Determination, by the electro-optical method, of the number of undamaged bacterial cells after extreme exposures

The results of electro-optical measurements can be used to determine rapidly how the number of intact cells decreases before and after the action of certain extreme factors. The experiments were conducted with suspensions of Escherichia coli and Bacillus thuringiensis cells subjected to the action of heat, toluene, ethanol, and freezing. Electro-optical measurements can make it possible to assay the relative number of cells with both lethal and sublethal damages.     

Purification and secondary structure determination of simian immunodeficiency virus p27

We have developed a novel method for the expression and purification of p27, the major core protein of simian immunodeficiency virus. Circular dichroism measurements of purified p27 were used to determine the relative amounts of alpha-helix, beta-sheet and unordered secondary structural elements. These empirically determined values appear to be inconsistent with previously published theoretical models based on homology comparisons.     

Synthesis of a thymidine phosphoramidite labelled with 13C at C6: relaxation studies of the loop region in a 13C labelled DNA hairpin.

A thymidine phosphoramidite labelled at C6 with 13C has been synthesized, and incorporated into a synthetic oligonucleotide, d(CGCGT*T*GT*T*CGCG), which adopts a hairpin conformation. NMR relaxation measurements indicate that internal motion may be present in the loop region of the oligonucleotide. The relaxation behavior of a the C6 carbon in a model compound, N,N-1,3 dimethylthymine is examined in detail as a function of magnetic field strength to determine relative contributions of various mechanisms to the relaxation. The relaxation behaviour of the labelled carbons in the oligonucleotide is discussed in relation to these measurements.     

A simple method to determine the water activity of ethanol-containing samples

The water activity (aw) of microbial substrates, biological samples, and foods and drinks is usually determined by direct measurement of the equilibrium relative humidity above a sample. However, these materials can contain ethanol, which disrupts the operation of humidity sensors. Previously, an indirect and problematic technique based on freezing-point depression measurements was needed to calculate the aw when ethanol was present. We now describe a rapid and accurate method to determine the aw of ethanol-containing samples at ambient temperatures. Disruption of sensor measurements was minimized by using a newly developed, alcohol-resistant humidity sensor fitted with an alcohol filter. Linear equations were derived from aw measurements of standard ethanol-water mixtures, and from Norrish's equation, to correct sensor measurements. To our knowledge, this is the first time that electronic sensors have been used to determine the aw of ethanol-containing samples. Copyright 1999 John Wiley & Sons, Inc.     

Conservation of the relative tRNA composition in healthy and cancerous tissues

Elongation in protein translation is strongly dependent on the availability of mature transfer RNAs (tRNAs). The relative concentrations of the tRNA isoacceptors determine the translation efficiency in unicellular organisms. However, the degree of correspondence of codons and the relevant tRNA isoacceptors serves as an estimator for translation efficiency in all organisms. In this study, we focus on the translational capacity of the human proteome. We show that the correspondence between the codon usage and tRNAs can be improved by combining experimental measurements with the genomic copy number of isoacceptor groups. We show that there are technologies of tRNA measurements that are useful for our analysis. However, fragments of tRNAs do not agree with translational capacity. It was shown that there is a significant increase in the absolute levels of tRNA genes in cancerous cells in comparison to healthy cells. However, we find that the relative composition of tRNA isoacceptors in healthy, cancerous, or transformed cells remains almost identical. This result may indicate that maintaining the relative tRNA composition in cancerous cells is advantageous via its stabilizing of the effectiveness of translation.     

