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H Karasuyama

Publications and source records attributed to H Karasuyama.

At least 19 recordsLinked to original sources
PubMed2000

The identification of a nonclassical cadherin expressed during B cell development and its interaction with surrogate light chain.

A 130-kDa glycoprotein (p130) has been found to be associated with surrogate light chain on pro- and pre-B I cells. Using peptide sequences obtained from purified p130 we have cloned its gene. The gene encodes a typical cadherin type 1 membrane protein with six extracellular cadherin domains (one pseudo domain) but lacking the catenin-binding site in its cytoplasmic part. Even without this catenin-binding site, p130 mediates Ca(2+)-dependent homotypic adhesion of cells. The interaction of p130 with surrogate light chain is confirmed by co-transfection and co-immunoprecipitation experiments. The expression of p130 is biphasic during the B cell development. Reverse transcriptase-polymerase chain reaction and flow cytometric analyses revealed that it is expressed on B220(+)c-Kit(+) pro-B and pre-B-I cells as well as on B220(+)CD25(-)IgM(+) immature and mature B cells but not on B220(+)CD25(+) pre-B-II cells. It is also expressed in fetal liver, at low levels in myeloid cells, and strongly in intestinal epithelial cells. In the spleen, p130-expressing cells are mainly localized in the marginal zone. We call this B lineage-, intestine-, liver- and leukocyte-expressed gene BILL-cadherin. The possible functions of BILL-cadherin in B cell development are discussed.

Amino Acid Sequence↗
PubMed2000

Ras mediates effector pathways responsible for pre-B cell survival, which is essential for the developmental progression to the late pre-B cell stage.

Ras is essential for the transition from early B cell precursors to the pro-B stage, and is considered to be involved in the signal cascade mediated by pre-B cell antigen receptors. To examine the role of p21(ras) in the late stage of B cell differentiation, we established transgenic mice (TG) expressing a dominant-inhibitory mutant of Ha-ras (Asn-17 Ha-ras) in B lineage cells at high levels after the early B cell precursor stage. Expression of p21(Asn-17) (Ha-ras) was associated with a prominent reduction in the number of late pre-B cells, but had little effect on proliferation of early pre-B cells. Inhibition of p21(ras) activity markedly reduced the life span of pre-B cells, due, at least in part, to downregulation of the expression of an antiapoptotic protein, Bcl-xL. Thus, the apparent role for p21(ras) activity in pre-B cell survival may explain the decreased numbers of late pre-B cells in Asn-17 Ha-ras TG. Consistent with this possibility, overexpression of Bcl-2 in Asn-17 Ha-ras TG reversed the reduction in the number of late pre-B cells undergoing immunoglobulin light chain gene (IgL) rearrangement and progressing to immature B cells. These results suggest that p21(ras) mediates effector pathways responsible for pre-B cell survival, which is essential for progression to the late pre-B and immature B stages.

Animals↗
PubMed2000

Genetic defect in human X-linked agammaglobulinemia impedes a maturational evolution of pro-B cells into a later stage of pre-B cells in the B-cell differentiation pathway.

Surrogate light chains (lambda 5/VpreB) are selectively expressed in early precursors of B cells. B-cell defects in X-linked agammaglobulinemia (XLA) are caused by mutations in the gene for Bruton's tyrosine kinase. To elucidate the nature of early B-lineage cells in bone marrow (BM), samples from 13 XLA patients and 24 healthy controls of different ages were comparatively analyzed using an antihuman VpreB monoclonal antibody. Expression of surrogate light (SL) and mu-heavy chains were examined after cell membrane permeabilization because they are mainly expressed in the cytoplasm of early B-lineage cells. A flow cytometric analysis of normal BM identified 5 discrete cell types of B cells: mu(-)SL(++) (pro-B [B-cell progenitor]), mu(low)SL(++) (pre-B1a), mu(low)SL(+) (pre-B1b), mu(low)SL(- )(pre-B2), and mu(high)SL(- )(B). The large cells, presumably in cycling states, were enriched in pre-B1a cells. The frequencies of B-lineage cells in BM were higher in young children, and declined with advancing age. In contrast, XLA showed a profound reduction in BM B-lineage cells. In XLA BM, an expansion of pro-B cells with some small pre-B1a cells was marked, but other cells were negligible. These observations illustrate a B-cell maturation defect in XLA as well as a normal human B-cell differentiation pathway. The results suggest that the genetic defect in XLA may impede the evolution of pro-B cells beyond the earlier pre-B stage into the later stage of pre-B cells in B-cell development. (Blood. 2000;96:610-617)

Adolescent↗
PubMed2000

Myopathy phenotype of transgenic mice expressing active site-mutated inactive p94 skeletal muscle-specific calpain, the gene product responsible for limb girdle muscular dystrophy type 2A.

