Characterization of the Herpesvirus saimiri Orf51 protein

Herpesvirus saimiri (HVS) is a gamma(2)-herpesvirus sharing genomic colinearity and a high degree of functional homology with HHV-8. To begin exploring the correlates of HVS infectivity and neutralization, we designed and implemented a new reporter assay. Using this assay, we could demonstrate that HVS neutralizing antibodies are present at high levels in naturally infected squirrel monkeys and are strongly induced after pathogenic, experimental infection of common marmosets. Further, we demonstrated that viral entry is influenced by cellular glycosaminoglycans and that, similar to HHV-8, soluble heparin is capable of blocking infectivity. We next cloned and characterized the positional homologue of HHV-8 K8.1, HVS Orf51. N-glycosidase F treatment indicates that like K8.1, Orf51 is a glycoprotein. Found in the viral particle, it localizes to the endoplasmic reticulum of expressing cells. Like K8.1, Orf51 could bind to agarose-conjugated heparin, implicating this molecule in viral attachment to cells. These studies provide the groundwork for additional experiments into the role that this protein may be playing in viral pathogenicity, persistence, and cell tropism.     

Tip, an Lck-interacting protein of Herpesvirus saimiri, causes Fas- and Lck-dependent apoptosis of T lymphocytes

Saimiriine herpesvirus-2 (Herpesvirus saimiri) transforms T lymphocytes, including human, to continuous growth in vitro. H. saimiri-induced transformation is becoming an important tool of T-cell biology, including studies of HIV replication. Two proteins of H. saimiri subgroup C, Tip and StpC, are essential for T-cell transformation. In spite of the important role of these proteins, their biological functions and the molecular mechanisms of their action remain insufficiently understood. To further elucidate the effects of Tip on T cells, we transduced T lymphocytes, using an efficient lentiviral gene transfer system, to express Tip in the absence of other H. saimiri proteins. Our results indicate that Tip specifically inhibits IL-2 production by human T lymphocytes. Furthermore, Tip promotes T-cell apoptosis, which appears to be the reason for the observed decrease in IL-2 production. Finally, the apoptotic effect of Tip in T cells is mediated by Fas and requires the presence of active Lck in the cell.     

p21WAF1 modulates NF-kappaB signaling and induces anti-apoptotic protein Bcl-2 in Tax-expressing rat fibroblast

Of the cell cycle-associated genes regulated by human T-cell leukemia virus type-1 (HTLV-1) Tax, cyclin-dependent kinase (CDK) inhibitor p21WAF1 is upregulated in HTLV-1-infected cells. Previously, we reported that p21WAF1 stimulated Tax-dependent NF-kappaB activation which influences a variety of cellular processes, including proliferation, differentiation, and apoptosis. In HTLV-1-infected cells, Tax is primarily involved in the constitutive activation of NF-kappaB signaling. Here, we demonstrate that p21WAF1 affects Tax-dependent NF-kappaB signaling by inducing p100/52, an NF-kappaB-related protein. W4, a Tax-transformed rat fibroblast cell line, exhibits the constitutive activation of NF-kappaB signaling, potentially mediated by overexpression of RelB. Ectopic expression of p21WAF1 in W4 cells, which lack endogenous expression due to methylation of the p21WAF1 promoter, induces the expression of p100/52. Bcl-2 expression was also upregulated by ectopic p21WAF1 in this cell line, suggesting that p21WAF1 plays an important role in the regulation of apoptosis by modulating NF-kappaB signaling in Tax-expressing rat fibroblasts. We also address the expression of NF-kappaB-related proteins in HTLV-1-infected cells.     

