Uptake of 12-HETE by human bronchial epithelial cells (HBEC): Effects on HBEC cytokine production

12-HETE, the major lipoxygenase end-product of platelets and macrophages, may be released in contact of bronchial epithelium in inflammatory diseases of the lung. We have studied the outcome of 12-HETE in presence of human bronchial epithelial cells (HBEC). When HBEC were incubated with [³H]12-HETE for 30 minutes, 27.5% of total radioactivity was found in HBEC and 72.5% in supernatants. Unesterified 12-HETE accounted for 22.4% of total radioactivity, 4.5% being recovered in phospholipids, preferentially in phosphatidylcholine and phosphatidylethanolamine. No incorporation in neutral lipids was detected. 72.9% of the incubated radioactivity was recovered in un identified metabolites. As 12-HETE has been shown to modulate the expression and production of various proteins, the consequence of the 12-HETE uptake on the release of GM-CSF and IL8 by HBEC was assessed. HBEC from control subjects were cultured for 24 hours with 12-HETE (10⁻⁹ to 10⁻⁷M) in the presence or absence of TNFα. Detectable amounts of both cytokines were released in the supernatant in basal conditions at 24hr, and TNFα increased significantly the release of GM-CSF. 12-HETE at 10⁻⁷M weakly but significantly decreased the TNF-induced release of GM-CSF from HBEC. Thus the uptake of 12-HETE could affect the epithelial cell function in some situations.     

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.     

Esterified, optical pure (3S, 3′S)-astaxanthin from flowers of Adonis annua

The quantitative carotenoid composition of the red flower petals of Adonis annua is reported. Optically pure (3S, 3′S)-astaxanthin occurs both as a diester (64% of total carotenoid) and as a monoester (11%). The optical purity was determined by hydrolysis of the natural esters in the absence of oxygen and subsequent HPLC analysis of the paren -ketol esterified with (−)-camphanic acid. All non-animal sources hitherto examined synthesize pure 3S,3′S- or 3R,3′R-isomers of astaxanthin, whereas marine animal sources contain mixtures of all three optical isomers, including the meso form.     

Microfluidic separation of (S)-ibuprofen using enzymatic reaction

This study was investigated for the enantioselective separation of (S)-ibuprofen using the ionic liquid in the microfluidic device. A stable and thin ionic liquid flow (ILF) was made by controlling the flow rate of the ILF in the microfluidic channel. In addition, coupling lipase as a biocatalyst with the ILF based on the microfluidic device showed the facilitative and selective transport of (S)-ibuprofen across the ILF, indicating successful optical resolution of a racemic mixture. Subsequently, the enantioselectivity was evaluated in the transport ratio (η) of (R)- and (S)-ibuprofen, the optical resolution ratio () and enantiomeric excess of (S)-ibuprofen (ee_S).     

Enzymatic formation and esterification of (S)-mandelonitrile

The (S)-selective hydroxynitrile lyase from Hevea brasiliensis (HbHNL) catalyzes the trans-cyanohydrin reaction (transcyanation). The equilibrium of this two-step reaction sequence is not favorable unless a large excess of acetone cyanohydrin (1) is used. Therefore, the coupling of this reaction with a follow-up reaction was investigated. It was established that the trans-cyanohydrin reaction could be performed in organic media, making it possible to couple it with a lipase-catalyzed acylation. Candida antarctica lipase B (CAL-B) shows a high selectivity (E=100) for (S)-mandelonitrile (4) and is, therefore, the ideal candidate for this type of multi-step one-pot reaction.     

Studies on the mechanism of formation of the 5S,12S-dihydroxy-6,8,10,14(E,Z,E,Z)-icosatetraenoic acid in leukocytes

