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Treatment of the triruthenium pentahydrido complex {Cp*Ru(μ-H)}3(μ3-H)2 (1; Cp* = η^5-C5Me5) with cycloalkene resulted in exclusive formation of the μ3-cycloalkyne complex {Cp*Ru(μ-H)}3(μ3-η²:η²(⊥)-CnH2n-4) (2a; n = 5, 2b; n = 6) as a result of vinylic C−H bond cleavage. VT NMR studies revealed fluxional behavior of the perpendicularly coordinated cycloalkyne ligand. Reaction of 2b with carbon monoxide afforded the 48-electron complex (Cp*Ru)3(μ-CO)(μ3-CO)(μ-H)(μ3-η²(∥)-C6H8) (3b) with a parallel-coordinated alkyne ligand.

Reaction of the triruthenium pentahydride complex {Cp*Ru(μ-H)}3(μ3-H)2 (1; Cp* = η⁵-C5Me5) with acetylene was investigated, in which the μ3-ethylidene−μ3-η²(∥)-ethyne complex {Cp*Ru(μ-H)}3(μ-CMeH){μ3-η²(∥)-CH&dbd;CH} (2) was produced via formation of the intermediary bis(μ-vinyl) complex {Cp*Ru(μ-H)}3(μ-η¹:η²-CH&dbd;CH2)2 (3). An α-C−H bond of the μ-ethylidene ligand of 2 underwent oxidative addition upon thermolysis to yield the μ3-ethylidyne−μ3-η²(∥)-ethyne complex (Cp*Ru)3(μ-H)2(μ3-CCH3){μ3-η²(∥)-CH&dbd;CH} (5), which showed fluxional behavior of both the μ3-ethyne and the hydride ligands. Further C−H bond cleavage of the μ3-ethylidyne ligand, affording the μ-vinylidene−μ3-η²(∥)-ethyne complex (Cp*Ru)3(μ-H)(μ3-C&dbd;CH2){μ3-η²(∥)-CH&dbd;CH} (6), was also investigated. Treatment of 5 with an excess amount of acetylene resulted in C−C bond formation at the Ru3 core, and the μ3-ethylidyne−μ3-η³-diruthenaallyl complex (Cp*Ru)3{μ3-η¹:η³:η¹-C(H)C(H)CCH3}(μ3-CCH3)(μ-H) (7) was obtained. The molecular structures of 5 and 7 were determined by single-crystal X-ray diffraction studies.

Somatic hypermutation is a key mechanism in generating Ig with higher affinities to antigen, a process known as affinity maturation. Using Igkappa transgenes, the 3' enhancer (kappaE3') has been shown to play an important role in introducing hypermutations. In order to identify the cis-acting elements that regulate hypermutagenesis, we have generated transgenic substrates containing mutations/deletions in the kappaE3' region. Here, we report that base substitutions in the kappaE3', either in the PU.1 or in the NF-EM5 binding motif, not only reduce the mutation rate but also disrupt the directed mutagenesis in the intrinsic hotspots of the Igkappa transgene.

During the development of lymphocytes, expression of the Ig genes is strictly regulated in a tissue-specific manner and in a time-ordered fashion. We have previously shown that the PU.1 binding motif in the Igκ 3′ enhancer (κE3′) and a novel Ets family protein other than PU.1 may be possibly involved in the control of V_κ-J_κ joining. In the attempt to isolate the novel Ets family protein, we have screened cDNA libraries with the yeast one-hybrid method and identified a new PU.1-related factor, Prf. This novel Ets family protein is shown to interact with the PU.1 binding sequences in various promoters and enhancers, including κE3′. It was found that expression of the prf gene is predominant in the B-lineage cells, with the exception of immature B cells. Since Prf does not exhibit functions of transcriptional activity, this novel protein may act as an antagonist against other Ets family proteins, e.g. PU.1 and Spi-B. Possible roles of Prf with respect to the B cell differentiation are discussed.

During the development of lymphocytes, expression of the Ig genes is strictly regulated in a tissue-specific manner and in a time-ordered fashion. We have previously shown that the PU.1 binding motif in the Igkappa 3' enhancer (kappaE3') and a novel Ets family protein other than PU.1 may be possibly involved in the control of V(kappa)-J(kappa) joining. In the attempt to isolate the novel Ets family protein, we have screened cDNA libraries with the yeast one-hybrid method and identified a new PU.1-related factor, Prf. This novel Ets family protein is shown to interact with the PU.1 binding sequences in various promoters and enhancers, including kappaE3'. It was found that expression of the prf gene is predominant in the B-lineage cells, with the exception of immature B cells. Since Prf does not exhibit functions of transcriptional activity, this novel protein may act as an antagonist against other Ets family proteins, e.g. PU.1 and Spi-B. Possible roles of Prf with respect to the B cell differentiation are discussed.

Lymphoid cell lines were isolated that were inducible for the expression of surface immunoglobulin by shift from 35.5 to 39.5 degrees C after infection of mouse bone marrow cells with a mutagen‐treated Abelson murine leukemia virus. Virus produced by one of the cell lines (ts49) transmitted the temperature‐sensitive phenotype to new lymphoid transformants as well as to NIH/3T3 cells. In addition, the tyrosine autophosphorylating activity of the p120gag‐abl protein synthesized in ts49‐transformed cells was found to be temperature‐sensitive. Shift experiments using ts49‐transformed lymphoid cells showed that at 39.5 degrees C they synthesize increased amounts of mu and kappa chain RNA and protein, and that they can be further induced to secrete IgM when treated with lipopolysaccharide.

