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T Furuno

Publications and source records attributed to T Furuno.

At least 19 recordsLinked to original sources
PubMed2006

The role of phosphatidylinositol 4-kinase type IIalpha in degranulation of RBL-2H3 cells.

OBJECTIVE AND DESIGN: We have studied the role of phosphatidylinositol 4-kinase IIalpha (PI4KIIalpha) in activation of rat basophilic leukemia (RBL-2H3) cells. MATERIALS AND METHODS: Antigen-mediated intracellular Ca(2+) concentration ([Ca(2+)](i)) increase and beta-hexosaminidase secretion were measured using RBL-2H3 cells stably expressing PI4KIIalpha-yellow fluorescent protein (YFP) or its kinase-deficient mutant PI4KIIalpha (K151A)-YFP. RESULTS: Neither PI4KIIalpha-YFP nor PI4KIIalpha (K151A)-YFP were distributed on the plasma membranes but on the exocytotic vesicles. The RBL-2H3 cells stably expressing PI4KIIalpha-YFP showed significantly enhanced beta-hexosaminidase secretion but not an increase in [Ca(2+)](i) after antigen stimulation. The cells with PI4KIIalpha (K151A)-YFP showed no change in the [Ca(2+)](i) increase nor degranulation. The promotion of secretion by PI4KIIalpha-YFP was not observed using co-stimulation with Ca(2+) ionophore and the protein kinase C activator, phorbol myristate acetate. CONCLUSIONS: These results suggest that PI4KIIalpha plays a role in the exocytotic process downstream of Ca(2+) signaling in antigen-mediated mast cell activation.

Animals↗
PubMed2005

Association between arterial stiffness and platelet activation.

Increased arterial stiffness is strongly associated with atherosclerosis, while platelet activation is an important trigger of thrombotic events in patients with atherosclerosis. However, little is known about the effect of arterial stiffness on platelet activation. We therefore investigated the association between arterial stiffness and platelet activation in 38 normal volunteers (20 men and 18 women) aged 23-77 years (mean = 49 +/- 15 years). Arterial stiffness was assessed by measuring brachial-ankle pulse wave velocity (ba-PWV) and heart-brachial PWV (hb-PWV). Flow cytometric analyses were performed to evaluate platelet activation by measuring surface expression of P-selectin and platelet-neutrophil complexes (PNC) before and after activation by ADP. We also calculated the difference between basal and stimulated states of P-selectin and PNC to assess platelet activation reserve. PWVs were significantly correlated with age and BP (r = 0.60-0.81). For platelet activation and activation reserve, correlations with age were less strong but remained significant (r = 0.36-0.61), with the exception of P-selectin (not significant, NS), and correlations with SBP were similar (r = 0.35-0.53). A significant correlation was found between PWVs and platelet activation (r = 0.43-0.74). Multiple regression analysis demonstrated significant correlations between platelet activation and reserve and PWVs (coefficient = 2.17-6.59), when both age and BP were adjusted for simultaneously. In conclusion, platelet activation was associated with arterial stiffness, suggesting that arterial stiffness may play an important role in thrombotic events.

Adult↗
PubMed2004

Association in Japanese patients between neuroleptic malignant syndrome and functional polymorphisms of the dopamine D(2) receptor gene.

A genetic predisposition to the development of neuroleptic malignant syndrome (NMS) has been suggested by clinical studies. Although the molecular basis of NMS is unclear, a dopaminergic blockade mechanism has been considered the main cause. We therefore investigated the association between NMS and three functional polymorphisms of the dopamine D(2) receptor (DRD(2)) gene: TaqI A, -141C Ins/Del, and Ser311Cys. Subjects included 32 Japanese patients, previously diagnosed with NMS, and 132 schizophrenic patients treated with neuroleptics without occurrence of NMS. Polymerase chain reaction and restriction fragment length polymorphism analyses were performed to determine each genotype. We found significant differences in genotypic and allelic frequencies of the -141C Ins/Del polymorphism between patients with and without NMS. The -141C Del allele was significantly more frequent in the NMS group (23.4 vs 11.7%, P=0.026). Similarly, the proportion of -141C Del allele carriers was significantly higher in the NMS group (40.6 vs 20.5%, P=0.022). No significant differences between the two groups were seen for allelic and genotypic frequencies of the TaqI A and Ser311Cys polymorphisms. This result suggests that the -141C Ins/Del polymorphism is likely to predispose toward the development of NMS, probably together with other unidentified factors.

