Involvement of a cellular glycolytic enzyme, phosphoglycerate kinase, in Sendai virus transcription.

In vitro mRNA synthesis of Sendai virus is almost entirely dependent on the addition of cellular proteins (host factors). Previous studies indicated that the host factor activity from bovine brain was resolved into at least two complementary fractions, one of which may be tubulin. In this study, the host factor activity that stimulates the transcription in the presence of tubulin was further purified from bovine brain. This fraction was found to contain at least two complementary factors, and one of them was purified to a single polypeptide chain with an apparent M(r) of 46,000 (p46). From the amino acid sequence, biochemical, and immunological analyses, p46 was identified as a glycolytic enzyme, phosphoglycerate kinase (PGK). Purified native PGK from rabbit and yeast, and a recombinant human PGK substituted for p46. Although, as previously suggested, tubulin was involved in the transcription initiation complex formation by being integrated into the complex, p46 and its complementary factor had little effect on the complex formation. On the other hand, when p46 and the complementary factor were added to the RNA chain elongation reaction from the isolated initiation complex formed with tubulin, mRNA synthesis was dramatically stimulated. The enzymatic activity per se of PGK did not seem to be required for its activity. West-Western blot analysis showed that PGK could directly interact with tubulin. These data suggest that PGK stimulates Sendai virus mRNA synthesis at the elongation step, probably through its interaction with tubulin in the initiation complex.

[Analysis of T cell epitopes on birch pollen allergen].

Allergy to birch pollen is common in the Northern Europe, North America, and in Japan, mainly in Hokkaido Island. Previously, we have reported the positive association of birch pollen allergy in HLA-DR9 antigen. We also have identified the recognition sites in 17kDa protein (Bet v1), known as the major allergen of birch pollen, by using T cell proliferation assay against the trypsin digested materials of the 17kDa protein. In this study, overlapping synthetic peptides correspond to the Bet v1 sequences were used to investigate the specificity of T cell responses in two HLA-DR9 positive patients. Three of the epitopes, residues 35-52, 75-92, and 102-119, were recognized by T cells from both patients. These three epitopes include HLA-DR9 binding motifs. Then we established T cell clone specific to p75-92 residue. 78-89 peptide was the core sequence of the epitope for the T cell clone contained the anchor residues characteristic to HLA-DR9. Within the core sequence, five residues were identified as critical for recognition on the basis of inhibition of the T cell proliferative response against singly substituted analogue peptides.

Epitope analysis of birch pollen allergen in Japanese subjects.

Birch pollen is a very common cause of nasal allergy (pollinosis) not only in Scandinavia, Europe, Canada, and the northern part of the United States but also in Hokkaido, Japan. We have previously reported a positive association between the HLA-DR9 phenotype and the development of birch pollen allergy in Japanese subjects. However, there is little information about T cell epitopes of birch pollen which are presented by HLA class II molecules other than HLA-DR9. Therefore, we analyzed the difference in T cell epitope usage in patients who had HLA-DR9 versus those who did not. Seven Japanese patients with birch pollinosis were studied. Some groups of peptides representing T cell epitopes (Betula verrucosa; Bet VI peptides, p7-33, p23-46, p138-160) appeared to be shared by the majority, while another peptide (Bet VI p72-95) was recognized predominantly by patients who expressed HLA-DR9 and/or HLA-DQ3 molecules. Moreover, seven T cell clones and eight T cell lines were generated from two patients who did not have HLA-DR9 or HLA-DQ3. Using some of these T cell clones/lines, we investigated the relationship between HLA class II molecules and antigenic peptides. One of these T cell clones recognized antigenic peptides in the context of the HLA-DQ1 molecule. To our knowledge, this is the first indication that the epitope on Bet VI can be presented by the HLA-DQ molecule.

An immunomodulating protein, Ling Zhi-8 (LZ-8) prevents insulitis in non-obese diabetic mice.

Ling Zhi-8 (LZ-8), a novel and recently discovered immunomodulatory protein having in vivo immuno-suppressive activity, was tested for in vivo effect against Type 1 (insulin-dependent) diabetes mellitus in the nonobese diabetic mouse, the disease having immunologically mediated aetiology in this animal. LZ-8 had mitogenic activity in vitro towards spleen cells of the non-obese diabetic mice as previously shown towards those of DBA/2 mice. Intraperitoneal administration of LZ-8 twice weekly into the mice (10.3-12.6 mg/kg body weight) from 4 weeks of age prevented insulitis and an almost normal number of insulin producing cells were observed. Extreme insulitis and reduction of the number of insulin producing cells were observed in the pancreata of the untreated non-obese diabetic mouse. No cumulative incidence of diabetes mellitus was observed in the LZ-8 treated group, while cumulative incidences of 70% and 60% were observed in an untreated group followed up to 42 weeks of age when the incidence of diabetes was defined as a plasma glucose level of greater than 11 mmol/l and as a urine glucose level of greater than 2+, respectively. T cell subset population analysis was performed to further investigate the action of LZ-8 on the non-obese diabetic mouse which revealed that LZ-8 treatment increased in L3T4'/Lyt-2+ ratio.