Untersuchungen zur Anreicherung von Ergosterol und Ubichinonen aus Lipid-Kohlenwasserstoff-Fraktionen

Adsorption on Silicagel was used for the enrichment of ergosterol and ubiquinone-9 from a lipid-hydrocarbon extract. The main components of the lipid-hydrocarbon extract were hydrocarbons of gas oil (B. p. 513 to 653 K)phosphatides, glycerides, and fatty acids. By adsorption on silicagel (1 part silicagel to 2–3 parts of lipid-hydrocarbon extract dissolved in hexane) were ergosterol and ubiquinone-9 enriched and nearly completely separarated from hydrocarbons and phosphatides, partly from glycerides and fatty acids. Ergosterol and ubiquinone-9 can be obtained by a simplified separation from the enriched fraction.     

Untersuchungen zur Anreicherung von Ubichinon-9 aus Lipid-Kohlenwasserstoff-Franktionen mittels Sepahdex LH-20

Sephadex LH-20 was tested for a potential application in the enrichment of ubiquinone-9 from a lipid-hydrocarbon-extract. The lipid-hydrocarbon-extract was isolated from yeast grown on gas oil (b. p. 240 to 380°C) as carbon-source. The main components of the lipid-hydrocarbon extract were phosphatides, glycerides, fatty acids, and hydrocarbons of gas oil. By use of Sephadex LH-20 and CHCl₃/CH₃OH (2:1)as eluent it is possible to concentrate the whole ubiquinone-9 in a special fraction both directly from the lipid-hydrocarbon-extract and from the aceton-soluble part of the extract The concentration of ubiquinone in the special fraction was ten times higher than in the raw-material and the hydrocarbons were separated completely. Ubiquinone-9 can be obtained by a simplified separation from the enriched fraction.     

Antitumor activity of the lipid fraction of the spores of an anaerobic bacterium <Emphasis Type="Italic">Clostridium butyricum</Emphasis>

Antitumor activity of the preparation of the lipid fraction of Clostridium butyricum spore extract was demonstrated in vivo on a transplantable mouse model of breast cancer. At a specific scheme of application, inhibition of tumor growth and improved survival dynamics compared to the control group were observed. Thin-layer chromatography (TLC) of the lipid fraction of the spore extract revealed, apart from a saturated hydrocarbon, cholesterol ester, cetyl palmitate, triacyl glycerol, and palmitic acid, also a phenolic lipid bound in a complex with a peptide component. Acetone extraction of the lipid pool revealed a phenolic lipid. According to the TLC and ¹H-NMR spectrum of the acetone extract of the lipid fraction of C. butyricum spores, the structure of the phenolic lipid was proposed, n -butyl benzoate substituted in the para position. The phenolic lipid is suggested to be responsible for the biological activity of the spore’s lipid fraction.     

Rôle of phagostimulants of cotton leaves in the feeding behaviour of Spodoptera littoralis

Petroleum ether extract of cotton leaves was the most attractive to the hatched larvae of Spodoptera littoralis, and it remained so even after removal of the chlorophyll and other pigments. Steam distillation of this filtrate gave a volatile oil which also proved highly attractive to the larvae. In this fraction, 12 components were identified by thin-layer chromatography. Of these components, α-terpineol, citronellol and α-pinene were the most attractive, whereas humulene and linalool were less attractive. The remaining non-volatile lipid fraction was also attractive to the larva. In this fraction the unsaponifiable matter contained the attractive ingredients. This fraction proved to be a hydrocarbon. The infrared spectrum and nuclear magnetic resonance showed only typical hydrocarbon bonds.     

Effect of stage of development on chemical yields in smooth sumac,Rhus glabra L.

Smooth sumac (Rhus glabra L.) has been identified as a potential source of polyphenols, oil, and polymeric hydrocarbon for the chemical industry. The effect of stage of development (prebud, full bud, full flower and seed set) on acetone extract (‘polyphenols’ and ‘oil’) and hexane extract (polymeric hydrocarbon) yields was studied in two Beltsville, Maryland (USA) populations in 1982 and 1983. Most extract was found in the leaves, but a substantial amount was also present in stem tissue. Leaf fraction acetone extract yields were generally highest at the full-flower stage and stem yields were generally highest at the seedset stage. Leaf fraction hexane extract yield was highest at the seed-set stage whereas stage of development had no consistent effect on stem fraction yields. For Population 2, there was a fairly strong inverse relationship between yields of leaf fraction acetone and leaf fraction hexane extract both years, and, for Population 1, a fairly strong direct relationship between yields of stem fraction acetone extract and stem fraction hexane extract in 1982. Data indicate that harvesting at the full-flower stage would maximize acetone extract yields, whereas harvesting at the seed-set stage would maximize hexane extract yields.     

