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Originally published In Press as doi:10.1074/jbc.M705366200 on September 28, 2007

J. Biol. Chem., Vol. 282, Issue 48, 34707-34718, November 30, 2007
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Identification of Dioxygenases Required for Aspergillus Development

STUDIES OF PRODUCTS, STEREOCHEMISTRY, AND THE REACTION MECHANISM*Formula

Ulrike Garscha{ddagger}, Fredrik Jernerén{ddagger}, DaWoon Chung§1, Nancy P. Keller§2, Mats Hamberg3, and Ernst H. Oliw{ddagger}4

From the {ddagger}Department of Pharmaceutical Bioscience, Uppsala Biomedical Center, SE-75124 Uppsala, Sweden, the §Department of Plant Pathology and the Department of Medical Microbiology and Immunology, University of Wisconsin, Madison, Wisconsin 53706, and the Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden

Received for publication, June 29, 2007 , and in revised form, September 18, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Aspergillus sp. contain ppoA, ppoB, and ppoC genes, which code for fatty acid oxygenases with homology to fungal linoleate 7,8-diol synthases (7,8-LDS) and cyclooxygenases. Our objective was to identify these enzymes, as ppo gene replacements show critical developmental aberrancies in sporulation and pathogenicity in the human pathogen Aspergillus fumigatus and the genetic model Aspergillus nidulans. The PpoAs of A. fumigatus and A. nidulans were identified as (8R)-dioxygenases with hydroperoxide isomerase activity, designated 5,8-LDS. 5,8-LDS transformed 18:2n-6 to (8R)-hydroperoxyoctadecadienoic acid ((8R)-HPODE) and (5S,8R)-dihydroxy-9Z,12Z-octadecadienoic acid ((5S,8R)-DiHODE). We also detected 8,11-LDS in A. fumigatus and (10R)-dioxygenases in both Aspergilli. The diol synthases oxidized [(8R)-2H]18:2n-6 to (8R)-HPODE with retention of the deuterium label, suggesting antarafacial hydrogen abstraction and insertion of molecular oxygen. Experiments with stereospecifically deuterated 18:2n-6 showed that (8R)-HPODE was isomerized by 5,8- and 8,11-LDS to (5S,8R)-DiHODE and to (8R,11S)-dihydroxy-9Z,12Z-octadecadienoic acid, respectively, by suprafacial hydrogen abstraction and oxygen insertion at C-5 and C-11. PpoCs were identified as (10R)-dioxygenases, which catalyzed abstraction of the pro-S hydrogen at C-8 of 18:2n-6, double bond migration, and antafacial insertion of molecular oxygen with formation of (10R)-hydroxy-8E,12Z-hydroperoxyoctadecadienoic acid ((10R)-HPODE). Deletion of ppoA led to prominent reduction of (8R)-H(P)ODE and complete loss of (5S,8R)-DiHODE biosynthesis, whereas biosynthesis of (10R)-HPODE was unaffected. Deletion of ppoC caused biosynthesis of traces of racemic 10-HODE but did not affect the biosynthesis of other oxylipins. We conclude that ppoA of Aspergillus sp. may code for 5,8-LDS with catalytic similarities to 7,8-LDS and ppoC for linoleate (10R)-dioxygenases. Identification of these oxygenases and their products will provide tools for analyzing the biological impact of oxylipin biosynthesis in Aspergilli.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The Aspergilli constitute a family of ascomycete fungi (1, 2). Several species are important human allergens, opportunistic pathogens, and producers of mycotoxins. Their spores are ubiquitous in the environment. Immunocompromised patients are particularly vulnerable to infections by Aspergillus fumigatus, causing farmer's lung disease and invasive aspergillosis (1, 2). Aspergilli are also plant pathogens and used as industrial microorganisms. Aspergillus nidulans is a model organism for studies of fungal biology (3). The genomes of nine Aspergillus sp. have now been fully or partly sequenced, which highlights their biological importance (2). One set of molecules known to be critical in Aspergillus developmental processes are a series of oxygenated fatty acids originally termed as psi5 factors.

Champe and co-workers (4, 5) showed in 1989 that A. nidulans oxidized 18:2n-6 and 18:1n-9 to psi factors, e.g. (8R)-HODE, (5S,8R)-DiHODE, and (8R)-HOME, which were identified as inducers of precocious sexual sporulation. Oxidation of polyunsaturated fatty acids to biologically active metabolites was well established in mammals and plants at that time, but this appears to be the first report of hormone-like activities of fungal oxylipins.

Oxidation of 18:2n-6 to (8R)-HODE and DiHODE was not restricted to A. nidulans. (8R)-HODE was originally discovered in Laetisaria arvalis (6, 7). (8R)-HODE is also produced by other fungi, e.g. Gaeumannomyces graminis (the take-all fungus of wheat), Magnaporthe grisea (the rice blast fungus), Leptomitus lacteus (the sewage fungus), and Agaricus bisporus (the field mushroom) (8–11). G. graminis and M. grisea also form (7S,8S)-DiHODE and the field mushroom (8R,11S)-DiHODE (8, 10, 11). The mechanism of biosynthesis of (8R)-HODE and (7S,8S)-DiHODE was determined in G. graminis (12–14). 18:2n-6 was oxidized to (8R)-HPODE by a heme-containing (8R)-DOX with hydroperoxide isomerase activity, 7,8-LDS. This enzyme abstracts the pro-S hydrogen at C-8 of 18:2n-6 and forms a carbon-centered radical, which reacts with O2 in an antaraficial way and forms (8R)-HPODE (15). A tyrosyl radical can be detected by EPR in this process (14). (8R)-HPODE is isomerized to (7S,8S)-DiHODE by suprafacial hydrogen abstraction and oxygenation at C-7 (16), catalyzed by a ferryl intermediate (PPIX Fe4+ = O) (14). Cloning and sequencing of 7,8-LDS revealed that this enzyme was a member of the MPO gene family, which also contains other fatty acid dioxygenases, notably cyclooxygenases and {alpha}-DOX (17–19).

