Actions of exogenous heparan sulfate and hyaluronic acid on growth and thymidine incorporation of normal and transformed human fibroblasts. A comparison with the effects of high cell density and low serum concentration and a warning against thymidine incorporation as a measure of DNA synthesis

Treatment of normal human (WI-38) cells with exogenous heparan sulfate (HS) reduced cell growth and incorporation of radio-isotope-labeled thymidine (TdR) into DNA. In spite that growth of their transformants (WI-38 CT-1) was enhanced by HS treatment, transformed cells also decreased in TdR incorporation thereby. This peculiar observation was explained by a reduction of TdR uptake, leading to a decrease in specific radioactivity of newly synthesized DNA. The changes in cell growth and TdR incorporation by HS treatment were revealed to be similar to the changes with increasing cell density rather than by serum starvation.     

Investigation of actin in Tetrahymena cells. A comparison with skeletal muscle actin by a devised two-dimensional gel electrophoresis method

Total protein constituents of Tetrahymena thermophila strain B1868 III were studied by two-dimensional agarose-polyacrylamide gel electrophoresis to detect actin among the constituents. In the attempts to prepare a whole-cell extract of Tetrahymena, it was found that protease activity in the extract was so high that high molecular components were quickly digested with the endogenous protease into small peptides unless the homogenization and heat-treatment in a sodium dodecylsulfate solution were performed within 5 s. It was eventually found that employment of 8 M guanidine hydrochloride (HCl) in the homogenization of cells perfectly prevented the degradation of protein components, even through a long preparation procedure. A devised two-dimensional agarose-polyacrylamide gel electrophoresis of the guanidine HCl extract gave a 'protein map' on which most proteins were located in their respective positions, including proteins with more than 200,000 mol. wt. Addition of rabbit skeletal muscle actin on the protein map revealed that no protein with isoelectric point and molecular weight identical with those of the actin was contained in the whole Tetrahymena extract, suggesting that Tetrahymena actin may have characteristics far different from those of skeletal muscle actin.     

Effect of polyamines on synthesis and degradation of guanosine 5'-diphosphate 3'-diphosphate

The effect of polyamines on the in vitro and in vivo synthesis and degradation on guanosine 5'-diphosphate 3'-diphosphate (ppGpp) has been studied in Escherichia coli. The presence of 2 mM spermidine lowered the optimal Mg2+ concentration for ppGpp formation from 17 mM to 11 mM. The formation of ppGpp in the presence of 2 mM spermidine and 11 mM Mg2+ was about 15% greater than that in the presence of 17 mM Mg2+. At a concentration of less than 11 mM Mg2+, spermidine was found to stimulate ppGpp formation greatly. Putrescine did not cause any effect. When a polyamine-requiring mutant of E. coli (EWH319) was starved for an amino acid by the addition of valine, spermidine stimulated ppGpp formation. The degradation of ppGpp was not influenced significantly by polyamines.     

Role of Light Intensity and Temperature in the Regulation of Hydrogen Photoproduction by the Marine Cyanobacterium Oscillatoria sp. Strain Miami BG7

The effects of several key environmental factors on the development and control of hydrogen production in the marine blue-green alga (cyanobacterium) Oscillatoria sp. strain Miami BG7 were studied in relation to the potential application of this strain to a bio-solar energy technology. The production of cellular biomass capable of evolving hydrogen gas was strongly affected by light intensity, temperature, and the input of ammonia as a nutrient. Depletion of combined nitrogen from the growth media was a prerequisite for the initiation of hydrogen production. Maximum hydrogen-producing capability coincided with the end of the linear phase of growth. Hydrogen production exhibited considerable flexibility to environmental extremes. The rate of production saturated at low light intensities (i.e., 15 to 30 μEinsteins/m² per s), and no photoinhibition was observed at high light intensity (i.e., 1,000 μEinsteins/m² per s). The upper temperature limit for production was 46°C. Above the light compensation point for O₂ evolution H₂ production was inhibited. However, this problem was alleviated by two related phenomena. (i) The capacity of cells to evolve oxygen deteriorated with increasing culture age and nitrogen depletion, and (ii) the ability of these cells to produce oxygen in closed anaerobic hydrogen production systems was temporally limited.     

