Biomedical subjects

S Shioda

Publications and source records attributed to S Shioda.

At least 37 records · Page 2Linked to original sources

PubMed2002

Immunocytochemical observation of ghrelin-containing neurons in the rat arcuate nucleus.

Ghrelin is a novel peptide that stimulates the release of growth hormone from the pituitary and is involved in hypothalamic feeding regulation. A pre-embedding immunostaining technique was used to study the ultrastructure and synaptic relationships of ghrelin-containing neurons in the rat arcuate nucleus (ARC). Ghrelin-like immunoreactive (ghrelin-LI) neurons were found in the ARC, and were especially abundant in its ventral part. At the electron microscopic level, ghrelin-LI neurons received afferent synapses from many unknown axon terminals. Ghrelin-LI products in the immunoreactive cell bodies, processes, and axon terminals were detected mainly in dense granular vesicles about 110 nm in diameter. Ghrelin-LI presynaptic axon terminals often made synapses with unknown immunonegative neurons. These results suggest that ghrelin acts to regulate food intake through synaptic connections in hypothalamic neuronal networks.

Animals↗

PubMed2002

Endomorphin-2 immunoreactivity in the cervical dorsal horn of the rat spinal cord at the electron microscopic level.

Endomorphin-2 is a newly discovered endogenous opioid peptide with high affinity and selectivity for the micro-opioid receptor, and potent analgesic activity, particularly in the spinal cord. Using immunoelectron microscopy, we examined the ultrastructure of the endomorphin-2-like immunoreactive processes and their synaptic relationships in the spinal cord. Endomorphin-2-like immunopositive dense-cored vesicles were observed in many axon terminals, and, in a few cases, were observed together with immunonegative dense-cored vesicles. Immunopositive axons with or without myelination were also observed. The endomorphin-2-like immunoreactive axon terminals formed synapses with both immunopositive and immunonegative processes. Most synapses were asymmetrical, but symmetrical synapses were also found. Examples of axo-dendritic, axo-somatic and axo-axonic contacts were observed. This first demonstration of the ultrastructure and synaptic relationships of endomorphin-2-like immunoreactive axon terminals in the spinal cord dorsal horn provides morphological evidence that this peptide functions as a transmitter regulating pain processes.

Animals↗

PubMed2002

Reciprocal synaptic relationships between orexin- and melanin-concentrating hormone-containing neurons in the rat lateral hypothalamus: a novel circuit implicated in feeding regulation.

OBJECTIVE: Both orexin (ORX)- and melanin-concentrating hormone (MCH) are expressed in different neurons in the lateral hypothalamic area (LH), and are considered to have common effects on stimulating food intake. There are no reports to demonstrate neural interactions between them at the ultrastructural level. We observed these neurons in the LH to evaluate the relationships between them. DESIGN: We used two different types of double immunostaining to reveal the ultrastructure of both the ORX- and MCH-containing neurons. A preembedding double immunostaining technique was used to study the synaptic relationships between the two kinds of neuron. RESULTS: The main new findings are as follows: 1) Both ORX- and MCH-containing neurons received other synaptic input and made synaptic input to other neurons; 2) Reciprocal synaptic relationships were observed between the ORX- and MCH-containing neurons. CONCLUSION: The ORX- and MCH-containing neurons in the lateral hypothalamic area may influence food intake through synapse with each other.

Animals↗

PubMed2001

Immunoelectron microscopic study of beta-endorphinergic synaptic innervation of GABAergic neurons in the dorsal raphe nucleus.

Using a preembedding double immunoreactive technique by immunostaining with antirat beta-endorphin and antisynthetic glutamic acid decarboxylase antisera sequentially, the synaptic relationships between beta-endorphinergic neuronal fibers and GABAergic neurons in the dorsal raphe nucleus of the rat were examined at the ultrastructural level. Although both beta-endorphin-like immunoreactive fibers and glutamic acid decarboxylase-like immunoreactive neurons can be found in the mediodorsal and medioventral parts of the dorsal raphe nucleus, the synapses between them were found only in the mediodorsal part. Most of the beta-endorphin-like immunoreactive neuronal fibers contained many dense-cored vesicles. The synapses made by beta-endorphin-like immunoreactive neuronal axon terminals on glutamic acid decarboxylase-like immunoreactive neurons were both symmetrical and asymmetrical, with the latter predominant, especially in the axo-dendritic synapses. Perikarya with beta-endorphin-like immunoreactivity were found only in the ventrobasal hypothalamus. These findings suggest the possibility that the beta-endorphin-producing neurons in the ventrobasal hypothalamus could influence GABAergic neurons in the dorsal raphe nucleus directly by synaptic relationships.

