ALBERT

Notes: Stable clones selected for resistance to tunicamycin (TM) have been isolated from Chinese Hamster Ovary (CHO) cells. The TMR phenotype is stable for more than nine months in the absence of the drug. The morphology of TMR mutant varies from epitheloid to abnormally elongate. The mutants do not display cross-resistance for ConA but are slightly cross-resistant to PHA. Biochemically labeled membrane proteins and glycoprotein of Vesicular stomatitis virus (VSV) grown in the TMR mutants revealed that the incorporation of radioactive glucosamine was markedly reduced in the mutants. The results indicate that TMR cells are a novel type of membrane mutant.

Notes: We have measured the turnover rate of ribosomal RNA in exponentially growing Tetrahymena thermophila cells, cells entering the plateau phase of growth, and nutrient-deprived (starved) cells. Ribosomal RNA is stable in cells in early log phase growth but it begins to turnover as the cells begin a deceleratory growth phase prior to entering a plateau state. Likewise, rRNA in cells transferred from early log phase growth to a starvation medium begins to be degraded immediately upon starvation. In both cases the degradation of rRNA exhibits biphasic kinetics. A rapid initial exponential degradation with a half time of nine and one-half hours lasting for six hours is followed by a slower exponential degradation with a half-life of 35 hours. When starved cells are transferred to fresh growth medium turnover of rRNA ceases. The evidence presented suggests that the alteration in degradation rate is a regulated process which is most likely independent of the cell cycle.

Notes: A variant endothelial cell type was found to arise spontaneously from cultures of bovine aortal endothelial cells. This variant showed no contact inhibition and overgrew confluent cultures of wild-type endothelial cells. Unlike other reported variants of this cell type produced by chemical mutagenesis or by withdrawal of polypeptide growth factor, this variant retained the capacity to synthesise factor VIII antigen, but showed no alteration from wild-type in capacity to adsorb platelets. The variant also had an increased capacity to bind FITC-conjugated con A to its surface.

Notes: The cell volume, cell water, intracellular ionic concentrations, and transmembrane potential of rat alveolar macrophages were determined. The measurements were made on cells which had been separated from the medium by centrifugation through dibutyl phthalate in order to greatly reduce the trapped extracellular space. The mean cell volume of the alveolar macrophages is 1,525 cubic microns and 72% of this volume is water. The intracellular fluid is high in Na+ (97 mM) and lower in K+ (50 mM) and the intracellular Cl- concentration is 64 mM. The transmembrane potential, as measured from the equilibrium distribution of tritiated triphenylmethyl phosphonium and by using the fluorescent probe, Di-S-C3(5), is approximately -37 millivolts. Neither Na+, K+, nor Cl- is distributed at equilibrium. However, the K+ permeability of alveolar macrophage membranes appears to be greater than Na+ permeability.

Notes: Cyclic AMP (cAMP) causes growth arrest in G1 and induction of cAMP phosphodiesterase and decrease of ornithine decarboxylase in S49 mouse lymphoma cells. Dibutyryl cAMP treatment of partially synchronized cells causes similar changes in activities of both enzymes, regardless of position in the cell cycle. This suggests that cAMP regulation of these enzymes is not mediated by growth perturbation.

Notes: A mutant of BHK cells (ts422E) temperature-sensitive for processing 32S rRNA to 28S rRNA (Toniolo et al., '73) also loses the ability to synthesize polyamines and 5.8S rRNA when shifted to the non-permissive temperature (39°). The activity of several enzymes not involved with polyamine synthesis, methylation of 32S rRNA, and small nuclear RNA production are apparently unaffected after at least 24 hours at 39°. When cultures are returned to the permissive temperature (33°), polyamine synthesizing capacity returns to normal as mature rRNA production resumes.

Notes: When replicating DNA is labeled sequentially with radioactive and density tracers and analyzed by equilibrium centrifugation, the fraction banding at heavier than normal density is inversely proportional to the rate of replication fork movement if there is a sharp transition from one tracer to the other on the newly synthesized chains (Painter and Schaefer, '69). Primate CV-1 DNA labeled for 5 to 30 minutes with 3H-dThd and then for three hours with BrdUrd in the presence of FdUrd bands in a bimodal distribution in alkaline CsCl, rather than in a continuous distribution with a skew toward heavier density seen when FdUrd is omitted and centrifugation is in neutral CsCl. The heavy density peak represents interspersion of both tracers in the DNA and is caused by slow transition from dThd to BrdUrd incorporation when the tracers are switched in the labeling medium. This may result from preferential uptake and incorporation of dThd over BrdUrd. Because of the interspersion, calculation of the rate of replication fork movement is inaccurate. Reversal of the labeling sequence with administration of the long density pulse before the radioactive pulse reduces the problem of interspersion. Using this sequence of labeling, estimates of the rate of fork movement of 0.36-0.38 μm/min are obtained when the 3H pulse time is long enough to allow accurate measurement of the fraction of heavy DNA. Analysis by fiber autoradiography yields a rate of 0.56 μm/min in the same cell line. If appropriate precautions are taken to minimize mixing of the two tracers in the precursor pool and to ensure that the fraction of heavy DNA is measured accurately, the hydrodynamic technique provides an objective method of measuring rate of fork movement that gives values only slightly lower than those obtained by autoradiography.

Notes: 1We studied the equilibrium distribution of Mg++ in the form of chloride and sulfate at two temperatures (5° and 25°C) in frog voluntary muscles. External Mg++ concentration was varied between 1.2 and 73.2 mM with the specific purpose of testing the diametrically opposed predictions of the membrane theory and the association-induction hypothesis.2There was a linear gain of Mg++ over the entire range of external Mg++ concentrations studied at both 5°C and 25°C. In a plot of intracellular Mg++ concentration in μmoles per gram of fresh muscle cells against extracellular Mg++ concentration, the slopes observed were 0.220 at 5°C and 0.0206 at 25°C.3The increase of external Mg++ from 1.2-73.2 mM at constant external K+ concentration (2.5 mM) had no discernible effect on intracellular K+ concentration, which remained constant at its normal levels in the vicinity of 90 μmoles/g/ fresh muscle cells.4We observed a similar rectilinear distribution of Mg++ in frog ovarian eggs. As in muscle tissues, no major alteration of intracellular K+ concentration results from increases of external Mg++ concentration from 1.2-73.2 mM.5With the rectilinear gain of Mg++, there was an entirely parallel gain of chloride in frog muscle cells. Indeed the slope of the Cl- curve and that of the Mg++ curve have essentially the same value. Thus Mg++ has entered the cell accompanied by chloride (and sulfate), rather than by exchange with other intracellular cations.6An increase of external K+ from 2.5 mM to 100 mM at a constant external Mg++ concentration depolarizes the muscle cell resting potential as shown by Ling and Gerard, ('50) and causes an increase of intracellular K+, doubling its normal concentration to 200 μmoles/g fresh muscle cells. Notwithstanding, the intracellular concentration of Mg++ remained totally unchanged from its normal value.7These findings profoundly disagree with the predictions of the Donnan theory of membrane equilibrium, according to which profound alteration of intracellular K+ concentration should follow exposure to high external Mg++ concentration, and vice versa. Furthermore, the postulation of a Mg++ pump is not feasible not only because of its energy requirements, but because it would also be inadequate to explain the lack of effect of varying external Mg++ concentration on the resting potential, the intracellular K+ concentrations, as well as the pattern of Cl- uptake totally different from that predicted by the membrane theory.8On the other hand, Mg++, K+, and Cl- distributions correlated completely with the predictions of the association-induction hypothesis, according to which Mg++ and K+, in a normal resting cell, are predominantly adsorbed on sites they do not share, hence there is no mutual interference. Saturating all the intracellular sites at an external concentration of 1.2 mM, the concentration of Mg++ in the muscle cells increased further only in the form of free Mg++ (accompanied by its anion) in the cell water.9The q-value for Mg++ in muscle cells at two different temperatures permits calculation of the thermodynamic parameters of the distribution of Mg++ salt in frog muscle cell water: a moderately favorable ΔH equal to -0.516 Kcal/mole, and an unfavorable entropy of 4.37 cal/degree/mole, showing an entropic cause for the exclusion of Mg++ from cell water.

Notes: Subcellular organelles from livers of rats three days prenatal to 50 weeks postnatal were separated on sucrose gradients. The peroxisomes had a constant density of 1.243 g/ml throughout the life of the animal. The density of the mitochondria changed from about 1.236 g/ml at birth to a constant value of 1.200 g/ml after two weeks.The peroxisomal and mitochondrial fatty acid β-oxidation and the peroxisomal and supernatant activities of catalase and glycerol-3-phosphate dehydrogenase were measured at each age, as well as the peroxisomal core enzyme, urate oxidase, and the mitochondrial matrix enzyme, glutamate dehydrogenase. All of these activities were very low or undetectable before birth. Mitochondrial glutamate dehydrogenase and peroxisomal urate oxidase reached maximal activities per g of liver at two and five weeks of age, respectively.Fatty acid β-oxidation in both peroxisomes and mitochondria and peroxisomal glycerol-3-phosphate dehydrogenase exhibited maximum activities per g of liver between one and two weeks of age before weaning and then decreased to steady state levels in the adult. Peroxisomal β-oxidation accounted for at least 10% of the total β-oxidation activity in the young rat liver, but became 30% of the total in the liver of the adult female and 20% in the adult male due to a decrease in mitochondrial β-oxidation after two weeks of age. The greatest change in β-oxidation was in the mitochondrial fraction rather than in the peroxisomes. At two weeks of age, four times as much β-oxidation activity was in the mitochondria as in the peroxisomal fraction. Peroxisomal glycerol-3-phosphate dehydrogenase activity accounted for 5% to 7% of the total activity in animals younger than one week, but only 1% to 2% in animals older than one week.Up to three weeks of age, 85% to 90% of the liver catalase was recovered in the peroxisomes. The activity of peroxisomal catalase per g of rat liver remained constant after three weeks of age, but the total activity of catalase further increased 2.5- to 3-fold, and all of the increased activity was in the supernatant fraction.

Notes: Leupeptin, chymostatin and antipain inhibited the degradation of long-lived proteins in cultured rat hepatocytes by 20-30%, probably by inhibiting lysosomal proteases:(1) Leupeptin and chymostatin decreased to a similar extent the degradation of an exogenous protein 125I-asialo fetuin, a process known to occur within lysosomes. (2) In extracts of cells treated with leupeptin, cathepsin B activity was inhibited by 35-50%. (3) Leupeptin, chymostatin and antipain inhibited proteolysis by homogenates of liver lysosomes but not by the supernatant fraction. These agents, however, do not appear to rapidly permeate the membrane of isolated lysosomes.Leupeptin, chymostatin and antipain did not inhibit the breakdown of short-lived normal cell proteins, and ones containing amino acid analogs. Even when the amount of abnormal proteins was increased, such that it comprised a large fraction of cell protein, the degradation of these polypeptides was still very rapid and not affected by these inhibitors. The pathway for the degradation of short-lived cell proteins thus appears distinct from that responsible for degradation of long-lived cell proteins. In accord with this conclusion, reduction of the temperature of cultures inhibited the breakdown of long-lived proteins to a much greater extent than it affected the breakdown of short-lived ones.Treatment of cultured hepatocytes with glucagon, or deprivation for serum or amino acids stimulated the degradation of the more stable cell proteins but did not affect the breakdown of 125I-asialo-fetuin. Under these conditions leupeptin and chymostatin inhibited the breakdown of long-lived cell proteins to the same extent as in control cultures. Thus, lysosomal enzymes seem to play an important role in protein breakdown both in fed hepatocytes and in cells where proteolysis is accelerated.

Notes: Three parameters involved in the production of new ribosomal RNA (rRNA) were measured in Tetrahymena thermophilia: (i) the rate of synthesis of the rRNA precursor, (ii) the rate of processing of the RNA precursor and rRNA intermediates and (iii) the efficiency of utilization of the rRNA precursor in producing mature ribosomal RNA. These parameters were measured in cells in exponential growth and in cells starved in a dilute salt solution. Growing cells synthesize rRNA 20 times faster and process rRNA precursors and intermediates 10 to 15 times more rapidly than do starved cells. Both utilize their rRNA precursors with an efficiency of one in converting them to mature rRNA.

Notes: Under conditions where a maximum stimulation of 3-O-methyl-glucose transport is observed, three thymocyte mitogens (concanavalin A, ionophore A23187 and hydrogen peroxide) cause cell rounding and a decrease in the density of intra-membrane particles on the plasma membrane. The early effects of mitogens on the thymocyte plasma membrane are similar to those of osmotic shock.

