bluuebunny/crossref_metadata_2025 huggingface dataset:
Contact inhibition of speed in 3T3 and its independence from postconfluence inhibition of cell division
Authors: Martz Eric
Published: February 2005

The average speed of nuclear translocation of 3T3 cells, recorded in a time‐lapse film of a perfused culture, was negatively correlated with the number of contacting cells, and, to a lesser degree, with the amount of a cell's perimeter in contact with other cells. When a cell was in contact with five or more other cells, its speed was reduced by 50%, on the average, although the variation in individual cell speed was considerable at each level of contact. A partial correlation analysis showed that any extracellular soluble factors governed by the local cell density had little or no effect on speed, relative to the prominent effect of the number of cell‐cell contacts, and hence that 3T3 cells display true contact inhibition of speed. This confirms the original demonstration by Abercrombie and Heaysman (1952), who studied chick embryo heart fibrpolasts. In our study, the relationships between average speed and age of the culture was such that a possible independent contribution of a time‐associated factor other than contact to the diminution in average speed, although not necessary to account for the data, could not be excluded.

The same intercellular contacts found to inhibit speed in this study were previously reported to cause no immediate prolongation of individual cell generation times, despite the fact that the filmed culture was undergoing so‐called “contact” inhibition of cell division. In the present study, moreover, no correlation was observed between the average speeds of individual cells and their generation times. Hence, postconfluence inhibition of cell division and contact inhibition of speed of cell movement seem to be independent phenomena.
The effect of environmental pH on the growth of normal and malignant cells
Authors: Eagle Harry
Published: February 2005

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.
Contact inhibition of division: Involvement of the electrical transmembrane potential
Authors: Cone Clarence D. , Tongier Max
Published: February 2005

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 Å 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.
The effect of helium and of hydrogen at high pressure on the cell division of Tetrahymena pyriformis W.
Authors: Macdonald A. G.
Published: February 2005

The rate of cell division of Tetrahymena growing in an observational high pressure vessel was measured at selected pressures of helium, hydrogen and at high hydrostatic pressure. Pressures greater than 100 atm reduced the rate of division, but the gases inhibited division to a lesser degree than pure hydrostatic pressure. Hydrogen's effect was distinguishable from that of hydrostatic pressure at 130 atm or more, while helium's effect appeared at 175 atm. These inert gases probably counteract the action of pressure by stabilising apolar pressure‐labile targets.
Dinoflagellate bioluminescence: A comparative study of invitro components
Authors: Schmitter Ruth E. , Njus David , Sulzman Frank M. , Gooch Van D. , Hastings J. W.
Published: February 2005

In vitro bioluminescence components of the dinoflagellates Gonyaulax polyedra, G. tamarensis, Dissodinium lunula, and Pyrocystis noctiluca were studied. The luciferases and luciferins of the four species cross‐react in all combinations. All of these species possess high‐molecular weight luciferases (200,000–400,000 daltons) with similar pH activity profiles. The active single chains of luciferases from the Gonyaulax species have a MW of 130,000 while those from P. noctiluca and D. lunula have a MW of 60,000. Extractable luciferase activity varies with time of day in the two Gonyaulax species, but not in the other two. A luciferin binding protein (LBP) can easily be extracted from the two Gonyaulax species (MW ˜ 120,000 daltons), but none could be detected in extracts of either D. lunula or P. noctiluca. Scintillons are extractable from all four species, but they vary in density and the degree to which activity can be increased by added luciferin. Although the biochemistry of bioluminescence in these dinoflagellates is generally similar, the observations that D. lunula and P. noctiluca apparently lack LBP and have luciferases with low MW single chains require further clarification.
Glucose metabolism of mammalian erythrocytes
Authors: Harvey John W. , Kaneko J. J.
Published: February 2005

Under physiological conditions, erythrocytes of the horse metabolized 638 ± 37 (± SE) nmoles glucose/ml cells/hr at 37°C compared to 942 ± 31 for the cat, 1,329 ± 44 for the dog, and 1,485 ± 43 for man. On an absolute basis, pentose phosphate metabolism was similar between species, with species differences in erythrocyte glucose utilization attributable to differences in Embden‐Meyerhof pathway metabolism. By examining pentose phosphate pathway recycling, it appears that some functional compartmentation exists within erythrocytes.
Magnesium deprivation reproduces the coordinate effects of serum removal or cortisol addition on transport and metabolism in chick embryo fibroblasts
Authors: Rubin H.
Published: February 2005

A variety of unrelated effectors stimulate or inhibit coordinately the same array of metabolic reactions in chick embryo fibroblasts, including the uptake of 2‐deoxy‐D‐glucose and uridine, and the incorporation of uridine and thymidine into acid insoluble material. The coordinate inhibition of these reactions by omission of serum or addition of cortisol is reproduced quantitatively by lowering the concentration of magnesium (Mg2+) in medium containing 0.2 mM Ca2+. The response times for the utilization of uridine and thymidine following the removal or addition of Mg2+ are similar to those which follow removal or addition of serum. The effect of serum on the incorporation of choline, which is not part of the coordinate response to unrelated effectors, is not reproduced by varying Mg2+ concentrations. The results support the hypothesis that the availability of Mg2+ within the cell plays a central role in the coordinate control of transport, metabolism and growth by external physiological effectors.
Isolation of UV‐sensitive clones from mouse cell lines by lederberg style replica plating
Authors: Kuroki Toshio , Miyashita Shigeyo Y.
Published: February 2005

