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S Gasparini

Publications and source records attributed to S Gasparini.

At least 37 records · Page 2Linked to original sources
PubMed1997

High-level production and isotope labeling of snake neurotoxins, disulfide-rich proteins.

The aim of this work was to produce and to label snake neurotoxins, disulfide-rich proteins. A mutant of a snake toxin, erabutoxin a, was used as a model. Its N-terminal part was fused to ZZ, a synthetic IgG-binding domain of protein A (B. Nilsson et al., 1987, Protein Eng. 1, 107-113), thus preventing degradation in the bacterial cytoplasm and providing a simple affinity-purification method on IgG Sepharose. A soluble fusion protein was obtained with a yield of 60 mg/L, corresponding to 20 mg/L toxin. The toxin moiety was folded on the column while the hybrid was still bound. The oxidoreducing conditions for the refolding were optimized and were found to be oxidative but with a need for reducing molecules. The concentration of the hybrid bound to the column could be increased up to 3.3 mg/ml without significantly altering the folding process. CNBr cleavage of the fusion protein followed by a purification step yielded about 2 mg of biologically active toxin mutant per gram of dry cell weight. This procedure was applied to produce 55 mg of a toxin uniformly labeled with 15N.

Binding, Competitive↗
PubMed1997

Action of the hyperpolarization-activated current (Ih) blocker ZD 7288 in hippocampal CA1 neurons.

The effects of ZD 7288, a "bradycardic" agent, in young rat hippocampal slices in vitro were studied. ZD 7288 (1-1000 microM) reduced the hyperpolarization-activated current (Ih) in CA1 pyramidal neurons by a voltage-independent blocking mechanism. Under current-clamp conditions, the bradycardic agent (10 microM) caused membrane hyperpolarization (by 5.9 +/- 0.5 mV) and a reduction of membrane conductance (by 17.9 +/- 4.1%). These data are consistent with the block of an inward current which is active at rest. The drug-induced hyperpolarization depressed the cell's excitability by increasing the threshold current necessary to induce firing. When the drug-induced hyperpolarization was compensated for by injection of a tonic depolarizing current, ZD 7288 caused a reduction of the inhibitory post-synaptic potential (IPSP) in EPSP-IPSP sequences. Since Cs+, another known blocker of Ih, is able to reverse long-term depression (LTD) of the CA3-CA1 synapse in hippocampal slices, we tested the effect of ZD 7288 on synaptic transmission. We found that ZD 7288 did not significantly modify LTD, suggesting that Cs+-induced inhibition of LTD maintenance is not directly related to block of Ih.

Animals↗
PubMed1997

Immune response prevalence to formaldehyde-human serum albumin molecular adduct in a healthy population.

Formaldehyde (F) covalently binds human serum albumin (HSA), giving rise to the molecular adduct, F-HSA, with F as a hapten. Humoral immune response to the adduct provides a biological marker of F exposure. A new indirect competitive enzyme immunoassay (displacement assay) using an in vitro conjugate F-HSA adduct with a 10:1 ratio was developed to titrate serum anti-F-HSA antibodies. 219 healthy subjects were examined using this assay. Examination of the data using multivariate regression analysis revealed a significant association between the F exposure marker and smoking variables in both smokers (p < 0.001) and ex-smokers (p < 0.01). No association was found between F exposure marker values and age, sex, residence, or profession. Except for the fact that the smoking variable subdivided the population into smokers, ex-smokers, and non-smokers, a significant association was found in non-smokers concerning professional exposure (p < 0.03). Results confirm that the Displacement Assay (sensitivity 96% and specificity 71%) is a useful method of evaluating F exposure, especially in large-scale public health monitoring. In order to evaluate the importance of other exposure sources, such as F pollution in indoor and outdoor air, an interesting follow up to our study would be an evaluation of a possible increase of anti-F-HSA immune response in non-smokers.

Antibody Formation↗
PubMed1997

Bronchoscopic biopsy techniques in the diagnosis and staging of lung cancer.

