Fluorescence microscopy demonstration of mitochondria in tissue culture cells using berberine]

The possibility of fluorescence microscopical examination of mitochondria in living animal cells using fluorochrome berberine sulphate is shown. At concentrations of 30--50 g per ml the chemical is accumulated selectively in mitochondria of living cells. The specificity of berberine sulphate accumulation in mitochondria was shown by comparative phase contrast and fluorescence microscopy. The advantages of the method is its high sensitivity and simplicity, especially when mitochondria can not be examined by the phase contrast microscopy.     

Argon laser micro-irradiation of mitochondria in rat myocardial cells in tissue culture

Three types of lesions produced by argon laser micro-irradiation of single mitochondria are described. A correlation between lesion severity and optical phase density and/or laser output power was observed. Cell survival was generally not affected by mitochondrial irradiation. As many as ten mitochondria were irradiated without cell death resulting. Absorption of the laser radiation was attributed to the natural chromophores, cytochromes c and c₁.     

Cytological studies of the entry of enzymes into cells in tissue culture

• 1.1. The uptake of enzymes under in vitro conditions in a selection of normal and malignant cell types has been investigated using chymotrypsin, a wheatgerm lipase preparation, and cytotoxic aliesterases isolated by column chromatography from the latter preparation.
• 2.2. Whereas chymotrypsin was not taken up by pinocytosis, uptake of the wheat-germ enzymes was very active in all the cell types studied, suggesting that pinocytosis was stimulated by a specific interaction of the wheat-germ enzymes with the cell.
• 3.3. An affinity of the wheat-germ enzymes for the cell membrane has been demonstrated, and possible explanations are considered.
     

Chemically facilitated microinjection of proteins into intact monolayers of tissue culture cells

Microinjection of physiologic quantities of macromolecules into tissue culture cells can facilitate the study of the biological effects of such macromolecules. In this communication, we describe a chemical technique which can be used to microinject proteins into monolayers of intact cells. Protein is loaded into erthrocyte ghosts, and the ghosts are then fused to the monolayer with polyethylene glycol 1000. Receipient cells can be injected with an efficiency of greater than 90% and contain an average of 3.8 X 10(6) microinjected molecules per cell. This technique circumvents certain problems encountered in virus-induced microinjection.     

Epithelial cells in tissue culture

In this review the characteristics of established renal and intestinal epithelial cell lines are described by summarizing the accumulated literature about specific properties retained by the cells in tissue culture. Furthermore, brief examples are given for the use of cultured epithelia as model systems to study epithelial transport and metabolic functions, epithelial cell polarity, and aspects of the differentiation and maturation of epithelia by physiological, biochemical and genetic, or cell and molecular biological approaches.     

The fate of nuclear RNA migrated into isolated mitochondria

Nuclear RNA migrating into isolated mitochondria under appropriate conditions may be reisolated intact. From electrophoretic evidence on polyacrylamide gels it is concluded that the size of the nuclear RNA species migrating into the mitochondria is approximately 9S.     

Calcium in myocardial tissue and isolated mitochondria from a teleost

A method is presented for isolating cardiac mitochondria from bony-fish. Calcium levels in ventricular whole tissue and isolated mitochondria of Gadus virens L. are determined by atomic absorption flame spectroscopy, and were found to be about 8 and 16 nmolCa/mg prot., respectively. In conclusion, the calcium concentration within the myocardial mitochondria in this species may be nearly three times higher than at the outside, and probably these structures serve as a "calcium sink". The results are compared with those previously reported for mammals.     

Energetics of Ehrlich ascites mitochondria: membrane potential of isolated mitochondria and mitochondria within digitonin-permeabilized cells

Ehrlich ascites tumour cells were treated with digitonin so that they became permeable for low-molecular-weight compounds but, at certain concentrations of digitonin, retained most of their cytoplasmic proteins. Respiration of mitochondria with exogenous substrates and their membrane potential could thus be measured in situ by means of oxygen electrode and tetraphenylphosphonium-sensitive electrode, respectively. The results were compared with data from similar measurements on mitochondria isolated from such digitonin-permeabilized cells. Isolated mitochondria and mitochondria in situ oxidized succinate at similar rates and developed membrane potential of comparable magnitude. Both preparations also exhibited an identical nonlinear relationship between resting state respiration (titrated with a respiratory inhibitor) and the membrane potential. In the cells permeabilized with low concentrations of digitonin (i.e., retaining most of cytoplasmic proteins) and suspended in medium containing NaCl and other major anions and cations at concentrations close to those in mammalian plasma, anaerobiosis did not produce a decrease in the mitochondrial membrane potential, which was collapsed only after a subsequent addition of oligomycin. In this medium, glucose had little effect on either respiration or the membrane potential.     

