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M Stefani

Publications and source records attributed to M Stefani.

At least 19 recordsLinked to original sources
PubMed2006

[Dental pulp metastasis from oral squamous cell carcinoma: a case report and a review of the literature].

A 61-year-old man was seen in the Oral Pathology Department with a three-month history of right mandibular pain. Clinical examination revealed a 3 cm mass involving the body and the angle of the right site of the mandible and palpable masses in the neck. The dental panoramic radiography showed on the right a radiolucenT area surrounding the lower second molar, extending to the mandible angle. Additionally, chest radiograph and routine laboratory analysis results were unremarkable. An incisional biopsy of the area, revealed histologically a well differentiated squamous cell carcinoma and after few days, the patient received a right hemimandibulectomy with dissection of submandibular salivary gland and laterocervical lymph nodes on the same side. The histology of surgical material confirmed the diagnosis of a differentiated squamous cell carcinoma G1, homolateral diffusion in the mandible bone and metastasis to the dental pulp of the second lower molar tooth and to one of 18 lymph nodes dissected. In this report is described a very rare metastasis to the dental pulp and literature review, in which only fifteen other cases are reported.

Carcinoma, Squamous Cell↗
PubMed2006

Oral metastasis of ciliary body melanoma. A case report.

Metastases of melanoma rarely occur in the oral cavity, and very few reports have been published. They are chiefly localized in the tonsil, tongue and lip, regardless of the primary site of the neoplasm. A 76-year-old woman presented a brownish berry-shaped floating neoformation at the upper lip, for which she was hospitalized at the Maxillofacial Surgery Unit, Italian Stomatologic Institute, Milan. Medical history revealed that 6 years previously, in 1998, she underwent enucleation of the right eye due to the presence of a melanoma of the ciliary body. The labial neoplasm was removed and at histological examination it was found to be a spindle cell melanoma with numerous melanophages containing granules of melanin. Both the spindle cells and the melanophages were strongly positive for HMB-45 and for S-100. Thus, the presence of melanic neoplasia at an unusual site together with the medical history of melanoma at the ciliary body, removed 6 years previously, indicated a diagnosis of labial metastasis of melanoma of the ciliary body and the patient was therefore transferred to the Oncology Unit for appropriate treatment.

Aged↗
PubMed2005

Right ventricular failure: physiology and assessment.

Right ventricular function can be altered in several disease states involving lungs and heart. Severe right ventricular dysfunction is a major determinant of outcome in such situations, and may strongly influence clinical management. The complex geometry of the right ventricle and the different physiology with respect to the left ventricle make the right ventricular failure difficult to define and assess. The response to increased afterload is the main determinant of right ventricle physiology in pathologic conditions. This consists of right ventricular hypertrophy and enlargement, with reduced coronary blood flow to the right ventricular wall, dilation of tricuspid annulus and displacement of interventricular septum. This latter change involves the left ventricular diastolic function, which is reduced by leftward septal shifting. In right ventricle myocardial ischemia and infarction the primum movens of altered right ventricular function is not an increase in afterload, but the ischemic involvement of the right ventricle, more often in the setting of an inferior acute myocardial infarction. The assessment of right ventricular failure is based on thermodilution by pulmonary artery catheter, contrast and radionuclide ventriculography, echocardiography, and magnetic resonance. Among these techniques, thermodilution and echocardiography play a relevant role in clinical scenarios, being readily available and feasible bedside.

Heart Failure↗
PubMed2004

Investigation of the effects of copper ions on protein aggregation using a model system.

Protein aggregation is a notable feature of various human disorders, including Parkinson's disease, Alzheimer's disease and many others systemic amyloidoses. An increasing number of observations in vitro suggest that transition metals are able to accelerate the aggregation process of several proteins found in pathological deposits, e.g. alpha-synuclein, amyloid beta (Abeta) peptide, beta(2)-microglobulin and fragments of the prion protein. Here we report the effects of metal ions on the aggregation rate of human muscle acylphosphatase, a suitable model system for aggregation studies in vitro. Among the different species tested, Cu(2+) produced the most remarkable acceleration of aggregation, the rate of the process being 2.5-fold higher in the presence of 0.1 mM metal concentration. Data reported in the literature suggest the possible role played by histidine residues or negatively charged clusters present in the amino acid sequence in Cu(2+)-mediated aggregation of pathological proteins. Acylphosphatase does not contain histidine residues and is a basic protein. A number of histidine-containing mutational variants of acylphosphatase were produced to evaluate the importance of histidine in the aggregation process. The Cu(2+)-induced acceleration of aggregation was not significantly altered in the protein variants. The different aggregation rates shown by each variant were entirely explained by the changes of hydrophobicity or propensity to form a beta structure introduced by the point mutation. The effect of Cu(2+) on acylphosphatase aggregation cannot therefore be attributed to the specific factors usually invoked in the aggregation of pathological proteins. The effect, rather, seems to be a general related to the chemistry of the polypeptide backbone and could represent an additional deleterious factor resulting from the alteration of the homeostasis of metal ions in cells.

