Biomedical subjects

H Green

Publications and source records attributed to H Green.

At least 199 records · Page 11Linked to original sources

PubMed1975

Effects of withdrawal of a serum stimulus on the protein-synthesizing machinery of cultured fibroblasts.

3T6 cells resting in medium containing 0.5% serum were stimulated to prepare for multiplication by the addition of medium containing 10% serum. After a number of hours, when the rate of preribosomal RNA synthesis, total RNA content (mainly ribosomal), and the cytoplasmic content of poly A (a measure of poly A(+) mRNA) were considerably elevated, the serum-rich medium was withdrawn, and the original medium replaced. The rate of preribosomal RNA synthesis began to drop within 30 minutes, but required a much longer time to fall to a new resting level. When the serum-rich medium was withdrawn after 12 hours of stimulation, the total RNA content required 12-18 hours to fall to the resting level, whereas cytoplasmic poly A content and the rate of protein synthesis declined more rapidly, reaching a new resting level within eight hours. During the 12 hours following withdrawal of the serum-rich medium an appreciable fraction of the cells initiated DNA synthesis. Presumably, the cellular preparations for DNA synthesis cannot be immediately reversed because of the inertial factors related to the protein-synthesizing machinery.

Blood↗

PubMed1975

Increasing content of poly A(+) mRNA of serum-stimulated cells in the absence of ribosome synthesis.

When 3T6 cells undergo a serum-induced transition from resting to growing state, the number of ribosomes and the amounts of mRNA increase as the cells prepare for DNA synthesis. We have examined the effect of preventing ribosome synthesis during this transition. When resting cells are stimulated to grow in the presence of 5-fluorouridine, mRNA accumulates normally during the first eight hours, though new ribosome formation is completely blocked by the drug. At later times, mRNA continues to accumulate, but at a reduced rate. The ratio of poly A(+) mRNA to rRNA increases from the value characteristic of resting 3T6 (1.8%) to that of growing 3T6 (2.7%) by five hours, and continues to increase to abnormally high values after this time. Although labelling of tRNA is not affected after brief exposure of cells to fluorouridine, the drug prevents the later accumulation of tRNA that ordinarily occurs following serum stimulation of resting cells. This failure of accumulation is not the result of increased lability of fluorinated tRNA, but is probably due to failure of the transcription rate of pre-tRNA to increase. It is possible that this effect might be due to a regulatory system coupling tRNA content to ribosome content. In cultures stimulated with serum in the presence of fluorouridine the rate of protein synthesis increases with poly A(+) mRNA content during the first eight hours; it then fails to increase further, possibly because ribosomes become rate-limiting.

Blood↗

PubMed1975

A human-mouse somatic hybrid line selected for human deoxycytidine deaminase.

A new selective medium has been developed for cells containing the enzyme deoxycytidine deaminase. This medium contains hypoxanthine, aminopterin, and 5-methyldeoxycytidine (HAM medium). To survive in the presence of the aminopterin, the cells must utilize deoxycytidine deaminase to convert the 5-methyldeoxycytidine to thymidine. The cells must also have thymidine kinase and hypoxanthine phosphoribosyltransferase. A mouse cell line deficient in deoxycytidine deaminase has been isolated from a deoxycytidine kinase-deficient line, using 5-bromodeoxycytidine as the selective agent. A hybrid line between this double mutant and a human diploid fibroblast was isolated in HAM medium. The hybrid line contains the chromosomes expected of a human-mouse hybrid. The deoxycytidine deaminase isozyme patterns on cellogel show that the human-mouse hybrid cell line produces an enzyme with an electrophoretic mobility intermediate between that of the human and that of the mouse.

Aminopterin↗

PubMed1975

An established preadipose cell line and its differentiation in culture. II. Factors affecting the adipose conversion.

