N-3 and n-6 fatty acid metabolism in undifferentiated and differentiated human intestine cell line (Caco-2)

Metabolism of n-6 and n-3 fatty acids in the undifferentiated and differentiated human adenocarcinoma colon cell line (Caco-2) was studied. In cells incubated with either 182n-6 or 183n-3, no significant amounts of long chain n-6 and n-3 metabolites were found. Incubation with either 183n-6 or 184n-3 raised significantly the levels of 203n-6 and 204n-3, respectively. In the undifferentiated cells, significant proportions of 203n-6 and 204n-3 were further 5-desaturated to form 204n-6 and 205n-3, respectively. Incubation with either 204n-6 or 205n-3 raised the levels of their direct elongation products, 224n-6 and 225n-3, respectively. Incubation with 224n-6 or 225n-3 increased the levels of 204n-6 and 205n-6. These results suggest that 6-desaturation in the Caco-2 cells is less active in comparision with elongation, 5-desaturation and retro-conversion. These enzymes were modulated by the state of differentiation, and appeared to be non-specific to n-3 and n-6 fatty acids. When cells were incubated with 183n-6 and 184n-3 concomitantly, the levels of incorporation of total n-6 fatty acids into cellular lipids were greater than those of the n-3 fatty acids, whereas the ratios of 20+22 carbon metabolites to 18-carbon precursor favored n-3 over n-6 fatty acids. These results suggest that n-3 and n-6 fatty acids were not metabolized identically in Caco-2 cells.     

Comparison of fatty acids of marine fungi using multivariate statistical analysis

Summary Ten obligate marine fungi have as their principal fatty acids 160, 180, 181n9 and 182n6. The fatty acids ranged from 14 to 22 carbons, completely dominated by those with even numbers of carbons. The amount of unsaturated fatty acids varied between 35% and 80%. Each isolate contained small amounts of the acids 183n3 and 204n6. Branched, hydroxy- or cyclic fatty acids were not detected. Multivariate statistical, i.e. principal component analysis, showed that all ten strains could be distinguished on the basis of their fatty acid composition. These results indicate that the marine fungi do not have an unusual fatty acid composition and suggest that chemometric, multivariate analysis might be employed to confirm taxonomic relationships among these organisms.     

Structure of the majorO-glycosidic oligosaccharide of monkey erythrocyte glycophorin

Sialic acids and the majorO-glycosidic oligosaccharide of glycophorin MK from monkey (Japanese monkey,Macaca fuscata) erythrocyte membranes were characterized.N-Glycolylneuraminic acid (neu5Gc) was found as the major sialic acid, which was confirmed by gas-liquid chromatography-mass spectrometry as the trimethylsilyl methyl ester. ThreeO-glycosidic oligosaccharide units were obtained from a tryptic glycopeptide that contained all of the carbohydrate units in glycophorin MK by mild alkaline borohydride/borotritide treatment. Carbohydrate analyses of the oligosaccharides revealed that they were composed of Neu5Gc, galactose andN-acetylgalactosaminitol in the molar ratios of 111 (trisaccharide), 211 (tetrasaccharide) and 111 (pentasaccharide). The content of oligosaccharide units was estimated to be 1125 for penta-, tetra- and trisaccharide, respectively, based on the yields, the molecular weight, and the number of oligosaccharide attachment sites in the amino-acid sequence. The tetrasaccharide was the major oligosaccharide and its structure was proposed to be Neu5Gc2-3Gal1-3[Neu5Gc2-6]GalNAcol.     

Isolation of EMS-induced mutants in Arabidopsis altered in seed fatty acid composition

Summary Mutants of Arabidopsis thaliana were identified by screening pedigreed M3 seed collections from EMS-treated plants for changes in fatty acid (FA) composition. The FA phenotypes of the most dramatic mutants are as follows: G30 and 1E5 (allelic) lack linolenic acid (183) and are elevated in linoleic acid (182); 4A5 is deficient in 182 and 183 and fourfold increased in oleic acid (181); 9A1 lacks all FAs > C18 and is twofold increased in 181; 1A9 is twofold increased in palmitic acid (160) and decreased by one-half in 181; 2A11 is two-to threefold increased in stearic acid (180) and decreased by one-half in 181. Based on segregation of F₂ selfed plants derived from crosses to wild type, all of these phenotypes are the result of single gene mutations.     

