A role for transportin in the nuclear import of adenovirus core proteins and DNA

Adenoviruses target their double-stranded DNA genome and its associated core proteins to the interphase nucleus; this core structure then enters through the nuclear pore complex. We have used digitonin permeabilized cell import assays to study the cellular import factors involved in nuclear entry of virus DNA and the core proteins, protein V and protein VII. We show that inhibition of transportin results in aberrant localization of protein V and that transportin is necessary for protein V to accumulate in the nucleolus. Furthermore, inhibition of transportin results in inhibition of protein VII and DNA import, whereas disruption of the classical importin alpha-importin beta import pathway has little effect. We show that mature protein VII has different import preferences from the precursor protein, preVII from which it is derived by proteolytic processing. While bacterially expressed glutathione S-transferase (GST)-preVII primarily utilizes the pathway mediated by importin alpha-importin beta, bacterially expressed GST-VII favours the transportin pathway. This is significant because while preVII is important during viral replication and assembly only mature VII is available during viral DNA import to a newly infected cell. Our results implicate transportin as a key import receptor for the nuclear localization of adenovirus core.     

The CTLA-4 and PD-1/PD-L1 Inhibitory Pathways Independently Regulate Host Resistance to Plasmodium-induced Acute Immune Pathology

The balance between pro-inflammatory and regulatory immune responses in determining optimal T cell activation is vital for the successful resolution of microbial infections. This balance is maintained in part by the negative regulators of T cell activation, CTLA-4 and PD-1/PD-L, which dampen effector responses during chronic infections. However, their role in acute infections, such as malaria, remains less clear. In this study, we determined the contribution of CTLA-4 and PD-1/PD-L to the regulation of T cell responses during Plasmodium berghei ANKA (PbA)-induced experimental cerebral malaria (ECM) in susceptible (C57BL/6) and resistant (BALB/c) mice. We found that the expression of CTLA-4 and PD-1 on T cells correlates with the extent of pro-inflammatory responses induced during PbA infection, being higher in C57BL/6 than in BALB/c mice. Thus, ECM develops despite high levels of expression of these inhibitory receptors. However, antibody-mediated blockade of either the CTLA-4 or PD-1/PD-L1, but not the PD-1/PD-L2, pathways during PbA-infection in ECM-resistant BALB/c mice resulted in higher levels of T cell activation, enhanced IFN-γ production, increased intravascular arrest of both parasitised erythrocytes and CD8⁺ T cells to the brain, and augmented incidence of ECM. Thus, in ECM-resistant BALB/c mice, CTLA-4 and PD-1/PD-L1 represent essential, independent and non-redundant pathways for maintaining T cell homeostasis during a virulent malaria infection. Moreover, neutralisation of IFN-γ or depletion of CD8⁺ T cells during PbA infection was shown to reverse the pathologic effects of regulatory pathway blockade, highlighting that the aetiology of ECM in the BALB/c mice is similar to that in C57BL/6 mice. In summary, our results underscore the differential and complex regulation that governs immune responses to malaria parasites.     

Insight into the role of sugars in bud burst under light in the rose

Bud burst is a decisive process in plant architecture that requires light in Rosa sp. This light effect was correlated with stimulation of sugar transport and metabolism in favor of bud outgrowth. We investigated whether sugars could act as signaling entities in the light-mediated regulation of vacuolar invertases and bud burst. Full-length cDNAs encoding two vacuolar invertases (RhVI1 and RhVI2) were isolated from buds. Unlike RhVI2, RhVI1 was preferentially expressed in bursting buds, and was up-regulated in buds of beheaded plants exposed to light. To assess the importance of sugars in this process, the expression of RhVI1 and RhVI2 and the total vacuolar invertase activity were further characterized in buds cultured in vitro on 100 mM sucrose or mannitol under light or in darkness for 48 h. Unlike mannitol, sucrose promoted the stimulatory effect of light on both RhVI1 expression and vacuolar invertase activity. This up-regulation of RhVI1 was rapid (after 6 h incubation) and was induced by as little as 10 mM sucrose or fructose. No effect of glucose was found. Interestingly, both 30 mM palatinose (a non-metabolizable sucrose analog) and 5 mM psicose (a non-metabolizable fructose analog) promoted the light-induced expression of RhVI1 and total vacuolar invertase activity. Sucrose, fructose, palatinose and psicose all promoted bursting of in vitro cultured buds under light. These findings indicate that soluble sugars contribute to the light effect on bud burst and vacuolar invertases, and can function as signaling entities.     

ER stress inhibits neuronal death by promoting autophagy

Endoplasmic reticulum (ER) stress has been implicated in neurodegenerative diseases but its relationship and role in disease progression remain unclear. Using genetic and pharmacological approaches, we showed that mild ER stress ("preconditioning") is neuroprotective in Drosophila and mouse models of Parkinson disease. In addition, we found that the combination of mild ER stress and apoptotic signals triggers an autophagic response both in vivo and in vitro. We showed that when autophagy is impaired, ER-mediated protection is lost. We further demonstrated that autophagy inhibits caspase activation and apoptosis. Based on our findings, we conclude that autophagy is required for the neuroprotection mediated by mild ER stress, and therefore ER preconditioning has potential therapeutic value for the treatment of neurodegenerative diseases.     

Detection and Quantification of Microparticles from Different Cellular Lineages Using Flow Cytometry. Evaluation of the Impact of Secreted Phospholipase A2 on Microparticle Assessment

Microparticles, also called microvesicles, are submicron extracellular vesicles produced by plasma membrane budding and shedding recognized as key actors in numerous physio(patho)logical processes. Since they can be released by virtually any cell lineages and are retrieved in biological fluids, microparticles appear as potent biomarkers. However, the small dimensions of microparticles and soluble factors present in body fluids can considerably impede their quantification. Here, flow cytometry with improved methodology for microparticle resolution was used to detect microparticles of human and mouse species generated from platelets, red blood cells, endothelial cells, apoptotic thymocytes and cells from the male reproductive tract. A family of soluble proteins, the secreted phospholipases A₂ (sPLA₂), comprises enzymes concomitantly expressed with microparticles in biological fluids and that catalyze the hydrolysis of membrane phospholipids. As sPLA₂ can hydrolyze phosphatidylserine, a phospholipid frequently used to assess microparticles, and might even clear microparticles, we further considered the impact of relevant sPLA2 enzymes, sPLA2 group IIA, V and X, on microparticle quantification. We observed that if enriched in fluids, certain sPLA₂ enzymes impair the quantification of microparticles depending on the species studied, the source of microparticles and the means of detection employed (surface phosphatidylserine or protein antigen detection). This study provides analytical considerations for appropriate interpretation of microparticle cytofluorometric measurements in biological samples containing sPLA2 enzymes.