Phytocystatins and their target enzymes-molecular cloning, expression and possible functions.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Biomedical subjects
Y Emori
Publications and source records attributed to Y Emori.
Effect of Z-100, an immunomodulator extracted from human type tubercle bacilli, on the pulmonary metastases of Lewis lung carcinoma in attempt to regulate suppressor T cells and suppressor factor, IL-4.
In the present study, anti-metastatic effect of Z-100 on the spontaneous pulmonary metastases of Lewis lung carcinoma (3LL) was examined in an attempt to regulate suppressor T cells. When Z-100 (10 mg/kg) was daily injected i.p. after 3LL inoculation, survival rate of these mice was increased significantly (p < 0.05). In addition, the number of pulmonary metastatic colonies of 3LL in Z-100-treated mice were significantly decreased by 38% at 21 days, as compared with that of control mice (p < 0.05). Along with the decrease of pulmonary metastases, suppressor cell activity was also gradually reduced in these mice, as compared with that of control mice. When splenic suppressor cells (5 x 10(7) cells) from 3LL-bearing mice were adoptively transferred into normal mice (recipients) just before inoculation of 3LL, the development of pulmonary metastases in recipients was significantly accelerated. However, splenocytes from 3LL-bearing mice treated with Z-100 did not affect the development of pulmonary metastasis. The potential to accelerate the metastasis of splenic mononuclear cells from 3LL-bearing mice was decreased significantly by the treatment with anti-Thy 1.2 monoclonal antibody (mAb), anti-Lyt 2.2 mAb or anti-CD 11b mAb followed by complement. IL-4 activity in the sera of 3LL-bearing mice was detected 15 days after tumor inoculation (13 pg/ml) and gradually increased (18 pg/ml) 20 days after tumor inoculation. However, when Z-100 (10 mg/kg) was daily injected i.p., IL-4 activity in sera was decreased significantly, and the IL-4 activity was not detected in these mice on day 20. These results suggest that Z-100 could inhibit the pulmonary metastases in 3LL-bearing mice through the inhibition of suppressor T cell activity and a possible candidate of its effector molecule, IL-4.
Adjuvants, Immunologic↗
GUST27 and closely related G-protein-coupled receptors are localized in taste buds together with Gi-protein alpha-subunit.
Gustatory, like olfactory signalling is probably mediated by seven-transmembrane receptors and coupling GTP-binding proteins (G proteins). We investigated the expression of a subset of these receptors and the Gi protein alpha-subunit by using their specific antibodies. Based on our previous finding that the mRNA for GUST27, one of these receptors, is expressed in rat lingual epithelia, we first prepared an antibody to the synthetic nonapeptide, H-Ser-Tyr-Ser-Gln-Ile-Ala-Ser-Ser-Leu-OH, which corresponds to the third intracellular domain of GUST27 and also to those of a subset of related receptors whose occurrence can be predicted by PCR. Immunohistochemical studies with rat circumvallate papillae indicated that the anti-GUST27 antibody reacted with many of the taste buds examined, with strong signals appearing in particular taste cells. We then carried out a similar immunohistochemical experiment with an antibody to the Gi protein alpha-subunit and found that this subunit is also expressed in taste buds as demonstrated in the case of gustducin and transducin. Taken together, these results strongly suggest that GUST27 and closely related receptors, as well as Gi alpha proteins, are involved in intracellular taste signal transduction.
Amino Acid Sequence↗
A cascade of degradative hydrolase activity contributes to hepatocyte necrosis during anoxia.
Calpain proteases contribute to hepatocyte necrosis during anoxia. Our aim was to ascertain the mechanism causing calpain activation during anoxia. In rat hepatocytes, a twofold increase in calpain activity occurred despite the lack of an increase in cytosolic Ca2+ concentration ([Ca2+]i). The increase in calpain activity was not associated with an increase in calpain mRNA or a decrease in calpastatin mRNA expression. Because phospholipid degradation products generated by phospholipases can activate calpains at physiological [Ca2+]i, we determined the effect of phospholipase inhibitors and activators on calpain activity. Pretreatment of hepatocytes with fluphenazine, a phospholipase inhibitor, decreased calpain activation and improved cell survival. Melittin, a phospholipase A2 activator, increased calpain activity and potentiated cell killing. Finally, phospholipid degradation preceded the increase in calpain activity. Thus the enhanced calpain activity occurring in hepatocytes during anoxia 1) is regulated at the posttranslational level and 2) appears to be dependent on phospholipase activity. These data suggest a novel cascade for degradative hydrolase activity during hepatocyte necrosis by anoxia with phospholipase-mediated activation of calpains.
Animals↗
Three-dimensional volume visualization of the in vivo human ocular lens showing localization of the cataract.
