Biosynthesis of the beta-diketones of barley spike epicuticular wax.

[1-¹⁴C]acetate and [2-¹⁴C]acetate were incorporated into the β-diketones of barley spike epicuticular wax via the peduncle. Utilizing column chromatography with dry copper acetate, the β-diketones were isolated and the labeling pattern in the hentriacontan-14, 16-dione determined after its degradation. A modified iodoform procedure was used to give myristic and palmitic acids. Radio-gas chromatography was then performed on the products of chemical α-oxidation of the separated fatty acids. This procedure, in effect, gave the specific activity of every carbon atom of hentriacontan-14,16-dione except carbon-1 to carbon-5 (from myristic acid) and carbon-27 to carbon-31 (from palmitic acid) for each labeled substrate. The specific activity of carbon-15 was determined by an indirect method. On the basis of these data it is suggested that the hentriacontan-14,16-dione is synthesized from the carbon-31 end of the molecule by elongation as follows. C₂ units are added, perhaps to a mixture of short chain precursors, to give a chain with 12 carbon atoms. This chain is then elongated to one with 16 carbon atoms so that the four added carbon atoms are uniformly labeled. Following this, the chain with 16 carbon atoms is elongated with C₂ units to give the complete molecule. Possibly some change in mechanism occurs in this last elongation process when the chain is 22 carbon atoms long. Barley spike wax β-diketones contain about 2% nonacosan-13, 15-dione which seems to be synthesized in an analogous manner.     

Growth and Potential Damage of Human Bone-Derived Cells Cultured on Fresh and Aged C60/Ti Films

Thin films of binary C₆₀/Ti composites, with various concentrations of Ti ranging from ~ 25% to ~ 70%, were deposited on microscopic glass coverslips and were tested for their potential use in bone tissue engineering as substrates for the adhesion and growth of bone cells. The novelty of this approach lies in the combination of Ti atoms (i.e., widely used biocompatible material for the construction of stomatological and orthopedic implants) with atoms of fullerene C₆₀, which can act as very efficient radical scavengers. However, fullerenes and their derivatives are able to generate harmful reactive oxygen species and to have cytotoxic effects. In order to stabilize C₆₀ molecules and to prevent their possible cytotoxic effects, deposition in the compact form of Ti/C₆₀ composites (with various Ti concentrations) was chosen. The reactivity of C₆₀/Ti composites may change in time due to the physicochemical changes of molecules in an air atmosphere. In this study, we therefore tested the dependence between the age of C₆₀/Ti films (from one week to one year) and the adhesion, morphology, proliferation, viability, metabolic activity and potential DNA damage to human osteosarcoma cells (lines MG-63 and U-2 OS). After 7 days of cultivation, we did not observe any negative influence of fresh or aged C₆₀/Ti layers on cell behavior, including the DNA damage response. The presence of Ti atoms resulted in improved properties of the C₆₀ layers, which became more suitable for cell cultivation.     

Identification by gas chromatography and mass spectrometry of lipids from the rat Harderian gland

Lipids from rat Harderian glands were extracted with ethyl acetate, hydrolysed with base and examined by gas chromatography (GC) and gas chromatography—mass spectrometry (GC—MS) as trimethylsilyl (TMS), [²H₉]TMS, methyl ester—TMS, picolinyl, nicotinate and nicotinylidene derivatives. The latter three derivatives were used to reveal the structures of the alkyl chains of fatty acids, alcohols and glycerol ethers, respectively. Forty-eight compounds were identified, representing about 97% of the total extracted lipids as measured by GC peak areas. The major constituents were fatty acids with chain lengths from 12 to 22 carbon atoms (mainly C₁₈ and C₂₀) and fatty alcohols (C₁₆ to C₂₆) derived from wax esters. Most of these acids and alcohols were unsaturated in the ω-7 position and were accompanied by smaller amounts of the saturated and ω-5 monounsaturated analogues. Glycerol ethers were also identified for the first time in this secretion; the ether chains contained from 14 to 19 carbon atoms (mainly 16) and were straight-chain saturated, unsaturated (ω-5 and ω-7) and branched (iso). The only sterol found was cholesterol amounting to 1.24% of the total extract.     

