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G Barone

Publications and source records attributed to G Barone.

At least 37 records · Page 2Linked to original sources
PubMed1999

The effects of polyols on the thermal stability of calf thymus DNA.

The effects on thermal denaturation of calf thymus DNA (ct-DNA) and its conformational changes induced by the presence in solution of different polyols, namely glycerol, i-erytritol, L( -- ) and D( + ) arabitol, D-mannitol, D-sorbitol and myo-inositol, have been investigated by means of differential scanning calorimetry (DSC) and circular dichroism (CD). By increasing the concentration of these additives a decrease in both the denaturation enthalpy (deltadH) and temperature of the maximum of the denaturation peak (Tmax) of DNA is observed. The values of these thermodynamic parameters depend on both the nature and concentration of the solute. The overall destabilization of DNA molecule has been related to the different capability of polyhydric alcohols to interact with the polynucleotide solvation sites replacing water and to the modification of the electrostatic interactions between the polynucleotide and its surrounding atmosphere of counterions. The particular behaviour of L( -- ) arabitol, which showed a much greater destabilizing ability compared to the other polyols, was further investigated and attributed to a direct more effective interaction with the double helix of DNA. CD spectra showed only a slight alteration of DNA-B structure in the presence of all the molecules here studied, except for L( -- ) arabitol where the DNA molecule seems to undergo a meaningful conformational change. The salt concentration dependence of DNA thermal stability in the presence of L( -- ) arabitol indicates a conformational change of polynucleotide towards a more extended conformation.

Animals↗
PubMed1999

The poor accuracy of indirect measurements of cadaveric donor kidney weights.

Reports that examined the issue of whether transplantation of inadequate nephron mass may be a risk factor for long-term allograft failure yielded conflicting results. One of the more accurate methods of estimating glomerular mass is kidney weight. Most of the clinical studies used body surface area (BSA) or kidney length as estimates of kidney weight. To test the hypothesis that indirect measures of kidney weight are accurate estimates of kidney weight, we compared the kidney weight of 41 consecutive cadaveric kidneys to donor BSA, dimensions measured with calipers at the time of transplantation, and dimensions supplied by the Organ Procurement Agency (OPA). Linear regression analysis was used with kidney weight as the dependent variable and BSA, kidney length, or kidney volume as the independent variable. Kidney length measured with calipers was also compared to kidney length supplied by the OPA. Kidney weight had the best correlation with kidney volume and kidney length determined by caliper measurements (r = 0.640 and 0.646, respectively). The regression analysis showed that the correlation of kidney weight with BSA was 0.487. The correlation of OPA-provided kidney length with kidney weights was poor (r = 0.410). The linear regression of caliper-measured kidney length versus OPA length yielded a slope of 0.360, instead of an ideal slope of 1. The assumption has been made that kidney weight or a surrogate of kidney weight has an excellent correlation with nephron mass. Some of the variability in studies that attempted to examine the effect of transplanted nephron mass on allograft outcome may be due to inaccurate estimates used for kidney weight. Our data suggest that surrogate measurements of kidney weight may not be accurate. We recommend that measured kidney weight should be used in studies examining the effect of donor renal mass on allograft outcomes.

Body Surface Area↗
PubMed1998

Guanidine-induced denaturation of beta-glycosidase from Sulfolobus solfataricus expressed in Escherichia coli.

Guanidine-induced denaturation of Sulfolobus solfataricus beta-glycosidase expressed in Escherichia coli, Sbetagly, was investigated at pH 6.5 and 25 degreesC by means of circular dichroism and fluorescence measurements. The process proved reversible when the protein concentration was lower than 0.01 mg mL-1. Moreover, the transition curves determined by fluorescence did not coincide with those determined by circular dichroism, and the GuHCl concentration corresponding at half-completion of the transition increased on raising the protein concentration in the range 0.001-0.1 mg mL-1. Gel filtration chromatography experiments showed that, in the range 2-4 M GuHCl, there was an equilibrium among tetrameric, dimeric, and monomeric species. These findings, unequivocally, indicated that the guanidine-induced denaturation of Sbetagly was not a two-state transition with concomitant unfolding and dissociation of the four subunits. A mechanism involving a dimeric intermediate species was proposed and was able to fit the experimental fluorescence intensity transition profiles, allowing the estimation of the total denaturation Gibbs energy change at 25 degreesC and pH 6.5. This figure, when normalized for the number of residues, showed that, at room temperature, Sbetagly has a stability similar to that of mesophilic proteins.