Direct mechanics assessment of elastic symmetries and properties of trabecular bone architecture

A method is presented to find orthotropic elastic symmetries and constants directly from the elastic coefficients in the overall stiffness matrix of trabecular bone test specimens. Contrary to earlier developed techniques, this method does not require pure orthotropic behavior or additional fabric measurements. The method uses high-resolution computer reconstructions of trabecular bone specimens as input for large-scale FE-analyses to determine all the 21 elastic coefficients in the overall stiffness matrix of the specimen, using a direct mechanics approach. An optimization procedure is then used to find the coordinate transformation that yields the best orthotropic representation of this matrix. The method is illustrated here relative to two trabecular bone specimens. The techniques developed here can be used to obtain a complete characterization of the mechanical properties of trabecular architecture. With the development of in vivo reconstruction techniques, even in vivo measurements will be possible.     

Biophysical Model of the Spatial Heterogeneity of Myocardial Flow

The blood flow in the myocardium has significant spatial heterogeneity. The objective of this study was to develop a biophysical model based on detailed anatomical data to determine the heterogeneity of regional myocardial flow during diastole. The model predictions were compared with experimental measurements in a diastolic porcine heart in the absence of vessel tone using nonradioactive fluorescent microsphere measurements. The results from the model and experimental measurements showed good agreement. The relative flow dispersion in the arrested, vasodilated heart was found to be 44% and 48% numerically and experimentally, respectively. Furthermore, the flow dispersion was found to have fractal characteristics with fractal dimensions (D) of 1.25 and 1.27 predicted by the model and validated by the experiments, respectively. This validated three-dimensional model of normal diastolic heart will play an important role in elucidating the spatial heterogeneity of coronary blood flow, and serve as a foundation for understanding the interplay between cardiac mechanics and coronary hemodynamics.     

Anticancer Metal Complexes: Synthesis and Cytotoxicity Evaluation by the MTT Assay

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; therefore their preparation should be conducted under an inert atmosphere. Evaluation of the anticancer activity of these complexes can be achieved by the MTT assay.The MTT assay is a colorimetric viability assay based on enzymatic reduction of the MTT molecule to formazan when it is exposed to viable cells. The outcome of the reduction is a color change of the MTT molecule. Absorbance measurements relative to a control determine the percentage of remaining viable cancer cells following their treatment with varying concentrations of a tested compound, which is translated to the compound anticancer activity and its IC₅₀ values. The MTT assay is widely common in cytotoxicity studies due to its accuracy, rapidity, and relative simplicity.Herein we present a detailed protocol for the synthesis of air sensitive metal based drugs and cell viability measurements, including preparation of the cell plates, incubation of the compounds with the cells, viability measurements using the MTT assay, and determination of IC₅₀ values.     

Streaming potential—a general affinity sensor

Electrokinetic phenomena, such as streaming potential or streaming current, have so far been used for studies of unspecific adsorption of ionic compounds on various materials. This paper shows that streaming potential measurements can also be used for studies of biospecific interactions.Several different interactions were studied, e.g. lectin-carbohydrate, IgG-Protein A. Regardless of the type of interaction an increasing change in streaming potential was obtained with increasing concentration of the interacting molecule. The authors also observed a correlation between streaming potential and affinity constants for carbohydrate-induced desorption of Concanavalin A from partially hydrolyzed Sephadex G50. In conclusion, it is shown that streaming potential measurements can be used to study molecular interactions, and to determine concentrations and relative binding constants of interacting molecules.     

Reliability of anthropometric methods and replicate measurements

The Spearman-Brown Prophesy formula, derived from psychometrics, may be used in anthropometric studies to describe the relationship between the intraclass reliability coefficient for a single measurement and the reliability resulting from the mean of replicate measurements. This theory may be applied to determine expected reliabilities of anthropometric protocols using replicate measurements and to determine the numbers of replicate measurements necessary to achieve desired levels of reliability.     

