Genetic analysis of autosomal and X-linked markers across a mouse hybrid zone

In this paper, we present results of the first comprehensive study of the introgression of both autosomal and sex-chromosome markers across the central European portion of the hybrid zone between two house mouse subspecies, Mus musculus musculus and M. m. domesticus. More than 1800 individuals sampled from 105 sites were analyzed with a set of allozyme loci (hopefully representing neutral or nearly neutral markers) and X-linked loci (which are assumed to be under selection). The zone center is best modeled as a single straight line independent of fine-scale local geographic or climatic conditions, being maintained by a balance between dispersal and selection against hybrids. The width (w) of the multilocus autosomal cline was estimated as 9.6 km whereas the estimate for the compound X-chromosome cline was about 4.6 km only. As the former estimate is comparable to that of the Danish portion of the zone (assumed to be much younger than the central European one), zone width does not appear to be related to its age. The strength (B) of the central barrier was estimated as about 20 km; with dispersal (sigma) of about 1 km/gen(1/2), this means effective selection (s*) is approximately 0.06-0.09 for autosomal loci and about 0.25 for X-linked loci. The number of loci under selection was estimated as N= 56-99 for autosomes and about 380 for X-linked loci. Finally, we highlight some potential pitfalls in hybrid zone analyses and in comparisons of different transects. We suggest that conclusions about parts of the mouse genome involved in reproductive isolation and speciation should be drawn with caution and that analytical approaches always providing some estimates should not be used without due care regarding the support or confidence of such estimates, especially if conclusions are based on the difference between these estimates. Finally, we recommend that analysis in two-dimensional space, dense sampling, and rigorous treatment of data, including inspection of likelihood profiles, are essential for hybrid zone studies.     

Angiopoietin-like protein 4: development, analytical characterization, and clinical testing of a new ELISA

The aim of our work was to develop an assay for the determination of angiopoietin-like protein 4 (Angplt4) in human blood, and to investigate its levels in healthy volunteers and donors suffer from metabolic syndrome. We developed and evaluated the sandwich ELISA method for the quantitative determination of human Angplt4 in serum samples. We conducted also the pilot study on individuals with metabolic syndrome or familiar hypercholesterolemia and healthy probands and measured blood pressure, waist circumference, Angplt4 serum levels, serum cholesterol, triglycerides, HDL-cholesterol, LDL-cholesterol, insulin, glucose, A-FABP and calculate BMI and QUICKI insulin sensitivity index. In the study on 30 healthy volunteers we demonstrated that sex or age is not the determinant for Angplt4 serum values. Furthermore, we tested 115 individuals with metabolic syndrome and found that probands with metabolic syndrome did not differ in Angplt4 values than healthy individuals from the first study (medians 8.7 vs. 8.1 ng/ml, p = 0.6). Individuals with metabolic syndrome did not differ in sex or age from healthy. Angplt4 values correlated with the HDL-cholesterol (r = -0.25; p < 0.01), FGF-21 (r = 0.23, p < 0.01), glucose (r = 0.17; p = 0.03), uric acid (r = 0.17; p = 0.49), lipocalin-2 (r = 0.23, p < 0.01), triacylglycerols (r = 0.25; p < 0.01) and number or characters of metabolic syndrome (r = 0.21; p < 0.01). No significant correlation was found between serum Angplt4 and BMI, WC or QUICKI. However, we performed stepwise regression and we found that Angplt4 was not an independent marker for metabolic syndrome. The patients from the metabolic syndrome group suffering diabetes mellitus (n = 83) did not differ in serum Angplt4 from the group of healthy patients, too. The pilot study supports the hypothesis about the role of Angplt4 as a new class of lipid metabolism modulator. Their values could be a new key predictors of metabolic syndrome. Further research is necessary to confirm our findings in individuals with dyslipidemia, obesity, coronary artery diseases and different medication in order to assess Angplt4 value as a risk predictor of accelerated atherosclerosis.     

A mitochondrial kinase complex is essential to mediate an ERK1/2-dependent phosphorylation of a key regulatory protein in steroid biosynthesis

