Update on treatment of lysosomal storage diseases.

Lysosomal storage diseases (LSDs) are a large group of disorders caused by a deficiency of specific enzymes responsible for the degradation of substances present in lysosomes. In the past few years, treatments for LSDs were non specific and could only cope with signs and symptoms of the diseases. A successful therapeutic approach to LSDs should instead address to the underlying causes of the diseases, thus helping the degradation of the accumulated metabolites in the various organs, and at the same time preventing their further deposition. One way is to see to an available source of the deficient enzyme: bone marrow transplantation, enzyme replacement therapy and gene therapy are based on this rationale. The purpose of substrate reduction therapy is to down regulate the formation of the lysosomal substance to a rate at which the residual enzyme activity can catabolize the stored and de novo produced lysosomal substrate. Chemical chaperone therapy is based on small molecules able to bind and stabilize the misfolded enzymes. This paper offers a historical overview on the therapeutic strategies for LSDs.

Enthalpic and entropic contributions to the mutational changes in the reduction potential of azurin.

The changes in the reduction potential of Pseudomonas aeruginosa and Alcaligenes denitrificans azurins following point mutations and residue ionizations were factorized into the enthalpic and entropic contributions through variable temperature direct electrochemistry experiments. The effects on the reduction enthalpy due to changes in the first coordination sphere of the copper ion, as in the Met121Gln and Met121His variants of A. denitrificans azurin, insertion of a net charge and alteration in the solvation properties and electrostatic potential in proximity of the metal site, as in the Met44Lys and His35Leu variants of P. aeruginosa azurin, respectively, and proton uptake/release in wild-type and mutated species could invariably be accounted for on the basis of simple coordination chemistry and/or electrostatic considerations. The concomitant changes in reduction entropy were found in general to contribute to the E degrees ' variation to a lesser extent as compared to the enthalpy changes. However, their effects were by no means negligible and in some instances were found to heavily contribute to (or even become the main determinant of) the observed change in reduction potential. Several lines of evidence indicate that the entropic effects are notably influenced by reduction-induced solvent reorganization effects. In particular, protein reduction tends to be favored on entropic grounds with increasing exposure of the copper site to the solvent. Moreover, enthalpy-entropy compensation phenomena are invariably observed when residue mutation or pH-induced conformational changes modify the solvent accessibility of the metal site or alter the H-bonding network in the hydration shell of the molecule. Therefore, in these cases, caution must be used in making predictions of E degrees ' changes simply based on Coulombic or coordination chemistry arguments.

Control of metalloprotein reduction potential: the role of electrostatic and solvation effects probed on plastocyanin mutants.

The changes in the thermodynamics of Cu(II) reduction for spinach plastocyanin induced by point mutations altering the electrostatic potential in proximity of the copper center were determined through variable temperature direct electrochemistry experiments. In particular, the functionally important surface residues Leu12 and Gln88 were replaced with charged and polar residues, and Asn38 was substituted with Asp. The mutational variations of the reduction enthalpy and entropy were analyzed with a QSPR (quantitative structure-property relationships) approach, employing global and local theoretical descriptors defined and computed on the three-dimensional protein structure. The correlations found are informative on how electrostatic and solvation effects control the E degrees ' values in this species through the combined effects on the reduction enthalpy and entropy. The changes in reduction enthalpy can be justified with electrostatic considerations. Most notably, enthalpy-entropy compensation phenomena play a significant role: the entropic effects due to the insertion of charged residues determine E degrees ' changes that are invariably opposite to those induced by the concomitant enthalpic effects. Therefore, the resulting E degrees ' changes are small or even opposite to those expected on simple electrostatic grounds. The mutational variation in the reduction entropy appears to be linked to the hydrogen bonding donor/acceptor character of the northern part of the protein, above the metal site, and to the electrostatic potential distribution around the copper site. Both properties influence the reduction-induced reorganization of the water molecules on the protein surface in the same region.

Isolation and characterization of two peroxidases from Cucumis sativus.

