Biomedical subjects

Mamoru Nakanishi

Publications and source records attributed to Mamoru Nakanishi.

At least 19 recordsLinked to original sources

PubMed2007

Characterization of biosurfactant-containing liposomes and their efficiency for gene transfection.

Recently we showed significance of biosurfactants in the field of non-viral vectors for gene transfection. There, a biosurfactant, mannosylerythritol lipid A (MEL-A), especially increased the efficiency of gene transfection mediated with cationic liposomes. However, the molecular mechanism has not been well-understood yet. Here, through the examination of the ability of cationic liposomes containing an MEL (MEL-A, MEL-B or MEL-C) for important transfectional processes of the DNA capsulation and the membrane fusion with anionic liposomes, we found that MEL-A-containing liposomes increased both processes, but that MEL-B and MEL-C-containing liposomes just increased either of them. The results indicated that these kinds of the physicochemical properties in MEL-A-containing liposomes are able to increase the efficiency of liposome-mediated gene transfection.

Anions↗

PubMed2006

Effects of Cot expression on the nuclear translocation of NF-kappaB in RBL-2H3 cells.

Cot is a serine/threonine protein kinase and is classified as a mitogen-activated protein (MAP) kinase kinase kinase. Overexpression of this protein has been shown to activate the extracellular signal-regulated kinase, the c-Jun N-terminal kinase, and the p38 MAP kinase pathways and to stimulate NF-AT and NF-kappaB-dependent transcription. Here we have shown that Cot kinase activity is intimately involved in the high affinity receptor for IgE (FcvarepsilonRI)-mediated nuclear translocation of NF-kappaB1 independent of NF-kappaB-inducing kinase (NIK) in rat basophilic leukemia (RBL-2H3) cells. A transfected green fluorescent protein-tagged NF-kappaB1 (GFP-NF-kappaB1) resided in the cytoplasm in RBL-2H3 cells and it remained in the cytoplasm even when Cot tagged with red fluorescent protein (Cot-RFP) was co-expressed. Western blotting analysis showed that IkappaB kinases (IKKs) were expressed in RBL-2H3 cells but NIK was not. GFP-NF-kappaB1 translocated from the cytoplasm to the nucleus after the aggregation of FcvarepsilonRI in Cot-transfected cells but not in kinase-deficient Cot-transfected cells. This finding gives a new insight into the role of Cot in the FcvarepsilonRI-mediated NF-kappaB activation in mast cells.

Active Transport, Cell Nucleus↗

PubMed2006

Flotillin-1 regulates IgE receptor-mediated signaling in rat basophilic leukemia (RBL-2H3) cells.

Cross-linking of high-affinity IgE receptors by multivalent Ag on mast cells (rat basophilic leukemia (RBL)-2H3) induces the phosphorylation of ITAM motifs of an IgE receptor by Src family tyrosine kinase, Lyn. The phosphorylation of IgE receptors is followed by a series of intracellular signals, such as Ca(2+) mobilization, MAPK activation, and degranulation. Therefore, Lyn is a key molecule in the activation of mast cells, but the molecular mechanisms for the activation of Lyn are still unclear. Recently, it is suggested that the localization of Lyn in lipid rafts is critical for its activation in several cell lines, although the precise mechanism is still unknown. In this study, we found that flotillin-1, which is localized in lipid rafts, is involved in the process of Lyn activation. We obtained flotillin-1 knockdown (KD)(2) rat basophilic leukemia (RBL)-2H3 cells, which express a low level of flotillin-1. In the flotillin-1 KD cells, we observed a significant decrease in Ca(2+) mobilization, the phosphorylation of ERKs, tyrosine phosphorylation of the gamma-subunit of IgE receptor, and IgE receptor-mediated degranulation. We also found that flotillin-1 is constitutively associated with Lyn in lipid rafts in RBL-2H3 cells, and Ag stimulation induced the augmentation of flotillin-1 binding to Lyn, resulting in enhancement of kinase activity of Lyn. These results suggest that flotillin-1 is an essential molecule in IgE receptor-mediated mast cell activation, and regulates the kinase activity of Lyn in lipid rafts.

Animals↗

PubMed2006

The plasma membrane shuttling of CAPRI is related to regulation of mast cell activation.

