Stable linear reaction oscillator

The linear chemical reaction system, described in a preceding paper, which showed undamped oscillations under certain conditions is reviewed in the light of the complete set of eigenvalues. Since one eigenvalue is found to be positive real, the system is extremely sensitive to the correct choice of initial conditions. A minute change in the “circuitry” of the system has the effect that all eigenvalues can be moved into the left complex half-plane (real part negative) including the imaginary axis. Thus, within a wide range, an arbitrarily chosen set of initial conditions will yield undamped sinusoidal oscillations after a short period of transient behaviour. Besides, the minimum number of steps in the feedback path is now three instead of five, making the original analogy to the electronic RC phase-shift oscillator even closer.     

State and Spectral Properties of Chloride Oscillations in Pollen

Pollen tube growth is a dynamic system expressing a number of oscillating circuits. Our recent work identified a new circuit, oscillatory efflux of Cl⁻ anion from the pollen tube apex. Cl⁻ efflux is the first ion signal found to be coupled in phase with growth oscillations. Functional analyses indicate an active role for Cl⁻ flux in pollen tube growth. In this report the dynamical properties of Cl⁻ efflux are examined. Phase space analysis demonstrates that the system trajectory converges on a limit cycle. Fourier analysis reveals that two harmonic frequencies characterize normal growth. Cl⁻ efflux is inhibited by the channel blocker DIDS, is stimulated by hypoosmotic treatment, and is antagonized by the signal encoded in inositol 3,4,5,6-tetrakisphosphate. These perturbations induce transitions of the limit cycle to new metastable states or cause system collapse to a static attractor centered near the origin. These perturbations also transform the spectral profile, inducing subharmonic frequencies, transitions to period doubling and tripling, superharmonic resonance, and chaos. These results indicate that Cl⁻ signals in pollen tubes display features that are characteristic of active oscillators that carry frequency-encoded information. A reaction network of the Cl⁻ oscillator coupled to two nonlinear feedback circuits that may drive pollen tube growth oscillations is considered.     

A novel approach for chemically deglycosylating O-linked glycoproteins. The deglycosylation of submaxillary and respiratory mucins.

A new approach for removing O-glycosidically linked carbohydrate side chains from glycoproteins is described. Periodate oxidation of the C3 and C4 carbons in peptide-linked N-acetylgalactosamine (GalNAc) residues generates a dialdehyde product which, under mild alkaline conditions, undergoes a beta-elimination which releases carbohydrate and leaves an intact peptide core. The pH and time dependence, and intermediates of the elimination, have been extensively followed by carbon-13 NMR spectroscopy and amino acid analysis using ovine submaxillary mucin (OSM) as the substrate. The deglycosylation of OSM is complete and provides apomucin in high yield with an amino acid composition identical to the starting material. Carboxymethylated OSM when deglycosylated by this method gives an apomucin with an apparent molecular weight of ca. 700 x 10(3). The molecular weight is the same as that calculated for the peptide core of the starting mucin, demonstrating the absence of peptide core cleavage. This contrasts with the use of trifluoromethanesulfonic acid (TFMSA), which generates apomucin products of lower molecular weights. Oligosaccharide side chains substituted at C3 of the peptide-linked GalNAc residue are resistant to the oxidation and elimination. Glycoproteins containing these more complex side chains can be deglycosylated by pretreatment with TFMSA under mild (0 degree C) conditions, which removes peripheral sugars (while leaving the peptide-linked GalNAc residue intact), followed by oxidation and beta-elimination. Studies on the deglycosylation of porcine submaxillary mucin and human tracheobronchial mucin indicate that this approach provides more efficient removal of carbohydrate and less peptide core degradation than a more vigorous (25 degrees C) treatment with TFMSA alone. 13C NMR spectroscopic studies and carbohydrate analysis of the deglycosylation intermediates of the human mucin indicate that certain sialic acid containing and N-acetylglucosamine-containing oligosaccharides have elevated resistance to TFMSA treatment at 0 degrees C. By the use of neuraminidase, repeated mild TFMSA treatments, and multiple oxidations and beta-eliminations, the human mucin can be nearly completely deglycosylated. It is expected that all mucins and most glycoproteins containing O-glycosidic linkages can be readily and nearly completely deglycosylated using this combined approach.     

