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971.
Multiple type I interferons (IFN-α/β) elicit Jak/Stat activation, rapid gene induction, and pleiotropic effects, such as differentiation, antiviral protection, and blocks in proliferation, which are dependent on the IFN subtype and the cellular context. To date, ligand- and receptor-specific molecular determinants underlying IFN-α/β differential activities or potencies have been well characterized. To analyze cellular determinants that impact subtype-specific potency, human fibrosarcoma U5A-derived clones, exhibiting a gradient of IFN sensitivity by virtue of increasing receptor levels, were monitored for Jak/Stat signaling, gene induction, cell cycle lengthening, and apoptosis. In cells with scarce receptors, IFN-β was more potent than IFN-α2 in antiproliferative activity, while the two subtypes were equipotent in all other readouts. Conversely, in cells with abundant receptors, IFN-α2 matched or even surpassed IFN-β in all readouts tested. Our results suggest that the differential activities of the IFN subtypes are dictated not only by the intrinsic ligand/receptor binding kinetics but also by the density of cell surface receptor components.A persistent question in the field of helically bundled cytokines concerns the molecular basis of intracellular signal activation following binding to cognate cell surface receptors. Typically, cytokine-induced dimerization of the receptor subunits is thought to trigger catalytic transactivation of the associated Jak tyrosine kinases. Phosphorylation of critical receptor tyrosine motifs by the activated Jak proteins allows recruitment and activation of downstream Stat effectors (25, 34). A clear distinction can be made between the short homodimeric Jak2-activating receptors, such as the growth hormone or the erythropoietin receptors, and the more complex heteromeric receptors. Among these latter is the type I interferon (IFN) receptor, a prototypic class 2 receptor, made of two subunits, each associated with a different Jak enzyme (29). IFNAR2 contains extracellularly two fibronectin III domains forming a well-defined cytokine binding module. The cytoplasmic region of IFNAR2 is 250 amino acids long, interacts with Jak1, and contains two principal Tyr-based Stat recruitment motifs (24, 35). IFNAR1 is made of a large ectodomain of four fibronectin III domains, not all involved in ligand binding, and a 100-amino-acid-long cytoplasmic region complexed with Tyk2 and subjected to ligand-induced ubiquitination driving receptor proteolysis (13, 14).A large array of IFNs (over a dozen α subtypes and one β subtype) bind to this ubiquitously expressed receptor complex to induce rapid gene expression programs that elicit measurable antiviral responses and cell growth inhibition as well as cell context-specific functional changes (4, 31). Several studies have reported on differential activities of type I IFNs, but no unique function has ever been attributed to a given subtype (see references in reference 29). Thus, a differential can be defined as a lack of correlation between two specific activities. For instance, depending on the cell system, IFN-α2 and IFN-β can exhibit equivalent antiproliferative potency or over a 100-fold difference in antiproliferative potency and nearly equipotency in antiviral activity. Since no overt differences are observed in the structure or stoichiometry of the ligand-receptor complex formed with different subtypes, the concordant view points to the way each IFN subtype engages the available receptors. Indeed, kinetic measurements of the interaction of IFN-α2 and IFN-β with receptor ectodomains have shown substantial differences. IFNAR2 represents the high-affinity subunit, toward which IFN-α2 exhibits nanomolar binding affinity and IFN-β exhibits ∼100 pM binding affinity. Conversely, IFNAR1 is the low-affinity subunit, toward which IFN-α2 exhibits micromolar affinity and IFN-β ∼50 nM affinity (19, 22). The contribution of the individual and combined affinities on ternary complex formation by either IFN subtype have been thoroughly studied (10, 26). However, how these dynamic parameters influence receptor function and translate into activation of Jak, recruitment of Stats and additional effectors, gene induction, and bioactivities remains ill defined.Rather than focusing on ligand and receptor determinants, here we investigated the relationship between receptor subunit levels and IFN-α2 versus IFN-β signaling and functional outcomes (IFN-α2/β differential potencies). Since we previously showed that no simple relationship between receptor levels and Jak/Stat signaling can be inferred by comparing different cell types (18), we have used a reductionist approach in a single cell type, from which we have engineered and studied clones expressing low or abundant receptor levels. We show that the density of receptors at the cell surface represents a critical determinant of the level of differential activity exhibited by two IFN subtypes.  相似文献   
972.
