CD4+ T cell clones specific for the human p97 melanoma-associated antigen can eradicate pulmonary metastases from a murine tumor expressing the p97 antigen.

p97 is a human tumor-associated Ag present on most melanoma cells that represents a possible target for immunologic attack. To evaluate the capacity of T cells reactive with this protein to promote elimination of melanoma cells expressing p97, a murine model was developed by transfecting a C3H/HeN melanoma with the p97 cDNA, generating p97-specific CD4+ T cells by in vivo immunization of C3H/HeN mice with a vaccinia/p97 recombinant virus followed by in vitro cloning with soluble p97 protein, and determining whether these CD4+ T cells could mediate rejection of pulmonary metastases. Characterization of the T cell clones demonstrated the presence of both I-Ak and I-Ek-restricted clones, although the majority of clones recognized p97 in the context of I-Ek. Analysis of clonal specificity using truncated p97 proteins revealed that at least three epitopes were immunogenic, and further studies with overlapping 15-amino acid peptides from a region of the p97 molecule defined by these truncated proteins identified an immunodominant epitope responsible for the majority of the I-Ek response. The T cell clones were not capable of directly recognizing the p97-expressing melanoma cells but responded to the tumor if syngeneic APC were present to process the tumor-derived p97 Ag. The therapeutic efficacy of these CD4+ T cell clones was evaluated in an adoptive therapy model in which mice bearing metastatic pulmonary lesions were treated by i.v. administration of the p97-specific cells. Despite the inability of the CD4+ clones to directly respond to or lyse the tumor cells, the clones were effective in promoting tumor eradication. In vitro studies demonstrated that this may have reflected secretion of lymphokines that activated macrophages to lyse the tumor. The results suggest that noncytolytic p97-specific CD4+ T cell clones can be effective in therapy of pulmonary melanoma metastases. Moreover, if human T cells reactive with the p97 protein could be generated, the expression of this tumor-associated Ag in melanoma cells might be adequate for such T cells to mediate a therapeutic antitumor response.     

The Pro to glycine mutation of staphylococcal nuclease simplifies the unfolding—folding kinetics

Kinetics of unfolding and refolding of a staphylococcal nuclease mutant, in which Pro¹¹⁷ is replaced by glycine, have been investigated by stopped-flow circular dichroism, and the results are compared with those for the wild-type protein. In contrast to the biphasic unfolding of the wild-type nuclease, the unfolding of the mutant is represented by a single-phase reaction, indicating that the biphasic unfolding for the wild-type protein is caused by cis-trans isomerization about the prolyl peptide bond in the native state. The proline mutation also simplifies the kinetic refolding. Importance of the results in elucidating the folding mechanism is discussed.     

Conversion of Tyrosine Hydroxylase to Stable and Inactive Form by the End Products

We have reported previously that tyrosine hydroxylase in the crude extract from rat striatum exists in the inactive form showing almost no activity at the physiological pH and that the inactive form is produced by the action of the end products of the enzyme, such as dopamine. The incubation of the enzyme with the end products resulted in not only the inactivation but also a remarkable stabilization of the enzyme. Catechols possessing amino groups but no negatively charged groups on the side chains (catecholamine-type catechols) were effective at a concentration as low as 10(-7) M in both the inactivation and stabilization of the enzyme. In contrast, catechols not possessing positively or negatively charged side chains (3,4-dihydroxyphenylethyleneglycol-type catechols) were ineffective at a concentration of 10(-7) M but effective at a concentration of 10(-6) M for both the inactivation and stabilization. Catechols possessing negatively charged groups (3,4-dihydroxyphenylacetic acid-type catechols) were ineffective even at a concentration of 10(-6) M. Thus, the end products of tyrosine hydroxylase appear to serve to keep the enzyme inactive and stable. The reaction mechanism of the conversion of the enzyme from the active/labile form to the inactive/stable form by dopamine was also investigated.     

Inner arm dynein ATPase fraction of sea urchin sperm flagella causes active sliding of axonemal outer doublet microtubule.

