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Four families in which asthma occurred in more than three members, and in which there were at least three children, were studied in terms of clinical symptoms, serum IgE levels determined by RIST and RAST, and HLA haplotypes. No association between clinical asthma and serum IgE level was found, but a close association between asthma and a particular HLA haplotype in a family was demonstrated. It was therefore postulated that a disease susceptibility gene closely linked to an HLA gene complex may play a role in the development of asthma. [ABSTRACT FROM AUTHOR]

Metaphors and sarcasm are precious fruits of our highly evolved social communication skills. However, children with the condition then known as Asperger syndrome are known to have difficulties in comprehending sarcasm, even if they possess adequate verbal IQs for understanding metaphors. Accordingly, researchers had employed a screening test that assesses metaphor and sarcasm comprehension to distinguish Asperger syndrome from other conditions with similar external behaviors (e.g., attention-deficit/hyperactivity disorder). This study employs a standardized test to evaluate recent large language models' (LLMs) understanding of nuanced human communication. The results indicate improved metaphor comprehension with increased model parameters; however, no similar improvement was observed for sarcasm comprehension. Considering that a human's ability to grasp sarcasm has been associated with the amygdala, a pivotal cerebral region for emotional learning, a distinctive strategy for training LLMs would be imperative to imbue them with the ability in a cognitively grounded manner.

Background: Due to its tumor-specific metabolic pathway characteristics, 5-aminolevulinic acid (5-ALA) is a natural amino acid widely used in cancer treatment. The current study, demonstrated that 5-ALA induced ferroptosis via glutathione peroxidase 4 (GPX4) and heme oxygenase 1 (HMOX1) and had an antitumor effect in esophageal squamous cell carcinoma (ESCC)., Methods: Expression of GPX4 and HMOX1 in pathologic specimens of 97 ESCC patients was examined, and prognostic analyses were performed. Real-time polymerase chain reaction (RT-PCR), RNA microarray, and Western blotting analyses were used to evaluate the role of 5-ALA in ferroptosis in vitro. In addition, this study used ferrostatin-1, a ferroptosis inhibitor, and a lipid peroxidation reagent against cell lines treated with 5-ALA. Finally, the role of 5-ALA was confirmed by its effect on an ESCC subcutaneous xenograft mouse model., Results: The study showed that upregulation of GPX4 and downregulation of HMOX1 were poor prognostic factors in ESCC. In an RNA microarray analysis of KYSE30, ferroptosis was one of the most frequently induced pathways, with GPX4 suppressed and HMOX1 overexpressed by 5-ALA treatment. These findings were verified by RT-PCR and Western blotting. Furthermore, 5-ALA led to an increase in lipid peroxidation and exerted an antitumor effect in various cancer cell lines, which was inhibited by ferrostatin-1. In vivo, 5-ALA suppressed GPX4 and overexpressed HMOX1 in tumor tissues and led to a reduction in tumor size., Conclusions: Modulation of GPX4 and HMOX1 by 5-ALA induced ferroptosis in ESCC. Thus, 5-ALA could be a promising new therapeutic agent for ESCC.

From the time of Thucydides in the 5th century BC, it has been known that specific recognition of pathogens and memory formation are critical components of immune functions. In contrast to the immune system of jawed vertebrates, such as humans and mice, plants lack a circulatory system with mobile immune cells and a repertoire of clonally distributed antigen receptors with almost unlimited specificities. However, without these systems and mechanisms, plants can live and survive in the same hostile environment faced by other organisms. In fact, they achieve specific pathogen recognition and elimination, with limited self-reactivity, and generate immunological memory, sometimes with transgenerational characteristics. Thus, the plant immune system satisfies minimal conditions for constituting an immune system, namely, the recognition of signals in the milieu, integration of that information, subsequent efficient reaction based on the integrated information, and memorization of the experience. In the previous report, this set of elements was proposed as an example of minimal cognitive functions. In this essay, I will first review current understanding of plant immunity and then discuss the unique features of cognitive activities, including recognition of signals from external as well as internal environments, autoimmunity, and memory formation. In doing so, I hope to reach a deeper understanding of the significance of immunity omnipresent in the realm of living organisms.

