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Biomedical subjects

A Bossi

Publications and source records attributed to A Bossi.

At least 19 recordsLinked to original sources
PubMed2006

Molecularly imprinted polymers for the recognition of proteins: the state of the art.

Molecular imprinting has proved to be an effective technique for the creation of recognition sites on a polymer scaffold. Protein imprinting has been a focus for many chemists working in the area of molecular recognition, since the creation of synthetic polymers that can specifically recognise proteins is a very challenging but potentially extremely rewarding objective. It is expected that molecularly imprinted polymers (MIPs) with specificity for proteins will find application in medicine, diagnostics, proteomics, environmental analysis, sensors and drug delivery. In this review, the authors provide an overview of the progress achieved in the decade between 1994 and 2005, with respect to the challenging area of MIPs for protein recognition. The discussion furnishes a comparative analysis of different approaches developed, underlining their relative advantages and disadvantages and highlighting trends and possible future directions.

Adsorption↗
PubMed2004

[Health education as prevention of alcoholism in young people].

The close correlation between road accidents and excessive consumption of alcoholic beverages, as well as the lowering of the minimal age for alcohol drinking, prompted the Authors to examine the acute and chronic effects of alcohol on the human organism; therefore, they analyze the metabolic effects of alcohol and, after having described it's few beneficial effects, they furnish some suggestions to avoid above all "the havoc of Saturday evening", outlining in detail the causes and progressive states of alcoholic intoxication. The Authors, in agreement with the current views of prevention, professed by the scientific community, emphasize information and health education as the main tools that guarantee primary prevention in young people who, as it is well-known, belong to the most vulnerable segments of the population. To such purpose they created a poster, to which a CD will follow, that could be distributed and discussed in schools with the purpose to encourage social debate and constructive involvement of students, teachers and their parents.

Accidents, Traffic↗
PubMed2003

Controlled enzyme-immobilisation on capillaries for microreactors for peptide mapping.

In the present paper, the covalent immobilisation of the digesting enzyme trypsin has been achieved through photo-immobilisation on a portion of a silica capillary, thus leading to the construction of a capillary electrophoretic (CE)-microreactor for peptide mapping. The CE-microreactor is characterised by being a single piece, thus ensuring no fluidic or electrical leakage. The enzyme was immobilised with a surface density of 15.8 microg/cm(2), the stability was high (80% after 38 days) and the rate of conversion was 0.2 ng/s. On-line protein mapping was tested with proteins of different dimensions, showing competitiveness in terms of time (completed map within 15 min) and exhaustive maps of small proteins. The results of the CE-microreactor and the potential to immobilise biocomponents easily on a desired portion of the capillary indicate further developments towards the construction of a variety of miniaturised enzymatic screening devices for high-throughput screening analysis.

Bradykinin↗
PubMed2002

Increased CD69 expression on peripheral blood eosinophils after specific inhalation challenge.

BACKGROUND: CD69 is a molecule expressed on human eosinophils after cytokine-activation. Different studies have described the eosinophil activation, evaluated by CD69 expression, at the site of an allergic inflammation. In this study we evaluated the expression of CD69 on peripheral blood eosinophils after a specific inhalation challenge (SIC), in order to better define the state of activation of peripheral blood eosinophils after exposure to sensitizers. METHODS: CD69 expression was evaluated by flow cytometry in nine asthmatic patients before and after a positive SIC with high or low molecular weight agents (pollens, house dust mites, Penicillia, isocyanates) and in 11 asthmatic patients who underwent an inhalation challenge with placebo. CD69 expression was evaluated at baseline, 120 min, and 240 min after the SIC or the placebo. RESULTS: Baseline (before challenge) CD69 expression was comparable between the group of SIC positive patients and the placebo group. CD69 expression on peripheral eosinophils significantly increased 240 min after the challenge in positive SIC patients compared to placebo. In patients with a positive SIC the percentage of peripheral blood eosinophils significantly decreased at 120 and 240 min after the inhalation challenge with respect to the baseline. CONCLUSION: CD69 expression on peripheral blood eosinophils is significantly increased in asthmatic patients after exposure to the sensitizing agent. These data show that the effects of a bronchial stimulation are also detectable on peripheral blood eosinophils.

