Factors affecting the kinetics of DNA reassociation in phenol–water emulsion at high DNA concentrations

DNA reassociation kinetics using the phenol emulsion reassociation technique (PERT) [Kohne, D. E., Levison, S. A. & Byers, M. J. (1977) Biochemistry 16 , 5329–5341] has been investigated at high DNA concentrations using an endonuclease S1 assay of reaction progress. Apparent second-order rate constants fall on two intersecting straight lines when presented as a function of DNA concentrations on a log–log plot. In the low DNA concentration range, the rate constants drop about 10-fold when concentration increases 1000-fold. In the high DNA concentration range, the rate constants drop more than 10-fold when concentration increases 10-fold. The slopes of these lines are the same in different solvents and at different temperatures. The intersection between the lines occurs when the available catalytic surface is saturated. At high DNA concentrations, high-complexity heterologous denatured DNA apparently competes 2–4 times better for the surface than homologous DNA because it does not participate in a reassociation reaction. Native and partially native DNA molecules cannot compete with single-stranded DNA for a saturated surface. At high DNA concentrations, reactions using PERT become dependent on the single-strand DNA length. Increasing length lowers reassociation rates.     

On the nonlinear relationship between the initial rates of dilution-induced microtubule disassembly and the initial free subunit concentration

We have examined the dilution-induced in vitro disassembly kinetics of bovine brain microtubules, initially at steady state, using a wider range of dilutions (2-100-fold) than previously employed. In contrast to earlier results, as well as to the simple nucleation-condensation model for microtubule formation, the initial rate of dimer loss from microtubule ends was not a linear function of the initial concentration of unpolymerized tubulin. Over a 2-20-fold dilution range, plots of the initial rate of dimer loss versus the initial unpolymerized tubulin concentration were approximately linear. However, at greater dilutions, rates of microtubule depolymerization increased nonlinearly. For example, between a 10-fold dilution and a 100-fold dilution, the initial rate of dimer loss for microtubule-associated protein-containing microtubules increased by 300%, rather than a maximum of 11% expected on the basis of a linear rate plot. The nonlinear response was observed for dimer loss from opposite microtubule ends separately and with microtubules containing and lacking associated proteins. Qualitatively similar results were obtained using a wide range of experimental protocols, from which we can reasonably exclude methodological artifact as a basis for the data. We can also reasonably exclude the dissociation of the high molecular weight microtubule-associated proteins 1 and 2 from the microtubules as an explanation for the nonlinearity of the rate plots. The nonlinearity of the rate plots indicates that kinetic constants obtained under nonsteady state conditions of extreme microtubule dilution may not describe the steady state condition accurately.     

基于因子分析的苜蓿叶片叶绿素高光谱反演研究

因子分析是一种能够将具有错综复杂关系的变量归结为少数几个综合因子的多变量统计分析方法,在降低数据维数的同时又可以保存足够的信息,这为处理信息量丰富但冗余较大的高光谱数据提供了一种有效方法。本文利用2010年9月23日采集的16个样点的苜蓿叶片反射率及叶绿素含量数据,采用因子分析方法,分别提取苜蓿叶片反射率光谱400～900nm,以及可见光400nm～760nm和近红外760～900nm光谱区的公共因子,分析因子载荷分布、载荷总量对公共因子与叶绿素含量相关性的影响。利用逐步回归法建立基于公共因子的叶片叶绿素反演模型,并将反演模型与光谱指数建立的模型进行对比。研究表明,1）公共因子与叶片叶绿素的相关性,在更大程度上是与该因子在各个波段上载荷分布有关,而不是总载荷量;2）对波谱进行分区建立的反演模型略优于全区因子分析建立的反演模型;3）与常用于叶片叶绿素含量反演的光谱指数CARI、MCARI、mND680、mND705、mSR705、TVI、DmSR、BGI、BRI相比,因子分析建立的叶绿素反演模型精度更高。     

Investigation of some theoretical models relating the concentrations of glucose and insulin in plasma

In this survey 24 possible theoretical models for the regulation of plasma glucose concentration by plasma insulin concentration have been proposed and tested. A set of data, consisting of plasma glucose concentrations at various times after an intravenous injection of glucose, was selected from the literature. An attempt was made to fit each model to the chosen data using a digital computer program and ten of these were successful. The behaviour of these ten models under different experimental conditions was compared, using a simulation technique, with further published data. At this stage six models were consistent with a steady state at the start or the end of an experiment, but only three were reasonably good at predicting all aspects of the chosen experiments. One was particularly good. Two other sets of data were taken from the literature and the six best models were fitted in order to calculate replicate values of the physiological parameters.     

