Tumors hamper the immunogenic competence of CD4+ T cell-directed dendritic cell vaccination

Dendritic cells loaded with tumor-derived peptides induce protective CTL responses and are under evaluation in clinical trails. We report in this study that prophylactic administration of dendritic cells loaded with a MHC class II-restricted peptide derived from a model tumor Ag (Leishmania receptor for activated C kinase (LACK)) confers protection against LACK-expressing TS/A tumors, whereas therapeutic vaccination fails to cure tumor-bearing mice. Although CD4+ T cell-directed dendritic cell vaccination primed effector-like (CD44(high)CD62L(low), IL-2(+), IFN-gamma(+)) and central memory-like lymphocytes (CD44(high)CD62L(high), only IL-2(+)) in tumor-free mice, this was not the case in tumor-bearing animals in which both priming and persistence of CD4+ T cell memory were suppressed. Suppression was specific for the tumor-associated Ag LACK, and did not depend on CD25+ T cells. Because T cell help is needed for protective immunity, we speculate that the ability of tumors to limit vaccine-induced CD4+ T cell memory could provide a partial explanation for the limited efficacy of current strategies.     

Coumarin-caged glycine that can be photolyzed within 3 microseconds by visible light

The synthesis and characterization of a new photolabile precursor of glycine (coumarin-caged glycine) are reported. The new compound is suitable for rapid chemical kinetic investigations of the membrane-bound neurotransmitter receptor activated by glycine. Unlike previously used caging groups for glycine, this precursor can be photolyzed rapidly and efficiently in the visible wavelength region. This allows the use of a relatively inexpensive light source. The alpha-carboxyl group of glycine was covalently coupled to the 7-(diethylamino)coumarin (DECM) caging group. The caged compound has a major absorption band with a maximum at 390 nm (epsilon390 = 13,900 M-1 cm-1). Photolysis was performed at wavelengths of >or=400 nm (epsilon400 = 12,400 M-1 cm-1). Under physiological conditions, DECM-caged glycine is water soluble and stable. In the visible wavelength region, it photolyzes rapidly to release glycine with a half-life of approximately 2.5 micrometers and a quantum yield of 0.12 +/- 0.01. The experimental results demonstrated that neither DECM-caged glycine nor its byproduct inhibits or activates human alpha1 glycine receptors expressed on the surface of HEK 293 cells.     

1-(5-Carboxy- and 5-carbamoylindol-1-yl)propan-2-ones as inhibitors of human cytosolic phospholipase A2alpha: bioisosteric replacement of the carboxylic acid and carboxamide moiety

Indole-5-carboxylic acids and -carboxamides with 3-aryloxy-2-oxopropyl residues in position 1 were previously reported to be potent inhibitors of cytosolic phospholipase A(2)alpha (cPLA(2)alpha) isolated from human platelets. In continuation of our attempts to develop novel cPLA(2)alpha inhibitors, a number of derivatives of these substances characterized by bioisosteric replacement of the carboxylic acid and carboxamide functionality, respectively, were prepared. The results of the biological evaluation of the obtained compounds enabled us to gain further insight into structural features critical for cPLA(2)alpha inhibition.     

Comparative analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42

Bacillus amyloliquefaciens FZB42 is a Gram-positive, plant-associated bacterium, which stimulates plant growth and produces secondary metabolites that suppress soil-borne plant pathogens. Its 3,918-kb genome, containing an estimated 3,693 protein-coding sequences, lacks extended phage insertions, which occur ubiquitously in the closely related Bacillus subtilis 168 genome. The B. amyloliquefaciens FZB42 genome reveals an unexpected potential to produce secondary metabolites, including the polyketides bacillaene and difficidin. More than 8.5% of the genome is devoted to synthesizing antibiotics and siderophores by pathways not involving ribosomes. Besides five gene clusters, known from B. subtilis to mediate nonribosomal synthesis of secondary metabolites, we identified four giant gene clusters absent in B. subtilis 168. The pks2 gene cluster encodes the components to synthesize the macrolactin core skeleton.     

Phylogenetic analysis of freshwater sponges provide evidence for endemism and radiation in ancient lakes

Morphologic and phylogenetic analysis of freshwater sponges endemic to lakes in Central Sulawesi, Siberia and South-East Europe is presented. We also analyzed several cosmopolitan sponge species from Eurasia and North America and included sponge sequences from public databases. In agreement with previous reports [Addis, J.S., Peterson, K.J., 2005. Phylogenetic relationships of freshwater sponges (Porifera, Spongillina) inferred from analyses of 18S rDNA, COI mtDNA, and ITS2 rDNA sequences. Zool. Scr. 34, 549-557], the metaniid sponge Corvomeyenia sp. was the most deeply branching species within a monophyletic lineage of the suborder Spongillina. Pachydictyum globosum (Malawispongiidae) and Nudospongilla vasta (Spongillidae), two morphologically quite distinct species from Sulawesi were found in a joint clade with Trochospongilla (Spongillidae) rendering Trochospongilla paraphyletic. Furthermore, Ochridaspongia sp., another Malawispongiidae, clustered far away from that clade, together with Ephydatia fluviatilis, making the latter family polyphyletic. The Lubomirskiidae endemic to Lake Baikal, Lubomirskia abietina, Baikalospongia bacillifera, B. intermedia, and Swartschewskia papyracea formed a well-supported clade that was most closely linked to the genus Ephydatia (99.9% identity over a total length of 2169 concatenated nucleotide positions). Our study indicates the frequent and independent origin of sponge species endemic to different freshwater ecosystems from a few cosmopolitan founder species. The highly specific primer sets newly developed here facilitate work on the molecular phylogeny and DNA barcoding of sponges.     

