Search

Significance Voltage sensing with genetically expressed optical probes is highly desirable for large-scale recordings of neuronal activity and detection of localized voltage signals in single neurons. Here we describe a method for a two-component (hybrid) genetically encodable fluorescent voltage sensing in neurons. The approach uses a glycosylphosphatidylinositol-tagged fluorescent protein (enhanced green fluorescent protein) that ensures the fluorescence to be specifically confined to the outside of the plasma membrane and D3, a voltage-dependent quencher. Previous hybrid genetically encoded voltage sensing approaches relied on a single quenching molecule, dipycrilamine (DPA), which is toxic, increases membrane capacitance, interferes with neurotransmitters, and is explosive. Our method uses a nontoxic and nonexplosive compound that performs better than DPA in all aspects of fluorescent voltage sensing., Voltage sensing with genetically expressed optical probes is highly desirable for large-scale recordings of neuronal activity and detection of localized voltage signals in single neurons. Most genetically encodable voltage indicators (GEVI) have drawbacks including slow response, low fluorescence, or excessive bleaching. Here we present a dark quencher GEVI approach (dqGEVI) using a Förster resonance energy transfer pair between a fluorophore glycosylphosphatidylinositol–enhanced green fluorescent protein (GPI-eGFP) on the outer surface of the neuronal membrane and an azo-benzene dye quencher (D3) that rapidly moves in the membrane driven by voltage. In contrast to previous probes, the sensor has a single photon bleaching time constant of ∼40 min, has a high temporal resolution and fidelity for detecting action potential firing at 100 Hz, resolves membrane de- and hyperpolarizations of a few millivolts, and has negligible effects on passive membrane properties or synaptic events. The dqGEVI approach should be a valuable tool for optical recordings of subcellular or population membrane potential changes in nerve cells.

The Bunyavirales order accommodates related viruses (bunyaviruses) with segmented, linear, single-stranded, negative- or ambi-sense RNA genomes. Their glycoproteins form capsomeric projections or spikes on the virion surface and play a crucial role in virus entry, assembly, morphogenesis. Bunyavirus glycoproteins are encoded by a single RNA segment as a polyprotein precursor that is co- and post-translationally cleaved by host cell enzymes to yield two mature glycoproteins, Gn and Gc (or GP1 and GP2 in arenaviruses). These glycoproteins undergo extensive N-linked glycosylation and despite their cleavage, remain associated to the virion to form an integral transmembrane glycoprotein complex. This review summarizes recent advances in our understanding of the molecular biology of bunyavirus glycoproteins, including their processing, structure, and known interactions with host factors that facilitate cell entry.

International audience; Mononegaviruses, such as Ebola virus, encode an L (large) protein that bears all the catalytic activities for replication/transcription and RNA capping. The C-terminal conserved region VI (CRVI) of L protein contains a K-D-K-E catalytic tetrad typical for 2'O methyltransferases (MTase). In mononegaviruses, cap-MTase activities have been involved in the 2'O methylation and N7 methylation of the RNA cap structure. These activities play a critical role in the viral life cycle as N7 methylation ensures efficient viral mRNA translation and 2'O methylation hampers the detection of viral RNA by the host innate immunity. The functional characterization of the MTase+CTD domain of Sudan ebolavirus (SUDV) revealed cap-independent methyltransferase activities targeting internal adenosine residues. Besides this, the MTase+CTD also methylates, the N7 position of the cap guanosine and the 2'O position of the n1 guanosine provided that the RNA is sufficiently long. Altogether, these results suggest that the filovirus MTases evolved towards a dual activity with distinct substrate specificities. Whereas it has been well established that cap-dependent methylations promote protein translation and help to mimic host RNA, the characterization of an original cap-independent methylation opens new research opportunities to elucidate the role of RNA internal methylations in the viral replication.

Currently, Salmonella enterica serovar Typhimurium (S. Typhimurium), is a major global public health problem, which has caused food-borne illnesses in many countries. Today, with the extensive use of antimicrobials, antimicrobial resistance is increasing at a serious rate in S. Typhimurium isolates. The present study sought the role of cysteine (Cys) auxotrophy on the resistance to quinolones and paraquat in S. Typhimurium. Cys auxotrophy was achieved by deleting either the cysDNC, cysJIH or cysQ loci. Deletion of these loci resulted in loss of susceptibility against nalidixic acid, levofloxacin, ciprofloxacin (CIP) and paraquat. Further studies with cysJIH mutant indicated increased expression of multi-antibiotic resistance genes marA and ramA, and consequently increased expression of efflux-pump systems. The cysJIH mutant presented a smaller increase of reactive oxygen species (ROS) in presence of paraquat or CIP. Expression of katG and sodA (expressing for a catalase and a superoxide dismutase, respectively) genes was increased in presence of paraquat in the cysJIH mutant; while expression of the superoxide dismutase gene sodB was decreased. These results indicate that deletion of cysDNC, cysJIH or cysQ genes of S. Typhimurium renders Cys auxotrophy along with decreased susceptibility in response to quinolone and paraquat. Overexpression of efflux-pump systems AcrB-TolC and SmvA-OmpD and antioxidant enzymes KatG and SodA could explain the mechanisms of antimicrobial resistance in the Cys auxotrophic mutants.

Benzimidazoles presenting intramolecular hydrogen bonding interactions have been normally used to better understand the role of H-bonding in biological processes. Here, we present an experimental and theoretical study of a new compound [2,4-di-tert-butyl-6-(3H-imidazo[4,5-c]pyridine-2-yl)phenol]; (B2), a benzimidazole derivate, exhibiting an intramolecular hydrogen bond. B2 was synthesized and characterized by its 1H, HHCOSY, FT-IR and mass spectra (EI-MS 323 M+). The electronic and optical properties of B2 were studied with theoretical calculations using density functional theory (DFT) and time-dependent DFT (TDDFT). B2 showed luminescent emission at room temperature in different solvents, with a large Stokes shift (e.g.; λex = 335 nm; λem = 510 nm in acetonitrile). Also, the quantum yield (φ = 0.21) and theoretical band emission are reported. We found that B2 exhibited a fluorescence emission at around 500 nm in ethanol and in acetonitrile that could be quenched by aqueous solutions of Hg(NO3)2 in the range of micro molar concentrations. Cyclic voltammetry in acetonitrile showed a strong anodic response due to a quasireversible process, with reduction and oxidation waves at −1.28 and −0.47 V vs. SCE. Regarding the biological properties, we assessed the antimicrobial activity of B2 in Salmonella enterica (bacteria), Cryptococcus spp. (yeast), Candida albicans (yeast), Candida tropicalis (yeast) and Botrytis cinerea (mold). To this end, we determined the minimal inhibitory concentration (MIC) (for bacteria and yeasts), the growth inhibition halos (for yeasts), and the inhibition of mycelial growth (for the mold). We observed that B2 exerted an antifungal effect against Cryptococcus spp. and Botrytis cinerea. In addition, due to its fluorescence properties, B2 has proven to be a suitable marker to observe bacteria (Salmonella enterica and an Escherichia coli derivative), yeasts (Candida albicans), and even human cells (SKOV-3 and HEK-293) by confocal microscopy.