Statistical techniques for modeling of <Emphasis Type="Italic">Corylus</Emphasis>, <Emphasis Type="Italic">Alnus</Emphasis>, and <Emphasis Type="Italic">Betula</Emphasis> pollen concentration in the air

Prediction of allergic pollen concentration is one of the most important goals of aerobiology. Past studies have used a broad range of modeling techniques; however, the results cannot be directly compared owing to the use of different datasets, validation methods, and evaluation metrics. The main aim of this study was to compare nine statistical modeling techniques using the same dataset. An additional goal was to assess the importance of predictors for the best model. Aerobiological data for Corylus, Alnus, and Betula pollen counts were obtained from nine cities in Poland and covered between five and 16 years of measurements. Meteorological data from the AGRI4CAST project were used as a predictor variables. The results of 243 final models (3 taxa \(\times\)  9 cities \(\times\) 9 techniques) were validated using a repeated k-fold cross-validation and compared using relative and absolute performance statistics. Afterward, the variable importance of predictors in the best models was calculated and compared. Simple models performed poorly. On the other hand, regression trees and rule-based models proved to be the most accurate for all of the taxa. Cumulative growing degree days proved to be the single most important predictor variable in the random forest models of Corylus, Alnus, and Betula. Finally, the study suggested potential improvements in aerobiological modeling, such as the application of robust cross-validation techniques and the use of gridded variables.     

Adenylate cyclase of bovine adrenal cortex plasma membranes. Divergence between corticotropin and fluoride combined effects with forskolin

The diterpene forskolin maximally stimulated bovine adrenal cortex adenylate cyclase activity 9-fold with a concentration producing half-maximum effect (ED50) of about 4 microM. The effects of forskolin and the fully active corticotropin fragment ACTH (I 24) were additive over nearly the whole range of concentration of both effectors, indicating separate and independent mechanisms of action. By contrast, 10 mM NaF blocked forskolin action in the nanomolar range of the diterpene concentration, while it allowed a partial stimulation by forskolin in the micromolar range. NaF thus reveals a heterogeneity of forskolin action in the adrenal cortex plasma membranes. Moreover, our data suggest that ACTH and NaF activation effects, both mediated by the stimulatory regulatory protein Gs, proceed through different mechanisms.     

Quantification of random motility and chemotaxis bacterial transport coefficients using individual-cell and population-scale assays

A number of individual-cell and population-scale assays have been introduced to quantify bacterial motility and chemotaxis. The transport coefficients reported in the literature, however, span several orders of magnitude, making it difficult to ascertain to what degree variations in bacterial species/strain, growth medium, growth and experimental conditions, and experiment type contribute to the reported differences in coefficient values. We quantified the random motility of Escherichia coli AW405 using the capillary assay, stopped-flow diffusion chamber (SFDC), and tracking microscope. We obtained good agreement for the random motility coefficient between these assays when using the same bacterial strain and consistent growth and experimental conditions. Chemotaxis of E. coli toward the attractant alpha-methylaspartate was quantified using the SFDC and capillary assay. Good agreement for the chemotactic sensitivity coefficient between the SFDC and the capillary assay was obtained across a limited attractant concentration range. Three different mathematical models were considered for analyzing capillary assay data to obtain a chemotactic sensitivity coefficient. These models differed by their treatment of the bacterial concentration in the chamber and the attractant concentration at the mouth. Results from our study indicate that the capillary assay, the most commonly used bacterial random motility and chemotaxis assay, can be used to accurately quantify bacterial transport coefficients over a limited range of attractant concentrations, provided experiments are performed carefully and appropriate mathematical models are used to interpret the experimental data.     

