Luca Calamai

Drought stress has contrasting effects on antioxidant enzymes activity and phenylpropanoid biosynthesis in Fraxinus ornus leaves: an excess light stress affair?

The experiment was conducted using Fraxinus ornus plants grown outside under full sunlight irradiance, and supplied with 100% (well-watered, WW), 40% (mild drought, MD), or 20% (severe drought, SD) of the daily evapotranspiration demand, with the main objective of exploring the effect of excess light stress on the activity of antioxidant enzymes and phenylpropanoid biosynthesis. Net CO₂ assimilation rate at saturating light and daily assimilated CO₂ were significantly smaller in SD than in WW and MD plants. Xanthophyll-cycle pigments supported nonphotochemical quenching to a significantly greater extent in SD than in MD and WW leaves. As a consequence, the actual efficiency of PSII (Φ(PSII)) was smaller, while the excess excitation-energy in the photosynthetic apparatus was greater in SD than in WW or MD plants. The concentrations of violaxanthin-cycle pigments relative to total chlorophyll (Chl(tot)) exceeded 200 mmol mol⁻¹ Chl(tot) in SD leaves at the end of the experiment. This leads to hypothesize for zeaxanthin a role not only as nonphotochemical quencher, but also as chloroplast antioxidant. Reductions in ascorbate peroxidase and catalase activities, as drought-stress progressed, were paralleled by greater accumulations of esculetin and quercetin 3-O-glycosides, both phenylpropanoids having effective capacity to scavenge H₂O₂. The drought-induced accumulation of esculetin and quercetin 3-O-glycosides in the vacuoles of mesophyll cells is consistent with their putative functions as reducing agents for H₂O₂ in excess light-stressed leaves. Nonetheless, the concentration of H₂O₂ and the lipid peroxidation were significantly greater in SD than in MD and WW leaves. It is speculated that vacuolar phenylpropanoids may constitute a secondary antioxidant system, even on a temporal basis, activated upon the depletion of primary antioxidant defences, and aimed at keeping whole-cell H₂O₂ within a sub-lethal concentration range.

Chemical Composition of Scent-Gland Secretions in an Old World Monkey (Mandrillus sphinx): Influence of Sex, Male Status, and Individual Identity

Primates are traditionally considered to be microsmatic, with decreased reliance on olfactory senses in comparison to other sensory modalities such as vision. This is particularly the case for Old World monkeys and apes (catarrhines). However, various lines of evidence suggest that chemical communication may be important in these species, including the presence of a sternal scent-gland in the mandrill. We investigated the volatile components of mandrill odor using gas chromatography-mass spectrometry. We identified a total of 97 volatile components in 88 swabs of the sternal gland secretion and 95 samples of sternal gland hair saturated with scent-gland secretion collected from 27 males and 18 females. We compared odor profiles with features of the signaler using principle components and discriminant function analyses and found that volatile profiles convey both variable (age, dominance rank in males) and fixed (sex, possibly individual identity) information about the signaler. The combination of an odor profile that signals sex, age, and rank with increased motivation to scent-mark and increased production of secretion in high-ranking males leads to a potent signal of the presence of a dominant, adult male with high testosterone levels. This may be particularly relevant in the dense Central African rain forest which mandrills inhabit. By contrast, we were unable to differentiate between either female cycle stage or female rank based on odor profiles, which accords with behavioral studies suggesting that odor signals are not as important in female mandrills as they are in males. The similarity of our findings to those for other mammals and in primates that are more distantly related to humans suggests a broader role for odor in primate communication than is currently recognized.

