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Daily fat and sugar intake has increased in Japan, while total energy intake has decreased. However, the number of type 2 diabetes mellitus patients has increased, and this often causes renal injury characterized by autophagic vacuoles. Although many studies with comparisons of high fat or sugar versus a normal macronutrient balanced diet have been reported, there are few studies that equalized calorie intake and body weights. In the current study, AIN93M diets (CONT group) with matching energy content with lard derived high saturated fat (LARD group), soybean oil derived unsaturated fat (SOY OIL group) and sucrose (SUCROSE group) were provided to compare their effects on renal morphology in streptozotocin-injected CD-1 mice without causing obesity. The number of renal tubular vacuoles was higher in SUCROSE and slightly higher in LARD compared with CONT mice, and was higher in LARD and SUCROSE compared with SOY OIL mice. Most of those vacuoles were LAMP1-positive, a marker of lysosomal autophagy. These results suggest that despite identical energy contents, diets with high sucrose or saturated fat compared to unsaturated fat may aggravate lysosomal renal injury in a non-obese, streptozotocin-induced model of diabetes mellitus.

Although monosodium glutamate (MSG) is classified as a causative substance of headache in the International Classification of Headache Disorders 3rd edition (ICHD-III beta), there is no literature in which causal relationship between MSG and headache was comprehensively reviewed. We performed systematic review of human studies which include the incidence of headache after an oral administration of MSG. An analysis was made by separating the human studies with MSG administration with or without food, because of the significant difference of kinetics of glutamate between those conditions (Am J Clin Nutr 37:194–200, 1983; J Nutr 130:1002S–1004S, 2000) and there are some papers which report the difference of the manifestation of symptoms after MSG ingestion with or without food (Food Chem Toxicol 31:1019–1035, 1993; J Nutr 125:2891S-2906S, 1995). Of five papers including six studies with food, none showed a significant difference in the incidence of headache except for the female group in one study. Of five papers including seven studies without food, four studies showed a significant difference. Many of the studies involved administration of MSG in solution at high concentrations (>2 %). Since the distinctive MSG is readily identified at such concentrations, these studies were thought not to be properly blinded. Because of the absence of proper blinding, and the inconsistency of the findings, we conclude that further studies are required to evaluate whether or not a causal relationship exists between MSG ingestion and headache.

Background The aim of the present study was to evaluate standard reference material (SRM) 1955 commutability as a reference material for serum folate using automated methods. We also designed so as to reduce the intermethod variability present in different automated methods. Methods Using a microbiological assay related to the ‘information value’ of SRM 1955 as a comparison method, we investigated the possibility of standardization for the assay values of serum folate as measured by the automated methods (Access, Centaur and Elecsys). In the assay of 50 patient sera by these automated methods, we corrected observed values by the SRM 1955 and compared with comparison values. Results The observed values of SRM 1955 Levels I, II and III were within or outside (but near) a 95% prediction interval obtained from patient sera by the automated methods. The normalized residuals obtained from SRM 1955 were within ±3.0 (in SD units), which enabled us to conclude that the SRM 1955 had a physicochemical characterization similar to native serum. Twelve patients were assessed as hypofolataemia (Conclusions The use of SRM 1955 would help to reduce the intermethod variability present in different automated methods for serum folate measurement.

We have hypothesized that a close correlation exists between tryptophan metabolism, exercise performance and mood. Before and after a 20-km run, a POMS test and serum l -kynurenine determination were performed. Serum l -kynurenine concentration was negatively correlated with running distance. After running, scores for Vigor and serum l -kynurenine concentration were correlated (r = 0.556); subjects with higher l -kynurenine concentrations tended to have higher scores for Vigor. In this study, we attempted to confirm this hypothesis. The present results suggest a close correlation between tryptophan metabolism, exercise performance and mood.

疾患の予防と治療において食の果たす役割は非常に重要である．しかし，一般の人々は現在非常に多く出回っている健康食品をどのように用いたら良いか理解するのが中々大変である．こんな状況下において厳しい生化学的教育を受ける人たちを対象に教育機関において「健康食品管理士」を養成する方法が考えられ具体化した．高度な生化学的教育を受けたこの健康食品管理士は今後の代替医療を発展させる重要な一因となってゆくことが期待される．

Calcium polycarbophil has been found to be useful in the treatment of irritable bowel syndrome. It is activated through transformation to polycarbophil by decalcification under the acidic conditions in the stomach. Polycarbophil absorbs water in the small intestine, where conditions are neutral, causing it to swell and form a gel.We encountered a patient in whom calcium polycarbophil was effective when given alone but developed diarrhea when it was administered together with lithium carbonate. When these two agents were taken separately, the diarrhea was relieved. In order to ascertain how lithium carbonate affected calcium polycarbophil, we carried out an in vitro experiment whose results showed that lithium carbonate caused an increase in pH. This inhibited decalcification, and reduced the extent of swelling of polycarbophil. These results suggest that the efficacy of calcium polycarbophil would be reduced when administered together with lithium carbonate.