Acid Anhydride Hydrolases↗
PubMed2004

Reduced compliance of left ventricle.

Diastolic function is essential for efficient systolic performance. A normal diastole allows left ventricle (LV) filling to occur under normal intracavitary pressure. It is an energy dependent process, as such affected by ischemia. Several factors influence diastolic function of the LV: the mitral valve area, the gradient between atrium and ventricle, LV relaxation and compliance, atrial compliance, the presence of sinus rhythm, the end-systolic volume. Echocardiography is the principal diagnostic tool to assess LV diastolic function noninvasively in clinical practice. Doppler evaluation allows to analyse each phase of LV diastole through measurement of transmitral and pulmonary veins flows velocities. Tissue Doppler echocardiography and color M-mode Doppler have also been introduced in the study of diastole. The use of echocardiography in the setting of diastolic dysfunction in ICU and operatory room has relevant implications in the management of haemodynamic instability, in vasoactive d rugs titration, in the detection of myocardial ischemia, and in performing prognostic stratification. These information can guide the management of cardiac patients undergoing cardiac and non cardiac surgery, in the perioperative phase, as well as in the management of critical ICU patients. On this basis evaluating the LV filling properties can contribute to improve the quality of treatments in such challenging situations.

Compliance↗
PubMed2001

Folding and aggregation are selectively influenced by the conformational preferences of the alpha-helices of muscle acylphosphatase.

The native state of human muscle acylphosphatase (AcP) presents two alpha-helices. In this study we have investigated folding and aggregation of a number of protein variants having mutations aimed at changing the propensity of these helical regions. Equilibrium and kinetic measurements of folding indicate that only helix-2, spanning residues 55-67, is largely stabilized in the transition state for folding therefore playing a relevant role in this process. On the contrary, the aggregation rate appears to vary only for the variants in which the propensity of the region corresponding to helix-1, spanning residues 22-32, is changed. Mutations that stabilize the first helix slow down the aggregation process while those that destabilize it increase the aggregation rate. AcP variants with the first helix destabilized aggregate with rates increased to different extents depending on whether the introduced mutations also alter the propensity to form beta-sheet structure. The fact that the first alpha-helix is important for aggregation and the second helix is important for folding indicates that these processes are highly specific. This partitioning does not reflect the difference in intrinsic alpha-helical propensities of the two helices, because helix-1 is the one presenting the highest propensity. Both processes of folding and aggregation do not therefore initiate from regions that have simply secondary structure propensities favorable for such processes. The identification of the regions involved in aggregation and the understanding of the factors that promote such a process are of fundamental importance to elucidate the principles by which proteins have evolved and for successful protein design.

Acid Anhydride Hydrolases↗
PubMed2001

Detection of two partially structured species in the folding process of the amyloidogenic protein beta 2-microglobulin.

beta 2-Microglobulin is a small, major histocompatibility complex class I-associated protein that undergoes aggregation and accumulates as amyloid deposits in human tissues as a consequence of long-term haemodialysis. The folding process of this amyloidogenic protein has been studied in vitro by diluting the guanidine hydrochloride-denatured protein in refolding buffer at pH 7.4 and monitoring the folding process by means of a number of spectroscopic probes that allow the native structure of the protein to be detected as it develops. These techniques include fluorescence spectroscopy, far and near-UV circular dichroism, 8-anilino-1-naphthalenesulfonic acid binding and double jump assays. All spectroscopic probes indicate that a significant amount of structure forms within the dead-time of stopped-flow measurements (<5 ms). The folding reaction goes to completion through a fast phase followed by a slow phase, whose rate constants are ca 5.1 and 0.0030 s(-1) in water, respectively. Unfolding-folding double jump experiments, together with the use of peptidyl prolyl isomerase, reveal that the slow phase of folding of beta 2-microglobulin is not fundamentally determined by cis/trans isomerisation of X-Pro peptide bonds. Other folding-unfolding double jump experiments also suggest that the fast and slow phases of folding are not related to independent folding of different populations of protein molecules. Rather, we provide evidence for a sequential mechanism of folding where denatured beta 2-microglobulin collapses to an ensemble of partially folded conformations (I(1)) which fold subsequently to a more highly structured species (I(2)) and, finally, attain the native state. The partially folded species I(2) appears to be closely similar to previously studied amyloidogenic forms of beta 2-microglobulin, such as those adopted by the protein at mildly acid pH values and by a variant with six residues deleted at the N terminus. Since amyloid formation in vivo originates from partial denaturation of beta 2-microglobulin under conditions favouring the folding process, the long-lived, partially structured species detected here might be significantly populated under some physiological conditions and hence might play an important role in the process of amyloid formation.

