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Biomedical subjects

Y Sakuma

Publications and source records attributed to Y Sakuma.

At least 19 recordsLinked to original sources
PubMed2001

Green fluorescent protein-transgenic rat: a tool for organ transplantation research.

The purpose of this study is to evaluate green fluorescent protein (GFP) transgenic rats for use as a tool for organ transplantation research. The GFP gene construct was designed to express ubiquitously. By flow cytometry, the cells obtained from the bone marrow, spleen, and peripheral blood of the GFP transgenic rats consisted of 77, 91, and 75% GFP-positive cells, respectively. To examine cell migration of GFP-positive cells after organ transplantation, pancreas graft with or without spleen transplantation, heart graft with or without lung transplantation, auxiliary liver and small bowel transplantation were also performed from GFP transgenic rat to LEW (RT1(1)) rats under a 2-week course of 0.64 mg/kg tacrolimus administration. GFP-positive donor cells were detected in the fully allogenic LEW rats after organ transplantation. These results showed that GFP transgenic rat is a useful tool for organ transplantation research such as cell migration study after organ transplantation without donor cell staining.

Animals↗
PubMed2001

Sex differences in the brain of goldfish: gonadotropin-releasing hormone and vasotocinergic neurons.

The differences between male and female behaviors are reflected in sexual dimorphism of brain structures and are found throughout the nervous system in a variety of vertebrates. The present study examined neurons immunolabeled for gonadotropin-releasing hormone and arginine vasotocin in the brain of the goldfish Carassius auratus to determine if these neurons are sexually dimorphic. There was no sex difference or influence of sex steroids on the neuronal volume and optical density of staining of arginine vasotocin neurons. Similarly, gonadotropin-releasing hormone neurons of the terminal nerve and midbrain tegmentum did not differ between sexually mature males, females and maturing females replaced with sex steroids with respect to distribution, numbers, optical density of staining, or gross morphology. In maturing females, testosterone specifically recruited additional preoptic gonadotropin-releasing hormone neurons to equal those in sexually mature individuals. Since estrogen had no effect, the influence of testosterone on gonadotropin-releasing hormone neuronal numbers appears to be independent of aromatization. Specifically, the preoptic gonadotropin-releasing hormone neuronal size was significantly larger in sexually mature males than females. 11-Ketotestosterone-replacement to ovariectomized maturing females induced male-typical secondary characters and male-type courtship behavior but did not masculinize the preoptic gonadotropin-releasing hormone neuronal size. Our results show that the sexually dimorphic preoptic gonadotropin-releasing hormone neuronal size is determined by factors (genetic) other than gonadal steroids. Further, we propose the hypothesis that phenotypic and behavioral sex differences need not be accompanied by structural differences in gonadotropin-releasing hormone and arginine vasotocin in the brain.

Animals↗
PubMed2001

Purinergic regulation of intracellular Ca2+ concentration of rat pituitary folliculo-stellate cells in primary culture.

Pituitary folliculo-stellate cells (FSCs) are glia-like cells in the anterior pituitary and are believed to modulate the activity of the pituitary endocrine cells. However, little is known what regulates the activity of FSCs. We hypothesized that ATP could act on FSCs, because ATP is coreleased with pituitary hormones from endocrine cells. To test this possibility, we examined the effect of ATP by measuring intracellular Ca2+ concentration [Ca2+]i of FSCs in primary culture. Both ATP and UTP increased the [Ca2+]i in a concentration-dependent manner in a range between 0.1 microM and 10 microM. The response was completely suppressed by thapsigargin, an inhibitior of endoplasmic reticulum Ca2+-ATPase, and was significantly suppressed by U-73122, an inhibitor of phospholipase C. The response was also suppressed by caffeine, a blocker of IP3 receptor, whereas that was not suppressed by ryanodine, an antagonist of ryanodine receptor. These results indicate that ATP increases [Ca2+]i of FSCs by activating phospholipase C via P2Y purinergic receptor and suggest that ATP would be one of paracrine factors to FSCs in the anterior pituitary.

Adenosine Triphosphate↗
PubMed2001

Acetylcholine increases intracellular Ca2+ in the rat pituitary folliculostellate cells in primary culture.

