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Amyloid β-protein (Aβ) oligomers are involved in the early stages of Alzheimer's disease (AD) and antibodies against these toxic oligomers could be useful for accurate diagnosis of AD. We identified the toxic conformer of Aβ42 with a turn at positions 22/23, which has a propensity to form toxic oligomers. The antibody 24B3, developed by immunization of a toxic conformer surrogate E22P-Aβ9-35 in mice, was found to be useful for AD diagnosis using human cerebrospinal fluid (CSF). However, it is not known how 24B3 recognizes the toxic conformation of wild-type Aβ in CSF. Here, we report the crystal structure of 24B3 Fab complexed with E22P-Aβ11-34, whose residues 16-26 were observed in electron densities, suggesting that the residues comprising the toxic turn at positions 22/23 were recognized by 24B3. Since 24B3 bound only to Aβ42 aggregates, several conformationally restricted analogs of Aβ42 with an intramolecular disulfide bond to mimic the conformation of toxic Aβ42 aggregates were screened by enzyme immunoassay. As a result, only F19C,A30homoC-SS-Aβ42 (1) bound significantly to 24B3. These data provide a structural basis for its low affinity to the Aβ42 monomer and selectivity for its aggregate form.

Characterization of amyloid β (Aβ) oligomers, the transition species present prior to the formation of Aβ fibrils and that have cytotoxicity, has become one of the major topics in the investigations of Alzheimer's disease (AD) pathogenesis. However, studying pathophysiological properties of Aβ oligomers is challenging due to the instability of these protein complexes in vitro. Here, we report that conformation-restricted Aβ42 with an intramolecular disulfide bond at positions 17 and 28 (SS-Aβ42) formed stable Aβ oligomers in vitro. Thioflavin T binding assays, nondenaturing gel electrophoresis, and morphological analyses revealed that SS-Aβ42 maintained oligomeric structure, whereas wild-type Aβ42 and the highly aggregative Aβ42 mutant with E22P substitution (E22P-Aβ42) formed Aβ fibrils. In agreement with these observations, SS-Aβ42 was more cytotoxic compared to the wild-type and E22P-Aβ42 in cell cultures. Furthermore, we developed a monoclonal antibody, designated TxCo-1, using the toxic conformation of SS-Aβ42 as immunogen. X-ray crystallography of the TxCo-1/SS-Aβ42 complex, enzyme immunoassay, and immunohistochemical studies confirmed the recognition site and specificity of TxCo-1 to SS-Aβ42. Immunohistochemistry with TxCo-1 antibody identified structures resembling senile plaques and vascular Aβ in brain samples of AD subjects. However, TxCo-1 immunoreactivity did not colocalize extensively with Aβ plaques identified with conventional Aβ antibodies. Together, these findings indicate that Aβ with a turn at positions 22 and 23, which is prone to form Aβ oligomers, could show strong cytotoxicity and accumulated in brains of AD subjects. The SS-Aβ42 and TxCo-1 antibody should facilitate understanding of the pathological role of Aβ with toxic conformation in AD.

Blood levels of the extracellular matrix protein nephronectin (Npnt), a protein critical for kidney development, are elevated in autoimmune experimental autoimmune encephalitis (EAE) mice, which are a model for multiple sclerosis. We found here that treatment with anti-Npnt antibody directed against the α8β1 integrin-binding site (Npnt-blocking antibody) inhibits EAE development. The selenium transporter selenoprotein P (SeP) was identified as a novel Npnt-binding partner. In EAE, Npnt induced SeP and glutathione peroxidase 1 (GPx1) expression, followed by reactive oxygen species (ROS) inhibition in CD4+ T cells; these changes were disturbed by Npnt-blocking antibody treatment, which also caused suppressed differentiation of interleukin (IL)-17-producing CD4+ T-helper cells (Th17s) and elevated differentiation of regulatory T cells (Tregs). Treatment of EAE mice with the ROS scavenger N-acetyl cysteine (NAC) blocked the Npnt-blocking antibody-induced decrease in Th17 differentiation and increase in Treg differentiation. In conclusion, the interaction between Npnt and SeP contributes to EAE development by regulating the Th17/Treg balance via the ROS level.

The extracellular matrix protein nephronectin (Npnt) is known to be critical for kidney development, but its function in inflammatory diseases is unknown. Here, we developed a new enzyme‐linked immunosorbent assay system to detect Npnt in various autoimmune diseases, which revealed that plasma Npnt levels are increased in various mouse autoimmune models. We also report that antibodies against the α8β1 integrin‐binding region of Npnt protect mice from anti‐type II collagen‐induced arthritis, suggesting that Npnt may be a potential therapeutic target molecule for the prevention of autoimmune arthritis., The role of extracellular matrix protein nephronectin (Npnt) in inflammatory diseases is unknown. In this study, we show that plasma Npnt levels are increased in various mouse autoimmune models and antibodies against the α8β1 integrin‐binding region attenuated the development of anti‐type II collagen‐induced arthritis in mice.