Bacterial regeneration of ammonium and phosphate as affected by the carbon:nitrogen:phosphorus ratio of organic substrates

The effect of carbonnitrogenphosphorus (CNP) ratio of organic substrates on the regeneration of ammonium and phosphate was investigated by growing natural assemblages of freshwater bacteria in mineral media supplemented with the simple organic C, N, and P sources (glucose, asparagine, and sodium glycerophosphate, respectively) to give 25 different substrate CNP ratios. Both ammonium and phosphate were regenerated when CN and NP atomic ratios of organic substrates were 101 and 161, respectively. Only ammonium was regenerated when CN and NP ratios were 101 and 10–201, respectively. On the other hand, neither ammonium nor phosphate was regenerated when CN and NP ratios were 151 and 51, respectively. In no case was phosphate alone regenerated. As bacteria were able to alter widely the CNP ratio of their biomass, the growth yield of bacteria appeared primarily dependent on the substrate carbon concentration, irrespective of a wide variation in the substrate CNP ratio.     

A DNA sequence of Drosophila melanogaster with a differential telomeric distribution

A DNA sequence (8–19T) of 2.3 kilobase pairs (kb) of Drosophila melanogaster was localized by in situ hybridization to the extreme ends of polytene chromosomes and to the chromocenter. The relative abundance of this sequence at the ends of polytene chromosomes X2L2R3L3R is 13.41.902.7. This differential distribution is probably due to different copy numbers at the individual telomeric regions. Restriction enzyme analysis of genomic DNA shows that 8–19T sequences are interspersed with other sequences. The clone 8–19T, which contains most of this interspersed repetitive sequence, is itself not internally repetitive but has a complex sequence composition. Some of these sequences are transcribed into poly(A)⁺RNA. We suggest that the ends of Drosophila chromosomes are of a complex arrangement with some sequences common to all ends.     

Interaction of free fatty acids with the erythrocyte membrane as affected by hyperthermia and ionizing radiation

The interference of hyperthermia and ionizing radiation, respectively, with the effects of capric (100), lauric (120), myristic (140), oleic (cis-181) and elaidic (trans-181) acids on the osmotic resistance of human erythrocytes was investigated. The results are summarized as follows: (A) not only at 37°, but also at 42° and 47°C lauric acid (120) represents the minimum chain length for the biphasic behaviour of protecting against hypotonic hemolysis at a certain lower concentration range and hemolysis promotion at subsequent higher concentrations; (B) with increasing temperatures the protecting as well as the hemolytic effects occur at lower concentrations of the fatty acids; (C) the increase of temperature promotes the extent of hemolysis and reduces the extent of protection against hypotonic hemolysis; (D) Gamma-irradiation of erythrocytes selectively affects the concentration of oleic acid at which maximum protection against hypotonic hemolysis occurs, without altering the minimum concentration for 100% hemolysis.     

Anaerobic biosynthesis of unsaturated fatty acids in the cyanobacterium,Oscillatoria limnetica

The mechanism for synthesis of monounsaturated fatty acids under aerobic and anaerobic conditions was studied in the facultative anaerobic cyanobacterium, Oscillatoria limnetica. The hexadecenoic acid (C161) of aerobically grown O. limnetica was shown to contain both the 7 (79%) and 9 (21%) isomers, while the octadecenoic (C181) acid was entirely the 9 acid. Incorporation of [2-¹⁴C] acetate into the fatty acids under aerobic conditions resulted in synthesis of the 7 and 9 C161 and the 9 C181. Synthesis of unsaturated fatty acids in the presence of DCMU required sulfide. Anaerobic incubations in the presence of DCMU and sulfide (less than 0.003% atmospheric oxygen) resulted in a two-fold increase in monounsaturated fatty acids of both 7 and 9 C161 and 9 and 11 C181. The synthesis of these isomers is characteristic of a bacterialtype, anaerobic pathway.Abbreviations DCMU 3(3,4-dichlorophenyl)-1,1-dimethylurea - MFA monounsaturated fatty acid     

Effect of L-triiodothyronine on liver microsomal Δ6 and Δ5 desaturase activity of male rats

