Effects of an inhibitor of phosphoenolpyruvate carboxylase on photosynthesis of the terrestrial forms of amphibious Eleocharis species

The leafless amphibious sedge Eleocharis vivipara develops culms with C₄ traits and Kranz anatomy under terrestrial conditions, but develops culms with C₃ traits and non-Kranz anatomy under submerged conditions. The culms of the terrestrial form have high C₄ enzyme activities, while those of the submerged form have decreased C₄ enzyme activities. The culms accumulate ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the mesophyll cells (MC) and the bundle sheath cells. The Rubisco in the MC may be responsible for the operation of the C₃ pathway in the submerged form. To verify the presence of the C₃ cycle in the MC, we examined the effects of 3,3-dichloro-2-(dihydroxyphosphinoylmethyl) -propenoate (DCDP), an inhibitor of phosphoenolpyruvate carboxylase (PEPC), on photosynthesis in culms of the terrestrial forms of E. vivipara and related amphibious species, E. baldwinii and E. retroflexa ssp. chaetaria. When 1 mM DCDP was fed via the transpiration stream to excised leaves, photosynthesis was inhibited completely in Fimbristylis dichotoma (C₄ control), but by only 20% in potato (C₃ control). In the terrestrial Eleocharis plants, the degree of inhibition of photosynthesis by DCDP was intermediate between those of the C₄ and C₃ plants, at 58–81%. These results suggest that photosynthesis under DCDP treatment in the terrestrial Eleocharis plants is due mainly to fixation of atmospheric CO₂ by Rubisco and probably the C₃ cycle in the MC. These features are reminiscent of those in C₄-like plants. Differential effects of DCDP on photosynthesis of the 3 Eleocharis species are discussed in relation to differences in the degree of Rubisco accumulation and C₃ activity in the MC. This revised version was published online in June 2006 with corrections to the Cover Date.     

Biochemical characterization of mouse microsomal prostaglandin E synthase-1 and its colocalization with cyclooxygenase-2 in peritoneal macrophages.

We cloned the cDNA for mouse microsomal prostaglandin (PG) E synthase-1 (mPGES-1) and expressed the recombinant enzyme in Escherichia coli. The membrane fraction containing recombinant mPGES-1 catalyzed the isomerization of PGH2 to PGE2 in the presence of GSH with K(m) values of 130 microM for PGH2 and 37 microM for GSH, a turnover number of 600 min(-1), and a k(cat)/K(m) ratio of 4.6 min(-1) microM(-1). Recombinant mPGES-1 was purified and used to generate a polyclonal antibody highly specific for mPGES-1. The antibody showed a single band on Western blotting of microsomal fractions from lipopolysaccharide-treated mouse peritoneal macrophages. Northern and Western blotting analyses revealed that mPGES-1 was induced together with cyclooxygenase-2 in mouse macrophages after treatment of the cells with lipopolysaccharide. Confocal immunofluorescence microscopy revealed that both mPGES-1 and cyclooxygenase-2 were colocalized in the lipopolysaccharide-treated macrophages. Taken together, these results demonstrate that mPGES-1 is an efficient downstream enzyme for the production of PGE2 in the activated macrophages treated by lipopolysaccharide.     

The human perforin gene is a direct target of STAT4 activated by IL-12 in NK cells

IL-12 activates STAT4 by inducing tyrosine phosphorylation, homo-dimerization, and nuclear translocation in NK cells and thereby stimulates proliferation and activation of these cells. The pore-forming protein perforin is a key effector protein for NK cell- and cytotoxic T lymphocyte-mediated cytolysis. Here we demonstrate that IL-12 induces the expression of the perforin gene in human NK cell line, NKL. Electrophoretic mobility shift assays using a probe containing two putative STAT-binding sequences located at -1085 and -1059 in the human perforin gene showed that STAT4 or STAT5 activated by IL-12 or IL-2, respectively, in NKL cells binds this region. Further analyses using various probes with or without mutated STAT-binding sequences showed that, although either of the two tandem STAT-binding sequences binds STAT4 weakly, the presence of both is required for significant binding of activated STAT4 and for formation of the STAT4-DNA-binding complex with lower electrophoretic mobility. Furthermore, mutation of either of the tandem STAT-binding sequences abolished the IL-12-induced activation of the perforin gene promoter in reporter gene assays. These results indicate that the IL-12-induced expression of the perforin gene in NK cells is directly regulated by STAT4, which binds, most likely as a homo-tetramer, to the tandem STAT-binding sequences in the perforin gene promoter.     

