Tumor-infiltrating lymphocytes (TIL) were tested for cytotoxicity against autologous tumor cells in a study utilizing a chemically induced cancer ofthe bladder (transitional cell carcinoma), -47, in inbred ACI/N rats. From tumors grown after subcutaneous implantation of -47 in the rats TIL were separated by density gradient centrifugation and incubated in plastic dishes for separation of non-adherent from adherent cells. The non-adherent cells were further fractionated into T and B cells by the panning method using anti-rat (')₂ antibody. The cell fractions were each added to -47 in culture to be assessed for antitumor effect by the crystal violet dye exclusion method and 3H-thymidine incorporation inhibition assay. Peripheral blood mononuclear cells (PBMC) were also tested as described above. TIL expressed significantly higher cytotoxicity against -47 with the mean % cytotoxicity of 56.6±.6% and 87.±.1% at /T ratios of 10:1 and 20:1, respectively, as compared to PBMC(.±.0% at /T 10:1) (P<0.001). The adherent cells, B and T cell fractions showed respective % cytotoxicity of 92.4±2.%, 57.±10.6% and .±.% at an /T ratio of 10:1. TIL pretreated with IFN or rIL-2 for 24or 48 hours did not exhibit any noticeably enhanced antitumor activity at an /T ratio of :1. Prevention of direct contact of -47 cells and TIL by an interposed Millipore membrane (0.45μm) resulted in an unequivocal reduction of antitumor effect. This finding clearly indicates that the direct contact with -47cells and TIL is necessary for TIL to manifest their cytotoxicity against the autologous tumor cells, suggesting that TIL exhibit autologous tumor killing activity through cell-mediated cytotoxicity.

抄録全体を表示

Four new sterols, , 6α-epoxy-(, 24R)-ergosta-(14), -diene-3β, -diol (1), (, 24R)-ergosta-, -diene-3β, , 6β, -tetrol (2), (, 24R)-ergosta-, (11), -triene-3β, , 6β-triol (3) and 3β, , 6β-trihydroxy-(, 24R)-ergost--en--one (4), have been isolated from the fruit bodies of Grifola frondosa (FR.) S. . GRAY (Polyporaceae)together with fourteen known ones (-18), of which two ( and 6) are reported for the first time from a natural source. The structures of these compounds were elucidated on the basis of spectral data.

抄録全体を表示

A full account is given of the first chemical synthesis of the antitumor antibiotic guanine -oxide () and its -substituted derivatives (24a-k and 26). Coupling of appropriate primary amines (17a-, g-k) with phanecyl bromide (16) produced, after treatment with HCl, the corresponding N-substituted phenacylamine hydrochlorides (18a-, g-k). A similar phenacylation of 4-amino-1-butanol (21) failed to give the desired compound 18, so that 21 was heated with 2-bromomethyl-2-phenyl-1, 3-dioxolane (20) at 150-155°C for 3h to furnish, after treatment with HCl, the amino ketal hydrochloride in 40% yield. Deketalization of with hot 2N aqueous HCl afforded 18 in % yield. Condensations of the free bases, generated in situ from the hydrochlorides 18a-l and 1N aqueous NaOH, with the chloropyrimidinone 6 were effected in aqueous EtOH at the boiling point for 20 min or at 25-30°C for 3-24h, giving the 6-phenacylamino-4-pyrimidinones 19a-l in 54-90% yields. On treatment with 2N aqueous NaOH at room temperature for 10-60 min, the nitropyrimidinones 19a-k cyclized to provide the -substituted guanine -oxides 24a-k in 61-% yields. A similar alkali-treatment of 19l failed to yield guanine -oxide (). However, removal of the -(arylmethyl) group from 24i-k was effected with conc, H₂SO₄ at room temperature for 1-3h in the presence of toluene, producing the target N-oxide in 56-89% yields. In the in vitro bioassay of antileukemic activity against murine L5178Y cells, none of the -substituted guanine -oxides (24a-k and 26) was more effective than the parent, natural N-oxide . Within this series, however, the benzyl analogues 24g-k with or without alkoxy functions were more cytotoxic, with IC₅₀'s of 13.0-48.0μg/ml, than the alkyl analogues 24a-.

抄録全体を表示

We screened the differentiation-inducing activities of 39 mushroom extracts from Akita prefecture, Japan, on the mouse osteoblastic cell line, MC3T3-1. Sixteen phosphate buffered saline (PBS), boiled PBS, 14 ethanol and 12 methanol extracts induced alkaline phosphatase (ALP) activities, an indicator of MC3T3-1 cell differentiation. The enzyme activities were markedly induced by extracts of Tricholoma auratum, and we isolated the active compound from methanol extracts of this mushroom. Physical data for the isolated active compound were identical to those for (,24R)-ergosta-,-diene-3β,,6β-triol (1). 1 induced ALP activities of MC3T3-1 cells and promoted cell proliferation. To investigate the relationships between the chemical structure and differentiation-inducing activity of the compound, ALP-inducing activities of MC3T3-1 cells by 1, ergosterol (2), ergocalciferol (3), cholesta-3β,,6β-triol (4), -dehydrocholesterol () and cholecalciferol (6) were tested. The enzyme activities of MC3T3-1 cells were increased 3.0-fold by 10 μM 1 and 2.4-fold by 10 μM 4. However, 2, 3, and 6 did not induce MC3T3-1 cell ALP activity at 0.1—10 μM. These results suggested that the OH groups at C- and/or C-6 of 1 and 4 played an important role in their differentiation-inducing activities on MC3T3-1 cells. Furthermore, 1 suppressed induction of MC3T3-1 cell apoptosis by serum starvation.