The Ca(2+)-promoted Ras inactivator (CAPRI), a Ras GTPase-activating protein, is involved in the inactivation of mitogen-activated protein kinase pathway. However, a precise role of CAPRI in immune responses is still unknown. Here we showed that overexpression of CAPRI suppresses antigen-induced degranulation and cytokine production in mast cells (RBL cells). Antigen elicited the translocation of CAPRI to the plasma membrane from the cytoplasm, which was concomitant with the increase in the intracellular Ca(2+) concentration. The nuclear import of extracellular signal-regulated kinase 2 (ERK2) occurred after the re-localization of CAPRI to the cytoplasm in the mast cells, suggesting that the early phase of ERK2 activation is eliminated. A mutant of GAP-related domain, CAPRI(R472S), showed a feeble translocation to the plasma membrane but did not affect the degranulation, ERK2 activation, and cytokine production. The results suggested that the translocation of CAPRI to the plasma membranes regulates crucially cellular responses in mast cells.

Animals↗

PubMed2006

Conditioned medium from feeder STO cells increases the attachment of mouse embryonic stem cells.

Mouse embryonic stem (ES) cell lines, which were established by culturing on feeder cells, have usually been cultured without feeder cells in the presence of leukemia inhibitory factor. However, proliferating rate of ES cells in the condition is often lower than that with feeder cells. Here, we found that conditioned medium (CM) from feeder cells (STO cells) increased the number of undifferentiated cells in a culture dish by promoting attachment of ES cells. The attached cells were increased in 4 h after replating ES cells in the presence of CM from STO cells, and they formed flat colonies composed of undifferentiated cells. This culture system with CM from feeder cells is useful in preparing a large number of well-defined ES cells. In addition, from present experiments we found that the dish double-coated with gelatin and CM was extremely useful for culturing ES cells.

Animals↗

PubMed2006

Effects of 2-methyl-1,4-naphtoquinone (menadione) on cellular signaling in RBL-2H3 cells.

Reactive oxygen species (ROS) are produced in various cells and affect many biologic processes. In this study, we examined the effects of 2-methyl-1,4-naphtoquinone (menadione; vitamin K3) on signal transduction in mast cells. Several lines of evidence suggest that H2O2 affects the antigen-induced responses in mast cells but its mechanism is not clearly understood. Unlike H2O2, menadione produces ROS only inside cells. Thus, it is possible to investigate the effects of ROS produced intracellularly. Pretreatment of mast cells (RBL-2H3) with menadione inhibited exocytotic secretion (degranulation) induced by antigen stimulation dose dependently. Menadione also inhibited the intracellular Ca2+ increase induced by antigen stimulation. Menadione did not inhibit the Ca2+ increase due to Ca2+ release from the intracellular calcium store in the absence of extracellular Ca2+, but inhibited the Ca2+ influx from the extracellular medium. These results suggest that reactive oxygen generated inside RBL cells by menadione inhibited degranulation by decreasing Ca2+ influx through the store operated Ca2+ channel on the plasma membrane.

Animals↗

PubMed2006

[Functional fluctuation].

The discovery of the double-helical structure of DNA, the elucidation of the genetic code, and the determination of the three-dimensional structure of several proteins are some of the outstanding achievements of biochemistry and life sciences in the latter half of the last century. Proteins play key roles in almost all the biological processes and the biological function of a protein depends on its conformation which is defined as the three-dimensional arrangement of the atoms of a molecule. The three-dimensional structure, however, is not rigid but fluctuated. Structural fluctuation plays an important role in bio-macromolecules. How about "functional fluctuation" in biological systems? The present review proposes that functional fluctuation is also very important for understanding the mechanism of supramolecules, biological processes in living cells, and the interaction between biological systems. This new theme is pretty well supported by our recent experiments for neuro-immune crosstalk, gene transfection with cationic liposomes, and cell signaling in embryonic stem cells.

Animals↗

PubMed2006

Comparison of HER2 gene amplification assessed by fluorescence in situ hybridization and HER2 protein expression assessed by immunohistochemistry in gastric cancer.

A monoclonal antibody to HER2 protein is widely used in the treatment of patients with HER2-overexpressing breast cancer and has also been found to exhibit antitumor activity in human gastric cancer cells that overexpress HER2. The purpose of this study was to evaluate the frequency of HER2 overexpression and concordance between the results for protein expression and gene amplification in both surgical and biopsy specimens of gastric cancer as assessed with two commercial kits, one for immunohistochemistry (IHC) and the other for fluorescence in situ hybridization (FISH). The specimens consisted of formalin-fixed, paraffin-embedded sections of biopsy specimens and surgically resected tumors from 200 cases of invasive gastric cancer that had been treated surgically at the National Cancer Center Hospital East. The lesions were analyzed with the IHC kit, and expression was graded by the United States Food and Drug Administration (FDA)-approved grading system. Gene amplification was evaluated by FISH. IHC revealed HER2 overexpression in 46 of the 200 (23%) cases. The FISH assay was technically successful in 199 cases (99.5%), and gene amplification was observed in 54 cases (27.1%). The concordance rate between the results obtained by IHC and FISH was 86.9%. The concordance rate between the findings in the surgically resected tumors and the 200 pre-treatment biopsy specimens was 88.7%. HER2 expression can be assessed in gastric cancer with a commercial kit as previously reported in breast cancer. Even small biopsy specimens were found to be suitable for evaluating gastric cancer for HER2 overexpression.