Seven UV‐sensitive clones were isolated from the mouse cell lines, FM3A and L5178Y, using Lederberg style replica plating. The UV‐sensitive clones were found at a frequency of 1 in 886 clones of FM3A and of 6 in 420 clones of L5178Y, after treatment with N‐methyl‐N′‐nitro‐N‐nitrosoguanidine, but no UV‐sensitive clones were obtained in the untreated controls. The Do values of freshly isolated clones were 10 to 21 ergs/mm2 (mainly 10 to 13 ergs/ mm2), while the original FM3A and L5178Y lines had Do values of 32 ergs/mm2. The UV‐sensitive clones were found to be rather stable for a period ranging from two to eight months after isolation. However, later some of them tended to lose their UV‐sensitivity, in terms of increase in Do values or n values. More stable UV‐sensitive subclones could be isolated from these populations by cloning using the replica plating method. Caffeine was found to potentiate the lethal action of UV‐irradiation on the UV‐sensitive clones as well as on the original FM3A cells.
Resistance of teratocarcinoma stem cells to infection with simian virus 40: Early events
Authors: Swartzendruber Douglas E. , Friedrich Thomas D. , Lehman John M.
Published: February 2005

Multipotential stem cells of a murine teratocarcinoma are resistant to typical infection with either polyoma virus (PV) or Simian virus 40 (SV40). Differentiated progeny of the stem cells are susceptible to infection in a manner identical to other mouse somatic cells, i.e., they are permissive for PV and nonpermissive for SV40. The early interactions between the stem cells (embryonal carcinoma or EC cells) and SV40 and PV were studied. Virions adsorbed to and penetrated into the cytoplasm and nucleus of EC cells, but did not induce expression of T antigen in the EC nuclei. Purified SV40 DNA was capable of inducing T antigen in differentiated teratocarcinoma cells but not in EC cells. Virus could not be rescued from EC cells previously exposed to SV40. The resistance of the stem cells to infection apparently involves a block in the infectious cycle after adsorption and penetration but before T antigen induction.
The stimulation of DNA synthesis by cytochalasin B in proliferative epidermal and dermal cells
Authors: Rothberg Simon , Nancarrow Grace E. , Church Virgene L.
Published: February 2005

Cytochalasin B influences a variety of cellular events that are associated with the contractile microfilament system and the formation of binucleate cells. Along with the formation of binucleate cells, cytochalasin B also causes an acceleration of cells from G1 to S in the cell cycle

By pulsing with cytochalasin B for 30 minutes and allowing for a previously established lag time (17.5 hours) a stimulation of thymidine incorporation into DNA of proliferative epidermal and dermal cells was found in both control and stripped epidermis

Autoradiographic analysis confirmed that the stimulation was due to an increased number of basal cells accelerated from G1 to S phase. A minimal number of binucleate basal cells, 1 in 300, was observed, which suggests that the stimulated synthesis is independent of binucleate cell formation. The amount of stimulation is maximum with cytochalasin B concentration pulse between 5γ and 30γ/ml

The results suggest a possible link in coupling cell membrane and surface events with subsequent increased cell nuclei synthetic activity.
Diamide effect on the hypertonic calcium uptake by rat red blood cells
Authors: Branca D. , Scutari G. , Siliprandi N.
Published: February 2005

Red blood cells of rat exhibit an enhanced hypertonic calcium uptake after incubation with diazenedicarboxylic acids bis (N,N‐dimethylamide) (diamide). Over the ranges reported in this paper the amount of membrane alteration is strongly and linearly dependent on the diamide concentration and on the osmolarity of the incubation medium. Treatment with 2,3‐dihydroxy‐1,4‐dithiolbutane (dithioerythritol or DTE), after diamide removal, restores red blood cells calcium intake to values similar to those of the control. The results indicate that the sinergic action of diamide and hypertonicity can oxidize some thiol groups essential for the cation barrier maintenance.
Mapping G1 phase by the structural morphology of the prematurely condensed chromosomes
Authors: Hittelman Walter N. , Rao Potu N.
Published: February 2005

The object of this study was to develop a map of G1 phase on the basis of the progressive changes taking place in the morphology of the prematurely condensed chromosomes as the cells traverse through G1 and then use this technique to determine the cell cycle location of normal and transformed cell populations in plateau phase. The morphology of the prematurely condensed chromosomes (PCC) of G1 cells in random populations was found to be highly variable. For a better understanding of the relationship between the morphology of the G1‐PCC and their position within G1 phase, synchronized populations of Chinese hamster ovary (CHO) cells in early, mid‐, and late G1 phase were fused with mitotic cells. Early G1 cells resulted in highly condensed G1‐PCC, while late G1 cells gave very extended G1‐PCC. Mid‐G1 cells resulted in PCC of intermediate condensation. To test the validity of these criteria for mapping the position of a cell in the cell cycle, synchronous G1 cell populations were treated with a variety of metabolic inhibitors. Cycloheximide and actinomycin D were shown to block cell in early G1 phase, while excess thymidine and hydroxyurea blocked cells in early S phase. The results presented here indicate that, upon reaching plateau phase, normal cell populations (BALB‐C mouse 3T3, human PA‐2, and WI 38) stop in early G1, while most cells in transformed cell lines (CHO, HeLa, and mouse SV‐3T3) accumulate in late G1.
Inhibition of glucose transport in human erythrocytes by benzylalcohol
Authors: Lacko L. , Wittke B. , Lacko I.
Published: February 2005

The inhibition of glucose transport by the non‐ionizable local‐anesthetic benzylalcohol is of the mixed type and independent of pH. The affinity of benzylalcohol to the free carrier is about three times larger than that to the carrier‐glucose complex.
The role of myonuclei in muscle regeneration: An in vitro study
Authors: Pullman William E. , Yeoh George C. T.
Published: February 2005

It is well established that during muscle regeneration, the satellite cells which are in a state of mitotic arrest, can initiate cell division to produce myoblasts which subsequently fuse to form myotubes. However, whether myonuclei, contained within damaged myotubes, or “freed” as a result of the trauma, play any role in muscle regeneration remains unresolved.

