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Biomedical subjects

H Aoyagi

Publications and source records attributed to H Aoyagi.

At least 55 records · Page 3Linked to original sources
PubMed1997

Helicobacter pylori vacuolating cytotoxin binds to the 140-kDa protein in human gastric cancer cell lines, AZ-521 and AGS.

To investigatie a potential mechanism of how Helicobacter pylori establishes infection, we purified a lot of vacuolating toxin (VacA) from supernatant of H. pylori ATCC49503 (tox+ strain 60190). We used an antibody which was prepared by immunizing rabbits with a synthetic peptide consisting of 16 amino acids reflecting a portion (Glu69-Arg83) of amino acid sequence of Vac A. VacA caused vacuoles in human gastric cancer cell lines AZ-521 AGS, and monkey kidney cell line COS-7, but not human promyeloblastic cell line HL-60. By immunoprecipitation analysis using anti VacA antibody, a biotinylated cell surface protein of 140kDa (p140) was precipitated only when the lysates of VacA-susceptible cells were incubated with VacA but not with inactivated VacA, indicating the association of p140 with VacA.

Animals↗
PubMed1997

Small-angle X-ray scattering study on CEL-III, a hemolytic lectin from Holothuroidea Cucumaria echinata, and its oligomer induced by the binding of specific carbohydrate.

Hemolytic lectin CEL-III from a marine invertebrate Cucumaria echinata forms an oligomer upon binding of specific carbohydrate such as lactose at high pH values and in the presence of high concentrations of salt. In this study, using small-angle X-ray scattering, we characterized CEL-III and its oligomer induced by the binding of lactose. The molecular mass of the oligomer was determined as 1019 kDa from its forward scattering value, compared with 47,490 Da for the monomer. This oligomer size is much larger than that estimated using SDS-polyacrylamide gel electrophoresis (SDS-PAGE, 270 kDa). The monomer has a 24.6 A radius of gyration and can be approximated by a rod which has a 20 A radius and a height of 75 A, while the oligomer has a 101.4 A radius of gyration. Together with the comparison of the radii of gyration and the forward scattering of the cross-section of the monomer and oligomer, it is suggested that in aqueous solution the oligomer comprises three or four molecules of a smaller unit which was observed by SDS-PAGE (270 kDa), held by a relatively weak interaction. The scattering profile also suggests that the oligomer has a hole in its central axis which might be associated with the formation of ion-permeable pores in the erythrocyte membrane by CEL-III during the hemolytic process.

Animals↗
PubMed1997

Diamond beam-position monitor for undulator radiation and tests at the Tristan Super Light Facility.

A photon beam-position monitor using a diamond foil as a position-sensitive device has been developed for use on insertion-device beamlines of third-generation synchrotron radiation facilities such as SPring-8, and was tested on the undulator beamline of the Tristan Super Light Facility at KEK. The beam test results show that the diamond monitor can be operated in a photoconductive mode like a semiconductor detector. It has a linear working range of about +/-1 mm and a position sensitivity of less than 3 mum. The stability of the monitor was confirmed by continuous operation under low photon beam intensity conditions.

Journal Article↗
PubMed1997

The enhancing effect of anionic alpha-helical peptide on cationic peptide-mediating transfection systems.

A peptide consisting of 12 amino acids including 3 glutamic acids (LAEL-LAEL-LAEL; 4(3)E) underwent pH-dependent conformational change from random coil to alpha-helix when the pH was decreased from 7.4 to 5.0 in the presence of egg PC. This alpha-helical 4(3)E had higher membrane-perturbation activity at acidic conditions compared with neutral conditions. When 4(3)E was incorporated with plasmid DNA-cationic peptide complex that utilizes an endocytosis pathway for uptake into cultured cells, high transfection efficiency was observed, indicating that 4(3)E can enhance the transfection activity of cationic peptide. It is likely that 4(3)E in the multi-complex of the plasmid DNA and the cationic peptide effectively disrupts the endosomal membrane and increases the population of the complex which could transfer to cytosol. The small lysosome-disruptive peptide is very probably useful as the enhancer molecule for the gene transfer techniques mediated by the endocytosis pathway.

Amino Acid Sequence↗
PubMed1997

Binding of cationic alpha-helical peptides to plasmid DNA and their gene transfer abilities into cells.