The effect of different doses of L-triiodothyronine (T₃) on the activity of 6 and 5 desaturases and lipid fatty acid composition was studied in liver microsomes of male rats. The activity of 6 and 5 desaturases was decreased 24 and 28%, respectively, in animals administered a daily intraperitoneal dose of 1000g T₃/100g body wt. for 5 days, whereas with 500g T₃/100g body wt. only 6 desaturase activity was decreased. On the other hand, no enzyme activity changed at a shorter period of hormone treatment. Changes in microsomal fatty acid composition did not seem to be a direct consequence of desaturation activity, since after 1 and 5 days of T₃ treatment, the concentrations of 18:2 (n-6) and 20:3 (n-6) decreased and only after 1 day that of 20:4 (n-6) increased in spite of unchanged or decreased 6 and 5 desaturase activities. Other factors than desaturation activity must be involved in fatty acid composition of thyroid hormonetreated rats, such as diet, membrane lipid synthesis and degradation, fatty acid turn-over and oxidation. (Mol Cell Biochem121: 149–153, 1993)     

Cerebral perfusion of metabolic inactivators: A new method for rapid fixation of labile lipid pools in brain

This paper describes a new method for the rapid fixation of labile lipid pools in the brain. Perfusion of the brain with 0.9% saline containing esterase inhibitors (p-bromphenacyl-bromide and diisopropyl fluorophosphate), an antioxidant (nordihydroguaiaretic acid) and a Ca²⁺ chelator (EDTA) resulted in a substantial reduction in the levels of free fatty acids, a biochemical marker for the degradation of labile membrane lipids. Levels of unesterified polyunsaturated fatty acids in whole brain were decreased by 90–96% as compared to levels in brains perfused with saline alone. Levels of docosahexaenoic acid approximated levels obtained after microwave irradiation. Unlike microwave irradiation, this perfusion technique perserves the cellular structure of the brain, thereby allowing subcellular fractionation with minimal postmortem changes in lipid pools. The release of arachidonic acid during isolation of the P₂ (synaptosomal) fraction was completely inhibited by the presence of the metabolic inactivators. The results of this study demonstrate a new and useful technique for the postmortem inactivation of enzymes responsible for the degradation of labile lipids in the brain. Further, the data underscore the key role of phospholipase A₂ and Ca²⁺ in mediating the release and accumulation of free fatty acids in the ischemic brain.Abbreviations 204 arachidonic acid - 226 docosahexaenoic acid - 160 palmitic acid - 180 stearic acid - 181 oleic acid - 182 linoleic acid - NDGA nordihydroguaiaretic acid - pBPB p-bromphenacylbromide - EDTA ethylenediamine-tetraacetic acid - DFP diisopropyl fluorophosphate - FFA free fatty acids - TLC thin layer chromatography - GLC gas liquid chromatography     

Inhibition by n-3 fatty acids of arachidonic acid metabolism in a primary culture of astroglial cells

Docosahexaenoic acid (226 n-3) was present in low concentrations in a primary culture of rat brain astroglial cells, when compared to brain cortex. We have thus supplemented these cells with this fatty acid and investigated the effects of its incorporation in cell phospholipids on the conversion of arachidonic acid, 204 n-6, through the cyclo and lipoxygenase pathways, after cell stimulation. Docosahexaenoic acid-enriched cells produced less thromboxane B₂ and 6-keto-Prostaglandin F₁ and markedly less 12-hydroxyeicosatetraenoic acid than unsupplemented cells, after stimulation with the Ca²⁺-ionophore A23187. The production of 15-hydroxyeicosatetraenoic acid from arachidonic acid was slightly increased in docosahexaenoic acid-supplemented cells. We have also supplemented these cells with eicosapentaenoic acid (205 n-3) and, in addition to accumulation of this fatty acid in cell phospholipids, we found elevation of 225 n-3 and some increment of 226, confirming that glial cells are able to convert eicosapentaenoic acid to the long chain, more unsaturated derivatives. In conclusion, n-3 fatty acids, when supplemented to glial cells, appear to modulate the arachidonic acid cascade and to be converted through the elongation and desaturation pathways.     

