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Biomedical subjects

Y Sakuma

Publications and source records attributed to Y Sakuma.

At least 55 records · Page 3Linked to original sources
PubMed2000

Crucial involvement of the EP4 subtype of prostaglandin E receptor in osteoclast formation by proinflammatory cytokines and lipopolysaccharide.

Prostaglandin E2 (PGE2) exerts its effects through the PGE receptor that consists of four subtypes (EP1, EP2, EP3, and EP4). Osteoclast formation in the coculture of primary osteoblastic cells (POB) and bone marrow cells was enhanced more by 11-deoxy-PGE1 (an EP4 and EP2 agonist) than by butaprost (an EP2 agonist) and other agonists, which suggests that EP4 is the main factor in PGE2-induced osteoclast formation. PGE2-induced osteoclast formation was not observed in the coculture of POB from EP4-deficient (EP4 k/o) mice and spleen cells from wild-type (w/t) mice, whereas osteoclasts were formed in the coculture of POB from w/t mice and spleen cells from EP4-k/o mice. In situ hybridization (ISH) showed that EP4 messenger RNA (mRNA) was expressed on osteoblastic cells but not on multinucleated cells (MNCs) in w/t mice. These results indicate that PGE2 enhances osteoclast formation through its EP4 subtype on osteoblasts. Osteoclast formation by interleukin 1alpha (IL-1alpha), tumor necrosis factor alpha (TNF-alpha), basic fibroblast growth factor (bFGF), and lipopolysaccharide (LPS) was hardly observed in the coculture of POB and bone marrow cells, both from EP4-k/o mice, which shows the crucial involvement of PG and the EP4 subtype in osteoclast formation by these molecules. In contrast, osteoclast formation by 1,25-hydroxyvitamin D3 (1,25(OH)2D3) was not impaired and that by parathyroid hormone (PTH) was only partially impaired in EP4-k/o mice, which may be related to the fact that EP4-k/o mice revealed no gross skeletal abnormalities. Because it has been suggested that IL-1alpha, TNF-alpha, bFGF, and LPS are involved in inflammatory bone loss, our work can be expected to contribute to an understanding of the pathophysiology of these conditions.

Animals↗
PubMed1999

Localization of curved DNA and its association with nucleosome phasing in the promoter region of the human estrogen receptor alpha gene.

We determined DNA bend sites in the promoter region of the human estrogen receptor (ER) gene by the circular permutation assay. A total of five sites (ERB-4 to -1, and ERB+1) mapped in the 3 kb region showed an average distance of 688 bp. Most of the sites were accompanied by short poly(dA) x poly(dT) tracts including the potential bend core sequence A2N8A2N8A2 (A/A/A). Fine mapping of the ERB-2 site indicated that this A/A/A and the 20 bp immediate flanking sequence containing one half of the estrogen response element were the sites of DNA curvature. All of the experimentally mapped bend sites corresponded to the positions of DNA curvature as well as to nucleosomes predicted by computer analysis. In vitro nucleosome mapping at ERB-2 revealed that the bend center was located 10-30 bp from the experimental and predicted nucleosome dyad axes.

Base Sequence↗
PubMed1999

Development and differentiation of gonadotropin hormone-releasing hormone neuronal systems and testes in the Japanese eel (Anguilla japonica).

In this study we investigated the relationship between the development of the olfactory, preoptic, and midbrain gonadotropin-releasing hormone (GnRH) neuronal systems and testicular differentiation in eels (Anguilla japonica) from embryonic stages through adulthood (5.4-50 cm body length). GnRH-synthesizing neuronal populations were first observed in the youngest fish ( approximately 5.0 cm) at the rostrobasal and caudalmost olfactory bulbs immunoreactive to a "promiscuous" (nonspecific) GnRH antiserum (635.5), and in the preoptic area and midbrain tegmentum immunoreactive to chicken GnRH II antiserum. The eel brains lacked salmon and seabream GnRH immunoreactivity. The evidence from our study suggests that the olfactory, preoptic, and midbrain GnRH populations have origins independent from those of proliferative periventricular zones within the brain. However, the olfactory GnRH neurons could have migrated out of the olfactory placodes during ages earlier than those observed in this study. Although all three GnRH neuronal populations contribute to pituitary innervation to some degree, the preoptic GnRH innervation was pronounced in the pituitary when primordial germ cells (animals approximately 5.0 cm) differentiated into male germ cells (animals 14-16 cm) and, therefore, an association can be assumed between preoptic GnRH expression and testicular differentiation in the Japanese eel.

