Arrest states in a set of mutants of rat 3Y1 cells temperature-sensitive for entering S phase

Four temperature-sensitive (ts) mutants of rat 3Y1 cells (3Y1tsD123, 3Y1tsF121, 3Y1tsG125, and 3Y1tsH203) are arrested at 39.8°C mainly with a 2N DNA content (temperature-arrested cells). The states of these cells were compared with findings in case of cells arrested at 33.8°C at saturation density (density-arrested cells), with regard to the ability to enter S phase after release from arrest or after serum stimulation at 39.8°C. With the 3Y1tsD123, the ts defect is an event which seems essential for the initiation of S phase and occurs after mitosis but not after release from the density arrest. The defect in 3Y1tsF121 related to the efficiency of utilization of serum component(s). In case of 3Y1tsG125, the state of temperature arrest appeared to locate between the state of density arrest and the beginning of S phase. There was no significant difference between the density- and the temperature-arrested cells, in case of 3Y1tsH203.     

Construction and Systematical Symmetric Studies of a Series of Supramolecular Clusters with Binary or Ternary Ammonium Triphenylacetates

Functions of clusters in nano or sub-nano scale significantly depend on not only kinds of their components but also arrangements, or symmetry, of their components. Therefore, the arrangements in the clusters have been precisely characterized, especially for metal complexes. Contrary to this, characterizations of molecular arrangements in supramolecular clusters composed of organic molecules are limited to a few cases. This is because construction of the supramolecular clusters, especially obtaining a series of the supramolecular clusters, is difficult due to low stability of non-covalent bonds compare to covalent bonds. From this viewpoint, utilization of organic salts is one of the most useful strategies. A series of the supramolecules could be constructed by combinations of a specific organic molecule with various counter ions. Especially, primary ammonium carboxylates are suitable as typical examples of supramolecules because various kinds of carboxylic acids and primary amines are commercially available, and it is easy to change their combinations. Previously, it was demonstrated that primary ammonium triphenylacetates using various kinds of primary amines specifically construct supramolecular clusters, which are composed of four ammoniums and four triphenylacetates assembled by charge-assisted hydrogen bonds, in crystals obtained from non-polar solvents. This study demonstrates an application of the specific construction of the supramolecular clusters as a strategy to conduct systematical symmetric study for clarification of correlations between molecular arrangements in supramolecules and kinds and numbers of their components. In the same way with binary salts composed of triphenylacetates and one kind of primary ammoniums, ternary organic salts composed of triphenylacetates and two kinds of ammoniums construct the supramolecular clusters, affording a series of the supramolecular clusters with various kinds and numbers of the components.     

Vertical distribution in and isolation of bacteria from Lake Vanda: an Antarctic lake

Vertical distribution of bacteria in Lake Vanda, an Antarctic meromictic lake, was examined by the acridine orange epifluorescence direct count method. Total bacteria were 10⁴–10⁵ cells · ml^–1 in the water at 55 m depth and above, and increased drastically to 10⁷ cells · ml^–1 in the bottom water. Filamentous or long rodshaped bacteria occurred at a high frequency in the upper layers, but in the bottom layers most bacteria were coccoidal or short rods. Mean bacterial cell volume in water of between 10 m and 60 m deep was fairly large compared with common bacterial populations in seawater and lake water. Aerobic heterotrophic bacteria were recovered from the water of a depth of 30 m and above, and were assumed to belong to Caulobacter. Viable heterotrophic bacteria were not recovered from the high salinity deep water by media prepared with the same deep water. Phototrophic purple non-sulphur bacteria were isolated by enrichment cultures from water at 55 m depth.     

