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Comparison of the sensitivity of human embryo kidney cells, HeLa cells, and WI38 cells for the primary isolation of viruses from the eye.

A comparison has been made of the efficiency of human embryo kidney (HEK) cills, HeLa cells, and WI38 cells for the isolation of viruses from the eyes of patients suffering from acute conjunctivitis or keratoconjunctivitis. From a total of 99 specimens 21 adenoviruses (serotypes 3, 4, 7, 8, and 13) were isolated in HEK cells, eight (serotypes 3 and 8) in HeLa cells, and four (serotype 3) in WI38 cells. Of the ten herpes simplex viruses isolated nine were recovered in HEK cells, seven in WI38 cells, and none in HeLa cells. The combination of HeLa cells and WI38 cells is not considered an adequate alternative to the difficult-to-obtain HEK cells for the isolation of viruses from the eye.

Adenoviridae

Properties of DNA ligase from uninfected and virus-infected HeLa cells.

HeLa cells contain a high M.W. form of DNA ligase which can be completely converted to a low M.W. form. Stokes radius, frictional ratio, sedimentation coefficient, molecular weight, pH dependence, and heat inactivation rate of the two forms have been studied. The major properties of the two forms of DNA ligase in HeLa cells (in particular molecular weights and pH dependence) resemble those of the "dimer" and "monomer" structures described in cultured human cells (Pedrali, G., Spadari, S., Ciarrocchi, G., Pedrini, M., Falaschi, A. (1973) Eur. J. Biochem., 39 343) . In synchronized HeLa cells, the DNA ligase shows a two fold increase during S phase and parallels the increase in the DNA synthesis rate. DNA ligase increases in parallel with viral DNA synthesis after infection of HeLa cells with vaccinia and Herpes virus but its cofactor requirements and physical properties (including the dimer leads to monomer conversion) are unchanged, suggesting that the newly formed ligase is not virus-coded.

HeLa Cells

Properties of beta-adrenergic receptors in untreated and butyrate-treated Hela cells.

HeLa cells contain receptors on their surface which are beta-adrenergic in nature. The binding of (-)-[3H]dihydroalprenolol is rapid, reversible, stereospecific and of relatively high affinity. The HeLa cells also contain an adenylate cyclase which is activated by (-)-isoproterenol greater than (-)-epinephrine greater than (-)-norepinephrine. The adenylate cyclase of HeLa is also activated by guanyl-5'-ylimidodophosphate (Gpp(NH)p), a nonhydrolyzable analogue of GTP. Inclusion of both (-)-isoproterenol and Gpp(NH)p leads to approximately additive rather than synergistic activation of adenylate cyclase. After treatment of HeLa cells with 5mM sodium butyrate there is an increase in the number of beta-adrenergic receptors, but not in their affinity, which is reflected in an increased ability of (-)-isoproterenol to activate adenylate cyclase. Other properties of the beta-adrenergic receptor including association and dissociation rates, temperature optimum of adenylate cyclase and response to Gpp(NH)p are relatively unaffected by butyrate pretreatment of the cells.

Adenylyl Cyclases

Heparan sulfate at the surface of HeLa cells.

HeLa cells, labeled with Na235SO4, release into the culture medium 35SO4 bound to plasma membrane vesicles next to 35SO4-glycoproteins and free 35SO4. Plasma membrane vesicles, experimentally produced by treatment with formaldehyde, contain 35SO4 and their surface can be stained with high iron diamine. Scanning of chromatograms of the trypsinate from labeled cells demonstrates radioactivity on the spot of heparan sulfate. It is concluded that HeLa cells synthesize heparan sulfate, which is incorporated at the plasma membrane and released by shedding of small vesicles.

Cell Membrane

Membrane isolation on polylysine-coated beads. Plasma membrane from HeLa cells.

HeLa cell plasma membranes have been purified after binding cells to polylysine-coated polyacrylamide beads. Cell attachment to beads and membrane recovery were maximal in a sucrose-acetate buffer, pH 5.0, at 25 degrees C. Measurements of ouabain-sensitive NaK-adenosine triphosphatase, membrane-bound 125I-wheat germ agglutinin, and chemical analyses showed that membranes on beads were of comparable or greater purity than membranes isolated by conventional methods. Because the isolation procedure is rapid (approximately 2.5 h), and produces membranes whose protoplasmic surfaces are fully exposed, it should be a useful supplement to standard isolation techniques.

