Analysis of Porphyromonas gingivalis PG27 by deletion and intragenic suppressor mutation analyses.

PG27 is required for secretion of virulence factor gingipains, and has recently been proposed as LptO, which is involved in O-deacylation of lipopolysaccharide. In the present study, a predicted 14 anti-parallel ß-strand structure of PG27 was ascertained. Deletion study showed that the region from Asp382 to the C-terminal His391 of PG27 is dispensable for the function of PG27. Analysis of C-terminal deletion mutants revealed that the region in strand S14 (Asn369-Gly385) is important for activity. Of the gingipain-defective mutants, ΔThr378-His391 and ΔPhe377-His391 produced amounts of PG27 comparable to those produced by wild-type cells, suggesting that Thr378-Phe381 contains essential residues for the function of PG27. In contrast, ΔPhe381-His391, ΔAla380-His391, ΔLeu379-His391 and ΔArg376-His391 produced no detectable PG27. The defects of the ΔAla380-His391 mutant were suppressed by changing either Ala346 or Ala359 of PG27 to valine. Importantly, Ala346 and Ala359 are located close to Leu379 in the structural model of PG27. A359V compensated for the instability of PG27, but not the gingipain-defective phenotypes, of other deletion mutants tested, suggesting that Ala380 and Phe381 of PG27 are important for the stability of PG27. Lastly, we found that the C-terminal region of PG27 may be located in the periplasm. Taken together, these findings fit well with a predicted ß-barrel structure model for PG27, and show that strand S14 is important for its function.

Protective effect of melatonin against alpha-naphthylisothiocyanate-induced liver injury in rats.

The protective effect of melatonin against alpha-naphthylisothiocyanate (ANIT)-induced liver injury with cholestasis was examined in rats injected once with the toxicant (75 mg/kg body weight (BW)). In rats injected with ANIT alone, liver injury with cholestasis did not occur 12 hr after the injection but appeared at 24 hr, judging from the serum levels of marker enzymes and components. When melatonin (10 or 100 mg/kg BW) was orally administered to the ANIT-injected rats at 12 hr after the injection, the administered indoleamine dose-dependently prevented the formation of liver injury with cholestasis. In rats injected with ANIT alone, serum lipid peroxide (LPO) concentration increased 24 hr after the injection, while liver LPO concentration increased 12 hr after the injection and further increased at 24 hr. Myeloperoxidase (MPO) activity, an index of tissue neutrophil infiltration, in the liver of the ANIT-injected rats increased 12 hr after the injection and further increased at 24 hr. The oral administration of melatonin (10 or 100 mg/kg BW) to the ANIT-injected rats attenuated the increases in serum and liver LPO concentrations and liver MPO activity found at 24 hr after the injection in a dose-dependent manner. These results indicate that orally administered melatonin at pharmacological doses protects against ANIT-induced liver injury with cholestasis in rats, and suggest that this protective effect of melatonin could be due to its antioxidant action and its inhibitory action against neutrophil infiltration in the liver of ANIT-injected rats.

Therapeutic effect of melatonin on carbon tetrachloride-induced acute liver injury in rats.

The therapeutic effect of melatonin on acute liver injury was examined in rats intoxicated with carbon tetrachloride (CCl4). Melatonin (10, 50, or 100 mg/kg body weight [BW]) was intraperitoneally administered to male Wistar rats 6 hr after intraperitoneal injection of CCl4 (1.6 g/kg BW) at which time an apparent liver injury occurred. This post-melatonin administration dose dependently prevented the progression of liver injury at 24 hr after CCl4 injection, judging from the levels of serum transaminases, indices of liver cell damage. Rats injected with CCl4 alone showed an increase in liver lipid peroxide (LPO) content and a decrease in liver reduced glutathione content at 6 and 24 hr after the injection. The post-melatonin administration dose dependently ameliorated both changes found at 24 hr after CCl4 injection. Rats injected with CCl4 alone showed an increase in liver triglyceride (TG) content and decreases in serum TG concentration and liver tryptophan 2,3-dioxygenase (TDO) activity, a marker of the inhibition of liver protein synthesis by CCl4, at 6 and 24 hr after the injection, and also a decrease in serum albumin concentration at 24 hr. The changes in serum TG, albumin concentration, liver TG content, and TDO activity found at 24 hr after CCl4 injection were not ameliorated by the post-administration of melatonin. The same administration of melatonin dose dependently reduced liver LPO content in CCl4-untreated rats. These results indicate that melatonin exerts a therapeutic effect on CCl4-induced acute liver injury in rats, possibly through its antioxidant action.

