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1.  Zn(II)-Chlorido Complexes of Phytohormone Kinetin and Its Derivatives Modulate Expression of Inflammatory Mediators in THP-1 Cells 
PLoS ONE  2013;8(6):e65214.
Kinetin (N6-furfuryladenine) belongs to a group of plant growth hormones involved in cell division, differentiation and other physiological processes. One of the possible ways to obtain biologically active compounds is to complex biologically relevant natural compounds to suitable metal atoms. In this work, two structural groups of Zn(II) complexes [Zn(Ln)2Cl2]·Solv (1–5) and [Zn(HLn)Cl3]·xLn (6–7); n = 1–5, Solv = CH3OH for 1 and 2H2O for 2; x = 1 for 6 and 2 for 7; involving a phytohormone kinetin and its derivatives (Ln) were evaluated for their ability to modulate secretion of tumour necrosis factor (TNF)-α, interleukin (IL)-1β and matrix metalloproteinase (MMP)-2 in a lipopolysaccharide (LPS)-activated macrophage-like THP-1 cell model. The penetration of the complexes to cells was also detected. The mechanism of interactions of the zinc(II) complexes with a fluorescent sensor N-(6-methoxy-8-quinolyl)-p-toluene sulphonamide (TSQ) and sulfur-containing biomolecules (l-cysteine and reduced glutathione) was studied by electrospray-ionization mass spectrometry and flow-injection analysis with fluorescence detection. The present study showed that the tested complexes exhibited a low cytotoxic effect on the THP-1 cell line (IC50>40 µM), apart from complex 4, with an IC50 = 10.9±1.1 µM. Regarding the inflammation-related processes, the Zn(II) complexes significantly decreased IL-1β production by a factor of 1.47–2.22 compared with the control (DMSO), but did not affect TNF-α and MMP-2 secretions. However, application of the Zn(II) complexes noticeably changed the pro-MMP-2/MMP-2 ratio towards a higher amount of maturated MMP-2, when they induced a 4-times higher production of maturated MMP-2 in comparison with the vehicle-treated cells under LPS stimulation. These results indicated that the complexes are able to modulate an inflammatory response by influencing secretion and activity of several inflammation-related cytokines and enzymes.
PMCID: PMC3670929  PMID: 23755195
2.  Importance of halide involving interactions at Hoogsteen sites in supramolecular architectures of some coordination metal complexes of N6-benzyl/furfuryl adenine 
Most of the benzyladenine and furfuryladenine derivatives inhibit tumor/cancer cell growth; their toxicity is lesser than the compounds used for the treatment of cancer now-a-days. Many cytokinin derivatives are tested for anticancer activity.
A series of transition metal complexes containing N6-benzyl/furfuryl aminopurines of formula [Mn(FAH)2(H2O)(Cl3)]2.Cl2(1), [Co(FAH)2(H2O)(Cl3)]2.Cl2(2), [Co(FAH)2(Cl4)]2 .[Co(FAH)2(H3O)(Cl3)].Cl2(3), [Ni(FAH)2(H2O)(Cl3)]2.Cl2. (H2O) (4), [Zn(BAH)Br3] (5) and [Cd2(BAH)2(μ-Br)4Br2]n(6) (where BAH and FAH benzyladeninium and furfuryladeninium cations respectively) have been synthesized and characterized. Crystal structures of (1-4) have similar distorted octahedral coordination geometry, while (5) and (6) have distorted tetrahedral geometry and octahedral geometries respectively. In (1-4) two halide ions and two cytokinin cations (BAH+/FAH+) are laterally coordinated to the metal ion. A water molecule and a halide ion are axially coordinated. But the coordination sphere of (5) consists of N7 coordinated benzyladeninium ion and three halide ions. The complex (6) is a coordination polymer bridged by bromide anions. A common notable feature in (1-4) is the presence of one or more lattice chloride anions. They help in a chain formation by N-H…Cl halide involving hydrogen bonding interactions in between the Hoogsteen site hydrogen.
The observed crystal structures emphasize the role of the halide ions in developing the supramolecular architectures by halide involving hydrogen bonding interactions. Also most of the reported cobalt cytokinin complexes possess tetrahedral coordination geometry, but some cobalt complexes have distorted octahedral coordination geometry, which are discussed and compared.
Graphical AbstractSupramolecular architectures of some coordination metal complexes of N6-benzyl/furfuryl adenine.
PMCID: PMC4228066  PMID: 25392712
N6-benzyladenine; N6-furfuryladenine; X-ray diffraction studies; Crystal structures; Supramolecular architectures
3.  Importance of halide involving interactions at Hoogsteen sites in supramolecular architectures of some coordination metal complexes of N6-benzyl/furfuryl adenine 
Most of the benzyladenine and furfuryladenine derivatives inhibit tumor/cancer cell growth; their toxicity is lesser than the compounds used for the treatment of cancer now-a-days. Many cytokinin derivatives are tested for anticancer activity.
A series of transition metal complexes containing N6-benzyl/furfuryl aminopurines of formula [Mn(FAH)2(H2O)(Cl3)]2.Cl2(1), [Co(FAH)2(H2O)(Cl3)]2.Cl2(2), [Co(FAH)2(Cl4)]2 .[Co(FAH)2(H3O)(Cl3)].Cl2(3), [Ni(FAH)2(H2O)(Cl3)]2.Cl2. (H2O) (4), [Zn(BAH)Br3] (5) and [Cd2(BAH)2(μ-Br)4Br2]n(6) (where BAH and FAH benzyladeninium and furfuryladeninium cations respectively) have been synthesized and characterized. Crystal structures of (1-4) have similar distorted octahedral coordination geometry, while (5) and (6) have distorted tetrahedral geometry and octahedral geometries respectively. In (1-4) two halide ions and two cytokinin cations (BAH+/FAH+) are laterally coordinated to the metal ion. A water molecule and a halide ion are axially coordinated. But the coordination sphere of (5) consists of N7 coordinated benzyladeninium ion and three halide ions. The complex (6) is a coordination polymer bridged by bromide anions. A common notable feature in (1-4) is the presence of one or more lattice chloride anions. They help in a chain formation by N-H…Cl halide involving hydrogen bonding interactions in between the Hoogsteen site hydrogen.