The biological importance of psi factors in the sporulation process of A. nidulans was further extended by Keller and co-workers (20–23). The genomes of A. nidulans and A. fumigatus were published in 2005 (24, 25). Keller and co-workers found with the aid of the 7,8-LDS sequence that both genomes contained three genes (ppoA, ppoB, and ppoC), which coded for putative fatty acid oxygenases of the MPO family with about 40% amino acid identity with 7,8-LDS (22). The exon-intron borders and the amino acid sequences of the gene transcripts could be deduced from sequence homology to 7,8-LDS, including homology to the presumed distal and proximal heme ligands of 7,8-LDS and the critical Tyr residue for catalysis.6 The deduced sequence of PpoA of A. nidulans was confirmed by cDNA analysis (23). Keller and co-workers (20–23) reported that deletion of these genes affected the ratio of asexual spores (conidia) to sexual spores (ascopores), the biosynthesis of (8R)-HODE, and mycotoxin production in A. nidulans. In addition to the impact on the sporulation process, deletion of these genes also led to alterations in virulence on host seed (20). Deletion of ppoA reduced formation of (8R)-HODE and increased the ratio of conidia to ascospores, whereas forced expression of ppoA had the opposite effect (23). Most recently, deletion of ppoB also increased conidia formation, whereas deletion of ppoC decreased conidia formation (20, 21). These results were recently extended to A. fumigatus. An initial study demonstrated that down-regulation of all three A. fumigatus ppo genes by RNA interference technology produced a hypervirulent strain (27). Further work showed that deletion of ppoC yielded a pleiotrophic phenotype with formation of aberrant conidia and increased virulence in a mouse model of aspergillosis.7 The biological effects of ppo gene loss in A. nidulans and A. fumigatus are summarized in Table 1.


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  		TABLE 1
Biological effects of ppo gene loss in Aspergillus sp.

 
Studies of recombinant 7,8-LDS suggested that (5S,8R)-DiHODE could be formed by an enzyme of A. nidulans with a closely related oxygenation mechanism (26). 7,8-LDS expressed in insect cells had similar properties as the native enzyme. 7,8-LDS expressed in Pichia pastoris oxygenated 18:2n-6 to (8R)-HPODE and (5,8R)-DiHODE and transformed exogenous (8R)-HPODE to (5,8R)-DiHODE (26). Mycelia and cell-free preparations of A. nidulans were found to oxidize 18:2n-6 to (8R)-HPODE and (5S,8R)-DiHODE and transformed (8R)-HPODE to (5S,8R)-DiHODE. In addition, (10R)-HODE was formed as a major metabolite under certain conditions (26, 29).

Fungi have been known to produce 10-HODE with R or S absolute configuration. The shiitake mushroom, Lentinula edodes, and the field mushroom form (10S)-HPODE, which can be transformed to an aroma compound, 1-octen-3-ol, or reduced to (10S)-HODE (30, 31). (10R)-HODE is formed by Epichlöe typhina (32), and this stereoisomer also predominates in G. graminis and A. nidulans (26, 29, 33). The corresponding (10R)-hydroperoxide has not been identified, and little is known about the mechanism of biosynthesis of (10R)-HODE.

The first aim of the present study was to examine the biosynthesis of oxylipins from 18:2n-6 by the human pathogen A. fumigatus. We next extended these studies to A. nidulans, as this has traditionally been used as a model organism (4). We found that both species transformed 18:2n-6 to (8R)-HPODE/(8R)-HODE, (5S,8R)-DiHODE, and (10R)-HPODE/(10R)-HODE. In addition, (8R,11S)-DiHODE was formed by A. fumigatus. The second aim was to determine the mechanism of biosynthesis of the Aspergillus metabolites and their relation to (8R)-HPODE. The third goal was to determine the stereochemical relation between hydrogen abstraction and oxygenation using stereospecifically deuterated 18:2n-6 at C-5, C-8, and C-11. These studies were consistent with expression of two enzymes, 5,8-LDS and (10R)-DOX, in A. nidulans and A. fumigatus, and a third enzyme, 8,11-LDS, in A. fumigatus. Our final goal was to link each of the genes to each of these enzymes by gene targeting. We report that ppoA likely codes for 5,8-LDS and ppoC for (10R)-DOX. The corresponding genes appear to occur in virtually all Aspergillus sp. sequenced so far.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
18:1n-9 (99%), 18:2n-6 (99%), 18:3n-3 (99%), 18:3n-6 (99%), and imidazole were from Merck. 18:2n-6 (94–96%) was from Carl Roth (Karlsruhe, Germany). [9,10,12,13-2H4]18:2n-6 (99%), 16:3n-3 (99%), 17:3n-3 (99%), 19:3n-3 (99%), 20:2n-6 (99%), 10-KODE (99%), and 10-ODA (99%) were obtained from Larodan (Malmö, Sweden). 16:1n-7 (99%), malt extract, and TNM were from Sigma. [(11S)-2H]18:2n-6 (>95%) and [(11R)-2H]18:2 (25%) were prepared as described (16), whereas [(8R)-2H]18:2n-6 and [(5S)-2H]18:2n-6 were synthesized as described in the Supplemental Material. The strains of Aspergillus sp. are summarized in Table 2 (cf. Refs. 20–23). A. fumigatus Fres. will be referred to as A. fumigatus, and this strain was a kind gift of Dr. Levenfors (MASE Laboratories, Sveriges Lantbruksuniversitet, Uppsala, Sweden (34)). Spores of A. fumigatus were isolated from mycelia on potato dextrose agar, harvested in 0.5% Bactopeptone (5 x 107 spores/ml), and kept at 4 °C. Solvents were HPLC grade from Merck and J. T. Baker Inc. Cartridges with C18 silica and silica (SepPak/C18 and SepPak, respectively) were from Waters. BW4AC was a kind gift from Wellcome Research Laboratories (Beckenham, UK), and stock solutions (10 mM) were made in ethanol. Zileuton was from Abbott. Paracetamol (acetaminophen) was obtained locally. (8R,11S)-DiHODE was obtained as described (11, 29).


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  		TABLE 2
Strains of Aspergillus sp. used in this study

 
Fungal Preparations
Fungal Growth—A. fumigatus and A. nidulans were grown in liquid media (1.5% malt extract) from spores or mycelia on agar in a rotary shaker (150 rpm) at 37 or 22 °C (dark or in laboratory light) for 3–10 days. Mycelia were harvested by filtration, washed with saline, and either used directly or blotted dry and ground to a fine powder in liquid nitrogen. A. fumigatus and A. nidulans were also grown in 9-cm plastic Petri dishes either in the dark or 50 cm under a fluorescent lamp (30 watts, Tru-lite fluorescent, Duro-test, Fairfield, NJ, with or without light-dark cycles) for a few days at 22 or 37 °C. Colonies were picked by forceps, blotted dry, and incubated with 18:2n-6.

