Involvement of group VI Ca2+-independent phospholipase A2 in protein kinase C-dependent arachidonic acid liberation in zymosan-stimulated macrophage-like P388D1 cells.

We investigated the possible involvement of group VI Ca2+-independent phospholipase A2 (iPLA2) in arachidonic acid (AA) liberation in zymosan-stimulated macrophage-like P388D1 cells. Zymosan-induced AA liberation was markedly inhibited by methyl arachidonoyl fluorophosphonate, a dual inhibitor of group IV cytosolic phospholipase A2 (cPLA2) and iPLA2. We found that a relatively specific iPLA2 inhibitor, bromoenol lactone, significantly decreased the zymosan-induced AA liberation in parallel with the decrease in iPLA2 activity, without an effect on diacylglycerol formation. Consistent with this, attenuation of iPLA2 activity by a group VI iPLA2 antisense oligonucleotide resulted in a decrease in zymosan-induced prostaglandin D2 generation. These findings suggest that zymosan-induced AA liberation may be, at least in part, mediated by iPLA2. A protein kinase C (PKC) inhibitor diminished zymosan-induced AA liberation, while a PKC activator, phorbol 12-myristate 13-acetate (PMA), enhanced the liberation. Bromoenol lactone suppressed the PMA-enhanced AA liberation without any effect on PMA-induced PKC activation. Down-regulation of PKCalpha on prolonged exposure to PMA also decreased zymosan-induced AA liberation. Under these conditions, the remaining AA liberation was insensitive to bromoenol lactone. Furthermore, the PKC depletion suppressed increases in iPLA2 proteins and the activity in the membrane fraction of zymosan-stimulated cells. In contrast, the zymosan-induced increases in iPLA2 proteins and the activity in the fraction were facilitated by simultaneous addition of PMA. Although intracellular Ca2+ depletion prevented zymosan-induced AA liberation, the translocation of PKCalpha to membranes was also inhibited. Taken together, we propose that zymosan may stimulate iPLA2-mediated AA liberation, probably through a PKC-dependent mechanism.     

Administration of fibroblast growth factor 2 in combination with bone marrow transplantation synergistically improves carbon-tetrachloride-induced liver fibrosis in mice

We previously reported that fibroblast growth factor 2 (FGF2) facilitated the differentiation of transplanted bone marrow cells (BMCs) into hepatocytes. Our earlier study also demonstrated that administration of FGF2 in combination with bone marrow transplantation (BMT) synergistically activated tumor necrosis factor-alpha signaling and significantly improved liver function and prognosis more than BMT alone. However, the way that it affected the extracellular matrix remained unclear. Here, we investigated the effect of FGF2 treatment together with BMT on liver fibrosis in mice treated with carbon tetrachloride (CCl₄). Transplantation of BMCs and concurrent treatment with FGF2 caused a statistically significant reduction in CCl₄-induced liver fibrosis that was accompanied by strong expression of matrix metalloproteinase 9 as compared with FGF2-only treatment or BMT alone. Moreover, in this process, the proliferation of bone-marrow-derived cells was accelerated without causing apoptosis. Thus, the administration of FGF2 in combination with BMT synergistically improves CCl₄-induced liver fibrosis in mice. This treatment has the potential of being an effective therapy for patients with liver cirrhosis. This study was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (nos. 16390211 and 16590597) and for translational research from the Ministry of Health, Labor and Welfare (H-trans-5 and H17-Special-015).     

Function of Mannan Chains of Yeast Repressible Acid Phosphatase on Its Enzymatic Properties

To find the function of the mannan chains covalently attached to yeast repressible acid phosphatase, the N-glycosidic carbohydrate chains were removed by endo-β-N-acetyl-glucosaminidase H under native conditions. Almost all of the N-glycosidic mannan chains were cleaved off by the glycosidase. The deglycosylated enzyme was shown to be a dimer structure as is the native enzyme. The deglycosylated enzyme retained enzyme activity, the same Km, and the same circular dichroism spectra as the native enzyme. These results indicate that the carbohydrate chains are not essential for maintaining the active enzyme structure, but the deglycosylated enzyme was shown to be more sensitive to acidic pH and high temperature.     