The observed crystal structures emphasize the role of the halide ions in developing the supramolecular architectures by halide involving hydrogen bonding interactions. Also most of the reported cobalt cytokinin complexes possess tetrahedral coordination geometry, but some cobalt complexes have distorted octahedral coordination geometry, which are discussed and compared.
Graphical AbstractSupramolecular architectures of some coordination metal complexes of N6-benzyl/furfuryl adenine.
PMCID: PMC4228066  PMID: 25392712
N6-benzyladenine; N6-furfuryladenine; X-ray diffraction studies; Crystal structures; Supramolecular architectures
4.  Superoxide Dismutase Mimics: Chemistry, Pharmacology, and Therapeutic Potential 
Antioxidants & Redox Signaling  2010;13(6):877-918.
Oxidative stress has become widely viewed as an underlying condition in a number of diseases, such as ischemia–reperfusion disorders, central nervous system disorders, cardiovascular conditions, cancer, and diabetes. Thus, natural and synthetic antioxidants have been actively sought. Superoxide dismutase is a first line of defense against oxidative stress under physiological and pathological conditions. Therefore, the development of therapeutics aimed at mimicking superoxide dismutase was a natural maneuver. Metalloporphyrins, as well as Mn cyclic polyamines, Mn salen derivatives and nitroxides were all originally developed as SOD mimics. The same thermodynamic and electrostatic properties that make them potent SOD mimics may allow them to reduce other reactive species such as peroxynitrite, peroxynitrite-derived CO3·−, peroxyl radical, and less efficiently H2O2. By doing so SOD mimics can decrease both primary and secondary oxidative events, the latter arising from the inhibition of cellular transcriptional activity. To better judge the therapeutic potential and the advantage of one over the other type of compound, comparative studies of different classes of drugs in the same cellular and/or animal models are needed. We here provide a comprehensive overview of the chemical properties and some in vivo effects observed with various classes of compounds with a special emphasis on porphyrin-based compounds. Antioxid. Redox Signal. 13, 877–918.
Manganese and Mn Complexes with Simple Ligands
SOD-like activity of manganese
The effects of manganese in vitro and in vivo
Porphyrin-Based SOD Mimics
Design of porphyrin-based SOD mimics
Anionic porphyrins, MnTBAP3− (MnTCPP3−), and MnTSPP3−
Neutral porphyrins
Stability of metalloporphyrins
Aerobic growth of SOD-deficient Escherichia coli
Bioavailability of Mn porphyrins
The effect of the length of the N-alkylpyridyl chains on in vivo efficacy of ortho isomers
The effect of the location of pyridinium nitrogens with respect to porphyrin meso position: meta vs. ortho vs. para isomeric Mn(III) N-alkylpyridylporphyrins
Mitochondrial accumulation of Mn porphyrins
Nuclear and cytosolic accumulation of Mn porphyrins
Intraperitoneal administration
Oral administration
Other modes of action
Superoxide reductase–like action
Peroxynitrite reducing ability
Reactivity toward HOCl
Reactivity toward H2O2
Prooxidative action of Mn porphyrins
Inhibition of redox-controlled cellular transcriptional activity
The effects of Mn porphyrins in suppressing oxidative-stress injuries in vitro and in vivo
General considerations
Central nervous system injuries
Subarachnoid hemorrhage
Spinal cord injury
Amyotrophic lateral sclerosis
Alzheimer's disease
Parkinson's disease
Cerebral palsy
Radiation injury
Breast cancer
Skin cancer
Prostate cancer
MnTE-2-PyP5+ + chemotherapy
MnTE-2-PyP5+ + radiotherapy
MnTE-2-PyP5+ + hyperthermia
Pain therapy: prevention of chronic morphine tolerance
Sickle-cell disease
Cardiac injury
Other ischemia–reperfusion injuries (renal, hepatic)
Lung injuries
Fe porphyrins
Ortho isomers of Fe(III) substituted pyridylporphyrins
Cu porphyrins
Co and Ni porphyrins
Porphyrin-Related Compounds: Biliverdins, Texaphyrins, and Corroles
Mn(III) biliverdin and its analogues
Mn Salen Compounds
SOD-like activity of Mn salens
Catalase-like activity of Mn salens
Reactivity toward other ROS/RNS
Mn salens in suppressing oxidative-stress injuries in vivo
Mn Cyclic Polyamines
SOD-like activity
Mn(II) cyclic polyamines in suppressing oxidative stress in vivo and in vitro
Nonmetal-Based SOD Mimics
SOD-like activity
The protective effects of fullerenes in vivo
SOD-like activity of nitroxides
Reactivity toward other ROS/RNS
The protective effects of nitroxides in vitro and in vivo
Other Compounds
Comparative Studies
PMCID: PMC2935339  PMID: 20095865
5.  Characterization and Dioxygen Reactivity of a New Series of Coordinatively Unsaturated Thiolate-Ligated Manganese(II) Complexes 
Inorganic chemistry  2012;51(12):6633-6644.
The synthesis, structural, and spectroscopic characterization of four new coordinatively unsaturated mononuclear thiolate-ligated manganese(II) complexes ([MnII(SMe2N4(6-Me-DPEN))](BF4) (1), [MnII(SMe2N4(6-Me-DPPN))](BPh4)•MeCN (3), [MnII(SMe2N4(2-QuinoPN))](PF6)•MeCN•Et2O (4), and [MnII(SMe2N4(6-H-DPEN)(MeOH)](BPh4) (5)) is described, along with their magnetic, redox, and reactivity properties. These complexes are structurally-related to recently reported [MnII(SMe2N4(2-QuinoEN))](PF6) (2) Dioxygen addition to complexes 1-5 is shown to result in the formation of five new rare examples of Mn(III) dimers containing a single, unsupported oxo bridge: [MnIII(SMe2N4(6-Me-DPEN)]2-(μ-O)(BF4)2•2MeOH (6), [MnIII(SMe2N4(QuinoEN)]2-(μ-O)(PF6)2•Et2O (7), [MnIII(SMe2N4(6-Me-DPPN)]2-(μ-O)(BPh4)2 (8), [MnIII(SMe2N4(QuinoPN)]2-(μ-O)(BPh4)2 (9), and [MnIII(SMe2N4(6-H-DPEN)]2-(μ-O)(PF6)2•2MeCN (10). Labeling studies show that the oxo atom is derived from 18O2. Ligand modifications, involving either the insertion of a methylene into the backbone, or the placement of an ortho substituent on the N-heterocyclic amine, are shown to noticeably modulate the magnetic and reactivity properties. Fits to solid-state magnetic susceptibility data show that the Mn(III) ions of μ -oxo dimers 6-10 are moderately antiferromagnetically coupled, with coupling constants (2J) that fall within the expected range. Metastable intermediates, which ultimately convert to μ-oxo bridged 6 and 7, are observed in low-temperature reactions between 1 and 2 and dioxygen. Complexes 3-5, on the other hand, do not form observable intermediates, thus illustrating the effect that relatively minor ligand modifications have upon the stability of metastable dioxygen-derived species.