"Nutraceuticals" are well-tolerated natural dietary compounds with drug-like properties that make them attractive as Alzheimer's disease (AD) therapeutics. Combination therapy for AD has garnered attention following a recent National Institute on Aging mandate, but this approach has not yet been fully validated. In this report, we combined the two most promising nutraceuticals with complementary anti-amyloidogenic properties: the plant-derived phenolics (-)-epigallocatechin-3-gallate (EGCG, an α-secretase activator) and ferulic acid (FA, a β-secretase modulator). We used transgenic mice expressing mutant human amyloid β-protein precursor and presenilin 1 (APP/PS1) to model cerebral amyloidosis. At 12 months of age, we orally administered EGCG and/or FA (30 mg/kg each) or vehicle once daily for 3 months. At 15 months, combined EGCG-FA treatment reversed cognitive impairment in most tests of learning and memory, including novel object recognition and maze tasks. Moreover, EGCG- and FA-treated APP/PS1 mice exhibited amelioration of brain parenchymal and cerebral vascular β-amyloid deposits and decreased abundance of amyloid β-proteins compared with either EGCG or FA single treatment. Combined treatment elevated nonamyloidogenic soluble APP-α and α-secretase candidate and down-regulated amyloidogenic soluble APP-β, β-C-terminal APP fragment, and β-secretase protein expression, providing evidence for a shift toward nonamyloidogenic APP processing. Additional beneficial co-treatment effects included amelioration of neuroinflammation, oxidative stress, and synaptotoxicity. Our findings offer preclinical evidence that combined treatment with EGCG and FA is a promising AD therapeutic approach.

Several plant-derived compounds have demonstrated efficacy in pre-clinical Alzheimer's disease (AD) rodent models. Each of these compounds share a gallic acid (GA) moiety, and initial assays on this isolated molecule indicated that it might be responsible for the therapeutic benefits observed. To test this hypothesis in a more physiologically relevant setting, we investigated the effect of GA in the mutant human amyloid β-protein precursor/presenilin 1 (APP/PS1) transgenic AD mouse model. Beginning at 12 months, we orally administered GA (20 mg/kg) or vehicle once daily for 6 months to APP/PS1 mice that have accelerated Alzheimer-like pathology. At 18 months of age, GA therapy reversed impaired learning and memory as compared with vehicle, and did not alter behavior in nontransgenic littermates. GA-treated APP/PS1 mice had mitigated cerebral amyloidosis, including brain parenchymal and cerebral vascular β-amyloid deposits, and decreased cerebral amyloid β-proteins. Beneficial effects co-occurred with reduced amyloidogenic and elevated nonamyloidogenic APP processing. Furthermore, brain inflammation, gliosis, and oxidative stress were alleviated. We show that GA simultaneously elevates α- and reduces β-secretase activity, inhibits neuroinflammation, and stabilizes brain oxidative stress in a pre-clinical mouse model of AD. We further demonstrate that GA increases abundance of a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10, Adam10) proprotein convertase furin and activates ADAM10, directly inhibits β-site APP cleaving enzyme 1 (BACE1, Bace1) activity but does not alter Adam10 or Bace1 transcription. Thus, our data reveal novel post-translational mechanisms for GA. We suggest further examination of GA supplementation in humans will shed light on the exciting therapeutic potential of this molecule.

To date, there is no effective Alzheimer's disease (AD)-modifying therapy. Nonetheless, combination therapy holds promise, and nutraceuticals (natural dietary compounds with therapeutic properties) and their synthetic derivatives are well-tolerated candidates. We tested whether combination therapy with octyl gallate (OG) and ferulic acid (FA) improves cognition and mitigates AD-like pathology in the presenilin-amyloid β-protein precursor (PSAPP) transgenic mouse model of cerebral amyloidosis. One-year-old mice with established β-amyloid plaques received daily doses of OG and FA alone or in combination for 3 months. PSAPP mice receiving combination therapy had statistically significant improved cognitive function versus OG or FA single treatment on some (but not all) measures. We also observed additional statistically significant reductions in brain parenchymal and cerebral vascular β-amyloid deposits as well as brain amyloid β-protein abundance in OG- plus FA-treated versus singly-treated PSAPP mice. These effects coincided with enhanced nonamyloidogenic amyloid β-protein precursor (APP) cleavage, increased α-secretase activity, and β-secretase inhibition. We detected elevated expression of nonamyloidogenic soluble APP-α and the α-secretase candidate, a disintegrin and metalloproteinase domain-containing protein 10. Correspondingly, amyloidogenic β-carboxyl-terminal APP fragment and β-site APP-cleaving enzyme 1 expression levels were reduced. In parallel, the ratio of β- to α-carboxyl-terminal APP fragment was decreased. OG and FA combination therapy strikingly attenuated neuroinflammation, oxidative stress, and synaptotoxicity. Co-treatment afforded additional statistically significant benefits on some, but not all, of these outcome measures. Taken together, these data provide preclinical proof-of-concept for AD combination therapy.

