Oxidative Stress-Responsive Apoptosis Inducing Protein (ORAIP) Plays a Critical Role in Dextran Sulfate Sodium-Induced Murine Model of Ulcerative Colitis.

Oxidative stress is implicated in the pathogenesis of various acute disorders including ischemia/reperfusion injury, ultraviolet/radiation burn, as well as chronic disorders such as dyslipidemia, atherosclerosis, diabetes mellitus, chronic renal disease, and inflammatory bowel disease (IBD). However, the precise mechanism involved remains to be clarified. We formerly identified a novel apoptosis-inducing humoral protein, in a hypoxia/reoxygenation-conditioned medium of cardiac myocytes, which proved to be 69th tyrosine-sulfated eukaryotic translation initiation factor 5A (eIF5A). We named this novel tyrosine-sulfated secreted form of eIF5A Oxidative Stress-Responsive Apoptosis-Inducing Protein (ORAIP). To investigate the role of ORAIP in a dextran sulfate sodium (DSS)-induced murine model of ulcerative colitis (UC), we analyzed the effects of in vivo treatment with anti-ORAIP neutralizing monoclonal antibody (mAb) on the DSS-induced disease exacerbation. The body weight in anti-ORAIP mAb-treated group was significantly heavier than that in a mouse IgG-treated control group on day 8 of DSS-treatment ((85.21 ± 1.03%) vs. (77.38 ± 2.07%); (mean ± SE0, n = 5 each, p < 0.01, t-test). In vivo anti-ORAIP mAb-treatment also significantly suppressed the shortening of colon length as well as Disease Activity Index (DAI) score ((5.00 ± 0.44) vs. (8.20 ± 0.37); (mean ± SE), n = 5 each, p < 0.001, t-test) by suppressing inflammation of the rectal tissue and apoptosis of intestinal mucosal cells. These data reveal the pivotal role of ORAIP in DSS-induced oxidative stress involved in an animal model of UC.

Oxidative stress-responsive apoptosis inducing protein (ORAIP) plays a critical role in doxorubicin-induced apoptosis in rat cardiac myocytes.

BACKGROUND: Oxidative stress is implicated in the pathogenesis of doxorubicin-induced apoptosis in cardiac myocytes. However, the precise mechanism remains uncertain. We identified an apoptosis-inducing humoral factor, in a conditioned medium from cardiac myocytes subjected to hypoxia/reoxygenation, to be 69th tyrosine-sulfated eukaryotic translation initiation factor 5A (eIF5A). We named this novel secreted form of eIF5A, Oxidative stress-Responsive Apoptosis Inducing Protein (ORAIP). We confirmed that ischemia/reperfusion, ultraviolet-irradiation, and ionizing radiation significantly increased plasma levels of ORAIP in vivo, supporting that secretion of ORAIP is specific to the oxidative stress. To investigate the role of ORAIP in doxorubicin-induced apoptosis of cardiac myocytes. METHODS: We analyzed plasma levels of ORAIP in rats treated with doxorubicin (10 mg/Kg) in vivo, and the effects of neutralizing anti-ORAIP monoclonal antibody (mAb) on doxorubicin-induced apoptosis of cardiac myocytes in vitro. RESULTS: The (mean ± SE) plasma ORAIP levels before doxorubicin administration were (13.7 ± 2.7) ng/mL, they markedly increased with peak levels ([178.6 ± 6.5] ng/mL, p < 0.00001, vs. before administration) at 20 to 60 min after doxorubicin administration, then gradually decreased to (118.0 ± 4.8) ng/mL at 120 min. Treatment with a neutralizing anti-ORAIP mAb significantly (nearly 50%) suppressed doxorubicin-induced apoptosis of cardiac myocytes. CONCLUSIONS: These data indicate that doxorubicin induces oxidative stress resulting in the strong expression of ORAIP in cardiac myocytes and marked secretion of ORAIP into peripheral circulation. This strongly suggests that ORAIP can be a novel sensitive biomarker as well as a possible therapeutic target for doxorubicin-induced cell injury in anti-cancer therapy.