Identification of a new member of the GLWamide peptide family: physiological activity and cellular localization in cnidarian polyps

KPNAYKGKLPIGLWamide, a novel member of the GLWamide peptide family, was isolated from Hydra magnipapillata. The purification was monitored with a bioassay: contraction of the retractor muscle of a sea anemone, Anthopleura fuscoviridis. The new peptide, termed Hym-370, is longer than the other GLWamides previously isolated from H. magnipapillata and another sea anemone, A. elegantissima. The amino acid sequence of Hym-370 is six residues longer at its N-terminal than a putative sequence previously deduced from the cDNA encoding the precursor protein. The new longer isoform, like the shorter GLWamides, evoked concentration-dependent muscle contractions in both H. magnipapillata and A. fuscoviridis. In contrast, Hym-248, one of the shorter GLWamide peptides, specifically induced contraction of the endodermal muscles in H. magnipapillata. This is the first case in which a member of the hydra GLWamide family (Hym-GLWamides) has exhibited an activity not shared by the others. Polyclonal antibodies were raised to the common C-terminal tripeptide GLWamide and were used in immunohistochemistry to localize the GLWamides in the tissue of two species of hydra, H. magnipapillata and H. oligactis, and one species of sea anemone, A. fuscoviridis. In each case, nerve cells were specifically labeled. These results suggest that the GLWamides are ubiquitous among cnidarians and are involved in regulating the excitability of specific muscles.     

Activation of the pyrrolysine suppressor tRNA requires formation of a ternary complex with class I and class II lysyl-tRNA synthetases

Monomethylamine methyltransferase of the archaeon Methanosarcina barkeri contains a rare amino acid, pyrrolysine, encoded by the termination codon UAG. Translation of this UAG requires the aminoacylation of the corresponding amber suppressor tRNAPyl. Previous studies reported that tRNAPyl could be aminoacylated by the synthetase-like protein PylS. We now show that tRNAPyl is efficiently aminoacylated in the presence of both the class I LysRS and class II LysRS of M. barkeri, but not by either enzyme acting alone or by PylS. In vitro studies show that both the class I and II LysRS enzymes must bind tRNAPyl in order for the aminoacylation reaction to proceed. Structural modeling and selective inhibition experiments indicate that the class I and II LysRSs form a ternary complex with tRNAPyl, with the aminoacylation activity residing in the class II enzyme.     

Difference in response by two cyprinid species to predatory threat from the nocturnal catfish Silurus asotus

Response to predators may not be identical between different prey species with different life histories and body sizes, particularly when the threat of predation is not great. To clarify this hypothesis, we introduced two prey species (10 Japanese dace, Tribolodon hakonensis, and 10 pale chub, Zacco platypus) into each experimental pond (in total, 8 ponds×4 trials) in which benthic algae had been allowed to grow. The presence or absence of Far Eastern catfish, Silurus asotus, and a refuge for prey fish was used to produce four treatments. The presence of catfish and/or a refuge did not affect either the feeding behavior or growth rate of Japanese dace. In contrast, when catfish were present and no refuge was available, the incidence of bottom feeding for pale chub greatly decreased. Pale chub growth rate was low when catfish were present and a refuge was available, indicating that pale chub spent more of their time in the refuge and lost opportunities of acquiring food. Japanese dace can reach a threshold size at which the prey are safe from predation, but pale chub cannot, and this may explain the differences in response to predators of the two species.     

Identification of elongation factor-1alpha as a Ca2+/calmodulin-binding protein in Tetrahymena cilia

Calmodulin (CaM) is known to be a ciliary component. However, the function of CaM in cilia or flagella has not been well understood. Immunoelectron microscopy using anti-CaM antibody showed that CaM was localized on the axonemal microtubules (MTs) and matrix of Tetrahymena cilia. To investigate the signal transduction of Ca(2+)/CaM in cilia, we performed Ca(2+)/CaM-affinity column chromatography in the membrane and matrix fraction. Elongation factor-1alpha (EF-1alpha) was identified as a Ca(2+)/CaM-binding protein in cilia. EF-1alpha is a highly conserved protein and functions in protein translation. In addition, EF-1alpha has been reported to interact with MTs and F-actin in several organisms. Immunoelectron microscopy showed that EF-1alpha was localized on the axonemal MTs. However, in immunoblot analysis, EF-1alpha was mainly extracted in the membrane and matrix fraction from the axonemal MTs by 1% Triton X-100 extraction. These results suggest that interaction between EF-1alpha and axonemal MTs is weak and sensitive to treatment with 1% Triton X-100 and that EF-1alpha mediates between axonemal MTs and CaM in the presence of Ca(2+). Moreover, EF-1alpha was also localized in cilia of Paramecium, suggesting that EF-1alpha functions as a target protein of Ca(2+)/CaM in ciliate cilia.