Human natural killer-1 (HNK-1) epitope, a highly-expressed glycan in the nervous system, is critical for normal synaptic plasticity and spatial learning. HNK-1 epitope modifies N-glycans on several neural glycoproteins, and also modifies O-mannosyl glycans. A branching enzyme for O-mannosyl glycans (GnT-IX, Core M2 synthase) exhibits brain-specific expression, and the product core M2 glycans are also limited to the brain. In a previous study, we showed that cuprizone-induced demyelination increased HNK-1-capped core M2 glycan expression, while GnT-IX deficiency ameliorated demyelination, suggesting that these glycans could be useful diagnostic markers for demyelination status and act as therapeutic targets. Nevertheless, a lack of appropriate detection tools hampered further analysis of HNK-1-capped O-mannosyl glycans. In the present study, we chemoenzymatically synthesized HNK-1-capped core M2 glycans for antibody production, and confirmed that the resulting immune sera reacted with HNK-1-capped core M2 glycans. We then examined several HNK-1-related antibodies, including the Cat-315 antibody, for reactions with HNK-1-capped core M2 glycans. Finally, we confirmed the increased HNK-1 epitope expression in demyelinated brains of cuprizone-fed mice.

Complex sulfide ores are typically mixtures of various sulfide minerals like sphalerite (ZnS), chalcopyrite (CuFeS2), galena (PbS), pyrite (FeS2), and barite (BaSO4) and processed by selective flotation to recover target-sulfide minerals like chalcopyrite and sphalerite. Some complex sulfide ores, however, contain anglesite (PbSO4), a mineral with relatively high solubility, that complicates selective flotation because its dissolution releases Pb2+, which ‘activates’ co-existing sulfide minerals (e.g., ZnS). Because both target and non-target sulfide minerals are recovered when flotation is non-selective, another flotation stage to recover target minerals in froth products is required. Aside from anglesite, co-existing gangue sulfide minerals like pyrite also complicate selective flotation because of their strong effects on the floatability of target-sulfide minerals via electrochemical interactions. In this study, the effects of pyrite on the floatability of lead-activated sphalerite were investigated using flotation tests and electrochemical techniques coupled with contact angle measurements. Moreover, factors important to lead-activated sphalerite floatability were elucidated in detail using dissolution experiments and X-ray photoelectron spectroscopy (XPS). Finally, a mechanism for lead-activated sphalerite depression by pyrite is proposed.

Amyloid precursor protein (APP) proteolysis is required for production of amyloid-β (Aβ) peptides that comprise β-amyloid plaques in the brains of patients with Alzheimer disease (AD). Here, we tested whether the experimental agent methylene blue (MB), used for treatment of methemoglobinemia, might improve AD-like pathology and behavioral deficits. We orally administered MB to the aged transgenic PSAPP mouse model of cerebral amyloidosis and evaluated cognitive function and cerebral amyloid pathology. Beginning at 15 months of age, animals were gavaged with MB (3 mg/kg) or vehicle once daily for 3 months. MB treatment significantly prevented transgene-associated behavioral impairment, including hyperactivity, decreased object recognition, and defective spatial working and reference memory, but it did not alter nontransgenic mouse behavior. Moreover, brain parenchymal and cerebral vascular β-amyloid deposits as well as levels of various Aβ species, including oligomers, were mitigated in MB-treated PSAPP mice. These effects occurred with inhibition of amyloidogenic APP proteolysis. Specifically, β-carboxyl-terminal APP fragment and β-site APP cleaving enzyme 1 protein expression and activity were attenuated. Additionally, treatment of Chinese hamster ovary cells overexpressing human wild-type APP with MB significantly decreased Aβ production and amyloidogenic APP proteolysis. These results underscore the potential for oral MB treatment against AD-related cerebral amyloidosis by modulating the amyloidogenic pathway.

The molecular chaperone heat shock protein 90 (Hsp90) is involved in the maturation and stabilization of a wide range of oncogenic client proteins for oncogenesis and malignant cell proliferation, which renders this protein a promising target in the development of cancer therapeutics. PU-H71 is a purine-scaffold Hsp90 inhibitor with less toxicity in normal cells than in cancer cells. In this study, we examined the in vitro radiosensitizing activity and molecular mechanisms of action of PU-H71 in human lung cancer cell lines. PU-H71 enhanced the sensitivity of the SQ-5 and A549 cancer cells to radiation. When the cancer cells were pre-treated with PU-H71, the repair of DNA double-strand breaks (DSBs) was markedly inhibited after irradiation compared with the cells that were not pre-treated with PU-H71, as evaluated by counting the foci of phosphorylated histone H2AX (γ-H2AX). We further demonstrated that post-irradiation, PU-H71 inhibited Rad51 foci formation, a critical protein for the homologous recombination pathway of DNA DSB repair. These data indicate that targeting Hsp90 with PU-H71 may be novel therapeutic strategy for radioresistant carcinomas.

