Tadahide Furuno

Membrane fusion plays an important role in gene transfection mediated by cationic liposomes

By confocal laser scanning microscopy (CLSM) we have studied the membrane fusion between cationic liposomes and the endosome membranes involved in gene transfection mediated by cationic liposomes. Antisense oligonucleotides were transferred by cationic liposomes with a cationic cholesterol derivative, cholesteryl‐3β‐carboxyamidoethylenedimethylamine (I). Cationic liposomes were made by a mixture of the derivative I and DOPE. The intracellular distribution of fluorescein‐conjugated antisense oligonucleotides (phosphorothioate) was studied by CLSM. The images showed that the antisense oligonucleotides were preferentially transferred into the nucleus of target cells (NIH3T3, COS‐7 and HeLa cells) by the liposomes with derivative I. However, their transfection was completely blocked by nigericin which was able to dissipate the pH gradient across the endosome membranes, although the liposome/DNA complex was found in the cytoplasm of the target cells. This was quite in contrast with the fluorescence images of the target cells treated with wortmannin, an inhibitor of endocytosis. The results suggest that at least two steps are effective for gene transfection mediated by the cationic liposomes with cationic cholesterol derivatives. One is the endocytosis of the liposome/DNA complex into the target cells and the other is the removal of antisense oligonucleotides (plasmid DNAs) from the complex in the endosomes. The latter step was preferentially preceded by the membrane fusion between the cationic liposomes and the endosome membranes at around pH 5.0.

Effect of zeta potential of cationic liposomes containing cationic cholesterol derivatives on gene transfection

Cationic liposomes are known to be useful tools for gene transfection. However, the relation between transfection efficiency and physicochemical properties of liposomes has not been well understood. Here, we synthesized eight cationic derivatives of cholesterol which contain a tertiary amino head group with a different spacer arm. Transfection of plasmid pSV2CAT DNA into cells was done by cationic liposomes made of a mixture of dioleoylphosphatidylethanolamine (DOPE) and each cationic cholesterol derivative. At the same time we measured zeta potential of cationic liposomes by laser Doppler spectroscopy. The present results indicated that zeta potentials of cationic liposomes were well related to transfection activity of pSV2CAT DNA. This suggested that zeta potential of cationic liposomes is one of important factors which control gene transfection.

Atomic force microscopy for studying gene transfection mediated by cationic liposomes with a cationic cholesterol derivative

Atomic force microscopy (AFM) was used for studying gene transfection mediated by cationic liposomes which contain a cationic cholesterol derivative with a different spacer arm. Cationic liposomes were made by a mixture of one of eight cationic cholesterol derivatives and 1,2‐dioleoyl‐sn‐glycero‐3‐phosphatidyl ethanolamine (DOPE). AFM images showed that vesicles made of the liposome/DNA complex had various diameters depending on each cationic cholesterol derivative with a different spacer arm. The results showed that the diameter of the liposome/DNA complex was well related to the transfection activity of plasmid pSV2CAT DNA to a cultured cell line (NIH3T3). From the results it was found that the vesicles with moderate diameters (from 0.4 to 1.4 μm) were moste effective for gene transfection of plasmid pSV2CAT DNA into the target cell. Neither smaller vesicles (<400 nm) nor larger vesicles (>1.4 μm) were adequate for gene transfection. As the gene transfection by the cationic liposomes was mostly inhibited by wortmannin, an inhibitor of endocytosis, it is suggested that the vesicles with moderate diameters were useful for gene transfection by endocytosis.

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 Ca2+ 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.

Enhanced Nerve–Mast Cell Interaction by a Neuronal Short Isoform of Cell Adhesion Molecule-1

Close apposition of nerve and mast cells is viewed as a functional unit of neuro-immune mechanisms, and it is sustained by trans-homophilic binding of cell adhesion molecule-1 (CADM1), an Ig superfamily member. Cerebral nerve–mast cell interaction might be developmentally modulated, because the alternative splicing pattern of four (a–d) types of CADM1 transcripts drastically changed during development of the mouse cerebrum: developing cerebrums expressed CADM1b and CADM1c exclusively, while mature cerebrums expressed CADM1d additionally and predominantly. To probe how individual isoforms are involved in nerve–mast cell interaction, Neuro2a neuroblastoma cells that express CADM1c endogenously were modified to express additionally either CADM1b (Neuro2a-CADM1b) or CADM1d (Neuro2a-CADM1d), and they were cocultured with mouse bone marrow-derived mast cells (BMMCs) and BMMC-derived cell line IC-2 cells, both of which expressed CADM1c. BMMCs were found to adhere to Neuro2a-CADM1d neurites more firmly than to Neuro2a-CADM1b neurites when the adhesive strengths were estimated from the femtosecond laser-induced impulsive forces minimally required for detaching BMMCs. GFP-tagging and crosslinking experiments revealed that the firmer adhesion site consisted of an assembly of CADM1d cis-homodimers. When Neuro2a cells were specifically activated by histamine, intracellular Ca2+ concentration was increased in 63 and 38% of CADM1c-expressing IC-2 cells that attached to the CADM1d assembly site and elsewhere, respectively. These results indicate that CADM1d is a specific neuronal isoform that enhances nerve–mast cell interaction, and they suggest that nerve–mast cell interaction may be reinforced as the brain grows mature because CADM1d becomes predominant.

