Biomedical subjects

M Stefani

Publications and source records attributed to M Stefani.

At least 37 records · Page 2Linked to original sources

PubMed1999

Determinants of psychiatric inpatient admission to general hospital psychiatric wards: an epidemiological study in a region of central Italy.

OBJECTIVE: To determine which factors contribute to the decision to admit individuals to psychiatric wards in general hospitals. METHOD: Data on 1,379 individuals undergoing psychiatric evaluation in eight emergency rooms in a region of central Italy were collected. A logistic regression analysis was used to evaluate the likelihood of psychiatric admission considering the independent effects of demographic, social, and clinical factors and of the history of psychiatric treatment. RESULTS: The adjusted odds ratio for psychiatric admission significantly increased with the following variables: severity of symptoms; presence of paranoid states and schizophrenic psychoses, affective psychoses and acute psychotic conditions (with neurotic disorders used as reference); a history of outpatient treatment; the presence of a staff member of a community mental health facility upon presentation at the emergency room; and the availability of beds in the psychiatric ward. CONCLUSION: The independent effect played by the presence of a staff member of a community mental health facility is of particular interest, suggesting the existence of a collaborative relationship between inpatient and outpatient services.

Adult↗

PubMed1999

Mutational analysis of acylphosphatase suggests the importance of topology and contact order in protein folding.

Muscle acylphosphatase (AcP) is a small protein that folds very slowly with two-state behavior. The conformational stability and the rates of folding and unfolding have been determined for a number of mutants of AcP in order to characterize the structure of the folding transition state. The results show that the transition state is an expanded version of the native protein, where most of the native interactions are partially established. The transition state of AcP turns out to be remarkably similar in structure to that of the activation domain of procarboxypeptidase A2 (ADA2h), a protein having the same overall topology but sharing only 13% sequence identity with AcP. This suggests that transition states are conserved between proteins with the same native fold. Comparison of the rates of folding of AcP and four other proteins with the same topology, including ADA2h, supports the concept that the average distance in sequence between interacting residues (that is, the contact order) is an important determinant of the rate of protein folding.

Acid Anhydride Hydrolases↗

PubMed1998

Structural characterization of the transition state for folding of muscle acylphosphatase.

The transition state for folding of a small protein, muscle acylphosphatase, has been studied by measuring the rates of folding and unfolding under a variety of solvent conditions. A strong dependence of the folding rate on the concentration of urea suggests the occurrence in the transition state of a large shielding of those groups that are exposed to interaction with the denaturant in the unfolded state (mainly hydrophobic moieties and groups located on the polypeptide backbone). The heat capacity change upon moving from the unfolded state to the transition state is small and is indicative of a substantial solvent exposure of hydrophobic groups. The solvent-accessibility of such groups in the transition state has also been found to be significant by measuring the rates of folding and unfolding in the presence of sugars. These rates have also been found to be accelerated by the addition of small quantities of alcohols. Trifluoroethanol and hexafluoroisopropanol were particularly effective, suggesting that stabilisation of local hydrogen bonds lowers the energy of the transition state relative to the folded and unfolded states. Finally, a study with a competitive inhibitor of acylphosphatase has provided evidence for the complete loss of ligand binding affinity in the transition state, indicating that specific long-range interactions at the level of the active site are not yet formed at this stage of the folding reaction. A model of the transition state for acylphosphatase folding, in which beta-turns and one or both alpha-helices are formed to a significant extent but in which the persistent long-range interactions characteristic of the folded state are largely absent, accounts for all our data. These results are broadly consistent with models of the transition states for folding of other small proteins derived from mutagenesis studies, and suggest that solvent perturbation methods can provide complementary information about the transition region of the energy surfaces for protein folding.

Acid Anhydride Hydrolases↗

PubMed1998

Quinazolines: combined type 3 and 4 phosphodiesterase inhibitors.