Therapeutic effect of Oren-gedoku-to extract on stress-induced acute gastric mucosal lesions in rats.

Oral administration of Oren-gedoku-to extract (TJ-15), a traditional Chinese herbal medicine for the therapy of gastric ulcers and gastritis, dose-dependently prevented the progression of acute gastric mucosal lesions in rats with water immersion restraint (WIR) stress. The preventive effect of TJ-15 on the lesion progression was stronger than that of Saiko-keishi-to extract (TJ-10) or Shigyaku-san extract (TJ-35), each of which is a traditional Chinese herbal medicine for the therapy of gastric ulcers and gastritis, when compared on the basis of a single dosage of each medicine for adults. This TJ-15 administration attenuated increases in gastric mucosal lipid peroxide concentration and xanthine oxidase and myeloperoxidase activities with the gastric mucosal lesion progression and recovered the decreased gastric mucosal non-protein SH concentration found at a progressed stage of the gastric mucosal lesions. These results indicate that TJ-15 exerts a therapeutic effect on WIR stress-induced acute gastric lesions in rats more strongly than TJ-10 or TJ-35, and suggest that the therapeutic effect of TJ-15 could be due to its preventive actions on lipid peroxidation and sulphydryl oxidation via oxygen free radicals generated by the xanthine-XO system and infiltrated neutrophils in the gastric mucosa and on neutrophil infiltration into the tissue.

Possible contribution of a decrease in serum albumin concentration to a low level of blood L-tryptophan in nephrotic rats.

In order to clarify whether or not a decrease in serum albumin concentration contributes to a low level of blood L-tryptophan (Trp) in nephrosis, blood Trp concentration at 30, 60, 90, and 120 min after oral administration of Trp (100 mumol/kg body weight) in the same rats injected once with puromycin aminonucleoside (PAN) (100 mg/kg body weight, i.p.), an inducer of nephrosis, was examined at different stages of nephrosis. The increase and decrease in blood Trp concentration after Trp administration were similar in the PAN-treated rats without nephrosis, the PAN-treated rats recovered from nephrosis, and untreated control rats. The maximum increase in blood Trp concentration at 30 min after Trp administration was lower in nephrotic rats than in control rats. In all rats treated with and without PAN, increased blood L-tryptophan concentrations at 30 min after L-tryptophan administration were positively correlated well with serum albumin concentrations (r = 0.88, p < 0.001). There was no difference in the intestinal absorption of the same dose of orally administered Trp between nephrotic and control rats. These results suggest that a decrease in serum albumin concentration may contribute to a low level of blood L-tryptophan in nephrosis.

Participation of xanthine-xanthine oxidase system and neutrophils in development of acute gastric mucosal lesions in rats with a single treatment of compound 48/80, a mast cell degranulator.

The participation of xanthine-xanthine oxidase and neutrophils in the development of acute gastric mucosal lesions was examined in rats injected once with compound 48/80, a mast cell degranulator. Gastric mucosal lesions appeared 0.5 hr after compound 48/80 injection and developed at 3 hr. The formation of gastric mucosal lesions at 0.5 hr after compound 48/80 injection was prevented by pretreatment with anti-neutrophil antiserum and NPC 14686, an antiinflammatory agent, but not with allopurinol, a xanthine oxidase inhibitor. The development of gastric mucosal lesions at 3 hr after compound 48/80 injection was prevented by pretreatment with anti-neutrophil antiserum, NPC 14686, or allopurinol. Increases in the activities of gastric mucosal xanthine oxidase and myeloperoxidase, an index of neutrophil infiltration, and the content of lipid peroxide occurred 0.5 hr after compound 48/80 injection, and these increases were enhanced at 3 hr. The increases in gastric mucosal myeloperoxidase activity and lipid peroxide content at 0.5 hr after compound 48/80 injection were attenuated by pretreatment with anti-neutrophil antiserum and NPC 14686, while only the increase in gastric mucosal xanthine oxidase activity at the same time point was arrested by allopurinol pretreatment. The increases in gastric mucosal xanthine oxidase and myeloperoxidase activities and lipid peroxide content at 3 hr after compound 48/80 treatment were attenuated by pretreatment with anti-neutrophil antiserum, NPC 14686, or allopurinol. When compound 48/80-injected rats were treated with allopurinol at 0.5 hr after compound 48/80 injection, the progression of gastric mucosal lesions at 3 hr after the injection was almost completely prevented with inhibition of the increases in gastric mucosal xanthine oxidase and myeloperoxidase activities and lipid peroxide content. These results indicate that in rats with a single compound 48/80 treatment neutrophils infiltrated into the gastric mucosa participated in the development of acute gastric mucosal lesions and that the xanthine-xanthine oxidase system in the gastric mucosa participated in the progression rather than the formation of the gastric mucosal lesions.

