Biomedical subjects

A Bossi

Publications and source records attributed to A Bossi.

At least 37 records · Page 2Linked to original sources

PubMed2000

Longitudinal distance standards of fetal growth. Intrauterine and Infant Longitudinal Growth Study: IILGS.

BACKGROUND: Most ultrasonographic fetal growth norms are derived from cross-sectional data or from longitudinal data treated as coming from cross-sectional studies, although only longitudinal models may detect particular aspects of fetal growth shape, such as peak of growth velocity. MATERIALS AND METHODS: The sample included 238 singleton normal pregnancies. All the fetal traits under study (biparietal diameter, occipito-frontal diameter, head circumference, femur diaphysis length and abdomen circumference) were measured according to the classical ultrasound techniques by highly trained operators. Individual growth profiles (made up of 5 to 9 measures) were taken at regular intervals between the 12th and the 40th week. Growth norms were traced by means of a two-stage linear model: (I) a 3-constant fetal growth function was fitted to each individual growth profile, (II) growth centiles were based upon the weighted mean and covariance matrix of the individual growth constants. RESULTS: Fetal growth curves show a sigmoid shape with a maximum slope (i.e. a peak growth velocity) which occurs earlier for head diameters (about 18 weeks), later for femur diaphysis length (20 weeks) and abdomen circumference (22 weeks). During intrauterine growth, all traits show a progressive increase in interindividual variability, which is more prominent for abdomen circumference. CONCLUSION: The mathematical model applied to a large sample of growth profiles provided a satisfactory description of the individual fetal development and its biological variability, and allowed the construction of longitudinal distance standards useful for clinical purposes.

Abdomen↗

PubMed2000

[Prevention focused on sexual behavior].

In Italy, studies on the sexual behaviour of young persons are rare, especially those conducted among samples that are representative of the same-age general population. This report presents the preliminary results of a knowledge, attitudes and practices survey on sexual behaviour among a representative sample of individuals 18-26 years of age. The study was begun in 1994, and as of 30 June 1997, 8533 (32%) of the 26,536 questionnaires sent so far have been completed: 3947 (46%) by males and 4586 (54%) by females. 70% of the males and 64% of the females reported that they are sexually active; the median age at first sexual experience is 18 years for both genders. The proportion of respondents who use condoms with occasional sexual partners is low, and only half of the respondents demonstrated an adequate level of knowledge regarding the transmission of HIV.

Adolescent↗

PubMed1999

Electrically immobilized enzyme reactors: bioconversion of a charged substrate. Hydrolysis Of penicillin G by penicillin G acylase.

The possibility of using the multicompartment immobilized enzyme reactor (MIER) in presence of a charged substrate is here explored. Penicillin G acylase is used to convert penicillin G (a free acid, with a pK of 2.6) into two charged products: phenyl acetic acid (PAA, with a pK of 4.2) and 6-aminopenicillanic acid (6-APA, a zwitterion with a pI of 3.6). The enzyme is trapped by an isoelectric mechanism in a chamber of the electrolyzer delimited by a pI 5.0 and a pI 9.0 amphoteric, isoelectric membranes. Under normal operating conditions (continuous substrate feeding in the presence of an electric field), only a low substrate conversion can be achieved, due to rapid electrophoretic transport of unreacted penicillin G out of the reaction chamber towards the anode. Excellent conversion rates (>96%) are obtained under a "doubly-discontinuous" operation mode: a time-lapse substrate feeding, accompanied by short times (4-8 min) of electric field interruption. The product of interest (6-APA, a precursor of semisynthetic penicillins), by virtue of its amphoteric nature, is trapped in a chamber delimited by a pI 3.5 membrane and a pI 5.5 membrane, adjacent to the reaction chamber on its anodic side. The other contaminant product (PAA) first accumulates in the same chamber and then progressively vacates it to collect in the anodic reservoir, leaving behind a pure 6-APA solution. In this operation mode, vanishing amounts of unreacted substrate (penicillin G) leave the reaction chamber to contaminate the adjacent, anodic chambers. A novel class of zwitterionic buffers is additionally reported, able to cover very thoroughly any pH value along the pH 3-10 interval: polymeric, zwitterionic buffers, synthesized with the principle of the Immobiline (acrylamido weak acids and bases) chemicals. Enhanced enzyme reactivity is found in this macromolecular buffers as compared to conventional ones.

