Biotransformation of geranyl acetate to geraniol during palmarosa (Cymbopogon martinii, Roxb. wats. var. motia) inflorescence development

Only immature palmarosa (Cymbopogon martinii, Roxb. wats. var. motia) inflorescence with unopened spikelets accumulated essential oil substantially. Geraniol and geranyl acetate together constituted about 90% of the palmarosa oil. The proportion of geranyl acetate in the oil decreased significantly with a corresponding increase of geraniol, during inflorescence development. An esterase enzyme activity, involved in the transformation of geranyl acetate to geraniol, was detected from the immature inflorescence using a gas chromatographic procedure. The enzyme, termed as geranyl acetate cleaving esterase (GAE), was found to be active in the alkaline pH range with the optimum at pH 8.5. The catalysis of geranyl acetate was linear up to 6 h, and after 24 h of incubation, 75% of the geranyl acetate incubated was hydrolyzed. The GAE enzymic preparation, when stored at 4 degrees C for a week, was quite stable with only 40% loss of activity. The physiological role of GAE in the production of geraniol during palmarosa inflorescence development has been discussed.     

Sucrose mobilization in relation to essential oil biogenesis during palmarosa (<Emphasis Type="Italic">Cymbopogon martinii</Emphasis> Roxb. Wats. var.<Emphasis Type="Italic">motia</Emphasis>) inflorescence development

Palmarosa inflorescence with partially opened spikelets is biogenetically active to incorporate [U-¹⁴C]sucrose into essential oil. The percent distribution of¹⁴C-radioactivity incorporated into geranyl acetate was relatively higher as compared to that in geraniol, the major essential oil constituent of palmarosa. At the partially opened spikelet stage, more of the geraniol synthesized was acetylated to form geranyl acetate, suggesting that majority of the newly synthesized geraniol undergoes acetylation, thus producing more geranyl acetate.In vitro development of palmarosa inflorescence, fed with [U-¹⁴C]sucrose, resulted in a substantial reduction in percent label from geranyl acetate with a corresponding increase in free geraniol, thereby suggesting the role of an esterase in the production of geraniol from geranyl acetate. At time course measurement of¹⁴CO₂ incorporation into geraniol and geranyl acetate substantiated this observation. Soluble acid invertase was the major enzyme involved in the sucrose breakdown throughout the inflorescence development. The activities of cell wall bound acid invertase, alkaline invertase and sucrose synthase were relatively lower as compared to the soluble acid invertase. Sucrose to reducing sugars ratio decreased till fully opened spikelets stage, concomitant with increased acid invertase activity and higher metabolic activity. The phenomenon of essential oil biosynthesis has been discussed in relation to changes in these physiological parameters.     

Antimicrobial action of palmarosa oil (Cymbopogon martinii) on Saccharomyces cerevisiae

The essential oil extracted from palmarosa (Cymbopogon martinii) has proven anti-microbial properties against cells of Saccharomyces cerevisiae. Low concentrations of the oil (0.1%) inhibited the growth of S. cerevisiae cells completely. The composition of the sample of palmarosa oil was determined as 65% geraniol and 20% geranyl acetate as confirmed by GC-FTIR. The effect of palmarosa oil in causing K(+) leakage from yeast cells was attributed mainly to geraniol. Some leakage of magnesium ions was also observed. Blocking potassium membrane channels with caesium ions before addition of palmarosa oil did not change the extent of K(+) ion leakage, which was equal to the total sequestered K(+) in the cells. Palmarosa oil led to changes in the composition of the yeast cell membrane, with more saturated and less unsaturated fatty acids in the membrane after exposure of S. cerevisiae cells to the oil. Some of the palmarosa oil was lost by volatilization during incubation of the oil with the yeast cells. The actual concentration of the oil components affecting the yeast cells could not therefore be accurately determined.     

