bluuebunny/crossref_metadata_2025 huggingface dataset:
Discrimination of a colony stimulating factor subclass by a specific receptor on a macrophage cell line
Authors: Das S. K. , Stanley E. R. , Guilbert L. J. , Forman L. W.
Published: February 2005

Utilizing the high affinity interactions between pure 125I‐L cell colony stimulating factor and its receptor(s) on the murine macrophage cell line J774, a murine radioreceptor assay (RRA) has been developed. The murine RRA selectively detects a colony stimulating factor (CSF) subclass (CSF‐1) previously defined by murine radioimmunoassay (RIA) (E.R. Stanley, Proc. Nat. Acad. Sci., USA, 76:2969–2973 ('79)). CSF‐1 stimulates macrophage production exclusively, and the occurrence of the CSF‐1 receptor(s) appears to be restricted to cells of the mononuclear phagocytic system (L.J. Guilbert and E.R. Stanley, J. Cell Biol. 85:153–160 ('80)). The murine CSF‐1 RRA failed to detect a variety of other CSF subclasses, growth factors, and hormones. In contrast to data obtained with the murine CSF‐1 RIA, human CSF‐1 (e.g., human urinary CSF) is detected by the mouse CSF‐1 RRA almost as sensitively as murine CSF‐1. In addition, there was an absolute correlation between CSF‐1 levels determined by murine CSF‐1 RRA and those determined by a human CSF‐1 RIA for a variety of human CSF‐1 sources. The murine CSF‐1 RRA is a sensitive (sensitivity 5 units or 1.0 femtomole of CSF‐1 protein), rapid, and highly specific assay for CSF‐1 in both murine and human sources.
Divergent effects of long acting cyclic nucleotides and lysine vasopressin on the release of matrix sulfated proteoglycans into the medium of fetal rat chondrocytes in monolayer culture
Authors: Miller R. P. , Erickson‐Lucas M.
Published: February 2005

Release of sulfated proteoglycans into the medium of fetal rat chondrocytes in monolayer culture was studied by contrasting the effects of 10% calf serum, long‐acting cyclic nucleotides (8 Br‐cAMP or DBcAMP), and lysine vasopressin (LVP). Eight hours after initiation of the experiment, the monolayer was pulsed for 2 hours with Na2[35SO], the radioactivity was chased, and the monolayer was reincubated for 6 hours with conditioned medium from replicate cultures. Immediately after labelling, the amount of newly synthesized sulfated proteoglycans was invariably higher in the insoluble matrix than in the medium compartment. Both additives selectively enhanced sulfate incorporation into chondroitin sulfate of the matrix when compared to serum controls, but only LVP stimulation caused increases in the medium. Remodeling (loss of cell layer and release into the medium at 6 hours) was suppressed by cAMP analogues and increased by LVP. This process was more active in cultures of lower cell density. Utilizing calibrated gel columns, no size difference of the glycosaminoglycans was found between the medium and cell layer compartments of the three treatment groups at the two time points. Because the cAMP analogues inhibit, while LVP stimulates cell division, our observations imply that the rate of degradation of the constraining matrix is increased when replication is favored, even when chondroitin sulfate synthesis is selectively stimulated.
Protein synthesis and the presence or absence of a measurable G1 in cultured chinese hamster cells
Authors: Liskay R. Michael , Kornfeld Bruce , Fullerton Paul , Evans Ronald
Published: February 2005

V79‐8 cells lack a measurable G1 interval under normal growth conditions. We found that partial inhibition of protein synthesis using low levels of cycloheximide (0.05 μ/ml) could induce a measurable G1 in these cells without any significant effects on S, G2, or M. In view of these findings, recessive mutants selected from the V79‐8 cell line, which each express G1, were analyzed for their rates of protein synthesis and degradation/loss. Three of the four mutants showed a decreased rate of protein synthesis sufficient to account for their G1 lengths. A fourth mutant, however, showed parental rates of both protein synthesis and degradation/loss. These results suggest not only that a G1 interval can be expressed as a result of a decreased rate of protein synthesis, but that other alterations (mutations) other than those simply affecting overall protein synthesis can result in the expression of a measureable G1 interval.
The effects of steel mutation on testicular germ cell differentiation
Authors: Nishimune Yoshitake , Haneji Tatsuji , Kitamura Yukihiko
Published: February 2005

The effects of artificial cryptorchidism and its surgical reversal on spermatogenesis were examined in germ cell mutant, S1/+ and wild type, +/+, mice. In cryptorchid testes no difference was found between S1/+ and +/+ mice in the number of undifferentiated type A spermatogonia. The activity of type A spermatogonia in mutant mice appeared normal as judged by its mitotic cell number and DNA synthesis. The surgical reversal of cryptorchidism resulted in regenerative differentiation of mature germ cells in both types of mice, but the pattern of cellular differentiation in the mutant testes was completely different from that of the wild type testes. At two steps of cellular differentiation, intermediate or type B spermatogonia and spermatid, the numbers of cells were much smaller in the S1/+ testes than those in the +/+ testes. The steel gene was therefore suggested to exert its effects on the differentiation of type A spermatogonia to intermediate or type B spermatogonia, on meotic division and/or the survival rate of these cells, but not on the undifferentiated type A spermatogonia or stem cells.
Endogenously produced CO2 induces stationary phase in tetrahymena cultures
Authors: Gillies R. J. , Deamer D. W.
Published: February 2005

