Simultaneous quantification of seven prostanoids using liquid chromatography/tandem mass spectrometry: the effects of arachidonic acid on prostanoid production in mouse bone marrow-derived mast cells

We have developed a method for the simultaneous estimation of the levels of the prostanoids 6-keto prostaglandin (PG) Flalpha, PGB2, PGD2, PGE2, PGF2(alpha), PGJ2, and thromboxane (TX) B2 in blood- or serum-containing medium using liquid chromatography-tandem mass spectrometry. These prostanoids and their deuterium derivatives, which were used as internal standards, were subjected to solid-phase extraction using Empore C18 HD disk cartridges and analyzed in the selected reaction-monitoring mode. A linear response curve starting at 10 pg of prostanoid/tube was observed for each prostanoid. The accuracy of the method was demonstrated with samples containing known amounts of the prostanoids. Furthermore, we used this method to analyze the prostanoids produced in mouse bone marrow-derived mast cells stimulated with arachidonic acid, which resulted in the production of PGD2, PGE2, PGF2alpha, and TXB2. The results suggest that this simultaneous quantification method is useful for the analysis of the production of biomedically important prostanoids.     

Phospholipase Cε, an Effector of Ras and Rap Small GTPases,Is Required for Airway Inflammatory Response in a Mouse Model of Bronchial Asthma

Background

Phospholipase Cε (PLCε) is an effector of Ras and Rap small GTPases and expressed in non-immune cells. It is well established that PLCε plays an important role in skin inflammation, such as that elicited by phorbol ester painting or ultraviolet irradiation and contact dermatitis that is mediated by T helper (Th) 1 cells, through upregulating inflammatory cytokine production by keratinocytes and dermal fibroblasts. However, little is known about whether PLCε is involved in regulation of inflammation in the respiratory system, such as Th2-cells-mediated allergic asthma.

Methods

We prepared a mouse model of allergic asthma using PLCε ^+/+ mice and PLCε ^ΔX/ΔX mutant mice in which PLCε was catalytically-inactive. Mice with different PLCε genotypes were immunized with ovalbumin (OVA) followed by the challenge with an OVA-containing aerosol to induce asthmatic response, which was assessed by analyzing airway hyper-responsiveness, bronchoalveolar lavage fluids, inflammatory cytokine levels, and OVA-specific immunoglobulin (Ig) levels. Effects of PLCε genotype on cytokine production were also examined with primary-cultured bronchial epithelial cells.

Results

After OVA challenge, the OVA-immunized PLCε ^ΔX/ΔX mice exhibited substantially attenuated airway hyper-responsiveness and broncial inflammation, which were accompanied by reduced Th2 cytokine content in the bronchoalveolar lavage fluids. In contrast, the serum levels of OVA-specific IgGs and IgE were not affected by the PLCε genotype, suggesting that sensitization was PLCε-independent. In the challenged mice, PLCε deficiency reduced proinflammatory cytokine production in the bronchial epithelial cells. Primary-cultured bronchial epithelial cells prepared from PLCε ^ΔX/ΔX mice showed attenuated pro-inflammatory cytokine production when stimulated with tumor necrosis factor-α, suggesting that reduced cytokine production in PLCε ^ΔX/ΔX mice was due to cell-autonomous effect of PLCε deficiency.

Conclusions

PLCε plays an important role in the pathogenesis of bronchial asthma through upregulating inflammatory cytokine production by the bronchial epithelial cells.     

Frequency of and Predictive Factors for Vascular Invasion after Radiofrequency Ablation for Hepatocellular Carcinoma

Background

Vascular invasion in patients with hepatocellular carcinoma (HCC) is representative of advanced disease with an extremely poor prognosis. The detailed course of its development has not been fully elucidated.

Methods

We enrolled 1057 consecutive patients with HCC who had been treated with curative intent by radiofrequency ablation (RFA) as an initial therapy from 1999 to 2008 at our department. We analyzed the incidence rate of and predictive factors for vascular invasion. The survival rate after detection of vascular invasion was also analyzed.

