Effects of cyclic AMP and butyrate on cell cycle,DNA, RNA,and purine synthesis of cultured astrocytes

Dibutyryl cyclic monophosphate (dBcAMP) has been shown to inhibit growth, and alter the morphology of astrocytes. However, the potential contribution of its hydrolytic product, butyrate, in inducing some of the changes that have been attributed to dBcAMP, is not clear. DNA, RNA, and purine synthesis were therefore studied in primary astrocyte cultures after 24 hours of exposure to varying concentrations of butyrate, dBcAMP, and agents that increase intracellular cAMP levels. Progression of cells through cell cycle was also studied by flow cytometry. Dibutyryl cAMP partially arrested cells in Go/G1 phase of cell cycle while sodium butyrate increased the percentage population of cells in G2/M phase. DNA synthesis and de novo purine synthesis were inhibited after treatment with dBcAMP, sodium butyrate, and various drugs that increase intracellular cAMP levels. RNA synthesis was increased with cAMP but was not affected by sodium butyrate. Our study shows that at millimolar concentrations, butyrate is capable of altering the cell cycle and inhibiting DNA synthesis in primary astrocyte cultures, in a manner that is similar although not identical to the effects of dBcAMP.     

Cryptic dispersal of Cyanidiophytina (Rhodophyta) in non-acidic environments from Turkey

Extremophiles - Cyanidiophytina are a group of polyextremophilic red algae with a worldwide, but discontinuous colonization. They are restricted to widely dispersed hot springs, geothermal...     

Crosstalk between keratinocytes and T lymphocytes via Fas/Fas ligand interaction: modulation by cytokines.

Apoptosis mediated by Fas/FasL interaction plays an important role during many inflammatory skin disorders. To estimate whether the expression of FasL, the ligand for Fas, might be regulated by cytokines we stimulated primary human keratinocytes with several pro- and anti-inflammatory cytokines. Keratinocytes cultured to subconfluence expressed FasL constitutively. Cells stimulated with the proinflammatory cytokines IL-1beta, TNF-alpha, IFN-gamma, and IL-15, respectively, increased significantly their intracellular as well as cell surface-bound FasL expression in a time- and dose-dependent manner. This cytokine-induced FasL expression was dependent on new protein synthesis. Despite enhanced expression of cell surface-bound FasL, no release of soluble FasL was measured in the cell supernatants determined by ELISA. Stimulation of the cells with IL-6, IL-10, IL-12, TGF-beta1, and GM-CSF did not modulate the constitutive FasL expression, but IFN-gamma-mediated FasL up-regulation was significantly diminished by IL-10 and TGF-beta1, respectively. Up-regulation of FasL on IFN-gamma-stimulated keratinocytes led to increased apoptosis within monolayers cultured for 48 h. Moreover, coculture experiments performed with Fas+ Jurkat T cells revealed that enhanced FasL expression on IFN-gamma-stimulated keratinocytes induced apoptosis in cocultured T cells, demonstrating that up-regulated FasL was functionally active. In summary, our data suggest the important regulatory role of cytokine-controlled Fas/FasL interaction in the cross-talk between keratinocytes and skin-infiltrating T cells for maintenance of homeostasis in inflammatory skin processes.     

Functional characterization of apolipoprotein E isoforms overexpressed in Escherichia coli.

Apolipoprotein (apo) E plays an important role in lipid metabolism, and the major isoforms of apoE (apoE2, apoE3, and apoE4) have significantly different metabolic effects. Apolipoprotein E4 is associated with a higher risk of both heart disease and Alzheimer's disease (AD). Patients homozygous for apolipoprotein E2 are predisposed to type III hyperlipoproteinemia, and apoE2 may be protective against AD. Structure/function studies have proved to be a useful tool in understanding how the different apoE isoforms result in different pathological consequences. As these studies continue, it is essential to have a reliable method to produce large quantities of apoE and mutants of apoE. We describe here a method of apoE production in Escherichia coli strain BL21(DE3). The cDNA from apoE isoforms was inserted into a pET32a vector with a T7 promoter and a fusion partner (thioredoxin). The T7 promoter results in high expression of an easily purified His-tagged fusion protein. A thrombin recognition site was positioned in the expression vector so that only two novel amino acids (Gly-Ser) are added to the amino terminus of apoE following the removal of thioredoxin. Approximately 20 mg of apoE is obtained from a 1-liter culture. The major isoforms of apoE produced with this system were extensively characterized for their ability to bind the low-density lipoprotein (LDL) receptor, for their characteristic lipid association preferences, and for their stability as measured by guanidine denaturation. The recombinant proteins behaved identically to plasma-derived apoE isoforms.     

Systematics,biogeography and evolution of the endemic Hemidactylus geckos (Reptilia,Squamata, Gekkonidae) of the Cape Verde Islands: based on morphology and mitochondrial and nuclear DNA sequences

