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Excessive expansion of Staphylococcus aureus is associated with several skin diseases, including atopic dermatitis (AD). Recently, we have demonstrated that washing skins with ultra-pure soft water containing little bivalent metal ions improved skin conditions of atopic subjects. In this study, we investigated the roles of calcium or magnesium on the proliferation of S. aureus both in vitro and in vivo. Depletion of calcium and magnesium in the culture medium significantly suppressed the expansion of S. aureus growth. When S. aureus, diluted with water containing calcium/magnesium at the concentration of medium-hard water (83·0 mg l-1 as CaCO3 ) or the one that contains little calcium/magnesium, was applied onto the tape-stripped skin of Hos:HR-1 mice, growth of S. aureus in water without those minerals on the skin was suppressed. These results suggest that depletion of both calcium and magnesium abrogate the proliferation of S. aureus not only in the culture system but also on the skin surface of mice. Since colonization of S. aureus on the skin is well-known to exacerbate AD symptoms, usage of ultra-pure soft water containing less calcium and magnesium may improve the skin condition through the suppression of S. aureus growth on the skin of patients with skin problems. Significance and impact of the study This study demonstrates the importance of calcium and magnesium for the colonization and growth of Staphylococcus aureus by using both in vitro culture systems and in vivo experiments on the murine skin. Our results indicate that the removal of these metal ions is probably beneficial for protecting the skin from S. aureus. Thus, using ultra-pure soft water without metal ions may improve the skin condition of patients with skin problems through the protection from S. aureus colonization.

The effects of probiotics on horses are still controversial. The present study was a randomized, double-blinded, placebo-controlled crossover study designed to evaluate the ability of probiotics to improve intestinal conditions in adult horses. Fermented probiotics were administered to 10 healthy adult geldings for 28 days. The clinical condition of the horses was monitored daily, and the blood and feces were biochemically analyzed every 14 days. In the probiotic-treated group, the concentration of carboxylic acids in the feces was increased at days 14 and 28. In contrast to the fecal pH in the control group, which increased at days 14 and 28, the fecal pH in the probiotic-treated group did not increase. Additionally, the relative amounts of enteropathogenic bacterial DNA were diminished in the probiotic-treated group. These results suggest that probiotic bacteria proliferated in the equine intestine. No instances of abnormal clinical conditions or abnormal values in blood tests were observed throughout the study. Oral administration of fermented probiotics may have the ability to improve the intestinal environment biochemically and microbiologically without the risk of adverse effects.

Aims Scedosporium apiospermum sometimes causes serious infectious diseases on the skin of immunodeficient subjects. Antifungal effects of fatty acid salts in soap against S. apiospermum were investigated under different water conditions. Methods and Results Ultrapure soft water (UPSW) was generated by the water softener with cation-exchange resin. The calcium and magnesium ions were replaced with sodium ions in UPSW. Scedosporium apiospermum was incubated with different fatty acid salts that constituted soap in distilled water (DW), tap water (TW) and UPSW. After incubation, the number of fungi was counted. Among the fatty acids, palmitic acid salt (C16) reduced the number of S. apiospermum. UPSW enhanced the antifungal effect of C16 on S. apiospermum. The absence of both calcium and magnesium ions and the existence of sodium chloride in UPSW were responsible for its antifungal effect. In addition, repeated short-term treatment with UPSW and C16 decreased the number of S. apiospermum. Conclusions Antifungal effects of C16 on S. apiospermum were demonstrated. Moreover, the use of UPSW promoted the antifungal effect of C16. Significance and Impact of Study This study provides the preventive method for diseases associated with S. apiospermum infection using novel palmitic acid soap in UPSW.

Ultrapure soft water (UPSW) is water in which calcium and magnesium ions have been replaced with sodium ions using a cation-exchange resin. We recently demonstrated that washing with soap and UPSW reduced the clinical severity of dermatitis and improved the skin barrier function in NC/NgaTnd mice, a murine model for human atopic dermatitis. The purpose of this pilot study was to evaluate the efficacy of shampoo treatment with UPSW for dogs with pruritus. Eleven dogs with pruritus were randomly assigned to two groups depending on whether they received weekly shampoo treatment with UPSW or tap water for 4 weeks. After a washout period, the treatment protocol was switched such that each dog received both treatments. The pre-treatment and post-treatment values of the following were compared: pruritus scores assessed by the owners; dermatitis scores recorded by an investigator; and transepidermal water loss (TEWL). Shampoo treatment with UPSW significantly decreased pruritus and dermatitis scores in the dogs, whereas shampoo treatment with tap water did not. In addition, shampoo treatment with UPSW, but not with tap water, significantly reduced TEWL in the dogs. Adverse events due to the treatment were not observed in the dogs. Furthermore, we found that topical application of UPSW for barrier-disrupted skin caused by tape stripping in healthy dogs decreased TEWL more rapidly than topical application of tap water. Our findings suggest that shampoo treatment with UPSW promotes skin barrier recovery and thus could be considered as a possible therapeutic option in the management of pruritus and dermatitis in dogs.