Alleviation of carbon tetrachloride-induced chronic liver injury and related dysfunction by L-tryptophan in rats.

We examined whether L-tryptophan (Trp) alleviates carbon tetrachloride (CCl4)-induced chronic liver injury and related dysfunction. Fifty rats were classified into four groups: the first (15 rats) served as the control; the second (10 rats) received subcutaneous injections of CCl4 (1.0 mL/kg) twice weekly for 8 weeks; the third (15 rats) received daily intraperitoneal injections of Trp (50 mg/kg) for 2 weeks after 6 weeks of CCl4 treatment; the fourth (10 rats) received both treatments. The activities of serum transaminases and the content of liver total hydroxyproline increased after 6 and 8 weeks of CCl4 treatment. The concentrations of serum albumin and liver protein and the in vitro activity of liver protein synthesis fell after 8 weeks of the treatment. Trp administration alleviated all these changes. At 6 and 8 weeks of CCl4 treatment the serum triglyceride concentration fell, whereas the liver triglyceride and lipid peroxide concentrations were elevated. Trp administration hardly affected these changes. These results indicate that Trp alleviates CCl4-induced chronic liver injury possibly by maintaining the activity of protein synthesis.

Cataract development in 12-month-old rats fed a 25% galactose diet and its relation to osmotic stress and oxidative damage.

We attempted to clarify the pattern of cataract development in 12-month-old rats fed a 25% galactose diet and to assess the relation of cataract development with osmotic stress and oxidative damage. In lenses of 12-month-old male Wistar rats fed a 25% galactose diet over an 8-month period, suture accentuation appeared at 6 months of galactose feeding and then opacities developed from the anterior subcapsular cortex toward the posterior subcapsular cortex, reaching the nuclear region at 8 months of galactose feeding. Increases in lens galactitol and lipid peroxide contents and a decrease in lens reduced glutathione content occurred at 4, 6 and 8 months of galactose feeding. The increase in lens lipid peroxide content and the decrease in lens reduced glutathione content were accelerated with an increase in feeding period, while the increase in lens galactitol content was decelerated. An increase in lens water content and a decrease in lens protein content occurred at 6 and 8 months of galactose feeding. The lens vitamin E content increased at 6 months of galactose feeding and this increase was concomitant with increases in serum vitamin E and total cholesterol concentrations. The serum lipid peroxide concentration increased at 4 and 6 months of galactose feeding. The present results indicate that in lenses of 12-month-old rats fed a 25% galactose diet, suture accentuation appears initially and then opacities develop from the anterior subcapsular cortex toward the posterior subcapsular cortex, finally reaching the nuclear region. These results also suggest that in the galactosemic aged rats, osmotic stress would mainly contribute to cataract formation, while oxidative damage could be linked to both cataract formation and progression, although an increase in lens vitamin E content occurs during the cataract development.

Preventive effect of topical vitamin E-containing liposome instillation on the progression of galactose cataract. Comparison between 5-week- and 12-week-old rats fed a 25% galactose diet.

