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Biomedical subjects

S Natori

Publications and source records attributed to S Natori.

At least 55 records · Page 3Linked to original sources
PubMed1999

Identification and characterization of a putative sevenless homologue in the malaria vector Anopheles gambiae.

Tyrosine kinase sequences were identified and characterized in Anopheles gambiae, the major vector of malaria in subsaharan Africa. One of these sequences has the characteristics expected for a homologue of the Drosophila sevenless gene, which is necessary for R7 photoreceptor cell fate determination in the developing compound eye. The putative Anopheles sevenless gene homologue is located in a telomeric region of the X chromosome and is expressed in the head of late larval and pupal stage mosquitoes. Identification of the Anopheles homologue of the sevenless gene is a first step towards the development of a dominant phenotypic marker that could be used for detecting transformed Anopheles mosquitoes in a wide variety of genetic backgrounds and, as such, could be used in the development of transgenic mosquitoes for the control of parasite transmission. Preliminary evidence for sevenless sequences were also found in DNA from blackfly, Mediterranean fruit fly and the honeybee.

Amino Acid Sequence↗
PubMed1999

A novel egg-derived tyrosine phosphatase, EDTP, that participates in the embryogenesis of Sarcophaga peregrina (flesh fly).

We have previously reported that cathepsin L mRNA is present in unfertilized eggs of Sarcophaga peregrina (flesh fly) as a maternal mRNA, which suggests that cathepsin L is required for embryogenesis. Now we have identified an egg protein, with a molecular mass of 100 kDa, that is extremely susceptible to cathepsin L digestion and which disappears rapidly as the embryos develop. We purified this protein to homogeneity, cloned its cDNA, and found that it contained a consensus sequence for the active site of tyrosine phosphatase. In fact this protein showed tyrosine phosphatase activity, indicating that it is a novel tyrosine phosphatase. The expression and subsequent disappearance of this protein, which we have named egg-derived tyrosine phosphatase (EDTP), may be indispensable for embryogenesis of Sarcophaga.

Amino Acid Sequence↗
PubMed1999

Molecular cloning and functional properties of two early-stage encapsulation-relating proteins from the coleopteran insect, Tenebrio molitor larvae.

Encapsulation is a major defensive reaction against foreign materials that are too large to be phagocytosed by individual hemocytes; however, the biochemical process of encapsulation is still obscure. To isolate and characterize the early-stage encapsulation-relating protein (ERP), we used the coleopteran insect, Tenebrio molitor larvae, injecting three differing kinds of bead or inserting pieces of surgical suture into the abdomen of T. molitor larvae. The resulting proteins from the injected beads or the inserted pieces of surgical suture were recovered 10 min after injection or insertion, and were analyzed on SDS/PAGE under reducing conditions. Four different proteins (86, 78, 56 and 48 kDa) were enriched compared with the crude hemolymph. Among them, we purified 56-kDa and 48-kDa ERPs to homogeneity and raised polyclonal antibodies against each protein. Immunoblotting analysis showed that the affinity-purified antibodies of the 56-kDa and 48-kDa ERPs cross-reacted with the 48-kDa and 56-kDa ERPs, respectively. Analysis of the cDNA of 56-kDa ERP consisted of 579 amino acid residues and showed a novel glutamine-rich protein. Positive clones of the 48-kDa ERP showed the same DNA sequence as 56-kDa ERP. Interestingly, the chemically determined N-terminal amino acid sequence and the three partial amino acid sequences of the 48-kDa protein were found in the 56-kDa ERP, suggesting that the 48 kDa ERP was produced by the cleavage of Arg101-Gly102 of the 56-kDa ERP by a limited proteolysis. Western blotting analysis showed that these ERPs were detected exclusively on membrane fractions of hemocytes. Also, when the early-stage encapsulated beads were coated with both the 56-kDa and 48-kDa ERP antibodies and re-injected into larvae, no further encapsulation reaction was observed. However, when the early-stage encapsulated beads were incubated with 56-kDa ERP antibody, 48-kDa ERP antibody or nonimmunized rabbit IgG and re-injected into larvae, further encapsulation did occur.

Amino Acid Sequence↗
PubMed1999

Activation of human neutrophils by a synthetic anti-microbial peptide, KLKLLLLLKLK-NH2, via cell surface calreticulin.

We previously reported that a synthetic anti-bacterial peptide, KLKLLLLLKLK-NH2 (L5), showed significant chemotherapeutic activity in methicillin-resistant Staphylococcus aureus-infected mice, and its ability to activate human neutrophils was related to its chemotherapeutic activity. In this study, we found that activation of neutrophils by L5 was inhibited by pertussis toxin, suggesting that GTP-binding protein (G-protein) participates in this process. We isolated an L5-binding protein, which turned out to be human calreticulin, with a molecular mass of 60 kDa from neutrophil membranes. From experiments using an anti-calreticulin antibody, we proposed that calreticulin is partly localized on the surface of neutrophils, and L5-bound calreticulin transmits a signal into cells via G-protein to activate neutrophils to generate superoxide anion.

