The cDNAs for two DNA binding proteins of BTE, a GC box sequence in the promoter region of the P‐450IA1(CYP1A1) gene, have been isolated from a rat liver cDNA library by using the BTE sequence as a binding probe. While one is for the rat equivalent to human Sp1, the other encodes a primary structure of 244 amino acids, a novel DNA binding protein designated BTEB. Both proteins contain a zinc finger domain of Cys‐Cys/His‐His motif that is repeated three times with sequence similarity of 72% to each other, otherwise they share little or no similarity. The function of BTEB was analysed by transfection of plasmids expressing BTEB and/or Sp1 with appropriate reporter plasmids into a monkey cell line CV‐1 and compared with Sp1. BTEB and Sp1 activated the expression of genes with repeated GC box sequences in promoters such as the simian virus 40 early promoter and the human immunodeficiency virus‐1 long terminal repeat promoter. In contrast, BTEB repressed the activity of a promoter containing BTE, a single GC box of the CYP1A1 gene that is stimulated by Sp1. When the BTE sequence was repeated five times, however, BTEB turned out to be an activator of the promoter. RNA blot analysis showed that mRNAs for BTEB and Sp1 were expressed in all tissues tested, but their concentrations varied independently in tissues. The former mRNA was rich in the brain, kidney, lung and testis, while the latter was relatively abundant in the thymus and spleen.(ABSTRACT TRUNCATED AT 250 WORDS)

The major proteolytic allergen derived from the house dust mite Dermatophagoides pteronyssinus, Der p1, is one of the most clinically relevant allergens worldwide. In the present study, we evaluate the contribution of the proteolytic activity and structure of a highly purified rDer p 1 to immune responses. Mice were i.p. immunized with three forms of rDer p 1 adsorbed to Alum: one enzymatically active, one treated with an irreversible cysteine protease-specific inhibitor, E-64, and one heat denatured. Immunization with E-64-treated or heat-denatured rDer p 1 elicited much less production of serum total IgE and not only rDer p 1-specific IgE but also IgGs compared with immunization with active rDer p 1. Assays for Ab-binding and its inhibition and structural analyses indicated that E-64-treated rDer p 1 retained its global structure and conformational B cell epitopes. A proliferative response and production of IL-5 by spleen cells restimulated with rDer p 1 were observed on immunization with the active rDer p 1 but not E-64-treated rDer p 1. The cells from mice immunized with heat-denatured rDer p 1 exhibited the highest levels of proliferation and production of IL-5 and IFN-γ. The results indicate that the proteolytic activity of the highly purified rDer p 1 crucially commits to the sensitization process, including both IgE and IgG responses. Additionally, we demonstrated immunogenic differences by functional or structural manipulations of the rDer p 1. The findings have implications for sensitization to this relevant allergen in humans and for the design of modified allergen-vaccines for future allergen-specific immunotherapy.

The first step of heme biosynthesis in animals is catalyzed by 5‐aminolevulinate synthase (ALAS), which controls heme supply in various tissues. To clarify the roles that the nonspecific isoform of ALAS (ALAS‐N) plays in vivo, we prepared a green fluorescent protein (GFP) knock‐in mouse line in which the Alas1 gene (encoding ALAS‐N) is replaced with a gfp gene. We found that mice bearing a homozygous knock‐in allele (Alas1GFP/GFP) were lethal by embryonic day 8.5, demonstrating that ALAS‐N is essential for early embryogenesis. Fluorescence microscopic and flow cytometric analyses of heterozygous mouse (Alas1+/GFP) tissues showed that the Alas1 expression level differs substantially in tissues; Alas1 is highly expressed in testis Leydig cells, exocrine glands (including submandibular and parotid glands), endocrine glands (such as adrenal and thyroid glands) and hematopoietic lineage cells (including neutrophils and eosinophils). Quantitative analyses of GFP mRNA and ALAS‐N mRNA in various tissues of Alas1+/GFP mice suggested that the destabilization of ALAS‐N mRNA was not uniform in the various tissues. These results thus lay bare that elaborate control of the endogenous heme supply operates in various mouse tissues through regulation of the ALAS‐N expression level and that this control is essential for heme homeostasis in animals.

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Mortality resulting from neuroblastoma in birth cohorts in both Sapporo City and the whole of Japan was investigated to evaluate the effects of a high-performance liquid chromatography (HPLC) mass screening program, targeting on 6 monthold infants. In Sapporo City, the non-HPLC screened cohort showed no reduction in mortality at 4 years of age compared with the pre-screening cohort. However, the HPLC screened cohort showed a reduction of 69% in mortality compared with the pre-screening cohort. On a nation-wide scale, there was a significant decline in mortality for the non-HPLC screened cohort compared with the pre-screening cohort; for the HPLC screened cohort for 1989-1991, there was also a reduction in mortality for children younger than 2 years of age. The incidence of neuroblastoma at 1-4 years of age in the HPLC cohort in Sapporo City was about half that in the pre-screening cohort, along with and probably because of an increasing incidence among infants in the same cohort. Our findings suggest that HPLC screening may detect some poor-prognosis neuroblastoma cases at early stages, thus providing for more favorable therapy. Int.