Amino Acid Substitution↗
PubMed2002

Nerve-mast cell (RBL) interaction: RBL membrane ruffling occurs at the contact site with an activated neurite.

Mast cell-neurite interaction serves as a model for neuroimmune interaction. We have shown that neurite-mast cell communication can occur via substance P interacting with neurokinin (NK)-1 receptors on the mucosal mast cell-like cell, the rat basophilic leukemia (RBL) cell. Neurite (murine superior cervical ganglia) and RBL cell [expressing the granule-associated antigen CD63-green fluorescent protein (GFP) conjugate] cocultures were established and stimulated with bradykinin (BK; 10 nM) or scorpion venom (SV; 10 pg/ml), both of which activate only neurites. Cell activation was assessed by confocal imaging of Ca2+ (cells preloaded with fluo 3), and analyses of RBL CD63-GFP+ granule movement were conducted. Neurite activation by BK or SV was followed by RBL Ca2+ mobilization, which was inhibited by an NK-1 receptor antagonist (NK-1 RA). Moreover, membrane ruffling was observed on RBL pseudopodial extensions in contact with the activated neurite, but not on noncontacting pseudopodia. RBL membrane ruffling was inhibited by NK-1 RA, but not NK-2 RA, and was accompanied by a significant increase in granule movement (0.13 +/- 0.04 vs. 0.05 +/- 0.01 microm/s) that was most evident at the point of neurite contact: many of the granules moved toward the plasmalemma. This is the first documentation of such precise (restricted to the membrane's contact site) transfer of information between nerves and mast cells that could allow for very subtle in vivo communication between these two cell types.

Animals↗
PubMed2002

A patient with treatment-resistant schizophrenia and cytochrome P4502D6 gene duplication.

We describe a patient with treatment-resistant schizophrenia who had a duplication in the cytochrome P450IID6 (CYP2D6) gene. This severely ill 71-year-old-woman had responded poorly to several neuroleptics. Molecular genetic study revealed CYP2D6 gene duplication, which results in excessive activity of CYP2D6 that metabolizes various commonly used neuroleptics. The mutation may have contributed to treatment resistance in this case.

Aged↗
PubMed2001

Structural study of DNA duplexes containing the (6-4) photoproduct by fluorescence resonance energy transfer.

Fluorescence resonance energy transfer (FRET) experiments have been performed to elucidate the structural features of oligonucleotide duplexes containing the pyrimidine(6-4)pyrimidone photoproduct, which is one of the major DNA lesions formed at dipyrimidine sites by UV light. Synthetic 32mer duplexes with and without the (6-4) photoproduct were prepared and fluorescein and tetramethylrhodamine were attached, as a donor and an acceptor, respectively, to the aminohexyl linker at the C5 position of thymine in each strand. Steady-state and time-resolved analyses revealed that both the FRET efficiency and the fluorescence lifetime of the duplex containing the (6-4) photoproduct were almost identical to those of the undamaged duplex, while marked differences were observed for a cisplatin-modified duplex, as a model of kinked DNA. Lifetime measurements of a series of duplexes containing the (6-4) photoproduct, in which the fluorescein position was changed systematically, revealed a small unwinding at the damage site, but did not suggest a kinked structure. These results indicate that formation of the (6-4) photoproduct induces only a small change in the DNA structure, in contrast to the large kink at the (6-4) photoproduct site reported in an NMR study.

Cisplatin↗
PubMed2001

Roles of long chain fatty acids and carnitine in mitochondrial membrane permeability transition.

