Changes in mRNA of Vigna mungo Cotyledons during Seed Germination

Quantitative and qualitative changes of mRNA in Vigna mungocotyledons during seed germination have been investigated. TotalRNA is higher in dry cotyledons and declines during germination.Poly(A)⁺ RNA also is present at a relatively high level in drycotyledons, increases slightly during the first day of germination,and then decreases. Polysomal RNA is very low in dry cotyledonsbut increases rapidly during the first day of germination, andthen declines. The translational activity of the mRNA in a wheatgerm cell-free system is low on day 0 but increases rapidlyon day 1 of germination. Two-dimensional gel electrophoresisof in vitro translation products reveals that many new peptidesare synthesized on day 1 of germination. Synthesis of most ofthese polypeptides continue throughout 5 days of germination. Change in the mRNA population during germination has been investigatedusing cDNA against poly(A)⁺ RNA from 3-day-old cotyledons. Withtotal RNA of day 3 and 5, the cDNA strongly hybridized withRNA similar in size to 25 S ribosomal RNA, but no specific bandsare detected with samples of day 0 or 1. With poly(A)⁺ RNA ofday 5 or 1, the cDNA tends to hybridize with RNAs of relativelysmall molecular size. Cordycepin and -amanitin prevent the increasein poly (A)⁺ RNA content and the appearance of new mRNAs duringthe first day of germination. ¹Present address: Division of Regulation of Macromolecular Function,Institute for Protein Research, Suita City, Osaka 565, Japan. (Received January 13, 1986; Accepted June 10, 1986)     

Change of Cu,Zn-superoxide dismutase activity of guinea pig lung in experimental asthma

Correlation between the level of reactive oxygen species (ROS) generated by airway inflammatory cells and superoxide dismutase (SOD) activity of pulmonary tissue during an asthma attach was investigated in a guinea pig model of allergic asthma. In addition, the influence of SOD inhibition by diethyldithiocarbamate (DDC, Cu-chelating agent) on the airway was investigated in terms of pulmonary function during an asthma attach. Relative to controls, the capacity of bronchoalveolar lavage fluid (BAL) cells to release ROS was significantly increased in guinea pigs sensitized with ovalbumin (OA) as the antigen, and significantly increased in guinea pigs with an asthma attack provoked by the inhalation of OA. SOD activity was increased significantly in the antigen-sensitized group. The asthma provocation group showed a tendency for increase in total SOD activity, compared with the sensitization group, whose increase was dependent on the increase in copper, zinc-SOD (Cu, Zn-SOD) activity. Pretreatment with DDC increased the severity and duration of the asthma attack. These results were indicated that Cu, Zn-SOD was closely involved in the asthma process, particularly in the scavenging of oxygen radicals secreted from BAL cells.     

Sphingosine 1-phosphate inhibits migration and RANTES production in human bronchial smooth muscle cells

Sphingosine 1-phosphate (S1P), a bioactive lipid mediator, has been shown to be increased in bronchoalveolar lavage fluid after allergen challenge in asthmatic patients. Here, we examined S1P actions and their intracellular signalings in cultured human bronchial smooth muscle cells (BSMCs). Expression of mRNAs of three subtypes of S1P receptors, including S1P(1), S1P(2), and S1P(3), was detected in BSMCs, and exposure of the cells to S1P inhibited platelet-derived growth factor (PDGF)-induced migration and tumor necrosis factor-alpha-induced RANTES production. S1P also inhibited PDGF-induced Rac1 activation, and dominant negative Rac1 inhibited PDGF-induced migration. On the other hand, dominant negative Galpha(q) attenuated the S1P-induced inhibition of RANTES production. Finally, an S1P(2)-selective antagonist, JTE-013, suppressed the S1P-induced inhibition of migration response and RANTES production. These results suggest that S1P attenuates cell migration by inhibiting a Rac1-dependent signaling pathway and decreases RANTES production by stimulating a Galpha(q)-dependent mechanism both possibly through the S1P(2) receptors.     