The (pro)renin receptor ((P)RR) plays a key role in the activation of the local renin-angiotensin system via interaction with renin and prorenin. A truncated form that is cleaved by furin, referred to as soluble (pro)renin receptor (s(P)RR), is secreted into the extracellular space. An accurate measurement of s(P)RR levels in vivo is an important issue in elucidating the roles of (P)RR in physiology and pathophysiology. To address this, we developed a sandwich ELISA that is applicable to human subjects. The standard curve of this ELISA showed a high linearity (125-8,000 pg/ml) with a correlation coefficient of >0.99. The recovery rate was approximately 90% in human blood and urine samples. The s(P)RR levels in plasma of healthy volunteers was in the range from 15.2 to 35.1 ng/ml (n = 20). Intra- and inter-assay coefficient of variations were less than 5.5% and 7.5%, respectively. It thus appears that this ELISA is a reliable tool for measuring s(P)RR levels in human subjects.

Objective.Although osteoarthritis (OA) is generally assessed using standard radiographic images in clinical practice, biochemical markers can be used to detect the disease and determine its severity. Osteopontin (OPN) is an extracellular matrix glycoprotein that is a potential inflammatory cytokine. Presence of the thrombin-cleaved form of OPN is well correlated with various inflammatory diseases. We examined whether thrombin-cleaved OPN in synovial fluid (SF) and synovium could be associated with the severity of knee OA.Methods.SF samples were obtained from 139 knees with OA. Thrombin-cleaved OPN product was determined using Western blotting. Levels of thrombin-cleaved and full-length OPN in SF were determined by ELISA. Synovium samples were analyzed by immunohistochemistry using an antibody specific to the thrombin-cleaved form.Results.Western blotting showed the presence of thrombin-cleaved OPN in SF from patients with advanced OA. Concentrations of OPN full-length in OA knees were not statistically different from those in controls (p = 0.134). In contrast, levels of OPN N-half were significantly higher in OA knees than in controls (p = 0.042). Statistically significant correlation was found between thrombin-cleaved OPN and disease severity by Kellgren-Lawrence grade 1, 2, 3, and 4 (R = 0.274, p < 0.001). Immunohistochemistry of the synovium showed stronger reactivity in samples from subjects with advanced OA.Conclusion.Local generation of thrombin-cleaved OPN was increased with greater OA severity.

Mesothelioma is an aggressive cancer often caused by chronic asbestos exposure, and its prognosis is very poor despite the therapies currently used. Due to the long latency period between asbestos exposure and tumor development, the worldwide incidence will increase substantially in the next decades. Thus, novel effective therapies are warranted to improve the prognosis. The ERC/mesothelin gene (MSLN) is expressed in wide variety of human cancers, including mesotheliomas, and encodes a precursor protein cleaved by proteases to generate C-ERC/mesothelin and N-ERC/mesothelin. In this study, we investigated the antitumor activity of C-ERC/mesothelin-specific mouse monoclonal antibody, 22A31, against tumors derived from a human mesothelioma cell line, ACC-MESO-4, in a xenograft experimental model using female BALB/c athymic nude mice. Treatment with 22A31 did not inhibit cell proliferation of ACC-MESO-4 in vitro; however, therapeutic treatment with 22A31 drastically inhibited tumor growth in vivo. 22A31 induced antibody-dependent cell-mediated cytotoxicity by natural killer (NK) cells, but not macrophages, in vitro. Consistently, the F(ab')(2) fragment of 22A31 did not inhibit tumor growth in vivo, nor did it induce antibody-dependent cell mediated cytotoxicity (ADCC) in vitro. Moreover, NK cell depletion diminished the antitumor effect of 22A31. Thus, 22A31 induced NK cell-mediated ADCC and exerted antitumor activity in vivo. 22A31 could have potential as a therapeutic tool to treat C-ERC/mesothelin-expressing cancers including mesothelioma.

Malignant mesothelioma is a highly aggressive tumor of the serosal cavity that arises from the mesothelial cells of the pleura, peritoneum, or pericardium. The immunohistochemical diagnosis of epithelioid mesothelioma from biopsy or surgically resected specimens has been actively pursued, using markers such as mesothelin. Several markers have indeed been helpful for confirming the diagnosis of mesothelioma and distinguishing between mesothelioma and adenocarcinoma. The authors have developed a novel mAb to human C-ERC/mesothelin, which performed well when used in western blotting, fluorescence-activated cell sorting, immunocytochemistry and immunohistochemistry, and which therefore will be useful in studying the molecular biology of mesothelin, in addition to improving the diagnosis and therapy of mesothelin-expressing cancers.