Polycationic reagents such as cationic lipids and poly-L-lysine are widely used for gene transfer into cells in vitro and show promise as vectors for in vivo gene therapy applications as nonviral gene transfer techniques. We have developed a novel transfection method using cationic amphiphilic alpha-helical oligopeptides with repeated sequences. Oligopeptide has the advantages of being easily designed and modified because of its simple structure. In this study, we synthesized five kinds of peptides of which the total chain length and the width of the hydrophobic region were changed. The binding of the peptides to plasmid DNA was evaluated by agarose gel electrophoresis. It was found that the long and/or hydrophobic peptides can strongly bind to the DNA. The formation of large aggregates with a 0.5-5-microm diameter, which consisted of the long peptides and the DNA, was observed by electron microscopy. The transfection abilities of the peptides were determined by the expression of luciferase from its cDNA in COS-7 cells. The long peptides showed high transfection abilities. As a result, it could be said that the transfection ability of these peptides was parallel to their ability to form aggregates with DNA. Furthermore, the transfection ability was increased by the addition of chloroquine in the transfection procedure. This result indicated that the internalization of the peptide-DNA aggregates would be mediated by the endocytosis pathway.

Animals↗
PubMed1997

Membrane interaction of synthetic peptides related to the putative fusogenic region of PH-30 alpha, a protein in sperm-egg fusion.

In order to investigate the relationship between structure and function of a putative fusogenic region of PH-30a, a protein active in sperm-egg fusion, two peptides, SFP22 and SFP23, whose sequences correspond to the residues 90-111 and 89-111 of PH-30 alpha, respectively, were chemically synthesized. An analog of SFP23, SFP23AA, which has an Ala-Ala sequence instead of the Pro-Pro sequence in SFP23, was also prepared. The CD study indicated that SFP22 and SFP23 mainly took a beta-structure in the presence of DPPC and DPPC/DPPG (3/1) vesicles, while SFP23AA showed an alpha-helical pattern though the alpha-helical content calculated was low (25-30%). alpha-Helical CD curve was observed for these peptides in trifluoroethanol. The membrane-perturbing activity of SFP22 and SFP23 was weaker than that of SFP23AA. On the other hand, the membrane-fusogenic activity of SFP22 and SFP23 to acidic phospholipid bilayers was much stronger than that of SFP23AA. All the peptides caused very weak cell lysis. These results are consistent with the reported speculation [Blobel, C. P. et al. (1992), Nature (London) 356, 248-252] that residues 90-111 of PH-30 alpha may be the fusogenic region and suggest that the Pro-Pro sequence is one of the important factors for holding the active secondary structure of the fusogenic region of PH-30 alpha in membranes.

ADAM Proteins↗
PubMed1997

Interaction of lipophilic peptides derived from mastoparan with phospholipid vesicles.

Various kinds of lipophilic peptides were prepared by acylation of an alpha-helical peptide, mastoparan, to investigate the effects of acyl groups on the interaction of peptides with phospholipid membranes. alpha-Helicity of the peptides was increased by introduction of long acyl groups. Acyl peptides showed different membrane-perturbation activities for neutral and acidic phospholipid vesicles, whereas a peptide with a dialkycarbamoyl group always exhibited a strong activity. High hemolytic activities were observed for the peptides with long acyls (single or double chain). These results indicate that lipophilic groups introduced to mastoparan contribute greatly to the interaction of the peptide with phospholipid membranes with lengthening of the acyl chain and that the structural character of the lipophilic group also influences the conformation of the peptide.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1997

[Heavy ion radiotherapy for lung cancer].

In our institute, heavy ion medical accelerator in Chiba (HIMAC) completed to be constructed October, 1994. Next year, we started to treat head & neck cancer firstly as a pilot study using carbon beam. Following the observation on the expected reaction of skin and mucosa membrane, we opened the protocol for lung cancer entitled "A phase I/II and II clinical trials of heavy iron particle radiotherapy for non-small cell carcinoma of the lung". Since then, Cabon beam possessing the well-balanced dual actions on cancer: efficient dose-localization and high RBE was given to 25 inoperable patients with stage 1 and 3 patients with stage IIIA as preoperative radiotherapy. The dose was raising from 59.4GyE, 64.5GyE and 72GyE by 10%. Tumor control of the patients who elapsed more than 6 months was evaluated in two of 4 patients (50%) given 59.4GyE, one of 4 (75%) given 64.5GyE and all of 17 (100%) given 72GyE. It appeared to be dose dependent. On the other hand, two of 28 patients (7%) showed acute radiation pneumonia treated with steroid hormone. However, the late effect of the lung was weaken with slight radiographic changes and with no manifestations. Now that heavy ion particle radiotherapy for lung cancer was revealed to be safe, we intend to attain the tumor control compatible with that of surgery.

