Treatment of splenic B lymphocytes and certain B-lineage cell lines with the mitogen lipopolysaccharide (LPS) and the lymphokine interleukin-4 (IL-4) induces expression of germ line immunoglobulin Ce transcripts and class switching to the CE gene. We show that LPS-plus-IL-4 induction of germ induce germ line E transcripts in freshly isolated B cells (13,14). The correlation between class switching and induction of germ line transcripts has raised the question of whether germ line transcription is an essential component of the switch process or a by-product of changes in chromatin structure prior to recombination. The former model implies the presence of inducible cis-acting elements that control germ line CH transcription. These elements would allow agents such as IL-4 to direct class switching by modulating germ line transcription at different CH loci. This model provides a mechanism by which different agents, through interacting effects on transcriptional activity, could combine to regulate class switching. If transcription played a mechanistic role in targeting recombination, one would also expect induction of germ line transcripts to occur at the transcriptional level. In this regard, IL-4 has been shown to induce a DNase I-hypersensitive site upstream of the yl switch region (5, 41). However, until now, no cis element controlling inducible germ line CH transcription been described. In this report, we demonstrate that LPS-plus-IL-4 (LPS/IL-4) treatment of a pre-B-cell line induces transcription of germ line E sequences. In addition, we identify a cis-acting element located near the transcriptional initiation site of germ line E transcripts that can contribute to this induction and describe several nuclear binding proteins that bind to this region. MATERIALS AND METHODSCell culture. The 18-81A20 Abelson murine leukemia virus (A-MuLV)-transformed cell line and M12.4.1 B-cell lymphoma cell line have been described previously (1, 15).

Recent work has shown that the ability of cytokines to direct immunoglobulin heavy chain class-switch recombination to particular heavy chain constant (C) region (CH) genes correlates with the induction of specific germ-line CH transcripts. To test the role of germ-line transcripts in class switching, we have used homologous recombination to mutate the immunoglobulin heavy chain locus of the 18.81A20 murine pre-B-cell line. In the parent cell line, the combination of interleukin-4 (IL-4) and lipopolysaccharide (LPS) induces germ-line E locus transcription prior to class switching to E. The heavy chain locus of the mutated cell line contains the immunoglobulin heavy chain enhancer and variable region gene promoter in place of the LPS/IL-4-responsive germ-line E promoter. The mutant cell line constitutively transcribes the E locus in the absence of IL-4. Strikingly, the mutant cell line also switches to E in the absence of IL-4. This result demonstrates that, at least in the 18.81A20 cell line, germ-line E transcription plays a direct role in class switching to the E locus. In addition, the ability to change the pattern of class switching by altering transcriptional activity indicates that transcription of germ-line CH is mechanistically important in regulation of class switching.Immunoglobulin heavy chain class switching is the process by which the constant region (C) of an antibody is changed, allowing the molecule to retain its antigen-binding specificity while changing its effector function. Most studies have found that class switching occurs by a recombination event between regions ofrepetitive DNA [termed switch (S) The molecular mechanism by which cytokines such as IL-4 direct class switching remains unknown. However, recent work has shown that the ability of cytokines to direct class-switch recombination to particular CH genes correlates with the induction of transcripts (termed "germ-line CH transcripts") that initiate upstream of the switch recombination sequences of the involved CH gene (reviewed in ref. 6). For example, concurrent treatment of murine splenic B lymphocytes or certain B-lineage cell lines with LPS and IL-4 induces expression ofgerm-line E transcripts at the transcriptional level before the production of IgE (7-9). Germ-line transcripts have been identified at almost all murine and human CH genes and are structurally conserved. Further evidence for the important role of germ-line CH transcription has been provided by the identification of cytokineresponsive cis elements around the initiation regions of certain CH transcripts (9)(10)(11).The correlation between germ-line CH transcription and subsequent recombination implicates transcription through specific S regions as a targeting mechanism for class switching. There are several models that could explain the mechanism by which germ-line transcription targets class switching (reviewed in ref. 6). One possibility is that there is a non-specific switch recombination machinery that is directed to specific regions by transcription, p...

In Ras cotransformation assays, Max exhibited a biphasic effect on Myc transformation activity. Cotransfection of low levels of Max expression plasmid stimulated Myc transformation activity, but cotransfection of high levels suppressed it. Mutations in the functionally undefined Max amino-and carboxy-terminal regions outside of the B/HLH/LZ motif partly separated these activities, suggesting various modes of Max regulation. We demonstrate that the Max protein is a nuclear protein in vivo and identify a carboxy-terminal region similar to nuclear localization signals whose integrity is necessary for efficient localization. Two mutants that delete amino-or carboxy-terminal consensus signals for casein kinase II (CKII) exhibited altered gel mobility and DNA-binding potential in vitro and showed modified transforming potential in the Ras cotransformation assay, suggesting that CKII or a CKII-related enzyme may regulate Max function in vivo. Our data suggest that both the ratio of Myc/Max hetero-oligomers to Max homo-oligomers and Max-specific regulation can contribute to determining the biological activity of Myc in vivo.

We have characterized the structure and expression of transcripts synthesized from the murine germline immunoglobulin gamma 3 heavy chain gene in certain B-lineage cells. The transcripts initiate upstream of the switch gamma 3 region, generating a 5' exon that is spliced to C gamma 3 exons. Expression of this germline transcript is induced when splenic B cells or A-MuLV-transformed pre-B cell lines are cultured in the presence of lipopolysaccharide (LPS). Addition of interleukin-4 (IL-4) to these lipopolysaccharide (LPS) cultures dramatically inhibits induction of the germline gamma 3 transcript. Induction of germline gamma 3 transcripts occurs before the increased accumulation of gamma 3-producing cells and VDJ-gamma 3 mRNA in cultures of splenic B cells. These data provide further evidence that germline CH transcriptional units are important components in the regulation of heavy chain class-switching. In addition, the pre-B cell lines that we describe represent the first example of permanent cell lines that regulate expression of the germline gamma locus in response to LPS plus IL-4 treatment in a manner analogous to normal B cells; therefore these lines should represent an excellent model system to further study the molecular mechanisms by which germline expression is regulated by these agents.

A series of aldose reductase inhibitors were prepared which were analogues of the potent, orally active inhibitor tolrestat (1). These compounds (5, 7, 9, and 10) have an extra substituent on one of the unoccupied positions on the naphthalene ring of 1. Primary amide prodrugs of several members from the series 5 and 7, namely 6 and 8, respectively, were also prepared. These compounds were evaluated in two in vitro systems: an isolated enzyme preparation from bovine lens to assess their intrinsic inhibitory activity and an isolated sciatic nerve assay to determine their ability to penetrate membranes of nerve tissue. These compounds were also evaluated in vivo as inhibitors of galactitol accumulation in the lens, sciatic nerve, and diaphragm of galactose-fed rats. In general, compounds in series 5, 7, 9, and 10 were potent inhibitors of bovine lens aldose reductase. 2-Halo-substituted analogues from the series 5, 7, and 9 exhibited high activity in the nerve of the 4-day-galactose-fed rat, and in several instances, the primary amide prodrug 8 enhanced the in vivo potency of the respective carboxylic acid 7. Two 2-fluoro-derivatives, 8a and 9a, had especially high activity in vivo and were chosen for additional studies. These compounds were found to be approximately equipotent to tolrestat in the sciatic nerve of the galactose-fed rat and the STZ rat, as judged by their ED50's in these assays. Although primary amide analogue 8a did not have intrinsic inhibitory activity toward aldose reductase, it was metabolized to an active form in vivo and also in vitro within the sciatic nerve.