A series of quinazolines has been prepared and evaluated for its ability to inhibit cyclic AMP phosphodiesterase type 3, type 4A, 4B and 4D. The most potent inhibitors showed IC50 values in the nanomolar range for type 3 and type 4 isoforms and bind with high affinity to the [3H]rolipram binding site. These quinazolines represent a new family of potent mixed PDE 3/4 inhibitors and are expected to have a therapeutic potential.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1998

Expression, purification and preliminary crystal analysis of the human low Mr phosphotyrosine protein phosphatase isoform 1.

The genes of the human low Mr phosphotyrosine protein phosphatase (PTPase) isoforms 1 (IF1) and 2 (IF2) were isolated by screening a human placenta cDNA library, cloned in pGEX and expressed in E. coli as fusion proteins with glutathione S-transferase. The recombinant proteins were purified by a rapid one-step procedure allowing each enzyme to purify with high final yield and specific activity. This result is important for IF1, whose purification from natural sources is difficult, due to precipitation propensity, thus hindering structural studies. The enzymes obtained showed kinetic parameters very similar to those previously determined for the enzymes purified by classical procedures from both human erythrocytes and rat liver. These recombinant enzymes can therefore be used in place of those purified from natural sources for every purpose. IF1 and IF2 crystals were also grown. IF1 crystals were X-ray-grade, diffracted to better than 2.4 A and were suitable for high resolution X-ray structure determination.

Cloning, Molecular↗

PubMed1998

Conformational stability of muscle acylphosphatase: the role of temperature, denaturant concentration, and pH.

The conformational stability (delta G) of muscle acylphosphatase, a small alpha/beta globular protein, has been determined as a function of temperature, urea concentration, and pH. A combination of thermally induced and urea-induced unfolding, monitored by far-UV circular dichroism, was used to define the conformational stability over a wide range of temperature. Through analysis of all these data, the heat capacity change upon unfolding (delta Cp) could be estimated, allowing the determination of the temperature dependence of the main thermodynamic functions (delta G, delta H, delta S). Thermal unfolding in the presence of urea made it possible to extend such thermodynamic analysis to examine these parameters as a function of urea concentration. The results indicate that acylphosphatase is a relatively unstable protein with a delta G(H2O) of 22 +/- 1 kJ mol-1 at pH 7 and 25 degrees C. The midpoints of both thermal and chemical denaturation are also relatively low. Urea denaturation curves over the pH range 2-12 have allowed the pH dependence of delta G to be determined and indicate that the maximum stability of the protein occurs near pH 5.5. While the dependence of delta G on urea (the m value) does not vary with temperature, a significant increase has been found at low pH values, suggesting that the overall dimensions of the unfolded state are significantly affected by the number of charges within the polypeptide chain. The comparison of these data with those from other small proteins indicates that the pattern of conformational stability is defined by individual sequences and not by the overall structural fold.

Acid Anhydride Hydrolases↗

PubMed1998

Sequence-specific recognition of peptide substrates by the low Mr phosphotyrosine protein phosphatase isoforms.

A number of phosphotyrosine-containing peptides derived from the PDGF receptor phosphorylation sites have been synthesised. The peptides were assayed as substrates of the two isoforms (IF1 and IF2) of the low Mr PTPase. The calculated k(cat), Km, and k(cat)/Km values indicate that only one peptide is best hydrolysed by IF2 (but not IF1), whose catalytic efficiency averages those previously reported for most PTPases (except the Yersinia enzyme). This peptide is the only one containing a couple of no bulky hydrophobic residues at the phosphotyrosine N-side. The determination of the same catalytic parameters in the presence of analogues of the best hydrolysed peptide in which one or both hydrophobic residues were replaced by Asp or Lys residues confirmed the importance of the hydrophobic cluster at the phosphotyrosine N-side for optimal enzymatic hydrolysis. These findings are discussed in the light of the known IF2 X-ray structure.

