Facilitation of NMDAR-independent LTP and spatial learning in mutant mice lacking ryanodine receptor type 3

To evaluate the role in synaptic plasticity of ryanodine receptor type 3 (RyR3), which is normally enriched in hippocampal area CA1, we generated RyR3-deficient mice. Mutant mice exhibited facilitated CA1 long-term potentiation (LTP) induced by short tetanus (100 Hz, 100 ms) stimulation. Unlike LTP in wild-type mice, this LTP was not blocked bythe NMDA receptor antagonist D-AP5 but was partially dependent on L-type voltage-dependent Ca2+ channels (VDCCs) and metabotropic glutamate receptors (mGluRs). Long-term depression (LTD) was not induced in RyR3-deficient mice. RyR3-deficient mice also exhibited improved spatial learning on a Morris water maze task. These results suggest that in wild-type mice, in contrast to the excitatory role of Ca2+ influx, RyR3-mediated intracellular Ca2+ ([Ca2+]i) release from endoplasmic reticulum (ER) may inhibit hippocampal LTP and spatial learning.     

Immunohistochemical localization of minor gangliosides in the rat central nervous system

We previously described the differential distribution of majorgangliosides (GM1, GD1a, GD1b, GT1b and GQ1b) in adult rat braindetected by specific antibodies (Kotani,M., Kawashima,I., Ozawa,I.,Terashima,T. and Tai,T. Glycobiology, 3, 137–146, 1993).We report here the distribution of minor gangliosides in theadult rat brain by an immunofluorescence technique with mousemonoclonal antibodies (MAbs). Ten MAbs (GMR6, GMB28, GMR11,GMR19, GMR2, GMR7, GGR51, AMR10, NGR54 and NGR53) that specificallyrecognize GM3, GM2, GT1a, GD3, O-Acdisialoganglioside, GD2,GM1b, GM4, IV³NeuAc     

Raman/absorption simultaneous measurements for cytochrome oxidase compound A at room temperature with a novel flow apparatus

A novel flow apparatus for continuously producing reaction intermediates of cytochrome oxidase was constructed and applied successfully to observe the transient absorption and resonance Raman spectra in its reaction with oxygen. Time-resolved difference absorption spectra in 500-650-nm region clearly indicated the formation of compound A upon photolysis of the fully reduced CO-bound form at 5 degrees C, and at this stage electrons were not transferred from cytochrome c to cytochrome oxidase. However, at the stage of formation of compound B, cytochrome c was oxidized. Resonance Raman spectra of these intermediates measured simultaneously with the absorption spectra are also reported.     

Intrapleural application of recombinant interleukin-2 in patients with malignant pleurisy due to lung cancer

Forty-three patients with malignant pleurisy due to lung cancer were entered into the trial to evaluate clinical efficacy of intrapleural instillation of recombinant interleukin-2 (RIL-2). Among 35 evaluable patients, serial cytological examinations of pleural effusion following the start of the treatment revealed disappearance of malignant cells in 26 (74%). Malignant cells were detected again in 7 of the 26, however, cytology remained negative in the other 19 patients for longer than 4 weeks. Pleural effusion disappeared roentogenographically in 13 of 35 evaluable patients. Additional 8 patients demonstrated marked decrease of pleural effusion. Complete response (CR) which means disappearance of both malignant cells and pleural effusion for longer than 4 weeks was obtained in 13 of the 35 patients (37%). No serious side effects were experienced in this trial. These results indicate that intrapleural RIL-2 is one of candidates to control intractable malignant pleurisy due to lung cancer.     

