Search PubMed⌕ Search

Biomedical subjects

E Koren

Publications and source records attributed to E Koren.

At least 73 records · Page 4Linked to original sources
PubMed1986

Characterization of a monoclonal antibody that binds equally to all apolipoprotein and lipoprotein forms of human plasma apolipoprotein B. I. Specificity and binding studies.

A stable mouse hybridoma cell line has been developed that produces monoclonal antibody to human plasma apolipoprotein B. This antibody was proven to be specific for apolipoprotein B immunoblotting and an enzyme immunoassay using apolipoprotein B and other apolipoproteins. The antibody bound with comparable affinities to soluble apolipoprotein B, chylomicrons, very-low-density (VLDL) and low-density lipoproteins (LDL). Coupled to agarose, this antibody allowed complete removal of apolipoprotein B-containing lipoproteins from normolipidemic, hypertriglyceridemic and hypercholesterolemic plasma. Desialyzation and deglycosylation had no effect on its binding to LDL. The described antibody had no effect on the receptor-mediated binding of radiolabeled LDL to the human hepatoma cells (HepG2) in culture. Analysis of 25 different samples of human plasma indicated identical expression of the corresponding epitope in these individuals. The described monoclonal antibody, most likely, binds to a rather stable domain of apolipoprotein B that is not altered by the interaction with lipids or polymorphism of the apolipoprotein B. We propose that this antibody be called 'Pan B' antibody.

Animals↗
PubMed1986

Characterization of a monoclonal antibody that binds equally to all apolipoprotein and lipoprotein forms of human plasma apolipoprotein B. II. Isolation of apolipoprotein B-containing lipoproteins from human plasma.

Monoclonal antibody ('Pan B' antibody) that binds equally to all major forms of human plasma apolipoprotein B was used in an immunoaffinity chromatography procedure to isolate apolipoprotein B-containing lipoproteins from hyperlipidemic human plasma. These lipoproteins were compared with lipoproteins in native plasma, with lipoproteins isolated by polyclonal antibodies and with lipoproteins isolated by the conventional ultracentrifugational method. Judged by the apolipoprotein and lipid composition, lipoproteins isolated with 'Pan B' antibody were virtually identical to those isolated by ultracentrifugation or polyclonal antibodies. Lipoproteins isolated by 'Pan B' antibody were comparable in size and shape to the lipoproteins in native plasma and to the lipoproteins isolated by polyclonal antibodies or ultracentrifugation. The immunoaffinity column with monoclonal 'Pan B' antibody retained all apolipoprotein B-containing lipoproteins and showed significantly higher capacity than polyclonal immunoaffinity column. The column with the highest capacity allowed the isolation from whole plasma of 0.144 mg of apolipoprotein B per ml of gel in less than 2 h.

Animals↗
PubMed1986

Quantitative determination of human plasma apolipoprotein A-II by a non competitive enzyme-linked immunosorbent assay.

A noncompetitive enzyme-linked immunosorbent assay (ELISA) for apolipoprotein A-II (ApoA-II) was developed. Microtiter plates were coated with affinity purified antibodies to ApoA-II. After incubation with human plasma, the amount of ApoA-II bound to the coated plate was determined with peroxidase-labeled antibodies to ApoA-II. When pure ApoA-II or delipidated reference plasma was used as standard, a single step delipidation was required in order to unmask some antigenic sites of ApoA-II. However, the underestimated ApoA-II values in untreated samples were shown to be corrected by using intact reference plasma as secondary standard. The average concentration of ApoA-II in normolipidemic plasma was 0.376 g/l.

Antibodies↗
PubMed1985

Evaluation of immunoaffinity chromatography for isolating human lipoproteins containing apolipoprotein B.

