Gastrointestinal digestion and absorption of Pen j 1, a major allergen from Kuruma prawn, Penaeus japonicus.

Tropomyosin had been identified as a major allergen in shrimp. The digestion and absorption of tropomyosin (Pen j 1) from kuruma prawn were investigated by ex vivo, in vitro, and in vivo techniques in order to elucidate the relationship between the allergenicity of the allergen and its gastrointestinal behavior. Pen j 1 transported the Caco-2 monolayer in a dose-dependent manner, and also enhanced the permeability of lucifer yellow, a marker of paracellular transportation, at high concentrations of the allergen. Studies with everted sacs revealed that Pen j 1 was rapidly degraded to small peptides (MW<3.5 kDa) and amino acids by intestinal proteases and absorbed from enterocytes. Furthermore, Pen j 1 orally administered to rats tended to remain in the stomach rather than in the small intestine, after which the allergen moved to the epithelial cells. These observations suggest that Pen j 1 may be absorbed via the gastric mucosa prior to its digestion in the intestines.

Investigation of multiple forms of Tri a Bd 27K, a major wheat allergen, by immunoblotting analysis.

Tri a Bd 27K, a major wheat allergen, is a glycoprotein. Tri a Bd 27K was found to occur in multiple forms by two-dimensional polyacrylamide gel electrophoresis and immunoblotting with a monoclonal antibody against the allergen. Furthermore, it was found that only Tri a Bd 27K components, which have N-linked glycan moieties with fucose residues, bound to IgE antibodies in the sera of wheat-sensitive patients.

Preparation and epitope mapping of a monoclonal antibody against Tri a Bd 27K, a major wheat allergen.

We produced a monoclonal antibody (mAb) as a probe for detection of Tri a Bd 27K, a major wheat allergen. The mAb recognized the allergen purified from wheat flour, and the epitope on the allergen to the mAb was determined to be amino acid sequence (154)VPWVVVDGKPL(164) of Tri a Bd 27K. Of the amino acid residues on the epitope, the amino acid residues responsible for the binding to the mAb were found to be W156, D160, G161, and P163.

Development of a method for the determination of gamma-aminobutyric acid in foodstuffs.

A method for the determination of gamma-aminobutyric acid (GABA) in foodstuffs has been developed by combination of its dinitrophenylation and high-performance liquid chromatography (HPLC) using norleucine as an internal standard. GABA was converted to its stable derivative with 1-fluoro-2,4-dinitrobenzene and the derivative was extracted with ether. After evaporation of the extract, the residue was dissolved in 0.1 M NaOH and the solution was subjected to reversed-phase HPLC with an elution system of a linear gradient of methanol in 10 mM Tris-HCl buffer (pH 6.0) and a detection system monitoring the absorbance of the effluent at 400 nm. The present method was shown to be utilized as a satisfactory method for the determination of gamma-aminobutyric acid in foodstuffs.

Molecular cloning and allergenicity of Pen j 1, a major allergen of kuruma prawn, Penaeus japonicus.

Tropomyosins have been identified as a common allergen in crustaceans, but their allergenicity is not well understood. In the present study, we isolated an allergen, Pen j 1, a tropomyosin from kuruma prawn Penaeus japonicus, and determined its N-terminal amino acid sequence. The cDNA encoding the allergen was cloned by 5'- and 3'-rapid amplification of cDNA ends (RACE), and was found to code for a protein which consists of 284 amino acid residues. Sequencing analyses indicated for the first time that mature tropomyosin is formed by the elimination of a leader peptide of nine amino acid residues. To elucidate the binding sites of IgE antibodies in the sera of shrimp-sensitive patients, various recombinant peptides were expressed in Escherichia coli as fusion proteins with glutathione S-transferase (GST), and the examined with regard to reactivity with IgE antibodies. The IgE-binding epitopes were found to locate over the whole sequence of the allergen, and the IgE antibodies in the sera were found to recognize strongly its C-terminal region.

Influence of cooking on anthocyanins in black rice (Oryza sativa L. japonica var. SBR).

The composition and thermal stability of anthocyanins in black rice (Oryza sativa L. japonica var. SBR) produced in California were investigated. Six anthocyanin pigments were identified and quantified by high performance liquid chromatography using photo diode-array detection (HPLC-PDA) and electrospray ionization mass spectrometry [LC-(ESI)MS/MS]. The predominant anthocyanins are cyanidin-3-glucoside (572.47 microg/g; 91.13% of total) and peonidin-3-glucoside (29.78 microg/g; 4.74% of total). Minor constituents included three cyanidin-dihexoside isomers and one cyanidin hexoside. Thermal stability of anthocyanins was assessed in rice cooked using a rice cooker, pressure cooker, or on a gas range. All cooking methods caused significant (P < 0.001) decreases in the anthocyanins identified. Pressure cooking resulted in the greatest loss of cyanidin-3-glucoside (79.8%) followed by the rice cooker (74.2%) and gas range (65.4%). Conversely, levels of protocatechuic acid increased 2.7 to 3.4 times in response to all cooking methods. These findings indicate that cooking black rice results in the thermal degradation of cyanidin-3-glucoside and concomitant production of protocatechuic acid.