ERC/mesothelin is expressed on the normal mesothelium and some cancers such as mesothelioma or ovarian carcinoma. A splicing isoform of ERC/mesothelin (known as SMRP), which has an 82-bp insertion and codes for a C-terminus with a hydrophilic, presumably soluble, tail instead of a GPI-anchoring signal, has been reported as a useful marker for the diagnosis of mesothelioma. However, the existence of SMRP has not yet been demonstrated in the serum of mesothelioma patients. To elucidate the existence of SMRP, we have established a new enzyme-linked immunosorbent assay (ELISA) system for SMRP. The ELISA study revealed that N- and C-ERC/mesothelin were detected in sera from mesothelioma patients, but not SMRP, even in these samples. This result showed that the SMRP detected with MESOMARK kit should be lack of soluble C-terminus and indistinguishable from C-ERC/mesothelin. Further study might be necessary to demonstrate the relationship between SMRP and mesothelin.

Background: Because mesothelioma initially progresses on the surface of the pleura and peritoneum without forming masses, it has been difficult to diagnose at an early stage. It would be very useful to identify a tumor marker that could be used for screening to enable more diagnoses to be made at an early, treatable stage. Materials and Methods: We had previously identified N-ERC/mesothelin as a potential biomarker for mesothelioma. In the current work, we used a newly developed ELISA system to gain data on N-ERC/mesothelin levels in various clinical settings. A total of 102 healthy volunteers were recruited. In addition, 39 patients were diagnosed with mesothelioma, 53 patients were diagnosed with diseases that should be distinguished from mesothelioma, and 201 subjects were diagnosed with asbestos-related nonmalignant diseases (including simple exposure to asbestosis) who were treated at any of the cooperating hospitals were enrolled. Results: Serum N-ERC/mesothelin levels measured by a new ELISA system showed that the median values from patients with mesothelioma were extremely high compared with levels obtained from other patients. Analysis in terms of histologic type showed that serum levels of N-ERC/mesothelin were elevated in epithelioid type mesothelioma, especially. In four important models of clinical settings, the sensitivity and specificity of N-ERC/mesothelin were about 71% to 90% and 88% to 93%, respectively. Conclusion: N-ERC/mesothelin is a very promising tumor marker for mesothelioma, especially epithelioid mesothelioma.

MYO18B is a class XVIII myosin, cloned as a tumor suppressor gene candidate. To investigate the mechanisms of MYO18B-dependent tumor suppression, MYO18B-interacting proteins were searched for by a yeast two-hybrid screen. HOMER2, a Homer/Ves1 family protein, was identified as a binding partner of MYO18B. These proteins co-localized in the regions of membrane protrusion and stress fiber, which are known as ones with filamentous actin-rich structures. Expression of HOMER2 enhanced the ability of MYO18B to suppress anchorage-independent growth. These results indicate that HOMER2 and MYO18B cooperate together in tumor suppression.

Vpr, an accessory gene product of HIV-1, has been reported in the plasma of HIV-1-positive patients, and exogenous Vpr induces the reactivation of viral production from latently infected cells and the apoptosis of T cells in vitro. These observations imply that Vpr is important in AIDS development, but the clinical relevance of the findings cannot be evaluated fully because the actual plasma Vpr concentration in HIV-1-positive patients is unknown. Here we generated two monoclonal antibodies against different portions of Vpr and successfully identified Vpr as a 14-kDa protein in HIV-1-positive patients. Semiquantitative analysis using a recombinant Vpr revealed that the concentration of Vpr in patient plasma was approximately 0.7 nM (10 ng/ml). Cross-sectional analysis of 52 HIV-1-positive patients revealed that the presence of Vpr detected in 20 patients was positively correlated with HIV-1 RNA copy number (p > 0.03), but not with the number of CD4(+) T cells. This is the first report demonstrating the actual amount of Vpr in HIV-1-positive patients, and the possible linkage of Vpr and viral titers indicates that it is important to continue to carry out the sequential analysis of Vpr, especially in clinical courses of HIV-1-positive patients. The threshold of viral titers, where Vpr appears in the patients' plasma, if present, contributes to better understanding the role of Vpr in AIDS pathogenesis.

Both osteopontin (OPN) and natural killer T (NKT) cells play a role in the development of immunological disorders. We examined a functional link between OPN and NKT cells. Concanavalin A (Con A)-induced hepatitis is a well-characterized murine model of T cell-mediated liver diseases. Here, we show that NKT cells secrete OPN, which augments NKT cell activation and triggers neutrophil infiltration and activation. Thus, OPN- and NKT cell-deficient mice were refractory to Con A-induced hepatitis. In addition, a neutralizing antibody specific for a cryptic epitope of OPN, exposed by thrombin cleavage, ameliorated hepatitis. These findings identify NKT cell-derived OPN as a novel target for the treatment of inflammatory liver diseases.

