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Biomedical subjects

A Pastore

Publications and source records attributed to A Pastore.

At least 73 records · Page 4Linked to original sources
PubMed1999

Surgical treatment of choanal atresia in CHARGE association: case report with long-term follow-up.

A patient affected by a multisystem malformation, the CHARGE association, is described. The choanal atresia is one of the stigmata characterizing this syndrome. The main anomalies of the association, the surgical correction (choanoplasty with endoscopic transnasal approach followed by the application of nasal stenting) and a comparison with different techniques are reported.

Abnormalities, Multiple↗
PubMed1999

Stage grouping of oropharyngeal cancer: evaluation of three systems by means of survival analysis.

Stage grouping is a method of summarizing multiple categories generated from the tumor, node, metastasis (TNM) classification system. Recently three different systems, the T and N integer score (TANIS), Hart, and 1997 Union Internationale Contre le Cancer systems, have been proposed. To verify their correlation to survival, a series of 64 patients affected by primary squamous cell carcinoma of the oropharynx was considered in this retrospective study. The data set was classified according to Union Internationale Contre le Cancer T stage and then grouped as recommended by the three systems. Results showed a crude survival rate of 28.13%. Univariate analysis by means of the logrank test yielded significant P values for the T and N integer score and Hart systems (0.0452 and 0.0179, respectively) and a borderline P value (0.0728) for the stratification based on the 1997 Union Internationale Contre le Cancer system. Multivariate analysis (Cox regression adjusted for age and sex) showed a significant correlation between the three staging systems and the mortality rate. Odds ratios were 1.36 (95% confidence interval, 1.12-1.66), 1.58 (95% confidence interval, 1.18-2.12), and 1.63 (95% confidence interval, 1.08-2.48) for the T and N integer score, Hart, and 1997 Union Internationale Contre le Cancer systems, respectively. The T and N integer score system showed the best statistical correlation, but a conclusive result could not be achieved because of the low number of patients in this study.

Adult↗
PubMed1999

The immunoglobulin-like modules Cepsilon3 and alpha2 are the minimal units necessary for human IgE-FcepsilonRI interaction.

Atopic allergy is a genetically determined immunodisorder that affects almost 20% of the population worldwide. Immediate symptoms of type I allergy are caused by the release of biologic mediators from effector cells induced by IgE-allergen complexes that cross-link the high-affinity receptor for IgE (FcepsilonRI). Chronic disease manifestations result from allergen-specific T-cell activation, a process that is enhanced when allergens are presented via FcepsilonRI-bound IgE. We report the baculovirus expression, as soluble recombinant proteins, of the minimal units required for human IgE and FcepsilonRI interaction: Cepsilon3 represents the third constant domain of the IgE heavy chain, and alpha2 is the membrane-proximal Ig-like module from FcepsilonRIalpha. Native overlay experiments showed binding of human FcepsilonRIalpha to recombinant Cepsilon3 and of natural or recombinant human IgE to recombinant alpha2. Moreover, recombinant Cepsilon3 inhibited binding of natural IgE antibodies to alpha2, and preincubation of human IgE with alpha2 inhibited anti-IgE-triggered histamine release from human basophils. Isolated Cepsilon3 and alpha2 can now be used for the molecular and structural analysis of the IgE-FcepsilonRI interaction, as well as for diagnostic and therapeutic applications.

Amino Acid Sequence↗
PubMed1998

Molecular and immunologic characterization of a highly cross-reactive two EF-hand calcium-binding alder pollen allergen, Aln g 4: structural basis for calcium-modulated IgE recognition.

