Potent estrogenic agonists bearing dicarba-closo-dodecaborane as a hydrophobic pharmacophore.
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Animals↗
Biomedical subjects
Y Yamakoshi
Publications and source records attributed to Y Yamakoshi.
[Biological activity of photoexcited fullerene].
Fullerene (C60, C70, etc.) is a third carbon allotrope discovered in 1985, and a great deal of attention has been focused on its physical and chemical properties in recent years. We are very interested in its biological properties for use fullerene as a pharmacophore. We first developed a method of solubilizing fullerene itself in water to perform in vitro biological screening. The concentrations of aqueous C60 and C70 solution with 5% poly(vinylpyrorridone) (PVP) are 400 and 200 micrograms/mL, respectively. By using aqueous fullerene solutions prepared in this manner, we have clarified a series of biological activities of fullerene, consisting of DNA-cleavage, hemolysis, cancer-initiation, and cell-toxicity under photoirradiation, and chondrogenesis and inhibition of glutathione S-transferase activity without photoirradiation. The biological activity of photo-excited fullerene was found to be promising, because fullerene is a highly efficient photo-sensitizer. We synthesized a C60 derivative with an acridine moiety as a DNA-chelating function and assessed its effective DNA-cleaving activity. What kind of active species is involved in the biological action of photo-excited fullerene is our next concerns. Two pathways have been reported for the photo-excitation of fullerene. The so-called Type II energy transfer pathway generates singlet oxygen (1O2), while the Type I electron transfer pathway gives a fullerene radical anion (C60.-, C70.-). In order to clarify the effective oxygen species actually responsible for the biological action of photo-excited fullerene, we performed DNA-cleaving tests and EPR spectroscopic analyses under several conditions. The results showed that the photo-induced biological activity of fullerene is not caused by 1O2, but by reduced oxygen species (O2.-, .OH) generated by the electron transfer reaction of C60.-, with molecular oxygen. Its specificity is thought to be mainly attributed to the high-reducible property of fullerene. Since the reductive activation of molecular oxygen by photo-excited fullerene was observed at physiological concentrations of NADH as the reductant, fullerene can be classified as an oxyl-radical-generating photosensitizer. Pharmaceutical application of fullerene to cancer photo-dynamic therapy appears promising.
Carbon↗
Immunocytochemical and immunochemical study of enamelins, using antibodies against porcine 89-kDa enamelin and its N-terminal synthetic peptide, in porcine tooth germs.
Enamelins comprise an important family of the enamel matrix proteins. Porcine tooth germs were investigated immunochemically and immunocytochemically using two antibodies: a polyclonal antibody raised against the porcine 89-kDa enamelin (89 E) and an affinity purified anti-peptide antibody against the porcine enamelin amino-terminus (EN). Immunochemical analysis of layers of immature enamel from the matrix formation stage detected immunopositive protein bands ranging from 10 kDa to 155 kDa in the outer layer enamel sample irrespective of the antibodies used. In contrast, the middle and inner enamel layer mainly contained lower molecular weight enamelins. In immunocytochemical analyses of the differentiation stage, 89 E stained enamel matrix islands around mineralized collagen fibrils of dentin, while EN stained both enamel matrix islands and stippled material. At the matrix formation stage, both antibodies intensely stained enamel prisms located in the outer layer. In the inner layer, 89 E moderately stained enamel matrix homogeneously, while EN primarily stained the prism sheath. The intense immunoreaction over the surface layer of enamel matrix at the matrix formation stage, following staining with 89 E and EN, disappeared by the end of the transition stage and the early maturation stage, respectively. The Golgi apparatus and secretory granules in the ameloblasts from the late differentiation stage to the transition stage were immunostained by both antibodies. These results suggest that expression of enamelin continues from late differentiation to the transition stage and the cleavage of N-terminal region of enamelin occurs soon after secretion. Some enamelin degradation products, which apparently have no affinity for hydroxyapatite crystals, concentrate in the prism sheaths during enamel maturation.
