Search PubMed⌕ Search

Biomedical subjects

M Nagaoka

Publications and source records attributed to M Nagaoka.

At least 37 records · Page 2Linked to original sources
PubMed2001

Design of novel zinc finger proteins: towards artificial control of specific gene expression.

In this review, we summarize design strategies for generating proteins with desired sequences such as long contiguous base pairs and diverse sequence specificities based on the nature of Cys(2)-His(2) zinc finger proteins. Recent progress towards artificial DNA binding proteins has been achieved by structure-based design processes and by selection strategies. Indeed, a multi-zinc finger protein with an 18 (or 27)-base pair address, and new zinc finger proteins for diverse DNA target sites (TATA-box and p53 binding site) have been created successfully. Such novel zinc finger proteins will probably be useful tools in molecular biology and potentially in human medicine.

Animals↗
PubMed2001

19-Oxygenation of C19-steroids with an A, B-ring enone structure, competitive inhibitors of estrogen biosynthesis, with human placental aromatase.

Aromatase is a cytochrome P-450 enzyme complex that catalyzes the conversion of androst-4-ene-3,17-dione (AD) to estrone through three sequential oxygenations of the 19-methyl group. To gain insight into the ability of AD isomers, 4-en-6-one 1a, 5-en-4-one 2a, and 5-en-7-one 3a, competitive inhibitors of aromatase with an A, B-ring enone structure to serve as a substrate, we incubated the three inhibitors separately with human placental aromatase in the presence of NADPH in air. The metabolites were analyzed as the methoxime-trimethylsilyl ethers by gas chromatography-mass spectrometry. All of the inhibitors were found to be oxygenated with aromatase to produce the corresponding 19-hydroxy derivatives 1b, 2b, and 3b with rates of 2.0, 51, and 0.3 pmol/min/mg protein, respectively. Only in the experiment with the 5-en-4-one steroid 2a, the production of the 19-oxo metabolite 2c was detected with a rate of 3.1 pmol/min/mg protein. The 19-oxygenation of steroid 2a, the best substrate for aromatase among the three, was kinetically determined to give the Vmax value of 40 pmol/min/mg protein and the Km value of 1.43 microM, respectively. The results reveal that a good inhibitor of aromatase is not essentially a good substrate for the enzyme in a series of the A, B-ring enone steroids.

Aromatase Inhibitors↗
PubMed2001

Gas chromatography-mass spectrometric determination of activity of human placental aromatase using 16alpha-hydroxyandrostenedione as a substrate.

Aromatization of 16alpha-hydroxyandrostenedione (16alpha-OH AD) with aromatase in human placental microsomes was studied by gas chromatography-mass spectrometry (GC-MS) using 12,4,6,6,9alpha,16beta,17alpha-2H7]estriol as an internal standard. 16alpha-OH AD was incubated with the microsomes in the presence of NADPH in air. The metabolite was extracted with ethyl acetate and treated with NaBH4. The reduced product, estriol, was isolated by Sep-Pak C18 cartridge and then analyzed as the tris(trimethylsilyl)ether by a GC-MS (EI mode). The production of estriol was dependent upon protein concentration and incubation time. Apparent Km and Vmax values of the microsomal aromatase for 16alpha-OH AD were 568 nM and 25.5 pmol/min/mg protein, respectively. In this assay, aromatase activity, estriol formation, could be determined at a level as low as 1 pmol/min/mg protein. Aromatase inhibitors, 4-hydroxy- and 6-oxo-androstenediones, prevented the estriol formation in a competitive manner with 25 and 30 nM of apparent Ki values, respectively.

Androstenedione↗
PubMed2001

Identification of 16alpha,19-dihydroxyandrostenedione in the serum of pregnant women by gas chromatography-mass spectrometry.

16alpha,19-Dihydroxyandrostenedione [16alpha,19-(OH)2 AD], a potential precursor of estriol, was identified in the serum of pregnant women by gas chromatography-mass spectrometry (GC-MS). Preliminary study indicated that the average serum concentration of 16alpha,19-(OH)2 AD in 10 normal pregnant women was 154.2+/-20 pg/ml. In contrast, the serum concentration of 16alpha,19-dihydroxydehydroepiandrosterone was less than the detection limit (30 pg/mI) of the GC-MS method.

Adult↗
PubMed2001

Effect of oligofucose derivatives on acetic acid-induced gastric ulcer in rats.