Animals↗

PubMed2001

ABCA3 is a lamellar body membrane protein in human lung alveolar type II cells.

The ABCA3 gene, of the ABCA subclass of ATP-binding cassette (ABC) transporters, is expressed exclusively in lung. We report here the cloning, molecular characterization, and distribution of human ABCA3 in the lung. Immunoblot analysis using the specific antibody reveals a 150-kDa protein in the crude membrane fraction of human lung. Immunohistochemical analyses of alveoli show that ABCA3 is expressed only in the type II cells expressing surfactant protein A. At the ultrastructural level, ABCA3 immunoreactivity was detected mostly at the limiting membrane of the lamellar bodies. Since members of the ABCA transporter family are known to be involved in transmembrane transport of endogenous lipids, our findings suggest that ABCA3 plays an important role in the formation of pulmonary surfactant in type II cells.

ATP-Binding Cassette Transporters↗

PubMed2001

Maf and Jun nuclear oncoproteins share downstream target genes for inducing cell transformation.

The Maf oncoprotein is a basic leucine zipper (bZip)-bearing transcriptional activator that recognizes the Maf recognition element (MARE) DNA sequence. In this study, we investigated the role of Maf's transactivation function in cell transformation. Replacement of the conserved amino terminus transactivator domain of Maf by a heterologous and stronger transactivator domain (the acidic transactivator domain of VP16) resulted in enhanced transformation of chicken embryo fibroblast cells. In contrast, the fusing of a transcriptional repressor domain (Sin3 interaction domain of Mxi1) with the whole Maf protein masked the transactivator function of Maf, which in turn inhibited its transforming activity. Furthermore, the leucine zipper domain of Maf, which defines its dimer-forming specificity, was exchangeable with that of GCN4 yeast protein in terms of its transactivating and cell transforming activities. Thus, heterodimer formation with other bZip factors is not required for Maf's ability to transform. These results together suggest that transactivation through MARE is necessary for Maf-induced transformation and that there exist downstream target gene(s) for transformation. Since the MARE sequence overlaps with the recognition element of another bZip oncoprotein Jun, we assessed whether Jun and Maf induce cell transformation through activating the same genes. We thus constructed a mutated version of Jun that has a GCN4 leucine zipper and lacks the transactivator domain. This mutant repressed the cell transformation not only by Jun but also by Maf. Thus, Maf and Jun share downstream target gene(s) that are involved in cell transformation.

Animals↗

PubMed2001

Orexin-a activates phospholipase C- and protein kinase C-mediated Ca2+ signaling in dopamine neurons of the ventral tegmental area.

The orexin-orexin receptor system has been implicated in the regulation of wakefulness/sleep states. Behavioral and psycho-stimulant effects of orexins have also been shown. Mesolimbic dopamine neurons in the ventral tegmental area (VTA) are implicated in the regulation of reward and wakefulness/sleep, In the present study, we examined the effect of orexin-A on cytosolic [Ca2+]i concentration ([Ca2+]) in the isolated rat VTA dopamine neurons. Orexin-A (10-12-10-8 M) concentration dependently increased [Ca2+]i in dopamine-containing neurons. The [Ca2+]i responses to orexin-A were inhibited under Ca2+-free conditions and by blockers of voltage-gated L- and N-type [Ca2+]i channels, nitrendipine and omega-conotoxin, respectively. The [Ca2+]i responses were also abolished by a phosphatidylcholine-specific phospholipase C inhibitor, D609, and a protein kinase C (PKC) inhibitor, calphostin C. A PKC activator, TPA, mimicked orexin-A in increasing [Ca2+]i. These results indicate that orexin-A increases [Ca2+]i in VTA dopamine neurons via phosphatidylcholine-specific PLC- and PKC-mediated activation of L- and N-type Ca2+ channels. This effect may serve as the mechanism by which orexin regulates wakefulness/sleep states and exerts its behavioral and psychostimulant effects.

