Alkaline phosphodiesterase I and alkaline phosphatase I in plasma membranes of herpes simplex virus type 1 transformed hamster cells

Plasma membrane extracts from Herpes simplex virus type 1 transformed hamster embryo fibroblasts were chromatographed on Lens culinaris lectin coupled to Sepharose (LcH-Sepharose) and analysed by dodecyl sulphate polyacrylamide gel electrophoresis. Coomassie blue-staining revealed two major protein bands with apparent molecular weights of 125 000 and of about 75 000–90 000. In plasma membranes isolated from these tumor cells prior labeled with [³H]fucose or [³H]glucosamine these bands contained the highest amounts of incorporated radioactivity. Separation by LeH-Sepharose-affinity chromatography as well as metabolic labeling clearly demonstrates their glycoprotein character. The 125 000 protein coincides with alkaline phosphodiesterase I activity with a K_m of 6 · 10^?4 M for TMP p-nitrophenyl ester and is competitively inhibited by UDP-N-acetylglucosamine. This enzymatic activity is also present in normal hamster embryo fibroblasts. Gel electrophoresis of the Lens culinaris lectin-binding glycoproteins from plasma membranes of normal hamster embryo fibroblasts additionally revealed a strong alkaline phosphatase activity represented by an apparent molecular weight of 150 000, while HSV₁ hamster tumor cells contain only a very weak activity of this enzyme activity. HSV-lytically infected cells, however, have unchanged levels of alkaline phosphatase activity, whereas alkaline phosphodiesterase activity increases slightly.     

Prostaglandin I2 upregulates the expression of anterior pharynx‐defective‐1α and anterior pharynx‐defective‐1β in amyloid precursor protein/presenilin 1 transgenic mice

Cyclooxygenase‐2 (COX‐2) has been recently identified to be involved in the pathogenesis of Alzheimer's disease (AD). Yet, the role of an important COX‐2 metabolic product, prostaglandin (PG) I₂, in the pathogenesis of AD remains unknown. Using human‐ and mouse‐derived neuronal cells as well as amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice as model systems, we elucidated the mechanism of anterior pharynx‐defective (APH)‐1α and pharynx‐defective‐1β induction. In particular, we found that PGI₂ production increased during the course of AD development. Then, PGI₂ accumulation in neuronal cells activates PKA/CREB and JNK/c‐Jun signaling pathways by phosphorylation, which results in APH‐1α/1β expression. As PGI₂ is an important metabolic by‐product of COX‐2, its suppression by NS398 treatment decreases the expression of APH‐1α/1β in neuronal cells and APP/PS1 mice. More importantly, β‐amyloid protein (Aβ) oligomers in the cerebrospinal fluid (CSF) of APP/PS1 mice are critical for stimulating the expression of APH‐1α/1β, which was blocked by NS398 incubation. Finally, the induction of APH‐1α/1β was confirmed in the brains of patients with AD. Thus, these findings not only provide novel insights into the mechanism of PGI₂‐induced AD progression but also are instrumental for improving clinical therapies to combat AD.     

Overexpression of CIN85 suppresses the growth of herpes simplex virus in HeLa cells

The adaptor protein CIN85 is widely distributed in different tissues and has three Src homology 3 (SH3) domains, a proline-rich region (PRR), and a coiled-coil domain. During studies on the function of CIN85, it was reported to form a complex with herpes simplex virus 1 (HSV-1) infected cell protein 0 (ICP0), which plays a key role in enabling viral replication. Here, we demonstrate that plaque formation by HSV-1 is reduced on HeLa cells expressing CIN85 ectopically. The PRR of CIN85 was found to be essential for the inhibition of virus growth, whereas the three SH3 domains were not required. CIN85 also suppressed HSV-1 growth in Chinese hamster ovary (CHO) cells expressing the receptor for herpes simplex virus entry (herpes virus entry mediator A; HVEM). However, immunoprecipitation experiments showed that CIN85 did not interact with HVEM directly, indicating that CIN85 is not involved in the HSV-1 cell-entry pathway, but rather in another downstream pathway. Collectively, our data indicate that CIN85 might play an important role in HSV-1 infection.     

