Human mesenchymal stem cell proliferation is regulated by PGE2 through differential activation of cAMP-dependent protein kinase isoforms

The conditions used for in vitro differentiation of hMSCs contain substances that affect the activity and expression of cyclooxygenase enzymes (COX1/COX2) and thereby the synthesis of prostanoids. hMSC constitutively produce PGE2 when cultivated in vitro. In this study we have investigated effects of PGE2 on proliferation of hMSC. We here demonstrate that one of the main control molecules in the Wnt pathway, GSK-3β, is phosphorylated at the negative regulatory site ser-9 after treating the cells with PGE2. This phosphorylation is mediated by elevation of cAMP and subsequent activation of PKA. Furthermore, PGE2 treatment leads to enhanced nuclear translocation of β-catenin, thus influencing cell proliferation. The presence of two PKA isoforms, types I and II, prompted us to investigate their individual contribution in PGE2-mediated regulation of proliferation. Specific activation of PKA type II with synthetic cAMP analogues, resulted in enhancement of proliferation. On the other side, we found that treatment of hMSC with high concentrations of PGE2 inhibited cell proliferation by arresting the cells in G₀/G₁ phase, an effect we found to be mediated by PKA I. Hence, the two different PKA isoforms seem to have opposing functions in the regulation of proliferation and differentiation in these cells.     

Filling the gap - COI barcode resolution in eastern Palearctic birds

Background

Many fish species experience long periods of fasting in nature often associated with seasonal reductions in water temperature and prey availability or spawning migrations. During periods of nutrient restriction, changes in metabolism occur to provide cellular energy via catabolic processes. Muscle is particularly affected by prolonged fasting as myofibrillar proteins act as a major energy source. To investigate the mechanisms of metabolic reorganisation with fasting and refeeding in a saltwater stage of Atlantic salmon (Salmo salar L.) we analysed the expression of genes involved in myogenesis, growth signalling, lipid biosynthesis and myofibrillar protein degradation and synthesis pathways using qPCR.

Results

Hierarchical clustering of gene expression data revealed three clusters. The first cluster comprised genes involved in lipid metabolism and triacylglycerol synthesis (ALDOB, DGAT1 and LPL) which had peak expression 3-14d after refeeding. The second cluster comprised ADIPOQ, MLC2, IGF-I and TALDO1, with peak expression 14-32d after refeeding. Cluster III contained genes strongly down regulated as an initial response to feeding and included the ubiquitin ligases MuRF1 and MAFbx, myogenic regulatory factors and some metabolic genes.

Conclusion

Early responses to refeeding in fasted salmon included the synthesis of triacylglycerols and activation of the adipogenic differentiation program. Inhibition of MuRF1 and MAFbx respectively may result in decreased degradation and concomitant increased production of myofibrillar proteins. Both of these processes preceded any increase in expression of myogenic regulatory factors and IGF-I. These responses could be a necessary strategy for an animal adapted to long periods of food deprivation whereby energy reserves are replenished prior to the resumption of myogenesis.     

New genes of Xanthomonas citri subsp. citri involved in pathogenesis and adaptation revealed by a transposon-based mutant library

Background  

Citrus canker is a disease caused by the phytopathogens Xanthomonas citri subsp. citri, Xanthomonas fuscans subsp. aurantifolli and Xanthomonas alfalfae subsp. citrumelonis. The first of the three species, which causes citrus bacterial canker type A, is the most widely spread and severe, attacking all citrus species. In Brazil, this species is the most important, being found in practically all areas where citrus canker has been detected. Like most phytobacterioses, there is no efficient way to control citrus canker. Considering the importance of the disease worldwide, investigation is needed to accurately detect which genes are related to the pathogen-host adaptation process and which are associated with pathogenesis.     

The serendipitous origin of chordate secretin peptide family members

Background  

The secretin family is a pleotropic group of brain-gut peptides with affinity for class 2 G-protein coupled receptors (secretin family GPCRs) proposed to have emerged early in the metazoan radiation via gene or genome duplications. In human, 10 members exist and sequence and functional homologues and ligand-receptor pairs have been characterised in representatives of most vertebrate classes. Secretin-like family GPCR homologues have also been isolated in non-vertebrate genomes however their corresponding ligands have not been convincingly identified and their evolution remains enigmatic.     