Antiphytovirale Aktivität von lipophilen Fraktionen aus der Hefe Lodderomyces elongisporus IMET H 128

Lipid extract isolated from biomasses of Lodderomyces elongisporus IMET H 128 grown on gas oil 7(B.p. 240–380°C) and fractions of the lipid extract (acetone soluble and fatty acid fractions) reduced the concentration of potato virus X in inoculated as well as in secondarily infected leaves markedly. The antiphytoviral inactive phosphatide fraction was converted into a fraction with high activity by partial hydrolysis with SO₂ as acting agent.     

On the localization of ubiquinone in phosphatidylcholine bilayers

The location of ubiquinone-10 in phospholipid bilayers was analyzed using a variety of physical techniques. Specifically, we examined the hypothesis that ubiquinone localizes at the geometric center of phospholipid bilayers. Light microscopy of dipalmitoylphosphatidylcholine at room temperature in the presence of 0.05-0.5 mol fraction ubiquinone showed two separate phases, one birefringent lamellar phase and one phase that consisted of isotropic liquid droplets. The isotropic phase had a distinct yellow color, characteristic of melted ubiquinone. [13C]NMR spectroscopy of phosphatidylcholine liposomes containing added ubiquinone indicated a marked effect on the 13C-spin lattice relaxation times of the lipid hydrocarbon chain atoms near the polar head region of the bilayer, but almost no effect on those atoms nearest the center of the bilayer. X-ray diffraction experiments showed that for phosphatidylcholine bilayers, both in the gel and liquid-crystal-line phases, the presence of ubiquinone did not change either the lamellar repeat period or the wide-angle reflections from the lipid hydrocarbon chains. In electron micrographs, the hydrophobic freeze-fracture surfaces of bilayers in the rippled (P beta') phase were also unmodified by the presence of ubiquinone. These results indicate that the ubiquinone which does partition into the bilayer is not localized preferentially between the monolayers, and that an appreciable fraction of the ubiquinone forms a separate phase located outside the lipid bilayer.     

Hemagglutinating Property of Haemophilus aegyptius

Extracts possessing the capacity to hemagglutinate normal human erythrocytes were recovered from Haemophilus aegyptius by treatment with either diethylene glycol or acetone. Antisera prepared against these extracts or the unextracted bacterial cell inhibited hemagglutination by homologous and heterologous antigens. Microgel diffusions indicated the presence of identical components in each extract as expressed by lines of identity between antisera to each fraction. The hemagglutinin was identified as a lipopolysaccharide, 42% lipid and 57% carbohydrate. The determination of 6% phosphorus in the lipid fraction identified it as containing phospholipid.     

On the localization of ubiquinone in phosphatidylcholine bilayers

The location of ubiquinone-10 in phospholipid bilayers was analyzed using a variety of physical techniques. Specifically, we examined the hypothesis that ubiquinone localizes at the geometric center of phospholipid bilayers. Light microscopy of dipalmitoylphosphatidylcholine at room temperature in the presence of 0.05–0.5 mol fraction ubiquinone showed two separate phases, one birefringent lamellar phase and one phase that consisted of isotropic liquid droplets. The isotropic phase had a distinct yellow color, characteristic of melted ubiquinone. [¹³C]NMR spectroscopy of phosphatidylcholine liposomes containing added ubiquinone indicated a marked effect on the ¹³C-spin lattice relaxation times of the lipid hydrocarbon chain atoms near the polar head region of the bilayer, but almost no effect on those atoms nearest the center of the bilayer. X-ray diffraction experiments showed that for phosphatidylcholine bilayers, both in the gel and liquid-crystal-line phases, the presence of ubiquinone did not change either the lamellar repeat period or the wide-angle reflections from the lipid hydrocarbon chains. In electron micrographs, the hydrophobic freeze-fracture surfaces of bilayers in the rippled (Pβ′) phase were also unmodified by the presence of ubiquinone. These results indicate that the ubiquinone which does partition into the bilayer is not localized preferentially between the monolayers, and that an appreciable fraction of the ubiquinone forms a separate phase located outside the lipid bilayer.     