Autosomal recessive retinitis pigmentosa locus RP28 maps between D2S1337 and D2S286 on chromosome 2p11-p15 in an Indian family

Retinitis pigmentosa (RP) is a group of clinically and genetically heterogeneous disorders characterised by night blindness, constriction of visual field, and dystrophic changes of the retina. Previous genetic studies have shown extensive allelic and non-allelic genetic heterogeneity of RP. Here we describe an Indian family with multiple consanguineous marriages and a total of four patients with autosomal recessive (AR) RP. The homozygosity mapping strategy was successfully used and indicated close linkage between the disease locus and D2S380, D2S441, D2S291, and D2S1394 with maximum lod scores between 1.51-3.07 at theta=0.00. The analysis of multiply informative meioses maps the locus (RP28) for ARRP in this family between D1S1337 and D2S286 on 2p11-p15. The involvement of visinin (VSNL1), a promising candidate gene assigned to chromosome 2p by previous studies, has been excluded by the absence of linkage.     

Inhibition of nuclear factor kappa B activation reduces Coxsackievirus B3 replication in lymphoid cells

Interactions between viral replication machineries and host cell metabolism display interesting information how certain viruses capitalize cellular pathways to support progeny production. Among those pathogens, Coxsackievirus B3 (CVB3) has been identified to manipulate intracellular signaling very comprehensively. Next to others, this human pathogenic virus causes acute and chronic forms of myocarditis, pancreatitis, and meningitis. Here, activation of nuclear factor kappa B (NFκB) signaling appears to be involved in successful infection. Viral replication is not restricted to solid organs but involves susceptible immune cells as well. In the present study, p65 phosphorylation as one aspect of NFκB activation and inhibition via BAY 11-7085 administration was analyzed in the context of CVB3 replication in lymphoid cells. During CVB3 infection, an up-regulation of p65 translation is detectable, which is accompanied by noticeable phosphorylation. Inhibition of NFκB signaling reduces viral replication in a dose- and time-dependent manner. Taken together, these results indicate that during CVB3 replication in human and murine lymphoid cells, NFκB signaling is activated and facilitates viral replication. Therefore, antiviral strategies to target such central cellular signaling pathways may represent potential possibilities for the development of new virostatica.     

Oxidative induction of pro-inflammatory cytokine formation by human monocyte-derived macrophages following exposure to manganese in vitro

Abstract

Manganese (as Mn²⁺), a superoxide dismutase mimetic, catalyzes the formation of the relatively stable membrane-permeable reactive oxygen species (ROS) hydrogen peroxide (H₂O₂), a mediator of intracellular redox signaling in immune and inflammatory cells. The goal of this study was to investigate the potential for Mn²⁺, via its pro-oxidative properties, to activate production of pro-inflammatory cytokines/chemokines IL-1β, IL-6, IL-8, IFNγ, TNFα, and G-CSF by human monocyte-derived macrophages in vitro. For these studies, the cells were isolated from peripheral blood mononuclear leukocytes and matured to generate a population of large CD14/CD16 co-expressing cells. The monocyte-derived macrophages were then exposed to bacterial lipopolysaccharide (LPS, 1?μg/ml) or MnCl₂ (25–100?μM)—alone or in combination—for 24?h at 37?°C, after which cell-free supernatants were analyzed using a multiplex cytokine assay procedure. Exposure of the cells to LPS caused modest statistically insignificant increases in cytokine production; MnCl₂ caused dose-related increases in production of all six cytokines (achieving statistical significance of p?<?0.0171–?<?0.0005 for IL-1β, IL-6, IL-8, IFNγ, and TNFα). In the case of LPS and MnCl₂ combinations, the observed increases in production of IL-1β, IL-6, IL-8, IFNγ, and G-CSF were greater than those seen with cells exposed to the individual agents. The Mn²⁺-mediated induction of cytokine production was associated with increased production of H₂O₂ and completely attenuated by inclusion of the H₂O₂-scavenger dithiothreitol, and partially by inhibitors of NF-κB and p38MAP kinase. The findings from the studies here help to further characterize the pro-inflammatory mechanisms that may underpin clinical disorders associated with excess exposure to Mn²⁺, particularly those disorders seen in the central nervous and respiratory systems.     