Palmitoyl-CoA (Pal-CoA) lowered the respiratory control ratio (RCR), and induced mitochondrial membrane permeability transition (MPT) and cytochrome c (Cyt. c) release from isolated rat liver mitochondria. L-Carnitine suppressed the Pal-CoA-induced dysfunction, MPT, and Cyt. c release of isolated mitochondria. This suppression was inhibited by cephaloridine, an inhibitor of carnitine uptake into mitochondria. Cyclosporin A (CsA), an inhibitor of MPT, and BSA also suppressed the Pal-CoA-induced MPT. In the presence of inorganic phosphate (P(i)), Ca2+-induced MPT was suppressed by BSA, L-carnitine, and chlorpromazine, an inhibitor of phospholipase A2. In the presence of a low concentration of Ca2+, 3,3',5-triiodothyronine, long chain fatty acids, salicylic acid, and diclofenac induced MPT by a mechanism that was suppressed by BSA, L-carnitine, or chlorpromazine. During the incubation of mitochondria on ice, their respiratory competence decreased; L-carnitine and BSA also prevented this decrease. Mitochondrial depolarization in pheochromocytoma PC12 cells was induced by either serum deprivation or arachidonic acid by a mechanism that was suppressed by acetyl-L-carnitine. These results indicate that some MPTs may be regulated by fatty acid metabolism and that the Pal-CoA-induced MPT plays an important role in the induction of apoptosis.

Animals↗
PubMed2001

Nuclear shuttling of mitogen-activated protein (MAP) kinase (extracellular signal-regulated kinase (ERK) 2) was dynamically controlled by MAP/ERK kinase after antigen stimulation in RBL-2H3 cells.

The mitogen-activated protein kinase (MAPK) cascade consists of the MAPK (extracellular signal-regulated kinase 2; ERK2) and its activator, MAPK kinase (MAP/ERK kinase; MEK). However, the mechanisms for activation of ERK2 have not been defined yet in cells. Here, we used fluorescent protein-tagged ERK2 and MEK to examine the localization of ERK2 and MEK in living rat basophilic leukemia (RBL-2H3) cells. ERK2 was mainly in the cytoplasm in resting cells but translocated into the nucleus after the ligation of IgE receptors. The import of ERK2 reached the maximum at 6--7 min, and then the imported ERK2 was exported from the nucleus. MEK mainly resided in the cytoplasm, and no significant MEK translocation was detected statically after ligation of IgE receptors. However, analysis of the dynamics of ERK2 and MEK suggested that both of them rapidly shuttle between the cytoplasm and the nucleus and that MEK regulates the nuclear shuttling of ERK2, whereas MEK remains mainly in the cytoplasm. In addition, the data suggested that the sustained calcium increase was required for the optimal translocation of ERK2 into the nucleus in RBL-2H3 cells. These results gave a new insight of the dynamics of ERK2 and MEK in the nuclear shuttling of RBL-2H3 cells after the ligation of IgE receptors.

Active Transport, Cell Nucleus↗
PubMed2001

No evidence of an association between CYP2D6 polymorphisms among Japanese and dementia with Lewy bodies.

Dementia with Lewy bodies (DLB) is the second most frequent degenerative dementia among the elderly, following Alzheimer-type dementia (ATD). An association of DLB with CYP2D6*4, one of the cytochrome P450IID6 (debrisoquine 4-hydroxylase; CYP2D6) gene polymorphisms, was reported previously, but this is controversial. Moreover, these reports have been restricted to Caucasian populations. Therefore, we compared frequencies of CYP2D6*3, *4, and *10 mutant alleles in 17 Japanese DLB patients to those among Alzheimer-type dementia (ATD) patients and healthy controls. Polymerase chain reaction amplification and restriction fragment length polymorphism analyses were used for genotyping. No significant difference of genotype or mutant allele frequencies was detected between DLB, ATD, and healthy controls. The present results do not support the suggestion that the CYP2D6 gene is related to DLB susceptibility, at least in the Japanese population.

Aged↗
PubMed2001

Dynamics of intracellular granules with CD63-GFP in rat basophilic leukemia cells.