In myogenic cultures, it is possible to obtain renewed myogenesis when initial cultures are sub‐cultured. The aim of this study, was to obtain evidence of the participation by myonuclei of primary cultures in myogenesis which occurs subsequently in secondary cultures. In culture, myonuclei can be labelled with H3‐thymidine and their ultimate fate, either as “free” myonuclei or myonuclei associated with disrupted myotubes can be followed unequivocally. Three types of experiments are performed: (i) Primary myogenic cultures containing only myotubes are subcultured. (ii) Primary myogenic cultures containing myotubes with labelled myonuclei are disrupted and subcultured. (iii) Primary myogenic cultures containing myotubes with unlabelled myonuclei are mixed with labelled mononucleated myogenic cells and sub‐cultured.

In all instances no evidence of myogenesis from myonuclei is obtained. It is concluded that myonuclei, which were rendered postmitotic during myogenesis, remain so when muscle is disrupted and cannot re‐enter the mitotic cycle.
Uridine transport and phosphorylation in mouse cells in culture: Effect of growth‐promoting factors, cell cycle transit and oncogenic transformation
Authors: Rozengurt Enrique , Mierzejewski Karl , Wigglesworth Noel
Published: February 2005

The rapid increase in uridine uptake produced by the addition of serum to quiescent cultures of fibroblasts is primarily caused by an enhanced rate of nucleoside phosphorylation. While quiescent and serum‐stimulated cells display identical initial rates of transport, they show a considerable change in the composition of the acid‐soluble pools labelled with [3H] uridine for five seconds. The radioactivity recovered in the phosphorylated pools increases 2‐, 3‐, 4‐ and 6‐fold after addition of serum to cultures of Swiss 3T3 cells, tertiary mouse embryo fibroblasts, Swiss 3T6 and Balb 3T3, cells respectively. Furthermore, insulin, a growth factor isolated from medium conditioned by SV40 BHK cells (FDGF) and epidermal growth factor (EGF) also stimulate uridine phosphorylation within minutes.

The initial rate of uridine uptake is 2‐ to 3‐fold faster in rapidly growing normal and Simian virus 40 or polyoma virus transformed 3T3 cells as compared to untransformed 3T3 cells in the quiescent state. When quiescent cultures of 3T3 or mouse embryo cells are stimulated to leave G1 and enter into DNA synthesis, transport increases several hours after addition of serum and apparently coincides with the S phase of the cell cycle. The results demonstrate that an increase in uridine phosphorylation is a rapid metabolic response elicited by growth‐promoting agents in a variety of cell types and that uridine transport and phosphorylation are independently regulated.
Karyology and tumorigenicity of a Simian virus 40‐transformed chinese hamster cell clone
Authors: Lehman John M. , Trevor Katrina
Published: February 2005

A pseudodiploid clone of Chinese hamster cells transformed in vitro with Simian virus 40 (SV40) was isolated from soft agar and injected into nude mice through three successive passages with a short in vitro cultivation between each animal inoculation. The original clone and the three subsequent tumor populations were characterized in regard to SV40 T antigen staining, modal chromosome number, and Giemsa‐banded karyotype. All tumor cell lines maintained the pseudodiploid mode, as well as the positive SV40 T antigen staining. Nonrandom chromosomal changes included loss of one of the X chromosomes, additions of abnormal variants of chromosomes No. 1 and No. 2, the appearance of unidentified marker chromosomes, and the loss of autosomes No. 5, No. 6, and No. 11. The deletion of one of the X chromosomes occurred with about the same frequency in all cell lines. Additions of abnormal chromosomes No. 1 and No. 2 tended to recur more often in the tumor cell lines than in the original clone. The appearance of marker chromosomes, as well as the loss of autosomes No. 5, No. 6, and No. 11 demonstrated a correlation with tumorigenicity. Yet, the three successive passages of the cells through the animal did not select for a tumor population with a single, homogeneous karyotype.
Cholera toxin stimulates division of 3T3 cells
Authors: Pruss Rebecca M. , Herschman Harvey R.
Published: February 2005

Cholera toxin was used in an attempt to inhibit epidermal growth factor stimulated 3T3 cell division. Instead, cholera toxin alone at low concentrations (10−10 M), was able to stimulate cell division and could augment EGF stimulated cell division. The mitogenic effect of cholera toxin can occur despite a dramatic increase in the intracellular levels of cAMP in 3T3 cells. Cholera toxin stimulated mitogenesis could not be mimicked by choleragenoid, the binding but inactive subunit of cholera toxin, or by other agents which elevate cAMP levels in 3T3 cells.
T98G: An anchorage‐independent human tumor cell line that exhibits stationary phase G1 arrest in vitro
Authors: Stein Gretchen H.
Published: February 2005