Oxidative stress-responsive apoptosis-inducing protein in patients with heterozygous familial hypercholesterolemia.

Oxidative stress, an inducer of apoptosis, plays a critical role in ischemia/reperfusion injury and atherosclerosis. We previously identified an apoptosis-inducing ligand, the post-translationally modified secreted form of eukaryotic translation initiation factor 5A (eIF5A), 'oxidative stress-responsive apoptosis-inducing protein' (ORAIP). In this study, we investigated the role of ORAIP in patients with heterozygous familial hypercholesterolemia (HeFH), a leading cause of premature cardiovascular disease. We analyzed plasma ORAIP and oxidized low-density lipoprotein (oxLDL) levels in 60 patients with HeFH (60% male, 57.0 ± 13.6 years of age) and 20 patients with LDL-C hypercholesterolemia (DL, 85% male, 64.1 ± 13.3 years of age). The coronary artery atherosclerosis from the patients with HeFH who had a coronary artery bypass graft was investigated by double immunostaining. The plasma ORAIP levels in the patients with HeFH were significantly elevated compared to those in the patients with DL (73.5 ± 46.0 vs. 48.3 ± 21.4 ng/mL, p = 0.0277). The plasma oxLDL levels in HeFH patients were also elevated (156.8 ± 65.2 vs. 123.7 ± 46.6 mg/dL, p = 0.0461) compared to those in DL patients and correlated with maxLDL-C levels (R = 0.4454, p = 0.00648). Double-immunostaining of ORAIP and oxLDL in the coronary artery from patients with HeFH who had a coronary artery bypass graft showed that ORAIP and oxLDL were colocalized with apoptotic vascular smooth muscle cells in the atherosclerotic plaque. ORAIP plays a role in the development of oxidative stress-induced atherosclerosis and may be an important therapeutic target for plaque rupture in patients with HeFH.

Oxidative stress-responsive apoptosis inducing protein (ORAIP) plays a critical role in cerebral ischemia/reperfusion injury.

Oxidative stress is known to play a critical role in the pathogenesis of various disorders, especially in ischemia/reperfusion (I/R) injury. We identified an apoptosis-inducing humoral factor and named this novel post translationally modified secreted form of eukaryotic translation initiation factor 5A (eIF5A) "oxidative stress-responsive apoptosis inducing protein" (ORAIP). The purpose of this study was to investigate the role of ORAIP in the mechanisms of cerebral I/R injury. Hypoxia/reoxygenation induced expression of ORAIP in cultured rat cerebral neurons, resulting in extensive apoptosis of these cells, which was largely suppressed by neutralizing anti-ORAIP monoclonal antibody (mAb) in vitro. Recombinant-ORAIP induced extensive apoptosis of cerebral neurons. Cerebral I/R induced expression of ORAIP in many neurons in a rat tandem occlusion model in vivo. In addition, we analyzed the effects of intracerebroventricular administration of neutralizing anti-ORAIP mAb on the development of cerebral infarction. Cerebral I/R significantly increased ORAIP levels in cerebrospinal fluid. Treatment with intracerebroventricular administration of neutralizing anti-ORAIP mAb reduced infarct volume by 72%, and by 55% even when started after reperfusion. These data strongly suggest that ORAIP plays a pivotal role and will offer a critical therapeutic target for cerebral I/R injury induced by thrombolysis and thrombectomy for acute ischemic stroke.

Oxidative Stress-Responsive Apoptosis Inducing Protein (ORAIP) Plays a Critical Role in High Glucose-Induced Apoptosis in Rat Cardiac Myocytes and Murine Pancreatic ß-Cells.