Bioreactors↗

PubMed1999

General experimental aspects of the use of isoelectric buffers in capillary electrophoresis.

Four acidic, isoelectric buffers, for peptide and protein separations, have been recently described and adopted in capillary zone electrophoresis: cysteic acid [Cys-A, isoelectric point (pI) 1.85], iminodiacetic acid (IDA, pI 2.23), aspartic acid (Asp, pI 2.77) and glutamic acid (Glu, pI 3.22). These four buffers allow to explore an acidic portion of the titration curves of macroions, covering about 1.6 pH units (from pH 1.85 to ca. 3.45), thus permitting resolution of compounds having coincident titration curves at a given pH value. Given the rather acidic pI values of these buffers, their long-term stability has been investigated, by monitoring pH and conductivity changes upon increasing storage times. When dissolved in plain water, all four buffers appear to give constant pH and conductivity readings up to 15 days; after that, the conductivity keeps steadily increasing in a similar fashion. The same parameters, when the same buffers are dissolved in 6 M urea, appear to be stable for only one week, with the conductivity progressively augmenting after this period. A similar behaviour is exhibited by histidine (pI 7.70), a neutral, isoelectric buffer adopted for separation of DNA fragments. By mass spectrometry, Cys-A shows minute amounts (ca. 1%) of a degradation product after ageing for 3 weeks; in the same time period, Glu is extensively degraded (20%). No degradation species could be detected in IDA and Asp solutions. It is additionally shown that the acidic buffers are not quite stationary in the electric field, but can be transported at progressively higher rates (according to the pI value) from the cathodic to the anodic vessel. This is due to the fact that, at their respective pI values, a fraction of the amphotere has to be negatively charged in order to provide counterions to the excess of protons due to bulk water dissociation. Guidelines are given for the proper use and storage of such buffers.

Buffers↗

PubMed1999

Capillary electrophoresis of peptides and proteins in acidic, isoelectric buffers: recent developments.

The use of isoelectric buffers in capillary zone electrophoresis is here reviewed. Such buffers allow delivery of very high voltage gradients (up to 1000 V/cm in relatively large bore capillaries, e.g. 75-100 microm I.D.), permitting separations of the order of a few minutes and thus conserving (in fact favouring) very high resolution due to minimal, diffusion-driven, peak spreading. Isoelectric Asp (pI 2.77 at 50 mM concentration and 25 degrees C) provides a medium of high resolving power for generating peptide maps. In difficult cases, of coincident titration curves, the pH can be moved up to higher values (e.g. pH 3.0 for 30 mM Asp) thus eliciting separation of unresolved peptides at pH 2.77. This was illustrated by running peptide maps of tryptic digests of human beta globin chains. Also imino diacetic acid (pI 2.33 at 50 mM concentration) allows generation of high resolution peptide maps. Isoelectric Asp, in presence of 7 M urea and 0.5% hydroxyethyl cellulose (Mn = 27 000 Da) is also the preferred medium for fast separation and analysis of storage proteins in cereals, such as gliadins in soft and durum wheat and zeins in maize. A solution of 50 mM iminodiacetic acid (pI 2.23) containing 7 M urea and 0.5% hydroxyethylcellulose (apparent pH 3.2) is effectively used as background electrolyte for fast separation of heme-free, denatured globin (alpha and beta) chains. In the presence of neutral to neutral amino acid substitutions, it is additionally shown that the inclusion of 3% surfactant (Tween 20) in the sample and background electrolyte induces the separation of the wild-type and mutant chains, probably by a mechanism of hydrophobic interaction of the more hydrophobic mutant with the detergent micelle, via a mechanism similar to 'micellar electrokinetic chromatography'.

Animals↗

PubMed1999

Analysis of cross-linked human hemoglobin by conventional isoelectric focusing, immobilized pH gradients, capillary electrophoresis, and mass spectrometry.

