ALBERT

Notes: A cell line was established in vitro from a spontaneous myeloid leukemia of SL strain mice. This cell line was cytologically identified as myeloblast, and its normal differentiation appeared to be completely blocked in mass culture. When the line cells were seeded in soft agar with a conditioned medium from normal cells, either macrophages or neutrophil granulocytes appeared from a single clone. The rate of formation of colonies containing differentiated cells always increased with an increase in the concentration of conditioned medium. The conditioned medium from this line cell was not as effective as was that from normal cells in inducing differentiation.

Notes: Considerable variability has been found in the yield of cells in batch cultures of Tetrahymena pyriformis grown axenically in 1% tryptone/0.05% yeast extract. This variability has been traced to the photolysis by visible light of the flavin mononucleotide and thiamine components of yeast extract.

Notes: Colony formation in vitro by mouse bone marrow cells following stimulation by human urine was analysed over a 7-day incubation period. There was a linear increase with time in the number of cell aggregates (clusters) developing in such plates. Early in the incubation period all clusters were granulocytic although later macrophage clusters developed. Although most fully developed colonies were composed of macrophages, mapping and transfer studies showed that at least half of these had initially arisen early in the incubation period as granulocytic clusters.

Notes: Maturation of bacterial viruses requires the formation of mature viral nucleic acid in the form found in progeny virus particles, the “packaging” of this viral nucleic acid within a capsule, and the assembly of “packaged” nucleic acid with the accessory structures necessary for infectivity. Recognition that the replicating viral nucleic acid is frequently in a form distinct from that found in mature particles has, in some instances, led to the postulation and formal description of presently unknown processes that must participate in the synthesis of the mature form. In at least some instances, packaging of the mature nucleic acid is clearly integrated with its synthesis.Examples involving the DNA bacteriophages T4, λ, and φ X174 and the RNA bacteriophages are presented to illustrate these points.

Notes: The development of a virus is programmed by a series of negative and positive controls which determine the timing and the segment on either of the two DNA strands (l or r) to be transcribed into specific messenger RNA's. Bacteriophage λ provides one of the most deeply studied systems for following the development of lysogenic viruses. In the lysogenic repressed state, only 2-4% of the λ genome is expressed. This pc-cI-rex region is transcribed leftward to produce a repressor protein which prevents any further transcription by blocking the oL and oR operators flanking the cI-rex operon (figs. 1, 2). This negative control is relieved by destruction of the repressor, and the result is the “induction” of viral development. The earliest post-induction or postinfection events are the leftward transcription of the pLoL N region from strand l and the rightward transcription mainly of the pRoR-x segment from strand r. The N product acts as a positive control, permitting a leftward transcription beyond gene N and a rightward transcription of genes cII-O-P and also Q. The int-xis system controls the excision of the λ genome, whereas the act of rightward transcription and the products of genes O and P initiate the replication of λ DNA. The product of gene Q, still another positive control, stimulates rightward transcription of the late genes which control the synthesis and assembly of the phage heads and tails as well as cell lysis. Among other types of negative control are the possible competition between the two divergent transcriptions originating in region x, the “antirepressor” effect of the x product, and the interference between the two convergent transcriptions which collide in the central b2 region. The majority of controls are based on protein-DNA interactions and can be modified by mutations. For instance, transcription can be rendered independent of negative repressor control either by constitutive, v, mutations which decrease or abolish the affinity of the o operators for the repressor or by insertion of new promoters-e.g., c17 or ric- on the “downstream” side of the operator. The need for the positive N and Q controls may also be obviated by mutations in the N- or Q-dependent promoter or terminator elements.The specific DNA structure within the controlling sites is not known. However, a remarkable coincidence was observed; namely, the occurrence of pyrimidine-rich clusters in those segments of the individual DNA strands acting as templates for RNA synthesis. This observation, which pertains to all studied DNA's, including those of phages T2, T3, T4, T5, T6, T7, λ, and φ 80, formed the basis for a proposal that implicates pyrimidine-rich clusters in the initiation, control and/or termination of transcription, and also in the determination of the preferred strand and, consequently, the orientation of transcription. General considerations regarding the possible role of the structural singularities, especially those represented by the pyrimidine clusters, in the bipartite structure of the recognition regions in DNA are discussed.

Notes: After the experimental verification of Crick's adaptor hypothesis for the role of tRNA, it became apparent that one of the most important of the protein-nucleic acid interactions occurs at the first step in protein synthesis, namely the amino acid activation reaction. It is here that a specific aminoacyl-tRNA synthetase must select, with high fidelity, a specific tRNA out of a large collection of molecules of similar size, shape, and overall composition. A mistake at this point, either by esterification of the wrong amino acid to the correct tRNA or by selection of the wrong tRNA, will inevitabley result in the insertion of an amino acid at an incorrect position in a growing polypeptide. Although there are known rules that dictate how one nucleic acid can recognize and interact with another nucleic acid, nothing is known regarding the mechanism by which a specific protein can recognize and interact with a specific nucleic acid. In order to gain some insight into the specific recognition between an aminoacyl-tRNA synthetase and its cognate tRNA, it became necessary to study the specific interaction with highly purified materials, preferably in gram quantities. An effort to do this for both the synthetases and the tRNA's was launched at the Oak Ridge National Laboratory about 6 years ago. Four high-resolution column chromatographic procedures have been developed in the ORNL Macromolecular Separations Program for the separation and production of highly purified species of tRNA's. An unexpected “spin-off” from this program is the analytical use of some of these systems to detect qualitative changes in the tRNA profile of cells as a consequence of virus infection, methionine starvation, and other metabolic alterations.Some examples of the heterologous interaction between aminoacyl-tRNA synthetases of one species with the tRNA's of another species, and some of the inherent dangers in the interpretation of such interactions, are considered.Finally, some speculations are made regarding the possible role of tRNA in regulation.

Notes: Several steps in the synthesis in vitro of infectious bacteriophage RNA can now be described. The reaction catalyzed by the Qβ RNA polymerase is known to involve several components, including the enzyme, host cell factors, Qβ RNA template, and the strand complementary to the Qβ RNA. The interaction of these components and the mechanims of the reaction appears to be considerably more complex than was proposed in earlier models.

Notes: Theoretical osmotic fragility curves were calculated and drawn by computer using the van't Hoff equation and the isotonic areas and volumes of 1000 individual erythrocytes. We studied the influence on the calculated curves of theoretically altering the fraction of the volume which was osmotically active from 50 to 70%, and of altering the permissible stretch before hemolysis from zero to 10%. With the two assumptions-that the membrane does not stretch before hemolysis, and that the osmotically active fraction of the cell volume is 0.58-it was possible to duplicate the general shape of the standard fragility curve; the exact NaCl concentration, however, at which there was 50% hemolysis was approximately 0.1 gm/100 ml higher than found in vitro. The calculated osmotic fragility curves can be made quantitatively similar to in vitro ones if the following statements are true: the osmotically active volume is 58%, the permissible stretch of the membrane without lysis is 6%, the cell membrane resists a slight osmotic pressure gradient of approximately 0.1 atmospheres, and hemolysis is an all or nothing phenomenon. This set of values for the relevant factors is sufficient but not unique in causing the superposition of the calculated and experimental curves. The frequency distribution of the cells according to the hemolytic salt concentrations (the sodium chloride concentration at which an individual cell just hemolyzes) was skewed positively and was leptokurtic for each of the seven normal subjects studied.

