bluuebunny/crossref_metadata_2025 huggingface dataset:
Spontaneous and agonist‐induced 86Rb efflux from rabbit aortic smooth muscle cells in culture: A comparison with fresh tissue
Authors: Martin W. , Gordon J. L.
Published: February 2005

Potassium efflux was measured in rabbit aortic strips and smooth muscle cells cultured from them by monitoring the release of isotope from preparations preloaded with 86Rb. The basal rate of 86Rb efflux from rabbit subcultured aortic smooth muscle cells was eightfold higher than from freshly isolated strips, but calculations of reuptake of isotope in the tissue indicated that the measured rate constant for efflux from aortic strips underestimated the true rate by about fourfold. The rate constant for efflux from freshly dispersed cells was less than half that of subcultured cells and remained unchanged for 5 days in culture. It then rose and by around day 10 had reached the value for subcultured cells. The increase in efflux coincided with the onset of cell division. The increased rate of efflux was accompanied by an increased rate of uptake so that the internal potassium content of the cells remained constant. Heparin decreased the efflux of 86Rb from subcultured cells to that of freshly isolated cells concomitant with a reduction in the rate of proliferation. The onset of cell division and increased basal efflux of potassium was associated with a loss of responsiveness to noradrenaline and histamine as assessed by monitoring 86Rb efflux, although depolarising solutions of potassium chloride were still able to elicit a response. Responsiveness to noradrenaline had histamine could be restored by the addition of heparin. These results suggest that the lack of responsiveness of subcultured cells is not due to irreversible dedifferentiation but to a reversible loss in proliferating cells of receptors for vasoactive agents or of a coupling mechanism between receptor occupation and ion gating.
Comparison of indirect probes of membrane potential utilized in studies of human neutrophils
Authors: Seligmann Bruce E. , Gallin John I.
Published: February 2005

Four indirect probes of membrane potential, triphenylmethylphosphonium ion (TPMP+), 3,3′dipentyloxacarbocyanine [di‐O‐C5(3)], 3,3′ dipentylindocarbocyanine [di‐I‐C5(3)], and 3,3′ dipropylthiodicarbocyanine [di‐S‐C3(5)] have been used to study neutrophil (PMN) activation. The data extend previous studies indicating that the cyanine dye di‐S‐C3(5) not only exhibits a different fluorescence response mechanism from di‐O‐C5(3) [and di‐I‐C5(3)] but also that the fluorescence of di‐S‐C3(5) is destroyed by reactive oxygen products produced by neutrophils following stimulation. When these aspects of the probes are taken into account, the interpretations of the results using all three cyanine dyes are identical. Studies with the isotope TPMP+ indicate that long incubations are necessary for PMN to fully equilibrate during which time the PMNs depolarize. Use of TPB−, to shorten the TPMP+ equilibration time, produces results identical with those obtained using the cyanine dyes. The cyanine dyes and TPMP+/TPB− are toxic to neutrophil functions although they do not cause cell death. Toxicity can be avoided by using low concentrations of di‐O‐C5(3) and di‐I‐C5(3) but cannot be avoided with di‐S‐C3(5) or TPMP+/TPB−. Using di‐O‐C5(3) with the fluorescence‐activated cell sorter, we demonstrate that heterogeneity of neutrophil responsiveness confuses the interpretation of studies characterizing the ionic basis of the fluorescence responses stimulated by certain stimuli. We conclude that some of the discrepancies currently reported in the literature using these probes are not due to inherent differences in the ability of the different probes to monitor the same event (i.e., PMN membrane potential) but instead are due to failure to correct for probe‐specific problems or response heterogeneity.
Glycosyl acceptors in intact and permeabilized normal and cystic fibrosis fibroblasts
Authors: Rudick Victoria L. , Rudick Michael J. , Jones Patricia M.
Published: February 2005

Using cell permeabilization, a technique which allows addition of exogenously supplied radiolabeled sugar nucleotides to serve as direct glycosyl donors, oligosaccharide biosynthesis was examined in fibroblasts obtained from normal and cystic fibrosis (CF) subjects. Incubation of logarithmically growing cells with either radiolabeled leucine or xylose has indicated that there was a difference in the synthetic rate between the cell types. Protein synthesis in normal cells made permeable with 50 m̈g/ml lysolecithin (LL) was demonstrated to be absent, and could not be induced to take place by adding exogenous components, including energy sources and amino acids, normally required for protein synthesis. Thus radiolabeled sugars were being added to peptide acceptors which were already present at the time of LL addition. Both permeable and intact fibroblasts were exposed to labeled UDP‐xylose, UDP‐galactose, and UDP‐glucuronic acid, all donors of mucopolysaccharide precursors. The uptake of xylose into protein was the same for both normal and CF cells, but permeable CF fibroblasts incorporated statistically greater amounts of sugar from UDP‐galactose and UDP‐glucuronic acid. Intact CF cells were also labeled using these two sugar nucleotides. Trypan blue exclusion indicated CF and normal fibroblasts were equally intact. This and the fact that preincubation of CF cells with the appropriate cold sugar nucleotide eliminated the differences in incorporation between the normal and CF cells suggested that CF fibroblasts had more cell surface acceptor than the normal cells.
Development of a serum‐free medium which supports the long‐term growth of human and nonhuman primate lymphoid cells
Authors: Brown Ronald L. , Griffith Richard L. , Neubauer Russell H. , Rabin Harvey
Published: February 2005

