Biomedical subjects

Masao Ishimoto

Publications and source records attributed to Masao Ishimoto.

12 recordsLinked to original sources

PubMed2006

Assessment of the importance of alpha-amylase inhibitor-2 in bruchid resistance of wild common bean.

Both alpha-amylase inhibitor-2 (alphaAI-2) and arcelin have been implicated in resistance of wild common bean (Phaseolus vulgaris L.) to the Mexican bean weevil (Zabrotes subfasciatus Boheman). Near isogenic lines (NILs) for arcelin 1-5 were generated by backcrossing wild common bean accessions with a cultivated variety. Whereas seeds of a wild accession (G12953) containing both alphaAI-2 and arcelin 4 were completely resistant to Z. subfasciatus, those of the corresponding NIL were susceptible to infestation, suggesting that the principal determinant of resistance was lost during backcrossing. Three independent lines of transgenic azuki bean [Vigna angularis (Willd.) Ohwi and Ohashi] expressing alphaAI-2 accumulated high levels of this protein in seeds. The expression of alphaAI-2 in these lines conferred protection against the azuki bean weevil (Callosobruchus chinensis L.), likely through inhibition of larval digestive alpha-amylase. However, although the seed content of alphaAI-2 in these transgenic lines was similar to that in a wild accession of common bean (G12953), it did not confer a level of resistance to Z. subfasciatus similar to that of the wild accession. These results suggest that alphaAI-2 alone does not provide a high level of resistance to Z. subfasciatus. However, alphaAI-2 is an effective insecticidal protein with a spectrum of activity distinct from that of alphaAI-1, and it may prove beneficial in genetic engineering of insect resistance in legumes.

Animals↗

PubMed2006

Protein disulfide isomerase family proteins involved in soybean protein biogenesis.

Protein disulfide isomerase family proteins are known to play important roles in the folding of nascent polypeptides and the formation of disulfide bonds in the endoplasmic reticulum. In this study, we cloned two similar protein disulfide isomerase family genes from soybean leaf (Glycine max L. Merrill cv. Jack) mRNA by RT-PCR using forward and reverse primers designed from the expressed sequence tag clone sequences. The cDNA encodes a protein of either 364 or 362 amino acids, named GmPDIS-1 or GmPDIS-2, respectively. The nucleotide and amino acid sequence identities of GmPDIS-1 and GmPDIS-2 were 68% and 74%, respectively. Both proteins lack the C-terminal, endoplasmic reticulum-retrieval signal, KDEL. Recombinant proteins of both GmPDIS-1 and GmPDIS-2 were expressed in Escherichia coli as soluble folded proteins that showed both an oxidative refolding activity of denatured ribonuclease A and a chaperone activity. Their domain structures were identified as containing two thioredoxin-like domains, a and a', and an ERp29c domain by peptide mapping with either trypsin or V8 protease. In cotyledon cells, both proteins were shown to distribute to the endoplasmic reticulum and protein storage vacuoles by confocal microscopy. Data from coimmunoprecipitation and crosslinking experiments suggested that GmPDIS-1 associates with proglycinin, a precursor of the seed storage protein glycinin, in the cotyledon. Levels of GmPDIS-1, but not of GmPDIS-2, were increased in cotyledons, where glycinin accumulates during seed development. GmPDIS-1, but not GmPDIS-2, was induced under endoplasmic reticulum-stress conditions.

Amino Acid Sequence↗

PubMed2006

Genetic improvement of the somatic embryogenesis and regeneration in soybean and transformation of the improved breeding lines.

Somatic embryos of soybean [Glycine max (L.) Merrill] have been used to generate transgenic plants by particle bombardment. The induction and proliferation of somatic embryos from immature cotyledons are dependent on the genotype of the cultivar. Whereas somatic embryogenesis and plant regeneration are inefficient in most cultivars, they are efficient in the cultivar Jack. We previously established a breeding line, QF2, by the integration of null mutations of each subunit of the major seed storage proteins glycinin and beta-conglycinin, but the embryogenic response of this line is insufficient to allow efficient transformation. We have now backcrossed QF2 to cultivar Jack in order to combine the null traits with competence for somatic embryogenesis. The backcrossed breeding lines selected on the basis of the absence of the major storage proteins exhibited an improved capacity for the induction and proliferation of somatic embryos compared with that of QF2. The induced somatic embryogenic tissue of these breeding lines was successfully used for the production of transgenic plants by particle bombardment. These results also indicate that somatic embryogenesis in soybean is genetically controlled and inherited in a manner independent of the null traits of the major seed storage proteins.