Because of high specificity, immunoaffinity chromatography is the most suitable procedure for the isolation of lipoprotein (LP) particles defined by their apolipoprotein (Apo) composition. The purpose of the present study was to describe immunosorber methodology and its application to the isolation of ApoB-containing lipoproteins from either plasma or isolated lipoprotein density classes. The exploration of various coupling procedures demonstrated that immunosorbers of highest capacity were obtained by cyanogen bromide activation of Sepharose. Among various dissociating agents tested, 3 M sodium thiocyanate was found to be the most effective desorbent for bound lipoproteins. Studies on the non-specific binding of serum albumin to several different immunosorbers showed a negligible retention (1.9%) of albumin. Good recoveries (80-98%) were obtained with all apolipoproteins tested with both anti-ApoA-I and anti-LP-B immunosorbers. By using the optimal experimental conditions, it was shown that the ApoB-containing lipoproteins retained by immunosorbers with antibodies to LP-B had chemical, physical and immunological properties similar, if not identical, to those of their corresponding parent density classes. The application of an alternative immunoaffinity chromatography procedure with serially connected immunosorbers with antibodies to apolipoproteins other than ApoB resulted in the isolation of LP-B, a lipoprotein containing ApoB as its sole protein constitutent. LP-B had chemical and physical properties very similar to those of subclass 2 of low-density lipoproteins (density 1.019-1.063 g/ml, flotation coefficient 0-12). Based on these studies, we suggest that immunoaffinity chromatography in combination with microanalytical procedures for quantification of lipids and apolipoproteins offers a powerful tool for the isolation and functional characterization of lipoprotein particles defined by their apolipoprotein composition.

Adsorption↗
PubMed1985

Quantitative determination of human plasma apolipoprotein A-I by a noncompetitive enzyme-linked immunosorbent assay.

A noncompetitive enzyme-linked immunosorbent assay (ELISA) for human plasma apolipoprotein A-I (ApoA-I) was developed. Microtiter plates were coated with purified antibodies to ApoA-I and blocked. Plasma samples from normolipidemic and hypertriglyceridemic subjects were added and ApoA-I was allowed to bind to coating antibodies. After washing, the amount of ApoA-I bound to microtiter plates was estimated with horseradish peroxidase-labeled antibodies to ApoA-I. A single step delipidization procedure was included to expose masked antigenic sites of ApoA-I in plasma. The average concentration of ApoA-I in plasma of normolipidemic subjects was 1.37 g/l. Recovery of ApoA-I added to plasma varied from 93-107%. Intra- and inter-assay coefficients of variations were 4 and 8%, respectively. The assay was also used for quantifying ApoA-I in lipoprotein density classes. There was a good correlation between this assay and electroimmunoassay (r = 0.84-0.92). The described sandwich ELISA is a specific, precise, sensitive and relatively simple method for measuring ApoA-I levels in human plasma.

Apolipoprotein A-I↗
PubMed1985

Immunologic and structural studies of the lupus/Sjögren's syndrome autoantigen, La/SSB, with a monoclonal antibody.

La/SSB is a small nuclear RNA protein against which precipitating autoantibodies are made in many patients with systemic lupus erythematosus or Sjögren's syndrome. The recent purification of La/SSB has made structural and immunologic studies possible. Consequently, a mouse hybridoma antibody (La1) was raised, after immunization and fusion, that reacted with bovine La/SSB. Results of inhibition tests with tissue extracts and fluorescent antinuclear antibody tests demonstrated that La1 reacted with bovine extracts and cells, but not with those from human, mouse, or rabbit sources. La1 reacted in Western blot and in an adapted anti-La/SSB enzyme-linked immunosorbent assay with only the 41-kD bovine La/SSB peptide and not with the smaller 29-kD bovine La/SSB peptide. RNA gels showed that La1 bound the La/SSB particle that contained the predominant La/SSB RNA species near 90 nucleotides as well as the minor RNA species, both of which were bound by the human autoimmune anti-La/SSB serum. A solid-phase assay for human autoimmune anti-La/SSB antibody using La1 was more sensitive for the detection of human anti-La/SSB than was a comparable assay using purified La/SSB, and showed that anti-La/SSB is present in nearly all Ro/SSA precipitin-positive sera. Thus, this study demonstrates that monoclonal antibody can be raised against La/SSB; that the protein moiety of bovine La/SSB differs from human, mouse, and rabbit at an epitope on the 41-kD La/SSB peptide; that the RNA bound to the La1-reactive particle was as heterogeneous as that binding the anti-La/SSB autoimmune serum; and that anti-Ro/SSA and anti-La/SSB are closely associated.

Animals↗
PubMed1985

Detection of lecithin: cholesterol acyltransferase (LCAT) in a human hepatoma cell line.

A human hepatoma cell line (HepG-2) was probed for the presence of lecithin: cholesterol acyltransferase (LCAT) using an antiserum to human plasma LCAT. Double immunodiffusion analysis using antiserum to human plasma LCAT revealed a single precipitin line in the sonicated cell homogenate. This precipitin line showed a reaction of identity with highly purified plasma LCAT. The presence of LCAT within the hepatoma cells was also confirmed by an immunofluorescence test. In contrast, the cell culture supernate showed a weak and inconsistent precipitin line. These data suggest that HepG-2 cells synthesize LCAT but secretion of the enzyme by these cells into the culture medium may be partially or totally impaired.

