Suppressors of T-cell receptor signaling Sts-1 and Sts-2 bind to Cbl and inhibit endocytosis of receptor tyrosine kinases

The ubiquitin (Ub) ligase Cbl plays a critical role in attenuation of receptor tyrosine kinase (RTK) signaling by inducing ubiquitination of RTKs and promoting their sorting for endosomal degradation. Herein, we describe the identification of two novel Cbl-interacting proteins, p70 and Clip4 (recently assigned the names Sts-1 and Sts-2, respectively), that inhibit endocytosis of epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor. Sts-1 and Sts-2 contain SH3 domains that interacted with Cbl, Ub-associated domains, which bound directly to mono-Ub or to the EGFR/Ub chimera as well as phosphoglycerate mutase domains that mediated oligomerization of Sts-1/2. Ligand-induced recruitment of Sts-1/Sts-2 into activated EGFR complexes led to inhibition of receptor internalization, reduction in the number of EGFR-containing endocytic vesicles, and subsequent block of receptor degradation followed by prolonged activation of mitogenic signaling pathways. On the other hand, interference with Sts-1/Sts-2 functions diminished ligand-induced receptor degradation, cell proliferation, and oncogenic transformation in cultured fibroblasts. We suggest that Sts-1 and Sts-2 represent a novel class of Ub-binding proteins that regulate RTK endocytosis and control growth factor-induced cellular functions.     

Distinct monoubiquitin signals in receptor endocytosis

Numerous cellular proteins are post-translationally modified by the addition of the small modifier protein ubiquitin (Ub). The functional consequences of the type of ubiquitination vary, such that polyubiquitinated proteins are targeted for degradation by the proteasome, whereas monoubiquitination is implicated in other cellular functions, including endocytic trafficking and DNA repair. The monoubiquitination of trafficking cargoes, such as receptors and associated proteins, as well as of endocytic accessory Ub-binding proteins, raises the question of the precise function of monoubiquitin signals in the endocytic route. Recent biochemical and genetic evidence shows that multiple monoubiquitination of epidermal growth factor and platelet-derived growth factor receptors provides trafficking and sorting tags that ensure receptor endocytosis and degradation, whereas monoubiquitination of accessory proteins might play a role in regulating their function as Ub-receptors in the endosome.     

Hybridization properties of support-bound oligonucleotides: the effect of the site of immobilization on the stability and selectivity of duplex formation

Four 12-mer oligodeoxyribonucleotide sequences were immobilized to uniformly sized (50 microm) polymer particles through C5-tethered thymine and N(4)-tethered cytosine bases at four different sites in each sequence. The effect of the site of immobilization on the efficiency and selectivity of hybridization of the particle-bound probes was quantified by a sandwich-type assay based on a time-resolved fluorometric measurement of an oligonucleotide probe labeled with a photoluminescent europium(III) chelate directly from the surface of a single particle. Immobilization through a base in the central part of the sequence was observed to destablize the duplex more markedly than tethering through a terminal base. The effect of a one-base mismatch on the duplex stability increased with the increasing distance from the site of immobilization.     

Plasmin activates pro-matrix metalloproteinase-2 with a membrane-type 1 matrix metalloproteinase-dependent mechanism

Membrane-type 1 matrix metalloproteinase (MT1-MMP) has been implicated as a physiological activator of progelatinase A (MMP-2). We previously reported that plasmin treatment of cells results in proMMP-2 activation and increased type IV collagen degradation. Here, we analyzed the role of MT1-MMP in plasmin activation of MMP-2 using HT-1080 cells transfected with MT1-MMP sense or antisense cDNA. Control, vector-transfected cells that expressed endogenous MT1-MMP, and antisense cDNA transfectants with very low levels of MT1-MMP did not activate proMMP-2. Conversely, cells transfected with sense MT1-MMP cDNA expressed high MT1-MMP levels and processed proMMP-2 to 68/66-kDa intermediate activation products. Control cells and MT1-MMP transfectants had much higher levels of cell-associated MMP-2 than antisense cDNA transfectants. Addition of plasmin(ogen) to control or MT1-MMP-transfected cells generated active, 62-kDa MMP-2, but was ineffective with antisense cDNA transfectants. The effect of plasmin(ogen) was prevented by inhibitors of plasmin, but not by metalloproteinase inhibitors, implicating plasmin as a mechanism for proMMP-2 activation independent of the activity of MT1-MMP or other MMPs. Plasmin-mediated activation of proMMP-2 did not result from processing of proMT1-MMP and did not correlate with alpha(v)beta(3) integrin or TIMP-2 levels. Thus, plasmin can activate proMMP-2 only in the presence of MT1-MMP; however, this process does not require the catalytic activity of MT1-MMP.     

