Time Course Of Reaction Research Articles

Optimization of the Solvent‐Free Lipase‐Catalyzed Synthesis of Fructose‐Oleic Acid Ester Through Programming of Water Removal

AbstractFructose oleate, an environmentally‐friendly biobased surfactant, was prepared using solvent‐free suspensions of saccharide in a mixture of acyl donor and monoester (the latter present at ≥5 wt% initially) continuously recirculated through a closed‐loop packed bed bioreactor (PBBR)‐based system at 53 °C, with the PBBR containing immobilized Rhizomucor miehei lipase (Lipozyme®IM, Novozymes, Franklinton, NC, USA). To replenish the acyl acceptor consumed during the time course of reaction, the medium was isolated, fructose added, and a suspension formed by rigorous stirring at 80 °C for 6 h followed by centrifugation to remove larger particles, with the placement of the acyl acceptor replenishment treatments during the time course of reaction were optimized. Water removal via free evaporation was augmented during the latter portion of the time course (using a molecular sieve packed column, N2 bubbling, vacuum pressure, or a combination of the latter two), with an optimal performance achieved when initiating N2 + vacuum ( removal rate) upon reaching 60% ester, to maintain the liquid‐phase water content near 0.40 wt%. When employing the above‐mentioned conditions, 92.6 wt% fructose oleate was produced within 132 h, yielding a productivity of .

Isolation and Structural Characterization of Acrylamide−Pyridoxamine Adducts

Pyridoxamine (PM) is an effective inhibitor of the formation of the carcinogen acrylamide (AA) from its precursors in low-moisture model systems. Although AA is widely assumed to act by scavenging carbonyl compounds, no alternative pathways have to date been explored. In this work, we found AA to directly react with PM in a low-moisture acrylamide-pyridoxamine model system heated at 140 °C for up to 40 min. The reaction products gave four major chromatographic peaks that were assigned to acrylamide-pyridoxamine adducts. Two of the adducts (AA-PM-1 and AA-PM-3) were selected for isolation and structural characterization with various spectroscopic (UV, fluorescence, IR, and NMR) and mass spectrometric techniques (MS, MS/MS). As shown by the proposed reaction scheme, PM can directly react with AA via Michael addition. The reaction involves a nucleophilic attack of the PM amine group on AA (an α,β-unsaturated carbonyl compound) to give adduct AA-PM-3, which was identified as 3-(((3-hydroxy-5-(hydroxymethyl)-2-methylpyridin-4-yl)methyl)amino)propanamide. However, AA-PM-3 further reacts with any additional AA present in the medium to give adduct AA-PM-1 identified as 3,3'-(((3-hydroxy-5-(hydroxymethyl)-2-methylpyridin-4-yl)methyl)azanediyl)dipropanamide. The time courses of these adduct formation reactions were studied in cookies supplemented with PM, where AA-PM-3 was found to be the predominant structure.

Impact of Anomalous Diffusion on Biochemical Kinetics

Biochemical reactions ultimately rely on the (repetitive) encounter of two or more molecules. Diffusive transport of the reaction partners not only determines the first encounter but also the subsequent time window during which the proteins stay near to each other and repeatedly collide. Experimental observations have highlighted that diffusion in crowded media like the cell's cytoplasm or the plasma membrane is strongly anomalous, i.e. the area that is explored by a particle grows less than linearly in time. We show here that anomalous diffusion can dramatically alter the time course of biochemical reactions. In general, the behavior deviates from the classical Smoluchowski result and shows signatures of anomalous, fractal-like kinetics. While single-step reactions are typically slowed down by anomalous diffusion, multi-step reactions (e.g. phosphorylation cascades) can also be enhanced. Hence, biochemical networks in living cells could have emerged in a way to deal with or even exploit anomalous subdiffusion.

