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Biomedical subjects

S Nagahara

Publications and source records attributed to S Nagahara.

At least 19 recordsLinked to original sources
PubMed2001

Collagen minipellet as a controlled release delivery system for tetanus and diphtheria toxoid.

The use of biodegradable polymer matrices as a single-dose vaccine delivery system was investigated using tetanus toxoid (TT) and diphtheria toxoid (DT). BALB/c mice were immunized with TT or DT in different formulations including individual, in minipellet and aluminum hydroxide (alum), and the antibody responses were monitored for 48 weeks. Antigens entrapped in minipellet elicited higher antibody responses compared to those obtained with individual antigens and antigens adsorbed to alum and the antibody levels remained elevated over 48 weeks. In addition, minipellet formulations induced the same subclasses of antibodies induced by alum formulations. These results raise the possibility to obtain optimal and long-lasting immune responses by a single administration of minipellet formulations.

Adjuvants, Immunologic↗
PubMed1996

Cell-substrate and cell-cell interactions differently regulate cytoskeletal and extracellular matrix protein gene expression.

In this study, cellular events during tissue formation were investigated at the mRNA level using the Northern blot technique. The levels of expression of mRNAs encoding specific proteins (beta-actin, fibronectin, and laminin) during tissue formation on tissue culture dishes were quantitatively assessed using a Northern blot technique with autoradiography. The level of beta-actin mRNA increased with incubation time and reached a maximal level near the confluent state, followed by reduced beta-actin mRNA expression at a later stage of tissue formation. The time course of beta-actin mRNA expression corresponded well to the time course of morphologic changes and cytoskeletal organization in adherent cells. Expression of the mRNAs encoding the extracellular matrix proteins fibronectin and laminin was initiated at the proliferation stage. After maximum expression levels of these two mRNAs were reached at the confluent stage, a gradual decrease in their expression levels was seen during long-term culture. Expression patterns of mRNAs encoding cytoskeletal and extracellular matrix proteins strongly depended on the type of artificial substrates used; a mRNA expression pattern similar to that observed during tissue formation on tissue culture dishes was observed on a cell-adhesive substrate during tissue formation, whereas reduced expression was seen during tissue formation on a less adhesive substrate. Thus, the dynamic changes occurring during tissue formation were quantified to investigate the roles of artificial substrates in tissue formation at the mRNA level.

Animals↗
PubMed1996

In situ detection of DNA fragmentation and expression of bcl-2 in human neuroblastoma: relation to apoptosis and spontaneous regression.

UNLABELLED: Spontaneous regression occurs in some cases of neuroblastoma, especially stage IVS. The incidence of neuroblastoma has been reported to be increasing since the mass screening program was introduced in Japan. This would indicate that the screening is detecting regressing tumors. However, the mechanism of regression is still unknown. To evaluate the hypothesis that the regression might be related to apoptosis, the authors examined apoptosis by in situ end-labeling of fragmented DNA and expression of the apoptosis-suppressing protein bcl-2. MATERIALS AND METHODS: One hundred eighteen neuroblastoma cases were available for examination. Eighty (67.8%) were detected by the mass screening program. Serial sections were cut from paraffin-embedded tumors. A modified TdT-mediated dUTP nick end-labeling (TUNEL) method was performed to detect apoptosis. Immunohistochemical analysis was performed to detect bcl-2 expression. RESULTS: In cases under 1 year of age or with a favorable clinical stage, the incidence of apoptosis was significantly high. Expression of bcl-2 was associated with N-myc amplification and unfavorable histology (Shimada classification). Tumors in patients under 1 year of age often had areas where cellularity was markedly decreased, and apoptosis was often observed while bcl-2 expression was reduced. In such cases, there was a negative correlation between occurrence of apoptosis and bcl-2 expression. This suggests that apoptosis may be related to spontaneous regression in neuroblastoma.

Age Factors↗
PubMed1993

Detection of specific base sequences in solution using DNA probes labeled with d- and/or 15N-substituted spin-labels.

