Biomedical subjects

E Koyama

Publications and source records attributed to E Koyama.

At least 19 recordsLinked to original sources

PubMed2007

Expression of HIF prolyl hydroxylase isozymes in growth plate chondrocytes: relationship between maturation and apoptotic sensitivity.

The overall goal of the current study was to examine the functional activity of the prolyl hydroxylases (PHDs) in maturing chondrocytes. Herein, we show for the first time that the PHDs are expressed in the maturing zone of the growth plate, and by a chondrocytic cell line. We determined if this protein and its substrate, hypoxia inducible factor (HIF)-1alpha, modulated the induction of apoptosis. Using a chondrocyte cell line that matured in culture, we inhibited HIF-1alpha expression using siRNA technology and pharmacologically blocked PHD activity. We noted that PHD suppression sensitized the cells to an apoptotic challenge with H(2)O(2). We next examined the interplay between the PHDs and HIF-1alpha by suppressing HIF-1alpha and blocking PHD activity. We noted reduced killing when the mature HIF-silenced cells were challenged with H(2)O(2). In contrast, there was limited change in the viability of immature cells. Based on these differences in chondrocyte susceptibility, it is concluded that HIF-1alpha sensitizes maturing cells to H(2)O(2)-mediated killing. We next determined if this change in the viability of the PHD-inhibited cells was linked to changes in activation of caspase-3. It was noted that there was a minimal change in enzyme activity of the PHD-inhibited HIF-1alpha suppressed cells. Finally, we found that as the chondrocytes mature, the activities of catalase and SOD were significantly reduced and that there was a decrease in the levels of Bcl-2 and Bcl(XL). This loss of protective activity together with the changes mediated by HIF would be expected to generate conditions that would favor the induction of chondrocyte apoptosis.

Amino Acids, Dicarboxylic↗

PubMed2003

In vitro metabolism of the glycosidic sweeteners, stevia mixture and enzymatically modified stevia in human intestinal microflora.

Stevia mixture, sweeteners extracted from the leaves of Stevia rebaudiana Bertoni, consists mainly of stevioside and rebaudioside A (glycosides of the diterpene derivative steviol). The aim of this study was to investigate human intestinal metabolism of stevia mixture and its alpha-glucose derivative (known in Japan as enzymatically modified stevia) by LC/MS/ESI analysis. Degradation was examined by incubating stevia mixture, enzymatically modified stevia, stevioside, rebaudioside A, alpha-monoglucosylstevioside, alpha-monoglucosylrebaudioside A and the aglycone, steviol with pooled human faecal homogenates (obtained from five healthy volunteers) for 0, 8 and 24 h under anaerobic conditions. Stevia mixture, enzymatically modified stevia, stevioside and rebaudioside A (0.2 mg/ml) were completely eliminated within 24 h, whereas no degradation of steviol (0.08 and 0.2 mg/ml) appeared to be found during the incubation period. Stevia mixture, stevioside and rebaudioside A appeared to be hydrolyzed to steviol by human intestinal microflora: this observation is consistent with previous rat metabolism studies. Similarly, enzymatically modified stevia appeared to be metabolized via stevia components and, finally, to steviol. This study suggests that there are apparently no species differences in intestinal metabolism of stevia mixture between rats and humans.

Adult↗

PubMed2003

Runx2 expression and action in chondrocytes are regulated by retinoid signaling and parathyroid hormone-related peptide (PTHrP).

