Biomedical subjects

K Homma

Publications and source records attributed to K Homma.

At least 55 records · Page 3Linked to original sources

PubMed1999

Glutathione depletion causes an uncoupling effect on retinal horizontal cells through oxidative stress.

To investigate a physiological role of glutathione in the horizontal cells of carp retina, the gap junctional intercellular communication between horizontal cells was studied using the techniques of intracellular recording of light-induced responses and coupling of the fluorescence dye Lucifer Yellow. Intravitreal injection of 2.5 micromol L-buthionine sulfoximine, an inhibitor of glutathione synthesis, induced a dramatic reduction (20% of control) of retinal glutathione level two days after treatment. The low level of glutathione continued for a further four to five days, and thereafter gradually recovered to about 40% (20 days after injection) and 70% (50 days after injection) of the control level. The spatial properties of the photopic L-type horizontal cell response were examined by enlarging the diameter of the central spot and peripheral annulus over the recording point. In normal retinas, the response amplitude of horizontal cells was monotonically enhanced as the diameter of the spot increased (0.5-4.0 mm) and correspondingly the dye diffusion area was wide, as the injected Lucifer Yellow normally diffused to several neighboring cells. Treatment with L-buthionine sulfoximine significantly altered the spatial properties of horizontal cells by increasing the response amplitude to central spots and slightly decreasing that to peripheral annuli, which were observed by four days after injection. It also restricted intracellular Lucifer Yellow to one or two cells. Accompanying the recovery of the cellular level of glutathione, the spatial properties and dye coupling of horizontal cells were restored to normal. A time lag (two days) of initiation in retinal glutathione depletion and alteration of spatial or dye coupling properties of horizontal cells is discussed, together with reactive oxygen species accumulation.

Animals↗

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

Formation of the myosin.ADP.gallium fluoride complex and its solution structure by small-angle synchrotron X-ray scattering.

In the presence of MgADP, a novel phosphate analogue of gallium fluoride (GaFn) forms a ternary complex with the myosin subfragment-1 (S-1), in the same way that has been previously reported with aluminum fluoride (AlF4-), beryllium fluoride (BeFn), scandium fluoride (ScFn), and vanadate (Vi), and this complex formation may mimic different states along the ATPase kinetic pathway. This novel complex has been characterized and compared with other complexes to ascertain whether it forms a transition-state analogue of myosin ATPase. The complex formed quickly, although several times slower than the BeFn complex. The half-life of the myosin.ADP.GaFn complex was about 50 h at 4 degreesC. The formation of the myosin.ADP.GaFn complex was accompanied by an increase in tryptophane fluorescence, similar to that observed upon the addition of ATP, but slightly lower than that of the M**.ADP.Pi complex. Upon addition of GaFn to acto-myosin.ADP, acto-myosin did not dissociate, and the S-1.ADP.GaFn complex was scarcely decomposed by actin, like the AlF4- and ScFn complexes but unlike the BeFn and Vi complexes. The conformations at the localized region of SH1, SH2, and RLR, which are very accessible to the binding of ATP, were studied by fluorescent labeling and chemical modification, and the results suggested that these conformations are very similar to that of the M**.ADP.Pi state. Small-angle X-ray solution scattering showed that the radius of gyration value decreases by about 3 A when S-1 forms an S-1.ADP.GaFn complex, suggesting that the shape of the complex becomes compact or rounded in shape, similar to that in the presence of ATP or complexes with other phosphate analogues, and thus mimics the myosin**.ADP.Pi state closely. The overall results may indicate that the complex mimics a somewhat different transient state from that of other complexes but has a similar global conformation along the ATPase kinetic pathway.

Actins↗

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

A new model rat with acute bronchiolitis and its application to research on the toxicology of inhaled particulate matter.