We have described here two distinct types of carrier-specific helper T cells which act independently and synergistically to augment the B-cell response to a hapten. They are separable by passage through a nylon wool column. The first type of helper T cell, which we designate as Th1, is nylon nonadherent, and can help the response of hapten-primed B cells only if the haptenic and carrier determinants are present on a single molecule (cognate interaction). The second type of helper T cell, Th2, adheres to the nylon wool column, and can help the B-cell response to a hapten coupled to a heterologous carrier upon stimulation with unconjugated relevant carrier (polyclonal interaction). The addition of a small number of Th2 to the mixture of Th1 and B cells significantly augmented the net response to the hapten carrier conjugate. Both Th1 and Th2 cells belong to the Lyt-1+,2-,3- subclass. Th1 has no detectable Ia antigen, whereas Th2 is killed by certain anti-Ia antisera and complement. The Ia antigen detected on Th2 was found to be controlled by a locus in the I-J subregion. The results clearly established the fact that there are two distinct pathways in the T- and B-cell collaboration, which involves two different subsets of carrier-specific helper T cells.

In pre-B cells, mu chains are expressed in association with "surrogate' L chains encoded by the lambda 5 and Vpre-B1 genes. In addition to their association with lambda 5 and Vpre-B1, mu chains in pre-B cells are associated with the products of the Vpre-B3 gene (formerly designated 8HS-20), which display a distinct association with mu chains and biochemical properties in terms of mol. wt, pI value and glycosylation. However, the mechanism of the generation of Vpre-B3 isoforms has been unknown. The present study indicates that the Vpre-B3 gene transcript underwent alternative RNA processing in normal B cells, in a pre-B cell lymphoma and in a mature B cell lymphoma, WEHI 231, that was transfected with the Vpre-B3 genomic clone. Vpre-B3 isoforms were expressed in a WEHI 231 cell line transfected with the Vpre-B3 genomic clone, comparable in biochemical nature to those expressed in a pre-B cell lymphoma. In contrast, expression of one of the isoforms was missing in a cell line transfected with the Vpre-B3 cDNA clone. These results suggest that Vpre-B3 isoforms with distinct biochemical characteristics are derived from alternatively processed Vpre-B3 mRNA.

The germinal center (GC) develops in secondary lymphoid tissues in response to thymus-dependent (TD) antigens. To investigate the molecular mechanism of B cell differentiation in GC, we enriched GC B cells from spleen of TD antigen-immunized wild-type and X-linked immunodeficient (XID) mice, and examined the differentiation of GC B cells into antigen-specific IgG1 antibody-forming cells (AFC) in response to anti-CD40 mAb and cytokines. A significant proportion of freshly purified GC B cells expressed receptors for IL-4 and IL-5. Anti-CD40 mAb sustained the viability of GC B cells and IL-4 co-operated with anti-CD40 mAb for further enhancement of the cell viability. Anti-CD40 mAb and IL-4 were essential for inducing differentiation of GC B cells into antigen-specific IgG1-AFC and IL-5 efficiently enhanced their differentiation. GC B cells with the xid mutation responded for proliferation to CD40 ligation to a lesser extent and for the IgG1-AFC response to anti-CD40 mAb together with IL-4, but they showed impaired responsiveness to IL-5, regardless of enhanced expression of IL-5R in response to anti-CD40 mAb and IL-4. These results suggest that anti-CD40 mAb, IL-4 and IL-5 play a critical role in the differentiation of mouse GC B cells. The GC B cells from XID mice show a functional defect with respect to IL-5-mediated differentiation.

A simple procedure to enrich the antigen (keyhole limpet hemocyanin, KLH)-specific suppressor T cell was described. The suppressor T cell from KLH)-immunized mice specifically bound to the KLH-coated Sephadex G-200 column at 37 degrees C, and was eluted from the column by cold (0-4 degrees C) medium. The helper T cell did not bind to the column under the identical condition. The suppressor T cell thus obtained had 100 times as potent suppressor activity as the original spleen cells in in vivo and in vitro secondary antibody responses against a hapten coupled to KLH. This procedure also enriched the cells bearing I-J determinants and Lyt-2,3 alloantigens, allowing us to study the phenotypic expressions on the suppressor T cell by direct serological procedures as well as by the use of the fluorescence activated cell sorter. Parallel functional and serological analyses indicated that the antigen-specific suppressor T cell belongs to a population of I-J+, Lyt-2+,3+ and Fc R- T cells.

An antigen-specific suppressive T-cell factor was extracted from physically disrupted thymocytes and spleen cells of mice that had been immunized with soluble protein antigens. The factor, when inoculated into syngeneic normal mice, could induce a significant suppression of IgG antibody response against a hapten coupled to the carrier protein by which the donor of the suppressor factor was immunized. The suppressor factor was found only effective in suppressing the antibody response of syngeneic or H-2 histocompatible recipients. The suppressive T-cell factor was removed by absorption with immunoadsorbent composed of the relevant antigen, but not with any of those of anti-immunoglobulin antibodies. The factor was successfully removed by alloantibodies with specificity for the K end (H-2K, I-A and I-B) of the H-2 complex of the donor strain, but not by those for the D end (I-C, SsSlp, and H-2D). The activity was removed by absorption with a heterologous antithymocyte serum. The mol wt of the suppression T-cell factor was between 35,000 and 60,000 as determined by Sephadex G-200 gel filtration. The suppressive T-cell factor was found to be a heat-liable protein.