Adult↗
PubMed2001

Surface-grafted molecularly imprinted polymers for protein recognition.

A technique for coating microplate wells with molecularly imprinted polymers (MIPs) specific for proteins is presented. 3-Aminophenylboronic acid was polymerized in the presence of the following templates: microperoxidase, horseradish peroxidase, lactoperoxidase, and hemoglobin, via oxidation of the monomer by ammonium persulfate. This process resulted in the grafting of a thin polymer layer to the polystyrene surface of the microplates. Imprinting resulted in an increased affinity of the polymer toward the corresponding templates. The influence of the washing procedure, template concentration, and buffer pH on the polymer affinity was analyzed. It was shown that the stabilizing function of the support and spatial orientation of the polymer chains and template functional groups are the major factors affecting the imprint formation and template recognition. Easy preparation of the MIPs, their high stability, and their ability to recognize small and large proteins, as well as to discriminate molecules with small variations in charge, make this approach attractive and broadly applicable in biotechnology, assays and sensors.

Animals↗
PubMed2001

Interaction of chicken liver basic fatty acid-binding protein with fatty acids: a 13C NMR and fluorescence study.

Two different groups of liver fatty acid-binding proteins (L-FABPs) are known: the mammalian type and the basic type. Very few members of this second group of L-FABPs have been characterized and studied, whereas most of the past studies were concerned with the mammalian type. The interactions of chicken liver basic fatty acid-binding protein (Lb-FABP) with 1-(13)C-enriched palmitic acid (PA) and oleic acid (OA) were investigated by (13)C NMR spectroscopy. Samples containing fatty acids (FA) and Lb-FABP at different molar ratios exhibited only a single carboxylate resonance corresponding to bound FA, and showed a binding stoichiometry of 1:1 both for PA and for OA. Fluorescence spectroscopy measurements yielded the same binding stoichiometry for the interaction with cis-parinaric acid [K(d) = 0.38(4) microM]. Competition studies between cis-parinaric acid and the natural ligands indicated a decreasing affinity of chicken Lb-FABP for PA, OA, and retinoic acid (RA). (13)C NMR proved that pH and ionic strength affect complex stability. The carboxyl signal intensity reversibly decreased upon lowering the pH up to 5. The pH dependence of the bound carboxyl chemical shift yielded an apparent pK(a) of 4.8. A decrease of the integrated intensity of the bound carboxylic signal in the NMR spectra was observed while increasing the chloride ion concentration up to 200 mM. This body of evidence indicates that the bound FA is completely ionized at pH 7.4, that its polar head is positioned in a solvent-accessible region, that a FA-protein strong ionic bond is not present, and that high ionic strength causes the release of the bound FA. The reported results show that, insofar as the number of bound ligands and its relative affinity for different FAs are concerned, chicken Lb-FABP is remarkably different from the mammalian liver FABPs, and, within its subfamily, that it is more similar to catfish Lb-FABP while it behaves quite differently from shark or axolotl Lb-FABPs.

Animals↗
PubMed2001

Do orientation effects contribute to the molecular weight dependence of the free solution mobility of DNA?

The free solution mobility of DNA increases with increasing molecular weight and then levels off and becomes constant at molecular weights above approximately 400 bp (Stellwagen, N. C., Gelfi, C., Righetti, P. G., Biopolymers 1997,42, 687-703). To investigate whether the increase in mobility could be attributed to an increased orientation of the larger DNA molecules in the electric field, the free solution mobility of DNA was measured by capillary electrophoresis as a function of electric field strength. Mixtures containing 20-, 118- and 422-bp DNA molecules, and 20-, 422- and 2116-bp DNAs, were studied. If the larger DNA molecules in each mixture were oriented by the electric field, their mobilities should increase with electric field strength faster than the mobility of the 20-bp oligomer, which is too small to be oriented by the electric fields used in this study. Instead, the ratios of the mobilities of the 118-, 422- and 2116-bp fragments to the mobility of the 20-bp oligomer were independent of electric field strength. Hence, orientation effects are not important for DNA molecules up to 2 kbp in size, in electric fields up to 500 V/cm in amplitude. An explanation is suggested.

