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M Ishimoto

Publications and source records attributed to M Ishimoto.

At least 19 recordsLinked to original sources
PubMed2005

Mapping of QTL associated with chilling tolerance during reproductive growth in soybean.

Low temperatures in summer bring about drastic reduction in seed yield of soybean [Glycine max (L.) Merr.]. To identify quantitative trait loci (QTL) associated with chilling tolerance during the reproductive growth in soybean, a recombinant inbred line (RIL) population consisting of 104 F(6)-derived lines was created from a cross between two cultivars, chilling-tolerant Hayahikari and chilling-sensitive Toyomusume. The RIL were genotyped with 181 molecular and phenotypic markers and were scored with regard to chilling tolerance, which was evaluated by comparison of seed-yielding abilities in two artificial climatic environments at chilling and usual temperatures. Three QTL were detected for chilling tolerance in seed-yielding ability. Two of them, qCTTSW 1 and qCTTSW 2, were mapped near QTL for flowering time, and the latter had an epistatic interaction with a marker locus located near another QTL for flowering time, where no significant QTL for chilling tolerance was detected. The analysis of an F(2) population derived from the cross between Hayahikari and an RIL of the Hayahikari genotype at all QTL for flowering time confirmed the effect of the third QTL, qCTTSW 3, on chilling tolerance and suggested that qCTTSW 1 was basically independent of the QTL for flowering time. The findings and QTL found in this study may provide useful information for marker-assisted selection (MAS) and further genetic studies on soybean chilling tolerance.

Acclimatization↗
PubMed2001

Purification and characterization of two alpha-amylase inhibitors from seeds of tepary bean (Phaseolus acutifolius A. Gray).

Two proteinaceous alpha-amylase inhibitors termed alphaAI-Pa1 and alphaAI-Pa2 were purified from seeds of a cultivated tepary bean (Phaseolus acutifolius A. Gray, cv. PI311897). The two inhibitors differed in their specificity towards alpha-amylases of insect pests such as bruchids, although neither showed any inhibitory activity against alpha-amylases of mammalian, bacterial or fungal origin. AlphaAI-Pa2 resembles two common bean inhibitors, alphaAI-1 and alphaAI-2, in several characteristics such as N-terminal amino acid sequences and oligomeric structure being composed of alpha and beta subunits. In contrast alphaAI-Pa1 is composed of a single glycopolypeptide with a molecular mass of 35 kDa, and its N-terminal amino acid sequence resembled that of seed lectins in tepary bean and common bean. The information on the two tepary bean alpha-amylase inhibitors may be useful not only for providing insight into critical structure for the specificity towards different alpha-amylase enzymes but also for enhancing insect resistance in crops.

Amino Acid Sequence↗
PubMed1999

Insecticidal activity of an alpha-amylase inhibitor-like protein resembling a putative precursor of alpha-amylase inhibitor in the common bean, Phaseolus vulgaris L.

alpha-Amylase inhibitor (alphaAI) in the common bean, Phaseolus vulgaris L., protects seeds from insect pests such as the cowpea weevil (Callosobruchus maculatus) and the azuki bean weevil (C. chinensis). Cultivars which lack alphaAI still show resistance to both bruchids. These cultivars have a glycoprotein that reacts with anti-alphaAI-1 antibodies. The glycoprotein with a molecular mass of 29 kDa (Gp29) was purified and the encoding gene was isolated. The primary structure of Gp29 is the same as alpha-amylase inhibitor-like protein (AIL) from which the encoding gene has already been isolated. AIL resembles a putative precursor of alphaAI, even though it does not form the active inhibitor. However, AIL has some inhibitory effect on the growth of C. maculatus but not C. chinensis. The presence of AIL alone is insufficient to explain the bruchid resistance of common bean cultivars lacking alpha-AI. Common bean seeds appear to contain several factors responsible for the bruchid resistance.

Amino Acid Sequence↗
PubMed1998

Genetic localization of a bruchid resistance gene and its relationship to insecticidal cyclopeptide alkaloids, the vignatic acids, in mungbean (Vigna radiata L. Wilczek).