We previously identified a novel apoptosis-inducing humoral factor in the conditioned medium of hypoxic/reoxygenated-cardiac myocytes. We named this novel post-translationally-modified secreted-form of eukaryotic translation initiation factor 5A Oxidative stress-Responsive Apoptosis-Inducing Protein (ORAIP). We confirmed that myocardial ischemia/reperfusion markedly increased plasma ORAIP levels and rat myocardial ischemia/reperfusion injury was clearly suppressed by neutralizing anti-ORAIP monoclonal antibodies (mAbs) in vivo. In this study, to investigate the mechanism of cell injury of cardiac myocytes and pancreatic ß-cells involved in diabetes mellitus (DM), we analyzed plasma ORAIP levels in DM model rats and the role of ORAIP in high glucose-induced apoptosis of cardiac myocytes in vitro. We also examined whether recombinant-ORAIP induces apoptosis in pancreatic ß-cells. Plasma ORAIP levels in DM rats during diabetic phase were about 18 times elevated as compared with non-diabetic phase. High glucose induced massive apoptosis in cardiac myocytes (66.2 ± 2.2%), which was 78% suppressed by neutralizing anti-ORAIP mAb in vitro. Furthermore, recombinant-ORAIP clearly induced apoptosis in pancreatic ß-cells in vitro. These findings strongly suggested that ORAIP plays a pivotal role in hyperglycemia-induced myocardial injury and pancreatic ß-cell injury in DM. ORAIP will be a biomarker and a critical therapeutic target for cardiac injury and progression of DM itself.

Plasma levels of oxidative stress-responsive apoptosis inducing protein (ORAIP) in rats subjected to physicochemical oxidative stresses.

Oxidative stress is known to play a pivotal role in the pathogenesis of various disorders including atherosclerosis, aging and especially ischaemia/reperfusion injury. It causes cell damage that leads to apoptosis. However, the precise mechanism has been uncertain. Recently, we identified an apoptosis-inducing humoral factor in a hypoxia/reoxygenated medium of cardiac myocytes. We named this novel post-translationally modified secreted form of eukaryotic translation initiation factor 5A (eIF5A) as oxidative stress-responsive apoptosis inducing protein (ORAIP). We developed a sandwich ELISA and confirmed that myocardial ischaemia/reperfusion markedly increased plasma levels of ORAIP. To investigate whether the role of ORAIP is common to various types of oxidative stress, we measured plasma ORAIP levels in rats subjected to three physicochemical models of oxidative stress including N2/O2 inhalation, cold/warm-stress (heat shock) and blood acidification. In all three models, plasma ORAIP levels significantly increased and reached a peak level at 10-30 min after stimulation, then decreased within 60 min. The (mean±S.E.M.) plasma ORAIP levels before and after (peak) stimulation were (16.4±9.6) and (55.2±34.2) ng/ml in N2/O2 inhalation, (14.1±12.4) and (34.3±14.6) ng/ml in cold/warm-stress, and (18.9±14.3) and (134.0±67.2) ng/ml in blood acidification study. These data strongly suggest that secretion of ORAIP in response to oxidative stress is universal mechanism and plays an essential role. ORAIP will be an important novel biomarker as well as a specific therapeutic target of these oxidative stress-induced cell injuries.

Glycosylation status of serum immunoglobulin G in patients with prostate diseases.

Occurrences of high values in patients with benign prostate disease and low values in patients with highly suspicious cancer have diminished the trustworthiness of prostate-specific antigen as an early diagnostic marker of prostate cancer. In the search for other complimentary markers, we focused on serum IgG from patients with prostate diseases as well as normal subjects. IgG purified from the sera of normal control subjects and patients with prostate diseases, was digested with peptide N-glycanase. Released glycans were quantified using MALDI-time of flight mass spectrometry. We report that N-linked (N-acetylhexosamine)2 (deoxyhexose)(mannose)3 (N-acetylglucosamine)2 was significantly increased in the IgG heavy chains of patients with prostate cancer compared with that of either benign prostatic disease patients or healthy subjects, whereas (hexose)(N-acetylhexosamine)2 (deoxyhexose)(mannose)3 (N-acetylglucosamine)2 was more abundant in the heavy chains of healthy subjects and benign prostatic disease patients. Thus, an absence of the terminal hexose of N-linked glycans has been closely connected to the progression of prostate cancer. Furthermore, surface plasmon resonance analyses have revealed that IgG from patients with prostate cancer has a decreased binding for Sambucus nigra lectin, compared with that from the benign prostatic disease patients or from normal subjects, suggesting lower levels of (N-acetylneuraminic acid)(α2-6)galactose/N-acetylgalactosamine groups in the N-linked glycans of patient IgG. Meanwhile, wheat germ agglutinin binding to IgG of the cancer group was significantly larger than that for the benign prostatic disease group but smaller than that for normal subjects. Our study indicates that the glycosylation changes in IgG can become useful diagnostic parameters for prostate cancer.

