Trans-Atlantic dispersal and phylogeography of Cerastium arcticum (Caryophyllaceae) inferred from RAPD and SCAR markers

Cerastium arcticum is an autogamous pioneer species with a distribution limited to the North Atlantic region. It has been suggested that such species must have survived in ice-free refugia on both sides of the Atlantic throughout the last, or even several, of the Pleistocene glaciations, because they lack special adaptations for long-distance dispersal. To address the possibility for recent trans-Atlantic dispersal of C. arcticum, we analyzed random amplified polymorphic DNA (RAPD) and sequence characterized amplified region (SCAR) differentiation among 26 populations of this high-polyploid species. Three SCAR markers were obtained that verified the main patterns identified in the RAPD analysis. Eighty-four multilocus RAPD phenotypes were observed in the 126 plants analyzed, based on 35 polymorphic markers. Multivariate analyses and analyses of molecular variance (AMOVAs) identified two highly divergent groups of populations: one arctic group (western and eastern Greenland, and the archipelagos of Svalbard and Franz Josef Land) and one nonarctic group (southern and northern Norway, and Iceland), indicating that C. arcticum is composed of two lineages with different evolutionary histories. However, there was little geographic structuring within each lineage, in spite of the fact that both lineages are disjunctly distributed across the Atlantic. Occurrence of very similar, in some cases even identical RAPD multilocus phenotypes on both sides of the Atlantic in this autogamous allopolyploid is most probably caused by postglacial dispersal. The present geographic distribution of C. arcticum may thus have been established after trans-Atlantic expansion from two Weichselian refugia, one for each evolutionary lineage. Unexpectedly, the level of intrapopulational variation increased towards the north. This may reflect that interpopulational migration is most extensive in the treeless arctic environment, where the species has a more continuous distribution than in the more southerly areas.     

Rate of intrachain contact formation in an unfolded protein: temperature and denaturant effects

We have measured the effect of temperature and denaturant concentration on the rate of intrachain diffusion in an unfolded protein. After photodissociating a ligand from the heme iron of unfolded horse cytochrome c, we use transient optical absorption spectroscopy to measure the time scale of the diffusive motions that bring the heme, located at His18, into contact with its native ligand, Met80. Measuring the rate at which this 62 residue intrachain loop forms under both folding and unfolding conditions, we find a significant effect of denaturant on the chain dynamics. The diffusion of the chain accelerates as denaturant concentration decreases, with the contact formation rate approaching a value near approximately 6x10(5) s(-1) in the absence of denaturant. This result agrees well with an extrapolation from recent loop formation measurements in short synthetic peptides. The temperature dependence of the rate of contact formation indicates an Arrhenius activation barrier, Ea approximately 20 kJ/mol, at high denaturant concentrations, comparable to what is expected from solvent viscosity effects alone. Although Ea increases by several kBT as denaturant concentration decreases, the overall rate of diffusion nevertheless increases. These results indicate that inter-residue energetic interactions do not control conformational diffusion in unfolded states, even under folding conditions.     

The "Goldilocks Effect" in Cystic Fibrosis: identification of a lung phenotype in the <Emphasis Type="Italic">cftr</Emphasis> knockout and heterozygous mouse

Background  

Cystic Fibrosis is a pleiotropic disease in humans with primary morbidity and mortality associated with a lung disease phenotype. However, knockout in the mouse of cftr, the gene whose mutant alleles are responsible for cystic fibrosis, has previously failed to produce a readily, quantifiable lung phenotype.     

Beta 1-subunits are required for regulation of coupling between Ca2+ transients and Ca2+-activated K+ (BK) channels by protein kinase C

Colonic myocytes have spontaneous, localized, Ins (1,4,5) trisphosphate (IP3) receptor-dependent Ca2+ transients that couple to the activation of Ca2+-dependent K+ channels and spontaneous transient outward currents (STOCs). We previously reported that the coupling strength between spontaneous Ca2+ transients and large conductance Ca2+ activated K+ (BK) channels is regulated by Ca2+ influx through nonselective cation channels and activation of protein kinase C (PKC). Here, we used confocal microscopy and the patch-clamp technique to further investigate the coupling between localized Ca2+ transients and STOCs in colonic myocytes from animals lacking the regulatory beta1-subunit of BK channels. Myocytes from beta1-knockout (beta1-/-) animals loaded with fluo 4 showed typical localized Ca2+ transients, but the STOCs coupled to these events were of abnormally low amplitude. Reduction in external Ca2+ or application of inhibitors of nonselective cation channels (SKF-96365) caused no significant change in the amplitude or frequency of STOCs. Likewise, an inhibitor of PKC, GF 109203X, had no significant effect on STOCs. Single-channel recording from BK channels showed that application of an activator (PMA) and an inhibitor (GF 109203X) of PKC did not affect BK channel openings in myocytes of beta1-/- mice. These data show that PKC-dependent regulation of coupling strength between Ca2+ transients and STOCs in colonic myocytes depends upon the interaction between alpha- and beta1-subunits.     