Notes: The effect of Ca+2 on the transport and intracellular distribution of Na+ and K+ in Ehrlich ascites tumor cells was investigated in an effort to establish the mechanism of Ca+2-induced hyperpolarization of the cell membrane. Inclusion of Ca+2 (2mM) in the incubation medium leads to reduced cytoplasmic concentrations of Na+, K+ and Cl- in steady state cells. In cells inhibited by ouabain, Ca+2 causes a 41% decrease in the rate of net K+ loss, but is without effect on the rate of net Na+ accumulation. Net K+ flux is reduced by 50%, while net Na+ flux is unchanged in the transport-inhibited cells. The membrane potential of cells in Ca+2-free medium (-13.9 ± 0.8 mV) is unaffected by the addition of ouabain. However, the potential of cells in Ca+2-containing medium (-23.3 ± 1.2 mV) declines in one hour after the addition of ouabain to values comparable to those of control cells (-15.2 ± 0.7 mV).The results of these experiment are consistent with the postulation that Ca+2 exerts two effects on Na+ and K+ transport. First, Ca+2 reduces the membrane permeability to K+ by 25%. Second, Ca+2 alters the coupling of the Na/K active transport mechanism leding to an electrogenic hyperpolarization of the membrane.

Notes: Freshly explanted human myeloma cells formed colonies of monoclonal plasma cells in soft agar in the presence of medium conditioned by the adherent spleen cells of mineral oil-primed BALB/c mice. The medium showed peak activity at a dilution of 1:4. 2-mercaptoethanol or monothioglycerol was necessary for colony formation. Other thiols tested were ineffective in promoting colony growth. Colony-forming cells adhered to nylon wool, but not glass beads or plastic dishes. The presence of E-rosetting cells was not required for myeloma colony formation. Antibody prepared against a human myeloma cell line, RPMI 8226, reduced colony formation. These studies demonstrate the usefulness of this bioassay for determining functional properties of the myeloma colony-forming cell.

Notes: Intracellular recordings were made from human oviduct smooth muscle maintained in cell culture. Solitary cells isolated from one another and cells in contact with one another retained electrical properties of smooth muscle in vivo. Membrane potential of solitary cells and connected cells was -35 mV. Connected cells formed electrotonic junctions which transmitted current from one cell to another. This current spread was responsible for differences in input resistance and time constant in solitary cells, 66 MΩ and 96 msec, compared to connected cells, 26 MΩ and 56 msec. All cells expressed delayed rectification to depolarizing current pulses. Some cells generated action potentials spontaneously or in response to intracellular current puleses. Action potentials were abolished by cobalt or by EGTA. Slow wave potentials, 5-20 mV in amplitude, occurred continuously once every 15 to 45 seconds in connected cells.

Notes: In quiescent confluent monolayers of WI-38 cells, the specific activity of the tRNA methyltransferases falls to 20% of the level found in log phase cells. When the resting cells are stimulated to proliferate by a change to fresh medium, the enzymes show a rapid rise in specific activity which correlates with early increases in the rate of tRNA synthesis. The specific activity of the enzymes continues to rise throughout the period of DNA synthesis, at the end of which it is somewhat higher than that of log phase cells. The increases in enzyme activity could be blocked by exposure of the stimulated cells to Actinomycin D (2μ/ml). The increases in activity were not equivalent for the different base-specific enzymes. The contribution of the N2-methylguanine specific enzyme remained relatively constant, while that of the N2,N2-dimethylguanine specific and 1-methyladenine specific enzymes doubled and tripled, respectively, by late S phase. The contributions of the 1-methylguanine and the 7-methylguanine specific enzymes fell to a few percent of the total by late S phase. This indicates non-coordinate variations in the expression of the different base-specific enzymes after stimulation of resting cells and may be related to altered isoaccepting tRNA profiles observed in resting and growing cells.

Notes: Brewer's yeast preparations influence glucose metabolism in vivo and in isolated tissues. We have studied the effect of a brewer's yeast extract on glucose metabolism and grwoth of rat hepatoma and human embryonic cells. Growth of the rat hepatoma cells was very much stimulated by the extract in a concentration-dependent manner. Glucose uptake was, on the other hand, appreciably inhibited, and lactate uptake completely abolished by the extract. Insulin stimulated cell growth and inhibited lactate uptake but did not affect the glucose level. Insulin and the extract had additive effects on growth and lactate uptake of the hepatoma cells. The inhibition by the brewer's yeast extract of glucose uptake was, however, antagonized by insulin. Niacin or Cr3+, which are suggested to be components of a “glucose tolerance factor” of brewer's yeast, did not affect growth or glucose and lactate uptake. The glucose uptake of the human embryonic cells was strongly inhibited by the brewer's yeast extract. Cell growth and lactate production were not influenced by the extract or by insulin; however, when both insulin and extract were present simultaneously, a slight stimulation of growth and inhibition of lactate production was observed. The results indicate that brewer's yeast can have appreciable direct effects on cells and that not all of these effects are “insulin-like”.

Notes: Agar cultures of CBA fetal liver, peripheral blood, yolk sac and adult marrow cells were stimulated by pokeweed mitogen-stimulated spleen conditioned medium. Two to ten percent of the colonies developing were mixed colonies, documented by light or electron microscopy to contain erythroid, neutrophil, macrophage, eosinophil and megakaryocytic cells. No lymphoid cells were detected. Mean size for 7-day mixed colonies was 1,800-7,300 cells.When 7-day mixed colonies were recloned in agar, low levels of colony-forming cells were detected in 10% of the colonies but most daughter colonies formed were small neutrophila and/or macrophage colonies. Injection of pooled 7-day mixed colony cells to irradiated CBA mice produced low numbers of spleen colonies, mainly erythroid in composition. Karyotypic analysis using the T6T6 marker chromosome showed that some of these colonies were of donor origin. With an assumed f factor of 0.2, the mean content of spleen colony-forming cells per 7-day mixed colony was calculated to vary from 0.09 to 0.76 according to the type of mixed colony assayed.The fetal and adult multipotential hemopoietic cells forming mixed colonies in agar may be hemopoietic stem cells perhaps of a special or fetal type.

Notes: Non-lethal concentrations of bromodeoxyuridine induce a 2- to 5-fold increase in the specific activity of alkaline phosphatase in HeLa subclone, S3G. Experiments employing 10-hour pulses of BRdU showed that 48 hours were required before induction commenced, and that maximal induction was attained by 96 hours. Under conditions in which DNA synthesis was prevented with hydroxyurea induction did not occur. Upon removal of hydroxyurea both DNA synthesis and induction were rapidly reestablished. Furthermore, experiments employing radiolabelled BRdU demonstrated that the kinetics of the induction process paralleled the incorporation of the analogue into cellular DNA.These results indicate that DNA synthesis, or some process intimately linked to DNA synthesis, is required for the induction of alkaline phosphatase, and suggest that the mode of the induction may be through the incorporation of the analogue into cellular DNA.

Notes: It has been postulated that superoxide dismutase (SOD) protects cells from free radical-induced damage. In these experiments SOD specific activity was measured as established human diploid cell lines from various donor ages progressed through their in vitro lifespans. Significant elevations in activity occurred during the in vitro lifespans of cells from fetal and newborn donors, but no change in activity was detected during the lifespan of cells from an adult donor. In addition, a direct relationship between enzyme activity and donor age was detected with the following relative activities: adult 〉 newborn 〉 fetal. The possible relationship between these findings and the free radical theory of aging is discussed.

Notes: A pseudodiploid clone of Chinese hamster cells transformed in vitro with Simian virus 40 (SV40) was isolated from soft agar and injected into nude mice through three successive passages with a short in vitro cultivation between each animal inoculation. The original clone and the three subsequent tumor populations were characterized in regard to SV40 T antigen staining, modal chromosome number, and Giemsa-banded karyotype. All tumor cell lines maintained the pseudodiploid mode, as well as the positive SV40 T antigen staining. Nonrandom chromosomal changes included loss of one of the X chromosomes, additions of abnormal variants of chromosomes No. 1 and No. 2, the appearance of unidentified marker chromosomes, and the loss of autosomes No. 5, No. 6, and No. 11. The deletion of one of the X chromosomes occurred with about the same frequency in all cell lines. Additions of abnormal chromosomes No. 1 and No. 2 tended to recur more often in the tumor cell lines than in the original clone. The appearance of marker chromosomes, as well as the loss of autosomes No. 5, No. 6, and No. 11 demonstrated a correlation with tumorigenicity. Yet, the three successive passages of the cells through the animal did not select for a tumor population with a single, homogeneous karyotype.

Notes: The incubation of human leukocytes with ascorbic acid increased chemotaxis of the cells. In addition, ascorbic acid promoted the assembly of intracellular polymorphonuclear leukocyte (PMN) microtubules as assessed by transmission electron microscopy. Prior incubation of the PMN with colchicine blocked the effect of ascorbic acid on promoting microtubule assembly. Not only did ascorbic acid promote the assembly of microtubules in vivo, but it enhanced the assembly of bovine brain tubulin into microtubules in vitro as quantitated by a glass-fiber filtration assay and by promotion of viscosity changes. The enhancement in leukocyte mobility by ascorbate at concentrations achievable in normal tissues correlates with its ability to assemble microtubule organelles.

Notes: The replication of mouse satellite DNA was delayed when synchronized 3T3 cells were exposed to low concentrations of hydroxyurea during S phase. It appears that the onset of satellite replication is not a time dependent event, but instead requires that a certain amount of main band DNA be synthesized first. Using hydroxyapatite chromatography and S1 nuclease digestion, a procedure was developed to quantitate the synthesis of both satellite and neighboring main band sequences. The replication kinetics of satellite determined by this method agree with previous estimates. Main band sequences adjacent to satellite appear to replicate in concert with satellite DNA. The results are discussed and related to the limitations of the techniques utilized.

Notes: Potassium influx and efflux were studied in human peripheral blood lymphocytes equilibrated over a wide range of external K+ levels. The absence of a net ion movement throughout the flux study was established, trapped space was measured with polyethylene glycol, and cells were separated from incubation media without exposure to any washing solution. There are both rapid and slow cellular fractions of 42K influx and efflux, with half-times of exchange of around 2 minutes, and 400 minutes, respectively. The rapid component is identical in magnitude to the smaller non-saturable component of cell K+, while the slow component is identified with the larger, sigmoidal, saturable component of cell K+ that was previously shown to follow a cooperative adsorption isotherm. These results support the association-induction hypothesis, which predicts (a) a rapid fraction of K+ flux due to equilibration of ion within cell water existing in a state of polarized multilayers, and (b) a slower component of K+ flux limited by adsorption onto, or desorption from, fixed anionic sites existing throughout the cell. K+ influx, as a function of external K+, showed a triphasic relation with a peak around 1 mM K+ex, then a trough around 4 mM K+ex, and then a gradual rise. This relation was readily explained, in terms of the association-induction hypothesis, by the cooperative interaction between, and ion occupancy of, fixed anionic sites that absorb K+ or Na+.

Notes: The glycosaminoglycans (GAG) of human cultured normal glial and malignant glioma cell lines were studied using 35S-sulphate or 3H-glucosamine as markers. 35S-labelled GAG were assayed by precipitation with cetylpyridinium chloride; 3H-labelled sulphated GAG and 3H-labelled hyaluronic acid were quantitated after separation on a DEAE-cellulose column. The net production of GAG and the distribution, composition and turnover of GAG were similar in all of the normal cell lines tested, but showed a great variability in the malignant cell lines. Most of the glioma cell lines produced more hyaluronic acid and less sulphated GAG than the normal cell lines, but exceptions were noted. The GAG of the trypsin susceptible (pericellular) pool of normal glial cells consisted mainly of heparan sulphate with only minor amounts of other GAG. The analogous material of most glioma cells showed hyaluronic acid as the major GAG. Material liberated by trypsin from EDTA-detached cells (membrane fraction) was enriched in heparan sulphate as compared to the entire pericellular pool. Substrate attached material (SAM) left with the plastic dish after EDTA treatment of normal cultures was rich in heparan sulphate, whereas SAM of glioma cells lacked heparan sulphate or showed greatly reduced amounts of this component. Release of newly synthesized GAG to the extracellular medium was a rapid process in the normal cells but was more or less delayed in the glioma cells. The extracellular medium of the malignant glioma cultures was consistently poor in dermatan sulphate, as compared to that of normal cultures.