Notes: The fraction of cell capable of division was determined for (1) population of the human diploid cell strains, WI38 after different numbers of subcultivations in vitro and (2) a single population of WI38 cells at intervals throughout its entire in vitro lifespan. In both cases the percentage of cells capable of division decreased with increasing age in tissue culture. The rate and the magnitude of the decrease is sufficient to account for the limited in vitro lifespan reported by other investigators. Furthermore, the decrease in the fraction of cells capable of division in similar in some respects of senescence among human populations.

Notes: A method is described which maintains viable erythroid cells in tissue culture for periods from nine to twenty days. These cells appear predominantly as small round cells with scanty cytoplasm. They synthesize both heme and globin and are relatively more numerous free in suspension than in the adherent monolayer. Ferritin isomorph may serve as a convenient marker in tissue culture of cells of erythroid origin, suggesting that such cells may persist despite a completely transformed appearance and a loss of the ability to produce hemoglobin.

Notes: The conformation of native double helical DNA is well-known, but it is possible that small regions occur within native DNA, undetectable by X-ray diffraction methods, which have different conformations. Model structures are the synthetic deoxypolynucleotides of defined sequence. Under the conditions used, DNA, poly d(A-T) • poly d(A-T), and poly d(T-G) • poly d(C-A) can all give similar X-ray diffraction patterns, whereas poly dA • poly dT, poly dI • poly dC, poly dG • poly dC, and poly d(T-C) • poly d(G-A) clearly differ from DNA. This led to the tentative hypothesis that those DNA's in which all purines are in one strand and all pyrimidines in the other differ in structure from those (such as native DNA) in which purines and pyrimidines alternate or are irregular. We now find that poly d(I-C) • poly d(I-C) does not fit the hypothesis and is a most unusual structure, having seven or eight base pairs per turn. Both molecular model building and circular dichroism studies suggest that it is a left-handed helix. A number of purified tRNA's have been crystallized. We have obtained, from unfractionated tRNA, crystalline “powder” X-ray diffraction patterns showing rings and spots to about 20 Å resolution. It is not clear whether cocrystallization has occurred, or whether there is fractional crystallization, though preliminary evidence favors the latter. Determination of the structure of crystalline tRNA has many features in common with protein crystallography, but there are a number of distinct differences.

Notes: All tRNA sequences so far known can be folded into a cloverleaf structure. Physical data and chemical reactions allow us to draw conclusions on secondary (cloverleaf) and tertiary structure. N-oxidation of adenosine to adenosine-1-N-oxide can be done with monoperphthalic acid in non-base-paired regions of polynucleotides and can be followed easily by changes in absorption of ultraviolet light. Thus this method can be used to determine the structure of tRNA's. A fingerprint of the N-oxidation product of tRNAyeastPhe reveals that all adenosine residues are protected except the 3′-terminal adenosine and the three adenosine residues in or adjacent to the anticodon. On this basis a conformation of tRNAyeastPhe is proposed. Similar tertiary structures can be constructed for the other tRNA's. In order to connect tertiary structure of a tRNA and recognition by its aminoacylating enzyme, the rate of aminoacylation, as a function of temperature, was measured. Neither changes in the anticodon nor specific changes at the 3′-terminal adenosine abolish aminoacylation. Single crystals of tRNAyeastPhe were obtained from aqueous solutions upon addition of various organic solvents.

Notes: Assemblies of protein molecules represent a fundamental level of biological organization. The dynamic behavior of these systems-including both the assembly process and functional rearrangements-may be accounted for by the specificity of the protein interactions, which depend on environmental conditions. Analysis of the self-assembly of virus particles has established that the design of an ordered structure can be built into the specific bonding properties of the constituent proteins. Any structure which can change its state of organization is, by definition, polymorphic. The distinctive aspect of polymorphism in protein structures, contrasted with nonliving states of matter, is that the molecular design has been selected to carry out a function and that this function is part of an integrated system. The differences in molecular conformation and arrangement in all polymorphic structures-for example, allosteric enzymes or ice crystals-depend on the intrinsic interaction properties of the molecules themselves. The structures of ice and water illustrate relations between specificity and polymorphism which are relevant to the form and function of protein assemblies.Two types of polymorphism can be distinguished: modal polymorphism, which is externally moderated, as in phase transitions between different crystals forms; and positional polymorphism, which is internally moderated, as in the different disposition of identical molecules within a single crystal lattice. Positional polymorphism, exemplified by the quasi-equivalent bonding of icosahedral virus coat proteins and the different arrangement of myosin and paramyosin at the center and polar portions of the bipolar filaments, results from specific interactions that are not compatible with a strictly equivalent packing of identical molecules. The structural rearrangements in muscle contraction and the switching between the oxy and deoxy forms of hemoglobin represent the formation of different structures in response to altered external conditions. The different structural states of many protein assemblies are characterized by conserved connections which may be regarded as providing the framework for functional rearrangements. The types of polymorphism displayed by hemoglobin, virus, and muscle proteins demonstrate the relevance of the simple view that the function of a protein is determined by the potential structures it can form.

Notes: Water exchange has been studied in ovarian and body cavity eggs of three Anuran species (Rana esculenta, R. pipiens and R. temporaria) with the isotope exchange method using the automatically recording diver balance. In order to estimate the rate of water diffusion in egg cytoplasm (D), a factor required to determine the rate of water permeation (= the exchange coefficient, E), three different chemical treatments (digitonin, ethanol and formaldehyde) have been used to remove the surface barrier to water flow. The obtained mean value of D, 5 × 10-6 cm2 sec-1, has been accepted as a close approximation of water diffusion in the egg cytoplasm. This value has been used to determine the exchange coefficients in the egg types of the above mentioned species. Comparisons have been made between the calculated values of E both within and between species, and some suggestions have been made concerning the influence of microvilli on the rate of water exchange. Although, on the average, water exchange proceeds at a greater rate in ovarian than in body cavity eggs, a measurable diffusion barrier at the surface can be demonstrated.

Notes: Cellular transformation was induced with avian myeloblastosis virus strain BAI-A (standard AMV) and with a strain of AMV containing subgroup B only. Cultures of muscle tissues from either chick embryo or day old chicks were used for this study. Results were similar in C/O and C/A cells. Leukemogenic virus was continuously produced by these transformed cultures.

Notes: In the presence of 1 mM ATP in the external medium, TA3 mouse ascites tumor cells showed a dramatic loss of potassium and gain of sodium down their respective concentration gradients. The volume changes detected by size discrimination with the Coulter counter have been adequately confirmed by densimetric techniques. Further, some experiments were so designed that net losses of both ions occurred and the cell shrank in response to ATP, a response which was predictable if the volume change was produced by a loss of cell water. We believe that ATP produces a major change in the passive permeability of the membrane to these ions and the effect may be due to a response of a contractile protein in the membrane to ATP.

Notes: Growth hormone, oxytocin, parathyroid hormone, prolactin and lysine vasopressin strongly stimulate mitotic activity in rat thymocyte populations maintained in vitro. These hormones have no mitotic effect on cells maintained in calcium-free medium. It is concluded that they stimulate mitosis only indirectly by sensitising the mitotically competent segment of a thymocyte population to the action of calcium. The stimulatory action of calcium itself is opposed by low concentrations of the mucopolysaccharide chondroitin sulphate. However, the inhibitory action of chondroitin sulphate can be overcome by growth hormone.A possible common mechanism of action of these hormones on mitotically competent cells is discussed.

