bluuebunny/crossref_metadata_2025 huggingface dataset:
Osmotic properties of a proliferating and differentiating line of cells from the bone marrow of the rat
Authors: Cicoria A. D. , Hempling H. G.
Published: February 2005

The effects of cellular differentiation on the osmotic properties of a proliferating line of bone marrow cells has been investigated. This population proliferated and differentiated into distinct maturation phases which have been separated quantitatively by velocity sedimentation at 1g in a Ficoll gradient.

Certain osmotic characteristics were evaluated. The volume of osmotically active water increased linearly with maturation. The osmotically inactive volume was variable during proliferation and maturation. Several correlations that concerned the exosmotic movement of water were noted, as follows: (1) membrane permeability to water increased with an increase in the diameter of the nucleus, (2) the ratio of osmotically active water to mean corpuscular volume increased with an increase in the diameter of the nucleus, (3) membrane permeability to water increased with an increase in the osmotically active water normalized to the mean cell volume, and (4) the heat of activation associated with the permeability of the membrane to water increased during maturation and varied inversely with the ratio b/MCV.

These results were used to assess the effects of cellular maturation on membrane function and the state of water in a maturing cell population. The permeability data suggest that charged groups on membrane proteins and phospholipids can vary the state of water in the membrane from a thermodynamically mobile state to an “ice‐like” state. These unusual properties may be a direct result of a dynamic and functional relationship between cellular water and active biological macromolecules.
Similarities between alkaline phosphatase and CA2+ ATPase activities in HeLa cells
Authors: Brahmacupta Prem , Melnykovych George
Published: February 2005

Parallel changes in the enzyme activities of Ca2+ ATPase and alkaline phosphatase were observed in HeLa cells. Both enzymes were inhibited to a similar degree by L‐phenylalanine, L‐tryptophan, and L‐leucine, while being relatively resistant to L‐homoarginine. Exposure to heat (56°C, 60°C, and 65°C) resulted in a loss of both enzyme activities. Both alkaline phosphatase and Ca2+ ATPase, when treated with EGTA, required Ca2+ for the restoration of activity. Cells grown in the presence of agents that affect alkaline phosphatase (dexamethasone, butyric acid, and hyperosmolar NaCl) showed similar changes in the activities of both enzymes.
Chemical and biological properties of a growth factor from human‐cultured osteosarcoma cells: Resemblance with platelet‐derived growth factor
Authors: Heldin Carl‐Henrik , Westermark Bengt , Wasteson Åke
Published: February 2005

A human osteosarcoma cell line, U‐2 OS, cultured under serumfree conditions, was shown to produce a growth factor (osteosarcoma‐derived growth factor, ODGF) for human‐cultured glial cells, fibroblasts, and other cells. ODGF, collected from the spent medium of 2 OS cultures, was purified by a sequence involving heparin‐Sepharose chromatography, hydrophobic chromatography, gel chromatography, and preparative gel electrophoresis in SDS. Purified ODGF, at a concentration of 3 ng/ml, elicited a mitogenic response in human glial cells equivalent to 50% of that afforded by human serum at a final concentration of 1%. The preparation was estimated to be > 50% pure.

The biological activity of ODGF resided in a cationic, relatively heat‐resistant, reduction‐susceptible protein with a molecular weight of 30,000 (by gel chromatography and SDS‐gel electrophoresis). The electrophoretic behaviour of radioiodinated ODGF suggested that the protein was composed of two different polypeptide chains (about 13,000‐14,000 and 16,000‐17,000 daltons, respectively) linked via disulphide bonds. The molecular makeup of ODGF was thus similar to that of platelet‐derived growth factor. 125I‐ODGF could be precipitated by an antibody to platelet‐derived growth factor, indicating that the two factors were immunologically related. Resemblance with platelet‐derived growth factor was also indicated by the finding that the latter (but not, e.g., fibroblast growth factor or epidermal growth factor) competed with 125I‐ODGF for binding to human‐cultured glial cells.
Decreased initiation of DNA synthesis in a temperature‐sensitive mutant of hamster cells
Authors: Eilen Eric , Hand Roger , Basilico Claudio
Published: February 2005

We have analyzed ongoing DNA replication in ts BN‐2, a dna− mutant of BHK‐21 cells (Nishimoto et al. '78). At the non‐permissive temperature of 39.5°C, inhibition of 3H‐thymidine into acid‐precipitable material begins 1 to 2 h after the cells are released from a block at the start of the S‐phase. The fraction of nuclei incorporating 3H‐thymidine is similar to that of wild‐type cells through the synchronized S‐phase of 8 h. Alkaline sucrose gradient analysis shows tht pulse‐labeled DNA from mutant cells is incorporated into high molecular weight material after 3 h at eithr the permissive or non‐permissive temperature. DNA fiber autoradiograms reveal that, at 39.5°C, the rate of replication fork movement is about 30% increased in the mutant as compared to wild‐type cells. In the mutant cells, however, the interval between adjacent initiation sites is increased and the relative frequency of initiation events is decreased at the restrictive temperature. The results indicate that there is a block to ongoing replication in ts BN‐2 at the level of initiation of synthesis on individual replication units; elongation of nascent chains is not inhibited.
Compartmentalized ATP pools produced from adenosine are nuclear pools
Authors: Rapaport Eliezer
Published: February 2005

