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Biomedical subjects

S Natori

Publications and source records attributed to S Natori.

At least 109 records · Page 6Linked to original sources
PubMed1995

Regulation of the expression of cathepsin B in Sarcophaga peregrina (flesh fly) at the translational level during metamorphosis.

Previously, we reported that cathepsin B of pupal hemocytes participates in dissociation of the fat body of Sarcophaga larvae at metamorphosis. In this study, we demonstrated that the amount of cathepsin B in hemocytes was controlled at the translational level, i.e. larval hemocytes stored a significant amount of untranslated cathepsin B mRNA. When the larvae pupated, translation of the mRNA commenced, resulting in accumulation of cathepsin B. Our results indicate that the 3'-untranslated region (3'-UTR) of this mRNA is necessary for the repression of its translation in a rabbit reticulocyte lysate. We detected the 3'-UTR binding protein in the lysate of larval hemocytes, but not in the pupal hemocytes. The role of this protein in the translation of cathepsin B mRNA is discussed.

Animals↗
PubMed1995

Molecular cloning of cDNA for sapecin B, an antibacterial protein of Sarcophaga, and its detection in larval brain.

A cDNA clone for sapecin B, an antibacterial protein of Sarcophaga, was isolated. This cDNA encoded a precursor protein of sapecin B consisting of a signal sequence (24 residues), prosegment (30 residues) and mature sapecin B (34 residues). Sapecin B was synthesized almost exclusively in the fat body when the larval body wall was injured, but the brain of naive larvae was also demonstrated to contain a significant amount of sapecin B. These findings suggested that sapecin B is a bifunctional protein.

Amino Acid Sequence↗
PubMed1995

Molecular cloning and nuclear localization of ATBP, a novel (A+T)-stretch-binding protein of Sarcophaga peregrina (flesh fly).

Previously, we purified a DNA-binding protein, named ATBP, from cultured Sarcophaga cells. This protein was found to bind specifically to (A+T) stretches that locate in the 5'-upstream region and the intron of the Sarcophaga lectin gene [Matsui, M., Kobayashi, A., Kubo, T. & Natori, S. (1994) Eur. J. Biochem. 219, 449-454]. Here, we report molecular cloning of a cDNA for ATBP. The cDNA encoded a protein with two zinc-finger motifs, an acidic domain, two glutamine-rich domains and a glutamine run. Immunofluorescence study revealed that ATBP was localized in the nuclei. Thus, ATBP is possibly a transcription factor for genes having (A+T) stretches in their promoter and/or enhancer regions.

Amino Acid Sequence↗
PubMed1995

Structure-function relationship of yeast S-II in terms of stimulation of RNA polymerase II, arrest relief, and suppression of 6-azauracil sensitivity.

The yeast S-II null mutant is viable, but the mutation induces sensitivity to 6-azauracil. To examine whether the region needed for stimulation of RNA polymerase II and that for suppression of 6-azauracil sensitivity in the S-II molecule could be separated, we constructed various deletion mutants of S-II and expressed them in the null mutant using the GAL1 promoter to see if the mutant proteins suppressed 6-azauracil sensitivity. We also expressed these constructs in Escherichia coli, purified the mutant proteins to homogeneity, and examined if they stimulated RNA polymerase II. We found that a mutant protein lacking the first 147 amino acid residues suppressed 6-azauracil sensitivity but that removal of 2 additional residues completely abolished the suppression. A mutant protein lacking the first 141 residues had activity to stimulate RNA polymerase II, whereas removal of 10 additional residues completely abolished this activity. We also examined arrest-relief activity of these mutant proteins and found that there is a good correlation between RNA polymerase II-stimulating activity and arrest-relief activity. Therefore, at least the last 168 residues of S-II are sufficient for expressing these three activities.

Antimetabolites↗
PubMed1995

Molecular cloning and sequencing of arylphorin-binding protein in protein granules of the Sarcophaga fat body. Implications of a post-translational processing mechanism.

Previously, we identified an arylphorin-binding protein of Sarcophaga peregrina (flesh fly) with a molecular mass of 120 kDa and suggested its participation in the selective uptake of arylphorin from the hemolymph into the pupal fat body at metamorphosis (Ueno, K., and Natori, S. (1984) J. Biol. Chem. 259, 12107-12111). This paper reports the isolation and sequencing of cDNA for the 120-kDa protein. This protein consists of 1146 amino acid residues. Immunoblotting and RNA blotting experiments revealed that this protein is present as two fragments of 76 kDa (695 residues) and 53 kDa (451 residues) in the larval fat body. When larvae pupate, the 120-kDa protein gene is further activated and the complete 120-kDa protein is synthesized without fragmentation. This suggests a novel mechanism for the production of the 120-kDa protein regulated by a proteinase depending upon the stage of development of Sarcophaga. All of these proteins were found to be localized in protein granules in the adipocytes.

Amino Acid Sequence↗
PubMed1995

Molecular characterization of an insect transferrin and its selective incorporation into eggs during oogenesis.

