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Biomedical subjects

F Regnier

Publications and source records attributed to F Regnier.

At least 19 recordsLinked to original sources
PubMed2000

Strategy for qualitative and quantitative analysis in proteomics based on signature peptides.

This paper describes a new analytical strategy for identifying proteins in concentration flux based on isotopic labeling peptides in tryptic digests. Primary amino groups in peptides from control and experimental samples were derivatized with acetate and trideuteroacetate, respectively. After mixing samples thus labeled from these two sources, the relative concentration of peptides was determined by isotope ratio analysis with MALDI and ESI mass spectrometry. More than a 100-fold difference in relative concentration could be detected. Simplification of complex tryptic digests prior to mass spectral analysis was achieved by selection of histidine-containing peptides with immobilized metal affinity sorbents or of glycopeptides by lectin columns. Because most of these peptides have sequences that are unique to a single protein, they are a signature of the protein from which they were derived; providing a facile route to protein analysis.

Chromatography, Affinity↗
PubMed1999

Microfabricated filters for microfluidic analytical systems.

Solvent and reagent filters were micromachined into quartz wafers using deep reactive ion etching to create a network of intersecting 1.5 x 10 microns channels. When placed at the bottom of reservoirs with a side exit, this channel network behaved as a lateral percolation filter composed of an array of cubelike structures one layer deep. Flow through these filters was driven by electroosmotic flow (EOF). Silanol groups at the walls of channels in the network provided the requisite charge to trigger EOF when voltage was applied laterally to the filter. Adsorption of cationic proteins in this silanol-rich matrix was controlled by the application of a polyacrylamide coating prepared by bonding N-hydroxysuccinimide (NHS)-activated poly(acrylic acid) to (gamma-aminopropyl)silane-derivatized filters. Subsequent reaction of residual NHS groups in the coating with 2-(2-aminoethoxy)ethanol provided channels of low charge density and adsorptivity. These lateral percolation filters were shown to be efficacious in filtering solvents containing a variety of particulate materials, ranging from dust to cells.

Adsorption↗
PubMed1999

Characterization of peroxidase:anti-peroxidase immune complexes by capillary zone electrophoresis and high-performance size-exclusion chromatography.

Determination of the molecular constituents of commercial peroxidase:anti-peroxidase (PAP) preparations is necessary for the proper interpretation of PAP applications based on competitive binding assay. Capillary zone electrophoresis with field 300 V/cm, 40 cm capillary length (20 cm effective length), and high-performance size exclusion chromatography equipped with Superose 12 HR10/30 column revealed that a PAP preparation used for Fc gamma receptor studies contained multiple sizes of immune complexes, an excess amount of free peroxidase, and little or no free anti-peroxidase antibody. The antibody:antigen ratios of the three major immune complex components were 2:2, 1:2, and 1:1. These techniques provide useful methods of qualitative, as well as quantitative analysis of PAP preparations.

Antigen-Antibody Complex↗
PubMed1998

Fabrication of nanocolumns for liquid chromatography.

This paper shows that in situ micromachining can be used to simultaneously position and define (i) support particles, (ii) convective transport channels, (iii) an inlet distribution network of channels, and (iv) outlet channels in multiple chromatography columns on a single quartz wafer to the level of a few tenths of a micrometer. Stationary phases were bonded to 5 x 5 x 10 microns collocated monolith support structures separated by rectangular channels 1.5 microns wide and 10 microns deep with a low degree of deviation of channel width between the top and bottom of channels. High aspect ratio microfabrication can only be achieved with deep reactive ion etching. The volume of a 150 microns x 4.5 cm column was 18 nL. Column efficiency was evaluated in the capillary electrochromatography (CEC) mode using rhodamine 123 and a hydrocarbon stationary phase. Plate heights in these columns were typically 0.6 micron in the nonretained and 1.3 microns in the retained modes of operation. Columns were designed to have identical mobile-phase velocity in all channels in an effort to minimize outgassing during operation. When the total lateral cross-sectional area of channels at all points along the separation axis is identical, linear velocity of the mobile phase in a CEC column should be the same. Columns were operated at atmospheric pressure.

Chromatography, Liquid↗
PubMed1998

Microfabricated liquid chromatography columns based on collocated monolith support structures.

There is great interest today in massively parallel analytical strategies as a way to accelerate the rate of discovery in biological research; among them being 'biochips' and 'laboratories-on-a-chip'. The concept in the 'chip' approach is that minaturization will allow large numbers of operations to be performed in parallel in a small space, as in electronics. Proceeding with the semiconductor analogy, this paper demonstrates that in situ micromachining can be used to simultaneously fabricate millions of micrometer size, particle like structures in multiple liquid chromatography columns on a single wafer. Reduction of this widely used bioanalytical tool to the nanoliter volume, parallel processing, chip format is a significant step toward laboratories-on-a-chip.

