Alternative magnetic field exposure metrics: relationship to TWA, appliance use, and demographic characteristics of children in a leukemia survival study.

The ongoing Childhood Leukemia Survival Study is examining the possible association between magnetic field exposure and survival of children with newly diagnosed acute lymphocytic leukemia (ALL). We report the results of the first year 24 h personal magnetic field monitoring for 356 US and Canadian children by time weighted average TWA and alternative exposure metrics. The mean TWA of 0.12 microT was similar to earlier personal exposure studies involving children. A high correlation was found between 24 h TWA and alternative metrics: 12 h day TWA, 12 night TWA, geometric mean, 95th percentile value, percentage time over 0.2 and 0.3 microT, and an estimate of field stability (Constant Field Metric). Two measures of field intermittency, rate of change metric (RCM) and standardized rate of change metric (RCMS), were not highly correlated with TWA. The strongest predictor of TWA was location of residence, with highest TWAs associated with urban areas. Residence in an apartment, lower paternal educational level, and residential mobility were also associated with higher TWAs. There were no significant differences in the appliance use patterns of children with higher TWA values. Children with the highest field intermittency (high RCM) were more likely to sit within 3 feet of a video game attached to the TV. Our results suggest that 24 h TWA is a representative metric for certain patterns of exposure, but is not highly correlated with two metrics that estimate field intermittency.     

Improved primer sequences for the mitochondrial ND1, ND3/4 and ND5/6 segments in salmonid fishes: application to RFLP analysis of Atlantic salmon

New specific primers for the mtDNA segments ND1, ND3/4 and ND5/6 designed from the rainbow trout sequence, improved PCR amplification for salmonid fishes. RFLP analysis revealed restriction site variation for all three segments in Atlantic salmon. Eleven haplotypes were detected in a screening of 30 individuals from four European populations.     

Simplified method for the collection, storage, and comet assay analysis of DNA damage in whole blood

Single-cell gel electrophoresis (comet assay) is one of the most common methods used to measure oxidatively damaged DNA in peripheral blood mononuclear cells (PBMC), as a biomarker of oxidative stress in vivo. However, storage, extraction, and assay workup of blood samples are associated with a risk of artifactual formation of damage. Previous reports using this approach to study DNA damage in PBMC have, for the most part, required the isolation of PBMC before immediate analysis or freezing in cryopreservative. This is very time-consuming and a significant drain on human resources. Here, we report the successful storage of whole blood in ~ 250 μl volumes, at − 80 °C, without cryopreservative, for up to 1 month without artifactual formation of DNA damage. Furthermore, this blood is amenable for direct use in both the alkaline and the enzyme-modified comet assay, without the need for prior isolation of PBMC. In contrast, storage of larger volumes (e.g., 5 ml) of whole blood leads to an increase in damage with longer term storage even at − 80 °C, unless a cryopreservative is present. Our “small volume” approach may be suitable for archived blood samples, facilitating analysis of biobanks when prior isolation of PBMC has not been performed.     

The use of a multipoint inoculation method to perform lysostaphin, lysozyme and glycerol-erythromycin tests for the differentiation of staphylococci and micrococci

A multipoint inoculation procedure for performing lysostaphin, lysozyme and glycerol-erythromycin tests for the separation of staphylococci from micrococci is described. The method enabled the tests to be carried out more rapidly and conveniently than with conventional methods.     

Evaluation and use of a diffusion-controlled sampler for determining chemical and dissolved oxygen gradients at the sediment-water interface

Field and laboratory evaluations were made of a simple, inexpensive diffusion-controlled sampler with ports on two sides at each interval which incorporates 0.2-m polycarbonate membrane to filter samples in situ. Monovalent and divalent ions reached 90% of equilibrium between sampler contents and the external solution within 3 and 6 hours, respectively. Sediment interstitial water chemical gradients to depths of tens of centimeters were obtained within several days after placement. Gradients were consistent with those determined from interstitial water obtained by centrifugation of adjacent sediment. Ten milliliter sample volumes were collected at 1-cm intervals to determine chemical gradients and dissolved oxygen profiles at depth and at the interface between the sediment and water column. The flux of dissolved species, including oxygen, across the sediment-water interface can be assessed more accurately using this sampler than by using data collected from benthic cores.     

