Emanuela Casali

Lipid peroxidation in tumour cells.

Several studies point to the existence of a disturbance in the metabolism of the reactive species of oxygen in cancer cells. Based on this evidence, and in particular on a characteristic behaviour of tumour membrane lipids, namely their growth-related resistance to oxy-radical-induced peroxidation, a sequence of events is outlined that could hypothetically drive the transformed cell to an uncontrolled proliferation. The proposed scheme is also conceived as a framework for further in vivo investigations of the complex biological phenomena of tumour cell growth and invasion in more integrated and kinetically controlled cellular systems.

Gramicidin a induces lysolecithin to form bilayers

Heat-induced association of Gramicidin A with lyso-lecithin micelles results in the formation of lipid bilayer structures. The capacity of the Gramicidin A peptide to transform the lysolecithin lipid structure from micelle to bilayer is considered in terms of molecular packing mechanisms and relevance to membrane processes in general. The resulting lipid-bilayer-packaged channel system has particular usefulness in c h a r a c t e r i z i n g channel structure and mechanism.

Biochemical assessment of red blood cells during storage by 1H nuclear magnetic resonance spectroscopy. Identification of a biomarker of their level of protection against oxidative stress

BACKGROUND Blood transfusion is an established therapeutic practice. The characteristics of blood components at different storage times are expected to affect the efficacy of transfusion therapy. Metabolic profiling by nuclear magnetic resonance (NMR) spectroscopy requires little or no sample treatment and allows identification of more than 50 soluble metabolites in a single experiment. The aim of this study was to assess the metabolic behaviour of red blood cells during 42 days of storage in blood bank conditions. MATERIALS AND METHODS Red blood cells (RBC), collected from eight healthy male donors, aged 25-50 years, were prepared as prestorage leukoreduced erythrocyte concentrates and stored under standard blood bank conditions. Samples taken at various storage times were separated in two fractions: the supernatant, recovered after centrifugation, and the red blood cell lysate obtained after protein depletion by ultrafiltration. The metabolic profile of the red blood cells was determined from analysis of (1)H-NMR spectra. RESULTS The red blood cell supernatant was studied to track the consumption of the preservative additives and to detect and quantify up to 30 metabolites excreted by the erythrocytes. The NMR spectra of the RBC lysate provided complementary information on some biochemical pathways and set the basis for building a time-dependent red blood cell metabolic profile. DISCUSSION We proved the analytical power of (1)H-NMR spectroscopy to study red blood cell metabolism under blood bank conditions. A potential biomarker able to provide information on the level of cellular oxidative stress protection was identified. Our data support the hypothesis that a more detailed knowledge of metabolic modifications during storage opens the way to the development of new and more effective protocols for red blood cell conservation and patient-oriented transfusion therapy.

Lipid Peroxidation in Cancer Cells: Chemical and Physical Studies

Our studies on the biochemical composition and the structural organization of smooth and rough endoplasmic reticulum isolated from Morris hepatomas 9618A and 3924A confirm the results obtained employing the total microsomal fraction. We have definitely established the following facts: (1) Tumor subcellular organelles exhibit the very low degree of peroxidizability that has been shown to be related to the growth rate of the tumor. (2) Associated with such a low susceptibility to peroxidation are (a) changed lipid composition of cellular membranes, whose content in polyunsaturated fatty acid is markedly decreased, and (b) changed static and dynamic properties of the membrane. Previously it was also found that cellular oxy-radical scavenging enzymes are markedly reduced. From these data, it is possible to infer that tumor membranes are altered structurally and functionally in part as the result of an oxy-radical-induced damage that occurs in vivo under conditions of oxygen toxicity. This seems to be supported by recent findings that the spontaneous increase in growth rate of the originally very slow-growing Morris hepatoma 9618A results also in the loss of cytochrome P-450 (an important intramembraneous propagator of lipid peroxidation) as well as of C20:4 and C22:6. Studies performed by GLC and GC-MS on the fatty acid residues of phospholipids of rat liver microsomes show the presence of C20:3-OH and C18:1-OH, but no hydroxyl derivatives of low molecular weight aldehydes. The hydroxyl derivatives of arachidonic acid and linoleic acid are present in much smaller amounts in the microsomes isolated from H9618A and H3924A.

The binding cavity of mouse major urinary protein is optimised for a variety of ligand binding modes

(15)N and (1)HN chemical shift data and (15)N relaxation studies have been used to characterise the binding of N-phenyl-naphthylamine (NPN) to mouse major urinary protein (MUP). NPN binds in the beta-barrel cavity of MUP, hydrogen bonding to Tyr120 and making extensive non-bonded contacts with hydrophobic side chains. In contrast to the natural pheromone 2-sec-butyl-4,5-dihydrothiazole, NPN binding gives no change to the overall mobility of the protein backbone of MUP. Comparison with 11 different ligands that bind to MUP shows a range of binding modes involving 16 different residues in the beta-barrel cavity. These finding justify why MUP is able to adapt to allow for many successful binding partners.