Incubation of peripheral blood leukocytes with arachidonic acid (and ionophore A23187) led to the formation of leukotriene B₄, Δ⁶-trans-leukotriene B₄, Δ⁶-trans-12-epi-leukotriene B₄, 5-hydroxy-icosatetraenoic acid, 12-hydroxy-icosatetraenoic acid and of 5S,12S-dihydroxy-6,8,10,14-(E,Z,E,Z)-icosatetraenoic acid (5S,12S-DiHETE). Incubation of leukocytes with leukotriene A₄ resulted in the formation of leukotriene B₄ and of its two Δ⁶-trans-isomers but not of the 5S,12S-DiHETE. ¹⁸O₂ labeling experiments have shown that the hydroxyl groups at C5 and C12 in the 5S,12S-DiHETE are derived from molecular oxygen. The tetraacetylenic analog of arachidonic acid was found to be a potent inhibitor of the formation of the 5S,12S-DiHETE whereas it potentiated the synthesis of the 5-hydroxy acid and of leukotriene B₄. Addition of the 12-hydroxy-icosatetraenoic acid to leukocytes, or of the 5-hydroxy-icosatetraenoic acid to a suspension of platelets caused the formation of the 5S,12S-DiHETE. It is concluded that the 5S,12S-DiHETE is not derived from leukotriene A₄ but is a product of the successive reactions of arachidonic acid with two lipoxygenases of different positional specificities.     

Production of (S)-styrene oxide by recombinant Pichia pastoris containing epoxide hydrolase from Rhodotorula glutinis

A recombinant yeast Pichia pastoris carrying the gene encoding epoxide hydrolase (EH) of Rhodotorula glutinis was constructed and used for producing (S)-styrene oxide by enantioselective hydrolysis of racemic mixtures of styrene oxides. The EH gene was obtained by PCR amplification of cDNA of R. glutinis and integrated into the chromosomal DNA of P. pastoris to express EH under the control of AOX promoter. The recombinant yeast has a high hydrolytic activity toward (R)-styrene oxide as 358 nmol min⁻¹ (mg cell)⁻¹, which is about 10-fold higher than that of wild type R. glutinis. When kinetic resolution was conducted by the recombinant yeast at a high initial epoxides concentration of 526 mM that constitutes an epoxide–water two-liquid phase, chiral (S)-styrene oxide with an enantiomeric excess (e.e.) higher than 98% was obtained as 36% yield (theoretical, 50%) at 16 h.     

Lipase-catalyzed production of optically active (S)-flurbiprofen in aqueous phase reaction system containing chiral succinyl β-cyclodextrin

The lipase-catalyzed production of optically active (S)-flurbiprofen was carried out in a dispersion reaction-system induced by chiral succinyl β-cyclodextrin (suβ-CD). The optimal reaction conditions were 500 mM (R,S)-flurbiprofen ethyl ester ((R,S)-FEE), 600 units of Candida rugosa lipase per 1 mmol of (R,S)-FEE, and 1000 mM suβ-CD at 37 °C for 72 h. An extremely high enantiomeric excess of 0.98 and conversion yield of 0.48 were achieved in the dispersed aqueous phase reaction system containing chiral suβ-CD added as a dispenser and chiral selector. The inclusion complex formability of the immiscible substrate (S)- and (R)-form of FEE with suβ-CD was compared using a phase-solubility diagram, DSC, and ¹H NMR. (S)-Isomer formed a more stable and selective inclusion complex with chiral suβ-CD. It was hydrolyzed much more selectively by lipase from C. rugosa, due to the selective structural modification through inclusion complexation with chiral suβ-CD.     

Preparation of (2S, 3R)-methyl-3-phenylglycidate using whole cells of Pseudomonas putida

Preparation of (2S, 3R)-methyl 3-phenylglycidate via enantioselective hydrolysis of racemic phenylglycidate was carried out using whole cells of Pseudomonas putida. Under optimal conditions (2S, 3R)-methyl-3-phenylglycidate could be got with ee value 99 and 48% chemical yield.     

Uptake, release and metabolism of docosahexaenoic acid (DHA, c22:6 omega 3) in human platelets and neutrophils

Exogenous DHA is converted by human platelets to 14- and 11- HDHE and by human neutrophils mainly to 7- HDHE . Human platelets prelabeled with 14C-DHA, 14C-EPA and 14C-AA and stimulated with thrombin release and metabolize DHA only in trace amounts as compared to EPA and AA. 14C-DHA is incorporated into the 2-position of platelet phospholipids and occurs predominantly in phosphatidylethanolamine. DHA and EPA were also incorporated by dietary means into phospholipids of platelets and neutrophils. In resting platelets free DHA as well as free AA and EPA are not detectable. In platelets stimulated ex vivo with thrombin DHA is not significantly released which is in contrast to EPA and AA. After stimulation, 14- HDHE is found only in trace amounts as compared to 12-HETE and 12- HEPE . In DHA enriched neutrophils formation of HDHEs cannot be demonstrated after stimulation with ionophore A 23187. We conclude that even after dietary enrichment of DHA in phospholipids of platelets and neutrophils the level of free DHA and/or formation of HDHEs might be too low to substantially affect arachidonic acid metabolism and related functions of these cells.     