The preventive effect of topical vitamin E-containing liposome instillation on the progression of galactose cataract was compared between 5-week- and 12-week-old Wistar rats fed a 25% galactose diet. Vitamin E-containing liposomes [LP(+VE)] and vitamin E-free liposomes [LP(-VE)] were prepared with dipalmitoylphosphatidylcholine and dioleoylphosphatidylcholine (7:3 w/w). Twice daily instillation of either LP(-VE) or LP(-VE) into both eyes of 5-week-old rats fed the galactose diet for 18 days (5WGR) and 12-week-old rats fed the galactose diet for 7 weeks (12WGR) at which time some vacuoles appeared in the lens cortical equator, was conducted for a period of 4 and 9 weeks, respectively. The severity of cataracts at the end of instillation was similar in 5WGR and 12WGR. Instillation of LP(+VE), but not LP(-VE), retarded cataract progression in 5WGR and 12WGR. In 12WGR, LP(-VE) instillation caused a transient retardation of the progression. In lenses of 5WGR and 12WGR, decreases in vitamin E and reduced glutathione contents and increases in lipid peroxide, galactitol, and water contents occurred at the onset of instillation. For 5WGR, a decrease in lens reduced glutathione content and increases in lens vitamin E, lipid peroxide, galactitol, and water contents occurred at the end of instillation. For 12WGR, decreases in lens reduced glutathione and vitamin E contents and increases in lens lipid peroxide, galactitol, and water contents occurred at the end of instillation. In sera of 5WGR and 12WGR, vitamin E concentration decreased at the onset of instillation increased at the end in 5WGR and was unchanged in 12WGR. In 5WGR, instillation of LP(+VE), but not LP(-VE), for 4 weeks prevented these changes except the changes in lens galactitol and water contents and serum vitamin E concentration. In 12WGR, instillation of LP(+VE), but not LP(-VE), for 9 weeks prevented these changes except the changes in lens galactitol and water contents and serum vitamin E concentration. These results indicate that topically instilled LP(+VE) can retard cataract progression in 5WGR and 12WGR, mainly by the antioxidative action of vitamin E contained in the instilled liposomes.

Modulation of stress-induced gastric mucosal lesions by exogenous L-arginine.

L-arginine, a nitric oxide (NO) precursor, can exert both ameriolative and deteriorative effects on gastric mucosal lesions. This study was designed to determine whether exogenous L-arginine modulates stress-induced gastric mucosal lesions through NO production by either constitutive NO synthase (cNOS) or inducible NO synthase (iNOS) in gastric mucosal tissues. In rats subjected to water immersion restraint stress over a 6-hour period, the concentration of gastric mucosal nitrite/nitrate, breakdown products of NO, increased with the development of gastric mucosal lesions and a decrease in cNOS activity and a drastic increase in iNOS activity in the gastric mucosal tissue. Preadministration of L-arginine (150 to 600 mg/kg intraperitoneally) attenuated the lesion development with prevention of increases in gastric mucosal nitrite/nitrate concentration and iNOS activity. In contrast, postadministration of L-arginine (150 to 600 mg/kg intraperitoneally) enhanced the lesion development with further increase in gastric mucosal nitrite/nitrate concentration. This deteriorative action of postadministration of L-arginine (300 mg/kg intraperitoneally) was prevented by pretreatment with aminoguanidine (100 mg/kg subcutaneously), a selective iNOS inhibitor, with inhibition of increases in gastric mucosal iNOS activity and nitrite/nitrate concentration. These results indicate that preadministered L-arginine protects against water immersion restraint stress-induced gastric mucosal lesions, possibly through restricted NO production by cNOS in gastric mucosal tissues, whereas postadministered L-arginine aggravates the stress-induced gastric mucosal lesions, possibly through excessive NO production by iNOS increasing in gastric mucosal tissues.

An association between lipid peroxidation and alpha-naphthylisothiocyanate-induced liver injury in rats.

An association between lipid peroxidation and alpha-naphthylisothiocyanate (ANIT)-induced liver injury was examined in rats injected once with the toxicant (75 mg/kg body weight). The severity of liver injury was estimated 12, 24, 48, and 72 h after ANIT injection. Liver injury appeared 24 h after ANIT injection, progressed at 48 h, and recovered at 72 h, judging from the serum levels of marker enzymes and components. Serum lipid peroxide (LPO) concentration increased 24 h after ANIT injection and further increased at 48 h, but this increase was attenuated at 72 h. In contrast, liver LPO content increased 12 h after ANIT injection and further increased 24 and 48 h, but this increase was attenuated at 72 h. Similarly, myeloperoxidase (MPO) activity, an index of neutrophil infiltration, in the liver tissue increased 12 h after ANIT injection and further increased at 24 and 48 h, but this increase was attenuated at 72 h. Either serum LPO concentration or liver LPO content was significantly correlated with liver MPO activity (r = 0.661 for serum LPO concentration; r = 0.585 for liver LPO content). These results suggest that lipid peroxidation might be associated with ANIT-induced liver injury in rats and that this lipid peroxidation might occur via oxygen radicals derived from neutrophils infiltrated into the liver tissue of ANIT-intoxicated rats.

