Cell cycle dependence of cytotoxicity and clastogenicity induced by treatment of synchronized human diploid fibroblasts with sodium fluoride

To study the cell cycle dependence of cytotoxicity and clastogenicity of sodium fluoride (NaF), synchronized human diploid fibroblasts were treated with NaF during different phases of the cell cycle and analyzed. Exponentially growing cells were synchronized by the following two procedures. (1) The cells were synchronized at G₀/G₁ phase by a period of growth in medium containing 1% serum (low serum medium). (2) The cells were synchronized at the G₁/S boundary by growth in low serum medium, followed by hydroxyurea treatment (Tsutsui et al., 1984a). Synchronized cells were treated with NaF for 3 h during the G₁ phase or G₂ phase, and for each of three 3-h periods during the S phase which lasted 9 h. Cytotoxicity, as determined by a decrease in colony-forming ability, was dependent upon the phase of the cell cycle during which NaF treatment was administered. The highest lethality was induced in when the cultures were treated with NaF during the second or third 3 h of S phase (middle or late S phase, respectively), or G₂ phase. Little lethality was observed in cultures in G₁ phase. Inducibility of chromosome aberrations of the cells following treatment with NaF was also dependent upon the phase of the cell cycle. A significant increase in the incidence of chromosome aberrations was observed only in cultures treated with NaF during early and / or middle S phases of cell cycle. These results suggest that cytotoxicity and clastogenicity of NaF to cultured human diploid fibroblasts are cell cycle dependent, and that the cells in early and middle S phases are more sensitive to the effects.     

Effects of hydrostatic pressure on the ultrastructure and leakage of internal substances in the yeast Saccharomyces cerevisiae

The structural damage to and leakage of internal substances from Saccharomyces cerevisiae 0–39 cells induced by hydrostatic pressure were investigated. By scanning electron microscopy, yeast cells treated at room temperature with pressuresbellw 400 MPa for 10 min showed a slight alteration in outer shape. Transmission electron microscopy, however, showed that the inner structure of the cell began to be affected, especially the nuclear membrane, when treated with hydrostatic pressure around 100 MPa at room temperature for 10 min; at more than 400–600 MPa, further alterations appeared in the mitochondria and cytoplasm. Furthermore, when high pressure treatment was carried out at — 20° C, the inner structure of the cells was severely damaged even at 200 MPa, and almost all of the nuclear membrane disappeared, although the fluorescent nucleus in the cytoplasm was visible by 4,6-diamidino-2-phenylindole (DAPI) staining. The structural damage of pressure-treated cells was accompanied by the leakage of internal substances. The efflux of UV-absorbing substances including amino acid pools, peptides, and metal ions increased with increase in pressure up to 600 MPa. In particular, amounts of individual metal ion release varied with the magnitude of hydrostatic pressures over 300 MPa, which suggests that the ions can be removed from the yeast cells separately by hydrostatic pressure treatment. Correspondence to: S. Shimada     

Seasonal variation in the physical properties of agar and biomass of Gracilaria sp. (chorda type) from Tosa Bay,southern Japan

Plants of Gracilaria sp.(chorda type), which grow along the coast of Uranouchi Inlet in Tosa Bay, southern Japan, showed the highest biomass in the summer (26 °C to 31 °C) and spring season (15.1 °C to 24.9 °C). Maximum biomass was 6952 g m^–2 in July, but gradually decreased in the autumn (30.5 °C in September to 20 °C in November) and winter (19.5 °C in December to 14.9 °C in February). Variation in yields and gel strength of the agars, were shown to depend on the time in the season. After alkali treatment (5% NaOH, 2 h) at three different temperatures (70, 80, and 90 °C), the agars showed gel strengths essentially that of commercial grade agars, with the best gel obtained at 80 °C. Maximum gel strength (1455 g cm^–2 of 1.5% agar gel) occurred in winter when the biomass and agar yield were low. Minimum gel strength was in spring. Gel strength was inversely correlated with agar yield, but was positively correlated with apparent viscosity. Maximum viscosity was 40 cP. in December. Gelling temperatures, pH of 1.5% agar gel, and moisture content in agars showed little variation.     

Immunohistochemical Detection of Sialyl LeX Antigen on Mucosal Langerhans Cells of Human Oral Mucosa following Neuraminidase Pretreatment

Histochemical assessment of selected carbohydrate sequences on Langerhans cells of human oral mucosa was made by combined use of enzyme digestion and immunostain-ing with monoclonal antibodies against specific carbohydrate structures. In both frozen sections and epithelial sheets without the enzyme pretreatment, mucosal Langerhans cells, identified by positive staining with anti-CD1a and HLA-DR antibodies, did not express any carbohydrate antigens on their surface. In contrast, following neuraminidase pretreatment of both types of material, the fucosylated type 2 chain (Le^X) became detectable on Langerhans cells, indicating that sialic acid is the terminal residue of this sequence. Other enzymes were ineffective in this apparent unmasking, and the staining patterns of the other related carbohydrate sequences (Le^y. Le^a, Le^b) remained unaffected by pretreatment with any of the enzymes used. These findings suggest that the mucosal Langerhans cells possess a unique carbohydrate chain, the sialyl fucosylated type 2 sequence (sialyl Le^X antigen).     

