Catalytic Cycling Research Articles

A large-bolus injection, but not a continuous infusion of sodium selenite improves outcome in peritonitis

A large-bolus injection, but not a continuous infusion of sodium selenite improves outcome in peritonitis. Shock32:140-146, 2009. To the Editor: We read with interest the article by Wang et al. (1) reporting beneficial effects of a large bolus of sodium selenite in a sheep model of septic shock. Administration of 2 mg sodium selenite as a bolus, but not a continuous infusion, delayed hypotension and lactic acidosis, alleviated microvascular alterations, lowered plasma IL-6, and prolonged survival in septic sheep. These findings are intriguing, being obtained at a dose of selenite known to promote pro-oxidant effects (1). Selenium exerts its biological activity through its incorporation into selenoproteins, including glutathione peroxidases and thioredoxin reductases, which function as antioxidant enzymes (2). Conversely, when present in excess for the needs of selenoprotein synthesis, selenium (mainly as selenite) becomes pro-oxidant via catalytic redox cycling (3, 4). Redox cycling agents accept one electron from various reductants, notably glutathione, and, in the presence of oxygen, are oxidized back to the original parent by donating this electron to oxygen, producing superoxide (·O2−). Repetition of the cycle builds up progressive oxidative stress by depleting reducing equivalents and by generating free radicals (5). Since oxidative stress represents a critical pathophysiological mechanism in septic shock (6), the benefits of a pro-oxidant dose of selenium appear paradoxical. It is here important to point out that oxidants and free radicals, beyond their direct cytotoxic potential, also exert multiple indirect effects by modulating redox-sensitive signal transduction pathways (7). A prototypical example is the transcription factor nuclear factor κB (NF-κB), a master regulator of inflammation, crucial for the coordinated transactivation of inflammatory mediators in sepsis (8). Nuclear factor κB is normally retained in the cytoplasm of resting cells, bound to an inhibitory IκB protein. Upon stimulation by cytokines or microbial components, NF-κB dissociates from IκB after IκB phosphorylation by a protein kinase complex termed IκB kinase (IKK), allowing NF-κB to translocate into the nucleus and activate target genes (9). Redox regulation of NF-κB is a complex and controversial issue. Although NF-κB can be activated in some cell types by oxidants through yet incompletely understood mechanisms, evidence is accumulating that redox stress can rather abrogate NF-κB activation by cytokines (9). As mentioned by Wang et al. (1), NF-κB-DNA binding can be disrupted through oxidation of essential thiols within NF-κB. We propose an alternate molecular mechanism, relying in the inactivation of IKK, the upstream kinase in the NF-κB pathway. Phosphorylation of serines 177/181 is required for IKK activation by cytokines, a mechanism that can be inhibited by redox modifications of interjacent cysteine 179: sulfhydryl oxidation or S-nitrosylation of cysteine 179 blocks IKK serine phosphorylation and activity (10). We (9) and others (11, 12) have thus shown that reactive oxygen and nitrogen species abrogated IKK activation and downstream signaling in response to cytokines. Such peculiar redox sensitivity of IKK may provide the molecular basis for the decreased inflammation elicited by the pro-oxidant doses of selenium in the study by Wang et al. (1). Future studies should be designed to precisely monitor the process of IKK-dependent NF-κB activation and its modulation by oxidants in sepsis, which might open the way to innovative anti-inflammatory strategies in this setting. Noureddine Loukili Lucas Liaudet University Hospital Lausanne, Switzerland

Fast ferrous heme–NO oxidation in nitric oxide synthases

During catalysis, the heme in nitric oxide synthase (NOS) binds NO before releasing it to the environment. Oxidation of the NOS ferrous heme-NO complex by O2 is key for catalytic cycling, but the mechanism is unclear. We utilized stopped-flow methods to study the reaction of O2 with ferrous heme-NO complexes of inducible and neuronal NOS enzymes. We found that the reaction does not involve heme-NO dissociation, but instead proceeds by a rapid direct reaction of O2 with the ferrous heme-NO complex. This behavior is novel and may distinguish heme-thiolate enzymes, such as NOS, from related heme proteins.