Two heme peroxidases of 35.2 and 36.5 kDa have been isolated from cucumber (Cucumis sativus) peelings and characterized through electronic and 1H NMR spectra in the pH range 3.5-10.5. Their spectroscopic and catalytic properties, which are closely similar, are characteristic of highly homologous isoenzymes. Both proteins, as isolated, exist as a mixture of two ferric forms containing a high-spin and a low-spin heme in an approximately 2:1 molar ratio. The latter form likely contains a hydroxide ion axially coordinated to the heme iron and is proposed to be the result of partial irreversible protein inactivation due to the purification procedure. Both proteins in the reduced form are fully high-spin. The high-spin ferric form is sensitive to two acid-base equilibria with apparent pKa values of approximately 5 and 8.5, which have been assigned to the distal histidine and the arginine adjacent to it, respectively. These equilibria also affect the catalytic activity and the interaction with inorganic anions such as azide and fluoride. The reactivity of both proteins is closely similar to that of other plant peroxidases, primarily horseradish peroxidase; however, they also show spectroscopic properties similar to those of cytosolic ascorbate peroxidase. Therefore, overall, these two species show molecular, spectroscopic and catalytic features which are rather peculiar among plant peroxidases.

Redox properties and acid-base equilibria of zucchini mavicyanin.

The reduction potential of mavicyanin isolated from zucchini peelings, which is a blue copper protein belonging to the subclass of the phytocyanins, has been determined through direct electrochemistry as a function of temperature and pH. The enthalpy and entropy changes accompanying protein reduction were found to be very similar with those determined previously for other phytocyanins and to differ remarkably from those of azurins and plastocyanins. This finding contributes to further characterize phytocyanins as a distinct cupredoxins family also on thermodynamic grounds and improves our understanding of how the reduction potential of these metal centers in proteins is modulated by coordinative and solvation properties. The E degrees' of mavicyanin is found to be sensitive to two acid-base equilibria at the extremes of pH. One occurs below pH 4, and is related to the protonation and detachment from the Cu(I) center of a histidine ligand. The other, observed above pH 8, causes a remarkable change in the electrostatic potential and/or the field strength around the copper.

Effects of nonspecific ion-protein interactions on the redox chemistry of cytochrome c.

The effects of the ionic atmosphere on the enthalpic and entropic contributions to the reduction potential of native (state III) beef heart cytochrome c have been determined through variable-temperature direct electrochemistry experiments. At neutral or slightly alkaline pH values, from 5 to 50 degrees C, the reduction enthalpy and entropy become less negative with decreasing ionic strength. The reduction entropy extrapolated at null ionic strength is approximately zero, indicating that, in the absence of the screening effects of the salt ions on the network of the electrostatic interactions at the protein-solvent interface, the solvation properties and the conformational flexibility of the two redox states are comparable. The moderate decrease in E degrees' observed with increasing ionic strength [DeltaE degrees'IS = (E degrees')I = 0.1 M - (E degrees')I = (0)M = -0.035 V at 25 degrees C], once the compensating enthalpic and entropic effects of the salt-induced changes in the hydrogen bonding within the hydration sphere of the molecule in the two redox states are factored out, results in being ultimately determined by the stabilizing enthalpic effect of the negatively charged ionic atmosphere on the ferri form. At pH 9, the ionic strength dependence of the reduction termodynamics of cytochrome c follows distinctive patterns, possibly as a result of specific binding of the hydroxide ion to the protein. A decrease in ionic strength at constant pH, as well as a pH increase at constant ionic strength, induces a depression of the temperature of the transition from the low-T to high-T conformer of cytochrome c, which suggests that a temperature-induced decrease in the pK(a) for a residue deprotonation is the key event of this conformational change.

Thermodynamics of the alkaline transition of cytochrome c.

The apparent equilibrium constant (Kapp) of the alkaline transition (AT) of beef heart cytochrome c, obtained from pH titrations of the current intensities in cyclic voltammetry experiments, has been measured as a function of the temperature from 5 to 65 degrees C, at different ionic strength (I = 0.01-0.2 M). The temperature profile of the pKapp values is biphasic and yields two distinct sets of DeltaH degrees 'AT and DeltaS degrees 'AT values below and above approximately 40 degrees C. In the low-temperature range, the process is endothermic and is accompanied by a small positive entropy change, while at higher temperatures it becomes less endothermic and involves a pronounced entropy loss. The temperature dependence of the transition thermodynamics is most likely the result of the thermal transition of native ferricytochrome c from a low-T to an high-T conformer which occurs at alkaline pH values at a temperature comparable with above (Ikeshoji, T., Taniguchi, I., and Hawkridge, F. M. (1989) J. Electroanal. Chem. 270, 297-308; Battistuzzi, G., Borsari, M., Sola, M., and Francia, F. (1997) Biochemistry 36, 16247-16258). Thus, it is apparent that the transitions of the two native conformers to the corresponding alkaline form(s) are thermodynamically distinct processes. It is suggested that this difference arises from either peculiar transition-induced changes in the hydration sphere of the protein or to the preferential binding of different lysines to the heme iron in the two temperature ranges. Extrapolation of the Kapp values at null ionic strength allowed the determination of the thermodynamic equilibrium constants (Ka) at each temperature, hence of the "true" standard thermodynamic parameters of the transition. The pKa value at 25 degrees C was found to be 8.0. A pKapp value of 14.4 was calculated for the alkaline transition of ferrocytochrome c at 25 degrees C and I = 0.1 M. The much greater relative stabilization of the native state in the reduced as compared to the oxidized form turns out to be almost entirely enthalpic in origin, and is most likely due to the greater affinity of the methionine sulfur for the Fe(II) ion. Finally, it is found that the Debye-Hückel theory fits the ionic strength dependence of the pKapp values, at least qualitatively, as observed previously for the ionic strength dependence of the reduction potential of this protein class. It is apparent that the increase in the pKapp values with increasing ionic strength is for the most part an entropic effect.

