Laura Guidoni

RNA-lipid complexes released from the plasma membrane of human colon carcinoma cells

Cultured cells from human colon adenocarcinoma spontaneously release structures which display an intense 31P NMR signal from RNA and mobile phospholipids. Furthermore, the DPH probe in the cell supernatant shows an intense fluorescence, thus indicating its insertion in lipid vesicles. The total membranes, prepared from the same cells, also release similar structures. The fatty acid chain signals from the mobile lipids, observable in the H NMR spectrum, and the fluorescence polarization of the DPH probe are strongly affected by RNAase digestion, thus indicating an association between RNA molecules and lipids. The enzymatic marker cytochrome c reductase was assayed to rule out possible contamination from endoplasmatic reticulum. A high alkaline phosphatase activity was instead found in the supernatant samples, thus indicating that the shed material is released by the plasma membrane.

1H-MRS lipid signal modulation and morphological and ultrastructural changes related to tumor cell proliferation

Changes in signal intensity of 1H‐MRS mobile lipids, mostly triglycerides, have been observed in cultured HeLa cells during exponential growth. Signals were intense in the first days after seeding and almost disappeared after a few days in culture. Choline‐based metabolites were not affected by growth. Treatment with the antitumor drug lonidamine, which blocks cell proliferation and cell progression through cycle, increased lipid signal intensity. Morphological changes in the organization of the cell surface were detected by scanning electron microscopy during exponential growth, and were confirmed by freeze fracture analysis. The observed metabolic and structural modifications during cell growth were correlated to cell cycle progression of HeLa cells, as high‐intensity lipid signals were typical of cells with a high percentage of S‐ and G2 + M‐phases, while cells with a high frequency in G1‐phase were characterized by mobile lipid signals of very low intensity. Magn Reson Med 42:248–257, 1999.

VARIATIONS IN CIRCULAR DICHROISM AND PROTON-NMR RELAXATION PROPERTIES OF MELITTIN UPON INTERACTION WITH PHOSPHOLIPIDS

Melittin, a 26 amino acid cationic peptide extracted from bee venom [ 11, is well known for its ability to interact with phospholipid structures, dramatically increasing the permeability to aqueous solutes of either natural or artificial membranes [2]. Upon interaction with melittin, the aliphatic chains of the phospholipid molecules seem to undergo a pronounced immobilization, as evidenced by infrared and spin label studies [3,4]. As for the melittin molecule, its only tryptophan residue moves, upon interaction with phospholipids, from an aqueous environment to a hydrophobic one, as judged from the blue shift of its fluorescence maximum [5,6]; the changes in fluorescence characteristics are however dependent on the phospholipid used [6]. No change in the infrared absorption or infrared dichroism spectra of the peptide has been evidenced 141. We describe here how the intrinsic circular dichroism of melittin and the NMR relaxation properties of its tryptophan protons are modified upon interaction with phospholipids.

Maximum entropy spectral analysis of 31P NMR signals from human cells

Abstract Two autoregressive methods, namely Burg and Yule-Walker algorithms, have been applied to 31 P NMR free induction decays of packed human tumor cells where the signalto-noise ratio was limited by short duration records. The comparison between FFT and MEM power spectral density points out some advantages of the latter analysis technique. In particular, very good resolution and sensitivity can be reached even with very short data records. The choice of the autoregression order is discussed.

The use of the maximum entropy method in NMR spectroscopy

Abstract The maximum entropy method can be used to process noisy NMR signals with great advantages in sensitivity and resolution. This method, though employed extensively in spectral analysis, demands some care in its use mainly with respect to the correct choice of the model order. In this paper, we indicate the procedure for its application to NMR data, choosing different data points, orders of model and signal filterings. The signals analyzed are from chemical and biological samples and are characterized by a fairly good signal-to-noise ratio.

Characterization of vesicles, containing an acylated oligopeptide, released by human colon adenocarcinoma cells: NMR and biochemical studies

RNA‐containing vesicles, recovered from the supernatant of high‐density cell samples of human colon carcinoma, produce a high‐resolution 1H NMR spectrum of lipids characterized by isotropic tumbling; these vesicles contain large amounts of triglycerides and cholesterol esters. Both findings have strict analogies to what is displayed by the proteolipid complexes isolated from the sera of tumor‐bearing patients [(1985) Proc. Natl. Acad. Sci. USA 82, 3455–3459; (1986) FEBS Lett. 203, 164–168]. Lipid analysis and enzymatic tests indicate that these vesicles are selected micromaps of plasma membranes, analogous to those that can be recovered from culture media in which tumor cells are grown [(1985) Dev. Biol. 3, 33–57]. Peculiar lipids, an acylated oligopeptide and a modified phospholipid, are also present in the vesicles.

