Synergistic activation of the Arabidopsis NADPH oxidase AtrbohD by Ca2+ and phosphorylation

Plant respiratory burst oxidase homolog (rboh) proteins, which are homologous to the mammalian 91-kDa glycoprotein subunit of the phagocyte oxidase (gp91(phox)) or NADPH oxidase 2 (NOX2), have been implicated in the production of reactive oxygen species (ROS) both in stress responses and during development. Unlike mammalian gp91(phox)/NOX2 protein, plant rboh proteins have hydrophilic N-terminal regions containing two EF-hand motifs, suggesting that their activation is dependent on Ca(2+). However, the significance of Ca(2+) binding to the EF-hand motifs on ROS production has been unclear. By employing a heterologous expression system, we showed that ROS production by Arabidopsis thaliana rbohD (AtrbohD) was induced by ionomycin, which is a Ca(2+) ionophore that induces Ca(2+) influx into the cell. This activation required a conformational change in the EF-hand region, as a result of Ca(2+) binding to the EF-hand motifs. We also showed that AtrbohD was directly phosphorylated in vivo, and that this was enhanced by the protein phosphatase inhibitor calyculin A (CA). Moreover, CA itself induced ROS production and dramatically enhanced the ionomycin-induced ROS production of AtrbohD. Our results suggest that Ca(2+) binding and phosphorylation synergistically activate the ROS-producing enzyme activity of AtrbohD.     

Regulation of divalent metal transporter expression in human intestinal epithelial cells following exposure to non-haem iron

A number of regulatory factors including dietary iron levels can dramatically alter the expression of the intestinal iron transporter DMT1. Here we show that Caco-2 cells exposed to iron for 4h exhibited a significant decrease in plasma membrane DMT1 protein, though total cellular DMT1 levels were unaltered. Following biotinylation of cell surface proteins, there was a significant increase in intracellular biotin-labelled DMT1 in iron-exposed cells. Furthermore, iron-treatment increased levels of DMT1 co-localised with LAMP1, suggesting that the initial response of intestinal epithelial cells to iron involves internalisation and targeting of DMT1 transporter protein towards a late endosomal/lysosomal compartment.     

Commensal relationship between a sheath-forming bacterium, Sphaerotilus natans, and a sheath-degrading bacterium, Paenibacillus sp

Paenibacillus sp. strain TB is capable of degrading the sheath prepared from a sheathed bacterium, Sphaerotilus natans. S. natans was able to grow alone on casamino acids but strain TB was not. Cocultivation of strain TB and S. natans was examined in a medium supplemented with casamino acids as a growth substrate. The growth of strain TB was observed when the sheath was supplied to the medium or in cocultivation with S. natans. The phospholipid amount reached a maximum after 24 h of cocultivation and subsequently kept almost the same level for 96 h. The sheath amount also reached a maximum after 24 h and then gradually declined. The cell concentration of strain TB increased throughout the cocultivation. By competitive PCR targeted for amplification of a part of 16S rDNA, the abundance ratio (S. natans/strain TB) of 6.7 was obtained at 72 h. Almost no growth of strain TB was detected in a coculture with a sheath-less mutant of S. natans. The evidence allows the conclusion that strain TB grew by utilizing the intact sheath in coculture with S. natans.     

Partial Male Sterility in Transgenic Tobacco Carrying Antisense and Sense PAL cDNA under the Control of a Tapetum-Specific Promoter

Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5 [EC] ) catalyzes thefirst step in the synthesis of phenylpropanoids. An antisenseor sense PAL cDNA of sweet potato under the control of a tapetum-specificpromoter of rice was introduced into tobacco. A reduction inpollen fertility was observed in two out of seventeen antisensePAL transformants and in six out of nineteen sense PAL transformants.The pollen fertility of these plants ranged from 8% to 60%.The distorted pollen grains that did not germinate lacked starchand flavonols. PAL activity in anthers at the microspore stagewas also reduced to some extent and the level of PAL activitywas positively correlated with the number of fertile pollengrains at the flowering stage. Although it was unclear how theantisense or sense transgene affected the PAL activity in anthers,our results clearly demonstrate that the PAL activity in theanther tapetum has a significant effect on the development ofmicrospores. (Received October 9, 1995; Accepted March 10, 1996)     

Novel α-Glucosidase from Aspergillus nidulans with Strong Transglycosylation Activity

Aspergillus nidulans possessed an α-glucosidase with strong transglycosylation activity. The enzyme, designated α-glucosidase B (AgdB), was purified and characterized. AgdB was a heterodimeric protein comprising 74- and 55-kDa subunits and catalyzed hydrolysis of maltose along with formation of isomaltose and panose. Approximately 50% of maltose was converted to isomaltose, panose, and other minor transglycosylation products by AgdB, even at low maltose concentrations. The agdB gene was cloned and sequenced. The gene comprised 3,055 bp, interrupted by three short introns, and encoded a polypeptide of 955 amino acids. The deduced amino acid sequence contained the chemically determined N-terminal and internal amino acid sequences of the 74- and 55-kDa subunits. This implies that AgdB is synthesized as a single polypeptide precursor. AgdB showed low but overall sequence homology to α-glucosidases of glycosyl hydrolase family 31. However, AgdB was phylogenetically distinct from any other α-glucosidases. We propose here that AgdB is a novel α-glucosidase with unusually strong transglycosylation activity.     

