Biomedical subjects

H Aoyagi

Publications and source records attributed to H Aoyagi.

At least 37 records · Page 2Linked to original sources

PubMed2000

Usefulness of PET with 11C-methionine for the detection of hilar and mediastinal lymph node metastasis in lung cancer.

UNLABELLED: We retrospectively evaluated the usefulness of PET with 11C-methionine (methionine PET) for the diagnosis of lymph node metastases in patients with lung cancer. METHODS: Methionine PET and CT were performed before surgical intervention in 41 patients with primary lung cancer. We evaluated individual lymph nodes by methionine PET along with corresponding CT images. The 11C-methionine accumulation of lymph nodes was assessed semiquantitatively by analysis of the tumor-to-muscle ratio (TMR) and was compared with CT and histological diagnoses. RESULTS: A total of 126 lymph nodes, 36 of which were histologically diagnosed as metastatic, were assessed by CT and methionine PET. The TMR in metastatic lymph nodes (n = 36) was 5.15+/-1.69, whereas that of nonmetastatic lymph nodes (n = 90) was 2.91+/-0.76; this difference was significant (P < 0.0001). The most adequate TMR cutoff value for diagnosis of metastasis based on the results of receiver operating characteristic curve analysis was 4.1. The positive and negative predictive values, sensitivity, specificity, and accuracy of methionine PET were 79.5%, 94.3%, 86.1%, 91.1%, and 89.7%, respectively, and were superior to those of CT (57.6%, P = 0.04; 81.7%, P = 0.008; 52.8%, P = 0.002; 84.4%, NS; and 75.4%, P = 0.002, respectively). All positive nodes that were shown to be true-positive by CT, and 12 of 17 false-negatives on CT were correctly diagnosed by PET. Ten of 14 lymph nodes that were false-positive on CT were also correctly diagnosed by PET. CONCLUSION: Methionine PET appears to be superior to CT for the diagnosis of lymph node metastasis in lung cancer patients. The high negative predictive value of methionine PET suggests that cases in which lymph nodes are enlarged on CT with negative PET analysis may be diagnosed as negative for metastasis.

Carbon Radioisotopes↗

PubMed1999

Activation of Helicobacter pylori VacA toxin by alkaline or acid conditions increases its binding to a 250-kDa receptor protein-tyrosine phosphatase beta.

Helicobacter pylori, a Gram-negative gastric bacterium, secretes VacA, a cytotoxin that causes vacuolar degeneration of susceptible cells. Velocity sedimentation analysis showed that treatment of VacA at alkaline pH led to disassembly of VacA oligomers, an observation reported previously for acid-treated VacA. Exposure of VacA to acid or alkali increased its binding to AZ-521 cells, as shown by indirect immunofluorescence and flow cytometry. Moreover, immunoprecipitates with polyclonal antibodies against VacA from AZ-521 cells previously exposed to acid- or alkali-treated VacA had a 250-kDa glycoprotein containing galactose-beta(1-3)-N-acetylgalactosamine and galactose-beta(1-4)-N-acetylglucosamine. p250, purified by chromatography on peanut agglutinin affinity and Superose 6 columns, contained N-terminal and internal amino acid sequences of YRQQRKLVEEIGWSYT and LIIQDHILEATQDDY, respectively. These sequences are identical to those of a receptor protein-tyrosine phosphatase (RPTPbeta/PTPzeta); in agreement, p250 reacted with anti-human RPTPbeta monoclonal antibody. Immunoprecipitation with anti-human RPTPbeta antibody of solubilized membrane preparations previously incubated with VacA or heat-inactivated VacA demonstrated that RPTPbeta bound native, but not denatured, VacA. Acidic and alkaline treatments were associated with activation of VacA and increased binding to the cell surface RPTPbeta.

Amino Acid Sequence↗

PubMed1999

Orientational behavior of phospholipid membranes with mastoparan studied by 31P solid state NMR.

