Phosphate release kinetics in calcareous grassland and forest soils in response to H+ addition

Phosphate release kinetics in soils are of global interest because sustainable plant nutrition with phosphate will be a major concern in the future. Dissolution of phosphate-containing minerals induced by a changing rhizosphere equilibrium through proton input is one important mechanism that releases phosphate into bioavailable forms. Our objectives were (i) to determine phosphate release kinetics during H+ addition in calcareous soils of the Schwäbische Alb, Germany, and to assess the influence of (ii) land-use type (grassland vs. forest) and (iii) management intensity on reactive phosphate pools and phosphate release rate constants during H+ addition. Phosphate release kinetics were characterized by a large fast-reacting phosphatepool, which could be attributed to poorly-crystalline calcium phosphates, and a small slow-reacting phosphate pool probably originating from carbonate-bearing hydroxylapatite. Both reactive phosphate pools—as well as total phosphate concentrations (TP) in soil—were greater in grassland than in forest soils. In organically fertilized grassland soils, concentrations of released phosphate were higher than in unfertilized soils, likely because organic fertilizers contain poorly-crystalline phosphate compounds which are further converted into sparingly soluble phosphate forms. Because of an enriched slow-reacting phosphate pool, mown pastures were characterized by a more continuous slow phosphate release reaction in contrast to clear biphasic phosphate release patterns in meadows. Consequently, managing phosphate release kinetics via management measures is a valuable tool to evaluate longer-term P availability in soil in the context of finite rock phosphate reserves on earth.

Hybrid stochastic simulations of intracellular reaction-diffusion systems.

With the observation that stochasticity is important in biological systems, chemical kinetics have begun to receive wider interest. While the use of Monte Carlo discrete event simulations most accurately capture the variability of molecular species, they become computationally costly for complex reaction-diffusion systems with large populations of molecules. On the other hand, continuous time models are computationally efficient but they fail to capture any variability in the molecular species. In this study a hybrid stochastic approach is introduced for simulating reaction-diffusion systems. We developed an adaptive partitioning strategy in which processes with high frequency are simulated with deterministic rate-based equations, and those with low frequency using the exact stochastic algorithm of Gillespie. Therefore the stochastic behavior of cellular pathways is preserved while being able to apply it to large populations of molecules. We describe our method and demonstrate its accuracy and efficiency compared with the Gillespie algorithm for two different systems. First, a model of intracellular viral kinetics with two steady states and second, a compartmental model of the postsynaptic spine head for studying the dynamics of Ca+2 and NMDA receptors.

The effect of ego depletion on sprint start reaction time

In the current study, we consider that optimal sprint start performance requires the self-control of responses. Therefore, start performance should depend on athletes' self-control strength. We assumed that momentary depletion of self-control strength (ego depletion) would either speed up or slow down the initiation of a sprint start, where an initiation that was sped up would carry the increased risk of a false start. Applying a mixed between- (depletion vs. nondepletion) and within- (before vs. after manipulation of depletion) subjects design, we tested the start reaction times of 37 sport students. We found that participants' start reaction times decelerated after finishing a depleting task, whereas it remained constant in the nondepletion condition. These results indicate that sprint start performance can be impaired by unrelated preceding actions that lower momentary self-control strength. We discuss practical implications in terms of optimizing sprint starts and related overall sprint performance.

Kinetics and kinematics of the tölt: effects of rider interaction and shoeing manipulations