An in vivo human lens containing a cataract has been visualized by volume rendering a transformed series of 60 rotated Scheimpflug digital images. The data set was obtained by rotating the Scheimpflug camera about the optic axis of the lens in 3-degree increments. The set of 60 Scheimpflug digital images were mathematically transformed into a new data set in which the images are oriented perpendicular to the optic axis of the eye. The transformed set of optical sections were first aligned to correct for eye movements during the data collection process, then rendered into a three-dimensional volume reconstruction with volume-rendering computer graphics techniques. The viewpoint and the transparency of the volume rendered in vivo human lens were varied in order to observe volume opacities in various regions of the lens. To help visualize lens opacities, the intensity of light scattering was pseudocolor-coded as an integral part of the three-dimensional volume rendering. Three-dimensional, pseudocolored volume rendering of the in vivo human ocular lens represents a new technique to visualize in vivo human cataracts.
Aged↗
Molecular cloning of a fish gene encoding a novel seven-transmembrane receptor related distantly to catecholamine, histamine, and serotonin receptors.
A genomic DNA fragment encoding a G protein-coupled seven-transmembrane receptor was isolated from Medaka fish, Oryzias latipes. The encoded protein is similar in sequence to other receptors including catecholamine, histamine and serotonin receptors. However, the similarity is much lower than those among members of these receptor subfamilies, thus suggesting this seven-transmembrane receptor to be an orphan receptor whose ligand has not yet been identified. Genomic Southern blot analysis suggested that the fish genome contains additional receptor genes related to the isolated gene, indicating that this novel receptor, possibly with its related receptors, might constitute a novel subfamily of the seven-transmembrane receptor superfamily.
Amino Acid Sequence↗
Cloning of cDNA and genomic DNA encoding fibroblast growth factor receptor-4 of Xenopus laevis.
We have isolated and characterized the cDNA and genomic DNA encoding fibroblast growth factor receptor-4 of Xenopus laevis (XFGFR-4). The gene encompassing the total coding sequence spans about 10 kb, consists of 17 exons, and has an organization very similar to those of mammalian genes encoding FGFR-1 and -2, except that the XFGFR-4 gene does not contain an alternative exon for the third immunoglobulin-like domain nor an internal poly(A)-addition site. Thus, XFGFR-4 appears not to generate multiple forms of mRNA, as are identified for the mammalian FGFR-1, -2 and -3 genes. The amino-acid sequence of XFGFR-4 shows high homology to other vertebrate FGFR-4 species, but the similarity was significantly lower than in the cases of FGFR-1 and -2. Northern blot analysis showed the XFGFR-4 mRNA to occur throughout X. laevis early embryogenesis in a profile different from those of X. laevis FGFR-1 and -2.
Amino Acid Sequence↗
A dual functional signal mediator showing RhoGAP and phospholipase C-delta stimulating activities.
We have cloned a novel regulator protein, p122, in the PLC-delta signalling pathway by screening a rat brain expression library with antiserum raised against purified phospholipase C-delta 1 (PLC-delta 1). This novel p122-RhoGAP binds to PLC-delta 1 and activates the phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolyzing activity of PLC-delta 1. As suggested by the deduced amino acid sequence, this regulator protein shows a similarity to the GTPase activating protein (GAP) homology region of Bcr and possesses GAP activity for RhoA, but not for Rac1; no guanine nucleotide exchange activity for RhoA and Rac1 was detected. These findings suggest that this novel RhoGAP is involved in the Rho signalling pathway, probably downstream of Rho activation, and mediates the stimulation of PLC-delta, which leads to actin-related cytoskeletal changes through the hydrolysis of PIP2, which binds to actin binding proteins such as gelsolin and profilin.
Amino Acid Sequence↗
A putative digestive cysteine proteinase from Drosophila melanogaster is predominantly expressed in the embryonic and larval midgut.
Plant seeds have biodefense systems for protection against insects. One of these systems may be based on the occurrence of phytocystatins, cysteine proteinase inhibitors of plant origin, that probably inhibit insect cysteine proteinases involved in digestive functions. To elucidate a molecular mechanism for this biodefense phenomenon, we isolated a gene encoding a putative digestive cysteine proteinase from Drosophila melanogaster, a suitable model species. The cloned genomic DNA fragment contained a sequence encoding a cysteine proteinase. The mature enzyme from the encoded protein, termed Drosophila cysteine proteinase-1, consisted of 218 amino acid residues. Drosophila cysteine proteinase-1 showed 67% similarity in its amino acid sequence to a lobster cysteine proteinase-3 contained in the digestive juice. This enzyme also showed significant similarities to cysteine proteinases of animal origin such as cathepsins H and L, and to proteinases of plant origin such as rice oryzains alpha and beta. In situ hybridization studies for the embryo showed that the mRNA for Drosophila cysteine proteinase-1 was predominantly expressed in the midgut. Larval alimentary organs, such as the salivary gland and the midgut including the gastric caeca, also expressed the mRNA at significant levels. These observations, suggesting that Drosophila cysteine proteinase-1 is a digestive cysteine proteinase which can be used as a model target of phytocystatins, will hopefully lead to new strategies for the regulation of pest insects.