The ability to grow lymphoid cells in serum‐free media affords the advantage of separately analyzing those components found to be involved in proliferation and differentiation. Iscove's medium (IMDM) supplemented with bovine serum albumin or casein, cholesterol, ferrous chloride, insulin, β‐mercaptoethanol, L‐α‐phosphatidylcholine, and transferrin supported the long‐term proliferation of a gibbon ape lymphoma T‐cell line, MLA144. These cells continue to produce Interleukin 2 (IL‐2, T‐cell growth factor) constitutively in the serum‐free medium. IL‐2‐dependent human T cells initiated and maintained in culture in serum‐free medium containing IL‐2 have continued to replicate for over 3 months with two population doublings every 3 to 4 days. A normal, IL‐2‐dependent marmoset T‐cell line, OH‐1, also proliferated on the serum‐free medium when supplemented with IL‐2. Several established primate B‐cell lines which do not require IL‐2 for growth were able to proliferate in the serum‐free medium. These B‐cell lines included B95‐8, an Epstein‐Barr virus (EBV)‐transformed marmoset cell line, HuCo/R‐H, a human cord B‐lymphocyte line transformed with EBV, and Namalwa, an EBV‐positive B‐cell line established from a Burkitt's lymphoma. B95‐8 cells grown on serum‐free medium showed high levels of EBV antigen‐positive cells after induction with 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA).
Serum‐free culture of PC13 murine embryonal carcinoma cells
Authors: Heath John K. , Deller M. Jane
Published: February 2005

The requirements for the serum‐free culture of PC13 murine embryonal carcinoma cells were determined. Supplementation of a 50:50 mixture of Dulbecco's modified Eagles medium and MCDB104 with transferrin (5 μg/ml), human high‐density lipoprotein (HDL) (100 μg/ml), and human low‐density lipoprotein (LDL) (50 μg/ml) supported growth comparable to that observed with 5% foetal calf serum. Media supplementation with lipoproteins apparently substitutes for the effects of insulin, desoctapeptide insulin (DOP), or multiplication‐stimulating activity (MSA) on EC cell multiplication. Clonal growth of PC13 EC cells in this serum‐free medium could only be achieved in the presence of suitable feeder cell monolayers. These observations demonstrate that PC13 EC cells do not have an absolute requirement for exogenous mitogens to support multiplication.
Induction of reverse transformation and normal cell cycle regulation by dibutyryl cAMP in a chemically transformed cell line
Authors: Wang Yong Chao , Rao Potu N.
Published: February 2005

The objective of this study was to determine whether N6, O2‐dibutyryl 3′,5′‐adenosine monophosphate (db‐cAMP)‐induced reverse transformation in a chemically transformed mouse cell line, AKR‐MCA, would restore normal cell cycle regulation, particularly with regard to their growth arrest in the early G1 period. The AKR‐MCA cells were grown to confluency in the presence or absence of db‐cAMP (0.5 mM) plus theophylline (1 mM). The confluent cultures were trypsinized and a portion of the cells were fused with mitotic HeLa cells to induce premature chromosome condensation, while the remaining cells were used to study the kinetics of initiation of DNA synthesis. The prematurely condensed chromosomes (PCC) of the control and the treated cultures were classified into G1, S, or G2 types on the basis of their morphology. The G1 PCC were further subclassified into six groups (+ 1‐ +6); +1 being the most condensed and +6 the most decondensed. The cyclic AMP (cAMP)‐treated cells exnibited better attachment to the culture dish, were blocked in early G1 period at confluency, and entered S phase about 4 h later than the control following subculturing. In contrast, a majority of cells in the control cultures were arrested in S phase at confluency. These data indicate that the db‐cAMP‐induced reverse transformation in AKR‐MCA cells at least partially restores normal cell cycle regulation in these chemically transformed cells.
Na+ K+ pump and passive K+ transport in large and small red cell populations of anemic high and low K+ sheep
Authors: Lauf P. K. , Valet G.
Published: February 2005

Reticulocytes, isolated by centrifugal elutriation from massively bled sheep and identified by cytometric techniques, were analyzed with respect to their cation transport properties. In sheep with genetically high K+ (HK) or low K+ (LK) red cells, two reticulocyte types were distinguished by conventional or fluorescence‐staining techniques 5–6 days after hemorrhage: Large reticulocytes as part of a newly formed macrocytic (M) erythrocyte population, and small reticulocytes present among the adult red cell population (volume population III of normal sheep blood, Valet et al., 1978). Although cellular reticulin disappeared within a few days, the M‐cell population persisted throughout weeks in the peripheral circulation permitting a transport study of in vivo maturation. At all times, M cells of LK sheep had lower K+ and higher Na+ contents than M cells of HK sheep. Regardless of the sheep genotypes, M cells apparently reduced their volume during their first days in circulation; however, throughout the observation period, they did not attain that characteristic for adult red cells. Both ouabain‐sensitive K+ pump and ouabain‐insensitive K+ leak fluxes were elevated in M cells of both HK and LK sheep. The increased K+ pump flux was mainly due to higher K+ pump turnover rather than to the modestly increased number of pumps as measured by [3H]ouabain binding. In contrast, small reticulocytes enriched from separated volume population III cells by a Percoll‐density gradient exhibited transport parameters close to their prospective mature HK or LK red cells. The data support the concept that the M cells derived from emergency reticulocytes while the small reticulocytes represented precursors of normal red cell maturation. The Na+ and K+ composition found in M cells of HK and LK sheep, respectively, suggest development of the LK steady state at or prior to the reticulocyte state, a finding consistent with that of Lee and Kirk (1982) on low K+ dog red cells.
Adhesion of endothelial cells to extracellular matrix proteins
Authors: Macarak E. J. , Howard P. S.
Published: February 2005