PMCID: PMC3422664  PMID: 22642272
6.  The landscape of cytokinin binding by a plant nodulin 
The crystal structures of complexes of M. truncatula nodulin 13 with four cytokinins, trans-zeatin, N 6-isopentenyladenine, kinetin and N 6-benzyladenine, show an unusual mode of dimerization of this PR-10-fold plant protein. The cytokinin-binding mode in the internal cavity of the protein is the same in each complex and resembles the pattern found in the cytokinin receptor protein.
Nodulation is an extraordinary symbiotic interaction between leguminous plants and nitrogen-fixing bacteria (rhizobia) that assimilate atmospheric nitrogen (in root nodules) and convert it into compounds suitable for the plant host. A class of plant hormones called cytokinins are involved in the nodulation process. In the model legume Medicago truncatula, nodulin 13 (MtN13), which belongs to the pathogenesis-related proteins of class 10 (PR-10), is expressed in the outer cortex of the nodules. In general, PR-10 proteins are small and monomeric and have a characteristic fold with an internal hydrophobic cavity formed between a seven-stranded antiparallel β-sheet and a C-terminal α-helix. Previously, some PR-10 proteins not related to nodulation were found to bind cytokinins such as trans-zeatin. Here, four crystal structures of the MtN13 protein are reported in complexes with several cytokinins, namely trans-zeatin, N 6-isopentenyladenine, kinetin and N 6-benzyladenine. All four phytohormones are bound in the hydrophobic cavity in the same manner and have excellent definition in the electron-density maps. The binding of the cytokinins appears to be strong and specific and is reinforced by several hydrogen bonds. Although the binding stoichiometry is 1:1, the complex is actually dimeric, with a cytokinin molecule bound in each subunit. The ligand-binding site in each cavity is formed with the participation of a loop element from the other subunit, which plugs the only entrance to the cavity. Interestingly, a homodimer of MtN13 is also formed in solution, as confirmed by small-angle X-ray scattering (SAXS).
PMCID: PMC3852650  PMID: 24311578
cytokinins; nodulin 13; Medicago truncatula; dimerization
7.  Structural and Magnetic Effects of meso-Substitution in Alkyl-Substituted Metalloporphyrinate π-Cation Radicals: Characterization of [Fe-(TalkylP.)(Cl)]SbCl6 (alkyl = ethyl and n-propyl) 
Inorganic chemistry  2010;49(17):8078-8085.
We report the preparation and characterization of two meso-alkyl substituted porphyrin π-cation radical derivatives, [Fe(TalkylP.)(Cl)]SbCl6 (alkyl = ethyl or propyl). Both complexes have been characterized by UV/vis/near-IR, IR and Mössbauer spectroscopy, temperature-dependent solid-state magnetic susceptibility measurements and X-ray structure determinations. All data for both oxidized species are consistent with the formulation of the complexes as ring-oxidized iron(III) porphyrin species. The molecular structures of the two five-coordinate species have the typical square-pyramidal coordination group of high-spin iron(III) derivatives. The crystal structures also reveal that the species form cofacial π–π dimers with lateral shifts of 1.44 Å and 3.22 Å, respectively, for the propyl and ethyl radical derivatives. Both radicals exhibit porphyrin cores with alternating bond distance patterns in the inner 16-membered ring. In addition, [Fe(TEtP.)(Cl)]SbCl6 and [Fe(TPrP.)(Cl)]SbCl6 have been characterized by temperature-dependent (6–300K) magnetic susceptibility studies, the best fitting of the temperature-dependent moments reveal strong coupling between iron spins and porphyrin radical, and a smaller magnitude of antiferromagnetic coupling between ring radicals, which are opposite to those found in the five-coordinate iron(III) OEP radicals. The differences in structure and properties of the cation radical meso-alkyl and β-alkyl derivatives possibly reflect differences in properties of a 1u- and a 2u-forming radicals.
PMCID: PMC2980783  PMID: 20799740
8.  Embedding the Ni-SOD mimetic Ni-NCC within a polypeptide sequence alters specificity of the reaction pathway 
Inorganic chemistry  2012;52(1):77-83.
The unique metal abstracting peptide (MAP) asparagine-cysteine-cysteine (NCC) binds nickel in a square planar 2N:2S geometry and acts as a mimic of the enzyme nickel superoxide dismutase (Ni-SOD). The Ni-NCC tripeptide complex undergoes rapid, site-specific chiral inversion to DLD-NCC in the presence of oxygen. Superoxide scavenging activity increases proportionally with the degree of chiral inversion. Characterization of the NCC sequence within longer peptides with absorption, circular dichroism (CD), and magnetic CD (MCD) spectroscopies and mass spectrometry (MS) shows that the geometry of metal coordination is maintained, though the electronic properties of the complex are varied to a small extent due to bis-amide, rather than amine/amide, coordination. In addition, both the Ni-tripeptides and Ni-pentapeptides have a −2 charge. The study here demonstrates that the chiral inversion chemistry does not occur when NCC is embedded in a longer polypeptide sequence. Nonetheless, the superoxide scavenging reactivity of the embedded Ni-NCC module is similar to that of the chirally inverted tripeptide complex, which is consistent with a minor change in reduction potential for the Ni-pentapeptide. Together, this suggests that the charge of the complex could affect the SOD activity as much as a change in primary coordination sphere. In Ni-NCC and other Ni-SOD mimics, changes in chirality, superoxide scavenging activity, and oxidation of the peptide itself all depend on the presence of dioxygen or its reduced derivatives (e.g., superoxide), and the extent to which each of these distinct reactions occurs is ruled by electronic and steric effects that emenate from the organization of ligands around the metal center.