Incubation of African green monkey kidney (BS‐C‐1) cells and mouse fibroblasts (3T6) in the presence of adenosine for 4 hours resulted in increases in the nuclear compartment pools of adenosine 5′‐triphosphate (ATP) and nuclear ATP/adenosine 5′‐diphosphate (ADP) ratios. Adenine and inosine, which yield increases in total cellular ATP pools and ATP/ADP ratios similar to those promoted by adenosine, do not produce similar increases in the nuclear compartment. Adenosine‐promoted increases in nuclear ATP pools were higher in the untransformed, serially propagated, BS‐C‐1 cells than in the spontaneously transformed 3T6 cells. Adenosine‐promoted compartmentalized ATP pools in primary chick embryo fibroblasts were reduced upon transformation of these cells with Rous sarcoma virus, resulting in free mixing of all the ATP pools synthesized from various salvage precursors. The growth regulatory properties of the nuclear compartment pools of adenine nucleotides is suggested by the big increases in nuclear ATPase and adenosine 5′‐monophosphate (AMP) demainase activities upon the entry of 3T6 cells into the S phase of their cycle. These enzymatic activities would tend to lower the nuclear ATP/ADP ratios and reduce the total adenine nucleotide pools in these nuclei respectively – conditions which were shown by earlier in vitro studies to be favorable to DNA replication.
Influence of proliferative rates and a system substrate availability on proline transport in primary cell cultures of the R3230AC mammary tumor
Authors: Gay Roger J. , Hilf Russell
Published: February 2005

Regulation of A system amino acid transport was studied in primary cultures of the R3230AC mammary adenocarcinoma. Higher rates of carrier‐mediated Na+‐dependent proline transport, vc, was decreased and was attributed to a two‐fold decrease in Vmax and a two‐fold increase in Km. When compared to cells grown in standard media (Eagle's minimal essential medium, MEM), cells grown in media supplemented with A system substrates (alanine, serine, glycine, and proline) demonstrated adaptive decreases in proline transport; the decrease was due to two‐fold reduction in Vmax, with no change in Km for proline. Even in the presence of preferred substrates for the A system, a density‐dependent decrease in proline transport was manifested. Both fast‐ and slow‐growing cultures maintained in MEM exhibited rapid increases in proline transport when switched to buffers devoid of amino acids; two‐fold increases in Vmax were seen within 4 hr, but Km was unchanged. This starvation‐induced adaptation was completely prevented by inclusion in the buffer of 10 mM proline, 0.1 mM ‐(methylamino)‐isobutyric acid (MetAIB) or 10 mM serine, whereas inclusion of the poorer A system substrate, phenylalanine (10 mM), had no effect. The effects of MetAIB to prevent starvation‐induced increases in proline transport were dose‐related, rapid, and reversible. Amino acid starvation‐induced increases in proline transport were partially blocked by cycloheximide or actinomycin D. Data were obtained demonstrating a temporal relationship between increasing intracellular [proline] and decreasing vc for proline uptake. In addition, efflux of proline from preloaded cells preceded the increase in initial rates of proline entry. Taken together, we concluded that: (1) A system transport in primary cultures of this mammary adenocarcinoma is regulated by cell density as well as by availability of A system substrates, but these two types of regulation are kinetically distinct; and (2) starvation‐induced enhancement of proline transport appears to be due to release from transinhibition, but may also involve a derepression‐repression type of mechanism.
Mammalian cells do not have a stringent response
Authors: Pollard J. W. , Lam T. , Stanners C. P.
Published: February 2005

A key attribute of the stringent response of bacteria is the rapid inhibition of ribosomal RNA synthesis mediated by unusual nucleotides in response to uncharged tRNA. The question as to whether mammalian cells show a stringent response analogous to that of bacteria was critically tested by the effective rapid amino acid starvation of both normal and transformed cells. Rapid starvation giving a high proportion of uncharged tRNA for leucine was produced within 7 minutes of expression of a nonleaky ts leucyl tRNA synthetase mutation in transformed CHO cells (tsH1) and in its normal growth control revertant (L‐73). To control for the effect of temperature alone, tsrevertants of tsH1 and L‐73 were included in the study, and to control for effects due simply to the inhibition of protein synthesis, the translational elongation inhibitor cycloheximide was used. In addition, rapid starvation for histidine was effected by incubation of both the CHO cell lines and of freshly explanted normal Chinese hamster embryo fibroblasts in histidine‐free medium containing high concentrations of histidinol.

The rate of preribosomal RNA synthesis and the extent of its maturation to mature rRNA was measured using (3H‐methyl) methionine as a donor of methyl groups during synthesis and methylation of pre‐rRNA. There was no effect on pre‐rRNA synthesis of the rapid generation of uncharged tRNA for 45 minutes for any of the cell types tested. A nonspecific inhibition of maturation of 18S rRNA and late (3 hour) inhibition of pre‐rRNA synthesis was observed, but could be mimicked by the inhibition of protein synthesis to comparable levels with cycloheximide. Less severe amino acid starvation resulting in a more physiological inhibition of protein synthesis to 30% also had no specific effect on pre‐rRNA synthesis and maturation.

Intracellular nucleotide pools were also examined for the appearance of unusual nucleotides such as guanosine tetraphosphate or pentaphosphate and for changes in the levels of normal nucleotides after severe amino acid starvation. No such changes could be detected.

