Genetic and molecular characterization of Skb15, a highly conserved inhibitor of the fission yeast PAK, Shk1

The p21-activated kinase, Shk1, is essential for viability, establishment and maintenance of cell polarity, and proper mating response in the fission yeast, Schizosaccharomyces pombe. Here we describe the characterization of a highly conserved, WD repeat protein, Skb15, which negatively regulates Shk1 in fission yeast. A null mutation in the skb15 gene is lethal and results in deregulation of actin polymerization and localization, microtubule biogenesis, and the cytokinetic machinery, as well as a substantial uncoupling of these processes from the cell cycle. Loss of Skb15 function is suppressed by partial loss of Shk1, demonstrating that negative regulation of Shk1 by Skb15 is required for proper execution of cytoskeletal remodeling and cytokinetic functions. A mouse homolog of Skb15 can substitute for its counterpart in fission yeast, demonstrating that Skb15 protein function has been substantially conserved through evolution.     

Ectomycorrhizal fungal diversity and community structure on three co-occurring leguminous canopy tree species in a Neotropical rainforest

? The ectomycorrhizal (ECM) symbiosis was historically considered restricted to the temperate zones, but recent studies have shown the importance of this symbiosis across the tropics. We examined ECM fungal diversity, host plant phylogeny and ECM host preferences in a rainforest dominated by the leguminous host plants Dicymbe corymbosa, Dicymbe altsonii and Aldina insignis. ? Ectomycorrhizal fungi were identified by internal transcribed spacer rDNA sequencing and host species were verified with chloroplast trnL sequencing. To test whether Dicymbe and Aldina represent independent gains of the ECM symbiosis, we constructed a Fabaceae phylogeny using MatK and trnL. We identified four independent ECM lineages within the Fabaceae. ? We detected a diverse community of 118 ECM species dominated by the /clavulina, /russula-lactarius, /boletus, and /tomentella-thelephora lineages. Ectomycorrhizal species in Agaricales, Atheliales and Polyporales may represent previously unrecognized tropical-endemic ECM lineages. Previous studies suggested that ECM fungi did not diversify in the tropics, but the /clavulina lineage appears to have a center of diversity in tropical South America. ? Dicymbe and Aldina represent independent gains of the ECM symbiosis in Fabaceae but their fungal symbionts showed no host preferences. Spatial factors are more important than hosts in structuring the ECM fungal community in this ecosystem.     

IL-converting enzyme/caspase-1 inhibitor VX-765 blocks the hypersensitive response to an inflammatory stimulus in monocytes from familial cold autoinflammatory syndrome patients

Familial cold autoinflammatory syndrome (FCAS) and the related autoinflammatory disorders, Muckle-Wells syndrome and neonatal onset multisystem inflammatory disease, are characterized by mutations in the CIAS1 gene that encodes cryopyrin, an adaptor protein involved in activation of IL-converting enzyme/caspase-1. Mutations in cryopyrin are hypothesized to result in abnormal secretion of caspase-1-dependent proinflammatory cytokines, IL-1beta and IL-18. In this study, we examined cytokine secretion in PBMCs from FCAS patients and found a marked hyperresponsiveness of both IL-1beta and IL-18 secretion to LPS stimulation, but no evidence of increased basal secretion of these cytokines, or alterations in basal or stimulated pro-IL-1beta levels. VX-765, an orally active IL-converting enzyme/caspase-1 inhibitor, blocked IL-1beta secretion with equal potency in LPS-stimulated cells from FCAS and control subjects. These results further link mutations in cryopyrin with abnormal caspase-1 activation, and support the clinical testing of caspase-1 inhibitors such as VX-765 in autoinflammatory disorders.     

Hemagglutinating and adherence properties of Staphylococcus saprophyticus: epidemiology and virulence in experimental urinary tract infection of rats

We studied hemagglutinating and adherence properties in Staphylococcus saprophyticus isolates originating from symptomatic urinary tract infections. 12/13 (92%) of strains adhered to Hep cells and 11/13 (85%) were able to agglutinate sheep erythrocytes. Adherence properties differed markedly between strains (P less than 0.0001). Two strains, one hemagglutinating and adherent and one negative for both properties were chosen for experimental urinary tract infections. Results indicate that presence of the hemagglutinin favours colonization of kidney tissue.     