B cell antigen receptor (BCR) signaling is positively and negatively regulated by various cell surface receptors such as CD19 and CD45. Functional analysis of these receptors has been performed using gene targeting technology, which is a valid approach to elucidate their functions. However, this type of analysis is restricted when multiple molecules are evaluated simultaneously. From a different perspective, synthetic biology provides a high degree of freedom for analyzing various molecules. Here we developed a system to reconstruct BCR signaling using the J558L myeloma cell line in combination with the protein-based Ca(2+) indicator YC3.60. BCR-reconstituted J558L cells harboring YC3.60 (J558Lμv11 cells) permitted monitoring of Ca(2+) mobilization. Reconstituting CD19 in J558Lμv11 cells resulted in detectable BCR-induced Ca(2+) mobilization but with kinetics different from that of CD45-expressing cells. Furthermore, we evaluated the validity of the J558L system by proteomic analysis of tyrosine-phosphorylated proteins after antigen stimulation. Identification of more than 100 BCR-induced tyrosine-phosphorylated proteins in J558Lμv11 cells revealed a similarity to that observed in B cells, and a novel member, non-receptor protein tyrosine kinase Fer, was found. Thus, this reconstruction system using J558L cells appeared to be valid for comprehensively investigating BCR signaling., (Copyright © 2013 Elsevier Inc. All rights reserved.)

SHP-1 plays an important role for the regulation of signaling from various hematopoietic cell receptors. In this study, we examined IL-3-induced cell proliferation and IL-3 depletion-induced apoptosis in bone marrow-derived mast cells (BMMC) established from motheaten (me) that lack SHP-1 expression, viable motheaten (me(v)) expressing phosphatase-deficient SHP-1, and wild-type (WT) mice. When BMMC were stimulated with IL-3, increased ERK activation was evident in resting state and sustained in me-BMMC relative to WT-BMMC. ERK is known to be involved in the regulation of cell proliferation and apoptosis in some cells. In accordance with sustained ERK activation, apoptosis was decreased in me- and me(v)-BMMC compared with WT-BMMC. In contrast to the predicted role of ERK as a pro-survival molecule, IL-3-induced cell proliferation was much lower in me- and me(v)-BMMC than WT-BMMC. Stimulation with lower concentration of IL-3 or addition of PD98059, a MEK inhibitor, to the culture resulted in the suppression of decreased apoptosis and cell proliferation in me- and me(v)-BMMC. Collectively, these results suggest that SHP-1 positively regulates IL-3-dependent mast cell proliferation and apoptosis by inhibiting ERK activity through its phosphatase activity. Furthermore, our results indicate that ERK would act as a negative regulator for cell proliferation and induce apoptosis when its activity is highly increased., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Modulation of mast-cell activation may provide novel ways to control allergic diseases. Here, we show that protein tyrosine phosphatase epsilon (PTPepsilon; Ptpre) plays key regulatory roles during mast-cell activation mediated by the high-affinity IgE receptor (FcepsilonRI). Bone marrow-derived mast cells (BMMC) from Ptpre(-/-) mice exhibited enhanced FcepsilonRI-induced Ca(2+) mobilization and mitogen-activated protein kinase (MAPK) (JNK and p38) activation, and showed corresponding enhancement of evoked degranulation and cytokine production, but not leukotriene production. Examination of proteins linking tyrosine kinase activation and Ca(2+) mobilization revealed that the absence of PTPepsilon leads to increased phosphorylation of the linker for activation of T cells and SH2 domain-containing leucocyte phosphoproteins of 76 kDa, but not Grb2-associated binder-2 (Gab2). Because Gab2 is considered to be situated downstream of Fyn kinase, we reasoned that Fyn may not be a target of PTPepsilon. In the event, Syk but not Lyn was hyperphosphorylated in PTPepsilon-deficient BMMC. Thus, PTPepsilon most likely exerts its effects at the level of Syk, inhibiting downstream events including phosphorylation of SLP-76 and linker of activated T cells and mobilization of Ca(2+). Consistent with the in vitro data, antigen- and IgE-mediated passive systemic anaphylactic reactions were augmented in Ptpre(-/-) mice. Given that the number of mast cells is unchanged in these mice, this observation most likely reflects alterations of mast cell-autonomous signalling events. These data suggest that PTPepsilon negatively regulates FcepsilonRI-mediated signalling pathways and thus constitutes a novel target for ameliorating allergic conditions.

