Yasuo Natori

Allergens in major crops

A great number of allergens have been found in foodstuffs. Many allergens in plant foods seem to be classified into a restricted number of groups such as storage proteins, structure proteins and defense-related proteins. This paper will review the present status of characterization of allergens in major crops (soybean, wheat, barley, rice and maize), and indicates attempts to develop hypoallergenic foodstuffs for the benefit of sensitive patients.

Purification, Characterization and Differentiation-Dependent Expression of a Perchloric Acid Soluble Protein from Rat Kidney

We have recently reported the presence of a novel perchloric acid soluble protein in rat liver (PSP1) that inhibits cell-free protein synthesis in a rabbit reticulocyte system. While studying the perchloric acid soluble proteins from different tissues of rats, we found that the kidney protein cross-reacted with antibody against the PSP1. In this investigation, we have purified a perchloric acid soluble protein from the rat kidney and studied its characterization and expression. The protein extracted from the postmitochondrial supernatant fraction with 5% perchloric acid was purified by ammonium sulfate fractionation and CM-Sephadex chromatography. By immunoscreening with the rabbit antisera against the PSP1, we detected a cDNA that contained an open reading frame of 411 bp, encoding a 137 amino-acid protein with a molecular mass of 14,149 daltons. The deduced amino acid sequence was completely identical with that of PSP1 from rat liver. The perchloric acid soluble protein from rat kidney (K-PSP1) also inhibited cell-free protein synthesis in the rabbit reticulocyte lysate system in a different manner than RNase A. Immunohistochemistry showed that the expression of K-PSP1 increased from fetal 17th day to postnatal 4th week, and it remained almost the same until the 7th week of postnatal age. Furthermore, the expression of K-PSP1 in the kidney of the nephrotic rat model was shown to be differentiation dependent. On the other hand, the expression of K-PSP1 in renal tumor cells was downregulated as compared with intact tissue. These results suggest that the expression of K-PSP1 is regulated in a differentiation-dependent manner in the kidney.

Vitamin B6 deficiency causes activation of RNA polymerase and general enhancement of gene expression in rat liver

The effect of vitamin B6 deficiency on the activity of RNA polymerase and expression of several mRNAs in rat liver was investigated. The activities of RNA polymerase I and II in the liver of vitamin B6‐deficient rats were found to be higher than the control rats by 30%. The expression of several mRNAs, including mRNAs for β‐actin and glyceraldehyde‐3‐phosphate dehydrogenase, and the content of poly(A)+ RNA were also increased in vitamin deficiency. These observations suggest that vitamin B6 influences gene expression in the liver, at least in part, by modulating the activity of RNA polymerase.

Effect of actinomycin D on turnover rate of messenger ribonucleic acid in rat liver

Abstract The administration of actinomycin D to rats was shown to cause a prolongation of the half-life of liver mRNA, by studying the kinetics of polysome breakdown after actinomycin treatment. This finding was confirmed by the study of the radioactive mRNA profiles obtained from control and actinomycin-treated rat livers.

Vitamin B6 suppresses growth and expression of albumin gene in a human hepatoma cell line HepG2

The effect of vitamin B6 on the growth of a human hepatoma cell line HepG2 in culture was studied. The growth of HepG2 cells and protein synthesis were almost completely inhibited in medium supplemented with 5 mM pyridoxine. Pyridoxal was as effective as pyridoxine, but pyridoxamine showed no inhibitory action. The growth inhibition of HepG2 cells by pyridoxine was accompanied by a marked inhibition of secretion of plasma proteins, particularly albumin. Northern blot analysis of albumin mRNA showed that pyridoxine caused a rapid decrease in the expression of albumin gene. The electron-microscopic examination of pyridoxine-treated HepG2 cells revealed a smoothing of nuclear membrane, a decrease in the number of nucleoli, and an appearance of aggregated heterochromatin structures. These morphological features are compatible with the depressed transcriptional activity in the pyridoxine-treated cells. The mechanism by which vitamin B6 exerts its inhibitory effect was discussed in terms of our recent finding that vitamin B6 modulates expression of albumin gene by inactivating tissue-specific DNA-binding proteins. Binding of pyridoxal phosphate with tissue-specific transcription factors may reduce the capacity of these factors to interact with the regulatory region of albumin gene, resulting in the inhibition of the gene expression.