PMCID: PMC3656465  PMID: 23214928
chiral inversion; Ni-NCC; MAP; nickel; bis-amide; nickel superoxide dismutase
9.  Superoxide Dismutase Structures, Stability, Mechanism and Insights into the Human Disease Amyotrophic Lateral Sclerosis from Eukaryotic Thermophile Alvinella pompejana 
Journal of molecular biology  2008;385(5):1534-1555.
Prokaroytic thermophiles supply stable human protein homologs for structural biology; yet, eukaryotic thermophiles would provide more similar macromolecules plus those missing in microbes. Alvinella pompejana is a deep-sea hydrothermal-vent worm that has been found in temperatures averaging as high as 68 °C, with spikes up to 84 °C. Here, we used Cu, Zn superoxide dismutase (SOD) to test if this eukaryotic thermophile can provide insights into macromolecular mechanisms and stability, by supplying better stable mammalian homologs for structural biology and other biophysical characterizations than those from prokaryotic thermophiles. Identification, cloning, characterization, X-ray scattering (SAXS) and crystal structure determinations show that Alvinella pompejana SOD (ApSOD) is super-stable, homologous, and informative. SAXS solution analyses identify the human-like ApSOD dimer. The crystal structure shows the active site at 0.99 Å resolution, plus anchoring interaction motifs in loops and termini accounting for enhanced stability of ApSOD versus human SOD. Such stabilizing features may reduce movements that promote inappropriate intermolecular interactions, such as amyloid-like filaments found in SOD mutants causing the neurodegenerative disease familial amyotrophic lateral sclerosis or Lou Gehrig’s disease. ApSOD further provides a long-sought SOD product complex at 1.35 Å resolution, suggesting a unified inner sphere mechanism for catalysis involving metal ion movement. Notably, this proposed mechanism resolves apparent paradoxes regarding electron transfer. These results extend knowledge of SOD stability and catalysis, and suggest that the eukaryote A. pompejana provides macromolecules highly similar to those from humans, but with enhanced stability more suitable for scientific and medical applications.
PMCID: PMC2669833  PMID: 19063897
Eukaryotic thermophile; thermostable proteins; superoxide dismutase; amyotrophic lateral sclerosis; amyloid filaments
10.  The impact of electrostatics in redox modulation of oxidative stress by Mn porphyrins: Protection of SOD-deficient E. coli via alternative mechanism where Mn porphyrin acts as a Mn-carrier 
Free radical biology & medicine  2008;45(2):201-210.
Understanding the factors that determine the ability of Mn porphyrins to scavenge reactive species is essential for tuning their in vivo efficacy. We present herein the revised structure-activity relationships accounting for the critical importance of electrostatics in the Mn porphyrin-based redox modulation systems and show that the design of effective SOD mimics (per se) based on anionic porphyrins is greatly hindered by inappropriate electrostatics. A new strategy for the β-octabromination of the prototypical anionic Mn porphyrins Mn(III) meso-tetrakis(p-carboxylatophenyl)porphyrin ([MnIIITCPP]3− or MnTBAP3−) and Mn(III) meso-tetrakis(p-sulfonatophenyl)porphyrin ([MnIIITSPP]3−), to yield the corresponding anionic analogues [MnIIIBr8TCPP]3− and [MnIIIBr8TSPP]3−, respectively, is described along with characterization data, stability studies, and their ability to substitute for SOD in SOD-deficient E. coli. Despite the MnIII/MnII reduction potential of [MnIIIBr8TCPP]3− and [MnIIIBr8TSPP]3− being close to the SOD-enzyme optimum and nearly identical to that of the cationic Mn(III) meso-tetrakis(N-methylpyridinium-2-yl)porphyrin (MnIIITM-2-PyP5+), the SOD activity of both anionic brominated porphyrins ([MnIIIBr8TCPP]3−, E½ = +213 mV vs NHE, log kcat = 5.07; [MnIIIBr8TSPP]3−, E½ = +209 mV, log kcat = 5.56) is considerably lower than that of MnIIITM-2-PyP5+ (E½ = +220 mV, log kcat = 7.79). This illustrates the impact of electrostatic guidance of O2•− toward the metal center of the mimic. With low kcat, the [MnIIITCPP]3−, [MnIIITSPP]3−, and [MnIIIBr8TCPP]3− did not rescue SOD-deficient E. coli. The striking ability of [MnIIIBr8TSPP]3− to substitute for the SOD enzymes in the E. coli model does not correlate with its log kcat. In fact, the protectiveness of [MnIIIBr8TSPP]3− is comparable to or better than that of the potent SOD mimic MnIIITM-2-PyP5+, even though the dismutation rate constant of anionic complex is 170-fold smaller. Analyses of the medium and E. coli cell extract revealed that the major species in the [MnIIIBr8TSPP]3− system is not the Mn complex, but the free-base porphyrin [H2Br8TSPP]4− instead. Control experiments with extracellular MnCl2 showed lack of E. coli protection, indicating that “free” Mn2+ cannot enter the cell at significant extent. We proposed herein the alternative mechanism where a labile Mn porphyrin [MnIIIBr8TSPP]3− is not a SOD mimic per se but carries Mn into the E. coli cell.
PMCID: PMC2614336  PMID: 18457677
Mn transport; Electrostatics; Escherichia coli; SOD mimics; Redox modulators; Mn porphyrins
11.  The experimental chemotherapeutic N6-furfuryladenosine (kinetin-riboside) induces rapid ATP depletion, genotoxic stress, and CDKN1A (p21) upregulation in human cancer cell lines 
Biochemical pharmacology  2008;77(7):1125-1138.
Cytokinins and cytokinin nucleosides are purine derivatives with potential anticancer activity. N6-furfuryladenosine (FAdo, kinetin-riboside) displays antiproliferative and apoptogenic activity against various human cancer cell lines, and FAdo has recently been shown to suppress tumor growth in murine xenograft models of human leukemia and melanoma. In this study, FAdo-induced genotoxicity, stress response gene expression, and cellular ATP depletion were examined as early molecular consequences of FAdo-exposure in MiaPaCa-2 pancreas carcinoma, A375 melanoma, and other human cancer cell lines. FAdo, but not adenosine or N6-furfuryladenine, displayed potent antiproliferative activity that was also observed in human primary fibroblasts and keratinocytes. Remarkably, massive ATP depletion and induction of genotoxic stress as assessed by the alkaline comet assay occurred within 60 to 180 minutes of exposure to low micromolar concentrations of FAdo. This was followed by rapid upregulation of CDKN1A and other DNA damage/stress response genes (HMOX1, DDIT3, GADD45A) as revealed by expression array and Western analysis. Pharmacological and siRNA-based genetic inhibition of adenosine kinase suppressed FAdo cytotoxicity and also prevented ATP-depletion and p21-upregulation suggesting the importance of bioconversion of FAdo into the nucleotide form required for drug action. Taken together our data suggest that early induction of genotoxicity and energy crisis are important causative factors involved in FAdo cytotoxicity.
