Human Parainfluenza Virus Type 1 C Proteins Are Nonessential Proteins That Inhibit the Host Interferon and Apoptotic Responses and Are Required for Efficient Replication in Nonhuman Primates

Recombinant human parainfluenza virus type 1 (rHPIV1) was modified to create rHPIV1-P(C−), a virus in which expression of the C proteins (C′, C, Y1, and Y2) was silenced without affecting the amino acid sequence of the P protein. Infectious rHPIV1-P(C−) was readily recovered from cDNA, indicating that the four C proteins were not essential for virus replication. Early during infection in vitro, rHPIV1-P(C−) replicated as efficiently as wild-type (wt) HPIV1, but its titer subsequently decreased coincident with the onset of an extensive cytopathic effect not observed with wt rHPIV1. rHPIV1-P(C−) infection, but not wt rHPIV1 infection, induced caspase 3 activation and nuclear fragmentation in LLC-MK2 cells, identifying the HPIV1 C proteins as inhibitors of apoptosis. In contrast to wt rHPIV1, rHPIV1-P(C−) and rHPIV1-CF170S, a mutant encoding an F170S substitution in C, induced interferon (IFN) and did not inhibit IFN signaling in vitro. However, only rHPIV1-P(C−) induced apoptosis. Thus, the anti-IFN and antiapoptosis activities of HPIV1 were separable: both activities are disabled in rHPIV1-P(C−), whereas only the anti-IFN activity is disabled in rHPIV1-CF170S. In African green monkeys (AGMs), rHPIV1-P(C−) was considerably more attenuated than rHPIV1-CF170S, suggesting that disabling the anti-IFN and antiapoptotic activities of HPIV1 had additive effects on attenuation in vivo. Although rHPIV1-P(C−) protected against challenge with wt HPIV1, its highly restricted replication in AGMs and in primary human airway epithelial cell cultures suggests that it might be overattenuated for use as a vaccine. Thus, the C proteins of HPIV1 are nonessential but have anti-IFN and antiapoptosis activities required for virulence in primates

Allosteric Modulators of Steroid Hormone Receptors : Structural Dynamics and Gene Regulation

Peer reviewedPublisher PD

The Effectiveness of RNAi in Caenorhabditis elegans Is Maintained during Spaceflight

PublishedJournal ArticleResearch Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov'tThis is the final version of the article. Available from Public Library of Science via the DOI in this record.BACKGROUND: Overcoming spaceflight-induced (patho)physiologic adaptations is a major challenge preventing long-term deep space exploration. RNA interference (RNAi) has emerged as a promising therapeutic for combating diseases on Earth; however the efficacy of RNAi in space is currently unknown. METHODS: Caenorhabditis elegans were prepared in liquid media on Earth using standard techniques and treated acutely with RNAi or a vector control upon arrival in Low Earth Orbit. After culturing during 4 and 8 d spaceflight, experiments were stopped by freezing at -80°C until analysis by mRNA and microRNA array chips, microscopy and Western blot on return to Earth. Ground controls (GC) on Earth were simultaneously grown under identical conditions. RESULTS: After 8 d spaceflight, mRNA expression levels of components of the RNAi machinery were not different from that in GC (e.g., Dicer, Argonaute, Piwi; P>0.05). The expression of 228 microRNAs, of the 232 analysed, were also unaffected during 4 and 8 d spaceflight (P>0.05). In spaceflight, RNAi against green fluorescent protein (gfp) reduced chromosomal gfp expression in gonad tissue, which was not different from GC. RNAi against rbx-1 also induced abnormal chromosome segregation in the gonad during spaceflight as on Earth. Finally, culture in RNAi against lysosomal cathepsins prevented degradation of the muscle-specific α-actin protein in both spaceflight and GC conditions. CONCLUSIONS: Treatment with RNAi works as effectively in the space environment as on Earth within multiple tissues, suggesting RNAi may provide an effective tool for combating spaceflight-induced pathologies aboard future long-duration space missions. Furthermore, this is the first demonstration that RNAi can be utilised to block muscle protein degradation, both on Earth and in space.This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, the Japan Society for the Promotion of Science, and “Ground-Based Research Announcement for Space Utilization” promoted by the Japan Space Forum. TE was supported by the Medical Research Council UK (G0801271). NJS was supported by the National Institutes of Health (NIH NIAMS ARO54342). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Amphiregulin Mediates Estrogen, Progesterone, and EGFR Signaling in the Normal Rat Mammary Gland and in Hormone-Dependent Rat Mammary Cancers

