scholarly journals Apolipoprotein B mRNA and lipoprotein secretion are increased in McArdle RH-7777 cells by expression of betaine-homocysteine S-methyltransferase

1999 ◽  
Vol 341 (3) ◽  
pp. 639-645 ◽  
Author(s):  
Mark P. SOWDEN ◽  
Heidi L. COLLINS ◽  
Harold C. SMITH ◽  
Timothy A. GARROW ◽  
Janet D. SPARKS ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Monoclonal Antibodies ◽  
Small Intestine ◽  
Apolipoprotein B ◽  
Rat Hepatocytes ◽  
Mrna Abundance ◽  
Mrna Editing ◽  
Apo B ◽  
Lipoprotein Secretion ◽  
Steady State Levels

The cDNA encoding rat betaine-homocysteine S-methyltransferase (BHMT) was isolated through production of monoclonal antibodies against protein fractions enriched with apolipoprotein B (apo B)-mRNA-editing complexes. BHMT mRNA was expressed predominantly in liver, and also in kidney, but not in small intestine. In stable McArdle RH-7777 (McA) cell lines expressing differing levels of BHMT, the editing efficiency of apo B mRNA was unchanged. Evaluation of apo B-mRNA expression revealed that steady-state levels were increased significantly and in parallel with BHMT protein expression. The highest levels of BHMT mRNA and BHMT enzyme activity expressed in stably transfected McA cells were comparable with those found in rat hepatocytes. In contrast to the changes in apo B-mRNA abundance, levels of other apolipoprotein-encoding mRNAs and several liver-specific and ubiquitously expressed mRNAs were unchanged by BHMT expression. In the cell line expressing the highest level of BHMT, apo B-containing lipoprotein secretion was increased, indicating utilization of increased endogenous message. Results suggest that apo B-mRNA abundance in McA cells is related to the expression of BHMT, an enzyme important in homocysteine metabolism.

Monoclonal antibodies can precipitate low-density lipoprotein. I. Characterization and use in determining apolipoprotein B.

1985 ◽  
Vol 31 (10) ◽  
pp. 1654-1658 ◽  
Author(s):  
S Marcovina ◽  
D France ◽  
R A Phillips ◽  
S J Mao
Keyword(s):  
Monoclonal Antibodies ◽  
Apolipoprotein B ◽  
Polyclonal Antibodies ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Radial Immunodiffusion ◽  
Enzyme Linked Immunosorbent Assay ◽  
Low Density ◽  
Apo B ◽  
Blotting Technique

Abstract We produced 20 mouse monoclonal antibodies against human plasma low-density lipoprotein (LDL). Individually they failed to precipitate LDL in agarose gel by the double-immunodiffusion technique; collectively they did, or as few as two combined monoclonal antibodies could do so. To mimic polyclonal antibodies in determination of apolipoprotein B (apo B) by radial immunodiffusion, a combination of four particular monoclonal antibodies (clones A, B, C, and D) was necessary. We characterized these four clones with respect to temperature dependency, affinity, total binding to 125I-labeled LDL, and specificity to the different species of apolipoprotein B. Two monoclonal antibodies (B and C) bound 100% of 125I-labeled LDL; clones A and D bound 80% and 87%, respectively. All four clones bound maximally to LDL at 4 degrees C. The affinity constants for clones A, B, C, and D were 0.6, 2.1, 3.8, and 2.3 X 10(9) L/mol, respectively. By the Western blotting technique, the four monoclonal antibodies all reacted with the species B-100 and B-74 of apolipoprotein B, and to various degrees with B-48 and B-26. Radial immunodiffusion (chi) and direct enzyme-linked immunosorbent assay (y) with a mixture of the four monoclonal antibodies gave almost identical results for 70 patients: y = 0.921 chi-2.58; r = 0.933.

Noncompetitive enzyme-linked immunoassay for apolipoprotein B in serum.