AIMS Skin colonization of Staphylococcus spp. critically affects the severity of dermatitis in humans and animals. We examined different types of fatty acid salts for their antibacterial activity against Staphylococcus spp. when used in ultrapure soft water (UPSW). We also evaluated their therapeutic effect on a spontaneous canine model of dermatitis. METHODS AND RESULTS UPSW, in which Ca(++) and Mg(++) were replaced with Na(+) , was generated using a water softener with cation-exchange resin. Staphylococcus aureus (Staph. aureus), Staphylococcus intermedius (Staph. intermedius), and Staphylococcus pseudintermedius (Staph. pseudintermedius) were incubated with various fatty acid salts in distilled water (DW) or UPSW and the number of bacteria was counted. Among the fatty acids, oleic acid salt and linoleic acid (LA) salt reduced the number of these bacteria. Also, UPSW enhanced the antibacterial effect of LA on Staph. spp. In spontaneously developed itchy dermatitis in companion dogs, shampoo treatment with liquid soap containing 10% LA in UPSW improved skin conditions. CONCLUSIONS LA salt showed antibacterial activity against Staph. spp. Treatment with soap containing LA with UPSW reduced clinical conditions in dogs with dermatitis. SIGNIFICANCE AND IMPACT OF THE STUDY Because colonization of Staph. spp. on the skin exacerbates dermatitis, the use of LA-containing soap in UPSW may reduce unpleasant clinical symptoms of the skin.

Five physiologically different strains of Acidithiobacillus ferrooxidans were isolated. Strains Funis 2-1, SUG 2-2, OK1-50 and NASF-1, respectively, are resistant to molybdenum, mercury, sulfite and 2,4-dinitrophenol (2,4-DNP). In contrast, strain AP19-3 is sensitive to all these toxic compounds. Cytochrome oxidase is one of the most important enzymes in the iron oxidation system of A. ferrooxidans . Cytochrome oxidase was purified from the five strains to an electrophoretically homogenous state to clarify the relationship between resistance and molecular structure of the enzyme. The apparent molecular weights of cytochrome oxidase β- and γ-subunits from Funis 2-1, SUG 2-2, OK1-50 and AP19-3 were 24 and 19 kDa, and those of NASF-1, 22.5 and 20 kDa, respectively. The apparent molecular weights of cytochrome oxidases α-subunits of the five strains ranged from 53 to 56 kDa. The N-terminal amino acid sequences of cytochrome oxidase β- and γ-subunits were nearly the same among the five strains except for the γ-subunits of AP19-3. The sequences of α-subunit from AP19-3, OK1-50 and Funis 2-1 differed markedly from each other. The N-terminal amino acid of SUG 2-2 α-subunit was blocked. These results indicate that A. ferrooxidans strains resistant to Mo 5+ , Hg 2+ , sulfite and 2,4-DNP have a cytochrome oxidase different from that of the sensitive strain AP19-3 with respect to apparent molecular size as well as N-terminal amino acid sequence.

Iron oxidase was purified from plasma membranes of a moderately thermophilic iron oxidizing bacterium strain TI-1 in an electrophoretically homogeneous state. Spectrum analyses of purified enzyme showed the existence of cytochrome a, but not cytochrome b and c types. Iron oxidase was composed of five subunits with apparent molecular masses of 46 kDa (alpha), 28 kDa (beta), 24 kDa (gamma), 20 kDa (delta), and 17 kDa (epsilon). As the molecular mass of a native enzyme was estimated to be 263 kDa in the presence of 0.1% n-dodecyl-beta-D-maltopyranoside (DM), a native iron oxidase purified from strain TI-1 seems to be a homodimeric enzyme (alpha beta gamma delta epsilon)(2). Optimum pH and temperature for iron oxidation were pH 3.0 and 45 degrees C, respectively. The K(m) of iron oxidase for Fe(2+) was 1.06 mM and V(max) for O(2) uptake was 13.8 micromol x mg(-1) x min(-1). The activity was strongly inhibited by cyanide and azide. Purified enzyme from strain TI-1 is a new iron oxidase in which electrons of Fe(2+) were transferred to haem a and then to the molecular oxygen.

