Biomedical subjects

P Facci

Publications and source records attributed to P Facci.

7 recordsLinked to original sources

PubMed2000

Kinetic and structural study of the interaction of myelin basic protein with dipalmitoylphosphatidylglycerol layers.

The interaction of myelin basic protein (MBP) with dipalmitoylphosphatidylglycerol films has been investigated by means of a microgravimetric gauge sensitive to the changes in load and structural modifications of the layer deposited onto its surface. Fourier transform infrared spectroscopy, circular dichroism, and x-ray diffraction have confirmed protein uptake by the lipid phase along with a global disordering effect onto the lipid alkyl chains and have shown a temporal evolution of the structure of water penetrating the lipid phase together with the protein. These effects are clearly related to the temporal variation of the microgravimetric gauge signal. Finally, measurements carried out on pre-annealed samples point out the role of mesoscopic morphology in determining the pathways through which MBP penetrates the lipid multilayer. The results obtained in our model system could be useful in clarifying the mechanisms of the myelinating and demyelinating processes that take place in the natural membrane.

Animals↗

PubMed1997

Thioredoxin from Bacillus acidocaldarius: characterization, high-level expression in Escherichia coli and molecular modelling.

The thioredoxin (Trx) from Bacillus acidocaldarius (BacTrx) was purified to homogeneity by anion-exchange chromatography and gel-filtration chromatography, based on its ability to catalyse the dithiothreitol-dependent reduction of bovine insulin disulphides. The protein has a molecular mass of 11577 Da, determined by electrospray mass spectrometry, a pI of 4.2, and its primary structure was obtained by automated Edman degradation after cleavage with trypsin and cyanogen bromide. The sequences of known bacterial Trxs were aligned at the active site: BacTrx has an identity ranging from 45 to 53% with all sequences except that of the unusual Anabaena strain 7120 Trx (37% identity). The gene coding for BacTrx was isolated by a strategy based on PCR gene amplification and cloned in a plasmid downstream of a lac-derived promoter sequence; the recombinant clone was used as the expression vector for Escherichia coli. The expression was optimized by varying both the time of cell growth and the time of exposure to the inducer isopropyl beta-d-thiogalactoside; expressed BacTrx represents approx. 5% of the total cytosolic protein. CD spectra and differential scanning calorimetry measurements demonstrated that BacTrx is endowed with a higher conformational heat stability than the Trx from E. coli. Nanogravimetry experiments showed a lower content of bound water in BacTrx than in E. coli Trx, and a transition temperature approx. 10 degrees C higher for BacTrx. The three-dimensional model of the oxidized form of BacTrx was constructed by a comparative molecular modelling technique, using E. coli Trx and Anabaena strain 7120 Trx as reference proteins. Increased networks of ion-pairs and shorter loops emerged as major features of the BacTrx structure compared with those of the template proteins. The findings are discussed in the light of the current knowledge about molecular determinants of protein stability.

Amino Acid Sequence↗

PubMed1997

Quartz balance DNA sensor.

Single-strand DNA-containing thin films were deposited onto quartz oscillators by the Langmuir-Blodgett technique towards the realization of a device capable of sensing the presence of the complementary DNA sequences which hybridize with the immobilized ones. DNA, once complexed with aliphatic amines, appears as a monolayer in a single-stranded form by X-ray small angle scattering. A quartz nanobalance is then utilized to monitor mass increment related to specific hybridization with a complementary DNA probe. The crystal quartz nanobalance, capable of high sensitivity, indeed appears capable of obtaining a prototype of a device capable of sensing the occurrence of particular genes or sequences in the sample under investigation. The validity of the nanogravimetric assay was confirmed by independent fluorescence measurements utilizing DAPI and a CCD camera.

Biosensing Techniques↗

PubMed1996

Room-temperature single-electron junction.

The design, realization, and test performances of an electronic junction based on single-electron phenomena that works in the air at room temperature are hereby reported. The element consists of an electrochemically etched sharp tungsten stylus over whose tip a nanometer-size crystal was synthesized. Langmuir-Blodgett films of cadmium arachidate were transferred onto the stylus and exposed to a H2S atmosphere to yield CdS nanocrystals (30-50 angstrom in diameter) imbedded into an organic matrix. The stylus, biased with respect to a flat electrode, was brought to the tunnel distance from the film and a constant gap value was maintained by a piezo-electric actuator driven by a feedback circuit fed by the tunneling current. With this set-up, it is possible to measure the behavior of the current flowing through the quantum dot when a bias voltage is applied. Voltage-current characteristics measured in the system displayed single-electron trends such as a Coulomb blockade and Coulomb staircase and revealed capacitance values as small as 10(-19) F.

Journal Article↗

PubMed1993

Thermal stability of protein secondary structure in Langmuir-Blodgett films.

The temperature dependence of the secondary structure of photosynthetic reaction centres from Rhodobacter sphaeroides in solution and in Langmuir-Blodgett film was studied by circular dichroism. It was shown that the secondary structure of the protein was not affected in Langmuir-Blodgett films by heating up to 200 degrees C, while in solution it was completely lost at 55 degrees C. Molecular order rather than decreased hydration degree was held responsible.

Circular Dichroism↗

PubMed1992

Fluorescence cytometry of microtubules and nuclear DNA during cell-cycle and reverse-transformation.

Synchronized CHO-K1 cells and their dibutyryl c-AMP treated counterparts have been characterized by means of static and flow fluorescence cytometry at the level of nuclear DNA and cytoplasmic microtubules. In order to confirm earlier findings on synchronized population, Carnoy fixed and hydrolyzed, several new findings are here reported at the level of single intact cell. The fluorescence intensity of DAPI-stained glutaraldehyde fixed 2C cells correlates well with the average absorbance of the corresponding Feulgen-stained cells, thereby appearing also to be a measure of chromatin condensation during the G1 phase. In the early part of G1, the drastic alteration in anti-beta tubulin immunostaining is shown to parallel microtubule depolymerization induced by calcium or colcemide. The known 1-2 h lengthening of the G1 period after reverse-transformation appears to correlate with a similar delay in the abrupt chromatin decondensation. The above results are discussed in terms of the role of microtubules and nuclear morphometry (and their coupling) in the control of cell cycle progression of transformed vs. fibroblast-like cells.

Animals↗

PubMed1991

Changes in DNA superhelical density monitored by polarized light scattering.

Linear and circular lambda-DNA at different ethidium bromide concentrations have been studied by means of polarized light scattering, namely the S14, S34, S33 and S13 elements of Mueller matrix. While S33 at low angle appears well correlated with the total light scattering evaluated by optical density measurements at 632.8 nm for linear and circular DNA of the same mass, the magnitude and slope of the S14, S34 and S13 signals display significant changes for the circular lambda-DNA depending on the degree of negative superhelical density as induced by the different ethidium bromide concentrations. At the same time, for linear lambda-DNA the signal remains invariant, making explicit for the differential scattering of polarized light the possibility to obtain additional information by its angular dependence. Strikingly also the effect of 0.2% glutaraldehyde versus ethanol fixation on the native lambda-DNA structural properties appears to confirm earlier findings by other well-established probes. Results are discussed in terms of first physical principles and of their potential bearings towards our understanding of the mechanism controlling gene expression.

Bacteriophage lambda↗