Mechanistic Studies of the Autoactivation of PAK2: A TWO-STEP MODEL OF CIS INITIATION FOLLOWED BY TRANS AMPLIFICATION

Protein kinase activation, via autophosphorylation of the activation loop, is a common regulatory mechanism in phosphorylation-dependent signaling cascades. Despite the prevalence of this reaction and its importance in biological regulation, the molecular mechanisms of autophosphorylation are poorly understood. In this study, we developed a kinetic approach to distinguish quantitatively between cis- and trans-pathways in an autocatalytic reaction. Using this method, we have undertaken a detailed kinetic analysis for the autoactivation mechanism of p21-activated protein kinase 2 (PAK2). PAK2 is regulated in vivo and in vitro by small GTP-binding proteins, Cdc42 and Rac. Full activation of PAK2 requires autophosphorylation of the conserved threonine, Thr(402), in the activation loop of its catalytic kinase domain. Analyses of the time courses of substrate reaction during PAK2 autoactivation suggest that autophosphorylation of Thr(402) in PAK2 obeys a two-step mechanism of cis initiation, followed by trans amplification. The unphosphorylated PAK2 undergoes an intramolecular (cis) autophosphorylation on Thr(402) to produce phosphorylated PAK2, and this newly formed active PAK2 then phosphorylates other PAK2 molecules at Thr(402) in an intermolecular (trans) manner. Based on the kinetic equation derived, all microscopic kinetic constants for the cis and trans autophosphorylation have been estimated quantitatively. The advantage of the new method is not only its usefulness in the study of fast activation reactions, but its convenience in the study of substrate effects on modification reaction. It would be particularly useful when the regulatory mechanism of the autophosphorylation reaction toward certain enzymes is being assessed.

Effect of membrane composition on lipid oxidation in liposomes

To study the effect of membrane composition on the oxidation of liposomes, different systems were prepared by adding one component at time to phosphatidylcholine (Epikuron 200). In particular, the effect of cholesterol and its ester, cholesterol stearate, on membrane structure and oxidation was studied. A first screening of the structure and net charge of the different preparation was made by means of z-potential and size measurements. Then the liposomes were oxidized by using a hydrophilic radical initiator, the (2,2-azobis(2-amidinopropane) hydrochloride, AAPH, which thermally decomposes to give a constant radical flux in water. The oxidation of liposomes, monitored by following the absorbance of the primary products of oxidation at 234nm, was shown to be dependent on the composition of the liposomal bilayer and so on its biophysical properties. In addition, size and z-potential measurements gathered in the time course of the peroxidation reaction, revealed that the oxidation induced a modification of the superficial characteristics of the membrane bilayer so as to change its charge at the shear plane (z-potential). This behaviour was shared by all liposomal preparations independent of the composition. The change in sizes of the different liposomal preparation, instead, followed different trends, being more stable both in control samples and in oxidized ones when cholesterol was present. From the analysis of the results, it can be concluded that cholesterol affects the oxidation induced by hydrophilic radical initiator of model membranes by changing the biophysical properties of the phospholipid bilayer. The rigidity induced by cholesterol at temperatures above the Tm makes the membrane more resistant to radical attack from an external aqueous phase and this in turn delays the start of the reaction. The decrease of z-potential of the liposomal particles induced by the oxidation process can be an important clue to understand the mechanisms involved in the etiology of important diseases.

Investigation of the Molecular Basis of High Oxygen Affinity Hemoglobin Brigham (α2Aβ2100 Pro→Leu) In a Subject with Familial Erythrocytosis

AbstractAbstract 4806A 40 year old female was seen at the NIH Clinical Center for polycythemia (Hb 16.3 g/dL) and recurrent pulmonary embolism. It was determined that she was a member of the original kindred with familial erythrocytosis due to the high oxygen affinity Hb Brigham first reported by Lokich et al (JCI, 1973;52:2060-7) where leucine replaces proline at amino acid 100 in the Hb beta globin chain. We sought to isolate and characterize the abnormal Hb in order to elucidate the mechanism by which the amino acid substitution causes a high affinity for oxygen. Fresh RBCs exhibited high oxygen affinity (P50 = 23.6 mmHg; a Hill coefficient of n=2.02) compared to that of fresh normal RBCs (P50 = 31.1 mmHg; n=2.1) in Hemox buffer, pH 7.4 at 37°C using the TCS automated Hemox analyzer. After 2, 3-diphosphoglycerate (DPG) depletion (24 hours incubation of RBCs at room temp) the RBCs exhibited a higher oxygen affinity, that decreased when inositol hexaphosphate (a 2, 3-DPG analogue) was electroporated into erythrocytes, indicating a normal allosteric response at the 2,3-DPG binding site. The Bohr effect (pH dependence of oxygen affinity) of stripped Hb Brigham was reduced by approximately 30% when compared to that of HbA0. Hb Brigham was separated from Hb A in this subject using strong cationic HPLC and the molecular composition of each protein was verified by tryptic peptide mapping and mass spectrometry of purified peptides. The purified fractions; F1 (HbA0) and FII (Hb Brigham) which represent approximately 60% and 40% of total Hb, respectively, were characterized using rapid mixing stopped-flow kinetics and compared to RBCs Brigham. The time course of the oxygen dissociation reaction from both (oxy) fractions in the presence of sodium dithionite were monophasic with apparent rate constants derived for FI (koff = 38 s-1) which is comparable to that of whole blood (koff = 37.9 s-1) and that of normal control HbAo (koff = 39.6 s-1). However, the rate constant for oxygen dissociation from FII was slightly reduced (koff = 33.9 s-1). To further characterize oxygen dissociation kinetics, carbon monoxide (CO) combination kinetics with the (deoxy) forms of each hemoglobin were also carried out. The kinetics of CO binding to deoxyHb were comparable between both purified fractions and whole blood (kon =0.22-0.23 mM-1 s-1). Unlike FI or control Hb A0, FII showed no change in its CO binding kinetics in the presence of increasing concentrations of IHP. We propose that the IHP-dependent difference in CO binding resulted from destabilization of the deoxy (tense) structure due to the β2 100 Pro→Leu substitution. Analysis of purified Hb Brigham, which was not previously possible, provides valuable insights into the contribution of this mutant to the overall oxygen affinity in this patient and may be useful in the design and evaluation of hemoglobin-based blood substitutes. Disclosures:No relevant conflicts of interest to declare.

