Targeted label-free quantitative analysis of secretory proteins from adipocytes in response to oxidative stress

Adipocytes are well known to release regulation factors associated with metabolic disorders. In particular, increased oxidative stress in adipocytes contributes to dysregulation of adipokine production. In this study, we applied relative quantitative proteomic analysis based on label-free multiple reaction monitoring (MRM) to discover biological changes of adipokines under oxidative stress. Among a total of 194 identified proteins, 8 proteins were selected and quantified between control and hydrogen peroxide (H₂O₂)-treated groups by label-free MRM quantification. The secretion levels of matrix metalloproteinase-2 (MMP-2), stromal cell-derived factor-1 (SDF-1, CXCL12), resistin, and complement factor D (CFD, adipsin) decreased, whereas the secretion levels of tissue inhibitor of metalloproteinase-2 (TIMP-2) and aldolase A increased. Here we suggest that our study with label-free quantitative analysis will contribute to the efficient quantitative analysis of targeted proteins in complex mixtures and specifically to a better understanding of changes of adipokines under oxidative stress.     

The control of nest climate in bumblebee (Bombus terrestris) colonies: interindividual variability and self reinforcement in fanning response

Interindividual variability in response to environmental stimuliis believed to have a major impact on collective behaviors insocial insects. The present study presents a detailed investigationof the variability in individual fanning behavior underlyingthe collective control of nest climate in bumblebee (Bombusterrestris) colonies. Four colonies were repeatedly exposedto increasing temperature and CO₂ levels. The response thresholdof each worker (defined as the mean stimulus intensity at whicha worker responded by fanning) was determined. Temperature responsethresholds of 118 workers and CO₂ response thresholds of 88workers were analyzed. Workers differed in their response thresholds.Some consistently responded to low stimulus intensities, othersconsistently responded to high stimulus intensities. No consistentcorrelation between temperature and CO₂ thresholds was foundwithin individuals. Response thresholds of fanning bees decreasedover successive trials, providing empirical support for theidea of specialization through individual threshold reinforcement.In addition to variability in individual response thresholds,workers of a colony differed in two other parameters of responsiveness:response probability (the probability of responding to a stimulusonce it exceeded an individual's response threshold) and responseduration (the persistency with which fanning was performed oncean individual responded). The results of the present study suggestthat response threshold, response probability and response durationare important independent parameters of individual responsivenessin the collective control of nest climate in bumblebee colonies.     

A quantitative analysis of the genetics of resting blood lactic acid levels in mice

Resting blood lactate levels were measured in inbred mouse strains, their F(1), and several of their segregating generations to determine whether the level of lactic acid is influenced by genetic factors. The inbred strains in each of the two sets used differed significantly from one another for this character. Only one strain showed a significant sex difference. The data could not be fully analyzed because of the failure to fulfill Mather's first criterion for an adequate scale. Nonallelic interactions, in particular, additive x dominance and dominance x dominance, were found to influence the generation means. Genotype x environment interaction was detected and eliminated by log transformation. Negative heterosis was exhibited by all but one noninbred generation.-The data suggest that genes influencing the character are dispersed between the parental lines and that interactions are predominantly of the duplicate kind. A buffering system by which lactate levels are kept at a minimum is proposed.     

Canalization effect in the coagulation cascade and the interindividual variability of oral anticoagulant response. a simulation Study

Background

The aim of the study was to find a simple intravenous glucose tolerance test (IVGTT) that can be used to estimate insulin sensitivity.

Methods

In 20 healthy volunteers aged between 18 and 51 years (mean, 28) comparisons were made between kinetic parameters derived from a 12-sample, 75-min IVGTT and the M_bw (glucose uptake) obtained during a hyperinsulinemic euglycemic glucose clamp. Plasma glucose was used to calculate the volume of distribution (V _d) and the clearance (CL) of the injected glucose bolus. The plasma insulin response was quantified by the area under the curve (AUC_ins). Uptake of glucose during the clamp was corrected for body weight (M_bw).

Results

There was a 7-fold variation in M_bw. Algorithms based on the slope of the glucose-elimination curve (CL/V _d) in combination with AUC_ins obtained during the IVGTT showed statistically significant correlations with M_bw, the linearity being r² = 0.63-0.83. The best algorithms were associated with a 25-75^th prediction error ranging from -10% to +10%. Sampling could be shortened to 30-40 min without loss of linearity or precision.

Conclusion

Simple measures of glucose and insulin kinetics during an IVGTT can predict between 2/3 and 4/5 of the insulin sensitivity.     

