Rodent-Borne Hantaviruses in Cambodia, Lao PDR, and Thailand

In order to evaluate the circulation of hantaviruses present in southeast Asia, a large scale survey of small mammal species was carried out at seven main sites in the region (Cambodia, Lao People's Democratic Republic, and Thailand). Small scale opportunistic trapping was also performed at an eighth site (Cambodia). Using a standard IFA test, IgG antibodies reacting to Hantaan virus antigens were detected at six sites. Antibody prevalence at each site varied from 0 to 5.6% with antibodies detected in several rodent species (Bandicota indica, B. savilei, Maxomys surifer, Mus caroli, M. cookii, Rattus exulans, R. nitidius, R. norvegicus, and R. tanezumi). When site seroprevalence was compared with site species richness, seropositive animals were found more frequently at sites with lower species richness. In order to confirm which hantavirus species were present, a subset of samples was also subjected to RT-PCR. Hantaviral RNA was detected at a single site from each country. Sequencing confirmed the presence of two hantavirus species, Thailand and Seoul viruses, including one sample (from Lao PDR) representing a highly divergent strain of Seoul virus. This is the first molecular evidence of hantavirus in Lao PDR and the first reported L segment sequence data for Thailand virus.     

Rapid and efficient clathrin-mediated endocytosis revealed in genome-edited mammalian cells

Clathrin-mediated endocytosis (CME) is the best-studied pathway by which cells selectively internalize molecules from the plasma membrane and surrounding environment. Previous live-cell imaging studies using ectopically overexpressed fluorescent fusions of endocytic proteins indicated that mammalian CME is a highly dynamic but inefficient and heterogeneous process. In contrast, studies of endocytosis in budding yeast using fluorescent protein fusions expressed at physiological levels from native genomic loci have revealed a process that is very regular and efficient. To analyse endocytic dynamics in mammalian cells in which endogenous protein stoichiometry is preserved, we targeted zinc finger nucleases (ZFNs) to the clathrin light chain A and dynamin-2 genomic loci and generated cell lines expressing fluorescent protein fusions from each locus. The genome-edited cells exhibited enhanced endocytic function, dynamics and efficiency when compared with previously studied cells, indicating that CME is highly sensitive to the levels of its protein components. Our study establishes that ZFN-mediated genome editing is a robust tool for expressing protein fusions at endogenous levels to faithfully report subcellular localization and dynamics.     

Natural antibody--complement dependent neutralization of bovine herpesvirus 4 by human serum

In contrast to most gammaherpesviruses, Bovine herpesvirus 4 (BoHV-4) has a broad range of host species both in vitro and in vivo. Several in vitro studies demonstrated that some human cell lines are sensitive or even permissive to BoHV-4. These observations led to the hypothesis that cross-species transmission of BoHV-4 could lead to human infections. In the present study, we investigate the sensitivity of BoHV-4 to neutralization by na?ve human sera in order to determine if humans exhibit innate anti-viral activities against this virus. Our results demonstrate that human sera from na?ve individuals, in contrast to the sera of na?ve subjects from various animal species, neutralize BoHV-4 efficiently. A series of complementary experiments were performed to unravel the mechanism(s) of this neutralization. The data obtained in this study demonstrates that human serum neutralizes BoHV-4 in a complement dependent manner activated by natural antibodies raised against the Galalpha1-3Galbeta1-4GlcNAc-R epitope expressed by bovine cells.     

Effects of the active metabolite of leflunomide, A77 1726, on cytokine release and the MAPK signalling pathway in human rheumatoid arthritis synoviocytes

Inflammatory cytokines or soluble factors are essential in the pathogenesis of rheumatoid arthritis (RA). Leflunomide is an effective disease modifying antirheumatic drug (DMARD) in RA. The objective of the present study was to evaluate for the first time the effects of A77 1726 on cytokine (interleukin (IL)-8, IL-10, IL-11 secretion and tumor necrosis factor-alpha soluble receptor I (sTNFRI)) shedding in human RA fibroblast-like synoviocytes (FLS). At 100 microM, we observed an increase in IL-10 secretion, a decrease in IL-11 release and no effect on sTNFRI shedding and IL-8 secretion in IL-1beta-stimulated human RA FLS. Furthermore, at this dose, our results also confirmed that A77 1726 decreased IL-6 and prostaglandin E2 (PGE2) synthesis while it increased IL-1 receptor antagonist secretion (IL-1Ra). The mitogen-activated protein kinases (MAPKs) represent an attractive target for RA because they can regulate cytokine expression. At 100 microM, the effect of A77 1726 on IL-10 and IL-11 secretion seemed to be associated with the status of p38 MAPK activation. Our results confirmed the immunoregulatory action of leflunomide in the cytokine network involved in RA pathogenesis. It could shift the balance from cytokine mediated inflammation to cytokine directed inhibition of the inflammatory process.     

Function of the SmpB tail in transfer-messenger RNA translation revealed by a nucleus-encoded form

Stalled bacterial ribosomes are freed when they switch to the translation of transfer-messenger RNA (tmRNA). This process requires the tmRNA-binding and ribosome-binding cofactor SmpB, a beta-barrel protein with a protruding C-terminal tail of unresolved structure. Some plastid genomes encode tmRNA, but smpB genes have only been reported from bacteria. Here we identify smpB in the nuclear genomes of both a diatom and a red alga encoding a signal for import into the plastid, where mature SmpB could activate tmRNA. Diatom SmpB was active for tmRNA translation with bacterial components in vivo and in vitro, although less so than Escherichia coli SmpB. The tail-truncated diatom SmpB, the hypothetical product of a misspliced mRNA, was inactive in vivo. Tail-truncated E. coli SmpB was likewise inactive for tmRNA translation but was still able to bind ribosomes, and its affinity for tmRNA was only slightly diminished. This work suggests that SmpB is a universal cofactor of tmRNA. It also reveals a tail-dependent role for SmpB in tmRNA translation that supersedes a simple role of linking tmRNA to the ribosome, which the SmpB body alone could provide.     

