bluuebunny/crossref_metadata_2025 huggingface dataset:
Sodium fluxes in human fibroblasts: Kinetics of serum‐dependent and serum‐independent pathways
Authors: Villereal Mitchel L.
Published: February 2005

Sodium influx in serum‐deprived human fibroblasts is by way of a pathway which shows saturation kinetics. A plot of initial Na influx versus [Na]o ([Na]i = 10 mM) gives a simple Michaelis‐Menten type of curve with a K1/2 = 70.0 ± 8.1 mM and a Vmax = 14.5 ± 1.9 /mol/g prot/min. A similar plot of initial Na influx versus [Na]o in the presence of 10% fetal bovine serum (FBS) gives a nonsatu‐rating curvilinear response which appears to be biphasic. A plot of the serum‐dependent Na influx versus [Na]o (obtained by subtracting the curve in the absence of FBS from the curve in the presence of 10% FBS) shows that there is a linear relationship between serum‐induced Na influx and external [Na]. At physiological Na concentrations, in the presence of FBS, the serum‐induced Na influx is equal to the amiloride‐sensitive Na flux, whereas in the absence of serum amiloride inhibits less than 10% of the Na influx. The effect of intracellular Na on Na flux was tested by preloading cells with Na in a digitoxin‐containing medium prior to measurement of Na flux. A plot of steady‐state Na exchange flux versus [Na]o ([Na]i ≈︁ [Na]o) in the absence of serum gives a curve that appears to saturate at approximately 100 mM Na (flux = 100 /μmol/g prot/min) and then declines with increasing [Na] (flux = 40 /μmol/g prot/min at 150 mM). In contrast to Na influx in control serum‐deprived cells, Na flux in Na‐loaded cells is dramatically inhibited by the presence of amiloride. Since the peak Na exchange flux of 100 μmol/g prot/min is greatly in excess of the Vmax for Na influx in control serum‐deprived cells and the enhanced Na flux is amiloride‐sensitive, elevating intracellular Na must somehow activate the amiloride‐sensitive Na transport system, which is normally only minimally active in the absence of serum.
Cellular regulatory role of leukotriene B4: Its effects on cation homeostasis in rabbit neutrophils
Authors: Sha'Afi R. I. , Naccache P. H. , Molski T. F. P. , Borgeat P. , Goetzl E. J.
Published: February 2005

We have found that exogenous leukotriene B4 modifies calcium homeostasis in rabbit neutrophils in a manner essentially analogous to that of the chemotactic peptide f‐Met‐Leu‐Phe. Leukotriene B4 causes a rapid and dose‐dependent increase in membrane permeability to calcium and a release of calcium from previously unexchangeable intracellular pool(s). The net result of these changes is to transiently elevate the intracellular level of exchangeable calcium. A stereoisomer of leukotriene B4 with greatly reduced secretory activity toward neutrophils (5S, 12S‐di HETE) is essentially without effect on the rate of 45Ca uptake at concentrations equal to those that produce near maximal enhancement by leukotriene B4. Leukotriene B4, in addition to its effects on calcium metablolism, also increases the rate of 22Na influx into rabbit neutrophils. The relationships between the action of leukotriene B4 on calcium homeostasis and the neutrophil‐directed activities of arachidonic acid and its lipoxygenase metabolites are discussed.
Regulation of the prereplicative changes in the synthesis and transport of messenger and ribosomal RNA in regenerating livers of normal and hypocalcemic rats
Authors: Walker P. Roy , Whitfield James F.
Published: February 2005

Partial hepatectomy induces increases in the synthesis of both messenger (poly(A)+) and ribosomal (poly(A)−) RNA, which precede the initiation of DNA synthesis. The increase in poly(A)+RNA, which commences soon after surgery and reaches a peak 1–3 hours later is particularly striking. Disruption of this early increase in poly(A)+RNA synthesis by colchicine (and other microtubule disrupters) or indomethacin results in a failure to initiate DNA synthesis. This suggests that prostaglandins and the microtubules are involved in the mechanism of proliferative activation. Hypocalcemia, which also prevents the initiation of DNA synthesis, has no effect on the prereplicative changes in either messenger or ribosomal RNA synthesis. These results help to define the critical stages of prereplicative development and give some insight into their regulation.
Cytochalasin inhibition of isolated rat gastric parietal cell function
Authors: Rosenfeld Gary C. , McAllister Ellen , Thompson W. Joseph
Published: February 2005

Submicrogram concentrations (0.04‐‐0.29 μM) of the microfilament disrupting agents cytochalasins D, E, and B (CD, CE, CB) were shown to inhibit secretagogue‐stimulated 14C‐aminopyrine accumulation (AP) in isolated rat gastric mucosal parietal cells. The microtubule disrupting agent colchicine had little influence on AP accumulation. Histamine‐ and dibutyryl cyclic AMP (DbcAMP)‐stimulated AP accumulation was inhibited with an order of potency CD > CE ≈︁ CB. CB inhibition of these secretagogue actions was, however only approximately 65‐‐70% of the maximal stimulated response, whereas CD and CE caused 100% inhibition. On the other hand, carbamylcholine‐stimulated AP accumulation was inhibited 100% by all cytochalasins tested with an order of potency CD ≈︁ CE > CB. These data are discussed in relation to acid secretagogue‐induced morphological changes involving actin filament organization in parietal cells.
Effects of thyroliberin (TRH) on cell proliferation and prolactin secretion by GH3/B6 rat pituitary cells: A comparison between serum‐free and serum‐supplemented media
Authors: Brunet N. , Rizzino A. , Gourdji D. , Tixier‐Vidal A.
Published: February 2005