High‐performance liquid chromatography/mass spectrometric and proton nuclear magnetic resonance spectroscopic studies of the transacylation and hydrolysis of the acyl glucuronides of a series of phenylacetic acids in buffer and human plasma

The use of high-performance liquid chromatography/mass spectrometry (HPLC/MS) and proton nuclear magnetic resonance ((1)H NMR) spectroscopy for the kinetic analysis of acyl glucuronide (AG) isomerisation and hydrolysis of the 1-β-O-acyl glucuronides (1-β-O-AG) of phenylacetic acid, (R)- and (S)-α-methylphenylacetic acid and α,α-dimethylphenylacetic acid is described and compared. Each AG was incubated in both aqueous buffer, at pH 7.4, and control human plasma at 37°C. Aliquots of these incubations, taken throughout the reaction time-course, were analysed by HPLC/MS and (1)H NMR spectroscopy. In buffer, transacylation reactions predominated, with relatively little hydrolysis to the free aglycone observed. In human plasma incubations the calculated rates of reaction were much faster than for buffer and, in contrast to the observations in buffer, hydrolysis to the free aglycone was a significant contributor to the overall reaction.A diagnostic analytical methodology based on differential mass spectrometric fragmentation of 1-β-O-AGs compared to the 2-, 3- and 4-positional isomers, which enables selective determination of the former, was confirmed and applied. These findings show that HPLC/MS offers a viable alternative to the more commonly used NMR spectroscopic approach for the determination of the transacylation and hydrolysis reactions of these AGs, with the major advantage of having the capability to do so in a complex biological matrix such as plasma.

Reversibility of object recognition but not spatial memory impairment following binge-like alcohol exposure in rats

Excessive alcohol use leads to neurodegeneration in several brain structures including the hippocampal dentate gyrus and the entorhinal cortex. Cognitive deficits that result are among the most insidious and debilitating consequences of alcoholism. The object exploration task (OET) provides a sensitive measurement of spatial memory impairment induced by hippocampal and cortical damage. In this study, we examine whether the observed neurotoxicity produced by a 4-day binge ethanol treatment results in long-term memory impairment by observing the time course of reactions to spatial change (object configuration) and non-spatial change (object recognition). Wistar rats were assessed for their abilities to detect spatial configuration in the OET at 1 week and 10 weeks following the ethanol treatment, in which ethanol groups received 9–15 g/kg/day and achieved blood alcohol levels over 300 mg/dl. At 1 week, results indicated that the binge alcohol treatment produced impairment in both spatial memory and non-spatial object recognition performance. Unlike the controls, ethanol treated rats did not increase the duration or number of contacts with the displaced object in the spatial memory task, nor did they increase the duration of contacts with the novel object in the object recognition task. After 10 weeks, spatial memory remained impaired in the ethanol treated rats but object recognition ability was recovered. Our data suggest that episodes of binge-like alcohol exposure result in long-term and possibly permanent impairments in memory for the configuration of objects during exploration, whereas the ability to detect non-spatial changes is only temporarily affected.