The herpes simplex virus DNA polymerase: Analysis of the functional domains

The structural and functional organization of the herpes simplex virus type I (HSV-1) DNA polymerase enzyme of strain ANG was studied by a combination of sequence and immunobiochemical analyses. Comparison of the HSV-1 ANG DNA polymerase sequence with those of pro- and eukaryotic DNA polymerases resulted in the allocation of eleven conserved regions within the HSV-1 DNA polymerase. From the analysis of all currently identified mutations of temperature-sensitive and drug-resistant HSV-1 DNA polymerase mutants as well as from the degree of conservancy observed, it could be deduced that the amino-acid residues 597–961, comprising the homologous sequence regions IV–IX, constitute the major structural components of the catalytic domain of the enzyme which should accommodate the sites for polymerizing and 3′-to-5′ exonucleolytic functions. Further insight into the structural organization was gained by the use of polyclonal antibodies responding specifically to the N-terminal, central and C-terminal polypeptide domains of the ANG polymerase. Each of the antisera was able to immunostain as well as to immunoprecipitate a viral polypeptide of 132 ± 5 kDa that corresponded well to the molecular mass of 136 kDa predicted from the coding sequences. Enzyme-binding and neutralization studies confirmed that both functions, polymerase and 3′-to-5′ exonuclease, are intimately related to each other, and revealed that, in addition to the sequences of the proposed catalytic domain, the very C-terminal sequences, except for amino-acid residues 1072–1146, are important for the catalytic functions of the enzyme, most likely effecting the binding to DNA.     

Involvement of cellular death signals in the reactivation of herpes simplex virus type 1 and lambda bacteriophage from a latent state

Herpes simplex virus (HSV) 1 has adapted to the human host through two modes of infection, the acute-transient infection that may cause diseases (such as encephalitis) and the latent state, which is a source for recurrent infection and disease. While much information has been gathered on the cellular and molecular concomitants of establishment and maintenance of HSV-1 latent state, the biological basis of viral reactivation is still unclear. Despite their obvious differences, HSV-1 and the bacterial temperate virus, the bacteriophage lambda, shares four distinct features that may help understand the viral latency phenomenon: (i) two modes of life cycle and a decision point to choose either latency (HSV-1) and lysogeny (bacteriophage lambda), or active replication, that results in cell destruction, (ii) establishment of lysogeny/latency of the respective virus is associated with protection from cell death, (iii) immunity/resistance to super-infection, (iv) agents that trigger mammalian and bacterial cell death also induce reactivation of both HSV-1 and lambda bacteriophage. Thus, despite their differences, these two viruses might display analogous mechanism(s) of reactivation. Based on clinical and experimental data, we propose in this hypothesis that while HSV-1 latency, like bacteriophage lambda lysogeny, is associated with protection from cell death and restriction to super-infection, viral reactivation from the latent state is triggered by exogenous stress signals that interfere with cellular viability and may eventually lead to cell death.     

Alzheimer’s disease-associated β-amyloid does not protect against herpes simplex virus 1 infection in the mouse brain

Alzheimer’s disease (AD) is a devastating fatal neurodegenerative disease. An alternative to the amyloid cascade hypothesis is that a viral infection is key to the etiology of late-onset AD, with β-amyloid (Aβ) peptides playing a protective role. In the current study, young 5XFAD mice that overexpress mutant human amyloid precursor protein with the Swedish, Florida, and London familial AD mutations were infected with one of two strains of herpes simplex virus 1 (HSV-1), 17syn+ and McKrae, at three different doses. Contrary to previous work, 5XFAD genotype failed to protect mice against HSV-1 infection. The region- and cell-specific tropisms of HSV-1 were not affected by the 5XFAD genotype, indicating that host–pathogen interactions were not altered. Seven- to ten-month-old 5XFAD animals in which extracellular Aβ aggregates were abundant showed slightly better survival rate relative to their wild-type (WT) littermates, although the difference was not statistically significant. In these 5XFAD mice, HSV-1 replication centers were partially excluded from the brain areas with high densities of Aβ aggregates. Aβ aggregates were free of HSV-1 viral particles, and the limited viral invasion to areas with a high density of Aβ aggregates was attributed to phagocytic activity of reactive microglia. In the oldest mice (12–15 months old), the survival rate did not differ between 5XFAD and WT littermates. While the current study questions the antiviral role of Aβ, it neither supports nor refutes the viral etiology hypothesis of late-onset AD.     

Pulsatilla decoction and its active ingredients inhibit secretion of NO,ET‐1, TNF‐α, and IL‐1α in LPS‐induced rat intestinal microvascular endothelial cells

To investigate the pharmacological mechanism of the traditional Chinese medicine, Pulsatilla decoction (PD), the levels of nitric oxide (NO), endothelin‐1 (ET‐1), tumor necrosis factor‐α (TNF‐α), and interleukin‐1α (IL‐1α) secreted by cultured rat intestinal microvascular endothelial cells (RIMECs) were determined after treatment with PD and its seven active ingredients, namely anemoside B₄, anemonin, berberine, jatrorrhizine, palmatine, aesculin, and esculetin. RIMECs were challenged with lipopolysaccharide (LPS) at 1 µg ml^?1 for 3 h and then treated with PD at 1, 5, and 10 mg ml^?1 and its seven ingredients at 1, 5, and 10 µg ml^?1 for 21 h, respectively. The results revealed that PD, anemonin, berberine, and esculetin inhibited the production of NO; PD, anemonin, and esculetin inhibited the secretion of ET‐1; PD, anemoside B₄, berberine, jatrorrhizine, and aesculin downregulated TNF‐α expression; PD, anemoside B₄, berberine, and palmatine decreased the content of IL‐1α. It showed that PD and its active ingredients could significantly inhibit the secretion of NO, ET‐1, TNF‐α, and IL‐1α in LPS‐induced RIMECs and suggested they would reduce inflammatory response via these cytokines. Copyright © 2009 John Wiley & Sons, Ltd.     