Notes: A system for fractionating populations of living cells by velocity sedimentation in the earth's gravitational field is described. The cells start in a thin band near the top of a shallow gradient of 3% to 30% fetal calf serum in phosphate buffered saline at 4°C. Cell separation takes place primarily on the basis of size and is approximately independent of cell shape. A sharply-defined upper limit, called the streaming limit, exists for the cell concentration in the starting band beyond which useful cell separations cannot be achieved. This limit, which varies with the type of cell being sedimented, can be significantly increased by proper choice of gradient shape. For sheep erythrocytes (sedimentation velocity of 1.6 mm/hour) it is 1.5 × 107 cells/ml. Measured and calculated sedimentation velocities for sheep erythrocytes are shown to be in agreement. The technique is applied to a suspension of mouse spleen cells and it is shown, using an electronic cell counter and pulse height analyzer, that cells are fractionated according to size across the gradient such that the sedimentation velocity (in mm/hour) approximately equals r2/4 where r is the cell radius in microns. Since cells of differing function also often differ in size, the system appears to have useful biological applications.

Notes: The progression of rat thynocytes (maintained in vitro) into mitosis is profoundly affected by the level of magnesium in the medium. Increasing the extracellular MgC12 concentration from 0 to 1.0 mM does not affect mitotic activity, but a further increase to 1.2 and 2.5 mM rapidly and strongly stimulates the flow of cells into mitosis. The level of extracellular magnesium also governs the mitogenic effectiveness of calcium ions. The mitogenic action of magnesium stems from an ability to stimulate the initiation of deoxyribonucleic acid synthesis.

Notes: Colony-forming cells (CFU), which have the general properties of hemopoietic “stem” cells, appear to be augmented in the mouse fetal liver from 12-18 days gestation and then decrease in the newborn. This finding suggests that few, if any, hemopoietic “stem” cells remain in the adult liver, an organ which appears to be unable to function erythropoietically, even at times of severe crises. In the spleen, and active adult as well as embryonic hematopoietic organ, the total number of CFU increases from 18 days gestation until at least 7 days after birth.Spleen and liver CFU augmentation seems to occur in cojunction with an analogous expansion of non-hematopoietic cells. The data suggests, in fact, that while there is an increase in the total number of liver CFU, there is also a dilution of liver CFU in the total cell population at successively later gestational ages.

Notes: The incorporation of tritiated thymidine and deoxycytidine into DNA of x-irradiated mammalian cells was studied. Both inhibition and stimulation were found due to pool changes rather than to effects on DNA synthesis, indicating that precursor uptake can be a misleading method to measure DNA synthesis rate.

Notes: A series of aldehydes of varying protein crosslinking strengths have been tested on intact and internally perfused crayfish axons. Non-crosslinking aldehydes have no effect, or cause a gradual decline in resting potential and overshoot with no widening of the spike. Strong crosslinking compounds, such as acrolein, crotonaldehyde, and glutaraldehyde, widen the action potential significantly while reducing its amplitude. Differences in the shapes of the resulting action potentials and accompanying impedance changes suggest that each crosslinking aldehyde exerts different effects on the axon. Glutaraldehyde, the strongest crosslinking agent tested, slows both rising and falling phases of the spike, and also of the impedance change, suggesting a prolongation of the transient increase in sodium conductance. The ability of protein crosslinking agents to alter excitability, and particularly to slow the various phases of the action potential, provides support for the hypothesis that a conformational change in a protein or protein-phospholipid complex is involved in excitation.

Notes: Growth of KB cells was inhibited by both spermine and spermidine, but the inhibition is reduced in conditioned medium. The amount of spermine required for 50% inhibition of plating varied according to the type of serum used with medium 199 (calf, fetal bovine, and horse; 0.55, 0.9, and 24 μg/ml respectively). The spermine oxidase activity of the three sera was calf 〉 horse 〉 fetal bovine, which is not the same ordering as was obtained for the inhibition. When the concentration of sera in the media was varied, the inhibition decreased as calf and fetal bovine sera concentration increased, whereas, with horse serum, an increase in serum concentration increased the inhibition. The opposite effects of increasing concentrations of the sera on the inhibition suggest that at least two factors are involved in the inhibition. A scheme which involves three factors (spermine oxidase, another enzyme and its activator) is postulated to account for the inhibitions and reversals observed. Spermine oxidase alone cannot account for the action of polyamine on cells.

Notes: Reduction in alkaline phosphatase activity was observed when HeLa S3 cells were grown in Puck's medium containing high concentrations of human serum. This effect was not seen with the enzyme of Chang liver 8A cells. The induction of increased alkaline phosphatase in HeLa S3 by prednisolone or by osmolality changes was not prevented by serum. The concentration of serum in the culture medium had no influence on acid phosphatase activity.

Notes: These studies have been conducted as a first step toward investigating the molecular mechanism by which DNA-dependent RNA polymerase recognizes a specific region of DNA. The DNA moieties are either bound to or freed from RNA polymerase. Results obtained so far indicate that the DNA moieties remaining as resistant segments after deoxyribonuclease digestion should be regarded as the binding regions on DNA. The results described here were carried out by use of E. coli and lambda DNA.

Notes: In cultures of differentiating chicken embryo muscle cells there is a steep decline in DNA polymerase activity which closely parallels the time of rapid cell fusion and the formation of multinucleated myotubes. The DNA polymerase activity remaining in the cultures is almost completely associated with single unfused cells. Cell fusion does not require a confluent culture and fusion capability appears to be severely reduced in the remaining single cells following an approximately ten hour time period during which the majority of fusion takes place. A model is presented to explain the observed kinetics of cell growth and cell fusion in vitro.

Notes: Synchronous cultures of HeLa cells obtained by selective detachment of mitoses were treated with high concentrations of thymidine. The inhibitor was added soon after completion of cell division and rates of cell enlargement and accumulation of DNA, RNA and protein were compared for untreated and thymidine-treated cultures at various points of the cell cycle. It was found that concentrations of thymidine which in randomly growing cultures inhibit the rate of cell division by more than 90% allowed a considerable degree of DNA synthesis and did not affect the rate of accumulation of RNA and protein, when applied to cells in the G1 phase of synchronous culture. Treated and untreated cells enlarged at the same rate throughout their life cycle. The results show that concentrations of thymidine commonly employed to produce cell synchrony do not arrest the cells at the G1-S boundary, but allow slow progress through S in respect to DNA synthesis, and near-normal progress towards G2 as regards RNA and protein accumulation and cell enlargement.

Notes: Survey of twelve mouse tissues revealed the presence of appreciable phenylalanine hydroxylase activity in the pancreas and kidney as well as the liver but in no other of the tissues tested. Single cell suspensions of mouse liver were prepared by use of tetraphenylboron. The enzyme activity of such suspensions was much more stable than that of liver extracts, and permitted determination of the Michaelis-Menten constant, the pseudo-first order reaction velocity constant on a cell-number basis, and the temperature coefficient and apparent activation energy of the enzyme activity. Possible applications of these methods to problems in cellular biology have been indicated.

Notes: The increased multiplication of converted chicken cells in the presence of limited amounts of serum results from more efficient utilization of multiplication-stimulating activity rather than increased binding or uptake of this activity.