Pituitary folliculostellate cells (FSCs) are thought to partially inhibit pituitary hormone secretion through a paracrine mechanism. In this process, one of the important questions is what factors regulate the function of FSCs. Because ACh is synthesized in and possibly released from the corticotrophs and lactotrophs, we examined whether FSCs respond to ACh by the method of Ca2+ imaging in primary cultured FSCs from male Wistar rats. ACh (30 nM-3 microM) increased intracellular calcium concentration ([Ca2+](i)) of FSCs in a concentration-dependent manner, with an initial rapid rise followed by a relatively sustained increase. The complete block of the response by atropine and pirenzepine suggests involvement of muscarinic receptors. Depletion of the stored Ca2+ by thapsigargin blocked the response completely. Blockers of phospholipase C, U-73122 and neomycin, suppressed significantly the rise of [Ca2+](i). These results suggest that ACh increases [Ca2+](i) in FSCs by activating phospholipase C, presumably through activation of M(1) receptors. The rise in [Ca2+](i) could trigger a variety of Ca2+-dependent cellular processes, including the synthesis and release of bioactive substances, which in turn act on endocrine cells.

Acetylcholine↗
PubMed2001

Two distinct populations of neurons expressing nitric oxide synthase mRNA in the female rat preoptic area: site specific changes induced by sex steroids.

Non-isotopic in situ hybridization histochemistry in the basal forebrain of gonadectomized juvenile female rats visualized neuronal nitric oxide synthase (nNOS) mRNA in two distinct cellular populations, one in the organum vasculosum of the lamina terminals (OVLT) and the other in the rostral preoptic area at the level of the anteroventral periventricular nucleus (rPOA). In the rPOA, digoxigenin-labeled nNOS mRNA positive cells were in close proximity to the cell body of gonadotropin-releasing hormone (GnRH) -immunoreactive neurons. In the OVLT, the labeled cells were in an area rich in GnRH fibers. In the frontal section of the rPOA, the labeled cells were distributed in an inverted V-shaped area over the third ventricle. Combined treatment with estradiol and progesterone caused a significant reduction in the number of nNOS mRNA positive cells in the inverted V-shaped area in the female rat rPOA. The treatment induced a luteinizing hormone surge at the time of sacrifice. In the OVLT, ovarian steroids had no effect on nNOS mRNA expression. The results indicate that nNOS mRNA expression in the rPOA is regulated by ovarian steroids in a site-specific manner.

Animals↗
PubMed2000

Neuronal activity in female rat preoptic area associated with sexually motivated behavior.

Single unit activities were recorded from 31 neurons in the preoptic area (POA) of female rats engaging in sexual interactions. Concurrent videotape recordings were used to establish a relationship between neuronal activity and particular behavioral events. In 14 of the 31 neurons, the firing rate changed in association with bouts of sexual activity. The remaining 17 fired with more variability regardless of episodes of sexual interactions. Peri-event histograms identified four types of neurons: type 1 (n=4) increased their firing rate when the female rats initiated proceptive behavior; type 2 (n=4) showed a brief activation when the male mounted; type 3 (n=4) fired in response to intromission, and type 4 (n=2) were inhibited prior to and throughout the display of lordosis reflex. Type 1 neurons fired at significantly higher rates during the solicitatory period, from the initiation of solicitatory locomotion to the male mounts. Their activity was suppressed when the males mounted successfully with intromission. Types 1-3 neurons were recorded from the transitional region between the medial and lateral POAs. Type 4 neurons were located more medially in the medial POA. Systemic injection of pimozide, a dopamine receptor blocker, diminished firing in type 1 neurons and abolished proceptivity. The firing pattern in type 1 neurons appeared to embody the motivational state of the animal with an implication for a consummatory value of penile intromission. Visceral or somatosensory inputs may be responsible for short bursts in types 2 and 3 neurons. Type 4 neurons behaved exactly as if they inhibit the execution of the lordosis reflex. The results showed separate sets of POA neurons each specifically associated with proceptive and receptive components of female rat sexual behavior.

Action Potentials↗
PubMed2000

Estrogen receptor alpha, but not beta, is expressed in the interneurons of the hippocampus in prepubertal rats: an in situ hybridization study.