Untangling climate and water chemistry to predict changes in freshwater macrophyte distributions

Forecasting changes in the distributions of macrophytes is essential to understanding how aquatic ecosystems will respond to climate and environmental changes. Previous work in aquatic ecosystems has used climate data at large scales and chemistry data at small scales; the consequence of using these different data types has not been evaluated. This study combines a survey of macrophyte diversity and water chemistry measurements at a large regional scale to demonstrate the feasibility and necessity of including ecological measurements, in addition to climate data, in species distribution models of aquatic macrophytes. A survey of 740 water bodies stratified across 327,000 square kilometers was conducted to document Characeae (green macroalgae) species occurrence and water chemistry data. Chemistry variables and climate data were used separately and in concert to develop species distribution models for ten species across the study area. The impacts of future environmental changes on species distributions were modeled using a range of global climate models (GCMs), representative concentration pathways (RCPs), and pollution scenarios. Models developed with chemistry variables generally gave the most accurate predictions of species distributions when compared with those using climate variables. Calcium and conductivity had the highest total relative contribution to models across all species. Habitat changes were most pronounced in scenarios with increased road salt and deicer influences, with two species predicted to increase in range by >50% and four species predicted to decrease in range by >50%. Species of Characeae have distinct habitat ranges that closely follow spatial patterns of water chemistry. Species distribution models built with climate data alone were insufficient to predict changes in distributions in the study area. The development and implementation of standardized, large‐scale water chemistry databases will aid predictions of habitat changes for aquatic ecosystems.     

Ion permeation in normal and batrachotoxin-modified Na+ channels in the squid giant axon

Na+ permeation through normal and batrachotoxin (BTX)-modified squid axon Na+ channels was characterized. Unmodified and toxin-modified Na+ channels were studied simultaneously in outside-out membrane patches using the cut-open axon technique. Current-voltage relations for both normal and BTX-modified channels were measured over a wide range of Na+ concentrations and voltages. Channel conductance as a function of Na+ concentration curves showed that within the range 0.015-1 M Na+ the normal channel conductance is 1.7-2.5-fold larger than the BTX-modified conductance. These relations cannot be fitted by a simple Langmuir isotherm. Channel conductance at low concentrations was larger than expected from a Michaelis-Menten behavior. The deviations from the simple case were accounted for by fixed negative charges located in the vicinity of the channel entrances. Fixed negative charges near the pore mouths would have the effect of increasing the local Na+ concentration. The results are discussed in terms of energy profiles with three barriers and two sites, taking into consideration the effect of the fixed negative charges. Either single- or multi-ion pore models can account for all the permeation data obtained in both symmetric and asymmetric conditions. In a temperature range of 5-15 degrees C, the estimated Q10 for the conductance of the BTX-modified Na+ channel was 1.53. BTX appears not to change the Na+ channel ion selectively (for the conditions used) or the surface charge located near the channel entrances.     

Principal component analysis of nonlinear chromatography

Principal component analysis (PCA) has been used for the modeling of nonlinear chromatography under overload conditions. A 10-fold range of crude erythromycin samples were loaded onto columns with different stationary-phase chemistries (2 polystyrene, 1 methacrylate) in direct proportion to the bed volumes. The elution profiles indicated slightly concave isotherms for the polystyrene supports and a convex Langmuirian isotherm for the methacrylic support used. The principal component models accounted for over 98% of the original variance in the data for all three columns and were able to give excellent models of complete chromatograms in the absence of first-principle models or physicochemical data. Correlations between sample mass and the principal component scores were made for each that were consistent for the column types despite the different geometries and stationary phases. Linear relationships with high correlation coefficients were observed when the scores of the same principal component were compared between columns. Such correlations offer considerable potential for modeling of nonlinear chromatography.     

Rapid measurement of binding constants and heats of binding using a new titration calorimeter

A new titration calorimeter is described and results are presented for the binding of cytidine 2'-monophosphate (2'CMP) to the active site of ribonuclease A. The instrument characteristics include very high sensitivity, rapid calorimetric response, and fast thermal equilibration. Convenient software is available for instrument operation, data collection, data reduction, and deconvolution to obtain least-squares estimates of binding parameters n, delta H degree, delta S degree, and the binding constant K. Sample through-put for the instrument is high, and under favorable conditions binding constants as large as 10(8) M-1 can be measured. The bovine ribonuclease A (RNase)/2'CMP system was studied over a 50-fold range of RNase concentration and at two different temperatures. The binding constants were in the 10(5) to 10(6) M-1 range, depending on conditions, and heats of binding ca. -15,000 cal/mol. Repeat determinations suggested errors of only a few percent in n, delta H degree, and K values over the most favorable concentration range.     

