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Biomedical subjects

L Masotti

Publications and source records attributed to L Masotti.

At least 55 records · Page 3Linked to original sources
PubMed1991

Identification of the peroxidation product hydroxystearic acid in Lewis lung carcinoma cells.

Whole cell lipids were extracted from the Lewis lung carcinoma in vitro line C108. The fatty acids were derivatized to methylesters in order to identify endogenous oxidized derivatives by gasmass spectroscopy. The presence of 9-hydroxystearic acid and 10-hydroxystearic acid was thus evidenced for the first time in cultured mammalian cells. Moreover a linear correlation was found between the concentration of these products expressed as percentage of total fatty acid methylesters and the cell density in tissue culture flasks. This finding suggests an involvement of hydroxystearic acid in cellular functions related to the cell density in monolayer cultures.

Animals↗
PubMed1991

Postnatal maturation of rat intestinal membrane: lipid composition and fluidity.

1. We studied the lipid composition and the fluidity of small intestine brush border membrane (BBM) of rats of different age: 'very young' (5-7 weeks old), 'young' (9 weeks old), 'adult' (30 weeks old) and 'old' (85 weeks old). 2. Fluorescence anisotropy, as assessed by 1,6-diphenyl-1,3,5-hexatriene probe (DPH), was increased from very young to adult rats. 3. In agreement with these results the lipid composition in adult animals showed a lower lipid/protein ratio (derived mainly from a lower content of total polar lipids) and an increase of cholesterol esters and sphingomyelin (SM) saturation index. 4. A marked decrease of the order parameter was observed in the 'old' group, accompanied by a decreased cholesterol/phospholipid ratio. 5. The percentage distribution of membrane phospholipids significantly changed during development, but the modifications were not correlated with the anisotropy of DPH.

Aging↗
PubMed1991

Oxy-radical metabolism and control of tumour growth.

1. The content of oxy-radical scavenging enzymes is decreased in Morris hepatomas in a fashion which is inversely related with the growth rate of the tumour. 2. Hepatoma microsomal membranes are more resistant than normal rat liver membranes to lipid peroxidation induced in vitro by organic hydroperoxides or superoxide radicals. 3. In tumour membranes the most relevant rate-limiting factor of peroxidation is the low availability of polyunsaturated fatty acids (PUFA). Besides lipids, some proteins (particularly cytochrome P-450) act as controlling factors of peroxidation. 4. Tumour microsomes are more ordered and less fluid than liver microsomes. The latter, exposed to superoxide radical attack, exhibit chemical (fatty acid composition) and physical (molecular order) properties that are similar to those of transformed cell membranes. 5. These data indicate an aberration in the oxy-radical metabolism of cancer cells, and a sequence of events is hypothesized that could drive the transformed cell towards uncontrolled proliferation.

Animals↗
PubMed1991

The role of the side chain in the antioxidant activity of ubiquinones.

The question has been addressed whether the side chain contributes to the antioxidant activity of Ubiquinones. The length, the chemical composition and structure of the chain have been considered. The effect of the actual concentration of the quinone in egg lecithin vesicles has been investigated by means of both UV spectroscopy and time resolved fluorescence quenching experiments of 12-AS. The results indicate that the antioxidant properties of the quinone do not seem to depend on the side chain.

Antioxidants↗
PubMed1990

Time-resolved fluorescence study of the neuron-specific phosphoprotein synapsin I. Evidence for phosphorylation-dependent conformational changes.

