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The green alga Chlorella zofingiensis can accumulate high level of oleic acid (OA, C18:1△(9)) rich oils in response to stress conditions. To understand the regulation of biosynthesis of fatty acid in particular OA at the molecular level, we cloned and characterized the stearoyl acyl carrier protein (ACP) desaturase (SAD) responsible for OA formation through desaturation of stearic acid (C18:0) from C. zofingiensis. Southern blot indicated that the C. zofingiensis genome contained a single copy of SAD, from which the deduced amino acid sequence shared high identity to the corresponding homologs from other microalgae and higher plants. The desaturation activity of SAD was demonstrated in vitro using C18:0-ACP as a substrate. Stress conditions such as high light (HL), nitrogen deficiency (N(-)), or combination of HL and N(-) (HL + N(-)) drastically up-regulated the transcripts of biotin carboxylase (BC, a subunit of ACCase) and SAD, and therefore induced considerably the cellular accumulation of total fatty acids including OA. Glucose (50 mM) gave rise to the similar up-regulation of the two genes and induction of fatty acid accumulation. The accumulation of intracellular reactive oxygen species was found to be associated with the up-regulation of genes. This is the first report of characterization of Chlorella-derived SAD and the results may contribute to understanding of the mechanisms involved in fatty acid/lipid biosynthesis in microalgae.

Phototherapy has been widely used in the treatment of neonatal hyperbilirubinemia. Recent reports, however, have indicated that fluorescent light may be toxic and mutagenic to mammalian cells. these findings suggest possible long-term side effects with the use of phototherapy. This study was undertaken to determine the effects of phototherapy on hepatic microsomal enzyme activity. The exposure of Sprague-Dawley and Gunn rats to special blue fluorescent light at an average irradiance of 1,200 microW/cm2 resulted in no significant changes in liver microsomal enzyme activity for aniline hydroxylase, p-nitroanisole-O-demethylase, ethylmorphine-N-demethylase, cytochrome c reductase or the quantity of cytochrome P-450. A significant decrease in aniline hydroxylase and p-nitroanisole-O-demethylase activity was observed when liver microsomes were exposed in vitro to special blue fluorescent light. Photoactivated bilirubin did not effect the activity of the mixed-function oxidase enzymes measured under the conditions of this study.

The authors studied the effects of the whole-body x-irradiation on the activity of delta-aminolevulinate synthase and heme oxygenase in the liver of Wistar rats. The activity of delta-aminolevulinate synthase decreased to 81-49% of normal by the 1st-3d day after irradiation in a dose of 7 Gy followed by partial normalization of the enzyme activity by the 5th-7th day. The activity of heme oxygenase was over 2 times as increased by the 5th-7th day following irradiation in a dose of 7 Gy. Irradiation in a dose of 5 Gy did not alter the activity of heme oxygenase and caused a negligible reduction in the activity of delta-aminolevulinate synthase. During the most pronounced decrease in the rate of heme synthesis in the liver of irradiated rats, there was an elevation in the level of "free" heme (measured by the degree of tryptophane pyrrolase saturation with heme). This attests to a possible lowering of the rate of heme utilization in the synthesis of heme. A possible role of the effects described in the irradiation-induced decrease in the content of cytochrome P-450 in the animals' liver.

A microsomal preparation from irradiated parsley cell cultures catalyses the NADPH and dioxygen-dependent hydroxylation of (S)-naringenin [(S)-5, 7, 4'-trihydroxyflavanone] to eriodictyol (5, 7, 3', 4'-tetrahydroxyflavanone). Dihydrokaempferol, kaempferol, and apigenin were also substrates for the 3'-hydroxylase reaction. In contrast prunin (naringenin 7-O-beta-glucoside) was not converted by the enzyme. The microsomal preparation, which also contains cinnamate 4-hydroxylase, did not catalyse hydroxylation of 4-coumaric acid to caffeic acid. 3'-Hydroxylase activity is partially inhibited by carbon monoxide in the presence of oxygen as well as by cytochrome c and NADP+. These properties suggest that the enzyme is a cytochrome P-450-dependent flavonoid 3'-monooxygenase. Pronounced differences in the inhibition of flavonoid 3'-hydroxylase and cinnamate 4-hydroxylase were found with EDTA, potassium cyanide and N-ethylmaleimide. Irradiation of the cell cultures led to increase of flavonoid 3'-hydroxylase activity with a maximum at about 23 h after onset of irradiation and subsequent decrease. This is similar to light-induction of phenylalanine ammonialyase and cinnamate 4-hydroxylase. In contrast, treatment of the cell cultures with a glucan elicitor from Phytophthora megasperma f. sp. glycinea did not induce flavonoid 3'-hydroxylase nor chalcone isomerase but caused a strong increase in the activities of phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, and NADPH--cytochrome reductase. The results prove that flavonoid 3'-hydroxylase and cinnamate 4-hydroxylase are two different microsomal monooxygenases.