Role of endogenous serotonin and histamine in the pathogenesis of gastric mucosal lesions in unanaesthetised rats with a single treatment of compound 48/80, a mast cell degranulator.

In unanaethetised rats with a single injection of compound 48/80, a mast cell degranulator (0.75 mg kg-1, i.p.), gastric lesions occurred with increased serum serotonin and histamine levels and reduced gastric mucosal blood flow at 0.5 h after the injection and developed at 3 h. Pretreatment with either cyproheptadine (a serotonin and histamine antagonist) or methysergide (a serotonin antagonist) prevented the formation of gastric mucosal lesions with attenuation of reduced gastric mucosal blood flow at 0.5 h after compound 48/80 injection, while pretreatment with either amitriptyline (a selective inhibitor of histamine release from mast cells), tripelennamine (a histamine H1-receptor antagonist), famotidine (a histamine H2-receptor antagonist) or cimetidine (a histamine H2-receptor antagonist) had no effect. Pretreatment with either cyproheptadine, methysergide, amitriptyline or tripelennamine prevented the development of gastric mucosal lesions at 3 h after compound 48/80 injection, while pretreatment with either famotidine or cimetidine had no effect. These results indicate that in unanaesthetised rats with a single compound 48/80 treatment, acutely released endogenous serotonin causes gastric mucosal lesions, while released endogenous histamine mainly contributes to the lesion development and that gastric acid plays little role in the pathogenesis of the compound 48/80-induced acute gastric lesions.

Preventive effect of teprenone on stress-induced gastric mucosal lesions and its relation to gastric mucosal constitutive nitric oxide synthase activity.

Recently, we demonstrated that teprenone, an anti-ulcer agent, exerts protective and preventive actions against water immersion restraint (WIR) stress-induced gastric mucosal lesions in rats both by inhibiting neutrophil infiltration into the gastric mucosal tissue and by preserving gastric mucus synthesis and secretion. In rats with WIR stress we have also found a decrease in gastric mucosal constitutive nitric oxide synthase (cNOS) activity and a drastic increase in gastric mucosal inducible nitric oxide synthase (iNOS) activity. The decrease in gastric mucosal cNOS activity is closely related to an increase in neutrophil infiltration into the gastric mucosa and a decrease in the level of gastric mucus. In this study of WIR-stressed rats, therefore, we examined whether the inhibitory actions of teprenone on neutrophil infiltration and decreases in mucus synthesis and secretion in the gastric mucosa of rats are related to the change in gastric mucosal cNOS activity during the development of gastric mucosal lesions. Pre-administration of teprenone (200 mg kg-1) prevented the decrease in gastric mucosal cNOS activity with attenuations of neutrophil infiltration into gastric mucosal tissues and decreased levels of gastric mucosal hexosamine, an index of gastric mucin, and adherent mucus in rats with 3 or 6 h of WIR stress. These preventive effects of teprenone on the gastric mucosal neutrophil infiltration and the decrease in gastric mucus levels in rats with WIR stress were completely reversed with inhibition of gastric mucosal cNOS activity by co-administration of NG-monomethyl L-arginine (L-NMMA), a non-selective NOS inhibitor. These results suggest that the inhibitory actions of teprenone on neutrophil infiltration and decreases in mucus synthesis and secretion in the gastric mucosa of rats with WIR stress are closely related to the maintenance of cNOS activity in the gastric mucosal tissue.

Contribution of serum albumin to the transport of orally administered L-tryptophan into liver of rats with L-tryptophan depletion.

The role of serum albumin in the transport of orally administered L-tryptophan (Trp) into rat tissues was examined using analbuminemic and Sprague-Dawley (SD) rats with and without alpha-methyl-DL-tryptophan (AMT)-induced Trp depletion. Trp was orally administered to rats 16 h after AMT or 0.85% NaCl administration, when liver tryptophan 2,3-dioxygenase and protein synthetic activities in AMT-treated rats were similar to those of 0.85% NaCl-treated rats. After oral Trp administration, regardless of the presence or absence of Trp depletion, free serum Trp concentrations were similar in the analbuminemic and SD rats, while total serum Trp concentrations were lower in analbuminemic rats than in SD rats. Although liver, brain, and muscle Trp concentrations after oral Trp administration under Trp depletion were lower in analbuminemic rats than in SD rats, the ratio of the liver Trp concentration in analbuminemic rats to that in SD rats was smaller than that of the brain or muscle Trp concentration. These results suggest that variations in serum albumin levels could affect the transport of orally administered Trp into the liver of rats with Trp depletion.