Synapsin I is a major nerve terminal-specific phosphoprotein. It consists of a hydrophobic head region containing one phosphorylation site for either cAMP-dependent protein kinase or Ca2+/calmodulin-dependent protein kinase I and of a basic and elongated tail region containing two phosphorylation sites for Ca2+/calmodulin-dependent protein kinase II. The steady-state emission spectrum of synapsin I was centered at 330 nm and was markedly red shifted upon denaturation, as expected for tryptophan residues segregated from the external aqueous environment in native conditions. Quenching studies showed a low accessibility of synapsin I tryptophans at low ionic strength which was further decreased by exposure to 200 mM NaCl but not significantly affected by phosphorylation. The intrinsic fluorescence of synapsin I was resolved into three major decay components with lifetimes of about 0.2, 3, and 7 ns. Upon phosphorylation of synapsin I on the tail sites, the spectra associated with the intermediate and long lifetimes were shifted to the red region, while the spectrum associated with the short lifetime was shifted to the blue region, in the absence of significant changes of the lifetimes. Phosphorylation of synapsin I on the head site was less effective. The anisotropy decay of synapsin I labeled with the long-living chromophore pyrene on Cys-223 was also analyzed. A shorter rotational correlation time was found for the tail phosphorylated form (corresponding to a Stokes radius of 41-42 A) than for the dephosphorylated or for the head phosphorylated form (corresponding to a Stokes radius of 60-63 A). The data suggest that phosphorylation of the tail sites induces changes in the conformation and hydrodynamic properties of synapsin I which may play a role in the regulation of the molecular interactions of synapsin I within the nerve terminal.

2-Naphthylamine↗
PubMed1990

Estimation of protein secondary structure from circular dichroism spectra: a critical examination of the CONTIN program.

The computer program CONTIN uses the Provencher and Glöckner procedure to calculate protein secondary structure from circular dichroism spectra. We have tested this program with peptides and proteins in which unfolding was either induced by denaturing treatment or was already present. Results indicate that the program does not clearly discriminate between the ordered and the unordered states of a protein.

Circular Dichroism↗
PubMed1989

An investigation on lipoperoxidation mechanisms in boar spermatozoa.

Aerobic incubation of washed boar spermatozoa in a heavy metal free medium at 37 degrees C results in a peroxidative breakdown of membrane phospholipids as revealed by malondialdehyde production. In the presence of iron ions, alone and with ascorbate, the amount of malondialdehyde produced increases noticeably. Alkoxy and lipoperoxy radicals are likely involved in these peroxidative processes, while OH. radical does not seem to be essential in the pathway of malondialdehyde formation.

Animals↗
PubMed1989

Conformation of bombesin in buffer and in the presence of lysolecithin micelles: NMR, CD, and fluorescence studies.

The conformation of the tetradecapeptide hormone bombesin has been studied in buffer and in the presence of lysolecithin micelles, using static and dynamic fluorescence, CD, and one- and two-dimensional nmr. The results obtained show that in buffer bombesin is present in an extended flexible chain, with no evidence for any ordered secondary structure. A marked change in the CD spectrum is observed changing from buffer to the lipid suspension. Concomitantly, the 1H-nmr spectrum of bombesin, in a D2O lipid dispersion, shows the persistence of resonances due to exchangeable protons and in similar conditions the fluorescence intensity increases. We think therefore that these results strongly support the hypothesis that bombesin interacts with the lipid phase, assuming ordered secondary structure. Finally, the marked dependence of tryptophan fluorescence quantum efficiency and order parameter from the hormone concentration in the presence of lysolecithin but not in buffer leads to the conclusion that bombesin can associate into the lipid matrix.

Bombesin↗
PubMed1989

Lipid peroxidation and physical properties of smooth and rough hepatoma microsomes.

Smooth and rough endoplasmic reticulum of two Morris hepatomas, the slow growing 9618A and the fast growing 3924A, have been isolated, and their biochemical composition, supramolecular organization, and response to the action of peroxidative agents have been studied. Cytochrome P450 content and lipid availability are the limiting factors of their peroxidizability. The hemoprotein content is reduced about 80% in hepatoma 9618A and is virtually absent in hepatoma 3924A. The peroxidizability decreases with increasing growth rate of the tumor. The protein, phospholipid, and cholesterol content, the fatty acid composition as well as the double bond index, and the saturated and unsaturated fatty acid content are reported. Differences have been found between normal liver and tumors and between the fractions within a given tumoral tissue. The molecular order, as determined by fluorescence anisotrophy decay of DPH, increases in total microsomes and in the smooth fraction going from liver 9618A to 3924A, whereas for the rough fraction it is the same in liver and hepatoma 9618A; in 3924A it increases of about 30%. Fluidity decreases in total microsomes going from liver to 3924A, to 9618A. In both the purified fractions it decreases with increasing deviation of the tumor.