A defect of the gene for p94 (calpain 3), a skeletal muscle-specific calpain, is responsible for limb girdle muscular dystrophy type 2A (LGMD2A), or 'calpainopathy', which is an autosomal recessive and progressive neuromuscular disorder. To study the relationships between the physiological functions of p94 and the etiology of LGMD2A, we created transgenic mice that express an inactive mutant of p94, in which the active site Cys129 is replaced by Ser (p94:C129S). Three lines of transgenic mice expressing p94:C129S mRNA at various levels showed significantly decreased grip strength. Sections of soleus and extensor digitorum longus (EDL) muscles of the aged transgenic mice showed increased numbers of lobulated and split fibers, respectively, which are often observed in limb girdle muscular dystrophy muscles. Centrally placed nuclei were also frequently found in the EDL muscle of the transgenic mice, whereas wild-type mice of the same age had almost none. There was more p94 protein produced in aged transgenic mice muscles and it showed significantly less autolytic degradation activity than that of wild-type mice. Although no necrotic-regenerative fibers were observed, the age and p94:C129S expression dependence of the phenotypes strongly suggest that accumulation of p94:C129S protein causes these myopathy phenotypes. The p94:C129S transgenic mice could provide us with crucial information on the molecular mech-anism of LGMD2A.

Animals↗
PubMed2000

Transgenic human lambda 5 rescues the murine lambda 5 nullizygous phenotype.

The human lambda 5 (hu lambda 5) gene is the structural homologue of the murine lambda 5 (m lambda 5) gene and is transcriptionally active in pro-B and pre-B lymphocytes. The lambda 5 and VpreB polypeptides together with the Ig mu H chain and the signal-transducing subunits, Ig alpha and Ig beta, comprise the pre-B cell receptor. To further investigate the pro-B/pre-B-specific transcription regulation of hu lambda 5 in an in vivo model, we generated mouse lines that contain a 28-kb genomic fragment encompassing the entire hu lambda 5 gene. High levels of expression of the transgenic hu lambda 5 gene were detected in bone marrow pro-B and pre-B cells at the mRNA and protein levels, suggesting that the 28-kb transgene fragment contains all the transcriptional elements necessary for the stage-specific B progenitor expression of hu lambda 5. Flow cytometric and immunoprecipitation analyses of bone marrow cells and Abelson murine leukemia virus-transformed pre-B cell lines revealed the hu lambda 5 polypeptide on the cell surface and in association with mouse Ig mu and mouse VpreB. Finally, we found that the hu lambda 5 transgene is able to rescue the pre-B lymphocyte block when bred onto the m lambda 5-/- background. Therefore, we conclude that the hu lambda 5 polypeptide can biochemically and functionally substitute for m lambda 5 in vivo in pre-B lymphocyte differentiation and proliferation. These studies on the mouse and human pre-B cell receptor provide a model system to investigate some of the molecular requirements necessary for B cell development.

Abelson murine leukemia virus↗
PubMed2000

Immunoglobulin beta signaling regulates locus accessibility for ordered immunoglobulin gene rearrangements.

