Antibody performance in western blot applications is context‐dependent

An important concern for the use of antibodies in various applications, such as western blot (WB) or immunohistochemistry (IHC), is specificity. This calls for systematic validations using well‐designed conditions. Here, we have analyzed 13 000 antibodies using western blot with lysates from human cell lines, tissues, and plasma. Standardized stratification showed that 45% of the antibodies yielded supportive staining, and the rest either no staining (12%) or protein bands of wrong size (43%). A comparative study of WB and IHC showed that the performance of antibodies is application‐specific, although a correlation between no WB staining and weak IHC staining could be seen. To investigate the influence of protein abundance on the apparent specificity of the antibody, new WB analyses were performed for 1369 genes that gave unsupportive WBs in the initial screening using cell lysates with overexpressed full‐length proteins. Then, more than 82% of the antibodies yielded a specific band corresponding to the full‐length protein. Hence, the vast majority of the antibodies (90%) used in this study specifically recognize the target protein when present at sufficiently high levels. This demonstrates the context‐ and application‐dependence of antibody validation and emphasizes that caution is needed when annotating binding reagents as specific or cross‐reactive. WB is one of the most commonly used methods for validation of antibodies. Our data implicate that solely using one platform for antibody validation might give misleading information and therefore at least one additional method should be used to verify the achieved data.     

Increased levels of plasma 8-EPI-PGF2α in patients with end stage renal failure

Summary

F₂-isoprostanes are a series of prostaglandin-F₂ like compounds specifically derived from peroxidation of arachidonic acid by a mechanism independent of the cyclooxygenase pathway. Of these, 8-epi PGF_2α is shown to be a potent vasoconstrictor. In this study, we have analysed plasma 8-epi PGF_2α as a marker of oxidative stress in patients with end stage renal failure (ESRF) undergoing haemodialysis (HD) or continuous ambulatory peritoneal dialysis (CAPD). Plasma F₂-isoprostanes were isolated by solid-phase extraction on a C₁₈ followed by an NH₂ cartridge. Quantitative analysis of the F₂-isoprostanes as pentafluorobenzyl (PFB) ester/trimethylsilyl (TMS) ether derivatives was carried out by gas chromatography-electron capture mass spectrometry. For 34 individuals with ESRF, the mean level of esterified 8-epi PGF_2α was 0.58 ± 0.22 M; range 0.21–1.16 nM. 8-epi PGF_2α concentration in the patient groups was markedly higher (P<0.0005 by separate variance t-test) than that of control subjects (n=15) 0.28 ± 0.17 nM; range 0.02–0.63 nM. There was no difference in levels of 8-epi PGF_2α in plasma from patients undergoing HD or CAPD, nor was there any association with age, plasma lipids or plasma creatinine. These data provide direct evidence of increased oxidative stress in individuals with ESRF. This marker should be useful in clinical studies examining the degree of oxidative stress in vivo and the therapeutic impact of antioxidants.     

Inactivation of thioredoxin f1 leads to decreased light activation of ADP‐glucose pyrophosphorylase and altered diurnal starch turnover in leaves of Arabidopsis plants

Chloroplast thioredoxin f (Trx f) is an important regulator of primary metabolic enzymes. However, genetic evidence for its physiological importance is largely lacking. To test the functional significance of Trx f in vivo, Arabidopsis mutants with insertions in the trx f1 gene were studied, showing a drastic decrease in Trx f leaf content. Knockout of Trx f1 led to strong attenuation in reductive light activation of ADP‐glucose pyrophosphorylase (AGPase), the key enzyme of starch synthesis, in leaves during the day and in isolated chloroplasts, while sucrose‐dependent redox activation of AGPase in darkened leaves was not affected. The decrease in light‐activation of AGPase in leaves was accompanied by a decrease in starch accumulation, an increase in sucrose levels and a decrease in starch‐to‐sucrose ratio. Analysis of metabolite levels at the end of day shows that inhibition of starch synthesis was unlikely due to shortage of substrates or changes in allosteric effectors. Metabolite profiling by gas chromatography–mass spectrometry pinpoints only a small number of metabolites affected, including sugars, organic acids and ethanolamine. Interestingly, metabolite data indicate carbon shortage in trx f1 mutant leaves at the end of night. Overall, results provide in planta evidence for the role played by Trx f in the light activation of AGPase and photosynthetic carbon partitioning in plants.     

