Apolipoprotein E-containing lipoproteins and their extracellular interactions with LRP1 affect LPS-induced inflammation.

The linkage between low-density lipoprotein receptor-related protein (LRP)1-mediated metabolism of apolipoprotein (apo) E-containing lipoproteins (apoE-LP) and the lipopolysaccharide (LPS)-induced inflammatory response contributes to the pathogenesis of sepsis; however, the underlying mechanisms are unclear. Therefore, in this study, the effects of apoE-LP and their constituents on the mRNA expression of interleukin (IL)-6 and LRP1 were evaluated using a culture system of human fibroblasts supplemented with LPS and apoE-containing emulsion particles (apoE-EP). The affinity of apoE-LP for LPS was examined using the interaction between fluorescence-labeled LPS and serum lipoprotein fractions. LPS-induced inflammation significantly upregulated the mRNA expression of IL-6 and LRP1. This upregulation was markedly suppressed by pre-incubation of LPS with apoE-EP or its constituents (apoE or EP). The suppressive effect of apoE-EP on IL-6 upregulation was attenuated in the presence of lactoferrin, an inhibitor of LRP1. The prepared apoE-EP and serum triglyceride-rich lipoproteins showed significant affinity for LPS. However, these affinities appeared to be lower than expected based on the extent to which IL-6 upregulation was suppressed by pre-incubation of LPS with apoE-EP. Overall, these results indicate that LPS-induced inflammation may be regulated by 1) the LPS-neutralizing effect of apoE-LP, 2) anti-inflammatory effect of apoE, and 3) LRP1-mediated metabolic pathways.

Recombinant γY278H Fibrinogen Showed Normal Secretion from CHO Cells, but a Corresponding Heterozygous Patient Showed Hypofibrinogenemia.

We identified a novel heterozygous hypofibrinogenemia, γY278H (Hiroshima). To demonstrate the cause of reduced plasma fibrinogen levels (functional level: 1.12 g/L and antigenic level: 1.16 g/L), we established γY278H fibrinogen-producing Chinese hamster ovary (CHO) cells. An enzyme-linked immunosorbent assay demonstrated that synthesis of γY278H fibrinogen inside CHO cells and secretion into the culture media were not reduced. Then, we established an additional five variant fibrinogen-producing CHO cell lines (γL276P, γT277P, γT277R, γA279D, and γY280C) and conducted further investigations. We have already established 33 γ-module variant fibrinogen-producing CHO cell lines, including 6 cell lines in this study, but only the γY278H and γT277R cell lines showed disagreement, namely, recombinant fibrinogen production was not reduced but the patients' plasma fibrinogen level was reduced. Finally, we performed fibrinogen degradation assays and demonstrated that the γY278H and γT277R fibrinogens were easily cleaved by plasmin whereas their polymerization in the presence of Ca2+ and "D:D" interaction was normal. In conclusion, our investigation suggested that patient γY278H showed hypofibrinogenemia because γY278H fibrinogen was secreted normally from the patient's hepatocytes but then underwent accelerated degradation by plasmin in the circulation.

The redox status of cysteine thiol residues of apolipoprotein E impacts on its lipid interactions.

Redox-mediated modulation of cysteine (Cys) thiols has roles in various pathophysiological functions. We recently found that formation of disulfide-linked complexes of apolipoprotein (apo) E3 prevented apoE3 from irreversible oxidation. In this report, the influence of modification of Cys thiols in apoE2 and apoE3 on interactions with lipids was investigated. The apoE redox status was examined by a band-shift assay using a maleimide compound, and interactions with lipids were evaluated by a kinetic assay using dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and non-denaturing polyacrylamide gel electrophoresis. A reduction in DMPC clearance activity of apoE2 and apoE3 but not apoE4 was observed. Although hydrogen peroxide-induced oxidation decreased the clearance activity of the isoforms, apoE2 showed the greatest residual activity. Both Cys thiol masking and dimerization decreased the activity of apoE2 and apoE3 but not apoE4. In contrast, apoAII preincubation markedly increased the activity (apoE2 > apoE3 > apoE4), in accordance with the formation of apoE-AII and apoAII-E2-AII complexes. ApoAII preincubation also reduced the particle size of apoE-DMPC liposome complexes, especially for apoE2. Redox-mediated modification of Cys thiols of apoE2 or apoE3, especially disulfide bond formation with apoAII, affects lipid metabolism and consequently may be responsible for the diverse isoform specificity of apoE.

