Biomedical subjects

Y Ohmiya

Publications and source records attributed to Y Ohmiya.

At least 19 recordsLinked to original sources

PubMed2006

Active-site properties of Phrixotrix railroad worm green and red bioluminescence-eliciting luciferases.

The luciferases of the railroad worm Phrixotrix (Coleoptera: Phengodidae) are the only beetle luciferases that naturally produce true red bioluminescence. Previously, we cloned the green- (PxGR) and red-emitting (PxRE) luciferases of railroad worms Phrixotrix viviani and P. hirtus[OLE1]. These luciferases were expressed and purified, and their active-site properties were determined. The red-emitting PxRE luciferase displays flash-like kinetics, whereas PxGR luciferase displays slow-type kinetics. The substrate affinities and catalytic efficiency of PxRE luciferase are also higher than those of PxGR luciferase. Fluorescence studies with 8-anilino-1-naphthalene sulfonic acid and 6-p-toluidino-2-naphthalene sulfonic acid showed that the PxRE luciferase luciferin-binding site is more polar than that of PxGR luciferase, and it is sensitive to guanidine. Mutagenesis and modelling studies suggest that several invariant residues in the putative luciferin-binding site of PxRE luciferase cannot interact with excited oxyluciferin. These results suggest that one portion of the luciferin-binding site of the red-emitting luciferase is tighter than that of PxGR luciferase, whereas the other portion could be more open and polar.

Animals↗

PubMed2004

Agrobacterium tumefaciens-mediated transformation of embryogenic tissue and transgenic plant regeneration in Chamaecyparis obtusa Sieb. et Zucc.

A genetic transformation procedure for Chamaecyparis obtusa was developed after co-cultivation of embryogenic tissues with disarmed Agrobacterium tumefaciens strain C58/pMP90, which harbours the sgfp (synthetic green fluorescent protein) visual reporter and nptII (neomycin phoshotransferase II) selectable marker genes. The highest transformation frequency was 22.5 independent transformed lines per dish (250 mg embryogenic tissue) following selection on kanamycin medium. Transgenic plantlets were regenerated through the maturation and germination of somatic embryos. The intensity of GFP fluorescence, observed under a fluorescence microscope, varied from very faint to relatively strong, depending on the transgenic line or part of the transgenic plant. The integration of the genes into the genome of regenerated plantlets was confirmed by Southern blot analysis.

Agrobacterium tumefaciens↗

PubMed2004

Cloning and characterization of the cDNA for the Brazilian Cratomorphus distinctus larval firefly luciferase: similarities with European Lampyris noctiluca and Asiatic Pyrocoelia luciferases.

Studies on firefly (Lampyridae) luciferases have focused on nearctic species of Photinus and Photuris and Euroasiatic species of Lampyris, Luciola, Hotaria, and Pyrocoelia. Despite accounting for the greatest diversity of fireflies in the world, no molecular studies have been carried out on the highly diverse genera from the neotropical region. Here we report the luciferase cDNA cloning for the larva of the Brazilian firefly Cratomorphus distinctus. The cDNA has 1978 bp and codes for a 547-residue-long polypeptide. Noteworthy, sequence comparison as well as functional properties show the highest degree of similarity with Lampyris noctiluca (93%) and Pyrocoelia spp. (91%) luciferases, suggesting a close phylogenetic relationship despite the geographical distance separating these species. The bioluminescence emission spectrum peaks at 550 nm and, as expected, is sensitive to pH, shifting to 605 nm at pH 6. The kinetic properties of the recombinant luciferase were similar to those of other firefly luciferases.

Animals↗

PubMed2001

Continuous measurement of targeted promoter activity by a secreted bioluminescence reporter, Vargula hilgendorfii luciferase.

The promoter activity of growth hormone (GH) was continuously monitored in rat pituitary adenoma cells (GH3) by a secreted bioluminescence reporter, Vargula hilgendorfii (Vh) luciferase. The sensitivity of the reporter was approximately 60-times higher than that of the firefly luciferase. GH3 cells were transfected with a plasmid containing a DNA sequence of the GH promoter (1.8 kb) and a full length of the Vh luciferase cDNA. Using the stable transformants, the Vh luciferase activity was monitored in the plate culture through the bioluminescence of Vh luciferase secreted into the culture medium. The reporter activity was well correlated with GH mRNA as well as GH when the GH promoter activity was activated by thyroid hormone. To develop a continuous monitoring system of the promoter, the reporter activity was sequentially measured in the perfusion system. When the promoter activity of the stable transformants was suppressed by a transcription inhibitor, the reporter activity and GH in the perfusate were simultaneously decreased. The Vh luciferase reporter is a sensitive and convenient tool for continuous and prolonged measurement of promoter activity in living cell culture systems.

