猫薄荷精油有效成分的提取及其对白纹伊蚊、淡色库蚊的驱避活性

采用同时蒸馏萃取及硅胶柱层析法从猫薄荷Nepeta cataria 植物中提取、分离制得驱蚊有效成分假荆芥内酯,并采用个体涂肤有效保护时间测定法及风洞空间驱避效果测试法, 对假荆芥内酯与避蚊胺(DEET)的驱蚊效果进行了对比试验。结果表明,400 g新鲜猫薄荷植株可制得精油5.6 g,其中假荆芥内酯含量为55%,Z,E-假荆芥内酯32%, E,Z-假荆芥内酯23%。室内标准药效评价结果表明,DEET对白纹伊蚊Aedes albopictus 及淡色库蚊Culex pipiens pallens有效保护时间分别为9.6±0.2 h与11.5±1.5 h,而假荆芥内酯仅为2.2±0.2 h与4.3±0.8 h。风洞定向行为测试结果表明,假荆芥内酯单独作用时,对蚊虫有微弱的引诱效果,但在人体气味及二氯甲烷L-乳酸存在时,可显著抑制这些引诱物对蚊虫的引诱效果,是优良的空间驱避剂;DEET对蚊虫无明显的引诱作用,对二氯甲烷L-乳酸溶液引诱剂也无明显的抑制效果,但可显著增强人体气味对蚊虫的引诱性。     

Quantification of nepetalactones in catnip (Nepeta cataria L.) by HPLC coupled with ultraviolet and mass spectrometric detection

Nepetalactones, the major chemical components of catnip (Nepeta cataria L.), were analysed by reversed-phase HPLC coupled with UV and MS detection. Two major nepetalactones, Z,E-nepetalactone and E,Z-nepetalactone, were successfully identified and quantified. The linearity range for Z,E-nepetalactone was determined as 0.00655-0.655 mg/mL with a correlation coefficient of 0.9999, and the linearity range of E,Z-nepetalactone was found to be 0.00228-0.456 mg/mL with a correlation coefficient of 0.9999, under UV detection at 228 nm. The linearity ranges were from 0.00164 to 0.0328 mg/mL, with a correlation coefficient of 0.9999, for ZE-nepetalactone and 0.00114-0.0228 mg/mL, with a correlation coefficient of 0.9999, for E,Z-nepetalactone by MS detection with selected ion monitoring of ion peak m/z 167. The MS detection was found to be more sensitive than UV detection and this method was validated as simple, reliable and sensitive for catnip nepetalactone analysis. This method can be used for identification and fingerprinting of catnip products.     

Catnip essential oil as a barrier to subterranean termites (Isoptera: Rhinotermitidae) in the laboratory

The essential oil of catnip, Nepeta cataria (Lamiacae) was evaluated for behavioral effects on two populations of subterranean termite, Reticulitermes flavipes (Kollar) and R. virginicus (Banks) (Isoptera: Rhinotermitidae). The catnip essential oil contained approximately 36:64 E,Z-nepetalactone and Z,E-nepetalactone, respectively. The time to 50% dissipation (DT50) of the isomers in sand was dependent on dose, and ranged from 5.7 to 12.6 d for the E,Z-isomer and 7.7-18.6 d for the Z,E-isomer. For R. flavipes, the 24-h topical LD50 value was approximately 8200 microg/g termite. The 24-h fumigation LC50 value for R. flavipes was between 36 and 73 microg/ml air, and the 7-d fumigation LC50 value was between 14 and 36 microg/ml air. Exposure of R. virginicus to treated sand resulted in a 24-h LC50 value (95% F.L.) of 84 (67.6, 112) microg/cm2 and a 7-d LC50 value of 21.1 (16.4, 26.8) microg/cm2; for R. flavipes these values were 63.2 (53.7, 73.9) and 44.4 (34.6, 58.1) microg/cm2, respectively. Vertical tunneling through treated sand was eliminated at 500 ppm for R. virginicus and at 250 ppm for R. flavipes. Horizontal tunneling was stopped at 250 ppm for R. virginicus and reduced at doses above 250 ppm for R. flavipes. Although tunneling ceased in these tests, mortality was not high, indicating that the termites avoided the treated sand. Efficacy of catnip oil was equivalent to other monoterpenoids reported in the literature.     

