Microbial Conversion of Arachidonic Acid to Arachidonyl Alcohol by a New <Emphasis Type="Italic">Acinetobacter</Emphasis> Species

Arachidonyl alcohol rarely occurs in natural oils. It can be used as a substrate for production of several ether lipids possessing beneficial functions. Although arachidonyl alcohol has been produced on a laboratory scale by the chemical reduction of arachidonic acid, it will be difficult to scale up this process for industrial application. The aim of this study was to develop a new bioprocess for converting arachidonic acid to arachidonyl alcohol. Screening was conducted using 11 wax ester- (esters of fatty acids and fatty alcohols) producing strains reported in our previous study, and a single-cell oil containing arachidonic acid. A new strain, Acinetobacter species N-476-2, most effectively converted arachidonic acid to arachidonyl alcohol, which accumulated inside the cells as a wax ester. GC–MS, FT–IR, and NMR analyses showed that this strain reduced the carboxyl group of 5-cis,8-cis,11-cis,14-cis-arachidonic acid to a hydroxyl group without altering the position or configuration of the double bonds; the product was identified as 5-cis,8-cis,11-cis,14-cis-arachidonyl alcohol. A time-course study of cultivation showed that the amount of arachidonyl alcohol produced by the strain after 4 days was 2.2 mg/mL culture. The bioprocess using Acinetobacter sp. N-476-2 can be applied to the large-scale production of arachidonyl alcohol.     

Structure and Function of Retinochrome

Retinochrome is a photo-isomerase of all-trans-retinal to the 11-cis-isomer for rhodopsin synthesis. This protein is probably folded into seven α-helices spanning the membranes. Upon irradiation, it is converted to metaretinochrome, via pre-lumi- and lumiretinochrome, which are stable under 50 and 250 K, respectively. A series of analogue studies revealed that the cyclohexene ring and 6-s-cis-twisted conformation are important for binding. Modification of the 14-position suggests close location of the 14-H to the counterion.     

Application of Artificial Pigments to Structure Determination and Study of Photoinduced Transformations of Retinal Proteins

The protonated Schiff bases of all-trans-retinal and its double bond isomers, 11-cis- and 13-cis-retinals, comprise the chromophores of bacteriorhodopsin, sensory rhodopsin, rhodopsin, and the Chlamydomonas photoreceptor pigment. Absorption of photons by these chromophores is directly responsible for the functioning of the various photopigments. Clarification of their extremely complex structures and mechanisms requires multidisciplinary collaboration. The study of synthetic retinal analogs and pigments reconstituted from analogs provides a powerful and indispensable tool for such clarification. This article summarizes the application of retinal analogs in the bioorganic, biophysical, and biochemical investigations of the various retinal pigments.     

2-trans,4-cis,7-cis-Decatrienal,the fishy off-flavor occurring in strongly autoxidized oils containing linolenic acid or ω 3,6,9, etc., fatty acids

A possible decomposition product of oils containing unsaturated fatty acids with the double bond pattern ω3,6,9 or ω3,6,9, etc., is 2-trans,4-cis,7-cis-decatrienal. It has therefore been investigated whether this trienal is present in strongly autoxidized linolenic acid, soybean, linseed and fish oils. Via a degassing technique the carbonyls from the samples of oils heated in a closed system under nitrogen were collected and separated as their 2,4-dinitrophenylhydrazones (DNPHs) by thin layer chromatography (TLC). The presence of 2-trans,4-cis,7-cis-decatrienal in the samples was positively established by TLC, UV absorption and mass spectroscopy of its DNPH. All analytical data established the complete identity of the isolated DNPH with a synthesized 2,4,7-decatrienal-DNPH. The free aldehyde obtained on hydrolysis of the alkatrienal-DNPH in question had the same flavor as authentic 2-trans,4-cis,7-cis-decatrienal, having a fish or rather a whale oil flavor.     

