Oxidation of hydroxycinnamic acid and hydroxycinnamyl alcohol derivatives by laccase and peroxidase. Interactions among p-hydroxyphenyl,guaiacyl and syringyl groups during the oxidation reactions

Fungal laccase oxidized derivatives of hydroxycinnamic acid. The rates decreased in the order sinapic acid > ferulic acid ≥p-coumaric acid. The laccase oxidized sinapyl alcohol faster than coniferyl alcohol. The rates of oxidation of the hydroxycinnamic acid derivatives by an isoenzyme of peroxidase from horseradish decreased in the order p-coumaric acid > ferulic acid ≥ sinapic acid. The peroxidase oxidized coniferyl alcohol much faster than sinapyl alcohol. The laccase and the peroxidase predominantly oxidized (a) ferulic acid in a reaction mixture that contained p-coumaric acid and ferulic acid, (b) sinapic acid in a mixture of p-coumaric acid plus sinapic acid, and (c) sinapic acid in a mixture of ferulic acid plus sinapic acid. In a reaction mixture that contained both coniferyl and sinapyl alcohols, both fungal laccase and horseradish peroxidase predominantly oxidized sinapyl alcohol. From these results, it is concluded (1) that the p-hydroxyphenyl radical can oxidize guaiacyl and syringyl groups and produce their radicals and (2) that the guaiacyl radical can oxidize the syringyl group under formation of its radical; and that (3) in both cases the reverse reactions are very slow.     

Detection of Monodehydroascorbic Acid Radical in Sulfite-Treated Leaves and Mechanism of its Formation

The aim of the present study is to detect the monodehydroascorbicacid (MDA) radical in broad bean (Vicia faba L.) leaves whichwere treated by vacuum-infiltration in Na₂SO₃ solution and subsequentcentrifugation (sulfite-treated leaves). When sulfite-treatedleaves were illuminated with white light, the electron spinresonance (ESR) signal of MDA radical was observed. The levelof the MDA radical depended on the concentration of sulfitethat was used for vacuum-infiltration and on the light intensityof illumination. The formation of the MDA radical in sulfite-treatedleaves was inhibited by DCMU or by replacement of air with N₂.Glycolaldehyde also inhibited the formation of MDA radical insulfite-treated leaves. Catalase activity was decreased by thesulfite treatment without affecting significantly the activitiesof ascorbate peroxidase (AA-POX) and of peroxidase which preferentiallyoxidizes phenolics (PhOH-POX). From these results, we concludethat the formation of the MDA radical in sulfite-treated leavesis catalyzed by peroxidases using the H₂O₂ which is generatedby photorespiration and the Mehler reaction. ¹On leave from the Center for Multidisciplinary Studies, Universityof Belgrade, Yugoslavia.     

Immunohistochemical Localization of Annetocin, an Earthworm Oxytocin-Related Peptide, and Identification and Ultrastructural Characteristics of the Annetocin-Secretory Cells in the Oligochaete Earthworm Eisenia foetida

Annetocin is an egg-laying-inducing oxytocin-related peptide which we have previously isolated from the earthworm, Eisenia foetida. Here we report the results of immunohistochemical and ultrastructural studies on annetocin-secretory cells in the earthworm. Annetocin-immunoreactive (IR) cell-somata were located mainly at the ventro-lateral side of the subesophageal ganglion. Only four annetocin-IR cells were seen in the cerebral ganglion. Some annetocin-IR cells displayed unipolar-like structure with a process directing to the core region (the neuropile) of the ganglion. Annetocin-IR fibers were also observed in the neuropile of the ventral ganglia and the ventral nerve cord between the 4th and the 30th segments including the clitellum, but not in the posterior segments (31-55th). The number of annetocin-IR fibers decreased from the 4th to the 30th segment. The annetocin-secretory cells were identified by the immunogold staining, and filled with gold-labeled vesicles, 200-250 nm in diameter, which included moderately electron dense material. The annetocin-secretory cells possessed a euchromatic nucleus, well-developed rough endoplasmic reticulum and Golgi apparatus. Some of the annetocin-secretory cells were found to form a neurohemal-like structure, where somata or fibers with loose glial investment came in contact with the coelomic space at the ventral side of the subesophageal ganglion. The results suggest that annetocin is a neuropeptide produced and secreted by the neuron in the cerebral and subesophageal ganglia of the earthworm.     