Sustained Water Oxidation by [Mn4O4]7+ Core Complexes Inspired by Oxygenic Photosynthesis

The bioinspired Mn-oxo cubane complex, [Mn(4)O(4)L(6)](+) 1b(+) (L = (p-MeO-Ph)(2)PO(2)), is a model of the photosynthetic O(2)-evolving complex. It is able to electro-oxidize water at 1.00 V (vs Ag/AgCl) under illumination by UV-visible light when suspended in a proton-conducting membrane (Nafion) coated onto a conducting electrode. Electrochemical measurements, and UV-visible, NMR, and EPR spectroscopies are interpreted to indicate that 1b(+) is the dominant electro-active species in the Nafion, both before and after catalytic cycling, and thus correlates closely with activity. The observation of a possible intermediate and free phosphinate ligand within the Nafion suggests a catalytic mechanism involving photolytic disruption of a phosphinate ligand, followed by O(2) formation, and subsequent reassembly of the cubane structure. Several factors that influence catalytic turnover such as the applied potential, illumination wavelength, and energy have been examined in respect of attaining optimum catalytic activity. Catalytic turnover frequencies of 20-270 molecules O(2) h(-1) catalyst(-1) at an overpotential of 0.38 V plus light (275-750 nm) and turnovers numbers >1000 molecules O(2) catalyst(-1) are observed. The 1b(+)-Nafion system is among the most active and durable molecular water oxidation catalysts known.

Binding to the Low-Density Lipoprotein Receptor Accelerates Futile Catalytic Cycling in PCSK9 and Raises the Equilibrium Level of Intramolecular Acylenzyme

Proprotein convertase subtilisin-kexin type 9 (PCSK9) binds to the low-density lipoprotein receptor (LDLR) on target cells and lowers the level of receptor by impeding its recycling. PCSK9 is self-processed to a complex of its prodomain and catalytic domain like a typical protein convertase, but it does not develop normal proteolytic activity. Instead, its propeptide remains complexed with the catalytic domain, and the C-terminal Gln152 of the prodomain occupies the active site like a substrate for peptide synthesis. To probe its latent catalytic activity, PCSK9 and its complex with the soluble LDLR extracellular domain were separately transferred into H218O, and time point samples were analyzed by peptide mapping with mass spectrometry to measure the rate and extent of incorporation of 18O into the Gln152 carboxylate. In free wild-type or D374Y mutant PCSK9, the t1/2 for exchange of 18O for both oxygens was near 5 min. This slow process progressed to completion, with the distribution of oxygen isotopes in the Gln152 carboxylate finally matching that in solvent. In contrast, exchange reached its final state in <30 s in LDLR-complexed D374Y mutant PCSK9, but approximately 40% of the molecules gave data indicating the presence of only one 18O atom in Gln152. With support from further experiments, this was attributed to hydrolysis of acylenzyme in H216O during preparations for digestion and indicated that PCSK9 complexed with LDLR contains approximately 40% intramolecular acylenzyme at equilibrium. The synthetic EGF-A domain of LDLR induced similar effects as the full-length receptor. The data suggest the existence of distinct conformational states in free and receptor-bound PCSK9.

