Translation of viral mRNA without active eIF2: the case of picornaviruses

Previous work by several laboratories has established that translation of picornavirus RNA requires active eIF2α for translation in cell free systems or after transfection in culture cells. Strikingly, we have found that encephalomyocarditis virus protein synthesis at late infection times is resistant to inhibitors that induce the phosphorylation of eIF2α whereas translation of encephalomyocarditis virus early during infection is blocked upon inactivation of eIF2α by phosphorylation induced by arsenite. The presence of this compound during the first hour of infection leads to a delay in the appearance of late protein synthesis in encephalomyocarditis virus-infected cells. Depletion of eIF2α also provokes a delay in the kinetics of encephalomyocarditis virus protein synthesis, whereas at late times the levels of viral translation are similar in control or eIF2α-depleted HeLa cells. Immunofluorescence analysis reveals that eIF2α, contrary to eIF4GI, does not colocalize with ribosomes or with encephalomyocarditis virus 3D polymerase. Taken together, these findings support the novel idea that eIF2 is not involved in the translation of encephalomyocarditis virus RNA during late infection. Moreover, other picornaviruses such as foot-and-mouth disease virus, mengovirus and poliovirus do not require active eIF2α when maximal viral translation is taking place. Therefore, translation of picornavirus RNA may exhibit a dual mechanism as regards the participation of eIF2. This factor would be necessary to translate the input genomic RNA, but after viral RNA replication, the mechanism of viral RNA translation switches to one independent of eIF2.     

Differentiation-dependent expression of proteins in brain endothelium during development of the blood-brain barrier

The blood-brain barrier is a specific property of differentiated brain endothelium. To study the differentiation of blood vessels in the brain, we have correlated the expression of a number of proteins in brain endothelial cells with the development of the blood-brain barrier in mouse, quail, and chick embryos. Using histochemical methods, alkaline phosphatase activity was found to be present in all species and appeared around embryonic Days 17 (mouse), 14 (quail), and 12 (chick). Butyrylcholinesterase activity was found in the mouse and quail but not the chick brain vasculature, and appeared around Days 17 (mouse) and 15 (quail). gamma-Glutamyltranspeptidase activity was demonstrated histochemically in mouse but not in chick and quail brain capillaries, beginning at Day 15. Transferrin receptor was localized on brain endothelium in all species by immunofluorescence methods using monoclonal antibodies. It appeared at Days 15 and 11 in mouse and chick embryonic brain, respectively. The staining of all markers in embryonic brain was compared with adult brain endothelium and the leptomeningeal blood vessels. The expression of these proteins was correlated with the development of the blood-brain barrier by studying the permeability of brain endothelium for the protein horseradish peroxidase during mouse embryogenesis. Vessels in the telencephalon were found to become impermeable around Day 16 of development. Taken together the results of previous investigations and those presented here, we conclude that a number of proteins are sequentially expressed in brain endothelial cells correlating in time with the formation of the blood-brain barrier in different species.     

Modulation of granulocyte-endothelium interactions by antileukoproteinase: inhibition of anti-type II collagen antibody-induced leukocyte attachment to the synovial endothelium

Antileukoproteinase (ALP) is a physiological inhibitor of granulocytic serine proteases that has been shown to have anti-inflammatory properties in addition to its antiproteolytic activity. On the basis of its potential to block anti-collagen type II (CII) antibody-induced arthritis (CAIA) and to suppress the conformational activation of β₂-integrins in leukocytes, the present study was undertaken to investigate its interference with leukocyte adherence to cytokine-activated endothelium. The potential of recombinant ALP to block the interactions of leukocytes with the endothelial lining was concomitantly investigated in vitro and in vivo. Thus, intravital fluorescence microscopic imaging of leukocyte rolling and firm adhesion to postcapillary venules were performed in the knee joints of DBA1/J mice after intravenous injection of anti-CII mAbs. An IL-1β-activated endothelial layer formed by a murine glomerular cell line (glEND.2) was used to assay the interaction with human leukocytes in vitro. Electromobility shift and luciferase reporter gene assays permitted the analysis of cytokine-induced activation of the NF-κB pathway. Fluorescence-activated cell sorting was applied to determine endothelial E-selectin expression. Leukocyte rolling and firm adhesion to the synovial endothelium in an early response to the anti-CII antibody transfer were significantly decreased in ALP-pretreated mice. Concomitantly, ALP suppressed the IL-1β-induced NF-κB activation and the upregulation of E-selectin expression in glEND.2 cells in vitro. These findings support the notion that the newly uncovered properties of ALP to interfere with cytokine signalling and upregulation of adhesion molecules in endothelial cells are likely to contribute to the therapeutic potential of ALP in immune-complex-induced tissue injury.     