ERK1/2 is known to be involved in hormone-stimulated steroid synthesis, but its exact roles and the underlying mechanisms remain elusive. Both ERK1/2 phosphorylation and steroidogenesis may be triggered by cAMP/cAMP-dependent protein kinase (PKA)-dependent and-independent mechanisms; however, ERK1/2 activation by cAMP results in a maximal steroidogenic rate, whereas canonical activation by epidermal growth factor (EGF) does not. We demonstrate herein by Western blot analysis and confocal studies that temporal mitochondrial ERK1/2 activation is obligatory for PKA-mediated steroidogenesis in the Leydig-transformed MA-10 cell line. PKA activity leads to the phosphorylation of a constitutive mitochondrial MEK1/2 pool with a lower effect in cytosolic MEKs, while EGF allows predominant cytosolic MEK activation and nuclear pERK1/2 localization. These results would explain why PKA favors a more durable ERK1/2 activation in mitochondria than does EGF. By means of ex vivo experiments, we showed that mitochondrial maximal steroidogenesis occurred as a result of the mutual action of steroidogenic acute regulatory (StAR) protein -a key regulatory component in steroid biosynthesis-, active ERK1/2 and PKA. Our results indicate that there is an interaction between mitochondrial StAR and ERK1/2, involving a D domain with sequential basic-hydrophobic motifs similar to ERK substrates. As a result of this binding and only in the presence of cholesterol, ERK1/2 phosphorylates StAR at Ser(232). Directed mutagenesis of Ser(232) to a non-phosphorylable amino acid such as Ala (StAR S232A) inhibited in vitro StAR phosphorylation by active ERK1/2. Transient transfection of MA-10 cells with StAR S232A markedly reduced the yield of progesterone production. In summary, here we show that StAR is a novel substrate of ERK1/2, and that mitochondrial ERK1/2 is part of a multimeric protein kinase complex that regulates cholesterol transport. The role of MAPKs in mitochondrial function is underlined.     

Involvement of the metabolic hormones leptin, ghrelin, obestatin, IGF-I and of MAP kinase in control of porcine oocyte maturation

The general aim of our in vitro experiments was to study the role of the metabolic hormones leptin, ghrelin, obestatin and IGF-I and mitogen-activated protein kinase (MAPK)-dependent intracellular mechanisms in the control of nuclear maturation of porcine oocytes. For this purpose, porcine oocytes were isolated from the ovary and cultured in the presence of leptin, ghrelin, obestatin, IGF-I, MAPK blocker PD98059 and the combinations of hormones with PD98059. Proportions of matured oocytes (at metaphase II of meiosis, determined by DAPI staining) and of oocytes containing MAPK/ERK1-2 (determined by immunocytochemistry) were measured before and after culture. It was observed that the majority of oocytes isolated from the ovary before culture were immature and did not contain visible MAPK, but some oocytes were mature, and the majority of these oocytes contained MAPK. Incubation of oocytes resulted in a significant increase in the proportion of matured oocytes and in the percentage of oocytes containing MAPK in both the matured and not matured groups. Addition of IGF-I to the culture medium increased the proportion of matured oocytes, addition of leptin decreased it, and ghrelin and obestatin did not oocyte maturation. Addition of hormones did not affect the expression of MAPK in either immature or mature oocytes. PD98059, when given alone, suppressed the maturation and accumulation of MAPK in both mature and immature oocytes. When given together with hormones, PD98059 was able to reduce the stimulatory effect of IGF-I, to invert the inhibitory action of leptin to stimulatory and to induce the stimulatory action of ghrelin and obestatin on meiosis. IGF-I, ghrelin and obestatin, but not leptin, when given together with PD98059, increased the accumulation of MAPK in both immature and mature oocytes. Association of nuclear maturation and expression of MAPK in oocytes before, but not after culture, as well as the prevention of oocyte maturation by MAPK blocker suggests the involvement of MAPK-dependent intracellular mechanisms in the promotion of reinitiation, but not completion of meiosis. The effect of hormonal additions on meiosis of oocytes suggests that IGF-I is a stimulator, leptin can be an inhibitor, while ghrelin and obestatin probably do not control oocyte maturation. The ability of PD98059 to modify the effect of hormones on oocyte maturation and on MAPK expression suggests possible interference of hormones and MAPK-dependent intracellular mechanisms in oocytes. However, no influence of hormones on MAPK and lack of association between action of hormones and PD98059 on MAPK and meiosis suggest that MAPK is probably not a mediator of effect of IGF-I, leptin, ghrelin and obestatin on porcine oocyte nuclear maturation.     

Secondary alcohol dehydrogenase catalyzes the reduction of exogenous acetone to 2-propanol in Trichomonas vaginalis

Secondary alcohols such as 2-propanol are readily produced by various anaerobic bacteria that possess secondary alcohol dehydrogenase (S-ADH), although production of 2-propanol is rare in eukaryotes. Specific bacterial-type S-ADH has been identified in a few unicellular eukaryotes, but its function is not known and the production of secondary alcohols has not been studied. We purified and characterized S-ADH from the human pathogen Trichomonas?vaginalis. The kinetic properties and thermostability of T.?vaginalis S-ADH were comparable with bacterial orthologues. The substantial activity of S-ADH in the parasite's cytosol was surprising, because only low amounts of ethanol and trace amounts of secondary alcohols were detected as metabolic end products. However, S-ADH provided the parasite with a high capacity to scavenge and reduce external acetone to 2-propanol. To maintain redox balance, the demand for reducing power to metabolize external acetone was compensated for by decreased cytosolic reduction of pyruvate to lactate and by hydrogenosomal metabolism of pyruvate. We speculate that hydrogen might be utilized to maintain cytosolic reducing power. The high activity of Tv-S-ADH together with the ability of T.?vaginalis to modulate the metabolic fluxes indicate efficacious metabolic responsiveness that could be advantageous for rapid adaptation of the parasite to changes in the host environment.     

