Sphingosine kinase 2 inhibitor SG-12 induces apoptosis via phosphorylation by sphingosine kinase 2

Sphingosine kinase (SPHK), which catalyzes the phosphorylation of sphingosine to generate sphingosine 1-phosphate, has two mammalian isotypes, SPHK1 and SPHK2. Both isozymes are promising anti-cancer therapeutic targets. In this report, we found that SG-12, a synthetic analogue of sphingosine that acts as a SPHK2 inhibitor, induces apoptosis via phosphorylation by SPHK2. The present results revealed the novel anti-cancer potential of a sphingosine analogue in the pathological setting where SPHK2 is upregulated.     

Identification and migration of primordial germ cells in Atlantic salmon,Salmo salar: Characterization of Vasa,Dead End,and Lymphocyte antigen 75 genes

No information exists on the identification of primordial germ cells (PGCs) in the super‐order Protacanthopterygii, which includes the Salmonidae family and Atlantic salmon (Salmo salar L.), one of the most commercially important aquatic animals worldwide. In order to identify salmon PGCs, we cloned the full‐length cDNA of vasa, dead end (dnd), and lymphocyte antigen 75 (ly75/CD205) genes as germ cell marker candidates, and analyzed their expression patterns in both adult and embryonic stages of Atlantic salmon. Semi‐quantitative RT‐PCR results showed that salmon vasa and dnd were specifically expressed in testis and ovary, and vasa, dnd, and ly75 mRNA were maternally deposited in the egg. vasa mRNA was consistently detected throughout embryogenesis while dnd and ly75 mRNA were gradually degraded during cleavages. In situ analysis revealed the localization of vasa and dnd mRNA and Ly75 protein in PGCs of hatched larvae. Whole‐mount in situ hybridization detected vasa mRNA during embryogenesis, showing a distribution pattern somewhat different to that of zebrafish; specifically, at mid‐blastula stage, vasa‐expressing cells were randomly distributed at the central part of blastodisc, and then they migrated to the presumptive region of embryonic shield. Therefore, the typical vasa localization pattern of four clusters during blastulation, as found in zebrafish, was not present in Atlantic salmon. In addition, salmon PGCs could be specifically labeled with a green fluorescence protein (GFP) using gfp‐rt‐vasa 3′‐UTR RNA microinjection for further applications. These findings may assist in understanding PGC development not only in Atlantic salmon but also in other salmonids. Mol. Reprod. Dev. © 2013 Wiley Periodicals, Inc.     

An Efficient Method for the Synthesis of [4–15N]Cytidine, 2′-Deoxy[4–15N]Cytidine, [6–15N]adenosine,and 2′-Deoxy [6–15N]adenosine Derivatives

Abstract

Nucleophilic substitution reactions of 4-azolyl-1 β-P-D-ribofuranosylpyrimidin-2(1H)-one and 6-azolyl-9-β-D-ribofuranosyl-9H-purine derivatives, which were converted from uridine and inosine, with [¹⁵N]phthalimide in the presence of triethylamine or DBU gave N ⁴-phthaloyl[4-¹⁵N]cytidine and N ⁶-phthaloyl[6-¹⁵N]- adenosine derivatives, respectively, in high yields. Similar reactions of those azolyl derivatives with succinimide afforded N ⁴-succinylcytidine and N ⁶-succinyladenosine derivatives in high yields. The corresponding 2′-deoxyribonucleosides were also synthesized efficiently through the same procedure.

     

Specific Targeting of a Naturally Presented Osteosarcoma Antigen,Papillomavirus Binding Factor Peptide,Using an Artificial Monoclonal Antibody

Osteosarcoma is a rare but highly malignant tumor occurring most frequently in adolescents. The prognosis of non-responders to chemotherapy is still poor, and new treatment modalities are needed. To develop peptide-based immunotherapy, we previously identified autologous cytotoxic T lymphocyte-defined osteosarcoma antigen papillomavirus binding factor (PBF) in the context of HLA-B55 and the cytotoxic T lymphocyte epitope (PBF A2.2) presented by HLA-A2. PBF and HLA class I are expressed in ∼90 and 70% of various sarcomas, respectively. However, the expression status of peptide PBF A2.2 presented by HLA-A2 on osteosarcoma cells has remained unknown because it is difficult to generate a specific probe that reacts with the HLA·peptide complex. For detection and qualification of the HLA-A*02:01·PBF A2.2 peptide complex on osteosarcoma cells, we tried to isolate a single chain variable fragment (scFv) antibody directed to the HLA-*A0201·PBF A2.2 complex using a naïve scFv phage display library. As a result, scFv clone D12 with high affinity (K_D = 1.53 × 10⁻⁹ m) was isolated. D12 could react with PBF A2.2 peptide-pulsed T2 cells and HLA-A2+PBF+ osteosarcoma cell lines and simultaneously demonstrated that the HLA·peptide complex was expressed on osteosarcoma cells. In conclusion, scFv clone D12 might be useful to select candidate patients for PBF A2.2 peptide-based immunotherapy and develop antibody-based immunotherapy.     

Pirfenidone inhibits fibrocyte accumulation in the lungs in bleomycin-induced murine pulmonary fibrosis

Background

Bone marrow-derived fibrocytes reportedly play important roles in the pathogenesis of idiopathic pulmonary fibrosis. Pirfenidone is an anti-fibrotic agent; however, its effects on fibrocytes have not been investigated. The aim of this study was to investigate whether pirfenidone inhibits fibrocyte pool size in the lungs of bleomycin-treated mice.

