Inhibition of ACh-stimulated exocytosis by NSAIDs in guinea pig antral mucous cells: autocrine regulation of mucin secretion by PGE2

The effects of indomethacin (IDM) and aspirin (ASA) on ACh (10 microM) -stimulated exocytotic events were studied in guinea pig antral mucous cells by using video optical microscopy. IDM or ASA, which inhibits cyclooxygenase (COX), decreased the frequency of ACh-stimulated exocytotic events by 30% or 60%, respectively. The extent of inhibition induced by ASA (60%) decreased by 30% when IDM or arachidonic acid (AA, the substrate of COX) was added. IDM, unlike ASA, appears to induce the accumulation of AA, which enhances the frequency of ACh-stimulated exocytotic events in ASA-treated cells. ONO-8713 (100 microM; an inhibitor of the EP1-EP4 prostaglandin receptors) and N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, HCl (H-89, 20 microM; an inhibitor of PKA) also decreased the frequency of ACh-stimulated exocytotic events by 60%. However, the supplementation of PGE(2) (1 microM) prevented the IDM-induced decrease in the frequency of ACh-stimulated exocytotic events. SC-560 (an inhibitor of COX-1) decreased the frequency of ACh-stimulated exocytotic events by 30%, but NS-398 (an inhibitor of COX-2) did not. Moreover, IDM decreased the frequency of exocytotic events stimulated by ionomycin, suggesting that COX-1 activity is stimulated by an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)). ACh and ionomycin increased PGE(2) release in antral mucosal cells. In conclusion, in ACh-stimulated antral mucous cells, an increase in [Ca(2+)](i) activates Ca(2+)-regulated exocytotic events and PGE(2) release mediated by COX-1. The released PGE(2) induces the accumulation of cAMP, which enhances the Ca(2+)-regulated exocytosis. The autocrine mechanism mediated by PGE(2) maintains the high-level mucin release from antral mucous cells during ACh stimulation.     

Systemic administration of lipopolysaccharide upregulates angiotensin II expression in rat renal tubules: immunohistochemical and ELISA studies

We investigated whether angiotensin II (AII) peptide is induced in the rat kidney under endotoxemic conditions. Immunohistochemistry revealed strong AII-like immunoreactivity in the renal tubules of rats given high-dose lipopolysaccharide (LPS; 1000 microg/kg) intraperitoneally (i.p.). AII-like immunoreactivity in renal tubules was slight at 1h after the LPS injection, but marked at 3 h. There were few signals in the kidney in saline-injected control rats. When injected at 0.1, 10, or 1000 microg/kg i.p., LPS-induced a dose-related increase in AII-like immunoreactivity in renal tubules that was unaffected by treatment with the prostaglandin-synthesis blocker indomethacin. ELISA measurement of the AII concentration in the whole kidney supported the above findings. These results suggest that systemically administered LPS induces AII peptide expression in renal tubules by a prostaglandin-independent mechanism.     

Effect of adrenomedullin administration on acetic acid-induced colitis in rats

Adrenomedullin (AM) administered intracolonically ameliorated the severity of acetic acid-induced colonic ulceration in rats. Ulcers were induced by subserosal injection of acetic acid into the colon. AM-treated group was administered 0.25–1.0 μg of AM in 0.5 ml of saline intracolonically once a day; the control group received only saline. AM administration dose-dependently and significantly reduced the size of the ulcerative lesions, the associated edema, and the infiltration of the affected area by inflammatory cells. AM also reduced tissue levels of interleukin-6, but not interferon-γ. AM reduces the severity of acetic acid-induced colitis in rats, probably by inhibiting the production and/or release of Th-2 cell-derived factors such as interleukin-6.     

Characterization of bacterium isolated from the sediment at coastal area of Omura Bay in Japan and several biological activities of pigment produced by this isolate