Flight and molecular modeling study on the response of codling moth, Cydia pomonella (Lepidoptera: Tortricidae) to (E,E)-8,10-dodecadien-1-ol and its geometrical isomers

In a previous study we have reported that both (E,Z)-8,10-dodecadienol (E,Z) and (Z,Z)-8,10-dodecadienol (Z,Z) isomers inhibit the attraction of male codling moth, Cydia pomonella L. when added to (E,E)-8,10-dodecadienol (E,E) while the (Z,E)-8,10-dodecadienol (Z,E) isomer induces slight increase in the number of males attracted to the pheromone source. In the present study, we have tested the behavioral activity of the individual geometrical isomers E,Z; Z,E and Z,Z. A few number of codling moth males flew to the Z,E-isomer while the other two isomers (i.e. E,Z and Z,Z) did not elicit any upwind orientation. Analysis of the flight behavior to the E,E- and Z,E-isomer showed significant differences in most of the flight parameters evaluated. Based on the biological observations and molecular modeling, we suggest that the behavioral activity of the Z,E-isomer is due to presence of specific receptors for this isomer on male antennae and not to its structural resemblance to the E,E-isomer. These results underline the importance of the Z,E-isomer in sex attraction of male codling moth.     

Insecticidal and Repellent Activities of the Essential Oil of Callistemon citrinus (Myrtaceae) Against Callosobruchus maculatus (F.) (Coleoptera: Bruchidae)

The essential oil of Callistemon citrinus (Curtis) leaves was extracted by hydro distillation and tested on female and male adults of Callosobruchus macullatus (F.) for insecticidal and repellent effects. GC-MS analysis was used to identify and quantify the volatile composition of the essential oil. Results showed that 1,8-cineole (34.2%) and α-pinene (29.0%) were the major components of the oil. Callistemon citrinus oil was found to be toxic to adult insects when applied by fumigation. Responses varied according to the gender of the insect and exposure time. LC₅₀ values were 12.88 and 84.4 μL.?L^?1 for males and females, respectively. An increase in exposure time from 3 to 24 h caused an increase in mortality from 50% to 100% in males and from 15.5% to 85.2% in females, at the highest concentration (500 μL.?L^?1). The essential oil also had a repellent effect against C. macullatus in a filter paper arena test. After 2 and 4 h, 86% and 94%, respectively, repellent effects were demonstrated at the highest concentration of 0.4 μL?.cm^?2. These observations suggest that C. citrinus essential oil may be usefully applied to control storage pests.     

Locomotor Behaviour of Blattella germanica Modified by DEET

N,N-diethyl-3-methylbenzamide (DEET) is the active principle of most insect repellents used worldwide. However, its toxicity on insects has not been widely studied. The aim of this work is to study the effects of DEET on the locomotor activity of Blattella germanica. DEET has a dose-dependent repellent activity on B. germanica. Locomotor activity was significantly lower when insects were pre-exposed to 700 µg/cm² of DEET for 20 or 30 minutes, but it did not change when pre-exposure was shorter. Locomotor activity of insects that were pre-exposed to 2.000 µg/cm² of DEET for 10 minutes was significantly lower than the movement registered in controls. No differences were observed when insects were pre-exposed to lower concentrations of DEET. A 30-minute pre-exposure to 700 µg/cm² of DEET caused a significant decrease in locomotor activity. Movement was totally recovered 24 h later. The locomotor activity measured during the exposure to different concentrations of DEET remained unchanged. Insects with decreased locomotor activity were repelled to the same extent than control insects by the same concentration of DEET. We demonstrated that the repellency and modification of locomotor activity elicited by DEET are non-associated phenomena. We also suggested that the reduction in locomotor activity indicates toxicity of DEET, probably to insect nervous system.     