[Electrophoretic Analysis of Serum Lipoproteins: Its Usefulness and Problems].

Analysis of serum lipoprotein fractions by electrophoresis (lipoprotein electrophoresis) reveals lipid metabolism disorders, and provides various types of bio-information which lead to an accurate diagnosis and effective treatment for dyslipidemia and related diseases (e.g., coronary artery disease or chronic kidney disease). In particular, lipoprotein electrophoresis enables us to define the phenotypes of dyslipidemia, and to detect abnormal lipoproteins, which are potent biomarkers of atherosclerotic disease. In addition, lipoprotein electrophoresis is an indispensable complement to other assay methods for serum lipid component measurement that have some limitations, such as a homogeneous assay for LDL-C. However, it appears to be underestimated regarding its clinical usefulness. Indeed, the fee for lipoprotein electrophoresis tests is too low. This review specifically discusses the clinical usefulness and problems of lipoprotein electrophoresis, with a special emphasis on cost.

Apolipoprotein E isoforms and their Cys-thiol modifications impact LRP1-mediated metabolism of triglyceride-rich lipoproteins.

The low-density lipoprotein (LDL) receptor-related protein (LRP)1 participates in the metabolism of apolipoprotein (apo) E-containing lipoproteins (apoE-LP). We investigated the effects of modifications of cysteine (Cys)-thiol of apoE on LRP1-mediated metabolism. Among the three isoforms, apoE2-LP exhibited the lowest affinity for LRP1 but was significantly catabolized, whereas apoE4-LP was sufficiently bound to LRP1 but showed the lowest catabolic capability. The reduction enhanced the binding and suppressed the catabolism of apoE3-LP, but had no effect on apoE2-LP. The formation of disulfide-linked complexes with apoAII suppressed binding, but enhanced the catabolism of apoE2-LP. Redox modifications of apoE-Cys-thiol may modulate the LRP1-mediated metabolism of apoE2- or apoE3-LP, but not apoE4-LP. The failure of this function may be involved in the pathophysiology of dyslipidemia.

The Asian project for collaborative derivation of reference intervals: (2) results of non-standardized analytes and transference of reference intervals to the participating laboratories on the basis of cross-comparison of test results.

BACKGROUND: The 2009 Asian multicenter study for derivation of reference intervals (RIs) featured: 1) centralized measurements to exclude reagent-dependent variations; 2) inclusion of non-standardized analytes (hormones, tumor makers, etc.) in the target; and 3) cross-check of test results between the central and local laboratories. Transferability of centrally derived RIs for non-standardized analytes based on the cross-check was examined. METHODS: Forty non-standardized analytes were centrally measured in sera from 3541 reference individuals recruited by 63 laboratories. Forty-four laboratories collaborated in the cross-check study by locally measuring aliquots of sera from 9 to 73 volunteers (average 22.2). Linear relationships were obtained by the major-axis regression. Error in converting RIs using the regression line was expressed by the coefficient of variation of slope b [CV(b)]. CV(b) <10% was set as the cut-off value allowing the conversion. The significance of factors for partitioning RIs was determined similarly as in the first report. RESULTS: Significant sex-, age-, and region-related changes in test results were observed in 17, 15, and 11 of the 40 analytes, respectively. In the cross-comparison study, test results were not harmonized in the majority of immunologically measured analytes, but their average CV(b)s were <10% except for total protein, cystatin C, CA19-9, free thyroxine, and triiodothyronine. After conversion, 74% of centrally derived RIs were transferred to each local laboratory. CONCLUSIONS: Our results point to the feasibility of: 1) harmonizing test results across different laboratories; and 2) sharing centrally derived RIs of non-standardized analytes by means of comparative measurement of a set of commutable specimens.

