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Biomedical subjects

K Miyajima

Publications and source records attributed to K Miyajima.

At least 37 records · Page 2Linked to original sources
PubMed1998

Mechanism of synergism between antimicrobial peptides magainin 2 and PGLa.

The antimicrobial peptides magainin 2 and PGLa, discovered in the skin of the African clawed frog, Xenopus laevis, exhibit marked synergism [Westerhoff, H. V., Zasloff, M., Rosner, J. L., Hendler, R. W., de Waal, A., Vaz Gomes, A., Jongsma, A. P. M., Riethorst, A., and Juretic, D., Eur. J. Biochem. 228, 257-264 (1995)], although the mechanism is not yet clear. They are believed to kill bacteria by permeabilizing membranes. In this study, we examined the interactions of these peptides in lipid bilayers. PGLa, like magainin 2, preferentially interacts with acidic lipids, forming an amphipathic helix. The peptide induces the release of a water-soluble dye, calcein, entrapped within liposomes. The coexistence of magainin 2 enhances membrane permeabilization, which is maximal at a 1:1 molar ratio. Fluorescence experiments using L18W-PGLa revealed that both peptides form a stoichiometric 1:1 complex in the membrane phase with an association free energy of -15 kJ/mol. Single amino acid mutations in magainin 2 significantly altered the synergistic activity, suggesting that precise molecular recognition is involved in complex formation. The complex as well as each component peptide form peptide-lipid supramolecular complex pores, which mediate the mutually coupled transbilayer transport of dye, lipid, and the peptide per se. The rate of pore formation rate is in the order complex >/= PGLa > magainin 2, whereas the pore lifetime is in the order magainin 2 > complex > PGLa. Therefore, the synergism is a consequence of the formation of a potent heterosupramolecular complex, which is characterized by fast pore formation and moderate pore stability.

Amino Acid Sequence↗
PubMed1998

Relationship of membrane curvature to the formation of pores by magainin 2.

Magainin 2, an antimicrobial peptide from the Xenopus skin, kills bacteria by permeabilizing the cell membranes. We have proposed that the peptide preferentially interacts with acidic phospholipids to form a peptide-lipid supramolecular complex pore, which allows mutually coupled transbilayer traffic of ions, lipids, and peptides, thus simultaneously dissipating transmembrane potential and lipid asymmetry [Matsuzaki, K., Murase, O., Fujii, N., and Miyajima, K. (1996) Biochemistry 35, 11361-11368]. In this paper, we examined the effect of membrane curvature strain on pore formation. Magainin effectively forms the pore only in phosphatidylglycerol bilayers at low peptide-to-lipid ratios, well below 1/100. In contrast, the permeabilization of phosphatidylserine, phosphatidic acid, or cardiolipin bilayers occurred at much higher peptide-to-lipid ratios (1/50 to 1/10) with some morphological change of the vesicles. The latter three classes of phospholipids are known to form hexagonal II structures under conditions of reduced interlipid electrostatic repulsions. Incorporation of phosphatidylethanolamine also inhibited the magainin-induced pore formation in the inhibitory order of dioleoylphosphatidylethanolamine > dielaidoylphosphatidylethanolamine. Addition of a small amount of palmitoyllysophosphatidylcholine enhanced the peptide-induced permeabilization of phosphatidylglycerol bilayers. Magainin greatly raised the bilayer to hexagonal II phase transition temperature of dipalmitoleoylphosphatidylethanolamine. These results suggest that the peptide imposes positive curvature strain, facilitating the formation of a torus-type pore, and that the presence of negative curvature-inducing lipids inhibits pore formation.

Amino Acid Sequence↗
PubMed1998

Streptomyces turgidiscabies sp. nov.