Identification of mouse mslp2 gene from EST databases by repeated searching, comparison, and assembling

The NPHS2 gene is expressed in podocytes and encodes the integral membrane protein called podocin, which is believed to play an important role in the renal function of glomerular filtration. Mutations in this gene can cause serious renal function disorders. In this study, we used data-mining techniques and bioinformatic tools to search for the mouse ortholog of the NPHS2-related gene. It might be valuable for future studies of renal diseases. We employed repeated cycles of searching, comparison, and assembling to extend the assembled EST sequences. The discovered gene sequence mslp2, an ortholog of the human SLP2 gene, was found to have a total length of 1253 bp with the amino acid coding region located in 32-1093 nt. It was further verified using the RT-PCR and RACE techniques to ensure its biological accuracy and then registered with the GenBank. When ClustalW was used for comparing the mslp2 and human SLP2 genes for similarities, the similarities were as high as 88% for nucleotide and 92% for amino acid sequences. In conclusion, we propose a method for rapid identification of the mouse ortholog gene from the human genome.     

机动车尾气诱发大鼠慢性阻塞性肺疾病并上调气道上皮细胞COX-2/PGE2/EP受体信号通路

目的：探究机动车尾气(MVE)长期暴露引起大鼠慢性阻塞性肺疾病(COPD)发生时,气道上皮细胞中环加氧酶2(COX-2)/前列腺素E₂(PGE₂)/E-前列腺素类激素(EP)受体信号通路成员的表达变化。方法：(1)动物实验：健康雄性SD纯系大鼠(SPF级)16只,随机分为2组：MVE暴露组(n=8)和空白对照(CTL)组(n=8)。采用MVE暴露6个月的方法建立COPD大鼠模型。造模结束后,使用Buxco小动物有创肺功能仪检测大鼠肺功能;肺组织切片行HE染色并评估肺组织病理变化;ELISA法检测大鼠支气管肺泡灌洗液(BALF)中炎症因子白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)和PGE₂的水平,评估大鼠肺部炎症情况;采用免疫荧光及Western blot法检测肺组织COX-2及EP受体蛋白水平;提取肺组织核蛋白,Western blot检测MVE对肺组织NF-κB核转位的影响。(2)细胞实验：采用MVE细颗粒物(PM2.5)标准品刺激人正常支气管上皮细胞BEAS-2B。ELISA法检测细胞培养液中PGE     

Synergism of a DNA and an RNA virus: enhanced tissue infiltration of the begomovirus Abutilon mosaic virus (AbMV) mediated by Cucumber mosaic virus (CMV)

Replication of the begomovirus Abutilon mosaic virus (AbMV) is restricted to phloem nuclei, generating moderate levels of virus DNA. Co-infection with Cucumber mosaic virus (CMV) evidently increased AbMV titers in Nicotiana benthamiana, tobacco, and tomato, resulting in synergistic symptom enhancement. In situ hybridization revealed that in double-infected leaves an increased number of nuclei contained elevated amounts of AbMV. Additionally, the begomoviral phloem-limitation was broken. Whereas CMV 3a movement protein-expressing tobacco plants did not exert any similar influence, the presence of CMV 2b silencing suppressor protein lead to enhanced AbMV titers and numbers of infected vascular cells. The findings prove that AbMV can replicate in nonvascular cells and represent the first report on a true synergism of an RNA/ssDNA virus combination in plants, in which CMV 2b protein plays a role. They indicate considerable consequences of mixed infections between begomo- and cucumoviruses on virus epidemiology and agriculture.     

IL-1beta expression in Int407 is induced by flagellin of Vibrio cholerae through TLR5 mediated pathway

Vibrio cholerae, a noninvasive enteric bacterium, causing inflammatory diarrheal disease cholera, is associated with the secretion of proinflamammatory cytokines including IL-1β in cultured epithelial cells. Incubation of Int407 with live V. cholerae resulted in increased IL-1β mRNA expression as early as 2 h of infection, reached a peak at 3.5 h and decreased thereafter. The identity of the effector molecule(s) is largely unknown. The bacterial culture supernatant showed IL-1β stimulating activity. An engineered aflagellate V. cholerae flaA mutant (O395FLAN) resulted in highly reduced level of IL-1β expression in Int407. The crude flagellar protein of V. cholerae as well as recombinant FlaA induced IL-1β expression in Int407. Infection of Toll-like receptor 5 (TLR5) transfected HeLa cells with O395FLAN showed reduced expression of IL-1β compared to wild-type. Unlike wild-type V. cholerae, O395FLAN did not activate the NF-κB while the recombinant flagellin could activate NF-κB. Finally, the mitogen activated protein kinases (ERK1 and 2, p38) were phosphorylated in wild-type and recombinant flagellin treated Int407 cells and inhibition of the p38 and ERK pathways significantly decreased the IL-1β response induced by wild-type V. cholerae as well as recombinant flagellin. Our data clearly indicate that flagellin of V. cholerae could induce IL-1β expression by recognizing TLR5 that activate NF-κB and MAP kinase in Int407.     

