Changes in the nuclear structure in the radiation-sensitive CHO mutant cell, xrs-5.

The structural organization of the cell nucleus was investigated by transmission electron microscopy in the radiosensitive Chinese hamster ovary (CHO) cell mutant, xrs-5 (D0 = 45 cGy), relative to parental K1 cells (D0 = 200 cGy). In 99% of all xrs-5 cells, the outer layer of the nuclear envelope was separated from the inner layer, while 96% of K1 cells had closely apposed layers. This separation of the inner and outer layers of the nuclear envelope in xrs-5 cells was not explained by an increased susceptibility of xrs-5 cells to osmotically induced changes because (1) xrs-5 cells retained the altered nuclear periphery even when several different fixation protocols were used and (2) xrs-5 cells were not more susceptible to cell lysis as measured by trypan blue dye exclusion or by the extracellular presence of lactate dehydrogenase. The difference in the morphological organization in the nuclear periphery of xrs-5 cells correlated with the radiation sensitivity of the cells; xrs-5 cells which spontaneously reverted to a radiation sensitivity similar to that of K1 cells also reverted to a nuclear morphology similar to that of K1 cells. The inner and outer layers of the nuclear envelope were retained in nuclear scaffolds isolated from K1 and xrs-5 cells, indicating that components of the nuclear periphery are part of the nuclear scaffold. These data show that xrs-5 cells have an altered nuclear periphery which correlates with the radiation sensitivity of the cells. The separation of the layers of the nuclear envelope may represent an altered template for repair of DNA damage at the nuclear scaffold and thus may play a role in the defective repair of X-ray-induced DNA double-strand breaks in xrs-5 cells.

Morphological and metabolic responses of embryonic hearts to administration of exogenous L-thyroxine.

Injection of 1 microgram L-thyroxine (T4) into the yolk sacs of embryonated chicken eggs at 3 to 6 days of incubation not only induced cardiomegaly but also instigated more rapid differentiation of the heart as an organ and of the individual myocytes per se. Myocytes showed evidence of responding to this dose of exogenous T4 as early as 5 to 6 days of incubation, even though endogenous T4 was not normally forthcoming (in amounts sufficient to provoke organ changes) until 11 to 12 days of incubation. By 7 days of incubation the hearts, conditioned by a single 1 microgram dose of T4, exhibited larger areas occupied by myofibrillar material than controls. Measurements, beginning at 9 days of incubation, indicated the presence of greater amounts of RNA, total non-lipid solids and total lipids. Early increases in DNA in T4-conditioned embryos, compared with controls, indicated that hypertrophic hearts had reached this condition, at least in part, by increased cell division. By 12 days of incubation, hearts pre-treated with T4 showed conversion of many mitochondria to vesicles resembling smooth endoplasmic reticulum. No evidence of classical sarcoplasmic reticulum was seen through hatching.

Effects of thyroxine on mitochondrial morphology and uptake of 35S in embryonic chicken livers.

Ultrastructural analyses of reactions of mitochondria in hepatocytes of chicken embryos to low levels of exogenous thyroxine (T4) reveal that such reactions (overall swelling accompanied by disruption of crest geometry) first take place at about 10 days of incubation, T4 having been administered on the 6th day. Physically altered mitochondria may be seen after 11-12 days of incubation but are no longer evident by 13 days. Correlated with the initial evidence of T4 effects on mitochondria at 10 days of incubation is a spurt in hepatocyte proliferation. The time correlation observed between T4 induced mitochondrial changes in morphology and abrupt increases in rates of cell proliferation, suggests that liver nuclear receptors for thyroxine are unavailable prior to 9-10 days of incubation. Golgi complexes within the hepatocytes appear to be especially active in the production of electron-opaque vesicles from at least the 8th day of incubation to 11-12 days. Uptake of 35S (probably into chondroitin sulphates) was found to be fifteen times greater on the 8th day of incubation than at 15 days. This correlates with the period of heightened activity of the Golgi complex. In livers exposed to T4 on the 6th day, uptake of 35S was higher on the 9th and 10th days of incubation as compared to controls.

Effects of chondroitin-4-sulphate on ultrastructure of ovarian granulosa cells.

Granulosa cells from medium sized porcine ovarian follicles exhibit inhibition of progesterone secretion after both 3 and 6 days of incubation in the presence of 2 mg chondroitin-4-sulphate (C-4-S)/ml. After 3 days of incubation some relatively small and undifferentiated granulosa cells are present in the above cultures. Six days following initiation of incubation in the presence of 2 mg C-4-S/ml many of the larger granulosa cells taken from medium sized follicles reveal lysis and/or resorption of material (probably lipoprotein) from their originally dense staining bodies as well as other indications of stress and degeneration.

Uptake of melanosomes and increased melanin formation by cultured melanoma cells after treatment with isolated melanosomes.

Melanosomes isolated from a subcutaneous Harding-Passey mouse melanoma and purified by density gradient centrifugation were labelled in vitro with 14C-tyrosine or 3H-dihydroxyphenylalanine in the melanin portion. Incubation of monolayer cultures of Harding-Passey melanoma cells during exponential growth phase (wherin cells contained relatively few melanosomes) with isolated and labelled melanosomes during a time-period of up to 3 days resulted in rapid cellular uptake of label (reaching a saturation level after about half a day). Following a lag period of several hours, the melanin content rose near-linearly in the course of 3 days. Comparison of curves of uptake of radioactivity and melanin concentration indicates that the latter rise is due primarily to newly formed melanin. Ultrastructural studies revealed a strikingly increased number of melanosomes in melanosome-treated cells. Some of these appeared to be the result of phagocytotic uptake. In fact, invagigations of the plasma membrane containing exogenous melanosomes were observed. Since most intracellular melanosomes were localized directly in the cytoplasmic matrix, dissolution of the phagosome membrane appeared to have taken place. Aggregates of melanosomes either surrounded by a membrane or free in the cytoplasm were also observed. These bodies might represent phagolysosomes or/and centres for the formation of new melanosomes. The combined biochemical and ultrastructural findings suggest stimulated melanogenesis induced by phagocytized melanosomes.

On formation of melanosomes in a cultured melanoma line.

Melanosomes are present in abundant numbers in HPM-73 melanoma cells maintained in surum-containing cultures. However, withholding of serum for 3 days caused severe retardation of development of these organelles. Addition of chondroitin-4-sulphate (0.4 mg/ml) to serum-containing culture media for 3 days resulted in greater numbers of premelanosomes and melanosomes relative to mitochondria. The same amount of chrondroitin-4-sulphate (0.4 mg/ml) added to serum-free cultures also resulted in an increased ratio of premelanosomes and melanosomes to mitochondria but, within the 3-day period many of these organelles did not attain the pattern of maturity evident in melanoma cells reared in serum-supplemented media. By exploitation of slowed rates of development of melanosomes in cultures (especially, but not exclusively, in serum-free samples) it was possible to examine various early developmental forms of these organelles. It was found that in this cell line mitochondria appear to be involved in the process of melanosome formation.