Secreted tyrosine sulfated-eIF5A mediates oxidative stress-induced apoptosis.

Oxidative stress plays a critical role in ischemia/reperfusion-injury, atherosclerosis, and aging. It causes cell damage that leads to apoptosis via uncertain mechanisms. Because conditioned medium from cardiac myocytes subjected to hypoxia/reoxygenation induces extensive apoptosis of cardiac myocytes under normoxia, we hypothesized that a humoral factor released from the hypoxic/reoxygenated cardiac myocytes mediates apoptosis. We identified an apoptosis-inducing humoral factor in the hypoxia/reoxygenation-conditioned medium. Here, we found that eIF5A undergoes tyrosine sulfation in the trans-Golgi and is rapidly secreted from cardiac myocytes in response to hypoxia/reoxygenation; then, eIF5A induces apoptosis by acting as a pro-apoptotic ligand. The apoptosis of cardiac myocytes induced by hypoxia/reoxygenation or ultraviolet irradiation was suppressed by anti-eIF5A neutralizing monoclonal antibodies (mAbs) in vitro. Myocardial ischemia/reperfusion (but not ischemia alone) markedly increased the plasma levels of eIF5A, and treatment with anti-eIF5A neutralizing mAbs significantly reduced myocardial injury. These results identify an important, novel specific biomarker and a critical therapeutic target for oxidative stress-induced cell injury.

Multi-sequential surface plasmon resonance analysis of haptoglobin-lectin complex in sera of patients with malignant and benign prostate diseases.

Screening for prostate cancer remains unsatisfactory. Recent studies have examined the cancer diagnostic/prognostic values of various acute phase proteins, such as haptoglobin. We describe here a novel method of surface plasmon resonance (SPR) based on multi-sequential analysis with SNA-1, AAL, and PHA-L(4) lectin, to estimate the glycosylation status of haptoglobin in sera of patients with prostate cancer (n=15), benign prostate disease (BPD) including benign prostatic hypertrophy (n=20), and normal subjects (n=11). The SPR-based analysis involves the use of anti-haptoglobin as ligand and dilution of the analyte to 1400-fold and filtration, followed by detection of the sugar chain by lectin solution. The normalized RU of lectin to haptoglobin represents the binding amount of lectin divided by that of haptoglobin. The normalized RU by SNA-1 of the prostate cancer group was significantly higher than those of the control and BPD group. SNA-1 detected NeuAcα2,6 in a biantennary sugar chain, whose content was the highest among the major glycoproteins in serum. Serum samples diluted about 7000-fold were subjected to microanalysis at 10 ng/µl and 10 µl/min for 4 min. The combination of SNA-1 and haptoglobin by SPR multi-sequential analysis offered the most accurate diagnosis of prostate cancer without any modification of serum glycoproteins.

Two cases of pregnancy involving conjoined twins, with details of management after opting for live birth.

Conjoined twinning is a unique complication of monochorionic pregnancy. This report describes the clinical findings in two cases of conjoined twins, and discusses their management. One case involved thoracopagus complicating a triplet pregnancy, and the other involved cephalothoracopagus, in which the outcome was intrauterine fetal death due to abruptio placentae after amniocentesis. Recent improvements in ultrasound imaging have facilitated the diagnosis of conjoined twins as early as the first trimester. Although many mothers opt to terminate pregnancy when conjoined twins are diagnosed, a few do not, as in the cases described. In such cases, pregnancy management, including accurate determination of the degree of organ fusion and psychological follow up, are important. On the basis of the two present cases, we present a systematic flow diagram for management of conjoined twin pregnancy from the time of diagnosis until delivery.

Globotriaosylceramide-expressing Burkitt's lymphoma cells are committed to early apoptotic status by rhamnose-binding lectin from catfish eggs.