Oscillatory Transpiration and Water Uptake of Avena Plants I. Preliminary Observations

Oscillations in transpiration and water uptake of individual, young oat plants have been studied. The free-running period of these oscillations was about 30 minutes. Conditions were reached under which the oscillations were sustained for about two days. Short perturbations were given to the transpiration oscillations, the perturbations consisting of a short time increase or decrease in the illumination. The phase shifts of the oscillations as well as the amplitude effects caused by these perturbations were investigated. Simultaneous recordings of transpiration and water uptake of a single plant showed that these functions were oscillating in phase. Both oscillations disappeared if the leaf was excised.     

The oxidation of tetrachloro-1,4-hydroquinone by microsomes and purified cytochrome P-450b. Implications for covalent binding to protein and involvement of reactive oxygen species

The enzymatic oxidation of tetrachloro-1,4-hydroquinone (1,4-TCHQ), resulting in covalent binding to protein of tetrachloro-1,4-benzoquinone (1,4-TCBQ), was investigated, with special attention to the involvement of cytochrome P-450 and reactive oxygen species. 1,4-TCBQ itself reacted very rapidly and extensively with protein (58% of the 10 nmol added to 2 mg of protein, in a 5-min incubation). Ascorbic acid and glutathione prevented covalent binding of 1,4-TCBQ to protein, both when added directly and when formed from 1,4-TCHQ by microsomes. In microsomal incubations as well as in a reconstituted system containing purified cytochrome P-450b, 1,4-TCHQ oxidation and subsequent protein binding was shown to be completely dependent on NADPH. The reaction was to a large extent, but not completely, dependent on oxygen (83% decrease in binding under anaerobic conditions). Inhibition of cytochrome P-450 by metyrapone, which is also known to block the P-450-mediated formation of reactive oxygen species, gave a 80% decrease in binding, while the addition of superoxide dismutase prevented 75% of the covalent binding, almost the same amount as found in anerobic incubations. A large part of the conversion of 1,4-TCHQ to 1,4-TCBQ is apparently not catalyzed by cytochrome P-450 itself, but is mediated by superoxide anion formed by this enzyme. The involvement of this radical anion is also demonstrated by microsomal incubations without NADPH but including the xantine/xantine oxidase superoxide anion generating system. These incubations resulted in a 1.6-fold binding as compared to the binding in incubations with NADPH but without xantine/xantine oxidase. 1,4-TCHQ was shown to stimulate the oxidase activity of microsomal cytochrome P-450. It is thus not unlikely that 1,4-TCHQ enhances its own microsomal oxidation.     

A mitochondrial oscillator dependent on reactive oxygen species

We describe a unique mitochondrial oscillator that depends on oxidative phosphorylation, reactive oxygen species (ROS), and mitochondrial inner membrane ion channels. Cell-wide synchronized oscillations in mitochondrial membrane potential (Delta Psi(m)), NADH, and ROS production have been recently described in isolated cardiomyocytes, and we have hypothesized that the balance between superoxide anion efflux through inner membrane anion channels and the intracellular ROS scavenging capacity play a key role in the oscillatory mechanism. Here, we formally test the hypothesis using a computational model of mitochondrial energetics and Ca(2+) handling including mitochondrial ROS production, cytoplasmic ROS scavenging, and ROS activation of inner membrane anion flux. The mathematical model reproduces the period and phase of the observed oscillations in Delta Psi(m), NADH, and ROS. Moreover, we experimentally verify model predictions that the period of the oscillator can be modulated by altering the concentration of ROS scavengers or the rate of oxidative phosphorylation, and that the redox state of the glutathione pool oscillates. In addition to its role in cellular dysfunction during metabolic stress, the period of the oscillator can be shown to span a wide range, from milliseconds to hours, suggesting that it may also be a mechanism for physiological timekeeping and/or redox signaling.     