A comparison of colicines K and V extracted from solid medium

Following the extraction of colicine K and colicine V from digest nutrient agar, the crude colicine was divided into 3 portions. Each portion was subjected to a primary treatment with either 30% chloroform, 90% acetone or 66% absolute alcohol. Aliquots of the active fractions obtained from each of these primary treatments were subsequently exposed to either of the other two extracants. It was noted that the best method for primary purification of both colicines was blending with chloroform but the results of subsequent fractionation differed. Colicine K was insoluble in 66% absolute alcohol whereas colicine V remained soluble in alcohol but was precipitated by 90% acetone.Mouse toxicity tests revealed that the toxic fraction of the crude colicine V was precipitated by 66% alcohol and that the non-toxic fraction, soluble in alcohol, was associated with the activity of the colicine. All the active fractions of crude colicine K were lethal for mice.We wish to express our appreciation to the various colleagues who were kind enough to send the colicinogenic or the indicator strains used in this investigation. This research was assisted in part by a grant HERT 215 from the Scottish Hospital Endowment Research Trust and partly by a grant from the Duff Fund. We are grateful to Mrs. P. Hercus for technical assistance.     

Cloning and functional expression of the dps gene encoding decaprenyl diphosphate synthase from Agrobacterium tumefaciens

A newly isolated gene from Agrobacterium tumefaciens (A. tumefaciens), which encoded a decaprenyl diphosphate synthase, was cloned in Escherichia coli (E. coli), and its nucleotide sequence was determined. DNA sequence analysis revealed an open reading frame of 1077 bp capable of encoding a 358-amino-acid protein with a calculated isoelectric point of pH 5.16 and a molecular mass of 38 960 Da. The primary structure of the enzyme shared significant homology with prenyl diphosphate synthases from various sources. The deduced amino acid sequence included oligopeptide DDxxD aspartate-rich domains conserved in the majority of prenyl diphosphate synthases. High levels of the active enzyme were expressed in the soluble fraction and were readily purified to homogeneity by Ni-NTA chromatography. E. coli JM109 harboring the dps gene produced ubiquinone-10 in addition to endogenous ubiquinone-8, while E. coli JM109 harboring the dps gene mutated on the DDxxD domain lost the ability to produce ubiquinone-10, which suggests that the A. tumefaciens dps gene is functionally expressed in E. coli and that it encodes a decaprenyl diphosphate synthase.     

Aryl hydrocarbon hydroxylase: Degradation of product due to contaminant in acetone

A contaminant present in reagent grade acetone causes degradation of fluorescent phenolic metabolites of benzo(a)pyrene as measured in the aryl hydrocarbon hydroxylase assay. Although the contaminant was not identified, its properties suggest that it is a relatively volatile organic material, possibly an oxidizing agent. The acetone may be readily purified by distillation.     

Stilbene derivatives from Gnetum gnemon Linn

Four stilbene derivatives, gnemonols K and L (resveratrol trimers), M (isorhapontigenin dimer), and gnemonoside K (glucoside of resveratrol trimer) together with eleven known stilbenoids and a lignan were isolated from the acetone, methanol and 70% methanol soluble parts of the root of Gnetum gnemon (Gnetaceae). The structures of the isolates were determined by spectral analysis. The antioxidant activity of the stilbenoids on lipid peroxide inhibition and super oxide scavenging activity were also investigated.     

Fluoranthene, a volatile mutagenic compound, present in creosote and coal tar

Creosote, a coal-tar distillation product, contains mutagens which are volatile at 37 degrees C. After distillation of creosote we found that these volatile mutagens were present in the distillation fraction with the highest boiling range (greater than 360 degrees C). The "volatile mutagenic activity" was connected with the presence of fluoranthene, a polycyclic aromatic hydrocarbon. Commercially available fluoranthene was positive in the so-called "taped-plate assay" (the test system used for the detection of volatile mutagens) towards the strains TA98 and TA100 in the presence of S9 mix. The tested creosote and coal tar contained fluoranthene in concentrations of 5.2 and 2.2%, respectively.     

Nonaprenyl-4-hydroxybenzoate transferase, an enzyme involved in ubiquinone biosynthesis, in the endoplasmic reticulum-Golgi system of rat liver

The properties and distribution of nonaprenyl-4-hydroxybenzoate transferase in rat liver were investigated with subcellular fractions, liver perfusion, and in vivo labeling with [3H]solanesyl-PP. In addition to some ubiquinone-9, only one labeled intermediate, i.e. nonaprenyl-4-hydroxybenzoate, was obtained. In the total microsomal fraction, the enzyme had a pH optimum of 7.5 and was completely inhibited by Triton X-100 and deoxycholate, but not by taurodeoxycholate and beta-octyl glucoside. Liver, kidney, and spleen demonstrated the highest activities of nonaprenyl-4-hydroxybenzoate transferase. Upon subcellular fractionation, high specific activities were found in smooth II microsomes and Golgi III vesicles. The enzyme was also found in lysosomes and plasma membranes, but only at low levels in rough and smooth I microsomes and mitochondria and not at all in peroxisomes and cytosol. When the product of the transferase reaction was used as a substrate in vitro and in a perfusion system, the only product obtained was end product ubiquinone-9. Although the transferase reaction was associated with the inner, luminal surface of microsomal vesicles, the terminal reaction(s) for ubiquinone-9 synthesis are found at the outer cytoplasmic surface. The results suggest that the major site for ubiquinone synthesis is the endoplasmic reticulum-Golgi system, which also participates in the distribution of ubiquinone-9 to other cellular membranes.     