Redox chemistry and acid-base equilibria of mitochondrial plant cytochromes c.

Mitochondrial cytochromes c from spinach, cucumber, and sweet potato have been investigated through direct electrochemical measurements and electronic and 1H NMR spectroscopies, under conditions of varying temperature and pH. The solution behaviors of these plant cytochromes closely resemble, but do not fully reproduce, those of homologous eukaryotic species. The reduction potentials (E0') at pH 7 and 25 degrees C are +0.268 V (spinach), +0.271 V (cucumber), and +0.274 V (sweet potato) vs SHE. Three acid-base equilibria have been determined for the oxidized proteins with apparent pKa values of 2.5, 4.8, and 8.3-8.9, which are related to disruption of axial heme ligation, deprotonation of the solvent-exposed heme propionate-7 and replacement of the methionine axially bound to the heme iron with a stronger ligand, respectively. The most significant peculiarities with respect to the mammalian analogues include: (i) less negative reduction enthalpies and entropies (Delta S0'rc and Delta H0'rc) for the various protein conformers [low- and high-T native (N1 and N2) and alkaline (A)], whose effects at pH 7 and 25 degrees C largely compensate to produce E degrees ' values very similar to those of the mammalian proteins; (ii) the N1 --> N2 transition that occurs at a lower temperature (e.g., 30-35 degrees C vs 50 degrees C at pH 7. 5) and at a lower pH (7 vs 7.5); and (iii) a more pronounced temperature-induced decrease in the pKa for the alkaline transition which allows observation of the alkaline conformer(s) at pH values as low as 7 upon increasing the temperature above 40 degrees C. Regarding the pH and the temperature ranges of existence of the various protein conformers, these plant cytochromes c are closer to bacterial cytochromes c2.

Evaluation of changing patterns in children poisonings and prevention.

Poisoning prevention strategies have to be modified according to the risk variations with age and time. Epidemiologic surveillance usefulness was confirmed by studying the 0-15 y-old children in the Trieste area from 1976-1987. Significant differences were observed in children under 5 due to an increase in caustic substance and home detergents poisoning and a decrease in analgesic-antipyretics ingestion. This was probably due to the introduction by law in our country of child-resistant containers and to a better awareness by parents. At 10 y-of-age and older, alcohol is the cause of most intoxications and more recently benzodiazepine poisonings have significantly increased. At this age poisoning results from deliberate ingestion in most cases; therefore education should be the main preventive strategy.

[Traffic-related injuries in children at Trieste, 1984-1986]. / Lesioni da traffico nel bambino a Trieste 1984-86.

Traffic-related injuries to children are poorly known in our country, in spite of their role in mortality and morbidity. Being that they are rather different from those reported in the adults, their study is useful in identifying risks and elaborating prevention strategies. This paper reports epidemiologic and clinical data on 1050 children under 16, referred to our hospital because of road traffic accidents. The study has been prospectively carried out during 1984-86; injuries severity was assessed according to the Abbreviated Injury Scale. Incidence rates resulted to increase with the age, highest values being observed for 15 year old children, due to motorcycle use. Patterns and severity of the injuries were significantly different according to the age and the group of injured subjects. Younger children as pedestrians and 14-15 y.o. motorcyclists resulted to be more involved than car passengers and bicyclists. At a telephone inquiry after 6-12 months sequelae resulted in 20-40% out of 482 children, according to different AIS Score.