The antigen receptor gene rearrangement at a given locus is tightly regulated with respect to cell lineage and developmental stage by an ill-defined mechanism. To study the possible role of precursor B cell antigen receptor (pre-BCR) signaling in the regulation of the ordered immunoglobulin (Ig) gene rearrangement during B cell differentiation, a newly developed system using mu heavy (H) chain membrane exon (microm)-deficient mice was employed. In this system, the antibody-mediated cross-linking of Igbeta on developmentally arrested progenitor B (pro-B) cells mimicked pre-BCR signaling to induce early B cell differentiation in vivo. Analyses with ligation-mediated polymerase chain reaction revealed that the Igbeta cross-linking induced the redirection of Ig gene rearrangements, namely, the suppression of ongoing rearrangements at the H chain locus and the activation of rearrangements at the light (L) chain locus. Upon the cross-linking, the kappaL chain germline transcription was found to be upregulated whereas the V(H) germline transcription was promptly downregulated. Notably, this alteration of the accessibility at the H and L chain loci was detected even before the induction of cellular differentiation became detectable by the change of surface phenotype. Thus, the pre-BCR signaling through Igbeta appears to regulate the ordered Ig gene rearrangement by altering the Ig locus accessibility.

Animals↗
PubMed2000

Deficiency of a STE20/PAK family kinase LOK leads to the acceleration of LFA-1 clustering and cell adhesion of activated lymphocytes.

Lymphocyte-oriented kinase (LOK) is a member of the STE20/p21-activated kinase (PAK) family and expressed predominantly in lymphoid organs. Generation of LOK-deficient mice revealed that the leukocyte-function-associated antigen (LFA-1)/intercellular adhesion molecules (ICAM)-mediated aggregation of mitogen-stimulated T cells was greatly enhanced in the absence of LOK. Though levels of total LFA-1 and ICAMs as well as the active form of LFA-1 on T cell blasts were comparable in the presence and absence of LOK, clustering of active LFA-1 detected by binding of soluble ICAM-1 was accelerated in the absence of LOK. These results suggest that LOK is potentially involved in the regulation of LFA-1-mediated lymphocyte adhesion.

Animals↗
PubMed2000

Ste20-like kinase (SLK), a regulatory kinase for polo-like kinase (Plk) during the G2/M transition in somatic cells.

BACKGROUND: Activation of the cyclin-dependent kinase cdc2-cyclin B1 at the G2/M transition of the cell cycle requires dephosphorylation of threonine-14 and tyrosine-15 in cdc2, which in higher eukaryotes is brought about by the Cdc25C phosphatase. In Xenopus, there is evidence that a kinase cascade comprised of xPlkk1 and Plx1, the Xenopus polo-like kinase 1, plays a key role in the activation of Cdc25C during oocyte maturation. In the mammalian somatic cell cycle, a polo-like kinase homologue (Plk1) also functions during mitosis, but a kinase upstream of Plk is still unknown. RESULTS: We show here that human Ste20-like kinase (SLK), which is a ubiquitously expressed mammalian protein related to xPlkk1, can phosphorylate and activate murine Plk1. During progression through the G2 phase of the mammalian cell cycle, the activity of endogenous SLK is increased. The amount of SLK protein is decreased in quiescent and differentiating cells. Treatment with okadaic acid induces a phosphorylation-dependent enhancement of SLK activity. CONCLUSIONS: We propose that SLK has a role in the regulation of Plk1 activity in actively dividing cells during the somatic cell cycle. SLK itself is suggested to be regulated by phosphorylation.

3T3 Cells↗
PubMed1999

Differential effect of an Ig mu transgene on development of pre-B cells in fetal and adult SCID mice.

Progression of pro-B lymphocytes to the pre-B stage depends on the expression of a pre-B cell receptor (pre-BCR), consisting of an Ig mu H chain, Ig surrogate light chain, and associated signal transducing chains. Mice that are unable to express a pre-BCR show an arrest of B cell development at the pro-B stage. Such is the case for severe combined immune deficient (SCID) mice in which mu chains are not made because of a defect in V(D)J recombination. When mu chains are made, as in SCID mice bearing a functional mu transgene, then B cell differentiation can proceed to the pre-B stage. However, as reported here, a mu transgene (M54) that promotes development of SCID pre-B cells in adult bone marrow fails to do so in fetal liver. We suggest that a pre-BCR containing the M54 mu chain cannot signal progression of pro-B cells to the pre-B stage in the fetal liver microenvironment.

Age Factors↗
PubMed1999

Molecular cloning of the human gene STK10 encoding lymphocyte-oriented kinase, and comparative chromosomal mapping of the human, mouse, and rat homologues.