Three groups of male Wistar rats were compared: one group received injections of Misonidazole (MISO) (200 mg/kg, i.p.), another group was treated by whole-body irradiation, and the third population received both treatments. Irradiation induced an important decrease of monooxygenase (O-demethylase) activity of hepatic microsomes seven days after the treatment. Cyt. P-450 levels hardly decreased, whereas lipid peroxidation was two-fold three days after irradiation. These different parameters were not modified neither after misonidazole treatment nor after association of irradiation and MISO regimen. The presence of oxygen in liver may explain that a radiosensitizer such as MISO does not increase irradiation damage on liver microsomes enzymes, oxygen preventive activation of MISO by radiation: the nitro groupment of MISO was not reduced in nitroso and amine compounds.

Light reversal of carbon monoxide inhibition of 2-acetylaminofluorene (AAF) N- and ring-hydroxylations is examined with liver microsomes from 3-methylcholanthrene (MC) pretreated hamsters. Photochemical action spectra indicate that carbon monoxide inhibition of ring-hydroxylation is readily reversed by light of 440 nm while that of N-hydroxylation is not reversed. These data suggest that these mixed function oxidations are catalyzed by CO combining enzymes with different properties.

Microsomal preparations from carrot (Daucus carota L.) cell suspension cultures catalyze the formation of trans-5-O-caffeoyl-D-quinate (chlorogenate) from trans-5-O-(4-coumaroyl)-D-quinate. trans-5-O-(4-Coumaroyl)shikimate is converted to about the same extent to trans-5-O-caffeoylshikimate. trans-4-O-(4-Coumaroyl)-D-quinate, trans-3-O-(4-coumaroyl)-D-quinate, trans-4-coumarate, and cis-5-O-(4-coumaroyl)-D-quinate do not act as substrates. The reaction is strictly dependent on molecular oxygen and on NADPH as reducing cofactor. NADH and ascorbic acid cannot substitute for NADPH. Cytochrome c, Tetcyclacis, and carbon monoxide inhibit the reaction suggesting a cytochrome P-450-dependent mixed-function monooxygenase. Competition experiments as well as induction and inhibition phenomena indicate that there is only one enzyme species which is responsibl for the hydroxylation of the 5-O-(4-coumaric) esters of both D-quinate and shikimate. The activity of this enzyme is greatly increased by in vivo irradiation of the cells with blue/uv light. We conclude that the biosynthesis of the predominant caffeic acid conjugates in carrot cells occurs via the corresponding 4-coumaric acid esters. Thus, in this system, 5-O-(4-coumaroyl)-D-quinate can be seen as the final intermediate in the chlorogenic acid pathway.

Mouse neuroblastoma cells in culture have been used as a model for the study of the mechanism by which activities of tyrosine hydroxylase (EC 1.14.3.a) are regulated in sympathetic tissue. The activity of tyrosine hydroxylase in cultured cells drops to barely detectable activities after 1 week and remains low for months in culture in the uncloned cell line of neuroblastoma. Activity in an adrenergic clone isolated from the uncloned line has about 20% of the activity of the fresh grated tumor cell. N(6), O(2')-dibutyryl adenosine 3':5'-cyclic monophosphate causes a concentration and time-dependent increase in enzyme activity in both the cloned and uncloned cell lines. Enzyme activity is elevated by other stable analogs of adenosine 3':5'-cyclic monophosphate, notably the N(6)-monobutyryl, 8-aminomethyl, and 8-methylthio derivatives of the cyclic nucleotide; by the inhibitor of cyclic nucleotide phosphodiesterase, papaverine; and by sodium butyrate. Changes in cell morphology and tyrosine hydroxylase activity are shown not to be necessarily related.