We propose a new indicator for diabetic control that shows the extent of glycation of hair protein (keratin), the glycation index (A₃₉₀ and A<412>) which is based on the ratio of glycated protein- to cystine-induced coloration, where A₃₉₀/A<412> represent each absorbance in the color reactions of gluycated protein and cystine in the hair protein. Samples can be quickly and non-invasively collected and easily stored. This index for the back and scalp hairs from hypercholesterolemic mice with hyperglycemia, diabetic rats and diabetic patients gave significantly higher values (2.0-6.0-fold) than those of normal subjects (p<0.01). The glycation indices (mean±S.D.) of hairs from diabetic and non-diabetic subjects were 3.00±0.96 (n=21) and 1.51±0.45(n=21) and 1.51±0.45 (n=30), respectively. These indices (y) correlated well with the levels of glycohemoglobin (HbA_1c, x) in diabetic and non-diabetic subjects : y=0.69x-2.03 (r=0.82, n=31, p<0.01). Within-run precision (reproducibility, CV) for the glycation indices of hairs from the three groups was 6.7-9.4% (n=10 each).The proposed glycation index of hair gave reasonable results for animals and humans with normo- and hyperglycemia, suggesting that it is reliable and can be diagnostically useful.

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The separation and characterization of vitamin D₃- and 25-hydroxyvitamin D₃-monoglucuronides, biliary metabolites obtained from rats dosed with D₃ and 25-hydroxyvitamin D₃ per os, respectively, were carried out by HPLC. The glucuronide fractions were obtained from bile specimens by the combined use of a Bond Elut C18 cartridge, for solid phase extraction, and a lipophilic gel (piperidinohydroxypropyl Sephadex LH-20), for ion-exchange chromatography. Each glucuronide was identified by comparison with an authentic sample in three ways : its chromatographic behavior, that of its fluorescent labeled derivative using 4-[4-(6-methoxy-2-benzoxazolyl)phenyl]-1, 2, 4-triazoline-3, 5-dione and data obtained following enzymatic hydrolysis using β-glucuronidase.

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Effects of biscoclaurine alkaloid cepharanthine on proliferation of skin cells were examined in vitro. The alkaloid was found to enhance attachment and cell layer formation of newborn rat skin epidermal cells in culture on type I collagen-coated Millipore filter, and to potentiate production of keratins in these cells. Spreading and growth of skin dermal fibroblasts were inhibited by cepharanthine in a dose-dependent manner, but the alkaloid-treated cells still retained the ability to synthesize type IV collagen. In culture of skin cells containing both epidermal and dermal cells, no growth of epidermal cells or over-growth of dermal fibroblasts was observed in medium without cepharanthine. In contrast, the presence of the alkaloid suppressed the growth of firoblasts and promoted the formation of epidermal cell layers accompanied by production of keratins. Thus, in comparison with the cells untreated with the alkaloid, cepharanthine-treated skin epidermal and dermal cells in vitro expressed in vivo-like characters.

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Basal cells from newborn rat skin epidermis were cultured on various culture matrixes, and the response of the cells to extracellular calcium was examined. When the cells were cultured on a type I collagen-coated Millipore filter to maintain clearance under the cultured cells as intact skin, the cells responded to a fluctuation in extracellular calcium and were detached from the collagen layer in a high calcium environment. In this case, protein synthesis of the cells increased calcium-dependently. In contrast, calcium-dependent protein synthesis of the cells was not detected in culture, neither on the plastic plate nor the type I collagen-coated plate. Moreover, in culture on a type I collagen-coated filter, the size of basal cells was unchanged for 2d. This should be consistent with the fact that morphological changes in cells are very gradual in vivo. On the other hand, when basal cells were cyltured either on a plastic plate or collagen-coated plate, the cells lost their normality; they were markedly enlarged for 2d of cylture.Thus, the responses of skin basal cells to extracellular calcium fluctuated according to the types of culture matrixes. In this case, keratin synthesis and a morphological change of the cells seems to be concerned with the clearance, which was established in the basal side of the cultured basal cells as intact skin.

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Cyclophilin 40 (CyP40) is a recently identified member of the cyclophilin family that may be a component of unactivated steroid receptor complexes. It consists of an N-half portion that is highly homologous to cyclophilin A and has peptidyl prolyl isomerase (PPIase) activity, and a C-half portion that resembles the C-terminal portion of FKBP52 (FK506 binding protein 52), another component of unactivated steroid receptor complexes. To better understand the structure and functional characteristics of this new class of cyclophilin, we have raised monoclonal antibodies against the C-half portion of human CyP40. Immunostaining with the antibodies showed its preferential localization in cytoplasm. One antibody cross-reacted with a 45kDa protein in yeast, suggesting high conservation throughout evolution. A CyP40-associated protein was isolated from rabbit reticulocyte lysate by means of an affinity resin, and was identified as hsp90. The C-half portion of CyP40 was necessary and sufficient for the interaction.