Hematological studies have revealed the importance of the visceral yolk sac (VYS) in the primitive erythropoiesis of mouse embryos at an early stage before day 12. We examined the possibility of the occurrence of extra-embryonic erythropoiesis at a stage later than embryonic day 12 by light and electron microscopic analyses. Surprisingly, a novel structure in the form of erythrocyte-like globules was observed in the VYS endodermal cells. They were consistently present in the VYS endodermal cells from embryonic day 12 until day 18 (birth is day 19), by immunocytochemical and enzyme histochemical analyses. They were immuno-positive for mouse erythrocyte antibody and also positive for the benzidine reaction showing the presence of hemoglobin. The erythrocyte-like globules were shown to be the erythrocytes present in the cytoplasm. These results indicated that erythropoiesis in the VYS endodermal cells continues from the early embryonic stage, as primitive erythropoiesis, until the late stage.

Rat cauda epididymal spermatozoa treated with α-chlorohydrin at concentrations of 0, 0.01, 0.1, and 1.0 under conditions that support in vitro fertilization were used to evaluate the effects of acrosomal status and sperm motility in predicting their fertilizing capacity. Acrosomal status was assessed by fluorescein isothiocyanate-conjugated concanavalin A (FITC-ConA) lectin assay in combination with supravital stain using calcein acetoxy methyl ester (CAM) and ethidium homodimer-1 (EthD-1) at 1, 3, and 5 h of incubation. Sperm motility was also examined with a computer assisted sperm analysis (CASA) system at the same time points as acrosomal status. The movement of spermatozoa treated with α-chlorohydrin at 0.01 mM was similar to the control spermatozoa over 5 h of incubation. The 0.1 mM treated spermatozoa showed progressive movement at 1 h of incubation, and low vigorous movement was seen after 3 h of incubation. The 1.0 mM treated spermatozoa showed low progression and low vigorous movement over 5 h of incubation. The FITC-ConA patterns of rat spermatozoa undergoing acrosome reaction were marked by the appearance of bright green fluorescence from the acrosomal region on their head, and were clearly distinguished from the degenerative acrosome loss in dead sperm. A time-related increase in the percentage of live acrosome lost sperm was observed in the control and 0.01 mM α-chlorohydrin treated groups, but a low percentage and no time-related increase in live acrosome lost sperm without degenerative acrosome loss in dead sperm were observed in the 0.1 and 1.0 mM treated groups. On the basis of these results, rat spermatozoa labeled with FITC-ConA combined with CAM and EthD-1 can have their acrosomal status clearly distinguished. The α-chlorohydrin treated spermatozoa were suppressed not only vigorous movement but also acrosome reaction, and the results suggested that the present staining procedure for acrosomal status can be a useful indicator for predicting spermatozoal fertilizing capacity.

Dual fluorescent staining (DFS) with calcein acetoxy methyl ester (CAM), which labels the cellular esterase activity that is a major component of energy metabolism in cellular mitochondria, and with ethidium homodimer-1 (EthD-1) was used to evaluate mitochondrial function and membrane integrity in rat spermatozoa. The spermatozoa stained by DFS could be classified into three different populations microscopically when excited at 490 nm after 60 min incubation. 1) Spermatozoa, which were stained with CAM alone and had maintained either mitochondrial function or membrane integrity, were identified as live during incubation. 2) Spermatozoa, which were stained with EthD-1 alone and had lost either mitochondrial function or membrane integrity, were identified as already dead at the beginning of incubation. 3) Spermatozoa, which were stained with both CAM and EthD-1 and had maintained mitochondrial function with membrane breached, were identified as having died during incubation. Two toxicological tests, an in vitro triton X-100 experiment and an in vivo nitrobenzene experiment, were done. All spermatozoa were immobilized and lost either mitochondrial function or membrane integrity by 1.0% triton X-100 treatment. Almost no motile sperm were found at 0.1% in the triton X-100 group and in the groups treated with 60 and 40 mg/kg/day of nitrobenzene, and these spermatozoa maintained their mitochondrial function but had their membrane breached. In conclusion, the DFS procedure, which uses CAM and EthD-1, can clearly and visually identify the population of viable and dead spermatozoa simultaneously by fluorescence microscopy in rats. This is a useful technique to characterize sperm status, which is determined by the mitochondrial function assessed by CAM and membrane integrity evaluated by EthD-1.

In order to clarify the cause of ommochrome deficiency in an albino strain of the terrestrial isopod, Armadillidium vulgare, levels of xanthommatin, 3-hydroxykynurenine, 3-hydroxyanthranilic acid and tryptophan in whole body extracts of the albino and the wild type individuals were determined together with enzyme activities of kynurenine-3-hydroxylase, kynureninase and tryptophan-2,3-dioxygenase. Xanthommatin could not be detected in the albinos. The levels of 3-hydroxykynurenine and 3-hydroxyanthranilic acid were determined by high-performance liquid chromatography (HPLC) with electrochemical detection and were markedly low in the albinos compared with the wild type individuals. In contrast to those, the tryptophan levels determined by HPLC with fluorescence detection did not differ significantly between the two phenotypes. In the albino A. vulgare, kynurenine-3-hydroxylase activity was lower and kynureninase activity was higher than in the wild type, although the differences were not statistically significant. Tryptophan-2,3-dioxygenase activity in the albinos was less than 10% that in the wild type. Thus, ommochrome deficiency in the albino A. vulgare is considered to be caused by the extremely low activity of tryptophan-2,3-dioxygenase.

