J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential

The spectral properties of a novel membrane potential sensitive probe (JC-1) were characterized in aqueous buffers and in isolated cardiac mitochondria. JC-1 is a carbocyanine with a delocalized positive charge. It formed under favorable conditions a concentration-dependent fluorescent nematic phase consisting of J-aggregates. When excited at 490 nm, the monomers exhibited an emission maximum at 527 nm and J-aggregates at 590 nm. Increasing concentrations of JC-1 above a certain concentration caused a linear rise in the J-aggregate fluorescence, while the monomer fluorescence remained constant. The membrane potential of energized mitochondria (negative inside) promoted a directional uptake of JC-1 into the matrix, also with subsequent formation of J-aggregates. The J-aggregate fluorescence was sensitive to transient membrane potential changes induced by ADP and to metabolic inhibitors of oxidative phosphorylation. The J-aggregate fluorescence was found to be pH independent within the physiological pH range of 7.15-8.0 and could be linearly calibrated with valinomycin-induced K+ diffusion potentials. The advantage of JC-1 over rhodamines and other carbocyanines is that its color altered reversibly from green to red with increasing membrane potentials. This can be exploited for imaging live mitochondria on the stage of a microscope.     

Immune interferon produced in vitro as a quantitative indicator of cell-mediated immunity.

The present study was constructed to provide some information on the possibility of utilizing immune interferon as a quantitative indicator of cell-mediated immunity and to clarify some of the nature of immune interferon-producing cells (IIPC). When spleen cells derived from L cell-sensitized mice were co-cultivated with L cells, interferon appeared in the culture fluid. It was shown by additional experiments that the cells responsible for immune interferon production in this system were T-lymphocytes assisted by macrophages. The pattern of kinetics of immune interferon production in vitro was similar to that of migration inhibitory factor or T cell-mediated cytotoxicity.     

Surplus cost as a life cycle impact indicator for fossil resource scarcity

Purpose

In life cycle impact assessment, various proposals have been made on how to characterise fossil resource scarcity, but they lack appropriateness or completeness. In this paper, we propose a method to assess fossil resource scarcity based on surplus cost, which is the global future cost increase due to marginal fossil resource used in the life cycle of products.

Methods

The marginal cost increase (MCI in US dollars in the year 2008 per kilogram per kilogram produced) is calculated as an intermediate parameter for crude oil, natural gas and coal separately. Its calculations are based on production cost and cumulative future production per production technique or country. The surplus cost (SC in US dollars in the year 2008 per kilogram) is calculated as an indicator for fossil resource scarcity. The SC follows three different societal perspectives used to differentiate the subjective choices regarding discounting and future production scenarios.

Results and discussion

The hierarchist perspective SCs of crude oil, natural gas, and coal are 2.9, 1.5, and 0.033 US$₂₀₀₈/GJ, respectively. The ratios between the indicators of the different types of fossil resources (crude oil/natural gas/coal) are rather constant, except in the egalitarian perspective, where contrastingly no discounting is applied (egalitarian 100:47:21; hierarchist 100:53:1.1; individualist 100:34:0.6). The ratio of the MCIs (100:48:1.0) are similar to the individualist and hierarchist SC ratios.

Conclusions

In all perspectives, coal has a much lower resource scarcity impact factor per gigajoule and crude oil has the highest. In absolute terms of costs per heating value (US dollars in the year 2008 per gigajoule), there are large differences between the SCs for each perspective (egalitarian > hierarchist > individualist).     

Standard line slopes as a measure of a relative matrix effect in quantitative HPLC-MS bioanalysis

A simple experimental approach for studying and identifying the relative matrix effect (for example "plasma-to-plasma" and/or "urine-to-urine") in quantitative analyses by HPLC-MS/MS is described. Using as a database a large number of examples of methods developed in recent years in our laboratories, the relationship between the precision of standard line slopes constructed in five different lots of a biofluid (for example plasma) and the reliability of determination of concentration of an analyte in a particular plasma lot (or subject) was examined. In addition, the precision of standard line slopes was compared when stable isotope-labeled analytes versus analogs were used as internal standards (IS). Also, in some cases, a direct comparison of standard line slopes was made when different HPLC-MS interfaces (APCI versus ESI) were used for the assay of the same compound, using the same IS and the same sample preparation and chromatographic separation conditions. In selected cases, the precision of standard line slopes in five different lots of a biofluid was compared with precision values determined five times in a single lot. The results of these studies indicated that the variability of standard line slopes in different lots of a biofluid [precision of standard line slopes expressed as coefficient of variation, CV (%)] may serve as a good indicator of a relative matrix effect and, it is suggested, this precision value should not exceed 3-4% for the method to be considered reliable and free from the relative matrix effect liability. Based on the results presented, in order to assess the relative matrix effect in bioanalytical methods, it is recommended to perform assay precision and accuracy determination in five different lots of a biofluid, instead of repeat (n=5) analysis in the same, single biofluid lot, calculate standard line slopes and precision of these slopes, and to use <3-4% slope precision value as a guide for method applicability to support clinical studies. It was also demonstrated that when stable isotope-labeled analytes were used as internal standards, the precision of standard line slopes in five different lots of a biofluid was 相似文献   