Humans↗
PubMed1996

[Bone marrow transplantation from an unrelated donor in patient with congenital heart diseases].

A 9-year-old with chronic myelogenous leukemia (CML) who received bone marrow transplantation from an unrelated donor (UBMT), is reported. He also suffered from congenital heart disease (CHD) consisting of corrected transposition of the great arteries and dextrocardia. The cardiac output was within normal limits. The conditioning regimen included busulfan, melphalan, ALG and TLI. Cyclophosphamide was not used because of it cardiotoxicity. The HLA-phenotype of the donor was identical with that of the patient. DNA typing showed was haplodentical DRB1. The patient is alive 18 months after the UBMT. This case showed that UBMT was possible in a CML patient with CHD, without congestive heart failure.

Adult↗
PubMed1995

[The diverse effects of hormonal therapeutic agents on the proliferations of human breast cancer cell lines, MCF-7 and its variants].

The author established three variant cell lines which were proliferative in the serum-free medium. These three cell lines were derived from MCF-7, a human breast cancer cell line. These 3 variants (MCF-S1, MCF-S2 and MCF-S3) proliferate estrogen (E2)-dependently, but the amounts of E2 receptor and progesterone receptor in the cells are different from each other among the variants. The author compared the effects of hormonal therapeutic agents, tamoxifen (TAM), 3-hydroxytamoxifen (3-OH-TAM), toremifene (TORE), and medroxyprogesterone acetate (MPA) on MCF-7 and the established variant cells. Although TAM showed little effect on the growth of any cell lines in the absence of E2, it considerably inhibited the growth of MCF-S2 cells and MCF-7 cells in the presence of 10(-10)M E2. TORE also provided no inhibitory effect at less than 10(-7)M concentration on any cells in the absence of E2, but it dose-dependently inhibited DNA synthesis of MCF-S2 cells in the presence of E2. On the other hand, although 3-OH-TAM markedly inhibited the growth of any cell lines at a concentration of less than 10(-6)M in the presence of E2, it dose-dependently inhibited the DNA synthesis of any cell lines in the absence of E2, and the effect was remarkable in the case of MCF-S2 or MCF-S3. MPA showed a tendency to increase the proliferation of any cells in the absence of E2, conversely in the presence of E2, and it slightly inhibited DNA synthesis at concentrations of over 10(-8)M. These results suggest that the effect of hormonal agents on breast cancer cells may depend on either hormonal levels or the characteristics of the cancer cells. The effect of the hormonal agents are diverse, and a hormonal agent which does not work well on one cancer cell population may work well on another population.

Breast Neoplasms↗
PubMed1995

Interaction of the fragments characteristic of bactenecin 7 with phospholipid bilayers and their antimicrobial activity.

Bactenecin 7 (Bac7), a cationic polypeptide from large granules of bovine neutrophils, exhibits antimicrobial activity mainly against Gram-negative bacteria. This peptide is characterized by high contents of Arg and Pro and by a unique sequence with an Arg-clustered region and three tandem repeats. In order to investigate the structure-function relationship of Bac7, two peptide fragments, which correspond to residues 1-17 (RRIRPRPPRLPRPRPRP, an Arg-clustered region) and 46-59 (LPFPRPGPRPIPRP, one of three tandem repeats), respectively, were synthesized. Circular dichroism (CD) measurements of the two fragments indicated that they took particular conformations, although these were not defined. These peptides can bind to acidic phospholipid bilayers without marked conformational changes. When acidic phospholipid bilayers entrapping 5,6-carboxyfluorescein were treated with the peptides, no trace of the dye leaked out, indicating that the peptides lack the ability to disrupt lipid membranes. Fragment 1-17 showed weak antimicrobial activity against several bacteria, but fragment 46-59 was almost inactive. The present results suggest that longer fragments or the entire molecule of Bac7 may be required for full expression of antimicrobial activity and that Bac7 may manifest its activity by a bacteriostatic rather than a bacteriolytic mechanism.

Amino Acid Sequence↗
PubMed1994

Two mode ion channels induced by interaction of acidic amphipathic alpha-helical peptides with lipid bilayers.