The lipogenic role of leucine in animal skeletal muscles

After incubation of rat, pig and cattle skeletal muscle homogenates with [U-14C]leucine, 80.4%, 37.0% and 57.0% of radioactivity was found in the proteins, 9.4%, 58.7% and 40.9% in the lipids, and 10.2%, 4.3% and 2.1% in 14CO2. This suggests that along-side with utilization in protein synthesis, leucine plays an essential role in lipid synthesis in muscle tissues of agricultural animals. The contribution of [U-14C]leucine to lipogenesis with substrates is greater than that of [U-14C]acetate and [U-14C]glucose in cattle skeletal muscles in vitro and greater than that of [U-14C]acetate in pig muscle. The CO2 production during oxidation of the [U-14C]leucine carbohydrate chain is higher than that during [U-14C]glucose and [U-14C]palmitate oxidation in skeletal muscles of rat and pig. In skeletal muscles of all animal species under study [U-14C]acetate is oxidized far more intensively than the other substrates tested.     

Changes in volatile constituents of Zingiber officinale rhizomes during storage and cultivation

The essential oil from the fresh rhizome of Zingiber officinale was characterized by the presence of acyclic oxygenated monoterpenes mainly composed of neral, geraniol, geranial and geranyl acetate. During storage the content of neral and geranial in the rhizome increased to ca 60% of the essential oil, while the content of geraniol and geranyl acetate decreased to an undetectable amount. The change resulted from the conversion of geranyl acetate into geraniol, geranial and neral, successively. The content of geranial and neral decreased to a small extent through cultivation of the stored rhizome, whereas a large quantity of geraniol and geranyl acetate occurred in the newly propagated fresh rhizome.     

The Substrate Utilization and Concentration of 14C Photosynthates in Citronella under Fe Deficiency

Changes in the utilization pattern of primary substrate, viz. [U-14C] acetate, 14CO2 and [U-14C] saccharose, and the contents of 14C fixation products in photosynthetic metabolites (sugars, amino acids, and organic acids) were determined in Fe-deficient citronella in relation to the essential oil accumulation. There was an overall decrease in photosynthetic efficiency of the Fe-deficient plants as evidenced by lower levels of incorporation into the sugar fraction and essential oil after 14CO2 had been supplied. When acetate and saccharose were fed to the Fe-deficient plants, despite a higher incorporation of label into sugars, amino acids, and organic acids, there was a lower incorporation of these metabolites into essential oils than in control plants. Thus, the availability of precursors and the translocation to a site of synthesis/accumulation, severely affected by Fe deficiency, is equally important for the essential oil biosynthesis in citronella.     

Coherent ontogenic dynamics of geraniol acetyltransferase activity and geranyl acetate concentration in flowers and leaves of aroma grass Cymbopogon martinii var. Motia

We have investigated the correlation between the concentration of geranyl acetate (GA) and acetyl CoA: geraniol acetyltransferase (GAAT) activity in palamarosa (Cymbopogon martini var. Motia) inflorescence and leaves at their different physiological stages. The results on GA concentration and the GAAT activity have been expressed on per gram fresh weight, per spikelet pair or leaf and per unit area of the phylloplane also incase of leaf. The percentage of geranyl acetate and geraniol in the volatile oil has also been considered. GA concentration was found to be highest in unopened floral spikelets and on the decline in fully open spikelets matching the trend of GAAT activity. Similarly, highest concentration of GA and maximum GAAT activity were found in leaves at mid-stage of development (stage II). The regression analysis curve between GA concentration (mg gFw⁻¹) and GAAT activity (IU 10⁻³ gFw⁻¹) gave an estimate of correlation coefficient (at 95% confidence) value of 0.79 for flowers and 0.92 for leaf. The results suggest that volatile ester (like geranyl acetate) synthesis in foliage and flowers of the aroma oil plant is controlled by the existent catalytic levels GAAT rather than the availability of geraniol. The study also indicates that the GAAT to be a good target to over-express for improvement of oil quality in terms of GA linked to fruit-fresh olfactory note of the oil.     