Nitrogen Powder of A. fumigatus and A. nidulans—A. nidulans was grown in liquid culture for 3 days at 37 °C (150 rpm), and A. fumigatus was grown for 24 h at 37 °C and then at room temperature (150 rpm) for 48 h. Mycelia (10–20 g) were harvested by filtration, washed with saline, and ground with liquid nitrogen to a fine powder, which was stored at –80 °C. The nitrogen powder was homogenized (glass-Teflon, 10 passes; 4 °C) in 10 volumes (w/v) of 0.1 mM KHPO4 buffer (pH 7.3), 2 mM EDTA, 0.04% Tween 20, centrifuged at 13, 000 x g (10 min, 4 °C), and used immediately for enzyme assay.

Enzyme Assays
Incubation with Mycelia—Mycelia (0.5–20 g) were incubated with 5 volumes (w/v) of 0.1 M NaBO3 buffer (pH 8.0 or 8.2) containing 18:2n-6 (0.5–1 mg/ml) for 5–6 h at 22 °C with shaking. The pH of the incubation buffer was typically above pH 7.3 at the end. The mycelia were separated from the incubation medium by filtration. Medium from large scale incubations was extracted with ethyl acetate and from small scale incubations on SepPak/C18. The ethyl acetate extract was dried (Na2SO4) and evaporated to dryness, and the products were purified by preparative TLC (ethyl acetate/hexane/acetic acid, 60:40:0.01) or by silicic acid chromatography. The latter was performed on a Sep/Pak cartridge or a short column with silica (Silicar CC-4, Mallinckrodt), which were eluted stepwise with increasing concentrations of diethyl ether (7, 25, and 50%) in hexane and finally with ethanol; 8- and 10-HPODE were eluted with 25% ether. The major metabolites were identified by LC-MS and by gas chromatography-MS analysis.

Incubation with Subcellular Fractions—An aliquot (0.5 ml) of nitrogen powder supernatant was incubated with 30–100 µM of 18:2n-6, 30–50 µM [2H]18:2n-6 or 15 µM (8R)-HPODE for 30–45 min on ice. The reaction was terminated with 0.5 ml of methanol, and the products were extracted on a cartridge of C18 silica (SepPak/C18, Waters) as described (29). In some experiments, 50 pmol of (13R)-[2H4]HODE was added as an internal standard, and TPP (10 µg) was added to reduce hydroperoxides to alcohols. The formation of (8R)-HODE and other oxylipins was quantified with help of the internal standard, (13R)-[2H4] HODE, as follows: 8-HODE, MS/MS analysis (m/z 295 -> full scan) with monitoring of m/z 157; (13R)-[2H4] HODE, MS/MS analysis (m/z 299 -> full scan) with monitoring of m/z 198; (5S,8R)-DiHODE, MS/MS analysis (m/z 173-> full scan); (8R,11S)-DiHODE, MS/MS analysis (m/z 213 -> full scan). Standard curves were prepared with 50 pmol of (13R)-[2H4]HODE and variable amounts of (8R)-HODE from a stock solution. The concentration of (8R)-HODE was determined by conversion to (8R,13R)-DiHODE by oxidation with manganese lipoxygenase (UV analysis) as described (29). Effects of drugs were assessed in duplicates with 100 µM 18:2n-6 as substrate.

LC-MS/MS Analysis
An ion trap mass spectrometer (LTQ, Thermo Fisher Scientific) was used with electrospray ionization and monitoring of negative ions. The pump for HPLC was from Thermo Fisher Scientific (Surveyor MS). The HPLC columns contained octadecyl silica (5-µm, 150 x 2 mm) and were usually eluted with methanol/water/acetic acid, 75:25:0.01 or 80:20:0.01, at 0.2–0.3 ml/min. The capillary temperature was 325 °C, and prostaglandin F1{alpha} was used for tuning. For analysis of deuterium labeling of products formed from [2H]18:2n-6, we used MS/MS analysis of m/z 295 and 311 (isolation width 6 atomic mass units) and studied with the zoom scan rate function over 10 atomic mass units centered at the daughter ions at m/z 157 (8-HODE), m/z 183 (10-HODE), m/z 173 (5,8-DiHODE), and at m/z 213 (8,11-DiHODE).

NP-HPLC-MS/MS was performed on silica with an analytical column (Kromasil-100SI; 250 x 2 mm, 5 µm, 100 Å), which was eluted with hexane/isopropyl alcohol/acetic acid, 95:5:0.05 or 93:7:0.95, using a Constametric 3200 pump (LDC). The effluent (0.3–0.7 ml/min) passed a photodiode array detector (5-cm path length, Surveyor PDA, Thermo Fisher Scientific). The effluent was then combined in a T junction with isopropyl alcohol/water (3:2; 0.3–0.5 ml/min) from a Surveyor MS pump and subjected to electrospray ionization in an ion trap mass spectrometer (LTQ, Thermo Fisher Scientific). The heated transfer capillary was set at 325 °C, the ion isolation width at 1.5 atomic mass units, and the collision energy at 25 (arbitrary scale). Prostaglandin F1{alpha} (100 ng per min) was infused for tuning. CP-HPLC analysis of 8-HODE and 10-HODE was performed as described (29). The effluent from the chiral columns was analyzed by MS/MS as described above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Oxylipin Biosynthesis by A. fumigatus
Mycelia of A. fumigatus, which had been growing in liquid culture at 37 °C in the dark, were incubated with 18:2n-6 at room temperature, and the products were analyzed by RP-HPLC-MS/MS. The profiles of oxylipins formed are shown in Fig. 1A. The products were identified by LC-MS/MS analysis, and the stereoisomers were analyzed by CP-HPLC-MS/MS and NP-HPLC-MS (cf. Refs. 29 and 35) and as discussed below. The two major dihydroxy fatty acids were (8R,11S)-DiHODE and (5S,8R)-DiHODE, and they eluted in this order. (8R,13)-DiHODEs were also noticed in some experiments. The latter eluted before 8,11-DiHODE on RP-HPLC and was likely formed from (8R,11S)-DiHODE by during the isolation procedure (29). MS/MS analysis showed that the main hydroxy fatty acid was (8R)-HODE, but ~10% (10R)-HODE was also detected (29), as judged from the reconstructed ion chromatograms of m/z 183 (10-HODE; –OOC-(CH2)6-CH=CH-CHO) and m/z 157 (8-HODE; –OOC-(CH2)6-CHO) during MS/MS analysis.