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The constitutive level of renal fatty acid hydroxylase was examined in ddY mice by measuring the activities of lauric acid ω- and (ω-1)-hydroxylase, (LA12H and La11H respectively). The activities of both LA12H and LA11H of male mice were significantly higher than those of female mice. This sex difference in renal lauric acid hydroxylase (LAH) activity exists in other strains of mice, including Balb/c, C57BL/6, C3H/HeN and C3H/HeJ. Renal LAH activities are at significantly low levels in both sexes of immature animals, but are sexually differentiated in the mature state. In male mice, orchiectomy caused a drastic decrease in renal LAH activities, and the activities were restored by testosterone treatment to above the level of the intact animal. In female mice, ovariectomy and estradiol treatment had no effect on the activities, but testosterone treatment caused an increase in the activities to the level of the intact male animal. These results suggested that testosterone is a key regulatory factor in the level of LAH activity in mouse kidney. The administration of dexamethasone and clofibrate affected the level of LAH activity. The above data are consistent with the level of CYP4A-related proteins (band H and L potein) measured by using anti-rat CYP4A1 antibodies. The antibodies inhibited both LA12H and LA11H activities. The level of band H protein was regulated by testosterone and dexamethasone. On the other hand, the level of band L protein was regulated by clofibrate. These results suggest that distinct modes of regulation exist in the level between band H and L protein in mouse kidney.

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Effects of epidermal growth factor (EGF) on the expression of α₂-adrenergic responses were examined in primary cultures of adult rat hepatocytes. The α₂-responses were assessed by the inhibition of the rate of forskolin-stimulated cAMP formation by the selective α₂-adrenergic agonists, oxymetazoline and UK-14304. Hepatocytes cultured with EGF (20 ng/ml) at a high cell density (1.0×10⁵/cm²) showed almost no response to the α₂-adrenergic agonists, oxymetazoline and UK-14304 (1-100 μM). In contrast, when cultured at a low cell density (3.3×10⁴ cells/cm²)with EGF, forskolin-stimulated cAMP production was inhibited by oxymetazoline and UK-14304 in a dose-dependent manner. The α₂-response was blocked by the α₂-antagonist yohimbine (10 μM). It was also reversed by treatment of hepatocytes with pertussis toxin (100 ng/ml). In addition, the effects of EGF on the appearance of α₂-responses were almost completely inhibited by treatment of the hepatocytes with genistein (10 μM) or cytochalasin B (10 μM). The α₂-response was abolished when cycloheximide (5 μM) was added to the cultures. These results demonstrate that when cultured at a low cell density with EGF, adult rat hepatocytes acquire a significant α₂-adrenergic response. The expression of this α₂-response is associated with de novo protein synthesis.

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The activities of microsomal phospholipase A₂ (PLA₂) and C (PLC) from mouse brain, heart and liver were determined using the substrate 1-palmitoyl-2-N-(4-nitrobenzo-2-oxa-1, 3-diazole amino caproyl-phosphatidyl-choline (NBD-PC), and the effects of chronic ethanol treatment (ethanol) as well as in vitro addition of various n-alcohols including ethanol on these activities were evaluated. Microsomal membrane fluidity was estimated by diphenylhexatriene anisotropy (γ). The microsomes from the brain and heart of ethanol-treated mice showed significantly higher PLA₂ activity than those from controls. The brains of ethanol group showed significantly higher PLC activity, while, the heart showed significantly lower PLC activity than those of controls. The microsomes from the brain and heart of ethanol-treated mice showed significantly reduced γ values compared to those of controls. The addition of ethanol in vitro to microsomes was found to increase PLC activity in these tissues, while it decreased PLA₂ activity in a dose-dependent manner. The other n-alcohols showed similar effects on PLA₂ and PLC activity in the liver microsomes, while decreases were observed the γ values in a dose-dependent manner. These result suggest that the change in the membrane fluidity associated with adition of alcohols is a prerequisite for the changes in PLA₂ and PLC activities. In addition, our findings suggest that these changes may play a major role in the cellular injury associated with chronic ethanol treatment in the mouse.