Amino Acid Sequence↗
PubMed1999

Enhanced resistance to bacterial diseases of transgenic tobacco plants overexpressing sarcotoxin IA, a bactericidal peptide of insect.

Sarcotoxin IA is a bactericidal peptide of 39 amino acids found in the common flesh fly, Sarcophaga peregrina. Many agronomically important bacteria in Japan are killed by this peptide at sub-micro molar levels, and the growth of tobacco and rice suspension cultured cells is not inhibited with less than 25 microM. Transgenic tobacco plants which overexpress the peptide, i.e. over 250 pmol per gram of fresh leaf, under the control of a high expression constitutive promoter showed enhanced resistance to the pathogens for wild fire disease (Pseudomonas syringae pv. tabaci) and bacterial soft rot disease (Erwinia carotovora subsp. carotovora).

Animals↗
PubMed1999

Two subunits of the insect 26/29-kDa proteinase are probably derived from a common precursor protein.

We previously identified the 26/29-kDa proteinase in the hemocytes of Sarcophaga peregrina (flesh fly) that appears to participate in elimination of foreign proteins in this insect [Eur. J. Biochem. 209, 939-944 (1992)]. Here, we report the cDNA cloning of this proteinase. The cDNA encodes a protein which includes both the 26- and 29-kDa subunit, strongly suggesting that the both subunits are derived from a single precursor protein. The 26- and 29-kDa subunit located at the amino-terminal and carboxyl-terminal of the precursor protein. The 29-kDa subunit itself appeared to be a proteinase, for this subunit had 52% sequence identity with Sarcophaga cathepsin L, while 26-kDa subunit had no significant similarity. We also showed that 26/29-kDa proteinase was insensitive to specific inhibitors of cathepsin L. These results indicate that this proteinase is a novel member of the papain family. We isolated similar cDNAs from Drosophila melanogaster and Periplaneta americana (cockroach), suggesting that this proteinase is conserved in a wide variety of insects and participates in their defense mechanisms.

Amino Acid Sequence↗
PubMed1999

Induction of Sarcophaga central nervous system remodeling by 20-hydroxyecdysone in vitro.

Proliferation and apoptosis of neural cells were found to be induced simultaneously when larval brains of Sarcophaga peregrina were cultured in the presence of 20-hydroxyecdysone (20-HE) for 24 h. The locations of proliferating cells and apoptotic cells in the brain hemispheres were different. The morphology of brains exposed to 20-HE for a short period proceeded to change sequentially when culture was continued for 2 days even in the absence of 20-HE. These changes mainly consisted of enlargement of the brain hemispheres and extension of the interval between two hemispheres, which closely paralleled the morphological changes of brains that occur in the early pupal stage, suggesting that ecdysteroid alone is sufficient to induce the remodeling of the central nervous system of holometabolous insects. Synthesis of a protein with a molecular mass of 66 kDa was shown to be selectively repressed when brains were cultured in the presence of 20-HE.

Animals↗
PubMed1999

Stage-specific inhibition of Xenopus embryogenesis by aprotinin, a serine protease inhibitor.

We examined the effects of various protease inhibitors on Xenopus laevis embryogenesis. Aprotinin, a serine protease inhibitor, was found to inhibit embryogenesis markedly, but other protease inhibitors had virtually no effect. The inhibitory effect of aprotinin was specific for embryos at the blastula or gastrula stage. These results suggest that an aprotinin-sensitive protease involved in embryonic development is secreted from the embryos or appears on the surface of embryonic cells at these stages. We found that various serine proteases are in fact secreted from the embryos with their development and that some of them are sensitive to aprotinin.

Amino Acid Sequence↗
PubMed1999

Different inhibitory effects of 5-S-glutathionyl-beta-alanyl-L-dopa (5-S-GA-L-D) analogues on autophosphorylation and substrate phosphorylation of Src protein tyrosine kinase.

Starting with 5-S-glutathionyl-beta-alanyl-L-dopa (1) and 5-S-glutathionyl-beta-alanyl-dopamine (2), a series of analogues with truncated glutathionyl and beta-alanyl-dopa moieties were synthesized, and their inhibitory effects on autophosphorylation and substrate phosphorylation reaction by c-Src and by epidermal growth factor receptor (EGFR) were evaluated. When the glutamyl residue was removed, the inhibitory effects on v-Src autophosphorylation decreased about 4- to 5-fold, and concomitant removal of the glutamyl and beta-alanyl residues resulted in a 40- to 60-fold decrease in the inhibition of v-Src autophosphorylation. On the other hand, these modifications had little effect on the inhibitory activity of substrate (Raytide) phosphorylation by c-Src. Interestingly, 5-S-cysteinyl dopamine inhibited the Src substrate phosphorylation reaction with comparable potency to that of genistein. Nonpeptide lipophilic derivatives had a similar inhibition on v-Src autophosphorylation but decreased inhibitory effects on substrate phosphorylation when compared to the lead compounds. Most compounds showed little effect on substrate phosphorylation by EGFR.