This study attempted to enhance the anti-ulcer activity of fucoidan from Cladosiphon okamuranus TOKIDA by chemical modification with a hydrophobic group. The suitable number of fucose residues in the effective compound was also clarified to obtain a compound of constant quality. Degraded fucoidans were coupled with several hydrophobic groups via Schiff bases, and their anti-ulcer activities were determined by acetic acid-induced ulcer models in rats. Size-fractionated oligofucose was also modified and assayed for anti-ulcer activity. Among the modified oligofucoses, only the oligofucose-dodecylaniline combination (OFDA) significantly promoted ulcer healing. The effective dose was 0.2 mg/kg/d. The most suitable number of fucose residues in the compound for the anti-ulcer activity was determined to be around 12. We succeeded in enhancing the anti-ulcer activity of Cladosiphon fucoidan by modification with dodecylaniline. The activity of this compound was comparable or greater than that of typical anti-ulcer agents. By determination of the optimal OF chain length for the anti-ulcer activity of OFDA, it became possible to obtain OFDA of constant quality.

Acetic Acid↗
PubMed2000

DNA-bending finger: artificial design of 6-zinc finger peptides with polyglycine linker and induction of DNA bending.

DNA structural changes such as bending play an important role in various biological reactions. Not only protein binding to its specific DNA sequence but also DNA bending induced by the protein is indispensable for unique gene expression. Therefore, an artificial protein that induces a DNA conformational change is interesting as a transcriptional regulator of a specific gene. We created 6-zinc finger proteins, Sp1ZF6(Gly)n (n = 4, 7, 10), by connecting two DNA binding domains of transcription factor Sp1 with flexible polyglycine peptide linkers, and their effects on DNA structure were compared with that of native 3-zinc finger Sp1(530-623). Gel electrophoretic methods revealed that Sp1ZF6(Gly)7 and Sp1ZF6(Gly)10 bind to two distal GC boxes and result in DNA bending. Evidently, the hydroxyl radical footprinting analysis demonstrated that hypersensitive cleavage was observed at the 5'-TA step in the intervening region bound by Sp1ZF6(Gly)7 or Sp1ZF6(Gly)10. The phasing assays strongly suggested that the induced DNA bending was directed toward the major groove and that Sp1ZF6(Gly)7 caused the most drastic directional change in DNA bending. Of special interest are the facts that the newly designed 6-finger peptides Sp1ZF6(Gly)7 and Sp1ZF6(Gly)10 can induce DNA bending at the intervening region of the two distal binding sites and that the linker length between two 3-zinc finger motifs has a crucial effect on the entire DNA-bending direction. Such DNA-bending fingers may be feasible for use as a gene expression regulator based on the structural change in DNA in the future.

Amino Acid Sequence↗
PubMed2000

Expression of kininogen, kallikrein and kinin receptor genes by rat cardiomyocytes.

To ascertain the existence of the kallikrein-kinin system in the heart, we have studied in vivo and in vitro whether rat cardiac tissue expresses kininogen, kallikrein and kinin receptor mRNAs. The reverse transcription-polymerase chain reaction demonstrated that the ventricular myocardium of adult male rats expressed mRNAs for T- and low-molecular-weight (L-) kininogens, tissue kallikreins such as true kallikrein and T-kininogenase, and bradykinin B2 receptor, but not those for high-molecular-weight kininogen and B1 receptor. Lipopolysaccharide (LPS; 0.5 mg/kg, i.v.) increased the levels of mRNA for T-kininogen at 12 h and the bradykinin B1 receptor at 24 h without affecting that for other components. All of these mRNAs for the kallikrein-kinin system were also detected in cultured cardiomyocytes derived from neonatal rat ventricles; dibutyryl cyclic AMP, LPS or inflammatory cytokines such as interleukin-1 and tumor necrosis factor, up-regulated mRNA expression of T-kininogen, T-kininogenase, or B1 receptor in these cells in vitro. These results suggest that there are two kinin-generating systems in rat myocardium comprising T-kininogen/T-kininogenase and L-kininogen/true kallikrein respectively, and that the former may be relatively important in inflammatory diseases or conditions in which cAMP levels increase in cardiomyocytes.

Animals↗
PubMed2000

Properties of fucoidan from Cladosiphon okamuranus tokida in gastric mucosal protection.

To elucidate the anti-ulcer potential of Cladosiphon fucoidan, anti-peptic activity, bFGF stabilizing activity and inflammatory properties of this and related substances were investigated. Anti-peptic activity was observed with this and other sulfated polysaccharides such as dextran sulfate, carrageenan, and Fucus fucoidan. However, non-sulfated polysaccharides such as mannan and dextran did not exert the anti-peptic activity. The loss of bFGF bioactivity was prevented by all sulfated polysaccharides tested except chondroitin sulfate, at pH 7.4 and at pH 4.0. At pH 2.0, only heparin protected the bFGF activity. The generation of superoxide by macrophages and PMNs was stimulated by dextran sulfate, carrageenan, and Fucus fucoidan, whereas Cladosiphon fucoidan, heparin and chondroitin did not. Dextran sulfate, carrageenan, and Fucus fucoidan also stimulated the secretion of TNFalpha from macrophages, while Cladosiphon fucoidan did not. Thus, Cladosiphon fucoidan is a sulfated polysaccharide without inflammatory action. These results suggest that Cladosiphon fucoidan is a safe substance with potential for gastric protection.