Amino Acid Sequence↗
Purification, characterization, and cloning of enamel matrix serine proteinase 1.
The maturation of dental enamel succeeds the degradation of organic matrix. Inhibition studies have shown that this degradation is accomplished by a serine-type proteinase. To isolate and characterize cDNA clones encoding this proteinase, we used two degenerate primer approaches to amplify part of the coding region using polymerase chain-reaction (PCR). First, we purified the proteinase from porcine transition-stage enamel matrix and characterized it by partial protein sequencing. The enzyme was isolated from the neutral soluble enamel extract by successive ammonium sulfate precipitations, hydroxyapatite HPLC, reverse-phase HPLC, DEAE ion exchange, and affinity chromatography with a Benzamidine Sepharose 6B column. The intact protein and lysylendopeptidase-generated cleavage products were characterized by amino acid sequence analyses. Degenerate oligonucleotide primers encoding two of the polypeptide sequences were synthesized. In a complementary strategy, degenerate oligonucleotide primers were designed against highly conserved active-site regions of chymotrypsin-like proteinases. Both approaches yielded PCR amplification products that served as probes for screening a porcine enamel organ epithelia-specific cDNA library. The longest full-length clone is 1133 nucleotides and encodes a preproprotein of 254 amino acids. We designate this protein enamel matrix serine proteinase 1 or EMSP1. The active protein has 224 amino acids, an isotope-averaged molecular mass of 24.1 kDa, and an isoelectric point of 6.0. Multiple-tissue Northern analysis indicates that EMSP1 is a tooth-specific protein. Gelatin enzymography shows a dramatic increase in EMSP1 activity in the transition-stage enamel matrix. EMSP1 is most homologous to kallikriens and trypsins.
Amelogenesis↗
Sites of asparagine-linked oligosaccharides in porcine 32 kDa enamelin.
The 32 kDa enamelin protein isolated from developing porcine enamel was previously shown to contain eight different asparagine-linked oligosaccharides. However, only three consensus attachment sites were evident in this protein. In this study, glycopeptides containing all three potential glycosylation sites (72-Asn, 79-Asn and 91-Asn) were purified from 32 kDa enamelin. The oligosaccharides were isolated from each glycopeptide following digestion with N-oligosaccharide glycopeptidase, labeled with 2-aminopyridine at the reducing ends, and then characterized by reverse phase HPLC. All three potential sites were found to be glycosylated heterogeneously (i.e., five biantennary complexes at 72-Asn, two biantennary complexes at 79-Asn, three triantennary complexes at 91-Asn), accounting for all eight oligosaccharides characterized previously. These results indicate that 32 kDa enamelin has a complex pattern of asparagine-linked glycosylation localized within a small region (20 residues) of the protein. The functional significance of this glycosylation remains to be established.
Animals↗
[Leiomyoma of the bladder: report of two cases].
We report 2 cases of leiomyoma of the urinary bladder. A 41-year-old female visited our hospital with the complaint of pollakisuria. A solid tumor of the urinary bladder was found by ultrasonography. A large shadow defect at the left-anterior wall was shown by drip infusion pyelography (DIP). Computed tomographic scan (CT) and magnetic resonance imaging (MRI) also revealed a large tumor. T1-weighted image revealed a homogeneous low intensity tumor and T2-weighted image disclosed heterogeneous low intensity tumor. Cystoscopy revealed a large submucosal tumor. Partial cystectomy was performed, and she has had neither recurrence nor metastasis for 36 months. A 32-year-old male was referred to our hospital with the complaint of macrohematuria. A solid tumor of the urinary bladder was found by ultrasonography. A shadow defect was not clearly detected by DIP. A large tumor was detected on the anterior wall by MRI. T1-weighted image showed a homogeneous low intensity tumor and T2-weighted image disclosed a high intensity tumor. Cystoscopy revealed a submucosal tumor on the anterior wall. Urine cytology did not suggest a malignancy. The biopsied specimens revealed only an inflammatory change in the mucosa. Partial cystectomy was carried out. He has had neither recurrence nor metastasis for 29 months. Histological diagnosis in both cases was leiomyoma of the urinary bladder.
