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Significance Stroke is the second leading cause of death and the most frequent cause of disability in adults. After stroke, most ischemic neurons die and a few neurons live, leading to brain dysfunction; yet, genes involved in both neuronal survival and death remain poorly understood. Here, we found that the activity-dependent transcription factor Npas4 is essential for acquisition of neuronal tolerance to ischemia. Moreover, a systematic search for Npas4-downstream genes identified Gem, which encodes Ras-related small GTPase that mediates neuroprotective effects of Npas4. Gem suppresses the membrane localization of voltage-gated Ca2+ channels to inhibit excess Ca2+ influx, thereby protecting neurons from excitotoxic death. Our findings suggest that Gem expression via Npas4 promotes neuroprotection and neuroplasticity in injured and healthy brains, respectively., Ischemic stroke, which results in loss of neurological function, initiates a complex cascade of pathological events in the brain, largely driven by excitotoxic Ca2+ influx in neurons. This leads to cortical spreading depolarization, which induces expression of genes involved in both neuronal death and survival; yet, the functions of these genes remain poorly understood. Here, we profiled gene expression changes that are common to ischemia (modeled by middle cerebral artery occlusion [MCAO]) and to experience-dependent activation (modeled by exposure to an enriched environment [EE]), which also induces Ca2+ transients that trigger transcriptional programs. We found that the activity-dependent transcription factor Npas4 was up-regulated under MCAO and EE conditions and that transient activation of cortical neurons in the healthy brain by the EE decreased cell death after stroke. Furthermore, both MCAO in vivo and oxygen-glucose deprivation in vitro revealed that Npas4 is necessary and sufficient for neuroprotection. We also found that this protection involves the inhibition of L-type voltage-gated Ca2+ channels (VGCCs). Next, our systematic search for Npas4-downstream genes identified Gem, which encodes a Ras-related small GTPase that mediates neuroprotective effects of Npas4. Gem suppresses the membrane localization of L-type VGCCs to inhibit excess Ca2+ influx, thereby protecting neurons from excitotoxic death after in vitro and in vivo ischemia. Collectively, our findings indicate that Gem expression via Npas4 is necessary and sufficient to promote neuroprotection in the injured brain. Importantly, Gem is also induced in human cerebral organoids cultured under an ischemic condition, revealing Gem as a new target for drug discovery.

The filament perforation model (FPM) in mice is becoming increasingly popular to elucidate the molecular pathogenesis of neuronal injury after subarachnoid hemorrhage (SAH). We evaluated brain MRI in a mouse FPM. A total of 28 male C57Bl/6J mice were used. Seventeen animals underwent SAH induction by FPM. In two animals, transient middle cerebral artery occlusion (MCAo) was induced. Nine mice served as controls. T1-weighted images (T1WI), T2-weighted images (T2WI), T2(∗)-weighted images (T2*WI) and apparent diffusion coefficient maps were acquired at day 0 and at various time points following SAH (range: day 1-6 after SAH). Cerebral blood flow (CBF) analysis by (14)C-iodoamphetamine ((14)C-IMP) autoradiography was conducted in nine animals. Hemorrhage could be best confirmed using T2*WI. The degree of hemorrhage varied. All animals evaluated for ⩾2days were hydrocephalic, which was best seen on T2WI. T2-hyperintensity of the corpus callosum and external capsule, indicating white matter (WM) injury, was present after SAH. Ventricle and WM injury volumes were statistically significantly higher at day 3 compared to day 0. Territorial ischemia was detectable in MCAo but not in SAH. Markedly hypointense cortical veins were visible in the hyperacute and delayed phase after SAH on T2*WI. The (14)C-IMP analysis indicated decreased CBF after SAH. MRI is feasible and useful in evaluating pathophysiological changes over time. T2*WI seems best for SAH detection and grading. The chronological change of hydrocephalus and WM injury could be analyzed. T2*WI illustrated specific signal changes of cortical veins, possibly caused by increased oxygen extraction fraction due to decreased CBF.

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.

