Fluid phase endocytosis of [125I]iodixanol in rat liver parenchymal, endothelial and Kupffer cells

Endocytosis of [125I]iodixanol was studied in vivo and in vitro in rat liver cells to determine fluid phase endocytic activity in different liver cells (hepatocytes, Kupffer cells and endothelial cells). The Kupffer cells were more active in the uptake of [l25I]iodixanol than parenchymal cells or endothelial cells. Inhibition of endocytic uptake via clathrin-coated pits (by potassium depletion and hypertonic medium) reduced uptake of [125I]iodixanol much more in Kupffer cells and endothelial cells than in hepatocytes. To gain further information about the importance of clathrin-mediated fluid phase endocytosis, the expression of proteins known to be components of the endocytic machinery was investigated. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, endothelial cells and Kupffer cells were found to express approximately fourfold more rab4, rab5 and rab7 than parenchymal cells, while clathrin was expressed at a higher level in endothelial cells than in Kupffer cells and hepatocytes. Using electron microscopy it was shown that liver endothelial cells contained approximately twice as many coated pits per membrane unit than the parenchymal and Kupffer cells, thus confirming the immunoblotting results concerning clathrin expression. Electron microscopy on isolated liver cells following fluid phase uptake of horseradish peroxidase (HRP) showed that HRP-containing organelles had a different morphology in the different cell types: In the liver endothelial cells HRP was in small, tubular endosomes, while in Kupffer cells HRP was mainly found in larger structures, reminiscent of macropinosomes. Parenchymal cells contained HRP in small vacuolar endosomes with a punctuated distribution. In conclusion, we find that the Kupffer cells and the endothelial cells have a higher pinocytic activity than the hepatocytes. The hepatocytes do, however, account for most of the total hepatic uptake. The fluid phase endocytosis in liver endothelial cells depends mainly on clathrin-mediated endocytosis, while the parenchymal cells have additional clathrin-independent mechanisms that may play an important role in the uptake of plasma membrane components. In the Kupffer cells the major uptake of fluid phase markers seems to take place via a macropinocytic mechanism.     

Variable region diversity of the Atlantic cod (Gadus morhua L.) immunoglobulin heavy chain

This study was undertaken to determine whether a lack of VH domain diversity could explain, in part, the failure of Atlantic cod to respond to immunization with the production of specific antibodies. The variability of cod VH regions was studied in 113 cDNA and 2 genomic clones. A fourth VH family and a second putative JH element were identified. The expressed VH repertoire showed a clear bias in the pattern of VH family utilization, with about 80% of the clones belonging to the VH-III family. Furthermore, the VH-III family was complex and could be subdivided into several subfamilies, while little variation was seen within the other families. The VH family bias gives a somewhat reduced variability of the VH gene region of cod, but not lower than that of the rabbit IgM repertoire. The H chain CDR3 region of cod was longer than that of trout, frog and mouse, and also highly variable in sequence, probably reflecting a relative importance of this region in cod. On the other hand, the CDR3 length variability was restricted, and this may reduce the diversity of the cod VH region.     

An approximation to the phylogeny of Sclerotinia and related genera

Phylogenies are constructed based on nuclear ribosomal internal transcribed spacer (ITS) DNA sequences from an ingroup consisting of 50 isolates representing 24 species of the discomycete family Sclerotiniaceae and an outgroup consisting of five related taxa of the same family. The ingroup taxa are: three Botrytis spp., two Botryotinia spp., one Ciborinia sp., one Dumontinia sp., one Grovesinia sp., six Myriosclerotinia spp., nine Sclerotinia spp. and one Sclerotium sp. The outgroup taxa are: one Ciboria sp., one Encoelia sp. and three Monilinia spp. The type species is included for all taxa except for Ciborinia and Encoelia. Several of the included taxa are important plant pathogens. The resulting phytogenies are discussed with regard to morphology, life history and taxonomy. A suspected relationship between Sclerotinia borealis and S. tetraspora , and Myriosclerotinia is rejected, while a suspected relationship between Ciborinia ciborium and Myriosclerotinia is strongly supported. Sclerotinia ulmariae , previously synonymized with Dumontinia tuberosa , is reinstated as an independent species of Dumontinia. Two new combinations, Dumontinia ulmariae and Myriosclerotinia ciborium , are proposed. The imperfectly known taxon Sclerotium cepivorum seems most closely related to Dumontinia. We conclude that Dumontinia , and Myriosclerotinia , as currently conceived, are monophyletic, and that Botryotinia along with Botrytis anamorphs probably also constitute a monophyletic lineage. The genus Sclerotinia is probably polyphyletic and characterized by symplesiomorphies rather than synapomorphies. Two putatively new taxa, Sclerotinia sp. 1, and Sclerotinia sp.2 are most closely related to S. minor, S. sclerotiorum and S. trifoliorum , and to S. borealis , respectively.     

