Kazuo Yoshihara

Photoisomerization of retinal at 13-ene is important for phototaxis of Chlamydomonas reinhardtii: Simultaneous measurements of phototactic and photophobic responses

A real-time automated method was developed for simultaneous measurements of phototactic orientation (phototaxis) and step-up photophobic response of flagellated microorganisms. Addition of all-trans retinal restored both photoresponses in a carotenoid-deficient mutant strain of Chlamydomonas reinhardtii in a dose-dependent manner. The phototactic orientation was biphasic with respect to both the light intensity and the concentration of retinal. All-trans retinal was more effective than 11-cis retinal to regenerate both photobehavioral responses. Analogs having locked 11-cis configurations and a phenyl ring in the side chain also induced photoresponses, although at concentrations more than two orders of magnitude higher than all-trans retinal. According to the present assay method, the responses were hardly detectable in cells incubated with retinal analogs in which the 13-ene was locked in either its trans or cis configuration. The results strongly suggest that the isomerization of the 13-14 double bond is important for photobehavioral signal transduction and that a single retinal-dependent photoreceptor controls both phototactic and photophobic responses.

Diversion of the sign of phototaxis in a Chlamydomonas reinhardtii mutant incorporated with retinal and its analogs

The blind mutant FN68 of the unicellular flagellate green alga Chlamydomonas reinhardtil is negatively phototactic in the presence of the native chromophore all‐trans retinal. In contrast, analog chromophores such as a ring‐acyclic retinal and those in which trans/cis isomerization about the C11–C12 double bond was blocked induced predominantly positive pho totaxis in the same strain under the same experimental conditions. These observations can be interpreted by assuming that the negative and the positive phototaxis is mediated distinctively by two rhodopsin species which differ in their affinities with the exogenous chromophores. However, a more reasonable explanation, which requires fewer assumptions, is that the sign of phototaxis depends on a delay in intracellular photosignal transduction. This novel view was deduced directly from the widely accepted Hypothesis [1980, Microbiol. Rev. 44, 572–630] on phototaxis mechanisms.

Evidence for Proton Transfer from Glu-46 to the Chromophore during the Photocycle of Photoactive Yellow Protein

Photoactive yellow protein (PYP) belongs to the novel group of eubacterial photoreceptor proteins. To fully understand its light signal transduction mechanisms, elucidation of the intramolecular pathway of the internal proton is indispensable because it closely correlates with the changes in the hydrogen-bonding network, which is likely to induce the conformational changes. For this purpose, the vibrational modes of PYP and its photoproduct were studied by Fourier transform infrared spectroscopy at −40 °C. The vibrational modes characteristic for the anionic p-coumaryl chromophore (Kim, M., Mathies, R. A., Hoff, W. D., and Hellingwerf, K. J. (1995)Biochemistry 34, 12669–12672) were observed at 1482, 1437, and 1163 cm−1 for PYP. However, the bands corresponding to these modes were not observed for PYPM, the blue-shifted intermediate, but the 1175 cm−1 band characteristic of the neutral p-coumaryl chromophore was observed, indicating that the phenolic oxygen of the chromophore is protonated in PYPM. A 1736 cm−1 band was observed for PYP, but the corresponding band for PYPM was not. Because it disappeared in the Glu-46 → Gln mutant of PYP, this band was assigned to the C=O stretching mode of the COOH group of Glu-46. These results strongly suggest that the proton at Glu-46 is transferred to the chromophore during the photoconversion from PYP to PYPM.

4-Hydroxyretinal, a new visual pigment chromophore found in the bioluminescent squid, Watasenia scintillans

The bioluminescent squid, Watasenia scintillans has three visual pigments. The major pigment, based on retinal (lambda max 484 nm), is distributed over the whole retina. Another pigment based on 3-dehydroretinal (lambda max approximately 500 nm) and the third pigment (lambda max approximately 470 nm) are localized in the specific area of the ventral retina just receiving the downwelling light. Visual pigment was extracted and purified from the dissected retina. The chromophores were then extracted and analyzed with HPLC, NMR, infrared and mass spectroscopy, being compared with the synthetic 4-hydroxyretinal. A new retinal derivative, 11-cis-4-hydroxyretinal, is identified as the chromophore of the third visual pigment of the squid.