A new bacterial species is described, for which the name Streptomyces turgidiscabies is proposed. This organism causes potato (Solanum tuberosum) scab in eastern Hokkaido, Japan; the lesions caused are distinctly erumpent. In culture, S. turgidiscabies is distinct from other scab-causing Streptomyces species, having flexuous spore chains and grey mass colour. The spores of this organism are cylindrical and smooth. Its cell walls contain the LL-diaminopimelic acid isomer, and its DNA G + C content is 71 mol%. S. turgidiscabies does not produce melanin or other diffusible pigments, does not grow on agar media at pH 4.0 or 37 degrees C, is positive for utilization of raffinose and inulin as a carbon source, and is sensitive to streptomycin (20 micrograms ml-1), penicillin G (10 IU ml-1), polymyxin B (15 micrograms ml-1), and thallium acetate (10 micrograms ml-1). The levels of DNA relatedness within S. turgidiscabies strains are high but relatedness between strains of this species and strains of S. acidiscabies, S. scabies, S. caviscabies, S. griseus, S. setonii and S. tendae are low. The type strain is SY9113T (= ATCC 700248T = IFO 16080T).

DNA, Bacterial↗
PubMed1998

Synthesis of Tn, sialyl Tn and HIV-1-derived peptide antigen conjugates having a lipid A analog as an immunoadjuvant for synthetic vaccines.

Conjugates (3-5) of Tn, sialyl Tn and HIV-1-derived peptide antigen with a N-tetradecanoyl L-serine-beta-alanine-containing D-glucosamine derivative, structurally related to lipid A, as an immunoadjuvant for the development of totally synthetic vaccines against cancers or HIV were synthesized. The mitogenic activity of compounds 3, 4 and 5 was stronger than that of lipid A analogs (1, 2).

Adjuvants, Immunologic↗
PubMed1998

Poly(ethylene glycol) derivative of cholesterol reduces binding step of liposome uptake by murine macrophage-like cell line J774 and human hepatoma cell line HepG2.

Liposome uptake by HepG2 human hepatoma cells was investigated in comparison with the uptake by J774 murine macrophage-like cells. HepG2 cells accumulated liposomes (egg yolk phosphatidylcholine (EPC)/Chol; 75/25, diameter 0.2 micron) at 37 degrees C comparably to J774 macrophage-like cells. Confocal microscopic observations revealed that J774 cells internalized EPC/Chol liposomes efficiently but HepG2 cells kept most of the liposomes bound on their plasma membrane surfaces. Poly(ethylene glycol) (PEG)-coated liposomes (0.2 micron) containing poly(ethylene glycol) cholesteryl ether (PEG-Chol) avoided cellular uptake at 37 degrees C by either cell line. In both cell lines, binding of PEG-coated liposomes was lower than that of EPC/Chol liposomes when incubation was carried out at 4 degrees C. To analyze the binding process at 37 degrees C, surface-bound liposomes were removed from the cells by pronase treatment. A reduction of the amount of bound-liposomes on cell surfaces was observed in the case of PEG-coated liposomes. Therefore, PEG-coating reduces direct binding of liposomes to the cell surfaces. The presence of apolipoprotein E (apoE) increased the uptake to EPC/Chol liposomes via its receptor in both cell lines. In contrast, cellular uptake of PEG-coated liposomes was not enhanced by treatment with apoE. Therefore, while apoE-mediated liposome uptake occurs in the case of EPC/Chol liposomes, it does not occur for PEG-coated liposomes; PEG-coating also inhibits protein-mediated binding to the cells. These results further imply that elusion from liver clearance of PEG-coated liposomes is not only due to the reduction of uptake by Kupffer cells but also by hepatocytes when liposomes are small enough to go through the fenestrates of the endothelial lining.

Animals↗
PubMed1998

[Load on the low back of teachers in kindergartens].

