Value-added products from wastewater reduce irrigation needs of Arundo donax energy crop.

Irrigation restrictions due to drought periods related to climate change, would affect different crops, especially to non-food crops. In this regard the effect of irrigation reduction should be studied in energy crops in order to obtain a sustainable bioenergy cropping system. Arundo donax, has been considered a crop with high water requirements, it has nevertheless been proven to be drought tolerant. However, there is a lack of knowledge on the effect of reduced irrigation combined with the use of different fertilizers. This work studied the combined effect of value-added products (VAPs) from wastewater (treated sewage sludge) or traditional inorganic fertilizers, and irrigation reduction in Arundo donax crop in a 2-year pot experiment. Plant biometric characteristics, chemical properties and biomass yield were studied as well as the effect of treatment on soil properties. Results showed that under reduced irrigation conditions, biomass production was reduced, especially during the second year. Organic treatments from sewage sludge minimize the effect of irrigation reduction. In these treatments, biomass yield for reduced irrigation was similar to that of the control treatment with irrigation at field capacity. For this reason, it is recommended to use VAPs from wastewater as organic amendments enabling water restriction with lower effect on Arundo production.

Assessing Arundo donax L. in vitro-tolerance for phytoremediation purposes.

Phytoremediation using high production crops could be an alternative for the recovery of metals polluted soils. In this sense, the Arundo donax L. energy crop has shown tolerance to moderate concentrations of heavy metals. The objective of this work was to test the tolerance of micropropagated plants of Arundo donax to increasing concentrations of cadmium, chromium, cooper, nickel and lead, in an in vitro culture medium. Biomass production and concentration of heavy metal in shoots and roots were analyzed. Results showed that heavy metals were accumulated mostly in subterranean organs. The increase in heavy metal concentration was dose dependent and not always follows a linear relationship. Arundo donax showed a broad tolerance to cadmium (0.5 mM), chromium (0.2 mM), cooper (2 mM), nickel (0.5 mM) and lead (1 mM). In relation to cooper, Arundo donax showed a hyperaccumulative potential. These results suggest the potential use of Arundo donax in the phytomanagement of polluted soils although further studies should be carried out using polluted soils.

New approach for rapid detection of known hemoglobin variants using LC-MS/MS combined with a peptide database.

The identification of hemoglobin (Hb) variants is usually performed by means of different analytical steps and methodologies. Phenotypic methods, such as gel electrophoresis and high performance liquid chromatography, are used to detect the different electrophoretic or chromatographic behaviors of hemoglobin variants in comparison to HbA0 used as a control. These data often need to be combined with mass spectrometry analyses of intact globins and their tryptic peptide mixtures. As an alternative to a 'step-by-step' procedure, we have developed a 'single step' approach for the identification of Hb variants present in biological samples. This is based on the microHPLC-ESI-MS/MS analysis of the peptide mixture generated by a tryptic digestion of diluted Hb samples and an in-house new database containing solely the variant tryptic peptide of known human Hb variants. The experimental results (full MS and MS/MS spectra) are correlated with theoretical mass spectra generated from our in-house-built variant peptide database (Hbp) using the SEQUEST algorithm. Simple preparation of samples and an automated identification of the variant peptide are the main characteristics of this approach, making it an attractive method for the detection of Hb variants at the routine clinical level. We have analyzed 16 different samples, each containing a different known variant of hemoglobin.

Transforming activity of the chimeric sequence formed by the fusion of collagen gene COL1A1 and the platelet derived growth factor b-chain gene in dermatofibrosarcoma protuberans.