Osteopontin (OPN) is a secreted protein that has been implicated in diverse physiological and pathological processes. OPN can bind to integrins, via GRGDS or SVVYGLR amino acid sequences, and to other cell surface receptors, and many of OPN's functions are likely mediated via cell adhesion and subsequent signaling. Here we developed and characterized a series of five monoclonal antibodies, raised to distinct internal peptide sequences of human OPN, and have used these sequence-specific reagents, along with the previously described anti-OPN monoclonal antibody mAb53, to map functional epitopes of OPN that are important to cell adhesion and migration. All antibodies were reactive with native as well as recombinant human OPN. One antibody (2K1) raised against the peptide VDTYDGRGDSVVYGLRS could inhibit RGD-dependent cell binding to OPN, with an efficacy comparable to that of mAb53. Furthermore, 2K1 could inhibit α9 integrin-dependent cell binding to OPN. The epitope recognized by 2K1 was not destroyed by thrombin digestion, whereas mAb53 has been shown to be unable to react with OPN following thrombin cleavage. The two distinct epitopes defined by 2K1 and mAb53 antibodies are closely related to the SVVYGLR cell-binding domain and the GLRSKS containing thrombin cleavage site, respectively, and are involved in cell binding and cell migration. J. Cell. Biochem. 84: 420–432, 2002. © 2001 Wiley-Liss, Inc.

Calcareous nannofossil biostratigraphy was studied for the Neogene marine Shimajiri Group distributed in Kume-jima Island, Ryukyu Islands, southwestern Japan. Out of 21 samples examined in this study, 14 samples yielded calcareous nannofossils. As was reported in a previous study, Zones CN9 and CN12 of Okada and Bukry (1980) were recognized in this study. In addition, Subzone CN11b (early Pliocene) was newly identified in the lower parts of the sequence where the ages were previously assigned to the late Miocene (upper part of Zone CN9 or older). This result shows the difficulty of establishing the stratigraphy of the subjected group based mainly upon lithotratigraphy, particularly in the lower part. Additional biostratigraphic data are needed to establish the reliable stratigraphy of the marine Shimajiri Group in Kume-jima Island.

In this study, we developed an isocenter verification device for use in proton therapy. Radiation and mechanical isocenters were verified for treatment equipment including room lasers, a digital radiography system and the beam axis of a rotational gantry. The special feature of this device is its ability to correlate the position of the three isocenters in one measurement and thus improve accuracy compared to the conventional method using three separate devices. The reproducibility of the method and the fluctuation of the position of the beam axis isocenter were both investigated using this device for almost a year. Monthly measurements of the isocenter position were acquired for two gantries and it was found that the fluctuation was +/- 0.10mm for the up-to-down direction and +/- 0.16mm for the right-to-left direction in Gantry 1 and was +/-0.14mm for the up-to-down direction and +/-0.18mm for the right-to-left direction in Gantry 2. We could be measured with a repeatability of +/-0.18 mm or less by using developed device for the relative positional relationship between each isocenters. Because we can confirm results in approximately 30 minutes, we can perform a quality control after a clinical practice.

SummarySyndecan-4 (ryudocan) is a cell-surface heparan sulfate proteoglycan, which plays an important role in a variety of biological functions including regulation of blood coagulation, cell adhesion, and cell growth. In this study, we measured plasma levels of syndecan-4 in patients with acute myocardial infarction using an enzyme-immunoassay, and found that they were extremely high, with a peak of average (10.5 ± 5.6 ng/ml, 2 weeks after onset), as compared with those in normal subjects (0.078 ± 0.030 ng/ml) (p < 0.001). We also observed a distinct expression of syndecan-4 in the repair region of the damaged cardiac tissues with infarction, but not in intact region, by immunohistochemical analysis. To clarify the mechanism of syndecan-4 induction, we investigated the hypoxia effect on its expression, and found that hypoxia treatment up-regulated the gene expression of syndecan-4 in various types of cells. Taken together, it is suggested that syndecan-4 is induced by hypoxia stimuli in ischemic heart tissues, and may function as a tissue-repair molecule via biological mediators such as heparin-binding growth factors.

