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Biomedical subjects

M Fujiwara

Publications and source records attributed to M Fujiwara.

At least 451 records · Page 25Linked to original sources
PubMed1989

A simple and sensitive determination for plasmalogen lysophosphatidylethanolamine in rabbit platelets.

Plasmalogen lysophosphatidylethanolamine (LPE) in rabbit platelets was quantitatively determined as glycero-3-phosphorylethanolamine (GPE) after treatment with 5% trichloroacetic acid at 20 degrees C for two hours. GPE was measured directly using a high performance liquid chromatography with monitoring the fluorescence of o-phthaldialdehyde/2-mercaptoethanol adducts. The assay was sensitive to 50 pmol of plasmalogen LPE and was linear over a 200-fold concentration range. While, 1-acyl LPE and phosphatidylethanolamine from bovine brain (containing about 50% plasmalogen) were hardly cleaved to GPE under the same conditions. These procedures were specific for plasmalogen LPE, and simpler and more sensitive in comparison with conventional analytical methods, e.g., two-dimensional thin layer chromatography with subsequent phosphorus assay. Employing the present method, plasmalogen LPE was found to increase rapidly and to decrease subsequently when rabbit platelets were stimulated by thrombin.

Animals↗
PubMed1989

Asparagine residue in the rho gene product is the modification site for botulinum ADP-ribosyltransferase.

We reported previously that the ADP-ribosyltransferase in C1 and D botulinum toxins specifically catalyzes ADP-ribosylation of an Mr 22,000 guanine nucleotide-binding protein and that this substrate named Gb (b = botulinum) has an amino acid sequence homologous to that deduced from the rho gene (Narumiya, S., Sekine, A., and Fujiwara, M. (1988) J. Biol. Chem. 263, 17255-17257). In this study we have determined the amino acid sequence at its ADP-ribosylation site. Purified substrate was [32P]ADP-ribosylated by C1 botulinum toxin and digested with trypsin. The radioactive peptides were isolated by reversed-phase high performance liquid chromatography and digested further either with protease V8, with proteases V8 and thermolysin, or with proline endopeptidase and thermolysin. By this procedure three radioactive peptides were obtained, and their amino acid sequences were X-Tyr-Val-Ala-Asp-Ile-Glu, X-Tyr, and Val-Phe-Glu-X-Tyr in which no amino acid peak was found in X. During the sequencing the radioactivity quantitatively adhered to the sequencing filter and was not eluted with either of the identified amino acid residues. Analysis of the protein without the ADP-ribosylation yielded the corresponding sequence as Thr-Val-Phe-Glu-Asn-Tyr which corresponds to Thr37-Tyr42 in the amino acid sequence deduced from the Aplysia rho gene. These results strongly suggest that the asparagine residue is the ADP-ribosylation site in the rho gene product. This ADP-ribose protein bond was stable in 0.5 M hydroxylamine at pH 7.5 at 37 degrees C for at least 5 h. The ADP-ribosylation of this protein affected neither its GTPase- nor its [35S]guanosine 5'-O-thiotriphosphate-binding activity.

Adrenal Medulla↗
PubMed1989

Interrelationship between Ca2+-dependent phospholipid base-exchange reaction and phospholipase D-like activity in bovine retina.

Endogenous substrates (phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine) for the Ca2+-dependent base-exchange reaction were investigated using bovine retinal microsomes. The amounts of the three bases, serine, ethanolamine and choline, released from the membranes and the amount of phosphatidic acid generated in the membranes were measured in the presence of Ca2+ with or without exogenous bases. When the membranes were incubated with Ca2+ alone, the three bases were liberated into the water-soluble fractions accompanied by accumulation of phosphatidic acid, suggesting the presence of Ca2+-dependent phospholipase D-like activity. When an exogenous base was added to the reaction mixture, the liberation of the other two bases increased slightly and the formation of phosphatidic acid decreased markedly. The exogenous base also stimulated the liberation of the same base from prelabeled phospholipids. Accompanying these changes, the exogenous base was incorporated into the membrane phospholipid. With respect to pH profile, time course and metal requirements, both the base incorporation and phospholipase D-like activity were quite similar. The amount of base incorporated generally agreed with both the decreased amount of phosphatidic acid formed and the increased amount of base released. These results suggest that, beside the base-exchange reaction, phospholipase D-like activity plays an important role in Ca2+-dependent base incorporation into bovine retinal membranes.

