Serum anti-DIDO1, anti-CPSF2, and anti-FOXJ2 antibodies as predictive risk markers for acute ischemic stroke.

BACKGROUND: Acute ischemic stroke (AIS) is a serious cause of mortality and disability. AIS is a serious cause of mortality and disability. Early diagnosis of atherosclerosis, which is the major cause of AIS, allows therapeutic intervention before the onset, leading to prevention of AIS. METHODS: Serological identification by cDNA expression cDNA libraries and the protein array method were used for the screening of antigens recognized by serum IgG antibodies in patients with atherosclerosis. Recombinant proteins or synthetic peptides derived from candidate antigens were used as antigens to compare serum IgG levels between healthy donors (HDs) and patients with atherosclerosis-related disease using the amplified luminescent proximity homogeneous assay-linked immunosorbent assay. RESULTS: The first screening using the protein array method identified death-inducer obliterator 1 (DIDO1), forkhead box J2 (FOXJ2), and cleavage and polyadenylation specificity factor (CPSF2) as the target antigens of serum IgG antibodies in patients with AIS. Then, we prepared various antigens including glutathione S-transferase-fused DIDO1 protein as well as peptides of the amino acids 297-311 of DIDO1, 426-440 of FOXJ2, and 607-621 of CPSF2 to examine serum antibody levels. Compared with HDs, a significant increase in antibody levels of the DIDO1 protein and peptide in patients with AIS, transient ischemic attack (TIA), and chronic kidney disease (CKD) but not in those with acute myocardial infarction and diabetes mellitus (DM). Serum anti-FOXJ2 antibody levels were elevated in most patients with atherosclerosis-related diseases, whereas serum anti-CPSF2 antibody levels were associated with AIS, TIA, and DM. Receiver operating characteristic curves showed that serum DIDO1 antibody levels were highly associated with CKD, and correlation analysis revealed that serum anti-FOXJ2 antibody levels were associated with hypertension. A prospective case-control study on ischemic stroke verified that the serum antibody levels of the DIDO1 protein and DIDO1, FOXJ2, and CPSF2 peptides showed significantly higher odds ratios with a risk of AIS in patients with the highest quartile than in those with the lowest quartile, indicating that these antibody markers are useful as risk factors for AIS. CONCLUSIONS: Serum antibody levels of DIDO1, FOXJ2, and CPSF2 are useful in predicting the onset of atherosclerosis-related AIS caused by kidney failure, hypertension, and DM, respectively.

Autologous antibody to src-homology 3-domain GRB2-like 1 specifically increases in the sera of patients with low-grade gliomas.

BACKGROUND: Glioma is the most common primary malignant central nervous system tumor in adult, and is usually not curable in spite of various therapeutic approaches. Clarification of the oncogenic process in its early stage is important for the diagnosis and effective therapy. METHODS: In the present study, we used the serological identification of antigens by recombinant cDNA expression cloning (SEREX) to explore the subtle changes of the protein expression in low-grade glioma. The levels of serum autoantibodies to the SEREX-identified glioma-related antigens were analyzed by ELISA, and the epitope site was identified using deletion mutants and overlap peptide array. Changes in the serum autoantibody levels were examined in the rat glioma model using C6 and 9 L glioma cell lines. RESULTS: We identified 31 glioma-related antigens by SEREX. Among them, the serum level of autoantibody to src-homology 3-domain GRB2-like 1 (SH3GL1) was significantly higher in patients with low-grade glioma than healthy volunteers or high-grade gliomas. The 10 amino-acids at the C-terminal were identified as the epitope site by the overlap peptide array and the ELISA using deletion mutants. The tissue expression of SH3GL1 protein increased in proportion to glioma progression. The rat glioma models confirmed the increase of anti-SH3GL1 autoantibody level in the early stage and the suppression in the late stage. CONCLUSION: SH3GL1 may be involved in the oncogenic process of gliomas and effectively elicit an autologous antibody response in low-grade gliomas. The immunological reaction to SH3GL1 would contribute to the establishment of a novel diagnostic and therapeutic target for gliomas.

