Relatively simple methodology for the determination of configuration of unsaturation of bacterial monounsaturated fatty acids: application to the unsaturates of Legionella spp.

Unsaturated fatty acids of known degree, position, and configuration of unsturation were esterified, and stereospecifically dihydroxylated at the double bond(s). cis-Hydroxylation was effected using Woodward's reagent (silver acetate/iodine/acetic acid), while trans-hydroxylation was effected using Fenton's reagent (hydrogen peroxide/ferrous sulfate/acetic acid). Diols derived from monounsaturated esters were recovered quantitatively, while tetraols derived from diunsaturates were lost, presumably during extensive washing. The stereospecifically dihydroxylated esters were analyzed by thin-layer chromatography on borate-impregnated silica gel plates, and by gas-liquid chromatography, on a nonpolar capillary column, as the trimethylsilyl, acetyl, n-butylboronyl, isopropylidene, and trifluoroacetyl derivatives. For each derivative, the erythro and threo diols were readily separable, and some resolution of positional isomers was observed. Thus, the cis/trans configuration, and in some instances, the position of unsaturation of the original monounsaturated fatty acid may be deduced. However, gas chromatography-mass spectrometry of appropriately derivatized diol esters is required for unambiguous determination of position of unsaturation in most cases. These reactions are simple, use readily available reagents, and require relatively little operator attention. Further, they do not require specialized apparatus, as do hydrogenation and ozonolysis, or potentially toxic chemicals, such as osmium tetroxide. This series of analyses was applied to the unsaturated non-hydroxylated fatty acids of Legionella species, which were shown to be monounsaturated and of the cis Δ9 family. Position of unsaturation was confirmed by gas chromatography-mass spectrometry.     

The secretion of inert gas into the swim-bladder of fish

The composition of the gas mixture secreted into the swim-bladders of several species of fish has been determined in the mass spectrometer. The secreted gas differed greatly from the gas mixture breathed by the fish in the relative proportions of the chemically inert gases, argon, neon, helium, and nitrogen. Relative to nitrogen the proportion of the very soluble argon was increased and the proportions of the much less soluble neon and helium decreased. The composition of the secreted gas approaches the composition of the gas mixture dissolved in the tissue fluid. A theory of inert gas secretion is proposed. It is suggested that oxygen gas is actively secreted and evolved in the form of minute bubbles, that inert gases diffuse into these bubbles, and that the bubbles are passed into the swim-bladder carrying with them inert gases. Coupled to a preferential reabsorption of oxygen from the swim-bladder this mechanism can achieve high tensions of inert gas in the swim-bladder. The accumulation of nearly pure nitrogen in the swim-bladder of goldfish (Carassius auratus) is accomplished by the secretion of an oxygen-rich gas mixture followed by the reabsorption of oxygen.     

Occurrence of N-acyl-L-homoserine lactones in extracts of some Gram-negative bacteria evaluated by gas chromatography-mass spectrometry

Acylated homoserine lactones (AHLs) are self-generated signal molecules that mediate population density-dependent gene expression (quorum sensing) in a variety of Gram-negative bacteria. These signal molecules diffuse from bacterial cells and accumulate in the medium as a function of cell growth. In selected foods AHLs contribute to product spoilage. As different bacterial species produce AHL analogs that differ in length of the N-acyl chain, ranging from 4 to 14 carbons and in the substitution at the C-3 position of the side chain (i.e., oxo or hydroxyl group), the suitability and applicability of a gas chromatography-mass spectrometry direct method for characterizing trace amounts of AHLs was evaluated using N-heptanoyl-homoserine lactone as internal standard. Crude cell-free supernatants of bacterial cultures of Aeromonas hydrophila, Aeromonas salmonicida, Pseudomonas aeruginosa, Pseudomonas fluorescens, Yersinia enterocolitica, and Serratia liquefaciens were screened for AHL production in selected ion monitoring mode, using the prominent fragment at m/z 143. The observed profiles of distinguishable N-acyl-homoserine lactones occurring in bacterial extracts were compared and discussed. The presence of a labile 3-oxo-hexanoylhomoserine lactone was evidenced but serious difficulties arose in estimating its concentration as thermal degradation occurs during the gas chromatographic separation. Its electron impact mass spectra was, however, given and interpreted.     