Adult↗

PubMed2005

Neurotrophic factors increase tumor necrosis factor-alpha-induced nuclear translocation of NF-kappaB in rat PC12 cells.

Neurotrophic factors regulate neuronal survival and differentiation and control neurite outgrowth by binding to tyrosine kinase receptors, the Trks, and a tumor necrosis factor (TNF) receptor-like molecule, p75 neurotrophin receptor. A proinflammatory cytokine, TNF, also affects survival and apoptotic death in neuronal cells. However, it is still unclear whether neurotrophic factors and TNF co-operate the intracellular signaling. Using green fluorescent protein-tagged NF-kappaB1 (GFP-NF-kappaB1), we examined here the effects of TNF-alpha and neurotrophic factors on the nuclear translocation of NF-kappaB in PC12 cells. TNF-alpha induced gradually the translocation of GFP-NF-kappaB1 from the cytoplasm to the nucleus within 60 min. Pretreatment of lactacystin which is a proteasome-specific inhibitor suppressed significantly the nuclear translocation of GFP-NF-kappaB1 after TNF-alpha stimulation. In addition, we found that co-stimulation of TNF-alpha and neurotrophic factors such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) increased greatly the nuclear translocation of GFP-NF-kappaB1 whereas neither NGF nor BDNF itself induced the translocation. These results suggested that there is a close correlation between the signaling pathways via TNF receptors and neurotrophin receptors for the NF-kappaB activation, and that NGF and BDNF enhance TNF-alpha-induced nuclear translocation of NF-kappaB.

Animals↗

PubMed2005

The spermatogenic Ig superfamily/synaptic cell adhesion molecule mast-cell adhesion molecule promotes interaction with nerves.

Nerve-mast cell interaction is involved in both homeostatic and pathologic regulations. The molecules that sustain this association have not been identified. Because synaptic cell adhesion molecule (SynCAM), alternatively named spermatogenic Ig superfamily (SgIGSF), is expressed on both nerves and mast cells and because it binds homophilically, this molecule may be a candidate. To examine this possibility, mast cells with or without SgIGSF/SynCAM were cocultured with superior cervical ganglion neurons that express SgIGSF/SynCAM, and the number of mast cells attached to neurites was counted. The attachment of mast cells with SgIGSF/SynCAM, i.e., bone marrow-derived mast cells (BMMC) from wild-type mice, was inhibited dose-dependently by blocking Ab to SgIGSF/SynCAM. Mast cells without SgIGSF/SynCAM, i.e., BMMC from microphthalmia transcription factor-deficient mice and BMMC-derived cell line IC-2 cells, were defective in attachment to neurite, and transfection with SgIGSF/SynCAM normalized this. When the nerves were specifically activated by scorpion venom, one-quarter of the attached IC-2 cells mobilized Ca(2+) after a few dozen seconds, and ectopic SgIGSF/SynCAM doubled this proportion. At points of contact between neurites and wild-type BMMC, SgIGSF/SynCAM was locally concentrated in both neurites and BMMC. SgIGSF/SynCAM on mast cells appeared to predominantly mediate attachment and promote communication with nerves.

Animals↗

PubMed2005

Complexin II facilitates exocytotic release in mast cells by enhancing Ca2+ sensitivity of the fusion process.

Recent studies have shown that soluble N-ethyl maleimide-sensitive factor attachment protein receptor (SNARE) proteins are involved in exocytotic release in mast cells as in neurotransmitter release. However, the roles of the proteins that regulate the structure and activity of SNARE proteins are poorly understood. Complexin is one such regulatory protein and is involved in neurotransmitter release, although ideas about its role are still controversial. In this study, we investigated the expression and role of complexin in the regulation of exocytotic release (degranulation) in mast cells. We found that complexin II, but not complexin I, is expressed in mast cells. We obtained RBL-2H3 cells that expressed a low level of complexin II and found that antigen-induced degranulation was suppressed in these cells. No significant changes in the Ca2+ response or expression levels of syntaxins and synaptotagmin were observed in knockdown cells. An immunocytochemical study revealed that complexin II was distributed throughout the cytoplasm before antigen stimulation. However, the distribution of complexin II changed dramatically with stimulation and it became localized on the plasma membrane. This change in the intracellular distribution was observed even in the absence of extracellular Ca2+, while exocytotic release was inhibited almost completely under this condition. The degranulation induced by phorbol 12-myristate 13-acetate and A23187 depended on the extracellular Ca2+ concentration, and its sensitivity to Ca2+ was decreased in knockdown cells. These results suggest that complexin II regulates exocytosis positively by translocating to the plasma membrane and enhancing the Ca2+ sensitivity of fusion machinery, although this translocation to the plasma membrane is not sufficient to trigger exocytotic membrane fusion.