Microbial Sensory Rhodopsins: Photochemistry and Function

The review covers recent progress on microbial sensory rhodopsins, visual pigment-like retinylidene photoreceptors that function in phototaxis by archaeons, such as Halobacterium salinarium, and by unicellular eukaryotic algae, such as Chlamydomonas reinhardtii. Six demonstrably different sensory rhodopsins are known in halophilic archaea. The best characterized is sensory rhodopsin I (SR-I), a color-sensitive receptor that relays attractant and repellent photosignals to a tightly bound transducer protein HtrI (halobacterial transducer for sensory rhodopsin I). New advances in the mechanism of signal transduction by the SR-I/HtrI complex from molecular-biological and biophysical approaches are summarized. Effects of HtrI on light-induced proton transfers in the receptor are discussed for their possible role in signaling. Current knowledge concerning the growing family of related archaeal sensory rhodopsins is presented. The evidence for a sensory rhodopsin in phototaxis by C. reinhardtii and other unicellular eukaryotic algae is reviewed. The molecular information is more limited than for the archaeal organisms, but the physiological information is rich and complex. Compelling data exist for a single retinal-containing receptor mediating both phototaxis and photophobic responses in C. reinhardtii. From retinal analog studies, the isomeric configuration and ring/chain conformation of the retinal in the receptor appear to be identical to those of the archaeal sensory rhodopsins. Also, photoisomerization from all-trans- to 13-cis-retinal appears to be the trigger for signaling, as in the archaeal pigments. Conflicting early studies suggesting an 11-cis-retinal chromophore and signaling without photoisomerization are analyzed and possible explanations for those reports are suggested. As a general conclusion, the microbial sensory rhodopsins provide an opportunity to explore photochemistry and protein/protein interaction in photosensory transduction in genetically tractable organisms.     

Properties and Photoactivity of Rhodopsin Mutants

A variety of spectroscopic and biochemical studies of recombinant site-directed mutants of rhodopsin and related visual pigments have been carried out. These studies have elucidated key structural elements common to visual pigments, such as a conserved disulfide bond. In addition, systematic analysis of the chromophore-binding pocket in rhodopsin and cone pigments has led to an improved understanding of the mechanism of the opsin-shift, and of particular molecular determinants underlying color vision in humans. Identification of conformational changes which occur upon rhodopsin photoactivation has been of particular recent concern. Assignment of these molecular alterations to specific regions in the receptor has been attempted by studying native opsin regenerated with synthetic retinal analogues or recombinant opsins regenerated with 11-cis-retinal. Individual molecular groups that undergo structural alterations during photoactivation have been identified. Analysis of particular mutant pigments in which specific groups are locked into their respective “on” or “off” states has provided a framework to identify determinants of the active conformation as well as the minimal number of intramolecular transitions required to switch between inactive and active conformations. A simple model for the active state of rhodopsin can be compared to structural models of its ground state to localize chromophore-protein interactions that may be important in the photoactivation mechanism.     

Prostaglandins of rat testis

The purpose of the study was to determine whether prostaglandins were present in mammalian testis, a tissue that has a large concentration of polyenoic fatty acids that are potential precursors of prostaglandins. Acid-soluble lipids of rat testis were extracted, purified, and fractionated by thin layer and column chromatographies.³H-Prostaglandins were added as internal reference standards to monitor recoveries and facilitate identification. Initial identification of prostaglandin species was done by chromatography. Further identification was done by elution of the prostaglandin zones followed by rechromatographies (both thin layer and column), measurements of UV absorption spectra, and by gas liquid chromatography. The results of these analyses indicate that prostaglandin E₁, 11α,15(S)-dihydroxy-9-oxo-β-trans-prostenoic acid; prostaglandin E₂, 11α-15(S)-dihydroxy-9-oxo-5-cis-13-trans-prostadienoic acid; and prostaglandin F₁, 9α,11α,15(S)-trihydroxy-13-trans-prostenoic acid occur in rat testicular tissue and that prostaglandin F_2α, 9α, 11α, 15(S)-trihydroxy-5-cis-13-trans-prostadienoic acid and prostaglandin E₂, 11α,15(S)-dihydroxy-9-oxo-5-cis-13-trans-prostadienoic acid may be the primary species of this tissue. Prostaglandin B₁, 15(S)-hydroxy-9-oxo-8(12),13-trans-prostadienoic acid and prostaglandin B₂, 15(S)-hydroxy-9-oxo-5-cis,8(12),13-trans-prostatrienoic acid also were detected, and some evidence was obtained for the presence of prostaglandin metabolites.     