A protein with a molecular mass of 65 kDa that was specifically taken up into eggs was purified from the hemolymph of adult female Sarcophaga peregrina flies. From cDNA analysis, this protein was shown to be a Sarcophaga transferrin. Unlike mammalian transferrin, the similarity between its N-terminal and C-terminal halves was only 19%, and it was suggested to conjugate one iron atom/molecule in its N-terminal half. Sarcophaga transferrin was found to transport iron ions into eggs during oogenesis and deliver them to another protein, thought to be ferritin. No significant activation of the transferrin gene was detected during embryogenesis, so probably maternal transferrin is used as an intercellular or intracellular iron-transporter during embryogenesis of this insect.

Amino Acid Sequence↗
PubMed1995

Mode of action of an antibacterial peptide, KLKLLLLLKLK-NH2.

Previously, we reported that a synthetic undecapeptide, KLKLLLLLKLK-NH2, and its D-enantiomer have potent bactericidal activities against both Gram-positive and Gram-negative bacteria. Here we examined the mode of action of KLKLLLLLKLK-NH2 with special reference to its effect on bacterial membranes. We found that both the outer and inner membrane of Escherichia coli become permeable to low molecular mass substances when treated with this peptide. Under these conditions, the bacteria lost the ability to synthesize ATP and to transport proline, suggesting that their electrochemical membrane potential was disrupted. This peptide appears to form numerous channels in bacterial membranes that interfere with membrane functions, resulting in cell death.

Adenosine Triphosphate↗
PubMed1995

Purification of low-molecular-weight GTP-binding proteins structurally related to MTG33, a mitochondrial GTP-binding protein of Ehrlich ascites tumor cells.

We purified a mitochondrial GTP-binding protein, MTG33, from a particulate fraction of Ehrlich ascites tumor cells [Takeda, S., Sagara, Y., Kita, K., Natori, S., and Sekimizu, K. (1993) J. Biochem. 114, 684-690]. In the present work, three GTP-binding proteins, p23A, p23B, and p26, were purified from the same material. The Kd values of p23A, p23B, and p26 for GTP were 19, 6.8, and 4.0 nM, respectively. Binding of [alpha-32P]GTP to these proteins was inhibited by GTP and GDP, but not appreciably by other nucleotides such as ATP, CTP, UTP, and GMP. p23A, p23B, and p26 hydrolyzed GTP to GDP as well as MTG33 did. Peptide mapping analyses revealed that these GTP-binding proteins share common primary structures with MTG33. The defined properties of the three proteins suggest structural and functional relations to MTG33, which is localized in mitochondria.

Animals↗
PubMed1995

Purification and cDNA cloning of an antifungal protein from the hemolymph of Holotrichia diomphalia larvae.

An antifungal protein (AFP), holotricin 3, was purified from the hemolymph of the coleopteran insect Holotrichia diomphalia larvae. Analysis of its cDNA showed that holotricin 3 is a novel Gly- and His-rich protein consisting of 84 amino acid residues. This protein was similar to AFP, an antifungal protein of Sarcophaga peregrina that was reported previously, in terms of molecular size and high content of Gly and His residues. However, no appreciable sequence similarity was detected between the two proteins.

Amino Acid Sequence↗
PubMed1995

A sapecin homologue of Holotrichia diomphalia: purification, sequencing and determination of disulfide pairs.

We purified and characterized a sapecin homologue, named holotricin 1, from the hemolymph of immunized larvae of a coleopteran insect, Holotrichia diomphalia. We determined its complete amino acid sequence and three disulfide pairs. Holotricin 1 consisted of 43 amino acid residues and showed potent antibacterial activity against gram-positive bacteria, but antibacterial activity against gram-negative bacteria was not obvious.

Amino Acid Sequence↗
PubMed1995

[Antimicrobial proteins of insect and their clinical application].

We isolated three antimicrobial proteins from the hemolymph of immunized Sarcophaga peregrina (flesh fly) larvae. These are sarcotoxin I, sapecin and antifungal protein (AFP). Sarcotoxin I and sapecin are highly specific for Gram-negative and Gram-positive bacteria, respectively. AFP was found to have fungicidal activity. Primary target for these antimicrobial proteins was assumed to be microbial membrane. We were able to identify a core undecapeptide of sapecin B. We modified this peptide and finally obtained KLKLLLLLKLK-NH2 and its D-enantiomer. These peptides were effective against various bacteria including MRSA. We found that MRSA-infected mine are cured by treating with these peptides.

Animals↗
PubMed1994

Novel synthetic antimicrobial peptides effective against methicillin-resistant Staphylococcus aureus.

Previously, we identified a core undecapeptide of sapecin B having antimicrobial activity. Based on the structure of this peptide, we systematically synthesized peptides consisting of terminal basic motifs and internal oligo-leucine sequences and examined their antimicrobial activities. Of these peptides, RLKLLLLLRLK-NH2 and KLKLLLLLKLK-NH2 were found to have potent microbicidal activity against Staphylococcus aureus, Escherichia coli, methicillin-resistant S. aureus and Candida albicans in liquid medium. We also synthesized the D-enantiomer of KLKLLLLLKLK-NH2. This enantiomer was resistant to tryptic digestion and persisted longer in the culture medium, showing greater antimicrobial activity than the original peptide.