Animals↗

PubMed2001

Brain responses to acute withdrawal in phenobarbital-dependent rats.

Heat shock proteins (HSP) such as HO-1 and HSP27 have been implicated as functioning in a protective manner against oxidative and physical stress. The objective of the current study was to determine the role of HSPs in drug-withdrawal stress induced in phenobarbital-dependent rats. Increased expression of HO-1 and HSP27 was observed in the hippocampus and the cerebral cortex of phenobarbital-withdrawn rats. Gene expression was measured by Northern and Western blot analyses and in situ hybridization. The induction of HO-1 mRNA was suppressed by the administration of the NMDA receptor antagonist, (+)-5-methyl-10,11-dihydro-5H-dibenzo (a,d) cyclohepten-5,10-imine (MK801). Despite significant upregulation of glutamatergic transmission, neuronal cell degeneration was not apparent. These findings suggest that the induction of HO-1 and HSP27 during withdrawal from phenobarbital dependence may play a role in protection against glutamate toxicity.

Animals↗

PubMed2001

Regulation of serotonin transporter gene expression in human glial cells by growth factors.

The aims of this study were to identify monoamine transporters expressed in human glial cells, and to examine the regulation of their expression by stress-related growth factors. The expression of serotonin transporter mRNA was detected by reverse transcriptase-polymerase chain reaction in normal human astrocytes, whereas the dopamine transporter (DAT) and the norepinephrine transporter (NET) were not detected. The cDNA sequence of the "glial" serotonin transporter in astrocytes was consistent with that reported for the "neuronal" serotonin transporter (SERT). Moreover, we also demonstrated SERT expression in glial fibrillary acidic protein-positive cells by immunocytochemical staining in normal human astrocytes. Serotonin transporter gene expression was also detected in glioma-derived cell lines (A172, KG-1-C and KGK). Addition of basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF) for 2 days increased serotonin transporter gene expression in astrocytes and JAR (human choriocarcinoma cell line). Basic fibroblast growth factor, but not epidermal growth factor, increased specific [3H]serotonin uptake in astrocytes in a time (1-4 days)- and concentration (20-100 ng/ml)-dependent manner. The expression of genes for basic fibroblast growth factor and epidermal growth factor receptors was detected in astrocytes. These findings suggest that the expression of the serotonin transporter in human glial cells is positively regulated by basic fibroblast growth factor.

Astrocytes↗

PubMed2001

Orexinergic innervation of POMC-containing neurons in the rat arcuate nucleus.

A pre-embedding double immunostaining technique was used to study the synaptic relationships between orexin-like immunoreactive axon terminals and preopiomelanocortin (POMC)-like immunoreactive neurons in the rat arcuate nucleus. Most of the synapses were axo-dendritic, while some axo-somatic synapses were also found. Both the axo-somatic and axodendritic synapses were symmetrical. In some cases the presynaptic orexin-like immunoreactive axon terminals contained a few large dense-cored vesicles. The results suggest that the orexinergic axon terminals in the arcuate nucleus may play an important role in the regulation of food intake via synapses through POMC neurons.

Animals↗

PubMed2001

Atp-binding cassette transporter ABC2/ABCA2 in the rat brain: a novel mammalian lysosome-associated membrane protein and a specific marker for oligodendrocytes but not for myelin sheaths.

We recently cloned a full-length cDNA of the rat ATP-binding cassette transporter 2 (ABC2, or ABCA2) protein, a member of the ABC1 (or ABCA) subfamily (-ABC1/ABCA1 is a causal gene for Tangier disease) and found it to be strongly expressed in the rat brain. In this study, we identified ABC2 as a lysosome-associated membrane protein that is being localized specifically in oligodendrocytes. The ABC2-immunolabeled cells were detected mainly in the white matter but were also scattered in gray matter throughout the whole brain. In addition, these cells were found to be colocalized with 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase) immunoreactivity when the marker antibody for oligodendrocytes was used. However, no such colocalization was observed with markers for other kinds of glial cells. Unlike the CNP antibody, which also intensely stains myelin sheaths in the white matter, ABC2 immunoreactivity was detected only in the cell bodies of oligodendrocytes. At the ultrastructural level, ABC2 immunoreactivity was detected mostly around lysosome and partly in Golgi apparatus by electron microscopy. This was confirmed by immunocolocalization of ABC2 and lysosomal markers in a neuroblastoma cell line. Immunoblotting analysis of ABC2 from the whole brain and the ABC2-transfected cell line revealed bands at approximately 260 kDa. The result of in situ hybridization with a riboprobe for ABC2 matched the results obtained from immunostaining. These findings strongly suggest that ABC2 is a specific marker for oligodendrocytes but not for myelinsheaths and that it is as a novel mammalian lysosome-associated membrane protein involved in myelinization or other kinds of metabolism in the CNS.