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We studied the effect of recombinant human basic fibroblast growth factor (bFGF) on wound healing in genetically diabetic mice. Wound closure after full-thickness excision of skin was markedly delayed in diabetic mice compared to normoglycemic mice. A single application of bFGF caused a marked acceleration of wound healing in a dose-dependent manner. There were to hypertrophic scars or unlimited granulation tissue formation in regenerated tissues treated with any doses of bFGF under histological examination. The repeated application of bFGF for 7d showed a bell-shaped dose-response in the rate of wound closure, and the optimal dose was as small as 0.2-2 μg per wound. Reduced angiogenesis and granulation tissue formation were observed in diabetic mice compared to normal mice, and bFGF treatment restored both responses to significant levels. The beneficial effect of bFGF on wound healing would be largely explained by enhanced angiogenesis and granulation tissue formation.

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Rats fed from weaning on semi-purified diets supplemented either with linoleate-rich safflower oil (S) or α-linolenate-rich perilla oil (P) were mated. Half of the progeny were weaned to the original diet of the dams (SS and PP), the other two groups were shifted to diets enriched in the other fatty acid (SP and PS). Brightness-discrimination learning ability was tested daily for 30 d beginning at 11 weels of age, with a bright light as the positive stimulus. The learning performance was inferior in the group fed the safflower diet through two generations (SS) as compared with groups fed the perilla diet through two generations (PP) or for which the diets were shifted at weaning (PS and SP). The docosahexaenoate content of brain phospholipids was significantly less in the SS group compared with the three other groups. After 30 d of the learning test, the effect of shifting the stimulus was tested for another 30 d, this time using a dim light as the positive stimulus. The learning performance was superior in the PP group to the SS group throughout the latter 30 sessions, the difference being even more obvious than during the first 30 d. These results indicate that the decrease in the discrimination-learning ability induced by α-linolenate deficiency is a relatively reversible process; both the docosahexaenoate content in brain and the learning performance were restored by supplementing α-linolenate after the weaning.

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4-Methylquinoline (4-MeQ) showed an extraordinarily potent mutagenicity when compared to quinoline and isomeric methylquinolines. The major metabolite of 4-MeQ was 4-hydroxymethylquinoline, which was not mutagenic under the assay condition employed. Deuteration of the methyl group of 4-MeQ resulted in a decrease in the amount of the hydroxymethyl metabolite and an increase in mutagenicity, indicating that hydroxylation of the substitutent methyl group is a detoxication process. A 3-chloro derivative of 4-MeQ was proven to be non-mutagenic. 4-Ethylquinoline, as well as 4-hydroxymethylquinoline, was much less mutagenic than 4-MeQ. Taking account of the structure-mutagenicity relationship, a possible mechanism is proposed for the potent mutagenic potential of 4-MeQ.

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We investigated the effect of human blood plasma lipoproteins on the hemolytic activity of Asp-hemolysin. Apolipoprotein B (apoB)-containing lipoproteins, such as low density lipoprotein (LDL) and very low density lipoprotein (VLDL), inhibit the activity of this hemolytic toxin. When Asp-hemolysin (15 μg) was incubated with 100 μg LDL (as protein), the hemolytic activity was inhibited by 90%. When 20 μg apoB was added, the hemolytic activity was almost completely inhibited. Furthermore, similar inhibition was obtained in the filtrates which were separated from the incubation mixture of Asp-hemolysin with LDL or apoB following ultrafiltration through a membrane with a molecular cutoff=100000. The current findings suggest that the inhibition by LDL is due to apoB binding to Asp-hemolysin.

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We previously reported that DNA single-strand breaks (ssb) induced by exposure to dimethylarsinic acid (DMAA) were enhanced by the presence of paraquat (PQ), a superoxide anion radical (O^-₂)-producing agent, in cultured human alveolar type II (L-132) cells in vitro. In the present study, we examined the effect of sequential exposure of the cells to PQ and then DMAA under conditions causing no ssb by each alone, and observed a remarkable occurrence of ssb. The result suggests that O^-₂ caused DNA damage, which became detectable as ssb by the treatment using DMAA. The DNA damage induced by the exposure to PQ alone was different from that by DMAA; PQ-induced damage was fully repaired after 24h, while DMAA-induced damage was repaired only partially, and aphidicolin, an inhibitor of repair-serving DNA polymerases α, δ and/or ε, inhibited only the latter repair but not the former. These findings indicate that the DMAA treatment may be an effective tool to reveal DNA damage induced by O^-₂, which to date has not been sufficiently clarified.