Preventive effect of Oren-gedoku-to (Huanglian-Jie-Du-Tang) extract on the development of stress-induced acute gastric mucosal lesions in rats.

The preventive effect of Oren-gedoku-to (Huanglian-Jie-Du-Tang) extract (TJ-15), a traditional Chinese herbal medicine for the therapies of gastric ulcers and gastritis, on the development of stress-induced acute gastric mucosal lesions was examined in rats with water immersion restraint (WIR) stress. Simultaneous p.o. administration of TJ-15 at a dose of 20, 100 or 250 mg/kg prevented dose-dependently gastric mucosal lesion development in rats subjected to WIR stress over a 6-h period. In the gastric mucosa of rats with WIR stress alone, lipid peroxide concentration and xanthine oxidase (XOD) and myeloperoxidase--an index of neutrophil infiltration--activities increased with lesion development, while nonprotein SH concentration decreased. The simultaneous administration of TJ-15 attenuated all these changes with gastric mucosal lesion development in a dose-dependent manner. These results indicate that simultaneously administered TJ-15 exerts a preventive effect on the development of WIR stress-induced acute gastric lesions in rats, and suggest that the preventive effect of TJ-15 could be due to its preventive actions on enhanced sulfhydryl oxidation and lipid peroxidation via oxygen free radicals generated by the xanthine-xanthine oxidase system and infiltrated neutrophils in the gastric mucosa and on neutrophil infiltration into the tissue.

Preventive effect of melatonin on the progression of carbon tetrachloride-induced acute liver injury in rats.

We examined the preventive effect of melatonin on the progression of carbon tetrachloride (CCl4)-induced acute liver injury in rats. In rats injected with CCl4 (1.0 ml/kg body weight), liver injury appeared 6 h after the injection and progressed at 24 h. This liver injury progression was prevented by treatment with melatonin (50 or 100 mg/kg body weight), which was conducted 6 h after CCl4 injection. An increase in liver lipid peroxide content and a decrease in liver reduced glutathione content occurred with the liver injury progression. These changes were prevented by the post-treatment of melatonin (50 or 100 mg/kg body weight). These results indicate that melatonin can prevent the progression of CCl4-induced acute liver injury through its antioxidant action.

Effect of albumin administration on L-tryptophan levels in the serum and tissues of rats with puromycin aminonucleoside-induced nephrosis.

We examined the effect of albumin administration on L-tryptophan (Trp) levels in the serum and tissues of rats with puromycin aminonucleoside (PAN)-induced nephrosis. PNA-injected rats showed increased urinary protein and serum non-esterified fatty acids levels and decreased serum albumin level at 8 days after the injection. The nephrotic rats showed a decrease in total serum Trp level and increases in free serum Trp and liver and kidney Trp levels but no change in urinary Trp level. At 5 min after intravenous injection of bovine serum albumin, the nephrotic rats had serum albumin concentration similar to the level of non-nephrotic rats and showed a recovery of decreased total serum Trp with the reduction of increased free serum Trp and liver Trp.

Cinnabarinic acid was formed in damaged mitochondria and its effect on mitochondrial respiration.

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 24 hr 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 10 microM.

Ommochrome genesis in an albino strain of a terrestrial isopod.

The contents of tryptophan (Trp) metabolites and the activities of the enzymes involved in ommochrome biosynthesis were measured in an albino strain of a terrestrial isopod Armadillidium vulgare. There was little difference between the Trp content in the albino mutant and that in the wild type, although the contents of 3-hydroxykynurenine (3-OH-Kyn), 3-hydroxyanthranilic acid (3-OH-AA) and xanthommatin in the albino were significantly lower than those in the wild type. Tryptophan 2,3-dioxygenase (TDO) activity in the albino was extremely low, while the activities of Kyn-3-hydroxylase and kynureninase did not differ significantly between the two phenotypes. The extremely low activity of TDO is probably one of main reasons why almost no ommochrome pigment is produced in the albino mutant.

Involvement of tryptophan metabolism in the body color of crustacea.

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.