Dopamine↗
PubMed1999

Inhibition effects of 5-S-glutathionyl-N-beta-alanyl-L-dopa analogues against Src protein tyrosine kinase.

Twelve analogues of the antibacterial phenolic peptide 5-S-glutathionyl-N-beta-alanyl-L-dopa (5-S-GA-L-D, 1) were synthesized via orthoquinone using tyrosinase. Several synthesized compounds inhibited the v-Src autophosphorylation tyrosine kinase reaction with an IC50 value comparable to that of herbimycin A. The inhibition of c-Src substrate phosphorylation was much less active than v-Src autophosphorylation inhibition. 5-S-GA-L-D (1) and its analogous competed with peptide substrate and non-compared with ATP. The analogues showed no effects on substrate phosphorylation by epidermal growth factor receptor (EGFR), and this selectivity is the most characteristic feature of the 5-S-GA-L-D and its analogues (1-12).

3T3 Cells↗
PubMed1998

Molecular cloning of cDNA for pro-phenol-oxidase-activating factor I, a serine protease is induced by lipopolysaccharide or 1,3-beta-glucan in coleopteran insect, Holotrichia diomphalia larvae.

Previously, we identified two pro-phenol oxidase-activating factors, named PPAF-I and PPAF-II, directly involved in the activation of the purified pro-phenol oxidase (pro-PO) from the hemolymph of the coleopteran, Holotrichia diomphalia larvae [Lee, S. Y., Kwon, T. H., Hyun, J. H., Choi, J. S., Kawabata, S. I., Iwanga, S, & Lee, B. L. (1998) Eur. J. Biochem. 254, 90-97]. Here, we report molecular cloning of cDNA for PPAF-I. Based on the sequence of the cloned cDNA, the PPAF-I gene appears to encode a member of serine protease zymogen consisting of 365 amino acid residues with a molecular mass of 40193 Da. The 109 amino acid residues preceding the amino-terminus Ile residue of the mature protein seem to constitute a prepro-sequence. The mature protein is a serine protease composed of 256 amino acids with a calculated molecular mass of 28009 Da. The overall structure is highly similar to that of Drosophila easter serine protease (42.9% identity), an essential serine protease zymogen for pattern formation in normal embryonic development. The locations of disulfide linkages in the pro-segment of PPAF-I were similar to those of Tachypleus proclotting enzyme and the mammalian neutrophil-derived defensin. Furthermore, [3H]diisopropylphosphate (iPr2P)-labeled PPAF-I was specifically produced from the crude preparation of PPAF-I zymogen by incubation with lipopolysaccharide or 1,3-beta-glucan, whereas [3H]iPr2P-labeled PPAF-I was not produced under the same conditions in the absence of these microbial polysaccharides. These results indicate that the pro-PO-activation system in H. diomphalia larvae may proceed with the activation of PPAF-I zymogen by microbial polysaccharides.

Amino Acid Sequence↗
PubMed1998

Chromogranin B (secretogranin I), a neuroendocrine-regulated secretory protein, is sorted to exocrine secretory granules in transgenic mice.

Chromogranin B (CgB, secretogranin I) is a secretory granule matrix protein expressed in a wide variety of endocrine cells and neurons. Here we generated transgenic mice expressing CgB under the control of the human cytomegalovirus promoter. Northern and immunoblot analyses, in situ hybridization and immunocytochemistry revealed that the exocrine pancreas was the tissue with the highest level of ectopic CgB expression. Upon subcellular fractionation of the exocrine pancreas, the distribution of CgB in the various fractions was indistinguishable from that of amylase, an endogenous constituent of zymogen granules. Immunogold electron microscopy of pancreatic acinar cells showed co-localization of CgB with zymogens in Golgi cisternae, condensing vacuoles/immature granules and mature zymogen granules; the ratio of immunoreactivity of CgB to zymogens being highest in condensing vacuoles/immature granules. CgB isolated from zymogen granules of the pancreas of the transgenic mice aggregated in a mildly acidic (pH 5.5) milieu in vitro, suggesting that low pH-induced aggregation contributed to the observed concentration of CgB in condensing vacuoles. Our results show that a neuroendocrine-regulated secretory protein can be sorted to exocrine secretory granules in vivo, and imply that a key feature of CgB sorting in the trans-Golgi network of neuroendocrine cells, i.e. its aggregation-mediated concentration in the course of immature secretory granule formation, also occurs in exocrine cells although secretory protein sorting in these cells is thought to occur largely in the course of secretory granule maturation.

