Superoxide production at phagosomal cup/phagosome through beta I protein kinase C during Fc gamma R-mediated phagocytosis in microglia

Protein kinase C (PKC) plays a prominent role in immune signaling. To elucidate the signal transduction in a respiratory burst and isoform-specific function of PKC during FcgammaR-mediated phagocytosis, we used live, digital fluorescence imaging of mouse microglial cells expressing GFP-tagged molecules. betaI PKC, epsilonPKC, and diacylglycerol kinase (DGK) beta dynamically and transiently accumulated around IgG-opsonized beads (BIgG). Moreover, the accumulation of p47(phox), an essential cytosolic component of NADPH oxidase and a substrate for betaI PKC, at the phagosomal cup/phagosome was apparent during BIgG ingestion. Superoxide (O(2)(-)) production was profoundly inhibited by G?6976, a cPKC inhibitor, and dramatically increased by the DGK inhibitor, R59949. Ultrastructural analysis revealed that BIgG induced O(2)(-) production at the phagosome but not at the intracellular granules. We conclude that activation/accumulation of betaI PKC is involved in O(2)(-) production, and that O(2)(-) production is primarily initiated at the phagosomal cup/phagosome. This study also suggests that DGKbeta plays a prominent role in regulation of O(2)(-) production during FcgammaR-mediated phagocytosis.     

Dietary resveratrol prevents the development of food allergy in mice

Background

Resveratrol is a bioactive polyphenol enriched in red wine that exhibits many beneficial health effects via multiple mechanisms. However, it is unclear whether resveratrol is beneficial for the prevention of food allergy. This study investigated whether resveratrol inhibited the development of food allergy by using a mouse model of the disease.

Methodology/Principal Findings

Mice fed standard diet or standard diet plus resveratrol were sensitized by intragastric administration of ovalbumin (OVA) and mucosal adjuvant cholera toxin (CT). Several manifestations of food allergy were then compared between the mice. The effects of resveratrol on T cells or dendritic cells were also examined by using splenocytes from OVA-specific T cell-receptor (TCR) transgenic DO11.10 mice or mouse bone marrow-derived dendritic cells (BMDCs) in vitro. We found that mice fed resveratrol showed reduced OVA-specific serum IgE production, anaphylactic reaction, and OVA-induced IL-13 and IFN-ã production from the mesenteric lymph nodes (MLNs) and spleens in comparison to the control mice, following oral sensitization with OVA plus CT. In addition, resveratrol inhibited OVA plus CT-induced IL-4, IL-13, and IFN-ã production in splenocytes from DO11.10 mice associated with inhibition of GATA-3 and T-bet expression. Furthermore, resveratrol suppressed the OVA plus CT-induced CD25 expression and IL-2 production in DO11.10 mice-splenocytes in association with decreases in CD80 and CD86 expression levels. Finally, resveratrol suppressed CT-induced cAMP elevation in association with decreases in CD80 and CD86 expression levels in BMDCs.

Conclusions/Significance

Ingestion of resveratrol prevented the development of a food allergy model in mice. Given the in vitro findings, resveratrol might do so by inhibiting DC maturation and subsequent early T cell activation and differentiation via downregulation of CT-induced cAMP activation in mice. These results suggest that resveratrol may have potential for prophylaxis against food allergy.     

Analysis of DNA double-strand break response and chromatin structure in mitosis using laser microirradiation

In this study the femtosecond near-IR and nanosecond green lasers are used to induce alterations in mitotic chromosomes. The subsequent double-strand break responses are studied. We show that both lasers are capable of creating comparable chromosomal alterations and that a phase paling observed within 1-2 s of laser exposure is associated with an alteration of chromatin as confirmed by serial section electron microscopy, DAPI, γH2AX and phospho-H3 staining. Additionally, the accumulation of dark material observed using phase contrast light microscopy (indicative of a change in refractive index of the chromatin) ～ 34 s post-laser exposure corresponds spatially to the accumulation of Nbs1, Ku and ubiquitin. This study demonstrates that chromosomes selectively altered in mitosis initiate the DNA damage response within 30 s and that the accumulation of proteins are visually represented by phase-dark material at the irradiation site, allowing us to determine the fate of the damage as cells enter G1. These results occur with two widely different laser systems, making this approach to study DNA damage responses in the mitotic phase generally available to many different labs. Additionally, we present a summary of most of the published laser studies on chromosomes in order to provide a general guide of the lasers and operating parameters used by other laboratories.     

Cytotoxic Alangium alkaloids from Alangium longiflorum

Seven alkaloids (1-7) were isolated from the stem bark of Alangium longiflorum. Compound 1, (-)-10-O-demethylisocephaeline, was isolated for the first time as a naturally occurring product from a plant source. All structures were elucidated by detailed spectroscopic analysis. Biological evaluation showed that 2, 10-O-demethylcephaeline, exhibited potent cytotoxic activity against human lung carcinoma (A549) and breast adenocarcinoma (MCF-7) with ED(50) values of 0.013 and 0.062 microM, respectively. The stereoisomer 1 was less potent than 2, and related compounds with different hydroxy/methoxy substitution patterns were also less potent or inactive. Thus, compound 2 merits attention as a cytotoxic lead for further study.     

Two enzyme-linked immunosorbent assay (ELISA) systems for N1,N8-diacetylspermidine and N1,N12-diacetylspermine using monoclonal antibodies

We obtained monoclonal antibodies against N(1),N(12)-diacetylspermine (DiAcSpm) and N(1),N(8)-diacetylspermidine (DiAcSpd), and developed two systems of competitive ELISA that utilize the antibodies and a common enzyme-labeled antigen to measure these di-acetylpolyamines. Cross-reactions with N(1)-acetylspermidine in the assay of DiAcSpm and with N8-acetylspermidine in the assay of DiAcSpd were as low as 0.26 and 0.6%, respectively, and were judged to be insignificant in clinical use for measuring urinary diacetylpolyamines. These assays were used to assess diurnal variations in diacetylpolyamine excretion in urine to show that the excretion of diacetylpolyamines after normalization for the concentration of creatinine is stable over a day with only minimal diurnal variation.     