In order to investigate the ion permeability and selectivity of ion channel formed by amphipathic alpha-helical peptides, we designed to synthesize an acidic peptide Ac(Leu-Ala-Glu-Leu)3NHCH3 (Glu-4(3)) and its channel property was compared with a basic peptides Arg-4(3) in which Glu in Glu-4(3) was replaced by Arg. Two modes of the conductance change were observed by the interaction of Glu-4(3) with planar lipid bilayers; a steady increase of the conductance with the elapse of time (mode 1) and a spike-like increase of the current (mode 2) appearing with a lag time and overlapping the model current increase. The application of negative membrane potential induced the mode 1 current and the lowering pH decreased it, suggesting that the mode 1 current is caused by slow insertion of Glu-4(3) into the lipid bilayer and then by forming certain unknown bundles like semichannels. Mode 2 was found to be consisted of channel type opened-close current with several different conductances and relatively short opening lifetimes. There was no ion selectivity in the mode 1 current, whereas the mode 2 current was cation selective. The peptide Arg-4(3) has formed a cation-selective ion channel but not shown such two mode current changes. The membrane potential formation experiment in liposomes using DiSC3(5) also showed the cation selectivity for Arg-4(3) and non-ion selectivity for Glu-4(3). The difference between Arg-4(3) and Glu-4(3) was also observed in conformational analysis by CD and in dye-release experiment from liposome. Such difference was discussed in terms of electrostatic interaction between peptides and lipid head groups.

Amino Acid Sequence↗
PubMed1994

Interactions of N-terminal fragments of groups I and II phospholipases A2 with phospholipid bilayers and their surface recognition properties.

In order to elucidate the roles of the N-terminal segments of groups I and II phospholipases A2 (PLA2s) which have been known to have alpha-helical structure and have been assumed to be involved in the water/lipid interface recognition site, the peptides corresponding to the N-terminal moieties of group I PLA2 (Naja naja atra) and group II PLA2s (Trimeresurus flavoviridis and Crotalus atrox) were synthesized and their interactions with model membranes were studied. Circular dichroism spectra showed that N-terminal peptides of both groups I and II PLA2s took alpha-helical structure in trifluoroethanol but no significant secondary structure in buffer (pH 8.0). In the presence of acidic liposomes, N-terminal fragments of group II PLA2s formed alpha-helical structure, while that of group I PLA2 remained unaffected. The hydrophobic moments showed that amphipathicities of N-terminal fragments of group II PLA2s are evidently larger than those of N-terminal fragments of group I PLA2s. The leakage of carboxyfluorescein from acidic liposomes was induced only with group II PLA2 peptides. Large blue shift and increase in intensity of tryptophan fluorescence were also observed for group II PLA2 peptides when interacting with acidic liposomes. Such difference in the modes of interactions with lipid bilayers between N-terminal peptides of groups I and II PLA2s appears to be due in large part to the difference in intrinsic alpha-helix forming properties of their amino acid sequences. It is inferred that N-terminal amphipathic alpha-helical structures of group I PLA2s are possibly formed by assistance of a neighboring chain bridged by Cys-11 and Cys-77.

Amino Acid Sequence↗
PubMed1994

Design and synthesis of amphipathic 3(10)-helical peptides and their interactions with phospholipid bilayers and ion channel formation.

It has been reported that a peptide corresponding to the S4 segment in sodium channel protein is able to form voltage-dependent cation-selective ion channels (Tosteson, M. T., Auld, D. S., and Tosteson, D. C. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 707-710). However, biological and other physical properties remain unexamined. In the present study, three peptides, H-(Ala-Arg-Leu)8-OH (ARL8), H-(Val-Arg-Leu)8-OH (VRL8), and H-(Leu-Arg-Leu)8-OH (LRL8) which were designed on the basis of the S4 segment and expected to form 3(10)-helix, were synthesized and examined with regard to conformational change by the interaction with membranes, membrane perturbation ability, ion channel formation, and antimicrobial activity. According to CD spectra, these peptides were found to form a 3(10)-helical structure in the presence of dipalmitoyl-DL-alpha-phosphatidylcholine/dipalmitoyl-DL-alpha- phosphatidylglycerol (3:1) liposomes. The experiment of the peptide-induced leakage of carboxyfluorescein from liposomes showed that all the peptides had a strong ability to perturb membranes. The peptides were able to form cation-selective ion channels in planar asolectin lipid bilayers. The conductances of the ion channels were small (approximately 2 picosiemens for VRL8 and LRL8 and approximately 23 picosiemens for ARL8), suggesting that the peptides produce narrow pores or wider pores with certain permeable barriers that are a portion of the whole channels. The differences in their conductances depend possibly on the sizes of the side chains of Ala, Val, and Leu residues. However, non of the peptides showed antimicrobial activity (minimum inhibitory concentrations, > 50 micrograms/ml). Here, we present the first evidence that the peptides can form 3(10)-helical structures with long chain lengths in a lipid bilayer environment.

