Phytochromes confer the photoperiodic control of flowering in rice (a short-day plant).

The photoperiodic sensitivity 5 (se5) mutant of rice, a short-day plant, has a very early flowering phenotype and is completely deficient in photoperiodic response. We have cloned the SE5 gene by candidate cloning and demonstrated that it encodes a putative heme oxygenase. Lack of responses of coleoptile elongation by light pulses and photoreversible phytochromes in crude extracts of se5 indicate that SE5 may function in phytochrome chromophore biosynthesis. Ectopic expression of SE5 cDNA by the CaMV 35S promoter restored the photoperiodic response in the se5 mutant. Our results indicate that phytochromes confer the photoperiodic control of flowering in rice. Comparison of se5 with hy1, a counterpart mutant of Arabidopsis, suggests distinct roles of phytochromes in the photoperiodic control of flowering in these two species.

[Spinal anesthesia in a patient with spina bifida occulta].

A 32-year-old woman with spina bifida occulta was scheduled for hemorroidectomy under spinal anesthesia. Preoperatively, computed tomography and magnetic resonance imaging (MRI) were performed. The MRI demonstrated the conus medul laris reaching the L 3 level and a lipoma connected with conus medullaris intrathecally. Spinal anesthesia was done successfully at the L 3-4 interspace using 0.3% dibucaine 1.2 ml with 5% glucose 0.8 ml. Postoperatively she showed no neurologic complications. With exact anatomical findings of MRI, spinal anesthesia can be safely performed for patients with spina bifida occulta.

A monoclonal antibody affects chromophore apoprotein interactions of phytochrome A.

A monoclonal antibody designated Mep-1 was raised against phytochrome A from pea (Pisum sativum L.). The binding of this antibody (class IgG1) to partially degraded phytochrome (114 kDa) caused a considerable increase in the far-red peak at the red-light-induced stationary state. The effect reached a plateau value when the antibody and phytochrome were present in approximately equimolar amounts. The dark transformation of the far-red-light-absorbing form to the red-light-absorbing form of the 114 kDa phytochrome was inhibited by the addition of the antibody. However, binding of the antibody to the undegraded 121 kDa phytochrome had no effects on the spectrum of the red-light-induced steady state. The site at which the antibody bound to phytochrome was determined to be between amino acid residues 256 and 383 of pea phytochrome A. This is the first report of a monoclonal antibody that enhances the far-red absorption of phytochrome in the red-light-induced photostationary state.

[Postoperative analgesic requirements after various operations].

The difference in postoperative analgesic requirements for operations involving 11 sites was assessed in 1100 patients. Pain relief was mainly attained by epidural administration of bupivacaine 0.25% on demand. Postoperative analgesic was required most frequently after thoracotomy, followed by gastrectomy with left diagonal approach, gastrectomy with median approach, operation for colon cancer, cholecystectomy and hysterectomy. After hip operation, periproctal operation, radical mastectomy, appendectomy and herniorrhephies, total analgesic requirement was similar and the minimum amount required. In conclusion, there was a high degree of correlation between the severity of the pain and the site of the operation.

[A fluoroscopic analysis of the catheters abnormally placed in the epidural space].

In order to know how the catheters are placed abnormally in the epidural space, we inserted the epidural catheters under fluoroscopy. The catheter was placed straightly upward when the epidural puncture was done in the center of epidural space. When the puncture site was nearly close to the side wall of epidural space, we could not insert catheter at all. When the tip of the needle was near the side wall and the inserting catheter slipped upward, catheter was placed straight. When the catheter wedged at the side wall, it made loop and curled or kinked. We thought that the cause of abnormal location of the catheters in epidural space is the right angle between the wall of the epidural space and the direction of catheter.

Phototransformation of pea phytochrome A induces an increase in alpha-helical folding of the apoprotein: comparison with a monocot phytochrome A and CD analysis by different methods.

The photoreversible conformational change associated with the Pr-->Pfr transformation of a dicot phytochrome A (Pisum sativum, pea) has been probed by circular dichroism (CD) studies. Three different CD analysis methods have been used to determine the secondary structure of pea phytochrome A in both Pr and Pfr forms. We have shown that the secondary structure of dicot pea phytochrome A is very similar to the structure of monocot oat phytochrome A which was determined earlier [Sommer & Song (1990) Biochemistry 29, 1943-1948]. As with oat phytochrome A, an increase in the alpha-helical folding of the apoprotein takes place when photochrome in the Pr form is phototransformed to the Pfr form. This conformational change might well be a general characteristic of all phytochrome A's.

