Biomedical subjects

S Natori

Publications and source records attributed to S Natori.

At least 91 records · Page 5Linked to original sources

PubMed1997

Purification and characterisation of cathepsin B mRNA 3'-untranslated-region-binding protein (CBBP), a protein that represses cathepsin B mRNA translation.

We reported that the 3'-untranslated region (3'-UTR) of cathepsin B mRNA of Sarcophaga peregrina (flesh fly) is necessary for the repression of its translation, and detected the 3'-UTR-binding protein in lysates of larval hemocytes in which cathepsin B mRNA translation was repressed [Yano, T., Kurata, S. & Natori, S. (1995) Eur. J. Biochem. 234, 39-43]. In this study, we purified the 3'-UTR-binding protein from an embryonic cell line of Sarcophaga. The purified protein (CBBP) was found to repress cathepsin B mRNA translation in a rabbit reticulocyte lysate. We found that the CBBP contents of the hemocytes did not change during metamorphosis, although the cathepsin B mRNA became translatable only at the pupal stage. Moreover, we found that pupal, but not larval hemocytes, contained a factor that inhibited the binding of CBBP to the 3'-UTR. A regulatory mechanism of cathepsin B expression in Sarcophaga hemocytes is discussed.

Animals↗

PubMed1997

Regeneration of Sarcophaga imaginal discs in vitro: implication of 20-hydroxyecdysone.

When the 3/4 sectors of leg imaginal discs of Sarcophaga were cultured in vitro in the presence of 2.5 x 10(-8) M 20-hydroxyecdysone, wound healing and restoration of their morphology occurred. This concentration of ecdysone was critical for wound healing and was 40 times lower than that necessary for inducing differentiation of imaginal discs in vitro. Lost positional values revealed by expression of the wingless gene were found to show partial recovery under these conditions. These results suggest that a low titer of ecdysone is essential for the regeneration of imaginal discs.

Amino Acid Sequence↗

PubMed1997

Molecular cloning of cDNA for Sarcophaga prolyl endopeptidase and characterization of the recombinant enzyme produced by an E. coli expression system.

A cDNA for prolyl endopeptidase (PEP) of Sarcophaga peregrina (flesh fly) was cloned and its sequence determined. The overall amino acid sequence identity between Sarcophaga and mammalian PEPs was 53%, indicating that these enzymes are structurally very similar. Northern blot hybridization revealed that the Sarcophaga PEP gene was activated significantly at the eversion stage of imaginal disc differentiation. We obtained recombinant PEP by expressing the cDNA in Escherichia coli. The recombinant and authentic enzymes showed almost identical characteristics, in terms of substrate specificities and sensitivities to inhibitors.

Amino Acid Sequence↗

PubMed1997

Nuclear localization and involvement in DNA synthesis of Sarcophaga prolyl endopeptidase.

A specific prolyl endopeptidase (PEP) inhibitor, ZTTA, selectively inhibited DNA synthesis by imaginal discs and cultured embryonic cells of Sarcophaga peregrina (flesh fly). PEP was found to be localized in restricted nuclear regions. Unfertilized eggs were shown to contain a maternal message for PEP and analysis of Sarcophaga embryos at blastdermal stage revealed PEP was localized exclusively in the nuclei. These results suggest that PEP participates in DNA synthesis by, and therefore cell proliferation, of insect cells. This is the first demonstration of a biological function of PEP.

Animals↗

PubMed1996

Identification of substrate proteins for cathepsin L that are selectively hydrolyzed during the differentiation of imaginal discs of Sarcophaga peregrina.

Previously, we showed that cathepsin L is essential for the differentiation of imaginal discs of the flesh fly [Homma, K. & Natori, S. (1994) J. Biol. Chem. 269, 15258-15264]. We have now identified imaginal disc proteins that are susceptible to digestion by cathepsin L and showed that they are selectively hydrolyzed during imaginal disc differentiation. Two of these proteins, with molecular masses of 210 and 200 kDa, were further characterized. Immunofluorescence studies suggested that they were intrinsic components of the basement membranes of various tissues. They were selectively hydrolyzed at the elongation stage of imaginal leg disc differentiation. Western blotting of embryos at various developmental stages showed that these proteins were only detected at the end of embryogenesis.