Animals↗

PubMed2001

Thr226 is a key residue for bioluminescence spectra determination in beetle luciferases.

The comparison of click beetle and railroadworm luciferases (pH-insensitive) with firefly luciferases (pH-sensitive) showed a set of conserved residues differing between the two groups which could be involved with the bioluminescence spectra pH sensitivity. The substitution C258V in Pyrocoelia miyako (Pml) firefly luciferase and V255C in Ragophthalmus ohbai railroad worm luciferase (Rol) had no effect on the bioluminescence spectra. Substitution of Thr226 in the green-light-emitting luciferases of Rol and Pyrearinus termitilluminans (Pyt) click beetle luciferases resulted in red-shifts (12 to 35 nm), whereas the substitution T226N in the red-light-emitting luciferase of Phrixothrix hirtus (PhRE) railroadworm resulted in a 10 nm blue-shift. In PmL the substitution N230S resulted in a typical red mutant (lambda(max) = 611 nm). The bioluminescence spectrum of all these luciferase mutants did not show altered pH-sensitivity nor considerably changed half-bandwidth in relation to the wild-type luciferases. Altogether present data suggest that Thr226 is an important residue for keeping active-site core in both groups of beetle luciferases. The mechanism for bioluminescence color determination between pH-sensitive and pH-insensitive luciferases could be different.

Amino Acid Sequence↗

PubMed2000

Evidence that endo-1,4-beta-glucanases act on cellulose in suspension-cultured poplar cells.

Suspension-cultured poplar (Populus alba) cells produce two distinct endo-1,4-beta-glucanases, one of which is released in the extracellular culture medium and the other localized in their walls. Two cDNA clones, PopCel1 and PopCel2, isolated from a poplar cDNA library, encode the extracellular and the wall-bound endo-1, 4-beta-glucanases, respectively, based upon deduced amino acid sequences. The products of these two genes contained domains conserved in endo-1,4-beta-glucanase (family 9) and showed 91.5% amino acid identity. The levels of both PopCel1 and PopCel2 mRNAs increased during the lag phase of growth and decreased rapidly during the linear phase. After the levels had decreased, they were again increased by addition of sucrose to the culture medium and further enhanced by the addition of 2,4-dichlorophenoxyacetic acid (2,4-D) in the presence of sucrose. The accumulation of the mRNAs was correlated with the solubilization of cello-oligosaccharides. Cello-oligosaccharides and xyloglucan were also solubilized from the wall preparations of poplar cells incubated with enzyme preparations from the extracellular culture medium and walls. An antibody against both PopCel proteins reduced the production of cello-oligosaccharides by the extracellular enzyme by 90% and that by the wall-bound enzyme by 55%, and also prevented xyloglucan solubilization. The results show that the accumulation of poplar endo-1,4-beta-glucanases is regulated indirectly by auxin in the presence of sucrose and can act on cellulose in suspension-cultured poplar cells.

2,4-Dichlorophenoxyacetic Acid↗

PubMed2000

Bioluminescence color determinants of Phrixothrix railroad-worm luciferases: chimeric luciferases, site-directed mutagenesis of Arg 215 and guanidine effect.

Chimeric proteins were produced using the green light-emitting luciferase of Phrixothrix vivianii (PxGr: lambda max = 548 nm) and the red light-emitting luciferase of Phrixothrix hirtus (PxRe: lambda max = 623 nm). Constructs containing residues 1-344 of the red light-emitting luciferase with residues 345-545 of the green light emitting one emitted red light (PxReGr; lambda max = 613 nm), while the reverse emitted green light (PxGrRe; lambda max = 552 nm). From these results we conclude that the region 1-344 determines the color of bioluminescence (BL) in railroad-worm luciferases, and that residues above 344 are not involved. The substitution R215S in the green light-emitting luciferase (PxGr) resulted in a approximately 40 nm redshift on the BL spectrum (lambda max = 585 nm) and an associated decrease of activity, whereas the same mutation in PxRe luciferase had little effect. Guanidine was shown to cause blueshifts in the BL spectra and stimulate the activity of the red-emitting luciferases (from lambda max = 623 to lambda max = 600 nm) and in PxGr R215S (from lambda max = 585 to lambda max = 560 nm) mutant luciferase, but not in the green-emitting luciferases, suggesting that guanidine can simulate positively charged residues involved in BL color determination.

Animals↗

PubMed1999

Cloning, sequence analysis, and expression of active Phrixothrix railroad-worms luciferases: relationship between bioluminescence spectra and primary structures.