磷脂酸含量测定法评价烟曲霉孢子对肺上皮细胞磷脂酶D活性的影响

目的 优化检测烟曲霉刺激A549细胞后磷脂酸（phosphotidic acid,PA）含量变化的方法,间接反应细胞内磷脂酶D（Phospholipase D,PLD）活性变化.方法 建立烟曲霉ATCC13073刺激肺上皮细胞模型;采用甲醇氯仿法提取胞内脂质;用改良的磷脂酸含量测定法测定PA标准品和细胞内PA水平变化规律.结果 PA标准品在5～ 250 μmol/L呈线性关系;经膨胀孢子刺激后,肺上皮细胞内PA含量显著升高,休眠孢子在这一过程中对肺上皮细胞内PA含量无明显作用.结论 改良的PA测量法能快速、稳定而有效地测定细胞内的PLD活性.烟曲霉膨胀孢子能显著激活肺上皮细胞内的PLD活性.     

A method for determining the positions of polar hydrogens added to a protein structure that maximizes protein hydrogen bonding.

An automated method for the optimal placement of polar hydrogens in a protein structure is described. This method treats the polar, side chain hydrogens of lysine, serine, threonine, and tyrosine and the amino terminus of a protein. The program, called NETWORK, divides the potential hydrogen-bonding pairs of a protein into groups of interacting donors and acceptors. A search is conducted on each of the local groups to find an arrangement which forms the most hydrogen bonds. If two or more arrangements have the same number of hydrogen bonds, the arrangement with the shortest set of hydrogen bonds is selected. The polar hydrogens of the histidyl side chain are specifically treated, and the ionization state of this residue is allowed to change, if this change results in additional hydrogen bonds for the local group. The program will accept Protein Data Bank as well as Biosym-format coordinate files. Input and output routines can be easily modified to accept other coordinate file formats. The predictions from this method are compared to known hydrogen positions for bovine pancreatic trypsin inhibitor, insulin, RNase-A, and trypsin for which the neutron diffraction structures have been determined. The usefulness of this program is further demonstrated by a comparison of molecular dynamics simulations for the enzyme cytochrome P-450cam with and without using NETWORK.     

Simplified method for purification of colostrum to obtain secretory component of immunoglobulin A, using secretory component as a reference protein in tracheal aspirate fluid

Many studies employ bronchoalveolar lavage fluid for assessment of biologically active substances secreted from the lung. However, investigators continue to search for a useful reference standard to correct for the inevitable but variable degree of dilution of this fluid. The glycoprotein, soluble secretory component of IgA, may serve as a valid reference protein. We report a simplified method for the purification of secretory component from colostrum. Soluble secretory component was isolated from human colostrum using serial centrifugation, size-exclusion fractionation and ion-exchange chromatography. Secretory component rich fractions were assayed by enzyme immunoassay. They were also evaluated for total amino acid content and distribution and sequence determination with satisfactory agreement with published results. We then demonstrated that soluble secretory component concentration in tracheal aspirate fluid did not correlate with either albumin or with total protein measured in the same samples. Therefore, we conclude that the secretory component of IgA serves as a useful reference marker because its use may avoid errors resulting from leakage of plasma proteins into epithelial lining fluid. Advantages of this method for establishing a standard for secretory component include ready availability of soluble secretory component, simplicity of the method and relative rapidity of the techniques.     

A simple and rapid spectrofluorimetric method for determining the pharmacokinetics and metabolism of rhaponticin in rat plasma,feces and urine using a cerium probe

Rhaponticin (RH) demonstrates a variety of pharmacological activities, including antitumor, antithrombotic and antioxidant effects. It is essential to establish a simple, rapid and reliable analytical method for determining the pharmacokinetics of RH. A simple cerium ion (Ce³⁺) probe method was developed and validated to determine RH in rat plasma, feces and urine. The fluorescence intensities of the cerium ion (Ce³⁺) were quenched by addition of RH, along with a remarkable red shift. Spectral data revealed that fluorescence quenching of Ce³⁺ by RH was due to the formation of a Ce³⁺–RH complex. Using to the Stern–Volmer equation, the binding parameters for interactions between Ce³⁺ and RH were obtained. Based on these, a rapid and simple spectrofluorimetric method was developed to determine the metabolism and pharmacokinetics of RH using a Ce³⁺ probe. The assay was linear over the concentration range of 0.11–9.52, 0.25–8.87 and 0.18–9.10 μM for plasma, feces and urine, respectivelyand RH recoveries were found to be 98.24 ± 0.8, 97.78 ± 1.2 and 97.54 ± 0.8% for plasma, feces and urine, respectively. The relative standard deviations were < 9.5%. The spectrofluorimetric method was simple and rapid for quantitative determination of RH and its metabolism, and was affordable for most laboratories because of the fluorescence spectroscopy and low equipment cost. These pharmacokinetic, bioavailability and metabolism studies of RH will provide helpful information for the development of suitable dosage forms and clinical references on rational administration. Copyright © 2013 John Wiley & Sons, Ltd.     