Animals↗

PubMed1996

Purification, characterization, and cDNA cloning of a galactose-specific C-type lectin from Drosophila melanogaster.

We purified a lectin from a pupal extract of Drosophila melanogaster. This lectin agglutinated trypsinized and glutaraldehyde-fixed bovine red blood cells in the presence of calcium or magnesium. The hapten sugar of this lectin was galactose. The molecular mass of the intact lectin was determined to be 41 kDa, and it comprised 14- and 17-kDa subunits. The 17-kDa subunit was shown to be a glycosylated form of the 14-kDa subunit. Analysis of the cDNA for this lectin revealed that the 14-kDa subunit consists of 163 amino acid residues and contains all residues conserved in various C-type lectins. It was suggested that the Drosophila lectin and Sarcophaga lectin share some properties and function similarly in defense and development, but probably they are not structural homologues.

Amino Acid Sequence↗

PubMed1996

Purification and characterization of N-beta-alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine, a novel antibacterial substance of Sarcophaga peregrina (flesh fly).

We purified a novel antibacterial substance from immunized adult Sarcophaga and determined its molecular structure to be N-beta-alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD). We synthesized 5-S-GAD enzymatically from N-beta-alanyl-3, 4-dihydroxyphenylalanine (beta-Ala-Dopa) and reduced glutathione (GSH). The antibacterial activity of 5-S-GAD was found to be due to its production of H2O2. This is a novel antibacterial mechanism as it differs from the mechanisms of known antibacterial peptides. Two possible roles of 5-S-GAD in insect immunity, suppression of bacterial growth and activation of a Rel family transcription factor, are proposed.

Animals↗

PubMed1996

Purification, characterization, and cDNA cloning of a novel growth factor from the conditioned medium of NIH-Sape-4, an embryonic cell line of Sarcophaga peregrina (flesh fly).

A growth factor from the conditioned medium of NIH-Sape-4, an embryonic cell line of the flesh fly, was purified to homogeneity. This growth factor, termed IDGF, stimulated the proliferation of NIH-Sape-4 cells in an autocrine manner; it was a homodimer of a protein with a molecular mass of 52 kDa, and its specific activity was comparable with those of mammalian growth factors. Immunoblotting experiments revealed that unfertilized mature eggs of the flesh fly contained this growth factor, a certain level of which was maintained throughout embryonic development. Analysis of cDNA for this growth factor showed that this factor is a novel protein consisting of 553 amino acid residues. No significant sequence similarity was found between this factor and other proteins except atrial gland granule-specific antigen of Aplysia californica.

Amino Acid Sequence↗

PubMed1996

Purification and cDNA cloning of the alcohol dehydrogenase of the flesh fly Sarcophaga peregrina. A structural relationship between alcohol dehydrogenase and a 25-kDa protein.

We purified to homogeneity two proteins with molecular masses of 25 kDa from the fat body of the Sarcophaga larva. One was alcohol dehydrogenase (ADH) and the other was a 25-kDa protein of which the genomic DNA had been cloned. We isolated the cDNA for ADH and determined its amino acid sequence. Amino acid sequence identity between ADH and the 25-kDa protein was 40%, indicating that they are structurally related proteins. The amount of ADH in Sarcophaga was almost constant through the larval stage to the adult stage, but the 25-kDa protein was detected only within a restricted period between the final larval instar and the early pupal stage.

Alcohol Dehydrogenase↗

PubMed1996

Chromogranin B (secretogranin I) promotes sorting to the regulated secretory pathway of processing intermediates derived from a peptide hormone precursor.

Chromogranin B (CgB, secretogranin I) is a widespread constituent of neuroendocrine secretory granules whose function is unknown. To determine whether CgB affects the sorting of peptide hormone and neuropeptide precursors to secretory granules, we overexpressed CgB in AtT-20 cells, which exhibit an only moderate capacity to sort proopiomelanocortin and proteolytic fragments derived therefrom. In mock-transfected AtT-20 cells, a substantial proportion of newly synthesized proopiomelanocortin and its two primary proteolytic products generated in the trans-Golgi network, the N-terminal 23-kDa fragment containing adrenocorticotropin and the C-terminal beta-lipotropin fragment, was secreted via the constitutive pathway. Two- to three-fold overexpression of CgB markedly reduced the constitutive secretion of the 23-kDa fragment, but not beta-lipotropin and tripled the amount of adrenocorticotropin generated and stored in secretory granules. Our results indicate the existence of neuroendocrine-specific helper proteins which promote the sorting from the trans-Golgi network to secretory granules of certain processing intermediates derived from peptide hormone and neuropeptide precursors and demonstrate that CgB functions as such.

