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K Adeli

Publications and source records attributed to K Adeli.

At least 37 records · Page 2Linked to original sources
PubMed1996

In vitro translation and translocation of apolipoprotein B in a cell-free system from HepG2 cells.

An mRNA-dependent cell-free system has been developed from HepG2 cells by hydrolysis of endogenous mRNA with micrococcal nuclease. When supplied with RNA extracted from HepG2 cells, the system synthesized liver specific proteins such as albumin and apolipoprotein B100. Significant amounts of microsomes were also detected in the lysate by measuring NADH-cytochrome c reductase activity and ultracentrifugation. Protease protection assays showed the capability of the HepG2 lysate to translocate newly-synthesized proteins such as apolipoprotein Al, albumin, and apoB into the microsomes as they were protected from digestion with exogenously added protease K, but not protected in the presence of protease K and Triton X-100. The system also proved to be very active toward translation of exogenous mRNAs as evidenced by efficient translation of brome mosaic virus RNA. The HepG2 translation-translocation system appears to provide a unique homologous system for studies on the biogenesis of liver specific proteins, particulary apoB100.

Apolipoproteins B↗
PubMed1996

Influence of DT diaphorase on quinone-mediated genotoxicity in human and fish cell lines.

The influence of the quinone-reducing enzyme, DT diaphorase [NAD(P)H: (quinone acceptor) oxidoreductase], on the genotoxicity of quinones was examined in two cell lines, namely a human hepatoma cell line, HepG2 and a brown bullhead fibroblast cell line, BB. The quinone-reductive characteristics of these two cell lines were examined using an acetylated cytochrome c reduction assay for enzymatic reductase activity. Subsequently, the influence of DT diaphorase on the genotoxicity of two model quinones, menadione (MND) and 9,10-phenanthrenequinone (PQ) was examined in an alkaline unwinding assay for DNA single-strand breaks. Results revealed that DT diaphorase was the predominant quinone reductase in cytosols of both cell lines, and that levels of specific DT diaphorase activity were generally equivalent in the two species. Despite these similarities, results revealed marked qualitative differences between the two species in terms of the influence of DT diaphorase on quinone-mediated genotoxicity. When pretreated with the DT diaphorase inhibitor, dicoumarol, HepG2 cells exhibited a marked exacerbation of genotoxicity in the presence of either MND or PQ, indicating protective influence of the enzyme. In contrast, quinone genotoxicity in BB cells was not affected by DT diaphorase inhibition, indicating the lack of a protective effect of DT diaphorase. This study illustrates the manner in which functionally analogous enzymes may have markedly distinct influences on xenobiotic toxicity in different cellular systems.

Animals↗
PubMed1996

Clinical laboratory investigation of the Sanofi ACCESS CK-MB procedure and comparison to electrophoresis and Abbott IMx.

This evaluation was undertaken to verify the application protocol for the CK-MB assay on the ACCESS Immunoassay Analyzer (Sanofi Diagnostics Pasteur, Chaska, MN). The results show that the ACCESS CK-MB assay total imprecision was 6.8% to 9.1%. Analytical linearity of the ACCESS CK-MB assay was excellent in the range of < 1-214 micrograms/L. A comparison of the ACCESS CK-MB assay with the IMx (Abbott Laboratories, Abbott Park, IL) method shows good correlation r = 0.990 (n = 108). Linear regression analysis yielded Y = 1.36X-0.3, Sx/y = 7.2. ACCESS CK-MB values also correlated well with CK-MB by electrophoresis with r = 0.968 (n = 132). The linear regression equation for this comparison was Y = 1.08X + 1.4, Sx/y = 14.1. The expected non-myocardial infarction range of CK-MB determined by the ACCESS system was 1.3-9.4 micrograms/L (mean = 4.0, n = 58). The ACCESS CK-MB assay would appear to be rapid, precise and clinically useful.

Adolescent↗
PubMed1995

Intracellular degradation of apolipoprotein B generates an N-terminal 70 kDa fragment in the endoplasmic reticulum.

