The "pro-drug" RibCys decreases the mutagenicity of high-LET radiation in cultured mammalian cells

We are carrying out studies aimed at reducing the mutagenic effects of high-LET 56Fe ions and 12C ions (56Fe ions, 143 keV/microm; 12C ions, 100 keV/microm) with certain drugs, including RibCys [2-(R,S)-D-ribo-(1',2',3',4'-tetrahydroxybutyl)-thiazolidine-4(R)-carboxylic acid]. RibCys, formed by condensation of L-cysteine with D-ribose, is designed so that the sulfhydryl amino acid L-cysteine is released intracellularly through nonenzymatic ring opening and hydrolysis leading to increased levels of glutathione (GSH). RibCys (4 or 10 mM), which was present during irradiation and for a few hours after, significantly decreased the yield of CD59- mutants induced by radiation in AL human-hamster hybrid cells. RibCys did not affect the clonogenic survival of irradiated cells, nor was it mutagenic itself. These results, together with the minimal side effects reported in mice and pigs, indicate that RibCys may be useful, perhaps even when used prophylactically, in reducing the mutation load created by high-LET radiation in astronauts or other exposed individuals.     

Formation of glycine conjugate and (-)-(R)-enantiomer from (+)-(S)-2-phenylpropionic acid suggesting the formation of the CoA thioester intermediate of (+)-(S)-enantiomer in dogs.

It has been proposed that the chiral inversion of the 2-arylpropionic acids is due to the stereospecific formation of the (-)-R-profenyl-CoA thioesters which are putative intermediates in the inversion. Accordingly, amino acid conjugation, for which the CoA thioesters are obligate intermediates, should be restricted to those optical forms which give rise to the (-)-R-profenyl-CoA, i.e., the racemates and the (-)-(R)-isomers. We have examined this problem in dogs with respect to 2-phenylpropionic acid(2-PPA). Regardless of the optical configuration of 2-phenylpropionic acid administered, the glycine conjugate was the major urinary metabolite and this was shown to be exclusively the (+)-(S)-enantiomer by chiral HPLC. Both (-)-(R)- and (+)-(S)-2-phenylpropionic acid were present in plasma after the administration of either antipode, and further evidence of the chiral inversion of both enantiomers was provided by the presence of some 25% of the opposite enantiomer in the free 2-phenylpropionic acid and its glucuronide excreted in urine after administration of (-)-(R)- and (+)-(S)-2-phenylpropionic acid. The (+)-(S)-enantiomer underwent chiral inversion to the (-)-(R)-antipode when incubated with dog hepatocytes. These data suggests that both enantiomers of 2-phenylpropionic acid are substrates for canine hepatic acyl CoA ligase(s) and thus undergo chiral inversion, but that the CoA thioester of only (+)-(S)-2-phenylpropionic acid is a substrate for the glycine N-acyl transferase. These studies are presently being extended to the structure and species specificity of the reverse inversion and amino acid conjugation of profen NSAIDs.     

Isolation and characterization of poly(A)-containing polyoma "early" and "late" messenger RNAs.

During late lytic infection of mouse kidney cell cultures polyoma 16S and 19S (late 19S RNA) were isolated by oligo(dT)-cellulose chromatography. Approximately 60-80% of total cytoplasmic polyoma RNA contained tracts of poly(A) which were retained by oligo(dT)-cellulose. Early in lytic infection when viral DNA synthesis and the production of capsid protein are blocked by the addition of 5-fluorodeoxyuridine, approximately 100% of polyoma "early" 19S RNA was quantitatively retained by oligo(dT)-cellulose indicating the presence of poly(A) tracts on most 19S mRNA molecules. In addition, 2 classes polyoma RNA, synthesized after the onset of cellular RNA synthesis under conditions where DNA synthesis is inhibited with 5-fluorodeoxyuridine, were found to contain tracts of poly(A). These species sedimenting at 16S and 19S in aqueous sucrose density gradients were also quantitatively retained by oligo (dT)-cellulose.     

