Relationship of Camphor Biosynthesis to Leaf Development in Sage (Salvia officinalis)

The camphor content of sage (Salvia officinalis L.) leaves increases as the leaves expand, and the increase is roughly proportional to the number of filled peltate oil glands which appear on the leaf surface during the expansion process. ¹⁴CO₂ is more rapidly incorporated into camphor and its direct progenitors in expanding leaves than in mature leaves, and direct in vitro measurement of the key enzymes involved in the conversion of geranyl pyrophosphate to camphor indicates that these enzymes, including the probable rate-limiting cyclization step, are at the highest levels during the period of maximum leaf expansion. These results clearly demonstrate that immature sage leaves synthesize and accumulate camphor most rapidly.     

Metabolism of Monoterpenes in Cell Cultures of Common Sage (Salvia officinalis) : Biochemical Rationale for the Lack of Monoterpene Accumulation

Leaves of common sage (Salvia officinalis) accumulate monoterpenes in glandular trichomes at levels exceeding 15 milligrams per gram fresh weight at maturity, whereas sage cells in suspension culture did not accumulate detectable levels of monoterpenes (<0.3 nanograms per gram fresh weight) at any stage of the growth cycle, even in the presence of a polystyrene resin trap. Monoterpene biosynthesis from [U-¹⁴C]sucrose was also virtually undetectable in this cell culture system. In vitro assay of each of the enzymes required for the sequential conversion of the ubiquitous isoprenoid precursor geranyl pyrophosphate to (+)-camphor (a major monoterpene product of sage) in soluble extracts of the cells revealed the presence of activity sufficient to produce (+)-camphor at a readily detectable level (>0.3 micrograms per gram fresh weight) at the late log phase of growth. Other monoterpene synthetic enzymes were present as well. In vivo measurement of the ability to catabolize (+)-camphor in these cells indicated that degradative capability exceeded biosynthetic capacity by at least 1000-fold. Therefore, the lack of monoterpene accumulation in undifferentiated sage cultures could be attributed to a low level of biosynthetic activity (relative to the intact plant) coupled to a pronounced capacity for monoterpene catabolism.     

Development and Essential Oil Content of Secretory Glands of Sage (Salvia officinalis)

Scanning electron microscopy of sage (Salvia officinalis L.) leaves confirmed the presence of two basic types of glandular trichomes consisting of a capitate stalked form containing a multicellular stalk and surmounted by a unicellular secretory head, and a capitate sessile form containing a unicellular stalk and unicellular, or multicellular, secretory head. In the latter type, secretory activity and filling of the subcuticular cavity may begin at virtually any stage of the division cycle affording fully developed glands containing from one to twelve cells in the secretory head. Gas liquid chromatographic analysis of the oil content of the most numerous gland species (capitate stalked, capitate sessile with one and with eight secretory cells) indicated only minor quantitative differences in essential oil composition. Thus, each gland type is capable of producing the four major monoterpene families (p-menthanes, pinanes, bornanes and thujanes) characteristic of sage.     

The Induction of Cytochrome P-450 and Ethanol Oxidation in Yarrowia lipolytica Cells

The study of the effect of different ethanol concentrations in the medium on the growth and activity of enzymatic systems involved in ethanol oxidation in Yarrowia lipolytica showed that the cultivation of yeast cells on 1 and 2% ethanol caused their rapid growth and a drastic increase in cell respiration and sensitivity to cyanide already in the first hours of cultivation. At the same time, during cultivation on 3, 4, and 5% ethanol, the growth and respiration of yeast cells were considerably suppressed. All of the ethanol concentrations studied induced the synthesis of cytochrome P-450, its dynamics in cells being dependent on the initial concentration of ethanol in the medium. When the initial concentration of ethanol was 1 and 2%, the content of cytochrome P-450 in cells steeply decreased after a short period of induction. However, when the initial concentration of ethanol in the medium was 4 to 5%, the content of cytochrome P-450 in cells was high throughout the cultivation period. The induction of cytochrome P-450 in cells preceded the induction of the NAD-dependent enzymes alcohol dehydrogenase and catalase, which, like cytochrome P-450, are also involved in ethanol oxidation by yeasts. The activity of catalase was higher in the yeast cells grown in the presence of 3 to 5% ethanol than in the cells grown in the presence of 1 and 2% ethanol. The roles played by cytochrome P-450, alcohol dehydrogenase, and catalase in ethanol oxidation by yeast cells are discussed.     