In order to evaluate the load on the low back of teachers in kindergartens, basic activity and working posture were analyzed for four teachers by means of video recording. The trunk inclination angle (TIA) was also measured continuously during full workshifts for 12 kindergarten teachers by means of an inclination monitor. The kindergarten teachers spent 67% of the workshift on activities in contact with children, "indoor group childcare", "indoor free playing", "outdoor childcare", "preparation and clearing away" and "help and care", and did not take a recess during the workshift. They spent 36% of the workshift in three working postures with the load on the low back, "standing bent forward", "squatting" and "kneeling". Cumulative time at a TIA of 20 degrees or more represented 43% of the workshift. The frequency of trunk-lifting from severe bending forward (TIA > 45 degrees) was 95 times/hr on average. A comparison of the kindergarten teachers and nursery teachers in 4-5 year age classes showed that the time distributions of basic activities were generally similar to each other. Although the time distributions of working postures were also similar, time spent "standing bent forward", "squatting" and "kneeling" was longer in the kindergarten teachers than in the nursery teachers. Cumulative time at a TIA of 45 degrees or more was significantly longer in the kindergarten teachers. Although the frequency of trunklifting was not significantly different, the kindergarten teachers tended to lift their trunk more frequently. The present study found that the load on the low back was considerably great in the kindergarten teachers.

Adult↗
PubMed1998

Effects of sphingomyelin and cholesterol on lipoprotein lipase-mediated lipolysis in lipid emulsions.

Lipoprotein lipase (LPL) hydrolyzes triacylglycerol (TG) of TG-rich lipoproteins. We investigated the effects of sphingomyelin (SM) and cholesterol (Chol) on the lipolysis of lipid emulsions by LPL using human apolipoprotein C-II (apoC-II) or plasma as an activator. Kinetic studies of the lipolysis rates clearly demonstrated that the primary effect of the activator on the LPL reaction was not to increase the affinity of LPL for the emulsion surface, but to enhance LPL catalytic activity. Incorporation of SM into the emulsion surface caused increases in Km(app) and decreases in Vmax(app), indicating that SM inhibited lipolysis by decreasing both affinity for substrates and catalytic activity of LPL. SM was also found to affect possible factors related to the lipolysis rates; that is, SM increased TG solubility in surface layers and decreased apoC-II binding to the emulsion surface. Interestingly, Chol did not affect the lipolysis rates even though it decreased TG solubility and apoC-II binding. These results indicated that neither TG solubility nor amount of apoC-II binding were determinate factors in LPL-mediated lipolysis under physiological conditions. Our results suggest that the content of SM in the lipoprotein surface plays an important role by controlling lipoprotein lipase-mediated lipolysis, and that cholesterol enrichment in the lipoprotein surface has no influence on lipolysis, but may affect other metabolic processes such as uptake by the liver through the selectivity of apolipoprotein binding.

Apolipoprotein C-II↗
PubMed1997

Cholesterol derivative of poly(ethylene glycol) inhibits clathrin-independent, but not clathrin-dependent endocytosis.

The effect of poly(ethylene glycol) cholesteryl ethers (PEG(n)-Chols) with two different numbers of units (n = 50 and 200) in the hydrophilic PEG moiety on cellular endocytic activity was studied on HT-1080 cells. The amphipathic molecules were soluble in aqueous solution. When fluorescein derivatives of PEG-Chols (one fluorescein at the distal end of PEG) were incubated with the cells in culture, the cellular fluorescence was localized at the plasma membrane level and in intracellular vesicles. Fluorescence quantification indicated that for the same external concentration, twice more FPEG(50)-Chol than FPEG(200)-Chol was associated with the cells under the same conditions. Regardless of the length of PEG moiety, PEG-Chols' interaction with cells reduced the endocytic internalization of a fluid phase marker, horseradish peroxidase (HRP) depending on the cell-associated amount. In contrast, internalization of 125I-labeled epidermal growth factor (EGF) through receptor-mediated endocytosis did not change upon incubation with PEG(50)-Chol. The effect of PEG(200)-Chol was also small, since EGF internalization showed a reduction of 10-20%, while at the same concentration as much as 80% of HRP uptake was inhibited. PEG(50)-Chol did not influence the internalization of a larger ligand, 125I-transferrin (Tfn). However, in the presence of PEG(200)-Chol, the uptake of 125I-Tfn decreased remarkably, and yet, PEG(200)-Chol has no influence on the binding and internalization of a monoclonal antibody directed toward the ectodomain of the Tfn-receptor. These results suggested that incorporation of PEG-Chols in the outer monolayer of the plasma membrane specifically inhibited clathrin-independent, but not clathrin-dependent endocytosis.