Fatty Acid Composition of Mitochondrial Membrane Lipids in Cultivated (Zea mays) and Wild (Elymus sibiricus) Grasses

The fatty acid composition of mitochondrial membrane lipids from seedlings of two grass species differing in their chilling tolerance, elymus (Elymus sibiricus) and maize (Zea mays), was studied by gas-liquid chromatography. An increased chilling tolerance of elymus, a perennial wild species, seems to be caused by a high content of unsaturated fatty acid residues in the total membrane lipids; linoleic (41.9%) and -linolenic (23.4%) acids predominated among these fatty acids. The contents of linoleic and -linolenic acids in the total lipids of maize membranes were 57.5% and 5.8%, respectively. Polar lipids of elymus mitochondrial membranes were characterized by about similar contents of linoleic (33.6%) and -linolenic (31.3%) acids. Linoleic acid (51.7%) predominated in maize mitochondrial polar lipids, whereas the -linolenic acid content was 9.0%. A high level of 3-desaturase activity in the mitochondrial membranes seems to be an important factor of elymus chilling tolerance. This high activity seems to be developed as an adaptation in the course of evolution of this species.     

Effect of nucleosides and nucleotides and the relationship between cellular adenosine 3′∶5′-cyclic monophosphate (cyclic AMP) and germ tube formation in Candida albicans

A yeast-mycelium (Y-M) transition in Candida albicans was induced by exogenous yeast extract, adenosine, adenosine 5-monophosphate (AMP), adenosine 5-diphosphate (ADP), adenosine 35 cyclic monophosphate (cAMP) and its analogue N⁶, O²-dibutyryl adenosine 35-cyclic monophosphate (dbcAMP) in defined liquid medium at 25°C. Adenosine 5-triphosphate (ATP) was found to delay germ tube formation in yeast cells, whereas the cAMP phosphodiesterase inhibitors, theophylline and caffeine, induced a Y-M transition. Intracellular and extracellular cyclic AMP levels increased during the yeast-mycelium transition and maximum levels of intracellular cyclic AMP coincided with maximum germ tube formation. Of the many inducers and inhibitors of germ tube and mycelium formation in C. albicans tested, including incubation at 37°C or in the presence of 1.5mM CaCl₂, the calmodulin inhibitor calmidazolium (R24571) added together with CaCl₂ induced the highest intra- and extracellular cyclic AMP levels. These results confirm the involvement of cyclic AMP in the yeast-mycelium transition of C. albicans.     

Developmental changes in the fatty acids of synaptic membrane phospholipids: Effect of protein malnutrition

The acyl-linked fatty acid composition of the major phospholipid species in rat cortical synaptic membranes was determined at various stages of development. For most species there was a decrease during development in the short chain saturated fatty acids, 140 and 160, an increase in 180 and 226 (n-3) and an increase in the ratio of 226 (n-3)/225 (n-6). Pups were protein deprived by feeding the dams a 12% casein diet as compared to the 24% casein control diet. Protein malnutrition markedly affected the composition of acyl-linked fatty acids in the synaptic membranes. The increases in the ratio of 226 (n-3)/225 (n-6) fatty acids were especially compromised.Abbreviations used (PC) Phosphatidylcholine - (PE) phosphatidylethanolamine - (EP) ethanolamine plasmalogen - (PS) phosphatidylserine - (PI) phosphatidylinositol - (SP) sphingomyelin - (t.l.c.) thin layer chromatography - (BHT) butylated-hydroxytoluene     

Das Fettsäurespektrum des Flußkrebses Orconectes limosus und seine Beeinflussung durch die Nahrung

