Showing papers in "Journal of Cellular Physiology in 1973"

A partially purified polypeptide fraction from rat liver cell conditioned medium with multiplication-stimulating activity for embryo fibroblasts.

Abstract: A polypeptide fraction with multiplication-stimulating activity for chicken and rat embryo fibroblasts was partially purified from serum-free medium conditioned by the growth of a line of rat liver cells. The specific multiplication-stimulating activity of this fraction was 27,000 times that of serum. The rat liver cell multiplication-stimulating activity had a molecular weight of approximately 10,000 daltons and was inactivated by mercaptoethanol and dithiothreitol. It had sulfation factor and non-suppressible insulin-like activities, but did not have anti-trypsin activity. The rat liver cell multiplication-stimulating activity resembled both multiplication-stimulating activity from calf serum and somatomedin.

Multiplication-stimulating activity for chicken embryo fibroblasts from rat liver cell conditioned medium: a family of small polypeptides.

Abstract: A partially purified multiplication-stimulating activity for chicken embryo fibroblasts in cell culture was isolated from rat liver cell conditioned medium (see preceding paper, Dulak and Temin, 1973). It has been analyzed by isoelectric focusing and by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Multiplication-stimulating activity resided in a family of at least four polypeptides which were similar in apparent molecular size, but different in electrical charge. These polypeptides have a specific activity of about 50,000 with respect to serum. One of them has been purified on a small scale to apparent homogeneity in a sodium dodecyl sulfate polyacrylamide gel.

A low molecular weight factor in lung-conditioned medium stimulating granulocyte and monocyte colony formation in vitro.

Abstract: Medium conditioned by excised whole lungs from endotoxin-injected C57BL mice was highly active in stimulating hemopoietic colony formation, particularly of granulocytic type, in agar cultures of mouse bone marrow cells. The colony stimulating factor (CSF) in this material had an α1-α2 electrophoretic mobility, was eluted from calcium phosphate gel by 0.04 M phosphate buffer and had an unusually low apparent S20W of 1.9. Sequestered polymor-phonuclear neutrophils were excluded as a major source of this CSF. The high specific activity and ease of preparation of lung conditioned medium make it valuable both for the large scale production of CSF and as a source of an unusual type of CSF.

The effect of environmental pH on the growth of normal and malignant cells

Abstract: Data have been presented with respect to the widely varying pH optima for the growth of a number of normal and transformed human, mouse, monkey, hamster, rabbit and rat cell lines. At that optimum, differences between normal and transformed cells with respect to maximum population densities tended to become less prominent. Other differences (serum requirement, long-term viability of cultures) were not demonstrably pH-dependent.

Stimulation of differentiation and proliferation of haemopoietic cells in vitro.

Abstract: A liquid culture system, for haemopoietic cells, has been developed using bone marrow cells alone, or co-cultures of thymus and bone marrow cells, inoculated into four ounce medical bottles. After several days growth, such cultures consisted of an attaching population of cells, forming discrete colonies, and a non-attaching population. In the (co-cultures) there was a 2 X enhancement of monolayer colony development compared with the combined total present in the (marrow alone) plus (thymus alone) cultures. Also, better maintenance of non-attaching cells was seen in the (co-cultures). Normal CFUS and CFUC were present in both the (marrow alone) and the (co-cultures) for at least 14 days. In the (marrow alone) cultures, granulocytes in all stages of development were present for the first week, but by 12 days the culture consisted mainly of mono-nuclear cells. In the (co-cultures), however, at 12 days more than 60% of the cells were granulocytes, in all stages of differentiation. (Co-cultures) established using lethally irradiated thymus cells were not able to support this prolonged myeloid differentiation. By feeding the (co-cultures) it was possible to maintain production of (granulocytic) cells for at least ten weeks, although no fully mature granulocytes were observed. After the second feeding, no CFUS were detectable, but variable numbers of agar colony forming cells (not classical CFUC) were present at least for ten weeks.

Adrenergic antagonists, and a possible link between the increase in cyclic adenosine 3',5'-monophosphate and DNA synthesis during liver regeneration.