Blood Proteins↗
PubMed1997

Membrane permeabilization mechanisms of a cyclic antimicrobial peptide, tachyplesin I, and its linear analog.

Tachyplesin I (T-SS), an antimicrobial peptide from Tachypleus tridentatus, has a cyclic antiparallel beta-sheet structure maintained by two disulfide bridges. The peptide effectively permeabilizes both bacterial and artificial lipid membranes. T-Acm, a linear analog peptide with the four SH groups protected by acetamidomethyl groups, exhibits a much weaker membrane-permeabilizing activity in spite of a greater disruption of the lipid organization [Matsuzaki, K., Nakayama, M., Fukui, M., Otaka, A., Funakoshi, S., Fujii, N., Bessho, K., & Miyajima, K. (1993) Biochemistry 32, 11704-11710]. To clarify the efficient permeabilization mechanism of T-SS, we studied the interactions of both peptides with liposomes and planar lipid bilayers. The cyclic peptide capable of spanning the bilayer (ca. 3 nm length) was found to form an anion-selective pore and translocate across the bilayer coupled with the pore formation. A cis-negative transmembrane potential facilitated the pore formation compared with the cis-positive potential. In contrast, the linear peptide failed to translocate. Instead, it impaired the membrane barrier by disrupting the lipid organization with morphological changes in the vesicles.

Amino Acid Sequence↗
PubMed1997

Interactions of an antimicrobial peptide, magainin 2, with outer and inner membranes of Gram-negative bacteria.

Magainin peptides, isolated from Xenopus skin, have broad spectra of antimicrobial activity and low toxicities to normal eukaryotic cells, thus being good candidates for therapeutic agents. The mechanism of action is considered to be the permeabilization of bacterial membranes. A number of studies using lipid vesicles have elucidated its molecular detail. However, their interactions with bacteria are not yet well understood. In this paper, we synthesized several magainin analogs with different charges (0 to +6) and hydrophobicities, and systematically studied their interactions with the outer and inner membranes of three species of Gram-negative bacteria (Escherichia coli, Acinetobacter calcoaceticus, Proteus vulgaris). The treatment of the E. coli cells with native magainin 2 (+4) immediately induced the efflux of the intracellular K+ ions and the cell death. A number of blebs were formed on the bacterial surface and the outer membrane became leaky. An increase in the peptide's positive charge enhanced the outer membrane permeabilization and the bactericidal activity. The cationic peptides also effectively permeabilized the inner membranes rich in acidic phospholipids, indicating the importance of electrostatic interactions. Substitution of Trp for Phe simultaneously increased the bactericidal activity and the hemolytic activity. A strategy to develop potent antimicrobial peptides was discussed on the basis of these results.

Acinetobacter↗
PubMed1997

Modulation of magainin 2-lipid bilayer interactions by peptide charge.