Notes: Mouse-human hybrid cells that preferentially segregate either mouse or human chromosomes were analyzed for their relative content of mouse and human rRNA genes and for their capacity to transcribe these genes. A distinctive Hind III restriction fragment containing 28S rRNA sequences was used to distinguish between mouse and human rDNA and a set of distinctive loop structures in the 45S pre-rRNA was used to distinguish between mouse and human gene transcripts. Our results indicate that the genes of only one species are transcriptionally active in these hybrid cells, even though both sets of genes are present.

Notes: Addition of insulin to nonproliferating serum-free cultures of secondary chicken embryo (CE) cells caused a 30% to 50% increase in cell number. Addition of any one of several glucocorticoids (dexamethasone, cortisol, or corticosterone) to the cultures two days before insulin addition increased the mitogenic effect of insulin by about twofold at each insulin concentration tested. This glucocorticoid stimulation of cell proliferation was “permissive” because in the absence of insulin glucocorticoids caused little increase in cell number (usually less than 15%). Glucocorticoids were maximally active at low concentrations (e.g., 10-10 M dexamethasone). Steroids without glucocorticoid activity were inactive over a wide range of concentrations. Glucocorticoids increased the mitogenic response to insulin largely by increasing the percentage of cells that insulin stimulated to synthesize DNA.The maximum mitogenic effect of insulin upon CE cells rapidly decreased after the cells were serially subcultured. After only nine population doublings (4 passages) in culture, the response to insulin was diminished by about 70%. The mitogenic effect of insulin plus dexamethasone declined similarly during serial subculture, and was always about twofold greater than the effect of insulin alone. The cells maintained their mitogenic responsiveness to serum as these responses decreased.In contrast to the growth promoting influence of glucocorticoids in the presence of insulin, glucocorticoids inhibited the mitogenic response of CE cells to serum. This result may resolve our above findings with reports that glucocorticoids inhibit the proliferation of CE cells.

Notes: Much controversy regarding the relationship between nutrients and serum in regulation of cell growth can be reconciled by recognizing that serum contains multiple factors which regulate different events in the cell cycle. Serum was fractioned into a platelet-derived growth factor (PDGF), which induces cells to become competent to synthesize DNA, and plasma which allows competent cells to traverse G0/G1 and enter the S phase. Nutrients are not required for the cellular response to PDGF; however amino acids are required for plasma to promote the entry of PDGF-treated, competent cells into S phase. The nutrient independent, PDGF-modulated, growth regulatory event (competence) is located 12 hours prior to the G1/S phase boundary in quiescent, density-arrested Balb/c-3T3 cells. The nutrient dependent, plasma-modulated event is located six hours prior to the G1/S phase boundary and corresponds in time to a plasma dependent growth arrest point. Moreover, plasma controls the concentration of amino acids required for DNA synthesis. Infection of density-arrested Balb/c-3T3 cells with SV40 overrides both the nutrient independent and the nutrient dependent growth regulatory events.

Notes: Iodinated proteins were degraded after injection into HeLa cells at first-order rates with half-lives varying from three hours for the trout nonhistone chromosomal protein, HMG-T, to 60 hours for whale myoglobin. Fluoresceinated-bovine serum albumin (fl-BSA) was degraded almost twice as fast as unmodified BSA. The rate of degradation of 125I-BSA was very similar in eight cell lines of mouse, human, monkey and rat origin. Microinjected proteins were analyzed on SDS-acrylamide gels after injection, and for BSA and immunoglobin G, all remaining intracellular 125I migrated at the molecular weight of the injected proteins. By contrast, more than 80% of the extracellular 125I chromatographed as iodotyrosine. With the exception of fl-BSA, which exhibited perinuclear accumulation in approximately one-half of the injected cells, autoradiography showed that throughout the period of study the injected proteins remained dispersed in the cytoplasm.

Notes: PGE1 elicited a slow, dose-dependent membrane depolarization with an increase in membrane conductance in the somatic cell hybrid TCX11. The ED 50 was 1-2 × 10-8 M with maximal responses at 1-5 × 10-7 M. Dopamine (DA) reversed the effect of PGE1 and caused the membrane potential and resistance to return to control levels. Chronic exposure of cells (measured in minutes) to DA alone would not cause this hyperpolarization. 5-HT was also tested and failed to consistently reverse the PGE1 effects. Chlorpromazine antagonized the effects of DA on the PGE1 response. The electrophysiological results reported here using TCX11 cells are discussed in light of previously reported biochemical results describing interactions of PGE1 and DA, and the electrophysiological effects of DA alone.

Notes: Mutants temperature-sensitive for growth have been isolated from the established line of Chinese hamster fibroblasts Wg1A. These mutants, together with the ones previously isolated by Roscoe et al. ('73), have been characterized with regard to their cell cycle properties. Most of them become arrested in the G1 phase of the cell cycle when incubated under restrictive conditions. By performing temperature shift experiments with synchronous cultures, the execution steps of most of the mutated functions have been located within the second half of the G1 phase.

Notes: Total cellular calcium levels do not change when 3T3-4a cells stop proliferating due to serum depletion, or when serum-arrested quiescent cells are incubated for up to 44 hours in calcium-deficient medium (∼10 μM Ca++). Upon stimulation with dialyzed serum cells enter S and progress through at least one cycle even at extremely low calcium levels in the culture medium (≥10 μM). Cells divide until a final cell density is attained which is proportional to the calcium concentration in the medium and cells reversibly arrest in G1. Cells which arrested in G1 in medium containing ≤26 μM Ca++ in the presence of excess serum can be stimulated to enter S in response to added calcium after a prereplicative phase of 14 to 16 hours. Serum does not affect 45Ca-uptake in these cells. Benzo[a]pyrene transformed 3T3 (BP3T3) cells have a 100-200 times lower Ca++-requirement than 3T3 cells but arrest in G1 at low Ca++ levels. In contrast, SV40-virus transformed 3T3 (SV3T3) cells that grow without restriction in monolayer cultures have even lower Ca++-requirements for growth than BP3T3 cells and have no Ca++-sensitive restriction point. Therefore, 3T3 and BP3T3 cells have retained the capacity to sense intracellular Ca++-pool sizes and to arrest in G1 at subthreshold cellular Ca++-levels.

Notes: Inosine triphosphate pyrophosphohydrolase from human erythrocytes was purified and characterized. The enzyme is highly specific for ITP and shows optimal activity in glycine buffer pH 9.6 and 50 mM MgCl2. The Km of the enzyme is 1.3 × 10-4, the Vmax = 1.2 × 10-9 and the Keq = 3.8 × 104. Human erythrocyte ITP pyrophosphohydrolase does not require SH compounds for activation. The enzyme is inhibited by Cd++, Co++, and Ca++ ions and by phydroxymercuribenzoate.

Notes: The ribonuclease (RNase) activity associated with the surface of cells (primarily the Vero line of Green monkey kidney) was assayed under conditions where all cells remained viable as members of colonies or monolayers. RNase activity associated with individual clones was revealed as clear zones of hydrolyzed (acid-soluble) RNA against an opaque background of acidprecipitated RNA in an isotonic agarose-yeast RNA overlay. High resolution assays for single- and double-stranded RNase were achieved by measuring the loss in activity of infectious Sindbis virus RNA, and of the antiviral state induced by poly(rI). poly(rC), respectively.Experiments using these assay procedures revealed that virtually all of the RNase activity associated with viable (intact) vertebrate cells in culture was contributed by the serum in the growth medium, and was superficially adsorbed to the cell surface, rendering it relatively easy to remove by extensive washing.A confluent monolayer of 2 × 106 unwashed Vero cells contained the equivalent of 500 ng of pancreatic RNase, representing about 5% of the total activity initially present in the serum of the growth medium. In terms of serum nuclease activity, only 0.45 ng equivalents of pancreatic RNase in 500 μl were required to destroy 50% of the activity of an infectious Sindbis virus RNA preparation in 15 minutes at 37°C.Cells grown in the absence of serum and cells washed about ten times had comparably low levels of RNase activity-about 100-fold less than unwashed cells grown in the presence of 6% calf serum.The isotonic yeast RNA:agarose overlay procedure may be useful to identify and isolate cell mutants with different endogenous levels, or binding capacities, of surface RNase activity.Operationally, these studies describe the assay of cell surface-associated RNase activity under conditions where all cells remain viable as members of individual colonies or monolayer populations. Specifically, the assay measures the solubilization of yeast RNA in an isotonic agarose mixture overlaying the cells. We describe its use to determine how much of the total RNase activity in viable cell monolayers or colonies is contributed by serum in the growth medium. In addition, we describe the results of two biological assays used as higher resolution probes for cell surface ribonuclease activity: (i) single-strand (ss) RNA, in the form of infectious RNA from Sindbis virus, and (ii) double-stranded(ds)-RNA, in the form of poly(rI). poly(rC) as an inducer of an interferon-mediated antiviral state.

Notes: A strain of nonsymbiotic A. proteus was infected with endosymbiotic bacteria isolated from another strain of amoeba which had become dependent on the symbionts after a few years of spontaneously established symbiosis. In the newly infected amoebae, the bacteria avoided digestion and multiplied at a faster rate than the hosts, reaching the maximum carrying number (about 42,000 per amoeba) in fewer than ten cell generations of the hosts. The experimentally infected amoebae were also examined under the electron microscope, and the development of bacteria-containing vesicles was followed. The results show that the infective bacteria that were initially harmful to host amoebae have become harmless and that they have changed in their mode of multiplication during the course of establishing a stable symbiosis with their hosts.

Notes: Removal of serum from BHK-21/C13 cells in culture results in a decline in thymidine incorporation extending over five days. Additional removal of any of several amino acids results in a rapid decrease in incorporation of thymidine to negligible levels by 24 hours. Replacement by complete medium then provokes a synchronous wave of DNA synthesis after only ten hours with DNA synthesis first increased at six hours. Starvation for glutamine results in a rapid decline in protein synthesis over the 24 hour period when DNA synthesis is falling. However, there is considerable degradation of total protein during this period, and RNA degradation is also greatly increased. Concurrently, synthesis of RNA falls to less than 10% of that in control cells.

Notes: Certain epithelial cell lines have morphologic, physiologic, biochemical and pharmacologic characteristics of transporting epithelia from intact organs. In this paper we show that dibutyryl cyclic AMP, 5′ AMP, adenosine and cyclic AMP phosphodiesterase inhibitors stimulate hemicyst formation by the dog kidney cell line MDCK. It is suggested that this effect is explained by elevation of intracellular cyclic AMP levels by means of an exogenous non-metabolizable source of cyclic AMP, phosphodiesterase inhibition or adenyl cyclase stimulation. Since hemicyst formation is in part due to transepithelial fluid transport, these findings raise the possibility that this fraction might be modulated by cAMP in an established cell line. We believe that cultured epithelial cells may provide an exploitable model system to investigate at the cellular and subcellular levels, the mechanism by which cyclic AMP modifies water and solute movements across epithelia.

Notes: Elemental concentrations in the cytoplasm and nucleus of post-natal mouse myocytes were measured at 2, 4, 8, 16, 32 days and in adults using electron probe X-ray microanalysis. Using an analysis of variance test, significant age dependent changes were found in intracellular potassium, sulfur, phosphorus, and chlorine concentations (mg/kg dry weight), while sodium and magnesium concentrations did not show significant changes. The application of t tests following linear regression analyses (age versus concentration for each element) did, however, give a significant slope for cytoplasmic sodium, potassium, sulfur, and phosphorus values. The findings correspond closely with an emission spectroscopy-titrimetric study of whole heart ventricle of the same developmental period (Hazelwood and Nichols, '70).