Human and bovine endothelial cells were used in a series of studies examining cell adhesion to extracellular matrix macromolecules. Collagen types I and III, an acetic acid extract of bovine lens capsule basement membrane, laminin, and fibronectin were demonstrated to affect, in varying degrees, cell adhesion, cell growth, and “sprout cell” formation in endothelial cell cultures. All the matrix macromolecules tested promoted endothelial cell adhesion, and the adhesion to the basement membrane extract was blocked by antibodies to fibronectin. When human endothelial cells were grown on defined matrices, the level of serum required for optimum growth was decreased, while bovine endothelium grown on defined matrices did not show sprout cell formation. These data suggest that endothelial cells are able to adhere to a wide variety of connective tissue components and that extracellular matrices significantly influence cell behavior.
Growth‐promoting effects of iron‐ and cobalt‐protoporphyrins on cultured embryonic cells
Authors: Verger Claude , Sassa Shigeru , Kappas Attallah
Published: February 2005

The effects of hemin (Fe‐protoporphyrin) and Co‐protoporphyrin on cellular growth have been investigated principally in cultured fibroblasts, but also in myoblasts and hepatocytes from chick embryos. In the presence of horse serum in the culture medium, which by itself did not stimulate cell growth appreciably, Co‐protoporphyrin stimulated cell attachment while hemin stimulated cell proliferation of fibroblasts. When Co‐protoporphyrin and hemin were added together, the most potent stimulation of cell growth, consisting of increases in cell attachment and rapid cell proliferation, was observed. These findings indicate that the two metalloporphyrins have differential and complementary effects on cellular growth in culture, with synthetic Co‐protoporphyrin principally affecting cellular attachment and Fe‐protoporphyrin stimulating cellular proliferation.
Evidence for monovalent phosphate transport in Ehrlich ascites tumor cells
Authors: Bowen Jesse W. , Levinson Charles
Published: February 2005

In an effort to determine whether the Na+‐dependent Pi transport system of Ehrlich ascites tumor cells exhibits specificity for H2PO4− or HPO4−2, Pi fluxes were determined by measuring 32PiPi self‐exchange. Three experimental approaches were employed. First, the effect of pH on steady‐state Pi transport at 0.5 and 5 mM was studied. Second, the relationship between Pi transport and Pi concentration (0.25–9.2 mM) at pH 5.6 and 7.9 was determined. Third, the dependence of Pi transport on [H2PO4−] (0.05–4.2 mM) at constant [HPO4−2] (0.5 mM), and the converse, [HPO4−2] (0.06–4.5 mM) at constant [H2PO4−] (0.5 mM), was evaluated. Ks (apparent half‐saturation constant) and Jmax (maximal transport rate) were calculated by two methods: weighted linear regression (WLR) and a nonparametric procedure. The dependence of Pi flux on pH indicates that optimum transport occurs at pH 6.9. Pi transport decreases as pH is reduced when extracellular Pi is either 0.5 or 5 mM. However, at pH 7.9, Pi flux is reduced only in 0.5 mM Pi. At pH 5.6, H2PO4− comprises 93% of the total Pi present, and the calculated Ks is 0.055 ± 0.026 mM (WLR). This is the same as the Ks determined from the initial phase of the flux vs. [H2PO4−] relationship (0.056 ± 0.020 mM). However, at pH 7.9 (where 94% of Pi is HPO4−2), the measured Ks is 0.58 ± 0.11 mM (WLR), which is ten times higher than at pH 5.6. This value is also five times greater than the Ks calculated from the flux vs. [HPO4−2] curve (0.106 ± 0.16 mM). Kinetic parameters calculated by the nonparametric method, though somewhat different, gave similar relative results. Taken together, these results support two conclusions: (1) H2PO4− is the substrate for the Na+‐dependent Pi transport system of the Ehrlich cell, and (2) H+ can inhibit Pi transport.
Elastin production by cultured calf pulmonary artery endothelial cells
Authors: Mecham Robert P. , Madaras Judy , McDonald John A. , Ryan Una
Published: February 2005

Calf pulmonary artery (CPA) endothelial cells synthesize and secrete soluble elastin when incubated in medium conditioned by arterial smooth muscle cells. Endothelial cell tropoelastin cross‐reacts with antiserum to bovine ligamentum nuchae elastin and comigrates on SDS‐PAGE with tropoelastins from fetal bovine ligamentum nuchae fibroblasts, aortic smooth muscle cells, and ear chondroblasts at an apparent molecular weight of 70,000. Endothelial cells synthesize only one‐third as much elastin as these other cell types, however. Approximately 80% of the elastin synthesized by endothelial cells in confluent culture is released into the culture medium. The remaining 20% remains associated with the cell layer and is readily extractable with dilute acetic acid as un‐cross‐linked, 70,000‐dalton tropoelastin. The addition of β‐aminopro‐pronitrile to culture medium did not alter the ratio of tropoelastin in the medium and cell layer, suggesting that cross‐linking of tropoelastin does not occur in culture.