Serum IgE was used to isolate a cDNA coding for a 9.4-kDa two EF-hand calcium-binding allergen, Aln g 4, from a lambda gt11 expression cDNA library constructed from alder (Alnus glutinosa) pollen. rAln g 4 was overexpressed in Escherichia coli and purified to homogeneity. It reacted with serum IgE from 18% of pollen-allergic patients (n = 122); shared IgE epitopes with homologous allergens present in tree, grass, and weed pollens; and thus belongs to a family of highly cross-reactive pollen allergens. Exposure of two E. coli-expressed rAln g 4 fragments comprising amino acids 1-41 and 42-85 to patients' IgE Abs, as well as to a rabbit antiserum raised against purified rAln g 4, indicated that most of the B cell epitopes reside in the N-terminal portion of the protein. IgE recognition of Aln g 4 was strongly modulated by the presence or absence of calcium. Circular dichroism analysis of rAln g 4 revealed that the protein consisted mostly of alpha helical secondary structure and possessed a remarkable thermal stability and refolding capacity, a property that was greatly reduced after calcium depletion. Circular dichroism analysis of the calcium-bound and apo form of rAln g 4 indicated that calcium-induced modulation of IgE binding could be due to changes in the protein conformation. Purified rAln g 4 elicited dose-dependent basophil histamine release and immediate type skin reactions in sensitized patients. It may hence be useful for allergy diagnosis and for specific immunotherapy.

Allergens↗
PubMed1998

The assembly of immunoglobulin-like modules in titin: implications for muscle elasticity.

Titin, a giant muscle protein, forms filaments that span half of the sarcomere and cover, along their length, quite diversified functions. The region of titin located in the sarcomere I-band is believed to play a major rôle in extensibility and passive elasticity of muscle. In the I-band, the titin sequence contains tandem immunoglobulin-like (Ig) modules intercalated by a potentially non-globular region. By a combined approach making use of small angle X-ray scattering and nuclear magnetic resonance techniques, we have addressed the questions of what are the average mutual orientation of poly-Igs and the degree of flexibility around the domain interfaces. Various recombinant fragments containing one, two and four titin I-band tandem domains were analysed. The small-angle scattering data provide a picture of the domains in a mostly extended configuration with their long axes aligned head-to-tail. There is a small degree of bending and twisting of the modules with respect to each other that results in an overall shortening in their maximum linear dimension compared with that expected for the fully extended, linear configurations. This shortening is greatest for the four module construct ( approximately 15%). 15N NMR relaxation studies of one and two-domain constructs show that the motions around the interdomain connecting regions are restricted, suggesting that titin behaves as a row of beads connected by rigid hinges. The length of the residues in the interface seems to be the major determinant of the degree of flexibility. Possible implications of our results for the structure and function of titin in muscles are discussed.

Connectin↗
PubMed1998

The three-dimensional structure of a type I module from titin: a prototype of intracellular fibronectin type III domains.

BACKGROUND: Titin is a huge protein ( approximately 3 MDa) that is present in the contractile unit (sarcomere) of striated muscle and has a key role in muscle assembly and elasticity. Titin is mainly composed of two types of module (type I and II). Type I modules are found exclusively in the region of titin localised in the A band, where they are arranged in a super-repeat pattern that correlates with the ultrastructure of the thick filament. No structure of a titin type I module has been reported so far. RESULTS: We have determined the structure of a representative type I module, A71, using nuclear magnetic resonance (NMR) spectroscopy. The structure has the predicted fibronectin type III fold. Titin-specific conserved residues are either located at the putative module-module interfaces or along one side of the protein surface. Several proline residues that contribute to two stretches in a polyproline II helix conformation are solvent-exposed and line up as a continuous ribbon extending over more than two-thirds of the module surface. Homology models of the type I module N-terminal to A71 (A70) and the double module A70-A71 were used to discuss possible intermodule interactions and their role in module-module orientation. CONCLUSIONS: As residues at the module-module interfaces are highly conserved, we speculate that similar interactions govern all of the interfaces between type I modules in titin. This conservation would lead to a regular multiple array of similar surface structures. Such an arrangement would allow arrays of contiguous type I modules to expose multiple proline stretches in a highly regular way and these may act as binding sites for other thick filament proteins.

Amino Acid Sequence↗
PubMed1998

Conformational stability studies of the pleckstrin DEP domain: definition of the domain boundaries.

Pleckstrin is the major substrate of protein kinase C in platelets. It contains at its N- and C-termini two pleckstrin homology (PH) domains which have been proposed to mediate protein-protein and protein-lipid interactions. A new module, called DEP, has recently been identified by sequence analysis in the central region of pleckstrin. In order to study this module, several recombinant polypeptides corresponding to the DEP module and N- and C-termini extended forms have been expressed. Using circular dichroism (CD) and nuclear magnetic resonance (NMR) techniques, the domain boundaries have been determined that yield a soluble and folded pleckstrin DEP domain. This comprises 93 amino acids with an alpha/beta fold in agreement with secondary structure predictions. Stability studies indicate that the regions surrounding the DEP domain do not contribute to its stability suggesting that the phosphorylation sites at S113, T114 and S117 are in an unstructured region. Identification of the regions of pleckstrin that are folded shall facilitate determination of its structure and function.

