Artificial selection for increased dispersal results in lower fitness

Dispersal often covaries with other traits, and this covariation was shown to have a genetic basis. Here, we wanted to explore to what extent genetic constraints and correlational selection can explain patterns of covariation between dispersal and key life‐history traits—lifespan and reproduction. A prediction from the fitness‐associated dispersal hypothesis was that lower genetic quality is associated with higher dispersal propensity as driven by the benefits of genetic mixing. We wanted to contrast it with a prediction from a different model that individuals putting more emphasis on current rather than future reproduction disperse more, as they are expected to be more risk‐prone and exploratory. However, if dispersal has inherent costs, this will also result in a negative genetic correlation between higher rates of dispersal and some aspects of performance. To explore this issue, we used the dioecious nematode Caenorhabditis remanei and selected for increased and decreased dispersal propensity for 10 generations, followed by five generations of relaxed selection. Dispersal propensity responded to selection, and females from high‐dispersal lines dispersed more than females from low‐dispersal lines. Females selected for increased dispersal propensity produced fewer offspring and were more likely to die from matricide, which is associated with a low physiological condition in Caenorhabditis nematodes. There was no evidence for differences in age‐specific reproductive effort between high‐ and low‐dispersal females. Rather, reproductive output of high‐dispersal females was consistently reduced. We argue that our data provide support for the fitness‐associated dispersal hypothesis.     

Influence of macroinvertebrate sample size on bioassessment of streams

In order to standardise biological assessment of surface waters in Europe, a standardised method for sampling, sorting and identification of benthic macroinvertebrates in running waters was developed during the AQEM project. The AQEM method has proved to be relatively time-consuming. Hence, this study explored the consequences of a reduction in sample size on costs and bioassessment results. Macroinvertebrate samples were collected from six different streams: four streams located in the Netherlands and two in Slovakia. In each stream 20 sampling units were collected with a pond net (25×25 cm), over a length of approximately 25 cm per sampling unit, from one or two habitats dominantly present. With the collected data, the effect of increasing sample size on variability and accuracy was examined for six metrics and a multimetric index developed for the assessment of Dutch slow running streams. By collecting samples from separate habitats it was possible to examine whether the coefficient of variation (CV; measure of variability) and the mean relative deviation from the “reference” sample (MRD; measure of accuracy) for different metrics depended only on sample size, or also on the type of habitat sampled. Time spent on sample processing (sorting and identification) was recorded for samples from the Dutch streams to assess the implications of changes in sample size on the costs of sample processing. Accuracy of metric results increased and variability decreased with increasing sample size. Accuracy and variability varied depending on the habitat and the metric, hence sample size should be based on the specific habitats present in a stream and the metric(s) used for bioassessment. The AQEM sampling method prescribes a multihabitat sample of 5 m. Our results suggest that a sample size of less than 5 m is adequate to attain a CV and MRD of ≤ 10% for the metrics ASPT (Average Score per Taxon), Saprobic Index and type Aka+Lit+Psa (%) (the percentage of individuals with a preference for the akal, littoral and psammal). The metrics number of taxa, number of individuals and EPT-taxa (%) required a multihabitat sample size of more than 5 m to attain a CV and MRD of ≤ 10%. For the metrics number of individuals and number of taxa a multihabitat sample size of 5 m is not even adequate to attain a CV and MRD of ≤ 20%. Accuracy of the multimetric index for Dutch slow running streams can be increased from ≤ 20 to ≤ 10% with an increase in labour time of 2 h. Considering this low increase in costs and the possible implications of incorrect assessment results it is recommended to strive for this ≤ 10% accuracy. To achieve an accuracy of ≤ 10% a multihabitat sample of the four habitats studied in the Netherlands would require a sample size of 2.5 m and a labour time of 26 h (excluding identification of Oligochaeta and Diptera) or 38 h (including identification of Oligochaeta and Diptera). Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Different kinetics of the regulation of respiration in permeabilized cardiomyocytes and in HL-1 cardiac cells. Importance of cell structure/organization for respiration regulation