Phrixothrix railroad-worms emit yellow-green light through 11 pairs of lateral lanterns along the body and red light through two cephalic lanterns. The cDNAs for the lateral lanterns luciferase of Phrixothrix vivianii, which emit green light (lambda max= 542 nm), and for the head lanterns of P. hirtus, which emit the most red-shifted bioluminescence (lambda max= 628 nm) among luminescent beetles, were cloned. Positive clones which emitted green (PvGR: lambda max= 549 nm) and red (PhRE: lambda max= 622 nm) bioluminescence were isolated. The lucifereases coded by PvGR (545 amino acid residues) and PhRE (546 amino acid residues) cDNAs share 71% identity. PvGR and PhRE luciferases showed 50-55% and 46-49% identity with firefly luciferases, respectively, and 47-49% with click-beetle luciferases. PhRE luciferase has some unique residues which replace invariant residues in other beetle luciferases. The additional residue Arg 352 in PhRE, which is deleted in PvGR polypeptide, seems to be another important structural feature associated with red light production. As in the case of other railroad-worms and click-beetle luciferases studied, Phrixothrix luciferases do not undergo the typical red shift suffered by firefly luciferases upon decreasing pH, a property which might be related to the many amino acid residues shared in common between railroad-worm and click-beetle luciferase.

Amino Acid Sequence↗

PubMed1999

[Synthesis of 18F-FDG with FDG MicroLab system: basic studies for clinical application].

We synthesized 18F-FDG by using an automated synthetic apparatus "FDG MicroLab" (GE Medical Systems) which produces 18F-FDG by a solid phase 18F-fluorination. Its quality and reproducibility were evaluated in order to assess feasibility of the apparatus for routine clinical production of 18F-FDG. For 5 consecutive 18F-FDG synthesis, target irradiation was carried out at 15 microA for 60 min. 18F-FDG was obtained in 50 min after EOB with an end-of-synthesis yield of 9.34 +/- 1.06 GBq. Radiochemical yield and radiochemical purity were 47 +/- 3% (decay corrected) and 98.0 +/- 0.5%, respectively. Other several quality control parameters tested conformed with "Standards of Compounds Labeled with Positron Nuclides" (RADIOISOTOPES, 44, 1995). Thus, the automated synthetic apparatus "FDG MicroLab" has proven to stably produce 18F-FDG with high yield and high purity. The apparatus is feasible for routine clinical production of 18F-FDG.

Chemical Phenomena↗

PubMed1997

Mutagenesis of firefly luciferase shows that cysteine residues are not required for bioluminescence activity.

Conflicting reports exist in the literature as to whether sulfhydryl groups are essential for firefly luciferase activity. Inactivation of Photinus pyralis luciferase with N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and site-directed mutagenesis demonstrate that the cysteine residues are not absolutely required for activity. However, loss of P. pyralis luciferase activity is observed when any of the 4 cysteine residues is replaced with serine.

Animals↗

PubMed1996

The structural origin of the color differences in the bioluminescence of firefly luciferase.

Six chimeric mutants between Hotaria parvula (lambda max = 568 nm) and Pyrocoelia miyako (lambda max = 550 nm) luciferases were reconstructed to determine the structural origin of the color differences in firefly luciferase. Based on light-emitting color, five chimeric luciferases could be divided into two groups: the three green-emitting mutants, classified as P. miyako luciferase, and the two yellow-emitting mutants, classified as H. parvula luciferase. Their common fragments between Val-209 and Ala-318 within each group contain the active site for the color differences.

Amino Acid Sequence↗

PubMed1996

Shining the light: the mechanism of the bioluminescence reaction of calcium-binding photoproteins.

The Ca2+-binding photoproteins from jellyfish have the unique ability to emit blue light in the presence of calcium ions but without molecular oxygen or any other cofactor. Although there is no crystallographic data on the structure of the photoprotein complex, structure-activity studies have elucidated many features of the complex and many aspects of the mechanism of the bioluminescence reaction.

Amino Acid Sequence↗

PubMed1996

[Risk factors in serologically diagnosed echinococcosis--a case control study in the Nemuro region of Hokkaido Prefecture].

To determine the risk factors for echinococcus infection, we conducted a case-control study in the Nemuro region of Hokkaido Prefecture. The cases included both clinically diagnosed patients with echinococcosis and registered persons who were seropositive. The control group was matched by residence, age (+/- 5 years) and sex. The data on the 99 cases and 430 controls were collected by mailed questionnaire. Because of the long latency of echinococcosis, we investigated the potential risk factors over two different periods-from 11-20 years before the time of registration, and from 10 years before the registration to the time of registration. For the 11-20 years before registration period, using well water as drinking water and cattle raising significantly increased the risk of echinococcosis. In the period 10 years before registration to the time of registration, hog raising also increased the risk. In contrast, drinking tap water decreased the risk. These results suggest the importance of a screening program, especially targeting people engaged in dairy farming and hog raising, as well as the need for extension of tap water services and health education on refuse disposal.