Bayesian modeling techniques (which accounted for imperfect detection) were used to assess changes in macrophyte assemblages in 58 wetlands along a typical salinity gradient in Western Victoria, Australia. By incorporating detectability into our predictions, an unbiased estimate was made of the relationship between salinity and both individual species occupancy and the expected number of species. When compared to the freshest wetlands, macrophyte species number was predicted to decrease by as much as 60–70% at conductivities of around 6.0 mS cm−1 (11% seawater), a value often considered the upper salinity tolerance for many freshwater aquatic plants. The model also predicted a 40–50% drop in species number at conductivities of around 1.5 mS cm−1 (3% seawater). It was also found that 25 out of 76 freshwater species were unlikely to occur at conductivities above 1.0 mS cm−1. Consequently, secondary salinisation of fresh non-riverine wetlands is highly likely to markedly and negatively impact upon non-riverine wetland macrophyte assemblages.  相似文献   
973.
Dendritic cell (DC) vaccines have emerged as a promising strategy to induce antitumoral cytotoxic T cells for the immunotherapy of cancer. The maturation state of DC is of critical importance for the success of vaccination, but the most effective mode of maturation is still a matter of debate. Whereas immature DC carry the risk of inducing tolerance, extensive stimulation of DC may lead to DC unresponsiveness and exhaustion. In this study, we investigated how short-term versus long-term DC activation with a Toll-like receptor 9 agonist influences DC phenotype and function. Murine DC were generated in the presence of the hematopoietic factor Flt3L (FL-DC) to obtain both myeloid and plasmacytoid DC subsets. Short activation of FL-DC for as little as 4 h induced fully functional DC that rapidly secreted IL-12p70 and IFN-α, expressed high levels of costimulatory and MHC molecules and efficiently presented antigen to CD4 and CD8 T cells. Furthermore, short-term activated FL-DC overcame immune suppression by regulatory T cells and acquired high migratory potential toward the chemokine CCL21 necessary for DC recruitment to lymph nodes. In addition, vaccination with short-term activated DC induced a strong cytotoxic T-cell response in vivo and led to the eradication of tumors. Thus, short-term activation of DC generates fully functional DC for tumor immunotherapy. These results may guide the design of new protocols for DC generation in order to develop more efficient DC-based tumor vaccines.  相似文献   
974.
Relapsing C. difficile disease in humans is linked to a pathological imbalance within the intestinal microbiota, termed dysbiosis, which remains poorly understood. We show that mice infected with epidemic C. difficile (genotype 027/BI) develop highly contagious, chronic intestinal disease and persistent dysbiosis characterized by a distinct, simplified microbiota containing opportunistic pathogens and altered metabolite production. Chronic C. difficile 027/BI infection was refractory to vancomycin treatment leading to relapsing disease. In contrast, treatment of C. difficile 027/BI infected mice with feces from healthy mice rapidly restored a diverse, healthy microbiota and resolved C. difficile disease and contagiousness. We used this model to identify a simple mixture of six phylogenetically diverse intestinal bacteria, including novel species, which can re-establish a health-associated microbiota and clear C. difficile 027/BI infection from mice. Thus, targeting a dysbiotic microbiota with a defined mixture of phylogenetically diverse bacteria can trigger major shifts in the microbial community structure that displaces C. difficile and, as a result, resolves disease and contagiousness. Further, we demonstrate a rational approach to harness the therapeutic potential of health-associated microbial communities to treat C. difficile disease and potentially other forms of intestinal dysbiosis.  相似文献   
975.
976.
Hsp90 is an essential molecular chaperone in the eukaryotic cytosol. Its function is modulated by cochaperones and posttranslational modifications. Importantly, the phosphatase Ppt1 is a dedicated regulator of the Hsp90 chaperone system. Little is known about Ppt1-dependent phosphorylation sites and how these affect Hsp90 activity. Here, we identified the major phosphorylation sites of yeast Hsp90 in its middle or the C-terminal domain and determined the subset regulated by Ppt1. In general, phosphorylation decelerates the Hsp90 machinery, reduces chaperone function in vivo, sensitizes yeast cells to Hsp90 inhibition and affects DNA repair processes. Modification of one particular site (S485) is lethal, whereas others modulate Hsp90 activity via distinct mechanisms affecting the ATPase activity, cochaperone binding and manipulating conformational transitions in Hsp90. Our mechanistic analysis reveals that phosphorylation of Hsp90 permits a regulation of the conformational cycle at distinct steps by targeting switch points for the communication of remote regions within Hsp90.  相似文献   
977.