Repellency of essential oils and their components to the human body louse, Pediculus humanus humanus

Five essential oils and nine of their components were compared to diethyl toluamide (DEET) for their repellent activity against the human body louse, Pediculus humanus humanus. The absolute or intrinsic repellency of the compounds was tested by applying the repellent to corduroy patches and comparing them with untreated patches. It was found that the most effective repellents were DEET and citronella, whose activity lasted at least 29 days. The activity of rosemary lasted at least 18 days and that of eucalyptus more than 8 days. The repellent activity of the oil components such as citronellal and geraniol lasted more than 15 and 8 days, respectively. DEET remained effective at a dilution of 1:32, geraniol at 1:8, citronella at 1:4 and rosemary and citronellal at 1:1. The comparative or standard repellency of the candidate repellents was examined with the aid of a new screening technique using hairs treated with ammonium bicarbonate which is attractive to lice. Using this technique it could be shown that the repellent activity of citronella and geraniol lasted 2 days and that of rosemary and citronellal for only one day. DEET was active for less than one day. Serial dilutions of these substances also revealed that citronella was the most potent repellent for lice, followed by citronellal, rosemary, geraniol and DEET. The differences however, were not significant.     

Bioactivity of essential oil of Litsea cubeba from China and its main compounds against two stored product insects

During our screening program for agrochemicals from Chinese medicinal herbs and wild plants, the essential oil of Litsea cubeba fruits was found to possess strong contact toxicity against the cigarette beetle Lasioderma serricorne adults and the booklouse Liposcelis bostrychophila, with LD₅₀ values of 27.33 μg/adult and 71.56 μg/cm², respectively, and also showed strong fumigant toxicity against the two stored product insects with LC₅₀ values of 22.97 and 0.73 mg/L, respectively. The essential oil obtained by hydrodistillation was investigated by GC MS. The main components of the essential oil were identified to be E-citral (geranial) (27.49%), Z-citral (neral) (23.57%) and d-limonene (18.82%) followed by β-thujene (3.34%), β-pinene (2.85%), α-pinene (2.57%), 6-methyl-5-hepten-2-one (2.40%) and linalool (2.36%). Citral (Z/E-citral), d-limonene, β-pinene, α-pinene and linalool were separated and purified by silica gel column chromatography and preparative thin layer chromatography, and further identified by means of physicochemical and spectrometric analysis. Citral and linalool showed strong contact toxicity against L. serricorne and L. bostrychophila (LD₅₀ = 11.76, 12.74 μg/adult and 20.15, 99.97 μg/cm², respectively) and fumigant toxicity against L. serricorne and L. bostrychophila (16.54, 18.04 mg/L air and 0.14, 0.71 mg/L air, respectively). Otherwise, citral, d-limonene and linalool were strongly repellent against the cigarette beetle L. serricorne as the essential oil whereas β-pinene and α-pinene exhibited weaker repellency against the cigarette beetle compared with the positive control, DEET. Moreover, except α-pinene and linalool, the other three compounds as well as the essential oil exhibited comparable repellency against the booklouse relative to DEET.     

Chemical Constituents and Activities of the Essential Oil from Myristica fragrans against Cigarette Beetle Lasioderma serricorne

Essential oil extracted from nutmeg seeds (Myristica fragrans Houtt .) by hydrodistillation was subjected to GC/MS and GC analysis. A total of 27 constituents were identified, of which eugenol (19.9%), methylisoeugenol (16.8%), methyleugenol (16.7%), sabinene (11.8%), and terpinen‐4‐ol (8.5%) were the major components. The essential oil was tested against Lasioderma serricorne for insecticidal and repellent activity, the LD₅₀ value at the end of 24 h exposure period was 19.3 μg/adult. Six active compounds were isolated by bioassay‐guided fractionation. They were identified as eugenol ( 1 ), methyleugenol ( 2 ), methylisoeugenol ( 3 ), elemicin ( 4 ), myristicin ( 5 ), and safrole ( 6 ). Among these isolates, 4 showed the strongest contact toxicity against L. serricorne adults with an LD₅₀ value of 9.8 μg/adult. Repellency of crude oil and active compounds were also determined. Compounds 1, 2, 4 , and 5 were strongly repellent against the cigarette beetle and exhibited the same level of repellency compared with the positive control, DEET. The results indicate that the essential oil of M. fragrans and its active constituents have potential for development as natural insecticides and repellents to control L. serricorne.     