Cloning and sequencing of cDNA encoding 4-aminobenzoate hydroxylase fromAgaricus bisporus

Abstract A cDNA clone encoding 4-aminobenzoate hydroxylase (EC 1.14.13.27) has been isolated using a probe prepared by PCR on the basis of partially determined amino acid sequences of the enzyme. The cDNA contained 1380-base pair open reading frame encoding 460 amino acid residues (Mr 50 974), 14-base pair 5′-untranslated region and 123-base pair 3′-untranslated region including a poly(A) tail of 20 nucleotides. All of the partially determined amino acid sequences were shown to be included in the deduced amino acid sequence. Homology analyses showed that the two regions on the enzyme share other flavoproteins such as salicylate hydroxylase and p-hydroxybenzoate hydroxylase.

Effect of vitamin B6 deficiency on the expression of glycogen phosphorylase mRNA in rat liver and skeletal muscle

The effect of vitamin B6 deficiency on the expression of glycogen phosphorylase mRNA in rat liver and skeletal muscle was investigated. The level of phosphorylase mRNA in the muscle of vitamin B6-deficient rats was reduced to 40% of that in the control rats. By contrast, the phosphorylase mRNA level was increased 5-fold in the liver of the deficient animals. It was also found that the expression of the β-actin gene, generally regarded as a ‘housekeeping’ gene, was unaffected by B6 deficiency in the muscle but was enhanced in the liver of the deficient animals. These observations suggest that vitamin B6 may modulate the transcriptional activation of the phosphorylase gene in a tissue-specific manner.

Gene expression and aging

Considerable amount of data has accumulated during the past few years showing several changes in gene expression as a function of age. However, the basic mechanism of aging still remains poorly understood. In this review, we have mainly analysed the data pertaining to the hypothesis that aging is associated with genetic instability and have attempted further to highlight the gaps that need to be bridged in order to have a clear picture of the aging phenomenon. Extensive investigations employing new and novel approaches are needed in future to elucidate the intricately interwoven patterns of molecular control that underlie the various aspects of gene expression during aging.

Metabolic turnover of messenger ribonucleic acid in rat liver

Abstract The experimental conditions are described for the selective digestion of hepatic polyribosome-associated mRNA by ribonuclease treatment. Comparison of the specific activities of high molecular weight RNA prepared from pulse-labeled polyribosomes before and after ribonuclease treatment made the quantitative estimation of activity in mRNA possible. By following the time-course change in the activities of mRNA, an average half-life of approx. 5 h for the mRNA of rat liver was obtained. The heterogeneity of the rates of turnover with respect to the size of mRNA was also indicated.

Purification and properties of an aminopeptidase from rat-liver cytosol

An aminopeptidase was purified from the rat-liver cytosolic fraction to apparent electrophoretic homogeneity. The enzyme is a monomeric protein of 95 kDa, having an isoelectric point of 4.9. Amino acid analyses indicate that the enzyme is rich in acidic amino acids and is poor in cysteine. The enzyme hydrolyzed a broad spectrum of amino acid beta-naphthylamides at a neutral pH. The enzyme also hydrolyzed di-, tri-, and oligopeptides, including physiologically active peptides such as enkephalins and Met-Lys-bradykinin. The enzyme was inhibited by metal-chelating agents, sulfhydryl-reactive reagents, N-P-tosyl-L-phenylalaninechloromethyl ketone, N-P-tosyl-L-lysinechloromethyl ketone, and puromycin but not by protease inhibitors of microbial origin. The enzyme was activated by the addition of Co2+ and sulfhydryl compounds. The aminopeptidase enhanced proteolysis when the enzyme was added to the protease assay system with purified rat-liver cytosolic neutral protease, suggesting the cooperative action of aminopeptidase in the overall process of protein degradation.