Animals↗
PubMed1989

Effect of in vivo exposure to hypoxia on muscarinic cholinergic receptor-coupled phosphoinositide turnover in the rat brain.

We examined the effects of a transient exposure of rats to severe (5% O2, 95% N2 for 30 min) or mild (10% O2, 90% N2 for 30 min) hypoxia and a chronic exposure to mild hypoxia (10% O2, 90% N2 up to 48 h) on carbachol-stimulated phosphoinositide (PI) turnover and [3H]quinuclidinyl benzilate (QNB) binding in 4 regions of the brain (cerebral cortex, striatum, hippocampus and cerebellum). When the rats were exposed to hypoxia transiently, significant changes were caused only by severe hypoxia. In all 4 regions examined, basal incorporation of [3H]inositol into inositol phosphates (IPs) in the absence of carbachol was significantly increased following transient severe hypoxia and remained high for 48 h. Carbachol stimulation of PI turnover (% of basal) was significantly enhanced only in the hippocampus. The increase in carbachol stimulation in the hippocampus was observed only after a 6-h exposure to room air following the severe hypoxia and lasted for at least 48 h. In Ca2+-free incubation medium, the increase in basal incorporation of [3H]inositol into IPs elicited by severe hypoxia was not observed but carbachol still had an enhanced stimulatory effect. Binding studies showed that maximum binding capacity (Bmax values) of [3H]QNB binding was significantly increased only in the hippocampus and only after a 6-h exposure to room air following the severe hypoxic exposure and remained increased for 48 h. Kd values showed no significant change. While a transient exposure of rats to mild hypoxia caused no significant change either in carbachol-stimulated PI turnover or in [3H]QNB binding parameters, a chronic exposure to mild hypoxia for more than 6 h caused a significant increase in [3H]QNB binding capacity localized in the hippocampus, which was accompanied by an enhanced stimulatory effect of carbachol on PI turnover. These changes observed with mild hypoxia, however, were transient and tended to return to control levels following a 48-h exposure. These results suggest an up-regulation of muscarinic cholinergic receptor-coupled PI turnover in the rat brain caused either by transient severe hypoxia or by chronic mild hypoxia.

Animals↗
PubMed1989

Expression of thromboxane A2 receptor in cultured human erythroleukemia cells and its induction by 12-O-tetradecanoylphorbol-13-acetate.

Using [125I]I-S-145-OH, a radiolabeled derivative of a thromboxane (TX) A2 receptor antagonist, we have studied the expression of the TXA2 receptor in several lines of cultured leukemia cells. Specific binding of the ligand was observed in cells of two human erythroleukemia cell lines, K562 and HEL. However, only negligible binding was seen in HL-60 human promyelocytic leukemia cells and L-1210 murine leukemia cells. Scatchard analyses revealed a curvilinear plot which indicated the existence of two classes of binding sites in these cells. The Kd and Bmax values of the high and low affinity bindings in HEL cells were 2.4 nM and 24 fmol/10(6) cells, and 58 nM and 360 fmol/10(6) cells, respectively. These values in K562 cells were 2.8 nM and 16 fmol/10(6) cells, and 18 nM and 46 fmol/10(6) cells, respectively. The addition of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) to the cultures of K562 and HEL cells caused a concentration- and time-dependent increase in the binding activity. TPA at 10(-8) M increased the Bmax values of both high and low affinity bindings approximately 3 times without significant change in their Kd values and these increases were inhibited by the addition of actinomycin D. Both classes of the binding in cells of each cell line were specifically displaced by several TXA2/prostaglandin (PG) H2 analogues, although the relative specificities to the analogues were different in the two classes. These results suggest that both HEL and K562 cells express the TXA2 receptor on their cell surface and TPA strongly induces this expression in these cells.