Amyloidosis↗
PubMed2001

Solution conditions can promote formation of either amyloid protofilaments or mature fibrils from the HypF N-terminal domain.

The HypF N-terminal domain has been found to convert readily from its native globular conformation into protein aggregates with the characteristics of amyloid fibrils associated with a variety of human diseases. This conversion was achieved by incubation at acidic pH or in the presence of moderate concentrations of trifluoroethanol. Electron microscopy showed that the fibrils grown in the presence of trifluoroethanol were predominantly 3-5 nm and 7-9 nm in width, whereas fibrils of 7-9 nm and 12-20 nm in width prevailed in samples incubated at acidic pH. These results indicate that the assembly of protofilaments or narrow fibrils into mature amyloid fibrils is guided by interactions between hydrophobic residues that may remain exposed on the surface of individual protofilaments. Therefore, formation and isolation of individual protofilaments appears facilitated under conditions that favor the destabilization of hydrophobic interactions, such as in the presence of trifluoroethanol.

Amyloid↗
PubMed2001

Reduction of the amyloidogenicity of a protein by specific binding of ligands to the native conformation.

It is known that human muscle acylphosphatase (AcP) is able, under appropriate conditions in vitro, to aggregate and form amyloid fibrils of the type associated with human diseases. A number of compounds were tested for their ability to bind specifically to the native conformation of AcP under conditions favoring denaturation and subsequent aggregation and fibril formation. Compounds displaying different binding affinities for AcP were selected and their ability to inhibit protein fibrillization in vitro was evaluated. We found that compounds displaying a relatively high affinity for AcP are able to significantly delay protein fibrillization, mimicking the effect of stabilizing mutations; in addition, the effectiveness of such outcome correlates positively to both ligand concentration and affinity to the native state of AcP. By contrast, the inhibitory effect of ligands on AcP aggregation disappears in a mutant protein in which such binding affinity is lost. These results indicate that the stabilization of the native conformation of amyloidogenic proteins by specific ligand binding can be a strategy of general interest to inhibit amyloid formation in vivo.

Acid Anhydride Hydrolases↗
PubMed2001

Clinical features associated with pre-hospital time delay in acute myocardial infarction.

BACKGROUND: The pre-hospital time delay in acute myocardial infarction (AMI) is still a challenging problem since for many patients there are long intervals between the onset of symptoms and the initiation of therapy. The aim of this study was to verify which, among several clinical variables, are associated with a prolonged pre-hospital time delay. METHODS: Five hundred and twenty-six unselected patients with AMI and consecutively admitted to three coronary care units were enrolled. The pre-hospital time delay was defined as the time interval from the onset of symptoms to admission to the coronary care unit. Clinical variables included: age, gender, body mass index, level of education, alcohol consumption, smoking habits, regular physical activity, history of hypertension, diabetes mellitus, history of coronary artery disease (documented history of angina and/or previous myocardial infarction), chronic atrial fibrillation, Q-wave AMI and off hours onset of symptoms. After univariate analysis, multivariable regression analysis was used. RESULTS: The mean age of the patients was 66.6 +/- 12.1 years and 28.7% were female. The median pre-hospital time interval was 200 min (95% confidence interval 60-1140). For 342 patients the pre-hospital time interval was < or = 6 hours and for 184 patients it was > 6 hours. Those variables which, at univariate analysis, were found to significantly influence the pre-hospital delay were analyzed using a multivariable regression model where the dependent variable was the pre-hospital time interval. Chronic atrial fibrillation (p = 0.010), a history of coronary artery disease (p = 0.017), diabetes (p = 0.016) and age > or = 70 years (p = 0.009) were found to be independently associated with a prolonged prehospital time interval. Similar results were obtained when considering only Q-wave AMI. As expected, the thrombolytic therapy rate was much lower in patients with a longer pre-hospital time delay. CONCLUSIONS: The present study shows that, in case of AMI, the time interval between the onset of symptoms and a patient's arrival to hospital is still far from being optimal. This is especially true for older patients with diabetes, a history of coronary artery disease or chronic atrial fibrillation. Cardiologists should be aware of this problem and should implement adequate educational strategies addressed to those patients at highest risk.