PMCID: PMC2656390  PMID: 19186174
kinetin; N6-furfuryladenosine; genotoxic stress; ATP depletion; CDKN1A; cancer
12.  Biochemical properties and in vivo effects of the SOD1 zinc binding site mutant (H80G) 
Journal of neurochemistry  2011;118(5):891-901.
This work presents the initial characterization of transgenic mice with mutations in a primary zinc binding residue (H80), either alone or with a G93A mutation. H80G;G93A SOD1 transgenic mice developed paralysis with motor neuron loss, and ubquitin inclusion-type rather than mitochondrial vacuolar pathology. Unlike G93A SOD1 related disease, the course was not accelerated by over-expression of CCS. H80G SOD1 transgenic mice did not manifest disease at levels of SOD1 transgene expressed.
The H80G mutation altered certain biochemical parameters of both human wild type SOD1 and G93A SOD1. The H80G mutation does not substantially change the age dependent accumulation of G93A SOD1 aggregates and hydrophobic species in spinal cord. However, both H80G;G93A SOD1 and H80G SOD1 lack dismutase activity, the ability to form homodimers, and cooperativity with CCS, indicating that their dimerization interface is abnormal. The H80G mutation also made SOD1 susceptible to protease digestion.
The H80G mutation alters the redox properties of SOD1. G93A SOD1 exists in either reduced or oxidized form, whereas H80G;G93A SOD1 and H80G SOD1 exist only in a reduced state. The inability of SOD1 with an H80G mutation to take part in normal oxidation-reduction reactions has important ramifications for disease mechanisms and pathology in vivo.
PMCID: PMC3155629  PMID: 21692800
Amyotrophic lateral sclerosis; motor neuron; Superoxide dismutase; copper; mitochondria; inclusions
13.  Identification of Human Monoclonal Antibodies Specific for Human SOD1 Recognizing Distinct Epitopes and Forms of SOD1 
PLoS ONE  2013;8(4):e61210.
Mutations in the gene encoding human SOD1 (hSOD1) can cause amyotrophic lateral sclerosis (ALS) yet the mechanism by which mutant SOD1 can induce ALS is not fully understood. There is currently no cure for ALS or treatment that significantly reduces symptoms or progression. To develop tools to understand the protein conformations present in mutant SOD1-induced ALS and as possible immunotherapy, we isolated and characterized eleven unique human monoclonal antibodies specific for hSOD1. Among these, five recognized distinct linear epitopes on hSOD1 that were not available in the properly-folded protein but were available on forms of protein with some degree of misfolding. The other six antibodies recognized conformation-dependent epitopes that were present in the properly-folded protein with two different recognition profiles: three could bind hSOD1 dimer or monomer and the other three were specific for hSOD1 dimer only. Antibodies with the capacity to bind hSOD1 monomer were able to prevent increased hydrophobicity when mutant hSOD1 was exposed to increased temperature and EDTA, suggesting that the antibodies stabilized the native structure of hSOD1. Two antibodies were tested in a G93A mutant hSOD1 transgenic mouse model of ALS but did not yield a statistically significant increase in overall survival. It may be that the two antibodies selected for testing in the mouse model were not effective for therapy or that the model and/or route of administration were not optimal to produce a therapeutic effect. Therefore, additional testing will be required to determine therapeutic potential for SOD1 mutant ALS and potentially some subset of sporadic ALS.
PMCID: PMC3629177  PMID: 23613814
14.  Regulatory and structural properties differentiating the chromosomal and the bacteriophage-associated Escherichia coli O157:H7 Cu, Zn Superoxide Dismutases 
BMC Microbiology  2008;8:166.
Highly virulent enterohemorrhagic Escherichia coli O157:H7 strains possess three sodC genes encoding for periplasmic Cu, Zn superoxide dismutases: sodC, which is identical to the gene present in non-pathogenic E. coli strains, and sodC-F1 and sodC-F2, two nearly identical genes located within lambdoid prophage sequences. The significance of this apparent sodC redundancy in E. coli O157:H7 has not yet been investigated.
We report that strains deleted of one or more sodC genes are less resistant than the wild type strain to a challenge with hydrogen peroxide, thus confirming their involvement in the bacterial antioxidant apparatus. To understand if the different sodC genes have truly overlapping functions, we have carried out a comparison of the functional, structural and regulatory properties of the various E. coli O157:H7 SodC enzymes. We have found that the chromosomal and prophagic sodC genes are differentially regulated in vitro. sodC is exclusively expressed in aerobic cultures grown to the stationary phase. In contrast, sodC-F1 and sodC-F2 are expressed also in the logarithmic phase and in anaerobic cultures. Moreover, the abundance of SodC-F1/SodC-F2 increases with respect to that of SodC in bacteria recovered from infected Caco-2 cells, suggesting higher expression/stability of SodC-F1/SodC-F2 in intracellular environments. This observation correlates with the properties of the proteins. In fact, monomeric SodC and dimeric SodC-F1/SodC-F2 are characterized by sharp differences in catalytic activity, metal affinity, protease resistance and stability.
Our data show that the chromosomal and bacteriophage-associated E. coli O157:H7 sodC genes have different regulatory properties and encode for proteins with distinct structural/functional features, suggesting that they likely play distinctive roles in bacterial protection from reactive oxygen species. In particular, dimeric SodC-F1 and SodC-F2 possess physico-chemical properties which make these enzymes more suitable than SodC to resist the harsh environmental conditions which are encountered by bacteria within the infected host.
PMCID: PMC2569942  PMID: 18828904
15.  Only one of a wide assortment of manganese-containing SOD mimicking compounds rescues the slow aerobic growth phenotypes of both Escherichia coli and Saccharomyces cerevisiae strains lacking superoxide dismutase enzymes1 
Journal of inorganic biochemistry  2007;101(11-12):1875-1882.
