Impact of DNA demethylation of the G0S2 gene on the transcription of G0S2 in squamous lung cancer cell lines with or without nuclear receptor agonists

We recently identified that DNA methylation of the G0S2 gene was significantly more frequent in squamous lung cancer than in non-squamous lung cancer. However, the significance of G0S2 methylation levels on cancer cells is not yet known. We investigated the effect of G0S2 methylation levels on cell growth, mRNA expression, and chromatin structure using squamous lung cancer cell lines and normal human bronchial epithelial cells. DNA methylation and mRNA expression of G0S2 were inversely correlated, and in one of the squamous lung cancer cell lines, LC-1 sq, G0S2 was completely methylated and suppressed. Overexpression of G0S2 in LC-1 sq did not show growth arrest or apoptosis. The G0S2 gene has been reported to be a target gene of all-trans retinoic acid and peroxisome proliferator-activated receptor agonists. We treated LC-1 sq with 5-Aza-2′-deoxycytidine, Trichostatin A, all-trans retinoic acid, Wy 14643, or Pioglitazone either alone or in combination. Only 5-Aza-2′-deoxycytidine restored mRNA expression of G0S2. Chromatin immunoprecipitation revealed that histone H3 lysine 9 was methylated regardless of DNA methylation or mRNA expression. In summary, mRNA expression of G0S2 was regulated mainly by DNA methylation in squamous lung cancer cell lines. When the G0S2 gene was methylated, nuclear receptor agonists could not restore mRNA expression of G0S2 and did not show any additive effect on mRNA expression of G0S2 even after the treatment with 5-Aza-2′-deoxycytidine.     

Induction of CD4+ murine natural killer T-like cells by immunization with syngeneic thymoma expressing embryonic alpha-fetoprotein

In embryo, before the establishment of acquired immunity, a variety of embryonic antigens like alpha-fetoprotein (AFP) are produced and secreted in the sera, which rapidly disappear after the birth. Such embryonic antigens sometimes reappear from various tumor cells and decrease in the case of remission, indicating embryonic antigens may alert immune system to control tumors. In the present study, to examine the evoked immune responses against the tumors expressing embryonic antigen, we administered AFP-gene-transfected EL4 cells into syngeneic C57BL/6 mice and established a killer line against the tumor cells. To our surprise, the killer line was CD4+ NK1.1+, natural killer T (NKT)-like cells and eliminated not only AFP-expressing EL4 but YAC-1 cells. Moreover, the established line uniformly expressed Vbeta11 and secreted IL-4, IL-10, IL-13, and IFN-gamma. In vivo inoculation of the line markedly reduced the tumor growth in SCID mice, suggesting novelty of the NKT-like line for tumor surveillance.     

KaiB functions as an attenuator of KaiC phosphorylation in the cyanobacterial circadian clock system

In the cyanobacterium Synechococcus elongatus PCC 7942, the KaiA, KaiB and KaiC proteins are essential for generation of circadian rhythms. We quantitatively analyzed the intracellular dynamics of these proteins and found a circadian rhythm in the membrane/cytosolic localization of KaiB, such that KaiB interacts with a KaiA-KaiC complex during the late subjective night. KaiB-KaiC binding is accompanied by a dramatic reduction in KaiC phosphorylation and followed by dissociation of the clock protein complex(es). KaiB attenuated KaiA-enhanced phosphorylation both in vitro and in vivo. Based on these results, we propose a novel role for KaiB in a regulatory link among subcellular localization, protein-protein interactions and post-translational modification of Kai proteins in the cyanobacterial clock system.     

Cytomorphologic features of a polymorphous low grade adenocarcinoma of the palate. A report of 2 cases with immunocytochemistry

BACKGROUND: Polymorphous low grade adenocarcinoma (PLGA) is a histologically low grade tumor of minor salivary gland origin. It is important to differentiate PLGA from other salivary gland tumors with myoepithelial differentiation, such as pleomorphic adenoma, adenoid cystic carcinoma and epithelial myoepithelial carcinoma. Here we report 2 cases of PLGA originating in the palate and describe the cytomorphologic and immunocytochemical features. CASES: The patients were a 55-year-old woman and a 63-year-old man. Both presented with a mass in the palate. Clinically the mass appeared malignant, and resection was performed. Cytologically the tumor cells were composed of sheet clusters, pseudopapillary epithelial clusters, naked cells and stromal components. Immunocytochemically the tumor cells showed strong expression of carcinoembryonic antigen (CEA) and vimentin. CONCLUSION: PLGA may be difficult to distinguish from other salivary gland tumors with myoepithelial differentiation. However, the cytopathologist should be aware of the distinctive cytomorphologic features of PLGA, demonstrating immunopositivity to CEA and vimentin.     