Phospholipid composition of liver in rats fed high levels of 13-cis retinoic acid

The composition of liver phospholipids was studied in rats fed for 4 weeks diets containing 0, 100 or 300 mg 13-cis retinoic acid per kg diet. There was a significant decrease in phosphatidylcholine content, whereas the levels of phosphatidylethanolamine were slightly increased in liver phospholipids of rats fed 13-cis retinoic acid. The fatty acid composition of total phospholipids, PC, PE, and PI+PS fractions revealed a general increase in the levels of 18∶2 and 20∶3ω6, whereas the levels of 20∶4ω6 and C₂₂ fatty acids were reduced in most of the hepatic phospholipids isolated from rats fed 13-cis retinoic acid containing diets. A decrease in the double-bond index of fatty acids was also observed in phospholipids of rats fed 13-cis retinoic acid. The data suggest that high levels of 13-cis retinoic acid may possibly be influencing the activities of microsomal desaturating and chain-elongating enzymes in the liver.     

Desaturation of positional and geometric isomers of monoenoic fatty acids by microsomal preparations from rat liver

A range ofcis- andtrans-monoenoic fatty acids was tested as substrates for desaturation in microsomal preparations from rat liver.Trans-monoenoic acids were generally desaturated in the Δ9 position to the same extent as stearic acid. Acids with Δ7-trans- and Δ11-trans-olefinic unsaturation produced Δ7-trans,9-cis- and Δ9-cis,11-trans-conjugated dienoic acids, respectively, but the Δ8-trans- and Δ10-trans-monoenoic acids did not give Δ8,9- or Δ9,10-allenes. Of thecis-monoenoic acids examined, only those with double bonds at or beyond the Δ14 position gave any measurable Δ9 desaturation. When Δ9 desaturation of long chain saturated acids was inhibited by adding sterculic acid, these saturated acids were desaturated at the Δ5 and Δ6 positions. Many of the monoenoic acids tested were also desaturated at the Δ5 and/or Δ6 positions, although the percentage conversions were always low. Δ9-cis,11-trans-, Δ9-cis,12-trans- and Δ9-cis,13-trans-dienoic acids, produced in situ by Δ9 desaturation of the corresponding monoenoic acids, were extensively desaturated in the Δ6 position. These results are discussed in terms of: (a) the various models proposed to explain the substrate specificities of the desaturases, and (b) the metabolism of unnatural fatty acids ingested from dietary sources.     

Incorporation ofall-trans-or 9-cis-β-carotene into mixed micellesin vitro

Various studies suggest thatall-trans- and 9-cis-β-carotene are absorbed in the intestine to different extents. The present study was undertaken to evaluate the degree ofin vitro incorporation of the two isomers into intestinal mixed micelles, which is an essential early step in the absorption process. The micelles were designed to simulate those present during fat digestion in the lumen of the human small intestine with respect to bile salts, lipids, pH and temperature. Solutions ofall-trans-and 9-cis-β-carotene at various ratios were added to the lipid mixture. A direct correlation was seen between the 9-cis-β-carotene level in the mixture and the degree of total β-carotene incorporation into micelles. An increased level ofall-trans-β-carotene incorporated into the micelles. In contrast, when carotene mixtures enriched with the 9-cis isomer were used, an increase in the level of total carotene in the solution was accompanied by a constant or even enhanced carotene incorporation. The results indicate that the differences in the absorption of β-carotene isomers might be the result of their different ability to be incorporated into the lipid micelles of the intestinal lumen. In addition, the results point toward the possibility that ingestion of 9-cis-β-carotene by humans may increase carotene availability.     

