Effect of sizofiran on regional lymph nodes in patients with head and neck cancer

The immunomodulating effects of preoperative sizofiran (SPG) administration on regional lymph nodes were studied in patients with stage III or IV head and neck cancer, by comparing the immunofunction of peripheral blood. The regional lymph nodes were dissected surgically, and freshly obtained mononuclear cells were studied to investigate the interleukin-2 (IL-2) production, the LAK and NK activities, and the quantitative analysis of the surface phenotype of the mononuclear cells. The results indicated that SPG enhanced immunological activities in the regional lymph nodes, as shown by increased IL-2 production and cytotoxic activities of the effector cells (NK, LAK), and increased helper T lymphocytes (CD4⁺) in the tumor-uninvolved lymph nodes. The immunofunction following SPG administration was attenuated, but was still augmented in the regional lymph nodes with metastases. Therefore, SPG was found to be a biologic response modifier to enhance the immunofunctions of the regional lymph node in patients with head and neck cancer.     

DNA-Binding Properties of the Antibody Specific for the Dewar Photoproduct of Thymidylyl-(3′′-5′′)-Thymidine

A monoclonal antibody (DEM-1) specific for the Dewar photoproduct is used for detection and quantification of photolesions in DNA. To help understand the molecular recognition of damaged DNA by the antibody protein, we have cloned and sequenced the variable region genes of DEM-1. We have also prepared Fab fragments of DEM-1 (DEM1Fab), and synthesized two kinds of 3′-biotinylated oligonucleotides of different lengths containing a central Dewar photoproduct of TpT to analyze the effects of the antigen size on the binding rates by means of surface plasmon resonance (SPR). Results obtained from SPR analyses suggest that DEM1Fab may recognize tetranucleotide unit as the epitope.     

Identification of miR-30e* Regulation of Bmi1 Expression Mediated by Tumor-Associated Macrophages in Gastrointestinal Cancer

Bmi1 is overexpressed in a variety of human cancers including gastrointestinal cancer. The high expression level of Bmi1 protein is associated with poor prognosis of gastrointestinal cancer patients. On the other hand, tumor-associated macrophages (TAMs) contribute to tumor growth, invasion, and metastasis by producing various mediators in the tumor microenvironment. The aim of this study was to investigate TAM-mediated regulation of Bmi1 expression in gastrointestinal cancer. The relationship between TAMs and Bmi1 expression was analyzed by immunohistochemistry and quantitative real-time PCR (qRT-PCR), and results showed a positive correlation with tumor-infiltrating macrophages (CD68 and CD163) and Bmi1 expression in cancer cells. Co-culture with TAMs triggered Bmi1 expression in cancer cell lines and enhanced sphere formation ability. miRNA microarray analysis of a gastric cancer cell line co-cultured with macrophages was conducted, and using in silico methods to analyze the results, we identified miR-30e* as a potential regulator of Bmi1 expression. Luciferase assays using miR-30e* mimic revealed that Bmi1 was a direct target for miR-30e* by interactions with the putative miR-30e* binding sites in the Bmi1 3′ untranslated region. qRT-PCR analysis of resected cancer specimens showed that miR-30e* expression was downregulated in tumor regions compared with non-tumor regions, and Bmi1 expression was inversely correlated with miR-30e* expression in gastric cancer tissues, but not in colon cancer tissues. Our findings suggest that TAMs may cause increased Bmi1 expression through miR-30e* suppression, leading to tumor progression. The suppression of Bmi1 expression mediated by TAMs may thus represent a possible strategy as the treatment of gastrointestinal cancer.     

IL-15 regulates CD8+ T cell contraction during primary infection

During the course of acute infection with an intracellular pathogen, Ag-specific T cells proliferate in the expansion phase, and then most of the T cells die by apoptosis in the following contraction phase, but the few that survive become memory cells and persist for a long period of time. Although IL-15 is known to play an important role in long-term maintenance of memory CD8+ T cells, the potential roles of IL-15 in CD8+ T cell contraction are not known. Using an adoptive transfer system of OT-I cells expressing OVA257-264/Kb-specific TCR into control, IL-15 knockout (KO) and IL-15 transgenic (Tg) mice followed by challenge with recombinant Listeria monocytogenes expressing OVA, we found that the survival of CD44+CD62L-CD127- effector OT-I cells during the contraction phase is critically dependent on IL-15. In correlation with the expression level of Bcl-2 in OT-I cells, the number of OT-I cells was markedly reduced in IL-15 KO mice but remained at a high level in IL-15 Tg mice during the contraction phase, compared with control mice. In vivo administration of rIL-15 during the contraction phase in IL-15 KO mice inhibited the contraction of effector OT-I cells accompanied by up-regulation of Bcl-2 expression. Furthermore, enforced expression of Bcl-2 protected the majority of effector OT-I cells from death in IL-15 KO mice after infection. These results suggest that IL-15 plays a critical role in protecting effector CD8+ T cells from apoptosis during the contraction phase following a microbial infection via inducing antiapoptotic molecules.     