Solid state 31P NMR spectroscopy was used to study the perturbing effect of the wasp venom peptide mastoparan (MP) on lipid bilayers composed of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG). The 31P chemical shift anisotropy of multilamellar vesicles decreased with increasing peptide concentration, indicating that MP interacts strongly and selectively with the charged DMPG head group. Macroscopically oriented MP-lipid samples between glass plates were studied by 31P NMR as a function of tilt angle. These spectra showed the coexistence of orientation-dependent lamellar signals as well as an isotropic peak, suggesting that MP can induce non-lamellar phases in DMPC/DMPG membranes.

Amino Acid Sequence↗

PubMed1999

Effect of amino acid substitution in amphiphilic alpha-helical peptides on peptide-phospholipid membrane interaction.

It was previously found that a cationic amphiphilic peptide, Ac-(Leu-Ala-Arg-Leu)3-NHCH3 (4(3)), caused the destabilization of a phospholipid membrane and showed strong antibacterial activity [Lee et al. Biochim. Biophys. Acta 1986; 862: 211-219]. In order to investigate the effect of changing alpha-helix propensity, hydrophobicity and basicity in 4(3) on the peptide conformation and activity, the 4(3) analogs, [Gly (or Val)6]4(3), [Gly (or Val)2,6]4(3), [Gly (or Val)2,6,10]4(3), [Gln3]4(3), [Gln3,7]4(3) and [Gln3,7,11]4(3) were synthesized. Except for [Val2,6]4(3) and [Val2,6,10]4(3), which mainly formed a beta-structure, other peptides formed an alpha-helix and showed moderate membrane-perturbing activity toward neutral and acidic lipid vesicles. All the peptides other than [Val2,6,10]4(3) and [Gln3,7,10]4(3) had the antibacterial activity comparable with that of 4(3). The relationship between the membrane-perturbing activity and the antibacterial activity was not always parallel. Conclusively, the Ala-->Val substitution in 4(3) causes the change of peptide conformation and the presence of a cationic amino acid residue is necessary for the antibacterial activity.

Amino Acid Sequence↗

PubMed1999

Interaction of bundled Ser-rich amphiphilic peptides with phospholipid membranes.

To investigate properties of hydrophilic bundled peptides and their interactions with phospholipid membranes, bundled peptides named [Trp2]- and [Trp12]-4alpha-46S9, which are composed of four fragments of amphiphilic 24-mer peptide, were designed and synthesized. Tryptophan (Trp) was introduced at the 2nd position from the N-terminal or at the centre (12th) of the helix to monitor the peptide-lipid interaction. Circular dichroism measurements indicated that the peptides had low alpha-helicities in a buffer solution (pH 7.4) and also in the presence of dipalmitoyl-DL-3-phosphatidylcholine (DPPC) vesicles. In the presence of DPPC/dipalmitoyl-DL-3-phosphatidylglycerol (DPPG) (3:1) vesicles, the measurement could not be taken because of turbidity induced by vesicle aggregation. Both peptides had moderate perturbation activity for both the neutral and acidic vesicles at 25 degrees C. The perturbation patterns at 50 degrees C were much different from those at 25 degrees C and the maximum activity reached 100% at a low peptide concentration. The results of the measurement of membrane fusion activity of peptides showed a similar tendency to that found in the perturbation experiment. A quenching experiment indicated that the Trp2 and Trp12 residues in [Trp2]- and [Trp12]-4alpha-46S9 were scarcely embedded in neutral lipid membranes.

Amino Acid Sequence↗

PubMed1999

Vacuolating cytotoxin purified from Helicobacter pylori causes mitochondrial damage in human gastric cells.