A variety of manganese-containing coordination compounds, frequently termed superoxide dismutase (SOD) mimics, have been reported to have SOD activity in vitro and to be effective at improving conditions related to increased oxidative stress in multicellular organisms. We tested the effectiveness of several of these compounds in substituting for authentic SOD enzymes in two simple systems – the prokaryote Escherichia coli and the single-celled eukaryote, Saccharomyces cerevisiae—where strains are available that completely lack cytoplasmic SOD activity and are thus significantly impaired in their ability to grow aerobically. Most of the compounds tested, including Euk-8 and Euk-134, manganese salen derivatives developed by Eukarion, M40403, a manganese complex of a bis(cyclohexylpyridine)-substituted macrocyclic ligand developed by Metaphore, and several manganese porphyrin derivatives, were ineffective in both systems. Only the manganese tetrapyridyl porphyrin complex MnTM-2-PyP and two close relatives were effective in rescuing aerobic growth of E. coli lacking SOD, and, in the case of sod1Δ yeast, only MnTM-2-PyP itself was fully effective. Surprisingly, several compounds reported to be beneficial in other in vivo model systems (Euk-8, Euk-134, M40403) were actually toxic to these organisms lacking SOD, although they had no effect on the wild type parent strains. Our results suggest the possibility that the beneficial effects of some of the so-called “SOD mimic drugs” may be due to some property other than in vivo superoxide dismutase activity.
PMCID: PMC3237304  PMID: 17723242
16.  Detection, Distribution and Characterization of Novel Superoxide Dismutases from Yersinia enterocolitica Biovar 1A 
PLoS ONE  2013;8(5):e63919.
Superoxide dismutases (SODs) cause dismutation of superoxide radicals to hydrogen peroxide and oxygen. Besides protecting the cells against oxidative damage by endogenously generated oxygen radicals, SODs play an important role in intraphagocytic survival of pathogenic bacteria. The complete genome sequences of Yersinia enterocolitica strains show presence of three different sod genes. However, not much is known about the types of SODs present in Y. enterocolitica, their characteristics and role in virulence and intraphagocytic survival of this organism.
Methodology/Principal Findings
This study reports detection and distribution of the three superoxide dismutase (sodA, sodB and sodC) genes in 59 strains of Y. enterocolitica and related species. The majority (94%) of the strains carried all three genes and constitutive expression of sodA and sodB was detected in 88% of the strains. Expression of sodC was not observed in any of the strains. The sodA, sodB and sodC genes of Y. enterocolitica were cloned in pET28a (+) vector. Recombinant SodA (82 kDa) and SodB (21 kDa) were expressed as homotetramer and monomer respectively, and showed activity over a broad range of pH (3.0–8.0) and temperature (4–70°C). SodA and SodB showed optimal activity at 4°C under acidic pH of 6.0 and 4.0 respectively. The secondary structures of recombinant SodA and SodB were studied using circular dichroism. Production of YeSodC was not observed even after cloning and expression in E. coli BL21(DE3) cells. A SodA− SodB− Escherichia coli strain which was unable to grow in medium supplemented with paraquat showed normal growth after complementation with Y. enterocolitica SodA or SodB.
This is the first report on the distribution and characterization of superoxide dismutases from Y. enterocolitica. The low pH optima of both SodA and SodB encoded by Y. enterocolitica seem to implicate their role in acidic environments such as the intraphagocytic vesicles.
PMCID: PMC3660340  PMID: 23704955
17.  MAPping the Chiral Inversion and Structural Transformation of a Metal-Tripeptide Complex having Ni-SOD Activity 
Inorganic chemistry  2011;50(6):2479-2487.
The metal abstraction peptide (MAP) tag is a tripeptide sequence capable of abstracting a metal ion from a chelator and binding it with extremely high affinity at neutral pH. Initial studies on the nickel-bound form of the complex demonstrate that the tripeptide asparagine-cysteine-cysteine (NCC) binds metal with 2N:2S, square planar geometry and behaves as both a structural and functional mimic of Ni superoxide dismutase (Ni-SOD). Electronic absorption, circular dichroism (CD), and magnetic CD (MCD) data collected for Ni-NCC are consistent with a diamagnetic NiII center. It is apparent from the CD signal of Ni-NCC that the optical activity of the complex changes over time. Mass spectrometry data show that the mass of the complex is unchanged. Combined with the CD data, this suggests that chiral rearrangement of the complex occurs. Following incubation of the nickel-containing peptide in D2O and back-exchange into H2O, incorporation of deuterium into non-exchangeable positions is observed, indicating chiral inversion occurs at two of the alpha carbon atoms in the peptide. Control peptides were used to further characterize the chirality of the final nickel-peptide complex, and DFT calculations were performed to validate the hypothesized position of the chiral inversions. In total, these data indicate Ni-SOD activity is increased proportionally to the degree of structural change in the complex over time, as cross-correlation between the change in CD signal and change in SOD activity reveals a linear relationship.
PMCID: PMC3059394  PMID: 21280586
Chiral inversion; Ni-NCC; Metal Abstraction Peptide (MAP); D-amino acid; D-Cys; square planar; peptide-metal complex; Ni-SOD; superoxide dismutase
18.  Amyotrophic Lateral Sclerosis Model Derived from Human Embryonic Stem Cells Overexpressing Mutant Superoxide Dismutase 1 
An in vitro familial amyotrophic lateral sclerosis (FALS) model was established from human ESCs overexpressing either a wild-type or a mutant SOD1 (G93A) gene, and the phenotypes and survival of the spinal motor neurons were evaluated. This model is expected to help unravel the disease mechanisms involved in the development of FALS and also lead to potential drug discoveries based on the prevention of neurodegeneration.
The generation of amyotrophic lateral sclerosis (ALS) disease models is an important subject for investigating disease mechanisms and pharmaceutical applications. In transgenic mice, expression of a mutant form of superoxide dismutase 1 (SOD1) can lead to the development of ALS that closely mimics the familial type of ALS (FALS). Although SOD1 mutant mice show phenotypes similar to FALS, dissimilar drug responses and size differences limit their usefulness to study the disease mechanism(s) and identify potential therapeutic compounds. Development of an in vitro model system for ALS is expected to help in obtaining novel insights into disease mechanisms and discovery of therapeutics. We report the establishment of an in vitro FALS model from human embryonic stem cells overexpressing either a wild-type (WT) or a mutant SOD1 (G93A) gene and the evaluation of the phenotypes and survival of the spinal motor neurons (sMNs), which are the neurons affected in ALS patients. The in vitro FALS model that we developed mimics the in vivo human ALS disease in terms of the following: (a) selective degeneration of sMNs expressing the G93A SOD1 but not those expressing the WT gene; (b) susceptibility of G93A SOD1-derived sMNs to form ubiquitinated inclusions; (c) astrocyte-derived factor(s) in the selective degeneration of G93A SOD1 sMNs; and (d) cell-autonomous, as well as non-cell-autonomous, dependent sMN degeneration. Thus, this model is expected to help unravel the disease mechanisms involved in the development of FALS and also lead to potential drug discoveries based on the prevention of neurodegeneration.
