Sucrose inhibits vascular smooth muscle cell proliferation/migration--protein synthesis is maintained.

We have previously shown that the onset of smooth muscle cell proliferation in tissue cultures is triggered independently of serum. The aim of the present study was to investigate if this process was affected by osmotic stress. Vascular explants from 8-month-old male rats were cultured under serum-free conditions using collagen I as migration substrate. Sucrose was added to the culture medium in concentrations varying from 1 to 3% (30-90 mOsM). Cell migration from aortic explants onto the culture dishes was totally inhibited at a sucrose concentration of 90 mOsM. A significant dose-dependent decline in proliferation was shown for cells in explants pulse labelled with 3H-thymidine. In contrast, pulse labelling with 35S-methionine revealed that protein synthesis was maintained in the presence of sucrose. The results indicate that osmotic pressure affects smooth muscle cell protein synthesis, proliferation and migration.

Autocrine/paracrine activator of cell proliferation: purification of a 4-6 kD compound with growth-factor-like effects in Tetrahymena thermophila.

Extracellular medium from cultures of Tetrahymena thermophila was collected, concentrated and fractionated by ultrafiltration and low-pressure column chromatography. The effect on proliferation of various fractions was tested on cultures of T. thermophila in synthetic medium at low initial cell densities (< 250 cells/ml). In unsupplemented cultures, cells failed to thrive and died before proliferation. Addition of the fraction isolated from the extracellular medium in the molecular weight range of 1-10 kD activated the cells to proliferate. The size of the main compound in this fraction, herein referred to as TPAF, was in the range of 4-6 kD as estimated by SDS-PAGE. Evidence of a dialysable factor produced by cells in the stationary phase inhibiting cell proliferation has also been found.

Effects of protein kinase C activators and staurosporine on protein kinase activity, cell survival, and proliferation in Tetrahymena thermophila.

Autocrine factors prevent cell death in the ciliate Tetrahymena thermophila, a unicellular eukaryote, in a chemically defined medium. At certain growth conditions these factors are released at a sufficient concentration by > 500 cells ml-1 to support cell survival and proliferation. The protein kinase C activators phorbol 12-myristate 13-acetate (PMA) or 1-oleyl 2-acetate glycerol (OAG) when added to 250 cells ml-1 supported cell survival and proliferation. In the presence of the serine and threonine kinase inhibitor staurosporine the cells died both at 250 cells ml-1 in cultures supplemented with either PMA or OAG, or at 2,500 cells ml-1. At 500 cells ml-1 PMA induced the in vivo phosphorylation of at least six proteins. The myelin basic protein fragment 4-14 was phosphorylated in vitro in crude extracts of a culture of 250,000 cells ml-1. Both the in vivo and the in vitro phosphorylation were inhibited by staurosporine.

Cell survival and multiplication. The overriding need for signals: from unicellular to multicellular systems.

There are clear similarities in the control mechanisms for cell survival and multiplication in the two eukaryotes, the ciliate Tetrahymena thermophila and the yeast, Saccharomyces cerevisiae. Cell multiplication in both organisms is activated by the same compounds (phorbol esters, diacylglycerol, tetrapyrroles, etc.). These compounds also affect cell multiplication and other activities in mammalian cell systems. This homology in control mechanisms in two distinct groups of unicellular eukaryotes on the one hand, and in cells from multicellular animals on the other, leads us to propose that these cytoplasmic control mechanisms for cell survival and multiplication originated in the unicellular eukaryotes.

Physiological studies on the effect of Ca2+ on the duration of the lag phase of Saccharomyces cerevisiae.

Cell multiplication and growth of Saccharomyces cerevisiae were followed in 2-ml test tubes containing Wickerham's synthetic medium or very dilute synthetic media supplemented in various ways. The ability of the cell cultures to leave the lag phase and enter the exponential phase of growth was investigated. Multiplication was assessed by microscopical observation. The results showed great differences in times required for the cultures to leave the lag phases and begin multiplication. In Wickerham's medium, all cultures grew well 6 h after inoculation. In the dilute medium, several days elapsed before all the cultures grew. These cultures went into exponential growth with approximately first order kinetics. In the unsupplemented medium, the 'half-lives' in the lag phase were about 28 h. Addition of either Ca2+ or Ca2+ plus A23187 (calcimycin) reduced the half-lives to 10 and 6 h, respectively. The doubling times in the exponential phases of growth were not shortened by these additions. We suggest that Ca2+ plays a crucial role as a signal to switch on the mode of cell proliferation in S. cerevisiae.

Survival of Tetrahymena thermophila at low initial cell densities. Effects of lipids and long-chain alcohols.

Cells of the ciliate Tetrahymena thermophila failed to establish cultures in lipid-free standard synthetic nutrient medium if the initial population density was 250 cells per ml or less. These cells died within 10 h, but were saved and formed dense cultures if their medium was supplemented with 10 micrograms per ml of either certain phospholipids, 1,2-di-, 1-monoglycerides, fatty acids, long-chain alcohols, or sterols. Cell multiplication was followed in cultures in which the standard synthetic medium was supplemented with a selection of the compounds listed above. It was observed that the cells in the supplemented cultures in their exponential phases of growth had about the same average doubling times as control cells starting multiplication at 10-fold higher initial cell densities in lipid-free medium. These cells have been grown for decades in lipid-free synthetic nutrient media at short (ca. two-three h) doubling times. Therefore lipids have been considered nutritionally non-essential for growth and multiplication of these cells. We propose that those compounds that rescue the cells at low cell densities act as "proliferation signals," sensu lato. This effect of lipids and long-chain alcohols has so far remained unnoticed.

Signalling in cell growth and death: adequate nutrition alone may not be sufficient for ciliates. A minireview.

The initial inoculum level of Tetrahymena in a chemically defined medium determines whether the cells are capable of exponential growth. Below 750 cells ml-1, the cells fail to go into exponential growth and will die within about 20 hours. By adding certain growth stimulants, death can be postponed and the cells begin to grow after a delay which depends on the intensity of the signal. The implication is that autocrine growth factor expression might be required for cells to grow, and that these stimulants either assist its production or lower the cell threshold to its action. The findings in Tetrahymena are reviewed, and the advantages of having a cell system in which all the components of the medium can be carefully controlled is recognised.

Porphyrin rings and phospholipids: stimulators of cloning efficiency in certain species of Tetrahymena.

Species of Tetrahymena, including T. vorax, T. thermophila, T. pyriformis, and T. pigmentosa, were tested for cloning efficiency in proteose peptone and in synthetic nutrient media to which were added hemin, protoporphyrin IX, chlorophyllin, or asolectin, an impure mixture of phospholipids. All species could be cloned with high efficiency in the crude media. In unsupplemented synthetic medium the cloning efficiencies were 0-10%, around 50%, around 50%, and 90-100% for T. thermophila, T. vorax, T. pyriformis, and T. pigmentosa, respectively. The first three were all stimulated to 90-100% by addition of the porphyrin or phospholipid compounds mentioned above. Uroporphyrin III and coproporphyrin I and III had no effect. We suggest that cells unable to form clones suffer from a lack of cellular energy. This situation may be alleviated by our additions, certain porphyrin rings may be built into cytochromes and phospholipids may be used as fuel. Thus, the synthetic media used so far for these ciliates have not been optimal.