Isamu Matsumoto

The Earliest Thymic Progenitors in Adults Are Restricted to T, NK, and Dendritic Cell Lineage and Have a Potential to Form More Diverse TCRβ Chains than Fetal Progenitors

T cell progenitors in the adult thymus (AT) are not well characterized. In the present study, we show that the earliest progenitors in the murine AT are, like those in fetal thymus (FT), unable to generate B or myeloid cells, but still retain the ability to generate NK cells and dendritic cells. However, AT progenitors are distinct from those in FT or fetal liver, in that they are able to produce ∼100 times larger numbers of T cells than progenitors in fetuses. Such a capability to generate a large number of T cells was mainly attributed to their potential to extensively proliferate before the TCRβ chain gene rearrangement. We propose that the AT is colonized by T/NK/dendritic cell tripotential progenitors with much higher potential to form diversity in TCRβ chains than FT progenitors.

Structures and contribution to the antigenicity of oligosaccharides of Japanese cedar (Cryptomeria japonica) pollen allergenCry j I: relationship between the structures and antigenic epitopes of plantN-linked complex-type glycans

The oligosaccharide structures ofCry j I, a major allergenic glycoprotein ofCryptomeria japonica (Japanese cedar, sugi), were analysed by 400 MHz1H-NMR and two-dimensional sugar mapping analyses. The four major fractions comprised a series of biantennary complex type N-linked oligosaccharides that share a fucose/xylose-containing core and glucosamine branches including a novel structure with a nongalactosylated fucosylglucosamine branch.Rabbit polyclonal anti-Cry j I IgG antibodies cross-reacted with three different plant glycoproteins having the same or shorter N-linked oligosaccharides asCry j I. ELISA and ELISA inhibition studies with intact glycoproteins, glycopeptides and peptides indicated that both anti-Cry j I IgGs and anti-Sophora japonica bark lectin II (B-SJA-II) IgGs included oligosaccharide-specific antibodies with different specificities, and that the epitopic structures against anti-Cry j I IgGs include a branch containing α1–6 linked fucose and a core containing fucose/xylose, while those against anti-B-SJA-II IgGs include nonreducing terminal mannose residues. The cross-reactivities of human allergic sera to miraculin andClerodendron Trichotomum lectin (CTA) were low, and inhibition studies suggested that the oligosaccharides onCry j I contribute little or only conformationally to the reactivity of specific IgE antibodies.

Diversities in animal vitronectins differences in molecular weight immunoreactivity and carbohydrate chains

Six animal plasma vitronectins, human, horse, porcine, bovine, rabbit and chicken vitronectins purified by a novel method using two successive heparin affinity columns, showed marked diversity in molecular weight, immunoreactivity and carbohydrate composition. Chicken vitronectin had a distinctly different amino acid composition from the mammalian vitronectins; and bovine vitronectin was the only one to contain N-glycolylneuraminic acid as well as N-acetylneuraminic acid. Binding studies with horseradish peroxidase-labelled lectins indicated that all the vitronectins contained complex-type, sialylated N-linked sugar chains and that only porcine vitronectin had a fucosylated sugar chain. D-Galactosamine determinations and binding studies with horseradish peroxidase-peanut lectin on native and asialovitronectins revealed that the mammalian vitronectins other than human vitronectin contained O-linked sugar chains with sialic acid, chicken vitronectin contained unsialylated chains, and human vitronectin contained neither. The results indicate that diversities in vitronectins are apparent in their molecular weights and glycosylations, especially in the number and structure of O-linked sugar chains.

Derivatization of epoxy-activated agarose with various carbohydrates for the preparation of stable and high-capacity affinity adsorbents: Their use for affinity chromatography of carbohydrate-binding proteins

Abstract Two types of affinity adsorbents for lectins were prepared by new simple procedures. Both types of adsorbents had high ligand concentration and chemically stable linkage between ligand and Sepharose 4B. Oligosaccharide ligands were coupled by reductive amination with sodium cyanoborohydride to amino-Sepharose 4B prepared by amination of epoxy-activated Sepharose 4B. The glycamyl-Sepharose 4B thus obtained had particularly high adsorption capacities for lectins; lactamyl-Sepharose 4B, 58 mg/l ml of gel for peanut lectin; maltamyl-Sepharose 4B, 146 mg/ml for concanavalin A; and tetra- N -acetylchitotetraamyl-Sepharose 4B, 36 mg/ml for wheat germ agglutinin. Hexosamine was coupled by the aid of carbodiimide to carboxyl-Sepharose 4B prepared by succinylation of amino-Sepharose 4B. Galactosamine-Sepharose 4B adsorbed 145 mg soybean agglutinin/l ml gel. The columns turned from a semitransparent white to a milky white as they were saturated with lectins.

Preparation of affinity adsorbents with toyopearl gels

Abstract The optimal conditions for the activation of Toyopearl by epichlorohydrin and subsequent immobilization of ligands were investigated. The optimal conditions for the activation were very different from those for agarose gel. The concentration of epoxy groups introduced was as high as 330 μmole per gram of wet gel for Toyopearl HW-55 and 150 μmole per gram of wet gel for Toyopearl HW-65 (diol type). Epoxy-activated Toyopearl was converted into amino and carboxyl derivatives and was subsequently coupled with various ligands. Glycamyl Toyopearl was prepared in a much shorter time (6 h) than agarose gel (800 h), because a higher reaction temperature could be used. The adsorbents obtained were successfully used for the affinity chromatography of lectin and trypsin. However, their adsorption capacities were lower than those of the agarose adsorbents prepared by the same methods.