Indexación: Scopus. Objectives: The aim was to study the role played by SmvA pump in the efflux of quaternary ammonium compounds (QACs) in Salmonella enterica serovar Typhimurium (Salmonella Typhimurium). Methods: Mutants in the smvA, acrB and tolC genes were constructed by the red swap method. P22 was used to transduce tolC to acrB and smvA mutant strains. The susceptibility of these strains to acriflavine and a variety of QACs was determined by MIC assays. Results: In comparison with the Salmonella Typhimurium wild-type strain, the smvA mutant was more susceptible to QACs than the acrB mutant strain. A tolC single mutant was more susceptible than an acrB mutant to QACs, acriflavine, ethidium bromide, malachite green and pyronin B. The tolC - acrB double mutant was as susceptible as the single tolC mutant to QACs. Additionally, the smvA mutant strain was more susceptible to acriflavine than the acrB mutant (MICs = 31.3 versus 125 mg/L, i.e. 4-fold). Finally, the tolC - smvA double mutant (3.9 mg/L) was approximately 10 times more susceptible to acriflavine than either smvA (31.3 mg/L) or tolC (31.3 mg/L) single mutants. Conclusions: It is the SmvA efflux pump, and not AcrB, that plays the major role in the efflux of acriflavine and other QACs from Salmonella Typhimurium. This apparently conflicting report is due to the fact that in Escherichia coli the smvA gene does not exist. Our results suggest that tolC and smvA genes encode components of two different efflux systems with overlapping specificities that work in parallel to export acriflavine and other QACs. © The Author 2008. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. https://academic-oup-com.recursosbiblioteca.unab.cl/jac/article/62/6/1273/771646

The transcriptional activator retinoic acid (RA) supports axonal regeneration of several neuronal cell populations in vitro, and it has been suggested that its receptor RARbeta2 may be used to support axonal regeneration in the adult mammalian spinal cord. We have previously shown that spinal cord injury induces activity of the RA synthesizing enzyme retinaldehyde dehydrogenase (RALDH)2 in NG2-positive cells. This report quantifies the increase of RALDH2 protein in the injured spinal cord and characterizes the RALDH2/NG2 expressing cells probably as a unique RA synthesizing subpopulation of activated oligodendrocyte precursors or "polydendrocytes". In the uninjured spinal cord low levels of RALDH2 are present in oligodendrocytes as well as in the meninges and in blood vessels. Following injury there is a significant increase in RALDH2 in these latter two tissues and, given that the RALDH2/NG2 positive cells are clustered in the same area, this implies that these are specific foci of RA synthesis. It is presumed that these cells release RA in a paracrine fashion in the region of the wound; however, the RALDH2/NG2-immunoreactive cells expressed the retinoid receptors RARalpha, RARbeta, RXRalpha and RXRbeta, suggesting that RA also serves an autocrine function.

Dimethyl fumarate (DMF) is a chemical compound which has been added to silica gel bags used for preserving leather products during shipment. DMF has recently been singled out due to its ability to induce a number of medical problems in people which touch products contaminated by it. Its use as a biocide has been recently made illegal in Europe. Two different extraction techniques, namely ultrasound-assisted extraction (UAE) and solid-phase microextraction (SPME), both coupled with gas chromatography/mass spectrometry were applied to the quantitative determination of DMF in silica gel. Linearity of the methods, reproducibility and detection limits were determined. The two methods were applied to the quantification of DMF in thirty-four silica gel samples used as anti-mould agents in different leather products sold in Italy, and the obtained results were statistically compared.

Indexación: Scopus. The Salmonella enterica tsx gene encodes a nucleoside-specific outer membrane channel. The Tsx porin is essential for the prototrophic growth of S. enterica sv. Typhi in the absence of nucleosides. RT-PCR analysis shows that the tsx gene is cotranscribed with an open reading frame unique to S. enterica, impX (STY0450), which encodes an inner membrane protein 108 amino acids in length, which is predicted to have only two transmembrane α-helices. Fusions of the lacZ gene to both tsx and impX reveal that the transcription of both genes is induced in the presence of adenosine. A null mutation in the S. Typhi impX gene suppresses the induced auxotrophy for adenosine or thymidine resulting from a tsx mutation and confers sensitivity to high concentrations of adenosine or thymidine. The ImpX protein, when tagged with a 3xFLAG epitope, is functional and associates with the inner membrane; impX mutants are defective in the export of 3H-radiolabeled thymidine. Taken together, these and other results suggest that the S. Typhi Tsx porin and ImpX inner membrane protein facilitate competing mechanisms of thymidine influx and efflux, respectively, to maintain the steady-state levels of internal nucleoside pools. https://academic.oup.com/genetics/article/173/1/25/6061595

The physiological reactions after spinal cord injury are accompanied by local synthesis of the transcriptional activator retinoic acid (RA). RA exerts its effects by binding to retinoic acid receptors (RAR) which heterodimerize with retinoid X receptors (RXR) and then act as ligand-activated transcription factors. To identify possible cellular targets of RA we investigated protein levels and cellular distribution of retinoid receptors in the rat spinal cord at 4, 7, 14 and 21 days after a contusion injury. In the nonlesioned spinal cord, immunoreactivity for RARalpha, RXRalpha, RXRbeta and RXRgamma was localized in the cytosol of neurons, that of RXRalpha and RXRbeta in astrocytes and that of RARalpha, RXRalpha and RXRgamma in some oligodendrocytes. After contusion injury RARalpha and all RXRs appeared in the cell nuclei of reactive microglia and macrophages. This nuclear staining began at 4 days, was most prominent at 7 and 14 days and had decreased at 21 days after injury. A similar nuclear translocation was also observed for the RARalpha, RXRalpha and RXRbeta staining in neurons situated around the border of the contusion. These observations suggest that RA participates as a signal for the physiological responses of microglia and neurons after CNS injury.

The nitric oxide (NO)-cyclic GMP (cGMP) signaling pathway is assumed to play an important role in processes underlying learning and memory. We used phosphodiesterase type 5 (PDE5) inhibitors to study the role of cGMP in object- and spatial memory. Our results and those reported in other studies indicate that elevated hippocampal cGMP levels are required to improve the memory performance of rodents in object recognition and passive avoidance learning, but not in spatial learning. The timing of treatment modulates the effects on memory and strongly supports a role for cGMP in early stages of memory formation. Alternative explanations for the improved memory performance of PDE5 inhibitors are also discussed. Immunocytochemical studies showed that in vitro slice incubations with PDE5 inhibitors increase NO-stimulated cGMP levels mainly in hippocampal varicose fibers. Reviewing the available data on the localization of the different components of the NO-cGMP signaling pathway, indicates a complex interaction between NO and cGMP, which may be independent of each other. It is discussed that further studies are needed, immunocytochemical and behavioral, to better understand the cGMP-mediated molecular mechanisms underlying memory formation.