Zusammenfassung Das Fettsäurespektrum von männlichen Flußkrebsen Orconectes limosus wurde gaschromatographisch analysiert. Die Fettsäuren zeigen eine spezifische Verteilung auf die Lipidklassen. Das Gesamtspektrum entspricht dem Schema eines Süßwassertieres im Winter mit den größten Fraktionen: C160, C181, C202 und C204 (zusammen rund 53% der Gesamtfettsäuren). In verschiedenen Fraktionen wurden ungeradzahlige und verzweigte Fettsäuren gefunden.Durch Verfüttern von Kartoffeln und Lebertran kann das Spektrum beeinflußt werden. Die Fettsäuren in der Mitteldarmdrüse, und in weniger starkem Ausmaß im Restkörper, haben sich nach viermonatiger Fütterung mit Lebertran qualitativ und quantitativ dessen Spektrum angepaßt. Nach siebenmonatiger Fütterung mit Kartoffeln zeigen Mitteldarmdrüse und Restkörper ein Spektrum, das dem eines unbehandelten Kontrollkrebses ähnelt und sowohl C182 als auch höher ungesättigte Fettsäuren enthält. Hierfür kommen zwei Deutungen in Frage: Entweder wird das Spektrum der Fettsäuren in Strukturlipiden aufrechterhalten, oder es erfolgt de novo Synthese einer C18:2^6,9-Fettsäure und deren Verlängerung analog dem von Wirbeltieren bekannten Weg.

---

The fatty acid composition in the crayfish, orconectes limosus, and the effect of nutrition

Summary The fatty acid composition in the male crayfish, Orconectes limosus, was analysed by gas-liquid chromatography. The fatty acids were found to have a specific distribution in different lipid classes. The composition corresponds to those of a fresh water animal in winter with the most important acids: C160, C181, C202, C204 (including about 53% of the total acids). In several lipid classes oddnumbered and branched chain fatty acids could be detected.Potatoe and fish-liver oil diets influence the fatty acid composition. After four months feeding with fish-liver oil the fatty acids in the hepatopancreas and to a lesser extent in the rest body show a similar spectrum as the fed oil. After feeding for seven months with potatoes hepatopancreas and rest body exhibit a fatty acid composition representative for the untreated animal, which contains C182 as well as higher unsaturated acids. These findings support the hypotheses that either the fatty acid composition in the structure lipids was maintained or that a C182^6,9-fatty acid was de novo synthesized and elongated analogous to the known vertebrate pathway.

---

---

Herrn Prof. Dr. K. Urich bin ich für die Anregung zu der vorliegenden Arbeit und für die Durchsicht des Manuskriptes zu großem Dank verpflichtet.     

Frequency-induced phase transitions in bimanual tapping

The stability of bimanual performance of the frequency ratios 38 and 58 was examined from the perspective of the sine circle map and the associated Farey mode-locking hierarchy. By gradually increasing movement frequency, abrupt transitions from the initial frequency ratios to other frequency ratios were induced. In general, transitions occurred to frequency ratios that were near the initial frequency ratio but lower in the Farey ordering and, hence, of higher stability in the sine circle map. A fair percentage of these transitions were to unimodularly related ratios. The transition routes from 38 and 58 remained largely unaffected by extensive practice of the lower-order ratios 25 and 35. Collectively, these results suggest that (i) bimanual tapping occurs in a domain in which frequency-locked states either overlap or are located sufficiently close to each other to make stochastic switching possible (coupling parameter K > 1 or close to 1); (ii) the overall stability of these frequency-locked states decreases as movement frequency increases (due to a decrease in K) and, consequently, (iii) the probability of transitions to nearby frequency ratios increases as movement frequency increases, due to the differential stability of the frequency locks.     

Circadian rhythms of corneal mitotic rate,retinal melatonin and immunoreactive visual pigments,and the effects of melatonin on the rhythms in the Japanese quail

We investigated circadian ocular rhythms in the Japanese quail, Coturnix coturnix japonica. The birds were placed under light-dark cycles (LD 1212), constant light (LL) and constant darkness (DD), and the retinas were dissected out at four-hour intervals throughout 24 h. Following measurements were performed. (1) Melatonin content in the retina was measured by radioimmunoassay. It was low in light and several folds higher in darkness under LD 1212. The rhythm continued in DD, but disappeared in LL. (2) Mitotic figures in the corneal epithelium were counted. Similar rhythms to the melatonin content were observed in the corneal mitotic rate with a slight phase delay. (3) The retinas were fixed at 4-h intervals and immunostained with anti-bovine rhodopsin serum and anti-chicken iodopsin monoclonal antibodies. The outer segments of photoreceptor cells were stained intensively throughout 24 h in LD 1212, LL and DD. In contrast, the stainability of the locus close to the outer limiting membrane where the Golgi apparatus exists changed diurnally. Scores showing the ratio of cells with positive staining indicated high values from 4 h after the onset of light to the beginning of dark phase under LD 1212. The values were high throughout 24 h in LL and intermediate or low in DD. (4) To investigate the effect of melatonin on the corneal mitotic rate and visual pigments at the Golgi region, melatonin was injected into one eye and saline into the contralateral eye. Melatonin induced a phase advance in the corneal mitotic rate under LD 1212, but did not induce a rhythm under LL. The ratio of photoreceptor cells with positive staining to anti-visual pigment antibodies at the Golgi region was not affected by melatonin injection.Abbreviations ANOVA analysis of variance - BSA bovine serum albumin - DD constant darkness - Io-mAb monoclonal antibodies against chicken iodospin - LD light-dark - LL constant light - mRNA messenger ribonucleic acid - PBS phosphate buffer solution - Rh-As antiserum against bovine rhodopsin - SCN suprachiasmatic nucleus - T transducin - T transducin      