We investigated the effects of vacuolating cytotoxin (VacA) prepared from Helicobacter pylori on the metabolism of gastric epithelial cells, AZ-521. VacA caused the ATP levels to decrease in a time-dependent manner; by approximately 20% in 6 h, 35% in 12 h and 50% in 24 h, at a concentration of 120 nM. This decrease was also dependent on the concentration of VacA. To evaluate the impairment of mitochondria by VacA, mitochondrial membrane potential was estimated by flow cytometric analysis using 3, 3'-dihexyloxacarbocyanine iodide as a substrate. VacA decreased membrane potential with the relative fluorescence intensity of AZ-521 cells in 6 h from 52+/-3 to 24+/-1. Treatment of the cells with bafilomycin A1, a specific inhibitor of vacuolar ATPase proton pump, showed no apparent effect on these changes in the levels of ATP and the mitochondrial membrane potential. Secondly, we estimated the effect of VacA on oxygen consumption. VacA inhibited oxygen consumption in AZ-521 cells: the levels of PO 2 in the medium of control cells decreased by 73% in 3 h and 37% in 6 h, whereas those in VacA-treated cells were 84% in 3 h and 59% in 6 h. Flow cytometric analysis showed the number of cells in the G0/G1 phase was increased by VacA. Taken together, VacA induced an inactivation of energy metabolism followed by mitochondrial damage, leading to impairment of the cell cycle in gastric epithelial cells.

Adenocarcinoma↗

PubMed1999

Development of an apparatus for monitoring protoplast isolation from plant tissues based on both dielectric and optical methods.

In order to develop a method allowing objective determination of the optimal conditions for the isolation of protoplasts, the process of protoplast isolation from plant tissues was quantitatively evaluated. First, a specialized spectrophotometer cuvette (working volume = 2.0 ml) was designed for the continuous monitoring of protoplast isolation from plant tissues based on the optical method. Homogeneous mixing of tissue sections and the protoplast suspension in the cuvette was accomplished by means of a magnetic bar. The cuvette was divided into upper and lower parts by a nylon mesh. Since tissue sections in the upper part could not pass through the mesh, they did not affect the optical path in the lower part, and only isolated protoplasts were able to move freely between the two parts. At the optimal agitation speed (200 rpm), mechanical damage to protoplasts of Catharanthus roseus did not occur. Increases in the protoplast concentration during their isolation from tissue sections (leaf and petal) could be continuously monitored by measuring the optical density (O.D.), making it possible to estimate the end of protoplast isolation. Degassing treatment of the tissues markedly enhanced protoplast isolation. In order to monitor the viable protoplast concentration, a larger specialized spectrophotometer cuvette (working volume = 25 ml) was developed which enabled simultaneous measurement of the permittivity and O.D. of the suspension to be carried out during protoplast isolation. Permittivity is a measure of the viable protoplast concentration, while the O.D. shows protoplast characteristics such as color. Using this large cuvette, the time courses of protoplast isolation from leaf and petal sections were monitored and large amounts of viable protoplasts were obtained. The protoplast isolation process after degassing treatment was described by a simple first-order reaction model and the viable protoplast isolation rate was quantitatively evaluated from the rate constant (k) on the basis of permittivity changes.

Journal Article↗

PubMed1999

Influence of lipophilic groups in cationic alpha-helical peptides on their abilities to bind with DNA and deliver genes into cells.

For the purpose of achieving gene transfer into cells mediated by peptides with a short chain length, we employed two kinds of amphiphilic alpha-helix peptides, mastoparan (INLK-ALAA-LAKK-IL-NH2) obtained from wasp venom and an alpha-helix model peptide (LARL-LARL-LARL-NH2). Furthermore, to strengthen the hydrophobicity of the peptide required for the formation of the aggregates with the DNA, we modified these peptides using several lipophilic groups, i.e. acyl groups with a single chain, a dialkylcarbamoyl group and a cholesteryloxycarbonyl group. We examined the ability of the peptides and their derivatives to bind and aggregate with plasmid DNA, the structural change in the peptides caused by binding with the DNA and the in vitro gene transfer abilities into COS-7 cells. As a result, mastoparan was found to acquire the DNA binding ability by introduction of the lipophilic group. The conformational change in the peptides depended on the hydrophobicity of the introduced acyl group. The DNA complex of most lipophilic mastoparan derivatives could be incorporated into the cells via the endocytosis pathway. In the case of the helix model peptide, the acyl group with a moderate chain length was required for the formation of the aggregate which is competent for incorporation into the cells. In this study, we succeeded in giving such short peptides sufficient gene transfer ability by modifying them with some lipophilic groups. However, the influence of the modification by the lipophilic groups on the formation of aggregates with DNA and the gene transfer ability depended on the structure of the peptide portion. These results indicate that consideration of total hydrophobicity balance is needed for the design of an efficient gene carrier peptide.