Cell Count

Potentiation of radiation effects in hypoxic HeLa cells.

HeLa cells were irradiated with two doses of gamma rays: the first dose was delivered under hypoxic conditions and the second dose was delivered under oxygenated conditions. Increased inactivation of cellular proliferative capacity as a function of intervening time was obtained when hypoxic conditions were maintained between doses. Hypoxic protection from ionizing irradiation can be counteracted by manipulating postirradiation conditions.

Cell Division

Chloramphenicol-induced loss of mitochondrial DNA polymerase activity in HeLa cells.

HeLa cells exposed to chloramphenicol for approximately one cell generation were found to contain a mitochondria-associated DNA polymerase with a significantly lower specific activity than that of control cells. This observation was not due to the presence of inhibitors in mitochondrial DNA polymerase preparations of chloramphenicol-treated cell cultures. In addition, there was no accumulation of a typical mitochondrial DNA polymerase in the post-mitochondrial supernatant of drug-treated cells.

Chloramphenicol

Effect of cytosine arabinoside triphosphate on the incorporation of 3H-thymidine in mitochondria isolated from HeLa cells.

HeLa cell mitochondria were allowed to incorporate 3H-thymidine in a cell free system and the effect of ethidium bromide, cytosine arabinoside and cytosine arabinoside triphosphate on the labeling of mitochondrial DNA was studied. The labeled products, isolated by sedimentation velocity in CsCl-ethidium bromide two-step gradients, showed similar sedimentation profiles as in vivo labeled mtDNA. Cytosine arabinoside triphosphate and ethidium bromide strongly inhibited the labeling of mitochondrial DNA, whereas cytosine arabinoside appeared to be much less effective. Tritiated deoxycytidine was found to be incorporated by isolated mitochondria, whereas cytosine arabinoside was shown to enter the mitochondrial acid-soluble pool but not to be incorporated in acid-insoluble form. These results are in agreement with the previously reported findings of in vivo experiments.

Arabinofuranosylcytosine Triphosphate

Effect of pentadecan-2-one on lipid metabolism in HeLa cells.

HeLa cells exposed to trace amounts of pentadecan-2-one showed changes in metabolism of 1(-14)C-palmitate. These changes consisted of an increased incorporation of radioactivity into the triglycerides and free fatty acids and a decreased 14C incorporation into the ether moiety of alk-1-enyl acyl phosphoglycerides. Chemical analysis of the several lipid fractions showed a threefold increase in triglyceride content but no change in the amount of alk-1-enyl acyl or diacyl phosphoglycerides in the treated cells. Pentadecan-2-one added to the culture medium apparently gains entrance to the cell since both pentadecan-2-one and pentadecan-2-ol were detected in the ketone-treated cells and their culture medium.

Fatty Acids, Nonesterified

Ingestion and survival of Y. pseudotuberculosis in HeLa cells.

HeLa cells were infected with Yersinia pseudotuberculosis for 0.5-3 h. Intracellular bacteria could then be demonstrated by three different techniques: viable count, fluorescent-antibody staining and electron microscopy. Most of the bacteria seemed to be viable, since there was a good positive correlation (0.94) between viable and fluorescent bacteria. The bacterial uptake seemed to be mediated by a phagocytic-like procedure. The intracellular bacteria seemed to reside in vacuoles some of which increased in size as a function of time. The kinetics of infection was studied after addition of 10(7) or 10(9) bacteria per cell culture (2 X 10(6) cells). After a lag period of about 30 min there was a linear increase of intracellular bacteria, and this uptake proceeded for 1-2 h until most of the bacteria were ingested or an upper limit of ingested bacteria was reached. The upper limit was calculated to be a mean of 60 per infected cell in the cell culture. More than 90% of the cells could be infected and a reasonable number of the bacteria survive in the cells for at least 3 days, as demonstrated by the viable-count technique. The bacteria-cell system may be used to study, for example, the effect of antibiotics or antibodies on intracellular bacteria and pathogenicity of intracellular diseases.