PH Effects on Iron-Catalyzed Oxidation using Fenton’s Reagent

This work extends investigations into the development and use of a kinetic model to simulate and improve the iron-catalyzed oxidation of organic compounds using Fenton's reagent. While a number of recent studies have successfully modeled the kinetics and species behavior in simple Fenton systems, none have extended and applied the model to examine the effect of operating parameters such as pH on treatment performance. The purpose of this work is to investigate the effect of pH in Fenton-based oxidation systems and to use kinetic modeling to gain insight into the reaction mechanism and speciation of the iron catalyst. Laboratory experiments were conducted across a range of starting concentrations of Fe(II) and H2O2 at pHs of 2.5, 3.0, and 4.0, both in the presence and absence of a target organic, formic acid (HCOOH). With minor modifications, the model presented is capable of accurately describing changes in Fe(II) concentrations over a wide range of reaction conditions and, provided account is taken of additional hydroxyl radical scavenging pathways, also accounts for the oxidation of formic acid over extended reaction times at all pHs considered. The use of composite values for rate constants of reactions involving weakly acidic species is shown to be appropriate, and analysis of the model reveals the catalytic role iron plays in the oxidation process. Experimental and simulated data at the different pHs highlights the effect the catalytic redox cycling of iron has on the performance and oxidation capacity of the Fenton system.

Substrate‐induced DNA strand misalignment during catalytic cycling by DNA polymerase λ

The simple deletion of nucleotides is common in many organisms. It can be advantageous when it activates genes beneficial to microbial survival in adverse environments, and deleterious when it mutates genes relevant to survival, cancer or degenerative diseases. The classical idea is that simple deletions arise by strand slippage. A prime opportunity for slippage occurs during DNA synthesis, but it remains unclear how slippage is controlled during a polymerization cycle. Here, we report crystal structures and molecular dynamics simulations of mutant derivatives of DNA polymerase λ bound to a primer–template during strand slippage. Relative to the primer strand, the template strand is in multiple conformations, indicating intermediates on the pathway to deletion mutagenesis. Consistent with these intermediates, the mutant polymerases generate single-base deletions at high rates. The results indicate that dNTP-induced template strand repositioning during conformational rearrangements in the catalytic cycle is crucial to controlling the rate of strand slippage.

Kinetic Modeling of the Oxidation of p-Hydroxybenzoic Acid by Fenton's Reagent: Implications of the Role of Quinones in the Redox Cycling of Iron

As Fenton (and Fenton-like) chemistry is increasingly implicated in a variety of areas and applications, an understanding of the mechanism and rates governing the system becomes relevant for a growing number of disciplines and purposes. In this work a kinetic model capable of describing species concentrations measured experimentally during the Fenton-mediated oxidation of p-hydroxybenzoic acid (pHBA) is presented and discussed. Experiments were conducted in the dark at low pH using reagent and substrate concentrations ranging from 100 microM to 2 mM. Analysis of the experimental and modeling results reveals that redox reactions between Fe and quinone or quinone-like compounds are essential for the model to qualitatively predict species concentration profiles observed in the laboratory. The quinone and quinone-like compounds generated as byproducts during the oxidation of pHBA act as reducing agents toward Fe(III), thereby assisting the redox cycling of Fe and increasing degradation of the target substrate. The experimental and kinetic modeling results presented highlight the role quinones play in the catalytic redox cycling of iron and the overall effect on the oxidative treatment performance of the system.

Subtle but variable conformational rearrangements in the replication cycle of Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) may accommodate lesion bypass

The possible conformational changes of DNA polymerase IV (Dpo4) before and after the nucleotidyl-transfer reaction are investigated at the atomic level by dynamics simulations to gain insight into the mechanism of low-fidelity polymerases and identify slow and possibly critical steps. The absence of significant conformational changes in Dpo4 before chemistry when the incoming nucleotide is removed supports the notion that the "induced-fit" mechanism employed to interpret fidelity in some replicative and repair DNA polymerases does not exist in Dpo4. However, significant correlated movements in the little finger and finger domains, as well as DNA sliding and subtle catalytic-residue rearrangements, occur after the chemical reaction when both active-site metal ions are released. Subsequently, Dpo4's little finger grips the DNA through two arginine residues and pushes it forward. These metal ion correlated movements may define subtle, and possibly characteristic, conformational adjustments that operate in some Y-family polymerase members in lieu of the prominent subdomain motions required for catalytic cycling in other DNA polymerases like polymerase beta. Such subtle changes do not easily provide a tight fit for correct incoming substrates as in higher-fidelity polymerases, but introduce in low-fidelity polymerases different fidelity checks as well as the variable conformational-mobility potential required to bypass different lesions.