Adult↗

PubMed1995

Sodefrin: a female-attracting peptide pheromone in newt cloacal glands.

A decapeptide called sodefrin was isolated from the abdominal gland of the cloaca of the male red-bellied newt, Cynops pyrrhogaster. The native peptide, as well as the synthetic one, had a female-attracting activity. Sodefrin was found in the apical portion of the epithelial cells of the abdominal gland. Sodefrin is apparently species specific because it did not attract females of Cynops ensicauda. This is the first amphibian pheromone to be identified and the first peptide pheromone identified in a vertebrate.

Amino Acid Sequence↗

PubMed1995

Cloning, expression and sequence analysis of cDNA for the luciferases from the Japanese fireflies, Pyrocoelia miyako and Hotaria parvula.

Cloning and sequence analysis of cDNA for the luciferases of Pyrocoelia miyako and Hotaria parvula were carried out (GenBank accession numbers L39928 and L39929, respectively). The amino acid sequence, deduced from the nucleotide sequence, showed P. miyako luciferase to consist of 548 amino acid residues with a molecular weight of 60,955, while the luciferase of H. parvula consisted of 548 amino acid residues with a molecular weight of 60,364. Pyrocoelia miyako luciferase showed 82.1% homology with the luciferase of Photinus pyralis and less than 70% homology with other firefly luciferases, whereas H. parvula luciferase showed 98%, 82.5% and 81.2% homology with the luciferases of Luciola mingrelica, Luciola lateralis and Luciola, cruciata respectively. Two regions in the enzymes were found to be highly conserved. The amino acid sequences were used to construct a phylogenetic tree, which showed that the fireflies could be divided into two groups.

Amino Acid Sequence↗

PubMed1995

Molecular evolution of the Ca(2+)-binding photoproteins of the Hydrozoa.

Alignment of the primary structures of the hydrozoan photoproteins, aequorin, mitrocomin, clytin and obelin showed very strong amino acid sequence identities. The Ca(2+)-binding sites of the proteins were found to be highly conserved. The Ca(2+)-binding sites were also homologous to the Ca(2+)-binding sites of other Ca(2+)-binding proteins. However, aequorin, mitrocomin, clytin and obelin differed from other Ca(2+)-binding proteins in that they contained a relatively large number of cysteine, tryptophan, histidine, proline and tyrosine residues, suggesting that these residues may have evolved as part of the light-emitting mechanism. Construction of a phylogenetic tree showed that aequorin, mitrocomin, clytin and obelin form a closely related group of proteins.

Aequorin↗

PubMed1995

Purification and properties of wall-bound endo-1, 4-beta-glucanase from suspension-cultured poplar cells.

A wall-bound endo-1, 4-beta-glucanase (EC 3.2.1.4) was obtained from a preparation of the cell walls of suspension-cultured poplar cells and purified to electrophoretic homogeneity by cation-exchange, hydrophobic, and gel-filtration chromatography. The molecular mass was estimated to be 47 kDa by SDS-PAGE and 48 kDa by gel filtration on Superdex 200 pg. The isoelectric point (pI) was 5.6. The purified enzyme catalyzed the endo-hydrolysis of carboxymethylcellulose with an optimal pH of 6.5, a Km of 1.2 mg ml-1, and a Vmax of 280 units. The purified enzyme specifically hydrolyzed the 1, 4-beta-glucosyl linkages of carboxymethylcellulose, phospho-swollen cellulose, lichenan, xylan and xyloglucan. The activity of the enzyme was strongly stimulated by cysteine-HCl. The N-terminal sequence of the enzyme was similar to that of an extracellular endo-1, 4-beta-glucanase found in suspension cultures of poplar cells and some homology was recognized to avocado fruit-ripening and bean abscission endo-1, 4-beta-glucanases.

Amino Acid Sequence↗

PubMed1995

[New sex pheromone isolated from the abdominal glands of male newt, Cynops pyrrhogaster].

Sodefrin, a novel decapeptide which attracts female newts, Cynops pyrrhogaster was isolated from the abdominal gland of the male of the same species. Synthetic sodefrin was tested for its activity with sexually active females and males and sexually inert females. It attracted only sexually active females. The effect of sodefrin was blocked by a bilateral nostril plugging with cotton balls soaked in melted vaseline. Immunoelectronmicroscopic study using antiserum against sodefrin revealed that sodefrin was located mainly in the secretory granules in the epithelial cells of the abdominal gland of the cloaca.

Abdomen↗