Role of ω-3 fatty acid desaturases in the regulation of the level of trienoic fatty acids during leaf cell maturation

Trienoic fatty acids, namely -linolenic acid and hexadecatrienoic acid, present in leaf lipids are produced by -3 fatty acid desaturases located in the endoplasmic reticulum and plastid membranes. The changes in the level of trienoic fatty acids during leaf maturation were investigated in wild-type plants of Arabidopsis thaliana (L.) Heynh. and in the fad7 mutant deficient in the activity of a plastid -3 desaturase. The levels of trienoic fatty acids increased in 26 °C- and 15 °C-grown wild-type plants with maturation of leaves. The increase in trienoic fatty acids was mainly due to galactolipids enriched in plastid membranes. In addition, the relative levels of trienoic fatty acids in major glycerolipids, including phospholipids enriched in the endoplasmic reticulum membranes, also increased with leaf maturation. By contrast, when the fad7 mutant was grown at 26 °C, the relative levels of trienoic fatty acids in individual lipids decreased with leaf maturation. The decreases in the levels of trienoic fatty acids, however, were alleviated when the fad7 mutant was grown at 15 °C. These results suggest that the plastid -3 desaturase plays a major role in increasing the levels of trienoic fatty acids with leaf maturation.Abbreviations 163 hexadecatrienoic acid - 183 -linolenic acid - DGD digalactosyldiacylglycerol - MGD monogalactosyldiacylglycerol - PC phosphatidylcholine - PE phosphatidylethanolamine - TA trienoic fatty acid - WT wild type - -3 refers to the position of the double bond from the methyl end of a fatty acid This research was supported in part by Grants-in-Aid for Scientific research (#07251214 and #06804050 to K.I.) from the Ministry of Education, Science and Culture, Japan, and by the research grant from Shorai Foundation.     

Production of a novel ω1-eicosapentaenoic acid by Mortierella alpina 1S-4 grown on 1-hexadecene

An arachidonic-producing fungus, Mortierella alpina 1S-4, was found to accumulate -unsaturated fatty acids of C-20 chain length together with 1-hexadecenoic acid, 1-octadecenoic acid and so on, when grown on 1-alkenes, i.e., 1-hexadecene and 1-octadecene. The results of mass spectroscopy and proton NMR showed that a C₂₀ polyunsaturated fatty acid (PUFA) is a novel cis-5,8,11,14,19-eicosapentaenoic acid (20:51). This PUFA was obtained at a yield of 0.13 mg/ml culture broth (2.8% of the fungal total fatty acid content) on cultivation of the fungus in a medium containing 4% (v/v) 1-hexadecene and 1% yeast extract at 28°C for 1 week. Investigation of the distribution of fatty acids showed that about 90% (by mol.) of the PUFA was present in the triglycerides and 10% was in the phospholipid fraction. About 70% of that found in the phospholipids was phosphatidylcholine (PC) and the value accounted for ca. 10% of the total fatty acid content. The formation of these -unsaturated fatty acids was presumed to occur through the arachidonic acid biosynthetic pathway (n-6 route).Abbreviations PUFA polyunsaturated fatty acid - EPA cis-5,8,11,14,17-eicosapentaenoic acid - TG triglycerides - PS phosphatidylserine - PC phosphatidylcholine Present address: Laboratory of Microbial Science, Institute for Fundamental Research, Suntory Ltd., Mishima-gun, Osaka 618, Japan     

Occurrence of Curtobacterium sp. possessing ω-cyclohexyl fatty acids in soil with zinc added

A bacterial strain, Curtobacterium sp., isolated from a soil with zinc added possessed -cyclohexyl fatty acids. -Cyclohexyl undecanoic acid made up 47% of the total fatty acids; it was the most abundant fatty acid in the strain grown in tryptone medium. 12-Methyl tetradecanoic acid (23%) and 14-methyl hexadecanoic acid (22%) were also major fatty acids. The proportion of -cyclohexyl undecanoic acid increased as the pH of the medium decreased and as the culture temperature increased.The bacteria grew almost normally in zinc-enriched medium, and -cyclohexyl undecanoic acid increased with zinc concentration. Zinc added to the medium was not abundant in the cell fraction, and the ratio of increase of zinc in the cells was not so high as in the culture medium. These results suggested that -cyclohexyl fatty acids are related to the zinc tolerance of the isolated strain, and that this tolerance depends on low permeability of the membrane to zinc.     

