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Biomedical subjects

R Ragone

Publications and source records attributed to R Ragone.

At least 37 records · Page 2Linked to original sources
PubMed1995

Hyperbaric oxygen, oxygen-ozone therapy, and rheologic parameters of blood in patients with peripheral occlusive arterial disease.

For many years, clinical practice has consolidated the use of both hyperbaric oxygen and oxygen-ozone therapy in the treatment of peripheral occlusive arterial disease (POAD). We investigated the influence of these treatments on hemorrheologic parameters that play an important role in the pathogenesis and the clinical course of arteriosclerosis. Two groups of 15 patients suffering from POAD, assigned at random either to a cycle of HBO therapy or O2-O3 therapy, were evaluated for blood viscosity, erythrocyte filterability, hematocrit value, fibrinogen concentration, and thrombin time. The O2-O3 therapy caused a significant increase of erythrocyte filterability and a significant decrease of blood viscosity. By contrast, HBO therapy did not produce any significant change. The increase of lipid peri-oxidation, proved by raised malonyldialdehyde plasma levels, seems a likely mechanism involved in the hemorrheologic effects of O2-O3 therapy.

Aged↗
PubMed1994

Determination of hydrophobic hydration in protein unfolding by an intrinsic reference state.

This paper describes a method for the evaluation of the unfolding heat capacity change of proteins by their amino-acid composition. The method hinges on a set of hydration heat capacity changes of amino acids extracted from the Protein Data Bank of crystallographic structures (Oobatake, M. and Ooi, T. (1988) J. Biochem. (Tokyo) 104, 433-439). This avoids problems linked to the choice of an arbitrary reference state. The published values have been normalized with respect to the total surface area of each amino-acid residue and related to the non-polar surface. The relationship found for amino acids allows a straightforward estimate of the unfolding heat capacity change of globular proteins. Predicted values for a large set of proteins fall within the experimental error. The devised algorithm shows that the unfolding heat capacity change depends on chain length and provides an explanation for the physical limits imposed upon this quantity.

Amino Acids↗
PubMed1994

Enthalpy-entropy balance and convergence temperatures in protein unfolding.

We find that isoenthalpic and isoentropic temperatures characterizing the unfolding of small globular proteins are linked by a simple relationship, which takes into account the occurrence of common values of specific unfolding enthalpy and entropy changes. The difference between these temperatures implies that the hydration effect favors protein folding over a quite large range of temperatures.

Models, Theoretical↗
PubMed1994

Referee bias.

Explore the source record for details and available documents.

Peer Review, Research↗
PubMed1994

In vitro cross-linking of calf lens alpha-crystallin by malondialdehyde.

The effect of malondialdehyde on structural features of bovine alpha-crystallin has been investigated by absorption and fluorescence spectroscopy as well as by far-UV circular dichroism. Experimental evidence suggests the occurrence of intermolecular cross-linking induced by malondialdehyde. This cross-linking does not seem to affect the tryptophan environment, as suggested by intrinsic protein fluorescence. On the contrary, the time dependence of far-UV dichroic activity indicates that the cross-linking is accompanied by a secondary structure change. The formation of high molecular mass aggregates, evidence by electrophoresis in denaturing conditions, leads to irreversible alpha-crystallin aggregation due to extensive intermolecular cross-linking. Since malondialdehyde is produced in vivo as a breakdown product of lipid peroxidation the possible involvement of this molecule in the pathological mechanism of cataract formation has been briefly discussed.

Animals↗
PubMed1992

Structural organization and stability of a thermoresistant domain generated by in vivo hydrolysis of the alpha-crystallin B chain from calf lens.

A protein fragment (M(r) approximately 9000) isolated from the cortex of nonpathological calf lenses has been structurally characterized. The polypeptide structure was well organized (39% alpha-helix, 33% beta-structure, and 28% remainder) according to the far-ultraviolet circular dichroism. The fluorescence was heterogeneous for the presence of two tryptophan classes. Structure perturbation by pH and denaturant revealed cooperative structural transitions which are characteristics of a globular organization. A single-step unfolding curve induced by Gdn-HCl (midpoint = 1.38 M Gdn-HCl) was monitored by emission maximum shift as well as by far-ultraviolet circular dichroism. This transition was analyzed as a two-state process. The standard free energy of unfolding in the absence of the denaturant, delta Go (H2O), was found to be 10.80 +/- 0.25 kJ/mol at 20 degrees C and pH 7.4. The fragment also shows an unusual thermal resistance. Its structure was unperturbed up to 90 degrees C according to the fluorescence and dichroism. This last property, its peculiar amino acid composition, and the sequence of a small segment are shared, among crystallins, only with the N-terminal region of the alpha-crystallin B chain. A search for proteolysis sites along the alpha-crystallin B chain sequence revealed that it possesses specific points for proteinase attack. These sites are particularly exposed and clustered in a very flexible region in the middle of the protein sequence. They are also well represented in the C-terminal extension of the molecule while a few are buried in the N-terminal region.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Effect of temperature on the propylamine transferase from Sulfolobus solfataricus, an extreme thermophilic archaebacterium. 1. Conformational behavior of the oligomeric enzyme in solution.