We conclude that although mammalian cells may have some biochemical reactions which respond to uncharged tRNA, they do not possess a macromolecular control system analogous to the stringent response of bacteria.
Human dermal microvascular endothelial cells in vitro: Effect of cyclic AMP on cellular morphology and proliferation rate
Authors: Davison P. M. , Karasek M. A.
Published: February 2005

Macrovascular endothelial cells isolated from the human umbilical vein and microvessel endothelium from the newborn foreskin dermis differ in their requirements for optimal growth in vitor. In the presence of 5 x 10−4 M dibutyryl cyclic AMP (Bt2cAMP), human dermal microvessel endothelial cell proliferation rate increased to give a cell number of 203% of control values by day 10 in culture. The cells retained their characteristic endothelial cell morphology, reached confluence, and could be serially passaged. Cells grown in the absence of Bt2cAMP did not proliferate readily and grew in a disorganized pattern. The effect of Bt2cAMP on microvascular endothelial cell proliferation rate and morphology could be duplicated by cholera toxin (CT) used together with isobutyl methyl‐xanthine (IMX). These agents were found to elevate intracellular levels of cyclic AMP in microvascular endothelium over 40‐fold. Human umbilical vein cells in culture failed to respond to either Bt2cAMP or CT together with IMX.

The growth‐promoting effect of dibutyryl cyclic AMP (Bt2cAMP) on human foreskin dermal microvascular endothelium in vitro is in marked contrast to the lack of response of human umbilical vein cells. These results provide further evidence of differences in the mechanisms that regulate macro and microvessel endothelial cell proliferation in vitro.
A unitary cause for the exclusion of NA+ and other solutes from living cells, suggested by effluxes of NA+, D‐arabinose, and sucrose from normal, dying, and dead muscles
Authors: Ling Gilbert N. , Walton Cheryl L. , Ochsenfeld Margaret M.
Published: February 2005

1. The effluxes of labeled Na+, D‐arabinose, and sucrose from normal muscle and muscle poisoned with low concentrations of iodoacetate were studied. The procedure involved repeated loading with isotope, followed by washing of the same muscle while still normal and at different states of dying. 2. The rates of Na+ efflux in both the fast and slow fraction remained either quite constant or showed some unpredictable, minor fluctuations. This was true for both Na+ and the two sugars studied, confirming earlier conclusions that the steady levels of these solutes were not maintained by pumps. 3. In all cases studied, the efflux curves showed at least two fractions. It is the fast‐exchanging fraction that steadily and consistently increased in magnitude as the muscles were dying, until finally the concentration of solute in this fraction reached and sometimes surpassed the labeled solute concentrations in the original labeled solutions in which the muscles were equilibrated. The slow fractions showed only a transient increase or none at all. These observations show that it is the fast fraction that represents solute dissolved in cell water and rate‐limited by passage through the cell surface and that the partial exclusion of Na+ and the sugars have a unitary cause—a reduced solubility in the cell water which in the presence of ATP exists in the state of polarized multilayers.
The membrane potential of Ehrlich ascites tumor cells: An evaluation of the null point method
Authors: Smith Thomas C. , Robinson Susan C.
Published: February 2005

The effects of valinomycin (25 pM) on the membrane potential and on initial, passive Na+ and K+ movements have been determined in Ehrlich ascites tumor cells. The membrane potential of steady‐state cells in a physiologic environment was – 23.2 mV. Addition of valinomycin induced a small, significant hyperpolarization (Vm = –29.6 mV) when averaged over the population tested. However, analyses of the response of individual cells to valinomycin showed two different potential effects: (1) the majority of cells hyperpolarized after treatment; but (2) a significant fraction depolarized when exposed to valinomycin. The Vm of steady‐state cells incubated in saline with K+ at concentrations of 21 mM or 75 mM was – 21.4 mV and –22.0 mV, respectively. Addition of valinomycin to these cells was without effect on Vm, thus establishing the “null point” responses. Only for cells incubated in saline with a K+ of 75 mM was there agreement between Vm and K+ equilibrium potential (Vk). Determinations of cellular Na+ and K+ showed that valinomycin induced net losses of K+ and gains of Na+ by cells incubated in either physiologic saline or saline with a K+ concentration of 21 mM. However, the celular K+ of cells incubated in saline with a K+ concentration of 75 mM was unaltered by valinomycin. There was a two‐ to threefold increase in K+ permeability of the cell membrane in the presence of valinomycin. These results are consistent with the existence of two null points in the membrane‐potential response to valinomycin: One is established when the membrane potential corresponds to Vk; the second occurs when the effects of valinomycin on K+ loss from the cell are exactly offset by its inhibition of active Na+ + K+ transport.
Distribution of the inducer of differentiation bis‐acetyl‐diaminopentane in murine erythroleukemia cells
Authors: Tsiftsoglou Asterios S. , Bhargava Kuldeep K. , Rittmann Lana S. , Sartorelli Alan C.
Published: February 2005