Characterization of<Emphasis Type="Italic"> Pseudomonas</Emphasis> sp. HK-6 cells responding to explosive RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine)

The cellular responses of Pseudomonas sp. HK-6 to explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) have been extensively analyzed in this study. The stress shock proteins, which might contribute to enhancing the cellular resistance to the cytotoxic effect of RDX, were induced at different concentrations of RDX used as a substrate for cell culture of Pseudomonas sp. HK-6. The proteins were identified as 70-kDa DnaK and 60-kDa GroEL by SDS-PAGE and Western blot using the anti-DnaK and anti-GroEL monoclonal antibodies. The stress shock proteins induced by RDX were found to increase in proportion to the RDX concentration used for this work. Analysis of membrane fatty acids of strain HK-6 following exposure to RDX showed that the amounts of dominant lipids 16:1 7c/15:0 iso 2OH, 16:0 and 18:1 7c/9t/12t decreased substantially or were not detected in the cells exposed to RDX, while amounts of lipids 10:0 iso, 14:1 5c/5t and 16:10 methyl increased dramatically. Scanning electron microcopy analyses revealed the presence of perforations and irregular rod shapes with wrinkled surfaces for cells treated with 0.135 mM RDX for 12 h, suggesting that RDX has a substantial cytotoxic impact on cells of strain HK-6.     

Food Quality Constraints in Daphnia: Interspecific Differences in the Response to the Absence of a Long Chain Polyunsaturated Fatty Acid in the Food Source

The effect of the long chain polyunsaturated fatty acid (PUFA) eicosapentaenoic acid (EPA, 20:53) on food quality for Daphnia galeata, Daphnia hyalina and a D. galeata×hyalina hybrid was examined. Somatic growth rates of all three species were determined when growing on EPA-free Scenedesmus obliquus and on S. obliquus which had been supplemented with free EPA prior to the growth experiments. Growth rates on S. obliquus were substantially higher in D. galeata(0.45day^–1) than in D. hyalina(0.27day^–1) and D. galeata×hyalina(0.31day^–1). Supplementary EPA increased growth of D. galeata and D. hyalina, but not of the hybrid. Hence within the D. galeata/hyalina complex species differ in their ability to cope with an EPA-free diet. Analysis of fatty acids revealed that tissue concentrations of 18:33, 18:43, 20:33 and 20:43 were higher in D. galeata than in D. hyalina which indicated higher rates of assimilation and biosynthesis of PUFAs in D. galeata. This was contrasted by lower tissue concentrations of 20:53 D. galeata than in D. hyalina which suggests that 20:53 is metabolised with growth. In the D. galeata×hyalina hybrid high PUFA assimilation and biosynthesis were associated with low growth rates which explains the finding that tissue concentrations of 20:53 were highest and that growth was not constrained by the availability of 20:53.     

Purification and characterization of hydroxycinnamoyl-coenzyme A: ω-hydroxypalmitic acid O-hydroxycinnamoyltransferase from tobacco (Nicotiana tabacum L.) cell-suspension cultures

