Mitsuko Usui

Molecular cloning of the second major allergen, Cry j II, from Japanese cedar pollen

Cloning of a cDNA from Cry j II, the second major allergen from Japanese cedar (Cryptomeria japonica) pollen, is described. An isolated Cry j II cDNA contained an open reading frame coding for 514 amino acid residues. The mature Cry j II protein consisted of 388 amino acid residues (R46—S433). According to a homology analysis, no amino acid sequence homology was observed between Cry j II and Cry j I, another major allergen. But Cry j II showed homology with polygalacturonase (PG) derived from tomato (40% identity) at the amino acid level. The sequence information can potentially be used to devise an effective course of immunotherapy for Japanese cedar pollinosis.

N-terminal amino acid sequence of a major allergen of Japanese cedar pollen (Cry j I)

A purified preparation of a major allergen of Japanese cedar pollen, sugi basic protein (SBP, Cry jI), was separated into 5 subtractions of 50‐45 kDa. All of the SBP subtractions were confirmed to be reactive to IgE antibodies from patients with Japanese cedar pollinosis, and also to mouse anti‐SBP monoclonal antibodies. The sequences of 20 N‐terminal amino acids of these 5 subtractions were found to be identical. Peptide mapping analyses of the SBP subtractions showed similar patterns, with some differences which might in part be due to the existence of an N‐linked carbohydrate chain. The N‐terminal amino acid sequence of SBP was identical to the reported sequence of an allergen of mountain cedar which vegetated in North America.

Antigenic Analyses of Sugi Basic Protein by Monoclonal Antibodies; I. Distribution and Characterization of B-Cell-Tropic Epitopes of Cry j I Molecules

Using 23 monoclonal antibodies raised against Sugi basic protein (SBP, major allergen of Japanese cedar pollen), composed of Cry j I and Cry j II, analyses of B-cell-tropic epitopes of Cry j I were performed. The following results were obtained. (1) As far as the mAbs were used, no major cross-reactive determinants were detected between Cry j I and Cry j II molecules. (2) 21 of the 23 mAbs were specific for Cry j I, and the anti-Cry j II mAbs were classified into four groups by their fine specificities, suggesting that Cry j I bears at least four antigenic determinant regions. (3) Cry j I molecules were found to take a monomeric form in solution and to display no repeating antigenic epitopes on their surfaces. (4) Some of the determinants seemed to be located in the interior of a Cry j I molecule, and when the Ag is coated on a plastic plate, the determinants become exposed on its surface. (5) Binding of human IgE antibodies to Cry j I and Cry j II was blocked by some of the obtained mAbs, suggesting that these epitopes recognized by the mAbs might have an important role in human allergic response against the cedar pollen.

Antigenic Analyses of Sugi Basic Protein by Monoclonal Antibodies: II. Detection of Immunoreactive Fragments in Enzyme-cleaved Cry j l

The 4 anti-Cry j I mAbs showing an epitope specificity different from each other, 046, 029, 026 and 027, were selected to analyze the structure of the antigenic determinant for each mAb on a Cry j I molecule. Immunoreactive fragments in enzyme-cleaved Cry j I were detected by means of the adsorption on the mAb column and of the binding to the mAbs on Elisa. The mAb 026 was found to be reactive to the fragments containing a Cry j I N-terminal region obtained by V8 protease or pepsin digestion, but not to those by lysylendopeptidase digestion. The mAb 027 was found to be capable of binding to the fragments containing a linear structure of Asn-Ala-Gly-Val-Leu-Thr-Cys-Ser-Leu-Ser-Lys, which were generated by V8 protease, lysylendopeptidase or pepsin digestion. Furthermore, the synthetic peptide Asn-Ala-Gly-Val-Leu-Thr-Cys-Ser- Leu-Ser-Lys-Arg could bind to 027, but not to 026, and could inhibit the binding of 027 to Cry j I or to its immunoreactive fragments. No fragments capable of reacting to the mAbs 046 and 029 could be found in this study, suggesting that 046 and 029 recognize a conformationally constituted epitope of Cry j I molecule which is destroyed by enzymatic cleavage. The epitope recognized by the mAbs 027 or 026 was found to be located in conformationally hidden parts of the molecule which was exposed to react to the mAbs only after the physicochemical or enzymatic treatment.

Growth of rat-mouse hybridoma cells in immunosuppressed hamsters: An easy and effective method to prepare monoclonal antibodies from heterohybridoma cell lines

Rat-mouse hybridoma cells producing anti-mouse IgE antibodies were intraperitoneally or subcutaneously inoculated into newborn or suckling hamsters receiving rabbit anti-hamster thymocyte globulin from the day of birth twice a week for at least 3 weeks. The hybridoma cells were found to grow in the abdominal cavity of the hamsters as ascites tumor or in subcutaneous tissue as solid tumor without loss of antibody-secreting activities. For the production of ascites, 2-week-old hamsters were preferable to newborn hamsters. In 3-week-old hamsters, the hybridoma cells could scarcely survive. The antibody titers of the ascites were determined to be 10(5)-10(6) in the ELISA and in the ability to neutralize the skin-sensitizing capacity of mouse IgE antibodies. The rat monoclonal antibodies were easily separated from ascites, serum or cell culture supernatant with affinity chromatography using Affigel protein A-Sepharose and anti-hamster IgG-Sepharose columns. The described method could be efficiently applicable for the proliferation of other hybridomas, such as human-human, human-mouse or hamster-mouse, etc.