Src homology region 2 domain-containing phosphatase 1 (SHP-1), a cytoplasmic protein tyrosine phosphatase, plays an important role for the regulation of signaling from various hematopoietic cell receptors. Although SHP-1 is shown to be a negative signal modulator in mast cells, its precise molecular mechanisms are not well defined. To elucidate how SHP-1 regulates mast cell signaling, we established bone marrow-derived mast cells from SHP-1-deficient motheaten and wild-type mice and analyzed downstream signals induced by cross-linking of high affinity IgE receptor, Fc epsilonRI. Upon Fc epsilonRI ligation, motheaten-derived bone marrow-derived mast cells showed enhanced tyrosine phosphorylation of Src homology region 2 domain-containing leukocyte protein of 76 kDa (SLP-76) and linker for activation of T cells, activation of mitogen-activated protein kinases and gene transcription and production of cytokine. Because the activity of Syk, responsible for the phosphorylation of SLP-76 and linker for activation of T cells, is comparable irrespective of SHP-1, both molecules might be substrates of SHP-1 in mast cells. Interestingly, the absence of SHP-1 expression disrupted the association between SLP-76 and phospholipase Cgamma, which resulted in the decreased phospholipase Cgamma phosphorylation, calcium mobilization, and degranulation. Collectively, these results suggest that SHP-1 regulates Fc epsilonRI-induced downstream signaling events both negatively and positively by functioning as a protein tyrosine phosphatase and as an adaptor protein contributing to the formation of signaling complex, respectively.

Despite the important role in the development and activation of T cells, NK cells, mast cells, and macrophages, the expression and function of SLP-76 in B cells have been largely unknown. Here we demonstrate that SLP-76 is expressed in all mouse B cell lines tested and in normal splenic B cells, and serves as an SHP-1 substrate. Dephosphorylation of SLP-76 by SHP-1 inhibits its association with Nck, down-regulating c-Jun N-terminal kinase (JNK) activation and exerting a positive effect on apoptosis. Knockdown of SLP-76 in WEHI-231 cells by small interfering RNA attenuated JNK activation, but showed little effects on extracellular signal-regulated kinase (ERK) or p38 activation. Although WEHI-231 does not express linker for activation of T cells (LAT), SLP-76 localizes in membrane fraction, which increases following B cell receptor (BCR) cross-linking. Further analyses revealed that SLP-76 complexed with Gads is associated with tyrosine-phosphorylated CD22 through the SH2 domains of SLP-76 and Gads. Given that SHP-1 binds to CD22 upon BCR ligation, our findings suggest that dephosphorylation of SLP-76 recruited to CD22 by SHP-1 inhibits BCR-induced JNK activation, dictating apoptosis.