Notes: Sarcoplasmic reticulum fragments, prepared from skeletal muscle homogenates, were found to consist of two major types when examined after negative staining. One type possessed 90 Å subunits and was thought to be of mitochondrial origin. The other had 35 Å subunits and ws presumably derived from the sarcoplasmic reticulum. Only the latter type accumulated visible calcium oxalate deposits inside the vesicle when they were exposed to a medium containing ATP, MgCl2, K2C2O4 and CaCl2. The calcium oxalate loaded vesicles were strikingly angular in shape and did not have tails. The calcium oxalate loaded vesicles had identical membrane subunit arrangement to inactive companion membranes and nonincubated controls; this suggested that the membrane subunits were not the critical structural requirement for calcium transport. A method was described whereby the calcium accumulating membranes could be purified 3- to 4-fold on the basis of the best previously used preparation procedures.

Notes: The lethal damage induced by the exposure of synchronized Chinese hamster cells to various concentrations of 5-fluoro-2′deoxyuridine (FUdR) was not selectively restricted to cells exposed during the period of DNA synthesis S. The colony survival fraction observed after treatment for one hour with 5 × 10-5 M FUdR was very low (0.0001-0.0003) whether the drug was administered during early G1, late G1, early S or in middle S. The survival of cells treated with the same concentration of FUdR during mitosis, however, was significantly higher (0.62) showing that mitotic cells were less sensitive to FUdR. Administration of 10-7M thymidine or “conditioned” medium for one hour reversed the lethal effect of FUdR or improved the survival, depending on the time after removal of the FUdR at which these substances were given.

Notes: Large numbers of thin-walled vesicles, 0.5 to 10 μ in diameter, can be formed by permitting a thinly spread layer of hydrated phospholipids to swell slowly in distilled water or an aqueous solution of nonelectrolytes. Electron micrographs and phospholipid analyses indicate that the walls consist of a single or a few bilayers. The vesicles can be centrifuged and resuspended in another medium, making them a useful system for studying permeability. The osmolarity of the solution in the interior of the vesicles can be estimated by immersion refractometry. The osmolarity of the internal aqueous phase is linearly related to the osmolarity of the external medium.

Notes: It has been shown that a dialyzable substance produced by normal and tumor cells can stimulate the growth of a myeloid, erythroid, and two lymphoid leukemias, and a sarcoma. The growth stimulation of the tumor cells was observed as an increase in cloning efficiency and number of cells per colony. Rat granulocytes stimulated the growth of mouse tumor cells as efficiently as a variety of mouse cells. The stimulating substance was found in conditioned medium, and it was produced by all the normal and tumor cells tested.

Notes: The rate of DNA synthesis in HeLa S3 cells, as measured by incorporation of C14-labeled thymidine, is strongly dependent on protein synthesis at all times during the S phase. The relation between the rate of DNA synthesis and the rate of protein synthesis is linear when measured two or three hours after reducing the rate of protein synthesis with either puromycin or cycloheximide. The effect is manifested rapidly, is found in both random and synchronized cultures, and is independent of the method of synchronization.

Notes: Nuclei from mature neutrophil granulocytes were prepared from peritonal exudates of goats. Fluorescein mercuric acetate was required to stabilize the nuclei and fix nucleoproteins. Following differential centrifugation and detergent treatment, electron microscopy showed the interlobar region to be free of cytoplasmic tabs. All of the DNA of the cell was recovered in the nucleus and 71% of the RNA. The DNA : RNA was 6 : 1 in the intact cells, and 9 : 1 in the isolated nuclei. Protein:DNA was 11 : 1 and 4 : 1 for cells and nuclei, respectively. Representative fractions of histones and tryptophan-rich acidic proteins were prepared with (asp + glu) : (lys + arg + hist) values averaging 0.7 and 1.4 respectively. Histones accounted for 30% of the nuclear proteins while the residual proteins contained the bulk of the cystinyl residues. Granulocytes were characterized by high glycine titers, from 8 to 18% of the nuclear proteins, and 70% of the total free amino acids of the cell.

Notes: When exogenous ATP is added to suspensions of TA3 ascites tumor cells suspended in Ca++ and Mg++ free media, a significant increase in cell volume can be measured. This increase is reversible upon addition of Ca++ and/or Mg++ back to the media.The enlargement of these cells is temperature sensitive and specific for ATP; no other nucleotides, EDTA or ouabain were effective. The evidence suggest that this phenomena may be due to an alteration in membrane permeability and that the regulation of membrane permeability is an energy dependent process.

Notes: Estradiol treatment of irradiated mice during repopulation of their spleens by endogenous hemopoietic cells reduced the number of myelocytic colonies and increased the numbers of erythropoietic and undifferentiated colonies. The inhibitory effects of the hormone on myelopoiesis were not dependent on stimulation of erythropoiesis, since they occurred in the absence of erythropoiesis in mice made polycythemic by hypertransfusion.Treatment of bone marrow donors with estradiol reduced the ability of their marrow cells to form spleen colonies, particularly reducing the proportion of myelopoietic colonies relative to the total number of colonies of all types. Conversely erythropoietic colonies, though reduced in absolute number, were increased in relative number. Such treatment also decreased the volume and cell content of the marrow cavity through stimulation of endosteal bone formation.Estradiol treatment of lethally irradiated recipient mice did not detectably alter the total numbers or types of hemopoietic spleen colonies formed in these animals from transplanted marrow cells; however, without estradiol treatment, myelopoietic colonies were so few and erythropoietic colonies so numerous that the effects of the hormones may have been undetectable by the methods employed.The sex of the donor or recipient mouse did not affect the numbers or types of colonies formed, suggesting that endogenous levels of estradiol were too low to exert effects dectectable by the methods used. However, since our mice were only 8 weeks old, the data do not exclude the possibility that older female mice, with higher levels of estradiol, would have differed from males in relative numbers of myelopoietic as compared with erythropoietic colonies.

Notes: Mature male rats were injected with radioiron, which labeled immature red cells that were in the stage of hemoglobin synthesis at the time. Washed cells were subjected to lysis by hypotonic saline at 14 intervals ranging from 1 to 85 hours after 59Fe injection. Lysis curves were determined colorimetrically (for the whole population) and by scintillation counting of 59Fe gamma rays (for the labeled population). Newly labeled rat red cells are much more resistant than mature erythrocytes. The mature curve of osmotic resistance is acquired about 67 hours after injection. The delivery of labeled cells continues for more than 36 hours, so the maturation of osmotic properties of a typical rat red cell takes about 30 hours from entry into the circulation. Washed dog cells behave in similar fashion but delivery continues for three days and the mature lysis curve does not appear until five days after labeling, so that maturation takes about two days. Preincubation of cells is requisite for the appearance of osmotic resistance, but the basis of the incubation effect is not yet known.

Notes: It has been shown by re-cloning of colonies formed in vitro from rat bone marrow cells, that normal granulocyte colonies can originate from single cells. No mixed macrophage (M) and granulocyte (G) colonies were obtained after re-cloning either M or G colonies. The results indicate, that clones of normal granulocytes and macrophages can be obtained in vitro, and that the mixed primary M and G colonies formed after seeding hematopoietic cells from animals presumably originate from a mixture of M and G cells.