Mismatch-Induced Conformational Distortions in Polymerase β Support an Induced-Fit Mechanism for Fidelity

Molecular dynamics simulations of DNA polymerase (pol) beta complexed with different incorrect incoming nucleotides (G x G, G x T, and T x T template base x incoming nucleotide combinations) at the template-primer terminus are analyzed to delineate structure-function relationships for aberrant base pairs in a polymerase active site. Comparisons, made to pol beta structure and motions in the presence of a correct base pair, are designed to gain atomically detailed insights into the process of nucleotide selection and discrimination. In the presence of an incorrect incoming nucleotide, alpha-helix N of the thumb subdomain believed to be required for pol beta's catalytic cycling moves toward the open conformation rather than the closed conformation as observed for the correct base pair (G x C) before the chemical reaction. Correspondingly, active-site residues in the microenvironment of the incoming base are in intermediate conformations for non-Watson-Crick pairs. The incorrect incoming nucleotide and the corresponding template residue assume distorted conformations and do not form Watson-Crick bonds. Furthermore, the coordination number and the arrangement of ligands observed around the catalytic and nucleotide binding magnesium ions are mismatch specific. Significantly, the crucial nucleotidyl transferase reaction distance (P(alpha)-O3') for the mismatches between the incoming nucleotide and the primer terminus is not ideally compatible with the chemical reaction of primer extension that follows these conformational changes. Moreover, the extent of active-site distortion can be related to experimentally determined rates of nucleotide misincorporation and to the overall energy barrier associated with polymerase activity. Together, our studies provide structure-function insights into the DNA polymerase-induced constraints (i.e., alpha-helix N conformation, DNA base pair bonding, conformation of protein residues in the vicinity of dNTP, and magnesium ions coordination) during nucleotide discrimination and pol beta-nucleotide interactions specific to each mispair and how they may regulate fidelity. They also lend further support to our recent hypothesis that additional conformational energy barriers are involved following nucleotide binding but prior to the chemical reaction.

Nanosize and bimodal porous polyoxotungstate–anatase TiO 2 composites: Preparation and photocatalytic degradation of organophosphorus pesticide using visible-light excitation

New and efficient heterogeneous photocatalytic materials, H 3PW 12O 40/TiO 2 and H 6P 2W 18O 62/TiO 2, with anatase crystalline phase, dual-pore structures (micro- and meso-porosity), and nanometer sizes (average particle size of 6 nm) were prepared by combination of the methods of sol–gel and hydrothermal treatment at a lower temperature (200 °C, 2 °C/min). Compositions, structures, physicochemical properties, and surface morphology of the composites were characterized by inductively coupled plasma atomic emission spectrum (ICP-AES), X-ray diffraction (XRD) patterns, UV diffuse reflectance spectroscopy (UV/DRS), 31P magic-angle spinning (MAS) NMR, Raman scattering spectra, nitrogen adsorption/desorption determination, and transmission electron microscopy (TEM), respectively. It indicated that the primary Keggin or Dawson structure remained intact after immobilization of the unit into TiO 2 network, and hydrogen bonding and chemical interactions between the unit and the anatase network existed in the composites. Both of the composites exhibited visible-light photocatalytic activity to degradation of an aqueous organophosphorus pesticide, parathion-methyl, and the deactivation of the catalysts was hardly observed after five times catalytic cycling.

