PubMed Health⌕ Search

Biomedical subjects

Y Emori

Publications and source records attributed to Y Emori.

At least 37 records · Page 2Linked to original sources
PubMed1999

Two subfamilies of olfactory receptor genes in medaka fish, Oryzias latipes: genomic organization and differential expression in olfactory epithelium.

We cloned and characterized two subfamilies of olfatory receptor (OR) genes from medaka fish (Oryzias latipes). Southern blot analysis showed that each of the two subfamilies, designated as subfamilies Y and E, consists of about five members, as usually observed for other vertebrate ORs. Analyses of the genomic clones encoding these members revealed that two members of subfamily Y and four members of subfamily E are tandemly reiterated in 15 and 22 kbp regions of the medaka genome, respectively. The members of each subfamily show very similar amino acid sequences, with similarities greater than 70%. However, the similarities to the sequences of other vertebrate ORs are lower. Members of subfamily Y show amino acid sequence similarities of ca. 30% to other fish ORs, including subfamily E members, as well as to mammalian ORs. On the other hand, members of subfamily E show sequence similarities of ca. 50% to other fish ORs and ca. 30% to mammalian ORs. Phylogenic analyses of various fish ORs, including medaka, catfish, and zebrafish ORs, indicate that the primary structures of fish ORs are diverse compared with those of mammalian ORs, which consist of much larger numbers of members. The expression patterns of subfamilies Y and E genes in the olfactory epithelium of adult medaka fish were examined by in situ hybridization, showing that the frequency of positive signals is different between the two subfamilies: about 2% of the olfactory neurons are positive to probes for members of subfamily Y, while less than 1% are positive to probes for members of subfamily E. These results indicate that each subfamily is under different transcriptional control.

Amino Acid Sequence↗
PubMed1999

Very young patient with peculiar squamous cell carcinoma of the lung.

A 20-year-old man with squamous cell carcinoma of the lung is described. Histology of the open lung biopsy specimen revealed squamous cell carcinoma with definite keratinization. Interestingly, the tumor cells were characterized by partial expression of CD34 antigen and neuroendocrine differentiation. The diagnosis was delayed in this case because of his young age. This delayed diagnosis resulted in rapid progression and short survival time. In our review of 667 cases of lung cancers at Okayama University Hospital, only 3 (0.4%) of them were under 30 years of age and also showed advanced stage and very poor prognosis. It is important to take note of lung cancer as a differential diagnosis to detect early-stage lung cancer in young patients when they present with abnormal shadow on chest radiograph.

Adult↗
PubMed1998

Molecular cloning and taste bud-specific expression of a novel cyclic nucleotide-gated channel.

Cyclic nucleotide-gated (CNG) channels serve as downstream targets of signaling pathways in vertebrate photoreceptor cells and olfactory sensory neurons. For taste signaling as well, a great deal of information is available predicting the presence of a CNG channel, but no report has been presented on its molecular entity. Here we report on molecular cloning and functional expression of a taste bud-specific CNG channel tentatively named CNGgust. Reverse transcriptase polymerase chain reaction (RT-PCR) primers were synthesized according to some amino acid sequences generally conserved in many CNG channels. RT-PCR was conducted using rat circumvallate papillary mRNA-derived cDNA as a template to obtain positive clones. A corresponding genomic DNA clone was then obtained by screening from a genomic DNA library. Dissecting the entire structure of this gene, we found that the encoding protein had an amino acid sequence similarity of 80% to each of retina and olfactory CNG channels. It was also found by immunostaining with a specific antibody that this gustatory CNG channel (CNGgust) is localized in the tongue and also expressed specifically on the pore side of each taste bud in the circumvallate papillae. Electrophysiological experiments demonstrated that CNGgust resided in a functional state. All these data suggest that CNGgust may be involved in taste signal transduction in sensory cells.

Amino Acid Sequence↗
PubMed1998

Identification of two alpha-subunit species of GTP-binding proteins, Galpha15 and Galphaq, expressed in rat taste buds.