Methods

Bleomycin (100 mg/kg) was infused with osmotic pumps into C57BL/6 mice, and pirfenidone (300 mg/kg/day) was orally administered daily for 2 wk. The lungs were removed, and single-cell suspensions were subjected to fluorescence-activated cell sorter (FACS) analysis to detect fibrocytes, which were defined as CD45 and collagen-I double-positive cells. Immunohistochemistry was performed on the lung specimens to quantify fibrocytes. Chemokines in the lung digests were measured with enzyme-linked immunosorbent assay. The effect of pirfenidone on alveolar macrophages was evaluated with bronchoalveolar lavage (BAL). In a therapeutic setting, pirfenidone administration was initiated 10 days after bleomycin treatment. For chemotaxis assay, lung fibrocytes were isolated with immunomagnetic selection (CD45-positive mesenchymal cells) after culture and allowed to migrate toward chemokines in the presence or absence of pirfenidone. Moreover, the effect of pirfenidone on the expression of chemokine receptors on fibrocytes was evaluated.

Results

Pirfenidone significantly ameliorated bleomycin-induced pulmonary fibrosis as assessed with quantitative histology and collagen measurement. Fibrocyte pool size in bleomycin-treated mice lungs was attenuated from 26.5% to 13.7% by pirfenidone on FACS analysis. This outcome was also observed in a therapeutic setting. Immunohistochemistry revealed that fibrocytes were significantly decreased by pirfenidone administration compared with those in bleomycin-treated mice (P = 0.0097). Increased chemokine (CC motif) ligand-2 (CCL2) and CCL12 production in bleomycin-treated mouse lungs was significantly attenuated by pirfenidone (P = 0.0003 and P < 0.0001, respectively). Pirfenidone also attenuated macrophage counts stimulated by bleomycin in BAL fluid. Fibrocyte migration toward CCL2 and chemokine (CC motif) receptor-2 expression on fibrocytes was significantly inhibited by pirfenidone in vitro.

Conclusions

Pirfenidone attenuated the fibrocyte pool size in bleomycin-treated mouse lungs via attenuation of CCL2 and CCL12 production in vivo, and fibrocyte migration was inhibited by pirfenidone in vitro. Fibrocyte inhibition is considered a mechanism of anti-fibrotic action of pirfenidone.     

Measurement of vitamin K in human liver by gradient elution high-performance liquid chromatography using platinum-black catalyst reduction and fluorimetric detection

A sensitive and precise method for measuring endogenous phylloquinone (K₁) and menaquinone (MK-n) in human liver was developed, based on gradient elution high-performance liquid chromatography using platinum-black catalyst reduction and fluorimetric detection. Subnanogram levels of vitamin K compounds in 1 g of liver specimen were detectable. We measured vitamin K concentrations in 38 human resected livers. K₁ and MK-4 to MK-13 were detected. The concentrations of MK-10 to MK-12 in livers with chronic hepatitis (n=10) and cirrhosis (n=22) were significantly lower than in normal livers (n=6). It is suggested that the decreased concentrations indicate functional damage of the hepatocytes.     

Myosin IIA regulates cell motility and actomyosin-microtubule crosstalk

Non-muscle myosin II has diverse functions in cell contractility, cytokinesis and locomotion, but the specific contributions of its different isoforms have yet to be clarified. Here, we report that ablation of the myosin IIA isoform results in pronounced defects in cellular contractility, focal adhesions, actin stress fibre organization and tail retraction. Nevertheless, myosin IIA-deficient cells display substantially increased cell migration and exaggerated membrane ruffling, which was dependent on the small G-protein Rac1, its activator Tiam1 and the microtubule moter kinesin Eg5. Myosin IIA deficiency stabilized microtubules, shifting the balance between actomyosin and microtubules with increased microtubules in active membrane ruffles. When microtubule polymerization was suppressed, myosin IIB could partially compensate for the absence of the IIA isoform in cellular contractility, but not in cell migration. We conclude that myosin IIA negatively regulates cell migration and suggest that it maintains a balance between the actomyosin and microtubule systems by regulating microtubule dynamics.     

The alternative pathway of complement activation is critical for blister induction in experimental epidermolysis bullosa acquisita

Epidermolysis bullosa acquisita is a subepidermal blistering disease associated with tissue-bound and circulating autoantibodies against type VII collagen, a major constituent of the dermal-epidermal junction. The passive transfer of Abs against type VII collagen into mice induces a subepidermal blistering disease dependent upon activation of terminal complement components. To further dissect the role of the different complement activation pathways in this model, we injected C1q-deficient, mannan-binding lectin-deficient, and factor B-deficient mice with rabbit Abs against murine type VII collagen. The development and evolution of blistering had a similar pattern in mannan-binding lectin-deficient and control mice and was initially only marginally less extensive in C1q-deficient mice compared with controls. Importantly, factor B-deficient mice developed a delayed and significantly less severe blistering disease compared with factor B-sufficient mice. A significantly lower neutrophilic infiltration was observed in factor B-deficient mice compared with controls and local reconstitution with granulocytes restored the blistering disease in factor B-deficient mice. Our study provides the first direct evidence for the involvement of the alternative pathway in an autoantibody-induced blistering disease and should facilitate the development of new therapeutic strategies for epidermolysis bullosa acquisita and related autoimmune diseases.