Amino Acid Sequence↗

PubMed1998

Studies on enzymatic activity and conformational stability of muscle acylphosphatase mutated at conserved lysine residues.

An oligonucleotide-directed mutagenesis study was carried out on the five acylphosphatase conserved lysine residues to assess their possible participation in enzyme active site formation and their contribution to the enzyme conformational stability. The study was designed to eliminate the ambiguity arising from the presence of a sulfate ion, an enzyme competitive inhibitor, bound to lysine 32 and 68 in the crystal structure of the erythrocyte isoenzyme. Furthermore, previous kinetic studies suggested the presence of residues with pKa=7.9 and 11, tentatively identified as two lysines. The kinetic parameters for the mutants under investigation are not significantly different from those of the wild-type enzyme, demonstrating that none of the lysine residues are involved in catalysis or in substrate binding. In addition, thermal and urea denaturation experiments performed by circular dichroism indicate that the mutated lysine residues do not play a significant role in the enzyme structural stabilization, as the destabilizing energy averages 1.40 kJ/mol. Such results are in agreement with those obtained with other proteins indicating that lysine residues make little contribution to the stability of the native structure.

Acid Anhydride Hydrolases↗

PubMed1997

Structure and function of the low Mr phosphotyrosine protein phosphatases.

Phosphotyrosine protein phosphatases (PTPases) catalyse the hydrolysis of phosphotyrosine residues in proteins and are hence implicated in the complex mechanism of the control of cell proliferation and differentiation. The low Mr PTPases are a group of soluble PTPases displaying a reduced molecular mass; in addition, a group of low molecular mass dual specificity (ds)PTPases which hydrolyse phosphotyrosine and phosphoserine/threonine residues in proteins are known. The enzymes belonging to the two groups are unrelated to each other and to other PTPase classes except for the presence of a CXXXXXRS/T sequence motif containing some of the catalytic residues (active site signature) and for the common catalytic mechanism, clearly indicating convergent evolution. The low Mr PTPases have a long evolutionary history since microbial (prokaryotic and eukaryotic) counterparts of both tyrosine-specific and dsPTPases have been described. Despite the relevant number of data reported on the structural and catalytic features of a number of low Mr PTPases, only limited information is presently available on the substrate specificity and the true biological roles of these enzymes, in prokaryotic, yeast and eukaryotic cells.

Amino Acid Sequence↗

PubMed1997

Structural and kinetic investigations on the 15-21 and 42-45 loops of muscle acylphosphatase: evidence for their involvement in enzyme catalysis and conformational stabilization.

The structural and catalytic importance of the 15-21 and 42-45 loop residues of the acylphosphatase muscular isoenzyme has been investigated by oligonucleotide-directed mutagenesis. Seven mutants involving conserved residues of the two loops have been prepared and characterized for structural, kinetic, and stability features by using different spectroscopic techniques and compared to the wild-type enzyme. The results are discussed in light of the crystal structure of the highly homologous common type acylphosphatase [Thunnissen et al. (1997) Structure 5, 69-79]. A differential role of the two loops has emerged: the 15-21 and the 42-45 loops appear mainly involved in active site formation and enzyme structural stabilization, respectively. These conclusions are supported by a strong impairment of the catalytic efficiency, in terms of enzymatic activity and substrate binding capability, for most of the 15-21 loop mutants. In particular, the Gly15Ala mutant is completely inactive and displays a native-like overall fold, indicating that the correct geometry of the 15-21 loop is an essential requisite for optimal enzymatic catalysis. Instead, the Gly45Ala mutant, though revealing unchanged catalytic properties, shows a considerably reduced conformational stability, as judged by circular dichroism and 1H NMR spectroscopy. This finding confirms previous results relative to Thr42 and Thr46 residues [Taddei et al. (1996) Biochemistry 35, 7077-7083] underlining the structural importance of the 42-45 loop as a linker for the two beta alpha beta units constituting the overall enzyme structure.

