Takahiro Adachi

Reduction of 14-16 kDa allergenic proteins in transgenic rice plants by antisense gene.

An antisense gene strategy was applied to suppress the 14–16 kDa allergen gene expression in maturing rice seeds. Gene constructs producing antisense RNAs of the 16 kDa allergen under the control of some rice seed‐specific promoters were introduced into rice by electroporation. Immunoblot and RNA blot analyses of the seeds from the transgenic rice plants using the allergen‐specific monoclonal antibody and a sequence‐specific antisense RNA probe demonstrated that the 14–16 kDa allergen proteins and their transcripts of the seeds from several transgenic lines were present in much lower in amounts than those of the seeds from parental wild‐type rice. The high levels of reduction observed were stably inherited in at least three generations.

Carboxy-terminal cytoplasmic domain of mouse butyrophilin specifically associates with a 150-kDa protein of mammary epithelial cells and milk fat globule membrane

A cDNA encoding mouse butyrophilin was obtained by reverse transcriptase-coupled polymerase chain reaction (RT-PCR) using poly (A)+ RNA from lactating mouse mammary gland as a template and screening a cDNA library with the RT-PCR-amplified fragment as a probe. DNA sequencing and computer analysis revealed that it has a rather long 3-untranslated sequence and that the carboxy-terminal cytoplasmic domain was well conserved between mouse and bovine butyrophilins. To elucidate the biological function of butyrophilin, the cytoplasmic region expressed as fusion protein with glutathione S-transferase (GST) was purified and incubated with the cell lysate of mouse mammary epithelial cell lines, COMMA-ID and HC11. A 150-kDa protein was shown to specifically associate with the cytoplasmic domain and the protein increased in amount when the cells were treated with basal medium supplemented with lactogenic hormones such as prolactin, insulin and glucocorticoid. N-terminal amino acid sequencing indicated that the protein is xanthine dehydrogenase/oxidase which has been cloned from mouse liver. Further, the cytoplasmic domain also bound xanthine dehydrogenase/oxidase from bovine milk fat globule membrane. These results suggest that butyrophilin might be physiologically associated with xanthine dehydrogenase/oxidase and might function in a complex form in milk fat secretion.

Gene structure and expression of rice seed allergenic proteins belonging to the α-amylase/trypsin inhibitor family

Genomic and two novel cDNA clones for rice seed allergenic protein (RA) belonging to the α-amylase/trypsin inhibitor family were isolated and their nucleotide sequences determined. Ten cysteine residues deduced from nucleotide sequences were completely conserved among three cDNA clones including a clone, RA17, reported previously. One genomic clone, λ4, contained two RA genes, RAG1 and RAG2. Although RAG1 was cloned at the 5′ portion only, two RA genes were arranged divergently. Nucleotide sequencing and DNA blotting analyses showed that RA are encoded by a multigene family consisting of at least four members. The transcriptional initiation site of RAG1 was localized at A, 26 bp upstream of the putative translational initiation codon, ATG, by the primer extension assay. The putative TATA box and CAAT box existed about 45 bp and 147 bp upstream of the transcription initiation site, respectively. A conserved sequence (ATGCAAAA) which was similar to the sequence (TGCAAAA) identified in rice glutelin promoters was observed in the 5′ region of the two genes. In addition, RNA blotting analyses provided that RA genes specifically expressed in ripening seed and their transcripts accumulated maximally between 15 and 20 days after flowering.

Nucleotide sequence of a cDNA clone encoding a major allergenic protein in rice seeds Homology of the deduced amino acid sequence with members of α‐amylase/trypsin inhibitor family

A cDNA clone of rice major allergenic protein (RAP) was isolated from a cDNA library of maturing rice seeds. The cDNA had an open reading frame (486 nucleotides) which coded a 162 amino acid residue polypeptide comprising a 27‐residue signal peptide and a 135‐residue mature protein of Mr 14,764. The deduced amino acid sequence of RAP showed a considerable similarity to barley trypsin inhibitor [1983, J. Biol. Chem. 258, 7998–8003] and wheat α‐amylase inhibitor [1981, Phytochemistry 20, 1781–1784].

Precursor-product relationship between chicken vitellogenin and the yolk proteins : the 40 kDa yolk plasma glycoprotein is derived from the C-terminal cysteine-rich domain of vitellogenin II