Activation of adenosine A3 receptor suppresses lipopolysaccharide-induced TNF-α production through inhibition of PI 3-kinase/Akt and NF-κB activation in murine BV2 microglial cells

Adenosine is an endogenous nucleoside that regulates many processes, including inflammatory responses, through activation of its receptors. Adenosine receptors have been reported to be expressed in microglia, which are major immune cells of brain, yet little is known about the role of adenosine receptors in microglial cytokine production. Thus, we investigated the effect of adenosine and adenosine A₃ receptor ligands on LPS-induced tumor necrosis factor (TNF-α) production and its molecular mechanism in mouse BV2 microglial cells. Adenosine and Cl-IB-MECA, a specific adenosine A₃ receptor agonist, suppressed LPS-induced TNF-α protein and mRNA levels. Moreover, MRS1523, a selective A₃ receptor antagonist, blocked suppressive effects of both adenosine and Cl-IB-MECA on TNF-α. We further examined the effect of adenosine on signaling molecules, such as PI 3-kinase, Akt, p38, ERK1/2, and NF-κB, which are involved in the regulation of inflammatory responses. Adenosine inhibited LPS-induced phosphatidylinositol (PI) 3-kinase activation and Akt phosphorylation, whereas it had no effect on the phosphorylation of p38 and ERK1/2. We also found that adenosine as well as Cl-IB-MECA inhibited LPS-induced NF-κB DNA binding and luciferase reporter activity. Taken together, these results suggest that adenosine A₃ receptor activation suppresses TNF-α production by inhibiting PI 3-kinase/Akt and NF-κB activation in LPS-treated BV2 microglial cells.     

Molecular mechanisms of lipopolysaccharide-induced cyclooxygenase-2 expression in human neutrophils: involvement of the mitogen-activated protein kinase pathway and regulation by anti-inflammatory cytokines

Neutrophils are an important cellular source of proinflammatory mediators, whose regulation may be of potential benefit for the treatment of a number of inflammatory diseases. However, the mechanisms of lipopolysaccharide (LPS)-induced neutrophil activation and its regulation by anti-inflammatory cytokines have not yet been fully elucidated. Recent studies have revealed that mitogen-activated protein kinases (MAPK) play a crucial role in the generation of proinflammatory mediators in some cell types. Therefore, we conducted this study to determine whether MAPK activation could be involved in prostaglandin E(2) (PGE(2)) production and cyclooxygenase (COX)-2 expression in LPS-stimulated human neutrophils. PD98059 (MEK1 inhibitor) and SB203580 (p38(MAPK) inhibitor) reduced PGE(2) production as well as COX-2 expression in LPS-stimulated neutrophils. In addition, both extracellular signal-regulated protein kinase (ERK) and p38(MAPK) were phosphorylated and activated in time- and dose-dependent manners. Since we previously showed that IL-10 and IL-4 similarly inhibited COX-2 expression in LPS-stimulated neutrophils, we next tested the effects of IL-10 and IL-4 on the phosphorylation and activation of both kinases. IL-10 inhibited the phosphorylation and activation of p38(MAPK), but not ERK. In addition, IL-4 caused a marginal inhibition in the activation of p38(MAPK). Taken together, these results suggest that both ERK and p38(MAPK) pathways are involved in LPS-induced COX-2 expression and PGE(2) production in neutrophils, and IL-10 and IL-4 inhibit neutrophil prostanoid synthesis by down-regulating the activation of p38(MAPK).     