Characterization of CIA-1, an Ambler class A extended-spectrum ß-lactamase from Chryseobacterium indologenes.

An Ambler class A ß-lactamase gene, bla(CIA-1), was cloned from the reference strain Chryseobacterium indologenes ATCC 29897 and expressed in Escherichia coli BL21. The bla(CIA-1) gene encodes a novel extended-spectrum ß-lactamase (ESBL) that shared 68% and 60% identities with the CGA-1 and CME-1 ß-lactamases, respectively. bla(CIA-1)-like genes were detected from clinical isolates. In addition to the metallo-ß-lactamase IND of Ambler class B, C. indologenes has a class A ESBL gene, bla(CIA-1), located on the chromosome.

Waldenström macroglobulinemia transforming from t (11;18) (q21;q21) -negative gastric MALT lymphoma after systemic dissemination.

AIM: We here describe the clinical course of a 70-year-old male patient with Waldenström macroglobulinemia (WM) putatively transformed from refractory mucosa-associated lymphoid tissue lymphoma (MALTL). METHODS: Immunological staining was performed on formalin-fixed, paraffin-embedded tissue sections, and M-protein and cryoglobulin were identified by immunofixation electrophoresis and the cold precipitation method. Chromosome translocation was analyzed by the G-banded karyotype, and API2/MALT1 fusion gene underwent fluorescent in situ hybridization. Multiplex polymerase chain reaction was performed to analyze the VH-JH or DH-JH rearrangements of the IGH gene. RESULTS: At diagnosis, the WM patient had monoclonal IgM with cryoglobulinemia and hyperviscosity syndrome. Eight years before developing WM, the patient experienced the onset of typical gastric MALT-L with H. pylori infection, but in spite of negative for chromosome translocation, t (11;18) and the successful eradication of H. pylori, the MALT-L relapsed repeatedly, and finally led to systemic metastasis. The lymphoma cells also infiltrated the large intestine and spleen. Immunoglobulin gene analyses of cellular clonality revealed that the same clone had been present in the stomach, bone marrow (BM) at the onset of MALT L, and in the BM at the diagnosis of WM. CONCLUSIONS: In this case, lymphoma developed as H. pylori-associated gastric MALT-L with negative for t (11;18), and might be transformed into MW during the systemic metastasis.

Pathophysiological investigation of the gastric surface mucous gel layer of patients with Helicobacter pylori infection by using immunoassays for trefoil factor family 2 and gastric gland mucous cell-type mucin in gastric juice.

BACKGROUND: The trefoil factor family (TFF) 2 protein is produced by gastric gland mucous cells (GMCs), and the secreted TFF2 shares a mucosal barrier function with GMC-type mucin. Recently, we presented an enzyme-linked immunosorbent assay (ELISA) method for measurement of GMC-type mucin in the gastric juice. AIMS: We aimed to develop an ELISA for TFF2 and to assess pathophysiological changes in the gastric surface mucous gel layer (SMGL) of patients with Helicobacter pylori infection. METHODS: The distribution of TFF2 and GMC-type mucin in the SMGL was immunohistochemically determined. The ELISA for TFF2 was based on a polyclonal goat antibody. Recombinant TFF2 was employed to prepare the calibrators. TFF2 and GMC-type mucin in the gastric juice in healthy individuals (n = 33) and patients with gastritis (n = 37), gastric ulcer (n = 16), and duodenal ulcer (n = 10) were assayed using ELISA. RESULTS: TFF2 and GMC-type mucin were immunohistochemically co-localized in the gastric SMGL and GMCs. The TFF2 levels in the patients were significantly higher than those in the healthy individuals. Further, the TFF2 levels in the H. pylori-positive patients were significantly higher than those in the H. pylori-negative patients, and decreased after the eradication of the infection. GMC-type mucin levels showed a tendency similar to that of TFF2 levels. CONCLUSIONS: The upregulation of TFF2 and GMC-type mucin secretion may reflect the response of the gastric mucosa to H. pylori-induced injuries. TFF2 and GMC-type mucin secreted into the SMGL may protect the gastric mucosa against H. pylori.