Adrenocorticotropic Hormone↗

PubMed1996

Spermatocyte-specific expression of the gene for mouse testis-specific transcription elongation factor S-II.

Previously, we characterized a rat cDNA for testis-specific transcription elongation factor S-II (SII-T1) (Q. Xu et al., J. Biol. Chem. 269, 3100-3103 (1994)). Here, we isolated a 335-bp fragment of the cDNA for mouse SII-T1, and used it to examine the expression of the SII-T1 gene in the testis by in situ hybridization. The results indicated that the SII-T1 gene is expressed exclusively in spermatocytes, showing no appreciable expression in spermatogonia, spermatids, or Leydig cells. RT-PCR experiments using testis RNA from W/Wv mutant mice also suggested that SII-T1 is a specific transcription elongation factor essential for spermatogenesis.

Amino Acid Sequence↗

PubMed1996

Molecular cloning of cDNA and analysis of expression of the gene for alpha-glucosidase from the hypopharyngeal gland of the honeybee Apis mellifera L.

Previously, we identified and purified an alpha-glucosidase with a molecular mass of 70 kDa from a homogenate of the hypopharyngeal gland of the older worker bee (forager bee) Apis mellifera L. (T. Kubo et al, J. Biochem., 1996, 119, 291-295). Here, we isolated and sequenced a cDNA for the alpha-glucosidase. The cDNA encoded a protein consisting of 650 amino acids, which had high sequence identity with fruit fly and mosquito possible maltase gene products. RT-PCR showed that the gene was expressed specifically in the hypopharyngeal gland of the forager bee.

Amino Acid Sequence↗

PubMed1996

Presence of the Periplaneta lectin-related protein family in the American cockroach Periplaneta americana.

We determined the partial amino acid sequences of Periplaneta lectin, which we had purified and characterized previously from the hemolymph of the American cockroach (Periplaneta americana) [Kubo T. and Natori S. (1987) Eur. J. Biochem. 168, 75-82]. Based on these sequences, we performed PCR and found that the cDNA library of the Periplaneta fat body contained many similar, but not identical, Periplaneta lectin-related cDNAs. Analysis of the cloned cDNAs suggested that Periplaneta has a protein family, of which the periplaneta lectin and LPS binding protein purified previously are members.

Amino Acid Sequence↗

PubMed1996

Change in the expression of hypopharyngeal-gland proteins of the worker honeybees (Apis mellifera L.) with age and/or role.

The roles of adult worker honeybees change with age; young workers nurse brood by secreting bee milk (royal jelly), and older workers forage for nectar and pollen and process nectar into honey. The electrophoretic profile of worker hypopharyngeal-gland proteins changes with age and/or role. Immunoblotting analysis using affinity-purified antibodies against three major proteins (50, 56, and 64 kDa) of the nurse-bee gland showed that they are synthesized selectively and secreted as bee-milk proteins. Immunofluorescence study showed that the proteins condense in the duct after secretion from acini. However, a major 70-kDa protein synthesized specifically in the forager-bee hypopharyngeal gland was identified as an alpha-glucosidase. Therefore, the hypopharyngeal gland seems to have two distinct states differentiated by synthesizing of different major proteins depending on the age-dependent role change.

Aging↗

PubMed1996

Transmethylation reaction is essential for Sarcophaga lectin gene activation.

Incubation of Sarcophaga fat bodies in buffered insect saline resulted in simultaneous activation of multiple defense protein genes [Sugiyama, H. and Natori, S. (1991) Eur. J. Biochem. 200, 495-500]. In this study, we demonstrated that the addition of both adenosine and homocysteine thiolactone to this medium abolished activation of these defense protein genes. As S-adenosyl-homocysteine, a potent inhibitor of the transmethylation reaction, is produced in the fat body under these conditions, methylation of a certain fat body protein seems to be a prerequisite for the selective activation of insect defense protein genes. Activation of the HSP70 gene was not interfered with S-adenosyl-homocysteine.