CD72 is a 45 kDa B cell-specific type II transmembrane protein of the C-type lectin superfamily. It was originally defined as a receptor-like molecule that regulates B cell activation and differentiation; however, its precise function remains unclear since more recent functional analyses, including a gene targeting study, suggest that CD72 may serve as a negative or a positive regulator of B cell signaling. In the present study, we analyzed the cell-autonomous function of CD72 in B cell receptor (BCR) signaling using CD72-deficient cells generated from mature BAL-17 cells. We found that BCR-mediated phosphorylation of CD19, Btk, Vav and phospholipase Cgamma2 and association of CD19 with phosphatidylinositol-3 kinase were impaired in CD72-deficient cells. Inositol trisphosphate synthesis was normally induced initially but ablated at 1 min of stimulation in CD72-deficient cells. In the event, Ca(2+) release from intracellular stores remained intact, though influx of extracellular Ca(2+) was severely impaired in CD72-deficient cells. Furthermore, BCR-evoked activation of mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase and c-Jun NH(2)-terminal kinase, and growth inhibition in BAL-17 cells were blocked in the absence of CD72. Significantly, these effects were largely reversed by re-expression of CD72. Thus, CD72 appears to exert a positive effect on BCR signaling pathways leading to Ca(2+) influx and MAPK activation, which in turn may determine the fate of BAL-17 cells.

CD45 is a key protein tyrosine phosphatase regulating Src-family protein tyrosine kinases (Src-PTKs) in lymphocytes; precisely how it exerts its effect remains controversial, however. We previously demonstrated that CD45 negatively regulates Lyn in the WEHI-231 B-cell line. Here we show that negative regulation by CD45 is physiologically significant in B cells and that some CD45 is constitutively associated with glycolipid-enriched microdomains (GEMs), where it inhibits Src-PTKs by dephosphorylating both the negative and the positive regulatory sites. Upon B-cell receptor (BCR) ligation, however, CD45 dissociates from GEMs within 30 seconds, inducing phosphorylation of 2 regulatory sites and activation of Src-PTKs, but subsequently reassociates with the GEMs within 15 minutes. Disruption of GEMs with methyl-beta-cyclodextrin results in abrogation of BCR-induced apoptosis in WEHI-231 cells, suggesting GEMs are critical to signals leading to the fate determination. We propose that the primary function of CD45 is inhibition of Src-PTKs and that the level of Src-PTK activation and the B-cell fate are determined in part by dynamic behavior of CD45 with respect to GEMs.

Src homology region 2 domain-containing phosphatase 1 (SHP-1) is a key mediator in lymphocyte differentiation, proliferation, and activation. We previously showed that B cell linker protein (BLNK) is a physiological substrate of SHP-1 and that B cell receptor (BCR)-induced activation of c-Jun NH(2)-terminal kinase (JNK) is significantly enhanced in cells expressing a form of SHP-1 lacking phosphatase activity (SHP-1-C/S). In this study, we confirmed that SHP-1 also exerts negative regulatory effects on JNK activation in splenic B cells. To further clarify the role of SHP-1 in B cells, we examined how dephosphorylation of BLNK by SHP-1 affects downstream signaling events. When a BLNK mutant (BLNK Delta N) lacking the NH(2)-terminal region, which contains four tyrosine residues, was introduced in SHP-1-C/S-expressing WEHI-231 cells, the enhanced JNK activation was inhibited. Among candidate proteins likely to regulate JNK activation through BLNK, Nck adaptor protein was found to associate with tyrosine-phosphorylated BLNK and this association was more pronounced in SHP-1-C/S-expressing cells. Furthermore, expression of dominant-negative forms of Nck inhibited BCR-induced JNK activation. Finally, BCR-induced apoptosis was suppressed in SHP-1-C/S-expressing cells and coexpression of Nck SH2 mutants or a dominant-negative form of SEK1 reversed this phenotype. Collectively, these results suggest that SHP-1 acts on BLNK, modulating its association with Nck, which in turn negatively regulates JNK activation but exerts a positive effect on apoptosis.