Abstract: The cyclic-AMP concentration in the liver remnant after 70% hepatectomy increases in a biphasic manner with peak values at 3 and 12 hours, and DNA synthesis begins at 18 hours. Propranolol (dl) injected at 30 minutes after surgery stopped the first wave of cyclic-AMP accumulation, but did not affect the second accumulation or the initiation of DNA synthesis. However, dl, propranolol injected at eight hours equally delayed (by 6 to 8 hours) the second wave of cyclic-AMP accumulation and the initiation of DNA synthesis. Propranolol (dl) did not affect DNA replication per se, since it was totally ineffective after the second wave of cyclic-AMP accumulation had passed and DNA synthesis had been initiated. Propranolol (dl) action was not due to a blockade of β-adrenergic receptors, since its d or l isomers were separately without effect, as were unrelated β-adrenergic blockers (Ku 1313 and M&B 17-803A). On the other hand, an activation of α-adrenergic receptors may be involved in the induction of hepatocyte proliferation, since α-adrenergic antagonists, such as phenoxybenzamine and phentolamine, both delayed and considerably reduced the second wave of cyclic-AMP accumulation and the subsequent initiation of DNA synthesis. It is concluded that the second wave of cyclic-AMP accumulation is somehow associated with the initiation of DNA synthesis.

The effect of ploidy on chemical mutagenesis in cultured Chinese hamster cells.

Abstract: The frequency of mutations induced by ethyl methane sulfonate was compared in a pseudodiploid Chinese hamster cell strain and in a tetraploid substrain derived from it. The frequency of reverse mutations from glycine auxotrophy to glycine independence was similar in the two strains, as expected for a dominant phenotype. Forward mutation to 6-thioguanine-resistance was 25 fold lower in the tetraploid as compared to the diploid strain. The resistant mutants lack hypoxanthine phosphoribosyl transferase activity and their resistant phenotype is recessive in somatic cell hybrids. A combination of chromosomal segregation and mutation could account for the frequency of these recessive drug-resistant mutants in the tetraploid population.

Electrical properties of normal and dysgenic mouse skeletal muscle in culture.

Abstract: Electrical properties of normal and dysgenic mouse skeletal muscle were studied by intracellular recording from embryonic cells developing in vitro. Passive membrane constants were determined from records of transmembrane potential responses to hyperpolarizing pulses of current using two types of analyses, assuming the tubes to be finite cylinders: the off transient and steady state analyses. The following properties of normal and dysgenic fibers were also studied. (a) membrane potentials (b) acetylcholine sensitivity (c) α-Bungarotoxin binding and (d) maximum rate of rise, overshoot and one-half fall time of the action potential. Rare electrotonic coupling between fibroblasts and myotubes was noted. An anomalous type of rectification Was observed in some fibers in which the transmembrane potential responses possessed under and overshoots. These responses may have affected the values of membrane constants as derived by the off transient analysis. In all parameters studied, including membrane constants derived by the steady state analysis, the cultured mouse cells resembled adult denervated mammalian muscle rather than innervated muscle. There were no differences between normal and dysgenic fibers with respect to any of the parameters studied. Dysgenic fibers did not contract although they displayed passive and active membrane properties like those in normal, non-dysgenic fibers.

Erythropoietic progenitors capable of colony formation in culture: state of differentiation.

Abstract: The differentiated state of mouse erythropoietic progenitor cells (CFU-E), detected by their ability to form erythropoietin-dependent colonies in vitro, has been investigated. Transfusion-induced plethora was found to reduce the population size of CFU-E in both spleen and femoral marrow, which indicates that a significant number of CFU-E arise by differentiation processes that are themselves erythropoietin-dependent. Individual spleen colonies were found to be heterogeneous in their content of CFU-E, and the numbers of CFU-E per colony were not correlated either positively or negatively with the numbers of granulocyte-macrophage progenitors (CFU-C) present in the same colonies. The absence of a negative correlation between CFU-E and CFU-C indicates that the erythropoietic and granulopoietic pathways of differentiation are not mutually exclusive within individual spleen colonies. The numbers of CFU-E per spleen colony were also found to vary independently of the numbers of pluripotent stem cells (CFU-S) per colony; in contrast, as found previously, the numbers of CFU-C and CFU-S per colony were positively correlated. These results indicate that more randomizing events separate CFU-E from CFU-S than separate CFU-C from CFU-S, and are consistent with the view that CFU-E occupy a position on the erythropoietic pathway of differentiation that is more remote from the pluripotent stem cells than is the corresponding position of CFU-C on the granulopoietic pathway.