Metabolism of Monoterpenes : Metabolic Fate of (+)-Camphor in Sage (Salvia officinalis)

The bicyclic monoterpene ketone (+)-camphor undergoes lactonization to 1,2-campholide in mature sage (Salvia officinalis L.) leaves followed by conversion to the β-d-glucoside-6-O-glucose ester of the corresponding hydroxy acid (1-carboxymethyl-3-hydroxy-2,2,3-trimethyl cyclopentane). Analysis of the disposition of (+)-[G-³H]camphor applied to midstem leaves of intact flowering plants allowed the kinetics of synthesis of the bis-glucose derivative and its transport from leaf to root to be determined, and gave strong indication that the transport derivative was subsequently metabolized in the root. Root extracts were shown to possess β-glucosidase and acyl glucose esterase activities, and studies with (+)-1,2[U-¹⁴C]campholide as substrate, using excised root segments, revealed that the terpenoid was converted to lipid materials. Localization studies confirmed the radiolabeled lipids to reside in the membranous fractions of root extracts, and analysis of this material indicated the presence of labeled phytosterols and labeled fatty acids (C₁₄ to C₂₀) of acyl lipids. Although it was not possible to detail the metabolic steps between 1,2-campholide and the acyl lipids and phytosterols derived therefrom because of the lack of readily detectable intermediates, it seemed likely that the monoterpene lactone was degraded to acetyl CoA which was reincorporated into root membrane components via standard acyl lipid and isoprenoid biosynthetic pathways. Monoterpene catabolism thus appears to represent a salvage mechanism for recycling mobile carbon from senescing oil glands on the leaves to the roots.     

Doxorubicin Overproduction in Streptomyces peucetius: Cloning and Characterization of the dnrU Ketoreductase and dnrV Genes and the doxA Cytochrome P-450 Hydroxylase Gene

Doxorubicin-overproducing strains of Streptomyces peucetius ATCC 29050 can be obtained through manipulation of the genes in the region of the doxorubicin (DXR) gene cluster that contains dpsH, the dpsG polyketide synthase gene, the putative dnrU ketoreductase gene, dnrV, and the doxA cytochrome P-450 gene. These five genes were characterized by sequence analysis, and the effects of replacing dnrU, dnrV, doxA, or dpsH with mutant alleles and of doxA overexpression on the production of the principal anthracycline metabolites of S. peucetius were studied. The exact roles of dpsH and dnrV could not be established, although dnrV is implicated in the enzymatic reactions catalyzed by DoxA, but dnrU appears to encode a ketoreductase specific for the C-13 carbonyl of daunorubicin (DNR) and DXR or their biosynthetic precursors. The highest DXR titers were obtained in a dnrX dnrU (N. Lomovskaya, Y. Doi-Katayama, S. Filippini, C. Nastro, L. Fonstein, M. Gallo, A. L. Colombo, and C. R. Hutchinson, J. Bacteriol. 180:2379–2386, 1998) double mutant and a dnrX dnrU dnrH (C. Scotti and C. R. Hutchinson, J. Bacteriol. 178:7316–7321, 1996) triple mutant. Overexpression of doxA in a doxA::aphII mutant resulted in the accumulation of DXR precursors instead of in a notable increase in DXR production. In contrast, overexpression of dnrV and doxA jointly in the dnrX dnrU double mutant or the dnrX dnrU dnrH triple mutant increased the DXR titer 36 to 86%.     

