Vitaliano Tugnoli

1H HR-MAS and genomic analysis of human tumor biopsies discriminate between high and low grade astrocytomas

We investigate the profile of choline metabolites and the expression of the genes of the Kennedy pathway in biopsies of human gliomas (n = 23) using 1H High Resolution Magic Angle Spinning (HR‐MAS, 11.7 Tesla, 277 K, 4000 Hz) and individual genetic assays. 1H HR‐MAS spectra allowed the resolution and relative quantification by the LCModel of the resonances from choline (Cho), phosphocholine (PC) and glycerophosphorylcholine (GPC), the three main components of the combined tCho peak observed in gliomas by in vivo 1H NMR spectroscopy. All glioma biopsies depicted a prominent tCho peak. However, the relative contributions of Cho, PC, and GPC to tCho were different for low and high grade gliomas. Whereas GPC is the main component in low grade gliomas, the high grade gliomas show a dominant contribution of PC. This circumstance allowed the discrimination of high and low grade gliomas by 1H HR‐MAS, a result that could not be obtained using the tCho/Cr ratio commonly used by in vivo 1H NMR spectroscopy. The expression of the genes involved in choline metabolism has been investigated in the same biopsies. High grade gliomas depict an upregulation of the β gene of choline kinase and phospholipase C, as well as a downregulation of the cytidyltransferase B gene, the balance of these being consistent with the accumulation of PC. In the low grade gliomas, phospholipase A1 and lysophospholypase are upregulated and phospholipase D is downregulated, supporting the accumulation of GPC. The present findings offer a promising procedure that will potentially help to accurately grade glioma tumors using 1H HR‐MAS, providing in addition the genetic background for the alterations of choline metabolism observed in high and low grade gliomas. Copyright

Effect of commercial lignosulfonate-humate on Zea mays L. metabolism.

Lignosulfonate-humate a and lignosulfonate-humate b, derived by an industrial process from lignin, were studied chemically and biologically, and their effects on maize metabolism compared with the responses induced by humic substances obtained from leonardite. Lignosulfonate-humate a and lignosulfonate-humate b elicited hormonelike activity and leonardite displayed giberellin properties. To improve our understanding of their biological action, lignosulfonate-humate a, lignosulfonate-humate b and leonardite were supplied to maize plants and their effect was studied on growth, nitrogen metabolism and photosynthesis. All products increased root and leaf growth. Glutamine-synthetase, glutamate-synthase enzyme activities and protein content were all increased. The treatments also increased chlorophyll content, glucose, fructose and rubisco enzyme activity, suggesting a positive role of lignosulfonate-humate a, lignosulfonate-humate b and leonardite in the photosynthetic process. In addition, an increase in phenol content was observed. In light of these results, being environmentally friendly products, lignosulfonate-humate a and lignosulfonate-humate b could be used to increase crop yield.

Spectroscopic characterization of soil organic matter in long-term amendment trials.

FT-IR, FT-Raman, and 1 H-NMR spectroscopies were applied to investigate molecular changes in soil organic matter (SOM) treated with different biomasses. The experiment consisted of several plots of soil treated over a 22-year period (from 1972 to 1994) with different amendments: cattle manure (CM), cow slurries (CS), and crop residues (CR). Samples taken from these plots were analyzed and compared with the unamended soil (C), which was used as the control sample. The samples were analyzed for both total organic carbon (TOC) and total nitrogen (N) content, both at the beginning of the study and after 22 years of treatments. The plots amended with CS and CR and the unamended plots showed a decrease in TOC and N after 22 years when compared with the control at the beginning of the experiment. However, the amended soil with CM showed a linear increase in TOC and N in relation to the control. The above mentioned spectroscopic procedures were employed to characterize both the unfractionated SOM and the low molecular weight (LMW) fraction extracted in an acidic medium. This spectroscopic analysis revealed that the composition of the soils amended with CM varied significantly in relation to the other amendments in the sense that the more aliphatic and aromatic moieties are resistant to the degradation. This is corroborated by the spectroscopic analysis of the LMW fraction, which is richer in small aromatic and aliphatic carboxylic acids in the soil amended with CM. The results shown in this work reveal that the SOM formed during the amendment with CM might be more resistant to the process of decomposition.