Contact inhibition of division: involvement of the electrical transmembrane potential.

Abstract: Measurements of simultaneous mitotic activity, electrical transmembrane potential (Em), and cell density levels in both 3T3 and Chinese hamster ovary (CHO) cell cultures reveal that a 5- to 6-fold increase in the Em level is associated with development of mitotic arrest at saturation densities. This rise occurs both in confluent monolayers and in interior areas of isolated colonies, and is independent of the rate at which confluence is attained. The Em rise is accompanied by a substantial decrease in intracellular Na. Electron microscopy of saturated CHO monolayer sections shows from 46 to 63% of the cell surfaces to be in close apposition (<300 A spacing). These results for contact inhibited cultures support the hypothesis that mitotic activity may be functionally coupled with the Em level and associated ionic concentration levels. It is suggested that contact inhibition of mitosis may result from a reduction in synthesis of mitogenically essential RNA following a decrease in intracellular Na produced by contact-induced alteration of surface ion-transport activity.

Hemoglobin synthesis in murine virus‐induced leukemic cells in vitro. III. Effects of 5‐bromo‐2′‐deoxyuridine, dimethylformamide and dimethylsulfoxide

Abstract: Erythroid differentiation of Friend leukemia cells is enhanced when the cells are grown for four days in the presence of dimethylsulfoxide (DMSO). Dimethylformamide (DMF) has a similar though less marked effect. 5-Bromo-2′-deoxyuridine (BUdR) (10−5M) inhibits both DMF- and DMSO-stimulated differentiation. For maximum inhibition, BUdR must be present during the first two days of growth, during which time DNA synthesis is maximal. The addition of BUdR after the third day has no effect. Since BUdR is incorporated into DNA and thymidine prevents BUdR inhibition of DMSO-stimulated differentiation, it is likely that BUdR acts by virtue of its incorporation into DNA. Although BUdR alone had little effect upon cell multiplication, in combination with DMSO, cell growth was inhibited up to 40%. Since the BUdR-inhibition of the DMSO effect was approximately 70%, it is unlikely that its effect on differentiation is due to selective killing of those cells which are stimulated to differentiate.

Calcium-dependent stimulation by a phorbol ester (PMA) of thymic lymphoblast DNA synthesis and proliferation.

Abstract: PMA (phorbol myristate acetate, i.e., 12-0-tetradecanoyl-phorbol-13-acetate) a tumor-promoting ester from croton oil, at its most effective concentration of 0.05 μg per milliliter, rapidly (within one hour) induces a large fraction of the lymphoblasts in suspended thymic lymphocyte populations to start making DNA, and these stimulated cells later progress into mitosis. This stimulatory PMA action is probably mediated by calcium because it disappears when calcium is omitted from the medium, and PMA strikingly increases the sensitivity of the lymphoblasts to calcium's stimulatory action.

Contact inhibition of what? An analytical review.

Abstract: Quite a number of phenomena having to do with cells' influences upon one another's movements have come to be regarded as expressions of “contact inhibition.” However, no single, central mechanism has been shown to underlie them all. Consequently, the term “contact inhibition” should not be used without operational modifiers. Inhibitions of individual cell movements imputed to be mediated by cell-cell contacts include inhibition of overlapping (which results in monolayering), of colony expansion, of cell speed (nuclear translocation), of ruffling, of orthogonal movement (proposed to explain spontaneous parallel alignment of cells), and of neighbor exchanges. The six inhibitions listed above are operationally distinct, and only two (overlapping and colony expansion) are known to result from a common mechanism. A seventh phenomenon, so-called “contact inhibition of cell division” (more operationally termed postconfluence inhibition of cell division) is in a separate category and is not considered here. Evidence eliminating action-at-a-distance is available only for the first three, and hence only these should at present be termed contact inhibitions. Inhibition of neighbor exchanges is yet hypothetical; at its extreme, it would immobilize cells in a confluent monolayer, but such immobilization has been found not to occur. Contact inhibition of overlapping, the most studied of the six, is not displayed by invasive cells with respect to normal cells; invasive tumor cells overlap freely upon normal cells, although not necessarily upon one another. Contact inhibition of overlapping, and its loss by invasive cells, can readily be interpreted, by means of the differential adhesion hypothesis, as consequences of cell-type-specific differences in cell-cell and cell-substratum “strengths of adhesion.” These strengths of adhesion are formulated as specific interfacial free energies, which are the only parameters of cellular adhesiveness that have been rigorously shown to determine equilibrium configurations of cell populations.

