The structural diversity of dermatan sulphate (DS), the major glycosaminoglycan component of mammalian skin, was investigated by examining different layers of porcine dermal tissue using ¹H-NMR and disaccharide compositional analysis by HPLC. Structural reporter signals were assigned using one-dimensional (1D) ¹H-NMR differential transient NOE and 1D totally correlated spectroscopy (TOCSY) spectra, measured at different probe temperatures. The results of these studies on 12 sliced layers (average thickness of 250 μm) of skin show that the content of glucoronic acid in DS decreases when moving from the outer surface of the skin to the inside, while the degree of sulfation of the C-2 hydroxy group of iduronate and the C-4 and C-6 hydroxy groups of N-acetylgalactosamine increases with depth. These results suggest that the utility of analysis of DS from various depths in porcine skin clearly show the origin of each sample, and might be useful for the quality control of these biological materials in clinical use.

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Laser scannins confocal fluorescence microscopic analysis provided a direct evidence of the internalization of non-permeant Lucifer Yellow and a dextran of MW 4400 into leukemia L1210 cells when the cells were vortex-stirred in the presence of a high molecular weight polyacrylic acid, A-119 (MW ca. 9×10⁶). The morphology of A-119-treated cells was probed by scanning electron microscopic analysis using 2% glutaraldehyde for fixation. The cells immediately after vortex-stirring with A-119 showed many blebs on the cell surface, indicative of local weakening of the plasma membrane. the blebby surface returned to normal within 5 min at 37°C, but not completely at 0°C even after 20 min. Several cultured cell lines, murine splenocytes, and Ehrlich ascites carcinoma cells were particularly susceptible to permeabilization by this method, although the efficiency varied from one type of cell to another.

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The absorption and distribution of ulinastatin following intra-articular administration of [¹²⁵I]ulinastatin to rabbits were determined and kinetic analysis using a multi-compartment model was performed. At 4h after administration, the content of radioactivity in the synovial fluid, which was comparable to that of the immunoreactive ulinastatin, was 14.51% of the dose and decreased in a biphasic manner. The highest level of radioactivity was observed in the synovial membrane, followed by the meniscus, ligament, cartilage and patella, the radioactivities of whcih also declined biphasically. After intra-articular administration, the plasma concentration of total radioactivity increased slowly and reached maximum at 4.3 h, and then declined slowly in a monophasic manner with a half-life of 10.8 h. The radioactivity of the high molecular weight fraction in plasma, which reached maximum at 1.7 h after administration and then declined with a half-life of 11.8 h, was consistent with the time curve for immunoreactive ulinastatin in the plasma through 24 h after the administration. Within 8 and 24 h after administration, respectively, 1.48 and 4.66% of the administered radioactivity were transferred to the lymphatic fluid. The pharmacokinetics of[¹²⁵I]ulinastatin after an intra-articular administration could be explained using a multi-compartment model in which a portion of the administered ulinastatin was absorbed via the lymphatic system. This finding suggested that ulinastatin was rapidly distributed and retained for a long period of time in the joint tissues. In addition, the pharmacokinetics of ulinastatin following intra-articular administration indicated typical flip-flop kinetics.

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Emetic and antiemetic effects of morphine were investigated in Suncus murinus. Subcutaneous (up to 30 mg/kg) or intracerebroventricular administration (50 μg) of morphine failed to cause emesis. However, pretreatment with morphine (s.c.) prevented the emesis induced by nicotine (10 mg/kg, i.p.), copper sulfate (40 mg/kg, p.o.), cisplatin(20 mg/kg, i.p.) and motion stimulus. These results suggest that morphine has only antiemetic potency and may block a common mechanism for the emetic reflex of suncus, because the antiemetic effects of the drug were exerted irrespective of the stimulus.

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It was found that the rectal temperature of rabbits restricted by a normal neck stock position dropped about 1.2-1.5°C when the reastriction position chenged from a normal restriction to a supine restriction position. This temperature change was also reversible. We studied the mechanisms of this phenomenon from a thermodynamic point of view and obtained the following results : 1) The rectal temperature of rabbit restricted in the supine position did not increase after the injection of lipopolysaccharide (LPS). 2) In the supine restricted rabbit, endogenous pyrogen which was induced by the injection of LPS to these rabbits appeared the same as the control. 3) Some pharmaceuticals(urethan, iproniazid, atropin and hexamethonium) did not show any influence on the drop of rectal temperature in either normal or supine restrictions. 4) The rectal temperature of shorn rabbits dropped more than that of normal rabbits in both normal and supine restrictions. 5) When the normal restriction position was changed to the supine and prone restriction positions, the mean surface temperature was also decreased. From these results, it was concluded that rabbit rectal temperature was changed with a change in the restriction position which could change the surface skin area.