The Wilms' tumor gene WT1 is overexpressed in most of human leukemias regardless of disease subtypes. To characterize the expression pattern of WT1 during normal and neoplastic hematopoiesis, we generated a knock-in reporter green fluorescent protein (GFP) mouse (WT1(GFP/+)) and assayed for WT1 expression in normal and leukemic hematopoietic cells. In normal hematopoietic cells, WT1 was expressed in none of the long-term (LT) hematopoietic stem cells (HSC) and very few (1%) of the multipotent progenitor cells. In contrast, in murine leukemias induced by acute myeloid leukemia 1 (AML1)/ETO+TEL/PDGFbetaR or BCR/ABL, WT1 was expressed in 40.5 or 38.9% of immature c-kit(+)lin(-)Sca-1(+) (KLS) cells, which contained a subset, but not all, of transplantable leukemic stem cells (LSCs). WT1 expression was minimal in normal fetal liver HSCs and mobilized HSCs, both of which are stimulated for proliferation. In addition, overexpression of WT1 in HSCs did not result in proliferation or expansion of HSCs and their progeny in vivo. Thus, the mechanism by which expansion of WT1-expressing cells occurs in leukemia remains unclear. Nevertheless, our results demonstrate that the WT1(GFP/+) mouse is a powerful tool for analyzing WT1-expressing cells, and they highlight the potential of WT1, as a specific therapeutic target that is expressed in LSCs but not in normal HSCs.

Enantiomeric pairs of mirror-image molecular structures are difficult to resolve by instrumental analyses. The human olfactory system, however, discriminates (−)-wine lactone from its (+)-form rapidly within seconds. To gain insight into receptor coding of enantiomers, we compared behavioural detection and discrimination thresholds of wild-type mice with those of ΔD mice in which all dorsal olfactory receptors are genetically ablated. Surprisingly, wild-type mice displayed an exquisite “supersensitivity” to enantiomeric pairs of wine lactones and carvones. They were capable of supersensitive discrimination of enantiomers, consistent with their high detection sensitivity. In contrast, ΔD mice showed selective major loss of sensitivity to the (+)-enantiomers. The resulting 108-fold differential sensitivity of ΔD mice to (−)- vs. (+)-wine lactone matched that observed in humans. This suggests that humans lack highly sensitive orthologous dorsal receptors for the (+)-enantiomer, similarly to ΔD mice. Moreover, ΔD mice showed >1010-fold reductions in enantiomer discrimination sensitivity compared to wild-type mice. ΔD mice detected one or both of the (−)- and (+)-enantiomers over a wide concentration range, but were unable to discriminate them. This “enantiomer odour discrimination paradox” indicates that the most sensitive dorsal receptors play a critical role in hierarchical odour coding for enantiomer identification.

Genomic analysis was performed for the murine odorant receptor (OR) genes. The MOR28 cluster on chromosome 14 was extensively studied. It contains six OR genes, MOR28, 10, 83, 29A, 29B and 30. The human homolog of this cluster is located on the human chromosome 14, and contains five OR genes, HOR28/10, 83, 29A, 29B and 30. Sequence comparison of these OR gene paralogs and orthologs suggests that the coding homologies are accounted for not only by recent gene duplication, but also by gene conversion among the coding sequences within the cluster. A possible role of gene conversion in the olfactory system is discussed in the context of the olfactory map.

The Wilms' tumor gene WT1 is overexpressed in leukemia and solid tumors and has an oncogenic role in leukemogenesis and tumorigenesis. However, precise regulatory mechanisms of WT1 overexpression remain undetermined. In the present study, microRNA-125a (miR-125a) was identified as a miRNA that suppressed WT1 expression via binding to the WT1-3'UTR. MiR-125a knockout mice overexpressed WT1, developed myeloproliferative disorder (MPD) characterized by expansion of myeloid cells in bone marrow (BM), spleen and peripheral blood, and displayed urogenital abnormalities. Silencing of WT1 expression in hematopoietic stem/progenitor cells of miR-125a knockout MPD mice by short-hairpin RNA inhibited myeloid colony formation in vitro. Furthermore, the incidence and severity of MPD were lower in miR-125a (-/-) mice than in miR-125a (+/-) mice, indicating the operation of compensatory mechanisms for the complete loss of miR-125a. To elucidate the compensatory mechanisms, miRNA array was performed. MiR-486 was occasionally induced in compete loss of miR-125a and inhibited WT1 expression instead of miR-125a, resulting in the cancellation of MPD occurrence. These results showed for the first time the post-transcriptional regulatory mechanisms of WT1 by both miR-125a and miR-486 and should contribute to the elucidation of mechanisms of normal hematopoiesis and kidney development.