Notes: The objective of the present study was to determine the effects of insulin on amphibian hepatocytes in primary culture. Hepatocytes were isolated from adult bullfrogs by collagenase perfusion and maintained as monolayers in serum-free medium. Cells cultured in the continuous presence of insulin exhibited a relatively constant rate of protein secretion over the first four to five days, whereas controls showed an almost three-fold decrease over the same time period. The decline in secreted proteins was equally represented in most exported proteins, except that serum albumin secretion showed twice as much of a decrease relative to the other proteins.The maintenance of protein secretion by insulin was the result of its effect on protein synthesis. The rate of protein synthesis was measured by the incorporation of (3H)-leucine into protein using culture medium containing 0.5 mM leucine, a condition where the specific radioactivity of leucyl-tRNA was shown to be equal to that of (3H)-leucine in the medium. Cultures maintained with insulin for 60 hours synthesized protein at two to three times the rate found in non-insulin treated controls whose rate of protein synthesis was first detectably decreased after nine hours of culture in the insulin-free medium.Sedimentation profiles of polyribosomes from hepatocytes maintained for 60 hours without insulin showed proportionately fewer ribosomes in large polysomes and more in monosomes and free ribosomal subunits than ribosomes from cells cultured with insulin. This result suggests that the decrease in protein synthesis found in the absence of insulin is due to a defect in initiation. Insulin does not exert its effect by regulating cellular levels of ATP; no change in ATP content was found in cells maintained with or without insulin.The results show that insulin maintains high levels of protein synthesis and secretion in amphibian hepatocytes. The hepatocytes in monolayer culture provide a system to study the molecular mechanisms involved in the translational control of protein synthesis by insulin.

Notes: The metachromatic fluorochrome acridine orange was used to differentially stain DNA and RNA in Chinese hamster ovary (CHO) cells and in mitogen-stimulated human lymphocytes during their progression through the cell cycle. Green and red fluorescence of individual cells, representing cellular DNA and RNA, respectively, was measured by flow cytometry.CHO cells were synchronized by selective detachment at mitosis. Their rate of progression through G1 and subsequently through S phase correlated with the content of stainable RNA. The mean duration of the G1 phase was 5.2 hours for cells with high RNA content (highest 25 percentile population) and 8.1 hours for cells with low RNA (lowest 25 percentile). The duration of S phase was 5.9 and 7.5 hours for high- and low-RNA, 25 percentile subpopulations, respectively.Lymphocytes synchronized at the G1/S boundary by hydroxyurea or 5-fluorodeoxyuridine showed extremely high intecellular variation with respect to content of stainable RNA. After release from the block they traversed S phase at rates linearly proportional to the content of stainable RNA. The duration of S phase was five hours for cells with high RNA-, six to nine hours for cells with moderate RNA- and up to 27 hours for cells with minimal RNA-content.The data suggest that the rate of progression the cell cycle of individual cells within a population may be correlated with the number of ribosomes per cell.

Notes: Stimulation of postconfluent Swiss 3T3 cells in serum-free medium with 4.3 mM Ca2+ results in marked increases in both released and cell-associated plasminogen activator (PA). Increased release of PA commenced approximately 10 to 12 hours post-stimulation and continued to increase steadily until 48 hours at which time the stimulated cells (4.3 mM Ca2+) released approximately 14 times more PA than control cells (1.8 mM Ca2+). Sr2+, like Ca2+, also stimulates PA synthesis/release either in the presence or in the absence of 1.8 mM Ca2+ whereas an excess of Mg2+ inhibits Ca2+ stimulation. Supranormal [Pi] in the medium stimulates PA synthesis/release in the presence of 1.8 mM Ca2+. Further, optimal stimulation by 4.3 mM Ca2+ requires a normal level of Pi (1.0 mM). Elevation of medium [Ca2+] or [Pi] results in an enhanced uptake of Ca2+. The facts that cycloheximide treatment completely abolishes the Ca2+ stimulatory effect and that an increase in cell associated PA precedes release indicate that PA release is coupled to synthesis of new PA. Ca2+ stimulation of PA synthesis/release also requires continuous energy production and RNA as well as protein synthesis. A hypothesis is proposed to explain the relationship between stimulation of PA production and its enhanced release from cells stimulated by elevated [Ca2+] or [Pi] in the media. The possibility that PA release may be an example of the phenomenon of membrane shedding as opposed to secretion is discussed.

Notes: Binding of either low density lipoprotein (LDL) or Concanavalin A (ConA) to actively growing vascular endothelial cells is associated with a redistribution of the appropriate cell surface receptor sites which form patches and caps. This receptor lateral mobility is greatly restricted when endothelial cells reach confluence and adopt the configuration of a cell monolayer composed of closely apposed and non-overlapping cells. In this case, although the cells still exhibit specific LDL binding to the appropriate cell surface receptor sites, neither the binding of LDL nor of ConA induces a receptor redistribution. The lack of LDL receptor redistribution correlates with a marked decrease in the rate of LDL internalization. In contrast, no such a density-dependent changes are observed in cell types which grow on top of each other and form multiple cell layers at confluence. Thus, neither LDL nor ConA induced cap formation in either sparse or confluent smooth muscle cell cultures and the same rate of LDL internalization is observed at both cell densities. Similarly, adsorptive endocytosis of cationized LDL (which enters the cell independently of the LDL receptor sites) was not correlated with a detectable receptor redistribution, nor was it significantly affected by changes in cell density and spatial organization.The formation of a confluent cell monolayer resting on an underlying basement membrane might therefore provide, via a change in membrane dynamics, a mechanism whereby the endothelium of large blood vessels can function as a protective barrier against the high circulating levels of LDL in plasma.

Notes: The three xanthine derivatives, caffeine, theophylline and 3-isobutyl-1-methyl-xanthine (IBMX) produced dose-dependent increases in cyclic AMP concentrations in HeLa cells after long term treatment. Only IBMX produced increases over the first 60 minutes, with a peak of approximately 5-fold control values five to 10 minutes after the addition of the drug. About four hours after the addition of either 0.67 or 1.0 mM IBMX there was a second peak in the concentration of cyclic AMP which was at least as large and usually larger than the peak observed at five to ten minutes. Neither caffeine nor theophylline increased cyclic AMP concentrations above control values until one hour after addition of the compounds, and there was no indication of a peak in the concentration at four hours. Between 24 and 72 hours, all three compounds produced elevations in cyclic AMP levels that were steadily maintained. At any given concentration, the order of potency was IBMX 〉 theophylline 〉 caffeine. If the xanthine derivatives were removed from the medium after 24 hours of treatment, the cyclic AMP concentrations fell to control levels within one hour. Treatment with 5-iodo-2′-deoxyuridine (IdUrd) or hydrocortisone alone did not change the levels of cyclic AMP, nor did the presence of these inducers of alkaline phosphatase activity alter the effects of the xanthine derivations on cyclic AMP concentrations. The data showed a significant correlation between the magnitude of the increase in cyclic AMP concentrations over the period from 24 to 72 hours and the degree of inhibition by the xanthine derivatives of the induction of alkaline phosphatase activity.

Notes: Hepatocytes were isolated by established procedures from freshly-excised livers of ovariectomized rats. Integrity of the cells was verified by DNA, protein, and calcium contents, and by dye exclusion. The cells also showed progressive increments in oxidation to 14CO2 of [26-14C]cholesterol during one to five hours' incubation. Analysis was undertaken of cellular reactivities toward estrogen and the hepatocarcinogen dibutylnitrosamine (DBN). Binding and retention of [3H]estradiol-17β (E2β) by isolated liver cells was specific for E2β, saturable, temperature-dependent, and maximal after 30-minute incubation. The apparent dissociation constant for the binding process at 22°C is 2 × 10-9 M, and the total number of binding-sites at saturation corresponds to approximately 3,400 E2β molecules per liver cell. To probe for steroid binding-sites at their external surfaces, cells were incubated 30 minutes with mounted 17β-estradiol-17-hemisuccinyl:albumin:nylon fibers. The covalentlyimmobilized estrogen (1 ng/mg albumin) was accessible for interaction with antiserum directed against 17β-estradiol-17-hemisuccinyl:albumin. Significant numbers of isolated liver cells were retained by estrogen-derivatized fibers at 22°C after extensive washes. Binding was markedly reduced by incubation at 4°C and by prior exposure to free E2β (× 10-8 M), but not to the relatively inert estradiol-17α (E2α). Fiber-bound cells could be dislodged by brief incubation in 150 mOsM saline with 2 × 10-7 M E2β or diethylstilbestrol, but not E2α, cortisol, progesterone, or testosterone, and recovered intact. Cells that had been retained by the fibers and those that were not adherent were collected and washed under identical conditions, then plated in serum-free, chemically-defined medium at 37°C. After 72 hours, specific binding of E2β by the fiber-binding cells during 30 minutes' incubation was 2.5-fold that of cells which had not bound the immobilized steroid. Similarly, stimulation of the oxidation to 14CO2 of [26-14C]cholesterol by E2β was greater in fiber-binding than in non-binding liver cells after three hours' incubation. In the absence of added mitogen, thymidine incorporation into macromolecular form (20 hours), and cell proliferation (48 hours) were significantly greater in fiber-binding cells as compared to non-binding hepatocytes. Moreover, in parallel experiments, when cells were exposed to 1 × 10-9 M estrogens or to 1 × 10-4 M nitrosamines to assess the capacities of these substances to increase basal thymidine incorporation, total DNA, and cell numbers, only those cells with estrogen-binding sites at their surfaces showed significant E2β- and DBN-induced increments in these parameters as compared with paired controls that had been treated with E2α or the noncarcinogen diphenylnitrosamine. These data indicate that the accessibility of hormone-binding components at the plasma membrane may contribute to the capacity of a given liver cell to respond to E2β, as well as to other known hepatocarcinogens.

Notes: The genetic approach to the problem of cellular growth control is limited by the availability of recessive mutations in cell lines which are capable of growth control in vitro. The CHO cell line has yielded many recessive mutations including, for example, tsH1, a temperature sensitive leucyl-tRNA synthetase mutant, which under non-permissive conditions rapidly shuts down protein synthesis and generates uncharged tRNA. Both CHO and tsH1 are transformed, however, and do not respond to environmental stimuli with the coordinated regulation of macromolecular processes observed in normal diploid fibroblasts. We describe here the isolation and characterization of growth control revertants obtained from both CHOwt and tsH1. The best of these GRC+L-73, isolated from tsH1, had 20 chromosomes, one less than tsH1, had normal fibroblastic morphology, would not grow in suspension, required high serum concentrations for growth, grew to relatively low cell densities at saturation in monolayer culture and showed a stationary phase characterized by arrest in a G1-like state with maintenace of high viability for several weeks. It is expected that this line as well as a ts revertant GRC+LR-73 will greatly facilitate the genetic investigation of growth control and, in particular, will help to elucidate the role of uncharged tRNA in the regulation of macromolecular synthesis in mammalian cells.

Notes: Treatment of adult rats with dexamethasone resulted in an increase in cardiac muscle weight but a decrease in skeletal muscle weight. The different response of skeletal and cardiac muscles to the glucocorticoid was also reflected by a dexamethasone-induced enhancement of myofibrillar protease activity in the gastrocnemius muscle and an inhibition of a similar proteolytic activity in the heart. Newborn rats also exhibit the same, tissue-specific response to the glucocorticoid hormone. Consequently, the difference between cardiac and skeletal muscle responsiveness to conditions of wasting was investigated in culture. Average rates of degradation of intracellular proteins were determined in cultured cells derived from rat skeletal and cardiac muscle by following the release of radioactivity from cells prelabelled with 14C-phenylalanine. The release of label into the TCA soluble medium as measured during 12 hours of incubation, conformed to a first-order reaction and both cell types were found to degrade intracellular proteins at a similar rate. After 12 hours of incubation in a complete Ham F-10 medium supplemented with serum approximately 18% of total cellular protein was degraded. Incubation in a minimal medium or serum-deprivation enhanced the average rate of proteolysis to a value of 29% degradation at 12 hours indicating that intracellular proteolysis in these cells is responding to nutritional deprivation by increased activity. However, addition of glucose (22.2 nM) or dexamethasone (10-6M) to the incubation medium failed to affect the rate of net protein degradation. Under no experimental condition could a difference be found between the proteolytic response of skeletal muscle cells to that of cardiac muscle cells and both cell types displayed similar changes in rates of protein degradation under various nutritional and hormonal conditions in culture. Thus, protein sparing in the heart of intact animals under catabolic conditions which enhance protein loss in skeletal muscle can probably not be ascribed to intrinsic differences in the direct response of cellular proteases to the tested hormones and nutrients. Rather, an extracellular factor(s) is apparently required for induction of the differential response of these tissues in the intact animal to protein wasting conditions. Alternatively, cells in culture might have lost the property of differential degradative response which operates in vivo.

Notes: In the past, it has been difficult to grow human diploid fibroblast cells at clonal densities. Newly devised cell culture media and rigorously controlled environmental conditions have greatly increased the ease with which such cells can be cloned. The present work was undertaken to determine whether, under appropriate conditions, diploid fibroblast cells from human embryonic lung, grow as well at clonal densities as in mass culture. The parameters studied were: (1) population doubling time, (2) in vitro proliferative capacity, (3) attachment, (4) percentage of non-dividing cells. In all cases essentially the same results were obtained for cultures at clonal densities and mass cultures. These results indicate that the behavior of these types of cells in clonal culture can be used to infer the behavior of individual cells and clones within a mass culture.