Diaspirin cross-linked hemoglobin (DCLHb), a hemoglobin-based oxygen carrier exhibiting near physiological oxygen binding capability and devoid of nephrotoxic side effects, was previously found, by gel permeation, reversed-phase high performance liquid chromatography (RP-HPLC) and mass spectrometry, to consist of ca. 94% cross-linked product (reacted on the Lys 99 of two alpha-chains), accompanied by ca. 6% cross-linked Hb, which also reacted on the Lys 132 and/or Lys-144 of the beta-chains and a small amount of intermolecularly cross-linked dimers. However, conventional isoelectric focusing in carrier ampholyte buffers (CA-IEF) gave an unexpected spectrum of four major, almost equally represented, pI species in the pH range of 6.82-7.01, a band of mid-intensity with a pI of 7.11, and two minor components with pls of 6.73 and 6.77. This extraordinary polydispersity was reevaluated by other surface charge probes, such as immobilized pH gradients (IPG) and capillary zone electrophoresis (CZE) of native and denatured globin chains. IPGs of DCLHb gave the expected spectrum of bands, consisting of a main component (92%) with pl 7.337 and three additional minor bands, with lower pIs, representing ca. 8% of the total. These data were in agreement with CZE profiles of native DCLHb, which resolved, in addition to the main DCLHb peak, 3-4 minor components representing ca. 10% of the total. Also, CZE of denatured, heme-free globin chains gave the expected pattern with only traces of minor, extrareacted species. The latter technique, in addition to resolving alpha- and beta-globin chains in a 1:1 ratio in control Hb, resolved a free beta- and the alpha-alpha-dimer in DCLHb. In a 1:1 mixture of control and DCLHb, three peaks were observed, eluting in the order alpha-, alpha-alpha- and beta-globin chains. The identity of the major DCLHb and of the minor species was ascertained by mass spectrometry.

Cross-Linking Reagents↗

PubMed1999

Soluble interleukin 2 receptor (sIL2R) in monitoring advanced lung cancer during chemotherapy.

STUDY OBJECTIVES: To evaluate the usefulness of measuring sIL2R for diagnostic and prognostic purposes and for monitoring disease during a 6-month period of chemotherapy, and to investigate the clinical significance of sIL2R serum concentrations. METHODS: The serum concentration of sIL2R, TPA and lymphocyte subsets CD4, CD8, CD25, CD16 were measured at diagnosis and then 1 and 6 months after the start of chemotherapy. PATIENTS: There were 39 patients (three females, 36 males; mean age 61.6 years) with lung cancer (LC), treated with chemotherapy and 22 control subjects (six females, 16 males; mean age 50.1 years) with non-neoplastic lung diseases. RESULTS: No significant differences in sIL2R serum concentrations were observed at diagnosis between the control and LC group or when comparing the different histotypes, disease stages (IIIa-b vs IV) and survival (survival < or = 12 vs > 12 months). On comparing the sequential variations of the examined parameters a significant increase in sIL2R (P < 0.007) after 1 and 6 months versus basal value was observed only in patients surviving less than 12 months and in those who did not respond to chemotherapy. Moreover a negative correlation was observed between sIL2R serum concentrations and CD25+ and CD16+ lymphocyte subsets. Evaluation of survival curves of patients with basal sIL2R > or < or = 700 U/ml showed a slightly lower survival rate in the former group. CONCLUSIONS: The present results, confirming the poor utility of sIL2R in the diagnostic phase of the disease, show its usefulness in prognostic evaluation and in the clinical surveillance of patients with advanced lung cancer submitted to polychemotherapy. In this case any variations in sIL2R serum levels are likely to relate to the spread of the neoplasia rather than to the host immune response.

Adenocarcinoma↗

PubMed1998

Production of D-phenylglycine from racemic (D,L)-phenylglycine via isoelectrically-trapped penicillin G acylase.

