Solubility, absorption, and anti-Helicobacter pylori activity of bismuth subnitrate and colloidal bismuth subcitrate: In vitro data Do not predict In vivo efficacy

Objectives. The aim of this study was to compare the dissolution, bioavailability, and anti– Helicobacter pylori activity of bismuth subnitrate and colloidal bismuth subcitrate. This could, first, provide insights into the mechanism of action of bismuth and, second, help to develop optimal therapeutic strategies.
Methods. Solubility and aquated size of bismuth species were determined in human gastric juice, while absorption into blood and urinary excretion of bismuth was determined in volunteers. Activity against H. pylori was determined in vitro in the presence and absence of antibiotics, while H. pylori eradication was compared in vivo.
Results. Bismuth from colloidal bismuth subcitrate was at least 10% soluble and ultrafilterable and was absorbed in volunteers (>0.5%), whereas that from bismuth subnitrate was insoluble and not absorbed (<0.01%). Colloidal bismuth subcitrate was active against H. pylori (mean inhibitory concentration, ≤12.5 μg/ml), while bismuth subnitrate was inactive (>400 μg/ml); neither was synergistic with antibiotics. With in vivo triple therapy, bismuth subnitrate was as effective as colloidal bismuth subcitrate in eradicating H. pylori (74% and 70% eradicated, respectively).
Conclusions. Colloidal bismuth subcitrate, unlike bismuth subnitrate, is partially soluble, absorbed in humans, and directly toxic to H. pylori in vitro. Surprisingly, however, these preparations had similar efficacy in vivo against H. pylori within triple therapy, suggesting that bismuth compounds may also exhibit indirect antimicrobial effects. We propose that this is an effect on the gastric mucus layer. Nonabsorbable bismuth compounds should be preferentially considered in bismuth-based therapies against H. pylori , as they would minimize toxicity while maintaining efficacy.     

Oxidative Modification of Low-Density Lipoprotein by the Human Hepatoma Cell Line HepG2

The human hepatoblastoma cell line HepG2 is a liver model commonly used for lipid metabolism studies. Numerous cell types have been found to oxidize low-density lipoprotein (LDL) but, to our knowledge, the effects of HepG2 cells on LDL have not been investigated. We found that LDL is modified by HepG2 cells through a peroxidative mechanism, as judged by an increase in TBARS content (which was prevented in the presence of the antioxidants vitamin E, 2, 6-di-tert-butyl-cresol and probucol), increased degradation by J774 macrophages, decreased internalization by MRC5 fibroblasts, and aggregation of apo B. Aspirin and allopurinol, which inhibit cyclooxygenase and xanthine-oxidase activities, respectively, had no effect on HepG2-induced LDL modification, and neither did catalase, which dismutates hydrogen peroxide; or mannitol, which scavenges hydroxyl radicals. In contrast, superoxide dismutase, a superoxide anion scavenger, and glutamate and threonine, which alter cellular cystine uptake, prevented LDL modifications, as did the removal of cysteine/cystine from the culture medium. Oxidation of LDL by HepG2 cells might thus involve superoxide anion production and/or thiol metabolism.     

Gene transfer into human hepatoma cells by receptor-associated protein/polylysine conjugates

