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Biomedical subjects

M Sekine

Publications and source records attributed to M Sekine.

At least 127 records · Page 7Linked to original sources
PubMed1989

Synthesis of N2, N2, 7-trimethylguanosine cap derivatives.

Several derivatives of N2,N2-7-trimethylguanosine (m3(2,2,7G)-cap, which was found at the 5' ends of small nuclear RNAs, were synthesized by use of S-phenyl N2,N2,7-trimethylguanosine 5'-phosphorothioate (PhSpm3(2,2,7)G) as a key intermediate. This compound was activated by iodine in the presence of phosphoric acid and diphosphoric acid to give N2,N2,7-trimethylguanosine-5'-diphosphate (ppm3(2,2,7)G) and 5'-triphosphate (ppm3(2,2,7)G), respectively. Similar reactions of PhSpm3(2,2,7)G with ADP and GDP gave capped dinucleoside triphosphates, m3(2,2,7)G5'pppA and m3(2,2,7)G5'pppG, respectively.

Adenosine Diphosphate↗
PubMed1989

Reinvestigation of deoxyribonucleoside phosphorothioites: synthesis and properties of deoxyribonucleoside-3' dimethyl phosphites.

Further investigation of the synthesis of deoxyribonucleoside-3' (t-butyl) O-(2-cyanoethyl) phosphorothioites as monomer building units for the phosphorothioite approach has led us to conclude that internucleotidic bond formation proceeded via bis(deoxyribonucleoside-3') (2-cyanoethyl) phosphite intermediates, which proved to be activated by iodine, rather than the mechanism previously reported. In connection with this study, deoxyribonucleoside-3' dimethyl phosphites were synthesized and detailed properties of them are also described.

Chemical Phenomena↗
PubMed1989

Chemical synthesis of the 24 RNA fragments corresponding to hop stunt viroid.

A general and practical synthetic method of oligoribonucleotides (10-20 mers) by using the cyanoethyl phosphoramidite approach was described. In this experiment 9-phenylxanthen-9-yl (Pix) and 9-(4-methoxy)phenylxanthen-9-yl (Mox) groups were employed for the 5'-hydroxyls and tetrahydropyranyl (Thp) group was used for the 2'-hydroxyl protecting groups. In addition, suitable acyl groups were introduced for the protection of the lactam functions of guanine and uracil moieties.

Base Sequence↗
PubMed1989

Pre-mRNA with a trimethylguanosine cap structure can be spliced efficiently in vitro.

A chemically synthesized 2,2,7-trimethylguanosine cap (TMG-cap) analogue was added to an in vitro pre-mRNA splicing system. A slight inhibition of the splicing was observed, but it was much less remarkable than that by a 7-methylguanosine cap analogue. This result does not favor a direct role of the TMG-cap structure of small nuclear RNAs in pre-mRNA splicing, and suggests that the TMG-cap does not effectively interact with the 7-methylguanosine cap binding factors. Using the SP6 in vitro transcription system and a TMG-cap analogue, we prepared a beta-globin transcript which has a TMG-cap at the 5' end. This TMG-capped transcript was spliced accurately and efficiently in vitro.

Binding, Competitive↗
PubMed1989

Pre-activation strategy for oligodeoxyribonucleotide synthesis using triaryloxydichlorophosphoranes in the phosphotriester method.

Triaryloxydichlorophosphoranes were tested as condensing agents for oligodeoxyribonucleotide synthesis in the phosphotriester method. Tris(2,4,6-tribromophenoxy)dichlorophosphorane (BDCP) was found to be a relatively stable crystalline material which could be used as a chemical reagent. A notable feature of the BDCP-promoted condensation reaction was studied by 31P-NMR. A small amount of BDCP compared to the conventional condensing agent was effective for the generation of active nucleotide intermediates and BDCP itself was quantitatively converted into an inert material, tris(2,4,6-tribromophenyl)phosphate (2). Thus, BDCP enabled us to separate the activation step from the condensation process in the phosphotriester method. This preactivation method was applied to the solid-phase synthesis.

Indicators and Reagents↗
PubMed1989

Two genes controlling the expression of extended globoglycolipids in mouse kidney are closely linked to each other on chromosome 19.