Chemotaxonomic study on Thymus villosus from Portugal

The composition of the essential oils of four populations of Thymus villosus subsp. lusitanicus (Boiss.) Coutinho from Portugal was investigated by GC and GC-MS. To study the chemical polymorphism the results obtained from GC analyses of the volatile oils from individual plants from four populations were submited to Principal Component and Cluster analyses. A comparision with the essential oil of T. villosus subsp. villosus, previously studied by us was done. Important differences with regard to the major constituents in these two taxa were found. Linalool, geranyl acetate, geraniol and terpinen-4-ol were the main components of the essential oils of T. villosus subsp. lusitanicus, whereas in the oil of T. villosus subsp. villosus p-cymene, myrcene and alpha-terpineol were the major ones. Although, both taxa showed chemical polymorphism, different types of essential oils were characterized in each one: linalool; linalool/ terpinen-4-ol/trans-sabinene hydrate; linalool/1,8-cineole; geranyl acetate/geraniol; geranyl acetate/geraniol/1,8-cineole in T. villosus subsp. lusitanicus and p-cymene/camphor/linalool; p-cymene/borneol; linalool/geraniol/geranyl acetate; alpha-terpineol/camphor/myrcene in T. villosus subsp. villosus. Thus, the two subspecies of T. villosus can be easely differenciated by the composition of their essential oils.     

Geranyl pyrophosphate synthase: characterization of the enzyme and evidence that this chain-length specific prenyltransferase is associated with monoterpene biosynthesis in sage (Salvia officinalis)

Cell-free homogenates from sage (Salvia officinalis) leaves convert dimethylallyl pyrophosphate and isopentenyl pyrophosphate to a mixture of geranyl pyrophosphate, farnesyl pyrophosphate, and geranylgeranyl pyrophosphate, with farnesyl pyrophosphate predominating. These prenyltransferase activities were localized primarily in the soluble enzyme fraction, and separation of this preparation on Sephadex G-150 revealed the presence of a partially resolved, labile geranyl pyrophosphate synthase activity. The product of the condensation reaction between [1-14C]dimethylallyl pyrophosphate and [1-3H]isopentenyl pyrophosphate was verified as [14C,1-3H]geranyl pyrophosphate by TLC isolation, enzymatic hydrolysis to geraniol, degradative studies, and the preparation of the crystalline diphenylurethane. The cis-isomer, neryl pyrophosphate, was not a product of the enzymatic reaction. By employing a selective tissue extraction procedure, the geranyl pyrophosphate synthase activity was localized in the leaf epidermal glands, the site of monoterpene biosynthesis, suggesting that the role of this enzyme is to supply the C10 precursor for the production of monoterpenes. Glandular extracts enriched in geranyl pyrophosphate synthase were partially purified by a combination of hydrophobic interaction chromatography on phenyl-Sepharose and gel permeation chromatography on Sephadex G-150. Substrate and product specificity studies confirmed the selective synthesis of geranyl pyrophosphate by this enzyme, which was also characterized with respect to molecular weight, pH optimum, cation requirement, inhibitors, and kinetic parameters, and shown to resemble other prenyltransferases.     

Volatiles from rhizomes of Rhodiola rosea L

Terpenes and aroma volatiles from rhizomes of Rhodiola rosea L. from Norway have been isolated by both steam distillation and headspace solid-phase micro-extraction coupled with gas chromatography and mass spectrometry analysis. The dried rhizomes contained 0.05% essential oil with the main chemical classes: monoterpene hydrocarbons (25.40%), monoterpene alcohols (23.61%) and straight chain aliphatic alcohols (37.54%). n-Decanol (30.38%), geraniol (12.49%) and 1,4-p-menthadien-7-ol (5.10%) were the most abundant volatiles detected in the essential oil, and a total of 86 compounds were identified in both the SD and HS-SPME samples. Geraniol was identified as the most important rose-like odour compound besides geranyl formate, geranyl acetate, benzyl alcohol and phenylethyl alcohol. Floral notes such as linalool and its oxides, nonanal, decanal, nerol and cinnamyl alcohol highlight the flowery scent of rose root rhizomes.     

Turnover of glucose and acetate coupled to reduction of nitrate, ferric iron and sulfate and to methanogenesis in anoxic rice field soil