Numerous studies have shown that prolactin (PRL) production by GH3 cells grown in serum supplemented media is regulated by several hormones including thyroliberin (TRH). The recent availability of hormonally defined, serum‐free media for the growth of GH3 cells has made it possible to determine the effect of TRH in absence of other prolactin regulating hormones. Here we demonstrate that transfer of GH3/B6 cells from serum‐supplemented medium to serum‐free media results in several important changes: (1) altered growth response to TRH, (2) altered cell attachment and morphology, (3) greatly reduced prolactin production, and (4) greater stimulation of prolactin production by TRH. After 4 days in serum‐free medium, TRH stimulates prolactin production by as much as 5‐fold instead of approximately 2‐fold in serum‐supplemented medium. Furthermore, this increased responsiveness to TRH in serum‐free medium is accompanied by a 10‐fold decrease in the ED50 for TRH (concentration needed for half‐maximal response) and paradoxically by a 2‐fold reduction in the number of high‐affinity TRH binding sites without significant change of their association constant.
Adhesive substrates for fibronectin
Authors: Klebe Robert J. , Bentley Kevin L. , Schoen Robert C.
Published: February 2005

In order to promote cell attachment, fibronectin must first undergo activation by a suitable substrate. In this study, 52 materials have been surveyed for their ability (a) to bind fibronectin, (b) to activate the cell‐adhesive property of fibronectin, and (c) to support the growth of cells. Many plastics, polysaccharides, metals, and ceramics were found to support cell growth as well as the fibronectin‐dependent attachment of cells. Several other substrates have been identified that were inactive in promoting either cell attachment or growth. Hydrophobic substrates were found to be active in fibronectin activation, whereas hydrophilic substrates were found to be inactive.

Since fibronectin binds to substrata of extremely varied chemical composition, it is clear that the binding of fibronectin to such substrata is nonspecific in nature. Since protein pretreatment of all substrata, except collagen and poly(L‐lysine), abolished the physical binding of fibronectin, the binding of fibronectin to artificial substrata is probably ascribable to a nonspecific hydrophobic protein‐substratum interaction. In contrast, several lines of evidence indicate that the interaction between fibronectin and collagen displays biological specificity.

Poly(hydroxyethylmethacrylate)(poly(HEMA)), which has previously been shown to be nonadhesive for cells, is demonstrated here to be unique in its inability to bind fibronectin. Addition of one part per million of an adhesive polymer to poly(HEMA) permits fibronectin binding to occur.
Hb Switching in neonatal cultures. Increase of Hb A synthesis in presence of an erythroid potentiating activity (EPA)
Authors: Testa Ugo , Vainchenker William , Guerrasio Angelo , Beuzard Yves , Breton‐Gorius Janine , Rosa Jean , Lusis A. J. , Golde David
Published: February 2005

The role of EPA (erythroid potentiating activity) on the growth and on the pattern of hemoglobin synthesis in erythroid colonies from human neonates was investigated. Conditioned medium from the Mo cell line was used as a source of EPA. The results have shown that the addition of Mo medium to cultures determined a significant enhancement of the number and size of BFU‐E and an increase of β chain synthesis. The acceleration of hemoglobin switching is not related to an amelioration of the maturation of the erythroid colonies when grown in the presence of Mo medium. The enhancement of Hb A synthesis induced by Mo medium can directly be related to its EPA, which may operate by two different mechanisms: (1) the recruitment of early erythroid progenitors already preprogrammed to synthesize prevalently β chains, or (2) the modulation of β and γ gene activity in cord blood BFU‐E. Some evidence suggests that the first mechanism does operate.
Effects of Ca ions on action potentials in immature cultured neurons from chick cerebral cortex
Authors: Mori Junko , Ashida Hiroshi , Maru Eiichi , Tatsuno Jiro
Published: February 2005

Action potentials evoked by depolarizing pulses were studied in immature cultured cerebral cortical neurons from chick embryos. The majority of action potentials were rather small, and they were still elicited in the presence of 10−7 gm/ml tetrodotoxin (TTX), but were almost completely abolished in Na+‐free solution or by 10−5 gm/ml TTX in Tyrode's solution. The elevation of external Ca2+ concentration not only increased the maximum rates of rise of action potentials in normal Tyrode's solution with and without low (10−7 gm/ml) TTX but also regenerated action potentials in high (10−5 gm/ml) TTX‐containing Tyrode's solution or in Na+‐free solution. These high Ca2+ effects were blocked by Mn2+ or Co2+. These results suggest that action potentials, which were predominantly Na‐dependent, are partially contributed by Ca ions in immature chick cerebral cortical neurons.
The relationship between the insulin content and inhibitory effects of bovine colostrum on protein breakdown in cultured cells
Authors: Ballard F. J. , Nield M. K. , Francis G. L. , Dahlenburg G. W. , Wallace J. C.
Published: February 2005

