Influence of extraction solvent (buffer, methanol, acetone) and time on the quantification of indoIe-3-acetic acid in plants

The effect of extraction solvent and time on the measured indole-3-acetic acid (IAA) level was investigated in plant materials having different contents of lAA-conjugates, Tissues from pine ( Pinus sylvestris L.). tobacco ( Nicotiana tabacum L.), and maize ( Zea mays L.) were extracted for 1–9 h with Na-phosphate buffer (pH 7.5). 80% methanol and 70% acetone. IAA was measured by combined gas chromatography-selected ion minitoring-mass spectromctry (GC-SIM-MS) with [¹³C₆]-IAA as an internal standard.
Extraction of maize seedlings with buffer gave a higher estimate of free IAA than did extraction with methanol or acetone, which produced similar values. The increase in free IAA after buffer extraction was paralleled by a stoichiometric decrease in lAA-ester conjugates, indicating that free IAA was formed during buffer extraction by hydrolysis of these conjugates, which are abundant in maize seedlings. The amount of hydrolysis during a 1-h extraction period was estimated to be ca 3% of the total lAA-ester pool. However, in the pine extraxylary tissues and tobacco in-ternodes which lack a significant lAA-ester pool, buffer extraction resulted in the same IAA estimate as extraction with the organic solvents, but produced a cleaner extract. For all the plant materials investigated, a 1-h extraction period was sufficient for equilibrating the internal standard with the endogenous IAA pool.     

Lack of discontinuous gas exchange in a tracheate arthropod, Leiobunum townsendi (Arachnida, Opiliones)

Abstract The discontinuous gas exchange cycle, characterized by stringent spiracular control and periods of near-zero external CO₂ emission separated by 'bursts' of CO₂ emission, has evolved independently in several taxa of tracheate arthropods. These include the hexapoda, diplopoda, and several arachnid taxa; ticks, pseudoscorpions and solphugids. This paper presents the first data on gas exchange kinetics in a harvestman (Arachnida; Opiliones). The experimental animal, Leiobunum townsendi Weed, from an arid area of the south-western United States, displayed a metabolic rate similar to those of other arthropods at 25 °C (129 ± 22 µW). Their CO₂ emission kinetics showed, when the animals were motionless, only minor variations about a mean value of 0.0217 ± 0.0037 mL/h ( n  = 6, mean body mass 86 mg). Expressed on an intra-recording basis, the coefficient of variation of CO₂ emission (= SD/MEAN), which is an index of short-term gas emission fluctuations and thus of spiracular control, had a mean value of only 0.082. In contrast, the coefficient of variation of animals employing a discontinuous gas exchange cycle is > 1.5. Gas exchange in opilionids, unlike the case with most other tracheate arthropods, may therefore be dominated by simple diffusion without a prominent role for wide modulations of spiracular conductance. Contributory to this conservative spiracular control strategy may be the weak degree of tracheation in opilionids, combined with circulating haemocyanin, which acts as both a transport medium and a buffering reservoir for respiratory gas exchange.     

Effects of water stress on gas exchange of field grown <Emphasis Type="Italic">Zea mays</Emphasis> L. in Southern Italy: an analysis at canopy and leaf level

Zea mays is cultivated in the Mediterranean regions where summer drought may lead to photoinhibition when irrigation is not available. In this work the response of maize to water stress was evaluated by gas exchange measurements at the canopy and leaf level. Leaf gas exchange was assessed before, during and after water stress, while canopy turbulent fluxes of mass and energy were performed on a continuous basis. In the early growth period, a linear increment of net ecosystem photosynthetic rate (P _NE) to incoming of photosynthetic photon flux density (PPFD) was found and net leaf photosynthetic rate (P _NL) showed the tendency to saturate under high irradiance. During water stress, the relationship between P _NE and PPFD became curvilinear and both P _NE and P _NL saturated in a range between 1,000 and 1,500 μmol (photons) m⁻² s⁻¹. Leaf water potential (ψ_l) dropped from −1.50 to −1.88 MPa during water stress, indicating that leaf and canopy gas exchanges were limited by stomatal conductance. With the restoration of irrigation, P _NE, P _NL and ψ_l showed a recovery, and P _NE and P _NL reached the highest values of whole study period. Leaf area index (LAI) reached a value of 3.0 m² m⁻². The relationship between P _NE and PPFD remained curvilinear and P _NE values were lower than those of a typical well-irrigated maize crop. The recovery in P _NE and P _NL after stress, and ψ_l values during stress indicate that the photosynthetic apparatus was not damaged while soil moisture stress after-effects resulted in a sub-optimal LAI values, which in turn depressed P _NE.     