Sex Pheromone of the French Black Cutworm Moth, Agrotis ipsilon (Lepidoptera: Noctuidae): Identification and Regulation of a Multicomponent Blend

The sex pheromone blend of a European strain of the black cutworm moth, Agrotis ipsilon (Hufnagel), was investigated. Chemical analyses of pheromone gland extracts from 4- to 8-day-old females showed that individual isolated glands contained only very small amounts of pheromone. High-resolution gas chromatography combined with mass spectrometry (GC–MS) analyses showed the presence of cis-7-dodecenyl acetate (Z7-12:Ac), cis-9-tetradecenyl acetate (Z9-14:Ac), cis-11-tetradecenyl acetate (Z11-14:Ac), cis-11-hexadecenyl acetate (Z11-16:Ac), and cis-11-hexadecenol (Z11-16:OH) in biologically active pheromone gland extracts. Removing 27-12: Ac, Z9-14:Ac, or Z11-16:Ac from the complete gland extract by GC trapping techniques strongly reduced the attractiveness of the pheromone blend tested in a wind tunnel. Lack of cis-5-decenyl acetate (Z5-10:Ac) or Z11-16:OH did not affect the blend attractiveness. Chemical and behavioral analyses showed that pheromone components are produced during photophase, at least 2 hr before lights off. Quantitative data showed that decapitation inhibited the production of Z7-12:Ac, Z9-14:Ac, Z11-14:Ac, Z11-16:Ac, and Z11-16:OH, but production in decapitated females was stimulated in response to injection of synthetic Heliothis zea PBAN (pheromone biosynthesis activating neuropeptide) or A. ipsilon brain–subesophageal (Br-SEG) homogenates. Moreover, upon injecting BR-SEG homogenates, other minor components were detected, which were tentatively identified as cis-8-dodecenyl actetate (Z8-12:Ac) and Z5-10:Ac. Our study demonstrated that Z11-l6:Ac is one of the main active components produced by the pheromone gland of this European population of A. ipsilon, in addition to Z7-12:Ac/Z9-14:Ac, which were investigated in a previous behavioral analysis. All these data strongly suggest that some polymorphism is present in pheromone communication in different strains of A. ipsilon.     

The Circadian Clock in the Retinal Pigment Epithelium Controls the Diurnal Rhythm of Phagocytic Activity

The diurnal peak of phagocytosis by the retinal pigment epithelium (RPE) of photoreceptor outer segments (POS) is under circadian control and believed that this process involves interactions from the retina and RPE. Previous studies have demonstrated that a functional circadian clock exists within multiple retinal cell types and RPE. Thereby, the aim of this study was to determine whether the clock in the retina or RPE controls the diurnal phagocytic peak and whether disruption of the circadian clock in the RPE would affect cellular function and the viability during aging. To that, we generated and validated an RPE tissue-specific KO of the essential clock gene, Bmal1, and then determined the daily rhythm in phagocytic activity by the RPE in mice lacking a functional circadian clock in the retina or RPE. Then, using electroretinography, spectral domain-optical coherence tomography, and optomotor response of visual function we determined the effect of Bmal1 removal in young (6 months) and old (18 months) mice. RPE morphology and lipofuscin accumulation was determined in young and old mice. Our data shows that the clock in the RPE, rather than the retina clock, controls the diurnal phagocytic peak. Surprisingly, absence of a functional RPE clock and phagocytic peak does not result in any detectable age-related degenerative phenotype in the retina or RPE. Thus, our results demonstrate that the circadian clock in the RPE controls the daily peak of phagocytic activity. However, the absence of the clock in the RPE does not result in deterioration of photoreceptors or the RPE during aging.     