Cytochrome P-450 in the sophorose-lipid-producing yeast Candida (Torulopsis) apicola

The appearance of cytochrome P-450 and of cytochrome oxidase aa₃ were determined in the sophorose lipid producing yeast Candida (Torulopsis) apicola IMET 43 747 grown on a mixture of glucose and n-hexadecane. Cytochrome P-450, detectable in both the logarithmic and the stationary growth phase was not repressed by glucose. At the end of the logarithmic growth phase the content of cytochrome P-450 was three- to fivefold increased, which was connected with initiation of sophorose lipid biosynthesis. After that it dropped to the basal level, which remained constant during sophorose lipid biosynthesis. Cytochrome P-450 from logarithmic cells was cross-reactive with an antibody derived against cytochrome P-450alk from C. tropicalis. With microsomal proteins of stationary cells no cross-reactivity was obtained. The microsomal hydroxylase system of stationary cells seem to be regulated by the carbohydrate used as carbon source. Correspondence to: R. K. Hommel     

Cytochrome P-450 from the Mesocarp of Avocado (Persea americana)

The microsomal fraction from the mesocarp of avocado (Persea americana) is one of few identified rich sources of plant cytochrome P-450. Cytochrome P-450 from this tissue has been solubilized and purified. Enzymatic assays (p-chloro-N-methylaniline demethylase) and spectroscopic observations of substrate binding suggest a low spin form of the cytochrome, resembling that in the microsomal membrane, can be recovered. However, this preparation of native protein is a mixture of nearly equal proportions of two cytochrome P-450 polypeptides that have been resolved only under denaturing conditions. Overall similarities between these polypeptides include indistinguishable amino acid compositions, similar trypsin digest patterns, and cross reactivity with the same antibody. The amino terminal sequences of both polypeptides are identical, with the exception that one of them lacks a methionine residue at the amino terminus. This sequence exhibits some similarities with the membrane targeting signal found at the amino terminus of most mammalian cytochromes P-450.     

Interactions of Avocado (Persea americana) Cytochrome P-450 with Monoterpenoids

The microsomal fraction of avocado (Persea americana) mesocarp is a rich source of cytochrome P-450 active in the demethylation of xenobiotics. Cytochrome P-450 from this tissue has been purified and well characterized at the molecular level (DP O'Keefe, KJ Leto [1989] Plant Physiol 89: 1141-1149; KR Bozak, H Yu, R Sirevag, RE Christoffersen [1990] Proc Natl Acad Sci USA 87: 3904-3908). Despite this extensive characterization, the role of the enzyme in vivo was not established. Optical and electron paramagnetic resonance binding studies described here suggest that the monoterpenoids, nerol and geraniol, are substrates of avocado cytochrome P-450 (spectral dissociation constant of 7.2 and 35 micromolar, respectively). Avocado microsomes have been shown to catalyze the hydroxylation of these monoterpenoids, and both nerol and geraniol have been shown to inhibit the activity of avocado cytochrome P-450 toward the artificial substrate 7-ethoxycoumarin, with nerol a competitive inhibitor of this activity.     

Effects of Hormones on Changes in Cytochrome P-450, Prolyl Hydroxylase,and Glycerol Phosphate Acyltransferase in Primary Monolayer Cultures of Parenchymal Cells from Adult Rat Liver

Previous studies have shown that isolation and primary culture of rat hepatocytes in a standard, chemically defined medium is associated with selective changes in microsomal function. These changes were found to be selectively sensitive to addition of hormones to the culture medium. The concentration of cytochrome P-450 declined dramatically during the first 24 hours of incubation. However, cytochrome P₁-450, a form of the hemoprotein induced by polycyclic aromatic hydrocarbons, was resistant to this change. Cytochrome P₁-450 levels selectively rose during the first ten hours in culture and, thereafter, declined at a less rapid rate than did the cytochrome P-450 in normal hepatocytes or in cells prepared from phenobarbital pretreated animals. Addition of dexamethasone to the medium at the time of cell plating partially prevented the fall of cytochrome P-450 and of ¹⁴C-heme in microsomes prepared from hepatocytes derived from rats given 5¹⁴[C]-δ-aminolevulinic acid. This suggests that the steroid decreases degradation of the hemoprotein. As compared to the loss of cytochrome P-450 in cultures of normal hepatocytes, the hemoprotein fell to lower levels in hepatocytes prepared from regenerated liver four days after partial hepatectomy. This result may be related to the accelerated formation of the monolayer in the cultures of regenerated hepatocytes. Both sn-glycerol-3-phosphate acyltransferase activity and glycerol kinase activity declined in the first 24 hours of culture. The fall in the latter enzyme was partially prevented by addition of estradiol. Collagen prolyl hydroxylase, a newly discovered microsomal constituent of the hepatocyte, rose slightly during the first 24 hours in culture. This change was augmented threefold by addition of insulin to the medium. We conclude that the present hepatocyte culture system with its attendant changes in functional phenotype may be useful in better defining the role of hormones in modulating metabolic processes in the liver.     