Effects of acetate on lipid metabolism in muscles and adipose tissues of type 2 diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats.

We have reported that orally administrated acetate contributed to suppression of lipogenesis in the liver and to reduction of lipid accumulation in the adipose tissue of Otsuka Long-Evans Tokushima Fatty (OLETF) rats. The aim of this study was to investigate the effect of acetate on skeletal muscle and adipose tissues. Treatment with acetate showed a higher rate of oxygen consumption and a smaller size of lipid droplets in white adipose and brown adipose tissues. An analysis by Northern blotting revealed that the transcripts of myoglobin and Glut4 genes in the abdominal muscle of the OLETF rats were increased by acetate treatment, while the transcripts of lipolytic genes increased in the white adipose and brown adipose tissues. It is possible that acetate has effects on lipid metabolism in the skeletal muscles and the adipose tissues, and has functions that work against obesity and obesity-linked type 2 diabetes.

Isolation and molecular cloning of a major wheat allergen, Tri a Bd 27K.

Tri a Bd 27K is the predominant allergen in wheat. In the present study, this allergen was purified to homogeneity from wheat flour. The N-terminal amino acid sequences of the purified allergen and the peptides obtained by its digestion, with trypsin were determined, and the allergen was shown to be a glycoprotein with an Asn-linked sugar moiety containing fucose residues. A cDNA encoding the allergen was obtained by polymerase chain reaction (PCR). The cDNA codes for a protein of 203 amino acid residues, with a molecular mass of 22,803 Da, that has two tentative sites glycosylated at Asn residues. Homology analysis suggested that the allergen might belong to a family of gamma-interferon-inducible thiol reductases. The cDNA was expressed as a fusion protein with glutathione S-transferase in Escherichia coli. However, unlike the allergen purified from wheat, recombinant Tri a Bd 27K was not immunoblotted with IgE antibodies in the serum of a wheat-sensitive patient.

Characterization of new 18-kDa IgE-binding proteins in beer.

The IgE-binding proteins in beer were examined by immunoblotting analysis with sera of patients sensitive to beer. Several proteins were immunoblotted with the sera, and among these, 18-kDa proteins were identified as new IgE-binding proteins in beer. Perhaps they originated from barley as a raw material.

Characterization of inhibitors of postprandial hyperglycemia from the leaves of Nerium indicum.

Nerium indicum is an India-Pakistan-originated shrub belonging to the oleander family. The ingestion of leaves of N. indicum before a meal is known to effect the lowering of postprandial glucose levels in Type II diabetic patients and this plant is now used as a folk remedy for Type II diabetes in some regions of Pakistan. In the present study, the hot-water extract of N. indicum leaves was found to reduce the postprandial rise in the blood glucose when maltose or sucrose was loaded in rats. It was also found that the extract strongly inhibited alpha-glucosidase, suggesting that the suppression of the postprandial rise in the blood glucose is due to the occurrence of some inhibitors of alpha-glucosidase in the leaves. We, therefore, tried to isolate the active principles from the leaf extract, using alpha-glucosidase-inhibitory activity as the index. Employing Sephadex G-15, silica gel and reversed-phase HPLC, we isolated two active compounds. The UV, mass and NMR spectrometric analyses established that the chemical structures of these compounds are 3-O-caffeoylquinic acid (chlorogenic acid) and its structural isomer, 5-O-caffeoylquinic acid. Both compounds were shown to inhibit alpha-glucosidases in a non-competitive manner. The authentic chlorogenic acid was found to suppress the postprandial rise in the blood glucose in rats and also inhibited the absorption of the glucose moiety from maltose and glucose in the everted gut sac system prepared from rat intestine. These results demonstrate that chlorogenic acid is one of the major anti-hyperglycemic principles present in the leaves of N. indicum. Furthermore, among polyphenol compounds tested, quercetin and catechins were shown to have strong inhibitory activity against alpha-glucosidase.

Improvement of obesity and glucose tolerance by acetate in Type 2 diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats.