The quantitative research of composite immune indicator for crustacean

Through analyzing the immunity indicators in recent crustacean research, two defects are pointed in comprehensive immunity evaluation, 1) the integrant indicators cannot comprehensively reflect the change of immunity, and 2) the conclusions that obtained from different indicators of immunity level cannot be compared objectively and scientifically. Basing on that, the paper firstly indicated that the immunity system could be regarded as a composite indicator. Secondly, the paper gave the specific definition of the composite immunity indicator (CII), and discussed the methods of calculation, especially provided two calculation methods of the weights, that is, the Analytic Hierarchy Process (AHP) and the Principal Component Analysis (PCA). Finally, examples were given to clarify the specific steps to compute the composite immunity indicator. The computing results gave the quantitative evaluation, which were in concordance with the existing conclusions.     

Comparison of relative mRNA quantification models and the impact of RNA integrity in quantitative real-time RT-PCR

Relative quantification in quantitative real-time RT-PCR is increasingly used to quantify gene expression changes. In general, two different relative mRNA quantification models exist: the delta-delta Ct and the efficiency-corrected Ct model. Both models have their advantages and disadvantages in terms of simplification on the one hand and efficiency correction on the other. The particular problem of RNA integrity and its effect on relative quantification in qRT-PCR performance was tested in different bovine tissues and cell lines (n = 11). Therefore different artificial and standardized RNA degradation levels were used. Currently fully automated capillary electrophoresis systems have become the new standard in RNA quality assessment. RNA quality was rated according the RNA integrity number (RIN). Furthermore, the effect of different length of amplified products and RNA integrity on expression analyses was investigated. We found significant impact of RNA integrity on relative expression results, mainly on cycle threshold (Ct) values and a minor effect on PCR efficiency. To minimize the interference of RNA integrity on relative quantification models, we can recommend to normalize gene expression by an internal reference gene and to perform an efficiency correction. Results demonstrate that innovative new quantification methods and normalization models can improve future mRNA quantification.     

Green fluorescent protein as a quantitative reporter of relative promoter activity in E. coli

Green fluorescent protein (GFP) has become a valuable tool for the detection of gene expression in prokaryotes and eukaryotes. To evaluate its potential for quantitation of relative promoter activity in E. coli, we have compared GFP with the commonly used reporter gene lacZ, encoding beta-galactosidase. We cloned a series of previously characterized synthetic E. coli promoters into GFP and beta-galactosidase reporter vectors. Qualitative and quantitative assessments of these constructs show that (a) both reporters display similar sensitivities in cells grown on solid or liquid media and (b) GFP is especially well suited for quantitation of promoter activity in cells grown on agar. Thus, GFP provides a simple, rapid and sensitive tool for measuring relative promoter activity in intact E. coli cells.     

Functional impact of manipulation on the relative orientation of human prolactin receptor domains

Hormone binding creates active receptor dimers for class 1 cytokine receptors; however, the detailed molecular mechanism by which these receptors are activated by their ligands is not well characterized, and it is unknown if these receptors share common mechanisms. A rotation model has been proposed for the activation of human erythropoietin receptor and human growth hormone receptor and is supported by evidence showing that additions of alanine at the junction of the transmembrane (TM) and intracellular (IC) domains and/or within the TM domain influenced receptor activities. This evidence suggests that alanine additions changed the relative orientations of the IC domains and their subsequent activation. We wished to determine if a similar mechanism was at play with human prolactin receptor (hPRLr). Up to four alanines were added between the TM and either the IC or extracellular (EC) domains to extend the TM helix and to rotate the IC or EC domains. Also, up to four glycines were placed between the TM and IC domains to provide increased flexibility between these two domains. Wild-type hPRLr or various mutant receptors were expressed in human embryonic kidney 293T cells that express endogenous Janus kinase 2. In the absence of human prolactin (hPRL), none of the alanine or glycine additions increased the level of receptor phosphorylation above that of wild-type hPRLr. In the presence of hPRL, both wild-type hPRLr and each of the mutant receptors were successfully phosphorylated. These data do not support a rotation mechanism for hPRLr activation or a requirement of a fixed spatial relationship between the TM and IC domains for hPRLr activation. In a second set of experiments, both wild-type hPRLr and either alanine- or glycine-extended receptors were coexpressed in 293T cells. In the absence of hPRL, there was no detectable phosphorylation of hPRLr. Such data do not support a piston movement between the hPRLr pair in their activation.     