High-performance liquid chromatographic determination of the mucoregulatory drug CO/1408 in rat plasma and urine.

A sensitive and selective high-performance liquid chromatographic (HPLC) method was developed for the determination of the mucoregulatory drug CO/1408 in plasma and urine. Samples containing an internal standard were prepared for analysis using a simple clean-up procedure based on Extrelut solid-phase extraction and chromatographed using a reversed-phase analytical column. Isocratic elution with a mobile phase consisting of 25 mM phosphate buffer (pH 2.5)-acetonitrile-methanol [85:10:5 or 87:9:4 (v/v) for plasma or urine analysis, respectively] was effected at a flow-rate of 0.8 ml/min. The eluate was monitored with an ultraviolet-visible variable-wavelength detector at 200 nm. The limit of quantification for the assay of CO/1408 was 80 ng/ml for plasma and 1 per 0.1 ml for urine samples. In spite of the high solubility of CO/1408 in water, the recovery from plasma and urine was very good and reproducible. The method was found to be applicable to pharmacokinetic studies of the drug in the rat.

Capillary gas chromatographic determination of epomediol in human plasma and urine using a rapid solid-phase extraction.

A simple and precise method for the quantitation of epomediol in human plasma and urine is described. Each biological sample is added with the internal standard and applied directly to an Extrelut-1 solid-phase column. After absorption the column is eluted with chloroform and the eluate is evaporated to dryness. The residue, reconstituted in ethanol, is analysed by capillary gas chromatography. No interferences from possible metabolites or endogenous constituents can be noted. The method has been applied to human pharmacokinetic studies: the results of a subacute administration to volunteers are presented.

[Evaluation of the usefulness of the motorcycle helmet in adolescents in Trieste]. / Valutazione dell'efficacia del casco da motociclista nell'adolescente triestino.

The effectiveness of the motorcycle helmet was assessed in 359 accidents involving adolescents under 16 in the Trieste area in 1985-88. Comparison of the 18 months preceding and following the introduction of the law revealed that helmet use significantly reduced the number and gravity (as objectively assessed using the Abbreviated Injury Scale) of cranial and facial injuries but made no significant difference to other areas. The 70-80% reduction in head injuries also significantly reduced the incidence of multiple injuries and hospital admissions. The effectiveness of the helmet demonstrated in this study primarily concerned with motorcycle accidents in urban areas suggests that it should now also be made obligatory for those currently exempt.

High-performance liquid chromatographic assay of nesosteine in human plasma and urine.

A high-performance liquid chromatographic method for the quantitation of the mucoactive drug sodium 2-(3-thiazolidinylcarbonyl)benzoate in plasma and urine was developed, involving liquid-liquid extraction under acidic conditions and reversed-phase chromatography using ultraviolet detection at 210 or 235 nm. The extraction efficiency, linearity, limit of detection, precision and accuracy were determined. Products of the main biotransformation of the drug, involving hydrolysis of the amidic bond, do not interfere. The method is selective, precise, reproducible and applicable to studies of the pharmacokinetic behaviour of the drug in humans. Pharmacokinetic parameters derived from six healthy subjects following acute intravenous administration of the drug (150 mg) are presented.

Repeated rescue of a Broviac catheter: fixation of exposed cuff by a skin flap.

A cutaneous infection exposed the cuff of a Broviac catheter employed for home-TPN in a 3-month-old child with ultra-short bowel syndrome. In order to avoid removal of the catheter, sepsis was abated by antibiotic administration through its lumen, then the exposed cuff was covered and fixed by a skin flap. The advantages proceeding from this sort of "emergency rescue" of the Broviac catheter have been: (1) to avoid a new cutdown in a child already submitted to several attempts of cannulation with sacrifice of major vessels; (2) to resume home total parenteral nutrition (TPN) in a short time, being the patient strictly dependent upon his parenteral intake and to spare a well-functioning catheter. Ten months after the last cuff covering by skin flap, the catheter is safely fixed in place and currently employed for home TPN.

Quantitation of hexaprazol in human plasma and urine by capillary gas chromatography with nitrogen-sensitive detection.

A selective and sensitive gas chromatographic assay for hexaprazol, a new antiulcer drug, in human plasma and urine has been developed. The method involves liquid-liquid extraction and capillary gas chromatography with nitrogen-sensitive detection. The limit of quantitation of plasma hexaprazol is ca. 25 ng/ml. The assay procedure permits the measurement of the levels of the unchanged drug following its clinical administration to humans.