Amino Acid Sequence↗
Calpain localization changes in coordination with actin-related cytoskeletal changes during early embryonic development of Drosophila.
Calpain, a calcium-dependent intracellular protease, was identified in Drosophila melanogaster. Drosophila calpain has an amino acid sequence highly homologous to those of mammalian calpains and exhibits a distinct domain structure consisting of cysteine protease and calcium-binding domains. Specific antibodies raised against a recombinant calpain fragment were used to identify the localization of calpain in developing Drosophila embryos. Calpain was first detected near the anterior pole and in posterior region of the embryo just after fertilization. The anterior calpain disappeared during the cleavage cycles. On the other hand, the posterior calpain moved to the posterior pole when polar buds were formed, and condensed just below the pole cells. At cleavage cycles 8 and 9, when nuclei reached the egg surface, calpain was localized between the nuclei at the surface beneath the precleavage furrows. Co-staining experiments with anti-actin antibody revealed that calpain condenses specifically at the edge of and between actin caps that underlie the plasma membrane immediately above each nucleus. These results indicate that calpain is involved in the dynamic changes in the embryonic cytoskeleton, especially actin-related structures, during early embryogenesis prior to cellularization.
Actins↗
Drosophila phospholipase C-gamma expressed predominantly in blastoderm cells at cellularization and in endodermal cells during later embryonic stages.
A Drosophila gene encoding a gamma-type isozyme of phosphoinositide-specific phospholipase C (PLC) was isolated and characterized. The gene, termed plc-gamma d, was mapped at position 14B-C of the X chromosome. The encoded protein, termed PLC-gamma D, contains X and Y regions, common to all known PLC isozymes. The two regions are split by a Z region that comprises two src homology 2 and one src homology 3 domains and is characteristic of gamma-type mammalian PLC (PLC-gamma 1 and -gamma 2). The deduced amino acid sequence of PLC-gamma D shows overall similarity to mammalian PLC-gamma s; no large deletion was observed except the short C-terminal extended region. In particular, the two split catalytic domains (X and Y regions) and the regulatory Z region including the src homology 2 and src homology 3 domains are well conserved. The mRNA is expressed throughout development, but expression is relatively higher during the embryonic stage, suggesting fundamental and important roles in both cell proliferation and differentiation. Distribution of the mRNA during embryogenesis, as analyzed by whole amount in situ hybridization, revealed that the mRNA emerges and reaches maximum levels at the cellular blastoderm stage and then decreases rapidly to a lower level. In later embryonic stages, invaginated anterior and posterior midgut primordia show high levels of mRNA expression, and fused midgut also maintains a high level of expression. In other tissues and cells, the mRNA was detected at lower levels. These results indicate that Drosophila PLC-gamma may be involved in universal cellular processes mediated possibly by receptor tyrosine kinases during embryogenesis and may also play specific roles during cellularization and midgut differentiation.
Amino Acid Sequence↗
A Drosophila homolog of human proto-oncogene ret transiently expressed in embryonic neuronal precursor cells including neuroblasts and CNS cells.
We have identified a Drosophila gene encoding a putative receptor tyrosine kinase by screening a genomic DNA library with a DNA probe for a Drosophila homolog of fibroblast growth factor receptors. The newly isolated gene codes for a transmembrane protein most similar in sequence to a mammalian proto-oncogene ret; thus, the gene was termed Dret. Dret mRNA is transcribed in very small amounts in the embryonic, larval, and pupal stages. Whole mount in situ hybridization experiments revealed that the mRNA is transiently expressed in neuroblasts in early embryos. In late embryos, Dret mRNA was detected in subpopulations of differentiating CNS and PNS cells. In addition, Dret expression was affected in neurogenic mutants. These results suggest that Dret can be considered as a functional homolog of mammalian ret and should play important roles in neurogenesis.
Amino Acid Sequence↗
Identification of a novel Drosophila gene encoding a Cdc2-related protein kinase.
We have identified a novel gene encoding a putative protein kinase from a Drosophila genomic library. The gene, about 2 kbp in length, consists of four exons and codes for a protein of 349 amino acid residues. The deduced sequence shows significant similarity to various kinases, especially to a subgroup of Ser/Thr kinases related to Cdc2 kinase; thus, the gene was termed Dcdrk (Drosophila cdc2-related kinase gene). Among the kinases examined, mammalian galactosyltransferase-associated 58 kDa protein kinase showed the highest homology (about 50% identity in the kinase domain) to Dcdrk kinase. Northern blot analysis revealed that the Dcdrk mRNA is expressed throughout development in nearly constant amounts. Moreover, a whole mount in situ hybridization experiment showed that the Dcdrk mRNA is ubiquitously distributed in almost all embryonic cells and tissues, suggesting a universal function of Dcdrk, possibly in cell cycle regulation.
