A new method to determine active enzyme distribution, effective diffusivity, rate constant for main reaction and rate constant for deactivation

A new method is presented to determine (1) the rate constant for the main reaction, (2) the rate constant for deactivation, (3) the effective diffusivity, and (4) the active enzyme distribution within a porous solid support by utilizing data of bulk substrate concentration versus time in a continuous stirred basket reactor. The method relies on an assumption of parallel deactivation mechanism with strong pore diffusional resistance with respect to substrate species. The data of hydrogen peroxide-immobilized catalase published in the literature are used to demonstrate the theory. A parameter determination procedure is also presented.     

A novel method of determination of the internal enzyme distribution within porous solid supports and the deactivation rate constant

This article presents a method for determining the rate constant for deactivation and the internal distribution of immobilized enzyme. This method makes use of the parallel deactivation process in a diffusion-controlled regime, in which the internal activity profile behaves like a penetration front. This front basically traces through the initial active enzymatic profile, and one can determine the internal profile and the rate constant for deactivation from the experimentally observable bulk concentration versus time. This method is applied to the experimental data of the system of hydrogen-peroxide-immobilized catalase on controlled pore glass and Si-Al particles.     

Inconsistency of the maximum parsimony method when the rate of nucleotide substitution is constant

The inconsistency of the maximum parsimony method is known to occur even when the rate of nucleotide substitution is constant. To understand why this inconsistency occurs, a mathematical study was conducted for the cases of five, six, and seven sequences. The results obtained indicate that this inconsistency occurs because the probability of occurrence of nucleotide configurations generated by one substitution on a short interior branch is often lower than that of configurations generated by more substitutions on other longer branches. The chance of occurrence of this event—or, the inconsistency of the maximum parsimony method—apparently increases as the number of sequences increases. The inconsistency may occur even when the extent of sequence divergence is relatively small. Correspondence to: M. Nei     

A new method for the determination of ligand dissociation rate constant of carboxyhemoglobin.

Concanavalin A (Con A) treatment of plasma membrane-enriched fractions from lactating mammary gland causes an activation of Mg⁺⁺-ATPase and an inactivation of 5′-nucleotidase. Both effects can be prevented by the presence of α-methylmannoside, and both exhibit cooperativity with Hill coefficients near 2. The cooperativity may arise from Con A effects on subunit interactions of the enzymes or by clustering of the enzyme molecules in the membrane, possibly induced by Con A. Investigations of these systems should be useful for developing an understanding of modes of action of Con A in complex phenomena.     

Robust estimation of the false discovery rate

MOTIVATION: Presently available methods that use p-values to estimate or control the false discovery rate (FDR) implicitly assume that p-values are continuously distributed and based on two-sided tests. Therefore, it is difficult to reliably estimate the FDR when p-values are discrete or based on one-sided tests. RESULTS: A simple and robust method to estimate the FDR is proposed. The proposed method does not rely on implicit assumptions that tests are two-sided or yield continuously distributed p-values. The proposed method is proven to be conservative and have desirable large-sample properties. In addition, the proposed method was among the best performers across a series of 'real data simulations' comparing the performance of five currently available methods. AVAILABILITY: Libraries of S-plus and R routines to implement the method are freely available from www.stjuderesearch.org/depts/biostats.     

A method for determining a rate constant for biexponential processes using data on dynamics of accumulating the end product of a reaction

A new method the rate constant determination of some biexponential processes is suggested. The method is based on the asymptotic solution of transcendental irrational equations, described such processes. This method can be used when kinetics of the final product of reaction is known. The values of the rate constants obtained by suggested method are precise, if the data used for calculation are also precise. In the other case methods of mathematical statistics should be used for evaluation of the slopes of kinetics curves in the semi-logarithmic coordinates.     