Composition of the essential oil of Lepidium meyenii (Walp)

The essential oil profile of maca (Lepidium meyenii) obtained from Lima, Peru, was examined. Steam distillates of the aerial parts of L. meyenii were continuously extracted with pentane and the pentane extracts analyzed by GC/MS. Retention indices and mass spectral data were used to identify 53 oil components. Phenyl acetonitrile (85.9%), benzaldehyde (3.1%), and 3-methoxyphenylacetonitrile (2.1%) were the major components of the steam distilled oil. The oil of L. meyenii was tested for phytotoxic, cyanobactericidal, and antitermite activity. The oil was selectively toxic towards the cyanobacterium Oscillatoria perornata compared to the green alga Selenastrum capricornutum, with complete growth inhibition at 100 microg/ml. Mortality of the Formosan subterranean termite, Coptotermes formosanus, was numerically, but not significantly, higher when held on filter paper treated with maca oil. At 1% (w/w), maca oil also appeared to act as a feeding deterrent to termites. Several minor components of the essential oil of maca including 3-methoxyphenylacetonitrile and benzylthiocyanate were significantly active against the Formosan termite. This is the first report on the essential oil composition of L. meyenii.     

41种植物精油对淡色库蚊的熏蒸活性

为发现具有较高杀蚊活性的精油, 推动植物精油类卫生杀虫剂的创制, 本研究采用三角瓶熏蒸法测定了17科41种植物精油对淡色库蚊Culex pipiens pallens雌成蚊的熏蒸活性, 并进一步采用密闭圆筒法验证其杀虫毒力大小。三角瓶熏蒸法测定表明, 在12 μL/L浓度下, 留兰香油（spearmint oil）、 薰衣草油（lavender oil）、 山苍子油（Litsea cubeba oil）等26种精油均有较强的熏蒸作用, KT₅₀小于15 min。该26种精油用密闭圆筒熏蒸法进一步测定表明, 在浓度为10 μL/L时, 冬青油（wintergreen oil）、 艾叶油（blumea oil）、 薄荷油（peppermint oil）、 桉叶油（Eucalyptus robusta oil）和蓝桉油（E. globulus oil）等植物精油熏蒸活性较强, 其KT₅₀分别为16.91, 21.20, 22.57, 18.43和19.48 min。结果证明冬青油、 艾叶油、 薄荷油、 桉叶油和蓝桉油等5种精油对淡色库蚊具有较强的熏蒸活性, 具备作为淡色库蚊防控剂开发的潜力, 值得进一步研究。     

Nepetalactones from essential oil of Nepeta cataria represent a stable fly feeding and oviposition repellent

The stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae), is one of the most serious pests to livestock. It feeds mainly on cattle and causes significant economic losses in the cattle industry. Standard stable fly control involving insecticides and sanitation is usually costly and often has limited effectiveness. As we continue to evaluate and develop safer fly control strategies, the present study reports on the effectiveness of catnip (Nepeta cataria L.) oil and its constituent compounds, nepetalactones, as stable fly repellents. The essential oil of catnip reduced the feeding of stable flies by >96% in an in vitro bioassay system, compared with other sesquiterpene-rich plant oils (e.g. amyris and sandalwood). Catnip oil demonstrated strong repellency against stable flies relative to other chemicals for repelling biting insects, including isolongifolenone, 2-methylpiperidinyl-3-cyclohexen-1-carboxamide and (1S,2'S)-2-methylpiperidinyl-3-cyclohexen-1-carboxamide. The repellency against stable flies of the most commonly used mosquito repellent, DEET, was relatively low. In field trials, two formulations of catnip oil provided >95% protection and were effective for up to 6 h when tested on cattle. Catnip oil also acted as a strong oviposition repellent and reduced gravid stable fly oviposition by 98%.     