Summary Sensory experience regulates the development of various brain structures, including the cortex, hippocampus, and olfactory bulb (OB). Little is known about how sensory experience regulates the dendritic spine development of OB interneurons, such as granule cells (GCs), although it is well studied in mitral/tufted cells. Here, we identify a transcription factor, Npas4 , which is expressed in OB GCs immediately after sensory input and is required for dendritic spine formation. Npas4 overexpression in OB GCs increases dendritic spine density, even under sensory deprivation, and rescues reduction of dendrite spine density in the Npas4 knockout OB. Furthermore, loss of Npas4 upregulates expression of the E3-ubiquitin ligase Mdm2 , which ubiquitinates a microtubule-associated protein Dcx. This leads to reduction in the dendritic spine density of OB GCs. Together, these findings suggest that Npas4 regulates Mdm2 expression to ubiquitinate and degrade Dcx during dendritic spine development in newborn OB GCs after sensory experience.

During the development of lymphocytes, expression of the Ig genes is strictly regulated in a tissue-specific manner and in a time-ordered fashion. We have previously shown that the PU.1 binding motif in the Igkappa 3' enhancer (kappaE3') and a novel Ets family protein other than PU.1 may be possibly involved in the control of V(kappa)-J(kappa) joining. In the attempt to isolate the novel Ets family protein, we have screened cDNA libraries with the yeast one-hybrid method and identified a new PU.1-related factor, Prf. This novel Ets family protein is shown to interact with the PU.1 binding sequences in various promoters and enhancers, including kappaE3'. It was found that expression of the prf gene is predominant in the B-lineage cells, with the exception of immature B cells. Since Prf does not exhibit functions of transcriptional activity, this novel protein may act as an antagonist against other Ets family proteins, e.g. PU.1 and Spi-B. Possible roles of Prf with respect to the B cell differentiation are discussed.

We have characterized genomic structure and transcription of a murine odorant receptor (OR) gene, MOR23. This gene has two distinct transcriptional initiation regions, which are differentially utilized in the olfactory and testicular cells. We have found that the MOR23 gene has an intron within the 5'-untranslated region upstream of the coding sequence. In olfactory cells, transcription of the gene is initiated in the region upstream of this intron, while in testicular cells, transcription starts within the intron. Differential initiation of transcription is discussed in the context of tissue-specific expression of the OR genes.

Visual Abstract, The glomerular map in the olfactory bulb (OB) is the basis for odor recognition. Once established during development, the glomerular map is stably maintained throughout the life of an animal despite the continuous turnover of olfactory sensory neurons (OSNs). However, traumatic damage to OSN axons in the adult often leads to dysosmia, a qualitative and quantitative change in olfaction in humans. A mouse model of dysosmia has previously indicated that there is an altered glomerular map in the OB after the OSN axon injury; however, the underlying mechanisms that cause the map distortion remain unknown. In this study, we examined how the glomerular map is disturbed and how the odor information processing in the OB is affected in the dysosmia model mice. We found that the anterior–posterior coarse targeting of OSN axons is disrupted after OSN axon injury, while the local axon sorting mechanisms remained. We also found that the connectivity of mitral/tufted cell dendrites is reduced after injury, leading to attenuated odor responses in mitral/tufted cells. These results suggest that existing OSN axons are an essential scaffold for maintaining the integrity of the olfactory circuit, both OSN axons and mitral/tufted cell dendrites, in the adult.

Clonogenic multiple myeloma (MM) cells reportedly lacked expression of plasma cell marker CD138. It was also shown that CD19(+) clonotypic B cells can serve as MM progenitor cells in some patients. However, it is unclear whether CD138-negative clonogenic MM plasma cells are identical to clonotypic CD19(+) B cells. We found that in vitro MM colony-forming cells were enriched in CD138(-)CD19(-)CD38(++) plasma cells, while CD19(+) B cells never formed MM colonies in 16 samples examined in this study. We next used the SCID-rab model, which enables engraftment of human MM in vivo. CD138(-)CD19(-)CD38(++) plasma cells engrafted in this model rapidly propagated MM in 3 out of 9 cases, while no engraftment of CD19(+) B cells was detected. In 4 out of 9 cases, CD138(+) plasma cells propagated MM, although more slowly than CD138(-) cells. Finally, we transplanted CD19(+) B cells from 13 MM patients into NOD/SCID IL2Rγc(-/-) mice, but MM did not develop. These results suggest that at least in some MM patients CD138-negative clonogenic cells are plasma cells rather than B cells, and that MM plasma cells including CD138(-) and CD138(+) cells have the potential to propagate MM clones in vivo in the absence of CD19(+) B cells.