X-ray equatorial diffraction studies on the cross-sectional structure of Salmonella flagella

X-ray diffraction studies have been made on the cross-sectional structure of the normal Salmonella flagella. Two approaches have been made: one based upon small-angle equatorial scatterings (2θ 3°) and the other upon moderate-angle angle equatorial diffractions (3° 2θ 10°).Analysis of small-angle scattering data gives the radius of gyration of the flagella as 68 Å. Cylindrically averaged electron density of the cross-section of the flagella is obtained by means of the Fourier-Bessel transformation method. The average radius of the flagella is about 65 Å.In the investigation of the moderate-angle diffraction pattern, validity is examined of the model that a flagellum consits annularly arranged strands, of which each has a cylindrically symmetric structure. Features of the pattern observed in the range of 3° < 2θ < 10° can be interpreted fairly well by this model. Average radii of the flagella obtained for the 11 and 13 strands models are close to that obtained by the analysis of the small-angle scattering data.     

Application of the Electron Microscope to the Cytochemical Peroxidase Reaction in Salamander Leukocytes

The present study has dealt with the localization by electron microscopy of the products of peroxidase reaction in neutrophil leukocytes in the subcapsular region of the livers of Triturus viridescens. Small pieces of liver tissue were fixed for 1 hour in buffered osmium tetroxide solution. After fixation they were divided into five groups: (a) Not treated with any reagent (control); (b) Treated for 4 minutes with the peroxidase reagent containing 0.3 per cent benzidine and 0.014 per cent (0.004 molar) hydrogen peroxide in 50 per cent alcohol; (c) Treated for 4 minutes with 0.3 per cent benzidine solution in 50 per cent alcohol alone (control); (d) Treated for 4 minutes with 0.014 per cent (0.004 molar) hydrogen peroxide in 50 per cent alcohol alone (control); (e) Treated for 5 minutes with pure methanol, washed in water, and treated for 4 minutes with the peroxidase reagent (inhibition test). Each group was then dehydrated and embedded in either methacrylate or epoxy resin. In electron micrographs, the reaction products of peroxidase activity were evidenced in the form of dense materials localized in the specific granules in the cytoplasm of the neutrophil leukocytes. Neither mitochondria nor any other particles showed increases in density. The specific granules showed no change of density in the control and inhibition tests. Paraffin-embedded tissues of the above mentioned five groups, when examined with the light microscope, revealed that the brown granules denoting a positive reaction appeared only in leukocytes of the tissue treated with the peroxidase reagent. Although much further work is necessary before definitive and constant results are to be expected, the possibility that the electron microscope may be applicable to peroxidase cytochemistry in leukocytes has been suggested by the present study.     

Crystallization of a complex between ribonuclease Ms and 3'-guanylic acid

The crystals of a complex between ribonuclease Ms, the extracellular ribonuclease from Aspergillus saitoi, and 3'-guanylic acid were obtained from 2-methyl-2,4-pentanediol solution by vapor diffusion technique in the hanging drop mode. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with dimensions a = 47.0 A, b = 62.8 A, c = 37.9 A. The crystals diffract strongly up to at least 2.0 A resolution.     

A temperature-sensitive osmophilic mutant of Zygosaccharomyces rouxii

Summary A temperature-sensitive osmophilic mutant of Zygosaccharomyces rouxii, OS15, was isolated, which required high salt or sugar concentration for growth above 30°C. Cell viability at 35°C in the presence of NaCl was higher than in the absence of NaCl, and a survival ratio of the mutant cells after incubation at 55°C was also higher in the presence of NaCl than NaCl-free condition. Furthermore, resistance to UV light, hygromycin B and geneticin was improved in the presence of NaCl. There was no difference between the parent and the mutant in fatty acid saturation and microscopic cell shape under NaCl condition.     

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.