Phytochemical constituents and inhibitory activity towards methicillin-resistant Staphylococcus aureus strains of Eryngium species (Apiaceae)

The chemical composition of the essential oils of Eryngium campestre, E. thorifolium, and E. creticum (Apiaceae), growing in the Aegean region of Turkey (Mount Sandras, Denizli), was determined by direct thermal desorption (DTD)-GC/MS analyses. A total of 49 components were identified in the oils, α-pinene and hexanal being the major compounds. The three essential oils were also tested for their inhibitory activity of nine different methicillin-resistant Staphylococcus aureus (MRSA) strains by the agar disc diffusion method. The anti-MRSA activity of E. thorifolium oil, the most active of the three oils, was comparable with those of the reference antibiotic vancomycin and oregano oil, although somewhat lower.     

Repellent,larvicidal and adulticidal activities of essential oil from Dai medicinal plant Zingiber cassumunar against Aedes albopictus

Zingiber cassumunar is an important plant used in traditional medicine and as a natural mosquito repellent. However, the compounds responsible for the repellent activity of the plant are still unknown. The aim of the study is to identify the components of Z. cassumunar essential oil that show repellent activity against Aedes albopictus. We also evaluated the larvicidal and adulticidal activities of Z. cassumunar essential oil against Ae. albopictus. In-cage mosquito repellent experiments showed that Z. cassumunar essential oil possessed moderate repellent activity with a minimum effective dose (MED) of 0.16 ± 0.01 mg/cm², compared to reference standard N,N-diethyl-3-methylbenzamide (DEET, 0.03 ± 0.01 mg/cm²). Bioassay-guided fractionation identified the major active compound of Z. cassumunar essential oil as (−)-terpinen-4-ol (1) (MED: 0.19 ± 0 mg/cm²). We also found that Z. cassumunar essential oil showed moderate larvicidal activity against first instar larvae of Ae. albopictus with a LC₅₀ (50% lethal concentration) of 44.9 μg/L after 24 h. Fumigation bioassays showed that Z. cassumunar essential oil exhibits moderate adulticidal activity against Ae. albopictus with a LC₅₀ of 5.44%, while (−)-terpinen-4-ol showed significant adulticidal activity with a LC₅₀ of 2.10% after 24 h. This study verifies that the Z. cassumunar essential oil has mosquito repellent activity, and that (−)-terpinen-4-ol is mainly responsible for this activity. Furthermore, this study provides scientific support for the folk usage of Z. cassumunar essential oil as mosquito repellent and indicates that Z. cassumunar essential oil and (−)-terpinen-4-ol can be used as plant-derived repellents and insecticides for mosquito control.     

Influence of trap color and host volatiles on capture of the emerald ash borer (Coleoptera: Buprestidae)

Field trapping assays were conducted in 2009 and 2010 throughout western Michigan, to evaluate lures for adult emerald ash borer, A. planipennis Fairmaire (Coleoptera: Buprestidae). Several ash tree volatiles were tested on purple prism traps in 2009, and a dark green prism trap in 2010. In 2009, six bark oil distillate lure treatments were tested against manuka oil lures (used in 2008 by USDA APHIS PPQ emerald ash borer cooperative program). Purple traps baited with 80/20 (manuka/phoebe oil) significantly increased beetle catch compared with traps baited with manuka oil alone. In 2010 we monitored emerald ash borer attraction to dark green traps baited with six lure combinations of 80/20 (manuka/phoebe), manuka oil, and (3Z)-hexenol. Traps baited with manuka oil and (3Z)-hexenol caught significantly more male and total count insects than traps baited with manuka oil alone. Traps baited with manuka oil and (3Z)-hexenol did not catch more beetles when compared with traps baited with (3Z)-hexenol alone. When compared with unbaited green traps our results show that (3Z)-hexenol improved male catch significantly in only one of three field experiments using dark green traps. Dark green traps caught a high number of A. planipennis when unbaited while (3Z)-hexenol was seen to have a minimal (nonsignificant) trap catch effect at several different release rates. We hypothesize that the previously reported kairomonal attractancy of (3Z)-hexenol (for males) on light green traps is not as obvious here because of improved male attractancy to the darker green trap.     

