Biomedical subjects

Nobuyuki Maruyama

Publications and source records attributed to Nobuyuki Maruyama.

At least 19 recordsLinked to original sources

PubMed2006

The C-terminal region of alpha' subunit of soybean beta-conglycinin contains two types of vacuolar sorting determinants.

In maturing seed cells, proteins that accumulate in the protein storage vacuoles (PSVs) are synthesized on the endoplasmic reticulum (ER) and transported by vesicles to the PSVs. Vacuolar sorting determinants (VSDs) which are usually amino acid sequences of short or moderate length direct the proteins to this pathway. VSDs identified so far are classified into two types: sequence specific VSDs (ssVSDs) and C-terminal VSDs (ctVSDs). We previously demonstrated that VSDs of alpha' and beta subunits of beta-conglycinin, one of major storage proteins of soybean (Glycine max), reside in the C-terminal ten amino acids. Here we show that both types of VSDs coexist within this region of the alpha' subunit. Although ctVSDs can function only at the very C-termini of proteins, the C-terminal ten amino acids of alpha' subunit directed green fluorescent protein (GFP) to the PSVs even when they were placed at the N-terminus of GFP, indicating that an ssVSD resides in the sequence. By mutation analysis, it was found that the core sequence of the ssVSD is Ser-Ile-Leu (fifth to seventh residues counted from the C-terminus) which is conserved in the alpha and beta subunits and some vicilin-like proteins. On the other hand, the sequence composed of the C-terminal three amino acids (AFY) directed GFP to the PSVs when it was placed at the C-terminus of GFP, though the function as a VSD was disrupted at the N-terminus of GFP, indicating that the AFY sequence is a ctVSD.

Amino Acid Sequence↗

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

[Clinical efficacy and cost benefit of oral antimicrobial prophylaxis with levofloxacin on neurological surgery].

OBJECTIVE: The clinical efficacy and cost benefit of oral antimicrobial prophylaxis with levofloxacin (LVFX) on neurosurgical operation were evaluated. PATIENTS AND METHODS: Fifty patients undergone burr hole drainage for chronic subdural hematoma were enrolled into this study. These patients had preoperative intravenous drip infusion of 1.0 g of cefazolin (CEZ), and were given oral 200 mg of LVFX twice a day for postoperative 6 days on their operations. Clinical effect on prophylaxis of postsurgical infection and surgical site infection as well as safeness of the drug were assessed. Their clinical course were retrospectively compared with another fifty patients as control group who had intravenous administration of CEZ for consecutive 7 days on same operation. RESULTS: There were no patients who had postsurgical infection and surgical site infection as well as drug related adverse effect, whereas three patients in control group suffered urinary infections or respiratory tract infection during their postsurgical course. CONCLUSIONS: Less invasive oral antimicrobial prophylaxis with LVFX is effective to prevent postoperative infections and surgical site infections, and attractive from the medical economic point of view.

Administration, Oral↗

PubMed2005

Design of genetically modified soybean proglycinin A1aB1b with multiple copies of bioactive peptide sequences.

The peptide IIAEK derived from beta-lactoglobulin has a hypocholesterolemic activity greater than that of beta-sitosterol. To create food proteins with multiple copies of this valuable peptide sequence, we introduced tandem multimers of the nucleotide sequence encoding the peptide into DNA regions corresponding to the five variable regions of soybean glycinin A1aB1b subunit, and expressed the mutants in Escherichia coli. The expression level and solubility of the five mutants, each containing four IIAEK sequences in each of the variable regions, were compared. Overall, the expression level and solubility of the mutants with four IIAEK sequences in the variable regions IV and V were the best followed by II > III > I. Further, introduction of the fifth IIAEK sequence to the variable region IV did not decrease expression level and solubility. Increasing the number of IIAEK to 7 and 10 slightly decreased expression level, while their solubility decreased to as low as 40 and 1%, respectively. Various mutations were combined to get a mutant containing as many IIAEK sequences as possible. Some of the resulting mutants were expressed in the soluble form. The mutant containing eight IIAEK from the combination of variable regions IV and V (IV-4 + V-4) showed the best balance of the expression level and solubility, followed by the combination of variable regions II and III (II-4 + III-4). The soluble fractions of these mutants were purified by hydrophobic, gel filtration and ion-exchange column chromatography. Yields of IIAEK peptide released by in vitro digestion with trypsin from both mutants were around 80%. This is the first report that a large amount of a physiologically active peptide could be introduced into food protein.

Amino Acid Sequence↗

PubMed2005

Evaluation of the solubility and emulsifying property of soybean proglycinin and rapeseed procruciferin in relation to structure modified by protein engineering.