Exposure of murine leukemia cells in culture to bis‐acetyl‐diaminopentane (BADP) caused erythroid maturation as measured by the accumulation of hemoglobin in treated cells. The appearance of differentiated cells in cultures exposed to BADP occurred 18 to 20 hours earlier than in those treated with dimethylsulfoxide (DMSO), a standard inducer of differentiation in this system. Studies with [3H]BADP indicated the occurrence of relatively rapid association of the inducer with cells, and subsequent linear accumulation. Fractionation of cellular components and measurement of radioactivity from BADP therein demonstrated that this agent preferentially associates with a fraction enriched for plasma membrane. In addition, [3H]BADP was capable of binding to the plasma membrane‐enriched fraction isolated from murine erythroleukemia cells as measured by gel filtration. These findings support the concept that interaction of inducers of murine erythroleukemia differentiation such as BADP with components of the surface membrane may be important in the cascade of events that lead to the erythroid maturation of these leukemic cells.
Inhibition of BALB/c‐3T3 cells in late G1: Commitment to DNA synthesis controlled by somatomedin C
Authors: Wharton Walker , Van Wyk Judson J. , Pledger W. J.
Published: February 2005

Methylglyoxal bis‐(guanylhydrazone) (mGBG) blocked the stimulation of DNA synthesis in quiescent, density‐inhibited BALB/c‐3T3 cells treated with platelet‐derived growth factor (PDGF) and platelet‐poor plasma (PPP). Competence formation produced by a transient exposure to PDGF was not effected by mGBG. In contrast, mGBG effectively inhibited the PPP‐stimulated progression of competent cells through the G1 phase of the cell cycle, although maximal inhibition was observed when mGBG was present during both the exposure to PDGF‐ and PPP‐supplemented media. When quiescent cells were treated with PDGF and PPP‐supplemented media in the presence of mGBG for 12–18 hours and the mGBG was then removed, cells entered the S phase after a 4 hour lag. The rate of entry into the S phase, but not the time necessary for the cells to progress from the mGBG block into the S phase, was dependent on the concentration of PPP present after removal of the mGBG. Either somatomedin C or insulin, but not epidermal growth factor, fibroblast growth factor, or PDGF were able to substitute for PPP in allowing cells to enter the S phase after the cells were released from the mGBG block. A marked inhibition of (3H)‐leucine incorporation in serum‐stimulated cultures was produced at mGBG concentrations which caused no decrease in the amount of (3H)‐uridine incorporated during a short (15 minute) pulse. The ability of hormones to allow cells to progress to the late G1 phase and become committed to DNA synthesis after a mGBG inhibition was not related to their ability to restore the normal rate of protein synthesis as determined by (3H)‐leucine incorporation.
Factors in the rat submaxillary gland that stimulate growth of cultured glioma cells: Identification and partial characterization
Authors: McClure D. B. , Ohasa S. , Sato G. H.
Published: February 2005

The effect of rat submaxillary extract on the growth of rat C6 glioma cells in serum‐free culture has been examined. Extracts (10–15 μg/ml) of submaxillary glands from both male and female rats markedly enhanced the growth of serum‐deprived C6 cells and, in combination with insulin, transferrin, and NIH‐LH (a source of fibroblast growth factor), were able to stimulate C6 cell growth to an extent comparable to that achieved with an optimal amount of fetal calf serum. The mitogenic activity of rat submaxillary extracts was found to be heat‐labile, acid‐stable, and partially inactivated by protease and 2‐mercaptoethanol. Under our assay conditions, biologically active preparations of purified mouse submaxillary gland epidermal growth factor (EGF) or nerve growth factor (NGF) were not mitogenic for C6 cells, nor was the mitogenic activity of rat submaxillary extracts inhibited by antiserum to these mouse submaxillary gland growth factors. These results suggest that the active component(s) of rat submaxillary extracts is unrelated to either EGF or NGF. The growth‐enhancing effect also appears unrelated to esteropeptidase activity present in these extracts since the mitogenic activity was unaffected by several protease inhibitors. Moreover, two purified mouse submaxillary gland arginylesteropeptidases, EGF‐binding protein and γ‐subunit of 7 S NGF, were unable to elicit a comparable growth response even when added to cell culture medium at unreasonably high concentrations.

The C6 cell mitogenic activity of crude submaxillary extracts could be separated into two biologically similar components by either gel filtration on Sephadex G‐100, preparative isoelectric focusing in a pH gradient of 3–10, or adsorption to DEAE‐cellulose followed by elution with a sodium chloride gradient. One of the active components was acidic in nature and had an apparent molecular weight of 40,000, while the other was near neutral in charge and possessed a molecular weight of approximately 20,000. The relationship between these two C6 cell mitogenic components and the rat submaxillary gland component responsible for stimulating Balb/c‐3T3 cell growth in serum‐free, factor supplemented medium (McClure et al., 1979, J. Cell Biol. 83: 96a) is also discussed.
The role of respiration in tumor cell transition from the noncycling to the cycling state
Authors: Olivotto Massimo , Paoletti Francesco
Published: February 2005

Resting Yoshida AH130 hepatoma cells, harvested at the plateau of tumor development in vivo, were recruited into the cycling state following transfer to an in vitro system whereby these cells were incubated in the autologous ascites plasma diluted with buffered saline and enriched with glucose. In this system, cell recruitment into the phase of DNA synthesis (S phase) strictly depends on the activity of the respiratory chain and is abolished by anaerobiosis as well as by antimycin A, although the intracellular levels of ATP and the rate of protein synthesis are practically unaffected by these treatments. Furthermore, 2,4‐dinitrophenol, at concentrations which uncouple the respiratory phosphorylation and hence enhance both glycolysis and oxygen consumption, does not hinder cell promotion into S phase. Thus, the absolute respiration dependence of cycling resumption by resting ascites cells does not seem to rely on respiratory ATP supply, but rather is linked to the electron flow through the respiratory chain.
Further studies of hormone‐sensitive sodium and potassium transport in red cells from developing chick embryos
Authors: Shanbaky Nabila M. , Wacholtz Mary C. , Sha'Afi Ramadan I.
Published: February 2005

