GAMYB controls different sets of genes and is differentially regulated by microRNA in aleurone cells and anthers

GAMYB is a component of gibberellin (GA) signaling in cereal aleurone cells, and has an important role in flower development. However, it is unclear how GAMYB function is regulated. We examined the involvement of a microRNA, miR159, in the regulation of GAMYB expression in cereal aleurone cells and flower development. In aleurone cells, no miR159 expression was observed with or without GA treatment, suggesting that miR159 is not involved in the regulation of GAMYB and GAMYB-like genes in this tissue. miR159 was expressed in tissues other than aleurone, and miR159 over-expressors showed similar but more severe phenotypes than the gamyb mutant. GAMYB and GAMYB-like genes are co-expressed with miR159 in anthers, and the mRNA levels for GAMYB and GAMYB-like genes are negatively correlated with miR159 levels during anther development. Thus, OsGAMYB and OsGAMYB-like genes are regulated by miR159 in flowers. A microarray analysis revealed that OsGAMYB and its upstream regulator SLR1 are involved in the regulation of almost all GA-mediated gene expression in rice aleurone cells. Moreover, different sets of genes are regulated by GAMYB in aleurone cells and anthers. GAMYB binds directly to promoter regions of its target genes in anthers as well as aleurone cells. Based on these observations, we suggest that the regulation of GAMYB expression and GAMYB function are different in aleurone cells and flowers in rice.     

In vitro culture technique for Cryptocaryon irritans, a parasitic ciliate of marine teleosts

A medium for the in vitro culture of Cryptocaryon irritans, which is an obligatorily parasitic ciliate of marine teleosts and causes 'white spot disease', was developed. The medium consisted of a layer of cultured fish cells (FHM), with an agarose gel layer covering the cell layer. The agarose gel contained 0.22% agarose, 10% fetal calf serum, 100 I.U. ml(-1) Penicillin G potassium and 100 microg ml(-1) streptomycin sulphate. Theronts of C. irritans transformed to trophonts and grew to 180 microm in mean length in the medium, although they gradually decreased in number. When trophonts fully developed in medium were transferred into seawater 4 d after inoculation, approximately 70% of them transformed to encysted tomonts and released theronts. When fish were challenged with theronts obtained from in vitro-raised parasites, approximately 40% of the theronts were recovered from fish, indicating comparative infectivity of in vitro-raised theronts to those of in vivo-raised theronts. This is the first report that C. irritans fully developed in vitro and its entire life cycle was completed without a host fish.     

Activation pattern of MAPK signaling in the hearts of trained and untrained rats following a single bout of exercise.

Since exercise training causes cardiac hypertrophy and a single bout induces mechanical stress to the heart, the present study aimed to characterize the activation patterns of multiple MAPK signaling pathways in the heart after a single bout of exercise or chronic exercises. The hearts of untrained rats received 5, 15, and 30 min of treadmill running exercise (Ex5 to Ex30) and rested for 0.5, 1, 3, 6, 12, and 24 h (PostEx0.5 to PostEx24) before subjecting them to the following different experiments. Activation of MAPKs (ERK, JNK, and p38) and MAPKKs (MEK1/2, SEK, and MKK3/6) increased immediately after acute exercise in a time-dependent manner, with ERK, JNK, and p38 peaking at Ex15, Ex15, and Ex30, respectively. Expression of immediate early genes (c-fos, c-jun, and c-myc) was augmented and activator protein-1 DNA binding activity was enhanced in untrained rats immediately after a single bout of exercise. The elevated levels of MAPKs declined to the resting levels within 24 h after exercise. In another set of experiments, following 4, 8, and 12 wk of exercise training, the rats exhibited significant cardiac hypertrophy by week 12. Activation of MAPKs in the 4-wk-trained rats increased after a 30-min single bout of exercise but decreased in the 8-wk group. Finally, the activity of MAPKs signaling in the 12-wk-trained rats exposed to an acute bout of exercise was unaltered. We conclude that exercise induces the activation of multiple MAPK (ERK, JNK, and p38) pathways in the heart, an effect that gradually declines with the development of exercise-induced cardiac hypertrophy.     

Lactic acid fermentation of potato pulp by the fungus Rhizopus oryzae

Production of molecules with toxic activity by genetically transformed symbiotic bacteria of pest insects may serve as a powerful approach to biological control. The symbiont, Enterobacter gergoviae, isolated from the gut of the pink bollworm (PBW), has been transformed to express Cyt1A, a cytolytic protein toxin lethal to mosquito and black fly larvae, as a model system. These transgenic bacteria might be used to spread genes encoding insecticidal proteins to populations of agricultural insects or as replacement for chemical insecticides such as malathion used in bait formulation to control specific insect pests, because of extreme public pressure against organophosphate pesticide spraying. Received: 27 November 2000 / Accepted: 29 December 2000     

Effects of chemically modified heparin on Chlamydia trachomatis serovar L2 infection of eukaryotic cells in culture