Chromatography, Gel↗
PubMed1998

Differential scanning calorimetry study of the thermodynamic stability of some mutants of Sso7d from Sulfolobus solfataricus.

Sso7d from the thermoacidophilic archaebacterium Sulfolobus solfataricus is a small globular protein with a known three-dimensional structure. Inspection of the structure reveals that Phe31 is a member of the aromatic cluster forming the protein hydrophobic core, whereas Trp23 is located on the protein surface and its side chain exposed to the solvent. The thermodynamic consequences of the substitution of these two residues in Sso7d have been investigated by comparing the temperature-induced denaturation of Sso7d with that of three mutants: F31A-Sso7d, F31Y-Sso7d, and W23A-Sso7d. The denaturation processes proved to be reversible for all proteins, and represented well by the two-state N if D transition model in a wide range of pH. All three mutants are less thermally stable than the parent protein; in particular, in the pH range of 5.0-7.0, the F31A substitution leads to a decrease of 24 degreesC in the denaturation temperature, the F31Y substitution to a decrease of 10 degreesC, and the W23A substitution to a decrease of 6 degreesC. A careful thermodynamic analysis of such experimental data is carried out.

Alanine↗
PubMed1998

Circular dichroism study of ribonuclease A mutants containing the minimal structural requirements for dimerization and swapping.

Four residues Pro19. Leu28, Cys31 and Cys32 proved to be the minimal structural requirements in determining the dimeric structure and the N-terminal segment swapping of bovine seminal ribonuclease, BS-RNase. We analyzed the content of secondary and tertiary structures in RNase A, P-RNase A, PL-RNase A, MCAM-PLCC-RNase A and MCAM-BS-RNase, performing near and far-UV CD spectra. It results that the five proteins have very similar native conformations. Thermal denaturation at pH 5.0 of the proteins. studied by means of CD measurements. proved reversible and well represented by the two-state N<==>D transition model. Thermodynamic data are discussed in the light of the structural information available for RNase A and BS-RNase.

Animals↗
PubMed1997

From ribonuclease A toward bovine seminal ribonuclease: a step by step thermodynamic analysis.

A proline, a leucine, and two cysteine residues, introduced at positions 19, 28, 31, and 32 of bovine pancreatic RNase A, i.e. the positions occupied by these residues in the subunit of bovine seminal RNase, the only dimeric RNase of the pancreatic-type superfamily, transform monomeric RNase A into a dimeric RNase, endowed with the same ability of BS-RNase of swapping its N-terminal segments. The thermodynamic consequences of the progressive introduction of these four residues into RNase A polypeptide chain have been studied by comparing the temperature- and urea-induced denaturation of three mutants of RNase A with that of a stable monomeric derivative of BS-RNase. The denaturation processes proved reversible for all proteins, and well represented by the two-state N<-->D transition model. The progressive introduction of the four residues into RNase A led to a gradual shift of the protein stability toward that characteristic of monomeric BS-RNase, which, in turn, is markedly less stable than RNase A with respect to both temperature- and urea-induced denaturation. On the other hand, the thermal stability of a dimeric active mutant of RNase A is found to approach that of wild-type seminal RNase.

Animals↗
PubMed1997

Effects of temperature and SDS on the structure of beta-glycosidase from the thermophilic archaeon Sulfolobus solfataricus.