Cytochrome P-450 and oxygen toxicity. Oxygen-dependent induction of ethanol-inducible cytochrome P-450 (IIE1) in rat liver and lung

The ethanol-inducible form of cytochrome P-450 (P-450IIE1) has previously been shown to exhibit an unusually high rate of oxidase activity with the subsequent formation of reactive oxygen species, e.g., hydrogen peroxide, and to be the main contributor of microsomal oxidase activity in liver microsomes from acetone-treated rats [Ekstr?m & Ingelman-Sundberg (1989) Biochem. Pharmacol. (in press)]. The results here presented indicate that oxygen exposure of rats causes an about 4-fold induction of P-450IIE1 in rat liver and lung microsomes. The induction in liver was not accompanied by any measurable increase in the P-450IIE1 mRNA levels, but the enhanced amount of P-450IIE1 accounted for 60% of the net 50% increase in the level of hepatic P-450 as determined spectrophotometrically. The induction of P-450IIE1 was maximal after 60 h of O2 exposure, and concomitant increases in the rates of liver microsomal CCl4-dependent lipid peroxidation, O2 consumption, NADPH oxidation, O2- formation, H2O2 production, and NADPH-dependent microsomal lipid peroxidation were seen. Liver microsomes from oxygen-treated rats had very similar properties to those of microsomes isolated from acetone-treated rats with respect to the P-450IIE1 content and catalytic properties, but different from those of thyroxine-treated animals. Treatment of rats with the P-450IIE1 inducer acetone in combination with oxygen exposure caused a potentiation of the NADPH-dependent liver and lung microsomal lipid peroxidation and decreased the survival time of the rats. The results reached indicate a role for cytochrome P-450 and, in particular, for cytochrome P-450IIE1 in oxygen-mediated tissue toxicity.     

Chemotype Diversity of Indigenous Dalmatian Sage (Salvia officinalis L.) Populations in Montenegro

To identify how many chemotypes of Salvia officinalis exist in Montenegro, the chemical composition of the essential oils of 12 wild‐growing populations was determined by GC‐FID and GC/MS analyses. Among the 40 identified constituents, the most abundant were cis‐thujone (16.98–40.35%), camphor (12.75–35.37%), 1,8‐cineol (6.40–12.06%), trans‐thujone (1.5–10.35%), camphene (2.26–9.97%), borneol (0.97–8.81%), viridiflorol (3.46–7.8%), limonene (1.8–6.47%), α‐pinene (1.59–5.46%), and α‐humulene (1.77–5.02%). The composition of the essential oils under study did not meet the ISO 9909 requirements, while the oils of populations P02–P04, P09, and P10 complied with the German Drug Codex. A few of the main essential‐oil constituents appeared to be highly intercorrelated. Strong positive correlations were observed between α‐pinene and camphene, camphene and camphor, as well as between cis‐thujone and trans‐thujone. Strong negative correlations were evidenced between cis‐thujone and α‐pinene, cis‐thujone and champhene, cis‐thujone and camphor, as well as between trans‐thujone and camphene. Multivariate analyses allowed the grouping of the populations into three distinct chemotypes, i.e., Chemotype A, rich in total thujones, Chemotype B, with intermediate contents of thujones, α‐pinene, camphene, and camphor and high borneol contents, and Chemotype C, rich in camphor, camphene, and α‐pinene. The chemotypes did not significantly differ in the total essential‐oil content and the cis/trans‐thujone ratio.     

