2-aminopurine inhibits lipopolysaccharide-induced nitric oxide production by preventing IFN-beta production

2-aminopurine (2-AP) is widely used as a specific inhibitor for double stranded-RNA dependent protein kinase (PKR). Here we report that 2-AP can inhibit lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production through the prevention of interferon (IFN)-beta production. 2-AP significantly inhibited NO production in LPS-stimulated RAW 264 murine macrophage cells. 2-AP also reduced the expression of IFN-beta and IFN-inducible genes, such as IFN-gamma-inducible protein (IP)-10 and immune-responsive gene (IRG)-1, and the inducible type of NO synthase (iNOS) mRNA in response to LPS. The addition of exogenous IFN-beta restored 2-AP-inhibited NO production in response to LPS. On the other hand, there was only partial inhibition by 2-AP of nuclear factor (NF)-kappaB activation, IL-6 mRNA expression and tumor necrosis factor (TNF)-alpha production. These results suggested that 2-AP inhibited LPS-induced IFN-beta production by preventing Toll/IL-1 receptor domain-containing adaptor-inducing IFN-beta (TRIF)-dependent signaling rather than myeloid differentiation factor (MyD) 88-dependent signaling, resulting in the inhibition of NO production.     

Infection of different cell lines of neural origin with subacute sclerosing panencephalitis (SSPE) virus

Measles virus is the causative agent of subacute sclerosing panencephalitis (SSPE). The viruses isolated from brain cells of patients with SSPE (called SSPE viruses) are defective in cell-free virus production in vitro. To investigate the cell tropism of three strains of SSPE virus (Osaka-1, Osaka-2, Osaka-3), SSPE virus-infected cell cultures were treated with cytochalasin D to prepare virus-like particles (CD-VLPs). All CD-VLPs formed syncytia after infection in CHO cells expressing CD150 but not in those expressing CD46. In addition, an antibody to CD46 did not block the infection of Vero cells by SSPE CDVLPs. The results were consistent with our previous suggestion that one or more unidentified receptors might be involved in the entry process. Infection with the CD-VLPs from three SSPE strains was further examined in different human cell lines, including those of neural origin, and was found to induce syncytia in epithelial cells (HeLa and 293T) as well as neuroblastoma cells (IMR-32 and SK-N-SH) with varying efficiency. SSPE CD-VLPs also infected glioblastoma cells (A172) and astrocytoma cells (U-251) but syncytial formation was rarely induced. These epithelial and neural cell lines were not permissive for the replication of wild-type MV. Together with our previous observations, these results suggest that the cell entry receptor is the major factor determining the cell tropism of SSPE viruses. Further studies are necessary to identify other viral and/or cellular factors that might be involved in the replication of SSPE virus in specific neural cells and in the brain.     

Role of p38 mitogen-activated protein kinase (MAPK) for vacuole formation in lipopolysaccharide (LPS)-stimulated macrophages

The role of p38 mitogen-activated protein kinase (MAPK) on vacuole formation in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells was examined. LPS definitely induced the formation of vacuoles in RAW 264.7 cells and SB202190 as a p38 specific inhibitor also induced slight vacuole formation. The simultaneous treatment with LPS and SB202190 induced many more vacuoles in RAW 264.7 cells than the treatment with LPS or SB202190 alone, and the vacuoles were extraordinarily large in size. On the other hand, an inactive inhibitor of p38 MAPK did not augment LPS-induced vacuole formation. Further, the inhibitors of other MAPKs and nuclear factor (NF)-kappaB pathways did not affect it. The extraordinarily large vacuoles in RAW 264.7 cells treated with LPS and SB202190 were possibly formed via fusion of small vacuoles. However, SB202190 did not augment vacuole formation in CpG DNA or interferon (IFN)-gamma-stimulated RAW 264.7 cells. The role of p38 MAPK in the vacuole formation in LPS-stimulated macrophages is discussed.     