Base Sequence↗
PubMed2001

Substitution of antibodies and receptors with molecularly imprinted polymers in enzyme-linked and fluorescent assays.

A new technique for coating microtitre plates with molecularly imprinted polymers (MIP), specific for low-molecular weight analytes (epinephrine, atrazine) and proteins is presented. Oxidative polymerization was performed in the presence of template; monomers: 3-aminophenylboronic acid (APBA), 3-thiopheneboronic acid (TBA) and aniline were polymerized in water and the polymers were grafted onto the polystyrene surface of the microplates. It was found that this process results in the creation of synthetic materials with antibody-like binding properties. It was shown that the MIP-coated microplates are particularly useful for assay development. The high stability of the polymers and good reproducibility of the measurements make MIP coating an attractive alternative to conventional antibodies or receptors used in enzyme linked immunosorbent assay (ELISA).

Antibodies↗
PubMed2001

Classification criteria and distinction between migraine and tension-type headache in children.

The International Headache Society (IHS) classification system (1988) was developed primarily for headache disorders in adults and its validity for paediatric age is currently under discussion; in 1995 Seshia et al. proposed a revision of the criteria for migraine without aura to make diagnostic criteria more applicable to children. The purposes of the current study were to: (1) compare the IHS classification with the Seshia proposal, (2) compare the children affected by migraine without aura (MO) with the children affected by tension headache (TH) as defined by Seshia, for characteristics which are not included in the classification. The patients are a series of 320 children (mean age 9.9, SD 2.6 years; 144 males, 176 females) with recurrent or chronic headaches referred to a headache clinic in Milan, Italy. Using the Seshia criteria instead of the IHS criteria a higher number of children were included in the MO category: bilateral pain and family history of migraine were the most important factors which allowed a shift of children into this category. However, with the Seshia classification there was no reduction in the number of unclassifiable children. The reason why some children could not be classified was a short duration of attacks; the majority of unclassifiable children were 6 years old or less. No relevant difference was found between children with MO and children with TH for the following variables: occurrence of attacks in the afternoon or evening after school, reduction of attacks during school holidays, full-time schooling, after-school activities on school days, disordered daily life. On the contrary children with MO when compared with those with TH showed a higher number of precipitating factors and for the following factors a significant difference was found: exposure to TV or a computer, sleep deficiency, and strong emotions. Furthermore, children with MO showed a greater severity of attacks.

Activities of Daily Living↗
PubMed2001

Follow-up of vertically HIV-1-infected long-surviving children.

This study describes the clinical, immunologic, and virological characteristics of 30 vertically human immunodeficiency virus type 1 (HIV-1)-infected children older than 8 years of age (long-survivors) before the introduction of protease inhibitors therapy. All of them were followed from birth. At the age of 8 years, 7 children presented no HIV-1-associated signs or only mild ones and only 5 had severe clinical manifestations (acquired immune deficiency virus [AIDS]). The remaining 18 children presented moderate signs with some immunodeficiency. The follow-up from 8 years of age (3.5 years on the average) showed that 6 children remained asymptomatic and were therefore defined as long-survivors nonprogressors (average, 13 years) and only 4 children developed AIDS. Progressive encephalopathy was the most striking clinical manifestation at follow-up and occurred in 6 children (always after immunodeficiency) with a polymorphic picture combining eye movement alterations, pyramidal signs and symptoms and mental deterioration. The majority of our long-survivors carried a virus with nonsyncytia-inducing phenotype, thus confirming its association with long survival. A switch to syncytia-inducing phenotype was observed only in 2 cases during the follow-up, but their clinical status did not change at follow-up.

Adolescent↗
PubMed2001

Automated protamine dose assay in heparin reversal management after cardiopulmonary by pass.

