Synaptophysin as a probable component of neurotransmission occurring in taste receptor cells.

Taste signal is received in taste buds and transmitted via sensory afferent nerves to the brainstem. Although a signaling pathway involving phospholipase C-beta2 has been shown to transduce taste signals of bitterness, sweetness and umami in taste receptor cells (Type II cells), these taste receptor cells appear to be different from the presynaptic cells (Type III cells) containing afferent synapses associated with nerve processes. To elucidate the neurotransmission system in the taste receptor cells expressing phospholipase C-beta2, we searched for candidate molecules involved in the neurotransmission, and identified synaptophysin. Synaptophysin was expressed in the taste receptor cells expressing phospholipase C-beta2, as well as in the presynaptic cells harboring synaptic structures with taste nerves and containing serotonin. Synaptophysin-immunoreactive signals were not limited to gustducin-positive bitter taste receptor cells, and sweet/umami taste receptor cells were indicated to also express synaptophysin. Expression of synaptophysin was already initiated 6 days after cell division, almost in synchrony with the initiation of phospholipase C-beta2 expression. Synaptophysin-containing cells co-expressed vesicular-associated membrane protein 2, a v-SNARE molecule which is important for exocytosis. In addition, majority of the synaptophysin-expressing cells also expressed cholecystokinin, a neuropeptide expressed in taste buds. These results suggest that the taste receptor cells have a neurotransmission system involving synaptophysin, which occurs alternatively or additionally to a recently shown hemichannel system.

Cytokeratin 14 is expressed in immature cells in rat taste buds.

We analyzed the differentiation of taste bud cells, by precisely describing expression profiles of cytokeratins (CKs) 8 and 14 in relation to those of marker molecules including label of 5-bromo-2'-deoxy uridine (BrdU) injected. In rat circumvallate papillae, cell division was observed at the basal layer of the epithelium expressing CK14 and located outside taste buds. The progenitor cells began to migrate toward the apical surface and maintained CK14 expression at 1 day after BrdU injection (day 1). On the other hand, a minor population of newly divided cells was infrequently incorporated into taste buds and also maintained CK14 expression at day 1. In taste buds, the conversion of CK subtypes occurred from CK14 to cytokeratin 8 (CK8) at day 2-3, showing the differentiation from immature cells expressing CK14 into mature or maturing cells expressing CK8. Functionally matured cells such as taste receptor cells expressing inositol triphospate receptor type 3 (IP(3)R3) never expressed CK14, suggesting that CK14 would be expressed only in immature cells. On the other hand, a small but distinct population of BrdU-positive cells still showed CK14 immunoreactivity in taste buds even at day 12, which might correspond to the cells that remain undifferentiated for a long period within taste buds.

Drosophila Calpain B is monomeric and autolyzes intramolecularly.

Drosophila calpains, Calpain A and Calpain B, show typical calpain domain structures similar to mammalian calpains. However, the small subunit of mammalian calpains, shown to be essential in both genetic and biochemical aspects, is absent in Drosophila calpains and is not required for enzymatic activity. How they compensate for the lack of small subunit is mostly unknown. Here we conducted experiments using recombinant Drosophila Calpain B for further characterization of the enzyme with particular focuses on two issues: possibility of homodimerization and mode of autolysis. The native molecular weight of Calpain B indicates that the active enzyme is primarily monomeric. Co-expression of two recombinant Calpain B proteins each with a unique affinity tag and a subsequent single round of affinity tag purification resulted in isolation of only one recombinant calpain type, suggesting there is no homodimeric interaction. Also the C-termini of Drosophila calpains lack many of the key hydrophobic residues considered to be important in the dimerization of mammalian calpains. Further, initial autolysis of Calpain B seems to occur intramolecularly, which supports the monomeric nature of Drosophila calpains. These results strongly suggest that dimerization is not an essential requirement for Drosophila calpains.

Depletion of hsp90beta induces multiple defects in B cell receptor signaling.

Hsp90 participates in many distinct aspects of cellular functions and accomplishes these roles by interacting with multiple client proteins. To gain insight into the interactions between Hsp90 and its clients, here we have reduced the protein level of Hsp90 in avian cells by gene targeting in an attempt to elicit the otherwise undetectable (because of the vast amount of cellular Hsp90) Hsp90-interacting proteins. Hsp90beta-deficient cells can grow, albeit more slowly than wild-type cells. B cell antigen receptor signaling is multiply impaired in these mutant cells; in particular, the amount of immunoglobulin M heavy chain protein is markedly reduced. Furthermore, serum activation does not promote ERK phosphorylation in Hsp90beta-deficient cells. These multifaceted depressive effects seem to be provoked independently of each other and possibly recapitulate the proteome-wide in vivo functions of Hsp90. Reintroduction of the Hsp90beta gene efficiently restores all of the defects. Unexpectedly, however, introducing the Hsp90alpha gene is also effective in restoration; thus, these defects might be caused by a reduction in the total expression of Hsp90 rather than by loss of Hsp90beta-specific function.