Redox index of Cys-thiol residues of serum apolipoprotein E and its diagnostic potential.

BACKGROUND: The redox modulation of Cys-thiol participates in various pathophysiological processes. We explored the proper index for estimating the redox status of Cys-thiol of serum apolipoprotein E (apoE), named "redox-IDX-apoE," which is necessary to understand the redox biology of age-related diseases. METHODS: The fractions of the reduced form (red-), reversible oxidized form (roxi-), and irreversibly oxidized form (oxi-) apoE in serum, obtained from the patients with no apparent disease (controls, n=192) and with atherosclerosis and type 2 diabetes (patients, n=16), were measured by a band-shift assay using a maleimide compound. Redox-IDX-apoE candidates were determined by calculating the values of these fractions and the total apoE concentration. RESULTS: Cys number of apoE significantly increased for the ratio of roxi-apoE to total-apoE (roxi/total) (E2/E3>E3/E3>E3/E4) but decreased for the ratios of red-apoE to roxi-apoE (red/roxi) and [red-apoE + oxi-apoE] to roxi-apoE ([red + oxi]/roxi) (E2/E3

A novel variant fibrinogen, AαE11del, demonstrating the importance of AαE11 residue in thrombin binding.

INTRODUCTION: We identified a novel heterozygous AαE11del variant in a patient with congenital dysfibrinogenemia. This mutation is located in fibrinopeptide A (FpA). We analyzed the effect of AαE11del on the catalyzation of thrombin and batroxobin and simulated the stability of the complex structure between the FpA fragment (AαG6-V20) peptide and thrombin. MATERIALS AND METHODS: We performed fibrin polymerization and examined the kinetics of FpA release catalyzed by thrombin and batroxobin using purified plasma fibrinogen. To clarify the association between the AαE11 residue and thrombin, we calculated binding free energy using molecular dynamics simulation trajectories. RESULTS: Increasing the thrombin concentration improved release of FpA from the patient's fibrinogen to approximately 90%, compared to the previous 50% of that of normal fibrinogen. Fibrin polymerization of variant fibrinogen also improved. In addition, greater impairment of variant FpA release from the patient's fibrinogen was observed with thrombin than with batroxobin. Moreover, the calculated binding free energy showed that the FpA fragment-thrombin complex became unstable due to the missing AαE11 residue. CONCLUSIONS: Our findings indicate that the AαE11 residue is involved in FpA release in thrombin catalyzation more than in batroxobin catalyzation, and that the AαE11 residue stabilizes FpA fragment-thrombin complex formation.

Cut-off values of serum IgG4 among three reagents, including a novel IgG4 reagent: a multicenter study.

Elevated serum IgG4 is a useful marker of IgG4-related disease (IgG4-RD) activity. However, there is no uniformity in the cut-off values of IgG4 among the various reagents. The aim of this study was to compare the measured and cut-off values of IgG4 assessed using three different reagents. This study enrolled 466 IgG4-RD and non-IgG4-RD patients who required measurement of serum IgG4 levels to diagnose or treat IgG4-RD. Serum IgG4 was measured using three reagents: N-assay LA IgG4 Nittobo (Nittobo), BS-NIA IgG4 (TBS), and N Latex IgG4 (Siemens). The values obtained using the three reagents were compared, and cut-off values were calculated for each. Although there was good correlation among the results with the three reagents, the measured and cut-off values were all different. The Nittobo values were 1.4 times the TBS values and the TBS values were almost half those of the Siemens values. ROC curve analysis showed cut-off values for the Nittobo, TBS, and Siemens reagents of 1.42, 1.31, and 2.38 g/L, respectively. The measured and cut-off values of serum IgG4 vary depending on the reagents used for the assay, although there is good correlation among the values measured by the three reagents.

Relationships between Cognitive Function and Odor Identification, Balance Capability, and Muscle Strength in Middle-Aged Persons with and without Type 2 Diabetes.