Sodium and potassium transport in the definitive series of chick embryo red cells changes significantly, both qualitatively and quantitatively, during maturation. Sodium efflux and potassium influx consist of three parts: a ouabain‐sensitive, a furosemide‐sensitive, and a ouabain‐furosemide‐insensitive component. In chick red cells of most ages, the ouabain‐sensitive and furosemide‐sensitive parts of the cation fluxes do not overlap. Cation transport in the more mature red cells is increased significantly by epinephrine, whereas cation transport in red cells from younger embryos is stimulated much less. This is a β‐adrenergic effect of epinephrine and is mediated by cyclic AMP. The relative lack of response in younger embryos is not due to the absence of β‐adrenergic receptor or the lack of production of cyclic AMP. Ouabain has no effect on the hormone‐sensitive sodium or potassium transport. On the other hand, furosemide nearly completely abolishes the effect of epinephrine. In addition, there is a good correlation between furosemide‐sensitive components of both sodium and potassium transport and the epinephrine‐sensitive component. Furosemide has no effect on cyclic AMP levels in the presence or absence of epinephrine. This suggests that furosemide may act directly on the cation transport system. In the red cells from younger embryos, furosemide‐sensitive units are present but cannot be fully activated by epinephrine. Therefore, the lack of the hormone effect on cation movements in these cells is consistent with the view that the appropriate units are present, but do not respond fully to intracellular cyclic AMP levels.
Use of an encapsulated fluorescent probe to measure intracellular PO2
Authors: Podgorski Gary T. , Longmuir Ian S. , Knopp James A. , Benson Douglas M.
Published: February 2005

The objectives of this investigation were to produce a reliable, senstive probe to measure intracellular PO2 with a high degree of resolution and to apply this technique to biological systems. A fluorescent molecule, pyrene dissolved in paraffin oil, was encapsulated in polyacrylamide to form a probe of nanometer dimensions. The quantitative and microscopic oxygen values were determined by analyzing the quenching of the fluorescence of the probe by oxygen, as displayed on a television monitor by a silicon‐intensified‐target camera. The nanocapsules had a sensitivity of approximately 1 mm PO2, a spatial resolution of 0.5 μm, and a temporal resolution of milliseconds. Calibrated nanocapsules within nonrespiring Amoeba proteus responded to ambient partial pressures of oxygen. At two different ambient partial pressures, nanocapsules engulfed by respiring amoebas indicated an intracellular PO2 28 mm Hg less than extracellular PO2. The capsules retained their sensitivity to oxygen for at least 8 months.
The effect of phorbol esters on the proliferation of C3H‐10T1/2 mouse fibroblasts: Consideration of both stimulatory and inhibitory effects
Authors: Tomei L. David , Cheney John C. , Wenner Charles E.
Published: February 2005

TPA stimulates cell cycle activation in both serum‐deprived and density‐inhibited cultures. The cells reestablish cycle arrest after no more than one generation, and addition of fresh drug produces no further response. However, cells freshly trypsinized can respond with a series of repetitive generations resulting in 3.5–4.0 population doublings over 72 hrs. In kinetic pulse experiments TPA enhanced 3H‐thymidine incorporation in densityinhibited cells stimulated by fresh serum but only after markedly suppressing incorporation 8–13 hrs after serum stimulation. When cells arrested by serum deprivation were pretreated with TPA, fresh serum stimulation led to initiation of 3H‐TdR incorporation 5 hrs earlier than untreated controls. However, TPA addition at the time of serum stimulation did not lead to a suppression at 8–13 hrs, whereas enhancement was observed during peak incorporation times regardless of whether the cells were pretreated with TPA during serum deprivation. The results support the concept that there can exist within G1 multiple states of responsiveness to phorbol esters. These pharmacologically induced states may be correlated with corresponding physiological states of the G1 phase of cell cycle.
Induction of polyamine limitation in Chinese hamster ovary cells by α‐methylornithine
Authors: Harada John J. , Porter Carl W. , Morris David R.
Published: February 2005

Chinese hamster ovary (CHO) cells in culture were limited for polyamines through the use of α‐methylornithine (αMO), a competitive inhibitor of ornithine decarboxylase. Initial exposure of the cells to the inhibitor caused growth rate and intracellular polyamine content to decline continuously. Reseeding the αMO‐treated cells into medium containing the inhibitor resulted in steady‐state (exponential) growth at cell densities below 5 × 103 cells/cm2, at a rate approximately twofold slower than untreated cells. Under these conditions, putrescine and spermidine were undetectable and spermine remained relatively constant at a level approximately half that found in untreated cells. Addition of exogenous putrescine elevated the polyamine content and stimulated the growth of αMO‐treated cultures. Thus, growth rate correlated with polyamine content in the αMO‐treated cells.