Sensory input has been shown to regulate development in a variety of species and in various structures, including the retina, cortex, and olfactory bulb (OB). Within the mammalian OB specifically, the development of dendrites in mitral/tufted cells is well known to be odor-evoked activity dependent. However, little is known about the developmental role of sensory input in the other major OB population of the GABAgenic interneurons, such as granule cells and periglomerular cells. Here, we identified, with DNA microarray andin situhybridization screenings, a trophoblast glycoprotein gene,5T4, whose expression in a specific subtype of OB interneurons is dependent on sensory input. 5T4 is a type I membrane protein, whose extracellular domain contains seven leucine-rich repeats (LRR) flanked by characteristic LRR-N-flanking and C-flanking regions, and a cytoplasmic domain.5T4overexpression in the newborn OB interneurons facilitated their dendritic arborization even under the sensory input-deprived condition. By contrast, both5T4knockdown with RNAi and5T4knockout with mice resulted in a significant reduction in the dendritic arborization of 5T4+granule cells. Further, we identified the amino acid sequence in the 5T4 cytoplasmic domain that is necessary and sufficient for the sensory input-dependent dendritic shaping of specific neuronal subtypes in the OB. Thus, these results demonstrate that 5T4 glycoprotein contributes in the regulation of activity-dependent dendritic development of interneurons and the formation of functional neural circuitry in the OB.

Murine embryonal carcinoma (EC) P19 cells, a tissue culture model of early embryonic development, failed to produce cytokines, such as interleukin-3 (IL-3), IL-4, granulocytemacrophage colony stimulating factor (GM-CSF) and interferon-beta (IFN-beta) at the mRNA level. Differentiation induced by retinoic acid (RA) released this repression to produce some cytokines. GM-CSF and IFN-beta genes were expressed in response to PMA/A23187, poly(I):poly(C), IL-1 alpha, forskolin, or LPS stimulation in differentiated P19 cells, whereas IL-3 and IL-4 genes were not expressed. To elucidate the mechanism of the GM-CSF gene induction after differentiation, we transfected a series of 5' deletion mutants of the mouse GM-CSF promoter fused to the bacterial CAT gene. The 740-bp fragment of the 5'-flanking region mediated the positive response. Deletion analysis revealed that the 5' boundary region of the DNA element required for activation lies between positions -95 and -84 and the region upstream of position -95 appears inhibitory. These results indicate that the maturation of the transcriptional machinery after differentiation results in the activation of the GM-CSF gene.

In a previous report,we reported that the induction of GM-GSF gene in differentiated P19 cells results from the maturation of the transcriptional machinery. Here, we identified a cis-DNA element which confers the activation of GM-CSF gene in response to PMA/A23 187 stimulation in differentiated state. Analysis of the 5′-flanking region between −113 and −60 revealed two elements responsible for promotion and one for inhibition, and the overall effects led to the activation of GM-CSF gene mainly through the sequence between −95 and −73. Using the oligonucleotide between −94 and −73 as a probe in gel retardation assays, we identified a DNA-binding protein, NF-GM-P19, the binding activity of which was induced after differentiation in response to PMA/A23 187 stimulation. These results indicate that the induction of GM-CSF gene after differentiation results from the maturation of the transcriptional machinery which recognizes the sequence between −95 and −73.

Expression of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene in T cells is activated by the combination of phorbol ester (phorbol myristate acetate) and calcium ionophore (A23187), which mimic antigen stimulation through the T-cell receptor. We have previously shown that a fragment containing bp -95 to +27 of the mouse GM-CSF promoter can confer inducibility to reporter genes in the human Jurkat T-cell line. Here we use an in vitro transcription system to demonstrate that a cis-acting element (positions -54 to -40), referred to as CLE0, is a target for the induction signals. We observed induction with templates containing intact CLE0 but not with templates with deleted or mutated CLE0. We also observed that two distinct signals were required for the stimulation through CLE0, since only extracts from cells treated with both phorbol myristate acetate and A23187 supported optimal induction. Stimulation probably was mediated by CLE0-binding proteins because depletion of these proteins specifically reduced GM-CSF transcription. One of the binding factors possessed biochemical and immunological features identical to those of the transcription factor AP1. Another factor resembled the T-cell-specific factor NFAT. The characteristics of these two factors are consistent with their involvement in GM-CSF induction. The presence of CLE0-like elements in the promoters of interleukin-3 (IL-3), IL-4, IL-5, GM-CSF, and NFAT sites in the IL-2 promoter suggests that the factors we detected, or related factors that recognize these sites, may account for the coordinate induction of these genes during T-cell activation.