The aim of this study was to investigate the mechanism of cellular regulation of mitochondrial respiration in permeabilized cardiac cells with clearly different structural organization: (i) in isolated rat cardiomyocytes with very regular mitochondrial arrangement, (ii) in HL-1 cells from mouse heart, and (iii) in non-beating (NB HL-1 cells) without sarcomeres with irregular and dynamic filamentous mitochondrial network. We found striking differences in the kinetics of respiration regulation by exogenous ADP between these cells: the apparent Km for exogenous ADP was by more than order of magnitude (14 times) lower in the permeabilized non-beating NB HL-1 cells without sarcomeres (25+/-4 microM) and seven times lower in normally cultured HL-1 cells (47+/-15 microM) than in permeabilized primary cardiomyocytes (360+/-51 microM). In the latter cells, treatment with trypsin resulted in dramatic changes in intracellular structure that were associated with 3-fold decrease in apparent Km for ADP in regulation of respiration. In contrast to permeabilized cardiomyocytes, in NB HL-1 cells creatine kinase activity was low and the endogenous ADP fluxes from MgATPases recorded spectrophotometrically by the coupled enzyme assay were not reduced after activation of mitochondrial oxidative phosphorylation by the addition of mitochondrial substrates, showing the absence of ADP channelling in the NB HL-1 cells. While in the permeabilized cardiomyocytes creatine strongly activated mitochondrial respiration even in the presence of powerful competing pyruvate kinase-phosphoenolpyruvate system, in the NB HL-1 cells the stimulatory effect of creatine was not significant. The results of this study show that in normal adult cardiomyocytes and HL-1 cells intracellular local restrictions of diffusion of adenine nucleotides and metabolic feedback regulation of respiration via phosphotransfer networks are different, most probably related to differences in structural organization of these cells.     

Analysis of naltrexone and its metabolite 6-ß-naltrexol in serum with high-performance liquid chromatography

ABSTRACT: BACKGROUND: Naltrexone has been proven to be an effective treatment option for the treatment of alcohol dependency. In this article we introduce a reliable and simple method developed for the simultaneous determination of naltrexone and 6-beta-naltrexol in human serum by using high-performance liquid chromatography (HPLC). FINDINGS: Liquid-liquid extraction with butyl acetate from basic solutions (pH 9) was chosen for extraction with nalorphine as an internal standard (IS). Analytes were back-extracted from organic solvent into perchloric acid. The acid extract was chromatographed by HPLC with a reverse-phase ODS-column and electrochemical detector. The mobile phase was a NaH2PO4-solution with acetonitrile as an organic modifier and octanesulphonic acid and tetraethylammonium hydrogen sulphate as ion-pair reagents. The recovery of the extraction method was 48 % for naltrexone and 75 % for 6-beta-naltrexol. The limit of quantification was 5.0 ng/ml for naltrexone and 1.0 ng/ml for 6-beta-naltrexol. The analysed concentrations of naltrexone differed from the theoretic concentrations by 0.7 to 2.3 % and those of 6-beta-naltrexol by 2.6 %. The relative standard deviation of within-day assay was from 0.9 to 5.7 % for naltrexone and from 0.8 to 4.2 % for 6-beta-naltrexol; for the between-day assay it was 5.7 % and 4.2 %, respectively. CONCLUSIONS: Our results indicate that the developed method is suitable for determination of naltrexone and 6-beta-naltrexol in human serum.     

Monospecific inhibitors show that both mannan-binding lectin-associated serine protease-1 (MASP-1) and -2 Are essential for lectin pathway activation and reveal structural plasticity of MASP-2

The lectin pathway is an antibody-independent activation route of the complement system. It provides immediate defense against pathogens and altered self-cells, but it also causes severe tissue damage after stroke, heart attack, and other ischemia reperfusion injuries. The pathway is triggered by target binding of pattern recognition molecules leading to the activation of zymogen mannan-binding lectin-associated serine proteases (MASPs). MASP-2 is considered as the autonomous pathway-activator, while MASP-1 is considered as an auxiliary component. We evolved a pair of monospecific MASP inhibitors. In accordance with the key role of MASP-2, the MASP-2 inhibitor completely blocks the lectin pathway activation. Importantly, the MASP-1 inhibitor does the same, demonstrating that MASP-1 is not an auxiliary but an essential pathway component. We report the first Michaelis-like complex structures of MASP-1 and MASP-2 formed with substrate-like inhibitors. The 1.28 Å resolution MASP-2 structure reveals significant plasticity of the protease, suggesting that either an induced fit or a conformational selection mechanism should contribute to the extreme specificity of the enzyme.     