Enantioselective hydrolysis of (R,S)-naproxen 2,2,2-trifluoroethyl ester in water-saturated solvents via lipases from Carica pentagona Heilborn and Carica papaya

A crude lipase prepared from Carica pentagona Heilborn latex was explored as an effective enantioselective biocatalyst for the hydrolytic resolution of (R,S)-naproxen 2,2,2-trifluoroethyl ester in water-saturated organic solvents. Comparisons of the enzyme performance with that from Carica papaya lipase indicated that both lipases showed low tolerance to the hydrophilic solvent and were inhibited by (S)-naproxen and 2,2,2-trifluoroethanol. Improvements on the enzyme activity and enantioselectivty were demonstrated when both lipases in partially purified forms were employed. By using the thermodynamic analysis, the enantiomeric discrimination was mainly driven by the difference of activation enthalpy for all reaction systems except for employing Carica papaya lipase as the biocatalyst for (R,S)-fenoprofen 2,2,2-trifluoroethyl thioester.     

Inhibitory action of soluble elastin on thromboxane B2 formation in blood platelets

Soluble elastin, prepared from insoluble elastin by treatment with oxalic acid or elastase, was found to inhibit the formation of thromboxane B₂ both from [1-¹⁴C]arachidonic acid added to washed platelets and from [1-¹⁴C]arachidonic acid in prelabeled platelets on stimulation with thrombin. In both systems, the formation of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) was accelerated. Oxalic acid-treated soluble elastin st 1 and 10 mg/ml inhibited the formation of thromboxane B₂ from exogenously supplied arachidonic acid 21 and 59%, respectively, and the formation of thromboxane B₂ in prelabeled platelets stimulated by thrombin 44 and 94%, respectively. These concentrations of elastin increased the formation of 12-HETE from exogenously supplied arachidonic acid about 3.4- and 7.3-times, respectively. Almost all the added arachidonic acid was converted to metabolites. In prelabeled platelets, soluble elastin at 1 and 10 mg/ml increased the formation of 12-HETE stimulated by thrombin about 1.3- and 2.8-times, respectively, and inhibited the thrombin-induced total productions of thromboxane B₂ (12-hydroxy-5,8,10-heptadecatrienoic acid (12-HETE) and free arachidonic acid by 26 and 25%, respectively. Elastase-treated digested elastin also inhibited the formation of thromboxane B₂ and stimulated the formation of 12-HETE in prelabeled platelets stimulated by thrombin. This inhibitory action of elastin was not replaced by desmosine. The level of cAMP in platelets was not affected by soluble elastin. Soluble elastin was also found to inhibit platelet aggregation induced by thrombin. However, the inhibitory action of soluble elastin on platelet aggregation cannot be explained by inhibition of thromboxane B₂ formation by the elastin.     

Modulation of Mucor miehei lipase properties via directed immobilization on different hetero-functional epoxy resins: Hydrolytic resolution of (R,S)-2-butyroyl-2-phenylacetic acid

Purified lipase from Mucor miehei (MML) has been covalently immobilized on different epoxy resins (standard hydrophobic epoxy resins, epoxy-ethylenediamine, epoxy-iminodiacetic acid, epoxy-copper chelates) and adsorbed via interfacial activation on octadecyl-Sepabeads support (fully coated with very hydrophobic octadecyl groups). These immobilized enzyme preparations were used under slightly different conditions (temperature ranging from 4 to 25 °C and pH values from 5 to 7) in the hydrolytic resolution of (R,S)-2-butyroyl-2-phenylacetic acid.

