Biomedical subjects
Publications and source records attributed to H Amos.
The growth of Ehrlich ascites tumor cells in Swiss mice was modified by the addition of certain naturally occurring sugars to the drinking water or by ip inoculation of mice after infection. D-Mannose, D-ribose, and D-glucosamine produced the most striking antitumor effects, increasing significantly the survival rate in mice so treated.
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The rate of D-glucose uptake by cells that had been deprived of sugar for 18-24h was consistently observed to be 15-20 times higher than that in control cells maintained for the same length of time in medium containing glucose. This increased rate of glucose transport by sugar-starved cells was due to a 3-5-fold increase in the Vmax. value of a low-affinity system (Km 1 mM) combined with an increase in the Vmax of a separate high-affinity system (Km 0.05-0.2 mM). The high-affinity system, which was most characteristic of starved cells, was particularly sensitive to low concentrations of the thiol reagent N-ethylmaleimide; 50% inhibition of uptake occurred at approx. 0.01 mM-N-ethylmaleimide. In contrast with the high-affinity system, the low-affinity system of either the fed cells or the starved cells was unaffected by N-ethylmaleimide. In addition to the increases in the rate of D-glucose transport, cells deprived of sugar had increased rates of transport of 3-O-methyl-D-glucose and 2-deoxy-D-glucose. No measurable high-affinity transport system could be demonstrated for the transport of 3-O-methylgucose, and N-ethylmaleimide did not alter the initial rate. Thus the transport of 3-O-methyglucose by both fed and starved cells was exclusively by the N-ethylmaleimide-insensitive low-affinity system. The low-affinity system also appeared to be the primary means for the transport of 2-deoxyglucose by fed and starved cells. However, some of the transport of 2-deoxyglucose by starved cells was inhibited by N-ethylmaleimide, suggesting that 2-deoxyglucose may also be transported by a high-affinity system. The results of experiments that measured transport kinetics strongly suggest that glucose can be transported by a least two separate systems, and 3-O-methylglucose and 2-deoxyglucose by one. Support for these interpretations comes from the analysis of the effects of N-ethylmaleimide and cycloheximide as well as from the results of competition experiments. The uptake of glucose is quite different from that of 2-deoxyglucose and 3-O-methylglucose. The net result of sugar starvation serves to emphasize these differences. The apparent de-repression of the transport systems studied presents an interesting basis for further studies of the regulation of transport in a variety of cells.
The rate at which chick embryo fibroblasts in primary or secondary culture transport glucose or 3-O-methyl glucose is strongly influenced by the presence of bicarbonate ion in the culture medium. Cells growing or maintained on glucose at physiologic concentration (5.5 mM) have an 8 to 10 fold higher rate of glucose uptake than their counterparts cultivated without bicarbonate. These cells also produce more lactate as a consequence of their more rapid intake of glucose. The hydrogen acceptors, methylene blue and dehydroascorbate added to the culture medium reduce the cell capacity to transport glucose and 3-O-methyl glucose to levels obtaining in the bicarbonate-free medium. There is a concomitant reduction in glucose utilized by cells during 24 hours and further reduction in lactate formed per molecule of glucose metabolized.
Substitution of galactose, xylose, or mannose for glucose in the growth medium of L cells or the addition of equal concentrations of the alternate sugars to glucose-containing medium results in marked morphologic changes. The changes are revealed by conventional staining for light microscopy and by scanning electron microscopy. L cells grow indefinitely on combinations of equal concentrations of glucose and galactose, xylose, or mannose. There appear to be no significant differences in growth rate on glucose compared to the combinations of sugars cited. Cells subcultured from glucose to the combinations while undergoing rapid multiplication show marked morphologic changes by light and scanning electron microscopy within 36 hr. Of particular note are the loss of microvilli; flattening of the cells; assumption of polygonal shape; prominence of nuclei and nucleoli; and changes in the structure and distribution of filopodia. Virtually all cells in the population exhibit the changes noted.
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Normal primary chick embryo fibroblast cultures product a nerve growth-promoting factor which cross-reacts with monospecfic antibody to pure male mouse submaxillary gland nerve growth factor (NGF). When taken together with the earlier demonstration that mouse L2 CELLS AND 3T3 cells also produce an NGF-like protein, these findings suggest that secretion of this factor may be a general property of fibroblast.
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After the addition of insulin to monolayers of chick fibroblasts previously incubated in serum-free medium, the rates of protein and RNA synthesis increase continuously during the first 8-10 h. Little stimulation of DNA synthesis or mitosis results with the addition of insulin alone in contrast to the addition of fresh serum which stimulates both markedly. The stimulation in RNA synthesis does not result from expansion of the nucleotide pool but is correlated with increases in RNA polymerase activity. All major classes of RNA are stimulated; processing of preribosomal RNA to 28S and 18S and the association of this mature RNA with ribosomes appear to occur normally. The kinetics of stimulation of 5S RNA differ from those of the synthesis of 4S and of ribosomal RNA. Insulin and serum appear to affect the synthesis or stability of certain transcripts differentially.
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Glucose entry, as measured by 5-min uptake into the acid-soluble fraction, is enhanced 15-30 times by long-term (12-24 hr) hexose starvation of chick fibroblasts. The rate of galactose accumulation in the cells increases only 5 times under the same conditions of starvation. Several carbon and energy sources that were tested for their effect on this "derepression" can be classified as: (i) those resembling glucose in blocking the "stimulation," (ii) those permitting full "derepression"; and (iii) those partially preventing the enhanced entry. Inhibitors of protein synthesis block enhancement under conditions otherwise conducive to it. We conclude that the glucose and galactose carrier systems are not identical, based largely on the asymmetric "repression" observed when glucose and galactose are compared as "repressors."
Incubation of HeLa cells at 24 degrees C results in the modification of the processing of pre-tRNA to tRNA. Both methylation and size reduction were shown to take place in vitro when purified pre-tRNA was subjected to processing in a cytoplasmic extract of HeLa cells The migration of pre-tRNA from the nucleus to the cytoplasm was not significantly altered at 24 degrees C
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Incubation of HeLa cells at suboptimal temperature has been used to study the synthesis of 45S ribosomal RNA precursor and the individual steps of the subsequent processing to 28S RNA. Below 20 degrees C no detectable 45S RNA is formed. The processing of 45S RNA to 32S RNA ceases around 15 degrees C, and the processing of 32S RNA to 28S RNA is inhibited near 25 degrees C. Prolonged incubation at reduced temperature results in further modification of the processing, resulting in the apparent accumulation of 41S RNA. The products of these reactions at reduced temperature appear normal in that the ribosomal RNA made at 27 degrees C can be isolated from functional polyribosomes in the cytoplasm after a short incubation at 37 degrees C.
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