Uptake of fatty acids by the yeasts, Saccharomyces uvarum and Saccharomycopsis lipolytica

Yeast cells take up exogenous fatty acids with subsequent rapid incorporation into glycerolipids. beta-Oxidation does not occur in Saccharomyces uvarum and is observed in Saccharomycopsis lipolytica only 2-5 min after addition of radioactively labeled fatty acid. Rates of fatty acid uptake are linear up to 30 s with S. lipolytica and up to 2 min with S. uvarum. The uptake kinetics are consistent with a dual mode of transport, comprising a saturable component with KT values in the range 10(-5)-10(-6) M, and apparently simple diffusion that predominates at high substrate concentrations. Kinetics of fatty acid permeation are independent of metabolic energy and membrane potential. At least two fatty acid carrier systems exist in both S. lipolytica and S. uvarum, one being specific for fatty acids with 12 and 14 C atoms, respectively, the other for C16 and C18 saturated or unsaturated fatty acids. Octanoic acid and decanoic acid are not taken up by S. lipolytica. Internalization of lauric acid and oleic acid by S. lipolytica cells is preceded by a rapid (less than 5 s) initial uptake which most likely represents irreversible adsorption. This phenomenon was not observed with heat-inactivated S. lipolytica cells or with viable S. uvarum. In azide-poisoned cells of S. lipolytica an up to 20-fold accumulation of unesterified fatty acid was observed within 30 s after the addition of substrate.     

Use of stable analogs of myosin ATPase intermediates for kinetic studies of the "weak" binding of myosin heads to F-actin.

It is known that ternary complexes of myosin subfragment 1 (S1) with ADP and the Pi analogs beryllium fluoride (BeFx) and aluminum fluoride (AlF4-) are stable analogs of the myosin ATPase intermediates M* x ATP and M** x ADP x Pi, respectively. Using kinetic approaches, we compared the rate of formation of the complexes S1 x ADP x BeFx and S1 x ADP x AlF4- in the absence and in the presence of F-actin, as well as of the interaction of these complexes with F-actin. We show that in the absence of F-actin the formation of S1 x ADP x BeFx occurs much faster (3-4 min) than that of S1 x ADP x AlF4- (hours). The formation of these complexes in the presence of F-actin led to dissociation of S1 from F-actin, this process being monitored by a decrease in light scattering. The light scattering decrease of the acto-S1 complex occurred much faster after addition of BeFx (during 1 min) than after addition of AlF4- (more than 20 min). In both cases the light scattering of the acto-S1 complex decreased by 40-50%, but it remained much higher than that of F-actin measured in the absence of S1. The interaction of the S1 x ADP x BeFx and S1 x ADP x AlF4- complexes with F-actin was studied by the stopped-flow technique with high time resolution (no more than 0.6 sec after mixing of S1 with F-actin). We found that the binding of S1 x ADP x BeFx or S1 x ADP x AlF4- to F-actin is accompanied by a fast increase in light scattering, but it does not affect the fluorescence of a pyrene label specifically attached to F-actin. We conclude from these data that within this time range a "weak" binding of the S1 x ADP x BeFx and S1 x ADP x AlF4- complexes to F-actin occurs without the subsequent transition of the "weak" binding state to the "strong" binding state. Comparison of the light scattering kinetic curves shows that S1 x ADP x AlF4- binds to F-actin faster than S1 x ADP x BeFx does: the second-order rate constants for the "weak" binding to F-actin are (62.8 +/- 1.8) x 10(6) M-1 x sec-1 in the case of S1 x ADP x AlF4- and (22.6 +/- 0.4) x 10(6) M-1 x sec-1 in the case of S1 x ADP x BeFx. We conclude that the stable ternary complexes S1 x ADP x BeFx and S1 x ADP x AlF4- can be successfully used for kinetic studies of the "weak" binding of the myosin heads to F-actin.     

Environmental pH as a factor in the competition between strains of the oral streptococci Streptococcus mutans, S. sanguis, and "S. mitior" growing in continuous culture

Strains of Streptococcus mutans (biotype 1), Streptococcus sanguis, and Streptococcus mitior have been grown in mixed continuous culture in a semidefined medium under glucose limitation at a growth rate of D = 0.1 h-1. The effect of varying the environmental pH on the proportions of the different populations within the community has been determined. Initially the populations were allowed to reach steady state at pH 7.0 when S. sanguis was dominant with S. mutans and "S. mitior" maintaining similar populations. The medium pH was then lowered in steps of 0.5 pH units from pH 7.0 to 4.5, and the community was grown at each step for at least 15 generations. Viable counts of each species were made at 24-h intervals. The population ratios established at pH 7.0 remained relatively stable when the environmental pH was set at pH 6.5. However, after the medium pH was lowered to 6.0 (days 18-27), the population of S. mutans began to increase and the S. mitior population began to decline. A further change was seen at pH 5.5 (days 27-34) when S. mutans became dominant, S. sanguis declined, and S. mitior was not detectable. At pH 4.5, both S. mutans and S. sanguis were reduced in numbers, but survived until the experimental run was terminated (44 days). Samples of culture fluid were taken throughout the experiment and analyzed for the presence of the acid products of glucose metabolism. The amounts of lactic acid produced by the community increased as the environmental pH was lowered.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipoxygenase metabolites of arachidonic and linoleic acids modulate the adhesion of tumor cells to endothelium via regulation of protein kinase C.