Estradiol is involved in the differentiation and plasticity of hippocampal neurons. In the CA1 region, estrogen treatment increases dendritic spines and synapse density on pyramidal cells. In the adult hippocampus, immunoreactivity for estrogen receptor alpha (ERalpha) has been reported in inhibitory interneurons, but neither in the pyramidal neurons nor in granule cells. Estrogens also mediate aspects of sexual differentiation of the hippocampus. To examine the possibility that an alteration in expression of the two types of estrogen receptors (ERalpha and ERbeta) in the hippocampus underlies different roles of estrogen and/or ERs during development and in adult life, we applied non-isotopic, digoxigenin (dig)-labeled, in situ hybridization histochemistry (ISHH) for the both ER forms and examined the distribution pattern of their messages in serial, frontal sections over the postnatal period and in the adult. ERalpha mRNA expression was found scattered throughout the hippocampus especially in the hilar region of the dentate gyrus, and in the strata radiatum and pyramidale in the cornus ammonis at postnatal days (PND) 14, 21 and 35. In the hilus of the dorsal hippocampus, the density of ERalpha-labelled cells was greater in the rostro-medial aspect, while less in the lateral and the caudal region. In the ventral hippocampus the signals for ERalpha mRNA were also found in relatively high density in the hilus. No significant sex difference in distribution and intensity of the ERalpha mRNA positive cells were detected. The hippocampal distribution of ERalpha mRNA expression at PND 14 remained the same on PND 21 and 35 and in adulthood. As reported for adults, ERalpha mRNA signals appear to be in interneurons of the hippocampus but neither in the pyramidal cells nor in the dentate granular cells based on their size and location. In contrast to the result of ERalpha, no clear signals for ERbeta mRNA were detected in the hippocampus across all ages examined, whereas they were clearly detected in the hypothalamus.

Amygdala↗
PubMed2000

Angiotensin II increases intracellular Ca(2+) concentration in folliculo-stellate cells of the rat anterior pituitary in primary culture.

It has been reported that pituitary gonadotrophs and lactotrophs contain angiotensin II (Ang II) and suggested that Ang II modulates hormone secretion from endocrine cells of the anterior pituitary through paracrine mechanism among the endocrine cells. However, there has been no report on the effect of Ang II on the folliculo-stellate cells (FSC) which are thought to play a regulatory role in the release of hormones from pituitary endocrine cells. We, therefore, examined the effect of Ang II on FCS in primary culture by Ca(2+) imaging technique. Certain proportion (42%) of FSC responded to 100 nM Ang II by increasing [Ca(2+)](i). In addition, Ang II elicited the Ca(2+) response in about 50% of the pituitary endocrine cells. The results indicate that Ang II functions as a paracrine factor among pituitary cells including FSC.

Angiotensin II↗
PubMed2000

Prostaglandin E(2) (PGE(2)) induces the c-fos and c-jun expressions via the EP(1) subtype of PGE receptor in mouse osteoblastic MC3T3-E1 cells.

This study examined which subtype(s) of PGE receptors is involved in the induction of c-fos and c-jun by PGE(2) in MC3T3-E1 cells. We also investigated the possibility that the induction of these genes is involved in the growth and differentiation of this cell line. PGE(2) dose-dependently induced c-fos and c-jun mRNA expressions in MC3T3-E1 cells. Of the PGE analogs, 17-phenyl-omega-trinor PGE(2) (EP(1) agonist) and sulprostone (EP(1)/EP(3) agonist) were far more potent than butaprost (EP(2) agonist) and 11-deoxy PGE(1) (EP(2)/EP(4) agonist) in inducing c-fos and c-jun mRNA expressions. Since MC3T3-E1 cells do not express the EP(3) subtype, these results suggest that PGE(2) induces c-fos and c-jun mRNA expressions through the EP(1) subtype of its receptor. In order to study the functional relevance of these protooncogenes, we then studied the effect of inhibition of their synthesis by the use of antisense oligonucleotide. Alkaline phosphatase (ALP) suppression by 17-phenyl-omega-trinor PGE(2) was reversed by antisense oligonucleotide for either c-fos or c-jun. These results suggest that PGE(2), via the EP(1) subtype of the PGE receptor, negatively modulates the transition from proliferation to the matrix maturation stage through the induction of c-fos and c-jun. However, antisense oligonucleotide for c-fos or c-jun did not alter the prostaglandin G/H synthase-2 mRNA expression induced by EP(1). Thus, it is possible that c-fos and c-jun inductions do not account for all the EP(1)-mediated PGE(2) actions in MC3T3-E1 cells.