BACKGROUND: To evaluate the impact of automated Protamine Dose Assay (PDA) performed with Hemochron 8000 (International Technodyne Company, Edison, NJ) on the management of heparin reversal after cardiopulmonary bypass (CPB). PDA was compared with empirical protamine to heparin ratio with regard to calculation of the protamine dose, and the sensitivity of PDA and ACT to residual circulating heparin after protamine administration was investigated too. METHODS DESIGN: prospective and randomized study. SETTING: cardiac surgical center of a General Hospital. PARTICIPANTS: 50 patients undergoing elective cardiac surgery with CPB. INTERVENTIONS: after CPB patients randomly received protamine according to our standard empirical ratio of 1 mg. protamine/100 U. heparin (group S, 24 patients), or to PDA result (group T, 26 patients) based on protamine titration method of determining circulating heparin. After protamine administration ACT and PDA were performed to assess heparin reversal and detect residual circulating heparin. Based on the PDA result, additional protamine was administered in both groups when required. MEASUREMENTS: in both groups basal and post-heparin ACT values, protamine doses, ACT and PDA after protamine administration were measured. RESULTS: The protamine dose was significantly lower (30%) in patients treated according to PDA. In 20% of patients showing normal ACT PDA revealed still circulating heparin, and additional protamine was required. In all other cases ACT and PDA both confirmed heparin reversal. CONCLUSIONS: PDA allowed us to administer a significantly lower amount of protamine. This can reduce incidence of adverse effects of over- and under-infusion of protamine. PDA also proved to be more sensitive than ACT in detecting residual circulating heparin after protamine administration.

Anticoagulants↗
PubMed2000

DNA and buffers: are there any noninteracting, neutral pH buffers?

The interaction of DNA with various neutral pH, amine-based buffers has been analyzed by free solution capillary electrophoresis, using a mixture of a plasmid-sized DNA molecule and a small DNA oligonucleotide as the reporter system. The two DNAs migrate as separate, nearly Gaussian-shaped peaks in 20-80 mM TAE (TAE, Tris-acetate-EDTA; Tris, tris[hydroxymethyl]aminomethane) buffer. The separation between the peaks gradually increases with increasing TAE buffer concentration because of differences in solvent friction between large and small DNA molecules. The two DNAs form complexes with the borate ions in TBE (Tris-borate-EDTA) buffer, with mobilities that depend on the DNA/borate ratio. In 45 mM TBE buffer, the two DNAs comigrate as a single sharp peak, with a mobility that is faster than either of the constituent DNAs in the same buffer. Hence, the mixed DNA-borate complex is stabilized by the binding of additional borate ions, possibly forming bridges between the different DNAs. The mixed DNA-borate complex is gradually dissociated into its component DNAs by increasing the TBE concentration, possibly because the borate binding sites become saturated at high buffer concentrations. Other neutral pH, amine-based buffers, such as Mops (3-[N-morpholino]propanesulfonic acid), Hepes (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]), Bes (N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid), Tes (N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid), and tricine (N-tris[hydroxymethyl]methylglycine) also form complexes with DNA, giving distorted peaks in the electropherograms. The combined results indicate that borate buffers and most neutral pH, amine-based buffers interact with DNA.

Acetates↗
PubMed2000

Capillary electrophoresis coupled to biosensor detection.

The present review highlights some modern aspects of biosensor revelation, a detection method which has already found a large number of applications in healthcare, food industry and environmental analysis. First, the concept of bio-recognition, which is at the heart of biosensor technology, is discussed, with emphasis on host-guest-like recognition mechanisms. This detection device has been successfully coupled, in its first applications, to chromatographic columns, which allow a high resolution of complex mixtures of analytes prior to interaction with the biosensing unit. The properties of the transducing elements, which should generate a signal (e.g., electrochemical, thermal, acoustic, optical) of proper intensity and of relative fast rise, are additionally evaluated and discussed. The review then focuses on potential applications of biosensing units in capillary electrophoresis (CE) devices. CE appears to be an excellent separation methodology to be coupled to biosensor detection, since it is based on miniaturized electrophoretic chambers, fast analysis times, complete automation in sample handling and data treatment and requires extremely small sample volumes. Although only a few applications of CE-based biosensors have been described up to the present, it is anticipated that this hyphenated technique could have a considerable expansion in the coming years.