Anti-A and anti-B activity in batches of different intravenous immunoglobulin products determined using a direct haemagglutination method.

The European Pharmacopoeia requires that manufacturers assess intravenous immunoglobulin (IVIG) products for antibodies against blood groups A and B using an indirect anti-globulin test (AGT). However, this method suffers from the disadvantage that the anti-globulin reagent may be neutralised by excess IgG and invalidate the data generated. In view of this, we have used a direct microtitre-based haemagglutination method to screen batches of IVIG products from five manufacturers for anti-A and anti-B, and compared the titres with those reported by the manufacturers. The range of reported titres varied 32-fold across the different products, whereas virtually all the direct method titres fell within a 4-fold range for each specificity. This indicated that the discrepancies in reported titres were due to inconsistencies in manufacturers' testing methodology and/or interpretation of results. Our finding that the anti-globulin reagent used to bring about agglutination of anti-A- or anti-B-sensitised erythrocytes in the AGT was neutralised by excess IgG at least down to a 1 in 8 dilution of IVIG (from 5% (w/v) IgG) casts serious doubts on the suitability of the AGT for testing high immunoglobulin concentration products.     

Studies on the neutralization of herpes simplex virus. VIII. Significance of viral sensitization for inactivation by complement.

Early and late IgG of rabbits immunized with herpes virus showed, respectively, 8-fold and 2-fold enhancement of neutralization endpoint in the presence of complement (C). Kinetic curve experiments employing an appropriate amount of virus revealed that both neutralization and sensitization followed first-order reaction, and each IgG possessed a certain range of concentration where neutralization was negligible while sensitization was marked. Dose responses of neutralization and sensitization velocities demonstrated that the C enhancement of late IgG was about 7-fold and that of early IgG more than 20-fold. These facts suggested that the IgGs contained two different entities of complement-requiring (CRN) and non-requiring neutralizing (N) antibodies at different proportions, only the former being responsible for sensitization. The different CRN: N ratios obtained by the endpoint and kinetic methods may mean either that the two antibodies differ in avidity for the virus or that the number of critical sites per virion for CRN antibody is greater than that for N antibody. In this interpretation, sensitization by CRN antibody as well as neutralization by N antibody is thought to result from attachment of a single antibody molecule to the viral critical site. Alternative explanations, ascribing the mechanism of neutralization to steric hindrance of critical sites or to multiple hit of those sites by antibody, were denied by analyses of the present data.     

A continuous spectrophotometric assay for the Bacillus cereus phospholipase C using a thiophosphate substrate analog: evaluation of assay conditions and chromogenic agents

A thiophosphate analog of dioctanoylphosphatidylcholine has been used as the substrate in a continuous spectrophotometric assay for the Bacillus cereus phospholipase C. The reaction has been monitored at 412 nm using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and at 324 nm using 4,4'-dithiopuridine (DTP) as the respective thiol-reactive chromogenic agents. An optimum pH 6.0 was determined for the phospholipase C-catalyzed reaction which was independent of the chromogen utilized. Although the reaction rates observed when DTP was used were increased over those seen with DTNB, the rates were insensitive to changes in the concentration of the chromogen normally used for the assay. The initial velocities were shown to be linearly dependent upon the amount of enzyme added over at least a 20-fold enzyme concentration range. The dependency of the initial velocity on the concentration of substrate showed a discontinuity at [S] = 40 microM when either DTP or DTNB was used. This was consistent with a value of 56 microM estimated for the substrate critical micelle concentration by an independent measurement. While the substrate data measured using DTP could not be fit to existing equations based on Michaelis-Menten kinetics, the data obtained using DTNB as the chromogen conformed with the model proposed by Wells for enzymes acting upon micelle-forming substrates (M. A. Wells (1974, Biochemistry 13, 2248-2257). This allowed for the estimation of monomer and micelle Michaelis-Menten parameters for the B. cereus phospholipase C-catalyzed reaction with a thiophosphate analog substrate.     

