Kinetic Heterogeneity Research Articles

The nature of the organoaluminum compound and the kinetic heterogeneity of active sites in lanthanide‐based diene polymerization

AbstractThe influence of the organoaluminum compound on the kinetic heterogeneity of active sites in lanthanide‐based diene polymerization was investigated. It was found that the heterogeneity of investigated catalytic systems was shown in the existence of four types of active centers. They were formed at the beginning of the polymerization process and produced macromolecules with lengths that were definite for each type of active center. The nature of the organoaluminum compound greatly influenced the kinetic activity of the polymerization centers. A method for analyzing the curves' maximums of distribution on kinetic activity and the change in kinetic activity of every type of active centers is proposed. This method allows calculating the concentration values for each type of active center separately. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 89: 674–685, 2003

Real-time NMR Kinetic Studies Provide Global and Residue-specific Information on the Non-cooperative Unfolding of the β-Trefoil Protein, Interleukin-1β

The interleukin-1β (IL-1β) structural motif is a β-trefoil super fold created by six two-stranded β-hairpins. Turns are thus particularly important in creating the topology and the arrangement of β-strands in this structural motif. In contrast to the signals observed in optical studies, real-time NMR kinetic investigations of the denaturant-induced unfolding of interleukin-1β provide direct, global, and residue-specific information on the structural nature of the unfolding reaction. Heterogeneity in the individual amino acid residue kinetics reveals a rugged unfolding landscape. The relative kinetic stability of native-like turns supports low temperature molecular dynamics predictions of turn-controlled unfolding.

KINETICS OF SOIL NITROGEN MINERALIZATION FROM UNDISTURBED AND DISTURBED SOIL

Knowledge of soil nitrogen (N) mineralization processes is essential for modeling soil processes in agriculture. Many authors have found discrepancies in N mineralization between disturbed and undisturbed samples. Nevertheless, most simulation models use a first-order kinetic model (exponential model) for all the layers under study, devised from studies using disturbed and superficial samples. The goal of the present study was to establish the best kinetic model to explain and predict N mineralization as affected by sample disturbance and soil depth in two soils of the semiarid region of Argentina. Disturbed (D: sieved <2 mm and quartz mixed) and undisturbed (UD) samples from two Haplustolls were subject to successive incubations and extractions to assess N mineralization rates. The amount of N mineralized in disturbed samples was up to 200% greater than undisturbed. Replicate D samples from the Torriorthentic had significantly higher variability than samples from the Torrifluventic. In the latter, UD samples had a kinetic heterogeneity that was not apparent in the D samples. These results suggest that the incubation-technique for D samples is ineffective for these soils.

Characterization of the Branched-Photocycle Intermediates P and Q of Bacteriorhodopsin

The bacteriorhodopsin branched-photocycle intermediates P and Q are studied with respect to chromophore isomeric content, photochemical origin, kinetic heterogeneity, and photoreversibility. These blue-shifted species are compared to products with similar spectroscopic properties generated via thermal denaturation. We observe that the thermal and photochemical species differ in both isomeric content and binding site environment. Sequential two-photon activation of glycerol suspensions of bacteriorhodopsin containing low concentrations of water (<15% v/v water/glycerol) form high yields of P state but almost no Q state. This observation is attributed to the role that water plays in the hydrolysis reaction that converts the P state to Q. Relatively large photostationary state populations of both intermediates can be generated in both 85% v/v glycerol suspensions and polyacrylamide gels. At ambient temperature, both intermediates can be fully converted back to bR with blue light. Chromophore extraction and HPLC analysis reveal that P, Q, and the spectrally similar thermal products have a 9-cis retinal chromophore. The thermal products are also found to contain small amounts of the 7-cis isomer. Time-resolved spectroscopy reveals that the P state is actually comprised of two components with maximum absorptivities at 445 and 525 nm. The molar absorptivities of the chromophore band maxima of the P and Q states in an 85% v/v glycerol/water suspension at pH 7 are ε(P445) = 47 000, ε(P525) = 39 000, and ε(Q) = 33 000 M-1 cm-1. Kinetic analyses support previous studies indicating that the P state is formed predominantly from the O state, which is consistent with the branched-photocycle model. However, other paths to the P state are possible, including direct excitation of the small population of blue membrane present in solution. We examine the hypothesis that the branched-photocycle evolved as a photochromic sunscreen for UVA and UVB protection.

