Identification of a new human lens UV filter compound.

A new UV filter compound, 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-diglucoside, has been identified in human lenses. The structure suggests that it is a further metabolic product of the second most abundant UV filter compound, 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-glucoside. Quantification studies on the new compound show that it decreases towards zero in both the nucleus and cortex as a function of age. The discovery of this novel disaccharide completes the identification of the major UV filter compounds present in the human lens.

Major changes in human ocular UV protection with age.

PURPOSE: Age-dependent human lens coloration may be explained by the binding of UV filters to crystallins. It has been proposed that glutathione may compete for reaction with UV filter degradation products and therefore protect crystallins from modification. To understand this process, UV filters were quantified together with oxidized and reduced glutathione in human lenses of varying age. METHODS: Lens tissues were homogenized in ethanol to extract the UV filters. Metabolites were quantified by HPLC and correlations between them in the nuclear and cortical regions of the lens were examined. RESULTS: The concentrations of the UV filters 3-hydroxykynurenine, kynurenine, and 3-hydroxykynurenine glucoside decreased linearly with age, with slightly lower levels in the nucleus than the cortex. 4-(2-Amino-3-hydroxyphenyl)-4-oxobutanoic acid glucoside was found in higher levels in the nucleus than the cortex and decreased slowly in both regions with age. Glutathionyl-3-hydroxykynurenine glucoside was present in higher concentrations in the nucleus, barely detectable in young lenses, but increased significantly after age 50. Reduced glutathione levels were lower in the nucleus and decreased in both regions with age, yet oxidized glutathione increased in the nucleus but remained constant in the cortex. CONCLUSIONS: Results are consistent with a predominantly nuclear origin for both 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid glucoside and glutathionyl-3-hydroxykynurenine glucoside. This is in accord with their proposed mechanism of formation, which involves an initial deamination of 3-hydroxykynurenine glucoside. This process is more pronounced in older lenses, possibly because of the barrier to diffusion. The barrier may also explain the increase in nuclear oxidized glutathione that is observed with age.

A new oleanane triterpenoid from Gordonia ceylanica.

The chemical investigation of the hexane extract of the stem bark of Gordonia ceylanica afforded 3beta-acetoxy-11alpha(2',3'-epoxyferulyloxy)-olean-13(18)-ene as a new natural product and alpha-spinasterol for the first time from Gordonia.

Two oleanane triterpenoids from gordonia ceylanica and their conversions to taraxarane triterpenoids.

Chemical investigation of the hot hexane extract of the stem bark of Gordonia ceylanica afforded two new oleanane triterpenoids, 3beta-acetoxy-11alpha, 13beta-dihydroxyolean-12-one and 3beta,11alpha-diacetoxy-13beta-hydroxyolean-12-one (2) The attempted acid hydrolysis of these two compounds resulted the dehydration and subsequent methyl group migration to afford the taraxarane triterpenoids 3beta,11alpha-dihydroxytaraxer-14-en-12-one (4) and 3beta-hydroxy-11alpha-acetoxytaraxer-14-en-12-one (5), respectively. These taraxaranes have not been previously reported.

Expression and purification of recombinant human indoleamine 2, 3-dioxygenase.

Indoleamine 2,3-dioxygenase, the first and rate-limiting enzyme in human tryptophan metabolism, has been implicated in the pathogenesis of many diseases. The human enzyme was expressed in Escherichia coli EC538 (pREP4) as a fusion protein to a hexahistidyl tag and purified to homogeneity in terms of electrophoretic and mass spectroscopic analysis, by a combination of phosphocellulose and nickel-agarose affinity chromatography. The yield of the fusion protein was 1.4 mg per liter of bacterial culture with an overall recovery of 56% from the crude extract. When the culture medium was supplemented with 7 microM hemin, the purified protein contained 0.8 mol of heme per mole of enzyme and exhibited an absorption spectrum consistent with the ferric form of hemoprotein. The pI value of the recombinant enzyme was 7.09 compared with 6.9 for the native enzyme. This was as expected from the addition of the hexahistidyl tag. Similar to the native enzyme, the recombinant enzyme required methylene blue and ascorbic acid for enzyme activity and oxidized not only l-tryptophan but also d-tryptophan and 5-hydroxy-l-tryptophan. The molecular activities for these substrates and their K(m) values were similar to those of the native enzyme, indicating that the addition of the hexahistidyl tag did not significantly affect catalytic activity. The recombinant protein can therefore be used to investigate properties of the native enzyme. This will aid the development of specific inhibitors of indoleamine 2,3-dioxygenase, which may be effective in halting disease progression.