Identification of sterols and alcohols produced by green algae of the generaChlorella andScenedesmus by means of gas chromatography—mass spectrometry

Sterols and alcohols isolated from a collection of industrially important fresh-water green algaeChlorella kessleri, Scenedesmus acuminatus, S. acutus andS. acutus var.Tomasclli cultivated heterotrophically or autotrophically under pilot plant conditions, were identified and quantified by gas chromatography — mass spectrometry. Previsously demonstrated sterols with double bonds in position 5 and 7 were detected. In addition, cholesta-5,7,22-trien-3 β-oI that had not yet been described in green algae was found inS. acutus. Alcohols were found only inC. kessleri cultivated under heterotrophic conditions. Some saturated and unsaturated alcohols were detected for the first time in green algae.     

The synthesis of a radioactive cytokinin with high specific activity

6-benzylaminopurine-[p-¹H-benzyl] at a specific activity of 10 Ci/mmol was synthesized by reacting p-bromo-6-benzylaminopurine with carrier-free tritium gas in the presence of 10% Pd/C. A radiochemical purity of 97% was obtained by a one-step purification of the tritiated reaction product using cellulose TLC. This simple procedure yields the highly active cytokinin, 6-benzylaminopurine, with tritium at near maximum specific activity in a known, stable position.     

Within-plant distribution of volatile compounds on the leaf surface of Flourensia cernua

We are using Flourensia cernua as a shrub model to study how terpenes affect livestock herbivory. Two experiments were conducted to examine distribution of volatile chemicals within a plant in an effort to minimize sample variability. In Experiment 1, leaves (current year's growth) were collected from 20 tarbush plants. Two leaders were sampled from each of three positions (outer canopy, subcanopy, and basal) in all four quadrants (based on ordinal direction). In Experiment 2, 10 leaders of current year's growth were removed from another 20 plants. Leaders were collected from the outer canopy of each quadrant and separated into thirds before removing leaves, thereby creating three leaf age categories. Volatile compounds were extracted with ethanol and analyzed with gas chromatography/mass spectrometry. Ninety-four chemicals (including 15 unknowns) were present on the leaf surface of F. cernua. Although 14 and 21 compounds differed (P < 0.05) among quadrants in Experiments 1 and 2, respectively, no consistent quadrant effect was detected in either study. Leaf position differed (P < 0.05) for 52 chemicals in Experiment 1 but outer canopy and subcanopy leaves differed for only 10 compounds. In Experiment 2, 63 compounds differed among leaf age categories. Immature leaves contained greater concentrations of 46 chemicals (P < 0.05) than intermediate or mature age categories, but intermediate and mature leaves differed for only seven compounds. Estimated total concentration (i.e., cumulative concentration of all compounds) was not affected by leaf position but varied among leaf age categories (P < 0.05; immature > intermediate > mature). Differences in leaf position were attributed about equally to mono- and sesquiterpenes, whereas leaf age differences were primarily due to sesquiterpenes. Leaf position and age both affect terpene concentration and sampling variability for tarbush. However, little difference was detected between subcanopy and canopy positions. Thus, by avoiding basal sprouts and sampling from the mid-point of current year's growth, sampling variation should be minimal.     