HDL2 can inhibit further oxidative modification of partially oxidized LDL.

AIM: To investigate whether HDL(2) can inhibit further oxidative modification of partially oxidized LDL (ox-LDL) by interrupting the chain oxidation reaction after lipid hydroperoxides (LOOH) formation. METHODS: Following incubation of LDL 400 microg protein/mL phosphate-buffered saline with Cu(2+) for 1.75 h (defined as 0 min), incubation was continued after adding HDL(2) 200 microg protein/mL or HDL(2) 800 microg protein/mL to give both ox-LDL+HDL(2) 200 microg protein/mL or ox-LDL+HDL(2) 800 microg protein/mL. As a control, ox-LDL 200 microg protein/mL and native LDL were prepared. Each sample was subjected to agarose gel electrophoresis and the LOOH in each sample was measured. RESULTS: When the electrophoretic mobility of native LDL was designated 1, the relative electrophoretic mobility (REM) of ox-LDL increased significantly over time. The REMs of ox-LDL+HDL(2) 800 microg protein/mL from 10 min to 9 h were significantly lower than the REM of ox-LDL at the respective times (p<0.01). LOOH of ox-LDL+HDL(2) 800 microg protein/mL at 1, 3, 6 and 9 h was significantly higher than LOOH in ox-LDL at the respective times (p<0.01). The results of ox-LDL+HDL(2) 200 microg protein/mL were almost the same but to a lesser extent than the results of ox-LDL+HDL(2) 800 microg protein/mL. CONCLUSION: The present findings suggest that HDL(2) can inhibit further oxidative modification of partially oxidized LDL by interrupting the chain oxidation reaction after LOOH formation in a concentration-dependent manner.

Increased receptor binding by bovine (b) TSH bound to monoclonal antibody to bTSHbeta-subunit.

TSH receptor (R) binding and cAMP production by bovine (b) TSH-bound to a monoclonal antibody (MoAb) or polyclonal antibody (Ab) to bTSH were examined, using TSH receptor (R) coating tube and porcine thyroid cells. (125) I-bTSH bound-to MoAbs to bTSH(alpha) or discontinuous type MoAb showed TSHR binding (10%) similar to intact (125) I-bTSH. TSHR binding was completely decreased (<2%) when (125) I-bTSH was bound by polyclonal Abs to bTSH(alpha) in Graves' patient or rabbit polyclonal Abs to bTSH. When either of the two MoAb (No. 1 and 2) to bTSH(beta) was bound to (125) I-bTSH, TSHR binding was 4 times higher (40%) compared to intact (125) I-bTSH. Binding of another MoAb (No. 3) caused no increased binding. TSHR binding of intact (125) I-bTSH was decreased from 10% to 2% by excess amounts of bTSH. Binding of (125) I-bTSH bound to MoAb to bTSH(beta) (No. 1 and 2) decreased from 40% to 30% by excess amounts of bTSH. When (125) I-bTSH bound-Fab of MoAb was used, the binding was reduced from 30 to 10% (No. 1) and from 25 to 6% (No. 2), respectively. In contrast, cAMP production by bTSH was decreased by pre-binding of all MoAbs and polyclonal Abs. Binding of (125) I-MoAb to bTSH (beta) to a synthetic peptide array of bTSH (beta) sequence was examined by the radioautography. The epitope of MoAb to bTSH(beta) was suggested to be LPK (beta 42-44) for No. 1, KLF (beta 39-41) for No. 2 and PKYA (beta 43-46) for No. 3, respectively, although the existence of discontinuous epitope could not be ruled out. The increased TSHR binding and the decreased cAMP production by bTSH bound to MoAbs may be due to the conformational change of TSH molecule or TSHR by binding of both bTSH and MoAb.

Mei4p coordinates the onset of meiosis I by regulating cdc25+ in fission yeast.