Signaling through the B cell antigen receptor (BCR) is negatively regulated by the SH2 domain-containing protein-tyrosine phosphatase SHP-1, which requires association with tyrosine-phosphorylated proteins for activation. Upon BCR ligation, SHP-1 has been shown to associate with the BCR, the cytoplasmic protein-tyrosine kinases Lyn and Syk, and the inhibitory co-receptors CD22 and CD72. How SHP-1 is activated by BCR ligation and regulates BCR signaling is, however, not fully understood. Here we demonstrate that, in the BCR-expressing myeloma line J558L mu 3, CD72 expression reduces the BCR ligation-induced phosphorylation of the BCR component Ig alpha/Ig beta and its cytoplasmic effectors Syk and SLP-65. Substrate phosphorylation was restored by expression of dominant negative mutants of SHP-1, whereas the SHP-1 mutants failed to enhance phosphorylation of the cellular substrates in the absence of CD72. This indicates that SHP-1 is efficiently activated by CD72 but not by other pathways in J558L mu m3 cells and that inhibition of SHP-1 specifically activated by CD72 reverses CD72-induced dephosphorylation of cellular substrates in these cells. Taken together, BCR-induced SHP-1 activation is likely to require inhibitory co-receptors such as CD72, and SHP-1 appears to mediate the negative regulatory effect of CD72 on BCR signaling by dephosphorylating Ig alpha/Ig beta and its downstream signaling molecules Syk and SLP-65.

TAMA300, an interferometric gravitational-wave detector with 300-m baseline length, has been developed and operated with sufficient sensitivity to detect gravitational-wave events within our galaxy and sufficient stability for observations; the interferometer was operated for over 10 hours stably and continuously. With a strain-equivalent noise level of h approximately 5x10(-21)/sqrt[Hz], a signal-to-noise ratio of 30 is expected for gravitational waves generated by a coalescence of 1.4M-1.4M binary neutron stars at 10 kpc distance. We evaluated the stability of the detector sensitivity with a 2-week data-taking run, collecting 160 hours of data to be analyzed in the search for gravitational waves.

Stimulation of B cell antigen receptor (BCR) may induce proliferation, differentiation, or apoptosis, depending upon the maturational stage of the cell and the presence or absence of signals transmitted via coreceptors. One such signal is delivered via CD40; for instance, ligation of CD40 rescues B cells from BCR-induced apoptosis. Here we show that, in contrast to WEHI-231 cells, CD40 ligation did not reverse BCR-induced growth inhibition in the BAL-17 mature B cell line and CD40 ligation itself inhibited proliferation. This inhibitory signaling was not observed in CD45-deficient cells. Further analyses demonstrate that transfection of dominant-negative form of SEK1 or treatment with SB203580 strongly reduced CD40-induced inhibition of BAL-17 proliferation, suggesting a requirement for c-Jun NH2-terminal kinase and p38 in CD40-induced inhibition of proliferation. Interestingly, CD40-initiated activation of c-Jun NH2-terminal kinase and p38 was enhanced and sustained in CD45-deficient cells, and these phenotypes were reversed by transfecting CD45 gene. However, CD40-mediated induction of cell surface molecules was not affected in CD45-deficient cells. Taken collectively, these results suggest that CD45 exerts a decisive effect on selective sets of CD40-mediated signaling pathways, dictating B cell fate.

In this study, we examined the contribution made by CD45 to B cell antigen receptor (BCR)-induced activation of mitogen-activated protein kinase (MAPK) family members. We found that CD45 negatively regulated BCR-induced c-Jun NH(2)-terminal kinase (JNK) and p38 activation in immature WEHI-231 cells, whereas in mature BAL-17 cells, CD45 positively regulated JNK and p38 activation and negatively regulated extracellular signal-regulated kinase activity. Furthermore, cooperative action of JNK and p38 dictated BCR-induced inhibition of growth. Thus, CD45 appears to differentially regulate BCR-induced activation of MAPK members, and can exert opposing effects on JNK and p38 in different cellular milieu, controlling the B cell fate.

We have identified the lymphocyte semaphorin CD100/Sema4D as a CD40-inducible molecule by subtractive cDNA cloning. CD100 stimulation significantly enhanced the effects of CD40 on B cell responses. Administration of soluble CD100 markedly accelerated in vivo antigen-specific antibody responses. CD100 receptors with different binding affinities were detected on renal tubular cells (K(d) = approximately 1 x 10(-9)M) and lymphocytes (K(d) = approximately 3 x 10(-7)M). Expression cloning revealed that the CD100 receptor on lymphocytes is CD72, a negative regulator of B cell responsiveness. CD72 thus represents a novel class of semaphorin receptors. CD100 stimulation induced tyrosine dephosphorylation of CD72 and dissociation of SHP-1 from CD72. Our findings indicate that CD100 plays a critical role in immune responses by the novel mechanism of turning off negative signaling by CD72.