Age Factors↗
PubMed2001

Conduction system in cardiac amyloidosis: two cases succumbed to cardiac arrest.

The present article reports the histological study of the conduction system in 2 cases of cardiac amyloidosis. The discrepant anatomical and clinical evidence yet confirmed the need for accurate ECG controls in all cases. Indeed, while in the first case evident lesions of the conduction system were revealed by surface ECGs, the second case did not exhibit significant ECG abnormalities but the right atrium and the His bundle showed slight fibro-amyloid involvement, as potential forerunners of high-risk arrhythmias.

Amyloidosis↗
PubMed2000

Stabilisation of alpha-helices by site-directed mutagenesis reveals the importance of secondary structure in the transition state for acylphosphatase folding.

The effects of stabilising mutations on the folding process of common-type acylphosphatase have been investigated. The mutations were designed to increase the helical propensity of the regions of the polypeptide chain corresponding to the two alpha-helices of the native protein. Various synthetic peptides incorporating the designed mutations were produced and their helical content estimated by circular dichroism. The most substantial increase in helical content is found for the peptide carrying five mutations in the second alpha-helix. Acylphosphatase variants containing the corresponding mutations display, to different extents, enhanced conformational stabilities as indicated by equilibrium urea denaturation experiments monitored by changes of intrinsic fluorescence. All the protein variants studied here refold with apparent two-state kinetics. Mutations in the first alpha-helix are responsible for a small increase in the refolding rate, accompanied by a marked decrease in the unfolding rate. On the other hand, multiple mutations in the second helix result in a considerable increase in the refolding rate without any significant effect on the unfolding rate. Addition of trifluoroethanol was found to accelerate the folding of the acylphosphatase variants, the extent of the acceleration being inversely proportional to the intrinsic rate of folding of the corresponding mutant. The trifluoroethanol-induced acceleration is far less marked for those variants whose alpha-helical structure is efficiently stabilised by amino acid replacements. This observation suggests that trifluoroethanol acts in a similar manner to the stabilising mutations in promoting native-like secondary structure. Analysis of the kinetic data indicates that the second helix is fully consolidated in the transition state for folding of acylphosphatase, whereas the first helix is only partially formed. These data suggest that the second helix is an important element in the folding process of the protein.

Acid Anhydride Hydrolases↗
PubMed2000

Initial denaturing conditions influence the slow folding phase of acylphosphatase associated with proline isomerization.

The folding kinetics of human common-type acylphosphatase (cAcP) from its urea- and TFE-denatured states have been determined by stopped-flow fluorescence techniques. The refolding reaction from the highly unfolded state formed in urea is characterized by double exponential behavior that includes a slow phase associated with isomerism of the Gly53-Pro54 peptide bond. However, this slow phase is absent when refolding is initiated by dilution of the highly a-helical denatured state formed in the presence of 40% trifluoroethanol (TFE). NMR studies of a peptide fragment corresponding to residues Gly53-Gly69 of cAcP indicate that only the native-like trans isomer of the Gly-Pro peptide bond is significantly populated in the presence of TFE, whereas both the cis and trans isomers are found in an approximately 1:9 ratio for the peptide bond in aqueous solution. Molecular modeling studies in conjunction with NMR experiments suggest that the trans isomer of the Gly53-Pro54 peptide bond is stabilized in TFE by the formation of a nonnative-like hydrogen bond between the CO group of Gly53 and the NH group of Lys57. These results therefore reveal that a specific nonnative interaction in the denatured state can increase significantly the overall efficiency of refolding.

Acid Anhydride Hydrolases↗
PubMed1999

Capillary zone electrophoretic analysis of positively charged poly(ethylene oxide) macromolecules using non-covalent polycation-coated fused-silica capillary and indirect UV detection.

Capillary zone electrophoresis was used to show the coupling between NH2-terminated poly(ethylene oxide) and oligomers of lactic acid activated by transforming carboxyl chain ends to acyl chloride ones. The demonstration was based on the use of fused-silica capillary physically modified by pre-adsorption of polycations in the reversed polarity mode. As poly(ethylene oxide) macromolecules are UV transparent, indirect UV detection was used. A creatinine solution at pH 4.8 was selected as background electrolyte. Commercially available polycations with different structures were tested. It was shown that the reversed electroosmosis could be modulated according to the structure of the polycation. The method was then applied to analyse a commercial alpha,omega-diamino poly(ethylene oxide), namely Jeffamine ED 600 characterised by a broad mass dispersion. Data showed that the method can detect and separate amino poly(ethylene oxide) of different structures. When applied to analyse post coupling products, no free NH2-terminated poly(ethylene oxide) segments were detected. Moreover, the method allowed detection of water-soluble oligomers generated by partial degradation of lactic segments during the reaction.