Different catalytic properties (activity, specificity, enantioselectivity) were found depending on the particular support used. For example, the epoxy-iminodiacetic acid-Sepabeads gave the most active preparation at pH 7 while, at pH 5, the ethylenediamine-Sepabeads was superior.

More interestingly, the enantiomeric ratio (E) also depends strongly on the immobilized preparation and the conditions employed. Thus, the octadecyl-MML preparation was the only immobilized enzyme derivative which exhibited enantioselectivity towards R isomer (with E values ranging from 5 at 4 °C and pH 7 to 1.2 at pH 5 and 25 °C).

The other immobilized preparations, in contrast, were S selective. Immobilization on iminodiacetic acid-Sepabeads afforded the catalyst with the highest enantioselectivity (E=59 under optimum conditions).     

Fed-batch production of (S)-flurbiprofen in lipase-catalyzed dispersed aqueous phase reaction system induced by succinyl β-cyclodextrin and its extractive purification

Optically active (S)-flurbiprofen was produced fed-batch-wisely in a lipase-catalyzed dispersed aqueous phase reaction system induced by succinyl β-cyclodextrin (suβ-CD). A highly concentrated 480 mM (S)-flurbiprofen, corresponding to 117.0 g/l, with an enantiomeric excess of 0.98 and conversion yield of 0.48 was obtained. (S)-Flurbiprofen produced in an inclusion complex form with suβ-CD was extractively purified using three-step procedures: decomplexation of (S)-flurbiprofen and residual (R)-flurbiprofen ethyl ester ((R)-FEE) using the ethyl acetate, dissolution of (S)-flurbiprofen from (R)-FEE using a sodium bicarbonate solution, and selective precipitation of (S)-flurbiprofen using 2-propanol. Consequently, an extremely high concentration of 420 mM (S)-flurbiprofen with an optical purity higher than 98% was recovered after purification.     

Synthesis of novel ageladine A analogs showing more potent matrix metalloproteinase (MMP)-12 inhibitory activity than the natural product

By employing a previously established synthetic scheme, the synthesis described in the title was carried out in order to explore the substituent effects in the pyrrole ring of ageladine A on MMP-12 inhibitory activity. It became evident that a halogen atom (Br or Cl) at the 2-position and an additional bromine atom at the 4-position are highly effective for improving the inhibitory activity. These studies led us to discover three novel ageladine A analogs (4a, c, o) showing more potent MMP-12 inhibitory activity than the natural product.     

Design of novel analogues with potent antibiotic activity based on the antimicrobial peptide, HP(2-9)-ME(1-12)

To develop novel antibiotic peptides useful as therapeutic drugs, a number of analogues were designed to increase the hydrophobic helix region either by Trp-substitution or net positive charge increase by Lys-substitution, from HP(2-9)-ME(1-12). The antibiotic activities of these peptides were evaluated using bacterial (Salmonella tryphimurium, Proteus vulgaris, Bacillus subtilis and Staphylococcus aureus), fungi (Saccharomyces cerevisiae, Trichosporon beigelii and Candida albicans), tumor and human erythrocyte cells. The substitution of Lys for Thr at position 18 and 19 of HP(2-9)-ME(1-12) (HM5) increased activity against Proteus vulgaris and fungal strains without hemolysis. In contrast, substitution of Trp for Lys and Thr at positions 2, 15 and 19 of HP(2-9)-ME(1-12), respectively (HM3 and HM4), decreased activity but increased hemolysis against human erythrocytes. This suggests that an increase in positive charge increases antimicrobial activity whereas an increase in hydrophobicity by introducing Trp residues at C-terminus of HP(2-9)-ME(1-12) causes a hemolytic effect. Circular dichroism spectra suggested that the alpha-helical structure of these peptides plays an important role in their antibiotic effect but that the alpha-helical property is not connected with the enhanced antibiotic activity.     