The aim of the present study was to establish a useful animal model that simulates humans sensitive to inhaled particulate matter (PM). We have developed a new rat model of acute bronchiolitis (Br) by exposing animals to NiCl2 (Ni) aerosols for five days. Three days following the Ni exposure, the animals developed signs of tachypnea, mucous hypersecretion, and bronchiolar inflammation which seemed to progress quickly during the fourth to fifth day. They recovered from lesions after four weeks in clean air. To assess the sensitivity of the Br rats to inhaled particles, two kinds of PM of respirable size were tested with doses similar to or a little higher to the recommended threshold limit values (TLVs) for the working environment in Japan. Titanium dioxide (TiO2 = Ti) was chosen as an inert and insoluble particles and vanadium pentoxide (V2O5 = V), as a representative soluble and toxic airborne material. The Br rats exposed to either Ti or V were compared the pathological changes in the lungs and the clearance of particles to those in normal control or Br rats kept in clean air. The following significant differences were observed in Br rats: 1. delayed recovery from pre-existing lesions or exacerbated inflammation, 2. reductions in deposition and clearance rate of inhaled particles with the progress of lesions. The present results suggest that Br rats are more susceptible to inhaled particles than control rats. Therefore, concentrations of particulate matter lower than the TLVs for Japan, which have no harmful effects on normal lungs, may not always be safe in the case of pre-existing lung inflammation.

Acute Disease↗

PubMed1998

Phosphatidylinositol-4-phosphate 5-kinase localized on the plasma membrane is essential for yeast cell morphogenesis.

Phosphatidylinositol 4,5-biphosphate (PtdIns(4,5)P2), an important element in eukaryotic signal transduction, is synthesized either by phosphatidylinositol-4-phosphate 5-kinase (PtdIns(4)P 5K) from phosphatidylinositol 4-phosphate (PtdIns(4)P) or by phosphatidylinositol-5-phosphate 4-kinase (PtdIns(5)P 4K) from phosphatidylinositol 5-phosphate (PtdIns(5)P). Two Saccharomyces cerevisiae genes, MSS4 and FAB1, are homologous to mammalian PtdIns(4)P 5Ks and PtdIns(5)P 4Ks. We show here that MSS4 is a functional homolog of mammalian PtdIns(4)P 5K but not of PtdIns(5)P 4K in vivo. We constructed a hemagglutinin epitope-tagged form of Mss4p and found that Mss4p has PtdIns(4)P 5K activity. Immunofluorescent and fractionation studies of the epitope-tagged Mss4p suggest that Mss4p is localized on the plasma membrane, whereas Fab1p is reportedly localized on the vacuolar membrane. A temperature-sensitive mss4-1 mutant was isolated, and its phenotypes at restrictive temperatures were found to include increased cell size, round shape, random distribution of actin patches, and delocalized staining of cell wall chitin. Thus, biochemical and genetic analyses on Mss4p indicated that yeast PtdIns(4)P 5K localized on the plasma membrane is required for actin organization.

Actins↗

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

Phosphatidic acid-dependent phosphorylation of a 29-kDa protein by protein kinase Calpha in bovine brain cytosol.

Activation of phospholipase D (PLD) is involved in receptor-mediated signal transduction responses. Signaling from PLD to a downstream molecule(s) appears to be mediated by the PLD product phosphatidic acid (PA). A target molecule(s) of PA, however, has not yet been identified. The present study sought to define such a target molecule(s) of PA. In bovine brain cytosol, proteins with apparent molecular weights of 29,000 (p29) and 32,000 (p32) were prominently phosphorylated in the presence of PA, but not in its absence, indicating that there is a PA-regulated protein kinase (PARK) in bovine brain that phosphorylates p29 and p32. One of these substrates, p29, was purified to near homogeneity. Its partial amino acid sequence was determined and found to be identical to that of a known brain-specific 25-kDa protein (p25). The purified p29 was also readily recognized by and immunoprecipitated with an anti-p25 antibody. These results suggest that p29 is very similar to or identical with p25. Using the purified p29 as a substrate, PARK was purified to near homogeneity. The purified PARK had an apparent molecular weight of 80,000, was strongly recognized by an antiprotein kinase C (PKC)alpha antibody, and was activated by phosphatidylserine (PS) as well as PA. The PA- and PS-stimulated PARK activity was extremely augmented by the presence of 1 microM free Ca2+. In the presence of 1 mM EGTA, phorbol 12-myristate 13-acetate activated PARK synergistically with PA or PS. Similar results were obtained with the purified recombinant PKCalpha. From these results, it is suggested that the PARK activity purified might be attributed to PKCalpha. In p25-depleted bovine brain cytosol, which was prepared by treatment of bovine brain cytosol with the anti-p25 antibody, PA-dependent phosphorylation of p29, but not p32, was almost completely eliminated. When PKCalpha in bovine brain cytosol was depleted by its precipitation with the anti-PKCalpha antibody, neither p29 nor p32 in this PKCalpha-depleted cytosol was phosphorylated in the presence of PA. These results indicate that in bovine brain cytosol PA activates PKCalpha, which, in turn, phosphorylates p29, which may be identical with p25.