Biosensing Techniques↗
PubMed2000

An assay for ascorbic acid based on polyaniline-coated microplates.

A technique for modification of the microtiter reader plates well with a polyaniline (PANI) film sensitive for ascorbic acid is presented. The principle of the analyte detection is based on monitoring the changes in optical absorption of the PANI film resulting from the reduction process initiated by ascorbic acid. The detection limit for ascorbic acid is 1 mg/L. Testing with real samples (soft drinks, fruit juices) gave good correlation of the method with iodimetric titration. High sensitivity, stability, and good reproducibility of the measurements make the proposed system an attractive alternative to traditional assays, used in medicine, ecology, and biotechnology.

Aniline Compounds↗
PubMed2000

Characterization of polymeric buffers for operating membrane-trapped enzyme reactors in an electric field.

A novel class of amphoteric, polymeric buffers, is described, consisting of grafting onto growing polyacrylamide chains weakly acidic and basic acrylamido-monomers (called Immobilines; protolytic groups as N-substituents on the nitrogen of the amido bond), for operating a membrane-immobilized enzyme reactor (MIER) in an electric field. With these soluble, polymeric buffers, it is possible to operate the membrane reactor at any optimum of pH activity, for any given enzyme, in the pH 3-10 scale. Such buffers, being amphoteric, are confined in the enzyme reaction chamber by the same isoelectric trapping mechanism. The best buffers were found to be those polymerized in presence of 9% neutral monomer (acrylamide) and containing 20 mM Immobiline as buffering ion. To decrease their viscosity in solution, the polymeric buffers are synthesized at high temperatures (70 degrees C) and in presence of a chain-transfer agent. The weight average molecular size in these conditions has been found to be ca. 200,000 Da. These buffers exhibited excellent performance in a variety of enzyme reactions in the MIER, such as in the case of penicillin G acylase and histidine decarboxylase and were found to greatly stabilize enzyme activity, permitting operation of the MIER over extended periods of time. As an example, in a penicillin G acylase reactor, >75% enzyme activity was maintained over a 10-d cycle of operation, while with conventional buffers more than 90% inactivation was experienced over the same period of time. This novel class of macromolecular, amphoteric buffers could also be exploited in other types of conventional bioreactors not based on an isoelectric trapping mechanism.

Bioreactors↗
PubMed2000

Crystallization of chicken liver (basic) fatty acid binding protein after purification in multicompartment electrolyzers with isoelectric membranes.

A preparation of chicken liver (basic) fatty acid binding protein was purified to homogeneity in multicompartment electrolyzers with isoelectric membranes. Large amounts of the isoelectric point (pI) 9.7 protein were collected into a compartment delimited by pI 8.8 and 11.0 membranes. The protein thus purified produced crystals which diffract to higher resolution than those obtained by purification via preparative isoelectric focusing (IEF) in soluble carrier ampholytes. In addition, a novel orthorhombic form with a different molecular packing was obtained. It is hypothesized that, when using conventional IEF, traces of carrier ampholytes could adhere to the protein, particularly in the hydrophobic ligand-binding pocket, rendering the interpretation of the electron density maps difficult. Multicompartment electrolyzers do not present this drawback, since they are based on insoluble buffering species.

Animals↗
PubMed2000

Capillary electrophoresis of peptides and proteins in isoelectric buffers: an update.

Capillary electrophoresis in acidic, isoelectric buffers is a novel methodology allowing fast protein and peptide analysis in uncoated capillaries. Due to the low pH adopted and to the use of dynamic coating with cellulose derivatives, silanol ionization is essentially suppressed and little interaction of macromolecules with the untreated wall occurs. In addition, due to the low conductivity of quasi-stationary, isoelectric buffers, high-voltage gradients can be applied (up to 800 V/cm) permitting fast peptide analysis with a high resolving power due to minimal diffusional peak spreading. Four such buffers are here described: cysteic acid (Cys-A, pI 1.85), iminodiacetic acid (IDA, pI 2.23), aspartic acid (Asp, pI 2.77) and glutamic acid (Glu, pI 3.22). A number of applications are reported, ranging from food analysis to the study of folding/unfolding transitions of proteins.

Animals↗