Biomedical subjects

Y Yamakoshi

Publications and source records attributed to Y Yamakoshi.

At least 19 recordsLinked to original sources

PubMed2006

The effect of the amelogenin fraction of enamel matrix proteins on fibroblast-mediated collagen matrix reorganization.

Enamel matrix proteins (EMP), extracted from developing porcine teeth, promote not only periodontal regeneration but also cutaneous wound healing presumably via the amelogenin fraction. Because it is unclear whether the effect of EMP can be ascribed to amelogenins, we compared EMP with recombinant amelogenin in the relaxed dermal equivalent (DE) in vitro model for early wound contraction. EMP and recombinant porcine amelogenin (rP172) at 1 mg/ml were incorporated into DEs composed of human dermal fibroblasts and a type I collagen matrix. The area reduction, as a measure of contraction, as well as fibroblast numbers and TGF-beta1 levels, were quantified over 7 days in culture in the presence of 10% foetal bovine serum. Both EMP and recombinant amelogenin increased contraction (p < 0.005) and fibroblast numbers (p < 0.005) compared with controls (acetic acid vehicle and 1mg/ml porcine serum albumin) and the positive control TGF-beta1 added at 10 ng/ml. Increased contraction with EMP and recombinant amelogenin was most pronounced after the first day of incubation and was associated with elevated (p < 0.005) TGF-beta1 levels in conditioned medium. In conclusion, the amelogenin component of EMP augmented fibroblast-driven collagen matrix remodelling, at least partially, by increasing the endogenous production of TGF-beta1. These effects of EMP/amelogenin may be beneficial for cutaneous wound healing.

Amelogenesis↗

PubMed2005

The onset of amelogenin nanosphere aggregation studied by small-angle X-ray scattering and dynamic light scattering.

Proteins with predominantly hydrophobic character called amelogenins play a key role in the formation of the highly organized enamel tissue by forming nanospheres that interact with hydroxyapatite crystals. In the present investigation, we have studied the temperature and pH-dependent self-assembly of two recombinant mouse amelogenins, rM179 and rM166, the latter being an engineered version of the protein that lacks a 13 amino acid hydrophilic C-terminus. It has been postulated that this hydrophilic domain plays an important role in controlling the self-assembly behavior of rM179. By small-angle X-ray and neutron scattering, as well as by dynamic light scattering, we observed the onset of an aggregation of the rM179 protein nanospheres at pH 8. This behavior of the full-length recombinant protein is best explained by a core-shell model for the nanospheres, where hydrophilic and negatively charged side chains prevent the agglomeration of hydrophobic cores of the protein nanospheres at lower temperatures, while clusters consisting of several nanospheres start to form at elevated temperatures. In contrast, while capable of forming nanospheres, rM166 shows a very different aggregation behavior resulting in the formation of larger precipitates just above room temperature. These results, together with recent observations that rM179, unlike rM166, can regulate mineral organization in vitro, suggest that the aggregation of nanospheres of the full-length amelogenin rM179 is an important step in the self-assembly of the enamel matrix.

Amelogenin↗

PubMed2005

Porcine amelogenin is expressed from the X and Y chromosomes.

Amelogenin is the major enamel matrix component in developing teeth. In eutherian mammals, amelogenin is expressed from the X chromosome only, or from both the X and Y chromosomes. Two classes of porcine amelogenin cDNA clones have been characterized, but the chromosomal localization of the gene(s) encoding them is unknown. To determine if there are sex-based differences in the expression of porcine amelogenin, we paired PCR primers for exons 1a, 1b, 7a, and 7b, and amplified enamel organ-derived cDNA separately from porcine males and females. The results show that exons 1a/2a and 7a are always together and can be amplified from both males (XY) and females (XX). Exons 1b/2b and 7b are also always paired, but can be amplified only from females. We conclude that porcine amelogenin is expressed from separate genes on the X and Y chromosomes, and not, as previously proposed, from a single gene with two promoters.

Amelogenin↗

PubMed2005

Enamel matrix derivative gel stimulates signal transduction of BMP and TGF-{beta}.