Identification of candidate genes involved in somatosensory functions of cranial sensory ganglia.

We have shown that the characteristics of tissue trees obtained by the hierarchical cluster analysis of DNA microarray data suggest the cellular expression patterns of genes in the gene clusters [J. Neurosci. Res. 74 (2003) 818]. We here identified three gene clusters containing 11 genes as a potential pool of candidate genes related to somatosensation in cranial structures such as the face, oral cavity and pharynx. To obtain the cellular expression profiles, eight genes other than three genes analyzed previously were subjected to in situ hybridization analysis. The results show that all of the 11 profiles are roughly similar and suggest that the positive cells are probably somatosensory neurons in two cranial sensory ganglia, the trigeminal and petrosal ganglia. The expression profiles and probable physiological functions of the 6 genes such as trkA, NaN and galanin suggest their direct involvement in specific somatosensory functions such as nociception. The function of another gene, calretinin, is putatively related to mechanosensation and proprioception. The roles of the remaining four genes, including aquaporin 1 and two EST clones, in sensory neurons are unknown, and may provide clues to understand the sensory function in TG and PG.

Identification of 24 genes and two pseudogenes coding for olfactory receptors in Japanese loach, classified into four subfamilies: a putative evolutionary process for fish olfactory receptor genes by comprehensive phylogenetic analysis.

Twenty-four olfactory receptor (OR) genes and two pseudogenes have been identified in the genome of Japanese loach (Misgurnus anguillicaudatus). The genes were classified into four subfamilies according to the similarity of the amino acid sequences. In each subfamily, members showed high sequence similarity not only to each other but also to orthologues of other fish species. The number of members in each OR subfamily was roughly estimated to be from 3 to 10 by genomic Southern blot analysis. The genes of all four OR subfamilies were shown to express on olfactory neurons of the olfactory epithelium by in situ hybridization analysis. Two major features of fish OR genes were found by comprehensive and comparative analyses on OR genes of Japanese loach and other fish species including catfish, zebrafish and pufferfish. First, the phylogenetic tree comprising of representative subfamily members suggests the existence of several prototype genes common to the genomes of many fish species. Second, when all members of orthologous subfamilies identified in each clade of the tree are integrated, the members of a single species comprise a monophyletic group. This means that 'intraspecies' sequence homology, that is, homology among paralogous genes of the same subfamily in a species, is higher than 'interspecies' homology, that is, homology between orthologous genes of different species. This suggests that the subfamily members of a species have evolved recently. Taken together, fish OR genes have evolved from a limited number of prototype genes common to most fish species, and several genes in a subfamily have diversely evolved in each species from each prototype.

Gene expression profiling of cranial sensory ganglia that transmit food intake stimuli.

Peripheral cranial sensory nerves projecting into the oral cavity receive food intake stimuli and transmit sensory signals to the central nervous system. They are derived from four cranial sensory ganglia, trigeminal, geniculate, petrosal, and nodose ganglia, each of which contains multiple kinds of sensory neurons with different cell morphologies and neuronal properties. We investigated the complex properties of these neurons from the viewpoint of gene expression using DNA microarrays. The 498 genes were selected from a total of 8,740 genes as showing tissue-dependent expression on the microarray by hierarchical cluster analysis, in which several genes known to be differentially expressed in cranial sensory ganglia are included. This suggests that DNA microarray cluster analysis revealed a number of characteristic genes for sensory neurons in these ganglia. Among the selected 498 genes, 44 genes are associated with neurotransmission, such as neuropeptides, their receptors, and vesicle transport, and 26 are ion channels regulating membrane potentials. The identification of a number of genes related directly to neural properties indicates that these sensory ganglia contain heterogeneous types of neurons with different neural properties.

DNA microarray cluster analysis reveals tissue similarity and potential neuron-specific genes expressed in cranial sensory ganglia.

Each of four cranial sensory ganglia, trigeminal, geniculate, petrosal, and nodose ganglia, contains multiple kinds of sensory neurons with different cell morphologies and neuronal properties that transmit information about sensory stimuli received peripherally. Here we analyze the complex properties of these neurons from the viewpoint of gene expression using DNA microarrays by cluster analysis. From a total of 8,740 genes, 498 genes were selected as showing tissue-dependent expression on the microarray by hierarchical cluster analysis, and their profiles indicated that, among the four sensory ganglia, the petrosal and trigeminal ganglia are intimately related. Tissue trees of 37 subclusters containing the 498 genes showed that the profiles of gene expression and the subclusters were classified into a smaller number of groups (18 groups) when information on the amounts of expression was added. In situ hybridization analysis of 21 genes selected from 13 different groups was carried out, and the gene expression patterns were classified into eight categories. The putative profiles postulated from the microarray data were essentially consistent with the patterns of expression at the cellular level as shown by in situ hybridization. In conclusion, from the overall analyses of gene expression by DNA microarray, we can identify a number of candidate genes showing neuron type-specific expression in the peripheral ganglia.