Because histamine receptor type I blockade attenuates allergic asthma, we asked whether complete neutralization of histamine by an arthropod-derived, high affinity histamine-binding protein (EV131) would prevent allergic asthma. Intranasal administration of EV131 given before Ag challenge in immunized mice prevented airway hyperreactivity by 70%, and abrogated peribronchial inflammation, pulmonary eosinophilia, mucus hypersecretion, and IL-4 and IL-5 secretion. Saturation with histamine abrogated the inhibitory effect of EV131 on bronchial hyperreactivity. The inhibitory effect of EV131 on bronchial hyperreactivity was comparable to that of glucocorticosteroids. These results demonstrate that histamine is a critical mediator of allergic asthma. Therefore, complete neutralization of histamine, rather than specific histamine receptor blockade, may have a profound effect on allergic asthma.

Eighty-five samples taken from polyethylene (PE) water/gas-ducts were pyrolyzed (from triplicate to 9-fold analyses) at 700 °C for 20 s in an N2 atmosphere using a platinum coil pyrolyzer and a tube cooled in liquid nitrogen. The pyrolyzed products were identified and quantified by gas chromatography/mass spectrometry (GC/MS), and subjected to discriminant analysis. The samples included intact materials and sections fractured both in-field and following laboratory tests. Statistical data treatment allowed the samples to be subdivided into three groups. These groups were consistent with a pattern that fits well with sample rating after a modified stress-cracking test based on pipe resistance to increasing internal pressure. Such results can be explained through the relationship between pyrolysis (PY) fingerprint and polymer structure, which is in turn known to be related to the lifetime of the material. Compared with semi-empirical mechanical tests which are seldom used by water companies because of their lengthy duration, the PY and GC/MS approach is faster, simpler, cost effective, and provides molecular data on the original samples. The statistical models based on PY and GC/MS can be a valuable tool for water/gas companies that need to check the quality of PE pipes and predict their lifetime prior to purchase and installation, thus, helping companies set appropriate quality standards to reduce the occurrence of failures along operating pipelines.

To overcome the inflammatory response in its host, the cattle-feeding, brown ear tick secretes histamine-binding proteins into the feeding site. These proteins are β-barrels with two internal binding sites: a high-affinity (H) site for histamine and a site (L) for which the natural ligand is unknown. Here we report a related protein (SHBP), secreted by a rodent- and cattle-feeding tick, that traps both histamine and serotonin. The histamine-binding H site is well conserved in SHBP, whereas residue changes in the L-like site are consistent with binding of the bulkier serotonin molecule. As histamine is a key inflammatory mediator in cattle, while serotonin takes on this role in rodents, the diversification of these tick proteins may reflect host adaptation

Item does not contain fulltext BACKGROUND: Although adenosine monophosphate activated protein kinase (AMPK) plays a crucial role in energy metabolism, a direct effect of AMPK modulation on thyroid function has only recently been reported, and much of its function in the thyroid is currently unknown. The aim of this study was to investigate the mechanism of AMPK modulation in iodide uptake. Furthermore, we wanted to investigate the potential of the AMPK inhibitor compound C as an enhancer of iodide uptake by thyrocytes. METHODS: The in vitro and in vivo effects of AMPK modulation on sodium-iodide symporter (NIS) protein levels and iodide uptake were examined in follicular rat thyroid cell-line cells and C57Bl6/J mice. Activation of AMPK by metformin resulted in a strong reduction of iodide uptake (up to sixfold with 5 mM metformin after 96 h) and NIS protein levels in vitro, whereas AMPK inhibition by compound C not only stimulated iodide uptake but also enhanced NIS protein levels both in vitro (up to sevenfold with 1 muM compound C after 96 h) and in vivo (1.5-fold after daily injections with 20 mg/kg for 4 days). We investigated the regulation of NIS expression by AMPK using a range of promoter constructs consisting of either the NIS promoter or isolated CRE (cAMP response element) and NF-kappaB elements, which are present within the NIS promoter. RESULTS: Metformin reduced NIS promoter activity (0.6-fold of control), whereas compound C stimulated its activity (3.4-fold) after 4 days. This largely coincides with CRE activation (0.6- and 3.0-fold). These experiments show that AMPK exerts its effects on iodide uptake, at least partly, through the CRE element in the NIS promoter. Furthermore, we have used AMPK-alpha1 knockout mice to determine the long-term effects of AMPK inhibition without chemical compounds. These mice have a less active thyroid, as shown by reduced colloid volume and reduced responsiveness to thyrotropin. CONCLUSION: NIS expression and iodine uptake in thyrocytes can be modulated by metformin and compound C. These compounds exert their effect by modulation of AMPK, which, in turn, regulates the activation of the CRE element in the NIS promoter. Overall, this suggests that the use of AMPK modulating compounds may be useful for the enhancement of iodide uptake by thyrocytes, which could be useful for the treatment of thyroid cancer patients with radioactive iodine.

Pyrolysis/gas chromatography/mass spectrometry (Py/GC/MS) was used to elucidate whether the chemical modifications produced by streptomycetes on wheat straw are related with the lignocellulose degrading enzymes produced by the microorganism on such substrate. Thus, Streptomyces sp. UAH 47 was grown on wheat straw under solid-state fermentation and periodically the major solubilisation product of this substrate (lignin–carbohydrate complex) and the residual wheat straw were pyrolysed. In parallel, different enzyme activities related with lignocellulose degradation were evaluated. The characteristic pyrolysis products detected by GC/MS from both solubilised lignocarbohydrate complex and residual wheat straw demonstrated an oxidative action of the microorganism on the lignin. Either oxidation or oxidation followed by cleavage of the C3 alkyl chain, from S and G units, mainly at the Cα and Cβ position could be detected. A correlation between the oxidation degree of lignin and both l -3,4-dihydroxyphenylalanine and 2,6-dimethoxyphenol peroxidases produced by the strain can be inferred. Moreover, a liberation of ferulic and p-coumaric acids from the substrate was detected by HPLC, which probably involves the action of ferulic and coumaric acid esterases secreted by the microorganism. These enzymes could be partially responsible for the different pattern in the relative area of 4-vinylguaiacol and 4-vinylphenol detected in all samples along the incubation time. At last, the pyrolysis data suggest a straight correlation between the relative area of the compounds derived from carbohydrates and the pattern of hemicellulolytic and cellulolytic enzymes produced by the strain.

