Tatsuo Iwasa

Enantioselective Diels–Alder Reaction of 1,2-Dihydropyridines with Aldehydes Using β-Amino Alcohol Organocatalyst

The enantioselective Diels-Alder reaction of 1,2-dihydropyridines with aldehydes using an easily prepared optically active β-amino alcohol catalyst was found to provide optically active isoquinuclidines, an efficient synthetic intermediate of pharmaceutically important compounds such as oseltamivir phosphate, with a satisfactory chemical yield and enantioselectivity (up to 96%, up to 98% ee). In addition, the obtained highly optically pure isoquinuclidine was easily converted to an optically active piperidine having four successive carbon centers.

G Protein α Subunit Genes in Octopus Photoreceptor Cells

Abstract The G protein messages in Octopus vulgaris photoreceptor were isolated and characterized with molecular biological techniques. Four classes of G protein α subunit cDNA were isolated from an octopus eye cDNA library: OvGαi, OvGαo, OvGαq and OvGαs. Northern blot analysis of octopus tissues showed abundant expression of OvGαq in the eye, and specific expression of OvGαo in neural tissues. OvGαi was expressed in all tissues studied. In situ hybridization revealed that OvGαq message was expressed in almost all photoreceptor cells. Based on these results, a possible phototransduction pathway in the octopus visual cells is discussed.

Heterotrimeric G protein subunits differentially respond to endoplasmic reticulum stress in Arabidopsis

Canonical heterotrimeric G proteins in eukaryotes are major components that localize at plasma membrane and transmit extracellular stimuli into the cell. Genome of a seed plant Arabidopsis thaliana encodes at least one Gα (GPA1), one Gβ (AGB1), and 3 Gγ (AGG1, AGG2 and AGG3) subunits. The loss-of-function mutations of G protein subunit(s) cause multiple defects in development as well as biotic and abiotic stress responses. However, it remains elusive how these subunits differentially express these defects. Here, we report that Arabidopsis heterotrimeric G protein subunits differentially respond to the endoplasmic reticulum (ER) stress. An isolated homozygous mutant of AGB1, agb1-3, was more sensitive to the tunicamycin-induced ER stress compared to the wild type and the other loss-of-function mutants of G protein subunits. Moreover, ER stress responsive genes were highly expressed in the agb1-3 plant. Our results indicate that AGB1 positively contributes to ER stress tolerance in Arabidopsis.

Photoreaction Cycle of Phoborhodopsin (Sensory Rhodopsin II) from Halobacterium salinarum Expressed in Escherichia coli

Phoborhodopsin (pR; also called sensory rhodopsin II, SRII) is a photoreceptor of negative phototaxis of halobacteria. The studies of photochemical properties of this pigment are not many because the amount of the pigment is small and the stability is low. Recently an expression system of phoborhodopsin from Halobacterium salinarum (called salinarum phoborhodopsin, spR; also HsSRII) in Escherichia coli and purification method has been developed (Mironova et al. [2005] FEBS Lett., 579, 3147–3151), which enables detailed studies on the photochemical properties of spR. In the present work, the photoreaction cycle of E. coli‐expressed spR was studied by low‐temperature spectroscopy and flash photolysis. Formations of K‐, M‐, O‐like intermediates and P480 were reconfirmed as reported previously. New findings are as follows. (1) The K‐like intermediate (P500) was a mixture of two photoproducts. (2) Formation of L‐like intermediate (P482) was observed by low‐temperature spectroscopy and flash photolysis at room temperature. (3) On long irradiation of spR at 20°C, formation of a new photoproduct P370 was observed and it decayed to the original spR in the dark with a decay half time of 190 min. Based on these results the similarities and dissimilarities between spR and ppR are discussed.

Anti-Bovine Rhodopsin Monoclonal Antibody Recognizing Light-Dependent Structural Change

Abstract The antigenic structure of the bovine rhodopsin molecule was investigated by using a bovine rhodopsin-specific monoclonal antibody designated Rh29. Competition assay with sealed intact disks and broken disks indicated that the antibody-binding region was localized in the intradiscal surface. An antigenic peptide obtained by a cyanogene bromide cleavage of rhodopsin was purified and determined as residues 2–39 in the amino acid sequence. Further analysis suggested that the antigenic determinant included at least residues 21–25. These results were consistent with the structural model for membrane topology of rhodopsin. The antigenicity of the rhodopsin was compared among several states. The antibody bound to both ammonyx LO-solubilized unbleached and bleached rhodopsin. In contrast, upon membrane-embedded rhodopsin, unbleached one was 100-times less antigenic than bleached one. The results suggested that the segment around the determinant of membrane-embedded rhodopsin should undergo a structural change upon absorption of light. Rh29 detected a band corresponding to bovine, porcine and octopus opsins in immunoblotting. Protein blot of crayfish rhabdome did not show any reactive band. These bands except for crayfish reacted with concanavalin A as well. The N-terminal structure may, therefore, conserved between mammal and erthropoda and diverge between them and cepharopoda.