Notes: RNA synthesis has been investigated in resting and growing cells of a culture of Scarlet rose. The rates of messenger RNA (mRNA) and ribosomal RNA (rRNA) synthesis are five- and ten-fold higher, respectively, in the growing culture. In stationary phase cultures, newly synthesized 26S and 18S rRNA do not appear in the cytoplasm in equimolar amounts. Rather, the 26S/18S ratio of [3H]-uridine labeled rRNA of stationary cells ranged from 0.9 to 1.3 while the ratio of the corresponding fraction from growing cells was 1.6 to 2.0. A similar result was obtained when cells were labeled with [3H-CH3] methionine. Pulse chase experiments demonstrated that the nascent pre-rRNA in resting cells could be chased into polysomes. These data are interpreted to indicate that a major part of the regulation of rRNA synthesis in stationary cells is at the level of the processing of the rRNA transcript.

Notes: Replicative activity of isolated chromatin from late passage cultured mouse cells has been compared to the activities of chromatin preparations from dividing and quiescent early passage cells. Rates of endogenous DNA synthesis are similar for chromatin from growing or resting cells but this activity is stimulated 2.5-fold in senescent cell chromatin. Chromatin from growing young cells copies exogenously added single stranded DNA at the highest efficiency. Chromatin of senescent cells copies this template at a lower rate and resting young cell chromatin replicates single stranded DNA at the lowest efficiency. Similar relative rates are obtained when activated DNA is copied by the various chromatin preparations. Total activity of DNA polymerase extracted by salt from chromatin is similar for dividing and quiescent young cells but the proportion of DNA polymerase β is higher in the latter. Elevated activities of DNA polymerases are extracted from chromatin of old cells. It is concluded, therefore, that chromatin-directed replication is differently arrested in nondividing senescent cells and in quiescent early passage cells. The possible regulatory mechanisms of DNA replication in quiescence and aging are discussed.

Notes: Evidence is presented which suggests the existence of at least two growth control points in cultured mammalian cells. These controls are in “parallel” rather than in “series.” It is suggested that in normal and some transformed cells the controls are coupled while in SV40 transformed cells and tumor cells they are uncoupled. One control is revealed by cytochalasin B (CB) treatment since in normal and some kinds of transformed cells, CB treatment results in the accumulation or the arrest of cells in G1. In this case, the control is normal or coupled. In DNA tumor virus transformed cells and many cultured tumor cells (excluding human glioblastomas) CB does not arrest cells although it prevents cytoplasmic division. Such cells continue DNA synthesis and nuclear division and become highly multinucleated. The effects of CB are not concentration dependent. Here the control is defective or uncoupled. Another control is revealed by caffeine treatment. Again, normal and some types of transformed cells are accumulated in G1 following caffeine treatment, while SV40 transformed cells and tumor cells continue DNA synthesis unabated. The effects of caffeine in arresting cells are concentration dependent. Similarly the control point revealed by caffeine is coupled in normal cells and altered or uncoupled in some SV40 transformed and tumor cells. Although CB and caffeine do not inhibit DNA synthesis in tumor and SV40 transformed cells when administered separately, they cause G1 accumulation when administered in combination. This observation can be explained by the possibility that some transformed cells can utilize alternate or parallel mechanisms to progress through G1 when one mechanism is blocked (CB or caffeine). When both are blocked the cells become arrested in G1. The human glioblastomas are unique among the cultured human tumors thus far examined since they are partially arrested by CB. However they are like other human tumors in that caffeine does not significantly affect DNA synthesis.

Notes: Measurements of plating efficiency, accumulation of metaphases and generation times have shown that fibroblast from patients with Fanconi anemia (FA) have decreased probability of completing a further division after successful mitosis. Thus FA cells show decreased growth rates and increased generation times. We have also measured the survival of FA fibroblasts and lymphoblasts after treatment with a variety of mutagens. All FA cells show an increased sensitivity to drugs such as MMC and psoralen plus long wave length UV which cause DNA interstrand crosslinks. FA strains show varying degrees of sensitivity to these drugs and the extent of this sensitivity seems to be characteristic of each patient. FA cells are equal to controls in their sensitivity to other alkylating agents such as ethyl methane sulfonate, N-methyl-N1-nitro-N-nitrosoguanidine and actinomycin D. Both the decreased growth and increased drug sensitivity may result from defect in DNA replication or repair.

Notes: We have tested the ability of [5′-32P]-deoxyribonucleoside monophosphates (dNMPs) to penetrate living mouse fibroblast L cells and human HeLa cells. Under the conditions of our experiments, small numbers of apparently intact dNMP molecules appeared to penetrate into the interior of L cells and be incorporated into DNA. This incorporation was not due to mycoplasma contamination nor to extracellular hydrolysis of the dNMPs followed by resynthesis inside the cell. Under these same conditions, penetration of HeLa cells by intact dNMPs did not occur to a significant extent. However, HeLa cells were capable of hydrolyzing extracellular dNMPs to Pi and deoxyribonucleosides at a much faster rate than L cells.These experiments provide a starting point for attempts to specifically label the DNA in intact, living eukaryotic cells with [32P]-dNMPs.

Notes: We have investigated the de novo synthesis of intermediates of purine nucleotides in 3T6 fibroblasts and determined the manner by which the activity of this pathway is increased in resting cells by the addition of fresh serum. Within 30 minutes after stimulation, 3T6 cells began to synthesize increased amounts of purines by the de novo pathway as measured by increased amounts of formylglycinamide ribonucleotide, a representative intermediate of this pathway. Within 15 minutes after serum-stimulation 3T6 cells exhibited a substantial increase in their capacity to synthesize ribose compounds, particularly in the form of 5-phosphoribosylpyrophosphate (PRPP). The availability of PRPP appeared to be limiting for the synthesis of purine nucleotides in resting fibroblasts, but not necessarily in serum-stimulated cells.The amount of the enzyme PRPP synthetase as measured in vitro remained constant for at least the first four hours. Therefore, a study was made of various compounds known to activate PRPP synthetase in vitro. No evidence was found that suggested involvement of concentrations of cyclic nucleotides or phosphate. Experiments with methylene blue, an artificial electron acceptor that stimulates the production of ribose 5-phosphate by the hexose monophosphate shunt, indicated that one of the immediate consequences of the addition of serum is increased cycling of the pyridine nucleotide coenzymes, NADP+ and NADPH, and that the rapid increase in formation of ribose compounds and, consequently, purine nucleotides was caused as a result of modulation by this coenzyme. The relative ration of ATP:ADP:AMP as well as their concentrations remain constant in resting and serum-stimulated cells under normal assay conditions. However, there was a substantial decrease in ATP concentrations with a corresponding increase in AMP concentration with methylene blue in the assay buffer. The production of AMP from ATP was 5-fold greater in the serum-stimulated than in the resting fibroblasts. The increased production of AMP is thus serum-dependent and may reflect a basic enzymatic function of proliferative as compared to resting cells.

Notes: Addition of ATP to medium surrounding intact, transformed 3T3 cells activates the formation of aqueous channels in the plasma membrane. This results in efflux of nucleotide pools and ions and entry into the cytosol of charged, phosphorylated species. In such permeabilized cells, glycolysis is totally dependent on the external addition of glucose, inorganic phosphate, ADP, K+, Mg2+ and NAD+ which restore lactic acid formation to levels found in untreated cells. As expected, such reconstitution of glycolytic activity is found to restore intracellular ATP levels. This is accompanied by sealing of the membrane channels so that efflux of nucleotide pools ceases. Pyruvate, a substrate for mitochondrial ATP synthesis, when provided along with ADP and inorganic phosphate also produces sealing of the membrane channels. On the other hand, reactivation of pentose phosphate shunt activity, which does not lead to ATP synthesis, does not induce restoration of the membrane permeability barrier. Furthermore, compounds which lower the internal ATP pool prevent sealing, and also render the plasma membrane more sensitive to external ATP (Roxengurt and Heppel, 1979). Sealing of aqueous channels following restoration of the internal ATP pool is associated with phosphorylation of the inner membrane surface, and is unaffected by inhibitors of protein synthesis, microfilament or microtubular assembly. These results indicate the probable role of intracellular ATP in the restoration and/or maintenance of an active membrane barrier against efflux of small molecules and ions in transformed 3T3 cells.

Notes: The relationship between cell density and the activity of 2′:3′-cyclic nucleotide 3′-phosphohydrolase (CNP), an enzyme believed to be specific to oligodendroglial cells and myelin in the brain, has been studied in cultured C-6 glioma cells. Over a 12-day period, the specific activity of CNP underwent a 4-fold increase in conjunction with an increase in the cell density (total protein/flask) and a decline in the growth rate of the cultures. In contrast, the specific activity of Na+, K+-ATPase was not influenced by cell density. Experiments with cultures seeded at different initial densities indicated that the increase in CNP activity coincided with the attainment of a specific cell density rather than with the length of time that the cells were maintained in culture. Arrest of cell proliferation in non-confluent C-6 cells by means of thymidine blockade was not sufficient to cause an increase in the activity of CNP; however, removal of serum from the culture medium resulted in a 3-fold induction of the enzyme in the absence of a high degree of cell contact. The induction of CNP in cells maintained in serum-free medium paralleled the development of a series of distinct morphological changes reminiscent of glial differentiation, which occurred within 48 hours after removal of the serum. Inhibition of protein synthesis by cycloheximide prevented the induction of CNP in serum-free cultures. The demonstration that an enhancement of an oligodendroglial characteristic in C-6 glioma cells can be obtained by growing the cells to high density or by removing serum from the medium, provides further support for the suggestion that these cells may be analogous to the glial stem cells present in the developing brain.

Notes: Extracts of submaxillary glands from two different strains of inbred mice were mitogenic for human endothelial cells in culture. The mitogenic activity of extracts from glands of males of the SWR/J and C57BL/10J strains were equivalent, and the growth stimulating effect was unrelated to renin or esteroproteolytic activity. Mitogenic activity in extracts from SWR/J females was less than that from males, and extracts from C57BL/10J females were inactive. The polypeptide growth factors, epidermal (EGF) and fibroblast (FGF) growth factors, also stimulated replication of endothelial cells. Cells from either umbilical arteries or veins responded to submaxillary extracts, EGF, or FGF with a similar increase in cell number, increase in protein and enhanced uptake of 3H-thymidine. The proliferative response was associated with decreased activity of angiotensin I converting enzyme which is localized on the endothelial surface. Nerve growth factor (NGF) was not mitogenic for endothelial cells. Extracts of submaxillary glands from male mice of either strain contained approximately 20 times more EGF than extracts from females, as determined by immunodiffusion. Mitogenic activity of the extracts was completely inhibited by antiserum to EGF, suggesting that the active component of these preparations is EGF.

Notes: Electron probe energy dispersive X-ray microanalysis was performed on freeze-dried tissue sections. The dry weight concentration of elements (mmole/kg dry weight) was measured in the cytoplasm of several cell types from adult mice and rats. This comparative investigation showed: (1) That the energy dispersive X-ray spectrum of element concentration from the cytoplasm of a specific cell type allows one to distinguish this specific cell type from other cell types with considerable accuracy. (2) That there is a relationship between the concentration of the various elements and the ultrastructural features of the cytoplasmic regions being analyzed. For example, areas rich in ribosomes are also rich in P, K and Mg. (3) These data support the idea that K is directly involved in the control of protein synthesis. The catalog of element concentrations in the cytoplasm of 13 cell types from both mice and rats should be of value to others who seek to answer various questions about these cell types.

Notes: A separation procedure has been developed for mouse splenic T and B lymphocytes which is based on their differential agglutination by wheat germ agglutinin (WGA). In the presence of 50-100 μg/ml of WGA, multicellular aggregates are formed which are enriched in B cells. These aggregates can be separated from monodisperse T cells by gravity sedimentation and subsequently dissociated into single cells by treatment with N-acetylglucosamine (NAG). Immunocytochemical analyses and mitogenic assays indicate approximately 10-15% cross contamination of the resultant B and T cell fractions. The separation procedure is not only convenient and rapid but also allows the simultaneous recovery of viable T and B cells from the same spleen preparation.