Amino Acid Sequence↗
PubMed1998

SH3 in muscles: solution structure of the SH3 domain from nebulin.

The huge modular protein nebulin is located in the thin filament of striated muscle in vertebrates and is thought to bind and stabilize F-actin. The C-terminal part of human nebulin is anchored in the sarcomeric Z-disk and contains an SH3 domain, the first of such motifs to be identified in a myofibrillar protein. We have determined the nebulin SH3 sequence from several species and found it strikingly conserved. We have also shown that the SH3 transcripts are constitutively expressed in skeletal muscle tissues. As the first step towards a molecular understanding of nebulin's cellular role we have determined the three-dimensional structure of the human nebulin SH3 domain in solution by nuclear magnetic resonance (NMR) spectroscopy and compared it with other known SH3 structures. The nebulin SH3 domain has a well-defined structure in solution with a typical SH3 topology, consisting of a beta-sandwich of two triple-stranded, antiparallel beta-sheets arranged at right angles to each other and of a single turn of a 310-helix. An additional double-stranded antiparallel beta-sheet in the RT loop bends over the beta-sandwich. The derived structure reveals a remarkable similarity with a distinct subset of SH3 domains, especially in the structural features of the exposed hydrophobic patch that is thought to be the site of interaction with polyproline ligands. On the basis of this similarity, we have modelled the interaction with an appropriate polyproline ligand and attempted to delineate the characteristics of the physiological SH3-binding partner in the Z-disk. Our results represent the first step in reconstructing the structure of nebulin and are expected to contribute to our understanding of nebulin's functional role in myofibrillar assembly.

Amino Acid Sequence↗
PubMed1998

A survey of the primary structure and the interspecies conservation of I-band titin's elastic elements in vertebrates.

Titin is a >3000-kDa large filamentous protein of vertebrate-striated muscle, and single titin molecules extend from the Z disc to the M line. In its I-band section, titin behaves extensible and is responsible for myofibrillar passive tension during stretch. However, details of the molecular basis of titin's elasticity are not known. We have compared the motif sequences of titin elastic elements from different vertebrate species and from different regions of the molecule. The I-band titin Ig repeats that are expressed in the stiff cardiac muscle and those that are tissue-specifically expressed in more elastic skeletal muscles represent distinct subgroups. Within the tissue-specifically expressed Ig repeats, a super-repeat structure is found. For the PEVK titin sequences, we surveyed interspecies conservation by hybridization experiments. The sequences of the titin gene which code for the C-terminal region of the PEVK domain are conserved in the genomes of a larger variety of vertebrates, whereas the N-terminal PEVK sequences are more divergent. Future comparisons of titin gene sequences from different vertebrates may improve our understanding of how titin contributes to species diversity of myofibrillar elasticity. Within one species, different classes of Ig repeat families may contribute to elastic diversity of the titin spring in different segments.

Amino Acid Sequence↗
PubMed1998

Evaluation of voice and speech following subtotal reconstructive laryngectomy.

Subtotal reconstructive laryngectomy (SRL) can be used to preserve voice in the treatment of selected laryngeal carcinomas. This study was designed to analyze both voice and speech results achieved after SRL in 14 male patients, aged from 48 to 73 years. Surgery was performed between 1983 and 1993. Fundamental frequencies, ranges of frequency, intensities, and intensity ranges were established using an S.I. 80 Philips AAC 600 Audio Active Comparative Language System. Five prolonged vowels and six phonetically balanced sentences were recorded on a tape positioned at a distance of 30 cm from the mouth of each patient during a 3-min recording time. The recorded material was then evaluated by a panel of ten trained listeners who were asked to consider the qualitative parameters and perceptual characteristics of voice and speech according to a scorecard modified from one devised by Voiers and Formigoni. Although a decrease was determined in Fundamental Frequency and intensity of the voice when compared to normal values, the quality and perception of speech were found to be satisfactory. The verbal message could be understood almost exactly by means of constant sonority, correct articulation and improved pneumophonic coordination. These values demonstrate that the new voice achieved after SRL is less sonorous and allows for understandable and socially acceptable speech.