The kinase Cdc2p is a central regulator of entry into and progression through nuclear division during mitosis and meiosis in eukaryotes. Cdc2p is activated at the onset of mitosis by dephosphorylation on tyrosine-15, the phosphorylation status of which is determined mainly by the kinase Wee1p and the phosphatase Cdc25p. In fission yeast, the forkhead-type transcription factor Mei4p is required for expression of many genes during meiosis, with mei4 mutant cells arresting before meiosis I. The mechanism of cell cycle arrest in mei4 cells has remained unknown, however. We now show that cdc25(+) is an important target of Mei4p in control of entry into meiosis I. Forced dephosphorylation of Cdc2p on tyrosine-15 thus induced meiosis I in mei4 mutant cells without a delay, although no spores were formed. We propose that Mei4p acts as a rate-limiting regulator of meiosis I by activating cdc25(+) transcription in coordination with other meiotic events.

Peptide array-based interaction assay of solid-bound peptides and anchorage-dependant cells and its effectiveness in cell-adhesive peptide design.

Peptide array, the designable peptide library covalently synthesized on cellulose support, was applied to assay peptide-cell interaction, between solid-bound peptides and anchorage-dependant cells, to study objective peptide design. As a model case, cell-adhesive peptides that could enhance cell growth as tissue engineering scaffold material, was studied. On the peptide array, the relative cell-adhesion ratio of NIH/3T3 cells was 2.5-fold higher on the RGDS (Arg-Gly-Asp-Ser) peptide spot as compared to the spot with no peptide, thus indicating integrin-mediated peptide-cell interaction. Such strong cell adhesion mediated by the RGDS peptide was easily disrupted by single residue substitution on the peptide array, thus indicating that the sequence recognition accuracy of cells was strictly conserved in our optimized scheme. The observed cellular morphological extension with active actin stress-fiber on the RGD motif-containing peptide supported our strategy that peptide array-based interaction assay of solid-bound peptide and anchorage-dependant cells (PIASPAC) could provide quantitative data on biological peptide-cell interaction. The analysis of 180 peptides obtained from fibronectin type III domain (no. 1447-1629) yielded 18 novel cell-adhesive peptides without the RGD motif. Taken together with the novel candidates, representative rules of ineffective amino acid usage were obtained from non-effective candidate sequences for the effective designing of cell-adhesive peptides. On comparing the amino acid usage of the top 20 and last 20 peptides from the 180 peptides, the following four brief design rules were indicated: (i) Arg or Lys of positively charged amino acids (except His) could enhance cell adhesion, (ii) small hydrophilic amino acids are favored in cell-adhesion peptides, (iii) negatively charged amino acids and small amino acids (except Gly) could reduce cell adhesion, and (iv) Cys and Met could be excluded from the sequence combination since they have less influence on the peptide design. Such rules that are indicative of the nature of the functional peptide sequence can be obtained only by the mass comparison analysis of PIASPAC using peptide array. By following such indicative rules, numerous amino acid combinations can be effectively screened for further examination of novel peptide design.

Capsaicin-induced, capsazepine-insensitive relaxation of the guinea-pig ileum.

The mechanisms underlying transient receptor potential vanilloid receptor type 1 (TRPV1)-independent relaxation elicited by capsaicin were studied by measuring isometric force and phosphorylation of 20-kDa regulatory light chain subunit of myosin (MLC(20)) in ileum longitudinal smooth muscles of guinea-pigs. In acetylcholine-stimulated tissues, capsaicin (1-100 microM) and resiniferatoxin (10 nM-1 microM) produced a concentration-dependent relaxation. The relaxant response was attenuated by 4-aminopyridine and high-KCl solution, but not by capsazepine, tetraethylammonium, Ba(2+), glibenclamide, charybdotoxin plus apamin nor antagonists of cannabinoid receptor type 1 and calcitonin-gene related peptide. A RhoA kinase inhibitor reduced the relaxant effect of capsaicin at 30 microM. Capsaicin and resiniferatoxin reduced acetylcholine- and caffeine-induced transient contractions in a Ca(2+)-free, EGTA solution. Capsaicin at 30 microM for 20 min did not alter basal levels of MLC(20) phosphorylation, but abolished an increase by acetylcholine in MLC(20) phosphorylation. It is suggested that the relaxant effect of capsaicin at concentrations used is not mediated by TRPV1, but by 4-aminopyridine-sensitive K(+) channels, and that capsaicin inhibits contractile mechanisms involving Ca(2+) release from intracellular storage sites. The relaxation could be explained by a decrease in phosphorylation of MLC(20).