Regulated apoB degradation in HepG2 cells occurs in the endoplasmic reticulum (ER), is catalyzed by an N-acetylleucylleucylnorleucinal (ALLN)-sensitive proteinase, and generates a specific 70 kDa fragment (Adeli, K., 1994, J. Biol. Chem. 269, 9166-9175) [corrected]. In the present report, we have characterized the 70 kDa fragment by immunoprecipitation of permeabilized HepG2 cells with a battery of monoclonal antibodies against various sites on the apoB molecule. N-Terminal monoclonal antibodies (1D1 and 2D8) were capable of binding to the 70 kDa fragment suggesting that this polypeptide is an N-terminal fragment of the intact apoB. Subcellular fractionation of permeabilized cells and carbonate extraction resulted in the detection of the 70 kDa fragment in the ER lumen. Endoglycosidase H treatment confirmed that the fragment is N-linked glycosylated. We hypothesize that the ALLN-sensitive proteinase which may be located on the luminal side of the ER membrane, catalyzes an initial cleavage of apoB near the N-terminus generating a 70 kDa fragment, which is then released into the ER lumen.

Animals↗
PubMed1995

Human bioassays to assess environmental genotoxicity: development of a DNA break bioassay in HepG2 cells.

OBJECTIVES: Increasing interest in environmental health issues has created a demand for improved methods for the assessment of pollutant effects on humans. Our laboratory has developed an in vitro assay for the quantification of genotoxicity, monitored as DNA single strand breaks (SSB), in the HepG2 human hepatoma cell line. DESIGN AND METHODS: This assay procedure, which is based upon alkaline unwinding and hydroxylapatite DNA chromatography, is both rapid and simple to perform. RESULTS: HepG2 cells responded to the standard mutagen, 4-nitroquinoline N-oxide, demonstrating SSB formation at concentrations above 0.1 mumol/L. Phenanthrene-9,10-quinone, a component of diesel exhaust, mediated SSB formation at concentrations above 250 nmol/L. Finally, an extract of contaminated sediment from the Great Lakes Basin mediated SSB formation in a dose-dependent manner. CONCLUSIONS: These results illustrate the utility of this human genotoxicity assay for future use in screening of environmental pollutants.

4-Nitroquinoline-1-oxide↗
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↗
PubMed1994

Regulated intracellular degradation of apolipoprotein B in semipermeable HepG2 cells.

A digitonin-permeabilized HepG2 cell system has been developed and used to characterize the pathway responsible for apolipoprotein B100 (apoB) degradation. Degradation of 35S-labeled apoB occurred after an initial lag period and resulted in the loss of approximately 75.8% of apoB after 2 h of chase. The degradation rate in permeabilized cells was slower than that in intact cells, but approximately the same percentage of apoB was lost over 2 h of chase. The loss of intact apoB in permeabilized cells coincided with the appearance of a number of degradation fragments, including 335- and 70-kDa fragments. The detection of a 70-kDa fragment was a sensitive indicator of degradation. ApoB degradation was inhibited at temperatures below 37 degrees C and maximally activated at 42 degrees C. Degradation was also pH-dependent, with inhibition at pH > or = 7.5. ApoB decay was stimulated by ATP supplementation but not GTP and was inhibited in the presence of energy inhibitors. Degradation was not significantly affected by cycloheximide. However, the stability of the 70-kDa fragment was prolonged with cycloheximide. Preincubation of cells with brefeldin A and nocodazole, as well as monensin, did not diminish the degradation of intact apoB or the appearance of the 70-kDa fragment, suggesting a lack of requirement for intracellular transport from the endoplasmic reticulum. Among the various protease inhibitors tested, degradation was most sensitive to N-acetylleucylleucylnorleucinal (ALLN), which abolished the generation of the 70-kDa fragment in a dose-dependent manner. ALLN-sensitive degradation of apoB was unaffected by the calcium ionophore, A23187. Interestingly, degradation of unglycosylated apoB, detected in tunicamycin-pretreated cells, occurred earlier, resulted in generation of additional fragments, and was largely uninhibited by ALLN. In summary, apoB degradation occurs in permeabilized HepG2 cells by a temperature- and pH-sensitive, pre-Golgi degradation system and is catalyzed by a calcium-independent, ALLN-sensitive protease. Specificity of the initial apoB cleavage may require the proper N-linked glycosylation of the protein in the endoplasmic reticulum.

Adenosine Triphosphate↗
PubMed1994

Human bioassays to assess environmental genotoxicity: development of a DNA repair assay in HepG2 cells.