We next examined whether growth conditions (fluorescent light/darkness, growth temperature, growth length) changed the oxylipin profile. The results are summarized in Fig. 1B. Temperature (22 versus 37 °C) had the greatest effect and increased the relative formation of (10R)-HODE in comparison with (8R)-HODE, whereas fluorescent light yielded inconsistent results (Fig. 1B). At 22 °C, (10R)-HODE was formed as a major metabolite (Fig. 1C); this difference between 22 and 37 °C remained after incubation of mycelia for 10 days with a relative increase in (10R)-HODE formation (cf. Fig. 1B). Steric analysis by CP-HPLC-MS/MS showed that 10-HODE, isolated from mycelia grown at 22 °C consisted of ~98% of the R stereoisomer (Fig. 1D; cf. Ref. 29). A. fumigatus formed (8R)-HODE with ~95% stereoselectivity (29).

The relative amounts of (8R,11S)- and (5S,8R)-DiHODE also appeared to be changed by temperature. The former was the main product at 22 °C and the latter at 37 °C (cf. Fig. 1, A and C).

NP-HPLC-MS/MS analysis of the product profiles at 22 °C revealed that 5,8-DiHODE formed by A. fumigatus had the same retention time as (5S,8R)-DiHODE of A. nidulans, whereas the 5S,8S and 5R,8R stereoisomers of 5,8-DiHODE elute 4–5 min later (26, 29). The 8S,11S and 8R,11R stereoisomers of 8,11-DiHODE eluted after ~8 min, whereas the 8S,11R and 8R,11S stereoisomers eluted after ~14 min. The 8,11-DiHODE formed by A. fumigatus eluted after ~14 min. As (8R)-HPODE is a precursor of both (5S,8R)-DiHODE and (8R,11S)-DiHODE (see below), we conclude that (5S,8R)- and (8R,11S)-DiHODE were formed by A. fumigatus (cf. Ref. 29).

Isolation and Identification of (8R)-HPODE and (10R)-HPODE
(8R)-HPODE was obtained in milligram amounts in some experiments by incubation of mycelia of A. fumigatus with 18:2n-6, as illustrated by LC-MS/MS analysis in Fig. 2A. The MS/MS/MS spectrum (m/z 311-> m/z 293 -> full scan) of 8-HPODE was as reported (35). (8R)-HPODE was then purified by silicic acid chromatography. The purity was assessed after reduction with TPP.


Figure 1
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  		FIGURE 1.
Identification by LC-MS/MS analysis of major oxylipins formed from 18:2 n-6 by mycelia of A. fumigatus. A, reconstructed ion chromatograms of oxylipins formed by A. fumigatus grown in liquid culture at 37 °C (dark). The mycelia were harvested and incubated with 18:2n-6 for 6 h at 22 °C. Top trace, total ion current (TIC); middle trace, MS/MS analysis (m/z 295 -> full scan) of HODE; bottom trace, MS/MS analysis (m/z 311 -> full scan) of DiHODE. The RP-HPLC column was eluted with methanol/water/acetic acid, 750:250:0.1. The major oxylipins are marked except 8,11-DiHODE, which eluted after 4.5 min. B, relative formation of (8R)-HODE and (10R)-HODE by mycelia in liquid culture, which were grown for 3 days at 37 °C (in dark or fluorescent light) and for 3 and 10 days at 22 °C (in dark or light). The traces show the reconstructed ion chromatograms (MS/MS analysis; m/z 295 -> full scan) of m/z 157 (8-HODE) and m/z 183 (10-HODE), normalized to the largest of the two peaks. The RP-HPLC column was eluted with methanol/water/acetic acid, 800:200:0.1. The area under the two peaks were measured by integration (XCalibur software) and used as relative measures of biosynthesis of 10- and 8-HODE. MA%, measured area of m/z 183 in percent of total area under m/z 157 and m/z 183. D, dark; L, light. C, reconstructed ion chromatograms of oxylipins formed by A. fumigatus grown in liquid media at 22 °C. Top trace, total ion current (TIC); middle trace, MS/MS analysis (m/z 295 -> full scan) of HODE; bottom trace, MS/MS analysis (m/z 311 -> full scan) of DiHODE. D, CP-HPLC analysis of 10-HODE produced by mycelia grown at 22 °C. Integration of the signal at m/z 183 suggested that 98% of R stereoisomer of 10-HODE was formed. The Reprosil Chiral-NR column was eluted with 3% isopropyl alcohol in hexane (0.25 ml/min).

 
Hydroperoxy fatty acids will be partly dehydrated to the corresponding keto fatty acids in the heated transfer line of the mass spectrometer (35, 36). We found that cell-free preparations (nitrogen powder) of mycelia of A. fumigatus transformed 18:2n-6 to 10-ODA, 10-KODE, and to 10-HPODE, as shown in Fig. 2B. 10-HPODE was identified by the signal at m/z 311 in full scan, which was associated with signals at m/z 183 (carboxylate anion of 10-ODA) and m/z 293 (carboxylate anion of 10-KODE). Incubation with [9,10,12,13-2H4]18:2n-6 yielded consistent MS/MS results. This suggested that (10R)-HPODE was transformed to 10-KODE and to 10-ODA in the heated transfer system of the mass spectrometer. In addition, (10R)-HPODE will also converted to 10-ODA and to 10-KODE during the incubation, whereas (8R)-HPODE was partly transformed to 8-KODE (cf. Fig. 2B). These transformations are catalyzed by metals and metalloproteins (37).

