bluuebunny/crossref_metadata_2025 huggingface dataset:
A membrane‐altered mutant cold‐sensitive for growth
Authors: Ling Victor
Published: February 2005

A colchicine‐resistant clone, CHRE5, has been isolated in a single step from a mutagenized culture of Chinese hamster ovary cells. Its resistance correlates with reduced colchicine permeability. At the same time, CHRE5 cells display a pattern of cross‐resistance to unrelated drugs similar to other membrane‐altered drug‐resistant mutants previously described (Ling and Thompson, 1974). However, CHRE5 cells also express a cold‐sensitivity for growth in that at 34° they do not double in number while at 38.5° they grow with a doubling time of about 22 hours. Employing synchronous cultures, the cold sensitive block in CHRE5 cells has been determined to be located prior to S in the G1 phase of the cell cycle. Mutant cells at 33.5° are not able to initiate DNA synthesis, however, cells already synthesizing DNA are able to complete the whole course of S. This cold sensitive block is reversed by shifting cells back to the higher temperature. Additonal clones with the CHRE5 phenotype have been isolated from non‐mutagenized cultures of wild‐type cells. Moreover, partial revertants of CHRE5 with increased ability to grow at 34° have been isolated and found to display increased colchicine sensitivity. These results are consistent with the hypothesis that the two phenotypes observed in CHRE5, namely, an altered plasma membrane (reduced drug permeability) and an altered ability to initiate DNA synthesis are the result of the same mutation.
Antagonistic effects of insulin and cortisol on coordinate control of metabolism and growth in cultured fibroblasts
Authors: Rubin H.
Published: February 2005

A variety of metabolic and biosynthetic pathways in chick embryo fibroblasts are stimulated coordinately by many unrelated exogenous agents. Three of the best characterized components of this coordinate response are the uptake of 2‐deoxy‐D‐glucose (2‐dGlc) and of uridine and the incorporation of thymidine into DNA. Insulin stimulates and cortisol inhibits the coordinate response. In cortisol‐treated cultures, as little as 10−3 units/ml of insulin may stimulate thymidine incorporation 4‐fold and 10−1 units/ml may stimulate as much as 40‐fold. The higher concentrations of insulin completely override the inhibitory effect of cortisol. They also cause about a 5‐fold stimulation of the uptake of 2‐dGlc and of uridine and a 2‐fold stimulation of proline incorporation into protein. The uptake rates of 2‐dGlc and uridine double within 30 minutes after addition of insulin to cortisol‐inhibited cultures, but the incorporation of thymidine only begins to increase markedly after a 4‐hour delay. When cortisol is added to cultures in the absence of insulin, the rates of uptake of 2‐dGlc and uridine begin to decrease within two hours, but the incorporation of thymidine remains constant for two hours before beginning to decrease. Deprivation of Mg2+ inhibits the accelerated coordinate response maintained by insulin, but does not further the inhibition induced by cortisol. Results with metabolic inhibitors indicate that the stimulation of 2‐dGlc and uridine uptake by insulin do not require RNA synthesis, and also suggest that they do not require protein synthesis. These and other findings can be explained by a model for coordinate control in which insulin increases and cortisol decreases the availability of Mg2+ for a wide spectrum of regulatory reactions in different metabolic pathways. In this model both hormones affect only the rates of ongoing reactions and do not instruct the cell to carry out specific new reactions unless the cell was predetermined to do so.
PHA stimulation of human lymphocytes during amino acid deprivation. Protein, RNA and DNA synthesis
Authors: Dauphinais Christiane , Waithe William I.
Published: February 2005

Resting lymphocytes are in the G0 phase of the cell cycle. Upon activation by PHA, they progress into G1 with accompanying increased protein and RNA synthesis, initiate DNA synthesis and divide. We have studied the kinetics of inhibition of macromolecular synthesis during activation in the absence of single amino acids. Three types of kinetics are observed. In the absence of tryptophan or isoleucine, stimulated lymphocytes show a normal increase in protein and RNA synthesis during the first 30 hours of stimulation, initiate DNA synthesis but are subsequently inhibited. In phenylalanine‐deficient medium, no DNA synthesis occurs in spite of a slight increase in protein synthesis. No increase in macromolecular synthesis is observed in medium lacking any one of the other essential amino acids (eg: lysine).

Our results indicate that the kinetics of macromolecular synthesis in tryptophan‐deficient medium is the result of a limited reserve of protein‐bound tryptophan which becomes exhausted after 30 hours. On the other hand, delayed inhibition of synthesis in isoleucine‐deficient medium probably reflects an initially low requirement for this amino acid followed by inhibition of the synthesis of isoleucine‐rich proteins involved in some late event of stimulation. Partial deprivation of lysine results in kinetics of protein synthesis similar to that in tryptophan‐ or isoleucine‐deficient media. The results indicate that the kinetics of macromolecular synthesis during activation of lymphocytes in the absence of an essential amino acid is a function of the quantitative requirement for that amino acid, at a given time during stimulation.