Adaptor Proteins, Vesicular Transport↗

PubMed2005

Live cell imaging to study signaling molecules in allergic reactions.

Mast cells are widely distributed throughout the body, predominantly near blood vessels and nerves, and express effector functions in allergic reactions, inflammatory diseases, and host defense. The activation of mast cells results in secretion of the preformed chemical mediators in their granules by a regulated process of exocytosis and leads to synthesis and secretion of lipid mediators and cytokines. Their soluble factors contribute to allergic inflammation. Mast cells are associated with hypersensitivity reactions, not only in the classical immunoglobulin E (IgE)-dependent mechanism but also in an IgE-independent manner. In particular, investigations of potential anatomical and functional interactions between mast cells and the nervous system have recently attracted great interest. To understand these molecular mechanisms in mast cell activation, the ability to visualize, track, and quantify molecules and events in living mast cells is an essential and powerful tool. Recent dramatic advances in imaging technology and labeling techniques have enabled us to carry out these tasks with high spatiotemporal resolution using confocal laser scanning microscopes, green fluorescent protein and its derivatives, and image analysis systems. Here we review our investigations of the dynamic processes of intracellular signaling molecules, cellular structure, and interactions with neurons in mast cells to provide basic and valuable information for allergy and clinical immunology using these new imaging methods.

Animals↗

PubMed2005

Calcium response and FcepsilonRI expression in bone marrow-derived mast cells co-cultured with SCG neurites.

Communication between nerves and mast cells is a prototypic demonstration of neuro-immune interaction. Numerous studies have shown that the stimulation of nerves (or addition of neurotransmitters) can evoke activation of mast cells, and that mast cell-derived mediators can influence neuronal activity. However, it is still unknown whether high affinity IgE receptors (FcepsilonRI) themselves are involved directly in the communication between nerves and mast cells. In the present experiments, we used an in vitro co-culture approach comprising interaction between immune (bone marrow-derived mast cells, BMMCs) and nerve cells (superior cervical ganglia, SCG) to solve the above problem. We found that the intracellular calcium ion concentration ([Ca2+]i) increased much more in BMMCs after antigen (DNP7-BSA) stimulation when they were associated with SCG neurites in the co-culture system. But the [Ca2+]i in BMMCs was less increased when they were not associated with the neurites. Further, the in vitro co-culture approach of BMMCs with SCG neurites for 3 d showed the increases of FcepsilonRI expression occurred on the plasma membranes of BMMCs which were attached to the neurites. On the contrary, N-cadherin molecules which localized on the interface between on the plasma membrane of BMMCs and SCG neurites did not increase with the co-culture for 3 d. All of these results indicated that co-culturing BMMCs with SCG neurites for 3 d promoted not only the calcium response but also the FcepsilonRI expression in BMMCs.

Animals↗

PubMed2004

Bone marrow-derived mast cells in mice respond in co-culture to scorpion venom activation of superior cervical ganglion neurites according to level of expression of NK-1 receptors.

In virtually all tissues of the body, mast cells are closely associated with nerve fibers, mostly of sensory origin. While mast cells can be activated by substance P, evidence for the involvement of NK-1 receptors is very limited. To study functional interactions between mast cells and peripheral nerves, bone marrow-derived mast cells (BMMC) and superior cervical ganglia (SCG) were co-cultured. Murine bone marrow-derived mast cells are homologues for mucosal mast cells and have recently been shown to express NK-1 receptors. Bi-directional interaction was studied using a fluorescent calcium indicator as an index of cellular activation. Scorpion venom, not affecting BMMC by itself, caused a rapid increase in neurite fluorescence subsequently followed by activation of the mast cell. The latter was inhibited by the NK-1 receptor antagonist SR140333, showing the direct involvement of substance P and its receptor in this co-culture system. Activation of BMMC seemed to be directly correlated with extent of NK-1 receptor expression. Immature c-kit positive cells not expressing NK-1 gave a negligible response to neurite activation. In addition, there was a maximum stimulation occurring when NK-1 expression exceeded 16% on BMMC after cytokine stimulation. Our findings show that the expression of NK-1 receptors appears to be important for nerve-mast cell communication.