Headspace-Solid Phase Microextraction Approach for Dimethylsulfoniopropionate Quantification in Solanum lycopersicum Plants Subjected to Water Stress

Dimethylsulfoniopropionate (DMSP) and dimethyl sulphide (DMS) are compounds found mainly in marine phytoplankton and in some halophytic plants. DMS is a globally important biogenic volatile in regulating of global sulfur cycle and planetary albedo, whereas DMSP is involved in the maintenance of plant-environment homeostasis. Plants emit minute amounts of DMS compared to marine phytoplankton and there is a need for hypersensitive analytic techniques to enable its quantification in plants. Solid Phase Micro Extraction from Head Space (HS-SPME) is a simple, rapid, solvent-free and cost-effective extraction mode, which can be easily hyphenated with GC-MS for the analysis of volatile organic compounds. Using tomato (Solanum lycopersicum) plants subjected to water stress as a model system, we standardized a sensitive and accurate protocol for detecting and quantifying DMSP pool sizes, and potential DMS emissions, in cryoextracted leaves. The method relies on the determination of DMS free and from DMSP pools before and after the alkaline hydrolysis via Headspace-Solid Phase Micro Extraction-Gas Chromatography-Mass Spectrometry (HS-SPME-GC-MS). We found a significant (2.5 time) increase of DMSP content in water-stressed leaves reflecting clear stress to the photosynthetic apparatus. We hypothesize that increased DMSP, and in turn DMS, in water-stressed leaves are produced by carbon sources other than direct photosynthesis, and function to protect plants either osmotically or as antioxidants. Finally, our results suggest that SPME is a powerful and suitable technique for the detection and quantification of biogenic gasses in trace amounts.

Determination of organic acids in urine by solid‐phase microextraction and gas chromatography–ion trap tandem mass spectrometry previous ‘in sample’ derivatization with trimethyloxonium tetrafluoroborate

A method for the determination of the organic acids directly in the urine employing derivatization with trimethyloxonium tetrafluoroborate as a methylating agent and sequential extraction by head space and direct immersion/solid phase microextraction is reported. Furoic acid, hippuric acid, methylhippuric acid, mandelic acid, phenylglyoxylic acid and trans, trans muconic acid contained in urine and proposed by the American Conference of Governmental Industrial Hygienists as biological exposure indices were determined after a fast and economically convenient preparation step and sensitive gas chromatography-ion trap-mass spectrometry/tandem mass spectrometry analysis. Urine is rather a complex sample and hence the acquisition method required specific GC-MS instrumentation capable of supporting the changeover, fully automated during a single chromatographic separation, from mass to tandem mass spectrometry and both chemical and electron ionization modes. The automation of the analytical method provides a number of advantages, including reduced analysis time for both routine analysis and method development, and greater reproducibility. The equilibrium and kinetics of this substances vs head space/direct immersion-solid phase microextraction were investigated and evaluated theoretically.

Interaction of catalase with montmorillonite homoionic to cations with different hydrophobicity: effect on enzymatic activity and microbial utilization

Abstract The exchange sites of montmorillonite (M) were made homoionic to calcium (Ca), hexadecyltrimethylammonium (HDTMA) or pyridinium (PY) cations. The clays were used as adsorbents for the enzyme, catalase (CA). Equilibrium adsorption and binding isotherms (i.e., after washing of the clay–CA complexes after adsorption at equilibrium until no CA was desorbed) were of the L 3 -type and fitted the Langmuir equation in the initial, but not in the later, portions of the isotherms. The amounts adsorbed and bound at the plateau, as well as the affinity, were higher for the hydrophobic clays (M–HDTMA and M–PY) as indicated by the Langmuir parameter, B max and K eq . In all three systems, there was additional adsorption after the initial plateau at higher concentrations of CA. In the case of M–Ca–CA this probably resulted from some penetration of CA into the interlayer spaces of the clay, as shown by X-ray diffraction analysis. No penetration of the interlayers was observed in the M–HDTMA–CA and M–PY–CA systems. The additional adsorption that occurred after the initial plateau in these systems may have resulted from the formation of multilayers of CA or from a change in the orientation of CA on these clay-organic surfaces, which may also have occurred in the M–Ca–CA system in addition to intercalation. Most of the CA adsorbed at equilibrium was bound on the clays (85–90%). Fourier-transform infrared difference spectra showed a shift in the Amide I and Amide II frequencies for only M–Ca–CA and M–PY–CA, which was consistent with the hypothesis of a conformational modification of the structure of CA on M–Ca and M–PY. The enzymatic activity of CA adsorbed at equilibrium on the three clays was lower than that of free CA and decreased in the order of M–Ca–CA>M–PY–CA>M–HDTMA–CA. As shown by the values of the overall first-order rate constant, K 1 , there was a further reduction in activity when CA was bound on the clays, especially on M–Ca. The pH optimum for the activity of CA remained essentially unchanged when adsorbed or bound on all clays. CA bound on the clay systems, except on M–PY–CA, was poorly utilized in comparison with the free enzyme as a sole source of carbon or nitrogen.