ATP-Binding Cassette Transporters↗

PubMed2001

A set of Hox proteins interact with the Maf oncoprotein to inhibit its DNA binding, transactivation, and transforming activities.

Maf oncoprotein is a basic-leucine zipper (bZip) type of transcriptional activator. Since many transcription factors are known to form functional complexes, we searched for proteins that interact with the DNA-binding domain of Maf using the phage display method and identified two homeodomain-containing proteins, Hoxd12 and MHox/Prx1/Phox1/Pmx1. Studies with mutants of Hox and Maf proteins showed that they associate through their DNA-binding domains; the homeodomain of Hox and the bZip domain of Maf, respectively. Reflecting the high similarity of the bZip domain, all other Maf family members tested (c-/v-Maf, MafB, MafK, MafF, and MafG) also associated with the Hox proteins. Pax6, whose homeodomain is relatively similar to MHox, also could interact with Maf. However, two other bZip oncoproteins, Fos and Jun, failed to associate with the Hox proteins, while a distantly related Hox family member, Meis1, could not interact with Maf. Through interactions with the bZip domain, the Hox proteins inhibited the DNA binding activity of Maf, whereas the binding of Hox proteins to their recognition sequences was not abrogated by Maf. We further showed that coexpression of the Hox proteins repressed transcriptional activation and transforming activity of Maf. These results suggested that the interaction of a set of Hox proteins with Maf family members may interfere not only with their oncogenicity but also with their physiological roles.

Amino Acid Sequence↗

PubMed2001

Reciprocal synaptic relationships between angiotensin II-containing neurons and enkephalinergic neurons in the rat area postrema.

A preembedding double immunostaining technique was used to study synaptic relationships between angiotensin-II-like immunoreactive and enkephalin-like immunoreactive neurons in the rat area postrema. The angiotensin-II-like immunoreactive neurons were detected by silver-gold intensification of the DAB reaction results while the enkephalin-like immunoreactive neurons were detected by simple ABC-DAB reaction. The synaptic relationships were reciprocal between the two neurons. Most of the synapses found between these two neurons were the presynaptic enkephalin-like immunoreactive axon terminals that made synapses on the angiotensin-II-like immunoreactive perikarya and dendrites. Both the axo-somatic and axo-dendritic synapses were symmetrical. However, although angiotensin-II-like immunoreactive axon terminals also made synapses on enkephalin-like perikarya and dendrites, the axo-somatic synapses were symmetrical, while the axo-dendritic synapses were asymmetrical. The present results confirm the presence of angiotensin-II-like immunoreactive neurons in the area postrema and suggest that these angiotensinergic neurons in the area postrema may play a role in the regulation of blood pressure via coordinated synaptic interactions with enkephalinergic neurons.

Angiotensin II↗

PubMed2001

Cosecretion of prolactin and growth hormone by dispersed pituitary cells of the adult bullfrog, Rana catesbeiana.

The coexistence of prolactin (PRL) and growth hormone (GH) was previously demonstrated in newly hatched bullfrog (Rana catesbeiana) tadpoles, whereas in adult bullfrogs, there were no cells containing both PRL and GH. However, a cell blot assay with enzymatically dispersed adult pituitary cells demonstrated the existence of cells secreting both PRL and GH. The number of cells secreting both PRL and GH was reduced by a protein synthesis inhibitor, cycloheximide, but not by an RNA synthesis inhibitor, actinomycin D. In situ hybridization and immunostaining of intact pituitary glands revealed the existence of GH mRNA in some of the PRL-immunoreactive cells and of PRL mRNA in some of the GH-immunoreactive cells. We propose that dispersion of the pituitary cells triggered the translation of GH mRNA in the PRL cells and/or of PRL mRNA in the GH cells.