Introduction . Compared to most equine horse breeds which are able to walk, trot and canter /gallop, the gait repertoire of the Icelandic horses additionally includes the lateral gait tölt and frequently also the pace. With respect to the tölt gait, special shoeing, saddling and riding techniques have been developed for Icelandic horses in order to enhance its expressiveness and regularity. Toes are left unnaturally long and heavy shoes and paddings, as well as weighted boots are used to enforce the individual gait predisposition. For the same reason, the rider is placed more caudally to the horse's centre of mass as compared to other riding techniques. The biomechanical impact of these methods on the health of the locomotor system has so far never been subject of systematic research. Objectives . The aims of the presented study are (1) to describe the kinetic and kinematic characteristics of the tölt performed on a treadmill, (2) to understand the mechanical consequences of shoeing manipulation (long hooves, weighted boots) on the loading and protraction movement of the limbs, as well as (3) to study the pressure distribution and effects on the gait pattern of 3 different saddle types used for riding Icelandic horses. Materials and methods . Gait analysis was carried out in 13 Icelandic horses at walk and at slow and medium tölting and trotting speeds on a high-speed treadmill instrumented for measuring vertical ground reaction forces as well as temporal and spatial gait variables. Kinematic data of horse, rider and saddle were measured simultaneously. Gait analysis was first carried out with high, long hooves (SH) without and in combination with weighted boots (ad aim (2)). Afterwards, horses were re-shod according to current horseshoeing standards (SN) and gait analysis was repeated (ad aims (1) and (2)). In a second trial, horses were additionally equipped with a pressure sensitive saddle mat and were ridden with a dressage-like saddle (SDres), an Icelandic saddle (Slcel) and a saddle cushion (SCush) in the standard saddle position (ad aim 3). Results and conclusions . Compared to trot at the same speed, tölting horses had a higher stride rate and lower stride impulses. At the tölt loading of the forelimbs was increased in form of higher peak vertical forces (Fzpeak) due to shorter relative stance durations (StDrel). Conversely, in the hindlimbs, longer StDrel resulted in lower Fzpeak. Despite the higher head-neck position at tölt, there was no measurable shift in weight to the hindlimbs. Footfall rhythm was in most horses laterally coupled at the tölt and frequently had a slight fourbeat and a very short suspension phase at trot; underlining the fact that performance of correct gaits in Icelandic horses needs special training. Gait performance as it is currently judged in competition could be improved using a shoeing with SH, resulting in a 21 ± 5 mm longer dorsal hoof wall, but also a weight gain of 273 ± 50 g at the distal limb due to heavier shoeing material. Compared to SN, SH led to a lower stride rate, a longer stride length and a higher, but not wider, forelimb protraction arc, which were also positively associated with speed. At the tölt, the footfall rhythm showed less tendency to lateral couplets and at the trot, the suspension phase was longer. However, on the long term, SH may have negative implications for the health of the palmar structures of the distal foot by increased limb impulses, higher torques at breakover (up to 20%); as well as peak vertical forces at faster speeds. Compared to the shoeing style, the saddle type had less influence on limb forces or movements. The slight weight shift to the rear with SCush and Slcel may be explained by the more caudal position of the rider relative to the horse's back. With SCush, pressure was highest under the cranial part of the saddle, whereas the saddles with trees had more pressure under the caudal area.

Kinetics and Mechanism of Oxygen Delignification

Considerable research has been conducted into the kinetics and selectivity of the oxygen delignification process to overcome limitation in its use. However most studies were performed in a batch reactor whereby the hydroxide and dissolved oxygen concentrations are changing during the reaction time in an effort to simulate tower performance in pulp mills. This makes it difficult to determine the reaction order of the different reactants in the rate expressions. Also the lignin content and cellulose degradation of the pulp are only established at the end of the experiment when the sample is removed from the batch reactor. To overcome these deficiencies, we have adopted a differential reactor system used frequently for fluid-solid rate studies (so-called Berty reactor) for measurement of oxygen delignification kinetics. In this reactor, the dissolved oxygen concentration and the alkali concentration in the feed are kept constant, and the rate of lignin removal is determined from the dissolved lignin content in the outflow stream measured by UV absorption. The mass of lignin removed is verified by analyzing the pulp at several time intervals. Experiments were performed at different temperatures, oxygen pressures and caustic concentrations. The delignification rate was found to be first order in HexA-free residual lignin content. The delignification rate reaction order in caustic concentration and oxygen pressure were determined to be 0.42 and 0.44 respectively. The activation energy was found to be 53kJ/mol. The carbohydrate degradation during oxygen delignification can be described by two contributions: one due to radicals produced by phenolic delignification, and a much smaller contribution due to alkaline hydrolysis. From the first order of the reaction and the pKa of the active lignin site, a new oxygen delignification mechanism is proposed. The number 3 carbon atom in the aromatic ring with the attached methoxyl group forms the lignin active site for oxygen adsorption and subsequent electrophic reaction to form a hydroperoxide with a pKa value similar to that of the present delignification kinetics. The uniform presence of the aromatic methoxyl groups in residual lignin further support the first order in lignin kinetics.

