Mitsuru Furusawa

Neutralization of diphtheria toxin in living cells by microinjection of antifragment A contained within resealed erythrocyte ghosts

When human erythrocytes suspended in phosphate-buffered saline (PBS) containing lgG were first dialyzed against a hypotonic solution and then dialyzed against PBS, lgG molecules were entrapped within resealed erythrocyte ghosts. The concentration of lgG inside the ghosts was about 33% of its concentration in the dialysis bag. With the aid of HVJ (Sendai virus), ghosts containing rabbit lgG antibody against fragment A of diphtheria toxin were fused with toxin-sensitive FL cells. The fused FL recipients were found to be resistant to the action of diphtheria toxin. Clones derived from the resistant recipient cells, however, became sensitive to the toxin again. Antifragment A neutralized the enzymic activity of isolated fragment A in vitro, but did not protect FL cells or rabbit skin against the complete toxin.

Chapter 5 Use of Erythrocyte Ghosts for Injection of Substances into Animal Cells by Cell Fusion

Publisher Summary HVJ (Sendai virus)-mediated cell fusion of cultured mammalian cells and mammalian erythrocytes results in transfer of hemoglobin to the cytoplasm of the cultured cells. It is considered that if erythrocytes containing a foreign substance were used in this system instead of intact erythrocytes, one could introduce the substance into the cells. By cell fusion of the ghosts and cultured cells, introduction of fluorescein isothiocyanate (FITC) into the cells was attained although the injection frequency was low. Subsequently, the technique for replacing contents of erythrocytes and succeeded in raising the fusion frequency has been improved. The injection procedure consists of two steps: the first step is to introduce foreign substance into erythrocyte ghosts and the second is to fuse the ghosts containing the substance with target cells using HVJ.

A simple microinjection technique not employing a micromanipulator.

Abstract A new microinjection method is presented in which a modified condenser is used instead of a conventional micromanipulator. The micropipette is located in a hole through the optical axis of the condenser lens of a normal microscope. The three-dimensional movement of the micropipette is simply controlled by manipulation of the adjusting screws controlling the movement of the condenser. In comparison with conventional microinjection instruments, the simplicity of this new instrument is outstanding.

Ebselen, a seleno-organic compound, protects against ethanol-induced murine gastric mucosal injury in both in vivo and in vitro systems

The inhibitory effect of the seleno-organic compound ebselen on ethanol-induced murine gastric mucosal injury was examined. In an in vivo study, absolute ethanol (50 microliters/mouse, oral) produced marked gastric mucosal necrosis along with hemorrhage or edema and elevations in both lipid peroxide and peptidoleukotriene levels in the fundic mucosa. Pretreatment with ebselen (30 and 100 mg/kg, oral) significantly prevented this gastric mucosal injury and, further, remarkably decreased the elevated lipid peroxide and peptidoleukotriene levels. In an in vitro study using a murine gastric surface mucous cell line GSM06, exposure to ethanol concentration dependently elicited cell damage (7.5-17.5% ethanol) and an increase in lipid peroxides without alterations in peptidoleukotrienes (15% ethanol). Addition of ebselen (10 and 100 microM) to this system (15% ethanol) significantly inhibited the cell damage and completely prevented the increase in lipid peroxide level. These results indicate that ebselen protects against murine gastric mucosal injury both in vivo and in vitro, and that this protection may be related at least in part to its inhibitory action on lipid peroxides.

Prostaglandin E1, an inducer of erythroid differentiation of friend erythroleukemia cells

Summary The effect of prostaglandin E 1 (PGE 1 ) on erythroid differentiation of Friend erythroleukemia cells were examined. Cultivation of the dimethylsulfoxide (DMSO)-inducible clones 745A and F4N in 1 × 10 −4 M PGE 1 resulted in significant numbers of hemoglobin and spectrin positive cells after 5 days. DMSO-resistant cell line, F4D5, which is not inducible for spectrin and heme (or hemoglobin) accumulation during DMSO treatment, responded to PGE 1 and became spectrin positive, but failed to synthesize hemoglobin. Neither cAMP, db-cAMP or theophylline had any effect.

Immuno‐Localization of DEAD Family Proteins in Germ Line Cells of Xenopus Embryos.

In order to investigate whether a vasa‐like protein is present in germ line cells of Xenopus, antibodies were produced which react specifically with synthetic oligopeptides of sequences from near the N‐ or C‐termini or with one including the DEAD box of the Drosophila vasa protein.

Specific arrest of spermatogenesis caused by apoptotic cell death in transgenic mice

Background: The c‐myc protooncogene has been implicated in the control of cell proliferation, differentiation and/or apoptosis in various cellular systems. However, the role of c‐myc in germ cell lineage is largely unknown.

Tumor promoters specifically and reversibly disturb development and behavior of Caenorhabditis elegans.

SummaryThe effect of phorbol ester tumor promoters on the development and behavior of a free-living soil nematode, Caenorhabditis elegans, was studied. When young developing C. elegans were grown on E. coli-seeded agar with low concentrations (0.1 μg/ml) of 12-0-tetradecanoyl-phorbol-13-acetate or phorbol-12,13-didecanoate, their growth was arrested. These tumor promoters reduced the brood size when gravid adults were treated and caused uncoordinated movement in animals treated at any stage of development. The effects of these tumor promoters on nematode development and behavior were partially reversible. The nonpromoting derivatives phorbol and 4α-phorbol-12,13-didecanoate showed no effect on the animals.

Germline‐specific Antigens Identified by Monoclonal Antibodies in the Nematode Caenorhabditis elegans1

Of 27 monoclonal antibodies identified to react, by indirect immunofluorescent antibody staining, with specific cells and tissues of the nematode Caenorhabditis elegans, we report here three monoclonal antibodies pertaining to the gonadal tissues. One antibody defines an antigen that is distributed over the entire embryo at earlier development and later becomes unique to the gonad, including mature oocytes. The antigens recognized by the other two are distributed asymmetrically in the posterior region of the fertilized eggs cytoplasm destined to become the germline precursor cell. Each antigen is successively segregated only to the germline precursor cells of the developing embryo and, postembryonically, is uniquely localized around the germline cell nuclei of the larvae and adults.

Cellular microinjection by cell fusion: technique and applications in biology and medicine.

Publisher Summary This chapter introduces the microinjection method using human erythrocytes and Sendai virus (HVJ), which is broadly usable in studies of biology at a cellular level. This technique can also be applied to medical treatments. The advantages of this technique are (1) intrinsically no limitation in the number of target cells subjected to microinjection and (2) the capability of performing a quantitative introduction of substance into each single cell. Despite these advantages, the present technique has an inevitable shortcoming in that intranuclear injection is impossible. The microinjection is carried out using a modified condenser of a normal light microscope. The micropipet is inserted and fixed in a straight perpendicular hole, which is made through the optical axis of the condenser lens. Delicate three-dimensional movement of the tip of the micropipet can be controlled by handling the adjusting screws for the movement of the condenser. This modified condenser can be used with a normal and an inverted microscope. Compared with conventional microinjection methods using a micromanipulator, this method has the considerable advantages of simplicity of handling and speed in performing microinjection or microsurgery using this new instrument.