Effect of catalase-specific inhibitor 3-amino-1,2,4-triazole on yeast peroxisomal catalase in vivo

3-Amino-1,2,4-triazole (3-AT) is known as an inhibitor of catalase to whose active center it specifically and covalently binds. Subcellular fractionation and immunoelectronmicroscopic observation of the yeast Candida tropicalis revealed that, in 3-AT-treated cells in which the 3-AT was added to the n-alkane medium from the beginning of cultivation, catalase transported into peroxisomes was inactivated and was present as insoluble aggregated forms in the organelle. The aggregation of catalase in peroxisomes occurred only in these 3-AT-treated cells and not in cells in which 3-AT was added at the late exponential growth phase. Furthermore, 3-AT did not affect the transportation of catalase into peroxisomes. The appearance of aggregation only in cells to which 3-AT was added from the beginning of cultivation suggests that, in the process of catalase transportation into yeast peroxisomes, some conformational change may take place and that correct folding may be inhibited by the binding of 3-AT to the active center of catalase. Accordingly, 3-AT will be an interesting compound for investigation of the transport machinery of the peroxisomal tetrameric catalase.     

Improvement of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) seed oil quality through introduction of a soybean microsomal omega-3 fatty acid desaturase gene

Microsomal omega-3 fatty acid desaturase is an essential enzyme in the production of the n-3 polyunsaturated fatty acid alpha-linolenic acid during the seed developing stage. We have constructed a chimeric gene consisting of a maize Ubi1-P-int and a soybean GmFAD3 cDNA, which was introduced into rice plants by Agrobacterium-mediated transformation. Ten transformants containing the chimeric gene were established and expression subsequently confirmed by Northern blotting. Furthermore, alpha-linolenic acid content of the T(1) seeds increased dramatically up to tenfold that of the control, and this phenotype was also stably inherited in the T(2) and T(3) progenies. These results demonstrate that the alpha-linolenic acid content of rice seed oil can easily be altered using the combination of a high-activity promoter and a GmFAD3 gene.     

Blue-light- and phosphorylation-dependent binding of a 14-3-3 protein to phototropins in stomatal guard cells of broad bean

Phototropins are blue-light (BL) receptor serine (Ser)/threonine kinases, and contain two light, oxygen, and voltage (LOV) domains, and are members of the PAS domain superfamily. They mediate phototropism, chloroplast movement, leaf expansion, and stomatal opening of higher plants in response to BL. In stomatal guard cells, genetic analysis has revealed that phototropins mediate activation of the plasma membrane H+-ATPase by phosphorylation and drive stomatal opening. However, biochemical evidence for the involvement of phototropins in the BL response of stomata is lacking. Using guard cell protoplasts, we showed that broad bean (Vicia faba) phototropins (Vfphots) were phosphorylated by BL, and that this phosphorylation of Vfphots reached to the maximum level earlier than that of the H+-ATPase. Phosphorylation of both Vfphots and H+-ATPase showed similar sensitivity to BL and were similarly suppressed by protein kinase and flavoprotein inhibitors. We found that a 14-3-3 protein was bound to Vfphots upon phosphorylation, and this binding occurred earlier than the H+-ATPase phosphorylation. Vfphots (Vfphot1a and Vfphot1b) were expressed in Escherichia coli, and phosphorylation sites were determined to be Ser-358 for Vfphot1a and Ser-344 for Vfphot1b, which are localized between LOV1 and LOV2. We conclude that Vfphots act as BL receptors in guard cells and that phosphorylation of a Ser residue between LOV1 and LOV2 and subsequent 14-3-3 protein binding are likely to be key steps of BL response in stomata. The binding of a 14-3-3 protein to Vfphot was found in etiolated seedlings and leaves in response to BL, suggesting that this event was common to phototropin-mediated responses.     

Amphibian in vitro heart induction: a simple and reliable model for the study of vertebrate cardiac development

Amphibian embryos are an excellent model system for analyzing the mechanisms of vertebrate cardiogenesis. Studies of heart development in Xenopus have, for example, revealed that the inductive interaction of the heart primordia with the adjacent underlying endoderm and dorsal lip starts at the early stages of gastrulation. However, the molecular basis of those early inductive events and the genes expressed during the early phases of heart differentiation remain largely unknown. Amphibian blastula embryos contain pluripotent cells in their ectodermal region, called the "animal cap," which fortunately can be exploited for understanding a variety of organogenesis processes. Despite an enormous potential for analysis, the use of this system in cardiogenesis research has languished due to a lack of information concerning appropriate culture methods. Herein we report conditions for generating an in vitro heart induction system and present evidence from two types of in vivo transplantations, that the cultured heart rudiment can develop and function in the adult organism. It is expected that the fundamental principles established in this model system will provide a versatile research platform for a variety of organ engineering projects, including modifying in vitro organ growth with exogenous components (e.g. various growth factors) and developing methods for preparing tissue for transplantation.     