Antigens, Plant↗

PubMed2006

A red fluorescent protein, DsRed2, as a visual reporter for transient expression and stable transformation in soybean.

Fluorescent proteins such as green fluorescent protein (GFP) from Aequorea victoria are often used as markers for transient expression and stable transformation in plants, given that their detection does not require a substrate and they can be monitored in a nondestructive manner. We have now evaluated the red fluorescent protein DsRed2 (a mutant form of DsRed from Discosoma sp.) for its suitability as a visual marker in combination with antibiotic selection for genetic transformation of soybean [Glycine max (L.) Merrill]. Transient and stable expression of DsRed2 in somatic embryos was readily detected by fluorescence microscopy, allowing easy confirmation of gene introduction. We obtained several fertile transgenic lines, including homozygous lines, that grew and produced seeds in an apparently normal manner. The red fluorescence of DsRed2 was detected by fluorescence microscopy without background fluorescence in both leaves and seeds of the transgenic plants. Furthermore, in contrast to seeds expressing GFP, those expressing DsRed2 were readily identifiable even under white light by the color conferred by the transgene product. The protein composition of seeds was not affected by the introduction of DsRed2, with the exception of the accumulation of DsRed2 itself, which was detectable as an additional band on electrophoresis. These results indicate that DsRed2 is a suitable reporter (even more suitable than GFP) for genetic transformation of soybean.

Animals↗

PubMed2006

Multiple vacuolar sorting determinants exist in soybean 11S globulin.

The sorting determinants of glycinin, a soybean (Glycine max) 11S globulin, which mediates protein targeting to the protein storage vacuole (PSV), were investigated in maturing soybean cotyledons by transient expression assays. A C-terminal stretch of 10 amino acids of A1aB1b, a glycinin group I subunit, was sufficient to direct green fluorescent protein (GFP) to the PSV. This peptide may correspond to a C-terminal vacuolar sorting determinant (ctVSD). Because functional inhibition of this putative ctVSD of A1aB1b did not block PSV sorting of A1aB1b, we used the three-dimensional structure of A1aB1b to identify candidates for a sequence-specific determinant (ssVSD). We found that the sequence downstream of disordered region 4 could direct GFP to the PSV and that Ile-297 is critical for sorting. However, functional inhibition of the ctVSD, combined with the Ile297Gly mutation, did not abolish the vacuolar sorting of A1aB1b, suggesting that A1aB1b has a third sorting determinant in addition to ctVSD and ssVSD. A glycinin group II subunit, A3B4, lacked a ctVSD but contained a VSD reminiscent of an ssVSD and an additional sorting determinant. We also demonstrate, by expression of dominant negative mutants of small GTPases and drug treatment experiments, that the trafficking of A1aB1b is COPII vesicle-dependent and wortmannin- and brefeldin A-sensitive.

ADP-Ribosylation Factor 1↗

PubMed2006

Molecular control of gene co-suppression in transgenic soybean via particle bombardment.

Molecular co-suppression phenomena are important to consider in transgene experiments. Embryogenic cells were obtained from immature cotyledons and engineered with two different gene constructs (pHV and pHVS) through particle bombardment. Both constructs contain a gene conferring resistance to hygromycin (hpt) as a selective marker and a modified glycinin (11S globulin) gene (V3-1) as a target. sGFP(S65T) as a reporter gene was, however, inserted into the flanking region of the V3-1 gene (pHVS). Fluorescence microscopic screening after the selection of hygromycin, identified clearly the expression of sGFP(S65T) in the transformed soybean embryos bombarded with the pHVS construct. Stable integration of the transgenes was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. Seeds of transgenic plants obtained from the pHV construct frequently lacked an accumulation of endogenous glycinin, which is encoded by homologous genes to the target gene V3-1. Most of the transgenic plants expressing sGFP(S65T) showed highly accumulation of glycinin. The expression of sGFP(S65T) and V3-1 inherits into the next generations. sGFP(S65T) as a reporter gene may be useful to increase the transformation efficiency of transgenic soybean with avoiding gene co-suppression.