As a consequence of a reciprocal translocation t(17;22)(q22;q13) and of supernumerary ring chromosomes derived from the t(17;22), a fusion between the platelet-derived growth factor b-chain (PDGF, c-sis proto-oncogene) and the collagen type 1A1 (COL1A1) genes has been recently described in dermatofibrosarcoma protuberans (DP), an infiltrating skin tumor (Simon et al., 1997). Although PDGFB has been implicated in transforming processes via autocrine and paracrine pathways, by the activation of the cognate receptor, no direct evidence of its involvement in neoplastic transformation of human tumours has been so far provided. In this report, we have tested the DNA from four DPs in the classical DNA transfection assay onto NIH3T3 fibroblast cell line. All the DNAs induced the formation of transformed foci in the transfected cultures whose derived cell lines were shown to contain a fused sequence comprising the human COL1A1 and PDGF genes. The relative breakpoint regions have been sequenced revealing that this gene fusion deleted exon 1 of PDGF and released the growth factor from its normal regulation. All the biochemical and biological assays were consistent with the model of an autocrine mechanism for NIH3T3 transformation by the human rearranged PDGFB gene involving the activation of the endogenous PDGF receptor.

Analysis of 10B antitumoral compounds by means of flow-injection into ESI-MS/MS.

Boron neutron capture therapy (BNCT) is a promising binary treatment for cancer. BNCT is based on the ability of the nonradioactive isotope (10)B to capture, with a very high probability, thermal neutrons. This nuclear reaction results in two particles (an alpha and a lithium nucleus). The particles have a high biological effectiveness, which is limited in tissue to approximately the diameter of one cell. If the reaction can be limited to a tumor cell, the physical characteristic opens up the possibility to selectively destroy cancer cells, while sparing the surrounding healthy tissue. Quality control of (10)B-containing compounds and their distribution at present are very important, and different analytical methods have been developed, such as time-of-flight secondary ion mass spectrometry (TOF-SIMS), electron energy loss spectrometry (EELS), prompt gamma analysis and inductively coupled plasma-optical emission spectrometry (ICP-OES). These methods allow the analyses of (10)B, but it is not possible to characterize the specific molecular compounds containing (10)B. For this reason, we propose a fast and quantitative method that permits the determination of closo-undecahydro-1-mercaptododecaborate (BSH) and (10)boron-phenylalanine (BPA) and their eventual metabolites. In particular, (10)B-containing compounds are detected by means of flow-injection electrospray tandem mass spectrometry (FI/ESI-MS/MS). This approach allows the identification of Boron compounds, BSH and BPA, using tandem mass spectrometry, and quantitative analysis is also possible (c.v. +/-4.7%; n = 5; linear range 10-10,000 ng/ml). Furthermore, (10)B-containing compounds were detected in actual biological sample (urine and plasma, diluted 10,000- and 1,000-fold, respectively) injecting a small volume (1 microl) of diluted samples.

Liquid chromatography/electrospray mass spectrometry of bioactive terpenoids in Ginkgo biloba L.

Standardized extracts of Ginkgo biloba leaves are mainly used in the treatment of peripheral and celebral circulation disorders, and also as a remedy against asthma, coughs, bladder inflammation, blenorrhagia and alcohol abuse. The leaf extracts contain biflavones, flavonol glycosides and terpene lactones. This paper reports a method based on liquid chromatography coupled with electrospray mass spectrometry for the analysis of terpenoids in G. biloba extracts. This method allows the rapid isocratic separation of underivatized ginkgolides (GA, GB, GC and GJ) and bilobalide at very low levels (10 pg on the column) and their quantitative detection by external standardization with relative standard deviations of 3 and 5% for intra- and inter-day analyses, respectively.

Electrospray ionization mass spectrometry of synthetic oligonucleotides using 2-propanol and spermidine

Oligonucleotides have become widely used tools in molecular biology and molecular diagnostics. Their parallel synthesis in large numbers and the increasing interest in microarray technology has raised the requirement for fast and informative analytical tools for their quality control. A direct injection electrospray ionization mass spectrometry (ESI-MS) technique based on the use of aqueous 2-propanol as running eluent, and spermidine (or triethylamine) as DNA modifiers, has been applied to analyze a large set of samples (about 200 synthetic oligonucleotides) ranging from 5 to 15 kDa (17-51mers) with good results in terms of sensitivity, suppression of sodium adduct formation, and speed of analysis. Copyright 2000 John Wiley & Sons, Ltd.