Activation of an innate immune response in the schistosome-transmitting snail Biomphalaria glabrata by specific bacterial PAMPs

Injection of crude lipopolysaccharide (LPS) from Escherichia coli into the hemocoel of Biomphalaria glabrata stimulates cell proliferation in the amebocyte-producing organ (APO). However, it is not known if mitogenic activity resides in the lipid A or O-polysaccharide component of LPS. Moreover, the possible role of substances that commonly contaminate crude LPS and that are known to stimulate innate immune responses in mammals, e.g., peptidoglycan (PGN), protein, or bacterial DNA, is unclear. Therefore, we tested the effects of the following injected substances on the snail APO: crude LPS, ultrapurified LPS (lacking lipoprotein contamination), two forms of lipid A, (diphosphoryl lipid A and Kdo₂-lipid A), O-polysaccharide, Gram negative PGN, both crude and ultrapurified (with and without endotoxin activity, respectively), Gram positive PGN, PGN components Tri-DAP and muramyl dipeptide, and bacterial DNA. Whereas crude LPS, ultrapurified LPS, and crude PGN were mitogenic, ultrapurified PGN was not. Moreover, LPS components, PGN components, and bacterial DNA were inactive. These results suggest that it is the intact LPS molecule which stimulates cell division in the APO.     

Activation of the Hog1p kinase in Isc1p-deficient yeast cells is associated with mitochondrial dysfunction, oxidative stress sensitivity and premature aging

The Saccharomyces cerevisiae Isc1p, an orthologue of mammalian neutral sphingomyelinase 2, plays a key role in mitochondrial function, oxidative stress resistance and chronological lifespan. Isc1p functions upstream of the ceramide-activated protein phosphatase Sit4p through the modulation of ceramide levels. Here, we show that both ceramide and loss of Isc1p lead to the activation of Hog1p, the MAPK of the high osmolarity glycerol (HOG) pathway that is functionally related to mammalian p38 and JNK. The hydrogen peroxide sensitivity and premature aging of isc1Δ cells was partially suppressed by HOG1 deletion. Notably, Hog1p activation mediated the mitochondrial dysfunction and catalase A deficiency associated with oxidative stress sensitivity and premature aging of isc1Δ cells. Downstream of Hog1p, Isc1p deficiency activated the cell wall integrity (CWI) pathway. Deletion of the SLT2 gene, which encodes for the MAPK of the CWI pathway, was lethal in isc1Δ cells and this mutant strain was hypersensitive to cell wall stress. However, the phenotypes of isc1Δ cells were not associated with cell wall defects. Our findings support a role for Hog1p in the regulation of mitochondrial function and suggest that constitutive activation of Hog1p is deleterious for isc1Δ cells under oxidative stress conditions and during chronological aging.     

The Drosophila IMD pathway in the activation of the humoral immune response

The IMD pathway signaling plays a pivotal role in the Drosophila defense against bacteria. During the last two decades, significant progress has been made in identifying the components and deciphering the molecular mechanisms underlying this pathway, including the means of bacterial sensing and signal transduction. While these findings have contributed to the understanding of the immune signaling in insects, they have also provided new insights in studying the mammalian NF-κB signaling pathways. Here, we summarize the current view of the IMD pathway focusing on how it regulates the humoral immune response of Drosophila.     

Antioxidant marine algae phlorotannins and radioprotection: A review of experimental evidence

Radiation has been widely used for cancer therapy in human medicine. However, the side effects of radiation are problematic and can limit its application. Radiation generates reactive oxygen species, leading to cell death via multiple signaling pathways. The blocking of certain signaling cascades using antioxidants represents a compensatory therapy of radiation-induced tissue injury. Although synthetic chemicals have been investigated in recent decades, anti-oxidants from natural resources have been searched for continuously. Among them, phlorotannins from marine algae, including Ecklonia cava, have been shown to protect cells from radiation-induced injury as well as oxidative stress. In the present review, the radioprotective capacity of phlorotannins derived from marine algae and the mechanisms involved are discussed.