Genotoxicity of 10 cigarette smoke condensates in four test systems: comparisons between assays and condensates

The particulate fraction of cigarette smoke, cigarette smoke condensate (CSC), is genotoxic in many short-term in vitro tests and is carcinogenic in rodents. However, no study has evaluated a series of CSCs prepared from a diverse set of cigarettes and produced with different smoking machine regimens in several short-term genotoxicity tests. Here we report on the genotoxicity of 10 CSCs prepared from commercial cigarettes that ranged from ultra-low tar per cigarette (< or =6.5 mg) to full flavor (>14.5 mg) as determined by the Federal Trade Commission (FTC) smoking regimen, a reference cigarette blended to be representative of a U.S. FTC-regimen low-tar cigarette, and experimental cigarettes constructed of single tobacco types. CSCs were tested in the presence of rat liver S9 in the Salmonella plate-incorporation assay using frameshift strains TA98 and YG1041; in micronucleus and comet assays in L5178Y/Tk(+/-) 7.3.2C mouse lymphoma cells, and in CHO-K(1) cells for chromosome aberrations. All 10 CSCs were mutagenic in both strains of Salmonella, and the rank order of their mutagenic potencies was similar. Their mutagenic potencies in Salmonella spanned 7-fold when expressed as rev/mug CSC but 158-fold when expressed as rev/mg nicotine; the range of genotoxic potencies of the CSCs in the other assays was similar regardless of how the data were expressed. All 10 CSCs induced micronuclei with a 3-fold range in their potency. All but one CSC induced DNA damage over a 20-fold range, and all but one CSC induced chromosome aberrations over a 4-fold range. There was no relation among the genotoxic potencies of the CSCs across the assays, and a qualitative advantage of the addition of the other assays to the Salmonella assay was not supported by our findings. Although consideration of nicotine levels may improve the relevance of the quantitative data obtained in the Salmonella and possibly comet assays, compensatory smoking habits and other factors may make the data from the assays used here have qualitative but not quantitative value in assessing risk of cigarette types and cigarette smoking to human health.     

Formation of pattern in regenerating tissue pieces of Hydra attenuata: II. Degree of proportion regulation is less in the hypostome and tentacle zone than in the tentacles and basal disc

The relative sizes of the various structures in Hydra attenuata were compared over a broad range of animal sizes to determine in detail the ability to regulate proportions during regeneration. The three components of the head, namely hypostome, tentacles, and tentacle zone from which the tentacles emerge, the body column, and the basal disc were all measured separately. Ectodermal cell number was used as the measure of size. The results showed that the basal disc proportioned exactly over a 40-fold size range, and the tentacle tissue proportioned exactly over a 20-fold size range. In contrast, the hypostome and tentacle zone proportioned allometrically. With decreasing size, the hypostome and tentacle zone became an increasing fraction of the animal at the expense of body tissue, and in the very smallest regenerates at the expense of tentacle tissue. In their current form, the reaction-diffusion models proposed for pattern regulation in hydra are not consistent with the data.     

Transfer Parameters—Are On-Site Data Really Better?

Transfer parameters for contaminants in the environment are notoriously variable. It is not unusual to measure a geometric standard deviation (GSD) of tenfold, so that the 5th and 95th percentiles are 10,000-fold apart. Despite many years of research, there remain large uncertainties, and often risk assessors will use on-site data for the transfer parameters. This Perspective assesses, using radionuclides as an example, whether a few site-specific measurements are more reliable than the more generic data compiled by researchers around the world. Using the example of plant/soil concentration ratios for several radionuclides, if one knew nothing about the soils and plants to be assessed, the GSD would be about 5.7-fold. If one had extensive knowledge about the soils and plants, the GSD would only diminish to about 3.2-fold. If a GSD of 3.2 is the level of residual natural variation to expect for plant/soil concentration ratios (the error term in a formal analysis of variance), then any on-site data that are within an order of magnitude of generic data could not be considered significantly different from the generic data. An appropriate conclusion in this case would be that unless on-site data are significantly different from the generic data, then the on-site data should not be used exclusively and the generic data should be considered suitable. The inherent variability of transfer parameters is so large that generic data may often be the best choice.     