Upon replacement of lysine, lymphocytes inhibited by lysine deficiency begin RNA and protein synthesis immediately and at a rate faster than that of unstimulated cultures to which PHA is added. They also initiate DNA synthesis earlier and therefore, are closer to the S phase than resting lymphocytes. It is concluded that lymphocytes stimulated in the absence of lysine are activated even though no overall increase in macromolecular synthesis is observed. Furthermore, the kinetics of DNA synthesis following reversal of inhibition by phenylalanine suggests that lymphocytes stimulated during phenylalanine deprivation become arrested at most six hours before S. These results indicate that amino acid deficiencies lead to arrest of activated lymphocytes at various stages of stimulation, depending on how stringent these deficiencies are.
Embryonal carcinoma cells (and their somatic cell hybrids) are resistant to infection by the murine parvovirus MVM, which does infect other teratocarcinoma‐derived cell lines
Authors: Miller Richard A. , Ward David C. , Ruddle Frank H.
Published: February 2005

Minute virus of mice (MVM), a non‐defective parvovirus, has been shown to infect cultures of non‐pluripotent differentiated teratocarcinoma‐derived cells, but pluripotent (and “nullipotent”) embryonal carcinoma cells derived from the same teratocarcinoma resist MVN infection. Somatic cell hybrids between an embryonal carcinoma line and Friend erythroblastic leukemia cells are also resistant to MVM, even though Friend cells are susceptible. Among three blastocyst‐derived lines tested, only one, a parietal yolk sac cell line, resists MVM infection. These results suggest that teratocarcinoma cultures may provide useful systems in which to study the cellular factors which mediate susceptibility to this teratogenic and oncolytic virus.
Measurements of contraction latencies to mechanical and electrical stimulation of the protozoan, Spirostomum ambiguum
Authors: Hamilton Thomas C. , Osborn Dustan
Published: February 2005

Measurements made on contraction latencies in Spirostomum suggest that mechanical stimulation causes contractions to be initiated by the release of small amounts of calcium from a store tightly coupled to the contractile apparatus. Contraction to electrical stimulation appears to result from the gross electrophoretic mobilization of large amounts of calcium from a loosely coupled store. Contraction latencies to mechanical stimulation were three milliseconds and were independent of stimulus strength, previous stimulation, and contraction probability. For 0.5‐millisecond biphasic electrical stimulation the contraction latencies varied widely. Latencies to initial contractions were dependent on stimulus strength: from 1.0 milliseconds for a stimulus that caused a 100% probability of contraction to 2.0 milliseconds for a stimulus that caused a 10% probability of contraction. Latencies of contraction to electrical stimulation were also dependent upon previous stimulation, lengthening to over 300 milliseconds after ten minutes of stimulation. Initial contraction latencies were not affected by previous stimulation to the other (electrical or mechanical) stimulus modality. Repeated electrical stimulation also reduced the animal's resting length and slowed the rate of post contraction re‐extension, whereas mechanical stimulation did not have these effects.
Effects of withdrawal of a mitogenic stimulus on progression of fibroblasts into S phase: Differences between serum and purified multiplication‐stimulating activity
Authors: Bolen Joe B. , Smith Gary L.
Published: February 2005

Multiplication‐stimulating activity (MSA) for chicken embryo fibroblasts was purified from serum‐free medium conditioned by the growth of a rat liver cell line. A comparison between calf serum and purified MSA was made regarding the regulation of the fibroblast cell cycle. Addition of serum or MSA to stationary, quiescent cells stimulates them to enter the DNA synthetic phase after a characteristic lag period. Exposure to serum for shorter periods of time will irreverisbly commit cells to continue through the cell cycle and initiate DNA replication in the absence of serum. In contrast, the withdrawal of purified MSA from the medium results in an abrupt halt in the progression of cells towards S phase. The results of labeled thymidine incorporation and autoradiographic experiments clearly indicate that the point at which cells become irreversibly committed to enter the DNA synthetic period is at or near the G1‐S boundary.

The abrupt decay of the stimulation upon withdrawal of purified MSA provides a unique opportunity to investigate the biochemistry of this discrete phase of the cell cycle.
Specificity of the requirements for magnesium and calcium in the growth and metabolism of chick embryo fibroblasts
Authors: Rubin H.
Published: February 2005

The rate of entry of chick embryo fibroblasts (CEF) into the S‐period of the cell cycle is reduced by lowering the external supply of Mg2+ below 0.2 mM. This slowdown, which is measured by the rate of incorporation of 3H thymidine into DNA, can be largely reversed by doubling or tripling the concentration of Ca2+ in the medium, normally 1.7 mM. The Ca2+‐induced stimulation is shown not to depend on contaminating traces of Mg2+ in the added Ca2+. The increase in cell number in the Ca2+‐stimulated cultures is delayed, possibly due to cell detachment. The effect of Ca2+ on thymidine incorporation can be simulated almost quantitatively by Sr2+. Ba2+ does not produce the effect, nor do any of the other cations tested. As little as 0.2 mM Mg2+ produces a full stimulation of thymidine incorporation in the absence of added Ca2+, and no substitute was found that is effective in the same concentration range. In short term experiments, i.e., 16 hours, even 5.0 mM Ca2+ cannot stimulate thymidine incorporation to the extent achieved with 0.2 mM Mg2+. Large amounts of Ca2+ or Sr2+ can accelerate the uptake of 2‐dGlc in Mg2+‐deprived cultures, but they are much less efficient than Mg2+ in this regard also. It is suggested that Mg2+ is the direct intracellular effector in controlling the diverse reactions of the coordinate response, and that Ca2+ and Sr2+ act indirectly by making Mg2+ available to participate in these reactions.
Effect of calcitonin on the phosphorylation of non‐histones from cultured bone cells
Authors: Bustos Sergio E. , Schuster George S. , Dirksen Thomas R. , McKinney Ralph V. , Lai David Y.‐L.
Published: February 2005