We cloned cDNAs for two G protein alpha-subunits belonging to the Galphaq family, each capable of activating PLCbeta, from rat tongue. One is a Galphaq in the narrow sense, and the other, termed rat Galpha15, is a rat counterpart of mouse Galpha15, sharing an amino acid sequence similarity of 94%. RT-PCR and Northern blot analysis demonstrated that rat Galpha15 and Galphaq were distinctly expressed in tongue epithelia containing taste buds. Immunostaining also showed that rat Galpha15, together with the Galphaq, was localized mainly in taste buds. These studies suggest the possibility that these two Galpha proteins function for taste signal transduction in sensory cells.

Amino Acid Sequence↗
PubMed1998

Functional expression and enzymatic properties of two Sitophilus zeamais cysteine proteinases showing different autolytic processing profiles in vitro.

To characterize in more detail the cathepsin L-like cysteine proteinases from Sitophilus zeamais (SCPs) cloned in our previous study [Matsumoto et al. (1997) J. Biochem. 121, 464-476], we established a system for their functional expression and purification using a glutathione S-transferase (GST) fusion gene vector from Escherichia coli. The proenzyme forms of two representative SCPs, proSCPc1 and proSCPg3, were expressed as GST-fusion proteins and purified on a glutathione Sepharose column. GST-proSCPc1 undergoes autoproteolytic cleavage into the mature form efficiently at acidic pH, and exhibits significant proteolytic activity toward various substrates including hemoglobin and Z-Phe-Arg-MCA. The enzymatic characteristics of the activated form of SCPc1 are similar to those of mammalian cathepsin L, but its pH optimum for the hydrolysis of hemoglobin is significantly lower. The other proSCP, GST-proSCPg3, which has a shorter COOH-terminal domain than SCPc1, undergoes almost no autolytic processing and shows only very slight proteolytic activity, although the other enzymatic characteristics of GST-proSCPg3 are similar to those of GST-proSCPc1.

Amino Acid Sequence↗
PubMed1998

Identification of taste-tissue-specific cDNA clones from a subtraction cDNA library of rat circumvallate and foliate papillae.

To identify genes specifically expressed in taste tissues, we constructed a subtraction cDNA library of epithelium of rat circumvallate and foliate papillae and carried out differential screening of this library. Dot blot analysis showed 46 out of 88 clones obtained by this method to be expressed in the epithelium of papillae. The cDNA inserts in these clones were sequenced and analyzed for similarity to entries the GenBank database. About 54.3% of the clones were known sequences, including the sequences of ebnerin, cytokeratin 18, and Na+,K+-ATPase, that were shown by in situ hybridization to be expressed in the circumvallate papillae. About 41.3% of the papillae-specific clones had no significant similarity to known sequences and are candidates for novel taste bud-specific marker genes.

Animals↗
PubMed1997

Taste buds have a cyclic nucleotide-activated channel, CNGgust.

Cyclic nucleotide-gated (CNG) channels have been characterized as important factors involved in physiological processes including sensory reception for vision and olfaction. The possibility thus exists that a certain CNG channel functions in gustation as well. In the present study, we carried out reverse transcription-polymerase chain reaction and genomic DNA cloning and characterized a CNG channel (CNGgust) as a cyclic nucleotide-activated species expressed in rat tongue epithelial tissues where taste reception takes place. Several types of 5'-rapid amplification of cDNA ends clones of CNGgust cDNA were obtained with various 5'-terminal sequences. As the CNGgust gene was a single copy, the formation of such CNGgust variants should result from alternative splicing. The encoded protein was homologous to known vertebrate CNG channels with 50-80% similarities in amino acid sequence, and particularly homologous to bovine testis CNG channel and human cone CNG channel with 82% similarities. CNGgust was functional when expressed in human embryonic kidney cells, where it opened upon the addition of cGMP or cAMP. Immunohistochemical analysis using an antibody raised against a CNGgust peptide demonstrated the channel to be localized on the pore side of each taste bud in the circumvallate papillae, with no signal observed for degenerated taste buds after denervation of the glossopharyngeal nerve. All these results, together with the indication that cyclic nucleotides play a role gustatory signaling pathway(s), strongly suggest the involvement of CNGgust in taste signal transduction.

Amino Acid Sequence↗
PubMed1997

Site-specific de-N-glycosylation of diglycosylated ovalbumin in hen oviduct by endogenous peptide: N-glycanase as a quality control system for newly synthesized proteins.