1984 ◽  
Vol 30 (1) ◽  
pp. 98-100 ◽  
Author(s):  
C Fievet ◽  
M Koffigan ◽  
D Ouvry ◽  
S Marcovina ◽  
Y Moschetto ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Human Plasma ◽  
Apolipoprotein B ◽  
Solid Phase ◽  
Minimum Detectable Concentration ◽  
Low Density ◽  
Apo B ◽  
Detectable Concentration ◽  
Sensitivity Specificity ◽  
Assay Conditions

Abstract We used a noncompetitive enzyme-linked immunoassay to measure apolipoprotein B (apo-B) concentration in human plasma. Goat anti-lipoprotein B immunoglobulins were adsorbed to the surface of polystyrene balls. After washing, this solid-phase antibody was incubated with antigen (plasma from normal or hyperlipoproteinemic fasting subjects), washed, and then incubated with peroxidase-labeled goat anti-lipoprotein B IgG. After a last washing, we measured the bound label, which provided a direct measurement of the antigen. Under optimized assay conditions, the minimum detectable concentration was 50 ng per assay. The assay may be used to measure apo-B in different lipoprotein fractions (low- or very-low-density) and yields values that compared favorably with those obtained by electroimmunoassay (r = 0.86). The assay offers several advantages over existing techniques: sensitivity, specificity, simplicity, avoidance of radioisotopes, and potential for use with monoclonal antibodies.

scholarly journals Synchronized synthesis and intracellular transport of serum albumin and apolipoprotein B in cultured rat hepatocytes as studied by double immunofluorescence.

1986 ◽  
Vol 34 (9) ◽  
pp. 1223-1230 ◽  
Author(s):  
G A Keller ◽  
C Glass ◽  
D Louvard ◽  
D Steinberg ◽  
S J Singer
Keyword(s):  
Serum Albumin ◽  
Apolipoprotein B ◽  
Intracellular Transport ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Cell System ◽  
Secretory Proteins ◽  
Transport Pathway ◽  
Apo B ◽  
Normal Rat

Synthesis and intracellular transport of two secretory proteins, serum albumin (SA) and apolipoprotein B (apo B) have been synchronized in primary cultures of normal rat hepatocytes to make possible immunocytochemical study of the transport pathway. Under appropriate conditions of cycloheximide treatment, synthesis of new protein was inhibited and, by double immunofluorescent labeling, the cells were found to be largely depleted of the SA and apo B previously synthesized. Re-initiation of protein synthesis led to sequential appearance of SA and apo B, first in the endoplasmic reticulum, then in the Golgi complex, and finally at the cell surface. These results indicate that it should be feasible to use this cell system for high-resolution investigation of the sequence of structures involved in intracellular transport of SA and apo B by corresponding immunolabeling experiments as observed by electron microscopy.

scholarly journals Insulin-mediated inhibition of apolipoprotein B secretion requires an intracellular trafficking event and phosphatidylinositol 3-kinase activation: studies with brefeldin A and wortmannin in primary cultures of rat hepatocytes

1996 ◽  
Vol 313 (2) ◽  
pp. 567-574 ◽  
Author(s):  
Janet D. SPARKS ◽  
Thuy L. PHUNG ◽  
Mary BOLOGNINO ◽  
Charles E. SPARKS
Keyword(s):  
Apolipoprotein B ◽  
Enzyme Inhibitor ◽  
Primary Cultures ◽  
Brefeldin A ◽  
Rat Hepatocytes ◽  
Phosphatidylinositol 3 Kinase ◽  
Apo B ◽  
Insulin Effect ◽  
Insulin Inhibition ◽  
Phosphatidylinositol 3

Insulin inhibition of the secretion of apolipoprotein B (apo B) was studied in primary cultures of rat hepatocytes by using brefeldin A (BFA), an inhibitor of protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus, and by using the phosphatidylinositol 3-kinase (PI 3-K) inhibitor wortmannin. Incubation of hepatocytes with BFA (10 μg/ml) for 1 h inhibited the subsequent secretion of apo B, albumin and transferrin for up to 3 h. BFA treatment resulted in the time-dependent accumulation in cells of [14C]leucine-labelled proteins and apo B. Under conditions where insulin decreased total apo B (cell plus secreted), BFA blocked the insulin-dependent effect. These results suggest that export of apo B from the ER is a prerequisite for the observed insulin effect. Treatment of hepatocytes with wortmannin for 20 min abolished insulin inhibition of apo B secretion, suggesting that the insulin effect on the apo B pathway involves activation of PI 3-K. Enzyme inhibitor studies indicate that chymostatin and (+)-(2S,3S)-3-[(S)-methyl-1-(3-m e t h y l b u t y l c a r b a m o y l) - b u t y l c a r b a m o y l] - 2-oxiranecarboxylate (E-64-c) partially block insulin effects on apo B compared with leupeptin, which had no discernible effect. The cell-permeable derivative of E-64-c, EST, and N-Ac-Leu-Leu-norleucinal (ALLN) were most effective in blocking insulin effects on apo B. These results suggest that insulin action on apo B in primary rat hepatocytes involves (1) vesicular movement of apo B from the ER; (2) activation of PI 3-K and (3) a cellular protease that is either a cysteine- or calcium-activated neutral protease.