Expression and immunogenicity of recombinant glycoprotein D of herpes simplex virus 1 in Drosophila S2 cells

Herpes simplex virus type 1 (HSV-1) is responsible for cold sores in the general population, but also contributes to the development of other more serious diseases in some circumstances. The viral glycoprotein D (gD) is essential for virus entry into host cells. In the present study, the Drosophila melanogaster Schneider 2 (S2) expression system (DES) was evaluated for the expression of recombinant gD1. The DNA sequences encoding the full-length gD1 (369aa, FLgD1) and a truncated gD1 form corresponding to the ectodomain (314aa, EgD1) were cloned into S2 expression vector pMT/BiP/V5-HisA to generate pMT-EgD1 and pMT-FLgD1, respectively. Two forms of gD1 gene were fitted with a hexahistidine tag to facilitate their purification. Cell populations expressing the highest gD1 levels were selected by using a limiting dilution assay. Western blot, flow cytometry (FACS), and confocal immunofluoresence assay demonstrated that the full-length form is restrained in the lipid membranes of the cell and the ectodomain form is secreted into the medium. Recombinant ectodomain gD1 was scaled up and purified from the culture medium using nickel nitrilotriacetic acid affinity chromatography, and a maximum production level of 56.8 mg/L of recombinant gD1 was obtained in a shake-flask culture of S2 cells after induction with 5 µM CdCl2 for 4 days. Mice were then immunized with recombinant purified gD1 and produced high titers of antibody measured by enzyme-linked immunosorbent assay (ELISA; 1:5,120,000) as well as high plaque neutralization titer (1:320). Overall, the data indicated that stable expression in S2 cells is a practical way of synthesizing gD1 for use in structural and functional studies in the further study.     

Regulation of interleukin‐1β by interferon‐γ is species specific,limited by suppressor of cytokine signalling 1 and influences interleukin‐17 production

Reports describing the effect of interferon‐γ (IFNγ) on interleukin‐1β (IL‐1β) production are conflicting. We resolve this controversy by showing that IFNγ potentiates IL‐1β release from human cells, but transiently inhibits the production of IL‐1β from mouse cells. Release from this inhibition is dependent on suppressor of cytokine signalling 1. IL‐1β and Th17 cells are pathogenic in mouse models for autoimmune disease, which use Mycobacterium tuberculosis (MTB), in which IFNγ and IFNβ are anti‐inflammatory. We observed that these cytokines suppress IL‐1β production in response to MTB, resulting in a reduced number of IL‐17‐producing cells. In human cells, IFNγ increased IL‐1β production, and this might explain why IFNγ is detrimental for multiple sclerosis. In mice, IFNγ decreased IL‐1β and subsequently IL‐17, indicating that the adaptive immune response can provide a systemic, but transient, signal to limit inflammation.     

Quercetin‐induced upregulation of human GCLC gene is mediated by cis‐regulatory element for early growth response protein‐1 (EGR1) in INS‐1 beta‐cells

The catalytic subunit of γ‐glutamylcysteine ligase (GCLC) catalyses the rate‐limiting step in the de novo synthesis of glutathione (GSH), which is involved in maintaining intracellular redox balance. GSH is especially important for antioxidant defense system since beta‐cells show intrinsically low expression of antioxidant enzymes. In the present study, we investigated the regulatory mechanisms by which quercetin, a flavonoid, induces the expression of the GCLC gene in rat pancreatic beta‐cell line INS‐1. Promoter study found that the proximal GC‐rich region (from ?90 to ?34) of the GCLC promoter contained the quercetin‐responsive cis‐element(s). The quercetin‐responsive region contains consensus DNA binding site for early growth response 1 (EGR1) at ‐67 (5′‐CGCCTCCGC‐3′) which overlaps with a putative Sp1 binding site. Electrophoretic mobility shift assay showed that an oligonucleotide containing the EGR1 site was bound to nuclear factors EGR1, Sp1, and Sp3. In the promoter analysis, mutation of EGR1 site significantly reduced the quercetin response, whereas mutation of Sp1 site decreased only the basal activity of the GCLC promoter. Additionally, the transient overexpression of EGR1 significantly increased basal activity of the GCLC promoter. Finally, we showed that quercetin potently induced both EGR1 mRNA and its protein levels without affecting the expression of Sp1 and Sp3 proteins. Therefore, we concluded that EGR1 was bound to GC‐rich region of the GCLC gene promoter, which was prerequisite for the transactivation of the GCLC gene by quercetin. J. Cell. Biochem. 108: 1346–1355, 2009. © 2009 Wiley‐Liss, Inc.