The conversion of gamma-glutamylcysteinylethyl ester (gamma-GCE) to glutathione in a reduced form (GSH) was examined using isolated rat hepatocytes pretreated with diethylmaleate, a GSH-depletor. Incubation of hepatocytes with 0.1 and 5.0 mM gamma-GCE (gamma-GCE-hepatocytes) over a 30-min period resulted in time-dependent increases in intracellular GSH and nonprotein-SH (NP-SH) concentrations. Hepatocytes incubated with 5.0 mM but not 0.1 mM GSH over a period of 30 min showed a time-dependent increase in intracellular GSH concentration. In the gamma-GCE-hepatocytes pretreated with bis-(p-nitrophenyl)phosphate (BNPP), a non-specific esterase inhibitor, an enhancement of intracellular GSH concentration was markedly reduced. gamma-GCE concentration in the gamma-GCE-hepatocytes with BNPP pretreatment was significantly higher than that in the cells without BNPP pretreatment, although there was no difference in the total amount of intracellular NP-SH, i.e., gamma-GCE, GSH, gamma-glutamylcysteine, cysteine ethyl ester, and cysteine between both gamma-GCE-hepatocytes. The present results indicate that gamma-GCE is transported into liver cells more easily than GSH itself, resulting in its conversion to GSH via esterase and glutathione synthetase within the cells.

The direct role of albumin in L-tryptophan transport into hepatocytes was studied by examining the effect of bovine serum albumin (BSA) on L-tryptophan uptake by isolated rat hepatocytes in comparison with that on the uptake of L-phenylalanine and L-leucine, which are known to be taken up into hepatocytes via the transport systems common to L-tryptophan. When the uptake rates of these amino acids were determined in an incubation medium containing BSA and at the same free-form substrate concentration, the rate of L-tryptophan uptake increased, while the rates of L-phenylalanine and L-leucine uptakes remained unchanged. L-Tryptophan was well bound to BSA, while L-phenylalanine and L-leucine were scarcely bound to the protein. BSA was clearly bound to the incubated cells. From the kinetic analysis of L-tryptophan uptake by hepatocytes with and without BSA, BSA was found to enhance the affinity between L-tryptophan and its transport site on the surface of liver cells. In hepatocytes pretreated with BSA, only L-tryptophan uptake was stimulated, in which the affinity between L-tryptophan and its transport site was enhanced. A significant amount of BSA was bound to the cells pretreated with the protein. When the effects of BSA co-addition and its pretreatment on the binding of L-tryptophan to plasma membranes prepared from rat livers were examined, the protein was found to promote the binding of L-tryptophan to the membranes. The present results suggest that albumin can facilitate L-tryptophan transport into hepatocytes by enhancing the affinity between the amino acid and its transport site present on the cell surface through its direct interaction with both the amino acid and the plasma membrane.

We examined the changes of phospholipase A 2 (PLA 2 ) activity and of reduced glutathione (GSH), Ca 2+ , and lipid peroxide (LPO) contents in isolated rat hepatocytes treated with anti-liver cell plasma membrane antiserum (ALPMA) following the development of immunological cell injury. Increases in PLA 2 activity and Ca 2+ content and a decrease in GSH content in the ALMA-treated hepatocytes occurred with an increase in release of alanine and asparatate transaminases from the cells. After these changes occurred, LPO level increased in the ALPMA-treated hepatocytes. These results suggest that in ALPMA-treated rat hepatocytes, depletion of intracellular GSH, which has a function of cell membrane stabilization, followed by increases in Ca 2+ level and PLA 2 activity primarily contributes to immunological cell injury and that stimulation of intracellular LPO production consequent of increased PLA 2 activity is secondarily contributory to the cell injury.

A new enzymatic assay method for ornithine carbamoyltransferase, OCT, activity was developed using ornithine:2-oxo-acid aminotransferase, OKAT, and 1-pyrroline-5-carboxylate dehydrogenase, P5CDH. The method is based on the reaction of L-ornithine formation from L-citrulline by OCT. The OCT activity was measured spectrophotometrically at 340 nm and the minimum detectable activity was about 4 units/L of serum. Results obtained by this method and by the chemical method correlated well. This method is superior to the chemical method, as it does not require boiling of the reaction mixture nor removal of interfering substances in the serum.