12(S)-hydroxyeicosatetraenoic acid (12[S]-HETE) and 13(S)-hydroxyoctadecadienoic acid (13[S]-HODE), lipoxygenase metabolites of arachidonic acid and linoleic acid, respectively, previously have been suggested to regulate tumor cell adhesion to endothelium during metastasis. Adhesion of rat Walker carcinosarcoma (W256) cells to a rat endothelial cell monolayer was enhanced after treatment with 12(S)-HETE and this 12(S)-HETE enhanced adhesion was blocked by 13(S)-HODE. Protein kinase inhibitors, staurosporine, calphostin C, and 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine, inhibited the 12(S)-HETE enhanced W256 cell adhesion. Depleting W256 cells of protein kinase C (PKC) with phorbol 12-myristate-13-acetate abolished their ability to respond to 12(S)-HETE. Treatment of W256 cells with 12(S)-HETE induced a 100% increase in membrane-associated PKC activity whereas 13(S)-HODE inhibited the effect of 12(S)-HETE on PKC translocation. High-performance liquid chromatographic analysis revealed that in W256 cells 12-HETE and 13-HODE were two of the major lipoxygenase metabilites of arachidonic acid and linoleic acid, respectively. Therefore, these two metabolites may provide an alternative signaling pathway for the regulation of PKC. Further, these findings suggest that the regulation of tumor cell adhesion to endothelium by 12(S)-HETE and 13(S)-HODE may be a PKC-dependent process.     

Biosynthesis of 1-alkenes in higher plants: stereochemical implications. A model study with Carthamus tinctorius (Asteraceae)

Odd numbered 1-alkenes, such as 1-pentadecene or 1,8,11,14-heptadecatetraene are formed from palmitic or linolenic acid by fragmentative decarboxylation. Incubation studies with germinating safflower (Carthamus tinctorius) and (2R,3R)-12-phenyl[2,3-2H2]dodecanoic acid, (2S,3S)-12-phenyl[2,3-2H2]dodecanoic acid, (2R)-12-phenyl[2-2H]dodecanoic acid and (2S)-12-phenyl[2-2H]dodecanoic acid instead of the natural alpha-linolenic acid precursor revealed the fragmentation to be an overall anti elimination of the 3-pro(S) hydrogen and the carboxyl group (anti-periplanar transition state geometry). Externally offered 3-hydroxy acids are not fragmented to 1-alkenes. The most probable mechanistic alternatives are in agreement with abstraction of the 3-pro(S) hydrogen as a radical followed by electron transfer and fragmentation, or transient insertion of oxygen into the 3-pro(S) C-H bond and subsequent fragmentation into an 1-alkene and CO2 after appropriate activation. The mechanism seems to be of general importance for the biosynthesis of vinylic substructures of natural products from oxygenated precursors.     

Kinetic and stoichiometric parameters estimation in a nitrifying bubble column through "in-situ" pulse respirometry

This article proposes a simple "in-situ" pulse respirometric method for the estimation of four important kinetic and stoichiometric parameters. The method is validated in a suspended biomass nitrifying reactor for the determination of (i) maximum oxygen uptake rate (OUR(ex)max), (ii) oxidation yield (f(E)), (iii) biomass growth yield (f(S)), and (iv) affinity constant (K(S)). OUR(ex)max and f(E) were directly obtained from respirograms. In the presented case study, a minimum substrate pulse of 10 mgNH(4) (+)-N L(-1) was necessary to determine OUR(ex)max which was 61.15 +/- 4.09 mgO(2) L(-1) h(-1) (5 repetitions). A linear correlation (r(2) = 0.93) obtained between OUR(ex)max and the biomass concentration in the reactor suggests that biomass concentration can be estimated from respirometric experiments. The substrate oxidation yield, f(E), was determined along 60 days of continuous operation with an average error of 5.6%. The biomass growth yield was indirectly estimated from the substrate oxidation yield f(E). The average obtained value (0.10 +/- 0.04 mgCOD mg(-1)COD) was in accordance with the f(S) estimation by the traditional COD mass balance method under steady-state conditions (0.09 +/- 0.01). The affinity constant K(S) was indirectly estimated after fitting the ascending part of the respirogram to a theoretical model. An average value of 0.48 +/- 0.08 mgNH(4) (+)-N L(-1) was obtained, which is in the range of affinity constants reported in the literature for the nitrification process (0.16-2 mgNH(4) (+)-N L(-1)).     