The influence of the nature of organoaluminum compound on kinetic heterogeneity of active sites in lanthanide-based diene polymerization

The influence of organoaluminum compound on kinetic heterogeneity of active sites in lanthanide-based diene polymerization was investigated. It was shown that heterogeneity of investigated catalytic systems was displayed in the existence of four types of active centers. They are formed at the beginning of the polymerization process and produce macromolecules with lengths that are definite for each type of active centers. The organoaluminum compound's nature greatly influences the kinetic activity of polymerization centers. The method of analysis of the curves' maximum distribution on kinetic activity and the change of kinetic activity of every active centers' type were proposed, which permits the calculation of values of concentration for each type of active center separately. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 87: 358–368, 2003

A novel dynamic model of hematopoietic stem cell organization based on the concept of within-tissue plasticity.

At present, no dynamic quantitative models of stem cell organization are available that fulfill all criteria of the prevalent functional definition of hematopoietic stem cells and, at the same time, provide a consistent explanation of cell kinetic and functional stem cell heterogeneity, reversibility of cellular properties, self-organized regeneration after damage, fluctuating activity and competition of stem cell clones, and microenvironment dependency of stem cell quality. To solve this problem, we propose a new, comprehensive model concept. A single cell-based stochastic model is described. It makes the novel concept of within-tissue plasticity operational. Within a range of potential options, individual cells may reversibly change their actual set of properties depending on the influence of the local growth environment. Stochastic switching between the growth environments introduces fluctuations that eventually generate heterogeneity. Extensive model simulations are compared with experimental data. Although stemness is not an explicit cellular model property, the system behavior is consistent with the functional definition of stem cells and explains a large set of experimental observations on stem cell function in vivo and in vitro on the level of cell populations and individual cells. Classic results such as the colony-forming unit spleen assay, as well as recent experimental observations on stem cell kinetics, individual clone tracking, and fluctuating clonal contribution, are discussed. This concept introduces a fundamentally new perspective on stem cell organization treating stemness not as an explicit cellular property but as the result of a dynamic process of self-organization. The model needs to be extended to incorporate lineage specification and tissue plasticity.

Kinetic Heterogeneity of an Experimental Tumour Revealed by BrdUrd Incorporation and Mathematical Modelling

In the present paper we propose a method of analysis of the cell kinetic characteristics of in vivo experimental tumours, that uses DNA–BrdUrd flow cytometry data at various times after the bromodeoxyuridine (BrdUrd) injection and mathematical modelling. The model of the cell population takes into account the cell–cell heterogeneity of the progression rate across cell cycle phases within the tumour, and assumes a strict correlation between the durations of S and G2M phases. The model also allows for a nonconstant DNA synthesis rate across S phase. In addition, the measurement process is modelled, considering the possibility of nonimpulsive labelling and providing a representation of the time course of the bivariate DNA–BrdUrd fluorescence distribution. Sequential DNA–BrdUrd distributions were obtained in vivo from a human ovarian carcinoma transplanted in mice and, for comparison, in vitro from a cell line of the same origin. From these data, that included the fractional density and the mean BrdUrd-fluorescence of BrdUrd-positive cells as a function of the DNA-fluorescence, kinetic parameters such as the potential doubling time (Tpot) and the mean and variance of the transit times in S and G2M phases, were estimated. This study revealed the presence of a substantial heterogeneity in S and G2M phases within the in vivo cell population and of a lower heterogeneity in the in vitro population. Moreover, our analysis suggests a nonnegligible effect of the BrdUrd pharmacokinetics in the in vivo cell labelling.