An acyltransferase hydroxycinnamoyl-Coenzyme A: -hydroxypalmitic acid O-hydroxycinnamoyltransferase (HHT; EC 2.3.1.-), which transfers hydroxycinnamic acids from hydroxycinnamoyl-CoA thioesters to several hydroxylated fatty acid derivatives, was characterized from tobacco (Nicotiana tabacum L. cv. Xanthi nc) cell-suspension cultures. It exhibited the same properties as the enzyme previously detected in wound-healing potato tuber discs (Lotfy et al., 1994, Phytochemistry 35: 1419–1424), and especially a marked specificity for -hydroxypalmitic acid and feruloyl-CoA. It was purified 300-fold to near homogeneity from late logarithmic-phase cell suspensions. The apparent molecular mass of the native protein was 55 kDa and its isoelectric point, estimated by electrofocusing, was 4.6. The purified enzyme conjugated ferulic acid to -hydroxypalmitic acid and to 1-tetradecanol, its main lipidic substrates, suggesting that the same enzyme probably synthesizes the different esters of 1-alkanols and of -hydroxy fatty acids which are formed in vitro.Abbreviations ABA abscisic acid - IEF isoelectric focusing - HHT hydroxycinnamoyl-Coenzyme A: -hydroxypalmitic acid O-hydroxycinnamoyltransferase - pI isoelectric point     

Production of poly(3-hydroxyalkanoates) from α, ω-alkanedioic acids and hydroxylated fatty acids by Alcaligenes sp.

Summary Poly(3-hydroxybutyrate) (P(3HB)) was produced from a series of , -alkanedioic acids of both even and odd carbon numbers by the Alcaligenes sp. AK201. In contrast, copolymers of 3HB and 3-hydroxyvalerate(P(3HB-co-3HV)) were produced from hydroxylated fatty acids of even carbon numbers such as 12-hydroxystearate and 2-hydroxyoctanoate. The biosynthetic pathways to poly(3-hydroxyalkanoates)(P(3HA)) are discussed.     

Similarities of omega gliadins from<Emphasis Type="Italic"> Triticum urartu</Emphasis> to those encoded on chromosome 1A of hexaploid wheat and evidence for their post-translational processing

The -gliadins encoded on chromosome 1 of the A genome were purified from Triticum aestivum L. (2n=6x=42, AABBDD) cv. Butte86, nullisomic 1D-tetrasomic 1A of cv. Chinese Spring (CS N1DT1A), and the diploid T. urartu (2n=2x=14, AA). Reverse-phase high-performance liquid chromatography combined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis of gliadin extracts from CS nullisomic-tetrasomic (NT) lines confirmed the assignment to chromosome 1A. The purified -gliadins were characterized by mass spectrometry and N-terminal sequencing. The 1A-encoded -gliadins were smaller than 1B- or 1D-encoded -gliadins. The N-terminal amino acid sequences for 1A -gliadin mature peptides were nearly identical to those for the T. urartu -gliadins and were more similar to 1D -gliadin sequences than to sequences for T. monococum -gliadins, barley C-hordeins, or rye -secalins. They diverged greatly from the N-terminal sequences for the 1B -gliadins. The data suggest that T. urartu is the A-genome donor, and that post-translational cleavage by an asparaginyl endoprotease produces those -gliadins with N-terminal sequences beginning with KEL.Communicated by J. Dvorak     

Restriction fragment analysis of the secalin loci of rye

Analyses of wheat/rye addition lines by Southern blotting confirmed the presence of sequences related to theSec 1, Sec 2, andSec 3 loci on chromosomes 1R and 2R. Comparison of the 1R and 2R addition lines allowed the identification of -secalin genes atSec 1 andSec 2, respectively, while -secalin and -secalin genes atSec 1 were discriminated by comparative hybridization with three probes: -secalin, total -secalin, and 3 -secalin. The high molecular weight (HMW) secalin genes atSec 3 were identified using a homologous HMW subunit probe from wheat. Gene copy numbers were estimated as about 40–60 for -secalins, 5–10 for -secalins, and 2 for HMW secalins. Comparison of individual plants of cv. Gazelle showed a high degree of polymorphism, particularly for sequences related to -secalins and HMW secalins.     

