PubMed Health⌕ Search

Biomedical subjects

A Bossi

Publications and source records attributed to A Bossi.

At least 55 records · Page 3Linked to original sources
PubMed1996

Steady-state two-dimensional maps of very alkaline proteins in an immobilized pH 10-12 gradient, as exemplified by histone types.

Two-dimensional steady-state patterns of histones are here reported for the first time. The first dimension run consists in nonlinear immobilized pH gradients, spanning the pH 10-12 range. The second dimension run is a standard SDS-PAGE in a constant concentration (15% T) gel slab, in presence of a 6% T stacking gel. All the histone fractions analysed (II-AS, VI-S, VII-S and VIII-S) exhibit pI values between pH 11 and 12. The M(r) values range from 13 to 32 kDa, with the heaviest distribution around 18 kDa. When running all different histone fractions in a single mixture, and analysing the 2-D gel by computerized image data acquisition, a total of 35 individual spots is detected.

Electrophoresis, Agar Gel↗
PubMed1996

Sample-streaks and smears in immobilized pH gradient gels.

In immobilized pH gradient (IPG) gel formulations as wide as pH 4-9, encompassing neutrality and containing the pK 7.0 acrylamido buffer as one of the buffering ions, smears are directly proportional to the total amount of the pK 7.0 species. At a total level of 10 mM pK 7.0 in these gel formulations, severe smears occur not only for mildly hydrophobic proteins (e.g., recombinant alcalase and termamylase) but also for the relatively hydrophilic pI marker proteins. Streaks and smears are essentially abolished in recipes devoid of the pK 7.0 compound or in formulations containing a maximum of 3 mM of this component. Although partitioning in water/n-octanol has shown the pK 7.0 acrylamido buffer to be quite hydrophobic (P = 0.5), the occurrence of smears could be to the presence of oligomers in some commercial preparations. Even when dissolved in n-propanol, some batches of acrylamido buffers might still contain oligomers, probably formed during the synthetic step.

Gels↗
PubMed1996

Investigation on minor degraded derivatives of the recombinant hirudin variant HM2 from Hirudinaria manillensis isolated by isoelectric focusing in multicompartment electrolyzers.

On isoelectric focusing in immobilized pH gradients (IPG) a preparation of recombinant hirudin from Hirudinaria manillensis, purified to homogeneity, was found to still contain a total of 5% minor components: three with higher pI values (pIs 4.10, 4.25 and 4.31), one with a lower pI value (pI 3.98) as compared with the main form (pI 4.03). Multicompartment electrolyzers with isoelectric membranes and micropreparative IPG gel slabs allowed the recovery of pure fractions of such minor components, which were further characterized by electrospray mass spectra, limited proteolysis, and sequence analysis. All four minor isoforms were found to be cleavage products of the parent, full-length hirudin molecule (molecular mass 6797 Da), as follows: the pI 4.31 (5032 Da) had lost sixteen amino acids from the N-terminus, the pI 4.25 (6212 Da) lacked five amino acids from the C-terminus, the pI 4.10 (2980 Da) was a cleavage product at residue Cys37, and the pI 3.98 (6610 Da) lacked the dipeptide Val-Ser at the N-terminus. Combining the extreme resolving power of IPGs with the high accuracy of mass spectra was found to be an attractive strategy in decoding post-synthetic modifications often encountered in r-DNA proteins.

Amino Acid Sequence↗
PubMed1996

Purification of glycopeptide antibiotics by isoelectric focusing in multicompartment electrolyzers with immobiline membranes.