Chemistry Techniques, Analytical↗
PubMed1998

Monoclonal antibody production with on-line harvesting and process monitoring.

A semi-automated system has been designed for on-line harvesting and monitoring of monoclonal antibody (mAb) production. [The antibody was directed against the peptide AGPAGTGKTTKDL.] Analytical and purification units were interfaced to the fermenter via a hollow fiber cartridge in which fermentation broth was continuously circulated through the lumen of the hollow fiber system. Permeate from the hollow fiber cartridge was pumped through either an analytical sampling loop or a preparative Protein G column where antibody species were captured. Switching between monitoring and harvesting was achieved by two 3-way toggle valves. Samples from the analytical sampling loop were transported to an analytical Protein G chromatography column for quantitation of all immunoglobulin G species in the fermenter. Data acquisition and processing was performed by the data system of the liquid chromatograph. All valves in the system except the two toggle valves were controlled by the liquid chromatograph. Antibody biosynthesis was monitored for the first 60 h of fermentation. Harvesting was initiated when mAb accumulated in the fermenter. Complete harvesting took approximately 90 h.

Amino Acid Sequence↗
PubMed1997

Multidimensional chromatography coupled with mass spectrometry for target-based screening.

The synthesis of structural analogs and the process of drug discovery have evolved dramatically through recent advances in solid-phase synthesis reagents and automated screening systems. As molecular diversity strategies emerge, the need for automated target-based selection of lead candidates becomes equally important. Multidimensional automated chromatographic techniques coupled to electrospray ionization mass spectrometry facilitate the selection process and provide maximum characterization information in a single screening run. The capture of tightly bound affinity leads by target biomolecules, followed by subsequent release and high-resolution separation with sensitive detection, significantly reduces the time required to identify and characterize lead compounds. This automated multidimensional chromatographic approach coupled with mass spectrometry, Selectronics, was used with several organic and natural libraries to demonstrate an automated target-based screening technique to select for high-affinity binders as potential lead compounds.

Amino Acid Isomerases↗
PubMed1996

Future potential of targeted component analysis by multidimensional liquid chromatography-mass spectrometry.

Multidimensional liquid chromatography (MDLC) may be used in either (i) the profiling mode where it is the objective to fractionate all components in a mixture or (ii) the targeted component mode in which it is the objective to determine specific analytes. This paper focuses on targeted component analysis from complex mixtures, addressing the critical operations of analyte selection and transport from the first to the second dimension. Although the physical operation of switching a component into the second dimension with computer controlled valving is simple, it is shown that changes in analyte retention time and peak width with column age and fouling are a serious problem. The analyte moves out of the preselected time window for valve switching and quantitation is compromised in the second dimension. It is proposed that a solution to the "drifting peak" phenomenon in targeted component analysis is to use binary mobility elution in the first dimension. Binary mobility refers to those systems, such as affinity chromatography, in which analyte mobility is generally either 0 or 1 relative to mobile phase velocity. Coupling these binary changes in analyte mobility in the first dimension with valve switching eliminates the "drifting peak" phenomenon. In addition, it is shown that a wide time window may be used in affinity separations without compromising the separation or accumulating contaminants. Several cases are described in which immunosorbents were used with reversed phase columns to provide quantitative targeted component analyses from complex mixtures.

Animals↗
PubMed1996

Automated analytical system for the examination of protein primary structure.

This paper describes an automated analytical system for the examination of protein primary structure in which (i) the target protein is first purified by immunoaffinity chromatography, (ii) subsequent chromatographic and chemical reaction steps in the sequencing process are directly coupled, (iii) buffer exchange between these unit operations is achieved while the protein is absorbed on a mixed bed of strong ion exchange sorbents, (iv) proteolysis occurs in an immobilized trypsin column having a 10-1000 fold-excess of enzyme, (v) the tryptic digest is directly transferred to a perfusion dilute capture column where it is concentrated and rapidly desalted, and (vi) peptides eluted from the dilute capture column and analytical microbore and capillary perfusion reversed-phase chromatography columns are analyzed by either single-stage mass spectrometry (MS) or tandem MS/MS. Protein structure variants were easily recognized, and in the case of hemoglobin (Hb) S, the site of variation from Hb A0 was verified.

Amino Acid Sequence↗
PubMed1996

Affinity-based screening of combinatorial libraries using automated, serial-column chromatography.

We have developed an automated serial chromatographic technique for screening a library of compounds based upon their relative affinity for a target molecule. A "target" column containing the immobilized target molecule is set in tandem with a reversed-phase column. A combinatorial peptide library is injected onto the target column. The target-bound peptides are eluted from the first column and transferred automatically to the reversed-phase column. The target-specific peptide peaks from the reversed-phase column are identified and sequenced. Using a monoclonal antibody (3E-7) against beta-endorphin as a target, we selected a single peptide with sequence YGGFL from approximately 5800 peptides present in a combinatorial library. We demonstrated the applicability of the technology towards selection of peptides with predetermined affinity for bacterial lipopolysaccharide (LPS, endotoxin). We expect that this technology will have broad applications for high throughout screening of chemical libraries or natural product extracts.