Characterization of glucose transport by cultured rabbit kidney proximal convoluted and proximal straight tubule cells.

Rabbit kidney proximal convoluted tubule (RPCT) and proximal straight tubule (RPST) cells were independently isolated and cultured. The kinetics of the sodium-dependent glucose transport was characterized by determining the uptake of the glucose analog alpha-methylglucopyranoside. Cell culture and assay conditions used in these experiments were based on previous experiments conducted on the renal cell line derived from the whole kidney of the Yorkshire pig (LLC-PK1). Results indicated the presence of two distinct sodium-dependent glucose transporters in rabbit renal cells: a relatively high-capacity, low-affinity transporter (V(max) = 2.28 +/- 0.099 nmoles/mg protein min, Km = 4.1 +/- 0.27 mM) in RPCT cells and a low-capacity, high-affinity transporter (V(max) = 0.45 +/- 0.076 nmoles/mg protein min, K(m) = 1.7 +/- 0.43 mM) in RPST cells. A relatively high-capacity, low-affinity transporter (V(max) = 1.68 +/- 0.215 nmoles/mg protein min, Km = 4.9 +/- 0.23 mM) was characterized in LLC-PK1 cells. Phlorizin inhibited the uptake of alpha-methylglucopyranoside in proximal convoluted, proximal straight, and LLC-PK1 cells by 90, 50, and 90%, respectively. Sodium-dependent glucose transport in all three cell types was specific for hexoses. These data are consistent with the kinetic heterogeneity of sodium-dependent glucose transport in the S1-S2 and S3 segments of the mammalian renal proximal tubule. The RPCT-RPST cultured cell model is novel, and this is the first report of sodium-dependent glucose transport characterization in primary cultures of proximal straight tubule cells. Our results support the use of cultured monolayers of RPCT and RPST cells as a model system to evaluate segment-specific differences in these renal cell types.

Structural relaxation, self-diffusion, and kinetic heterogeneity in the two-dimensional lattice Coulomb gas

We present Monte Carlo simulation results on the equilibrium relaxation dynamics in the two-dimensional lattice Coulomb gas, where finite fractions f of the lattice sites are occupied by positive charges. In the case of high-order rational values of f close to the irrational number 1-g [g identical with (square root of 5-1)/2 is the golden mean], we find that the system exhibits, for a wide range of temperatures above the first-order transition, a glassy behavior resembling the primary relaxation of supercooled liquids. Single-particle diffusion and structural relaxation show that there exists a breakdown of proportionality between the time scale of diffusion and that of structural relaxation analogous to the violation of the Stokes-Einstein relation in supercooled liquids. Suitably defined dynamic cooperativity is calculated to exhibit the characteristic nature of dynamic heterogeneity present in the system.

Control of heme reactivity by diffusion: structural basis and functional characterization in hemoglobin mutants.

The effect of mutagenesis on O(2), CO, and NO binding to mutants of human hemoglobin, designed to modify some features of the reactivity that hinder use of hemoglobin solutions as blood substitute, has been extensively investigated. The kinetics may be interpreted in the framework of the Monod-Wyman-Changeux two-state allosteric model, based on the high-resolution crystallographic structures of the mutants and taking into account the control of heme reactivity by the distal side mutations. The mutations involve residues at topological position B10 and E7, i.e., Leu (B10) to Tyr and His (E7) to Gln, on either the alpha chains alone (yielding the hybrid tetramer Hbalpha(YQ)), the beta chains alone (hybrid tetramer Hbbeta(YQ)), or both types of chains (Hb(YQ)). Our data indicate that the two mutations affect ligand diffusion into the pocket, leading to proteins with low affinity for O(2) and CO, and especially with reduced reactivity toward NO, a difficult goal to achieve. The observed kinetic heterogeneity between the alpha(YQ) and beta(YQ) chains in Hb(YQ) has been rationalized on the basis of the three-dimensional structure of the active site. Furthermore, we report for the first time an experiment of partial CO binding, selective for the beta chains, to high salt crystals of the mutant Hb(YQ) in the T-state; these crystallographic data may be interpreted as "snapshots" of the initial events possibly occurring on ligand binding to the T-allosteric state of this peculiar mutant Hb.