"In vivo" (35S)methionine interaction with rat liver tRNA

As part of a study to characterize the methionine role in tumorigenesis, we report that methionine sulfur interacts with rat liver tRNA "in vivo" (35S) radioactivity remained associated to the nucleic acid after a number of treatments, including tRNA deacylation. Similar data were obtained after administration of (methyl-3H) methionine, while no comparable tRNA labelling was detected when the aminoacid labelled in the aliphatic chain was given. Hplc analysis of (35S) tRNA enzymic hydrolysate showed two unidentified UV-absorbing radioactive peaks. NMR spectra of these two peaks did not reveal any thiomethyl group.     

Synthesis of four stereoisomers of 1,4-thiazane-3-carboxylic acid 1-oxide via the asymmetric transformation (combined isomerization-preferential crystallization) of 1,4-thiazane-3-carboxylic acid

In order to synthesize four stereoisomers of 1,4-thiazane-3-carboxylic acid 1-oxide (TCA SO), (S)-1,4thiazane-3-carboxylic acid [(S)-TCA], which is one of the precursors, was prepared by the asymmetric transformation (combined isomerization-preferential crystallization) of (RS)-TCA. This asymmetric transformation was used (2R, 3R)-tartaric acid [(R)-TA] as a resolving agent and salicylaldehyde as the epimerization catalyst in propanoic acid at 110 degrees C to afford a salt of (S)-TCA with (R)-TA in 100% de with a yield of over 90%. Optically pure (S)-TCA was obtained by treating the salt with triethylamine in methanol in a yield of over 80%, based on (RS)-TCA as the starting material. In addition, asymmetric transformation of (R)-TCA gave (S)-TCA in a yield of 60-70%. (S)-TCA was oxidized by hydrogen peroxide in dilute hydrochloric acid to selectively crystallize (1S, 3S)-TCA.SO. (1R, 3S)-TCA SO of 70% de from the filtrate was allowed to form a salt with (R)-TA after a treatment with triethylamine to give (1R, 3S)-TCA SO as a single diastereoisomer. (1R, 3R)- and (1S, 3R)-TCA.SO were also prepared by starting from (R)-TCA that had been synthesized from L-cysteine.     

Metabolism of [6]-gingerol in rats

The metabolic fate of [6]-gingerol, one of the active constituents of Zingiber officinale Roscoe, was investigated using rats. The bile of rats orally administered [6]-gingerol was shown to contain a major metabolite (1) by HPLC analysis. Although the metabolites derived from [6]-gingerol were not detected in the urine, the ethyl acetate extract of the urine after enzymatic hydrolysis was shown to contain six minor metabolites (2-7). Their structures were determined to be (S)-[6]-gingerol-4'-O-beta-glucuronide (1), vanillic acid (2), ferulic acid (3), (S)-(+)-4-hydroxy-6-oxo-8-(4-hydroxy-3-methoxyphenyl) octanoic acid (4), 4-(4-hydroxy-3-methoxyphenyl)butanoic acid (5), 9-hydroxy [6]-gingerol (6) and (S)-(+)-[6]-gingerol (7) based on spectroscopic and chemical data. The total cumulative amount of 1 excreted in the bile and 2-7 in the urine during 60 h after the oral administration of [6]-gingerol were approximately 48% and 16% of the dose, respectively. The excretion of 2-7 in the urine decreased after gut sterilization. On the other hand, the incubations of [6]-gingerol with rat liver showed the presence of 9-hydroxy [6]-gingerol, gingerdiol (8), and (S)-[6]-gingerol-4'-O-beta-glucuronide (1). These findings suggest that the gut flora and enzymes in the liver play an important part in the metabolism of [6]-gingerol.     