Influence of dilution rate on the acidogenic phase products distribution during two-phase lactose anaerobiosis

Acidogenic fermentation of lactose was carried out in a continuous stirred reactor with a mixed anaerobic culture. From the variation of the reactor products with pH and dilution rate two possible carbon flow schemes were proposed for the reaction. In both schemes the carbon flow from pyruvate to butyrate and lactate was assumed to occur in parallel. A change in gas composition and in product concentrations at dilution rates between 0.1 and 0.15 h(-1) for pH levels between 4.5 and 6.0 was ascribed to a shift in microbial population. To clarify the mechanism radiotracer tests were made using [U-(14)C]-butyrate, [2-(14)C]-propionate and [U-(14)C]-lactate to determine the path of carbon flow during acidogenesis of lactose using a mixed culture. At a dilution rate between 0.1 and 0.15 h(-1) and pH from 4.5 to 6.0 a rise in the lactate concentration in the product was shown to be due to a microbial population shift which disabled the conversion of lactate to other intermediary metabolites. It was also found that the flow of carbon from pyruvate to butyrate and lactate occurred by parallel pathways. Also, in the presence of hydrogen reducing methanogens, lactate was almost completely converted to acetate and not propionate. Butyrate was found to be converted to acetate at a slow rate as long as hydrogen reducing methanogens were present. The role played by propionibacteria in this lactose acidogenic eocosystem was minor. From the carbon flow model it can be concluded that lactate is the most suitable marker for optimizing an acidogenic reactor in a two-phase biomethanation process.     

Preparation of liposomes using the phase reversal method without sonication

A simple procedure for preparation of liposomes based on the phase reversal method but differing from the original technique by the absence of ultrasonic treatment, has been developed. Using this method, the effects of NaCl concentration used for the preparation of liposomes from a mixture of neutral and charged lipids, on the inner liposome volume were studied. The experimental data suggest that NaCl concentration may be regarded as a basic criterion determining the size of liposomes in many routine techniques used for the preparation of liposomes from a mixture of neutral and charged lipids.     

A method for estimating bicarbonate buffering of lactic acid during constant work rate exercise

A method to estimate the CO₂ derived from buffering lactic acid by HCO₃ ^– during constant work rate exercise is described. It utilizes the simultaneous continuous measurement of O₂ uptake ( O₂) and CO₂ output ( CO₂), and the muscle respiratory quotient (RQ_m). The CO₂ generated from aerobic metabolism of the contracting skeletal muscles was estimated from the product of the exercise-induced increase in O₂ and RQ_m calculated from gas exchange. By starting exercise from unloaded cycling, the increase in CO₂ stores, not accompanied by a simultaneous decrease in O₂ stores, was minimized. The total CO₂ and aerobic CO₂ outputs and, by difference, the millimoles (mmol) of lactate buffered by HCO₃ ^– (corrected for hyperventilation) were estimated. To test this method, ten normal subjects performed cycling exercise at each of two work rates for 6 min, one below the lactic acidosis threshold (LAT) (50 W for all subjects), and the other above the LAT, midway between LAT and peak O₂ [mean (SD), 144 (48) W]. Hyperventilation had a small effect on the calculation of mmol lactate buffered by HCO₃ ^– [6.5 (2.3)% at 6 min in four subjects who hyperventilated]. The mmol of buffer CO₂ at 6 min of exercise was highly correlated (r = 0.925, P < 0.001) with the increase in venous blood lactate sampled 2 min into recovery (coefficient of variation = ±0.9 mmol·l^–1). The reproducibility between tests done on different days was good. We conclude that the rate of release of CO₂2 from HCO₃ ^– can be estimated from the continuous analysis of simultaneously measured CO₂, O₂, and an estimate of muscle substrate.     