Characterization of B16 melanoma-specific cytotoxic T lymphocytes

A B16 melanoma-specific CD8⁺ T cell line (AB1) was established from the spleen cells of C57BL/6 mice cured of B16 melanoma with interleukin (IL)-12 treatment. The AB1 line exclusively used T cell receptor V_β11. The AB1 cells exhibited a cytolytic activity against both syngeneic B16 melanoma and allogeneic P815 mastocytoma, whereas a cold inhibition assay revealed specificity of the AB1 cells against B16 melanoma. Their lostability to kill a class I loss variant of B16 melanoma was restored by the transfection of H-2K^b gene. In addition, their interferon (IFN)-γ production was significantly suppressed by the addition of anti-H-2K^b monoclonal antibody, and RT-PCR analysis showed that the AB1 line expressed the mRNA encoding IFN-γ, but not IL-4 or IL-10. The experiment using synthetic peptides of tyrosinase-related protein-2 (TRP-2) revealed that the AB1 cells could recognize TRP-2_181–188 peptide. Moreover, the AB1 cells showed an in vivo antitumor effect against established pulmonary metastases of B16 melanoma. Overall, these results indicate that the Tc1-type V_β11 ⁺ AB1 cells exert an antitumor activity against syngeneic B16 melanoma through recognition of TRP-2_181–188 peptide in an H-2K^b-restricted manner. Received: 4 June 1998 / Accepted: 21 July 1998     

Role of Macrophages in Acute Murine Cytomegalovirus Infection

It has been recognized that macrophages play an important role in controlling virus infection in experimental animal models. To evaluate the role of macrophages in acute murine cytomegalovirus infection, macrophages in the spleen and the liver were eliminated by an intravenous injection of liposomes containing a cytolytic agent, dichloromethylene diphosphonate. The depletion of macrophages led to a significant increase of virus titer in the spleen and lungs in both susceptible BALB/c and resistant C57BL/6 mice during the first three days after intravenous infection. In the spleen, the increase of virus titer in macrophage-depleted BALB/c mice was much greater than that in NK cell-depleted mice. These results suggest that macrophages contribute to protection mainly by the mechanisms which are independent of NK cells during the first three days after infection. The increase of virus titer in macrophage-depleted C57BL/6 mice was as great as that in NK cell-depleted mice because of the high contribution of NK cells to protection in C57BL/6 mice. In the liver in both strains of mice, the effects of macrophage depletion on virus titer were not as much as those in the spleen and lungs. Furthermore, the local depletion of peritoneal macrophages resulted in a great increase of virus titer in the spleen at three days after intraperitoneal infection. We conclude that macrophages greatly contribute to decreasing the virus load in some organs possibly through either or both intrinsic and extrinsic mechanisms in the early phase of primary infection with murine cytomegalovirus.     

Induction of Interleukin 2-Responsiveness in Thymocytes of the Transgenic Mice Carrying lck-Transgene

The role of lck gene in T cell proliferation and differentiation was investigated with transgenic mice carrying human lck cDNA whose expression was regulated by the promoter of mouse H-2K^b and the enhancer element of mouse IgH. RNase protection assay revealed that the lck transgene was expressed in the thymus and spleen, whereas immunoblot analysis demonstrated that amounts of p56^lck in freshly isolated lymphoid organs were almost equal between transgenic mice and negative littermates. Cell-surface marker analyses of the thymocytes and peripheral lymphocytes revealed no remarkable difference between both groups. Notable finding is that the thymocytes from transgenic mice showed a significant proliferative response to the stimulation with IL-2, but not the thymocytes from negative littermates. Further analysis revealed that CD4⁺8^– single positive thymocytes proliferated in response to IL-2. While surface expression levels of IL-2Rα and IL-2Rβ of these CD4⁺8^– thymocytes from transgenic and control mice were almost equal before stimulation with IL-2, the expression of IL-2Rβ was induced only in transgenic thymocytes after stimulation with IL-2. Immunoblot analysis demonstrated that the expression of p56^lck of transgenic thymocytes was not down-reguated at 4 hr after stimulaion with IL-2, whereas p56^lck of control ones were not detectable any more at 4 hr after stimulation with IL-2. Moreover, in vitro kinase assay substantiated such unchanged expression of p56^lck in the thymocytes from transgenic mice: the kinase activities of p56^lck did not decrease in thymocytes from transgenic mice after stimulation with IL-2, while kinase activities of control ones were significantly down-regulated by stimulation of IL-2. These results suggested that a significant proliferative response found in the thymocytes from lck-transgenic mice after the stimulation with IL-2 was caused by a constitutive expression of p56^lck in these thymocytes even after the stimulation. Our findings, therefore, support a possibility that p56^lck may play a role in the IL-2R-mediated signaling system in CD4⁺8^– thymocytes.     

