Seikagaku Biobusiness Corporation

Tokyo, Japan

Seikagaku Biobusiness Corporation

Tokyo, Japan
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Kawabe K.,Ritsumeikan University | Kawabe K.,Japan National Institute of Biomedical Innovation | Tateyama D.,Japan National Institute of Biomedical Innovation | Toyoda H.,Ritsumeikan University | And 13 more authors.
Glycobiology | Year: 2013

We have generated a monoclonal antibody (R-10G) specific to human induced pluripotent stem (hiPS)/embryonic stem (hES) cells by using hiPS cells (Tic) as an antigen, followed by differential screening of mouse hybridomas with hiPS and human embryonal carcinoma (hEC) cells. Upon western blotting with R-10G, hiPS/ES cell lysates gave a single but an unusually diffuse band at a position corresponding to >250 kDa. The antigen protein was isolated from the induced pluripotent stem (iPS) cell lysates with an affinity column of R-10G. The R-10G positive band was resistant to digestion with peptide N-glycanase F (PNGase F), neuraminidase, fucosidase, chondrotinase ABC and heparinase mix, but it disappeared almost completely on digestion with keratanase, keratanase II and endo-β-galactosidase, indicating that the R-10G epitope is a keratan sulfate. The carrier protein of the R-10G epitope was identified as podocalyxin by liquid chromatography/ mass spectrometry (LC/MS/MS) analysis of the R-10G positive-protein band material obtained on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). The R-10G epitope is a type of keratan sulfate with some unique properties. (1) The epitope is expressed only on hiPS/ES cells, i.e. not on hEC cells, unlike those recognized by the conventional hiPS/ES marker antibodies. (2) The epitope is a type of keratan sulfate lacking oversulfated structures and is not immunologically crossreactive with high-sulfated keratan sulfate. (3) The R-10G epitope is distributed heterogeneously on hiPS cells, suggesting that a single colony of undifferentiated hiPS cells consists of different cell subtypes. Thus, R-10G is a novel antibody recognizing hiPS/ES cells, and should be a new molecular probe for disclosing the roles of glycans on these cells. © The Author 2012.

Hintze J.P.,Saint Louis University | Tomatsu S.,Saint Louis University | Fujii T.,Sapporo IDL | Montano A.M.,Saint Louis University | And 5 more authors.
Biomarker Insights | Year: 2011

Background and aim: Mucopolysaccharidosis IVA (MPS IVA) leads to skeletal dysplasia through excessive storage of chondroitin-6-sulfate and keratan sulfate (KS). KS is synthesized mainly in cartilage and released into circulation, making it a critical biomarker for MPS IVA to evaluate clinical course and effectiveness of therapies. Therefore, an accurate and sensitive method is required to measure KS levels. Material and methods: Using sandwich ELISA and liquid chromatography tandem mass spectrometry (LC/MS/MS) assays, we measured KS levels in blood and urine from MPS IVA patients and healthy controls to evaluate comparability of results. Blood (patients, n = 110; controls, n = 364) and urine (patients, n = 103; controls, n = 326) specimens were obtained. Results: Plasma and urine KS measurements in patients were age-dependent and higher than age-matched controls. We observed a moderate correlation (r = 0.666; P, 0.001) between urine KS measurements and a weak correlation (r = 0.333; P = 0.002) between plasma KS measurements by ELISA and LC/MS/MS methods in patients. No correlation was found between plasma KS measurements in controls. The difference between KS measurements assayed by LC/MS/MS and ELISA was greater in controls than in patients. A moderate correlation between blood and urine KS measurements in the same individual was observed. Conclusion: These findings indicate that both methods to measure blood and urine KS are suitable for diagnosis, monitoring therapies, and longitudinal assessment of the disease course in MPS IVA, but the LC/MS/MS method measures over 10 times more KS present in body fluids. © the author(s), publisher and licensee Libertas Academica Ltd.

Nagaoka I.,Juntendo University | Suzuki K.,Juntendo University | Murakami T.,Juntendo University | Niyonsaba F.,Juntendo University | And 2 more authors.
International Journal of Molecular Medicine | Year: 2010