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ES46.5K, a novel esterase from mouse hepatic microsomes (Watanabe K., et al., Biochem. Mol. Biol. Int., 31, 25-30 (1993)), catalyzed hydrolysis of phthalate esters. ES46.5K and mouse hepatic microsomes hydrolyzed diethyl-, dibutyl-, diisobutyl-, dioctyl- and diethylhexyl phthalates, whereas dicyclohexyl- and diphenyl phthalates having ring structure were not hydrolyzed by the enzymes. V_max (μmol/min/mg protein)/K_m (μM) ratios of ES46.5K for diethyl-, dibutyl-, diisobutyl-, dioctyl- and diethylhexyl phthalates were 291, 2786, 565, 51 and 57, respectively, while those of microsomes were 0.58, 0.83, 1.71, 0.05 and 1.10, respectively. The hydrolytic activity of ES46.5K was inhibited by diisopropylfluorophosphate and bis-p-nitrophenylphosphate. These results suggest that ES46.5K has high catalytic activity for phthalate esters and some role in the metabolism of phthalate esters in mice.

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A new 5-HT₃ receptor ligand, CP2289, was synthesized and pharmacologically tested. Although CP2289 inhibited the Bezold-Jarisch reflex, it contracted the excised ileal muscle of mice, rats and guinea pigs. This response may reflect a partial agonist character of CP2289 in the gut. In vivo antiemetic and gastric emptying tests gave similar results.

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The benzene extract of Hibiscus rosa sinensis flowers was administered intraperitoneally at the dose levels of 125 and 250 mg/kg body weight to adult mice and resulted in an irregular estrous cycle with prolonged estrus and metestrus. An increase in the atretic follicles and the absence of corpora lutea indicate the antiovulatory effect of the extract. The extract also showed estrogenic activity in immature mice by early opening of the vagina, premature cornification of the vaginal epithelium and an increase in uterine weight. Therefore the antiovulatory effect may be due to an imbalance in the hormonal environment, as there may be an increase in the endogenous secretion of estrogen by atretic follicles, and also to the estrogencity of the flower extract.

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In this study, we examined the contribution of lignin-like materials in lower molecular weight (MW) fractions from the hot water extract of Bupleuri Radix (Bupleurum chinense) (HWE-BR) for their immunopharmacological activities. Mitogenic activity was detected in all the fractions of MW ranges : lower than 1.0 kDa, 1.0-3.5 kDa, 3.5-10 kDa, and 10-50 kDa. After NaClO₂ treatment of these subfractions, UV spectra, ESR spectra, mitogenic activities on murine B-cells, and the activity of inducing nitric oxide in RAW 264.7 cells were significantly reduced, suggesting that lignin-like polyphenolic substance(s) of various MW might take part in these activityes. The intensity of ESR spectra and mitogenic activities were stronger in higher MW subfractions, thus the content of stable radical species and/or the degrees of polymerization would be important for their immunopharmacological activities.

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In order to develop convenient and reproducible methods for the identification of Ginseng drugs at a DNA level, PCR-Restriction fragment length polymorphism (PCR-RFLP) and Mutant allele specific amplification (MASA)analyses were applied, based on differences of the 18S rRNA gene sequence among three Panax species. The PCR product of each species on the 18S rRNA gene was digested with the restriction enzymes Ban II and Dde I. Each fragment gave unique electrophoretic profiles for each epecies (PCR-RFLP analysis). The extracted DNA of each species was amplified by PCR using a designed species-specific oligonucleotide primer. The expected size of the fragments corresponding to each species were detected only when the optimum temperature and reaction time for annealing and extension were established (MASA analysis). These two analytical methods were carried out on three Ginseng drugs and the same results an in their original plants were obtained. The results suggest that PCR-RFLP and MASA analyses under the established conditions are convenient for identifying three Ginseng drugs. Moreover, to insure completion of the identification, a partial sequence of the plastid gene matK was determined in addition to the 18S rRNA gene. The gene sequences of three Panax species were of 1259 base pairs and that of P. quinquefolius was different from the other two at nucleotide position 102.

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The cDNA encoding cycloartenol synthase [EC 5.4.99.8] has been isolated from pea seedling by an efficient PCR using sets of degenerate primers based on the highly conserved sequences of the known 2, 3-oxidosqualene cyclase cDNAs. The obtained cDNA contains a 2271-bp open reading frame and is encoding a predicted protein of 757 amino acids with high homology (81%) to Arabidopsis thaliana cycloartenol synthase. The PCR-amplified open reading frame (ORF) has been inserted into pYES2, an expression vector in yeast, under the control of galactose-inducible promoter. Significant cycloartenol synthase activity has been found in the homogenate of the yeast transformed with the plasmid containing PCR-amplified ORF.

