Fulvia Greco

Application of NMR microscopy to the morphological study of the silkworm, Bombyx mori, during its metamorphosis.

Zero (ZQ) and double (DQ) quantum 2D chemical shift selective and spin-echo 3D NMR imaging at microscopy resolution, has been applied to the morphological study of silkworm, Bombyx mori, during its metamorphosis. Attention had been focused on the evolution of the internal structure of the insect during its postembryonal life occurring through the larval, pupal and adult development. A major objective of this work was the characterization of the silk glands, responsible for the synthesis and secretion of fibroin and sericin, through the changes of distribution and mobility of water, by imaging the water protons during postembryonal life stages. Moreover, alanine deriving from silk gland proteins was imaged during the last life stage of Bombyx mori.

Application of NMR microscopy to the histochemistry study of olives (Olea europaea L.)

1H multi-echo, chemical shift selective and 3D NMR imaging at microscopy resolution, as well as CPMAS13C NMR spectroscopy have been applied to the study of oil and water distribution in olives, with particular attention to the seed reserve substances and their mobilization during fruit maturation.

Poly(amidoamine)s carrying TEMPO residues for NMR imaging applications

An amphoteric bio-compatible and stealth-like poly (amidoamine) named ISA23 was obtained from 2,2-bis(acrylamido)acetic acid (BAC) and 2-methylpiperazine. The partial substitution of 4-amino-2,2,6,6-tetramethylpiperidin-1-oxyl (Amino-TEMPO) for 2-methylpiperazine as co-monomer gave two new PAA-TEMPO conjugates based on ISA23: ISA23-TEMPO1 and ISA23-TEMPO2 with 10 and 40% TEMPO-carrying units per polymer chain, respectively. In this study, a thorough NMR characterization of ISA23 polymer together with NMR and ESR characterizations of the ISA23-TEMPO derivatives and a preliminary evaluation of their potential as NMR labels for imaging applications are reported. Relaxivity measurements performed on ISA23-TEMPO1 and ISA23-TEMPO2 showed relaxivities of 0.4 and 1.8 mM−1 s−1, respectively, indicating that the PAA-TEMPO adducts have a definite potential as NMR imaging contrast agents. This was confirmed by preliminary magnetic resonance imaging (MRI) determinations.

Magnetic resonance imaging of molecular transport in living morning glory stems

MRI was applied to investigate the transport pathways in Morning Glory plant stems. The study was carried out on living plants without affecting their integrity. The architecture of a dicotyledonous plant was deeply characterized: the root system structure and the vascular bundle location were identified, the presence of central voids caused by cell maturation and loss were observed in the stem. Molecular transport components were recognized, by observing the concentration profile of a tracer, which changed with time after its absorption by the plant roots. MRI analysis revealed the presence of an axial transport as the progress of the tracer front through the vascular bundles and a radial molecular transport from the vascular bundles toward the surface of the stem. As a result, the tracer molecular transport formed the parabolic tracer front (PTF). A model was built up through the analysis of the PTF that consisted of an axial front at the peak position and a radial front at the width of the parabolic tail. PTF analysis revealed differences between the tracer transport velocities in the axial and the radial directions in the plant stem. The model revealed that the width of the parabolic tail reflected the magnitudes of diffusion and permeation of the tracer in the plant stem.

Solid state and microscopy NMR study of the chemical constituents of Afzelia cuanzensis seeds.

Multi-echo, chemical shift selective, and 3D NMR imaging at microscopy resolution, and CP/MAS 13C NMR spectroscopy have been applied to the chemistry study of oil in oil-rich seeds of Afzelia cuanzensis, a tropical plant belonging to the Leguminosae taxum Caesalpinoideae or Caesalpinaceae.

Effects of temperature and extracellular pH on metabolites: kinetics of anaerobic metabolism in resting muscle by 31P- and 1H-NMR spectroscopy.

SUMMARY Environmental stress, such as low temperature, extracellular acidosis and anoxia, is known to play a key role in metabolic regulation. The aim of the present study was to gain insight into the combined temperature-pH regulation of metabolic rate in frog muscle, i.e. an anoxia-tolerant tissue. The rate of exergonic metabolic processes occurring in resting isolated muscles was determined at 15°C and 25°C as well as at extracellular pH values higher (7.9), similar (7.3) and lower (7.0) than the physiological intracellular pH. 31P and 1H nuclear magnetic resonance spectroscopy high-resolution measurements were carried out at 4.7 T in isolated frog (Rana esculenta) gastrocnemius muscle during anoxia to assess, by means of reference compounds, the concentration of all phosphate metabolites and lactate. Intra- and extracellular pH was also determined. In the range of examined temperatures (15-25°C), the temperature dependence of anaerobic glycolysis was found to be higher than that of PCr depletion (Q10=2.3). High-energy phosphate metabolism was confirmed to be the initial and preferential energy source. The rate of phosphocreatine hydrolysis did not appear to be affected by extracellular pH changes. By contrast, independent of the intracellular pH value, at the higher temperature (25°C) a lowering of the extracellular pH from 7.9 to 7.0 caused a depression in lactate accumulation. This mechanism was ascribed to the transmembrane proton concentration gradient. This parameter was demonstrated to regulate glycolysis, probably through a reduced lactate efflux, depending on the activity of the lactate-H+ co-transporter. The calculated intracellular buffer capacity was related to intra- and extracellular pH and temperature. At the experimental extracellular pH of 7.9 and at a temperature of 15°C and 25°C, calculated intracellular buffering capacity was 29.50 μmol g-1 pH unit-1 and 69.98 μmol g-1 pH unit-1, respectively.