Diffusion of small molecules in disordered media: study of the effect of kinetic and spatial heterogeneities

The diffusion of small molecules in disordered media has been studied by employing kinetic Monte Carlo (KMC) simulations and the time-dependent effective medium approximation (EMA). The simulations were conducted in a cubic lattice, to the bonds of which were assigned rate constants governing the elementary jump events, according to a prescribed probability distribution function. Different distributions with a variance ranging from a very small value, representative of a homogeneous medium, to a very large value, representative of a highly disordered, heterogeneous medium, were studied. It was found that the variance of the distribution of rate constants has a profound effect on the diffusion process, giving rise to an anomalous, non-Fickian regime at short time scales. The higher the variance of the distribution, the longer the duration of the anomalous regime and the smaller the value of the diffusion coefficient in the long-time, Fickian regime. The EMA-based calculations are in excellent quantitative agreement with the simulation findings, particularly for distributions of not too high variance. Simulations were also performed on spatially correlated lattices, consisting of domains within each of which the rate constants assume similar values. Spatial correlations were found to strongly influence the diffusion process at short time scales, prolonging the duration of the anomalous regime; at long time scales, however, spatially correlated lattices are characterized by the same diffusivity as uncorrelated ones with the same rate constant distribution.

Ac voltammetry studies of electron transfer kinetics for a redox couple attached via short alkanethiols to a gold electrode

Abstract Ac voltammetry (ACV) is used to determine the chain length dependence of electron transfer kinetics between gold bead electrodes and ruthenium redox centers attached to the electrode surface via short alkanethiols. The pentaamminepyridine ruthenium redox centers are attached to pre-assembled monolayers of mercaptoalkanecarboxylic acids (HS(CH 2 ) m COOH, m =5, 7, 10). Equations for faradaic admittance of strongly adsorbed, non-interacting electroactive species are fitted to the experimental faradaic admittance data. Kinetic heterogeneity is suggested to be a probable reason for the apparent increase in the standard rate constant ( k s ) and decrease in the total coverage of redox centers ( θ total ) as the perturbation frequency increases. Because of the frequency dependence of k s and because of limitations in the correction for uncompensated resistance, faradaic admittance at a fixed phase angle of 70° is chosen to compare k s for different chain lengths. The plot of ln( k s ) versus m (the number of methylene units) is linear with a slope of −1.2±0.1 per CH 2 .

A New Look at Proliferation

Assessment of proliferation has failed to make an impact on clinical radiotherapy. In this review, a different view of proliferation, its organization and its implication for radiotherapy are presented. Cell kinetic heterogeneity is present in all tumours and understanding and measuring this heterogeneity might reveal insight and tools to predict the mode of failure and repopulation during different schedules of radiotherapy and chemotherapy.

A quantitative assessment of heterogeneity for surface-immobilized proteins.

Many biotechnological applications use protein receptors immobilized on solid supports. Although, in solution, these receptors display homogeneous binding affinities and association/dissociation kinetics for their complementary ligand, they often display heterogeneous binding characteristics after immobilization. In this study, a fluorescence-based fiber-optic biosensor was used to quantify the heterogeneity associated with the binding of a soluble analyte, fluorescently labeled trinitrobenzene, to surface-immobilized monoclonal anti-TNT antibodies. The antibodies were immobilized on silica fiber-optic probes via five different immobilization strategies. We used the Sips isotherm to assesses and compare the heterogeneity in the antibody binding affinity and kinetic rate parameters for these different immobilization schemes. In addition, we globally analyzed kinetic data with a two-compartment transport-kinetic model to analyze the heterogeneity in the analyte-antibody kinetics. These analyses provide a quantitative tool by which to evaluate the relative homogeneity of different antibody preparations. Our results demonstrate that the more homogeneous protein preparations exhibit more uniform affinities and kinetic constants.