IL-2 intratumoral immunotherapy enhances CD8+ T cells that mediate destruction of tumor cells and tumor-associated vasculature: a novel mechanism for IL-2

Therapeutic use of IL-2 can generate antitumor immunity; however, a variety of different mechanisms have been reported. We injected IL-2 intratumorally (i.t.) at different stages of growth, using our unique murine model of mesothelioma (AE17; and AE17 transfected with secretory OVA (AE17-sOVA)), and systematically analyzed real-time events as they occurred in vivo. The majority of mice with small tumors when treatment commenced displayed complete tumor regression, remained tumor free for >2 mo, and survived rechallenge with AE17 tumor cells. However, mice with large tumors at the start of treatment failed to respond. Timing experiments showed that IL-2-mediated responses were dependent upon tumor size, not on the duration of disease. Although i.t. IL-2 did not alter tumor Ag presentation in draining lymph nodes, it did enhance a previously primed, endogenous, tumor-specific in vivo CTL response that coincided with regressing tumors. Both CD4(+) and CD8(+) cells were required for IL-2-mediated tumor eradication, because IL-2 therapy failed in CD4(+)-depleted, CD8(+)-depleted, and both CD4(+)- and CD8(+)-depleted C57BL/6J animals. Tumor-infiltrating CD8(+) T cells, but not CD4(+) T cells, increased in association with a marked reduction in tumor-associated vascularity. Destruction of blood vessels required CD8(+) T cells, because this did not occur in nude mice or in CD8(+)-depleted C57BL/6J mice. These results show that repeated doses of i.t. (but not systemic) IL-2 mediates tumor regression via an enhanced endogenous tumor-specific CTL response concomitant with reduced vasculature, thereby demonstrating a novel mechanism for IL-2 activity.     

Functional characterization and expression analysis of members of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family from Drosophila melanogaster

Here we report the cloning and functional characterization of eight members of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase gene family from Drosophila melanogaster (polypeptide GalNAc transferase = pgant1-8). Full-length cDNAs were isolated from a Drosophila embryonic library based on homology to known ppGaNTases. Alignments with characterized mammalian isoforms revealed strong sequence similarities between certain fly and mammalian isoforms, highlighting putative orthologues between the species. In vitro activity assays demonstrated biochemical transferase activity for each gene, with three isoforms requiring glycosylated substrates. Comparison of the activities of Drosophila and mammalian orthologues revealed conservation of substrate preferences against a panel of peptide and glycopeptide substrates. Furthermore, Edman degradation analysis demonstrated that preferred sites of GalNac addition were also conserved between certain fly and mammalian orthologues. Semi-quantitative PCR amplification of Drosophila cDNA revealed expression of most isoforms at each developmental stage, with some isoforms being less abundant at certain stages relative to others. In situ hybridization to Drosophila embryos revealed specific staining of pgant5 and pgant6 in the salivary glands and pgant5 in the developing hindgut. Additionally, pgant5 and pgant6 expression within the egg chamber was restricted to the follicle cells, cells known to be involved in egg formation and subsequent embryonic patterning. The characterization reported here provides additional insight into the use of this model system to dissect the biological role of this enzyme family in vivo during both fly and mammalian development.     

Fast and efficient synthesis of a novel homologous series of L-fucosylated trisaccharides using the Helix pomatia alpha-(1-->2)-L-galactosyltransferase

The alpha-(1-->2)-L-galactosyltransferase from the albumen gland of the vineyard snail Helix pomatia exhibits high alpha-(1-->2)-L-fucosyltransferase activity and can be used to transfer L-fucose from GDP-L-fucose to terminal, non-reducing D-galactose residues of an oligosaccharide, thus providing facile access to a range of H-antigen-containing oligosaccharides. The enzymatic glycosylation was applied here on a milligram scale to a series of disaccharide acceptor substrates. Apparently the site of interglycosidic linkage between the terminal and subterminal acceptor sugar units is of little or no consequence. The homologous series of trisaccharides thus produced were fully characterised by NMR analysis of their peracetates.