Mitochondrial trifunctional protein deficiency associated with recurrent myoglobinuria in adolescence

A 23-year-old man with recurrent myoglobinuria had low muscle-free carnitine levels and deficient fasting ketogenesis. Urinary organic acid analysis showed large amounts of C6-C14 3-hydroxydicarboxylic acids. Mitochondrial trifunctional protein (TP), harboring long-chain enoyl-coenzyme A (CoA) hydratase, long-chain 3-hydroxyacyl-CoA dehydrogenase, and long-chain 3-ketoacyl-CoA thiolase showed markedly decreased activity in fibroblasts. On immunoblot analysis, the TP content of his fibroblasts was less than 2% that of the control cells. TP deficiency can be a life-threatening disorder with early infantile onset, but it can also present in adolescence with recurrent myoglobinuria.

Pilot study of gas chromatographic–mass spectrometric screening of newborn urine for inborn errors of metabolism after treatment with urease

Gas chromatographic-mass spectrometric (GC-MS) techniques for urinary organic acid profiling have been applied to high-risk screening for a wide range of diseases, mainly for inborn errors of metabolism (IEM), rather than to low-risk screening or mass screening. Using a simplified procedure with urease-pretreatment and the GC-MS technique, which allows simultaneous determination of organic acids, amino acids, sugars and sugar acids, we performed a pilot study of the application of this procedure to neonatal urine screening for 22 IEM. Out of 16,246 newborns screened, 11 cases of metabolic disorders were chemically diagnosed: two each of methylmalonic aciduria and glyceroluria, four of cystinuria, and one each of Hartnup disease, citrullinemia and alpha-aminoadipic aciduria/alpha-ketoadipic aciduria. The incidence of IEM was thus one per 1477, which was higher than the one per 3000 obtained in the USA in a study targeting amino acids and acylcarnitines in newborn blood spots by tandem mass spectrometry. Also, 227 cases were found to have transient metabolic abnormalities: 108 cases with neonatal tyrosinuria, 99 cases with neonatal galactosuria, and 20 cases with other transient metabolic disorders. Two hundred and thirty-eight cases out of 16,246 neonates (approximately 1/68) were thus diagnosed using this procedure as having either persistent or transient metabolic abnormalities.

Levels of annexin IV and V in the plasma of pregnant and postpartum women

Annexin (Anx) V is pivotal in the maintenance of pregnancy by preventing the activation of blood coagulation. The homology of the amino acid sequence between Anx IV and Anx V is highest in Anx family proteins. However, little is known about the roles of Anx IV in pregnancy. The aim of this study is to clarify the roles of circulating Anx IV and Anx V in normal pregnancy. Subjects were non-pregnant women (n = 50), 120 pregnant women, and maternal subjects just after delivery (n = 53) or postpartum (n = 67). Anx IV in the plasma of non-pregnant women was at a concentration 20 times that of Anx V. The plasma levels of Anx IV suddenly increase after delivery, but Anx V levels remain low during this period. Anx IV and Anx V exert similar levels of anticoagulant activity. Anx IV protein was expressed on the basal surface of syncytiotrophoblasts; Anx V protein, on the apical surface of syncytiotrophoblasts. These results suggest that Anx IV enters the maternal bloodstream just after delivery and might play a role in preventing disseminated intravascular coagulopathy, and that Anx V helps to prevent clotting in the placenta during pregnancy.

Four-hydroxyphenylpyruvic acid oxidase deficiency with normal fumarylacetoacetase: a new variant form of hereditary hypertyrosinemia.

Summary: Enzymatic studies on the liver of an infant are described-a case of hypertyrosinemia without hepatic dysfunction. His parents were siblings and the mother had hypertyrosinemia. Excessive amounts of 4-hydroxyphenylpyruvic acid (pHPP), 4-hydroxyphenylacetic acid (pHPL), and 4-hydroxyphenylacetic acid (pHPA) were found to be excreted in the patients urine as well as in the urine of the mother and the inhibitor of porphobilinogen synthetase was not found. Soluble tyrosine aminotransferase (s-TAT), separated from that of the mitochondrial form (m-TAT) by DE 52 column chromatography, was normal in the patients liver, both quantitatively and qualitatively. The activities of fumarylacetoacetase in the patients liver and in the peripheral leucocytes from the parents were normal. The activity of pHPP oxidase in the patients liver was approximately 5% of the control and the enzyme had a high Km value for pHPP (controls: 0.06 ± 0.01 mM, patient: 0.23 ± 0.03 mM). From these results, the patient was thought to be different from previously described types of tyrosinemia and perhaps representative of a new variant form.This is the first report concerning 4-hydroxyphenylpyruvic acid oxidase deficiency alone. Mild metal retardation and mild hypertyrosinemia may be offered as typical clinical features of the disease.

Successful treatment by direct hemoperfusion of coma possibly resulting from mitochondrial dysfunction in acute valproate intoxication.

: Purpose: We evaluated the efficacy of direct hemoperfusion (DHP) for treatment of acute valproate (VPA) intoxication and speculate on the biochemical perturbations that suggest a mechanism of coma induced by VPA overdose.