Insecticidal activities of methyleugenol and β-asarone,from the herbal medicines Saishin and Sekishōkon,and other alkoxy-propenyl-benzene derivatives against the cigarette beetle <Emphasis Type="Italic">Lasioderma serricorne</Emphasis> (Coleoptera: Anobiidae)

The principal insecticidal compounds from the herbal medicines Saishin, the root of Asarum sieboldii Miquel, and Sekishōkon, the rhizome of Acorus gramineus Soland, were isolated by successive silica gel column chromatography and high performance liquid chromatography. The active components, which work against larvae of the cigarette beetle, Lasioderma serricorne (F.), were identified as methyleugenol (4-allyl-1,2-dimethoxybenzene) and β-asarone {1,2,4-trimethoxy-5-[(Z)-prop-1-enyl]benzene} by gas chromatography and gas chromatography/mass spectrometry analyses. These two compounds share some structural features, such as a benzene ring with o-dimethoxy groups and a propenyl group in the opposite position. Consequently, the insecticidal activities of 20 structurally related compounds were tested to evaluate their structure–activity relationship. We found myristicin (5-allyl-1-methoxy-2,3-methylenedioxybenzene) exhibited comparable insecticidal activity to methyleugenol and β-asarone, but the other tested compounds were less active. The lack of insecticidal activity of compounds with a hydrogen, hydroxy, or acetoxy substituent in place of one methoxy group indicates that o-dimethoxy groups are essential for insecticidal activity. The position and configuration of a double bond in the propenyl side chain affected the toxicity, but there was a lack of consistency in the structure–activity relationship for this.     

Average thickness of the gas vesicle wall in Anabaena flos-aquae.

The average thickness of the layer of protein which forms the wall of the gas vesicles in Anabaena flos-aquae was estimated from measurements of their density and geometry. The volume of the gas space in a purified gas vesicle suspension was determined from the contraction which occurred when the gas vesicles were collapsed by pressure. The volume of the protein in the same sample was calculated from its dry weight and density. From knowledge of the geometry of the average gas vesicle the thickness of the protein layer, 1.54 nm, was then calculated. By a similar method the thickness of the Microcystis gas vesicle wall, 1.62 nm, was calculated from data published by others. The average thickness of the protein layer is, as expected, slightly less than the stacking periodicity of collapsed gas vesicle walls indicated by X-ray diffraction studies.Anabaena gas vesicles with a mean length of 494 nm have an average density of 0.119 mg μl^?1 1 mg of protein is present in gas vesicles having a, total volume of 8.43 μl and a gas space of 7.67 μl. Suspensions of isolated gas vesicles with a gas space concentration of 1 μl ml^?1 give a pressure-sensitive optical density, E_1cm (500 nm) of 2.72, but gas vacuoles in cells give a smaller value.     

A hydrocarbon-oxidizing acidophilic thermotolerant bacterial association from sulfur blocks

A stable bacterial association isolated from a sulfur block sample of the Astrakhan gas processing complex was able to utilize n-alkanes as the sole carbon and energy source at low pH. Hydrocarbon-dependent growth occurred at pH 1.6–5.5 (optimum at pH 2.5) and 20–50°C (optimum at 30–35°C). Analysis of the 16S rRNA gene fragments isolated from the total DNA of the enrichment by PCR-DGGE revealed the nucleotide sequences most closely related to extreme acidophilic chemolithotrophs Acidithiobacillus thiooxidans and Sulfobacillus sp. (98–99% similarity) and the sequences exhibiting high similarity to those of slowly growing actinobacteria Mycobacterium europaeum and M. parascrofulaceum (98%). Capacity of any of these organisms for hydrocarbon oxidation has not been reported previously. The taxonomic position of the 16S rRNA gene fragments from the enrichment culture suggests that this bacterial association is a unique microbial community, in which development of acidophilic hydrocarbon-oxidizing bacteria is mediated by a localized pH decrease in the sulfur blocks resulting from elemental sulfur oxidation due to massive development of chemolithotrophic sulfur-oxidizing bacteria.     