Animals↗
PubMed2000

Ultrasonographic examination of de Quervain's disease.

In order to assess the usefulness of ultrasonography in the preoperative evaluation of de Quervain's disease, we retrospectively analyzed the ultrasonographic findings in 32 patients, and compared these findings with operative findings. Ultrasonography identified a septum between the abductor pollicis longus and extensor pollicis brevis tendons in 26 patients and showed absence of the septum in 6 patients. During surgery, the presence of the septum was confirmed in 27 patients, and was absent in 5. Our results demonstrated the usefulness of preoperative ultrasonography in correctly detecting anatomic abnormalities in patients with de Quervain's disease.

Adult↗
PubMed2000

Artificial zinc finger peptides: creation, DNA recognition, and gene regulation.

Proteins control most biological reactions and the disorder of their expression level causes many diseases. The advent of genomic sequencing and the availability of the complete sequences of several genomes provide new opportunities to study biology and to develop therapeutic strategies through specific modulation of the transcription of target genes. Therefore, regulation of the transcription level by "artificial repressors" is of special importance. Of the DNA-binding motifs that have been manipulated by design or selection, Cys2-His2 zinc finger proteins have demonstrated the greatest potential for manipulation into general and specific transcription factors. Of special interest is the feature that this family of proteins has modular structures and can recognize a diverse set of DNA sequences in a sequence-specific manner. Therefore, zinc finger motifs offer an attractive framework for the design of novel DNA binding proteins, and such a DNA binding protein would be expected to possess a unique binding sequence with high specificity and affinity. Principally, two approaches have been taken to the design of artificial zinc finger proteins. One is a selection strategy via phage display methods to alter the recognition sequence, and another is a structure-based linking strategy to extend the length of a DNA recognition sequence. Such novel zinc finger peptides (or proteins) offer great promise for genome-specific transcription switches in the near future.

Amino Acid Sequence↗
PubMed2000

The engineering, structure, and DNA binding properties of a novel His4-type zinc finger peptide.

We have created a novel His4-type zinc finger protein (H4Sp1) engineered by Cys-->His mutations of the Cys2His2-type zinc finger in transcription factor Sp1. The CD and NMR studies reveal that the His4 domain has Zn(II)-dependent folding properties and similar secondary structures to wild-type Cys2His2 domain. The DNA binding experiments demonstrate that H4Sp1 can bind DNA in a specific way. The present artificial peptide H4Sp1 will provide valuable information about the interaction between a metallopeptide and DNA.

Amino Acid Sequence↗
PubMed1999

19-oxygenations of 3-deoxy androgens, potent competitive inhibitors of estrogen biosynthesis, with human placental aromatase.

Aromatase is a cytochrome P450 enzyme complex that catalyzes the conversion of androst-4-ene-3,17-dione (AD) to estrone through three sequential oxygenations of the 19-methyl group. To gain insight into the ability of 3-deoxy derivative of AD, compound 1, and its 5-ene isomer 4, which are potent competitive inhibitors of aromatase, to serve as a substrate, we studied their 19-oxygenation by human placental aromatase and the metabolites isolated were analyzed by gas chromatography-mass spectrometry. Inhibitors 1 and 4 were found to be oxygenated with aromatase to produce the corresponding 19-hydroxy derivatives 2 and 5 and 19-oxo derivatives 3 and 6 as well as the 17beta-reduced 19-hydroxy compounds 7 and 8. Kinetic studies indicated that the 5-ene steroid 4 was surprisingly a good substrate for the aromatase-catalyzing 19-oxygenation with the V(max) value of 45 pmol/min per mg prot which was approx. four times higher than that of the other. The relative K(m) value for steroids 1 and 4 obtained in this study is opposite from the relative K(i) value obtained previously in the inhibition study. The results reveal that there is a difference between a binding suitable for serving as an inhibitor of aromatase and a binding suitable for serving as a substrate of the enzyme in the 3-deoxy steroid series and the C-3 carbonyl group of AD is essential for a proper binding as a substrate to the active site of aromatase.

Androgens↗
PubMed1999

Common modulation of motor unit pairs during slow wrist movement in man.