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Low-fat conventional diets supplemented with 5 or 10% vegetable oils were fed to stroke-prone spontaneously hypertensive rats (SHR-SP9) from weaning and the mean survival times were determined. A 1% aqueous sodium chloride solution was used as drinking water throughout the experiments. In four separate experiments, the rapeseed oil group showed a significantly shorter mean survival time. The relative mean survival times were 50-59% (rapeseed oil group), 78-106% (soybean oil group) and 86% (microbial oil group) as compared with the group fed perilla oil (100%). The group which received 4-fold diluted rapeseed oil exhibited a significantly shorter survival time as compared with the group receiving soybean oil. Although the feeding experiments were performed under very simple and restricted conditions, these results suggest that the rapeseed oil prepared for human use contains a factor (s) which is toxic to SHR-SP rats.

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The effect of plant phenolics, including flavonoids and green tea polyphenolics, on hydroxyl radical was examined by a common method using an electron spin resonance (ESR) technique with 5, 5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trapping agent. The intensity of the ESR signals of DMPO-OH adduct formed by the interaction of DMPO with Fenton reagent was reduced in the presence of each phenolic in a dose-dependent manner. However, the decrease in the intensity of the signals was due partly to the enhanced disappearance of the spin adduct by the phenolics, as has been previously shown. This spin trapping method was unreliable for evaluation of the effect of the phenolics against hydroxyl radical. Hydroxyl radical induced-DNA single-strand breaks may be a better index for evaluation of the activity of the phenolics regarding hydroxyl radical. The effect of the phenolics on DNA single-strand breaks induced by Fenton reagent was examined. While sesamol and esculetin were inhibitory, most polyphenolics, especially (-)-epigallocatechin (EGC) and (-)-epigallocatechin gallate (EGCG), were rather stimulatory. The results indicate that sesamol and esculetin scavenged hydroxyl radical, and EGC and EGCG generated hydroxyl radical under the conditions where hydroxyl radical was generating.

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Cytotoxic activities of 20 steroidal glucosides obtained from Solanum genera plants were examined against various cell lines to provide new evidence as follows : 1) As regarding the sugar linkage, the glucosides possessing a β-chacotriosyl moiety were the most effective and the presence of the terminal rhamnosyl residue was required for exhibiting strong activity. 2) As for the aglycone, the glycosides carrying a spirostanol aglycone showed the strongest activity and even an aglycone without a sugar linkage presented considerably strong activity.

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Hepatoprotective effect of celosian, an acidic polysaccharide isolated from the water extract of the seed of Celosia argentea, was investigated using chemical and immunological liver injury models. Celosian inhibited the elevation of serum enzyme (GPT, GOT, LDH) and bilirubin levels on carbon tetrachloride (CCl₄) -induced liver injuries in rat. In addition, the hepatoprotective effect of celosian was also observed in this model of liver injury by histopathological findings. Moreover, celosian suppressed rises in GPT or mortality on fulminant hepatitis induced by D-galactosamine/lipopolysaccharide (D-GalN/LPS) or Propionibacterium acnes/LPS in mice. These findings suggested that celosian is an active component in protection against chemical and immunological hepatitis and the activity was found to be a dose dependent.Celosian showed a concentration dependent inhibitory effect on lipid peroxide (LPO) generation in vitro. Though celosian did not reduce the release of tumor necrosis factor-α (TNF-α), it protected against recombinant human TNF-α (rhTHF-α)-induced liver injury in D-galactosamine sensitized mice.

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Inhibitory activity against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation in mice was observed in the methanol extract of Chlorella vulgaris, a green alga. The hexane soluble fraction obtained from the methanol extract exhibited marked inhibitory activity from which were isolated two Δ^{5, 7}-sterols (ergosterol and 7-dehydroporiferasterol), two Δ^{5, 7, 9(11)}-sterols [9(11)-dehydroergosterol and 7, 9(11)-bisdehydroporiferasterol], two 5α, 8α-epidioxy-Δ⁶-sterols (ergosterol peroxide and 7-dehydroporiferasterol peroxide), and a 7-oxo-Δ⁵-sterol (7-oxocholesterol), among others. The Δ^{5, 7}-sterols, 5α, 8α-epidioxy-Δ⁶-sterols and 7-oxo-Δ⁵-sterol inhibited TPA-induced inflammation in mice. The 50% inhibitory dose of these compounds for TPA-induced inflammation was 0.2-0.7 mg/ear. Furthermore, ergosterol peroxide markedly inhibited the tumor-promoting effect of TPA in 7, 12-dimethylbenz[a]anthracene-initiated mice.

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Total DNA was extracted from the leaves of Atractylodes lancea DE CANDOLLE, A. ovata DE CANDOLLE and A. japonica KOIDZUMI ex KITAMURA of various origins and hybridized with digoxigenin-labeled rice ribosomal DNA after digestion with eight different restriction endonucleases. The resulting restriction fragment length polymorphism (RFLP) profiles allowed us to distinguish the three Atractylodes species when DNA was digested with SacI. Although atractylon was detected in the rhizomes of some of the cultivated strains of A. lancea, their RFLP profiles clearly indicate that these plants are not hybrids of A. ovata or A. kaponica.RFLP analysis also revealed the presence of intraspecific variation in DNA sequence of rRNA locus among A. lancea as well as A. japonica.

