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The green tea gallocatechins, (-)-epigallocatechin-3-O-gallate (EGCG) (IC(50) = 0.69 microM), (-)-gallocatechin-3-O-gallate (GCG) (IC(50) = 0.67 microM), (-)-epicatechin-3-O-gallate (ECG) (IC(50) = 1.3 microM), and theasinensin A (IC(50) = 0.13 microM), were found to be potent and selective inhibitors of rat squalene epoxidase (SE), a rate-limiting enzyme of cholesterol biogenesis. On the other hand, flavan-3-ols without galloyl group at C-3 did not show significant enzyme inhibition. It was demonstrated for the first time that the cholesterol lowering effect of green tea may be attributed to their potent SE inhibition activities. Inhibition kinetics revealed that EGCG inhibited SE in noncompetitive (K(I) = 0.74 microM), and non-time-dependent manner. The potent enzyme inhibition would be caused by specific binding to the enzyme, and by scavenging reactive oxygen species required for the monooxygenase reaction., (Copyright 2000 Academic Press.)

A novel putative SR protein, designated cisplatin resistance-associated overexpressed protein (CROP), has been cloned from cisplatin-resistant cell lines by differential display. The N-half of the deduced amino acid sequence of 432 amino acids of CROP contains cysteine/histidine motifs and leucine zipper-like repeats. The C-half consists mostly of charged and polar amino acids: arginine (58 residues or 25%), glutamate (36 residues or 16%), serine (35 residues or 15%), lysine (30 residues, 13%), and aspartate (20 residues or 9%). The C-half is extremely hydrophilic and comprises domains rich in lysine and glutamate residues, rich in alternating arginine and glutamate residues, and rich in arginine and serine residues. The arginine/serine-rich domain is dominated by a series of 8 amino acid imperfect repetitive motif (consensus sequence, Ser-Arg-Ser-Arg-Asp/Glu-Arg-Arg-Arg), which has been found in RNA splicing factors. The RNase protection assay and Western blotting analysis indicate that the expression of CROP is about 2-3-fold higher in mRNA and protein levels in cisplatin-resistant ACHN/CDDP cells than in host ACHN cells. CROP is the human homologue of yeast Luc7p, which is supposed to be involved in 5'-splice site recognition and is essential for vegetative growth.

Interleukin (IL) 12 has been shown to elicit tumor regression when this cytokine induces the migration of T cells to tumor sites. The present study investigates the role of a peritumoral stromal reaction in IL-12-induced T-cell migration. In the CSA1M and OV-HM tumor models, IL-12 treatment induced tumor regression that is associated with T-cell migration. Neither T-cell migration nor tumor regression was observed in the Meth A and MCH-1-A1 models. Stromal tissue containing neovascular blood vessels developed at the peritumoral area of the former two IL-12-responsive tumors but not at the peritumoral area of the latter two IL-12-unresponsive tumors. The significance of stroma development was investigated using a pair of tumor models (CSA1M and a subline derived from CSA1M designated the CSA1M variant), both of which exhibit the same tumor immunogenicity. In contrast to the parental CSA1M cell line, the variant cell line was not responsive to IL-12, and neither stroma development nor T-cell migration was observed, even after IL-12 treatment. Histological analyses revealed that the parental cell line had peritumoral stroma with intrastromal vessels but only a few vessels in tumor parenchyma, whereas the variant cell line showed no stroma but had abundant vasculature in the tumor parenchyma. Most importantly, only stromal vessels in the parental tumors expressed detectable and enhanced levels of vascular cell adhesion molecule 1 (VCAM-1)/ intercellular adhesion molecule 1 (ICAM-1) before and after IL-12 treatment, respectively. In contrast, parenchymal vasculature in the variant cell line failed to express VCAM-1/ICAM-1 even after IL-12 treatment. When transferred into recipient tumor-bearing mice, IL-12-stimulated T cells from the parental CSA1M-bearing or the variant CSA1M-bearing mice migrated into the parental but not into the variant tumor mass. Together with our previous finding that T-cell migration depends on the VCAM-1/ICAM-1 adhesive interactions, the present results indicate a critical role for peritumoral stroma/stromal vasculature in the acceptance of tumor-infiltrating T cells that is a prerequisite for IL-12-induced tumor regression.

Five new euglobals possessing the phloroglucinol-monoterpene structure, euglobals G8-G12, together with a known euglobal-IIc were isolated from the hexane fraction of the methanol extract of the leaves of Eucalyptus grandis. Euglobal-G8 is an adduct of formyl-isovaleroyl-phloroglucinol and gamma-terpinene whereas -G9, -G10 and -G11 have the same phloroglucinol moiety fused with alpha-terpinene, while Euglobal-G12 has terpinolene fused with the same phloroglucinol moiety. The structures of these compounds were elucidated on the basis of spectral evidences. Biomimetic synthesis of euglobals suggests that these compounds are derived biogenetically by the Diels-Alder type cycloaddition of the corresponding terpenes with an ortho-quinone methide generated from grandinol.

Unfractionated spleen cells taken from tumor-bearing mice contained tumor-primed T cells which produced lymphokines such as IFN-gamma and IL-2 through collaboration with antigen-presenting cells (APC) binding tumor antigens when cultured in vitro. Here, we investigated the regulatory mechanisms underlying IFN-gamma production by T-APC interactions. Elimination of B cells from a splenic population of tumor-bearing mice resulted in enhanced IFN-gamma production. Adding B cells back into cultures down-regulated IFN-gamma production to almost the same levels as those induced by unfractionated spleen cells. IL-2 production was not enhanced by B cell depletion, but rather was significantly suppressed. IFN-gamma-selective up-regulation was due to an enhancement of IL-12 production because IL-12 was detected in B cell-depleted cultures and enhanced IFN-gamma production was prevented by addition of anti-IL-12 mAb or anti-CD40 ligand (CD40L) mAb capable of inhibiting CD40L-induced IL-12 production. These results indicate that B cells interfere with IFN-gamma production induced through interactions between anti-tumor T cells and APC, and this suppressive effect is based on the capacity of CD40+ B cells to down-regulate the CD40L-induced IL-12 production by APC.