OBJECTIVE: Runx2 (also known as Cbfa1) is a transcription factor required for chondrocyte maturation and osteoblast differentiation. While there is information on the regulation of its expression during osteogenesis, much less is known about it during cartilage maturation. Here we asked whether Runx2 expression and function are affected by retinoic acid (RA) and parathyroid hormone-related peptide (PTHrP), which represent an important stimulator and inhibitor of chondrocyte maturation, respectively. DESIGN: We first cloned and characterized Runx2 expressed by chick chondrocytes (cRunx2). We then constructed expression vectors of cRunx2 and a dominant-negative form (DN-cRunx2) and determined their effects on chondrocyte maturation in culture before and during retinoid and PTHrP treatment. RESULTS: cRunx2 showed similar transactivation activity to that of its mammalian counterparts although it has a very short QA domain and lacks a small portion of the PST domain. cRunx2 over-expression stimulated chondrocyte maturation, as indicated by increases in alkaline phosphatase activity (APase), mineralization, and type X collagen and MMP-13 expression, and by maintenance of Indian hedgehog (Ihh) expression. RA treatment stimulated cRunx2 gene expression and boosted its pro-maturation effects. PTHrP treatment blocked Runx2 expression and its pro-maturation effects. Over-expression of DN-cRunx2 inhibited maturation and even prevented RA from exerting its pro-maturation role. CONCLUSIONS: As previously indicated by mammalian studies, cRunx2 has chondrocyte pro-maturation activity. Its expression and roles are favorably modulated by retinoid signaling but are completely inhibited by PTHrP. A model integrating cRunx2 with PTHrP, Ihh and retinoid signaling and operating during skeletogenesis is proposed.

Animals↗

PubMed2003

Over- and ectopic expression of Wnt3 causes progressive loss of ameloblasts in postnatal mouse incisor teeth.

Intercellular signaling is essential for the development of teeth during embryogenesis and in maintenance of the continuously growing incisor teeth in postnatal rodents. WNT intercellular signaling molecules have been implicated in the regulation of tooth development, and the Wnt3 gene shows specific expression in the enamel knot at the cap stage. We demonstrate here that Wnt3 also is expressed in specific epithelial cell layers in postnatal incisor teeth. To begin to delineate the functions of Wnt3 in developing and postnatal teeth, we determined the effects of over- and ectopic expression of Wnt3 in the tooth epithelium of mice carrying a keratin 14-Wnt3 transgene. Expression of the transgene caused a progressive loss of ameloblasts from postnatal lower incisor teeth. Loss of ameloblasts may be due to defective proliferation or differentiation of ameloblast precursors, progressive apoptosis of ameloblasts, or loss of ameloblast stem cells.

Ameloblasts↗

PubMed2003

Roles of FGF-10 on the development of diathrodial limb joints.

OBJECTIVES: Members of the fibroblast growth (FGF) family of signaling proteins are known to play important roles in limb skeletal patterning and in chondrocyte proliferation and maturation. Recent work from this laboratory showed that FGF members are expressed in limb developing joints. Thus, the present project focused on what roles these proteins may have in joint development. METHODS: Heparin-coated beads precoated with recombinant FGF-10 or GDF-5 were implanted around incipient proximal and distal joints of digits 3 and 4 in Day 6-8 chick limb buds in organ culture. Specimens were processed for whole mount in situ hybridization using antisense riboprobes encoding chick GDF-5 and FGF-10 or for histology analysis at indicated time points. RESULTS: Whole mount in situ hybridization revealed that FGF-10 is expressed, and its transcripts are present, during interzone formation. Gain-of-function experiment revealed that exogenous FGF-10 caused down-regulation of expression of FGF-10 as well as GDF-5. In specimens continuously treated with exogenous FGF-10, joint formation was markedly impaired and often resulted in fusion of contiguous cartilaginous phalanges. CONCLUSIONS: The study provides evidence for the first time that FGF-10 is expressed during joint development in addition to FGF-2 and FGF-4. The precise roles of these signaling molecules will require further work. However, it is possible to speculate that these proteins, singly or in concert, may favor proliferation of mesenchymal cells during interzone formation. Our data also show that prolonged treatment with exogenous FGF-10 leads to joint impairment and fusion. Similar defects were observed previously when other joint-associated proteins were experimentally manipulated, indicating that a fine balance among distinct regulatory molecules is needed for normal joint formation.

Animals↗

PubMed2001

Evidence for intermolecular interaction between sulfonium and sulfide sulfur atoms and its application to synthesis of cyclic bis(disulfide) dimer.

