B cells are essential for murine mammary tumor virus transmission, but not for presentation of endogenous superantigens.

Murine mammary tumor viruses (MMTVs) are retroviruses that encode superantigens capable of stimulating T cells via superantigen-reactive T cell receptor V beta chains. MMTVs are transmitted to the suckling offspring through milk. Here we show that B cell-deficient mice foster nursed by virus-secreting mice do not transfer infectious MMTVs to their offspring. No MMTV proviruses could be detected in the spleen and mammary tissue of these mice, and no deletion of MMTV superantigen-reactive T cells occurred. By contrast, T cell deletion and positive selection due to endogenous MMTV superantigens occurred in B cell-deficient mice. We conclude that B cells are essential for the completion of the viral life cycle in vivo, but that endogenous MMTV superantigens can be presented by cell types other than B cells.

Role of tyrosine phosphorylation of HS1 in B cell antigen receptor-mediated apoptosis.

The 75-kD HS1 protein is highly tyrosine-phosphorylated during B cell antigen receptor (BCR)-mediated signaling. Owing to low expression of HS1, WEHI-231-derived M1 cells, unlike the parental cells, are insensitive to BCR-mediated apoptosis. Here, we show that BCR-associated tyrosine kinases Lyn and Syk synergistically phosphorylate HS1, and that Tyr-378 and Tyr-397 of HS1 are the critical residues for its BCR-induced phosphorylation. In addition, unlike wild-type HS1, a mutant HS1 carrying the mutations Phe-378 and Phe-397 was unable to render M1 cells sensitive to apoptosis. Wild-type HS1, but not the mutant, localized to the nucleus under the synergy of Lyn and Syk. Thus, tyrosine phosphorylation of HS1 is required for BCR-induced apoptosis and nuclear translocation of HS1 may be a prerequisite for B cell apoptosis.

B cell adaptor containing src homology 2 domain (BASH) links B cell receptor signaling to the activation of hematopoietic progenitor kinase 1.

The B cell adaptor containing src homology 2 domain (BASH; also termed BLNK or SLP-65), is crucial for B cell antigen receptor (BCR)-mediated activation, proliferation, and differentiation of B cells. BCR-mediated tyrosine-phosphorylation of BASH creates binding sites for signaling effectors such as phospholipase Cgamma (PLCgamma)2 and Vav, while the function of its COOH-terminal src homology 2 domain is unknown. We have now identified hematopoietic progenitor kinase (HPK)1, a STE20-related serine/threonine kinase, as a protein that inducibly interacts with the BASH SH2 domain. BCR ligation induced rapid tyrosine-phosphorylation of HPK1 mainly by Syk and Lyn, resulting in its association with BASH and catalytic activation. BCR-mediated activation of HPK1 was impaired in Syk- or BASH-deficient B cells. The functional SH2 domain of BASH and Tyr-379 within HPK1 which we identified as a Syk-phosphorylation site were both necessary for interaction of both proteins and efficient HPK1 activation after BCR stimulation. Furthermore, HPK1 augmented, whereas its kinase-dead mutant inhibited IkappaB kinase beta (IKKbeta) activation by BCR engagement. These results reveal a novel BCR signaling pathway leading to the activation of HPK1 and subsequently IKKbeta, in which BASH recruits tyrosine-phosphorylated HPK1 into the BCR signaling complex.

IL-5 receptor-mediated tyrosine phosphorylation of SH2/SH3-containing proteins and activation of Bruton's tyrosine and Janus 2 kinases.

Interleukin 5 (IL-5) induces proliferation and differentiation of B cells and eosinophils by interacting with its receptor (IL-5R) which consists of two distinct polypeptide chains, alpha and beta (beta c). Although both IL-5R alpha and beta c lack a kinase catalytic domain, IL-5 is capable of inducing tyrosine phosphorylation of cellular proteins. We investigated the role of IL-5R alpha in tyrosine phosphorylation of molecules involved in IL-5 signal transduction, using an IL-5-dependent early B cell line, Y16 and transfectants expressing intact or mutant IL-5R alpha together with intact beta c. The results revealed that the transfectants expressing truncated IL-5R alpha, which entirely lacks a cytoplasmic domain, together with beta c, showed neither protein-tyrosine phosphorylation nor proliferation in response to IL-5. This confirms that IL-5R alpha plays a critical role in protein-tyrosine phosphorylation which triggers cell growth. IL-5 stimulation results in rapid tyrosine phosphorylation of beta c and proteins containing Src homology 2 (SH2) and/or SH3 domains such as phosphatidyl-inositol-3 kinase, Shc, Vav, and HS1, suggesting their involvement in IL-5-mediated signal transduction. IL-5 stimulation significantly enhanced activities of Janus 2 and B cell-specific Bruton's tyrosine kinases (JAK2 and Btk) and increased the tyrosine phosphorylation of JAK2 kinase. These results and recent data on signaling of growth factors taken together, multiple biochemical pathways driven by tyrosine kinases such as JAK2 and Btk are involved in IL-5 signal transduction.