Stable storage conditions of Immobiline chemicals for isoelectric focusing.

Most of the problems connected with the use of the Immobiline chemicals (a set of six, non-amphoteric, acrylamido buffers having pK values in the pH 3.5-9.5 interval) can be attributed to the alkaline species (with pK values 6.2, 7.0, 8.5 and 9.3). These compounds, to varying degrees are subjected to two degradation pathways: (a) hydrolysis of the amido bond, producing free acrylic acid and a diamine, the latter unable to be incorporated into the polyacrylamide matrix; (b) spontaneous auto-polymerization, producing a number of oligomers up to n-mers, able to aggregate and precipitate large proteins. Storage of their water solutions as frozen aliquots, a method widely employed, only partially alleviates the problem. Addition of trace-amounts of inhibitors, as lately adopted by the manufacturer, could only reduce the problem of auto-polymerization, but not block the hydrolysis of the amido bond. A new solution has been found, which abolishes both phenomena: storage in n-propanol. As demonstrated by gas chromatography, HPLC analyses and two-dimensional separations of complex samples, storage in organic solvent completely abolishes both hydrolysis and auto-polymerization and allows production of highly reproducible focusing patterns.

Cell kinetics of melanocytes in common and dysplastic nevi and in primary and metastatic cutaneous melanoma.

The 3H-thymidine labelling (LI) and mitotic (MI) indexes were calculated in 29 cutaneous melanocytic lesions: 6 common nevi (CN), 11 dysplastic nevi, subclassified as nevi with architectural atypia (NAA = 4) and nevi with cyto-architectural atypia (NCAA = 7), 2 melanomas in situ (MIS), 4 invasive superficial spreading melanomas (IM) and 6 metastatic melanomas (MM). The LI mean values resulted to be: CN = 0.23%, NAA = 0.98%, NCAA = 1.79%, MIS = 5.75%, IM = 5.16%, MM = 3.80%. In CN, NAA, NCAA and MIS, these values were calculated at epidermal level; in IM and MM at dermal level. At dermal level, the LI mean values of CN, NAA and NCAA were: 0.20%, 0.20%, 0.23% respectively. The MI mean value was close to 0 in CN, NAA, NCAA, MIS; 0.18% in IM, 0.16% in MM. Confirming a low proliferative activity in CN and a high activity in melanomas (MIS, IM, MM), the results showed that dysplastic nevi (NAA, NCAA) had a proliferative activity intermediate between common nevi and melanomas. The lesions with melanocytic atypia (NCAA) resulted to have a higher proliferative activity than those without this histological feature (NAA).

Electrospray characterization of selected medicinal plant extracts.

Extracts of selected medicinal plants were examined by electrospray mass spectrometry (ESI-MS). This technique allowed identification of the main components of each extract, thereby providing a typical finger-print of the examined plants. More specifically, anthocyanins (Vaccinium myrtillus), isoflavones (Glycine max, soybean), flavonol-glycosides and terpenes (Ginkgo biloba), triterpenes (Centella asiatica), caffeoyl-quinic acids (Cynara scolymus, artichoke), ginsenosides (Panax ginseng), catechins (Camellia sinensis, green tea) and flavones and flavanones (Propolis) were detected rapidly at levels in the range of 0.1-1 microg/ml, using 0.2-1 mg/ml of each medicinal plant extract.

LC-APCI-MS/MS analysis of urinary 8-hydroxy-2'-deoxyguanosine.

8-Hydroxy-2'-deoxyguanosine (8OHdG) is regarded as an important biomarker of oxidative DNA damage and it may be estimated by using different techniques in various biological matrices, most notably DNA and urine. In the case of DNA, artifactual oxidation may take place during the isolation of DNA, its hydrolysis and possible derivatization (as for GC-MS), invalidating the measurement of 8OHdG. Therefore, the direct analysis of 8OHdG excreted into urine was preferred. Interferences from the urine matrix were excluded by applying LC-APCI-MS/MS in the multiple reaction monitoring (MRM) mode. The abundant fragment ion at m/z 168 arising from 8OHdG was monitored in the urine sample of volunteers supplemented with tomato concentrate for different times. The procedure allowed the detection of levels of 8OHdG as low as 1 ng/ml in urine sample.