Since the discovery of odorant receptors (ORs) in rodents, most ORs have remained orphan receptors. Even for deorphanized ORs in vitro, their in vivo properties are largely unknown. Here, we report odor response profiles of two highly homologous mouse ORs, MOR29A and MOR29B, both in vivo and in vitro. The BAC transgenic mouse was generated, in which olfactory sensory neurons (OSNs) expressing the transgenes MOR29A and MOR29B were differently tagged with IRES-gapECFP and IRES-gapEYFP, respectively. MOR29A- and MOR29B-expressing OSN axons converged on separate but nearby loci on the dorsal surface of the olfactory bulb (OB). Optical imaging of intrinsic signals in the OB identified five different phenyl ethers as candidate ligands for MOR29B. Based on in vitro calcium imaging with the isolated OSNs and luciferase assay with heterologous cells, only guaiacol and vanillin were found to be potent agonists for MOR29A and MOR29B. Because of its accessible glomerular locations in the dorsal OB and defined odor response profiles both in vivo and in vitro, the MOR29A/29B tagging mouse will serve as an excellent tool for studying both odor-signal processing and neural circuitry in the OB.

Activation of T cells is initiated by the recognition of antigen on antigen presenting cells to exert the effector functions in immune and inflammatory responses. Two types of helper T cell (Th) clones (Th1 and Th2) are defined on the basis of different patterns of cytokine (lymphokine) secretion. They determine the outcome of an antigenic response toward humoral or cell-mediated immunity. Although lymphokine genes are coordinately regulated upon antigen stimulation, they are regulated by the mechanisms common to all as well as those which are unique to each gene. For most lymphokine genes, a combination of phorbol esters (phorbol 12-myristate 13 acetate, PMA) and calcium ionophores (A23187) is required for their maximal induction. Yet phorbol ester alone or calcium ionophore alone produce several lymphokines. The production of the granulocyte-macrophage colony stimulating factor (GM-CSF) is completely dependent on the two signals. We have previously found a cis-acting region spanning the GM-CSF promoter region (positions -95 to +27) that confers inducibility to reporter genes in transient transfection assays. Further analysis identified three elements required for efficient induction, referred to as GM2, GC-box and conserved lymphokine element (CLE0). GM2 defines a binding site for protein(s) whose binding is inducible by PMA. One protein, NF-GM2 is similar to the transcription factor NF-kB. GC-box is a binding site for constitutively bound proteins. CLEO defines a binding site for protein(s) whose optimum binding is stimulated by PMA and A23187. Viral trans-activators such as Tax (human T cell leukemia virus-1, HTLV-1) and E2 (bovine papilloma virus, BPV) proteins are other agents which activate lymphokine gene expression by bypassing T cell receptor (TCR) mediated signaling. The trans-activation domain of E2 and Tax is interchangeable although they have no obvious sequence homology between them. The viral trans-activators appear to target specific DNA binding protein such as NF-kB and Sp1 to cis-acting DNA site and promote lymphokine gene expression without TCR-mediated stimulation.

The upstream region of the mouse granulocyte macrophage colony stimulating factor (GM-CSF) gene between positions -95 and -73 is required for phorbol-12-myristate-13-acetate (PMA)- and calcium ionophore (A23187)-inducible transcriptional activity in vivo. To study the mechanism of GM-CSF gene activation in T cells, nuclear extracts from non-stimulated and PMA/A23187-stimulated Jurkat cells were used in DNA binding assays. DNA mobility shift assays with wild type and mutant oligonucleotides revealed that this region contains at least two DNA binding motifs. One is the binding sequence GGTAGTTCCCC (positions -91 to -81), recognized by NF-GM2 (nuclear factor of GM-CSF 2), and the other is a GC-rich sequence (GC-box). NF-GM2, induced in Jurkat cells by PMA/A23187 stimulation, effectively competed with DNA containing the NF-kappa B binding sequence, suggesting that it has NF-kappa B-like activity. By UV cross-linking analysis, three cross-linked bands, corresponding to Mr 165, 70, and 60 kd for NF-GM2, and 110 and 130 kd for A1 and A2, respectively, were identified. Transfection experiments showed that activation of the GM-CSF gene in response to PMA/A23187 stimulation was abolished by point mutations in either the GC-box or the NF-GM2 site; these mutations also abolished binding of the respective proteins. These results indicate that constitutive (GC-box binding factor) and inducible (NF-GM2) factors regulate the GM-CSF promoter co-operatively in a PMA/A23187-inducible manner in vivo.

