Identification of specific binding sites for leukotriene C4 in human fetal lung

Specific leukotriene C₄ (LTC₄) binding sites were identified in membrane preparations from human fetal lung. Specific binding of [³H]-LTC₄ represented 95 percent of total binding, reached steadystate within 10 minutes and was rapidly reversible upon addition of excess unlabeled LTC₄. Binding assays were performed at 4°C under conditions which prevented metabolism of [³H]-LTC₄ (80 mM serineborate, 10 mM cysteine, 10 mM glycine). Under these conditions, greater than 95 percent of the membrane bound radioactivity, as analyzed by high performance liquid chromatography, co-eluted with the LTC₄ standard. Computer-assisted analyses of saturation binding data showed a single class of binding sites with a dissociation constant (K_d) of 26 + 6 nM and a density (B_max) of 84 ± 18 pmol/mg protein. Pharmacological specificity was demonstrated by competition studies in which specific binding of [³H]-LTC₄ was displaced by LTC₄ and its structural analogs with inhibition constants (K_j) of 10 to 30 nM, whereas LTD₄, diastereoisomers of LTD₁, LTE₄ and the end organ antagonist FPL 55712 were 150 to 700 fold less potent competitors than LTC₄. These results provide evidence for specific, reversible, saturable, high affinity binding sites for [³H]-LTC₄ in human fetal lung membranes.     

Studies on the Mechanism of Strengthening Fish Gel by Basic Amino Acids

An electro-energizing fermentation (E-E F) method has been developed. In this method, a direct electrical current is applied to a microbial culture to accelerate the reductive metabolism of microorganisms or to impart profitable effects to microbial cells. This E-E F method was applied to l-glutamic acid fermentation by Brevibacterium flavum No. 2247. When glucose was used as a substrate, the addition of 0.01 mm neutral red (NR), redox dye (electron carrier), to the fermentation broth at the beginning of cultivation was effective for l-glutamate (l-Glu) production. A direct current of 200~300 μA/cm² at 1.5 V was applied through out the cultivation of this bacterium. This resulted in about a 10% increase in yield of l-Glu.     

Identification of the sex pheromone of Ostrinia palustralis

By means of gas chromatography with electroantennographic detection, gas chromatography-mass spectrometry and a series of bioassays, (E)-11-tetradecenyl acetate (E11-14:OAc) and (Z)-11-tetradecenyl acetate (Z11-14:OAc) at a ratio of 99:1 were identified as female sex pheromone components of Ostrinia palustralis. The average amounts of E11- 14:OAc and Z11-14:OAc in a single sex pheromone gland were 37.2±24.4 ng and 0.3±0.2 ng, respectively. In a wind-tunnel bioassay, the binary blend of E11- and Z11-14:OAc elicited the same male behavioral responses as did virgin females.     

Facile syntheses for (5Z,9Z)-5,9-hexadecadienoic acid, (5Z,9Z)-5,9-nonadecadienoic acid, and (5Z,9Z)-5,9-eicosadienoic acid through a common synthetic route

The delta 5,9 fatty acids (5Z,9Z)-5,9-hexadecadienoic acid, (5Z,9Z)-5,9-nonadecadienoic acid, and (5Z,9Z)-5,9-eicosadienoic acid were synthesized for the first time in four steps (9-12% overall yield) starting from commercially available 2-(2-bromoethyl)-1,3-dioxolane. The synthetic approach provided enough material to corroborate the structure and stereochemistry of (5Z,9Z)-5,9-nonadecadienoic acid which was recently identified in the flowers of Malvaviscus arboreus (Malvaceae). The novel phospholipids 1-hexadecanoyl-2-[(5Z,9Z)-5,9-eicosadienoyl]-sn-glycer o-3-phosphocholine and 1-octadecanoyl-2-[(5Z,9Z)-5,9-eicosadienoyl]-sn- glycero-3-phosphocholine were also synthesized from commercially available L-alpha-phosphatidylcholine (egg yolk) and characterized by positive ion electrospray mass spectrometry. These are the first examples of unsymmetrical phospholipids with saturated fatty acids at the sn-1 position and delta 5,9 fatty acids at the sn-2 position.     