Nicotiana chloroplast genome

Summary Using the existing restriction map and probes from wheat and pea ct-DNA, seven protein genes have been localized in the chloroplast genome of N. tabacum. On the clock-like map, the location of each gene is indicated by its time zone: the 15.2 kD polypeptide of the cytochrome b/f complex at 315, cytochrome f at 430, LS of RuBPCase at 450, both and subunits of ATP synthase at or near 500, proton-translocating subunit of ATP synthase at 820, subunit of ATP synthase at 840 and the 32 kD protein at 930. The genome organization of Nicotiana chloroplast DNA is similar to spinach.     

Molecular mapping of a new gene Rrs4 CI 11549 for resistance to barley scald (Rhynchosporium secalis)

Doubled haploid (DH) progeny from a cross between the scald susceptible barley (Hordeum vulgare L.) cultivar Ingrid and the resistant accession CI 11549 (Nigrinudum) was evaluated for resistance in the pathogen Rhynchosporium secalis (Oudem) J.J. Davis. Two linked and incompletely dominant loci confer resistance CI 11549 against isolate 4004. One is an allele at the complex Rrs1 locus on chromosome 3H close to the centromere; the other is located 22 cM distally on the long arm. The latter locus is designated Rrs4. In BC₃-lines into Ingrid from CI 2222 (another Nigrinudum) resistance seems governed by one locus close to the telomeric region of chromosome 7H, probably allelic to Rrs2. In neither case did we find any trace of the recessive gene rh8 reported to be present in Nigrinudum. Various resistance donors of Ethiopian origin designated as Nigrinudum, Jet or Abyssinian were identical to a great extent with respect to markers, but differed in resistance to different isolates of scald or in barley yellow dwarf virus (BYDV) resistance. The implications for their use as differentials in scald tests and screening of germplasm collections are discussed.     

Volume-sensitive taurine transport in fish erythrocytes

Summary Taurine plays an important role in cell volume regulation in both vertebrates and invertebrates. Erythrocytes from two euryhaline fish species, the eel (Anguilla japonica) and the starry flounder (Platichthys stellatus) were found to contain high intracellular concentrations of this amino acid ( 30 mmol per liter of cell water). Kinetic studies established that the cells possessed a saturable high-affinity Na⁺-dependent -amino-acid transport system which also required Cl^– for activity (apparentK _m (taurine) 75 and 80 m;V _max 0.85 and 0.29 mol/g Hb per hr for eel (20°C) and flounder cells (10°C), respectively. This -system operated with an apparent Na⁺/Cl^–/taurine coupling ratio of 211. A reduction in extracellular osmolarity, leading to an increase in cell volume, reversibly decreased the activity of the transporter. In contrast, low medium osmolarity stimulated the activity of a Na⁺-independent nonsaturable transport route selective for taurine, -amino-n-butyric acid and small neutral amino acids, producing a net efflux of taurine from the cells. Neither component of taurine transport was detected in human erythrocytes. It is suggested that these functionally distinct transport routes participate in the osmotic regulation of intracellular taurine levels and hence contribute to the homeostatic regulation of cell volume. Volume-induced increases in Na⁺-independent taurine transport activity were suppressed by noradrenaline and 8-bromoadenosine-3, 5-cyclic monophosphate, but unaffected by the anticalmodulin drug, pimozide.