Administration of recombinant interleukin 12 (IL-12) induces tumor regression that is associated with T-cell infiltration in the OV-HM ovarian carcinoma and CSA1M fibrosarcoma models. After confirming the blocking of regression by injection of anti-IFN-gamma monoclonal antibody (mAb), we investigated the mechanisms underlying the requirement of IFN-gamma in T-cell migration and tumor regression. T-cell migration was inhibited by injection of anti-IFN-gamma mAb to OV-HM tumor-bearing mice prior to IL-12 treatment. We examined, using the lymphoid cell migration assay, whether IFN-gamma is required for enhancing the migratory capacity of T cells or the T cell-accepting potential of tumor masses during IL-12 treatment. Spleen cells from IL-12-treated or untreated OV-HM-bearing mice were stained in vitro with a fluorescein chemical and transferred i.v. into OV-HM-bearing mice that were not treated with IL-12. Migration of donor cells was quantitated by counting the number of fluorescent cells on cryostat sections of tumor masses from recipient mice. Compared to spleen cells from OV-HM-bearing mice that were not treated with IL-12, enhanced migration was observed for cells from IL-12-treated OV-HM-bearing mice. Anti-IFN-gamma pretreatment of donor mice before IL-12 treatment did not reduce the migratory capacity of T cells, whereas migration was markedly inhibited in recipient mice injected with anti-IFN-gamma. Anti-IFN-gamma pretreatment decreased vascular cell adhesion molecule-1 (VCAM-1)-/intercellular adhesion molecule-1 (ICAM-1)-positive blood vessels at tumor sites. Consistent with this, migration was also inhibited by treatment of recipient mice with either anti-VCAM-1 or anti-ICAM-1 mAb. In contrast to the OV-HM model, T-cell migration was not affected in the CSA1M model following preinjection of anti-IFN-gamma mAb. In this model, VCAM-1-/ICAM-1-positive blood vessels existed even after anti-IFN-gamma treatment, although tumor regression was completely inhibited. These results indicate that IFN-gamma plays two distinct roles in expressing the antitumor efficacy of IL-12: one is to support the T-cell acceptability of tumor masses, and the other is to mediate the antitumor effects of migrated T cells.

1. The biotransformation of 1-(3,4-dichlorobenzyl)-5-octylbiguanide (OPB-2045), a new potent biguanide antiseptic, was investigated in male beagle dogs. Urinary and faecal excretion of unchanged compound and metabolites were studied following a single subcutaneous injection of 14C-labeled compound at a dose of 1 mg/kg. 2. Four urinary metabolites were structually identified using synthetic standards and/or spectral data as 3,4-dichlorobenzoic acid, 6-[5-(3,4-dichlorobenzyl)-1-biguanidino] hexanoic acid (DM-210), 4-[5-(3,4-dichlorobenzl)-1-biguanidino] butanoic acid (DM-212) and 5-[5-(3,4-dichlorobenzyl)-1-biguanidino] pentanoic acid (DM-213). 3. The predominant radioactive substances in the excreta were DM-213 and DM-210 at 26.1% and 25.5%, respectively, of the dose. No unchanged compound was detected in the urine, and in the faeces it was only 2% of the dose.

Five new oleanane-type saponins, polygalasaponins XLII-XLVI, along with two known saponins were isolated from the roots of Polygala glomerata Lour. The structures of polygalasaponins XLII-XLVI were elucidated as 3-O-beta-D-glucopyranosyl presenegenin 28-O-beta-D-xylopyranosyl-(1-->4)-alpha-L-rhamnopyranosyl- (1-->2)-{4-O-[(E)-3,4-dimethoxycinnamoyl]}-beta-D-fucopyranosyl ester, 3-O-beta-D-glucopyranosyl presenegenin 28-O-beta-D-galactopyranosyl-(1-->4)-beta-D-xylopyranosyl-(1-->4)- alpha-L-rhamnopyranosyl-(1-->2)-[alpha-L-arabinopyranosyl- (1-->3)]-[4-O-(E)-p-methoxycinnamoyl]-beta-D-fucopyranosyl ester, 3-O-beta-D-glucopyranosyl presenegenin 28-O-beta-D-galactopyranosyl-(1-->4)-beta-D-xylopyranosyl-(1-->4)- alpha-L-rhamnopyranosyl-(1-->2)-[beta-D-glucopyranosyl- (1-->3)]-{4-O-[(E)-3,4-dimethoxycinnamoyl]}-beta-D-fucopyranosyl ester, 3-O-beta-D-glucopyranosyl presenegenin 28-O-beta-D-galactopyranosyl-(1-->4)-beta-D-xylopyranosyl-(1-->4)- alpha-L-rhamnopyranosyl-(1-->2)-[6-O- acetyl-beta-D-glucopyranosyl-(1-->3)]-{4-O- [(E)-3,4-dimethoxycinnamoyl]}-beta-D-fucopyranosyl ester, 3-O-beta-D- glucopyranosyl presenegenin 28-O-beta-D-galactopyranosyl-(1-->4)- beta-D-xylopyranosyl-(1-->4)-alpha-L-rhamnopyranosyl- (1-->2)-[6-O-acetyl-beta-D-glucopyranosyl-(1-->3)]-{4-O-[(Z)-3, 4-dimethoxycinnamoyl]}-beta-D-fucopyranosyl ester, respectively, on the basis of spectroscopic and chemical evidence.