Wheat-straw treatment with a Mn2+-oxidizing peroxidase from the ligninolytic fungus Pleurotus eryngii was investigated using pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS). Changes in the ratio between p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) lignin units were found, consistent with a degradation of S units and increase of the H content after enzymatic treatment. However, the most noticeable modification was the significant decrease of lignin phenolic content as revealed by Py-GC-MS of methylated straw. It was shown that 10 U of peroxidase per mg of straw decreased the amount of phenolic H units from 31% in the control to 3% in the treated straw, that of G units from 40 to 4%, and completely removed the small amount of phenolic S units present in wheat straw. The same tendency to decrease lignin phenolic content was observed when lower enzyme doses were used. These results provide conclusive evidence for modification of lignin during in vitro treatment of a natural lignocellulosic substrate with a ligninolytic peroxidase.

The organic structures in sapropels sampled from two cores obtained at known locations beneath the southern Black Sea have been characterised. Fluorescence petrography shows the sapropels to occur as layers of impure alginite, ∼50 μm thick, within Unit 2 of the sediments. Solid state 13 C NMR indicates the bulk chemical structures to be very similar to those in an immature Type 1 kerogen (lamosite) oil shale with an aromaticity of ∼0.2. Consistent with the immaturity of the sapropels, which are between 3000 and 7000 years old, temperature programmed reduction showed aliphatic and aromatic sulphides to be the major organic sulphur forms. Alkanes formed from phytoplankton lipids, alkyl benzenes, alkyl naphthalenes and some phenols dominated the mix of volatile compounds identified by pyrolysis-gas chromatography/mass spectrometry. About half of the sapropels remained as an involatile, tarry residue after pyrolysis. The structure of the sapropels is consistent with their formation resulting from marine phytoplankton with only small terrigeneous inputs. Future catagenesis may be expected to decarboxylate the lipids, increase the aromaticity and to dry and compress the muds to form a source rock.

Native populations of Origanum vulgare L. from the Liguria and Emilia regions of northern Italy were analysed for essential oil content and composition. Morphological characters suggested that the Ligurian samples belonged to the ssp. viride. The essential oil content of inflorescences ranged from less than 5 mg g ˇ1 in the samples from Emilia, to more than 50 mg g ˇ1 in some Ligurian samples, values similar to those reported for ssp. hirtum from southern Italy. Sixty-four compounds were identified in the essential oil. The samples were allotted to three main groups on the basis of oil composition: the first group had a high content of components belonging to the carvacrol/thymol biosynthetic pathway; the second was characterized by a diAerent sesquiterpene composition and a high linalool content; and the third, including the two samples of ssp. vulgare from Emilia, was characterized by the presence of abundant sesquiterpenes. The high essential oil content of the samples studied, and the presence of some particular morphological characters suggest that the taxonomy of O. vulgare, especially in its western distribution area, needs further investigation. Linalool types, although often not considered as ‘typical oregano’, may be useful in promoting wider utilization of the biodiversity of this species. # 2000 Annals of Botany Company

Some fractions of beer-factory wastewaters represent an important environmental concern owing to their high content of polyphenols and dark-brown color. The capacity of Coriolopsis gallica to preferentially degrade lignin has been successfully applied in our laboratory to the biotreatment and decolorization of paper-industry effluents. In this work, the ability of this white-rot fungus to degrade high-tannin-containing wastewaters is evaluated. Under all the conditions studied, effluent decolorization and chemical oxygen demand reduction achieved by C. gallica at day 12 of incubation were close to 50 and 65%, respectively. No adhesion of dark color to the fungal mycelium was observed suggesting that decolorization could be ascribed to C. gallica degradation systems. Mycelium dry-weight values showed that C. gallica is tolerant to relatively high tannin content present in the effluent samples. In the sample containing the highest effluent concentration (60% v/v), dry-weight values suggested an inhibition of fungal growth at day 6 of incubation and a further adaptation of the fungus to the stressing tannin effect at day 12 of fungal treatment. Pyrolysis/gas chromatography/mass spectrometry results showed a decrease of polyphenols pyrolysis products, mainly phenol and guaiacol, with the incubation time. All these results indicate the potential use of C. gallica in bioremediation of tannin-containing industrial wastewaters and in other applications where a reduction in polyphenols content is required.

The analysis of organic pollutants in drinking water is a topic of wide interest, reflecting on public health and life quality. Many different methodologies have been developed and are currently employed in this context, but they often require a time-consuming sample pre-treatment. This step affects the recovery of the highly volatile compounds. Trace analysis of volatile organic pollutants in water can be performed 'on-line' by membrane inlet mass spectrometry (MIMS). In MIMS, the sample is separated from the vacuum of the mass spectrometer by a thin polymeric hollow-fibre membrane. Gases and organic volatile compounds diffuse and concentrate from the sample into the hollow-fibre membrane, and from there into the mass spectrometer. The main advantages of the technique are that no pre-treatment of samples before analysis is needed and that it has fast response times and on-line monitoring capabilities. This paper reports the set-up of the analytical conditions for the analysis of volatile organohalogen compounds (chloroform, bromoform, bromodichloromethane, chlorodibromomethane, tetrachloroethylene, trichloroethylene, 1,1,1-trichloroethane, and carbon tetrachloride). Linearity of response, repeatability, detection limits, and spectra quality are evaluated.

Milled-wood lignin (MWL) from Carpinus betulus, Eucryphia cordifolia, Picea abies, Pinus sp. and Bambusa sp., Kraft lignin from Fagus sylvatica and Eucalyptus globulus, and alkali lignin and hemicellulose-linked lignin from Triticum aestivum, were investigated with respect to their composition in phenolic and etherified phenylpropanoid H (p-hydroxyphenyl), G (guaiacyl) and S (syringyl) units. For this purpose, a methodology based on lignin permethylation, followed by pyrolysis-gas chromatography/mass spectrometry, and quantitation of marker compounds (H-, G- and S-type vinylphenols and their methylated derivatives) in single-ion chromatograms, was developed. The phenolic content in the samples analyzed ranged from 2% of total units in hemicellulose-linked lignin to near 70% in Kraft lignins. Softwood MWL showed higher amounts of phenolic units than MWL from annual plants and hardwoods. It was found that the phenolic content of MWL from the Austral tree species E. cordifolia was unexpectedly high for a hardwood lignin. The significance of this finding in terms of lignin degradability by white-rot fungi, of biotechnological interest, is discussed. Copyright # 1999 John Wiley & Sons, Ltd.