Catalytic Cycling in β-Phosphoglucomutase: A Kinetic and Structural Analysis,

Lactococcus lactis beta-phosphoglucomutase (beta-PGM) catalyzes the interconversion of beta-d-glucose 1-phosphate (beta-G1P) and beta-d-glucose 6-phosphate (G6P), forming beta-d-glucose 1,6-(bis)phosphate (beta-G16P) as an intermediate. Beta-PGM conserves the core domain catalytic scaffold of the phosphatase branch of the HAD (haloalkanoic acid dehalogenase) enzyme superfamily, yet it has evolved to function as a mutase rather than as a phosphatase. This work was carried out to identify the structural basis underlying this diversification of function. In this paper, we examine beta-PGM activation by the Mg(2+) cofactor, beta-PGM activation by Asp8 phosphorylation, and the role of cap domain closure in substrate discrimination. First, the 1.90 A resolution X-ray crystal structure of the Mg(2+)-beta-PGM complex is examined in the context of previously reported structures of the Mg(2+)-alpha-d-galactose-1-phosphate-beta-PGM, Mg(2+)-phospho-beta-PGM, and Mg(2+)-beta-glucose-6-phosphate-1-phosphorane-beta-PGM complexes to identify conformational changes that occur during catalytic turnover. The essential role of Asp8 in nucleophilic catalysis was confirmed by demonstrating that the D8A and D8E mutants are devoid of catalytic activity. Comparison of the ligands to Mg(2+) in the different complexes shows that a single Mg(2+) coordination site must alternatively accommodate water, phosphate, and the phosphorane intermediate during catalytic turnover. Limited involvement of the HAD family metal-binding loop in Mg(2+) anchoring in beta-PGM is consistent with the relatively loose binding indicated by the large K(m) for Mg(2+) activation (270 +/- 20 microM) and with the retention of activity found in the E169A/D170A double loop mutant. Comparison of the relative positions of cap and core domains in the different complexes indicated that interaction of cap domain Arg49 with the "nontransferring" phosphoryl group of the substrate ligand might stabilize the cap-closed conformation, as required for active site desolvation and alignment of Asp10 for acid-base catalysis. Kinetic analyses of the specificity of beta-PGM toward phosphoryl group donors and the specificity of phospho-beta-PGM toward phosphoryl group acceptors were carried out. The results support a substrate induced-fit mechanism of beta-PGM catalysis, which allows phosphomutase activity to dominate over the intrinsic phosphatase activity. Last, we present evidence that the autophosphorylation of beta-PGM by the substrate beta-G1P accounts for the origin of phospho-beta-PGM in the cell.

Intermolecular Conformational Coupling and Free Energy Exchange Enhance the Catalytic Efficiency of Cardiac Muscle SERCA2a following the Relief of Phospholamban Inhibition

Activation of cardiac muscle sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) by beta1-agonists involves cAMP- and PKA-dependent phosphorylation of phospholamban (PLB), which relieves the inhibitory effects of PLB on SERCA2a. To investigate the mechanism of SERCA2a activation, we compared the kinetic properties of SERCA2a expressed with (+) and without (-) PLB in High Five insect cell microsomes to those of SERCA1 and SERCA2a in native skeletal and cardiac muscle SR. Both native SERCA1 and expressed SERCA2a without PLB exhibited high-affinity (10-50 microM) activation of pre-steady-state catalytic site dephosphorylation by ATP, steady-state accumulation of the ADP-sensitive phosphoenzyme (E1P), and a rapid phase of EGTA-induced phosphoenzyme (E2P) hydrolysis. In contrast, SERCA2a in native cardiac SR vesicles and expressed SERCA2a with PLB lacked the high-affinity activation by ATP and the rapid phase of E2P hydrolysis, and exhibited low steady-state levels of E1P. The results indicate that the kinetic differences in Ca2+ transport between skeletal and cardiac SR are due to the presence of phospholamban in cardiac SR, and not due to isoform-dependent differences between SERCA1 and SERCA2a. Therefore, the results are discussed in terms of a model in which PLB interferes with SERCA2a oligomeric interactions, which are important for the mechanism of Ca2+ transport in skeletal muscle SERCA1 [Mahaney, J. E., Thomas, D. D., and Froehlich, J. P. (2004) Biochemistry 43, 4400-4416]. We propose that intermolecular coupling of SERCA2a molecules during catalytic cycling is obligatory for the changes in Ca2+ transport activity that accompany the relief of PLB inhibition of the cardiac SR Ca2+-ATPase.