A beta-Galactosidase from Radish (Raphanus sativus L.) Seeds

A basic β-galactosidase (β-Galase) has been purified 281-fold from imbibed radish (Raphanus sativus L.) seeds by conventional purification procedures. The purified enzyme is an electrophoretically homogeneous protein consisting of a single polypeptide with an apparent molecular mass of 45 kilodaltons and pl values of 8.6 to 8.8. The enzyme was maximally active at pH 4.0 on p-nitrophenyl β-d-galactoside and β-1,3-linked galactobiose. The enzyme activity was inhibited strongly by Hg²⁺ and 4-chloromercuribenzoate. d-Galactono-(1→4)-lactone and d-galactal acted as potent competitive inhibitors. Using galactooligosaccharides differing in the types of linkage as the substrates, it was demonstrated that radish seed β-Galase specifically split off β-1,3- and β-1,6-linked d-galactosyl residues from the nonreducing ends, and their rates of hydrolysis increased with increasing chain lengths. Radish seed and leaf arabino-3,6-galactan-proteins were resistant to the β-galase alone but could be partially degraded by the enzyme after the treatment with a fungal α-l-arabinofuranosidase leaving some oligosaccharides consisting of d-galactose, uronic acid, l-arabinose, and other minor sugar components besides d-galactose as the main product.     

Induction of tumoricidal macrophages from bone marrow cells of normal mice or mice bearing a colony-stimulating-factor-producing tumor

Summary Nonadherent cells of the bone marrow of C3H/HeN mice were incubated for 3 days with the culture supernatant of an L-929 cell line containing macrophage-colony-stimulating factor. Approximately, 70% of the cells became phagocytic, adherent to plastic dishes and positive for nonspecific esterase staining. The adherent cells exhibited a weak tumoricidal activity against MM48 syngeneic mammary carcinoma cells, and the cytotoxicity was strongly augmented by the addition of bacterial lipopolysaccharide to the cytotoxicity assay. The cytotoxicity induced by lipopolysaccharide was also shown to be mediated by Thy1.2^– and asialo-GM1⁺ cells, and was abrogated by the addition of carrageenan. Macrophage-colony-stimulating-factor-producing (D66) and nonproducing (A23) variants were separated from the MM48 tumor line in in vitro culture following limiting dilution. There was no difference between these two variants in either the in vitro growth rate or the susceptibility to macrophage-mediated cytotoxicity. C3H/HeN mice inoculated i.p. with D66 survived longer than did those inoculated i.p. with A23. C3H/HeN mice bearing D66 or A23 as an ascitic form were given i.p. injections of Nocardia rubra cell wall skeleton (N-CWS). N-CWS significantly prolonged the survival period of mice bearing D66, whereas it exhibited no apparent antitumor effect on mice bearing A23. The increase in the cell number of D66 in the peritoneal cavity was significantly retarded, compared with that of A23. In contrast, the number of peritoneal macrophages increased more in D66-bearing mice than in A23-bearing mice. The increase in the peritoneal macrophage number was further augmented by an i.p. injection of N-CWS. Peritoneal macrophages of D66-bearing mice exhibited apparent tumoricidal activity against MM48 tumor cells in the presence of lipopolysaccharide, and the cytotoxicity was significantly augmented by i.p. injection of N-CWS. On the other hand, the responsiveness of peritoneal macrophages to lipopolysaccharide was found to be poor in A23-bearing mice and the tumoricidal activity was only weakly augmented by N-CWS. These results strongly suggest that M-CSF plays an important role not only in the maturation of macrophage progenitors but also in the induction and the accumulation of activated macrophages. Abbreviations used: M-CSF, macrophage-colony-stimulating factor; NABMC, nonadherent bone marrow cells; CM, conditioned medium; NK, natural killer; N-CWS, Nocardia rubra cell-wall skeleton     

Synergistic induction of lymphokine (IL-2)-activated killer activity by IL-2 and the polysaccharide lentinan,and therapy of spontaneous pulmonary metastases