Adenosine triphosphate-evoked currents in cultured dorsal root ganglion neurons obtained from rat embryos: desensitization kinetics and modulation of glutamate release

Sensory neurons express purinergic P2X receptors on their central and peripheral terminals as well as their cell bodies. ATP activation of these receptors drives action potential firing and glutamate release with potentially important consequences for sensory function. Here we show ATP-gated currents activated in cultured embryonic dorsal root ganglion neurons have heterogeneity of time-courses comparable to those observed in different subpopulations of acutely dissociated adult dorsal root ganglion neurons. The distribution of time-courses across the population of cultured neurons is strongly influenced by culture conditions. Heterogeneity in ATP current kinetics occurs even though immunocytochemical staining reveals a relatively homogeneous and widespread expression of the P2X2 and P2X3 subunits. We show that the time-courses of ATP-gated currents recorded at the cell bodies are mirrored by the time-courses of transmitter release from the dorsal root ganglion nerve terminals, indicating similar P2X receptor properties on the soma and their associated terminals. Our results illustrate a functional heterogeneity of P2X receptor-mediated currents that is strongly influenced by external factors. This heterogeneity in current kinetics may have implications for neuronal function as it constrains the time-course of ATP-mediated modulation of neurotransmitter release at sensory nerve terminals.

Heterogeneity and finite size effects in the dynamics of supercooled liquids

After brief review of our recent simulation studies on kinetic heterogeneity in supercooled liquids, we report finite-size effects observed in computer simulations by decreasing system size from N = 10 4 to 10 3 and 108. Although no significant deviation is detected in the static pair correlation functions, it is demonstrated that the structural a relaxation in smaller systems become considerably slower than that in larger systems for temperatures below T x at which the size of the cooperative particle motions becomes comparable to the unit cell length of the small system. Also the effects of confinements in planar walls are reported.

Photo-oxidation of thick isotactic polypropylene films I. Characterisation of the heterogeneous degradation kinetics

Photo-oxidation kinetics of thick isotactic polypropylene films have been compared to thermal-oxidation kinetics of thin films, and noticeable differences have been found. The non-classical kinetic trend of the former can be described as a three step evolution: a typical induction/autoacceleration POOH build-up at the beginning, an intermediate slower POOH content increase and, finally, a gentler POOH increase, which can be better described by quadratic functions of the oxidation time than by a linear dependence. In addition, a series of oscillations appearing from the beginning of the photo-oxidation are found. This kinetic heterogeneity is suggested to be related to the progression of the oxidation to the inside of the strips. However, the FTIR analysis of the evolution of the POOH band position in both photo and thermal oxidation, enables the observed shape changes to be associated to kinetic stages.

Multicompartment analysis of gadolinium chelate kinetics: blood-tissue exchange in mammary tumors as monitored by dynamic MR imaging.

The blood-tissue exchange kinetics of gadopentetate were studied in 49 malignant and benign mammary tumors. Signal enhancement was monitored simultaneously in the aorta and in tumor for 10.5 minutes after the beginning of a 1 minute i.v. infusion of the contrast medium (CM). Kinetic analysis was based on a model with two compartments for systemic pharmacokinetics and up to three kinetically distinct compartments for tumor. Kinetic heterogeneity, ie, two or more compartments with different exchange rate constants in a given tumor, was found in 85% of carcinomas, 38% of fibroadenomas, and 14% of mastopathic tumors. The within-tumor average of CM exchange rates was 1.22 (0.62-1.65) min(-1) in carcinomas, 0.38 (0.26-0.60) min(-1) in fibroadenomas, and 0.16 (0. 12-0.20) min(-1) in mastopathies (median and interquartile distances). The area under the signal enhancement-time curve of the aorta varied 4.5-fold between individuals. It is concluded that individual CM kinetics in arterial blood should be taken into account when CM exchange rates between blood and tumor are to be determined and that a kinetic model for potentially malignant tumors should allow for kinetic heterogeneity.