Turnover of glucose and acetate in the presence of active reduction of nitrate, ferric iron and sulfate was investigated in anoxic rice field soil by using [U-(14)C]glucose and [2-(14)C]acetate. The turnover of glucose was not much affected by addition of ferrihydrite or sulfate, but was partially inhibited (60%) by addition of nitrate. Nitrate addition also strongly reduced acetate production from glucose while ferrihydrite and sulfate addition did not. These results demonstrate that ferric iron and sulfate reducers did not outcompete fermenting bacteria for glucose at endogenous concentrations. Nitrate reducers may have done so, but glucose fermentation may also have been inhibited by accumulation of toxic denitrification intermediates (nitrite, NO, N(2)O). Addition of nitrate resulted in complete inhibition of CH(4) production from [U-(14)C]glucose and [2-(14)C]acetate. However, addition of ferrihydrite or sulfate decreased the production of (14)CH(4) from [U-(14)C]glucose by only 70 and 65%, respectively. None of the electron acceptors significantly increased the production of (14)CO(2) from [U-(14)C]glucose, but all increased the production of (14)CO(2) from [2-(14)C]acetate. Uptake of acetate was faster in the presence of either nitrate, ferrihydrite or sulfate than in the unamended control. Addition of ferrihydrite and sulfate reduced (14)CH(4) production from [2-(14)C]acetate by 83 and 92%, respectively. Chloroform completely inhibited the methanogenic consumption of acetate. It also inhibited the oxidation of acetate, completely in the presence of sulfate, but not in the presence of nitrate or ferrihydrite. Our results show that, besides the possible toxic effect of products of nitrate reduction (NO, NO(2)(-) and N(2)O) on methanogens, nitrate reducers, ferric iron reducers and sulfate reducers were active enough to outcompete methanogens for acetate and channeling the flow of electrons away from CH(4) towards CO(2) production.     

本姜子属及山胡椒属的平行演化

本文阐述樟科木姜子属及山胡椒属两者在形态、分布、起源及演化等方面有许多共同之处,不仅在属内具有平行演化的情况,而且各自平行演化成单花木姜子属 Dodecade－nia和单花山胡椒属Iteadaphne。同时两者又分别自4药室的拟擦术属Parasassafras和2药室的黄脉擦术属 sinosassafras演化而来。本文将单花木姜子亚属 Litsea subg．Uniflos Yang et P．H．Huang归人单花木姜子属,并把 Litsea monantha Yang et P．H．Hu－ang一种作为单花木姜子（原变种）Dodecadenia grandiflora Nees var．grandiflora的新异名,恢复了单花山胡椒属Iteadaphne Bl.,并将香面叶Lindera caudata（Nees）Hook·f．重新组合归入该属为Ⅰ．caudata（Nees）H．W．Li,comb．nov。此外,本文还发表了一个新属即黄脉擦木屑 Sinosassafras H．W．Li,gen．nov.,它具有2药室花药而不同于其平行演化的拟擦本属。     

A geraniol-synthase gene from Cinnamomum tenuipilum

Geraniol may accumulate up to 86-98% of the leaf essential oils in geraniol chemotypes of the evergreen camphor tree Cinnamomum tenuipilum. A similarity-based cloning strategy yielded a cDNA clone that appeared to encode a terpene synthase and which could be phylogenetically grouped within the angiosperm monoterpene synthase/subfamily. After its expression in Escherichia coli and enzyme assay with prenyl diphosphates as substrates, the enzyme encoded by the putative C. tenuipilum monoterpene synthase gene was shown to specifically convert geranyl diphosphate to geraniol as a single product by GC-MS analysis. Biochemical characterization of the partially purified recombinant protein revealed a strong dependency for Mg2+ and Mn2+, and an apparent Michaelis constant of 55.8 microM for geranyl diphosphate. Thus, a new member of the monoterpene synthase family was identified and designated as CtGES. The genome contains a single copy of CtGES gene. Expression of CtGES was exclusively observed in the geraniol chemotype of C. tenuipilum. Furthermore, in situ hybridization analysis demonstrated that CtGES mRNA was localized in the oil cells of the leaves.     

Anomeric specificity of D-glucose phosphorylation and oxidation in human erythrocytes

1. In human erythrocytes, alpha-D-[U-14C]glucose is more efficiently oxidized than beta-D-[U-14C]glucose at a low concentration of the hexose (0.1 mM), but not so at higher glucose concentrations. 2. This unexpected situation may be attributable in part to the lower Km of hexokinase for alpha- than beta-D-glucose, this difference in affinity compensating for the higher maximal velocity found with the beta- rather than alpha-anomer. 3. A contributive role for aldose reductase in the anomeric control of D-glucose 6-phosphate circulation in the pentose phosphate pathway should not be ruled out, since aldose reductase inhibitors decrease the production of 14CO2 by erythrocytes exposed to D-[U-14C]glucose. 4. Nevertheless, the essential role of hexokinase in such an anomeric control is supported by the finding that, in the presence of menadione, which augments considerably D-[U-14C]glucose oxidation but fails to affect D-[5-3H]glucose utilization, the anomeric alpha/beta ratio in 14CO2 production from D-[U-14C]glucose follows, at increasing concentrations of the hexose, the same pattern as that found for its phosphorylation.     