Composition of fatty acids triacylglycerols and unsaponifiable matter in Calophyllum calaba L. oil from Guadeloupe

The composition of the kernel oils of two Calophyllum species (Calophyllum calaba L. and Calophyllum inophyllum L.) was investigated. The physico-chemical properties and fatty acid composition of the kernel oils were examined. In two species, oleic acid C18:1 (39.1-50%) is the dominating fatty acid followed by linoleic acid C18:2 (21.7-31.1%) as the second major fatty acid. Stearic C18:0 (13.4-14.3%) and palmitic C16:0 (11-13.7%) acids are the major saturates. The oils contains an appreciable amount of unsaturated fatty acids (70.8-73.10%). Most of the fatty acids are present as triacylglycerol (76.7-84%), twenty one triacylglycerols are detected with predominantly unsaturated triacylglycerols. The total unsaponifiable content, its general composition and the identity of the components of the sterol and tocopherol fractions are presented. In both species, analysis of the unsaponifiable fractions revealed the preponderance of phytosterols, mainly stigmasterol (35.8-45.1%) and beta-sitosterol (41.1-43.1%). Among the eight tocopherols and tocotrienols present in two species, variations exist; alpha-tocopherol (183 mg/kg) is the main tocopherol in Calophyllum calaba L. and Delta-tocotrienol (236 mg/kg) is the dominant tocotrienol in Calophyllum inophyllum L.     

Determination of dichloromethane, trichloroethylene and perchloroethylene in urine samples by headspace solid phase microextraction gas chromatography-mass spectrometry

A method for the determination of volatile chlorinated hydrocarbons, namely dichloromethane (DCM), trichloroethylene (TCE), and perchloroethylene (PCE), in urine samples was developed using headspace solid phase microextraction (HS-SPME) gas chromatography-mass spectrometry (GC-MS). HS-SPME was performed using a 75 microm Carboxen-polydimethylsiloxane fiber. Factors, which affect the HS-SPME process, such as adsorption and desorption times, stirring, salting-out effect, and temperature of sampling have been evaluated and optimized. The highest extraction efficiency was obtained when sampling was performed at room temperature (22 degrees C), from samples saturated with salt and under agitation. Linearity of the HS-SPME-GC-MS method was established over four orders of magnitude and the limit of detection was 0.005 microg/l for all the compounds. Precision, calculated as %R.S.D. at three different concentration levels, was within 1-8% for all intra- and inter-day determinations. The method was applied to the quantitative determination of TCE and PCE in human urine samples from exposed (TCE, n=5; median, 9.32 microg/l and PCE, n=39; median, 0.58 microg/l) and non-exposed individuals (n=120; median concentrations, 0.64, 0.22 and 0.11 microg/l for DCM, TCE and PCE, respectively. In addition, two cases of acute accidental exposure to DCM are reported, and the elimination kinetics in blood and urine was followed up. The calculated half-lives of urinary and blood DCM were, respectively, 7.5 and 8.1 h for one subject and 3.8 and 4.3 h for the other.     

Structural changes in a binary mixed phospholipid bilayer of DOPG and DOPS upon saposin C interaction at acidic pH utilizing P and H solid-state NMR spectroscopy

Saposin C (Sap C) is known to stimulate the catalytic activity of the lysosomal enzyme glucosylceramidase (GCase) that facilitates the hydrolysis of glucosylceramide to ceramide and glucose. Both Sap C and acidic phospholipids are required for full activity of GCase. In order to better understand this interaction, mixed bilayer samples prepared from dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylserine (DOPS) (5:3 ratio) and Sap C were investigated using ²H and ³¹P solid-state NMR spectroscopy at temperatures ranging from 25 to 50 °C at pH 4.7. The Sap C concentrations used to carry out these experiments were 0 mol%, 1 mol% and 3 mol% with respect to the phospholipids. The molecular order parameters (S_CD) were calculated from the dePaked ²H solid-state NMR spectra of Distearoyl-d70-phosphatidylglycerol (DSPG-d70) incorporated with DOPG and DOPS binary mixed bilayers. The S_CD profiles indicate that the addition of Sap C to the negatively charged phospholipids is concentration dependent. S_CD profiles of 1 mol% of the Sap C protein show only a very slight decrease in the acyl chain order. However, the S_CD profiles of the 3 mol% of Sap C protein indicate that the interaction is predominantly increasing the disorder in the first half of the acyl chain near the head group (C1-C8) indicating that the amino and the carboxyl termini of Sap C are not inserting deep into the DOPG and DOPS mixed bilayers. The ³¹P solid-state NMR spectra show that the chemical shift anisotropy (CSA) for both phospholipids decrease and the spectral broadening increases upon addition of Sap C to the mixed bilayers. The data indicate that Sap C interacts similarly with the head groups of both acidic phospholipids and that Sap C has no preference to DOPS over DOPG. Moreover, our solid-state NMR spectroscopic data agree with the structural model previously proposed in the literature [X. Qi, G.A. Grabowski, Differential membrane interactions of saposins A and C. Implication for the functional specificity, J. Biol. Chem. 276 (2001) 27010-27017] [1].     