Localisation of Ig heavy chain mRNA positive cells in Atlantic cod (Gadus morhuaL.) tissues; identified byin situhybridisation

Localisation of immunoglobulin heavy chain (IgH) producing cells was determined in sections from head kidney, spleen, thymus, gills, gut, skin, heart and liver from the Atlantic cod. In general, IgH mRNA positive cells were detected in all organs examined and were mainly located to the connective tissue surrounding the vascular system in these organs. In the head kidney and spleen, IgH mRNA positive cells appeared as single distributed cells or as dense clusters, whereas in the thymus only single distributed positive cells were observed. The percentage of Ig heavy chain mRNA positive (plasma) cells in the head kidney, spleen and thymus was estimated at about 1% of the total cell mass. The number of IgH mRNA positive cells was lower than this in all the other organs examined.     

Novel selective thiazoleacetic acids as CRTH2 antagonists developed from in silico derived hits. Part 1

Structure–activity relationships of three related series of 4-phenylthiazol-5-ylacetic acids, derived from two hits emanating from a focused library obtained by in silico screening, have been explored as CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells) antagonists. Several compounds with double digit nanomolar binding affinity and full antagonistic efficacy for human CRTH2 receptor were obtained in all subclasses. The most potent compound was [2-(4-chloro-benzyl)-4-(4-phenoxy-phenyl)-thiazol-5-yl]acetic acid having an binding affinity of 3.7 nM and functional antagonistic effect of 66 nM in a BRET and 12 nM in a cAMP assay with no functional activity for the other PGD2 DP receptor (27 μM in cAMP).     

Phosphorylation of eIF4E Confers Resistance to Cellular Stress and DNA-Damaging Agents through an Interaction with 4E-T: A Rationale for Novel Therapeutic Approaches

Phosphorylation of the eukaryotic translation initiation factor eIF4E is associated with malignant progression and poor cancer prognosis. Accordingly, here we have analyzed the association between eIF4E phosphorylation and cellular resistance to oxidative stress, starvation, and DNA-damaging agents in vitro. Using immortalized and cancer cell lines, retroviral expression of a phosphomimetic (S209D) form of eIF4E, but not phospho-dead (S209A) eIF4E or GFP control, significantly increased cellular resistance to stress induced by DNA-damaging agents (cisplatin), starvation (glucose+glutamine withdrawal), and oxidative stress (arsenite). De novo accumulation of eIF4E-containing cytoplasmic bodies colocalizing with the eIF4E-binding protein 4E-T was observed after expression of phosphomimetic S209D, but not S209A or wild-type eIF4E. Increased resistance to cellular stress induced by eIF4E-S209D was lost upon knockdown of endogenous 4E-T or use of an eIF4E-W73A-S209D mutant unable to bind 4E-T. Cancer cells treated with the Mnk1/2 inhibitor CGP57380 to prevent eIF4E phosphorylation and mouse embryonic fibroblasts derived from Mnk1/2 knockout mice were also more sensitive to arsenite and cisplatin treatment. Polysome analysis revealed an 80S peak 2 hours after arsenite treatment in cells overexpressing phosphomimetic eIF4E, indicating translational stalling. Nonetheless, a selective increase was observed in the synthesis of some proteins (cyclin D1, HuR, and Mcl-1). We conclude that phosphorylation of eIF4E confers resistance to various cell stressors and that a direct interaction or regulation of 4E-T by eIF4E is required. Further delineation of this process may identify novel therapeutic avenues for cancer treatment, and these results support the use of modern Mnk1/2 inhibitors in conjunction with standard therapy.     

Extrapair paternity and genetic diversity: the white‐throated dipper Cinclus cinclus

Passerine bird species vary considerably in the frequency of extrapair paternity, but the factors causing this variation are not well understood. There is some comparative evidence that extrapair paternity is associated with the population level of genetic diversity, but there is no consensus of how genetic diversity should be measured and compared across species or populations. Here we report a low frequency of extrapair paternity (2% extrapair offspring) in a Norwegian population of the white‐throated dipper Cinclus cinclus, which shows strong signs of reduced genetic diversity. We encountered difficulties in constructing a robust parentage analysis system for the species, largely due to consistently low polymorphism levels in 100 heterologous microsatellite markers. Furthermore, single‐nucleotide polymorphisms (SNPs) were almost absent in intron sequences in 10 nuclear genes (>5 kb) that are much more polymorphic in other species. Hence, our results seem consistent with the genetic diversity hypothesis that predicts a low frequency of extrapair paternity in species with low genetic diversity. Heterologous microsatellite markers are generally unsuitable for interspecific comparisons of genetic diversity as they show strong phylogenetic dependency in polymorphism levels. We suggest that SNP rates at homologous nuclear introns, like those presented here, can provide a useful method for obtaining unbiased estimates of genome‐wide genetic diversity across populations and species.