Takeshi Ide

A cDNA clone encoding an IgE-binding protein from Brassica anther has significant sequence similarity to Ca2+-binding proteins

Thirteen cDNA clones encoding IgE-binding proteins were isolated from expression libraries of anthers of Brassica rapa L. and B. napus L. using serum IgE from a patient who was specifically allergic to Brassica pollen. These clones were divided into two groups, I and II, based on the sequence similarity. All the group I cDNAs predicted the same protein of 79 amino acids, while the group II predicted a protein of 83 amino acids with microheterogeneity. Both of the deduced amino acid sequences contained two regions with sequence similarity to Ca2+-binding sites of Ca2+-binding proteins such as calmodulin. However flanking sequences were distinct from that of calmodulin or other Ca2+-binding proteins. RNA-gel blot analysis showed the genes of group I and II were preferentially expressed in anthers at the later developmental stage and in mature pollen. The recombinant proteins produced in Escherichia coli was recognized in immunoblot analysis by the IgE of a Brassica pollen allergic patient, but not by the IgE of a non-allergic patient. The cDNA clones reported here, therefore, represent pollen allergens of Brassica species.

Immunodiagnosis of human sparganosis mansoni by micro-chemiluminescence enzyme-linked immunosorbent assay

We compared a microcolorimetric enzyme-linked immunosorbent assay (colorimetric ELISA) and a microchemiluminescence enzyme-linked immunosorbent assay (chemiluminescence ELISA) for the detection of specific immunoglobulin G (IgG) in the serum of 9 patients with sparganosis mansoni and 9 healthy controls. The chemiluminescence ELISA was able to measure serum levels of specific IgG over a far wider range than the colorimetric assay, and its detection limit was at least 10-fold lower. An additional 5 sera from sparganosis patients and 5 more from healthy controls, together with sera from 28 patients with other parasitic diseases, were also examined by the chemiluminescence ELISA. All 14 patients with sparaganosis mansoni showed high levels of chemiluminescence (21,302 +/- 18,907 counts per second [cps]). All sera from the 14 healthy controls (1580 +/- 569 cps) and sera from 27 of the 28 patients with other parasites (4 with taeniasis saginata [1767 +/- 501 cps], 11 with diphyllobothriasis latum [1479 +/- 501 cps], 13 with cysticercosis cellulosae [2376 +/- 1437 cps]) showed chemiluminescence levels lower than those of any of the sparganosis mansoni patients. The exception was a patient with cysticercosis (5980 cps), who may have had a dual infection with Cysticercus cellulosae and Sparganum mansoni. Thus, the chemiluminescence ELISA demonstrated high sensitivity and specificity for human sparganosis mansoni.

Electrochemical detection of reducing carbohydrates produced by the transferase action of yeast debranching enzyme on maltosaccharides

Abstract A sensitive method for the detection of maltosaccharides up to maltoheptaose is based on an electrochemical detector using bis(1,10-phenanthroline)-copper(II) in the post column reaction after h.p.l.c. on an amino-bonded column. This method has been used for the analysis of the maltosyl and maltotriosyl transferase action of the yeast debranching enzyme with maltosaccharides as the substrates. The smallest donor substrate for maltosyl transfer was maltotetraose, and maltopentaose, maltohexaose, and maltoheptaose were donor substrates for both maltosyl and maltotriosyl transfers. Maltosyl residues were transferred in preference to maltotriosyl residues from maltopentaose, but maltotriosyl residues were transferred preferentially from maltohexaose and maltoheptaose. Maltotriose is an acceptor but not a donor of maltosyl and maltotriosyl transfers.

Purification and characterization of α-glucosidases produced by Saccharomyces in response to three distinct maltose genes

alpha-Glucosidases or maltases (EC 3.2.1.20) were purified to electrophoretic homogeneity from a respective strain of Saccharomyces cerevisiae which carries a single MAL gene, either MAL alpha, MAL beta, or MAL gamma, using gluconate-Sepharose affinity chromatography and isoelectrofocusing. Of these maltases, two types of maltase were obtained from the MAL gamma strain, the pI values of which were 5.6 and 5.9. From the MAL alpha and MAL beta strain was obtained only one type of maltase with the pI at 5.6 which was identical to one of the maltases from the MAL gamma strain. These four maltases possessed the same properties, except for pI. They were monomers with molecular weights of between 66 000 and 67 000. With regard to the substrate specificity, they hydrolyzed maltose and sucrose exclusively but not alpha-methylglucoside nor maltooligosaccharide. They did not differ in immunological properties.

Accumulation of Japanese cedar pollen allergen, Cry j 1, in the protein body I of transgenic rice seeds using the promoter and signal sequence of glutelin GluB-1 gene

Recombinant Cry j 1, a Japanese cedar pollen allergen, was produced in rice seeds for potential use for oral immunotherapy. Cry j 1 cDNA was divided into two parts, an N-terminal half and a C-terminal half, and each was fused downstream to glutelin GluB-1 gene containing sequences of the promoter, 5′ untranslated region and signal peptide. A gene for green fluorescent protein was also fused to the 3′ end of the Cry j 1 fragment. Recombinant Cry j 1 of up to 16.6 μg per mg total protein of the seeds was expressed in transgenic rice seeds. Although the recombinant Cry j 1 was expected to be accumulated in protein body II because of the employment of glutelin signal peptide, it was demonstrated to be accumulated exclusively in protein body I. The recombinant Cry j 1 was not shown to react with IgE of allergic patients, indicating the reduction of the risk of anaphylactic reaction. These results demonstrate that the transgenic rice seeds with the recombinant Cry j 1 would be useful for the study of oral immunotherapy.