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Two lignans, asarinin (6) and xanthoxylol (7), were isolated from the radix of Asiasarum heterotropoides var. mandshuricum, which consist of a kampo prescription, Shouseiryu-to, as inhibitors of Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). These lignans also exhibited remarkable inhibitory effects on a two-stage carcinogenesis test of mouse skin and pulmonary tumors. Furthermore, it was confirmed that these hydrophobic lignans dissolved in the water decoction of Shouseiryu-to, and these lignans might be among the active consituents of this kampo prescription in terms of its anti-tumor-promoting activity.

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Experimental rat models (5-week-old Sprague-Dawley rats) with hyperlipemia were prepared by feeding high-cholesterol feed containing sodium cholate and casein as a protein source. Dried maitake (Grifola frondosa) powder was mixed with the basic high-cholesterol feed and the serum lipids were periodically measured. Values of cholesterol, triglyceride and phospholipid in serum of rats in the maitake-feed group were suppressed by 0.3-0.8 times those in animals fed the basic feed, the latter values being close to those in rats given normal feed. The value of high density lipoprotein (HDL)-cholesterol in serum which is generally reduced by the ingestion of high-cholesterol feed remained the level it was at the beginning of the experiment. Weights of extirpated liver and epididymal fat-pads were significantly less (0.6-0.7 times) than those in the basic feed group, indicating that maitake inhibits lipid accumulation in the body. Liver lipids were also measured and the values were found to be decreased by maitake administration as true of serum lipid, suggesting maitake has an anti-liver lipid activity. Measurement of the amount of total cholesterol and bile acid in feces showed, the ratio of cholesterol-excretion had increased 1.8 times and bile acid-excretion 3 fold by maitake treatment. From these results, it is believed that maitake helps to improve the lipid metabolism as it inhibits both liver lipid and serum lipid which are increased by the ingestion of high-fat feed.

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The fate of trafermin (recombinant human basic fibroblast growth factor) was examined after intravenous administration of its iodinated form to rats. Autoradiography at 5 and 30 min after the injection showed that ¹²⁵I-trafermin is localized specifically in the fenestrated endothelium through binding to heparan sulfate proteoglycans (HSPG) in liver, kidney, adrenal, spleen, hypophysis and bone marrow. Metabolites in the organs were examined at 5 min and 24 h after the injection. More than 73% of radioactivity in liver and kidney was extractable at either time point, and a large majority of the extracted radioactivity was heparin-binding. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the substantial radioactivity recovered from liver and kidney can commonly be attributed to a peptide with the same molecular weight as the intact trafermin (B-1, 17.7 kDa) and only three truncated metabolites (B-2, 15.0 kDa; B-3, 7.2 kDa; B-4, 4.2 kDa). Because no truncated metabolites were found in serum, these metabolites seem to be produced inherently in liver and kidney. Although they all retained heparin-binding capacity, only B-1 and B-2 exhibited a stimulatory effect on proliferation of endothelial cells, and these bioactive peptides disappeared completely from liver within a day, indicating a rapid inactivation process in the organs. Taken together with the morphological evidence on autoradiography, it seems most likely that the injected trafermin could be inactivated in sinusoidal endothelial cells, probably through a well-known internalization mechanism of the basic fibroblast growth factor-HSPG complex.

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The effect of membrane surface potential of the apical side on the intracellular uptake of ionic compounds was investigated using the human colon adenocarcinoma cell line (Caco-2). The transepithelial transport of indolepropionic acid and tryptamine was consistent with the uptake behavior shown by rat intestinal brush-border membrane (BBM) vesicles. Imipramine, which diminished the negative charge of the membrane surface (for both Caco-2 and BBM), acted to increase the uptake of the anionic compounds, inodolepropionic acid and ceftibuten, and to decrease that of tryptamine (cationic compound) by both the Caco-2 monolayer and the intestinal BBM vesicles at a pH of 7.5. These results suggest that the effects of membrane surface potential on the permeability of ionic compounds were detectable on the Caco-2 cell line as well as the BBM vesicles. On the other hand, the inhibition of H⁺-linked transport and the stimulation of the surface change-regulated uptake of ceftibuten have occurred simultaneously on the Caco-2 cell line in the presence of imipramine. It seems that the membrane surface charge (negative) plays an important role in the transport process of ionic compounds across the intestinal epithelium.