Branched Chain Amino Acid Metabolism in the Biosynthesis of Lycopersicon pennellii Glucose Esters

Lycopersicon pennellii Corr. (D'Arcy) an insect-resistant, wild tomato possesses high densities of glandular trichomes which exude a mixture of 2,3,4-tri-O-acylated glucose esters that function as a physical impediment and feeding deterrent to small arthropod pests. The acyl moieties are branched C₄ and C₅ acids, and branched and straight chain C₁₀, C₁₁, and C₁₂ acids. The structure of the branched acyl constituents suggests that the branched chain amino acid biosynthetic pathway participates in their biosynthesis. [¹⁴C]Valine and deuterated branched chain amino acids (and their oxo-acid derivatives) were incorporated into branched C₄ and C₅ acid groups of glucose esters by a process of transamination, oxidative decarboxylation and subsequent acylation. C₄ and C₅ branched acids were elongated by two carbon units to produce the branched C₁₀-C₁₂ groups. Norvaline, norleucine, allylglycine, and methionine also were processed into acyl moieties and secreted from the trichomes as glucose esters. Changes in the acyl composition of the glucose esters following sulfonylurea herbicide administration support the participation of acetohydroxyacid synthetase and the other enzymes of branched amino acid biosynthesis in the production of glucose esters.     

Substrate specificity of acyl-lipid Δ9-desaturase from <Emphasis Type="Italic">Prochlorothrix hollandica</Emphasis> cyanobacterium producing myristoleic acid

Chlorophyll b-containing cyanobacterium Prochlorothrix hollandica is characterized by a high content of esterified fatty acids (FA) with 14 and 16 carbon atoms in the membrane lipids. Depending on the conditions of cultivation, the relative amount of myristic (C_14:0) and myristoleic (C_14:1) acids can reach 35%, and palmitic (С_16:0) and palmitoleic (С_16:1) acids can reach 60% of the sum of all fatty acids in cells. Monounsaturated FAs are represented by C_14:1, and C_16:1 with an olefinic bond presumably located in the Δ⁹ position. We cloned the gene of acyl-lipid Δ9-desaturase, desC1, from Prochlorothrix hollandica and characterized its specificity to the length of the substrate using the heterologous expression in Escherichia coli cells adding C_14:0 or stearic (C_18:0) acids as exogenous substrates. The results show that DesC1 Δ⁹ desaturase generates olefinic bonds in the FAs with a length of 14 to 18 carbon atoms with an approximately equal efficiency. This indicates that the length of the FA chain in P. hollandica is determined by the activity of the FA synthase, and the chain is desaturated at the Δ⁹ position nonspecifically relatively to its length.     

Identification of In-Chain-Functionalized Compounds and Methyl-Branched Alkanes in Cuticular Waxes of Triticum aestivum cv. Bethlehem

In this work, cuticular waxes from flag leaf blades and peduncles of Triticum aestivum cv. Bethlehem were investigated in search for novel wax compounds. Seven wax compound classes were detected that had previously not been reported, and their structures were elucidated using gas chromatography-mass spectrometry of various derivatives. Six of the classes were identified as series of homologs differing by two methylene units, while the seventh was a homologous series with homologs with single methylene unit differences. In the waxes of flag leaf blades, secondary alcohols (predominantly C₂₇ and C₃₃), primary/secondary diols (predominantly C₂₈) and esters of primary/secondary diols (predominantly C₅₀, combining C₂₈ diol with C₂₂ acid) were found, all sharing similar secondary hydroxyl group positions at and around C-12 or ω-12. 7- and 8-hydroxy-2-alkanol esters (predominantly C₃₅), 7- and 8-oxo-2-alkanol esters (predominantly C₃₅), and 4-alkylbutan-4-olides (predominantly C₂₈) were found both in flag leaf and peduncle wax mixtures. Finally, a series of even- and odd-numbered alkane homologs was identified in both leaf and peduncle waxes, with an internal methyl branch preferentially on C-11 and C-13 of homologs with even total carbon number and on C-12 of odd-numbered homologs. Biosynthetic pathways are suggested for all compounds, based on common structural features and matching chain length profiles with other wheat wax compound classes.     

