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Biomedical subjects

C Gelfi

Publications and source records attributed to C Gelfi.

At least 91 records · Page 5Linked to original sources
PubMed1994

Capillary zone electrophoresis in polymer networks of polymerase chain reaction-amplified oligonucleotides: the case of congenital adrenal hyperplasia.

The use of capillary zone electrophoresis (CZE) in polymer networks for the analysis of an 8 bp (base pair) deletion in congenital adrenal hyperplasia was investigated. Separations were performed in Tris-borate-EDTA buffer (pH 8.3) containing 6% liquid linear polyacrylamide as a sieving dynamic matrix and 10 microM ethidium bromide for improving DNA fragment separation. Easy analysis and detection of the 127 and 135 bp amplified fragments was accomplished. The capillary column can be used for > 50 analyses before degradation and loss of resolution. The results are comparable to those obtained by gel-slab zone electrophoresis in a 12%T, 4%C polyacrylamide matrix. The sensitivity, by simple UV absorption at 254 nm, is similar to that obtained in gel slabs by dye intercalation staining.

Adrenal Hyperplasia, Congenital↗
PubMed1994

Detection of point mutations by capillary electrophoresis in liquid polymers in temporal thermal gradients.

A new and fast method is described for detection of point mutations in polymerase-chain reaction (PCR)-amplified DNA, based on capillary electrophoresis in sieving liquid polymers in presence of temporal thermal gradients. The background electrolyte contains a constant amount of denaturing agent (e.g., 6 M urea) and the DNA fragments are injected in a constant-temperature plateau below the melting temperature (Tm). After loading, a temperature ramp is activated (typically from 0.2 to 0.6 degrees C/min, according to the melting profiles of the DNA duplexes under investigation) with resultant branching of homo- and heteroduplexes at different times along the migration path. In the case of individuals heterozygous for a point mutation, the expected four-band pattern is obtained. The temperature gradient is not produced externally, via circulating coolant and a thermostat, but is generated internally by using a dedicated computer program able to calculate the precise inner temperature under given electric conditions. The method is applied to the identification of three point mutations located in exon 17b (R1066H, R 1066C, F1052V) and of two polymorphisms located in exon 14a (V868V, T854T) of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

Adenine↗
PubMed1994

Capillary zone electrophoresis of polymerase chain reaction-amplified DNA fragments in polymer networks: the case of GATT microsatellites in cystic fibrosis.

In cystic fibrosis (CF), the most common mutation, delta F508 (a three-base-pair deletion) accounts for ca. 70% of mutations in the worldwide population. The majority of other mutations (more than 350 reported so far to the Genetic Analysis Consortium) have been detected in single cases, thus rendering quite cumbersome a molecular diagnostic approach for the identification of CF chromosomes. As an alternative, linkage analysis based on intragenic polymorphism can be useful for prenatal diagnosis and CF-carrier detection, provided that the heterozygosity of the allelic forms is very high. For this purpose, DNA microsatellites, consisting of two to epta nucleotide repeat clusters, displaying a high degree of polymorphism, are being increasingly used as markers in linkage studies. Two main allelic forms, one hexameric (111 bp) and one heptameric (115 bp), of a tetranucleotide (GATT) repeat polymorphism, at the junction of intron IVS6a and exon 6b, have been amplified by PCR technology. These two alleles can be separated in a 10-20% T polyacrylamide gradient gel and detected by ethidium bromide staining. As an alternate procedure, these two fragments are efficiently separated by capillary zone electrophoresis in a viscous solution of 6%T linear polyacrylamide and detected by their intrinsic absorbance at 254 nm.

Alleles↗
PubMed1990

DNA sequencing in HydroLink matrices: extension of reading ability to greater than 600 nucleotides.

All the systems for optimizing DNA sequencing published so far have introduced modifications regarding: (i) linearization of band migration via ionic strength gradients or wedge-shaped gels; (ii) automatization of band reading via introduction of fluorescent probes; (iii) direct blotting analysis; (iv) pulsed electric fields and (v) discontinuous buffer systems. In all these systems, DNA sequence reading with an accuracy of ca. 98% rarely exceeds a length of 350 bases. We have chosen, in order to increase the reading ability of a single gel, to manipulate the characteristics of the gel matrix. The Seq-HydroLink gel formation here reported allows optimal reading, from a single gel run, of at least 600 bases. In order to guarantee this reading ability in a single run, the upper and lower ends of the ladder are time-resolved, i.e. the same sample is applied to the gel matrix at three different time intervals. The present system represents an increase of at least 30% in reading ability as compared with any type of polyacrylamide gel formulation so far reported.

Bacteriophages↗
PubMed1989

Monoclonal antibodies directed to the calcium-free conformation of human protein S.