Characteristics of [125I]ω-conotoxin labeling using bifunctional cross linker DSP in crude membranes from chick brain

Characteristic of [¹²⁵I]-conotoxin (-CgTX) labeling using bifunctional cross linker (dithio bis[succinimidyl propionate]: DSP) was systematically investigated in crude membranes from chick whole brain. [¹²⁵I]-CgTX specifically labeled 216 kDa as a main and 236 kDa as a minor bands in the crude membranes under non-reduced condition, but not labeled under reduced condition. We investigated the effect of various Ca channel antagonists on [¹²⁵I]-CgTX labeling with DSP in detail, and found that there is a strong correlation between the effects of Ca channel antagonists on [¹²⁵I]-CgTX labeling of the 216 kDa band and specific [¹²⁵I]-CgTX binding. These results suggest that labeling of the 216 kDa band under non-reduced condition with [¹²⁵I]-CgTX using DSP involves the specific binding sites of [¹²⁵I]-CgTX, perhaps including one of the neuronal N-type Ca channel subunits in the crude membranes.     

Isolation of mutants ofLactobacillus bacterias,grown in skim milk,that resist fatty acid inhibition

The effect of saturated fatty acids from 60 to 160 and oleic acid was determined onLactobacillus leichmannii growing in skim milk. The growth of this strain was markedly inhibited by fatty acids from 80 to 120 but not by straight chain fatty acids greater than 130 or less than 70 and oleate. Laurate was the fatty acid with the highest bactericidal effect. Similar results, with little changes depending on strains, were obtained withL. casei, L. plantarum, L. bulgaricus, L. lactis, L. helveticus. Mutants from theseLactobacillus organisms, resistant to fatty acid inhibition, were isolated by a recycling selection procedure. These mutants exhibited high levels of oxidation for laurate. The presence of 2 mM of this compound in the skim milk culture increased the fatty acid oxidation activity four to ten times higher than was exhibited by the parent strains. The practical implications of these observations are discussed.     

Formation ofα,ω-dodecanedioic acid andα,ω-tridecanedioic acid from different substrates by immobilized cells of a mutant ofCandida tropicalis

Summary The metabolic formation of either,-dodecanedioic acid or,-tridecanedioic acid from the individual n-alkane, n-alcohol, n-monoacid and,-diol with corresponding carbon chain length using K-carrageenan entrapped mutants S76 ofCandida tropicalis was studied. The immobilized cells of S76 could also directly produce-hydroxy acid and,-dioic acid from,-diol. With n-alcohol and n-monoacid as substrate, the amount of-hydroxy acid and,-dioic acid produced was also a function of the incubation time.The results demonstrated that in the immobilized cells of S76 the formation of,-dioic acid from n-alcohol can also run both via n-monoacid and via,-diol as well as in the normal cells of S76.     

Cultivation of the agarophyte Gelidium pristoides in Algoa Bay,South Africa

Biofilms were allowed to develop on wooden slides of the River Red Gum (Eucalyptus camaldulensis Dehnh., Myrtaceae) submerged in two billabongs of south-eastern Australia. The slides were placed in the photic zone and the aphotic zone, and the biofilms sampled after eight week's growth over the summer of 1989–1990 and winter of 1990. Bacterial numbers, estimated with epifluorescence microscopy, ranged from 4–78 × 10⁶ cells cm^–2. Bacteria were more abundant in the photic zone than the aphotic zone, and more abundant in summer than winter. Fewer than 0.5% of the bacteria could be cultivated on nutrient agar plates. Concentrations of phospholipids ranged from 8–79 ng cm^–2, which corresponded to bacterial abundances of 2–17 × 10⁶ cells cm^–2. Fifty five phospholipid fatty acids (PLFA) were identified, of which 16:0 (13–29% of total PFLA) was the most common. Other abundant PFLA included 16:17c (6–28%), 18:26 (3–16%), 18:33 (4–12%), 18:19c (3–5%), 18:l7c (5–11%) and 18:0 (2–8%). Minor PLFA included 14:0, i and a 15:0, 15:0, 16:l5c, 16:113c, 18:36, 18:43, 20:46 and 20:53. The PLFA profiles of the biofilms were quite different from those of the sediments and plankton. There was a clear distinction between the PLFA profiles of summer and winter biofilms, but less evidence for unequivocal site or light-regime effects.     