Protein degradation in ten mammalian cell lines is markedly inhibited by small amounts of bovine colostrum. This response is consistent with the growth‐promoting activity of colostrum that has been reported previously. Fractionation of colostrum on DEAE cellulose showed that most of the inhibitory activity against protein breakdown in H35 cells coeluted with insulin. Insulin concentrations in different batches of bovine colostrum ranged from 0.67 nM to 5.7 nM, approximately 100‐fold higher than in blood. The sensitivity of protein breakdown in H35 or MH1C1 hepatoma lines to these colostrum samples was proportional to their insulin concentrations and could largely be accounted for by the amount of insulin present. Removal of insulin from colostrum by means of a protein A‐anti‐insulin antibody affinity column was accompanied by a loss of the ability of colostrum to inhibit protein breakdown in H35 or MH1C1 cells. However, in IMR90 fibroblasts, a cell line with a similar sensitivity to colostrum as the two hepatomas but very insensitive to insulin, protein breakdown was still inhibited by the insulin‐free colostrum. These results suggest that, whereas the effect of bovine colostrum in H35 or MH1C1 cells is actually a response to insulin, different growth factors in colostrum account for the inhibition of protein breakdown in other cell lines.
Changes of ornithine decarboxylase activity and polyamine content upon differentiation of mouse NB‐15 neuroblastoma cells
Authors: Chen Kuang Yu , Presepe Vincent , Parken Nancy , Liu Alice Y.‐C.
Published: February 2005

The possible functions of ornithine decarboxylase (ODC) and polyamines in the differetiation of mouse NB‐15 neuroblastoma cells were investigated by examining the changes of these parameters in the differentiaton and nondifferentiating NB‐15 cells over a 5‐day culture period. Differentiation of NB‐15 cells was induced by the addition of dibutyryl cyclic AMP and 3‐isobutyl‐1‐methylxanthin (IBMX) to the growth medium and was monitored by neurite outgrowth, increase of acetylcholinesterase (AChE), and RI cAMP‐binding protein. Plating of NB‐15 cells in fresh serum‐containing growth medium was accompanied by rapid growth and a marked increase of ODC activity, this early increase of ODC activity was attenuated, both in duration and in magnitude, in the differentiating cells. The spermidine content of the differentiating neuroblastoma cell was significantly lower than that of the nondifferentiating cells. In the fully differentiated neuroblastoma cells, the ODC activity and spermidine content were lower than that of the undifferentiated cells by approximately 15‐fold and five‐fold, respectively. Based on these results it is proposed that changes of polyamine metabolism may be of significance in the differentiation of mouse neuroblastoma cells.
Cytoplasts can transfer factor(s) that stimulate quiescent fibroblasts to enter S phase
Authors: Mercer W. Edward , Schlegel Robert A.
Published: February 2005

Cytoplasts prepared from fibroblasts arrested by hydroxyurea were fused with serum‐arrested, quiescent fibroblasts. In contrast to unfused mon‐nucleated cells in the same culture, a sizable fraction of these cybridoids entered S phase in the absence of extracellular serum stimulation. Because DNA synthesis commenced only after a considerable lag following fusion, it was concluded that the cytoplasts contain factors which initiate the progression of quiescent cells toward S phase.
Selective inhibition of DNA synthesis by a protein released from spleen cells
Authors: Miyata Shohei , Kihara Hirozi K.
Published: February 2005

Spleen cells cultured release proteins into the culture medium, some of which shows both binding activity to single stranded DNA (ss‐DNA) and inhibitory activity to 3H‐thymidine incorporation into cells. The inhibitory protein of DNA synthesis was purified to near homogeneity by ammonium sulfate precipitation, ss‐DNA‐cellulose affinity chromatography, and gel filtration chromatography. The molecular weight of the major band of this protein, as estimated by sodium dodecyl‐sulfate gel electrophoresis, was approximately 14,000. The effect of the protein was compared with various cell lines and primary cell culture, and it was found that the protein strongly inhibited DNA synthesis of HeLa, L, and HEp2 cells, but only inhibited to a minor extent synthesis of Ehrlich cells and primary culture of mouse embryo.
Fibroblasts from Retinoblastoma patients: Enhanced growth in fetal calf serum and a normal response to ionizing radiation
Authors: Kossakowska A. E. , Gallie B. L. , Phillips R. A.
Published: February 2005

Retinoblastoma is a rare malignant eye tumor found almost exclusively in young children. In 30% of cases, the tumor is bilateral and is inherited as an autosomal dominant trait. In such patients, all of the cells in the body must carry the mutation predisposing to retinoblastoma. To search for the expression of the gene in cells outside the retina, we have studied several in vitro properties of skin fibroblasts from patients with bilateral retinoblastoma. Measurement in low concentrations of fetal calf serum of the initial growth rate and the plating efficiency show that fibroblasts from retinoblastoma donors grow significantly better than those from normal donors. However, we were unable to confirm the results of other investigators that fibroblasts from donors with bilateral retinoblastoma are unusually sensitive to ionizing radiation. In family studies, skin fibroblasts from normal siblings had the same radiation sensitivity as fibroblasts from sibling with retinoblastoma.
Stimulation of thymidine kinase activity by chloramphenicol in Naegleria
Authors: Corff Sondra , Yuyama Shuhei , Young Paul G.
Published: February 2005