Src homology region 2 (SH2) domain-containing phosphatase-1 (SHP-1) is a cytosolic protein tyrosine phosphatase containing two SH2 domains in its NH2 terminus. That immunological abnormalities of the motheaten and viable motheaten mice are caused by mutations in the gene encoding SHP-1 indicates that SHP-1 plays important roles in lymphocyte differentiation, proliferation, and activation. To elucidate molecular mechanisms by which SHP-1 regulates BCR-mediated signal transduction, we determined SHP-1 substrates in B cells using the substrate-trapping approach. When the phosphatase activity-deficient form of SHP-1, in which the catalytic center cysteine (C453) was replaced with serine (SHP-1-C/S), was introduced in WEHI-231 cells, tyrosine phosphorylation of a protein of about 70 kDa was strongly enhanced. Immunoprecipitation and Western blot analyses revealed that this protein is the B cell linker protein (BLNK), also named SH2 domain leukocyte protein of 65 kDa, and that upon tyrosine phosphorylation BLNK binds to SHP-1-C/S in vitro. In vitro kinase assays demonstrated that hyperphosphorylation of BLNK in SHP-1-C/S-expressing cells was not due to enhanced activity of Lyn or Syk. Furthermore, BCR-induced activation of c-Jun NH2-terminal kinase was shown to be significantly enhanced in SHP-1-C/S transfectants. Taken collectively, our results suggest that BLNK is a physiological substrate of SHP-1 in B cells and that SHP-1 selectively regulates c-Jun NH2-terminal kinase activation.

Protein tyrosine phosphatase delta (PTPdelta) is a receptor-type PTP expressed in the specialized regions of the brain including the hippocampal CA2 and CA3, B lymphocytes and thymic medulla. To elucidate the physiological roles of PTPdelta, PTPdelta-deficient mice were produced by gene targeting. It was found that PTPdelta-deficient mice were semi-lethal due to insufficient food intake. They also exhibited learning impairment in the Morris water maze, reinforced T-maze and radial arm maze tasks. Interestingly, although the histology of the hippocampus appeared normal, the magnitudes of long-term potentiation (LTP) induced at hippocampal CA1 and CA3 synapses were significantly enhanced in PTPdelta-deficient mice, with augmented paired-pulse facilitation in the CA1 region. Thus, it was shown that PTPdelta plays important roles in regulating hippocampal LTP and learning processes, and that hippocampal LTP does not necessarily positively correlate with spatial learning ability. To our knowledge, this is the first report of a specific PTP involved in the regulation of synaptic plasticity or in the processes regulating learning and memory.

The immunoreceptor tyrosine-based inhibition motif (ITIM) is found in various membrane molecules such as CD22 and the low-affinity Fc receptor for IgG in B cells and the killer cell-inhibitory receptor and Ly-49 in NK cells. Upon tyrosine phosphorylation at the ITIMs, these molecules recruit SH2 domain-containing phosphatases such as SH2-containing tyrosine phosphatase-1 and negatively regulate cell activity. The B cell surface molecule CD72 carries an ITIM and an ITIM-like sequence. We have previously shown that CD72 is phosphorylated and recruits SH2-containing tyrosine phosphatase-1 upon cross-linking of the Ag receptor of B cells (BCR). However, whether CD72 modulates BCR signaling has not yet been elucidated. In this paper we demonstrate that expression of CD72 down-modulates both extracellular signal-related kinase (ERK) activation and Ca2+ mobilization induced by BCR ligation in the mouse B lymphoma line K46micromlambda, whereas BCR-mediated ERK activation was not reduced by the ITIM-mutated form of CD72. Moreover, coligation with CD72 with BCR reduces BCR-mediated ERK activation in spleen B cells of normal mice. These results indicate that CD72 negatively regulates BCR signaling. CD72 may play a regulatory role in B cell activation, probably by setting a threshold for BCR signaling.