Stimulation of density-inhibited cell cultures by insulin

Abstract: Cell proliferation in density-inhibited chick embryo cell cultures was induced by microgram quantities of insulin, neuraminidase, trypsin or papain. Other proteins tested, including albumin, fetuin, ribonuclease and hyaluronidase were inactive except in very high concentrations (> 100 μg/ml). The insulin chick embryo model was selected for detailed analysis of the initiation of proliferation. Insulin insolubilized by conjugation with Sepharose particles was also active, but only in so far as it was released in soluble form from the particles. This was measured by a radioimmunoassay. Under the conditions giving maximal cell proliferation less than 0.002-0.2% of insulin was taken up by the cells. This suggests that an interaction of insulin with the cell surface only is sufficient to stimulate the cells. Insulin released the density-inhibited cells from G1 phase to produce an almost synchronous wave of proliferation. The following sequence of events was characteristic of the cells after stimulation by insulin: an early increase in sugar uptake and decrease in leucine uptake, increase in cell volume, stimulation of RNA and protein synthesis, increase in thymidine uptake, DNA synthesis, mitosis and cell division.

Evidence for similar biochemical requirements for bioluminescence among the coelenterates.

Abstract: We present evidence that the chemical requirements among all the bioluminescent coelenterates that have been examined are very similar or identical to those already described for Renilla by Cormier and associates Components required for luminescence in Renilla were also found in a number of bioluminescent coelenterates examined such as Aequorea, Obelia, Cavernularia, Ptilosarcus, Stylatula, Acanthoptilum, Parazoanthus and Mnemiopsis Depending on the organism these include one or more of the following: luciferyl sulfate, luciferase, and luciferin sulfokinase These isolated components were found to be indistinguishable from those found in Renilla as evidenced by their reactivity in the Renilla bioluminescent system, by the spectral characteristics of the isolated luciferyl sulfates, by the molecular weights of the luciferases, and by the colors of the bioluminescence produced in vitro

Characteristics of calcium accumulation by lymphocytes and alterations in the process induced by phytohemagglutinin.

Abstract: Calcium uptake by normal human lymphocytes was found to be a saturable process which was competitively inhibited by manganese indicating the existence of a carrier-mediated mechanism for calcium uptake. Exchange diffusion was not observed, Phytohemagglutinin (PHA) significantly stimulated calcium uptake within minutes after treatment. The increased uptake was attributed to a decreased Km for the proposed membrane carrier rather than to an increased Vmax. Also PHA did not stimulate a normally unused exchange diffusion process, nor did it affect calcium efflux. Uptake by both unstimulated and PHA-treated lymphocytes was not influenced by magnesium or by cycloheximide or actinomycin D.

The effects of inhibitors of RNA and DNA synthesis on protein synthesis and polysome levels in mouse L-cells.

Abstract: Metabolic inhibitors of RNA synthesis, such as actinomycin D and MPB (2-mercapto-1-(β-4-pyridethyl) benzimidazole) have often been used to test the possibility that transcription is required for some cellular process or to measure the „half-life” of messenger RNA (mRNA). The basic assumption of this work has been that the primary effect of these inhibitors is on transcription alone, and that any effect on translation is secondary to the inhibition of mRNA synthesis. This assumption has been tested by examining the effects of these inhibitors at different doses on various parameters of the transcriptional and translation processes in mouse L-cells in culture, i.e., the rates of RNA, DNA, and protein synthesis; the level and size of cytoplasmic polysomes; the rates of polypeptide elongation and termination, and the role of peptide chain initiation in protein synthesis. The results indicate that the basic assumption is not correct since these inhibitors affect protein synthesis by inhibiting the rate of initiation and not the level of mRNA. Another implication of the experiments is that the average mRNA in L-cells has a half-life of at least one cell generation (16–18 hours), and that earlier values from experiments using actinomycin are underestimates. Cordycepin (3′-deoxyadenosine) which inhibits the production of ribosomal and messenger RNA also affects protein synthesis at the level of initiation. In contrast, two inhibitors of DNA synthesis (hydroxyurea and cytosine arabinoside) inhibit protein synthesis and the level of polysomes by some other mechanism.