32P‐uptake into non‐histones from bone cell cultures was selectively stimulated in the presence of calcitonin. Comparison of the control and experimental radioactivity profiles of non‐histones fractionated by SDS gel electrophoresis showed that, in response to calcitonin stimulation, there was a 2‐ to 3‐fold increase in the specific activity associated with non‐histone proteins in the molecular weight range of 10,000 to 45,000 daltons while that of bands between 50,000 to 200,000 decreased.
Serum stimulation of human fibroblasts: Effect on non‐histones of nuclear ribonucleoprotein and chromatin
Authors: Augenlicht Leonard H. , Lipkin Martin
Published: February 2005

In quiescent human fibroblasts stimulated to proliferate by fresh medium plus 15% serum, no changes were seen in the incorporation of 3H‐tryptophan into the protein of nuclear ribonucleoprotein during the first three hours following re‐feeding. This was in contrast to non‐histone chromosomal proteins where the incorporation increased by 90% within ten minutes. The density of the formaldehyde fixed nuclear ribonucleoprotein in CsCl was 1.43–1.44 g/ml and this also did not change following stimulation. The electrophoretic profile of the proteins of nuclear ribonucleoprotein on SDS gels exhibited a predominant band corresponding to a molecular weight of 44,000 closely trailed by a band at 47,000 and other bands at higher molecular weight. This pattern was not altered by serum stimulation and the same was true for the more complex electrophoretic profile of the chromatin proteins. Following a 10‐minute pulse of 3H‐tryptophan at ten minutes after stimulation, there was a selective increase in the labeling of non‐histone chromosomal protein of molecular weight 59,000; no change was seen in the labeling of any protein of nuclear ribonucleoprotein.
Thrombin potentiates the mitogenic response of cultured fibroblasts to serum and other growth promoting agents
Authors: Zetter Bruce R. , Sun Tung‐Tien , Chen Lan Bo , Buchanan John M.
Published: February 2005

Thrombin may stimulate the proliferation of resting fibroblasts by itself or by potentiating the mitogenic effects of other growth stimulating agents. When added alone to a dense quiescent culture of chick embryo fibroblasts in either a serum‐free or a low serum medium, thrombin stimulates these cells to proliferation at a rate comparable to that seen with 5% serum. In the case of rabbit corneal fibroblasts neither fibroblast growth factor nor thrombin is particularly effective as a growth stimulant when added alone but exhibited a pronounced synergism on cell proliferation when present together. Established cell lines in the resting state, including 3T3, SV‐3T3, 3T6, BHK‐21 and 3T3 injected with avian sarcoma virus strain B77 (B77‐3T3), are not responsive to thrombin alone. When the serum concentration in the medium equals or exceeds 2%, thrombin potentiates the mitogenic response of 3T3 cells to serum factors. With the exception of 3T3 and SV3T3 cells, the other cell lines show a potentiation of growth when thrombin is added to a low‐serum (0.5%) medium containing epidermal growth factor and prostaglandin F2α. The addition of thrombin to cultures of B77‐3T3 cells growing in the presence of epidermal growth factor and prostaglandin F2α does not increase the initial growth rate of these cells but increases significantly the final cell density of these cultures.
A retarded rate of DNA replication and normal level of DNA repair in Werner's syndrome fibroblasts in culture
Authors: Fujiwara Yoshisada , Higashikawa Toshiaki , Tatsumi Mariko
Published: February 2005

We investigated the cloning efficiency, DNA repair, and the rate of DNA replication in the skin fibroblasts from patients with Werner's syndrome (WS) of an autosomal recessive premature aging disease. Five WS strains exhibited normal levels of sensitivity toward X‐ray and UV killings and repair of X‐ray induced single strand breaks of DNA (rejoining) and UV damage to DNA (unscheduled DNA synthesis). The sedimentation of newly synthesizing DNA in alkaline sucrose gradients demonstrated a characteristic feature that only the elongation rate of DNA chains, estimated by the molecular weight increase, was significantly slower during early passages in WS cells than in normal Hayflick Phase II fibroblasts. In addition, plating efficiencies as well as the replicative potentials of five WS strains were more limited than those of normal cells under the identical culture conditions. It seems therefore that at least in the WS cells tested, the slow rate of DNA replication may be more related to the shortened lifespan and enhanced cell death, as manifestation of premature senescence at the cellular level, than be the DNA repair ability.
Increased messenger RNA from protein synthesis inhibited human fibroblasts
Authors: Maroun L. E. , Miller E. T.
Published: February 2005

Numerous reports have demonstrated that specific protein synthesis in response to specific inducers is markedly stimulated by a simultaneous brief exposure to protein synthesis inhibitors such as cycloheximide. This phenomenon is known as “superinduction” and is most often attributed to the accumulation of cytoplasmic messenger RNA during the inhibition period. Messenger RNA, as defined by rapid labeling, oligo (dt)‐cellulose binding, and cell free protein synthesis stimulation was measured in cycloheximide treated human fibroblasts. In spite of a consistent 40% decrease in total polysomal 3H‐uridine labeled RNA, a 1.5‐ to 2‐fold increase in extractable mRNA was observed. These data provide direct evidence that protein synthesis inhibition stimulates the appearance of cytoplasmic mRNA and/or completely blocks its degradation and, are consistent with the hypothesis that mRNA accumulation partly underlies the superinduction phenomena.
Selective lethal effect of thymidine on human and mouse tumor cells
Authors: Lee S. S. , Giovanella B. C. , Stehlin J. S.
Published: February 2005

Human cell lines derived from a melanoma and a colon carcinoma, and cultures of human melanocytes and intestinal epithelial cells, as well as a mouse mesenchymal non‐neoplastic cell line and a malignant subline of the same have been quantitatively studied in tissue culture for their sensitivity to thymidine. All three tumor lines produced solid tumors when injected into nude thymus‐deficient mice. No tumors were obtained by injecting cells of the human normal long‐term cultures or of the non‐neoplastic mouse line.