It has been shown that Emdogain Gel (Emd-Gel) containing enamel matrix proteins promotes biomineralization, such as osteogenesis and cementogenesis, during the regeneration of periodontal tissues. However, the growth factors involved in these activities of Emd-Gel remain unclear. In this study, Emd-Gel was fractionated into 22 sub-fractions by size exclusion chromatography. The osteoinductive factors, TGF-beta and BMP, were examined by a specific luciferase reporter gene assay. In the unfractionated Emd-Gel, TGF-beta-like activity was detected, while BMP activity was not. In contrast, in the fractionated Emd-Gel samples, TGF-beta-like activity was detected from fractions 8 to 13, and BMP-like activity was detected from fractions 4 to 6. Also, it was confirmed that the BMP-like activity in Emd-Gel was inhibited by authentic TGF-beta1 and TGF-beta-like activity. These results indicate that Emd-Gel contains both TGF-beta- and BMP-like growth factors that contribute to the induction of biomineralization during periodontal regeneration.

Animals↗

PubMed2003

Enamelin and autosomal-dominant amelogenesis imperfecta.

Dental enamel forms as a progressively thickening extracellular layer by the action of proteins secreted by ameloblasts. The most abundant enamel protein is amelogenin, which is expressed primarily from a gene on the X-chromosome (AMELX). The two most abundant non-amelogenin enamel proteins are ameloblastin and enamelin, which are expressed from the AMBN and ENAM genes, respectively. The human AMBN and ENAM genes are located on chromosome 4q13.2. The major secretory products of the human AMELX, AMBN, and ENAM genes have 175, 421, and 1103 amino acids, respectively, and are all post-translationally modified, secreted, and processed by proteases. Mutations in AMELX have been shown to cause X-linked amelogenesis imperfecta (AI), which accounts for 5% of AI cases. Mutations in ENAM cause a severe form of autosomal-dominant smooth hypoplastic AI that represents 1.5%, and a mild form of autosomal-dominant local hypoplastic AI that accounts for 27% of AI cases in Sweden. The discovery of mutations in the ENAM gene in AI kindreds proved that enamelin is critical for proper dental enamel formation and that it plays a role in human disease. Here we review how enamelin was discovered, what is known about enamelin protein structure, post-translational modifications, processing by proteases, and its potentially important functional properties such as its affinity for hydroxyapatite and influence on crystal growth in vitro. The primary structures of human, porcine, mouse, and rat enamelin are compared, and the human enamelin gene, its structure, chromosomal localization, temporal and spatial patterns of expression, and its role in the etiology of amelogenesis imperfecta are discussed.

Amelogenesis↗

PubMed2002

Pig amelogenin gene expresses a unique exon 4.

The pig amelogenin gene was isolated from a Lambda genomic library, and a 6.3 kb SalI/XbaI restriction fragment, inclusive of exons 3 through 7, was subcloned into a plasmid vector. DNA sequencing revealed two putative exon 4 sequences. The derived amino acid sequence of exon 4a, KSGRWGARLTAFVSSVQ, had previously been identified in a 190-amino-acid amelogenin isoform by protein sequencing. Exon 4b encoded the peptide DLYLEAIRIDRTAF, which is homologous to exon 4-encoded segments reported for human, mouse, and rat. Oligonucleotides from both of these exons were used to amplify cDNA generated from developing teeth. Amplification products were analyzed by agarose gel electrophoresis, cloned, and characterized by DNA sequencing. Exon 4a was found in transcripts encoding amelogenin isoforms having 190 and 73 amino acids. Exon 4b was found only in apparent splicing intermediates that retained intron 3, but was not detected in any final mRNA transcripts. Pig amelogenin having apparent molecular mass of 23 kD were isolated from the enamel matrix and characterized by mass spectrometry. Two mass values, 18,512.5, and 18,571.2 Da, were measured that match the values predicted for the 162-amino-acid cleavage product of the 173-amino-acid amelogenin, and the 165-amino-acid cleavage product of the 190-amino-acid amelogenin, which includes 17 amino acids encoded by exon 4a. We conclude that the pig amelogenin gene expresses a unique exon 4 that is not homologous to, or evolved from, the exon 4 segment expressed in humans and rodents.

Alternative Splicing↗

PubMed2002

Odontoblasts enhance the maturation of enamel crystals by secreting EMSP1 at the enamel-dentin junction.

The temporal expression patterns and activity distributions of enamelysin and EMSP1, which are the major proteinases in immature enamel, were characterized. Extracellular matrix fractions from developing porcine incisors, individually comprised of predentin, dentin, and four secretory-stage enamel samples, including the highly mineralized enamel (HME) at the enamel-dentin junction (EDJ), were isolated, and their resident proteinases were identified by zymography. Soft-tissue fractions, which included cells from the extension site of enamel formation (ESEF), secretory- and maturation-stage ameloblasts, and odontoblasts, were characterized histologically and by RT-PCR for their expression of enamelysin and EMSP1. A significant finding was that EMSP1, expressed by odontoblasts, concentrates in the HME, but is not detected in predentin or dentin. We conclude that odontoblasts deposit EMSP1 via their cell processes into the deepest enamel layer, which facilitates the hardening of this layer and contributes significantly to the functional properties of the EDJ.

