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Biomedical subjects

A Bonincontro

Publications and source records attributed to A Bonincontro.

At least 19 recordsLinked to original sources
PubMed2001

Intrinsic structural differences between "tight couples" and Kaltschmidt-Wittmann ribosomes evidenced by dielectric spectroscopy and scanning microcalorimetry.

Measurements of dielectric spectroscopy (DS) and microcalorimetry (differential scanning calorimetry (DSC)) of Escherichia coli 70S, 50S and 30S were performed on particles prepared according either to the "classical" twice NH(4)Cl-washed ribosomes, also known as loose couples (LC), or to the "tight couples" preparative protocol (TC). Results show that 70S particles prepared according to the two different protocols exhibit different structural properties. Two subsequent relaxation processes occur in both samples as measured by DS. However, in LC ribosomes the first one is shifted towards a lower frequency with a higher dielectric increment. This is suggestive of a more extensive exposure of RNA to the solvent and of an overall more relaxed structure. The smaller LC subunit exhibits only one relaxation while the TC 30S shows two dielectric dispersions as well as 70S. No substantial differences were evidenced in either 50S species. Two typical melting peaks were observed by DSC both in LC and TC 70S as well as in 50S. Thermograms obtained from the TC 30S show a single well structured peak while LC particles produce a large unstructured curve. On the basis of these results we conclude that TC 70S particles are more compact than LC ribosomes and that in the former ones the rRNA is less exposed to the solvent phase. Furthermore 30S particles obtained from TC show a more stable structure with respect to LC 30S. We conclude that the 30S subunit gives a major contribution to the compact character of the whole TC 70S. These differences might be related to the intrinsic and well documented functional difference between the two ribosome species.

Calorimetry, Differential Scanning↗
PubMed2001

pH-dependent local structure of ferricytochrome c studied by x-ray absorption spectroscopy.

We have studied, using x-ray absorption spectroscopy by synchrotron radiation, the native state of the horse heart cytochrome c (N), the HCl denatured state (U(1) at pH 2), the NaOH denatured state (U(2) at pH 12), the intermediate HCl induced state (A(1) at pH 0.5), and the intermediate NaCl induced state (A(2) at pH 2). Although many results concerning the native and denatured states of this protein have been published, a site-specific structure analysis of the denatured and intermediate solvent induced states has never been attempted before. Model systems and myoglobin in different states of coordination are compared with cytochrome c spectra to have insight into the protein site structure in our experimental conditions. New features are evidenced by our results: 1) x-ray absorption near edge structure (XANES) of the HCl intermediate state (A(1)) presents typical structures of a pentacoordinate Fe(III) system, and 2) local site structures of the two intermediate states (A(1) and A(2)) are different.

Absorptiometry, Photon↗
PubMed2001

Rotational and translational dynamics of lysozyme in water-glycerol solution.

In this paper, we report a study of the effect of solvent viscosity on both translational and rotational dynamics of a simple model protein: the egg white lysozyme. For this, we investigated the dynamical properties of lysozyme in mixtures water-glycerol by means of parallel measurements of photon correlation spectroscopy (PCS) and dielectric spectroscopy at radiofrequencies (DS). In the framework of the Debye-Stokes-Einstein theory, the translational and rotational coefficients allow an estimation of hydrodynamic radius of the protein. A decoupling between translational and rotational dynamics, observed as a different estimation of hydrodynamic radius, is reported in the literature for some systems. In order to ascertain if this effect is present also in our sample, we performed PCS and DS measurements on lysozyme-water-glycerol solutions. The content of glycerol was in the range of 0-70% w/w, with a solvent viscosity from 0.9 to about 10 cpoise, and the protein concentration was up to 20 mg ml(-1). The average sizes of lysozyme, obtained by the two methods, are remarkably different at high protein concentrations. However, the values of hydrodynamic radius extrapolated to infinite dilution are coincident and independent of glycerol. These results indicate that the diffusive behavior of lysozyme in the water-glycerol mixture is coherent with the Debye-Stokes-Einstein hydrodynamic model.