Reduction and partial degradation mechanisms of naphthylaminesulfonic azo dye amaranth by <Emphasis Type="Italic">Shewanella decolorationis</Emphasis> S12

Reduction and biodegradation mechanisms of naphthylaminesulfonic azo dye amaranth using a newly isolated Shewanella decolorationis strain S12 were investigated. Under anaerobic conditions, amaranth was reduced by strain S12, and a stoichiometric amount of two reduction products RP-1 and RP-2 were generated. UV/visible spectrophotometric and high performance liquid chromatography (HPLC) analysis indicated that RP-1 and RP-2 were 1-aminenaphthylene -4-sulfonic acid and 1-aminenaphthylene-2-hydroxy-3, 6-disulfonic acid. The result strongly supports a mechanism of azo dye reduction by the process via the reductive cleavage of the azo bond to form corresponding aromatic amines. The result of HPLC analyses revealed that these aromatic amines were not able to be mineralized by strain S12 under anaerobic conditions. But after re-aeration of the decolorized culture, RP-2 was mineralized completely by this microorganism, but the consumption of RP-1 was not observed. Ames test showed that amaranth had mutagenic but no cytotoxic potential. The mutagenic potential was relieved after the anaerobic treatment with strain S12 as the mutagenic effect of the two reduction products from amaranth was not detected by Ames test. Thus, the ability of strain S12 to reduce and partially mineralize the naphthylaminesulfonic azo dye efficiently was demonstrated, which can potentially be used to biodegrade and detoxify wastewater containing azo dyes using an alternating anaerobic/aerobic treatment procedure.     

Steps in the conversion of alpha-ketosuberate to 7-mercaptoheptanoic acid in methanogenic bacteria

The biosynthetic steps involved in the conversion of alpha-ketosuberate to 7-mercaptoheptanoic acid were studied in cell-free extracts of methanogenic bacteria. The pathway was established by measuring the incorporation of stable isotopically labeled precursors into the S-methyl ether methyl ester derivative of the enzymatically generated 7-mercaptoheptanoic acid by using gas chromatography-mass spectrometry (GC-MS). Quantitation of the 7-mercaptoheptanoic acid produced in the incubations with the substrates was accomplished by using an internal standard of 6-mercaptohexanoic acid. [4,4,6,6-2H4]-2-Oxosuberic acid, [7-2H]-7-oxoheptanoic acid, [2-2H]-2(RS)-(5-carboxypentyl)thiazolidine-4(R)-carboxylic acid, and S-(6-carboxyhexyl)cysteine were each shown to be converted to 7-mercaptoheptanoic acid. Incubation of cell extracts with a mixture of 2(RS)-(5-carboxypentyl)thiazolidine-4(R)-carboxylic acid and [2-2H]-2-(RS)-(5-carboxypentyl)-[34S]thiazolidine-4(R)-carboxylic acid showed that both 34S and 2H are incorporated into the 7-mercaptoheptanoic acid but only after separation of the cysteine from the [7-2H]-7-oxyheptanoic acid portion of the molecule. Furthermore, the sulfur from the cysteine was incorporated into the thiol only after its elimination from the cysteine and subsequent mixing with an unlabeled sulfur source which had a molecular weight of sufficient size that it was excluded from Sephadex G-25. Hydrogen sulfide was found to supply the sulfur for the production of the 7-mercaptoheptanoic acid in a reaction that was shown to obtain its reducing equivalents from hydrogen via an F420-dependent hydrogenase.     

Preparation of (<Emphasis Type="Italic">R</Emphasis>)-(−)-mandelic acid and its derivatives from racemates by enantioselective degradation with a newly isolated bacterial strain <Emphasis Type="Italic">Alcaligenes</Emphasis> sp. ECU0401

An enantioselective mandelate-degrading bacterium, Alcaligenes sp. ECU0401, was newly isolated from soil. By fed-batch culture, (R)-(-)-mandelic acid was successfully prepared in a 5-L fermenter with 32.8% isolated yield and >99.9% enantiomeric excesses (e.e.) from totally 3.04% (w/v) of racemic mandelic acid after 99 h of biotransformation. The optimal reaction pH and temperature were 6.5 and 30 degrees C, respectively. Using the resting cell as a biocatalyst for asymmetric degradation of racemic mandelic acid and chloro-substituted derivatives thereof, (R)-(-)-mandelic acid, (R)-(-)-o-chloromandelic acid, (S)-(+)-m-chloromandelic acid and (S)-(+)-p-chloromandelic acid were recovered with high analytic yields and excellent enantiomeric excesses (e.e. > 99.9%). (R)-(-)-Mandelic acid could also be obtained after 12 h of biotransformation with 41.5% isolated yield and >99.9% e.e.     

Synthesis of enantiopure non-natural alpha-amino acids using tert-butyl (2S)-2-[bis-(tert-butoxycarbonyl)amino]-5-oxopentanoate as key-intermediate:the first synthesis of(S)-2-amino-oleic acid.