Fragmentation and dimerization of copper-loaded prion protein by copper-catalysed oxidation

Prion protein consists of an N-terminal domain containing a series of octapeptide repeats with the consensus sequence PHGGGWGQ and a C-terminal domain composed of three alpha-helices and two short beta-strands. Several studies have shown that the N-terminal domain binds five Cu2+ ions. In the present study, we have investigated copper-catalysed oxidation of a recombinant mouse prion protein, PrP23-231. The copper-loaded PrP23-231 was found to be carbonylated by incubation with dopamine. Besides the formation of carbonyls, a cross-linked species with the dimeric size and C-terminally truncated species were generated. These reactions were retarded in the presence of Cu+- and Cu2+-specific copper chelators, catalase, and SOD (superoxide dismutase), but not in the presence of various bivalent metal ions. Together, these results indicate that the copper bound to prion protein undergoes catalytic cycling in the presence of catecholamines and causes the oxidation of the protein.

Distinct roles of copper in bimetallic copper–rhodium three-way catalysts deposited on redox supports

Copper (4.7%)–rhodium (0–2000ppm) catalysts deposited on three supports with different oxygen storage capacity (OSC) were tested under three-way catalytic cycling conditions using a low frequency and large composition fluctuations. The reducibility by hydrogen was also studied for all the catalysts in order to assess their OSC. Alumina (Al), ceria–alumina (CeAl) and ceria–zirconia (CeZr) were selected as supports. Both copper and rhodium metals favour the reduction of CeAl and CeZr at low temperature. The catalytic activity of rhodium in CO, NO and C3H6 conversion in presence of oxygen is less influenced by the oxygen mobility of the support. However the OSC of the supports allow to attenuate or even suppress the effects of the composition fluctuations and thus improves the conversion at high temperatures. For monometallic copper catalysts, copper participates in the regulation of the oxidant/reducer ratio and is determinant if the support OSC is insufficient. Moreover, the interaction between copper and the mobile oxygen of the support greatly favours the CO oxidation at low temperature, whereas it has less influence on C3H6 oxidation and disfavours the NO reduction at low temperature. No synergetic effect was observed for the bimetallic CuRh catalysts. In this case, the activity is ruled by the metal or the association metal-support, which is the most active in each temperature range. The association “copper-support exhibiting mobile oxygen” is the most active for CO conversion. NO reduction depends mainly on the rhodium content, especially at low temperature, and C3H6 conversion is a little improved by rhodium addition.

Structural insights into DNA polymerase beta deterrents for misincorporation support an induced-fit mechanism for fidelity.

DNA polymerases generally select the correct nucleotide from a pool of structurally similar molecules to preserve Watson-Crick base-pairing rules. We report the structure of DNA polymerase beta with DNA mismatches situated in the polymerase active site. This was achieved by using nicked product DNA that traps the mispair (template-primer, A-C or T-C) in the nascent base pair binding pocket. The structure of each mispair complex indicates that the bases do not form hydrogen bonds with one another, but form a staggered arrangement where the bases of the mispair partially overlap. This prevents closure/opening of the N subdomain that is believed to be required for catalytic cycling. The partially open conformation of the N subdomain results in distinct hydrogen bonding networks that are unique for each mispair. These structures define diverse molecular aspects of misinsertion that are consistent with the induced-fit model for substrate specificity.

Interaction between common folate polymorphisms and B-vitamin nutritional status modulates homocysteine and risk for a thrombotic event