Notes: The cytotoxic effect of high, as well as low, oxygen tension of proliferation and metabolism of Low line cells in culture is reversible even after several days of exposure provided the cells are returned to 95% air + 5% CO2 environment. This suggests that the activity of certain mechanisms within the cells may have been altered or in other ways inhibited by the abnormal environments but are quite rapidly regenerated once the adverse condition is removed. The cells tolerate a low O2 exposure for at least 20 days while continuous exposure to high O2 atmosphere results in degeneration and death after 7-10 days. Both glucose utilization and lactic acid production are elevated in cultures exposed to either low or high O2 tensions, although they are markedly higher in the latter condition. When cell so exposed are returned to an air + 5% CO2 atmosphere, rate of glucose uptake and lactic acid formation soon approaches that found in control cultures.

Notes: Histologic examination of the spleens in RFM/Un mice killed 6 to 9 days after 350 to 800 R whole-body x-irradiation revealed hemopoietic colonies, the numbers of which decreased exponentially with increasing radiation dose. In such animals, myelocytic colonies were the predominant type on the sixth to the eighth day. However, they decreased in number with time, being fewer than erythropoietic colonies by the ninth day after irradiation. In C57BL mice, erythropoietic colonies were relatively more numerous, markedly predominating on both the eighth and the thirteenth days.RFM/Un mice injected with nonirradiated syngeneic bone marrow cells within 24 hours after 750 R developed colonies, predominantly of erythropoietic and undifferentiated types, the numbers of which were proportional to the numbers of marrow cells injected. The number of colonies formed from exogenous marrow cells increased slightly between the sixth and ninth days after inoculation, possibly because of a greater likelihood of counting them due to an increase in their size.

Notes: The rate of attachment to the substratum was found to be a strain specific character of certain large free-living amoebae. Two strains, which differed significantly in this respect, were compared with regard to the rate of attachment of enucleated cytoplasm, nuclear-cytoplasmic hybrids and amoebae injected with cytoplasm from another strain. It was found that both the nucleus and the cytoplasm of the more rapidly attaching strain were able to transmit this character to amoebae of the more slowly attaching strain. The possible mechanisms underlying attachment and transmission of the rate of attachment are discussed.

Notes: The transition of suspension cultures of Novikoff rat hepatoma cells from the exponential to the stationary phase is accompanied by decreases of over 90% in the rates of synthesis of RNA, DNA and protein, a 90% loss of the apparent DNA-dependent RNA polymerase activity of the cells, and a disaggregation of the polyribosomes with a concomitant accumulation of 80 S and 110 S ribosomal structures. The cells also attain a minimum content of DNA, RNA and protein and a minimum size. Upon dilution of stationary phase cultures with fresh medium, the rate of protein synthesis begins to increase immediately and this correlates with a rapid reformation of the polyribosomes. The initial re-formation of polyribosomes is little affected by the presence of actinomycin D. RNA polymerase activity also begins to increase immediately after dilution and an increase in rate of RNA synthesis becomes apparent shortly thereafter. The increase in polymerase activity is inhibited by treating the cells with puromycin or actidione. Cell division commences only 9-13 hours after dilution and the rate of DNA synthesis begins to increase about midway through the lag period. During the lag period the average cellular content of protein increases about 80% and that of RNA and DNA about 30%. These increases are accompanied by a marked increase in the average size of the cells.Upon continued incubation of stationary phase cultures, the cells become irreversibly damaged physiologically before gross morphological damage becomes apparent. The irreversible physiological damage is recognized by the fact that the cells fail to recover when suspended in fresh medium.

Notes: Spleen or lymph node cells of C57BL/6 origin or both were mixed with subcellular material from Balb/c tissue for several hours at 37°C. The subcellular material was then washed out (by repeated centrifugation) with PBS and the immune cells were placed either in diffusion chambers in the peritoneal cavity of Swiss mice or in Petri dishes with C57BL/6 embryo cell monolayer (feeder layer) and incubated in a thermostat at an atmosphere of 5% CO2. After 4 to 5 days these cells were added to target Balb/c embryo cells in Petri dishes. The first control was done by sensitiziing the cells with syngeneic material, the second control by adding the immune cells to syngeneic target embryo cells. The sensitized cells mixed with allogeneic embryo cells produced a positive reaction in the form of agglutination around the target cells and destruction of these cells. In controls the immune cells were seldom found and were randomly distributed.

Notes: The respective importance of mitochondria and of sarcoplasmic reticulum in the uptake and maintenance of Ca++ by the isolated rat diaphragm has been compared. Diaphragms were incubated at 30° in conditions optimal for Ca++ uptake either by isolated mitochondria or by sarcoplasmic reticulum: more Ca++ was taken up from the “mitochondrial” medium. For maximal uptake, Pi and Mg++ were necessary; substitution of NaCl and KC1 with sucrose had no effect on the uptake. The uptake was markedly inhibited by uncouplers of oxidative phosphorylation, by respiratory inhibitors, and by lowering the temperature of the incubation medium to 0°; it was not affected by oligomycin, aurovertin, DCCD, nor by inhibitors of Ca++ transport in the isolated sarcoplasmic reticulum (ergotamine, ergobasinine, caffeine). The lack of effect of caffeine was not due to lack of penetration into the muscle. Permeability barriers for ergotamine and ergobasinine could not be excluded. The maintenance of Ca++ by the diaphragm was optimal in a medium contaming Pi and Mg++. Uncoupling agents and respiratory inhibitors accelerated the rate and extent of release of Ca++ by the diaphragm. Lowering the temperature of the incubation medium to 0°, or addition of oligomycin, aurovertin, DCCD, had no effect on the release. The release of Ca++ was also unaffected by ergotamine, ergobasinine, caffeine. The results suggest a role for mitochondria in the uptake and maintenance of Ca++ by the isolated diaphragm.

Notes: Crayfish giant axons remain viable following internal perfusion with a mixture of fluoride and citrate salts. The relative favorability of various internal anions, and the dependence of resting and action potentials on internal cations are both similar to results on internally perfused squid axons. TEA widens the falling phase of the spike only from inside the axon, while DDT is active from either side of the membrane. Records of impedance changes show that effects of TEA and DDT on components of ionic conductances are similar to those found in other axons by voltage clamp measurements.Tannic acid perfused internally at a concentration of the order of 10 μM produces spontaneous activity, and a progressive increase in spike width. After 30 minutes, action potentials are “cardiac” type and are up to several minutes in duration. Records of impedance changes, and data from rapid changes in external ionic concentrations, suggest that the plateau phase of the spike is due to a maintained increase in sodium conductance. Since tannic acid is capable of crosslinking proteins and “rigidifying” protein monolayers, it is suggested that its effects on the axon may be the result of an interference with a conformational change in a membrane protein or protein-phospholipid complex during excitation.

Notes: In Tetrahymena pyriformis, mating type I, variety 1, cycloheximide rapidly and completely inhibited incorporation of 14C-L-leucine into protein. Actinomycin D (25 μg per ml) inhibited incorporation of 14C-uracil into cold-TCA-insoluble material, after a 5-10 minute lag. Frequently a subsequent decline in the amount of radioactivity was observed. Protein synthesis continued in actinomycintreated cultures for a variable time after cessation of RNA synthesis.Oral development was affected by cycloheximide virtually immediately, and by actinomycin D after a 10-15 minute lag. Cells affected by either drug before the onset of oral membranelle formation were permanently arrested in the stomatogenic field phase. Cells affected in the early and middle stages of membranelle formation completed development of membranelles, but did not invariably complete cell division. Cycloheximide, when added at the beginning of membranelle formation, brought about arrest or resorption of membranelles after they were completed. Actinomycin did not elicit resorption, but sometimes brought about blockage during cell division. Cells affected by either drug after membranelles were fully formed (and cell division was just beginning) completed oral development, nuclear divisions, and cell division. These results suggest that concurrent RNA and protein synthesis are essential for the initiation but not for the completion of membranelle differentiation. The results also suggest that a specific messenger RNA(s) with a very short half-life is required for the synthesis of proteins involved in the initiation of membranelle differentiation.