A total of 1854 bp of mitochondrial DNA (669 bp of cytochrome b (cyt b) and 386 bp of 12S rRNA), and 804 bp of a nuclear gene (RAG2) were investigated in endemic Hemidactylus from eight Cape Verde Islands, and used to explore their phylogeny, biogeography and evolution. Maximum‐likelihood, maximum‐parsimony and Bayesian analyses based on mtDNA revealed four well‐supported clades with uncorrected genetic divergences of 7.8–12.4% in the cyt b plus 12S rRNA genes, which were also supported by nuclear DNA. A population from the southern island of Fogo is the most divergent in both molecules and morphology and is described as Hemidactylus lopezjuradoi sp. n., and the populations on Sal and Boavista are also assigned species status as H. boavistensis. Although divergent in their DNA, the clade on S. Nicolau and that in the north‐western islands are morphologically similar and both are assigned to H. bouvieri for the present. Hemidactylus b. razoensis from Raso is genetically similar to H. b. bouvieri and differs only in its smaller body size. A molecular clock suggests that the ancestor of the endemic Hemidactylus of the Cape Verde Islands colonized the archipelago approximately 10 ± 2.48 Mya, perhaps reaching the north‐eastern islands first. The H. lopezjuradoi lineage separated soon after, and the north‐western islands were colonized progressively but slowly, S. Nicolau probably being reached first, then S. Vicente and islands on the same bank, and finally Sto. Antão, which is likely to have been colonized less than 1 Mya. Hemidactylus boavistensis is abundant on the arid islands where it occurs, but H. bouvieri appears to have been uncommon at least since it was described 130 years ago, and the same may be true of H. lopezjuradoi sp. n. The impact of introduced H. angulatus and H. mabouia on the endemic Hemidactylus of the Cape Verde Islands is not clear, but the discovery of substantial genetic diversity in endemic Cape Verde Hemidactylus means that the conservation requirements of the group should be reassessed.     

Comparison of environmental sampling methods for detecting Salmonella in commercial laying flocks in the UK

Aims: To investigate the performance of the Salmonella National Control Programme (NCP) sampling/testing methods in laying flocks of domestic fowl. Methods and Results: Eighty‐five visits were made to 69 flocks representative of the main production systems (cage, barn and free‐range) infected with Salmonella. In each visit, three methodologies were compared: (i) the European Union (EU) baseline survey method (five faeces and two dust samples); (ii) an in‐house (Veterinary Laboratories Agency, VLA) ‘wet’ method that involved collecting 10 dust and 10 faeces samples into jars with buffered peptone water; and (iii) a method involving two samples of pooled faeces and one of dust (cultured as one sample of each type), which has been adopted for the NCP for laying flocks across the EU. Conclusions: The ‘wet’ method was the most sensitive, and the NCP the least, although individual NCP samples were the most sensitive ones. Significance and Impact of the Study: The apparent lower sensitivity of the NCP method may be compensated by repeated sampling of flocks (twice during rear and several times during lay). Sampling using VLA methodology should be advocated for farms aiming to disclose low‐level Salmonella before restrictions on the sale of eggs from Salmonella Enteritidis or Salmonella Typhimurium‐infected flocks are in place.     

Phylogenetic analysis of the pPT23A plasmid family of Pseudomonas syringae

The pPT23A plasmid family of Pseudomonas syringae contains members that contribute to the ecological and pathogenic fitness of their P. syringae hosts. In an effort to understand the evolution of these plasmids and their hosts, we undertook a comparative analysis of the phylogeny of plasmid genes and that of conserved chromosomal genes from P. syringae. In total, comparative sequence and phylogenetic analyses were done utilizing 47 pPT23A family plasmids (PFPs) from 16 pathovars belonging to six genomospecies. Our results showed that the plasmid replication gene (repA), the only gene currently known to be distributed among all the PFPs, had a phylogeny that was distinct from that of the P. syringae hosts of these plasmids and from those of other individual genes on PFPs. The phylogenies of two housekeeping chromosomal genes, those for DNA gyrase B subunit (gyrB) and primary sigma factor (rpoD), however, were strongly associated with genomospecies of P. syringae. Based on the results from this study, we conclude that the pPT23A plasmid family represents a dynamic genome that is mobile among P. syringae pathovars.     

Enantioseparation by ligand-exchange using particle-loaded monoliths: capillary-LC versus capillary electrochromatography

Particle-loaded monoliths containing a polymethacrylamide backbone were prepared by suspending a silica-based chiral phase in the mixture of the monomers followed by in-situ polymerization in the capillary. As chiral selector l-4-hydroxyproline chemically bonded to 3 microm silica particles was used following the separation principle of ligand-exchange. Electrolytes containing Cu(II) ions were used. Amino acid enantiomers were separated by capillary-LC and CEC, whereby the latter showed the better resolution properties. For the chiral separation of alpha-hydroxy acids the EOF was reversed by copolymerizing diallyldimethylammonium chloride instead of vinylsulfonic acid as charge providing agent. Short columns of 6 cm were found to be sufficient in the case of CEC for baseline separations of amino acids with alpha values up to 5.     

Nitrous oxide emissions from drained organic forest soils––an up-scaling based on C:N ratios

Total emissions of N₂O from drained organic forest soils in Sweden were estimated using an equation linking the C:N ratio of the soil to N₂O emissions. Information on soil C:N ratios was derived from a national database. It was estimated that the emissions from Histosols amount to 2,820 tonnes N₂O a⁻¹. This is almost five times the value calculated for the same soils using the method suggested by the Intergovernmental Panel on Climate Change: 580 tonnes N₂O a⁻¹. The higher value in the present study can mainly be explained by improved accuracy of estimates of N₂O emissions from nutrient-rich soils, including former agricultural soils. In Sweden, in addition to 0.94 Mha of drained Histosols, there are 0.55 Mha of other types of drained organic soils. The annual emissions from these soils were estimated to amount to 1,890 tonnes of N₂O. The total emission value calculated for drained organic forest soils was thus 4,700 tonnes N₂O a⁻¹, which, if added, would increase the current estimate of the Swedish anthropogenic N₂O source strength by 18%. Of these emissions, 88% occur from sites with C:N ratios lower than 25. The exponential relationship between C:N ratio and N₂O emissions, in combination with a scarcity of data, resulted in large confidence intervals around the estimates. However, by using the C:N ratio-based method, N₂O emission estimates can be calculated from a variable that is readily available in databases. Also, the recent findings that there are exceptionally large emissions of N₂O from the most nitrogen-rich drained organic forest soils are taken into account.