Reconstructing sibling relationships in wild populations

Reconstruction of sibling relationships from genetic data is an important component of many biological applications. In particular, the growing application of molecular markers (microsatellites) to study wild populations of plant and animals has created the need for new computational methods of establishing pedigree relationships, such as sibgroups, among individuals in these populations. Most current methods for sibship reconstruction from microsatellite data use statistical and heuristic techniques that rely on a priori knowledge about various parameter distributions. Moreover, these methods are designed for data with large number of sampled loci and small family groups, both of which typically do not hold for wild populations. We present a deterministic technique that parsimoniously reconstructs sibling groups using only Mendelian laws of inheritance. We validate our approach using both simulated and real biological data and compare it to other methods. Our method is highly accurate on real data and compares favorably with other methods on simulated data with few loci and large family groups. It is the only method that does not rely on a priori knowledge about the population under study. Thus, our method is particularly appropriate for reconstructing sibling groups in wild populations.     

Capture of flowing human neutrophils by immobilised immunoglobulin: roles of Fc-receptors CD16 and CD32

We investigated capture and activation of flowing human neutrophils through their Fc-receptors, FcRgammaIIIB (CD16) and FcRgammaIIA (CD32). Immobilised platelets bearing murine monoclonal antibody against glycoprotein IIbIIIA were able to capture and activate flowing neutrophils. The activation response was inhibited by antibody blockade of neutrophil CD32. However, capture only occurred efficiently at wall shear stress below 0.1 Pa if platelet P-selectin was blocked. If neutrophils were perfused over immobilised human IgG, many adhered at 0.025 or 0.05 Pa, but not at 0.1 Pa. Adhesion was reduced by blockade of CD16 or CD32, but blockade of CD16 had the greater effect. When neutrophils were perfused over a combination of purified P-selectin and IgG, blockade of CD16 and CD32 inhibited activation of captured cells. Immunoglobulin deposited in tissue could capture and activate slow-flowing neutrophils. It might also potentiate inflammatory responses at higher stress if presented along with selectins. The dominant FcR for capture of neutrophils was CD16, but with murine antibody, CD32 played a greater role.     

Extracellular calcium sensing in rat aortic vascular smooth muscle cells

Extracellular calcium (Ca(2+)(o)) can act as a first messenger in many cell types through a G protein-coupled receptor, calcium-sensing receptor (CaR). It is still debated whether the CaR is expressed in vascular smooth muscle cells (VSMCs). Here, we report the expression of CaR mRNA and protein in rat aortic VSMCs and show that Ca(2+)(o) stimulates proliferation of the cells. The effects of Ca(2+)(o) were attenuated by pre-treatment with MAPK kinase 1 (MEK1) inhibitor, as well as an allosteric modulator, NPS 2390. Furthermore, stimulation of the VSMCs with Ca(2+)(o)-induced phosphorylation of ERK1/2, but surprisingly did not cause inositol phosphate accumulation. We were not able to conclusively state that the CaR mediates Ca(2+)(o)-induced cell proliferation. Rather, an additional calcium-sensing mechanism may exist. Our findings may be of importance with regard to atherosclerosis, an inflammatory disease characterized by abnormal proliferation of VSMCs and high local levels of calcium.     

Calcium receptor is functionally expressed in rat neonatal ventricular cardiomyocytes

Both intra- and extracellular calcium play multiple roles in the physiology and pathophysiology of cardiomyocytes, especially in stimulus-contraction coupling. The intracellular calcium level is closely controlled through the concerted actions of calcium channels, exchangers, and pumps; however, the expression and function(s) of the so-called calcium-sensing receptor (CaR) in the heart remain less well characterized. The CaR is a seven-transmembrane receptor, which, in response to noncovalent binding of extracellular calcium, activates intracellular effectors, including G proteins and extracellular signal-regulated kinases (ERK1/2). We have shown that cultured neonatal cardiomyocytes express the CaR messenger RNA and the CaR protein. Furthermore, increasing concentrations of extracellular calcium and a type II CaR activator "calcimimetic" caused inositol phosphate (IP) accumulation, downregulated tritiated thymidine incorporation, and supported ERK1/2 phosphorylation, suggesting that the CaR protein is functionally active. Interestingly, the calcimimetic induced a more rapid ERK1/2 phosphorylation than calcium and left-shifted the IP concentration-response curve for extracellular calcium, supporting the hypothesis that CaR is functionally expressed in cardiac myocytes. This notion was underscored by studies using a virus containing a dominant-negative CaR construct, because this protein blunted the calcium-induced IP response. In conclusion, we have shown that the CaR is functionally expressed in neonatal ventricular cardiomyocytes and that the receptor activates second messenger pathways, including IP and ERK, and decreases DNA synthesis. A specific calcium-sensing receptor on cardiac myocytes could play a role in regulating cardiac development, function, and homeostasis.     