Resonance Raman spectra of the intermediates in phototransformation of large phytochrome: deprotonation of the chromophore in the bleached intermediate.

Resonance Raman (RR) scattering from type A large phytochrome of pea was measured at cryogenic as well as ambient temperatures to determine an intermediate in which deprotonation of the chromophore takes place. The RR bands of the red-absorbing (Pr) and far-red-absorbing forms (Pfr) of large pea phytochromes at ambient temperatures are almost the same in their frequencies as those of the intact form reported previously (Mizutani et al., 1991). The RR spectrum of large phytochrome excited at 364 nm at -120 degrees C, where Pr and a photointermediate, I700 (= lumi-R), are trapped, showed a strong band at 1625 cm-1 with a shoulder at 1648 cm-1 in the C C=C stretching region. The shoulder disappeared, and a new band appeared at 1597 cm-1 upon raising the temperature to -80 degrees C, where transformation from I700 to meta-Ra proceeds. The RR spectra remained unchanged until -10 degrees C, indicating that the RR spectra of meta-Rb and meta-Rc are close to that of meta-Ra, and we call them comprehensively the bleached intermediate, Ibl. At ambient temperatures where photo-steady-states among a few species are attained, strong RR bands were observed at 1625 and 1599 cm-1 upon excitation at 364 nm under simultaneous far-red illumination, and the 1599-cm-1 band was appreciably intensified under simultaneous red- instead of far-red illumination. By comparison of these spectra with those at low temperatures, the 1625- and 1599-cm-1 bands were reasonably assigned to Pr and Ibl, respectively. A chemically prepared model of the bleached form, Pbl, also gave a prominent band at 1599 cm-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultraviolet resonance Raman spectra of pea intact, large, and small phytochromes: differences in molecular topography of the red- and far-red-absorbing forms.

Ultraviolet resonance Raman (UV RR) spectra excited at 244 nm were observed for pea intact, large, and small phytochromes at pH 7.8. Raman bands assignable to Trp residues dominated the UV RR spectra. The intensity ratios of Trp W7 doublet bands, I(1358)/I(1342), of all three phytochromes in the red light-absorbing form (Pr) were almost the same as that of an aqueous Trp solution, indicating that most of the six and four Trp residues in the 59-kDa chromophoric and the C-terminal 59-kDa nonchromophoric domains, respectively, reside in hydrophilic microenvironments in Pr. This ratio increased under red light illumination, where photoequilibria are attained between Pr and the far-red-absorbing form (Pfr) for intact and small phytochromes and among Pr, a bleached intermediate (Ibl), and Pfr for large phytochromes. The increase of the intensity ratio was most prominent for small phytochromes. These observations suggest that the microenvironments around some Trp residues become more hydrophobic due to conformational changes induced by phototransformation from Pr to Ibl and that the hydrophobicity increase occurs mainly in the chromophoric domain. Among the six Trp residues in the chromophoric domain, Trp365 and Trp567 are likely candidates for those involved in this hydrophobicity increase. The intensity distribution of the amide I band shows little beta-sheet in both Pr and Pfr of the intact, large, and small phytochromes and indicates that alpha-helices and nonregular structure are less populated in the chromophoric domain than in the N-terminal 6-kDa segment and the C-terminal nonchromophoric domain.

Preventative effect of PGE1 for postoperative liver damage.

The efficacy of a low dose of PGE(1)-use on the postoperative liver damage was evaluated. PGE(1) was infused in with the mean rate of 0.026 microg.kg(-1).min(-1) during surgical procedure to 93 patients under GO-enflurane anesthesia (the PG). Serum GOT, GPT and total bilirubin (TBIL) values measured before, at the end of (End) and 3 days (3d) after the operation were compared to those obtained from 43 patients without PGE(1) administration (the control). This dose of PGE(1) did not change blood pressure and heart rate, but slightly decreased Pa(O)(2). In patients with preoperative normal values of GOT, GPT and TBIL, increases in GOT, GPT and TBIL observed at End in the PG were significantly lower than those in the control (31.9 vs 72.2 IU, 25.9 vs 61.9 IU, 0.68 vs 0.83 mg.dl(-1), respectively). GOT, GPT and TBIL at 3d significantly increased in both groups, and these levels were identical between the two groups. In patients with preoperative abnormal values, only GOT at End increased in both groups, while no significant difference between the PG and the control group was noted. GOT at 3d and GPT at End and 3d did not significantly changed in either group. These results suggest that the low dose of PGE(1) administered during an operation prevents the development of postoperative liver damage, but does not treat the damaged hepatic cells.