In the present study, the possible association of thymidine kinase (TK) with mitochondria in Naegleria was investigated by treating growing and differentiating cells with chloramphenicol (CAP), an inhibitor of mitochondrial protein synthesis. In some systems, CAP causes an overproduction of mitochondrial proteins coded for in the nucleus. The present results show that in growing Naegleria, CAP stimulates a dramatic increase in TK activity while growth and division is gradually inhibited. CAP does not stabilize the enzyme in vivo or in vitro. The stimulation is cycloheximide (CHI)‐sensitive and specific since nucleoside phosphotransferase activity does not increase. In cells stimulated to differentiate, CAP does not prevent differentiation or the expected decrease in TK activity. Using polyacrylamide gel electrophoresis, a comparison of TK in mitochondrial and postmitochondrial fractions of CAP‐treated and untreated cells was made. Results suggest some processing of the enzyme, resulting in a slight change in electrophoretic mobility. No mitochondrial TK was found. The stimulation of a cytoplasmic enzyme by CAP suggests a form of mitochondrial control of nuclear transcription for other than mitochondrial proteins. DNA synthesis in CAP‐treated cells was not stimulated, suggesting (since TK and DNA synthesis are usually tightly coupled) an uncoupling of these two events, most likely, at the beginning of the S phase.
6‐Deoxy‐D‐glucose and D‐xylose: Analogs for the study of D‐glucose transport by mouse 3T3 cells
Authors: Romano Antonio H. , Connell Nancy D.
Published: February 2005

6‐Deoxy‐D‐glucose and D‐xylose, structural homomorphs of D‐glucose that lack a 6‐hydroxyl group or a 6‐hydroxymethyl group, respectively, are transported efficiently by mouse 3T3 cells, with good affinity and high specificity for the D‐glucose transport system. Since these analogs lack the 6‐hydroxyl group, which is the site of phosphorylation of glucose by hexokinase, they are taken up and are recoverable from cells in an unchanged state. Thus, 6‐deoxy‐D‐glucose and D‐xylose offer advantages as transport substrates over 2‐deoxy‐D‐glucose, which is phosphorylated by intercellular hexokinases, and 3‐O‐methyl‐D‐glucose, which shows a lower specificity for the D‐glucose transport system.
Effects of cycloheximide and actinomycin D on the amino acid transport system of tetrahymena
Authors: Blum J. J.
Published: February 2005

Tetrahymena pyriformis were grown in axenic culture to late logarithmic and stationary phases, resuspended in an inorganic medium, and the rates of transport of α‐aminoisobutyric acid (AIB) and of the decarboxylation of L‐[1‐14C]leucine and L‐[1‐14C]tyrosine were measured. There was a rapid loss of each of these measures of amino acid transport in both late log phase and stationary phase cells. Addition of actinomycin D to the washed cells caused a small increase in the rate of loss of capacity to decarboxylate tyrosine and leucine. Addition of cycloheximide to the washed cells caused a reduction in the rates of loss of capacity to transport AIB and to decarboxylate leucine and tyrosine except that in late log phase cells cycloheximide markedly increased the rate of loss of capacity to decarboxylate leucine. When cells that had been pretreated with chlorpromazine to reduce their amino acid transport capacity were washed and resuspended in proteose peptone the capacity to decarboxylate tyrosine and leucine increased to control values within 1.5 hours. Addition of actinomycin D reduced the rate of recovery of transport capacity, but addition of cycloheximide caused transport capacity to decrease further. These results raised the possibility that there were two amino acid transport systems in this cell. The finding that AIB and N‐methylaminoisobutyrate are both taken up by Tetrahymena, the latter at one‐eighth the rate of the former, but that neither one alters the rate of uptake of the other provides preliminary support for this possibility. The present results further suggest that the transport system(s) has a short lifetime and that the balance between rate of synthesis and rate of loss of the transport system is controlled in part by the presence of exogenous amino acids.
Synergistic growth stimulation of corneal fibroblasts by components of mesodermal growth factor from murine submaxillary glands
Authors: Haraguchi Kenneth H. , Knox Robert J. , Weimar Virginia L. , Anderson Ronald A.
Published: February 2005

Recycled mesodermal growth factor (R‐MGF) is a pool of proteins of 26,000 molecular weight obtained by recycling gel chromatography of male murine submaxillary gland extracts. R‐MGF strikingly accelerates corneal stromal wound healing in vivo, fibroblast growth and migration in cultured corneal buttons and is shown here to stimulate stromal fibroblast growth and division in tissue culture. Chromatographic fractionation of R‐MGF has yielded several components, none of which has a greater biological potency than the parent R‐MGF. In contrast, two components, MGF‐I and ‐II, when recombined synergistically stimulate fibroblast response in tissue culture and organ culture in excess of those obtained with the parent R‐MGF. Three MGF components (I, III, and IV) have been purified and are inactive at 10–15 m̈g/ml in organ culture but potently stimulate fibroblast responses when combined in pairs containing 7.5 m̈g of each component. The striking synergism in organ culture suggests that the stimulation of wound healing by R‐MGF in vivo may also reflect synergistic action of more than one R‐MGF component. Procedures for isolating gram quantities of R‐MGF and for the purification of different R‐MGF components by ion exchange chromatography are detailed.
Glycosaminoglycan changes involved in polymorphonuclear leukocyte activation in vitro
Authors: Vannucchi Simonetta , Fibbi Gabriella , Cappelletti Renzo , Rosso Mario Del , Chiarugi Vincenzo
Published: February 2005

The changes of cell coat and endocellular glycosaminoglycans associated with adhesion of polymorphonuclear leukocytes to tissue culture dishes were studied by electrophoresis on cellulose acetate. Polymorphonuclear leukocytes are coated by chondroitin 4 sulphate, while undersulphated chondroitin 4 sulphate and heparan sulphate are present in the cytoplasm. Upon adhesion polymorphonuclear leukocytes shed into the culture medium chondroitin 4 sulphate and concomitantly hyaluronic acid and heparan sulphate are exposed at the cell surface and shed into the culture medium. The role of these compounds in polymorphonuclear leukocyte physiology is discussed.
Inhibition of the serum‐dependent, amiloride‐sensitive sodium transport pathway in human fibroblasts by extracellular divalent cations
Authors: Villereal Mitchel L.
Published: February 2005