The gene (Gsl-5) controlling the expression of GL-Y (Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-6(Gal beta 1-3)Gb4Cer) in mouse kidney was suggested to be located near Ea-4 on mouse chromosome 19 by the results of glycolipid analysis of BXD/Ty recombinant inbred strains (Sekine et al. [1987] J. Biochem. 101, 563-568). In this study, Gsl-5 was mapped on mouse chromosome 19. Among 133 backcross progeny produced on mating between DBA/2 mice and (WHT/Ht x DBA/2)F1 mice, 10 recombinants between Lyt-1 and Gsl-5 were detected, indicating that Gsl-5 is located at 7.5 +/- 2.3 centimorgans (cM) from Lyt-1. While among 154 backcross progeny produced on mating between DBA/2 and (DBA/2 x Mus musculus castaneus)F1 mice, 39 recombinants between Got-1 and Gsl-5 were obtained, indicating that the distance between Got-1 and Gsl-5 is 25.3 +/- 3.5 cM and that Gsl-5 is telomeric to Lyt-1. In the latter mating experiment, we detected 3 recombinants between Gsl-5 and the gene (Gsl-6) controlling the expression of the Z1 ganglioside (NeuGc alpha 2-3Gal beta 1-3Gb4Cer) among the 154 backcross mice. These results indicate that these two genes, Gsl-5 and Gsl-6, are closely linked to each other, being 1.9 +/- 1.1 cM apart. This is the report of evidence that two genes controlling the expression of carbohydrates in glycoconjugates are closely linked and the first to suggest that some genes controlling the expression of carbohydrates may be clustered.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

High-performance liquid chromatography-mass spectrometry of glycosphingolipids: I. Structural characterization of molecular species of GlcCer and IV3 beta Gal-Gb4Cer.

A method of high-performance liquid chromatography-fast atom bombardment mass spectrometry (HPLC/FAB/MS) for the structural characterization of glycosphingolipids was developed, which involves a frit interface between the HPLC and the MS. The molecular species of glucosylceramide (GlcCer) purified from the spleen of a patient with Gaucher's disease and galactosylglobotetraosylceramide (IV3 beta Gal-Gb4Cer) from mouse kidney were analyzed using this system on a reversed-phase column, with methanol containing 1% glycerol as the elution solvent. The injection of 1 microgram of GlcCer gave the mass spectra of seven major molecular species, the pseudo-molecular ion for each of the seven molecular species being observed at m/z 698, 726, 754, 782, 808, 796, and 810, respectively. The injection of 200 pg of synthetic N-stearoyl glucosylsphingosine (d18:1) gave a clear peak with the single ion monitoring method detecting the pseudo-molecular ion at m/z 726. The injection of 5 micrograms of IV3 beta Gal-Gb4Cer gave the mass spectra of six major molecular species, the pseudo-molecular ions being observed at m/z 1,489, 1,471, 1,515, 1,497, 1,517, and 1,499. This report deals with a new HPLC/FAB/MS system, which was successfully applied to the structural characterization of the molecular species of neutral glycosphingolipids, and the system is a quite promising for development into a quantitative method for glycosphingolipids with high sensitivity and specificity.

Animals↗
PubMed1989

Molecular cloning of a gene for indole-3-acetamide hydrolase from Bradyrhizobium japonicum.

A pLAFR1 cosmid genomic library of wild-type Bradyrhizobium japonicum J1063 was constructed. A cosmid clone designated pBjJ4, containing a 26-kilobase (kb) DNA insert, was identified as being able to confer the ability to convert alpha-naphthaleneacetamide acid on B. japonicum J1B7 Rifr, which cannot perform this conversion. The gene coding for the enzyme that converts alpha-naphthaleneacetamide to alpha-naphthaleneacetic acid was localized in the 3.5-kb region of pBjJ4 by recloning in plasmid pSUP202. The gene coding for the enzyme was also mapped by Tn5 insertion mutagenesis to a region of ca. 2.3 kb. When the gene was placed behind the lacZ promoter and used to transform Escherichia coli, a high level of expression of indole-3-acetamide hydrolase activity was found. Since there have been no reports of this activity in E. coli, we have thus confirmed that the gene cloned here is a structural gene for indole-3-acetamide hydrolase and have designated it as the bam (Bradyrhizobium amidehydrolase) gene. Southern hybridization with the central region of the bam gene indicated that a high degree of similarity exists among the bam gene, the iaaH gene from Pseudomonas savastonoi, and the tms-2 gene from Agrobacterium tumefaciens. The result suggests that there is a common origin for the gene that encodes the enzyme that catalyzes the biosynthesis of indoleacetic acid.

Amidohydrolases↗
PubMed1989

Oligoribonucleotide synthesis by use of the 2-(methylthio)-phenylthiomethyl (MPTM) group.