Behaviour of males in relation to the female sex pheromone in the parasitoid wasp, Anisopteromalus calandrae (Hymenoptera: Pteromalidae)

Male sexual behaviour in Anisopteromalus calandrae was observed and experimentally analyzed. The female secretes a sex pheromone which releases the wing vibration of males. The female sex pheromone, and not the visual stimulus, is essential for the recognition by males of the nearby presence of a female. This pheromone which is secreted from glands situated in abdomen of the female, is already released since pupal stage, but the male is attracted most effectively by the female still under the reed of the beans. Copulation takes place immediately after the female comes out of the bean.

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Résumé Le comportement sexuel mâle chez Anisopteromalus calandrae a été observé et expérimentalement analysé. La femelle émet la phéromone sexuelle qui déclenche la vibration des ailes chez le mâle. La phéromone sexuelle, et non le stimulus visuel, est essentielle pour que le mâle reconnaisse la présence voisine d'une femelle. Cette phéromone, sécrétée par des glandes situées dans l'abdomen de la femelle, est émise dès le stade nymphal, et le mâle est effectivement attiré par les femelles encore incluses dans le pois. L'accouplement a lieu aussitôt que la femelle sort dehors.

     

Fish-egg predation by the small clingfish <Emphasis Type="Italic">Pherallodichthys meshimaensis</Emphasis>(Gobiesocidae) on the shallow reefs of Kuchierabu-jima Island,southern Japan

Synopsis We observed predation of demersal fish eggs by the clingfish Pherallodichthys meshimaensis on the shallow reefs of Kuchierabu-jima Island, southern Japan. The processes of egg predation varied with the target species. For targeting of eggs of the combtooth blenny Istiblennius edentulus, up to 65 individuals of the clingfish gathered around the I. edentulus nest hole. Some individuals succeeded in intruding into the hole and fed on eggs while the egg-guarding male of I. edentulus temporarily left the nest. For targeting of eggs of the triplefin blenny Helcogramma obtusirostris, solitary clingfish individuals closely approached spawning females of H. obtusirostris at nests on open surfaces of rocks. Most clingfish contained only fish eggs in their diets, and these fish-egg eaters had larger body sizes than individuals that mainly fed on harpacticoid crustacea. Thus, P. meshimaensis may change its feeding habits when growing to an obligate fish-egg eater targeting demersal egg spawners.     

A novel protein specifically interacting with Homer2 regulates ubiquitin-proteasome systems

Homer family proteins are encoded by three genes, homer1, 2 and 3. Most of these proteins are expressed constitutively in nervous systems and accumulated in postsynaptic regions. However, the functional significance of these proteins, especially the significance of the distinction among the proteins encoded by homer1, 2 and 3, is still obscure. In the present study, we isolated a cDNA clone encoding a novel protein by two-hybrid system screening using the C-terminal half of Homer2b as the bait. This protein, termed 2B28, has 297 amino acid residues and contains three major domains: a UBA domain, a coiled-coil region, and a UBX domain. When expressed in HEK293T cells, 2B28 showed colocalization with uniquitin and enhanced the expression levels of IkappaB or Homer1a proteins, which are known to be degraded by proteasomes, indicating that 2B28 is involved in ubiquitin-proteasome functions. 2B28 specifically interacted and colocalized with Homer2 proteins, but not with Homer1 proteins. So far, we have identified no counterpart of 2B28 for Homer1 experimentally or in the protein databases. These results suggest that the specific interaction of 2B28 with Homer2 may play a role in regulation of protein degradation by ubiquitin-proteasome systems and that this function may be specific to Homer2 proteins among Homer family proteins.     

Discovery of 1,2,3,4-tetrahydropyrimido[1,2-a]benzimidazoles as novel class of corticotropin releasing factor 1 receptor antagonists

A new class of corticotropin releasing factor 1 (CRF₁) receptor antagonists characterized by a tricyclic core ring was designed and synthesized. Novel tricyclic derivatives 2a–e were designed as CRF₁ receptor antagonists based on conformation analysis of our original 2-anilinobenzimidazole CRF₁ receptor antagonist. The synthesized tricyclic derivatives 2a–e showed CRF₁ receptor binding activity with IC₅₀ values of less than 400?nM, and the 1,2,3,4-tetrahydropyrimido-[1,2-a]benzimidazole derivative 2e was selected as a lead compound with potent in vitro CRF₁ receptor binding activity (IC₅₀?=?7.1?nM). To optimize the pharmacokinetic profiles of lead compound 2e, we explored suitable substituents on the 1-position and 6-position, leading to the identification of compound 42c-R, which exhibited potent CRF₁ receptor binding activity (IC₅₀?=?58?nM) with good oral bioavailability (F?=?68% in rats). Compound 42c-R exhibited dose-dependent inhibition of [¹²⁵I]-CRF binding in the frontal cortex (5 and 10?mg/kg, p.o.) as well as suppression of locomotor activation induced by intracerebroventricular administration of CRF in rats (10?mg/kg, p.o.). These results suggest that compound 42c-R successfully binds CRF₁ receptors in the brain and exhibits the potential to be further examined for clinical studies.