Expression of AGR2 in non small cell lung cancer

We aimed to evaluate immunohistochemically the expression of the human Anterior Gradient-2 (AGR2), a gene which has recently been proposed as an oncogene for lung carcinoma development, in non small cell lung cancer and to correlate the findings to clinico-pathological data including patient survival. 95 cases of NSCLC were immunostained using a polyclonal AGR2 antibody and statistical analyses were applied to test for prognostic and diagnostic associations. AGR2 was expressed in 66.3% of cases, preferentially adenocarcinomas. There were no relevant associations with clinico-pathological parameters. A prognostic value of AGR2 could not be demonstrated neither in multivariate nor in univariate analyses. Interestingly, this is the first study to demonstrate AGR2 expression in squamous cell carcinomas. Although a prognostic value of AGR2 seems unlikely further studies are warranted to investigate the biological role of AGR2 in NSCLC and its differential expression according to histology.     

Structure-activity relationships of novel N-acyloxy-1,4-dihydropyridines as P-glycoprotein inhibitors

Series of novel N-acyloxy-1,4-dihydropyridines have been synthesized and evaluated as P-glycoprotein inhibitors in an in vitro assay to estimate their potential to act as multidrug resistance modulators in cancer cells. Structure-activity relationships are discussed and prove a significant and regiospecific influence of certain functional groups.     

454 Genome sequencing of Pseudoperonospora cubensis reveals effector proteins with a QXLR translocation motif

Pseudoperonospora cubensis is a biotrophic oomycete pathogen that causes downy mildew of cucurbits, a devastating foliar disease threatening cucurbit production worldwide. We sequenced P. cubensis genomic DNA using 454 pyrosequencing and obtained random genomic sequences covering approximately 14% of the genome, thus providing the first set of useful genomic sequence information for P. cubensis. Using bioinformatics approaches, we identified 32 putative RXLR effector proteins. Interestingly, we also identified 29 secreted peptides with high similarity to RXLR effectors at the N-terminal translocation domain, yet containing an R-to-Q substitution in the first residue of the translocation motif. Among these, a family of QXLR-containing proteins, designated as PcQNE, was confirmed to have a functional signal peptide and was further characterized as being localized in the plant nucleus. Internalization of secreted PcQNE into plant cells requires the QXLR-EER motif. This family has a large number of near-identical copies within the P. cubensis genome, is under diversifying selection at the C-terminal domain, and is upregulated during infection of plants, all of which are common characteristics of characterized oomycete effectors. Taken together, the data suggest that PcQNE are bona fide effector proteins with a QXLR translocation motif, and QXLR effectors are prevalent in P. cubensis. Furthermore, the massive duplication of PcQNE suggests that they might play pivotal roles in pathogen fitness and pathogenicity.     

Molecular evidence for a fourth species within the Isotoma viridis group (Insecta, Collembola)

For identification of single species within the Isotoma viridis group, we present polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) as a fast and efficient DNA-based molecular method. We used five PCR primers amplifying the cytochrome oxidase II (COII) region (760 bp) of the mitochondrial DNA. The sequences clearly separated four species ( I. viridis , I. riparia , I. anglicana and I. caerulea ) out of samples from Norway, Sweden, Germany and Switzerland. Examination of genetic variation and phylogenetic relationship did not support the separation of two colour pattern forms of I. viridis into distinct species. For RFLP, several restriction enzymes were tested for their ability to produce not only species-specific restriction fragment patterns but to discriminate more than one species per enzyme used with as few cleavage sites as possible. Such a design should render a clear fragment pattern when performing a double digest. These demands appear to be fulfilled best by the combination of the restriction enzymes Mfe I, Nci I and one of Aci I, Bst EII, Nde I, or Sfc I. From the enzymes tested in a previous study, Ase I proved to be reliable, whereas Mbo I can no longer be recommended.     

