Alternariol induces abnormal nuclear morphology and cell cycle arrest in murine RAW 264.7 macrophages.

The mycotoxin alternariol (AOH), a frequent contaminant in fruit and cereal products, is known to induce DNA damage with subsequent cell cycle arrest. Here we elucidated the effects of AOH on stages of cell cycle progression using the RAW 264.7 macrophage model. AOH resulted in an accumulation of cells in the G2/M-phase (4N). Most cells exhibited a large G2 nucleus whereas numbers of true mitotic cells were reduced relative to control. Both cyclin B1 and p-cdc2 levels increased, while cyclin B1 remained in the cytoplasm; suggesting arrest in the G2/M transition point. Remarkably, after exposure to AOH for 24h, most of the cells exhibited abnormally shaped nuclei, as evidenced by partly divided nuclei, nuclear blebs, polyploidy and micronuclei (MN). AOH treatment also induced abnormal Aurora B bridges, suggesting that cytokinesis was interfered within cells undergoing karyokinesis. A minor part of the resultant G1 tetraploid (4N) cells re-entered the S-phase and progressed to 8N cells.

Cbl escapes Cdc42-mediated inhibition by downregulation of the adaptor molecule betaPix.

The Pix/Cool proteins are involved in the regulation of cell morphology by binding to small Rho GTPases and kinases of the Pak family. Recently, it has been shown that betaPix/Cool-1 associates with the ubiquitin ligase Cbl, which appears to be a critical step in Cdc42-mediated inhibition of epidermal-growth-factor-receptor (EGFR) ubiquitylation and downregulation. Here we show that the SH3 domain of betaPix specifically interacts with a proline-arginine motif (PxxxPR) present within the ubiquitin ligase Cbl and Pak1 kinase. Owing to targeting of the same sequence, Cbl and Pak1 compete for binding to betaPix. In this complex, Cbl mediates ubiquitylation and subsequent degradation of betaPix. Our findings reveal a double feedback loop in which the Cdc42/betaPix complex blocks Cbl's ability to downregulate EGFR, while Cbl in turn promotes degradation of betaPix in order to escape this inhibition. Such a relationship provides a mechanism to fine-tune the kinetics of RTK endocytosis and degradation depending on the pool of active Cdc42 and the duration of EGFR signaling.

Distress, quality of life and strategies to 'keep a good mood' in patients with carcinoid tumours: patient and staff perceptions.

Patients with carcinoid tumours have reported a relatively good health-related quality of life (HRQoL) (European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-C30, EORTC QLQ-C30), and low levels of anxiety and depression (Hospital Anxiety and Depression Scale, HADS). The aim was to test the validity of these results. Data were gathered through interviews with 19 patients and 19 staff. Participants were asked about disease and treatment related distress, important aspects of quality of life and strategies to 'keep a good mood'. Patients were interviewed about themselves and staff were interviewed about a certain patient. Data were analysed by content analysis. Identified aspects of distress and quality of life were referred to an emotional, a physical and a social dimension. Most aspects of distress were of a physical character whereas most aspects of quality of life were of a social character. Several aspects of emotional distress not included in the EORTC QLQ-C30 and/or the HADS were identified.

Homeobox gene Cdx1 regulates Ras, Rho and PI3 kinase pathways leading to transformation and tumorigenesis of intestinal epithelial cells.

The Cdx1 homeobox gene encodes for an intestine-specific transcription factor involved in the control of proliferation and differentiation of epithelial cells. Although it has been indicated that Cdx1 may act as a proto-oncogene in cultured fibroblasts, its direct role in the regulation of intestinal tumorigenesis has not been demonstrated. Here we show that expression of Cdx1 in an intestinal epithelial cell line (IEC-6) induces anchorage-independent growth in soft agar and promotes the formation of adenocarcinoma in vivo. The phenotype of Cdx1-induced tumors was exacerbated when IEC-6/Cdx1 cells were injected together with matrigel containing mitogens and extracellular matrix components. These changes were correlated with an increase in the GTP-bound form of Ras, modulation of Cdc42 and Rho-A activities, and accumulation of phosphatidyl inositol 3 (PI3) kinase products. Moreover, combined inhibition of Ras/Rho and PI3 kinase signaling by synthethic inhibitors blocked colony formation of IEC-6/Cdx1 cells in soft agar. Taken together, these results demonstrate a direct involvement of Cdx1, and its collaboration with Ras, Rho and PI3 kinase pathways, in transformation and tumorigenesis of intestinal epithelial cells.