Recently we discovered a bacterial strain (MS-02-063) that produces large amounts of red pigment from coastal area of Nagasaki Prefecture, Japan. Comparative 16S rDNA gene sequencing analysis revealed that strain MS-02-063 was phylogenetically closely related to gamma-proteobacterium Hahella sp. MBIC 3957 that produces prodigiosin. However, some physiological and biochemical differences between strain MS-02-063 and Hahella sp. MBIC 3957 were observed. The red pigment (RP-063) produced by this isolate was highly purified from the culture supernatant. It was speculated that RP-063 might be prodigiosin-like pigment in physical properties and biological activities such as antibacterial and cytotoxic activity. Antibacterial activity of RP-063 was examined by an agar dilution method. The results indicated that RP-063 showed antibacterial activity for specific for pathogenic gram-positive bacteria such as Staphylococcus aureus. The potency of antibacterial activity against S. aureus was nearly equal to those of tetracycline. Moreover, RP-063 showed inhibition of the superoxide generation by 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated mouse macrophage RAW 264.7 cell line. Prodigiosin members have a wide variety of biological properties, including anticancer and antimalarial, etc. Especially, potent immunosuppressive properties have been reported for prodigiosin members with the mechanism of action different from that of the other well known immunosuppressors in atopic dermatitis therapy such as cyclosporin A, FK506 and rapamycin. It is suggested that RP-063 may be able to arrest the inflammation caused by superantigens secreted from S. aureus, which colonized skin on atopic dermatitis as well as suppression of activated lymphocyte proliferation and superoxide generation from leucocytes.     

Biochemical screening of stable dinucleosomes using DNA fragments from a dinucleosome DNA library

The dinucleosome is an informative unit for analysis of the higher-order chromatin structure. DNA fragments forming stable dinucleosomes were screened from a dinucleosome DNA library after the reconstitution of nucleosomes in vitro and digestion with micrococcal nuclease. Reconstituted dinucleosomes showed a diversity of sensitivity to micrococcal nuclease, suggesting that the biochemical stability of a dinucleosome depends, in part, on the DNA fragments. The DNA fragments after the screening were classified into three groups represented by clones bf10, af14 and af32 according to the sensitivity to micrococcal nuclease. Mapping of the nucleosome boundaries by Southern blotting of the DNA after restriction digestion and by primer extension analysis showed that each nucleosome position of clone af32 was fixed. Analysis of reconstituted dinucleosomes using mutant DNA fragments of clone af32 revealed a unique property characteristic of a key nucleosome, given that the replacement of a DNA fragment corresponding to the right nucleosome position resulted in marked sensitivity to micrococcal nuclease, whereas the replacement of the other nucleosome fragment had almost no effect on sensitivity as compared to the original af32 construct. The mutant construct in which the right nucleosome was removed showed multiple nucleosome phases, suggesting that the right nucleosome stabilized first each mononucleosome and then the dinucleosome. An oligonucleotide bending assay revealed that the DNA fragment in the right nucleosome included curved DNA, suggesting that the positioning activity of the nucleosome was attributed to its DNA structure. These results suggest that information for forming stable dinucleosome is embedded in the genomic DNA and that a further characterization of the key nucleosome is useful for understanding the building up of the chromatin structure.     

Mumps virus V protein antagonizes interferon without the complete degradation of STAT1

Mumps virus (MuV) has been shown to antagonize the antiviral effects of interferon (IFN) through proteasome-mediated complete degradation of STAT1 by using the viral V protein (T. Kubota et al., Biochem. Biophys. Res. Commun. 283:255-259, 2001). However, we found that MuV could inhibit IFN signaling and the generation of a subsequent antiviral state long before the complete degradation of cellular STAT1 in infected cells. In MuV-infected cells, nuclear translocation and phosphorylation of STAT1 and STAT2 tyrosine residue (Y) at 701 and 689, respectively, by IFN-beta were significantly inhibited but the phosphorylation of Jak1 and Tyk2 was not inhibited. The transiently expressed MuV V protein also inhibited IFN-beta-induced Y701-STAT1 and Y689-STAT2 phosphorylation, suggesting that the V protein could block IFN-beta-induced signal transduction without the aid of other viral components. Finally, a substitution of an alanine residue in place of a cysteine residue in the C-terminal V-unique region known to be required for STAT1 degradation and inhibition of anti-IFN signaling resulted in the loss of V protein function to inhibit the Y701-STAT1 and Y689-STAT2 phosphorylation.     