Acid Anhydride Hydrolases↗

PubMed1997

Insights into acylphosphatase structure and catalytic mechanism.

Acylphosphatase is one of the smallest enzymes known (about 98 amino acid residues). It is present in organs and tissues of vertebrate species as two isoenzymes sharing over 55% of sequence homology; these appear highly conserved in differing species. The two isoenzymes can be involved in a number of physiological processes, though their effective biological function is not still certain. The solution and crystal structures of different isoenzymes are known, revealing a close packed protein with a fold similar to that shown by other phosphate-binding proteins. The structural data, together with an extended site-directed mutagenesis investigation, led to the identification of the residues involved in enzyme catalysis. However, it appears unlikely that these residues are able to perform the full catalytic cycle: a substrate-assisted catalytic mechanism has therefore been proposed, in which the phosphate moiety of the substrate could act as a nucleophile activating the catalytic water molecule.

Acid Anhydride Hydrolases↗

PubMed1997

Effect of liver cirrhosis on the systemic availability of naltrexone in humans.

BACKGROUND/AIMS: Naltrexone is a competitive opiate antagonist with high hepatic extraction. It is used for detoxification treatment for heroin addicts and has been proposed as a possible treatment of pruritus in cholestasis. Such patients are likely to have impaired liver function, underscoring the need to understand the pharmacokinetic behavior of naltrexone in liver disease. These studies were undertaken to evaluate the effect of liver cirrhosis on the plasma time-course of naltrexone. METHODS: A total of 18 patients were investigated: seven migraine patients with normal liver function regarded as controls and 11 patients with liver cirrhosis (six with decompensated disease and five with preserved liver function). A bolus of 100 mg of naltrexone was administered orally in the morning, after an overnight fast. Blood samples were taken in basal conditions and at fixed intervals, up to 24 h after administration. Serum levels of naltrexone and of its major active metabolite, 6 beta-naltrexol, were assayed by reversed-phase HPLC analysis. RESULTS: In control subjects, circulating concentrations of naltrexone were always much lower than those of 6 beta-naltrexol (area under the curve: naltrexone, 200 +/- 97 ng/ml x 24 h; 6 beta-naltrexol, 2467 +/- 730 ng/ml x 24 h, p < 0.01). In severe cirrhosis serum levels of 6 beta-naltrexol increased more slowly, so that circulating levels of naltrexone during the first 2-4 h after drug intake were higher than those of 6 beta-naltrexol (6 beta-naltrexol/naltrexone ratio at 2 h: controls, 10.91 +/- 4.80; cirrhosis, 0.39 +/- 0.18, p < 0.01). The area under the curve for naltrexone (1610 +/- 629 ng/ml x 24 h) was significantly greater than in controls, whereas that for 6 beta-naltrexol (2021 +/- 955 ng/ml x 24 h) was not significantly different. Patients with compensated cirrhosis showed an intermediate pattern. No differences in elimination half-life of the two drugs were detected among the groups. CONCLUSIONS: Our data suggest the occurrence of important changes in the systemic availability of naltrexone and 6 beta-naltrexol in liver cirrhosis; such alterations are consistent with lesser reduction of naltrexone to 6 beta-naltrexol and appear to be related to the severity of liver disease. This must be considered when administering naltrexone in conditions of liver insufficiency.

Adult↗

PubMed1997

Structural, catalytic, and functional properties of low M(r), phosphotyrosine protein phosphatases. Evidence of a long evolutionary history.

