular Dichroism , Ferrocyanides , Fluorescence , Molecular Structure , Polydeoxyribonucleotides/chemistry , Thermodynamics , Transition Temperature
15.
PLoS One ; 7(6): e39156, 2012.
Article in English | MEDLINE | ID: mdl-22723953

ABSTRACT

Mammalian DNA polymerase δ (Pol δ), a four-subunit enzyme, plays a crucial and versatile role in DNA replication and DNA repair processes. We have reconstituted human Pol δ complexes in insect cells infected with a single baculovirus into which one or more subunits were assembled. This system allowed for the efficient expression of the tetrameric Pol δ holoenzyme, the p125/p50 core dimer, the core+p68 trimer and the core+p12 trimer, as well as the p125 catalytic subunit. These were isolated in milligram amounts with reproducible purity and specific activities by a highly standardized protocol. We have systematically compared their activities in order to gain insights into the roles of the p12 and p68 subunits, as well as their responses to PCNA. The relative specific activities (apparent k(cat)) of the Pol δ holoenzyme, core+p68, core+p12 and p125/p50 core were 100, 109, 40, and 29. The corresponding apparent K(d)'s for PCNA were 7.1, 8.7, 9.3 and 73 nM. Our results support the hypothesis that Pol δ interacts with PCNA through multiple interactions, and that there may be a redundancy in binding interactions that may permit Pol δ to adopt flexible configurations with PCNA. The abilities of the Pol δ complexes to fully extend singly primed M13 DNA were examined. All the subassemblies except the core+p68 were defective in their abilities to completely extend the primer, showing that the p68 subunit has an important function in synthesis of long stretches of DNA in this assay. The core+p68 trimer could be reconstituted by addition of p12.


Subject(s)
Baculoviridae/genetics , DNA Polymerase III/genetics , DNA Polymerase III/metabolism , Baculoviridae/metabolism , Calpain/metabolism , DNA Polymerase III/isolation & purification , DNA Replication/physiology , Enzyme Activation , Gene Expression , HEK293 Cells , HeLa Cells , Holoenzymes/genetics , Holoenzymes/isolation & purification , Holoenzymes/metabolism , Humans , Multienzyme Complexes/chemistry , Multienzyme Complexes/metabolism , Plasmids/genetics , Polydeoxyribonucleotides/metabolism , Proliferating Cell Nuclear Antigen/metabolism , Protein Stability , Protein Subunits/genetics , Protein Subunits/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism
16.
Inorg Chem ; 50(21): 10667-72, 2011 Nov 07.
Article in English | MEDLINE | ID: mdl-21958310

ABSTRACT

Efficient intercalation of a luminescent Ir(III) complex exclusively made of polypyridine ligands in natural and synthetic biopolymers is reported for the first time. The emission of the complex is largely enhanced in the presence of [poly(dA-dT)(2)] and strongly quenched in the presence of [poly(dG-dC)(2)]. By comparing the emission decays in DNA and in synthetic polynucleotides, it is proposed that the emission quenching of the title compound by guanine residues in DNA is no longer effective over a distance of four dA-dT base pairs.


Subject(s)
DNA/metabolism , Intercalating Agents/chemical synthesis , Iridium/chemistry , Molecular Probes/chemical synthesis , Pyridines/chemistry , Biopolymers/chemistry , Biopolymers/metabolism , Circular Dichroism , Intercalating Agents/analysis , Intercalating Agents/metabolism , Ligands , Luminescence , Models, Molecular , Molecular Probes/analysis , Molecular Probes/metabolism , Poly dA-dT/chemistry , Poly dA-dT/metabolism , Polydeoxyribonucleotides/chemistry , Polydeoxyribonucleotides/metabolism , Polymerization , Polynucleotides/chemistry , Polynucleotides/metabolism
17.
FEBS J ; 278(18): 3447-62, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21787377

ABSTRACT

The occurrence of DNA architectural proteins containing two functional domains derived from two different architectural proteins is an interesting emerging research theme in the field of nucleoid structure and function. Mycobacterium tuberculosis HupB, unlike Escherichia coli HU, is a two-domain protein that, in the N-terminal region, shows broad sequence homology with bacterial HU. The long C-terminal extension, on the other hand, contains seven PAKK/KAAK motifs, which are characteristic of the histone H1/H5 family of proteins. In this article, we describe several aspects of HupB function, in comparison with its truncated derivatives lacking either the C-terminus or N-terminus. We found that HupB binds a variety of DNA repair and replication intermediates with K(d) values in the nanomolar range. By contrast, the N-terminal fragment of M. tuberculosis HupB (HupB(MtbN)) showed diminished DNA-binding activity, with K(d) values in the micromolar range, and the C-terminal domain was completely devoid of DNA-binding activity. Unlike HupB(MtbN) , HupB was able to constrain DNA in negative supercoils and introduce negative superhelical turns into relaxed DNA. Similarly, HupB exerted a robust inhibitory effect on DNA strand exchange promoted by cognate and noncognate RecA proteins, whereas HupB(MtbN), even at a 50-fold molar excess, had no inhibitory effect. Considered together, these results suggest that synergy between the N-terminal and C-terminal domains of HupB is essential for its DNA-binding ability, and to modulate the topological features of DNA, which has implications for processes such as DNA compaction, gene regulation, homologous recombination, and DNA repair.


