Pathological apoptosis by xanthurenic acid, a tryptophan metabolite: activation of cell caspases but not cytoskeleton breakdown.

BACKGROUND: A family of aspartate-specific cysteinyl proteases, named caspases, mediates programmed cell death, apoptosis. In this function, caspases are important for physiological processes such as development and maintenance of organ homeostasis. Caspases are, however, also engaged in aging and disease development. The factors inducing age-related caspase activation are not known. Xanthurenic acid, a product of tryptophan degradation, is present in blood and urine, and accumulates in organs with aging. RESULTS: Here, we report triggering of apoptotic key events by xanthurenic acid in vascular smooth muscle and retinal pigment epithelium cells. Upon exposure of these cells to xanthurenic acid a degradation of ICAD/DFF45, poly(ADP-ribose) polymerase, and gelsolin was observed, giving a pattern of protein cleavage characteristic for caspase-3 activity. Active caspase-3, -8 and caspase-9 were detected by Western blot analysis and immunofluorescence. In the presence of xanthurenic acid the amino-terminal fragment of gelsolin bound to the cytoskeleton, but did not lead to the usually observed cytoskeleton breakdown. Xanthurenic acid also caused mitochondrial migration, cytochrome C release, and destruction of mitochondria and nuclei. CONCLUSIONS: These results indicate that xanthurenic acid is a previously not recognized endogenous cell death factor. Its accumulation in cells may lead to accelerated caspase activation related to aging and disease development.

Xanthurenic acid provokes formation of unfolded proteins in endoplasmic reticulum of the lens epithelial cells.

The role of xanthurenic acid in a cell is unknown, but it is suspected to provoke several diseases. This study shows that accumulation of xanthurenic acid in the lens epithelial cells leads to an overexpression of endoplasmic reticulum (ER) resident stress chaperones proteins, glucose-regulated protein (Grp94), and calreticulin. Both chaperones proteins are overexpressed in the presence of unfolded proteins. A formation of the unfolded protein in the presence of xanthurenic acid may take place due to covalent binding of xanthurenic acid to protein. Grp94 is responsible for scavenging of the unfolded proteins. The results suggest that Grp94 scavenged xanthurenic acid-modified proteins, and for this reason become preferentially yellow-stained in the presence of yellow xanthurenic acid. Such a modified Grp94 is weakly recognized by anti-Grp94 antibody. An end point of the xanthurenic acid accumulation in the cell is the cell death. In conclusion xanthurenic acid can lead to cell pathology.

Xanthurenic acid derivative formation in the lens is responsible for senile cataract in humans.

BACKGROUND: The tryptophan degradation pathway leads to NAD production via 3-hydroxykynurenine. Kynurenine aminotransferase (KAT) transforms 3-hydroxykynurenine into xanthurenic acid. In this study, we measured the activity of KAT in human lenses and studied the consequences of xanthurenic acid formation METHODS: KAT activity was determined by the method of Tobes. Fluorescence spectroscopy and SDS-PAGE were used for the protein studies. Thin-layer chromatography and infrared and fast atom bombardment spectrometry were used for substance characterization. RESULTS: The KAT activity was detected in senile cataratous lenses, but was absent in the young lenses. Xanthurenic acid at physiological pH exists in equilibrium with its tautomeric form reported by us as oxo-xanthurenic acid (OXA), which is oxidized to di-oxoxanthurenic acid (DOXA), a naphthoquinone-like substance. The incubation of DOXA with crystallins in a solution of physiological pH led to crystallin crosslinking and formation of conjugates with glutathione. CONCLUSIONS: Xanthurenic acid is formed in human lenses. Its tautomerization and oxidation leads to a naphthoquinone-like substance, DOXA. DOXA provoked formation of conjugates with glutathione and crosslinking of crystallins. Thus, KAT activity seems to be the initial event in senile cataract formation in humans.