Sensitive fluorimetric method for the determination of putrescine,spermidine and spermine by high-performance liquid chromatography and its application to human blood

A fast and sensitive method for the determination of putrescine, spermidine and spermine by high-performance liquid chromatography is described. These compounds are converted to their fluorescent dansyl derivatives and are separated by a reversed-phase chromatographic system (Micropak CH-10) with water and acetonitrile as mobile phase. The sensitivity of the method is 30 pmoles.The application of the method to the determination of polyamines in blood is described. It was found that most of the polyamines circulating in blood are localized in the erythrocytes, their content in normal human blood being spermidine 14.1 ± 3.1, and spermine 8.4 ± 2.8 nmoles/ml packed erythrocytes. Putrescine is not present in normal human erythrocytes. The polyamine level in serum is less than 0.1 nmole/ml.The polyamine content of the erythrocytes from patients with malignant neoplasm was significantly elevated.     

A combined method for determining inhibition type, kinetic parameters, and inhibition coefficients for aerobic cometabolism of 1,1,1-trichloroethane by a butane-grown mixed culture.

A combined method for determining inhibition type, kinetic parameters, and inhibition coefficients is developed and presented. The method was validated by applying it to data obtained from batch kinetics of the aerobic cometabolism of 1,1,1-trichloroethane (1,1,1-TCA) by a butane-grown mixed culture. The maximum degradation rates (k(max)) and half-saturation coefficients (K(s)) were independently determined in single compound tests, and compared with those obtained from inhibition tests. The inhibition type was determined using direct linear plots at various substrate and inhibitor concentrations. Kinetic parameters (k(max) and K(s)) and inhibition coefficients (K(ic) and K(iu)) were determined by nonlinear least squares regression (NLSR) fits of the inhibition model determined from the direct linear plots. Initial guesses of the kinetic parameters for NLSR were determined from linearized inhibition equations that were derived from the correlations between apparent maximum degradation rates (k(app)(max)) and/or the apparent half-saturation coefficient (K(app)(s)) and the k(max), K(s), and inhibitor concentration (I(L)) for each inhibition equation. Two different inhibition types were indicated from the direct linear plots: competitive inhibition of 1,1,1-TCA on butane degradation, and mixed inhibition of 1,1,1-TCA transformation by butane. Good agreement was achieved between independently measured k(max) and K(s) values and those obtained from both NLSR and the linearized inhibition equations. The initial guesses of all the kinetic parameters determined from linear plots were in the range of the values estimated from NLSR analysis. Overall the results show that use of the direct linear plot method to identify the inhibition type, coupled with initial guesses from linearized plots for NLSR analysis, results in an accurate method for determining inhibition types and coefficients. Detailed studies with pure cultures and purified enzymes are needed to further demonstrate the utility of this method.     

A comparison of the different methods available for determining BCG-macrophage interactions in vitro, including a new method of colony counting in broth

Abstract Different methods of determining BCG viability based on colony forming unit (CFU) counting and radio-isotope labelling were comparatively assessed. These included radio-isotope labelling with [³H]uracil, [³H]uridine, [³H]glycerol, and CFU counting, by both agar plate dilution, and microcolony counting in broth. The sensitivity ranges of the different techniques were determined in both macrophage-free and macrophage-treated systems and used to assess the anti-mycobacterial potential of human monocyte-derived macrophages following BCG infection.     

Development and evaluation of a sensitive,Diffusive Gradients in Thin-Films (DGT) method for determining microcystin-LR concentrations in freshwater and seawater

A Diffusive Gradients in Thin-Films (DGT) passive sampling technique was developed for microcystin-LR (MC-LR), one of the most common and toxic microcystins. Three types of resins (HP20, SP700, and XAD18) were evaluated for MC-LR uptake kinetics, capacities, and extraction efficiencies and simple procedures were developed for determining MC-LR concentration in binding disc extracts by Adda-ELISA (U.S. EPA Method 546). The XAD18-DGT/Adda-ELISA method had a 7-d deployment time detection limit of ≈0.05 μg/L and capacity of >250 μg/L of MC-LR in water samples which encompass U.S. EPA and WHO advisory concentrations for drinking and recreational waters. The XAD18-DGT/Adda-ELISA method determined time-averaged MC-LR concentrations in waters with wide ranging pH (4.9–8.3) and ionic strength (0.04–0.8 M) under well-stirred and quiescent conditions with 90–101% accuracy. In addition to high sensitivity and accuracy, the method is simple, inexpensive, and applicable for determining MC-LR and related MCs concentrations in waterbodies with wide ranging chemical characteristics and hydrodynamic conditions.     