Geographical variation in the early regeneration process of Siebold's Beech (Fagus crenata BLUME) in Japan

The causes and timing of seed death in early regeneration process of Siebold's beech (Fagus crenata Blume) was studied at 15 sites along a snowfall gradient in Japan, in order to clarify why the seedling density of the species has geographic difference remarkably. Seed production did not significantly differ along the snowfall gradient. Pre-dispersal seed mortality by insect damage was higher at sites with light snowfall than at sites with heavy snowfall, but this only seemed to be a minor factor influencing the population. A large proportion of the viable nuts that fall in autumn ware killed in winter before germination. Winter mortality was much higher at sites with thin snow cover than that at sites with thick snow cover, and this factor was strongly correlated with the geographic variation of seedling regeneration probability. There was little seed mortality by winter desiccation. The main factor contributing to the geographic difference seemed to be a seed predation by rodents in winter. Deep snow cover may reduce the success of rodents finding seeds in winter. Thus the observed relationship between snowpack depth and early mortality may be due to an indirect effect through the process of seed predation.p>     

Thyroid hormone binding protein contains glycosylation site binding protein activity

Several lines of evidence provided by other workers indicate that within the same species thyroid hormone binding protein, the beta-subunit of prolyl hydroxylase, and protein disulfide isomerase are the same protein. We sought to determine if glycosylation site binding protein, a lumenal protein of the endoplasmic reticulum, also has the same primary structure. To accomplish this the level of glycosylation site binding protein (GSBP) activity, measured by photolabeling with a glycosylation site peptide probe, was carried out in preparations of 3T3 cells and in E. coli transformed with human thyroid hormone binding protein cDNA. The results strongly support the idea that GSBP is identical to these other lumenal proteins of the endoplasmic reticulum.     

Relationship of large and small invertases in Saccharomyces: mutant selectively deficient in small invertase.

A mutant strain of Saccharomyces cerevisiae (D10-ER1) has been isolated after a two-step mutagenesis of strain 4059-358D (SUC 1) using ethyl methane sulfonate. Cells of this new strain produced a level of total invertase equaling that of 4059 but contained only trace amounts of the small, internal, aglycan form of the enzyme (less than 0.1% of total in D10-ER1 compared with 6% in 4059). When D10.ER1 was crossed with an invertase-hyperproducing strain dgr3 (SUC3), progeny were isolated (HZ400-5A and HZ400-2C) in which levels of total invertase had at least quadrupled. The percentage of small invertase, however, remained insignificant. Levels of small invertase in strain HZ400-5A were determined by affinity chromatography on conconavalin A-Sepharose, gel permeation chromatography, and isopycnic centrifugation in CsCl. The large invertase of the SUC1 yeasts described here was found to contain a form apparently greater in size than the large invertase of the SUC2 strain FH4C; this probably reflects a higher content of carbohydrate. The overall results of this study do not support a direct structural relationship between large and small invertases. The implications on invertase biosynthesis and structure are discussed.     

Separation of Soybean Sterols by Florisil Chromatography and Characterization of Acylated Steryl Glucoside

Florisil column chromatography was demonstrated to be effective in differentiation between different forms of sterols. Sterols of ground soybeans are in four forms, free, ester, and free and acylated glucosides, as analyzed on acetone extracts. In soybean oil foots, steryl ester is present in negligibly small amount. The acylated steryl glucosides were isolated from oil foots in a crystalline state. A chemical structure, steryl 6-acyl d-glucoside, was assigned to the compound, and its probable identity with the glucosides reported by Lepage is discussed. The acylated glucoside preparation was shown to be heterogeneous in composition, carrying palmitic, stearic, oleic, linoleic and linolenic acids as the main acyl moieties and campesterol, stigmasterol and β-sitosterol as steryl moieties. The presence of the three sterols is common to three other forms of sterols.     