Anguilla↗
PubMed1999

C-Type natriuretic peptide/guanylate cyclase B system in rat osteogenic ROB-C26 cells and its down-regulation by dexamethazone.

There is recent evidence that natriuretic peptides are important regulators of bone and cartilage, although they were originally identified as the cardiac hormones causing natriuresis and hypotension. Three members of natriuretic peptide family are known: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). The biologically active receptors for these peptides are particulate guanylate cyclases; the two known types are GC-A and GC-B. ANP and BNP have high affinities for GC-A, and CNP is the preferred ligand for GC-B. In this paper we report the results of our study of the expression and possible role(s) of natriuretic peptides in the ROB-C26 cell, which is an osteogenic cell line with multiple potentials for differentiating into myoblast, osteoblast, and adipocyte. ROB-C26 cells produced cGMP in response to natriuretic peptides at both their basal state and after enhanced differentiation into osteoblast which was induced by bone morphogenetic protein [(BMP)-2]. CNP was far more potent than ANP in cGMP production. In contrast, enhanced differentiation into adipocyte by dexamethasone resulted in the marked decrease in their responsiveness to natriuretic peptides. Although the messages for GC-A and GC-B were demonstrated by Northern blot analysis at both the basal stage and after BMP treatment, they were down-regulated after dexamethasone treatment. The presence of CNP was shown by RT-PCR and immunohistochemistry in ROB-C26 cells. C3H10T1/2, which is another and more primitive mesenchymal cell line, also produced cGMP in response to CNP, and less potently to ANP. Culturing ROB-C26 cells with CNP or 8-bromo cGMP decreased [(3)H]thymidine uptake and slightly increased the message for alkaline phosphatase, which is a marker for osteoblast differentiation. These results suggest that the CNP/GC-B system is preferentially expressed in the cells of osteogenic lineage and their expression is down-regulated with differentiation into adipocyte lineage. The CNP/GC-B system is likely to be an autocrine/paracrine regulator of osteoblast growth and differentiation.

Alkaline Phosphatase↗
PubMed1999

Expression and secretion of a biologically active mouse sonic hedgehog protein by the methylotrophic yeast Pichia pastoris.

We have successfully secreted the amino-terminal functional domain of mouse sonic hedgehog protein (SHH) into culture fluid using a yeast Pichia pastoris expression system. A cDNA fragment encoding the amino-terminal domain of mouse SHH was inserted downstream of the Saccharomyces cerevisiae alpha-mating factor secretion signal. The DNA fragment was introduced into the host genome by the spheroplast transformation method. Transformants were selected based on their resistance to G418: His+ transformants which showed resistance to over 8 mg G418/ml were selected and analyzed for determination of the plasmid copy number. One His+ clone which has eight copies of the expression cassette per genome was cultured in minimal medium deficient for histidine, and further cultured in buffered medium supplemented with methanol which activates the AOX1 promoter. SDS-PAGE analysis indicated efficient expression and secretion of mouse SHH into culture fluid. The yield of secreted SHH was estimated to be 50 micrograms/ml. Purified protein was assayed for biological activity and found to activate the transcription of the Patched genes (Ptc-1 and Ptc-2) encoding receptors for SHH.

Animals↗
PubMed1999

Effects of short-acting and long-acting loop diuretics on heart rate variability in patients with chronic compensated congestive heart failure.