Cell separation by velocity sedimentation of postnatal mouse cerebellum.

Abstract: Trypsinized cells of newborn mouse cerebellum have been separated by velocity sedimentation at unit gravity in shallow gradients of Ficoll. The two main technical difficulties were formation of gels around the dissociated cells and clumping of cells before and during the sedimentation procedure. These were solved by adding DNase to the dissociation medium and with holding serum, respectively. Proliferating cells of the external granular layer separated according to size differences in the cell generation cycle. Identification of Purkinje or other early-forming neurons was made by labeling them with 3H-thymidine on their birthdays. Many of the fractions contain viable cells capable of aggregating in culture.

The temperature dependence of the exchange transport of glucose in human erythrocytes

Abstract: The temperature dependence of the uptake of glucose by exchange transport is investigated at two pH values (7.5 and 5). The Arrhenius's energy of activation, the heat of activation, the free energy of activation and the entropy of activation are calculated. The parameters are different at pH 7.5 and 5. For both pH levels the heat of activation and the entropy of activation change abruptly at 20°C. This finding leads us to assume that at this temperature a change in the structure of the membrane takes place.

Purine excretion by mammalian cells deficient in adenosine kinase.

Abstract: An adenosine kinaseless (AK−) mutant of the mouse fibroblast line 3T6 has been obtained in cell culture by evolution of resistance to 6-thio-methylpurine ribonucleoside and tubercidin. The mutant excretes purines (xanthine and hypoxanthine) into the culture medium. Human or mouse cells lacking hypoxanthine-guanine phosphoribosyl transferase (HPT−) excrete increased amounts of purines, but a human cell mutant lacking both HPT and AK excretes considerably more hypoxanthine. The difference in hypoxanthine excretion between the HPT− mutant and the HPT− AK− mutant originates from the adenosine normally reutilized through the activity of adenosine kinase. The activity of adenosine kinase is essential to retard the adenosine cycle and to prevent cellular loss of purines.

Conditionally lethal mutations in Chinese hamster cells. I. Isolation of a temperature-sensitive line and its investigation by cell cycle studies.

Abstract: The isolation of a temperature sensitive cell line from the Chinese hamster line CCL39 of the American Type Culture Collection is described. At the nonpermissive temperature (39°C) the cells become attached to the surface of tissue culture dishes, but no microscopically observable colonies are formed upon prolonged incubation. Exposure to the high temperature for more than 24 hours leads to an almost complete loss in viability. A karyotypic analysis showed that this new line has lost one of the medium-sized metacentric chromosomes, although no proof is available so far to show that this loss is not simply coincidental. In nonsynchronized cultures transferred to 39°C DNA synthesis stops first, RNA synthesis shortly thereafter, while protein synthesis (turnover) continues for a longer time. After such a shift the cell number increases by less than 15% as measured with the Coulter counter. Studies with synchronized cultures give the following results: (1) one round of DNA synthesis can occur at 39°C when the cells are released from serum starvation or a hydroxyurea block, or when mitotic cells are placed at 39°C; (2) the entry of cells into metaphase of mitosis at 39°C is almost normal when the preceding time interval at 39°C is only eight hours (release of cells from G1/S boundary), but considerably reduced when the cells spend an additional 12 to 15 hours at 39°C in G1 (release from serum starvation). Infection by SV40 virus temporarily induces DNA synthesis after it has come to a stop at the nonpermissive temperature, but cells permanently transformed by SV40 still exhibit the temperature-sensitive phenotype.

