Regulation of OPA1-mediated mitochondrial fusion by leucine zipper/EF-hand-containing transmembrane protein-1 plays a role in apoptosis

Carboxyl-terminal modulator protein (CTMP) is a tumor suppressor-like binding partner of Protein kinase B (PKB/Akt) that negative regulates this kinase. In the course of our recent work, we identified that CTMP is consistently associated with leucine zipper/EF-hand-containing transmembrane-1 (LETM1). Here, we report that adenovirus-LETM1 increased the sensitivity of HeLa cells to apoptosis, induced by either staurosporine or actinomycin D. As shown previously, LETM1 localized to the inner mitochondrial membrane. Electron-microscopy analysis of adenovirus-LETM1 transduced cells revealed that mitochondrial cristae were swollen in these cells, a phenotype similar to that observed in optic atrophy type-1 (OPA1)-ablated cells. OPA1 cleavage was increased in LETM1-overexpressing cells, and this phenotype was reversed by overexpression of OPA1 variant-7, a cleavage resistant form of OPA1. Taken together, these data suggest that LETM1 is a novel binding partner for CTMP that may play an important role in mitochondrial fragmentation via OPA1-cleavage. (C) 2009 Elsevier Inc. All rights reserved

C/EBP alpha Is Up-regulated in a Subset of Hepatocellular Carcinomas and Plays a Role in Cell Growth and Proliferation

BACKGROUND & AIMS: C/EBP alpha (cebpa) is a putative tumor suppressor. However, initial results indicated that cebpa was up-regulated in a subset of human hepatocellular carcinomas (HCCs). The regulation and function of C/EBP alpha was investigated in HCC cell lines to clarify its role in liver carcinogenesis. METHODS: The regulation of C/EBP alpha expression was studied by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blotting, immunohistochemistry, methylation-specific PCR, and chromatin immunoprecipitation assays. C/EBP alpha expression was knocked-down by small interfering RNA or short hairpin RNA. Functional assays included colony formation, methylthiotetrazole, bromodeoxyuridine incorporation, and luciferase-reporter assays. RESULTS: Cebpa was up-regulated at least 2-fold in a subset (approximately 55%) of human HCCs compared with adjacent non tumor tissues. None of the up-regulated samples were positive for hepatitis C infection. The HCC cell lines Hep3B and Huh7 expressed high, PLC/PRF/5 intermediate, HepG2 and HCC-M low levels of C/EBP alpha, recapitulating the pattern of expression observed in HCCs. No mutations were detected in the CEBP alpha gene in HCCs and cell lines. C/EBP alpha was localized to the nucleus and functional in Hep3B and Huh7 cells; knocking-down its expression reduced target-gene expression, colony formation, and cell growth, associated with a decrease in cyclin A and CDK4 concentrations and E2F transcriptional activity. Epigenetic mechanisms including DNA methylation, and the binding of acetylated histone H3 to the CEBP alpha promoter-regulated cebpa expression in the HCC cells. CONCLUSIONS: C/EBP alpha is up-regulated in a subset of HCCs and has growth-promoting activities in HCC cells. Novel oncogenic mechanisms involving C/EBP alpha may be amenable to epigenetic regulation to improve treatment outcomes.

PI3K beta Plays a Critical Role in Neutrophil Activation by Immune Complexes

Neutrophils are activated by immunoglobulin G (IgG)-containing immune complexes through receptors that recognize the Fc portion of IgG (Fc gamma Rs). Here, we used genetic and pharmacological approaches to define a selective role for the beta isoform of phosphoinositide 3-kinase (PI3K beta) in Fc gamma R-dependent activation of mouse neutrophils by immune complexes of IgG and antigen immobilized on a plate surface. At low concentrations of immune complexes, loss of PI3K beta alone substantially inhibited the production of reactive oxygen species (ROS) by neutrophils, whereas at higher doses, similar suppression of ROS production was achieved only by targeting both PI3K beta and PI3K delta, suggesting that this pathway displays stimulus strength-dependent redundancy. Activation of PI3K beta by immune complexes involved cooperation between Fc gamma Rs and BLT1, the receptor for the endogenous proinflammatory lipid leukotriene B-4. Coincident activation by a tyrosine kinase-coupled receptor (Fc gamma R) and a heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor (BLT1) may provide a rationale for the preferential activation of the beta isoform of PI3K. PI3K beta-deficient mice were highly protected in an Fc gamma R-dependent model of autoantibody-induced skin blistering and were partially protected in an Fc gamma R-dependent model of inflammatory arthritis, whereas combined deficiency of PI3K beta and PI3K delta resulted in near-complete protection in the latter case. These results define PI3K beta as a potential therapeutic target in inflammatory disease.