A moderately thermophilic bacterium, strain TI-1, produces H2S outside of the cells when grown at 45 degrees C on Fe(2+)-medium (pH 1.8) containing elemental sulfur and L-glutamic acid. A newly identified sulfur reductase was present in the cytosol of this strain and was purified to an electrophoretically homogeneous state from strain TI-1. The apparent molecular weight of sulfur reductase was 86,000 by gel filtration and 48,000 by SDS-PAGE, so the enzyme was a homodimer. The enzyme was most active at pH 9.0 and 60 to 70 degrees C, and it catalyzed the reduction of 1 mol of elemental sulfur with 1 mol of NADH to give 1 mol of H2S and 1 mol of NAD+. Elemental sulfur was a specific electron acceptor of this enzyme. Thiosulfate, sulfite, and tetrathionate were not electron acceptors, but inhibited sulfur reductase activity. NADPH was not used as an electron donor.

Among seventy five strains of iron-oxidizing bacteria obtained from natural environments, only one strain, Thiobacillus ferrooxidans Funis 2-1, grew on Fe2+-medium with 1.25 mm of sodium molybdate (Mo6+). In contrast, T. ferrooxidans AP19-3, the representative of Mo sensitive strains, could not grow on Fe2+-medium with 1.0mm of sodium molybdate. By comparing the levels of inhibition of iron oxidase and cytochrome c oxidase by Mo6+ or Mo5+, it was found that Mo5+ but not Mo6+ is an actual inhibitor for the iron oxidation enzyme system, especially for cytochrome c oxidase. Cytochrome c oxidase of Funis 2-1 was more resistant to Mo5+ than AP19-3. Mo5+, compared to Mo6+, strongly binds to both cells and the plasma membrane of T. ferrooxidans. Funis 2-1 cells showed a lower binding activity to Mo6+ or Mo5 + compared to AP19-3. Cytochrome c oxidase of T. ferrooxidans has been known to catalyze the oxidation of not only reduced mammalian cytochrome c but also Mo5+ . Mo5+-oxidizing activities measured with intact...

The moderately thermophilic iron-oxidizing bacterium strain TI-1, which lacks enzyme systems involved in CO 2 fixation, grows at 45°C in Fe 2+ medium supplemented with yeast extract to give a maximum cell growth of 1.0 × 10 8 cells per ml, but does not grow in Fe 2+ medium without yeast extract. To elucidate the physiology of the strain, a synthetic medium was developed. It was found that the best synthetic medium was Fe 2+ -6AA, containing Fe 2+ , salts, and the following six l -amino acids: alanine, aspartic acid, glutamic acid, arginine, serine, and histidine. In this medium, strain TI-1 showed a maximum cell growth of 10 × 10 8 cells/ml. The six amino acids in the Fe 2+ -6AA medium were used not only as a carbon source but also as a source of nitrogen. Inorganic nitrogen sources, such as ammonium ion, hydrazine, hydroxylamine, nitrite, and nitrate, were not used as a sole source of nitrogen, but rather strongly inhibited the utilization of the six amino acids at 1 mM. In the Fe 2+ (10 mM)-6AA medium supplemented with 21 mM Fe 3+ , reduction of Fe 3+ to Fe 2+ that was dependent on the added amino acids was observed, suggesting another role of the amino acids in the growth of strain TI-1. Washed, intact cells of strain TI-1 had the activity to reduce Fe 3+ to Fe 2+ .