Behavioral and electrophysiological responses of Tetropium fuscum (Coleoptera: Cerambycidae) to pheromone and spruce volatiles

The brown spruce longhorn beetle, Tetropium fuscum (F.), is an invasive wood-boring species in eastern Canada. Gas chromatographic/electroantennographic (GC/EAD) analyses of Norway and red spruce volatiles detected a number of consistent EAD-active responses to compounds that are known to be stress-induced in spruce. The effects of these EAD-active compounds on various aspects of adult behavior were tested. In two-choice olfactometer assays, a monoterpene spruce blend, (R)-(-)-linalool, (3Z,6E)-α-farnesene, (E)-β-farnesene and spruce essential oil were attractive to both sexes. However, when they were combined with the male-produced pheromone (fuscumol), they elicited a sex-specific response: females were significantly attracted to combinations of fuscumol plus either (3Z,6E)-α-farnesene, (E)-β-farnesene and spruce essential oil but males were not. Fuscumol alone was unattractive to either sex in the olfactometer. Males exposed to fuscumol, (3Z,6E)-α-farnesene, or a combination of both, but not (E)-β-farnesene, were more likely to engage in the pheromone calling posture relative to controls. Both the monoterpene spruce blend and spruce essential oil elicited significantly greater trap capture of both sexes of T. fuscum in the presence of fuscumol and ethanol than (3Z,6E)-α-farnesene or (R)-(-)-linalool, which did not elicit trap capture alone or in combination with fuscumol. The data support the hypothesis that stress-induced sesquiterpene components, such as (3Z,6E)-α-farnesene, are important for mediating close-range attraction and behavior in T. fuscum while the monoterpene components are important for long-range processes (trap capture).     

Preparation of Four Geometric Isomers of (11E)-4,6,11-Hexadecatrienal and Their Effect toward Male Eri-Silk Moths

Four geometric isomers of (11E)-4,6,11-hexadecatrienal were prepared, and their pheromone activity towards male eri-silk moths was evaluated. The EAG activity of each isomer was determined by the EAG-GLC method in order of increasing activity to be (4Z,6E,11E)- and (4E,6E,11E)?? (4E,6Z,11E) ?? (4Z,6Z,11E)-hexadecatrienal.     

Arena size, hole density, and capture of Oryzaephilus surinamensis (Coleoptera: Silvanidae) in grain probe traps

The relationship of size of test arena, number of holes in a grain probe trap body and capture of the sawtoothed grain beetle, Oryzaephilus surinamensis (L.), was determined in simulated field tests conducted in an outdoor screen enclosure exposed to natural temperature fluctuations. Polyvinylchloride (PVC) probe bodies were attached to electronic sensor heads, and insect captures were recorded electronically using an electronic grain probe insect counter (EGPIC) system. In comparisons among PVC probe trap bodies with 60, 132, 252, and 492 holes, tested at 18 insects per kilogram in 4.5, 17, and 40 kg of soft wheat in cylindrical arenas (10.2, 20.3, and 30.5 cm in diameter, respectively), number of holes in the probe trap body had no effect on insect capture, but percentage of insects recovered was indirectly related to size of the test arena. Periodicity of insect capture was determined using the time-stamp data that were recorded by the EGPIC system. Circadian rhythm was observed in the periodicity of the capture that corresponded to foraging activity peaks documented for sawtoothed grain beetles, with activity peaks occurring early in the scotophase. There were shifts in times of peak activity among the different test arena sizes that corresponded to differences in temperature in the grain mass. Increases in both temperature and contact between insects and grain probe in the smallest arenas resulted in higher capture of sawtoothed grain beetles. This research documents additional important factors when evaluating capture of sawtoothed grain beetles in grain probe traps.     