Ameloblasts↗

PubMed2002

Amelogenin gene expression in porcine odontoblasts.

Amelogenin is the major organic component in the enamel matrix of developing teeth and plays an important role in enamel biomineralization. Amelogenin has been reported to be a specific secretory product of ameloblasts. In this study, we examined amelogenin gene expression in various cell layers prepared from a porcine permanent tooth germ using reverse transcription-polymerase chain-reaction (RT-PCR). Amelogenin amplification products were detected only in the secretory ameloblast layer after 20 cycles of PCR. After 30 cycles of PCR, amelogenin amplification products were detected in secretory and maturation-stage ameloblasts and in odontoblasts. The relative levels of amelogenin gene expression in secretory and maturation-stage ameloblasts and odontoblasts were determined. Secretory ameloblasts expressed over 1000 times the level of amelogenin mRNA found in odontoblasts. Amelogenin gene expression in odontoblasts was confirmed in an erupted porcine permanent first molar, which has no ameloblasts. Amelogenin PCR amplification products were identified from 4 different alternatively spliced transcripts in the ameloblast samples, and the same spliced forms were detected in the odontoblast samples.

Ameloblasts↗

PubMed2001

Immunoblot detection and expression of enamel proteins at the apical portion of the forming root in porcine permanent incisor tooth germs.

There have been many immunohistochemical studies of enamel proteins during root formation. In the present article, the detection and expression of enamel proteins in tissue samples prepared from the apical portion of the forming root (APFR) in porcine permanent incisor tooth germs were studied. Amelogenin, enamelin, and sheathlin were detected by immunoblot analysis, but only in small amounts. The detection of their derivatives indicated their degradation. It is, at present, unclear as to which proteinases are involved in these degradations, because activity of enamel matrix serine proteinase 1 and enamelysin was not detected on gelatin and casein zymograms. The expression of enamel proteins was also proved in the APFR sample by the detection of polymerase chain reaction products of their cDNAs, and this may be related to cells of fragmentized Hertwig's epithelial root sheath. Amelogenin expression was not greater than that of enamelin and sheathlin. It was different from the expression pattern of secretory ameloblasts involved in enamel matrix formation. These results suggest that the amelogenins found in the APFR do not form a three-dimensional structure of amelogenin micelles, which has been proposed for the secretory enamel matrix structure. In this case, the enamel proteins could spread out easily following degradation into the matrix of future cementum. Some of their derivatives may play a role in the formation of the cementum.

Amelogenin↗

PubMed2001

Calcium binding of enamel proteins and their derivatives with emphasis on the calcium-binding domain of porcine sheathlin.

Dental enamel is believed to form by the transfer of ions from solution, primarily calcium, phosphate, hydroxyl and carbonate, to the surface of solid-state mineral. Such precipitation phenomena can be controlled by regulating the degree of saturation of the solution with respect to the potential solid phases that can form. The concentration of free calcium is the factor that most affects the degree of saturation for calcium hydroxyapatite, and its buffering by calcium-binding proteins has been proposed as the mechanism that determines the enamel mineral structure. In this study, Stains-all staining was used to identify and isolate calcium-binding proteins from the enamel matrix, and determine their structures and association constants for calcium. Proteolytic cleavage fragments derived from the C-terminus of sheathlin, having apparent molecular weights of 13, 15, 27 and 29 kDa, were characterized by amino-terminal protein sequencing, amino acid analysis, and sugar, phosphate and sulphate determinations. Sheathlin C-terminal cleavage products were shown to have no N-linked glycosylations or phosphorylated amino acids, but Pro(350) was hydroxylated, and there was one sulphated O-linked glycosylation at Thr(386), containing galactose and N-acetylgalactosamine. The calcium-binding association constants for enamel proteins ranged from a high of 1.2 x 10(4) M(-1) to a low of 4.4x10(1) M(-1). The relative strengths of binding in order of decreasing affinity were: 13 and 15 kDa calcium-binding domain of sheathlin >27 and 29 kDa calcium-binding proteins >32 kDa enamelin >89 kDa enamelin >6.5 kDa, 25 kDa, 23 kDa, 20 kDa, 13 kDa, 5.3 kDa amelogenins. It is concluded that if enamel proteins have similar calcium-binding properties in vivo as have been measured in vitro, they would tend to buffer the free calcium ion concentration in enamel fluid.