On the basis of the remote Pummerer reaction of p-bis(alkylthio)-aromatic S-oxides, the intermolecular interaction between the sulfonium and sulfide sulfur atoms is described. (1) In marked contrast to the Pummerer reaction of 1b-d(3) with (CF(3)CO)(2)O (J. Org. Chem. 1999, 64, 3190-3195), the reaction of 3,3',5,5'-tetramesityl-4-(trideuteriomethylsulfinyl)-4'-(methylthio)biphenyl (1a-d(3)) as a sterically hindered analogue of 1b gave only 2a-d(2). (2) Both reactions of the two unsymmetrical regioisomers of 1-(ethylthio)-4-(methylthio)benzene S-oxide (5a and 5b) with (CF(3)CO)(2)O afforded a mixture of the mono-Pummerer products 6a and 6b, the bis-Pummerer product 7, and the bis-sulfide 8 in a similar ratio. The quenching at the initial stage of both reactions produced 5a, 5b, 8, and the bis-sulfoxide 10 in a similar ratio. These results indicate the equilibrium in the intermolecular interaction between the sulfur atoms. (3) The reaction of the p-bis(benzylthio)-aromatic S-oxide 16 with (CF(3)SO(2))(2)O gave the cyclic bis(disulfide) dimer 17 for the diphenyl sulfide and diphenylmethane spacers or the cyclic tetrakis(disulfide) tetramer 19 for the benzene and biphenyl spacers via the debenzylation of an intermolecular dithia dication. The cyclic bis(dithia dication) dimer A resulting from the intermolecular interaction between the sulfonium and sulfide sulfur atoms is proposed as an intermediate throughout the present reactions.

Journal Article↗

PubMed2001

Development of stratum intermedium and its role as a Sonic hedgehog-signaling structure during odontogenesis.

Stratum intermedium is a transient and subtle epithelial structure closely associated with inner dental epithelium in tooth germs. Little is known about its development and roles. To facilitate analysis, we used bovine tooth germs, predicting that they may contain a more conspicuous stratum intermedium. Indeed, early bell stage bovine tooth germs already displayed an obvious stratum intermedium with a typical multilayered organization and flanking the enamel knot. Strikingly, with further development, the cuspally located stratum intermedium underwent thinning and involution, whereas a multilayered stratum intermedium formed at successive sites along the cusp-to-cervix axis of odontogenesis. In situ hybridization and immunohistochemistry showed that stratum intermedium produces the signaling molecule Sonic hedgehog (Shh). Maximal Shh expression was invariably seen in its thickest multilayered portions. Shh was also produced by inner dental epithelium; expression was not constant but varied with development and cytodifferentiation of ameloblasts along the cusp-to-cervix axis. Interestingly, maximal Shh expression in inner dental epithelium did not coincide with that in stratum intermedium. Both stratum intermedium and inner dental epithelium expressed the Shh receptor Patched2 (Ptch2), an indication of autocrine signaling loops. Shh protein, but not RNA, was present in underlying dental mesenchyme, probably resulting from gradual diffusion from epithelial layers and reflecting paracrine loops of action. To analyze the regulation of Shh expression, epithelial and mesenchymal layers were separated and maintained in organ culture. Shh expression decreased over time, but was maintained in unoperated specimens. Our data show for the first time that stratum intermedium is a highly regulated and Shh-expressing structure. Given its dynamic and apparently interactive properties, stratum intermedium may help orchestrate progression of odontogenesis from cusp to cervix.

Animals↗

PubMed2001

Nineteen-hour variation of postural sway, alertness and rectal temperature during sleep deprivation.

Nineteen-hour variation of postural sway, alertness and rectal temperature during sleep deprivation were studied. Alertness decreased gradually at night and remained low until morning. Postural sway in the eyes-closed condition increased during early morning. In six of the eight subjects the greatest sway was observed during the 3-h period when rectal temperature was at its minimum. It is suggested that unbearable sleepiness during sleep deprivation will give rise to measurable impairment of postural balance especially during the time zone of temperature nadir.

Adult↗

PubMed2001

Effects of bright light at lunchtime on sleep of patients in a geriatric hospital I.

The effects of lunchtime bright light exposure in patients of a geriatric hospital were investigated. Ten inpatients (six women and four men; mean age +/- SD: 81.2 +/- 8.8 years) with sleep disturbances were studied for 9 weeks. Nurses performed daily ratings for sleep-wakefulness disturbances. Approximately 8000 lx bright light exposure was performed for 3 weeks in the light therapy room. Before and after exposure, ocular function was evaluated. Clinical ratings of sleep-wakefulness improved in eight patients. The score of difficulty in falling asleep and drowsiness in the morning declined during the light exposure. The score of drowsiness in the afternoon decreased during the post-light exposure. Post-exposure ocular disturbances were not found.