Metabolism of organic compounds in anaerobic,hydrothermal sulphate-reducing marine sediments

Previous studies of hot (>80 degrees C) microbial ecosystems have primarily relied on the study of pure cultures or analysis of 16S rDNA sequences. In order to gain more information on anaerobic metabolism by natural communities in hot environments, sediments were collected from a shallow marine hydrothermal vent system in Baia di Levante, Vulcano, Italy and incubated under strict anaerobic conditions at 90 degrees C. Sulphate reduction was the predominant terminal electron-accepting process in the sediments. The addition of molybdate inhibited sulphate reduction in the sediments and resulted in a linear accumulation of acetate and hydrogen over time. [U-14C]- acetate was completely oxidized to 14CO2, and the addition of molybdate inhibited 14CO2 production by 60%. [U-14C]-glucose was oxidized to 14CO2, and this was inhibited when molybdate was added. When the pool sizes of short-chain fatty acids were artificially increased, radiolabel from [U-14C]-glucose accumulated in the acetate pool. L-[U-14C]-glutamate, [ring-14C]-benzoate and [U-14C]-palmitate were also anaerobically oxidized to 14CO2 in the sediments, but molybdate had little effect on the oxidation of these compounds. These results demonstrate that natural microbial communities living in a hot, microbial ecosystem can oxidize acetate and a range of other organic electron donors under sulphate-reducing conditions and suggest that acetate is an important extracellular intermediate in the anaerobic degradation of organic matter in hot microbial ecosystems.     

The role of phosphoenolpyruvate carboxykinase in muscle alanine synthesis.

3-Mercaptopicolinic acid (3-MPA) is reportedly a specific inhibitor of phosphoenolpyruvate (PEP) carboxykinase and has hitherto been used accordingly to elucidate the metabolic role of PEP carboxykinase in vitro and in vivo. We show that 3-MPA has multiple effects on intermediary metabolism in hemidiaphragms from 40 h-starved rats. It decreases the release of lactate + pyruvate and alanine in hemidiaphragms provided with no added substrate or with valine, leucine or isoleucine. Moreover, irrespective of the substrate provided (none, valine, leucine, isoleucine, glucose, acetate, oleate), 3-MPA decreases the [lactate]/[pyruvate] ratio. 3-MPA is without effect on 14CO2 production from [U-14C]valine, [1-14C]valine, [1-14C]leucine, [U-14C]isoleucine or [1-14C]oleate, but stimulates 14CO2 production from [U-14C]glucose and [1-14C]pyruvate and inhibits 14CO2 production from [1-14C]acetate. Glycolytic flux (measured as 3H2O formation from [5-3H]glucose) is stimulated by 3-MPA. It is concluded that 3-MPA has site(s) of actions other than PEP carboxykinase and that the putative role of PEP carboxykinase in alanine synthesis de novo in skeletal muscle from tricarboxylic acid-cycle intermediates and related amino acids requires reappraisal.     

Metabolism of L-(U-14C)valine, L-(U-14C)leucine, L-(U-14C)histidine and L-(U-14C) phenylalanine by the isolated perfused lactating guinea-pig mammary gland.

1. The incorporation of L-[U-14C]leucine, L[U-14C]histidine and L-[U-14C]phenylalanine into casein secreted during perfusion of isolated guinea-pig mammary glands was demonstrated. 2. The extent of incorporation of label into casein residues was consistent with their being derived from free amino acids of the perfusate plasma. 3. The mean transit time of the amino acids from perfusate into secreted casein was approx. 100 min. 4. Whereas radioactive histidine and phenylalanine were incorporated solely into milk protein, radioactivity from [U-14C]valine was also transferred to CO2 and to an unidentified plasma component, and from [U-14C]leucine to plasma glutamic acid. 5. Evidence from experiments with [U-14C]phenylalanine suggests that, as in rats, but in contrast with ruminant species, guinea-pig mammary tissue does not possess phenyl alanine hydroxylase activity. 6. The results are discussed in relation to the possible role of essential amino acid catabolism in the control of milk-protein synthesis.     