Depression and excitation induced in mice by two geometric isomers of (+)13,14-didehydro-20-methyl-carboprostacyclin, FCE 22177 and FCE 22176. Comparison with effects on blood pressure and platelet aggregation

Two geometric isomers of a stable derivative of PGI2 (13,14-didehydro-20-methyl-carbo PGI2), FCE 22177 (5E) and FCE 22176 (5Z), have been injected i.v. in mice (5-400 mcg/Kg) to investigate the effects at CNS level (Irwin's test). The synthetic mixture of the two isomers (5E:5Z = 7:3) was also tested. Isomers 5E and 5Z induced opposite effects: 5E (similar to PGI2) was depressive, 5Z induced stimulation. The mixture showed a strong depressive symptomatology. The two isomers and their mixture induced motor incoordination and impaired the performance of mice in the rotarod test (200 mcg/Kg i.v.). Mice were depressed after 5E and mixture; whilst 5Z induced excitation, in accordance with Irwin's test. 5E and 5Z showed analogous effects on mean blood pressure (hypotension) and heart rate (increase) in normotensive rats, but 5E was 7 times more active than 5Z. The two isomers showed analogous inhibitory effect on guinea-pig platelet aggregation in vitro, but 5E was 30 times more active than 5Z. In conclusion, the two geometric isomers have similar effects on blood pressure, heart rate and platelet aggregation, even if differing quantitatively. In contrast, 5E and 5Z have opposite effects at CNS level on motor function and behaviour. The data suggest the presence of two different sites of action for PGI2 in CNS (Ca2+- and adenylate cyclase-linked?). The two geometric isomers, 5E and 5Z, may be useful to study the PGI2 receptor sites in different tissues.     

10,20-Methanorhodopsins: (7E,9E,13E)-10,20-methanorhodopsin and (7E,9Z,13Z)-10,20-methanorhodopsin. 11-cis-locked rhodopsin analog pigments with unusual thermal and photo-stability

Synthesis of the retinal analog, 10,20-methanoretinal (R6), where the 11Z conformation is locked in a six-membered ring, yielded four stereoisomers (7E,9E,13E, 7E,9E,13Z, 7E,9Z,13E and 7E,9Z,13Z). These four isomers were separated by straight-phase isocratic HPLC and identified by 1H-NMR and NOE analysis. All isomers smoothly recombined with bovine opsin at a relatively high rate (5-10% of that of the natural chromophore 11Z-retinal). The corresponding 13E and 13Z isomers yielded identical analog pigments, probably due to rapid thermal isomerization around the C13 = C14 double bond. The (7E,9E)-isomers produced a pigment with maximal absorbance at 510 nm, while the pigment produced from the (7E,9Z)-isomers had maximal absorbance at 494 nm. Based upon kinetic considerations, the chromophore structure in the 510-nm-absorbing pigment should be (7E,9E,13E), i.e. equivalent to 11Z-retinal and rhodopsin, while the chromophore structure in the 494-nm-absorbing pigment should be (7E,9Z,13Z), i.e. equivalent to (9Z,11Z,13Z)-rhodopsin, an isorhodopsin analog. In analogy to the 11-cis-locked rhodopsin analogs Rh5 and Rh7, the 510-nm-absorbing pigment, (7E,9E,13E)-10,20-methanorhodopsin, was dubbed Rh6 and the 494-nm-absorbing pigment. (7E,9Z,13Z)-10,20-methanorhodopsin, was dubbed Iso6. The opsin shift of Rh6 (2660 cm-1) is practically identical to that of rhodopsin itself (2650 cm-1). Rh6 and Iso6 are nearly as stable as rhodopsin towards hydroxylamine and solubilization in detergent solution and could be easily purified and reconstituted into proteoliposomes by established procedures. Due to the 11-cis-lock, Rh6 is much less photolabile (bleaching rate less than 1%) than rhodopsin, but it is not completely photostable, probably since photoisomerization around the C7 = C8, C9 = C10 and C13 = C14 bonds is allowed. Illumination of either Rh6 or Iso6 does not generate the common photointermediates but instead produces a complex pattern of photochemical transitions, which during continuous illumination leads to the same final steady state, absorbing at 498 nm. This process is accompanied by a slow but steady loss of pigment, probably due to hydrolytic release of chromophore, which is markedly accelerated in the presence of hydroxylamine. In a physiological assay (light-dependent activation of rod cGMP phosphodiesterase) Rh6 is only marginally active and this probably reflects conformational changes accompanying the above-mentioned photochemical transitions. This supports the concept that normal rhodopsin-based phototransduction requires 11Z to all-E isomerization.(ABSTRACT TRUNCATED AT 400 WORDS)