Stepwise glycosidation was adopted for the construction of glycyrrhetic acid β-glycosides (27-30) having β(1→2)-linked disaccharides such as 2-O-β-D-flucuronopyranosyl-β-D-glucopyranose, 2-O-β-D-glucuronopyranosyl-β-D-galactopyranose, 2-O-β-D-glucopyranosyl-β-D-glucuronopyranose and 2-O-β-D-galactopyranosyl-β-D-glucuronopyranose. In the first glycosidation, 2-O-trichloroacety-β-D-pyranosyl chlorides (9-11) were utilized as starting sugar derivatives to react with methyl glycyrrhetinate (5) : Glycosidation of 5 with 9 and 10 gave β- and α-monoglycosides (12) and (13), and (15) and (16), respectively. Treatment of the β-glycoside 12 and 15 with ammonia-saturated ether gave products (14) and (17), respectively. The glycosidation of 5 with 11 followed by treatment with ammonia-saturated ether gave compounds (18) and (19), respectively. The second step glycosidations of 14 and 17 with methyl 2, 3, 4-tri-ο-acetyl-α-D-glucuronatopyranosyl bromide (20) gave diglycoside derivatives (23) and (24), respectively, and that of 18 with 2, 3, 4, 6-tetra-O-acetyl-α-D-glucopyranosyl bromide (21) and -α-D-galactopyranosyl bromide (22) gave deglycoside derivatives (25) and (26), respectively. The removal of the protecting groups of 23-26 gave diglycosides 27-30, respectively, having a β-D-glucuronopyranose (β-D-glcUA) as one of two sugar components in the molecules. The cytoprotective effects of the synthesized glycosides 27-30 on carbon tetrachloride (CCl4)-induced hepatotoxicity in vivo were compared with deglycosides 31-33 having only neutral sugar components, and naturally occurring glycyrrhizin (34) having two acidic sugar components (β-D-glcUA). While glycosides 31-33 had no cytoprotective effect, glycosides 27-30 showed potent effects. Especially, 27 and 28, having a β-D-glcUA as the terminal sugar component, were more effective meterials against hepatic injury than glycyrrhizin 34.

We examined the inhibitory effects of “group A saponin” and “group B saponin” fractions, which were extracted and separated from soybean seed hypocotyls, on water-soluble 2, 2′-azobis(2-amidinopropane) (AAPH)- and lipid-soluble 2, 2′-azobis(2, 4-dimethylvaleronitrile)(AMVN)-initiated lipid peroxidation reactions that were conducted with mouse liver microsomes. The simultaneous addition of the group A saponin fraction dose-dependently inhibited AAPH- or AMVN-initiated lipid peroxidation in microsomes more strongly than that of the group B saponin fraction. The group A saponin fraction inhibited the AAPH-initiated lipid peroxidation with a lag phase, while it immediately blocked the AMVN-initiated lipid peroxidation. The group A saponin fraction inhibited microsomal AAPH-initiated lipid peroxidation even when added to the reaction mixture after the lag phase period. Microsomes pretreated with the group A saponin fraction showed inhibition of AAPH-initiated lipid peroxidation with a prolonged lag phase, and the saponin fraction-pretreated microsomes showed inhibition of the AMVN-initiated lipid peroxidation in which a lag phase was found. These results indicate that in mouse liver microsomes, the group A saponin fraction from soybean seed hypocotyls, which is present outside and/or near the microsomal membranes, inhibits AAPH-initiated lipid peroxidation by inhibiting the initiation and propagation of this reaction, while it prevents microsomal AMVN-initiated lipid peroxidation mainly by inhibiting the propagation of this reaction. In addition, the present results indicate that the group A saponin fraction can inhibit AAPH- or AMVN-initiated lipid peroxidation in mouse liver microsomes by its presence within the membranes and/or by binding to them.

Glycyrrhetic acid glycosides (1-7) having beta(1----2)-linked disaccharides such as 2-O-beta-D-glucopyranosyl-beta-D-galactopyranose, 2-O-beta-D-galactopyranosyl-beta-D-galactopyranose, 2-O-beta-D-glucuronopyranosyl-beta-D-galactopyranose, 2-O-beta-D-glucopyranosyl-beta-D-glucuronopyranose, 2-O-beta-D-galactopyranosyl-beta-D-glucuronopyranose, 2-O-beta-D-galactopyranosyl-beta-D-glucopyranose, 2-O-beta-D-glucuronopyranosyl-beta-D-glucopyranose, respectively, were synthesized by stepwise construction; from glycyrrhetic acid monoglycosides to the diglycosides. The cytoprotective activities of the glycosides 1-7 and 2-O-(beta-D-glucopyranosyl)-beta-D-glucopyranosyl-11-oxoolean-12-e n-30-oate (8) were compared with natural occurring glycyrrhizin (9). Among these glycosides 1-8, glycosides 3 and 7 having beta-D-glucuronopyranose (glcUA) as the only terminal sugar component were more effective materials against hepatic injury than glycyrrhizin 9.

It is well known that avian yolk sac is involved in both primitive and definitive erythropoiesis during embryonic development. Definitive erythropoiesis is first detected at about 4-5 days incubation and its maximum activity is reached between day 10 and 15 of incubation, ending between days 18 and 20 of incubation. We confirmed the definitive erythropoietic foci in the chicken yolk sac throughout the 5th to 19th day of incubation by histochemical light and electron microscopy. The definitive erythropoietic foci were observed in the yolk sac endodermal layer from day 5 until day 19, just before hatching. Ultrastructurally, definitive erythropoietic foci were observed extravascularly in the yolk sac endodermal cell layer in direct contact with the vitellolysis zone. These findings provide a basis for clarifying definitive erythropoiesis in vertebrates.