Establishment and characterization of a human gestational choriocarcinoma cell line (HOCC)

Human gestational choriocarcinoma cell line (HOCC) was established from the mouse graft of choriocarcinoma. The HOCC cells were spindle or polygonal in shape and multi-nucleated giant cells, showing neoplastic and pleomorphic features. The cell proliferated stably, and the population doubling time was about 32 hours. The chromosome numbers showed a wide distribution of aneuploidy, the mode was in hypertriploid range and the marker chromosomes were recognized in the several generations. Heterotransplantation was easy, and subcutaneous transplantation of 1 X 10(7) cells in nude mouse formed a tumor composed of choriocarcinoma. It is most noteworthy characteristic of the cell line that it produced human chorionic gonadotropin (hCG) in an in vitro culture system and in vivo in nude mice.     

Quantifying determinants of ozone detoxification by apoplastic ascorbate in peach (Prunus persica) leaves using a model of ozone transport and reaction

Ascorbate in leaf apoplast (ASC_apo) reacts with ozone (O₃) and thereby reduces O₃ flux reaching plasmalemma (F_pl). Some studies have shown significant protection of cells from O₃ by ASC_apo, while others have questioned its efficacy. Hypothesizing that the protection by ASC_apo depends on other variables, we quantified determinants of O₃ detoxification with a model of O₃ transport and reaction in apoplast. The model determines ascorbic acid concentration in apoplast (AA_apo) using measured values of O₃ concentration (c_o), leaf tissue ascorbic acid concentration (AA_leaf), cell wall thickness (L₃), apoplastic pH (pH_apo), and stomatal conductance (G_sw). We compared the measured and model‐estimated AA_apo in leaves of peach (Prunus persica) grown in open‐top chambers under non‐filtered air (NF) and elevated (EO₃: NF + 80 ppb) O₃ concentrations. The estimated AA_apo in individual leaves agreed well with the measured values (R² = .91). Analyses of the simulation results yielded the following findings: (a) The efficacy of O₃ reduction with ASC_apo as quantified by fractional reduction (?₃) of O₃ flux at the surface of plasmalemma (F_pl) was lowered from 70% in NF to 40% in EO₃ due to the reduction of L₃. The EO₃ reduced AA_apo, but the lower G_sw and L₃ in EO₃ increased AA_apo resulting in no significant change in AA_apo due to EO₃. ?₃ can be calculated with measured values of AA_apo and L₃, and F_pl can be estimated with the measurement‐based ?₃. (b) When c₀ is increased, F_pl increased curvilinearly with the increase of F_st: nominal O₃ flux via stomatal diffusion, exhibiting apparent threshold on F_st. The deviation of F_pl from F_st became greater when L₃, pH_apo, and AA_leaf were increased. The quantification of ?₃ and F_pl using leaf traits shall facilitate the understanding of the mechanisms of differential plant sensitivity to O₃ and improve quantification of the O₃ impacts on plants.     

A role of heparin-binding epidermal growth factor-like growth factor in cardiac remodeling after myocardial infarction

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is known to induce cell growth in various cell types via transactivation of epidermal growth factor receptor (EGFR). To investigate the involvement of HB-EGF and EGFR in cardiac remodeling after myocardial infarction (MI), we examined the expressions of mRNA and protein in rat hearts 6 weeks after MI-induction. Where increased expressions of HB-EGF mRNA and protein were observed, infarcted myocardium was replaced by extracellular matrix and interstitial fibroblasts. EGFR mRNA and protein expression did not show significant changes in sham-operated heart tissues, non-infarcted region, and infarcted region. In vitro study demonstrated that HB-EGF mRNA was expressed mainly in cultured fibroblasts rather than in myocytes. We suggest that the interaction between HB-EGF and EGFR transactivation is closely related to the proliferation of cardiac fibroblasts and cardiac remodeling after MI in an autocrine, paracrine, and juxtacrine manner.     