Media concentration of total soluble CO2 increases with culture age of Tetrahymena pyriformis. CO2 is a weak acid and is capable of acidifying intracellular pH (pHi). Changes in pHi have been demonstrated to affect cell metabolism and growth in many systems. For these reasons, we investigated whether the concentrations of CO2 produced in vitro were sufficient to affect cell proliferation and pHi in Tetrahymena. In this study, we used DMO to mimic the weak acid properties of CO2. DMO is freely permeable to membranes in its uncharged form and has a pKa similar to that of CO2/HCO. In addition, it has the advantages of being metabolically inert and non‐volatile. At concentrations similar to endogenously produced CO2, DMO acidifies pHi and arrests culture growth. In addition, procedures are described which decrease the media CO2 concentrations in both growing and non‐growing cultures. These conditions lead to increased maximum culture density at stationary phase. The data indicate that, under our conditions, accumulation of CO2 in the culture leads to cessation of growth, probably through elimination of transmembrane pH gradients, which are necessary for regulation of metabolism and growth.
Correlation between growth rate, cell density, and intracellular concentrations of cyclic nucleotides in chemsostat cultures of mouse L1210 cells
Authors: Tovey Michael G. , Rochette‐Egly Cécile , Castagna Monique
Published: February 2005

Constant intracellular concentration of both adenosine 3′,5′ cyclic monophosphate (cyclic AMP) and guanosine 3′,5′ cyclic monophosphate (cyclic GMP) were obtained when mouse leukemia L1210 cells were cultivated under steady‐state conditions in the chemostat. When L1210 cells were maintained at a constant growth rate in the chemostat, the mean steady‐state intracellular concentration of cyclic AMP decreased with increasing cell density, while the concentration of cyclic GMP remained unchanged. When cell growth rate was increased by three‐fold, independently of cell density, in the chemostat, then the mean steady‐state intracellular concentration of cyclic GMP increased by 3.6‐fold, whereas that of cyclic AMP increased by only 22%. The ratio of cyclic AMP to cyclic GMP was found to decrease with increasing cell growth rate in the chemostat. Our results show that a close correlation exists between the steady‐state intracellular concentrations of cyclic nucleotides and changes in both cell growth rate and cell density in chemostat cultures of L1210 tumour cells.
Intracellular degradation of hemoglobin transferred into fibroblasts by fusion with red blood cells
Authors: Hendil Klavs B.
Published: February 2005

Hamster fibroblast protein and rabbit hemoglobin were labelled by incubation of fibroblasts (BHK21) or reticulocytes with [3H]leucine. Alternatively, human or rabbit hemoglobin was labelled by carbamoylation of erythrocytes with K14CNO. The labelled hemoglobins were introduced into fibroblasts by virus‐mediated fusion between the blood cells and fibroblasts. The hemoglobins became uniformly distributed throughout the cytoplasm.

Degradation was assessed from release of acid‐soluble radioactivity into the medium. Radioactivity from [14C]‐carbamoylhemoglobin was released as carbamoylvaline and homocitrulline, and these compounds were not metabolized or reincorporated by the cells.

Intermediate degradation products could not be detected.

The degradation of hemoglobin followed first‐order kinetics. The half‐life of both carbamoylated and native rabbit hemoglobin in hamster fibroblasts was 28 h, and the half‐life of carbamoylated human hemoglobin was about 150 h in fibroblasts from hamster (BHK21), mouse (Balb/3T3), and man (MRC 5), corresponding to that of the more stable endogenous proteins.

Phenylhydrazine increased the intracellular degradation of carbamoylated human hemoglobin about 13 times, whereas the degradation of endogenous proteins was little affected. Hemoglobin was degraded in homogenates at 31% h−1 at pH 5 and 0.3% h−1 at pH 7.4. Phenylhydrazine increased these rates to 45% h−1 and 9.7% h−1, respectively.

Growing hamster fibroblasts, which are brought into quiescence by serum deprivation or by high culture density, increase the degradation of endogenous protein and of hemoglobin in parallel.
Cell surface molecules of friend erythroleukemias: Decrease in T200 glycoprotein expression after induction
Authors: Hyman Robert , Trowbridge Ian , Cunningham Kristie
Published: February 2005

Monoclonal antibodies against the Thy‐1 and T200 glycoproteins were used to study the expression of cell surface molecules on mouse hematopoietic cell lines. Friend erythroleukemias express T200 glycoprotein but do not express significant amounts of Thy‐1 glycoprotein on their cell surface. The rate of T200 glycoprotein synthesis in maximally‐induced Friend erythroleukemia 745.6 cells is less than 10% that in noninduced cells, although total protein synthesis shows only a twofold decline and induced cells express 2‐6‐fold less T200 glycoprotein on their surface compared to noninduced cells. T200 glycoprotein expression is reduced in a variant cell line obtained by selection for growth in dimethylsulfoxide, showing that the reduction in T200 glycoprotein synthesis characteristic of induced cells is an event that can be dissociated from commitment and hemoglobin synthesis. Analysis of T200 glycoprotein negative cell lines, isolated by cytotoxic immunoselection against T200 glycoprotein, indicates that the presence of T200 glycoprotein on the cell surface is not necessary for induction of hemoglobin synthesis and terminal differentiation of Friend erythroleukemias.
Phorbol ester tumor promoters block the transition from the early to the heme‐dependent late program of friend cell differentiation
Authors: Mager Dixie , Bernstein Alan
Published: February 2005