A new in vivo model for studying luteolysis was developed in sheep to provide a convenient method for collecting corpora lutea for molecular, biochemical, and histological analysis during a procedure that mimics natural luteolysis. It was found that the infusion of prostaglandin F(2α) (PGF(2α)) at 20 μg/min/h into the systemic circulation during the mid luteal phase of the cycle allowed sufficient PGF(2α) to escape across the lungs and thus mimic the transient 40% decline in the concentration of progesterone in peripheral plasma seen at the onset of natural luteolysis in sheep. Additional 1h-long systemic infusions of PGF(2α), given at physiological intervals, indicated that two infusions were not sufficient to induce luteolysis. However, an early onset of luteolysis and estrus was induced in one out of three sheep with three infusions, two out of three sheep with four infusions, and three out of three sheep with five infusions. Reducing the duration of each systemic infusion of PGF(2α) from 1h to 30 min failed to induce luteolysis and estrus even after six systemic infusions indicating that, not only are the amplitude and frequency of PGF(2α) pulses essential for luteolysis, but the actual duration of each pulse is also critical. We conclude that a minimum of five systemic pulses of PGF(2α), given in an appropriate amount and at a physiological frequency and duration, are required to mimic luteolysis consistently in all sheep. The five pulse regimen thus provides a new accurate in vivo model for studying molecular mechanisms of luteolysis.  相似文献   
978.
High performance liquid chromatography?Cmass spectrometry (HPLC?CMS) technique, employing a hybrid triple quadrupole/linear ion trap (QqQ/LIT) mass analyzer, was used for comprehensive metabolomic fingerprinting of several fruit juices types, prepared from expensive (orange) or relatively low-priced (apple, grapefruit) fruits. Following the automated data mining and pre-treatment step, the suitability of the multivariate HPLC?CMS metabolomic data for authentication, i.e., classification of fruit juice and adulteration detection, was assessed with the use of advanced chemometric tools (principal component analysis, PCA, and linear discrimination analysis, LDA). The LDA classification model, constructed and validated employing a highly variable samples set, was able to reliably detect 15% addition of apple or grapefruit juice to orange juice. In the final stage of this study, high performance liquid chromatography?Cquadrupole?Cquadrupole-time-of-flight mass spectrometry (HPLC?CQqTOFMS) measurements were performed in order to obtain data for identification of pre-selected marker compounds using elemental formula calculation and online databases search.  相似文献   
979.
980.
Head and neck squamous cell carcinoma (HNSCC) has the potential for early metastasis and is associated with poor survival. Ano1 (Dog1) is an established and sensitive marker for the diagnosis of gastrointestinal stromal tumors (GIST) and has recently been identified as a Ca(2+) activated Cl(-) channel. Although the ANO1 gene is located on the 11q13 locus, a region which is known to be amplified in different types of human carcinomas, a detailed analysis of Ano1 amplification and expression in HNSCC has not been performed. It is thus still unclear how Ano1 contributes to malignancy in HNSCC. We analyzed genomic amplification of the 11q13 locus and Ano1 together with Ano1-protein expression in a large collection of HNSCC samples. We detected a highly significant correlation between amplification and expression of Ano1 and showed that HNSCC patients with Ano1 protein expression have a poor overall survival. We further analyzed the expression of the Ano1 protein in more than 4'000 human samples from 80 different tumor types and 76 normal tissue types and detected that besides HNSCC and GISTs, Ano1 was rarely expressed in other tumor samples or healthy human tissues. In HNSCC cell lines, expression of Ano1 caused Ca(2+) activated Cl(-) currents, which induced cell motility and cell migration in wound healing and in real time migration assays, respectively. In contrast, knockdown of Ano1 did not affect intracellular Ca(2+) signaling and surprisingly did not reduce cell proliferation in BHY cells. Further, expression and activity of Ano1 strongly correlated with the ability of HNSCC cells to regulate their volume. Thus, poor survival in HNSCC patients is correlated with the presence of Ano1. Our results further suggest that Ano1 facilitates regulation of the cell volume and causes cell migration, which both can contribute to metastatic progression in HNSCC.  相似文献   
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