The growth of reseeded. αMO‐treated cells became nonexponential at a density (5 × 103 cells/cm2) far below that at which untreated cells departed from exponential growth (1 × 105 cells/cm2). Medium obtained from high density, αMO‐treated cultures inhibited the growth of cells at low density in the presence of αMO. Doubling the concentration of the defined components of conditioned medium did not markedly affect its capacity to inhibit growth. However, dialysis completely removed the inhibitory activity from conditioned medium. The results imply that a low molecular weight inhibitor of growth is produced by polyamine‐limited cells. This is a variable that must be controlled in studies with polyamine‐limited animal cells.

Morphological studies indicated that subcellular organelles, including mitochondria, were largely unaffected by treatment with αMO. The maintenance of mitochondrial integrity in the presence of αMO demonstrates that the swelling of mitochondria observed previously in cells treated with methylglyoxal bis(guanylhydrazone) was not due to polyamine limitation. αMO‐treated cells did, however, accumulate numerous cytoplasmic vacuoles. The identity of these vacuoles and their relationship to cellular physiology is not yet understood.
Suppression of the adipose conversion of 3T3 cells by acidified serum
Authors: Kuri‐Harcuch Walid , Green Howard
Published: February 2005

The adipose conversion of cultured 3T3‐F442A cells is strongly inhibited if the fetal bovine serum of the culture medium is briefly acidified before it is used. The inhibitory factor is a polypeptide with an apparent molecular weight of 24,000, and is inactivated by pronase or trypsin. Cells grown to confluence in the presence of this factor do not become spherical or accumulate triglyceride; they also do not increase the activity of their triglyceride‐synthesizing enzymes. The factor suppresses adipose conversion even in the presence of untreated serum. Once adipose conversion has begun in the absence of the inhibitory factor, subsequent addition of the factor does not arrest the conversion.
Coordinate regulation of gluconeogenesis by the glucocorticoids and glucagon: Evidence for acute and chronic regulation by glucagon
Authors: Kletzien Rolf F. , Weber Charles A. , Stumpo Deborah J.
Published: February 2005

The coordinate regulation of gluconeogenesis by the glucocorticoids and glucagon in primary cultures of adult rat liver parenchymal cells has been studied. The results suggest that glucagon stimulation of glucose production from 3‐carbon precursors is composed of at least two components which the glucocorticoids differentially affect. Glucagon treatment of hepatocytes results in an immediate increase in glucose production which is not blocked by cycloheximide and occurs in the absence of any detectable increase of phosphoenolpyruvate carboxykinase activity. This component appears to be regulated by a post‐translational mechanism and involves redirection of carbon flow from glycolysis to gluconeogenesis. The second component is characterized by the need for long‐term glucagon treatment. This increase in glucose production can be blocked by cycloheximide and is correlated with an increase in phosphoenolpyruvate carbooxykinase activity. The reaction that is accelerated by long‐term glucagon incubation is located prior to the triose‐phosphate level since long‐term incubation with glucagon fails to increase glucose production from dihydroxyacetone any more than does short‐term incubation. It is suggested that phosphoenolpyruvate carboxykinase rather than amino acid transport is the key pacemaker reaction in the long‐term incubation since the direction and magnitude of the response for glucocorticoid and glucagon stimulation of glucose production is the same whether alanine or lactate is used as the 3‐carbon precursor. The glucocorticoids exhibit an additive effect on glucagon‐stimulated glucose production for the first component whereas they amplify the second component.
Failure of hydrocortisone or growth factors to influence the senescence of fibroblasts in a new culture system for assessing replicative lifespan
Authors: Didinsky James B. , Rheinwald James G.
Published: February 2005

It has been reported that the replicative lifespan of human fibroblasts can be substantially extended by supplementing the growth medium with hydrocortisone or increased levels of serum proteins. These observations have been made only on cell populations transferred many times at high cell density, and cumulative population doublings have been recorded, rather than a more direct measure of cell division potential. We have measured the replicative potential of human fibroblasts cultured so as to avoid conditions of high cell density, medium depletion, and departure from exponential growth. Two fetal lung and two newborn foreskin fibroblast strains were serially passaged in the presence or absence of hydrocortisone (HC), epidermal growth factor (EGF), and fibroblast growth factor (FGF) until they senesced. At each passage cells were plated at densities sufficiently low that colony‐forming efficiency could be calculated. We determined cumulative population doublings and also estimated the number of cell generations attained under each condition. FGF caused small but possibly significant changes, while HC and EGF failed to substantially alter replicative lifespan. The reported effect of HC on the doubling potential of fetal lung fibroblasts is therefore not an inevitable action of this hormone on the senescence mechanism, but may instead depend for its apparent activity on the passage regimen used. The fibroblast's insensitivity to EGF as a modulator of replicative potential, as compared with the keratinocyte, whose lifespan can be tripled by EGF, implies that the mechanisms limiting the replicative potential of these two cell types are not identical.
Murine embryonal carcinoma hybrids: Decreased ability to spontaneously differentiate as a dominant trait
Authors: Oshima Robert G. , McKerrow James , Cox David
Published: February 2005

