EP2 receptor mediated cAMP release is augmented by PGF2α activation of the FP receptor via the calcium-calmodulin pathway

Prostaglandins exert their effects on target cells by coupling to specific G protein-coupled receptors (GPCRs) that are often co-expressed in the same cells and use alternate and in some cases opposing intracellular signaling pathways. This study investigated the cross-talk that influences intracellular signaling and gene expression profiling in response to co-activation of the EP2 and FP prostanoid receptors in Ishikawa cells stably expressing both receptors (FPEP2 cells). In this study we show that in FPEP2 cells, PGF alone does not alter adenosine 3′,5′-cyclic monophosphate (cAMP) production, but in combination with Butaprost enhances EP2 receptor mediated cAMP release compared to treatment with Butaprost alone. PGF-mediated potentiation of cAMP release was abolished by antagonism of the FP receptor, inhibition of phospholipase C (PLC) and inositol phosphate receptor (IP3R) whereas inhibition of protein kinase C (PKC) had no effect. Moreover, inhibition of calcium effectors using calmodulin antagonist (W7) or Ca²⁺/calmodulin-dependent kinase II (CaMK-II) inhibitor (KN-93) abolished PGF potentiation of Butaprost-mediated cAMP release. Using siRNA molecules targeted against the adenylyl cyclase 3 (AC3) isoform, we show that AC3 is responsible for the cross-talk between the FP and EP2 receptors. Using gene array studies we have identified a candidate gene, Spermidine/N1-acetyltransferase (SAT1), which is regulated by this cAMP mediated cross-talk. In conclusion, this study demonstrates that co-activation of the FP and EP2 receptors results in enhanced release of cAMP via FP receptor-Gα_q-Ca²⁺-calmodulin pathway by activating calcium sensitive AC3 isoform.     

Synthesis and in vitro sodium channel blocking activity evaluation of novel homochiral mexiletine analogs

New chiral mexiletine analogs were synthesized in their optically active forms and evaluated in vitro as use‐dependent blockers of skeletal muscle sodium channels. Tests carried out on sodium currents of single muscle fibers of Rana esculenta demonstrated that all of them exerted a higher use‐dependent block than mexiletine. The most potent analog, (S)‐3‐(2,6‐dimethylphenoxy)‐1‐phenylpropan‐1‐amine (S)‐( 5 ), was six‐fold more potent than (R)‐Mex in producing a tonic block. As observed with mexiletine, the newly synthesized compounds exhibit modest enantioselective behavior, that is more evident in 3‐(2,6‐dimethylphenoxy)butan‐1‐amine ( 3 ). Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Synthesis of a novel tricyclic 1,2,3,4,4a,5,6,10b-octahydro-1,10-phenanthroline ring system and CXCR4 antagonists with potent activity against HIV-1

Stereorandom and diastereoselective syntheses of a novel 1,2,3,4,4a,5,6,10b-octahydro-1,10-phenanthroline ring system are described. Derivatives of all four diastereomers were prepared and isolated in >98% ee. The pure enantiomers were compared in order to determine the preferred absolute and relative configuration required for optimal anti-HIV activity. Anti-HIV potency and pharmacokinetic properties of the newly synthesized tricyclic octahydrophenanthroline inhibitors are presented and comparisons are made to previously reported bicyclic (8S)-N-methyl-5,6,7,8-tetrahydro-8-quinolinamine analogs.     

Combining two Meishan F2 crosses improves the detection of QTL on pig chromosomes 2, 4 and 6

Background

In pig, a number of experiments have been set up to identify QTL and a multitude of chromosomal regions harbouring genes influencing traits of interest have been identified. However, the mapping resolution remains limited in most cases and the detected QTL are rather inaccurately located. Mapping accuracy can be improved by increasing the number of phenotyped and genotyped individuals and/or the number of informative markers. An alternative approach to overcome the limited power of individual studies is to combine data from two or more independent designs.

Methods

In the present study we report a combined analysis of two independent design (a French and a Dutch F2 experimental designs), with 2000 F2 individuals. The purpose was to further map QTL for growth and fatness on pig chromosomes 2, 4 and 6. Using QTL-map software, uni- and multiple-QTL detection analyses were applied separately on the two pedigrees and then on the combination of the two pedigrees.

Results

Joint analyses of the combined pedigree provided (1) greater significance of shared QTL, (2) exclusion of false suggestive QTL and (3) greater mapping precision for shared QTL.

Conclusions

Combining two Meishan x European breeds F2 pedigrees improved the mapping of QTL compared to analysing pedigrees separately. Our work was facilitated by the access to raw phenotypic data and DNA of animals from both pedigrees and the combination of the two designs with the addition of new markers allowed us to fine map QTL without phenotyping additional animals.     