The PTPase family comprises a number of classes of functionally and structurally unrelated enzymes; it represents an important component of the protein-tyrosine phosphorylation/dephosphorylation machinery, which regulates the level of tyrosine phosphorylation of a number of intracellular proteins. A wealth of recently reported data indicates growing interest in a group of PTPases characterized by low (near 20 kDa) molecular weight and high sequence homology (low M(r), PTPases). These enzymes are present in organisms spanning the philogenetic scale, from prokaryotes to yeast and mammals. The sequence homology of the low M(r), PTPases with other classes of PTPases is limited to the active site sequence CXXXXXRS/T, containing the Cys and Arg residues involved in enzyme catalysis found in all PTPases. The X-ray structural data of three enzymes belonging to different classes of PTPases, a bovine liver low M(r), PTPase isoenzyme, PTP1B, and Yersinia PTPase, show that all these enzymes maintain the same active site and overall catalytic mechanism, though displaying different chain foldings and topologies, supporting convergent evolution. Limited findings on the in vivo function of the low M(r), PTPases are presently available; however, an involvement of the mammalian enzymes in the membrane growth factor receptor signal transduction is emerging. The distribution of these enzymes in philogenetically distant unicellular and multicellular organisms supports their participation in important cell functions.

Amino Acid Sequence↗

PubMed1997

Analgesic drug taking: beliefs and behavior among headache patients.

OBJECTIVE: To explore beliefs and behavior with respect to analgesic drug taking in headache patients. To compare episodic headache to chronic headache sufferers. METHODS: A consecutive series of 280 headache patients, newly admitted to the Headache Center of the University of Modena, all referred by their general practitioner, were asked to fill out a brief questionnaire, specially compiled for this survey. The questionnaire invited patients to indicate how they themselves thought they should best cope with their headache, and how they actually did so in practice. RESULTS: The majority of our patients had a positive attitude towards the over-the-counter analgesics, which they believed to be more adequate than prescription drugs for acute treatment of their headache. They handled analgesics very carefully, believing it correct to take the drug only when the pain became unbearable, if it was not possible for them to stop work. Chronic headache patients tended to consume more prescription drugs than episodic headache sufferers. Furthermore, the majority of chronic sufferers, as opposed to episodic sufferers, took the analgesic even when not at work. CONCLUSIONS: The use of over-the-counter drugs is considered the best way to treat acute headache even by subjects suffering from severe idiopathic headache and seeking professional care in specialized clinics. Prescribed analgesics are underused by patients with serious episodic headache, which is precisely the group for which they are principally intended.

Adolescent↗

PubMed1996

Looking for residues involved in the muscle acylphosphatase catalytic mechanism and structural stabilization: role of Asn41, Thr42, and Thr46.

Asn41, Thr42, and Thr46 are invariant residues in both muscle and erythrocyte acylphosphatases isolated so far. Horse muscle acylphosphatase solution structure suggests their close spatial relationship to Arg23, the main substrate binding site. The catalytic and structural role of such residues, as well as their influence on muscle acylphosphatase stability, was investigated by preparing several gene mutants (Thr42Ala, Thr46Ala, Asn41Ala, Asn41Ser, and Asn41Gln) by oligonucleotide-directed mutagenesis. The mutated genes were cloned and expressed in Escherichia coli, and the mutant enzymes were purified by affinity chromatography and investigated as compared to the wild-type enzyme. The specific activity and substrate affinity of Thr42 and Thr46 mutants were not significantly affected. On the contrary, Asn41 mutants showed a residual negligible activity (about 0.05-0.15% as compared to wild-type enzyme), though maintaining an unchanged binding capability of both substrate and inorganic phosphate, an enzyme competitive inhibitor. According to the 1H nuclear magnetic resonance spectroscopy and circular dichroism results, all mutants elicited well-constrained native-like secondary and tertiary structures. Thermodynamic parameters, as calculated from circular dichroism data, demonstrated a significantly decreased stability of the Thr42 mutant under increasing temperatures and urea concentrations. The reported results strongly support a direct participation of Asn41 to the enzyme catalytic mechanism, indicating that Asn41 mutants may well represent a useful tool for the investigation of the enzyme physiological function by the negative dominant approach.

Acid Anhydride Hydrolases↗

PubMed1996

C-terminal region contributes to muscle acylphosphatase three-dimensional structure stabilisation.