Notes: A temperature sensitive mutant of Rous sarcoma virus (tsNY68) was used to obtain cultures of quiescent virus-infected chicken embryo fibroblasts arrested by serum starvation at the non-permissive temperature. Upon shift to the permissive temperature, these cells enter the replicative cell cycle as evidenced by increases in 2-deoxyglucose uptake, 3H-thymidine incorporation and percent labeled nuclei. These changes occur in the absence of serum and the cells become morphologically transformed within eight to ten hours after the temperature shift. Entry into the S phase temporally resembles that of normal quiescent fibroblasts stimulated with serum. This experimental system was used to examine the proliferative response of transformed cells to serum and purified multiplication-stimulating activity (MSA) during the transition from the resting to the growing state. Data are presented which show that the presence of serum in the medium enhances the proliferative response of quiescent infected cells shifted to the permissive temperature over those shifted in the absence of serum. In contrast, the presence of MSA has no additional effect on the response exhibited by infected cells shifted to the permissive temperature in serum-free medium. Labeled MSA binding experiments show that this lack of response is not due to a loss of MSA receptors on the cell surface since transformed cells are still capable of binding MSA at the same level as normal cells. The results are consistent with the hypothesis that the set of biochemical events initiated by MSA in normal cells are turned on in infected cells shifted to the permissive temperature by the activation of the src gene product.

Notes: Protein synthesis in differentiated MOPC-21 and MPC-11 mouse myeloma cells was studied to determine the basis for the differences in the temperature and actinomycin D sensitivity of translation between non-differentiated mouse L-cells and differentiated rabbit reticulocytes. The temperature dependence of total protein synthesis was similar to that of L-cells and reticulocytes, being biphasic in Arrhenius plots with apparent activation energies of approximately 25 and 42 kcal/mol, above and below 25°C. The dependence of the secretion process was different since it was not biphasic, having a single activation energy of about 22 kcal/mol. Myeloma polysomes were like L-cell polysomes in their response to lower temperature and reached a minimum level of 50% at 15°C. This response was also found for the specific polysomes synthesizing the IgG H- and L-chains. In the presence of actinomycin D, myeloma polysomes declined exponentially with a half-life of ∼6 hours. These two L-cell-like responses were not found in reticulocytes. Translation of both the IgG mRNAs and the non-IgG mRNAs was reduced by lower temperatures and actinomycin D, even though the L-chain mRNA was slightly more resistant, suggesting that this mRNA is slightly more efficient. The results of these experiments suggest that the translational differences between L-cells and reticulocytes are not mRNA dependent, but are cell type differences.

Notes: Cytotoxicity and membrane permeability alterations induced by the polyene macrolide antibiotics filipin (FIL) and pimaricin (PIM) have been compared in parental intraspecific and interspecific somatic cell hybrids. B82 (mouse) and B1 (hamster) cells were found to be more resistant than RAG (mouse) parental cells to both polyene macrolides as indicated by 24-hour survival, 72-hour viability, and growth rate. Analysis of both intraspecific and interspecific somatic cell hybrids indicated that polyene macrolide resistance was being expressed even in the presence of the polyene macrolide-sensitive (RAG) genome. Where one of the two parental cell types is relatively polyene macrolide resistant, the use of specific polyene macrolides may prove efficacious as half-selective agents in cell hybridization.

Notes: Enhanced uptake of amino acids is frequently associated with hyperplasia in cultured cells; we have recently shown (Merrill et al., '77) that this is true of the stimulation of rat myoblast proliferation in culture by Temin's Multiplication Stimulating Activity (MSA). In many cases the asymmetric distribution of Na+ and K+ across the cell membrane profoundly affects uptake of certain amino acids, so we investigated the possibility that enhanced Na+-dependent AIB transport was the result of an MSA-induced increase in K+ accumulation. MSA stimulated the rate of uptake of the potassium analog 86Rb+ 15-25% within ten minutes; this rate remained elevated for at least seven hours. Effects were limited to the ouabain-sensitive component of Rb+ uptake. (Our simultaneous measurements of 86Rb+ and 42K+ uptake demonstrated that Rb+ provides a useful qualitative but not an exact quantitative index of K+ uptake.) The stimulation of K+ uptake by MSA did not cause a large increase in total cellular K+ of the myoblasts; after five hours, MSA-treated cells contained only about 20% more K+ than did corresponding controls (1.21 ± 0.02 vs. 1.01 ± 0.02 μmoles/mg protein, respectively). To investigate whether this small increase in K+ content could be amplified by the cell to account for the 50-150% stimulation of AIB uptake, we preincubated cells with ouabain for various times and then measured total cell K+ and AIB uptake in the same culture dishes. Under conditions in which the MSA-stimulated increase of total cell K+ was prevented by ouabain, a substantial stimulation of AIB uptake was still observed. We conclude that MSA stimulation of AIB transport is independent of increased accumulation of K+ in rat myoblasts.

Notes: Conditions for in vitro growth of mononuclear phagocytes from newborn hamster liver and lung were studied. In the primary cultures of liver and lung, round cells outgrew and frequently floated off into the culture medium. They were separated from fibroblast-like cells adherent to plastic by collecting the medium. The round cells were identified as mononuclear phagocytes on the criteria of phagocytic capacity of heat-killed bacteria and IgG-coated erythrocytes, fine cell structure and cytochemistry. The phagocytes that had not been activated previously proliferated for about ten generations in F12 medium supplemented with 10% fetal calf serum depending on a growth factor produced by hamster brain, liver or lung cells. Without the factor, the cells quickly cytolysed. Mononuclear phagocytes from blood had the same characteristics of growth and cytochemistry, but had fewer IgG receptors at the cell surface than similar cells from the liver and lung.The effects of a variety of chemical compounds on the growth of the liver and lung cells were studied. Insulin stimulated their growth by 20-30%, but was not replaceable for the growth factor. Glucocorticoids, dexamethasone and hydrocortisone, inhibited the growth of the phagocyates at the physiological concentrations: 3 × 10-9 M and 2 × 10-8 M for 50% inhibition, respectively. Indomethacin, non-steroid anti-inflammatory reagent, at 10-8 M to 10-6 M gave no effect. Choleragen that increases the intracellular cyclic AMP level, inhibited the growth at a concentration as low as 5 pg/ml. These data suggest that the growth of mononuclear phagocytes is controlled not only by a growth factor produced by other cells but also by glucocorticoids.

Notes: Epidermal growth factor (EGF) at nanomolar concentrations stimulated DNA synthesis in confluent, serum-starved cultures of calf aorta and human uterine smooth muscle cells. Stimulation of DNA synthesis in lens epithelial cells was studied for comparison. L and D-ascorbic acid potentiated the effect of serum and EGF on DNA synthesis in calf aorta cells. In contrast L-ascorbic acid had minimal potentiating effect with serum and no effect with EFG present along with serum on DNA synthesis in human uterine smooth muscle and rabbit lens epithelial cells. EGF and ascorbic acid increased cell number when added to stationary phase cultures. Specific binding of 125I-labelled EGF to smooth muscle cells was demonstrated. Receptor concentration in calf-aorta smooth muscle cells was higher in dense cultures compared to sparse cultures. The time course of binding and dissociation of 125I-labelled EGF was similar in “dense” and “sparse” cultures.Human uterine smooth muscle cells in culture exhibited a finite lifespan. There was no stimulation of DNA synthesis in response to serum and EGF in cells of high population doubling level (PDL); although 125I-labeled EGF binding was higher in old cells (high PDL) compared to young cells (low PDL). This increase in binding was shown to be due to changes in the concentration of receptors without changes in their affinity for EGF.

Notes: The effect of estrogen stimulation in vitro on the electrical properties of vascular smooth muscle (VSM), and the concentration of estrogen receptors in VSM were measured in isolated coronary arteries. Microelectrode measurements of the dog coronary artery membrane potential (Em) showed quiescent values of -51 millivolts (mV) and an input resistance (rin) of 10 megohms. Addition of diethylstibestrol (DES) at 10-6 M hyperpolarized the membrane to -64 mV and reduced in resistance (rin) to 5 megohms within 15 minutes. Extrapolation of the Em vs. log [K]o curve to zero potential gave similar values of [K]i of around 170 mM in both normal and DES treated muscles suggesting that the DES induced hyperpolarization is not due to increased Na-K pump activity. The 0.5% ethanol vehicle alone had no effect on the membrane potentials. Tetraethylammonium ion (TEA) induced action potentials in the previously quiescent tissue. When DES was applied in the presence of TEA, the membrane potential increased and the action potential were abolished. Scatchard analysis of the estrogen receptor binding demonstrated both a high and a low affinity receptor for estrogen in the VSM. These data indicate that DES hyperpolarizes the VSM cells by a mechanism other than an increased Na-K pump activity. The mechanism of this increased Em may be due to factors which increase K+ conductance either mediated directly through estrogen interaction with its cytosolic receptors or through some unidentified second mechanism.

Notes: The relationship of cell surface changes to proliferative decline of human diploid fibroblasts was investigated using the concanavalin A-mediated red blood cell adsorption assay. The amount of the red blood cells adsorbed to human diploid fibroblasts via concanavalin A increased continously from the early phases of cell passage up through cell senescence, while the amount of 3H-concanavalin A binding did not change to a significant extent. The red blood cell adsorption is not a function of cell cycle phase and time spent in culture. Cocultivation of young cells with old cells also did not affect the adsorption capacity of respective cells. Thus, the concanavalin A-mediated red blood cell adsorption can be expected to serve as a new cell surface marker for aging in vitro. Using this marker, it was revealed that transient cell size or 3H-thymidine incorporating capacity do not have a direct relationship with the division age of a cell. Small rapidly dividing cells in old populations resemble large slowly dividing or nondividing cells of the same populations and differ from small rapidly dividing cells in young populations, in terms of cell surface properties.

Notes: We have found that tryptose phosphate broth (TPB) prevents the inhibitory effect of chloramphenicol (CAM) on the cell proliferation of chick embryo fibroblasts. Study of growth parameters indicated that no lag or adaptation period appeared necessary for TPB-exposed chick cell populations to grow in the presence of CAM suggesting that a particular cell type was not selected. TPB did not prevent the inhibitory effect of CAM on the mitochondrial protein-synthesizing system. This was supported by cytochrome oxidase activity measurements, studies on the incorporation of 35S-methionine into mitochondrial proteins, electron microscopic observation of alterations in mitochondrial structure. Oxygen consumption was reduced by 95% and cyanide, 2-4-dinitrophenol, and salicylhydroxamic acid do not significantly affect the residual respiration. Analyses of reduced-minus-oxidized-cytochrome spectra of CAM-treated chick cells demonstrate the disappearance of the absorption bands of cytochromes aa3, b559, c1, and c. The presence of a type b cytochrome with maxima at 552 and 557 nm was observed. The results obtained indicate that long-term cultures of CAM-treated chick embryo cells cultivated in the presence of TPB grow with mitochondria devoid of a functional respiratory chain.

Notes: Decreasing the K+ concentration of the medium from 5 mM to 0.59 mM decreased the K+ content of chick embryo fibroblasts to 22% of control values and increased the Na+ content to 820% of control values. The alteration of monovalent cation content occurred within two hours but had no effect on the rate of DNA synthesis, as measured by 3H-thymidine incorporation, for at least 16 hours. By decreasing the Na+ concentration in the medium, a 50% reduction in cellular Na+ could be obtained with no effect on thymidine incorporation. Since these changes in cellular Na+ and K+ are much larger than any known to occur under physiological conditions but have no effect on thymidine incorporation, we conclude that Na+ and K+ do not play a critical role in determining multiplication rate.Addition of 1.8 mM EGTA to cells in media containing 1.7 mM Ca2+ and 0.8 mM Mg2+ inhibited thymidine incorporation and sharply decreased cellular K+ and increased cellular Na+ content. However, there was no reduction in total cellular Ca2+ levels. Likewise, decreasing the Ca2+ concentration of the medium below 0.01 mM inhibited thymidine incorporation, decreased cellular K+ and Mg2+, and increased cellular Na+ but did not affect total cellular Ca2+ levels. Inhibition of DNA synthesis, therefore, could not be correlated with changes in cellular Ca2+ levels.