Notes: Four clonal adrenal tumor cell lines which exhibit biochemical lesions in the ACTH-stimulated steroidogenic pathway have been isolated. Two of these cell lines, designated Y-6 and OS3, appear to contain their lesions at points proximal to cyclic AMP formation in the ACTH-stimulated steroidogenic pathway. Growth of Y-6 and OS3 as tumors in isogenic mice results in a restoration of ACTH sensitivity in both cell lines by mechanisms which do not appear to involve selection or fulfillment of specific nutritional requirements. Growth of Y-6 and OS3 as tumors in heterogenic mice results in restoration of ACTH sensitivity in Y-6 but not in OS3, suggesting that the biochemical lesions in these cell lines are at different loci. Two other cell lines, designated OS1 and OS4, possess biochemical lesions in the steroidogenic pathway beyond the formation of cyclic AMP and before the formation of pregnenolone. Growth of OS1 and OS4 as tumors in isogenic mice results in the repair of the biochemical lesions in these cells distal to cyclic AMP formation in the ACTH-stimulated steroidogenic pathway. The four cell lines described are potentially useful in elucidating the mechanism of action of ACTH in adrenal cells as well as in determining the factors required for maintaining differentiated function in cultured cells.

Notes: L cells were cultivated in test medium which contained 14C-sodium acetate, and the amount of labeled digitonin-precipitable sterol was assayed in medium and cells. Increasing concentrations of whole serum in the medium had two effects: depressed cellular synthesis and enhanced release of synthesized sterol from the cells. In experiments with delipidized serum containing unesterified cholesterol, cellular sterol synthesis decreased as free cholesterol concentration in the medium increased. In other experiments using medium containing increasing lecithin concentration and no exogenous sterol, the concentration of lecithin markedly influenced the distribution of synthesized sterol between the cells and the medium which then directly influenced the amount of sterol synthesized. These experiments indicate that cell sterol synthesis is regulated by internal levels of free sterol. This, in turn, is a function of cellular sterol flux which is regulated by the concentration and composition of serum lipoprotein in the medium.

Notes: RNA in the peripheries of various populations of lymph node cells (LNC) has been evaluated by measuring the electrophoretic mobilities of cells, before and after treatment with active or inactivated ribonucleases. Three different populations of LNC were studied: (1) “resting” normal age control LNC; (2) “syngeneic” LNC from irradiated (C3H × C57BL)F1 or C3H mice four to six days following transplantation of syngeneic spleen cells; such cells were progeny of lymphopoietic progenitor cells of the spleen; and (3) “allogeneic” LNC from irradiated (C3H × C57BL)F1 mice four to six days after grafting C3H (parental) spleen cells; such cells were progeny of lymphopoietic progenitor cells, but also alloantigen-sensitive cells of the spleen which proliferate in response to the host's alloantigens (a “graft-versus-host” immunological reaction). Whereas the normal LNC had no detectable peripheral RNA, the allogeneic and syngeneic LNC did, i.e., ribonuclease reduced their mean electrophoretic mobilities by 13.6 and 9.2 per cent, respectively. Since both allogeneic and syngeneic LNC had peripheral RNA, no specific correlation could be made with immunological activity. 3H-uridine and 14C-thymidine incorporation into lymph nodes was greatest in allogeneic, intermediate in syngeneic and least in age control lymph nodes, indicating a “population shift” in the spleen cell chimeras toward relatively immature, rapidly proliferating cells, which had a relatively high rate of RNA synthesis. Thus, rapidly proliferating lymphoid cells do have RNA in their peripheries, but its relation to specific immunological function has yet to be ascertained.

Notes: Functionally active 30S ribosomes can be reconstituted in vitro from 16S RNA and a mixture of 30S ribosomal proteins under certain defined conditions. Our previous studies on the specificity and kinetics of reconstitution are summarized and discussed. The reconstitution reaction is first-order with respect to formation of active 30S ribosomes. The rate-limiting reaction is probably unimolecular, and it represents the structural rearrangement of an intermediate. Presumed reconstitution intermediates, or RI particles, have been isolated from reconstitution mixtures incubated at low temperature. It has been concluded that the reconstitution takes place in stepwise fashion: 16S ribosomes. New experimental results that show the highly cooperative nature of the assembly reaction are described; the number of sites, per RNA chain, which can bind ribosomal proteins independently from each other (i.e., without cooperativity) is at most two to three. Finally, another approach to the study of ribosome assembly in vivo is described. It utilizes cold-sensitive E. coli mutants that are defective in ribosome biosynthesis at low temperature and accumulate incomplete “intermediate” ribosomal particles.

Notes: The apparently inconsistent reports on flagellar ATPase properties may be resolved by elimination of adenylate kinase from the system. Removal of the adenylate kinase from alkaline M/2 KCl extracts of bull sperm flagella yields a spermosin-ATPase which liberates only the terminal phosphate of ATP. In any case spermosin is preferentially activated by calcium. However, combination of spermosin with flactin (e.g., by addition of digitonin and MgCl2 to the extraction medium) produces an ATPase much more highly activated by magnesium. But flactospermosin has so far resisted purification from its adenylate kinase contaminant. In divalent cation activation, pH optima and nature of ATP hydrolysis, the flagellar contractile protein system closely parallels the muscle system.

Notes: T4 bacteriophage infection of E. coli B cells induces the formation of 4S RNA molecules that specifically hybridize with T4 DNA. The T4 4S RNA extracted from the hybrid was found to contain pseudouridylic acid, suggesting that some of this RNA might have amino acid acceptor function. In order to study the amino acid acceptor capacity of the T4 4S RNA, two procedures were devised. The first one involved the isolation and purification of specific 4S RNA-DNA hybrids in a manner that avoided the use of RNase and permitted the extraction of biologically active tRNA from the hybrid. It was found that a significant fraction of the T4 4S RNA isolated by this method had amino acid acceptor activity. This was shown by assaying with a mixture of 15 14C-labeled amino acids or with [14C]leucine alone.In the second method, T4 N-acetyl[3H]aminoacyl-tRNA was prepared in order to stabilize the aminoacyl-tRNA ester bond. T4 N-acetyl[3H]leucyl-tRNA was incubated with T4 DNA in the presence of 50% formamide at 30°C. Sephadex G-200 chromatography revealed that a significant fraction of the N-acetyl[3H]leucyl-tRNA hybridized with the T4 DNA. Another procedure involved the hybridization of N-acetyl[3H]-aminoacyl-tRNA with T4 DNA at 70°C in a citrate buffer in the absence of formamide. The annealing mixture also contained a 20-fold excess of uncharged E. coli tRNA. The hybrid-containing solution was loaded onto nitrocellulose filters and treated with T1 RNase. At this point, the T4 tRNA was found to contain leucine-, arginine-, isoleucine-, and possibly tyrosine-specific chains.