Acetate has been found to have an inhibitory effect on the activity of carbohydrate-responsive element-binding protein (ChREBP) in cultured hepatocytes, this being a transcription factor that regulates several genes required for the conversion of glucose to fatty acids in the liver. The aim of this study was to investigate whether an oral administration of acetate would contribute to reducing lypogenic genes and protecting against obesity. We orally injected 5.2 mg/kg BW of acetate to obesity-linked type 2 diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats. The treatment with acetate showed a marked reduction in lipid accumulation in the adipose tissue, protection against accumulation of fat in the liver, and improved glucose tolerance. An analysis by Northern blotting revealed that the transcripts of several lipogenic genes in the liver of OLETF rats were decreased by the acetate treatment. On the basis of those results, it was indicated that acetate was a potential compound to improve obesity and obesity-linked type 2 diabetes.

Effect of a hypolipidemic drug, Di (2-ethylhexyl) phthalate, on mRNA-expression associated fatty acid and acetate metabolism in rat tissues.

Di (2-ehtylhexyl) phthalate (DEHP) is a peroxisome proliferator and a drug having a hypolipidemic effect. The body-weight change of rats treated with DEHP was lower than that of rats in an untreated control group. Expressions of long-chain acyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase, which are involved in fatty acid oxidation and acetate formation in mitochondria, showed an increase in the liver and testes of rats treated with DEHP. The expression of acetyl-CoA synthetase 1 was significantly decreased in the testes and relatively decreased in the liver, while the expression of acetyl-CoA synthetase 2 was significantly increased in the heart. Furthermore, the expressions of acetyl-CoA carboxylase in heart and testes showed a tendency to decrease. From these results, it is suggested that DEHP-treatment increased fatty acid oxidation and acetate formation in liver and testes, and that acetate utilization was increased in peripheral tissues such as the heart.

Acetate generation in rat liver mitochondria; acetyl-CoA hydrolase activity is demonstrated by 3-ketoacyl-CoA thiolase.

Acetate has been found as an endogenous metabolite of beta-oxidation of fatty acids in liver. In order to investigate the regulation of acetate generation in liver mitochondria, we attempted to purify a mitochondrial acetyl-CoA hydrolase in rat liver. This acetyl-CoA-hydrolyzing activity in isolated mitochondria was induced by the treatment of rats with di(2-ehtylhexyl)phthalate (DEHP), a peroxisome proliferator which induces expression of several peroxisomal and mitochondrial enzymes involved in beta-oxidation of fatty acids. The purified enzyme was 43-kDa in molecular mass by SDS/PAGE. Internal amino acid sequencing of this enzyme revealed that it was identical with mitochondrial 3-ketoacyl-CoA thiolase, suggesting that this enzyme has two kinds of activities, 3-ketoacyl-CoA thiolase and acetyl-CoA hydrolase activities. Kinetic studies clearly indicated that this enzyme had the both activities and each activity was inhibited by the substrates of the other activity, that is, 3-ketoacyl-CoA thiolase activity was inhibited by acetyl-CoA, on the other hand, acetyl-CoA hydrolase activity was inhibited by acetoacetyl-CoA in a competitive manner. These findings suggested that acetate generation in liver mitochondria is a side reaction of this known enzyme, 3-ketoacyl-CoA thiolase, and this enzyme may regulate its activities depending on each substrate level.

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.

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.

Purification and partial characterization of acetyl-coA synthetase in rat liver mitochondria.

Acetyl-CoA synthetase (AceCS), which catalyzes the activation of acetate to produce acetyl-CoA, was found to have a much greater Km value for acetate in liver mitochondria than that in the heart mitochondria of rats, indicating that two different types of AceCS are located in the liver and heart mitochodria. Recently, Fujino et al. reported that mouse heart mitochondrial AceCS, designated AceCS2, was expressed in a wide range of tissues, however, it was apparently absent from the liver. In this study, liver mitochondrial AceCS activity, but not heart AceCS2, was greatly induced in di(2-ethylhexyl)phthalate (DEHP)-treated rats. We purified and characterized the rat liver mitochondrial AceCS. The molecular mass of the enzyme estimated by SDS-PAGE was -58 kDa, which was quite different from that of the heart mitochondrial enzyme, AceCS2. The calculated Km value for the acetate of the partially purified liver enzyme was much greater, being about 100 times that of heart enzyme, AceCS2.

Identification of a wheat allergen, Tri a Bd 36K, as a peroxidase.

A 36-kDa allergen, Tri a Bd 36K, was purified from wheat albumin and characterized. The protein was similar to barley peroxidase BP-1 both in its amino acid sequence and peroxidase activity. The enzyme seemed to contain L-fucose and D-mannose and the glycan moiety reacted with IgE antibodies in a patient's serum.