A direct assessment of the effects of environmental chemicals on human health has been hampered by the lack of suitable experimental systems. We have recently employed a human liver cell line (HepG2) to assess the biological effects of pollutants at both cellular and DNA levels. A Neutral Red dye uptake assay was used to assess potential cytotoxic effects of xenobiotics. DNA damage was quantified using an unscheduled DNA synthesis assay that measures repair that is induced following exposure to genotoxic compounds. HepG2 cells responded to the known mutagens, 4-nitroquinoline N-oxide and methylmethane sulfonate, both in the Neutral Red assay for cytotoxicity and two DNA repair assays for genotoxicity (monitored autoradiographically or by liquid scintillation counting). The HepG2 DNA repair and cytotoxicity assays also responded to an extract (containing polycyclic aromatic hydrocarbons) of sediment obtained from a polluted site in the Great Lakes. Results indicate that this system can be deployed further to assess potential cyto- and genotoxicity of pollutants. The development of human cell culture assays is a critical step towards a full assessment of the risk that such pollutants pose to human health.

4-Nitroquinoline-1-oxide↗
PubMed1993

Expression of cathepsin B and aryl hydrocarbon hydroxylase activities, and of apolipoprotein B in human hepatoma cells maintained long-term in a serum-free medium.

We have established the human hepatoma cell line, HepG2, in a defined, serum-free medium. These cells were maintained and studied over a 100-generation period (i.e. 10 serial transfers). Cells maintained in serum-free medium exhibited growth parameters (i.e. saturation density, efficiency of plating, and population doubling time) similar to those obtained with HepG2 cells maintained in serum-supplemented medium. Serum-free cells were also similar to their serum-supplemented counterparts with respect to the expression of cathepsin B activity and the induction of aryl hydrocarbon hydroxylase by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Significantly, HepG2 cells maintained in serum-free conditions also retained the ability to synthesize and secrete proteins, including the liver plasma protein, apo-lipoprotein B. These results indicate that the serum-free medium used in this study supports the long-term growth and maintenance of human hepatoma, HepG2, cells in culture. Inasmuch as these cells retain phenotypes, including differentiated markers previously reported for their serum-supplemented counterparts, they may provide a more reliable, standardized culture system to study the expression, secretion, and regulation of proteins during biological and pathologic processes.

Apolipoproteins B↗
PubMed1993

Hormonal regulation of human apolipoprotein E gene expression in HepG2 cells.

1. Hormonal regulation of apolipoprotein E (apoE) gene expression by insulin and thyroid hormone was studied in a human hepatoma cell line, HepG2. 2. Changes at the mRNA level, mRNA translation, in vivo synthesis and secretion were monitored. 3. Both insulin and triiodothyronine were found to have no significant effect on apoE mRNA levels. 4. Insulin treatment caused an inhibition of: (a) the in vitro translation of endogenous apoE mRNA in a HepG2 cell-free system (25%), and (b) the incorporation of radioactivity into newly-synthesized apoE in an in vivo pulse-chase labeling experiment (32%). 5. Interestingly, apoE secretion rate was found to be significantly reduced with insulin (84%) suggesting that a major portion of newly-synthesized apoE may be shunted into a degradative pathway. 6. Using a similar experimental approach, triiodothyronine showed no significant effect on the rate of apoE synthesis or translation (6-15% decrease), however a slight reduction (20%) in secretion rate was shown. 7. Overall, apoE gene expression does not appear to be influenced by triiodothyronine significantly but is modulated by insulin at the translational and post-translational level.

Apolipoproteins E↗
PubMed1993

Development of an amplified enzyme-linked immunosorbent assay for sensitive measurement of apolipoprotein B in plasma and tissue culture media.

We have developed a non-competitive sandwich enzyme-linked immunosorbent assay for the quantitation of apolipoprotein B, which utilizes the biotin-avidin amplification system. All components were optimized with respect to either concentration or incubation time. The assay uses microtitre wells as a solid phase and polyclonal, affinity-purified capture antibody. The amplification system was composed of hydroxy-succinimide biotin conjugated to an affinity-purified anti-apolipoprotein B IgG and either ExtrAvidin-alkaline phosphatase or streptavidin-alkaline phosphatase. The within-run precision (n = 10) of the apolipoprotein B control (1.39 g/l) was 3%, while the between-run precision of the assay (n = 6) for 9 consecutive assays was determined to be 8%. A sensitivity of 3 ng was attained, with a mean analytical recovery of 110%. Comparison of the assay with an established immunoturbidometric method resulted in a correlation of 0.92 using patient plasma samples (n = 19). The use of the biotin-avidin amplification system provides the sensitivity required for measuring apolipoprotein B in tissue culture media samples and circumvents the many problems associated with the direct conjugation of enzymes to antibodies. Minimal amounts of reagents are required and the assay can be performed in 5 h, making it both economical and practical for clinical laboratories.