Eleven triterpenoidal saponins having oleanolic acid or hederagenin as the aglycone and two flavonoidal glycosides were isolated from the leaves of Aralia elata SEEM. (Araliaceae). The structures of four new saponins (1, 2, 3, and 4) were established to be 3-O-α-L-rhamnopyranosyl(1→2)-α-L-arabinopyranosyl-hederagenin 28-O-β-D-xylopyranosyl-(1→6)-β-D-glucopyranosyl ester, 3-O-α-L-rhamnopyranosyl(1→2)-α-L-arabinopyranosyl-oleanolic acid 28-O-β-D-xylopyranosyl(1→6)-β-D-glucopyranosyl ester, 3-O-β-D-glucopyranosyl(1→3)-α-L-rhamnopyranosyl(1→2)-α-L-arabinopyranosyl-hederagenin 28-O-β-D-glucopyranosyl(1→6)-β-D-glucopyranosyl ester, and 3-O-β-D-glucopyranosyl(1→3)-α-L-rhamnopyranosyl(1→2)-α-L arabinopyranosyl-hederagenin, respectively, on the basis of spectroscopic and chemical evidence.

Light and electron microscopic examination of first-trimester and term human placental tissues were performed to identify erythrocytes containing hemoglobin in the villous trophoblast cell layer. Erythrocytes were not identified in chorionic villous epithelium at week 7 of gestation. These cells first appeared in the villous cytotrophoblast at week 8, and continued to be present in the villous cytotrophoblast until week 9, as shown by benzidine staining. At week 12 gestation, a cluster of erythrocytes was present in a villous syncytial sprout. At 40 and 41 weeks gestation, erythrocytes were located in the villous cytotrophoblast cell layer. Electron microscopic observations focused on the cytoplasm of villous cytotrophoblast at week 8, the syncytial sprout at week 12 and the cytotrophoblast cell layer at term, confirmed the presence of erythrocytes at an extravascular location, as observed by light microscopy.

We have already reported that exercise activates kynurenine pathway. But, the mechanism for this activation by exercise is still unclear. Kynurenine is metabolized to NAD, which is an essential factor for energy metabolism. In this study, exercise on treadmill was loaded to rats until all-out and tryptophan metabolites and tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) activities were determined in the blood, livers, and macrophages, respectively, in the exercise-loaded rats. The mean values of serum tryptophan concentration decreased from 92.6 +/- 6.0 nmol/ml to 52.4 +/- 10.2 nmol/ml (p0.05) just after treadmill load. The serum kynurenine concentration had increased from 2.06 +/- 0.25 nmol/ml to 3.08 +/- 0.62 nmol/ml (p0.005). And whole blood NAD concentration increased from 68.8 +/- 14.6 nmol/ml to 77.9 +/- 19.1 nmol/ml (p0.005). These results showed that exercise activated the kynurenine pathway of tryptophan metabolism and made NAD which will be concerned with energy metabolism in mitochondria. Tryptophan-NAD pathway was initiated by cleavage of indole ring of tryptophan by TDO in the liver and IDO in many organs. We had also found that the exercise increase IDO activity of macrophages, but not TDO activity.

Background: Serum ornithine carbamoyltransferase is a diagnostic marker of hepatic disorders due to its localization in periportal mitochondria. Methods: We have developed a new method for the determination of serum ornithine carbamoyltransferase. It is based on the reverse reaction of ornithine carbamoyltransferase, using ornithine-ketoacid aminotransferase, ∆1-pyrroline-5-carboxylate dehydrogenase and glutamate dehydrogenase, which together convert citrulline through ornithine to glutamate. The glutamate is then quantitatively measured using glutamate oxidase and Trinder's reagent. Results: The results obtained by this method agreed well with those obtained using the diacetylmonoxime method as a gold standard [correlation coefficient (r) = 0·973 P1-pyrroline-5-carboxylate) were eliminated by the initial futile reaction. The new method appears to be more accurate at low levels of ornithine carbamoyltransferase activity than the diacetylmonoxime method. Conclusions: Here we report a new method for serum ornithine carbamoyl-transferase assay which might be useful for clinical diagnosis of hepatic disorders, including hepatic cancer.

The mouse visceral yolk sac (VYS) is widely known to play an important role as erythropoietic tissue during embryonic periods. Mouse VYS from embryonic days 9 to 12 was examined by light microscopy, electron microscopy and histochemical analysis with benzidine to detect the presence of hemoglobin with special reference to the development of VYS, the disappearance of the blood islands in VYS, and the appearance of a novel structure in the form of erythrocyte-like globules in VYS endodermal cells. The villous appearance of VYS became complicated by the development of VYS endodermal cells. The blood islands positive for the benzidine reaction were light microscopically detected on embryonic days 9, 10, and 11. They disappeared on embryonic day 12, however. Erythrocyte-like globules positive for the benzidine reaction were not observed in VYS endodermal cells on embryonic days 9, 10, and 11, but then appeared on embryonic day 12, by light and electron microscopy. Erythrocyte-like globules in VYS endodermal cells, which appear after the disappearance of blood islands in VYS, may participate in erythropoiesis during embryonic development.