Notes: Human α-L-fucosidase, purified from placenta, was taken up from the culture medium by skin fibroblasts from patients with fucosidosis (α-L-fucosidase deficiency). The rate of uptake was low (uptake coefficient = 6 × 10-4 ml.mg-1.h-1). Intracellular α-L-fucosidase activity was directly proportional to enzyme in the medium up to an activity of at least 40 nmoles/min/ml. No evidence for saturation of specific cell-surface receptors was seen. However, uptake was reduced by 75% by 1 mM mannose-6-phosphate and by 50% by 1 mM glucose-6-phosphate, suggesting that uptake may be mediated by a receptor recognising a phosphorylated sugar or an analagous compound. Enzyme taken up by the cells was most active in subcellular fractions enriched with lysosomes and had an isozyme pattern, by isoelectric focusing, identical to that of the original enzyme preparation.Fucosidosis fibroblasts were shown to accumulate low molecular-weight, fucose-containing compounds to a level several times greater than control cells. This stored material was eluted from Sephadex G-25 as an asymmetrical peak with an elution volume of approximately twice the void volume of the column. Addition of placental α-L-fucosidase to the culture medium of fucosidosis fibroblasts prevented excessive accumulation of fucose-containing material and accelerated the breakdown of material accumulated prior to enzyme uptake.

Notes: The stability of both rapidly and slowly degraded proteins in wild type CHO cells is similar to that in three ts aminoacyl-tRNA synthetase mutants at both permissive and non-permissive temperatures, although the degree of tRNA charging in the synthetase mutants differs considerably with temperature. These results indicate that the altered rate of protein breakdown seen under a variety of physiological conditions in eukaryotic systems is not mediated by uncharged tRNA.

Notes: The manganese content of the egg and embryo of the Medaka, Oryzias latipes was determined by activation analysis. A remarkable increase in the amount of manganese in the egg was observed within one hour after fertilization. The rate of increase was reduced by the gastrula stage and the concentration of manganese remained unchanged at a later stage. The accumulation of manganese by the Oryzias egg was discussed in relation to the effect of manganese on respiratory enzyme systems.

Notes: Growth of a human leukemic T-cell line (CEM C7) in 10-6 M dexamethasone results in inhibition of growth and rapid loss of cell viability after a delay of approximately 18 to 24 hours. Analysis of dexamethasonetreated cells by flow-microfluorometry showed that they were arrested in the G1 phase of the cell cycle. Loss of cell viability began at the same time as G1 accumulation was first detectable, and 20% of all cells were found to be blocked in G1 at this time suggesting that loss of viability and G1 arrest were coincident events. Half-maximal and maximal effects on both viability and G1 arrest after 48 hours in steroid were nearly identical with respect to steroid concentration and corresponded to half-maximal and full occupancy of glucocorticoid specific receptor by hormone, consistent with a glucocorticoid receptor mediated mechanism for both phenomena. Most non-viable cells were arrested in G1, and accumulation of cells in G1 was irreversible; removal of steroid in the presence of colcemid did not result in a decreased fraction of G1 cells. Furthermore, dexamethasone treatment did not protect cells against the effects of 33258 Hoechstamplified killing of bromodeoxyuridine substituted cells exposed to light. These results show that dexamethasone arrests these leukemic cells in G1 and strongly suggest that dexamethasone-treated cells are killed upon entry into G1.

Notes: During synchronous differentiation of embryonic chick muscle cells in culture, the Na-dependent uptake of an amino acid analog, α-amino isobutyric acid (AIB) undergoes an abrupt, transient increase. The increase in AIB uptake is concomitant with the rapid fusion of mononucleated myoblasts, and precedes the accumulation of muscle-specific proteins. Subsequently, Nadependent AIB transport diminishes markedly during postfusional differentiation of myotubes. The rate of AIB uptake is increased by insulin both before and after myoblast fusion. This stimulation by insulin is restricted to the Nadependent component of total AIB uptake but is apparently not the result of insulin-mediated increase in the trans-membrane Na gradient.

Notes: The characterization of a temperature-sensitive Chinese hamster cell mutant has been continued with the aim of localizing the apparent defect in glycoprotein synthesis (Tenner et al., 1977). Although the mutation is lethal, a demonstration of the ability of the mutant cells to support proliferation of Mengo virus at the nonpermissive temperature indicates that the general metabolic processes of the cells remain intact at a time when glycoprotein synthesis is severely depressed. A quantitative study of protein synthesis on membrane-associated polysomes suggests that the synthesis of the polypeptide portion of the glycoproteins at 40.8°C may be normal. The investigation of lipidsaccharide molecules which have been implicated in the formation and transfer of the oligosaccharide „core„ to polypeptide acceptors shows that mutaant cells at the nonpermissive temperature are capable of synthesizing these lipid saccharides normally, and that the pool of the dolichyl oligosaccharide is maintained at a constant level independent of the temperature. The rate of formation of the lipid-oligosaccharide, however, is reduced in intact mutant cells at the nonpermissive temperature. Further investigations show this decreased rate to be the result of an increased half life of the lipid-oligosaccharide at 40.8°C. These data indicate that the temperature-sensitive step in glycoprotein biosynthesis is the transfer of the oligosaccharide core from the lipid-oligosaccharide intermediates to the nascent polypeptide chain. The data presented also provide evidence that the lipid-saccharide intermediates, previously described mainly in in vitro systems, are in fact involved in the glycosylation of a majority, if not all, of the mannose-containing glycoproteins in intact, growing hamster cells.

Notes: The involvement of post-transcriptional mechanisms in the determination of the frequency distribution of messenger RNA sequences has been studied in cloned mouse embryo cells in culture. Hybridization kinetic experiments between poly(A)-containing RNA and complementary DNA have been used to study the alterations in frequencies which occur in those nuclear poly(A)-containing RNA sequences which are conserved in cytoplasmic polyribosomes. Sequences adjacent to nuclear poly(A) tracts are present in a much narrower frequency distribution in the nucleus than in polysomes, with a large proportion of the nuclear sequences present in an average frequency of about one molecule per cell. Few nuclear sequences appear to be present in more than ten copies per cell. A minimum of 70% of these sequences are also found in polyribosomal RNA but in greatly altered frequencies. Abundant sequences which comprise a major fraction of the poly(A)-containing polyribosomal RNA are derived from a small fraction of the nuclear poly(A)-adjacent RNA sequences. Very few nuclear poly(A)-adjacent sequences are present in a frequency characteristic of high abundance polysomal sequences. Conversely, poly(A)-containing polyribosomal RNA appears to contain few sequences which are present in as low a frequency as the majority of nuclear poly(A)-adjacent sequences. These observations suggest that post-transcriptional mechanisms play a major role in determining the steady-state frequency of polyribosome-associated messenger RNAs.

Notes: Intermittent compressive (IC) forces (96 mm Hg, 0.3 Hz) inhibit by 35-60% the serum stimulated increase in ornithine decarboxylase activity (ODC) in chick embryo epiphyseal cartilage cells and rat chondrosarcoma cells. IC had no effect on mouse fibroblast L-cells ODC. The dose-response pattern of the IC effect indicated an all-or-none response with a threshold at 80 mm Hg, a pressure roughly equivalent to the in vivo weight bearing force. The km of the cartilage cell ODC, measured at four hours, was about 0.1 mM and was not affected by IC. The Vmax, on the other hand, was significantly reduced by IC which is consistent with less enzyme or non-competitive inhibition. IC also produced a significant increase in cAMP levels in both cartilage explants and isolated cells in the presence and absence of serum and a significant reduction in 3H-thymidine incorporation into DNA. The findings show that cellular cAMP, on one hand, and ODC and DNA synthesis, on the other hand, change in opposite directions following exposure to serum and/or IC. Investigation of the IC effect on DNA synthesis in serum-deprived synchronized cartilage cells revealed that IC reduced the number of cells going into S but did not lengthen the G1 phase. Exposure to IC early in G1 (0-13 hours) produced the full effect, whereas IC application between 13 to 24 hours (pre S) had no effect. IC had no effect on 3H-thymidine incorporation in L-cells.

Notes: Changes in amino acid transport activity by system A (a Na+-dependent agency with affinity for a discrete group of neutral amino acids) caused by the addition of serum to serum-deprived cultured chick embryo fibroblasts have been evaluated by measurements of 14C-labeled L-proline and α-methylaminoisobutyric acid uptake under conditions approaching initial entry rates. Dialysed serum was as effective as undialysed serum in stimulating amino acid transport. This effect was inhibited by 7 μM cycloheximide, by 80 nM actinomycin D and by 40 μM cordycepin, but not by 0.3 mM cytosine arabinoside. Cultured avian fibroblasts previously incubated in a cycloheximide-containing medium (phase of inhibited translation) in the presence of serum, subsequently exhibited a net increase of proline transport activity when transferred to a medium containing actinomycin D (phase of inhibited transcription). Omission of serum in the cycloheximide-phase prevented the increase of transport activity during subsequent incubation in the actinomycin D-phase; omission of serum in the actinomycin D-phase allowed a shorter and less pronounced increase of transport activity than in the presence of serum. Additions of actinomycin D or cycloheximide slightly increased the rate of decay of amino acid transport caused by serum withdrawal. These observations suggest that in cultured avian fibroblasts, serum modulates the activity of transport system A by a mechanism acting at the transcription level.

Notes: The temperature sensitive leucyl-tRNA synthetase mutant tsHl and two revertants have been compared to the parental Chinese hamster ovary cells with respect to the effects of amino acid concentrations in the medium on growth. Elevating the leucine concentration 30- or 100-fold allowed tsHl to grow exponentially at 38.5°C, normally the nonpermissive temperature. Partial revertants that had recovered some enzyme activity required smaller supplements for growth. Measurements of the leucine pools indicated that they respond directly to the extracellular leucine concentration and may mediate the effect. Use of combinations of amino acids confirmed that isoleucine has a similar though weaker effect on tsHl and identified an even weaker protection by valine. The triple combination of leucine, isoleucine and valine was a much more efficient medium supplement and three times normal concentrations of these amino acids supported growth of tsHl at 38.5°C. It is postulated that they are acting at their respective aminoacyl-tRNA synthetases to help stabilize a complex which also contains the mutant leucyl-tRNA synthetase. The pool size measurements also showed that the leucine pools of tsHl and a revertant increased 2-fold more in a response to increased temperature than those of WT. It is suggested that this is a regulatory response to low leucyl-tRNA synthetase activity and is important in determining growth phenotypes.

Notes: Nine spontaneous and seven ethyl methanesulfonate induced revertants of the Chinese hamster ovary cell line mutant (tsHl), which possesses a temperature sensitive leucyl-tRNA synthetase, were isolated and characterized with respect to growth rate, leucyl-tRNA synthetase activity and thermolability, intracellular leucine pool size, and rRNA content. Although most revertants had increased leucyl-tRNA synthetase activity, and of those tested, all but one had increased thermostability, each appears to be unique. One revertant may be an intergenic suppressor since it appears to contain an elevated level of tsHl-like synthetase. There was no evidence for any of the revertants having increased rRNA and tRNA contents, however, many showed leucine pools two to three times larger than wild type cells. Since similar increases have been observed in tsHl cells they are believed to result from regulation of leucine pool size by the leucyl-tRNA synthetase and are of a magnitude sufficient to affect significantly the growth of revertants at 38.5°C.

Notes: Fibroblasts were isolated from keloid, normal skin, and normal scar and maintained in tissue culture for four passages. Growth kinetics were the same for all groups on days 2 through 12. However, the rate of collagen synthesis per fibroblast was greater in keloid derived cells than any controls at all growth phases. Keloid fibroblasts have an autonomous capacity to synthesize collagen at a significantly increased level in vitro, which may explain in part why these lesions are characterized by increased collagen deposition.

Notes: Friend erythroleukemic cells, which grow continuously in tissue culture, resemble in many respects early precursors of mouse erythrocytes. To determine whether or not the membranes of these cells exhibit the rapid and selective exchange of chloride, a specialized feature of the mature erythrocyte membrane, anion fluxes were compared in Friend cells and mouse erythrocytes. The chloride flux in Friend cells at 37°C was about 800-fold lower than in mouse erythrocytes (extrapolated from data at lower temperatures). This difference could not be accounted for by the somewhat lower chloride concentration in Friend cells relative to erythrocytes. Comparison of chloride and sulfate fluxes revealed that the Friend cells had over a 1,000-fold lower selectivity for chloride versus sulphate than did the mouse red cells. The temperature dependence of chloride fluxes in Friend cells corresponded to an Arrhenius activation energy of 17.9 kcal/mol, in contrast to over 30 kcal/mol for mature red cells. The chloride flux in Friend cells was also 10-fold less sensitive to the inhibitor, furosemide, than was the flux in mature red cells. The selective chloride exchange system of the mature erythrocyte therefore does not seem to be functional at the stage represented by the Friend cell, and must appear at some later stage of erythroid maturation.

