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Biomedical subjects

R Arai

Publications and source records attributed to R Arai.

At least 37 records · Page 2Linked to original sources
PubMed2000

Schizosaccharomyces pombe rho2p GTPase regulates cell wall alpha-glucan biosynthesis through the protein kinase pck2p.

Schizosaccharomyces pombe rho1(+) and rho2(+) genes are involved in the control of cell morphogenesis, cell integrity, and polarization of the actin cytoskeleton. Although both GTPases interact with each of the two S. pombe protein kinase C homologues, Pck1p and Pck2p, their functions are distinct from each other. It is known that Rho1p regulates (1,3)beta-D-glucan synthesis both directly and through Pck2p. In this paper, we have investigated Rho2p signaling and show that pck2 delta and rho2 delta strains display similar defects with regard to cell wall integrity, indicating that they might be in the same signaling pathway. We also show that Rho2 GTPase regulates the synthesis of alpha-D-glucan, the other main structural polymer of the S. pombe cell wall, primarily through Pck2p. Although overexpression of rho2(+) in wild-type or pck1 delta cells is lethal and causes morphological alterations, actin depolarization, and an increase in alpha-D-glucan biosynthesis, all of these effects are suppressed in a pck2 delta strain. In addition, genetic interactions suggest that Rho2p and Pck2p are important for the regulation of Mok1p, the major (1-3)alpha-D-glucan synthase. Thus, a rho2 delta mutation, like pck2 delta, is synthetically lethal with mok1-664, and the mutant partially fails to localize Mok1p to the growing areas. Moreover, overexpression of mok1(+) in rho2 delta cells causes a lethal phenotype that is completely different from that of mok1(+) overexpression in wild-type cells, and the increase in alpha-glucan is considerably lower. Taken together, all of these results indicate the presence of a signaling pathway regulating alpha-glucan biosynthesis in which the Rho2p GTPase activates Pck2p, and this kinase in turn controls Mok1p.

Cell Wall↗
PubMed2000

Fluorolabeling of antibody variable domains with green fluorescent protein variants: application to an energy transfer-based homogeneous immunoassay.

A site-specific and efficient fluorolabeling of antibody variable regions with green fluorescent protein (GFP) variants and its application to an energy transfer-based homogeneous fluoroimmunoassay (open sandwich FIA) were attempted. Two chimeric proteins, Trx-V(H)-EBFP and Trx-V(L)-EGFP, consisting of V(H) and V(L) fragments of anti-hen egg lysozyme (HEL) antibody HyHEL-10 and two GFP color variants, EBFP and EGFP, respectively, were designed to be expressed in cytoplasm of trxB - mutant Escherichia coli as fusions with thioredoxin from E.coli The mixture of two proteins could be purified with HEL-affinity chromatography, retaining sufficient intrinsic fluorescence and binding activity to HEL. A significant increase in fluorescence resonance energy transfer (FRET) dependent on HEL concentration was observed, indicating the reassociation of the V(H) and V(L) domains of these chimeric proteins due to co-existing antigen. With this open sandwich FIA, an HEL concentration of 1-100 microg/ml could be non-competitively determined. The assay could be performed in a microplate format and took only a few minutes to obtain a sufficient signal after simple mixing of the chimeric proteins with samples. This represents the first demonstration that the FRET between GFP variants is applicable to homogeneous immunoassay.

Base Sequence↗
PubMed2000

Highly repetitive elements from Chinese bitterlings (genus Rhodeus, Cyprinidae).

We have isolated and characterized several highly repetitive DNA elements from two species of Chinese bitterlings, Rhodeus atremius suigensis and R. ocellatus ocellatus. They comprise a partly interspersed and partly tandem repetitive family of about 1.0 to 1.3 kb in length. Individual elements showed considerable length variation, but genomic Southern blotting revealed two major length groups. Their restricted presence of these elements among related species and relative copy number differences indicated rapid change of genome structure in this group of fish. The isolated elements may be useful landmarks for further chromosomal studies.

Animals↗
PubMed1999

Calretinin and calbindin-D28k in dopaminergic neurons of the rat midbrain: a triple-labeling immunohistochemical study.