Overal reaction rate and stoichiometry for thermal decomposition of hydrazine

An asymptotic analysîs of the Eberstein-Glassman kinetic mechanlsm for the thermal décomposition of hydrazine is carried out. It is shown that at températures near 800°K and near 1000°K,and for hydrazine molar fractions of the order of unity, 10-2 the entire kinetics reduces to a single, overall reaction. Characteristic times for the chemical relaxation of ail active, intermediate species produced in the décomposition, and for the overall reaction, are obtained. Explicit expressions for the overall reaction rate and stoichiometry are given as functions of température, total molar concentration (or pressure)and hydrazine molar fraction. Approximate, patched expressions can then be obtained for values of température and hydrazine molar fraction between 750 and 1000°K, and 1 and 10-3 respectively.

Structural kinetics of transcription activation at the malT promoter of Escherichia coli by UV laser footprinting

We have studied the kinetics of transcriptional initiation and activation at the malT and malTp1 promoters of Escherichia coli using UV laser footprinting. Contrary to previous studies and because of the very rapid signal acquisition by this technique, we can obtain structural information about true reaction intermediates of transcription initiation. The consequences of adding a transcriptional activator, the cAMP receptor protein/cAMP complex (CRP), are monitored in real time, permitting us to assign specific interactions to the activation of discrete steps in transcription initiation. Direct protein–protein contacts between CRP and the RNA polymerase appeared very rapidly, followed by DNA melting around the −10 hexamer. CRP slightly increased the rate of this isomerization reaction but, more importantly, favored the establishment of additional contacts between the DNA upstream of the CRP binding site and RNA polymerase subsequent to open complex formation. These contacts make a major contribution to transcriptional activation by stabilizing open forms of the promoter complex, thereby indirectly accelerating promoter escape. The ensemble of the kinetic, structural signals demonstrated directly that CRP exerts most of its activating effects on the late stages of transcriptional initiation at the malT promoter.

Proton uptake by bacterial reaction centers: The protein complex responds in a similar manner to the reduction of either quinone acceptor

In bacterial photosynthetic reaction centers, the protonation events associated with the different reduction states of the two quinone molecules constitute intrinsic probes of both the electrostatic interactions and the different kinetic events occurring within the protein in response to the light-generated introduction of a charge. The kinetics and stoichiometries of proton uptake on formation of the primary semiquinone QA− and the secondary acceptor QB− after the first and second flashes have been measured, at pH 7.5, in reaction centers from genetically modified strains and from the wild type. The modified strains are mutated at the L212Glu and/or at the L213Asp sites near QB; some of them carry additional mutations distant from the quinone sites (M231Arg → Leu, M43Asn → Asp, M5Asn → Asp) that compensate for the loss of L213Asp. Our data show that the mutations perturb the response of the protein system to the formation of a semiquinone, how distant compensatory mutations can restore the normal response, and the activity of a tyrosine residue (M247Ala → Tyr) in increasing and accelerating proton uptake. The data demonstrate a direct correlation between the kinetic events of proton uptake that are observed with the formation of either QA− or QB−, suggesting that the same residues respond to the generation of either semiquinone species. Therefore, the efficiency of transferring the first proton to QB is evident from examination of the pattern of H+/QA− proton uptake. This delocalized response of the protein complex to the introduction of a charge is coordinated by an interactive network that links the Q− species, polarizable residues, and numerous water molecules that are located in this region of the reaction center structure. This could be a general property of transmembrane redox proteins that couple electron transfer to proton uptake/release reactions.

Determination of the binding sites of the proton transfer inhibitors Cd2+ and Zn2+ in bacterial reaction centers