In this study, the mechanism of inhibition of differentiation of Friend erythroleukemia cells by the phorbol ester tumor promoter, 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), has been examined. These studies indicate that some early events associated with Friend cell differentiation, including an early change in 86Rb+ transport and a decrease in cell volume, still occur in the presence of TPA. However, several late events in the program of Friend cell differentiation, including the induction of heme synthesis and the loss of proliferative capacity, are inhibited by TPA. These effects of TPA can be reversed by hemin, which alone does not induce Friend cells to differentiate. The addition of hemin to cultures grown in the presence of inducer plus TPA for several days results in the rapid restoration of hemoglobin synthesis, and also causes a parallel decrease in colony‐forming ability. These results suggest that tumor promoters may inhibit only heme‐dependent events, rather than the entire program of Friend cell differentiation.
Modulation of the G0 to S phase transit time by insulin: Potential involvement of protein phosphorylation
Authors: Kletzien Rolf F. , Day Philip
Published: February 2005

BHK fibroblasts can be growth arrested by incubation in low serum (0.1%) medium. Growth is initiated by incubating cells in high serum (10%) medium. We have found that if the quiscent cells in low serum medium are incubated with insulin, the G0 to S transit time is decreased by two to six hours when serum (10%) is added back to the culture. The effect of insulin treatment of quiescent cells on the cellular phosphoprotein profile was examined. It was found that insulin stimulated the phosphorylation of a 96,000 dalton cytosol protein. This protein is also intensely phosphorylated in proliferating cells and may be one of the critical intracellular events to occur when a cell initiates growth.
Comparative aspects of oxidative metabolism of neutrophils from human blood and guinea pig peritonea: Magnitude of the respiratory burst, dependence upon stimulating agents, and localization of the oxidases
Authors: Badwey J. A. , Curnutte J. T. , Robinson J. M. , Lazdins J. K. , Briggs R. T. , Karnovsky M. J. , Karnovsky M. L.
Published: February 2005

We have compared the subcellular sites of H2O2 and presumably also superoxide‐(O2−) production, and certain aspects of metabolic responses (O2 consumption, O2− production) of stimulated neutrophils from human blood and those elicited into guinea pig peritonea. Stimulation was accomplished with either opsonized zymosan or phorbol‐12‐myristate‐13‐acetate (PMA). Striking quantitative differences were observed between these cell types with regard to the increased respiration and O2− production observed during stimulation. These differences were most apparent when opsonized zymosan served as the stimulating agent. They were minimized when the soluble stimulating agent, PMA, was used. With either stimulus, the subcellular sites of H2O2 production were the same for both types of neutrophils, i.e., the plasmalemma and phagosomal membranes. No H2O2 production could be detected cytochemically in the absence of stimulation.

Treatment of both unstimulated human blood and elicited guinea pig peritoneal neutrophils with the nonpenetrating, covalently linking reagent, p‐diazobenzenesulfonic acid, failed to diminish O2− production upon subsequent stimulation, in contrast to a previous report. These data are discussed in terms of the possible cytological arrangements of the respiratory enzyme(s), and the different modes of stimulation of neutrophil metabolism by various agents. Ancillary data on elicited mouse peritoneal neutrophils are presented.
Stimulation of autophosphorylation of liver cell membrane proteins by calcium and partial hepatectomy
Authors: MacManus J. P. , Whitfield J. F.
Published: February 2005

Partial hepatectomy in the rat stimulated the phosphorylation of three proteins (Mr 100,000, 48,000, and 35,000) in the plasma membranes of the proliferatively activated cells of the liver remnant. The autophosphorylation of these plasma membrane proteins began to rise about 8 hours after surgery, peaked at 14 hours, and then returned to the original low level by 24 hours. This increase in autophosphorylation was not evident in isolated plasma membranes from other proliferatively activated liver cells, such as those in fetal liver or hepatomas. The protein kinase responsible for the phosphorylation of the three membrane proteins was activated by calcium but appeared both cyclic‐AMP‐ and calmodulinindependent.
Amphotericin B resistance is recessive in Chinese hamster hybrid cells
Authors: Hidaka Katsuhiko , Akiyama Shin‐Ichi , Kuwano Michihiko
Published: February 2005

Previous studies from our laboratory have shown that Chinese hamster V79 cells mutated to high level resistance to amphotericin B have a lower cellular level of cholesterol, the target molecule for the polyene antibiotic. Two amphotericin B‐resistant (AMBR) mutants were each hybridized to their parental amphotericin B‐sensitive (AMBS) V79 cells. All the hybrids derived from AMBR/AMBS fusions were as sensitive to polyene antibiotics (amphotericin B, filipin, and pimaricin) as AMBS/AMBS hybrids. The AMBR/AMBS hybrids were found to contain cholesterol per phospholipids that is comparable to those in AMBS or AMBS/AMBS. The analysis of hybrids formed between mutant and wild‐type cells thus indicated that resistance to amphotericin B is a recessive marker, and that the cellular level of cholesterol is compensated in the AMBS/AMBR hybrids. Hybrids of AMBR and AMBR cells were all resistant, so that the three AMBR mutants all fell into a single complementation group.
Aging in vitro and D‐glucose uptake kinetics of diploid human fibroblasts
Authors: Cremer T. , Werdan K. , Stevenson A. F. G. , Lehner K. , Messerschmidt O.
Published: February 2005