In order to enhance the sensitivity and the accuracy of the detection by the DNA probe method in which spin-labeled oligonucleotides are used as probes, 4-amino-2,2,6,6-tetramethylpiperidine-15N-oxyl-d16 (4-amino-TEMPO-15N-d16) and 4-amino-2,2,6,6-tetramethylpiperidine-14N-oxyl-d16 (4-amino-TEMPO-14N-d16) were incorporated into the phosphate linkage of oligonucleotides by the hydrogen phosphonate method and these DNA probes were used for the detection of target DNAs in solution. The intensities of the electron paramagnetic resonance (EPR) signals of the oligonucleotides labeled with 4-amino-TEMPO-14N-d16 and 4-amino-TEMPO-15N-d16 were 3-fold and 4-fold larger than that of the oligonucleotide labeled with 4-amino-TEMPO-14N, respectively. Also, the EPR lines of these labeled oligonucleotides do not overlap each other, allowing the detection of two different regions in the same target DNA by the single EPR measurement.

Base Sequence↗
PubMed1993

Oligonucleotides site-specifically spin-labeled at 5'-terminal or internucleotide linkage and their use in gene analyses.

Spin-labeled oligonucleotides (S-probes) were synthesized and examined as DNA probes to monitor hybrid formation. TEMPO was introduced either at the internucleotide linkage of 5'-terminus (Type 1) or at the 5'-terminal hydroxyl group (Type 2) and both types of S-probes were used in this study. The presence of target DNA was detected in solution by EPR spectroscopy for both types of S-probes. Hybridization of the S-probes resulted in notable broadening of EPR line width, accompanied by a decrease in the EPR signal height ratio for I(-1)/I(0).I(-1)/I(0) of S-probes having no spacer between oligonucleotide and TEMPO decreased more markedly than that of S-probes with a spacer, indicating that TEMPO should be introduced to an oligonucleotide directly to monitor hybrid formation. When M13mp8 single-stranded DNA with or without an EcoRI recognition site was selected as a target DNA, hybrid formation was detected only for DNA containing EcoRI site in solution using spin-labeled oligonucleotides.

Bacteriophage M13↗
PubMed1992

Interaction of antisense DNA with nucleic acids/proteins.

In order to study interaction of various types of labeled antisense DNAs were prepared. Fluorescein and 2,2,6,6-tetramethypiperidine-N-oxyl were the label molecules, which were introduced to 5'-end of oligonucleotides and their analogs. Interactions of labeled antisense DNAs with nucleic acids or proteins such as HSA, HIG and TF, were studied by UV, fluorescence depolarization spectroscopy, and ESR spectroscopy. Hybrid formation of antisense DNAs with oligonucleotides in solution could be monitored by the increase in fluorescence anisotropy (r) and by intensity change in ESR spectra. When phosphorothioate type antisense molecules anchoring fluorescein (F-OPT) were mixed with proteins, r drastically increased, whereas ODN slightly increased. These results suggest that OPTs have much more affinity for proteins than ODNs.

Blood Proteins↗
PubMed1991

Fluorescent-labeled oligonucleotide probes: detection of hybrid formation in solution by fluorescence polarization spectroscopy.

Fluorescein-labeled oligonucleotides as DNA-probes were synthesized and used to monitor hybrid formation, namely to detect DNA or oligonucleotide sequence in solution. The introduction of fluorescein to oligonucleotides was carried out by oxidation of a hydrogen phosphonate linkage with ethylenediamine or hexamethylenediamine as a tether and by a subsequent labeling of the primary amine moiety by FITC. Fluorescence anisotropy, r, was adopted as an index to monitor the behavior of F-probe in solution. An increase in the anisotropy was observed upon an increase in the chain-length of F-probe. When F-Probe formed a hybrid with its complementary oligonucleotide in solution, the r value increased compared to that of F-Probe itself. These observations clearly indicate that measurements of r in solution will readily lead to the monitoring of the presence of a hybrid in solution. Consequently, it is promising to detect a certain nucleic acid sequence in solution using fluorescent-labeled oligonucleotides.

Chromatography, High Pressure Liquid↗
PubMed1991

Characterization of antisense DNA derivatives having stereoisomeric linkages.