Modeling of Glucose-Induced cAMP Oscillations in Pancreatic β Cells: cAMP Rocks when Metabolism Rolls

Recent advances in imaging technology have revealed oscillations of cyclic adenosine monophosphate (cAMP) in insulin-secreting cells. These oscillations may be in phase with cytosolic calcium oscillations or out of phase. cAMP oscillations have previously been modeled as driven by oscillations in calcium, based on the known dependence of the enzymes that generate cAMP (adenylyl cyclase) and degrade it (phosphodiesterase). However, cAMP oscillations have also been reported to occur in the absence of calcium oscillations. Motivated by similarities between the properties of cAMP and metabolic oscillations in pancreatic β cells, we propose here that in addition to direct control by calcium, cAMP is controlled by metabolism. Specifically, we hypothesize that AMP inhibits adenylyl cyclase. We incorporate this hypothesis into the dual oscillator model for β cells, in which metabolic (glycolytic) oscillations cooperate with modulation of ion channels and metabolism by calcium. We show that the combination of oscillations in AMP and calcium in the dual oscillator model can account for the diverse oscillatory patterns that have been observed, as well as for experimental perturbations of those patterns. Predictions to further test the model are provided.     

Oxidation of Methanol,Formaldehyde and Formate by a Candida Species

1. The oxidation of methanol to carbon dioxide by Candida N–16 grown on methanol was investigated. The presence of enzymes which catalyze the following reaction was found in the cell-free extract of the yeast employed; CH₃OH→HCHO→HCOOH→CO₂. 2. Methanol was oxidized to formaldehyde by an alcohol oxidase. The reaction was as follows; CH₃OH+O₂→HCHO+H₂O₂. The alcohol oxidase was crystallized after purification by ammonium sulfate-precipitation and column chromatography using DEAE-Sephadex A-50. A prosthetic group of the enzyme was proved to be FAD. The enzyme possessed a broad specificity for alcohols such as methanol, ethanol, n-propanol, n-butanol and n-amylalcohol. The enzyme was inducibly formed only by the addition of methanol. 3. The oxidation of formaldehyde to formate was catalyzed by a NAD-linked dehydrogenase dependent on GSH. 4. Formate was oxidized by a NAD-linked dehydrogenase. 5. Catalase was also found in the extract, and methanol was chemically oxidized by the reaction of catalase and hydrogen peroxide which was generated by the alcohol oxidase system. 6. The oxidation pathway from methanol to carbon dioxide was also found in other methanol-utilizing yeasts such as Candida N-17, Saccharomyces H-1 and Torulopsis M-1.     

Synchronization in two interacting oscillatory systems.

Nonlinear phenomena arising from the interaction of two oscillating systems of chemical reactions are studied experimentally. The system of two connected flow-through continuous stirred tank reactors (cells) with controlled exchange of reaction mixture is used. The Belousov reaction (oxidation of malonic acid by bromate in sulphuric acid with ceric/cerous ions as catalyst) served as model system. The frequency of oscillations was controlled by change of the reaction temperature. Phenomena such as synchronization of oscillations at a common frequency, synchronization at multiples of a common frequency, rhythm splitting and amplitude amplification were observed, depending on the degree of interaction and the differences in the original oscillation frequencies. Mathematical modelling of the above phenomena failed, probably due to insufficient knowledge of a kinetic model.     

Sustained oscillations in glycolysis: an experimental and theoretical study of chaotic and complex periodic behavior and of quenching of simple oscillations

We report sustained oscillations in glycolysis conducted in an open system (a continuous-flow, stirred tank reactor; CSTR) with inflow of yeast extract as well as glucose. Depending on the operating conditions, we observe simple or complex periodic oscillations or chaos. We report the response of the system to instantaneous additions of small amounts of several substrates as functions of the amount added and the phase of the addition. We simulate oscillations and perturbations by a kinetic model based on the mechanism of glycolysis in a CSTR. We find that the response to particular perturbations forms an efficient tool for elucidating the mechanism of biochemical oscillations.     