Natural killer (NK) and lymphokine-activated killer (LAK) cell functions from healthy dogs and 29 dogs with a variety of spontaneous neoplasms

To investigate natural killer (NK) and lymphokine-activated killer (LAK) cell functions from 10 healthy dogs and 29 dogs with a variety of spontaneous neoplasms, large granular lymphocytes (LGLs) from blood samples were separated by a 58.5% Percoll density gradient. LGLs were stimulated with a low dose of recombinant human interleukin 2 (rhIL-2) for 7 days. Cytotoxicity of effector cells against the susceptible CTAC cell line was measured before and after stimulation. Compared with those before stimulation, the percentage of LGLs after stimulation with rhIL-2 was found to be significantly increased (P<0.01) in both dogs with tumors and controls. However, the increase was significantly higher in control animals, indicating a defect in proliferation ability of NK cells in canine tumor patients. After stimulation with rhIL-2, lymphokine-activated killer (LAK) cell activity in dogs with tumors was significantly lower (P<0.01) when compared with controls. Reduced cytotoxicity of rhIL-2–activated NK cells in dogs with tumors seems to be attributable to the presence of a diminished proliferative capacity of NK cells and a decreased ability of LAK cells to lyse target cells. Further knowledge of the precise function of IL-2–activated NK cells in dogs with tumors may help to optimize new and therapeutically beneficial treatment strategies in canine and human cancer patients. Our findings suggest that the dog could also serve as a relevant large animal model for cancer immunotherapy with IL-2.     

Complex determinants in human immunodeficiency virus type 1 envelope gp120 mediate CXCR4-dependent infection of macrophages

Host cell range, or tropism, combined with coreceptor usage defines viral phenotypes as macrophage tropic using CCR5 (M-R5), T-cell-line tropic using CXCR4 (T-X4), or dually lymphocyte and macrophage tropic using CXCR4 alone or in combination with CCR5 (D-X4 or D-R5X4). Although envelope gp120 V3 is necessary and sufficient for M-R5 and T-X4 phenotypes, the clarity of V3 as a dominant phenotypic determinant diminishes in the case of dualtropic viruses. We evaluated D-X4 phenotype, pathogenesis, and emergence of D-X4 viruses in vivo and mapped genetic determinants in gp120 that mediate use of CXCR4 on macrophages ex vivo. Viral quasispecies with D-X4 phenotypes were associated significantly with advanced CD4+-T-cell attrition and commingled with M-R5 or T-X4 viruses in postmortem thymic tissue and peripheral blood. A D-X4 phenotype required complex discontinuous genetic determinants in gp120, including charged and uncharged amino acids in V3, the V5 hypervariable domain, and novel V1/V2 regions distinct from prototypic M-R5 or T-X4 viruses. The D-X4 phenotype was associated with efficient use of CXCR4 and CD4 for fusion and entry but unrelated to levels of virion-associated gp120, indicating that gp120 conformation contributes to cell-specific tropism. The D-X4 phenotype describes a complex and heterogeneous class of envelopes that accumulate multiple amino acid changes along an evolutionary continuum. Unique gp120 determinants required for the use of CXCR4 on macrophages, in contrast to cells of lymphocytic lineage, can provide targets for development of novel strategies to block emergence of X4 quasispecies of human immunodeficiency virus type 1.     

Preferential vastus medialis oblique activation achieved as a treatment for knee disorders

The purpose of this study was to compare the effect of an open-stance cycling protocol (OSCP) with the traditional cycling foot position (TCFP) for preferential vastus medialis oblique (VMO) muscle activation, measured by surface electromyography (SEMG), and preferential VMO activation as defined by achieving significantly increased VMO/VL (vastus lateralis muscle) ratio values. Forty subjects of both sexes participated, 18 symptomatic with patellofemoral pain and 22 control subjects; ages ranged from 18 to 60 years (mean = 28.7 +/- 8 years). The OSCP and TCFP were ridden in randomized order while SEMG recordings were taken of the VMO and VL muscles, collecting the mean of peak amplitudes to calculate VMO/VL ratio values. The SEMG readings were taken 4 times per testing session with randomized resistance and a consistent cycling cadence of 85 rpm. The OSCP displayed preferential VMO activation for all subject groups (F = 40.47, p = 0.0001), and this study revealed a protocol that effectively treats patellofemoral pain.     