Results

During a mean follow-up period of 4.5 years, 6075 nodules including primary and recurrent lesions were treated by RFA. Vascular invasion was observed in 97 patients. The rate of vascular invasion associated with site of original RFA procedure was 0.66% on a nodule basis. The incidence rates of vascular invasion on a patient basis at 1, 3, and 5 years were 1.1%, 5.9%, and 10.4%, respectively. Univariate analysis revealed that tumor size, tumor number, alpha-fetoprotein (AFP), des-gamma-carboxy prothrombin (DCP), and Lens culinaris agglutinin-reactive fraction of alpha-fetoprotein were significant risk predictors of vascular invasion. In multivariate analysis, DCP was the most significant predictor for vascular invasion (compared with a DCP of ≤100 mAu/mL, the hazard ratio was 1.95 when DCP was 101–200 mAu/mL and 3.22 when DCP was >200 mAu/mL). The median survival time after development of vascular invasion was only 6 months.

Conclusion

Vascular invasion occurs during the clinical course of patients initially treated with curative intent. High-risk patients may be identified using tumor markers.     

High-Sensitivity Real-Time Imaging of Dual Protein-Protein Interactions in Living Subjects Using Multicolor Luciferases

Networks of protein-protein interactions play key roles in numerous important biological processes in living subjects. An effective methodology to assess protein-protein interactions in living cells of interest is protein-fragment complement assay (PCA). Particularly the assays using fluorescent proteins are powerful techniques, but they do not directly track interactions because of its irreversibility or the time for chromophore formation. By contrast, PCAs using bioluminescent proteins can overcome these drawbacks. We herein describe an imaging method for real-time analysis of protein-protein interactions using multicolor luciferases with different spectral characteristics. The sensitivity and signal-to-background ratio were improved considerably by developing a carboxy-terminal fragment engineered from a click beetle luciferase. We demonstrate its utility in spatiotemporal characterization of Smad1–Smad4 and Smad2–Smad4 interactions in early developing stages of a single living Xenopus laevis embryo. We also describe the value of this method by application of specific protein-protein interactions in cell cultures and living mice. This technique supports quantitative analyses and imaging of versatile protein-protein interactions with a selective luminescence wavelength in opaque or strongly auto-fluorescent living subjects.     

Polycation liposome-mediated gene transfer in vivo

The polycation liposome (PCL), a recently developed gene transfer system, is simply prepared by a modification of liposomes with cetylated polyethylenimine (PEI), and shows remarkable transgene efficiency with low cytotoxicity. In the present study, we investigated the applicability of PCLs for in vivo gene transfer, since the PCL-mediated transgene efficiency was found to be maintained in the presence of serum. PCLs composed of dioleoylphosphatidylethanolamine (DOPE) with 5 mol% cetyl PEI (PEI average mr. wt. 1800), were superior for transfection to those of dipalmitoylphosphatidylcholine (DPPC) and cholesterol (2:1 as molar ratio) with 5 mol% cetyl PEI in vitro, although the latter PCLs were more efficient for gene transfer in vivo. PCL-DNA complexes were injected into mice via a tail or the portal vein, with the DNA being a plasmid encoding green fluorescent protein (GFP) or luciferase; and the expression was monitored qualitatively or quantitatively, respectively. Tail vein injection resulted in high expression of both GFP and luciferase genes in lung, and portal vein injection resulted in high expression of both genes in the liver. Concerning the gene delivery efficiency, the PCL was found to be superior to PEI or cetyl PEI alone. The optimal conditions for in vivo transfection with PCLs were also examined.     

Cyclin D1 overexpression perturbs DNA replication and induces replication-associated DNA double-strand breaks in acquired radioresistant cells

Fractionated radiotherapy (RT) is widely used in cancer treatment, because it preserves normal tissues. However, repopulation of radioresistant tumors during fractionated RT limits the efficacy of RT. We recently demonstrated that a moderate level of long-term fractionated radiation confers acquired radioresistance to tumor cells, which is caused by DNA-PK/AKT/GSK3β-mediated cyclin D1 overexpression. The resulting cyclin D1 overexpression leads to forced progression of the cell cycle to S-phase, concomitant with induction of DNA double-strand breaks (DSBs). In this study, we investigated the molecular mechanisms underlying cyclin D1 overexpression-induced DSBs during DNA replication in acquired radioresistant cells. DNA fiber data demonstrated that replication forks progressed slowly in acquired radioresistant cells compared with corresponding parental cells in HepG2 and HeLa cell lines. Slowly progressing replication forks were also observed in HepG2 and HeLa cells that overexpressed a nondegradable cyclin D1 mutant. We also found that knockdown of Mus81endonuclease, which is responsible for resolving aberrant replication forks, suppressed DSB formation in acquired radioresistant cells. Consequently, Mus81 created DSBs to remove aberrant replication forks in response to replication perturbation triggered by cyclin D1 overexpression. After treating cells with a specific inhibitor for DNA-PK or ATM, apoptosis rates increased in acquired radioresistant cells but not in parental cells by inhibiting the DNA damage response to cyclin D1-mediated DSBs. This suggested that these inhibitors might eradicate acquired radioresistant cells and improve fractionated RT outcomes.     