Sodium influx in serum‐deprived human fibroblasts in a nominally Ca‐free, Mg‐free medium is significantly higher (17.8 ± 1.9 μmole/g prot/min) than that measured in a medium containing 1.8 mM Ca and 1 mM Mg (10.9 ± 0.7 μmole/g prot/min), and is stimulated dramatically (44.1 ± 6.1 μmole/g prot/min) by the addition of 10% fetal bovine serum (FBS), suggesting that an enhanced influx of Ca ions is not a necessary condition for serum activation of the amiloride‐sensitive Na influx pathway. The addition of 2 mM ethylenediaminetetraacetic acid (EDTA) to serum‐deprived cells in a low Ca, low Mg medium also results in a dramatic stimulation of Na influx (40.4 ± 3.7 μmole/g prot/min), while the addition of EDTA to cells assayed in a low Ca, low Mg medium in the presence of FBS has no significant effect on Na influx (45.3 ± 4.1 μmole/g prot/min). Thus, the stimulatory effects of FBS and EDTA are not additive. Kinetic analysis in the presence of varying amiloride concentrations indicate that the EDTA‐stimulated Na influx occurs via the amiloride‐sensitive Na pathway. The activation of Na influx in cells rinsed free of Ca and Mg Can be readily reversed by the addition of Ca or Mg to the assay medium. The Ca concentration required to give 50% inhibition of Na influx is 52 ± 7.6 μM (n = 3) for cells assayed in serum‐free medium and 272 ± 29 μM (n = 3) for cells assayed in the presence of 10% FBS. At physiological Ca concentrations (1.8 mM) the Na influx is maximally inhibited by Ca both in the presence and absence of serum. Since Na influx in 1.8 mM Ca medium is 2.5‐fold higher in the presence of serum than in its absence, these data suggest that the serum‐induced change in the K, for Ca modulation of the amiloride‐sensitive Na transport pathway is not sufficient to explain the serum stimulation of Na influx in human fibroblats.
High intracellular pH accompanies mitotic activity in murine lymphocytes
Authors: Gerson Donald F. , Kiefer Hansruedi
Published: February 2005

Intracellular pH is crucial to the control of many cellular processes such as glycolysis, protein synthesis, and cell division. To study the relation between intracellular pH and mitotic activity in actively dividing Con A‐ or LPS‐stimulated splenic lymphocytes, a method was developed to determine intracellular pH using a fluorescence‐activated cell sorter. The new method uses the pH‐sensitive fluorochrome, 4‐methylumbelliferone. Results obtained with it not only correspond qualitatively with the results obtained using 14C‐dimethyloxazolidinedione (DMO) but also clearly show the active and inactive subpopulations. The intracellular pH of mitogen‐stimulated murine lymphocytes increases from pH 7.15 to pH 7.45 when the population has greatest mitotic activity. The intracellular pH of three virus‐transformed lymphocyte cell lines is higher by ∼ 0.5 pH unit when the cells are in exponential growth compared to stationary phase.
Ribosomal proteins are synthesized preferentially in cells commencing growth
Authors: Tushinski Robert J. , Warner Jonathan R.
Published: February 2005

Mouse 3T3 cells, in stationary phase because of serum deprivation, have only half the ribosome content of growing cells. Furthermore, the proportion of protein synthesis devoted to ribosomal proteins is only half that in growing cells. On addition of serum the synthesis of each ribosomal protein increases threefold, demonstrating the coordination of the synthesis of the ribosomal proteins. Half that increase is due to a general increase in total protein synthesis; half is due to a differential increase in ribosomal protein synthesis. The latter is abolished by a concentration of actinomycin D which blocks only ribosomal RNA transcription. The results are discussed with reference to a general hypothesis of growth regulation proposed by Stanners et al. (1979).
The use of flurescent dyes to measure membrane potentials: A response
Authors: Smith Thomas C.
Published: February 2005

The use of fluorescent cyanine dyes to estimate membrane potential in cell suspensions has been considered. Several problems related to the application of the dyes have been reviewed. These problems include: (1) alteration of the membrane potential (Em) and factors involved in establishing Em by the dyes themselves, (2) the effect of altered energy metabolism on the fluorescent response of the dyes and on Em, and (3) calibration of dye fluorescence. Recent reports that advocate the use of the fluorescent dyes are misleading.
Transcription of chick genes by mammalian RNA polymerase II in chick erythrocyte‐mammalian cell heterokaryons
Authors: Zuckerman Steven H. , Linder Stig , Ringertz Nils R.
Published: February 2005

The introduction of chick erythrocyte nuclei into mammalian cell cytoplasms results in their reactivation as evidenced by the de novo transcription of chick genes and the synthesis of both globin and constitutive proteins. In the present study, chick erythrocytes have been fused to L6 rat myoblasts and to alphaamanitin‐resistant variants of L6 to determine whether the chick or the mammalian RNA polymerase II was responsible for transcription of chick genes. Heterokaryons formed by fusing chick erythrocytes with alpha‐amanitin‐resistant L6 myoblasts synthesize both chick globin and chick constitutive proteins in the continued presence of 5 μg/ml alpha amanitin ten days postfusion. Both the synthesis of globin and other chick polypeptides occurs at levels comparable to those observed for untreated heterokaryons. Synthesis occurs under conditions in which insignificant chick RNA polymerase II activity can be detected irv wild‐type heterokaryons by autoradiography. These results demonstrate that RNA polymerase II is one of the mammalian proteins that is selectively taken up by the chick nucleus during reactivation in the presence of alpha amanitin. Furthermore, the mammalian RNA polymerase II alone can account for the transcription of both differentiation specific and constitutive genes in the chick nucleus.
Effects of urea treatment on the divalent cation‐independent cell aggregation of 3T3MIT fibroblasts
Authors: Wright Thomas C. , Robinson John M. , Karnovsky Morris J.
Published: February 2005