The tumor‐producing lines showed a greater sensitivity to the lethal effects of high concentrations of thymidine than their non‐tumor‐producing counterparts. Less than 23% of the tumor cells survived 72 hours in the presence of 1 mg/ml of thymidine, in contrast to 60% or more of the non‐tumor cells.

Colony formation was much more inhibited by thymidine and the differential between normal and tumor cells was even more pronounced. Tumor cells which also were treated for 72 hours with 1 mg/ml of thymidine and then plated in fresh medium formed very few colonies. If the plating efficiency of the untreated controls is considered as 100%, 4.3% or less of the treated tumor cells formed colonies, in contrast to 33% or more of the non‐tumor cells.
Cell surface glycosaminoglycans: Identification and organization in cultured human embryo fibroblasts
Authors: Vogel Kathryn G. , Kelley Robert O.
Published: February 2005

A morphologically detectable cell coat, composed of glycoprotein, glycolipid, and glycosaminoglycan, is present on the external surface of most vertebrate cells. We have invetigated the composition and organization of glycosaminoglycans in the cell coat of cultured human embryo fibroblasts by labeling cells with 3H‐glucosamine and Na235SO4 and subsequently treating cultures with specific enzymes. Components released were identified by chromatography and specific enzymatic digestion. In situ incubation with leech hyaluronidase (4 μg/ml) removed only hyaluronic acid from the cell surface whereas testicular hyaluronidase (0.5 mg/ml) removed both hyaluronic acid and chondroitin sulfate. Trypsin (0.1 mg/ml) released a large mass of glycopeptides in addition to hyaluronic acid, chondroitin sulfate, and heparan sulfate. The affinity of the cell coat for the cationic dye, ruthenium red, was reduced by leech hyaluronidase treatment. Sequential enzyme digestions of the cell surface showed that hyaluronic acid could be removed without the concomitant or subsequent release of sulfated glycosaminoglycans, suggesting that the hyaluronic acid is not a structural backbone for glycosaminoglycan complexes of the external cell surface.
Proliferative response of human diploid fibroblasts to intermittent light exposure
Authors: Parshad Ram , Sanford Katherine K.
Published: February 2005

Three‐ to four‐hour exposure to fluorescnt light, one to three times weekly, reproducibly enhanced the proliferation rate of human diploid fibroblasts. This enhancement was observed in WI‐38 and a line from whole embryo mince at late population doubling level (PDL) as well as in a line from adult skin at early PDL. Single or multiple exposures of short duration stimulated proliferation, whereas exposures of long duration were cytotoxic. This proliferative response is reversible, and is mediated through the culture medium, Dulbecco Vogt's supplemented with 10% fetal bovine serum. Apparently light produces some mitogenic substance(s) in the culture medium that accumulates in the cells and is toxic or growth‐stimulatory depending on its concentration per cell. Another possibility is that light produces in the medium both cytotoxic and growth‐stimulatory substances.
Renal sugar transport in the winter flounder IV. Effect of CA2+ on sugar transport in teased renal tubules
Authors: Kleinzeller Arnost , Dubyak George R.
Published: February 2005

The electrolyte distribution and sugar uptake by teased renal tubules of winter flounder (Pseudopleuronectes americanus) was studied using incubation media with 1.4 mM Ca2+ (controls) and without Ca2+. The omission of Ca2+: (a) produced some cellular swelling, and increase in cell Na+ and loss of K+; (b) had no effect on the extracellular propylene‐glycol space; (c) increased the uptake of non‐metabolizable methyl‐α‐D‐glucoside by the tissue: whereas in controls the equilibrium tissue/medium ratio (T/M) for the Na+‐independent uptake of the sugar was 0.71 ± 0.03 (SEM, n = 9), in Ca2+‐free media the T/M rose in 1.18 ± 0.06. The increase in sugar uptake seen in absence of Ca2+ was abolished by absence of Na+ (Li+‐media), 0.5 mM ouabain and 0.5 mM phlorizin; (d) produced an increase in the galactose phosphate level without affecting that of free sugar; (e) decreased the active uptake of 2‐deoxy‐D‐galactose by the tubules. These results were analyzed on the basis of available information on the transport characteristics of the sugars at the luminal and antiluminal cell faces. It is suggested that absence of Ca2+ increases the permeability of the intercellular junctions, thus permitting sugars to enter from the external medium into the tubular lumen. Methyl‐α‐D‐glucoside and D‐galactose can then be taken up into the cells by brush‐border localized active processes, whereas 2‐deoxy‐D‐galactose is not reabsorbed at this cellular face. At 1.4 mM Ca2+, the intercellular junction appears to be relatively impermeable to sugars and the transport properties in teased tubules reflect then events predominantly localized at the antiluminal cell face.
Release of a factor during early stages of contact between hamster sperm and eggs in vitro
Authors: Hartmann John F. , Hutchinson Cameron F.
Published: February 2005