Epicuticular hydrocarbons of Drosophila pseudoobscura (Diptera; Drosophilidae) Identification of unusual alkadiene and alkatriene positional isomers

Epicuticular hydrocarbons of Drosophila pseudoobscura were analyzed by gas chromatography and mass spectrometry. Methyl-branched alkanes and alkadienes were the predominant hydrocarbons, with lesser amounts of monoenes (14%) and trienes (9%) also present. Alkanes (49%) were mostly odd carbon number 2-methylalkanes (C₂₅–C₃₁). Alkadienes (27%) were odd carbon number components (C₂₅–C₃₃), with the (Z,Z)-5,9-isomer predominating. Monounsaturated hydrocarbons were a mixture of 5-, 7-, 9-, 11-, 13-, 14- and 15-isomers containing 25–33 carbon atoms. The major alkatriene components contained 29–31 carbon atoms and were either 5,19,17- or 5,9,19-isomers. Sodium[1-¹⁴C]acetate was incorporated into each class of hydrocarbon and into each of the major alkadienes.     

Fatty acids and long-chain bases of gangliosides of human gastrointestinal mucosa

The fatty acid and long-chain base composition of five major gangliosides from human stomach and small and large intestine mucosa were analyzed with gas chromatography. All the gangliosides greatly resembled each other in the fatty acid pattern. The main fatty acids were C_16:0, C_18:0 and C_24:0. No hydroxy fatty acids could be detected. In all the gangliosides 4-sphingenine was the predominant long-chain base (70–75%). About 15% of the long-chain bases had 20 carbon atoms in their chain. No trihydroxy long-chain bases could be detected.     

Long-chain and very long-chain methyl-branched alcohols and their acetate esters in pupae of the tobacco hornworm,Manduca sexta

Four homologous series of very long-chain methyl-branched alcohols (VLMA, C₃₈ to >C₄₄) were found in the internal lipids of developing male pupae of the tobacco hornworm, Manduca sexta, both as free alcohols and as acetate esters. The four major homologous series, with carbon chain backbones of 36–44 carbon atoms, consisted of a monomethyl, two dimethyls and a trimethyl-branched alcohol series. The major alcohol of each homologous series (with the corresponding alkane obtained by reduction in parentheses) was identified as 24-methyltetracontan-1-ol (17-methyltetracontane), 24,28-dimethyltetracontan-1-ol (13,17-dimethyltetracontane), 22,34-dimethyloctatriacontan-1-ol (5,17-dimethyloctatriacontane) and 22,26,34-trimethyloctatriacontan-1-ol (5,13,17-trimethyloctatriacontane). The minor components of the VLMA had backbones with an odd number of carbon atoms (37, 39, 41 and 43). Methyl branches of the minor components were identified on the 18- and 14,18-positions when numbered from the alkyl end of the chain. Also identified were minor amounts of long-chain methyl-branched alcohols (LMA, C₂₅ to C₃₂). The major components in the “wax ester” TLC fraction were acetate esters of the LMA and VLMA.     

Enzymatic fractionation of carbon isotopes by phosphoenolpyruvate carboxylase from c(4) plants

The carbon atoms of glucose and malate in C₄ plants are 2 to 3‰ enriched in ¹²C with respect to atmospheric CO₂; whereas these intermediates in C₃ plants are 15 to 18‰ enriched with ¹²C with respect to atmospheric CO₂. The enzymatic synthesis of malate from phosphoenolpyruvate and bicarbonate in preparations of leaves of Sorghum bicolor, Haygrazer result in a carbon isotope fractionation of about 3‰. The enzymatic synthesis of phosphoglyceric acid from ribulose 1,5-diP and CO₂ in these preparations (contaminated with carbonic anhydrase) at 24 C and 37 C result in a carbon isotope fractionation of 33.7‰ and 18.3‰, respectively. These data are consistent with the conclusion that the small enrichment of ¹²C in the carbon atoms of malate and glucose (with respect to atmospheric CO₂) in leaves of Sorghum bicolor, Haygrazer occurs at the phosphoenolpyruvate carboxylase step.     

