Effect of temperature and cell density on cellular protein content in temperature-sensitive mutants of rat 3Y1 diploid fibroblasts

Summary We examined cellular protein content in four temperature-sensitive (ts) mutants of rat 3Y1 fibroblasts (3Y1tsD123, 3Y1tsF121, 3Y1tsG125, and 3Y1tsH203) under various conditions of culture that affect cell proliferation. When proliferation of the ts mutants was inhibited at a nonpermissive temperature (39.8°C) in the G₁ phase, prominent accumulation of cellular protein occurred in three mutants (3Y1tsF121, 3Y1tsG125, and 3Y1tsH203) but not in 3Y1tsD123. The over-accumulation of protein at 39.8°C in the former three mutants was inhibited at high cell densities. At low cell densities there was an upper limit in the protein accumulation at 39.8°C. When the three mutants, proliferation-arrested at high cell densities at 33.8°C, were replated sparsely in fresh medium and shifted to 39.8°C, proliferation was completely inhibited whereas over-accumulation of protein occurred. These results indicating dissociation of protein accumulation and cell proliferation suggest that the two events are regulated by different mechanisms. This work was supported in part by a Grant-in-Aid for Encouragement of Young Scientists (1984) to K. Y. from the Ministry of Education, Science, and Culture, Japan.     

The Effect of Ageing on Bud Differentiation in the Moss, Physcomitrium sphaericum

In the moss Physcomitrium sphaericum, we examined the numberof buds per filament, the position of buds, and the ratio ofbud-differentiated filaments when treated with cytokinin, inrelation to the increase in the number of cells per filament. When filaments of a young protonema were treated with cytokinin,many filaments did not differentiate buds. As the number ofcells in a filament increased, both the mean number of budsper filament and the ratio of bud-differentiated filaments increased.However, the position of bud differentiation was unaffectedby application of cytokinin. A higher concentration of cytokininincreased the mean number of buds per filament and the ratioof bud-differentiated filaments. The relationship between cytokinin, ageing of filaments andthe ability to differentiate buds is discussed. (Received June 17, 1985; Accepted September 9, 1985)     

Reduction of Aluminum Toxicity by Addition of a Conditioned Medium from Aluminum-Tolerant Cells of Carrot

The toxic effect of aluminum (Al) on the growth of Carrot cells(SO-l) decreased to a greater degree with addition of a mediumconditioned by Al-tolerant carrot cells (TA-l) than with a mediumconditioned by SO-l cells. The toxic effect of Al was reducedgreatly by adding an acidic fraction of the conditioned media,but little or not at all by a neutral or basic fraction. Offour organic acids detected in the acidic fraction, the majorone was citric acid which was present in a much greater amountin the conditioned medium of TA-l cells than in that of SO-lcells. The toxic effect of Al was reduced by adding citric or malicacid instead of the conditioned medium, but not by succinicor fumaric acid. Chelating abilities of the organic acids wereevaluated by shifts in their titration curves, and were foundto be closely correlated with the detoxification effects. Thus,the Al tolerance of TA-l cells may in fact be due to the chelatingeffect of citric acid which is abundantly released into themedium by the Al-tolerant carrot cells. (Received July 9, 1984; Accepted November 22, 1984)     

Inactivation and sedimentation of mercury from organomercurials by Klebsiella pneumoniae

Abstract A susceptibility of 63 clinical isolates of Klebsiella pneumoniae to inorganic and organic mercuric compounds was determined. 18 of them were found to be resistant to fluorescein mercuric acetate (FMA) and merbromin (MB). Moreover, all the resistant strains inactivate the antibacterial effect of FMA. The changes in the amount of organic mercury at the time of inactivation of the drug and the structures of the end products were examined in detail with the plasmid-bearing strain JK9 and its transconjugants of Escherichia coli .
The results showed that FMA was inactivated by an intracellular enzyme produced inducively and was degraded to fluorescein (sodium salt, uranine), which led to the sedimentation of metallic mercury. The discovery of the genes conferring inducible organic mercury-inactivating enzymes determined by plasmids was the next step and their application in the recovery of metallic mercury from organomercurials is now imminent.     