Receptor-associated protein (RAP) is a ligand for all members of low-density lipoprotein (LDL) receptor families. RAP is internalized into cells via receptor-mediated endocytic trafficking, making it an attractive mechanism for efficient gene delivery. In this study, we have developed a gene delivery system using RAP as a targeting ligand. A RAP cDNA lacking a C-terminal heparin-binding domain was amplified by polymerase chain reaction (PCR) from a human liver cDNA library and was reamplified by using a primer containing a cysteine codon at its carboxyl end to facilitate its conjugation to polylysine (polyK). RAP was purified using a bacterial expression system and coupled to poly-D-lysine (PDL) or poly-L-lysine (PLL) of average MW 50 kDa via the heterobifunctional cross-linker SPDP. Using fluorescence-labeled RAP ligand, cellular uptake of the transfection complexes into HepG2 cells was shown to be highly efficient and more specific to PDL-conjugated RAP compared with PLL-conjugated one. Plasmid DNA containing a luciferase reporter gene was condensed with either RAP-PDL or RAP-PLL. In vitro transfection into HepG2 cells with RAP-PDL conjugate resulted in significantly higher luciferase expression levels in comparison to either nonconjugated PDL, or RAP-PLL, or LipofecAMINE/DNA complexes in the presence of 10% fetal bovine serum. Luciferase expression was inhibited by the addition of excess RAP. Treatment of the cells with Lovastatin, which inhibits HMG-Co reductase and increases expression of LDL receptor, stimulates luciferase expression, suggesting that the gene delivery is specifically mediated by LDL receptor. Thus, RAP-PDL conjugates have the potential to be used as a new nonviral gene delivery vector.     

Overexpression of cysteine dioxygenase reduces intracellular cysteine and glutathione pools in HepG2/C3A cells

Cysteine levels are carefully regulated in mammals to balance metabolic needs against the potential for cytotoxicity. It has been postulated that one of the major regulators of intracellular cysteine levels in mammals is cysteine dioxygenase (CDO). Hepatic expression of this catabolic enzyme increases dramatically in response to increased cysteine availability and may therefore be part of a homeostatic response to shunt excess toxic cysteine to more benign metabolites such as sulfate or taurine. Direct experimental evidence, however, is lacking to support the hypothesis that CDO is capable of altering steady-state intracellular cysteine levels. In this study, we expressed either the wild-type (WT) or a catalytically inactivated mutant (H86A) isoform of CDO in HepG2/C3A cells (which do not express endogenous CDO protein) and cultured them in different concentrations of extracellular cysteine. WT CDO, but not H86A CDO, was capable of reducing intracellular cysteine levels in cells incubated in physiologically relevant concentrations of cysteine. WT CDO also decreased the glutathione pool and potentiated the toxicity of CdCl(2). These results demonstrate that CDO is capable of altering intracellular cysteine levels as well as glutathione levels.     

Enhanced bismuth digestive absorption in rats by some sulfhydryl compounds: nmr study of complexes formed

In a preliminary paper [Therapie 34, 397 (1979), we showed that cysteine enhances bismuth digestive absorption in rats. In this paper, we have studied in rats the effects of various thiol compounds (mercaptopropionic acid, penicillamine, cysteine, homocysteine, 2-mercaptoethylamine, mercaptoethane) and nonthiol compounds (methionine, serine, alanine) orally administered on the absorption and elimination of bismuth also given orally. Bismuth was measured in blood and urine by electrothermal atomic absorption spectrophotometry.All of the thiol substances, and particularly cysteine, homocysteine, and mercaptopropionic acid, have considerably enhanced bismuth absorption and elimination: whereas, nonthiol substances have had no effect. Moreover, the acute toxicity of bismuth was enhanced when bismuth was given as a complex with cysteine (LD₅₀ = 156± 20 mg/kg).Studies by nmr spectroscopy of interactions between bismuth and these organic compounds have shown that bismuth induces an important chemical shift of the protons of the alpha carbon of the sulfhydrile group. Mainly, studies of ¹³C and ¹⁵N have confirmed this fact. The selectivity of such a complexation, in our pH conditions, may be tentatively explained on the ground of hard and soft acid and base (HSAB) theory.We have suggested that an increase in the concentration of thiol compounds in the gas-trointestinal tract arising from food, or more probably from microorganism synthesis, could be an explanation for human encephalopathies.     