Development of measures for limiting negative impacts of the «Atomic¼ lake on population and environment.

In arid climate, economic activities at the territory of the "Atomic" lake is one of the topical issues for the Semipalatinsk test site (STS). Hence, the boundaries of areas with radionuclides contamination, which correspond to the level of radioactive wastes at the territory adjacent to the "Atomic" lake of STS, are to be determined. The territory around the lake is used for cattle breeding and the water of the "Atomic" lake that is the one large water source is used for livestock watering. It is important to develop measures that will limit possible negative impact on population and environment. In results of the conducted research were developed measures consisting of remediation and access limitation to the stockpile of soils with contamination level corresponding to the level of radioactive waste (RW).

Stage-specific expression of DNasegamma during B-cell development and its role in B-cell receptor-mediated apoptosis in WEHI-231 cells.

Here, we describe the non-redundant roles of caspase-activated DNase (CAD) and DNasegamma during apoptosis in the immature B-cell line WEHI-231. These cells induce DNA-ladder formation and nuclear fragmentation by activating CAD during cytotoxic drug-induced apoptosis. Moreover, these apoptotic manifestations are accompanied by inhibitor of CAD (ICAD) cleavage and are abrogated by the constitutive expression of a caspase-resistant ICAD mutant. No such nuclear changes occur during oxidative stress-induced necrosis, indicating that neither CAD nor DNasegamma functions under necrotic conditions. Interestingly, the DNA-ladder formation and nuclear fragmentation induced by B-cell receptor ligation occur in the absence of ICAD cleavage and are not significantly affected by the ICAD mutant. Both types of nuclear changes are preceded by the upregulation of DNasegamma expression and are strongly suppressed by 4-(4,6-dichloro-[1, 3, 5]-triazin-2-ylamino)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-benzoic acid (DR396), which is a specific inhibitor of DNasegamma. Our results suggest that DNasegamma provides an alternative mechanism for inducing nuclear changes when the working apoptotic cascade is unsuitable for CAD activation.

The cis-acting regulatory elements of immunoglobulin heavy chain gene involved in enhanced immunoglobulin production after lipopolysaccharide (LPS) stimulation.

In the present study, the transient expression of the human immunoglobulin heavy chain gene (HIG1) was analyzed in a mouse pre-B-cell line, 70Z/3, after LPS stimulation. HIG1 gene and its recombinant plasmids were transfected by the calcium phosphate method into 70Z/3 cells and the cells were stimulated with lipopolysaccharide (LPS) 48 hr after DNA transfection. The amounts of human heavy chain gene products greatly increased in 70Z/3 cells after LPS stimulation, but the increment was diminished by deletion of the heavy chain gene enhancer element (MluI-HpaI fragment) in the J-C intron from the HIG1 gene. The gene, delta 3, which contained the 5' promoter region and the rearranged VDJ region of HIG1 but lacked the enhancer element, was weakly transcribed in 70Z/3 cells after LPS stimulation. Insertion of the enhancer element into the delta 3 gene greatly enhanced the transcription of the VDJ gene. The highest enhancement of the VH gene transcription rate was obtained when the 3' half of the enhancer element was ligated to the delta 3 gene. The present data suggest that the 3' half of the enhancer element of the heavy chain gene may play an important role in the enhanced production of immunoglobulin which is induced with LPS stimulation.

Establishment of an embryonic stem (ES) cell line derived from a non-obese diabetic (NOD) mouse: in vivo differentiation into lymphocytes and potential for germ line transmission.

A non-obese diabetic (NOD) mouse-derived embryonic stem (ES) cell line has been stably maintained in an undifferentiated state with a characteristic ES cell-like morphology, expressing the stem cell marker alkaline phosphatase, and displaying a normal diploid karyotype. After injecting the NOD-ES cells into blastocysts, chimeric mice were obtained. Small but significant numbers of lymphocytes expressed the NOD-derived MHC allele. When a chimeric mouse was mated with C57BL/6 mice, an agouti mouse was obtained, having the NOD-derived H-2 I-A(beta)g7 haplotype. Thus, an NOD-ES cell line which can differentiate into lymphocytes with potential for germ line transmission was successfully established.