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The uptake of fractionated [³H]heparin was examined to elucidate the uptake mechanism in isolated rat Kupffer cells. The equilibrium binding of fractionated [³H]heparin to Kupffer cells was concentration-dependent with the dissociation constant of 5.7 nM and the maximum binding capacity of 1.5 pmol/10⁶ cells. Several ligands of scaveger receptors inhibited the binding of fractionated [³H]heparin to Kupffer cells competitively and also the internalization of heparin, suggesting the involvement of scavenger receptors in the uptake of fractionated [³H]heparin. Fractionated [³H]heparin was also suggested to be internalized according to first order kinetics with the apparent internalization rate constant of 0.010 min^-1. Lowering temperature from 37 to 4°C reduced the fraction internalized from 33% to 6% without affecting the total association, while the fraction internalized at 25°C was comparable with that at 37°C. Metabolic inhibitors (2, 4-dinitrophenol and rotenone), an inhibitor of receptor-mediated and adsorptive endocytosis of polypeptides (phenylarsine oxide) and phagocytosis inhibitors (cytochalasine B and colchicine) did not inhibit the internalization of fractionated [³H]heparin. As known inhibitors of receptor-mediated and adsorptive endocytosis of polypeptides and phagocytosis did not affect the uptake of fractionated heparin, the scavenger receptor-mediated uptake is suggested to be ATP-independent and differene from receptor-mediated and adsorptive endocytosis of polypeptides and phagocytosis, although for temperature dependency it showed the typical characteristics of receptor-mediated endocytosis.

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A prescription of Chinese herbal medicine, tentatively named P-19, was examined for its inhibitory effect and its mechanism using an experimental model of nephropathy induced by purified snake venom proteinase, Ac₁-proteinase (Ac₁-P). The treated mice were injected with 0.1 ml of crude extract of P-19 intraperitoneally every other day beginning 2 d before to 1 week after the injection of Ac₁-P. The non-treated mice were injected with saline instead of the medicine P-19. The physiological condition and histopathological observation of the mice at one week after Ac₁-P injection were better in the treated group than in the non-treated group. This indicates that P-19 inhibited the production of glomerular lesions induced in mice by Ac₁-P. The physiological condition and histopathological changes in the mice were better with P-19 treatment than with P-3 treatmnt. Differences in the mechanism of action between the crude extract of P-3 and P-19 are not only in diuretic action but also in the changes in the glomerular basement membrane. On the basis of spectrophotometric studies, phenolic carboxylates were confirmed to be contained in the crude extract of P-19, having a different chemical structure of caffeic acid, which is the effective component in P-3.Immunohistochemical observation revealed a difference between the groups. In the non-treated mice, deposits of the venom were clearly observed in the glomerular tuft and Bowman's capsule, corresponding to the histopathological changes, within 2.5 min after the injection of Ac₁-P. In the treated mice, the deposits were indistinct in the Bowman's capsule. The difference was considered to be caused by changes in the glomerular basement membrane after P-19 treatment.

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We examined which cytochrome P-450 (P-450) species other than CYP1A participates in the oxidative metabolism of theophylline (TP) in mouse hepatic microsomes. Among the three metabolic pathways of TP, only 8-hydroxylation was selectively enhanced by acetone, a potent inducer of CYP2E. We assumed that two P-450 populations with different metabolic ability were involved in this metabolic process, and kinetic analyses revealed that the enhancement was due to the induction of a high-capacity P-450 population. The 8-hydroxylation at a substrate concentration, where most of the total activity was attributed to the catalysis of the high-capacity phase, was markedly impaired by CYP2E inhibitors such as 4-methylpyrazole and aminoacetonitrile, whereas the N-demethylations were little affected by these agents. The activity of TP 8-hydroxylation was significantly correlated with that of p-nitrophenol hydroxylation, a probe for CYP2E, in untreated microsomes. The activities of these oxidative reactions were modified to a similar degree by known enzyme inhibitors with a range of inhibitory potencies and affinity for P-450 isoforms. On the other hand, a relationship between TP N-demethylations and p-nitrophenol hydroxylation was not apparent, but there was a nehavioral similarity between the two types of N-demethylations. The results indicated that TP 8-hydroxylation, which accounts for a large portion of TP oxidations, involves CYP2E, and that its N-demethylations are mediated by a common or closely similar P-450 species distinct from CYP2E.

