Mamoru Nakanishi

Expression of chimeric receptor composed of immunoglobulin-derived V regions and T-cell receptor-derived C regions

Chimeric genes composed of immunoglobulin (Ig)-derived variable (V) regions and T-cell receptor (TCR)-derived constant (C) regions were constructed. The VL and VH genes showing anti-phosphorylcholine (PC) activity were used in this study. Two pairs of chimeric genes, VL-C beta and VH-C alpha genes, and VL-C alpha and VH-C beta genes, were inserted into an expression vector containing both Ecogpt and neo genes, and transfected into EL4 cells. Cells which express both chimeric receptor molecules were established. The activity of the transformants to the antigen was examined by using stopped-flow fluorometry. An increase in the concentration of cytoplasmic calcium ion was observed after addition of Staphylococcus pneumoniae R36A bacteria grown in the choline-containing medium which express PC molecules, but not after the PC-negative bacteria grown in the ethanolamine-containing medium.

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.

LOX Index, a Novel Predictive Biochemical Marker for Coronary Heart Disease and Stroke

BACKGROUND Lectin-like oxidized LDL receptor 1 (LOX-1) is implicated in atherothrombotic diseases. Activation of LOX-1 in humans can be evaluated by use of the LOX index, obtained by multiplying the circulating concentration of LOX-1 ligands containing apolipoprotein B (LAB) times that of the soluble form of LOX-1 (sLOX-1) [LOX index = LAB x sLOX-1]. This study aimed to establish the prognostic value of the LOX index for coronary heart disease (CHD) and stroke in a community-based cohort. METHODS An 11-year cohort study of 2437 residents age 30-79 years was performed in an urban area located in Japan. Of these, we included in the analysis 1094 men and 1201 women without history of stroke and CHD. We measured LAB and sLOX-1 using ELISAs with recombinant LOX-1 and monoclonal anti-apolipoprotein B antibody and with 2 monoclonal antibodies against LOX-1, respectively. RESULTS During the follow-up period, there were 68 incident cases of CHD and 91 cases of stroke (with 60 ischemic strokes). Compared with the bottom quartile, the hazard ratio (HR) of the top quartile of LOX index was 1.74 (95% CI 0.92-3.30) for stroke and 2.09 (1.00-4.35) for CHD after adjusting for sex, age, body mass index, drinking, smoking, hypertension, diabetes, non-HDL cholesterol, and use of lipid-lowering agents. Compared with the bottom quartile of LOX index, the fully adjusted HRs for ischemic stroke were consistently high from the second to the top quartile: 3.39 (95% CI 1.34-8.53), 3.15 (1.22-8.13) and 3.23 (1.24-8.37), respectively. CONCLUSIONS Higher LOX index values were associated with an increased risk of CHD. Low LOX index values may be protective against ischemic stroke.

Fluctuation of the lysozyme structure: II. Effects of temperature and binding of inhibitors☆

Abstract The kinetics of the hydrogen-deuterium exchange reaction in hen egg-white lysozyme (muramidase) have been followed by infrared absorption measurement in aqueous solutions at various temperatures. The kinetics have also been followed in solution with oligomers of N-acetyl- d -glucosamine which are bound to this protein in a specific manner. It was found that, in every case, 34% of the total peptide hydrogen atoms exchange relatively slowly at the rate of a first-order reaction. From the rate constant determined, and on the basis of the reaction mechanism shown in a previous paper (Nakanishi et al., 1972b), an estimate was made of the fluctuation amplitude of the lysozyme molecule, i.e. the amount of unfolded form D of this molecule in each solution. There are two types of unfolded (D) form found, D1 and D2. In the temperature range of 25 to 50 °C, the D1 form predominates. In this temperature range, the abundance ratio [ D 1 ] [N ]of the unfolded (D1) versus native (N) forms is of the order of 10−6 to 10−5, and the enthalpy and entropy differences of the D1 and N forms are ΔH = 2.2 kcal/mol and ΔS = −19 e.u., respectively. The entropy decrease in the N →D1 process has been attributed to a localization of the broken hydrogen bonds in the molecule. In the 65 to 85 °C range, on the other hand, the D2 form predominates; here, ΔH = 127 kcal/mol and ΔS = 364 e.u. and the amount of the D2 form is much greater than that of D1 form in the lower temperature range. The oligosaccharide inhibitors always suppress the fluctuation, and the efficiency of the suppression is found to be in the following order: monomer

Cationic cholesterol with a hydroxyethylamino head group promotes significantly liposome-mediated gene transfection

A novel cationic cholesterol derivative with a hydroxyethylamino head group, cholesteryl‐3β‐carboxyamidoethylene‐N‐hydroxyethylamine (II), has been synthesized and used for liposome‐mediated gene transfection. The cationic liposomes containing the derivative (II) facilitated greatly pSV2CAT gene transfection into mouse NIH3T3 and L929 cells in the absence of serum. The transfection efficiency was much higher than those by the cationic liposomes containing cationic derivatives with a dialkylamino head group (I, III or IV). Further, the efficiency by the cationic liposomes with the derivative (II) was not so much decreased in the presence of serum. This suggested that a novel cationic cholesterol derivative (II) should be very promising in liposome‐mediated gene transfection of plasmid and antisense DNA into target cells.

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.

Fluctuation of the lysozyme structure

Abstract The kinetics of hydrogen-deuterium exchange in hen egg-white lysozyme (muramidase) has been followed in aqueous solutions of various pH values and in solutions with various concentrations of lithium chloride, by an infrared absorption measurement. It was found that, in every case, 34% of the total peptide hydrogen atoms exchange relatively slowly with a rate of a first-order reaction. This amount corresponds to 44 peptide groups per molecule, and this is equal to the number of peptide NH-groups which are found to be involved in hydrogen bonds with the carbonyls of other peptide groups in the lysozyme molecule in the crystalline state. Each rate constant determined is in good agreement with the value expected from two simple assumptions. (1) The scheme of the isotope exchange reaction is N ⇌ D → D ∗ (⇌ N ∗ ), where N is the native form of the molecule, D a denatured (unfolded) form, and ∗ indicates the deuterated products. (2) The N ⇌ D fluctuation rate is much higher than the rate of the isotope exchange reaction D → D ∗ . It has been shown that the N ⇌ D transition postulated here is the same as that which can be followed by circular dichroism measurement and by some other physical measurements. The effect of lithium chloride on the exchange reaction rate is solely attributable to the change in the N ⇌ D equilibrium caused by the salt, whereas the effect of pH (in the 5 to 8 range) is wholly ascribed to the catalytic action of the OH− anion on the D → D ∗ reaction rate. From the deuterium exchange rate observed, an effective value of the mole fraction of the D form is estimated to be 3 × 10−6in the solution with no lithium chloride at 20 °C and of pH = 5 to 8.