<Emphasis Type="Italic">Alkalimonas amylolytica</Emphasis> gen. nov., sp. nov., and<Emphasis Type="Italic"> Alkalimonas delamerensis</Emphasis> gen. nov., sp. nov., novel alkaliphilic bacteria from soda lakes in China and East Africa

Two related novel alkaliphilic and slightly halophilic bacteria are described. They are strain N10 from Lake Chahannor in China and strain 1E1 from Lake Elmenteita in East Africa. Both strains are strictly aerobic, heterotrophic, alkaliphilic, mesophilic, and require NaCl for growth. The optimal conditions for growth were at pH 10–10.5 and 2–3% (w/v) NaCl. Cells of both strains were Gram-negative, rod-shaped, non-spore-forming, and motile with a single polar flagellum. Cellular fatty acids in both strains were predominantly saturated and mono-unsaturated straight-chain fatty acids (16:0, 16:17c and 18:17c). The major isoprenoid quinone of both strains was Q8. The major polar lipids are phosphatidylglycerol, diphosphatidylglycerol, phosphatidylglycerol phosphate and phosphatidylethanolamine. The guanine plus cytosine (G+C) content of the DNA was 52.5 mol% and 55.4 mol%, respectively. Phylogenetic analysis revealed that the two strains formed a distinct lineage within the gamma-3 subclass of the Proteobacteria. The strains shared a 16S rDNA sequence similarity of 96.1% and showed less than 93.7% of sequence similarity to any other known species. Based on polyphasic data, the two strains were differentiated from currently recognized genera and represent a new genus, Alkalimonas gen. nov., with two species, Alkalimonas amylolytica sp. nov. (type strain is N10^T = AS 1.3430) and Alkalimonas delamerensis sp. nov. ( type strain is 1E1^{P, T} = CBS 391.94). The GenBank accession numbers for the 16S rRNA gene sequence of strains N10 and 1E1 are AF250323 and X92130, respectively.Communicated by K. Horikoshi     

Peptide modification by incorporation ofα-trifluoromethyl substituted amino acids

Summary Metabolic stabilization of pharmacologically active peptides can be achieved by incorporation of sterically hindered non-natural amino acids, e.g. C ^, -disubstituted amino acids.-Trifluoromethyl substituted amino acids, a subclass of C ^, -disubstituted amino acids, also fulfil this requirement while featuring additional properties based on the electronic influence of the fluorine substituents.This review summarizes the results concerning the stability of peptides containing-TFM amino acids towards proteolysis by-chymotrypsin. Furthermore, configurational effects of-TFMAla on the proteolytic stability of peptides are explained using empirical force field calculations. The influence of-TFMAla incorporation on the secondary structure of selected tripeptide amides is compared to the effects exerted by its fluorine-free analogue, aminoisobutyric acid.Finally, results on metabolic stabilization and biological activity of modified thyrotropin releasing hormone are interpreted.     

A novel fungal ω3-desaturase with wide substrate specificity from arachidonic acid-producing Mortierella alpina 1S-4

A filamentous fungus, Mortierella alpina 1S-4, is capable of producing not only arachidonic acid (AA; 20:4n-6) but also eicosapentaenoic acid (EPA; 20:5n-3) below a cultural temperature of 20°C. Here, we describe the isolation and characterization of a gene (maw3) that encodes a novel 3-desaturase from M. alpina 1S-4. Based on the conserved sequence information for M. alpina 1S-4 12-desaturase and Saccharomyces kluyveri 3-desaturase, the 3-desaturase gene from M. alpina 1S-4 was cloned. Homology analysis of protein databases revealed that the amino acid sequence showed 51% identity, at the highest, with M. alpina 1S-4 12-desaturase, whereas it exhibited 36% identity with Sac. kluyveri 3-desaturase. The cloned cDNA was confirmed to encode the 3-desaturase by its expression in the yeast Sac. cerevisiae. Analysis of the fatty acid composition of the yeast transformant demonstrated that 18-carbon and 20-carbon n-3 polyunsaturated fatty acids (PUFAs) were accumulated through conversion of exogenous 18-carbon and 20-carbon n-6 PUFAs. The substrate specificity of the M. alpina 1S-4 3-desaturase differs from those of the known fungal 3-desaturases from Sac. kluyveri and Saprolegnia diclina. Plant, cyanobacterial and Sac. kluyveri 3-desaturases desaturate 18-carbon n-6 PUFAs, Spr. diclina 3-desaturase desaturates 20-carbon n-6 PUFAs and Caenorhabditis elegans 3-desaturase prefers 18-carbon n-6 PUFAs as substrates rather than 20-carbon n-6 PUFAs. The substrate specificity of M. alpina 1S-4 3-desaturase is rather similar to that of C. elegans 3-desaturase, but the M. alpina 3-desaturase can more effectively convert AA into EPA when expressed in yeast. The M. alpina 1S-4 3-desaturase is the first known fungal desaturase that uses both 18-carbon and 20-carbon n-6 PUFAs as substrates.     