Suppression of Murine IgE Responses with Ovalbumin-Pullulan Conjugates: Comparison of the Suppressive Effect of Different Conjugation Methods and Different Molecular Weights of Pullulan

Ovalbumin (OVA)-pullulan conjugates were made using four different conjugation methods and eight different molecular weights of pullulan ranging from 4,200 to 600,000. Pretreatment of mice by the administration of conjugates made by using cyanulic chloride or cyanogen bromide and pullulan of molecular weight 40,000 or more, anti-OVA IgE antibody response was suppressed completely and anti-OVA IgM and IgG antibody response was enhanced. In contrast, by the administration of conjugates made by using an oxidation or thiol activation method, only partial suppression of anti-OVA IgE antibody response was achieved and no enhancement of anti-OVA IgM and IgG antibody production was observed.

A new approach to make toxoid eliciting good IgM and/or IgG but little IgE antibody responses

We have found that the induction of IgE antibody response is suppressed selectively and antigen-specifically, whereas IgM and IgG antibody responses are enhanced in mice pretreated with an antigen conjugated with pullulan, a linear polymer of glucose [1]. Even when the conjugate is repeatedly injected into mice, the IgE antibody response is very poor but the IgM and IgG responses are well elicited as compared with those to the antigen immunized with alum adjuvant. These findings led us to the assumption that a toxin when conjugated with pullulan (or other polysaccharides) can be used as a toxoid; this is because the toxin cannot manifest its toxicity since the binding site of the toxin to a cell surface receptor is hindered stereochemically by the pullulan moiety of the conjugate [2]. The experiments revealing that toxins conjugated with pullulan are detoxicated and can suppress the induction of IgE antibody while enhancing the IgM and IgG antibody responses to the toxins will be briefly described here. Fifty mg of pullulan (345 000 daltons), dissolved in 24 ml of phosphate-buffered saline (PBS, pH 7.0), was cooled in an ice bath. Cyanuric chloride (20 mg) in 1 ml of cooled N,N-dimethylformamide was added to the solution. The mixture was stirred in the bath for 4h, keeping the pHat 7. Then, 12500 Lf(ca. 59.3 mg protein) of tetanus toxin in PBS were added to the mixture, which was stirred at 37 ~ for another 72 h. To the mixture was added 2 mg of glycine to stop the reaction of cyanuric chloride. It was then dialyzed against 0.05 M Tris-HC1 buffer, pH 7.5, overnight. The resulting mixture was chromatographed on DEAE sephacel equilibrated with the same buffer. The conjugate of toxin to pullulan was eluted gradually by 0.05M Tris-HC1 buffer containing NaC1 of increasing concentrations. The eluate was monitored by cellulose acetate membrane electrophoresis. The fractions by which the electrophoretic mobility was much smaller than that of toxin or of formalinized toxoid were pooled. The conjugation of toxin to pullulan was confirmed by simultaneous staining with nigrosin and with Schiffs reagent. The resulting preparation has practically no toxicity against mice. Two Lb per mouse of the preparation was injected subcutaneously to groups of 5 mice several times at an interval of 1-4 weeks. The sera taken at various intervals were titrated for the IgE antibody by passive cutaneous anaphylaxis (PCA) using Sprague-Dawley rats and for the IgM and/or IgG antibodies by passive hemagglutination (PHA). A representative data is illustrate in Fig. 1 a. The IgE antibody to tetanus toxin was well suppressed; but IgM and/or IgG antibodies responded fairly well compared with control groups which were injected in the same way with the traditional toxoid plus alum adjuvant or with plain toxoid. The habu (Trirnerusus flavoviridis) venom conjugated with pullulan by the same method revealed no toxicity in the rabbit skin hemarrhage test. The conjugate elicited the enhancement of IgM and/or IgG antibody responses with the suppression of IgE antibody response (Fig. 1 b). These results suggest that the conjugation IgE anti-tetanus toxin (PCA) 40 80 120 160 I I I I

IgA- and IgG-enhancing but IgE-suppressing characteristics of lipopolysaccharide or cholesterol-ovalbumin conjugate intubated to mice

on, the root hair was usually on the apical side of the cell in the still culture after centrifugation. On the basis of the above experiments it seenas, so far as the present material is concerned, that the apex of the root epidermal celt is determined as the site of root hair formation in the cell not by gravity but by the cellular polarity, and this polarity is related neither to localization of the vacuole nor to movable cytoplasm, but is controlled by the stable cortex or cell membrane as reported in some other plants [4, 5].

Nonspecific binding of irrelevant antibodies to the complexes of proteinic antigen and it's antibody

tinguished (Fig. 1 a). The assembly of both chromosome complements together at metaphase has been observed frequently in this study and this we claim must be due to fusion. Generally mitosis in heterokaryocytes was observed to occur synchronously, even when the nuclei were not fused. Here, continuous division of these unfused nuclei led to the formation of chimerical colonies. In addition to our analysis of metaphases in the heterokaryocytes, we also observed heterokaryocytes at anaphase. At this stage we could distinguish adjacent sets of Petunia and Atropa chromosomes which are presumably a result of the formation of a common or a split spindle (Fig. lb). These observations provide evidence in support of further division of the two chromosome complements with a common spindle mechanism which subsequently leads to hybrid callus and ultimately hybrid plants. The authors are grateful to Dr. M.M. Yeoman for assistance during the preparation of these manuscripts and to Miss W. Hofeditz for excellent technical assistance.