Penicillin G acylase (PGA) is exploited for producing pure D-phenylglycine from a racemate mixture, via an acylation reaction onto a cosubstrate, the ester methyl-4-hydroxyphenyl acetate. The reaction, when carried in a batch, is severely hampered by the reverse process, by which the product, 4-hydroxyphenylacetyl-(L)phenyl glycine, upon consumption of L-phenylglycine, is converted by the enzyme back into free substrate and 4-hydroxyphenyl acetic acid via lysis of the amido bond. To prevent this noxious reaction, a multicompartment electrolyzer with isoelectric membranes (MIER) is used as enzyme reactor, operating in an electric field. PGA is trapped between pI 5.5 and pI 10.5 membranes, together with an amphoteric, isoelectric buffer (lysine). As the 4-hydroxyphenylacetyl-(L)phenyl glycine product is formed, it vacates the reaction chamber by electrophoretic transport and is collected close to the anode, in a chamber delimited by pI 2.5 and 4.0 membranes. The same fate occurs to the free acid 4-hydroxyphenyl acetic acid, formed upon spontaneous (and enzyme-driven) hydrolysis of the methyl ester in the reaction chamber. These combined processes leave behind, in the enzyme reaction chamber, the desired product, pure D-phenylglycine. The advantages of the MIER reactor over batch operations: the consumption of the L-form in the racemate is driven to completion and the enzyme is kept in a highly stable form, maintaining 100% activity after one day of operation, during which time the PGA enzyme, in the batch reactor, has already lost >75% catalytic activity.

Electrophoresis, Capillary↗

PubMed1998

Radiological findings when very small lung volumes are irradiated in breast and chest wall treatment.

Acute pneumonitis following breast irradiation is a rare and transient phenomenon that can be easily managed by drugs. The aim of this study is to evaluate late sequelae on lung, after postoperative radiotherapy (RT) for breast cancer. We were concerned with investigating late radiological findings when very small lung volumes are involved in the irradiated volume. We studied 28 consecutive patients. They underwent clinical examination and all staging procedures before surgery, evaluation of pulmonary function with spirometry, postoperative chest x-ray and high resolution computed tomography (HRCT) of the lung before RT. Clinical examinations were usually performed every 3 months after RT. A second chest x-ray, HRCT and spirometry were carried out after nearly 7 months from the end of RT. We estimated the irradiated lung volume by measuring the area of the lung surface enclosed by the 50% isodose (LA50) in each profile. We found a significant correlation between LA50 and the score of radiological findings after RT. No correlations were found between other factors (i.e., adjuvant chemotherapy, age, weight, smoking) and lung fibrosis. No woman developed radiation pneumonitis syndrome or respiratory symptoms. Our results indicate that irradiation of the breast and/or chest wall is well tolerated if treatment planning is done accurately. The fibrosis likelihood is strongly correlated to the irradiated lung volume. The use of tangential fields limits radiological changes that can be detected only by HRCT examination and are not associated with clinical symptoms.

Adult↗

PubMed1998

An isoelectrically trapped enzyme reactor operating in an electric field.

Membrane enzyme reactors constitute an attempt at integrating catalytic conversion, product separation and/or concentration and catalyst recovery into a single operation. Whereas conventional membrane reactors confine an enzyme, in a free form, to one side of a membrane by size exclusion, electrostatic repulsion, or physical or chemical immobilization onto an intermediate support (gel, liposome), the membrane reactor here described is shown to operate under an entirely new principle: enzyme confinement into an isoelectric trap located in a multicompartment electrolyzer operating in an electric field. Two isoelectric membranes, having pI values encompassing both the enzyme pI and the pH of its optimum of activity, act by continuously titrating the enzyme trapped inside, thus preventing it from escaping the reaction chamber. Charged products generated by the enzyme catalysis are continuously electrophoretically transported away from the reaction chamber and collected into other chambers stacked either towards the cathodic or anodic sides. In a urease reactor, ammonia is continuously harvested towards the cathode, thus allowing >95% substrate consumption with maintenance of enzyme integrity over much longer time periods than in a batch reactor. In a trypsin reactor, casein is digested and biologically active peptides are continuously harvested in a pure form into appropriate isoelectric traps. In a third example, pure D-phenylglycine is produced from a racemate mixture, via an acylation reaction onto a cosubstrate (the ester methyl-4-hydroxyphenyl acetate), brought about by the enzyme penicillin G acylase.