Three-dimensional solution structure of an archaeal FKBP with a dual function of peptidyl prolyl cis-trans isomerase and chaperone-like activities

Here we report the solution structure of an archaeal FK506-binding protein (FKBP) from a thermophilic archaeum, Methanococcus thermolithotrophicus (MtFKBP17), which has peptidyl prolyl cis-trans isomerase (PPIase) and chaperone-like activities, to reveal the structural basis for the dual function. In addition to a typical PPIase domain, a newly identified domain is formed in the flap loop by a 48-residue insert that is required for the chaperone-like activity. The new domain, called IF domain (the Insert in the Flap), is a novel-folding motif and exposes a hydrophobic surface, which we consider to play an important role in the chaperone-like activity.     

New Preclinical Antimalarial Drugs Potently Inhibit Hepatitis C Virus Genotype 1b RNA Replication

Background

Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a global health problem. Although new triple therapy (pegylated-interferon, ribavirin, and telaprevir/boceprevir) has recently been started and is expected to achieve a sustained virologic response of more than 70% in HCV genotype 1 patients, there are several problems to be resolved, including skin rash/ageusia and advanced anemia. Thus a new type of anti-HCV drug is still needed.

Methodology/Principal Findings

Recently developed HCV drug assay systems using HCV-RNA-replicating cells (e.g., HuH-7-derived OR6 and Li23-derived ORL8) were used to evaluate the anti-HCV activity of drug candidates. During the course of the evaluation of anti-HCV candidates, we unexpectedly found that two preclinical antimalarial drugs (N-89 and its derivative N-251) showed potent anti-HCV activities at tens of nanomolar concentrations irrespective of the cell lines and HCV strains of genotype 1b. We confirmed that replication of authentic HCV-RNA was inhibited by these drugs. Interestingly, however, this anti-HCV activity did not work for JFH-1 strain of genotype 2a. We demonstrated that HCV-RNA-replicating cells were cured by treatment with only N-89. A comparative time course assay using N-89 and interferon-α demonstrated that N-89-treated ORL8 cells had more rapid anti-HCV kinetics than did interferon-α-treated cells. This anti-HCV activity was largely canceled by vitamin E. In combination with interferon-α and/or ribavirin, N-89 or N-251 exhibited a synergistic inhibitory effect.

Conclusions/Significance

We found that the preclinical antimalarial drugs N-89 and N-251 exhibited very fast and potent anti-HCV activities using cell-based HCV-RNA-replication assay systems. N-89 and N-251 may be useful as a new type of anti-HCV reagents when used singly or in combination with interferon and/or ribavirin.     

Liposome Vector Containing Biosurfactant-Complexed DNA as Herpes Simplex Virus Thymidine Kinase Gene Delivery System

For injectable-sized liposome complexed with DNA (lipoplexes) with high transfection efficiency of genes, we initially prepared small-sized liposomes by addition of biosurfactant. For selectivity of gene expression, the thymidine kinase (MK-tk) gene controlled by midkine was used for herpes simplex virus thymidine kinase (HSV-tk) gene therapy. Liposomes composed of 3([N-(N′,N′–dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), L-dioleoylphosphatidylethanolamine (DOPE), and a biosurfactant, such as β-sitosterol β-D-glucoside (Sit-G) for Sit-G-liposomes and mannosylerythrytol lipid A (MEL) for MEL-liposomes, produced about 300-nm-sized lipoplexes. Sit-G- and MEL-liposomes showed higher transfection efficiency of the luciferase marker gene and thymidine kinase activity in the presence of serum in the cells. The treatment with transfection of MK-tk gene by Sit-G-liposome and injection of ganciclovir significantly reduced tumor growth in a solid tumor model, compared with that by Sit-G-liposome alone. This finding suggested that Sit-G-liposome is a potential vector for HSV-tk gene therapy.     

Effects of gestational exposure to 1.95‐GHz W‐CDMA signals for IMT‐2000 cellular phones: Lack of embryotoxicity and teratogenicity in rats

The present study was designed to evaluate whether gestational exposure to an EMF targeting the head region, similar to that from cellular phones, might affect embryogenesis in rats. A 1.95‐GHz wide‐band code division multiple access (W‐CDMA) signal, which is one applied for the International Mobile Telecommunication 2000 (IMT‐2000) system and used for the freedom of mobile multimedia access (FOMA), was employed for exposure to the heads of four groups of pregnant CD(SD) IGS rats (20 per group) for gestational days 7–17. The exposure was performed for 90 min/day in the morning. The spatial average specific absorption rate (SAR) for individual brains was designed to be 0.67 and 2.0 W/kg with peak brain SARs of 3.1 and 7.0 W/kg for low (group 3) and high (group 4) exposures, respectively, and a whole‐body average SAR less than 0.4 W/kg so as not to cause thermal effects due to temperature elevation. Control and sham exposure groups were also included. At gestational day 20, all dams were killed and fetuses were taken out by cesarean section. There were no differences in maternal body weight gain. No adverse effects of EMF exposure were observed on any reproductive and embryotoxic parameters such as number of live (243–271 fetuses), dead or resorbed embryos, placental weights, sex ratios, weights or external, visceral or skeletal abnormalities of live fetuses. Bioelectromagnetics 30:205–212, 2009. © 2008 Wiley‐Liss, Inc.