Induction of new proteins by gamma interferon in cultured human keratinocytes.

Human epidermal keratinocytes incubated with recombinant gamma interferon (r-IFN-gamma) show, on two-dimensional gel electrophoresis, both the appearance of new proteins and the loss of others. Among [35S]methionine-labeled proteins, which are induced in an actinomycin- or alpha-amanitin-sensitive manner, is a prominent group with an apparent relative molecular mass of 53,000 and pI of 5.3-5.8. The synthesis of these proteins continues for at least 4 days in the presence of gamma interferon (IFN-gamma). Over the concentration range tested, up to 670 pM, there is no inhibition of protein synthesis, so the appearance of these proteins cannot be explained by overall inhibition of protein synthesis. Furthermore, at 4 pM we found only minor inhibition of DNA (21%) and RNA (29%) synthesis. Half-maximal induction of the prominent 53 kD proteins occurs at an interferon concentration of 0.8-3.5 pM which may be compared with a range of 1.5-30 pM for HLA-DR induction. The same prominent proteins are also induced by type I interferons. The 53 kD protein complex appears to consist of at least 4 different proteins, one of which is phosphorylated and another one of which is not induced in fibroblasts treated with IFN-gamma. We could obtain no evidence that the proteins were related by glycosylation. The presence of these proteins provides a sensitive means of identifying keratinocytes responding to interferons. Lack of these proteins in normal epidermis indicates that interferon does not play a major role in the control of keratinocyte behavior in sound skin.

Exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA) induces the synthesis of histidine-rich protein (filaggrin) in monolayer cultures of rat keratinocytes.

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent tumor promoter, on epidermal differentiation has been studied by investigating the appearance of the epidermal differentiative marker protein, histidine-rich protein (HRP), in monolayer cultures of rat keratinocytes grown in low-calcium (0.1 mM) medium. Monoclonal antibody, 3F6-6, raised against the 60K HRP (HRPII) derived from the epidermis of the newborn rat was utilized to detect HRP in cultured keratinocytes. As a major component of keratohyalin in the cells of the granular layer in epidermis, HRP is not normally found in a monolayer culture of rat keratinocytes containing a high population of proliferating cells. However, when these cultures were exposed to 10 ng/ml of TPA, DNA and protein synthesis were decreased by up to 95% and 60%, respectively, and the cells in the monolayer culture became HRP-positive within 18-24 h. TPA has been shown to cause a withdrawal of the basal cells from the cell cycle and a commitment of these cells to an accelerated rate of terminal differentiation. Exposure to TPA at the same time that the calcium level of the monolayer cultures was raised to 2.0 mM in order to allow stratification resulted in positive immunostaining for HRP much earlier and much more intensively than in the absence of TPA. TPA also enhanced stratification of these cultures; however, the positive expression of HRP preceded the stratification. These observations support the view that the induction of terminal differentiation occurs independently of stratification and that the process of terminal differentiation (maturation) of basal cells precedes and triggers the movement of these cells to a suprabasal position (stratification).

Macromolecular synthesis is required for stimulation of estrogen synthetase activity by dibutyryl cyclic AMP plus theophylline in choriocarcinoma cell culture.

Estrogen secretion and estrogen synthetase (aromatase) activity are stimulated in human trophoblast cells (JAr line) after addition of 1 mM dibutyryl cyclic AMP plus 1 mM theophylline (dbT) to the growth medium. The data given here show that (a) the aromatase specific activity in homogenized cells increases linearly over a 96 hr incubation period after addition of dbT; (b) addition of inhibitors of macromolecular synthesis, cycloheximide or actinomycin D, to the culture medium at the time of addition of dbT abolishes the stimulation of aromatase activity; (c) mixing dbT-grown cells, containing increased aromatase activity, with control cells does not result in an aromatase specific activity higher than the expected average, suggesting that dbT-grown cells do not contain a factor present in excess which serves to stimulate aromatase in control cells; and (d) NADPH, included in vitro in the aromatase assay or incubated with the cells for 48 hr as well as being present in the aromatase assay, has no stimulatory effect on aromatase specific activity in homogenized cells.

A temperature-sensitive mutant showing two defective functions.