Phosphorylation of HS1, GAP-associated p190 and a novel GAP-associated p60 protein by cross-linking of Fc gamma RIIIA.

Human Fc gamma receptor IIIA (hFc gamma RIIIA) cDNA was introduced into mouse macrophage/monocyte cell line P388D1, and several stable cell clones expressing hFc gamma RIIIA were isolated. This facilitated the study of the biological function of Fc gamma RIIIA in monocytes/macrophages. The cloned cells showed the high phagocytic activity mediated by hFc gamma-RIIIA, while the original P388D1 cells did not. In order to examine the phosphorylation of proteins involved in hFc gamma RIIIA signal transduction, these receptors were stimulated by cross-linking. The cross-linking of hFc gamma RIIIA induced a rapid increase in tyrosine phosphorylation of several proteins, including PLC-gamma 1, Syk, HS1, and p21rasGAP-associated p190 and p60 proteins. Immunoblotting with a polyclonal antibody specific for the GAP-associated p62 protein, which was originally found in fibroblasts and is homologous with an RNA-binding protein, revealed that the p60 phosphorylated after cross-linking of hFc gamma RIIIA seemed to represent a novel GAP-associated protein unrelated to the known GAP-associated p62 protein, which was also present in the P388D1 cells.

[Studies of physiological functions of cytokines by generating mutant mice through the transgenic and the gene-targeting technology].

Molecular cloning of gene coding for cytokines, growth factors and their receptors have facilitated an accumulation of findings regarding the function of their products. So far, many kinds of functions of such molecules in vitro have been identified. To clarify the physiological roles of such molecules in vivo a variety of cytokine-transgenic mice and a few cytokine-gene targeted mice have been generated. Many of the phenotypes expressed by these animals are as expected from the in vitro assay data, but some are unexpected. In this short article, some of the phenotypes reported are introduced and their significance is discussed.

[Function of HS1 protein in B-cell antigen-receptor signaling and induction of apoptosis].

Cross-linking of antigen receptor complex on the surface of B cells induces activation of the associated protein-tyrosine kinases (PTKs) which, in turn, phosphorylate and activate a variety of intracellular proteins, some of those are known to transmit the signal to further downstream. In some circumstances, such signals eventually result in apoptosis, rather than proliferation, of the cells. The programmed B-cell death is thought to be crucial in developmental selection of functional cells, self-tolerance, regulation of immune response, and so on. Molecular mechanisms of the intracellular signaling pathways leading to the apoptosis are still obscure. HS1, a hematopoietic-lineage specific protein, is a major substrate of the antigen-receptor associated PTKs. The biochemical and predicted structural features suggest HS1 actively involves in the signal transduction pathway from the antigen receptors and may directly regulate a gene transcription. In this review article, discussed are the roles of non-receptor type tyrosine kinases and HS1 in the pathway leading to apoptosis of B cells, recently evidenced by cell- and mouse-genetics.

Targeted disruption of mu chain membrane exon causes loss of heavy-chain allelic exclusion.

Burnet's clonal selection theory suggests that each B lymphocyte is committed to a single antibody specificity. This is achieved by a programme of somatic rearrangements of the gene segments encoding antibody variable (V) regions, in the course of B-cell development. Evidence from immunoglobulin-transgenic mice and immunoglobulin-gene-transfected transformed pre-B cells suggest that the membrane form of the immunoglobulin heavy (H) chain of class mu (microns), expressed from a rearranged H-chain (IgH) locus, may signal allelic exclusion of the homologous IgH locus in the cell and initiation of light (L)-chain gene rearrangement in the Ig kappa loci. We report here that targeted disruption of the membrane exon of the mu chain indeed results in the loss of H-chain allelic exclusion. But, some kappa chain gene rearrangement is still observed in the absence of micron expression.

B cell development regulated by gene rearrangement: arrest of maturation by membrane-bound D mu protein and selection of DH element reading frames.

