Biomedical subjects

C Labeur

Publications and source records attributed to C Labeur.

44 records · Page 3Linked to original sources

PubMed1990

Abetalipoproteinemia with an ApoB-100-lipoprotein(a) glycoprotein complex in plasma. Indication for an assembly defect.

Patients with autosomal recessive abetalipoproteinemia (ABL) lack in their plasma all lipoproteins containing apolipoprotein (apo)B-100 or B-48. Previous studies have suggested that this is due to the complete absence of apoB. We have investigated whether such patients (n = 10) are able to secrete the lipoprotein(a) (Lp(a] glycoprotein (apo(a] which, in normal plasma, exists as a complex with low density lipoproteins containing apoB-100 (Lp(a) lipoprotein). All 10 patients had reduced but detectable apo(a) levels in plasma (mean, 0.49 mg/dl; range, 0.2-2.03 mg/dl) but no Lp(a) lipoprotein. However, we also detected small amounts (0.2-2.8 mg/dl) of apoB in all patients with ABL. The apoB in the ABL patients had the size of apoB-100 and occurred as a lipid-poor complex with the Lp(a) glycoprotein in a fraction of density 1.22 g/ml. This material may represent partially assembled Lp(a) lipoprotein. There was also uncomplexed apo(a) and apoB-100 in the ABL plasma. The distribution and relative concentration of both proteins in the density fraction greater than 1.06 g/ml varied among patients. The data suggest that in ABL, the assembly of apoB-containing lipoproteins is defective and that apoB-100 may be secreted without its full lipid complement when complexed with apo(a).

Abetalipoproteinemia↗

PubMed1990

Immunological assays of apolipoproteins in plasma: methods and instrumentation.

A number of immunological techniques--radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), electroimmunoassay, radial immunodiffusion, and a variety of immunoprecipitin assays--have been used to quantify apolipoproteins in plasma. This paper outlines their technical details and discusses their major advantages and drawbacks. The most sensitive procedures, RIAs and ELISAS, are best suited to quantifying those apoproteins found in low concentration in plasma. Immunoturbidimetric assays, on the other hand, which are readily automated, are being widely used to quantify apolipoproteins A-I and B. Apolipoprotein quantification is complicated by the interaction of the proteins with lipids, which can often mask their antigenic determinants. This problem may be circumvented by pretreatment of the samples, by selection of appropriate standards, or by the use of polyclonal or monoclonal antibodies that interact with permanently exposed epitopes on the lipoproteins' surfaces. Our proposed methods for measurement of the individual apolipoproteins give consideration to these approaches.

Apolipoproteins↗

PubMed1989

Lipoprotein(a) quantified by an enzyme-linked immunosorbent assay with monoclonal antibodies.

This new, sensitive, specific "sandwich"-type enzyme-linked immunosorbent assay (ELISA) for quantifying lipoprotein(a) [Lp(a)] in human serum and in ultracentrifugal lipoprotein fractions is based on use of a monoclonal antibody raised against apolipoprotein(a) as coating protein and a polyclonal antibody, raised against either apo B or against Lp(a) and conjugated with peroxidase, for detection of bound Lp(a). Mean intra- and interassay CVs for assay of 16 samples were 3.0% and 5.6%, respectively. Sample pretreatment with urea did not enhance Lp(a) immunoreactivity, and treatment with nonionic detergents decreased binding to the monoclonal antibody. Results correlated well (r = 0.99, n = 38) with those by radial immunodiffusion (RID). The ELISA assay, however, detects amounts corresponding to Lp(a) contents of 10 to 1000 mg/L in plasma samples diluted 1000-fold, compared with 100-500 mg/L for RID. For 92 normolipidemic subjects, the mean Lp(a) concentration was 120 (SD 130) mg/L. In patients undergoing coronary angiography, Lp(a) concentrations increased with the severity of the disease but were not correlated with either HDL cholesterol, triglycerides, apo A-I, or apo B, and only weakly with plasma cholesterol and apo A-II. These two correlations were even weaker in normal subjects, and only the correlation with total cholesterol was valid. Lp(a), measured at birth and at seven days and six months, steadily increased with age. This assay is well suited for measuring Lp(a) in plasma and in lipoprotein fractions and also for screening programs evaluating this significant genetic risk factor for the development of atherosclerosis.

Adult↗

PubMed1986

Immunogold-silver staining of lymphocyte surface antigens on cells in suspension and in lymph node cryostat sections.

An immunogold-silver staining (IGSS) technique for the light microscopical detection of leucocyte cell surface antigens in cell suspensions and cryostat sections is described. The specimens were first incubated with monoclonal mouse antibodies and then with colloidal gold-labelled goat anti-mouse antibodies. They were then immersed in a physical developer, counterstained and mounted. In light microscopy, the tissue architecture and the cellular morphology were well preserved. Positive cells showed dark granules on their surface membranes. Optimal labelling conditions were determined. This method proved to be a reliable tool for the enumeration of T-cells and their subsets in peripheral blood. The dense labelling permitted the use of panoptic counterstains like May-Grünwald-Giemsa or Wright's stain. This IGSS technique was used to determine the distribution of the T- and B-cell subsets in cryostat sections of reactive lymph nodes. The sensitivity of the method was comparable with that of immunofluorescence microscopy for cell suspensions and that of the biotin-avidin-peroxidase technique for tissue sections. Immunogold-silver staining was combined with enzyme cytochemistry. In dark-field or epipolarization microscopy the labelling appeared as bright granules on a dark background. With its dense granular membrane labelling and its good morphology IGSS is an ideal method for the study of particular cell types in mixed cell suspensions. In addition, it could be a general method for the detection of cell surface antigens in all kinds of cells and tissues.