Structure-function studies of PANDER, an islet specific cytokine inducing cell death of insulin-secreting beta cells

PANDER (pancreatic derived factor, FAM3B) is a novel cytokine, present in insulin secretory granules, that induces apoptosis of alpha and beta cells of mouse, rat, and human islets in a dose- and time-dependent manner, and may be implicated in diabetes. PANDER has the predicted secondary structure of 4 alpha-helical bundles with an up-up-down-down topology, and two disulfide bonds. Eleven mutated PANDERs were constructed and expressed in beta-TC3 cells to identify the essential region of PANDER involved in beta-cell death. Beta-cell function was assessed by assays of cell viability and insulin secretion. Based on quantitative real-time RT-PCR all mutant PANDERs had similar mRNA expression levels in beta-TC3 cells. Immunoblotting showed that ten of eleven mutant PANDER proteins were synthesized and detected in beta-TC3 cells. A mutant PANDER with no signal peptide, however, was not expressed. Truncation of helix D alone caused a 40-50% decrease in PANDER's activity, while truncation of both helices C and D resulted in a 75% loss of activity. In contrast, truncation of the N-terminus of PANDER (helix A, the loop between helices A and B, and the first two cysteines) had no effect on PANDER-induced beta-cell death. The third and fourth cysteines of PANDER, C91 and C229, were shown to form one disulfide bond and be functionally important. Finally, the region between Cys91 and Phe152 constitutes the active part of PANDER, based on the demonstration that mutants with truncation of helix B or C caused decreased beta-cell death and did not inhibit insulin secretion, as compared to wild-type PANDER. Hence, helices B and C and the second disulfide bond of PANDER are essential for PANDER-induced beta-cell death.     

Effective dephosphorylation of Src substrates by SHP-1

The protein-tyrosine phosphatase SHP-1 is a negative regulator of multiple signal transduction pathways. We observed that SHP-1 effectively antagonized Src-dependent phosphorylations in HEK293 cells. This occurred by dephosphorylation of Src substrates, because Src activity was unaffected in the presence of SHP-1. One reason for efficient dephosphorylation was activation of SHP-1 by Src. Recombinant SHP-1 had elevated activity subsequent to phosphorylation by Src in vitro, and SHP-1 variants with mutated phosphorylation sites in the C terminus, SHP-1 Y538F, and SHP-1 Y538F,Y566F were less active toward Src-generated phosphoproteins in intact cells. A second reason for efficient dephosphorylation is the substrate selectivity of SHP-1. Pull-down experiments with different GST-SHP-1 fusion proteins revealed efficient interaction of Src-generated phosphoproteins with the SHP-1 catalytic domain rather than with the SH2 domains. Phosphopeptides that correspond to good Src substrates were efficiently dephosphorylated by SHP-1 in vitro. Phosphorylated "optimal Src substrate" AEEEIpYGEFEA (where pY is phosphotyrosine) and a phosphopeptide corresponding to a recently identified Src phosphorylation site in p120 catenin, DDLDpY(296)GMMSD, were excellent SHP-1 substrates. Docking of these phosphopeptides into the catalytic domain of SHP-1 by molecular modeling was consistent with the biochemical data and explains the efficient interaction. Acidic residues N-terminal of the phosphotyrosine seem to be of major importance for efficient substrate interaction. Residues C-terminal of the phosphotyrosine probably contribute to the substrate selectivity of SHP-1. We propose that activation of SHP-1 by Src and complementary substrate specificities of SHP-1 and Src may lead to very transient Src signals in the presence of SHP-1.