Structural basis for pH-dependent alterations of reaction specificity of vertebrate lipoxygenase isoforms

Lipoxygenases have been classified according to their specificity of fatty acid oxygenation and for several plant enzymes pH-dependent alterations in the product patterns have been reported. Assuming that the biological role of mammalian lipoxygenases is based on the formation of specific reaction products, pH-dependent alterations would impact enzymes' functionality. In this study we systematically investigated the pH-dependence of vertebrate lipoxygenases and observed a remarkable stability of the product pattern in the near physiological range for the wild-type enzyme species. Site-directed mutagenesis of selected amino acids and alterations in the substrate concentrations induced a more pronounced pH-dependence of the reaction specificity. For instance, for the V603H mutant of the human 15-lipoxygenase-2 8-lipoxygenation was dominant at acidic pH (65%) whereas 15-H(p)ETE was the major oxygenation product at pH 8. Similarly, the product pattern of the wild-type mouse 8-lipoxygenase was hardly altered in the near physiological pH range but H604F exchange induced strong pH-dependent alterations in the positional specificity. Taken together, our data suggest that the reaction specificities of wild-type vertebrate lipoxygenase isoforms are largely resistant towards pH alterations. However, we found that changes in the assay conditions (low substrate concentration) and introduction/removal of a critical histidine at the active site impact the pH-dependence of reaction specificity for some lipoxygenase isoforms.     

Role of Arg403 for thermostability and catalytic activity of rabbit 12/15-lipoxygenase

12/15-Lipoxygenases (12/15-LOX) have been implicated in inflammatory and hyperproliferative diseases but the numerous aspects of structural biology of these enzymes are far from clear. Early mutagenesis data and structural modeling of enzyme–substrate complexes suggested that Arg403, which is localized at the entrance of the putative substrate binding pocket, might interact with the fatty acid carboxylic group. On the other hand, side-chain of Arg403 is a part of an ionic network with the residues of α2-helix, which undergoes pronounced conformation changes upon inhibitor binding. To explore the role of Arg403 for catalysis in more detail we exchanged positively charged Arg403 to neutral Leu and quantified structural and functional consequences of the alteration at the site of mutation using fluorometric techniques. We found that a loss of electrostatic interaction between Arg403 and negatively charged amino acid residues of α2-helix has only minor impact on protein folding, but partially destabilized the tertiary structure of the enzyme. We hypothesize that interaction of Arg403 with the substrate's carboxylate might be involved in a complex mechanism triggering conformational changes of the α2-helix, which are required for formation of the catalytically competent dimer r12/15-LOX complex at pre-catalytic stages.     

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 and Characterization of a Pressure-induced Gel from Freeze-concentrated Milk

A process was developed to produce a characteristic milk gel. Raw and market milk samples were freeze-concentrated using bacterial ice nuclei. The concentrates were kept at 5°C and compressed at 300–600 MPa for 5 min. The combination of the freeze concentration and the pressurization gave a milk gel without adding any gelling agents. The addition of sugar at 10% to the concentrated milk improved its gel strength and viscoelasticity. The gel was characterized by a phase transition at about 62–75°C.     

A rapid miniature thin layer chromatography system for analysis of prostaglandins an lipoxygenase products

We have developed a miniature thin layer chromatography system for rapidly identifying the major arachidonate metabolites in a radiolabeled form elaborated by cells or tissues. This system separates the total spectrum of cyclooxygenase products and several of the most commonly found lipoxygenase pathway metabolites, while retaining the fine resolution of larger-scale and more time-consuming procedures. It requires less than one hour for extraction of metabolites, chromatography, and counting.     