Animals↗
PubMed1989

Effects of hypoxia on noradrenaline release and neuronal reuptake in isolated rabbit thoracic aortic strips.

To clarify the effects of hypoxia on stimulus-evoked noradrenaline release and on neuronal reuptake of the released noradrenaline, we examined the effects of hypoxia on contraction responses of rabbit thoracic aortic strips to transmural electrical stimulation and on the stimulation-evoked overflow of total [3H] and [3H]noradrenaline from the strips prelabelled with [3H]noradrenaline. This was done in the presence or absence of an inhibitor of neuronal uptake (cocaine). In a medium equilibrated with a gas mixture of 95% O2/5% CO2 (control), cocaine doubled the stimulation-evoked overflow of total [3H] and [3H]noradrenaline; there was a concomitant increase (130%) in contractions to electrical stimulation. At 0% O2 (95% N2/5% CO2, hypoxia), cocaine had no significant effects on either the stimulation-evoked overflow of total [3H] and [3H]noradrenaline or contractions. In the absence of the drug, hypoxia decreased the stimulation-evoked overflow of total [3H] and [3H]noradrenaline to 47% and 43%, respectively, of the control values, whereas these values were 31% and 28%, respectively, after exposure to cocaine. The inhibition by hypoxia of contraction responses to electrical stimulation was greater in the presence of cocaine than in its absence. These results show that hypoxia inhibits both noradrenaline release evoked by a given stimulus and neuronal uptake.

Animals↗
PubMed1989

Effects of specific alpha-adrenoceptive agents on extraneuronal uptake (uptake2) of isoproterenol in perfused rat heart.

Effects of specific alpha-adrenoceptive agents (alpha 1-agonist, alpha 1-antagonist, alpha 2-agonist and alpha 2-antagonist) on the extraneuronal accumulation of 3H-isoproterenol in the perfused rat heart were examined. The extraneuronal accumulation of 3H-isoproterenol in the hearts perfused with 3H-isoproterenol (10(-6)M) under COMT inhibition by tropolone (10(-4)M) was about 6 times higher than that of intact COMT. The increase in the accumulation by COMT inhibition was regarded as 100% and the effects of specific alpha-adrenoceptive agents on the accumulation was evaluated. alpha 1-agonists, methoxamine and phenylephrine, did not affect the accumulation. alpha 1-antagonists, prazosin, bunazosin and YM-12617, significantly decreased the accumulation of 3H-isoproterenol and these IC50 values were 2 x 10(-6)M, 3.5 x 10(-6)M and 2.3 x 10(-5)M, respectively. alpha 2-agonists, clonidine and guanabenz, significantly reduced the accumulation and these IC50 values were 3.4 x 10(-5)M and 2.9 x 10(-7)M, respectively. The alpha 2-antagonist, yohimbine, did not affect the accumulation. The present experiments clearly demonstrated that the tested alpha 1-antagonists and alpha 2-agonists inhibited uptake2 in rat heart but the tested alpha 1-agonists and an alpha 2-antagonist did not inhibit it.

Adrenergic alpha-Agonists↗
PubMed1989

In vivo changes in brain catecholamine release from rat hypothalamus following olfactory bulbectomy.