Cellular and transcriptional responses of wheat during compatible and incompatible race‐specific interactions with Puccinia striiformis f. sp. tritici

The initial stages of Puccinia striiformis f. sp. tritici (the causal agent of yellow rust in wheat) infection triggered a hypersensitive cell death (HCD) response in both compatible and Yr1-mediated incompatible interactions, although the response was earlier and more extensive in the incompatible interaction. Later stages of fungal development were only associated with an HCD response in the incompatible interaction, the HCD response being effectively suppressed in the compatible interaction. Cell autofluorescence was seen in mesophyll cells in direct contact with fungal infection hyphae (primary HCD) and in adjacent mesophyll cells (secondary HCD), indicating the activation of cell-to-cell signalling. Microarray analysis identified a number of defence-related transcripts implicated in Yr1-mediated resistance, including classical pathogenesis-related (PR) transcripts and genes involved in plant cell defence responses, such as the oxidative burst and cell wall fortification. A quantitative reverse transcriptase-polymerase chain reaction time course analysis identified a number of defence-related genes, including PR2, PR4, PR9, PR10 and WIR1 transcripts, associated with the latter stages of Yr1-mediated resistance. A meta-analysis comparison of the Yr1-regulated transcriptome with the resistance transcriptomes of the race-specific resistance gene Yr5 and the race-nonspecific adult plant resistance gene Yr39 indicated limited transcript commonality. Common transcripts were largely confined to classic PR and defence-related genes.

Glutamates 78 and 122 in the Active Site of Saccharopine Dehydrogenase Contribute to Reactant Binding and Modulate the Basicity of the Acid-Base Catalysts

Saccharopine dehydrogenase catalyzes the NAD-dependent oxidative deamination of saccharopine to give l-lysine and alpha-ketoglutarate. There are a number of conserved hydrophilic, ionizable residues in the active site, all of which must be important to the overall reaction. In an attempt to determine the contribution to binding and rate enhancement of each of the residues in the active site, mutations at each residue are being made, and double mutants are being made to estimate the interrelationship between residues. Here, we report the effects of mutations of active site glutamate residues, Glu(78) and Glu(122), on reactant binding and catalysis. Site-directed mutagenesis was used to generate E78Q, E122Q, E78Q/E122Q, E78A, E122A, and E78A/E122A mutant enzymes. Mutation of these residues increases the positive charge of the active site and is expected to affect the pK(a) values of the catalytic groups. Each mutant enzyme was completely characterized with respect to its kinetic and chemical mechanism. The kinetic mechanism remains the same as that of wild type enzymes for all of the mutant enzymes, with the exception of E78A, which exhibits binding of alpha-ketoglutarate to E and E.NADH. Large changes in V/K(Lys), but not V, suggest that Glu(78) and Glu(122) contribute binding energy for lysine. Shifts of more than a pH unit to higher and lower pH of the pK(a) values observed in the V/K(Lys) pH-rate profile of the mutant enzymes suggests that the presence of Glu(78) and Glu(122) modulates the basicity of the catalytic groups.

Assessment of Michaelis–Menten parameters by analysis of single time courses of enzyme-catalyzed reactions

Assessment of Michaelis–Menten parameters by analysis of single time courses of enzyme-catalyzed reactions