Growth of nontransformed 3T3MIT fibroblasts in media containing 200 mM urea leads to the rapid acquisition of the transformed adhesive phenotype as evidenced by an increased rate of divalent cation‐independent cell aggregation. The increased rate of divalent cation‐independent cell aggregation of urea treated 3T3MIT cells shares many properties with the high rate of aggregation of transformed cells including a sensitivity to treatment with trypsin or hyaluronidase and a reduction in the presence of exogenously added hyaluronic acid. Reversal of the urea‐induced increase in aggregation occurs within 24 hours in the absence of urea and can be blocked by 0.2 μg/ml cycloheximide. In the presence of cycloheximide, low rates of aggregation can be restored by the addition of urea‐conditioned supernatents. The results of these experiments suggest that the loss of an aggregation‐inhibitory activity during growth in media containing 200 mM urea is responsible for the increased rate of divalent cation‐independent cell aggregation. After removal of this aggregation‐inhibitory activity, the normally lowly adhesive 3T3MIT cells become phenotypically transformed with regards to the rate of divalent cation‐independent cell aggregation.
Amiloride, protein synthesis, and activation of quiescent cells
Authors: Lubin Martin , Cahn Frederick , Coutermarsh Bonita A.
Published: February 2005

Amiloride is known to inhibit both influx of sodium ions and activation of quiescent cells by growth factors. The coincidence of these effects has been cited to support the proposal that influx of sodium ions acts as a mitogenic signal. Although it was noted that amiloride inhibited protein synthesis, this was attributed to an action on transport of amino acids, particularly those coupled to sodium fluxes. We find, however, that amiloride directly inhibits polypeptide synthesis in a reticulocyte lysate. In Swiss 3T3 cells, concentrations of amiloride and of cycloheximide that are nearly matched in their degree of inhibition of protein synthesis, produce about the same degree of inhibition of transit of cells from G0 to S. Inhibition of protein synthesis is sufficient to explain the effect of amiloride on mitogenesis; the drug, therefore, is not suitable for testing the hypothesis that sodium influx is a mitogenic signal.
Intracellular distribution of transglutaminase activity during rat liver regeneration
Authors: Remington John A. , Russell Diane Haddock
Published: February 2005

Transglutaminase and ornithine decarboxylase activities have been assayed at intervals after partial hepatectomy in regenerating liver cells fractionated to obtain nuclear, cytoplasmic‐particulate, and cytoplasmic‐soluble fractions. Ornithine decarboxylase activity, localized entirely in the cytoplasmic fractions, undergoes a dramatic induction during the first 4 h after partial hepatectomy and remains elevated. This induction is very sensitive to inhibition by cycloheximide and actinomycin D, as previously reported. Transglutaminase activity is localized in both the cytoplasm and the nucleus with the highest specific activity in the nucleus. Nuclear transglutaminase activity approximately doubles in the first 2 h of liver regeneration, apparently as a result of a translocation of enzyme from the cytoplasm to the nucleus. Inhibitor studies indicate that the translocation is not dependent upon protein or RNA synthesis. In the first 2 h, actinomycin D slightly activates transglutaminase activity in the cytoplasmic‐particulate and nuclear fractions. Only at 4 h after the onset of regeneration do actinomycin D and cycloheximide show some inhibition of transglutaminase activity indicating de novo synthesis at this time. A broad increase of transglutaminase activity occurs from hours 12–16 to hour 32 after partial hepatectomy in the nuclear and cytoplasmic‐particulate fraction. These data suggest the existence of a function for transglutaminase in the nucleus of rat liver cells.
Properties and fate of plasma fibronectin bound to the tissue culture substratum
Authors: Haas Robert , Culp Lloyd A.
Published: February 2005

Plasma fibronectin (pFn) is a serum protein which, when adsorbed to a glass or plastic substratum, mediates the adhesion of fibroblasts in culture. We have studied some of the details of its adsorption and subsequent fate. By using 125l‐labeled pFn, we show that a substratum incubated with pFn adsorbs approximately 0.4 μg/cm2 pFn (a monomolecular layer), and one incubated with medium containing serum adsorbs approximately 7 ng/cm2 pFn (a 12‐fold enrichment relative to a random selection of the soluble proteins). SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE) suggests the bound serum proteins (eluted with SDS) are primarily BSA and β‐globulins. The bound pFn adheres so tightly, though, that most resists elution, as assayed (1) with pFn radioiodinated before binding, (2) with pFn radioiodinated after binding, or (3) by the cell spreading activity of the bound pFn retained after SDS treatment. Under culture conditions, there is a continuous “turnover” of substratum‐bound pFn: soluble pFn can bind to a serum‐coated substratum, while bound pFn is gradually removed by incubation with serum proteins. The presence of fibroblasts increases the rate of this removal several‐fold. By SDS‐PAGE the material removed (as well as that eluted from the substratum with SDS after cell detachment) is intact pFn or large (possibly proteolytically generated) fragments. Thus, pFn binds preferentially to the tissue culture substratum, but can be removed subsequently by the combined action of cells and other serum proteins.
Complementary recessive 25‐hydroxycholesterol‐resistant somatic cell mutants – assay of 25‐hydroxycholesterol binding activity
Authors: Sinensky M. , Logel J. , Torget R.
Published: February 2005