Cinnamates↗

PubMed2004

Structure-physicochemical function relationships of soybean glycinin at subunit levels assessed by using mutant lines.

Glycinin is a hexameric protein composed of five kinds of subunits. The subunits are classified into two groups, group I (A1aB1b, A1bB2, and A2B1a) and group II (A3B4 and A5A4B3). We purified four mutant glycinins composed of only group I subunits (group I-glycinin), only group II subunits (group II-glycinin), only A3B4 (A3B4-glycinin), and only A5A4B3 (A5A4B3-glycinin) from mutant soybean lines. The physicochemical properties of these glycinin samples were compared with those of the normal glycinin (11S) composed of five kinds of subunits. The thermal stabilities (as measured by thermal denaturation midpoint temperatures) of 11S, group I-glycinin, and group II-glycinin were similar to each other, although that of A3B4-glycinin was significantly lower than those of the others. The orders of aromatic and aliphatic surface hydrophobicities were the same: A3B4-glycinin > group II-glycinin > A5A4B3-glycinin > 11S > group I-glycinin. The solubility of 11S as a function of pH at mu = 0.5 was governed by that of group I-glycinin and followed this order at acidic pH: 11S = group I-glycinin > A3B4-glycinin > group II-glycinin = A5A4B3-glycinin. The order of emulsifying abilities was A5A4B3-glycinin > group II-glycinin > A3B4-glycinin > 11S > group I-glycinin. This order was consistent with that of the length of their hypervariable regions. Except for this relationship, there was no significant relationship among the other physicochemical properties of the mutant glycinins.

Chemical Phenomena↗

PubMed2004

8S globulin of mungbean [Vigna radiata (L.) Wilczek]: cloning and characterization of its cDNA isoforms, expression in Escherichia coli, purification, and crystallization of the major recombinant 8S isoform.

Three isoforms of the cDNA of the major 8S globulin of mungbean, 8Salpha, 8Salpha', and 8Sbeta, were isolated, cloned, and characterized. The cDNA sequences of 8Salpha, 8Salpha', and 8Sbeta had open reading frames of 1362, 1359 or 1362, and 1359 bp, respectively, which code for 454, 453 or 454, and 453 amino acids corresponding to molecular weights of 51 973, 51 627 or 51 758, and 51 779, respectively. Homology in terms of cDNA and amino acid sequences was 91-92% between 8Salpha and 8Salpha', 87% between 8Salpha and 8Sbeta, and 86-88% between 8Salpha' and 8Sbeta. The signal peptide was found to be 1-25, 1-24 or 25, and 1-23 for 8Salpha, 8Salpha', and 8Sbeta, respectively, using the signalP website (Nielsen, H.; Engelbrecht, J.; Brunak, S.; von Heijne, G. Protein Eng. 1997, 10, 1-6). The propeptide was determined to be IVHREN. A single site for glycosylation (N-X-S/T) was observed about 90 amino acids from the C terminus. Homology between mungbean 8S isoforms and other 7-8S proteins ranged from 45 to 68% within members of the legume family and 29 to 34% for crops of different species. The major isoform 8Salpha was expressed in Escherichia coli and purified by successive ammonium sulfate fractionation, hydrophobic interaction, and Mono Q column chromatography. The recombinant 8Salpha, but not the native form, was successfully crystallized producing rhombohedral crystals.

Amino Acid Sequence↗

PubMed2004

The composition of newly synthesized proteins in the endoplasmic reticulum determines the transport pathways of soybean seed storage proteins.

