Teruhiko Iino

Expression analysis of the aldo-keto reductases involved in the novel biosynthetic pathway of tetrahydrobiopterin in human and mouse tissues.

Tetrahydrobiopterin (BH(4)) acts as a cofactor of the aromatic amino-acid hydroxylases, and its deficiency may result in hyperphenylalaninemia (HPA) and decreased production of the neurotransmitters. BH(4) is synthesized by sepiapterin reductase (SPR) from 6-pyruvoyl-tetrahydropterin (PPH(4)). A patient with SPR deficiency shows no HPA; however, an SPR knockout mouse exhibits HPA. We have reported on the SPR-unrelated novel biosynthetic pathway from PPH(4) to BH(4) (salvage pathway II) in which 3alpha-hydroxysteroid dehydrogenase type 2 and aldose reductase work in concert. In this study, we performed the expression analysis of both proteins in humans and wild-type mice. The results of expression analysis indicated that salvage pathway II worked in human liver; however, it did not act in human brain or in mouse liver and brain. For this reason, a patient with SPR deficiency may show progressive neurological deterioration without HPA, and SPR knockout mice may exhibit HPA and abnormal locomotion activity.

Purification and properties of gut arginase from earthworm Pheretima communissima

Abstract 1. 1. Arginase from Pheretima communissima was purified about 10,000-fold and its properties were compared with those of the same enzyme in other organisms. 2. 2. The molecular weight of the enzyme, as determined by gel filtration on Sephadex G-100, was about 25,000, which is substantially less than that of the subunits of oligomeric arginases. 3. 3. The arginase was extremely labile, losing 90% of its original activity by dialysis against a Mn2+-free buffer for 4 hr. Seventy percent of its original activity was recovered following treatment with 1 mM MnCl2 at 37°C for 20 min. 4. 4. The K m values for l -arginine were 8.5 mM at pH 9.5 and 54 mM at pH 7.5, respectively and the activity was competitively inhibited by l -ornithine and l -lysine, and caused a mixed-type inhibition by l -leucine, l -isoleucine and l -valine at both pHs.

Molecular cloning and characterization of a cDNA encoding a novel cuticle protein in the silkworm, Bombyx mori

We have cloned the full length of a novel cDNA named Bombyx mori cuticle protein that contains an AlaAlaProAla/Val-repeat (BMCPA) from a cDNA library of integument in the larval silkworm. Both a typical tandem repeat (A-A-P-A/V) for cuticle protein and a unique tandem repeat with Ser, Ala, Gly, Pro, Val, Tyr and Thr were observed in the predicted amino acid sequence of the cDNA encoding BMCPA. Approximately 80% of the amino acids in BMCPA were composed of Ser, Ala, Gly, Pro, Val and Tyr. Northern-hybridization analysis indicated that BMCPA mRNA is expressed only in the larval epidermis and that the expression pattern of the BMCPA gene in the developmental stage was observed mainly at the larval stage. We propose BMCPA may be a novel component of cuticle, and may play an important role in the integument of the larval silkworm.

A Comparative Study of Urinary Xanthopterin and Neopterin in Liver Diseases

By adsorption to activated charcoal, various pteridine derivatives in human urine are oxidized to xanthopterin. Following this oxidation, xanthopterin in urine from healthy subjects and from patients with liver diseases was assayed by high performance liquid chromatography. The mean values for xanthopterin in healthy subjects were 532 +/- 116 mumol/mol creatinine (mean +/- SD) in males and 585 +/- 153 mumol/mol creatinine in females; the difference was statistically significant (p < 0.01). Xanthopterin concentrations in patients with liver disease were significantly higher than those in normal subjects. When compared with urinary neopterin, which is a marker of activated cell immunity, xanthopterin was significantly increased even in fatty liver disease. These findings suggest that increased concentrations of urinary xanthopterin in liver diseases reflect not only the status of activated cell-mediated immunity, but also injury to liver cells.

Roles of a 20 kDa Protein Associated with a Carbocycle-Forming Enzyme Involved in Aminoglycoside Biosynthesis in Primary and Secondary Metabolism

2-Deoxy-scyllo-inosose (DOI) synthase participates in the biosynthesis of 2-deoxystreptamine (DOS)-containing aminoglycoside antibiotics. The enzyme is expected to be of industrial use, because it converts a sustainable resource (glucose 6-phosphate) into carbocycle (DOI), which easily aromatizes to yield catechol. In the present study, we clarified the physiological role of a non-catalytic 20 kDa protein, BtrC2, associated with DOI synthase from the butirosin-producer Bacillus circulans. Based on the results of complementation analysis using btrC2 disruptant and western analysis against catalytic 40 kDa protein (BtrC) and BtrC2, it is suggested that BtrC2 has two functions. It is involved in the vitamin B6 biosynthesis in primary metabolism, like homologous protein (Pdx2) in Bacillus subtilis. Additionally, it takes part in butirosin biosynthesis as stabilizer of DOI synthase by forming a heterodimer with BtrC, and qualifies B. circulans for stable and constant production of butirosin for long periods.

Urinary and Serum Pteridines in Liver Diseases

Summary The aim of this mini-review is to show the usefulness of pteridines as clinical markers. We assayed urinary neopterin and xanthopterin levels in liver disease. Total pterins were oxidized by active carbon to yield xanthopterin. Urinary neopterin levels were elevated in patients with chronic active hepatitis and in those with chronic persistent hepatitis but not in patients with alcohol-induced liver disease or in those with non-alcoholic fatty liver. Urinary xanthopterin levels were elevated in chronic active hepatitis and alcohol- induced liver disease, but not in chronic persistent hepatitis and non-alcoholic fatty liver. Urinary neopterin levels were high in infectious diseases involving activated cell-mediated immunity, and increased urinary xanthopterin reflects liver-cell damage. We also compared plasma neopterin values in anti-hepatits C virus antibody (anti-HCV) -positive or - negative donors with elevated alanine aminotransferase (ALT). Plasma neopterin values of anti-HCV-negative donors with elevated ALT were the same as those of healthy groups. These results show that the ALT elevation in anti-HCV-negative donors is attributable mainly to steatosis due to excess body weight and/or alcohol consumption, rather than viral infection. During the course of these studies, we were also able to identify esophageal carcinoma in the very early stage by measuring urinary pteridine levels. Our results indicate that pteridines are clinically useful indicators. In particular, neopterin may be used as a means of screening for unknown viral infection.

An example of the detection of an esophageal carcinoma in its very early stage by urinary xanthopterin determination.

Neopterin (NP) has been used as a biochemical marker of the activated state of cell-mediated immunity and to monitor and screen for some clinical disorders1–4. In previous papers 5–6, we reported that xanthopterin (XP) also reflects the same type of immune status as well as various types of liver disease. In the course of our study on the determination of urinary pteridines, one of the authors (M.T.) happened to find that his urinary XP level was higher than the normal range. Although his other tumor indicators such as CEA, SCC and CA19-9 showed normal values, an abnormality was found in the squamous cells of his esophagus after endoscopic inspection. In the present paper we compare longitudinal changes in his urinary excretion levels of XP and NP for a period of eleven months before and after surgery.