By use of a rapid technique, initial rates of D‐glucose transport were obtained during the lifespan in vitro of a commercially available strain of human embryo lung fibroblasts (Flow 2000). The apparent Km of the D‐glucose carrier did not change during senescence in vitro: x̄ = 1.8 mM (range 1.3–2.3) in phase II, x̄ = 1.8 mM (range 1.5–2.2) in phase III. Transport rates remained constant in stationary phase II cultures, which had completed between 30% and 80% of their replicative lifespan. A wide variation, however, was observed in terminally differentiated cells (phase III), which showed a two‐ to threefold increase in average cell size and protein content. In some senescent cultures, glucose transport calculated on a per cell basis was also two‐to threefold increased, while it was strongly decreased (‐75%) in others. When calculated per unit of cell water, protein, and surface area, respectively, transport rates in phase III cultures ranged from values established for stationary phase II cultures down to very low values. Detaching cells flushed off from senescent cultures did not show measurable rates of glucose transport into the inulin impermeable cell space. Present evidence argues against the idea that an impairment of D‐glucose transport might precede loss of replicative potential in aging human fibroblasts. Instead our data indicate that the transport capacity of cell membrane finally decreases during postreplicative senescence in terminally differentiated cells.
Development of capping ability during differentiation of HL‐60 human promyelocytic leukemia cells
Authors: Brown William J. , Norwood Cynthia F. , Smith R. Graham , Snell William J.
Published: February 2005

Developmental changes in cell surface and cytoskeletal elements have been studied in human promyelocytic leukemia cells (line HL‐60) which differentiate into functionally mature myeloid cells when grown in dimethyl sulfoxide (DMSO)‐supplemented medium. Both differentiated and undifferentiated HL‐60 cells bind fluorescent concanavalin A (F‐Con A) in a diffuse pattern over the entire cell surface. As with normal neutrophils, pretreatment of the differentiated HL‐60 cells with colchicine before incubation with Con A causes the formation of large cytoplasmic protrusions over which the lectin associates into a cap. On the other hand, similarly treated undifferentiated HL‐60 cells do not form the cytoplasmic protuberances and are unable to cap the Con A. Transmission electron microscopy reveals that the number and distribution of microtubules and microfilaments change during differentiation. Thus, developing myeloid cells undergo important alterations in the structure and function of the cytoskeleton as they differentiate into mature phagocytes.
Control and virus‐transformed baby hamster kidney cells resistant to ethidium bromide. II. Membrane properties
Authors: Soslau Gerald , Conover Thomas E. , Schneider Richard F. , Zavodny Paul J.
Published: February 2005

Aspects of membrane stucture and functions were studied in ethidium bromide resistant cells. Submitochondrial particles were solubilized and electrophoresed. The gel patterns, representing mitochondiral membrane proteins, demonstrated qualitative and quantitative alterations in mitochondrial preparations derived from virus‐transformed cells and ethidium bromide resistant cells as compared to the control cells. The plasma membrane glycoproteins were labelled by the sodium borohydride method. The glycoporteins were released with Triton X‐100 and electrophoresed. Fluorograms of the gels demonstratred some marked differences between the ethidium bromide resistant cells and their parental strain. The observed alterations in the membrane glycoproteins did not result in altered glucose transport properties or in the elution patterns of plasma membrane glycopeptides as analyzed by Sephadex G‐50 chromatography. Dye uptake and binding studies with intact parental and drug resistant cells and their isolated mitochondria demonstrated no alteration of the membrane permeability or the number of binding sites for ethidium bromide. Similar results were also obtained with a cyanine dye. This latter finding was significant in that it permitted one to exclude dye exclusion as a mechanism for ethidium bromide resistance.
Further evidence for an inhibitor of proliferation elaborated by normal human fibroblasts in culture: Partial characterization of the inhibitor
Authors: Strobel‐Stevens Janna D. , Lacey James C.
Published: February 2005

We present evidence that extends our earlier preliminary report on the stimulatory effect of saline washes on confluent cells in culture (Lacey et al., '77). That work suggested that an inhibitory substance was being removed by the washes. The present work suggests that the inhibitor is in the 10,000–30,000 MW range, is reversibly bound, is cationic and is also a protease inhibitor. It is heat stable, but is apparently degraded with time in our experimental systems.
Calcium transport and cellular distribution in quiescent and serum‐stimulated primary cultures of bone cells and skin fibroblasts
Authors: Eilam Y. , Szydel N.
Published: February 2005

Primary cultures of bone cells and skin fibroblasts were examined for their Ca++ content, intracellular distribution and Ca++ fluxes. Kinetic analysis of 45Ca++ efflux curves indicated the presence of three exchangeable Ca++ compartments which turned over at different rates: a “very fast turnover” (S1), a “fast turnover” (S2), and a “slow turnover” Ca++ pool (S3). S1 was taken to represent extracellular membrane‐bound Ca++, S2 represented cytosolic Ca++, and S3 was taken to represent Ca++ sequestered in some intracellular organelles, probably the mitochondria. Bone cells contained about twice the amount of Ca++ as compared with cultured fibroblasts. Most of this extra Ca++ was localized in the “slow turnover” intracellular Ca++ pool (S3). Serum activation caused the following changes in the amount, distribution, and fluxes of Ca++: (1) In both types of cells serum caused an increase in the amount of Ca++ in the “very fast turnover” Ca++ pool, and an increase in the rate constant of 45Ca++ efflux from this pool, indicating a decrease in the strength of Ca++ binding to ligands on cell membranes. (2) In fibroblasts, serum activation also caused a marked decrease in the content of Ca++ in the “slow turnover” Ca++ pool (S3), an increase in the rates of Ca++ efflux from the cells to the medium, and from S3 to S2, as well as a decrease in the rate of influx into S3. (3) In bone cells the amount of Ca++ in S3 remained high in “serum activated” cells, the rate of efflux from S3 to S2 increased, and the rate of influx into S3 also increased. The rate of efflux from the cells to the medium did not change. The results suggest specific properties of bone cells with regard to cell Ca++ presumably connected with their differentiation. Following serum activation we investigated the time course of changes in the amount of exchangeable Ca++ in bone cells and fibroblasts, in parallel with measurements of 3H‐thymidine incorporation and cell numbers. Serum activation caused a rapid decrease in the content of cell Ca++ which was followed by a biphasic increase lasting until cell division.
Inhibition of growth of proline‐requiring Chinese hamster ovary cells (CHO‐K1) resulting from antagonism by a system amino acids
Authors: Curriden Scott , Englesberg Ellis
Published: February 2005

