Rotaviral Enterotoxin Nonstructural Protein 4 Targets Mitochondria for Activation of Apoptosis during Infection

Viruses have evolved to encode multifunctional proteins to control the intricate cellular signaling pathways by using very few viral proteins. Rotavirus is known to express six nonstructural and six structural proteins. Among them, NSP4 is the enterotoxin, known to disrupt cellular Ca²⁺ homeostasis by translocating to endoplasmic reticulum. In this study, we have observed translocation of NSP4 to mitochondria resulting in dissipation of mitochondrial membrane potential during virus infection and NSP4 overexpression. Furthermore, transfection of the N- and C-terminal truncated NSP4 mutants followed by analyzing NSP4 localization by immunofluorescence microscopy identified the 61–83-amino acid region as the shortest mitochondrial targeting signal. NSP4 exerts its proapoptotic effect by interacting with mitochondrial proteins adenine nucleotide translocator and voltage-dependent anion channel, resulting in dissipation of mitochondrial potential, release of cytochrome c from mitochondria, and caspase activation. During early infection, apoptosis activation by NSP4 was inhibited by the activation of cellular survival pathways (PI3K/AKT), because PI3K inhibitor results in early induction of apoptosis. However, in the presence of both PI3K inhibitor and NSP4 siRNA, apoptosis was delayed suggesting that the early apoptotic signal is initiated by NSP4 expression. This proapoptotic function of NSP4 is balanced by another virus-encoded protein, NSP1, which is implicated in PI3K/AKT activation because overexpression of both NSP4 and NSP1 in cells resulted in reduced apoptosis compared with only NSP4-expressing cells. Overall, this study reports on the mechanism by which enterotoxin NSP4 exerts cytotoxicity and the mechanism by which virus counteracts it at the early stage for efficient infection.     

Nation-wide litter fall data from 21 forests of the Monitoring Sites 1000 Project in Japan

This data paper reports litter fall data collected in a network of 21 forest sites in Japan. This is the largest litter fall data set freely available in Japan to date. The network is a part of the Monitoring Sites 1000 Project launched by the Ministry of the Environment, Japan. It covers subarctic to subtropical climate zones and the four major forest types in Japan. Twenty-three permanent plots in which usually 25 litter traps were installed were established in old-growth or secondary natural forests. Litter falls were collected monthly from 2004, and sorted into leaves, branches, reproductive structures and miscellaneous. The data provide seasonal patterns and inter-annual dynamics of litter falls, and their geographical patterns, and offer good opportunities for meta-analyses and comparative studies among forests.     

Severe inhibition of in vitro cardiomyogenesis in mouse embryonic stem cells ectopically expressing EGAM1C homeoprotein

Embryoid bodies were prepared from mouse embryonic stem cells expressing exogenous EGAM1C to analyze their ability to differentiate toward terminally differentiated cell types. The generation of cardiomyocytes was severely suppressed in Egam1c transfectants without upregulation of Nkx2-5, a crucial gene for cardiomyogenesis. These results indicate that EGAM1C is capable of affecting terminal differentiation in mouse embryonic stem cells.     

Sublingual vaccination with fusion protein consisting of the functional domain of hemagglutinin A of Porphyromonas gingivalis and Escherichia coli maltose-binding protein elicits protective immunity in the oral cavity

This study demonstrated that sublingual immunization with a fusion protein, 25k-hagA-MBP, which consists of a 25-kDa antigenic region of hemagglutinin A purified from Porphyromonas gingivalis fused to maltose-binding protein (MBP) originating from Escherichia coli as an adjuvant, elicited protective immune responses. Immunization with 25k-hagA-MBP induced high levels of antigen-specific serum IgG and IgA, as well as salivary IgA. High level titers of serum IgG and IgA were also induced for almost 1 year. In an IgG subclass analysis, sublingual immunization with 25k-hagA-MBP induced both IgG1 and IgG2b antibody responses. Additionally, numerous antigen-specific IgA antibody-forming cells were detected from the salivary gland 7 days after the final immunization. Mononuclear cells isolated from submandibular lymph nodes (SMLs) showed significant levels of proliferation upon restimulation with 25k-hagA-MBP. An analysis of cytokine responses showed that antigen-specific mononuclear cells isolated from SMLs produced significantly high levels of IL-4, IFN-γ, and TGF-β. These results indicate that sublingual immunization with 25k-hagA-MBP induces efficient protective immunity against P. gingivalis infection in the oral cavity via Th1-type and Th2-type cytokine production.     