In ion trap mass spectrometry, collisional experiments are easily performed by precursor ion selection and software controlled operations. Collisional activation can be achieved by two different approaches, resonant and non-resonant excitation. Different results are usually obtained by the two approaches. Four related compounds of biological interest (lignin monomers), i.e. guaiacol (2-methoxyphenol), syringol (2,6-dimethoxyphenol), 4-allyl syringol (2,6-dimethoxy-4-allyl-phenol) and 4-syringaldehyde (4-hydroxy-3,5-dimethoxybenzaldehyde) have been studied by gas chromatography/tandem mass spectrometry. Sample concentration affects both single ion storage and collisional spectra of these compounds. The use of the two collisional approaches leads to different collision induced dissociation spectra of molecular species. The present work attempts to rationalize the observed behaviour. © 1998 John Wiley & Sons, Ltd.

Pyrolysis/gas chromatography/mass spectrometry (PY/GC/MS) was used to characterize the pericarp fiber of grain samples of 6 spelt (Triticum spelta L.) along with 3 different bread wheats plus 7 spelt–wheat crosses. Absolute amounts of lignin and polysaccaride pyrolysis products were quantified as μg mg−1 of sample using an internal standard method. The pyrolysis fragments ascribed to the polysaccharide fraction were significantly more abundant in spelt whereas wheats contained the highest percentage of lignin-derived pyrolysis fragments. Discriminant analysis differentiated spelt, spelt–wheat crosses and wheat. Thus, among possible options for fiber analysis, PY/GC/MS proved to be a reproducible and reliable analytical tool, which can be easily applied to the screening and characterization of cereal fiber. © 1998 John Wiley & Sons, Ltd.

Hulled wheat grain is used mainly in the alternative or health food market. Most of the suggested beneficial effects of these cereals are due to specific characteristics of their fibre. Pyrolysis/gas-chromatography/mass-spectrometry (PY/GC/MS) has proved to be a reproducible analytical tool for fibre analysis applicable to the analysis of cereal fibre. Pericarp fibre of 10 emmer and einkorn wheat landraces were analysed by PY/GC/MS and compared to a test durum wheat variety. The pyrolysis fragments derived from the polysaccharide fraction were significantly more abundant in emmers than in the other genotypes, whereas the highest percentage of lignin-derived pyrolysis fragments was detected in durum wheat. The genotype groups (winter and spring emmers, einkorn and durum wheat) were satisfactorily discriminated on the basis of the molecular composition of the fibre, whereas a discrimination within groups was not possible due to the limited number of samples available. The results suggest that the native emmer genetic material may represent a source of high-value dietary fibre. Breeding should aim at preserving such characters. © 1998 Society of Chemical Industry

Twenty-four steam-distilled samples of essential oils from inflorescences from Origanum vulgare ssp. hirtum (Link) Ietswaart growing wild in Calabria, southern Italy, were analyzed by gas chromatography and gas chromatography/mass spectrometry. A total of 56 compounds were identified. The main components of the essential oil were thymol and carvacrol, while their biogenetic precursors, p-cymene and γ-terpinene, were the most aboundant monoterpenes. The relative amounts of the two main constituents were comparable to the literature data on this species. Four chemotypes were identified in Calabria on the basis of the phenolic content, i.e., thymol, carvacrol, thymol/carvacrol, and carvacrol/thymol chemotypes. The first chemotype was the most frequent. A significant variability of composition possibly correlated with the individual genotypes was observed. Keywords: Oregano; Origanum vulgare ssp. hirtum; essential oil composition; GC/MS

Cellular excitability of CA1 neurons from a kindled focus in the rat hippocampus is persistently increased. The changes in the underlying voltage-dependent sodium current were characterized under whole-cell voltage-clamp conditions. We compared sodium currents in acutely isolated CA1 neurons from kindled rats with those in matched controls, one day and five weeks after cessation of kindling stimulations. The sodium current in CA1 neurons was tetrodotoxin sensitive and inactivated completely with two time-constants. In 97 cells from control rats, the current evoked at -20 mV consisted of a fast-inactivating component of 3.8 +/- 0.2 nA which decayed with a time-constant of 1.0 +/- 0.1 ms, and a slow-inactivating component of 1.2 +/- 0.1 nA with a time-constant of 3.6 +/- 0.1 ms. The potential of half-maximal inactivation was -72.2 +/- 1.0 mV for the fast-inactivating component and -63.2 +/- 1.0 mV for the slow-inactivating component. The time-constant of recovery at -80 mV was 14.1 +/- 0.4 ms for the fast-inactivating component and 9.3 +/- 0.4 ms for the slow-inactivating component. One day after kindling, the voltage dependence of inactivation of the slow-inactivating and the fast-inactivating component was shifted in the depolarizing direction (3.2 +/- 1.3 and 3.0 +/- 1.3 mV, respectively). The voltage dependence of recovery from inactivation was shifted in the same direction. Five weeks after kindling, the shift in voltage dependence of inactivation was (3.3 +/- 1.2 and 2.9 +/- 1.2 mV, respectively) and was accompanied by a 20% increase in sodium current amplitude. The voltage-dependent activation was not different after kindling. The changes in sodium current inactivation will increase the number of channels available for activation and may enhance the maximum firing rate. This implies that the changes in sodium current inactivation will contribute to the enhanced excitability of pyramidal neurons observed after kindling.

The possible differential biodegradation of wheat straw performed by different Streptomyces strains was investigated on solid-state fermentation conditions. The modified lignin polymer named acid-precipitable polymeric lignin (APPL), harvested with NaOH or water, from wheat straw transformed by selected Streptomyces strains was tentatively characterized by pyrolysis-gas chromatography-mass spectrometry (PY/GC/MS). For comparative analyzes, lignin-carbohydrate-protein complexes obtained from uninoculated wheat straw were used as controls. Significant differences in the p-hydroxyphenyl: guaiacyl: syringyl(H: G: S) and in the syringyl guaiacyl ( S G ) ratios of the APPLs extracted with water from wheat straw transformed by the strains Streptomyces UAH 23, Streptomyces UAH 52 and Streptomyces viridosporus T7 A were detected compared with the control. While S. UAH 23 produced a preferential degradation of the non-condensed Slignin units, a decrease in the condensed G-units was produced byS. UAH 52 and S. viridosporus T7 A. From the results obtained it can be concluded that PY/GC/MS is a suitable technique to establish differences in the composition of the main product of solubilization of lignocellulose by different Streptomyces strains. Nevertheless for chemical characterization of this polymer, it is important to select the method to be used to extract the APPL from degraded residues in order to prevent chemical modifications of the substrate as occurred when NaOH was used which make difficult to attribute the changes observed to the microbial activity.