The presence or absence of a highly negatively charged extension region in beta-conglycinin (J. Agric. Food Chem. 1999, 47, 5278) and the length of a highly negatively charged variable region IV in glycinin (J. Agric. Food Chem. 2004, 52, 8197) are important determinants of solubility and emulsifying property. To examine the effects of the variable region IV from proglycinin A1aB1b and A3B4 and of the extension region from beta-conglycinin alpha' (alpha'ext) on solubility and emulsifying properties in detail, several mutants of proglycinin, procruciferin, and beta-conglycinin were designed and prepared in Escherichia coli. Nine out of 10 mutants were expressed at high levels in E. coli and shown to be homotrimer similar to the wild types as assessed by gel filtration. The position of the introduced negatively charged region as well as the amino acid composition were demonstrated to affect solubility at mu = 0.08. All of the proglycinin, procruciferin, and beta-conglycinin mutants with the alpha'ext in the C-terminus, especially the proglycinin mutant, exhibited excellent emulsifying ability and emulsion stability. These indicate that improvement of emulsifying properties by insertion of the alpha'ext in the C-terminus may be generally applicable to seed globulins.

Antigens, Plant↗

PubMed2005

Structure-function relationships of soybean proglycinins at subunit levels.

Glycinin consists of five kinds of subunits, group I (A1aB1b, A1bB2, and A2B1a) and group II (A3B4 and A5A4B3). cDNAs for individual subunits were cloned by reverse transcription-polymerase chain reaction method and expressed in Escherichia coli using pET vector. The recombinant proglycinins were purified by ammonium sulfate fractionation and column chromatography in the form of homotrimers. Physicochemical properties such as molecular dimensions, solubility, surface hydrophobicity, thermal stability, and emulsifying ability of individual proglycinins were studied. Molecular dimensions were proportional to molecular size for all proglycinins except A2B1a. Solubility was intrinsic to each proglycinin. At the ionic strength of 0.5, all proglycinins except A1aB1b showed a very low solubility at acidic pH, but A1aB1b was soluble to higher than 60%. At ionic strength 0.08, all proglycinins exhibited isoelectric precipitation, although A2B1a and A1bB2 were not completely insoluble. The order of emulsifying ability (A1bB2 < A2B1a < A5A4B3 < A3B4 < or = A1aB1b) was not of the same for surface hydrophobicity (A5A4B3 < A1aB1b < or = A3B4 < A1bB2 < A2B1a) and thermal stability (A1bB2 << A2B1a < or = A5A4B3 < A3B4 < or = A1aB1b).

Calorimetry, Differential Scanning↗

PubMed2005

Molecular analysis and physicochemical properties of electrophoretic variants of wild soybean Glycine soja storage proteins.

Cultivated soybeans (Glycine max) are derived from wild soybeans (Glycine soja) and can be crossed with them to produce fertile offspring. The latter exhibit greater genetic variation than the former, suggesting a possibility that wild soybeans contain storage proteins with properties different from and better than those of cultivated soybeans. To identify a wild soybean suitable for breeding a new soybean cultivar, we analyzed seed proteins from 390 lines of wild soybeans by electrophoresis. We found some lines containing electrophoretic variants of glycinin and beta-conglycinin subunits: one line containing a small alpha' subunit of beta-conglycinin and two and five lines containing small A3 and large A4 polypeptides of glycinin, respectively. Beta-Conglycinin and glycinin containing such variant subunits exhibited solubility and emulsifying ability similar to those of the predominant types of wild and cultivated soybeans. Glycinins containing small A3 and large A4 gave a shoulder derived from the start of denaturation at a temperature 4 degrees C lower than that of glycinin from the predominant types of wild and cultivated soybeans, although their thermal denaturation midpoint temperatures were very similar to each other. Cloning and sequencing of the predominant and variant subunit cDNAs revealed that the small alpha' and the small A3 lacked 24 amino acid residues in the extension region and four amino acid residues in the hypervariable region, respectively, and that the large A4 did not have an insert corresponding to the difference in the electrophoretic mobility but Arg279 and Gln305 were replaced by glutamine and histidine, respectively, in the hypervariable region. These suggest that small differences even in the hypervariable region can affect the thermal stability, as well as the electrophoretic mobilities, of the proteins.

Amino Acid Sequence↗

PubMed2005

Epilepsy and variation in the frontal lobe artery.

A patient with a rare variation of fronto-orbital artery (FOA) that developed generalized tonic and clonic seizures is reported. The epilepsy focus was in her left frontal region, where blood was supplied by the contralateral fronto-orbital artery. The region was vulnerable to ischemic changes due to a decrease in blood CO2 gas caused by an increase in endogenous progesterone in the luteal period. The anomaly illustrates an important mechanism of ischemia in epilepsy.