We have assessed the relationship between homocysteine, its thiol metabolites, specific folate coenzymes, and vitamin B 12 according to the two main functionally relevant genotype–genotype categories that maintain the balance between homocysteine transsulphuration to cysteine, and homocysteine remethylation via folate dependent methionine biosynthesis, namely 2756A → G-MS/66A → G-MSR and 677C → T-MTHFR/1298A → C-MTHFR. We examined 152 individuals who were being treated for either thromboembolic (TE) or non-thromboembolic (non-TE) events. χ 2 test for linear trend in odds ratio provides reasonable evidence for an altered risk of thromboembolism within the range of compound MS/MSR genotypes encountered (wt/wt → recessive/recessive) ( p⩽0.05), but not within the same range of MTHFR/MTHFR genotypes. Logistic regression analysis of the risk for a TE event gave OR=0.49 (95% CI, 0.26–0.92; p=0.026) for 2756A → G-MS, OR=1.08 (95% CI, 0.65–1.78) for 66A → G-MSR, OR=1.19 (95% CI, 0.69–2.06) for 677C → T-MTHFR and OR=0.98 (95% CI, 0.52–1.85) for 1298A → C-MTHFR. When genotypes were examined individually, one-way ANOVA showed only 677C → T-MTHFR ( p=0.005 [TE]) and 2756A → G-MS ( p=0.005 [non-TE] and p=0.0006 [all subjects]) influence homocysteine. One-way ANOVA also showed that MTHFR/MTHFR compound genotype significantly influences TE homocysteine distribution ( p=0.044), but no other variable. In MS/MSR, homocysteine distribution is not significantly affected in TE subjects, but approaches significance in non-TE individuals ( p=0.062). However, the increased power obtained when all subjects are analysed demonstrates a significant influence of MS/MSR upon homocysteine distribution ( p=0.008). Other significant influences of MS/MSR were on total cellular 5-methyl-H 4folate in non-TE subjects ( p=0.042) and vitamin B 12 in TE subjects ( p=0.018). Given the central role of vitamin B 12 in MS/MSR activity, 5-methyl-H 4folate and homocysteine were also looked at by vitamin B 12 quartile, independent of genotype: Vitamin B 12 quartile significantly affected homocysteine distribution in TE ( p=0.013) but not non-TE individuals, with no effect on 5-methyl-H 4folate distributions. Similarly, the prevalence of clinical phenotypes ( p=0.013) and of ‘high risk’ 2756A → G-MS wildtypes ( p=0.039) was associated with the disposition of homocysteine/B 12 in TE but not non-TE subjects. Overall, results indicate compound MS/MSR genotype is associated with risk for a TE event. This may be related to variation in activity of the functional enzymes coded for by polymorphic forms of compound MS/MSR, resulting in altered catalytic cycling of methylcobalamin/cob(I)alamin, which in turn influences Hcy (and total 5-methyl-H 4folate). The effect on vitamin B 12 is greater in TE than non-TE subjects. The compound MTHFR/MTHFR genotype also influences variation in Hcy in TE subjects, but seemingly without the same level of mediation by vitamin B 12. These results are consistent with accepted paradigms and offer a plausible explanation for the effect and interaction of specific SNPs in the TE phenotype. The biological implications of the limited number of MTHFR/MTHFR mutant alleles that can coexist, usually no more than two, may be explained by the serious consequences to folate status that these genotype combinations precipitate. We show that lowering of all folate 1-C pools occurs in the rare ct/cc compound genotype, except for the 5,10-methenyl-H 4folate pool, which expands. 5,10-methenyl-H 4folate is the immediate product of 5,10-methylene-H 4folate, which is likely diverted away from methionine biosynthesis via the aberrant MTHFR enzyme. Consequences for the methylation cycle may be severe, and in most cases lethal for the developing embryo, where methylation is required for dozens of critical processes, but particularly for maintaining DNA methylation patterns that are now known to regulate the expression of half the complement of human genes via CpG islands located in the 5 ′ promotor region, or within the first few exons of the gene.

The anticancer drug-DNA complex: femtosecond primary dynamics for anthracycline antibiotics function.