Seven new sucrose and oligosaccharide esters, glomeratoses A-G, together with four known compounds, 3-O-[(E)-3,4,5-trimethoxycinnamoyl]-beta-D-fructofuranosyl-(2-->1) - (6-O-benzoyl)-alpha-D-glucopyranoside, 3-O-(E)-sinapoyl-beta-D-fructofuranosyl-(2-->1)-[6-O-(E)-sinapo yl]-alpha-D- glucopyranoside, tenuifoliside C and reiniose G were isolated from the roots of Polygala glomerata. Glomeratoses A-G were elucidated as 3-O-[(E)-3,4,5-trimethoxycinnamoyl]-beta-D-fructofuranosyl-(2-->1) -alpha-D- glucopyranoside, 3-O-(E)-sinapoyl-beta-D-fructofuranosyl-(2-->1)-[6-O-(E)-p- coumaroyl]-alpha-D-glucopyranoside, 3-O-[(E)-3,4,5-trimethoxycinnamoyl]-beta-D-fructofuranosyl-(2-->1) -[6-O-(E)- p-coumaroyl]-alpha-D-glucopyranoside, 3-O-[(E)-3,4,5-trimethoxycinnamoyl]-beta-D-fructofuranosyl-(2-->1) -[6-O-(E)- 3,4,5-trimethoxycinnamoyl]-alpha-D-glucopyranoside, 1-O-{6-O-[3-O-(E,E)-(beta, beta'-bis-sinapoyl)-beta-D-fructofuranosyl]}-alpha- D-glucopyranoside intramolecular ester, 1-O-(E)-p-coumaroyl-(3-O-benzoyl)-beta-D- fructofuranosyl-(2-->1)-[beta-D-glucopyranosyl-(1-->2)]-[6-O-acetyl-beta -D- glucopyranoysl-(1-->3)]-[4-O-(E)-feruloyl]-(6-D-acetyl)-alph a-D- glucopyranoside, 1-O-(E)-p-coumaroyl-(3-O-benzoyl)-beta-D-fructofuranosyl-(2-->1)-[ beta-D- glucopyranosyl-(1-->2)]-[6-O-acetyl-beta-D-glucopyranoside-(1-->3)]-{4-O - [4-O-beta-D-glucopyranosyl-(E)-feruloyl]}-[6-O-(E)-p-coumaroyl+ ++]-alpha- D-glucopyranosyl, respectively, on the basis of chemical and spectral evidence.

1. The metabolism of carteolol, a beta-adrenoceptor blocking drug, was investigated in male Sprague-Dawley rat liver microsomes. 2. The formation of 8-hydroxycarteolol was the principal metabolic pathway of carteolol in vitro and followed Michaelis-Menten kinetics with a K(m) = 11.0 +/- 5.4 microM and a Vmax = 1.58 +/- 0.64 nmol/min/nmol P450 respectively (mean +/- SD, n = 5). Eadie-Hofstee plot analysis of carteolol 8-hydroxylase activity confirmed single-enzyme Michaelis-Menten kinetics. 3. The cytochrome P450 isoforms involved in 8-hydroxylation of carteolol were investigated using selective chemical inhibitors and polyclonal anti-P450 antibodies. Quinine (Ki = 0.06 microM) and quinidine (Ki = 2.0 microM), selective inhibitors of CYP2D1, competitively inhibited 8-hydroxycarteolol formation. Furthermore, only anti-human CYP2D6 antibody inhibited this reaction. 4. These results suggest that carteolol is metabolized to 8-hydroxycarteolol by CYP2D1. The K(m) of carteolol for CYP2D1 in male rat liver microsomes was much greater than those of propranolol or bunitrolol, indicating that carteolol has a lower affinity for CYP2D1 compared with these other beta-adrenoceptor blocking drugs.

Administration of recombinant interleukin-12 (rIL-12) into CSA1M fibrosarcoma-bearing mice results in complete regression of growing tumors. This tumor regression is associated with massive lymphoid cell infiltration to tumor sites and is completely blocked by injection of anti-interferon-gamma (IFN-gamma) monoclonal antibody (mAb). We investigated whether anti-IFN-gamma mAb exerts its suppressive effect on tumor regression by blocking the IL-12-induced lymphoid cell migration to tumor sites or by inhibiting the secondary effects of IFN-gamma produced by infiltrating cells. Injection of anti-IFN-gamma mAb to CSA1M-bearing mice before IL-12 treatment prevented the induction of tumor regression, whereas this treatment affected only marginally the infiltration of lymphoid cells to tumor masses. In accordance with this, IFN-gamma mRNA was expressed inside tumor masses by infiltrating cells after IL-12 therapy irrespective of whether anti-IFN-gamma mAb was injected. However, anti-IFN-gamma mAb treatment almost completely abrogated the in situ expression of inducible nitric oxide synthase (iNOS) as well as IFN-inducible protein-10 (IP-10) genes as examples of IFN-gamma-inducible genes. Immunohistochemical analyses also revealed that the expression of iNOS protein was completely inhibited by anti-IFN-gamma injection. These results suggest that the implementation of in situ IFN-gamma activity and its secondary induction of anti-tumor pathways such as iNOS and IP-10 expression are important processes in the IL-12-induced tumor regression.

Five new oligosaccharide polyesters, fallaxoses A-E, along with four known ones, reiniose D, senegose G, tenuifolioses C and P, were isolated from the roots of Polygala fallax. Fallaxoses A-E were elucidated as 3-O-{4-O-[beta-D-glucopyranosyl-(1-->4)- alpha-L-rhamnopyranosyl]-feruloyl}-beta-D-fructofuranosyl- (2-->1)-(4,6-di-O-benzoyl)-alpha-D-glucopyranoside, 3-O-{4-O-[beta-D-glucocopyranosyl-(1-->3)-(2-O-acetyl)- alpha-L-rhamnopyranosyl]-feruloyl}-beta-D-fructofuranosyl-(2-->1)- (4, 6-di-O-benzoyl)-alpha-D-glucopyranoside, 1-O-p-coumaroyl-(3-O-benzoyl)-beta-D-fructofuranosyl-(2-->1)- [beta-D-glucopyranosyl-(1-->2)]-[6-O-acetyl-beta-D-glucopyranosyl- (1-->3)]-(4-O-p-coumaroyl)-alpha-D-glucopyranoside, 1-O-p-coumaroyl-(3-O-benzoyl)-beta-D-fructofuranosyl-(2-->1)- [beta-D-glucopyranosyl-(1-->2)]-[6-O-acetyl-beta-D-glucopyranosyl-(1-->3 )]-(4-O-feruloyl)-alpha-D-glucopyranoside, 1-O-feruloyl-(3-O-benzoyl)-beta-D-fructofuranosyl-(2-->1)- [beta-D-glucopyranosyl-(1-->2)]-[beta-D-glucopyranosyl- (1-->3)-(6-O-acetyl)-beta-D-glucopyranosyl-(1-->3)]- (6-O-feruloyl)-alpha-D-glucopyranoside, respectively, by spectroscopic and chemical means.