LOK is a new and unique member of the STE20 family with serine/threonine kinase activity, and its expression is restricted mostly to lymphoid cells in mice. We cloned the cDNA encoding the human homologue of LOK. The amino acid sequence deduced from the cDNA shows a high similarity to that of mouse LOK, with 88% identity as a whole. The kinase domains at the N-terminus and the coiled-coil regions at the C-terminus are particularly conserved, showing 98% and 93% identity, respectively. Western blot analysis with mouse LOK-specific antibody detected 130 000 Mr LOK proteins in human and rat lymphoid cell lines and tissues. The gene encoding the LOK (STK10/Stk10) gene was mapped by fluorescence in situ hybridization to chromosome 5q35.1 in human, chromosome 11A4 in mouse, and chromosome 10q12.3 in rat. By virtue of polymorphic CA repeats found in the 3' untranslated region of the mouse Stk10 gene, the Stk10 locus was further pinpointed to chromosome 11 between D11Mit53 and D11Mit84, using the intersubspecific backcross mapping panel. These results established STK10 as a new marker of human chromosome 5 to define the syntenic boundary of human chromosomes 5 and 16 on mouse chromosome 11.

Amino Acid Sequence↗
PubMed1999

Establishment of antigen-specific IgE transgenic mice to study pathological and immunobiological roles of IgE in vivo.

We have established transgenic mice that carry the genes coding for heavy and light chains of TNP-specific IgE. They produced high titers of TNP-specific IgE (20-40 microg/ml in serum) and their mast cells were heavily loaded with IgE. The level of FcepsilonRI expression on their mast cells was 6-8 times higher than that in non-transgenic littermates. The expression of low-affinity IgE receptor FcepsilonRII (CD23) on splenic B cells was also 6-8 times higher in the transgenic mice. Consistent with this, substantial amounts of IgE were detected on B cells in the transgenic mice. When challenged with i.v. administration of the corresponding antigen, the transgenic mice exhibited systemic anaphylactic symptoms such as a drastic drop of body temperature and extravasation of administered dye. Biphasic (immediate and delayed) ear swelling response was also elicited in a TNP-specific manner by epicutaneous antigen challenge without any prior sensitization. Thus, IgE produced in the transgenic mice was found to be biologically active to induce both local and systemic allergic reactions in vivo upon the challenge of the corresponding antigen. Taken together, the antigen-specific IgE transgenic mice established for the first time in this study appear to provide an attractive model system to study the pathological roles of IgE in acute and chronic phases of allergic inflammation as well as their immunobiological roles in vivo. They may also be useful to develop novel therapeutic strategies for atopic disorders.

Anaphylaxis↗
PubMed1999

Lambda5 is required for rearrangement of the Ig kappa light chain gene in pro-B cell lines.

Lambda5 associates with V(pre-B) to form the surrogate light (L) chain. The phenotype of lambda5 knockout mice showed severe impairment of B cell development from pro-B to immature B cell stages. To investigate the function of the surrogate L chain at this stage, we restored expression of lambda5 to lambda5-deficient pro-B cell lines which were established from bone marrow cells of lambda5 knockout mice in the presence of IL-7 and a stromal cell line. Some of these lines are severely impaired in B cell development from pro-B to immature B cell stages as is seen in vivo in lambda5 knockout mice. Restoration of lambda5 protein by retroviral-mediated gene transfer into established lambda5-deficient pro-B cell lines induced rearrangement of the Ig kappa L chain genes after removal of IL-7 from the culture. Immunoprecipitation revealed that the restored lambda5 in the cell line is coupled with V(pre-B) to form the surrogate L chain. The results demonstrate that formation of a complete surrogate L chain, consisting of both lambda5 and V(pre-B), stimulates efficient rearrangement of the kappa L chain genes.

Animals↗
PubMed1998

Flow cytometric diagnosis of the cell lineage and developmental stage of acute lymphoblastic leukemia by novel monoclonal antibodies specific to human pre-B-cell receptor.