Tryptophan 171 in Pharaonis phoborhodopsin (sensory rhodopsin II) interacts with the chromophore retinal and its substitution with alanine or threonine slowed down the decay of M-and O-intermediate

Pharaonis phoborhodopsin (ppR), also called pharaonis sensory rhodopsin II, NpSRII, is a photoreceptor for the photophobic response of Natronomonas pharaonis. Tryptophan 182 (W182) of bacteriorhodopsin (bR) is near the chromophore retinal and has been suggested to interact with retinal during the photoreaction and also to be involved in the hydrogen‐bonding network around the retinal. W182 of bR is conserved in ppR as tryptophan 171 (W171). To elucidate whether W171 of ppR interacts with retinal during the photoreaction and/or is involved in the hydrogen‐bonding network as in bR, we formed W171‐substituted mutants of ppR, W171A and W171T. Our low‐temperature spectroscopic study has revealed that the substitution of W171 to Ala or Thr resulted in the stabilization of M‐ and O‐intermediates. The stability of M and absorption spectral changes during the M‐decay were different depending on the substituted residue. These findings suggest that W171 in ppR interacts with retinal and the degree of the interaction depends on the substituted residues, which might be rate determining in the M‐decay. In addition, the involvement of W171 in the hydrogen‐bonding network is suggested by the O‐decay. We also found that glycerol slowed the decay of M and not of O.

Heterotrimeric G Protein α and β Subunit Genes of the Ascidian, Halocynthia roretzi

In order to investigate G protein-coupling signal transduction system of ascidians in developmental stages and in embryonic neural system, we have cloned G protein α and β subunit genes and studied their spatial and temporal expression patterns in the ascidian, Halocynthia roretzi. Five different cDNA clones of G protein α subunit were isolated from a cDNA library of ascidian larvae. The deduced amino acid sequences of three of them (HrGiα, HrGqα, and HrGsα) showed high homology to those of α subunits of human Gi Gq, and Gs, respectively. The other two (HrGnα and HrGxα) was supposed to define a novel subfamily within the Gj and Gq families, respectively. Northern blot analysis revealed that messages of all these G protein α subunits were found during embryogenesis and in adult organs. Whole-mount in situ hybridization of tadpole larvae showed a distinct spatial expression pattern of the each α subunit gene.

Photocycle of Sensory Rhodopsin II from Halobacterium salinarum (HsSRII): Mutation of D103 Accelerates M Decay and Changes the Decay Pathway of a 13-cis O-like Species

Aspartic acid 103 (D103) of sensory rhodopsin II from Halobacterium salinarum (HsSRII, or also called phoborhodopsin) corresponds to D115 of bacteriorhodopsin (BR). This amino acid residue is functionally important in BR. This work reveals that a substitution of D103 with asparagine (D103N) or glutamic acid (D103E) can cause large changes in HsSRII photocycle. These changes include (1) shortened lifetime of the M intermediate in the following order: the wild‐type > D103N > D103E; (2) altered decay pathway of a 13‐cis O‐like species. The 13‐cis O‐like species, tentatively named Px, was detected in HsSRII photocycle. Px appeared to undergo branched reactions at 0°C, leading to a recovery of the unphotolyzed state and formation of a metastable intermediate, named P370, that slowly decayed to the unphotolyzed state at room temperature. In wild‐type HsSRII at 0°C, Px mainly decayed to the unphotolyzed state, and the decay reaction toward P370 was negligible. In mutant D103E at 0°C, Px decayed to P370, while the recovery of the unphotolyzed state became unobservable. In mutant D103N, the two reactions proceeded at comparable rates. Thus, D103 of HsSRII may play an important role in regulation of the photocycle of HsSRII.

Analysis of Surface Acoustic Wave Propagation Velocity in Biological Function-Oriented Odor Sensor

We describe the measurement of surface acoustic wave (SAW) velocities to identify five odorant molecules by using a SAW device system. We derive a new frequency equation for SAWs propagating in the SAW device system with Cynops pyrrhogaster lipocalin (Cp-Lip1) protein, in order to identify five odorant molecules, R-limonene (R-Lim), ethyl butyrate (Eth), 2-isobutylthiazole (Iso), benzophenone (Ben), and 2-acetylthiazole (Ace). We developed a method to identify these odorant molecules combined with the Cp-Lip1 odorant-binding protein. Our frequency equation can satisfactorily predict different odorant molecules in the Cp-Lip1 SAW device. Moreover, our data suggest that the propagation velocity of the SAWs mostly relate to the density and concentration of the Cp-Lip1 odorant molecule mixtures. At the same sample concentration, the propagation velocity depends on the density. For the same odorant molecule, the propagation velocity decreases with increasing concentration.

Effect of Concentration on Surface Acoustic Wave Velocity in A Rayleigh-type Biosensor

To predict the propagation velocity of different concentrations of bovine serum albumin (BSA) in a Rayleigh-type biosensor, we derived a new calculation method for a Rayleigh surface acoustic wave (SAW) device. The method can calculate propagation velocities of leaky Rayleigh waves and Rayleigh waves when liquid is placed in the groove of a Rayleigh-type SAW device. Different concentration of BSA proteins were used to evaluate the effect of these concentrations. In this study, we used different concentrations of BSA and a Rayleigh-type SAW device consisted of grooves of 0.25 mm width and 0.26 mm height and grooves of 0.40 mm width and 0.25 mm height, quartz as the substrate and interdigital transducers (IDT) with a 25.00 mm distance. The frequency of the Rayleigh-type SAW device was set to 157.6 MHz. Our data suggests that the propagation velocity of SAWs depends mainly upon BSA protein concentration and the size of groove. At the same concentration of BSA, the propagation acoustic wave velocity of BSA depends on the size of groove. The results showed the 0.40 mm width-0.25 mm height groove was more suitable than the 0.25 mm width-0.26 mm height groove for studying the propagation velocity of wave with liquid in a Rayleigh-type SAW device. With the same groove, the propagation velocity decreased when the concentration of BSA protein increased.