A temperature sensitive growth mutant, 13B11 derived from CHO-K1 has been partially characterized. Upon shift to the nonpermissive temperature (39 degrees C) DNA synthesis slows down, but it is partly resumed after prolonged incubation at 39 degrees C. The radioautographic results suggest that reduction in the rate of DNA synthesis is mainly due to decrease in the proportion of DNA synthesizing cells and that increase in that rate observed afterwards is due to appearance of cells that enter S. Synchronized mutant cells incubated at 39 degrees C from the beginning of G1 enter S with the delay of 6 hours. By incubating the cells at 39 degrees C for a restricted period during the G1 phase, some processes in late G1 are found to be susceptible to the high temperature. Mutant cells shifted up in the middle of S performed cell division whereas cells up-shifted in G1 phase did not, although about a half of these cells divided when they were shifted down in the presence of hydroxyurea. The analysis of DNA content of the cells cultured at 39 degrees C for more than one generation time, revealed the accumulation of nuclei containing DNA in amount of almost 4C level. Accordingly in this mutant the process necessary for cell division is also temperature sensitive. Considering the frequency of the appearance of spontaneous revertants (2 X 10(16)), the apparent two lesions of this mutant might be ascribed to the single mutation.

Biochemical study of minosaminomycin in relation to the kasugamycin group antibiotics.

Minosaminomycin is structurally related to kasugamycin and inhibits protein synthesis in mycobacteria. It also inhibits phage f2 RNA-directed protein synthesis in a cell-free system of Escherichia coli by 50% at 2 x 10(-7) M. It is 100-times more potent than kasugamycin in this system. At 10(-7) M minosaminomycin inhibits EF-T dependent binding of aminoacyl-tRNA to ribosomes by 50%. This effect is markedly diminished if minosaminomycin is added to the assay system after a brief incubation of ribosomes with mRNA. Like kasugamycin, minosaminomycin preferentially inhibits the initiation of protein synthesis directed by phage f2 RNA in vitro and does not cause miscoding. Ribosomes from kasugamycin-resistant mutants Ksg A and Ksg C were as sensitive to minosaminomycin as those from each parent strain. In spite of the strong inhibitory activity of minosaminomycin manifested in cell-free systems of E. coli, this compound inhibits the growth of the organism itself only slightly. This discrepancy could be ascribed to impermeability, as E. coli cells with modified permeability show greater sensitivity to minosaminomycin. There is no indication that the antibiotic is activated in E. coli cells. On the basis of these results, the structural features of these antibiotics is inactivated in E. coli cells. On the basis of these results, the structural features of these antibiotics essential for interaction with ribosomes and for permeability into the cells are discussed.

Time sequence of nuclear pore formation in phytohemagglutinin-stimulated lymphocytes and in HeLa cells during the cell cycle.

The time sequence of nuclear pore frequency changes was determined for phytohemagglutinin (PHA)-stimulated human lymphocytes and for HeLa S-3 cells during the cell cycle. The number of nuclear pores/nucleus was calculated from the experimentally determined values of nuclear pores/micro(2) and the nuclear surface. In the lymphocyte system the number of pores/nucleus approximately doubles during the 48 hr after PHA stimulation. The increase in pore frequency is biphasic and the first increase seems to be related to an increase in the rate of protein synthesis. The second increase in pores/nucleus appears to be correlated with the onset of DNA synthesis. In the HeLa cell system, we could also observe a biphasic change in pore formation. Nuclear pores are formed at the highest rate during the first hour after mitosis. A second increase in the rate of pore formation corresponds in time with an increase in the rate of nuclear acidic protein synthesis shortly before S phase. The total number of nuclear pores in HeLa cells doubles from approximately 2000 in G(1) to approximately 4000 at the end of the cell cycle. The doubling of the nuclear volume and the number of nuclear pores might be correlated to the doubling of DNA content. Another correspondence with the nuclear pore number in S phase is found in the number of simultaneously replicating replication sites. This number may be fortuitous but leads to the rather speculative possibility that the nuclear pore might be the site of initiation and/or replication of DNA as well as the site of nucleocytoplasmic exchange. That is, the nuclear pore complex may have multiple functions.