Adult↗

PubMed2005

Unusual variant of the anterior cerebral artery.

A rare abnormality of the A1 segment of the anterior cerebral artery (ACA) is reported. The right ACA bifurcated into two parts at the middle point of the A1 segment, and these segments did not rejoin. The superior right A1 segment connected with the left A1 and formed a single pericallosal artery. The inferior right A1, from which the right ophthalmic artery originated, had no connection with the left A1.

Adult↗

PubMed2005

A novel enzyme-linked immunosorbent assay for quantification of soybean beta-conglycinin, a major soybean storage protein, in soybean and soybean food products.

Soybean (Glycine max L.) storage proteins are composed of two major components, beta-conglycinin and glycinin, corresponding to 7S and 11S globulins, respectively. Recently, soybean beta-conglycinin (7S globulin) has been reported to show beneficial functions in animals and human. To date, there is no method for the precise quantification of soybean beta-conglycinin in processed food products or soybean seeds. We report here a novel method for this purpose. At first, antibodies specifically reactive to the subunits of beta-conglycinin were prepared. And then, a direct enzyme-linked immunosorbent assay (ELISA) for the quantification of soybean beta-conglycinin in processed foods and seeds was developed. In this assay, the sample was treated with sodium dodecyl sulfate sample buffer followed by dilution with phosphate-buffered saline. The diluted samples were poured and coated onto an ELISA plate and reacted with rabbit anti-beta-conglycinin antibody and peroxidase-labeled anti-rabbit IgG. Finally, the bound peroxidase-labeled antibody was detected by colorimetric reaction. By using this system, it has been possible to measure soybean beta-conglycinin concentrations in several processed food products. In addition, this simple quantification ELISA system was demonstrated to be adaptable for the quantification of beta-conglycinin contents of various soybean cultivars.

Animals↗

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

Identification of the 23-kDa peptide derived from the precursor of Gly m Bd 28K, a major soybean allergen, as a new allergen.

One of the major soybean allergens, Gly m Bd 28K, is suggested to be biosynthesized as a preproprotein form, which would be composed of a signal peptide, Gly m Bd 28K and the C-terminal peptide (the 23-kDa peptide). However, the 23-kDa peptide has never been characterized. In the present study, we prepared a monoclonal antibody (mAb) against a recombinant 23-kDa peptide expressed in Escherichia coli to detect the 23-kDa peptide in soybean. Several proteins were detected by immunoblotting with the mAb. All of the proteins were shown to have the identical N-terminal amino acid sequence, suggesting that the proteins correspond to the C-terminal part of the Gly m Bd 28K precursor. Furthermore, Gly m Bd 28K and the 23-kDa peptide were observed to come out at the 21st day after flowering and to locate in the crystalloid part of protein storage vacuoles in growing cotyledons. Some of the 23-kDa peptides were shown to be glycoproteins with an N-linked glycan moiety and exhibited the binding to IgE antibodies in the sera of patients sensitive to soybean. The binding of the peptides to IgE antibodies was suggested to be predominantly dependent on their glycan moiety. This study proves the occurrence of the 23-kDa peptide in soybean and that it is a new allergen.

Allergens↗

PubMed2004

Effects of protein engineering of canola procruciferin on its physicochemical and functional properties.

The primary structure of Brassica napus procruciferin 2/3a was engineered to elucidate structure-function relationships and to improve the functionality of cruciferin. The following mutants were constructed: (1) C287T, (2) DeltaII, variable region II was deleted; (3) C287T/DeltaII, mutation involving (1) and (2); (4) DeltaIV + A1aIV; and (5) DeltaIV + A3IV, variable region IV was replaced with variable region IV containing many charged residues from soybean glycinin A1aB1b and A3B4 subunits. Differential scanning calorimetry analysis revealed that the A1aIV region has a more favorable interaction with the procruciferin molecule than does A3IV as well as the original regions. On the basis of heat-induced precipitation analysis, it was concluded that replacement of the free cysteine residue with threonine (C287T) and insertion of charged regions (DeltaIV + A1aIV and DeltaIV + A3IV) could lead procruciferin to form soluble aggregates after heating. Low solubility was observed in mutants DeltaIV + A3IV, DeltaII, and C287T/DeltaII, especially between pH 4 and 6 at mu = 0.08, but not in DeltaIV + A1aIV, indicating that the number of acidic amino acid residues and the high number of glutamine residues are important factors for solubility at mu = 0.08. None of the mutants showed any improvements in emulsifying ability, indicating that destabilization and addition of the hydrophilic region are not effective for emulsification. The insertion of the A1aIV region in procruciferin made the molecule more susceptible to alpha-chymotrypsin.