Aged↗
PubMed1998

Common mutation in methylenetetrahydrofolate reductase. Correlation with homocysteine and other risk factors for vascular disease.

A common mutation in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene results in elevated homocysteine levels and, presumably, in increased atherosclerotic risk. We evaluated serum homocysteine levels, MTHFR genotype, and a panel of variables in a sample of 155 middle-aged Italian subjects (mean age 38.1 years). Biometrical, hematological, and biochemical variables (including serum folate and vitamin B12) and lifestyle characteristics were investigated. MTHFR genotype was studied by polymerase chain reaction. The frequency of the genotype Val/Val (homozygosity for the mutant allele) was 16.13%. The Val/Val genotype was associated with increased levels of homocysteine; no differences among genotypes were seen in individuals with folate or vitamin B12 levels at or above the median values. In multivariate analysis, MTHFR genotype was an independent predictor of homocysteine levels in both biochemical and non biochemical regression models. Sex and diastolic blood pressure emerged as non biochemical variables independently associated with homocysteine. Apart from cofactors, uric acid was the only biochemical variable independently associated with homocysteine, particularly in subjects with Val/Val genotype. The observed parallel increases in homocysteine and uric acid levels in subjects with thermolabile MTHFR warrant further investigation.

Adult↗
PubMed1998

Purification and characterization of a novel alpha-class glutathione transferase from human liver.

The importance of glutathione transferases (GST) as a major group of detoxification enzymes is well known. The human liver possesses these enzymes in high concentration and in a multiplicity of forms. We describe here a novel glutathione transferase isoenzyme isolated from liver using glutathione affinity chromatography, DEAE-sepharose and Mono-Q ion-exchange chromatography. The isoenzyme is a dimer of approximately 25 kDa with a blocked N-termini. Its kinetic and immunological properties indicate that it belongs to the alpha-class of GSTs. Its isoelectric point (8.0) is closely related to GST alpha (pI 7.8) and GST beta (pI 8.2) reported previously. More than 70% of the amino-acid sequence of this isoenzyme has been determined by automated Edman degradation procedure. The results suggest that this isoenzyme (which we term GST 8.0) may be a heterodimer of two, closely related, novel alpha-class GST subunits. Comparisons between the amino acid sequences of these two novel alpha-class subunits with those of the other alpha-class GST subunits already known indicate changes in a number of different residues localized in the electrophilic binding site. Further studies are needed to establish whether such differences are due to allelic polymorphism of the enzyme or to the existence of additional genes for alpha-class GSTs in human liver. These results are consistent with previous data which suggest that a multitude of different GSTs, especially of alpha class, are present in the human liver providing this tissue with an efficient mechanism of protection against xenobiotic and endogenous compounds.

Adult↗
PubMed1998

Calcium-binding allergens: from plants to man.

Calcium-binding proteins contain a variable number of motifs, termed EF-hands, which consist of two perpendicularly placed alpha-helics and an inter-helical loop forming a single calcium-binding site. Due to their ability to bind and transport calcium as well as to interact with a variety of ligands in a calcium-dependent manner, they fulfill important biological functions in eukaryotic cells. After parvalbumin, a three EF-hand fish allergen, calcium-binding allergens were discovered in pollens of trees. grasses and weeds and, recently, as autoallergens in man. Although only a small percentage of atopic individuals displays IgE reactivity to calcium-binding allergens, these allergens may be important because of their ability to cross-sensitize allergic individuals. Confrontation and stability++ as well as IgE recognition of calcium-binding allergens greatly depend on the presence of protein-bound calcium ions. It is thus likely that hypoallergenic derivatives of calcium-binding allergens can be engineered by recombinant DNA technology for immunotherapy++ of sensitized patients.

Allergens↗
PubMed1998

Parotid gland carcinoma: analysis of prognostic factors.