Peptide antibiotics possess potent antimicrobial activities against invading micro-organisms and contribute to the innate host defense. Antimicrobial α-defensin human neutrophil peptides (HNPs) not only exhibit potent bactericidal activities against Gram-negative and -positive bacteria but also function as immunomodulatory molecules by inducing cytokine and chemokine production, as well as inflammatory and immune cell activation. Neutrophil is a critical effector cell in host defense against microbial infection, and its lifespan is regulated by various pathogen- and host-derived substances. Here, in order to further evaluate the role of HNPs in innate immunity, we investigated the action of HNPs-1 to -3 on neutrophil apoptosis. Neutrophil apoptosis was assessed using human blood neutrophils based on the morphological changes. Of note, HNP-1 most potently suppressed neutrophil apoptosis among HNPs-1 to -3, accompanied by the downregulation of truncated Bid (a pro-apoptotic protein), the upregulation of Bcl-xL (an anti-apoptotic protein), and the inhibition of mitochondrial membrane potential change and caspase 3 activity. It should be noted that, a selective P2Y6 antagonist, MRS2578, abolished the suppression of neutrophil apoptosis elicited by HNP-1 as well as UDP (a P2Y6 ligand). Collectively, these observations suggest that HNPs, especially HNP-1, can not only destroy bacteria but also modulate (suppress) neutrophil apoptosis via the P2Y6 signaling pathway. The suppression of neutrophil apoptosis results in the prolongation of their lifespan and could be advantageous for the host defense against bacterial invasion.

Fujii M.,Aichi Cancer Center Research Institute | Fujii M.,Nagoya University | Fujii M.,Aichi Hospital | Yusa A.,Aichi Cancer Center Research Institute | And 15 more authors.
Glycoconjugate Journal | Year: 2010

The expressions of heparan sulfate glycosaminoglycans (HSGAGs) in breast carcinoma specimens from 60 patients were immunohistochemically investigated using monoclonal antibodies (mAbs) that recognized different epitopes of the glycan structure. Cytoplasmic expression of GlcA-GlcNH 3 + on HSGAG was detected in carcinomas at high frequency (58.3%) using mAb JM403, whereas it was almost undetectable in normal breast ducts. This cytoplasmic expression was confirmed using confocal laser scanning microscopy. The expression of JM403 antigen in invasive carcinomas significantly correlated with nuclear atypia score (p∈=∈0.0004), mitotic counts score (p∈=∈0.0018), nuclear grade (p∈=∈0.0061) and the incidence of metastasis to axillary lymph nodes (p∈=∈0.0061). Furthermore, its expression was significantly correlated with the Ki67-labeling index in 55 invasive carcinomas (p∈<∈0.05) as well as in 26 non-invasive carcinomas (5 non-invasive carcinomas and 21 non-invasive carcinomas that were observed in individual invasive carcinomas) (p∈<∈0.005). Interestingly, the JM403 antigen GlcA-GlcNH 3 + was also expressed in the cytoplasm of normal crypt epithelial cells where Ki67 protein was expressed in the cell nuclei in the proliferative compartment of the human small intestines. To date, HSGAGs have generally been found to exist on cell surface membranes and in extracellular matrices as components of HS proteoglycans, and the negatively-charged sulfated domains on HSGAGs are considered to be important for their functions. However, our present findings indicate that the cytoplasmic expression of the JM403 antigen GlcA-GlcNH 3 + on positively charged, non-sulfated HSGAG may be involved in cell proliferation and associated with increased degrees of malignancy. The unordinary carbohydrate antigen of GlcA-GlcNH 3 + on HSGAGs recognized by mAb JM403 may represent a novel proliferative biomarker for highly malignant mammary carcinomas. © 2010 Springer Science+Business Media, LLC.

Hosoda H.,Juntendo University | Tamura H.,Seikagaku Biobusiness Corporation | Kida S.,Tokyo University of Agriculture | Nagaoka I.,Juntendo University
Life Sciences | Year: 2011

Aims: Triggering receptor expressed on myeloid cells (TREM)-1 is expressed in macrophages, and functions as an amplifying molecule in inflammatory responses. TREM-1 is constitutively expressed in macrophage, and upregulated by bacterial components, such as lipopolysaccharide (LPS). In this present study, we investigated the regulatory mechanism for the basal and LPS-induced transcription of mouse TREM-1 gene in mononuclear cells using RAW264.7 macrophage-like cells. Main methods: To elucidate the potential role of cis-acting elements in the basal and LPS-induced transcription of mouse TREM-1 gene, the luciferase vector containing the promoter with 5′ deletion and adenine substitution mutants was transfected into RAW264.7 cells and incubated in the absence or presence of LPS. To further identify the transcription factor(s), gel shift/supershift analysis was performed. Key findings: The CRE (cAMP response element) and NF-κB-1 (a distal NF-κB site) in the mouse TREM-1 promoter are positively and negatively regulating the basal TREM-1 transcription via the interaction with C/EBPα and NF-κB p50/p50 homodimer, respectively. In addition, the CRE and NF-κB-1 likely participate in the LPS-induced upregulation of TREM-1 promoter activity possibly via the interaction with phosphorylated CREB and NF-κB p65/p50 heterodimer. Furthermore, the AP-1-1 (a distal AP-1 site) is likely to be involved in the LPS-induced TREM-1 transcription via the interaction with phosphorylated c-fos/c-jun. Significance: The present study has demonstrated for the first time the detailed mechanism for the basal and LPS-induced expression of TREM-1, an amplifying molecule in inflammation. © 2011 Elsevier Inc. All Rights Reserved.

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