The F9 murine embryonal carcinoma (ec) cell line does not differentiate spontaneously at high frequency either in vitro or in vivo. A derivative of this variant ec cell line resistant to ouabain and thioguanine was fused with the PSA1 ec cell line, which retains the ability to differentiate to a variety of cell types, either in vitro or in vivo. Hybrids were selected in media containing ouabain, hypoxanthine, aminopterin, and thymidine at a frequency of approximately 10−3. Authentic hybrids were chosen by screening for the expected hybrid DNA content by flow microfluorometry and the lack of parental cell contamination in appropriate selective media. After injection into strain 129 mice, five of eight independently isolated hybrids formed tumors composed exclusively of embryonal carcinoma cells. The remaining three hybrids of this cross caused tumors composed of greater than 95% embryonal carcinoma cells but with small foci of differentiated tissues as well. Like the F9‐derived parental line, less than 10 percent of the clones, formed by the hybrids after six days of growth, contained cells that secreted plasminogen activator. Since control hybrids derived from the fusion of either two F9 derivatives or two pluripotent cell lines resembled their parental cell lines with regard to the types of tumors formed and their differentiation in vitro, it appears that the F9 phenotype is dominant to the pluripotent phenotype in early passage embryonal carcinoma hybrids.
The serum‐free growth of cultured cells in bovine colostrum and in milk obtained later in the lactation period
Authors: Steimer Kathelyn Sue , Packard Robert , Holden David , Klagsbrun Michael
Published: February 2005

Seven established cell lines, including both epithelial cells and fibroblasts (MDCK, Vero, CV‐1, NRK, 3T3, F2408, and NIL8) and four early passage cell strains (bovine articular chondrocytes, bovine smooth muscle cells, human foreskin fibroblasts, and rat embryo cells) were cultured in serum‐free medium supplemented with milk obtained 1 day after birth (colostrum) or 80 days after birth (older milk). MDCK, Vero, CV‐1, NRK, and 3T3 grew readily in colostrum and attained saturation densities ranging from 22% to 63% of that in serum. There was no growth of F2408, NIL8, or the early passage strains in bovine colostrum. None of the 11 cell cultures grew in older milk. The temporal dependence of growth in milk was examined in detail using MDCK cells. Growth equivalent to that in serum occurred in 3% colostrum and in 15% milk obtained 2 days after birth. Milk obtained 3 days and 10 days after birth was not effective as a growth supplement for MDCK cells at any concentration. Those cells, unable to grow in colostrum or in older milk, could be induced to grow if culture dishes were precoated with fibronectin. In addition to fibronectin, it was necessary in some cultures to supplement colostrum or older milk with insulin and/or transferrin in order to achieve growth. In the presence of fibronectin and appropriate factors, the final saturation density attained in colostrum or older milk ranged from 25% to 100% of that in serum. The fibronectin contents of bovine colostrum and milk were determined. The fibronectin level of colostrum was found to be approximately 5% of bovine serum. There was no detectable fibronectin in the 80‐day‐old milk.
Biochemical properties of differentiation factors for murine myelomonocytic leukemic cells in organ conditioned media — separation from colony‐stimulating factors
Authors: Nicola Nicos A. , Metcalf Donald
Published: February 2005

A number of different organs and tissues from untreated or endotoxin‐injected mice were surveyed for their ability to produce conditioned medium that could induce differentiation of the myelomonocytic leukemic cell line WEHI‐3B. All organs were able to do this but were much more effective from endotoxin‐injected animals. Production of the differentiation factor (DF) was dependent on protein synthesis and occurred throughout the 7‐day incubation period. DF produced by the different organs in vitro and found in vivo in endotoxin serum were indistinguishable from each other by several fractionation procedures but could be distinguished from the majority of granulocyte‐macrophage and macrophage colony‐stimulating factors (GM‐ and M‐CSFs). DF was precipitated by 55% ammonium sulfate whereas CSF required 85% saturation. DF did not bind to Concanavalin A‐Sepharose whereas CSF did. DF eluted later than CSF from Phenyl‐Sepharose columns. The dissociated molecular weight of DF was 21,000‐29,000 and was not greatly affected by neuraminidase treatment. There was no evidence that DF was glycoprotein in nature although this cannot yet be ruled out. Although the DF activity could be clearly separated from the bulk of GM‐CSF and M‐CSF activities, there was always residual CSF activity associated with the DF. This CSF was distinct from GM‐ and M‐CSF in its biochemical properties, in the fact that it only stimulated a subset of colony‐forming cells and that upon dilution it stimulated exclusively granulocytic colonies to develop. In two steps (ammonium sulfate precipitation and Phenyl‐Sepharose chromatography) it was possible to selectively enrich the differentiation activity over the CSF activity by 21‐fold.
Cellular mutations and drug resistance probed by herpes simplex virus
Authors: Campisi Judith , Pardee Arthur B.
Published: February 2005

The growth of herpes simplex virus type 1 (HSV‐1), monitored by plaque assay, was inhibited by the cellular antimetabolites thioguanine (TG), cytosine arabinoside (AraC), methotrexate (MTX), and 5‐fluorodeoxyuridine (5‐FUdR). These results suggested a rapid means for assaying cellular drug sensitivity, based on the ability of infected cells to support viral replication. We have explored the feasibility of a virus‐mediated assay for cellular metabolic function in two model systems.

Using an immunofluorescence assay to assess viral growth, we found that all of the antimetabolites tested were effective in diminishing HSV‐1 specific fluorescence in human fibroblasts. However, a DNA‐damaging agent, bleomycin, was lethal to cells but was completely ineffective in reducing viral fluorescence. HSV‐1 growth was markedly decreased by TG in a normal human fibroblast strain, FS‐2. In contrast, a Lesch‐Nyhan strain (LNF), resistant to TG owing to its genetic defect, showed no suppression of viral growth in the presence of TG. The drug's effect on viral fluorescence closely paralleled its effect on cellular colony forming ability and rate of cellular DNA synthesis.