Immunofluorescent staining of confluent endothelial cell cultures with antielastin serum demonstrated elastin occurring as a web‐like network of fine filaments extending throughout the extracellular space. The fibrous elastin was different in organization and distribution from fibers stained with antifibronectin serum, which were localized primarily beneath the cell layer and in regions of cell‐cell contact. Extracellular matrix remaining after solu‐bilization of cellular material with Triton X‐100 stained positive for fibronectin, but not for elastin.
Renewal inhibition of human mammary cell growth in vitro: Cortisol and the recruitment of cells to terminal differentiation
Authors: McGrath Charles M. , Soule Herbert D.
Published: February 2005

Cortisol and insulin stimulated exponential growth of normal human mammary epithelium in short‐term monolayer culture. The response of cells depended on their organization into “growth units” on the surface of the culture dish; single cells did not respond. Growth of cells in the units ceased after only 3–4 doublings, ending in terminal differentiation. The 3–4 divisions that occurred in response to insulin and cortisol and that resulted in terminal differentiation, were not inhibited by short‐range signals normally transmitted at population confluence. When growing above confluence density in response to hormones, cells reduced volume to accommodate the “terminal differentiation” divisions while still largely preserving a monolayer. The longevity of populations of normal cells (9–14 divisions), which occurred in 3 or 4 passages, exceeded the average longevity of individual cells in one passage (3–4 divisions). This disparity between real and apparent longevity was due to the inhibition of growth of divisional cells within growth units, which occurred in concert with terminal differentiation of other cells in these units. Inhibited cells could be recruited to undergo terminal differentiation divisions in response to cortisol and insulin, but only when the growth units were disrupted and terminally differentiated cells were eliminated, which occurred at subculture. We refer to the inhibition of growth that occurs in growth units as “renewal inhibition” to distinguish it from population‐wide “confluence inhibition” and to emphasize three other aspects: (1) it occurred in terminally differentiating growth units; (2) it occurred in the continued presence of an inductive hormonal stimulus for differentiative growth; and (3) it conserved less differentiated cells for recruitment to terminal differentiation. There are parallels between renewal inhibition in vitro and the signals that restrain growth of mammary cells in “growth buds” in vivo to preserve their capacity for multiple cycles of secretory differentiation. Differences in the behavior of normal and malignant breast cells in vitro suggest that renewal inhibition, rather than confluence inhibition, may be an important locus of growth control alteration in malignant transformation.
Regulation of low‐density lipoprotein metabolism by cell density and proliferative state
Authors: Kenagy R. , Bierman E. L. , Schwartz S.
Published: February 2005

Stimulation of the proliferation of human skin fibroblasts by platelet‐derived growth factor increased the binding and degradation of low‐density lipoproteins at cell densities of 2000–30,000 cells/cm2. Binding and degradation of low‐density lipoprotein was an inverse function of cell density in both proliferating and quiescent cells, indicating that the effect of cell density on the LDL receptor has proliferation‐dependent and proliferation‐independent components. The effect of medium conditioned by confluent fibroblasts on LDL metabolism was tested to determine if the effects of cell density on LDL metabolism might be mediated by cellular secretion products. Fibroblast‐conditioned medium increased LDL metabolism, suggesting secretion products do not mediate these effects of cell density. These data indicate that regulation of the low‐density lipoprotein receptor is not a simple on/off response to growth stimulation, but is responsive to extracellular cues such as cell density.
Possible involvement of cGMP in the control of tyrosine aminotransferase degradation in rat hepatocytes
Authors: Strinden Sarah Taylor , Stellwagen Robert H.
Published: February 2005

Addition of theophylline to primary cultures of rat hepatocytes in which tyrosine aminotransferase had been preinduced with dexamethasone caused a further increase in specific activity of the enzyme. This increase was due in part to a reduction in the rate of tyrosine aminotransferase degradation that began about 2 hr after theophylline was added. The level of cGMP also increased with a similar time lag following the addition of theophylline. The concentration of theophylline which produced the above effects (1 mM) did not alter the rate of general protein degradation in hepatocytes. Addition of 8‐bromo‐cGMP (0.5 mM) resulted in an immediate reduction in the rate of tyrosine aminotransferase degradation and in an increase in the activity of the enzyme. Treating hepatocytes with MnCl2 (0.9 mM) caused an elevation of cGMP and a concomitant slowing of tyrosine aminotransferase degradation without changing the level of cAMP significantly. These results suggest an inverse relationship between the level of cGMP and the rate of tyrosine aminotransferase degradation in hepatocytes.
Associations of small nuclear RNAs in human cells
Authors: Smith James H. , Eliceiri George L.
Published: February 2005

Several observations have been made about the associations of small nuclear RNAs (snRNAs) in human cells. When nuclear RNA was extracted with phenol and chloroform under standard nondenaturing conditions, the proportion of the nuclear snRNA content that cosedimented with high molecular weight RNA was very low. These results do not support the proposal that it is a large percentage of the cellular snRNA content that is involved in relatively stable base‐paired interactions with heterogeneous nuclear RNA at any given time. The various small nuclear ribonucleoprotein particles (snRNPs), in which the snRNAs are found in the cell, appear to differ substantially in their sedimentation rates under conditions of physiological ionic strength. Using anti‐RNP and anti‐Sm antibodies to analyze various subcellular fractions, we found that most, if not all, of the U1 snRNA cellular content is associated with the polypeptide(s) bearing the RNP determinant (in interphase and mitotic cells) and with the polypeptide(s) carrying the Sm determinant (in mitotic cells).
12–0‐tetradecanoylphorbol‐13‐acetatea and concanavalin a enhance glucose uptake in thymocytes by different mechanisms
Authors: Nordenberg Jardena , Stenzel Kurt H. , Novogrodsky Abraham
Published: February 2005