Comparison of fatty acid content and DNA homology of the filamentous gliding bacteriaVitreoscilla,Flexibacter, Filibacter

DNA hybridization experiments showed that there was a high degree of homology amongVitreoscilla strains but not with DNA fromFilibacter limicola. Flexibacter spp were much more heterogeneous indicating a low genetic similarity. These results were also reflected in the membrane fatty acids of the bacteria. TheVitreoscilla strains were very similar with the 16:17c fatty acid being dominant. The membrane fatty acids ofF. limicola were dominated by a15:0 and a17:0 components which provided additional support for its relatedness to the genusBacillus. There was much greater diversity in the fatty acid patterns of theFlexibacter spp.F. aurantiacus, F. ruber andF. elegans shared the common dominant fatty acids 16:17c with theVitreoscilla strains, but this was replaced by the 16:16c acid inF. flexilis. F. ruber was distinguished by the absence of branched odd-chain monounsaturated fatty acids andF. elegans by the dominance of the -OH i15:0 acid. Precise determination of fatty acid double bond positions and geometry are essential for correct interpretation of increasingly complex ecological and taxonomic data sets.     

Fatty acid content in wild type and WZSL mutant of Euglena gracilis

The effects of growth conditions on fatty acid profilewere examined in the photosynthetic wild type and inthe spontaneous non-photosynthetic WZSL mutant of theunicellular flagellate Euglena gracilis. Inthe light, the amount of polyunsaturated fatty acids(PUFAs) is higher in the wild type than in the mutant,independent of the carbon source. Among importantPUFAs, linolenic acid (18:3 3) is present inhigh amount only in wild type cells grown in the lightwith any of the tested carbon sources. The content ofother PUFAs, such as arachidonic acid (20:46), EPA (20:5 3) and DHA (22:63), is not correlated with the presence oflight or chloroplasts.The main effect of the dark in both strains is tolower the content of PUFAs and mono-unsaturated fattyacids and to increase the content of saturated fattyacids with all the carbon sources.     

Average phase difference theory and 1∶1 phase entrainment in interlimb coordination

The dynamics of coupled biological oscillators can be modeled by averaging the effects of coupling over each oscillatory cycle so that the coupling depends on the phase difference between the two oscillators and not on their specific states. Average phase difference theory claims that mode locking phenomena can be predicted by the average effects of the coupling influences. As a starting point for both empirical and theoretical investigations, Rand et al. (1988) have proposed d/dt= — K sin ), with phase-locked solutions =arcsin( /K), where is the difference between the uncoupled frequencies and K is the coupling strength. Phase-locking was evaluated in three experiments using an interlimb coordination paradigm in which a person oscillates hand-held pendulums. was controlled through length differences in the left and right pendulums. The coupled frequency _c was varied by a metronome, and scaled to the eigenfrequency _v of the coupled system K was assumed to vary inversely with _c. The results indicate that: (1) and K contribute multiplicatively to (2) =0 or = regardless of K when =0; (3) 0 or regardless of when K is large (relative to ); (4) results (1) to (3) hold identically for both in phase and antiphase coordination. The results also indicate that the relevant frequency is _c/_v rather than _c. Discussion high-lighted the significance of confirming =arcsin(/K) for more general treatments of phase-locking, such as circle map dynamics, and for the 11 phase-entrainment which characterizes biological movement systems.