Phosphatidylinositol stimulates phosphorylation of protein components I and II in rod outer segments of frog photoreceptors

We studied the effect of phosphoinositides on the phosphorylation of endogenous proteins in the soluble fraction of the frog photoreceptor rod outer segments (ROS). Phosphatidylinositol (PI) stimulated the phosphorylation of two low molecular weight proteins, components I and II (12 and 11 kDa) which are known to be the preferential substrates of the cyclic GMP (cGMP)-dependent protein kinase in the ROS. Polyphosphoinositides (PPI) specifically inhibited the PI-dependent phosphorylation of these two components. On the other hand, PPI stimulated the phosphorylation of 38, 48 and 52 kDa proteins in the absence of PI. These data suggest that PI and PPI may function in the ROS by regulating the phosphorylation of some enzymes or regulator proteins in the transduction mechanism in the ROS.     

Calcium- and Calmodulin-Dependent Phosphorylation of Diphosphoinositide in Acetylcholine Receptor-Rich Membranes from Electroplax of Narke japonica

The phosphorylation of phosphoinositides in the acetylcholine receptor (AChR)-rich membranes from the electroplax of the electric fish Narke japonica has been examined. When the AChR-rich membranes were incubated with [gamma-32P]ATP, 32P was incorporated into only two inositol phospholipids, i.e., tri- and diphosphoinositide (TPI and DPI). Even after the alkali treatment of the membrane, AChR-rich membranes still showed a considerable DPI kinase activity upon addition of exogenous DPI. It is likely that the 32P-incorporation into these lipids was realized by the membrane-bound DPI kinase and phosphatidyl inositol (PI) kinase. Such a membrane-bound DPI kinase was activated by Ca2+ (greater than 10(-6) M), whereas the PI kinase appeared to be inhibited by Ca2+. The effect of Ca2+ on the DPI phosphorylation was further enhanced by the addition of ubiquitous Ca2+-dependent regulator protein calmodulin. Calmodulin antagonists such as chlorpromazine (CPZ), trifluoperazine (TFP), and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibited the phosphorylation of DPI in the AChR-rich membranes. It is suggested that the small pool of TPI in the plasma membrane is replenished by such Ca2+- and calmodulin-dependent DPI kinase responding to the change in the intracellular Ca2+ level.     

Stable and unstable phase of memory in classically conditioned fly, Phormia regina: Effects of nitrogen gas anaesthesia and cycloheximide injection

This study examined the effects of nitrogen anaesthesia and cycloheximide injection on memory of the classically-conditioned fly, Phormia. 1 M NaCl solution was given to each fly as a conditioning stimulus and 0.5 M sucrose solution was the unconditioned stimulus that induced the proboscis extension response. The training period was as short as 2 min and testing was usually carried out 2 hr later. At varying times (0–60 min) between training and testing, flies were anaesthetized with nitrogen gas for 25 sec. When flies were anaesthetized immediately after training the effect of nitrogen gas was the greatest and few flies showed any conditioned response, but the sensitivity of memory to nitrogen gas declined as the interval between training and nitrogen treatment became longer, and such treatment had no effect on memory when the interval was longer than 30 min. The effect on memory of cycloheximide, an inhibitor of peptide bond synthesis, was also investigated. The injection of cycloheximide (0.37 μg) immediately after training diminished the memory, but when given 1 hr after training it had no effect on memory. These results show that the memory in Phormia has two phases, stable and an unstable phase, like long-term and short-term memory in vertebrates.     

Properties of membrane-bound α-glucosidases: Possible sugar receptor protein of the blowfly, Phormia regina

Previously reported PII-type α-glucosidase located in the precipitate of the labellar homogenate of the blowfly Phormia regina was solubilized by sodium deoxycholate (DOC) and further separated into three isozymes with different molecular weight: PII-M (mol. wt 9 × 10⁴). PII-D (mol. wt 2 × 10⁵) and PII-T (mol. wt 8 × 10⁵) by molecular sieve chromatography on Biogel P-300 or Ultragel AcA-34. These three isozymes had almost the same K_m's and relative values of V_m's for several substrates, suggesting that they had the same common active site.PII-D and PII-T are more strongly embedded in the membrane than PII-M, because the proportion of PII-D and PII-T was much increased when the remaining glucosidase in the precipitate after the first solubilization was reextracted by DOC. A large peak of α-glucosidase isozyme P-IV which preferentially hydrolyze sucrose eluted just after P-II (soluble P-II) when the supernatant fraction of the labellar homogenate was chromatographed on DEAE-Sephadex A-50. P-IV was scarcely present in the precipitate fraction.Soluble P-II had the same mol. wt as PII-M and had similar properties to PII-M except for the ratio of V_m's.A large proportion of PII-D was contained in the well washed labellar integuments, a preparation rich in labellar chemosensilla. It suggests that most of the insoluble α-glucosidase contained in the dendrite in labellar chemosensilla is PII-D. PII-D (and PII-T) are possible sites of the pyranose receptor molecule because their properties and localization agree well with those of the receptor.     