Indoleamine 2,3-dioxygenase: antioxidant enzyme in the human eye.

BACKGROUND: Indoleamine 2,3-dioxygenase (IDO) is the first enzyme of the tryptophan degradation pathway. IDO is an antioxidant enzyme because it is a direct scavenger of superoxide radicals. In this study, we measured the activity of IDO in the human eye. METHODS: IDO was detected in the protein extract of human retina, iris/ciliary body, and lens. The products formed were measured using HPLC with electrochemical detection. Enzyme activity was expressed as the quantity of kynurenine and 3-hydroxykynurenine formed. RESULTS: IDO activity in the retina extract was 51.5 (+/-10) nmol/g tissue/h, and kynurenine formation was detected. In the iris/ ciliary body, IDO activity was 191.8 (+/49) nmol/g tissue/h, and both kynurenine and 3-hydroxy-kynurenine were formed from tryptophan. In the extract of lens cortex only 3-hydroxykynurenine was formed from tryptophan. IDO activity was 351 (+/-67.3) nmol/g tissue/h. CONCLUSION: Free tryptophan is degradated in the human eye by IDO, an antioxidative enzyme. IDO may be an antioxidant mechanism in the eye.

3-hydroxykynurenine transamination leads to the formation of the fluorescent substances in human lenses.

The levels of alanine, aspartate and glutamine transaminase increase considerably in some diseases. We measured the activity of these enzymes and of the transaminase of 3-hydroxykynurenine, an aminoacid, which acts as a UV lens filter. Alanine and glutamine transaminases (carboxypeptidase) were not detected in normal and cataractous human lenses, and aspartate transaminase was found only in the cortex of normal lenses. 3-hydroxykynurenine transaminase was not found in lenses from persons below thirty years of age, but was found in lenses at about fifty years of age, and in cataractous lenses. Transamination of 3-hydroxykynurenine leads to the formation of xanthurenic acid and its derivatives. These substances appear to be responsible for the increase of lens fluorescence during cataract development.

Deamination of 3-hydroxykynurenine in bovine lenses: a possible mechanism of cataract formation in general.

BACKGROUND: 3-Hydroxykynurenine, a metabolite of tryptophan, acts as UV filter in the human lens. In this study, we looked for this substance and its metabolites in young and old bovine lenses, because of their possible role in the formation of cataract. METHODS: The substances were detected by HPLC analysis. The fluorescent substance formed from 3-hydroxykynurenine was characterized by thin-layer chromatography followed by reaction with ninhydrin, UV and fluorescence spectrum analysis, and atom bombardment for molecular mass determination. The kynurenine aminotransferase activity was determined by the method of Tobes. RESULTS: 3-Hydroxykynurenine was detected at concentrations of 0.07, 0.19, and 1.14 micrograms/g of tissue in the bovine iris/ciliary body, retina, and transparent bovine lenses respectively. 3-Hydroxykynurenine was deaminated in old bovine eyes but not in calf eyes. In old eyes, kynurenine aminotransferase activity was 2.7, 3.5, and 9.6 mumol/g of tissue per h in retina, iris/ciliary body, and lens respectively. CONCLUSION: The deamination of 3-hydroxykynurenine resulted in the formation of a fluorescent substance which was identified as oxidized xanthurenic acid. This substance, accumulating in the bovine lens and interacting with lens proteins, could induce cataract formation.

Indoleamine 2,3-dioxygenase activity in the aqueous humor, iris/ciliary body, and retina of the bovine eye.