BACKGROUND: We investigated the effects of a short-acting loop diuretic (furosemide) and a long-acting loop diuretic (azosemide) on heart rate variability, fluid balance, and neurohormonal responses in patients with mild to moderate chronic congestive heart failure. METHODS: Nineteen patients with mild to moderate chronic congestive heart failure received furosemide (40 to 60 mg/day) or azosemide (60 to 90 mg/day) for 5 days in a crossover manner. We performed time-domain and frequency-domain analyses of 24-hour Holter electrocardiographic recordings to assess heart rate variability. RESULTS: The 24-hour urinary sodium excretion was similar during the furosemide and azosemide treatment periods but was significantly greater in the first 2 hours after drug administration during furosemide treatment. Plasma renin activity and the hematocrit level increased and high-frequency power significantly decreased 2 hours after the administration of furosemide only. The standard deviation of all normal R-R intervals and the root mean square of successive differences in the R-R interval were lower with furosemide than with azosemide (P <.05). CONCLUSIONS: Furosemide, a short-acting loop diuretic, has a greater influence on heart rate variability and fluid balance than azosemide, a long-acting loop diuretic, in patients with mild to moderate chronic congestive heart failure.

Blood Pressure↗
PubMed1999

Sensory requirements for noncontact penile erection in the rat.

Physical contact is not necessary for male rats to display penile erection when placed in the vicinity of females in estrus (noncontact erection, NCE). The modality and reception of sensory stimuli for the induction of NCE were analyzed. Olfactory bulbectomy eliminated NCE, and destruction of olfactory mucosa by zinc sulfate solution caused a significant suppression of NCE, whereas removal of the vomeronasal organ had no effect. Fresh urine collected from estrous females, but not fresh feces, induced NCE. Neither devocalization of estrous females by cutting the inferior laryngeal nerve nor deprivation of visual cues by placing an opaque barrier between the subjects and stimulus females disrupted NCE. The results indicate that olfactory cues emitted by estrous females induce NCE via the olfactory epithelium. Contrary to what had been suggested by previous experiments, vomeronasal inputs are not indispensable for the induction of NCE.

Acoustic Stimulation↗
PubMed1999

The effect of GHRP-6 on the intracellular Na+ concentration of rat pituitary cells in primary culture.

The objective of the present study was to further investigate the ionic mechanism of the action of GHRP-6 on male rat pituitary cells in culture. A synthetic hexapeptide, GHRP-6 stimulates the secretion of growth hormone both in vivo and in vitro. It is generally accepted that Ca2+ and protein kinase C but not cAMP are involved in the signal transduction pathway of the action of GHRP-6. Ca2+-influx through voltage-gated Ca2+ channels and mobilization of internal stored Ca2+ are thought to be responsible for an increase in cytosolic Ca2+ concentration. For activation of the voltage-gated Ca2+ channels, however, it is not determined whether the membrane Na+ permeability plays a role. To answer this question, we measured intracellular Na+ concentration of the pituitary cells with ion imaging technique. We found that GHRP-6 increased [Na+]i; the Na+ response depended on the presence of extracellular Na+ and was blocked by Gd3+, known as a blocker of nonselective cation channels but not by tetrodotoxin, a blocker of the voltage-gated Na+ channel; thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ ATPase, had no effect on the response; Ca2+ chelating agent, BAPTA had no inhibitory effect on the response; ouabain, an inhibitor of Na+-K+ ATPase, did not block the rise in [Na+]i induced by GHRP-6; somatostatin, which hyperpolarizes the cells by activating K+ channels, suppressed the response. These data clearly showed that GHRP-6 increased [Na+]i in the rat pituitary cells including somatotrophs. The rise in [Na+]i is likely to be due to an increase in the membrane Na+ permeability which should depolarize the cells, thereby activating the voltage-gated Ca2+ channels. This process leads to an influx of Ca2+ and subsequent increase in [Ca2+]i which results in an exocytotic release of GH.

Animals↗
PubMed1999

In vitro determination of oxidation of atmospheric tritium gas in vegetation and soil in Ibaraki and Gifu, Japan.