In order to clarify the role of the inner arms of the axoneme in sperm flagellar movement, we prepared an ATPase fraction (12S) from the outer arm-depleted axonemes of sea urchin sperm flagella. When both arm-depleted axonemes were incubated with the 12S ATPase, they exhibited the sliding disintegration of outer doublet microtubules. Electron microscopy revealed that the ATPase rebound to the original inner arm sites of the axoneme. Therefore, it is quite likely that the 12S ATPase is one of the components of the inner arms. We referred to it as "inner arm dynein".     

Mechanism in 1,25(OH)2D3-induced suppression of helper/suppressor function of CD4/CD8 cells to immunoglobulin production in B cells

On the basis of previous data that 1,25(OH)2D3 suppressed both helper and suppressor activities of CD4 and CD8 cells in the pokeweek mitogen-stimulated culture, we examined the further effect of 1,25(OH)2D3 on both cells to define how 1,25(OH)2D3 is involved in the deterioration of their functions. 1,25(OH)2D3 suppressed the pokeweed mitogen and phytohemagglutinin-induced DNA synthesis of CD4 and CD8 cells. The suppression by 1,25(OH)2D3 of DNA synthesis was caused by a time lag in reaching maximal response. 1,25(OH)2D3 also suppressed interleukin-2 production of CD4 and CD8 cells. 1,25(OH)2D3 did not, however, affect their interleukin-2 receptor expression detected within 24 hr after phytohemagglutinin stimulation. In addition, 1,25(OH)2D3 failed to suppress DNA synthesis of CD4 and CD8 cells when cultured with a large amount of interleukin-2. Suppression by 1,25(OH)2D3 of proliferation and interleukin-2 production in CD4 and CD8 cells would bring about the decrease of their helper or suppressor functions by inhibiting their expansion or maturation.     

Alteration of plasmid DNA-mediated transformation and mutation induced by covalent binding of benzo[a]pyrene- 7,8-dihydrodiol-9,10-oxide in Escherichia coli

Plasmid-mediated transformation and mutagenesis induced by (±)-trans- benzo[a]pyrene-7,8-dihydrodiol-9,10-oxide (BP-DEI) in recipient Escherichia coli (E. coli) have been studied. Because plasmid DNA is used, the system is entirely free from direct toxic effects of BP-DEI on the recipient cells. Plasmid pK0482 DNA, which has two dominant genes, β-lactamase (amp-r) and galactokinase (galK) was modified with BP-DEI prior to its transformation of E. coli N99, AB1157, AB2463(recA^?) and AB1886(uvrA^?). Transformants were selected by ampicillin resistance and mutations were analyzed simultaneously by the altered expression of the galK gene. (1) Approx. 3 molecules of BP-DEI per molecule of pK0482 DNA decreased the transformation efficiency to 37% in AB1157 and the mutation frequency in this strain was proportional to the amount of BP-DEI covalently bound to pK0482 DNA. (2) In AB1886(uvrA^?) a 37% transformation efficiency was produced by only 1 molecule of BP-DEI per molecule of pK0482 DNA, and the mutation frequency in this strain was higher than in AB1157. (3) In AB2463(recA^?), the transformation efficiency was similar to that obtained with AB1157, but mutagenesis was clearly suppressed. (4) Polyacrylamide gel patterns of restriction digests of the pK0482 mutated at the galK gene were indistinguishable from those of the unmutated plasmid DNA.     

Establishment of Autoantibody-Producing Cell Lines from Peripheral Blood Lymphocytes of Patients with Systemic Lupus Erythematosus

Autoantibody-producing B cell lines were established from peripheral blood lymphocytes of patients with systemic lupus erythematosus. Peripheral blood lymphocytes obtained from five of seven patients were successfully transformed by Epstein-Barr virus. Two of four established B lymphoblastoid cell lines examined in this study produced anti-nuclear factor antibodies and one of them produced anti-single-stranded DNA and anti-double-stranded DNA antibodies. These results indicate that B cell clones committed to self antigens are transformed by Epstein-Barr virus and continue to produce autoantibodies. In order to establish a monoclonal autoantibody-producing B cell line, the cells were cloned by a limiting dilution method. The data suggest that it is possible to establish a monoclonal autoantibody-producing B cell line by the combination of transformation of B cells by Epstein-Barr virus and extensive cloning.