Synthesis, excited state properties, and dynamic structural change of photoresponsive dendrimers

This article reviews recent advances in research of the dendrimer synthesis in connection with the design, synthesis and reaction of photoresponsive dendrimers. Dendrimers with photoreversible stilbene cores undergo mutual cis-trans isomerization in organic solvents to give photostationary state mixture of cis and trans isomers. Even the fourth generation (G4) stilbene dendrimer with molecular weight as high as 6500 underwent cis-trans photoisomerization within the lifetime of the excited singlet state. The large dendron group surrounding the photoreactive core may affect the excited state properties of the core to increase the efficiency of photoisomerization and/or decrease the fluorescence efficiency. The photochemistry of stilbene dendrimers with various types of dendron groups, and azobenzene dendrimers is discussed. Furthermore, recent advance of dendrimer syntheses and the possibility of their application to construct photoresponsive large molecules are discussed.     

Novel Azocoumarin Derivatives—Synthesis and Characterization

The paper presents synthesis and characterization of nine new thiazolyl-(phenyldiazenyl)-2H-chromen-2-one dyes. The impact of substituent structure in thiazole ring in the synthesized azocoumarin derivatives on electrochemical properties, photoisomerization process and photovoltaic response was examined. The dyes were electrochemically active and undergo reduction and oxidation processes. They showed low electrochemically estimated energy band gap in the range of 1.71–2.13 eV. Photoisomerization process of the synthesized molecules was studied in various solvents such as ethanol, chloroform and N,N-dimethylformamide (DMF) upon the UV illumination. It was found that novel azodyes showed reversible trans-cis-trans isomerization and exhibited long thermal back to the trans form, that was even 7 days in DMF. Selected azocoumarin were molecularly dispersed in polystyrene for preparation of guest-host azopolymer systems to study the cis-trans thermal isomerization of obtained dyes in solid state. The photovoltaic activity of the azochromophores was tested in bulk-heterojunction solar cells. They acting as weak donors in device with structure ITO/PEDOT:PSS/dye:PC70BM/Al. No photovoltaic response of cells with azocoumarin derivatives bearing 4-fluorobenzene, 3,4-dichlorobenzene, or 4-(1-adamantyl) unit was found. Additionally, dye which showed the best activity was examined in three-component solar cells ITO/PEDOT:PSS/PTB7:PC70BM:dye/PFN/Al.     

On the existence of cis/trans peptide mixtures in poly(N-methylglycine)

Unperturbed dimensions have been computed for poly(trans-N-methylglycine), poly(cis-N-methylglycine) and poly(cis/trans-N-methylglycine) by a Monte Carlo simulation technique using potential energy calculations. Computed characteristic ratios for the above polypeptides vary within a narrow range supporting the view that both cis and trans units could be present in the polymer in different proportions under different solvent conditions.     

cis-5,cis-9,cis-12-octadecatrienoic and some unusual oxygenated acids inXeranthemum annuum seed oil

Seed oil ofXeranthemum annuum (family Compositae) contains a number of unusual fatty acids in addition to palmitic, stearic, oleic, linoleic and linolenic. These acids includecis-5,cis-9,cis-12-octadecatrienoic, 5%;cis-9-l,10-l-epoxyoctadecanoic, 3%;cis-9-l,10-l-epoxy-cis-12-octadecenoic (coronaric), 8%; andcis-12-d,13-d-epoxy-cis-9-octadecenoic (vernolic), 2%; as well as a mixture of two hydroxy acids, 11%. The absolute configurations of the two 9,10-epoxy acids are established for the first time. Presented in part at the AOCS Meeting in Cincinnati, October 1965. No. Utiliz. Res. Dev. Div., ARS, USDA.     

Biosynthesis of conjugated linoleic acid in humans

This paper deals with the reanalysis of serum lipids from previous studies in which deuterated fatty acids were administered to a single person. Samples were reanalyzed to determine if the deuterated fatty acids were converted to deuterium-labeled conjugated linoleic acid (CLA, 9c, 11t-18∶2) or other CLA isomers. We found 11-trans-octadecenoate (fed as the triglyceride) was converted (Δ9 desaturase) to CLA, at a CLA enrichment ofca. 30%. The 11-cis-octadecenoate isomer was also converted to 9c, 11c-18∶2, but at <10% the concentration of the 11t-18∶1 isomer. No evidence (within our limits of detection) for conversion of 10-cis-or 10-trans-octadecenoate to the 10,12-CLA isomers (Δ12 desaturase) was found. No evidence for the conversion of 9-cis, 12-cis-octadecadienoate to CLA (via isomerase enzyme) was found. Although these data come from isomerase enzyme) was found. Although these data come from four single human subject studies, data from some 30 similar human studies have convinced us that the existence of a metabolic pathway in one subject may be extrapolated to the normal adult population.     