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Intestinal absorption and first-pass elimination of 2', 3'-dideoxynucleosides (ddNs), including 3'-azido-3'-deoxythymidine (AZT), 2', 3'-dideoxyinosine (DDI) and 2', 3'-didehydro-3'-deoxythymidine (D4T), following oral administration was investigated in rats. Enzymatic degradation of ddNs in rat intestinal washing and in the intestinal homogenate showed them to be stable in the washing with half lives of more than 140 h, whereas degradation of DDI in the intestinal homogenate was more than ten times as rapid as those of AZT and D4T. Intestinal absorption was studied in three segments of the rat intestine (duodenum, jejunum and colon) using an in situ closed-loop method. The area under plasma ddN concentration curve (AUC) and the residual percent of dose 1 h after dosing indicated a greater absorption of AZT and D4T in the upper intestinal tract than in the colon, very poor absorption of DDI in all segments, and considerable absorption of AZT in the colon. The AUC and the mean residence time (MRT) of ddNs following four different routes (intracenous : i.v., intra portal vein : i.p.v., intra duodenal : i.d. and intra gastric : i.g.) were measured using the in vivo multiple sites of input method in rats. AZT and D4T were rapidly absorbed from the gastrointestinal tract and their bioavabilaility was more than 90%. DDI was less absorbed (33.02%) following i.d. administration compared with AZT and D4T. This poor absorption of DDI was partly attributable to its metabolism in the intestine.

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As an efficient method for estimating the amount orally absorbed, the fraction of D-xylose absorbed in rats was estimated from the ratio of the fecally excreted proportion of D-xylose dose to that of polyethylene glycol (PEG) 4000 as a nonabsorbable marker (D-xylose/PEG 4000 ratio). The D-xylose/PEG 4000 ratio was demonstrated to be independent of the fecal excretion of D-xylose and PEG 4000 (or sampling period), suggesting that it can represent the fraction of the total dose remaining. This result was consistent with the theoretical prediction based on the assumptions that every small fraction of dose behaves in the same manner with regard to absorption and transit through the absorption site (small intestine), and that the gastrointestinal transit of PEG 4000 is identical with that of D-xylose. The fraction of D-xylose absorbed was estimated to be 95.9% by subtracting the remaining fraction (D-xylose/PEG 4000 ratio) from unity (100%). The D-xylose/PEG 4000 ratio, or drug/marker ratio in general, can be determined before fecal excretion is completed. Thus it can provide an efficient way for estimating the fraction of a drug absorbed that is stable in the gastrointestinal tract, such as D-xylose. Although this proposed method of estimating a fraction is generally not applicable unless a given drug is proved to be stable in the gastrointestinal tract, it can be an effcient screening method to identify poorly absorbable drugs. It is especially useful in basic studies and preclinical tests in laboratory animals.

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Nitric oxide (NO) is an important effector molecule on antimicrobial and antitumor effects of macrophages. (1→3)-β-D-Glucan (β-glucan) is well known to show various immunopharmacological effects such as antimicrobial effect and antitumor effect by activating various points of host defense mechanisms. This paper deals with NO synthetic activity or peritoneal macrophage (PM) induced by β-glucan administration in mice. The activity was determined by measuring NO concenration in PM culture by Griess reagent after 24 or 48h in vitro culture. Administration (i.p. or i.v.) of a branched soluble (1→3)-β-D-glucan, grifolan (GRN), from Grifola frondosa enhanced NO synthesis of PM dose and time dependently. The activity was abrogated by the addition of N^G-monomethyl-L-arginine (L-NMMA) in vitro. The most significant activity was observed at 3-7d after the administration of GRN (250 μg/mouse). PM from all strains of ICR, C3H/HeN, C3H/HeJ, BALB/c, BALB/c nu/nu, C57BL, and AKR mice showed significant activity by GRN administration. Among β-glucans tested, SSG and OL-2, highly branched soluble glucans, and a particulate β-glucan, zymosan, showed similar activity. Addition of GRN directly to in vitro RAW 264.7 or proteose peptone induced peritoneal macrophage (PP-PEC) cultue could not enhance NO synthesis. However, NO synthesis of PP-PEC was enhanced in vitro by addition of GRN in the presence of interferon γ (IFNγ). Gene expression of IFNγ mRNA in the liver and PEC were enhanced in GRN administered mice assessed by reverse transcriptase assisted PCR (RT-PCR) method. These facts strongly suggested that β-glucan has capacity to enhance NO synthesis of PM in vivo through IFNγ mediated mechanism.

