Nicotine suppresses tunicamycin-induced, but not thapsigargin-induced, expression of GRP78 during ER stress-mediated apoptosis in PC12 cells

We previously reported that nicotine protected against tunicamycin (Tm)-induced ER stress-mediated apoptosis, but not thapsigargin (Tg)-induced apoptosis in PC12 cells. In the present study, we report that the expression of glucose-regulated protein 78 (GRP78) was suppressed by nicotine in Tm-treated PC12 cells. Interestingly, the GRP78 expression was not changed by nicotine in Tg-treated cells. Moreover, nicotine reduced the activation of caspase-12 in Tm-treated cells, but not in Tg-treated cells. These results suggest that nicotine prevented Tm-induced ER stress-mediated apoptosis by attenuating an early stage of Tm-induced ER stress. It was possible that the suppression of GRP78 expression by nicotine was achieved through the suppression of the Ire1-XBP1 and/or ATF6 pathways. We observed that nicotine suppressed the Tm-induced, but not Tg-induced, splicing of XBP1 mRNA, and also suppressed the Tm-induced, but not Tg-induced, production of cleaved ATF6 in PC12 cells. These results indicate that the suppression of Ire1-XBP1 and ATF6 pathways contributes to the suppression of GRP78 expression by nicotine in Tm-treated PC12 cells, suggesting that nicotine suppresses a common step upstream of both the Ire1-XBP1 and ATF6 pathways which are required for the expression of GRP78 during Tm-induced ER stress.     

Production of bioactive compounds based on phylogeny in the genus Penicillium preserved at NBRC

Penicillium strains (n=394) preserved at NBRC (the NITE Biological Resource Center) were compared as to groupings (11 species-clusters) based on phylogeny and the production of bioactive compounds. The strains in two clusters, of which P. chrysogenum and P. citrinum are representative, showed higher rates of positive strains with multi-biological activities.     

Spatial variations in xylem sap flux density in evergreen oak trees with radial-porous wood: comparisons with anatomical observations

To estimate whole-tree water use when employing sap flow measurements, integration of the sap flux density (F _d) over the sapwood area is needed. Accordingly, it is necessary to obtain information on the characteristics of stem water transportation such as spatial variations in F _d and the active xylem area in the stem cross-section. Although evergreen oak trees with radial-porous wood represent a major component of secondary forests in western Japan, detailed information on their stem water transportation characteristics remains unclear. In the present study, we used the heat dissipation method (Granier method) to conduct measurements of azimuthal and radial variations in the F _d of Quercus glauca Thunb. ex Murray, a representative evergreen broad-leaved tree in western Japan. Further, by analyzing the anatomy of the xylem structure, we examined why F _d varies spatially in the stem cross-section. By using a dye solution injected into a radial hole bored into the tree trunk, we confirmed that the entire stem is hydroactive. We also compared the spatial variations in F _d and water conductivity per xylem area (K _s) which were estimated by using the observed vessel diameters and their density over the stem cross-section and Hagen–Poiseuille’s law. Azimuthal and radial variations in F _d reached about 60 and 50% of the maximum values, respectively, and could be explained by spatial variation in K _s. As a result, we obtained statistical parameters describing the spatial variation in F _d in Q. glauca and determined that whole-tree water use estimated from measurements in one direction had at most ±20% potential errors for studied trees.     

Myristoylation of neutrophil proteins and their biological characteristics

We have studied protein acylation in neutrophils of guinea pigs using [3H]myristate. A large number of neutrophil proteins were acylated with exogenously added myristic acid. The myristoylation was detected on 110, 77, 56, 54, 52, 42, and 37 kDa proteins. These myristoylations were stronger in peripheral blood than in peritoneal cells. Myristic acid was found to be covalently linked by an amid bond to these proteins since the proteins were resistant to boiling, chloroform/methanol and hydroxylamine treatment. Most myristoylated proteins appeared to be associated with the membrane fraction, while some of the proteins such as 77 kDa one was distributed also in the cytoplasm and translocated from the cytoplasm to the plasma membrane by stimulation. Lysozyme was myristoylated in vitro by the N-hydroxysuccinimide ester of myristic acid. The myristoylated lysozyme had an ability to be associated with phospholipid liposomes, and the membrane-associated lysozyme became a substrate of the rat brain Ca2+- and phospholipid dependent protein kinase (protein kinase C). These results indicate that myristoylation in neutrophil proteins may have an important role in metabolic regulation through their membrane association.     

Effect of calcium ion on fatty acid-induced generation of superoxide in guinea pig neutrophils

When phospholipases of plasma membranes are activated by certain stimuli, unsaturated fatty acids are liberated. Because unsaturated fatty acids enhance the transmembrane movement of calcium ions, the fatty acids released may modulate intracellular calcium homeostasis in various cells, including neutrophils. To determine the physiological function of these unsaturated fatty acids, we studied the effects of various fatty acids on superoxide generation and on changes in intracellular calcium contents of guinea pig neutrophils. Some unsaturated fatty acids, arachidonate and linoleate, stimulated the rate of superoxide generation concomitant with the increase in the amount of intracellular calcium. In contrast, the saturated fatty acid, myristate, stimulated the generation of superoxide without affecting the content of intracellular calcium. The stimulating actions of arachidonate and myristate were increased dramatically by the presence of a low concentration (1 microM) of extracellular calcium ion. The rate of superoxide generation in fatty acid-treated neutrophils was inhibited by chlorpromazine, an inhibitor of such calcium-binding proteins as C-kinase. These and other observations suggest that liberated unsaturated fatty acids increase the amount of intracellular calcium and enhance C-kinase activity also that the increased activity of the enzyme is involved in the chain of events leading to the stimulation of superoxide generation in fatty acid-treated neutrophils.     