Plays of Difference

Review-essay

Service User and Carer Involvement in Role-Plays to Assess Readiness for Practice

Service user and carer involvement in social work education is now well established since its inception as a compulsory requirement in the social work curriculum in the United Kingdom in 2003. Since then, there have been many examples of how such involvement has been approached by education providers. Nevertheless, one of the key obstacles and challenges in this field continues to centre on the need to achieve non-tokenistic user involvement which cements the engagement of service users and carers at the heart of social work education. This paper describes one such initiative where service user and carer colleagues in a university in Northern Ireland have been actively involved in the assessment of first year social work students’ preparation for their first period of practice learning. The paper presents the background to this initiative explaining how the project unfolded; the detailed preparations that were involved and the evidence gathered from evaluations undertaken with the students, service users and carers, and academic colleagues who were all involved. We believe that the findings from this project can contribute to the advancement of existing knowledge in the field in exploring and recommending creative methodologies for user involvement in social work education.

The lipopolysaccharide outer core of Yersinia enterocolitica serotype O:3 is required for virulence and plays a role in outer membrane integrity

Lipopolysaccharide (LPS) of Yersinia enterocolitica O:3 has an inner core linked to both the O-antigen and to an outer core hexasaccharide that forms a branch. The biological role of the outer core was studied using polar and non-polar mutants of the outer core biosynthetic operon. Analysis of O-antigen- and outer core-deficient strains suggested a critical role for the outer core in outer membrane properties relevant in resistance to antimicrobial peptides and permeability to hydrophobic agents, and indirectly relevant in resistance to killing by normal serum. Wild-type bacteria but not outer core mutants killed intragastrically infected mice, and the intravenous lethal dose was approximately 10(4)-fold higher for outer core mutants. After intragastric infection, outer core mutants colonized Peyer's patches and invaded mesenteric lymph nodes, spleen and liver, and induced protective immunity against wild-type bacteria. In mice co-infected intragastrically with an outer core mutant-wild type mixture, both strains colonized Peyer's patches similarly during the first 2 days, but the mutant was much less efficient in colonizing deeper organs and was cleared faster from Peyer's patches. The results demonstrate that outer core is required for Y. enterocolitica O:3 full virulence, and strongly suggest that it provides resistance against defence mechanisms (most probably those involving bactericidal peptides).

Elevated NRD1 metalloprotease expression plays a role in breast cancer growth and proliferation

Understanding the molecular etiology of cancer and increasing the number of drugs and their targets are critical to cancer management. In our attempt to unravel novel breast-cancer associated proteins, we previously conducted protein expression profiling of the MCF10AT model, which comprises a series of isogenic cell lines that mimic different stages of breast cancer progression. NRD1 expression was found to increase during breast cancer progression. Here, we attempted to confirm the relevance of NRD1 in clinical breast cancer and understand the functional role and mechanism of NRD1 in breast cancer cells. Immunohistochemistry data show that NRD1 expression was elevated in ductal carcinoma in situ and invasive ductal carcinomas compared with normal tissues in 30% of the 26 matched cases studied. Examination of NRD1 expression in tissue microarray comprising >100 carcinomas and subsequent correlation with clinical data revealed that NRD1 expression was significantly associated with tumor size, grade, and nodal status (P <0.05). Silencing of NRD1 reduced MCF10CA1h and MDA-MD-231 breast-cancer-cell proliferation and growth. Probing the oncogenic EGF signaling pathways revealed that NRD1 knock down did not affect overall downstream tyrosine phosphorylation cascades including AKT and MAPK activation. Instead, silencing of NRD1 resulted in a reduction of overall cyclin D1 expression, a reduction of EGF-induced increase in cyclin D1 expression and an increase in apoptotic cell population compared with control cells.

Activation of the Wnt pathway plays a pathogenic role in diabetic retinopathy in humans and animal models

Although Wnt signaling is known to mediate multiple biological and pathological processes, its association with diabetic retinopathy (DR) has not been established. Here we show that retinal levels and nuclear translocation of beta-catenin, a key effector in the canonical Wnt pathway, were increased in humans with DR and in three DR models. Retinal levels of low-density lipoprotein receptor-related proteins 5 and 6, coreceptors of Wnts, were also elevated in the DR models. The high glucose-induced activation of beta-catenin was attenuated by aminoguanidine, suggesting that oxidative stress is a direct cause for the Wnt pathway activation in diabetes. Indeed, Dickkopf homolog 1, a specific inhibitor of the Wnt pathway, ameliorated retinal inflammation, vascular leakage, and retinal neovascularization in the DR models. Dickkopf homolog 1 also blocked the generation of reactive oxygen species induced by high glucose, suggesting that Wnt signaling contributes to the oxidative stress in diabetes. These observations indicate that the Wnt pathway plays a pathogenic role in DR and represents a novel therapeutic target.