Silurus asotus (catfish) egg lectin (SAL) has a strong affinity to Gal alpha-linked carbohydrate chains of not only glycoproteins but also glycosphingolipids such as globotriaosylceramide (Gb3). SAL uniformly bound to surfaces of Gb3-expressing (Gb3+) Burkitt's lymphoma cells, while Gb3 molecules were interspersed on the surfaces of Gb3+ cells. After a short period of treating Raji and Daudi cells with SAL, each cell size was 10 and 25% smaller than that of untreated cells, respectively. Treatment of Gb3+ cells with SAL caused an increase in binding of annexin V, however, neither caspase activation nor DNA fragmentation was observed after treatment with SAL for 22 h. Since SAL did not induce cell death in Gb3+ cells, SAL may function as an inducer of early apoptotic signal. We have revealed that SAL did not bind to D-threo-1-phenyl-2-decanoylamino-3-morphorino-1-propanol (D-PDMP)-treated Raji cells, and no cell shrinkage was observed in Gb3-deficient Raji cells treated with SAL, indicating that Gb3 localized in the glycosphingolipid-enriched microdomain (GEM) was involved in SAL-induced cell shrinkage through activation of voltage-gated potassium channel Kv1.3, and that the glycoprotein ligands on Gb3-deficient Raji cells treated with SAL were not included in this phenomenon. These results suggest that SAL leads the cells to early apoptotic status via binding to Gb3 existing in GEM, and that this binding is a prerequisite condition to induce early stage of apoptosis.

Haptoglobin-beta chain defined by monoclonal antibody RM2 as a novel serum marker for prostate cancer.

In our previous study, monoclonal antibody RM2, established toward the glycosyl epitope, reflected grade of malignancy of prostate cancer cells whereas RM2 reactivity to benign glands was negative or weak. RM2 reactivity was also detected in stroma, suggesting the glycoprotein RM2 recognizes could be released into the bloodstream. Then, we explored RM2 reactivity to sera of early prostate cancer. We compared RM2 reactivity to sera between 62 patients with early prostate cancer and 43 subjects with benign prostatic disease, and examined RM2 reactivity before and after radical prostatectomy in 15 patients by Western blotting. We also examined RM2 reactivity to sera of the other urogenital cancers. RM2 reactivity was significantly enhanced on a serum glycoprotein with molecular mass approximately 40 kDa, hereby termed GPX, in the patients with early prostate cancer when compared with those with benign prostatic disease (p < 0.0001). Setting an appropriate cutoff level, RM2 reactivity to GPX for detection of prostate cancer had sensitivity of 87% and specificity of 84%, respectively. Furthermore, the level of RM2 reactivity significantly decreased after radical prostatectomy (p = 0.006). However, increased RM2 reactivity to GPX was also observed in the other urogenital cancers. The proteomics approach identified GPX as haptoglobin-beta chain and RM2 showed preferential reactivity toward haptoglobin-beta chain derived from prostate cancer when compared with polyclonal anti-haptoglobin antibody. Haptoglobin-beta chain defined by RM2 is a novel serum marker that may be useful for detection of early prostate cancer when coupled with prostate-specific antigen because it is not specific to prostate cancer.

Detection of Carcinoembryonic Antigens Using a Surface Plasmon Resonance Biosensor.

Carcinoembryonic antigen (CEA) is an oncofoetal cell-surface glycoprotein that serves as an important tumor marker for colorectal and some other carcinomas. In this work, a CEA immunoassay using a surface plasmon resonance (SPR) biosensor has been developed. SPR could provide label-free, real-time detection with high sensitivity, though its ability to detect CEA in human serum was highly dependent on the analytical conditions employed. We investigated the influences of various analytical conditions including immobilization methods for anti-CEA antibody and composition of sensor surface on the selective and sensitive detection of CEA. The results show that anti-CEA antibody immobilized via Protein A or Protein G caused a large increase in the resonance signal upon injection of human serum due to the interactions with IgGs in serum, while direct covalent immobilization of anti-CEA antibody could substantially reduce it. An optimized protocol based on further kinetic analysis and the use of 2nd and 3rd antibodies for the sandwich assay allowed detecting spiked CEA in human serum as low as 25 ng/mL. Furthermore, a self-assembled monolayer of mixed ethylene-glycol terminated alkanethiols on gold was found to have a comparable ability in detecting CEA as CM5 with thick dextran matrix and C1 with short flat layer on gold.