Bruchid resistance, controlled by a single dominant gene (Br) in a wild mungbean accession (TC1966), has been incorporated into cultivated mungbean (Vigna radiata). The resistance gene simultaneously confers inhibitory activity against the bean bug, Riptortus clavatus Thunberg (Hemiptera: Alydidae). The resultant isogenic line (BC20 generation) was characterized by the presence of a group of novel cyclopeptide alkaloids, called vignatic acids. A linkage map was constructed for Br and the vignatic acid gene (Va) using restriction fragment length polymorphism (RFLP) markers and a segregating BC20F2 population. By screening resistant and susceptible parental lines with 479 primers, eight randomly amplified polymorphic DNA (RAPD) markers linked to Br were identified and cloned for use as RFLP probes. All eight RAPD-based markers, one mungbean, and four common bean genomic clones were effectively integrated around Br within a 3.7-cM interval. Br was mapped to a 0.7-cM segment between a cluster consisting of six markers and a common bean RFLP marker, Bng110. The six markers are closest to the bruchid resistance gene, approximately 0.2 cM away. The vignatic acid gene, Va, cosegregated with bruchid resistance. However, one individual was identified in the BC20F2 population that retained vignatic acids in spite of its bruchid susceptibility. Consequently, Va was mapped to a single locus at the same position as the cluster of markers and 0.2 cM away from Br. These results suggest that the vignatic acids are not the principal factors responsible for bruchid resistance in V. radiata but will facilitate the use of map-based cloning strategies to isolate the Br gene.

Alkaloids↗
PubMed1997

Molecular characterization of a bean alpha-amylase inhibitor that inhibits the alpha-amylase of the mexican bean weevil Zabrotes subfasciatus.

Cultivated varieties of the common bean (Phaseolus vulgaris L.) contain an alpha-amylase inhibitor (alpha AI-1) that inhibits porcine pancreatic alpha-amylase (PPA; EC 3.2.1.1) and the amylases of certain seed weevils, but not that of the Mexican bean weevil, Zabrotes subfasciatus. A variant of alpha AI-1, called alpha AI-2, is found in certain arcelin-containing wild accessions of the common bean. The variant alpha AI-2 inhibits Z. subfasciatus alpha-amylase (ZSA), but not PPA. We purified alpha AI-2 and studied its interaction with ZSA. The formation of the alpha AI-2-ZSA complex is time-dependent and occurs maximally at pH 5.0 or below. When a previously isolated cDNA assumed to encode alpha AI-2 was expressed in transgenic tobacco seeds, the seeds contained inhibitory activity toward ZSA but not toward PPA, confirming that the cDNA encodes alpha AI-2. The inhibitors alpha AI-1 and alpha AI-2 share 78% sequence identity at the amino acid level and they differ in an important region that is part of the site where the enzyme binds the inhibitor. The swap of a tripeptide in this region was not sufficient to change the specificity of the two inhibitors towards their respective enzymes. The three-dimensional structure of the alpha AI-1/PPA complex has just been solved and we recently obtained the derived amino acid sequence of ZSA. This additional information allows us to discuss the results described here in the framework of the amino acid residues of both proteins involved in the formation of the enzyme-inhibitor complex and to pinpoint the amino acids responsible for the specificity of the interaction.

Amino Acid Sequence↗
PubMed1997

Structural characterization of an alpha-amylase inhibitor from a wild common bean (Phaseolus vulgaris): insight into the common structural features of leguminous alpha-amylase inhibitors.

The primary structures of two subunits of an alpha-amylase inhibitor (alpha AI-2) from a wild common bean (Phaseolus vulgaris) were revealed by a comparison of the amino acid sequence previously deduced from the nucleotide sequence with the amino- and carboxyl-terminal amino acid sequences determined by conventional methods. The polypeptide molecular weight of alpha AI-2 obtained by the light-scattering technique, considered together with the sequence molecular weights revealed for the subunits, indicated that alpha AI-2 has the subunit stoichiometry of an alpha 2 beta 2 complex. These structural features were closely similar to those recently elucidated for a white kidney bean (P. vulgaris) alpha-amylase inhibitor, which is quite different in the inhibitory specificity from alpha AI-2. The post-translational processing of the precursor glycoproteins to form the tetrameric structure appeared to require an Arg residue close to the processing site. Further, the proper associations of the subunits into the tetrameric structures seemed to be strictly controlled by a few amino acids on the subunit interfaces.