Adult↗
Immunochemical and immunohistochemical study of the 27- and 29-kDa calcium-binding proteins and related proteins in the porcine tooth germ.
Our previous report identified 27- and 29-kDa calcium-binding proteins in porcine immature dental enamel. In this study we revealed that the N-terminal amino acid sequences of the two proteins were identical: LLANPXGXIPNLARGPAGRSRGPPG. The sequence matches a portion of the amino acid sequence of the porcine sheath protein, sheathlin. Porcine tooth germs were investigated immunochemically and immunohistochemically using specific antibodies raised against synthetic peptide that included residues 13-25 of this sequence. The affinity-purified antibodies reacted with several proteins extracted from newly formed immature enamel in immunochemical analyses, especially protein bands migrating at 62, 35-45, 29, and 27 kDa in SDS-polyacrylamide gels. The largest protein detected was a weak band near 70 kDa. In immunochemical analyses of proteins extracted from the inner (old) immature enamel, the antibody reacted faintly with the 27- and 29-kDa proteins. In immunohistochemical preparations, the Golgi apparatus and secretory granules of the secretory ameloblast, and the surface layer of immature enamel showed immunoreactivity. The immunoreactivity of immature enamel just beneath the secretory face of the Tomes' process was intense. No immunoreactivity was found in the Golgi apparatus of the maturation ameloblast. These results suggest that the 70-kDa protein, whose degradation might be very fast, is the parent protein of the 27- and 29-kDa proteins.
Ameloblasts↗
Sheathlin: cloning, cDNA/polypeptide sequences, and immunolocalization of porcine enamel sheath proteins.
Sheath proteins designate low-molecular-weight non-amelogenin enamel polypeptides and their parent protein, which concentrate in the sheath space separating rod and inter-rod enamel (Uchida et al., 1995). Two porcine sheath proteins, with apparent molecular weights of 13 and 15 kDa, are characterized by protein sequencing. The primary structures of these polypeptides match a portion of the derived amino acid sequences of clones isolated from a porcine enamel organ epithelia-specific cDNA library. Sheath protein RNA messages differ by the inclusion or deletion of a 45-nucleotide segment and by the use of three alternative polyadenylation/cleavage sites. The secreted proteins are 395 and 380 residues in length, with molecular masses of 42,358 and 40,279 Daltons and calculated isoelectric points of 6.3 and 6.7, respectively. Polyclonal antibodies were raised against a synthetic peptide having the sheathlin-specific sequence EHETQQYEYSGGC. Immunohistochemistry with this antibody demonstrates that the protein encoded by the sheathlin cDNA is preferentially localized in the sheath space. We propose that the porcine sheath proteins and their proteolytic cleavage products be designated "sheathlin".
Amino Acid Sequence↗
Cloning and characterization of porcine enamelin mRNAs.
Dental enamel forms by matrix-mediated biomineralization. The components of the developing enamel matrix are generally specific for that matrix. The primary structures of three enamel proteins-amelogenin, tuftelin, and sheathlin (ameloblastin/amelin)-have been derived from cDNA sequences. Here we report the cloning and characterization of mRNA encoding a fourth enamel protein: enamelin. The longest porcine enamelin cDNA clone has 3907 nucleotides, exclusive of the poly(A) tail. The primary structure of the secreted protein is 1104 amino acids in length. Without post-translational modifications, the secreted protein has an isotope-averaged molecular mass of 124.3 kDa and an isoelectric point of 6.5. Polymerase chain-reaction phenotyping of enamelin cDNA suggests that porcine enamelin transcripts are not alternatively spliced and use a single polyadenylation/cleavage site. Immunohistochemical and Western blot analyses with an affinity-purified antipeptide antibody specific for the enamelin carboxyl terminus demonstrate that enamelin is synthesized and secreted by secretory-phase ameloblasts. The parent protein is a 186-kDa glycoprotein that concentrates along the secretory face of the ameloblast Tomes' process. Intact enamelin and proteolytic cleavage products containing its carboxyl terminus are limited to the most superficial layer of the developing enamel matrix, while other enamelin cleavage products are observed in deeper enamel.