Involvement of very long fatty acid-containing lactosylceramide in lactosylceramide-mediated superoxide generation and migration in neutrophils.

The neutral glycosphingolipid lactosylceramide (LacCer) forms lipid rafts (membrane microdomains) coupled with the Src family kinase Lyn on the plasma membranes of human neutrophils; ligand binding to LacCer activates Lyn, resulting in neutrophil functions, such as superoxide generation and migration (Iwabuchi and Nagaoka, Lactosylceramide-enriched glycosphingolipid signaling domain mediates superoxide generation from human neutrophils, Blood 100, 1454-1464, 2002 and Sato et al. Induction of human neutrophil chemotaxis by Candida albicans-derived beta-1,6-long glycoside side-chain-branched beta glycan, J. Leukoc. Biol. 84, 204-211, 2006). Neutrophilic differentiated HL-60 cells (D-HL-60 cells) express almost the same amount of LacCer as neutrophils. However, D-HL-60 cells do not have Lyn-associated LacCer-enriched lipid rafts and lack LacCer-mediated superoxide-generating and migrating abilities. Here, we examined the roles of LacCer molecular species of different fatty acid compositions in these processes. Liquid chromatography-mass spectrometry analyses revealed that the very long fatty acid C24:0 and C24:1 chains were the main components of LacCer (31.6% on the total fatty acid content) in the detergent-resistant membrane fraction (DRM) from neutrophil plasma membranes. In contrast, plasma membrane DRM of D-HL-60 cells included over 70% C16:0-LacCer, but only 13.6% C24-LacCer species. D-HL-60 cells loaded with C24:0 or C24:1-LacCer acquired LacCer-mediated migrating and superoxide-generating abilities, and allowed Lyn coimmunoprecipitation by anti-LacCer antibody. Lyn knockdown by siRNA completely abolished the effect of C24:1-LacCer loading on LacCer-mediated migration of D-HL-60 cells. Immunoelectron microscopy revealed that LacCer clusters were closely associated with Lyn molecules in neutrophils and C24:1-LacCer-loaded D-HL-60 cells, but not in D-HL-60 cells or C16:0-LacCer-loaded cells. Taken together, these observations suggest that LacCer species with very long fatty acids are specifically necessary for Lyn-coupled LacCer-enriched lipid raft-mediated neutrophil superoxide generation and migration.

Glycosylation status of haptoglobin in sera of patients with prostate cancer vs. benign prostate disease or normal subjects.

We studied chemical level and glycosylation status of haptoglobin in sera of patients with prostate cancer, as compared to benign prostate disease and normal subjects, with the following results. (i) Haptoglobin level was enhanced significantly in sera of prostate cancer. (ii) Sialylated bi-antennary glycans were the dominant structures in haptoglobins from all 3 sources, regardless of different site of N-linked glycan. The N-linked glycans at N184 were exclusively bi-antennary, and showed no difference between prostate cancer vs. benign prostate disease. (iii) Tri-antennary, N-linked, fucosylated glycans, carrying at least 1 sialyl-Lewis(x/a) antenna, were predominantly located on N207 or N211 within the amino acid 203-215 sequence of the beta-chain of prostate cancer, and were minimal in benign prostate disease. Fucosylated glycans were not observed in normal subjects. A minor tri-antennary N-linked glycan was observed at N241 of the beta-chain in prostate cancer, which was absent in benign prostate disease. (iv) None of these N-linked structures showed the expected presence of disialylated antennae with GalNAcbeta4(NeuAcalpha3)Galbeta3(NeuAcalpha6)GlcNAcbetaGal, or its analogue, despite cross-reactivity of prostate cancer haptoglobin with monoclonal antibody RM2. (v) Minor levels of O-glycosylation were identified in prostate cancer haptoglobin for the first time. Mono- and disialyl core Type 1 O-linked structures were identified after reductive beta-elimination followed by methylation and mass spectrometric analysis. No evidence was found for the presence of specific RM2 or other tumor-associated glycosyl epitopes linked to this O-glycan core. In summary, levels of haptoglobin are enhanced in sera of prostate cancer patients, and the N-glycans attached to a defined peptide region of its beta-chain are characterized by enhanced branching as well as antenna fucosylation.