Allergens↗

PubMed2004

Structure of the core region of the soybean beta-conglycinin alpha' subunit.

The crystal structure of the core region of the alpha' subunit (alpha(c')) of soybean beta-conglycinin has been determined at 2.3 A resolution. alpha(c') was superimposed on the known crystal structure of the beta-conglycinin beta subunit with a small root-mean square deviation of 0.77 A, which is consistent with the high sequence identity of 75.5% between alpha(c') and the beta subunit. It is known that the thermal stability of the beta subunit is higher than that of the alpha' subunit and that their thermal stabilities are conferred by highly homologous core regions. Comparisons of the three-dimensional structures and primary sequences between alpha(c') and the beta subunit suggest that five factors account for this difference between subunits as regards the difference in thermal stability: (i) the total cavity volume is larger in alpha(c'), (ii) the cluster of charged residues at the intermonomer interface is smaller in alpha(c') and alpha(c') lacks the intermonomer salt bridge of the beta subunit, (iii) the solvent-accessible surface is more hydrophobic in alpha(c'), (iv) there are fewer proline residues in alpha(c') and (v) a loop region between helix 3 and strand J' in alpha(c') is more flexible owing to the insertion of five additional residues. Although more hydrogen bonds were found in alpha(c'), this difference should be more than compensated for by the combined contributions of these other factors.

Amino Acid Sequence↗

PubMed2004

Optimal designing of beta-conglycinin to genetically incorporate RPLKPW, a potent anti-hypertensive peptide.

Previously, we introduced the RPLKPW sequence, a highly potent hypotensive peptide designed based on ovokinin (2-7), into three homologous sites in the soybean beta-conglycinin alpha' subunit by site-directed mutagenesis. The modified protein expressed in Escherichia coli reduced blood pressure of spontaneously hypertensive rats (SHRs) after oral administration at a dose of 10 mg/kg, which suggested about 30% of the introduced peptide was released in vivo. In this study amino acid residues around the RPLKPW sequence were optimized with a use of synthetic peptides to facilitate release of RPLKPW by gastrointestinal proteases. Then, fourth RPLKPW was also introduced into the extension domain of the protein. The newly modified protein, which was produced in E. coli, significantly lowered blood pressure in SHRs at a dose of 2.5 mg/kg 4 h after oral administration. Furthermore, we produced an extension domain that corresponds to residues 1-143 of the modified alpha' subunit containing four RPLKPW sequences by introducing a termination codon. The minimum effective dose of the modified extension domain was 1.0 mg/kg, which is 1/2000 that of ovalbumin.

Administration, Oral↗

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↗

PubMed2004

Gelling properties of soybean beta-conglycinin having different subunit compositions.

The effects of protein concentration, and heating temperature and time on the gelling properties of soybean beta-conglycinin (7S globulins) lacking the alpha or alpha' subunit were compared with those of 7S containing all three subunits (alpha, alpha', and beta) to determine whether each subunit contributes equally. In most of the conditions, the relative order of gel hardness was alpha'-lacking > 7S > alpha-lacking. From Fourier transform infrared studies, the secondary structure change after heating was very similar among the three samples; thus, the secondary structural change is not the reason for the differences in gel hardness. By using scanning electron microscopy, we observed differences in strand thickness and the density of the gel network among the three samples. These differences correlated well with the differences in gel hardness.

Antigens, Plant↗

PubMed2004

Soybean glycinin A1aB1b subunit has a molecular chaperone-like function to assist folding of the other subunit having low folding ability.

Soybean (Glycine max L.) glycinin is composed of five subunits which are classified into two groups (group I: A1aB1b, A1bB2, and A2B1a; group II: A3B4 and A5A4B3). All the common soybean cultivars contain both group I and II subunits (Maruyama, N. et al., Phytochemistry, 64, 701-708 (2003)). The biosynthesis of group I starts earlier compared with that of the A3B4 subunit during seed development (Meinke, D.W. et al., Planta, 153, 130-139 (1981)). We have revealed that group I A1aB1b was mostly expressed as a soluble protein, but that A3B4 was expressed mainly as an insoluble protein in Escherichia coli under the same expression conditions; namely, A1aB1b had higher folding ability than A3B4. We therefore assumed that A1aB1b assists folding of group II subunits like a molecular chaperone does. In order to ascertain this, A1aB1b and A3B4 were co-expressed in E. coli. All of the expressed proteins of A3B4 were recovered in a soluble fraction. To confirm this result, we also co-expressed A1aB1b with modified A3B4 versions having extremely low folding ability. All expressed modified A3B4 versions were soluble. These results clearly suggest that A1aB1b has a molecular chaperone-like function in their folding.

Cloning, Molecular↗