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We prepared an anti-idiotype (Id) abtibody against leptospirosis. Serum from rabbit immunized with mohoclonal antibody (MAb) LW2, which reacted to the main protective antigen prepared from Leptospira interrogans serovar lai, inhibited agglutination of the organism by MAb LW2. The immune rabbit serum was applied to a column coupled with normal mouse IgG as a ligand (first colume), and the unbound fraction eluted was applied to a column coupled with MAb LW2 as a ligand (second column). The bound fraction (anti-Id antibody) eluted from the second column inhibited the binding of MAb LW2 to sonicated leptospiral cells in ELISA. Mice produced antibodies against Leptospira by intraperitoneal immunization with the anti-Id antibody at doses of 2 μg/mouse or more. Hamsters were protected by immunization with the anti-Id antibody at doses of 2 and 20 μg/hamster from the lethal infection of Leptospira. This is the first report concerning the use of an anti-Id antibody against leptospirosis.

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Two kinds of dosage forms (tablets and retarded capsules) of furosemide (F) were compared in vitro dissolution profile and in vivo absorption studies. The dissolution of F from retarded capsules was extremely restricted in the first fluid of the JP XII disintegration test (within 0.8%), while the dissolution of F from tablets and retarded capsules in the second fluid of JP XII disintegration test were both complete.Metabolite specific assay of F showed F, conjugation of F with glucuronic acid (FG) and acyl migration isomers of FG (FG-iso) in urine or plasma. The mean cumulative urinary excretion of F following administration of the tablets during 24 h was twice that of retarded capsules. The mean area under the plasma concentration-time curve (AUC) of F following administration of tablets was 1.5 times that of retarded capsules. The mean cumulative urine volume during 24 h, however, was not significantly different between the two dosage forms. Clockwise hysteresis relationships between the diuretic response and the urinary excretion rate of F was observed after administration of retarded capsules. A straight relation between logarithm of the diuresis and logarithm of the urinary excretion of F was observed after maximum excretion rate of F following administration of both dosage forms.

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The effect of sesamol and 20 related compounds on the lipid peroxidation of liposomes induced by Fe²⁺, on the lipid peroxidation of rat liver microsomes induced by CCl₄ or NADPH and on the lipid peroxidation of mitochondria induced by ascorbate/Fe²⁺ were demonstrated. Consequently, sesamol and related compounds, such as 3-methoxy-4-hydroxyquinone, isosafrol, isoeugenol, eugenol, 3, 4-methylenedioxyaniline, catechol. hydroxyhydroquinone, 3, 4-dimethoxyaniline and caffeic acid, echibited powerful inhibitory effects on the lipid peroxidation system investigated. In particular, isoeugenol was the most powerful inhibitor among all the sesamol-related compounds tested on the lipid peroxidation system. In addition, 1, 2-methylenedioxybenzene, ferulic acid, and 3, 4-methylenedioxynitrobenzene were also effective on the lipid peroxidation system of liposomes induced by Fe²⁺. The correlation between the structures of sesamol-related compounds and their inhibitory effect is discussed.

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The effects of the continuous intravenous infusion of diltiazem on plasma urate levels and the urinary excretion of urate were examined in urethane anesthetized oxonate-loaded rats. The intravenous infusion of diltiazem (4 or 10 μg/rat/min) caused a gradual decrease in blood pressure, a gradual increase in renal blood flow and a transient increase in glomerular filtration rate. This infusion also caused diuresis, natriuresis, uricosuria and definite hypouricemia. Our results show that diltiazem infused i.v. has uricosuric and hypouricemic effects.

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To elucidate the molecular basis of the induction of peroxisomal enzymes, transcriptional regulation of the acyl-CoA oxidase (AOX) gane by dehydroepiandrosterone sulfate (DHEAS) was examined using primary cultures of rat hepatocytes. Incubation of the hepatocytes with 0.1 mM DHEAS resulted in a progressive increase in the cellular AOX mRNA level, which after 24 h reached a 5.6-fold higher level than that in the control cells treated with vehicle alone. However, this mRNA induction was completely inhibited by α-amanitin, an RNA polymerase II inhibitor. When examined by the nuclear run-on transcription assay, the rate of de novo synthesis of AOX mRNA was 3- to 4-fold higher in the hepatocytes exposed to DHEAS than in the control cells, even after 3 h. These results indicate that DHEAS acts directly on hepatocytes to activate the transcription of the AOX gene, which leads to the marked induction of AOX in the cell.