TDAG8 is a proton-sensing and psychosine-sensitive G-protein-coupled receptor

T cell death-associated gene 8 (TDAG8) has been reported to be a receptor for psychosine. Ovarian cancer G-protein-coupled receptor 1 (OGR1) and GPR4, G-protein-coupled receptors (GPCRs) closely related to TDAG8, however, have recently been identified as proton-sensing or extracellular pH-responsive GPCRs that stimulate inositol phosphate and cAMP production, respectively. In the present study, we examined whether TDAG8 senses extracellular pH change. In the several cell types that were transfected with TDAG8 cDNA, cAMP was markedly accumulated in response to neutral to acidic extracellular pH, with a peak response at approximately pH 7.0-6.5. The pH effect was inhibited by copper ions and was reduced or lost in cells expressing mutated TDAG8 in which histidine residues were changed to phenylalanine. In the membrane fractions prepared from TDAG8-transfected cells, guanosine 5'-O-(3-thiotriphosphate) binding activity and adenylyl cyclase activity were remarkably stimulated in response to neutral and acidic pH. The concentration-dependent effect of extracellular protons on cAMP accumulation was shifted to the right in the presence of psychosine. The inhibitory psychosine effect was also observed for pH-dependent actions in OGR1- and GPR4-expressing cells but not for prostaglandin E(2)- and sphingosine 1-phosphate-induced actions in any pH in native and sphingosine 1-phosphate receptor-expressing cells. Glucosylsphingosine and sphingosylphosphorylcholine similarly inhibited the pH-dependent action, although to a lesser extent. Psychosine-sensitive and pH-dependent cAMP accumulation was also observed in mouse thymocytes. We concluded that TDAG8 is one of the proton-sensing GPCRs coupling to adenylyl cyclase and psychosine, and its related lysosphingolipids behave as if they were antagonists against protein-sensing receptors, including TDAG8, GPR4, and OGR1.     

Mechanism and Role of High Density Lipoprotein-induced Activation of AMP-activated Protein Kinase in Endothelial Cells

The upstream signaling pathway leading to the activation of AMP-activated protein kinase (AMPK) by high density lipoprotein (HDL) and the role of AMPK in HDL-induced antiatherogenic actions were investigated. Experiments using genetic and pharmacological tools showed that HDL-induced activation of AMPK is dependent on both sphingosine 1-phosphate receptors and scavenger receptor class B type I through calcium/calmodulin-dependent protein kinase kinase and, for scavenger receptor class B type I system, additionally serine-threonine kinase LKB1 in human umbilical vein endothelial cells. HDL-induced activation of Akt and endothelial NO synthase, stimulation of migration, and inhibition of monocyte adhesion and adhesion molecule expression were dependent on AMPK activation. The inhibitory role of AMPK in the adhesion molecule expression and monocyte adhesion on endothelium of mouse aorta was confirmed in vivo and ex vivo. On the other hand, stimulation of ERK and proliferation were hardly affected by AMPK knockdown but completely inhibited by an N17Ras, whereas the dominant-negative Ras was ineffective for AMPK activation. In conclusion, dual HDL receptor systems differentially regulate AMPK activity through calcium/calmodulin-dependent protein kinase kinase and/or LKB1. Several HDL-induced antiatherogenic actions are regulated by AMPK, but proliferation-related actions are regulated by Ras rather than AMPK.     