RE‐EXAMINATION OF THE DOUBLE BOND POSITIONS IN ALKENONES AND DERIVATIVES: BIOSYNTHETIC IMPLICATIONS1

By derivatizing with OsO₄, we determined the double bond positions of all the diunsaturated alkenones present in different haptophytes and demonstrated that the positions in C₃₇–C₄₀ homologs occur at a fixed carbon number from the carbonyl group, a finding contrary to earlier speculation. These data have allowed us to recognize three “families” of alkenones, for which we propose different biosynthetic pathways. The proposed biosynthesis of classical C₃₇–C₄₀ alkenones starts with acetyl‐ or propionyl‐SCoA and involves classical chain elongation steps with malonyl‐ and methylmalonyl‐SCoA affording alkanoyl‐ACP intermediates, which undergo subsequent Δ^{14, 21}‐desaturation and decarboxylation reactions. Unusual diunsaturated shorter‐chain (C₃₅ and C₃₆) ketones now being produced for yet unknown physiological reasons by our specimen of Emiliania huxleyi (Lohm). Hay et Mohler strain CCMP1742. These compounds exhibit ω15,22 and ω16,23 double bonds, which are at a shorter distance (two carbon atoms) from the carbonyl group than the higher homologs, implying that their biosynthesis must involve an additional chain‐shortening step after Δ^{14, 21}‐desaturation of alkanoyl‐ACP. Alkenones exhibiting a very unusual double bond spacing (three methylene groups instead of five) were detected in Holocene Black Sea sediments, in particulate matter collected in the Ligurian Sea and in Gephyrocapsa oceanica strain JB‐02. The formation of these compounds seems to be linked to the biosynthesis of monounsaturated alkenones previously detected in several haptophytes. Our work demonstrates the value of determining the double bond locations of alkenones whenever possible.     

Use of sodium dodecyl sulphate for stimulation of biodegradation of n-alkanes without residual contamination by the surfactant

The capacity of a range of aliphatic alkanes (C₆–C₁₆), intermediates of n-decane oxidation and sodium dodecyl sulphate (SDS) to induce decane-mineralization activity in the cells of Pseudomonas C12B was compared with that for n-decane. The comparison on quantitative basis had two serious limitations: low solubility of tested inducers in aqueous solutions and their toxicity to bacterial cells. Carbon chain length and the presence of hydroxyl group were the important factors for induction activity. However, presence of hydroxyl groups at both ends of alkyl chain prevented the induction of decane-mineralization activity. Good induction activity by SDS was caused either by the presence of free end of alkyl chain, or by bacterial hydrolysis of sulphate group to yield alcohol, which in turn served as true inducer. The presence of SDS in the culture medium with n-decane as main source of carbon and energy accelerated the growth of Pseudomonas C12B. SDS disappeared from the culture medium in early stages of cultivation suggesting preferential degradation by the bacterium, while the consumption of n-decane was accelerated. This may be associated with the capacity of SDS to induce decane-mineralization system in Pseudomonas C12B and/or with the ability of SDS to stimulate the surface attachment of competent bacteria resulting in the close proximity of the cells with alkane droplets, and thus, enhanced breakdown of the hydrocarbon pollutant.     

An inspection into the class of bis(alkyne)-undecacarbonyl-quadro-tetraruthenium complexes. Crystal structure and dynamic behaviour of Ru4(CO)11 (μ4, η2-MeC2Ph)2

The reactions of the butterfly complex Ru₄(CO)₁₂(MeC₂Ph) with several alkynes give the quasiplanar derivatives Ru₄(CO)₁₁(MeC₂Ph)(Alkyne) in almost quantitative yields.The structure of Ru₄(CO)₁₁(MeC₂Ph)₂ has been determined by X-ray methods. Crystals are monoclinic, space group C2/c, with Z = 4 in a unit cell of dimensions a 22.383(16), b 9.048(8), c 18.268(12) Å, β = 127.25(4)°. The structure has been solved from diffractometer data by Patterson and Fourier methods and refined by full-matrix least-squares to R = 0.034 for 1420 observed reflections. The complex, having an imposed C₂ symmetry, presents a tetranuclear metal cluster in which the Ru atoms are in a tetrahedrally-distorted square arrangement. Ten carbonyls are terminal and one symmetrically bridges an edge of the cluster. Each of the two alkyne ligands is σ-bonded to two Ru atoms on the opposite vertices of the cluster and π-bonded to the other two. The organometallic cluster has a Ru₄C₄ core in which the metal and carbon atoms occupy the vertices of a triangulated dodecahedron.     