Wild-type Wilms tumor gene WT1 is highly expressed not only in hematopoietic malignancies, including leukemia and myelodysplastic syndromes (MDS), but also in various kinds of solid tumors. Human cytotoxic T lymphocytes (CTLs) which could specifically lyse WT1-expressing tumor cells with HLA class I restriction were generated in vitro. We have also demonstrated that mice immunized with the WT1 peptide or WT1 cDNA rejected challenges by WT1-expressing tumor cells and survived with no signs of auto-aggression to normal organs which physiologically expressed WT1 in prophylactic and therapeutic models. Furthermore, we and others detected IgM and IgG WT1 antibodies in the patients with hematopoietic malignancies, indicating that WT1 protein was highly immunogenic, and that immunoglobulin class-switch-inducing WT1- specific cellular immune responses were elicited in the patients. CD8+ WT1-specific CTLs were also detected in peripheral blood or tumor-draining lymph nodes of cancer patients. These results provided us with the rationale for elicitation of CTL responses targeting the WT1 product for cancer immunotherapy. On the basis of the findings mentioned above, we performed a phase I clinical trial of WT1 peptide cancer vaccine for the patients with malignant neoplasms. These results strongly suggested that WT1 peptide cancer vaccine had efficacy in the clinical setting, because clinical responses, including reduction of leukemic blast cells or regression of tumor masses, were observed after the WT1 vaccination in patients with hematopoietic malignancies or solid cancers. The power of TAA-derived cancer vaccine may be enhanced by combination with stronger adjuvants, helper peptide, or conventional treatments such as molecular-target-based drugs.

Vertebrate odorant receptor (OR) genes are divided phylogenetically into two distinct classes: the fish-like class I and the terrestrial-specific class II. In the present study, we systematically analysed mouse class I OR genes (42 subfamilies) to elucidate the expression profiles in the olfactory epithelium (OE) and the projection sites of their olfactory sensory neurons (OSNs) in the olfactory bulb (OB). In situ hybridization (ISH) revealed that most class I OR genes (36 subfamilies) were expressed in the dorso-medial zone (zone 1) of the OE. Furthermore, there appeared to be no significant differences in the distributions of OSNs expressing class I genes within zone 1. These results indicate that there is a clear boundary between zone 1 and non-zone 1 areas in the OE. Some class I ORs are known to possess ligand specificity for aliphatic acids, aldehydes and alcohols. Our ISH analysis has revealed that OSNs expressing the class I ORs in zone 1 tend to converge their axons on a cluster of glomeruli in an antero-dorsal domain that is assumed to be involved in responses to the aliphatic compounds on the OB.

In rodents, the olfactory epithelium (OE) can be divided into four topographically distinct zones, and each member of the odorant receptor (OR) gene family is expressed only in one particular zone. To study the functional significance of the zonal structure of the OE, we searched for genes expressed in a zone-specific manner by using the differential display method. Among the clones isolated from the rat OE, we characterized a novel olfactory protein termed O-MACS, a member of the medium-chain acyl-CoA synthetase family. The o-macs gene encodes a protein of 580 amino acids, sharing 56–63% identity with other MACS family proteins. RT-PCR analysis demonstrated that the o-macs gene is expressed only in the OE, unlike other MACS family genes. In situ hybridization revealed that the o-macs transcripts are present in the neuronal cell layer of olfactory sensory neurons (OSNs) as well as in the supporting and basal cell layers in the most dorso-medial area (zone 1) of the OE. Developmental analysis revealed that the o-macs gene is already expressed on embryonic day 11.5, before the onset of the OR gene expression, in a restricted area within the rat olfactory placode. Recombinant O-MACS protein tagged with c-Myc and His6 demonstrated an acyl-CoA synthetase activity for fatty acid activation, and protein localization to mitochondria like other MACS family proteins. The present study indicates that this novel protein may play important roles in processing odorants in a zone-specific manner, or the zonal patterning of the OE during development.

We have studied the projection of olfactory sensory neurons (OSNs), during the developmental and regeneration processes, using the transgenic mouse carrying the differently tagged odorant receptor genes, MOR28. We have found that the axon terminals of the two sets of MOR28-positive OSNs, one expressing the lacZ tag and the other expressing the green fluorescent protein gene, are dispersed and intermingled at early developmental or regeneration stages. Projection areas become more distinct and separated at later stages, however, two sets of axon fibers are not typically bundled or segregated during pathfinding. It appears that segregation of axons mainly occurs when they target at the olfactory bulb to form the glomerular structure.