The 2-(methylthio)phenylthiomethyl (MPTM) group was developed as a new type of 2'-hydroxyl protecting group in oligoribonucleotide synthesis. The building monomer units of uridine and cytidine for the phosphotriester approach were synthesized from 2'-O-(1,3-benzodithiol-2-yl)-3',5'-O- (1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)uridine and successfully utilized for the synthesis of CpUpG.

Indicators and Reagents↗
PubMed1989

[Distribution of cone pedicle and superficial capillary. A morphological study on the macular region of normal human retina].

The distribution and arrangement of cone pedicle (CP) and peri-foveolar superficial capillary net works (SC) in the macular region was studied in two eyes obtained from a 24-year-old male and a 34-year-old female, light and electron microscopically. CP and SC were not seen in the central area of the macular region. Both the CP-free zone and SC-free area were ellipsoid in form and the horizontal dimensions were larger than 140 microns and 425 microns in one case and 360 microns and 630 microns in the other. The dimensions of the SC-free area measured 200 microns and 500 microns in the first case and 520 microns and 650 microns in the second. The para-foveolar circle formed by CP- and SC-free zones were concentric and the SC circle was about 50 microns larger in radius than that of CP.

Adult↗
PubMed1988

Monoclonal antibodies that bind to galactosylgloboside (SSEA-3 antigen).

The 3-fucosyllactosamine (3-FL) antigenic determinant, Gal(beta 1-4)[Fuc(alpha 1-3)]-GlcNAc-R, is very immunogenic in BALB/c mice. A panel of monoclonal antibodies was raised against a novel glycosphingolipid that contains a terminal 3-FL structure (structure 1): (formula: see text) Surprisingly, most of these antibodies reacted with the internal galactosylgloboside structure rather than with the terminal 3-FL epitope. The specificities of two of these antibodies, 5A3 and 8A7, were analyzed by enzyme-linked immunosorbent assay and immunostaining procedures, and compared with MC631 (anti-SSEA-3), the only other monoclonal antibody known to bind to galactosylgloboside. 5A3 and 8A7 bound equally well to structure 1 and to galactosylgloboside. These antibodies also bound to structure 1 from which the terminal galactose, or the galactose and fucose, had been removed, but not to globoside or sialylated galactosylgloboside. In contrast, MC631 did not bind to structure 1 but, as described previously, did bind to globoside and sialylated galactosylgloboside. Galactosylgloboside is a developmentally regulated antigen of mouse embryos, human teratocarcinomas, and mouse embryonic brain. Antibodies 5A3 and 8A7 will complement MC631 in the analysis of the distribution and regulation of galactosylgloboside.

Antibodies, Monoclonal↗
PubMed1988

A single autosomal gene controlling the expression of the extended globoglycolipid carrying SSEA-1 determinant is responsible for the expression of two extended globogangliosides.

Two extended globogangliosides, designated as Z1 and Z2, were purified from the kidney of DBA/2 mice. By means of GLC, 1H-NMR spectroscopy, negative-ion fast atom bombardment mass spectrometry, methylation analysis, and enzymatic digestion, the structures of Z1 and Z2 were determined to be NeuGc alpha 2-3Gal beta 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-Cer and NeuGc alpha 2-8NeuGc alpha 2-3Gal beta 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-Cer, respectively. Since Z1 and Z2 were not detectable in the kidney of C57BL/10 and 6, BALB/c, and WHT/Ht mice, the mode of genetic control on Z1 and Z2 expression was examined by mating experiments between C57BL/10 or BALB/c and DBA/2. The results indicated that the expression of Z1 and Z2 is a recessive phenotype and that DBA/2 mice carry a single autosomal recessive gene. In the previous paper, we reported that DBA/2 mice do not express GL-Y (Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-6(Gal beta 1-3)Gb4Cer) but express GL-X (Gal beta 1-3Gb4Cer) in the kidney (J. Biochem. 101, 553-562 (1987)), and that a single autosomal defective gene responsible for the defective GL-Y expression was identified by genetic analysis (J. Biochem. 101, 563-568 (1987)).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Tris(2,4,6-tribromophenoxy)dichlorophosphorane as a condensing agent: a new type of activation of phosphodiester components in the phosphotriester method.

The title compound (BDCP) was found to be effective for the rapid internucleotidic bond formation in the presence of 3-nitro-1,2,4-triazole (NT). Mechanism of the condensation reaction was elucidated by means of 31P-NMR. It was proved that there was no active species but the desired nucleotide derivatives, phosphorochloridate and phosphorylnitrotriazole, in the reaction mixture. Thus, the side-reaction of excess condensing agent in the conventional phosphotriester method was overcome.

Indicators and Reagents↗