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DNA strand breakage induced in cultured cells by bleomycin (BLM) was remarkably enhanced in the presence of glycerol or polyvinyl alcohol which produces an enhancement effect of BLM cytotoxicity, byt not in the presence of alcohols such as methanol, ethanol, or polyethylene glycol which do not produce enhanced cytotoxicity. The comet assay, a useful analytical method for evaluating DNA strand breakage, clearly demonstrated that combinations of BLM and polyhydric alcohols induced serious DNA damage in the whole cell populations compared with those induced by treatment with BLM alone.The comet assay also successfully showed distributions of cell populations with various degrees of DNA damage. These results suggested that an increase in DNA damage induced in the presence of polyhydric alcohols might be responsible for the enhancement of BLM cytotoxicity.

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The freeze-dried ternary formulations of meclizine (MZ, an anti-motion sickness drug), prednisolone (PRED, an anti-inflammatory drug) and norfloxacin (NFLX, an anti-microbial drug) which are poorly water-soluble and are low bioavailability drugs, were prepared using egg albumin and olive oil. The powder X-ray diffractions, the dissolution rate and the bioavailabilities in vivo of these formulations were studied in comparison with each drug alone. By forming ternary formulations of these drugs, the dissolution rates of the drugs from the formulations were significantly improved compared with each drug alone. The results of their powder X-ray diffraction measurements showed that these drugs in the ternary formulations presented in an amorphous form, indicating increased dissolution rates. On the other hand, the plasma concentrations of theses drugs increased significantly after oral administration in formulations to rats, except for the NFLX formulation, and the areas under the concentration-time curves (AUC) of the ternary formulations of MZ, PRED and NFLX were 2.1, 1.6 and 1.3 times those of the drugs alone, respectively. From these results, it was proven that formulations consisting of egg albumin, olive oil and poorly water-soluble drugs were useful preparations for improving the drug's disadvantageous pharmaceutical properties.

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Isaria japonica YASUDA was cultured in a liquid medium, and its culture fluid (IJCE) was tested for stimulatory activity to humoral antibody production. IJCE significantly enhanced the production of anti-sheep red blood cell (SRBC) plaque forming cells (PFC) by oral ingestions at 10 and 30 mg/kg/d for 4 consecutive days, either before or after SRBC challenge. It also recovered the reduction of anti-SRBC PFC response and number of spleen cells caused by treatment with 5-fluorouracil. It is suggested that IJCE is a promising source for an immunomodulating tool or medicine.

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The effect of ginseng on brain glucose metabolism has been determined in normal and transient cerebral ischemic rat using the autoradiographic [¹⁴C]2-deoxyglucose method. Ginseng had no effect on local cerebral glucose utilization (LCGU) in normal rat. By contrast, in rats subjected to 30 min of four vessel occlusion, a significant reduction of LCGU was shown in parts of the cortex and striatum in comparison to the control group. One-week administration of ginseng at 200 mg/kg/d before the occlusion produced an improvement of LCGU compared with the untreated group. These findings indicate that ginseng has some protective effects against brain damage in transient global cerebral ischemia.

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The biotransformation of oxazepam by Bifidobacterium bifidum was studied. The major metabolite was purified by chromatographic methods and found to be desmethyldiazepam using NMR, IR and other physicochemical data.

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Nitric oxide (NO) readily makes corresponding complexes not only with ferrous iron but also with ferric iron. However, NO-ferric complexes of many heme proteins were unstable, while horseradish peroxidase formed the very stable NO-ferric porphyrin complex with a shift of the Soret band of the absorption wavelength from 396.5 nm to 420.0 nm. The concentration of NO in aqueous media could be monitored by measuring the absorption changes, and the detection limit was 10 nM. The simple procedure is convenient for concentration determination of NO solution.

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The feeding of cholesterol-enriched diet for 2 weeks was enough to reduce nitric oxide (NO), prostaglandin E₂ (PGE₂) and interleukin-1 (IL-1) productions in thioglycollate-ellicited murine macrophages. Although not showing anti-hypercholesterolemic action against ICR mice, Shosaikoto, a Kampo medicine, partially prevented the reduction of NO and IL-1 productions induced by the feeding of cholesterol-enriched diet, and completely released the reduction of PGE₂ production. These data suggest that the malfunction of macrophage induced by hypercholesterolemia may contribute to early atherogenesis and that Shosaikoto retains macrophage function to preveny the development of atherosclerosis, even though serum cholesterol is markedly increased.

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Hepatoprotective activity guided chemical analyses led to the isolation of two dicaffeoyl quinic acid derivatives, methyl 3, 4-di-O-caffeoyl quinate (1) and 3, 4-di-O-caffeoyl quinic acid (2) from water extract of propolis, and their structures were determined by the use of 2D NMR. These compounds were stronger antihepatotoxic agents than glucyrrhizin.

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