Improved strain distribution patterns of SMXA recombinant inbred strains by microsatellite markers.

To develop SMXA recombinant inbred (RI) strains as more valuable genetic resources, 302 microsatellite (Mit) loci were added to the strain distribution patterns (SDP) reported previously. The improved SDP were constructed in a total of 1085 loci containing 484 Mit markers, 571 restriction landmark genomic scanning (RLGS) spot markers and 30 others. This substantially improved SDP can be freely accessed on our homepage (http://www.med.nagoya-u.ac.jp/sisetu/SDP.htm).     

Effect of triethylenepentaminehexaacetic acid on the renal damage in cadmium-treated Syrian hamsters

Cadmium (Cd)-induced nephropathy was treated by triethylene-pentaminehexaacetic acid (TTHA) in male Syrian hamsters. Hamsters injected three times a week with 3 mg/kg body wt CdCl₂ showed proteinuria, urinaryN-acetyl-β-d-inglucosaminidase (NAG), and fractional excretion of sodium (FENa) when compared to saline-injected control. Cd-treated hamsters injected ip with TTHA 10 mg/kg body wt five times a week showed reduction of renal damage, including reductions in urinary protein (from 6.7±2.2 to 4.3±0.5 mg/d) and NAG (0.17±0.06 to 0.04±0.02 U/d). Urinary excretion of Cd was significantly increased (from 87±51.3 to 3052±1485 mg/L) by TTHA administration. Cd concentration in renal cortical tissue was slightly reduced (26.4±3.0 to 21.8±2.7 mg/g. protein). Excretion of malondialdehyde (MDA) was increased only in Cd-injected hamsters (to 2.1±1.6 nM/L), and elevated MDA in renal cortical tissue was not reduced by the administration of TTHA (1041±105 vs 1104±358 nM/g protein). Glutathione (GSH) concentration in the renal cortex was significantly elevated after Cd administration and further increased after TTHA administration (5.5±2.1 to 9.8±2.0 μg/50 mg protein). There were no marked effects on creatinine clearance (Ccr) and hematocrit. Moreover, renal morphological changes were improved significantly by treatment with TTHA. We demonstrated the efficacy of TTHA in the treatment of Cd-induced nephropathy in hamsters. Although the precise mechanism of the TTHA effects on Cd-induced nephropathy has not been elucidated, it might involve GSH reducing the elevated MDA concentration in renal tissue.     

Accumulation of glutamate by osmotically stressed Escherichia coli is dependent on pH.

In the present study, we measured the accumulation of glutamate after hyperosmotic shock in Escherichia coli growing in synthetic medium. The accumulation was high in the medium containing sucrose at a pH above 8 and decreased with decreases in the medium pH. The same results were obtained when the hyperosmotic shock was carried out with sodium chloride. The internal level of potassium ions in cells growing at a high pH was higher than that in cells growing in a neutral medium. A mutant deficient in transport systems for potassium ions accumulated glutamate upon hyperosmotic stress at a high pH without a significant increase in the internal level of potassium ions. When the medium osmolarity was moderate at a pH below 8, E. coli accumulated gamma-aminobutyrate and the accumulation of glutamate was low. These data suggest that E. coli uses different osmolytes for hyperosmotic adaptation at different environmental pHs.     

Production of a lipopeptide antibiotic, surfactin, by recombinant Bacillus subtilis in solid state fermentation

Production of a lipopeptide antibiotic, surfactin, in solid state fermentation (SSF) on soybean curd residue, Okara, as a solid substrate was carried out using Bacillus subtilis MI113 with a recombinant plasmid pC112, which contains lpa-14, a gene related to surfactin production cloned at our laboratory from a wild-type surfactin producer, B. subtilis RB14. The optimal moisture content and temperature for the production of surfactin were 82% and 37 degrees C, respectively. The amount of surfactin produced by MI113 (pC112) was as high as 2.0 g/kg wet weight, which was eight times as high as that of the original B. subtilis RB14 at the optimal temperature for surfactin production, 30 degrees C. Although the stability of the plasmid showed a similar pattern in both SSF and submerged fermentation (SMF), production of surfactin in SSF was 4-5 times more efficient than in SMF. (c) 1995 John Wiley & Sons, Inc.