Tumor growth and metastasis are angiogenesis-dependent processes initiated and regulated by a number of cytokines. Vascular endothelial growth factor (VEGF) is a potent angiogenic protein with a selective mitogenic effect on vascular endothelial cells. Osteopontin (OPN) induces endothelial cell migration and upregulates endothelial cell migration induced by VEGF. To clarify the cooperative role of VEGF and OPN in tumor angiogenesis, we stained VEGF, OPN, and CD34 immunohistochemically in 87 cases of stage I non-small cell lung cancer (adenocarcinoma, 55, and squamous cell carcinoma, 32). Of the 87 patients studied, 27 patients had postoperative relapse and 60 patients did not. VEGF was found in 34 of 55 cases of adenocarcinomas and 14 of 32 squamous cell carcinomas, and OPN was found in 30 of 55 adenocarcinomas and 10 of 32 squamous cell carcinomas. In adenocarcinoma, microvessel counts of VEGF-positive and OPN-positive tumors were significantly higher than VEGF-negative and OPN-negative tumors, respectively, whereas in squamous cell carcinoma they were not. More importantly, patients with VEGF- and OPN-positive stage I lung adenocarcinoma had significantly worse prognosis as compared with other groups. Cooperation of OPN is important in VEGF-mediated tumor angiogenesis in stage I lung adenocarcinoma.

Objectives: We have previously reported that the level of leucine-rich alpha-2-glycoprotein (LRG) expression is specifically increased in cerebrospinal fluid (CSF) of idiopathic normal pressure hydrocephalus (INPH). The objective of this study is to examine the localization of LRG – the cerebral areas where it is expressed.

Objective Cerebrospinal fluid (CSF) shunting can improve symptoms of elderly patients' idiopathic normal pressure hydrocephalus (iNPH). However, adjunctive means for confirming the diagnosis remain unavailable. We have previously reported the specific increase of leucine-rich alpha-2-glycoprotein (LRG) in iNPH CSF, and the present study investigates its potential clinical applications. Methods We performed CSF tap test (TT) on 90 patients (mean age 73.4 years) and shunting in 52 patients (mean age 73.5 years), evaluating symptom improvement and higher cerebral functions—mini-mental state examination (MMSE) and Frontal Assessment Battery (FAB) before and 12 months after shunting. LRG and tau protein concentrations in TT CSF were simultaneously measured using enzyme-linked immunosorbent assay. We then compared the predictive value of these concentrations with TT results regarding successful shunting outcomes. Results Positive combinations of TT and LRG concentrations of 67 ng/ml or higher, gave 81.6% sensitivity and 78.6% specificity. Therefore we used LRG (67 ng/ml) and tau (200 pg/ml) cut-off values, dividing patients into four groups. In group A (LRG ≥ 67 ng/ml and tau

Objective.Osteopontin (OPN) is an extracellular matrix glycoprotein that has been recognized as a potential inflammatory cytokine. The function of OPN is modulated by protease digestion, and a thrombin-cleaved form of OPN is involved in the pathogenesis of various inflammatory disorders. We examined thrombin-cleaved OPN products in synovial fluid from patients with rheumatoid arthritis (RA) and osteoarthritis (OA).Methods.Synovial fluid samples were obtained from knees of 20 patients with RA and 111 patients with OA. Thrombin-cleaved OPN product was determined using Western blotting. Levels of thrombin- cleaved and full-length OPN in synovial fluid were determined by ELISA. Synovia were analyzed by immunohistochemistry using an antibody specific to the thrombin-cleaved form.Results.Immunoblotting showed the presence of thrombin-cleaved OPN in synovial fluid from patients with RA and OA. ELISA results showed no difference between concentrations of full-length OPN in the synovial fluid of RA and OA patients; however, thrombin-cleaved OPN concentrations in RA synovial fluid samples were roughly 30-fold higher compared with OA samples (p < 0.001). Synovial fluid concentrations of thrombin-cleaved OPN in RA did not correlate with C-reactive protein levels. Immunohistochemistry of the synovium showed stronger reactivity in RA than in OA samples.Conclusion.Local generation of thrombin-cleaved OPN was increased in RA joints. Thrombin-cleaved OPN may be a useful biochemical marker of RA.

In order to address whether galectin-3 in the sera and fine needle aspirates serve as a diagnostic marker distinguishing between benign and malignant thyroid nodules, we developed an enzyme-linked immunosorbent assay. We quantified galectin-3 in fine needle aspirates from a series of 118 patients with thyroid nodules and serum galectin-3 from another series of 46 patients, which were compared with final histology after thyroidectomy. Relative galectin-3 value (ng/mg), defined as galectin-3 concentration (ng/ml) divided by total protein concentration (mg/ml) in fine needle aspirates, was significantly higher in papillary carcinoma than in the other thyroid entities. There was no significant difference in serum galectin-3 level among patients with thyroid nodules and healthy individuals. Accordingly, relative galectin-3 value allows a definitive diagnosis of papillary carcinoma independent of cellular morphology, whereas serum galectin-3 does not serve as a marker for papillary carcinoma.