Isolation from the microsomal fraction of rat liver of a subfraction highly enriched in uncoated endocytic vesicles with high H+-ATPase activity and a 50 kDa phosphoprotein

A subcellular fraction, highly enriched in uncoated vesicles (UCV) with high H+-ATPase (EC 3.6.1.34) activity, was isolated from the crude microsomal fraction of rat liver homogenates by discontinuous sucrose gradient centrifugation. The UCV fraction, recovered at the interface of sucrose density 1.08 and 1.10 g/ml, was shown morphologically to be a mixture of small, smooth-surfaced univesicular and a few multivesicular structures. A permeable anion (e.g. chloride) was required for internal acidification, indicating an electroneutral proton pump. Specific inhibitors of anion transport (pyridoxal 5'-phosphate and 4-acetamide-4'-isothiocyanostilbene-2,2'-disulfonic acid) totally inhibit proton translocation. The proton pump activity was insensitive to oligomycin, but was completely inhibited by about 5 microM of the tridentate bathophenanthroline chelate of Fe(II). The activity was also inhibited 100% by low concentrations of the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone, the proton conduction inhibitor N,N'-dicyclohexylcarbodiimide and the ionophore monensin. The UCV fraction contained 2 proteins of Mr 50000 (major) and 54000 (minor) which were phosphorylated by an endogenous cyclic nucleotide- and Ca2+-independent protein kinase.     

Persistence of duplicated PAC<Subscript>1</Subscript> receptors in the teleost, <Emphasis Type="Italic">Sparus auratus</Emphasis>

Background:  

Duplicated genes are common in vertebrate genomes. Their persistence is assumed to be either a consequence of gain of novel function (neofunctionalisation) or partitioning of the function of the ancestral molecule (sub-functionalisation). Surprisingly few studies have evaluated the extent of such modifications despite the numerous duplicated receptor and ligand genes identified in vertebrate genomes to date. In order to study the importance of function in the maintenance of duplicated genes, sea bream (Sparus auratus) PAC₁ receptors, sequence homologues of the mammalian receptor specific for PACAP (Pituitary Adenylate Cyclase-Activating Polypeptide), were studied. These receptors belong to family 2 GPCRs and most of their members are duplicated in teleosts although the reason why both persist in the genome is unknown.     

Draft Genome Sequence of the Methane-Oxidizing Bacterium Methylococcus capsulatus (Texas)

Methanotrophic bacteria perform major roles in global carbon cycles via their unique enzymatic activities that enable the oxidation of one-carbon compounds, most notably methane. Here we describe the annotated draft genome sequence of the aerobic methanotroph Methylococcus capsulatus (Texas), a type strain originally isolated from sewer sludge.     

Nano-biolistics: a method of biolistic transfection of cells and tissues using a gene gun with novel nanometer-sized projectiles

Background

Biolistic transfection is proving an increasingly popular method of incorporating DNA or RNA into cells that are difficult to transfect using traditional methods. The technique routinely uses 'microparticles', which are ~1 μm diameter projectiles, fired into tissues using pressurised gas. These microparticles are efficient at delivering DNA into cells, but cannot efficiently transfect small cells and may cause significant tissue damage, thus limiting their potential usefulness. Here we describe the use of 40 nm diameter projectiles - nanoparticles - in biolistic transfections to determine if they are a suitable alternative to microparticles.

Results

Examination of transfection efficiencies in HEK293 cells, using a range of conditions including different DNA concentrations and different preparation procedures, reveals similar behaviour of microparticles and nanoparticles. The use of nanoparticles, however, resulted in ~30% fewer damaged HEK293 cells following transfection. Biolistic transfection of mouse ear tissue revealed similar depth penetration for the two types of particles, and also showed that < 10% of nuclei were damaged in nanoparticle-transfected samples, compared to > 20% in microparticle-transfected samples. Visualising details of small cellular structures was also considerably enhanced when using nanoparticles.

Conclusions

We conclude that nanoparticles are as efficient for biolistic transfection as microparticles, and are more appropriate for use in small cells, when examining cellular structures and/or where tissue damage is a problem.     

Assessing horizontal transfer of nifHDK genes in eubacteria: nucleotide sequence of nifK from Frankia strain HFPCcI3

The structural genes for nitrogenase, nifK, nifD, and nifH, are crucial for nitrogen fixation. Previous phylogenetic analysis of the amino acid sequence of nifH suggested that this gene had been horizontally transferred from a proteobacterium to the gram-positive/cyanobacterial clade, although the confounding effects of paralogous comparisons made interpretation of the data difficult. An additional test of nif gene horizontal transfer using nifD was made, but the NifD phylogeny lacked resolution. Here nif gene phylogeny is addressed with a phylogenetic analysis of a third and longer nif gene, nifK. As part of the study, the nifK gene of the key taxon Frankia was sequenced. Parsimony and some distance analyses of the nifK amino acid sequences provide support for vertical descent of nifK, but other distance trees provide support for the lateral transfer of the gene. Bootstrap support was found for both hypotheses in all trees; the nifK data do not definitively favor one or the other hypothesis. A parsimony analysis of NifH provides support for horizontal transfer in accord with previous reports, although bootstrap analysis also shows some support for vertical descent of the orthologous nifH genes. A wider sampling of taxa and more sophisticated methods of phylogenetic inference are needed to understand the evolution of nif genes. The nif genes may also be powerful phylogenetic tools. If nifK evolved by vertical descent, it provides strong evidence that the cyanobacteria and proteobacteria are sister groups to the exclusion of the firmicutes, whereas 16S rRNA sequences are unable to resolve the relationships of these three major eubacterial lineages.