To elucidate the penetrability of carteolol, a beta-adrenoceptor antagonist (beta-blocker) into the brain of rats, intracerebral and serum concentrations of the compound were determined in male rats receiving single or repetitive oral administration of carteolol hydrochloride at 30 mg/kg. The time-course of the intracerebral concentration of carteolol following single IV administration of the compound at 10 and 30 mg/kg was also studied in male rats. A high-performance liquid chromatography method was used to determine the intracerebral and serum concentrations. Following single oral dosing, the intracerebral concentration of carteolol reached a maximum of 0.074 microgram/g at 2 h postdosing and declined with a half-life of 3.7 h, and the Cmax and AUC of carteolol in the brain were 12.5% and 19.8% of those in serum. The intracerebral and serum concentrations of carteolol were determined in male rats receiving repetitive oral dosing of the compound once daily for 7 days. The concentration of carteolol in the brain and serum at 1 h postdosing varied within a range of 0.059-0.091 microgram/g and 0.321-0.443 microgram/ml, respectively, throughout the dosing period, showing no changes in the penetrability of the compound into the brain due to repeated dosing. The concentration of carteolol in the brain and serum increased in a dose-dependent manner in rats receiving a single IV administration of the compound. The elimination half-life of carteolol in the serum and brain was 0.6-0.8 h and 1.3-1.7 h, respectively, in rats following single IV dosing of the compound. The half-life in the brain was about twice as long as that in the serum. The brain to serum concentration ratio was 0.306:0.499. From the above results, it was concluded that carteolol is distributed from the circulation to the brain with low penetrability.

The NO-generating abilities of aromatic N-nitroso compounds (nitrosoureas, nitrosamides and nitrosamines), and N-acetyl-S-nitroso-DL-penicillamine at ambient temperature were compared by employing the Griess reaction. 3,3-Dibenzyl-1-(4-tolyl)-1-nitrosourea showed the greatest NO-generating ability among the tested compounds. The NO-generating ability of the aromatic N-nitrosoureas and N-nitrosamides was greater than that of the N-nitrosamines, presumably reflecting differences in electrostatic repulsion between the carbonyl oxygen and nitroso oxygen in these compounds. In addition, a conjugative effect between the aromatic ring carbon and neighboring nitrogen influences the NO-generating ability; the conjugative effect in the case of N-nitrosoureas and N-nitrosamides having an ortho-alkyl substituted aromatic ring, or N-nitrosamines having a bulky N-group, such as tert-butyl, is decreased by an increase in steric hindrance around the nitroso group. The N-NO bond then becomes more stable. The NO-generating ability was related to the reciprocal of the ID50 value for growth inhibition of cultured L-5178 Y cells by the aromatic N-nitroso compounds. On the other hand, NO production from the aliphatic N-nitroso compounds was not observed under our conditions, and these N-nitroso compounds did not show effective cytotoxic activity.

We investigated the localization of nitric oxide synthase in the pancreas of the dog in comparison to the rat by the methods of immunocytochemistry using antineuronal type nitric oxide synthase serum and histochemistry using NADPH-diaphorase activity. In both species, the most intense staining was observed in neuronal cell bodies and fibers in the pancreas and nitric oxide synthase immunoreactivity was completely colocalized with NADPH-diaphorase activity. However, there were differences of the distribution between the two species. In the dog pancreas, immuno- and NADPH-diaphorase-positive nerve fibers were numerous around pancreatic ducts and moderate around the arteries and the acini but few in the islets. In contrast, in the rat pancreas, immuno- and diaphorase-positive fibers were fewer around the pancreatic ducts and acini and more abundant in the islets. The expression ratio of NADPH-diaphorase in intrapancreatic ganglion cell bodies that were scattered in the interlobular connective tissue was low to moderate (28.1% in the right lobe, 49.5% in the left lobe) in the dog, while the ratio in rat pancreas was very high in both lobes of the pancreas (about 86%). Except for neuronal staining, weak NADPH-diaphorase-positive reactions were detected in the vascular endothelial cells of the pancreas in both species. In rat islet cells, weak neuronal type nitric oxide synthase immunoreactivity was observed; however, in dog islet cells, no immunoreactivity was detected. These results suggest that nitric oxide in the pancreas is derived from vascular endothelium and neuronal tissue in both species and that the neuronal nitrergic regulation of the exocrine and endocrine pancreas is different between the species.

In the previous paper, we reported the differentiation inducing activities of lignoids from Arctium Fructus (the fruits of Arctium lappa L., Compositae) against mouse myeloid leukemia cells (M1). We reinvestigated the active components of this extract and isolated three new dilignans. Furthermore, structure modifications were carried out using the most active lignan (arctigenin, 1) and its structure-activity relationship was investigated. Its aliphatic esters were more effective in inducing the differentiation of M1 cells than its aromatic esters. Especially, n-decanoate, which was the most active derivative, induced more than half of the M1 cells into phagocytic cells at a concentration of 2 microM.