Both estrogen (E) and progesterone (P) are implicated in the etiology of human breast cancer. Defining their mechanisms of action, particularly in vivo, is relevant to the prevention and therapy of breast cancer. We investigated the molecular and cellular mechanisms of E and/or P-induced in vivo proliferation, in the normal rat mammary gland and in hormone-dependent rat mammary cancers which share many characteristics with the normal human breast and hormone-dependent breast cancers. We show that E+P treatment induced significantly greater proliferation in both the normal gland and mammary cancers compared to E alone. In both the normal gland and tumors, E+P-induced proliferation was mediated through the increased production of amphiregulin (Areg), an epidermal growth factor receptor (EGFR) ligand, and the activation of intracellular signaling pathways (Erk, Akt, JNK) downstream of EGFR that regulate proliferation. In vitro experiments using rat primary mammary organoids or T47D breast cancer cells confirmed that Areg and the synthetic progestin, R5020, synergize to promote cell proliferation through EGFR signaling. Iressa, an EGFR inhibitor, effectively blocked this proliferation. These results indicate that mediators of cross talk between E, P, and EGFR pathways may be considered as relevant molecular targets for the therapy of hormone-dependent breast cancers, especially in premenopausal women

Inhibition of the Progesterone Nuclear Receptor during the Bone Linear Growth Phase Increases Peak Bone Mass in Female Mice

Augmentation of the peak bone mass (PBM) may be one of the most effective interventions to reduce the risk of developing osteoporosis later in life; however treatments to augment PBM are currently limited. Our study evaluated whether a greater PBM could be achieved either in the progesterone nuclear receptor knockout mice (PRKO) or by using a nuclear progesterone receptor (nPR) antagonist, RU486 in mice. Compared to their wild type (WT) littermates the female PRKO mice developed significantly higher cancellous and cortical mass in the distal femurs, and this was associated with increased bone formation. The high bone mass phenotype was partially reproduced by administering RU486 in female WT mice from 1–3 months of age. Our results suggest that the inhibition of the nPR during the rapid bone growth period (1–3 months) increases osteogenesis, which results in acquisition of higher bone mass. Our findings suggest a crucial role for progesterone signaling in bone acquisition and inhibition of the nPR as a novel approach to augment bone mass, which may have the potential to reduce the burden of osteoporosis

Non-traditional platinum compounds for improved accumulation, oral bioavailability, and tumor targeting

The five platinum anticancer compounds currently in clinical use conform to structure–activity relationships formulated (M. J. Cleare and J. D. Hoeschele, Bioinorg. Chem., 1973, 2, 187–210) shortly after the discovery that cis-diamminedichloroplatinum(II), cisplatin, has antitumor activity in mice. These compounds are neutral platinum(II) species with two am(m)ine ligands or one bidentate chelating diamine and two additional ligands that can be replaced by water through aquation reactions. The resulting cations ultimately form bifunctional adducts on DNA. Information about the chemistry of these platinum compounds and correlations of their structures with anticancer activity have provided guidance for the design of novel anticancer drug candidates based on the proposed mechanisms of action. This article discusses advances in the synthesis and evaluation of such non-traditional platinum compounds, including cationic and tumor-targeting constructs.National Cancer Institute (U.S.) (Grant CA34992

Regulation of the IGFBP-5 and MMP-13 genes by the microRNAs miR-140 and miR-27a in human osteoarthritic chondrocytes

<p>Abstract</p> <p>Background</p> <p>MMP-13 and IGFBP-5 are important factors involved in osteoarthritis (OA). We investigated whether two highly predicted microRNAs (miRNAs), miR-140 and miR-27a, regulate these two genes in human OA chondrocytes.</p> <p>Methods</p> <p>Gene expression was determined by real-time PCR. The effect of each miRNA on IGFBP-5 and MMP-13 expression/production was evaluated by transiently transfecting their precursors (pre-miRNAs) and inhibitors (anti-miRNAs) into human OA chondrocytes. Modulation of IGFBP-5, miR-140 and miR-27a expression was determined upon treatment of OA chondrocytes with cytokines and growth factors.</p> <p>Results</p> <p>IGFBP-5 was expressed in human chondrocytes with its level significantly lower (p < 0.04) in OA. Five computational algorithms identified miR-140 and miR-27a as possible regulators of MMP-13 and IGFBP-5 expression. Data showed that both miRNAs were expressed in chondrocytes. There was a significant reduction (77%, p < 0.01) in miR-140 expression in OA compared to the normal chondrocytes, whereas miR-27a expression was only slightly decreased (23%). Transfection with pre-miR-140 significantly decreased (p = 0.0002) and with anti-miR-140 significantly increased (p = 0.05) IGFBP-5 expression at 24 hours, while pre-miR-27a did not affect either MMP-13 or IGFBP-5. Treatment with anti-miR-27a, but not with anti-miR-140, significantly increased the expression of both MMP-13 (p < 0.05) and IGFBP-5 (p < 0.01) after 72 hours of incubation. MMP-13 and IGFBP-5 protein production followed the same pattern as their expression profile. These data suggest that IGFBP-5 is a direct target of miR-140, whereas miR-27a down-regulates, likely indirectly, both MMP-13 and IGFBP-5.</p> <p>Conclusion</p> <p>This study is the first to show the regulation of these miRNAs in human OA chondrocytes. Their effect on two genes involved in OA pathophysiology adds another level of complexity to gene regulation, which could open up novel avenues in OA therapeutic strategies.</p