Amino Acid Sequence↗

PubMed1999

Characterization of the interaction of hemolytic lectin CEL-III from the marine invertebrate, Cucumaria echinata, with artificial lipid membranes: involvement of neutral sphingoglycolipids in the pore-forming process.

The hemolytic lectin, CEL-III, is a Ca2+-dependent, galactose/N-acetylgalactosamine-specific lectin purified from the marine invertebrate, Cucumaria echinata (Holothuroidea). After binding to specific carbohydrates on the erythrocyte surface, CEL-III forms ion-permeable pores by oligomerizing in the membrane, which leads to colloid osmotic rupture of the cells. When incubated with liposomes composed of total lipids from the human erythrocyte membrane, CEL-III efficiently induced the leakage of carboxyfluorescein (CF) trapped in the vesicles, suggesting the presence of its receptor in the membrane lipids. The rate of CF-leakage increased with increasing temperature, although the hemolytic activity of CEL-III had been found to be much higher at lower temperatures (around 10 degrees C). Identification of the receptor for CEL-III was performed by examining the ability of individual lipids from human erythrocytes to induce CF-leakage from DOPC-liposomes. As a result, the most effective receptor was found to be lactosyl ceramide (LacCer), while globoside (Gb4Cer) also showed slight induction of CF-leakage. On the other hand, a binding assay involving CEL-III-horseradish peroxidase conjugate indicated that CEL-III exhibits similar affinity for LacCer and Gb4Cer, suggesting that the structure or length of the carbohydrate portion of sphingoglycolipids is also relevant as to their ability to induce CF-leakage in addition to their affinity. Electron micrographs of CEL-III-treated liposomes revealed that CEL-III induced considerable morphological changes in the vesicles, while a clearly distinguishable oligomeric structure of the protein was not observed.

Animals↗

PubMed1999

Carbohydrate-dependent hemolytic activity of the conjugate composed of a C-type lectin, CEL-I, and an amphiphilic alpha-helical peptide, 4(3)-beta Ala2.

A lectin-cationic peptide conjugate, 4(3)-CEL-I, was prepared from an invertebrate C-type lectin, CEL-I, and an amphiphilic alpha-helical peptide, 4(3)-beta Ala2 [Ac-(Leu-Ala-Arg-Leu)3-beta Ala2]. When 4(3)-CEL-I was incubated with rabbit erythrocytes, hemolysis was observed, especially at basic pH. Inhibition experiment using some carbohydrates suggested that hemolytic activity of 4(3)-CEL-I was caused by the interaction between 4(3)-beta Ala2 portion in the conjugate and the lipid bilayer after binding to the carbohydrate chains on the cell surface by the lectin activity of CEL-I.

Animals↗

PubMed1999

[Infectious complications in patients with ulcerative colitis].

Several infectious disease play a role in altering the clinical course of ulcerative colitis(UC). Such infections are considered to be opportunistic infections. Especially cytomegalovirus(CMV) infection is a famous exacerbating factor. We describe a case of fulminant ulcerative colitis with methicillin-resistant staphylococcus aureus (MRSA) infection. A 55-year-old man was admitted to the hospital because of yellowish watery diarrhea. Antibiotics were administered, but the patient's condition was not improved. On the 9th hospital day, a stool culture yielded MRSA. On the 12th hospital day the patient developed toxic megacolon. Colonoscopic examination revealed the characteristic signs of UC. He underwent the emergency total colectomy and ileostomy. We emphasize in this article that MRSA infections should be recognized as an exacerbating factor of UC.

Colitis↗

PubMed1998

Present status and performance of SPring-8 front ends.