Animals↗

PubMed2004

Biosurfactant MEL-A dramatically increases gene transfection via membrane fusion.

Biosurfactants, which are surface-active compounds produced by microorganisms growing on water-insoluble substrates, have many biological activities. We studied here three different biosurfactants, mannosylerythritol lipid (MEL) or 4-O-[(4',6'-di-O-acetyl-2',3'-di-O-alkanoyl)-beta-D-mannopyranosyl] meso-erythritol (MEL-A), 4-O-[(6'-O-acetyl-2',3'-di-O-alkanoyl)-beta-D-mannopyranosyl] meso-erythritol (MEL-B) and 4-O-[(4'-O-acetyl-2',3'-di-O-alkanoyl)-beta-D-mannopyranosyl] meso-erythritol (MEL-C). MEL-A enhanced the efficiency of gene transfection by cationic liposomes, but MEL-B and MEL-C did not. We also studied the localization of FITC-conjugated antisense DNAs (15-mer oligonucleotides; phosphorothioate) in the target cells by confocal laser scanning microscopy (CLSM). The FITC-conjugated antisense oligonucleotides were temporarily on the plasma membrane of the target cells, thereafter they were transferred into the nucleus of the target cells. In the case of MEL-B and MEL-C, such localization of DNA was not observed both in the plasma membrane and in the nucleus. The results obtained by CLSM images were in good agreement with the transfection efficiency. This suggests that MEL-A induces the membrane fusion between the target cells and the cationic liposomes, accelerating the efficiency of gene transfection dramatically.

Animals↗

PubMed2004

N-cadherin plays a role in the synapse-like structures between mast cells and neurites.

Animals↗

PubMed2004

Relationships of nutrient intake and lifestyle-related factors to serum folate and plasma homocysteine concentrations in 30-69 year-old Japanese.

We studied non-hospitalized 30-69 y-old Japanese subjects to ascertain the influences of a 677C-T methylene-tetrahydrofolate reductase (MTHFR) genotype, nutritional intake and lifestyle-related factors on plasma homocysteine (Hcys) and serum folate concentrations. Hcys was higher and serum folate was lower in males than in females (p < .01). The Hcys concentration was higher in the VV group than in the AA and AV groups for both males and females. However, a relatively low serum folate concentration of 18 +/- 7 nmol/L was found in the entire male group as compared with 22 +/- 10 nmol/L in all females. In the female subjects, serum folate concentrations differed among MTHFR genotypes, being lowest in the VV group. In all male subjects, log folate intake per 1,000 kcal was a significant positive predictor of log serum folate concentration (p < 0.01), while in females the log vitamin C intake per standard body weight was a significant positive variable (p < 0.001) predicting the log serum folate concentration. Smokers had significantly lower serum folate concentrations, regardless of dietary folate intake. High folate and vitamin C consumptions, appears to be beneficial to normal and heterozygous MTHFR genotype subjects for maintaining serum folate concentrations. Even a 400 microg daily intake of folate might be less than what is needed, especially for homozygous MTHFR subjects and smokers, to maintain an adequate serum folate concentration.

Adult↗

PubMed2003

The effects of ITIM-bearing FcgammaRIIB on the nuclear shuttling of MAP kinase in RBL-2H3 cells.

We have studied the effects of ITIM-bearing FcgammaRIIB2 on the FcepsilonRI-dependent nuclear shuttling of mitogen-activated protein (MAP) kinase (ERK2) in rat basophilic leukemia (RBL-2H3) cells. The cross-linking of FcepsilonRI elicited the sustained increase of the intracellular calcium ion concentration ([Ca(2+)](i)) and the translocation of ERK2 from the cytoplasm to the nucleus. The import of ERK2 to the nucleus reached the maximum at 6-7 min, thereafter ERK2 was exported within 30 min. The co-clustering of FcepsilonRI and FcgammaRIIB2 increased the [Ca(2+)](i) and induced the import of ERK2. However, the calcium increase was transient and ERK2 was rapidly exported to the cytoplasm. In addition, the phosphorylation of ERK2 and the production of TNF-alpha were decreased in case of co-clustering of FcepsilonRI and FcgammaRIIB2. This suggested that the co-clustering negatively control the production of pro-inflammatory cytokines through the suppression of nuclear shuttling of ERK2.

Animals↗