Complementation analysis of recessive 25‐hydroxycholesterol‐resistant mutants of the CHO‐KI cell shows the existence of at least two complementation groups, one of which is missing a binding activity for 25‐hydroxycholesterol. Both complementation groups are shown to be refractory to inhibition of cellular HMG‐CoA reductase activity and in the inhibition of biosynthesis of this enzyme by 25‐hydroxycholesterol.
Glycopeptide hormone production by cultured human diploid fibroblasts
Authors: Milsted Amy , Day Debra‐Lynn , Cox Rody P.
Published: February 2005

Human diploid fibroblasts in culture were examined for production of glycopeptide hormones. Forty‐one percent of the strains produced human chorionic gonadotropin (hCG) under normal growth conditions. Constitutive hCG synthesis was apparently unrelated to donor age, length of time in culture, or number of passages. Follicle stimulating hormone (FSH) was not found in any strain investigated. Only one cell strain produced free α‐chains of glycopeptide hormones. Hydroxyurea (HU) at a concentration of 1 mM mediated a small, statistically significant increase in hCG production (p < 0.01) in all constitutive strains, but had no effect on non‐hCG‐producing fibroblast strains. Sodium butyrate (Bu) was effective in increasing hCG synthesis in only one constitutive strain, derived from a newborn foreskin. HU treatment had no apparent effect on cell structure. All Bu‐treated strains, both those producing hCG and the nonproducers, showed morphological alterations; cells were flattened and they contained ordered arrays of refractile granules. It is suggested that hCG synthesis in cultured human diploid fibroblasts may result from a localized chromosomal event in which the loci responsible for this hormone are activated. Human diploid fibroblasts in culture are shown to be amenable to the study of gene expression and its modulation.
Stimulation of dome formation in MDCK kidney epithelial cultures by inducers of differentiation: Dissociation from effects on transepithelial resistance and cyclic AMP levels
Authors: Thomas S. Randall , Schultz Stanley G. , Lever Julia E.
Published: February 2005

Changes in transepithelial electrical resistance and cyclic nucleotide levels were monitored accompanying chemical induction of domes in a clonal subline of MDCK kidney epithelial cells. Confluent cell monolayers grown on nitrocellulose filters exhibited a relatively high mean transepithelial resistance (387 ohms · cm2). Hexamethylene bisacetamide, a potent inducer of dome formation (Lever, 1979b), stimulated significantly increased transmonolayer resistance as well as elevated levels of intracellular cyclic AMP. By contrast, dimethylformamide, an equally potent inducer of dome formation in MDCK cells, did not appreciably alter either resistance values or cyclic nucleotide levels. These results suggest that induction of dome formation in epithelial cell cultures by compounds generally known as inducers of differentiation may involve multiple and separate mechanisms.
Regulation of neutral amino acid transport in hepatocytes isolated from adrenalectomized rats
Authors: Kilberg M. S. , Vida T. A. , Barber E. F.
Published: February 2005

The present report shows that System A‐mediated 2‐aminoisobutyric acid (AIB) uptake is elevated in hepatocytes Isolated from adrenalectomized rats when they are compared to control cells. Although System ASC activity also shows this perturbation, Systems N, β, L1, and L2 are unaffected. Transport of AIB in both cell types is stimulated by dexamethasone, insulin, and glucagon, yet the hepatocytes from the adrenalectomized rats are much less responsive to these hormones. This apparent decrease in competence is seen for adaptive regulation of System A as well. The in vitro addition of dexamethasone to the hepatocytes from the adrenalectomized animals does not restore fully their ability to respond to hormones or amino acid deprivation. These effects are observed even after the cells have been held in primary culture for 24 hr. The simultaneous addition of glucagon and dexamethasone to either cell type resulted in stimulation of transport to rates significantly greater than the sum of the increases produced by the two hormones when added separately. In contrast, insulin and dexamethasone were additive in their effects rather than synergistic. These results suggest that hepatocytes from adrenalectomized rats are less competent than control cells with respect to regulation of neutral amino acid transport, including stimulation by insulin or amino acid starvation, two processes which appear not to depend on glucocorticoid for maximal response.
Permselectivity changes in malaria (Plasmodium falciparum) infected human red blood cell membranes
Authors: Kutner S. , Ginsburg H. , Cabantchik Z. I.
Published: February 2005

The development of the malaria parasite Plasmodium falciparum in human red blood cells induces parasite‐dependent perturbations in the permselectivity properties of the host cell membrane. The changes appear as parasites develop from ring to the trophozoite stage and persist during schizogony. In the present work we assessed the permeability changes of the infected cells to anionic substances by the use of radioactive and fluorescent probes. Our data show that (i) covalent binding probes, such as diisothiocyano ditritiostilbene disulfonic acid [3H]H2DIDS, which are virtually impermeant to normal red blood cells, became markedly permeant to trophozoites and schizonts, as evidenced by high labeling of intracellular hemoglobin; (ii) permeation of the fluorescent anion transport substrate NBD‐taurine, measured in the efflux mode, was very rapid and substantially enhanced in parasitized exythrocytes, as compared with noninfected cells; (iii) this efflux could not be blocked by H2DIDS, which is a specific inhibitor of anion transport in normal red blood cells; (iv) permeation of anionic probes was temperature dependent (Ea:11 ± 1 kcal/mole); and (v) could be blocked by nonspecific transport inhibitors that are known to interact with membrane lipids. The appearance of a new permeation pathway in the host cell membrane of trophozoites is associated with structural modification of the host cell membrane matrix.
The adhesive glycoprotein laminin is an agglutinin
Authors: Kennedy Dorothy W. , Rohrbach David H. , Martin George R. , Momoi Takashi , Yamada Kenneth M.
Published: February 2005