Journal Article↗
PubMed2000

Structural stability of ribosomes subjected to RNase treatment evidenced by dielectric spectroscopy and differential scanning microcalorimetry.

Previous studies from our laboratory demonstrated the existence of at least two levels of structural complexity in E. coli 70S ribosomes. Ribosomal RNA seems to be principally involved in the overall stability of these structures. In this paper we present an investigation of ribosomes subjected to treatment with RNase. The study is based on both differential scanning microcalorimetry and dielectric spectroscopy. In the thermograms obtained on treated ribosomes only the low temperature peak of the two typical denaturation events observed in native ribosomes, is promptly eliminated by the enzyme treatment. Dielectric spectroscopy measurements carried out on the same samples indicate an alteration of the dielectric behavior previously shown to consist of two subsequent relaxation processes. In fact, only the low frequency relaxation is affected by the treatment. The second one, observed at higher frequency, remains unaltered. The same effect on the dielectric parameters is observed if the ribosome particles are heated and then cooled prior to measurement. These results are consistent with the idea that two different structures are present within the ribosome. One is very stable and withstands both temperature and RNase treatment while the second is promptly abolished by both treatments. Data presented here strongly suggest that the RNA domains exposed to the solvent play a fundamental role in the stability of the 3-D structure of the ribosome particle.

Calorimetry, Differential Scanning↗
PubMed2000

Critical analysis of the impedance method for the evaluation of permittivity and conductivity of the plasma membrane.

We report a critical analysis of a typical method of dielectric spectroscopy consisting in impedance measurements as a function of frequency. Experimental data were obtained by measuring impedance on human erythrocyte suspensions. Since these cells do not have a nucleus they represent an ideal material for the application of the well established single shell model. This allows the evaluation of permittivity and conductivity of the plasma membrane. We discuss the influence on the reliability of results of parameters such as fractional volume, average dimensions and membrane thickness of cells.

Cell Membrane Permeability↗
PubMed1998

Differential stability of E. coli ribosomal particles and free RNA towards thermal degradation studied by microcalorimetry.

We investigated the thermal degradation of E. coli ribosomes by differential scanning microcalorimetry. The 70S particles show two distinctive and irreversible peaks upon thermal degradation. Free rRNA in solution produces, on the contrary, an unstructured denaturation profile. The thermal analysis of 50S particles shows a profile substantially identical to that observed in 70S, while 30S particles produce an unstructured denaturation pattern. Therefore the thermal behavior of the 70S particle is essentially attributable to the denaturation of the 50S subunit. Our data validate previous observations that the 50S has a more rigid structure as compared to 30S, which behaves as a 'floppy' particle. In addition our data suggest that protein/RNA interactions play a significant role to stabilize three-dimensional structures of the ribosome.

Calorimetry, Differential Scanning↗
PubMed1997

A study of the dielectric properties of E. coli ribosomal RNA and proteins in solution.

The permittivity of ribosomal proteins and ribosomal RNA (rRNA) in solution was measured in the range 100 kHz to 1 GHz at four different temperatures (5, 15, 25 and 35 degrees C). The experimental dielectric relaxation was analysed by the Cole-Cole equation and, from the best-fit parameters, the average values of the dipole moment and molecular radius of the proteins were obtained. The activation enthalpy was calculated from an Arrhenius plot of the relaxation time. The energy involved in the dielectric polarization of free proteins has a magnitude of about one hydrogen bond. The data on RNA were analysed according to the Mandel model. This analysis allowed the calculation of the "subunit b" as defined by Mandel. This parameter is dependent on the temperature and therefore the relaxation time does not follow the Arrhenius law. Our data thus show that, in solution, the rRNA structure is thermally rather unstable and highly flexible.

Bacterial Proteins↗
PubMed1997

Effects of magnesium and temperature on the conformation and reassociation of Escherichia coli and Sulfolobus solfataricus ribosomes.