Aged↗

PubMed2001

Effects of bright light at lunchtime on sleep in patients in a geriatric hospital II.

Inpatients with sleep disturbances in a geriatric hospital received 1 h of exposure to approximately 8000 lx bright light per day for 3 weeks. Polysomnogram was recorded for four female patients. Two (aged 68 and 87 years) were non-demented patients with weak cataracts and the other two (aged 92 and 93 years) were demented patients with severe cataracts. Electroencephalogram results showed that light exposure decreased the proportion of Stage W, while increased the proportion of Stage 2, and these effects continued for at least 3 weeks after the cessation of light exposure. These results suggest that exposure to bright light is effective in improving the disturbed sleep of patients.

Aged↗

PubMed2001

The role of ERG (ets related gene) in cartilage development.

OBJECTIVE: Based on function and developmental fate, cartilage tissue can be broadly classified into two types: transient (embryonic or growth-plate) cartilage and permanent cartilage. Chondrocytes in transient cartilage undergo terminal differentiation into hypertrophic cells, induce cartilage-matrix mineralization, and eventually disappear and are replaced by bone. On the other hand, chondrocytes in permanent cartilage do not differentiate further, do not become hypertrophic, and persist throughout life at specific sites, including joints and tracheal rings. While many studies have described differences in structure, matrix composition and biological characteristics between permanent and transient cartilage, it is poorly understood how the fates of permanent and transient cartilage are determined. Previous studies demonstrated that chondrocytes isolated from permanent cartilage have the potential to express markers of the mature hypertrophic phenotype once grown in culture, suggesting that cell hypertrophy is an intrinsic property of all chondrocytes and must be actively silenced in permanent cartilage in vivo. These silencing mechanisms, however, are largely unknown. In this paper, we first review nature of chondrocytes in transient and permanent cartilages and then report the cloning and characterization of a novel variant of ets transcription factor chERG, hereafter called C-1-1, which might be involved in regulation of permanent cartilage development. DESIGN: For cloning of a novel variant of chERG (C-1-1), we isolated RNA from the cartilaginous femur or tibiotarsus of Day 17 chick embryos and processed it for reverse transcription-polymerase chain reaction (RT-PCR) with the primers from sequences upstream and downstream of the 81 and 72 bp segments alternatively-spliced in mammals. For investigation of function of chERG and C-1-1, we over-expressed chERG or C-1-1 in cultured chick chondrocytes or the developing limb of chick embryo using a retrovirus (RCAS) system, and examined the phenotype changes in the infected chondrocytes or the infected limb elements. RESULTS: C-1-1 is an alternative and novel variant lacking the 27 amino acids segment of chERG that has been reported previously. C-1-1 is preferentially expressed in developing articular cartilage, whereas chERG is preferentially expressed in growth plate cartilage. Growth of articular chondrocytes in culture was accompanied by decreasing C-1-1 expression after several passages, while expression of hypertrophic markers increased. Expression of C-1-1 in cultured chondrocytes inhibited cell hypertrophy, alkaline phosphatase activity, and cartilage matrix mineralization. In contrast, over-expression of chERG promoted chondrocyte maturation and mineralization. CONCLUSION: Our data demonstrate for the first time that chERG and C-1-1 play distinct roles in skeletogenesis and may have crucial roles in the development and function of transient and permanent cartilages.

Animals↗

PubMed2001

[Direct identification of mycobacteria from the positive cultures in mycobacteria growth indicator tube (MGIT)].