Layer-type complexes consisting of VOSO4 or VOPO4 and aliphatic alcohols

The intercalates of unhydrated VOSO₄ and VOPO₄ with alcohols (C₁ to C₈) have been prepared and characterized by their compositions, basal spacings and thermal stabilities. X-ray powder studies show that the layered structures of VOSO₄ and VOPO₄ lattices are maintained in these complexes. The alcohol molecules are penetrated into van der Waal's gaps of the host lattices and form bimolecular layers. The observed alternation of the basal spacing increments with the number of carbon atoms of the linear aliphatic chain is explained from the arrangement of these chains.     

Cellular fatty acid composition ofCorallococcus coralloides

The fatty acid composition of five strains ofCorallococcus coralloides and three reference species ofMyxococcus were determined by gas-liquid chromatography. Methyl esters of fatty acid containing from 12 to 22 carbon atoms were identified. The major fatty acids present were C₁₅ and C₁₇ saturated branched chain, and both C₁₆ saturated and unsaturated straight chain acids. The C₁₇ saturated branched and straight chain acids, which were in valuable concentration in species ofMyxococcus, were not, however, detected in all strains ofC. coralloides. The application of these results in the distinction ofC. coralloides from the genusMyxococcus is discussed.     

A structure-function correlation for fatty acids inSaccharomyces cerevisiae

A relationship which distinguishes those fatty acids that support the growth of oxygen-deprivedSaccharomyces cerevisiae from those that do not was found. To function properly, a long chain of saturated carbon atoms appears to require interruption by an appropriate chemical group such that only a maximum number of contiguous saturated carbon atoms is present anywhere in the chain. A double bond was found to serve as an interrupting group, and for 19cis-unsaturates studied, ranging from C _14:1-Δ⁹ to C_22:6-Δ^{4,7,10,13,16,19}, the number of saturated C atoms was 9. The chain length and the position and number of double bonds had no influence in determining whether the acids were active or inactive except as these structural features related to the interruption rule. Although less extensively examined, a hydroxyl group ortrans-double bond also appeared to act as an interrupting group with allowed numbers of 9 and 7, respectively, for saturated C atoms. Oxygen deprivation did not result in a shift to shorter chain length of the saturates formed, and the presence of unsaturates did not prevent biosynthesis of saturates.     

Polyester Biosynthesis Characteristics of Pseudomonas citronellolis Grown on Various Carbon Sources, Including 3-Methyl-Branched Substrates

Forty-two different carbon sources were tested for the polyester synthesis of a citronellol-utilizing bacterium, Pseudomonas citronellolis (ATCC 13674). These included linear C₂ to C₁₀ monocarboxylic acids, C₃ to C₁₀ dicarboxylic acids, saccharides, α,ω-diols, hydrocarbons, and 3-methyl-branched substrates such as 3,7-dimethyl-6-octen-1-ol (citronellol), 3-methyl-n-valerate, 3-methyl-1-butanol, and 3-methyladipate. Isolated polymers were characterized by gas chromatography, infrared spectroscopy, ¹H- or ¹³C-nuclear magnetic resonance spectroscopy, ¹H-¹³C heteronuclear correlation spectroscopy (¹H-¹³C COSY), ¹H-¹H homonuclear COSY, and differential scanning calorimetry. Polyesters from nine monocarboxylic acids and two related carbon sources could be metabolically divided into three groups. The first group of C₂ to C₄ carbon sources resulted in copolyesters composed of 61 to 70 mol% 3-hydroxydecanoate, 23 to 33 mol% 3-hydroxyoctanoate, 3.6 to 9.0 mol% 3-hydroxy-5-cis-dodecenoate, and 1.8 to 2.6 mol% 3-hydroxy-7-cis-tetradecenoate. Carbon sources in group II (C₇ to C₁₀) produced copolyesters composed of 3-hydroxyacid monomer units with the same number of carbon atoms as the substrate (major constituent) and monomer units with either two less or two more carbons. Negligible amounts of 3-hydroxy-5-cis-dodecenoate and 3-hydroxy-7-cis-tetradecenoate were detected in copolyesters from this group. Copolyesters from group III (C₅ and C₆) had a monomer unit distribution that could be said to be between those of groups I and II. In addition, a novel copolyester, poly(3-hydroxy-7-methyl-6-octenoate-co-3-hydroxy-5-methylhexanoate), was synthesized when grown on citronellol. The ¹H-¹³C heteronuclear COSY spectrum for monomer unit II revealed that both methylene and isopropyl groups, proximately connected in series to a single chiral center, had magnetically diastereotopic natures.     