Nine new oleanane-type saponins, polygalasaponins XXXIII--XLI, along with seven known saponins were isolated from the roots of Polygala fallax HEMSL. Polygalasaponins XXXIII-XLI were elucidated as 3-O-beta-D-glucopyranosyl presenegenin 28-O-beta-D-xylopyranosyl-(1-->4)-alpha-L-rhamnopyranosyl- (1-->2)-(4-O-acetyl)-beta-D-fuco-pyranosyl ester, 3-O-beta-D-glucopyranosyl presenegenin 28-O-beta-D-galactopyranosyl-(1-->4)-beta-D-xylopyranosyl- (1-->4)-alpha-L-rhamnopyranosyl-(1-->2)-(4-O-acetyl)-beta-D- fucopyranosyl ester, 3-O-beta-D-glucopyranosyl presenegenin 28-O-beta-D-galactopyranosyl-(1-->4)-beta-D-xylopyranosyl-(1-->4)- alpha-L-rhamnopyranosyl-(1-->2)-(3,4-di-O-acetyl)-beta-D-fucopyranosyl ester, 3-O-beta-D-glucopyranosyl presenegenin 28-O-beta-D-galactopyranosyl-(1-->4)-beta-D-xylopyranosyl-(1-->4)- [(5-O-acetyl)-beta-D-apiofuranosyl-(1-->3)]-alpha-L-rhamnopyranosy l- (1-->2)-(3,4-di-O-acetyl)-beta-D-fucopyranosyl ester, 3-O-beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranosyl presenegenin 28-O-beta-D-xylopyranosyl-(1-->4)-alpha-L-rhamnopyranosyl-(1-->2)- (3-O-acetyl)-beta-D-fucopyranosyl ester, 3-O-beta-D-glucopyranosyl- (1-->2)-beta-D-glucopyranosyl presenegenin 28-O-beta-D-galactopyranosyl-(1-->4)-beta-D-xylopyranosyl- (1-->4)-alpha-L-rhamnopyranosyl-(1-->2)-(4-O-acetyl)-beta-D- fucopyranosyl ester, 3-O-beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranosyl presenegenin 28-O-beta-D-xylopyranosyl-(1-->4)-alpha-L-rhamnopyranosyl-(1-->2)- [alpha-L-rhamnopyranosyl-(1-->3)]-(4-O-acetyl)-beta-D-fucopyranosyl ester, 3-O-beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranosyl presenegenin 28-O-beta-D-galactopyranosyl-(1-->4)-beta-D-xylopyranosyl-(1-->4)- [beta-D-apiofuranosyl-(1-->3)]-alpha-L-rhamnopyranosyl-(1-->2)- (3,4-di-O-acetyl)-beta-D-fucopyranosyl ester and 3-O-beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranosyl presenegenin 28-O-beta-D-galactopyranosyl-(1-->4)-beta-D-xylopyranosyl-(1-->4)- [(5-O-acetyl)-beta-D-apiofuranosyl-(-->3)]-alpha-L-rhamnopyranosyl - (1-->2)-(3,4-di-O-acetyl)-beta-D-fucopyranosyl ester, respectively, on the basis of spectroscopic and chemical evidence.

Five new oleanane-type saponins, polygalasaponins XXVIII-XXXII, along with one known saponin, polygalasaponin XXIV, and one known acylated sucrose, tenuifoliside C, were isolated from the root of Polygala japonica. The structures of these new compounds were elucidated as 3-O-beta-D-glucopyranosyl pesenegenin 28-O-beta-D-xylopyranosyl (1-->4)-alpha-L-rhamnopyranosyl (1-->2)-beta-D-fucopyranosyl ester, 3-O-beta-D-glucopyranosyl presenegenin 28-O-beta-D-galactopyranosyl (1-->5)-beta-D-apiofuranosyl (1-->4)-beta-D-xylopyranosyl (1-->4)-alpha-L-rhamno-pyranosyl (1-->2)-beta-D-fucopyranosyl ester, 3-O-beta-D-glucopyranosyl presenegenin 28-O-beta-D-galactopyranosyl (1-->4)-beta-D-xylopyranosyl (1-->4)-alpha-L-rhamnopyranosyl (1-->2)-[4-O-p-methoxycinnamoyl]-[beta-D-glucopyranosyl (1-->3)]-beta-D-fucopyranosyl ester, 3-O-beta-D-glucopyranosyl presenegenin 28-O-alpha-L-arabinopyranosyl (1-->3)-beta-D-xylopyranosyl (1-->4)-[beta-D-apiofuranosyl (1-->3)]-alpha-L-rhamnopyranosyl (1-->2)-[4-O-3,4,5-trimethoxycinnamoyl]-beta-D-fucopyranosyl ester, 3-O-beta-D-glucopyranosyl persenegenin 28-O-alpha-L-arabinopyranosyl (1-->3)-beta-D-xylopyranosyl (1-->4)-[beta-D-apiofuranosyl (1-->3)-alpha-L-rhamnopyranosyl (1-->2)-[4-O-p-methoxycinnamoyl]-[alpha-L-rhamnopyranosyl (1-->3)-beta-D-fucopyranosyl ester, respectively, on the basis of spectroscopic and chemical evidence.

Recent advances in the diagnosis of gastroesophageal varices are reviewed in this paper. Endoscopy, angiography, ultrasound, and ultrasound Doppler are now employed in the diagnosis of varices. As for endoscopic diagnosis, new criteria for the evaluation of gastroesophageal varices have been applied. Angiography is now not only used to demonstrate the varices and collateral circulations in portal hypertension but also used in the embolization therapy for large gastric varices or intractable esophageal varices. Ultrasound and color Doppler are now widely used in the diagnosis of portal hypertension because of its noninvasiveness and conveniency. Furthermore, in its use in endoscopy, varical vessels and blood flow in the wall of the esophagus and the stomach can be directly demonstrated. Recent advances in these diagnostic modalities will made it possible to diagnose gastroesophageal varices and evaluate the pathophysiology in patients with portal hypertension more precisely.

Six pregnane glycosides and three cardenolides were isolated as differentiation inducers using mouse myeloid leukemia (Ml) cells from Periplocae Radicis Cortex (bark of Periploca sepium BGE., Asclepiadaceae). The cardenolides showed much higher activities than the pregnane glycosides. Besides these nine compounds, commercially available cardenolides were tested for their differentiation inducing activities using Ml cells. Digitoxin and digoxin induced Ml cells into phagocytic cells, but others did not. In the presence of 1 nM of actinomycin-D, the activity of steroid glycosides was enhanced against Ml cells.

