Biomedical subjects

R Nishi

Publications and source records attributed to R Nishi.

At least 19 recordsLinked to original sources

PubMed2002

Cerebral endothelial cells are a major source of adrenomedullin.

Adrenomedullin is a peptide hormone with multifunctional biological properties. Its most characteristic effects are the regulation of circulation and the control of fluid and electrolyte homeostasis through peripheral and central nervous system actions. Although adrenomedullin is a vasodilator of cerebral vasculature, and it may be implicated in the pathomechanism of cerebrovascular diseases, the source of adrenomedullin in the cerebral circulation has not been investigated thus far. We measured the secretion of adrenomedullin by radioimmunoassay and detected adrenomedullin mRNA expression by Northern blot analysis in primary cultures of rat cerebral endothelial cells (RCECs), pericytes and astrocytes. We also investigated the expression of specific adrenomedullin receptor components by reverse transcriptase-polymerase chain reaction and intracellular cAMP concentrations in RCECs and pericytes. RCECs had approximately one magnitude higher adrenomedullin production (135 +/- 13 fmol/10(5) cells per 12 h; mean +/- SD, n = 10) compared to that previously reported for other cell types. RCECs secreted adrenomedullin mostly at their luminal cell membrane. Adrenomedullin production was not increased by thrombin, lipopolysaccharide or cytokines, which are known inducers of adrenomedullin release in peripheral endothelial cells, although it was stimulated by astrocyte-derived factors. Pericytes had moderate, while astrocytes had very low basal adrenomedullin secretion. In vivo experiments showed that adrenomedullin plasma concentration in the jugular vein of rats was approximately 50% higher than that in the carotid artery or in the vena cava. Both RCECs and pericytes, which are potential targets of adrenomedullin in cerebral microcirculation, expressed adrenomedullin receptor components, and exhibited a dose-dependent increase in intracellular cAMP concentrations after exogenous adrenomedullin administration. Antisense oligonucleotide treatment significantly reduced adrenomedullin production by RCECs and tended to decrease intraendothelial cAMP concentrations. These findings may suggest an important autocrine and paracrine role for adrenomedullin in the regulation of cerebral circulation and blood-brain barrier functions. Cerebral endothelial cells are a potential source of adrenomedullin in the central nervous system, where adrenomedullin can also be involved in the regulation of neuroendocrine functions.

Adrenomedullin↗

PubMed2001

Adrenomedullin regulates blood-brain barrier functions in vitro.

Adrenomedullin (AM) is an important vasodilator in cerebral circulation, and cerebral endothelial cells are a major source of AM. This in vitro study aimed to determine the AM-induced changes in blood-brain barrier (BBB) functions. AM administration increased, whereas AM antisense oligonucleotide treatment decreased transendothelial electrical resistance. AM incubation decreased BBB permeability for sodium fluorescein (mol. wt 376 Da) but not for Evan's blue albumin (mol. wt 67 kDa), and it also attenuated fluid-phase endocytosis. AM treatment resulted in functional activation of P-glycoprotein efflux pump in vitro. Our results indicate that AM as an autocrine mediator plays an important role in the regulation of BBB properties of the cerebral endothelial cells.

Adrenomedullin↗

PubMed2001

Cell death and neuronal replacement during formation of the avian ciliary ganglion.

Programmed cell death is a prominent feature of embryonic development and is essential in matching the number of neurons to the target tissues that are innervated. Although a decrease in neuronal number which coincides with peripheral synaptogenesis has been well documented in the avian ciliary ganglion, it has not been clear whether cell death also occurs earlier. We observed TUNEL-positive neurons as early as stage 24, with a large peak at stage 29. This cell death at stage 29 was followed by a statistically significant (P < 0.0001) decrease in total neuron number at stage 31. The total number of neurons was recovered by stage 33/34. This suggested that dying neurons were replaced by new neurons. This replacement process did not involve proliferation because bromodeoxyuridine applied at stages 29 and 31 was unable to label neurons harvested at stage 33/34. The peak of cell death at stage 29 was increased 2.3-fold by removal of the optic vesicle and was reduced by 50% when chCNTF was overexpressed. Taken together, these results suggest that the regulation of neuron number in the ciliary ganglion is a dynamic process involving both cell death and neural replacement from postmitotic precursors prior to differentiation and innervation of target tissues.

Animals↗

PubMed2001

Combined transcriptome and proteome analysis as a powerful approach to study genes under glucose repression in Bacillus subtilis.