Reproducibility of geometrical acquisition of intra-thoracic organs of children on CT scans

This paper analyses geometry of intra-thoracic organs from computed tomography (CT) scans performed on 20 children aged from 4 months to 16 years. A set of two measurements on lungs and heart were performed by the same observer. A third set was performed by a second observer. Thus, the intra- and inter-observer relative deviation of measurements was analysed. Multiple regressions were used in order to study the relationship between the CT properties (scanner, voltage, dose, pixel size, slice increment) and the relative deviation of measurements. There is a very low systematic intra- and inter-observer bias in measurements except for the volume of the heart. None of the CT data properties has a significant influence on the relative deviation of measurement. In the present paper, the measurements and 3D reconstruction protocol described can be applied to characterise the growth of the intra-thoracic organs.     

Reproducibility of geometrical acquisition of intra-thoracic organs of children on CT scans

This paper analyses geometry of intra-thoracic organs from computed tomography (CT) scans performed on 20 children aged from 4 months to 16 years. A set of two measurements on lungs and heart were performed by the same observer. A third set was performed by a second observer. Thus, the intra- and inter-observer relative deviation of measurements was analysed. Multiple regressions were used in order to study the relationship between the CT properties (scanner, voltage, dose, pixel size, slice increment) and the relative deviation of measurements. There is a very low systematic intra- and inter-observer bias in measurements except for the volume of the heart. None of the CT data properties has a significant influence on the relative deviation of measurement. In the present paper, the measurements and 3D reconstruction protocol described can be applied to characterise the growth of the intra-thoracic organs.     

Muscle geometry affects accuracy of forearm volume determination by magnetic resonance imaging (MRI)

Upper extremity musculoskeletal modeling is becoming increasingly sophisticated, creating a growing need for subject-specific muscle size parameters. One method for determining subject-specific muscle volume is magnetic resonance imaging (MRI). The purpose of this study was to determine the validity of MRI-derived muscle volumes in the human forearm across a variety of muscle sizes and shapes. Seventeen cadaveric forearms were scanned using a fast-spoiled gradient echo pulse sequence with high isotropic spatial resolution (1mm(3) voxels) on a 3T MR system. Pronator teres (PT), extensor carpi radialis brevis (ECRB), extensor pollicis longus (EPL), flexor carpi ulnaris (FCU), and brachioradialis (BR) muscles were manually segmented allowing volume to be calculated. Forearms were then dissected, muscles isolated, and muscle masses obtained, which allowed computation of muscle volume. Intraclass correlation coefficients (ICC(2,1)) and absolute volume differences were used to compare measurement methods. There was excellent agreement between the anatomical and MRI-derived muscle volumes (ICC = 0.97, relative error = 12.8%) when all 43 muscles were considered together. When individual muscles were considered, there was excellent agreement between measurement methods for PT (ICC = 0.97, relative error = 8.4%), ECRB (ICC = 0.93, relative error = 7.7%), and FCU (ICC = 0.91, relative error = 9.8%), and fair agreement for EPL (ICC = 0.68, relative error = 21.6%) and BR (ICC = 0.93, relative error = 17.2%). Thus, while MRI-based measurements of muscle volume produce relatively small errors in some muscles, muscles with high surface area-to-volume ratios may predispose them to segmentation error, and, therefore, the accuracy of these measurements may be unacceptable.     

Integrated function of a kinetic proofreading mechanism: steady-state analysis testing internal consistency of data obtained in vivo and in vitro and predicting parameter values

Experimental measurements of the kinetic mechanism involving isoleucyl-tRNA synthetase proofreading valyl-tRNAIle in Escherichia coli have been incorporated into the conventional Michaelis-Menten model for this system. The model was subjected to a detailed mathematical analysis in the steady state. The results of this analysis provide an excellent illustration of the value of integrating fragmentary data into a model of the intact system. (1) Such integration provides a rigorous test for consistency of the individual measurements. For the above synthetase system, the published experimental data were found to be internally inconsistent. (2) Such integration predicts which experimental data are most suspect. In this case, one of the three most questionable measurements, the isoleucine pool size in vivo, was found upon reexamination to be in error by 10-15-fold. Correction of this error produced a self-consistent set of parameter values. (3) The integrated analysis provides predictions for various parameter values. In many cases, these predictions provide estimates for parameter values that are difficult to determine directly or that have yet to be measured experimentally. (4) A sensitivity analysis provides an indication of the relative importance of various parameter values and, hence, an indication of where future experimental effort might be focused most profitably.     