The cardiovascular effects of FK664, [6-(3, 4-dimethoxyphenyl)-1-ethyl-4-mesitylimino-3-methyl-3, 4-dihydro-2 (1H)-pyrimidinone], were examined in both in vitro and in vivo preparations. FK664 is an orally effective noncatechol and nonglycoside cardiotonic agent. FK664 has vasodilating activity not only in resistance vessels but also in capacitance vessels in both in vitro and in vivo studies (venodilation). This venodilating effect of FK664 has been observed at much lower concentrations than those required to produce an inotropic response. FK664 is effective in the conscious dog at 1 mg/kg (2.5 μmol/kg) orally and in a dog model of congestive heart failure at 3.2 μg/kg/min (7.8 nmol/kg/min), intravenously. The vasodilating and positive-inotropic activities of FK664 are mainly due to an increase in cyclic adenosine 3′,5′-monophosphate (cyclic AMP) levels due to inhibition of cyclic AMP phosphodiesterase. Moreover, FK664 does not affect the balance between myocardial oxygen supply and demand due to a significant increase in coronary blood flow, though the agent does increase myocardial oxygen consumption. In conclusion, the agent produces potent preload reduction and mild cardiac stimulation. This unique pharmacological profile may be beneficial in the management of congestive heart failure. © 1993 Wiley-Liss, Inc.

This paper describes the structure and characteristics of granules which were unexpectedly formed in anaerobic-oxic activated sludge process. The activated sludge granules had prominent characteristics of sedimentation and agglomeration : sedimentation velocity was 13-15cm·min-1 ; sludge volume for 30 minutes was 6% ; sludge volume index was 27ml·g-1. The surface structure of the granules was densely agglomerated by various bacteia, mainly rod type bacteria. Some parts of its surface were covered by extracellular polymer, and other parts showed the complex structure with multi-layers. By the quantitical analysis of chemical compositions of the granules, the contents of Fe, Zn and Ca in the granules were found to be more than those of activated sludge. The microflora of granules was characterized using quinone profile. In the granules, Rhodoquinone-8 (RQ-8) of 1.8mol% was detected, while little RQ-8 was found in activated sludge. This result suggested that bacteria containing RQ-8 would participate in formation of the granules.

Ultrapure soft water (UPSW) is water in which calcium and magnesium ions have been replaced with sodium ions using a cation-exchange resin. We recently demonstrated that washing with soap and UPSW reduced the clinical severity of dermatitis and improved the skin barrier function in NC/NgaTnd mice, a murine model for human atopic dermatitis. The purpose of this pilot study was to evaluate the efficacy of shampoo treatment with UPSW for dogs with pruritus. Eleven dogs with pruritus were randomly assigned to two groups depending on whether they received weekly shampoo treatment with UPSW or tap water for 4 weeks. After a washout period, the treatment protocol was switched such that each dog received both treatments. The pre-treatment and post-treatment values of the following were compared: pruritus scores assessed by the owners; dermatitis scores recorded by an investigator; and transepidermal water loss (TEWL). Shampoo treatment with UPSW significantly decreased pruritus and dermatitis scores in the dogs, whereas shampoo treatment with tap water did not. In addition, shampoo treatment with UPSW, but not with tap water, significantly reduced TEWL in the dogs. Adverse events due to the treatment were not observed in the dogs. Furthermore, we found that topical application of UPSW for barrier-disrupted skin caused by tape stripping in healthy dogs decreased TEWL more rapidly than topical application of tap water. Our findings suggest that shampoo treatment with UPSW promotes skin barrier recovery and thus could be considered as a possible therapeutic option in the management of pruritus and dermatitis in dogs.

A moderately thermophilic iron-oxidizing bacterium strain TI-1 has been shown to have a unique enzyme system that produces hydrogen sulfide (H2S) extracellularly under acidic conditions. The optimal culture conditions for H2S production were determined to apply the bacterium to the treatment of acidic wastewater containing heavy metals. The production of H2S by the cells was dependent on l -glutamic acid, ferrous sulfate and elemental sulfur in the medium. The optimal culture medium for H2S production contained 0.3% l -glutamic acid, 1.7% ferrous sulfate, and 1.0% elemental sulfur and the optimal pH and temperature were 4.0 and 55°C, respectively. Under the optimal culture conditions, strain TI-1 produced H2S continuously at a rate of 0.2 μmol/ml/d for 14 d.