AIM: We investigated the relationship between cognitive function and olfactory and physical functions in middle-aged persons with and without type 2 diabetes (T2D) to examine the potential of olfactory and physical functions as biomarkers for early cognitive impairment. METHODS: Enrolled were 70 T2D patients (age 40 to <65 y) and 81 age-matched control participants without diabetes. Cognitive function was assessed by the Montreal Cognitive Assessment (MoCA), Trail Making Test parts A and B (TMT-A/-B), Wisconsin Card Sorting Test (WCST), Quick Inventory of Depressive Symptomatology Self-Report (QIDS), and Starkstein Apathy Scale (SAS). Multiple linear regression analyses were performed. RESULTS: Odor identification was an independent determinant shown in the results of the TMT-A in the entire participant group and was independently associated with the MoCA and TMT-B in the T2D group. Balance capability assessed with a stabilometer was independently associated with all cognitive function tests except for QISD and SAS in the entire participant group and the T2D group and was independently associated with TMT-A in the control group. Knee extension strength was independently associated with the SAS in the entire participant group and the T2D group. CONCLUSIONS: Odor identification, balance capability, and knee extension strength were potential markers for cognitive decline in middle-aged persons with T2D.

Evaluation of a novel serum IgG4 assay and determination of reference interval for the Japanese population.

BACKGROUND: IgG4-related disease (IgG4-RD) is a new syndrome characterized by elevated serum IgG4 concentration and tissue infiltration of IgG4-positive plasma cells. Here, we evaluated the analytical performance of a new IgG4 assay reagent featuring a wide dynamic range, highly specific monoclonal antibody, and the reversed passive latex agglutination assay and determined the IgG4 reference interval (RI) for the Japanese population. METHODS: Performance evaluations were conducted on precision, linearity, sensitivity, interference, and method comparison with The Binding Site (TBS) and Siemens reagents. The RI was derived by the parametric method from 619 apparently healthy Japanese 18 to 65 years of age. RESULTS: Between-day precisions ranged from 1.99 to 5.52 CV%. Linearity was confirmed up to 5.0 g/l. The limit of quantitation was 0.085 g/l. Interfering substances did not significantly influence values. Method comparison among the 3 reagents yielded correlation coefficients between 0.973 and 0.988. Values for the new reagent matched those of TBS reagent except at a higher concentration range, where reactivity dissociated. The RI was 0.11-1.21 g/l without distinction by sex and age. CONCLUSION: The novel IgG4 assay reagent demonstrated satisfactory analytical performance for clinical use. Because of matched value with TBS reagent at low concentrations, it is possible to use the IgG4-RD cut-off value determined by TBS reagent.

[Characterization of sialo-apolipoprotein E in human cerebrospinal fluid].

Apolipoprotein E (apoE) in human cerebrospinal fluid (CSF) is extensively sialylated, and the sialylation of certain proteins is known to modulate their biological function. We investigated the effects of a sialic acid moiety of apoE on the apoE-Abeta interaction using the surface plasmon resonance assay. Further, we established a method for the determination of the sialic acid content of CSF apoE, and carried out a more detailed characterization of CSF apoE-containing lipoproteins. The binding avidity of asialo-apoE3 was approximately 50% of that of sialo-apoE3 (p<0.0001). The binding avidity of the apoE-containing lipoproteins for Abeta reduced after neuraminidase treatment. The sialic acid levels in the CSF apoE-containing lipoprotein fractions were 5.3 +/- 1.3% of the total CSF sialic acid, and were correlated with the CSF apoE concentrations. However, the ratios of sialic acid to apoE were inversely proportional to the CSF lipid concentrations. The lipoprotein particle sizes were larger when the ratios of sialic acid to CSF apoE were greater. The sialic acid moiety of the apoE molecules affects the interaction of apoE with Abeta and the formation of apoE-containing lipoprotein particles. The posttranslational modification of apoE, such as the presence of sialic acid moieties, may be involved in the regulation of lipid transport to the brain.