Osteoblast cytoskeletal modulation in response to compressive stress at physiological levels

In this study we assessed activities of antioxidant enzymes, lipid peroxidation end-products, and nitric oxide (NO) levels in women with postmenopausal osteoporosis (PMO). Relationship between oxidative stress parameters and NO levels with bone mineral density (BMD) and clinical variables influencing bone mass and health related quality of life measures was also investigated in women with PMO. Postmenopausal women (n = 87), aged 40–65, without previous diagnosis or treatment for osteoporosis and independent in daily living activities were included. BMD was measured at the lumbar spine and proximal femur using dual-X-ray absorptiometry (DXA). Erythrocyte catalase (CATe) enzyme activity, erythrocyte and plasma enzyme activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and lipid peroxidation end-product malondialdehyde (MDA) and nitrite/nitrate levels, by product of NO were studied. A total of 23 healthy non-porotic women were included as controls. Women with PMO had significantly lower erythrocyte CATe enzyme activity and higher erythrocyte malondialdehyde (MDAe) and erythrocyte nitric oxide (NOe) levels in comparison to controls whereas erythrocyte SODe and GSH-Px enzyme activity was similar. In plasma, osteporotic women had significantly higher SOD enzyme activity and higher MDA levels whereas similar GSH-Px enzyme activity and NO levels compared to non-porotic controls. Significant correlation was found between erythrocyte SODe, CATe enzyme activity and NOe levels with proximal femur BMD. Some of the quality of life scores as pain, mental, and social functions correlated with antioxidant enzyme activities and NO levels. Consequently, oxidative stress markers may be an important indicator for bone loss in postmenopausal women. Further researches assessing the oxidative stress markers and NO in bone tissue and changes with anti-osteoporotic drugs would be valuable to better understand the role of free radicals, antioxidants, and NO in the regulation of bone mass.     

Sources of interindividual variation in the quantitative levels of apolipoprotein B in pedigrees ascertained through a lipid clinic.

The quantitative level of apolipoprotein (apo) B associated with low-density lipoprotein (LDL) varies among individuals within the population. This variation in level of the LDL receptor ligand appears to have predictive value, and may have an etiologic role, in coronary artery disease. Complex segregation analysis was used to compare eight different models of transmission. This study confirms the existence of allelic variations at a single genetic locus with large effects on the interindividual variation in the level of the serum apo B associated with LDL. This is the first study to consider the possible effects of inherited polymorphic variation in the apo E molecule when analyzing the components of variation in apo B associated with LDL. Our analyses suggest that the common alleles coding for the apo E polymorphism act independently of the unmeasured single-gene locus characterized by this study.     

Principal component analysis of quantitative trait loci for immune response to adenovirus in mice

Data on the duration of transgene expression in the liver, the presence of cytotoxic T lymphocytes (CTLs) against adenovirus, and serum cytokines from 18 strains of C57BL/6 x DBA/2 (B x D) recombinant inbred mice were analyzed. Our aim was to detect quantitative trait loci (QTLs) that may have causal relationship with the duration of adenovirus-mediated transgene expression in the liver. Information from beta-galactosidase (LacZ) expression; CTL production; and serum levels of gamma interferon, tumor necrosis factor-alpha, and interleukin-6 30 days after intravenous injection of liver LacZ were summarized by principal component analysis and analyzed using maximum likelihood interval mapping implemented in the QTL cartographer software. Two principal component (PC) scores explained 82.5% of the phenotypic variance in the original variables and identified QTLs not identified by analysis of individual traits. The distribution of original variables among PCs was such that variables in PC1 were predominantly cytokines with little CTL response whereas LacZ and CTL were the predominant contributors to PC2 with practically no contribution from cytokines. PC1 was significantly associated with two QTLs on chromosomes 7 and 9 located at 57.5 cM and 41.01 cM, respectively. Five QTLs were significantly associated with PC2 on chromosomes 12 (23.01 and 31.01 cM) and 15 (29.21, 36.01, and 56.31 cM). These results illustrate the use of principal component analysis in mapping QTLs using multiple correlated traits.     

Individual variability in the stress response of C57BL/6J male mice correlates with trait anxiety

Stress strongly alters the physiology and behavior of some individuals, while others are little or not affected. The causes of this individual variability have remained unknown. Here, we hypothesize that epigenetically induced levels of trait anxiety predict the stress response of individual mice in a genetically homogeneous population. Inbred C57BL/6 male mice were selected for their latency to freely enter from their home cage into an unfamiliar arena and classified as having high or low levels of trait anxiety. Mice were then exposed to acute stress (1-h olfactory contact with a rat) or control conditions. After 24 h, acute stress enhanced state anxiety measured in the elevated-plus maze test only in mice previously classified as having high levels of trait anxiety. This anxiogenic effect of acute stress was paralleled by enhanced novelty-induced plasma corticosterone secretion and increased messenger RNA (mRNA) expression for glucocorticoid and mineralocorticoid receptors in the hippocampus. No effects of acute stress were observed in mice classified as having low levels of trait anxiety. Under unstressed control conditions, mice only differed in basal levels of hippocampal mRNA for the glucocorticoid receptor, which were higher in mice with high trait anxiety than in mice with low trait anxiety. In summary, inbred C57BL/6 mice display a remarkably high interindividual variability in their trait anxiety that predicts the behavioral and neuroendocrine response to an acute stressor, indicating that expression of extremely different coping strategies can develop also between genetically identical individuals.     