Spectroscopic study (DRIFT, SERS and 1H NMR) of peat, leonardite and lignite humic substances

Abstract Diffuse reflectance infrared Fourier transform, surface-enhanced Raman and proton nuclear magnetic resonance spectroscopies were applied to investigate the structure of humic acids (HA) extracted from peat (P–HA), leonardite (Le–HA) and lignite (Li–HA) samples. The combined use of these techniques has shown a specific pattern of functional groups for each sample. P–HA was characterised by a greater content of oxygenate (COOH, C–OH in carbohydrates and phenols) and aliphatic groups. Le–HA and Li–HA showed a lower content of sugar-like components and polyethers. On the other hand, the aromatic structures were ubiquitous in all samples, although the different composition in Le–HA and Li–HA could be employed to identify and distinguish the HA in these two kinds of humic materials.

Superoxide dismutase in gastric adenocarcinoma: is it a clinical biomarker in the development of cancer?

Abstract Gastric cancer is the second most common cancer worldwide. The involvement of reactive oxygen species (ROS) in the pathogenesis of gastric malignancies is well known. Many human tumours have shown significant changes in the activity and expression of superoxide dismutase (SOD), which might be correlated with clinical–pathological parameters for the prognosis of human carcinoma. The aim of this study is the detection of MnSOD and CuZnSOD activity and their expression in gastric adenocarcinoma and healthy tissues. Gastric samples (adenocarcinoma and healthy tissues) harvested during endoscopy or resected during surgery were used to determine MnSOD and CuZnSOD activity and expression by spectrophotometric and Western blotting assays. The total SOD activity was significantly higher (p<0.05) in healthy mucosa with respect to gastric adenocarcinomas. No differences were found in MnSOD activity and, on the contrary, CuZnSOD activity was significantly lower (p<0.001) in cancer samples with respect to normal mucosa. The rate of MnSOD/CuZnSOD activity in adenocarcinoma was over ninefold higher than that registered in healthy tissues (p<0.05). Moreover, in adenocarcinoma MnSOD activity represented the 83% of total SOD with respect to healthy tissues where the ratio was 52% (p<0.001). On the contrary, in cancer tissues, CuZnSOD activity accounted for only 17% of the total SOD (p<0.001 if compared with the values recorded in normal mucosa). After immunoblotting, MnSOD was more expressed in adenocarcinoma with respect to normal mucosa (p<0.001), while CuZnSOD was similarly expressed in adenocarcinoma and healthy tissues. The SOD activity assay might provide a specific and sensitive method of analysis that allows the differentiation of healthy tissue from tumour tissue. The MnSOD to CuZnSOD activity ratio, and the ratio between these two isoforms and total SOD, presented in this preliminary study might be considered in the identification of cancerous from healthy control tissue.

Biochemical Alterations from Normal Mucosa to Gastric Cancer by Ex vivo Magnetic Resonance Spectroscopy

Background and Aims: The metabolic profile and morphologic aspects of normal and pathologic human gastric mucosa were studied. The aim of the present research was the application of ex vivo high-resolution magic angle spinning magnetic resonance spectroscopy (HR-MAS MRS) to the human gastric tissue to get information on the molecular steps involved in gastric carcinogenesis and the identification of biochemical markers useful for the development of in vivo MRS methodologies to diagnose gastric pathologies in clinical situations. Methods: Twelve normal subjects, five with autoimmune atrophic gastritis, five with Helicobacter pylori infection, and five with adenocarcinoma were examined. Ten biopsies were taken during endoscopy from each patient. Specimens from carcinoma were also obtained during gastrectomy. Of the 10 biopsies, 4 were used for histologic evaluation, 4 were fixed in glutaraldehyde and processed for transmission and scanning electron microscopy, and 2 were immersed in liquid nitrogen and stored at −85°C for monodimensional and bidimensional ex vivo HR-MAS MRS analysis. Results: Ex vivo HR-MAS MRS identified glycine, alanine, free choline, and triglycerides as possible molecular markers related to the human gastric mucosa differentiation toward preneoplastic and neoplastic conditions. Ultrastructural studies of autoimmune atrophic gastritis and gastric adenocarcinoma revealed lipid accumulations intracellularly and extracellularly associated with a severe prenecrotic hypoxia and mitochondria degeneration. Conclusions: This is the first report of synergic applications of ex vivo HR-MAS MRS and electron microscopy in studying the human gastric mucosa differentiation. This research provides useful information about some molecular steps involved in gastric carcinogenesis. The biochemical data obtained on gastric pathologic tissue could represent the basis for clinical applications of in vivo MRS. (Cancer Epidemiol Biomarkers Prev 2008;17(6):1386–95)