Animals↗

PubMed1998

A unique loop contributing to the structure of the ATP-binding cleft of skeletal muscle myosin communicates with the actin-binding site.

Actin binding to skeletal muscle myosin subfragment-1 (S1) increases the dissociation rate of reaction products from the myosin ATPase site; conversely, ATP binding facilitates dissociation of complexed acto-S1. However, details of the molecular mechanism by which the ATP- and actin-binding sites communicate with each other is still obscure. We present evidence that the effect of actin is mediated by a conformational change in the loop containing amino acids from 677 to 689 [loop M (677-689)], a segment of the 20-kDa tryptic fragment that contributes to the structure of the ATP-binding cleft. Initially, a fluorescent ADP analogue, methylanthranyloyl-8-azido-ADP (Mant-8-N3-ADP), was covalently crosslinked to loop M (Mant-S1), perhaps at Lys 681. Actin-activated Mg2+-ATP hydrolysis by Mant-S1 was accelerated approximately 6 times over that by unmodified S1, suggesting that the ATPase site is not blocked by the ADP analogue crosslinked in the loop M (677-689). Nevertheless, analysis of Mant-group fluorescence polarization and acrylamide-induced quenching showed the crosslinked probe to be entrapped within the ATP-binding cleft at a location where Mant-group rotational mobility was hindered, and where it was relatively inaccessible to the solvent. Exposing Mant-S1 to Mg2+-ATP and/or actin elicited similar decreases in fluorescence polarization, indicating increased rotational mobility of the Mant-group and movement of crosslinked Mant-8-N3-ADP to a less hindered position. Stern-Volmer quench curves showed that Mant-8-N3-ADP was translocated to a site where it was more accessible to dissolved quencher, perhaps outside the ATP-binding cleft. Since actin does not bind to the ATPase site, actin-induced translocation of Mant-8-N3-ADP crosslinked to loop M (677-689) probably results from a conformational change in loop M (677-689). These results suggest that loop M acts as a signal transducer mediating communication between the ATP- and actin-binding sites.

Actins↗

PubMed1998

Conformational changes at the highly reactive cystein and lysine regions of skeletal muscle myosin induced by formation of transition state analogues.

Myosin forms stable ternary complexes with Mg2+-ADP and phosphate analogues of aluminum fluoride (AlF4-), beryllium fluoride (BeFn), and scandium fluoride (ScFn). These complexes are distinct from each other and may mimic different transient states in the ATPase cycle [Maruta et al. (1993) J. Biol. Chem. 268, 7093-7100]. Regions of skeletal muscle myosin containing the highly reactive residues Cys 707 (SH1), Cys 697 (SH2), and lysine 83 (RLR) dramatically alter their local conformation when myosin hydrolyzes ATP, and these changes may reflect formation of a series of transient intermediates during ATP hydrolysis. We used the fluorescent probes 4-fluoro-7-sulfamoylbezofurazan, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid, and trinitrobenzene-sulfonate, which bind to SH1, SH2, and RLR, respectively, to examine differences in local conformations within myosin.ADP.phosphate analogue (BeFn, Vi, AlF4-, and ScFn) complexes. It was observed that the ternary complexes had SH1 conformations similar to those seen on S-1 in the presence of ATP. In contrast, local conformations in the SH2 and RLR regions of S-1.ADP.BeFn were different from those in corresponding regions of S-1.ADP.AlF4- or ScFn. These results suggest that SH1 and SH2 move distinctly during ATP hydrolysis and that the local conformations of the SH2 and RLR regions more sensitively reflect different transient states.