The structural response of the ribosomes of the extremely thermophilic archaeon Sulfolobus solfataricus was analysed and compared to that of the mesophilic (E. coli) ribosomes by assaying ethidium bromide (EB) binding to the native 70S particles as a function of magnesium concentration. We found that the thermophilic ribosomes bound more EB than their mesophilic counterparts; on the other hand, inhibition of EB binding by Mg2+ ions was more effective in the E. coli 70S particle. In Sulfolobus, the separated 30S and 50S subunits and the 70S particle bound the drug in a similar fashion, whereas the E. coli 70S had a reduced number of binding sites with respect to the subunits. Light scattering measurements as a function of Mg2+ concentration were carried out at various temperatures to study the interaction between the ribosomal subunits from the thermophilic and the mesophilic bacteria. As expected, the association of ribosomal subunits in E. coli was magnesium dependent and could be observed also at low temperature. By contrast, the interaction between Sulfolobus ribosomal subunits was obligatorily dependent upon both magnesium ions and a temperature of at least 80 degrees C, close to the physiological optimum for cell growth (87 degrees C).

Binding Sites↗
PubMed1997

Apoptosis dependent decrease of the intramembrane ion traffic in cultured mouse fibroblasts shown by conductivity dispersion.

We have investigated the intramembranal ion traffic in apoptotic 3T6 cells in culture. Apoptosis was induced by various treatments, such as serum deprivation, high density growth and hydrogen peroxide at subnecrotic doses. Cell death was assessed by nucleosomal DNA fragmentation, single cell electrophoresis, immunofluorescence and histological staining. To study the modifications of membrane structure and function, we adopted a well established biophysical strategy based on the measurement of the electrical conductivity of cell suspensions, as a function of the frequency of the electrical field applied to the sample. A comparison between the conductivity of normal and apoptotic cell suspensions shows that programmed cell death causes a decrease of membrane conductivity which indicates a diminished intramembranal ion traffic. Our results strongly suggest that one of the early events in the triggering of apoptosis is represented by an overall reduction of plasma membrane function. Finally, our results are in agreement with the idea that the nucleus is not the sole target of the apoptotic process.

Animals↗
PubMed1996

Electrical conductivity dispersion as a probe of membrane modifications in mouse polyomavirus infected cells in culture.

In this report we investigate the inhibition of membrane conductivity, due to the murine polyomavirus infection in permissive cells in culture. We define experimental conditions to have reproducible results and demonstrate that the intensity of the effects on the cell membrane, depends upon the virus titer used in the infection. Finally, the virus dependent effects disappear if the infection is performed in the presence of a drug that inhibits polymavirus DNA replication.

Animals↗
PubMed1995

Effect of thymine dimer introduction in a 21 base pair oligonucleotide.

It is well known that the pyrimidine dimers are the main damage produced by UV radiation on the DNA structure. However, while studies on the photoproduct structure have been carried out extensively, uncertainties still exist on the implication that a single damaging event has on the overall conformation. In particular, the extension of the damage influence on the polynucleotide chain is a matter of debate. This problem is especially important to understanding some steps of the repair mechanisms. In this study we performed a chemical-physical characterization of 21 base pair oligonucleotides containing a single thymine dimer in one strand. Thermodynamic parameters were determined by means of thermal denaturation experiments, and static fluorescence measurements were performed to unequivocally define the primary structure-conformation relationship in this specific case. We used hydroxyl radicals, produced by means of gamma-irradiation of the sample solution, to detect fine structure changes. Our data show that the introduction of a single thymine dimer might cause only a slight distortion of the helix geometry, as judged by the evaluation of the enthalpic and the entropic terms and by the small changes observed in the binding of ethidium bromide to DNA. The modifications in the sugar phosphate backbone subsequent to the damaging event are especially evident, near the thymine dimer, toward the 5'-end direction in the strand containing the dimer.

Base Sequence↗
PubMed1993

Effects of magnesium ions on ribosomes: a fluorescence study.