Caseins↗

PubMed1998

Use of a Hepta-tyr glycopeptide antibiotic as chiral selector in capillary electrophoresis.

A new glycopeptide antibiotic, MDL 63,246 (Hepta-tyr), of the teicoplanin family, has been evaluated in capillary electrophoresis for the resolution of chiral compounds of pharmaceutical and environmental interest. Electrophoretic separations were carried out in a polyacrylamide-coated capillary using the partial filling-counter current mode with aqueous-organic buffers in the pH range 4-6. Experimental parameters affecting resolution, such as antibiotic concentration, buffer pH, organic modifier type and capillary temperature, were studied. The Hepta-tyr antibiotic exhibited a high enantiorecognition capability towards the studied compounds at very low concentrations (1-2 mg/mL). The optimum experimental conditions were achieved by using a buffer at pH 5 containing acetonitrile at 25 degrees C.

Anti-Bacterial Agents↗

PubMed1998

Interphysician variability in defining the planning target volume in the irradiation of prostate and seminal vesicles.

With the improvements in treatment accuracy the risk exists of over-reliance on the capability of the physician to estimate the extent of the tumour. We assessed the variability between six radiotherapists in defining the planning target volume (PTV) on CT slices for three prostate tumour cases. Percentage differences between measured volumes and mean values calculated for each case ranged from -53.64 to +60.48% (SD 36.00%). There is a considerable variation in delineating the PTV, both in the cranio-caudal direction and in the in-slice extension of the areas drawn on each slice (standard deviations ranged from 0.35 to 2.64 cm2). We also checked the uncertainty in the shape and position of the contours on each CT image. The analysis was performed on three slices of one test case. As we expected, the uncertainty seems largest for seminal vesicle slices and smallest for prostatic apex slices. These results endorse the need for uncertainty analysis of all departmental processes in order to define a detailed protocol and consequently to minimize the interphysician differences in PTV delineations.

Analysis of Variance↗

PubMed1997

Isoelectric focusing in immobilized pH gradients: an update.

The latest trends on isoelectric focusing (IEF) in immobilized pH gradients (IPG) are here reviewed. The major advances on IPG technologies have been made when interfacing this technique with sodium dodecyl sulfate-polyacrylamide gel electrophoresis to produce two-dimensional (2-D) maps. Previous 2-D maps were routinely performed using conventional IEF as a first dimension, which typically resulted in poor reproducibility of spot position. With IPGs, correlation between experimental and calculated protein pI values is as good as +0.01 to 0.02 pH units. A new software has also been released, permitting easy calculation and optimization of linear, concave and convex exponential gradients, even in very complex recipes utilizing all ten Immobiline chemicals. It has also been proven that IPGs can be interfaced with mass spectrometry, thus obtaining a novel 2-D map with the best of pI measurements in the first dimension coupled with the best of mass determination in the second dimension. Recently, it has been shown that IPGs can be exploited to charter forbidden grounds, with the creation of non-linear pH gradients covering the extreme alkaline pH 10-12 gradient. In such basic regions, excellent steady-state patterns of histones and subtilisin mutants have been reported. Different families of histones could be mapped not only in this pH 10-12 interval, but also in 2-D maps exploiting this very alkaline gradient in the first dimension. Although the IPG technique is now a trouble-free, user-friendly technique, some annoying artefacts, producing severe protein smears and precipitation, were very recently reported, but found to be linked to some commercial Immobiline preparations containing up to 5% oligomers. Better quality control on the part of the company producing such chemicals should eliminate even this last source of troubles.

Computer Simulation↗

PubMed1997

Protein purification in multicompartment electrolyzers with isoelectric membranes.