Both HDL3 and HDL2 exert a powerful anti-oxidative and protective effect against acceleration of oxidative modification of LDL by ascorbic acid.

The objective of the present study was to establish whether high-density lipoprotein 3 (HDL3) or high-density lipoprotein 2 (HDL2) might show an anti-oxidative effect on the acceleration of the oxidative modification of low-density lipoprotein (LDL) by ascorbic acid from measurement of the agarose gel electrophoretic mobility of LDL. LDL was incubated without adding transitional-metal ions for 48 or 96 h in phosphate-buffered saline (PBS) alone, with ascorbic acid (20 microg/mL), or with both ascorbic acid (20 microg/mL) and HDL3 (200 microg protein/mL). The LDL autoxidation occurred in PBS alone. Although ascorbic acid significantly suppressed oxidative modification of LDL after incubation for 48 h, the opposite was true after 96 h. However, since the anti-oxidative ability of HDL2 shows a weaker tendency than that of HDL3, both HDL3 and HDL2 significantly inhibited this acceleration of oxidative modification of LDL by ascorbic acid as assessed by electrophoretic mobility. If there is an augmented oxidative modification of LDL due to ascorbic acid in vivo, HDL3 or HDL2 may thus have an important role in inhibiting this ascorbic acid-accelerated oxidation of LDL.

Autoantigenicity of DFS70 is restricted to the conformational epitope of C-terminal alpha-helical domain.

Autoantibodies against DFS70 (Dense Fine Speckles 70) are found in 30% of Japanese atopic dermatitis patients, and less frequently in patients with other diseases. We have recently reported that they are also seen in 11% of hospital workers, but in only approximately 2% of patients with systemic rheumatic disease. In this study, in order to investigate the possible pathological role of anti-DFS70 antibodies, fine epitope mapping was carried out using 93 anti-DFS70 autoantibody-positive sera. Immunoblotting using overlapping peptides failed to reveal major linear epitopes. Western blotting using various truncated proteins showed a strikingly uniform epitope distribution on a suspected tertiary structure expressed by DFS70(349-435). Some sera showed reactivity only in an immunoprecipitation assay using an in vitro translated DFS70. Circular dichroism analysis revealed that DFS(349-435) contains an approximately 40% alpha-helical conformation, while an overlapping, non-antigenic peptide is composed of random coiled structures. The skewed single major epitope enabled us to establish a highly quantitative ELISA for the epitope region. Antibody titers showed no significant differences between the diseased group and healthy individuals. We propose that anti-DFS70 antibody may be a natural autoantibody, which might modify or reflect the inflammatory process of various disorders.

Angiotensin II inhibitory peptide found in the receptor sequence using peptide array.

Peptide array consisting of hundreds of peptides spatially addressed and synthesized on a cellulose membrane support was used to screen ligand-inhibitory peptides. As a model, angiotensin II (Ang II), a significant peptide related to the treatment of cardiovascular diseases, was chosen as the target ligand. Peptide arrays covering the Ang II receptor type 1 sequence were prepared, and peptide domains with high affinity to the Ang II fluorescein conjugate were investigated. The peptide (VVIVIY) within the first transmembrane region exhibited the highest affinity to Ang II. The synthesized soluble VVIVIY peptide had an 84% inhibitory effect on Ang II-induced aorta contraction. These results indicate that our screening strategy utilizing peptide array is an effective approach for the peptide drug development.

Taurochenodeoxycholic acid induced biphasic hepatotoxicity in isolated perfused rat liver: roles of Ca2+ and calpain.