CHO‐K1 requires proline for growth. Two proline‐independent revertants were isolated—K1‐J and K1‐6. CHO‐K1 pro− is much more sensitive than the pro+ cell lines to inhibition of growth by addition to the medium of amino acids and amino acid analogues that are transported through the A system. In contrast, pro+ cells are as sensitive as, or in some cases slightly more sensitive than, pro− cells to glycine, basic amino acids, and to amino acids that are mainly transported by the L system. The A system analogue α(methylamino) isobutyric acid (MAIB) in low concentrations reacts competitively with proline to regulate the growth of pro− cells, yielding a Ki for MAIB of 0.56 mM. CHO‐K1 and K1‐6 transport proline at the same initial rate and are equally sensitive to the inhibition of proline transport by alanine. Alanine and MAIB inhibit proline transport strongly and similarly in CHO‐K1. Thus although these compounds inhibit the transport of proline by both cell types to the same extent, pro+ cells are immune to the effect of this starvation since they are able to synthesize their own proline. We also describe a secondary inhibition caused by high A system amino acid concentrations that affects both pro− and pro+ cells.
Collagen gels as a matrix for haemopoiesis
Authors: Lanotte Michael , Schor Seth , Dexter T. Michael
Published: February 2005

We have shown that collagen gel can be used as a culture matrix for the cloning of granulocyte/macrophage progenitor cells (CFU‐C), the production of foci of marrow stromal cells and the maintenance of stem cell proliferation, differentiation and the production of CFU‐C. Since collagen is a physiological matrix and allows the simultaneous growth of a variety of cellular elements, the system should prove useful for examining the role of cell/cell interactions and regulatory molecules involved in haemopoiesis.
Amino acid transport in human lymphocytes: Distinctions in the enhanced uptake with PHA treatment or amino acid deprivation
Authors: Segel George B. , Lichtman Marshall A.
Published: February 2005

Human blood lymphocytes increase their concentrative uptake of amino acids when treated with plant lectins such as phytohemagglutinin or when exposed to an amino acid deficient environment (adaptation). Previous studies of the PHA effect have been conducted principally with α‐aminoisobutyric acid (AIB). We have studied the transport characteristics of naturally occurring amino acids by PHA‐treated lymphocytes. These studies have been conducted in the absence and presence of added carrier amino acid to determine whether an in‐crease in transport after PHA treatment is mediated by the same mechanism that occurs during adaptation to a low amino acid environment.

PHA stimulated the uptake of AIB, alanine, and proline 1.5‐ to 2‐fold after 4 hours' exposure. AIB and proline, but not alanine transport, also increased when lymphocytes were in an amino acid‐deficient medium. When lymphocytes were in an amino acid‐deficient medium with PHA, the increase in uptake induced by PHA was superimposed on the increase that occurred in response to an amino acid‐deficient medium. Also, PHA stimulated a delayed increase (16–20 hours) in the transport of leucine, whose uptake does not adapt to an amino acid‐deficient medium. These data suggest that PHA and amino acid deprivation stimulated rate increases in amino acid transport by separate mechanisms.
Effects of dexamethasone and dibutyryl cyclic AMP on polyamine synthesizing enzymes in mouse lymphoma cells
Authors: Russell Diane Handdock , Haddox Mari K. , Gehring Ulrich
Published: February 2005

S49.1 Lymphoma cells were arrested in G1 phase of the cell cycle when treated with either 1 μM dexamethasone (Dex) or 0.5 mM N6, O2‐dibutyryl cyclic adenosine 3′ :5′ ‐monophosphate (Bt2cAMP) plus 0.2 mM theophylline. However, the two agents had markedly different effects on aspects of polyamine and cyclic nucleotide metabolism within the arrested cells. Bt2cAMP had an early and pronounced inhibitory effect on ornithine decarboxylase (ODC) activity causing a decrease to 40% of control within 1 h. However, there was no significant inhibition of ODC activity in the Dex‐treated cells until 4 h of exposure, at which time ODC activity was reduced to approximately 60% of the control value. Sadenosyl‐L‐methionine decarboxylase (SAMD) activity was reduced by both agents, Bt2cAMP having the more pronounced inhibitory effect. The activity of SAMD was reduced to 40% of control after 10 h of Dex, whereas Bt2cAMP reduced the activity to approximately 25% of control within 4 h. Intracellular polyamine pools were decreased rapidly in Dex‐treated cells but not in those exposed to Bt2cAMP. Bt2cAMP decreased the amount of type I (PKI) and type II (PKII) cyclic AMP‐dependent protein kinase (cAMP‐PK) activity to 30% of control or less within 2 h. In contrast, Dex had very little effect on either PKI or PKII until 24 h, when cell viability was affected. The specific activity of both PKI and PKII remained significantly decreased in cells exposed to Bt2cAMP for 6 h and then resuspended in fresh medium. The rapid decrease in ODC activity in response to Bt2cAMP and the slow recovery after washout may be due to the marked decreases in total PKI and PKII activities. Dex, which had no effect on PKI and PKII specific activities, only slowly inhibited ODC activity and recovery of enzyme activity was rapid upon resuspension in fresh medium. These data further stress the importance of the maintenance of the cellular protein kinase pools in the regulation of the recovery time to growth inhibition in response to naturally occurring steroids and second messengers.
Hexose transport in normal and SV40‐transformed human endothelial cells in culture
Authors: Corkey Richard F. , Corkey Barbara E. , Gimbrone Michael A.
Published: February 2005