To clarify the role of nitric oxide (NO) in the pancreas, we investigate the localization of NADPH-diaphorase activity and neuronal nitric oxide synthase (nNOS) immunoreactivity in the pancreas of the rat and dog. NADPH-diaphorase activity and nNOS immunoreactivity were identical in the neuronal element of both species. NADPH-diaphorase activity and nNOS immunoreactivity were localized in neurons and endothelial cells of vascular system. Numerous NADPH-diaphorase positive and nNOS immunoreactive fibers were observed around pancreatic ducts and arteries. A moderate number of NADPH-diaphorase positive and nNOS immunoreactive fibers were observed surrounding the acini of dog pancreas, but there were few of these structures in rat pancreas. The islets of the rat pancreas contained a moderate number of NADPH-diaphorase positive and nNOS immunoreactive fibers. However in the dog, these positive fibers were not detected inside the islets. In the rat pancreas, 85% of the ganglion cells showed NADPH-diaphorase staining. In the dog, however, 30-50% of the ganglion cells demonstrated NADPH-diaphorase activity. Although NADPH-diaphorase histochemistry did not show any positive staining in the islet cells of pancreas in either species, NOS immunocytochemical method demonstrated weak positive staining in the rat islet cells. These results indicate that NO may play an important role for the neuronal regulation of pancreatic exocrine and endocrine activities in both species, but in the species-specific manner.

Nine new oleanane-type saponins polygalasaponins XI-XIX were isolated from the aerial part of Polygala japonica. The structures of these compounds were established on basis of spectroscopic and chemical evidence.

By means of NADPH-diaphorase (NADPH-d) histochemistry and nitric oxide synthase (NOS) immunohistochemistry, we demonstrate that considerable numbers of NADPH-d-positive neurons are distributed throughout the canine superior cervical ganglion (SCG). These neurons also show NOS immunoreactivity. This finding indicates that NADPH-d histochemistry, a simple and reliable technique, can be used as a reliable marker of NOS activity in the sympathetic innervation of canine head and neck. The present findings suggest that the participation of nitric oxide in the SCG differs greatly between species.

Nitric Oxide (NO), which was initially identified as an endothelium-derived relaxing factor, has recently been demonstrated to be a neuronal messenger in central and peripheral nervous systems. In the present study, we examined the possibility of NO producing neurons in teh intermediolateral (IML) cell collum of the thoracic spinal cord (Th) project to the superior cervical ganglion (SCG). First, we observed the NADPH-diaphorase-positive/nitric oxide synthase (NOS)-immunoreactive neurons of the IML and the dorsal part of the central canal at the level of Th1-Th3, and numerous fiber-stainings in the superior cervical ganglion. Second, after injecting WGA-HRP (wheat germ agglutinin-horse radish peroxidase complex), a retrograde neuronal tracer, into the SCG, and developing WGA-immunohistochemistry and the NADPH-diaphorase histochemistry in the same sections, we detected double-labeled neurons in the IML. These findings provide evidence that sympathetic preganglionic NO producing neurons directly innervate to the SCG.

Ten new oleanane-type saponins, polygalasaponins I-X, along with two known saponins, bayogenin-3-O-beta-D-glucopyranoside and lobatoside B were isolated from the aerial part of Polygala japonica Houtt. The structures of these compounds were established on the basis of spectroscopic and chemical evidence.

The methanol extract of the aerial part of Andrographis paniculata Nees showed potent cell differentiation-inducing activity on mouse myeloid leukemia (M1) cells. From the ethyl acetate-soluble fraction of the methanol extract, six new diterpenoids of ent-labdane type, 14-epi-andrographolide (3), isoandrographolide (4), 14-deoxy-12-methoxyandrographolide (7), 12-epi-14-deoxy-12-methoxyandrographolide (8), 14-deoxy-12-hydroxyandrographolide (9) and 14-deoxy-11-hydroxyandrographolide (10) as well as two new diterpene glucosides, 14-deoxy-11,12-didehydroandrographi-side (12) and 6'-acetylneoandrographolide (14), and four new diterpene dimers, bis-andrograpolides A (15), B (16), C (17) and D (18), were isolated along with six known compounds. The structures of the diterpenoids were determined by means of spectral methods. Some of these compounds showed potent cell differentiation-inducing activity towards M1 cells.

A method was developed for determining the activity of acetyl-CoA:1-alkyl-sn-glycero-3-phosphocholine acetyltransferase (EC 2.3.1.67), a key enzyme in the biosynthesis of platelet-activating factor (PAF, 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine). The assay involves measurement of the radioactivity in the trichloroacetic acid (TCA)-precipitated complex of radioactive product and albumin after incubation of 1-alkyl-sn-glycero-3-phosphocholine and [3H]acetyl-CoA with rat spleen microsomes or membrane fractions of human polymorphonuclear leukocytes (PMNs). The radioactive product associated with the precipitate was identified as PAF using an ultrahigh-sensitivity TV camera system after extraction and separation by TLC. This TCA method was then used to screen the components of crude preparations that inhibited acetyltransferase activity. Major components from the cortex of Magnoliae (magnolol and honokiol), which have anti-inflammatory and anti-bacterial actions, inhibited the acetyltransferase activity in rat spleen microsomes (IC50, 150 and 150 microM, respectively) and membrane fractions of human PMNs (IC50, 70 and 60 microM, respectively). The inhibitory action of magnolol and honokiol was reversible, and similar to or higher than that of nordihydroguaiaretic acid. PAF production in human PMNs stimulated by the ionophore A23187 was also suppressed dose dependently by magnolol and honokiol. These activities may be relevant to the claimed therapeutic effects of the extract from Magnoliae cortex.

In connection with the study of differentiation inducers from plants, the methanol extract of Condurango Cortex (bark of Marsdenia condurango REICH, Asclepiadaceae) was investigated to examine its differentiation-inducing activity towards mouse myeloid leukemia (M1) cell line. Six pregnane glycosides, including three new compounds, were isolated as differentiation inducers. Each of the six active glycosides has three or four deoxylated sugars which are well-known to occur in Asclepiadaceae plants. M1 cells were differentiated into phagocytic cells by these glycosides, and they were found to be more effective than their aglycones. Condurangoglycosides A (7) and C (8), having a cinnamoyl group in their aglycones, were the most potent differentiation inducers and M1 cells became phagocytic cells after 24 h treatment with these compounds.