1H NMR detects different metabolic profiles in glioblastoma stem-like cells

The metabolic profiles of glioblastoma stem‐like cells (GSCs) growing in neurospheres were examined by 1H NMR spectroscopy. Spectra of two GSC lines, labelled 1 and 83, from tumours close to the subventricular zone of the temporal lobe were studied in detail and compared with those of neural stem/progenitor cells from the adult olfactory bulb (OB‐NPCs) and of the T98G glioblastoma cell line. In both GSCs, signals from myoinositol (Myo‐I), UDP‐hexosamines (UDP‐Hex) and glycine indicated an astrocyte/glioma metabolism. For line 1, the presence of signals from N‐acetyl aspartate, GABA and creatine pointed to a neuronal fingerprint. These metabolites were almost absent from line 83 spectra, whereas lipid signals, absent from normal neural lineages, were intense in line 83 spectra and remained low in those of line 1, irrespective of apoptotic fate. Spectra of OB‐NPC cells displayed strong similarities with those from line 1, with low lipid signals and clearly detectable neuronal signals. In contrast, the spectral profile of line 83 was more similar to that of T98G, displaying high lipids and nearly complete absence of the neuronal markers. A mixed neural–astrocyte metabolic phenotype with a strong neuronal fingerprint was therefore found in line 1, while an astrocytic/glioma‐like metabolism prevailed in line 83. We found a signal assigned to the amide proton of N‐acetyl galactosamine in GSC lines and in OB‐NPC spectra, whereas it was absent from those of T98G cells. This signal may be related to a stem‐cell‐specific protein glycosylation pattern and is therefore suggested as a marker of cell multipotency.

Prony-Burg method for NMR spectral analysis

Abstract A new procedure, the Prony-Burg method (PBM), has been applied to process 31 P NMR signals in the time domain (the free induction decays). As in the standard Prony method, it is assumed that the model for the signal is a sum of damped sinusoids. Our approach makes use of Burgs algorithm to calculate the autoregressive coefficients of the homogeneous equation associated with the model and the final prediction error criterion for the determination of the autoregression order. The spectral parameters, i.e., frequencies, damping factors, amplitudes, and phases, are then retrieved via polynomial rooting and singular value decomposition. The stability of the PBM is studied as a function of the signal-tonoise ratio of the free induction decay. The results indicate good spectral fidelity and stability so long as the signal-to-noise ratio is not very low.

Characterization of 1H NMR detectable mobile lipids in cells from human adenocarcinomas

Magnetic resonance spectroscopy studies are often carried out to provide metabolic information on tumour cell metabolism, aiming for increased knowledge for use in anti‐cancer treatments. Accordingly, the presence of intense lipid signals in tumour cells has been the subject of many studies aiming to obtain further insight on the reaction of cancer cells to external agents that eventually cause cell death. The present study explored the relationship between changes in neutral lipid signals during cell growth and after irradiation with gamma rays to provide arrest in cell cycle and cell death. Two cell lines from human tumours were used that were differently prone to apoptosis following irradiation. A sub‐G1 peak was present only in the radiosensitive HeLa cells. Different patterns of neutral lipids changes were observed in spectra from intact cells, either during unperturbed cell growth in culture or after radiation‐induced growth arrest. The intensities of triglyceride signals in the spectra from extracted total lipids changed concurrently. The increase in lipid peak intensities did not correlate with the apoptotic fate. Modelling to fit the experimental data revealed a dynamic equilibrium between the production and depletion of neutral lipids. This is observed for the first time in cells that are different from adipocytes.

Multiexponential relaxation in Fricke agarose gels: implications for NMR dosimetry

NMR relaxation times T1 and T2 of agarose and Fricke agarose gels have been measured in the range 17-51 MHz. The analysis of the spin echo curves indicates a multiexponential behaviour, characterized by three components, at all the examined frequencies. The relative T2 values, ranging from a few to a hundred milliseconds, can be attributed to different species of water molecules present in the gel. Two of these components are characterized by relaxation rates R2a and R2b, more sensitive than R1 to gamma irradiation, the sensitivity S being S(R1) = 0.066 s-1 Gy-1, S(R2a) = 0.088 s-1 Gy-1, S(R2b) = 0.17 s-1 Gy-1. The three T2 values decrease as a function of frequency, but no gain in dose sensitivity is obtained by changing the working frequency in the examined range. The relaxivity of agarose gels containing ferrous or ferric ions has also been measured and found to be different from those of the corresponding solutions in the absence of agarose. Thus it was possible to estimate the irradiation yield from three independent parameters, R1, R2a and R2b. No effect of the dose rate or of the source energy has been observed for any of these parameters.