Serum dependent growth of primary cultured differentiated fetal rat hepatocytes in arginine-deficient medium.

Abstract: Fetal rat hepatocytes in primary monolayer cultures multiply in arginine-deficient medium. Both the “recovery efficiency” and the final cell density of the cultured cells are proportional to the concentration (0–15%, v/v) of dialyzed fetal bovine serum in the medium. Stationary-phase cells divide again following addition of fresh serum to the culture. After two to three generations of growth, the chromosome number of these cells remains diploid [2N = 42]. Cross-feeding (of a subpopulation of arginine-requiring liver-derived cells by parenchymal arginine-synthesizing cells) and cellular degradation of various serum proteins do not account for sources of arginine required for cell multiplication in this culture system. Because these cultured hepatocytes utilize ornithine for arginine biosynthesis, and because ornithine enhances the rate and the amount of cell multiplication, it is more likely that the multiplying cells are parenchymal arginine-synthesizing hepatocytes. At least two classes of serum factors are required for the growth of cultured fetal rat hepatocytes: one stimulates cell multiplication; the other is required for cellular survival and/or attachment to the culture dish. These factors have been partially separated by fractionation with ammonium sulfate; they are non-dialyzable, heat labile, and sensitive to changes in pH.

Isolation and characterization of revertant cell lines. 3. Isolation of density-revertants of SV40-transformed 3T3 cells using colchicine

Abstract: Treatment of the SV40 transformed 3T3 cell line SV101 with colchicine permits the isolation of polyploid revertant sublines Which have lower saturation densities than SV101. These low saturation density lines have also reverted to a high serum requirement for growth, and are unable to form colonies in methocel. Normal SV40 has been recovered from these revertants. 3T3 cells are more resistant to colchicine than SV3T3 cells at all cell densities. Colchicine revertants do not display a 3T3-like resistance to colchicine at low density, but do survive colchicine at confluent cell densities, presumably due to their increased contact inhibition.

Isolation and characterization of revertant cell lines. IV. Direct selection of serum-revertant sublines of SV40-transformed 3T3 mouse cells.

Abstract: SV101, the SV40-transformed subline of the mouse fibroblast line 3T3, is both serum- and density transformed, since it grows in both 1% and 10% calf serum, and grows beyond confluence in 10% calf serum. Negative selection at low cell density in 1% calf serum or in 10% agamma-depleted serum permits direct recovery of serum-revertant sublines of SV101. These sublines are unable to grow in 1% calf serum. Although negative selection at high cell density in 10% calf serum is known to permit recovery of density-revertant sublines of SV101, most density-revertants are not serum-revertant. However, all serum-revertants isolated so far are density-revertant as well.

A temperature-sensitive function in a Chinese hamster line affecting DNA synthesis.

Abstract: A temperature-sensitive (ts) mutant of Chinese hamster cells (K12) is blocked at a step late in G1, required for the initiation of DNA synthesis. The ts lesion is recessive in interspecific hybrids.

Deoxyglucose transport by uninfected, murihe sarcoma virus-transformed, and murine leukemia virus-infected mouse cells

Abstract: The initial rates of deoxy-D-glucose transport by cultures of growing and density-inhibited mouse embryo cells and lines of mouse cells transformed spontaneously or after infection by murine leukemia virus or murine sarcoma virus were investigated as a function of the deoxyglucose concentration. The apparent Km for deoxyglucose transport was about the same for all types of cells (1–2 mM). The Vmax of secondary cultures of mouse embryo cells decreased from 6 nmoles/106 cells/minute for sparse cultures to less than 1 nmole/106 cells/minute for density-inhibited cultures. The Vmax was about the same whether estimated in monolayer culture or in suspensions of cells dispersed by treatment with trypsin. The Vmax for deoxyglucose transport by the established cells, whether transformed spontaneously or by virus infection, was 4 to 25 times higher than that for density-inhibited mouse embryo cells and was independent of the cell density of the cultures. Deoxyglucose transport was competitively inhibited by Cytochalasin B, Persantin, glucose and 3-O-methyl-D-glucose and the apparent Ki values of inhibition were similar for the mouse embryo cells and the various cell lines. Similarly, the sensitivity of the glucose transport systems to inactivation by p-chloromercuribenzoate was about the same for all types of cells. The results suggest that the glucose transport system of the normal mouse embryo cells and the cells of the various established lines is qualitatively the same, but that the number of functional transport sites differs for the various cell lines and decreases markedly in mouse embryo cells with an increase in cell density of the cultures.