SPring-8 front ends have a novel structure which makes it easy to rearrange them and exchange the components. The structure has a common support for all the components except X-ray beam-position monitors and lead collimators. The alignment of the common support as well as the components was carried out with an accuracy of 0.25 mm in the vertical and horizontal directions. Replaceable pumping systems have also been placed on the common support and have achieved a vacuum of 2 x 10(-8) Pa at the upstream part of the front ends without synchrotron radiation. During the commissioning with synchrotron radiation, the pumping systems displayed good pumping-down characteristics. Commissioning has been successfully performed for four standard in-vacuum X-ray undulators and three bending-magnet-beamline front ends up to July 1997. Measurements of temperature rise show that absorber, pre-slits and XY slits can handle the anticipated maximum heat load at a ring current of 100 mA.

Journal Article↗

PubMed1998

Electronics for SPring-8 X-ray beam monitors.

A sensitive current-measuring system is required to construct a highly sensitive X-ray beam-position monitor (XBPM). A current-voltage converter (I/V) which can measure currents between 0.1 nA and 10 mA was designed, and the signal processing system of the XBPM was constucted using this I/V. This system was used in beamline commissioning. Beam-position data standard deviations of sigma approximately 3 micro m for the bending-magnet beamline, and sigma(x) approximately 3 micro m and sigma(y) approximately 1 micro m for the insertion-device beamline were obtained during the beamline commissioning.

Journal Article↗

PubMed1998

Importance of hydrophobic region in amphiphilic structures of alpha-helical peptides for their gene transfer-ability into cells.

It has been showned that cationic alpha-helical peptides can be useful as nucleic acid-carrier molecules for gene transfer into cells. In order to investigate the significancemake sure of importance of the hydrophobic region in amphiphilic peptides in relation to their transfection ability, we have employed five kinds of peptides with a systematically varied hydrophobic-hydrophilic balance in the amphiphilic structures, and have evaluated the relationship between the structure and the gene transfer ability of the peptides into COS-7 cells. The peptides with a large hydrophobic region took alpha-helical structures, formed large aggregates and showed high transfection efficiency. Their high efficiency can be explained on the basis of their ability to form stable aggregates which can be internalized by endocytosis and remain resistant to digestion in lysosomal vesicles. Furthermore, it was suggested that the hydrophobic region of peptides plays an important role in the disruption of the endosomal membrane, which ca prevent the degradation of DNA in lysosomal vesicles. When peptides do not have so strong membrane-disruptive activity, but form aggregates which can be incorporated by endocytosis, the transfection efficiency can be recovered by the addition of an endosome-disruptive peptide.

Amino Acid Sequence↗

PubMed1998

Interaction of alpha-helical peptides with phospholipid membrane: effects of chain length and hydrophobicity of peptides.

To investigate the interaction of amphiphilic alpha-helical peptides with phospholipid membranes, we synthesized Ac-(Leu-Ala-Arg-Leu)3-NHCH3 (4[3]) and three derivatives, in which the chain length and the size of the hydrophobic region of the peptides were different from each other. These peptides formed an alpha-helical structure in the presence of vesicles. In the membrane-perturbation measurement, only 43 showed a strong membrane-perturbation activity below phase-transition temperature (25 degrees C), but above phase-transition temperature (50 degrees C), most peptides showed similar strong activities. On the other hand, in membrane-fusion measurement the long peptides, e.g., Ac-(Leu-Ala-Arg-Leu)3-(Leu-Arg-Ala-Leu)3-NHCH3, had strong activities at low peptide concentrations at 25 degrees C. The present study indicated a parallel relationship did not always exist between membrane fusion and perturbation caused by peptides.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1998

Structure and property of model peptides of proline/arginine-rich region in bactenecin 5.