Background Hardness in water is consisted with mineral salts, including calcium (Ca++) and magnesium (Mg++). The mineral salts react with soap to form an insoluble precipitate known as metallic soap (soap scum). Since metallic soap remains tightly on the skin and is hard to rinse off, it may become one of irritants that exacerbate dermatitis. In this study, we used cation-exchange resin to prepare ultra-pure soft water (UPSW) excluding both Ca++ and Mg++, and investigated effect of UPSW rinsing on skin conditions in subjects with atopic dermatitis. Furthermore, antigenic activity of metallic soap was investigated by in vivo experiments. Methods Stratum corneum was collected from arms of healthy volunteers who rinsed soap in tap water or UPSW, and the quantity of remained soap scum was determined with a gas chromatography. After 4 weeks of bathing in UPSW, the water content of the stratum corneum and transepidermal water loss (TEWL) of volunteers with mild atopic dermatitis were measured. With atopic NC/Tnd mice, a model for atopic dermatitis, we attempted to confirm results obtained from atopic volunteers. Plasma total IgE was measured by an ELISA after immunization of mice with metallic soap. Results On skins rinsed in UPSW, soap was disappeared immediately and remained metallic soap was significantly reduced when compared to that on skins rinsed in tap water. In skins of atopic volunteers who used UPSW for bath, the water content in stratum corneum was increased and TEWL was decreased. Skin dryness and itch scores were reduced in most volunteers. After washed with soap and rinsed in UPSW for 2 weeks, severe dermatitis of NC/Tnd mice were reduced as well as TEWL. On the other hand, dermatitis in NC/Tnd mice rinsed in tap water became worse. Plasma total IgE was increased in mice that were immunized with metallic soap. Conclusions UPSW protected skins from residue of metallic soap. Metallic soap has antigenic activity and increased plasma IgE levels. Clinical symptoms and the skin barrier function were improved by the use of soap with UPSW. UPSW is beneficial for the skin of patients with atopic dermatitis.

Wet heat inactivation of lipopolysaccharide (LPS) from E. coli Serotype 055:B5 was investigated. The LPS solutions were heated at study temperatures ranging from 78 degrees C to 175 degrees C, and were assayed using the Limulus Amebocyte Lysate (LAL) test. Plots of the log of the amount of endotoxin remaining versus heating time showed biphasic decreases. The initial slopes are associated with a faster rate of decrease to about a 0.5-log unit reduction, and were followed by a slower, linear rate of decline in the secondary slopes. The curves were applied to the biexponential model expressed by the equation (Et = E1e-klt + E2e-k2t). The LPS inactivation rates (k1 and k2) each conformed to their own Arrhenius equation. Therefore, the processes required to achieve the desired level of LPS inactivation can be obtained by mathematical means.

A novel moderately thermophilic iron-oxidizing bacterium was isolated from a coal dump in Iizuka City, Japan, and designated strain TI-1. Strain TI-1 was a Gram negative, non-spore-forming, and rod-shaped bacterium, which had a optimum temperature and pH for growth at 48°C and 2.2, respectively. The mean G+C content of the DNA was 56.2 mol%. Strain TI-1 required yeast extract for growth and did not have the ability to fix carbon dioxide as a carbon source, indicating that the strain is not chemolithoautotroph. When grown on Fe2+ (1.7%)-yeast extract medium, Fe2+ was oxidized concomitantly with cell growth. The optimum pH and temperature for Fe2 + oxidation by washed intact cells of TI-1 were 2.8 and 50°C, respectively. The properties of strain TI-1 shown above, such as the G+C mol content of the DNA, the requirement of yeast extract for growth, and the inability to fix carbon dioxide, suggest that strain TI-1 belongs to a new type of mederately thermophilic, acidophilic iron-oxidizing bacterium.

Iron oxidase was purified from plasma membranes of a moderately thermophilic iron oxidizing bacterium strain TI-1 in an electrophoretically homogeneous state. Spectrum analyses of purified enzyme showed the existence of cytochrome a, but not cytochrome b and c types. Iron oxidase was composed of five subunits with apparent molecular masses of 46 kDa (alpha), 28 kDa (beta), 24 kDa (gamma), 20 kDa (delta), and 17 kDa (epsilon). As the molecular mass of a native enzyme was estimated to be 263 kDa in the presence of 0.1% n-dodecyl-beta-D-maltopyranoside (DM), a native iron oxidase purified from strain TI-1 seems to be a homodimeric enzyme (alpha beta gamma delta epsilon)(2). Optimum pH and temperature for iron oxidation were pH 3.0 and 45 degrees C, respectively. The K(m) of iron oxidase for Fe(2+) was 1.06 mM and V(max) for O(2) uptake was 13.8 micromol x mg(-1) x min(-1). The activity was strongly inhibited by cyanide and azide. Purified enzyme from strain TI-1 is a new iron oxidase in which electrons of Fe(2+) were transferred to haem a and then to the molecular oxygen.