When cells of the established preadipose line 3T3-L1 enter a resting state, they accumulate triglyceride and convert to adipose cells. The adipose conversion is brought about by a large increase in the rate of triglyceride synthesis, as measured by the incorporation rate of labeled palmitate, acetate, and glucose. In a resting 3T3 subline which dose not undergo the adipose conversion, the rate of triglyceride synthesis from these precursors is very low, and similar to that of growing 3T3-L1 cells, before their adipose conversion begins. If 3T3-L1 cells incorporate bromodeoxyuridine during growth, triglyceride synthesis does not increase when the cells reach a stationary state, and triglycerides do not accumulate. As would be expected from their known actions on tissue adipose cells, lipogenic and lipolytic hormones and drugs affect the rate of synthesis and accumulation of triglyceride by 3T3-L1 cells, but in contrast to bromodeoxyuridine, these modulating agents do not seem to affect the proportion of cells which undergoes the adipose conversion. Insulin markedly increases the rate of synthesis and accumulation of triglyceride by fatty 3T3-L1 cells, and produces a related increase in cell protein content. Of 20 randomly selected clones isolated from the original 3T3 stock, 19 are able to convert to adipose cells. The probability of such a conversion varies greatly among the different clones, in most cases being much lower than for 3T3-L1; but once the conversion takes place, the adipose cells produced from all of the 19 clones appear similar. The adipose conversion would seem to depend on an on-off switch, which is on with a different probability in different clones. This probability is quasistably inherited by the clonal progeny.

Acetates↗

PubMed1975

Changes in RNA in relation to growth of the fibroblast. III. Posttranscriptional regulation of mRNA formation in resting and growing cells.

The proportion of hnRNA processed into cytoplasmic mRNA in resting and growing 3T3 and 3T6 cells has been investigated by measuring the efficiency of transfer of nuclear poly(A) to the cytoplasm. When nuclear poly(A) is either labeled continuously with precursor or pulse labeled and then chased with cordycepin, growing cells transfer approximately twice as much of the poly(A) from the nucleus to the cytoplasm as do resting cells. When cells undergo a serum-induced transition from the resting to growing state, the efficiency of poly(A) transfer is increased to that characteristic of growing cells by 3 hr after the addition of the serum. On the other hand, the proportion of hnRNA which is polyadenylated and the total nuclear poly(A) content are the same in resting and growing cells. It appears that the principal means by which the cell regulates its poly(A)+ mRNA content in the growing and resting states is by regulating the efficiency which nuclear poly(A)+ RNA is converted to cytoplasmic mRNA.

Adenine↗

PubMed1975

Formation of a keratinizing epithelium in culture by a cloned cell line derived from a teratoma.

From a transplantable mouse teratoma it has been possible to derive an established keratinizing cell line (XB) which grows well in cultures containing lethally irradiated 3T3 fibroblasts at the correct density. Single cells of the keratinizing line grow into colonies each consisting of a stratified squamous epithelium. The keratinizing nature of the colonies has been demonstrated by specific staining with Rhodanile blue, and by light and electron microscopy of sections through the colonies. A function of fibroblasts appears to be a strict requirement for keratinization and an important though less strict requirement for cell growth. The fibroblast function can be carried out by medium harvested from 3T3 cultures. It is possible to detect keratinizing colonies in primary cultures of disaggregated teratoma cells combined with 3T3 cells. Such colonies appeared in cultures of a transplantable teratoma with an overall frequency of 6 X 10(-6) of the cells plated. Nonkeratinizing colonies of cells with otherwise very similar appearance were about 10 fold more abundant. Since both the keratinizing and the related nonkeratinizing colonies can be identified in the living state, it is possible to isolate them from the primary cultures.

Cell Differentiation↗

PubMed1975

Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells.

Human diploid epidermis epidermal cells have been successfully grown in serial culture. To initiate colony formation, they require the presence of fibroblasts, but proliferation of fibroblasts must be controlled so that the epidermal cell population is not overgrown. Both conditions can be achieved by the use of lethally irradiated 3T3 cells at the correct density. When trypsinized human skin cells are plated together with the 3T3 cells, the growth of the human fibroblasts is largely suppressed, but epidermal cells grow from single cells into colonies. Each colony consists of keratinocytes ultimately forming a stratified squamous epithelium in which the dividing cells are confined to the lowest layer(s). Hydrocortisone is added to the medium, since in secondary and subsequent subcultures it makes the colony morphology more oderly and distinctive, and maintains proliferation at a slightly greater rate. Under these culture conditions, it is possible to isolate keratinocyte clones free of viable fibroblasts. Like human diploid fibroblasts, human diploid keratinocytes appear to have a finite culture lifetime. For 7 strains studied, the culture lifetime ranged from 20-50 cell generations. The plating efficiency of the epidermal cells taken directly from skin was usually 0.1-1.0%. On subsequent transfer of the cultures initiated from newborns, the plating efficiency rose to 10% or higher, but was most often in the range of 1-5% and dropped sharply toward the end of their culture life. The plating efficiency and culture lifetime were lower for keratinocytes of older persons.

Age Factors↗

PubMed1975