The reaction center (RC) from Rhodobacter sphaeroides couples light-driven electron transfer to protonation of a bound quinone acceptor molecule, QB, within the RC. The binding of Cd2+ or Zn2+ has been previously shown to inhibit the rate of reduction and protonation of QB. We report here on the metal binding site, determined by x-ray diffraction at 2.5-Å resolution, obtained from RC crystals that were soaked in the presence of the metal. The structures were refined to R factors of 23% and 24% for the Cd2+ and Zn2+ complexes, respectively. Both metals bind to the same location, coordinating to Asp-H124, His-H126, and His-H128. The rate of electron transfer from QA− to QB was measured in the Cd2+-soaked crystal and found to be the same as in solution in the presence of Cd2+. In addition to the changes in the kinetics, a structural effect of Cd2+ on Glu-H173 was observed. This residue was well resolved in the x-ray structure—i.e., ordered—with Cd2+ bound to the RC, in contrast to its disordered state in the absence of Cd2+, which suggests that the mobility of Glu-H173 plays an important role in the rate of reduction of QB. The position of the Cd2+ and Zn2+ localizes the proton entry into the RC near Asp-H124, His-H126, and His-H128. Based on the location of the metal, likely pathways of proton transfer from the aqueous surface to QB⨪ are proposed.

Identification of the proton pathway in bacterial reaction centers: Inhibition of proton transfer by binding of Zn2+ or Cd2+

The reaction center (RC) from Rhodobacter sphaeroides converts light into chemical energy through the light induced two-electron, two-proton reduction of a bound quinone molecule QB (the secondary quinone acceptor). A unique pathway for proton transfer to the QB site had so far not been determined. To study the molecular basis for proton transfer, we investigated the effects of exogenous metal ion binding on the kinetics of the proton-assisted electron transfer kAB(2) (QA−•QB−• + H+ → QA(QBH)−, where QA is the primary quinone acceptor). Zn2+ and Cd2+ bound stoichiometrically to the RC (KD ≤ 0.5 μM) and reduced the observed value of kAB(2) 10-fold and 20-fold (pH 8.0), respectively. The bound metal changed the mechanism of the kAB(2) reaction. In native RCs, kAB(2) was previously shown to be rate-limited by electron transfer based on the dependence of kAB(2) on the driving force for electron transfer. Upon addition of Zn2+ or Cd2+, kAB(2) became approximately independent of the electron driving force, implying that the rate of proton transfer was reduced (≥ 102-fold) and has become the rate-limiting step. The lack of an effect of the metal binding on the charge recombination reaction D+•QAQB−• → DQAQB suggests that the binding site is located far (>10 Å) from QB. This hypothesis is confirmed by preliminary x-ray structure analysis. The large change in the rate of proton transfer caused by the stoichiometric binding of the metal ion shows that there is one dominant site of proton entry into the RC from which proton transfer to QB−• occurs.

Structural basis for heterogeneous kinetics: Reengineering the hairpin ribozyme

The RNA cleavage reaction catalyzed by the hairpin ribozyme shows biphasic kinetics, and chase experiments show that the slow phase of the reaction results from reversible substrate binding to an inactive conformational isomer. To investigate the structural basis for the heterogeneous kinetics, we have developed an enzymatic RNA modification method that selectively traps substrate bound to the inactive conformer and allows the two forms of the ribozyme-substrate complex to be separated and analyzed by using both physical and kinetic strategies. The inactive form of the complex was trapped by the addition of T4 RNA ligase to a cleavage reaction, resulting in covalent linkage of the 5′ end of the substrate to the 3′ end of the ribozyme and in selective and quantitative ablation of the slow kinetic phase of the reaction. This result indicates that the inactive form of the ribozyme-substrate complex can adopt a conformation in which helices 2 and 3 are coaxially stacked, whereas the active form does not have access to this conformation, because of a sharp bend at the helical junction that presumably is stabilized by inter-domain tertiary contacts required for catalytic activity. These results were used to improve the activity of the hairpin ribozyme by designing new interfaces between the two domains, one containing a non-nucleotidic orthobenzene linkage and the other replacing the two-way junction with a three-way junction. Each of these modified ribozymes preferentially adopts the active conformation and displays improved catalytic efficiency.