赤松毛虫性信息素次要组分的鉴定——组分、电生理和田间效果

用带有极性和非极性毛细柱的气相色谱(GC)分析赤松毛虫Dendrolimus spectabilis性信息素腺体提取物,发现腺体中除含有已鉴定的性信息素(顺,反)-5,7-十二碳二烯醇(Z5,E7-12∶OH)外,还有微量的(顺,反)-5,7-十二碳二烯醛(Z5,E7-12∶Ald)和(顺,反)-5,7-十二碳二烯乙酸酯(Z5,E7-12∶Oac),三种成分以100∶5-6±5-4∶3-2±1-8的比例存在。使用气相色谱-质谱选择性离子检测法（GC-MS-SIM）分析赤松毛虫腺体提取物,发现腺体中确实含有微量的Z5,E7-12∶Oac和痕量的Z5,E7-12∶Opr。赤松毛虫腺体提取物的气相色谱和触角电位检测联用（GC-EAD）分析发现只有Z5,E7-12∶OH能激起EAD反应,然而使用较高剂量的标准化合物进行GC-EAD分析发现Z5,E7-12∶OH、Z5,E7 12∶Oac和(顺,反)-5,7-十二碳二烯丙酸酯(Z5,E7-12∶Opr)均能刺激起EAD反应,而Z5,E7-12∶Ald则不能。触角电位（EAG）剂量-反应关系研究表明,当剂量变化范围在0.01～1 μg时,雄虫触角对Z5,E7-12∶OH最敏感,对Z5,E7-12∶Oac和Z5,E7-12∶Opr次之。田间试验表明,由Z5,E7-12∶OH, Z5,E7-12∶Oac和Z5,E7-12∶Opr配制的三组分诱芯,其诱蛾量显著高于由Z5,E7-12∶OH组成的单组分或是它与其乙酸酯或丙酸酯组成的两组分诱芯,当Z5,E7-12∶OH,Z5,E7-12∶Oac和Z5,E7-12∶Opr的比例为100∶3∶25时,诱蛾效果最佳。在上述三组分混合物中加入一定量的Z5,E7-12∶Ald,则对诱蛾有明显的抑制作用。上述事实表明,Z5,E7-12∶Oac和Z5,E7-12∶Opr是赤松毛虫性信息素的两种次要组分,而Z5,E7-12∶Ald则是信息素的抑制剂。     

New Amides from Buckwheat Seeds (Fagopyrum esculentum Moench)

Two new amino acid amides which yield in acid hydrolysis isomeric hydroxybenzylamines and amino acids have been isolated from the achenes of Fagopyrum esculentum Moench. One of them called BN-II is composed of salicylamine and allo-4-hydroxy-l-glutamic acid, and the other, BN-III, p-hydroxybenzylamine and l-glutamic acid. These coupled compounds link one another to form an amide respectively. Finally the structures of BN-II and BN-III were determined to be N⁵-(2′-hydroxybenzyl)-allo-4-hydroxy-l-glutamine and N⁵-(4′-hydroxybenzyl)-l-glutamine respectively from their chemical and spectrometry properties.     

Novel derivatives of chitosan and their antifungal activities in vitro

Three kinds of Schiff bases of carboxymethyl chitosan (CMCTS) were prepared, and their antifungal activities were assessed according to Jasso de Rodríguez's method. The results indicated that 2-(2-hydroxybenzylideneamino)-6-carboxymethylchitosan (HNCMCTS) and 2-(5-chloro-2-hydroxybenzylideneamino)-6-carboxymethylchitosan (HCCMCTS) had better inhibitory effects than those of chitosan or CMCTS against Fusarium oxysporium f. sp. vasinfectum, Alternaria solani, and Valsa mali.     

Identification and functional characterization of sex pheromone receptors in beet armyworm Spodoptera exigua (Hübner)

In moths, males can detect a distinct blend of several pheromone components by specialized olfactory receptor neurons (ORNs) on the antennae. Four candidate pheromone receptors (PR) with seven transmembrane domains were identified by homology cloning from the antennae of Spodoptera exigua (Sexi). Phylogenetic analyses reveal that all four odorant receptors (OR) belong to pheromone receptor subtypes. Expression patterns revealed that PRs were male-specific in the antenna except for SexiOR11, which was female antenna-biased. Functional analyses of these PRs were conducted using heterologous expression in Xenopus oocytes. SexiOR13 and SexiOR16 were all broadly activated by multiple pheromone components. SexiOR13 responded robustly to the critical pheromone component, Z9, E12-14:OAc and the minor pheromone component, Z9-14:OAc at a concentration of 10^?4 M. Dose-response studies indicate that SexiOR13 was approximately 4 times more sensitive to Z9,E12-14:OAc (EC50 = 3.158 × 10^?6 M) compared to Z9-14:OAc (EC50 = 1.203 × 10^?5 M). While, SexiOR16 responded robustly to the secondary pheromone component Z9-14:OH with high sensitivity (EC50 = 9.690 × 10^?7 M). However, similar tests of the five pheromones with SexiOR6 and SexiOR11 failed to elicit any response. These results provide basic knowledge to further advance research on the molecular mechanisms of pheromone reception.     