Amino Acid Sequence↗
PubMed1994

Purification, characterization, and cDNA cloning of procathepsin L from the culture medium of NIH-Sape-4, an embryonic cell line of Sarcophaga peregrina (flesh fly), and its involvement in the differentiation of imaginal discs.

A cysteine proteinase that preferentially hydrolyzes carbobenzyloxy-Phe-Arg-MCA (MCA, methylcoumaryl-7-amide) was purified from the culture medium of NIH-Sape-4 cells. The molecular mass of this enzyme was easily changed from 50 to 35 kDa, without appreciable loss of enzyme activity. From the analysis of its cDNA, this enzyme was concluded to be a procathepsin L of Sarcophaga. Only procathepsin L was found in the medium, while the mature 35-kDa cathepsin L was found exclusively in the cells, indicating that the cells have a mechanism to secrete procathepsin L selectively. An antibody against procathepsin L was found to inhibit the differentiation of imaginal discs cultured in vitro in the presence of 20-hydroxyecdysone. In fact imaginal discs were shown to secrete procathepsin L into the culture medium. These results suggest that procathepsin L is an essential proteinase for differentiation of imaginal discs. Northern blotting analysis revealed that unfertilized eggs contain a significant amount of mRNA for the procathepsin L as a maternal mRNA.

Amino Acid Sequence↗
PubMed1994

Synthesis and characterization of sapecin and sapecin B.

Two insect defencins, sapecin and sapecin B, were chemically synthesized to confirm their structure and antibacterial activity and also to examine the possibility that these peptides bind to the same site on the large conductance calcium-activated potassium channel as charybdotoxin. Both synthetic peptides showed the same antibacterial activity as native sapecins, indicating that the synthetic peptides folded correctly in the chemical synthesis. Synthetic sapecins did not show an inhibitory effect on [125I]charybdotoxin binding to rat brain synaptic membranes, suggesting that sapecin B recognizes a different binding site from that of charybdotoxin despite the similar structural motif.

Amino Acid Sequence↗
PubMed1994

Characterization of the antimicrobial peptide derived from sapecin B, an antibacterial protein of Sarcophaga peregrina (flesh fly).

Sapecin B, an antibacterial protein of Sarcophaga peregrina, was divided into four peptides. A hendecapeptide derived from its helix region was found to have comparable antibacterial activity with that of the complete protein. This peptide had a much wider spectrum of antimicrobial activity than that of sapecin B, exhibiting activity on not only Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative), but also some yeasts, including Candida albicans. The peptide was shown to bind to liposomes containing acidic phospholipids and cause release of entrapped glucose, suggesting that its primary site of action is the bacterial membrane. Its antimicrobial activity could be increased by substituting various amino acid residues for hydrophobic and/or basic ones.

Amino Acid Sequence↗
PubMed1994

Cloning and functional expression of poly(ADP-ribose) polymerase cDNA from Sarcophaga peregrina.

A cDNA spanning the entire coding region for poly(ADP-ribose) polymerase (PARP) of Sarcophaga peregrina was isolated and the nucleotide sequence was determined. The longest open reading frame encodes a polypeptide of 996 amino acid residues with a molecular mass of 113,033 Da. The similarities to the human PARP in amino acid sequence were relatively low in the DNA-binding and auto-modification domains, but very high in the C-terminal catalytic domain: identity of amino acids is 34% in the N-terminal DNA-binding domain (residues 1-369), 27% in the auto-modification domain (residues 370-507), and 56% in the C-terminal NAD-binding domain (residues 508-996). Two zinc-fingers (C-X2-C-X28-H-X2-C and C-X2-C-X31-H-X2-C)2 and a basic region in the N-terminal DNA-binding domain recognized in other PARP are conserved. Downstream of the basic region, another cysteine-rich motif (C-X2-C-X13-C-X9-C), a putative zinc-finger, was found to be well conserved in the PARP of Sarcophaga, Drosophila and human. A leucine-zipper motif (L-X6-L-X6-L-X6-L) which was found in the auto-modification domain of Drosophila PARP, is disrupted in the Sarcophaga enzyme: the second leucine is replaced by proline, and the third leucine by valine. Full-length cDNA for Sarcophaga PARP was cloned into an expression plasmid and expressed in Escherichia coli. A lysate of E. coli cells containing expressed protein reacted with antibody against Sarcophaga PARP, and PARP activity was detected. Thus, we conclude that isolated cDNA encodes a functional Sarcophaga PARP cDNA.

Amino Acid Sequence↗
PubMed1994

Inhibition of the Ca(2+)-activated K(+)-channel by sapecin B, an insect antibacterial protein.

Sapecin is an antibacterial protein of the flesh fly and sapecin B is its homologue structurally similar to charybdotoxin of scorpion venom, which is known to be a K+ channel inhibitor. We found that, like charybdotoxin, sapecin B inhibits part of the voltage pulse-induced K+ currents of rat cerebellar Purkinje cells. We suggest that this effect is due to inhibition of the Ca(+)-activated K+ channel. Probably, sapecin B is a naturally occurring K+ channel inhibitor as well as an antibacterial protein.

Animals↗