An immunodominant domain in adenovirus type 2 early region 1A proteins.

Six independent rat hybridoma cell lines producing monoclonal antibodies to human subgroup C adenovirus early region 1A (E1A) proteins were isolated. Competition binding experiments revealed that each of the monoclonal antibodies was directed against the same epitope or overlapping cluster of epitopes on the E1A proteins. Viral E1A deletion mutants and deleted forms of E1A proteins expressed in Escherichia coli were used to localize the antibody recognition sites to sequences between amino acids 23 and 120, encoded within the first exon of the E1A gene. Similarly, polyclonal antisera raised against the trpE-E1A fusion protein, as well as against the native, biologically active E1A protein, were also directed primarily against this immunodominant region.     

Inhibin activity and secretion of gonadotropin during the period of follicular maturation

To evaluate the relative contributions of the ovarian inhibin and estradiol-17 beta (E) on the regulation of FSH secretion, inhibin and E in ovarian venous plasma (OVP) and FSH and LH in peripheral plasma were simultaneously measured using superovulating rats with special reference to follicular maturation. By the transplantation of a pituitary gland from adult male rats under the kidney capsule between 1100 and 1200 hr on diestrus-1 in cyclic rats, superovulation was successfully induced on the morning of the next estrus without any additional treatment with human chorionic gonadotropin (hCG). The number of maturing follicles capable of ovulating in response to hCG significantly increased at 12 hours after the grafting as compared with sham-operated controls and further increases occurred until the afternoon of proestrus. In the superovulating rat, first and second surges of FSH were completely blocked and an LH surge was also partially suppressed during the periovulatory period when surges of FSH and LH were normally observed in controls. Contents of FSH as well as LH in the animal's own pituitary gland were suppressed significantly after the grafting as compared with controls. A marked increase in inhibin activity in OVP of rats with a pituitary transplant occurred concomitantly with an increase in the number of follicles capable of ovulating whereas E levels in OVP did not so. Inhibin activity in OVP at each point was much higher in the pituitary grafted rats than in controls but this was not true for E levels. These results suggest that ovarian inhibin derived from the maturing follicles rather than E may be a primary factor for regulation of FSH secretion, and high levels of endogenous inhibin can suppress synthesis of LH as well as FSH in the pituitary gland of the female rat.     

Estrogen-mediated induction of a vitellogenin-specific nonhistone chromatin protein in the male chicken liver

Summary Non-histone chromatin proteins prepared from the livers of estrogen-treated and nontreated male chickens were compared by reverse-phase high performance liquid chromatography (RP-HPLC), followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results revealed that the hormone-treated male liver chromatin contained a specific protein corresponding to the vitellogenin-specific protein previously purified from the liver of egg-laying hens (Nakayama 1985). The chicken protein, purified further by gel-filtration high performance liquid chromatography (GF-HPLC), showed specific binding activity to DNA fragments carrying a part of the vitellogenin gene. On the basis of similarities in the elution patterns from GF-HPLC and RP-HPLC as well as in the mobility on SDS-PAGE, we concluded that this hormone-induced protein in the male chicken liver was identical to the vitellogenin-specific protein identified in the hen liver, and assumed it to be a specific regulatory protein for the vitellogenin gene expression. The amino acid composition of this chicken protein has been determined.     

A Study on Erythrocyte Membrane Plasmalogen in Myotonic Dystrophy

Phospholipid classes that included plasmalogens of erythrocyte membranes in seven myotonic dystrophy (MyD) patients and seven normal controls were analyzed by HPLC. No significant difference in phospholipid classes was found between patients with MyD and normal controls, but there was a visible difference in peak profiles of compounds of the phosphatidylethanolamine class. In the study of plasmalogens, we used two preparation methods: exposure to HCl and deacylation with mild alkaline. The area ratio of the plasmalogen form to the diacyl form in the phosphatidylethanolamine class of MyD erythrocyte membranes was significantly lower than that of normal controls. Fatty acid analyses showed that fatty acids of both phosphatidylethanolamine subclasses have less unsaturation in MyD.     

Effects of Mutations of Rad50, Rad51, Rad52, and Related Genes on Illegitimate Recombination in Saccharomyces Cerevisiae

To examine the mechanism of illegitimate recombination in Saccharomyces cerevisiae, we have developed a plasmid system for quantitative analysis of deletion formation. A can1 cyh2 cell carrying two negative selection markers, the CAN1 and CYH2 genes, on a YCp plasmid is sensitive to canavanine and cycloheximide, but the cell becomes resistant to both drugs when the plasmid has a deletion over the CAN1 and CYH2 genes. Structural analysis of the recombinant plasmids obtained from the resistant cells showed that the plasmids had deletions at various sites of the CAN1-CYH2 region and there were only short regions of homology (1-5 bp) at the recombination junctions. The results indicated that the deletion detected in this system were formed by illegitimate recombination. Study on the effect of several rad mutations showed that the recombination rate was reduced by 30-, 10-, 10-, and 10-fold in the rad52, rad50, mre11, and xrs2 mutants, respectively, while in the rad51, 54, 55, and 57 mutants, the rate was comparable to that in the wild-type strain. The rad52 mutation did not affect length of homology at junction sites of illegitimate recombination.     