Notes: Partially purified preparations of two protein factors, one of which is stable at 50°C (FIs) and the other unstable at 50°C (FIu), are required for the GTP-dependent, mRNA-directed binding of aminoacyl-tRNA to ribosomes, as well as for polypeptide synthesis in the presence of a third factor, FII. Both FIs and FIu are required for maximal interaction with GTP to form a GTP•protein complex that subsequently interacts with aminoacyl-tRNA, but not with deacylated tRNA or with N-(substituted)-aminoacyl-tRNA, to form an aminoacyl-tRNA•GTP•protein complex. A mixture of FIs and FIu also interacts with GDP to form a GDP•protein complex; however, no subsequent interaction with aminoacyl-tRNA is observed. In addition to aminoacyl-tRNA and GTP, Mg2+ and NH4+ are required for the formation of the aminoacyl-tRNA•GTP•protein complex. Although both protein factors, FIs and FIu, are required for the formation of this complex, only the heat-labile protein, FIu, is a component of the complex. Very little dissociation of the GTP moiety of the complex occurs in the presence of Mg2+, and no detectable exchange is observed with GTP, GDP, or Pi. In contrast, appreciable dissociation of the aminoacyl-tRNA from the GTP•protein occurs even in the presence of Mg2+, and exchange with other aminoacyl-tRNA's can be readily demonstrated. In the absence of Mg2+, complete dissociation of both the GTP and the aminoacyl-tRNA from the protein occurs. Evidence has been obtained to demonstrate that the aminoacyl-tRNA•GTP•protein complex is an intermediate in the GTP-dependent binding of aminoacyl-tRNA to ribosomes. The binding of the aminoacyl-tRNA to the ribosome occurs with the concomitant formation of Pi and a GDP • protein complex. Incorporation of the bound aminoacyl-tRNA into polypeptide requires additional GTP and the third transfer factor, FII.

Notes: The concentration of neutrophils and megakaryocytes was determined in the marrow of anemic mice of genotype W/Wv and their normal (+/+) litter mates. In all groups studied, the humerus of W/Wv mice contained significantly less neutrophils and megakaryocytes than did normal animals. Blood neutrophil concentration was less in all groups of W/Wv mice but in only one group which was the youngest group studied, did this value differ significantly from normal.The blood and marrow neutrophil response to endotoxin was similar in W/Wv and “+/+” animals. This suggests that the neutrophilic system of W/Wv mice responds to this stimulus in a relatively normal manner, much as their erythroid system responds to hypoxia, and androgens.

Notes: The net negativity of the surfaces of Ehrlich ascites cells was reduced by treating them with either neuraminidase or ribonuclease. Neither enzyme treatment affected the Na+ or K+ content of the cells, before or after cooling at 4°C. Experiments with K42 revealed a reduction (9.5 to 17%) in unidirectional K+-fluxes following incubation with neuraminidase, but no change after ribonuclease-treatment. Our data suggest that surface anionic sites associated with RNA and sialic acid moieties are not of major quantitative importance in regulating either intracellular Na+ and K+ concentrations, or unidirectional transmembrane K+-flux. Our results do not enable us to determine whether ion-binding to anionic sites at the electro-kinetic surface is not an essential prerequisite to transmembrane movement, or whether it is essential, but occurs through the 40% of cell surface net negativity which is unaffected by ribonuclease- and neuraminidase-treatment.

Notes: During embryonic tooth formation, interactions between epithelial and mesenchymal cells results in the formation of a metachromatic interface or extracellular matrix. The cervical or germinative region of this epidermal organ system is populated by an increasing gradient of cellular differentiation and an extracellular matrix which is the progenitor for subsequent dentine organic matrix formation. Embryonic rabbit tooth primordia can be maintained in culture enabling kinetic studies of labeled precursor incorporation. Autoradiographs of tooth organ cultures continusly incubated with labeled uridine for periods up to eight hours, demonstrated initial cellular incorporation with subsequent transfer of 2% of the grain density to the extracellular matrix by four hours. The grain density was removed by ribonuclease treatment. No incorporation of tritiated thymidine into the matrix was observed. The incorporation of C14-uridine during organ culture was inhibited by actinomycin D. Micrurgy was employed to isolate the extracellular matrix free of adherent cells. Electron microscopy demonstrated membrane-bound, electron dense bodies within the matrix, presumably cytoplasmic extensions. No cells per se were observed on the isolated matrix. Several experimental criteria suggested that uridine incorporation into the extracellular matrix was regulated by epithelial and mesenchymal cells. Phenol extraction procedures of labeled cervical matrices demonstrated an ultraviolet absorption maximum at 260 μU. Both spectrophotometric determinations and orcinol assays found RNA to be 0.4-0.5% of the cervical extracellular matrix.These results are interpreted to indicate that RNA is a component of the metachromatic extracellular matrix during epithelio-mensenchymal interactions associated with tooth formation. The functional significance of these observations is premature at this time.

Notes: Uridine kinase activity measured in cell-free extracts of Novikoff rat hepatoma cells grown in suspension culture fluctuates about 10 fold during the growth cycle of the cells. Maximum specific activity (units/106 cells) is observed early in the exponential phase and then decreases progressively until the stationary phase. The rate of incorporation of uridine into the acid-soluble pool by intact cells fluctuates in a similar manner and both the rate of uridine incorporation by intact cells and the uridine kinase actvity of the cells increase several fold before cell division commences following dilution of stationary phase cultures with freshmedium. Regardless of the stage of growth, uridine is rapidly phosphorylated to the triphosphate level by the cells.The rates of incorporation of uridine into the nucleotide pool and into RNA by intact cells fluctuate in a similar manner during the growth cycle. However, evidence is presented that indicates that alterations in the rate of incorporation of uridine into RNA are not simply due to changes in the rate of phosphorylation of uridine, but are regulated independently.Inhibition of protein synthesis by treating cells with puromycin or actidione causes a marked inhibition of incorporation of uridine into RNA, but has little effect on the phosphorylation of uridine to UTP for several hours. Thus the depression of incorporation of uridine into RNA probably reflects a decrease in the rate of RNA synthesis as a result of inhibition of protein synthesis. Inhibition of RNA synthesis by treating cells with actinomycin D does not affect the rate of conversion of uridine to UTP and thus results in the accumulation of labeled UTP in treated cells.

Notes: Dietary zinc deficiency has been shown to decrease the in vivo incorporation of thymidine into the nuclear DNA of liver parenchymal cells in the rat. A single injection of 100 μg of zinc was sufficient to rapidly reverse the effect of zinc deficiency on the synthesis of DNA.

Notes: Randomly growing cultures of HeLa-S3 cells were subjected to synchronizing treatment with excess thymidine. Unbalanced growth occurred during the thymidine treatment, but the abnormal cellular composition was found to revert to control levels so that at the time of the first mitosis following a 12-24 hour treatment with the inhibitor the cellular DNA, RNA and protein content and cell size were close to control values. However, an alkaline deoxyribonuclease which attacks denatured DNA was still found to be significantly elevated at this time. The results argue against the use of thymidine as a synchrony producing agent.

Notes: The system previously described for inducing single gene mutations in Chinese hamster cells has been extended to produce additional auxotrophic mutants. An improved method for quantitating the efficiency of single gene mutation to specific auxotrophies has been developed. Mutagenesis in the forward direction has been measured after treatment of these cells with ethyl methanesulfonate, N-methyl-N1-nitro-N-nitrosoguanidine, hydroxylamine, an acridine mustard (ICR-191), caffeine and ultraviolet- and X-irradiation. For each agent, the single cell survival curve and the efficiency of chromatid breakage and rearrangement were measured. Similar measurements were also carried out with a water-soluble carcinogen N-nitrosomethylurea, which was shown to be effective in producing auxotrophic, somatic mutations. These results offer promise of illuminating the relationships between cell killing, chromosomal aberration, single gene mutations and carcinogenesis produced by various agents. The methods described can be used in routine testing of drugs, food additives, and environmental pollutants for mutagenic action in mammalian cells in vitro.