We used 2D protein gel electrophoresis and DNA microarray technologies to systematically analyze genes under glucose repression in B:acillus subtilis. In particular, we focused on genes expressed after the shift from glycolytic to gluconeogenic at the middle logarithmic phase of growth in a nutrient sporulation medium, which remained repressed by the addition of glucose. We also examined whether or not glucose repression of these genes was mediated by CcpA, the catabolite control protein of this bacterium. The wild-type and ccpA1 cells were grown with and without glucose, and their proteomes and transcriptomes were compared. 2D gel electrophoresis allowed us to identify 11 proteins, the synthesis of which was under glucose repression. Of these proteins, the synthesis of four (IolA, I, S and PckA) was under CcpA-independent control. Microarray analysis enabled us to detect 66 glucose-repressive genes, 22 of which (glmS, acoA, C, yisS, speD, gapB, pckA, yvdR, yxeF, iolA, B, C, D, E, F, G, H, I, J, R, S and yxbF ) were at least partially under CcpA-independent control. Furthermore, we found that CcpA and IolR, a repressor of the iol divergon, were involved in the glucose repression of the synthesis of inositol dehydrogenase encoded by iolG included in the above list. The CcpA-independent glucose repression of the iol genes appeared to be explained by inducer exclusion.

Amino Acid Sequence↗

PubMed2001

Chick ciliary neurotrophic factor is secreted via a nonclassical pathway.

In contrast to mammalian ciliary neurotrophic factors (CNTFs), chick CNTF is secreted, although it lacks an N-terminal signal. We determined that a 52 aa region of chick CNTF containing an internal hydrophobic domain could direct secretion of rat CNTF. Using a stable cell line that overexpressed chick CNTF, we found that chick CNTF immunoreactivity was punctate throughout the cytosol. Cellular fractionation confirmed chick CNTF to be protected by vesicles. Chick CNTF did not colocalize with fibronectin, calreticulin, wheat germ agglutinin binding sites, or with transferrin receptor. The distribution of chick CNTF was altered neither by brefeldin A nor by chloroquine treatment. Although the punctate pattern of chick CNTF immunoreactivity was not due to reuptake, chick CNTF could be found in a cellular compartment labeled after a brief incubation with dextran microbeads. When synthesized in vitro, chick CNTF did not translocate into microsomes. We conclude that chick CNTF is secreted via a nonclassical pathway.

Animals↗

PubMed2001

Isolation and characterization of two genes encoding ubiquitin fused to a ribosomal protein of 53 amino acids in rice.

We isolated and determined the nucleotide sequences of two genes encoding ubiquitin fused to a ribosomal protein, Ub-CEP52, from rice (Oryza sativa L.). The deduced amino-acid sequences of the two genes were found to be completely identical. The N-terminal region of 76 residues corresponds to ubiquitin, and the C-terminal region of 53 residues corresponds to ribosomal protein L40. A putative TATA-like sequence, a polypyrimidine sequence, and a similar sequence to telo-box were found in the promoter regions of the two genes. Furthermore, the putative tRNA(Pro) gene was found in the 5'-upstream region of one of them.

Amino Acid Sequence↗

PubMed2000

Histone H1 variants play individual roles in transcription regulation in the DT40 chicken B cell line.

Thirty-nine of the 44 chicken histone genes are located in a major gene cluster of 110 kb, the others being distributed in four separate regions. All 6 H1 genes, which are present in the cluster and encode different variants, are expressed in the DT40 chicken B cell line, at levels ranging from about 5 to 40%. To clarify differences in the natures of these H1 variants, using gene-targeting techniques, we generated a series of DT40 mutants, which are devoid of each of the 5 H1 genes, respectively. Analyses of six H1-deficient mutants, comprising the latter five and a previously generated H1-deficient mutant, revealed that the protein patterns on 2D-PAGE were definitely different from each other, indicating that each H1 variant plays an individual role in the transcription regulation of specific genes in DT40 cells.

Alleles↗

PubMed2000

Charcoal hemoperfusion in the treatment of phenytoin overdose.

In the case of phenytoin, a drug that is generally highly protein bound, there is a lack of consensus on the use of charcoal hemoperfusion in cases of overdose. We performed charcoal hemoperfusion on a phenytoin-overdosed patient to assess the effectiveness of this treatment. The plasma concentrations of total and free phenytoin fell rapidly, from 40.0 microg/mL and 3.6 microg/mL to 16.2 microg/mL and 1.5 microg/mL, respectively, after 3 hours of hemoperfusion. The total phenytoin elimination half-life was 3.9 hours. The fraction of protein-bound phenytoin was constant (90.8% +/- 0.5%) before, during, and after the procedure. The relations between the in vitro protein binding and adsorption of phenytoin to activated charcoal were also examined. Interestingly, bound phenytoin was found to dissociate from plasma proteins in the presence of activated charcoal and subsequently became adsorbed to the activated charcoal. Considering that phenytoin is bound to albumin with a large number of binding sites (n = 6) and a small binding constant (K = 6 x 10(3/)mol/L), the extent of adsorption to activated charcoal may depend on the magnitude of the binding constant of the drug to plasma proteins. The current results suggest that charcoal hemoperfusion is effective for the removal of drugs that bind to plasma proteins with a low binding constant.