Convenient method for determining the absolute configuration of chiral alcohols with racemic 1H NMR anisotropy reagent,M(alpha)NP acid: use of HPLC-CD detector

A convenient method for determining the absolute configuration of chiral secondary alcohols using the racemic NMR anisotropy reagent, (+/-)-2-methoxy-2-(1-naphthyl)propionic acid [(+/-)-M(alpha)NP acid], and an HPLC-CD detector was developed. The method was successfully applied to some chiral alcohols derived from (-)-alpha-santonin.     

Factors determining the host plant range of the phytophagous mite, Tetranychus urticae (Acari: Tetranychidae): a method for quantifying host plant acceptance

The host plant acceptance of the phytophagous mite Tetranychus urticae was experimentally quantified. Host plant acceptance is described as the proportion of adult females settling on the test plant on which they have been placed. On the other hand, the host plant suitability of T. urticae on different plant species is expressed as the mean number of eggs produced by the females within 5 days (hereafter 'fecundity'). An inbred T. urticae line was tested with regard to host plant acceptance and fecundity on 11 potential host plants. These two variables were positively correlated across host plants; host plant species on which the fecundity was low were also those on which females settled less readily compared to host plants with high fecundity. The characteristics of host plant acceptance of the T. urticae are discussed in light of their potential food resource under natural conditions.     

Vocal individuality in the roding calls of Woodcock Scolopax rusticola and their use to validate a survey method

The Woodcock Scolopax rusticola is a difficult species to survey in the breeding season because of its cryptic plumage and, with the exception of courtship flights, secretive behaviour. Sparse data from general bird surveys prohibit reliable estimation of population sizes and trends, and a new species-specific method to provide baseline information on population status and to enable reliable future monitoring is required. Counts of displaying, or 'roding', males at dawn or dusk potentially provide such a method but their value is unclear because it is impossible for an observer to distinguish different birds. We examined the vocal individuality of 39 roding males and were able to attribute calls to individuals correctly in 95% of cases on the basis of five parameters measured from spectrograms. Using calls, we determined the number of individual males at 43 sites. Roding activity differed between males at one site, with the two most active birds accounting for, on average, 55 and 27% of passes. There was no evidence of sequential roding by different males. We quantified the relationship between numbers of males and numbers of passes of roding birds during a 1-h period at dusk. This relationship was not affected by region, month, habitat or woodland size class. We conclude that counts of roding males provide a suitable index for monitoring populations of this species.     

Design of enhanced agonists through the use of a new virtual screening method: application to peptides that bind class I major histocompatibility complex (MHC) molecules

A new screening procedure is described that uses docking calculations to design enhanced agonist peptides that bind to major histocompatibility complex (MHC) class I receptors. The screening process proceeds via single mutations of one amino acid at the positions that directly interact with the MHC receptor. The energetic and structural effects of these mutations have been studied using fragments of the original ligand that vary in length. The results of these docking studies indicate that the mutant affinity ranking of long peptides can be practically reproduced with a screening approach performed using fragments of six residues. Fragments of four and five residues could mimic, in some cases, the structural arrangement of the side chains of the full-length peptide. We have compared the structural and energetic results of the docking calculations with experimental data using three unrelated ligand peptides that differ greatly in their affinity for the MHC complex. Analysis of the affinity of the fragments led to the identification of three important parameters in the construction of fragments that mimic the structural and energetic properties of the full-length ligand: the length of the fragment; its intermolecular energy; and the number and localization, internal or terminal, of the anchor residues. The results of this new peptide-design methodology have been applied to suggest new peptides derived from the MUC1-8 peptide that could be used as murine vaccines that trigger the immune response through the MHC class I protein H-2K(b).     

A method for determining the relative effect of ligands on A-T and G-C base pairs in DNA: applications to metal ions, protons and two amino acids.

A new method is described for the study of specific interactions of low-molecular ligands with the base pairs of DNA. This method is based on the comparative analysis of melting temperature changes in DNAs of different GC-content in the presence of low molecular weight ligands. In this paper, the method is applied to Mn2+, Ni2+, Co2+ ions, deprotonation, amino acids, beta-alanine and gamma-aminobutyric acid (gamma-ABA). Differences in Tm are affected not only by the changes of relative stability of AT- and GC-pairs, but also by other factors. A theoretical analysis of the sequence specificity of low-molecular ligands on the base pairs in DNA molecules characterized by a high degree of sequence heterogeneity is also presented.