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PubMed1995

A competitive chemiluminescent immunoassay for the sensitive measurement of apolipoprotein B100.

OBJECTIVES: We have developed a competitive chemiluminescent immunoassay for the sensitive measurement of apolipoprotein B100 (apoB) and conducted a preliminary evaluation of the method. DESIGN AND METHODS: In the assay, apoB-rabbit immunoglobulin G (IgG) conjugate competes with apoB in the sample for binding to acridinium N-hydroxysuccinimide labelled anti-apoB antibody. Goat anti-rabbit-IgG immobilized to magnetic particles is used to separate the apoB-rabbit IgG conjugate bound to the labelled anti-apoB antibody. Chemiluminescence, measured using a Ciba Corning Magic-Lite chemiluminometer, is inversely proportional to the concentration of apoB in the sample. RESULTS: The assay is completed in 1-2 h. Within-run imprecision (n = 5) determined at 10, 100, and 200 micrograms/L of apoB was found to be 4.8%, 2.4%, and 3.5%, respectively. The between-run imprecision (n = 5) at the same concentrations of apoB was determined to be 14.2%, 9.5%, and 7.7%, respectively. The proposed assay showed reasonable correlation (r = 0.86) with a commercially available immunoturbidimetric method. The lower limit of detection was 2.2 micrograms/L (4.0 pmol/L). CONCLUSIONS: This assay may be useful in tissue culture studies where sensitive measurement of secreted and intracellular concentrations of apoB are required. Our preliminary evaluation of the assay appears to confirm is usefulness.

Acridines↗
PubMed1995

Regulation of apolipoprotein B biogenesis in human hepatocytes: posttranscriptional control mechanisms that determine the hepatic production of apolipoprotein B-containing lipoproteins.

OBJECTIVES: Hepatic overproduction of apolipoprotein B (apoB)-containing lipoproteins appears to be a common cause of hyperlipoproteinemia in humans. Patients with overproduction states secrete denser cholesterol ester-rich lipoprotein particles which are highly atherogenic. The formation of apoB particles involves a very complex process that requires the coordinated synthesis and assembly of apoB, triglycerides, cholesterol esters, phospholipids, and other components. ApoB expression is an important prerequisite for the assembly and secretion of apoB particles. Evidence to date appears to suggest that apoB expression is regulated posttranscriptionally. ApoB secretion rate is determined at the levels of apoB translocation into the endoplasmic reticulum (ER) as well as degradation within the ER. RESULTS AND HYPOTHESIS: Based on available data, we postulate that the rate of apoB particle secretion is determined at the critical point where newly-synthesized apoB interacts with core lipids, particularly triglycerides. The supply of these lipids determines the rate of translocation of the apoB molecule across the ER membrane and into the ER lumen. Lipidation of apoB facilitates its proper folding, its assembly into a lipoprotein particle, and its extracellular secretion. In the absence of lipids, apoB is misfolded resulting in the abortion of ER translocation and subsequent degradation by an apoB specific protease. CONCLUSIONS: The balance between intracellular degradation and extracellular secretion determines the rate at which the human liver secretes apoB particles.

Animals↗
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Behaviorism↗
PubMed1995

Effects of adenosine receptor agonists and antagonists on physostigmine-induced yawning.

The effect of adenosine receptor agonists and antagonists on physostigmine-induced yawning was investigated in intact or cannulated rats. Intraperitoneal (i.p.) or intracerebroventricular (i.c.v.) administration of physostigmine to rats induced yawning dose dependently. I.p. or i.c.v. treatment of the animals with atropine, theophylline, 5-N-ethylcarboxamidoadenosine (NECA) or N6-cyclohexyladenosine reduced the yawning induced by i.p. injection of physostigmine. I.p. administration of theophylline decreased the yawning induced by i.c.v. injection of physostigmine. The inhibitory action of N6-cyclohexyladenosine (i.p.) also was decreased by 8-phenyltheophylline (i.p.) pretreatment. It is concluded that yawning induced by a central cholinergic mechanism and a central adenosine mechanism interacts with the cholinergic-induced behaviour.

Adenosine↗
PubMed1985

Biogenesis of liver delta-aminolevulinate synthase. The role of cAMP in the induction.

In primary cultures of chick embryo hepatocytes pulse labeled with [35S]methionine, immunochemical analyses indicated that adenosine 3':5'-cyclic monophosphate (cAMP) did not affect either the rate of production or the maturation of delta-aminolevulinate synthase (ALA synthase). In addition, allylisopropylacetamide caused a slight drop in intracellular cAMP while testosterone caused the levels of cAMP to rise to 260% of the basal levels measured in hepatocytes in culture. Thus the results of this study did not indicate a direct short-term role for cAMP in the regulation of production of ALA synthase.