Summary Spleen cells of C57BL/6N mice bearing lung metastases were induced to the cytotoxic state by subcutaneous injection of recombinant human interleukin-2 (IL-2) at a minimum dose of 5×10⁴ U/mouse three times a day for 3 consecutive days. A single intraperitoneal injection of lentinan alone at concentrations of up to 10 mg/kg body weight did not render spleen cells cytotoxic to P-29 cells, but a combination of subthreshold doses of these agents (5×10⁴ U/ml IL-2 and 5 mg/kg lentinan) induced significant in vivo lymphokine-activated killer activity in spleen cells of tumor-bearing mice. Similarly, spleen cells from mice treated i.p. with lentinan became cytotoxic on in vitro treatment with IL-2. The in vitro responsiveness of spleen cells to IL-2 was maximal 3 days after i.p. injection of lentinan. Synergism between IL-2 and lentinan was also observed in mice bearing spontaneous lung micrometastases: neither IL-2 (<5×10⁴ U/mouse) nor lentinan (<2.5 mg/kg) alone had a therapeutic effect, but multiple injections of IL-2 with a single injection of lentinan resulted in significant inhibition of spontaneous pulmonary metastases. From these results we conclude that IL-2 and lentinan in combination are more effective than either one alone for inducing destruction of pulmonary metastases.     

Effector mechanism of human monocyte-mediated cytotoxicity: role of a new tumor cytotoxic factor distinct from interleukin 1 and tumor necrosis factorα

Human blood monocytes activated to the tumoricidal state were previously found to release a factor(s) responsible for tumor cell killing. The activity of the tumor cytotoxic factor(s) (TCF) was determined by release assay of radioactivity from human A375 melanoma cells. On fractionation of the supernatant of activated monocytes by Ultrogel AcA34 and TSK-G3000SW gel chromatographies two major peaks of the material with TCF activity with MWs of 30,000 and 15,000, called TCF-I and TCF-11, respectively were obtained. TCF-II could be neutralized by polyclonal anti-IL-1 antiserum, but anti-IL-1 antiserum did not neutralize either factor. TCF-I was separated by ampholine column electrofocusing into three major fractions with TCF activity at pI 5, 6 and 6.8, named TCF-1, TCF-1 and TCF-1, respectively. The cytotoxic and IL-1 activities of TCF-1 were neutralized by anti-IL-1 serum, whereas those of TCF-1 and TCF-1 were not completely neutralized by anti-IL-1 or anti-IL-1 antiserum. On DEAE ion-exchange chromatography (TSK DEAE 5PW) TCF-I gave two peaks with TCF activity (TCF-I1 and TCF-I2). TCF-I1 was slightly neutralized by anti-TNF antibody, but TCF-I2 was not affected by antisera against IL-1 and IL-1, or anti-TNF antibody, thus ruling out the possibility that tumor necrosis factor (TNF) might be involved in tumor cell killing mediated by TCF-I2. These results indicate that human monocyte-mediated cytotoxicity against human A375 melanoma cells is mediated in part by a tumor cytotoxic factor (TCF; MW, 30,000; pI 6), differing from IL-1 and TNF.     

Changes in allele‐specific association of histone modifications at the imprinting control regions during mouse preimplantation development

Allele‐specific association of histone modification is observed at the regulatory region of imprinted genes and has been suggested to work as an epigenetic marker for monoallelic gene expression, along with the allelic CpG methylation of DNA. Although the parent‐origin‐specific epigenetic status in imprinted genes is thought to be established during preimplantation development, little is known about the allelic specificity of histone modifications during this period because of the limited volume of material available for analysis. In this study, we first revealed the allelic enrichment of histone modifications and variant histones at the imprinting control regions (ICRs) of four‐cell to blastocyst stage preimplantation embryos by using carrier chromatin immunoprecipitation and sequence polymorphism analysis of immunoprecipitated DNA. We found relative enrichment of histone H3 lysine 9 dimethylation at the imprinted alleles of ICRs and obtained the results suggesting that histone modifications at ICRs are established during a late preimplantation stage. genesis, 47:611–616, 2009. © 2009 Wiley‐Liss, Inc.