Ser-Ala and Ser-Ala-Ser-Ala C-terminus elongated (delta+2 and delta+4, respectively) and two C-terminus deleted (delta-2 and delta-3) muscle acylphosphatase mutants were investigated to assess the catalytic and structural roles of the C-terminal region. The kinetic analysis of these mutants shows that the removal of two or three C-terminal residues reduces the catalytic activity to 7% and 4% of the value measured for the wild-type enzyme, respectively; instead, the elongation of the C-terminus does not significantly change the enzyme behaviour. 1H Nuclear magnetic resonance spectroscopy indicates that all mutants display a native-like fold though they appear less stable, particularly delta-2 and delta-3 mutants, as compared to the wild-type enzyme. Such destabilisation of the C-terminal modified mutants is further confirmed by urea inactivation experiments. The results here presented account for an involvement of the C-terminal region in the stabilisation of the three-dimensional structure of acylphosphatase, particularly at the active-site level. Moreover, a participation of the C-terminal carboxyl group to the catalytic mechanism can be excluded.

Acid Anhydride Hydrolases↗

PubMed1996

Properties of Cys21-mutated muscle acylphosphatases.

Cys21 is an invariant residue in muscle acylphosphatases, but is absent in the erythrocyte isozymes. To assess the importance of this residue in the muscle isozymes for catalytic, structural, and stability properties, two gene mutants have been prepared by oligonucleotide-directed mutagenesis and expressed in Escherichia coli cells; in these mutants, the codon for Cys21 was replaced by those for Ser and Ala, respectively. The two mutant enzymes, purified by immunoaffinity chromatography, showed kinetic and structural properties similar to those of the wild-type recombinant enzyme; however, the specific activity of the two mutants, especially that of the C21A mutant, was lower. The urea and thermal stabilities of the mutant enzymes were reduced with respect to those of the wild-type form, contrary to the susceptibility to inactivation by mercuric ions. The reported data support the possibility that Cys21 is involved in the stabilization of the enzyme active-site conformation.

Acid Anhydride Hydrolases↗

PubMed1996

[Interstitial photocoagulation with laser in the treatment of liver metastasis].

Interstitial laser photocoagulation (ILP) causes tumor necrosis with local hyperthermia produced by laser light energy. We treated with US-guided ILP 14 patients (7 men and 7 women; mean age: 67 years) and 20 metastases: 9 of them were < 3 cm in max. diameter and 11 were > 3 cm (mean diameter: 2.9 cm); 14 metastases were from colon carcinoma, 5 from breast cancer and 1 from lung cancer. ILP was performed with 300 and 600 microns quartz fiberoptic guides advanced in 21-18G Chiba needles and a continuous-wave Nd: YAG laser with 1064 nm wavelength. We used single expositions of 5-6 minutes with an irradiation power of 5 watts and scheduled 3 treatment sessions, performing CT scans and biopsies at the end of each session. The extent of induced necrosis was classified as follows on the basis of CT findings: grade 1 = 100% necrosis; grade 2 = necrosis > 50%; grade 3 = necrosis < 50%. The average follow-up was 6 months. After the 3 scheduled treatment sessions, CT showed grade 1 necrosis in all the lesions < 3 cm in diameter and in 4/9 (44%) lesions > 3 cm and grade 2 and 3 necrosis in the remaining cases (necrosis > 50% in 95% of the lesions and 92% of the patients). The cytologic findings were in agreement with CT results in all grade 2 and 3 cases, but in one grade 1 necrosis cytology showed residual viable tumor. To conclude, ILP is a safe and well-tolerated procedure. Maximum efficacy was observed in the lesions < 3 cm, while lesion volume was markedly reduced in the lesions > 3 cm. US is a useful tool in the real-time monitoring of this procedure and CT is the most accurate imaging technique to assess treatment efficacy.

Aged↗