抄録全体を表示

Six new cembranoids sarcophytol P (3), R (4), S (), K (), (11), and T (13) were isolated from the soft coral Sarcophyton glaucum. Sarcophytol P (3) was shown to be the 20-hydroxy derivative of the major component sarcophytol A (1), and afforded the cyclization product 6 in CHCl_3 at room temperature, in a same way as in 1. Sarcophytols R (4) and S () were correlated to 1, by conversion of its R,R and S,S epoxide derivatives. Sarcophytol K() was a 13, 14-dihydroxycembranoid having a 1,3Z-diene moiety. The absolute configuration of and its 1Z,3- and 1Z,3Z-isomers sarcophytol B (2) and J (10) were determined by circular dichroism study of their bis-p-dimethylaminobenzoate derivatives. Sarcophytols (11) and T (13) were 1- and 1,3Z-isomers of 1. Compound 11 showed characteristic broadening of the ^1H-NMR chemical shifts, due to the restricted conformational interconversion. Using the three cembranoids sarcophytols (11, 1,3), N (15, 1Z,3Z) and T (13,1,3Z), spontaneous autoxidation-cyclization, in CHCl_3,was examined, in order to compare the stereochemical course of the reaction with that of 1 (1Z,3), which affords trans-fused bicyclo[.3.0]tetradecene systems. The 1,3Z-isomer 13 gave the same cyclization product 18, as in the reaction of 1, even though it is isomeric at C-1,3. The 1Z,3Z isomer 15 gave 19, in consequence of the reversed geometry at C-3 of 15. The 1,3-isomer (11) gave the bicyclic product , having an antipodal fusion as compared with 19. This was confirmed by PCC oxidation of 19 and , giving enantiomeric ketones 23 and 24 respectively. The 1(14)-epoxide 26 was shown to be the immediate precursor of , and acounted for the inversion of the geometry at C-1 of the cyclization product. Compound 26 is isomeric with the epoxide 17 derived from 1. The epoxide 17 is the postulated precursor in the conversion of 13 to 18. It is noteworthy that the mode of antipodal fusion of the cyclopentane rings, in 18 and , was controlled by the chirality of the epoxy rings. The C-14 hydroxyl group participates in the transannular cyclization, but was found not to be the requisite functional group for the reaction. Similar treatment of cembrene C (30), the parent hydrocarbon of 1, also reacted in CHCl_3 giving the bicyclic product 32.

抄録全体を表示

A full account is given of the chemical behavior observed for , -dialkyladeninium salts (16). On treatment with boiling 1N aqueous NaOH for 60min, 16a, b, d, (X=I), 16c (X=Br), and 16 (X=ClO₄) rearranged to isomeric N⁶, -dialkyladenines (21a-) in 50-91% yields. Treatment of the salts with 0.N aqueous Na₂CO₃ at room temperature for 30-90min or with Amberlite CG-400 (OH^-) in H₂O at room temperature gave the ring-opened derivatives a- (in the trans-formamide form) in 56-83% yields, and rate constants for the ring-opening reactions of 16a, b, d-g (X=ClO₄) and 16c (X=Br) leading to a-g were determined in H₂O at pH . and ionic strength 0.50 at 25°C. Cyclization of a with NaH in AcNMe₂ at room temperature or with boiling 1N aqueous NaOH produced 21a in % or 72% yield, respectively.In solution, the trans-formamides seemed to transform slowly into the cis-formamides 23, attaining equilibria. The existence of such an equilibrium in D₂O or Me₂SO-d₆ at 25°C or in H₂O at pH . and ionic strength 0.50 at 25°C was kinetically confirmed in the case of a, and the mechanism of the rearrangement of 16 to 21 through is discussed on the basis of the above kinetic results and Deslongchamps' theory of stereoelectronic control. On treatment with NaBH₄ in MeOH at room temperature, 16a (X=I) furnished the , -dihydro derivative 28 (% yield), which slowly decomposed in H₂O at 60°C to give a in 49% yield.The , -dialkyladeninium salts (16) were found to be obtainable from N'-alkoxy-1-alkyl--formamidoimidazole-4-carboxamidines () through an alternative synthetic route : Alkylations of with alkyl halides in HCONMe₂ in the absence of base, followed by hydrogenolysis of the N'-alkoxy group and cyclizatio (or vice versa) produced 16 in acceptable yields. In order to interpret the proton nuclear magnetic resonance spectrum of a, the 2-deuterated species 26 was also synthesized from 24 via 25 and 27.