Three novel monoclonal antibodies (MoAbs) have been established that recognize distinct epitopes of a human pre-B-cell receptor (pre-BCR) composed of a mu heavy (muH) chain and a lambda5/VpreB surrogate light (SL) chain. HSL11 reacts with lambda5 whereas HSL96 reacts with VpreB. Intriguingly, HSL2 does not bind to each component of the pre-BCR but does bind to the completely assembled pre-BCR complex. Flow cytometric analyses with cytoplasmic staining of a panel of human cell lines showed that HSL11 and HSL96 specifically stained cell lines derived from the pro-B and pre-B-cell stages of B-cell development. In contrast, HSL2 stained exclusively cell lines derived from the pre-B-cell stage. These results prompted us to explore the possibility of clinical application of these MoAbs for the determination of the cell lineage and developmental stage of acute lymphoblastic leukemia (ALL). Whereas none of mature B-lineage ALLs (B-ALLs), T-lineage ALLs (T-ALLs), and acute myeloid leukemias analyzed were stained in the cytoplasm with these three MoAbs, the vast majority of non-B- and non-T-ALLs (53 out of 56 cases) were found positive for either lambda5, Vpre-B, or both in their cytoplasm. Among these 53 cytoplasmic SL chain-positive ALLs, 19 cases were also positive for cytoplasmic muH chain, indicative of pre-B-cell origin. Interestingly, 6 out of these 19 pre-B-ALL cases were found negative for cytoplasmic staining with HSL2. From these results, we propose a novel classification of B-ALL in which five subtypes are defined on the basis of the differential expression of SL chain, muH chain, pre-BCR, and light chain along the B-cell development.

Antibodies, Monoclonal↗
PubMed1998

Identification of Bach2 as a B-cell-specific partner for small maf proteins that negatively regulate the immunoglobulin heavy chain gene 3' enhancer.

Maf family transcription factors are important regulators in various differentiation systems. Putative Maf recognition elements (MAREs) are found in the 3' enhancer region of the immunoglobulin heavy chain (IgH) gene. These elements are bound in B-cell extracts by a heterodimeric protein complex containing both Bach2 and a small Maf protein. Analysis of normal hematopoietic cells revealed that Bach2 is specifically expressed in B cells. Bach2 is abundantly expressed in the early stages of B-cell differentiation and turned off in terminally differentiated cells. Bach2 acts together with MafK as a negative effector of the IgH 3' enhancer and binds to the co-repressor SMRT (silencing mediator of retinoid and thyroid receptor). Hence the Bach2-small-Maf heterodimer may represent the first example of a B-cell lineage, and of a developmental stage-restricted negative effector of the MARE in the IgH 3' enhancer region.

B-Lymphocytes↗
PubMed1998

Antigen receptor engagement turns off the V(D)J recombination machinery in human tonsil B cells.

The germinal center (GC) is an anatomic compartment found in peripheral lymphoid organs, wherein B cells undergo clonal expansion, somatic mutation, switch recombination, and reactivate immunoglobulin gene V(D)J recombination. As a result of somatic mutation, some GC B cells develop higher affinity antibodies, whereas others suffer mutations that decrease affinity, and still others may become self-reactive. It has been proposed that secondary V(D)J rearrangements in GCs might rescue B cells whose receptors are damaged by somatic mutations. Here we present evidence that mature human tonsil B cells coexpress conventional light chains and recombination associated genes, and that they extinguish recombination activating gene and terminal deoxynucleotidyl transferase expression when their receptors are cross-linked. Thus, the response of the recombinase to receptor engagement in peripheral B cells is the opposite of the response in developing B cells to the same stimulus. These observations suggest that receptor revision is a mechanism for receptor diversification that is turned off when antigen receptors are cross-linked by the cognate antigen.

Animals↗
PubMed1998

Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9.

Caspases are essential components of the mammalian cell death machinery. Here we test the hypothesis that Caspase 9 (Casp9) is a critical upstream activator of caspases through gene targeting in mice. The majority of Casp9 knockout mice die perinatally with a markedly enlarged and malformed cerebrum caused by reduced apoptosis during brain development. Casp9 deletion prevents activation of Casp3 in embryonic brains in vivo, and Casp9-deficient thymocytes show resistance to a subset of apoptotic stimuli, including absence of Casp3-like cleavage and delayed DNA fragmentation. Moreover, the cytochrome c-mediated cleavage of Casp3 is absent in the cytosolic extracts of Casp9-deficient cells but is restored after addition of in vitro-translated Casp9. Together, these results indicate that Casp9 is a critical upstream activator of the caspase cascade in vivo.

Animals↗