Electron and proton transfer on the acceptor side of the reaction center in chromatophores of Rhodobacter capsulatus: evidence for direct protonation of the semiquinone state of QB.

1. The absorption changes associated with the formation of P+QBred (QBred stands for the semiquinone state of the secondary quinone acceptor) were investigated in chromatophores of Rhodobacter capsulatus. Marked modifications of the semiquinone spectrum were observed when the pH was lowered from 7 to 5. These modifications match those expected for a complete conversion of QBred from the anionic state QB- at pH 7 to the neutral protonated state QBH at pH 5. Similar modifications were observed in chromatophores from Rb. sphaeroides, but not in purified reaction centers from Rb. capsulatus, suggesting that the environment of the reaction center (native membrane vs detergent micelle) is the crucial parameter. 2. The recombination reaction P+QBred --> PQB was investigated as a function of pH. No particular kinetic heterogeneity was observed at low pH, showing that QBH remains mostly bound to the reaction center. The rate constant reaches a minimum value of 0.08 s-1 at pH 6, suggesting that the direct route for recombination prevails in chromatophores below this pH, instead of the usual pathway via QA-. 3. The proton uptake caused by QBred is about 1 below pH 7 and decreases at higher pH. It is suggested that the pH dependence of the conversion of QB- to QBH, occurring in a range where the uptake is constant, cannot be accommodated by a purely electrostatic model, but probably involves a conformational change. 4. The kinetics of the electron-transfer reaction QA-QB-->QAQBred were investigated. A 2-fold acceleration was observed between pH 7 and pH 5 (t1/2 approximately 30 and 15 microseconds, respectively). A fast (<<10 microseconds) unresolved phase appears to be present at both pHs. The second electron-transfer QA-QBred-->QAQBH2 proceeds with a similar rate as the first electron transfer (15-30 microseconds phase). Consequences for the rate-limiting step are discussed. 5. The carotenoid shift, indicative of the membrane potential, displays a rising phase concomitant with the QA-QB-->QAQBred electron transfer. Its relative extent is markedly increased at pH 5, with part of the kinetics occurring during the unresolved fast phase. 6. The extent of the electrochromic shift of bacteriopheophytin around 750 nm associated with formation of QBred decreases toward acidic pH, reflecting the charge compensation due to proton uptake and the formation of neutral QBH.

Multiple pathways on a protein-folding energy landscape: kinetic evidence.

The funnel landscape model predicts that protein folding proceeds through multiple kinetic pathways. Experimental evidence is presented for more than one such pathway in the folding dynamics of a globular protein, cytochrome c. After photodissociation of CO from the partially denatured ferrous protein, fast time-resolved CD spectroscopy shows a submillisecond folding process that is complete in approximately 10(-6) s, concomitant with heme binding of a methionine residue. Kinetic modeling of time-resolved magnetic circular dichroism data further provides strong evidence that a 50-microseconds heme-histidine binding process proceeds in parallel with the faster pathway, implying that Met and His binding occur in different conformational ensembles of the protein, i.e., along respective ultrafast (microseconds) and fast (milliseconds) folding pathways. This kinetic heterogeneity appears to be intrinsic to the diffusional nature of early folding dynamics on the energy landscape, as opposed to the late-time heterogeneity associated with nonnative heme ligation and proline isomers in cytochrome c.