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Based on the antibacterial activity of 9-phenylnonylamine (pC9a) against Escherichia coli (ATCC29522) and Staphylococcus aureus (ATCC25923), we have further tested the inhibitory ability of the growth of the bacteria by (±)1-(4-aminobutyl)-6-benzylindane (PM2) and (±)1-benzyl-6-(4-aminobutyl) indane (PM3), that is, two kinds of 1, 6-disubstituted indanes. In an in vivo assay, they showed almost the same antibacterial activities against the bacteria as pC9a, as well as that of magainin 2 analogs (i.e., the peptides MSI-78 and 87-ISM), except in the case of 87-ISM against S. aureus. At the MIC (minimum inhibitory concentration) values, however, their killing rate of E.coli is actually quicker than pC9a. This indicates that an indane scaffold, used as a template to mimic a part of the α-helical structure of magainin 2, can accelerate the killing rate. At present, however, it is unknown wherther either the hydrophobicity or the α-helical structure, or both, of the indane scaffold is involved in accelerating the rate. Moreover, these two indanes also showed stronger antibacterial activity against two strains of Helicobacter pylori (ATCC43526, ATCC43579) than either pC9a or magainin 2 related peptides.

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Magainin 2 is an antimicrobial peptide isolated from the skin of Xenopus laevis. We have tested the antibacterial activities of normal and reversed magainin 2 analogs against two strains of Helicobacter pylori (ATCC 43526, ATCC 43579), compared with those against Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923). Among these analogs, MSI-78A showed the strongest activity against H. pylori. The MIC (minimum inhibitory concentration) values were almost the same as those against E. coli and S. aureus. No or lesser activity was observed in all the reversed peptides compared to the corresponding normal magainin 2 analogs. Based on the CD (circular dichroism) measurement, the more active peptide tends to show a higher α-content. The positively-charged five amino acids (KILKK) positioned at the C terminus on the amphipathical α-helical structure play important roles in exerting the strong activity against H. pylori. This indicates that the net charge of the cell surface in H. pylori may be more negative than that of E. coli, though both strains belong to the same genus.

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In order to characterize the pharmacological role of the crude polysaccharide fraction in kampo-hozai, we chose 4 kinds of kampo-hozai, Shosaiko-to, Daisaiko-to, Hachimi-jio-gan and Hochu-ekki-to, and studied the effects of their crude polysaccharide fractions on nitric oxide (NO) production by 3% thioglycollate-induced murine peritoneal macrophage. Oral administration of these fractions for 7 d augmented lipopolysaccharide (LPS, 0.1-10 μg/ml)-induced NO production by peritoneal macrophages. These results suggest the possibility that a crude polysaccharide fraction affect the macropharge function in most kampo-hozai.

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The effect of absorption enhancers on the small and large intestinal absorption of drug in rats was examined using an in vitro modified Ussing chamber method, and the results were compared with those from an in situ absorption experiment. Phenol red was chosen as a model drug, while the absorption enhancers used were sodium glycocholate (Na-GC), sodium taurocholate (Na-TC), sodium deoxycholate (Na-DC), EDTA, sodium salicylate (Na-Sal), sodium caprate (Na-Cap), diethyl maleate (DEM) and N-lauryl-β-D-maltopyranoside (LM), all used at a concentration of 20 mM. This modified Ussing chamber method showed that Na-DC and EDTA were the most effective absorption enhancers in the small intestine, whereas Na-DC, EDTA and LM were the most effective absorption enhancers in the large intestine. A good correlation exists between the area under the curve (AUC) (in situ loop model) and the cumulative amount of phenol red absorbed (in vitro medified Ussing chamber method). These results indicated that the in vitro modified Ussing chamber method can be used to evaluate the effects of various absorption enhancers in the intestine.

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We established a high performance liquid chromatography (HPLC) method for the simultaneous determination of the lactone and carboxylate forms of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of the antitumor drug irinotecan (CPT-11), in rat plasma. Plsma samples were pretreated with chilled MeOH and zinc sulfate to precipitate protein, and were then directly injected into the HPLC system. Chromatography was carried out with a Puresil C18 column (particle size 5 μm), and the mobile phase consisted of 0.1 M ammonium acetate buffer (pH 5.5) and acetonitrile (70/30, v/v) containing 20 mM of tetra-n-pentylammonium bromide. The column effluent was monitored with a spectrofluorometer (excitation wavelength 380 nm, emission wavelength 540 nm). The method was valid for SN-38 lactone (5-2500 ng/ml) and carboxylate (5-1000 ng/ml).

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The dnaA gene of Staphylococcus aureus has now been cloned and sequenced. The deduced amino acid sequence of Staphylococcus aureus DnaA protein is 62% and 39% identical to those of the DnaA protein from Bacillus subtilis and Escherichia coli, respectively.Expression of the dnaA gene of Staphylococcus aureus in Escherichia coli caused lethality in an oriC-dependent manner.

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