Cytochrome P-450(17alpha) in beta-cells of rat pancreas and its local steroidogenesis

We have found cytochrome P-450(17alpha) in the islets of Langerhans of rat pancreas. Its existence coincided with that of insulin and demarcated those of glucagon and somatostatin, demonstrating the localization in beta-cells. The enzyme has not only 17alpha-hydroxylase activity but also lyase one, which is a prerequisite for androgen biosynthesis. The pancreatic microsomes converted progesterone mainly to androstenedione with a minor production of 17alpha-hydroxyprogesterone. Due to a low activity of the built-in lyase, cytochrome P-450(17alpha) requires a sufficient electron-transfer from P-450 reductase or presence of an activator to promote the C-C bond cleavage. In beta-cells, P-450 reductase was abundant and could efficiently transfer electrons to P-450(17alpha). Actually, inhibition with anti-P-450 reductase or limitation of NADPH preferentially reduced the lyase activity. Androstenedione was accumulated when its further metabolism was suppressed. We also found localization of cytochrome P-450scc and 3beta-hydroxysteroid dehydrogenase in beta-cells. These results indicate that the immediate substrate for androgen formation, progesterone, is intracellularly produced and is converted mainly to androstenedione with support by an efficient electron supply from P-450 reductase. The product was supposed to be further metabolized to the reduced derivatives such as testosterone, 5alpha-androstanedione, and dihydrotestosterone, which would act as local steroids in the islets of Langerhans.     

Cytochrome P-450 human-2 (P-450IIC9) in mephenytoin hydroxylation polymorphism in human livers: differences in substrate and stereoselectivities among microheterogeneous P-450IIC species expressed in yeasts

The cDNA of a P-450 human-2 and the two other closely related cDNAs, MP-8 (two deduced amino acids substituted) and lambda hPA6 (two deduced amino acids deleted) were expressed in Saccharomyces cerevisiae cells, and their catalytic and chemical properties were compared to identify which cDNA encodes a major S-mephenytoin 4'-hydroxylase in human livers. In immunoblots, P-450 human-2 cDNA-derived protein in yeasts was stained at the position identical with P-450 human-2 purified from liver and a major protein in microsomes of 19 Japanese livers. MP-8- and lambda hPA6-derived proteins were immunostained at positions near, but distinct from P-450 human-2, and were not detected in those 19 livers. All three proteins expressed in yeasts catalyzed hydroxylation of mephenytoin, hexobarbital, benzo[a]pyrene and tolbutamide, although the rates of the hydroxylation of most of the drugs by P-450 human-2 were higher than those of the two others. In addition, these expressed proteins showed clear differences in the hydroxylation of chiral substrates: P-450 human-2 catalyzed the hydroxylation of S-mephenytoin five times faster than that of the R-enantiomer. Similar high enantioselectivities were also observed on the hydroxylation of R- and S-hexobarbital. However, MP-8- and lambda hPA6-derived proteins catalyzed hydroxylation of these two drugs with less or almost no stereoselectivity. These results indicate that only a few amino acid alterations cause dramatic changes in both the chemical and catalytic properties of P-450 human-2.     

Regulation of Oleoresinosis in Grand Fir (Abies grandis) (Coordinate Induction of Monoterpene and Diterpene Cyclases and Two Cytochrome P450-Dependent Diterpenoid Hydroxylases by Stem Wounding)