In productively rearranged murine VH-DH-JH genes (encoding immunoglobulin heavy chain variable regions), the DH elements are preferentially used in one particular reading frame (RF1), although the recombination breakpoints at the DH-JH border vary. Despite this variability, the bias of RF usage is not due to cellular selection by antigen but is quantitatively established at the stage of DH-JH rearrangement: RF3 is counterselected on the basis of stop codons. RF2 allows the expression of a truncated mu chain (D mu protein) from most DH-JH joints. Using B cells in which the membrane exon of the mu chain is disrupted by homologous recombination on one of the two homologous chromosomes, we obtain evidence that membrane-bound D mu signals arrest of differentiation, presumably by preventing VH-DHJH joining. In addition to RF3 and RF2 counterselection, promotion of DH-JH joining in areas of sequence homology further enforces RF1 usage.

A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin mu chain gene.

Of the various classes of antibodies that B lymphocytes can produce, class M (IgM) is the first to be expressed on the membrane of the developing cells. Pre-B cells, the precursors of B-lymphocytes, produce the heavy chain of IgM (mu chain), but not light chains. Recent data suggest that pre-B cells express mu chains on the membrane together with the 'surrogate' light chains lambda 5 and V pre B (refs 2-7). This complex could control pre-B-cell differentiation, in particular the rearrangement of the light-chain genes. We have now assessed the importance of the membrane form of the mu chain in B-cell development by generating mice lacking this chain. We disrupted one of the membrane exons of the gene encoding the mu-chain constant region by gene targeting in mouse embryonic stem cells. From these cells we derived mice heterozygous or homozygous for the mutation. B-cell development in the heterozygous mice seemed to be normal, but in homozygous animals B cells were absent, their development already being arrested at the stage of pre-B-cell maturation.

The human HCLS1 gene maps to chromosome 3q13 by fluorescence in situ hybridization.

The human HS1 gene (HCLS1, hematopoietic cell-specific Lyn substrate 1) expressed in human hematopoietic cells encodes a major substrate of protein-tyrosine kinase, p75HS1. This intracellular protein is involved in the signal transduction pathways that initiate at the antigen receptors of both B and T lymphocytes. Fluorescence in situ hybridization using a 2.0-kb cDNA and an 8.0-kb genomic DNA clone of HCLS1 as probes revealed that the gene maps to 3q13.

Notch signaling suppresses IgH gene expression in chicken B cells: implication in spatially restricted expression of Serrate2/Notch1 in the bursa of Fabricius.

The bursa of Fabricius is a central organ for chicken B cell development and provides an essential microenvironment for expansion of the B cell pool and for generation of a diversified B cell repertoire. We report here that genes encoding the Notch family of transmembrane proteins, key regulators of cell fate determination in development, are differentially expressed in the bursa of Fabricius: Notch1 is expressed in medullary B cells located close to the basement membrane-associated epithelium (BMAE). In contrast, a Notch ligand, Serrate2, is expressed exclusively in the BMAE, which surrounds bursal medulla. A basic helix-loop-helix-type transcription factor, Hairy1, a downstream target of Notch signaling, is expressed in the bursa coordinately with Notch1 and Serrate2 and an immature B cell line, TLT1, which expresses both Notch1 and Serrate2. Furthermore, stable expression of a constitutively active form of chicken Notch1 or Notch2 in a B cell line results in a down-regulation of surface IgM expression, which is accompanied by the reduction of IgH gene transcripts. Transient reporter assay with the human IgH gene intronic enhancer reveals that an active form of Notch1 inhibits the IgH enhancer activity in chicken B cells, suggesting that Notch-mediated signals suppress the IgH gene expression via influencing the IgH intronic enhancer. These findings raise the possibility that the local activation of Notch1 in a subset of B cells by Serrate2 expressed in BMAE may influence the cell fate decision that is involved in B cell differentiation and selection inside the bursa.

Cell cycle arrest and apoptosis induced by Notch1 in B cells.

Notch receptors play various roles for cell fate decisions in developing organs, although their functions at the cell level are poorly understood. Recently, we found that Notch1 and its ligand are each expressed in juxtaposed cell compartments in the follicles of the bursa of Fabricius, the central organ for chicken B cell development. To examine the function of Notch1 in B cells, a constitutively active form of chicken Notch1 was expressed in a chicken B cell line, DT40, by a Cre/loxP-mediated inducible expression system. Remarkably, the active Notch1 caused growth suppression of the cells, accompanied by a cell cycle inhibition at the G(1) phase and apoptosis. The expression of Hairy1, a gene product up-regulated by the Notch1 signaling, also induced the apoptosis, but no cell cycle inhibition. Thus, Notch1 signaling induces apoptosis of the B cells through Hairy1, and the G(1) cell cycle arrest through other pathways. This novel function of Notch1 may account for the recent observations indicating the selective inhibition of early B cell development in mice by Notch1.