Four mouse hybridomas secreting monoclonal antibodies specific for human protein S (PS) have been generated. The antibodies, all of the IgG1 subclass, were designated S2, S3, S8, and S10. In a fluid phase radioimmunoassay, the binding of monoclonal antibodies to PS was about 30% greater in the presence of EDTA and totally inhibited in presence of Ca2+. Using the same technique, we performed displacement curves of 125I-labeled PS by purified PS, thrombin-cleaved PS, normal plasma, plasma from a patient on warfarin therapy, and plasma from a patient with no free PS and only PS bound to C4b-binding protein. The slopes of the curves show that the monoclonal antibodies reacted equally with all the tested forms of PS indicating that the antigenic site(s) to which the monoclonal antibodies are directed are present and exposed in free and bound PS, in thrombin-cleaved PS, and in the coumarin form of the protein. Each EDTA-dependent antibody, immobilized on Sepharose 4B-CNBr was used to purify PS from the barium citrate-absorbed, ammonium sulphate-soluble fraction of plasma. The fraction eluted from the immunoabsorbent with a buffer containing 4 mmol/l CaCl2 and analysed by SDS-PAGE, contained two bands, one migrating with conventionally purified PS and the other with purified C4b-binding protein. Homogeneous PS was obtained by chromatography of the barium citrate adsorbate on a DEAE-Sephadex column. The protein peak containing the bulk of PS was subsequently applied to the immunoadsorbent and eluted with 4 mmol/l CaCl2.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Immobilized pH gradients: effect of salts, added carrier ampholytes and voltage gradients on protein patterns.

Salts formed from strong acids and bases (e.g. NaCl, Na2SO4, Na2HPO4), present in a protein sample applied to an immobilized pH gradient (IPG) gel, induce protein modification (oxidation of iron moiety in hemoglobin) already at low levels (5 mM) and irreversible denaturation (precipitation) at higher levels (greater than 50 mM). This effect is due to production of strongly alkaline cationic and strongly acidic anionic boundaries formed by the splitting of the salt's ion constituents, as the protein zone is not and can not be buffered by the surrounding gel until it physically migrates into the gel matrix. Substitution of "strong" salts in the sample zone with salts formed by weak acids and bases, e.g.. Tris-acetate, Tris-glycinate, Good's buffers such as (N-[2-acetamido]-2-iminodiacetic acid (ADA), (2-[(2-amino-2-oxoethyl)-amino] ethanesulfonic acid (ACES), (3-[N-morpholino]propane sulfonic acid (MOPS), essentially abolishes both phenomena, oxidation and irreversible denaturation. Suppression of "strong" salt's effects is also achieved by adding, to the sample zone, carrier ampholytes in amounts proportional to the salt present (e.g. by maintaining a salt: carrier ampholytes molar ratio of at least 1:1). This suppression is due to the strong buffering power of the added carrier ampholytes, able to counteract drastic pH changes in the two moving boundaries. A reduction of these deleterious effects of strong salts is also achieved when the IPG run is performed at low voltage for a prolonged time (4 h at 500 V instead of only 1 h at 500 V, before switching to high-voltage settings). Guidelines are given for trouble-free IPG operations.

Ampholyte Mixtures↗
PubMed1988

Isoenzyme analysis of lichen algae in immobilized pH gradients.

A base for a modern species' concept of chlorococcal algae can be obtained not by morphological analysis, but by biochemical characters, e.g. isoenzyme banding patterns. From isolated lichen algae of the genus Trebouxia de Puymaly a set of five such enzymes has been studied by isoelectric focusing in immobilized pH gradients (IPG): phosphoglucomutase, phosphoglucose isomerase, malate dehydrogenase, mannitol dehydrogenase and leucine aminopeptidase. The first four are resolved into isoforms in a pH 4-7 IPG interval, while the last one is analyzed in an IPG pH 3.5-5 span. The patterns are specific for distinct populations, inter- and intraspecifically varying in dependence from their geographical distribution or the lichen species from which they have been isolated. Their limited heterogeneity (one to four isoforms) suggests that they are the products of specific genes rather than artefacts of the extraction procedure or the IPG analysis. Sharp isozyme patterns can only be obtained in a mixed-bed, carrier-ampholyte (CA)-IPG gel and by anodic application, suggesting that the recently proposed mechanism of hydrophobic protein-IPG matrix interaction (Electrophoresis, 1987, 8, 62-70) is fully operative here. As an additional mechanism, it is proposed that, in some cases, CA might simply act, when added to an IPG gel, by buffering, in the transient state, the sample zone before the protein migrates from the liquid phase into the IPG matrix.