Hen ovalbumin (OVA) is known to exist as a singly N-glycosylated form with a glycan chain on Asn-292 in egg white. Previous studies showed that di-N-glycosylated form of OVA [Di-OVA; CHO-Asn-292/CHO-Asn-311 (CHO, N-glycan chain)], which has two N-glycan chains on Asn-292 and Asn-311, was expressed only transiently in hen oviduct. Di-OVA was not found in egg white, suggesting that this form cannot be secreted normally and may possibly be converted to mono-N-glycosylated OVA (CHO-Asn-292/Asp-311) by the action of peptide:N-glycanase (PNGase) during synthesis and secretion. In this study, we have identified the putative PNGase activity in the homogenate of hen oviduct, purified 1,000-fold, and designated as PNGase HO. We examined the reactivity of Di-OVA to PNGase HO and found that this enzyme site-specifically cleaved off the glycan chain at Asn-311 to convert Di-OVA into the mono-N-glycosylated form (CHO-Asn-292/Asp-311). In contrast, this enzyme was found not to act on the mono-N-glycosylated OVA (CHO-Asn-292/Asn-311) found in egg white when it was tested as a substrate. The present findings support our view that de-N-glycosylation catalyzed by PNGase may be involved in quality control of newly synthesized proteins by converting its diglycosylated form into the mono-N-glycosylated form that can be secreted. However, the alternative possibility that de-N-glycosylation may trigger cytosolic degradation of the aberrantly glycosylated glycoprotein cannot be ruled out.

Amidohydrolases↗
PubMed1997

Immunological analysis of two calpain-like Ca2+-dependent proteinases from lobster striated muscles: relationship to mammalian and Drosophila calpains.

Lobster skeletal muscles contain four Ca2+-dependent cysteine proteinases (CDPs I, IIa, IIb, and III) that degrade myofibrillar proteins. Lobster CDPs share many properties with calpains from vertebrate tissues, but differ in native mass and subunit composition. Recently, cDNAs encoding a calpain-like protein (Dm-calpain; 91.5 or 94 kDa) have been isolated from fruit fly, Drosophila melanogaster. To further clarify the relationship between invertebrate CDPs and mammalian calpains, antibodies specific for mu-, m-, p94 (nCL-1), and Dm-calpains and lobster CDP IIb (native M(r) 195,000, subunit M(r) 95,000) were used in immunoblots to test for antigenic cross-reactivity. No common epitopes were found between CDP IIb and vertebrate calpains. However, polyclonal antibodies to CDP IIb cross-reacted strongly with a C-terminal 70-kDa portion of Dm-calpain expressed in Escherichia coli. Conversely, polyclonal antibodies to Dm-calpain recognized CDP IIb. A second CDP, CDP IIa (native M(r) 125,000), was partially purified from lobster muscle; enzyme activity coeluted with a 60-kDa polypeptide using anion-exchange chromatography. The 60-kDa protein reacted with a polyclonal antibody raised against a 20-amino acid peptide sequence found around the catalytic cysteine residue of mu- and m-calpains, but not with antibodies raised against other regions of mu- or m-calpain or with the anti-CDP IIb antibody. These results suggest that (1) the CDP IIb is the homolog of Drosophila calpain in crustaceans and (2) the active site regions of CDP IIa and mu- and m-calpains are similar.

Amino Acid Sequence↗
PubMed1997

Z-100, a polysaccharide-rich preparation extracted from the human type Mycobacterium tuberculosis, improves the resistance of Meth-A tumor-bearing mice to endogenous septic infection.