Cooperative influence of thiolate ligands on the bio-relevant coordination chemistry of bismuth

The widespread use of bismuth compounds (e.g., bismuth subsalicylate, colloidal bismuth subcitrate) in medicine for over 200 years is founded on empirical observations, and definitive chemical mechanisms associated with the bioactivity of these compounds are not understood. The thiophilic nature of bismuth is a strong indication that sulfur-containing biological molecules are likely preliminary targets for bismuth. Using electrospray ionization mass spectrometry (ESI-MS), we have discovered a dramatic cooperative influence of thiolate ligands on the formation of bismuth complexes containing other biologically significant non-thiolate moieties. Reactions of Bi(NO3)3 with L-cysteine, 3-mercaptopropionic acid or 2-mercaptoethylamine, together with citric acid provide the first evidence of bismuth-citrate complexes in the gas phase. Analogously, reactions of Bi(NO3)3 with L-cysteine, together with other amino acids, reveal a wide range of new biologically relevant complex ions of bismuth that provide insight into the bioactivity of bismuth.     

Nifuratel-containing initial anti-Helicobacter pylori triple therapy in children

BACKGROUND: Proton pump inhibitor-containing triple therapy with amoxicillin and metronidazole is recommended as initial treatment of Helicobacter pylori in childhood. However, eradication rate with this "classic" regimen is relatively low in Russia. AIM: To evaluate empiric nifuratel, amoxicillin, and bismuth triple therapy for H. pylori gastritis in childhood. MATERIALS AND METHODS: Pediatric outpatients with H. pylori-associated chronic gastritis who underwent endoscopy for dyspeptic symptoms received the combination of bismuth subcitrate (8 mg/kg/day, q.d.s.), nifuratel (30 mg/kg/day, q.d.s.), and amoxicillin (50 mg/kg/day, q.d.s.) for 10 days. H. pylori status was determined before and after the treatment (in 4-6 weeks) by modified Giemsa staining. RESULTS: Seventy-three children (48 boys, 25 girls, age range 9-14) were entered. H. pylori was eradicated in 63 patients (86%; 95% confidence interval: 76.6-93.2; intention-to-treat and per protocol). There were no serious adverse reactions and were no withdrawals due to any side-effects. All of side-effects were self-limiting (dark stools, urine discoloration, blackening of the tongue, and others). CONCLUSIONS: The combination of nifuratel, bismuth subcitrate, and amoxicillin was an effective and tolerable regimen for H. pylori eradication.     

Role of reactive oxygen species generated by NADPH oxidase in the mechanism of activation of K+-Cl--cotransport by N-ethylmaleimide in HepG2 human hepatoma cells

K⁺-Cl^--cotransport (KCC) is ubiquitously present in all cells, and plays an essential role in ion and volume regulation. In this study we investigated the role of reactive oxygen species (ROS) in regulation of KCC in HepG2 human hepatoblastoma cells. N-ethylmaleimide (NEM), a KCC activator, induced Cl^--dependent K⁺ efflux, which was markedly prevented by KCC inhibitors (calyculin-A, genistein and BaCl₂), indicating that KCC is activated by NEM in the HepG2 cells. Treatment with NEM also induced a sustained increase in the level of intracellular ROS assessed by 2',7'-dichlorofluorescein flourescence. Antioxidants, N-acetyl cysteine or N,N'-diphenyl-p-phenylenediamine significantly inhibited both ROS generation and KCC activation induced by NEM. The NEM-induced ROS production was significantly suppressed by inhibitors of NADPH oxidase (diphenylene iodonium, apocynin and neopterine). These inhibitors also significantly inhibited the NEM-induced KCC activation. Taken together, these results suggest that ROS generated by NADPH oxidase may mediate the NEM-induced activation of KCC in human hepatoma cells.     