Preparative purification of proteins under isoelectric conditions is reviewed, with particular regard to novel equipment, a multicompartment electrolyzer with isoelectric membranes, which can capture any desired protein into an isoelectric trap as the sole, ultra-pure component. This novel machine is based on the Immobiline chemistry, i.e. the novel generation of non-amphoteric buffers, based on the chemistry of acrylamides, which can be insolubilized onto polyacrylamide supports. After a description of the instrument and of its performance, a number of protein purification protocols are described, leading to truly homogeneous (by the most stringent criterion of surface charge) protein fractions. Such a high charge purity has been found to be often a fundamental prerequisite for the growth of protein crystals. Interfacing the electrolyzer with mass spectrometry has permitted the decoding of the structure of minor components generated from a parental molecule, especially ones having a higher pI. It was found that these species were often generated either by proteolytic cleavage or by the formation of a trisulphide bridge between two Cys residues. A unique application of the electrolyzer is finally described: its use as an immobilized enzyme reactor under an electric field. The performance of this reaction is outstanding, in that the kinetic parameters of the immobilized enzyme are identical to those of a free enzyme form.

Amino Acid Sequence↗

PubMed1997

Isoelectric focusing in immobilized pH gradients: recent analytical and preparative developments.

Isoelectric focusing in immobilized pH gradients (IPG), covering both analytical and preparative aspects, is here reviewed. An extensive introduction covers the development of the technique from its inception in 1982 to present day methodology, with particular emphasis on the development of computer programs able to calculate and optimize linear and nonlinear pH gradients, spanning as much as 9 pH units, from a mixture of as many as 10 different buffering ions and titrants. The unique resolving power of IPGs is illustrated with the resolution of fetal globin chains differing by an Ala/Gly substitution in residue 75, this bringing about a minute difference in pI value of only 0.001 pH units. IPG runs, performed under denaturing conditions, allow an excellent correlation between experimental and theoretical protein pIs, to the extent that outliers were found to be polypeptide chains which had undergone post-synthetic modifications. The IPG methodology allows easy interfacing with mass spectrometry, due to the fact that proteins eluted from an IPG gel are isoionic as well as isoelectric, and thus are not contaminated by any buffer ion. The review ends with an excursus on preparative aspects of IPGs: a novel apparatus, based on the principle of isoelectric, buffering membranes, allows pilot-scale purification of r-DNA proteins to extreme purity, with recovery in a liquid vein. Isoelectric membranes have a selectivity based on a continuous titration process, and thus act as isoelectric traps for individual protein species. This same preparative apparatus can be used as a novel immobilized enzyme reactor, with superior performance compared to conventional types of reactors.

Amino Acid Sequence↗

PubMed1997

Generation of peptide maps by capillary zone electrophoresis in isoelectric iminodiacetic acid.

Capillary zone electrophoresis in stationary, isoelectric buffers is a novel method for generating peptide maps of protein digests. The buffer system developed is composed of iminodiacetic acid (IDA), whose physico-chemical parameters were found -- by theoretically modeling and experimental verification -- to be: pI 2.23 (at 100 mM concentration), pK1 = 1.73 and pK2 = 2.73 (no attempts were made at measuring the pK of the primary amino group, since such a low pI value would be compatible with any pK value of the basic group, down to as low as pK 5.5). IDA is compatible with most hydro-organic solvents, including trifluoroethanol (TFE), up to at least 40% v/v, typically used for modulating peptide mobility. In naked capillaries, a buffer comprising 50 mM IDA, 10% TFE and 0.5% hydroxyethylcellulose (HEC) allows generation of peptide maps with high resolution, reduced transit times and no interaction of even large peptides with the wall. However, the best background electrolyte was found to be a solution of 50 mM IDA in 0.5% HEC and 6-8 M urea, one of the best solubilizers of proteins and peptides known. In this last electrolyte system, peptide maps of beta-casein digests (known to contain also very large peptides, up to 6000 Da) could be generated with excellent resolution and half the transit times as compared with the standard buffer adopted in peptide analysis (80 mM phosphate buffer, pH 2.0). IDA thus appears to be another valid isoelectric buffer system, operating in a different pH window (pH 2.33 in 50 mM IDA) as compared to the other amphotere previously adopted (50 mM Asp, pH 2.77) for the same kind of analysis.

Buffers↗

PubMed1997

Capillary zone electrophoresis of oligonucleotides and peptides in isoelectric buffers: theory and methodology.