Ampholyte Mixtures↗
PubMed1987

Hydrophobic interaction between alkaline immobilines and ferritin during isoelectric focusing in immobilized pH gradients.

It has been found that three alkaline Immobilines (out of seven weak acids and bases used to generate immobilized pH gradients), having pK values of 6.2, 7.0 and 9.3, act as cross-linking agents, aggregating and precipitating out of solution ferritin and other large macromolecules (e.g., from serum and tissue extracts) present in body fluids and human biopsies. All the acidic Immobilines (pK 3.6, 4.4 and 4.6) and the basic species of pK 8.5 appear to be unreactive. The three precipitin Immobilines mimic cationic detergents, acting on the basis of two different principles at the opposite extremes, by ionic interaction at one end and by hydrophobic bonding at the other end of the molecule. The ionic type of interaction was clearly demonstrated, owing to its sensitivity to pH extremes and to progressively increasing ionic strength. The hydrophobic interaction in the region of the double bond (Immobilines are N-substituted acrylamido acids and bases) was deduced on the basis of the following observations: (a) oxidation of the double bond with introduction of a vicinal diol totally inhibited ferritin aggregation; (b) addition of SH groups to the double bond increased protein precipitation and (c) the protein-Immobiline aggregates were found to be sensitive to alkyl-substituted ureas (especially ethyl- and propylurea), which are known to bind to hydrophobic regions of proteins, and insensitive to urea, which is known to split only hydrogen bonds. Interestingly, neutral and zwitterionic detergents were unable to split the Immobiline-ferritin complexes, suggesting that their large micelles could not have access to the tightly packed Immobiline cross-linking region.

Chemical Phenomena↗
PubMed1987

Diffusion coefficients of proteins in carrier ampholyte versus immobiline gels.

The apparent diffusion coefficients of proteins in carrier ampholyte isoelectric focusing (CA-IEF) and in immobilized pH gradients (IPGs) are strongly dependent on the amount of buffering ions present in the system. However, whereas in CA-IEF increased levels of ampholytes facilitate diffusion, in IPGs they strongly quench it. It is concluded that a protein in an IPG matrix is isoelectric but not isoionic, in the sense that it forms a salt with the surrounding ions bound to the polyacrylamide matrix. This salt formation is beneficial as it greatly increases protein solubility at the pI. It is suggested that, when performing zymograms in situ, the IPG gel should contain at least twice the standard amount of Immobiline, so as to keep sharp enzyme bands even with prolonged incubation periods.

Animals↗
PubMed1987

Amphoteric, isoelectric immobiline membranes for preparative isoelectric focusing.

Amphoteric, isoelectric agarose membranes, as devised by Martin and Hampson [Martin, A.J.P. and Hampson, F. (1978) J. Chromatogr. 159, 101-110], are found unsuitable for blocking electroendosmosis in multi-compartment electrolysers during preparative isoelectric focusing, due to the poor and highly unpredictable incorporation of carboxyls and amino groups on the polysaccharide moiety. New, polyacrylamide-based membranes are described, containing as buffers and titrants the Immobiline chemicals used to produce immobilized pH gradients. These new membranes are supported on both faces by a non-woven polypropylene cloth, a material exhibiting minimal adsorption properties for proteins. Due to the extensively developed Immobiline technology, membranes with highly predictable isoelectric points, well-defined buffering capacity and conductivity can be synthesized at any pH value along the pH 3-10 scale. They are effective in blocking electroendosmosis even when the delta pH on either side of the membrane is as high as 1.5 pH unit.

Acrylic Resins↗
PubMed1987

Purification of human hemoglobin valence intermediates by preparative immobilized pH gradients.

We compare three separation techniques for preparative purposes, i.e. ion-exchange chromatography on CM-cellulose, conventional isoelectric focusing in polyacrylamide gel slabs and immobilized pH gradients. The biological system used to test the three methods is a solution containing four hemoglobin (Hb) valence intermediates, i.e. metHb, oxyHb, (alpha + beta O2)2 and (alpha O2 beta +)2. The delta pI between the two valence intermediates is 0.04 pH units. Immobilized pH gradients give the best performance in terms of resolving power, total amount of protein which can be loaded and retention of biological activity by the protein (the latter assessed by determination of CO dissociation rates).

Carboxyhemoglobin↗
PubMed1987

Isoelectric focusing in immobilized pH gradients in the pH 10-11 range.