The mechanism eliciting mouse-killing behavior (muricide), induced by bilateral olfactory bulbectomy, has been shown to involve the brain noradrenergic system; this is because muricide is specifically inhibited by the drugs which potentiate the activity of catecholaminergic neurons such as tricyclic antidepressants. Our previous reports also demonstrated that the hypothalamic noradrenaline (NA) contents increased in the rats which exhibited muricide. To further examine the hypothalamic noradrenergic function in muricide, a push-pull perfusion technique was applied for direct measurement of NA release from the lateral (LH) and ventromedial (VMH) hypothalamus in freely moving rats. Subsequently, the perfusates, including catecholamines and their metabolites were measured by means of high-performance liquid chromatography with electrochemical detection (HPLC-ECD). Three days after olfactory bulbectomy, 67% of the rats elicited muricide and NA release from LH tended to decrease. Moreover, 7 days after olfactory bulbectomy, most of the rats elicited muricide and NA release from LH was significantly decreased, but not from VMH. On the other hand, dopamine (DA) release from VMH without LH conversely increased on the 7th day after olfactory bulbectomy. These results suggest that the dysfunction of the noradrenergic system caused by the decrease in NA release from LH played an important role for the incidence of muricide.

Animals↗
PubMed1989

Relative importance of the dopaminergic system in haloperidol-catalepsy and the anticataleptic effect of antidepressants and methamphetamine in rats.

The mechanisms involved in haloperidol (HPD)-catalepsy in rat and the effect of antidepressants and methamphetamine (MA) were studied. HPD-catalepsy, as measured by high bar test, lasted for 6-8 min. MA, imipramine (IMP), nomifensine (NOM) and mianserin (MIAN) reduced the duration of catalepsy on IP injection. Electrolytic lesion of the caudate-putamen (CP) and nucleus accumbens (ACC) extensively reduced HPD-catalepsy. Microinjection of MA and NOM into ACC had a similar effect. In the medial amygdala and CP, only MA displayed anticataleptic activity. Zimelidine did not reduce the duration of catalepsy. These results suggest that dopaminergic systems play a key role in mediating HPD-catalepsy and the anticataleptic activity of MA and NOM.

Animals↗
PubMed1989

Delta 9-tetrahydrocannabinol facilitates striatal dopaminergic transmission.

We examined the effects of delta 9-tetrahydrocannabinol (THC) on striatal dopaminergic neurons in rats. THC inhibited the uptake of 3H-dopamine (DA) into striatal synaptosomes. THC facilitated the release of endogenous DA but not dihydroxyphenylacetic acid (DOPAC) from striatal slices. The concentration of DA in the dorsolateral striatum was reduced by THC. We propose that THC may stimulate nigrostriatal dopaminergic neurotransmission mainly by inhibiting uptake of DA and by facilitating release of DA.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1989

Alpha-adrenoceptors in human and animal cerebral arteries: alterations after sympathetic denervation and subarachnoid hemorrhage.

Vasospasm of cerebral arteries in patients with subarachnoid hemorrhage frequently presents severe clinical problems resulting from cerebral ischemia, but the pathogenesis of vasospasm is still poorly understood. The contractile response of human cerebral arteries to noradrenaline is larger than the responses in other species. Neurogenic factors controlling brain circulation may play an important role in pathological conditions such as subarachnoid hemorrhage. Motohatsu Fujiwara and colleagues review species variations of alpha-adrenoceptors in cerebral arteries and their alterations after surgical sympathectomy. They compare these changes to those occurring in human cerebral arteries following subarachnoid hemorrhage and discuss their relationship to vasospasm.

Animals↗
PubMed1989

Activities of dipeptidyl peptidases in BXSB mice and MRL/lpr mice with lupus erythematosus-like syndrome.

Activities of peptidases were examined in tissues of male BXSB and male MRL/Mp-lpr/lpr (MRL/lpr) mice which are animal models of human systemic lupus erythematosus. Female BXSB and male MRL/+ + mice without histopathological changes were used as controls. Activity of DPP II in the spleen, kidney, and liver showed an increase at 13 and 20 weeks of age, while that of DPP IV was decreased at 20 weeks of age, as compared to control mice. The ratio of DPP II/DPP IV activities in the tissues was significantly increased and these findings agree with our previous results in the tissues of NZB mice and in the serum of patients with lupus erythematosus, underscoring the importance of hydrolytic enzymes in the pathogenesis of autoimmune diseases.