Magainin 2, an antimicrobial peptide from Xenopus skin, assumes an amphiphilic helix when bound to acidic phospholipids, forming a pore composed of a dynamic, peptide-lipid supramolecular complex [Matsuzaki et al. (1996) Biochemistry 35, 11361-11368]. Upon the disintegration of the pore, a fraction of the peptide molecules stochastically translocates across the bilayer (Matsuzaki, et al., 1995). In order to investigate the effects of peptide charge on the magainin 2-lipid bilayer interactions, we synthesized four magainin 2 analogs with different charges (0-6+). MG0: K10E, K11E, F12W-magainin 2. MG2+: K10E, F12W-magainin 2. MG4+: F12W-magainin 2. MG6+: F12W, E19Q-magainin 2 amide. An increase in charge resulted in a stronger binding of the peptide to the negatively charged membranes, suggesting that electrostatic attractions play a crucial role in the binding process. The helical stability in a trifluoroethanol/buffer mixture was decreased with increasing positive charge because of electrostatic repulsions between the closely spaced positive side chains, whereas the helicity in the lipid bilayer was much higher and appeared to be independent of the peptide charge. However, enhanced repulsions between the highly positively charged helices destabilized the pore. Therefore, the efficiency of the most basic peptide (MG6+) to translocate across the bilayer was the greatest by virtue of the short life span of its pore and the very tight membrane binding. The charge distribution of wild-type magainin 2 was found to be so designed as to exhibit the maximal lytic activity by simultaneously achieving a strong binding and a moderate pore stability.

Amino Acid Sequence↗
PubMed1997

Pore formation and translocation of melittin.

Melittin, a bee venom, is a basic amphiphilic peptide, which mainly acts on the lipid matrix of membranes, lysing various cells. To elucidate the molecular mechanism, we investigated its interactions with phospholipid vesicles. The peptide formed a pore with a short lifetime in the membrane, as revealed by the release of an anionic fluorescent dye, calcein, from the liposomes. Our new double-labeling method clarified that the pore size increased with the peptide-to-lipid ratio. Upon the disintegration of the pore, a fraction of the peptides translocated across the bilayer. The pore formation was coupled with the translocation, which was proved by three fluorescence experiments recently developed by our laboratory. A novel model for the melittin pore formation was discussed in comparison with other pore-forming peptides.

Dextrans↗
PubMed1997

An estimation of craniofacial growth in the untreated Class III female with anterior crossbite.

The literature has little to say regarding the normal growth and development of untreated individuals with Class III malocclusion or anterior crossbite. In part, this paucity of information is because of the relatively low prevalence of these characteristics in European-American populations and the need, recognized by the lay public and health professionals, for treatment of these conditions. Given the absence of true longitudinal data, this study attempts to estimate the growth of the untreated individual with Class III malocclusion and anterior crossbite by evaluating large samples of untreated subjects at distinct developmental stages. Initially the morphologic characteristics of 2074 Japanese female patients who had anterior crossbite were evaluated cephalometrically before treatment. On the basis of the cephalometric analysis, all subjects who did not have a Class III molar relationship were excluded from further analysis, leaving a sample of 1376. The subjects then were classified into seven groups (120-256 subjects per group) according to Hellman's stages of dental development. Descriptive statistics for 28 measurements were calculated. The results of this study imply that the maxilla in Japanese females maintains a retruded relationship to the cranial base and does not become less retrusive with time. In contrast, the mandible is protrusive even in the late deciduous dentition and becomes more protrusive with time, making the discrepancy between the upper and lower jaws progressively more severe. Dental compensations in both arches become increasingly evident as development progresses, and the underlying skeletal and dentoalveolar imbalances also are reflected in the soft tissue profile.

Adolescent↗
PubMed1997

Sustained release of flufenamic acid from a drug-triacetyl-beta-cyclodextrin complex.

