Novel metabolic routes during the oxidation of hydroxylated aromatic acids by the yeast Arxula adeninivorans.

AIM: To complete our study on tannin degradation via gallic acid by the biotechnologically interesting yeast Arxula adeninivorans as well as to characterize new degradation pathways of hydroxylated aromatic acids. METHODS AND RESULTS: With glucose-grown cells of A. adeninivorans, transformation experiments with hydroxylated derivatives of benzoic acid were carried out. The 12 metabolites were analysed and identified by high performance liquid chromatography and GC/MS. The yeast is able to transform the derivatives by oxidative and nonoxidative decarboxylation as well as by methoxylation. The products of nonoxidative decarboxylation of protocatechuate and gallic acid are substrates for further ring fission. CONCLUSION: Whereas other organisms use only one route of transformation, A. adeninivorans is able to carry out three different pathways (oxidative, nonoxidative decarboxylation and methoxylation) on one hydroxylated aromatic acid. The determination of the KM-values for protocatechuate and gallic acid in crude extracts of cells of A. adeninivorans cultivated with protocatechuate and gallic acid, respectively, suggests that the decarboxylation of protocatechuate and gallic acid may be catalysed by the same enzyme. SIGNIFICANCE AND IMPACT OF THE STUDY: This transformation pathway of protocatechuate and gallic acid via nonoxidative decarboxylation up to ring fission is novel and has not been described so far. This is also the first report of nonoxidative decarboxylation of gallic acid by a eukaryotic micro-organism.

[The influence of VEGF inhibitors on corneal endothelium after injection into the anterior chamber in a porcine eye model]. / Einfluss von VEGF-Inhibitoren auf das Hornhautendothel bei Injektion in die Vorderkammer am Modell des Schweineauges.

BACKGROUND: The injection of antiangiogenic agents, such as ranibizumab (Lucentis®) and bevacizumab (Avastin®) into the anterior chamber of the eye represents a suitable alternative for treating neovascular glaucoma by reducing intraocular pressure. OBJECTIVES: As the antiangiogenic substances are in direct contact with the sensitive corneal endothelium, the aim of this study was to show the effects of intracameral injection of ranibizumab and bevacizumab on this cell layer. METHODS: Each injection consisted of 50 µl containing either ranibizumab (0.5 mg/0.05 ml), bevacizumab (1.25 mg/0.05 ml) or triamcinolone containing benzyl alcohol (2 mg/0.05 ml) which was used as the control group. These compounds were injected into the anterior chamber of pig eyes. Afterwards the corneas were dissected, fixed, examined by a scanning electron microscopy and evaluated according to a specified score. Assessment of the endothelium was carried out by evaluating the condition of microvilli, cell borders, cell surface and cell pattern. The findings were compared to untreated corneas and those injected with 50 µl of balanced salt solution (BSS). RESULTS: The corneal endothelium exposed to the antiangiogenic substances showed only minor changes in comparison to the controls treated only with BSS. Also seen during this research was the irreversible cell damage in the control group using triamcinolone. CONCLUSION: Ranibizumab and bevacizumab have no damaging effects on the corneal endothelium when used in the anterior chamber. They can be administered as an intracameral injection for the treatment of rubeotic secondary glaucoma. Triamcinolon containing benzyl alcohol causes severe damage to the endothelial cells of the cornea by direct contact.

[Scanning electron microscopic characteristics of lamellar keratotomies using the Femtec femtosecond laser and the Zyoptix XP microkeratome. A comparison of quality]. / Vergleich des Femtec Femtosekundenlasers und des Zyoptix XP Mikrokeratoms. Rasterelektronenmikroskopische Gegenüberstellung lamellärer Keratotomien.