Constitutive OX40/OX40 ligand interaction induces autoimmune-like diseases

The interaction between OX40 and OX40 ligand (OX40L) is suggested to provide T cells with an effective costimulatory signals during T cell-APC interaction. To examine the in vivo effect of constitutive OX40/OX40L interaction during immune regulation, we report the establishment of OX40L-transgenic (OX40L-Tg) mice that constitutively express OX40L on T cells. Markedly elevated numbers of effector memory CD4(+) T cells, but not CD8(+) T cells, were observed in the secondary lymphoid organs of OX40L-Tg mice. Upon immunization with keyhole limpet hemocyanin in the absence of adjuvant, profound T cell proliferative responses and cytokine productions were seen in the OX40L-Tg mice as compared with wild-type mice. Furthermore, in OX40L-Tg mice administrated with superantigen, this constitutive OX40/OX40L interaction on CD4(+) T cells completely prevented normal in vivo clonal T cell deletion. Interestingly, OX40L-Tg mice on the C57BL/6 background spontaneously developed interstitial pneumonia and inflammatory bowel disease that was accompanied with a significant production of anti-DNA Ab in the sera. Surprisingly, these diseases were not evident on the OX40L-Tg mice on the BALB/c strain. However, such inflammatory diseases were successfully reproducible in recombination-activating gene (RAG)2-deficient mice upon transfer of OX40L-Tg CD4(+) T cells. Blockade of OX40/OX40L interaction in the recipient RAG2-deficient mice completely prevented disease development. The present results orchestrated in this study indicate that OX40/OX40L interaction may be a vital link in our understanding of T cell-mediated organ-specific autoimmunity.     

Effect of diazinon exposure on antioxidant reactions in the silkmoth,Bombyx mori

We investigated the effects of exposure to diazinon, an organophosphate insecticide, on the expression of antioxidant and detoxification factors in the silkmoth. Exposure to diazinon resulted in induction of mRNA encoding manganese containing superoxide dismutase (SOD) and omega‐class glutathione S‐transferases (GST), whereas no changes were observed in catalase, other class of GST and acetylcholinesterase. Liquid chromatography showed that the amount of reactive oxygen species was increased, whereas the amount of glutathione was decreased in the silkmoth fat body after exposure to diazinon. These results suggest that SOD and omega‐class GSTs can contribute to organophosphate resistance in Lepidopterans.     

Recent Fluctuations in Distribution and Biomass of Cold and Warm Temperature Species of Laminarialean Algae at Cape Ohma, Northern Honshu, Japan

The Cape Ohma region of Shimokita Peninsula, the northernmost point of Honshu Island, Japan, is subject to both the warm Tsugaru Current and the cold Kurile Current. As a result, the Laminarialean flora includes both cold temperature species (Laminaria japonica Areschoug, Kjellmaniella crassifolia Miyabe and Costaria costata (C. Agardh) Saunders) and warm temperature species (Undaria peterseniana (Kjellman) Okamura, Ecklonia stolonifera Okamura), as well as Undaria pinnatifida (Yendo) Okamura, which is distributed in both waters. The frequency of occurrence (as a measure of distribution) and the biomass of these species were recorded in June 1976 (at 50 points in depths between 8–30 m), July 1988 (192 points, 2.5–25 m) and July 2001 (78 points, 2.5–25 m). Comparison of these data revealed a decrease in cold temperature species and an increase in warm temperature species from 1976 or 1988 to 2001. Long-term data of seawater temperature measured at 5 m depth near the study site showed that mean temperatures in the middle of winter (late January to February) in 1989–2000 were 0.9–1.1 ^∘C higher than those in 1980–1988. Higher seawater temperatures in the last decade appear to have affected the frequency of occurrence and biomass of the Laminarialean species along the coasts of Cape Ohma. This result supported our previous conclusion that 1 ^∘C higher mean seawater temperature in late January caused a decrease in the biomass of L. japonica (by ca. 64%) along the same coast.