Inhibition of human neutrophil arachidonate 5-lipoxygenase by 6, 9-deepoxy-6, 9-(phenylimino)- Δ 6, 8-prostaglandin I1 (U-60257)

6, 9-Deepoxy-6, 9-(phenylimino)-Δ 6, 8-prostaglandin I₁ (U-60257) and its methyl ester (U-56467) are selective inhibitors of leukotriene C and D biosynthesis both $\text{in}$$\text{vitro}$ and $\text{in vivo}$. In this study, we demonstrated that the principal site of inhibition may be arachidonate 5-lipoxygenase, the initial enzyme of leukotriene biosynthesis. U-60257 and its methyl ester block LTB₄ synthesis in human peripheral neutrophils with an ID₅₀ of 1.8 and 0.42 μM respectively. This inhibitory action of U-60257 on neutrophil 5-lipoxygenase can be reduced or reversed by a high concentration of exogenous arachidonic acid. U-60257 at 100 μM has no apparent effect on the following enzymes. 1) cyclooxygenase of sheep vesicular gland or human platelets; 2) 12-lipoxygenase of human platelets and 3) soybean 15-lipoxygenase. Thus, we conclude that U-60257 and its methyl ester potent and selective inhibitors of arachidonate 5-lipoxygenase.     

Degradation of 1,3-dichloropropene by a soil bacterial consortium and Rhodococcus sp. AS2C isolated from the consortium

A bacterial consortium capable of degrading the fumigant 1,3-D ((Z)- and (E)-1,3-dichloropropene) was enriched from an enhanced soil. This mixedculture degraded (Z)- and (E)-1,3-D only in the presence of a suitable biodegradable organic substrate, such as tryptone, tryptophan, or alanine. After 8 months of subculturing at 2- to 3-week intervals, a strain of Rhodococcus sp. (AS2C) that was capable of degrading 1,3-D cometabolically in the presenceof a suitable second substrate was isolated. (Z)-3-chloroallyl alcohol (3-CAA) and (Z)-3-chloroacrylic acid (3-CAAC), and (E)-3-CAA and (E)-3-CAAC were the metabolites of (Z)- and (E)-1,3-D, respectively. (E)-1,3-D was degraded faster than (Z)-1,3-D by the strain AS2C and the consortium. AS2C also degraded (E)-3-CAA faster than (Z)-3-CAA. Isomerization of (E)-1,3-D to (Z)-1,3-D orthe (Z) form to the (E) form did not occur.     

Sex pheromone analysis and effective attraction of males of the cabbage webworm,Hellula undalis,inhabiting the Mekong Delta of Vietnam

Hellula undalis is a harmful insect pest of green mustard in the Mekong Delta of Vietnam. In order to establish a tool for a sustainable pest control program, the sex pheromone of H. undalis inhabiting the Mekong Delta was examined. GC-EAD and GC–MS analyses of pheromone gland extracts from the virgin females elucidated three new components, (Z)-11-tetradecenyl acetate (Z11-14:OAc), (Z)-11-hexadecenal (Z11-16:Ald), and (11E,13E)-11,13-hexadecadien-1-ol, in addition to the known pheromone component (11E,13E)-11,13-hexadecadienal (E11,E13-16:Ald). Double bond positions of the two monoenyl components were determined by GC–MS analysis of the pheromone extract treated with dimethyl disulfide. On the other hand, GC–MS analysis of the female body extract detected the unsaturated hydrocarbon (3Z,6Z,9Z)-3,6,9-tricosatriene (Z3,Z6,Z9-23:H). Field examinations of their synthetic compounds indicated the significant role of E11,E13-16:Ald as a major component and a clear synergistic effect of the two monoenyl compounds as a minor component. Although the 3:3:7 mixture of Z11-14:OAc, E11-16:Ald, and E11,E13-16:Ald captured the largest number of males among the tested mixtures, the activity was still quite a bit lower than that of virgin females. However, the 3:3:7:1 mixture, which was prepared by adding a small amount of Z3,Z6,Z9-23:H to the 3:3:7 ternary lure, succeeded in attracting males more powerfully than the females did. This strong synergistic effect was not observed when the triene was added to unmixed E11,E13-16:Ald, indicating important roles of not only the triene but also the two monoenyl compounds as natural pheromone components.     