A gene frequency model for QTL mapping using Bayesian inference

Background

Information for mapping of quantitative trait loci (QTL) comes from two sources: linkage disequilibrium (non-random association of allele states) and cosegregation (non-random association of allele origin). Information from LD can be captured by modeling conditional means and variances at the QTL given marker information. Similarly, information from cosegregation can be captured by modeling conditional covariances. Here, we consider a Bayesian model based on gene frequency (BGF) where both conditional means and variances are modeled as a function of the conditional gene frequencies at the QTL. The parameters in this model include these gene frequencies, additive effect of the QTL, its location, and the residual variance. Bayesian methodology was used to estimate these parameters. The priors used were: logit-normal for gene frequencies, normal for the additive effect, uniform for location, and inverse chi-square for the residual variance. Computer simulation was used to compare the power to detect and accuracy to map QTL by this method with those from least squares analysis using a regression model (LSR).

Results

To simplify the analysis, data from unrelated individuals in a purebred population were simulated, where only LD information contributes to map the QTL. LD was simulated in a chromosomal segment of 1 cM with one QTL by random mating in a population of size 500 for 1000 generations and in a population of size 100 for 50 generations. The comparison was studied under a range of conditions, which included SNP density of 0.1, 0.05 or 0.02 cM, sample size of 500 or 1000, and phenotypic variance explained by QTL of 2 or 5%. Both 1 and 2-SNP models were considered. Power to detect the QTL for the BGF, ranged from 0.4 to 0.99, and close or equal to the power of the regression using least squares (LSR). Precision to map QTL position of BGF, quantified by the mean absolute error, ranged from 0.11 to 0.21 cM for BGF, and was better than the precision of LSR, which ranged from 0.12 to 0.25 cM.

Conclusions

In conclusion given a high SNP density, the gene frequency model can be used to map QTL with considerable accuracy even within a 1 cM region.     

Gene expression profiling of early intervertebral disc degeneration reveals a down-regulation of canonical Wnt signaling and caveolin-1 expression: implications for development of regenerative strategies

Introduction

Early degeneration of the intervertebral disc (IVD) involves a change in cellular differentiation from notochordal cells (NCs) in the nucleus pulposus (NP) to chondrocyte-like cells (CLCs). The purpose of this study was to investigate the gene expression profiles involved in this process using NP tissue from non-chondrodystrophic and chondrodystrophic dogs, a species with naturally occurring IVD degeneration.

Methods

Dual channel DNA microarrays were used to compare 1) healthy NP tissue containing only NCs (NC-rich), 2) NP tissue with a mixed population of NCs and CLCs (Mixed), and 3) NP tissue containing solely CLCs (CLC-rich) in both non-chondrodystrophic and chondrodystrophic dogs. Based on previous reports and the findings of the microarray analyses, canonical Wnt signaling was further evaluated using qPCR of relevant Wnt target genes. We hypothesized that caveolin-1, a regulator of Wnt signaling that showed significant changes in gene expression in the microarray analyses, played a significant role in early IVD degeneration. Caveolin-1 expression was investigated in IVD tissue sections and in cultured NCs. To investigate the significance of Caveolin-1 in IVD health and degeneration, the NP of 3-month-old Caveolin-1 knock-out mice was histopathologically evaluated and compared with the NP of wild-type mice of the same age.

Results

Early IVD degeneration involved significant changes in numerous pathways, including Wnt/β-catenin signaling. With regard to Wnt/β-catenin signaling, axin2 gene expression was significantly higher in chondrodystrophic dogs compared with non-chondrodystrophic dogs. IVD degeneration involved significant down-regulation of axin2 gene expression. IVD degeneration involved significant down-regulation in Caveolin-1 gene and protein expression. NCs showed abundant caveolin-1 expression in vivo and in vitro, whereas CLCs did not. The NP of wild-type mice was rich in viable NCs, whereas the NP of Caveolin-1 knock-out mice contained chondroid-like matrix with mainly apoptotic, small, rounded cells.

Conclusions

Early IVD degeneration involves down-regulation of canonical Wnt signaling and Caveolin-1 expression, which appears to be essential to the physiology and preservation of NCs. Therefore, Caveolin-1 may be regarded an exciting target for developing strategies for IVD regeneration.     

SIMPLE: implementation of recommendations from international evidence-based guidelines on caesarean sections in the Netherlands. Protocol for a controlled before and after study

Background

Caesarean section (CS) rates are rising worldwide. In the Netherlands, the most significant rise is observed in healthy women with a singleton in vertex position between 37 and 42 weeks gestation, whereas it is doubtful whether an improved outcome for the mother or her child was obtained. It can be hypothesized that evidence-based guidelines on CS are not implemented sufficiently. Therefore, the present study has the following objectives: to develop quality indicators on the decision to perform a CS based on key recommendations from national and international guidelines; to use the quality indicators in order to gain insight into actual adherence of Dutch gynaecologists to guideline recommendations on the performance of a CS; to explore barriers and facilitators that have a direct effect on guideline application regarding CS; and to develop, execute, and evaluate a strategy in order to reduce the CS incidence for a similar neonatal outcome (based on the information gathered in the second and third objectives).