Adult↗

PubMed1999

Increase of vasopressin mRNA in the hypothalamus of inbred polydipsic mice.

The genetically inbred polydipsic mice, STR/N strain, are characterized by extreme polydipsia and polyuria without arginine vasopressin (AVP) deficiency. The expression of AVP gene in the hypothalamus of polydipsic and non-polydipsic mice was examined by Northern blot analysis and in situ hybridization histochemistry. Northern blot analysis revealed that the total amount of AVP mRNA in the hypothalamus of the STR/N mice was approximately three-fold of that in the control, ICR mice. In situ hybridization histochemistry showed that the signals of AVP mRNA in the paraventricular (PVN) and supraoptic nuclei (SON) of the STR/N were stronger than those in the ICR. Although AVP gene transcripts were detected in the anteroventral parts of the PVN (avPVN) in the STR/N, there was a few AVP transcripts in the same area (avPVN) in the ICR. There were no differences in plasma osmolality and hematocrit between STR/N and ICR mice. These results suggest that upregulation of AVP mRNA in the hypothalamus of STR/N may be involved in the central mechanism responsible for the polydipsia in genetically polydipsic mice.

Animals↗

PubMed1999

Temporal modulation of cytokine expression following focal cerebral ischemia in mice.

There is increasing evidence that the inflammatory response plays an important role in CNS ischemia. The murine model of focal ischemia, however, remains incompletely characterized. In this study we examined expression of several cytokines and the vascular adhesion molecule E-selectin, in order to characterize the molecular events following stroke in the C57BL/6J mouse. Using a multi-probe RNAse protection assay (RPA), mRNA for 19 cytokines was analyzed following permanent and transient occlusion of the middle cerebral artery in mice. In addition, samples from the same mice were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) to evaluate E-selectin mRNA expression levels. Several cytokine mRNAs showed a similar expression pattern in both permanent and transient CNS ischemia while others showed a temporal expression pattern that was dependent on the type of stroke. For both models, mRNA levels of TNFalpha rose early (4 h) followed by IL-6 (10-18 h) and a comparatively late increase (96 h) in TGFbeta1. IL-1alpha, IL-1beta and IL-1ra levels showed a model dependent shift in temporal expression. Reperfusion appeared to delay the induction of these cytokines. Temporal changes in cytokine mRNA expression in the mouse CNS occur following ischemic damage. Our findings demonstrate the utility and power of multi-probe RPA for evaluation of changes in cytokine mRNA levels. Moreover, this study is, to our knowledge the first to show temporal changes in cytokine mRNA in mouse cerebral ischemia, forming a basis for further exploration of the roles of these cytokines in modulating ischemic neuronal damage in this model.

Animals↗

PubMed1998

Development of the avian iris and ciliary body: mechanisms of cellular differentiation during the smooth-to-striated muscle transition.

The avian iris and ciliary body undergoes a transition from smooth-to-striated muscle during embryonic development. Using antibodies specific for smooth muscle-specific alpha-actin and myosin heavy chain, we confirm that a smooth-to-striated muscle transition occurs between E8 and E17 in both iris and ciliary body of the chick. To study the mechanisms regulating the transition in muscle type, we analyzed the fate of quail clones derived from E7 iris cells. When cells were cloned alone, 45/71 colonies differentiated into smooth muscle and 10/71 became striated muscle. None of the colonies were mixed with respect to muscle phenotype, indicating a lack of pluripotent stem cells. Furthermore, clones giving rise to nonstriated muscle could not be forced to incorporate into myotubes when cocultured with chick myocytes. Clones grown in coculture with chick embryo fibroblasts or E11 iris cells had very high cloning efficiencies (>98%). Significantly more clones differentiated into striated muscle when cocultured with E11 cells (60/156) than when cocultured with fibroblasts (29/108). This was due to an increased recruitment of undifferentiated cells into striated muscle, rather than a change in the percentage of cells differentiating into smooth muscle. In vivo and in vitro, various smooth and striated muscle-specific markers including contractile proteins, acetylcholine receptor subtypes, and transcription factors were colocalized in cells. Although our data argue against a multipotent stem cell for smooth and striated muscle cells, they cannot exclude a role for transdifferentiation. Cumulatively these results suggest that both smooth muscle and migratory myoblasts contribute to the development of myotubes in the avian iris and that this process is regulated in a non-cell-autonomous fashion by locally generated signals.