The mechanism of glucose entry into human vascular endothelial cells was studied in monolayer cultures of normal (primary) and virally (SV40) transformed umbilical vein endothelium. Radioisotopic uptake studies with the glucose analogues 2‐deoxy‐D‐glucose, and 3‐O‐methyl‐D‐glucose, and the nonmetabolizable stereoisomer L‐glucose, indicated the presence of a saturable, stereospecific hexose carrier mechanism in both cell types. In other experiments with D‐glucose and 3‐O‐methyl‐D‐glucose, the phenomenon of countertransport was demonstrable. Hexose transport was not affected by KCN, dinitrophenol, or ouabain, but was inhibited by phloretin and phlorizin in a pattern consistent with facilitated diffusion. Kinetic constants were obtained for both 2‐deoxy‐D‐glucose and 3‐O‐methyl‐D‐glucose uptake. Similar Km values (range, 3.3–4.7 mM) were noted with normal and transformed cells, whereas the apparent Vmax was 0.56 nmol/μ1 cytosol/minute for primary cells and 1.7–2.5 nmol/μ cytosol/minute for transformed cells. Under standard culture conditions, as well as following 18 hours of serum deprivation, insulin at concentrations up to 10−5 M did not appear to influence hexose uptake in either cell type. Metabolism of 14C(U)‐D‐glucose to 14CO2 also was not stimulated by insulin. The presence of an insulin‐insensitive, facilitated transport system for glucose in vascular endothelium has relevance for glucose metabolism in this tissue, and potentially for the association of certain vascular diseases (e.g., diabetic microangiopathy, atherosclerosis) with altered glucose homeostasis.
Induction of Macrophage DNA Synthesis by phorbol esters
Authors: Hamilton J. A. , Dientsman S. R.
Published: February 2005

Tumor‐promoting phorbol esters, such as 12‐0‐tetradecanoyl‐phorbol‐13‐acetate (TPA), stimulate mouse peritoneal exudate macrophages to undergo DNA synthesis without added macrophage growth factor (MGF). Resident peritoneal macrophages do not respond in this manner. Plasminogen activator (PA) levels of both exudate and resident peritoneal macrophages are elevated by TPA. Thus the phorbol esters appear to mimic the action(s) of MGF and may be useful in understanding the events involved in macrophage and precursor cell growth responsiveness.
Calcium spikes in cultured human reticular cells from peritoneal exudates
Authors: Gallin Elaine K.
Published: February 2005

Intracellular recordings of cultured human peritoneal exudate cells reveal that cells within the culture exhibit an active depolarizing response to injected currents which can reach positive potentials and resemble slow spikes. The cells exhibiting spikes are similar to the reticular cells described by Stuart and Davidson (1971a,b) in that they are esterase(+), acid phosphatase(+), and internalize colloidal carbon but not opsonized red blood cells. The active depolarizing response is unaffected by either decreasing the external sodium concentration or by adding tetrodotoxin (3 × 10−5 M), whereas increasing the external calcium concentration increases both the spike amplitude and rate of rise, and the addition of cobalt (3 mM) blocks the response. Addition of barium increases the duration and amplitude of the spikes but reduces the afterhyperpolarization. The data indicate that cultued human reticular cells from the peritoneal cavity exhibit a calcium spike.
Effect of serum step‐down on protein metabolism and proliferation kinetics of NHIK 3025 cells
Authors: Rønning Øystein W. , Lindmo Tore , Pettersen Erik O. , Seglen Per O.
Published: February 2005

Human NHIK 3025 cells growing exponentially in 30% or 3% serum had population doubling times of 19.1 and 27.6 hours, respectively. These values were equal to the calculated protein doubling times (17.6 and 26.5 hours, respectively), showing that the cells were in balanced growth at both serum concentrations. Stepdown from 30% to 3% serum reduced the rate of protein synthesis within 1–2 hours, from 5.7% hour to 4.3% hour, while the rate of protein degradation was unchanged (1.7%/hour). In cells synchronized by mitotic selection from an exponentially growing population, the median cell cycle durations in 30% and 3% serum were 17.2 and 23.6 hours, respectively, which were also in good agreement with the protein doubling times. The median G1 durations were 7.1 and 9.6 hours, respectively. Thus the duration of G1 relative to the total cell cycle duration was the same in the two cases. Complete removal of serum for a period of 3 hours resulted in a 3‐hour prolongation of the cell cycle regardless of the time after mitotic selection at which the serum was removed. For synchronized cells, the rate of entry into both the S phase and into the subsequent cell cycle were reduced in 3% serum as compared to 30% serum, the former rate being significantly greater than the latter at both serum concentrations. Our results thus indicate that these cells are continuously dependent upon serum throughout the entire cell cycle.
Variants of 3T3 cells lacking mitogenic response to the tumor promoter tetradecanoyl‐phorbol‐acetate
Authors: Butler‐Gralla Edith , Herschman Harvey R.
Published: February 2005