Induced mutagenic effects in the nucleotide excision repair deficient Drosophila mutant mus201(D1), expressing a truncated XPG protein

Defects in nucleotide excision repair (NER) as defined by the UV sensitivity of xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD) patients has lead to the identification of most of the genes involved: XPA through XPG, CSA and CSB. Whereas XP patients often show an increased risk for skin cancer after exposure to sunlight, this is not the case for patients with CS and TTD. Several CS patients have been shown to carry a defect in the XPG gene. The XPG, a structure specific endonuclease makes the incision 3' of damage and is also involved in the subsequent 5'incision during the NER process. In addition, XPG plays a role in the removal of oxidative DNA damage. The Drosophila XPG gene was isolated and based on the molecular defect of a spontaneous (insertion) and an EMS induced mutant, it was shown that a mutated XPG is responsible for the Drosophila mutagen-sensitive mutants mus201. One of these mutants, mus201(D1) has been used extensively in studies of the effects and mechanisms of many chemical mutagens as well as X-rays. The results of these studies are discussed in the light of the finding that mus201p is the Drosophila homologue of XPG.     

Cyclothiazide binding to functionally active AMPA receptor reveals genuine allosteric interaction with agonist binding sites

The agonist, [3H](-)[S]-1-(2-amino-2-carboxyethyl)-5-fluoro-pyrimidine-2,4-dione ([3H](S)F-Willardiine) binding to functional alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors of resealed plasma membrane vesicles and nerve endings freshly isolated from the rat cerebral cortex displayed two binding sites (K(D1)=33+/-7 nM, B(MAX1)=1.6+/-0.3 pmol/mg protein, K(D2)=720+/-250 nM and B(MAX2)=7.8+/-4.0 pmol/mg protein). The drug which impairs AMPA receptor desensitisation, 6-chloro-3,4-dihydro-3-(2-norbornene-5-yl)-2H-1,2,4-benzothiadiazine-7-sulphonamide-1,1-dioxide (cyclothiazide, CTZ) fully displaced the [3H](S)F-Willardiine binding at a concentration of 500 microM. In the presence of 100 microM CTZ (K(I(CTZ))=60+/-6 microM), both the antagonist [3H]-1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo(F)quinoxaline-7-sulfonamide ([3H]NBQX: K(D)=24+/-4 nM, B(MAX)=12.0+/-0.1 pmol/mg protein) and the high-affinity agonist binding showed similar affinity reduction ([3H](S)F-Willardiine: K(D)=140+/-19 nM, B(MAX)=2.9+/-0.5 pmol/mg protein; [3H]NBQX: K(D)=111+/-34 nM, B(MAX)=12+/-3 pmol/mg protein). To disclose structural correlates underlying genuine allosteric binding interactions, molecular mechanics calculations of CTZ-induced structural changes were performed with the use of PDB data on extracellular GluR2 binding domain dimeric crystals available by now. Hydrogen-bonding and root mean square (rms) values of amino acid residues recognising receptor agonists showed minor alterations in the agonist binding sites itself. Moreover, CTZ binding did not affect dimeric subunit structures significantly. These findings indicated that the structural changes featuring the non-desensitised state could possibly occur to a further site of the extracellular GluR2 binding domain. The increase of agonist efficacy on allosteric CTZ binding may be interpreted in terms of a mechanism involving AMPA receptor desensitisation sequential to activation.     

Knee kinematics in medial arthrosis. Dynamic radiostereometry during active extension and weight-bearing

We studied the kinematics of the knee during weight-bearing active extension in 14 patients with medial osteoarthrosis (OA) and in 10 controls using dynamic radiostereometry. Between 50 degrees and 20 degrees of extension the OA knees showed decreased internal tibial rotation corresponding to less posterior displacement of the lateral femoral flexion facet center. The midpoint between the two tips of the tibial intercondylar eminence occupied a more posterior position within the range of motion analyzed. The observed changes were similar to those previously recorded in chronic tear of the anterior cruciate ligament. Patients with medial arthrosis of the knee joint show a specific and abnormal pattern of joint motion.