CD63 is located on the basophilic granule membranes in resting basophils, mast cells, and platelets, and is also located on the plasma membranes of the cells. We constructed a CD63-GFP (green fluorescent protein) plasmid and introduced it into rat basophilic leukemia (RBL-2H3) cells to observe the movements of CD63 on degranulation. The movements of CD63-GFP were studied in living RBL cells by confocal laser scanning microscopy (CLSM). CD63-GFP, in which GFP was conjugated to the C-terminus of CD63, was located on both the granule membranes and the plasma membranes of RBL cells. The diameter of the fluorescent granules in the cytoplasm varied from 0.5 to 1.5 microm. Before antigen stimulation most granules with CD63-GFP hardly moved in RBL cells. However, after antigen stimulation the plasma membranes ruffled violently and the granules moved dramatically. They reached the plasma membranes in a few minutes and fused with them instantaneously. Analysis of the movement of each granule provided a new insight into the elementary process of degranulation. The velocity of the granule movement toward the plasma membranes on antigen stimulation was calculated to be 0.1+/-0.02 microm/s. This shows that the granules are able to reach the plasma membranes in 2 or 3 min if the diameter of the cells is 20 microm.

Animals↗
PubMed2001

Calcium signals in rat basophilic leukemia (RBL-2H3) cells primed with the neuropeptide substance P.

Communication between nerves and mast cells is a prototypic demonstration of neuroimmune interaction. We have recently shown that direct nerve-mast cell cross-talk can occur in the absence of an intermediary transducing cell and that the neuropeptide substance P is an important mediator of this communication. Here we study the calcium signals in rat basophilic leukemia cells (RBL-2H3; mucosal-type mast cells) primed with substance P. RBL cells responded only slightly to stimulation with compound 48/80, however they responded to the stimulation when the cells had been primed with substance P (0.5 microM) for one week. The present results provide a foundation to study the neuroimmune cross-talk in a co-culture system.

Animals↗
PubMed2001

Bi-directional relationship of in vitro mast cell-nerve communication observed by confocal laser scanning microscopy.

Communication between nerves and mast cells is a prototypic demonstration of neuro-immune interaction. Recently, we used an in vitro co-culture approach comprising cultured murine superior cervical ganglia (SCG) and rat basophilic leukemia (RBL) cells to study this interaction. Previously, we concentrated mainly on the activation signal from neurites to mast cells (RBL). However, it is proposed that mast cell-nerve communication is not a one-sided relationship but a bi-directional one. In the present work, we studied the communication from mast cells to neurites. We observed that binding of anti-IgE receptor antibodies to mast cells increases calcium ion concentration [Ca2+]i in SCG neurites. This indicates that mast cell-nerve communication is bi-directional. Confocal fluorescence microscopic images indicated that [Ca2+]i in neurites increased after an increase of [Ca2+]i in mast cells. The lag-time of neurite activation was several times longer than that of mast cell activation. The correlation coefficient between the lag-times for mast cell and nerve activation was calculated to be 0.81. In addition, the fluorescence images showed that calcium signals in SCG neurites were able to extend to a long distance (100-200 microm) from the site where mast cells (RBL) attached to neurites.

Animals↗
PubMed2001

Role of calpain in human sperm activated by progesterone for fertilization.

We evaluated the activation of mu-calpain in progesterone-activated human sperm. Semen collected from fertile donors with informed consent was liquefied and subjected to percoll gradient centrifugation. After exposure to different concentrations of progesterone, the samples were used for immunostaining, SDS-PAGE and Western blot analysis. An increase of the intracellular free calcium concentration in the sperm following the addition of progesterone was observed using fura-2 AM. Immunostaining using an antibody against active mu-calpain produced 6 distinct staining patterns: (1) the acrosome, (2) an equatorial segment, (3) the whole head, (4) the neck, (5) the neck and tail or (6) unstained sperm. After addition of progesterone, the predominant type changed from the neck type (90%) to the neck and tail type (79%). Western blot analysis using a pro-mu-calpain and a mu-calpain domain III antibody revealed autodigestion of mu-calpain, indicating activation by progesterone. Using calpain-specific inhibitors it was shown that calpain activation contributes to sperm motility as well as to the acrosome reaction. These results suggest the possibility that activation of mu-calpain in human sperm by progesterone plays an important role in fertilization.

Blotting, Western↗
PubMed2000

Atomic force microscopy to study direct neurite-mast cell (RBL) communication in vitro.