Resonance Raman study on intact pea phytochrome and its model compounds: evidence for proton migration during the phototransformation.

Resonance Raman (RR) scattering from intact pea phytochrome was observed in resonance with the blue band at ambient temperature. The relative populations of the red-light-absorbing form (Pr) and far-red-light-absorbing form (Pfr) under laser illumination were estimated from the absorption spectra. The most prominent RR band of Pr obtained by 364-nm excitation under 740-nm pumping exhibited a frequency shift between H2O and D2O solutions, but that of Pfr obtained by 407-nm excitation under 633-nm pumping did not, indicating a distinct difference in a protonation state of their chromophores. Since the protonation level of a whole molecule of intact phytochrome remains unchanged between Pr and Pfr, this observation indicates migration of a proton from the chromophore of Pr to the protein moiety of Pfr. As model compounds, octaethylbiliverdin (OEBV-h3), its deuterated and 15N derivatives, and their protonated forms were also studied with both RR and 1H and 15N NMR spectroscopies. The RR spectrum of the protonated form, for which the protonation site was determined to be C-ring pyrrole nitrogen by NMR, displayed a deuteration shift corresponding to that of Pr, suggesting a similar protonated structure for the pyrrolic rings of Pr. The RR spectral difference between OEBV-h3 and OEBV-d3 and that between H2O and D2O solutions of Pfr suggested that the N-H protons of the A-, B-, and D-rings of intact phytochrome are replaced with deuterons in D2O. A role of the 7-kDa segment of phytochrome is discussed on the basis of RR spectral differences between the intact and large phytochromes.

Formation of aggregates of tryptic fragments derived from the carboxyl-terminal half of pea phytochrome and localization of the site of contact between the fragments by amino-terminal amino acid sequence analysis.

Limited trypsinization of large pea (Pisum sativum cv. Alaska) phytochrome and subsequent size-exclusion chromatography (SEC) in 0.1 M Na phosphate, pH 7.8, yielded a high-molecular-mass aggregate of tryptic fragments of phytochrome. Further SEC in 0.1 M Tris-HCl, pH 7.5, plus various concentrations of NaSCN, indicated that the tryptic-fragment complex contained an aggregate of 7 fragments of molecular mass from 38 to 55 kDa. The amino-terminal sequence of each fragment was determined from the samples electroblotted from sodium dodecylsulfate polyacrylamide gels onto polyvinylidene difluoride membranes, in order to localize the various fragments on the phytochrome polypeptide chain. All of the 7 fragments in the aggregate were found to be derived from the carboxyl-terminal half of phytochrome. A portion of the polypeptide chain (from Ala-752 to Arg-1000) common to all the tryptic fragments has been assigned as the site(s) of contact of the fragments. The tryptic-fragment complex, as well as large phytochrome itself, has been shown by SEC to dissociate to monomers in 2 M NaSCN. The result indicates that the main force involved in maintaining the complex and in contacts between monomers of phytochrome is non-ionic in nature. Relationship between the contact site(s) of the tryptic-fragment complex and large phytochrome monomer is discussed.

Primary photoprocesses of phytochrome. Picosecond fluorescence kinetics of oat and pea phytochromes.

The primary photoprocesses of etiolated oat and pea phytochromes (Pr forms) are diffusion-modulated by the microscopic viscosity within the chromophore pocket. The chromophore pocket is preferentially accessible to glycerol but not to Ficoll. Glycerol preferentially retarded the rate (rate constant ca. 1-2 X 10(10) s-1) of the initial reaction from the Qy excited state of phytochrome, whereas it increased the long fluorescence lifetime (nanosecond) component that can be attributed to either an emitting intermediate or to modified/conformationally heterogeneous phytochrome populations. The picosecond time-resolved fluorescence spectra of different phytochrome preparations (i.e., full-length vs 6/10-kDa NH2-terminus truncated forms of phytochromes from monocot and dicot plants) revealed no significant differences. The spectra in the picosecond time scale showed no spectral shifts, but at longer time scales of up to approximately 1.90 ns, significant blue spectral shifts were observed. The shifts were more in the truncated than in the full-length pea phytochrome. Comparison of the fluorescence decay data and the picosecond time-resolved fluorescence spectra suggests differences in conformational flexibility/heterogeneity among the preparations of the monocot vs dicot phytochromes and the full-length native vs the amino terminus truncated phytochromes.