Triacetyl-beta-cyclodextrin (TA-beta-CyD), a hydrophobic cyclodextrin derivative that is insoluble in water, was used to form a complex with flufenamic acid (FA). Complexes of FA with TA-beta-CyD (FA-TA-beta-CyD) at various molar ratios (1:1, 1:2, 1:3) were prepared by a kneading method, using ethanol as a solvent. FA-TA-beta-CyD complex formation was demonstrated by differential scanning calorimetry and powder X-ray diffractometry. The release rate of FA from the FA-TA-beta-CyD complexes was measured in both the Japanese Pharmacopoeia XII 1st fluid pH 1.2 and isotonic phosphate buffer pH 6.8. The release rate of FA from the FA-TA-beta-CyD complexes in the isotonic phosphate buffer pH 6.8 was significantly retarded compared to the release rate of FA from the FA-glucose mixture. After 1 h, 100% of the drug was released from the FA-glucose mixture and 10-25% was released from the complexes. When either the powder of the FA-glucose mixture or the FA-TA-beta-CyD mixture was administered directly into the intraduodenal lumen in rats, the plasma concentration of FA reached a maximum level within 40 min after administration. On the other hand, when the FA-TA-beta-CyD complexes were administered into the intraduodenal lumen, the plasma concentration of FA did not show a sharp peak, but remained at a plateau level (10-18 microg/ml) for 6-8 h. An increased mean residence time of FA following FA-TA-beta-CyD complexes administration was observed; however, the AUC(0-10) for the FA-TA-beta-CyD complexes showed no significant difference from that for the FA-TA-beta-CyD mixture. These results indicate that TA-beta-CyD may serve as a hydrophobic carrier in sustained-release preparations of FA. The drug-TA-beta-CyD complexes may therefore be useful in oral administration to achieve prolonged action and reduced side effects.

Absorption↗
PubMed1997

High-efficiency entrapment of superoxide dismutase into cationic liposomes containing synthetic aminoglycolipid.

A monofatty acid ester of glucosamine (PGlcN) was synthesized to provide liposomal membranes with a positive charge, and the trapping efficiency of negatively charged substances (superoxide dismutases, SODs) into cationic liposomes containing PGlcN or stearylamine (SA) prepared by various methods was compared to find the most efficient trapping methods. We demonstrated that cationic liposomes, which were prepared in a buffer of low ionic strength containing sorbitol by a simple hydration method, could entrap a large amount of negatively charged SODs which retained their activity, as compared with cationic liposomes prepared in a buffer of high ionic strength. We also showed a reverse-phase evaporation method entrapped a large amount of SODs. However, SODs were inactivated during the preparation; therefore, this method was not suitable to entrap the enzyme. Freeze-thaw method induced the formation of cationic liposomes which were smaller than extruded liposomes and could entrap the SODs in a buffer of low ionic strength. Dehydration-rehydration method with a buffer of low ionic strength also entrapped a large amount of SODs, indicating that the integrity of liposomes was lost in the lipid bilayer after freeze-drying and the SODs were entrapped in the reconstruction of liposomes during rehydration. These findings showed that the hydration method based on electrostatic attraction with a buffer of low ionic strength was simple and the most effective for entrapping SODs without loss of their activity.

Cations↗
PubMed1997

Giant hepatic angiomyolipoma associated with disseminated intravascular coagulation.

We report a case of giant hepatic angiomyolipoma in a 68-year-old woman who had an increase in the fibrinolytic activity concomitant with disseminated intravascular coagulation (DIC). The presence of the tumor was confirmed by ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI) of the abdomen and the selective arteriography of the liver via the superior mesenteric artery. Following treatment with heparin and gabexate mesilate, abnormal hemostatic values were corrected. Furthermore, the surgical removal of the huge hepatic angiomyolipoma completely normalized the alternations of the clotting system. These findings suggest that giant hepatic angiomyolipoma was profoundly associated with DIC.

Aged↗
PubMed1997

PF1092A, B and C, new nonsteroidal progesterone receptor ligands produced by Penicillium oblatum. II. Physico-chemical properties and structure elucidation.

The structures of PF1092A (1), B (2) and C (3), new nonsteroidal progesterone receptor ligands produced by Penicillium oblatum, were elucidated by spectroscopic analyses. These compounds possess an eremophilane-type sesquiterpene carbon skeleton and differ only in that 1 and 2 are different monoacetates of 3. The absolute configurations of 1-3 were determined by single crystal X-ray diffraction analysis of the 4-bromobenzoyl ester of PF1092A and by measuring the optical rotations of the acetylation products of these compounds.

Furans↗