In a review of the bronchoscopic biopsy techniques currently available for the diagnosis and staging of lung cancer, the author individually analyses the sampling instruments and procedures used in cancer of the central airways, peripheral lung lesions, and in the study of hilar and mediastinal lymph nodes. With regard to central bronchial lesions, data concerning diagnostic yield, the advantages and limits of forceps biopsy, brushing, washing and transbronchial needle aspiration are reported. With the integration of two or more of the sampling methods, a cytohistological diagnosis can be obtained in almost all cases. In the field of peripheral lung cancer, the diagnostic possibilities of the transbronchial approach are described. The experience of the Regional Hospital of Ancona, Italy on 1,027 patients affected by peripheral pulmonary nodules or masses is reported. The biopsy technique is based on the integration of transbronchial (using forceps biopsy and transbronchial needle aspiration) and percutaneous approaches, and on a team approach with the close co-operation of a pulmonologist, a radiologist and a cytopathologist, all simultaneously present in the diagnostic suite during the procedures. On the basis of the results obtained, the author suggests that, in peripheral pulmonary lesions, the transbronchial approach should generally be performed before the percutaneous needle aspirate, especially in patients who are candidates for surgery. The transbronchial approach has the advantages of allowing an examination of the tracheobronchial tree and staging of lymph nodes with a lower incidence of complications. In addition, the diagnostic yield of transbronchial needle aspiration in the study of hilar and mediastinal lymph nodes is analysed. This method, if positive, plays a major role in the staging of lung cancer and makes it possible to avoid unnecessary surgical procedures. Knowledge of the advantages and limits of different sampling instruments and procedures, and of their integration, is essential to optimize the diagnostic management of each patient with lung cancer. The goal is to maximize the diagnostic possibilities whilst minimizing risk and reducing costs.

Biopsy, Needle↗
PubMed1997

[Virtual bronchoscopy: the correlation between endoscopic simulation and bronchoscopic findings].

PURPOSE: We carried out a preliminary clinical validation of 3D spiral CT virtual endoscopic reconstructions of the tracheobronchial tree, by comparing virtual bronchoscopic images with actual endoscopic findings. MATERIALS AND METHODS: Twenty-two patients with tracheobronchial disease suspected at preliminary clinical, cytopathological and plain chest film findings were submitted to spiral CT of the chest and bronchoscopy. CT was repeated after endobronchial therapy in 2 cases. Virtual endoscopic shaded-surface-display views of the tracheobronchial tree were reconstructed from reformatted CT data with an Advantage Navigator software. Virtual bronchoscopic images were preliminarily evaluated with a semi-quantitative quality score (excellent/good/fair/poor). The depiction of consecutive airway branches was then considered. Virtual bronchoscopies were finally submitted to double-blind comparison with actual endoscopies. RESULTS: Virtual image quality was considered excellent in 8 cases, good in 14 and fair in 2. Virtual exploration was stopped at the lobar bronchi in one case only; the origin of segmental bronchi was depicted in 23 cases and that of some subsegmental branches in 2 cases. Agreement between actual and virtual bronchoscopic findings was good in all cases but 3 where it was nevertheless considered satisfactory. The yield of clinically useful information differed in 8/24 cases: virtual reconstructions provided more information than bronchoscopy in 5 cases and vice versa in 3. Virtual reconstructions are limited in that the procedure is long and difficult and needing a strictly standardized threshold value not to alter virtual findings. Moreover, the reconstructed surface lacks transparency, there is the partial volume effect and the branches < or = 4 pixels phi and/or meandering ones are difficult to explore. CONCLUSIONS: Our preliminary data are encouraging. Segmental bronchi were depicted in nearly all cases, except for the branches involved by disease. Obstructing lesions could be bypassed in some cases, making an indication for endoscopic laser therapy. Future didactic perspectives and applications to minimally invasive or virtual reality-assisted therapy seem promising, even though actual clinical applications require further studies.

Adult↗
PubMed1995

Integration of transbronchial and percutaneous approach in the diagnosis of peripheral pulmonary nodules or masses. Experience with 1,027 consecutive cases.

A study to evaluate the usefulness of the integration of the transbronchial and percutaneous approaches in the diagnosis of peripheral pulmonary nodules or masses (PPN/M) was conducted. The authors used both procedures, performed by a single diagnostic team, a pulmonologist, radiologist, and cytopathologist, who were all simultaneously present in the radiologic suite during the maneuvers. From January 1985 to June 1993, under fluoroscopic guidance, the authors performed 557 transbronchial pulmonary biopsies (TBPB), 483 transbronchial needle aspirations (TBNA), and 652 percutaneous needle aspirations (PCNA) on 1,027 consecutive patients referred because of a PPN/M (mean diameter, 3.5 cm; range, 0.8 to 8 cm). The procedure used was as follows: (1) bronchoscopy with exploration of the upper airways and bronchial tree, followed by TBNA and immediate cytologic assessment (ICA); (2) at least three TBPB; (3) if TBNA was diagnostic, the procedure was stopped; if not, a second pass with the needle was performed and then the bronchoscope was removed; (4) if the second TBNA was not diagnostic, PCNA with ICA was performed up to a maximum of three needle passes. Diagnostic sensitivity for malignant lesions was as follows: 53.9% for TBPB, 69.3% for TBNA, 75.4% for TBPB and TBNA together, 93.2% for PCNA, and 95.2% overall. The percentage of benign nodules correctly defined was 41.4% for TBPB, 17.4% for TBNA, 45.8% for PCNA, and 59.5% overall. Examination of the upper airways and bronchial tree was positive for lesions endoscopically visible in 12.6% of cases. The authors' experience shows that transbronchial and percutaneous approaches must be considered complementary and that their integrated use not only increases diagnostic yield but also permits important information to be obtained for disease staging. The creation of teams able to use both approaches with the cytopathologist present for ICA should be encouraged to optimize the diagnostic management of PPN/M with a reduction in diagnostic and hospitalization time and consequent cost saving.