Cytoplasm

Structure and ribonucleoprotein staining of kinetochores of colchicine-treated HeLa cells.

HeLa cells treated with colchicine show a lengthening of kinetochores lacking an inner layer. Combined EDTA- and ribonuclease treatment (BERNHARD 1969) indicate the presence of ribonucleoprotein in the dense outer layer as well as in the surrounding fine fibrillar mass. Reconstructions from serial sections reveal a band-like enlargement of these layers around the primary constriction during the inhibition of mitosis by colchicine.

Centromere

Induction of functional beta-adrenergic receptors in HeLa cells.

HeLa cells contain beta-adrenergic receptors that are characterized by specific binding of I[3H]dihydroalprenolol, increased 3':5'-cyclic AMP production in intact cells after incubation with l-isoproterenol, and increased adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity in the presence of l-isoproterenol. After cells were cultured with butyrate, the number of beta-adrenergic receptors, cyclic AMP production in intact cells, and adenylate cyclase activation by l-isoproterenol were increased severalfold over those of untreated cells. The increase involved the induction of synthesis of new receptor molecules with identical affinities for l-[3H]-dihydroalprenolol; all three processes were blocked by cycloheximide and actinomycin D. This induction was relatively specific for butyric acid and only the closely related short-chain fatty acids, propionic and valeric acids, were capable of partially inducing the same effect. In contrast to induction of beta-adrenergic binding sites, there was no increase in basal or fluoride-activated adenylate cyclase activity, indicating that the beta-adrenergic receptor and adenylate cyclase and different molecules that may be controlled separately.

Adenylyl Cyclases

Phosphorylation of ribosomal protein S6 in suspension cultured HeLa cells.

HeLa cell ribosomal protein S6, and the increase in its phosphorylation level that occurs after resuspending cells in fresh medium plus serum, were studied using two-dimensional gel electrophoresis. The maximum level of S6 phosphorylation occurs about 2 h after adding fresh medium and seum to cells that have been allowed to grow to high density; this results in an almost complete shift of the spot representing S6 in two-dimensional polacrylamide gels to a new location. Mixing experiments showed that the differences in the level of phosphorylation occur in vivo and are not an artifact of in vitro sample preparation. This method of stimulating S6 phosphorylation provides a convenient system for studying the functional significance of the phenomenon. Only one other ribosomal protein was detectably phosphorylated using [32P]-labeling and autoradiography of dried two-dimensional gels. The level of phosphorylation of this protein, L14, does not change after serum stimulation.

Electrophoresis, Polyacrylamide Gel

Effects of tetraacetyl-bis-dehydroascorbic acid, a derivative of ascorbic acid, on Ehrlich cells and HeLa cells (human carcinoma cells).

Derivatives of ascorbic acid were synthesized, and the studies were made on their effects in Ehrlich ascites carcinoma cells, in regard to the inhibition and the prolongation of survival time as well as on the morphological degeneration in HeLa cells. In a model infection study carried out by using tetraacetyl-bis-dehydroascorbic acid in dd mice infected with Ehrlich cells, it was proved that the prolongation of survival time was nearly double in comparison to the control group mice. Also, it was noted that hypertrophy due to abdominal dropsy and body weight were reduced much more than in the control group. From these results, the inhibiting effect of tetraacetyl-bis-dehydroascorbic acid was confirmed. While in the case of DHA and other derivatives, almost no inhibition and prolongation of survival time were observed. As for HeLa cells in a tissue culture, tetraacetyl-bis-DHA, in a dosage of 125-250 mug/ml, demonstrated definitely its morphological degeration. After 125 mug/ml of tetraacetyl-bis-DHA was added to a tissue culture solution of HeLa cells, the cells were washed and recultured. No growth of the cells was observed. Consequently, this substance was confirmed to be anti-HeLa substance with a low toxicity.

Animals

Contrast of Glycogenesis and protein synthesis in monkey kidney cells and HeLa cells infected with Chlamydia trachomatis lymphogranuloma venereum.