Identification of beta and gamma subunits of laminins localized in the basement membrane of rat circumvallate papillae.

Taste bud cells have elongated shape and are anchored to the basement membrane. Here we analyzed the subunits of laminin, a major component of the basement membrane, in circumvallate papillae of rat tongue. Two beta subunits, beta1 and beta2, were detected by RT-PCR, of which the beta2 subunit was immunohistochemically identified as a major component of the basement membrane. The gamma1 subunit was also immunohistochemically identified as a major component. Profiles of these two laminin subunits were nearly the same as that obtained by an anti-laminin antibody, indicating that laminins of the basement membrane in the papillae contain beta2 and gamma1 as major beta and gamma subunits, respectively. As potential receptors for these laminin ligands, integrin subunits were analyzed by RT-PCR. Three integrin beta subunit species, beta1, beta4, and beta5, were shown to be expressed in the epithelium of the circumvallate papillae by RT-PCR among the five species examined.

Plant seed cystatins and their target enzymes of endogenous and exogenous origin.

Cystatins are protein inhibitors of cysteine proteinases of the papain family, and those of animal origin have long been studied from medical and physiological aspects. In the meantime, oryzacystatin cloned from rice seeds in 1987 was recognized as the first well-defined cystatin of plant origin. Cloning studies followed to disclose various plant cytstatins including those of corn and soybean origin, their similarities to and differences from animal cystatins being analyzed in detail. Plant seed cystatins are now understood as factors controlling germination by inhibition of endogenous cysteine proteinases. They can also recognize insect midgut proteinases as exogenous target enzymes to control. This paper discusses chemical and phytophysiological relationships between cystatins and their targets.

Ligand-dependent activation of breathless FGF receptor gene in Drosophila developing trachea.

Spatially and temporally regulated activity of Branchless/Breathless signaling is essential for trachea development in Drosophila. Early ubiquitous breathless (btl) expression is controlled by binding of Trachealess/Tango heterodimers to the btl minimum enhancer. Branchless/Breathless signaling includes a Sprouty-dependent negative feedback loop. We show that late btl expression is a target of Branchless/Breathless signaling and hence, Branchless/Breathless signaling contains a positive feedback loop, which may guarantee a continuous supply of fresh receptors to membranes of growing tracheal branch cells. Branchless/Breathless signaling activates MAP-kinase, which in turn, activates late btl expression and destabilizes Anterior-open, a repressor for late btl expression. Biochemical and genetic analysis indicated that the minimum btl enhancer includes binding sites of Anterior-open.

Endo-beta-N-acetylglucosaminidase, an enzyme involved in processing of free oligosaccharides in the cytosol.

Formation of oligosaccharides occurs both in the cytosol and in the lumen of the endoplasmic reticulum (ER). Luminal oligosaccharides are transported into the cytosol to ensure that they do not interfere with proper functioning of the glycan-dependent quality control machinery in the lumen of the ER for newly synthesized glycoproteins. Once in the cytosol, free oligosaccharides are catabolized, possibly to maximize the reutilization of the component sugars. An endo-beta-N-acetylglucosaminidase (ENGase) is a key enzyme involved in the processing of free oligosaccharides in the cytosol. This enzyme activity has been widely described in animal cells, but the gene encoding this enzyme activity has not been reported. Here, we report the identification of the gene encoding human cytosolic ENGase. After 11 steps, the enzyme was purified 150,000-fold to homogeneity from hen oviduct, and several internal amino acid sequences were analyzed. Based on the internal sequence and examination of expressed sequence tag (EST) databases, we identified the human orthologue of the purified protein. The human protein consists of 743 aa and has no apparent signal sequence, supporting the idea that this enzyme is localized in the cytosol. By expressing the cDNA of the putative human ENGase in COS-7 cells, the enzyme activity in the soluble fraction was enhanced 100-fold over the basal level, confirming that the human gene identified indeed encodes for ENGase. Careful gene database surveys revealed the occurrence of ENGase homologues in Drosophila melanogaster, Caenorhabditis elegans, and Arabidopsis thaliana, indicating the broad occurrence of ENGase in higher eukaryotes. This gene was expressed in a variety of human tissues, suggesting that this enzyme is involved in basic biological processes in eukaryotic cells.

Changes in cell morphology and cell-to-cell adhesion induced by extracellular Ca2+ in cultured taste bud cells.

Cell morphology and cell-to-cell adhesion of taste bud cells were significantly altered by extracellular Ca2+ during in vitro culture. Under high Ca2+ condition (above 0.5 mM), the cells were tightly associated with each other and formed packed aggregates. Under low Ca2+ condition (below 0.1 mM), the cells were dispersed and had an elongated shape. These two forms were reversible and specifically dependent on Ca2+. The results indicate that extracellular Ca2+ regulates cell shape and cell-to-cell adhesion of taste bud cells.