Derivation and induction of the differentiation of animal ES cells as well as human pluripotent stem cells derived from fetal membrane

We succeeded in the derivation and maintenance of pluripotent embryonic stem (ES) cells from equine and bovine blastocysts. These cells expressed markers that are characteristics of mouse ES cells, namely, alkaline phosphatase, stage-specific embryonic antigen 1, STAT 3 and Oct 4. We confirmed the pluripotential ability of these cells, which were able to undergo somatic differentiation in vitro to neural progenitors and to endothelial or hematopoietic lineages. We were able to use bovine ES cells as a source of nuclei for nuclear transfer and we generated cloned cattle with a higher frequency of pregnancies to term than has been achieved with somatic cells. On the other hand, we established human fetal membrane derived stem cell lines by the colonial cloning techniques using MEMalpha culture medium containing 10 ng/ml of EGF, 10 ng/ml of LIF and 10% fetal bovine serum (FBS). These cells appeared to maintain normal karyotype in vitro and expressed markers characteristics of stem cells. Furthermore, these cells contributed to the formation of chimeric murine embryoid bodies and gave rise to all three germ layers in vitro. Results from animal ES cells and human fetal membrane derived stem cells clearly demonstrate that these cells might be used for providing different types of cells for regenerative medicine as well as used for targeted genetic manipulation of the genome.     

Involvement of Ca/calmodulin-dependent protein kinases in mycelial growth of the basidiomycetous mushroom, Coprinus cinereus

Although multifunctional Ca²⁺/calmodulin-dependent protein kinases (CaM-kinases) are widely distributed in animal cells, the occurrence of CaM-kinases in the basidiomycetous mushroom has not previously been documented. When the extracts from various developmental stages from mycelia to the mature fruiting body of Coprinus cinereus were analyzed by Western blotting using Multi-PK antibodies, which had been generated to detect a wide variety of protein serine/threonine kinases (Ser/Thr kinases), a variety of stage-specific Ser/Thr kinases was detected. Calmodulin (CaM) overlay assay using digoxigenin-labeled CaM detected protein bands of 65 kDa, 58 kDa, 46 kDa, 42 kDa, and 38 kDa only in the presence of CaCl₂, suggesting that these bands were CaM-binding proteins. When the CaM-binding fraction was prepared from mycelial extract of C. cinereus by CaM-Sepharose and analyzed with Multi-PK antibodies, two major immunoreactive bands corresponding to 65 kDa and 46 kDa were detected. CaM-binding fraction, thus obtained, exhibited Ca²⁺/CaM-dependent protein kinase activity toward protein substrates such as histones. These CaM-kinases were found to be highly expressed in the actively growing mycelia, but not in the resting mycelial cells. Mycelial growth was enhanced by the addition of CaCl₂ in the culture media, but inhibited by the addition of EGTA or trifluoperazine, a potent CaM inhibitor. This suggested that CaM-dependent enzymes including CaM-kinases play crucial roles in mycelial growth of basidiomycete C. cinereus.     

Molecular cloning and expression of the gene encoding family 19 chitinase from Streptomyces sp. J-13-3

The gene encoding chitinase from Streptomyces sp. (strain J-13-3) was cloned and its nucleotide structure was analyzed. The chitinase consisted of 298 amino acids containing a signal peptides (29 amino acids) and a mature protein (269 amino acids), and had calculated molecular mass of 31,081 Da. The calculated molecular mass (28,229 Da) of the mature protein was almost same as that of the native chitinase determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometer. Comparison of the encoded amino acid sequences with those of other chitinases showed that J-13-3 chitinase was a member of the glycosyl-hydrolase family 19 chitinases and the mature protein had a chitin binding domain (65 amino acids) containing AKWWTQ motif and a catalytic domain (204 amino acids). The J-13-3 strain had a single chitinase gene. The chitinase (298 amino acids) with C-terminal His tag was overexpressed in Escherichia coli BL21(DE3) cells. The recombinant chitinase purified from the cell extract had identical N-terminal amino acid sequence of the mature protein in spite of confirmation of the nucleotide sequence, suggesting that the signal peptide sequence is successfully cut off at the predicted site by signal peptidase from E. coli and will be a useful genetic tool in protein engineering for production of soluble recombinant protein. The optimum temperature and pH ranges of the purified chitinase were at 35-40 degrees C and 5.5-6.0, respectively. The purified chitinase hydrolyzed colloidal chitin and trimer to hexamer of N-acetylglucosamine and also inhibited the hyphal extension of Tricoderma reesei.