A simple method to obtain absolute quantitation of lignin pyrolysis fragments by on-line pyrolysis–gas chromatography–mass spectrometry (Py–GC–MS) is proposed. Three compounds were tested as internal standards, i.e. 1,3,5-tri-tert.-butylbenzene, 1,2,4-benzenetricarboxylic acid trimethyl ester and 1,3,5-trimethoxybenzene. The characteristic of the proposed internal standards is that they vaporize in the hot pyrolysis interface during the 3-min equilibration and focus on the top of the GC column, thus avoiding loss of internal standard due to thermal fragmentation. The linearity and reproducibility of response of the standards over a range of concentrations are reported. 1,3,5-Tri-tert.-butylbenzene was selected as the most appropriate among the tested standards to be used in Py–GC–MS of lignin. The correction factors for the main lignin pyrolysis fragments were calculated and a practical application of the proposed method to the analysis of a wheat straw sample is discussed.

Pyrolysis/gas chromatography/mass spectrometry (PY/GC/MS) and high-performance liquid chromatography with electrochemical detection (HPLC−ED) were carried out on stover samples from ten commercial ...

Lignin biodegradation is an oxidative process involving the action of fungal enzymes and the subsequent chemical reactions of aromatic radicals formed. Aromatic acid release has been reported during biodegradation of lignocellulosic materials, suggesting oxidative degradation of side chains in lignin units. In the present study, wheat lignin degradation by two basidiomycetes was investigated by pyrolysis coupled to GC/MS after sample permethylation. In this way, analysis of phenolic and etherified lignin units, yielding C4-OMe and C4-OH compounds respectively, was performed. The amount of etherified residues of carboxy-free vanillic and syringic acids was found to be very low in sound straw. However, the molar abundances of these acid residues increased strongly during fungal degradation of lignin. For example, each of them attained 8% of total lignin-derived compounds recovered after pyrolysis of straw treated with Pleurotus eryngii (after 50% lignin degradation). These findings support Ca-Cs cleavage as one of the most frequent reactions during lignin biodegradation, and constitute evidence for in situ cleavage of lignin side chains during fungal attack to this macromolecule. © 1997 by John Wiley & Sons, Ltd.

Stovers of five maize hybrids harvested at various growth stages were subjected to pyrolysis/gas chromatography/mass spectrometry (PY/GC/MS) and conventional analyses for fiber and protein, i.e. Van Soest's neutral detergent fiber (NDF) and Kjeldahl crude protein (CP). The aim of the experiments was to find pyrolysis markers of the growth stage and to ascertain what degree of correlation exists between analytical pyrolysis and conventional analyses of fiber and crude protein. Correlation coefficients higher than 0.90 in absolute values were found for some pyrolysis fragments and NDF, CP and growth stage. Such fragments included lignin, carbohydrates and protein thermal decomposition products. In conclusion, PY/GC/MS can monitor chemical changes in the main components of maize stover during maturation with the advantage over the conventional assays NDF and CP of providing molecular information and of being faster and much simpler.

Pyrolysis/gas chromatography/mass spectrometry (PY/GC/MS) was applied to the characterization of paper industry effluents subjected to various treatments, such as ultrafiltration and lignin precipitation. A number of pyrolysis products which can be ascribed to lignin and polysaccharides, as well as nitrogen containing compounds and elemental sulphur (S8) were identified in the pyrograms. The comparison of the relative distribution of p-hydroxyphenyl (H), guaiacyl (G), syringyl (S) lignin units and polysaccharide markers in the various effluent fractions showed higher contents of polysaccharides and H lignin units in the acid-soluble fractions and in the ultrafiltration permeate, whereas the acid-insoluble fraction and the ultrafiltration concentrate were richer of lignin with G and S monomeric units.

Alkalilignin samples obtained from untreated paper-mill effluent and effluent decolorized by Streptomyces UAH 30 and Streptomyces UAH 51 were analyzed by pyrolysis/gas chromatography/mass spectrometry. The molecular composition of the samples and the chemical modifications occurring in the lignin units were studied in order to know the mechanism of decolorization carried out by these microorganisms. The pyrolysis data showed a notable reduction in the lignin pyrolysis products of alkalilignins in effluent decolorized by Streptomyces strains. Moreover, chemical modifications, such as oxidation and shortening of the side chain of the phenyl–propyl moieties of lignin were also detected.

Pyrolysis-gas chromatography /mass spectrometry (Py-GC/MS) and Py-GC/MS in the presence of tetramethylammonium hydroxide (TMAH) (pyrolysis methylation-GC/MS) have been used to characterize humin of a representative agricultural soil. Analyses have been performed on humin de-ashed by treatment with HCl-HF (HU) and the results have been compared with those obtained from the corresponding humic acid (HA). Both semiquantitative conventional pyrolysis and pyrolysis methylation agreed in showing that the pyrolytic behaviour of HU is quite different from that of HA, the mass spectrometry pyrograms of the former being characterized by a larger amount of carbohydrates and aliphatic compounds. Pyrolysis/methylation of HU produced methyl esters of aliphatic and aromatic carboxylic acids, methylated carbohydrate derivatives and methyl pyrroles. Peaks diagnostic of the presence of glucose and cellulose have been proposed when pyrolysis is performed in the presence of TMAH.

The essential oil of leaves and berries of Vitex agnus-castus L. (Verbenaceae) growing wild in Calabria, southern Italy, was analyzed by gas chromatography/mass spectrometry. A total of 38 compounds (including two coeluting pairs) were identified (95.8 and 66.1% in leaf and berry oil, respectively); 6 compounds were tentatively identified and 7 compounds remained unknown. All the unknown and tentatively identified compounds eluted in the final part of the chromatograms and were particularly abundant in the berry oil. The main components of the essential oil were 1,8-cineole, sabinene, α-pinene, β-farnesene, β-caryophyllene and α-terpinenyl acetate. The relative amounts of two main constituents namely sabinene and β-farnesene, versus 1,8-cineole and the whole sesquiterpenic fraction in leaves were more comparable to the data reported in the literature for a Dalmatian V. agnus-castus than to the composition of an African sample. The berry essential oil of the present samples was characterized by a large content of sesquiterpenes, diterpenes and other unknown high-molecular-weight compounds (62.1%) compared to the leaf oil (15.3%) and to the literature data regarding the Dalmatian and African types.

Lignin is one of the components, with cellulose and hemicellulose, of cell walls in all terrestrial plants. Lignin is an amorphous, three-dimensional, polyphenolic molecule charged with the function of giving strength and impermeability to the plant structure. The determination of its molecular weight and structure is still problematic owing to the difficulties in isolation of lignin from the other cell wall constituents. The present work reports the experimental conditions for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the characterization of different types of natural lignins and of one synthetic lignin. A nitrogen laser (λ=337 nm) was used for ion desorption and sinapinic acid (3,5-dimethoxy-4-hydroxycinnamic acid) was chosen as the best matrix for lignin analysis. Data on the molecular weight distribution of oligomers in lignin and information on the monomeric constituents of different types of lignin are shown.