Role of Ascorbate in Detoxifying Ozone in the Apoplast of Spinach (Spinacia oleracea L.) Leaves

Both reduced and oxidized ascorbate (AA and DHA) are present in the aqueous phase of the extracellular space, the apoplast, of spinach (Spinacia oleracea L.) leaves. Fumigation with 0.3 [mu]L L-1 of ozone resulted in ozone uptake by the leaves close to 0.9 pmol cm-2 of leaf surface area s-1. Apoplastic AA was slowly oxidized by ozone. The initial decrease of apoplastic AA was <0.1 pmol cm-2 s-1. The apoplastic ratio of AA to (AA + DHA) decreased within 6 h of fumigation from 0.9 to 0.1. Initially, the concentration of (AA + DHA) did not change in the apoplast, but when fumigation was continued, DHA increased and AA remained at a very low constant level. After fumigation was discontinued, DHA decreased very slowly in the apoplast, reaching control level after 70 h. The data show that insufficient AA reached the apoplast from the cytosol to detoxify ozone in the apoplast when the ozone flux into the leaves was 0.9 pmol cm-2 s-1. The transport of DHA back into the cytosol was slower than AA transport into the apoplast. No dehydroascorbate reductase activity could be detected in the apoplast of spinach leaves. In contrast to its extracellular redox state, the intracellular redox state of AA did not change appreciably during a 24-h fumigation period. However, intracellular glutathi-one became slowly oxidized. At the beginning of fumigation, 90% of the total glutathione was reduced. Only 10% was reduced after 24-h exposure of the leaves to 0.3 [mu]L L-1 of ozone. Necrotic leaf damage started to become visible when fumigation was extended beyond a 24-h period. A close correlation between the extent of damage, on the one hand, and the AA content and the ascorbate redox state of whole leaves, on the other, was observed after 48 h of fumigation. Only the youngest leaves that contained high ascorbate concentrations did not exhibit necrotic leaf damage after 48 h.     

Changes in peroxidase activity and in peroxidase isozymes in carrot callus

Activity of soluble peroxidase and its isozyme patterns in carrot ( Daucus carota L.) callus after excision and transfer to fresh Murashige and Skoog (MS) culture medium was investigated. The activity decreased markedly until day 1 and then increased gradually during days 7–10 at room temperature. The rapid initial decrease of the activity was also observed at low temperature (2°C) as well as in the presence of cycloheximide. However, the subsequent increase in the peroxidase activity after day I was slower at low temperature than at room temperature, and was not detected in the presence of cycloheximide. Activity of catalase decreased slightly within 4 days and cycloheximide enhanced the decrease of the activity. Two cationic and one anionic peroxidase isozymes disappeared or decreased markedly within the first day and one cationic and anionic peroxidase recovered 3–6 days after excision.     

Phenolic Components of Brown Scales of Onion Bulbs Produce Hydrogen Peroxide by Autooxidation

2 O₂ when the brown scales were suspended in water. Brown components isolated from the brown scales also transformed molecular oxygen into H₂O₂. During the autooxidation process, absorbance in the visible region was increased. On acid hydrolysis of the brown fraction, 2,4,6-trihydroxyphenylglyoxylic acid, 3,4-dihydroxybenzoic acid and the quinone form of benzoic acid were detected. In addition, glucose was detected as a sugar. 3,4-Dihydroxybenzoic acid was preferentially oxidized during autooxidation of the brown fraction. One of the oxidation products was the quinone form. Stable electron spin resonance (ESR) signals were detected in the brown fraction. New ESR signals appeared on oxidation of the brown fraction by hexacyanoferrate (III). One of the newly formed radicals seemed to have a 3,4-dihydroxyphenyl group. Based on these results, possible structures, mechanism of H₂O₂ formation and biological significance of the brown components are discussed. Received 11 April 2001/ Accepted in revised form 3 August 2001     

Transformation of ethyl alcohol to ethyl nitrite in acidified saliva: possibility of its occurrence in the stomach

After taking alcoholic beverages, the ethanol is mixed with saliva and then gastric juice. As pH of gastric juice is around 2, the ethanol might be transformed to ethyl nitrite in the stomach by reacting with salivary nitrite. In this study, reactions between ethanol and nitrite in acidified saliva were investigated. The result indicates that nitrite in acidified saliva reacted with ethanol producing ethyl nitrite. It is discussed that ethyl nitrite might be formed in the stomach after drinking alcoholic beverages and that the ethyl nitrite might function as a donor of NO in intestinal and gastric tissues.     