Tryptophan interactions of gramicidin A′ channels in lipids: a time-resolved fluorescence study

The evolution of the incorporation of cation transport channels into lysolecithin micelles by gramicidin A was followed by measuring the ns time-resolved fluorescence of the tryptophan residues. In all samples, the tryptophan fluorescence could be resolved into three exponentially decaying components. The three decay times ranged from 6 to 8 ns, 1.8 to 3 ns, and 0.3 to 0.8 ns, depending on the emission wavelength. The fractional fluorescence of each component changed with incubation time. The long lifetime component had a reduced contribution to the total fluorescence while the short decay time component increased. The fluorescence spectra could be resolved into three distinct fluorescent components having maxima at 340 nm, 330 nm and 323 nm after 90 min of incubation, and 335 nm, 325 nm and 320 nm after 24 h of incubation. These maxima were, respectively, associated with the long, medium and short decay components. The fluorescence decay behaviour was interpreted as representing three families of tryptophans, the short lifetime component being due to a stacking interaction between tryptophan residues. The variation with incubation time suggests a two-step process in the channel-lipid organization. The first is associated with the conformational change of the polypeptide as it takes up a left-handed helical head-to-head dimer structure in the lipid. The second step is proposed to involve changes originating from membrane assembly and intermolecular interactions between channels as they form hexameric clusters.

O2−dependent lipid peroxidation does not affect the molecular order in hepatoma microsomes

Microsomal membranes from rat liver and from the fast‐growing Morris hepatoma 3942A have been peroxidized to different extents and the order parameter of the membranes measured by fluorescence depolarization of the probe 1,6‐diphenyl‐1,3,5‐hexatriene. The data have been analysed by applying a mathematical approach that takes into account simultaneously static and dynamic fluorescence parameters. It appears that tumour membranes are more ordered than the control and their order parameter does not increase with greater exposure to the action of O− 2 radicals in contrast to liver membranes. The fatty acid composition of the membrane lipids has been studied under different experimental conditions and correlated to the behaviour of the physical parameter.

In Search of a Vaccine for Mouse Allergy: Significant Reduction of Mus m 1 Allergenicity by Structure-Guided Single-Point Mutations

Background: Mouse urinary proteins are relevant allergens from mice urine. We used the recombinant protein Mus m 1 as an allergen model to identify if, by altering Mus m 1 architecture via single-point mutations, we could effectively modify its allergenicity. Methods: Based on structural considerations, we synthesized two single-point mutants, Mus m 1-Y120A and Mus m 1-Y120F, which were expected to harbor large structural alterations. Circular dichroism and fluorescence analysis showed significant conformational rearrangements of the aromatic side chains in the internal cavity of Mus m 1-Y120A when compared to Mus m 1-Y120F and Mus m 1. Evaluation of the allergenic potential of the recombinant molecules was performed in vitro with both immunochemical approaches and assays based on the measurement of basophil degranulation. Moreover, to assess the integrity of the T cell epitopes and as an in vitro measure of immunogenicity, we tested the reactivity of T lymphocytes from subjects allergic to mouse urine against proteins and synthetic peptides encompassing the immunodominant linear epitope containing the mutation. Results: We found that the selected point mutation was able to modulate the protein allergenicity, and to severely impair the recognition of Mus m 1 by IgE, while T cell reactivity was fully maintained. Conclusions: In silico predicted, minimum selected structural modifications allowed to design one protein with reduced allergenicity and preserved immunogenicity. Structurally guided mutations can direct the design of proteins with reduced allergenicity which can be used as vaccines for a safer and more effective immunotherapy of allergic disorders.

A comparative study of the effect of leukoreduction and pre-storage leukodepletion on red blood cells during storage

Blood transfusion is a fundamental therapy in numerous pathological conditions. Regrettably, many clinical reports describe adverse transfusions drawbacks due to red blood cells alterations during storage. Thus, the possibility for a blood bank to ameliorate the quality of the erythrocyte concentrates units is crucial to improve clinical results and reduce transfusion adverse occurrences. Leukodepletion is a pre-storage treatment recognized to better preserve the quality of red blood cells with respect to leukoreduction. Aim of this work is to unravel the biochemical and biophysical basis that sustain the good clinical outcomes associated to the use of leukodepleted erythrocytes units. Erythrocytes concentrates were prepared as leukoreduced (n = 8) and pre-storage leukodepleted (n = 8) and then studied during 6 weeks in blood bank conditions. Overall, the data indicate that leukodepletion not only provide red blood cells with an appropriate amount of nutrients for a longer time but also selects red blood cells characterized by a more resilient plasma membrane fit to prolong their viability. We believe these results will stimulate new ideas to further optimize the current storage protocols.

Influence of hydroxystearic acid on in vitro cell proliferation

Lipid peroxidation products have recently been proposed among the possible regulators of tumour cell growth. According to our current working hypothesis, the greatly diminished content of such products in tumour cells might relieve the inhibition of cell growth thus leading to uncontrolled proliferation. Hydroperoxy- and hydroxy derivatives of long chain fatty acids have been identified and determined in normal and tumour cells. Among these, hydroxystearic acid (HSA) has been shown to have a different cytostatic and cytotoxic effect when administered to murine lung carcinoma cells or to human colon tumour cells. It interferes with cell cycle kinetics, blocking the murine cells in G2-M and the human ones in G0-G1. The molecular target of HSA in both cell lines has been shown to be the cdc2 kinase complex. The results so far obtained in tumour as long as in normal highly proliferating cells do not exclude a potential future use of this class of compounds as selective anti tumour drugs.