Maize stovers collected every 14 days over an 84-day growth period were subjected to high-performance liquid chromatography with electrochemical detector (HPLC-ED) and pyrolysis/gas chromatography/mass spectrometry (PY/GC/MS) in order to monitor changes in the phenolic composition. Prior to HPLC-ED analyses, ground samples were sequentially extracted with (i) methanol, (ii) 0.1 M sodium hydroxide and (iii) 2 M sodium hydroxide in the presence of nitrobenzene to separate, respectively, free phenolic monomers, alkali-labile phenolic monomers and alkali-resistant lignin. In turn, solution (ii) was treated with alkaline nitrobenzene to obtain (iv) alkali-labile lignin. Pyrolysis was carried out on ground native samples by using a platinum heated filament pyrolyser. Increases in the absolute phenolic concentrations in the residues of 0.1 M sodium hydroxide extraction and in the ratio of alkali-resistant lignin vs total lignin were observed by HPLC-ED during the first 28-42 days of maturation, reaching a steady level in the remaining maturation period. A linear increase of syringyl units vs guaiacyl units was for found the alkali-resistant lignin fraction over the entire period of maturation. Similar trends were showed by PY/GC/MS with regard to relative lignin content and syringyl/guaiacyl ratio. Both techniques showed their usefulness to gauge changes in the phenolic composition during the lignification process.

Aspartame (N- l -α-aspartyl- l -phenylalanine 1-methyl ester) was determined by high-performance liquid chromatography with electrochemical detection. Aspartame, which is electrochemically inactive, was made oxidizable in the range 0.1–1.1 V after post-column irradiation at 254 nm. A detection limit of 0.5 mg/l (signal-to-noise ratio 3:1) was attained using a coulometric detector with the working cell set at 0.8 V and a C6 column (150 × 4.6 mm I.D.) operated under isocratic conditions with 0.1% perchloric acid-methanol (85:15, v/v) as the eluent at a flow-rate of 1 ml/min. A linear response for aqueous solutions of aspartame in the range 1–20 mg/l and a 5% standard deviation for five replicate injections were obtained. The method was applied to the determination of aspartame in two diet colas and a pharmaceutical product. Chromatograms and a hydrodynamic voltammogram are shown.

The phenolic composition of 10 samples of maize stovers collected every 7 days after blooming was determined by high-performance liquid chromatography with electrochemical detection (HPLC-ED) and pyrolysis/gas chromatography/mass spectrometry (PY/GC/MS). An increase of the p-hydroxycinnamic acids (i.e., p-coumaric acid and ferulic acid) with maturation was observed by HPLC-ED of mild alkaline extracts. Likewise, vanillin and syringaldehyde, i.e., the typical aldehydes constituting lignin, showed an increase with maturation in the alkali-soluble lignin fraction, as determined by HPLC-ED after nitrobenzene oxidation. As to PY/GC/MS, a less marked variation in the typical lignin units was observed with ripening. The ratios between syringyl and guaiacyl lignin units as determined by both analytical techniques were higher, but of the same order, than those determined previously in a set of maize stovers sampled over a wider range of maturation. Keywords: Maize stover; phenolics; lignin; high-performance liquid...

Pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS) was applied in order to monitor the extent of chemical modification of lignin units after seven-day treatment of paper-industry wastewaters using two ligninolytic fungi. Py-GC-MS after the effluent treatment with Ganoderma australe revealed an increase in the lignin compounds, in which oxidation and shortening of the side chain of lignin molecules have occurred. In addition, no significant alkalilignin weight-loss nor reduction of lignin pyrolysis products were detected. On the other hand, these chemical transformations of lignin units were not observed after the effluent treatment with Phanerochaete chrysosporium. However, a remarkable reduction of lignin pyrolysis products and a large alkalilignin weight-loss were observed. The percentages of decolorization attained at day seven of culture were similar for both fungi. These results are possibly due to the different lignin-degrading mechanisms and/or to the earlier expression of the ligninolytic system of P. chrysosporium, under the experimental conditions assayed. Effluent decolorization could not be attributed to adsorption on the mycelia, since no significant quantity of alkalilignin was found attached to the mycelia of either fungi.

Two native Chilean woods (Eucryptia cordifolia Cav. and Laurelia philippiana Looser) and their corresponding sound and degraded milled wood lignin preparations, were characterized by pyrolysis-gas chromatography/mass spectrometry. The main goal of this work was to study the chemical changes in a lignin molecule during the natural fungal decay. A higher proportion of syringyl (S) than guaiacyl (G) units was detected in the native wood of E. cordifolia (S/G ratio of 3.9), whereas L. philippiana native wood showed similar amounts of S and G units (S/G ratio of 0.9). The modified Bjorkman method used here proved to be a suitable procedure for removing almost all the carbohydrate fraction of wood, with only minor modifications (demethylations) to the resulting purified lignin. The alterations affecting the propane chain of lignin units (C 3 -alkyl chain oxidations and shortenings) observed in L. philippiana after Phlebia chrysocrea (Berk. and Curt.) decay were similar to these described in E. cordifolia by Ganoderma australe (Fr.), except that the former were only detected in S-lignin units. Additionally, in L. philippiana some demethylation reactions were also suggested, although oxidations were not observed in the C β -position. Both fungi produced a decrease in lignin content, independent of the different S/G ratio of the studied woods. After the brown-rot fungus attack the lignin content did not change. Although this brown-rot microorganism does not substantially degrade lignin, it is able to produce some minor chemical alterations: demethylations and oxidations of lignin side-chains

The thermal decomposition products of aspartame at temperatures ranging from 350 to 950 °C were determined by pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS), using a quartz sample holder, placed in a platinum coil filament pyrolyzer, and an ion trap detector mass spectrometer. Fragments typical of the amino acid constituents of aspartame, along with other aspartame-diagnostic fragments, were determined in the pool of pyrolysis fragments. Pyrolyses were replicated for aspartame samples ranging from 0.2 to 1.3 mg and the reproducibility of the results was determined. Samples were pyrolyzed as they were or in the presence of tetramethylammonium hydroxide in order to achieve thermally assisted hydrolysis methylation (THM). The results obtained from the Py-GC/MS and THM-GC/MS of table-top sweeteners and a soft drink containing aspartame are discussed in view of using such a technique as a rapid tool to determine aspartame content in food products.