Carcinoma, Hepatocellular↗
PubMed1984

Presence of B-100 in rat mesenteric chyle.

Molecular forms of apolipoprotein B (ApoB) were studied in the rat intestinal chyle by SDS-polyacrylamide gel electrophoresis, immunoblotting and immunodiffusion. Time studies on intestinal chyle showed the presence of B-100 in all the samples analyzed within 3 hr after drawing. However, the analyses repeated on day 2 or day 3 revealed disappearance of B-100 and appearance of B-48. Addition of 3 mM EDTA, 10 mM diisopropylfluorophosphate, 5 mM chloroquine and 10 mM epsilon-amino caproic acid slowed down but could not prevent the disappearance of B-100. Chylomicrons isolated from chyle in the presence of preservatives immediately after drawing displayed B-100 as a major and B-48 as a minor ApoB form. However, repeatedly washed chylomicrons or those isolated from chyle 18-24 hr after drawing showed B-48 as the only ApoB present. These results suggest that rat intestine synthesizes B-100 which is quickly converted to smaller molecular form.

Animals↗
PubMed1984

Catabolism of human low density lipoproteins by human hepatoma cell line HepG2.

The mechanism of hepatic catabolism of human low density lipoproteins (LDL) by human-derived hepatoma cell line HepG2 was studied. The binding of 125I-labeled LDL to HepG2 cells at 4 degrees C was time dependent and inhibited by excess unlabeled LDL. The specific binding was predominant at low concentrations of 125I-labeled LDL (less than 50 micrograms protein/ml), whereas the nonsaturable binding prevailed at higher concentrations of substrate. The cellular uptake and degradation of 125I-labeled LDL were curvilinear functions of substrate concentration. Preincubation of HepG2 cells with unlabeled LDL caused a 56% inhibition in the degradation of 125I-labeled LDL. Reductive methylation of unlabeled LDL abolished its ability to compete with 125I-labeled LDL for uptake and degradation. Chloroquine (50 microM) and colchicine (1 microM) inhibited the degradation of 125I-labeled LDL by 64% and 30%, respectively. The LDL catabolism by HepG2 cells suppressed de novo synthesis of cholesterol and enhanced cholesterol esterification; this stimulation was abolished by chloroquine. When tested at a similar content of apolipoprotein B, very low density lipoproteins (VLDL), LDL and high density lipoproteins (HDL) inhibited the catabolism of 125I-labeled LDL to the same degree, indicating that in HepG2 cells normal LDL are most probably recognized by the receptor via apolipoprotein B. The current study thus demonstrates that the catabolism of human LDL by HepG2 cells proceeds in part through a receptor-mediated mechanism.

Binding, Competitive↗
PubMed1982

Isolation and characterization of simple and complex lipoproteins containing apolipoprotein F from human plasma.

Apolipoprotein F (ApoF), one of the minor apolipoproteins in human plasma, has been recently isolated and partially characterized [Olofsson, S.O., McConathy, W.J., & Alaupovic, P. (1978) Biochemistry 17, 1032-1036]. In the present work, the interaction of ApoF with other apolipoproteins and lipids in human plasma was studied. By the successive use of immunosorbers specific for ApoF, apolipoprotein A-II (ApoA-II) and apolipoprotein A-I (ApoA-I), three different ApoF-containing lipoproteins were isolated from normolipidemic fasting human plasma. Their apolipoprotein content was determined by double immunodiffusion against monospecific antisera to all known serum apolipoproteins, electroimmunoassay, crossed immunoelectrophoresis, and polyacrylamide gel electrophoresis. Their lipid composition was determined by thin-layer chromatography. The three ApoF-containing lipoproteins were identified as LpF:A-I:A-II (lipoprotein containing ApoF, ApoA-I, and ApoA:II), LpF:A-I (lipoprotein containing ApoF and ApoA-I), and LpF (lipoprotein containing only ApoF). LpF:A-I:A-II was found to contain ApoF, ApoA-I, and ApoA-II in an apparent 2:1:1 molar ratio. Its lipid moiety was characterized by cholesterol ester (45%) and free cholesterol (28%) as the predominant lipids. LpF contained only ApoF, and in its major lipid components were also cholesterol esters (63%) and free cholesterol (21%). It is suggested that ApoF-containing lipoproteins may be involved in transport and/or esterification of cholesterol.