The influence of tuber mineral element composition as a function of geographical location on acrylamide formation in different Italian potato genotypes

BACKGROUND The present study was aimed at examining the effect of tuber mineral composition, distinctive for geographical location, on the expression of acrylamide precursors in three potato genotypes (Arinda, Rossa di Cetica and Sieglinde) in three Italian potato cultivation regions (Puglia, Sicily and Tuscany). RESULTS Sucrose and amino acids were not correlated with acrylamide formation. In contrast, reducing sugars, limiting with respect to the principal amino acid precursor asparagine, were positively correlated with acrylamide. From analysis of variance, both acrylamide and reducing sugars were not significant for variety but highly significant for location, with higher levels occurring in all three varieties cultivated in Tuscany, followed by Sicily and Puglia respectively. Reducing sugars were negatively correlated with K and Ca and positively correlated with Zn and Cu. Neither N nor P was correlated with reducing sugar content. Path analysis, a statistical technique distinguishing causation and correlation between variables, was implemented to provide additional insight on the interactions between mineral elements and reducing sugars under open field conditions. The variation in reducing sugars in all three varieties was shown to be attributable to Zn. CONCLUSION Cultivation location has a significant impact on the composition of tuber mineral elements and, as a result, on genotype in the expression of reducing sugars. The negative correlation between Zn and K and the variation in reducing sugar content attributed to the element Zn, which is particularly available in acid soils, are important factors warranting future research aimed at reducing acrylamide formation from an agronomic perspective.

Increase in the Level of Proinflammatory Cytokine HMGB1 in Nasal Fluids of Patients With Rhinitis and its Sequestration by Glycyrrhizin Induces Eosinophil Cell Death

Objectives The nuclear protein high mobility group protein box 1 (HMGB1) is a proinflammatory mediator that belongs to the alarmin family of proinflammatory mediators, and it has recently emerged as a key player in different acute and chronic immune disorders. Several lines of evidence demonstrate that HMGB1 is actively released extracellularly from immune cells or passively released from necrotic cells. Because of the ability of HMGB1 to sustain chronic inflammation, we investigated whether the protein is present in nasal fluids of patients with different forms of rhinitis. Methods HMGB1 levels were evaluated in nasal fluids of healthy subjects or rhinitis patients who were treated or not treated with different treatments. Results We report that the level of HMGB1 was significantly increased in nasal fluids of patients with allergic rhinitis, patients with NARES (nonallergic rhinitis with eosinophiliac syndrome), as well as patients with polyps. We also found that a formulation containing the HMGB1-binding compound glycyrrhizin (GLT) reduced the HMGB1 content in nasal fluids of rhinitis patients to an extent similar to that with nasal budesonide treatment. We also found that among the cultured human leukocyte populations, eosinophils released higher amounts of HMGB1. Based on the ability of HMGB1 to sustain eosinophil survival and the ability of GLT to inactivate HMGB1, we report that GLT selectively killed cultured eosinophils and had no effect on neutrophils, macrophages, and lymphocytes. Conclusion Collectively, these data underscore the role of HMGB1 in rhinitis pathogenesis and the therapeutic potential of GLT formulations in treatment of chronic inflammatory disorders of the nasal mucosa.

