Modulation of apoptosis by V protein mumps virus

<p>Abstract</p> <p>Background</p> <p>The Urabe AM9 vaccine strain of mumps virus contains two variants of V protein: VWT (of HN-A1081 viral population) and VGly (of HN-G1081). The V protein is a promoting factor of viral replication by blocking the IFN antiviral pathway.</p> <p>Findings</p> <p>We studied the relationship between V protein variants and IFN-α2b-induced apoptosis. V proteins decrease activation of the extrinsic IFN-α2b-induced apoptotic pathway monitored by the caspase 8 activity, being the effect greater with the VWT protein. Both V proteins decrease the activity of caspase 9 of the intrinsic apoptotic pathway. In a system without IFN, the VWT and VGly proteins expression promotes activation of caspases 3 and 7. However, when the cellular system was stimulated with IFN-α, this activity decreased partially. TUNEL assay shows that for treatment with IFN-α and ibuprofen of cervical adenocarcinoma cells there is nuclear DNA fragmentation but the V protein expression reduces this process.</p> <p>Conclusions</p> <p>The reduction in the levels of caspases and DNA fragmentation, suggesting that V protein, particularly VWT protein of Urabe AM9 vaccine strain, modulates apoptosis. In addition, the VWT protein shows a protective role for cell proliferation in the presence of antiproliferative signals.</p

Interaction of mumps virus V protein variants with STAT1-STAT2 heterodimer: experimental and theoretical studies

<p>Abstract</p> <p>Background</p> <p>Mumps virus V protein has the ability to inhibit the interferon-mediated antiviral response by inducing degradation of STAT proteins. Two virus variants purified from Urabe AM9 mumps virus vaccine differ in their replication and transcription efficiency in cells primed with interferon. Virus susceptibility to IFN was associated with insertion of a non-coded glycine at position 156 in the V protein (VGly) of one virus variant, whereas resistance to IFN was associated with preservation of wild-type phenotype in the V protein (VWT) of the other variant.</p> <p>Results</p> <p>VWT and VGly variants of mumps virus were cloned and sequenced from Urabe AM9 vaccine strain. VGly differs from VWT protein because it possesses an amino acid change Gln₁₀₃Pro (Pro¹⁰³) and the Gly¹⁵⁶insertion. The effect of V protein variants on components of the interferon-stimulated gene factor 3 (ISGF3), STAT1 and STAT2 proteins were experimentally tested in cervical carcinoma cell lines. Expression of VWT protein decreased STAT1 phosphorylation, whereas VGly had no inhibitory effect on either STAT1 or STAT2 phosphorylation. For theoretical analysis of the interaction between V proteins and STAT proteins, 3D structural models of VWT and VGly were predicted by comparing with simian virus 5 (SV5) V protein structure in complex with STAT1-STAT2 heterodimer. <it>In silico </it>analysis showed that VWT-STAT1-STAT2 complex occurs through the V protein Trp-motif (W¹⁷⁴, W¹⁷⁸, W¹⁸⁹) and Glu⁹⁵residue close to the Arg⁴⁰⁹and Lys⁴¹⁵of the nuclear localization signal (NLS) of STAT2, leaving exposed STAT1 Lys residues (K⁸⁵, K⁸⁷, K²⁹⁶, K⁴¹³, K⁵²⁵, K⁶⁷⁹, K⁶⁸⁵), which are susceptible to proteasome degradation. In contrast, the interaction between VGly and STAT1-STAT2 heterodimer occurs in a region far from the NLS of STAT2 without blocking of Lys residues in both STAT1 and STAT2.</p> <p>Conclusions</p> <p>Our results suggest that VWT protein of Urabe AM9 strain of mumps virus may be more efficient than VGly to inactivate both the IFN signaling pathway and antiviral response due to differences in their finest molecular interaction with STAT proteins.</p

Maintaining RNA integrity in a homogeneous population of mammary epithelial cells isolated by Laser Capture Microdissection

Background: Laser-capture microdissection (LCM) that enables the isolation of specific cell populations from complex tissues under morphological control is increasingly used for subsequent gene expression studies in cell biology by methods such as real-time quantitative PCR (qPCR), microarrays and most recently by RNA-sequencing. Challenges are i) to select precisely and efficiently cells of interest and ii) to maintain RNA integrity. The mammary gland which is a complex and heterogeneous tissue, consists of multiple cell types, changing in relative proportion during its development and thus hampering gene expression profiling comparison on whole tissue between physiological stages. During lactation, mammary epithelial cells (MEC) are predominant. However several other cell types, including myoepithelial (MMC) and immune cells are present, making it difficult to precisely determine the specificity of gene expression to the cell type of origin. In this work, an optimized reliable procedure for producing RNA from alveolar epithelial cells isolated from frozen histological sections of lactating goat, sheep and cow mammary glands using an infrared-laser based Arcturus Veritas LCM (Applied Biosystems®) system has been developed. The following steps of the microdissection workflow: cryosectioning, staining, dehydration and harvesting of microdissected cells have been carefully considered and designed to ensure cell capture efficiency without compromising RNA integrity.[br/] Results: The best results were obtained when staining 8 μm-thick sections with Cresyl violet® (Ambion, Applied Biosystems®) and capturing microdissected cells during less than 2 hours before RNA extraction. In addition, particular attention was paid to animal preparation before biopsies or slaughtering (milking) and freezing of tissue blocks which were embedded in a cryoprotective compound before being immersed in isopentane. The amount of RNA thus obtained from ca.150 to 250 acini (300,000 to 600,000 μm2) ranges between 5 to 10 ng. RNA integrity number (RIN) was ca. 8.0 and selectivity of this LCM protocol was demonstrated through qPCR analyses for several alveolar cell specific genes, including LALBA (α-lactalbumin) and CSN1S2 (αs2-casein), as well as Krt14 (cytokeratin 14), CD3e and CD68 which are specific markers of MMC, lymphocytes and macrophages, respectively.[br/] Conclusions: RNAs isolated from MEC in this manner were of very good quality for subsequent linear amplification, thus making it possible to establish a referential gene expression profile of the healthy MEC, a useful platform for tumor biomarker discovery

A preliminary study of mercury exposure and blood pressure in the Brazilian Amazon

BACKGROUND: Fish is considered protective for coronary heart disease (CHD), but mercury (Hg) intake from fish may counterbalance beneficial effects. Although neurotoxic effects of methylmercury (MeHg) are well established, cardiovascular effects are still debated. The objective of the present study was to evaluate blood pressure in relation to Hg exposure and fish consumption among a non-indigenous fish-eating population in the Brazilian Amazon. METHODS: The study was conducted among 251 persons from six communities along the Tapajós River, a major tributary of the Amazon. Data was obtained for socio-demographic information, fish consumption, height and weight to determine body mass index (BMI), systolic and diastolic blood pressure, and Hg concentration in hair samples. RESULTS: Results showed that overall, systolic and diastolic blood pressure, were relatively low (mean: 113.9 mmHg ± 14.6 and 73.7 mmHg ± 11.0). Blood pressure was significantly associated with hair total Hg (H-Hg), age, BMI and gender. No association was observed between fish consumption and blood pressure, although there were significant inter-community differences. Logistic regression analyses showed that the Odds Ratio (OR) for elevated systolic blood pressure (≥ 130 mmHg) with H-Hg ≥ 10 μg/g was 2.91 [1.26–7.28], taking into account age, BMI, smoking, gender and community. CONCLUSION: The findings of this preliminary study add further support for Hg cardiovascular toxicity