SUMMARYMastermind (Mam) is one of the elements of Notch signaling, a system that plays a pivotal role in metazoan development. Mam proteins form transcriptionally activating complexes with the intracellular domains of Notch, which are generated in response to the ligand-receptor interaction, and CSL DNA-binding proteins. In mammals, three structurally divergent Mam isoforms (MamL1, MamL2 and MamL3) have been identified. There have also been indications that Mam interacts functionally with various other transcription factors, including the p53 tumor suppressor, -catenin and NF-B. We have demonstrated previously that disruption of MamL1 causes partial deficiency of Notch signaling in vivo. However, MamL1-deficient mice did not recapitulate total loss of Notch signaling, suggesting that other members could compensate for the loss or that Notch signaling could proceed in the absence of Mam in certain contexts. Here, we report the generation of lines of mice null for MamL3. Although MamL3-null mice showed no apparent abnormalities, mice null for both MamL1 and MamL3 died during the early organogenic period with classic pan-Notch defects. Furthermore, expression of the lunatic fringe gene, which is strictly controlled by Notch signaling in the posterior presomitic mesoderm, was undetectable in this tissue of the double-null embryos. Neither of the single-null embryos exhibited any of these phenotypes. These various roles of the three Mam proteins could be due to their differential physical characteristics and/or their spatiotemporal distributions. These results indicate that engagement of Mam is essential for Notch signaling, and that the three Mam isoforms have distinct roles in vivo.

Abstract. The aim of this study was to assess whether modulated electro-hyperthermia (mEHT) can induce an abscopal effect and thereby enhance the antitumor effects of immunotherapy. We used an intratumoral dendritic cell (DC) injection and mEHT to treat C3H/He mice inoculated with squamous cell carcinoma SCCVII cells in the left leg, and we assessed the whole body antitumor effects. Tumors were examined every two or three days in order to assess growth inhibition. The tumor-draining lymph nodes were removed to enable flow cytometric analysis of CD3 + and CD8 + cells, whereas immunohistochemistry was used to assess CD8, S100 and Foxp3 expression in the tumors. Additionally, GP96 expression in the tumors from the different treatment groups was measured. In the control group, the mean tumor volume was larger than that in other groups. These results indicated that the combination therapy of an intratumoral DC injection and mEHT evoked systemic antitumor activity. A larger number of CD3 + and CD8 + cells were detected by flow cytometric analysis in the DC plus mEHT treatment group. Tumor tissue immunostaining showed that CD8 and S100 were more strongly expressed in the DC plus mEHT treatment group, although Foxp3 expression was much higher in the control group. The GP96 gene expression level in the mEHT group was significantly different from the expression level in the control group. An abscopal effect may be induced by mEHT, and the effect of immunotherapy with DCs was strongly enhanced by the overexpression of GP96. GP96 is thought to be one of the molecules explaining the abscopal effect. Direct intratumoral administration of DCs and mEHT may be a feasible future treatment strategy.

Polyamines play important roles in cell growth mainly through their interaction with RNA. We have previously reported that polyamines stimulate the synthesis of oligopeptide-binding protein OppA in Escherichia coli and the formation of Ile-tRNA in rat liver (Igarashi, K., and Kashiwagi, K. (2000) Biochem. Biophys. Res. Commun. 271, 559 -564). These effects involve an interaction of polyamines with the bulged-out region of double-stranded RNA in the initiation region of OppA mRNA and in the acceptor stem of rat liver tRNA Ile . In this study, the effects of polyamines on E. coli OppA synthesis and rat liver Ile-tRNA formation were compared using OppA mRNA and tRNA Ile with or without the bulged-out region of doublestranded RNA. The results indicate that the bulged-out region is involved in polyamine stimulation of OppA synthesis and IletRNA formation. A selective structural change by spermidine in the bulged-out region of double-stranded RNA was confirmed by circular dichroism.Polyamines (putrescine, spermidine, and spermine) are essential for normal cell growth due to effects mainly at the level of translation (1-3). It is known that polyamines bind preferentially to double-stranded RNA rather than single-stranded RNA and double-stranded DNA (4). Indeed, polyamines were found mostly in polyamine-RNA complexes when measured in rat liver, bovine lymphocytes, and Escherichia coli (5, 6). We reported previously that polyamines have not only a sparing effect on the Mg 2ϩ requirement of polyphenylalanine and globin synthesis but also a stimulatory effect that cannot be fulfilled by any amount of Mg 2ϩ in the absence of polyamines (7,8). It has also been reported that polyamines enhance, at the level of translation, the synthesis of several kinds of proteins that are important for cell growth in E. coli (3, 9 -11). We propose that a group of genes whose expression is enhanced by polyamines at the level of translation be referred to as the "polyamine modulon" (3).There are three different mechanisms underlying polyamine stimulation of the synthesis of various members of the polyamine modulon. First, polyamine stimulation of protein synthesis can occur when a Shine-Dalgarno sequence in the mRNA is obscure or is distant from the AUG initiation codon. In this case, polyamines cause structural changes in a region of the Shine-Dalgarno sequence and the AUG initiation codon of the mRNA, facilitating formation of the initiation complex, and examples include OppA, a periplasmic substrate-binding protein of the oligopeptide uptake system; FecI factor ( 18 ), for transcription of the iron transport operon; Fis, a global regulator of transcription of some growth-related genes, including those for rRNA and some tRNAs; RpoN factor ( 54 ), for transcription of genes for nitrogen metabolism; and H-NS, a transcription factor of many kinds of mRNAs, including ribosomal protein mRNAs and flagellar protein mRNAs. By a second mechanism, polyamines enhance the inefficient initiation codon UUG (or GUG)-dependent fMet-tRNA binding to Cya (or...