Amino Acid Sequence↗
Internal tissue displacement measurement based on ultrasonic wave Doppler signal digital detection and its application to fetal movement monitoring.
We have developed a fetal movement monitoring system based on small displacement measurement of internal tissues. When ultrasonic pulses are transmitted to the fetus, the reflected ultrasonic waves which have a Doppler frequency shift due to the fetal movements are detected by using an ultrasonic pulsed Doppler technique. In this paper, we propose a displacement measurement method for internal tissues which is based on the Doppler signal digital detection technique. In the method, the received ultrasonic RF signals are sampled with a sampling frequency of four times higher than the centre frequency of the ultrasonic waves; the Doppler frequency shift signals are derived using digital signal processing. From the detected signals, the internal displacements are estimated using the arc-tangent method. The basic algorithm of the detection method has already been used in the area of blood flow sensing, however, we apply the algorithm to the displacement measurement of internal tissues. The comparison between the proposed method and the conventional method is presented. The fetal movement quantitative monitoring system based on the method which has been constructed is shown.
Algorithms↗
Automated fetal breathing movement detection from internal small displacement measurement.
We have previously proposed a method to measure small displacements of internal tissue which is based on an ultrasonic pulsed Doppler technique. The Doppler frequency shift of the ultrasonic waves due to the tissue movement is detected using a modified quadrature detector, and the displacement of the internal tissue is estimated by using an arctangent method. Based on the algorithm, we have constructed a quantitative monitoring system for fetal breathing movement. The fetal breathing movement is a rhythmic movement of the fetal diaphragm and it is recorded as a nearly sinusoidal displacement with a repetition frequency from 0.5 Hz to 2 Hz. In this paper, an automated fetal breathing movement detection algorithm for the displacement data observed by using the small displacement measurement system is proposed. This method uses a modified autocorrelation technique for detecting the displacement due to the fetal breathing movement. The method is applied to five fetuses of late pregnancy and the result is compared with the manual detection.
Algorithms↗
Transvaginal sonographic orientation detection system using ceramic gyroscopes.
Use of three-dimensional ultrasonograms in daily clinical medicine requires the development of data accumulation techniques and image reconstruction techniques . We describe an image position and orientation detection technique using small piezoelectric vibratory gyroscopes. Such gyroscopes, of the same type that is used in an automobile navigation system, produce angular velocity by Coriolis force. After attaching three gyroscopes to an ordinary transvaginal probe, we attempted to detect image orientation to assist transvaginal scanning and to reconstruct three-dimensional projection images. Three-dimensional images were reconstructed by simple white and black inversion thresholding technique. Using free hand transvaginal scanning, the direction of the transvaginal ultrasound beam was displayed with B-mode ultrasonography. Using a personal computer, three-dimensional images of follicles and fetuses were successfully reconstructed within a few minutes. Our system provides easy understanding of transvaginal sonographic images and potentially might allow ordinary ultrasound machines to handle three-dimensional images in a day-to-day clinical practice.
Equipment Design↗
Carbohydrate moieties of porcine 32 kDa enamelin.
The structures of asparagine-linked oligosaccharides of porcine 32 kDa enamelin are reported. The oligosaccharides were released by N-oligosaccharide glycopeptidase digestion, and the reducing ends of the oligosaccharides were derivatized with a fluorescent reagent, 2-aminopyridine. The pyridylamino oligosaccharides were separated into eight kinds of oligosaccharides. The structures of these oligosaccharides were determined by a combination of a sequential exoglycosidase digestion and a two-dimensional sugar mapping technique. The oligosaccharides consisted of fucose, galactose, mannose, N-acetylglucosamine, and N-acetylneuraminic acid, and were classified into two groups according to their core-sugar chain structures; one was a biantennary-type and the other was a triantennary-type oligosaccharide. The variation of the oligosaccharides in each of these groups was caused by the differences in the number, the site, and the mode of linkage of N-acetylneuraminic acid to the core-sugar chains.