EilA, a HilA-like regulator in enteroaggregative Escherichia coli

Enteroaggregative Escherichia coli (EAEC) is increasingly recognized as a diarrhoeal pathogen in developing and industrialized countries. Most EAEC virulence factors thus far described are encoded on virulence plasmid pAA, yet recent completion of the EAEC genome has suggested the presence of additional factors encoded on chromosomal islands. Previous reports have recognized the presence of a type III secretion system (T3SS), designated ETT2, at the glyU locus of prototype EAEC strain 042, along with possible T3SS effectors at the selC locus. The selC locus was also noted to harbour homologues of Salmonella enterica regulator HilA and of invasin from Yersinia spp., yet previous publications suggested that these loci may be silent. Here, we show that the genes of the selC locus are present inconsistently among a collection of well-characterized EAEC strains. Notably, however, there was perfect correlation between the presence of hilA-homologue eilA and predicted Yersinia invasin homologue gene eaeX. We hypothesized that if expressed, the putative gene product EilA would contribute to EAEC virulence in part by activation of the T3SS and its effectors. An eilA mutant was constructed in EAEC strain 042, and complementation was achieved by cloning the eilA gene under control of an arabinose-dependent promoter. In this system, we observed expression of at least seven genes to be affected by expression of eilA, either directly or indirectly: selC locus genes eipB, eipC, eipD, eicA and eaeX (renamed here air), as well as glyU ETT2 genes eivF and eivA. Notably, the eilA mutant was shown to be less adherent to epithelial cells in culture and to form less abundant biofilms than the isogenic parent. These effects were recapitulated in the air mutant, suggesting that the predicted outer membrane protein product of the air gene is involved as an accessory adhesin and aggregin of EAEC, coexpressed with the T3SS. Our data suggest that the T3SS of EAEC and presumed effectors located on different chromosomal islands may be coordinately activated by EilA, which also activates the genetically linked high molecular weight bacterial surface protein Air. Contributions of this new putative virulence-related regulon in EAEC may include adherence, aggregation, and as yet uncharacterized roles for the T3SS.     

Inhibition of Na+-Taurocholate Co-transporting Polypeptide-mediated Bile Acid Transport by Cholestatic Sulfated Progesterone Metabolites

Sulfated progesterone metabolite (P4-S) levels are raised in normal pregnancy and elevated further in intrahepatic cholestasis of pregnancy (ICP), a bile acid-liver disorder of pregnancy. ICP can be complicated by preterm labor and intrauterine death. The impact of P4-S on bile acid uptake was studied using two experimental models of hepatic uptake of bile acids, namely cultured primary human hepatocytes (PHH) and Na⁺-taurocholate co-transporting polypeptide (NTCP)-expressing Xenopus laevis oocytes. Two P4-S compounds, allopregnanolone-sulfate (PM4-S) and epiallopregnanolone-sulfate (PM5-S), reduced [³H]taurocholate (TC) uptake in a dose-dependent manner in PHH, with both Na⁺-dependent and -independent bile acid uptake systems significantly inhibited. PM5-S-mediated inhibition of TC uptake could be reversed by increasing the TC concentration against a fixed PM5-S dose indicating competitive inhibition. Experiments using NTCP-expressing Xenopus oocytes confirmed that PM4-S/PM5-S are capable of competitively inhibiting NTCP-mediated uptake of [³H]TC. Total serum PM4-S + PM5-S levels were measured in non-pregnant and third trimester pregnant women using liquid chromatography-electrospray tandem mass spectrometry and were increased in pregnant women, at levels capable of inhibiting TC uptake. In conclusion, pregnancy levels of P4-S can inhibit Na⁺-dependent and -independent influx of taurocholate in PHH and cause competitive inhibition of NTCP-mediated uptake of taurocholate in Xenopus oocytes.     

Bile Acid-Induced Arrhythmia Is Mediated by Muscarinic M2 Receptors in Neonatal Rat Cardiomyocytes

Background

Intrahepatic cholestasis of pregnancy (ICP) is a common disease affecting up to 5% of pregnancies and which can cause fetal arrhythmia and sudden intrauterine death. We previously demonstrated that bile acid taurocholate (TC), which is raised in the bloodstream of ICP, can acutely alter the rate and rhythm of contraction and induce abnormal calcium destabilization in cultured neonatal rat cardiomyocytes (NRCM). Apart from their hepatic functions bile acids are ubiquitous signalling molecules with diverse systemic effects mediated by either the nuclear receptor FXR or by a recently discovered G-protein coupled receptor TGR5. We aim to investigate the mechanism of bile-acid induced arrhythmogenic effects in an in-vitro model of the fetal heart.

Methods and Results

Levels of bile acid transporters and nuclear receptor FXR were studied by quantitative real time PCR, western blot and immunostaining, which showed low levels of expression. We did not observe functional involvement of the canonical receptors FXR and TGR5. Instead, we found that TC binds to the muscarinic M₂ receptor in NRCM and serves as a partial agonist of this receptor in terms of inhibitory effect on intracellular cAMP and negative chronotropic response. Pharmacological inhibition and siRNA-knockdown of the M₂ receptor completely abolished the negative effect of TC on contraction, calcium transient amplitude and synchronisation in NRCM clusters.

Conclusion

We conclude that in NRCM the TC-induced arrhythmia is mediated by the partial agonism at the M₂ receptor. This mechanism might serve as a promising new therapeutic target for fetal arrhythmia.