Polymorphonuclear leukocyte-inhibitory factor of Bordetella pertussis. III. Inhibition of Arthus reaction and peritoneal infiltration of PMN

In vivo biologic effects of the polymorphonuclear leukocyte-inhibitory factor (PIF) of Bordetella pertussis were tested by using two experimentally induced inflammatory processes in mice. The intravenous injection of a partially purified extract from phase I bacteria strongly inhibited the glycogen-induced peritoneal infiltration of polymorphonuclear leukocytes (PMN) and the Arthus reactions, whereas little inhibitory activity was found in the extract from phase III bacteria. The activity was localized in the outer membrane of phase I bacteria, as was the in vitro PIF activity, and the two activities gave the same behavior in DEAE-cellulose chromatography. Therefore the observed suppression of inflammatory processes in mice is probably due to the inhibitory action of PIF on the function of PMN in vivo.     

Does genomic variation in a foundation species predict arthropod community structure in a riparian forest?

Understanding how genetic variation within a foundation species determines the structure of associated communities and ecosystem processes has been an emerging frontier in ecology. Previous studies in common gardens identified close links between intraspecific variation and multispecies community structure, and these findings are now being evaluated directly in the complex natural ecosystem. In this study, we examined to what extent genomic variation in a foundation tree species explains the structure of associated arthropod communities in the field, comparing with spatial, temporal and environmental factors. In a continuous mixed forest, arthropods were surveyed on 85 mature alders (Alnus hirsuta) in 2 years. Moreover, we estimated Nei's genetic distance among the alders based on 1,077 single nucleotide polymorphisms obtained from restricted‐site‐associated DNA sequencing of the alders’ genome. In both years, we detected significant correlations between genetic distance and dissimilarity of arthropod communities. A generalized dissimilarity modelling indicated that the genetic distance of alder populations was the most important predictor to explain the variance of arthropod communities. Among arthropod functional groups, carnivores were consistently correlated with genetic distance of the foundation species in both years. Furthermore, the extent of year‐to‐year changes in arthropod communities was more similar between more genetically closed alder populations. This study demonstrates that the genetic similarity rule would be primarily prominent in community assembly of plant‐associated arthropods under temporally and spatially variable environments in the field.     

Two Distinct Populations of Primary Cytotoxic Cells Infiltrating into Allografted Tumor Rejection Sites: Infiltration of Macrophages Cytotoxic against Allografted Tumor Precedes That of Multiple Sets of Cytotoxic T Lymphocytes with Distinct Specificity to Alloantigens

It has been reported that the rejection of tumor allografts is mainly mediated by cytotoxic T lymphocytes (CTLs). Here, we characterized the cytotoxic effector cells of C57BL/6 (B6; H-2^b) mice infiltrating into the rejection site of the i.p. allografted Meth A fibrosarcoma (or P815 mastocytoma) cells of H-2^d origin. Two types of cytotoxic cells (i.e., CD8⁺ CTLs and macrophages (Mφs)) were identified by flow cytometric fractionation of the infiltrates or by specific in vitro elimination of cells either with antibody (Ab)-coated beads or with an Ab-plus complement. Of particular interest, these effector cells showed distinct and unique target specificities. First, the CTLs were inactive against transplanted tumor (e.g., Meth A) cells, whereas they were cytotoxic against donor-related concanavalin A (Con A) blasts as well as CTLL-2 (H-2^b) cells transfected with a class I gene of H-2^d origin. A cold target competition assay suggested that the CTLs were composed of multiple sets of T cells, each of which specifically recognized different allo-antigens. Second, the Mφs lysed the allografted tumor cells but were inert toward the Con A blasts and the CTLL-2 transfectants. Unexpectedly, the infiltration of Mφs preceded the infiltration of CTLs by several days during the course of rejection. These results indicate that two distinct populations of unique cytotoxic cells (i.e., CTLs and Mφs) are induced in the allografted tumor rejection site, and that the infiltration of cytotoxic Mφs responsible for rejection precedes that of the CTLs cytotoxic against cells expressing donor-related allo-antigens.     

Cytochemical detection of endogenous peroxidase in the acinar cells of the hamster submandibular gland

The presence of endogenous peroxidase activity in the hamster submandibular gland was investigated cytochemically by light and electron microscopy using diaminobenzidine methods. After fixation of tissue with 2% paraformaldehyde--2.5% glutaraldehyde and incubation in a DAB reaction medium containing 0.01% H₂O₂, the peroxidase reaction product was localized in the nuclear envelope, the cisternae of the endoplasmic reticulum, secretory granules and the Golgi apparatus in both the acinar and granular duct cells of the submandibular gland. This is in contrast to earlier investigators who failed to detect peroxidase activity in acinar cells of the hamster submandibular gland and reported that peroxidase is localized only in the granular duct cells. The discrepancy may be caused by differences in experimental procedures. It is suggested that fixation of tissue with a high concentration of glutaral dehyde and incubation in a DAB reaction medium containing a high concentration of H₂O₂ inhibits the peroxidase activity of acinar cells in the hamster submandibular gland This revised version was published online in November 2006 with corrections to the Cover Date.