Oleoresin (pitch) is a defensive secretion composed of monoterpene olefins (turpentine) and diterpene resin acids (rosin) that is produced in grand fir (Abies grandis Lindl.) stems in response to wounding. Monoterpene and diterpene biosynthesis are coordinately induced in wounded stems as determined by monitoring the activity of monoterpene and diterpene cyclases, as well as two cytochrome P450-dependent diterpenoid hydroxylases involved in the formation of ([mdash])-abietic acid, the principal resin acid of this species. The activity of these enzymes reaches maximum levels that are 5- to 100-fold higher than those of nowwounded control stems 10 d after wounding and this is followed by a synchronous decline. The increase in biosynthetic activity is consequently followed by the accumulation of a viscous mass of resin acids, with the loss of the volatile monoterpenes, at the site of injury. The observed coordinate induction of monoterpene olefin and abietic acid bio-synthesis and the results of oleoresin analysis are consistent with the role of the volatile monoterpenes as a solvent for the mobilization and deposition of resin acids at the wound site to seal the injury with a rosin barrier after the evaporation of the turpentine. The last step of resin acid biosynthesis is catalyzed by an operationally soluble aldehyde dehydrogenase that is not inducible by wounding but seemingly is expressed constitutively at a high level. In vivo [14C]acetate feeding and resin analysis indicate that this enzyme is not efficiently coupled to the earlier steps of the pathway.     

Cytochrome P-450 omega-hydroxylase: a potential O(2) sensor in rat arterioles and skeletal muscle cells

The purposes of this study were to 1) further evaluate the possible role that vasoconstrictor metabolites of cytochrome P-450 (CYP) omega-hydroxylase plays in O(2)-induced constriction of arterioles in the rat skeletal muscle microcirculation, 2) determine whether omega-hydroxylases are expressed in rat cremaster muscle, and 3) determine whether the enzyme is located in the parenchyma or the arterioles. O(2)-induced constriction of third-order arterioles in the in situ cremaster muscle of Sprague-Dawley rats was significantly inhibited by the CYP inhibitors N-methyl-sulfonyl-12,12-dibromododec-11-enamide (DDMS; 50 microM) and 17-octadecynoic acid (ODYA; 10 microM). Immunoblot analysis with antibody raised against CYP4A protein indicated the presence of immunoreactive proteins in the cremaster muscle and in isolated arterioles and muscle fibers from this tissue. However, the molecular mass of the immunoreactive proteins was 85 kDa instead of the expected 50--52 kDa for CYP4A omega-hydroxylase isolated from rat liver or kidney. Treatment of the cremaster muscle with deglycosidases shifted the bands to the expected range which indicates that these proteins are likely glycosylated in skeletal muscle. Immunohistochemistry revealed intense staining of both muscle fibers and microvessels in the cremaster muscle. The results of this study indicate that O(2) sensing in the skeletal muscle microcirculation may be mediated by CYP4A omega-hydroxylases in both arterioles and parenchymal cells.     

杨树细胞色素P450类固醇单加氧酶(CYP90)基因的克隆与分析

拟南芥的CPD基因编码一种与植物油菜素内酯(brassinosteroids,BRs)生物合成有关的细胞色素P450类固醇单加氧酶(CYP90 )。为探讨油菜素内酯这类新型植物激素在多年生木本植物中生物合成及作用的分子机理,以拟南芥CPD基因的一个cDNA片段为探针,从一种杂交杨 (Populustremula×tremudelois)的cDNA文库中分离出一个长 1 442bp的cDNA片段,然后再以这个cDNA的 5′区为探针,从这种杂交杨的基因组文库中分离出一个长 1 900bp的基因组DNA(gDNA)片段。测序结果表明,这段cDNA的 5′区与这段gDNA的 3′区重合; 由这段cDNA和gDNA组成的读框编码一个由 476个氨基酸组成的分子量为 63kD的蛋白质。该蛋白与拟南芥CYP90的同源性为 78 32%,比后者仅长 4个氨基酸,在所有已知的功能结构域,其中包括与BR生物合成密切相关的类固醇结合位点,也具有较高的同源性,表明CPD基因在一年生的草本和多年生的木本植物之间具有很高的保守性。系统树分析还表明,CYP90蛋白与番茄和玉米的矮化基因产物、鱼的all trans retinoicacid4 hydroxy lase及微澡青菌(Synechocystissp. )的细胞色素P450在进化上有较密切的联系。