Regeneration of <Emphasis Type="Italic">Robinia</Emphasis><Emphasis Type="Italic">pseudoacacia</Emphasis> riparian forests after clear-cutting along the Chikumagawa River in Japan

Regeneration processes of riparian Robinia pseudoacacia forests after clear-cutting were investigated through dendroecological and microsatellite polymorphism analyses. Age determination of regenerated R. pseudoacacia trees based on the dendroecological analysis revealed that forests regenerating after clear-cutting were composed of trees that mostly established within a few years after clear-cutting. This suggests that the stimulus to form new shoots was evoked by clear-cutting but faded within a few years. Genet identification via the microsatellite polymorphism analysis showed that ramet trees belonging to the same genet were distributed in a cluster. Almost all trees regenerated asexually through new ramet formation on the cut stumps and residual horizontal roots after clear-cutting. AMOVA with microsatellite markers showed that among- and within-population variations contributed 6 and 94% to the total variation, respectively, suggesting that R. pseudoacacia trees in the forests were initially established from seeds dispersed randomly from mother trees in a wide area.     

Organizer-like reticular stromal cell layer common to adult secondary lymphoid organs

Mesenchymal stromal cells are crucial components of secondary lymphoid organs (SLOs). Organogenesis of SLOs involves specialized stromal cells, designated lymphoid tissue organizer (LTo) in the embryonic anlagen; in the adult, several distinct stromal lineages construct elaborate tissue architecture and regulate lymphocyte compartmentalization. The relationship between the LTo and adult stromal cells, however, remains unclear, as does the precise number of stromal cell types that constitute mature SLOs are unclear. From mouse lymph nodes, we established a VCAM-1(+)ICAM-1(+)MAdCAM-1(+) reticular cell line that can produce CXCL13 upon LTbetaR stimulation and support primary B cell adhesion and migration in vitro. A similar stromal population sharing many characteristics with the LTo, designated marginal reticular cells (MRCs), was found in the outer follicular region immediately underneath the subcapsular sinus of lymph nodes. Moreover, MRCs were commonly observed at particular sites in various SLOs even in Rag2(-/-) mice, but were not found in ectopic lymphoid tissues, suggesting that MRCs are a developmentally determined element. These findings lead to a comprehensive view of the stromal composition and architecture of SLOs.     

Muscle-specific RING finger-1 interacts with titin to regulate sarcomeric M-line and thick filament structure and may have nuclear functions via its interaction with glucocorticoid modulatory element binding protein-1

Signaling from receptor tyrosine kinases (RTKs)* requires the sequential activation of the small GTPases Ras and Rac. Son of sevenless (Sos-1), a bifunctional guanine nucleotide exchange factor (GEF), activates Ras in vivo and displays Rac-GEF activity in vitro, when engaged in a tricomplex with Eps8 and E3b1-Abi-1, a RTK substrate and an adaptor protein, respectively. A mechanistic understanding of how Sos-1 coordinates Ras and Rac activity is, however, still missing. Here, we demonstrate that (a) Sos-1, E3b1, and Eps8 assemble into a tricomplex in vivo under physiological conditions; (b) Grb2 and E3b1 bind through their SH3 domains to the same binding site on Sos-1, thus determining the formation of either a Sos-1-Grb2 (S/G) or a Sos-1-E3b1-Eps8 (S/E/E8) complex, endowed with Ras- and Rac-specific GEF activities, respectively; (c) the Sos-1-Grb2 complex is disrupted upon RTKs activation, whereas the S/E/E8 complex is not; and (d) in keeping with the previous result, the activation of Ras by growth factors is short-lived, whereas the activation of Rac is sustained. Thus, the involvement of Sos-1 at two distinct and differentially regulated steps of the signaling cascade allows for coordinated activation of Ras and Rac and different duration of their signaling within the cell.     

Synip phosphorylation is required for insulin-stimulated Glut4 translocation

Previously we identified an unusual potential dual Akt/protein kinase B consensus phosphorylation motif in the protein Synip (RxKxRS(97)xS(99)) with serine 99 as a unique Akt2, but not Akt1 or for Akt3, substrate phosphorylation site. Although we have previously reported that serine 99 to phenylalanine (S99F-Synip) resulted in a constitutive inhibition of insulin-stimulated Glut4 translocation, a recent report indicated that Synip serine 99 to alanine mutant (S99A-Synip) failed to inhibit insulin-stimulated Glut4 translocation [H. Sano, S. Kane, E. Sano, G.E. Lienhard, Synip phosphorylation does not regulate insulin-stimulated GLUT4 translocation, Biochem. Biophys. Res. Commun. 332 (2005) 880-884]. To address this apparent discrepancy, we have now examined the S99A-Synip mutant and find that this mutant behaves essentially identical to S99F-Synip in that overexpression inhibits insulin-stimulated Glut4 translocation and is incapable of undergoing insulin-stimulated Syntaxin4 dissociation. These data are consistent with Synip serine 99 phosphorylation required for insulin-stimulated Glut4 translocation.     

Chemical and genetic diversity of Ligularia latihastata and Ligularia villosa in Yunnan Province of China

The chemical constituents of the root extracts and the nucleotide sequences of the atpB-rbcL intergenic region of Ligularia latihastata and L. villosa, collected in northwestern Yunnan Province, were studied. In the twelve collected samples of L. latihastata, two major benzofurans, 5,6-dimethoxy-2-(1-methylethenyl)-1-benzofuran (1) and euparin (2) were detected as major components. The minor compound (2R*,3S*)-5-acetyl-2,3-dihydro-6-hydroxy-2-(1-methylethenyl)-1-benzofuran-3-yl (2Z)-2-[(acetoxy)methyl]but-2-enoate (4) was found to be susceptible to artifact formation upon extraction with EtOH. The intra-specific diversity in chemical composition of the samples was small, but the diversity in the atpB-rbcL sequence was fairly large. Compounds 1 and 2 were also found in the three collected samples of L. villosa, indicating that the two species are chemically close to each other, in agreement with morphological taxonomy.