Mesothelioma is a type of malignant tumor that most commonly arises from the pleural or peritoneal membrane and is usually associated with previous exposure to asbestos. In humans, ERC/ mesothelin is expressed on the normal mesothelium and in some cancers such as mesothelioma or ovarian carcinoma. Recently, several enzyme-linked immunosorbent assay (ELISA) systems for ERC/mesothelin have been developed, the reported usefulness of which has been assessed and demonstrated as a diagnostic tool. However, the basic roles or physiological functions of, and relationship between, ERC/mesothelin and asbestos exposure– mediated carcinogenesis remain to be resolved. In order to elucidate the precise mechanism, animal models of mesothelioma are desperately needed. In this study, we consider the development of a novel specific ELISA system for not only rat N-ERC/mesothelin but also C-ERC/mesothelin, and the first data on the presence of rat ERC/ mesothelin in the body fluids of rat malignant mesothelioma– bearing nude mice. The transplanted mice have revealed the higher concentrations of rat N-ERC/mesothelin in the blood and ascites than C-ERC/mesothelin. We hope these novel ELISA systems are useful in the rat model system to clarify the mechanism of asbestosinduced carcinogenesis and to develop new effective drugs for mesothelioma. (Cancer Sci 2008; 99: 666–670)

By representational difference analysis, we previously identified the rat Erc (Expressed in renal carcinoma) gene that was more abundantly expressed in the renal carcinoma tissues of Eker rats than in the rat normal kidney. In this study, we raised antibodies against the amino-terminal portion of the rat Erc, and demonstrated the existence of a ∼30-kDa secretory form in the supernatant of cultured cells derived from rat renal carcinoma. The enzyme-linked immunosorbent assay (ELISA) system using these antibodies detected high concentrations of this form in the sera of Eker rats bearing renal carcinomas, and in the sera of rats transplanted with mesothelioma cells. Mesothelin, a human homolog of the rat Erc, was recently reported to be a serum marker of malignant mesothelioma. The prognosis of mesothelioma is poor and there is no effective treatment at present. There are several rat model systems of mesothelioma that may be promising tools in the development of an antimesothelioma treatment. We hope our ELISA to detect the soluble form of rat Erc/Mesothelin is useful in the rat model system to exploit the antimesothelioma therapy to be used in human cases. (Cancer Sci 2007; 98: 659–664)

We have developed a novel enzyme-linked immunosorbent assay (ELISA) system for the detection of N-ERC/mesothelin in the serum of mesothelioma patients and have begun to examine its clinical usefulness. N-ERC/mesothelin is a 31-kDa protein that forms the N-terminal fragment of the full-length 71-kDa ERC/mesothelin protein, and is physiologically secreted into the blood of mesothelioma patients where it can be detected using our sandwich ELISA containing two antibodies (rabbit polyclonal anti-ERC/mesothelin antibody-282 and mouse monoclonal antibody 7E7). Our ELISA system has thus far detected much higher serum levels of N-ERC/mesothelin in mesothelioma patients than in healthy controls or patients with other lung or pleural diseases. In conclusion, N-ERC/mesothelin is a promising candidate tumor marker for mesothelioma.

We investigated the role of osteopontin (OPN) in interleukin-12 (IL-12) production from peripheral blood mononuclear cells (PBMCs) stimulated withPenicillium marneffei. Kinetic studies showed that OPN synthesis preceded that of IL-12 at both mRNA and protein levels when PBMCs were cocultured withP. marneffei. Treatment with anti-OPN monoclonal antibodies (MAb) significantly suppressed IL-12 secretion. Furthermore, native OPN induced a profound level of synthesis of IL-12 from noninfected PBMCs. The major cellular source of OPN was monocytes, because depletion of CD14+cells resulted in the abrogation of such production. We also examined the regulatory role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in OPN secretion fromP. marneffei-stimulated PBMCs. Neutralizing anti-GM-CSF MAb significantly reduced OPN secretion, and treatment with this cytokine induced OPN production from both infected and noninfected PBMCs. Finally, antagonists against the mannose receptor but not the β-glucan receptor almost completely abrogated the production of OPN. Our results demonstrated that OPN secreted from monocytes is involved in the production of IL-12 from PBMCs after stimulation withP. marneffeiand that OPN production is regulated by GM-CSF. Our results also indicated the possible involvement of the mannose receptor as a signal-transducing receptor for triggering the secretion of OPN byP. marneffei-stimulated PBMCs.

We have generated synthetic peptides corresponding to various portions of human osteopontin (OPN) and have immunized rabbits and mice with these peptides to generate polyclonal and monoclonal antibodies specific to human OPN. We then generated six distinct sandwich enzyme-linked immunoabsorbent assay (ELISA) systems by using different pairs of polyclonal and monoclonal antibodies against human OPN. These systems allowed us to detect not only various isoforms and truncated forms of recombinant OPN, but also the glycosylated form of native urinary OPN. Most importantly, tumor-derived OPN was differentially detected by the six ELISA systems. The ELISA systems that we have developed will be useful for clarifying the functional roles for OPN in vivo in various physiologic and pathologic conditions.