Determination of sunscreen agents by micellar electrokinetic chromatography.

The separation of UV-A and UV-B sunscreens by micellar electrokinetic chromatography has been studied. The optimized method, which involves the presence of an anionic surfactant (sodium dodecyl sulphate) and an organic modifier in the background electrolyte, was applied to determine these sunscreens in cosmetic products. Identification was achieved by "on-line" UV spectra. Recovery was in the range of 88-92% and the lower limit of detection was 0.15 mg ml-1.

Proliferative activity in normal colon mucosa and tumor tissue: clinical implications.

The study deals with the analysis of proliferative activity in colon carcinomas and adjacent normal appearing mucosa, evaluated with in vitro 3H-Thymidine and autoradiography. In the colonic mucosa no significant differences in 3H-Thymidine Labelling Index (TLI) were observed in relation to the distance of the sample from the neoplasia. The distribution of S-phase cells along the crypt length is low at the bottom, increases rapidly with a maximum within the lower 25% and decreases in the highest positions. When the proliferative activity is increased there is the possibility of expanding the proliferative compartment towards the luminal region of the crypt. The division of the crypt into 5 parts makes it possible to identify 2 different patterns: the first with a very high TLI in the lower fifth, then a sharp decrease and without labelled cells in the highest parts; the second with labelled cells present also in the luminal fifth. These 2 aspects are characteristic of specimens with the lowest and the highest TLI values respectively. The analysis of TLI in colo-rectal cancers shows that cell kinetics parameters are not related to clinical and histopathological features such as sex, age, Dukes and TNM stages and grade of differentiation.

Improved procedure for the preparation of DNA restriction fragments suitable for sequencing.

A rapid and integrated procedure was developed for the preparation of small DNA restriction fragments (< or = 1000 bp) starting from a large cosmid (35,000 bp) containing exogenous DNA. The process is based on restriction enzymatic digestion followed by HPLC separation and fractions collection. All DNA fragments are separated in a single run, detected "on-line" by UV absorption, and straightforward collected with very high recovery. Small fragments can be directly subjected to the sequence procedure, whereas those larger than 1000 bp are redigested with a second enzyme, the fractionated subfragments are separated, ligated to plasmid vector, and sequenced. A human genomic cosmid of 35,000 bp (26H7) has been chosen as a model.

Direct characterization of caffeoyl esters with antihyaluronidase activity in crude extracts from Echinacea angustifolia roots by fast atom bombardment tandem mass spectrometry.

Fast atom bombardment (FAB-MS) and fast atom bombardment tandem mass spectrometry (FAB-MS/MS) techniques (negative ions) have been successfully applied for identification of the constituents responsible for the antihyaluronidase activity of Echinacea angustifolia roots, whose extracts are widely employed for the adjuvant therapy of chronic inflammatory diseases. Crude extracts from different solvents were tested for antihyaluronidase activity, and those with the greatest inhibitory action (the ethylacetate, butylacetate and chloroform fractions, IC50 0.44, 0.50 e 0.62 mg/ml) were directly analyzed by MS. Full scan mass spectra produced intense molecular anions: collisional activation of these resulted in tandem mass spectra rich in significant product ions. Four main caffeoyl conjugates were detected and identified by tandem mass spectrometry (daughter and parent ion mode): 2,3-O-dicaffeoyltartaric acid (chicoric acid) and 5-O-dicaffeoylquinic acid (cynarine) and 2-O-caffeoyltartaric acid (caffaric acid) in the ethylacetate fraction. Among these caffeoyl conjugates, chicoric and caftaric acids had the greatest antihyaluronidase activity: IC50 = 0.42 and 0.61 mM, while the IC50 of cynarine and chlorogenic acid were 1.85 and 2.25 mM.