To quantify the rate of oxidation of tritium gas (referred to as HT) to tritiated water in the environment, various woody and herbaceous plant leaves and roots, mosses and lichens taken from a forest and fields in Ibaraki prefecture, and a forest in Toki, Gifu prefecture, were investigated as to their ability to oxidize atmospheric HT in in vitro experiments. The HT oxidation activity in vegetation was compared with that in the surrounding surface soil (0-5 cm in depth). The rate of oxidation of HT in woody plant leaves including pine needles was extremely low, only about 1/10000-1/1000 that in the surface soil, as well as in herbaceous plant leaves with some exceptions (Phalaris arundinacea and Vaccinium smallii), whereas the rate in mosses and lichens was 50-500 times that in pine needles. The HT oxidation activity in roots of several plants including Phalaris arundinacea, Pieris japonica and Lespedeza homoloba was quite high and comparable to that in the surrounding surface soil. These results suggest that mosses, lichens and the leaves or roots of particular plants with high HT oxidation activity can be used to monitor the accidental release of HT into the environment.

Atmosphere↗
PubMed1998

Neurons synthesizing gonadotropin-releasing hormone mRNA subtypes have multiple developmental origins in the medaka.

The origins of the different populations of gonadotropin-releasing hormone (GnRH)-containing neurons in the brains of two genotypes (HO4C; HNI-II) of medaka Oryzias latipes were analyzed at different stages of development (day 1 after fertilization through adulthood), by using oligonucleotide probes specific to salmon-, seabream-, and chicken II-GnRH mRNA and antisera against specific GnRH peptides. Between the two genotypes, there was no difference in the site and time of GnRH expression or the final pattern of GnRH neuronal organization. In the adult fish of both sexes, salmon GnRH mRNA and peptide-containing neurons were seen in the terminal nerve ganglia (nucleus olfactoretinalis; NOR) and chicken II-GnRH mRNA and peptide-containing neurons in the midbrain tegmentum. GnRH cells at the base of the olfactory placode (1-2 cells) and in the midbrain tegmentum were first seen in 1-day-old fish of both genotypes. On day 15, lightly immunoreactive GnRH cells were seen in the NOR of only HNI genotype. By day 30, GnRH expression in the NOR and in the midbrain was prominent. GnRH cells along the basal olfactory bulb and basal telencephalon were occasionally seen in animals 30 days or older. This developmental study shows differential distribution of salmon and chicken II-GnRH mRNA subtypes and emphasizes their separate embryonic origins from the olfactory apparatus (salmon-GnRH) and the ependymal cells of the third ventricle (chicken II-GnRH). The absence of preoptic GnRH hybridization signals, immunoreactivity and the lack of GnRH fibers in the pituitary suggests that the preoptic GnRH neurons are distinct from the olfactory derived-terminal nerve GnRH neurons, and that the GnRH neurites reported in the pituitary of teleost must be of preoptic origin.

Animals↗
PubMed1998

Inhibitors of acyl-CoA:cholesterol O-acyltransferase. 3. Discovery of a novel series of N-alkyl-N-[(fluorophenoxy)benzyl]-N'-arylureas with weak toxicological effects on adrenal glands.

A series of N-alkyl-N-[(fluorophenoxy)benzyl]-N'-arylureas were prepared and evaluated for their ability to inhibit intestinal acyl-CoA:cholesterol O-acyltransferase and to inhibit accumulation of cholesteryl esters in macrophages in vitro. In vivo hypocholesterolemic activity was assessed in cholesterol-fed rats by oral administration as a dietary admixture and/or by gavage in a PEG400 vehicle. Modification of the alkyl substituent on the N'-aryl moiety and on the urea nitrogen significantly influenced macrophage assay in vitro. Toxicological study revealed a distinct relationship between macrophage assay and the toxicity observed in adrenal glands of rabbits treated with representatives of this series of compounds. Investigations utilizing the macrophage assay as an indicator for adrenal toxicity led to the identification of compounds 1g (FR190809) and 1k (FR186485, or FR195249 as its hydrochloride salt) as potent, nonadrenotoxic, orally efficacious ACAT inhibitors irrespective of the administration method.