To study the mutually exclusive expression of odorant receptor (OR) genes, we generated transgenic mice that carried the murine OR gene MOR28. Expression of the transgene and the endogenous MOR28 was distinguished by using two different markers, beta-galactosidase and green fluorescent protein (GFP), respectively. Double staining of the olfactory epithelium revealed that the two genes were rarely expressed simultaneously in individual olfactory neurons. A similar exclusion was also observed between differently tagged but identical transgenes integrated into the same locus of one particular chromosome. Although allelic inactivation has been reported for the choice between the maternal and paternal alleles, this is the first demonstration of mutually exclusive activation among non-allelic OR gene members with identical coding and regulatory sequences. Such an unusual mode of gene expression, monoallelic and mutually exclusive, has previously been shown only for the antigen-receptor genes of the immune system.

The Na+-K+ co-transporter HKT1, first isolated from wheat, mediates high-affinity K+ uptake. The function of HKT1 in plants, however, remains to be elucidated, and the isolation of HKT1 homologs from Arabidopsis would further studies of the roles of HKT1 genes in plants. We report here the isolation of a cDNA homologous to HKT1 from Arabidopsis (AtHKT1) and the characterization of its mode of ion transport in heterologous systems. The deduced amino acid sequence of AtHKT1 is 41% identical to that of HKT1, and the hydropathy profiles are very similar.AtHKT1 is expressed in roots and, to a lesser extent, in other tissues. Interestingly, we found that the ion transport properties of AtHKT1 are significantly different from the wheat counterpart. As detected by electrophysiological measurements, AtHKT1 functioned as a selective Na+ uptake transporter inXenopus laevis oocytes, and the presence of external K+ did not affect the AtHKT1-mediated ion conductance (unlike that of HKT1). When expressed in Saccharomyces cerevisiae, AtHKT1 inhibited growth of the yeast in a medium containing high levels of Na+, which correlates to the large inward Na+ currents found in the oocytes. Furthermore, in contrast to HKT1, AtHKT1 did not complement the growth of yeast cells deficient in K+ uptake when cultured in K+-limiting medium. However, expression ofAtHKT1 did rescue Escherichia colimutants carrying deletions in K+ transporters. The rescue was associated with a less than 2-fold stimulation of K+uptake into K+-depleted cells. These data demonstrate that AtHKT1 differs in its transport properties from the wheat HKT1, and that AtHKT1 can mediate Na+ and, to a small degree, K+ transport in heterologous expression systems.

We have studied the interaction between recombination signal sequences (RSSs) and protein products of the truncated forms of recombination-activating genes (RAG) by gel mobility shift, DNase I footprinting, and methylation interference assays. Methylation interference with dimethyl sulfate demonstrated that binding was blocked by methylation in the nonamer at the second-position G residue in the bottom strand and at the sixth- and seventh-position A residues in the top strand. DNase I footprinting experiments demonstrated that RAG1 alone, or even a RAG1 homeodomain peptide, gave footprint patterns very similar to those obtained with the RAG1-RAG2 complex. In the heptamer, partial methylation interference was observed at the sixth-position A residue in the bottom strand. In DNase I footprinting, the heptamer region was weakly protected in the bottom strand by RAG1. The effects of RSS mutations on RAG binding were evaluated by DNA footprinting. Comparison of the RAG-RSS footprint data with the published Hin model confirmed the notion that sequence-specific RSS-RAG interaction takes place primarily between the Hin domain of the RAG1 protein and adjacent major and minor grooves of the nonamer DNA.

The cis-acting region, GM-kappa B/GC-box (positions -95 and -73), within the murine GM-CSF gene promoter is required for maximal induction by stimulation with phorbol-12-myristate acetate (PMA) and calcium ionophore (A23187) in T cells. GM-kappa B defines a binding site for NF-kappa B, and GC-box defines a binding site for three (A1, A2, B) constitutive proteins. We report here that three copies of the GC-box can functionally compensate for the GM-kappa B/GC-box region, suggesting that the GC-motif can function independently of the GM-kappa B motif. The major GC-box binding activity A1 was purified and identified as the transcription factor Sp1. We show that depletion of Sp1 (A1) from nuclear extracts specifically decreases in vitro transcription activity on GM-CSF templates. Since the human GM-CSF promoter has a base difference within the GC-box, we speculate that this may explain why the human promoter is weak and that an upstream enhancer is required for the induction of the human GM-CSF gene.