Luteinising hormone,follicle stimulating hormone and gonadotropin releasing hormone immunoreactivity in two insects: Locusta migratoria migratoroides R&F and Sarcophaga bullata (Parker)

Summary

Materials immunologically related to luteinising hormone (LH), follicle stimulating hormone (FSH) and the gonadotropin releasing hormone (GnRH) were localised in cerebral tissue of Locusta migratoria and Sarcophaga bullata by means of the peroxidase-antiperoxidase method. Several polyclonal and a monoclonal antisera were used. From the third larval instar a positive reaction was observed in cells located in several parts of the brain. Each antiserum reacted with a constant number of well defined cells and nerve fibers. No differences between sexes were observed.     

HABITAT ACOUSTICS OF A NEOTROPICAL LOWLAND RAINFOREST

ABSTRACT

The acoustic characteristics of an Amazonian lowland rain forest study site in southern Venezuela was analysed to determine environmental constraints upon acoustic communication. Signal degradation was measured by conducting transmission experiments at different heights above ground level. Measurements of ambient noise served to determine possible communication distances for various times of day, heights above ground level and frequencies. “Sound windows” for acoustic long-range communication were found for low frequencies, calling heights in the midstorey and calling in the morning or during the night. Sound attenuation was affected by height and frequency but not by time of day. Background noise varied remarkably with time of day and frequency and had a greater impact on communication distance than signal attenuation.     

Nocturnal passerine migration without tailwind assistance

Nocturnal passerine migrants could substantially reduce the amount of energy spent per distance covered if they fly with tailwind assistance and thus achieve ground speeds that exceed their airspeeds (the birds’ speed in relation to the surrounding air). We analysed tracking radar data from two study sites in southern and northern Scandinavia and show that nocturnally migrating passerines, during both spring and autumn migration, regularly travelled without tailwind assistance. Average ground and airspeeds of the birds were strikingly similar for all seasonal and site‐specific samples, demonstrating that winds had little overall influence on the birds’ resulting travel speeds. Distributions of wind effects, measured as (1) the difference between ground and airspeed and (2) the tail/headwind component along the birds’ direction of travel, showed peaks close to a zero wind effect, indicating that the migratory flights often occurred irrespective of wind direction. An assessment of prevailing wind speeds at the birds’ mean altitude indicated a preference for lower wind speeds, with flights often taking place in moderate winds of 3–10 m/s. The limited frequency of wind‐assisted flights among the nocturnal passerine migrants studied is surprising and in clear contrast to the strong selectivity of tailwinds exhibited by some other bird groups. Relatively high costs of waiting for favourable winds, rather low probabilities of occurrence of tailwind conditions and a need to use a large proportion of nights for flying are probably among the factors that explain the lack of a distinct preference for wind‐assisted flights among nocturnal passerine migrants.     

Subfossil chironomid assemblages in deep,stratified European lakes: relationships with temperature,trophic state and oxygen

1. The distributions of subfossil remains of chironomid larvae in 28 large, deep and stratified lakes in Europe were examined in surface sediments along a latitudinal transect ranging from northern Sweden to southern Italy. 2. Canonical correspondence analysis (CCA) showed that summer surface water and July air temperature, as well as total phosphorus (TP) concentrations, hypolimnetic oxygen availability and conductivity were statistically significant (P < 0.05) explanatory variables explaining between 11 and 14% of the variance in the chironomid data. 3. Owing to the spatial scale covered by our study, many environmental variables were covarying. Temperature, TP concentration and oxygen availability were positively or negatively correlated with the first axis of a detrended correspondence analysis (DCA) of chironomid assemblages, suggesting that climatic and trophic conditions influenced profundal chironomid assemblages either in a direct (food and oxygen) or in an indirect (temperature) way. Parameters related to local environmental conditions, lake morphology and bedrock geology, such as organic matter content of the sediment, maximum lake depth, Secchi depth and pH, were not significant in explaining the distribution of chironomid assemblages in our study lakes. 4. The strong relationship between chironomid assemblages and summer temperature may be related to the covariation of temperature with parameters, such as nutrient and oxygen availability, known to affect chironomid assemblages in deep, stratified lakes. However, summer temperature explained a statistically significant proportion of the variance in the chironomid assemblages even when effects of oxygen availability and TP concentrations were partialled out. This suggests that summer temperature has an effect on chironomid assemblages in deep lakes, which is not related to its covariation with trophic state. 5. The potential of fossil chironomid analysis for quantitatively reconstructing past nutrient conditions in deep, stratified lakes was examined by calculating the Benthic Quality Index (BQI) based on subfossil chironomids and by comparing BQI values with observed TP concentrations. BQI was linearly related to log‐transformed TP. Applying this relationship to fossil chironomid assemblages from Lake Päijänne (Finland) produced a TP reconstruction in agreement with measured TP during the period 1970–1990, demonstrating that this approach can provide quantitative estimates of past nutrient concentrations in deep, stratified lakes.