Autoantibodies directed to novel components of the PM/Scl complex, the human exosome

The autoantigenic polymyositis/scleroderma (PM/Scl) complex was recently shown to be the human homologue of the yeast exosome, which is an RNA-processing complex. Our aim was to assess whether, in addition to targeting the known autoantigens PM/Scl-100 and PM/Scl-75, autoantibodies also target recently identified components of the PM/Scl complex. The prevalence of autoantibodies directed to six novel human exosome components (hRrp4p, hRrp40p, hRrp41p, hRrp42p, hRrp46p, hCsl4p) was determined in sera from patients with idiopathic inflammatory myopathy (n = 48), scleroderma (n = 11), or the PM/Scl overlap syndrome (n = 10). The sera were analyzed by enzyme-linked immunosorbent assays and western blotting using the affinity-purified recombinant proteins. Our results show that each human exosome component is recognized by autoantibodies. The hRrp4p and hRrp42p components were most frequently targeted. The presence of autoantibodies directed to the novel components of the human exosome was correlated with the presence of the anti-PM/Scl-100 autoantibody in the sera of patients with idiopathic inflammatory myopathy (IIM), as was previously found for the anti-PM/Scl-75 autoantibody. Other clear associations between autoantibody activities were not found. These results further support the conception that the autoimmune response may initially be directed to PM/Scl-100, whereas intermolecular epitope spreading may have caused the autoantibody response directed to the associated components.     

Convergent evolution of cysteine residues in sperm protamines of one genus of marsupials, the Planigales

A characteristic feature of the sperm P1 protamines of eutherian mammals is the constant presence of six to nine cysteine residues per molecule. During spermiogenesis these residues become oxidized to form a three-dimensional network of disulfide bridges between, and within, protamine molecules in the sperm chromatin. This covalent cross linking strongly stabilizes eutherian sperm nuclei. In contrast, protamines sequenced from teleost fish, birds, monotremes, and marsupials all lack cysteine residues and their sperm nuclei, without the stabilizing cross links, are easily decondensed in vitro. We have now found that one genus of tiny, shrewlike dasyurid marsupials, the Planigales, possess P1 protamines containing five to six cysteine residues. These residues appear to have evolved since the divergence of Planigales from other members of the family Dasyuridae, such as the marsupial mouse, Sminthopsis crassicaudata. We believe this constitutes a case of convergent evolution in a subfamily of dasyurid marsupials toward the cysteine-rich eutherian form of sperm protamine P1.      

Evaluation of corn gluten meal with urea as a source of supplementary nitrogen for growing calves and lambs

Three growth trials and a metabolism study were conducted to evaluate corn gluten meal—urea (CGM—urea) combinations compared to a urea control as sources of supplementary nitrogen for growing calves and lambs fed on diets based on corn cobs.In the first growth study cattle were fed individually twice daily while in the sheep growth study and the second cattle growth study, animals were fed in groups. The average daily gain and feed conversion were similar for animals given the soya bean meal (SBM) and CGM—urea supplements. Feed intakes were similar for all treatments except for growth study 1. Nitrogen retention and dry matter and protein digestibility did not differ significantly between the SBM and CGM—urea supplemented groups. Urea consistently gave inferior results in all the experiments. These results suggest that CGM—urea combination is essentially equal to SBM in supporting growth of calves and lambs.     

Lead compounds discovered from libraries

Lead compounds with the potential to progress to viable drug candidates have been identified from libraries using several strategies. These include rapid screening of large diverse collections, thematic libraries, project-directed libraries, and three-dimensional molecular models of corporate databases. There have been numerous success stories, including the identification of several clinical candidates.     

1,2,3,4-Tetrahydroisoquinolinyl sulfamic acids as phosphatase PTP1B inhibitors

High-throughput screening of the P&GP corporate repository against several protein tyrosine phosphatases identified the sulfamic acid moiety as potential phosphotyrosine mimetic. Incorporation of the sulfamic acid onto a 1,2,3,4-tetrahydroisoquinoline scaffold provided a promising starting point for PTP1B inhibitor design.     

Co-Depletion of Cathepsin B and uPAR Induces G0/G1 Arrest in Glioma via FOXO3a Mediated p27Kip1 Upregulation

Background

Cathepsin B and urokinase plasminogen activator receptor (uPAR) are both known to be overexpressed in gliomas. Our previous work and that of others strongly suggest a relationship between the infiltrative phenotype of glioma and the expression of cathepsin B and uPAR. Though their role in migration and adhesion are well studied the effect of these molecules on cell cycle progression has not been thoroughly examined.

