The aggregation of high affinity IgE receptors (Fcɛ receptor I [FcɛRI]) on mast cells is potent stimulus for the release of inflammatory and allergic mediators from cytoplasmic granules. However, the molecular mechanism of degranulation has not yet been established. It is still unclear how FcɛRI-mediated signal transduction ultimately regulates the reorganization of the cytoskeleton and how these events lead to degranulation. Here, we show that FcɛRI stimulation triggers the formation of microtubules in a manner independent of calcium. Drugs affecting microtubule dynamics effectively suppressed the FcɛRI-mediated translocation of granules to the plasma membrane and degranulation. Furthermore, the translocation of granules to the plasma membrane occurred in a calcium-independent manner, but the release of mediators and granule–plasma membrane fusion were completely dependent on calcium. Thus, the degranulation process can be dissected into two events: the calcium-independent microtubule-dependent translocation of granules to the plasma membrane and calcium-dependent membrane fusion and exocytosis. Finally, we show that the Fyn/Gab2/RhoA (but not Lyn/SLP-76) signaling pathway plays a critical role in the calcium-independent microtubule-dependent pathway.

Mucosal epithelial M cells provide an efficient portal of entry for microorganisms. Initially defined by their irregular microvilli and abundant transcytotic channels in the avian bursa of Fabricius, M cells also are found in the lymphoid follicle-associated epithelium of the mammalian appendix, Peyer's patches, and other mucosal surfacelymphoid interfaces. We describe here a previously unrecognized cathelicidin gene in chickens, chCATH-B1, that is expressed exclusively in the epithelium of the bursa of Fabricius. Like the mature peptides of previously identified cathelicidins, the carboxyl-terminal peptide of chCATH-B1 has broad antimicrobial activity against Gram-positive and Gram-negative bacteria. chCATH-B1 expression is restricted to the secretory epithelial cell neighbors of the M cells, whereas its mature peptide is transported to become concentrated on the fibrillar network surrounding basolateral surfaces of the M cells that overlie the bursal lymphoid follicles. We conclude that chCATH-B1 is well placed to serve a protective antimicrobial role at the M cell gateway.antimicrobial peptides ͉ follicle-associated epithelium ͉ innate immunity ͉ bursa of Fabricius

Commitment of the germ cell lineage during embryogenesis depends on zygotic gene expression in mammals, but little is known about the signaling molecules required for germ cell formation. Here we show that the intracellular signaling molecule SMAD1, acting downstream of bone morphogenetic protein (BMP) receptors, is required for the commitment of germ cell lineage from epiblast in early mouse embryos. Smad1 homozygous mutant embryos (Smad1-/-) were generated by in-frame insertion of lacZ gene into an exon of the Smad1 gene. Most of the Smad1-/- embryos contained no primordial germ cells (PGCs) and had short allantois, while histological analysis and in situ hybridization for the mesoderm marker genes revealed that early mesoderm induction was normal in those embryos. Smad1 expression was observed in epiblast and in visceral endoderm during gastrulation, while only a few alkaline phosphatase-positive PGCs at 7.5 and 8.5 days post coitum (E7.5 and E8.5) expressed Smad1. Phosphorylated SMAD proteins were localized in the proximal region of epiblast at E6.0-6.5, where the progenitors of PGCs and of allantois reside. Single-cell reverse transcription-polymerase chain reaction analysis revealed that the expression of Smad1, -5 and -8 were sporadic and mutually independent in proximal epiblast cells. We also found that BMP4-induced differentiation of PGCs from epiblast in vitro was fully dependent on the existence of phosphorylated SMAD1. These results indicate that SMAD1 signaling possesses a critical and non-redundant function in the initial commitment of the germ cell lineage.

Pre-B-cell leukemia spontaneously develops in BLNK-deficient mice, and pre-Bcell acute lymphoblastic leukemia cells in children often lack BLNK protein expression, demonstrating that BLNK functions as a tumor suppressor. However, the mechanism by which BLNK suppresses pre-B-cell leukemia, as well as the identification of other genetic alterations that collaborate with BLNK deficiency to cause leukemogenesis, are still unknown. Here, we demonstrate that the JAK3/STAT5 signaling pathway is constitutively activated IntroductionIn early B-cell development, successful rearrangement of the immunoglobulin (Ig) heavy (H) chain gene in progenitor B cells results in surface expression of H chain in the form of a complex with VpreB and 5, called the pre-B-cell receptor (pre-BCR), resulting in differentiation to the pre-B-cell stage. Transient surface expression of the pre-BCR triggers rapid cell-cycle progression, thereby forming a large pre-B-cell population, and ultimately promoting development toward the small pre-B-cell and immature B-cell stages. 1,2 Pre-B cells in the absence of signals derived from the pre-BCR undergo apoptotic cell death. 3 Signal transduction from the pre-BCR requires recruitment and activation of the Syk tyrosine kinase. 4 Activated Syk phosphorylates several downstream signaling elements, including BLNK (also known as SLP-65 or BASH).BLNK is a pivotal adapter protein in signal transduction from the pre-BCR and BCR. BLNK contains multiple tyrosine phosphorylation sites that provide binding sites for key signaling proteins such as PLC␥, Btk, and Vav. 5 BLNK gene mutations cause a complete block in B-cell development at the pro-B-cell to pre-Bcell transition in humans. 6 In BLNK-null mutant mice the developmental block is partial, resulting in the accumulation of pre-BCR ϩ large pre-B cells in the bone marrow and a reduction of mature B cells in the periphery. 7 We and others previously reported that 5% to 10% of BLNK Ϫ/Ϫ mice spontaneously develop pre-B-cell leukemia at 4 to 20 weeks of age. [7][8][9] Pre-B-cell-derived acute lymphoblastic leukemia (pre-B-ALL) is the most common type of leukemia in children. 10 Interestingly, one study reported that 50% of the pediatric B-ALL cases they investigated had lost BLNK protein expression, 11 although other studies reported a lower frequency. 12,13 Thus, it has been proposed that BLNK functions as a tumor suppressor, but the molecular mechanisms by which it exerts tumor suppressor activity are still unknown. Because tumorigenesis is a multistep process requiring sequential changes in various genes, 14 it is unlikely that BLNK deficiency is sufficient to initiate leukemogenesis.Combined deficiency of BLNK and Btk results in a more severe developmental block at the pre-B-cell stage 15 and a higher incidence of pre-B-cell leukemia compared with mice deficient in either gene alone, [7][8][9]16 suggesting that the developmental block is one of the tumor-promoting factors. However, mice that cannot express the pre-BCR, such as MT or RAG-deficient mice, exhibi...

The pre-B cell receptor triggers expansion and differentiation of pre-B cells (the pre-B cell transition), as well as inhibition of V(H) to DJ(H) recombination (allelic exclusion). The latter also accounts for counter-selection of pro-B cells expressing Dmu protein (Dmu selection). However, the signaling pathways responsible for these events remain poorly defined. Here we show complete arrest of B cell development at the pre-B cell transition in BASH/CD19 double mutant mice, indicating partial redundancy of the two B cell-specific adaptors. Allelic exclusion remained intact in the double mutant mice, whereas Dmu selection was abolished in BASH mutant mice. Thus, distinct signals are required for these events. In addition, both mutant mice succumbed to pre-B cell leukemia, indicating that BASH and CD19 contribute to tumor suppression.