This article is available online at http://www.jlr.org Arachidonic acid (AA) is a precursor of eicosanoids, including prostaglandins, thromboxanes, and leukotrienes, playing an important role in several physiological functions ( 1 ). The biosynthesis of these AA metabolites occurs mainly through the activation of phospholipase A2 (PLA2) in response to a wide variety of stimuli such as cytokines, growth factors, and neurotransmitters ( 2 ). PLA2 catalyzes hydrolysis of the sn-2 position of glycerophospholipids to release free AA. Mammalian cells have structurally diverse forms of PLA2 including secretory PLA2, Ca 2+ -independent PLA2, and cytosolic PLA2 (cPLA2) ( 3, 4 ). Among these PLA2s, the 85 kDa cPLA2, specifi cally cPLA2 ␣ , is highly selective for glycerophospholipids containing AA. cPLA2 ␣ is regulated mainly by an increase in the intracellular Ca 2+ concentrations ([Ca 2+ ]i) and by the phosphorylation on serine residues by mitogen-activated protein kinase (MAPK) ( 3, 4 ). The binding of Ca 2+ to the C2 domain of cPLA2 ␣ triggers translocation of cPLA2 ␣ from the cytosol to the perinuclear region including the Golgi apparatus, endoplasmic reticulum (ER), and nuclear envelope. cPLA2 ␣ can be phosphorylated at Ser 505 , Ser 515 , and Ser 727 , which increases its intrinsic enzymatic activity 2-to 3-fold in vitro ( 5-8 ). Abstract

Glioblastoma (GBM) is the most common malignant brain tumor, and infiltrates into the surrounding normal brain tissue. Induction of a tumor-specific immune response is one of the best methods to obtain tumor-specific cytotoxicity. Photodynamic therapy (PDT) is known to effectively induce an antitumor immune response. We have developed a clinically translatable nanoparticle, liposomally formulated phospholipid-conjugated indocyanine green (LP-iDOPE), applicable for PDT. This nanoparticle accumulates in tumor tissues by the enhanced permeability and retention effect, and releases heat and singlet oxygen to injure cancer cells when activated by near infrared (NIR) light. We assessed the effectiveness of the LP-iDOPE system in brain using the rat 9L glioblastoma model. Treatment with LP-iDOPE and NIR irradiation resulted in significant tumor growth suppression and prolongation of survival. Histopathological examination showed induction of both apoptosis and necrosis and accumulation of CD8+ T-cells and macrophages/microglia accompanied by marked expressions of heat shock protein-70 (HSP70), which was not induced by mild hyperthermia alone at 45° C or an interleukin-2-mediated immune reaction. Notably, the efficacy was lost in immunocompromised nude rats. These results collectively show that the novel nanoparticle LP-iDOPE in combination with NIR irradiation can efficiently induce a tumor-specific immune reaction for malignant gliomas possibly by inducing HSP70 expression which is known to activate antigen-presenting cells through toll-like receptor signaling.