Thymidine kinase‐deficient HSV‐1 (TK−HSV‐1) did not grow in a normal mouse fibroblast line (A31) in the presence of 5‐FUdR. However, a TK− derivative of the A31 line allowed full production of the TK−HSV‐1 antigens at low to moderate doses of 5‐FUdR.

Two potential applications for this assay are the prenatal diagnosis of some genetic disorders and the rapid detection of drug resistant populations in tumor specimens. Toward these ends, we demonstrated that human fibroblasts from patients with the hereditary disorder Xeroderma pigmentosum (group A) were easily distinguished from normal human fibroblasts by their inability to support the growth of UV‐irradiated HSV‐1. We also investigated the effects of TG upon HSV‐1 fluorescence in two human tumor cell lines isolated from head and neck squamous cell carcinomas (SCC‐15) and (SCC‐25). Whereas TG was effective in reducing viral fluorescence in SCC‐15 cells, it was only marginally so in SCC‐25 cells. These latter cells showed the greater resistance to TG by growth and isotope incorporation experiments.
Effect of H+ on the kinetics of Na+‐dependent amino acid transport in ehrlich ascites tumor cells: Evidence for H+ as an alternative substrate
Authors: Smith Thomas C. , Robinson Susan C.
Published: February 2005

The effects of H+ on the kinetics of α‐aminoisobutyric acid (AIB) influx in Ehrlich ascites tumor cells have been investigated at different external Na+ concentrations. Elevation of [H+] in the presence of both high (154 mEq/l) and low (10 mEq/l) external Na+ leads to decreases in the maximum influx (J) and increases in the apparent Michaleis‐Menten constant (K) for influx of AIB. In the virtual absence of external Na+ (0.96 ± 0.04 mEq/l), alterations in [H+] are without measurable effect on AIB flux. Furthermore, addition of AIB (10 mM) to cell suspensions (pH 5.90) stimulates H+ uptake by the cells in either the presence or absence of Na+. The data are consistent with two kinetic models for Na+‐dependent amino acid transport: an order bireactant (Na+‐binding necessary before AIB binding) system or a random bireactant system. Both models require that H+ serve as an alternative substrate for Na+. The consistency of the models was tested by fit to data from the present study (not used to evaluate the kinetic parameters) and by prediction of the pH dependence of Na+‐dependent amino acid transport compared to earlier studies.
Decreased epidermal growth factor binding in cells growth arrested in G1 by nutrient deficiency
Authors: Robinson Robert A. , Volkenant Mary E. , Ryan Robert J. , Moses Harold L.
Published: February 2005

Previous studies have shown that the nontransformed AKR‐2B mouse embryo derived cell line may growth arrest by two separate mechanisms in the G1 phase of the cell cycle‐growth factor deficiency arrest (G0) and low molecular weight nutrient deficiency arrest. An examination of epidermal growth factor (EGF) receptors under the different resting or growth conditions has shown that rapidly growing cells or cells arrested due to growth factor deficiency have the expected amount of 125I‐EGF binding with approximately 105 receptors per cell being present in G0 arrested cells. In contrast, cells arrested due to nutrient deficiency show a reduction in 125I‐EGF binding to 10‐‐20% of that observed under the other conditions. This effect appears to be due to decreased receptor number and not to a change in the affinity of the receptor. Stimulation of DNA synthesis by nutrient replenishment causes a tenfold increase in EGF binding 20 hours later, with some increase in binding being detectable as early as six hours. The increase in binding is inhibited by cycloheximide and actinomycin D. This suggests that new mRNA synthesis as well as increased protein synthesis is required for the increase in EGF binding.
Membrane transport properties differ following return of serum‐deprived versus Ca++‐deprived human fibroblasts to a proliferative state
Authors: Tupper Joseph T. , Ryals William T. , Bodine Peter V.
Published: February 2005

Human lung fibroblasts (W138) can be brought to a quiescent state by removal of serum from the medium or by lowering of the extracellular Ca++. Upon return of Ca++ or serum, the cells enter the G1 phase and progress to S within 15–18 hours. Since multiple G1 phase blocks have been demonstrated, we wished to determine whether the Ca++ and serum block were equivalent since previous data suggested that these two medium components may act at a common point in the initiation of proliferation. We have evaluated the membrane transport of 86Rb, 3‐O‐methylglucose, AIB, and cycloleucine following stimulation of quiescent cells by Ca++ or serum. Serum stimulation results in large increases in the influx of all the substances tested. These increases are prevented if Ca++ is absent upon serum stimulation or they are rapidly diminished following Ca++ removal. In contrast, Ca++ stimulation of Ca++‐deprived cells causes little or no enhancement of any of the transport systems, yet the cells progress to S phase in a manner similar to serum‐stimulated cells. These results indicate that the Ca++ and serum G0 or G1 block are not equivalent and that the serum‐induced change in transport of these components does not appear necessary for successful G1 phase progression. Furthermore, the data suggest that the sequence in which Ca++ or serum are presented to the cells alters the ability of Ca++ to modulate the transport systems. Quiescent cells which are exposed to Ca++ prior to serum possess a Ca++ modulation of several transport systems. Cells which are exposed to Ca++ subsequent to serum do not appear to possess this Ca++ regulation.