Acetylgalactosamine↗

PubMed2001

Potent estrogen agonists based on carborane as a hydrophobic skeletal structure. A new medicinal application of boron clusters.

BACKGROUND: Carboranes (dicarba-closo-dodecaboranes) are a class of carbon-containing polyhedral boron-cluster compounds having remarkable thermal stability and exceptional hydrophobicity. Applications of the unique structural and chemical properties offered by icosahedral carboranes in boron neutron capture therapy have received increasing attention over the past 30 years. However, these features of carboranes may allow another application as a hydrophobic pharmacophore in biologically active molecules that interact hydrophobically with receptors. RESULTS: We have designed candidate estrogen-receptor-binding compounds having carborane as a hydrophobic skeletal structure and synthesized them. The most potent compound bearing a carborane cage exhibited activity at least 10-fold greater than that of 17beta-estradiol in the luciferase reporter gene assay. Estrogen receptor-alpha-binding data for the compound were consistent with the results of the luciferase reporter gene assay. The compound also showed potent in vivo effects on the recovery of uterine weight and bone loss in ovariectomized mice. CONCLUSION: Further development of the potent carborane-containing estrogenic agonists described here, having a new skeletal structure and unique characteristics, should yield novel therapeutic agents, especially selective estrogen receptor modulators. Furthermore, the suitability of the spherical carborane cage for binding to the cavity of the estrogen receptor-alpha ligand-binding domain should provide a basis for a similar approach to developing novel ligands for other steroid receptors.

Animals↗

PubMed2000

Clinical evaluation of sonoelasticity measurement in liver using ultrasonic imaging of internal forced low-frequency vibration.

The aim of this study is to evaluate liver elasticity noninvasively. We have already proposed an ultrasonic imaging system that can reconstruct vibration maps inside tissue under forced mechanical vibration. With this system, shear elastic properties of soft tissue can be evaluated as vibration velocities. Theoretically, these velocities increase with the increase of tissue elasticity. Sonoelasticity imaging was performed on 236 patients with chronic hepatitis and liver cirrhosis, and 50 healthy volunteers. The average of the velocities was 598.8 +/- 151.7 cm/s in healthy volunteers, 984.4 +/- 362.5 cm/s in chronic hepatitis and 1189.0 +/- 411.7 cm/s in liver cirrhosis. The average velocity of Child C group was statistically faster than those of Child A and B groups. Fibrotic rate from biopsy specimens statistically had the strongest positive correlation with velocities. With our system, the degree of liver fibrosis and function can be estimated objectively and noninvasively.

Adult↗

PubMed2000

Interpolation of missing part of human follicle border on ultrasonic B-mode image by iterative revision.

In clinical infertility treatments, assessment of ovarian follicle growth by ultrasonography is important. In order to measure the geometrical characteristics of the human follicle, such as the area of the cross-sectional image and the volume inside the follicle, a method based on manual tracing of the follicle contour from the ultrasonic B mode image is widely used. However, the observable ultrasonic B mode images are sometimes imperfect and some parts of the follicle border are missing due to the existence of the acoustic shadow. In this paper, a method that interpolates the missing part of the follicle border from the known part is proposed. This method uses a priori information of the follicle, which is usually known in actual cases: (1) the follicle's surface is so smooth that its border is assumed to be a smooth closed curve; and (2) the position of the follicle's center is roughly predicted in advance in the ultrasonic B-mode image. In the proposed method, the missing part of the human follicle border is interpolated from the known part by applying an iterative revision so as to satisfy the smoothness condition of the follicle. This method is also applied to three-dimensional image reconstruction of the human follicle.

Algorithms↗

PubMed2000

Dependence of estrogenic activity on the shape of the 4-alkyl substituent in simple phenols.

The ability of certain chemicals to mimic the effects of natural steroid hormones and their potential to disrupt the delicate balance of the endocrine system in animals has attracted much interest in recent years. Alkylphenolic chemicals have been reported to be weakly estrogenic. Estrogen receptor (ER) binding is primarily the result of interaction of the receptor with both a phenolic residue, and a hydrophobic pharmacophore. We have prepared and screened various phenols having a bulky 4-alkyl group, which may interact hydrophobically with the receptor, for estrogenic activity by using a previously described reporter gene assay employing COS-1 cells transfected with rat ER alpha-expression plasmid and an appropriate reporter plasmid. Some of the tested compounds, such as 4-(1-adamantyl)phenol and 4-cycloalkylphenols, exhibited much more potent activity than the typical estrogenic alkylphenol, 4-tert-octylphenol.

