Hirofumi Nishizumi

Impaired proliferation of peripheral B cells and indication of autoimmune disease in lyn-deficient mice.

The Src family protein-tyrosine kinase Lyn associates physically with the BCR and has been suggested to play an important role in BCR-mediated signaling. Studies with lyn-/- mice showed that the number of B cells decreased by half in their peripheral tissues. In addition, these B cells do not respond normally to a number of stimuli, including BCR cross-linking and CD40 ligand. Induction of tyrosine phosphorylation on a variety of cellular proteins, such as Vav, Cbl, and HS1, upon BCR cross-linking was also abolished in these B cells. Despite the impaired BCR-mediated signaling, concentrations of IgM and IgA in sera were remarkably elevated, and production of autoantibodies was detected in lyn-/- mice. Histological study showed splenomegaly and enlargement of lymph nodes that became evident with age in the mutant mice. The spleen contained significant number of plasma cells as well as unusual lymphoblast-like cells carrying Mac1 antigen and cytoplasmic IgM. These cells spontaneously secreted a large amount of IgM in vitro. Finally, significant number of lyn-/- mice show glomerulonephritis, an indication of autoimmune disease. From these data, we conclude that Lyn plays a role in signal transduction for not only clonal expansion and terminal differentiation of peripheral B cells but also elimination of autoreactive B cells.

Deletion of the core-H region in mice abolishes the expression of three proximal odorant receptor genes in cis

We have previously reported that a 2.1-kb homology (H) sequence, conserved between mouse and human, regulates the odorant receptor (OR) gene MOR28 in transgenic mice. Here, we narrowed down the essential sequences of the H to a core of 124 bp by using a transient expression system in zebrafish embryos. Transgenic experiments in mice demonstrated that the core-H sequence is sufficient to endow expression of the MOR28 minigene. Deletion and mutation analyses of the core-H region revealed two homeodomain sequences to be essential for the H enhancer activity. Targeted deletion of the core-H abolished expression of three proximal OR genes, MOR28, MOR10, and MOR83, in cis, indicating the presence of another locus control region/enhancer in the downstream region, that regulates four distal OR genes in the same MOR28 cluster. In the heterozygous mice, the H− phenotype of the mutant allele was not rescued by the wild-type H+ allele in trans.

Agonist-Independent GPCR Activity Regulates Anterior-Posterior Targeting of Olfactory Sensory Neurons

G-protein-coupled receptors (GPCRs) are known to possess two different conformations, active and inactive, and they spontaneously alternate between the two in the absence of ligands. Here, we analyzed the agonist-independent GPCR activity for its possible role in receptor-instructed axonal projection. We generated transgenic mice expressing activity mutants of the β2-adrenergic receptor, a well-characterized GPCR with the highest homology to odorant receptors (ORs). We found that mutants with altered agonist-independent activity changed the transcription levels of axon-targeting molecules--e.g., Neuropilin-1 and Plexin-A1--but not of glomerular segregation molecules--e.g., Kirrel2 and Kirrel3--thus causing shifts in glomerular locations along the anterior-posterior (A-P) axis. Knockout and in vitro experiments demonstrated that Gs, but not Golf, is responsible for mediating the agonist-independent GPCR activity. We conclude that the equilibrium of conformational transitions set by each OR is the major determinant of expression levels of A-P-targeting molecules.

Expression and antigenicity of human herpesvirus 8 encoded ORF59 protein in AIDS-associated Kaposi's sarcoma

Human herpesvirus 8 (HHV‐8, Kaposis sarcoma‐associated herpesvirus, KSHV) is a new herpes virus isolated from patients with AIDS‐associated Kaposis sarcoma (AIDS‐KS). The ORF59 protein of HHV‐8 has recently been shown to encode a processivity factor (PF‐8) for HHV‐8‐encoded DNA polymerase. By immunoscreening a cDNA library derived from the HHV‐8‐infected cell line TY‐1, ORF59 antigen was identified in AIDS‐KS patients. Immunoblotting revealed that recombinant ORF59 protein reacted with sera from patients with AIDS‐KS. Enzyme‐linked immunosorbent assay (ELISA) using ORF59‐recombinant protein as the antigen revealed that 7 of 22 (31.8%) AIDS‐KS patients and 6 of 263 (2.2%) Japanese HIV‐negative patients or healthy blood donors were positive for anti‐ORF59 antibodies. Immunohistochemistry using anti‐ORF59 rabbit antibodies revealed that this protein was expressed in some of the tumor cells found in KS tissues and that ORF59 protein was detected in 11 of 22 (50%) AIDS‐KS tissues. In situ hybridization indicated that some of KS tumor cells were positive for HHV‐8 T1.1 mRNA in the same specimen. These data suggest that ORF59 is one of the HHV‐8 encoded antigens in patients with AIDS‐KS and also indicated that viral replication occurred in some of KS tumor cells. J. Med. Virol. 59:346–355, 1999.

Regulation of antigen-receptor gene assembly in hagfish

Variable lymphocyte receptors (VLRs) are antigen receptors in the jawless vertebrates lamprey and hagfish. VLR genes are classified into VLRA and VLRB, and lymphocytes expressing VLRA are T‐cell‐like, whereas those expressing VLRB are B‐cell‐like in the sea lamprey. Diverse VLR genes are assembled somatically in lymphocytes; however, how the assembly is regulated is still largely unknown. Here, we analyse VLR gene assembly at the single‐cell level in the inshore hagfish (Eptatretus burgeri). Each lymphocyte assembles and transcribes only one type of VLR gene, either VLRA or VLRB. In general, monoallelic assembly of VLR was observed, but diallelic assembly was found in some cases—in many of which, one allele was functional and the other was defective. In fact, all VLR‐assembled lymphocytes contained at least one functional VLR gene. Together, these results indicate a feedback inhibition of VLR assembly and selection of VLR‐positive lymphocytes.