5-Aminolevulinate Synthetase↗
PubMed1990

Secretion of apolipoprotein B in serum-free cultures of human hepatoma cell line, HepG2.

We have developed a defined medium which can maintain efficient growth of HepG2 cells sustaining the synthesis of a variety of plasma proteins including apolipoprotein B. This defined system was used to investigate long-term effects of insulin, estrogen, triiodothyronine, cholesterol, and oleate on the growth pattern of HepG2 cells and secretion rate of apolipoprotein B. Oleate and triiodothyronine caused significant increases in secretion of apolipoprotein B. The stimulatory effect of triiodothyronine was only observed after long (6 days) exposure of cells to the hormone. In contrast, insulin caused up to a 4-fold decrease in the secretion rate of apolipoprotein B during the early growth periods. This inhibitory effect appeared to be partially abolished after 6 days. Our data suggest that some important questions on regulation of apolipoprotein B expression can be addressed by the long-term culture of HepG2 cells in defined medium.

Apolipoproteins B↗
PubMed1988

Maturation of embryonic chick liver delta-aminolevulinate synthase: precursor pools and regulation by intra-cellularly produced heme.

1. Immunoblot analyses were carried out to determine the relative distributions of delta-aminolevulinate synthase (ALA synthase) in mitochondrial and cytosol fractions prepared from embryos at different times after injections with allylisopropylacetamide (AIA). 2. The results indicated that the molecular mass of mature ALA synthase (Mr 65,000) increased with time in mitochondria. 3. At no time was the precursor form (Mr 75,000) of the enzyme detected either in mitochondria or in the cytosol. 4. In primary cultures of hepatocytes, where the increased production of ALA synthase had been induced with AIA, addition of delta-aminolevulinic acid (ALA) and Fe2(SO4)3 into the culture medium completely blocked the processing of the precursor form of the enzyme. 5. On the other hand, the addition of ALA together with deferoxamine mesylate into the medium had no detectable effect on the maturation of ALA synthase in the hepatocytes. 6. The results indicated: first, that upon induction of porphyria the pools of pre-ALA synthase in liver are relatively low in chick embryos when compared with those in other organisms; and second, that increased heme production by the hepatocytes caused the inhibition of processing of the precursor form of ALA synthase.

5-Aminolevulinate Synthetase↗
PubMed1988

Properties of chicken erythrocyte delta-aminolevulinate synthase.

A procedure is described for preparing a fraction highly enriched for chicken blood delta-aminolevulinate synthase (ALA-S) using animals recovering from acetylphenylhydrazine-induced anemia. 1. Blood cells collected from chickens recovering from anemia were disrupted by nitrogen cavitation, and the mitochondrial fraction was prepared from the cell homogenates. ALA-S was released then from mitochondria by sonication and isolated by a procedure involving gel filtration chromatography on Sephadex G-150, fractionation with ammonium sulfate, ion exchange chromatography on DEAE-Sephacel, and preparative isoelectric focusing. 2. Electrophoretic analyses under denaturing conditions indicated that the final ALA-S preparation was particularly enriched from a 62,200 Da polypeptide. The enzyme eluted from Sephadex G-200 with an equivalent molecular weight of 115,000; this suggested that active ALA-S was a dimer. 3. ALA-S was most active in the pH range of 7.0-8.0, with an apparent KM of 13 microM for succinyl-CoA and of 4.0 mM for glycine. The activity was inhibited 50% by 30 microM hemin.

5-Aminolevulinate Synthetase↗
PubMed1989

Coordinate elevations of liver delta-aminolevulinate synthase and cytochrome P-450 RNA by phenobarbital in chicken embryos: the effects of heme.

1. The role of heme in the coordinate elevations of liver delta-aminolevulinate (ALA) synthase activity and microsomal cytochrome P-450 concentration induced by phenobarbital (PB) was investigated in the chicken embryo. 2. Eighteen day old chicken embryos were given PB, and the changes in liver content of PB-inducible cytochrome P-450 RNA and of ALA synthase RNA were determined at different times after exposure to the drug. 3. The concentrations of both types of RNA increased rapidly after PB administration, and by 9 hr the level of ALA synthase RNA was 55-fold higher than control and that of cytochrome P-450 RNA was 7-fold higher than normal. 4. While the rate of increase in ALA synthase activity paralleled closely that of the enzyme's RNA concentration, the rate of increase of spectrally active cytochrome P-450 concentration in microsomes lagged behind that of the apoprotein's RNA by several hours. 5. To test whether heme depletion was responsible for the coordinate inductions of the two enzymes, embryos were loaded with ALA 2 hr before exposure to PB. 6. The protocol led to a drop in the PB-inducible ALA synthase RNA concentration and to an increase in that of cytochrome P-450 RNA, measured 6 hr after drug administration. 7. In primary cultures of hepatocytes, hemin in the culture medium caused a modest drop in ALA synthase RNA concentration but had a variable effect on that of cytochrome P-450 RNA in cells incubated with PB for 9 hr.(ABSTRACT TRUNCATED AT 250 WORDS)