Animals↗

PubMed2000

New estrogenic antagonists bearing dicarba-closo-dodecaborane as a hydrophobic pharmacophore.

We have designed and synthesized estrogen antagonists bearing dicarba-closo-dodecaborane (carborane) as a hydrophobic pharmacophore based on the structure of 1-(4-hydroxyphenyl)-1,12-dicarba-closo-dodecaborane, a potent estrogen agonist that we reported previously. Compounds with a long alkyl chain bearing an amide moiety on the carborane skeleton (6, 7) showed estrogen antagonistic activity in a luciferase reporter gene assay using COS-1 cells transfected with a rat ER alpha-expression plasmid and as an appropriate reporter plasmid.

Animals↗

PubMed2000

[Reactive species responsible for biological actions of photoexcited fullerenes].

Fullerene (C60, C70, etc.) is an effective photosensitizer and its utilization as a pharmacophore for photo-chemotherapy of tumors has received considerable attention. We developed a method to solubilize fullerenes into water with polyvinylpyrrolidone (PVP) as a detergent. By using thus prepared aqueous fullerene solutions, we have clarified a series of biological activities of fullerene under photoirradiation which include DNA-cleavage, hemolysis, mutagenicity, cancer-initiation, and cell-toxicity. A newly synthesized C60 derivative with an acridine moiety as a DNA-chelating function showed much more effective DNA-cleaving activity in the presence of NADH. Visible-light irradiation of PVP-solubilized C60 in water in the presence of NADH as a reductant and molecular oxygen resulted in the formation of O2.-, which was detected by the EPR spin-trapping method. Formation of O2.- was also evidenced by the direct observation of a characteristic signal of O2.- by the use of a low-temperature EPR technique at 77 K. On the other hand, no formation of 1O2 was observed by the use of TEMP as a 1O2 trapping agent. No near-IR luminescence of 1O2 was also observed in the aqueous C60/PVP/O2 system. These results suggest that photoinduced bioactivities of the PVP-solubilized fullerene are caused not by 1O2, but by reduced oxygen species (O2.-, .OH) which are generated by the electron-transfer reaction of C60.- with molecular oxygen.

Animals↗

PubMed1999

Estrogenic antagonists bearing dicarba-closo-dodecaborane as a hydrophobic pharmacophore.

Dicarba-closo-dodecaboranes (carboranes), which have spherical geometry and hydrophobicity, are applicable as a hydrophobic pharmacophore of biologically active molecules. We have designed and synthesized estrogenic antagonists based on the structure of the potent agonist 1-hydroxymethyl-12-(4-hydroxyphenyl)-1,12-dicarba-closo-d odecaborane, which we have developed. The compounds showed potent antagonistic activity in luciferase reporter gene assay using COS-1 cells transfected with rat ER alpha-expression plasmid and an appropriate reporter plasmid.

Animals↗

PubMed1999

Human prostate CYP3A5: identification of a unique 5'-untranslated sequence and characterization of purified recombinant protein.

We have isolated a cDNA clone coding for CYP3A5 from a human prostate cDNA library. The human prostate CYP3A5 cDNA had a unique 5'-untranslated sequence, suggesting that the prostate specific regulation of CYP3A5 is different from liver. Hybridization screening using a human genomic BAC library yielded four positive clones, two of which were shown to contain the unique 5'-untranslated sequence by Southern blot analysis. The CYP3A5 recombinant protein expressed in Escherichia coli using the pCWOri expression vector was purified to an almost electrophoretically homogeneous state with a specific content of 4.4 nmol of P450/mg of protein. This P450 exhibited 6beta-hydroxylation activity toward both testosterone and progesterone. No polar metabolite of 5alpha-dihydrotestosterone (DHT) was detected. The apparent K(m) values for testosterone and progesterone 6beta-hydroxylation were 143 and 114 microM, respectively, with V(max) values of 0.48 and 0. 21 nmol/min/nmol of P450, respectively. This is the first report that a particular form of P450, CYP3A5, has been isolated from human prostate and that the purified recombinant protein of CYP3A5 has been shown to be active in the metabolism of sex hormones.

5' Untranslated Regions↗