Two cis-acting elements GM-kappa B/GC-box and CLE0, of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene are required for maximal induction in Jurkat T cells by costimulation with phorbol-12-myristate acetate (PMA) and Ca2+ ionophore (A23187). The GM-kappa B sequence is recognized by NF-kappa B, which is mainly induced by PMA. The CLE0 sequence interacts with factors, related to a PMA-induced AP-1 and a PMA/A23187-induced NF-AT. We examined whether signal transducing components in T cells can activate transcription of the GM-CSF gene. Cotransfection of NF-kappa B (p50/p65)- or AP-1 (c-Jun/c-Fos)-expression vectors into Jurkat cells with a luciferase reporter containing the GM-CSF promoter did not stimulate transcription from the GM-CSF promoter. In contrast, cotransfection with a combination of NF-kappa B and AP-1 significantly augmented transcription from the GM-CSF promoter containing the GM-kappa B/GC-box and the CLE0 (AP-1/NF-AT). Expression of a constitutively active calcineurin (CN), a Ca2+/calmodulin-dependent protein phosphatase, potentiated by two fold the transcriptional activation by NF-kappa B/AP-1. Both constitutively active forms of CN and protein kinase C (PKC) synergistically activated transcription from the GM-CSF promoter. These results suggest that cooperation among NF-kappa B-, AP-1- and NF-AT-binding sequences is required for induction of the GM-CSF gene through PKC- and Ca2+-signaling pathways downstream of T cell activation.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2) are produced by stimulation with phorbol-12-myristate acetate (PMA) and calcium ionophore (A23187) in human T cell leukemia Jurkat cells. The expression of GM-CSF and IL-2 is inhibited by immunosuppressive drugs such as cyclosporin A (CsA) and FK506. Earlier studies on the IL-2 gene expression showed that overexpression of calcineurin (CN), a Ca2+/calmodulin-dependent protein phosphatase, can stimulate transcription from the IL-2 promoter through the NF-AT-binding site. In this study, we obtained evidence that transfection of the cDNAs for CN A (catalytic) and CN B (regulatory) subunits also augments transcription from the GM-CSF promoter and recovers the transcription inhibited by CsA. The constitutively active type of the CN A subunit, which lacks the auto-inhibitory and calmodulin-binding domains, acts in synergy with PMA to activate transcription from the GM-CSF promoter. We also found that the active CN partially replaces calcium ionophore in synergy with PMA to induce expression of endogenous GM-CSF and IL-2. By multimerizing the regulatory elements of the GM-CSF promoter, we found that one of the target sites for the CN action is the conserved lymphokine element 0 (CLE0), located at positions between -54 and -40. Mobility shift assays showed that the CLE0 sequence has an AP1-binding site and is associated with an NF-AT-like factor, termed NF-CLE0 gamma. NF-CLE0 gamma binding is induced by PMA/A23187 and is inhibited by treatment with CsA. These results suggest that CN is involved in the coordinated induction of the GM-CSF and IL-2 genes and that the CLE0 sequence of the GM-CSF gene is a functional analogue of the NF-AT-binding site in the IL-2 promoter, which mediates signals downstream of T cell activation.

SummaryIn the mouse olfactory system, the anatomical locations of olfactory sensory neurons (OSNs) roughly correlate with their axonal projection sites along the dorsal-ventral (D-V) axis of the olfactory bulb (OB). Here we report that an axon guidance receptor, Neuropilin-2 (Nrp2), and its repulsive ligand, Semaphorin-3F (Sema3F), are expressed by OSNs in a complementary manner that is important for establishing olfactory map topography. Sema3F is secreted by early-arriving axons of OSNs and is deposited at the anterodorsal OB to repel Nrp2-positive axons that arrive later. Sequential arrival of OSN axons as well as the graded and complementary expression of Nrp2 and Sema3F by OSNs help to form the topographic order along the D-V axis.

We identified a stomatin-related olfactory protein (SRO) that is specifically expressed in olfactory sensory neurons (OSNs). The mousesrogene encodes a polypeptide of 287 amino acids with a calculated molecular weight of 32 kDa. SRO shares 82% sequence similarity with the murine stomatin, 78% withCaenorhabditis elegansMEC-2, and 77% withC. elegansUNC-1. Unlike other stomatin-family genes, thesrotranscript was present only in OSNs of the main olfactory epithelium. Nosroexpression was seen in vomeronasal neurons. SRO was abundant in most apical dendrites of OSNs, including olfactory cilia. Immunoprecipitation revealed that SRO associates with adenylyl cyclase type III and caveolin-1 in the low-density membrane fraction of olfactory cilia. Furthermore, anti-SRO antibodies stimulated cAMP production in fractionated cilia membrane. SRO may play a crucial role in modulating odorant signals in the lipid rafts of olfactory cilia.