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Biomedical subjects

M Yamaizumi

Publications and source records attributed to M Yamaizumi.

At least 19 recordsLinked to original sources
PubMed1991

Identification of a specific protein factor defective in group A xeroderma pigmentosum cells.

A protein factor which corrects the defect in xeroderma pigmentosum cells belonging to complementation group A (XP-A cells) was detected in a cell extract prepared from calf thymus. The activity of this factor was measured as the amount of unscheduled DNA synthesis (UDS) reappearing in UV-irradiated XP-A cells after microinjection of the extract. The native molecular mass of this factor was estimated to be 80 kDa by gel-filtration and 25 kDa by glycerol gradient centrifugation. The activity was, however, recovered at a position corresponding to 43 kDa after renaturation on an SDS-PAGE gel. The isoelectric point was determined to be approximately 7.5 by measuring the activity after renaturation on an IEF gel. These values were obtained with a partially purified sample. A spot corresponding to these values was detected on two-dimensional gel electrophoresis with a highly purified sample recovered from an SDS-PAGE gel. The purified protein stimulated UDS specifically in the XP-A cells and endowed the cells with a normal level of UV-resistance. The XP-A cells injected with the factor also showed a normal level of UDS after treatment with either 4HAQO or psoralen plus UV-A. This factor (XP-A complementing factor; XP-ACF) may be involved in the repair of DNA damage induced by various agents.

Animals↗
PubMed1989

Microinjection of T4 endonuclease V produced by a synthetic denV gene stimulates unscheduled DNA synthesis in both xeroderma pigmentosum and normal cells.

A structural gene for T4 endonuclease V was constructed by ligating synthetic oligonucleotides. The endonuclease V was overproduced in E. coli under control of the E. coli tryptophan promoter and purified to apparent homogeneity. The product had comparable DNA glycosylase and apurinic/apyrimidinic (AP) endonuclease activities to the natural enzyme in vitro. When this endonuclease V was microinjected into the cytoplasm of xeroderma pigmentosum (XP) cells of complementation group A, B, C, D, F, G or H, unscheduled DNA synthesis (UDS) above the residual level was detected in all the cells at a dose of about 10(3) molecules following UV irradiation. The gain numbers of UDS in these XP cells increased with increase in the dose of enzyme and reached a plateau at the normal cell level on introduction of about 10(4) molecules. Introduction of more enzyme into either XP cells or normal human cells did not increase the grain number under regular labelling conditions (2.5 h, 37 degrees C). In normal mouse cells, introduction of the enzyme increased the grain number more than 4-fold under the same conditions during at least 8.5 h following UV irradiation. Furthermore, with a labelling time of 30 min, the enzyme more than doubled the grain number even in normal human cells.

Animals↗
PubMed1987

Reversible inhibition of protein import into the nucleus by wheat germ agglutinin injected into cultured cells.

The importance of glycoproteins located in the nuclear envelope in nuclear transport was tested by microinjection of karyophilic proteins into the cytoplasm of cultured human cells together with various lectins. Wheat germ agglutinin (WGA) blocked the nuclear transport of nucleoplasmin, a nuclear protein of Xenopus laevis oocytes, and of nonnuclear proteins conjugated with a synthetic peptide containing the nuclear localization signal sequence for simian virus 40 (SV40) large T antigen. Its inhibitory activity persisted for about 1 h after its injection into the cells and then gradually decreased. Export of at least some kinds of RNA from the nucleus seemed not to be affected by WGA even when import of the proteins into the nucleus was completely blocked (within 1 h after WGA injection). Moreover, WGA did not inhibit the passive diffusion of fluorescein isothiocyanate (FITC)-dextran (average Mr 17,900) into the nucleus. Wistaria floribunda agglutinin (WFA), concanavalin A (Con A), and lentil lectin did not block nuclear transport. These results indicate that WGA specifically blocks active protein import, but not passive diffusion of materials into the nucleus.

Antigens, Polyomavirus Transforming↗
PubMed1986

Microinjection of partially purified protein factor restores DNA damage specifically in group A of xeroderma pigmentosum cells.

Microinjection of cell extracts prepared from both human placenta and HeLa cells into xeroderma pigmentosum (XP) cells of complementation group A restores unscheduled DNA synthesis (UDS) in these cells after UV irradiation [de Jonge, A., Vermeulen, W., Klein, B. & Hoeijmakers, J. (1983) EMBO J. 2, 637-641]. These cells also showed normal resistance to UV irradiation. The half-life of the factors in the cell extracts corresponding to the UDS activity (factor A) was 14 hr in XP cells of group A, and the maximal level of UDS was exerted 2 hr after microinjection. The factors were sensitive to protease treatment but not to RNase treatment and were found to be approximately equal to 160 and approximately equal to 90 kDa by gel filtration. These two fractions of the factor(s) acted specifically in XP cells of complementation group A among complementation groups A, B, C, D, F, G, and probably E and H.

DNA Repair↗
PubMed1979

Reconstitution of lipid vesicles associated with HVJ (Sendai virus) sikes. Purification and some properties of vesicles containing nontoxic fragment A of diphtheria toxin.