Ca2+-induced phase separation in phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine mixed membranes.

Ca2+-induced phase separation in phosphatidylserine/phosphatidylethanolamine and phosphatidylserine/phosphatidylethanolamine/phosphatidylcholine model membranes was studied using spin-labeled phosphatidylethanolamine and phosphatidylcholine and compared with that in phosphatidylserine/phosphatidylcholine model membranes studied previously. The phosphatidylethanolamine-containing membranes behaved in qualitatively the same way as did phosphatidylserine/phosphatidylcholine model membranes. There were some quantitative differences between them. The degree of phase separation was higher in the phosphatidylethanolamine-containing membranes. For example, the degree of phase separation in phosphatidylserine/phosphatidylethanolamine membranes containing various mole fractions of phosphatidylserine was 94--100% at 23 degrees C and 84--88% at 40 degrees C, while the corresponding value for phosphatidylserine/phosphatidylcholine membranes was 74--85% at 23 degrees C and 61--79% at 40 degrees C. Ca2+ concentration required for the phase separation was lower for phosphatidylserine/phosphatidylethanolamine than that for phosphatidylserine/phosphatidylcholine membranes; concentration to cause a half-maximal phase separation was 1.4 . 10(-7) M for phosphatidylserine-phosphatidylethanolamine and 1.2 . 10(-6) M for phosphatidylserine/phosphatidylcholine membranes. The phase diagram of phosphatidylserine/phosphatidylethanolamine membranes in the presence of Ca2+ was also qualitatively the same as that of phosphatidylserine/phosphatidylcholine except for the different phase transition temperatures of phosphatidylethanolamine (17 degrees C) and phosphatidylcholine (-15 degrees C). These differences were explained in terms of a greater tendency for phosphatidylethanolamine, compared to phosphatidylcholine, to form its own fluid phase separated from the Ca2+-chelated solid-phase phosphatidylserine domain.

Proton-induced phase separation in phosphatidylserine/phosphatidylcholine membranes.

Effects of ph and ionic strength on phosphatidylserine/phosphatidylcholine mixed membranes prepared on Millipore filter pore surfaces have been studied using spin-labeled phosphatidylcholine. Lowering pH at constant ionic strength and lowering ionic strength at constant pH caused a lateral reorganization of the membrane. The trigger was protonation of the serine carboxyl group which caused solidification of phosphatidylserine molecules in the membrane, leaving a fluid phase consisting mainly of phosphatidylcholine. The appearent pK for the proton-induced phase separation was measured in a wide range of salt concentrations. The ionic strength dependence was satisfactorily explained based on the electrostatic free energy of proton in the field of membrane surface potential. The Gouy-Chapman theory gave a good approximation for the surface potential. The surface pK of phosphatidylserine and phosphatidic acid vesicles was directly measured in various salt concentrations by 31P-NMR and the results confirmed validity of the Gouy-Chapman-type analysis. The lateral reorganization was triggered by electrostatic interaction but the bulk of the stabilization energy for the structural changes would be the gains in intermolecular van der Waals energy due to closer packing of phosphatidylserine on solidification.

Disappearance of calcium-induced phase separation in phosphatidylserine-phosphatidylcholine membranes caused by protonation and by electric current.

Disappearance of Ca2+-induced phase separation in phosphatidylserine-phosphatidylcholine membrane has been studied under several conditions by monitoring electron spin resonance spectrum of spin-labeled phosphatidylcholine. The membranes were prepared in Millipore filters. Electron micrographs of the pre parations showed formation of multilayered structures lined on the pore surface. The phase separation was disappeared when the membrane was soaked in non-buffered salt solution (100 ml KCl, pH 5.5). It was markedly contrasting that when the bathing salt solution was buffered no disappearance was observed. Disappearance of the phase separation was also observed when the Ca2+-treated membrane was transferred to acidic salt solutions (less than or equal to pH 2.5) or to low ionic strength media (less than or equal to mM) buffered at pH 5.5, and then to the buffered salt solution (100 mM KCl, pH 5.5). These are due to replacement of Ca2+ by proton, proton-induced separation, followed by disappearance of the phase separation in the buffered salt solution. Biological significance of the competition between Ca2+ and proton for the phase separation or domain formation in the membranes was emphasized.