New drying process for lactic bacteria based on their dehydration behavior in liquid medium

This study describes the different stages of optimization in an original drying process for lactic acid bacteria that allows the retrieval of dried samples of Lactobacillus plantarum with maximum viability. The process involves the addition of casein powder to bacterial pellets, followed by mixing and then air-drying in a fluidized bed dryer. The effects on bacterial viability of the a(w) of the casein powder and the kinetics of a(w) variation in the fluidized bed dryer are considered. These parameters were first studied in a water-glycerol solution and the results were then applied to the drying process. Data from the study in liquid medium were reliable in the fluidized drying stage, insofar as optimal viability was achieved for similar dehydration times (16-50 min in liquid medium, and 30 min in the fluidized bed dryer). However, when the powder was mixed rapidly with bacteria, the level of destruction differed from that observed in liquid medium. Viability was up to 70% when the a(w) of water-glycerol was 0.55, whereas it was only 2.1% when the a(w) of the casein-bacterial mix was 0.64. The predictive capacity of dehydration in liquid medium is discussed with regard to the permeability of cells to external solutes. The new process allowed 100% survival of L. plantarum after complete drying (final a(w) < 0.2). However, when used for the desiccation of L. bulgaricus, these parameters achieved a viability of less than 10%.     

The hippocampal cholinergic neurostimulating peptide, the N-terminal fragment of the secreted phosphatidylethanolamine-binding protein, possesses a new biological activity on cardiac physiology

Phosphatidylethanolamine-binding protein (PEBP), alternatively named Raf-1 kinase inhibitor protein, is the precursor of the hippocampal cholinergic neurostimulating peptide (HCNP) corresponding to its natural N-terminal fragment, previously described to be released by hippocampal neurons. PEBP is a soluble cytoplasmic protein, also associated with plasma and reticulum membranes of numerous cell types. In the present report, using biochemistry and cell biology techniques, we report for the first time the presence of PEBP in bovine chromaffin cell, a well described secretion model. We have examined its presence at the subcellular level and characterized this protein on both secretory granule membranes and intragranular matrix. In addition, its presence in bovine chromaffin cell and platelet exocytotic medium, as well as in serum, was reported showing that it is secreted. Like many other proteins that lack signal sequence, PEBP may be secreted through non-classic signal secretory mechanisms, which could be due to interactions with granule membrane lipids and lipid rafts. By two-dimensional liquid chromatography-tandem mass spectrometry, HCNP was detected among the intragranular matrix components. The observation that PEBP and HCNP were secreted with catecholamines into the circulation prompted us to investigate endocrine effects of this peptide on cardiovascular system. By using as bioassay an isolated and perfused frog (Rana esculenta) heart preparation, we show here that HCNP acts on the cardiac mechanical performance exerting a negative inotropism and counteracting the adrenergic stimulation of isoproterenol. All together, these data suggest that PEBP and HCNP might be considered as new endocrine factors involved in cardiac physiology.     

The potyviral virus genome-linked protein VPg forms a ternary complex with the eukaryotic initiation factors eIF4E and eIF4G and reduces eIF4E affinity for a mRNA cap analogue

The virus protein linked to the genome (VPg) of plant potyviruses is a 25-kDa protein covalently attached to the genomic RNA 5' end. It was previously reported that VPg binds specifically to eIF4E, the mRNAcap-binding protein of the eukaryotic translation initiation complex. We performed a spectroscopic study of the interactions between lettuce eIF4E and VPg from lettuce mosaic virus (LMV). The cap analogue m7GDP and VPg bind to eIF4E at two distinct sites with similar affinity (K(d) = 0.3 microm). A deeper examination of the interaction pathway showed that the binding of one ligand induces a decrease in the affinity for the other by a factor of 15. GST pull-down experiments from plant extracts revealed that VPg can specifically trap eIF4G, the central component of the complex required for the initiation of protein translation. Our data suggest that eIF4G recruitment by VPg is indirectly mediated through VPg-eIF4E association. The strength of interaction between eIF4E and pep4G, the eIF4E-binding domain on eIF4G, was increased significantly by VPg. Taken together these quantitative data show that VPg is an efficient modulator of eIF4E biochemical functions.     

Four Pathogenic Candida Species Differ in Salt Tolerance

The virulence of Candida species depends on many environmental conditions, including extracellular pH and concentration of alkali metal cations. Tests of the tolerance/sensitivity of four pathogenic Candida species (C. albicans, C. dubliniensis, C. glabrata, and C. parapsilosis) to alkali metal cations under various growth conditions revealed significant differences among these species. Though all of them can be classified as rather osmotolerant yeast species, they exhibit different levels of tolerance to different salts. C. parapsilosis and C. albicans are the most salt-tolerant in general; C. dubliniensis is the least tolerant on rich YPD media and C. glabrata on acidic (pH 3.5) minimal YNB medium. C. dubliniensis is relatively salt-sensitive in spite of its ability to maintain as high intracellular K⁺/Na⁺ ratio as its highly salt-tolerant relative C. albicans. On the other hand, C. parapsilosis can grow in the presence of very high external NaCl concentrations in spite of its high intracellular Na⁺ concentrations (and thus lower K⁺/Na⁺ ratio) and thus resembles salt-tolerant (halophilic) Debaryomyces hansenii.