Time course of vasovagal syncope with whole blood donation

Background Improving the safety of the donation experience will reduce donor injuries and increase donations, donation frequency and donor satisfaction. Understanding the physiology of donor reactions supports selection of effective interventions to reduce risk.Aims The examination of the time course of donor vasovagal syncopal reactions (VVS) to determine when most reactions occur supports the development of appropriate theories about cause of the majority of the reactions and the application of interventions to reduce the risk of a significant number of reactions.Materials and Methods A database of more than 900 000 donor registrations and more than 500 000 whole blood donation events was examined for the time course of VVS reactions by reviewing the onset time of the reactions. The donor experience was divided into three periods based on the theory that the risk of reaction depends on different factors in each of the periods. The time course was analyzed using frequency distribution counts stratified by donation status and separately by gender.Results There were 956 766 registrations, 554 513 attempted whole blood donations, 536 907 complete donations and 17 606 incomplete donations. The mean draw time of a complete donation was 8.21 (95% CI, 8.2–8.21) min and for an incomplete donation was 8.91 (95% CI, 8.8–9.01) min. The reaction rate for Period 1 (before venipuncture) was 0.045/1000 registrations; for Period 2 (venipuncture to 4 min post‐venipuncture) and Period 3 (4 min post venipuncture to last reported VVS reaction ‐265 min), the reaction rate was 3.5/1000 for women and 1.5/1000 for men. There was a steady increase in the reaction rate during phlebotomy and the rate peaked at the time of needle removal. After needle removal there were peaks in the VVS rate at 5 and 9 min. The reaction rate declined steadily thereafter.Discussion The peak reaction rates occur within a time period of 10 min beginning 1 min before needle removal. The reactions which have the most significant risk of donor injury occur later than this and often occur after the donor leaves the donation site. Approximately 10% of VVS reactions in male donors and 25% of VVS reactions in female donors occur more than 15 min after the needle is removed. The latest VVS reaction was reported to have begun at about 4.5 h after needle withdrawal.Conclusion It is our thought that preventive interventions for reactions which occur during phlebotomy might be slightly different from the preventive measure aimed at reducing risk to donors after assuming upright posture. Once the donor stands up, relative hypovolemia plays a greater role and interventions should be aimed at preventing changes in blood pressure due to hypovolemia during this period.

ChemInform Abstract: Isotopic Exchange of Oxygen Between Proton-Exchanged Zeolites and Water.

The time courses of the title reaction were analyzed by using a closed circulation system. It was found that there are three kinds of framework oxygen, OI, OII, and OIII, that is, easily, moderately, and hardly exchanging oxygens. The respective rate constants of the exchange reaction, k1, k2, and k3, were almost constant, or independent of the zeolite structures and silica/alumina ratios. In contrast, the numbers of OI and OII (n1 and n2) decreased with increasing silica/alumina ratios, whereas that of OIII (n3) increased. n1 + n2 and n3 are approximately in agreement with the numbers of bridging oxygens of Si−O−Al and Si−O−Si, respectively. Next, IR measurement was applied to confirm the origin of the active framework oxygens. Upon the exchange reaction on H-MFI-23, one acidic OH band at 3612 cm-1 readily shifted to lower frequency while the silanol band at 3735 cm-1 and ν(Si−O−T (T = Si or Al)) bands at 1219 and 1092 cm-1 shifted slightly. The 18O contents in the bands, evaluated by the deconvolution o...

ChemInform Abstract: Evaluation of Time Course and Inter-Relationship of Inflammatory Mediators in Experimental Inflammatory Reaction

Inflammatory signs, such as heat, redness, swelling and pain, have been described from the Greek era. In these phenomena various endogenous active substances, i.e., inflammatory mediators, could cause and manifest vascular dilatation, a vascular permeability increase and sensitization of pain receptors, etc. In order to evaluate the roles of inflammatory mediators, we have studied the time courses of inflammatory reaction along with detection of various active substances directly or indirectly in the experimental animal model of pleurisy, such as rat carrageenin-induced, and zymosan-induced pleurisy. These pleurisies showed almost similar time courses of pleural exudate accumulation and neutrophil migration. However, mediators detected in the exudates of such pleurisies were different; in carrageenin-induced pleurisy bradykinin and prostacyclin (PGI2) caused exudate formation, while zymosan-induced pleurisy showed early degradation of mast cells and activation of complements, followed by an increase in platelet activating factor (PAF). In both pleurisies TNF alpha, IL-1, IL-6 and CINC (cytokine-induced neutrophil chemoattractant) appeared similarly in the exudates to cause chemoattractant for neutrophils. TNF alpha and IL-1 could stimulate to produce IL-6 and IL-8. While prostaglandins may regulate cytokine production via a cellular cAMP-dependent mechanism. Thus one should consider the time for application of anti-inflammatory drugs, such as cyclooxygenase inhibitor, indomethacin, since it causes increases in TNF alpha and IL-1 production by reducing PGI2 and prostaglandin E2 (PGE2) levels. In conclusion, inflammatory reaction has its own automatic regulation mechanism through complex cross talks between inflammatory mediators.