A case of neonatal lupus erythematosus presenting delayed dilated cardiomyopathy with circulating autoantibody to annexin A6.

Patients with neonatal lupus erythematosus (NLE) often have congenital heart block with or without heart failure and are born to mothers who have anti-SS-A and/or anti-SS-B antibodies. NLE has been considered to result from the placental transmission of maternal autoantibodies into the fetal circulation causing myocardial damage. We report a case of NLE with congenital heart block who had undergone pacemaker implantation at the age of 17, and then developed dilated cardiomyopathy (DCM) at the age of 19, which is much later than in most other cases. The patient's mother was positive for anti-SS-A and anti-SS-B antibodies, whereas the patient was negative for both anti-SS-A and anti-SS-B antibodies. There were some autoantibodies against cell surface antigens of cardiac myocytes in the serum from the patient, and annexin A6 was identified as one of the autoantigens. This is the first report demonstrating that annexin A6 is involved in the myocardial injury in patients with NLE. The results indicate that inhibition of annexin A6 function may prevent autoantibody-mediated myocardial injury in at least some cases of DCM.

Dihydroorotate dehydrogenase arises from novel fused gene product with aspartate carbamoyltransferase in Bodo saliens.

The ACT-DHOD gene in the kinetoplastid Bodo saliens encodes aspartate carbamoyltransferase and dihydroorotate dehydrogenase, the second and fourth enzymes of pyrimidine biosynthesis. Although the single mRNA species yielded a 70-kDa ACT-DHOD protein, Western blotting with anti-DHOD-peptide antibody showed a major band of 35-kDa and minor bands. In-gel digestion and liquid chromatography-tandem mass (MS/MS) spectrometry showed that the 35-kDa band contained DHOD-specific polypeptides and an ACT-specific polypeptide, suggesting the occurrence of independent DHOD and ACT. Immunoprecipitation and MS/MS analysis identified a 70-kDa ACT-DHOD and a 35-kDa DHOD independently, and the N-terminal amino acid of 35-kDa DHOD was blocked. In vitro processing assay showed that recombinant ACT-DHOD was decreased by the B. saliens lysate, accompanying the appearance of 35-kDa DHOD and 35-kDa ACT. These results indicate that fused ACT-DHOD is the precursor to mature DHOD. Large amount of 35-kDa DHOD in B. saliens is discussed from a viewpoint of its physiological roles.

Mass spectrometric identification and quantification of glycosyl flavonoids, including dihydrochalcones with neutral loss scan mode.

We developed a strategy for determination and quantification of glycosyl flavonoids using liquid chromatography-triple quadrupole mass spectrometry with neutral loss scan at 15 and 30eV collision energy in the positive ion mode. The fragmentation patterns of glycosyl flavonoids at 15 and 30eV showed that fragmentation of sugar moiety depended on the type of glycosidic bond to aglycone, the site of C-glycosylation, and the type of aglycone. C-Glycosyl dihydrochalcones especially stood out because they produced [M+H-162](+) even at 15eV such as O-glycoside in spite of C-glycoside. C-Glycosides were classified according to (i) the intensity ratio A of [M+H-150](+) to [M+H-120](+) at 30eV and (ii) the intensity ratio B of [M+H-120](+) at 15eV to one at 30eV. The 8-C-glycosides were A<1 and B<1, the 6-C-glycosides were A>1 and B<1, and the C-glycosyl dihydrochalcones were A>1 and B>>1. Therefore, the intensity ratios of the neutral loss scan of 120 and 150Da at 30eV and those of 120, 162, and 308Da at 15eV allowed sequential distinction among these three types of C-glycosides as well as between O- and C-glycosides. Our method was applied for analysis of Rooibos tea, and the identified glycosides could be quantified specifically by the selected reaction monitoring method.