Apolipoproteins B↗
PubMed1992

Thyroid hormone modulates apolipoprotein B gene expression in HepG2 cells.

We have investigated the modulation of apolipoprotein B gene expression in HepG2 cells by thyroid hormone. ApoB secretion rate in serum-free media was found to be significantly increased in the presence of the hormone in long-term cultures (48 h, 37%). This stimulatory effect was dose-dependent. The mechanisms underlying the stimulatory effect of triiodothyronine on apoB production were investigated. Triiodothyronine increased apoB mRNA levels by about 25-36% as determined by slot- and Northern-blot analysis of total RNA. ApoB synthesis rate was also found to be increased both in in vivo pulse-chase experiments (61%) and in in vitro translation studies (54.5%). Despite the 54.5-61% increase in apoB synthesis with triiodothyronine, only a 30% increase in apoB secretion was noted suggesting that part of the increase in the intracellular apoB pool may be lost by degradation. Overall, apoB gene expression appears to be modulated by thyroid hormone at both transcriptional and posttranscriptional levels.

Apolipoproteins B↗
PubMed1992

Apolipoprotein B gene polymorphism and plasma lipids and lipoproteins in a Canadian Caucasian population.

We have investigated the frequency of Hind III DNA polymorphism of the human apolipoprotein B gene in a Canadian Caucasian population with coronary artery disease, as documented by angiography, and a healthy control population. Patients had significantly (p < 0.05) higher levels of cholesterol, triglycerides, LDL-cholesterol, apolipoprotein B, and lower level of apoAI compared to the controls. Restriction fragment-length polymorphism analysis detected nine hybridizable fragments denoted as H1 to H9. The H1, H2, H3, and H7 alleles were polymorphic. The [H4-H9] genotype seems to be the normal genotype within the population studied since it was detected in 69% of the control group. The [H1-H9] genotype was most frequently observed in the patients (frequency = 0.68). We were unable to strongly associate any of the alleles or genotypes detected with the changes in lipids. The additional alleles observed in the patient group may indicate possible mutations at the 3' end of the apolipoprotein B gene locus.

Adult↗
PubMed1992

Purification and characterization of an extracellular thiol-containing serine proteinase from Thermomyces lanuginosus.

An extracellular protease produced by the filamentous fungus Thermomyces lanuginosus has been purified and characterized. The results indicate that the enzyme, which we have called humicolin, is a thiol-containing serine protease with a molecular mass of 38,000 kilodaltons. Secretion of humicolin, which is glycosylated, is tightly regulated by protein substrates. Kinetic characterization has revealed that humicolin activity is highly dependent upon the deprotonation of a group with a pKa of 6.6 and that the enzyme has a specificity for phenylalanine in the P1 position of the substrate.

Hydrogen-Ion Concentration↗
PubMed1992

Insulin modulation of human apolipoprotein B mRNA translation: studies in an in vitro cell-free system from HepG2 cells.

Insulin modulation of apolipoprotein B gene expression was studied at the translational level by the use of a cell-free translation system from a hepatoma cell-line, HepG2. Extracts of HepG2 cells lysed with lysolecithin were found to have high in vitro protein synthesizing activity utilizing endogenous mRNA. The level of peptide chain initiation was high, as suggested by a significant inhibition of translation by edeine. The translation products of endogenous mRNA in HepG2 cell-free lysate were probed with anti-apolipoprotein B antibodies to investigate its synthesis. A 550 kilodalton (kDa) polypeptide was selected by a polyclonal antibody, as well as a monoclonal antibody, against the C-terminal end of apolipoprotein B molecule. This in vitro synthesized polypeptide was also found to compare well in size with the in vivo product. The HepG2 lysate was also shown to efficiently synthesize in vitro a number of other proteins including albumin, apolipoprotein E, apolipoprotein A1, and actin. The in vitro synthesis of polypeptides as large as 500 kDa was unexpected and has not previously been demonstrated in a cell-free system. The HepG2 translation system was used to investigate the effect of insulin on the in vitro translation of apolipoprotein B. Lysates prepared from HepG2 cells treated with insulin were found to have lower translational activity (by an average of 52.3%) for apolipoprotein B compared with lysates from control untreated cells. In vitro synthesis of actin and apolipoprotein E were unaffected under these conditions. The insulin-stimulated decline in in vitro apolipoprotein B synthesis was not due to a change in apolipoprotein B mRNA levels as determined by slot- and Northern-blot analyses, suggesting that the inhibitory effect of insulin may be exerted partly at the level of apolipoprotein B mRNA translation.

Actins↗