Glycinin (11S) and beta-conglycinin (7S) are major storage proteins in soybean (Glycine max L.) seeds and accumulate in the protein storage vacuole (PSV). These proteins are synthesized in the endoplasmic reticulum (ER) and transported to the PSV by vesicles. Electron microscopic analysis of developing soybean cotyledons of the wild type and mutants with storage protein composition different from that of the wild type showed that there are two transport pathways: one is via the Golgi and the other bypasses it. Golgi-derived vesicles were observed in all lines used in this study and formed smooth dense bodies with a diameter of 0.5 to several micrometers. ER-derived protein bodies (PBs) with a diameter of 0.3-0.5 microm were observed at high frequency in the mutants containing higher amount of 11S group I subunit than the wild type, whereas they were hardly observed in the mutants lacking 11S group I subunit. These indicate that pro11S group I may affect the formation of PBs. Thus, the composition of newly synthesized proteins in the ER is important in the selection of the transport pathways.

Biological Transport, Active↗

PubMed2003

Accumulation of high levels of free amino acids in soybean seeds through integration of mutations conferring seed protein deficiency.

Soybean ( Glycine max [L.] Merr.) seeds are rich in protein, most of which is contributed by the major storage proteins glycinin (11S globulin) and beta-conglycinin (7S globulin). Null mutations for each of the subunits of these storage proteins were integrated by crossbreeding to yield a soybean line that lacks both glycinin and beta-conglycinin components. In spite of the absence of these two major storage proteins, the mutant line grew and reproduced normally, and the nitrogen content of its dry seed was similar to that for wild-type cultivars. However, protein bodies appeared underdeveloped in the cotyledons of the integrated mutant line. Furthermore, whereas free amino acids contribute only 0.3-0.8% of the seed nitrogen content of wild-type varieties, they constituted 4.5-8.2% of the seed nitrogen content in the integrated mutant line, with arginine (Arg) being especially enriched in the mutant seeds. Seeds of the integrated mutant line thus appeared to compensate for the reduced nitrogen content in the form of glycinin and beta-conglycinin by accumulating free amino acids as well as by increasing the expression of certain other seed proteins. These results indicate that soybean seeds are able to store nitrogen mostly in the form of either proteins or free amino acids.

Amino Acids↗

PubMed2002

A lipase-inhibiting protein from lipoxygenase-deficient soybean seeds.

A lipase-inhibiting protein was isolated from lipoxygenase (LOX)-deficient soybean seeds. The molecular mass of the protein was 56.0-kDa and the N-terminal amino acid was blocked. The protein was identified by peptide mass fingerprinting in combination with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. The masses of the lysyl endopeptidase-digested peptides of the 56.0-kDa inhibiting protein were almost identical to the calculated masses of the theoretically predicted lysyl endopeptidase-treated peptides of beta-amylase from soybean seed. In a previous paper (Biosci. Biotechnol. Biochem., 62, 1498-1503, 1998), we reported that LOX-1, an isozyme of soybean seed LOX, inhibited hydrolysis of soybean oil by pancreatic lipase. Purified beta-amylase also inhibited lipase activity, although the magnitude of inhibition was weaker than that by LOX-1. Thus, there are at least two lipase-inhibiting proteins, one is a LOX and the other is a beta-amylase, in soybean seed.

Amino Acid Sequence↗

PubMed2002

Reproducible transformation in two grain legumes--soybean and azuki bean--using different systems.

Two plasmid vectors were introduced into soybean (Glycine max (L.) Merr.) and azuki bean (Vigna angularis Willd. Ohwi and Ohashi) using different transformation systems. Azuki bean epicotyl explants were prepared from etiolated seedlings and co-cultivated with Agrobacterium tumefaciens for 2 days. Adventitious shoots were developed from the callus of the explants on a regeneration medium containing hygromycin, and the shoots were excised and transferred to a rooting medium containing hygromycin at the same concentration. Rooting shoots were transferred to soil and grown in a glass-house to produce viable seeds. PCR analysis confirmed clearly the presence of the hpt gene in most of the azuki beans regenerated under hygromycin selection. A soybean embryogenic suspension culture was generated from immature cotyledons, and used for the introduction of plasmids by particle bombardment. Hygromycin-resistant embryogenic clones were isolated after 8 weeks of hygromycin selection, and then the green clones were matured on the differentiation medium. After desiccation, the embryos were germinated on the rooting medium, and the plants were transferred to soil in a glass-house. More than 50% of the regenerated soybean plants tolerant to hygromycin yielded the hpt fragment on PCR analysis. The azuki bean transformants were obtained more rapidly and with higher efficiency than the soybean transformants.

Fabaceae↗