We have already reported that exercise activates kynurenine pathway. But, the mechanism for this activation by exercise is still unclear. Kynurenine is metabolized to NAD, which is an essential factor for energy metabolism. In this study, exercise on treadmill was loaded to rats until all-out and tryptophan metabolites and tryptophan 2,3dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) activities were determined in the blood, livers, and macrophages, respectively, in the exercise-loaded rats. The mean values of serum tryptophan concentration decreased from 92.6/6.0 nmol/ml to 52.4/10.2 nmol/ml (p

Adriamycin (ADR), an anticancer drug which induces testicular toxicity, was administrated to Slc:SD male rats at doses of 0.5, 1.0, and 2.0 mg/kg intravenously once a week for 4 weeks. The males treated with ADR were mated with untreated females, and sperm analyses (motion, count, and morphology) were performed. Sperm motion was analyzed by Hamilton-Thorne Sperm analyzer (HTM-IVOS) to investigate the useful parameters. Copulated females were necropsied at Day 13 of gestation, and reproductive status was evaluated. In ADR-treated groups, the testicular weight was dose-dependently decreased. Associated with this decrease was a depletion of the number of spermatogonia noted histopathologically at all dosage levels. Sperm morphological abnormalities, which were classified as tailless sperm and/or no-hook head sperm, were increased in both the 1.0 and 2.0 mg/kg groups. The males treated with ADR at 1.0 and 2.0 mg/kg had a decreased number of sperms per cauda epididymis. In sperm motion analysis, decreases in the percentage of motile sperm, percentage of progressive sperm, and sperm velocity (straight line velocity and curvilinear velocity) were noted at 2.0 mg/kg. Impaired fertility was noted at 2.0 mg/kg in the form of decreased numbers of implantations and live embryos, and an increased number of pre-implantation losses. In conclusion, ADR induced deterioration of sperm motion and sperm content, which were responsible for the adverse effect on male fertility. The most sensitive indicators to detect male reproductive toxicity induced by ADR were testicular weight and histopathological findings in the testis. Among the parameters generated by HTM-IVOS, the percentage of motile sperm, the percentage of progressive sperm, and sperm velocity are useful for assessing male fertility.

The terrestrial isopod, Armadillidium vulgare is usually grey or black in color, however, red ones are occasionally found in the field. This is caused by the mutation of the ommochrome genesis in the integument. We focused our experiments on the mechanism of pigment genesis in which tryptophan metabolism had been expected to be different from the grey or black wild types. We obtained the result that 3-hydroxyanthranilic acid content was significantly higher in the red phenotype than in the wild type, and kynureninase activity was also higher in the red phenotype.

Tryptophan administration aggravates experimental mouse liver injury caused by carbon tetrachloride when 3-hydroxyanthranilic acid concentration elevates in serum. Tryptophan metabolism is changed by macrophages in injured liver. 3-Hydroxyanthranilic acid may be oxidized to cinnabarinic acid by injured mitochondria in the liver aggravating the state of injured liver. Mitochondria prepared from the liver 24hr after CCl4 treatment have lost their ability of respiratory control. In consequence, 3-hydroxyanthranilic acid is oxidized to cinnabarinic acid by incubation with these mitochondria, whereas 3-hydroxykynurenine is not. Thus, formed cinnabarinic acid is able to inhibit completely the mitochondrial respiratory control at concentration of 10μM.

Kynurenine pathway of tryptophan makes a lot of physiological active substances, such as quinolinate, NAD and so on, suggesting that kynurenine itself may play a very important role physiologically.

The recovering effect of betaine (derivative of choline) on carbon tetrachloride (CCl4)-injured liver was investigated by oral and intraperitoneal administration of betaine at 24 h after acute CCl4 intoxication. The effect of betaine was estimated by the activity of alanine aminotransferase (ALT) released into the serum, histological score, and the incorporation of S-phase indicator (5-bromo-2-deoxyuridine/5-fluoro-2'-deoxyuridine; BrdU/FdU). A significant decrease in the activity of serum ALT was observed by the intraperitoneal (3 mg/kg body) or oral (15 mg/kg body) administration of betaine. Furthermore, an increase in the uptake of BrdU into the hepatocyte nuclear DNA and a reduction in liver necrosis after the oral treatment of betaine (15 mg/kg body) was observed. From these results, the administration of betaine showed a significant effect in recovering CCl4-injured liver.

Ethanolamine, a positively charged hydrophilic component of the phosphatidylethanolamine (PE) which is also a precursor of phosphatidylcholine (PC), enhances the repair processes 24 h after carbon tetrachloride (CCl4) intoxication of mouse liver. Ethanolamine quickly reduced aminotransferase activity released in the serum, increased the hepatocellular nuclear BrdU uptake, a marker of S phase, and the epidermal growth factor (EGF) receptor content in the liver injured with CCl4. Thus, exogenous administration of ethanolamine may function as a liver proliferating factor in Ccl4 intoxicated mouse liver.