Tea catechins inhibit cholesterol oxidation accompanying oxidation of low density lipoprotein in vitro

Endogenous oxidized cholesterols are potent atherogenic agents. Therefore, the antioxidative effects of green tea catechins (GTC) against cholesterol oxidation were examined in an in vitro lipoprotein oxidation system. The antioxidative potency of GTC against copper catalyzed LDL oxidation was in the decreasing order (-)-epigalocatechin gallate (EGCG)=(-)-epicatechin gallate (ECG)>(-)-epicatechin (EC)=(+)-catechin (C)>(-)-epigallocatechin (EGC). Reflecting these activities, both EGCG (74%) and ECG (70%) inhibited the formation of oxidized cholesterol, as well as the decrease of linoleic and arachidonic acids, in copper catalyzed LDL oxidation. The formation of oxidized cholesterol in 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH)-mediated oxidation of rat plasma was also inhibited when the rats were given diets containing 0.5% ECG or EGCG. In addition, EGCG and ECG highly inhibited oxygen consumption and formation of conjugated dienes in AAPH-mediated linoleic acid peroxidative reaction. These two species of catechin also markedly lowered the generation of hydroxyl radical and superoxide anion. Thus, GTC, especially ECG and EGCG, seem to inhibit cholesterol oxidation in LDL by combination of interference with PUFA oxidation, the reduction and scavenging of copper ion, hydroxyl radical generated from peroxidation of PUFA and superoxide anion.     

On the mechanism of nitrobenzene liquid membrane oscillators containing hexadecyltrimethylammonium bromide

The oscillatory behaviour of a liquid membrane oscillator was investigated in order to contribute to the oscillation mechanism at the molecular level. The chosen system involved nitrobenzene as liquid membrane containing a constant amount of picric acid. The aqueous donor phase contained the cationic surfactant, hexadecyltrimethylammonium bromide, and ethanol. The aqueous acceptor phase was made up by sucrose solution. It was established that the oscillations take place exclusively at the aqueous acceptor phase/membrane interface. Three stages mechanism of the oscillation (I--induction period, II--first peak formation, III--creation of the first peak) together with appropriate processes were proposed. The molecular events provoking the oscillations of electric potential difference between the two aqueous phases concern: 1) the diffusion of hexadecyltrimethylammonium bromide and ion pairs formed by cation of the surfactant and the picrate anion to the vicinity of the membrane/acceptor phase interface, 2) sudden adsorption of these ion-pairs at this interface in non-catalytic and autocatalytic steps, 3) desorption of ion pairs from the a/m interface to the acceptor phase. It is shown by numerical simulations that the proposed mechanism may account for the observed oscillations and for the species distribution throughout the system as found experimentally.     

Studies on methanol — oxidizing yeast

Oxidation of methanol, formaldehyde and formic acid was studied in cells and cell-free extract of the yeast Candida boidinii No. 11Bh. Methanol oxidase, an enzyme oxidizing methanol to formaldehyde, was formed inducibly after the addition of methanol to yeast cells. The oxidation of methanol by cell-free extract was dependent on the presence of oxygen and independent of any addition of nicotine-amide nucleotides. Temperature optimum for the oxidation of methanol to formaldehyde was 35 degrees C, pH optimum was 8.5. The Km for methanol was 0.8mM. The cell-free extract exhibited a broad substrate specificity towards primary alcohols (C1--C6). The activity of methanol oxidase was not inhibited by 1mM KCN, EDTA or monoiodoacetic acid. The strongest inhibitory action was exerted by p-chloromercuribenzoate. Both the cells and the cell-free extract contained catalase which participated in the oxidation of methanol to formaldehyde; the enzyme was constitutively formed by the yeast. The pH optimum for the degradation of H2O2 was in the same range as the optimum for methanol oxidation, viz. at 8.5. Catalase was more resistant to high pH than methanol oxidase. The cell-free extract contained also GSH-dependent NAD-formaldehyde dehydrogenase with Km = 0.29mM and NAD-formate dehydrogenase with Km = 55mM.     