Two ligninolytic fungi, Coriolopsis gallica and Paecilomyces variotii, attained elevated percentages of decolorization (48 and 85% respectively after seven days) when they were assayed to decolorize effluent from the paper industry. The species P. variotii produced a very high alkalilignin loss of the treated effluent with respect to the control (78%). However, a significant proportion of effluent alkalilignin was found attached to the fungal biomass. Py-GC/MS analyses revealed that this alkalilignin fraction was constituted mainly of lignin-derived compounds. This fact indicates that although some transformation of the lignin compounds occurred in the effluent after treatment, the adhesion to the mycelium in the decolorization process with P. variotii is an important factor for consideration. In the case of C. gallica, the alkalilignin loss was also very elevated (77%), and the Py-GC/MS also showed alterations in the lignin units. The proportion of lignin products remarkably decreased, and no significant amounts of lignin-related compounds were found attached to the mycelium of this fungus, the major compound being 1,6-anhydro-β- d -glucopyranose, whose origin could probably be ascribed to the fungal cell-wall polysaccharide. Therefore, effluent decolorization was due to the degradation and not to adsorption by the mycelium. This is especially important for future biotechnological applications. Py-GC/MS demonstrated potential for monitoring the extent of structural modification of lignin during biological treatments, being a suitable technique for the study of paper industry waste-water treatment.

Catechin and ellagic acid, two basic constituents of important natural polyphenols such as condensed tannins and ellagitannins, were subjected to thermally-assisted hydrolysis–methylation in the presence of tetramethylammonium hydroxide (25% aq.). The reaction was carried out at 600°C for 5 s using a platinum filament pyrolyzer which, in turn, was connected to a gas chromatograph/ion trap detector mass spectrometer. The permethylated compounds determined in the pool of the reaction products suggest that the reaction occurring is different from a simple methylation of free hydroxyls after thermal degradation and entails thermal fission of different bonds than those involved in conventional pyrolysis or chemical hydrolysis as the main decomposition pathway. The mass spectra of the permethylated hydrolysis products of catechin and ellagic acid are of practical use in the analysis of plant materials containing condensed tannins and ellagitannins.

Pyrolysis/gas chromatography/mass spectrometry (PY/GC/MS) of As4S6 (orpiment), As4S4 (realgar), As4O6 (arsenolite), NaH2AsO4 (sodium di-H-orthoarsenate) was performed using a platinum-filament pyrolyzer heated at 700°C for 10 s and coupled to a capillary gas chromatograph/ion trap mass spectrometer. As4 was observed as a pyrolysis fragment in the pyrograms of all compounds. Pyrolysis of NaH2AsO4 equivalent to 100 ng of As, produced a strong As4 chromatographic peak. As4S3 and As4S4 were detected as pyrolysis products of As4S6 (orpiment). As4S4 (realgar) and As4O6 (arsenolite) were detected as intact molecules after pyrolysis. By contrast, intact As4S6 was not detected after pyrolyses in the range 400–700°C. As4 was detected as an impurity in the painting layer of an Egyptian mummy. The mass spectra of such arsenic compounds, none of which is included in the NIST library of spectra, are reported. Advantages and limitations of PY/GC/MS as a technique for the qualitative detection of arsenic inorganic compounds are discussed.

Oak and chestnut cask staves were planed to a depth of one centimeter, collecting a sample every millimeter. The samples were ground and subjected to pyrolysis/gas chromatography/mass spectrometry in order to determine their composition. Thirty-six compounds were identified, including a series of typical lignin phenolics commonly considered responsible for wine taste. All ions found in a previous pyrolysis/mass spectrometry study of oak wood and related to good quality whisky were confirmed in the mass spectra of the pyrolysis fragments specific for polysaccharides and lignin recorded in the present work. Compared to chestnut wood, oak wood showed significantly higher lignin and syringyl unit content and less variability as to lignin composition with depth.

The use of pyrolysis/gas chromatography/mass spectrometry (PY/GC/MS) to determine the molecular composition of lignocellulose is reviewed and the technique illustrated with applications on some novel systems. Constituents and structural arrangement of lignocellulosic polymers in plant cell wall are briefly described. The basic principles of pyrolysis and the main advantages and disadvantages of PY/GC/MS are discussed. PY/GC/MS analyses of (a) paper industry effluents, (b) agricultural by-products subjected to biological processes of delignification and (c) recycled paper are presented as examples of the amount of information that PY/GC/MS can provide on lignin classification and monitoring of delignification treatments as well as on the fingerprinting of lignocellulosic materials. The ion trap detector mass spectra of 56 pyrolysis products of lignocellulose, including compounds of phenolic, furanic and pyranic nature, are shown.

The main goal of this work was to identify the mechanisms responsible for carbapenem resistance in 61 Chilean clinical isolates of Enterobacteriaceae (Enterobacter spp., Serratia marcescens, Morganella morganii, Escherichia coli and Klebsiella pneumoniae) with reduced susceptibility to at least one carbapenem (ertapenem, imipenem or meropenem). All of the isolates were analysed for the presence of carbapenemases, extended spectrum β-lactamases (ESBLs), AmpC enzymes and outer-membrane proteins. None of the isolates exhibited carbapenemase activity nor did they have any of the carbapenemase genes that were screened for. Most of the 61 strains produced at least one ESBL and/or one AmpC enzyme and either lost their porins or had altered porins according to sequence analysis. The distribution of ESBLs and AmpC enzymes was different among the species studied. Resistance in K. pneumoniae and E. coli isolates was associated with ESBLs; in M. morganii isolates, resistance was attributed to overexpression of an AmpC enzyme; and in Enterobacter spp. isolates, resistance was associated with both types of enzymes. In K. pneumoniae isolates, porin integrity was more a determinant of carbapenem resistance than the presence of ESBLs, whereas in isolates of Enterobacter spp., M. morganii and S. marcescens, the presence of an overexpressed AmpC enzyme was associated with higher imipenem and meropenem MIC values. Therefore, carbapenem resistance in Chilean isolates is not due to true carbapenemases but rather to a combination of porin loss/alteration and β-lactamase activity. The fact that carbapenemases were not detected in this study is unique, given that many countries in the region have already reported the presence of these enzymes.

Different wheat straw fractions representative of industrial and laboratory-scale alkaline pulping processes have been studied by pyrolysis—gas chromatography/mass spectrometry (Py-GC/MS). For comparative purposes, a milled straw lignin from wheat straw was also studied. The pyrolytic patterns were suitable for estimating the extent to which lignin-derived material is present in the hemicellulose preparations, or hemicellulose-derived products contributed to the alkalilignin fractions. The results reflect the different efficiency of the treatments to remove lignin and carbohydrate during alkaline pulping of Gramineae straws. The virtual lack of carbohydrate-derived pyrolysis products was observed only in the alkalilignin preparation obtained with the highest NaOH concentration studied (1 M) at 30°C for 12 h. The pyrolysis data showed that the guaiacyl fragments were dominant in the lignin associated to hemicellulose fractions, suggesting structural relationships between hemicellulose and lignin through guaiacyl type units. This was complementary to the large amounts of syringyl units that remained in the alkalilignin preparations. Although Py-GC/MS provides markers for the major classes of cell wall components, such as lignin and structural polysaccharides, it does not yield fragments which could be ascribed with certainty to cinnamic acids, which are abundant and play an important role in cereal straws as lignin—carbohydrate linkages.