Animals↗

PubMed2001

PACAP activates PKA, PKC and Ca(2+) signaling cascades in rat neuroepithelial cells.

Several studies have reported that the PAC(1) receptor (PAC1-R), the specific receptor for PACAP, is expressed at early developmental stages. Here, we describe that the cytosolic Ca(2+) concentration ([Ca(2+)](i)) was increased by PACAP, but not VIP, in a concentration range from 10(-12) to 10(-8) M via the PAC(1)-R in isolated single cells from the rat neural fold. This activation of the cells by PACAP was mimicked by agonists and inhibited by antagonists of the cAMP/PKA and PLC/PKC cascades. These data indicate that PACAP/PAC(1)-R is linked to [Ca(2+)](i) signaling via two G-protein-coupled protein kinase pathways and may thereby play an important role in early neurodevelopment.

Animals↗

PubMed2001

Characterization of mouse GBP28 and its induction by exposure to cold.

OBJECTIVE: To investigate whether the expression of the novel adipose tissue-specific protein GBP28 in adipose tissue and serum are altered in mice under a variety of conditions. DESIGN: Mice were fed a high-fat diet for 4 weeks, fasted for 48 h or exposed at 4 degrees C. SUBJECTS: C57BL/6J mouse, male, 4--6 weeks old. MEASUREMENTS: GBP28 mRNA, GBP28 protein, blood glucose, insulin and fad pad weight of the mice. RESULTS: We first confirmed that the mouse has GBP28 and its characteristics are the same as human GBP28. Serum concentration and mRNA levels of GBP28 significantly increased in the mice exposed to cold. CONCLUSION: GBP28 may play a role in homeostasis, regulating body temperature and basal metabolic rate in response to changing environmental conditions. International Journal of Obesity (2001) 25, 75-83

Adiponectin↗

PubMed2001

Application and modification of in situ RT-PCR for detection and cellular localization of PAC1-R splice variant mRnas in frozen brain sections.

Many important biopolymers such as neurotransmitters, modulators, transporters and receptors are expressed in discrete regions of the brain or other tissues, and they often occur at extremely low concentrations; therefore, a sensitive detection system is required to map their distribution. To study the precise distribution patterns of the splice variants of the PAC1 receptor, which specifically binds pituitary adenylate cyclase-activating polypeptide (PACAP) with affinity in the nano- or picomolar range, we have applied an in situ reverse transcription-polymerase chain reaction (RT-PCR) technique in frozen tissue sections. We describe here a modified protocol using a single rTth enzyme, which can synthesize cDNA from RNA, then PCR amplifying it in a single reaction mixture by varying the times and temperatures of a thermal cycler. The primer pairs were the same as those used in the solution phase RT-PCR that had been used to obtain the expected bands of the amplified products previously. A nonradioactive labeling system with digoxigenin conjugated with peroxidase or fluorescence for signal detection was compared. The gene expression of two PAC1-R splice variants in the rat motor nucleus is first reported here.

Animals↗

PubMed2001

Developmental origin of the rat adenohypophysis prior to the formation of Rathke's pouch.

In amphibians, it has already been shown that the adenohypophysis originates from the anterior neural ridge. During the migration and morphogenesis of this organ, the anterior neural ridge transiently forms a Rathke's pouch-like structure by attaching itself to the rostral tip of the foregut, and finally gives rise to the adenohypophysis by detaching from the foregut and becoming connected to the infundibulum of the hypothalamus. In order to identify the origin of the adenohypophyseal cells in mammalian embryos prior to the formation of Rathke's pouch (RP), we labeled the rostral end of the neural plate and the adjacent area focally with DiI at the open neurula stage (9.5 dpc). After a 48-hours culture of the whole embryos, strongly labeled cells were detected in the RP only when DiI was applied to a small area situated just anterior to the rostral end of the neural plate. By explanting the labeled RP for a further 7 days, we confirmed immunohistochemically that the labeled cells developed into the secretory cells of the adenohypophysis. The developmental origin of the adenohypophysis is identified for the first time in the early mammalian embryo before the formation of RP.

Animals↗