The iron-oxidizing activity of a moderately thermophilic iron-oxidizing bacterium, strain TI-1, was located in the plasma membrane. When the strain was grown in Fe2+ (60 mM)-salts medium containing yeast extract (0.03%), the plasma membrane had iron-oxidizing activity of 0.129 mumol O2 uptake/mg/min. Iron oxidase was solubilized from the plasma membrane with 1.0% n-octyl-beta-D-glucopyranoside (OGL) containing 25% (v/v) glycerol (pH 3.0) and purified 37-fold by a SP Sepharose FF column chromatography. Iron oxidase solubilized from the plasma membrane was stable at pH 3.0, but quite unstable in the buffer with the pH above 6.0 or below 1.0. The optimum pH and temperature for iron oxidation were 3.0 and 55 degrees C, respectively. Solubilized enzyme from the membrane showed absorption peaks characteristic of cytochromes a and b. Cyanide and azide, inhibitors of cytochrome c oxidase, completely inhibited iron-oxidizing activity at 100 microM, but antimycin A, 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) and myxothiazol, inhibitors of electron transport systems involved with cytochrome b, did not inhibit enzyme activity at 10 microM. The absorption spectrum of the most active enzyme fraction from SP Sepharose FF column chromatography (4.76 mumol O2 uptake/mg/min) compared with lower active fractions from the chromatography (0.009 and 2.10 mumol O2 uptake/mg/min) showed a large alpha-peak of cytochrome a at 602 nm and a smaller alpha-peak of cytochrome b at 560 nm. The absorption spectrum of pyridine ferrohemochrome prepared from the most highly purified enzyme showed an alpha-peak characteristic of heme a at 587 nm, but not the alpha-peak characteristic of heme c at 550 nm. The cytochrome a, but not cytochrome b, in the most highly purified enzyme fraction was reduced by the addition of ferrous iron at pH 3.0, indicating that electrons from Fe2+ were transported to cytochrome a, but not cytochrome b. These results strongly suggest that cytochrome a, but not cytochromes b and c, is involved in iron oxidation of strain TI-1.

The effects of FK664, a novel positive inotropic agent, and enoximone on pentobarbital-induced heart failure were compared in dog heart-lung preparations. Both FK664 and enoximone improved the cardiac function curve in a dose-dependent manner and restored it to the control level at drug concentrations of 1 microgram/ml and 10 micrograms/ml, respectively. Therefore, the cardiotonic potency of FK664 appears to be 10 times that of enoximone. These agents were almost equal in force-rate separation of cardiac effect. Neither of the agents produced arrhythmia at any dose tested. These results suggest that FK664 may be a potent cardiotonic agent for the treatment of heart failure.

Enantiomeric isomers of 1-oxaspiro[2.8]undecone derivative (10), which are key compounds to synthesize nine-membered ring diterpenoids, were synthesized stereoselectivity from optically active monoterpene, limonene, via anionic oxy-Cope rearrangement reaction

Epoxidation of alnus-5 (10)-en-3β-yl acetate (3) with m-chloroperbenzoic acid gave a 5α, 10α-epoxide (4), which was subjected to the BF3·OEt2-catalyzed backbone rearrangement to afford multiflora-5, 8-dien-3β-yl acetate (6) as a main product. Hydrogenation of the diene (6) followed by hydrolysis gave isomultiflorenol (1a).

Epoxidation of alnus-5(10)-en-3β-yl acetate with m-chloroperbenzoic acid gave a 5α,10α-epoxide, treatment of which with boron trifluoride etherate afforded multiflora-5,8-dien-3β-yl acetate. On hydrogenation and hydrolysis, the diene gave isomultiflorenol.

Epoxidation of alnus-5-en-3β-yl acetate with m-chloroperbenzoic acid gave a 5β,6β-epoxide as a main product together with a 5α,6α-epoxide as a minor product. Treatment of the α-epoxide with BF3·OEt2 afforded a 1(10),5-diene and a mixture of 6α-hydroxy-12- and -18-ene derivatives. The 12-ene was converted into daturadiol (=olean-12-ene-3β,6β-diol).

Epoxidation of alnus-5-en-3β-yl acetate with m-chloroperbenzoic acid gave a 5β,6β-epoxide as a main product together with a 5α,6α-epoxide as a minor product. Treatment of the α-epoxide with BF3·OEt2 afforded a 1(10),5-diene and a mixture of 6α-hydroxy-12- and -18-ene derivatives. The 12-ene was converted into daturadiol (=olean-12-ene-3β,6β-diol).