Oxalic acid biosynthesis and oxalacetate acetylhydrolase activity in Streptomyces cattleya

In addition to producing the antibiotic thienamycin, Streptomyces cattleya accumulates large amounts of oxalic acid during the course of a fermentation. Washed cell suspensions were utilized to determine the specific incorporation of carbon-14 into oxalate from a number of labeled organic and amino acids. L-[U-14C]aspartate proved to be the best precursor, whereas only a small percentage of label from [1,5-14C]citrate was found in oxalate. Cell-free extracts catalyzed the formation of [14C]oxalate and [14C]acetate from L-[U-14C]aspartate. When L-[4-14C]aspartate was the substrate only [14C]acetate was formed. The cell-free extracts were found to contain oxalacetate acetylhydrolase (EC 3.7.1.1), the enzyme that catalyzes the hydrolysis of oxalacetate to oxalate and acetate. The enzyme is constitutive and is analogous to enzymes in fungi that produce oxalate from oxalacetate. Properties of the crude enzyme were examined.     

Ketone body utilization for energy production and lipid synthesis in isolated rat brain capillaries

Isolated brain capillaries from 2-month-old rats were incubated for 2 h in the presence of [3-14C]acetoacetate, D-3-hydroxy[3-14C]butyrate, [U-14C]glucose, [1-14C]acetate or [1-14C]butyrate. Labelled CO2 was collected as an index of oxidative metabolism and incorporation of label precursors into lipids was determined. The rate of CO2 production from glucose was slightly higher than from the other substrates. Interestingly, acetoacetate was oxidized at nearly the same rate as glucose. This shows that ketone bodies could be used as a source of energy by brain capillaries. Radiolabelled substrates were also used for the synthesis of lipids, which was suppressed by the addition of albumin. The incorporation of [U-14C]glucose in total lipids was 10-times higher than that from other precursors. However, glucose labelled almost exclusively the glycerol backbone of phospholipids, especially of phosphatidylcholine. Ketone bodies as well as glucose were incorporated mainly into phospholipids, whereas acetate and butyrate were mainly incorporated into neutral lipids. The contribution to fatty acid synthesis of various substrates was in the following order: butyrate greater than or equal to acetate greater than ketone bodies greater than or equal to glucose. All precursors except glucose were used for sterol synthesis. Glucose produced almost exclusively the glycerol backbone of phospholipids.     

Insulin stimulates glucose metabolism via the pentose phosphate pathway in Drosophila Kc cells

Drosophila melanogaster has become a prominent and convenient model for analysis of insulin action. However, to date very little is known regarding the effect of insulin on glucose uptake and metabolism in Drosophila. Here we show that, in contrast to effects seen in mammals, insulin did not alter [(3)H]2-deoxyglucose uptake and in fact decreased glycogen synthesis ( approximately 30%) in embryonic Drosophila Kc cells. Insulin significantly increased ( approximately 1.5-fold) the production of (14)CO(2) from D-[1-(14)C]glucose while the production of (14)CO(2) from D-[6-(14)C]glucose was not altered. Thus, insulin-stimulated glucose oxidation did not occur via increasing Krebs cycle activity but rather by stimulating the pentose phosphate pathway. Indeed, inhibition of the oxidative pentose phosphate pathway by 6-aminonicotinamide abolished the effect of insulin on (14)CO(2) from D-[U-(14)C]glucose. A corresponding increase in lactate production but no change in incorporation of D-[U-(14)C]glucose into total lipids was observed in response to insulin. Glucose metabolism via the pentose phosphate pathway may provide an important source of 5'-phosphate for DNA synthesis and cell replication. This novel observation correlates well with the fact that control of growth and development is the major role of insulin-like peptides in Drosophila. Thus, although intracellular signaling is well conserved, the metabolic effects of insulin are dramatically different between Drosophila and mammals.