Subject(s)
Bacterial Proteins/chemistry , DNA, Superhelical/metabolism , Histones/chemistry , Mycobacterium tuberculosis/metabolism , Protein Interaction Domains and Motifs , Rec A Recombinases/metabolism , Recombination, Genetic , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Carrier Proteins/chemistry , Carrier Proteins/genetics , Carrier Proteins/isolation & purification , Carrier Proteins/metabolism , Cross-Linking Reagents/chemistry , DNA, Bacterial/chemistry , DNA, Bacterial/metabolism , DNA, Superhelical/chemistry , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/isolation & purification , DNA-Binding Proteins/metabolism , Dimerization , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Escherichia coli Proteins/isolation & purification , Escherichia coli Proteins/metabolism , Histones/genetics , Histones/isolation & purification , Histones/metabolism , Kinetics , Mutant Proteins/chemistry , Mutant Proteins/isolation & purification , Mutant Proteins/metabolism , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/isolation & purification , Peptide Fragments/metabolism , Polydeoxyribonucleotides/chemistry , Polydeoxyribonucleotides/metabolism , Protein Stability , Rec A Recombinases/antagonists & inhibitors , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Transcription Factors/chemistry , Transcription Factors/genetics , Transcription Factors/isolation & purification , Transcription Factors/metabolism
19.
J Enzyme Inhib Med Chem ; 25(5): 629-34, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20429779

ABSTRACT

The biological evaluation of carbocyclic minor groove binders 1-6 is described. The cytotoxicity of the obtained compounds was tested on MDA-MB-231 breast cancer cells. The mechanism of action of compounds 1-6 was studied employing the topoisomerase I/II inhibition assay and ethidium displacement assay using pBR322. Determination of association constants was done using calf thymus DNA, T4 coliphage DNA, poly(dA-dT)(2), and poly(dG-dC)(2). The effect of compounds 1-6 on the amidolytic activity of plasmin, trypsin, thrombin, and urokinase was also examined.


Subject(s)
Aniline Compounds/metabolism , Aniline Compounds/pharmacology , Benzamides/metabolism , Benzamides/pharmacology , DNA/metabolism , Serine Proteinase Inhibitors/metabolism , Serine Proteinase Inhibitors/pharmacology , Topoisomerase I Inhibitors/metabolism , Topoisomerase I Inhibitors/pharmacology , Topoisomerase II Inhibitors/metabolism , Topoisomerase II Inhibitors/pharmacology , Bacteriophage T4 , Binding, Competitive , Cell Line, Tumor , Cell Proliferation/drug effects , DNA, Superhelical/metabolism , DNA, Viral/metabolism , Drug Design , Ethidium/metabolism , Humans , Inhibitory Concentration 50 , Kinetics , Plasmids , Polydeoxyribonucleotides/metabolism
20.
J Biol Inorg Chem ; 15(4): 515-32, 2010 May.
Article in English | MEDLINE | ID: mdl-20087612

ABSTRACT

The interaction of the Cu(II) drugs CuL(NO(3)) and CuL'(NO(3)) (HL is pyridine-2-carbaldehyde thiosemicarbazone and HL' is pyridine-2-carbaldehyde 4N-methylthiosemicarbazone, in water named [CuL](+) and [CuL'](+)) with [poly(dA-dT)](2), [poly(dG-dC)](2), and calf thymus (CT) DNA has been probed in aqueous solution at pH 6.0, I = 0.1 M, and T = 25 degrees C by absorbance, fluorescence, circular dichroism, and viscosity measurements. The results reveal that these drugs act as groove binders with [poly(dA-dT)](2), with a site size n = 6-7, whereas they act as external binders with [poly(dG-dC)](2) and/or CT-DNA, thus establishing overall electrostatic interaction with n = 1. The binding constants with [CuL'](+) were slightly larger than with [CuL](+). The title compounds display some cleavage activity in the presence of thiols, bringing about the rupture of the DNA strands by the reactive oxygen species formed by reoxidation of Cu(I) to Cu(II); this feature was not observed in the absence of thiols. Mutagenic assays performed both in the presence and in the absence of S9 mix, probed by the Ames test on TA 98, TA 100, and TA 102, were negative. Weak genotoxic activity was detected for [CuL](+) and [CuL'](+), with a significative dose-response effect for [CuL'](+), which was shown to be more cytotoxic in the Ames test and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell proliferation assays. Methylation of the terminal NH(2) group enhances the antiproliferative activity of the pyridine-2-carbaldehyde thiosemicarbazones.


Subject(s)
Copper/chemistry , DNA/metabolism , Organometallic Compounds/chemistry , Organometallic Compounds/metabolism , Poly dA-dT/metabolism , Polydeoxyribonucleotides/metabolism , Thiosemicarbazones/chemistry , 3-Mercaptopropionic Acid/metabolism , Animals , Base Sequence , Cattle , Cell Line , DNA/genetics , DNA Breaks/drug effects , Dithiothreitol/metabolism , Glutathione/metabolism , Hydrogen-Ion Concentration , Mutagenicity Tests , Organometallic Compounds/pharmacology , Oxidation-Reduction , Poly dA-dT/genetics , Polydeoxyribonucleotides/genetics , Spectrum Analysis , Temperature , Viscosity
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