The anthracycline-DNA complex, which is a potent agent for cancer chemotherapy, has a unique intercalating molecular structure with preference to the GC bases of DNA, as shown by Rich's group in studies of single-crystal x-ray diffraction. Understanding cytotoxicity and its photoenhancement requires the unraveling of the dynamics under the solution-phase, physiological condition. Here we report our first study of the primary processes of drug function. In a series of experiments involving the drug (daunomycin and adriamycin) in water, the drug-DNA complexes, the complexes with the four nucleotides (dGTP, dATP, dCTP, and dTTP), and the drug-apo riboflavin-binding protein, we show the direct involvement of molecular oxygen and DNA base-drug charge-separation-the rates for the reduction of the drug and dioxygen indicate the crucial role of drug/base/O(2) in the efficient and catalytic redox cycling. These dynamical steps, and the subsequent reactions of the superoxide product(s), can account for the photoenhanced function of the drug in cells, and potentially for the cell death.

Quinoid redox cycling as a mechanism for sustained free radical generation by inhaled airborne particulate matter

The health effects of airborne fine particles are the subject of government regulation and scientific debate. The aerodynamics of airborne particulate matter, the deposition patterns in the human lung, and the available experimental and epidemiological data on health effects lead us to focus on airborne particulate matter with an aerodynamic mean diameter less than 2.5 μm (PM2.5) as the fraction of the particles with the largest impact in health. In this article we present a novel hypothesis to explain the continuous production of reactive oxygen species produced by PM2.5 when it is deposited in the lung. We find PM2.5 contains abundant persistent free radicals, typically 1016 to 1017 unpaired spins/gram, and that these radicals are stable for several months. These radicals are consistent with the stability and electron paramagnetic resonance spectral characteristics of semiquinone radicals. Catalytic redox cycling by semiquinone radicals is well documented in the literature and we had studied in detail its role on the health effects of cigarette smoke particulate matter. We believe that we have for the first time shown that the same, or similar radicals, are not confined to cigarette smoke particulate matter but are also present in PM2.5. We hypothesize that these semiquinone radicals undergo redox cycling, thereby reducing oxygen and generating reactive oxygen species while consuming tissue-reducing equivalents, such as NAD(P)H and ascorbate. These reactive oxygen species generated by particles cause oxidative stress at sites of deposition and produce deleterious effects observed in the lung.

Contrasting effects of catecholic and O-methylated tetrahydroisoquinolines on hydroxyl radical production

Tetrahydroisoquinolines (TIQs) are intraneuronal, catecholamine-derived alkaloids that have been implicated in the etiology of Parkinson’s disease and in alcohol related disorders. The in vitro production of the cytotoxic hydroxyl radical ( ⋅OH) was recorded during the autoxidation of salsolinol (SAL) and salsolinol-1-carboxylic acid (SAL-1C), but not when these two catecholic TIQs were oxidized by tyrosinase. Significantly higher levels of the radical were produced when these catecholic TIQs were incubated with ⋅OH generating complexes, or with chelated iron. In contrast, mono- O-methylated TIQs such as salsoline (SLN) and salsoline-1-carboxylic acid (SLN-1C) did not generate ⋅OH during autoxidation or when incubated with chelated iron or tyrosinase. Radical production by ⋅OH-generating complexes was reduced in the presence of O-methylated TIQs. The neurotoxicity of TIQs may result from their propensity to autoxidize and generate reactive quinoids and ensuing oxygen radicals. The functional significance of the replacement of a hydroxyl group attached to C-7 of SAL or SAL-1C with a methoxyl group remains to be determined. This single structural modification may prevent mono- O-methylated TIQs from participating in catalytic redox cycling reactions that would otherwise augment ⋅OH production. If true, then O-methylation and other cellular mechanisms that circumvent the autoxidation of catecholamine-derived TIQs may reduce the likelihood of these substances forming cytotoxic quinoids and influencing endogenous ⋅OH-generating reactions.

Relationship between cytochrome P450 catalytic cycling and stability: fast degradation of ethanol-inducible cytochrome P450 2E1 (CYP2E1) in hepatoma cells is abolished by inactivation of its electron donor NADPH‒cytochrome P450 reductase

Relationship between cytochrome P450 catalytic cycling and stability: fast degradation of ethanol-inducible cytochrome P450 2E1 (CYP2E1) in hepatoma cells is abolished by inactivation of its electron donor NADPH‒cytochrome P450 reductase