OBJECTIVE: To demonstrate the qualities and compare the typical features of cut surfaces and cut edges created by the Femtec femtosecond laser and the Zyoptix XP microkeratome, using scanning electron microscope (SEM) pictures. METHODS: Lamellar keratotomies were performed using a femtosecond laser (40 kHz) or a microkeratome on freshly enucleated porcine eyes (n=16, eight each per technique). After special preparation, SEM images were taken to evaluate the qualities of the cut surfaces and cut edges. Therefore, special criteria were involved, including relief and homogeneity of the surface and sharpness of the cut edges. RESULTS: Surfaces created by microkeratome cuts were very homogenous. Concerning surface relief, nearly no irregularities occurred. Cut edges showed a flat, serrated course from the epithelial layer to the stroma of the cornea. The edges were sharp and easily visible. After preparation using the femtolaser, the surface showed many rips in the tissue, leading to irregularities. Nevertheless, the cut edges were very sharp and entered the corneal layer straight at 90 degrees . CONCLUSIONS: A comparison of the two systems shows that the microkeratome creates a more homogenous cut surface. The need for preparation after automated cutting with the femtosecond laser leads to irregularities on the cut surface. The cut edges of both systems tested here differ concerning their angles on entering the tissue. With regard to the sharpness of the cuts, the qualitative aspect is nearly similar, although the cut edges of the microkeratome are serrated. Because the microkeratome-cut edge has a flatter course, the wound area might be bigger. Cut edges with the steepness produced by the femtosecond laser could be an advantage for repositioning the flap after LASIK. If excimer laser ablation is performed later, the flap bed created by the femtosecond laser could be disadvantageous.

Hydroxylation of biphenyl by the yeast Trichosporon mucoides.

Hydroxylation of biphenyl by the dibenzofuran-degrading yeast Trichosporon mucoides SBUG 801 was studied. Glucose-grown cells degraded 40% of the biphenyl added within the first 24 h of incubation. The first step in the biotransformation pathway was the monohydroxylation of the biaryl compound to produce 2-, 3-, and 4-hydroxybiphenyl. Further oxidation produced seven dihydroxylated intermediates; the second hydroxyl group was added either on the aromatic ring already hydroxylated or on the second ring. Of all metabolites, 2,5-dihydroxybiphenyl accumulated in the supernatant in the highest concentration. The initial hydroxylation favors the 4-position to produce 4-hydroxybiphenyl, which is subsequently hydroxylated to form 3,4-dihydroxybiphenyl. When biphenyl was replaced as a substrate by 4-hydroxybiphenyl, further hydroxylation of the intermediate 3,4-dihydroxybiphenyl resulted in 3,4,4'-trihydroxybiphenyl. Incubation of T. mucoides with biphenyl and 18O2 indicated a monooxygenase-catalyzed reaction in the oxidation of biphenyl. The hydroxylation was inhibited by 1-aminobenzotriazole and metyrapone, known cytochrome P450 inhibitors. These results are very similar to those observed in the biotransformation of biphenyl in mammals.

Novel ring cleavage products in the biotransformation of biphenyl by the yeast Trichosporon mucoides.

The yeast Trichosporon mucoides, grown on either glucose or phenol, was able to transform biphenyl into a variety of mono-, di-, and trihydroxylated derivatives hydroxylated on one or both aromatic rings. While some of these products accumulated in the supernatant as dead end products, the ortho-substituted dihydroxylated biphenyls were substrates for further oxidation and ring fission. These ring fission products were identified by high-performance liquid chromatography, gas chromatography-mass spectrometry, and nuclear magnetic resonance analyses as phenyl derivatives of hydroxymuconic acids and the corresponding pyrones. Seven novel products out of eight resulted from the oxidation and ring fission of 3,4-dihydroxybiphenyl. Using this compound as a substrate, 2-hydroxy-4-phenylmuconic acid, (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)acetic acid, and 3-phenyl-2-pyrone-6-carboxylic acid were identified. Ring cleavage of 3,4,4'-trihydroxybiphenyl resulted in the formation of [5-oxo-3-(4'-hydroxyphenyl)-2,5-dihydrofuran-2-yl]acetic acid, 4-(4'-hydroxyphenyl)-2-pyrone-6-carboxylic acid, and 3-(4'-hydroxyphenyl)-2-pyrone-6-carboxylic acid. 2,3,4-trihydroxybiphenyl was oxidized to 2-hydroxy-5-phenylmuconic acid, and 4-phenyl-2-pyrone-6-carboxylic acid was the transformation product of 3,4,5-trihydroxybiphenyl. All these ring fission products were considerably less toxic than the hydroxylated derivatives.