Methodology/Principal Findings

Cathespin B and uPAR single and bicistronic siRNA plasmids were used to downregulate these molecules in SNB19 and U251 glioma cells. FACS analysis and BrdU incorporation assay demonstrated G0/G1 arrest and decreased proliferation with the treatments, respectively. Immunoblot and immunocyto analysis demonstrated increased expression of p27^Kip1 and its nuclear localization with the knockdown of cathepsin B and uPAR. These effects could be mediated by αVβ3/PI3K/AKT/FOXO pathway as observed by the decreased αVβ3 expression, PI3K and AKT phosphorylation accompanied by elevated FOXO3a levels. These results were further confirmed with the increased expression of p27^Kip1 and FOXO3a when treated with {"type":"entrez-nucleotide","attrs":{"text":"Ly294002","term_id":"1257998346","term_text":"LY294002"}}Ly294002 (10 µM) and increased luciferase expression with the siRNA and {"type":"entrez-nucleotide","attrs":{"text":"Ly294002","term_id":"1257998346","term_text":"LY294002"}}Ly294002 treatments when the FOXO binding promoter region of p27^Kip1 was used. Our treatment also reduced the expression of cyclin D1, cyclin D2, p-Rb and cyclin E while the expression of Cdk2 was unaffected. Of note, the Cdk2-cyclin E complex formation was reduced significantly.

Conclusion/Significance

Our study indicates that cathepsin B and uPAR knockdown induces G0/G1 arrest by modulating the PI3K/AKT signaling pathway and further increases expression of p27^Kip1 accompanied by the binding of FOXO3a to its promoter. Taken together, our findings provide molecular mechanism for the G0/G1 arrest induced by the downregulation of cathepsin B and uPAR in SNB19 and U251 glioma cells.     

Directionality of individual kinesin-5 Cin8 motors is modulated by loop 8, ionic strength and microtubule geometry

Kinesin-5 motors fulfil essential roles in mitotic spindle morphogenesis and dynamics as slow, processive microtubule (MT) plus-end directed motors. The Saccharomyces cerevisiae kinesin-5 Cin8 was found, surprisingly, to switch directionality. Here, we have examined directionality using single-molecule fluorescence motility assays and live-cell microscopy. On spindles, Cin8 motors mostly moved slowly (～25 nm/s) towards the midzone, but occasionally also faster (～55 nm/s) towards the spindle poles. In vitro, individual Cin8 motors could be switched by ionic conditions from rapid (380 nm/s) and processive minus-end to slow plus-end motion on single MTs. At high ionic strength, Cin8 motors rapidly alternated directionalities between antiparallel MTs, while driving steady plus-end relative sliding. Between parallel MTs, plus-end motion was only occasionally observed. Deletion of the uniquely large insert in loop 8 of Cin8 induced bias towards minus-end motility and affected the ionic strength-dependent directional switching of Cin8 in vitro. The deletion mutant cells exhibited reduced midzone-directed motility and efficiency to support spindle elongation, indicating the importance of directionality control for the anaphase function of Cin8.     

SOCS-1 inhibition of type I interferon restrains Staphylococcus aureus skin host defense

The skin innate immune response to methicillin-resistant Staphylococcus aureus (MRSA) culminates in the formation of an abscess to prevent bacterial spread and tissue damage. Pathogen recognition receptors (PRRs) dictate the balance between microbial control and injury. Therefore, intracellular brakes are of fundamental importance to tune the appropriate host defense while inducing resolution. The intracellular inhibitor suppressor of cytokine signaling 1 (SOCS-1), a known JAK/STAT inhibitor, prevents the expression and actions of PRR adaptors and downstream effectors. Whether SOCS-1 is a molecular component of skin host defense remains to be determined. We hypothesized that SOCS-1 decreases type I interferon production and IFNAR-mediated antimicrobial effector functions, limiting the inflammatory response during skin infection. Our data show that MRSA skin infection enhances SOCS-1 expression, and both SOCS-1 inhibitor peptide-treated and myeloid-specific SOCS-1 deficient mice display decreased lesion size, bacterial loads, and increased abscess thickness when compared to wild-type mice treated with the scrambled peptide control. SOCS-1 deletion/inhibition increases phagocytosis and bacterial killing, dependent on nitric oxide release. SOCS-1 inhibition also increases the levels of type I and type II interferon levels in vivo. IFNAR deletion and antibody blockage abolished the beneficial effects of SOCS-1 inhibition in vivo. Notably, we unveiled that hyperglycemia triggers aberrant SOCS-1 expression that correlates with decreased overall IFN signatures in the infected skin. SOCS-1 inhibition restores skin host defense in the highly susceptible hyperglycemic mice. Overall, these data demonstrate a role for SOCS-1-mediated type I interferon actions in host defense and inflammation during MRSA skin infection.     

The phylogenetic position of an <Emphasis Type="Italic">Armillaria</Emphasis> species from Amami-Oshima,a subtropical island of Japan,based on elongation factor and ITS sequences

An undetermined Armillaria species was collected on Amami-Oshima, a subtropical island of Japan. The phylogenetic position of the Armillaria sp. was determined using sequences of the elongation factor-1α (EF-1α) gene and the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) of ribosomal DNA (rDNA). The phylogenetic analyses based on EF-1α and ITS sequences showed that this species differs from known Japanese taxa of Armillaria. The sequences of this species and A. novae-zelandiae from Southeast Asia were contained in a strongly supported clade, which was adjacent to a well-supported sister clade containing A. novae-zelandiae from Australia and New Zealand.