Design and initial characterization of the SC-200 proteomics standard mixture

High-throughput (HTP) proteomics studies generate large amounts of data. Interpretation of these data requires effective approaches to distinguish noise from biological signal, particularly as instrument and computational capacity increase and studies become more complex. Resolving this issue requires validated and reproducible methods and models, which in turn requires complex experimental and computational standards. The absence of appropriate standards and data sets for validating experimental and computational workflows hinders the development of HTP proteomics methods. Most protein standards are simple mixtures of proteins or peptides, or undercharacterized reference standards in which the identity and concentration of the constituent proteins is unknown. The Seattle Children's 200 (SC-200) proposed proteomics standard mixture is the next step toward developing realistic, fully characterized HTP proteomics standards. The SC-200 exhibits a unique modular design to extend its functionality, and consists of 200 proteins of known identities and molar concentrations from 6 microbial genomes, distributed into 10 molar concentration tiers spanning a 1,000-fold range. We describe the SC-200's design, potential uses, and initial characterization. We identified 84% of SC-200 proteins with an LTQ-Orbitrap and 65% with an LTQ-Velos (false discovery rate?=?1% for both). There were obvious trends in success rate, sequence coverage, and spectral counts with protein concentration; however, protein identification, sequence coverage, and spectral counts vary greatly within concentration levels.     

Nucleotide aggregation in aqueous solution. A multicomponent self-diffusion study

The self-aggregation of the mononucleotides (AMP, CMP, GMP and UMP) and caffeine up to their solubility limit in 2H2O has been monitored through self-diffusion measurements, using the Fourier transform NMR pulsed-gradient spin-echo self-diffusion technique. The data were iteratively fitted to a number of aggregation models. It was concluded that the best agreement between simulations and experiment for the mononucleotides was obtained for a 'semi-isodesmic', indefinite aggregation model (also known as a Type III SEK or cooperative indefinite self-association model), where the first (dimerization) aggregation constant is a magnitude lower than those for the higher aggregation steps. Typical values were 0.4 and 6 l mol-1, respectively. Under these conditions, the main fraction of solute is monomeric throughout the concentration range and the distribution of higher oligomers is very broad. Caffeine self-aggregation is clearly different and is consistent with several aggregation models. The mixed aggregation of caffeine (at a low total concentration) and the mononucleotides was successfully monitored in an extension of the basic study. It was found that caffeine binding to mononucleotide aggregates increases in the series UMP, CMP, GMP and AMP.     

Biosorption of nickel(II) ions by baker's yeast: kinetic, thermodynamic and desorption studies

In this study, the biosorption of nickel(II) ion on deactivated protonated yeast was investigated as a function of temperature at different initial metal ion concentrations. The effect of temperature on the sorption was more significant at lower nickel(II) ion concentrations compared to higher concentrations. The protonated yeast biomass exhibited the highest nickel(II) ion uptake capacity at 27 degrees C at an initial nickel(II) ion concentration of 400mg/l and an initial pH of 6.75. The biosorption capacity decreased from 9.8 to 9.3mg/g at an initial nickel(II) ion concentration of 400mg/l, while at a lower initial concentration of 100mg/l, it decreased from 8.2 to 4.9 mg/g, as the temperature was increased from 27 degrees C to 60 degrees C. The equilibrium data fit better to the Freundlich and Redlich-Peterson isotherm models compared to the Langmuir model in the concentration range studied (10-400mg/l). Kinetic models applied to the sorption data at different temperatures showed that nickel(II) ion uptake process followed the pseudo-second order rate model and the adsorption rate constants decreased with increasing temperature. The activation energy of biosorption (Ea) was determined to be -13.3 kJ/mol using the pseudo-second order rate constants. The results indicated that the biosorption of nickel(II) ion on to baker's yeast was spontaneous and exothermic in nature. Desorption studies revealed that the protonated yeast biomass can be regenerated using 0.1N HCl and reused.