An attempt was made to isolate differentiation inducers from Aurantii Nobilis Pericarpium and the fruit peel of Citrus reticulata Blanco (Rutaceae). Twenty-seven kinds of flavones, including five new flavones, were isolated after repeated chromatography from methanol extracts of these plants and their structures were established, from their physicochemical data, to be highly methoxylated flavones. Each compound, except for two flavone glucosides, showed the differentiation inducing activity toward mouse myeloid leukemia cells (M1), and the cells came to have phagocytic activity. Furthermore, differentiation inducing activity was tested using human acute promyelocytic leukemia cell line (HL-60).

A precursor of biphenomycin A in mixed culture of Streptomyces griseorubiginosus No. 43708 with Pseudomonas maltophilia No. 1928 was isolated and characterized. The structure of the precursor, designated biphenomycin C was determined to be a peptide which is composed of biphenomycin A and arginylserine residue (Fig. 1), on the basis of chemical and spectroscopic evidence.

Production of biphenomycin A by Streptomyces griseorubiginosus 43708 was stimulated by a mixed culture with a partner strain, Pseudomonas maltophilia 1928. This stimulatory effect on biphenomycin A accumulation by the mixed culture was caused by the enzyme activity which strain 1928 possessed. It is suggested that in a mixed culture strain 43708 produces a precursor of biphenomycin A in culture broth and that strain 1928 converts the precursor to biphenomycin A.

Differentiation-inducing activity of over 180 extracts of crude drugs and plants was tested using mouse myeloid leukemia cell line (M1). The methanol extracts of clove (Syzygium aromaticum Merrill et Perry, Myrtaceae) showed remarkable induction of differentiation of M1 cells into macrophage-like cells. From the extract, oleanolic acid (1) and crategolic acid (2) were isolated as the active components. We also tested other triterpenes, such as oleananes, ursanes and dammaranes, to investigate the structure-activity relationship. Some triterpene aglycones showed differentiation-inducing activity, but triterpene glycosides showed little activity. Furthermore, the differentiation-inducing activity of these triterpene compounds was tested against human acute promyelocytic leukemia cell line (HL-60).

The effects of food restriction on immune function was investigated in germfree (GF) and specific pathogen-free (SPF) mice. They were maintained from five weeks of age under either full-fed or food-restricted conditions to 4.5 grams per day (equivalent to approximately 80% of full-fed intake) of a commercial diet. Longest survival rate was attained in food-restricted SPF mice followed by food-restricted GF, full-fed GF, and full-fed SPF animals. Food-restricted GF mice showed shorter survival rate than their SPF counterparts. This result suggests that food restriction may be just as effective as GF status for extending life span. Immune function declined significantly with age in full-fed groups of GF and SPF mice. In both food-restricted GF and SPF mice, mitogenic response to concanavalin A or lipopolysaccharide and antibody response to sheep red blood cells were lower early in life and became higher later in life as compared with full-fed mice. Hence, the maintenance of effective immunological function until old age may be the reason for food-restricted groups to live slightly longer than full-fed groups.

An investigation was undertaken to study the effects of germfree (GF) status and mild food restriction on life span in GF and specific pathogen-free (SPF) male ICR mice either full-fed (ad libitum) or on a restricted diet of 4.5 grams per day (equivalent to approximately 80% of full-fed intake) from five-week-old. The mean life span of the full-fed SPF and GF mice was 75.9 and 88.9 weeks respectively, while the mean life span of the food-restricted SPF and GF mice was 117.5 and 109.6 weeks, respectively. Mice in both GF and SPF food-restricted groups were characterized by lower body weight and increased survival. These findings suggest that the cessation of growth may be importantly and perhaps causally related to longevity. The GF mice survived longer than the SPF mice, but the combination of GF status with food restriction did not seem to extend life span more than food restriction alone.

In mice infected with a lethal dose of murine cytomegalovirus (MCMV) the serum immunoglobulin (Ig) levels and the Ig-bearing cells in the spleen dropped to barely detectable levels 2 days after infection. In mice with acute but non-lethal MCMV infection, the serum IgM was twice and the IgG 32 times that of the uninfected controls by Day 8 of infection; the numbers of spleen cells bearing IgM and the IgG subclasses (IgG1, IgG2a, IgG2b, IgG3) were also greatly increased. In the asymptomatically infected group, serum IgM remained unchanged but the IgG increased to 16 times that of uninfected controls by Day 11 of infection; the numbers of spleen cells bearing IgM and IgG subclasses were also increased, although to a lesser extent than in the acute, non-lethally infected mice. In the latter two groups, serum IgA and IgA-bearing cells in the spleen did not alter significantly. Complement-requiring neutralizing antibodies to MCMV were detected 8 days post infection.

The immunological response of spleen cells was examined during murine cytomegalovirus (MCMV) infection of germfree (GF) mice. GF mice were more susceptible to MCMV infection than specific pathogen-free (SPF) mice. Recovery from the acute phase of MCMV infection was delayed in GF mice as compared to SPF mice. During MCMV infection, the immune response of GF mice, measured in terms of mitogen and of antibody response to sheep erythrocytes, was more markedly depressed and the recovery from the depression was more delayed than in SPF mice. In MCMV-infected mice, natural killer (NK) cell activity was increased by day 1 and 2 post-infection, with similar kinetics in GF and SPF mice, suggesting that NK cells may be exerting the most potent antiviral effect during the early stage of MCMV infection.

The effects of concurrent murine cytomegalovirus (MCMV) and Klebsiella pneumoniae infections were studied in germfree (GF) mice. The mice received sublethal doses, 5 x 10(5) pfu, of MCMV. K. pneumoniae was injected in doses of 40 to 100 cfu, which by itself killed 0-33% of GF mice. When K. pneumoniae was given to GF mice infected with MCMV, the mortality increased up to 100%, with distinct enhancement persisting until day 10 of the MCMV infection. The virus titer in various organs did not change after superinfection with K. pneumoniae, while the viable counts of K. pneumoniae in organs remained remarkably high until death, suggesting the cause of death to be severe generalized infection by the bacteria. When compared to specific pathogen-free (SPF) mice, GF mice were more susceptible to both MCMV and K. pneumoniae infection, had higher titers of the virus for longer periods in various organs, and showed extension in the duration of enhanced mortality by the bacteria. Histopathologically, the spleen and liver were found to be the most severely affected tissues, more so in GF than in SPF mice, with recovery from the changes being slower in the GF animals.