DNA synthesis and mitosis in a temperature sensitive Chinese hamster cell line.

Abstract: Viability, DNA synthesis and mitosis have been followed in the temperature sensitive Chinese hamster cell mutant K12 under permissive and non-permissive conditions. On incubation at 40°C cells retained their ability to form colonies at 33°C for 15 to 20 hours, but viability was lost gradually during the following 20 hours. When random cultures of K12 were shifted to 40°C the rate of DNA synthesis was normal for three to four hours but then decreased markedly, reaching 95% inhibition after 24 hours. Under the same conditions mitosis was inhibited after 15 hours. If cultures which had been incubated at 40°C for 16 hours were placed at 33°C the rate of DNA synthesis increased five hours after the shift down and mitosis 18 hours after. These results can be interpreted on the assumption that K12 at 40°C is unable to complete a step in the cell cycle which is essential for DNA synthesis and which occurs three to four hours before the start of S at 33°C.

Isolation of temperature sensitive mammalian cells by selective detachment

Abstract: Temperature sensitive cells have been isclated from Syrian and Chinese hamster cells using a method based on selective detachment from a glass substrate. The Syrian hamster isolates occurred at a high frequency (about 1 in 103) and reverted rapidly; polyoma virus transformation conferred on cells the ability to grow, perhaps abnormally, in agar suspension. A slightly modified isolation technique was applied to Chinese hamster cultures and resulted in the isolation of at least one mutant (from a starting population of 5 × 108 cells) with a spontaneous reversion rate of less than one in 6 × 107. Treatment of the mutant with ethyl methane sulphonate induced reversion. It was concluded that selective detachment provided a useful method for the isolation of conditional lethal mutants of mammalian cells.

The effect of endotoxin on murine stem cells.

Abstract: Previous studies showed that after 5 μg of Salmonella typhosa endotoxin there was an increase in colony stimulating factor temporally related to a fall in murine marrow in vitro colony forming cells (CFC) This was followed by differentiation along the marrow granulocytic pathway The present studies showed that after 5 μg of endotoxin the peripheral blood CFC fell by approximately 50% at one hour, rose to a level ten fold that of control at six hours and then returned to control values by 48 hours There was a progressive increase in the number of splenic CFC to ten fold that of control from 24 to 72 hours after endotoxin These data imply a migration of CFC from the marrow to the spleen along with an in-situ increase in splenic CFC Thus, either migration or differentiation may explain the fall in marrow CFC after endotoxin Spleen colony forming units (CFU) in the marrow were measured by a transplantation technique and the transplantation fraction (f Fx) determined A decrease in marrow CFU at 24 hours after endotoxin was secondary to a change in the f Fx from 111% to 76% There was however, an increased percentage of CFU in DNA synthesis in the interval of 6–48 hours after endotoxin, as judged by the hydroxyurea technique As the marrow CFC fell within 20 minutes of endotoxin administration, the data suggest the CFC may be affected initially and that changes in the generative cycle of the CFU may be of a secondary nature

Trichodermin, a possible inhibitor of the termination process of protein synthesis.

Abstract: The mode of action of the antibiotic, trichodermin, on yeast cells has been investigated. Trichodermin specifically inhibits protein synthesis and, during the in vivo inhibition of protein synthesis, ribosomes remain in polyribosomes rather than shifting to monoribosomes. This observation suggests that trichodermin inhibits either an elongation step or a termination step of protein biosynthesis. These two possibilities were distinguished by comparing the action of trichodermin with that of cycloheximide, a known elongation inhibitor, upon the reformation of polyribosomes during recovery from a block in polypeptide chain initiation. Cycloheximide slows the recovery of polyribosomes from monoribosomes following a block in polypeptide chain initiation whereas trichodermin enhances the recovery of polyribosomes. This observation is interpreted to mean that trichodermin primarily inhibits the termination step of protein biosynthesis.