The transmembrane electrochemical gradient of Na+ as driving force for methanol oxidation in Methanosarcina barkeri

A sodium ion gradient (inside low) across the cytoplasmic membrane of Methanosarcina barkeri was required for methanogenesis from methanol. This could be concluded from the following results. (a) Inhibition of the Na+/H+ antiporter by K+ or amiloride led to an inhibition of methanogenesis from methanol. (b) Upon addition of the sodium ionophore monensin the Na+ gradient was abolished and at the same time methanogenesis from methanol was inhibited. (c) Methanogenesis was impaired when the Na+ gradient had the opposite orientation (inside high). All these inhibitory effects were not observed when H2 was present in addition to methanol indicating that the oxidation of methanol to CO2 was driven by a sodium-motive force. In accordance with this, a methanol-dependent influx of Na+ and a corresponding decrease of the membrane potential could be observed, when the Na+/H+ antiporter was inhibited by amiloride. This influx was indicative of the presence of a Na+ transport system which was functional when the oxidation of methanol had to be driven, but was not functional when H2 was present for reduction of methanol to methane.     

Effect of dynamic channelling on the transient-state kinetics of coupled enzyme reactions.

Evidence is presented to show that the presence of a channelled reaction branch in a coupled two-enzyme reaction leads to a slower overall relaxation of the system towards steady state following perturbations of the reaction conditions. A predominant part of the total reaction flux change resulting from perturbations may be governed by transients that are more rapid in the presence than in the absence of a channel, but only under specific conditions that are not likely to be of biological relevance.     

Oscillations in energy metabolism

Organisation of mitochondrial metabolism is a quintessential example of a complex dissipative system which can display dynamic instabilities. Several findings have indicated that the conditions inducing instabilities are within the physiological range and that mild perturbations could elicit oscillations. Different mathematical models have been put forth in order to explain the genesis of oscillations in energy metabolism. One model considers mitochondria as an organised network of oscillators and indicates that communication between mitochondria involves mitochondrial reactive oxygen species (ROS) production acting as synchronisers of the energy status of the whole population of mitochondria. An alternative model proposes that extramitochondrial pH variations could lead to mitochondrial oscillations. Oscillatory phenomena in energy metabolism have also been investigated in vivo on the basis of ³¹P magnetic resonance spectroscopy (MRS) measurements of phosphocreatine post-exercise recovery in human and animal skeletal muscle. The corresponding results provide experimental evidences about the role exerted by cytosolic pH on oscillations. Finally a new simple non-linear mathematical model describing the overall chemical reaction of phosphocreatine recovery predicting oscillatory recovery pattern under certain experimental conditions is presented and discussed in the light of the experimental results reported so far.     

Are circadian oscillators structurally unstable?

The common belief is that all biological oscillations are of limit cycle type. It is shown in this article that the phase response curves simulated on a two-species Lotka-Volterra linear (i.e. non-limit cycle type) oscillator, do look similar to those obtained by experimental methods by different workers. The form of the phase response curves, the existence of singularities and the mirror-image symmetry of opposite perturbations are modelled on the Lotka-Volterra system. The study, which is strongly indicative of the possibility that the underlying oscillator (or oscillators) is (are) not structurally stable, also indicates the necessity of designing critical experiments, capable of distinguishing between limit cycle and non-limit cycle oscillators, since the single-pulse phase resetting does nothing to distinguish between them.     

The modelling of the glycolytic oscillations in the potential of the oxidative-reductive status of the brain tissue in waking and anesthetized rats]

It was found that chemical hypoxia created by intraperitoneal injection of potassium cyanide (5-7 mg/kg) induced in both waking and anaesthetized (pentobarbital, 40 mg/kg) albino rats a significant decrease in the brain redox state potential (E) monitored with platinum electrodes. This decrease could be accompanied by a generation in some brain points of local chains of gradually damped quasisinusoidal E oscillations. Such oscillations were more expressed in waking than in anaesthetized animals. The frequency range of these oscillations was 4-7 cycles/min. This is the range of overlapping frequency ranges characteristic for the high level of vigilance (5-20 cycles/min) and slow-wave sleep and drowsiness (1.5-6 cycles/min). The amplitude of the observed oscillations was close to the maximal amplitude of the brain E oscillations characteristic for the high level of vigilance (up to several mV). The obtained evidence favors our suggestion that behavior-related E oscillations are formed by the oscillations in the redox balance of glycolysis. The similarity of the normal physiological oscillations and those simulated by us under abnormal conditions suggest a certain common mechanism of their generation.