A sensitive bioassay was devised which detected the release of a factor resulting from contact between the surfaces of sperm and zona pellucida of the golden hamster in vitro. This assay is based upon the ability of the factor to induce premature binding between the gametes. Release of the factor occurred in a dose‐dependent manner as a function of increasing concentration of sperm, but only after they were capacitated, i.e., subjecting sperm to those conditions which endow them with the ability to penetrate the egg. The factor was released, in what appeared to be pulses of activity, throughout the 40‐minutes prepenetration period, and this release culminated in a large pulse which was rapidly terminated soon after penetration began. The factor was also detected following contact with homologous zonae pellucidae from which the vitellus had been mechanically removed. Thus, factor release and cessation of its release occurred independently of the vitellus. When hamster eggs or isolated zonae were replaced by those of the mouse, the factor was not detected even though hamster sperm attached to them; nor was it recovered when isolated zonae or eggs of the hamster were treated with trypsin before exposure to sperm. Factor release and penetration of eggs were inhibited in a similar manner as a function of increasing concentrations of trypsin. This finding and the observation that the factor was not detected when the sperm were not capacitated, and therefore incapable of penetration, suggests that a relationship may exist between factor release and penetration.
Relationship of heat labile glucose‐6‐phosphate dehydrogenase and multiple molecular forms of the enzyme in senescent human fibroblasts
Authors: Duncan Michael R. , Dell'Orco Robert T. , Guthrie Patrick L.
Published: February 2005

Human fibroblasts derived from newborn foreskin and designated CF‐3 were assayed for heat labile glucose‐6‐phosphate dehydrogenase (G6PD) when they had grown to confluency, as well as when they were arrested in an essentially nonmitotic state. Under both culture conditions there was an increase in heat labile G6PD (up to 25% of the total activity) as cells progressed through their in vitro lifespan; however, arrested cells exhibited less heat labile G6PD than did comparable growth controls (5–10% decrease). Acrylamide gel electrophoresis of crude G6PD preparations revealed three distinct bands of enzymatic activity. One of the bands was tentatively identified as the dimeric form of the enzyme and another as the tetrameric form. The tetrameric form was more heat sensitive, and the percent to the total activity of this form increased as the cells became senescent. The percent of total activity of the tetrameric form was comparable to the percent heat liable enzyme assayed at a given population doubling level. These results indicated that the observed increase in heat lability of G6PD with age may be due to a shift in equilibrium to the tetrameric form of the enzyme rather than the synthesis of aberrant enzyme molecules.
Membrane transport by guinea pig peritoneal exudate leukocytes: Effect of phagocytosis on hexose and amino acid transport
Authors: Straus David C. , Imhoff John G. , Bonventre Peter F.
Published: February 2005

Short term, carrier mediated transport of D‐glucose, L‐leucine and L‐lysine by guinea pig peritoneal macrophages was characterized. Analysis of the amino acid transport demonstrated two‐limbed double reciprocal plots suggesting two transport systems for each amino acid. The low concentration limb of the curves established a Km of 0.1 mM for L‐leucine and 0.05 mM for L‐lysine; Vmax values were 2.0 and 2.85 nmole/mg protein/90 seconds, respectively. Leucine and lysine were shown to be competitive inhibitors of each other. Further competition studies revealed that other amino acids also had affinity for these carriers. Amino acid transport was found to be sensitive to sulfhydryl active compounds. Colchicine treatment of peritoneal macrophages did not inhibit the transport of the amino acids tested. Preloading macrophages with latex beads or heat‐killed staphylocci by phagocytosis stimulated 2‐deoxy‐D‐glucose (2‐dOG) uptake markedly, but had no measurable effect on amino acid transport. Although total transport of 2‐dOG increased in post‐phagocytic macrophages, the kinetics of the system were not altered significantly. The Km for both pre‐ and post‐phagocytic transport of 2‐dOG was shown to be 1.2 mM and the Vmax was shown to increase from a pre‐phagocytic value of 20 nmoles/mg protein/90 seconds to a post‐phagocytic 27 nmoles/mg protein/90 seconds. Phagocytosis of heat‐killed staphylococci by guinea pig polymorphonuclear leukocytes (PMNs), however, did not cause an augmentation in hexose transport in the cells. The presence of colchicine during phagocytosis did not alter subsequent uptake of amino acids by the macrophages.
Transformed BHK cells exhibiting normal or subnormal sugar uptake
Authors: Moolten F. L. , Moolten D. N. , Capparell N. J.
Published: February 2005