Quantitative analysis of anaerobic metabolism in resting anoxic muscle by 31P and 1H MRS

31P and 1H MRS high resolution measurements at 4.7 T were carried out in isolated frog (Rana esculenta) gastrocnemius muscle during anoxia to assess, using reference compounds, the concentration of high energy phosphates (approximately P = phosphocreatine (PC) plus adenosinetriphosphate (ATP)), inorganic phosphate (P(i)), phosphomonoesters (PME) and lactate (La): Two sets of measurements were performed, with (p) and without (up) muscle IAA poisoning and the time course of the metabolite concentration changes was described. The rate of phosphocreatine hydrolysis during the first phase of anaerobiosis, when no lactate is accumulated in either case, appears to be greater in p than in up preparations. This finding can be explained with the sizeable accumulation of phosphomonoesters (PME) in the former. The efficiency of anaerobic glycolysis, i.e. the approximately P/La ratio, recalculated taking into account also PME changes, was found to be 1.48 +/- 0.28, a value higher than that obtained by previous chemical measurements and close to the maximum stoichiometric approximately P/La value. Hence, the in vivo substrate of glycolysis, in the resting anoxic frog gastrocnemius, appears to be almost exclusively glycogen.

Structural and Nutritional Properties of Pasta from Triticum monococcum and Triticum durum Species. A Combined 1H NMR, MRI, and Digestibility Study

The aim of the present study was to characterize the structure of two different types of pasta, namely Triticum turgidum ssp. durum (cv. Saragolla) and Triticum monococcum ssp. monococcum (cv. Monlis), under different processing conditions. MRI analysis and NMR spectroscopy (i.e., T1 and T2 NMR relaxation times and diffusion parameters) were conducted on pasta, and (1)H NMR spectroscopic analysis of the chemical compounds released by pasta samples during the cooking process was performed. In addition, starch digestibility (enzimatically determined) was also investigated. The NMR results indicated that Saragolla pasta has a more compact structure, ascribed to pasta network and in particular to different technological gluten properties, that mainly determine the lower ability of Monlis pasta in binding water. These results correlate well with the lower rate of starch hydrolysis measured for Monlis pasta compared to Saragolla when both are dried at high temperature.

1H-NMR studies of native and fragmented subtilisin Carlsberg

NMR studies have been carried out on subtilisin Carlsberg in order to identify the sharp resonances observed in the proton spectra of the enzyme dissolved in aqueous solution. NMR spectra, obtained with the combination of spin-echo and selective excitation sequences, from both the native and inactivated protein, enabled us to assign sharp signals to subtilisin fragments derived from enzymatic autolysis (monitored by high-performance size-exclusion chromatography), rather than to mobile segments of the intact protein.

A quantitative method to assess muscle tissue oxygenation in vivo by monitoring 1H nuclear magnetic resonance myoglobin resonances.

A nuclear magnetic resonance (NMR) method was implemented to assess in vivo oxygenation levels by a quantitative determination of the 1H NMR myoglobin (Mb) resonances. The proximal His-F8 NdeltaH at 70-90 ppm and Val-E11 gammaCH3 resonance at -2.8 ppm, reflecting deoxygenated (deoxy-Mb) and oxygenated (met-Mb) states, were alternately recorded. The method was developed in vitro choosing a couple of NMR sequences that could each maximize the signal-to-noise ratio (SNR) while avoiding baseline rolling and suppressing the water signal. Two quantitative calibration methods were implemented for deoxy- and met-Mb samples (0.1-1 mM), respectively. The respective limit of detection (LOD) and limit of quantification (LOQ) were 0.015 and 0.05 mM for met-Mb and 0.013 and 0.042 mM for deoxy-Mb. Sequences and calibration curves were employed in vivo in Arenicola marina to obtain, for the first time, an accurate measurement of oxy- and deoxy-Mb actual concentrations. In Arenicola, the peaks at approximately 87 and -2.7 ppm, reflecting the deoxy- and oxy-Mb states, respectively, were alternately recorded during increasing hypoxia. The deoxy-Mb concentrations were obtained from the calibration curve. The oxy-Mb concentrations were calculated from the calibration of met-Mb because it was proved that oxy- and met-Mb gave the same NMR molar response. From oxy- and deoxy-Mb concentrations, the intracellular oxygen partial pressure (PiO2) trend was determined.