Antigens, Surface↗

PubMed1986

Potential of immunogold-silver staining for the study of leukocyte subpopulations as defined by monoclonal antibodies.

The potential of immunogold-silver staining for study of leukocyte subpopulations, as defined by monoclonal antibodies in cell suspensions, was examined. The cells were labeled in suspension as described for immunogold staining. Cytocentrifuge preparations of the suspensions were then immersed in a physical developer. By light microscopy, cells reacting with the monoclonal antibodies showed dark granules on their surface membrane. The morphology of the cells, as revealed by a panoptic counterstain, was comparable with that seen in routine cell smears for differential counts. The numbers of T-cells, T-helper/inducer cells, and T-cytotoxic/suppressor cells counted by this method in normal peripheral blood were nearly identical to those identified by immunogold staining and immunofluorescence microscopy in the same cell suspensions. The good morphological delineation also made possible rapid and accurate identification of particular leukocyte subsets in complex cell suspensions. Atypical lymphocytes from patients with infectious mononucleosis displayed the surface phenotype of activated T-cytotoxic/suppressor cells. Different maturation stages of neoplastic cells in patients with acute myeloid leukemia showed differences in surface antigen expression. Immunological detection of cell surface antigens could be combined with cytochemical staining of intracellular enzymatic activities. Finally, the labeling could be performed on cells prefixed on glass slides.

Adult↗

PubMed1986

An immunogold-silver staining method for detection of cell-surface antigens in light microscopy.

An immunogold-silver staining technique for detection of cell-surface antigens in cell suspensions was developed. Leukocyte cell suspensions were first incubated with monoclonal antibodies directed against cell-surface antigens and then with colloidal gold-labeled goat anti-mouse antibodies. Cytocentrifuge preparations of the cell suspensions were immersed in a physical developer containing silver lactate and hydroquinone as reducing substance. The preparations were then counterstained and mounted. In light microscopy, cells reacting with the monoclonal antibodies showed dark granules on their surface membrane. An optimal morphology, as revealed by a May-Grünwald-Giemsa counterstain, permitted accurate cell identification. The labeling was influenced by the gold particle diameter and the concentration of the gold reagents, by the duration of incubation in the physical developer, and by the composition and temperature of this medium. The T-cell subsets enumerated with this method in the peripheral blood of normal adults were identical to those found with other methods. The sensitivity of the technique was comparable with that of immunofluorescence microscopy. This immunogold-silver staining procedure proved to be a reliable tool for detection of cell-surface antigens in light microscopy.

Antibodies, Monoclonal↗

PubMedDate not supplied

Apoptosis induced in neuronal cells by C-terminal amyloid beta-fragments is correlated with their aggregation properties in phospholipid membranes.

A number of findings suggest that lipophilic monomeric Abeta peptides can interact with the cellular lipid membranes. These interactions can affect the membrane integrity and result in the initiation of apoptotic cell death. The secondary structure of C-terminal Abeta peptides (29-40) and the longer (29-42) variant have been investigated in solution by circular dichroism measurements. The secondary structure of lipid bound Abeta (29-40) and (29-42) peptides prepared at different lipid/peptide ratio's, was investigated by ATR-FTIR spectroscopy. Finally, the changes in secondary structure (i.e. the transition of alpha-helix to beta-sheet) of the lipid bound peptides were correlated with the induction of neurotoxic and apoptotic effects in neuronal cells. The data suggest that the C-terminal fragments of the Abeta peptide induce a significant apoptotic cell death, as demonstrated by caspase-3 measurements and DNA laddering, with consistently a stronger effect of the longer Abeta (29-42) variant. Moreover, the induction of apoptotic death induced by these peptides can be correlated with the secondary structure of the lipid bound amyloid beta peptides. Based on these observations, it is proposed that membrane bound aggregated Abeta peptides (produced locally as the result of gamma-secretase cleavage) can accumulate and aggregate in the membrane. These membrane bound beta-sheet aggregated amyloid peptides induce neuronal apoptotic cell death.

Amyloid beta-Peptides↗

PubMedDate not supplied

Contribution of the hydrophobicity gradient to the secondary structure and activity of fusogenic peptides.

Fusogenic peptides belong to a class of helical amphipathic peptides characterized by a hydrophobicity gradient along the long helical axis. According to the prevailing theory regarding the mechanism of action of fusogenic peptides, this hydrophobicity gradient causes the tilted insertion of the peptides in membranes, thus destabilizing the lipid core and, thereby, enhancing membrane fusion. To assess the role of the hydrophobicity gradient upon the fusogenic activity, two of these fusogenic peptides and several variants were synthesized. The LCAT-(57-70) peptide, which is part of the sequence of the lipolytic enzyme lecithin cholesterol acyltransferase, forms stable beta-sheets in lipids, while the apolipoprotein A-II (53-70) peptide remains predominantly helical in membranes. The variant peptides were designed through amino acid permutations, to be either parallel, perpendicular, or to retain an oblique orientation relative to the lipid-water interface. Peptide-induced vesicle fusion was monitored by lipid-mixing experiments, using fluorescent probes, the extent of peptide-lipid association, the conformation of lipid-associated peptides and their orientation in lipids, were studied by Fourier Transformed Infrared Spectroscopy. A comparison of the properties of the wild-type and variant peptides shows that the hydrophobicity gradient, which determines the orientation of helical peptides in lipids and their fusogenic activity, further influences the secondary structure and lipid binding capacity of these peptides.

Amino Acid Sequence↗