Glycogen metabolism of monkey kidney (LLC-MK-2) cells and HeLa 229 cells infected with a Chlamydia trachomatis lymphogranuloma venereum 440 L (LGV) was studied. The growth cycle of LGV in both host cells was similar; however, a greater number of infectious organism developed intracellularly and were released into the medium during LGV infection of HeLa 229 cells than MK-2 cells. A rapid infection accompanied by a high rate of glycogen synthesis and a short period of accumulation was found in GeLa 229 cells infected with LGV. LGV infected MK-2 cells started to accumulate glycogen about the same time as HeLa 229 cells; however, the rate of glycogen synthesis was lower and the period of accumulation was longer. The LGV agent grew in cycloheximide-treated cells in the absence of host cell protein synthesis. Protein synthesis associated with LGV throughout the developmental cycle was similar in both cell types and could be abolished by chloramphenicol. The continued synthesis of glycogen in the presence of cycloheximide suggested that the synthesis of glycogen was directed by the organism in both MK-2 cells and HeLa 229 cells.

Cell Line

Mitochondrial growth and division during the cell cycle in HeLa cells.

The growth and division of mitochondria during the cell cycle was investigated by a morphometric analysis of electron micrographs of synchronized HeLa cells. The ratio of total outer membrane contour length to cytoplasmic area did not vary significantly during the cell cycle, implying a continuous growth of the mitochondrial outer membrane. The mean fraction of cytoplasmic area occupied by mitochondrial profiles was likewise found to remain constant, indicating that the increase in total mitochondrial volume per cell occurs continuously during interphase, in such a way that the mitochondrial complement occupies a constant fraction( approximately 10-11(percent)) of the volume of the cytoplasm. The mean area, outer membrane contour length, and axis ratio of the mitochondrial profiles also did not vary appreciably during the cell cycle; furthermore, the close similarity of the frequency distributions of these parameters for the six experimental time-points suggested a stable mitochondrial shape distribution. The constancy of both the mean mitochondrial profile area and the number of mitochondrial profiles per unit of cytoplasmic area was interpreted to indicate the continuous division of mitochondria at the level of the cell population. Furthermore, no evidence was found for the occurrence of synchronous mitochondrial growth and division within individual cells. Thus, it appears that, in HeLa cells, there is no fixed temporal relationship between the growth and division of mitochondria and the events of the cell cycle. A number of statistical methods were developed for the purpose of making numerical estimates of certain three-dimensional cellular and mitochondrial parameters. Mean cellular and cytoplasmic volumes were calculated for the six time-points; both exhibited a nonlinear, approx. twofold increase. A comparison of the axis ratio distributions of the mitochondrial profiles with theoretical distributions expected from random sectioning of bodies of various three-dimensional shapes allowed the derivation of an "average" mitochondrial shape. This, in turn, permitted calculations to be made which expressed the two-dimensional results in three-dimensional terms. Thus, the estimated values for the number of mitochondria per unit of cytoplasmic volume and for the mean mitochondrial volume were found to remain constant during the cell cycle, while the estimated number of mitochondria per cell increase approx. twofold in an essentially continuous manner.

Cell Division

Studies on the interaction between coxsackievirus A9 and HeLa cells. I. Plaque-forming ability of coxsackievirus A9 in HeLa cell cultures.

Most of the coxsackievirus A9 (CA 9 virus) including the prototype strain formed plaques in HeLa cell monolayers under agar overlay, although they showed little or no cytopathogenicity under fluid medium. These viruses were isolated or passaged in primary cynomolgus monkey kidney (MK) cell cultures, and the infectivity of any strain in terms of plaque-forming units was much higher in MK cells than in HeLa cells, even after plaque purification of the virus in HeLa cell cultures. CA 9 virus contained in the original throat swabs as well as some clones obtained by plaque purification in MK cells failed to form plaques in HeLa cells, but virus preparations obtained after several undiluted passages through MK cells included plaque-formers in HeLa cells, suggesting that such plaque (HeLa)-forming viruses may have developed at a certain rate during multiplication of the original non-plaque (HeLa)-forming virus population in MK cells. Out of four lines of HeLa cells examined, two, including a clonal line S3, failed to support plaque formation by CA 9 virus.

Agar