Animals↗
PubMed1998

A novel lectin from Sarcophaga. Its purification, characterization, and cDNA cloning.

A novel C-type lectin that agglutinates rabbit red cells was purified from NIH-Sape-4 cells derived from the flesh fly (Sarcophaga peregrina), and its cDNA was isolated. This lectin, named granulocytin, appeared to be a trimer of a 20-kDa subunit consisting of 151 amino acid residues. The gene for granulocytin was activated in third instar larvae, and its expression was enhanced when the larval body wall was injured. In third instar larvae, granulocytin was found to be synthesized by hemocytes and secreted into the hemolymph. The molecular mass and gene expression patterns of granulocytin were very similar to those of Drosophila lectin that we reported previously (Haq, S., Kubo, T., Kurata, S., Kobayashi, A., and Natori, S. (1996) J. Biol. Chem. 271, 20213-20218). However, these two lectins showed amino acid identities of 20% at most, and no significant hapten sugar for granulocytin was identified.

Adipose Tissue↗
PubMed1998

Antibacterial activity of a novel 26-kDa serine protease in the yellow body of Sarcophaga peregrina (flesh fly) pupae.

We have reported a novel serine protease produced by Sarcophaga peregrina (Nakajima et al., J. Biol. Chem. 272 (1997) 23805-23810). This 26-kDa protease showed antibacterial activity against several bacteria. This activity was an intrinsic characteristic of the enzyme protein and not directly related to its protease activity, because treating the 26-kDa protease with diisopropyl fluorophosphate had no appreciable effect on its antibacterial activity. Unlike bovine trypsin, the 26-kDa protease interacted with acidic phospholipids, suggesting that its antibacterial activity is attributable to interaction with bacterial membranes.

Animals↗
PubMed1998

Preferential expression of the gene for a putative inositol 1,4,5-trisphosphate receptor homologue in the mushroom bodies of the brain of the worker honeybee Apis mellifera L.

A gene expressed preferentially in the mushroom bodies of the brain of the worker honeybee Apis mellifera L. was identified by the differential display method and its cDNA was isolated. The cDNA fragment of 534 bp (clone A1) contained an open reading frame encoding 177 amino acid residues having 78, 72, 70, 59 and 55% sequence identities with the inositol 1,4,5-trisphosphate (IP3) receptors of Drosophila melanogaster, Xenopus laevis and humans (types 1, 2 and 3), respectively, suggesting that it encodes a putative IP3 receptor homologue of the honeybee. In situ hybridization revealed that the gene encoding clone A1 was expressed preferentially in the mushroom bodies and not in the optic lobes, antennal lobes and central bodies; in the mushroom body, it was expressed strongly in the large type Kenyon cells and weakly in the small type Kenyon cells. Reverse transcription polymerase chain reaction analysis showed that the gene was expressed strongly in the head and weakly in the antennae, legs, thorax, and abdomen. These results suggest that the A1 gene product plays a crucial role in neural transmission in the mushroom bodies of the worker bee brain.

Amino Acid Sequence↗
PubMed1998

Cholestasis confers resistance to the rat liver mitochondrial permeability transition.

BACKGROUND & AIMS: Bile salts can cause hepatocyte death by inducing the mitochondrial permeability transition (MPT). However, the slow progression of human cholestatic liver diseases suggests that hepatocytes adapt to resist the MPT. Bcl-x, a protein, and increased mitochondrial cardiolipin, a membrane lipid, elevate the threshold for the MPT. Our aims were to determine if liver mitochondria become resistant to the MPT during cholestasis and, if so, if the resistance is mediated by Bcl-x and/or increased cardiolipin. METHODS: Hepatocytes and liver mitochondria were obtained from bile duct-ligated (BDL) rats and sham-operated rats (control). RESULTS: After addition of glycochenodeoxycholate (GCDC), the magnitude of the MPT was reduced in mitochondria from BDL rats vs. controls. Although Bcl-xL was not increased, mitochondrial cardiolipin content was significantly greater in BDL rats vs. controls. Cell viability was also increased in hepatocytes from BDL rats vs. controls after treatment with GCDC. Feeding BDL rats a fatty acid-deficient diet prevented the increase in mitochondrial cardiolipin content; mitochondria and hepatocytes from these rats were susceptible to the MPT and hepatocellular death by GCDC. CONCLUSIONS: These data suggest that an increase in mitochondria cardiolipin content occurs during cholestasis as an adaptive phenomenon to resist cell death by the MPT.

Animals↗