抄録全体を表示

In the first part of the present work the interaction of glycophorin with dimyristoylphosphatidylcholine (DMPC) is studied by freeze fracture electron microscopy, densitometry, calorimetry, and 90° static light scattering. An exothermic lipid/protein interaction energy of W_P=190 kJ•mol^-1 was found by application of the well known Van Laar relation for the displacement of the freezing point and the Gibbs-Duhem relationship. Secondly, the effects of Ca²⁺ on the lipid/protein interaction were studied. Following Ca²⁺ addition a remarkable decoupling of the interaction of the glycophorin head group with the bilayer surface was revealed by densitometry and gold-labeling electron microscopy. It is estimated that about 80% of lipid once disturbed by the adsorption of glycophorin head groups is decoupled after addition of Ca²⁺. Thirdly, the selective interaction of glycophorin with binary lipid mixtures was studied, including the mixtures of DMPC with dimyristoylphosphatidylserine (DMPS) and dilauroylphosphatidylcholine (DLPC), and the mixture of dipalmitoylphosphatidylcholine (DPPC) with DLPC.

抄録全体を表示

Eight new sterols, , -epidioxy-(, 24R)-23-methylergosta-6, -dien-3β-ol (1), 3β, , -trihydroxy-(, 24R)-23-methylergosta-, -dien-6-one (2), 3β, , -trihydroxy-(24S)-ergost--en-6-one (3), 3β, , , 14α-tetrahydroxy-(, 24R)-ergosta-, -dien-6-one (4), (, 24R)-ergosta-, -diene-3β, , 6α, -tetrol (), , -epidioxy-3β-hydroxy-(, 24R)-ergosta-, -dien-6-one (6), , -epidioxy-3β-hydroxy-(24S)-ergost--en-6-one () and , 6α-epoxy-(, 24R)-ergosta-, -diene-3β, , 14α-triol (), have been isolated from five edible mushrooms, Lentinus edodes, Flammulina velutipes, Hypsizigus marmoreus, Pleurotus ostreatus and Pholiota nameko together with fifteen known ones (-23), of which two (16 and 17) are reported for the first time from a fungal source. The structures of these new compounds were elucidated on the basis of their spectral data.

抄録全体を表示

抄録全体を表示

The effect of dietary vitamin on ,12-dimethylbenz [α] an thracene-induced mammary carcinogenesis in rats and on the lipid peroxidation due to DMBA was investigated. Female Sprague-Dawley rats were given iv injections of 2 mg of ,12-DMBA at both 56 and 62 days of age. Feeding of control diet (included 20 mg of vitamin per kg) or diet included 235 mg of vitamin per kg (YES-group) was initiated at 2 week before the first carcinogen injection and was terminated at 120 days postcarcinogen. Control rats had 97% incidence of mammary carcinoma, whereas there was 71% incidence of cancer in YES-groups (P>0.05). Similarly, the average number of cancers per rat was significantly reduced in YES-groups (.2 versus 3.; P<0.01), and latency of cancer appearance was prolonged in comparison to con trols. Serum lipoperoxide level were significantly decreased in YES-groups compared to controls (.88versus 10.26; P<0.05). Serum vitamin leveles were significantly elevated in YES-groups in comparison to controls (1. versus 0.; P<0.001). In the groups, in which were switched from control diet to vitamin supplemented diet and vice versa at the time of days of age, there were a % and 89% incidence of mammary cancer and average number of cancer per rat were 6. and 4. respectively.
The effects of vitamin on lipid peroxidation due to DMBA were studied. It was found that malonaldehyde (MDA), one of the most important products of lipid peroxidation, increased suddenly in the liver, mammary fat pad and serum with the treatment of DMBA. But, excess increase of MDA was significantly suppressed in the rats fed on vitamin diet. Furthermore, temporal depression of serum vitamin was induced by the administration of DMBA in both the control group and high vitamin group, being significantly (P<0.02) marked in a high vitamin group.
It was co ncluded that inhibitory effects of vitamin on DMBA-induced mammary carcinogenesis might be caused by the inhibition of lipid peroxidation with vitamin , and lipid peroxidation might play an important role in DMBA-induced mammary tumorigenesis in rats.

抄録全体を表示

Six new sterols, , 6α;, -diepoxy-(, 24R)-ergost--ene-3β, -diol (1), , 6α;, -diepoxy-(, 24R)-ergost--ene-3β, -diol (2), , 6α-epoxy-(, 24R)-ergosta-, -diene-3β, -diol (3), (, 24R)-23-methylergosta-, -diene-3β, , 6β-triol (4), (, 24R)-23-methulergosta-, -diene-3β, , 6β, -tetrol () and (24S)-ergost--ene-3β, , 6β, -tetrol (6), have been isolated from seven mushrooms, Amanita pantherina, Amantia virgineoides, Lactarius piperatus, Lyophyllum shimeji, Tricholoma portentosum, Hypsizigus marmoreus and Lentinula edodes together with eighteen known ones (-24). The structures of these new compounds were elucidated on the basis of their spectral data.

抄録全体を表示