The effect of temperature on the molecular structure of propylamine transferase from Sulfolobus solfataricus has been investigated. Sulfolobus solfataricus is an extreme thermophilic archaebacterium with an optimum living condition at 90 degrees C. The enzyme is an oligomeric (trimer) protein of molecular mass 112 kDa. The frictional ratio for the native protein suggests an irregularly shaped compact globular structure. The protein matrix is well organized as suggested by far ultraviolet circular dichroism at 25 degrees C (18% alpha helix, 43% beta structure, 19% beta bends and 20% unordered: root mean square = 7). Structural effects of temperature were investigated over 25-85 degrees C. The protein retains its quaternary structure in this temperature range. A highly reversible subtle conformational transition was detected by numerous structure-dependent techniques over 40-50 degrees C, with a midpoint centered at 45 degrees C. Functional data also support this view. In fact, two enzyme forms, characterized by different catalytic properties, are present in solution. The Arrhenius plot suggests the occurrence of two different activation-energy-dependent processes, one at a temperature higher and one at a temperature lower than 45 degrees C. The transition has been considered as a molecular switch between two protein populations at equilibrium with different functional and structural properties, temperature modulated. A physiological role for the molecular switch has also been postulated. The protein also shows some subtle and reversible spectroscopic changes around 75 degrees C. The molecular basis of the thermophilic nature of this enzyme seems to reside in its capability to dynamically couple catalytic and structural events to the thermal properties of the ambient medium.

Amino Acids↗
PubMed1992

Effect of temperature on the propylamine transferase from Sulfolobus solfataricus, an extreme thermophilic archaebacterium. 2. Denaturation and structural stability.

The thermal stability of propylamine transferase from Sulfolobus solfataricus, an extreme thermophilic archaebacterium, has been characterized thermodynamically by a Van't Hoff analysis. Conformational transitions induced by guanidine hydrochloride, as well as by temperature, have been linked together in a scheme involving six equilibria, which arise from both dissociation and unfolding. The mechanism by which the protein achieves thermal stabilization is quite unusual. It is driven by a conformational equilibrium between two forms of different stability. The stability of each form towards denaturation is characterized by a specific temperature dependence. The low-temperature form, indicated as 'form A', is stable over 12-89 degrees C. Its stability maximum is 36.8 kJ/mol at 50 degrees C. 'Form B', which is populated at higher temperature, spans the interval 28-146 degrees C. Its stability maximum is 71.6 kJ/mol at 87 degrees C. A possible explanation for the mechanism underlying this behaviour is discussed assuming that two major terms contribute to stability, i.e. hydrophobic interactions arising from burying of the accessible surface residues as well as conformational entropy. The thermal stabilization of the enzyme seems to depend on effects related to both an overall increase of flexibility and a concomitant decrease of the area buried upon folding. In this regard proteins from extreme thermophilic organisms appear to be a useful model to shed new light on the general problem of protein stability.

Enzyme Stability↗
PubMed1990

Molecular organization and structural stability of beta s-crystallin from calf lens.

beta s-Crystallin has been purified to homogeneity. Its structural features and conformational behavior have been studied in solution. Protein secondary structure was estimated by curve fitting of far-UV circular dichroism spectra, which gave 16% alpha-helix, 45% beta-sheet, 12% bends, and 27% remainders. This result indicates that the structural organization of beta s-crystallin is reasonably similar to that of other beta and gamma family members. A comparison assessed between beta s- and gamma 2-crystallin by the use of predictive methods (flexibility and volume plots) reveals that the two proteins differ in respect to their local flexibility and packing, although they show similar overall organization. The interdomain and the C-terminal regions were found to be more flexible in beta s-crystallin. This finding can be explained by the presence of smaller amino acid residues within these structural districts. The location of one out of four tryptophans, i.e., Trp-162, in a flexible and exposed region of the protein was found to be the origin of the fluorescence heterogeneity. In fact, the fluorescence emission maximum of the native protein, centered at 328 nm, is due to two emitting centers, whose emission maxima are located at 323 and 330 nm, respectively, as evidenced by acrylamide quenching of fluorescence. The effect of perturbing agents, such as pH and guanidine hydrochloride, on the conformational behavior of beta s has also been evaluated by numerous spectroscopic techniques. The range of pH stability was between 6.5 and 8.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

FAST (Flexible Analysis by Software Tool) and CHAMP (CHemico-physical AMinoacidic Parameter data bank): a new tool to investigate protein structure.

A flexible package designed to study protein structure is described. The package is devoted to the analysis of protein sequences by drawing structural profiles of specific structure-related amino acid parameters. An Aminoacidic Parameters Data Bank (CHAMP) containing 32 different series of physico-chemical parameters of amino acids is available. Sequences can be loaded from any ASCII format data bank or from keyboard. The program possesses a routine which enables easy updating of the protein data bank and CHAMP Data Bank. FAST reads statistical correlations between two plots in order to identify structural similarities. Plots can be printed, saved or used for correlation, comparison or graph overlap by using common spreadsheets (e.g. Lotus 123). Plots can be smoothed by a running mean or a running median. The program also has a special feature--a global flexibility analysis of proteins. The package runs on IBM or compatibles and requires DOS 3.0 or later.