Biogenesis and speciation of nascent apoA-I-containing particles in various cell lines

It is generally thought that the large heterogeneity of human HDL confers antiatherogenic properties; however, the mechanisms governing HDL biogenesis and speciation are complex and poorly understood. Here, we show that incubation of exogenous apolipoprotein A-I (apoA-I) with fibroblasts, CaCo-2, or CHO-overexpressing ABCA1 cells generates only alpha-nascent apolipoprotein A-I-containing particles (alpha-LpA-I) with diameters of 8-20 nm, whereas human umbilical vein endothelial cells and ABCA1 mutant (Q597R) cells were unable to form such particles. Interestingly, incubation of exogenous apoA-I with either HepG2 or macrophages generates both alpha-LpA-I and prebeta1-LpA-I. Furthermore, glyburide inhibits almost completely the formation of alpha-LpA-I but not prebeta1-LpA-I. Similarly, endogenously secreted HepG2 apoA-I was found to be associated with both prebeta1-LpA-I and alpha-LpA-I; by contrast, CaCo-2 cells secreted only alpha-LpA-I. To determine whether alpha-LpA-I generated by fibroblasts is a good substrate for LCAT, isolated alpha-LpA-I as well as reconstituted HDL [r(HDL)] was reacted with LCAT. Although both particles had similar V(max) (8.4 vs. 8.2 nmol cholesteryl ester/h/microg LCAT, respectively), the K(m) value was increased 2-fold for alpha-LpA-I compared with r(HDL) (1.2 vs. 0.7 microM apoA-I). These results demonstrate that 1) ABCA1 is required for the formation of alpha-LpA-I but not prebeta1-LpA-I; and 2) alpha-LpA-I interacts efficiently with LCAT. Thus, our study provides direct evidence for a new link between specific cell lines and the speciation of nascent HDL that occurs by both ABCA1-dependent and -independent pathways.     

Comparison of HepG2 feeder cells generated by exposure to gamma-rays, X-rays, UV-C light or mitomycin C for ability to activate 7,12-dimethylbenz[a]anthracene in a cell-mediated Chinese hamster V79/HGPRT mutation assay

The cell-mediated Chinese hamster V79/HGPRT mutagenicity assay is an established in vitro testing method. Although gamma-irradiated human HepG2 hepatoma cells have been used recently for chemical activation, an alternative is now needed due to scheduled retirement of the available gamma-source. X-irradiation, 254 nm UV-C light and mitomycin C were examined as possible HepG2 mitotic inhibitors, and treated cells compared for activation of 7, 12-dimethylbenz[a]anthracene (DMBA). In colony-forming assays, V79 and HepG2 cells differed in sensitivity to DMBA, with V79 survival declining sharply between 1-2.5 microM (LD50=1.75 microM) while HepG2 survival decreased gradually, beginning at 0.01 microM DMBA (LD50=0.045 microM). When HepG2 feeder cells generated by each method were included in V79/HGPRT mutation assays, activation of 1 microM DMBA was found to vary according to the mitotic inhibitor used, with mutation frequencies decreasing in the order 4000 rads gamma-rays>25 microg/ml mitomycin C>4000 rads X-rays>25 J/m2 UV-C light. Only assays containing gamma-irradiated HepG2 cells generated an increase (2-3-fold) in mutation frequency when DMBA exposure was extended from 24 to 48 h. The effect of HepG2 preincubation with either Aroclor 1254 or DMBA on feeder cell activation of DMBA was also assessed using concentrations of Aroclor 1254 (10 microg/ml) or DMBA (1.0 microM) which were found to produce optimum induction of ethoxyresorufin-O-deethylase (EROD) activity (3.1-fold and 2-fold increases, respectively). Compared to results obtained with uninduced HepG2 cells, assays incorporating HepG2 cells activated by either Aroclor 1254 or DMBA produced slightly increased V79/HGPRT mutation frequencies after 24 h of exposure to mutagen; however, a 48 h incubation with mutagen in the presence of HepG2 preincubated with either Aroclor 1254 or DMBA resulted in higher mutation frequencies regardless of the mitotic inhibitor treatment. EROD activity was also induced 1.4-fold following exposure of HepG2 cells to mitomycin C alone. Although gamma-irradiation remains the treatment of choice for producing metabolically active HepG2 feeder cells, comparison of the alternatives tested suggests that mitomycin C would be a convenient and suitable replacement.     