Effects of interleukin-12 on in vitro culture with interleukin-2 of regional lymph node lymphocytes from lung cancer patients

 In the present study, we carried out a functional analysis of regional lymph node lymphocytes (RLNL) from patients with lung cancer after in vitro activation by interleukin-2 (IL-2) and interleukin-12 (IL-12). IL-12 (100 U/ml) enhanced both the proliferation and cytotoxic activity of RLNL in a culture with low doses of IL-2 (5 – 10 JRU/ml). After comparing an RLNL culture with a low dose of IL-2 alone, a higher proportion of CD8⁺ cells and CD56⁺ cells and a lower proportion of CD4⁺ cells were found in the culture with both IL-12 and a low dose of IL-2. Such a combination of the cytokines effectively activated RLNL in terms of the expression of IL-2 receptors. In the culture condition of IL-12 and a low dose of IL-2, a synergistic effect was observed in the production of such cytokines as interferon γ, tumor necrosis factor α (TNFα), and TNFβ, as well as in tumor cytotoxicity. However, the addition of IL-12 inhibited the cytotoxicity of RLNL in the culture with a high dose of IL-2 (100 JRU/ml). This inhibition is considered to be partially due to the endogenous production of TNFα by lymphocytes, because the neutralization of TNFα bioactivity partially restored the cytotoxic activities of RLNL. Furthermore, in the presence of hydrocortisone, IL-12 synergistically enhanced the cytotoxic activity of RLNL cultured with a high dose of IL-2. These results provide useful information about the improvement of adoptive immunotherapy against cancer using RLNL. Received: 2 February 1996 / Accepted: 30 July 1996     

Specific antitumor activity of tumor-infiltrating lymphocytes expanded first in a culture with both anti-CD3 monoclonal antibody and activated B cells and then in a culture with interleukin-2

In order to expand tumor-infiltrating lymphocytes (TIL) efficiently and in order to use them for immunotherapy, we utilized lipopolysaccharide-activated B cells (LPS blasts) as costimulatory-signal-providing cells in an in vitro culture system. TIL, prepared from subcutaneously inoculated B16 melanoma, failed to expand when cultured with anti-CD3 monoclonal antibody (mAb) alone followed by a low dose of interleukin(IL)-2. In contrast, such TIL did expand efficiently in culture with both anti-CD3 mAb and LPS blasts followed by culture with IL-2. These findings suggest that the presence of LPS blasts in the initial culture was essential for the cell expansion. The expansion of TIL was partially blocked by the addition of CTLA4 Ig, which is an inhibitor of costimulatory molecules such as CD80 and CD86, and was almost blocked by the addition of anti-(Fc receptor II)mAb. These findings thus indicate that such molecules, in conjunction with the receptor on the LPS blasts, participate in the efficient expansion of TIL. The B16-derived TIL, which expanded in our culture system, were predominantly CD8+T cells and showed a higher level of cytolytic activity against B16 melanoma than either lymphokine-activated killer cells or TIL cultured with a high dose of IL-2. In addition, the in vitro expanded B16-derived TIL produced interferon , but not IL-4, in response to B16 melanoma. What is more important, the adoptive transfer of such TIL had a significant antitumor effect against pulmonary metastasis in B16 melanoma, even without the concurrent administration of IL-2. Collectively, our results thus indicate the therapeutic efficacy of the protocol presented here for antitumor immunotherapy with TIL.This work was supported in part by a grant from the Ministry of Education, Science and Culture     

Typing of Verotoxins by DNA Colony Hybridization with Poly- and Oligonucleotide Probes,a Bead-Enzyme-Linked Immunosorbent Assay,and Polymerase Chain Reaction

To identify the type of Verotoxins (VT) produced by Verocytotoxin-producing Escherichia coli (VTEC), a sensitive bead-enzyme-linked immunosorbent assay and polymerase chain reaction with common and specific primers to various VTs (VT1, VT2, VT2vha, VT2vhb, and VT2vp1) were developed. Together with colony hybridization tests with oligo- and polynucleotide probes, these methods were applied to VTEC isolates to type the VT produced. The toxin types of 26 of 37 strains were identified, but the reaction profiles in assays of the remaining 11 strains suggested the existence of new VT2 variants. The application of these identification procedures may be useful as a tool for clinical and epidemiological studies of VTEC infection.