Using CD45-deficient clones from the immature B cell line, WEHI-231, we previously demonstrated that CD45 selectively dephosphorylates the Src-family protein tyrosine kinase Lyn and inhibits its kinase activity. To further define the mechanisms of CD45 action on Lyn, we metabolically labeled Lyn from CD45-positive and -negative WEHI-231 cells and analyzed cyanogen bromide fragments by SDS-PAGE analysis. Phosphoamino acid analysis confirmed that Lyn is tyrosine phosphorylated with little serine or threonine phosphorylation. In CD45-negative cells, two bands at 8.2 and 4.1 kDa were phosphorylated in the absence of B cell Ag receptor (BCR) ligation. The 8.2-kDa band corresponded to a fragment containing the positive regulatory site (Tyr397), as assessed by its size and its phosphorylation in an in vitro kinase assay. The 4.1-kDa band was phosphorylated by COOH-terminal Src kinase, suggesting that it contains the COOH-terminal negative regulatory site (Tyr508). CD45 was also shown to dephosphorylate autophosphorylated Lyn in vitro. Thus, CD45 dephosphorylates not only the negative but also the positive regulatory tyrosine residues of Lyn. Furthermore, coimmunoprecipitations using anti-Igalpha Ab demonstrated that Lyn associated with the resting BCR was constitutively phosphorylated and activated in CD45-negative cells. In the parental cells, both regulatory sites were phosphorylated on BCR ligation. Taken collectively, these results suggest that CD45 keeps both BCR-associated and total cytoplasmic pools of Lyn in an inactive state, and a mechanism by which Lyn is activated by relative reduction of CD45 effect may be operative on BCR ligation.

Signaling events leading to B cell growth or apoptosis are beginning to be unravelled, but detailed information is still lacking. To identify signaling molecules involved in B cell antigen receptor (BCR)-initiated pathways, we used the immature B cell line, WEHI-231, to investigate protein tyrosine phosphatases (PTP) whose expression was modulated by BCR ligation. Among the PTP cloned by reverse transcription-PCR, mRNA expression of the proline-, glutamic acid-, serine- and threonine-rich (PEST) domain phosphatase (PEP) was selectively elevated 3.1-fold within 3 h after anti-IgM antibody stimulation. In contrast, expression of another PEST domain phosphatase, PTP-PEST, was unaffected. Western blot analysis revealed that 71% of PEP was located in the cytosolic fraction, while 29% was in the membrane fraction. To examine the direct contribution made by PEP to BCR-initiated signal transduction, we transfected an antisense PEP cDNA into WEHI-231 cells. Two stable clones were established in which PEP expression was reduced by 34% and 47%, respectively. Strikingly, BCR-mediated inhibition of DNA synthesis was significantly rescued in the clones, and G1 phase cell cycle arrest and apoptosis were almost completely ablated. Considered collectively, these results indicate that PEP is a positive, crucial regulator in determining B cell fate triggered by BCR engagement.

Activation signals of lymphocytes are negatively regulated by the membrane molecules carrying the immunoreceptor tyrosine-based inhibition motif (ITIM). Upon tyrosine phosphorylation, ITIMs recruit SH2-containing phosphatases such as SHP-1, resulting in down-modulation of cell activation. We showed that the cytoplasmic domain of the CD72 molecule carries an ITIM and is associated in vitro with SHP-1 upon tyrosine phosphorylation. Moreover, cross-linking of B cell Ag receptor (BCR) enhances both tyrosine phosphorylation of CD72 and association of CD72 with SHP-1 in B cell line WEHI-231. These results indicate that CD72 recruits SHP-1 upon tyrosine phosphorylation induced by BCR signaling, suggesting that CD72 is a negative regulator of BCR signaling.