Kinetics of DNA Unwinding by the RecD2 Helicase from Deinococcus radiodurans

RecD2 from Deinococcus radiodurans is a superfamily 1 DNA helicase that is homologous to the Escherichia coli RecD protein but functions outside the context of RecBCD enzyme. We report here on the kinetics of DNA unwinding by RecD2 under single and multiple turnover conditions. There is little unwinding of 20-bp substrates by preformed RecD2-dsDNA complexes when excess ssDNA is present to trap enzyme molecules not bound to the substrate. A shorter 12-bp substrate is unwound rapidly under single turnover conditions. The 12-bp unwinding reaction could be simulated with a mechanism in which the DNA is unwound in two kinetic steps with rate constant of k(unw) = 5.5 s(-1) and a dissociation step from partially unwound DNA of k(off) = 1.9 s(-1). These results indicate a kinetic step size of about 3-4 bp, unwinding rate of about 15-20 bp/s, and low processivity (p = 0.74). The reaction time courses with 20-bp substrates, determined under multiple turnover conditions, could be simulated with a four-step mechanism and rate constant values very similar to those for the 12-bp substrate. The results indicate that the faster unwinding of a DNA substrate with a forked end versus only a 5'-terminal single-stranded extension can be accounted for by a difference in the rate of enzyme binding to the DNA substrates. Analysis of reactions done with different RecD2 concentrations indicates that the enzyme forms an inactive dimer or other oligomer at high enzyme concentrations. RecD2 oligomers can be detected by glutaraldehyde cross-linking but not by size exclusion chromatography.

Kinetic Analysis of the Three-step Steroid Aromatase Reaction of Human Cytochrome P450 19A1

Cytochrome P450 19A1 (P450 19A1), the aromatase, catalyzes the conversion of androgens to estrogens through a sequential three-step reaction, generating 19-hydroxy and 19-aldehyde intermediates en route to the product estrogen. A procedure for the heterologous expression and purification of P450 19A1 in Escherichia coli was developed (k(cat) of 0.06 s(-1) for the conversion of androstenedione to estrone). Binding of the substrate and intermediates show low micromolar dissociation constants and are at least two-step processes. Rates of reduction of the iron were fast in the presence of substrate, either intermediate, or product. P450 19A1 is a distributive rather than a processive enzyme, with the sequential reaction allowing free dissociation of the intermediates as revealed by pulse-chase experiments. Conversion of androstenedione to estrone (under single turnover conditions) generated a progress curve showing changes in the concentrations of the substrate, intermediates, and product. A minimal kinetic model containing the individual rate constants for the steps in P450 19A1 catalysis was developed to globally fit the time course of the overall reaction, the dissociation constants, the two-step ligand binding, the distributive character, the iron-reduction rates, and the steady-state conversion of the 19-hydroxy androstenedione and 19-aldehyde androstenedione intermediates to estrone.

Kinetics of DNA unwinding by the RecD2 helicase from Deinococcus radiodurans

RecD2 from Deinococcus radiodurans is a superfamily 1 DNA helicase that is homologous to the E. coli RecD protein but functions outside the context of RecBCD enzyme. We report here on the kinetics of DNA unwinding by the D. radiodurans RecD2 under single‐ and multiple‐turnover conditions. There is little unwinding of 20 bp substrates by preformed RecD2‐dsDNA complexes when excess ssDNA is present to trap enzyme molecules not bound to the substrate, using substrates with a 5′‐ssDNA overhang or a forked end. A 12 bp substrate is unwound rapidly under single‐turnover conditions. The unwinding time course can be simulated with a mechanism in which the DNA is unwound in two kinetic steps with rate constant = 5.5 sec1, kunw and a dissociation step from partially unwound DNA of koff = 1.9 sec1. These results indicate a kinetic step size of about 3 – 4 bp, an unwinding rate of about 15 – 20 bp/sec, and low processivity. The reaction time course data with the 20 bp substrates, determined under multiple‐turnover conditions, can be simulated with a four‐step mechanism and rate constant values very similar to those for the 12 bp substrate. Analysis of reactions done at several RecD2 protein concentrations indicates that the enzyme forms an inactive dimer or other oligomeric form at high enzyme concentrations.

Direct Ubiquitination of β-Catenin by Siah-1 and Regulation by the Exchange Factor TBL1