The effects of temperature and SDS on the three-dimensional organization and secondary structure of beta-glycosidase from the thermophilic archaeon Sulfolobus solfataricus were investigated by CD, IR spectroscopy and differential scanning calorimetry. CD spectra in the near UV region showed that the detergent caused a remarkable change in the protein tertiary structure, and far-UV CD analysis revealed only a slight effect on secondary structure. Infrared spectroscopy showed that low concentrations of the detergent (up to 0.02%) induced slight changes in the enzyme secondary structure, whereas high concentrations caused the alpha-helix content to increase at high temperatures and prevented protein aggregation.

Bacterial Proteins↗
PubMed1997

Safety and tolerability of cyclosporine microemulsion versus cyclosporine: two-year data in primary renal allograft recipients: a report of the Neoral Study Group.

BACKGROUND: The new microemulsion formulation of cyclosporine (CsA-ME) is more bioavailable than cyclosporine (CsA) in de novo renal transplant patients. Therefore, it was of interest to compare the safety profile of each formulation in such patients. METHODS: In a multicenter, double-blind, parallel-group study, 101 renal transplant recipients were randomized after transplantation to receive either CsA (n=50) or CsA-ME (n=51) capsules twice daily for 2 years. Of these patients, 54 (CsA, n=26; CsA-ME, n=28) completed 1 year of the study and entered the second-year, double-blind extension. Initial dose at the time of transplantation was 5 mg/kg b.i.d.; doses were titrated to target trough levels. METHODS: The mean (+/- SD) doses at the end of 2 years were 4.6 +/- 1.8 and 3.8 +/- 1.1 mg/kg per day for CsA- and CsA-ME-treated patients, respectively. The mean (+/- SD) CsA trough levels at end point were 187 +/- 63 and 210 +/- 95 ng/ml for CsA- and CsA-ME-treated patients, respectively. At least one adverse event was reported by 25/26 (96%) of CsA- and 27/28 (96%) of CsA-ME-treated patients. No patient discontinued the study because of adverse events. No deaths occurred during the study. Renal function, as measured by serum creatinine levels, and blood pressure were comparable over time in both treatment groups. CONCLUSIONS: There was no significant difference in safety and tolerability between CsA- and CsA-ME-treated kidney recipients for 2 years after transplantation.

Adolescent↗
PubMed1997

Thermodynamic analysis of the effect of selective monodeamidation at asparagine 67 in ribonuclease A.

Selective deamidation of proteins and peptides is a reaction of great interest, both because it has a physiological role and because it can cause alteration in the biological activity, local folding, and overall stability of the protein. In order to evaluate the thermodynamic effects of this reaction in proteins, we investigated the temperature-induced denaturation of ribonuclease A derivatives in which asparagine 67 was selectively replaced by an aspartyl residue or an isoaspartyl residue, as a consequence of an in vitro deamidation reaction. Differential scanning calorimetry measurements were performed in the pH range 3.0-6.0, where the unfolding process is reversible, according to the reheating criterion used. It resulted that the monodeamidated forms have a different thermal stability with respect to the parent enzyme. In particular, the replacement of asparagine 67 with an isoaspartyl residue leads to a decrease of 6.3 degrees C of denaturation temperature and 65 kJ mol-1 of denaturation enthalpy at pH 5.0. These results are discussed and correlated to the X-ray three-dimensional structure of this derivative. The analysis leads to the conclusion that the difference in thermal stability between RNase A and (N67isoD)RNase A is due to enthalpic effects arising from the loss of two important hydrogen bonds in the loop containing residue 67, partially counterbalanced by entropic effects. Finally, the influence of cytidine-2'-monophosphate on the stability of the three ribonucleases at pH 5.0 is studied and explained in terms of its binding on the active site of ribonucleases. The analysis makes it possible to estimate the apparent binding constant and binding enthalpy for the three proteins.

Amides↗
PubMed1997

DSC studies on bovine serum albumin denaturation. Effects of ionic strength and SDS concentration.