T98 and T98G are two related cell lines that were derived from a human glioblastoma multiforma tumor. T98G has almost twice as many chromosomes as T98, suggesting that it is a polyploid variant of T98. Three aspects of control of cellular proliferation were studied in T98 and T98G cells in comparison to WI‐38 normal human diploid cells. WI‐38 cells have the following properties: (1) they can undergo only a limited number of population doublings in vitro; (2) they cannot proliferate without anchorage; and (3) they become arrested in G1 phase under stationary phase conditions. T98 cells differ from normal cells in all three of these properties, as do many other transformed cell lines. However, the derivative of T98, namely T98G, expresses an unique combination of normal and transformed aspects of the control of cellular proliferation. T98G cells are like normal cells in that they become arrested in G1 phase under stationary phase conditions, yet they also exhibit the transformed characteristics of anchorage independence and immortality. Thus, T98G cells demonstrate that transformation to immortality and anchorage independence can exist without concomitant loss of the normal mechanism for G1 arrest in response to stationary phase conditions. This result supports the hypothesis that each of these three aspects of control of cellular proliferation can be altered independently. Partially transformed cell lines, such as T98G, should be useful for sorting out the biochemical changes associated with transformation in each of these aspects.
Repair and survival after UV in quiescent and proliferating Microtus agrestis cells: Different rates of incision and different dependence on DNA precursor supply
Authors: Collins Andrew R. S. , Johnson Robert T.
Published: February 2005

Cultured cells of Microtus agrestis, the common field vole, perform unscheduled DNA synthesis after UV irradiation. They respond to incubation with a DNA synthesis inhibitor (1‐β‐D‐arabinofuranosylcytosine) following UV in ways typical of cells capable of excision repair, with reduced survival and an accumulation of breaks in pre‐existing DNA.

Microtus cells irradiated with UV in a quiescent pre‐S‐phase state are more sensitive to UV than are proliferating cells, in terms of survival. Adding DNA precursors (deoxyribonucleosides), and—in the case of proliferating cells—growing in complete rather than dialysed serum, enhance UV survival.

Quiescent cells show a higher rate of endonucleolytic incision of DNA after UV than do proliferating cells.

The balance between incision (producing single‐strand DNA breaks) and repair DNA synthesis (leading to rejoining of breaks) is shifted by the addition of deoxyribonucleosides, which suggests that DNA precursor supply is a rate‐limiting factor in repair. The lower survival of quiescent cells (in the absence of added deoxyribonucleosides) may be due to insufficient precursor supply to meet the demands of the high incision rate.
Steroid hormone toxicity in human fibroblasts does not correlate with high affinity receptor content
Authors: Breslow J. L. , Epstein J. , Forbes G. B. , Fontaine J. H.
Published: February 2005

Human diploid skin fibroblasts derived from normal individuals and those with the testicular feminization syndrome (TFM) have been shown to be killed to the same degree by dihydrotestosterone in spite of the absence of high affinity cellular androgen receptors in the TFM fibroblasts. Furthermore, several different normal fibroblast strains from various anatomical sites all showed similar amounts of androgen‐induced cytotoxicity even though their respective receptor contents differed by as much as ten‐fold. These results suggest that steroid‐induced cytotoxicity in human fibroblasts in not correlated with receptor content, unlike murine lymphoid cells in which the receptor content has been shown to be closely related to their ability to survive hormone exposure.
Characteristics of concanavalin A‐resistant Chinese hamster ovary cells and certain revertants
Authors: Cifone M. A. , Hynes R. O. , Baker R. M.
Published: February 2005

Clones of Chinese hamster ovary (CHO) cells were isolated by single‐step selection for resistance to killing by Concanavalin A (ConA) and certain cellular and membrane properties were examined. The ConA‐resistant isolates were only about 2‐fold more resistant than wild type cells to the selecting lectin, but exhibited pleiotropic temperature‐sensitivity for growth, markedly altered morphology and adherence, and significant differences in susceptibility to other agents such as colchicine. Two revertants to full temperature‐resistance were isolated from different ConA‐resistant mutants. One revertant clone had reacquired wild type sensitivity to ConA while the other revertant remained ConA‐resistant. The two series of wild type, ConA‐resistant, and temperature revertant clones were analyzed for altered mobility of cell surface glycoproteins using lactoperoxidase/125I and galactose oxidase/[3H]borohydride labelling procedures. The ConA‐resistant clones showed increased mobility on polyacrylamide gels of three classes of labelled proteins, in the molecular weight ranges 225,000, 200,000, and 130,000 daltons. These changes persisted in the temperature‐revertant that remained ConA‐resistant, while two of the altered protein classes were restored to wild type mobility in the revertant that regained ConA‐sensitivity. Cell hybridization experiments indicated that the temperature‐sensitive phenotypes of different ConA‐resistant isolates are recessive and noncomplementing, implying that the same gene is affected in each case. The reversions to temperature resistance appear to be recessive suppressor mutations in different genes.
Tuftsin‐macrophage interaction: Specific binding and augmentation of phagocytosis
Authors: Bar‐Shavit Zvi , Stabinsky Yitzhak , Fridkin Mati , Goldman Rachel
Published: February 2005