NOX5 variants are functionally active in endothelial cells

NADPH oxidases have been identified as sources of reactive oxygen species (ROS) in vascular cells. In addition to the initially described enzyme containing gp91phox (NOX2), several homologues to NOX2 have been identified. Whereas NOX1, NOX2, and NOX4 are expressed in endothelial cells, a functional role of NOX5 containing additional N-terminal calcium-binding domains of varying sequences has not been reported in these cells. NOX5 protein was found in the endoplasmic reticulum of human microvascular endothelial cells (HMEC-1) and in the vascular wall. HMEC-1 cells expressed NOX5beta and NOX5delta as well as a variant lacking calcium-binding domains (NOX5S). NOX5beta and NOX5S increased basal ROS levels. Ionomycin exclusively enhanced NOX5beta-mediated ROS production. Although p22phox, when overexpressed, interacted with both NOX5 proteins, it was not essential for NOX5-mediated ROS production. NOX5 proteins stimulated endothelial cell proliferation and the formation of capillary-like structures whereas depletion of NOX5 by siRNA prevented these responses to thrombin. These data show that endothelial cells express different NOX5 variants including NOX5S lacking calcium-binding domains. NOX5 proteins are functional, promoting endothelial ROS production, proliferation, and the formation of capillary-like structures and contribute to the endothelial response to thrombin. These findings suggest that NOX5 variants play a novel role in controlling ROS-dependent processes in the vasculature.     

Endogenous retroviral insertion in Cryge in the mouse No3 cataract mutant

No3 (nuclear opacity 3) is a novel congenital nuclear cataract in mice. Microsatellite mapping placed the No3 locus on chromosome 1 between D1Mit480 (32cM) and D1Mit7 (41cM), a region containing seven crystallin genes; Cryba2 and the Cryga-Crygf cluster. Although polymorphic variants were observed, no candidate mutations were found for six of the genes. However, DNA walking identified a murine endogenous retrovirus (IAPLTR1: ERVK) insertion in exon 3 of Cryge, disrupting the coding sequence for gammaE-crystallin. Recombinant protein for the mutant gammaE was completely insoluble. The No3 cataract is mild compared with the effects of similar mutations of gammaE. Quantitative RT-PCR showed that gammaE/F mRNA levels are reduced in No3, suggesting that the relatively mild phenotype results from suppression of gammaE levels due to ERVK insertion. However, the severity of cataract is also strain dependent suggesting that genetic background modifiers also play a role in the development of opacity.     

Environmental adaptation in Chinook salmon (Oncorhynchus tshawytscha) throughout their North American range

Landscape genomics is a rapidly growing field with recent advances in both genotyping efficiency and statistical analyses that provide insight towards local adaptation of populations under varying environmental and selective pressure. Chinook salmon (Oncorhynchus tshawytscha) are a broadly distributed Pacific salmon species, occupying a diversity of habitats throughout the northeastern Pacific with pronounced variation in environmental and climate features but little is understood regarding local adaptation in this species. We used a multivariate method, redundancy analysis (RDA), to identify polygenic correlations between 19 703 SNP loci and a suite of environmental variables in 46 collections of Chinook salmon (1956 total individuals) distributed throughout much of its North American range. Models in RDA were conducted on both rangewide and regional scales by hierarchical partitioning of the populations into three distinct genetic lineages. Our results indicate that between 5.8 and 21.8% of genomic variation can be accounted for by environmental features, and 566 putatively adaptive loci were identified as targets of environmental adaptation. The most influential drivers of adaptive divergence included precipitation in the driest quarter of the year (Rangewide and North Coastal Lineage, anova P = 0.002 and 0.01, respectively), precipitation in the wettest quarter of the year (Interior Columbia River Stream‐Type Lineage, anova P = 0.03), variation in mean diurnal range in temperature (South Coastal Lineage, anova P = 0.005), and migration distance (Rangewide, anova P = 0.001). Our results indicate that environmental features are strong drivers of adaptive genomic divergence in this species, and provide a foundation to investigate how Chinook salmon might respond to global environmental change.     

Comparative assessment of the sensitivity of oilseed rape and wheat to limited water supply

The drought‐sensitivity of oilseed rape (OSR, Brassica napus cv. SW Landmark) was investigated, using the more widely studied crop species wheat (Triticum aestivum cv. Tybalt) as a benchmark. The water relations of OSR and wheat were compared in lysimeter and controlled environment experiments to test the hypothesis that the growth of OSR is restricted to a greater extent by soil drying than wheat and to determine whether the greater sensitivity results from differences in root or shoot traits. Plants were grown, with or without irrigation, in 1.2 m tall lysimeters packed with a sandy clay loam soil. The experiment was conducted in an open‐sided glasshouse to encourage air flow and to resemble a field environment as far as possible; plant population densities were equivalent to commercial crops. Irrigated OSR (evapo)transpired more water than wheat (498 vs. 355 mm), but had a comparable water use efficiency (WUE; 4.1 vs. 4.4 g DW mm^?1 H₂O). Oilseed rape showed a greater reduction in above‐ground growth (52% vs. 21%) and a smaller increase in WUE (27% vs. 45%) when water was withheld. Oilseed rape also responded to soil drying at a lower soil moisture deficit than wheat; transpiration rates fell below the potential of irrigated plants when plant available water remaining in the soil profile declined from 54 to 23% compared to 38 to 9% for wheat. The root hydraulic conductivity of young OSR plants, measured on root surface area basis, was about twice that of wheat, and was comparable on a root length basis. The results show that OSR was more sensitive to a restricted water supply than the benchmark species wheat and that the greater sensitivity resulted from differences in shoot, rather than root, characteristics. The root system of OSR was at least as efficient as wheat at extracting water from soil.