The effect of Z-100, an immunomodulatory arabinomannan extracted from Mycobacterium tuberculosis, on cecal ligation and puncture (CLP)-induced sepsis in mice bearing Meth-A fibrosarcoma was investigated. When normal BALB/c mice were subjected to the CLP procedure, their mortality rate was 17%. On the other hand, an increased mortality was observed in tumor-bearing mice subjected to CLP 10 days after tumor inoculation, and then all mice died when tumor-bearing mice were subjected to CLP 20 days after tumor inoculation. However, the increased percent mortality was decreased by 50% when these mice were injected intraperitoneally with a 10 mg/kg dose of Z-100. When splenocytes (5 x 10(7) cells), obtained from Meth-A tumor-bearing mice 20 days after tumor inoculation, were transferred intravenously to normal mice (recipient mice), mortality of these recipient mice were increased by 62% as compared with that of the control (22%). However, no increased mortality (25%) was observed in recipient mice which were transferred with splenocytes from tumor-bearing mice injected intraperitoneally with Z-100 (10 mg/kg). In addition, suppressor cell activity was demonstrated in splenocytes from Meth-A tumor-bearing mice at 20 days after tumor inoculation using one-way mixed lymphocyte reaction. However, the suppressor cell activity was significantly decreased by the intraperitoneal administration of a 10 mg/kg dose of Z-100 (p < 0.01). The increase of mortality in recipient mice by adoptive transfer of mononuclear cells (MNCs) from tumor-bearing mice was not detected when these MNCs were treated with anti-Thy 1.2 monoclonal antibody (mAb), anti-Lyt 2.2 mAb or anti-CD11b mAb, but an increase was seen with anti-Lyt 1.2 mAb or anti-immunoglobulin antiserum treated MNCs. These results suggest that the suppressor cells affect the mortality of CLP-induced sepsis and Z-100 may have a therapeutic activity against opportunistic infections in immunocompromised hosts through the regulation of suppressor T-cells.

Adjuvants, Immunologic↗
PubMed1997

Characterization of a gene family encoding cysteine proteinases of Sitophilus zeamais (maize weevil), and analysis of the protein distribution in various tissues including alimentary tract and germ cells.

We have identified and characterized a gene family comprising at least four genes encoding cathepsin L-like cysteine proteinases (SCPs) in Sitophilus zeamais, Coleoptera. A cDNA clone corresponding to a major mRNA species of the gene family in adult insects codes for a cysteine proteinase (CP) of 338 amino acid residues, and two genomic clones code for similar CPs of 331 residues whose COOH-terminal structures are seven amino acids shorter. These three SCPs show high sequence similarity to one another as well as to other insect and mammalian cathepsin L-like proteinases. A polyclonal antibody raised against bacterially expressed SCP was used as a probe to examine the molecular forms and distribution of the enzyme. SCP exists in both proenzyme and mature forms in larvae, pupae, and adults, and the proenzyme converts in vitro into the mature form at acidic pH. Immunohistochemical analysis showed that SCP is present in several tissues including alimentary organs and germ cells. In alimentary organs, SCP is distributed in the gastric caeca, but not the midgut. SCP is also present in genital organs, especially in oocytes and nurse cells, where it exists at high levels. These results indicate that SCP plays a variety of physiological roles including a role in food digestion.

Amino Acid Sequence↗
PubMed1997

Identification of endogenous substrates for Drosophila calpain from a salt-extracted fraction of Drosophila ovaries.

Drosophila calpain (Dm-calpain) produced in Escherichia coli has a distinct Ca2+-dependent activity. By using a recombinant Dm-calpain, we searched for its substrates occurring in Drosophila ovaries, where Dm-calpain is expressed. Among a number of major proteins, several proteins in a salt-extracted fraction were selectively degraded by Dm-calpain in a Ca2+-dependent manner. The major substrates were identified by microsequencing the lysylendopeptidase-digested proteins. Three ribosomal proteins, the L5, L7, and L8 subunits of the 60S ribosome, were found to be potential Dm-calpain substrates. In addition, the alpha subunit of elongation factor-1 (EF-1alpha), a multi-functional protein involved in both protein synthesis and cytoskeletal regulation, was shown to be cleaved by Dm-calpain into several distinct fragments when expressed as a GST-fusion protein. Endogenous EF-1alpha in ovary extracts was also shown by western blot analysis to be similarly degraded. These observations suggest that Dm-calpain may regulate protein synthesis and cytoskeletal structure through its degradative or processing activity.

Amino Acid Sequence↗
PubMed1996

A water channel closely related to rat brain aquaporin 4 is expressed in acid- and pepsinogen-secretory cells of human stomach.