Larval,megalopal and early juvenile development in Pagurus granosimanus (Stimpson, 1859) (Decapoda: Anomura: Paguridae) reared in the laboratory

Summary

The larval, megalopal and early juvenile stages of Pagurus granosimanus are described, illustrated and compared with other North Pacific species of the genus. Morphologically, the zoeae of P. granosimanus appear most similar to the Japanese P. brachiomastus in the majority of characters, but share the endopodal setation of the third maxilliped with a second Japanese species, P. pectinatus. The megalopae of P. granosimanus are unlike those of other North Pacific species in having 5+5 marginal setae on the telson, rather than the customary 4+4, or less frequent 3+3. Comparison of juvenile characters is limited to pleopodal changes among the regional species for which data are available. P. granosimanus is unusual in undergoing complete pleopodal loss at the second crab stage with return of left pleopods in the fourth stage.     

MHC Multimer-Guided and Cell Culture-Independent Isolation of Functional T Cell Receptors from Single Cells Facilitates TCR Identification for Immunotherapy

Adoptive therapy using T cells redirected to target tumor- or infection-associated antigens is a promising strategy that has curative potential and broad applicability. In order to accelerate the screening process for suitable antigen-specific T cell receptors (TCRs), we developed a new approach circumventing conventional in vitro expansion-based strategies. Direct isolation of paired full-length TCR sequences from non-expanded antigen-specific T cells was achieved by the establishment of a highly sensitive PCR-based T cell receptor single cell analysis method (TCR-SCAN). Using MHC multimer-labeled and single cell-sorted HCMV-specific T cells we demonstrate a high efficacy (approximately 25%) and target specificity of TCR-SCAN receptor identification. In combination with MHC-multimer based pre-enrichment steps, we were able to isolate TCRs specific for the oncogenes Her2/neu and WT1 even from very small populations (original precursor frequencies of down to 0.00005% of CD3⁺ T cells) without any cell culture step involved. Genetic re-expression of isolated receptors demonstrates their functionality and target specificity. We believe that this new strategy of TCR identification may provide broad access to specific TCRs for therapeutically relevant T cell epitopes.     

Detection of a HindIII restriction fragment length polymorphism in the human phenol sulfotransferase (STP) locus

The gene encoding human phenol-preferring phenol sulfotransferase (STP) has been cloned and mapped to chromosome 16p. A HindIII RFLP in this gene is described.     

Identification of PSME3 as a novel serum tumor marker for colorectal cancer by combining two-dimensional polyacrylamide gel electrophoresis with a strictly mass spectrometry-based approach for data analysis

The purpose of this study was to identify and validate novel serological protein biomarkers of human colorectal cancer (CRC). Proteins from matched CRC and adjacent normal tissue samples were resolved by two-dimensional gel electrophoresis. From each gel all spots were excised, and enveloped proteins were identified by MS. By comparison of the resulting protein profiles, dysregulated proteins can be identified. A list of all identified proteins and validation of five exemplarily selected proteins, elevated in CRC was reported previously (Roessler, M., Rollinger, W., Palme, S., Hagmann, M. L., Berndt, P., Engel, A. M., Schneidinger, B., Pfeffer, M., Andres, H., Karl, J., Bodenmuller, H., Ruschoff, J., Henkel, T., Rohr, G., Rossol, S., Rosch, W., Langen, H., Zolg, W., and Tacke, M. (2005) Identification of nicotinamide N-methyltransferase as a novel serum tumor marker for colorectal cancer. Clin. Cancer Res. 11, 6550-6557). Here we describe identification and initial validation of another potential marker protein for CRC. Comparison of tissue protein profiles revealed strong elevation of proteasome activator complex subunit 3 (PSME3) expression in CRC tissue. This dysregulation was not detectable based on the spot pattern. The PSME3-containing spot on tumor gels showed no visible difference to the corresponding spot on matched control gels. MS analysis revealed the presence of two proteins, PSME3 and annexin 4 (ANXA4) in one and the same spot on tumor gels, whereas the matched spot contained only one protein, ANXA4, on control gels. Therefore, dysregulation of PSME3 was masked by ANXA4 and could only be recognized by MS-based analysis but not by image analysis. To validate this finding, antibody to PSME3 was developed, and up-regulation in CRC was confirmed by Western blot analysis and immunohistochemistry. Finally by developing a highly sensitive immunoassay, PSME3 could be detected in human sera and was significantly elevated in CRC patients compared with healthy donors and patients with benign bowel disease. We propose that PSME3 be considered a novel serum tumor marker for CRC that may have significance in the detection and in the management of patients with this disease. Further studies are needed to fully assess the potential clinical value of this marker candidate.