Expression of human immunodeficiency virus type 1 Gag protein precursor and envelope proteins from a vesicular stomatitis virus recombinant: high-level production of virus-like particles containing HIV envelope.

Recombinant vesicular stomatitis viruses have been developed as high-level expression vectors which serve as effective vaccine vectors in animals (Roberts et al., 1998, J. Virol. 72, 4704-4711; Roberts et al., 1999, J. Virol. 73, 3723-3732). Here we show that two genes can be expressed simultaneously from a single, live-attenuated VSV recombinant. The genes used encode the Pr55(gag) protein precursor of HIV-1 (1.7-kb gene) and an HIV-1 envelope (Env) protein (2.4 kb gene). Our results show that VSV can accommodate up to a 40% increase in genome size with only a threefold reduction in virus titer. Recombinants expressing the Pr55(gag) protein precursor with or without Env protein produced abundant HIV virus-like particles (VLPs) in addition to bullet-shaped VSV particles. HIV Env protein expressed from a VSV recombinant also expressing Gag was specifically incorporated into the HIV VLPs but not into the VSV particles. In contrast, VSV G protein was found in both VSV particles and in HIV VLPs. Such VSV/HIV recombinants producing HIV VLPs with Env protein could be an effective source of HIV-like particles inducing both cellular and antibody-mediated immunity to HIV-1.

Identification, expression, and immunogenicity of Kaposi's sarcoma-associated herpesvirus-encoded small viral capsid antigen.

We describe a recombinant antigen for use in serologic tests for antibodies to Kaposi's sarcoma (KS)-associated herpesvirus (KSHV). The cDNA for a small viral capsid antigen (sVCA) was identified by immunoscreening of a library prepared from the BC-1 body cavity lymphoma cell line induced into KSHV lytic gene expression by sodium butyrate. The cDNA specified a 170-amino-acid peptide with homology to small viral capsid proteins encoded by the BFRF3 gene of Epstein-Barr virus and the ORF65 gene of herpesvirus saimiri. KSHV sVCA was expressed from a 0.85-kb mRNA present late in lytic KSHV replication in BC-1 cells. This transcript was sensitive to phosphonoacetic acid and phosphonoformic acid, inhibitors of herpesvirus DNA replication. KSHV sVCA expressed in mammalian cells or Escherichia coli or translated in vitro was recognized as an antigen by antisera from KS patients. Rabbit antisera raised to KSHV sVCA expressed in E. coli detected a 22-kDa protein in KSHV-infected human B cells. Overexpressed KSHV sVCA purified from E. coli and used as an antigen in immunoblot screening assay did not cross-react with EBV BFRF3. Antibodies to sVCA were present in 89% of 47 human immunodeficiency virus (HIV)-positive patients with KS, in 20% of 54 HIV-positive patients without KS, but in none of 122 other patients including children born to HIV-seropositive mothers and patients with hemophilia, autoimmune disease, or nasopharyngeal carcinoma. Low-titer antibody was detected in three sera from 28 healthy subjects. Antibodies to recombinant sVCA correlate with KS in high-risk populations. Recombinant sVCA can be used to examine the seroepidemiology of infection with KSHV in the general population.

Expression of additional genes in a vector derived from a minimal RNA virus.

Previous studies have shown that expression of the vesicular stomatitis virus (VSV) glycoprotein (G) from a Semliki Forest virus (SFV) RNA replicon results in the production of propagating infectious particles that we call minimal viruses. These minimal viruses consist of vesicles containing VSV G protein that bud from the plasma membrane and trap the infectious SFV G RNA, but they do not contain other viral structural proteins. The cell binding and membrane fusion activity of the VSV G protein allow minimal viruses to propagate in tissue culture cells. To determine if these minimal viruses could be used to express foreign genes, we added a second SFV promoter and a multiple cloning site downstream of the VSV G gene. We report here expression of three different proteins from this modified, minimal virus vector. Although expression of each foreign, unselected gene was lost rapidly from the vector upon passaging, it was possible after the initial transfection to derive stocks of infectious particles that could be used to infect multiple additional cultures and transfer protein expression efficiently. When cells were infected with these minimal viruses, host protein synthesis was shut off and the foreign protein and VSV G proteins were the major proteins expressed in the infected cells. Both were expressed at similar levels and accumulated to about 1-2% of total cell protein.