Amino Acid Sequence↗
PubMed1995

Complications of percutaneous liver biopsy in infants and children.

UNLABELLED: In this study, 144 consecutive percutaneous liver biopsies performed with a 1.6 mm Menghini needle, during a 2-year period were reviewed. All the children were aged under 15 years, 57 patients less than 1 year and 87 more than 1 year. All biopsies were adequate and the mean number of portal tracts examined was 17.6 per biopsy (14.3 in patients weighing less than 10 kg and 19.1 in the others). There were no deaths and we observed only bleeding complications. In patients with normal coagulation (128 cases), 1 bleeding requiring transfusion occurred; and in patients with abnormal coagulation (16 cases), we observed 2 bleeding cases requiring transfusion. CONCLUSION: Percutaneous liver biopsy can be performed with 1.6 mm needles in children. For increased safety, ultrasound-guided biopsies are recommended.

Adolescent↗
PubMed1995

[Early manifestations of Tangier disease].

BACKGROUND: The diagnosis of Tangier disease in childhood is based on the specific aspect of tonsils or by screening relatives of affected subjects. CASE REPORT: A moderately enlarged liver associated with splenomegaly was found upon routine physical examination of a 3 month-old breast-fed boy, born in Turkey from consanguineous parents. Laboratory studies disclosed moderate increase in serum alanine aminotransferase activity (ALAT 52 UI/l, N < 30). The diagnosis of Tangier disease was confirmed by studies of plasma cholesterol and apolipoprotein A. By 8 months of age, the patient had enlarged orange tonsils. Small cervical, axillary and inguinal lymphadenopathies were present. The tonsilar and adenoidal tissues were removed at 18 months of age because the patient suffered from chronic airway obstruction. Colonoscopic examination revealed tiny flat orange spots, 1 to 2 mm in diameter, scattered throughout the rectosigmoidal and colonic mucosa. Survey of the family led to the discovery of one sister, with asymptomatic apolipoprotein Al deficiency and a normal sister, while the parents were heterozygotes for Tangier disease. CONCLUSION: Enlarged liver associated with a moderate level in serum aminotransferase may be an early manifestation of Tangier disease in infants. Rectosigmoidal and colonic lesions may be convenient for biopsy when tonsillectomy is not indicated.

Hepatomegaly↗
PubMed1994

Effect of protein adsorption on the surface charge characteristics of aluminium-containing adjuvants.

The effect of adsorbing two model proteins, bovine serum albumin and lysozyme, on the point of zero charge of aluminium-containing vaccine adjuvants was studied. At physiological pH, the adsorption of the negatively charged albumin (isoelectric point = 5.0) by aluminium hydroxide adjuvant (point of zero charge = 11.1) resulted in a decrease in the point of zero charge. In contrast, the adsorption of positively charged lysozyme (isoelectric point = 9.6) by the negatively charged aluminium phosphate adjuvant (point of zero charge = 5.0) resulted in an increase in the point of zero charge. The surface charge characteristics of the aluminium-containing adjuvant dominated at low protein coverage. In contrast, the surface charge characteristics of the adsorbed protein dominated at high protein coverage. Therefore, the physicochemical properties of the antigen-adjuvant complex and not the adjuvant alone should be considered during vaccine preparation.

Adjuvants, Immunologic↗
PubMed1993

Treatment with lenograstim (glycosylated recombinant human granulocyte colony-stimulating factor) and orthotopic liver transplantation for glycogen storage disease type Ib.

A 7 1/2-year-old patient with glycogenosis type lb had chronic neutropenia, recurrent bacterial infections, and severe hypoglycemia in spite of continuous nasogastric feeding. She was treated with lenograstim (glycosylated recombinant human granulocyte colony-stimulating factor) and orthotopic liver transplantation. Absolute neutrophil counts and platelet counts rapidly reached normal values, and infectious episodes were reduced. She resumed oral feeding after transplantation and had no subsequent hypoglycemic episodes.

Adjuvants, Immunologic↗
PubMed1986

[Biosynthetic growth hormone and the opinions of pediatricians. The form of the problem after a regional survey].

An enquiry was undertaken with 35 pediatricians in the area of Bordeaux, concerning the attitudes induced by the future availability of a synthetic growth hormone obtained by genetic engineering and which might allow for treatment of a greater number of children presenting with important growth retardation. Biosynthetic growth hormone is perceived as a innovative technologic advance. However, the pediatricians questioned showed prudence as to the risks of excesses as to indications.

Attitude of Health Personnel↗