The mechanism and inhibitors of Chlamydia trachomatis serovar L2 infection of eukaryotic host cells were studied using a tissue culture model infection system. Potent inhibition of infectivity was observed when elementary bodies (EBs) were exposed to heparin or when HeLa 229 cells were treated with heparinase. No significant inhibition was seen the other way around. The same potent inhibition was observed when EBs were exposed to chemically 2-O-desulfated heparin (2-ODS heparin), which is composed of repeating disaccharide units of IdoA-GlcNS(6S), but not when exposed to chemically 6-ODS heparin or completely desulfated and N-resulfated heparin, which is composed of repeating disaccharide units of IdoA(2S)-GlcNS or IdoA-GlcNS, respectively. The inhibitory effects of 2-ODS heparin could be seen only with oligosaccharides longer than dodecasaccharides. The mutant Chinese hamster ovary (CHO) cell line 677, which is deficient in the biosynthesis of heparan sulfate, was less sensitive to C. trachomatis infection than were wild-type CHO cells. F-17 cells, deficient in 2-O-sulfation of heparan sulfate, had the same sensitivity to infection as wild-type CHO cells did. These data suggest that infection of host cells by EBS results from the specific binding of ligand molecules with affinity for heparin on the EB surface to heparan sulfate proteoglycans on the host cell surface. This binding may depend on host cell heparan sulfate chains that are 6-O-sulfated and longer than dodecasaccharides. The 2-ODS heparin oligosaccharides may be a potential agent for the prevention of C. trachomatis infection.     

Energetics and volume changes of the intermediates in the photolysis of octopus rhodopsin at a physiological temperature

Enthalpy changes (Delta H) of the photointermediates that appear in the photolysis of octopus rhodopsin were measured at physiological temperatures by the laser-induced transient grating method. The enthalpy from the initial state, rhodopsin, to bathorhodopsin, lumirhodopsin, mesorhodopsin, transient acid metarhodopsin, and acid metarhodopsin were 146 +/- 15 kJ/mol, 122 +/- 17 kJ/mol, 38 +/- 8 kJ/mol, 12 +/- 5 kJ/mol, and 12 +/- 5 kJ/mol, respectively. These values, except for lumirhodopsin, are similar to those obtained for the cryogenically trapped intermediate species by direct calorimetric measurements. However, the Delta H of lumirhodopsin at physiological temperatures is quite different from that at low temperature. The reaction volume changes of these processes were determined by the pulsed laser-induced photoacoustic method along with the above Delta H values. Initially, in the transformation between rhodopsin and bathorhodopsin, a large volume expansion of +32 +/- 3 ml/mol was obtained. The volume changes of the subsequent reaction steps were rather small. These results are compared with the structural changes of the chromophore, peptide backbone, and water molecules within the membrane helixes reported previously.     

Analysis of reductant supply systems for ferredoxin-dependent sulfite reductase in photosynthetic and nonphotosynthetic organs of maize

Sulfite reductase (SiR) catalyzes the reduction of sulfite to sulfide in chloroplasts and root plastids using ferredoxin (Fd) as an electron donor. Using purified maize (Zea mays L.) SiR and isoproteins of Fd and Fd-NADP(+) reductase (FNR), we reconstituted illuminated thylakoid membrane- and NADPH-dependent sulfite reduction systems. Fd I and L-FNR were distributed in leaves and Fd III and R-FNR in roots. The stromal concentrations of SiR and Fd I were estimated at 1.2 and 37 microM, respectively. The molar ratio of Fd III to SiR in root plastids was approximately 3:1. Photoreduced Fd I and Fd III showed a comparable ability to donate electrons to SiR. In contrast, when being reduced with NADPH via FNRs, Fd III showed a several-fold higher activity than Fd I. Fd III and R-FNR showed the highest rate of sulfite reduction among all combinations tested. NADP(+) decreased the rate of sulfite reduction in a dose-dependent manner. These results demonstrate that the participation of Fd III and high NADPH/NADP(+) ratio are crucial for non-photosynthetic sulfite reduction. In accordance with this view, a cysteine-auxotrophic Escherichia coli mutant defective for NADPH-dependent SiR was rescued by co-expression of maize SiR with Fd III but not with Fd I.     

Different combinations of Notch ligands and receptors regulate V2 interneuron progenitor proliferation and V2a/V2b cell fate determination

The broad diversity of neurons is vital to neuronal functions. During vertebrate development, the spinal cord is a site of sensory and motor tasks coordinated by interneurons and the ongoing neurogenesis. In the spinal cord, V2-interneuron (V2-IN) progenitors (p2) develop into excitatory V2a-INs and inhibitory V2b-INs. The balance of these two types of interneurons requires precise control in the number and timing of their production. Here, using zebrafish embryos with altered Notch signaling, we show that different combinations of Notch ligands and receptors regulate two functions: the maintenance of p2 progenitor cells and the V2a/V2b cell fate decision in V2-IN development. Two ligands, DeltaA and DeltaD, and three receptors, Notch1a, Notch1b, and Notch3 redundantly contribute to p2 progenitor maintenance. On the other hand, DeltaA, DeltaC, and Notch1a mainly contribute to the V2a/V2b cell fate determination. A ubiquitin ligase Mib, which activates Notch ligands, acts in both functions through its activation of DeltaA, DeltaC, and DeltaD. Moreover, p2 progenitor maintenance and V2a/V2b fate determination are not distinct temporal processes, but occur within the same time frame during development. In conclusion, V2-IN cell progenitor proliferation and V2a/V2b cell fate determination involve signaling through different sets of Notch ligand–receptor combinations that occur concurrently during development in zebrafish.