We compared the transformation of (10R)-HPODE to 10-ODA by subcellular fractions of A. fumigatus with a heat-inactivated control without detecting significant hydroperoxide lyase activity. The MS/MS spectra of 10-KODE and 10-ODA are shown in Fig. 3, A and B. The MS/MS spectrum of 10-KODE (m/z 293 -> full scan) showed signals, inter alia, at m/z 275 (A–-18), m/z 249 (A–-44), m/z 233, and in the lower mass range at m/z 199, m/z 155, m/z 153, m/z 139, and m/z 137. Authentic 10-KODE yielded an identical MS/MS spectrum. The corresponding MS/MS spectrum of 10-[9,12,13-2H3]KODE, obtained by incubation with [9,10,12,13-2H4]18: 2n-6, showed signals, inter alia, at m/z 277 and m/z 278, m/z 252, m/z 201, and in the lower mass range at m/z 155 and 156, and at m/z 138 and 139.

The MS/MS spectrum of 10-ODA (m/z 183 -> full scan) showed signals at m/z 165 (A–-18), m/z 155 (A–-28, unidentified, possibly loss of CO), and m/z 139 (A–-44), and the spectrum of authentic 10-ODA was identical. The corresponding MS/MS spectrum of 10-ODA, derived from an incubation with [9,10,12,13-2H4]18:2n-6, showed major signals at m/z 167 and 166, m/z 157 and 156, m/z 140 and 141. This spectrum suggested that 10-[9,10-2H2,]ODA was formed.


Figure 2
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  		FIGURE 2.
Biosynthesis of (8R)-HPODE and (10R)-HPODE by A. fumigatus. A, LC-MS/MS analysis (m/z 311 -> full scan) of metabolites formed from 18:2n-6 by mycelia in 0.1 M NaBO3 buffer. The amount of (8R)-HPODE varied from trace amounts to undetectable (cf. Fig. 1, A and B), but in some experiments (8R)-HPODE was obtained as a major metabolite. The MS/MS/MS spectrum (m/z 311 -> m/z 293 -> full scan) of (8R)-HPODE was as reported (35). B, demonstration of biosynthesis of (10R)-HPODE, 10-ODA, and 10-KODE from 18:2n-6 by nitrogen powder preparation of mycelia of A. fumigatus. The products were analyzed for 10-ODA (m/z183->fullscan; toptrace) and for the carboxylate anions of (8R)- and (10R)-HPODE (m/z 311), as shown in the middle reconstructed ion chromatograms. Hydroperoxides decompose in the heated transfer system of the mass spectrometer to keto fatty acids. MS/MS analysis of KODE (m/z 293 -> full scan) showed that the major peaks of 8-KODE and 10-KODE co-eluted with (8R)-HPODE and (10R)-HPODE, respectively. Pre-formed 10-KODE and 8-KODE eluted before and after the two hydroperoxides.

 
Mechanism of Biosynthesis of DiHODE and HPODE
Cell-free preparations of A. fumigatus transformed (8R)-HPODE to (5S,8R)-DiHODE and (8R,11S)-DiHODE, but (8R)-HPODE did not increase the biosynthesis of (10R)-HODE (Fig. 4). We conclude that (8R)-HPODE is transformed into the two diols by hydroperoxide isomerases, whereas (10R)-HODE is apparently formed directly from 18:2n-6.

The reaction mechanism of the (8R)- and (10R)-DOX and the hydroperoxide isomerases were studied with stereospecifically deuterated 18:2n-6 as substrates for oxygenases/hydroperoxide isomerases of cell-free preparations of A. fumigatus. The results are summarized in Table 3.


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  		TABLE 3
Isotopic composition of stereospecifically deuterated linoleic acids and their oxygenated products formed by A. fumigatus

 


Figure 3
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  		FIGURE 3.
MS/MS spectra of 10-KODE and 10-ODA. A, MS/MS analysis of 10-KODE (m/z 293 -> full scan). Formation of some fragments is suggested by the inset of the carboxylate anion of 10-KODE. B, MS/MS analysis of 10-ODA (m/z 183 -> full scan). The inset shows the carboxylate anion of 10-ODA. C, MS/MS analysis of 10-HODE isolated from an incubation with [(8R)-2H]18: 2n-6 with low collision-induced fragmentation. The signals at m/z 184.2 are mainly due to the monodeuterated fragment –OOC-(CH2)6-C2H=CH-CHO, and at m/z 296.3 to the monodeuterated carboxylate anion.

 
A. fumigatus metabolized [(8R)-2H]18:2n-6 to (8R)-HODE with retention of the pro-R hydrogen at C-8, and this deuterium atom was also retained in (5S,8R)-DiHODE, (8R,11S)-DiHODE, and (10R)-HODE(Fig.3C). Transformation of [(11S)-2H]18:2n-6 to (8R,11S)-DiHODE occurred with loss of the label, whereas [(11R)-2H]18: 2n-6 was oxidized to (8R,11S)-DiHODE with retention of the deuterium atom. [(5S)-2H]18:2n-6 was oxidized to (5S,8R)-DiHODE with retention of the deuterium label; as a consequence of the nomenclature rules, the pro-R hydrogen at C-5 of (8R)-HPODE was retained in the biosynthesis of (5S,8R)-DiHODE.

These results suggest that biosynthesis of (8R)-HPODE occurs by abstraction of the pro-S hydrogen at C-8 and antarafacial insertion of O2, whereas biosynthesis of (10R)-HPODE occurs by abstraction of the hydrogen at C-8, double bond migration to C-8 and C-9, and antarafacial dioxygenation at C-10 of the planar structure of C-8 to C-10. The hydroperoxidase activities, leading to hydroxylation reactions at C-5 and C-11, apparently occur by abstraction of the pro-S hydrogens at C-5 and C-11 of (8R)-HPODE and suprafacial insertion of oxygen.

Effects of Inhibitors on Oxylipin Biosynthesis
TNM causes nitration of tyrosine residues and inhibits 7,8-LDS, possibly by blocking formation of the tyrosyl radical (14), whereas BW4AC is a redox inhibitor of lipoxygenases and 7,8-LDS (13, 38). The effects of these drugs on biosynthesis of 10-HODE and 8-HODE are shown in Fig. 5. TNM (100 and 30 µM) reduced biosynthesis of (8R)-HODE and (10R)-HODE from 100 µM 18:2n-6 by 89 and 69% and by 81 and 43%, respectively. BW4AC (30 and 100 µM) had little effect on (8R)-HODE biosynthesis, but BW4AC (100 µM) reduced biosynthesis of (10R)-HODE by 58%. Zileuton, which is a 5-lipoxygenase inhibitor, paracetamol (100 µM), and sodium salicylate (1 mM) did not change the oygenation of 18:2n-6 by cell-free preparations of A. fumigatus (data not shown).