Electrochemistry↗
PubMed1999

The low Mr phosphotyrosine protein phosphatase behaves differently when phosphorylated at Tyr131 or Tyr132 by Src kinase.

The low molecular weight phosphotyrosine protein phosphatase (LMW-PTP) is phosphorylated by Src and Src-related kinases both in vitro and in vivo; in Jurkat cells, and in NIH-3T3 cells, it becomes tyrosine-phosphorylated upon stimulation by PDGF. In this study we show that pp60Src phosphorylates in vitro the enzyme at two tyrosine residues, Tyr131 and Tyr132, previously indicated as the main phosphorylation sites of the enzyme, whereas phosphorylation by the PDGF-R kinase is much less effective and not specific. The effects of LMW-PTP phosphorylation at each tyrosine residue were investigated by using Tyr131 and Tyr132 mutants. We found that the phosphorylation at either residue has differing effects on the enzyme behaviour: Tyr131 phosphorylation is followed by a strong (about 25-fold) increase of the enzyme specific activity, whereas phosphorylation at Tyr132 leads to Grb2 recruitment. These differing effects are discussed on the light of the enzyme structure.

3T3 Cells↗
PubMed1999

Designing conditions for in vitro formation of amyloid protofilaments and fibrils.

We have been able to convert a small alpha/beta protein, acylphosphatase, from its soluble and native form into insoluble amyloid fibrils of the type observed in a range of pathological conditions. This was achieved by allowing slow growth in a solution containing moderate concentrations of trifluoroethanol. When analyzed with electron microscopy, the protein aggregate present in the sample after long incubation times consisted of extended, unbranched filaments of 30-50 A in width that assemble subsequently into higher order structures. This fibrillar material possesses extensive beta-sheet structure as revealed by far-UV CD and IR spectroscopy. Furthermore, the fibrils exhibit Congo red birefringence, increased fluorescence with thioflavine T and cause a red-shift of the Congo red absorption spectrum. All of these characteristics are typical of amyloid fibrils. The results indicate that formation of amyloid occurs when the native fold of a protein is destabilized under conditions in which noncovalent interactions, and in particular hydrogen bonding, within the polypeptide chain remain favorable. We suggest that amyloid formation is not restricted to a small number of protein sequences but is a property common to many, if not all, natural polypeptide chains under appropriate conditions.

Acid Anhydride Hydrolases↗
PubMed1999

Thermodynamics and kinetics of folding of common-type acylphosphatase: comparison to the highly homologous muscle isoenzyme.

The thermodynamics and kinetics of folding of common-type acylphosphatase have been studied under a variety of experimental conditions and compared with those of the homologous muscle acylphosphatase. Intrinsic fluorescence and circular dichroism have been used as spectroscopic probes to follow the folding and unfolding reactions. Both proteins appear to fold via a two-state mechanism. Under all the conditions studied, common-type acylphosphatase possesses a lower conformational stability than the muscle form. Nevertheless, common-type acylphosphatase folds more rapidly, suggesting that the conformational stability and the folding rate are not correlated in contrast to recent observations for a number of other proteins. The unfolding rate of common-type acylphosphatase is much higher than that of the muscle enzyme, indicating that the differences in conformational stability between the two proteins are primarily determined by differences in the rate of unfolding. The equilibrium m value is markedly different for the two proteins in the pH range of maximum conformational stability (5. 0-7.5); above pH 8.0, the m value for common-type acylphosphatase decreases abruptly and becomes similar to that of the muscle enzyme. Moreover, at pH 9.2, the dependencies of the folding and unfolding rate constants of common-type acylphosphatase on denaturant concentration (mf and mu values, respectively) are notably reduced with respect to pH 5.5. The pH-induced decrease of the m value can be attributed to the deprotonation of three histidine residues that are present only in the common-type isoenzyme. This would decrease the positive net charge of the protein, leading to a greater compactness of the denatured state. The folding and unfolding rates of common-type acylphosphatase are not, however, significantly different at pH 5.5 and 9.2, indicating that this change in compactness of the denatured and transition states does not have a notable influence on the rate of protein folding.

Acid Anhydride Hydrolases↗