Zur Gewinnung von Ubichinon-10 aus Acetobacter methanolicus IMET B 346

Mixtures of acetone/water (93 : 7 ··· 90 : 10, v/v) are favoured solvents for the extraction of ubiquinone-10 from Acetobacter methanolicus IMET B 346. Using these solvent mixtures ubiquinone-10 was extracted nearly completely. Other lipids were extracted partially. Extracts were obtained by optimal conditions with a ubiquinone content > 5%.     

Potent inhibitors of glucagon-stimulated adenylate cyclase associated with serum lipoprotein particles

Lipoprotein fractions from some individuals have inhibitory effects on rat liver adenylate cyclase. Precipitation of the lipoprotein fractions with acetone released an inhibitory factor, which was soluble in acetone-H2O (3:1, v/v). The inhibition was greater against glucagon-stimulated activity than against basal activity. Acetone extraction increased the potency of inhibition. All three lipoprotein fractions, i.e., very low, low, and high density lipoproteins, released some inhibitory component after acetone extraction. The inhibitor was concentrated in the lipoprotein fractions, since acetone extraction of plasma did not release an inhibitor. The acetone extract from the very low density lipoprotein was the most inhibitory. This material was further purified and partially characterized. The inhibitor had a molecular mass of about 500. It was inhibitory at micromolar concentrations. The material was sufficiently hydrophobic to migrate in normal-phase thin-layer chromatography (TLC). Nuclear magnetic resonance results indicated that it was not a polar lipid. There were several different inhibitory factors that were separable by TLC. The sequestration of these inhibitors into lipoproteins reduced their effectiveness in inhibiting the action of counter-regulatory hormones, such as glucagon.     

Note: Antioxidant properties of lipid fractions from Deinococcus radiophilus strain UWO1055

Lipids of the radio-resistant bacterium Deinococcus radiophilus were tested for their antioxidant properties. The crude lipid extract showed a significant antioxidant effect in linoleic acid emulsion. The crude extract was separated to polar and non-polar lipid fractions. The non-polar fraction showed an antioxidant effect in both suspensions and emulsions of linoleic acid, and inhibition of oxidation in a β-carotene emulsion. Lipids of the non-polar fraction were separated and their antioxidant activity was determined in a β-carotene emulsion; the lipid that was marked NP9 showed the highest antioxidant effect. Lipid NP9 inhibited oxidation in a β-carotene emulsion in the concentration range of 5–51 ppm. It is suggested that the antioxidant activity of lipids of D. radiophilus contribute to its radio-resistance.     

The use of tetrahydrofuran for delipidation and water solubilization of brain proteolipid proteins.

Tetrahydrofuran, an efficient total lipid solvent, was found to precipitate chloroform-methanol soluble proteins from CM (chloroform-methanol, 2:1) lipid extracts of bovine and rat white and grey matter. To total lipid extracts containing proteolipid proteins 4 volumes of tetrahydrofuran are added; the precipitate is centrifuged and washed once with the same solvent. The product contains all the protein and about 1 to 2% of the lipids of the original lipid extract. It is insoluble in CM; solubility in CM is restored by addition of acetic acid or of the lipids recovered from the tetrahydrofuran-soluble fraction It is also soluble in glacial acetic acid and this solution can be diluted either with CM or with distilled water.     

Identification of sn-2-omega-hydroxycarboxylate-containing phospholipids in a lipid extract from bovine brain

Phospholipids having both a long-chain acyl (palmitoyl or stearoyl) and a short-chain hydroxycarboxylyl (C3-C9) residue were identified by GC-MS in a fraction with PAF-like activity from a bovine brain lipid extract. The hydroxyl group in the hydroxycarboxylate residue was determined to be at the omega-position by comparison of the mass spectra of the tert-butyl-dimethylsilyl derivatives of these compounds with those of synthetic hydroxybutyrate-containing phosphatidylcholines. The co-existence of short-chain hydroxycarboxylate-, monocarboxylate- and dicarboxylate-containing phospholipids in the bovine brain lipid extract suggested that these compounds were formed by peroxidation of membrane phospholipids, especially phosphatidylcholines.