The uptake of deoxyglucose was compared in BHK cells and in DMN4B cells, a conditionally transformed line of BHK cells which exhibits transformed behavior at 38.5° but not at 32°. At 32°, DMN4B cells took up deoxyglucose more slowly than BHK cells, reflecting a higher Km for uptake of this sugar. When both cell lines were grown at 38.5°, the Km for DMN4B cells was reduced to a level only slightly greater than for BHK cells, and deoxyglucose uptake became similar in the two cell lines. Growth in glucose‐free medium for 22 hours stimulated deoxyglucose uptake in both BHK and DMN4B cells; under these conditions, uptake was equal in the two cells lines, both at 32° and 38.5°. Glycolysis, as measured by lactic acid production, was slower in DMN4B than BHK cells, but in contrast to deoxyglucose uptake, this difference was observed at 38.5° rather than 32°. The observation that the subnormal deoxyglucose uptake of DMN4B cells in the untransformed state (32°) can be normalized by growth at 38.5°, a temperature permissive for transformation, suggests that membrane changes facilitating sugar uptake, which have been found in other transformed cells, are associated with transformation in DMN4B cells as well. However, the failure of uptake to exceed normal in these cells indicates that their transformed behavior is not attributable to excessive sugar uptake per se.
Differences in surface membrane ecto‐ATPase and ecto‐AMPase in normal and malignant cells I. Decrease in ecto‐ATPase in myeloid leukemic cells and the independent regulation of ecto‐ATPase and ecto‐AMPase
Authors: Weiss Benny , Sachs Leo
Published: February 2005

The hydrolysis of ATP and AMP by enzymes located on the external side of the plasma membrane (ecto‐ATPase and ecto‐AMPase) was studied in mouse myeloid leukemic cells, normal early myeloid cells, and normal mature granulocytes and macrophages. Nine clones of myeloid leukemic cells were used belonging to three groups that differ in their ability to be induced to differentiate by the differentiation‐inducing protein MGI. These three groups consisted of MGI+D+ that can be induced to undergo complete differentiation, MGI+D+ that can be induced to partially differentiate and MGI−D− with no induction of differentiation. The ecto‐ATPase activity of normal early myeloid cells was similar to that of normal mature granulocytes and macrophages and higher than that of any of the leukemic cells. Among the leukemic cells, the MGI−D− cells had the highest level of ecto‐ATPase activity.

The behaviour of ecto‐AMPase differed from that of ecto‐ATPase. Some MGI−D− clones had a higher ecto‐AMPase activity than normal cells and MGI+D− and MGI+D+ cells showed no detectable activity. Neither the ecto‐ATPase nor ecto‐AMPase activities changed after induction of differentiation in normal early myeloid or MGI+D+ leukemic cells. The results indicate that the myeloid leukemic cells had a decreased ability to hydrolyse external ATP, that there can be an independent regulation of ecto‐ATPase and ecto‐AMPase and that neither of these enzyme activities changed during differentiation.
Identification of a chromosome that controls malignancy in chinese hamster cells
Authors: Bloch‐Shtacher Navah , Sachs Leo
Published: February 2005

A chromosome that controls malignancy in Chinese hamster cells has been identified by analysis of the Giemsa banding pattern of a malignant cell line transformed by simian virus 40 (SV40), non‐malignant revertants from this line, segregants from the revertants that were again malignant and a cell line transformed by methylcholanthrene. The malignant cell line transformed by SV40 was near diploid and had gained additional material of chromosome 3. Revertants with a suppression of malignancy and malignant segregants from these revertants were near triploid. The malignant segregants also showed a gain of material of chromosome 3 compared to the non‐malignant revertants from which they were derived. Malignancy of these cells was associated with the ability to form colonies in agar. Cells of a line transformed by methylcholanthrene were malignant, formed almost no colonies in agar and the only chromosome change from the normal diploid chromosome banding complement was the addition of a long arm of chromosome 3. The results indicate that chromosome 3 carries gene(s) that control malignancy in Chinese hamster cells in cell lines transformed by a viral or a chemical carcinogen and that malignancy was induced in both cell types by an increase of these genes.
Variation in potassium transport properties of mouse 3T3 cells as a result of subcultivation
Authors: Tupper Joseph T.
Published: February 2005

Unidirectional potassium influx and the fraction of this influx sensitive to ouabain, an inhibitor of the (Na + K) activated ATPase, have been evaluated as a function of subcultivation of the 3T3 and SV40 transformed 3T3 cell. Total and ouabain‐sensitive K influx change little over approximately 50 passages of the transformed 3T3 cell. In contrast, these components of K influx increase nearly 5‐fold over a similar number of passages of the 3T3 cell. During early passages total and ouabain‐sensitive K influx of the 3T3 cell are below that of the SV40 3T3 cell on a per cell volume basis. At later passages the magnitude of these components of K transport exceed those found in the SV40 3T3 cell. Previous studies have reported the ouabain‐sensitive uptake of K and the levels of (Na + K) activated ATPase as being higher, lower or equivalent in the 3T3 versus transformed 3T3 cell. The present data suggest these differences may result from the degree to which the cells were passaged at the time of the experiments. Evaluation of previous studies substantiates this conclusion.
Altered biochemical properties of mitochondria in mouse mammary epithelial cells during primary culture
Authors: White Michael T. , Nandi S.
Published: February 2005