The ability of the AccuProbe (Kyokuto) to identify mycobacteria directly from the positive cultures in an automatic detector for mycobacteria (BACTEC MGIT960, Becton Dickinson) was evaluated. Sputum samples were collected from patients with suspected mycobacteriosis between February and April 1999 and conventionally incubated in MGIT960 (37 degrees C for 42 days). The MGIT-positive cultures were successively incubated for several days and were directly identified with the AccuProbe. Monomycobacterial strains were detected from 93 (93.9%) of the 99 sputum samples and polymycobacterial strains were detected from 6 sputum samples (6.1%). Viable cell counts in the positive cultures in MGIT960 were determined using Middlebrook 7H10 agar. The cell counts of Mycobacterium tuberculosis and Mycobacterium avium-intracellulare complex (MAC) varied greatly among the individual samples; the former, 3.8 x 10(2)-2.5 x 10(6) cfu/ml and the latter 1.5 x 10(3)-1.9 x 10(8) cfu/ml. The great differences in the cell counts were observed among these samples. Although the cultures in MGIT were estimated as positive, the early stage of bacterial growth might be used as the samples for the cell counting. By this method, 96 of the total 101 cases were successfully identified as follows: M. tuberculosis 57 cases (56.4%) and MAC 39 cases (38.6%). In conclusion, the present results indicate that the direct identification of mycobacteria from the positive cultures in MGIT960 using AccuProbe is useful for a rapid diagnosis in microbiological testing for mycobacteriosis which has tended to increase in recent years.

Culture Media↗

PubMed2000

Transcription factor ERG variants and functional diversification of chondrocytes during limb long bone development.

During limb development, chondrocytes located at the epiphyseal tip of long bone models give rise to articular tissue, whereas the more numerous chondrocytes in the shaft undergo maturation, hypertrophy, and mineralization and are replaced by bone cells. It is not understood how chondrocytes follow these alternative pathways to distinct fates and functions. In this study we describe the cloning of C-1-1, a novel variant of the ets transcription factor ch-ERG. C-1-1 lacks a short 27-amino acid segment located approximately 80 amino acids upstream of the ets DNA binding domain. We found that in chick embryo long bone anlagen, C-1-1 expression characterizes developing articular chondrocytes, whereas ch-ERG expression is particularly prominent in prehypertrophic chondrocytes in the growth plate. To analyze the function of C-1-1 and ch-ERG, viral vectors were used to constitutively express each factor in developing chick leg buds and cultured chondrocytes. We found that virally driven expression of C-1-1 maintained chondrocytes in a stable and immature phenotype, blocked their maturation into hypertrophic cells, and prevented the replacement of cartilage with bone. It also induced synthesis of tenascin-C, an extracellular matrix protein that is a unique product of developing articular chondrocytes. In contrast, virally driven expression of ch-ERG significantly stimulated chondrocyte maturation in culture, as indicated by increases in alkaline phosphatase activity and deposition of a mineralized matrix; however, it had modest effects in vivo. The data show that C-1-1 and ch-ERG have diverse biological properties and distinct expression patterns during skeletogenesis, and are part of molecular mechanisms by which limb chondrocytes follow alternative developmental pathways. C-1-1 is the first transcription factor identified to date that appears to be instrumental in the genesis and function of epiphyseal articular chondrocytes.

Alkaline Phosphatase↗

PubMed2000

Temporal order of sleepiness, performance and physiological indices during 19-h sleep deprivation.

Nineteen-hour variation of subjective sleepiness, performance and physiological indices were assessed during sleep deprivation. Longitudinal data of each index had its characteristic curve through which the values changed from day level to night level. A comparison of the time when each curve crossed its mid-range (50% value of its range) showed that those of subjective sleepiness and heart rate were significantly earlier than those of tracking error and coefficient of variation of R-R interval (CV(R-R)), P<0.01. That of rectal temperature was located at between 1:00 and 4:00 am. These temporal relationships were reproducible under two lighting conditions. These results will be useful in considering the occurrence of human errors by night-time workers in the early morning.

Adult↗

PubMed2000

Effect of short duration morning bright light in elderly men: sleep structure.

Sleep structure was measured in five healthy elderly men in their homes. The subjects were exposed to bright light (6000 lx) for 30 min in the morning or instructed to sit in front of a desktop lighting device without light. Relative to the control conditions, bright light exposure significantly decreased time in bed and the number of awakenings. Rapid eye movement sleep was significantly fragmented by stage 1 sleep in the control condition compared with the bright light condition. These findings indicate that a short duration of morning bright light changes sleep structure and is effective in maintaining sleep.

Aged↗

PubMed2000

Hedgehog proteins stimulate chondrogenic cell differentiation and cartilage formation.