A general method for the synthesis of enantiopure non-natural alpha-amino acids is described. The key intermediate tert-butyl (2S)-2-[bis(tert-butoxycarbonyl)amino]-5-oxopentanoate was obtained from l-glutamic acid after suitable protection and selective reduction of the gamma-methyl ester group by DIBALH. Wittig reaction of this chiral aldehyde with various ylides led to a variety of delta,epsilon-unsaturated alpha-amino acids. This methodology was applied to the synthesis of (S)-2-amino-oleic acid.     

Experimental studies on the growth and usnic acid production in "lichen"Usnea ghattensis in vitro

The study was aimed to optimize the culture conditions for the production of usnic acid in the cultured cell aggregates composed of symbionts in lichen Usnea ghattensis in vitro. The cultured lichen tissue composed of symbionts appeared after about 2-3 weeks of inoculation in water-agar and malt-yeast extract (MYE) media and shown the production of usnic acid after 2-3 months of inoculation. However, the growth of symbionts was strongly affected by different culture conditions. The addition of excess carbon and nitrogen sources in the media has significantly enhanced the growth as well as usnic acid content. The cultured symbionts in MYE medium having 4% sucrose, 4% polyethyl glycol (PEG) gave 7.63 g dry biomass with 3.9 microg usnic acid/g dry biomass. In water-agar medium having 4% sucrose and 4% PEG gave 3.08 g dry biomass with 1.11 microg usnic acid/g dry biomass. The positive effects of medium on the growth of symbionts and the production of usnic acid are seemed to be due to nutritional factors.     

Synthesis and thrombolytic activity of pseudopeptides related to fibrinogen fragment

Two kinds of linkers consisting of 3-(S)-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid, ARPAK, GRPAK and QRPAK were synthesized. The thrombolytic activities in vivo indicated that the coupling position of 3-(S)-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid in the peptides effected on the potencies significantly. When the C-terminal of the peptides was amidated by 3-(S)-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid, the thrombolytic potency of the peptides was enhanced or kept. When the N-terminal of the peptides was acylated by 3-(S)-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid, however, the thrombolytic effect of the peptides was banished. The expected specific beta II'-turn conformation and the stability to trypsin in the pseudopeptides with 3-(S)-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid in its C-terminal may be responsible for the enhanced thrombolytic potency.     

Formation of 14,15-hepoxilins of the A(3) and B(3) series through a 15-lipoxygenase and hydroperoxide isomerase present in garlic roots.

We report herein for the first time the formation by freshly grown garlic roots and the structural characterization of 14,15-epoxide positional analogs of the hepoxilins formed via the 15-lipoxygenase-induced oxygenation of arachidonic acid. These compounds are formed through the combined actions of a 15(S)-lipoxygenase and a hydroperoxyeicosatetraenoic acid (HPETE) isomerase. The compounds were formed when either arachidonic acid or 15-HPETE were used as substrates. Both the "A"-type and the "B"-type products are formed although the B-type compounds are formed in greater relative quantities. Chiral phase high performance liquid chromatography analysis confirmed the formation of hepoxilins from 15(S)- but not 15(R)-HPETE, indicating high stereoselectivity of the isomerase. Additionally, the lipoxygenase was of the 15(S)-type as only 15(S)-hydroxyeicosatetraenoic acid was formed when arachidonic acid was used as substrate. The structures of the products were confirmed by gas chromatography-mass spectrometry of the methyl ester trimethylsilyl ether derivatives as well as after characteristic epoxide ring opening catalytically with hydrogen leading to dihydroxy products. That 15(S)-lipoxygenase activity is of functional importance in garlic was shown by the inhibition of root growth by BW 755C, a dual cyclooxygenase/lipoxygenase inhibitor and nordihydroguaiaretic acid, a lipoxygenase inhibitor. Additional biological studies were carried out with the purified intact 14(S), 15(S)-hepoxilins, which were investigated for hepoxilin-like actions in causing the release of intracellular calcium in human neutrophils. The 14,15-hepoxilins dose-dependently caused a rise in cytosolic calcium, but their actions were 5-10-fold less active than 11(S), 12(S)-hepoxilins derived from 12(S)-HPETE. These studies provide evidence that 15(S)-lipoxygenase is functionally important to normal root growth and that HPETE isomerization into the hepoxilin-like structure may be ubiquitous; the hepoxilin-evoked release of calcium in human neutrophils, which is receptor-mediated, is sensitive to the location within the molecule of the hydroxyepoxide functionality.