Selective importation of RNA into isolated mitochondria from Leishmania tarentolae

All mitochondrial tRNAs in kinetoplastid protozoa are encoded in the nucleus and imported from the cytosol. Incubation of two in vitro-transcribed tRNAs, tRNA(Ile)(UAU) and tRNA(Gln)(CUG), with isolated mitochondria from Leishmania tarentolae, in the absence of any added cytosolic fraction, resulted in a protease-sensitive, ATP-dependent importation, as measured by nuclease protection. Evidence that nuclease protection represents importation was obtained by the finding that Bacillus subtilis pre-tRNA(Asp) was protected from nuclease digestion and was also cleaved by an intramitochondrial RNase P-like activity to produce the mature tRNA. The presence of a membrane potential is not required for in vitro importation. A variety of small synthetic RNAs were also found to be efficiently imported in vitro. The data suggest that there is a structural requirement for importation of RNAs greater than approximately 17 nt, and that smaller RNAs are apparently nonspecifically imported. The signals for importation of folded RNAs have not been determined, but the specificity of the process was illustrated by the higher saturation level of importation of the mainly mitochondria-localized tRNA(Ile) as compared to the level of importation of the mainly cytosol-localized tRNA(Gln). Furthermore, exchanging the D-arm between the tRNA(Ile) and the tRNA(Gln) resulted in a reversal of the in vitro importation behavior and this could also be interpreted in terms of tertiary structure specificity.     

Irradiation of cells in tissue culture

Summary Cultures of human amnion were employed to check the hypothesis that cell strains with heteroploid chromosome counts regularly produce giant cells within 12 days following treatment with 2000 r and 4000 r of gamma irradiation from a cobalt source, while this response has not been obtained from primary cultures whose cells were presumed to be diploid.The giant cell reaction not only was obtained from two transfer passage lines of a well-established amnion strain developed at Berkeley (No A 185-21C-26 and No A 185-21C-45) but was also found for a 20-day second passage culture of amnion. Since this line has continued to reproduce at a rapid rate, it is presumed to have assumed the features of a typical strain within the period of observation. This impression was reinforced by the finding that the chromosome number for 32 cells fixed on the 35th day had a modal value of 67.In contrast, both untrypsinized and trypsinized spindle cells in primary cultures as well as unaltered epithelial elements which had not been subcultured gave no evidence of giant cell formation 12 days after exposure to 2000 r and 4000 r from a Cobalt⁶⁰ source.These data lend evidence that giant cell formation is related to the chromosomal constitution of the irradiated elements.This research was supported by funds provided under Contract AF 18(600)-1263 with the School of Aviation Medicine, USAF, Randolph Air Force Base, Texas.Fellow of the Instituto de Alta Cultura and the Fundacão Calouste Gulbenkian of Lisbon, Portugal.Tobacco Industry Research Committee Fellow.     

Irradiation of cells in tissue culture

Summary Strain cultures of the cervical carcinoma HeLa (Puck-clone), human fetal intestinal epithelium (Henle) and adult human skin (NCTC clonal 2414) were used in Rose chambers for gamma irradiation at 2000 r and 4000 r from a Cobalt⁶⁰ source.Phase contrast, time-lapse cinematographic records generally made from one to 5 days following irradiation yielded a total of more than 6000 feet of 16 mm film records for analysis.Cell enlargement was regularly observed. Telo-reduplication, including a second division is reported. Multinucleation arising from cells with single and multiple nuclei producing one or two daughter cells with numerous micronuclei was found for all three strains.It is believed that these mitotic anomalies represented a quantitative rather than a qualitative difference between irradiated and control cultures.The method permits an accurate assessment of the divisional potentialities of living cells during long periods of life in vitro under experimental conditions.This research was supported by the USAF under Contract No. AF 18(600)-1263, monitored by the School of Aviation Medicine, USAF, Randolph Air Force Base, Texas.Captain, USAF (MSC).