Adenosine Triphosphatases↗

PubMed1997

Chemotherapeutic activity of synthetic antimicrobial peptides: correlation between chemotherapeutic activity and neutrophil-activating activity.

The chemotherapeutic activity of three synthetic antibacterial peptides was investigated. KLKLLLLLKLK-NH2 and its D-enantiomer showed significant chemotherapeutic activity in MRSA-infected mice, whereas KLKLLLKLK-NH2, which showed the highest antibacterial activity among them in vitro, was found to have almost no ability to prevent MRSA infection. These results suggest that the antibacterial activity of peptides assessed in vitro does not necessarily correlate with their chemotherapeutic activity. We found that KLKLLLLLKLK-NH2 and its D-enantiomer, but not KLKLLLKLK-NH2, have the ability to activate human neutrophils to produce superoxide, suggesting that the prevention of MRSA infection by these peptides is not simply due to their direct bactericidal activity but to augmentation of the systemic defense mechanism mediated by neutrophils.

Animals↗

PubMed1997

Probes for narcotic receptor-mediated phenomena. 25. Synthesis and evaluation of N-alkyl-substituted (alpha-piperazinylbenzyl)benzamides as novel, highly selective delta opioid receptor agonists.

A series of N-alkyl- and N,N-dialkyl-4-[alpha-[(2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl] benzyl]-benzamides were synthesized and evaluated for binding affinities at mu, delta, and kappa opioid receptor subtypes. Several compounds (2e,f,h,i,m) strongly bound to the delta receptor with IC50 values in the nanomolar range. On the other hand, the binding affinities of these compounds for the mu and kappa receptors were in the micromolar or greater range indicating excellent delta opioid receptor subtype selectivities. In this series, two important structure-activity relationships were found for the delta receptor binding affinity. First, the spatial orientation of the alpha-benzylic position influenced the affinities with the alpha R derivatives 2a-n generally showing more than 10-fold greater affinity than the alpha S derivatives 3a-n. Second, the binding affinities were strongly influenced by the number of alkyl substituents on the amide nitrogen. N-Monoalkylbenzamide derivatives 2b-d showed lower affinity than N,N-dialkylbenzamide derivatives 2e-n, and the N-unsubstituted benzamide derivative 2a had the lowest affinity for the delta receptor in the series. The dramatic effect of the amide group substitution pattern on the binding affinity for the delta receptor strongly suggests that the amide function is an important structural element in the interaction of this series of compounds at the delta receptor. Selective compounds in this series were examined for binding affinity in cloned human mu and delta receptors. The results obtained generally paralleled those from the rat brain binding assay. Compounds 2e,f with potent delta binding affinities and high delta selectivities were shown to be delta agonists with high selectivity by studies in the guinea pig ileum (GPI) and mouse vas deferens (MVD) preparations. Compound 2f was the most selective compound in the rat brain and GPI/MVD assays with 1755- and 958-fold delta vs mu selectivity, respectively.

Alkylation↗

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↗

PubMed1997

Efficient synthesis and gastric (H+/K+)-ATPase-inhibitory activity of 2-aryl-4,5-dihydro-1H-thieno[3,2-e]benzimidazoles.

A series of 2-aryl-4,5-dihydro-1H-thieno[3,2-e]benzimidazoles (1, 2) was prepared by condensation of 5-acylamino-4,5,6,7-tetrahydrobenzo[b]thiophen-4-ones (9, 10) with ammonium acetate under azeotropic reaction conditions. Various congeners, N-methyl and N-phenyl analogues (3-5), 4,5-dihydro-1H-thieno[2,3-e]benzimidazoles (6), 4,5-dihydro-1H-thieno[2,3-g]benzoxazoles (7), and 4,5-dihydro-1H-thieno[2,3-g]benzothiazoles (8), were also prepared. Several compounds in this series were shown to be K(+)-competitive inhibitors of the gastric (H+/K+)-ATPase and more potent inhibitors than SK&F-96067, 3-butyryl-8-methoxy-4-(2-tolylamino)quinoline, on pentagastrin-stimulated acid secretion in chronic gastric fistula rats after intraduodenal administration.

Animals↗