Human pharynx microflora as the indicator of resistance of human organisms

The analysis of the results of 220 microbiological investigations of the aerobic microflora of the pharynx mucosa in males made it possible to divide the pharynx microbiocenosis of adults into normocenosis of the I, II, III orders and dysbacteriosis of the I, II, III degrees. The use of this division for the determination of the reaction of nonspecific resistance was proposed. The possibility of using it for the diagnostics of health disturbances of sportsmen-servicemen developing due to strains characteristic for modern sport and military service, was shown.     

Multiple antibiotic resistance of heterotrophic bacteria in the littoral zone of Lake Shira as an indicator of human impact on the ecosystem

Resistance to Ampicillin and Kanamycin displayed by heterotrophic bacteria isolated in Summer and in Spring from the littoral and the central parts of Lake Shira (a therapeutic lake in the Khakasia Republic, Russia) has been investigated. It has been found that in Summer, human and animal microflora featuring multiple antibiotic resistance (to Ampicillin and Kanamycin) predominates in all the studied stations of the littoral zone of the lake. In Spring, concentrations of bacteria featuring multiple antibiotic resistance decrease significantly and bacteria sensitive to antibiotics predominate in the lake. Emergence of multiple antibiotic resistance in bacteria of Lake Shira is caused by the input of allochthonous bacteria into the lake; this feature of heterotrophic bacteria of Lake Shira can be used to monitor the impact on the ecosystem made by health resorts.     

The relative effectiveness of human plasma glutathione peroxidase as a catalyst for the reduction of hydroperoxides by glutathione

To reveal clues to the function of human plasma glutathione peroxidase (GPx), we investigated its catalytic effectiveness with a variety of hydroperoxides. Comparisons of hydroperoxides as substrates for plasma GPx based on the ratio ofV _max /K _m were blocked by the limited solubility of the organic hydroperoxides, which prevented kinetic saturation of the enzyme at the chosen glutathione concentration. Therefore, we compared the hydroperoxides by the fold increase in the apparent first-order rate constants of their reactions with glutathione owing to catalysis by plasma GPx. The reductions of aromatic and small hydrophobic hydroperoxides (cumene hydroperoxide,t-amyl hydroperoxide,t-butyl hydroperoxide, paramenthane hydroperoxide) were better catalyzed by plasma GPx than were reductions of the more “physiological” substrates (linoleic acid hydroperoxide, hydrogen peroxide, peroxidized plasma lipids, and oxidized cholesterol).     

Depression screening as a quality indicator

Objective Although practice guidelines for depression screening are evidence based, with their development relying on reviews of controlled studies, their adaptation and use as quality indicators have not been subject to rigorous study. This paper will therefore review the evidence supporting this practice.Methods A rational evaluation was carried out on both controlled studies and other sources of evidence related to the technical, clinical and policy assumptions underlying the use of depression screening guidelines as quality indicators.Results 1) Technical assumptions: depression screening could be used as a quality indicator. Current information technology does not allow accurate determination of who would benefit from being screened, whether they actually were screened, or the optimal percentage that should be screened. 2) Clinical assumptions: depression screening would improve outcomes. The evidence suggests that although depression screening might increase the diagnosis of depression, depressed patients so recognised tend to be less ill, less in need of treatment, or less likely to benefit from treatment, while screening, in the absence of other interventions, does not improve outcomes. 3) Policy assumptions: depression screening should be a focus of quality improvement. However, relative to other preventative measures, depression screening is a low priority. It does not meet usual cost-effectiveness criteria. There are more robust interventions for depression (i.e. collaborative care) that could be a focus of quality improvement efforts.Conclusion Although routine depression screening may be an acceptable practice guideline, its use as a quality measure is not supported.