Sonic hedgehog (Shh) and Indian hedgehog (Ihh) are important regulators of skeletogenesis, but their roles in this complex multistep process are not fully understood. Recent studies have suggested that the proteins participate in the differentiation of chondrogenic precursor cells into chondrocytes. In the present study, we have tested this possibility more directly. We found that implantation of dermal fibroblasts expressing hedgehog proteins into nude mice induces ectopic cartilage and bone formation. Immunohistological and reverse-transcription polymerase chain reaction (RT-PCR) analyses revealed that the ectopic tissues derived largely if not exclusively from host cells. We found also that treatment of clonal prechondrogenic RMD-1 and ATDC5 cells in culture with Ihh or recombinant amino half of Shh (recombinant N-terminal portion of Shh [rShh-N]) induced their differentiation into chondrocytes, as revealed by cytoarchitectural changes, Alcian blue staining and proteoglycan synthesis. Induction of RMD-1 cell differentiation by Ihh or rShh-N was synergistically enhanced by cotreatment with bone morphogenetic protein 2 (BMP-2) but was blocked by cotreatment with fibroblast growth factor 2 (FGF-2). Our findings indicate that hedgehog proteins have the ability to promote differentiation of chondrogenic precursor cells and that their action in this process can be influenced and modified by synergistic or antagonist cofactors.

Animals↗

PubMed2000

Development of articular cartilage: what do we know about it and how may it occur?

Articular cartilage has a fundamental role in joint function. While much is known about its structure, organization and biomechanical properties, there is a very poor understanding of how articular chondrocytes develop during embryogenesis and acquire the unique ability to organize and maintain the articular tissue. Given that articular cartilage forms in close juxtaposition with the joint, here we review past studies on limb joint determination and morphogenesis and more recent studies on a number of factors thought to have roles in joint and epiphysis development. These factors include: the homeobox gene Barx-1; the bone morphogenetic protein (BMP) family member GDF-5; the growth factors HGF and PTHrP; and the transcription factor ERG. We summarize current thinking on how these factors participate in joint development and how some of these factors may influence development and behavior of epiphyseal chondrocytes. We also describe pertinent recent studies from our laboratories on ERG and the newly-identified alternatively spliced variant C-1-1, and finally propose a sequela of events that may subtend the process of determination and emergence of articular chondrocytes during limb synovial joint development.

Animals↗

PubMed1999

Matrix GLA protein is a developmental regulator of chondrocyte mineralization and, when constitutively expressed, blocks endochondral and intramembranous ossification in the limb.

Matrix GLA protein (MGP), a gamma-carboxyglutamic acid (GLA)-rich, vitamin K-dependent and apatite-binding protein, is a regulator of hypertrophic cartilage mineralization during development. However, MGP is produced by both hypertrophic and immature chondrocytes, suggesting that MGP's role in mineralization is cell stage-dependent, and that MGP may have other roles in immature cells. It is also unclear whether MGP regulates the quantity of mineral or mineral nature and quality as well. To address these issues, we determined the effects of manipulations of MGP synthesis and expression in (a) immature and hypertrophic chondrocyte cultures and (b) the chick limb bud in vivo. The two chondrocyte cultures displayed comparable levels of MGP gene expression. Yet, treatment with warfarin, a gamma-carboxylase inhibitor and vitamin K antagonist, triggered mineralization in hypertrophic but not immature cultures. Warfarin effects on mineralization were highly selective, were accompanied by no appreciable changes in MGP expression, alkaline phosphatase activity, or cell number, and were counteracted by vitamin K cotreatment. Scanning electron microscopy, x-ray microanalysis, and Fourier-transform infrared spectroscopy revealed that mineral forming in control and warfarin-treated hypertrophic cell cultures was similar and represented stoichiometric apatite. Virally driven MGP overexpression in cultured chondrocytes greatly decreased mineralization. Surprisingly, MGP overexpression in the developing limb not only inhibited cartilage mineralization, but also delayed chondrocyte maturation and blocked endochondral ossification and formation of a diaphyseal intramembranous bone collar. The results show that MGP is a powerful but developmentally regulated inhibitor of cartilage mineralization, controls mineral quantity but not type, and appears to have a previously unsuspected role in regulating chondrocyte maturation and ossification processes.

1-Carboxyglutamic Acid↗