The glycoprotein laminin appears to function in the attachment of various epithelial cells to basement membranes. We examined whether its putative cell‐adhesive activity could be analyzed in a simple, one‐component model system—the agglutination of erythrocytes. Laminin is a potent agglutinin of aldehyde‐fixed sheep and human erythrocytes, with half‐maximal agglutination of 0.8 μg/ml in a standard hemagglutination assay. Inhibitors of this hemagglutinating activity include gangliosides and certain charged phospholipids. The spectrum of molecules is similar but not identical to inhibitors of the hemagglutinating activity of the adhesive glycoprotein fibronectin. Laminin is much less biologically active in three other assays for fibronectin biological activity involving cell spreading on tissue culture substrates, attachment of fibroblastic cells to type I collagen, and restoration of normal morphology to transformed fibroblasts. The adhesive glycoproteins laminin and fibronectin therefore differ markedly in biological activities in several specific adhesion assays; however, they resemble one another in binding to heparin, collagen, and cell surfaces and in their agglutinin activity.
Transepithelial transport in cell culture: Bioenergetics of Na‐, D‐glucose‐coupled transport
Authors: Sanders Martin J. , Simon Lawrence M. , Misfeldt Dayton S.
Published: February 2005

The renal cell line LLC‐PK1 contransports Na and D‐glucose from the apical to the basolateral side of the cell monolayer, and the short‐circuit current (Isc) measures the net amount of Na transported. Under conditions of maximal cotransport, the addition of phlorizin or removal of Na rreversibly decreased oxygen consumption by one‐hal. In the absence of glycolytic substrates, α‐methyl‐D‐glucoside stimulated Isc and oxygen consumption, although the Isc came to a steady state 50% less than when glycolytic substrates were present. The addition of other aerobic substrates did not increase Isc; however, when non‐contransported glycolytic substrates were introduced the Isc returned to a maximum with an associated fall in oxygen consumption and increased lactate production. Thus, in the absence of glycolytic substrates aerobic ATP formation may be rate‐limiting for Na, D‐glucose contansport. For this epithelium glycolysis makes an impotant contribution to the provision of energy or transport. Oxygen consumption does not correlate well with Isc and is not a good measured off the energy used in transport.
Glucose uptake and ornithine decarboxylase activity in tetradecanoyl phorbol acetate non‐proliferative variants
Authors: Butler‐Gralla Edith , Herschman Harvey R.
Published: February 2005

The potent tumor promoter 12‐0‐tetradecanoyl phorbol‐13‐acetate (TPA) is alos an excellent mitogen for 3T3 cells. We have previously isolated two independent variants, 3T3‐TNR‐2 and 3T3‐TNR‐9, that are unable to divide in response to TPA (Butler‐Gralla and Herschman, 1981). We have now tested tow components of the pleiotypic response, elevation of 2‐deoxyglucose uptake and ornithine decarboxylase induction, in these cells. Basal levels of 2‐deoxyglucose uptake were nearly tenfold higher in confluent 3T3‐TNR‐2 and 3T3‐TNR‐9 cells than in 3T3 cells. In contrast, basal ornithine decarboxylase levels were five‐ to tenfold lower in the variants. TPA stimulation of 2‐deoxyglucose uptake was as great in absolute terms in the variant cell lines as that of 3T3 cells but was only half that observed with serum. TPA was unable to induce any elevation of ornithine decarboxylase in 3T3‐TNR‐9 cells. treated with TPA, the maximal specific activity in the variant was less than the unstimulated value for 3T3 cells.
Mouse teratocarcinoma cells resistant to aphidicolin and arabinofuranosyl cytosine: Isolation and initial characterization
Authors: Aizawa Shinichi , Loeb Lawrence A. , Martin George M.
Published: February 2005

With the long‐range goals of elucidating the biochemical genetics and the phenotypic expression, both in vitro and in vivo, of mutator strains of mammalian cells, we have isolated and partially characterized a series of drug resistant mutants from a subline of a feeder‐dependent parental mouse teratocarcinoma line (PSA‐1) known to be capable of chimerizing host blastocyts via the injection of inner cell mass. Two series of stable mutants were isolated—one (Aphr) selected on the basis of resistance to aphidicolin (Aph), a specific inhibitor of DNA polymerase‐α, and the other (AraCr) selected by resistance to arabinofuranosyl cytosine, an analogue of cytosine. Irrespective of the method of selection, most of the 32 mutants isolated were resistant to both agents, although to different degrees, and with variations in growth characteristics, deoxynucleoside sensitivities, and sensitivities to mutagens (5‐bromodeoxyuridine, N‐methyl‐N′‐nitro‐N‐nitrosoguanidine and ultraviolet light). A protocol of multi‐step selection provided stable mutants highly resistant to aphidicolin and only mildly resistant to AraC, even in the absence of prior mutagenesis, and is thus recommended as an approach to the isolation of candidate mutator strains of multipotent teratocarcinomas.