Double-blind comparison of absorbable colloidal bismuth subcitrate and nonabsorbable bismuth subnitrate in the eradication of Helicobacter pylori and the relief of nonulcer dyspepsia

Background. Bismuth is widely used for the eradication of H. pylori , especially in developing countries, although there are concerns over its neurotoxicity. Whether bismuth has to be absorbed in humans to act against H. pylori is not known. In this study, we compared "absorbable" (colloidal bismuth subcitrate) and "nonabsorbable" (bismuth subnitrate) bismuth as part of triple therapy in the eradication of H. pylori.
Materials and Methods. A double-blind, randomized, placebo-controlled trial was carried out with 120 H. pylori –positive patients with nonulcer dyspepsia. Group CBS + Ab (n = 35) received colloidal bismuth subcitrate (one tablet qds), amoxicillin (500 mg qds), and metronidazole (400 mg tds). Group BSN + Ab (n = 35) received bismuth subnitrate (two tablets tds) and the same antibiotics. Group Ab (n = 35) received placebo bismuth (two tablets tds) and the antibiotics. Group BSN (n = 15) received bismuth subnitrate (two tablets tds) and placebo antibiotics. Bismuth was taken for 4 weeks and the antibiotics for the first 2 weeks. H. pylori eradication, side effects, compliance, pre- and post-treatment symptom scores, and bismuth absorption were assessed.
Results. H. pylori eradication was 69%, 83%, 31%, and 0% in CBS + Ab, BSN + Ab, Ab, and BSN, respectively. Side effects, compliance, and symptom relief were similar in all groups, but blood bismuth levels were significantly greater in CBS + Ab than the other three groups.
Conclusion. The efficacy of bismuth-based therapies as part of triple therapy in the eradication of H. pylori is unrelated to absorption. Hence, the use of effective but poorly absorbed bismuth preparations should be encouraged for bismuth-based eradication therapies.     

Different Involvement of Promoter Methylation in the Expression of Organic Cation/Carnitine Transporter 2 (OCTN2) in Cancer Cell Lines

Organic cation/carnitine transporter 2 (OCTN2) is responsible for the cellular uptake of the antineoplastic agent, oxaliplatin. Epigenetic modification is a possible mechanism of altered drug-transporter expression in cancers, leading to altered efficacy of chemotherapeutic drugs. However, the mechanisms governing OCTN2 regulation are not completely understood. In this study, the low levels of OCTN2 in HepG2 and LS174T cells were elevated by the demethylating reagent, decitabine (DCA). To further reveal the epigenetic mechanism of down-regulation of OCTN2, we found that Region-1 within the OCTN2 promoter (spanning −354 to +85) was a determinant of OCTN2 expression in a luciferase reporter assay. Moreover, methylation-specific PCR (MSP) and bisulfite genomic sequencing showed that the degree of individual methylated CpG sites within this region was inversely correlated with the levels of OCTN2 in different cancer cells. Application of DCA to HepG2 and LS174T cells reversed the hypermethylation status of the OCTN2 promoter and increased OCTN2 expression, enhancing cellular uptake of oxaliplatin. Thus, we identified that promoter methylation is responsible for epigenetic down-regulation of OCTN2 in HepG2 and LS174T cells. Given the essential role of OCTN2 in cancer cell uptake of chemotherapeutics, and thus treatment efficacy, pretreatment with a demethylating reagent is a possible strategy for optimizing pharmacotherapies against cancers.     