Oxygenation of C16–C20 Fatty Acids—The transformation of unsaturated C16–C20 fatty acids by the fatty acid oxygenases of A. fumigatus is summarized in Table 4. (10R)-DOX apparently oxidized 16:1n-8, 18:1n-1(cis), 18:2n-6, and 18:3n-3. Several fatty acids were oxidized at their C-8 carbon and the corresponding hydroperoxide was apparently transformed to 5,8- and 8,11-diols. This seemed to require a saturated carbon chain from the carboxyl group to the first double bond of 7 or 9 carbons.


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  		TABLE 4
Oxygenation of unsaturated fatty acids by dioxygenases and hydroperoxide isomerases of A. fumigatus

The products were identified by MS/MS analysis of the carboxylate anions (A- -> full scan) and by detection of characteristic fragments.

 


Figure 4
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  		FIGURE 4.
Transformation of (8R)-HPODE by A. fumigatus. The relative abundance of (8R,11S)-DiHODE, (5S,8R)-DiHODE, and (10R)-HODE was estimated with the aid of the internal standard in incubations with (open box) or without (8R)-HPODE (black box). Mean ± S.D. of three experiments.

 
The LC-MS/MS analysis showed that absence of double bonds in the n-6 position changed the fragmentation pattern. MS/MS analysis of 10-HODE showed a characteristic fragment at m/z 183 (–OOC (CH2)6-CH=CH-CHO), whereas 10-HOME showed a characteristic intense signal at m/z 155 (–OOC (CH2)6-CH=CH2).

Effects of Deletion of ppoA and ppoC on Oxylipin Biosynthesis by A. fumigatus AF293
A. fumigatus AF293 formed (8R)-H(P)ODE, (5S,8R)-DiHODE, and (10R)-H(P)ODE as major metabolites. We could not detect biosynthesis of 8,11-DiHODE with certainty, but we cannot exclude that traces of this metabolite could be formed. Deletion of ppoA resulted in complete loss of biosynthesis of (5S,8R)-DiHODE, as judged from RP-HPLC analysis, and deletion of ppoC led to diminished biosynthesis of (10R)-HODE (Table 5). Deletion of ppoB had little effect on oxylipin formation and transformed 18:2n-6 in the same way as A. fumigatus AF293.


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  		TABLE 5
Relative abundance of characteristic ions during LC-MS/MS analysis of 5S, 8R-DiHODE, 8R-HODE, and 10R-HODE formed by A. fumigatus AF293 and its two mutants {Delta}ppoA and {Delta}ppoC

 
Oxygenation of Fatty Acids by A. nidulans
In agreement with the original report by Champe and co-workers (4, 5), mycelia of A. nidulans grown at 37 °C transformed 18:2n-6 to (8R)-HODE and (5S,8R)-DiHODE as major products. In addition, we also noticed biosynthesis of (8R)-HPODE and (10R)-HODE (29). Biosynthesis of (10R)-HPODE was confirmed in cell-free preparations of A. nidulans, as described above for A. fumigatus, by identification of 10-ODA, 10-KODE, and the products formed from (10R)-HPODE by decomposition in the mass spectrometer (10-ODA and 10-KODE).


Figure 5
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  		FIGURE 5.
Effects of TNM and BW4AC on the (8R)- and (10R)-DOX activities of A. fumigatus. The relative abundance of the integrated signal intensities at m/z 183 (10-HODE, black bars) and m/z 157 (8-HODE, white bars) were set to 100% in control incubations of nitrogen powder of A. fumigatus, and the drug effects are given in percent of these values (mean ± S.D.).

 
[(8R)-2H]18:2n-6 was oxidized by nitrogen powder of A. nidulans to (8R)-HODE, (10R)-HODE, and (5S,8R)-DiHODE with retention of the deuterium label. We also confirmed that [(5S)-2H]18:2n-6 was oxidized to (5S,8R)-DiHODE with retention of the deuterium label in analogy with oxidation by 5,8-LDS of A. fumigatus.

Effects of Deletion of ppoA and ppoC of A. nidulans
The transformations of 18:2n-6 by the wild type and two mutants are summarized in Table 6.


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  		TABLE 6
Relative abundance of characteristic ions of 5,8-DiHODE, 8-HODE, and 10-HODE formed by A. nidulans and its two mutants {Delta}ppoA and {Delta}ppoC

 
Wild Type and {Delta}ppoB—These two strain appeared to oxidize 18:2n-6 to the same spectrum of metabolites, e.g. (8R)-H(P)ODE, (10R)-H(P)ODE, and (5S,8R)-DiHODE, although the relative amounts differed. NP-HPLC of the hydroxy fatty acids formed by reduction with TPP did not reveal any qualitative difference in formation of hydroxy fatty acids between the wild type and {Delta}ppoB. Biosynthesis of (8R,11S)-DiHODE could not be detected.

{Delta}ppoA—This strain had completely lost the capacity to form (5S,8R)-DiHODE, and the biosynthesis of (8R)-HODE was strongly reduced (Table 6). The main metabolite of 18:2n-6 was (10R)-HODE. The (10R)-DOX activity appeared to increase when the mycelia were grown at room temperature compared with 37 °C (Table 6). Steric analysis showed that 8-HODE contained some excess of the 8R stereoisomer (65%), suggesting enzymatic biosynthesis, possibly related to (10R)-DOX activity. Incubation of nitrogen powder of {Delta}ppoA with 18:2n-6 led to identification of 10-ODA, 10-KODE, and 10-HPODE (Fig. 6), and products formed from [9,10,12,13-2H4]18:2n-6 yielded consistent results. Steric analysis showed that 10-HODE consisted mainly of the 10R stereoisomer (Fig. 7A). We conclude that ppoA may code for 5,8-LDS.