Acetylcholine↗

PubMed1998

Activin A and follistatin influence expression of somatostatin in the ciliary ganglion in vivo.

An important developmental question concerns whether neurotransmitter phenotype is an inherent property of neurons or is influenced by target tissues. This issue can be addressed in the avian ciliary ganglion (CG) which contains two cholinergic populations, ciliary and choroid neurons, that differentially express the peptide cotransmitter, somatostatin. The present study tests the hypothesis that differences in the level of expression of activin A and its endogenous inhibitor follistatin in CG neuron target tissues are responsible for selective expression of somatostatin in choroid neurons. Intraocular injection of activin A or follistatin (300 ng injected at E10/E11) in cultured embryos resulted in a 39% increase or a 23% decrease, respectively, in somatostatin-positive neurons relative to controls. Chorioallantoic membrane application of follistatin (1 microgram daily from E7 to E13) reduced somatostatin positive neurons by 54%. Neuron number, size, and target tissue morphology were unaffected by these treatments. Together with our previous studies, these data suggest that activin A and follistatin are target-derived molecules that regulate neuropeptide phenotype in the ciliary ganglion.

Activins↗

PubMed1998

Development of the avian iris and ciliary body: the role of activin and follistatin in coordination of the smooth-to-striated muscle transition.

Although general principles have been established in the regulation of vetebrate organogenesis, the specific molecules responsible for such signaling are just being identified. We have studied differentiation in the avian iris and ciliary body which undergoes a transition from smooth to striated muscle. Using heterochronic cocultures, we have found that striated muscle differentiation in pretransition (E8) cells is induced by midtransition (E11) cells through a secreted and soluble activity. In addition, contact-mediated mechanisms among pretransition cells prevented precocious striated muscle differentiation. We have tested the role of activin and its antagonist follistatin, as candidate regulators of this muscle transition. Activin induced smooth muscle differentiation while repressing striated muscle development. Conversely, follistatin promoted the emergence of striated muscle, while inhibiting smooth muscle differentiation. Significantly, secreted follistatin activity was found to increase during the smooth-to-striated muscle transition. Moreover, the striated muscle inducing activity from midtransition iris and ciliary body cell conditioned medium was depleted with an activin-affinity column which binds follistatin. These results suggest that activin and follistatin coordinate differentiation in the avian iris and ciliary body.

Activins↗

PubMed1998

Overexpression of ciliary neurotrophic factor in vivo rescues chick ciliary ganglion neurons from cell death.

Ciliary ganglion (CG) neurons undergo target-dependent cell death during embryonic development. Although ciliary neurotrophic factor (CNTF) was identified in vitro by its ability to support the survival of chick CG neurons, its function as a target-derived neurotrophic factor has been questioned by those working on mammalian-derived forms of CNTF. We have purified and cloned a chicken CNTF [chCNTF; formerly growth-promoting activity (GPA)] that is expressed in CG targets during the period of cell death and is secreted by cells transfected with chCNTF. In the present study we used a retroviral vector, RCASBP(A), to overexpress chCNTF in CG target tissues. Elevation of chCNTF biological activity three- to fourfold in the embryonic eye rescued an average of 31% of the neurons that would have normally died in vivo. In some individuals, nearly all of the neurons were rescued. ChCNTF had no effect on the number of neurons observed prior to cell death, nor were there any deleterious effects of either viral infection or overexpression of CNTF. These results show that chCNTF is able to function in vivo as a trophic factor for CG neurons, and suggest that limited availability of trophic support is one of the factors regulating CG neuron survival during development.

Animals↗

PubMed1997

Opposing effects of activin A and follistatin on developing skeletal muscle cells.

Activin and the activin-binding protein follistatin modulate a variety of biological processes and are abundant at sites of muscle development. Activin and follistatin were expressed in developing chick pectoral muscle in vivo and in primary cell culture. Addition of recombinant activin inhibited muscle development in a dose-dependent manner as measured by the number of nuclei in myosin heavy chain positive cells and creatine phosphokinase activity. Conversely, follistatin potentiated muscle development. The effects of activin were found to be distinct from those of the related protein transforming growth factor (TGF) beta1. Muscle development was repressed by activin at all time points investigated and did not recover with the removal of activin following a limited exposure. In contrast, while myogenic differentiation in TGFbeta1 was initially repressed, muscle marker expression recovered to control levels--even in the continued presence of TGFbeta1. Fibroblast growth factor (FGF) had little effect on inhibiton of muscle development caused by activin A. However, inhibition of development produced by TGFbeta increased with increasing concentrations of FGF. Finally, early expression of myoD and myf5 mRNA by muscle cultures in the presence of activin and follistatin was analyzed. Activin-treated cultures expressed reduced myoD and myf5 levels at 1.5 days after plating. Myf5 levels in follistatin-treated cultures were elevated, but, surprisingly, these cultures showed a reduction in myoD levels. These data suggest that endogenously expressed activin and follistatin are important modulators of muscle development.