The localization of the local anesthetic tetracaine in phospholipid vesicles: A fluorescence quenching and resonance energy transfer study

Fluorescence quenching and resonance energy transfer have been used to determine the localization of the local anesthetic tetracaine in vesicles composed of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) as a function of both temperature and ionic strength. The fluorescence behaviour of tetracaine in vesicles can be attributed to its different partition coefficients in acid and basic solution, in gel phase and fluid phase vesicles, respectively. Using both steady-state and time-resolved fluorescence measurements we show that a saturable binding rather than a partitioning model holds for the interaction of tetracaine with gel phase bilayers. The relative quenching efficiencies of the series of n-AS dyes depend on the phase state of the bilayer and suggest a deeper incorporation of tetracaine in fluid phase than in gel phase membranes. Resonance energy transfer measurements support the view that tetracaine is incorporated predominantly in the region of the 9-AS chromophore in DMPC-bilayers.     

Myocardial infarct size measurement in the mouse chronic infarction model: comparison of area- and length-based approaches.

Efficacy of potential treatments for myocardial infarction (MI) is commonly assessed by histological measurement of infarct size in rodent models. In experiments involving an acute MI setting, measurement of the infarcted area in tissue sections of the left ventricle is a standard approach to determine infarct size. This approach has also been used in the chronic infarct setting to measure infarct area several weeks post-MI. We tested the hypothesis that, because wall thinning is known to occur in the chronic setting, the area measurement approach would be less appropriate. We compared infarct measurements in tissue sections based on 1) infarct area, 2) epicardial and endocardial infarct arc lengths, and 3) midline infarct arc length. Infarct sizes from all three measurement approaches correlated significantly with left ventricular ejection fraction and wall motion abnormality. However, the infarct size values derived from the area measurement approach were significantly smaller than those from the other two measurement approaches, and the range of values obtained was compressed 0.4-fold. The midline method allowed detection of the expected size differences between infarcts of variable severity resulting from proximal vs. distal ligation of the coronary artery. Segmental infarct size was correlated with segmental wall motion abnormality. We conclude that both area- and length-based measurements can be used to determine relative infarct size over a wide range of severity, although the area-based measurements are substantially more compressed due to wall thinning, and that the estimation of infarct midlines is a simple, reliable approach to infarct size assessment.     

Effects of chemistry, manufacturing and concentration of the dye basic fuchsin regarding its use in quantitative cytophotometry

Four separately manufactured preparations of basic fuchsin were compared to determine the effects of concentration, chemistry, and the manufacturing process for their quantitative value in the nuclear Feulgen reaction. In order to make the necessary comparisons, the two wavelength method of quantitative cytophotometry was employed to analyse each stain application regarding DNA measurements. Chicken erythrocytes, and myxamoebae and plasmodia of Didymium iridis were employed as experimental tissues. Results indicated that all four preparations of the stains yielded acceptable and valid quantitative data in relative DNA values. Differing manufacturers and dye concentrations had no appreciable effect on relative quantitative measurements. However, the maximum staining intensity was affected by differences both in the chemical structure of the individual stains and by the products from various manufacturers. The maximum dye intensity and accurate quantitative absolute values for DNA measurement were best obtained by the use of basic fuchsin having the same colour index (Cl) 42510 as that manufactured by Fisher Scientific Incorporated.     