Phosphorus nutrition-mediated effects of arbuscular mycorrhiza on leaf morphology and carbon allocation in perennial ryegrass

The aim of this work was to disentangle phosphorus status-dependent and -independent effects of arbuscular mycorrhizal fungus (AMF) on leaf morphology and carbon allocation in perennial ryegrass (Lolium perenne). To this end, we assessed the P-response function of morphological components in mycorrhizal and nonmycorrhizal plants of similar size. AMF (Glomus hoi) stimulated relative P-uptake rate, decreased leaf mass per area (LMA), and increased shoot mass ratio at low P supply. Lower LMA was caused by both decreased tissue density and thickness. Variation in tissue density was almost entirely caused by variations in soluble C, while that in thickness involved structural changes. All effects of AMF were indistinguishable from those mediated by increases in relative P-uptake rate through higher P-supply rates. Thus the relationships between relative P-uptake rate, leaf morphology and C allocation were identical in mycorrhizal and nonmycorrhizal plants. No evidence was found for AMF effects not mediated by changes in plant P status.     

Organization of functional domains in the docking protein p130Cas

The docking protein p130Cas becomes phosphorylated upon cell adhesion to extracellular matrix proteins, and is thought to play an essential role in cell transformation. Cas transmits signals through interactions with the Src-homology 3 (SH3) and Src-homology 2 domains of FAK or v-Crk signaling molecules, or with 14-3-3 protein, as well as phosphatases PTP1B and PTP-PEST. The large (130kDa), multi-domain Cas molecule contains an SH3 domain, a Src-binding domain, a serine-rich protein interaction region, and a C-terminal region that participates in protein interactions implicated in antiestrogen resistance in breast cancer. In this study, as part of a long-term goal to examine the protein interactions of Cas by X-ray crystallography and nuclear magnetic resonance spectroscopy, molecular constructs were designed to express two adjacent domains, the serine-rich domain and the Src-binding domain, that each participate in intermolecular contacts dependent on protein phosphorylation. The protein products are soluble, homogeneous, monodisperse, and highly suitable for structural studies to define the role of Cas in integrin-mediated cell signaling.     

Functional fluo-3/AM assay on P-glycoprotein transport activity in L1210/VCR cells by confocal microscopy

Multidrug resistance (MDR) phenotype of L1210/VCR cell line, acquired by selection for vincristine (VCR), is predominantly mediated by P-glycoprotein (Pgp). Calcein/AM (Cal) was recently described as a fluorescent substrate for Pgp and may be used for measuring of transport activity of Pgp. Expression of Pgp in the cells prevents them to be loaded with the fluorescent marker. To detect the activity of Pgp, verapamil (Ver) or cyclosporine A (CsA) has to be used as Pgp inhibitors. Multidrug resistance protein (MRP), another drug efflux pump, may be inhibited by probenecid (Pro), i.e, the inhibitor of a wide variety of anion transporters. Ver, but not Pro, is able to induce the loading of L1210/CR cells by Cal that is measurable by fluorescence-activated cell sorter (FACS). Another dye, fluo-3/AM (F-3), has a similar behaviour like Cal. Using confocal microscopy we have proved that L1210/VCR cells, in contrast to parental sensitive cells, are not loaded with F-3. Marking of cells with the dye can be achieved using inhibitors of Pgp like Ver or CsA but not by Pro. These results indicate that F-3 is usable for detection of Pgp function in various MDR tissue cells.     

In vitro efficacy of new antiprotozoals against Philasterides dicentrarchi (Ciliophora, Scuticociliatida)

Philasterides dicentrarchi is a histiophagous ciliate that causes severe losses in turbot and sea bass farming. This study investigated the in vitro efficacy against P. dicentrarchi of 85 newly synthesized compounds and 12 commercial compounds, of which 2 are fluoroquinolones (norfloxacine and lomefloxacine) with known antibacterial activity. Seventeen of the newly synthesized compounds (2 naphthyridines, 2 pyridothienodiazines and 13 pyridothienotriazines) and the fluoroquinolone norfloxacin showed good activity. The most promising compound was the pyridothienotriazine 12k, with activity similar to that of the salicylanilides niclosamide and oxiclozanide (MLC 0.8 mg l(-1) in PBS, 1.5 mg l(-1) in seawater; MLC = minimum 24 h lethal concentration).