BACKGROUND/AIMS: We examined taurochenodeoxycholic acid-induced hepatotoxicity with reference to Ca2+ and calpain involvement, intracellular bile acid content, and zone specificity in isolated perfused rat liver. METHODOLOGY: Taurochenodeoxycholic acid or chenodeoxycholic acid was infused into the portal vein and lactate dehydrogenase release, a marker of hepatocyte injury, in the effluent and bile acid output were measured in the presence and absence of either nickel, a membranous Ca2+ channel blocker, or calpain inhibitor in isolated perfused rat liver. RESULTS: Taurochenodeoxycholic acid induced a significant and transient increase (first peak; 4 min) and subsequent time- and dose-dependent elevation in lactate dehydrogenase release which was proportional to accumulated bile acids in the liver. Although the first peak was significantly suppressed by pretreatment with nickel, the subsequent release was not reduced. Lactate dehydrogenase release at 15, 20, and 25 min was significantly suppressed by the calpain inhibitor. Numbers of damaged hepatocytes stained with trypan blue were significantly increased in the periportal region (zone 1) compared with the pericentral region (zone 3) and these cells were consistently stained with anti-calpain antibody. CONCLUSIONS: Taurochenodeoxycholic acid causes both transient damage and subsequent increasing hepatotoxicity which are respectively dependent on Ca2+ influx via membranous Ca2+ channels and calpain, with the periportal region being more susceptible.

Interaction of protein-bound polysaccharide (PSK) with smooth muscle myosin regulatory light chain.

The interaction of a protein-bound polysaccharide (PSK) isolated from Basidiomycetes with smooth muscle myosin components was evaluated by limited digestion, urea/glycerol gel electrophoresis, affinity chromatography and overlay assay using a peptide array. PSK was bound to the regulatory light chain (RLC) of myosin, but not to the essential light chain. The binding to PSK was definitely observed for unphosphorylated RLC, compared to phosphorylated one. From the amino acid sequence of the RLC, 490 peptides were synthesized on a cellulose membrane. Overlay assays showed that the PSK-binding on the molecule of RLC were localized in the N- and C-terminal basic regions and these sites were conserved in RLC from the human smooth muscle and nonmuscle cells.

Calpain is involved in the HIV replication from the latently infected OM10.1 cells.

Treatment of OM10.1 cells latently infected with human immunodeficiency virus type 1 (HIV-1) with phorbol ester and calcium ionophore (A23187) induced virus replication which was blocked by N-Ac-Leu-Leu-norleucinal (ALLnL), a calpain inhibitor I, and not by lactacystin, a specific proteasome inhibitor. When the purified NF-kappa B/I kappa B complex was treated with mu-calpain, the specific DNA-binding activity was demonstrated by using electrophoretic mobility shift assay in vitro. This effect of mu-calpain was inhibited by ALLnL and calpastatin and not by lactacystin. In fact, we found that mu-calpain efficiently degraded I kappa B alpha. Furthermore, our Western blotting analysis has revealed that mu-calpain cleaves I kappa B alpha at its N-terminal and C-terminal regions that were previously reported to be involved in the interaction with NF-kappa B p65. These observations indicate that in monocyte/macrophage cells calcium signaling is involved in NF-kappa B activation through activation of calpain and thus calpain inhibitors may be effective in inhibiting the activation of latently infected HIV.

HDL3 exerts a more powerful antiperoxidative and protective effect against peroxidative modification of LDL than HDL2 does.

The objective of the present study was to establish that HDL3 has a greater antiperoxidative effect on the peroxidative modification of LDL than HDL2 has. The protective influence of HDL subfractions on this form of LDL modification was examined in samples by measuring the concentration of thiobarbituric acid-reactive substance (TBARS), as well as the electrophoretic mobility of LDL. LDL was incubated for 96 hours in phosphate-buffered saline (PBS) alone, without the addition of transition-metal ions or in the presence of PBS and HDL2 or HDL3. All samples were subjected to agarose gel electrophoresis. Both HDL2 and HDL3 significantly inhibited peroxidative modification of LDL, as assessed by electrophoretic mobility, but this effect was much more pronounced with HDL3. HDL3 may play a more important role than HDL2 in the prevention of atherosclerosis in vivo by more effectively inhibiting LDL peroxidation.