The effects of the tumor promoter 12–0‐tetradecanoylphorbol‐13‐acetate (TPA) and the plant lectin concanavalin A (Con A) on glucose uptake in murine thymocytes were studied. TPA induces a rapid dose‐dependent increase in the uptake of 2‐deoxyglucose and in the transport of 3–0‐methylglucose. Con A also elicits a time‐ and dose‐dependent enhancement of 2‐deoxyglucose uptake. The effect of Con A, however, is less pronounced. The effect of combined treatment of thymocytes with Con A and TPA is not additive. Cytochalasin B completely inhibits the basal, as well as TPA‐ and Con A‐enhanced, 2‐deoxyglucose uptake. Dexamethasone markedly inhibits basal 2‐deoxyglucose uptake, but is less inhibitory to enhanced 2‐deoxyglucose uptake induced by TPA and Con A. The effect of TPA on 2‐deoxyglucose uptake and 3–0‐methylglucose transport is refractory to inhibition by isobutyl methylxanthine, dibutyryl cyclic AMP, and ethyleneglycol tetraacetic acid. These agents markedly inhibit the enhancement of 2‐deoxyglucose (2‐DOG) uptake by Con A. p‐Bromophenacyl bromide, an inhibitor of phospholipase A2, also selectively inhibits Con A enhancement of 2‐DOG uptake. Taken together, the results suggest that Con A and TPA exert their stimulatory effect on glucose uptake by different activating mechanisms, but they may share a final common transport pathway.
Effects of polyamines on cyclic AMP‐mediated stimulation of amino acid transport in isolated rat hepatocytes
Authors: Auberger Patrick , Samson Michel , Cam Ginette Le , Cam Alphonse Le
Published: February 2005

The effects of natural polyamines on cyclic AMP‐mediated stimulation of amino acid transport in isolated rat hepatocytes were analyzed. Despite the fact that polyamines could directly compete with α‐aminoisobutyric acid (AIB) for uptake, preincubation of hepatocytes with polyamines did not significantly alter basal AIB transport. The stimulatory effect of glucagon or cyclic AMP analogs was differently affected by polyamines, since it was reduced in the presence of spermine and, inversely, potentiated by spermidine, putrescine, and cadaverine. Dose‐dependence analysis showed that half maximal and maximal effects occurred with 2–3 and 6–10 mM external concentrations, respectively. None of the polyamine effects could be ascribed to transstimulation or transinhibition of amino acid uptake. The inhibitory effect exerted by spermine correlated its capacity to inhibit [3H]‐leucine incorporation into proteins partially. The potentiating effect of the other polyamines did not result from stabilization of newly synthesized carrier proteins. Instead, the increase in Vmax of the high affinity transport component suggested that more carriers became available, presumably because polyamines facilitated their synthesis by interacting directly with one or several steps controlled by cyclic AMP.

Polyamines appear to represent a new class of factors capable of modulating the cyclic AMP‐mediated stimulation of amino acid transport, in hepatocytes.
Growth effect of lithium on mouse mammary epithelial cells in serum‐free collagen gel culture
Authors: Tomooka Yasuhiro , Imagawa Walter , Nandi Satyabrata , Bern Howard A.
Published: February 2005

The effect of lithium on the growth of mammary epithelial cells from adult virgin and midpregnant BALB/c or BALB/cfC3H mice was tested in a serum‐free collagen gel culture system. The serum‐free medium consisted of a 1:1 mixture of Ham's F12 and Dulbecco's Modified Eagle's medium supplemented with insulin, transferrin, cholera toxin, epidermal growth factor (EGF), and bovine serum albumin fraction V (BSA V). A multifold increase in cell number occurred during 10–12 days of culture in this medium. In dose‐response studies in which the concentration of each component of this serum‐free medium was varied in turn, the addition of LiCL (10 mM) enhanced growth at most concentrations of each factor. However, LiCL could not enhance growth in the absence of insulin or BSA V, but could replace EGF. The optimal concentration of LiCl was 5–10 mM; higher concentrations (20–80 mM) were toxic. KCl (1–10 mM) when added to the serum‐free medium slightly stimulated growth; the addition of NaCl to the medium had little effect on growth. LiCl did not enhance the growth of cells from spontaneous mammary tumors of BALB/cfC3H mice.
Myocytes and fibroblasts exhibit functional synergism in mixed cultures of neonatal rat heart cells
Authors: Schroedl Nancy A. , Hartzell Charles R.
Published: February 2005

Cardiac cells obtained from neonatal rat heart contain a mixed population of cell types that can be enriched in culture in either myocytes or fibroblast‐like cells. A metabolic comparison of mixed heart cell cultures with enriched cultures of the same age‐in‐culture and initial cell density showed that mixed cultures used glucose more rapidly than either enriched myocytes or fibroblasts. Mixed cultures were shown to respond to deprivation of insulin and of serum with decreases in the rate of glucose usage and decreases in the protein content of cells, whereas enriched cultures did not respond in the expected manner to insulin deprivation. Mixed, 11‐day‐old cells also exhibited greater increases in cellular protein and greater resistance to the stress of starvation than enriched cultures. Palmitate usage, however, was similar in all cultures examined. We conclude that mixed cultures may serve as a better model system to study cardiac metabolism and to monitor the effects of drugs and hormones on the neonatal myocardium. In addition, it is clear from our results that myocytes and fibroblastic‐like cells coexist in a metabolically functional synergism.
The presence of a soluble interphotoreceptor retinol‐binding protein (IRBP) in the retinal interphotoreceptor space
Authors: Pfeffer B. , Wiggert B. , Lee L. , Zonnenberg B. , Newsome D. , Chader G.
Published: February 2005