Notes: The pattern of photodynamic damage of pig erythrocyte and rat brain microsomal ATPases and erythrocyte acetylcholinesterases has been studied, using rose bengal as photosensitizer. Of these enzymes the Na+-K+-Mg2+-ATPase, believed to be associated with active transport, is very much more sensitive to damage than are the Mg2+-ATPase and the erythrocyte acetylcholinesterase. Earlier photoxidative studies of the haemolysis of erythrocytes have shown that ion movements occur in two phases which are dependent on the duration of exposure to light and it is suggested that these are correlated with the differential sensitivity of these membrane enzymes.The ATPases of the brain microsomal preparation were more sensitive to photodynamic damage during preincubation at 0°C, i.e. in the absence of substrate. Raising the preincubation temperature to 37°C protected both enzymes, the inactivation of the Na+-K+-Mg2+-ATPase being markedly reduced. The presence of substrate during pre-incubation at 0°C also protects both enzymes, especially the Mg2+-ATPase. These interacting effects of temperature and substrate are compared with the known different temperature sensitivities of these two enzymes.

Notes: The growth enhancing effect of media conditioned by cells from established lines (BHK and L60) is comparable to that of media conditioned by cells of primary origin (chick embryo). However, the properties of the conditioned media from these two systems show marked differences: the growth enhancing factors in the former are dialyzable and heat-stable, in contrast to the non-dialyzable and heat-labile factors in the latter.Media conditioned for only four hours by BHK or L60 cells stimulated cell growth. Amino acid analyses revealed that non-essential amino acids had appeared in these conditioned media.To verify this as the metabolic basis of conditioning by cells from established lines, media containing dialyzed serum were supplemented with each of six non-essential amino acids, and assayed on BHK and L60 at various population densities. Serine was the most stimulatory and alanine the most inhibitory of the amino acids tested.Mixed supplementation of the medium showed that when low levels of alanine and serine were added simultaneously, cell growth was enhanced but any increase in the level of alanine required an increase in the level of serine also to achieve growth stimulation.

Notes: The effect of cell crowding on DNA synthesis (incorporation of 3HTdR and 32PO4) was studied by an improved method in monolayers of secondary cells and established cell lines, either normal or transformed by viruses or carcinogens. The method was based mainly on pulse labeling of cultures of cells a few hours after their seeding in equal numbers onto areas of different size in identical dishes, a condition which ensured equal physiological conditions and different degrees of crowding of cells.DNA synthesis was hardly inhibited in crowded monolayers of secondary chick, mouse and hamster embryo cells. The incorporation of radioactive thymidine and phosphate into DNA of cell lines such as BHK 21, 3T3/SV40 and L929 was strongly inhibited. An SV40-transformed line of hamster kidney cells (HKT7) synthetized DNA equally well in sparse as in crowded monolayers. In lines of human amnion (FL) and BHK 21 cells which were more extensively studied the degree of inhibition of DNA synthesis was inversely proportional to their density.Autoradiography after 3HTdR pulse-labeling indicated that the same proportion of cell nuclei were labeled in sparse and in crowded cultures. The extent of labeling (number of grains per nucleus) was lower in crowded cultures of those cells that also showed inhibition of incorporation of this label as measured by scintillation. The inhibition is thus expressed in retardation of DNA synthesis in cells in S phase rather than arresting it in a larger percentage of cells.

Notes: Inhibition of cellular respiration by treatment with the nonionic detergent Triton WR-1339 was found to be related to the cytotoxic response of cell to the surfactant. Respiration of sensitive cell lines (AV-3 and HeLa) markedly inhibited by Triton concentrations as low as 125 μgm/ml. Conditionally sensitive lines (BHK-21 and L-929) were affected by 500 μgm/ml while the respiration of insensitive cultures (primary rat and chick embryo cells) was unaffected by this concentration. Macrocyclon, a cyclic analogue of Triton, failed to alter the respiration rate of any of the above cell cultures.The levels of isocitric and succinic dehydrogenases in sensitive and conditionally sensitive cells were depressed within 2 hours after treatment with 500 μgm/ml of Triton was initiated and by 6 hours the activity was only 25% of the untreated controls. Similar results were obtained with mitochondrial preparations from these cells. Enzyme levels in insensitive cells were unaffected by Triton treatment.Mitochondrial damage was the most striking characteristic noted in treated cells examined by electron microscopy. The mitochondria were quite distorted and had lost most of their cristae formation. This mitochondrial damage was seen in all cell types examined although the rate at which it occurred varied. With sensitive cultures, damage was pronounced within 6 hours after the addition of Triton while mitochondria from conditionally sensitive cells were not grossly affected until 48 hours and they appeared to repair the damage following the removal of Triton.

Notes: The effects of cycloheximide on synchronized Tetrahymena pyriformis strain GL-C were investigated at concentrations ranging from 0.01 to 10 μg/ml. The initial inhibition of protein synthesis was nearly total (〉85%) at 1 μg/ml and above, partial (50-80%) at 0.2 to 0.05 μg/ml, and slight (〈30%) at 0.02 μg/ml. Eventual recovery of protein synthesis to a rate approaching that of the controls took place at concentrations of 1 μg/ml and less. When the drug was added before a “transition point” at 55 minutes after the end of the synchronizing treatment (EST), cell division was blocked by 10 μg/ml, and delayed at concentrations of 1 μg/ml or less. The duration of delay was related to the degree of initial inhibition, and to the time required for recovery of protein synthesis; it also depended on the time after EST at which the drug was added. At a given concentration, maximum division delay was observed just prior to the “transition point;” this maximum delay was correlated with resorption of differentiating oral primordia, followed by the appearance of new primordia. The lesser delays observed at earlier times were correlated with temporary blockage of development of primordia in the “stomatogenic field” stage. Resumption of oral primordium development was, in both cases, temporally correlated with a substantial recovery of protein synthesis. After the “transition point,” cell division, and completion of oral development, was delayed slightly at the lower concentrations, and more substantially at 1 and 10 μg/ml, with some division-arrest at the latter concentration. Except for the recovery phenomenon, the developmental responses elicited by cycloheximide were similar to those observed earlier with puromycin.The bearing of these findings on the mechanism of synchronization in Tetrahymena is considered in the Discussion.

Notes: Mouse bone marrow cells in suspension were separated into a number of fractions on the basis of cell density by equilibrium density gradient centrifugation, or on the basis of cell size by velocity sedimentation. After each type of separation, the cells from the various fractions were assayed for their ability to form macroscopic spleen colonies in irradiated recipient mice, and for their ability to form colonies in a cell culture system. The results from either separation technique demonstrate that cells in some fractions formed more colonies in vivo than in the culture system, while cells in other fractions formed more colonies in culture than in the spleen. The results of control experiments indicate that this separation of the two types of colony-forming cells was not an artifact of the separation procedures. From these experiments it was concluded that the population of cells which form colonies in culture under the conditions used is not identical to the population of cells detected by the spleen colony assay.