Detection of nonsterol isoprenoids by HPLC-MS/MS

Isoprenoids constitute an important class of biomolecules that participate in many different cellular processes. Most available detection methods allow the identification of only one or two specific nonsterol isoprenoid intermediates following radioactive or fluorescent labeling. We here report a rapid, nonradioactive, and sensitive procedure for the simultaneous detection and quantification of the eight main nonsterol intermediates of the isoprenoid biosynthesis pathway by means of tandem mass spectrometry. Intermediates were analyzed by HPLC-MS/MS in the multiple reaction monitoring mode using a silica-based C₁₈ HPLC column. For quantification, their stable isotope-labeled analogs were used as internal standards. HepG2 cells were used to validate the method. Mevalonate, phosphomevalonate, and the six subsequent isoprenoid pyrophosphates were readily determined with detection limits ranging from 0.03 to 1.0 μmol/L. The intra- and interassay variations for HepG2 cell homogenates supplemented with isoprenoid intermediates were 3.6-10.9 and 4.4-11.9%, respectively. Under normal culturing conditions, isoprenoid intermediates in HepG2 cells were below detection limits. However, incubation of the cells with pamidronate, an inhibitor of farnesyl pyrophosphate synthase, resulted in increased levels of mevalonate, isopentenyl pyrophosphate/dimethylallyl pyrophosphate, and geranyl pyrophosphate. This method will be suitable for measuring profiles of isoprenoid intermediates in cells with compromised isoprenoid biosynthesis and for determining the specificity of potential inhibitors of the pathway.     

Specific gene transfer mediated by lactosylated poly-L-lysine into hepatoma cells.

Plasmid DNA/glycosylated polylysine complexes were used to transfer in vitro a luciferase reporter gene into human hepatoma cells by a receptor-mediated endocytosis process. HepG2 cells which express a galactose specific membrane lectin were efficiently and selectively transfected with pSV2Luc/lactosylated polylysine complexes in a sugar dependent manner: i) HepG2 cells which do not express membrane lectin specific for mannose were quite poorly transfected with pSV2Luc/mannosylated polylysine complexes, ii) HeLa cells which do not express membrane lectin specific for galactose were not transfected with pSV2Luc/lactosylated polylysine complexes. The transfection efficiency of HepG2 cells with pSV2Luc/lactosylated polylysine complexes was greatly enhanced either in the presence of chloroquine or in the presence of a fusogenic peptide. A 22-residue peptide derived from the influenza virus hemagglutinin HA2 N-terminal polypeptide that mimics the fusogenic activity of the virus, was selected. In the presence of the fusogenic peptide, the luciferase activity in HepG2 cells was 10 fold larger than that of cells transfected with pSV2Luc/lactosylated polylysine complexes in the presence of chloroquine.     

脱氢表雄酮调节HepG2/GFP-HBx细胞p16基因甲基化及其与细胞周期和凋亡的相关性

目的探讨HBx与p16基因甲基化的关系,研究脱氢表雄酮（DHEA）对p16基因甲基化以及细胞周期和细胞凋亡的调节作用。方法以HepG2、HepG2/GFP和HepG2/GFP-HBx三种细胞为材料,采用MTT法检测细胞生长;流式细胞术检测细胞周期和凋亡率;MSP-PCR检测p16基因甲基化水平。比较分析HBx与p16基因启动子甲基化、DHEA与各检测指标的关系。结果HepG2/GFP-HBx细胞与HepG2细胞和HepG2/GFP细胞相比,细胞的增殖速度提高（P〈0.05）,G0/G1期细胞减少,S期细胞增多（P〈0.05）,凋亡率降低（P〈0.05）;HepG2/GFP-HBx细胞的p16基因启动子呈现高水平甲基化,HepG2和HepG2/GFP细胞呈现高水平非甲基化。100μmol/L的DHEA使三种细胞的增殖速度降低（P〈0.05）,G0/G1期细胞增加,S期细胞减少（P〈0.05）,凋亡率提高（P〈0.05）。DHEA可下调HepG2/GFP-HBx细胞的p16基因启动子甲基化水平,但对HepG2和HepG2/GFP细胞的p16基因启动子甲基化水平不产生影响。结论HBx引起肝癌细胞p16基因甲基化,并缩短细胞周期抑制细胞凋亡;DHEA可明显下调HBx引起的p16基因甲基化水平,延长细胞周期促进细胞凋亡,在无HBx基因整合的情况下,DHEA对肝癌细胞生长、细胞周期和凋亡的影响不通过p16基因甲基化的途径实现。     