We used triple-labeling immunohistochemistry in rat midbrain sections to identify dopaminergic neurons that contain either one or both of the calcium-binding proteins, calretinin (CR) and calbindin-D28k (CB). Midbrain dopaminergic neurons were immunohistochemically labeled for tyrosine hydroxylase (TH), CR, and CB. In the substantia nigra pars compacta (SNC), TH+/CR+/CB+ cells were clustered in two regions: the dorsal tier of the rostral SNC and the medial part of the intermediate SNC. The ventral tier of the rostral SNC mainly comprised both TH+/CR+/CB- and TH+/CR-/CB- cells. The lateral part of the intermediate SNC and the caudal SNC primarily consisted of TH+/CR-/CB- cells. Throughout the extent of the SNC, approximately half of the TH+ neurons were stained for neither CR nor CB, while the remaining TH+ populations were labeled for CR and/or CB. Throughout the ventral tegmental area, TH+/CR+/CB+ cells, TH+/CR+/CB- cells, TH+/CR-/CB+ cells, and TH+/CR-/CB- cells were found generally scattered, though the TH+/CR-/CB- cells were dominant in number. In the substantia nigra pars lateralis, interfascicular nucleus, and caudal linear nucleus, more than half of the TH+ cells were stained for both CR and CB. In the retrorubral field, two-thirds of the TH+ neurons contained neither protein. The present findings suggest that the SNC can be divided into subcompartments based on the distribution of dopaminergic neurons that contain calcium-binding proteins. Furthermore, because CR and CB likely contribute to calcium homeostasis by buffering intracellular calcium concentrations, midbrain dopaminergic neurons containing one or both of these calcium-binding proteins may have a higher calcium-buffering capacity than those lacking the two proteins.

Animals↗
PubMed1999

Histochemical study of dopamine-degrading monoamine oxidase activity in dopaminergic neurons of rat brain.

We examined whether dopamine-degrading activity of monoamine oxidase (MAO) is present in dopaminergic neurons of the rat brain. We employed a double-labeling procedure combining immunohistochemistry for tyrosine hydroxylase (TH) and enzyme histochemistry for MAO activity using dopamine as a substrate. The following dopaminergic cell groups were examined: A16 (glomerular layer of the olfactory bulb), A14 (hypothalamic periventricular region), A13 (zona incerta), A12 (arcuate nucleus), A11 (periventricular gray matter of the caudal thalamus), A10 (ventral tegmental area), A9 (substantia nigra pars compacta, SNC) and A8 (retrorubral nucleus). Although no MAO activity was detected in any of the TH-immunoreactive dopaminergic neurons, strong dopamine-degrading MAO activity was found in TH-positive neurons in the locus coeruleus (LC) (i.e., noradrenergic neurons). Our results indicate that dopamine-degrading MAO activity is very low in dopaminergic neurons compared to the MAO activity in LC noradrenergic neurons.

Animals↗
PubMed1999

Monoamine oxidase in the intermediolateral nucleus of the thoracic spinal cord of the rat. A histochemical study.

We examined monoamine oxidase (MAO) activity in the intermediolateral nucleus (IML) of the rat thoracic spinal cord by histochemistry with tyramine as a common substrate for both MAO types A and B. Light microscopy showed MAO activity in neuronal cell bodies, processes, and varicosities. Electron microscopic examination showed both MAO-positive and -negative neuronal cell bodies. In the stained cell bodies, histochemical reaction products were localized in the cytoplasm showing a selective association with mitochondrial outer membranes. MAO-positive axon terminals were often found in contact with MAO-negative neurons but only occasionally with MAO-positive neurons. MAO histochemistry in the IML was also performed using serotonin (a MAO type A preferential substrate) and beta-phenylethylamine (a MAO type B preferential substrate). Light microscopy identified MAO activity for serotonin in a plexus of varicosities but not in any neuronal cell bodies. The activity for beta-phenylethylamine was detected frequently in neuronal cell bodies but rarely in varicosities. Our findings indicate that two groups of IML neurons can be chemically distinguished, one contains MAO type B while the other lacks both MAO types A and B. In addition, many axon terminals contain MAO type A but only a few fibers include MAO type B in the IML.

Adrenergic Fibers↗
PubMed1999

Tyrosine hydroxylase and aromatic L-amino acid decarboxylase do not coexist in neurons in the human anterior cingulate cortex.

Immunoreactivity for aromatic L-amino acid decarboxylase (AADC), the second step dopamine-synthesizing enzyme, was found immunohistochemically in neurons of the human anterior cingulate cortex (ACC). Most of these neurons were located in layers V and VI and subcortical white matter; a small number were occasionally found in layer III. Double immunohistochemistry for tyrosine hydroxylase (TH: the first step dopamine-synthesizing enzyme) and AADC revealed that no neuronal cell bodies in the ACC were doubly immunostained for TH and AADC, suggesting that these TH-only- or AADC-only-immunoreactive neurons were not dopaminergic. AADC neurons in the human ACC might transform L-DOPA to dopamine, droxidopa to noradrenaline, and/or 5-hydroxytryptophan to serotonin.