Various biochemical properties of mitochondria isolated from primary monolayer cultures of mammary epithelial cells from mid‐pregnant or hormonally stimulated mice were examined daily for seven or eight days. When compared with mitochondria from mammary glands of mid‐pregnant animals, the specific activities of several mitochondrial enzymes were greatly reduced in cells after seven days in culture. There was a 5‐ to 6‐fold reduction in the specific activities of cytochrome oxidase, succinate dehydrogenase and α glycerophosphate oxidase while malate dehydrogenase and adenylate kinase activities were 2‐ to 3‐fold lower. The reduction in mitochondrial enzyme activities was gradual and related to the length of time the cells were in culture. Progressive changes were also seen in the electrophoresis profiles of mitochondrial proteins in SDS‐urea polyacrylamide gels. Mitochondria isolated from 1‐, 2‐, 3‐ and 8‐day cell cultures showed a continuous reduction in the relative amounts of several mitochondrial polypeptides in the gel profiles. Addition of 35S‐methionine to cell cultures for short and long periods indicated that mitochondrial protein synthesis continued throughout the 8‐day culture period. However, the synthesis of several particular polypeptides was reduced progressively during the culture period. These studies indicate that mouse mammary epithelial cells have a lowered capacity for respiratory metabolism as a result of specific mitochondrial alterations which might be associated with the general loss of differentiated morphology by those cells during monolayer culture.
Embryonic diapause in the marsupial Macropus eugenii. Stimulation of nuclear RNA polymerase activity in the blastocyst during resumption of development
Authors: Moore G. P. M.
Published: February 2005

During the breeding season from January to June, female wallabies which are suckling a young animal in the pouch may carry a dormant embryo (lactational quiescence). Removal of the pouch young during this period results in a resumption of embryo development. In the latter half of the year, the embryo will not reactivate after removing the suckling young (seasonal quiescence). In this situation, development resumes spontaneously in late December or may be induced prematurely by progesterone treatment. The response of the genome of quiescent macropod blastocysts was studied during the early period of growth. Changes in the transcriptional activity of the embryo cells were measured by assay for endogenous RNA polymerases. Embryos actively synthesized RNA during both lactational and seasonal quiescence. Termination of seasonal quiescence resulted in increases in RNA polymerase activities within the nucleolus and nucleoplasm of the cell. This occurred on the day following the summer solstice, December 22, 1974, in animals captured in the wild, or within 48 hours of administration of progesterone. Embryos which were induced to resume development during the breeding season also showed increases in nucleolar and nucleo‐plasmic polymerase activities within five days of removal of suckling young from the pouch. In all situations, the response of the nucleolar enzymes was greater than that of the nucleoplasmic enzymes. This is in agreement with other observations of the regulation of gene activity in growth‐stimulated cells.
ATP, trehalose, glucose and ammonium ion localization in the two cell types of Dictyostelium discoideum
Authors: Wilson Jeanne B. , Rutherford Charles L.
Published: February 2005

Ultra‐microfluorometric techniques were adapted to follow several compounds related to energy metabolism through the developmental cycle of Dictyostelium discoideum. Each compound (ATP, trehalose, glucose, and ammonium ion) was found to be present in stalk and/or spore cells. The accumulation of NH4+ was interpreted as an indication of protein degradation, a source of energy in this organism. During the early stages of differentiation NH4+ was localized only in prestalk cells. However, it accumulated in spore cells during culmination such that levels were comparable in the two cells types by the end of development. Trehalose, an energy source for germinating spores, was found in both cell types but was preferentially degraded in stalk cells late in development. Glucose, the degradation product of trehalose, was localized in prestalk cells and varied inversely with trehalose levels. ATP was not localized in a specific cell type during development. However, ATP declined in stalk cells at an earlier stage of development.
The role of butyrate in the reverse transformation reaction in mammalian cells
Authors: Storrie Brian , Puck Theodore T. , Wenger Leonor
Published: February 2005

The reverse transformation reaction of Chinese hamster ovary cells from compact, epithelial‐like, randomly growing, heavily knobbed, lectin reactive cells into stretched, tighly adherent, smooth‐surfaced, lectin resistant, fibroblast‐like cells normally elicited by dibutyryl cAMP can be produced to its complete extent by N6‐monobutyryl cAMP or 8‐bromo‐cAMP. O2‐monobutyryl cAMP is ineffective as is cAMP itself in the absence of an inhibitor of phosphodiesterase activity. In the presence of a phosphodiesterase inhibitor, cAMP is fully effective. These results indicate that the role of the butyryl groups of dibutyryl cAMP and, especially, the N6‐butyryl, in the reverse transformation raction is protection of the cAMP analogue from degradation.

Butyrate at concentrations of about 1 mM does produce a response which to some extent mimics that of cAMP analogues. The cells, however, fail to assume a fibroblastic‐like shape, but rather become flattened. The butyrate effect is much slower and less readily reversible than that evoked by cAMP analogues. Butyrate produces an approximately 2‐fold increase in intracellular cAMP levels. These results are consistent with the hypothesis that butyrate effects, in part, are mediated by cAMP.
Inhibition of spermidine and spermine synthesis leads to growth arrest of rat embryo fibroblasts in G1
Authors: Rupniak H. T. , Paul D.
Published: February 2005