Intrathecal adenosine A1 receptor agonist attenuates hyperalgesia without inhibiting spinal glutamate release in the rat

The analgesia effects of intrathecal adenosine A₁ receptor agonist, R-PIA, on the hyperalgesia and CSF-glutamate release after formalin injection into the rat paw were evaluated. R-PIA significantly and dose-dependently attenuated increases in flinching behavior, and this attenuating effect was reversed by the adenosine A₁ receptor antagonist, aminophylline. Morphine blocked flinchs, however MK-801 partially abolished. The increase in CSF-glutamate release evoked by formalin stimulation was inhibited by morphine but not by either R-PIA or MK-801. These findings suggest that the intrathecal adenosine A₁ receptor agonist provokes analgesic effect via the postsynaptic action independent of an effect upon spinal glutamate release.     

New method for synthesizing the intermediates to 5-HETE from yeast-mediated reduction products by employing Baeyer-Villiger oxidation with complete retention of enantiomeric excess

(R) and (S)-Aldehydes 2, which are intermediates for the synthesis of (5R) and (5S)-HETE, were respectively synthesized from the yeast-mediated reductive products, hydroxy ester 3 and cis-lactone 4, through Baeyer-Villiger oxidation with complete retention of enantiomeric excess.     

The mechanism of the stabilization of the hexagonal II (HII) phase in phosphatidylethanolamine membranes in the presence of low concentrations of dimethyl sulfoxide

Dimethyl sulfoxide (DMSO), a water-miscible organic solvent, has been used as a cryoprotectant for cells. It is known that DMSO stabilizes the HII phase of phosphatidylethanolamine (PE) membranes rather than the Lalpha phase, while most other water-miscible organic solvents such as acetone and ethanol destabilize the HII phase. To elucidate the mechanism for this stabilizing effect of DMSO on the HII phase, we have investigated its effects on the structures and physical properties of PE membranes. X-ray diffraction data indicated that dipalmitoleoylphosphatidylethanolamine (DPOPE) membranes in H2O at 20 degrees C were in the Lalpha phase and that an Lalpha to HII phase transition occurred at X=0.060 (mole fraction of DMSO) in water/DMSO mixtures. As the DMSO concentration increased, the basis vector length of the dioleoylphosphatidylethanolamine (DOPE)/ 16 wt% tetradecane membrane and also of the DPOPE/ 16 wt% tetradecane membrane in the HII phase decreased, suggesting that the spontaneous curvature of these membranes increased. We have also investigated the effects of DMSO on the physical properties of the PE membranes, and compared them with those of acetone. As the DMSO concentration increased, the excimer to monomer fluorescence intensities of pyrene-phosphatidylcholine in the PE membranes decreased, indicating that the membrane fluidity decreased, and also the generalized polarization value of the Laurdan fluorescent probe in the DPOPE membrane increased, indicating that the polarity of the membrane interface decreased. On the other hand, acetone had the opposite effects to DMSO. The interaction free energy between the membrane surface segments and solvent increased with an increase in DMSO concentration. It decreased the amount of solvent in the membrane interface, inducing an increase in the spontaneous curvature. This can reasonably explain the effects of DMSO on the phase stability and the physical properties of the membranes.     

Dynamic localization and function of Bni1p at the sites of directed growth in Saccharomyces cerevisiae

Formin homology (FH) proteins are implicated in cell polarization and cytokinesis through actin organization. There are two FH proteins in the yeast Saccharomyces cerevisiae, Bni1p and Bnr1p. Bni1p physically interacts with Rho family small G proteins (Rho1p and Cdc42p), actin, two actin-binding proteins (profilin and Bud6p), and a polarity protein (Spa2p). Here we analyzed the in vivo localization of Bni1p by using a time-lapse imaging system and investigated the regulatory mechanisms of Bni1p localization and function in relation to these interacting proteins. Bni1p fused with green fluorescent protein localized to the sites of cell growth throughout the cell cycle. In a small-budded cell, Bni1p moved along the bud cortex. This dynamic localization of Bni1p coincided with the apparent site of bud growth. A bni1-disrupted cell showed a defect in directed growth to the pre-bud site and to the bud tip (apical growth), causing its abnormally spherical cell shape and thick bud neck. Bni1p localization at the bud tips was absolutely dependent on Cdc42p, largely dependent on Spa2p and actin filaments, and partly dependent on Bud6p, but scarcely dependent on polarized cortical actin patches or Rho1p. These results indicate that Bni1p regulates polarized growth within the bud through its unique and dynamic pattern of localization, dependent on multiple factors, including Cdc42p, Spa2p, Bud6p, and the actin cytoskeleton.