Isolation of tetraploid clones with high efficiency from diploid 3Y1 rat fibroblasts treated with sodium butyrate

Summary Sodium butyrate causes proliferation arrest with a G2 (4C) DNA content and induces formation of tetraploid cells upon removal of the inhibitor, in rat 3Y1 diploid fibroblasts. We isolated tetraploid clones from the butyrate-treated 3Y1 cells with high efficiency; among 21 clones randomly isolated, 5 were pure diploid, 7 were mainly tetraploid with a small contaminating diploid population, and 7 were pure tetraploid. Among the pure tetraploid clones, two showed doubled chromosome numbers with slightly broader distributions than that seen in parental 3Y1 cells. Butyrate further induced polyploid formation in the tetraploid cells thus produced, but octaploid cells that resulted could not be maintained for prolongeed, cultivation. We found no difference between the tetraploid and the (parental and parallel isolated) diploid clones in terms of colony-forming ability, proliferation rate, and sensitivity to density-dependent inhibition of proliferation. These results suggest that doubling of chromosome number by itself does not cause a change in proliferation property. The tetraploid clones had lower average saturation densities possibly due to enlargement of cell size represented by higher cellular protein content.     

Continuous requirement of cAMP for pre-spore differentiation in Dictyostelium discoideum

Abstract Using a shaking culture system, we have previously shown that both cell contact and cAMP are required for pre-spore differentiation in Dictyostelium discoideum [2]. In the present study, cAMP was removed from the medium by the use of a hydrolysing enzyme after cells had formed agglomerates. This treatment left the agglomerates unchanged, but caused a rapid decrease in the activity of UDP galactose transferase, a pre-spore-specific enzyme. This result indicates that cAMP is required even after agglomerate formation to maintain pre-spore differentiation.     

A Highly Sensitive Enzyme Immunoassay for Mouse β Nerve Growth Factor

Abstract: A sensitive two-site enzyme immunoassay system for mouse β nerve growth factor (NGF) was developed, based on the sandwiching of the antigen between anti-mouse β NGF antibody IgG coated to a polystyrene tube and anti-mouse β NGF antibody Fab'-linked β- d -galactosidase (β- d -galactoside hydrolase, EC 3.2.1.23). This method has the following advantages: (a) the procedures are simple and rapid compared to bioassay or two-site radioimmunoassay; (b) antibody Fab'-β- d -galactosidase complex is more stable than ¹²⁵I-labeled antibody; (c) purified β NGF is detectable at a concentration as low as 10 pg/ml. Our enzyme immunoassay was used to examine the levels of NGF in some tissues of mice. The submaxillary gland contained a high concentration of NGF. However, other tissues, such as the heart, brain, and skeletal muscle, and serum did not contain detectable NGF. These results support recent findings by other investigators that NGF was not found in the organs/tissues other than the submaxillary gland of mice.     

Changes in the Amounts of Ribulose Bisphosphate Carboxylase Synthesized and Degraded during the Life Span of Rice Leaf (Oryza sativa L.)

In situ synthesis and degradation of ribulose bisphosphate carboxylase(RuBPCase) were studied quantitatively in the 12th leaf bladeof the rice plant during the life span of the leaf. Levels ofRuBPCase protein were determined by rocket immunoelectrophoresis.The amounts of RuBPCase synthesized and degraded were estimatedusing ¹⁵N tracer. RuBPCase was scarcely recognized in the leaf when the tip ofthe leaf had just emerged from the 1 lth leaf sheath. Then itincreased rapidly and reached its maximum content a week afterthe completion of leaf expansion. At this time RuBPCase accountedfor 56% of the soluble leaf protein N (26% of the total leafN). The total amount of RuBPCase synthesized up to this timewas about 90% of the amount synthesized throughout the leaf'slife. Degradation of RuBPCase started about the time when it reachedthe maximum content and proceeded at a faster rate during senescencethan that of the remaining soluble protein. When the leaf hadsenesced completely, it contained little measurable RuBPCasealthough the total leaf N was about 30% of the maximum level.These results clearly suggest that RuBPCase is a major N componentwhich is used as remobilized N for the growth of young tissues. Influx and efflux of N and the synthesis and degradation ofRuBPCase are discussed in relation to leaf age. (Received February 18, 1983; Accepted June 16, 1983)     

Caprolactam,a light-promoted growth inhibitor in sunflower seedlings

A neutral growth inhibitor, isolated from methanolic extracts of sunflower seedlings, was characterized by spectral data as caprolactam. Light-grown se