The binding of [3H]tuftsin to normal and in vivo stimulated mouse peritoneal macrophage populations was studied at 22°C. The [3H]tuftsin binding to thioglycollate‐stimulated macrophages was shown to be rapid and saturable, with an equilibrium dissociation constant (KD) (calculated from a Scatchard plot) of 5.3 × 10−8 M. The calculated number of binding sites per macrophage amounts to approximately 72,000. Binding competition studies with unlabelled tuftsin yielded a KD of 5.0 × 10−8 M. [3H] [N‐Acetyl‐Thr1]tuftsin, an inactive analog of tuftsin, failed to bind specifically to thioglycollatestimulated macrophages. [N‐Acetyl‐Thr1]tuftsin and the tripeptide [Des‐Arg4]tuftsin failed to compete for tuftsin binding sites, while [D‐Arg4]tuftsin, an analog with small tuftsin‐like activity, exhibited a low degree of inhibition of [3H]tuftsin binding. Thus a rather high degree of specificity is involved in the binding of the tetrapeptide.

Normal as well as six different macrophage populations induced by stimulation with thioglycollate, concanavalin‐A, starch, mineral oil, glucan and Bacillus Calmette Guerrin (BCG), exhibited a similar degree of binding of [3H]tuftsin. Corynebacterium parvum (CP)‐stimulated macrophages, on the other hand, showed a 6‐ to 10‐fold‐lower capacity for tuftsin binding.

See Note added in proof on p. 62.

Under similar experimental conditions, mouse fibroblast and lymphocyte preparations revealed no detectable specific binding.

Tuftsin augmented the phagocytic response of normal and stimulated macrophages assessed both for phagocytosis mediated via the Fc‐receptor and via non‐specific receptors. CP‐stimulated macrophages did not exhibit an increased phagocytic response upon treatment with tuftsin.
Long acting cAMP analogues enhance sulfate incorporation into matrix proteoglycans and suppress cell division of fetal rat chondrocytes in monolayer culture
Authors: Miller R. P. , Husain M. , Lohin S.
Published: February 2005

The relationship between replication and the synthesis of matrix sulfated proteoglycans was investigated with fetal rat chondrocytes grown in monolayer culture. The effect of N6 O2′ dibutyryl adenosine 3′, 5′ cyclic monophosphate (DBcAMP), adenosine 3′, 5′ cyclic monophosphate (cAMP), 8 Bromo adenosine 3′, 5′ cyclic monophosphate (8 Br‐cAMP), sodium butyrate and hydroxyurea was examined. Between 0.05 and 0.5 mM DBcAMP, a dose related inhibition of cell division and stimulation of [35SO] incorporation into matrix proteoglycans was demonstrated. At the higher concentrations of DBcAMP, cell division was completely inhibited and the enhancement of [35SO] incorporation into matrix proteoglycans ranged between 40 and 120% (P < 0.01). Utilizing 14C‐glucosamine and photometric determination of proteoglycans with Alcian Blue, it was demonstrated that the increase in sulfate incorporation reflected enhanced accumulation of extracellular matrix. The effects of DBcAMP were mimickled by 8 Br‐cAMP, suggesting they were mediated by the adenylyl cyclase system. cAMP (0.05‐0.5 mM), sodium butyrate (0.1‐0.5 mM) and hydroxyurea (0.5‐5 mM) partially or fully inhibited cell division, but either failed or only slightly enhanced sulfate incorporation. The enhanced sulfated proteoglycan deposition promoted by DBcAMP began 8 to 12 hours after serum stimulation, its onset occurred prior to thymidine incorporation and the effect persisted for 28 hours. Determination of cell volume demonstrated an increase in size of DBcAMP treated chondrocytes between 8 and 12 hours, coincident with the onset of increased sulfate incorporation. These results are consistent with a model where matrix sulfated proteoglycan deposition by chondrocytes is mediated by intracellular cAMP levels and occurs in the G1 phase of the cell cycle.
Postnatal development of peroxisomal and mitochondrial enzymes in rat liver
Authors: Krahling Jeffrey B. , Gee Robert , Gauger John A. , Tolbert N. E.
Published: February 2005

Subcellular organelles from livers of rats three days prenatal to 50 weeks postnatal were separated on sucrose gradients. The peroxisomes had a constant density of 1.243 g/ml throughout the life of the animal. The density of the mitochondria changed from about 1.236 g/ml at birth to a constant value of 1.200 g/ml after two weeks.

The peroxisomal and mitochondrial fatty acid β‐oxidation and the peroxisomal and supernatant activities of catalase and glycerol‐3‐phosphate dehydrogenase were measured at each age, as well as the peroxisomal core enzyme, urate oxidase, and the mitochondrial matrix enzyme, glutamate dehydrogenase. All of these activities were very low or undetectable before birth. Mitochondrial glutamate dehydrogenase and peroxisomal urate oxidase reached maximal activities per g of liver at two and five weeks of age, respectively.

Fatty acid β‐oxidation in both peroxisomes and mitochondria and peroxisomal glycerol‐3‐phosphate dehydrogenase exhibited maximum activities per g of liver between one and two weeks of age before weaning and then decreased to steady state levels in the adult. Peroxisomal β‐oxidation accounted for at least 10% of the total β‐oxidation activity in the young rat liver, but became 30% of the total in the liver of the adult female and 20% in the adult male due to a decrease in mitochondrial β‐oxidation after two weeks of age. The greatest change in β‐oxidation was in the mitochondrial fraction rather than in the peroxisomes. At two weeks of age, four times as much β‐oxidation activity was in the mitochondria as in the peroxisomal fraction. Peroxisomal glycerol‐3‐phosphate dehydrogenase activity accounted for 5% to 7% of the total activity in animals younger than one week, but only 1% to 2% in animals older than one week.