This work analyzed the thermal denaturation process of defatted bovine serum albumin (BSA). DSC measurements were performed on changing the pH, the ionic strength and the sodium dodecyl sulfate (SDS) concentration. These data have been compared with those previously obtained by us and other authors. The purpose of these measurements was to study the correlation between the three-dimensional organization of BSA native protein structure and its thermodynamic stability and to clarify the non-covalent interactions between the globular proteins and amphipathic molecules. These measurements have shown that the thermal denaturation is always irreversible regardless of pH, ionic strength and SDS concentration. The nature of the irreversible process superimposed on the protein unfolding is discussed. The strong stabilizing effect of NaCl on the BSA native structure has been found for the range 0-1.0 M. It is worth noting that the calorimetric curves, confined to the pH region studied, could not be represented by a two-state transition model; they were deconvoluted as the sum of two independent two-state transitions. These transitions were correlated to the domain structure of BSA. Sodium dodecyl sulfate has a net stabilizing effect up to a molar ratio of 10:1 (ligand to protein). In this range of concentrations the presence of SDS cause a biphasic profile of excess heat capacity. A simple thermodynamic model was developed in attempt to reproduce the experimental DSC profiles and collect information regarding the binding equilibrium of SDS.

Binding Sites↗
PubMed1997

Interaction with D-glucose and thermal denaturation of yeast hexokinase B: A DSC study.

DSC measurements have been performed on the monomeric form of yeast hexokinase B in the absence and presence of increasing concentrations of D-glucose. The hexokinase, in the absence of D-glucose, at both pH 8.0 and 8.5, shows reproducible calorimetric profiles characterized by the presence of two partially overlapped peaks. These can be ascribed to the presence of two structural domains in the native conformation of the enzyme, that possess different thermal stabilities and are denatured more or less independently. In the presence of saturating and increasing concentrations of D-glucose, the shape of the DSC profiles dramatically changes, since a single well-shaped peak is present. The binding of D-glucose enhances the interaction between the two lobes, as evidenced by the shrinking of the protein in overall dimensions, and gives rise to DSC profiles resembling those of a single domain protein. To deconvolve the DSC curves we considered a denaturation model consisting of two sequential steps with three macroscopic states of the protein and the binding of D-glucose only to the native state. We carried out two-dimensional nonlinear regression of the excess heat capacity surface constructed with the experimental DSC curves. This approach allows the calculation of a unique set of thermodynamic parameters characterizing both the thermal denaturation of hexokinase, and the binding equilibrium between D-glucose and the enzyme. It was found that the association constant is 9,800+/-1,500 M(-1) at pH 8.0. The binding of D-glucose is entropy-driven, since the binding enthalpy is zero. This finding is rationalized by a thermodynamic cycle for the association of two molecules in aqueous solution.

Calorimetry, Differential Scanning↗
PubMed1997

A reassessment of the molecular origin of cold denaturation.

The existence of cold denaturation is now firmly demonstrated by its direct observation for several globular proteins in aqueous solution. But the physico-chemical explanation of this intriguing phenomenon is still unsatisfactory. In this paper we deepen our understanding of cold denaturation by taking advantage of the theoretical model developed by Ikegami and using thermodynamic data on the transfer to water of liquid N-alkyl amides. The analysis leads to the conclusion that the presence of water is fundamental to determine the existence of cold denaturation due to its strong energetic interaction with the amino acid residues previously buried in the protein's interior.

Cold Temperature↗
PubMed1996

Temperature-induced denaturation of ribonuclease S: a thermodynamic study.

In this paper the thermal denaturation of ribonuclease S, the product of mild digestion of ribonuclease A by subtilisin, is deeply investigated by means of DSC and CD measurements. It results that at whatever pH in the range 4-7.5 the process if fully reversible but not well represented by the simple two-state N<-->D transition. Actually, a two-state model that considers both unfolding and dissociation, NL<-->D + L*, well accounts for the main features of the process: the tail present in the low-temperature side of DSC peaks and the marked dependence of denaturation temperature on protein concentration. This mechanism is strictly linked to the exact stoichiometry of RNase S. An excess of the protein component of RNase S, the so-called S-protein, shifts the system toward a more complex behavior, that deserves a separate treatment in the accompanying paper [Graziano, G., Catanzano, F., Giancola, C., & Barone, G. (1996) Biochemistry 35, 13386-13392]. The thermodynamic analysis leads to the conclusion that the difference in thermal stability between RNase S and RNase A is due to entropic effects, i.e., a greater conformational flexibility of both backbone and side chains in RNase S. The process becomes irreversible at pH 8.0-8.5, probably due to side-reactions occurring at high temperature. Finally, the influence of phosphate ion on the stability of RNase A and RNase S at pH 7.0 is studied and explained in terms of its binding on the active site of ribonuclease. The analysis enables us to obtain an estimate of the apparent association constant and binding enthalpy also.