The Kinetics of Zeaxanthin Formation Is Retarded by Dicyclohexylcarbodiimide1

The de-epoxidation of violaxanthin to antheraxanthin (Anth) and zeaxanthin (Zeax) in the xanthophyll cycle of higher plants and the generation of nonphotochemical fluorescence quenching in the antenna of photosystem II (PSII) are induced by acidification of the thylakoid lumen. Dicyclohexylcarbodiimide (DCCD) has been shown (a) to bind to lumen-exposed carboxy groups of antenna proteins and (b) to inhibit the pH-dependent fluorescence quenching. The possible influence of DCCD on the de-epoxidation reactions has been investigated in isolated pea (Pisum sativum L.) thylakoids. The Zeax formation was found to be slowed down in the presence of DCCD. The second step (Anth → Zeax) of the reaction sequence seemed to be more affected than the violaxanthin → Anth conversion. Comparative studies with antenna-depleted thylakoids from plants grown under intermittent light and with unstacked thylakoids were in agreement with the assumption that binding of DCCD to antenna proteins is probably responsible for the retarded kinetics. Analyses of the DCCD-induced alterations in different antenna subcomplexes showed that Zeax formation in the PSII antenna proteins was predominantly influenced by DCCD, whereas Zeax formation in photosystem I was nearly unaffected. Our data support the suggestion that DCCD binding to PSII antenna proteins is responsible for the observed alterations in xanthophyll conversion.

Kinetics of self-assembling microtubules: an "inverse problem" in biochemistry.

Experimental time series for a nonequilibrium reaction may in some cases contain sufficient data to determine a unique kinetic model for the reaction by a systematic mathematical analysis. As an example, a kinetic model for the self-assembly of microtubules is derived here from turbidity time series for solutions in which microtubules assemble. The model may be seen as a generalization of Oosawa's classical nucleation-polymerization model. It reproduces the experimental data with a four-stage nucleation process and a critical nucleus of 15 monomers.

Potentiation of proton transfer function by electrostatic interactions in photosynthetic reaction centers from Rhodobacter sphaeroides: First results from site-directed mutation of the H subunit.

The x-ray crystallographic structure of the photosynthetic reaction center (RC) has proven critical in understanding biological electron transfer processes. By contrast, understanding of intraprotein proton transfer is easily lost in the immense richness of the details. In the RC of Rhodobacter (Rb.) sphaeroides, the secondary quinone (QB) is surrounded by amino acid residues of the L subunit and some buried water molecules, with M- and H-subunit residues also close by. The effects of site-directed mutagenesis upon RC turnover and quinone function have implicated several L-subunit residues in proton delivery to QB, although some species differences exist. In wild-type Rb. sphaeroides, Glu L212 and Asp L213 represent an inner shell of residues of particular importance in proton transfer to QB. Asp L213 is crucial for delivery of the first proton, coupled to transfer of the second electron, while Glu L212, possibly together with Asp L213, is necessary for delivery of the second proton, after the second electron transfer. We report here the first study, by site-directed mutagenesis, of the role of the H subunit in QB function. Glu H173, one of a cluster of strongly interacting residues near QB, including Asp L213, was altered to Gln. In isolated mutant RCs, the kinetics of the first electron transfer, leading to formation of the semiquinone, QB-, and the proton-linked second electron transfer, leading to the formation of fully reduced quinol, were both greatly retarded, as observed previously in the Asp L213 --> Asn mutant. However, the first electron transfer equilibrium, QA-QB <==> QAQB-, was decreased, which is opposite to the effect of the Asp L213 --> Asn mutation. These major disruptions of events coupled to proton delivery to QB were largely reversed by the addition of azide (N3-). The results support a major role for electrostatic interactions between charged groups in determining the protonation state of certain entities, thereby controlling the rate of the second electron transfer. It is suggested that the essential electrostatic effect may be to "potentiate" proton transfer activity by raising the pK of functional entities that actually transfer protons in a coupled fashion with the second electron transfer. Candidates include buried water (H3O+) and Ser L223 (serine-OH2+), which is very close to the O5 carbonyl of the quinone.

Longevity and the genetic determination of collagen glycoxidation kinetics in mammalian senescence.

A fundamental question in the basic biology of aging is whether there is a universal aging process. If indeed such a process exists, one would expect that it develops at a higher rate in short- versus long-lived species. We have quantitated pentosidine, a marker of glycoxidative stress in skin collagen from eight mammalian species as a function of age. A curvilinear increase was modeled for all species, and the rate of increase correlated inversely with maximum life-span. Dietary restriction, a potent intervention associated with increased life-span, markedly inhibited glycoxidation rate in the rodent. On the assumption that collagen turnover rate is primarily influenced by the crosslinking due to glycoxidation, these results suggest that there is a progressive age-related deterioration of the process that controls the collagen glycoxidation rate. Thus, the ability to withstand damage due to glycoxidation and the Maillard reaction may be under genetic control.