{Delta}ppoC—This strain had reduced the capacity to form (10R)-HODE (Table 6), and steric analysis of the small amounts of 10-HODE formed by this mutant showed that it was racemic (Fig. 7B). We conclude that ppoC codes for linoleate (10R)-DOX.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have studied three previously uncharacterized fatty acid oxygenases of the human pathogen A. fumigatus and extended the results to the model organism, A. nidulans. (10R)-DOX and 5,8-LDS were found in both species and 8,11-LDS in A. fumigatus. We have studied their reaction mechanisms by aid of stereospecifically deuterated 18:2n-6 and conclusively identified the genes of 5,8-LDS and (10R)-DOX by gene targeting of ppoA and ppoC. The genes are homologous and belong to the MPO family along with 7,8-LDS, cyclooxygenases, and {alpha}-DOX. Our results suggest that diol synthases may have a common reaction mechanism.


Figure 6
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  		FIGURE 6.
Oxygenation of 18:2n-6 by nitrogen powder of the A. nidulans mutant {Delta}ppoA. The products were analyzed for 10-ODA (m/z 183 -> full scan; top trace) and for the carboxylate anions of (8R)- and (10R)-HPODE (m/z 311), as shown in the middle reconstructed ion chromatograms, and (10R)-HPODE was detected only. Hydroperoxides decompose in the heated transfer system of the mass spectrometer to keto fatty acids. MS/MS analysis of KODE (m/z 293 -> full scan; bottom ion chromatogram) showed that the major peak of 10-KODE co-eluted with (10R)-HPODE, respectively. Preformed 10-KODE and 10-ODA eluted before (10R)-HPODE on RP-HPLC.

 


Figure 7
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  		FIGURE 7.
CP-HPLC analysis of 10-HODE formed by the A. nidulans mutants {Delta}ppoA and {Delta}ppoC. A, steric analysis of 10-HODE formed by {Delta}ppoA. B, steric analysis of 10-HODE formed by {Delta}ppoC.

 
The three Aspergillus oxygenases catalyzed abstraction of the pro-S hydrogen at C-8 of 18:2n-6. Antarafacial insertion of O2 occurred either at C-8 with formation of (8R)-HPODE by the two diol synthases or, after double bond migration, at C-10 with formation of (10R)-HPODE by (10R)-DOX (Fig. 8). This steric course is in agreement with the antarafacial relationship between hydrogen abstraction and oxygen insertion found for most lipoxygenases and for cyclooxygenases-1 and -2 (39). A few dioxygenase reactions deviate from this rule. Manganese lipoxygenase catalyzes suprafacial formation of linoleic acid 11- and 13-hydroperoxides (40), and the major {alpha}-DOX of tobacco and Arabidopsis produces {alpha}-hydroperoxides with retention of absolute configuration at C-2 (19). Furthermore, (10S)-DOX from the field mushroom (A. bisporus) catalyzes formation of (10S)-HPODE in a reaction that involves suprafacial stereochemistry.8 In the next step (Fig. 8), (8R)-HPODE was transformed to (5S,8R)-DiHODE or to (8R,11S)-DiHODE. Studies with stereospecifically deuterated 18:2n-6 at positions 5 and 11 suggested that these diols were formed by suprafacial hydrogen abstraction and oxygenation; cytochromes P450s catalyze aliphatic hydroxylation by the same mechanism (39). The results are also summarized in Table 3. The oxygenation mechanism of 5,8- and 8,11-LDS is similar to 7,8-LDS of G. graminis (14).

(10R)-HPODE partly decomposed to 10-ODA and 10-KODE during incubation (37), and it was also reduced to (10R)-HODE (Figs. 3B and 6). We could not detect transformation to diols. 10-ODA and 10-KODE were also formed during the LC-MS/MS analysis of (10R)-HPODE, presumably in the heated capillary. The transformation of (10R)-HPODE to 10-ODA appeared to be mainly nonenzymatic, as it was noted to a similar extent in heat-inactivated controls.

The genes of 5,8-LDS and (10R)-DOX were identified by gene targeting. Deletion of ppoA of both Aspergilli resulted in complete loss of biosynthesis of (5S,8R)-DiHODE and to biosynthesis of only small amounts of (8R)-H(P)ODE, whereas (10R)-HPODE now was formed as the major metabolite. Steric analysis showed that 8-HODE from these ppoA mutants contained moderate excess of the R stereoisomer. Deletion of ppoC resulted in almost complete loss of biosynthesis of (10R)-H(P)ODE, and the small amounts of 10-HODE formed was a racemic mixture of R and S stereoisomers. The changes in product formation in the ppoA and ppoC mutants (Tables 4 and 5) may not only be due to loss of the particular gene in question but also to a release of feedback inhibition observed by previous transcript analysis of ppo expression in ppo mutants (8, 10). We conclude that ppoA codes for 5,8-LDS and ppoC for (10R)-DOX in both species. Table 7 summarizes these findings. The two 5,8-LDS enzymes can be aligned with 78% and the two (10R)-DOX enzymes with 66% amino acid identity. Whether 8-HODE can be formed by (10R)-DOX as a minor product or by other fungal enzymes awaits further studies.


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  		TABLE 7
Overview of genes of A. nidulans and A. fumigatus with homology to 7,8-LDS

 
Interestingly, the biosynthesis of (10R)-HPODE was augmented in both species grown at 22 °C compared with 37 °C. Deletion of ppoC in A. fumigatus augments the survival of this mutant in a mouse model of invasive aspergillosis.7 Whether biosynthesis of (10R)-HPODE will affect the virulence of A. fumigatus may merit further investigation, as little is known about its virulence factors. In contrast, loss of ppoC had no effect on virulence in the A. nidulans/seed interaction. However, a double mutant deleted in both ppoC and ppoA showed a decrease in virulence on peanut seed (20). These results suggest possible different roles of oxylipins dependent on the host/fungal pathosystem.


Figure 8
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  		FIGURE 8.
Overview over the reaction mechanism of 5,8-LDS, 8,11-LDS, and (10R)-DOX. In the first step marked (i), the pro-S hydrogen at C-8 of 18:2n-6 is abstracted with formation of a carbon-centered radical by all three enzymes, 5,8-LDS, 8,11-LDS, and (10R)-DOX. Oxygen is then inserted at C-8 in an antarafacial way with formation of (8R)-HPODE by the (8R)-DOX activities of 8,11-LDS and 5,8-LDS. (8R)-HPODE is isomerized to (8R,11S)-DiHODE by 8,11-LDS, to (5S,8R)-DiHODE by 5,8-LDS, or reduced to (8R)-HODE, as indicated in the top right column. Alternatively, the 9–10 double bond migrates to position 8–9, and oxygen is inserted in the carbon-centered radical at C-10 in an antarafacial way in relation to the C-8 to C-10 structural element with formation of (10R)-HPODE by (10R)-DOX. (10R)-HPODE can be reduced to (10R)-HODE and decomposed to 10-KODE and 10-ODA by metal complexes (37), as indicated in the bottom right column.

