Cloning and linkage analysis of Neisseria gonorrhoeae DNA methyltransferases.

We have cloned DNA methyltransferases (MTases) from various strains of Neisseria gonorrhoeae. Each of these clones represents a single specificity, indicating that the multiple gonococcal MTase specificities are encoded by monospecific MTases. The DNAs of five strains (FA5100, F62, MS11, Pgh3-2, and WR302) were digested with NheI, SpeI, or NheI plus SpeI and subjected to pulsed-field gel electrophoresis. The DNA MTase clones were used to probe Southern blots of these pulsed-field gels to determine whether the MTase genes are linked and whether there are strain-to-strain differences. The results indicate that none of these genes are closely linked, but variable hybridization patterns indicate that there exist restriction fragment length polymorphisms between the strains tested. Most of the chromosomal regions containing these restriction fragment length polymorphisms are clustered in regions containing gonococcal genes known or suspected to antigenically vary via genetic recombination.     

The Effect of 2,4,6-Trinitro-m-Cresol on Cation and Anion Transport in Sheep Red Blood Cells

2,4,6-Trinitro-3-methyl-phenol (trinitrocresol, H⁺TNC^-) was found to inhibit anion and stimulate cation movements across the membranes of both high potassium (HK) and low potassium (LK) sheep red blood cells. The concentration of TNC^- required to inhibit SO₄ ^- and Cl^- efflux (10^-5-10^-3 M) was less than that required to increase Na⁺ and K⁺ leakage (10^-3-10^-2 M). Both the inhibition of anion and stimulation of cation permeation were reversed if TNC^- was washed from the red cells. The cation leak caused by TNC^- was much greater at 0° and 37°C than at room temperature (23°C). In sheep red cells, TNC^- was found to be about 20 times more effective than salicylate and about 40 times more effective than thiocyanate in increasing cation leak. TNC^- also inhibited the ouabain-sensitive potassium influx.     

Characteristics of Chloride Transport in Human Red Blood Cells

The efflux of chloride-36 from human erythrocytes under steady-state conditions is a saturable process that is competitively inhibited by bicarbonate and noncompetitively inhibited by acetate. This chloride self-exchange flux is reversibly dependent on the pH of the medium between 5.7 and 9.6 with a maximum flux at pH 7.8. The increase in chloride flux between pH 5.7 and 7.8 is inexplicable by the fixed charge hypothesis. The interpretations are made that chloride transport in human erythrocytes is carrier mediated, that bicarbonate utilizes the same transport mechanism, and that the mechanism can be titrated with hydrogen ions into less functional forms for chloride transport.     

The 5' untranslated region of protein kinase Cdelta directs translation by an internal ribosome entry segment that is most active in densely growing cells and during apoptosis

Protein kinase Cdelta (PKCdelta) is a member of the PKC family of phospholipid-dependent serine/threonine kinases and is involved in cell proliferation, apoptosis, and differentiation. Previous studies have suggested that different PKC isoforms might be translationally regulated. We report here that the 395-nt-long 5' untranslated region (5' UTR) of PKCdelta is predicted to form very stable secondary structures with free energies (deltaG values) of around -170 kcal/mol. The 5' UTR of PKCdelta can significantly repress luciferase translation in rabbit reticulocyte lysate but does not repress luciferase translation in a number of transiently transfected cell lines. By using a bicistronic luciferase reporter, we show that the 5' UTR of PKCdelta contains a functional internal ribosome entry segment (IRES). The activity of the PKCdelta IRES is greatest in densely growing cells and during apoptosis, when total protein synthesis and levels of full-length eukaryotic initiation factor 4G are reduced. However, the IRES activity of the 5' UTR of PKCdelta is not enhanced during serum starvation, another condition shown to inhibit cap-dependent translation, suggesting that its potency is dependent on specific cellular conditions. Accumulating data suggest that PKCdelta has a function as proliferating cells reach high density and in early and later events of apoptosis. Our studies suggest a mechanism whereby PKCdelta synthesis can be maintained under these conditions when cap-dependent translation is inhibited.     

Targeted optical injection of gold nanoparticles into single mammalian cells

We present an all optical technique for the targeted delivery of single 100 nm diameter gold nanoparticles into a specified region of the interior of an individual mammalian cell through a combination of optical tweezing and optical injection. The internalisation of the nanoparticle is verified by confocal laser scanning microscopy and confocal laser scanning reflectance microscopy. This represents the first time that nano sized particles have been tweezed and optically injected into mammalian cells using only light, and provides a novel methodology for internalising nanosphere based biosensors within specific intracellular regions of a mammalian cell. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Gene duplications at the chemokine locus on mouse chromosome 4: multiple strain-specific haplotypes and the deletion of secondary lymphoid-organ chemokine and EBI-1 ligand chemokine genes in the plt mutation