Syntheses of water-soluble [60]fullerene derivatives and their enhancing effect on neurite outgrowth in NGF-treated PC12 cells

Water-soluble [60]fullerene (C₆₀) derivatives were synthesized to examine their bioactivities. PC12 cells were used as a model of nerve cells and the bioactivities of synthesized C₆₀ derivatives together with some reported ones were tested. Among the compounds tested, C₆₀/(γ-CyD)₂, C₆₀-bis(γ-CyD) (5) containing C₆₀-mono(γ-CyD) (5′), and C₆₀/PVP were sufficiently soluble in water and showed an enhancing effect on the neurite outgrowth of NGF-treated PC12 cells.     

Hatch opening and closing on oxygenation and deoxygenation of C60 bathysphere

The structures and stabilities of C₆₀O_n, where n=1-6, 9, have been calculated using the AM1 Hamiltonian and the program mopac 6.0, and by the density functional technique B3LYP/6-31G* at the AM1 geometry using Gaussian 98. Modes of oxygen addition considered were ethers, epoxides, ketones and ketenes. It is confirmed that in C₆₀O a carbon-carbon bond on a pent-hex edge is replaced by an ether linkage. For higher levels of oxygen addition the replacement of a carbon-carbon bond by two ketone groups becomes important. Particularly stable structures are formed if the additions are on the same, or adjacent, C₆ rings of the C₆₀ structure where the oxygen atoms cooperate in opening up large holes in the molecule. Molecules of greatest stability have a mixture of ether oxygen atoms on pent-hex edges and diketone additions on hex-hex edges. A particularly important stable structure is the mixed ether/ketone isomer of C₆₀O₆ in which a hinged C₅O₂ hatch lid containing two ketone oxygen atoms is opened revealing a hatch 4-5 Å in diameter. An even larger hole of approximately 6 Å in diameter is opened up in a particularly stable isomer of C₆₀O₉ which contains three ether and six ketone groups. Removal of the oxygen atoms from these structures and reoptimisation leads to hatch closing and C₆₀ is reformed.     

The relationship between the structure of plastoquinone derivatives and their biological activity in Photosystem II of spinach chloroplasts

The relationship between the structure of reconstituted plastoquinone derivatives and their ability to recover the Hill reaction was investigated by extraction and reconstitution of lyophilized chloroplasts from spinach, followed by monitoring DCIP photoreduction at 600 nm. The results show that: It is not essential that the plastoquinone side chain be an isoprenoid or a phytol; the activity increases with increasing length of the side chain up to 13–15 carbon atoms; for chains longer than 15 carbon atoms, the activity is practically constant. Lipophilic groups (such as -Br) in the side chain increased the activity, hydrophilic groups (such as -OH) decreased the activity. Conjugated double bonds in the side chain decreased the activity greatly, but non-conjugated double bonds had almost no effect on the activity, indicating a requirement of flexibility of the side chain. The activity is decreased in the order of PQ, UbiQ and MQ, showing a large effect of the ring structure.Abbreviations DCIP 2,6-dichlorophenolindophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Q_A primary electron acceptor in PS II reaction centers - Q_B secondary electron acceptor in PS II reaction centers - PQ _n plastoquinones with an isoprenoid side chain (n, number of the isoprenoid units in the side chain) - PQ-n synthetic plastoquinones with alkyl side chain (n, number of the carbon atoms in the alkyl side chain) - PQ-n synthetic plastoquinones with a double bond in the alkyl side chain - UQ _n ubiquinones with an isoprenoid side chain (n, number of the isoprenoid units in the side chain) - UQ-n synthetic ubiquinones with alkyl side chain (n, number of the carbon atoms in the akyl side chain) - MQ-n 2-alkyl-1,4-naphthoquinone (n, number of the carbon atoms in the alkyl side chain)