Symbiotic <Emphasis Type="Italic">Chlorella variabilis</Emphasis> strain, 1 N,can influence the digestive process in the host <Emphasis Type="Italic">Paramecium bursaria</Emphasis> during early infection

The association between the ciliate Paramecium bursaria and symbiotic Chlorella spp. is mutually beneficial. However, this relationship is facultative mutualism because both the host and the symbiotic algae can grow by themselves. This association is easily re-established by mixing the two species together. Following algal mixing, some algae become enclosed in the digestive vacuole membrane of the paramecia to which both acidosomes and lysosomes fuse. To establish endosymbiosis, some algae acquire temporal resistance to the host lysosomal enzymes in the digestive vacuoles. We examined whether the algae influence the differentiation of the host digestive process using LysoSensor staining to evaluate the acidification of the digestive vacuoles. Furthermore, to assess lysosomal fusion with the digestive vacuole, Gomori’s staining was conducted. Acidification and lysosomal fusion occurred later in digestive vacuoles containing living algae than in those containing boiled algae or latex spheres. This phenomenon was observed when the living algae were maintained under a constant light condition. These results suggest that the algae release some unknown factor in response to light exposure, and the factor may be associated with the alteration of the host digestive process, indicating that the living algae can influence the host digestive processes during early algal infection.     

Tyramine and β-phenylethylamine,from fermented food products,as agonists for the human trace amine-associated receptor 1 (hTAAR1) in the stomach

The aromatic amines tyramine and β-phenylethylamine are abundant in fermented foods. Recently, a family of human trace amine-associated receptors (hTAARs) was discovered that responds to these compounds. This study examined the expression of hTAAR genes in five human organs. Among them, the stomach expressed hTAAR1 and hTAAR9. Interestingly, more hTAAR1 was expressed in the pylorus than in the other stomach regions. The CRE-SEAP reporter assay revealed that only hTAAR1 functioned as a G_s-coupled receptor in response to tyramine and β-phenylethylamine stimulation. The β-phenylethylamine-mediated hTAAR1 activity could be potentiated using 3-isobutyl-1-methylxanthine. These data suggest that tyramine and β-phenylethylamine in fermented foods act at hTAAR1 as agonists in the pylorus of stomach.     

Production of nitric oxide-derived reactive nitrogen species in human oral cavity and their scavenging by salivary redox components

Nitrite is reduced to nitric oxide (NO) in the oral cavity. The NO generated can react with molecular oxygen producing reactive nitrogen species. In this study, reduction of nitrite to NO was observed in bacterial fractions of saliva and whole saliva. Formation of reactive nitrogen species from NO was detected by measuring the transformation of 4,5-diaminofluorescein (DAF-2) to triazolfluorescein (DAF-2T). The transformation was fast in bacterial fractions but slow in whole saliva. Salivary components such as ascorbate, glutathione, uric acid and thiocyanate inhibited the transformation of DAF-2 to DAF-2T in bacterial fractions without affecting nitrite-dependent NO production. The inhibition was deduced to be due to scavenging of reactive nitrogen species, which were formed from NO, by the above reagents. The transformation of DAF-2 to DAF-2T was faster in bacterial fractions and whole saliva which were prepared 1-4 h after tooth brushing than those prepared immediately after toothbrushing. Increase in the rate as a function of time after toothbrushing seemed to be due to the increase in population of bacteria which could reduce nitrite to NO. The results obtained in this study suggest that reactive nitrogen species derived from NO are continuously formed in the oral cavity and that the reactive nitrogen species are effectively scavenged by salivary redox components in saliva but the scavenging is not complete.