With the synthesis of a new, strongly basic Immobiline (pK 10.3 at 10 degrees C) it has been possible to formulate a new pH 10-11 recipe for focusing very alkaline proteins, not amenable to fractionation with conventional isoelectric focusing in carrier ampholyte buffers. In this formulation, water is added as an acidic Immobiline having pK = 14 and a unit molar concentration (or with a pK = 15.74 and standard 55.56 molarity) since around pH 11 its buffering power becomes significant. The gel contains a 'conductivity quencher', i.e. a density gradient incorporated in the matrix, with the dense region located on the cathodic side (pH 11) for (a) smoothing the voltage gradient on the separation cell and (b) reducing the anodic electrosmotic flow due to the net positive charge acquired by the matrix at pH 11 (1 mM excess protonated amino groups to act as counterions to the 1 mm OH- groups in the bulk water solution generated by the local value of pH 11). Excellent focusing is obtained for such alkaline proteins as lysozyme (pI 10.55), So-6 (a leaf protein, pI 10.49), cytochrome c (pI 10.45) and ribonuclease (pI 10.12).

Computer Simulation↗
PubMed1987

Isoelectric protein purification by orthogonally coupled hydraulic and electric transports in a segmented immobilized pH gradient.

A new method is described for preparative protein purification, based on isoelectric focusing on immobilized pH gradients. The principle is entirely new, as it is based on keeping the protein of interest isoelectric, in a flow-chamber, and focusing the impurities in the Immobiline gel. For this, a hydraulic flow is coupled orthogonally to an electric flow, sweeping away the non-isoelectric impurities from the recycling chamber. The sample flow-chamber is built in the centre of the apparatus, and is coupled to an upper and lower segment of an immobilized pH gradient. The protein to be purified is kept isoelectric in the flow-chamber and prevented from leaving it by arranging for the extremities of the immobilized pH gradient, forming the ceiling and the floor of this chamber, to have isoelectric points just higher (e.g. +0.05 pH units, on the cathodic side) and just lower (e.g. -0.05 pH units, on the anodic side) than the known pI of the species of interest. Macromolecules and small ions leave the flow chamber at a rate corresponding to a first order reaction kinetics (the plot of log C vs. time being linear). In general, for macromolecules, 12 h of recycling under current allow removal of 95% impurities. After 24 h of recycling, the protein of interest is more than 99.5% pure. The recoveries are very high (approaching 100%) as the sample under purification never enters the Immobiline gel and thus does not have to be extracted from a hydrophilic matrix, as typical of preparative gel electrophoresis.

Adult↗
PubMed1987

Serum alkaline phosphatase isoenzymes in hepatobiliary disorders resolved by use of immobilized pH gradients.

This new method for fractionating alkaline phosphatase isoforms in hepatobiliary disorders is based on isoelectric focusing on a mixed-type polyacrylamide support containing an immobilized pH gradient with a superimposed carrier-ampholyte gradient. The high-Mr alkaline phosphatase forms typical of hepatobiliary disease (greater than 1 mega-dalton), which cannot migrate into the Immobiline gel, are disaggregated in zwitterionic detergents (the most effective being sulfobetaine 3-12)--20 g/L in the sample, 5 g/L in the gel--suggesting that they are still complexed with membrane fragments or that they tend to aggregate spontaneously in solution. These isoforms focus in the pI 5-6 range (while alkaline phosphatases in normal serum focus in the pI 4-5 interval) in immobilized pH gradients, but behave as strongly acidic components by agarose isoelectric focusing in the presence of carrier ampholytes, suggesting that they are strongly complexed with the latter. On treatment with neuraminidase, the low-pI isoforms in normal serum focus in the pI 5-6 range typical of the hepatobiliary isoforms, suggesting that the latter are poorly glycosylated. By a second-dimension run, in a porosity gradient, followed by activity staining, all alkaline phosphatase forms that have entered the Immobiline gel in the first dimension (normal forms and high-Mr species) exhibit the same Mr (ca. 140,000 Da), suggesting that no new chains are synthesized in hepatobiliary disorders.

Alkaline Phosphatase↗
PubMed1986

Electroblot and immunoperoxidase staining for rapid screening of the abnormalities of the multimeric structure of von Willebrand factor in von Willebrand's disease.

A new electrophoretic method is described for rapid screening of abnormalities of the multimeric structure of von Willebrand factor in von Willebrand's disease. The method is based on the transfer of the separated proteins from agarose gels onto nitrocellulose foils followed by immunoperoxidase staining. It has the advantage of not requiring radio-iodinated antibodies and reduces the working time for the entire procedure from 5-6 days to 3 days. Electroblotting followed by immunoperoxidase staining differentiates patients with intact multimeric structure from those without intermediate and/or large multimers. The more subtle defects of the inner structure of the smallest multimers found in patients with type II von Willebrand's disease can also be identified. A potential disadvantage of electroblotting and immunoperoxidase staining is the lesser sensitivity of this technique, which results in the detection of a smaller number of multimers (11-12 bands) than by autoradiography without transfer onto nitrocellulose (16-17 bands).

Autoradiography↗