Screening of Saccharomyces cerevisiae strains with respect to anaerobic growth in non-detoxified lignocellulose hydrolysate

A microplate screening method was used to assess anaerobic growth of 12 Saccharomyces cerevisiae strains in barley straw, spruce and wheat straw hydrolysate. The assay demonstrated significant differences in inhibitor tolerance among the strains. In addition, growth inhibition by the three hydrolysates differed so that wheat hydrolysate supported growth up to 70%, while barley hydrolysate only supported growth up to 50%, with dilute-acid spruce hydrolysate taking an intermediate position.     

Distinct footprints of TCR engagement with highly homologous ligands

T cell receptor engagement promotes proliferation, differentiation, survival, or death of T lymphocytes. The affinity/avidity of the TCR ligand and the maturational stage of the T cell are thought to be principal determinants of the outcome of TCR engagement. We demonstrate in this study that the same mouse TCR preferentially uses distinct residues of homologous peptides presented by the MHC molecules to promote specific cellular responses. The preference for distinct TCR contacts depends on neither the affinity/avidity of TCR engagement (except in the most extreme ranges), nor the maturity of engaged T cells. Thus, different portions of the TCR ligand appear capable of biasing T cells toward specific biological responses. These findings explain differences in functional versatility of TCR ligands, as well as anomalies in the relationship between affinity/avidity of the TCR for the peptide/MHC and cellular responses of T cells.     

Inbreeding depression in perennial Lychnis viscaria (Caryophyllaceae): effects of population mating history and nutrient availability

We studied inbreeding depression in a perennial plant, Lychnis viscaria, in three populations differing in their inbreeding history and population size by measuring several traits at two nutrient levels over the plant's life cycle. The observed levels of inbreeding depression (cumulative inbreeding depression, from -0.057 to 0.629) were high for a plant with a mixed mating system. As expected, the population with a low level of isozyme variation expressed the least inbreeding depression for seed germination. Highest inbreeding depression for germination was found in the largest and genetically most variable population. No clear differences between populations in expression of inbreeding depression in the later life stages were found. The population level inbreeding depression varied with the nutrient conditions and among populations and life stages, but we found no evidence that inbreeding depression increased with lower nutrient availability. These results emphasize the importance of measuring inbreeding depression under several environmental conditions and over life stages.     

Effect of nutrient enrichment on bacterioplankton biomass and community composition in mesocosms in the Archipelago Sea, northern Baltic

Effects of nutrient enrichment on the biomass and communitycomposition of heterotrophic bacteria and picocyanobacteriawere studied in large (42 m³) mesocosms in the brackish-waterArchipelago Sea (Baltic Sea) in late summer 2000 using cellcounts and denaturing gradient gel electrophoresis (DGGE) ofpolymerase chain reaction (PCR)-amplified 16S rRNA gene fragments.The identity of the major DNA bands was determined by sequencing.The obtained sequences were related to - and -proteobacteria,actinobacteria, verrucomicrobia and cyanobacteria. Nitrogenand phosphorus additions increased the biomasses of heterotrophicbacteria and picocyanobacteria and caused significant changesin their community composition judging from the DGGE bandingpatterns. Most verrucomicrobial bands had their highest relativeintensity in the control treatment and their lowest in the highernutrient addition treatment, whereas most Synechococcus-relatedbands had their lowest relative intensity in the lower nutrientaddition treatment. The responses of proteobacteria and actinobacteriawere more variable. The presence of both freshwater and marinesequences among the closest relatives to our sequences highlightsthe intermediate character of the Archipelago Sea between afreshwater and truly marine environment.     

Black currant seed oil and fish oil supplements differ in their effects on fatty acid profiles of plasma lipids, and concentrations of serum total and lipoprotein lipids, plasma glucose and insulin

European diets provide a suboptimal intake of eicosapentaenoic (20:5n3) and docosahexaenoic (22:6n3) acids, which are derived mainly from fish oils. The present study indicates that black currant seed oil, which contains 14.5% alpha-linolenic (18:3n3), 12.6% gamma-linolenic (18:3n6), 47.5% linoleic (18:2n6) and 2.7% stearidonic (18:4n3) acids, could potentially serve as alternative to fish oil as a n3 fatty acid source. Fifteen healthy females participated in a randomized, double-blind, crossover study including two 4-week periods with either 3 g/day of black currant seed oil or 2.8 g/day of fish oil separated by a 4-week washout period. The results show that black currant seed oil supplementation increased the proportion of 18:3n6 in triacylglycerols (TAG) and cholesteryl esters (CE), and that of dihomo-gamma-linolenic (20:3n6) in TAGs, CEs and glycerophospholipids (GPL) (P<.05). Proportion of 18:3n6 was higher (P<.05) after black currant seed oil than after fish oil in TAGs and CEs, and that of 20:3n6 in TAGs, CEs and GPLs. Black currant seed oil supplementation caused only minor changes in the proportions of 20:5n3 or 22:6n3. Serum levels of LDL cholesterol were lower (P<.05) after black currant seed oil compared to fish oil. Plasma glucose concentration decreased during the fish oil supplementation (P<.05).