Amino Acid Sequence↗
PubMed1994

Ion channel activity of a synthetic peptide with a primary structure corresponding to the presumed pore-forming region of the voltage dependent potassium channel.

A 26-mer peptide of which the sequence contains the presumed pore forming region of the Shaker K+ channel (H5 region) was chemically synthesized. The peptide was found to interact and penetrate lipid membranes based on the fluorescence of Trp residues of the peptide in the presence and absence of liposomes. The secondary structure and the ion channel forming ability of the peptide were measured by CD spectroscopy and by a planar bilayer technique, respectively. The secondary structure of the peptide was composed of a mixture of an alpha-helix, beta-sheet, beta-turn, and a random coil. The content of beta-sheet structure was increased by the presence of liposomes. In planar bilayers, the peptide formed anion-selective ion channels with a larger conductance than that of the native Shaker K+ channel. These results suggest that the H5 region of the Shaker K+ channel can penetrate into lipid bilayers and form ion channel structures by itself, but it requires other structural components to reproduce the native characteristics of the K+ channel.

Amino Acid Sequence↗
PubMed1994

[Possibility of local control of breast cancer by microwave tissue coagulator (MTC)].

The tissue injury caused by microwave tissue coagulator (MTC) was assessed clinically and pathologically in a case of advanced breast cancer with skin invasion. On condition that the output was 10 watts and radiation lasted for 90 seconds at one time, temperature on breast skin was measured after each radiation. The maximum temperature was over 60 degrees C at the needle electrode and over 42 degrees C at 10 mm from the electrode after 21 radiations. From assessment of the resected specimen after operation, the following results were obtained. The mechanism of tissue injury by MTC consists of direct heat injury and infarct by circulatory disturbance. The extent of injury from the needle electrode is approximately 10 mm in the former mechanism and 15 mm in the latter. In conclusion, MTC is useful for the therapy of local recurrence of the breast cancer because of effectiveness in control of tumor-oozing and occurrence of tumor necrosis.

Breast Neoplasms↗
PubMed1993

Effect of salts on conformational change of basic amphipathic peptides from beta-structure to alpha-helix in the presence of phospholipid liposomes and their channel-forming ability.

A synthetic model peptide, H-(Leu-Al alpha-Arg-Leu)3-(Leu-Arg-Al alpha-Leu)3-OH (4(6)) can form ion channels in planar lipid bilayers by taking an amphipathic alpha-helix (Agawa, Y., Lee, S., Ono, S., Aoyagi, H., Ohno, M., Taniguchi, T., Anzai, K. and Kirino, Y. (1991) J. Biol. Chem. 266, 20218-20222). For further study of ion channels formed by this type of peptides, we planned to synthesize [Trp1]-4(6)(Ser) and [Trp12]-4(6)(Ser) in which a hydrophilic amino acid, Ser, was introduced in several positions of 4(6) instead of hydrophobic ones. This modification was expected to decrease the ability of membrane perturbation and to simplify various current levels of the channel observed for 4(6). Furthermore, additional Trp was introduced to the N-terminus or position 12 to monitor the lipid-peptide interaction. CD study showed that both peptides formed a random structure in buffer, but an alpha-helix in the presence of egg PC and a beta-structure in egg PC/egg PG (3:1). Moreover, addition of NaCl to the acidic liposomes induced the conformational transition in the peptide from beta-structure to alpha-helix. Salt-induced conformational transition in the presence of acidic liposomes was discussed in terms of membrane binding and ion-channel formation in planar lipid bilayer. Despite introduction of hydrophilic residues instead of hydrophobic residues in 4(6), the peptide showed nearly the same dye-release ability from egg PC- egg PG liposomes as 4(6). [Trp12]-4(6)(Ser) was able to form cation-selective ion channels with two levels of conductance (mainly 250 and occasionally 125 pS) in asolectin planar lipid bilayer, suggesting that appropriate orientation of hydrophobic and hydrophilic residues in amphipathic peptide can simplify channel current levels.

Amino Acid Sequence↗