The potent tumor promoter 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA) can stimulate quiescent, nonproliferating 3T3 cells to reenter the cell cycle and divide. We have previously used a slection technique developed in our laboratory to isolate variant cell lines which no longer divide in response to epidermal growth factor. We have now utilized the same selection procedure to isolate, from 3T3 cells, two variant cell lines, TNR‐2 and TNR‐9, which retain growth control and divide in response to elevated serum or fibroblast growth factor, but which do not respond to TPA. The variants do not incorporate precursors into DNA in response to TPA, demonstrating that the cells do not enter the S phase of the cell cycle. The TPA nonresponsive variant TNR‐2 cannot respond to epidermal growth factor; TNR‐9 responds to this mitogen. TNR‐2 variant cells, which do not respond to EGF, do not bind 125I‐EGF. TPA can modulate 125I‐EGF binding to TNR‐9 cells in a manner similar to its action on parental 3T3 cells. This TPA‐induced alteration of EGF binding indicates that TNR‐9 cells still interact with TPA, despite their inability to mount a mitogenic response.
Neutral amino acid transport during the cell cycle of cultured human lymphocytes
Authors: Glassy Mark C. , Furlong Clement E.
Published: February 2005

Neutral amino acid (isoleucine, leucine, and valine) transport was monitored in synchronous populations of WIL‐2 cells, a diploid human lymphoblastoid B cell line. Cells were synchronized by either the double thymidine block technique or by density‐dependent arrest in G1. Cells synchronized by these methods showed up to a 66% decrease in amino acid transport upon progressing from the early S phase through the G2 and M phases with a concomitant increase in transport during the G1 phase. The lowest activities of amino acid transport in synchronized cells were observed during the G2 and M phases.
Studies on a human melanoma cell line: Effect of cell crowding and nutrient depletion on the biophysical and kinetic characteristics of the cells
Authors: Sheridan J. W. , Simmons R. J.
Published: February 2005

In an investigation of the changes that occur in cultured neoplastic cells as they outgrow their supply of nutrient, MM96 human melanoma cells were found to diminish in size and to proliferate more slowly. These changes were accompanied by a moderate increase in the proportion of cells with a G1‐like DNA content. When replated under favorable conditions, many of these cells gradually resumed active proliferation. Continuing adverse culture conditions led to a continued fall in cell size, loss of reproductive viabllity, and finally to rapid cell death. Simultaneous buoyant‐density and velocity‐sedimentation‐fractionation experiments showed that cells from exponential cultures were moderately dense and rapidly sedimenting, cells from postexponential cultures were less dense and much more slowly sedimenting, and dye‐excluding cells from reproductively nonviable, late postexponential cultures were of widely variable though generally high density, and were moderately rapidly sedimenting. Although neither fractionation method resulted in significant enrichment of clonogenic cells, depletion was seen at both extremes of both types of profile. Cells fractionated by velocity were sorted according to DNA content and hence location in the cell cycle. The relationship between sedimentation rate and cell‐cycle location was reflected in the continuous thymidine labeling patterns of the separated cells. Study of these patterns suggested that cycle durations lengthened as crowding increased and nutrient became depleted, and shortened upon reseeding at low density into fresh medium.
Cell population kinetics of the mouse lens epithelium
Authors: Rafferty Nancy S. , Rafferty Keen A.
Published: February 2005

The dividing lens epithelium of 8‐week‐old CF1 mice consists of a monocellular layer of about 31,000 cells and does not include the postmitotic cells of the meridional rows and another postmitotic zone of seven cell positions' width immediately anterior to the rows. The latter two populations contain approximately 3,600 and 9,000 cells, respectively, for a total of 44,000 cells in the entire lens epithelium. Autoradiographic analysis based upon mitotic index and cell cycle times indicates that the epithelium produces 207 new lens fibers a day. Throughout the 20‐day period of study, labeled cells appeared almost entirely as pairs following a single dose of 3H‐thymidine and clusters of labeled nuclei were not seen. Moreover, the number of labeled cells dropped only slowly with time, as did the grain counts. These observations indicate that logarithmic division “cascade” does not occur in the lens.

The dividing cell population consists largely of a slowly cycling stem cell group, dividing once about every 17–20 days, and consisting of some 5,000 cells. A subpopulation may exist which undergoes two rapid consecutive divisions before becoming postmitotic, but this is too small to make a significant contribution to lens fiber production. Four days are required to transit the postmitotic zone, and an additional 43 or so are needed to transit the meridional rows and differentiate into anucleate lens fibers. Data from other laboratories indicate that the entire process, from mitosis to final differentiation, requires about 4 months. Hence, most of this time is spent in migration of nondividing cells.
Partial characterization of a mitogenic factor with somatomedin‐like activity produced by culture WI‐38 human fibroblasts
Authors: Atkison Paul R. , Bala R. Marvin
Published: February 2005