[Crucial roles of Helicobacter pylori infection in the pathogenesis of gastric cancer and gastric mucosa-associated lymphoid tissue (MALT) lymphoma].

Helicobacter pylori (H. pylori) is a gram-negative helical rod that colonizes human gastric mucosa. Its discovery has opened up new opportunities regarding the understating and management of gastrointestinal disorders. In humans, infection with H. pylori has been established as a major cause of chronic gastritis and peptic ulcer, and is important in the pathogenesis of gastric cancer and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Bacterial and host factors determine the outcome of H. pylori infection. The eradication of H. pylori can, therefore, contribute to the treatment and prevention of these diseases. H. pylori infection plays a critical role in gastric carcinogenesis through two major pathways: the indirect action of H. pylori on gastric epithelial cells through inflammation, and the direct action of the bacteria on epithelial cells through the induction of protein modulation and gene mutation. MALT lymphoma is a common low grade B-cell lymphoma arising from a background of chronic inflammatory disease at a number of mucosal sites. Those originating in the stomach are causatively linked to H. pylori infection, and eradication of the bacterium with antibiotics leads to the long-term complete regression of lymphoma. t (11;18)/API2-MALT1 and t(1;14)/IGH-BCL10 are specifically associated with the gastric MALT lymphoma entity, and the oncogenic products of these translocations have been shown to target a common molecular pathway, i.e., the nuclear factor-kappaB pathway. This paper reviews recent advances in our understanding of the association of H. pylori infection with gastric cancer and gastric MALT lymphoma and the molecular genetics underlying tumor development.

[Functional analysis of heterozygous plasma dysfibrinogens derived from two families of gammaArg275Cys and three families of gammaArg275His, and haplotype analysis for these families].

We have identified five heterozygous dysfibrinogenemias, two families with variant fibrinogen gammaArg275Cys (CGC > TGC; Matsumoto III and Sendai) and three families with gammaArg275His (CGC > CAC; Otsu II, Iida, and Shizuoka), from PCR-amplified DNA fragments and direct sequence analysis. gammaArg275 is the most important residue in fibrinogen for the so-called "D-D interface" in protofibril elongation. We compared the functions of plasma fibrinogen purified from affected family members with gammaArg275Cys and gammaArg275His. Both fibrinogens showed markedly impaired thrombin-catalyzed fibrin polymerization in comparison with normal controls. The degree of impairment of gammaArg275Cys fibrinogen was greater than that of gammaArg275His. These results were consistent with the fibrinogen concentration ratio (thrombin time method/immunological method). That is, the ratio of gammaArg275Cys was significantly lower than that of gammaArg275His. Moreover, scanning electron microscopy indicated significantly thicker fibers in fibrin clots made from gammaArg275Cys than in those of normal controls or gammaArg275His, and abnormal bundles with tapered ends. Factor XIIIa-catalyzed cross-linking of the fibrinogen gamma-chain (in the absence of thrombin) showed a similar delay for gammaArg275Cys and gammaArg275His. We report markedly impaired fibrin polymerization of gammaArg275Cys compared to gammaArg275His, and speculate that the difference is due to the disulfide-linked Cys in gammaArg275Cys, as we have already demonstrated for plasma and recombinant mutant fibrinogens. These results also indicate that an amino acid substitution of gammaArg275 disrupts D:D interactions in fibrin fiber formation. Furthermore haplotype analysis for three families with gammaArg275His suggested that founder of Iida family might be different from that of Otsu II or Shizuoka family.

[Clinical assessment of novel ChromID ESBL agar plates for detection of ESBL producers in the family Enterobacteriaceae].