The use of isoelectric buffers in capillary zone electrophoresis is reviewed. Such buffers allow application of extremely high voltage gradients (up to 1000 V/cm in relatively high bore capillary, e.g. 75 to 100 microm internal diameter), permitting separations of the order of a few minutes and thus favoring high resolution due to minimal, diffusion-driven peak spreading. The fundamental properties of ampholytes are first discussed, such as buffering power (beta) as a function of delta pK, i.e. of the distance between the pI value and neighboring protolytic groups. The highest possible relative beta value (= 2) is obtained for amphoteres possessing a delta pK = 0.6, a condition not met by existing amphoteric species. A novel parameter for ampholyte evaluation is then proposed, namely the beta/lambda ratio, i.e. the ratio between the beta power and conductivity at the pI value. It is additionally shown that the pI is not a constant value, but depends on ampholyte concentration in solution. In addition, at constant concentration, the theoretical pI can change as a function of delta pK. Isoelectric His and, to a lesser extent, Lys have been found to offer unique separations of oligonucleotides in sieving liquid polymers. In the absense of sieving media, isoelectric Asp, in presence of 7 M urea (apparent pH 3.77), permits unique separations of oligonucleotides having the same length but different nucleotide composition. Isoelectric Asp (pI 2.77 at 50 mM concentration) provides a medium of high resolving power for generating peptide maps. In difficult cases, of coincident titration curves, the pH can be moved up to higher values (e.g. pH 3.0 for 30 mM Asp) thus eliciting separation of unresolved peptides at pH 2.77. This was illustrated by running peptide maps of tryptic digests of human beta-globin chains. Also imino diacetic acid (pI 2.33 at 50 mM concentration) allows generation of high resolution peptide maps.

Buffers↗

PubMed1996

Accelerated radiation therapy for locally advanced squamous cell carcinomas of the oral cavity and oropharynx selected according to tumor cell kinetics--a phase II multicenter study.

PURPOSE: A Phase II multicenter trial testing an accelerated regimen of radiotherapy in locally advanced and inoperable cancers of the head and neck, in patients selected on the basis of 5-bromo-2-deoxyuridine/DNA flow cytometry-derived tumor potential doubling time (Tpot). METHODS AND MATERIALS: From September 1992 to September 1993, 23 patients consecutively diagnosed to have locally advanced, inoperable carcinomas of the oral cavity and the oropharynx, with Tpot of < or = 5 days, received an accelerated radiotherapy regimen (AF) based on a modification of the concomitant boost technique: 2 Gy/fraction once a day, delivered 5 days a week up to 26 Gy, followed by 2 Gy/fraction twice a day, with a 6-h interval, one of the two fractions being delivered as a concomitant boost to reduced fields, up to 66 Gy total dose (off-cord reduction at 46 Gy), shortening the overall treatment time to 4.5 weeks. A contemporary control group of 46 patients with Tpot of >5 days or unknown was treated with conventional fractionation (CF): 2 Gy/fraction once a day, 5 days a week, up to 66 Gy in 6.5 weeks, with fields shrinkage after 46 Gy. RESULTS: All patients completed the accelerated regimen according to protocol and in the prescribed overall treatment time. Immediate tolerance was fairly good: 65% of the patients in the AF group experienced Grade 3 mucositis vs. 45% in the CF group (p = n.s.). Symptoms related to mucosal reactions seemed to persist longer in AF than in CF patients. The crude proportion of mild (Grades 1 and 2) late effects on skin (p < 0.01) and salivary glands (p < 0.05) was higher in AF than in CF patients, although these reactions did not exceed the limits of tolerance. Three patients in the AF and 1 in the CF arm experienced a late Grade 4 bone complication. Actuarial estimates of severe (Grades 3 and 4) late complications showed a 2-year hazard of 33.3% in the AF arm and 49.7% in CF (p = NS). The actuarial 2-year local control rate of the AF patients was 49.4%, while actuarial 2-year overall survival for the same patients was 43.5%. CONCLUSION: The results suggested that this accelerated regimen is worth testing in a controlled randomized trial to compare different accelerated schedules. Our findings also confirmed the 5-bromo-2-deoxyuridine/DNA flow cytometry technique as a suitable method of evaluating tumor cell kinetics in multicenter clinical studies, on condition that all measurements are carried out by one most experienced laboratory.

Adult↗