Cell Adhesion Molecule 1 Is a Novel Pancreatic–Islet Cell Adhesion Molecule That Mediates Nerve–Islet Cell Interactions

BACKGROUND & AIMS Cell adhesion molecule 1 (CADM1), mediates nerve-mast cell attachment and communication through homophilic binding. An immunohistochemical screen showed that CADM1 is expressed in pancreatic islets. Here, we determined the cell types expressing CADM1 and examined its role in nerve-islet cell interactions. METHODS Immunohistochemistry and double-staining immunofluorescence were performed on murine and human pancreases and on islet cell tumors (ICTs). alphaTC6 cells, a murine alpha cell line, were cultured on neurite networks of superior cervical ganglia. Neurite-alphaTC6 cell attachment and communication were examined after nerves were activated specifically by scorpion venom. RESULTS CADM1 was expressed on the plasma membrane in all 4 major types of islet cells, alpha, beta, D, and pancreatic polypeptide in human beings, but primarily in alpha cells in mice. In cocultures, alphaTC6 cell to neurite attachment was inhibited dose-dependently by an anti-CADM1 function-blocking antibody. In response to scorpion venom-evoked nerve activation, 36% of the alphaTC6 cells mobilized Ca(2+), and introduction of a CADM1-targeting small interfering RNA reduced the fraction of responding cells to 7%. In 21 human ICTs, CADM1 was present in the plasma membrane of 7, and the others were negative for CADM1. Six of the CADM1-expressing tumors were functional hormonally, whereas all but 2 of the CADM1-negative tumors were nonfunctional (P = .0032). CONCLUSIONS CADM1 is a novel islet cell adhesion molecule mediating nerve-islet cell interactions. The strong correlation between CADM1 expression and hormonally functional phenotypes suggests that CADM1 is involved in hormone secretion from ICTs.

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.

Nuclear translocation of green fluorescent protein-nuclear factor κB with a distinct lag time in living cells

A highly fluorescent mutant form of the green fluorescent protein (GFP) has been fused to the human nuclear factor κB (NF‐κB) p50 and p105 (p50/IκBγ), a precursor protein of NF‐κB p50. GFP‐p50 and GFP‐p105 were expressed in monkey COS‐7 cells and human HeLa cells. Translocation of these chimeric proteins was observed by confocal laser scanning microscopy. GFP‐p50 (without IκBγ) in the transfected cells resided in the nucleus. On the other hand, GFP‐p105 (GFP‐p50 with IκBγ) localized only in the cytoplasm before stimulation and translocated to the nucleus with stimulant specificity similar to that of native NF‐κB/IκB. In addition, the translocation of NF‐κB to the nucleus had a distinct lag time (a quiescent time) in the target cells. The lag time lasted 10–20 min after stimulation with hydrogen peroxide or tumor necrosis factor α. It was suggested that this might be due to the existence of a limiting step where NF‐κB is released from NF‐κB/IκB by the proteasome.

Neurotrophic factors increase tumor necrosis factor-α-induced nuclear translocation of NF-κB 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.

Receptor-mediated calcium signal playing a nuclear third messenger in the activation of antigen-specific B cells.

We have studied receptor-mediated calcium signals in antigen-specific B cells (trinitrophenol-specific B cell clone, TP67.21) using a confocal fluorescence microscope with an argon ion laser (488 nm) and a He-Cd laser (325 nm). Confocal fluorescence images of fluo-3 loaded B cells, excited by an argon ion laser, became much brighter and more nonhomogeneous than those before antigen stimulation. Time-dependent fluorescence changes in intensities were abrupt and quite similar to the patterns of the intracellular calcium ion concentration [Ca2+]i observed by a conventional fluorescence microscope using fura-2. From the morphological patterns of the calcium images, the parts of the bright fluorescence seemed to belong to the nucleus in B cells. To confirm the above events we measured the confocal fluorescence images of the nucleus. From the fluorescence images of co-loaded Hoechst 33342 (a DNA-specific fluorescent probe), which excited by a He-Cd laser, the brighter parts of the fluo-3 fluorescence intensities were identified to the nucleus in B cells. This suggested the possibility that the increased intranuclear calcium ions may play a nuclear third messenger in B cells.