Adult↗
PubMed1999

Noradrenaline-degrading activity of monoamine oxidase is localized in noradrenergic neurons of the locus coeruleus of the rat.

We found intense monoamine oxidase (MAO) activity in rat locus coeruleus (LC) neurons by means of a histochemical method using noradrenaline as a substrate. This MAO activity was abolished by clorgyline, a specific inhibitor of MAO type A. Fluorescence immunohistochemistry for tyrosine hydroxylase (TH) combined with MAO histochemistry revealed intense MAO activity in virtually all TH-immunoreactive LC neurons (i.e. noradrenergic neurons). The results indicate that noradrenaline produced in LC neurons might be degraded by MAO type A activity.

Animals↗
PubMed1999

Overproduction of elongation factor 1alpha, an essential translational component, causes aberrant cell morphology by affecting the control of growth polarity in fission yeast.

BACKGROUND: Elongation factor 1alpha (EF1alpha), an essential component of the eukaryotic translational machinery, has been shown to possess various biochemical and biological activities, including F-actin-binding and -bundling, microtubule- severing, and the activity of making fibroblasts highly susceptible to transformation. However, our understanding of the biological significance of EF1alpha with respect to these various biochemical or biological activities remains limited. Here we report the identification of EF1alpha-encoding genes as genes whose over-expression causes aberrant cell morphology in fission yeast. RESULTS: Overproduction of EF1alpha caused aberrant cell morphology-elliptic, curved or branched-and growth defects in yeast cells at high temperatures. EF1alpha-overproducing cells showed a supersensitivity to the actin inhibitor cytochalasin D and to the tubulin inhibitor thiabendazole. Genetic analyses using cdc mutants suggested that excess EF1alpha disturbed the establishment and the maintenance of growth polarity in the G1 phase by pre- venting the localization of F-actin to the polarized growing site and the organization of microtubules. Results from DNase I column chromatography indicated that EF1alpha was bound to G-actin. Indeed, the fission yeast actin was immunoprecipitated along with EF1alpha. Moreover, the temperature sensitivity caused by the overproduction of EF1alpha was restored by co-overproduction of actin. CONCLUSIONS: Fission yeast EF1alpha has the ability to alter the cell morphology of yeast by affecting the control of actin and microtubule cytoskeletons.

Actins↗
PubMed1999

Calbindin D28k and calretinin in oxytocin and vasopressin neurons of the rat supraoptic nucleus.A triple-labeling immunofluorescence study

The aim of the present study was to examine quantitatively whether two calcium-binding proteins, calbindin D28k and calretinin, are localized in oxytocin and vasopressin neurons of the supraoptic nucleus of the male rat. We used a triple-labeling immunofluorescence method with a confocal laser scanning microscope. Of the oxytocin-labeled cells, 70% were stained for both calbindin D28k and calretinin, 15% were stained for only calbindin D28k, 13% were stained for only calretinin, and 2% were stained for neither protein. Of the vasopressin-labeled cells, 73% were stained for neither calbindin D28k nor calretinin, 21% were stained for only calbindin D28k, 4% were stained for only calretinin, and 2% were stained for both proteins. Calbindin D28k and calretinin have been shown previously to contribute to calcium homeostasis by buffering [Ca(2+)](i). Therefore, these findings suggest that most of the oxytocin neurons may have a higher Ca(2+)-buffering capacity than most of the vasopressin neurons.

Journal Article↗
PubMed1999

Calbindin D28k and calretinin in oxytocin and vasopressin neurons of the rat supraoptic nucleus. A triple-labeling immunofluorescence study.

The aim of the present study was to examine quantitatively whether two calcium-binding proteins, calbindin D28k and calretinin, are localized in oxytocin and vasopressin neurons of the supraoptic nucleus of the male rat. We used a triple-labeling immunofluorescence method with a confocal laser scanning microscope. Of the oxytocin-labeled cells, 70% were stained for both calbindin D28k and calretinin, 15% were stained for only calbindin D28k, 13% were stained for only calretinin, and 2% were stained for neither protein. Of the vasopressin-labeled cells, 73% were stained for neither calbindin D28k nor calretinin, 21% were stained for only calbindin D28k, 4% were stained for only calretinin, and 2% were stained for both proteins. Calbindin D28k and calretinin have been shown previously to contribute to calcium homeostasis by buffering [Ca2+]i. Therefore, these findings suggest that most of the oxytocin neurons may have a higher Ca(2+)-buffering capacity than most of the vasopressin neurons.