Amino Acid Sequence↗
[She was a nurse and my student in writing class].
Explore the source record for details and available documents.
Education, Nursing↗
Molecular cloning and expression of cDNA encoding rat skeletal muscle cytosolic sialidase.
We have isolated a cDNA clone encoding the cytosolic sialidase of rat skeletal muscle. Degenerate oligonucleotides, based on amino acid sequence data for the purified enzyme, were used as primers to amplify fragments of the gene from rat skeletal muscle cDNA by the polymerase chain reaction. The amplified cDNA fragment was then applied as probe to screen a rat skeletal muscle cDNA library. The longest cDNA clone thus isolated was incomplete at the 5'-end, and therefore an amplified cDNA from the 5'-end portion of the gene was further generated by polymerase chain reaction. These two cDNAs were used to construct a cDNA encoding the entire sequence of rat sialidase. The composite sequence encodes an open reading frame of 379 amino acids that include all sequenced peptides. Although the deduced amino acid sequence is not largely similar to those of bacterial and parasite sialidases, it contains two Asp blocks, the conserved sequence of the sialidases from these microorganisms. When the cDNA was inserted into an expression vector followed by transformation in Escherichia coli, sialidase activity appeared in the cell extract. The sialidase could be completely immunoprecipitated by antiserum against the cytosolic sialidase of rat skeletal muscle.
Amino Acid Sequence↗
Molecular cloning of cDNAs for two Xenopus proteasome subunits and their expression in adult tissues.
Proteasome, a large protein complex with ATP-dependent protease activities, is composed of non-identical but closely related multi-subunits. Using cDNAs for rat proteasome subunits as probes, we obtained three cDNA clones for two Xenopus proteasome subunits from ovary cDNA library. The primary structures of the three cDNAs showed high homology to the corresponding proteasome subunits of other mammalian species (above 90%) and also considerable homology to those of Drosophila and yeast. These results indicate that the sequences of proteasome subunits are well conserved during evolution. Northern blot hybridization revealed that RNAs for the newly isolated subunits (XC8 and XC9) and the previously isolated subunit (XC3) occur at very high levels in testis and ovary, at moderately high levels in lung, skin kidney and spleen, and at low levels in liver, stomach and muscle. It was also shown that relative amounts of the mRNAs for the three subunits are similar in all the adult tissues examined. From these results, we concluded that the expression of the genes for the three subunits (XC3 XC8 and XC9-1) takes place in a roughly coordinated manner in different adult tissues.
Amino Acid Sequence↗
Distinct expression of two Drosophila homologs of fibroblast growth factor receptors in imaginal discs.
The expression of two Drosophila homologs of FGF receptors (DRF1 and DRF2) in imaginal discs was studied. DFR1 mRNA was observed in several imaginal discs, whereas DFR2 mRNA was not detected. DFR1 expression in the wing and leg discs took place in probable myoblasts in a pattern similar to that of twist, a mesodermal gene. The mRNA was also detected in the morphogenetic furrow and its posterior region of the eye disc and around the proliferation center of the brain. These results suggest that DFR1 is involved in the development of mesodermal and neuronal cells constituting the adult body.
Animals↗
Primary structure and cell-type specific expression of a gustatory G protein-coupled receptor related to olfactory receptors.
We have reported on the partial structures of a multigene family encoding GTP-binding protein (G protein)-coupled, seven-transmembrane receptors expressed in the tongue (Abe, K., Kusakabe, Y., Tanemura, K., Emori, Y., and Arai, S. (1993) FEBS Lett. 316, 253-256). Here we describe a full-length cDNA clone encoding a tongue cell-type specific receptor. The encoded protein consists of 312 amino acid residues. In overall structure, the protein is similar to known G protein-coupled, seven-transmembrane receptors such as an olfactory receptor (56% identity) but is significantly different in part, particularly in NH2-terminal extracellular and COOH-terminal cytoplasmic domain structures. Northern analysis showed that the mRNA for this protein is expressed only in the epithelium of the tongue, not in other organs. In situ hybridization experiments clearly indicated that the mRNA is expressed exclusively on the tongue apical surface, not on the reverse side of the tongue nor in its muscle layer. Expression was also detected in the taste buds and surrounding cellular tissues of the fungiform and circumvallate papillae. It is suggested that this gustatory receptor structurally related to olfactory receptors may be a candidate for a taste receptor.
Amino Acid Sequence↗