PMCID: PMC3659703  PMID: 23197818
Embryonic stem cells; Experimental models; Neuron; Astrocytes
19.  The Hypersensitivity Reaction of Tomatoes Resistant to Meloidogyne incognita: Reversal by Cytokinins 
Journal of Nematology  1969;1(1):55-61.
Initiation of larval growth, induction of cell necrosis, and gall formation in the host were measured as criteria of resistance or susceptibility of tomato seedlings to the root-knot nematode, Meloidogyne incognita (Kofoid &White) Chitwood. Seedlings grown at 27 C on water agar containing additions were scored 3 or 4 days after infection.
In the absence of exogenous plant growth regulatory substances, approximately 73% of larvae that entered roots of susceptible plants showed growth, none induced necrosis and nearly all induced gall formation. In roots of a resistant variety, only 4% of the larvae grew, 88% induced necrosis of host cells, and only 29% induced galls. Exogenously supplied cytokinins shifted the response of the resistant plants toward the susceptible reaction. Exogenous kinetin at 0.4 and 0.8 µmolar allowed 55 and 57% of the nematodes to grow, reduced the incidence of necrosis to 32 and 31%, and increased gall formation to 73 and 65%. Three additional cytokinins, Zeatin, 6-(γ,γ-dimethylallylamino)purine, and 6-benzylaminopurine produced effects similar to kinetin. Exogenous indoleacetic acid, gibberellic acid, adenine, guanine, uracil, thymine, cytidine, and 6-methylaminopurine neither increased the percentage of larvae which grew nor decreased the extent of host cell necrosis.
PMCID: PMC2617794  PMID: 19325655
20.  Molecular cloning, characterization, and expression in Escherichia coli of iron superoxide dismutase cDNA from Leishmania donovani chagasi. 
Infection and Immunity  1994;62(2):657-664.
A cDNA corresponding to superoxide dismutase (SOD; EC was isolated from a Leishmania donovani chagasi (L. d. chagasi) promastigote cDNA library, using PCR with a set of primers derived from conserved amino acids of manganese SODs (MnSODs) and iron SODs (FeSODs). Comparison of the deduced amino acid sequences with previously reported SOD amino acid sequences revealed that the L. d. chagasi 585-bp open reading frame had considerable homology with FeSODs and MnSODs. The highest homology was shared with prokaryotic FeSODs. The coding region of L. d. chagasi SOD cDNA has been expressed in fusion with glutathione-S-transferase, using an Escherichia coli mutant, QC779, lacking both MnSOD and FeSOD genes (sodA and sodB). Staining of native polyacrylamide gels for SOD activity of Leishmania crude lysate and the recombinant SOD revealed that both had SOD activity that was inactivated by 5 mM hydrogen peroxide but not by 2 mM potassium cyanide, which is indicative of FeSOD. The recombinant enzyme also protected E. coli mutant QC779 from paraquat toxicity. This indicated that the glutathione-S-transferase peptide does not interfere with the in vivo and in vitro activities of the recombinant SOD. Cross-species hybridization showed that FeSOD is highly conserved in the Leishmania genus. Interestingly, the hybridization pattern of the FeSOD gene(s) coincided with other classification schemes that divide Leishmania species into complexes. The cloning of FeSOD cDNA may contribute to the understanding of the role of SODs in Leishmania pathogenesis.
PMCID: PMC186154  PMID: 8300222
21.  The binding characteristics of isoniazid with copper–zinc superoxide dismutase and its effect on enzymatic activity 
Isoniazid (INH) is front-line anti-tuberculosis (TB) drugs, which are usually prescribed to TB patients for a total period of 6 months. Antituberculosis drug-induced hepatotoxicity (ATDH) is a serious adverse reaction of TB treatment. It is reported that INH-induced hepatotoxicity is associated with oxidative stress. Superoxide dismutase (SOD, EC is the key enzyme for the protection of oxidative stress, which catalyzes the removal of superoxide radical anion, thereby raising the need to better understand the interaction between INH and SOD.
The experimental results showed that the fluorescence intensity of Cu/Zn-SOD regularly decreased owing to form a 1:1 INH-SOD complex. According to the corresponding association constants (KSV) between INH and SOD obtained from Stern–Volmer plot, it is shown that values of KA are 1.01 × 104, 5.31 × 103, 3.33 × 103, 2.20 × 103 L · mol−1 at four different temperatures, respectively. The binding constants, binding sites and the corresponding thermodynamic parameters (ΔH, ΔG and ΔS) were calculated. A value of 3.93 nm for the average distance between INH and chromophore of Cu/Zn-SOD was derived from Förster theory of non-radiation energy transfer. The conformational investigation showed that the presence of INH resulted in the microenvironment and conformational changes of Cu/Zn-SOD. In addition, Effects of INH on superoxide dismutase activity was examined.
The results show that the hydrogen bonding and van der Waals forces play major roles in stabilizing the 1:1 INH-SOD complex. After addition of INH during the range of the experiment, the conformation and microenvironment of Cu/Zn-SOD are changed, but the activity of Cu/Zn-SOD is not changed.
PMCID: PMC3679938  PMID: 23738738
Superoxide dismutase; Isoniazid; Interaction; Antioxidation
22.  Mono- and Dinuclear Iron Complexes of Bis(1-methylimidazol-2-yl)ketone (bik): Structure, Magnetic Properties and Catalytic Oxidation Studies 
Inorganic chemistry  2011;50(19):9243-9255.