Amidohydrolases↗
Porcine amelogenins.
Amelogenins were extracted from the thin outer layer of porcine secretory enamel and purified by gel filtration and reverse-phase HPLC. The results of amino acid sequencing of the purified porcine amelogenins indicated the presence of at least four prototype amelogenins translated from alternatively spliced transcripts. The results of mass spectroscopy of the CNBr-cleaved peptides derived from the 25 kDa amelogenin indicated that porcine 25 kDa amelogenin is neither phosphorylated nor glycosylated.
Amelogenin↗
Foetal heart rate in early pregnancy by detection of optimally estimated displacements with pulsed Doppler signals.
Foetal heart rate (FHR) in the early stage of pregnancy can be optimally estimated by a simple system combining a conventional pulsed Doppler device with a microcomputer (CPU). The input signals for FHR have been obtained as low frequency components of foetal Doppler signals. These components are less than 50 Hz and are directly transferred from the conventional pulsed Doppler device to a microcomputer as radio-frequency signals by a specially designed I/O unit. The low frequency components have been converted into displacement signals by the continuous calculation of an arc-tangent function combined with five channel recordings from the foetal cardiac area, each separated by 2 mm on the same echo line. After selecting the optimal displacement data line, the FHR is measured by the proposed algorithm of non-parametric frequency estimation and/or the conventional autocorrelation method. The algorithm draws attention to the improved frequency selectivity by multiplication with reference signals, whose frequency is automatically optimized by the standard FHR rate determined by the length of pregnancy. The FHR determined from the displacement signals by this algorithm has provided a more optimally controlled selectivity than with the presently used autocorrelation method, especially in those pregnancies of less than 8 weeks gestation. Clinical application of this system allowed a complete determination of foetal heart rate from 6 weeks of gestation by a transvaginal approach and from 7 weeks by transabdominal procedures. In some specific cases, with prominent foetal components, it was possible from 5 weeks 2 days by a vaginal, and 6 weeks by an abdominal procedure.
Algorithms↗
Numerical analysis of quasiperiodic perturbations for the Alfvén wave.
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Journal Article↗
New method for tracking fetal breathing movements using real-time pulsed Doppler ultrasonographic displacement measurement.
To develop a clinical device for monitoring FBM with a simplified mechanism, the multichannel pulsed Doppler device was achieved by modifying the ultrasonographic module and probe of a cardiotocograph. A new algorithm of signal processing with a high-accuracy displacement estimation technique produced real-time and continuous displacement calculations. In vivo measurements demonstrated that real-time output produced commendable quality in FBM tracking. The displacement of a few hundred micrometers because of FBM was measured successfully. The results suggest that quantitative assessments of FBM should be possible with this technique.
Female↗
[Advanced system for real-time fetal breathing movements tracking using pulsed Doppler ultrasound].
Utilization of the fetal behavioral information in clinical medicine requires the development of an automated monitoring technique. As previously reported, the off-line signal processing of the low frequency Doppler signals by high accuracy displacement estimation made it possible to measure fetal breathing movements (FBM). However, there were several problems to solve before clinical application. We propose an advanced system that provides real-time calculation. The multi-channel pulsed doppler device was newly developed by modifying the Doppler ultrasound module of the cardiotocograph (CTG). A new signal processing algorithm was designed to produce real-time measurements. To improve the resolution, the backing module was attached to the probe. On-line signal processing produced a successful real-time and continuous calculation of displacement inside tissue. By means of an on-line 2/3 Hz high pass filter, the very low frequency noises and the artifacts derived from maternal respiration etc. were removed. The signal/noise ratio was obviously increased and the waves of fetal tissue movements due to FBM were easily recognized in real-time LCD outputs. Although there is a limitation in measuring accuracy as far as using modified CTG units, the results demonstrate that our system has commendable quality in FBM tracking.
Computer Systems↗