Alkaline Phosphatase↗
PubMed2000

Organization and expression of two Arabidopsis DREB2 genes encoding DRE-binding proteins involved in dehydration- and high-salinity-responsive gene expression.

In plants, a cis-acting element, DRE/CRT, is involved in ABA-independent gene expression in response to dehydration and low-temperature stress. To understand signal transduction pathways from perception of the dehydration stress signal to gene expression, we characterized a gene family for DRE/CRT-binding proteins DREB2A and DREB2B in Arabidopsis thaliana. Northern analysis showed that both genes are induced by dehydration and high-salt stress. Organ-specific northern analysis with gene-specific probes showed that these genes are strongly induced in roots by high-salt stress and in stems and roots by dehydration stress. The DREB2A gene is located on chromosome 5, and DREB2B on chromosome 3. We screened an Arabidopsis genomic DNA library with cDNA fragments of DREB2A and DREB2B as probes, and isolated DNA fragments that contained 5'-flanking regions of these genes. Sequence analysis showed that both genes are interrupted by a single intron at identical positions in their leader sequence. Several conserved sequences were found in the promoter regions of both genes. The beta-glucuronidase (GUS) reporter gene driven by the DREB2 promoters was induced by dehydration and high-salt stress in transgenic Arabidopsis plants.

Arabidopsis↗
PubMed2000

p73: structure and function.

Alteration of the p53 tumor suppressor gene is a common, if not general, observation in human malignant tumors. p73 Is a novel member of the p53 family at chromosome 1p36.3, at which locus frequent defects are seen in many tumors including neuroblastoma. Besides structural similarities, the fact that p73 functions in the regulation of the cell cycle and apoptosis promotes the expansion of the research field concerning p53-associated tumor progression. In this paper, we review the structure and function of p73 as well as the mutational status in various human tumors. In addition, possibilities for new therapeutic applications with p73 for cancer cell control are discussed.

Apoptosis↗
PubMed2000

The effects of monotherapy or combined therapy with an angiotensin converting enzyme inhibitor following initial treatment with calcium channel blockers on residual cardiovascular abnormalities.

The effects of sequential therapy with angiotensin-converting enzyme inhibitor (cilazapril) on left ventricular (LV) mass, LV diastolic function, and carotid artery distensibility were evaluated in 90 hypertensive patients whose blood pressure controlled below 140/90 mmHg with a calcium channel blocker monotherapy. The possibility of predicting the efficacy of cilazapril based on evaluation of biochemical and genetic markers of the renin-angiotensin system was examined. Before cilazapril therapy, LV diastolic function and carotid artery distensibility were significantly impaired in 32 patients with residual LV hypertrophy compared with patients without LV hypertrophy. Cilazapril improved the LV mass in these patients with LV hypertrophy and improved LV diastolic function in a subset of 20 patients with elevated plasma renin activity. Patients with residual LV hypertrophy accompanied by cardiovascular functional abnormalities. Subsequent treatment with cilazapril significantly improved LV morphology and function in those with residual LV hypertrophy or elevated plasma renin activity.

Aged↗
PubMed2000

Impaired bone resorption by lipopolysaccharide in vivo in mice deficient in the prostaglandin E receptor EP4 subtype.

In a previous study we showed that the involvement of EP4 subtype of the prostaglandin E (PGE) receptor is crucial for lipopolysaccharide (LPS)-induced osteoclast formation in vitro. The present study was undertaken to test whether EP4 is actually associated with LPS-induced bone resorption in vivo. In wild-type (WT) mice, osteoclast formation in vertebrae and tibiae increased 5 days after systemic LPS injection, and urinary excretion of deoxypyridinoline, a sensitive marker for bone resorption, statistically increased 10 days after injection. In EP4 knockout (KO) mice, however, LPS injection caused no significant changes in these parameters throughout the experiment. LPS exposure for 4 h strongly induced osteoclast differentiation factor (ODF) mRNA expression in primary osteoblastic cells (POB) both from WT and EP4 KO mice, and this expression was not inhibited by indomethacin, suggesting prostaglandin (PG) independence. LPS exposure for 24 h further induced ODF expression in WT POB, but not in EP4 KO POB. Indomethacin partially inhibited ODF expression in WT POB, but not in EP4 KO POB. These data suggest that ODF is induced both PG dependently and PG independently. LPS exposure for 24 h induced slightly greater osteoclastgenesis inhibitory factor (OCIF) mRNA expression in EP4 KO than in WT POB. These findings suggest that the reduced ODF expression and apparently increased OCIF expression also are responsible for the markedly reduced LPS-induced osteoclast formation in EP4 KO mice. Our results show that the EP4 subtype of the PGE receptor is involved in LPS-induced bone resorption in vivo also. Since LPS is considered to be largely involved in bacterially induced bone loss, such as in periodontitis and osteomyelitis, our study is expected to help broaden our understanding of the pathophysiology of these conditions.