Methods

An independent expert panel of Dutch gynaecologists and midwives will develop a set of quality indicators on the decision to perform a CS. These indicators will be used to measure current care in 20 hospitals with a population of 1,000 women who delivered by CS, and a random selection of 1,000 women who delivered vaginally in the same period. Furthermore, by interviewing healthcare professionals and patients, the barriers and facilitators that may influence the decision to perform a CS will be measured. Based on the results, a tailor-made implementation strategy will be developed and tested in a controlled before-and-after study in 12 hospitals (six intervention, six control hospitals) with regard to effectiveness, experiences, and costs.

Discussion

This study will offer insight into the current CS care and into the hindering and facilitating factors influencing obstetrical policy on CS. Furthermore, it will allow definition of patient categories or situations in which a tailor-made implementation strategy will most likely be meaningful and cost effective, without negatively affecting the outcome for mother and child.

Trial registration

http://www.clinicaltrials.gov: NCT01261676     

A Pseudo-Atomic Model for the Capsid Shell of Bacteriophage Lambda Using Chemical Cross-Linking/Mass Spectrometry and Molecular Modeling

Bacteriophage lambda is one of the most exhaustively studied of the double-stranded DNA viruses. Its assembly pathway is highly conserved among the herpesviruses and many of the bacteriophages, making it an excellent model system. Despite extensive genetic and biophysical characterization of many of the lambda proteins and the assembly pathways in which they are implicated, there is a relative dearth of structural information on many of the most critical proteins involved in lambda assembly and maturation, including that of the lambda major capsid protein. Toward this end, we have utilized a combination of chemical cross-linking/mass spectrometry and computational modeling to construct a pseudo-atomic model of the lambda major capsid protein as a monomer, as well as in the context of the assembled procapsid shell. The approach described here is generalizable and can be used to provide structural models for any biological complex of interest. The procapsid structural model is in good agreement with published biochemical data indicating that procapsid expansion exposes hydrophobic surface area and that this serves to nucleate assembly of capsid decoration protein, gpD. The model further implicates additional molecular interactions that may be critical to the assembly of the capsid shell and for the stabilization of the structure by the gpD decoration protein.     

Five new species of dicyemid mesozoans (Dicyemida: Dicyemidae) from two Australian cuttlefish species, with comments on dicyemid fauna composition

Five new species of dicyemid mesozoans in two genera are described from two Australian cuttlefish species, Sepia apama Gray (giant Australian cuttlefish) and S. novaehollandiae Hoyle (nova cuttlefish): Dicyema coffinense n. sp. from S. apama collected from Coffin Bay, South Australia (SA), Australia; D. koinonum n. sp. from S. apama and S. novaehollandiae collected from Gulf St Vincent (GSV) and Spencer Gulf (SG), SA, Australia; D. multimegalum n. sp. from S. apama collected from Cronulla and North Bondi, New South Wales, Australia; D. vincentense n. sp. from S. novaehollandiae collected from GSV, SA, Australia; and Dicyemennea spencerense n. sp. from S. novaehollandiae and S. apama collected from SG, SA, Australia. Totals of 51 S. apama and 27 S. novaehollandiae individuals were examined, of which all except for four S. apama were infected by at least one dicyemid species. Dicyemid parasites were also observed in host individuals that were held in tanks for 2–3 months prior to examination, including nematogen-exclusive infections, leading to questions about persistence of dicyemids after host death and the mechanism responsible for the switch between a nematogen phase and a rhombogen phase. Variations in host size, calotte shape and collection locality are explored as predictors of differences in observed composition of the parasite fauna. In particular, dicyemid parasite fauna varied with host collection locality. As these parasites are highly host-species specific, their use as biological tags to assess cephalopod population structure using a combined morphological and molecular approach is discussed. This study increases the number of dicyemid species described from Australian cephalopods from five to ten, and from 117 to 122 species described worldwide.     

Early ontogeny of the mackerel icefish, Champsocephalus gunnari, in the southern Scotia Arc

During the early ontogeny of fishes, the timing and duration of key events such as larval hatching and the switch from endogenous to exogenous feeding largely determine the offspring viability and survival. The aim of the present study was to investigate the life history traits of the early larvae of the mackerel icefish, Champsocephalus gunnari, collected in summer south of the South Shetland Islands in the Bransfield Strait and north of Elephant Island. Through the analysis of sagittal otolith microstructure, we assessed the timing and duration of egg incubation, larval hatching, first exogenous feeding, rate of yolk resorption and body growth rate. Compared to populations living further north (i.e. around South Georgia and Kerguelen Islands), mackerel icefish in the southern Scotia Arc exhibits longer egg incubation (lasting 90–120 days from winter to summer) and delayed hatching time spread over a relatively short period lasting 26 days between January and February. The first exogenous feeding takes place between 13 and 24 days after hatching still in the presence of the yolk-sac, indicating a prolonged mixed feeding afterward. The specific growth rate or daily percentage change in size (G) was 1.9 % SL day^?1, corresponding to a daily growth rate at mean size of 0.31 mm day^?1. While showing significant differences in early life history traits across their geographical distribution, C. gunnari populations share a common strategy, spawning a small number of large eggs that hatch in relatively large-sized larvae, at a time which may be independent of the timing of pack-ice retreat and onset of the production cycle.