Acetylation↗
PubMed1998

Testosterone differentially regulates expression of GnRH messenger RNAs in the terminal nerve, preoptic and midbrain of male tilapia.

The purpose of the present study was to examine the regulation of three molecular variants of gonadotropin-releasing hormone (GnRH)-encoding mRNAs by testosterone in the male tilapia Oreochromis niloticus. Tilapias castrated for two weeks were injected intraperitoneally with sesame oil or 5 microgram/g testosterone for 7 days. In situ hybridization histochemistry was performed using 35S-labelled 30-mer antisense oligonucleotide probes complementary to exon two (bases 1-30) of salmon-, seabream-, and chicken II-GnRH. Computerized image analysis was performed to quantify GnRH mRNA expression in the terminal nerve ganglia (nucleus olfactoretinalis) and in individual cells of the preoptic area and the midbrain tegmentum. Testosterone treatment significantly elevated terminal nerve salmon-GnRH mRNA, reduced preoptic seabream-GnRH mRNA but had no effect on midbrain chicken II-GnRH mRNA levels. The total number and size of preoptic and midbrain GnRH mRNA-containing neurons or the total volume of the terminal nerve ganglia in testosterone-treated animals did not differ significantly from oil-treated animals. The midbrain chicken II-GnRH neurons are not targets of testosterone. These results demonstrate for the first time differential regulation of subpopulations of GnRH neurons with molecular diversity and different topography.

Animals↗
PubMed1998

Inhibitors of acyl-CoA:cholesterol O-acyltransferase. 2. Identification and structure-activity relationships of a novel series of N-alkyl-N-(heteroaryl-substituted benzyl)-N'-arylureas.

A series of N-alkyl-N-(heteroaryl-substituted benzyl)-N'-arylurea and related derivatives represented by 2 and 3 have been prepared and evaluated for their ability to inhibit acyl-CoA:cholesterol O-acyltransferase in vitro and to lower plasma cholesterol levels in cholesterol-fed rats in vivo. Among these novel compounds, the type 3 series was superior. A pyrazol-3-yl group on the N-benzyl group of this trisubstituted urea (i.e. 3, Ar1 = pyrazol-3-yl) was identified as a heteroaromatic ring providing a good profile of biological activity. As a result of optimization of the combination with the N-alkyl group (R) and N-aryl group (Ar3), compound 3aq (FR186054) was identified as a new, orally efficacious ACAT inhibitor, which exhibited potent in vitro ACAT inhibitory activity (rabbit intestinal microsomes IC50 = 99 nM) and excellent hypocholesterolemic effects in cholesterol-fed rats, irrespective of administration mode (ED50 = 0.046 mg/kg dosed via the diet, ED50 = 0. 44 mg/kg administered by gavage in PEG400 vehicle). Moreover, a toxicological study revealed compound 3aq to be nontoxic to the adrenal glands of dogs when tested at a single dose of 10 mg/kg po.

Administration, Oral↗
PubMed1998

Natriuretic peptide regulation of endochondral ossification. Evidence for possible roles of the C-type natriuretic peptide/guanylyl cyclase-B pathway.

The natriuretic peptide family consists of three structurally related endogenous ligands: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). The biological actions of natriuretic peptides are thought to be mediated through the activation of two guanylyl cyclase (GC)-coupled receptor subtypes (GC-A and GC-B). In this study, we examined the effects of ANP and CNP, which are endogenous ligands for GC-A and GC-B, respectively, on bone growth using an organ culture of fetal mouse tibias, an in vitro model of endochondral ossification. CNP increased the cGMP production much more potently than ANP, thereby resulting in an increase in the total longitudinal bone length. Histological examination revealed an increase in the height of the proliferative and hypertrophic chondrocyte zones in fetal mouse tibias treated with CNP. The natriuretic peptide stimulation of bone growth, which was mimicked by 8-bromo-cGMP, was inhibited by HS-142-1, a non-peptide GC-coupled natriuretic peptide receptor antagonist. The spontaneous increase in the total longitudinal bone growth and cGMP production was also inhibited significantly by HS-142-1. CNP mRNA was expressed abundantly in fetal mouse tibias, where no significant amounts of ANP and BNP mRNAs were detected. A considerable amount of GC-B mRNA was present in fetal mouse tibias. This study suggests the physiologic significance of the CNP/GC-B pathway in the process of endochondral ossification.