Mannose-specific lectin from the mushroom Hygrophorus russula

A lectin was purified from the mushroom Hygrophorus russula by affinity chromatography on a Sephadex G-50 column and BioAssist S cation exchange chromatography and designated H. russula lectin (HRL). The results of sodium dodecyl sulfate-polyaclylamidegel electrophoresis, gel filtration and matrix-assisted laser desorption ionization time-of-flight mass spectrometry of HRL indicated that it was composed of four identical 18.5?kDa subunits with no S-S linkage. Isoelectric focusing of the lectin showed bands near pI 6.40. The complete sequence of 175 amino acid residues was determined by amino acid sequencing of intact or enzyme-digested HRL. The sequence showed homology with Grifola frondosa lectin. The cDNA of HRL was cloned from RNA extracted from the mushroom. The open reading frame of the cDNA consisted of 528?bp encoding 176 amino acids. In hemagglutination inhibition assay, α1-6 mannobiose was the strongest inhibitor and isomaltose, Glcα1-6Glc, was the second strongest one, among mono- and oligosaccharides tested. Frontal affinity chromatography indicated that HRL had the highest affinity for Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc, and non-reducing terminal Manα1-6 was essential for the binding of HRL to carbohydrate chains. The sugar-binding specificity of HRL was also analyzed by using BIAcore. The result from the analysis exhibited positive correlations with that of the hemagglutination inhibition assay. All the results suggested that HRL recognized the α1-6 linkage of mannose and glucose, especially the Manα1-6 bond. HRL showed a mitogenic activity against spleen lymph cells of an F344 rat. Furthermore, an enzyme-linked immunosorbent assay showed strong binding of HRL to human immunodeficiency virus type-1 gp120.     

Investigation of 7-benzylidenenaltrexone derivatives as resistance reverser for chloroquine-resistant Plasmodium chabaudi

Derivatives of 7-benzylidenenaltrexone (BNTX), which was recently reported to be an effective chloroquine (CQ)-resistance reverser, were synthesized and evaluated for their CQ-resistance reversing activities. The synthesized derivatives showed CQ-resistance reversing effects. They also reacted with glutathione (GSH) both enzymatically and chemically, and inhibited glutathione reductase activity. 7-Benzyl derivative, which was obtained by reduction of the olefin group in α,β-unsaturated ketone structure of BNTX, also exhibited CQ-resistance reversing effect, but its potency was significantly lower than that of BNTX. These outcomes suggested that the decrease in GSH level could be one of the mechanisms of CQ-resistance reversing effects induced by BNTX derivatives.     

Effect of persistent activation of phosphoinositide 3-kinase on heart

AimsInsulin/insulin-like growth factor-1 (IGF-1) signaling plays an important role in many biological processes. The class IA isoform of phosphoinositide 3-kinase (PI3K) is an important downstream effector of the insulin/IGF-1 signaling pathway. The aim of this study is to examine the effect of persistent activation of PI3K on gene expression and markers of cellular senescence in murine hearts.Main methodsTransgenic mice expressing a constitutively active PI3K in a heart-specific manner were analyzed at the ages of 3 and 20 months. Effects of persistent activation of PI3K on gene expression were comprehensively analyzed using microarrays.Key findingsUpon comprehensive gene expression profiling, the genes whose expression was increased included those for several heat shock chaperons. The amount and nuclear localization of a forkhead box O (FOXO) protein was increased. In addition, the gene expression of insulin receptor substrate-2 decreased, and that of phosphatase and tensin homolog deleted on chromosome ten (PTEN) increased, suggesting that the persistent activation of PI3K modified the expression of molecules of insulin/IGF-1 signaling. The expression of markers of cellular senescence, such as senescence-associated beta-galactosidase activity, cell cycle inhibitors, proinflammatory cytokines, and lipofuscin, did not differ between old wild-type and caPI3K mice.SignificanceThe persistent activation of PI3K modified the expression of molecules of insulin/IGF-1 signaling pathway in a transgenic mouse line. Markers of cellular senescence were not changed in the aged mutant mice.     

Evaluation of unbound free heme in plant cells by differential acetone extraction

Heme functions not only as a prosthetic group of hemoproteins but also as a regulatory molecule, suggesting the presence of 'free' heme. Classically, total non-covalently bound heme is extracted from plant samples with acidic acetone after removal of pigments with basic and neutral acetone. Earlier work proposed that free heme can be selectively extracted into basic acetone. Using authentic hemoproteins, we confirmed that acidic acetone can quantitatively extract heme, while no heme was extracted into neutral acetone. Meanwhile, a certain amount of heme was extracted into basic acetone from hemoglobin and myoglobin. Moreover, basic acetone extracted loosely bound heme from bovine serum albumin, implying that the nature of hemoproteins largely influences heme extraction into basic acetone. Using a highly sensitive heme assay, we found that basic and neutral acetone can extract low levels of heme from plant samples. In addition, neutral acetone quantitatively extracted free heme when it was externally added to plant homogenates. Furthermore, the level of neutral acetone-extractable heme remained unchanged by precursor (5-aminolevulinic acid) feeding, while increased by norflurazon treatment which abolishes chloroplast biogenesis. However, changes in these heme levels did not correlate to genomes uncoupled phenotypes, suggesting that the level of unbound free heme would not affect retrograde signaling from plastids to the nucleus. The present data demonstrate that the combination of single-step acetone extraction following a sensitive heme assay is the ideal method for determining total and free heme in plants.