Palmitoleic (16:1 cis-9) and cis-Vaccenic (18:1 cis-11) Acid Alter Lipogenesis in Bovine Adipocyte Cultures

Our objectives were to: (1) confirm elongation products of palmitoleic acid (16:1 cis-9) elongation in vitro using stable isotopes and (2) evaluate if exogenous supplementation of palmitoleic acid, elongation products, or both are responsible for decreased desaturation and lipogenesis rates observed with palmitoleic acid supplementation in bovine adipocytes. Stromal vascular cultures were isolated from adipose tissue of two beef carcasses, allowed to reach confluence, held for 2?days, and differentiated with a standard hormone cocktail (day?0). On day?2, secondary differentiation media containing 1 of 4 fatty acid treatments [0?μM fatty acid (control), or 150?μM palmitic (16:0), palmitoleic, or cis-vaccenic (18:1 cis-11)] was added for 4?days. On day?6, cells were incubated with [¹³C] 16:1, [¹³C] 2, or [¹³C] 18:0 to estimate elongation, lipogenic, and desaturation rates using gas chromatography-mass spectrometry. Enrichment of [¹³C] 18:1 cis-11 confirmed 18:1 cis-11 is an elongation product of 16:1. Additionally, [¹³C] label was seen in 20:1 cis-13 and cis-9, cis-11 CLA. Synthesis of [¹³C] 16:0 from [¹³C] 2 was reduced (P?<?0.05) in palmitoleic acid and cis-vaccenic acid-treated compared with control cells following 36?h incubation. By 12?h of [¹³C] 18:0 incubation, cells supplemented with palmitoleic acid had reduced (P?<?0.05) [¹³C] 18:1 cis-9 compared with all other treatments. Gene expression and fatty acid results support isotopic data for lipogenesis and desaturation. Therefore, palmitoleic acid is actively elongated in vitro and its elongation product, cis-vaccenic acid, can also reduce lipogenesis. However, inhibition of desaturation can be directly attributed to palmitoleic acid and not its elongation products, 18:1 cis-11 or 20:1 cis-13.     

Isomers of hexadecenoic and hexadecadienoic acids in <Emphasis Type="Italic">Androsace septentrionalis</Emphasis> (Primulaceae) seed oil

Seeds of Androsace septentrionalis of the genus Androsace (tribus Primuleae) from the plant family Primulaceae were studied for their oil content and FA composition. The seed oil of A. septentrionalis was found to contain two unusual FA rarely occurring in plants: 11-cis-hexadecenoic acid (16∶1Δ11c or 16∶1n−5) and 9-cis,12-cis-hexadecadienoic acid (16∶2Δ9c,12c or 16∶2n−4). It also contained an unusually high amount (21.4%) of 9-cis-hexadecenoic acid (palmitoleic acid; 16∶1Δ9c or 16∶1n−7), i.e., at a level higher than that of oleic acid, in addition to common FA. Compared with most plant seed oils, at 3.8% the level of 18∶1Δ11c (or 18∶1n−7) also was elevated. The nonidentity of the Androsace 16∶2-acid with the 16∶2-acid, which is very typical for Ranunculus spp., as well as its identity with the 16∶2-acid typically found in Asclepiadaceae was established by co-chromatography. The structure and composition of the constituent FA of A. septentrionalis were also determined by various chromatographic methods (TLC, Ag⁺-TLC, capillary GLC) and spectroscopic methods (IR, GC-MS). The significant deviation of the Androsace FA pattern from that of other Primuleae, indicating a separate phylogenetic position of Androsace, is discussed.