 
Both A. nidulans and A. fumigatus contain a third gene (ppoB, see Table 2 and Table 7) that might code for fatty acid oxygenases. Alignment suggests that these genes may code for proteins with less than 40% amino acid identity and may not be closely related. Loss of ppoB had no discernible effect on virulence in A. fumigatus7 but had in A. nidulans a large effect on increasing virulence on seed.7 Disruption of ppoB did not influence oxylipin biosynthesis in this study. Recent mRNA analysis shows A. nidulans ppoB to be a pathogenesis-induced gene,9 which may explain why changes in oxylipin profile were not observed in this strain in the present study. A. fumigatus AF293 may only produce traces of (8R,11S)-DiHODE, whereas A. fumigatus Fres. formed this metabolite as one of the major products. In analogy, Aspergillus clavatus contains three genes with homology to the three oxygenase genes of A. fumigatus, and this fungus also expresses 5,8-LDS, 8,11-LDS, and (10R)-DOX activity.10 It is possible that the PpoB of A. fumigatus forms 8,11-LDS, a topic of further studies.

The MPO family contains fatty acid dioxygenases with hydrogen abstraction by a tyrosyl radical as a common feature. The homology of oxygenases of A. nidulans and A. fumigatus include the distal and proximal heme His ligands and the catalytically important Tyr residue of cyclooxygenases. In agreement with this oxygenation mechanism, TNM (30–100 µM) inhibited the (8R)- and (10R)-DOX activities of A. fumigatus, possibly by interfering with the oxygenation mechanism by nitration of Tyr residues.

What are the structural differences between (8R)-DOX and (10R)-DOX? As discussed above, a Tyr radical formed by both groups of enzymes likely abstracts the pro-S hydrogen at C-8 of 18:2n-6, but oxygen then reacts either at C-8 or at C-10 of 18:2n-6. It is known from lipoxygenase biochemistry that mutation of a single amino acid can change the position of oxygenation (26). It is therefore of interest to determine the conserved differences in the primary sequences of 5,8-LDS and (10R)-DOX and to compare them with homologous positions in cyclooxyenases. Replacement of Ser-530 of cyclooxygenase-1 with threonine or acetylation of the corresponding Ser residue of cyclooxygenase-2 with aspirin shifted the position of oxygenation of 20:4n-6 from C-11 to C-15 (28). Cyclooxygenase-1 has conserved Tyr-348 and Val-349 residues, which are important for substrate positioning and for the cyclooxygenase reaction (28). Replacement of Val-349 with a larger (leucine) or smaller residue (alanine) increased the oxygenation at C-15 and C-11, respectively (28). There are putative oxygenases with close similarity to PpoA (5,8-LDS) and to PpoC ((10R)-DOX) in several species of Aspergilli. Sequence alignments of the 5,8-LDS and the (10R)-DOX groups reveal several conserved differences. The corresponding Tyr-Val residues are conserved in all known or deduced 7,8-LDS and all 5,8-LDS sequences, whereas (10R)-DOX and their putative analogues have Tyr-Leu in this position. Replacement of Val-330 in 7,8-LDS with a smaller residue, alanine, abolished activity (26, 28). It will be of interest to study the effect of replacement with a larger residue, e.g. V330L.

In summary, we have identified novel oxygenases and oxylipins in two Aspergilli sp. Our results indicate that diol synthases and (10R)-DOX have fundamental catalytic and structural properties in common. The genes of these diol synthases and (10R)-DOX of A. nidulans and A. fumigatus have been deleted, and the resulting phenotypes can now be interpreted in the light of our report.


   FOOTNOTES
 
* This work was supported in part by Vetenskapsrådet Grant 03X-06523, Formas Grant 222-2005-1733, and The Lars Hierta Memorial Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental text and additional references. Back

1 Present address: Dept. of Plant Pathology and Microbiology, 2132 Texas A & M University, College Station, TX 77843. Back

2 Recipient of National Science Foundation Grant MCB-0236393 and the National Research Initiative Competitive Grant 2005-35201-15350 from the United States Department of Agriculture Cooperative State Research, Education, and Extension Service. Back

3 Supported by the Swedish Research Council for Environment, Agricultural Sciences, and Spatial Planning Project 229-2004-833. Back

4 To whom correspondence should be addressed: Division of Biochemical Pharmacology, Dept. of Pharmaceutical Biosciences, Uppsala University, P.O. Box 591, SE-75124 Uppsala, Sweden. Tel.: 46-18-4714455; Fax: 46-18-552936; E-mail: Ernst.Oliw{at}farmbio.uu.se.

5 The abbreviations used are: psi, precocious sexual inducer; CP, chiral phase; DiHODE, dihydroxyoctadecadienoic acid; DOX, dioxygenase; HODE, hydroxyoctadecadienoic acid; HOME, hydroxyoctadecenoic acid; HPODE, hydroperoxyoctadecadienoic acid; KODE, ketooctadecadienoic acid; LC-MS/MS, liquid chromatography-tandem mass spectrometry; LDS, linoleate diol synthase; NP, normal phase; MPO, myeloperoxidase; ODA, 10-oxy-8E-decenoic acid; Ppo, psi producing oxygenase; ppo, gene coding for Ppo; RP-HPLC, reversed phase-high pressure liquid chromatography; TPP, triphenylphosphine; TNM, tetranitromethane. Back

6 U. Garscha and E. H. Oliw, submitted for publication. Back

7 D. Chung, unpublished data. Back

8 M. Hamberg and E. Oliw, unpublished observations. Back

9 D. Tsitsigiannis and N. P. Keller, submitted for publication. Back

10 F. Jernerén, U. Garscha, M. Hamberg, and E. Oliw, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Drs. V. Laszlo and G. Csaba (Semmelweis University, Hungary) for the generous gift of Tetrahymena pyriformis.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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