Adolescent↗
PubMed1994

On the site by which alpha-dendrotoxin binds to voltage-dependent potassium channels: site-directed mutagenesis reveals that the lysine triplet 28-30 is not essential for binding.

We constructed a synthetic gene encoding the published amino acid sequence of DTx from Dendroaspis angusticeps, a ligand of voltage-dependent postassium channels that facilitates neurotransmitter release. We expressed it in Escherichia coli as a fusion protein secreted in the culture medium. The recombinant DTx was generated in vitro by chemical treatment and recovered as two isoforms. One of them (rDTx), like the venom toxin, has an N-terminal pyroglutamate whereas the other (rQDTx) has a free N-terminal glutamine. Chromatographic differences between rDTx and natural DTx led us to re-examine the amino acid sequence of natural DTx. In contrast to what was previously published, position 12 was an Asp and not Asn. Despite this difference, rDTx and DTx had similar toxicity in mice and binding affinity to synaptosomes, suggesting that residue 12 is not important for DTx function. Nor is the N-terminal residue implicated in DTx function since rDTx and rQDTx also had similar biological activities. We also synthesized and expressed a mutant of the DTx gene in which the lysine triplet 28-30 was changed into Ala-Ala-Gly. The two resulting recombinant isoforms exhibited only small decreases in biological activity, excluding the possibility that the positively charged lysine triplet 28-30 of DTx is directly involved in the toxin functional site.

Amino Acid Sequence↗
PubMed1994

The low molecular weight protein which co-purifies with alpha-latrotoxin is structurally related to crustacean hyperglycemic hormones.

LMWP is the low molecular weight protein which copurifies with alpha-latrotoxin, the main neurotoxin from the black widow venom. It contains 70 residues and three disulfides. We found that its primary structure, including its 6 half-cystines, can be aligned with the amino acid sequences of crustacean hyperglycemic hormones (CHHs) which contain 72-73 residues and three disulfides. To further investigate this structural relationship, we produced a recombinant analog of LMWP in which the unique Met was changed in Leu (LMWPM35L). LMWPM35L was produced as a folded fusion protein in the periplasm of Escherichia coli and was generated in vitro by treating the fusion protein with cyanogen bromide. We showed that LMWPM35L and CHHs have an identical disulfide pairing pattern and possess some alpha-helical structure, as deduced from a comparison of their circular dichroism spectra. In addition, LMWPM35L and CHHs are consensually predicted to possess a helical structure within the region 13-17. Together, the data indicate that CHHs are structurally related to LMWPM35L and presumably also to LMWP. Finally, preliminary studies showed that LMWPM35L is not toxic to mice and does not form channels in lipid bilayers, two well-known properties of alpha-latrotoxin preparations.

Amino Acid Sequence↗
PubMed1993

Production of recombinant notechis 11'2L, an enzymatically active mutant of a phospholipase A2 from Notechis scutatus scutatus venom, as directly generated by cleavage of a fusion protein produced in Escherichia coli.

We have constructed an expression vector to produce, in Escherichia coli, a fusion protein containing successively two IgG binding domains from staphyloccocal protein A, a nine-amino-acid linker peptide terminating in a methionine residue and the phospholipase A2 notechis 11'2L, an isoform of notexin of Notechis scutatus scutatus venom. Notechis 11'2L is a mutant of the naturally occurring notechis 11'2 [Bouchier, C., Boyot, P., Tesson, F., Trémeau, O., Bouet, F., Hodgson, D., Boulain, J. C. & Ménez, A. (1991) Eur. J. Biochem. 202, 493-500] in which Met8 has been replaced by Leu. The fusion protein was recovered in the periplasmic extract with a yield of 0.25 mg/l culture. It was hydrolyzed with cyanogen bromide, yielding a protein having the molecular mass, amino acid composition and N-terminal sequence of notechis 11'2L. Notechis 11'2L and the wild notechis 11'2 displayed identical circular dichroic spectra and shared similar enzymatic, myotoxic and antigenic properties, suggesting that the recombinant notechis 11'2L was directly generated in a correctly folded form.

Base Sequence↗
PubMed1991

Identification of structurally and functionally important histidine residues in cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae.

Cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae is an alpha 2 dimer (alpha, Mr 63,000), each alpha containing 12 histidines. The covalent incorporation of 6-7 mol of diethyl pyrocarbonate per monomer corresponded to complete enzyme inactivation. This inactivation was reversed by hydroxylamine hydrolysis which regenerates free histidine (and tyrosine) while leaving the carbethoxy group still attached to the epsilon-amino group of lysine. Three histidines, one tyrosine, and four lysines were the main targets of the reagent. Site-directed mutagenesis was also tried to replace each of these modified residues. Given the unstability of the carbethoxy-imidazole bond, the nine histidines that were not modified by diethyl pyrocarbonate were mutated too. For these experiments, the enzyme was expressed in Escherichia coli by using a vector bearing the structural gene in which the first 13 codons were replaced by the first 14 of the CII lambda gene. This substitution had no effect on the kinetic parameters. The combined results of chemical modification and site-directed mutagenesis show that one histidine seems to be part of the active site while two others play an important structural role. On the other hand, labeled lysines and tyrosine are nonessential residues. These results are discussed in light of two recent articles establishing the existence of a second family of aminoacyl-tRNA synthetases devoid of the HIGH and KMSKS consensus sequences and containing no Rossmann's domain in their three-dimensional structures.

Amino Acid Sequence↗
PubMed1990

New indices of arterial oxygen status: an experience on 168 subjects.

We investigated the repeatability and the clinical usefulness of assessing several parameters related to oxygen status in arterial blood. pH, PaCO2, PaO2, oxygen saturation, total hemoglobin concentration, and fractions of carboxy- and methemoglobin were measured in arterial blood. Applying a new algorithm, other parameters were calculated from the above mentioned, among which total oxygen concentration, half saturation tension, 2,3-DPG concentration, uncompensated mixed venous oxygen tension, and cardiac oxygen compensation factor. In 12 subjects we performed three simultaneous determinations of these indices with a good repeatability of the measures. Then, we examined the same parameters in 92 subjects with normal PaO2, 52 patients with respiratory failure (38 normocapnic and 14 hypercapnic), 9 of which were monitored to follow the behaviour of the measures with time according to variations of the clinical status, and in 12 subjects before and after exercise. We found that important alterations in oxygen transport and delivery may be present even when oxygen tension in arterial blood is normal. Furthermore, oxygen status of arterial blood in respiratory failure may be affected by several factors, whose knowledge is essential for a proper treatment. Our data suggest that such an approach to the evaluation of oxygen status offers interesting clinical perspectives.

2,3-Diphosphoglycerate↗
PubMed1987

Does activity of pulmonary sarcoidosis depend on disease duration? A correlation between bronchoalveolar lavage, scintigraphic, radiologic, and physiologic parameters and time of onset of the disease.

In pulmonary sarcoidosis the alveolitis is thought to precede and modulate granuloma formation and fibrosis. Thus, patients with early disease may have a more active pulmonary process, while patients with long standing sarcoidosis are thought to have fibrotic, stabilized lesions. The aim of the present study is to correlate radiographic stage, alveolitis activity, and respiratory function changes with the length of the disease. For this purpose 116 patients with biopsy-proven untreated pulmonary sarcoidosis have been divided in three groups according to time of onset of the illness: group A (48 patients with sarcoidosis duration less than or equal to 3 months), group B (25 patients with sarcoidosis duration greater than 3 less than or equal to 6 months), and group C (43 patients with sarcoidosis duration greater than 6 months less than or equal to 3 years). We found a progression with time from roentgenographic stage I to III, but no correlation was present between the disease duration and T lymphocytes of BAL, results of gallium lung scans, and respiratory function values. Our results suggest that the activity of alveolitis and the derangement of respiratory function in pulmonary sarcoidosis are unrelated to the time of onset of the disease.

Adolescent↗
PubMed1986

Evaluation of Duovent in the prevention of exercise-induced asthma.

Exercise-induced asthma (EIA) is a frequent symptom in asthmatic patients. The study of drugs which inhibit EIA is very important because EIA seriously limits the life of the patients. In addition, this study could allow further insights to be gained into the pathogenesis of the phenomenon. The aim of the present study is to evaluate the duration of the protective effect of fenoterol alone or in combination with ipratropium bromide administered 5 h before exercise. For this purpose we studied 12 asthmatic patients (7 males and 5 females, mean age 23, range 7-41 years) with EIA, in clinical and functional stable state. Respiratory function parameters were measured before and 5 h after the administration of fenoterol 400 or 200 micrograms, or fenoterol 200 micrograms plus ipratropium bromide 80 micrograms (Duovent), or placebo in randomized order. Then, such parameters were measured at 5, 15, 30, and 60 min after exercise, which consisted of free running up and down the stairs. The results show that, at 5 h after the administration of each drug, the protective action against EIA is only partial. However, the intense bronchodilation afforded by the 'active' drugs, and particularly by 400 micrograms of fenoterol, keeps post-exertional respiratory parameters above the baseline values.

Adolescent↗
PubMed1983

[IgM myeloma].

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Antineoplastic Combined Chemotherapy Protocols↗