Conditioned serum‐free medium (CSFM) obtained from WI‐38 human fibroblasts was found to contain a mitogenic factor(s) with somatomedin (SM)‐like activity. Treatment of the cells with cycloheximide eliminated the SM‐like activity in CSFM, suggesting that these cells produce and release the activity. Gel filtration revealed that the fibroblast SM‐like activity (FSLA) had a molecular size near 45,000. Isoelectric focusing of this FSLA yielded 2 bands of SM activity with pIs of 4.7 and 6.1, and corresponding molecular sizes of ∼29,000 and 16,500, respectively. The FSLA obtained by gel filtration revealed parallel dose response curves with a basic SM in a SM radioreceptor and radioimmunoassay and stimulated: (1) 35So4 uptake by hypophysectomized rat cartilage; (2) (U‐14C) glucose oxidation is isolated rat adipocytes; and (3) (3H) thymidine uptake and cell division in these same WI‐38 fibroblasts. Out studies indicate that this FSLA and basic SM are similar but not identical.
Aminoimidazole carboxamide ribonucleoside toxicity: A model for study of pyrimidine starvation
Authors: Thomas Cornelius B. , Meade Jean C. , Holmes Edward W.
Published: February 2005

Aminoimidazole carboxamide ribonucleoside (AIC‐R), a purine precursor, has biphasic effects on the growth of Chinese hamster fibroblasts. At 200 μM AIC‐R cell growth is almost completely arrested, while at 50 and 700 μM AIC‐R cell growth is comparable to that observed in the absence of nucleoside. The growth inhibition produced by AIC‐R is the consequence of inhibition of the orotate phosphoribosyltransferase‐orotidylic decarboxylase (OPRT‐ODC) reactions, as evidenced by a 87% reduction in the intracellular concentrations of UTP and CTP, accumulation of orotate in the medium, and restoration of normal growth by inclusion of 100 μM uridine in the medium. Inhibition of pyrimidine nucleotide synthesis at 200 μM AIC‐R is associated with an 82% reduction in the intracellular concentration of PP‐ribose‐P and a 150% increase in the concentration of purine nucleotides. Restoration of cell growth to a normal rate at 700 μM AIC‐R—a condition under which PP‐ribose‐P remains depressed and purine nucleotide concentrations are also depressed (40% of control)—and absence of toxicity at 50 μM AIC‐R—a condition under which purine nucleotide concentrations are increased by 150% and PP‐ribose‐P concentration is normal—suggest that the inhibition of OPRT‐ODC observed at 200 μM AIC‐R is caused by the combination of the reduction in PP‐ribose‐P and increase in purine nucleotides. These studies provide a better understanding of the control of the OPRT‐ODC reactions in the cell and provide additional insight into the basis of pyrimidine starvation induced by purine nucleosides.
Epstein‐Barr virus in nontumorigenic and tumorigenic nasopharyngeal carcinoma (NPC) somatic cell hybrids
Authors: Staczek John , Steplewski Zenon , Weinmann Roberto , Klein George , Koprowski Hilary
Published: February 2005

Somatic cell hybrids between mouse fibroblasts and human cells derived from nasopharyngeal carcinoma (NPC) biopsies or NPC tumors propagated in nude mice were examined for the expression of the Epstein‐Barr nuclear antigen (EBNA), retention of Epstein‐Barr viral (EBV) DNA, and tumorigenicity in nude mice. In all hybrids the expression of EBNA correlated with the detection of EBV‐DNA. After more than 2 years in culture, the hybrids examined retained similar amounts of EBV‐DNA when compared to previously published data. Retention of EBV‐DNA did not correlate with the presence of any particular human chromosome. Use of either rodent cell lines, clone 1D or IT‐22, did not affect the retention nor loss of EBV‐DNA. For tumorigenicity studies, NPC cells were fused with IT‐22 cells and injected into nude mice. Tumor formation did not depend on the presence or absence of EBNA and detectable EBV‐DNA sequences; tumorigenicity in these studies could not be correlated with the presence of any particular human chromosome or the origin of the NPC biopsy.
Carbon dioxide efflux accompanies release of fertilization acid from sea urchin eggs
Authors: Gillies R. J. , Rosenberg M. P. , Deamer D. W.
Published: February 2005

“Fertilization acid” is released from sea urchin eggs upon fertilization and decreases the pH of the surrounding seawater. In bicarbonate‐free artificial seawater flushed with nitrogen gas, the pH shift still occurs but returns to the original value in a few minutes, suggesting that the released acid is volatile. A likely candidate for a volatile acid is carbon dioxide released from the eggs. Therefore, the total CO2 content of seawater was measured pre‐ and post‐fertilization and was found to be correlated stoichiometrically with released proton equivalents, leading to the conclusion that fertilization acid is largely carbon dioxide. Manometric analysis of cell extracts and ashed eggs suggest that the carbon dioxide may be stored in the unfertilized egg as an inorganic carbonate.
Mechanisms controlling the kinetics in proliferation and differentiation of populations of mouse myeloid leukemic cells in vitro
Authors: Hayashi Moriaki , Okabe‐Kado Junko , Hozumi Motoo
Published: February 2005

The kinetics of differentiation and proliferation of clone cells (B24) of mouse myeloid leukemic Ml cells in vitro were studied by quantitative determination of cellular morphology. B24 cells were induced to differentiate into only macrophagelike cells by an inducer of differentiation in conditioned medium (CM) of embryo cells. During cell differentiation, the ratio of the area of the nucleus to that of the cell (N.C.R.) decreased from about 55% to 10%. Decrease in the N.C.R. was used as an index of cell differentiation in analysis of the kinetics of differentiation of cells treated with various concentrations of CM. The results showed that the process of differentiation was promoted by increasing the concentration of CM, and that the transition of the cells from the undifferentiated state to the differentiated state occurred in a stochastic manner. Comparison of these morphometric results with those of autoradiography showed that the labeling index of the cells decreases gradually in association with decrease in the N.C.R. of the cells from 50% to 30%. A stochastic model for the kinetics of proliferation and differentiation of the cells simulated the experimental observations on the production of differentiated cells.