Animals↗
PubMed1999

Injection of 6-hydroxydopamine into the ventral tegmental area suppresses the increase in arterial pressure during REM sleep in the rat.

We have examined the effect of injection of 6-hydroxydopamine (6-OHDA) into the ventral tegmental area (VTA) on the changes in arterial blood pressure (AP) and heart rate (HR) during the transition from non-rapid eye movement (NREM) sleep to REM sleep. The 6-OHDA-treated rats showed suppression of the increase of AP and HR during REM sleep and of theta frequency in the cortical electroencephalogram (EEG) during wakefulness (W) and REM sleep. It is suggested that midbrain dopaminergic neurons are involved in the control of AP and HR during REM sleep and in the EEG theta activity.

Analysis of Variance↗
PubMed1998

Monoamine oxidase activity in noradrenaline neurons of the locus coeruleus of the rat. A double-labeling histochemical study.

The aim of the present study is to examine whether noradrenergic neurons of the locus coeruleus (LC) of the rat contain monoamine oxidase (MAO) activity. Sections were processed initially for MAO enzyme histochemistry using tyramine as a substrate, followed by fluorescence immunohistochemistry for tyrosine hydroxylase (TH). In the LC, virtually all TH-immunoreactive neurons (i.e., noradrenergic neurons) were also positive for MAO. No MAO activity was found in any TH-negative neurons. Neurons in the LC have previously been shown to form dopamine during noradrenaline biosynthesis and to produce serotonin from exogenously administered l-5-hydroxytryptophan. Moreover, dopamine- and serotonin-degrading MAO activity has also been found in LC neurons. Therefore, our results indicate that MAO activity is localized within noradrenergic neurons in the LC and is likely involved in the degradation of dopamine that is endogenously synthesized, and also in the elimination of serotonin that is produced from exogenous precursors.

Animals↗
PubMed1998

Dopamine-degrading activity of monoamine oxidase is not detected by histochemistry in neurons of the substantia nigra pars compacta of the rat.

Monoamine oxidase (MAO) activity was examined in neurons of the substantia nigra pars compacta (SNC) of the rat using a histochemical method, and compared to MAO activity in neurons of the locus coeruleus (LC) and dorsal raphe nucleus (DR). Using dopamine as a substrate, dopamine-degrading MAO activity was not detected in any SNC neurons, although LC and DR neurons were intensely stained for this activity. We further examined MAO activity in these neurons using other substrates, including serotonin (an MAO type A preferential substrate), beta-phenylethylamine (an MAO type B preferential substrate), and tyramine (a substrate common to both MAO types A and B). As for dopamine, no SNC neurons were stained for MAO activity using any of these other substrates. In contrast, LC neurons were intensely stained when either serotonin or tyramine was used, and DR neurons were darkly stained when either beta-phenylethylamine or tyramine was used. The lack of evidence of MAO activity in the SNC is surprising given that there are densely packed tyrosine hydroxylase (TH)-immunoreactive neurons in the SNC (i.e., dopaminergic neurons). By comparison, in the LC and DR the distribution patterns of the MAO-stained neurons were similar to those of TH-immunolabeled neurons (i.e., noradrenergic neurons) and serotonin-immunoreactive neurons, respectively. Our results suggest that dopamine-degrading MAO activity and MAO types A and B activities in SNC dopamine neurons are very low compared to MAO activity in LC noradrenaline neurons and in DR serotonin neurons.

Animals↗
PubMed1998

Do all of human midbrain tyrosine hydroxylase neurons synthesize dopamine?

We examined whether all of human midbrain tyrosine hydroxylase (TH) neurons substantially synthesize dopamine (DA) using dual labeling immunohistochemical technique of TH and aromatic L-amino acid decarboxylase (AADC). In the substantia nigra, besides many neurons doubly stained for TH and AADC, neurons stained only for TH and only for AADC (D-neurons [C.B. Jaeger, D.A. Ruggiero, V.R. Albert, T.H. Joh, D.J. Reis, Immunocytochemical localization of aromatic l-amino acid decarboxylase, in: A. Björklund, T. Hökfelt (Eds.), Handbook of Chemical Neuroanatomy, Classical Transmitters in the CNS, Vol. 2, Part 1, Elsevier, Amsterdam, 1984, pp. 387-408.]) were identified. In the ventral tegmental area, dually labeled neurons and TH-only-positive neurons were found. It is indicated that the number of midbrain TH neurons does not reflect the exact number of DA neurons.

Adult↗