Following growth stimulation of rat embryo fibroblast (REF) cells previously arrested in G1 by serum deprivation, there occurs a large increase in the synthesis of the polyamines putrescine, spermidine and spermine. Methylglyoxal bis(guanylhydrazone) (MGBG), a potent inhibitor of S‐adenosylmethionine decarboxylase can block the accumulation of both spermidine and spermine over a period of several days. Under such conditions REF cells treated with MGBG will approximately double in number and then become growth‐arrested again predominantly in the G1 phase of the cell cycle. REF cells therefore appear to contain sufficient spermidine and spermine to progress through one cell cycle before the intracellular levels of these polyamines is reduced sufficiently to arrest growth in the absence of continued polyamine synthesis. Limitation of intracellular polyamine levels is therefore not the mechanism by which deprivation of serum growth factors arrests cell growth. While continued growth is nevertheless dependent on polyamine synthesis, this cell type is capable of limited proliferation in its absence. Addition of spermidine or spermine to MGBG‐arrested REF cells results in a rapid resumption of proliferation demonstrating that either polyamine can fulfill the role played by these polyamines in the growth process. Low levels of spermidine and spermine therefore arrest this cell type at a resriction point in G1 at which it is decided whether the intracellular level of these polyamines is sufficiently high to enable a cell to enter into and complete a new cell cycle. This polyamine‐sensitive restriction point is considered to be analogous to the restriction point(s) in G1 at which serum and nutrient limitation act.
Isolation of high‐rate DNA synthetic cells by Con A chromatography
Authors: Wallenbrock Mark A. , Killion Jerald J.
Published: February 2005

The separation of L1210 cells with columns of Con A‐derivatized nylon was investigated. Most of the cells bound to the column irreversibly. The binding was lectin‐specific. Cells were pulse labeled with 3H‐thymidine and applied to Con A columns. Those cells not binding the columns were enriched in incorporated thymidine compared to the unseparated population. Data is presented which suggests that a small, synchronized fraction of cells synthesizing DNA at a high rate is reduced in Con A‐nylon affinity. It is proposed that L1210 cell DNA synthesis is not uniform in rate and that changes in this rate are related to changes in the ability of cells to bind Con A‐nylon.
Stimulation of DNA synthesis in human fibroblasts by thrombin
Authors: Pohjanpelto Pirkko
Published: February 2005

Earlier work showed that thrombin stimulates proliferation of human fibroblasts in serumfree medium. This work demonstrates (1) that thrombin has to be present during most or all of the G1 period to ensure maximal DNA synthesis, (2) that DNA synthesis increases about three hours later after thrombin than after serum treatment, (3) that both thrombin and serum activate transport of uridine, D‐2‐deoxy‐glucose and putrescine, (4) that thrombin is able to increase 3H‐thymidine incorporation also in SV40 transformed human fibroblasts, in HeLa cells and in two continuous monkey cell lines.
Epidermal growth factor induced membrane changes in 3T3 cells
Authors: Aharonov Aharon , Vlodavsky Israel , Pruss Rebecca M. , Fox C. Fred , Herschman Harvey R.
Published: February 2005

Epidermal growth factor (EGF) is a mitogen for Swiss 3T3 cells. Short incubation periods with physiological concentrations of EGF induced increased binding of Swiss 3T3 cells to Con A‐coated nylon fibers. This effect was not induced in an EGF non‐responsive 3T3 variant, in the transformed murine XC cells or in Swiss SV3T3 cells. The increase in Con A fiber‐binding seems to be specific for EGF, since it was not observed in response to insulin, prostaglandin F2α or a higher serum concentration, which also initiate cell division of confluent quiescent 3T3 cells.

EGF also reduced Con A‐mediated hemadsorption to 3T3, but had no effect on hemadsorption by the EGF non‐responsive 3T3 variant. There was no change in the number of Con A‐receptors on 3T3 cells after EGF treatment. Binding to WGA‐coated fibers and WGA‐mediated hemadsorption were not effected by preincubation with EGF.
Effect of low dose rate irradiation on the division potential of cells in vitro. IV. Embryonic and adult human lung fibroblast‐like cells
Authors: Macieira‐Coelho A. , Diatloff C. , Billard M. , Fertil B. , Malaise E. , Fries D.
Published: February 2005

Early and late passage human embryonic lung fibroblasts were compared with early passage adult lung fibroblasts with regards to their survival (number of population doublings), after low dose rate ionizing radiation. It was found that early passage embryonic cells are quite resistant to this type of radiation. Late passage embryonic and early passage adult fibroblasts are more sensitive to ionizing radiations. The results suggest that cell aging is accompanied by an increased sensitivity to low dose rate ionizing radiation and favor the idea that aging in vitro, expressed as a function of the fibroblast division potential, is correlated with aging in vivo.
The capping of lymphocytes and other cells, studied by an improved method for immunofluorescence staining of frozen sections
Authors: Bourguignon Lilly Y. W. , Tokuyasu K. T. , Singer S. J.
Published: February 2005

Experiments have been carried out on the capping by lectins and antibodies of surface receptors of mouse splenic T and B lymphocytes and other cells, in which the surface distribution of the lectin or antibody, and the intracellular distribution of myosin or actin, were determined on the same cells by a double fluorescence technique. For this purpose, a general method for intracellular staining was developed which is intended to preserve sensitive antigens and fragile ultrastructural elements. The method involves mild formaldehyde fixation of the cells or tissues, infusion with concentrated sucrose, rapid freezing, and the preparation of frozen sections thinner than 2 μm thickness. The immunofluorescent or other appropriate fluorescent reagents are then applied to the thawed section. In the present experiments, intracellular actin was detected using a fluorescent staining method based on the interaction of F‐actin with heavy meromyosin, while intracellular myosin was detected by an indirect immunofluorescence procedure. Our findings were that the formation of a cap by each of the lectins or antibody reagents was always accompanied by a concentration of myosin and actin directly under the cap. These and other results suggest that capping is an active process in which actin and myosin participate directly in the formation of all caps. This proposal carries important new implications for the molecular mechanism of capping.