The enzyme indoleamine 2,3-dioxygenase (IDO), which uses free oxygen radicals to cleave the pyrrole ring of indoleamines and give kynurenamines, has previously been found in most tissues, but not in the eye. In this study, IDO activity was measured in post-mortem bovine eyes using Yamazaki's method with L-tryptophan as substrate. Because of the physiological importance of IDO in the protection against free oxygen radical damage, a search was conducted to find this enzyme in the eye. Products of tryptophan degradation by IDO, the kynurenine and 3-hydroxyanthranilic acid were detected and measured in the aqueous humor, iris/ciliary body, and the retina by high-performance liquid chromatography (HPLC) coupled with electrochemical detection. IDO activity was 3.2, 9.0 and 10 nmol/mg protein per h for the aqueous humor, iris/ciliary body, and retina, respectively. These findings suggest that, because of its scavenger properties, IDO is involved in the protection of the eye where, because of its transparency, free radicals are formed not only in the normal oxidation process, but also photochemically.

The ciliary body--the third organ found to synthesize indoleamines in humans.

Indoleamines are associated with circadian rhythms in pineal gland and retina. Because the ciliary epithelium has an embryonic origin similar to that of pineal gland and retina, and intraocular pressure shows circadian variations, indoleamines were searched for in aqueous humor and ciliary body in humans. In aqueous humor, serotonin, 6-hydroxymelatonin, and melatonin were simultaneously detected and measured using high-performance liquid chromatography with electrochemical detection. The concentration was 48.7 +/- 10.9 ng/ml for serotonin, 0.47 +/- 0.8 ng/ml for melatonin, and 13.9 +/- 7.7 ng/ml for 6 hydroxymelatonin. In ciliary bodies from freshly enucleated human eyes, tryptophan, 5-hydroxytryptophan, serotonin, and 5-hydroxyindoleacetic acid were detected using high-performance liquid chromatography with simultaneous fluorescence- and electrochemical detection. Finally, the enzymatic activities of arylalkylamine N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT), enzymes indispensable in the synthesis of melatonin, were measured. The NAT activity was 273 +/- 25 pmol/mg protein/hour and that of HIOMT, 13520 +/- 50 pmol/mg protein/hour in ciliary body. Comparison of these activities (NAT versus HIOMT) permits the suggestion that NAT is a limiting enzyme in serotonin metabolism in this tissue. These findings indicate that a circadian rhythm of indoleamines exists in human aqueous humor and that the human ciliary body is the third organ, after the pineal gland and the retina, found to synthesize indoleamines in humans.

Preliminary investigation of the structure of antibiotics produced by highly active mutants of Streptomyces griseus NRRL 3851 and Streptomyces lactamdurans NRRL 3802.

Streptomyces griseus NRRL 3851 and Streptomyces lactamdurans NRRL 3802 are well known as producers of cephamycins A, B and C, respectively. Antibiotics produced by their highly active mutants: S. griseus G-12 (termed X-12-1 and X-12-2) and S. lactamdurans L-2 (termed X-2) have been isolated and found to belong also to the cephamycin family. Their IR spectra revealed beta-lactam absorption at 1750 cm-1 and their 1H NMR spectra demonstrated the presence of protons attached to 7-methoxycephem skeleton and, in addition, signals attributable to one vinyl proton and four aromatic protons. The chemical shift of the aromatic protons indicated meta substitution in the benzene ring of the antibiotic X-12-1, and para substitution in the benzene ring of the antibiotics X-12-2 and X-2. The product of acid hydrolysis of X-2 was shown to be identical with p-hydroxy-alpha-methoxycinnamic acid.

Influence of medium composition on the production of cephamycins by S. griseus and S. lactamdurans and the characterization of their growth.

The process of cephamycin biosynthesis by S. griseus and S. lactamdurans strains was investigated. The antibiotic activities of both strains in thirteen media elaborated in our laboratory were compared with those in nine media described in the literature. The best medium was that one which contained 20 g/dcm3 of glucose and 30 g/dcm3 of corn steep liquor. In this medium after a two stage shaken culture fermentation both strains showed specific growth rate from 0.027 to 0.034 h-1 and biomass yield about 5 g of dry weight per 1 dcm3. Under these conditions the highest activity was achieved after 96 hours of the fermentation.