The purification of small glycopeptides (a Hepta-Tyr of the teicoplanin family, exhibiting broad activity against highly glycopeptide-resistant enterococci) by isoelectric focusing in multicompartment electrolyzers with buffering, isoelectric membranes, is described. The main obstacle to such a preparative technique, in common with all focusing methodologies, is the poor solubility of the analyte at the pI value with resultant precipitation and coprecipitation of all impurities with the main fraction. A good solubilizing power was obtained in hydro-organic solvents, particularly a mixture of 6 M urea and 20-25% trifluoroethanol. Best results, however, were obtained with mixtures of 8 M urea and zwitterionic detergents, notably the 3-[(3-cholamidopropyl)dimethylammonia]-1-propanesulfonate (CHAPS) family. A unique behavior of the peptide was found in concentration gradients of CHAPS: solubility increases up to 3.5% CHAPS, but the curve shows a maximum and then solubility decreases again at 5% CHAPS. In mixtures of 8 M urea and 3.5% CHAPS, sample loads of 500 up to 1000 mg Hepta-Tyr could be purified in a single run, with recoveries > 90% and purity in excess of 99%. The main glycopeptide fraction (pI 8.56) was collected into an isoelectric trap delimited by pI 8.46 and pI 8.65 membranes. Attempts at purifying the glycopeptide by most known reversed phase-high performance liquid chromatography (RP-HPLC) techniques failed completely.

Anti-Bacterial Agents↗
PubMed1996

Purification of thermamylase in multicompartment electrolyzers with isoelectric membranes: the problem of protein solubility.

The main alpha-amylase from Bacillus licheniformis (called thermamylase because of its resistance to high temperatures, 90 degrees C) has been subjected to purification by isoelectric focusing in multicompartment electrolyzers with isoelectric membranes. The enzyme tended to precipitate, producing severe smears in proximity of its pI value (7.18). Solubility could not be ameliorated by any of the known means typically adopted in isoelectric focusing and compatible with enzyme activity, such as addition of neutral and zwitterionic surfactants (e.g., Nonidet, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate, up to 2%), mixed hydro-organic solvents (glycerol, ethylene glycol, propylene glycol) and addition of zwitterions unable to form micelles, such as taurine. However, addition of sugars, notably saccharose, sorbitol, and, to a lesser extent, sorbose, greatly improved protein solubility in the pI proximity. The improvement was dramatic if these sugars were admixed with 0.2 M taurine. Additionally, the increment of solubility (which occurred when reaching a level of 40% of the different sugars) was accompanied by a large pI shift, typically reducing the pI value by as much as 0.4 pH units (e.g., from a pI of 7.18 in the absence of additives to a pI of 6.80 in presence of a mixture of 40% sucrose and 0.2 M taurine, the best solubilizer in all the series investigated). This apparent pI shift was not due to a change of pH gradient caused by the presence of additives, since pH measurements in the absence as well as presence of additives gave identical results. The results are explained by the theory of Timasheff and Arakawa on stabilization of protein structure by solvents: sugars (at ca. 1 M concentration) and zwitterions such as taurine belong to class I stabilizers, characterized by negative binding to proteins and by increasing the surface tension of water. As a result, the protein is in a state of "superhydration", which might prevent binding to Immobilines in the gel matrix and might alter some pKs on the protein surface. In solutions of 40% saccharose and 0.2 M taurine, thermamylase could be successfully purified to a single isoelectric and isoionic band in the multicompartment electrolyzer.

Bacillus↗
PubMed1996

Fish species identification by isoelectric focusing of parvalbumins in immobilized pH gradients.

Isoelectric focusing in immobilized pH gradients (IPG) is reported for unequivocal identification of fish species. Three orders of fishes have been analyzed: salmons, flat and cod fishes. In each family, four closely related species have been analyzed. For clear-cut species identification, two strategies have been adopted: (i) to perform IPGs in very narrow (1 pH unit and less) acidic gradients, typically spanning the pH 4-5 range, where fewer proteins are present and the pattern is much clearer; (ii) to focus the analysis on the parvalbumins, since this protein class if highly species-specific and resistant to heat. Thus, not only fresh muscle could be analyzed, but also boiled fish samples. In all cases unambiguous determination of each species could be performed, either by simple visual band inspection or, in the most difficult cases, by densitometric evaluation of the Coomassie-blue stained profiles. The analysis was performed in extracts of single species and also in mixtures of the most closely related species.

Animals↗
PubMed1996

Fourier-transform infrared conformational study of bovine insulin in surfactant solutions.