A new, gentle technique has been developed for washing of the retinal interphotoreceptor space (IPS) to obtain soluble components of the extracellular matrix (ECM). Using this method, we have determined that the major soluble coustituent of monkey IPS is a 146,000 Mr glycoprotein, which binds [3H]retinol, sediments on sucrose gradients at 7S and has an Rf of 0.42 on native gel electrophoresis. Using size‐exclusion high performance liquid chromatography, the apparent molecular weight of the native protein was calculated to be 250,000 daltons. In contrast to previous studies, no 15,000‐dalton cellular retinol‐binding protein (CRBP) or 33,000‐dalton cellular retinaldehydebinding protein (CRALBP) was observed in the IPS wash, indicating that these proteins are probably not involved in retinol transport between retina and pigment epithelium (PE). In the supernatant fraction of retinal homogenates that contains soluble intracellular proteins as well as extracellular constituents, the 146,000 Mr protein was closely associated with a 93,000 Mr protein that could be separated on SDS‐gel electrophoresis; the 93,000 Mr protein was not found in the IPS wash. The 146,000 Mr interphotoreceptor retinol‐binding protein (IRBP) may function in extracellular retinol transport in the IPS.
Uridine kinase molecular species and uridine uptake in some variants of rat hepatomas
Authors: Fulchignoni‐Lataud Marie‐Claude , Roux Jeanne M.
Published: February 2005

In a family of clonal lines derived from the Reuber H 35 rat hepatoma, four electrophoretically distinct molecular forms of uridine kinase (UK I, II, III, and IV) have been characterized. They are the same as those found in foetal rat liver. Different UK profiles occur in these cell lines, and no strict correlation could be established between the state of differentiation of the cells and the form of UK expressed. A clone of somatic hybrid cells between line p4 (form 1 only) and Fu5‐5 (forms II, III, and IV) that does not express form I indicates that p4 cells may lack a factor controlling the polymerization of form I. This variety of clonal cell lines was used to study the uptake and phosphorylation of labeled uridine. The results suggest a relationship between the UK form present and the rate uridine phosphorylation by the intact cells.
Secretory activity of hamster tracheal explants and isolated tracheal epithelial cells and the effects of cystic fibrosis serum
Authors: Rudick Victoria L. , Wooten Marie W. , Rudick Michael J.
Published: February 2005

Serum from cystic fibrosis patients has been shown by scanning electron microscopy to cause release of large quantities of mucus from the cultured tracheal rings of 3–4‐month‐old male Golden Syrian hamsters. In order to study this phenomenon on single cells, an epithelial (HTE) cell culture has been established from the hamster tracheal rings using the cell rescue method of Goldman and Baseman (1980a, In Vitro, 16:313). The cells were demonstrated to be epithelial by histochemical staining and immunofluorescent detection of laminin. Proteins secreted by HTE cells were partially characterized and shown to consist, at least in part, of acidic glycoproteins. The proteins were precipitated by addition of buffered alcian blue (AB) to the cell‐free medium under conditions in which all of a polyanionic protein [3H]‐labeled mucin, was precipitated without carrier. [14C] galactosamine‐labeled AB precipitate was β‐eliminated and, after neutralization and centrifugation, the material in the supernatant was sized by chromatography on a calibrated Bio‐Gel P2 column. The label eluted with a molecular weight close to a disaccharide. HTE cells pulse‐‐labeled for 1.0 hr with [3H] leucine or [14C] galactosamine secreted increasing amounts of labeled glycoprotein during the chase. Sodium dodecylsulfate‐polyacrylamide gel electrophoresis and fluorography of labeled AB precipitates revealed three major bands, two with molecular weights greater than 100 kd. Secretion was stimulated by retinoate (50% increase), but not by retinol. Exposure of HTE cells to whole sera from cystic fibrosis patients resulted in heightened secretion rates as compared to results obtained with normal sera. Heterozygote sera produced secretion rates intermediate between the two extremes.
Human megakaryocytic progenitors (CFU‐M) assayed in methylcellulose: Physical characteristics and requirements for growth
Authors: Kimura Hideo , Burstein Samuel A. , Thorning David , Powell Jerry S. , Harker Laurence A. , Fialkow Philip J. , Adamson John W.
Published: February 2005

The basic culture requirements and several physical characteristics were defined for megakaryocytic colony‐forming cells (CFU‐M) from normal human marrow growing in methylcellulose. Ficoll‐hypaque separated mononuclear cells from human, marrow gave rise to megakaryocytic colonies in the presence of normal human plasma and phytohemagglutinin‐stimulated leukocyte‐conditioned medium (PHA‐LCM). Their identity as megakaryocytic colonies was confirmed by immunofluorescence staining with a monoclonal antibody to human factor VIII antigen and by electron microscopy of individually harvested colonies. Demonstration of the single‐cell origin of the colonies was provided by analysis of the glucose‐6‐phosphate dehydrogenase (G‐6‐PD) enzyme type of individually harvested colonies grown from a G‐6‐PD heterozygote. The colonies grew best in heparinized or citrated plasma as opposed to serum. Detailed studies suggested that platelet‐release products were responsible for this difference. Tritiated thymidine suicide studies showed that the percentage of CFU‐M in DNA synthesis was 23 ± 8% (n = 10). The modal velocity sedimentation rate of CFU‐M was 4.9 ± 0.6 mm/hr (n = 4) while that of concurrently studied granulocyte/macrophage colony‐forming cells (CFU‐GM) was 5.7 ± 0.5 mm/hr. Examination of the PHA‐LCM dose‐response characteristics suggested the presence in the conditioned medium of an inhibitor to megakaryocyte colony growth which was partially removed by chromatography of the medium on Sephadex G‐100. The resulting conditioned medium increased the cloning efficiency for CFU‐M compared with that with crude PHA‐LCM (15.3 ± 7.0 and 8.2 ± 5.3/105 marrow cells, respectively).
A new assay for factors that regulate the synthesis of granulocyte differentiation proteins in vitro
Authors: Evans Warren H. , Wilson Shirley M. , Alvarez Vernon
Published: February 2005