Animals↗
PubMed1998

Synthesis, X-ray crystal structure, and biological activity of FR186054, a novel, potent, orally active inhibitor of acyl-CoA:cholesterol O-acyltransferase (ACAT) bearing a pyrazole ring.

The synthesis, single crystal X-ray structural analysis, and biological activity of FR186054 (2c), a new, potent, orally efficacious inhibitor of acyl-CoA:cholesterol O-acyltransferase (ACAT), containing a pyrazole ring are described. This compound displayed excellent in vitro efficacy, irrespective of dosing method, indicating superior characteristics compared to other ACAT inhibitors.

Administration, Oral↗
PubMed1998

Quantitative in situ hybridization of three gonadotropin-releasing hormone-encoding mRNAs in castrated and progesterone-treated male tilapia.

We investigated the effects of castration and progesterone administration on the three gonadotropin-releasing hormone (GnRH)-encoding mRNAs in sexually mature male tilapia Oreochromis niloticus. In situ hybridization histochemistry was performed using 35S-labeled antisense oligonucleotide probes complementary to salmon-, seabream-, and chicken II-GnRH cDNAs to quantify cellular GnRH mRNA expression in the terminal nerve ganglia (nucleus olfactoretinalis), preoptic area, and midbrain tegmentum of animals castrated for 2 weeks and injected intraperitoneally with sesame oil or progesterone. Castration significantly elevated salmon-GnRH mRNA but not seabream- or chicken II-GnRH mRNA levels. Progesterone treatment had no effect on salmon-, seabream-, or chicken II-GnRH mRNA levels. Comparisons between intact, castrated, and progesterone-treated animals showed no change in the total volume of nucleus olfactoretinalis, cell sizes, and total numbers of cells expressing GnRH mRNA within the midbrain and preoptic area. These results demonstrate that salmon-GnRH but not seabream- or chicken II-GnRH-synthesizing neurons are under a gonadal steroid negative feedback control and that progesterone might not be the main hormone regulating the three GnRH-encoding mRNAs in the male tilapia.

Animals↗
PubMed1998

Prostaglandin E2 (PGE2) autoamplifies its production through EP1 subtype of PGE receptor in mouse osteoblastic MC3T3-E1 cells.

Prostaglandin E2 (PGE2) is known to autoamplify its production in the osteoblasts through the induction of prostaglandin G/H synthase-2 (PGHS-2), which is the inducible form of the rate-limiting enzyme in PG synthesis, PGHS. To elucidate the cellular mechanism mediating this process, we have employed the PGE2 analogs, which are specific agonists for four subtypes of PGE receptor, and studied the potency of these analogs to induce PGHS-2 mRNA in mouse osteoblastic MC3T3-E1 cells. The induction was mainly observed by 17-phenyl-omega-trinor PGE2 (EP1 agonist) and sulprostone (EP3/EP1 agonist), but not by butaprost (EP2 agonist) or 11-deoxy PGE1 (EP4/EP2 agonist). Since EP3 subtype was undetectable in MC3T3-E1 cells, these data indicate that PGHS-2 mRNA induction is mediated through EP1 subtype of PGE receptor in MC3T3-E1 cells. PGE2 production determined by radioimmunoassay was also increased by 17-phenyl-omega-trinor PGE2 and sulprostone. The autoamplification of PGE2 production is considered to be important in elongating the otherwise short-lived PGE2 action in certain physiological conditions such as mechanical stress and fracture healing, as well as the pathological inflammatory bone loss. The observations in the present study provide us with the better understanding of these processes.

3T3 Cells↗