scholarly journals Developmental Regulation of Apolipoprotein B mRNA Editing Is an Autonomous Function of Small Intestine Involving Homeobox Gene Cdx1

2002 ◽  
Vol 278 (9) ◽  
pp. 7600-7606 ◽  
Author(s):  
Amy P. Patterson ◽  
Zhigang Chen ◽  
Deborah C. Rubin ◽  
Virginie Moucadel ◽  
Juan Lucio Iovanna ◽  
...  
Keyword(s):  
Small Intestine ◽  
Apolipoprotein B ◽  
Developmental Regulation ◽  
Homeobox Gene ◽  
Mrna Editing ◽  
Autonomous Function

scholarly journals Inhibition of apolipoprotein B net synthesis and secretion from cultured rat hepatocytes by the calcium-channel blocker diltiazem

1989 ◽  
Vol 263 (2) ◽  
pp. 411-415 ◽  
Author(s):  
T C Kwong ◽  
J D Sparks ◽  
D J Pryce ◽  
J F Cianci ◽  
C E Sparks
Keyword(s):  
Molecular Mass ◽  
Apolipoprotein B ◽  
Channel Blocker ◽  
Rat Hepatocytes ◽  
Acetate Incorporation ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Apo B ◽  
Cellular Lipids ◽  
Mass Form

1. The effect of the Ca2+-channel blocker diltiazem on hepatic apolipoprotein B (apo B) synthesis and secretion was studied in 12-18 h cultures of collagenase-dispersed rat hepatocytes. 2. The presence of diltiazem in the medium decreased apo B secretion by hepatocytes in a concentration-dependent manner. At 25 microM, diltiazem inhibited apo B secretion by approx. 36%, but there was no evidence of intracellular accumulation of apo B. 3. The inhibition of apo B secretion by hepatocytes was significantly correlated with cell-associated diltiazem (r = 0.72, P less than 0.01). 4. The rate of apo B secretion remained linear over 16 h even in the presence of 50 microM-diltiazem. 5. At diltiazem concentrations in the medium which were inhibitory for apo B secretion, [14C]acetate incorporation into cellular lipids and [35S]methionine incorporation into protein were enhanced. 6. Diltiazem inhibited the secretion of the apo B variants with a preferential inhibition of the higher-molecular-mass form of apo B (apo BH) over the lower-molecular-mass form (apo BL) at diltiazem concentrations in the medium greater than 25 microM. 7. Together, these results suggest that Ca2+ may play an important role in the synthesis and secretion of apo B-containing lipoproteins.

Monoclonal antibodies can precipitate low-density lipoprotein. II. Radioimmunoassays with single and combined monoclonal antibodies for determining apolipoprotein B in serum of patients with coronary artery disease.

1985 ◽  
Vol 31 (10) ◽  
pp. 1659-1663 ◽  
Author(s):  
S Marcovina ◽  
B A Kottke ◽  
S J Mao
Keyword(s):  
Coronary Artery Disease ◽  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Coronary Artery ◽  
Apolipoprotein B ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Low Density ◽  
Apo B ◽  
Artery Disease

Abstract We have established four lines of monoclonal antibodies against human low-density lipoproteins (LDL) that, mixed in equal proportions, can precipitate LDL in gel and so can be used for apolipoprotein (apo) B determination in plasma. One monoclonal antibody (clone A), with a relatively low binding affinity to LDL (ka = 0.6 X 10(9) L/mol) and recognizing only two species of apo B, significantly underestimated the concentration of apo B in 74 patients with and 27 without coronary artery disease (CAD). High-affinity monoclonal antibody C (Ka = 3.8 X 10(9) L/mol), which recognized all four apo B species, gave the same value for apo B as determined with the mixture of monoclonal antibodies. The latter results (by radioimmunoassay, y) correlated well with those by radial immunodiffusion (chi): y = 0.994 chi + 0.003 (r = 0.987). The CAD patients showed an increased concentration of apo B as compared to the angiographically documented CAD-negative patients. Except for the values determined by clone B (p = 0.07), the increase was statistically significant (p = 0.002-0.018) for values determined by use of the other clones or their mixture.

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