Morphological and molecular aspects of granulocyte differentiation can be studied concomitantly using liquid cultures of immature granulocytes in conjunction with a newly developed high‐performance liquid chromatographic (HPLC) assay for differentiation proteins. Immature granulocytes, isolated from guinea pig bone marrow by Ficoll density centrifugation, were placed in liquid cultures and incubated for periods up to 1 week. In the presence of 10% dialyzed, normal guinea pig serum, these cells were almost all converted to mature granulocytes, whereas at serum concentrations below 1% mostly macrophages were formed. Cell multiplication does not appear to be necessary for granulocyte maturation in this culture system. The data also show that morphological maturation in vitro is accompanied by the formation of all the major membrane and secondary granule differentiation proteins detected by the HPLC assay in extracts of mature granulocytes formed in vivo. The techniques described here should facilitate the isolation and purification of the factors in normal serum that control the induction of synthesis of these differentiation markers.
Localisation of Alanine uptake by cultured renal epithelial cells (LLC‐PK1) to the basolateral membrane
Authors: Sepulveda Francisco V. , Pearson Jeremy D.
Published: February 2005

Alanine uptake by LLC‐PK1 cells has previously been demonstrated to be almost exclusively sodium dependent. We here confirm that when the cells are grown on an impermeable substratum there is a marked fall in uptake as confluence is reached. By applying an autoradiographic technique to visualize transported alanine, it is clear, however, that even in subconfluent cultures there is marked cellular inhomogeneity with regard to uptake, which takes place predominantly in those cells at the periphery of growing islands and not those at the interior. In contrast, when cells are grown on permeable substrata, a uniform distribution of silver grains is found. In two other types of experiment, we found that when confluent cell monolayers on an impermeable support were treated briefly with a chelating agent or suspended by mechanical treatment, there was a marked increase per cell in sodium‐dependent alanine uptake and in ouabain‐sensitive potassium uptake. We conclude that the apparent fall in alanine uptake as cells reach confluence on an impermeable support is due to masking of transport sites, which are predominantly, if not exclusively, located at the basolateral membrane.
Coordinate regulation of glycolysis by hypoxia in mammalian cells
Authors: Robin Eugene D. , Murphy Brian J. , Theodore James
Published: February 2005

The impact of hypoxic exposure on the activities of all 11 glycolytic enzymes was studied in cell culture into mammalian cells—mouse lung macrophages and L8 rat skeletal muscle cells. During hypoxic exposure, the measured activity of all glycolytic enzymes increased, establishing coordinate regulation. Three nonglycolytic cytoplasmic enzymes showed no change in activity under the same conditions, suggesting a specific mechanism. Hypoxia appears to increase the activities of all glycolytic enzymes whether rate‐limiting or not, presumably increasing adenosine triphosphate availability despite decreased O2 supply.
Aluminum lons stimulate DNA synthesis in quiescent cultures of Swiss 3T3 and 3T6 cells
Authors: Smith Jeffrey Bingham
Published: February 2005

Micromolar concentrations of Al3+ are shown to be strongly mitogenic for quiescent cultures of Swiss 3T3 and 3T6 cells. Al3+ caused a striking shift in the dose‐response curve for the effect of fetal bovine serum on 3H‐thymidine incorporation. In the absence of serum the mitogenic effect of aluminum was greatly potentiated by insulin or cholera toxin, but not epidermal growth factor or 12–0‐tetradecanoyl‐phorbol‐13‐acetate. The stimulation of DNA synthesis was maximal by 15–20 μMM Al3+. Al3+ at 100 μMM had no inhibitory effect on DNA synthesis. Al3+ had no significant effect on cellular cyclic adenosine monophosphate in the presence or absence of insulin or an inhibitor of cyclic nucleotide phosphodiesterases.
Uptake of epidermal growth factor into a lysosomal enzyme‐deficient organelle: Correlation with cell's mitogenic response and evidence for ubiquitous existence in fibroblasts
Authors: Miskimins W. Keith , Shimizu Nobuyoshi
Published: February 2005

The internalization process following cell surface receptor binding by epidermal growth factor (EGF) was studied. It was found that EGF is taken up into a dense, membranous organelle. This organelle is deficient in lysosomal enzyme activity and is biochemically dissimilar to the major lysosomal fraction. The uptake of EGF by this organelle was demonstrated in three different cell types representing three different species. Each of these cell types is highly responsive to the mitogenic action of EGF. These results indicate that EGF is endocytosed and delivered to a dense, possibly nonlysosomal, organelle which is ubiquitous in fibroblasts. Furthermore, we demonstrate a close, positive correlation between EGF uptake into this fraction and the ability of cells to respond to the mitogen. A negative relationship between uptake into the subcellular fraction containing lysosomal enzymes and EGF‐stimulated DNA synthesis was observed. Using numerous incubation conditions no exceptions to the correlation between EFG uptake into the lysosomal enzymedeficient fraction and EGF‐induced DNA synthesis were observed. These results suggest a role for this dense organelle in the production of a mitogenic signal.
Oxygen dependence of cellular metabolism: The effect of O2 tension on gluconeogenesis and urea synthesis in isolated rat hepatocytes
Authors: Kashiwagura Tadashi , Wilson David F. , Erecińska Maria
Published: February 2005