Interferences of suspended clay fraction in protein quantitation by several determination methods

Seven current methods of protein quantitation, Bradford (standard, micro, and 590/450 nm ratio), Lowry, bicinchoninic acid (BCA), UV spectrophotometry at 280 nm, and Quant-iT fluorescence-based determination, were compared with regard to their susceptibility to interferences due to the presence of suspended and not easily detectable clay particles. Bovine serum albumin (BSA) and Na-Wyoming montmorillonite were selected as model protein and reference clay, respectively. Protein-clay suspension mixtures were freshly prepared for each assay to simulate supernatants not completely centrifuged in batch sorption/kinetic experiments. Seven fixed increasing levels of clay (0.0, 0.00725, 0.0145, 0.029, 0.058, 0.145, 0.435 mg ml(-1)) were mixed with different levels of BSA in an appropriate range for each assay. To ascertain the interfering effect of different levels of clay, the theoretical concentrations of BSA were plotted against the estimated BSA concentrations of the samples, as obtained from the calibration curve of each method. A correct quantitation of the BSA concentration not influenced by clay would be described by a regression line with slope (b) not significantly different from 1 and an intercept (a) not significantly different from zero. At the lowest clay levels (0.00725 mg ml(-1)) a significant interference was evident for Bradford micro, Bradford 590/450, UV, and fluorescence. The three methods (Bradford standard, Lowry, and BCA) that seemed to show the better performances in the presence of clay after this first screening step also underwent an ANCOVA analysis, with the measured BSA concentrations as dependent variable and the clay concentrations as covariate. The Bradford standard and BCA methods were affected by a clay-dependent interference on BSA quantitation. The Lowry assay was the only method that gave correct estimates of BSA concentrations in the presence of any of the clay levels tested.

Interactions of two acetanilide herbicides with clay surfaces modified with Fe(III) oxyhydroxides and hexadecyltrimethyl ammonium

Abstract The adsorption at 22° and 5°C of two acetanilide herbicides (Alachlor and Metolachlor) on montmorillonite saturated with HDTMA+ cation or coated with Fe(III) oxyhydroxides (modified clays) was studied. The isotherms were of L, S and C type depending on the type of the adsorbent. The calculated values of isosteric adsorption heat (ΔH) and desorption experiments suggest that weak bonds take place. FT-IR analyses showed that both molecules are adsorbed on clays by hydrogen bond between the C-O group and OH groups of Fe(III) oxyhydroxy cation coating the clay. Also on HDTMA-Montmorillonite the C-O group is involved in the adsorption but with lower strenght. Alachlor is only partially decomposed to 2-chloro-2′,6′-diethylacetanilide when the dirty clays complexes are heated at 70°C for 18 h. X-ray analyses showed that both herbicides were unable to penetrate into the inter-layers of the clays.

Chemical disguise of myrmecophilous cockroaches and its implications for understanding nestmate recognition mechanisms in leaf-cutting ants

BackgroundCockroaches of the genus Attaphila regularly occur in leaf-cutting ant colonies. The ants farm a fungus that the cockroaches also appear to feed on. Cockroaches disperse between colonies horizontally (via foraging trails) and vertically (attached to queens on their mating flights). We analysed the chemical strategies used by the cockroaches to integrate into colonies of Atta colombica and Acromyrmex octospinosus. Analysing cockroaches from nests of two host species further allowed us to test the hypothesis that nestmate recognition is based on an asymmetric mechanism. Specifically, we test the U-present nestmate recognition model, which assumes that detection of undesirable cues (non-nestmate specific substances) leads to strong rejection of the cue-bearers, while absence of desirable cues (nestmate-specific substances) does not necessarily trigger aggression.ResultsWe found that nests of Atta and Acromyrmex contained cockroaches of two different and not yet described Attaphila species. The cockroaches share the cuticular chemical substances of their specific host species and copy their host nest’s colony-specific cuticular profile. Indeed, the cockroaches are accepted by nestmate but attacked by non-nestmate ant workers. Cockroaches from Acromyrmex colonies bear a lower concentration of cuticular substances and are less likely to be attacked by non-nestmate ants than cockroaches from Atta colonies.ConclusionsNest-specific recognition of Attaphila cockroaches by host workers in combination with nest-specific cuticular chemical profiles suggest that the cockroaches mimic their host’s recognition labels, either by synthesizing nest-specific substances or by substance transfer from ants. Our finding that the cockroach species with lower concentration of cuticular substances receives less aggression by both host species fully supports the U-present nestmate recognition model. Leaf-cutting ant nestmate recognition is thus asymmetric, responding more strongly to differences than to similarities.