Up to three weeks of age, 85% to 90% of the liver catalase was recovered in the peroxisomes. The activity of peroxisomal catalase per g of rat liver remained constant after three weeks of age, but the total activity of catalase further increased 2.5‐ to 3‐fold, and all of the increased activity was in the supernatant fraction.
Membrane potential measurements during rat liver regeneration
Authors: Wondergem Robert , Harder David R.
Published: February 2005

Membrane potential was measured in perfused rat liver and was shown to increase from −33 ± 1.0 mV in livers from normal rats to −50 ± 1.1 mV in livers from rats 12 hr after partial hepatectomy. The hyperpolarization of the membrane in regenerating liver was no longer evident after perfusion with 1 mM ouabain for 5 min. Ouabain had a small (4 mV) depolarizing effect on membrane potential in normal liver. The potential measured in normal and regenerating liver decreased as a function of the external potassium concentration above 5 mM; however, the potential was more electronegative in regenerating liver compared to normal liver at all values of external potassium concentration, and the differences in potential between the two kinds of cells did not decrease at higher concentrations of external potassium. Thus, a plot of membrane potential vs external potassium concentration resulted in approximately parallel curves for the two different cell types. We conclude that hyperpolarization of the liver cell membrane is an early event during rat liver regeneration and results from an electrogenic Na‐K pump.
Pharmacological evidence for cell surface control of oral regeneration in Stentor coeruleus
Authors: Maloney Michael S.
Published: February 2005

This study suggests that membrane perturbations can affect oral morphogenesis in Stentor, possibly by a mechanism involving calcium ions. Exposure of regenerating Stentor to micromolar concentrations of the membrane active local anesthetics dibucaine, tetracaine, or procaine greatly delayed the progress of oral regeneration. In the case of tetracaine and dibucaine the greatest delays were observed in the early stages of regeneration prior to stage 4, when the majority of essential synthetic activity is occurring. The effects of dibucaine were generally readily reversible upon removal of the cells from the drug, with some residual effects occurring at higher dibucaine concentrations. Regenerating cells in the presence of dibucaine and excess extracellular calcium were not delayed, suggesting that the effects of dibucaine were reversible by calcium ions. The effects of tetracaine were not reversible by calcium ions, however. Exposure of regenerating cells to medium either lacking in, or containing an excess of, extracellular calcium had no effect on the time required to complete oral regeneration. The plant lectin, phytohemagglutinin, can also delay oral regeneration. The possible implications of these findings on the control of oral regeneration are discussed.
The growth requirements of SV40 virus transformed Balb/C‐3T3 cells in serum‐free monolayer culture
Authors: Rockwell G. A. , Sato G. H. , McClure D. B.
Published: February 2005

The growth requirements of SV40 transformed Balb/c‐3T3 cells have been studied in the absence of serum. For growth in serum‐free medium, the cells require (i) insulin, (ii) transferrin, and (iii) cis‐unsaturated fatty acids added in combination with fatty acid free bovine serum albumin. The growth rate, saturation density, and morphology of cells grown in this serum‐free medium are the same as those of cells grown in serum supplemented medium. This mixture also supports the growth of SV40 transformed Swiss‐3T3 cells and SV40 transformed primary mouse embryo cells, but does not support the growth of untransformed Balb/c‐3T3 cells.

The addition of fibronectin to this mixture allows routine subculture, repeated passage, and indefinite propagation of SV40 transformed Balb/c‐3T3 cells. Cells grown in this medium for a period of two months retain their ability to induce tumors when injected into athymic nude mice.
Formation of sulfhydryl groups in the culture medium by human diploid fibroblasts
Authors: Bannai Shiro , Ishii Tetsuro
Published: February 2005

When human diploid fibroblasts IMR‐90 are cultured in routinely used medium (Eagle's basal medium supplemented with 10% fetal calf serum), sulfhydryl compounds appear in the medium. The major component of these sulfhydryl compounds is cysteine, and it is shown that a part of medium cystine is converted into cysteine by the cells. It is also shown that the sulfhydryl groups of serum albumin, which are masked and barely detectable before the culture, are restored. Probably cysteine formed by the cells reacts with serum albumin to give rise to the protein sulfhydryl groups via sulfhydryl–disulfide exchange reactions. Total sulfhydryl concentrations in the medium are maintained in a considerable level throughout the culture, and a possible physiological function of these sulfhydryl groups is discussed.
Facilitated transport of uracil and 5‐fluorouracil, and permeation of orotic acid into cultured mammalian cells
Authors: Wohlhueter Robert M. , McIvor R. Scott , Plagemann Peter G. W.
Published: February 2005