The murine paucity of lymph node T cell (plt) mutation leads to abnormalities in leukocyte migration and immune response. The causative defect is thought to be a loss of secondary lymphoid-organ chemokine (SLC) expression in lymphoid tissues. We now find that the plt defect is due to the loss of both SLC and EBI-1 ligand chemokine (ELC) expression in secondary lymphoid organs. In an examination of the plt locus, we find that commonly used inbred mouse strains demonstrate at least three different haplotypes. Polymorphism at this locus is due to duplications of at least four genes, three of them encoding chemokines. At least two cutaneous T cell-attracting chemokine (CTACK), three SLC, and four ELC genes or pseudogenes are present in some haplotypes. All haplotypes share a duplication that includes two SLC genes, which demonstrate different expression patterns, a single functional ELC gene, and an ELC pseudogene. The plt mutation represents a deletion that includes the SLC gene expressed in secondary lymphoid organs and the single functional ELC gene, leaving only an SLC gene that is expressed in lymphatic endothelium and an ELC pseudogene. This lack of CCR7 ligands in the secondary lymphoid organs of plt mice provides a basis for their severe abnormalities in leukocyte migration and immune response.     

Rapid transfer of low copy-number episomal plasmids fromSaccharomyces cerevisiae to Escherichia coli by electroporation

A simple and reproducible method for transferring low copy-number episomal plasmids from yeast toEscherichia coli has been developed. Although slightly more time-consuming than direct transfer methods, which are effective with high copy number plasmids, the method is significantly faster than methods that require purification of yeast DNA. Plasmid DNA is released from yeast cells during brief treatments involving grinding with glass beads and heating. The treated yeast are cooled, electrocompetentE. coli is added, the mixture is electroporated, and transformants are selected using standard conditions forE. coli electrotransformation. The procedure typically yields sufficient transformants for most applications.     

PPARalpha /gamma ragaglitazar eliminates fatty liver and enhances insulin action in fat-fed rats in the absence of hepatomegaly

Peroxisome proliferator-activated receptor (PPAR)alpha and PPARgamma agonists lower lipid accumulation in muscle and liver by different mechanisms. We investigated whether benefits could be achieved on insulin sensitivity and lipid metabolism by the dual PPARalpha/gamma agonist ragaglitazar in high fat-fed rats. Ragaglitazar completely eliminated high-fat feeding-induced liver triglyceride accumulation and visceral adiposity, like the PPARalpha agonist Wy-14643 but without causing hepatomegaly. In contrast, the PPARgamma agonist rosiglitazone only slightly lessened liver triglyceride without affecting visceral adiposity. Compared with rosiglitazone or Wy-14643, ragaglitazar showed a much greater effect (79%, P < 0.05) to enhance insulin's suppression of hepatic glucose output. Whereas all three PPAR agonists lowered plasma triglyceride levels and lessened muscle long-chain acyl-CoAs, ragaglitazar and rosiglitazone had greater insulin-sensitizing action in muscle than Wy-14643, associated with a threefold increase in plasma adiponectin levels. There was a significant correlation of lipid content and insulin action in liver and particularly muscle with adiponectin levels (P < 0.01). We conclude that the PPARalpha/gamma agonist ragaglitazar has a therapeutic potential for insulin-resistant states as a PPARgamma ligand, with possible involvement of adiponectin. Additionally, it can counteract fatty liver, hepatic insulin resistance, and visceral adiposity generally associated with PPARalpha activation, but without hepatomegaly.     

Applying Circuit Theory for Corridor Expansion and Management at Regional Scales: Tiling,Pinch Points,and Omnidirectional Connectivity

Connectivity models are useful tools that improve the ability of researchers and managers to plan land use for conservation and preservation. Most connectivity models function in a point-to-point or patch-to-patch fashion, limiting their use for assessing connectivity over very large areas. In large or highly fragmented systems, there may be so many habitat patches of interest that assessing connectivity among all possible combinations is prohibitive. To overcome these conceptual and practical limitations, we hypothesized that minor adaptation of the Circuitscape model can allow the creation of omnidirectional connectivity maps illustrating flow paths and variations in the ease of travel across a large study area. We tested this hypothesis in a 24,300 km² study area centered on the Montérégie region near Montréal, Québec. We executed the circuit model in overlapping tiles covering the study region. Current was passed across the surface of each tile in orthogonal directions, and then the tiles were reassembled to create directional and omnidirectional maps of connectivity. The resulting mosaics provide a continuous view of connectivity in the entire study area at the full original resolution. We quantified differences between mosaics created using different tile and buffer sizes and developed a measure of the prominence of seams in mosaics formed with this approach. The mosaics clearly show variations in current flow driven by subtle aspects of landscape composition and configuration. Shown prominently in mosaics are pinch points, narrow corridors where organisms appear to be required to traverse when moving through the landscape. Using modest computational resources, these continuous, fine-scale maps of nearly unlimited size allow the identification of movement paths and barriers that affect connectivity. This effort develops a powerful new application of circuit models by pinpointing areas of importance for conservation, broadening the potential for addressing intriguing questions about resource use, animal distribution, and movement.