Apolipoproteins↗
PubMed1980

The possibilities of scintigraphic visualization of surfactants in the lung of the rat.

Incorporation of radioactive iodine into the surfactant system of the rat lung has been studied. Specially prepared particles labelled by 125I-oleic acid or 131I-oleic acid were injected intravenously and the distribution of radioiodine in the body of the animals was observed at various time intervals by means of a gamma camera and a gamma counter. The results showing appreciable accumulation of isotope in the lungs and the surfactant phospholipids, constitute a basis for the development of a new scintigraphic method for studying metabolic functions and morphology of the lung.

Animals↗
PubMed1979

Triglyceride-poor very low density lipoprotein in human serum.

The chemical and physical properties of very low density lipoproteins, isolated from the pool of the sera of 60 persons with high pre-beta and normal triglyceride and cholesterol concentrations, have been studied. These very low density lipoproteins, a designated as triglyceride-poor very low density lipoproteins, consist of 20.5% phospholipids, 30.8% free cholesterol, 15% cholesterol esters and 33.7% triglycerides. Their protein content consists of 54.5% apo B, 26% apo A, 11.5% apo E and only 8% apo C, so they differ from any serum lipoprotein described until now. Triglyceride-poor very low density lipoproteins consist of spherical particles 300-450 A in diameter as revealed by electron microscopy.

Cholesterol↗
PubMed1979

Mechanism of liquefaction of the human ejaculate. I. Changes of the ejaculate proteins.

The spontaneous liquefaction of the human ejaculate in vitro was studied by macroscopic inspection, disc electrophoresis of the ejaculate proteins, free amino acid and nitrogen determinations and by electron microscopy. Liquefaction occurred in three phases. Phase 1 was characterized by the macroscopic solubilization of the gelatinous material and by disappearance of its granular ultrastructure. Zn++, Hg++ and Cu++ completely inhibited the first step of the liquefraction, which could be reversed by Na2EDTA. The factor(s) involved is most probably a proteolytic enzyme(s) which is tightly incorporated into the gelatinous material. In Phase 2 of liquefaction solubilized proteins were degraded to peptides and in Phase 3 these peptides were degraded to amino acids. Factor(s) responsible for these two steps of liquefaction could be removed by washing the gelatinous material.

Adult↗
PubMed1979

Mechanism of liquefaction of the human ejaculate. II. Role of collagenase-like peptidase and seminal proteinase.

Collagenase-like peptidase and seminal proteinase were isolated from human testis and human seminal plasma. The effects of both enzymes upon proteins isolated from the human ejaculate were studied. Both enzymes degraded ejaculate proteins. The data suggest that collagenase-like peptidase is responsible for the first, and seminal proteinase for the second, phase of human ejaculate liquefaction in vitro.

Chromatography, Gel↗
PubMed1977

The metalloenzymic nature of collagenase-like peptidase of the rat testis.

Collagenase-like peptidase, an enzyme degrading synthetic collagenase substrate (PZ-pentapeptide), was purified from rat testes and its properties were examined. Its activity was strongly inhibited by chelating agents, such as EDTA and 1,10-phenanthroline. By chelation and exhaustive dialysis it was possible to obtain this enzyme in its inactive, metal-free form. The activity of the metal-free enzyme was partly recovered by treatment with zinc or manganese ions, while a combined zinc and manganese treatment resulted in complete recovery of enzyme activity.

Animals↗
PubMed1976

Calcium-binding protein in bull seminal vesicle secretion and seminal plasma.

A protein which showed high affinity for calcium ions was isolated from bull seminal vesicle secretion and seminal plasma. Its calcium-binding activity depended on the ionic strength and pH of the medium. The dissociation constant was 7-7 X 10(-7) M and there were 14 binding sites per protein molecule. The molecular weight of calcium-binding protein from bull seminal vesicle secretion, estimated by the gel filtration method, was 110,000. The protein may be involved in the regulation of the calcium ion level in seminal plasma.

Animals↗
PubMed1975

The coagulation of insoluble and basic proteins from rat siminal vesicle secretion with vesiculase: influence of collagenase-like peptidase from rat testis.

Proteins from the secretion of rat seminal vesicles were fractionated to yield (i) insoluble protein, (ii) basic proteins (soluble in the presence of urea) and (iii) neutral plus acidic proteins. The first two are necessary for the formation in vitro of a coagulum which is very similar to the copulatory plug. Collagenase-like peptidase from rat testis degrades both protein fractions and prevents coagulum formation.

Amides↗