5-Aminolevulinate Synthetase↗
PubMed1989

Regulation of biogenesis of liver delta-aminolevulinate synthase: effects of structural modifications of heme on the enzyme's RNA.

1. The aim of this study was to determine the effects of several metallo-porphyrins, derived by modifications of heme, on the concentration delta-aminolevulinate (ALA) synthase RNA in hepatocytes. 2. Primary cultures of chick embryo hepatocytes were incubated with allylisopropylacetamide (AIA) for 5 hr in the presence and absence of each metallo-porphyrin (10 microM). At the end of each incubation, total RNA was isolated from the cells and analyzed for ALA synthase-specific RNA by solution hybridization. 3. The concentration of ALA synthase RNA increased 7.3 fold in hepatocytes incubated with AIA alone. The AIA-induced elevations in the enzyme's RNA were blocked partially and equally in cells. incubated with zinc- or with iron-protoporphyrin IX. The block was greater in cells incubated with cobalt-protoporphyrin IX. 4. Modifications of the side chains of the porphyrin ring at positions 2 and 4, giving mesoporphyrin IX and deuteroporphyrin IX, changed the effectiveness of the iron- and the cobalt-porphyrins to limit the AIA-induced increase in ALA synthase RNA. The modifications did not affect the capacities of the zinc-porphyrins to inhibit the rise in RNA. 5. In conclusion, the effect of a given metallo-porphyrin on liver ALA synthase RNA following side chain modification depended on the coordinated metal.

5-Aminolevulinate Synthetase↗
PubMed1989

Characterization of chicken liver L-alanine:4,5-dioxovaleric acid aminotransferase.

1. A procedure is described for purifying the enzyme L-alanine:4,5-dioxovaleric acid aminotransferase (DOVA transaminase) from chicken liver. The enzyme catalyzes a transamination reaction between L-alanine and 4,5-dioxovaleric acid (DOVA), yielding delta-aminolevulinic acid (ALA). 2. In cell fractionation studies, DOVA transaminase activities were detected in mitochondria and in the post-mitochondrial supernatant fraction from liver homogenates. 3. For the mitochondrial enzyme, any of most L-amino acids could serve as a source for the amino group transferred to DOVA, but L-alanine appeared the preferred substrate. At pH 7.0, the enzyme had an apparent Km of 60 microM for DOVA and of 400 microM for L-alanine. 4. The enzyme was purified from disrupted mitoplasts in three steps: chromatography on DEAE-Sephacel, gel filtration through Sephadex G-150, and chromatography on hydroxyapatite. The yield was approx. 100 micrograms of enzyme protein per 10 g wet wt of liver. 5. The purified enzyme had a subunit mol. wt of 63,000 as determined by gel electrophoresis under denaturing conditions. 6. The activity of DOVA transaminase was also measured in embryonic chicken liver, and based on activity, the enzyme's capacity to produce ALA was significantly greater than that of ALA synthase. Unlike ALA synthase, however, DOVA transaminase activity did not increase in liver mitochondria of chicken embryos exposed for 18 hr to two potent porphyrogenic agents.

Animals↗
PubMed1990

Induction of cytochrome P-450 RNA by porphyrogenic agents: on the nature of the coordinate induction with delta-aminolevulinate synthase in tissues of the chicken embryo.

1. We determined by cDNA-RNA solution hybridization analyses that in ovo administration of allylisopropylacetamide in combination with diethyl 1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate increased the concentrations of cytochrome P-450 RNA in liver, kidney, and intestine of 18-day-old chicken embryos. 2. Similarly, the administration of testosterone to embryos caused elevations in the cytochrome P-450 RNA levels in liver and kidney. 3. The increases in cytochrome P-450 RNA concentrations occurred only in those tissues where elevations in delta-aminolevulinate (ALA) synthase activity and mRNA content were measured (liver, kidney and intestine) but not in tissues where the activity and RNA levels of ALA synthase did not change (heart, brain, lung). 4. The increases in the concentrations of the cytochrome P-450 RNA were not affected by loading embryos with ALA and FeCl3 at the time of administration of the inducers.

5-Aminolevulinate Synthetase↗