Fluorescence intensity measurements of ethidium bromide (EB) bound to ribosomal RNA (rRNA) in suspensions of 30S and 50S subunits, of 70S ribosomal particles and of protein-free extracted rRNA are presented. Changes in the intercalation of EB reflect changes in conformation and degree of exposure of rRNA. The effect of removal of magnesium ions on the binding of EB is compared in protein-free rRNA and in ribosomal particles by a Scatchard plot analysis. In free ribosomal RNA the number of bound EBs do not depend on magnesium content, only the association constant is affected. In intact 70S particles and both in the separated 50S and 30S subunits the presence of magnesium greatly reduces binding of EB and no saturation of the fluorescence intensity with rRNA concentration is observed, preventing a Scatchard plot analysis. Removal of magnesium restores a strong EB intercalation. Then magnesium ions induce a conformational change in the 70S particles as well as in the separated subunits. The different behavior of the free-rRNA and of the ribosomal particles indicates that ribosomal proteins are relevant to the structural changes induced by magnesium ions. The comparison of the number of excluded sites and of the association constant in the 30S, 50S subunits and in the 70S particles indicates that even without Mg2+ ions the two subunits still interact, at variance with the commonly shared opinion that subunits dissociation takes place at low magnesium concentration.

Edetic Acid↗
PubMed1993

Influence of defects on the electrophoretic, thermodynamic and dielectric properties of a 21 base pair DNA in solution.

The thermodynamic and dielectric properties of a 21 base pair DNA have been evaluated and compared with those of samples with some defects. In particular, fragments in which the absence of a phosphate group (nick) or of two nucleotides (gap) causes chain interruptions were studied. Measurements of ultraviolet absorption as a function of temperature at different oligomer concentrations and at various ionic strengths were performed. Dielectric spectroscopy at radiofrequencies (1 MHz-1 GHz) was applied on aqueous solutions of the duplexes at 5 degrees C, where the solutes are thermally stable. Dielectric dispersions with 30-40 MHz characteristic frequencies were defined. The results of melting experiments indicate a thermal destabilization of the oligomers containing the defects. Electrophoretic data and the dielectric results show that the conformations of the nicked and control samples are very similar, while the oligomer with a gap is more compact with a different charge distribution at the ends.

Base Composition↗
PubMed1992

Differences in membrane electrical properties between C3H 10T1/2 mouse embryo fibroblasts and their ionizing radiation and chemically transformed counterparts.

Membrane electrical properties of mouse embryo fibroblasts and their ionizing radiation and chemically transformed counterparts were investigated using dielectric relaxation measurements in the radio frequency range. This determination is possible because, in the radio frequency range, suspensions of cells in an electrolyte buffer show a conductivity dispersion due to interfacial polarization. An analysis of the experimental data based on a "single-shell" model showed that conductivity and permittivity of the membranes of both radiation and chemically transformed fibroblasts were lower than in normal cells. In addition, the conductivity of the cytoplasm was higher in both transformed cell types than in the normal mouse fibroblasts. We discuss the significance of these findings in view of the possible structural and functional modifications brought about by the process of neoplastic transformation.

Animals↗
PubMed1991

Radiofrequency dielectric spectroscopy of ribosome suspensions.

Dielectric measurements on different ribosome suspensions were carried out in the frequency range from 10 kHz to 1 GHz. In intact ribosomes two dispersions were detected: one around 100 kHz and the other one in the MHz region. In separated ribosomal subunits and in ribosomes resuspended in a buffer with no magnesium ions (relaxed ribosomes) only the MHz dispersion was observed. Electrical conductivities of the samples at 1 kHz were also measured. The temperature dependence of the two dispersions was investigated and a tentative attribution was proposed.

Electric Conductivity↗
PubMed1991

Temperature dependence of DNA dielectric dispersion at radiofrequency.

We have studied the dielectric behavior of DNA aqueous solutions at various ionic strengths and in the presence of the specific DNA ligand ethidium bromide, in the frequency range 1 MHz-1 GHz, at different temperatures ranging from 5 to 40 degrees C. The activation enthalpies of the dielectric relaxations studied were obtained by Arrhenius plots of In(tau T)-1 vs. T-1. The results are consistent with a counterion fluctuation model as previously developed by Mandel and colleagues.

DNA↗