Regulation of Acid Phosphatase Synthesis in Baker’s Yeast Protoplast by Phosphate Compounds

The regulation of acid phosphatase synthesis by various phosphate compounds was examined in Baker’s yeast protoplasts. Synthesis was repressed by inorganic phosphate and phosphomonoesters. Phosphomonoesters were hydrolysed by a small amount of non-specific acid phosphatase present in the protoplast membrane. The inorganic phosphate that was liberated and incorporated into protoplasts probably repressed acid phosphatase synthesis. Phosphodiesters, such as 3′, 5′-cyclic AMP, 3′, 5′-cyclic CMP and 3′, 5′-cyclic GMP, promoted acid phosphatase synthesis. The effect of 3′, 5′-cyclic AMP was not to overcome hexose repression, because high hexose did not repress acid phosphatase synthesis. 3′, 5′-cyclic AMP did not overcome repression of the enzyme synthesis by inorganic phosphate. From these observations 3′, 5′-cyclic nucleotides probably had some effect on the yeast acid phosphatase-synthesizing system but the exact role of the nucleotides is obscure.     

Within-branch heterogeneity of the light environment and leaf temperature in a Fagus crenata crown and its significance for photosynthesis calculations

On days with clear skies in late August 2002 diurnal changes in the within-branch heterogeneity of photosynthetic photon flux density at the leaf surface (PPFD_s) and leaf temperature (T _leaf) were measured at natural leaf orientations in the upper and lower layers of a Fagus crenata crown. The PPFD_s and T _leaf measurements were converted to branch photosynthesis rates (P _B; μmol s⁻¹) using a photosynthetic model proposed by Farquhar et al. (Planta 149:78–90, 1980), an empirical stomatal conductance model suggested by Leuning et al. (Plant Cell Environ 18:339–335, 1995), and the total leaf area of the branches. To evaluate the importance of the variation in PPFD_s and T _leaf on photosynthesis calculations, P _B calculated with the observed variation in PPFD_s and T _leaf was compared with estimates, based on the average (variation-free) values of PPFD_s and T _leaf, respectively. In both the layers, daily total P _B values obtained with T _leaf averaging were very close to those obtained with no averaging because of the weak inflection of the net photosynthesis rate (P _n) to T _leaf curves in the observed T _leaf ranges (24.4–36.5 and 21.9–29.1°C in the upper and lower layers, respectively) and relatively small variation in within-branch T _leaf at each time of day. This finding applied across potential climate conditions on fine days in August (T _leaf range of 19.4–41.5 and 16.9–34.1°C in the upper and lower layers, respectively) and when the spatial scale was increased from branch to leaf layer, which increased the maximum variation in within-branch T _leaf from 7.8 to 9.5°C and 4.5 to 5.5°C in the upper and lower layers, respectively. In contrast, averaging PPFD_s caused 25–50% and 41–90% overestimation of daily total P _B in the upper and lower layers, respectively, due to the sharp curvature in the PPFD_s response curve to P _n, and relatively large variation in within-branch PPFD_s. Further, it led to overestimation of midday depression of P _B in the upper layer, possibly because branch structural acclimation to incident light was neglected. Our results indicate that averaged values of T _leaf could be used for the estimation of carbon gain at layer scale throughout August, but spatial variations in PPFD_s need to be considered in detail for reliable estimates of carbon gain.     

Continuous high expression of XBP1 and GRP78 is important for the survival of bone marrow cells in CCl4-treated cirrhotic liver

We have previously shown that infusion of bone marrow cells (BMC) improves CCl₄-induced cirrhosis. However, it is unclear why the injected BMC are resistant to CCl₄ damage and subsequently improve the local microenvironment in damaged liver. To analyze the cellular phenomena involved in this process, we studied the damaged liver using electron microscopy. We found that CCl₄ caused rough endoplasmic reticula to swell in hepatocytes. To analyze the gene expression patterns associated with this process, we conducted PCR-selected suppressive subtractive hybridization. We found that expression levels of HSP84, HSP40, and XBP1 differed markedly between control liver and liver infused with BMC. Immunohistochemical staining revealed that expression levels of HSP84 and HSP40 were markedly higher in the early phase of differentiation immediately after BMC infusion, but decreased over time. XBP1 expression remained high during the late phase, and GRP78 expression increased with XBP1 activation. We also found that GFP-positive BMC expressed XBP1 and GRP78. XBP1 and GRP78 are associated with ER stress. Thus, continuous high XBP1 and GRP78 expression might be essential for the survival and proliferation of BMC in a CCl₄-induced persistent liver damage environment.