The dependencies of gluconeogenesis and urea synthesis on oxygen concentration were measured in suspensions of isolated rat hepatocytes and compared with the O2 dependence of cellular energy supply (reduction of cytochrome c, respiratory rate, mitochondrial [NAD+]/[NADH], lactate production, and [ATP]/[ADP][Pi]). As the oxygen concentration was decreased, production of both glucose and urea declined; the changes were observable at 20 μM oxygen and below, with the apparent Km values for both processes of near 5 μM. The similar dependence of gluconeogenesis and urea synthesis on oxygen concentration indicates that the two pathways have equal access to the cellular ATP supply, i.e., there is no evidence that either pathway is preferentially turned off to spare ATP for the other. The cellular energy state had an oxygen dependence similar to that of glucose and urea synthesis. It is suggested that the behavior of gluconeogenesis and urea production is a reflection of homeostatic regulation of cellular metabolism which is designed to respond to changes in [ATP]/[ADP][Pi].
Advance toward S phase and retreat toward deeper “GO” states in resting 3Y1 cells with environmental changes
Authors: Zaitsu Hirokazu , Kimura Genki
Published: February 2005

To elucidate conditions which affect the lag time for resting cells to enter S phase after serum stimulation, we used a wild‐type 3Y1 rat fibroblast line and four temperature‐sensitive mutants of 3Y1 (3Y1tsD123, 3Y1tsF121, 3Y1tsG125, and 3Y1tsH203). Among these five lines, in only tsG125 cells was there an obviously prolonged lag time with increase in time in resting state at 33.8°C. The resting wild‐type 3Y1 cells, preexposed to 39.8°C, also showed a prolongation of lag time. The prolongation in tsG125 had a certain limit. Preexposure to 39.8°C before serum stimulation accelerated such prolongation in tsG125 to its limit, but did not change the limit, per se. Resting tsG125 cells stimulated by serum at 39.8°C, did not enter S phase, yet they did advance toward S phase. When they were kept at 39.8°C, they retreated toward a deeper resting state (“GO”) with time. These retreats correlated with the decrease in stimulating activity in the culture media. About 20% of the resting tsG125 cells stimulated by serum at 39.8°C were committed to enter S phase, when the extent of commitment was examined at 33.8°C. Most of the tsG125 cells committed at 33.8°C did not enter S phase, when the extent of commitment was examined at 39.8°C. More cells were committed after stimulation at 33.8°C than at 39.8°C, when the test was done at 33.8°C. We suggest that resting cells may be reversibly changed within range of resting states, in either direction, that is, advance toward S phase or retreat toward deeper “GO”. These changes may be determined by alterations in the balance between synthesis and decay of the preparedness for the initiation of DNA synthesis caused by cellular response to environmental changes (e.g., medium activity, temperature, etc.). The ts defect in tsG125 may affect the cell cycle progression, both before and after commitment by serum.
Cell growth and differentiation in vitro in mouse macrophages transformed by a tsA mutant of simian virus 40. II. Changes in the distribution of DNA content during the reversible transition between macrophage and nonmacrophage states in the cultures of tsA 640‐transformed macrophages
Authors: Tanigawa Takahiko , Okuda Atsuyuki , Takayama Hisao , Yamada Koji , Takagi Atsushi , Kimura Genki
Published: February 2005

Cultures of mouse macrophage cell lines transformed by wild‐type or the tsA640 mutant of simian virus 40 (SV40) show a reversible phenotypic transition between the nonmacrophage (proliferating phase) and the macrophage (stationary phase) states (Takayama, 1980; Tanigawa et al., 1983). Distribution of DNA content in the cultures of the tsA640‐transformed macrophage lines in the process of the phenotypic transition was determined by flow cytometry. Taking the mean DNA content of mouse peritoneal macrophages as 1 unit in the scale of fluorescence intensity in the flow cytogram, the transformed macrophages showed, at 33°C, two peaks, one located around the 1.0‐unit position (peak 1.0) and the other around the 1.6‐unit position (peak 1.6), and a plateau distribution continuing to 3.2 units. Peak 1.0 was predominant in the stationary‐phase culture, whereas peak 1.6 was predominant in the proliferating‐phase culture. Almost the entire population of the strictly resting culture, which was obtained by culturing the stationary‐phase culture for a further 5 days at nonpermissive temperature (39°C), was phagocytic, and had accumulated at peak 1.0. Cells in peak 1.0 moved to peak 1.6 and to higher positions, after the strictly resting culture was sparsely reseeded and incubated at 33°C. In contrast, the DNA content distribution of the successively proliferating cells, which were obtained by repeated passage of an extensively proliferating culture and none of which were phagocytic, was similar to that of proliferating hypotetraploid BALB/c3T3 fibroblasts with a G1 peak at 1.6 unit followed by a plateau containing S‐ and G2‐phase cells. The peak 1.0 cell population appeared from the recloned population of the successively proliferating cells in company with the restoration of the culture condition‐dependent phagocytic ability when cocultured with primary macrophages. Each peak in the flow cytogram reflected fairly well DNA content per cell as determined by other methods.