The mode of permeation of uracil, 5–fluorouracil, and orotic acid into cells has been investigated in four established cell lines (Novikoff rat hepatoma, P388 mouse leukemia, mouse L, and Chinese hamster ovary cells) in attempts to assess the rate‐determining step(s) in their incorporation into the nucleotide pool and nucleic acids. Uracil and 5–fluorouracil shared a saturable transport system (Km = 5 to 15 mM) capable of rapid equilibration of these substrates across the cell membrane (t1/2 at 25 in first‐order range of concentration = 25 to 58 sec). Thus it seems unlikely that transport is limiting the incorporation hypoxanthine. Only the non‐ionized form of fluorouracil was a substrate for the transporter; exclusion of charged pyrimidines may explain why orotate was not a substrate at physiological pH. Orotate permeated the cell membrane much more slowly (t1/2 = 2890 to 6930 sec); its permeation was apparently non‐mediated and rate‐determining in the conversion of extracellular orotate to intracellular nucleotides.
Adhesion sites of murine fibroblasts on cold insoluble globulin‐adsorbed substrata
Authors: Murray Ben A. , Ansbacher Riva , Culp Lloyd A.
Published: February 2005

The attachment and detachment behavior of three mouse fibroblast cell lines adhering to plastic tissue culture substrata coated with the serum protein cold‐insoluble globulin (CIg) resembles that seen on the usual serumcoated substrata. The transformed cell line SVT2 spreads more extensively on the CIg‐coated than on the serum‐coated substratum, while the nontransformed Balb/c 3T3 line and concanavalin A‐selected “revertant” of SVT2 are equally well spread on both substrata. In all three cases, immunofluorescence microscopy using antibodies to CIg suggests that the cells are more tightly apposed to the CIg‐coated substratum than to the serum‐coated substratum.

Substrate‐attached material (SAM), which contains cell‐substratum adhesion sites and which is left after EGTA‐mediated detachment of cells, is enriched for cell surface fibronectin and glycosaminoglycans (GAG). When cells are seeded onto CIg‐coated substrata rather than serum‐coated substrata, there is an increased deposition of GAG but a comparable deposition of cellular proteins. The protein distribution of the two types of SAM are identical as analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, including fibronectin content. This indicates that substratum‐bound CIg cannot functionally substitute for cell surface fibronectin in these adhesion sites. Analysis of the GAG deposited on CIg‐coated substrata reveals that hyaluronate and the chondroitins are increased to a much greater extent than heparan sulfate; however, the ratio of hyaluronate to the various chondroitin species is invariant. These data provide further evidence that hyaluronate and the chondroitins are deposited in adhesion sites in well‐defined stoichiometric proportions, possibly as supramolecular complexes, and that CIg may mediate adhesion of cells in the serum layer by binding to GAG‐containing proteoglycans.
Effects of ouabain and ortho vanadate on transport‐related properties of the LLC‐PK1 renal epithelial cell line
Authors: Mullin James M. , Diamond Leila , Kleinzeller Arnost
Published: February 2005

Uptake of α‐methyl‐D‐glucoside (AMG) by LLC‐PK1 cells is inhibited by the uncoupler p‐trifluoro‐methoxyphenyl‐hydrazone (FCCP) and by the absence of extracellular Na+, indicating that the transport system is energy‐ and Na+ ‐dependent. We have previously demonstrated that transport of AMG by LLC‐PK1 cells proceeds against a concentration gradient and is phlorizin‐sensitive (Mullin et al., '80). Uptake of AMG was also inhibited by ouabain (OUA) but not by ortho‐vanadate (VAN). Rubidium uptake also was affected by OUA but not by VAN. VAN, however, caused collapse of the three‐dimensional domes of confluent LLC‐PK1 monolayers much more rapidly and thoroughly than OUA. Since domes are presumably dependent upon the Na+ pump, yet VAN is not acting on transport‐related functions of the OUA‐sensitive (Na+ + K+)‐ATPase, we hypothesize a direct effect of VAN on the water permeability of these cells. We also suggest that OUA does not act on these cells until domes collapse in normal course, and access of the OUA to the extracellular surface of antiluminal membranes is then achieved.
Construction of viable mouse‐human hybrid cells by nuclear transplantation
Authors: Hightower Margaret J. , Lucas Joseph J.
Published: February 2005

Viable interspecies cytoplasmic‐nuclear hybrid cells were constructed by fusion of karyoplasts prepared from the highly tumorigenic A9 mouse fibroblast cell line and cytoplasts prepared from the Detroit 532 normal human diploid cell strain. The identity of the hybrid cells was ascertained using a variety of morphological, immunological, and genetic criteria, including: nuclear pattern of staining with the fluorochrome Hoechst 33258, appearance of the actin‐myosin containing cytoskeleton, presence of fibronectin, and resistance to azaguanine and diphtheria toxin. About 90% of the hybrid cells were viable, that is, capable of division. Changes in the morphology of the hybrid cells, apparently nuclear directed, were observed before cell division occurred. Using the techniques described here, large numbers of interspecies hybrid cells suitable for many types of biochemical analyses can be routinely produced.
A hemopoietic cell line dependent upon a factor in pokeweed mitogen‐stimulated spleen cell conditioning medium
Authors: Hasthorpe Suzanne
Published: February 2005

A continously growing cell line (FMP1.1) has been isolated, which is dependent upon a factor in PWCM for both growth and survival. FMP1.1 appears to be a mast cell, since IgE receptors are present and their graunles react specifically with a mast cell granule stain. The factor in PWCM may be a glycoprotein and has biochemical properties in common with PWCM factors, which stimulate at least four other lineages of hemopoietic colonyforming cell. This line provides a pure population of cells as a model for studying molecular events of hemopoietic precursor cell growth and differentiation.
Shedding of mitotic cells from the surface of multicell spheroids during growth
Authors: Landry Jacques , Freyer James P. , Sutherland Robert M.
Published: February 2005