Extended-Spectrum beta-Lactamase (ESBL)-producers in the family Enterobacteriaceae are recognized worldwide as nosocomial pathogens, however it is difficult to screen them in the routine laboratory processing. ChromID ESBL agar newly developed for screening ESBL-producing Enterobacteriaceae was released in Japan in April, 2007. We evaluated the clinical assessment of ChromID ESBL agar in routine microbiology laboratory. The 47 strains investigated were clinical isolates belonging to the family Enterobacteriaceae with the MICs of cefpodoxime greater than 2 mug/ml. The 27 ESBL-producers examined were comprising of 19 Escherichia coli, 3 Klebsiella oxytoca, 1 Citrobacter freundii, 3 Enterobacter cloacae, and 1 S. marcescens (ESBL group) and 20 ESBL non-producers consiating of 5 K. oxytoca, 1 Proteus mirabilis, 1 P. vlugaris, 2 Serratia marcescens, 8 C. freundii, 2 Enterobacter cloacae, and 1 E. aerogenes (non-ESBL group). Characterization of beta-lactamase genes was carried out by use of polymerase chain reaction. As the results, the sensitivity and the specificity of ChromID ESBL agar plates after incubation for 18 hours was 100% and 20%, respectively. It should be noted that the values of specificity was extremely low compared with those of the sensitivity. These findings clearly suggested that in cases of utilizing ChromID ESBL agar plates, it should be important to consider its characteristic properties, as even the ESBL-non-producers could grow on these media only when they were resistant to CPDX.

Redox equilibrium of serum apolipoprotein E3: a buffering effect of disulfide-linked complexes against oxidative stress on apolipoprotein E3-containing lipoproteins.

Reversible redox modification of cysteine thiols is crucial for protecting proteins from irreversible detrimental change. However, the physiological significance of the redox modification of apolipoprotein (apo) E is unclear. Here, we hypothesized that the disulfide-linked complexes of apoE3 corresponding to the representative reversible-modified apoE3 play a protective role against oxidative stress. The effects of disulfide bond formation on oxidative stress on apoE3 were evaluated with a band-shift assay. Maleimide-labeled apoE3 and unlabeled apoE3 were defined as the reduced (r)-apoE3 and non-reduced (nr)-apoE3 forms, respectively. Hydrogen peroxide-induced oxidation decreased for reduced-form apoE (r-apoE3) but increased for nr-apoE3. Induction of apoE3-AII complex formation with excess of apoAII markedly suppressed the oxidative stress-induced increase in nr-apoE3 (P<0.001) and enhanced homodimer formation. The apoE3-AII complex was more dominant in high-density lipoprotein (HDL) than in very low-density lipoprotein. Under oxidative stress, HDL showed a significant decrease, rather than an increase, in nr-apoE3 levels with a concomitant significant increase in apoE3-AII levels (P<0.005). This finding suggests that the majority of nr-apoE3 in HDL exists in a reversible oxidized form. The apoE3-AII complex, formed from the reversible oxidized apoE3, is beneficial for maintaining the redox equilibrium of apoE3 by preventing the modification of apoE3 to its irreversible oxidized form. The apoE3-AII complex may be possibly implicated in the pathophysiology of various apoE-related diseases.

Sialic acid moiety of apolipoprotein E3 at Thr(194) affects its interaction with beta-amyloid(1-42) peptides.

BACKGROUND: The interaction between apolipoprotein (apo) E and beta-amyloid (Abeta) is associated with the development of Alzheimer's disease (AD); however, the details remain unknown. ApoE in cerebrospinal fluid is extensively sialylated, and sialylation of certain proteins are known to modulate biological function. We investigated the effects of a sialic acid moiety of apoE on the apoE-Abeta interaction. METHODS: We prepared normal apoE3 and its mutant (Thr(194) --> Ala) and analyzed their interactions with Abeta(1-42) by using the surface plasmon resonance (SPR) assay. In addition, we performed the SPR assay by using apoE-containing lipoproteins treated with neuraminidase. We also assessed the effect of the mutation on the interaction of apoE3 with liposomes. RESULTS: The binding avidity of the mutant apoE3(#) was approximately 50% that of normal apoE3 (p<0.0001). The binding avidity of the apoE-containing lipoproteins for Abeta(1-42) reduced after neuraminidase treatment. CONCLUSIONS: We suggest that AD development is controlled not only by the apoE isoforms but also by the posttranslational modifications in apoE, such as those in the sialic acid moieties, which are abundant in apoE derived from the brain.