Impairment of N-methyl-D-aspartate receptor-controlled motor activity in LYN-deficient mice.

The N-methyl-D-aspartate (NMDA) receptor, an ionotropic glutamate receptor, is implicated in motor activity that is regulated in the striatum and nucleus accumbens of the brain. A Src family kinase Lyn is highly expressed in striatum, cortex, thalamus, and cerebellum in the brain. Here we show that spontaneous motor activity is suppressed in lyn-/- mice. S.c. injection of methylphenidate, which causes accumulation of dopamine in synapses, reveals that dopaminergic pathway is normal in lyn-/- mice. After blocking the NMDA receptor, motor activity of lyn-/- mice increased to the same level as that of wild type mice. Therefore, the NMDA receptor-mediated signaling is enhanced in lyn-/- mice, indicating that Lyn regulates the NMDA receptor pathway negatively. Intriguingly, the activity of protein kinase C (PKC), an enzyme regulated downstream of NMDA receptors, is increased in lyn-/- mice. The present data suggest that the NMDA receptor signal that is enhanced in the absence of Lyn suppresses the motor activity, probably through inhibition of dopaminergic pathway at striatum. We conclude that Lyn contributes to coordination of motor activity through regulation of the NMDA pathway. It appears that this negative regulation involves suppression of downstream signaling of NMDA receptor such as those mediated by PKC.

A major allogenic leukocyte antigen in the agnathan hagfish

All vertebrates, from jawless fish to mammals, possess adaptive immune systems that can detect and inactivate non-self-antigens through a vast repertoire of antigen receptors. Unlike jawed vertebrates, the hagfish utilizes variable lymphocyte receptors (VLRs) that are unrelated to immunoglobulin molecules but are diversified by copy-choice gene conversion mechanism. Here, we report that hagfish VLRs react with allogenic leukocyte antigens but not with self-antigens. We found that a highly polymorphic membrane protein, NICIR3, is recognized by VLRs as an allogenic leukocyte antigen (ALA). In a serological cross-reactivity test, a close correlation was observed between the amino acid differences in the protein sequences and the VLR cross-reactivities. This leukocyte antigen was predominantly expressed in phagocytic leukocytes, where it was associated with phagocytosed protein antigens. These findings suggest that a polymorphic leukocyte antigen, NICIR3/ALA, plays a pivotal role in jawless vertebrate adaptive immunity.

Developmental regulation of neural map formation in the mouse olfactory system

In the mouse olfactory system, various odorants are detected by approximately 1000 different odorant receptors (ORs) expressed in the olfactory sensory neurons (OSNs). It is well established that each OSN expresses only one functional OR gene in a monoallelic manner. Furthermore, OSN axons expressing the same OR converge to a set of glomeruli in the olfactory bulb (OB). During embryonic development, a coarse map is formed by the combination of two genetically programmed processes. One is OR‐independent axonal projection along the dorsal‐ventral (D‐V) axis, and the other is OR‐dependent projection along the anterior‐posterior (A‐P) axis. D‐V projection is regulated by the anatomical location of OSNs within the olfactory epithelium (OE), whereas A‐P projection is instructed by expressed OR molecules using cyclic adenosine monophosphate (cAMP) signals. After birth, the map is further refined in an activity‐dependent manner by its conversion from a continuous to a discrete map through segregation of glomerular structures. Here, we summarize recent progress from our laboratory in understanding neural map formation in the mouse olfactory system.

Neuropilin-2 is required for the proper targeting of ventral glomeruli in the mouse olfactory bulb

Recent evidence shows that olfactory sensory neurons expressing a given odorant receptor (OR) are not necessarily confined to one of four zones, rather arranged in an overlapping manner in the olfactory epithelium (OE). In this study, in situ hybridization of OE sections with the OR probes indicated that the OR genes, the mRNAs of which were detected in an array of glomeruli on olfactory bulb (OB) along the anterodorsal/posteroventral (AD/PV) axis, are expressed in subareal zones within the most ventral zone, zone 4, along the dorsomedial/ventrolateral (DM/VL) axis. We also found that Neuropilin-2 (Nrp2) is expressed in a DM-low to VL-high gradient within zone 4 of OE. Furthermore, in Nrp2 mutant mice, we observed multiple glomeruli for zone 4 ORs in OB. These results suggest that the graded expression of Nrp2 in OE is required for the proper targeting of ventral glomeruli along the AD/PV axis in OB.

BET, a novel neuronal transmembrane protein with multiple EGF-like motifs

Using the signal sequence trap method, we have identified a cDNA clone coding for a type I transmembrane protein, BET, with 10 epidermal growth factor (EGF) motifs in the extracellular domain. In situ hybridization revealed that the bet mRNA is specifically expressed in the mitral/tufted cells in the olfactory bulb, Purkinje cells in the cerebellum, and pyramidal cells in the hippocampus. Using polyclonal antibodies, we have demonstrated that the BET protein is highly glycosylated and is localized in patches in the dendrites and in the somata of neurons. Since the predicted structure of BET shares many similarities with the Notch ligands, this novel protein may play crucial roles in establishing the neuronal networks in the olfactory system, cerebellum, and hippocampus. BET is the first transmembrane protein containing only multiple EGF-like repeats specifically expressed in the brain.