Spatial variability in the response of lower trophic levels after carp exclusion from a freshwater marsh

Large common carp (Cyprinus carpio >30 cm) wereexcluded from a turbid, eutrophic coastal marsh of Lake Ontario with theconstruction of a fishway at the outlet. The marsh was sampledintensively for 2 seasons prior to (1993, 1994) and following (1997,1998) carp exclusion to study changes in water quality and shifts incommunity structure of phytoplankton and zooplankton. Samples werecollected from May to September in three habitats: open water, vegetated(cattail beds) and sewage lagoon. In the first year after carpexclusion, mean seasonal water turbidity decreased at all sites by49–80%; this was accompanied by growth of submergentplants in shallow, sheltered areas including the vicinity of cattails atthe vegetated site. This drop in turbidity was not significant in thesecond year after exclusion at the open water and lagoon sites, withturbidity levels declining by only 26–54% of1993–1994 values; only the vegetated site showed a sustaineddecrease in turbidity and persistent growth of submergent plants. At thevegetated site, increased clarity was concurrent with a significantreduction in edible algal biomass and an increased representation oflarge zooplankton grazers and substrate-associated cladocerans. At theopen water site, a spring clear-water phase was evident during the firstyear of exclusion and this coincided with the unusual appearance of alarge population of Daphnia. Compared to the other sites, thelagoon remained relatively turbid throughout the study. Results of thisstudy indicate that the response of lower trophic levels tobiomanipulation was variable from site-to-site and contributed to theco-existence of two alternative states in the marsh. In vegetated areas,water clarity was maintained by a positive feedback system betweenzooplankton and submergent macrophytes in the first 2 years followingexclusion. We suggest that both benthivore removal (to reducebioturbation) and planktivore reductions (to produce top down effects)were required to produce clear water and allow submersed macrophytegrowth. Although carp removal likely contributed to a 45%reduction in turbidity, an unusual climactic event in 1997, resulting indelayed fish spawning in the marsh, temporarily reduced zooplanktivoryand favoured zooplankton grazing-induced water clarity improvements.     

Cangrelor increases the magnitude of platelet inhibition and reduces interindividual variability in clopidogrel-pretreated subjects

BackgroundInadequate platelet inhibition despite aspirin and clopidogrel therapy during and after a percutaneous coronary intervention is associated with an impaired clinical outcome. Cangrelor, a direct and reversible P2Y₁₂ inhibitor that is currently in development, has the potential to achieve higher levels of inhibition of ADP-induced platelet aggregation than clopidogrel. The aim of the present study was to compare the magnitude of platelet inhibition in clopidogrel-pretreated patients before and after the in vitro addition of a subtherapeutic dose of cangrelor. MethodsBlood samples were drawn from patients pretreated with clopidogrel and aspirin who were undergoing elective percutaneous coronary intervention (n=39). Platelet function analysis with ‘classical’ light transmittance aggregometry (both peak and late aggregation [at 6 min]) was performed before and after the in vitro addition of cangrelor (0.25 μmol/l) to platelet-rich plasma (PRP). After an incubation period of five minutes, platelet aggregation was induced by 5 and 20 μmol/l ADP. ResultsThe in vitro addition of 0.25μmol/l cangrelor to the PRP from clopidogrel-treated subjects resulted in an additional reduction in ADP-induced platelet aggregation. For ADP concentrations of 5 and 20 μmol/l, peak aggregation showed a decrease of 75 and 85%, respectively (p<0.001 for both), while late aggregation was almost completely diminished (p=0.003 and p<0.001, respectively). Furthermore, the interindividual variation in inhibition of ADP-induced platelet aggregation by clopidogrel was greatly reduced after the addition of cangrelor. ConclusionWe demonstrate that the in vitro addition of even a subtherapeutic dose of cangrelor to the PRP of clopidogrel-pretreated patients results in an additional reduction of ADP-induced platelet aggregation. Moreover, cangrelor was able to diminish the interindividual variation observed in clopidogrel-inhibited platelet aggregation. (Neth Heart J 2009;17:195–8.)     

The comparison of levels of anxiety and stress response in mice selected for low and high brain weight

Emotionality, anxiety and stress reactivity were studied in mice selected for large (LW) and small (SW) brain weight. The open-field test, startle reaction, cross-maze, tail suspension and slippery funnel tests were used. It was shown that the SW mice are more active in the open field, more anxious and predisposed to the stereotyped behavior and passive reactions in stress situations. The relationship between the studied traits and brain weight and their influence on cognitive behavior are discussed.     

Effects of fluoxetine on the immunosuppressive response to stress in mice

Mice exposed to a chronic auditory Stressor and treated with fluoxetine (5 mg/kg) showed a reduction in stress-induced suppression of thymus and spleen cellularity, and in peripheral T lymphocyte population. The blastogenic response of spleen lymphoid cells and the delayed type hypersensitivity response (DTH) to sheep red blood cells (SRBC) were also assessed and fluoxetine was found to partially reverse the inhibitory effect of stress on both parameters.