Triterpenoid saponins stimulate the sugar taste receptor cell through a G protein-mediated mechanism in the blowfly,Phormia regina

The blowfly has taste chemosensilla on the labellum. The sensory receptor cells in the chemosensillum are highly specialized for the tastes of sugar, salt and water, respectively. Previously we introduced chromosaponin I (CSI) and glycyrrhizin (GL), as sweet substances for the blowfly, Phormia regina. Application of these triterpenoid saponins induced feeding responses as well as impulses of the sugar taste receptor cell in the LL-type sensillum at a much lower concentration than that of sucrose. In the present paper, we show the involvement of G protein-mediated cascade in the CSI- and GL-responses as well as in sugar responses. CSI activates the sugar signal transduction cascade after penetrating through the membrane. On the other hand, GL exerts dual effects to stimulate the sugar signal transduction possibly by activating it inside the cell and also by interacting with the pyranose sugar receptor site. A non hydrolyzable G protein inhibitor guanosine 5′-O-(2-thiodiphosphate), GDPβS, markedly decreased the responses of the sugar receptor cell to the two triterpenoid saponins as well as the response to sucrose and fructose. These results suggest that CSI and GL are direct activators of G protein.     

Plasmacytoid dendritic cells regulate Th cell responses through OX40 ligand and type I IFNs

Dendritic cells (DCs) show a functional plasticity in determining Th responses depending on their maturational stage or on maturational signals delivered to the DCs. Human plasmacytoid DCs (PDCs) can induce either Th1- or Th2-type immune responses upon exposure to viruses or IL-3, respectively. In this study we have investigated the Th-polarizing capacity of PDCs after short (24-h) or long (72-h) culture with stimuli and have assessed the expression and function of OX40 ligand (OX40L) in PDC-mediated Th polarization in addition to type I IFN-dependent responses. IL-3-treated PDCs expressed OX40L, but produced almost no IFN-alpha in response to T cell stimulation (CD40 ligand or T cell interaction), resulting in the preferential priming of Th2 cells through OX40L-dependent mechanisms. Meanwhile, PDCs were rapidly endowed by viral infection (Sendai virus) with a high potency to develop IFN-gamma-producing Th cells depending on their capacity to residually produce IFN-alpha. Although Sendai virus-stimulated PDCs simultaneously expressed OX40L in their maturational process, the Th1-inducing effect of endogenous type I IFNs may overcome and thus conceal the OX40L-dependent Th2 responses. However, during maturation in response to Sendai virus over the longer 72-h period, the expression level of OX40L was up-regulated, whereas the residual IFN-alpha-producing ability was down-regulated, and consequently, the PDCs with prolonged Sendai virus stimulation induced Th2 responses to some extent. Thus, PDCs have the distinct means to dictate an appropriate response to environmental stimuli.     

Chromosaponin I stimulates the sugar taste receptor cells of the blowfly, Phormia regina

Chromosaponin I (CSI), a gamma-pyronyl-triterpenoid saponin isolated from pea and other leguminous plants, stimulates the growth of roots in a variety of plants. In the present work, we introduce CSI as a sugar taste substance for the blowfly, Phormia regina. The blowfly has taste chemosensilla on the labellum. The sensory receptor cells in the chemosensillum are highly specialized for the tastes of sugar, salt and water, respectively. Application of CSI induced the feeding response of blowflies including full proboscis extension. CSI also induced impulses of the sugar taste receptor cell in the LL-type sensillum. The optimum concentration of CSI in these responses was 0.1 mM which is much lower than that of sucrose. Based on the comparison of dose-response relationships, CSI is 100 times more effective than sucrose in stimulating the sugar taste receptor cells. CSI-induced impulses appeared after a significant latency compared with sucrose. As far as we know, this is the first report describing that a natural saponin induces sugar responses in insects. CSI is a unique saponin because of its bifunctional property in plants and insects.