Animals↗
PubMed1989

Normal sonographic findings of the infant temporal lobe in coronal sections.

Coronal sonograms of the temporal lobe of the infant brain were obtained using a real time sector scanner with a 5.0 MHz transducer probe. The precise normal anatomy in the coronal sections was determined by correlating with corresponding brain specimens. It was revealed that the spatial arrangement of structures in the medial part of the temporal lobe, i.e. the hippocampal formation and the inferior horn, varied in the three different planes. In the plane through the anterior part of the temporal lobe, these structures were seen as a horizontal lamellar configuration, whereas in the plane through the middle part these structures were depicted as a typical interfolded arrangement. However, the hippocampal formation was barely visible in the plane through the posterior part.

Brain↗
PubMed1989

T-cell clones with L3T4-positive or Lyt-2-positive phenotypes responding to mutant MHC class II antigen and inducing graft versus host reaction.

Two types of T cell clones responding to mutant major histocompatibility class II antigen (Iabm12) were established from spleen cells of C57BL/6 mice: one was L3T4-positive and the other Lyt-2-positive. These two types of clones carried functionally different properties. Lyt-2+ clones were absolutely dependent on exogenous interleukin-2 for their proliferation, whereas some L3T4+ clones secreted interleukin-2 and proliferated autonomously. Both types of clones had cytotoxic activities to bm12 target cells, and Lyt-2+ clones showed stronger activities than L3T4+ clones. Lyt-2+ clones induced induration in situ, whereas the L3T4+ clones induced ulcerative reaction when injected intradermally into mice. Histologically, the L3T4+ clones caused necrosis of the epidermis or upperdermis, while the Lyt-2+ clones induced infiltration of small round cells through the epidermis to the subcutaneous tissues and caused thickening of the epidermis. These characteristic reactivities might be due to a difference in lymphokines produced by each type of T cell subset in response to Iabm12 antigen.

Animals↗
PubMed1989

Responsiveness of T cells to mutant major histocompatibility complex class I antigen. II. Role of stimulator-type accessory cells with reference to interleukin 1 production.

Mechanisms of the activation of T cells responding to major histocompatibility complex (MHC) class I antigen were investigated with special reference to interleukin 1 (IL-1) production from stimulator-type accessory cells. For this purpose, we used mainly fractionated Lyt-2+T cells of C57BL/6 (B6) mice as responder cells and irradiated spleen cells or those deprived of adherent cells of B6.C-H-2bm1 (bm1) mice as stimulator cells. Lyt-2+ T cells of B6 mice proliferated in the presence of irradiated whole spleen cells of bm1 mice but did not to Sephadex G-10 column-passed bm1 spleen cells. The unresponsiveness in the latter case was overcome by the supplement of recombinant IL-1 and/or IL-2 in the culture medium. These interleukins were shown to promote the proliferative response of B6 Lyt-2+ T cells in the presence of stimulator-type T or B cells. Both interleukins also facilitated the generation of cytotoxic T cells from B6 Lyt-2+ cells to H-2Kbm1 antigen in the mixed lymphocyte culture deficient in stimulator-type accessory cells. IL-1 was shown to enhance the expression of IL-2 receptor on the responding Lyt-2+ T cells as assessed by flow cytometry. IL-1 binding to responding T cells were also assayed by means of iodinated IL-1 and was shown to increase significantly on responding Lyt-2+ cells. Overall results indicate that accessory cells might play dual roles in the activation of Lyt-2+ T cells responding to allogeneic MHC class I antigen: direct presentation of the antigen to responder T cells and production of IL-1. Both signals are essentially required for Lyt-2+ T cells responding to allogeneic MHC class I antigen to initiate proliferation and also to differentiate into cytotoxic T cells.

Animals↗