A facile method for the conversion of oximes to ketones and aldehydes by the use of activated MnO2

Abstract Activated manganese dioxide (MnO 2 ) was found to be an efficient oxidant in the conversion of oximes to carbonyl compounds. The utility of this method in synthesis was demonstrated by the conversion of galactose to its acyclic aldose derivative 5.

Typhasterol (2-deoxycastasterone): a new plant growth regulator from cat-tail pollen

Abstract Typhasterol, a new plant growth promoting substance from Typha latifolia L. was determined to be (22 R ,23 R ,24 S )-3a,22,23-trihydroxy-24-methyl-5α-cholestan-6-one.

Phosphatidyl inositol-phospholipase C in squid photoreceptor membrane is activated by stable metarhodopsin via GTP-binding protein, Gq

Phosphatidyl inositol-phospholipase C (PI-PLC) in squid retina was studied by immunoblotting and its activities were determined using [3H]phosphatidyl inositol bisphosphate ([3H]PIP2) as substrate. PI-PLC activity was found mostly in soluble fraction when the retina homogenate was treated with 400 mM KCl, but was associated with rhabdomal membranes under low salt conditions (20 mM Hepes). A protein with apparent molecular mass of 130kD was recognized by an antibody against PLC beta 4/norp A in both 400 mM KCl soluble and rhabdomal membrane fractions. A 42 kD protein recognized by antibody against the C-terminus of Gq alpha was also present in these two fractions. GTP gamma S stimulated only the PI-PLC activity associated with membrane and was magnesium dependent. PI-PLC activity was found to be (i) highly dependent upon calcium concentrations, (ii) enhanced by GTP but not by other nucleotides, and (iii) significantly stimulated by light at lower concentrations of GTP gamma S. The stimulation by light was still observed when irradiated membrane was incubated at 10 degrees C for 10 min and then mixed with GTP gamma S. These results suggest that stable metarhodopsin stimulates a PLC beta 4/norp A-like enzyme via a G-protein, Gq.

Revisions of the absolute configurations of C-8 methyl groups in dehydroiridodiol, neonepetalactone, and matatabiether from Actinidia polygama miq

Abstract The absolute configurations of C-8 Me groups in dehydro-iridodiol, neonepetalactone, and matatabiether isolated from the cat- and lacewmg-attracting plant Actinidia polygama Miq. were revised to the S configurations on the basis of chemical transformations and unambiguous syntheses.

Facile synthesis of 6-hydroxyindole-3-acetic acid: On the structure of the aromatic subunit of nephilatoxin-1∼6

Abstract A facile synthesis of 6-hydroxyindole-3-acetic acid 1a, which is the proposed aromatic subunit of NPTX-1∼6, is described. Radical cyclization of isonitrile 2 successfully afforded 9 in high yield. The aromatic subunit of NPTX-1∼6 was confirmed as 4-hydroxyindole-3-acetic acid 12 by comparison of the 1H-NMR spectra with those of authentic 4- and 6-hydroxyindole-3-acetic acids.

A putative binding protein for lipophilic substances related to butterfly oviposition

A unique protein of 23 kDa (Jf23) was found in the tarsus of the female swallowtail butterfly, Atrophaneura alcinous. Jf23 has 38% identity with a bilin‐binding protein, which was found in the cabbage butterfly, Pieris brassicae, and which has two consensus sequences in common with the members of the lipocalin family, suggesting that it is a binding protein for lipophilic ligands. Western blot analysis showed that Jf23 was expressed only in the female, and not in the male. Electrophysiological response of the female tarsi was stimulated by methanolic extract of their host plant, Dutchmans pipe (Aristolochia debilis). The stimulated response was depressed by the presence of Jf23 antiserum. These results suggest that Jf23 is one of the chemosensory signaling proteins, which plays one or more roles in female butterfly oviposition.