Phosphorylation mimicking mutations of ALOX5 orthologs of different vertebrates do not alter reaction specificities of the enzymes

5-Lipoxygenase (ALOX5) plays a key role in the biosynthesis of pro-inflammatory leukotrienes whereas 15-lipoxygenases (ALOX15) have been implicated in the formation of pro-resolving eicosanoids (lipoxins, resolvins). Recently, it has been suggested that a phosphorylation mimicking mutant (Ser663Asp) of a stabilized variant of human ALOX5 exhibits dominant arachidonic acid 15-lipoxygenase activity (> 95%). To test whether similar alterations in the reaction specificity can also be observed for ALOX5 orthologs of other species we expressed wildtype and phosphorylation mimicking mutants (Ser271Asp, Ser523Asp, Ser663Asp, Ser663Glu) of human, mouse and zebrafish ALOX5 in pro- and eukaryotic overexpression systems and characterized their reaction specificities. We found that neither of the phosphorylation mimicking mutants produced significant amounts of 15-hydroperoxyeicosatetraenoic acid and the 5-lipoxygenation/15-lipoxygenation ratio for all wildtype and mutant enzyme species was lower than 100:2. Taken together, this data suggest that phosphorylation of native ALOX5 orthologs of different vertebrates may not induce major alterations in the reaction specificity and thus may not inverse their biological activity.     

Sex pheromone components of the leafroller moth Pandemis cerasana

Female-tip washings of the leafroller moth, Pandemis cerasana, were found to contain (E)-11-tetradecenyl acetate, (Z)-11-tetradecenyl acetate, (E)-11-tetradecenol, (Z)-11-tetradecenol and tetradecyl acetate, based on chemical analysis and electroantennogram tests. The relative amounts of these compounds in the gland were ca. 64:21:10:3:2 in the order named. Only (E)-11 and (Z)-11-tetradecenyl acetate were required for attraction of males to trap dispensed in the ratio 3:1, respectively.     

A two-component sex attractant for males of the geometrid moth Alsophila quadripunctata

The binary mixture of the hydrocarbons (3Z,6Z,9Z)-3,6,9-nonadecatriene and (6Z,9Z)-6,9-nonadecadiene is a sex attractant for males of Alsophila quadripunctata Esp. (Lepidoptera, Geometridae). Triene/diene ratios from 1:1 to 1:0.01 attracted males in the field, the ratios 1:1 and 1:0.7 were most effective. The triene on its own also attracted males, but in significantly lower numbers than the 1:1 mixture. In a dosage test the highest dose, 1800 g of 1:0.8 [triene:diene] mixture applied to rubber caps was most effective. This is the first report of a geometrid sex attractant with field activity containing more than one component.

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Zusammenfassung Die binäre Mischung der Kohlenwasserstoffe (3Z,6Z,9Z)-3,6,9-Nonadecatrien [ZZZ3,6,9-19:H] und (6Z,9Z)-6,9-Nonadecadien [ZZ6,9-19:H] in den Verhältnissen 1:1 bis 1:0.01 erwies sich im Freiland als Sexuallockstoff für Männchen von Alsophila quadripunctata (Geometridae, Lepidoptera). Dabei besaß die 1:1-Mischung die beste Fängigkeit, das Nonadecatrien allein lockte schlechter, während das C₁₉-Alkadien unwirksam war. Ein Dosiswirksamkeitstest ergab bei Köderbeladungen von 1.8 mg (Trien:Dien + 1:0.8) die besten Fangergebnisse. Im Verlauf der Untersuchungen wurden weitere zwölf geometrische-oder positionsisomere C₁₉-Polyenkohlenwasserstoffe getestet, wobei keine der geprüften Verbindungen Attraktant-oder Inhibitorwirkung zeigte.

     

Synthesis of (3E, 5Z)-3,5-Dodecadienylacetate,the Sex Pheromone of Phtheochroa cranaodes (Lepidoptera: Tortricidae)

(3E, 5Z)-3,5-Dodecadienyl acetate, the female sex pheromone of Phtheochroa cranaodes, was regio and stereo-selectively synthesized from 1-octyne and (E)-4-bromo-3-buten-1-ol by using Pd(PPh₃)₄, CuI and piperidine to afford the enyne (5). Further elaboration afforded the target pheromone. The synthetic pheromone was identified with the natural product by its MS and IR, data GLC retention time and biological activity.