The histologic variety of parotid gland carcinomas, their different natural history, and the peculiar anatomy of the parotid region can make prognosis and therapeutic strategy quite controversial. The present study was designed to evaluate those prognostic factors able to affect the long-term results in a group of 167 consecutively treated parotid epithelial malignancies. The continuous or discrete covariants considered as potential prognostic factors are age, sex, histotype, grading, TNM and pTNM classification, facial nerve involvement, type of surgery on the tumor site and on nodes, facial nerve resection, and postoperative radiotherapy. All the material has been statistically analyzed and the results have been compared with the principal data published. According to the analysis, the most relevant prognostic factors in parotid gland carcinomas appear to be pTN staging, tumor grading, facial nerve involvement, and local extension. These factors could reliably predict the patient's chance for survival, and thus influence the therapeutic strategy.

Adolescent↗
PubMed1998

Fully automated assay for total homocysteine, cysteine, cysteinylglycine, glutathione, cysteamine, and 2-mercaptopropionylglycine in plasma and urine.

We describe a 6-min HPLC method to measure the total concentrations of the most important thiols in plasma and urine--cysteine, homocysteine, cysteinylglycine, and glutathione--as well as the concentrations in plasma and urine, respectively, of cysteamine and 2-mercaptopropionylglycine, two compounds used to treat disorders of cysteine metabolism. Precolumn derivatization with bromobimane and reversed-phase HPLC were performed automatically by a sample processor. Throughput was up to 100 samples in 24 h. The within-run CV ranged from 0.9% to 3.4% and the between-run CV ranged from 1.5% to 6.1%. Analytical recovery was 97-107%, with little difference between plasma and urine samples. The detection limit was approximately 50 nmol/L for all the analytes studied. Thiol concentrations were determined in the plasma of 206 healthy donors and in the urine of 318 healthy donors distributed for age and sex. Mean values of plasma cysteine and homocysteine were significantly lower in infants (ages, <1 y) compared with other age groups (P <0.005). In adults, mean plasma homocysteine values were higher in males than in females (9.2 vs 6.7 micromol/L, P <0.0001) and in the 6- to 10-year-old group (P <0.05). Mean values for glutathione and cysteinylglycine were not sex- and age-dependent. In urine, both cysteine and homocysteine showed a wide range of variation.

Adolescent↗
PubMed1997

Conversion of the major birch pollen allergen, Bet v 1, into two nonanaphylactic T cell epitope-containing fragments: candidates for a novel form of specific immunotherapy.

A novel approach to reduce the anaphylactic activity of allergens is suggested. The strategy makes use of the presence of conformational immunoglobulin E (IgE) epitopes on one of the most common allergens. The three dimensional structure of the major birch pollen allergen, Bet v 1, was disrupted by expressing two parts of the Bet v 1 cDNA representing amino acids 1-74 and 75-160 in Escherichia coli. In contrast to the complete recombinant Bet v 1, the fragments showed almost no allergenicity and exhibited random coil conformation as analyzed by circular dichroism. Both nonanaphylactic fragments induced proliferation of human Bet v 1-specific T cell clones, indicating that they harbored all dominant T cell epitopes and therefore may be considered as a basis for the development of a safe and specific T cell immunotherapy.

Allergens↗
PubMed1997

When a module is also a domain: the rôle of the N terminus in the stability and the dynamics of immunoglobulin domains from titin.

In the course of a structural study of titin, a giant modular protein from muscle, we have reported that N-terminal extension of immunoglobulin-like (Ig-like) domains from titin stabilizes this fold. In order to investigate the structural basis of such an effect, we have solved the structure of NEXTM5, which has six amino acids added to the sequence of M5, a domain for which full structure determination has been previously achieved. In the present work, the structures and the dynamics of M5 and NEXTM5 are compared in the light of data collected for these and other titin domains. In NEXTM5, three out of the six added residues are structured and pack against the nearby BC and FG loops. As a consequence, three new backbone hydrogen bonds are formed with the B strand, extending the A strand by two residues and decreasing the exposed surface area of the loops. Additional contacts which involve the side-chains give rise to a remarkable pH dependence of the stability. Interestingly, no correlation is observed on the NMR time-scale between the overall dynamics of the extended domain and its increased stability. The most noticeable differences between the two constructs are localised around the N terminus, which becomes more rigid upon extension. Since a similar pattern of contacts is observed for other domains of the immunoglobulin I-set, our results are of general relevance for this protein family. Our work might also inspire a more rational approach to the investigation of domain boundaries and their influence on module stability.

Algorithms↗