Beta-catenin is a key component of the Wnt signaling pathway that functions as a transcriptional co-activator of Wnt target genes. Upon UV-induced DNA damage, beta-catenin is recruited for polyubiquitination and subsequent proteasomal degradation by a unique, p53-induced SCF-like complex (SCF(TBL1)), comprised of Siah-1, Siah-1-interacting protein (SIP), Skp1, transducin beta-like 1 (TBL1), and adenomatous polyposis coli (APC). Given the complexity of the various factors involved and the novelty of ubiquitination of the non-phosphorylated beta-catenin substrate, we have investigated Siah-1-mediated ubiquitination of beta-catenin in vitro and in cells. Overexpression and purification protocols were developed for each of the SCF(TBL1) proteins, enabling a systematic analysis of beta-catenin ubiquitination using an in vitro ubiquitination assay. This study revealed that Siah-1 alone was able to polyubiquitinate beta-catenin. In addition, TBL1 was shown to play a role in protecting beta-catenin from Siah-1 ubiquitination in vitro and from Siah-1-targeted proteasomal degradation in cells. Siah-1 and TBL1 were found to bind to the same armadillo repeat domain of beta-catenin, suggesting that polyubiquitination of beta-catenin is regulated by competition between Siah-1 and TBL1 during Wnt signaling.

The flitting of electrons in complex I: A stochastic approach

A stochastic approach based on the Gillespie algorithm is particularly well adapted to describe the time course of the redox reactions that occur inside the respiratory chain complexes because they involve the motion of single electrons between the individual unique redox centres of a given complex. We use this approach to describe the molecular functioning of the peripheral arm of complex I based on its known crystallographic structure and the rate constants of electron tunnelling derived from the Moser and Dutton phenomenological equations. There are several possible electrons pathways but we show that most of them take the route defined by the successive sites and redox centres: NADH+ site – FMN – N3 – N1b – N4 – N5 – N6a – N6b – N2 – Q site. However, the electrons do not go directly from NADH towards the ubiquinone molecule. They frequently jump back and forth between neighbouring redox centres with the result that the net flux of electrons through complex I (i.e. net number of electrons reducing a ubiquinone) is far smaller than the number of redox reactions which actually occur. While most of the redox centres are reduced in our simulations the degree of reduction can vary according to the individual midpoint potentials. The high turnover number observed in our simulation seems to indicate that, in the whole complex I, one or several slower step(s) follow(s) the redox reactions involved in the peripheral arm. It also appears that the residence time of FMNH• and SQ• (possible producers of ROS) is low (around 4% and between 1.6% and 5% respectively according to the values of the midpoint potentials). We did not find any evidence for a role of N7 which remains mainly reduced in our simulations. The role of N1a is complex and depends upon its midpoint potential. In all cases its presence slightly decreases the life time of the flavosemiquinone species. These simulations demonstrate the interest of this type of model which links the molecular physico-chemistry of the individual redox reactions to the more global level of the reaction, as is observed experimentally.

Dynamic retinal vessel response to flicker in age-related macular degeneration patients before and after vascular endothelial growth factor inhibitor injection

Retinal vessel responses to flickering light are different in various systemic and ocular diseases and can be improved after successful therapy. We investigated retinal vessel response to flickering light in age-related macular degeneration (AMD) patients before and after treatment with a single intravitreal bevacizumab (Avastin(®) ) injection. In 10 patients with exudative AMD [age: median (1.quartile; 3.quartile) 76.0 (73.5; 80.0) years], retinal vessel reactions were examined by Dynamic Vessel Analyser (DVA) before and 3 months after a single intravitreal application of bevacizumab (1.25 mg). A baseline measurement was followed by three consecutive monochromatic flicker stimulations (530-600 nm, 12.5 Hz, 20 seconds). Temporal retinal vessel reaction was analysed and compared with the reaction in healthy controls. Mean arterial dilation at the end of flicker was not different in all groups. For veins this parameter amounted to: pre-treatment, 2.6 (1.7; 3.9)%; post-treatment, 2.9 (2.4; 4.0)%; control, 4.3 (3.2; 5.7)%; significant: pre-treatment - control (Dunnett's procedure, p < 0.05). Maximal dilation occurred in arteries at: pre-treatment, 17.5 (14.8; 32.5) seconds; post-treatment, 18.0 (16.6; 30.6) seconds; control, 14.5 (10.8; 17.3) seconds. Both AMD groups were slower (p < 0.05): in veins at 17.0 (14.5; 20.0) seconds, 12.8 (8.6; 14.8) seconds and 18.5 (17.1; 19.9) seconds, respectively; significant post-treatment - control (p < 0.05). In the post-treatment AMD group arterial constriction after stimulation occurred more slowly compared with the control group (p < 0.05). Dynamic retinal arterial and venous reactions to flickering light are altered in AMD compared with controls. Three months after a single injection of a vascular endothelial growth factor inhibitor, the investigated retinal dynamic vascular parameters were not altered in our study.