Fatty acids ofLactobacillus bulgaricus determined by gas chromatography — Mass spectrometry

Fatty acids (in the form of pyrrolidinides) ofLactobacillus bulgaricus were identified by gas chromatography—mass spectrometry using two columns differing in the polarity of the stationary phase. In addition to the described fatty acids (primarily the most abundant vaccenic acid), we detected nine fatty acids that have not yet been described in the genusLactobacillus (all of them minority compounds) and in four of them we determined the position of the double bond. The occurrence of vaccenic acid was related to other sources of positional isomers of octadecenoic acids (parsley seed - petroselmic acid; olive oil — oleic acid).     

Chemical Attraction of Conspecifics in <Emphasis Type="Italic">Folsomia candida</Emphasis> (Collembola)

Chemical substances excreted from individuals are regarded as chemo-sensory signals that induce aggregation in Collembola. However, chemical or physiological evidence is required to identify the chemical composition of signals responsible for aggregation behavior. Present study applied solvent extraction and gas chromatograph-mass spectrometer-computer technology to separate and identify the chemical compounds in cuticle extracts from the model Collembola species, Folsomia candida. The occurrence of aggregation behavior was confirmed by conspecific attractiveness using a trap bioassay. Eight chemical compounds were obtained from F. candida’s cuticle solvent. The trap assay detected fatty acid combinations from the body extracts of F. candida that induced attractiveness behavior among conspecific individuals and small molecules with higher concentration induced repellent. However, single sensillum recordings of two thin-wall chemical receptors at the second segment of the distal position of the antenna did not receive any positive responses to fatty acids, either single or in combination. The way F. candida used fatty acid combinations as chemical signal for conspecific aggregating was discussed.     

Ethane in pine needles preventing the feeding of the beetle, Monochamus alternatus

Since the remarkable repulsion activity for Monochamus alternatus of a gas from freshly ground needles of Pinus densiflora was observed by feeding tests, volatile components in the gas were submitted for further feeding tests. Among six components, five were known monoterpenic hydrocarbons, all of which showed a relatively low activity. The most abundant residual volatile in the gas was ethane, whose presence in gymnosperms has not been reported so far. Ethane showed a strong repulsion activity and was proved to be present also in the gases from other conifer needles of nine species but in less quantity than in the gas from the needles of P. densiflora. However, the order of repulsion due to various conifer needles was found to be roughly consistent with the order of ethane concentration in the gases from the respective needles. Saturated hydrocarbons with straight-chain C₅ to C₁₀ were also shown to be active for the beetle.     

Steroid metabolism in human and boar testis tissue. Steroid concentrations and the position of the sulfate group in steroid sulfates

The steroid composition of and steroid conjugation in human cadaver and boar testes were investigated by analyzing the endogenous steroids. Gas-liquid chromatography and gas chromatography-mass spectrometry were used to identify the steroids and to determine the position of the sulfate group in sulfate conjugates. For the latter purpose, the steroids were first acetylated and subsequently solvolyzed and converted to trimethylsilyl ethers.In addition to the compounds previously identified as endogenous components, human testis was also found to contain progesterone, 17_α-hydroxyprogesterone and 20_α-hydroxy-4-pregnen-3-one in the free steroid fraction and 3_β-hydroxy-17_α-5-pregnen-20-one in the monosulfate fraction.     

Exchange of oxime functions: a useful reaction in GC-ms analysis of steroids

Simple and various O-substituted oxime derivatives of steroids may be interconverted by an acid-catalyzed reaction of the oxime with the appropriate substituted hydroxylamine hydrochloride in pyridine. For example, unsubstituted oximes of common hormonal steroids with keto groups in positions 3, 17, and 20 are converted into O-methyloximes by reaction of the oximes with 5 mg of methoxyamine hydrochloride in 50 μl of pyridine for 1 hr at 100°C. The reaction involves cleavage of the carbon-nitrogen bond and follows pseudo-first-order kinetics in the presence of excess reagent. The conversion of an oxime with a small O-substituent into one with a larger O-substituent was faster than the reverse reaction at the C-3 position. The rate of conversion depends on the position of the oxime function and decreases in the order: C-3 > C-20 > C-17 > C-11. The proximity and the configuration of other functional groups also influence the rates. Thus, determination of reaction rates with the aid of gas chromatography-mass spectrometry may be used to determine the positions of functional groups in steroids. The reactions are likely to be applieable to other groups of compounds possessing keto or aldehyde functions.     