Roles of Arabidopsis AtREV1 and AtREV7 in translesion synthesis

Plants have mechanisms for repairing and tolerating detrimental effects by various DNA damaging agents. A tolerance pathway that has been predicted to be present in higher plants is translesion synthesis (TLS), which is catalyzed by polymerases. In Arabidopsis (Arabidopsis thaliana), however, the only gene known to be involved in TLS is the Arabidopsis homolog of REV3, AtREV3, which is a putative catalytic subunit of Arabidopsis DNA polymerase zeta. A disrupted mutant of AtREV3, rev3, was previously found to be highly sensitive to ultraviolet-B (UV-B) and various DNA damaging agents. REV1 and REV7 are thought to be components of translesion synthesis in plants. In this study, we identified the Arabidopsis homologs of REV1 and REV7 (AtREV1 and AtREV7). Several mutants carrying disrupted AtREV1 and AtREV7 genes were isolated from Arabidopsis T-DNA-inserted lines. An AtREV1-disrupted mutant, rev1, was found to be moderately sensitive to UV-B and DNA cross-linkers. A rev1rev3 double mutant, like rev3, showed high sensitivity to UV-B, gamma-rays, and DNA cross-linkers. An AtREV7-disrupted mutant, rev7, was possibly sensitive to cis-diamminedichloroplatinum(II), a kind of DNA cross-linker, but it was not sensitive to acute UV-B and gamma-ray irradiation. On the other hand, the aerial growth of rev7, like the aerial growth of rev1 and rev3, was inhibited by long-term UV-B. These results suggest that a TLS mechanism exists in a higher plant and show that AtREV1 and AtREV7 have important roles in tolerating exposure to DNA-damaging agents.     

Post-translational regulation of CND41 protease activity in senescent tobacco leaves

The degradation of chloroplast proteins is an important occurrence in the mobilization of nutrients from senescing leaves to reproductive organs during senescence. Recently, we proved that tobacco CND41 protease is involved in Rubisco degradation and the translocation of nitrogen during senescence. In this study, we show the post-translational regulation of CND41 protease. Using very specific antibodies that were prepared against CND41-specific peptide (anti-Val 186 to Ser 206), immunoblot analysis clearly indicated a change in the accumulation and processing of CND41 during the maturation of leaves in whole plants. The developmental modification of CND41 was also observed in transgenic tobacco with constitutive expression of CND41 under cauliflower mosaic virus 35S promoter. Further studies of seedlings under senescence induced by combined treatment with nitrogen-starvation and high sucrose confirmed that the processing of CND41 was important for protease activity and senescence. A possible mechanism for the regulation of CND41 activity is discussed.     

Production of infectious hepatitis C virus in cell culture

Hepatitis C virus (HCV) is a major public health problem, infecting an estimated 170 million people worldwide. Current therapy for HCV-related chronic hepatitis is based on the use of interferon. However, virus clearance rates are insufficient. Investigations to develop the anti-viral therapy or to understand the life cycle of this virus have been hampered by the lack of viral culture systems. We isolated the JFH-1 strain from a patient with fulminant hepatitis, and the JFH-1 subgenomic replicon could replicate efficiently in culture cell without adaptive mutation. Recently, we developed the HCV infection system in culture cells with this JFH-1 strain. The full-length JFH-1 RNA was transfected into Huh7 cells. Subsequently, viral RNA efficiently replicated in transfected cells and viral particles were secreted. Furthermore, secreted virus displayed infectivity for naive Huh7 cells. This system provides a powerful tool for studying the viral life cycle and constructing anti-viral strategies.     

Endoplasmic reticulum stress induces the phosphorylation of small heat shock protein, Hsp27

There are several reports describing participation of small heat shock proteins (sHsps) in cellular protein quality control. In this study, we estimated the endoplasmic reticulum (ER) stress-induced response of Hsp27 and alphaB-crystallin in mammalian cells. Treatment targeting the ER with tunicamycin or thapsigargin induced the phosphorylation of Hsp27 but not of alphaB-crystallin in U373 MG cells, increase being observed after 2-10 h and decline at 24 h. Similar phosphorylation of Hsp27 by ER stress was also observed with U251 MG and HeLa but not in COS cells and could be blocked using SB203580, an inhibitor of p38 MAP kinase. Other protein kinase inhibitors, like G?6983, PD98059, and SP600125, inhibitors of protein kinase C (PKC), p44/42 MAP kinase, and JNK, respectively, were without major influence. Prolonged treatment with tunicamycin but not thapsigargin for 48 h caused the second induction of the phosphorylation of Hsp27 in U251 MG cells. Under these conditions, the intense perinuclear staining of Hsp27, with some features of aggresomes, was observed in 10%-20% of the cells.