姜黄素通过下调P-糖蛋白和热休克蛋白70逆转耐热肝癌细胞的阿霉素耐受性

探讨姜黄素对耐热肝癌细胞 (HepG2/TT) 阿霉素耐受性的逆转作用及其机制．用MTT检测细胞活力,PI染色流式细胞术检测细胞凋亡,高效液相色谱法检测细胞内阿霉素的积累,Western blot检测细胞P-糖蛋白 (P-glycoprotein,P-gp)、热休克蛋白70 (heat shock protein 70, Hsp70) 和caspase-3 的表达．耐热肝癌细胞HepG2/TT能耐受阿霉素引起的细胞毒性和 凋亡;姜黄素在5、10和20 μmol/L时,能浓度依赖性地降低阿霉素对HepG2/TT 细胞的IC50,增强阿霉素对HepG2/TT 细胞的凋亡诱导作用．耐热肝癌细胞HepG2/TT 与非耐热肝癌细胞HepG2比较,其P-gp和Hsp70 的表达水平明显增高; 10 μmol/L姜黄素处理24 h 后,HepG2/TT细胞P-gp和Hsp70的表达水平显著下降．HepG2/TT 细胞内阿霉素的积累低于HepG2细胞;10 μmol/L姜黄素处理 3 h后,HepG2/TT 细胞内阿霉素的积累明显增加．HepG2/TT细胞能抑制阿霉素激活 caspase-3;10 μmol/L姜黄素处理24 h后,阿霉素对 HepG2/TT细胞caspase-3的激活作用增强．上述结果表明,姜黄素能逆转耐热肝癌细胞HepG2/TT的阿霉素耐受性,其机制可能与其下调P-gp和Hsp70的表达,进而促进阿霉素激活caspase-3 有关．     

On the hepatic mechanism of HDL assembly by the ABCA1/apoA-I pathway

The mechanism for the assembly of HDL with cellular lipid by ABCA1 and helical apolipoprotein was investigated in hepatocytes. Both HepG2 cells and mouse primary culture hepatocytes produced HDL with apolipoprotein A-I (apoA-I) whether endogenously synthesized or exogenously provided. Probucol, an ABCA1 inactivator, inhibited these reactions, as well as the reversible binding of apoA-I to HepG2. Primary cultured hepatocytes of ABCA1-deficient mice also lacked HDL production regardless of the presence of exogenous apoA-I. HepG2 cells secreted apoA-I into the medium even when ABCA1 was inactivated by probucol, but it was all in a free form as HDL production was inhibited. When a lipid-free apoA-I-specific monoclonal antibody, 725-1E2, was present in the culture medium, production of HDL was suppressed, whether with endogenous or exogenously added apoA-I, and the antibody did not influence HDL already produced by HepG2 cells. We conclude that the main mechanism for HDL assembly by endogenous apoA-I in HepG2 cells is an autocrine-like reaction in which apoA-I is secreted and then interacts with cellular ABCA1 to generate HDL.     

Autodegradation of protein disulfide isomerase

Protein disulfide isomerase (PDI) and its degradation products were found in HepG2, COS-1, and CHO-K1 cells. Whether or not the products were formed through autodegradation of PDI was examined, since PDI contains the CGHC motif, which is the active center of proteolytic activity in ER-60 protease. Commercial bovine PDI was autodegraded to produce a trimmed PDI. In addition, human recombinant PDI also had autodegradation activity. Mutant recombinant PDIs with CGHC motifs of which cysteine residues were replaced with serine or alanine residues were prepared. However, they were not autodegraded, suggesting the cysteine residues of motifs are necessary for autodegradation.