1. The activity of 36 pairs of single motor units were recorded with intramuscular wire electrodes from m. extensor carpi radialis while subjects performed slow wrist extension and flexion movements. Periods of steady position holding were interposed between movements. 2. The discharge trains from pairs of motor units were analysed statistically in the time and frequency domains. During extension movements, when the muscle recorded from was the agonist, coherence between motor units was significant below 12 Hz, with a peak at 6-12 Hz in 30 of 36 pairs (83 %). The magnitude of coherence decreased during position holding compared to movements in 26 pairs, while the difference in average firing rate was small. 3. During movements, but not during position holding, coherence estimates between single motor units and acceleration showed a significant peak at 6-12 Hz in 56 out of 62 motor units, suggesting that a modulation of motor unit discharge contributed to angular acceleration at these frequencies. Common motor unit modulation was present at 3 Hz as well, although the coupling between motor unit activity was weaker than at 6-12 Hz. 4. It is concluded that a 6-12 Hz common modulation of agonist motor units is a distinguishing feature of slow voluntary wrist movements, extending the previously established notion of an 8-10 Hz rhythmic organization of slow finger movements to more proximal limb segments. It is suggested that the 6-12 Hz input is specific for movements and is normally absent or much weaker during steady maintenance of position or force.

Acceleration↗
PubMed1999

Expression of kininogen genes by rat cardiomyocytes.

To determine the existence of the kallikrein-kinin system in the heart, we have studied in vitro and in vivo whether rat heart expresses kininogens (KGNs). The reverse transcription-polymerase chain reaction (RT-PCR) for KGN mRNAs demonstrated that the cardiac tissue of adult male rats expresses T-KGN mRNA but not high-molecular-weight (H-) KGN mRNA. An intravenous injection of lipopolysaccharide (LPS) resulted in a significant increase in T-KGN mRNA levels of rat heart within 12 h. Polyacrylamide gel electrophoresis of cDNA products generated by RT-PCR from heart mRNA using primers specific for either T- or low-molecular-weigh (L-) KGN revealed that rat heart expressed not only T-KGN gene but also L-KGN gene, and that LPS injection exclusively stimulated the expression of T-KGN but not of L-KGN gene. T-KGN mRNA was also detected in cultured myocytes derived from fetal rat heart, and the expression was markedly enhanced by an addition of LPS to cultures. These results demonstrated that rat cardiomyocytes are the source of T- and L-KGNs but not of H-KGN, and that their expression of T-KGN mRNA is stimulated by LPS, probably via LPS-receptor CD14.

Animals↗
PubMed1999

Structural study of fucoidan from Cladosiphon okamuranus TOKIDA.

A structural study was carried out on a fucoidan isolated from the brown seaweed Cladosiphon okamuranus. The polysaccharide contained fucose, glucuronic acid and sulfate in a molar ratio of about 6.1 : 1.0 : 2.9. The results of Smith degradation showed that this polysaccharide has a linear backbone of 1-->3-linked alpha-fucopyranose with a half sulfate substitution at the 4-positions, and a portion of the fucose residues was O-acetylated. The data obtained from partial acid hydrolysis, a methylation analysis and NMR spectra indicated that the alpha-glucuronic acid residue is linked to the 2-positions of the fucose residues, which were not substituted by a sulfate group. These results indicated that the average structure of this fucoidan is as follows: -[(-->3Fuc-4(+/-OSO3-)alpha1-)5-->3[GlcA alpha1-->2]Fuc alpha1-]n-. (Half of each fucose residue was sulfated. One O-acetyl ester was present in every 6 fucose residues.)

Molecular Structure↗
PubMed1999

3-Geranyl-4-hydroxy-5-(3'-methyl-2'-butenyl)benzoic acid as an anti-inflammatory compound from Myrsine seguinii.

Bioassay-guided isolation of anti-inflammatory compounds from the methanol extract of Myrsine seguinii yielded an anti-inflammatory compound (1). The structure of compound 1 was elucidated to be 3-geranyl-4-hydroxy-5-(3'-methyl-2'-butenyl)benzoic acid on the basis of its spectroscopic data. Compound 1 strongly suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation on mouse ears at a dose of 500 micrograms (inhibitory effect (IE): 65%). The acetate and the methyl ether of 1 showed moderate activity at a 500-microgram application, with IE 38% and 27%, respectively. However, the methyl ester and the dimethyl derivative of 1 did not show activity at the same dose. The related compounds of 1, o-, m- and p-hydroxybenzoic acids also did not exhibit notable activity. These results indicate that the carboxylic acid and lipophilic terpene moieties of 1 were significant structural features for anti-inflammatory activity.

Animals↗