A mixture of HVJ (Sendai virus) spike proteins, the nontoxic fragment A of diphtheria toxin, lecithin, and cholesterol was solubilized in sucrose solution containing a nonionic neutral detergent. The liposomal vesicles which formed on removal of the detergent by dialysis were purified by gel filtration and centrifugation on a sucrose gradient. The resulting purified vesicles had hemagglutinating activity, hemolytic activity and, after solubilization, the enzymic activity of fragment A. The vesicles had no cell fusion activity. Electron microscopy showed that both the outside and inside of membranes of the vesicles were associated with the spikes. When the vesicles were freeze-fractured, no large aggregates of particles were seen on either face. Such fragment A-containing lipid vesicles (liposomes) with HVJ spikes bound to mamalian cell membrane and released their fragment A into the cytoplasm causing cell death. Neither fragment A-containing liposomes without spikes nor empty liposomes with spikes were toxic.

Cell Fusion↗
PubMed1978

Reconstitution of hybrid toxin from Fragment A of diphtheria toxin and a subunit of Wistaria floribunda lectin.

A hybrid protein was prepared in which Fragment A of diphtheria toxin was linked to a monovalent subunit of the lectin from Wistaria floribunda seeds by a disulfide bridge. The hybrid molecule was reconstituted effectively from a reduced mixture of Fragment A and the lectin subunit by oxidation with o-phenanthroline and CuSO4. The monovalent hybrid protein was partially purified and found to be toxic to L cells. The toxicity of the hybrid protein on the cells was blocked by the addition of either N-acetyl-D-galactosamine, which specifically binds to the lectin, or by anti-Fragment A antibody.

Acetylgalactosamine↗
PubMed1978

One molecule of diphtheria toxin fragment A introduced into a cell can kill the cell.

Erythrocyte ghosts containing a known number of molecules of purified fragment A of diphtheria toxin with a constant amount of FITC-BSA as a fluorescence marker were prepared by dialyzing a mixture of erythrocytes and these substances against hypotonic solution. These substances were then introduced into diphtheria toxin-resistant mouse L cells by virus-mediated cell fusion of the cells with the ghosts, and mononuclear recipients that has fused with only one erythrocyte ghost were separated in a flourescence-activated cell sorter (FACS) on the basis of their cell size and fluorescence intensity. After separation, the viability of cells containing known numbers of fragment A was examined by measuring colony-forming ability. The results demonstrated that a single molecule of fragment A was sufficient to kill a cell. This fact was confirmed by introduction into cells of fragment A from an immunologically related mutant toxin, CRM 176 (fragment A176); this has a completely functional fragment B region, but in cell extracts, the enzymic activity of its fragment A is about 10 fold less than that of wild toxin. The cytotoxicity of CRM 176 is about two hundredths of that of the wild-type (Uchida, Pappenheimer and Greany, 1973). As expected, about 100-200 fold excess of fragment A-176 was needed to kill the cells.

Cell Survival↗
PubMed1978

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.

Antitoxins↗
PubMed1978

An attempt to separate mononuclear cells fused with human red blood cell-ghosts from a cell mixture treated with HVJ (Sendai virus) using a fluorescence activated cell sorter (FACS II).

Nucleated cells (Ehrlich ascites tumor cells or L strain cells) and human red blood cells (RBC)-ghosts were mixed and fused by ultraviolet-inactivated HVJ (Sendai virus). The cell mixture was stained with FITC conjugated anti-RBC ghost antiserum and then applied to FACS II apparatus. The apparatus sorted mononuclear cells fused with RBC-ghosts from the cell mixture on the basis of both the light scattering and fluorescence profiles. When the same procedure was carried out on a mixture containing cells and intact human RBC, the cells sorted by this method were cells into which hemoglobin had been injected. The sorted cells were capable of forming colonies in culture. This sorting method may be useful for collecting cells in which macromolecules have been injected artificially by fusion of RBC-ghosts enclosing macromolecules.

Cell Fusion↗
PubMed1978

Quantitative introduction of a given macromolecule into cells by fusion with erythrocyte ghosts using a fluorescence activated cell sorter.

FITC-conjugated bovine serum albumin (FITC-BSA) molecules were quantitatively introduced into human erythrocyte ghosts by gradual hemolysis. When the ghosts and L cells were fused with UV-inactivated HVJ (Sendai virus), FITC-BSA was introduced into the cytoplasm of the L cells and fluorescence could be observed inthe cells with a fluorescence microscope. A mixture of L cells and ghosts was introduced into a fluorescence activated cell sorter (FACS), which could separate the mononuclear cells on the basis of their light-scattering profile. Four distinct populations of mononuclear cells were found by fluorescence analysis. These populations were separated from the cell mixture and found to correspond to cells fused with one, two and three ghosts and unfused cells. After separation, the cells from each population could form colonies in culture. As a given macromolecule can be quantitatively introduced into erythrocyte ghosts with the FITC-BSA, after fusion of these ghosts with cells, this sorting method is useful for separating cells containing a definite number of macromolecules.

Cell Fusion↗