Animals↗

PubMed1996

Selective interaction of synthetic antimicrobial peptides derived from sapecin B with lipid bilayers.

By measuring carboxyfluorescein leakage from liposomes and the increase in membrane current through planar lipid bilayer membranes, we examined the capacities of a series of low-molecular-weight cationic amphiphilic peptides derived from the alpha-helix domain of sapecin B for membrane-perturbation and ion-channel formation. Some of these peptides strongly interact with membranes containing acidic phospholipids and phosphatidylethanolamine, with a very negative potential, which are characteristic of the Escherichia coli membrane, in parallel with their antimicrobial activity. In contrast, they do not interact with membranes which predominantly contain choline phospholipids and cholesterol in their outer leaflets, with a slightly negative potential, all of which are characteristic of eukaryotic membranes, thereby providing a molecular basis for their selective toxicity. Membranes doped with these peptides are as permeable to inorganic phosphates as to chloride ions and are far more permeable to cations. The loss of inorganic phosphates may damage bacterial cells due to rapid depletion of cytoplasmic ATP. Examination of the structure-activity relationships of a series of derived peptides in their interaction with a model of the E. coli membrane confirmed the necessity of cationic amphiphilicity for the peptides to attack the bacterial membrane and to exhibit antimicrobial activity.

Adenosine Triphosphate↗

PubMed1996

Purification, characterization, and cDNA cloning of ABP-2 (arylphorin gene-specific binding protein-2) that specifically binds to the ABP-1-binding sequence in the arylphorin gene of Sarcophaga peregrina.

Previously, we demonstrated that ABP-1 (arylphorin gene-specific binding protein-1), which is suggested to be the transcriptional activator of the arylphorin gene of Sarcophaga peregrina, is present in NIH-Sape-4 cells, which do not express arylphorin. As well as ABP-1, these cells were found to contain another protein (ABP-2) that probably binds to the same sequence as that to which ABP-1 binds [Adachi, N., Kubo, T., and Natori, S. (1993) J. Biochem. 114, 55-60]. We purified ABP-2 from a nuclear extract of NIH-Sape-4 cells and compared its DNA-binding activity with that of ABP-1. Both ABP-1 and ABP-2 were found to bind to the same sequence in the arylphorin gene with the same affinity and stability, but an ABP-2-specific hypersensitive site was detected by DNase I footprinting analysis. Analyses of proteolytic fragments suggested that both ABP-1 and ABP-2 have Zn fingers showing high similarity with that of AEF-1, a transcriptional repressor of the Drosophila melanogaster alcohol dehydrogenase gene that binds to a sequence very similar to that binding ABP-1 and ABP-2. We isolated a candidate cDNA for ABP-2, and the protein it encoded contained nine Zn fingers and regions rich in alanine, glutamine, serine/threonine, glycine, histidine, and asparagine.

Amino Acid Sequence↗

PubMed1996

Secretory protein 7B2 response to oral glucose loading and intravenous glucagon injection in patients with diabetes mellitus.

Serum 7B2 concentrations in control subjects and patients with diabetes mellitus were measured following a 75 g oral glucose load and following intravenous glucagon infusion. In response to oral glucose, serum 7B2 levels increased in the controls (n = 10) and in the diabetic patients (n = 7). The increment of the serum 7B2 level was smaller in the diabetic patients than the controls. During the 75 g oral glucose tolerance test (75g OGTT), serum 7B2 levels were significantly positively correlated with serum C-peptide levels. In contrast, following intravenous glucagon infusion, serum 7B2 levels increased only in diabetic patients treated with oral hypoglycemic agents (n = 20) and did not increase in controls (n = 5): the group having the highest insulin secretion activity in the present study, nor in diet or insulin-treated diabetic patients. No correlation between serum 7B2 levels and serum CPR levels was observed in the intravenous glucagon infusion study. These data suggest that an extra-pancreatic source which produces the observed serum 7B2 increase following oral glucose intake can not be excluded and that 7B2 may not be secreted concomitantly with insulin from the pancreatic beta cell in response to intravenous glucagon injection.

Administration, Oral↗