Notes: Cell division in fertilized sea urchin eggs was reversibly inhibited when the ketoaldehyde phenyl glyoxal (PG) at a concentration of 0.1 mM was added to eggs for ten minutes prior to the formation of the mitotic spindle. We investigated whether inhibition of mitosis was due to PG binding to the cell surface (as previously suggested by Stein and Berestecky, '74) or to some intracellular effect. When 14C-PG was added to eggs, label was readily taken up into the egg cytoplasm; very little label was associated with the egg surface. In the cytoplasm PG combined with equimolar amounts of reduced glutathione (GSH), decreasing the levels of cellular GSH to less than 15% of normal and accounting for at least 50% of the PG taken up by eggs. The concentrations of oxidized and protein-bound glutathione were unaffected by PG treatment. We showed that glyoxalase enzymes were present in sea urchin eggs and were capable of metabolizing the PG-GSH complex, thereby restoring GSH to normal levels after PG was removed from the sea water. Though some other effect of PG cannot be ruled out, the major fate of PG in eggs was to combine with GSH, and the transient decrease in GSH which resulted could lead to inhibition of mitosis. While other reports (Nath and Rebhun, '76; Oliver et al., '76) have shown that reagents which oxidize GSH disrupt microtubule-related events, our results showed that such inhibition could be caused by decreased GSH levels alone.

Notes: Follicle stimulating hormone (FSH) stimulates “colony formation” by immature rat Sertoli cells in primary culture. “Colony formation” involves cell aggregation. Consequently, the involvement of cell surface glycoproteins in cell aggregation was investigated by treatment of dissociated 10-day rat testis cells with sodium metaperiodate, glucosamine, various lectins, tunicamycin, and puromycin. Treatment of control cultures with 5 μM glucosamine stimulated cell aggregation; however, glucosamine did not affect FSH-stimulated cultures. Treatment of dissociated testis cells with 5 μM sodium metaperiodate, 10 μg/ml castor bean agglutinin (ricin), or 2.5 μg/ml horseshoe crab agglutinin inhibited FSH stimulation of cell aggregation. A similar inhibition of cell aggregation was observed following addition of 10 μg/ml puromycin or tunicamycin to culture media from 0- to 18-hours incubation. Treatment with soybean agglutinin, concanavalin A, or wheat germ agglutinin had no effect. The galactose-specific lectins, Ricin, Ricinus communis agglutinin I, and Bendeirea simplicifolia agglutinin, inhibit the FSH stimulation of 3H-aminoacid incorporation as well as cell aggregation in 24-hour cultres. The inhibition of cell aggregation by sodium metaperiodate treatment was reversed with 5 μM sodium borohydride reduction. Sodium metaperiodate treatment did not alter cell viability (as assayed with trypan blue dye exclusion), did not alter cell attachment, nor significantly decrease 125I-FSH binding by cultured testis cells. The results suggest that FSH stimulation of cell aggregation by immature rat Sertoli cells requires cell surface glycoprotein interactions. Furthermore, the specificity of lectin inhibition suggests that glycoproteins with terminal galactose and sialic acid residues are required for the FSH induction of cell aggregation.

Notes: Activities related to Na-K transport were measured in cell cultures of ground squirrel kidney cortex in order to compare these cells with those of intact kidney and of continuous cell lines. A microsomal preparation containing plasma membrane Na,K-ATPase from fresh kidney showed twice the activity of a similar preparation from 72-hour cultured cells. Na,K-ATPase of homogenates of 72-hour cells showed one-third to one-fourth the specific activity of that from 6-hour cultured cells. The associated K-dependent phosphatase activity also declined as a function of time in culture. The ouabain-sensitive influx of K into 6-hour cultured cells was twice as great as the K influx into 72-hour cells. The number of sites binding 3H-ouabain in intact cultured cells declined 81% on a cell protein basis between 6 and 72 hours in culture. This decline in ouabain binding sites was relatively greater than that of K influx, so that the K turnover number increased over this same time period.The decline in ouabain-sensitive K influx during culture was complementary to an increase in furosemide-sensitive K influx. Measurements of unidirectional and net K fluxes showed that there were three components of K influx into 3-day cultured cells: ouabain-sensitive Na:K exchange, furosemide-sensitive K:K exchange, and K diffusion. In the 6-hour cultures, however, there was no furosemide-sensitive K:K exchange.Thus, after three days in culture ground squirrel kidney cells lose a feature characteristic of the original parent cells (high Na,K-ATPase activity), and gain a feature common to many undifferentiated cultured cells (furosemidesensitive K:K exchange).

Notes: We have studied the effects of theophylline treatment on pigmentation characteristics and growth of two B16 melanoma cell lines, HFH-18 and P/140. Cell counts of control and theophylline-treated cultures confirmed that the drug inhibits cell growth. Light and electron microscope cytochemistry with the L-dopa reaction indicated that the two cell lines differ in their ability to transfer Golgi-associated tyrosinase to developing premelanosomes. The results of these experiments, considered with results of electrophoretic analyses and activity measurements by the Pomerantz method, also provide evidence that increased tyrosinase synthesis occurs in response to theophylline treatment. In addition, results indicate that theophylline induces changes in the rate of synthetic or degradative posttranslational modification of tyrosinase. Measurements of intracellular cyclic AMP levels by radioimmunoassay in control cultures and in theophylline- and α-MSH-treated cultures were made. Although the hormone induced spectacular increases in cyclic AMP levels, theophylline produced no detectable change. These results indicate that theophylline differs from α-MSH because theophylline-induced changes in pigmentation may not require the participation of intracellular cyclic AMP.

Notes: Phorbol esters stimulate 2-deoxy-D-glucose (DG) uptake in rodent and human cell cultures. The potent tumor promoting agent, 12-0-tetradecanoyl phorbol-13 acetate (TPA), induces a 12-fold stimulation in confluent 3T3 cells and a 2.5-fold stimulation in HeLa cells. When a series of macrocyclic diterpenes are assayed, their relative potencies in stimulating DG uptake in 3T3 cells correlate with other known biologic effects of these compounds. On a molar basis, TPA is a much more potent stimulator of DG transport than insulin or epidermal growth factor. In HeLa cells, the ED50 value of the TPA effect is 0.2 nM. The increase in DG uptake occurs immediately after the addition of TPA, reaches a maximum at 90 minutes, persists for at least three hours after removal of TPA from the medium, and is temperature dependent. The stimulation is not inhibited by cycloheximide or actinomycin D. As in control cells, DG uptake in TPA treated cells is inhibited by p-hydroxymercuribenzoate, phloridzin, cytochalasin B, and dexamethasone. Although the precise mechanism is not known, evidence is presented that the TPA stimulation of DG uptake is due to enhanced transport of the sugar rather than to effects of intracellular metabolism. The enhanced transport may be secondary to a more generalized change in membrane structure.

Notes: Three cell lines of mouse erythroleukemia transformed by Friend virus (FLC), namely 745, F4-1, and 3BM-78, were grown for six days in the absence or in the presence of 1.5% (v/v) dimethylsulfoxide (DMSO) and compared cytochemically for naphtol-AS D-chloroacetate esterase (E), alkalinephosphatase (AP), myeloperoxidase (MP) and periodic acid Schiff (PAS) reaction activity. In the absence of inducer only 1-2% of slightly E positive cells could be found. E positivity greatly increased in 3BM-78 and F4-1 but poorly in 745 cells, after treatment with DMSO. Unlike E reaction, AP and MP reactions were positive in about 5% 3BM-78 and F4-1 cells without DMSO, but there were no positive cells after DMSO treatment. All three lines were always PAS negative. Hemoglobin synthesis (benzidine staining) was intensively induced by DMSO in all three lines. Morphologically after DMSO treatment, FLC matured displaying characteristics of basophilic megaloblastoid cells. The emergence of specific esterase activity, a marker of granulocytes, in FLC differentiating along the erythroid pathway, suggests that in these leukemia cells the genetic determinants for leukopoietic differentiation are retained and capable of being expressed phenotypically.

Notes: The potential of nanomelic chondrocytes to synthesize chondroitin sulfate was investigated by providing the mutant cells with p-nitrophenyl-β-D-xyloside, a compound which acts as an artificial acceptor for glycosaminoglycan synthesis. Under these conditions the synthesis of chondroitin sulfate in nanomelic and normal chondrocytes is comparable. The chondroitin sulfate synthesized by the mutant is indistinguishable in molecular size and composition from that synthesized by similarly treated normal chondrocytes.

Notes: The equilibrium distribution of 5,5-dimethyloxazoladine 2,4-dione (DMO) between intra- and extracellular volume was used to estimate intracellular pH (pHi) in Tetrahymena pyriformis. In control experiments, DMO was found to equilibrate rapidly in response to a pH gradient. Under normal growth conditions, pHi was constant over a finite range of external pH, being maintained near pH 7.1 over the external pH range 5.25 to 7.3. This same range of external pH was also optimal for growth. pHi was monitored during the cell cycle of a synchronous population of T. pyriformis GL. The cells were synchronized either by starvation/refeeding or heat shock. Under both conditions, there were two alkaline shifts of approximately 0.4 pH units per cell cycle. These shifts in pH retained a constant remporal relationship to S phase and were not affected by changes in the time, duration, or magnitude of cytokinesis.

Notes: Methods for the induction of an exudate of polymorphonuclear neutrophilic leukocytes (PMN) in the peritoneal cavity of C57BL, BALB/c, SJL and CBA mice were analysed. Peritoneal exudates in male mice were highly enriched for PMN (80-90%) three hours after a single injection of calcium caseinate whereas eosionophils comprised less than 1% of the exudate population. Female mice were a less satisfactory source of PMN because the proportion of eosinophils in the exudate was variable. Purification of PMN from peritoneal exudate cells was performed on the basis of light scattering using a Becton-Dickinson cell sorter or by density gradient centrifugation with graded polyvinylpyrroliodone-coated silica particles (Percoll). Both techniques yielded approximately 97% pure PMN preparations. Electrophoretic analysis of the PMN proteins revealed an abundance of lactoferrin and actin, but several other proteins were also present in high concentrations. Proteolytic degradation of several high molecular weight proteins (〉90,000) was prevented by the addition of phenylmethylsulphonyl fluoride (PMSF) and ethylene diamine tetracetic acid (EDTA). Surface iodination, using diphenyl, tetrachloroglycouril (IODO-DEN), indicated that there were six tyrosine-containing proteins present on the external cell membrane. The apparent molecular weights of these surface proteins ranged from 185,000 to 90,000 and the major 125I-labeled protein had an apparent molecular weight of 90,000. Neither actin nor lactoferrin was labeled with 125I unless cell viability was lost during the iodination procedure. Standard conditions for labeling the cell surface only, required low iodide and IODO-GEN concentrations. Biosynthetic labeling of PMN using 35S-methionine increased the sensitivity of detection for most of the proteins, but some of the granule storage proteins (such as lactoferrin) were not effectively labeled within three hours.

Notes: Clones of Chinese hamster ovary (CHO) cells were isolated by single-step selection for resistance to killing by Concanavalin A (ConA) and certain cellular and membrane properties were examined. The ConA-resistant isolates were only about 2-fold more resistant than wild type cells to the selecting lectin, but exhibited pleiotropic temperature-sensitivity for growth, markedly altered morphology and adherence, and significant differences in susceptibility to other agents such as colchicine. Two revertants to full temperature-resistance were isolated from different ConA-resistant mutants. One revertant clone had reacquired wild type sensitivity to ConA while the other revertant remained ConA-resistant. The two series of wild type, ConA-resistant, and temperature revertant clones were analyzed for altered mobility of cell surface glycoproteins using lactoperoxidase/125I and galactose oxidase/[3H]borohydride labelling procedures. The ConA-resistant clones showed increased mobility on polyacrylamide gels of three classes of labelled proteins, in the molecular weight ranges 225,000, 200,000, and 130,000 daltons. These changes persisted in the temperature-revertant that remained ConA-resistant, while two of the altered protein classes were restored to wild type mobility in the revertant that regained ConA-sensitivity. Cell hybridization experiments indicated that the temperature-sensitive phenotypes of different ConA-resistant isolates are recessive and noncomplementing, implying that the same gene is affected in each case. The reversions to temperature resistance appear to be recessive suppressor mutations in different genes.