Animals↗
PubMed1988

Fluorescence spectral resolution of myelin basic protein conformers in complexes with lysophosphatidylcholine.

The structure of (Deibler) myelin basic protein in solution and in a lysolecithin++ lipid complex has been studied by using the emission properties of the single tryptophan residue of the protein (Trp-115). The studies have been carried out using both static and time-resolved fluorescence techniques. Relative to the free protein, the lipid bound myelin basic protein showed a twofold increase in fluorescence intensity and a marked blue-shift in the emission maximum wavelength. The multiexponential fluorescence decays and the decay associated spectra indicated that the protein exists in at least three different conformations both in buffer and in lipids. Fluorescence polarization and acrylamide quenching experiments showed that the tryptophan containing region of the protein is embedded in the lipid matrix. The binding of the protein to the lipid appears to be comparable with that predicted for the interaction of amphipathic helices with nonpolar lipids.

Animals↗
PubMed1988

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.

Animals↗
PubMed1988

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.

Animals↗
PubMed1987

DAPI-pUC8 complex: a tool to investigate biological effects of nucleic acid-drug interaction.

A complex consisting of pUC8, a 1.8 Md plasmid, and 4'-6-diamidino-2-phenylindole, a DNA-binding agent, has been performed in vitro under different conditions of ionic strength, and used to transform competent cells. A strong interference with the plasmid-coded activities, related to the P/D ratio where at the DNA-drug complex was formed, was shown to occur. Since the compound does not inhibit the uptake process neither affects plasmid activity once dissociated at high ionic strength, it is likely to be acting from inside the cell while still in the form of a DNA-adduct. This system is proposed as a useful tool to investigate the effects on target genes of drugs endowed with DNA sequence specificity.

Circular Dichroism↗
PubMed1987

Two-dimensional echocardiographic imaging: in vitro comparison of conventional and dynamically focused annular array transducers.

Quantitative two-dimensional echocardiography has been adversely affected by a tendency for underestimation of cross-sectional areas of cardiac chambers, a difficulty that might be ameliorated by recent advances in imaging technology. To determine if this were so, we measured echocardiographic cross-sectional areas of 25 formalin-fixed animal left ventricular (LV) sections in vitro using conventional 13 mm and 15 mm diameter fixed-focused single element transducers, and a 15 mm diameter dynamically focused annular array transducer at 3 different distances between myocardial slice and transducer (2 cm, 6 cm and 10 cm) and compared the 2-dimensional echocardiographic areas to the corresponding anatomic cross-sectional areas of the same hearts. LV total and cavity area were measured by computer assisted planimetry of videotaped echo images, performed blinded to the transducer used, and photographed anatomic slices; LV myocardial area was derived by subtraction. Comparison of two-dimensional echocardiographic total, myocardial, and cavity areas with corresponding anatomic measurements showed excellent correlation for each transducer at all depths (r = 0.97 to 0.98 for total area; r = 0.98 to 0.99 for cavity area; r = 0.93 to 0.97 for myocardial area). For total and myocardial cross-sectional areas, the slope of the relation between echographic and anatomic areas did not differ significantly from unity, but for LV cavity area this was achieved only by the dynamically focused transducer. In contrast, the conventional 13 mm transducer significantly underestimated larger LV cavity areas in both the near and middle fields (slopes = 0.90 and 0.91, respectively) and the 15 mm transducer yielded slopes from 0.86 to 0.91 in all fields.(ABSTRACT TRUNCATED AT 250 WORDS)

Echocardiography↗
PubMed1986

Conformational flexibility of the hormonal peptide bombesin and its interaction with lipids.

The conformational flexibility of the tetradecapeptide hormone bombesin has been studied using circular dichroism and fluorescence of its single tryptophan residue. The spectral changes observed indicate that the peptide changed from a random flexible coil in solution to a helical structure in lysolecithin micelles and dimyristoylphosphatidylserine vesicles. The tryptophan residue in the lipid complexes was located in a hydrophobic environment. The interaction with lipids was shown to involve both hydrophobic and electrostatic forces.

Bombesin↗
PubMed1986

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.

Gramicidin↗
PubMed1986

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 O2 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.

Animals↗