During the growth of EMT6/Ro mammary tumor multicell spheroids, a large number of cells are shed into the suspension medium. The rate of cell shedding was 218 cells per square millimeter of spheroid surface per hour, or up to 1.5% of the total spheroid cell content per hour. Shed cells had a clonogenic capacity equal to that of exonential monolayer cultures and were further characterized by volume distribution, mitotic index, flow cytoflurometry, and autoradiography. The results indicated that cells are released from the spheroid surface at mitosis, presumably due to a loosening of the cell‐to‐cell attachment during this cycle phase. These mitotic cells, when placed in monolayer culture, attached and grew synchronously with a cell cycle time of about 13 hours. Shed cells kept in suspension culture had a similar cell cycle time, but these cells reaggregated immediately after mitosis. The results indicated that cell shedding and reaggregation both occur near the time of mitosis and are intrinsic factors regulating the initiation and subsequent growth of multicell spheroids. Although these studies were done with spheroids cultured in vitro, shedding of mitotic cells may play an important role in the in vivo process of metastasis.
The accumulation of three specific proteins related to glucose‐regulated proteins in a temperature‐sensitive hamster mutant cell line K12
Authors: Lee Amy Shiu
Published: February 2005

A temperature‐sensitive mutant K12 isolated from an established line of Chinese hamster fibroblasts has been identified as a cell‐cycle mutant blocked in G1 (Roscoe et al., '73a, b). While characterizing the phenotype of K12 in more detail, we observed that K12 cells are highly sensitive to the concentration of glucose in the culture medium as well as the temperature of incubation. Although overall cellular metabolism remains intact for at least 24 hours after shifting the cells to the nonpermissive temperature (40.5°C), the incorporation of 3H‐leucine into three specific cellular proteins of molecular weights 94, 78, and 58 K daltons is greatly increased. We have analyzed the K12 proteins by two‐dimensional, isoelectric focusing (IEF) sodium dodecyl sulfate (SDS) gel electrophoresis and found that the set of proteins which is overproduced by K12 cells at 40.5°C is the same as that produced at 35.0°C in medium depleted of glucose or supplemented with 10 mM glucosamine. In addition, the increased synthesis of these proteins cannot be suppressed by the addition of N‐acetylglucosamine to the medium. The relation ship of these proteins to the glucose‐regulated proteins in the chick embryo fibroblast system (Shiu et al., '77) is discussed.
The biotin requirement of HeLa cells
Authors: Dakshinamurti K. , Chalifour L. E.
Published: February 2005

We have examined the effect of alterations in the biotin content of the medium on the growth, viability, biotin content, and the activities of biotin‐dependent and biotin‐independent enzymes of the HeLa cells. The inclusion in the growth medium of avidin, which almost irreversibly binds with biotin (Kd, 10−15 M), results in an increase in cellular biotin content and biotin enzyme activity over that seen when the cells are grown in a biotin‐depleted medium. The addition of avidin‐bound biotin to the growth medium led to a forty‐fold increase in cellular biotin when compared to the inclusion of an equivalent amount of free biotin in the medium. HeLa cells are able to internalize avidin‐bound biotin. Biotin is released from this complex to function as the prosthetic group of biotin enzymes. HeLa cells do have a nutritional requirement for biotin.
Cellular responses to stress: Comparison of a family of 71–73‐kilodalton proteins rapidly synthesized in rat tissue slices and canavanine‐treated cells in culture
Authors: Hightower Lawrence E. , White Fredric P.
Published: February 2005

Cultured rat embryo cells exposed to the L‐arginine analogue L‐canavanine rapidly accumulated a major 71 kilodalton polypeptide and several minor ones (110, 95, 88, and 78 kilodaltons). Canavanine‐treated cultures contained elevated levels of translatable mRNA encoding P71, and the stimulated synthesis of this protein was blocked by actinomycin D, suggesting that P71 is inducible. Rat embryo cells maintained under routine culture conditions synthesized only trace amounts of P71; however, they accumulated an abundant 73 kilodalton protein that was closely related to P71. No kinetic evidence of a precursor‐product relationship between P73 and P71 was found. The peptide map of P71 from cultured cells was identical to the map of proteins with the same electrophoretic mobility isolated from incubated slices of rat telencephalon. Previous studies (White, ′80a, b, c) have shown that the latter proteins are rapidly synthesized by cells associated with cerebral microvessels in incubated brain slices, but are not detectable in vivo. Herein we present evidence that the synthesis of P71 is not unique to brain slices. Incubated slices of heart, lung, thymus, kidney, spleen, and liver all accumulated an abundant 71 kilodalton size class. The peptide maps of P71 obtained from brain, heart, lung, and thymus tissue were similar. The stimulated synthesis of P71 in brain, heart, and lung slices was inhibited strongly by the addition of actinomycin D at the start of incubation. The 71–73 kilodalton proteins of canavanine‐treated rat embryo cells and incubated slices from seven different organs were compared in detail on two‐dimensional poly‐acrylamide gels. Eight charge variants were detected in extracts of lung, spleen, and thymus tissue, four in liver and heart, three in kidney, and two different pairs of variants in extracts of brain tissue and cultured cells. The possible significance of the rapid synthesis of a similar small set of proteins in tissue slices and cultured cells in response to a variety of physical, chemical, and biological stimuli is discussed in terms of cellular responses to traumatic injury and metabolic stress.