We have reported that administration of tryptophan (Trp) usually cures hepatic injury caused by carbontetrachrolide but occasionally causes aggravation of it and the serum concentration of kynurenine (Kyn) in hepatic injured mouse increases even though the low activity of tryptophandioxygenase (TDO) in hepatic injury. We will discuss that the accel¬eration of Kyn generation would be caused by macrophage(MP) and some Trp metabolites may play an important role in both the cure of hepatic injury or aggravation of it.

Glycosylations of 3-O-(2,3,4-tri-O-acetyl-alpha-L-rhamnopyranosyl(1--2)- 3,4-di-O-acetyl-alpha-L-arabinopyranosyl)-23-O-acetylhederageni n (15) with mono- (16), di- (17) and trisaccharide bromide (18) gave the bisdesmoside peracetates 19, 20 and 22, respectively, which were treated with 5% KOH in MeOH to give the bisdesmosides 25-27. Hydrolysis of the glycosides 6 and 9 having beta-D-glucopyranose as a terminal sugar component with beta-glucosidase in acetate buffer (pH 4.7) gave compounds 28 and 29, respectively. Cytoprotective effects of the synthesized triterpenoidal saponins against CCl4-induced hepatic injury were compared with those of saponins isolated from the leaves of Aralia elata Seem. (Araliaceae) using isolated hepatocytes from rat liver. Although the monodesmosides 1-4 having neutral sugar components only at the O-3 position on the aglycones showed no cytoprotective effect, bisdesmosides having sugar components at both the O-3 and O-28 positions on the aglycones had potent effects, even when the species of the sugar components were different. The bisdesmosides 10, 11, and 27 having five monosaccharides in the molecules exhibited the most potent cytoprotective effects.

The fact that a considerable amount of kynurenine (Kyn) is species-specifically accumulated in rat fur was discovered in 1960 by Ishiguro and co-workers (Hotta et al., 1960). The cause of this phenomenon has been exhaustively investigated in our laboratory, but no adequate explanation has been obtained so far.

In adult rats, about 10% of total plasma L-tryptophan (Trp) (ca. 100 μM) exists in a free form (not albumin-bound) under physiological conditions (Madras et al., 1974). We have demonstrated that Trp uptake into adult rat hep-atocytes occurs mainly via a trypsin-sensitive high-affinity transport component (Km – 14 μM) under physiological conditions (Saito et al., 1986). We have also shown that Trp uptake into adult rat hepatocytes via the high-affinity transport component is closely related to liver tryptophan 2,3-dioxy-genase (TPO) activity (Saito et al., 1987). However, rat liver TPO activity is known to appear rapidly about two weeks after birth (Franz and Knox, 1967). In addition, the proportion of free Trp, which can be utilized readily in tissues, in the plasma of suckling rats has been shown to be high compared to that of young and adult rats (Sarna et al., 1970). Taking these findings into account, it is suggested that Trp transport into suckling rat liver is different from that into young and adult rat liver. In order to elucidate the characteristics of Trp transport into suckling rat liver, we have examined the changes of liver TPO activity and Trp levels not only in serum and liver but also brain and muscle during development, using male rats aged 10 to 42 days. We also compared the mode of Trp uptake into hepatocytes between suckling and adult rats using isolated hepatocytes.

Many kinds of organic anions such as long-fatty acids, bilirubin, bile acids and hormones, bind to albumin in the plasma. L-Tryptophan (Trp) also binds to albumin in the plasma, and this property is quite different from those of other amino acids (Macmenamy and Oncley, 1958). Recent studies on the transport of organic anions into liver cells using perfused rat liver have suggested that ligands tightly bound to albumin rather than unbound li-gands are transferred into liver cells readily, and that hepatic uptake of ligands is primarily mediated by the direct interactions between albumin-ligand complexes and the surface of liver cells (Weisigar et al., 1981).

In order to characterize the system of L-tryptophan (TRP) transport into liver during the growing period of 10 to 42 days, the changes of tryptophan 2,3-dioxygenase (TDO) activity, levels of serum, liver, brain, and muscle TRP, and the rate and mode of TRP uptake into isolated hepatocytes were examined in male Wistar rats. Liver TDO activity increased rapidly at 16 days of age. A marked and rapid decrease in free serum TRP level occurred before weanling, while a small decrease in total serum TRP level was found after weanling. The change of liver TRP level was similar to that of free serum TRP level and correlated well. There was a significant inverse correlation between liver TDO activity and either free serum TRP level or liver TRP level. A rapid change in TRP level did not occur in brain and muscle during the growing period. The concentrations of brain and muscle TRP correlated better with those of total serum TRP than with those of free serum TRP. The rate of TRP uptake into hepatocytes isolated from rats aged 10 days was lower than that from rats aged 21 and 42 days. The former hepatocytes were lacking in a high-affinity saturable transport component for TRP uptake which was present in the latter ones. The present results indicate that a great change in the system of TRP transport into liver occurs in growing rats, and that in suckling rats a high level of free serum TRP contributes to the efficient transport of the amino acid into the liver.