Antibacterial compounds from Carissa lanceolata R.Br.

The dichloromethane extract of the wood of Carissa lanceolata R.Br. (Apocynaceae) afforded the eudesmanes carissone, dehydrocarissone and carindone. This is the first account of carissone being isolated from the wood of C. lanceolata, and of carindone being isolated from this Carissa species. Dehydrocarissone has not been isolated previously from any Carissa species. The antibacterial activity of these natural products were examined against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. All three compounds showed activity, with dehydrocarissone and carindone having a minimum inhibitory concentration less than 0.5 mg/ml against S. aureus and E. coli.

UV filter compounds in human lenses: the origin of 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-beta-D-glucoside.

PURPOSE: To investigate UV filter synthesis in the human lens, in particular the biosynthetic origin of the second most abundant UV filter compound, 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-beta-D-glucoside. METHODS: Human lenses were analyzed by high-performance liquid chromatography (HPLC) after separate incubation with 3H-tryptophan (3H-Trp), beta-benzoylacrylic acid, D,L-alpha-amino-beta-benzoylpropionic acid, or D,L-3-hydroxykynurenine O-beta-D-glucoside. The effect of pH on the model compound D,L-alpha-amino-beta-benzoylpropionic acid and D,L-3-hydroxykynurenine O-beta-D-glucoside was also investigated. RESULTS: UV filters were not detected in fetal lenses, despite a 5-month postnatal lens displaying measurable levels of UV filters. In adults no radiolabel was incorporated into 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-beta-D-glucoside after 3H-Trp incubations. Beta-benzoylacrylic acid was readily reduced in lenses. D,L-alpha-amino-beta-benzoylpropionic acid and D,L-3-hydroxykynurenine O-beta-D-glucoside slowly deaminated at physiological pH and were converted to beta-benzoylpropionic acid and 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-beta-D-glucoside, respectively, after lens incubations. CONCLUSIONS: UV filter biosynthesis appears to be activated at or near birth. Compounds containing the kynurenine side chain slowly deaminate, and in the lens, the newly formed double bond is rapidly reduced. These findings suggest that 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-beta-D-glucoside is derived from L-3-hydroxykynurenine O-beta-D-glucoside through this deamination-reduction process. The slowness of the deamination presumably accounts for the absence of incorporation of radiolabel from 3H-Trp into 4(2-amino-3-hydroxyphenyl)4-oxobutanoic acid O-beta-D-glucoside.

Regulation of indoleamine 2,3-dioxygenase, the first enzyme in UV filter biosynthesis in the human lens. Relevance for senile nuclear cataract.

3-Hydroxykynurenine (3OHKyn), the precursor of UV filters in human lens, is highly autooxidizable, generates H2O2, and binds to lens proteins, yielding a tanned/yellow product resembling senile nuclear cataractous materials. Thus, if 3OHkyn can be shown to be the causative agent in cataract, it may be possible to prevent the disease by lowering the level of 3OHKyn. To this end, indoleamine 2,3-dioxygenase, the first enzyme in UV filter synthesis, was studied using lens epithelial cell lines. The results indicated that the IDO expression is mediated by IFN-gamma. Immuno-suppressants which inhibit production of IFN-gamma may act as anti-cataract agents. Another way to lower the level of 3OHKyn is to use specific inhibitors for IDO. A recombinant human IDO was expressed to develop the inhibitors.