Assessing transpiration in the tussock grass Stipa tenacissima L.: the crucial role of the interplay between morphology and physiology

Plant transpiration has a key role on both plant performance and ecosystem functioning in arid zones, but realistic estimates at appropriate spatial-temporal scales are scarce. Leaf and tiller morphology and crown architecture were studied together with leaf physiology and whole plant water balance in four individual plants of Stipa tenacissima of different sizes to determine the relative influence of processes taking place at different spatial and temporal scales on whole plant transpiration. Transpiration was estimated in potted plants by leaf-level gas exchange techniques (infrared gas analyzer and porometer), by sap flow measurements, and by integrating leaf physiology and crown architecture with the 3-D computer model Yplant. Daily transpiration of each individual plant was monitored using a gravimetric method, which rendered the reference values. Leaves on each individual plant significantly varied in their physiological status. Young and green parts of the leaves showed five times higher chlorophyll concentration and greater photosynthetic capacity than the senescent parts of the foliage. Instantaneous leaf-level transpiration measurements should not be used to estimate plant transpiration, owing to the fact that extrapolations overestimated individual transpiration by more than 100%. Considering leaf age effects and scaling the estimates according to the relative amount of each foliage category reduced this difference to 46% though it was still significantly higher than gravimetric measurements. Sap flow calculations also overestimated tussock transpiration. However, 3-D reconstruction of plants with Yplant and transpiration estimates, considering both the physiological status and the daily pattern of radiation experienced by each individual leaf section within the crown, matched the gravimetric measurements (differences were only 4.4%). The complex interplay of leaf physiology and crown structure must be taken into account in scaling up plant transpiration from instantaneous, leaf-level measurements, and our study indicates that transpiration of complex crowns is easily overestimated.     

Comparative craniofacial variation in Navajo Indians and North American Caucasians

Landmarks digitized from lateral cephalometric radiographs of 107 Navajo Indians between 10 and 12 years of age were analyzed to determine coefficients of variation or standard deviations for 38 cephalometric measurements. These values were compared with the same measures of variation for identical measurements on North American whites derived from the Michigan and Philadelphia Growth Studies. For the majority of variables, there were no differences between groups. Variation for the genetically and environmentally isolated Navajo Indians was the same as that of the highly diverse Caucasian samples. However, measurements of upper, lower, and total anterior facial height (N-ANS, ANS-Me, and N-Me, respectively) indicate that these features are substantially less variable in Navajo Indians relative to the Michigan and Philadelphia populations.     

Assessing transpiration in the tussock grass Stipa tenacissima L.: the crucial role of the interplay between morphology and physiology

Plant transpiration has a key role on both plant performance and ecosystem functioning in arid zones, but realistic estimates at appropriate spatial-temporal scales are scarce. Leaf and tiller morphology and crown architecture were studied together with leaf physiology and whole plant water balance in four individual plants of Stipa tenacissima of different sizes to determine the relative influence of processes taking place at different spatial and temporal scales on whole plant transpiration. Transpiration was estimated in potted plants by leaf-level gas exchange techniques (infrared gas analyzer and porometer), by sap flow measurements, and by integrating leaf physiology and crown architecture with the 3-D computer model Yplant. Daily transpiration of each individual plant was monitored using a gravimetric method, which rendered the reference values. Leaves on each individual plant significantly varied in their physiological status. Young and green parts of the leaves showed five times higher chlorophyll concentration and greater photosynthetic capacity than the senescent parts of the foliage. Instantaneous leaf-level transpiration measurements should not be used to estimate plant transpiration, owing to the fact that extrapolations overestimated individual transpiration by more than 100%. Considering leaf age effects and scaling the estimates according to the relative amount of each foliage category reduced this difference to 46% though it was still significantly higher than gravimetric measurements. Sap flow calculations also overestimated tussock transpiration. However, 3-D reconstruction of plants with Yplant and transpiration estimates, considering both the physiological status and the daily pattern of radiation experienced by each individual leaf section within the crown, matched the gravimetric measurements (differences were only 4.4%). The complex interplay of leaf physiology and crown structure must be taken into account in scaling up plant transpiration from instantaneous, leaf-level measurements, and our study indicates that transpiration of complex crowns is easily overestimated.