The newly synthesized dinuclear complex [FeIII2(μ-OH)2(bik)4](NO3)4 (1) (bik, bis(1-methylimidazol-2-yl)ketone) shows rather short Fe···Fe (3.0723(6) Å) and Fe–O distances (1.941(2)/1.949(2) Å) compared to other unsupported FeIII2(μ-OH)2 complexes. The bridging hydroxide groups of 1 are strongly hydrogen bonded to a nitrate anion. The 57Fe isomer shift (δ = 0.45 mm s−1) and quadrupole splitting (ΔEQ = 0.26 mm s−1) obtained from Mössbauer spectroscopy are consistent with the presence of two identical high-spin iron(III) sites. Variable temperature magnetic susceptibility studies revealed antiferromagnetic exchange (J = 35.9 cm−1 and = JS1·S2) of the metal ions. The optimized DFT geometry of the cation of 1 in the gas phase agrees well with the crystal structure, but both the Fe···Fe and Fe-OH distances are overestimated (3.281 and 2.034 Å, respectively). The agreement in these parameters improves dramatically (3.074 and 1.966 Å) when the hydrogen-bonded nitrate groups are included, reducing the value calculated for J by 35%. Spontaneous reduction of 1 was observed in methanol, yielding a blue [FeII(bik)3]2+ species. Variable temperature magnetic susceptibility measurements of [FeII(bik)3](OTf)2 (2) revealed spin crossover behavior. Thermal hysteresis was observed with 2, due to a loss of co-crystallized solvent molecules, as monitored by thermogravimetric analysis. The hysteresis disappears once the solvent is fully depleted by thermal cycling. [FeII(bik)3](OTf)2 (2) catalyzes the oxidation of alkanes with t-BuOOH. High selectivity for tertiary C-H bond oxidation was observed with adamantane (3°/2° value of 29.6); low alcohol/ketone ratios in cyclohexane and ethylbenzene oxidation, a strong dependence of total turnover number on the presence of O2, and a low retention of configuration in cis-1,2-dimethylcyclohexane oxidation were observed. Stereoselective oxidation of olefins with dihydrogen peroxide yielding epoxides was observed under both limiting oxidant and substrate conditions.
PMCID: PMC3221465  PMID: 21902227
23.  Metalloporphyrin Mixed-Valence π-Cation Radicals: [Fe(oxoOEC./2)(Cl)]2SbCl6, Structure, Magnetic Properties, and Near-IR Spectra 
Inorganic chemistry  2011;50(18):9114-9121.
The preparation and characterization of a mixed-valence π-cation radical derivative of an iron(III) oxochlorinato complex is reported. The new complex has been synthesized by the one-electron oxidation of a pair of [Fe(oxoOEC)(Cl)] molecules to form the dimeric cation [Fe(oxoOEC)(Cl)]+2. The cation has been characterized by an X-ray analysis, Mössbauer spectroscopy, UV-vis and near-IR spectroscopy, and magnetic susceptibility measurements from 6–300 K. The crystal structure shows that the two rings have a smaller overlap area that those of the formally related nickel and copper octaethylporphyrinateethylporphinate derivatives, reflecting the larger steric congestion at the periphery in part of the oxochlorin rings. The Mössbauer data is consistent with two equivalent iron(III) centers. The unpaired electron is delocalized over the two oxochlorin rings and mediates a strong antiferromagnetic interaction between the high-spin iron(III) centers.
PMCID: PMC3171574  PMID: 21809820
24.  Characterization of a Novel SOD-1(G93A) Transgenic Mouse Line with Very Decelerated Disease Development 
PLoS ONE  2010;5(11):e15445.
Amyotrophic Lateral Sclerosis (ALS) is a fatal motoneuron disease, characterized by progressive weakness, muscle wasting and death ensuing 3–5 years after diagnosis. The etiology of ALS is complex and therapeutic approaches rely mostly on transgenic animal models with SOD-1 mutations. Most frequently employed is a mouse line transgenic for SOD-1 (SOD-1 Tg) that contains a point mutation at amino acid position 93 (G->A), present in patients suffering from a familial form of amyotrophic lateral sclerosis. Here we report on a SOD-1 (G93A) Tg mouse line with abnormally delayed onset of disease and prolonged survival. This phenotype arose spontaneously in our colony of the classic SOD-1 (G93A) line. We found that the copy number of the SOD-1 transgene was drastically decreased. We established a new breeding colony, the SOD-1 (G93A)PS line (PS for prolonged survival) where the phenotype is stably inherited for 4 generations now. The mice develop symptoms at an age of approximately 12 months and die at 15 months of age. The delayed development of disease may more closely mimic human pathophysiology, and studying drug effects in this model may yield added confidence for potential efficacy of ALS drug candidates.
PMCID: PMC2978730  PMID: 21102999
25.  Differential localization and potency of manganese porphyrin superoxide dismutase-mimicking compounds in Saccharomyces cerevisiae 
Redox Biology  2014;3:1-6.
Cationic Mn(III) porphyrin complexes based on MnTM-2-PyP are among the most promising superoxide dismutase (SOD) mimicking compounds being considered as potential anti-inflammatory drugs. We studied four of these active compounds in the yeast Saccharomyces cerevisiae, MnTM-2-PyP, MnTE-2-PyP, MnTnHex-2-PyP, and MnTnBu-2-PyP, each of which differs only in the length of its alkyl substituents. Each was active in improving the aerobic growth of yeast lacking SOD (sod1∆) in complete medium, and the efficacy of each mimic was correlated with its characteristic catalytic activity. We also studied the partitioning of these compounds between mitochondria and cytosol and found that the more hydrophobic members of the series accumulated in the mitochondria. Moreover, the degree to which a mimic mitigated the sod1Δ auxotrophic phenotype for lysine relative to its auxotrophic phenotype for methionine depended upon its level of lipophilicity-dependent accumulation inside the mitochondria. We conclude that localization within the cell is an important factor in biological efficacy in addition to the degree of catalytic activity, and we discuss possible explanations for this effect.
Graphical abstract
•Cellular distribution of Mn porphyrin SOD mimics correlates with their lipophilicity.•Higher lipophilicity directs Mn porphyrin SOD mimics to the mitochondria.•In sod1∆ yeast, SOD mimics in mitochondria increased rescue of Lys, not Met auxotrophy.•A mitochondrial target is involved in the sod1∆-dependent Lys auxotrophy.
PMCID: PMC4299968  PMID: 25462059
Oxidative stress; Lysine synthesis; Methionine synthesis; Mitochondria; Mn(III) N-alkylpyridylporphyrin; SOD mimic

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