Oral administration of adenovirus strain K87 to BALB/c nude mice resulted in viral proliferation in the intestinal tract up to around week 6 at which point replication was suppressed. In other words, the host acquired resistance. However, this resistance was temporary and the viral infection persisted over a long period with repeated periods of proliferation and resistance. That the appearance of this resistance is the result of infecting mice with the virus and is not due to age difference per se was made clear through experimentation with nude mice of different age groups. However, it was indicated that increase in age is involved in the decreased rate of reproliferation following initial suppression. No evidence of the virus was obtained from any other organ throughout the infection. Furthermore, throughout the persistent infection, even during the aforementioned periods of resistance, no neutralizing antibody was detected from sera, intestinal wall or intestinal content. When spleen cells from BALB/c heterozygous littermate mice was transferred to the nude mice, an earlier onset of antiviral resistance was seen than in nude mice without the transfer, and this was accompanied by a rise in neutralizing antibody titer. From these results, it is believed that the resistance characteristic of nude mice infected by mouse adenovirus is dependent on some factor other than the neutralizing antibody invoked resistance exhibited by euthymic mice.

The cytopathic effects of extracts of pharyngeal cells from patients with IgA nephropathy on fibroblasts were determined. Autoradiographical analysis of antigens in the fibroblasts was also described. Freeze and thawed extracts of pharyngeal cells obtained from patients with IgA nephropathy, other glomerular diseases and healthy adults were cultured with fibroblasts such as Vero or Hel cells at 37 degrees C for 1 or 2 weeks. The cytopathic effects of fibroblasts were examined with a light microscope. In addition, the eluate obtained from renal tissues of IgA nephropathy was labeled with iodine-125 by the chloramine-T method. These cultured fibroblasts were incubated with iodine-125-labeled eluate from the same or other patients with IgA nephropathy. The degree of cytopathic effects of extracts of pharyngeal cells from patients with IgA nephropathy on Vero or Hel cells was significantly increased compared with that from patients with other glomerular diseases or healthy adults without glomerular diseases. The cytopathic effects of such fibroblasts were not inhibited by filtration of extracts of pharyngeal cells obtained from patients with IgA nephropathy. It was shown that the antibodies eluted from renal tissues of patients with IgA nephropathy specifically bound with the nuclear regions of such fibroblasts. It was suggested that some antigenic substances might exist in the epithelial cells of the upper respiratory tracts of some patients with IgA nephropathy, and that such antigens were able to be transferred to cultured fibroblasts.

Tooth movement in orthodontic treatment is achieved by a combination of mechanical and physiologic forces acting on the teeth and their supporting structures to effect the biologic changes that cause teeth to move through bone. Traditional orthodontic appliances using wires, springs, and elastic forces inevitably cause some degree of discomfort and pain. In the present study, a new magnetized edgewise bracket was designed. The magnet works to move teeth mesiodistally and is not merely a mechanical force. It is well adapted to the biophysical system of tooth and bone interrelationships. Therefore, it works effectively, gives less discomfort, and produces less stress for the patient. The new magnetic appliance provides a force system that approaches the ideal orthodontic requirements.

Streptomyces griseoflavus, a bicozamycin-producing wild type strain and its high-producing one derived from it by multiple (greater than 15) mutagenic treatments, were analyzed physiologically and biochemically. The high-producing strain was characterized by: (1) An increased pool size of amino acids including leucine and isoleucine, precursors for bicozamycin synthesis, (2) an earlier and greater accumulation of intracellular ppGpp, (3) a more accentuated decrease in GTP pool size, (4) a higher specific activity of ornithine transcarbamylase which produces citrulline, (5) an increased ability to form aerial mycelium, and (6) an increased resistance to its own antibiotic. We propose that (1), (2) and (4) may be responsible for the high yields of bicozamycin and, possibly, of some other antibiotics produced by Streptomyces sp.

Germfree F344 rats developed severe arthritis with 100% incidence after a single intradermal inection of either squalane containing 0.5 mg of heat-killed Mycobacterium bovis BCG or a water-in-oil emulsion containing 0.2 mg of peptidoglycan derived from Staphylococcus epidermidis. Conventional F344 rats developed less-severe arthritis with 20% incidence for heat-killed BCG and 0% incidence for peptidoglycan. Specific-pathogen-free rats showed an intermediate susceptibility between germfree and conventional rats. Interestingly, both unimmunized specific-pathogen-free and conventional rats. but not unimmunized germfree rats, showed weak delayed-type hypersensitivity reactions to peptidoglycans derived from either S. epidermidis or Lactobacillus plantarum, suggesting that a bacterial flora may furnish a stimulus for induction of cell-mediated immunity to ubiquitous bacterial peptidoglycans. It is thus possible that although a bacterial flora is not necessary for development of adjuvant arthritis, it may have some suppressive effect on the development of the disease in specific-pathogen-free and conventional F344 rats, possibly through modulation of the immune response.

Biphenomycin A, C23H28N4O8, and biphenomycin B, C23H28N4O7, were isolated from the cultured broth of Streptomyces griseorubiginosus No. 43708. The antibiotics are active in vitro and in vivo against bacteria, and are especially potent against Gram-positive bacteria. The acute toxicity of biphenomycin A is very low in mice.

We tried to isolate pathogenic viruses from specimens of patients with Kawasaki disease. Blood clots and sera, spinal fluid, throat swabs, stool and urine from 24 patients with Kawasaki disease were studied. The specimens were inoculated into HEL cells and Vero cells. The cells were observed for one month, but no cytopathic effect (CPE) occurred. Blind passage was performed, but no degeneration of the second series of cells was observed. Fluorescent antibody indirect methods to determine viral antigens in cells inoculated with patients' specimens was also negative.

New vasodilators, designated WS-1228 A and B have been discovered in the culture filtrate of a strain of Streptomyces aureofaciens. The active compounds were purified by column chromatography with Diaion HP-20 and silica gel, and finally separated from each other by high performance liquid chromatography. They were obtained as pale yellow crystals and their molecular formulae were both C11H17N3O.