Induction of hepatic injury by hepatitis C virus-specific CD8+ murine cytotoxic T lymphocytes in transgenic mice expressing the viral structural genes

In the present study, we generated killer cells specific for hepatitis C virus (HCV) structural protein by re-stimulation of immune spleen cells from H-2(d) haplotype transgenic (Tg) mice, expressing the core, E1, E2, and NS2 genes of HCV regulated by the Cre/loxP switching system. The generated killer cells were conventional CD8(+)L(d) class-I MHC molecule-restricted cytotoxic T lymphocytes (CTLs) and specific for the HCV E1 structural protein. Because the CTLs could also kill hepatocytes from the Tg mice expressing HCV structural proteins in vitro, we attempted to transfer those CTLs intravenously into interferon regulatory factor-1 (IRF-1) negative, CD8-deficient Tg mice representing the HCV structural genes on hepatocytes to examine whether the inoculated CD8(+) CTLs can eliminate hepatocytes expressing the HCV genes in vivo. We observed an elevation of serum ALT level as well as damage of the liver tissue histologically. To our knowledge, this is the first demonstration to show that HCV-specific CD8(+) CTLs specifically attack hepatocytes expressing the HCV structural proteins both in vitro and in vivo.     

Toll-like receptor 4-MD-2 complex mediates the signal transduction induced by flavolipin, an amino acid-containing lipid unique to Flavobacterium meningosepticum

Flavolipin, an amino acid-containing lipid isolated from Flavobacterium meningosepticum, induces many immune responses. It has been shown that flavolipin does not induce an immune response of macrophages derived from C3H/HeJ mice, which possess a point mutation in Toll-like receptor 4 (TLR4). To determine whether TLR4 or the molecular complex of TLR4 and TLR4 association molecule MD-2 mediates the flavolipin signal, flavolipin responsiveness was examined by measuring NF-kappaB activation in Ba/F3 cells and Ba/F3 transfectants expressing TLR4 or both TLR4 and MD-2. Flavolipin-induced NF-kappaB activation was detected in the cells expressing both TLR4 and MD-2, but not in the other cells. Expression of CD14 in the transfectant expressing both TLR4 and MD-2 increased the sensitivity to flavolipin. Furthermore, flavolipin stereoisomers were chemically synthesized, and their abilities to induce NF-kappaB activation were examined. (R)-Flavolipin, in which the configuration of the lipid moiety is R, induced NF-kappaB activation via the TLR4-MD-2 complex, but (S)-flavolipin did not. In this study, we demonstrated the involvement of TLR4-MD-2 and CD14 in flavolipin signaling and the importance of the (R)-configuration of the flavolipin lipid moiety for the induction of an immune response via TLR4-MD-2.     

Correlation between X-ray diffraction measurements of cellulose crystalline structure and the susceptibility to microbial cellulase

Chemical and physical treatments of cotton cellulose have been studied in order to elucidate the relationship between the degree of crystallinity of cellulose and the susceptibility of cellulose to cellulase. Cotton cellulose powder was treated with the following solvents: 60% H₂SO₄, Cadoxen, and DMSO-p -formaldehyde. The dissolved celluloses were recovered at high yield of over 97% by addition of nine volumes of cold acetone. X-ray diffraction for measurements of relative crystallinity showed that the crystalline structure of cellulose declined in quantity and perfection by the dissolving treatment and changed to an amorphous form that is highly susceptible to enzymatic hydrolysis. These reprecipitated celluloses were hydrolyzed almost completely within 48 hr by Aspergillus niger cellulase containing mainly 1,4-β-glucan glucanohydrolase (EC 3.2.1.4), without action of 1,4-β-glucan cellobiohydrolase (EC 3.2.1. 91). On the other hand, cryo-milled cellulose (below 250 mesh) still had a crystalline structure, was resistant to cellulase, and gave a low percentage of saccharification. These results indicate that in pure cellulose there are good correlations between x-ray diffractograms and susceptibility to microbial cellulase.