Activity and Differential Induction of Chitinase Isozymes in Soybean Cultivars Resistant or Susceptible to Root-knot Nematodes

Host physiological events in relation to infestation by parasitic nematodes are not well documented. Soybean plant responses to Meloidogyne incognita infestation were compared to resistant (Bryan) and susceptible (Brim) cultivars at 0, 1, 3, 10, 20, and 34 days after infestation (DAI). The resistant cultivar had higher chitinase activity than the susceptible cultivar at every sample time beginning at 3 DAI. Results from isoelectric focusing gel electrophoresis analyses indicated that three acidic chitinase isozymes with isoelectric points (pIs) of 4.8, 4.4, and 4.2 accumulated to a greater extent in the resistant compared to the susceptible cultivar following challenge. SDS-PAGE analysis of root proteins revealed that two proteins with molecular weights of approximately 31 and 46 kD accumulated more rapidly and to a higher level in the resistant than in the susceptible cultivar. Additionally, three major protein bands (33, 22, and 20 kD) with chitinase activity were detected with a modified SDS-PAGE analysis in which glycolchitin was added into the gel matrix. These results indicate that higher chitinase activity and early induction of specific chitinase isozymes may be associated with resistance to root-knot nematode in soybean.     

Comparison of Methods for Evaluation of the Bactericidal Activity of Copper-Sputtered Surfaces against Methicillin-Resistant Staphylococcus aureus

Bacteria can survive on hospital textiles and surfaces, from which they can be disseminated, representing a source of health care-associated infections (HCAIs). Surfaces containing copper (Cu), which is known for its bactericidal properties, could be an efficient way to lower the burden of potential pathogens. The antimicrobial activity of Cu-sputtered polyester surfaces, obtained by direct-current magnetron sputtering (DCMS), against methicillin-resistant Staphylococcus aureus (MRSA) was tested. The Cu-polyester microstructure was characterized by high-resolution transmission electron microscopy to determine the microstructure of the Cu nanoparticles and by profilometry to assess the thickness of the layers. Sputtering at 300 mA for 160 s led to a Cu film thickness of 20 nm (100 Cu layers) containing 0.209% (wt/wt) polyester. The viability of MRSA strain ATCC 43300 on Cu-sputtered polyester was evaluated by four methods: (i) mechanical detachment, (ii) microcalorimetry, (iii) direct transfer onto plates, and (iv) stereomicroscopy. The low efficacy of mechanical detachment impeded bacterial viability estimations. Microcalorimetry provided only semiquantitative results. Direct transfer onto plates and stereomicroscopy seemed to be the most suitable methods to evaluate the bacterial inactivation potential of Cu-sputtered polyester surfaces, since they presented the least experimental bias. Cu-polyester samples sputtered for 160 s by DCMS were further tested against 10 clinical MRSA isolates and showed a high level of bactericidal activity, with a 4-log₁₀ reduction in the initial MRSA load (10⁶ CFU) within 1 h. Cu-sputtered polyester surfaces might be of use to prevent the transmission of HCAI pathogens.     

Performance deficits of NK1 receptor knockout mice in the 5-choice serial reaction-time task: effects of d-amphetamine, stress and time of day

Background

The neurochemical status and hyperactivity of mice lacking functional substance P-preferring NK1 receptors (NK1R-/-) resemble abnormalities in Attention Deficit Hyperactivity Disorder (ADHD). Here we tested whether NK1R-/- mice express other core features of ADHD (impulsivity and inattentiveness) and, if so, whether they are diminished by d-amphetamine, as in ADHD. Prompted by evidence that circadian rhythms are disrupted in ADHD, we also compared the performance of mice that were trained and tested in the morning or afternoon.

Methods and Results

The 5-Choice Serial Reaction-Time Task (5-CSRTT) was used to evaluate the cognitive performance of NK1R-/- mice and their wildtypes. After training, animals were tested using a long (LITI) and a variable (VITI) inter-trial interval: these tests were carried out with, and without, d-amphetamine pretreatment (0.3 or 1 mg/kg i.p.). NK1R-/- mice expressed greater omissions (inattentiveness), perseveration and premature responses (impulsivity) in the 5-CSRTT. In NK1R-/- mice, perseveration in the LITI was increased by injection-stress but reduced by d-amphetamine. Omissions by NK1R-/- mice in the VITI were unaffected by d-amphetamine, but premature responses were exacerbated by this psychostimulant. Omissions in the VITI were higher, overall, in the morning than the afternoon but, in the LITI, premature responses of NK1R-/- mice were higher in the afternoon than the morning.

Conclusion

In addition to locomotor hyperactivity, NK1R-/- mice express inattentiveness, perseveration and impulsivity in the 5-CSRTT, thereby matching core criteria for a model of ADHD. Because d-amphetamine reduced perseveration in NK1R-/- mice, this action does not require functional NK1R. However, the lack of any improvement of omissions and premature responses in NK1R-/- mice given d-amphetamine suggests that beneficial effects of this psychostimulant in other rodent models, and ADHD patients, need functional NK1R. Finally, our results reveal experimental variables (stimulus parameters, stress and time of day) that could influence translational studies.