Amino Acids↗
PubMed1989

Flexibility plot of proteins.

The flexibility plot of a protein lies on the observation that amino acid residues with the highest turn potential, i.e. located in highly mobile regions of protein surface, also possess the smallest volumes as well as the lowest hydrophobicities. The plot is generated by shifting a five residue window along the protein sequence and calculating the value of the hydrophobicity-volume product for consecutive quintuplets of amino acid residues. The concomitant occurrence of small volumes and low hydrophobicities results in very deep minima. A threshold value has also been introduced in order to discriminate significant minima. To substantiate the interpretation that the selected minima actually indicate very flexible segments of a protein (loops, turns, etc.), we have compared plots obtained for model proteins (lysozyme, myoglobin, ribonuclease, trypsin, thermolysin and T4 lysozyme) with X-ray thermal factors profiles available for the same proteins. When compared to thermal profiles, the majority of flexible segments evidenced by our plots have been found to be in agreement with regions characterized by high thermal factors. Results have also been discussed in the light of local organization possessed by examined proteins.

Amino Acids↗
PubMed1988

Presence of nontryptophan fluorophores specifically bound to gamma 2-crystallin.

alpha-, beta-, and gamma-crystallins have been purified from nonpathological lenses of calves. The pure proteins have been examined for nontryptophan fluorescence and fluorescent compounds have been found specifically bound to gamma 2-crystallin. The protein has been unfolded by 6 M guanidine hydrochloride (Gdn-HCl) and a separation of the fluorescent compounds has been obtained by gel chromatography in the presence of 6 M Gdn-HCl. The spectroscopic features (absorbance, fluorescence) of the protein returned to normal following removal of the chromophores. The low-molecular-weight separated fluorescent compounds have been fractionated and extracted from the Gdn-HCl solution by ethyl acetate. TLC chromatography has shown the presence of kynurenine, 3-OH-kynurenine, and free tryptophan. These data suggest that direct involvement of the intrinsic protein tryptophans in the photochemical processes leading to formation of fluorescent compounds has to be excluded. Free tryptophan and intrinsic metabolic factors are probably more relevant in determining the cataractous insult.

Animals↗
PubMed1988

New graphic representation of structural parameters of proteins.

A computer program is described that is designed to make the visual inspection of classical plots of protein properties (e.g. hydrophobicity, volume, etc.) as a function of sequence easier. An algorithm written in BASIC language has been used in order to generate a pseudo-tridimensional representation of the desired protein property. The data utilized by the program are arithmetic averages of the selected parameter obtained by using a five-residue window as a shuttle along the given amino acid sequence.

Algorithms↗
PubMed1987

Unfolding pathway of myoglobin: effect of denaturants on solvent accessibility to tyrosyl residues detected by second-derivative spectroscopy.

The effects of denaturants on the solvent accessibility to tyrosyl residues of apomyoglobin have been examined by means of second-derivative spectroscopy in the near-ultraviolet. Three apomyoglobins, i.e., sperm whale, horse, and tuna, were selected because of the different distribution of tyrosyl residues in their primary structure. The results are consistent with the occurrence of two independent consecutive events in the guanidine-induced denaturation pattern of apomyoglobin. The first event, which is responsible for the lack of the ability to bind the heme, has been proved to involve conformational changes in both the domains, i.e., segments 1-79 and 80-153, identified in the myoglobin molecule. However, the conformational changes are not of the same type. In fact, the solvent accessibility to tyrosine HC2 is increased probably because of a partial unfolding of the 80-153 domain. Conversely, the solvent accessibility to tyrosine B2 is decreased, thus indicating that a refolding occurs in some region of the N-terminal moiety (1-79 domain) of the molecule.

Animals↗
PubMed1984

Determination of tyrosine exposure in proteins by second-derivative spectroscopy.

The mutual interference between the second-derivative bands of tyrosine and tryptophan in proteins has been evaluated in terms of the ratio r between two peak to peak distances. The r values have been found to be not only related to the tyrosine/tryptophan ratio but also dependent on the polarity of the medium in which tyrosyl residues are embedded. The results obtained on purified proteins have been found consistent with the available X-ray information and with the existing solvent perturbation data.

Acetylation↗
PubMedDate not supplied

Fluorescence studies on the binding between 1-47 fragment of cholecystokinin receptor CCK(A)-R(1-47) and nonsulfated cholecystokinin octapeptide CCK8.

The interaction between the 1-47 N-terminus fragment of the cholecystokinin receptor and the nonsulfated cholecystokinin octapeptide, CCK8, is monitored by fluorescence emission. Quenching of the fluorescence intensities is observed on binding. Dissociation constants calculated by these data are in the same submicromolar range as found for the binding of linear CCK8 analogues to B-type receptors. Although detailed structural information cannot be obtained, fluorescence emission is more sensitive than other techniques and permits fast detection of receptor-ligand interaction.

Amino Acid Sequence↗