Amino Acid Sequence↗
PubMed1997

Purification and properties of beta-fructofuranosidase from Clostridium perfringens.

beta-Fructofuranosidase [EC 3.2.1.26] in Clostridium perfringens was induced in the presence of sucrose and suppressed in the presence of glucose or maltose. The enzyme seems to be present in protoplasm in a soluble state. The beta-fructofuranosidase from C. perfringens cells grown on sucrose was purified by ammonium sulfate precipitation. DEAE-cellulose chromatography, Sephadex G-150 gel filtration, and hydroxylapatite chromatography to a homogeneous state. The molecular weight was 37,000 by gel filtration using Sephadex G-150 and by SDS-polyacrylamide gel electrophoresis. The amino acid composition is not much different from those of other microorganisms, but the Glx content was a little higher. The enzyme was inhibited by heavy metals, such as Hg2+, Cu2+, and Ag+, as well as pCMB; the activity was restored by incubating with mercaptoethanol. Fructose and amines including Tris and aniline had inhibitory effects.

Amino Acids↗
PubMed1996

Protective mechanism of the Mexican bean weevil against high levels of alpha-amylase inhibitor in the common bean.

Alpha-amylase inhibitor (alpha AI) protects seeds of the common bean (Phaseolus vulgaris) against predation by certain species of bruchids such as the cowpea weevil (Callosobruchus maculatus) and the azuki bean weevil (Callosobruchus chinensis), but not against predation by the bean weevil (Acanthoscelides obtectus) or the Mexican bean weevil (Zabrotes subfasciatus), insects that are common in the Americas. We characterized the interaction of alpha AI-1 present in seeds of the common bean, of a different isoform, alpha AI-2, present in seeds of wild common bean accessions, and of two homologs, alpha AI-Pa present in seeds of the tepary bean (Phaseolus acutifolius) and alpha AI-Pc in seeds of the scarlet runner bean (Phaseolus coccineus), with the midgut extracts of several bruchids. The extract of the Z. subfasciatus larvae rapidly digests and inactivates alpha AI-1 and alpha AI-Pc, but not alpha AI-2 or alpha AI-Pa. The digestion is caused by a serine protease. A single proteolytic cleavage in the beta subunit of alpha AI-1 occurs at the active site of the protein. When degradation is prevented, alpha AI-1 and alpha AI-Pc do not inhibit the alpha-amylase of Z. subfasciatus, although they are effective against the alpha-amylase of C. chinensis. Alpha AI-2 and alpha AI-Pa, on the other hand, do inhibit the alpha-amylase of Z. subfasciatus, suggesting that they are good candidates for genetic engineering to achieve resistance to Z. subfasciatus.

Amino Acid Sequence↗
PubMed1994

cDNA sequence and deduced primary structure of an alpha-amylase inhibitor from a bruchid-resistant wild common bean.

alpha-Amylase inhibitor-2 (alpha AI-2), a seed storage protein present in a bruchid-resistant wild common bean (Phaseolus vulgaris), inhibits the growth of bruchid pests. The authors isolated and determined the sequence of an 852 nucleotide cDNA, designated as alpha ai2, and found it to contain a 720 base open reading frame (ORF). This ORF encodes a 240 amino-acid alpha AI-2 polypeptide 75.8% identical with alpha-amylase inhibitor-1 (alpha AI-1) and 50.6-55.6% with arcelin-1, phytohemagglutinin (PHA)-L and PHA-E of common bean. The high degree of sequence homology suggests that there is an evolutionary relationship among these genes.

Amino Acid Sequence↗
PubMed1992

The enhancement of specific antibody production rate in glucose- and glutamine-controlled fed-batch culture.

The concentration effects of certain amino acids (Asp, Ile, Leu, Lys, Met, Val, Phe and Gln which were highly consumed during cultivation), and glucose on cell growth and antibody productivity were investigated using dish culture. From these experiments, it was found that only glutamine enrichment enhanced the specific antibody production rate. The other amino acids described above did not affect either the specific growth rate or specific antibody production rate. Thus we investigated the quantitative effects of glutamine concentration in the range of 0.4-33.3 mmol.l-1 on kinetic parameters in fed-batch culture which kept both glucose and glutamine concentration constant. As a result the specific growth rate decreased with increase in glutamine concentration in the range larger than 20 mmol.l-1. The specific antibody production rate had a maximum value at about 25 mmol.l-1 glutamine concentration.

Animals↗
PubMed1989

Rubredoxin from Clostridium perfringens: complete amino acid sequence and participation in nitrate reduction.