An enzyme immunosensor was constructed for the determination of human chorionic gonadotropin (HCG), which is a hormone and an important diagnostic measure of pregnancy. An antibody to HCG was immobilized to a membrane. The antibody-bound membrane was placed onto an oxygen probe so as to react with HCG either specifically or selectively. Catalase, which catalyzes the decomposition of H2O2 into H2O and O2, was used to label HCG. Nonlabeled HCG to be assayed and catalase-labeled HCG were competitively reacted with the membrane-bound antibody of the sensor to form an antigen-antibody complex on the membrane surface. After the removal of nonspecifically adsorbed HCG, the sensor was contacted with a H2O2 solution. The membrane-adsorbed catalase enzymatically generated oxygen with a resulting increase in cathodic current of the sensor. The HCG concentration was determined from the initial rate of the current increase. The enzyme immunosensor was applied to the determination of HCG in the concentration range of 2 × 10−2 to 102 IU/ml.

The formation of cinnabarinate in the presence of manganese ions and catalase was investigated spectrophotometrically. The absorption peak of cinnabarinate at 460 nm appeared only in a reaction system containing manganese ions and catalase. If catalase was omitted, a new absorption peak at 360 nm was observed while the absorption peak of cinnabarinate reached a plateau. Furthermore, in the presence of hydrogen peroxide, the absorption spectrum of cinnabarinate changed; catalase suppressed this absorption change. We conclude that in the course of cinnabarinate formation in the presence of manganese ions hydrogen peroxide is produced, which decomposes cinnabarinate. Catalase prevents the accumulation of hydrogen peroxide, which results in the steady increase of cinnabarinate. Cinnabarinate formation by manganese ions shows an initial lag phase. This lag phase disappeared by preincubating 3-hydroxyanthranilate under aerobic conditions. Incubation of 3-hydroxyanthranilate resulted in the generation of superoxide anions. When both manganese ions and superoxide anions were present, the lag phase of cinnabarinate formation disappeared. In the process of cinnabarinate formation, manganese ions serve to dismutate superoxide anions, as does superoxide dismutase; manganese (II) ions were oxidized to manganese (III) ions by superoxide anions. From these results we have proposed a mechanism of cinnabarinate formation catalysed by manganese ions.

An immuno responsive membrane was prepared by immobilizing Wasserman antigen to acetylcellulose. The antigen-binding membrane was used in developing an immunosensor for determining specifically corresponding antibody in serum. The senor is characterized by electrochemical determination of the antigen-antibody complex formed at the membranesoluti on interface. Experimental data on the applicability of the sensor to serology tests for syphilis are given as well as an outline of a possible mechanism for evaluating the electrical potential difference across the antigen-binding membrane.

An immuno sensor for determining specific protein has been developed. The measuring system is characterized by the potentiometric determination of potential difference across an antigenbinding membrane. A lipid antigen containing cardiolipin, phosphatidyl choline, and cholesterol was immobilized to an acetylcellulose membrane. The membrane-bound antigen retained the immunochemical reactivity to Wassermann antibody. The assymmetric membrane potential developed was dependent on the concentration of the antibody.

We have found an enzyme system that catalyzes the conversion of L-tryptophan to L-kynurenine, presumably via L-formylkynurenine, in soluble and insoluble fractions of rat skin. The enzymatic activity was stimulated by hematin, ascorbate, and catalase, but not by methylene blue. Highest activity was located in the skin of the dorsal posterior region and lowest activity in the abdominal region. The activity in plucked (depilated) skin was only about 25% of that obtained from unplucked (depilated) tissue of the same region. D-Tryptophan, 5-hydroxytryptophan, and tryptamine were not degraded by the skin enzyme and the Km for L-tryptophan determined with the crude enzyme was 1 microM. The decycling activity of rat skin and liver for L-tryptophan began to be stimulated after birth and reached the highest level at 6 weeks. But, 1 week later, most of the skin activity suddenly disappeared and the low level continued at least until 12 weeks. In contrast, the hepatic enzyme did not change so drastically. These findings suggest that an enzyme that catalyzes L-tryptophan to L-kynurenine via L-formylkynurenine is present in rat skin.

An enzyme immunosensor has been developed for assaying human immunoglobulin G (IgG). The sensor is composed of an oxygen sensoring system and an antibody-binding membrane. The assay procedure involves the competitive immunochemical reaction of the membrane-bound antibody with nonlabeled and catalase-labeled IgG and the electrochemical determination of membrane-bound catalase activity. The analytical result is directly displayed by the output current of the sensor. The sensor exhibited an excellent performance in monitoring specifically human IgG.

このL-Ky.調製法は従来の方法に比較して, 操作が簡単で, 極めて高収量に得られる.又このL-Ky.硫酸塩は2,3の化学的性状から極めて高純度なものであることを確認した.そして十分に生化学的実験に供することができる物質である., L-kynurenine was isolated in a pure crystalline form from rat hair. L-kynurenine which was extracted from rat hair at hot water was separeted with Sephadex G-10 and crystallized from ethanol. The rat hair was very useful for a simplified preparation of L-kynurenine, for hair of 30g yielded 120mg.