Binding Sites↗
PubMed1996

The pharmacokinetics of a microemulsion formulation of cyclosporine in primary renal allograft recipients. The Neoral Study Group.

This study was a randomized, double-blind, 12-week comparison of the pharmacokinetics, safety, and tolerability of two cyclosporine (CsA) formulations, cyclosporine emulsion capsules and oral solution for microemulsion and cyclosporine, in the postoperative management of renal transplant patients. Of the 101 patients, aged 18 to 65, who entered the study, 89 were evaluable for pharmacokinetics. Initial dosage was 10 mg/kg per day, administered twice daily in two equal doses. Dosages were adjusted to achieve target CsA concentrations. The pharmacokinetic (PK) parameters (dose-normalized) of greatest interest were maximum blood concentration (C(max)/dose), time to reach maximum concentration (t(max), area under the blood concentration-vs.-time curve (AUC/dose), and trough blood concentrations (Co h/dose). The relative CsA bioavailabilty was found to be significantly enhanced with cyclosporine emulsion compared with cyclosporine with a 16% to 31% increase in AUC and a 32% to 42% increase in C(max). Intrapatient variability of PK parameters was significantly lower with cyclosporine emulsion than with cyclosporine for AUC, C(oh), t(max), and C(max) in many instances. This indicates a more consistent, rapid, and more complete total absorption of CsA. Despite higher CsA C(max) levels and AUCs with cyclosporine emulsion, safety and tolerability (detailed in a parallel report) were comparable to those of cyclosporine. The PK advantages of cyclosporine emulsion over cyclosporine are either independent of food conditions or possibly reflective of more consistent absorption of CsA with cyclosporine emulsion. The findings suggest that de novo use of cyclosporine emulsion may simplify and improve management of organ transplant recipients and that the PK advantages of cyclosporine emulsion may translate into clinical benefits.

Adolescent↗
PubMed1996

The safety and tolerability of cyclosporine emulsion versus cyclosporine in a randomized, double-blind comparison in primary renal allograft recipients. The Neoral Study Group.

A 12-week, randomized, double-blind, multicenter pharmacokinetics study was conducted to compare the clinical safety and tolerability of cyclosporine capsules and oral solution for microemulsion and cyclosporine in 101 primary renal transplant recipients Cyclosporine emulsion has more complete absorption and improved bioavailability compared with cyclosporine, and dosing of both cyclosporine formulations was adjusted to achieve comparable whole-blood trough levels. Mean serum creatinine values were higher in the cyclosporine emulsion group at baseline, 8, and 12 weeks (P<0.05). The incidence of acute rejection was similar in both treatment groups although fewer patients required monoclonal antibody therapy in the cyclosporine group (31% vs. 82%, respectively). Despite the increased bioavailability of cyclosporine emulsion, no significant differences in the incidence of adverse events were observed; the safety, tolerability, and efficacy of cyclosporine emulsion and cyclosporine were comparable.

Administration, Oral↗
PubMed1996

Structural investigation of the polysaccharide fraction from the mucilage of Diceroaryum zanguebaricum Merr.

The polysaccharide fraction from the mucilage of Dicerocaryum zanguebaricum (Pedaliaceae) appears to be mainly constituted of a chemically homogeneous polysaccharide. By NMR and chemical degradative methods its structure appeared to consist of alternate-->4)-beta-D-GlcpA-(1--> and -->2)-alpha-D-Man p-(1-->units. Single branch units of beta-D-Xyl p and alpha-D-Gal p are linked to the O-3 positions of Man p and a significant number of Glc pA residues.

Africa↗