Evaluation of the transpiration rate of lotus using the stem heat-balance method

To reveal the mechanism of transpiration by hydrophytes in the field, it is necessary to evaluate the transpiration rate without the effect of the evaporation from the water surface. In order to test the suitability for evaluating the transpiration rate of lotus (Nelumbo nucifera Gaertn.) leaves in the field, stem heat-balance method was applied and the obtained sap-flow rate was compared with the transpiration rate measured by weighing and with the overall canopy evapotranspiration rate by means of the eddy covariance technique. The transpiration rate estimated with the sap-flow measurements showed good agreement with that obtained from the weighing method. Lotus has many air canals in its petiole to carry oxygen-rich air to the rhizome and methane- and carbon dioxide-rich air back to the atmosphere, but there was little effect of the mass flow of air through these canals on the sap-flow rates. In the field observations, the canopy evapotranspiration rate (0.28 mm h⁻¹ at maximum) was nearly equal to the sum of the transpiration rate from all sunlit leaves (0.30 mm h⁻¹), and the contribution of the transpiration from shaded leaves and evaporation from the water surface was considered to be minor in the seasons when the leaves were fully developed. Evaluation of bulk leaf conductance revealed that the conductance in the leaf boundary layer of lotus could be low (ca. 0.23 mol m⁻² s⁻¹) because of its large leaf area. The low conductance in the leaf boundary layer would increase leaf temperature, which, in turn, would generate air circulation within the plant's ventilation system. Because there was a linear relationship between transpiration rate and the leaf-to-air vapor-pressure deficit, with no apparent maximum, high vapor-pressure deficits (3.4 kPa at maximum) did not appear to cause significant stomatal closure in lotus plants. The stomata of lotus leaves play a role as air inlets to carry oxygen-rich air to the rhizome, so their low sensitivity would help to increase air intake.     

Estimation of bumblebee queen dispersal distances using sibship reconstruction method

Dispersal ability is a key determinant of the propensity of an organism to cope with habitat fragmentation and climate change. Here we quantify queen dispersal in two common bumblebee species in an arable landscape. Dispersal was measured by taking DNA samples from workers in the spring and summer, and from queens in the following spring, at 14 sites across a landscape. The queens captured in the spring must be full sisters of workers that were foraging in the previous year. A range of sibship reconstruction methods were compared using simulated data sets including or no genotyping errors. The program Colony gave the most accurate reconstruction and was used for our analysis of queen dispersal. Comparison of queen dispersion with worker foraging distances was used to take into account an expected low level of false identification of sister pairs which might otherwise lead to overestimates of dispersal. Our data show that Bombus pascuorum and B. lapidarius queens can disperse by at least 3 and 5 km, respectively. These estimates are consistent with inferences drawn from studies of population structuring in common and rare bumblebee species, and suggest that regular gene flow over several kilometres due to queen dispersal are likely to be sufficient to maintain genetic cohesion of ubiquitous species over large spatial scales whereas rare bumblebee species appear unable to regularly disperse over distances greater than 10 km. Our results have clear implications for conservation strategies for this important pollinator group, particularly when attempting to conserve fragmented populations.     

Studies on the catalytic rate constant of ribosomal peptidyltransferase

A detailed kinetic analysis of a model reaction for the ribosomal peptidyltransferase is described, using fMet-tRNA or Ac-Phe-tRNA as the peptidyl donor and puromycin as the acceptor. The initiation complex (fMet-tRNA X AUG X 70 S ribosome) or (Ac-Phe-tRNA X poly(U) X 70 S ribosome) (complex C) is isolated and then reacted with excess puromycin (S) to give fMet-puromycin or Ac-Phe-puromycin. This reaction (puromycin reaction) is first order at all concentrations of S tested. An important asset of this kinetic analysis is the fact that the relationship between the first order rate constant kobs and [S] shows hyperbolic saturation and that the value of kobs at saturating [S] is a measure of the catalytic rate constant (k cat) of peptidyltransferase in the puromycin reaction. With fMet-tRNA as the donor, this kcat of peptidyltransferase is 8.3 min-1 when the 0.5 M NH4Cl ribosomal wash is present, compared to 3.8 min-1 in its absence. The kcat of peptidyltransferase is 2.0 min-1 when Ac-Phe-tRNA replaces fMet-tRNA in the presence of the ribosomal wash and decreases to 0.8 min-1 in its absence. This kinetic procedure is the best method available for evaluating changes in the activity of peptidyltransferase in vitro. The results suggest that peptidyltransferase is subjected to activation by the binding of fMet-tRNA to the 70 S initiation complex.     