We isolated a cDNA clone encoding a water channel protein, aquaporin ( AQP), from human stomach. The encoded protein consisted of 323 amino acid residues, containing six putative transmembrane domains. The protein was designated human aquaporin 4 (hAQP4) because of its 94% sequence similarity to rat brain AQP4. Expression of hAQP4 cRNA in Xenopus oocytes resulted in a significant increase in osmotic water permeability, indicating that this protein functions as a water channel. Northern blot analysis demonstrated a strong signal of hAQP4 mRNA in brain, lung, and skeletal muscle as well as in stomach. Immunohistochemical experiments with human stomach tissues showed that hAQP4 as a protein is expressed mainly in cells located in the glandular portion of the fundic mucosa. These include chief cells which secrete pepsinogen and parietal cells which secrete hydrochloric acid. These results strongly indicate that hAQP4 is a principal factor involved in the osmotic regulation of pepsinogen and acid secretion in the stomach.

Amino Acid Sequence↗
PubMed1996

Molecular cloning and functional expression of the alpha1A-adrenoceptor of Medaka fish, Oryzias latipes.

A genomic DNA encoding a subtype adrenoceptor (AR) was cloned from Medaka fish, Oryzias latipes, using an oligonucleotide probe corresponding to the consensus sequence of mammalian alpha-AR and beta-AR. The gene spans at least 9kbp, and the coding region consists of two exons split by an intron of 7.2 kbp located at the same position as those of mammalian alpha1B-AR genes. The gene encodes 470 amino acid residues, the sequence of which shows the highest similarity to that of mammalian alpha1A-AR (61%) and significant but lower similarities to other alpha-AR and beta-AR proteins (31-45%), indicating that the gene encodes a Medaka homolog of alpha1A-AR. To characterize the encoded protein, the mRNA was synthesized in vitro and injected into Xenopus oocytes. As a result, the oocytes responded to 100 nM epinephrine evoking a Ca2 + -dependent C1- current in the order of microamperes, which was not observed for oocytes injected with water alone. The response was reversibly inhibited by an alpha1-selective antagonist, WB4101 (2-[2,6-dimethoxphenoxyethyl]aminomethyl)-1,4-benzodioxane). Similar experiments using several adrenergic agonists revealed that Medaka alpha1A-AR responds to the following agonists in the order: epinephrine > or = (-)norepinephrine > oxymetazoline > or = methoxamine, which is similar to the responses of rat alpha1A receptor expressed in COS cells. The results indicate that fish contains adrenoceptor systems similar to those of mammals in terms of primary structure and pharmacological properties.

Amino Acid Sequence↗
PubMed1996

Hepatocellular carcinoma cells resist necrosis during anoxia by preventing phospholipase-mediated calpain activation.

Although hepatocellular carcinoma (HCC) cells are more resistant to anoxic injury than normal hepatocytes, the mechanisms responsible for this differential sensitivity remain obscure. Because enhanced calpain protease activity contributes to hepatocyte necrosis, we tested the hypothesis that HCC cells resist anoxia by preventing calpain activation. Cell viability in two rat HCC cell lines (N1S1 and McA-RH7777 cells) was fourfold greater compared to rat hepatocytes after 4 h of anoxia. Although calpain activity increased twofold in rat hepatocytes during anoxia, no increase in calpain activity occurred in HCC cells. Western and Northern blot analysis revealed greater or equivalent expression of calpains and calpastatin in HCC cells compared to hepatocytes. Because increases in cytosolic free Ca++ (Cai++) and phospholipid degradation products regulate calpains in vitro, we measured Cai++ and phospholipid degradation. Ca++i did not change in any cell types during 60 min of anoxia. In contrast, phospholipid degradation was fourfold greater in hepatocytes compared to HCC cells. Melittin, a phospholipase A2 activator, increased calpain activity and cell necrosis in all cell types; melittin-induced cell necrosis was ameliorated by a calpain protease inhibitor. In summary, these data demonstrate for the first time 1) calpain activation without a measureable increase in Ca++i, 2) phospholipase-mediated calpain activation in hepatocytes and HCC cells, and 3) the adaptive mechanism responsible for the resistance of HCC cells to anoxia-an inhibition of phospholipid-mediated calpain activation. Interruption of phospholipase-mediated calpain activation may be a therapeutic strategy for preventing anoxic cell injury.

Adenosine Triphosphate↗