Communication between nerves and mast cells is a prototypic demonstration of neuroimmune interaction. We used an in vitro co-culture approach comprising cultured murine superior cervical ganglia (SCG) and rat basophilic leukemia (RBL-2H3) cells. Atomic force microscopy (AFM) showed how neurites attached to a pseudopodium or a cell body of an RBL cell. After stimulation of SCG neurites with bradykinin or scorpion venom, RBL cells attached to neurites spread and flattened, and several discharged granules (0. 5-1.0 microm in diameter) were found on the surface of the RBL cells. A neurokinin (NK)-1 receptor (i.e. substance P receptor) antagonist prevented the RBL degranulation. The results showed that activation of the SCG neurites with bradykinin or scorpion venom was able to elicit degranulation in RBL cells which were attached to neurites.

Animals↗
PubMed2000

Hormone binding induces rapid proteasome-mediated degradation of thyroid hormone receptors.

The thyroid hormone 3,3',5-triiodo-l-thyronine (T3) is essential for growth, differentiation, and development. Its biological activities are mediated by T3 nuclear receptors (TRs). At present, how T3 regulates TR proteins and the resulting functional consequences are still unknown. Immunofluorescence analyses of endogenous TR in the growth hormone-producing GC cells showed that the T3-induced rapid degradation of TR was specifically blocked by lactacystin, a selective inhibitor of the ubiquitin-proteasome degradation pathway. Immunoblots demonstrated that the transfected TRbeta1 was ubiquitinated and that the ubiquitination was T3 independent. Studies with a series of truncated TRbeta1 showed that the hormone-binding domain was sufficient for the T3-induced rapid degradation of TRbeta1 by the proteasome degradation pathway. T3 also induced rapid degradation of TRbeta2 and TRalpha1. In contrast, the stability of the non-T3-binding TRalpha2 and naturally occurring TRbeta1 mutants that do not bind T3 was not affected by T3 treatment, indicating that hormone binding to receptor was essential for the degradation of the wild-type receptors. In the presence of proteasome protease inhibitors, the levels of both total and ubiquitinated TRbeta1 protein increased, yet T3-dependent transcriptional activation and the expression of the growth hormone gene were diminished, suggesting that proteasome-mediated degradation played a novel role in modulating transcriptional activation by TR. The present study reveals a role of T3 in modulating the functions of TR by regulating its receptor level via the ubiquitin-proteasome degradation pathway.

Animals↗
PubMed2000

Effect of angiotensin-converting enzyme inhibitor (enalapril or imidapril) on ventilation during exercise in patients with chronic heart failure secondary to idiopathic dilated cardiomyopathy.

Eighteen patients with heart failure were studied to clarify whether angiotensin-converting enzyme inhibitor treatment improves excess ventilation during exercise. Treatment with angiotensin-converting enzyme inhibitors had a beneficial effect on excess ventilation during exercise, without significant improvement in exercise capacity in patients with moderate congestive heart failure.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed2000

Lack of association in Japanese patients between neuroleptic malignant syndrome and a debrisoquine 4-hydroxylase genotype with low enzyme activity.

Decreased activity of debrisoquine 4-hydroxylase (CYP2D6), which participates in hepatic metabolism of several frequently used neuroleptics and antidepressants, is inherited as an autosomal recessive trait through polymorphic CYP2D6 gene alleles. In eastern Orientals, a C --> T substitution at nucleotide 188 (Pro34Ser) is primarily responsible for decreased ability to metabolize CYP2D6 substrates. We therefore studied a possible association between neuroleptic malignant syndrome (NMS) and the C188T mutation. We examined the frequency of the C188T mutation by polymerase chain reaction and restriction fragment length polymorphism analysis in 36 Japanese patients previously diagnosed with NMS and 107 neuroleptic-treated schizophrenic patients with no NMS history. The C188T allele frequency was 0.417 in NMS patients and 0.463 in patients without NMS. No significant allele or genotype associations were observed. We cannot conclude that low CYP2D6 activity genotype causes susceptibility to NMS in Japanese patients.

Adult↗