EPA or DHA Supplementation Increases Triacylglycerol, but not Phospholipid, Levels in Isolated Rat Cardiomyocytes

It is well recognized that a high dietary intake of long-chain polyunsaturated fatty acids (LC-PUFA) has profound benefits on health and prevention of chronic diseases. In particular, in recent years there has been a dramatic surge of interest in the health effects of n-3 LC-PUFA derived from fish, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids. Notwithstanding, the metabolic fate and the effects of these fatty acids once inside the cell has seldom been comprehensively investigated. Using cultured neonatal rat cardiomyocytes as model system we have investigated for the first time, by means of high-resolution magic-angle spinning nuclear magnetic resonance (HR-MAS NMR) spectroscopy in combination with gas chromatography (GC), the modification occurring in the cell lipid environment after EPA and DHA supplementation. The most important difference between control and n-3 LC-PUFA-supplemented cardiomyocytes highlighted by HR-MAS NMR spectroscopy is the increase of signals from mobile lipids, identified as triacylglycerols (TAG). The observed increase of mobile TAG is a metabolic response to n-3 LC-PUFA supplementation, which leads to an increased lipid storage. The sequestration of mobile lipids in lipid bodies provides a deposit of stored energy that can be accessed in a regulated fashion according to metabolic need. Interestingly, while n-3 LC-PUFA supplementation to neonatal rat cardiomyocytes causes a huge variation in the cell lipid environment, it does not induce detectable modifications in water-soluble metabolites, suggesting negligible interference with normal metabolic processes.

Biochemical characterization of human renal tumors by in vitro nuclear magnetic resonance

Abstract This study reports an in vitro magnetic resonance spectroscopy characterization of healthy renal parenchyma, renal cell carcinomas and oncocytomas. In vitro 1 H NMR measurements allow in-depth biochemical characterization of human healthy and neoplastic renal tissues. Some metabolites with an osmotic activity are considered markers of physiological renal function. Moreover, the HPLC technique was applied to investigate the amino acidic profile of these tissues: some amino acids appear to have statistic significance.

A spectroscopic investigation of captopril and the Cu(II)–captopril system

Abstract A Raman and IR study of captopril (CpSH), a synthetic derivative of l -proline, and the Cu(II)–CpSH system at different pHs and metal/ligand ratios was carried out. The vibrational spectra suggested disulphide formation (CpSSCp) by the reduction of Cu(II) to Cu(I) and allowed the identification of the sites involved in metal coordination. Various complexes can be formed and the nature of the predominant species depends mainly on the pH. At pH 10 CpSH gives rise to two monomeric complexes with different structures depending on the metal/ligand ratio, whereas at acid pH a water-insoluble polymeric species predominates.

Imidazole Ring Opening of 7-Methylguanosine at Physiological pH

Abstract It is shown by carbon-13 NMR that the only product of the imidazole ring-opening of 7-methyl-guanosine at physiological pH is 2-amino-6-hydroxy-5-N-methylformamido-4-(N-β-ribofurano-sylamino)pyrimidine and that, contrary to previous results, the hydrolysis of the glycosylic bond at pH=7.2 is a much slower process than the ring opening. The ring opened formamidopyrimidine derivative is a very mobile molecule existing under different conformations depending on the solvent, and in water it is capable of giving different kinds of aggregates. Possible biochemical implications of these results are discussed.