Temporal Variation in Seed Predation by Insects in a Population of Syagrus romanzoffiana (Arecaceae) in Santa Catarina Island, SC, Brazil

Insect seed predation may vary depending on seed production. The present study considers the hypothesis that the rates of seed predation tend to be smaller in years of higher fruit production. Thus, we monitored the production of fruits and predation of seeds of the palm Syagrus romanzoffiana over 2?years in the Atlantic Forest (Parque Municipal da Lagoa do Peri, Florianópolis, SC, Brazil), between July 2006 and June 2008. Plots of 0.25?m² were fitted under 20 mother plants and fruits were monthly collected for assessment of abundance and seed predation. There was variation in fruit production between the 2?years and among reproductive plants. Predation rates were high and occurred in the predispersal phase by the Curculionidae Revena rubiginosa Boheman, Anchylorhynchus aegrotus Fahraeus, and Anchylorhynchus variabilis Gyllenhal. Seed predation by these species of Anchylorhynchus is first registered in the present study. In average, about 60% of the seeds monthly produced in the population tend to escape insect predation in year of high or low production, becoming available for recruitment. The predation rate was not related to the amount of fruits produced per reproductive plant. Also, different than expected, there was a positive relation between the rates of seed predation and the total of fruits produced monthly on the plots. Thus, no evidence for the satiation of insect seed predators was found in this study with S. romanzoffiana.     

Induction of scavenger receptor class B type I is critical for simvastatin enhancement of high-density lipoprotein-induced anti-inflammatory actions in endothelial cells

Changes in plasma lipoprotein profiles, especially low levels of high-density lipoprotein (HDL), are a common biomarker for several inflammatory and immune diseases, including atherosclerosis and rheumatoid arthritis. We examined the effect of simvastatin on HDL-induced anti-inflammatory actions. HDL and sphingosine 1-phosphate (S1P), a bioactive lipid component of the lipoprotein, inhibited TNF alpha-induced expression of VCAM-1, which was associated with NO synthase (NOS) activation, in human umbilical venous endothelial cells. The HDL- but not S1P-induced anti-inflammatory actions were enhanced by a prior treatment of the cells with simvastatin in a manner sensitive to mevalonic acid. Simvastatin stimulated the expression of scavenger receptor class B type I (SR-BI) and endothelial NOS. As for S1P receptors, however, the statin inhibited the expression of S1P(3) receptor mRNA but caused no detectable change in S1P(1) receptor expression. The reconstituted HDL, a stimulator of SR-BI, mimicked HDL actions in a simvastatin-sensitive manner. The HDL- and reconstituted HDL-induced actions were blocked by small interfering RNA specific to SR-BI regardless of simvastatin treatment. The statin-induced expression of SR-BI was attenuated by constitutively active RhoA and small interfering RNA specific to peroxisome proliferator-activated receptor-alpha. Administration of simvastatin in vivo stimulated endothelial SR-BI expression, which was accompanied by the inhibition of the ex vivo monocyte adhesion in aortas from TNF alpha-injected mice. In conclusion, simvastatin induces endothelial SR-BI expression through a RhoA- and peroxisome proliferator-activated receptor-alpha-dependent mechanism, thereby enhancing the HDL-induced activation of NOS and the inhibition of adhesion molecule expression.     

Involvement of proton-sensing receptor TDAG8 in the anti-inflammatory actions of dexamethasone in peritoneal macrophages

Dexamethasone (DEX), a potent glucocorticoid, increased the expression of T-cell death associated gene 8 (TDAG8), a proton-sensing G protein-coupled receptor, which is associated with the enhancement of acidic pH-induced cAMP accumulation, in peritoneal macrophages. We explored the role of increased TDAG8 expression in the anti-inflammatory actions of DEX. The treatment of macrophages with either DEX or acidic pH induced the cell death of macrophages; however, the cell death was not affected by TDAG8 deficiency. While DEX inhibited lipopolysaccharide-induced production of tumor necrosis factor-α, an inflammatory cytokine, which was independent of TDAG8, at neutral pH, the glucocorticoid enhanced the acidic pH-induced inhibition of tumor necrosis factor-α production in a manner dependent on TDAG8. In conclusion, the DEX-induced increase in TDAG8 expression is in part involved in the glucocorticoid-induced anti-inflammatory actions through the inhibition of inflammatory cytokine production under the acidic pH environment. On the other hand, the role of TDAG8 in the DEX-induced cell death is questionable.