Hydroxylated jasmonic acid and related compounds from Botryodiplodia theobromae

From the culture filtrate of the fungus Botryodiplodia theobromae five hydroxylated cyclopentane fatty acids of the jasmonic acid type were isolated and identified as (11 S -(-)-hydroxyjasmonic acid; (11R)-(-)-hydroxyjasmonic acid; (-)-12-hydroxyjasmonic acid; (-)-8ξ-hydroxyjasmonic acid; (-)-3-oxo-2-(1ξ-hydroxy-2Z-pentenyl)cyclopent-1-yl-butyric acid; (-)-3-oxo-2(4ξ-hydroxy-2Z-pentenyl)cyclopent-1-yl-butyric acid. In addition, the corresponding hydroxylated iso-jasmonic acid analogues were found as minor constituents. During silica gel chromatography 11,12-didehydrojasmonic acid, 11ξ-acetoxyjasmonic acid, 3-oxo-2-(4ξ-acetoxy-2Z-pentenyl)cyclopent-1-yl-butyric acid 3-oxo-2-(2Z,4-pentadienyl)cyclopent-1-yl-butyric acid were formed as artefacts.     

Growth inhibitory activity of extracts and compounds from Cimicifuga species on human breast cancer cells

The purpose of this report is to explore the growth inhibitory effect of extracts and compounds from black cohosh and related Cimicifuga species on human breast cancer cells and to determine the nature of the active components. Black cohosh fractions enriched for triterpene glycosides and purified components from black cohosh and related Asian species were tested for growth inhibition of the ER⁻ Her2 overexpressing human breast cancer cell line MDA-MB-453. Growth inhibitory activity was assayed using the Coulter Counter, MTT and colony formation assays.Results suggested that the growth inhibitory activity of black cohosh extracts appears to be related to their triterpene glycoside composition. The most potent Cimicifuga component tested was 25-acetyl-7,8-didehydrocimigenol 3-O-β-d-xylopyranoside, which has an acetyl group at position C-25. It had an IC₅₀ of 3.2 μg/ml (5 μM) compared to 7.2 μg/ml (12.1 μM) for the parent compound 7,8-didehydrocimigenol 3-O-β-d-xylopyranoside. Thus, the acetyl group at position C-25 enhances growth inhibitory activity.The purified triterpene glycoside actein (β-d-xylopyranoside), with an IC₅₀ equal to 5.7 μg/ml (8.4 μM), exhibited activity comparable to cimigenol 3-O-β-d-xyloside. MCF7 (ER⁺Her2 low) cells transfected for Her2 are more sensitive than the parental MCF7 cells to the growth inhibitory effects of actein from black cohosh, indicating that Her2 plays a role in the action of actein. The effect of actein on Her2 overexpressing MDA-MB-453 and MCF7 (ER⁺Her2 low) human breast cancer cells was examined by fluorescent microscopy. Treatment with actein altered the distribution of actin filaments and induced apoptosis in these cells.These findings, coupled with our previous evidence that treatment with the triterpene glycoside actein induced a stress response and apoptosis in human breast cancer cells, suggest that compounds from Cimicifuga species may be useful in the prevention and treatment of human breast cancer.     

(S)-雌马酚对肠道菌群的体外调控作用及其可代谢性研究

[目的]研究(S)-雌马酚对人体肠道菌群的体外调控作用和人体肠道菌群对(S)-雌马酚的代谢衍生作用。[方法]采用人体肠道菌群体外批量发酵、细菌16S rRNA基因高通量测序、气相色谱、液相色谱和质谱等检测(S)-雌马酚与人体肠道菌群体外相互作用。[结果]体外添加(S)-雌马酚对总体人肠道菌群结构和短链脂肪酸产量影响不明显。添加0.45 mmol/L (S)-雌马酚组与对照组相比,未检测到相对丰度发生显著变化的细菌;添加0.90 mmol/L (S)-雌马酚组与对照组相比,显著增加了肠杆菌科(Enterobacteriaceae)等条件致病菌的相对丰度,减少了潜在益生菌粪球菌属(Coprococcus)的比例。代谢分析发现,发酵培养液中(S)-雌马酚的浓度降低了约15%−30%,推测可能被微生物进一步降解或衍生修饰。[结论]从体外调控肠道菌群的角度判断,0.45 mmol/L (S)-雌马酚相对较安全,而0.90 mmol/L (S)-雌马酚可能会破坏肠道菌群平衡。(S)-雌马酚可以被人体肠道菌群进一步代谢,其特定代谢产物的结构与功能及其体内生物安全性有待进一步研究。