Tatsuzo Oka

Diabetes and glucose transporter gene expression in rat small intestine

The expressions of Na(+)-dependent glucose transporter (SGLT1) and five facilitative glucose transporter genes (GLUT1-5) in the small intestine of streptozotocin (STZ)-induced diabetic rats were examined by RNA blotting analysis. The transcripts of SGLT1 mRNA gave bands of 4.5 Kilobases (Kb) and 2.8Kb (very faint band). The levels of SGLT1 mRNA were significantly increased in 30- and 60-day STZ rats, but not changed in acute diabetic rats (2- to 10- day STZ rats). The GLUT2 mRNA levels changed in parallel with the D-galactose transport activity, being increased about 4-fold in 5-day STZ rats. The transcripts of GLUT5 mRNA gave three bands of 5.1Kb, 2.8Kb and 2.OKb, whose levels were significantly reduced in 30- and 60-day STZ rats. These results suggest that the facilitative glucose transporter (GLUT2), in addition to the Na(+)-dependent glucose transporter (SGLT1), may play an important role in intestinal glucose transport in diabetic rats.

Developmental changes in intestinal glucose transporter mRNA levels

Developmental changes in glucose transporter mRNA levels in the jejunum of rats of different ages were examined by using slot blot RNA analysis. The level of SGLT1 mRNA did not change significantly through life. The GLUT5 mRNA level was highest in 10-day-old rats and then decreased reaching the adult level by day 20 after birth. The GLUT2 mRNA level was low in rats of 5 and 10 days old, but then increased progressively reaching the adult value by day 25 after birth. These results indicate that the expressions of intestinal facilitative glucose transporter genes change markedly in the third week after birth.

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.

The mechanism of action of the cytotoxic lectin from Phoradendron californicum: The RNA N‐glycosidase activity of the protein

A toxic lectin from Phoradendron californicum (PCL) was found to inactivate catalytically 60 S ribosomal subunits of rabbit reticulocytes, resulting in the inhibition of protein synthesis. To study the mechanism of action of PCL, rat liver ribosomes were treated with the toxin and the extracted rRNA was treated with aniline. A fragment containing about 450 nucleotides was released from the 28 S rRNA. Analysis of the nucleotide sequence of the fragment revealed that the aniline‐sensitive phosphodiester bond was between A4324 and G4325 of the 28 S rRNA. These results indicate that PCL inactivates the ribosomes by cleaving an N‐glycosidic bond at A4324 of 28 S rRNA in the ribosomes as does ricin A‐chain.

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.

Modulation of albumin gene expression by amino acid supply in rat liver is mediated through intracellular concentration of pyridoxal 5′-phosphate

Abstract Rats were nourished by infusion of total parenteral nutrition solutions containing 0% or 3.3% amino acids for 7 days. The level of albumin mRNA in the liver of amino acid-infused rats was found to be several fold higher than that in the liver of amino acid-depleted rats. Expression of albumin gene is known to be regulated by tissue-specific transcription factors such as HNF-1 and C EBP . We determined the binding activities of liver nuclear extracts to the HNF-1- and C EBP -binding sites by gel mobility-shift assay and found that the activities of the extract prepared from liver of amino acid-infused rats were greater than those of amino acid-depleted rats. In view of our recent finding that vitamin B6 modulates albumin gene expression through inactivation of tissue-specific transcription factors by direct interaction with pyridoxal phosphate, we determined the intracellular concentrations of vitamin B6 derivatives. We found that the concentration of pyridoxal phosphate in the liver of amino acid-infused rats was decreased to almost half of that of amino acid-depleted rats, whereas the concentration of pyridoxamine phosphate was increased in the opposite direction. These observations suggest that an increase in albumin mRNA level in the liver of amino acid-infused rats may be caused by a decrease in intracellular concentration of pyridoxal phosphate, which in turn relieves inactivation of tissue-specific transcription factors.