To obtain conformational data on the monomeric form of insulin, which is believed to be the physiologically active form of the hormone, insulin in sodium dodecyl sulfate solution was studied by Fourier-transform infrared (FTIR) spectroscopy and circular dichroism, and results were compared with those obtained with des(B23-30) octapeptide insulin (DOI) and dimeric insulin in buffer. The FTIR amide I band (1600-1700 cm-1) was examined, and a quantitative evaluation of the secondary structure fractions of the various conformations showed less of a beta-sheet component for both insulin in SDS and DOI in buffer than for insulin in buffer, corresponding to a lack of monomers binding to form dimers. At the concentrations used for FTIR (> or = 2 mg/mL), the CD spectra of insulin in SDS and DOI in buffer were qualitatively identical but different from that of insulin in buffer, which is associated at these concentrations. The CD spectrum pattern of insulin in very dilute solution (80 nM), where it is prevalently monomeric, is very similar to that of monomeric insulin in SDS, which suggests that the conformation of the hormone in the two cases is very similar.

Animals↗
PubMed1996

Fetal growth velocity: kinetic, clinical, and biological aspects.

With the aim of determining fetal growth kinetics, prenatal data were analysed which had been longitudinally collected in the framework of a perinatal growth survey. The sample comprised 238 singleton normal pregnancies, selected in Genoa and Turin (between 1987 and 1990), and repeatedly assessed by ultrasound scans (five to nine per pregnancy). Five morphometric traits were considered: BPD (biparietal diameter), OFD (occipitofrontal diameter), HC (head circumference), FDL (femur diaphysis length) and AC (abdomen circumference). Growth rate seemed to increase in the early part of the second trimester, and decrease subsequently: velocity peaks were steeper and earlier for head diameters and circumference (about 18 weeks) than for femur length (20 weeks) and abdomen circumference (22 weeks). Velocity standards were traced using a longitudinal two-stage linear model: this ensures unbiased description of the shape of the growth curve, even when growth kinetics are asynchronous, and efficient estimation of the outer centiles--the most useful for diagnostic purposes.

Abdomen↗
PubMed1995

Fractionation of carrier ampholytes in multicompartment electrolyzers with isoelectric membranes.

Multicompartment electrolyzers with isoelectric membranes can be successfully utilized for the preparation of carrier ampholytes of narrow pH ranges from commercially available wider pH intervals. An example is given on the preparation of a 0.6 pH unit (pH 6.7-7.3) range from a standard pH 6-8 interval. A 10% Ampholine solution is focused in an electrolyzer equipped with the following isoelectric membranes: pI 6.0, 6.7, 7.3 and 8.0. The narrow pH 6.7-7.3 cut can be efficiently utilized for base-line separation and quantitation of hemoglobin A from its glycated form, Hb A1c. This analysis is important for the screening and follow up of diabetic patients. The advantage of multicompartment electrolyzers are: (i) precision in the preparation of narrow pH cuts, due to the presence of membranes with defined pI values; (ii) ability to perform in both small- and large-scale operations and (iii) absence of contaminants leaching from granulated supports, as typical of some previous techniques.

Chemical Fractionation↗
PubMed1995

An ICRU 50 radiotherapy treatment chart.

We illustrate a radiotherapy treatment chart elaborated to fulfil the necessity for clarity in reporting information about radiotherapeutic treatment. The schematic configuration of the chart results from the experience and the cooperation of physicists, physicians and technicians, and an effort has been made to satisfy Levels 2/3 of the ICRU 50 recommendations. The chart has been divided into four sections corresponding to different kinds of information: a cover sheet, a section containing data about the treatment planning geometry and the console parameters adopted, a section showing dosimetric data, and a section showing treatment data. The chart seems to give a good level of accuracy in reporting treatment plan information.

Humans↗
PubMed1995

Scintigraphic assessment of pituitary adenomas and several diseases by indium-111-pentetreotide.

The aim of this study was to identify the specific somatostatin receptors expressed by several tumors, utilizing 111In-octreotide, a long acting somatostatin analogue. We studied two different groups of patients: the first group was composed of 21 patients suffering from different pituitary adenomas, while the second group consisted of 12 patients affected by several different neoplasms. In vivo scintigraphy showed pentetreotide receptors only in large GH-secreting adenomas and several macroadenomas. Concerning the endocrine activity of the adenomas, 111In-octreotide showed a good sensibility in detecting GH-secreting tumors. In the second group, 111In-octeotride proved to be a good diagnostic tool to show carcinoid lesions which were missed by other complementary methodologies.