Isolation of 2-methyl branched unsaturated very long fatty acids from marine sponge Halichondria panicea and identification of them by GC-MS and NMR

In order to study the biomarker fatty acids of symbionts in the marine sponge Halichondria panicea, purification and structural identification of two new 2-methyl branched monoenoic very long fatty acids (2-Me-24:1 n-7 and 2-Me-26:1 n-9) were performed for the first time. These acids amounted to 7.1% of total sponge FAs, but our attempts to determine their structures by one-step GC-MS analysis were unsuccessful because of low yields of the correspondent N-acyl pyrrolidide derivatives. Silver-ion thin-layer chromatography isolated enriched fractions of monoenoic fatty acids extracted from the sponge. Further purification of unknown fatty acid methyl esters was carried out by reversed-phase high-performance liquid chromatography. Determination of the chain length, degree and position of unsaturations was achieved by gas chromatography-mass spectrometry on methyl esters and dimethyldisulfide adducts. Structures, position of methyl substitution, and double bonds cis isomery were confirmed by 1H nuclear magnetic resonance.     

Ontogeny of hemocyanin in the ovoviviparous cockroach Blaptica dubia suggests an embryo-specific role in oxygen supply

For a long time it had been assumed that specific oxygen transport proteins are absent in insects. Only recently it has been demonstrated that hemocyanins occur in the hemolymph of many ametabolous and hemimetabolous insect taxa, but not in the Eumetabola (Hemiptera + Holometabola). Therefore, the loss of respiratory hemocyanin in insects is not correlated with the evolution of an efficient tracheal system. The specific contribution of hemocyanin to oxygen supply in insects, however, has remained uncertain. Here we investigate the stage-specific expression of hemocyanin in the ovoviviparous cockroach Blaptica dubia (Blattaria), which consists of two distinct subunit types (Hc1 and Hc2). Employing quantitative real-time RT-PCR and Western blotting, we showed that the expression of hemocyanin is restricted to late embryos, thus being detectable also in whole female extracts and oothecae. Hemocyanin protein is also present in 1st instar nymphs, but not in later developmental stages. The ontogeny of hemocyanin in cockroaches is distinct from that known from Zygentoma and Plecoptera, in which hemocyanin occurs in both nymphal and adult stages. Our findings suggest a specific role of hemocyanin in embryonic cockroaches, which may be related to an enhanced oxygen supply in the oothecae. For some reason, the fundamental physiological changes associated to the evolution of holometaboly have made hemocyanin unnecessary.     

Cryoprotection in dampwood termites (Termopsidae, Isoptera)

In contrast to the majority of the Order, the dampwood termites of the family Termopsidae found in colder regions can experience frost and snow, either in cool temperate areas at high latitudes (45°), or alpine areas at high elevations (>1000 m). This suggests that dampwood termites are adapted to cold climates. We investigated this hypothesis in two dampwood termites, Porotermes adamsoni Froggatt and Stolotermes victoriensis Hill. We measured nest temperatures and atmospheric temperatures of their alpine habitat during winter, and measured survival and recovery at subzero temperatures. We also determined the minimum temperature at which these species remain active and the LT50 values. We used a novel gas chromatographic strategy to examine eight metabolites from individuals of both species collected in winter and summer to identify possible cryoprotectants. Both P. adamsoni and S. victoriensis had significantly higher levels of trehalose, a known cryoprotectant, in winter than in summer; in addition S. victoriensis also had higher levels of unsaturated fatty acid ligands in winter than in summer, consistent with patterns observed for cold adaptation in other organisms. These results are the first to reveal that dampwood termites are adapted to cold climates and use trehalose and unsaturated lipids as cryoprotectants.