The isomer separation of oligonucleoside phosphoramidates (OPA) by RPLC was studied. All stereoisomers of OPA-tetramer (dATCG) modified with isopropylamine could be separated and they showed different CD spectra each other. OPA-decamer (dGGGCATCGTC) modified with 3-amino-1-propanol could be separated into five fractions. Each fraction was found to have different ability to form hybrids with its complementary oligonucleotide, indicating that it is possible to exclude stereoisomers which can hardly bind to target nucleic acids.

Amides↗
PubMed1990

Structural analysis of nucleic acids by labeled oligonucleotides.

In this report, the characterization of labeled oligonucleotides was discussed from the view points of base sequence analysis and structural analysis of nucleic acids in solution. Oligonucleotides site specifically spin labeled with TEMPO and fluorescent labeled with fluorescein were prepared and used for those analyses. The changes of ESR lines and rotational correlation time (tau) of the spin labeled oligonucleotide (S-probe) were dependent on the base sequence of S-probe, diastereoisomers, and the manner of hybridization. These results suggest that the conformation of the hybrid largely affected the local mobility of TEMPO and that tau value of S-probe reflected the local structure of the hybrid. When S-probe which was complementary to a single strand region of 5S RNA, was mixed with 5S RNA, tau value largely changed, indicating that the S-probe could form hybrid with 5S RNA in solution. Similar results were also obtained in the fluorescence depolarization analysis using fluorescent labeled oligonucleotide (F-probe). These results suggest that S-probe and F-probe are capable for the recognition of the secondary structure of 5S RNA in solution and useful for the analysis of the secondary structure of other nucleic acids in solution.

Base Sequence↗
PubMed1989

A study on spin-labelled oligonucleotide synthesis and its electron spin resonance behavior in solution.

An oligonucleotide spin-labelled with 4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl (4-amino-TEMPO) at the internucleotide bond (d-Tp(L)TpTpTpT) prepared by oxidation of the pentanucleotide containing the H-phosphonate diester (d-Tp(H)TpTpTpT) in the presence of 4-amino-TEMPO, was separated and identified by high-performance, reverse-phase liquid chromatography combined with detection by electron spin resonance spectroscopy. This spin-labelled oligonucleotide produced a triplet with the slightly broadened M1 = -1 ESR component, while a triplet with almost equal intensities was obtained from the spin-label. The M1 = -1 component from the labelled oligonucleotide was further broadened in the presence of poly(A) which forms a complementary double strand with this molecule.

Chromatography, Liquid↗
PubMed1989

ESR behaviour of spin-labelled oligoDNAs.

Oligodeoxyribonucleotides (oligoDNAs) were spin-labelled at the 5'-end internucleotide linkage, directly or through the spacers of different lengths, with TEMPO and the labelled oligoDNAs were explored for the ESR spectroscopical behaviour in solution. Oxidation of H-phosphonate intermediates in CCl4 solutions of TEMPO and diamines was taken for the introduction of TEMPO and spacers, respectively. ESR lines of TEMPO attached to oligoDNAs, directly or through spacers, were already broadened compared to those of free TEMPO, and the broadening decreased with increasing chain length of the spacer and increased with increase in the chain length of the oligoDNAs. The ESR lines of the oligoDNA labelled directly were further broadened in the presence of a DNA complementary to the labelled oligoDNA. In addition, the broadened lines were further broadened in the presence of a larger DNA. This observation implies that spin labelling at the internucleotide linkage is useful in the DNA probe method to avoid B/F separation.

Base Sequence↗
PubMed1983

Study on hydroxamic acids and their urease inhibitory potency by quantum chemistry calculation.

The electronic structures of 34 hydroxamic acids [R-(CONHCH2(n-CONHOH, R = aromatic or aliphatic, n = 1 or 0] were calculated by the INDO method and their urease inhibitory potencies were discussed in terms of the calculated electronic parameters and molar refraction. The charge distribution of -CONHOH residue which has been expected as a functional group for inhibition slightly be affected by the change of R - moiety and by the presence or absence of the -CONHCH2- residue. The best improved regression equation indicated that the inhibitory potency of hydroxamic acids was parabolically varied with the molar refraction and that the increase of the inhibitory potency by the presence of -CONHCH2- residue was explained by the variation of the charge density of a carbon atom directly bonding the -CONHOH group.

Chemical Phenomena↗