Activins↗

PubMed1996

Expression of a chicken ciliary neurotrophic factor in targets of ciliary ganglion neurons during and after the cell-death phase.

Ciliary ganglion (CG) neurons, like other neuronal populations, become dependent on their targets for survival during development. We have previously purified and cloned a secreted ciliary neurotrophic factor that was called growth-promoting activity (GPA). We report here the expression and purification of a highly active form of recombinant GPA, the preparation of GPA-specific polyclonal and monoclonal antibodies, and the use of these antibodies to investigate the cellular location and timing of GPA expression in tissues innervated by CG neurons. Virtually all of the trophic activity in extracts of embryonic eyes could be depleted by GPA-specific antibodies. GPA-like immunoreactivity was found in both targets of the CG: the arterial vasculature of the choroid layer and the ciliary body of the eye. In the choroid layer, GPA was localized to smooth muscle cells surrounding the choroid arteries. Staining in the choroid layer was first detectable at embryonic day (E) 10, or about 2 days after cell death has begun in the ganglion, then increased in intensity through E19. Quantification of trophic activity from whole eye extracts at various ages showed a small increase in activity observed between E9 and E12 and at least a 10-fold increase between E12 and E18. The presence of GPA protein in target cells of CG neurons during the specific developmental period when these neurons undergo cell death is consistent with its proposed function as a target-derived ciliary neurotrophic factor.

Animals↗

PubMed1996

Identification of functional receptors for ciliary neurotrophic factor on chick ciliary ganglion neurons.

Ciliary neurotrophic factor and an avian homolog, growth promoting activity, are members of the cytokine/neurokine family of trophic factors and have been proposed to function as survival and developmental factors for ciliary ganglion neurons in vivo. Here we identify for the first time functional receptors for ciliary neurotrophic factor and growth promoting activity on cultured ciliary ganglion neurons. [(125)I]Rat ciliary neurotrophic factor binding studies indicate that rat ciliary neurotrophic factor and growth promoting activity bind to these receptors with a single affinity, while human ciliary neurotrophic factor recognizes both a high- and low-affinity site. Comparison of the relative potency of human ciliary neurotrophic factor and avian growth promoting activity in biological assays indicates that growth promoting activity is three to five times more active in promoting survival and in regulating acetylcholine receptors. The binding of ciliary neurotrophic factor is specific, sensitive to phosphatidylinositol-specific phospholipase C and partially inhibited by leukemia inhibitory factor, but not inhibited by other members of the human neurokine family, including interleukin-6, interleukin-22 and oncostatin M. Cross-linking of [(125)I]rat ciliary neurotrophic factor to ciliary neurons results in the specific labeling of three proteins with estimated molecular masses of 153,000, 81,000 and 72,000. Only the 81,000 molecular weight component is released from the cells after treatment with phosphatidylinositol-specific phospholipase C, suggesting a membrane attachment via a glycosylphosphatidylinositol linkage. Stimulation with ciliary neurotrophic factor or growth promoting activity, but not by other neurokines, results in the rapid tyrosine phosphorylation of a 90,000 molecular weight protein that is inhibited by pretreatment with phosphatidylinositol-specific phospholipase C. In conclusion, we report here the pharmacological and functional properties of ciliary neurotrophic factor receptors on embryonic ciliary ganglion neurons. These results provide the means for elaborating the molecular mechanisms of ciliary neurotrophic factor action and understanding its physiological role in a defined neuronal population.

Animals↗

PubMed1996

Autonomic and sensory neuron cultures.

This chapter has provided a rather detailed protocol for the dissection, dissociation, and culture of autonomic and sensory neurons from the chicken embryo. These protocols are by no means absolute. Many other laboratories that routinely culture these neurons may use techniques that differ significantly from the ones detailed in this chapter. All of the protocols described in this chapter can also be applied to quail embryos, which develop more rapidly but are of comparable size to chicken embryos until about E9. The list of suppliers for the various reagents described in these protocols is also limited. Many other vendors of cell culture products are probably equally reliable.

Animals↗