Kinetics of recombination of photoseparated charges in Rhodobacter sphaeroides reaction centers analyzed by relaxation rate constant distribution

We studied the kinetics of dark recombination of charges photoseparated between the bacteriochlorophyll dimer (P) and the quinone acceptors of the photosynthetic reaction centers (RCs) of Rhodobacter sphaeroides. The time of light activation was varied from 1 to 60 s. The relaxation rate constant distribution was computed from experimental kinetic curves of charge recombination, using our original method whereby relaxation curves are approximated by a set of Gaussian-like peaks in the time domain that correspond to different conformational sub-states of RCs. With increasing photoactivation exposure, some peaks shift toward longer relaxation times. A ‘bifurcation’ of the slower peak is observed at exposures of 20–30 s. The phenomenon is interpreted as evidence of a conformational transition induced by separated charges in the RC structure.     

Phylogenetic reconstruction of weevil superfamily Curculionoidea (coleoptera) using the SYNAP method

Phylogenetic relationships within the superfamily Curculionoidea were reconstructed. Autapomorphies of the superfamily Curculionoidea include more or less pronounced snout, clubbed antennae, and partially sclerotized or completely membranous male tergite 9. Weevil families can be divided into three groups. The first group includes the most primitive family Nemonychidae. The second group includes nine families (Anthribidae, Belidae, Oxycorynidae, Eccoptarthridae, Allocorynidae, Rhynchitidae, Attelabidae, Ithyceridae, and Brentidae). The third (“higher”) group includes six families (Brachyceridae, Cryptolaryngidae, Dryophthoridae, Curculionidae, Scolytidae, and Platypodidae).     

Reverse flow in roots of Sesbania rostrata measured using the constant power heat balance method

This investigation was performed to examine qualitatively and quantitatively the reverse flow in partially dried roots of Sesbania rostrata using the constant power heat balance method. First, a semi-empirical technique for estimating sheath conductance of sap-flow sensors without assuming that sap flow is zero at night was proposed. Sap flow measured with the heat balance method was compared with water uptake as measured by a potometric method. Sap flow was overestimated by 56·1% for a 3·3-mm-diameter root, and by 40·0% for 6·1 mm and 33·3% for 8·8 mm roots. However, high correlation coefficients between the rates of water uptake and sap flow demonstrated that calibration would provide reliable values for root sap flow. To detect reverse flow, a split root experiment was conducted using a S. rostrata plant with its root system divided between dry and wet compartments. Daily sap flow of the drying compartment declined whereas that in ‘wet’ root increased, suggesting that the decrease in water uptake by ‘dry’ roots was offset by the ‘wet’ roots. Reverse flow was observed at night in the root on the dry side of the container when the soil water potential was less than –0·30 MPa. The total amount of water released into the soil during the night period was estimated to be 22·5 g.     

A new graphic method for determining the rate constant for affinity of a ligand for its receptor]

A new method of determination of the equilibrium constant for a ligand binding to acceptor and evaluation of the number of binding sites on the acceptor molecules (or cells) is suggested. The method is simpler, more convenient, and more precise than Klotz's or Scatchard's method.     

Multilayer neural networks and function reconstruction by using a priori knowledge

This paper proposes a new methodology to approximate functions by incorporating a priori information. The relationship between the proposed scheme and multilayer neural networks is explored theoretically and numerically. This approach is particularly interesting for the very relevant class of limited spectrum functions. The number of free parameters is smaller if compared to Back-Propagation Algorithm opening the way for better generalization results.