Bactenecin 5 (Bac 5), a cationic antibacterial peptide, contains a repeating region of Arg-Pro-Pro-X (X = hydrophobic residue). To investigate the structure and property of a Pro/Arg-rich region, we synthesized a series of repeating peptides, Ac-(Arg-Pro-Pro-Phe)n-NHCH3 (n = 2, 4, 6, 8 and 10) (PR2, PR4, PR6, PR8 and PR10) as models. The circular dichroism (CD) study suggested that the peptides with longer repeats, PR6, PR8 and PR10, formed a conformation similar to poly(proline)-II in aqueous solution. The CD spectra did not change in the presence of dipalmitoyl-DL-alpha-phosphatidylcholine (DPPC), but they changed in the presence of DPPC/ dipalmitoyl-DL-3-phosphatidylglycerol (DPPG). The gamma-helix, which is very similar in conformation to the poly(proline)-II helix, had the lowest energy conformation for the peptides by energy calculations. Peptides PR6, PR8 and PR10 caused slight leakage of fluorescent dye entrapped in DPPC vesicles, and in the presence of DPPC/DPPG, these peptides showed a considerable level of dye-leakage activity. In contrast, the shorter peptides PR2 and PR4 showed no activity. The same tendency was found in measurements of membrane-fusion activity. Judging from these results, the repeating region of Bac 5 may make a framework to hold a conformation resembling the poly(proline)-II structure in aqueous solution. In addition, this region may interact with acidic lipids, resulting in a change in conformation of the peptide.

Amino Acid Sequence↗

PubMed1998

Chemical modification of the hemolytic lectin CEL-III by succinic anhydride: involvement of amino groups in the oligomerization process.

CEL-III is a Ca(2+)-dependent lectin from a marine invertebrate, Cucumaria echinata, which shows strong hemolytic activity toward human and rabbit erythrocytes. After binding to carbohydrate receptors, CEL-III oligomerizes in the erythrocyte membrane to form ion-permeable pores, leading to the colloid osmotic rupture of the cells. Since hemolysis was greatly increased in the alkaline pH, especially above pH 9, involvement of amino groups of CEL-III in its hemolytic activity was evaluated using chemical modification by succinic anhydride. After modification of 7 amino groups per protein molecule, the hemolytic activity of CEL-III was reduced to 23% of the native protein, but hemagglutinating and carbohydrate-binding activities were only slightly affected even after modification of 14 amino groups. A circular dichroism spectrum of modified CEL-III showed almost no change in the secondary structure from that of the native protein, indicating that the decrease of hemolytic activity was not caused by partial unfolding of the protein. Immunoblotting analysis of the erythrocyte membrane treated with modified CEL-III showed a decrease in the formation of CEL-III oligomer in the membrane in parallel with the decrease in hemolytic activity. These results suggest that amino groups of CEL-III are involved in its oligomerization in the cell membrane, and their modification leads to inactivation of the protein without much influence on the carbohydrate-binding activity.

Amino Acids↗

PubMed1998

[Radiotherapy in the treatment of brain metastasis from small cell lung carcinoma: study on appropriately controlled dose].

Fifty-two brain metastatic lesions occurring in 20 patients with small cell lung carcinoma (SCLC) were irradiated, and then the relationship between tumor size, dose and control was clinically and histopathologically studied. Lesions of 8 mm in diameter and those of 10 mm in diameter were determined to be controllable by irradiation at about 38 Gy and 42 Gy, respectively. According to size-dependent curative minimum doses, the lesions could be divided by the 10 Gy/ 5fr/wk method into controlled and non-controlled groups with the curve expressed as dose (Gy) = 15.27 log10 [tumor volume (mm3)] +0.6. Based on these results, tumors of 2 mm and 3 mm in diameter were estimated to be controllable at 14 and 20 Gy, respectively. Thus prophylactic cranial irradiation (PCI) in the treatment of SCLC was thought to be not always necessary if early treatment of small metastatic lesions, detected by Gd-DTPA-enhanced MRI could be achieved. The optimal interval between follow-up examinations was thought to be 1 month during the first two years after the diagnosis of SCLC, and then 3 months after that. In addition, the omission of PCI can save many patients who do not actually require PCI from suffering its adverse effects.

Aged↗