Chicken vitellogenin, a serum lipoprotein specific for laying hens, has been thought to be proteolytically cleaved into the heavy and light chain lipovitellins and phosvitin, the major yolk granule proteins, during or after transportation into oocyte. In this study, another proteolytic product of vitellogenin has newly been isolated from the beta-livetin fraction of yolk plasma. It is a yolk glycoprotein of 40 kDa (YGP40) with asparagine-linked carbohydrate chain(s) recognized by Concanavalin A and castor bean lectin (RCA-I), and it is identified as a C-terminal cysteine-rich fragment of the major vitellogenin (vitellogenin II), the cysteine-rich domain homologous to D2 region of von Willebrand factor. Another yolk plasma glycoprotein of 42 kDa is suggested to be one of the proteolytic products of the minor vitellogenin (vitellogenin I). Both 40 kDa and 42 kDa glycoproteins were shown to be present in growing oocytes but absent in laying hens serum. Limited proteolysis of vitellogenin II with cathepsin D produced a 40 kDa protein with reactivity to anti-YGP40 antibody. Gel filtration analysis of vitellogenin II digested with cathepsin D showed that YGP40 dissociated from lipovitellin-phosvitin complex after the proteolytic cleavage. These results suggest that after incorporation from serum via a specific receptor vitellogenin II is cleaved in the oocyte into four fragments, heavy and light chain lipovitellins, phosvitin and YGP40, and that YGP40 is released into the yolk plasma before or during compartmentation of lipovitellin-phosvitin complex into the yolk granule.

Asymmetric beta-boration of alpha,beta-unsaturated carbonyl compounds promoted by chiral rhodium-bisoxazolinylphenyl catalysts.

Chiral rhodium-bisoxazolinylphenyl acetate complexes exhibited high catalytic activity for the beta-boration of alpha,beta-unsaturated carbonyl compounds with bis(pinacolato)diboron in the presence of sodium t-butoxide with enantioselectivity up to 97%.

Stage specific expression of milk fat globule membrane glycoproteins in mouse mammary gland: comparison of MFG-E8, butyrophilin, and CD36 with a major milk protein, β-casein.

Abstract The expression of mouse milk fat globule membrane (MFGM) glycoproteins, MFG-E8, butyrophilin, CD36 was analyzed by Northern blot analyses. MFG-E8 and butyrophilin mRNAs were specifically detected in the mammary gland of lactating mice, whereas CD36 mRNA was detected in the heart and lung as well as in the mammary gland of lactating mice. The mRNAs of the three MFGM glycoproteins accumulated at mid-lactation were about 2–10-times as much as those of the early and late gestation stages, whereas β-casein mRNA accumulation was dramatically increased; the mRNA at mid-lactation was no less than 40-times as much as that before lactation. In mouse mammary epithelial cell lines, HC11 and COMMA-1D, only a slight or almost no enhancement for the expression of MFG-E8, butyrophilin and CD36 mRNAs was induced simply by the treatment with the lactogenic hormones such as prolactin, insulin and dexamethasone, whereas the β-casein mRNA expression was remarkably enhanced only by that treatment. Furthermore, while the β-casein protein was constantly detected in milk throughout the lactation stage, the content of MFG-E8 and butyrophilin proteins increased during the lactation with an increase in the milk fat content. These results suggest that the stage-specific expression of milk fat globule membrane glycoproteins in mammary epithelial cells is regulated in a similar but not necessarily identical mechanism to that of a major milk protein, β-casein.

Cloning of the rice seed α-globulin-encoding gene: sequence similarity of the 5'-flanking region to those of the genes encoding wheat high-molecular-weight glutenin and barley D hordein

A genomic clone encoding the rice endosperm major globulin (alpha-globulin) with an apparent molecular mass of 26 kDa was isolated, and its nucleotide (nt) sequence and transcription start point (tsp) were determined. The tsp was identical to that of the gene encoding the wheat high-molecular-weight (HMW) glutenin subunit. The consensus -300 element and an A + T-rich sequence exist upstream from the TATA box in the 5-flanking region. A nt sequence of about 130 bp in the 5-flanking region was found to be markedly homologous to those of the genes encoding the wheat HMW glutenin subunit and barley D hordein.

Characterization of a novel rice bZIP protein which binds to the α-globulin promoter

Many plant basic leucine-zipper (bZIP) proteins have been isolated several of which have been shown to play a role in seed-specific gene expression. We isolated a novel bZIP protein (REB) gene encoding 425 amino acid residues from rice endosperm, which is similar to Opaque-2 heterodimerizing protein (OHP) of maize. The gene product, termed REB, contains Pro- and Gly-rich regions at its N terminus, followed by the typical basic and leucine-repeat regions. Recombinant REB binds to the region from -754 to -562 in the α-globulin gene promoter, but not to promoters of other major storage genes such as glutelin, prolamin and albumin. The 5′ region of the α-globulin gene possesses three binding sites for REB, which were determined as GCCACGT(A/C)AG, by using synthetic oligonucleotides. A Super-shift assay using anti-REB antibody suggested that REB is a major DNA-binding protein for the α-globulin gene promoter in rice endosperm.

Intermolecular Antiselective and Enantioselective Reductive Coupling of Enones and Aromatic Aldehydes with Chiral Rh(Phebox) Catalysts

The intermolecular reductive coupling reaction of cyclopent-2-enone and aromatic aldehydes was realized by chiral rhodium-(bisoxazolinyl)phenyl catalysts, Rh(Phebox-Ph)(OAc)(2)(H(2)O), with diphenymethylsilane as a hydride donor to give the corresponding beta-hydroxyketones in high anti-selectivity (up to 96%) with high enantioselectivity (up to 93%).