Adenoma↗
PubMed1994

Focusing of alkaline proteases (subtilisins) in pH 10-12 immobilized gradients.

Isoelectric focusing in very alkaline immobilized pH gradients (IPG) was adopted for checking the purity and assessing the pI value of two strongly alkaline proteases: Savinase and Durazym. The first enzyme (known to be the most alkaline) contains 5 Asp, 5 Glu, 7 His, 7 Tyr, 5 Lys, no Cys and 8 Arg residues and should have a theoretical pI of 9.7. Yet, when focused in a pH 9-11 IPG interval, it was lost in the cathodic compartment. After repeated attempts at creating even more alkaline pH intervals, a pH 10-12 IPG range was finally optimized and proved successful in focusing both enzymes midway between the two electrodic compartments. The pI of Savinase was measured as 11.15 +/- 0.15; that of Durazym as 10.95 +/- 0.20 and that of the pI marker cytochrome c as 10.6 +/- 0.17. Both enzymes (and a number of minor components in each preparation) were proven to be active by an in situ zymogram consisting of a casein/agar overlay. The discrepancy between theoretical and experimental pI values could not be fully reconciled: when correcting for pK values of amino acids in proteins at 10 degrees C, instead of the tabulated values at 25 degrees C, the pI should increase to a value of 10. Differential UV spectra showed that ca. 1/2 Tyr are buried in the protein interior and are thus unable to contribute to surface charge. This further increases the pI value by 0.3 pH units to a value of 10.3, still quite removed from the experimentally assessed pI value (in the gel, at 10 degrees C) of 11.15.

Amino Acid Sequence↗
PubMed1994

Evaluation of most frequent errors in daily compilation and use of a radiation treatment chart.

Between 1 March and 30 April (1994) we recorded the errors detected by the physician, the radiographer or the physicist during prescription, preparation and execution phases of 227 treatment plans. The radiation treatment modalities used were the following: (i) single or opposed fields, moulded or not; and (ii) multiple fields or kinetic techniques. The total number of sessions performed is 1613 with the cobalt unit and 2131 with the linear accelerator (total, 3744). The total number of wrong data is 155, consisting of 24/227 (10.5%) in compilation, 22/3744 (0.58%) in execution and 109/3744 (2.9%) in registration phases. The number of missing data is 140, consisting of 10/227 (4.4%) in compilation, 9/3744 (0.2%) in execution and 121/3744 (3.2%) in registration phases. Wrong data of compilation, even if in high rate (10.5%), were all found during the same compilation phase or at the first treatment, so that they did not alter the exactness of the treatment plan. Wrong and missing data, found in the registration phase (2.9% and 3.2%, respectively), depend on the repetition of daily treatment and on the registration of data on the chart after having digitized them on the display.

Cobalt Radioisotopes↗
PubMed1993

[The irradiated volume in stage III non-small-cell carcinoma of the lung. Comparison of outdated and new techniques].

March 1988 through April 1992, three hundred and ninety-six patients affected with bronchogenic carcinoma were treated at the Department of Radiation Oncology of the S. Anna Hospital, Como, Italy. A hundred and ten patients presenting stage-III non-small-cell lung carcinoma were evaluable. All evaluable patients underwent radiation therapy alone, with either palliative or curative purposes. Two main periods can be distinguished: in period A, before June 30th, 1990, treatment planning included conventional techniques, with no simulators; the patients were treated with opposing anteroposterior fields only. In period B, after July 1st, 1990, either the simulator alone was used or a simulator, a CT unit and a treatment planning computer system were combined; anteroposterior opposing fields or multiportal technique were used. Median overall survival was 10 months. Independent of treatment goals, the irradiated volume was markedly different in the patients treated in period A than in those treated in period B. A marked and statistically significant increase in survival was observed in group B. Survival also increased in patients treated with doses > 40 Gy, but only if treatment planning had used adequate technology and accuracy. To conclude, better survival can be achieved only by improving treatment accuracy and quality.

Adult↗