Studies of serum-free medium for the generation of lymphokine-activated killer cells

We examined a serum-free medium (designated as TYI 101) for the generation of lymphokine-activated killer (LAK) cells from human lymphocytes, regional lymph node lymphocytes (RLNL) and peripheral blood lymphocytes (PBL). TYI 101 medium consisted of, in addition to nutrient mixture, transferrin, insulin, fetuin, sodium selenite, 2-mercaptoethanol, o-phosphorylethanolamine, chick egg yolk and porcine kidney extract. These hormones were effective for supporting RLNL proliferation as assessed by (³H)-thymidine uptake. When human lymphocytes from two different sources were cultivated with recombinant interleukin 2 (rIL-2) in TYI 101 medium, LAK activity was generated. In cultures of PBL from a healthy donor, LAK cells were generated in TYI 101 medium as efficiently as in RPMI 1640 medium supplemented with 10% human AB-type serum (RPMI-AB). In cultures of RLNL from lung cancer patients, LAK activity obtained in TYI 101 medium was about sixty-five percent of that in RPMI-AB. However, the addition of a small amount of AB-type serum improved the generation of LAK activity, LAK cell expansion, and cell viability in TYI 101 medium. We conclude that TYI 101 medium can be used for the generation of LAK cells from human lymph node lymphocytes with supplementation of none or only a reduced amount of human serum.Abbreviations IMDM Iscove's Modification of Dulbecco's Medium - rIL-2 recombinant Interleukin - LAK Lymphokine-Activated Killer - RLNL Regional Lymph Node Lymphocytes - PBL Pheripheral Blood Lymphocytes - PBS Phosphate-Buffered Saline - RBC Red Blood Cells - RPMI-AB RPMI 1640 medium supplemented with 10% human AB-type serum Address for offprints: Tsukuba Research Laboratory, Japan Synthetic Rubber Co., Ltd., 25 Miyukigaoka, Tsukuba-shi, Ibaraki, 305 Japan     

A regimen of surgical adjuvant immunotherapy for cancer with interleukin 2 and lymphokine-activated killer cells

We designed a unique regimen of adoptive immunotherapy with lymphokine-activated killer (LAK) cells and recombinant interleukin 2 (rIL-2) for application with surgical adjuvant therapy of cancer. The regimen features the prolonged (6 consecutive days) s.c. administration of low-dose rIL-2 and the transfer ofex vivo generated LAK cells from regional lymph node lymphocytes, obtained at the time of surgical operation. According to this regimen, 5 patients with primary lung cancer received immunotherapy about 2 weeks after surgery (pulmonary lobectomy). Clinical toxicities included fever(5/5), fatigue(5/5), slight(< 5%) weight gain(5/5), increase of pleural effusion at the lobectomy site(2/5), and edema formation(1/5). All toxicities reversed within 4 days after the completion of therapy. Rebound lymphocytosis after therapy ranged from 2.4 to 5.5-fold (mean, 4.3-fold) over the baseline. Peripheral blood lymphocytes obtained during this lymphocytosis exhibitedin vitro LAK activity in 4 of 5 patients. Thus, the regimen is considered to be well-tolerable and immunologically active in regard to the postoperative state of the patients.     

Vertical distribution of organic constituents in an Antarctic lake: Lake Vanda

Vertical distribution of organic constituents, i.e. total organic carbon (TOC), extractable organic carbon with ethyl acetate (EOC), hydrocarbons, phytol, sterols, fatty acids and phenolic acids in Lake Vanda was studied to elucidate their features in relation to the stratification of lake water and the distribution of lake organisms. The concentrations of TOC, EOC and sterols increased with depth and attained the maximum values of 25 and 1.5 mgC l^–1 and 1.4 g l^–1 in the bottom, respectively, while those of fatty acids showed the maximum value of 61 g l^–1 at a depth of 55.4 m, along with the highest value of the ratio of unsaturated (UC₁₆, uC₁₈) to saturated (C₁₆, C₁₈) acids (8.5) and with the highest carbon preference index (35). Hydrocarbons were only found in the bottom layers (60.4 and 65.9 m) and bottom sediment. These results suggest strongly that the vertical distribution of lake organisms and their activity are quite different due to depth. In the bottom warm anoxic layers the degradation of organic materials must have occurred significantly and thus refractory organic materials should be concentrated.