Microbial Hydroxylation of 1,4-Cineole

Microorganisms were examined for their potential to hydroxylate the oxygenated monoterpene 1,4-cineole. Using gas chromatography and thin-layer chromatography, screening experiments revealed that hydroxylation at position 2 was the most commonly observed microbial transformation reaction. In most microorganisms, the predominant alcohol metabolite was the 2-endo-alcohol isomer. Preparative-scale incubations were conducted in order to isolate and characterize microbial transformation products by comparison of proton nuclear magnetic resonance, mass spectrometry, and chromatography profiles with those of cineole standards. Streptomyces griseus yielded 8-hydroxy-1,4-cineole as the major hydroxylation product together with 2-exo- and 2-endo-hydroxy-1,4-cineoles.     

Regio- and stereoselective hydroxylation of 10-undecenoic acid with a light-driven P450 BM3 biocatalyst yielding a valuable synthon for natural product synthesis

We report herein the selective hydroxylation of 10-undecenoic acid with a light-activated hybrid P450 BM3 enzyme. Under previously developed photocatalytic reaction conditions, only a monohydroxylated product is detected by gas chromatography. Hydroxylation occurs exclusively at the allylic position as confirmed from a synthesized authentic standard. Investigation into the stereochemistry of the reaction indicates that the R enantiomer is obtained in 85% ee. The (R)-9-hydroxy-10-undecenoic acid obtained enzymatically is a valuable synthon en route to various natural products further expanding the light-activated P450 BM3 biocatalysis and highlighting the advantages over traditional methods.     

Effects of geometrical design on hydrodynamic and mass transfer characteristics of a rectangular-column airlift bioreactor

Gas holdup and gas–liquid mass transfer coefficients were measured in a 21-L rectangular-column airlift bioreactor with aspect ratio of 10 and working volumes ranging from 10 to 16 L. The effect of the bottom and top clearances was investigated using water and mineralized CMC solutions and covering a range of effective viscosity from 0.02 to 0.5 Pa s and surface tension from 0.065 to 0.085 N m⁻¹. The gas holdup and mass transfer results were successfully correlated using expressions derived via dimensional analysis. The separator gas holdup was found to be similar to the total gas holdup in the airlift bioreactor. The downcomer gas holdup (ɛ_d) increased two-fold when the bottom clearance (h_b) was increased from 0.014 to 0.094 m while the top clearance (h_t) had no effect. Increasing h_b decreased the mass transfer by 50% compared to 31% when the top clearance (h_t/D_hr) was increased. It was found that the gas–liquid separator diameter ratio (D_hs/D_hc) exerted the maximal influence of over 65% on mass transfer as compared to both clearances.     

Gas transport during high-frequency ventilation: the significance of direct wash-out

Gas transport during high-frequency oscillation was investigated in vitro using CO₂ elimination from the lung surrogate as a measure of gas transport efficiency. The length of the connecting tube between the piston pump and the three-port connector did not affect gas transport efficiency if the oscillatory volume (V_DEL) was constant; inserting an additional tube between the three-port connector and the endotracheal tube decreased gas transport efficiency dramatically. In contradistinction, increasing V_DEL caused a steep rise in gas transport efficiency as soon as V_DEL surpassed the volume of the tubes connecting the lung surrogate with its surroundings. As gas transport effiency was found to be very sensitive to the net oscillatory volume, i.e. V_DEL minus the volume of the tubes connecting the lung and the surroundings, direct wash-out was considered to be an effective gas transport mechanism during high frequency oscillation. Two preliminary experiments on dogs allowed us to substantiate this hypothesis in vivo.