Animals↗
PubMed2000

Thyroid hormone and estrogen regulate brain region-specific messenger ribonucleic acids encoding three gonadotropin-releasing hormone genes in sexually immature male fish, Oreochromis niloticus.

The present study was undertaken to determine whether T3, estrogen, and 11-ketotestosterone could alter a specific population of GnRH-containing neurons, as indicated by a change in messenger RNA (mRNA) levels in sexually immature male tilapia, Oreochromis niloticus. Two weeks after castration, fish were assigned to four treatment groups. One group served as the control (sesame oil); a single ip injection of (T3; 5 microg/g), estradiol benzoate (EB; 5 microg/g), or 11-ketotestosterone (KT; 5 microg/g) was administered to the remaining three groups. Twenty-four hours after the injection, brains were collected and processed for in situ hybridization histochemistry using 35S-labeled 30-mer antisense oligonucleotide probes complementary to the GnRH-coding region of chicken II, salmon, and seabream GnRH. Computerized image analysis was performed to quantify mRNA concentrations, neuronal numbers, and neuronal size of the terminal nerve-nucleus olfactoretinalis, preoptic, and midbrain GnRH neurons. KT had no effect on any of the above neuronal parameters examined for salmon or seabream GnRH. Neither T3, EB, nor KT was effective to induce changes in midbrain chicken GnRH II mRNA concentrations, neuronal numbers, and neuronal size, indicating that an as yet unknown regulatory mechanism may operate midbrain GnRH neurons. T3 specifically suppressed the concentration of terminal nerve salmon GnRH mRNA, and EB significantly increased preoptic seabream GnRH neuronal numbers. These results are consistent with the hypothesis that thyroid hormone, by suppressing terminal nerve GnRH expression, promotes inhibition of sexual maturation. Furthermore, the failure of KT, a nonaromatizable androgen, to influence preoptic GnRH neurons emphasizes that an estrogenic pathway, at the onset of sexual maturation, is responsible for the recruitment of additional preoptic GnRH neurons that are fundamental to reproduction and behavior.

Animals↗
PubMed2000

Perineal musculature and its innervation by spinal motoneurons in the male rabbit: effects of testosterone.

Striated muscles in the perineum, which include the ischiocavernosus (IC), bulbocavernosus (BC), and levator ani (LA), were identified in an attempt to understand motor regulation of penile erection in the male rabbit. The IC surrounded the corpus spongiosum of the penis whereas the BC attached to the dorsum of the penis at the midline. The LA encircled the rectum and attached to the base of the penis. This anatomy suggested that the IC plays the primary role in the erection in the rabbit, whereas the BC may cause flips of the erected penis. Spinal motoneurons that innervate the IC were identified by retrograde labeling by horseradish peroxidase (HRP). As in other mammals, spinal labels from the IC appeared ipsilaterally in the ventral horn that encompassed the sixth lumbar (L6) and the first sacral segments. HRP injections into the BC labeled a small number of cells bilaterally at the same spinal levels. Rabbits are peculiar in having the IC motoneurons scattered among other motoneurons, unlike the rat and other rodents that have their IC motoneurons aggregated to form a spinal nucleus. Castration caused significant decreases in both the wet weight of IC muscles and the size of IC motoneurons within 2 weeks. Testosterone supplement following castration maintained the IC muscle weight and the neuronal size. Neither castration nor testosterone supplement induced changes in the number of IC motoneurons.

Animals↗
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↗