The complete primary structure of rubredoxin (Rd) isolated from Clostridium perfringens was sequenced to be: MKKFICDVCGYIYDPAVGDPDNGVEPGTEFKDIPDDWVCPLCGVDKSQFSETEE. The sequence was highly homologous to that of C. pasteurianum Rd but was different at 13 sites out of the total 54 amino acid residues (76% homology). It contained 1 Fe atom, 4 cysteine residues, and no labile sulfur, had a molecular weight of 6,056, and shared the general properties of classical anaerobic Rds. The pI was 4.4. The Rd was reduced with NADH in the presence of a specific NAD(P)H oxidoreductase preparation from the bacterium. The Km value of nitrate reductase for Rd as an electron-donor was 12 microM, a value comparable to that of the 13 microM for ferredoxin (Fd). These results taken together provide additional support for its role as the electron carrier in the nitrate reductase system [Seki, S., Ikeda, A., and Ishimoto, M. (1988) J. Biochem. 103, 583-584].

Amino Acid Sequence↗
PubMed1988

Rubredoxin as an intermediary electron carrier for nitrate reduction by NAD(P)H in Clostridium perfringens.

The NAD(P)H-dependent nitrate reductase system in Clostridium perfringens was reconstituted with rubredoxin (Rd), nitrate reductase (NaR), and an unadsorbed fraction, on a DEAE-cellulose column, of the extract (designated as fraction A), under nitrogen gas. Ferredoxin in place of Rd was not effective as an electron carrier in this reconstituted system. NAD(P)H-dependent nitrate reducing activity was also obtained by replacing fraction A with ferredoxin-NADP+ reductase from spinach. We propose the following scheme for the electron transfer in this NAD(P)H dependent nitrate reduction system. NAD(P)H----NAD(P)H-Rd reductase----Rd----NaR----NO3-.

Clostridium perfringens↗
PubMed1988

Introduction and expression of the interleukin 2 receptor (Tac) gene in hematopoietic stem cells with retrovirus vectors.

Retrovirus vectors provide an efficient carrier for introducing a gene into hematopoietic stem cells although expression of the inserted gene is not always successful. We constructed and compared three retrovirus vectors which carried cDNA encoding the light chain (Tac) of the interleukin 2 receptor under the control of different promoters; long terminal repeat (LTR) of murine retroviruses, the early promoter of simian virus 40 (SV40) and the promoter of the class I antigen gene of the major histocompatibility complex. We made three constructs containing these promoters. A first construct did not contain any additional promoter but LTR. A second and a third constructs contained the SV40 and the class I antigen gene promoters, respectively, in addition to LTR. The LTR of retrovirus vectors is derived from MoMuLV except that the U3 region of the 3'LTR of the third construct is derived from myeloproliferative sarcoma virus (MPSV). The second and third constructs were used for infection of bone marrow stem cells as the first construct was less efficient in expression of the interleukin 2 receptor in fibroblasts. Hematopoietic stem cells infected with the recombinant viruses were transplanted into lethally irradiated mice, and the expression of the transduced gene in hematopoietic progenitor cells was analyzed. Analysis of RNA isolated from spleen colonies showed that substantial amounts of interleukin 2 receptor mRNA were made by the construct containing the class I gene promoter and MPSV LTR. However, we could not detect any transcripts from the constructs containing MoMuLV LTR and SV40 early region promoter.

Animals↗
PubMed1987

Studies on nitrate reductase of Clostridium perfringens. IV. Identification of metals, molybdenum cofactor, and iron-sulfur cluster.

Nitrate reductase of Clostridium perfringens was purified by an improved method using immuno-affinity chromatography. The purified preparation contained Mo, Fe, and acid-labile sulfide; the Mo content was 1 mol per mol and the Fe 3.7 mol per mol of the enzyme. The inactive enzyme obtained from cells grown in the presence of tungstate did not hold Mo but contained 1 mol of W. The content of Fe was not increased. The presence of molybdenum cofactor in the nitrate reductase was indicated by the formation of molybdopterin form A in the oxidation of the enzyme by iodine and by the complementation of NADPH-nitrate reductase with the heart-treated enzyme in the extract of Neurospora crassa nit-1. The Clostridium nitrate reductase had an absorption maximum at 279 nm and shoulders at 320, 380, 430, and 520 nm. This enzyme seems to contain an iron sulfur cluster since the reduced enzyme showed decreased absorption in visible region. The CD spectrum of the enzyme has a positive peak at 425 nm and negative ones at 310, 360, and 595 nm. It was compared with the CD spectrum of ferredoxin (2Fe-2S or 4Fe-4S cluster) and the nitrate reductase of Plectonema boryanum.

Chromatography, Affinity↗