bluuebunny/crossref_metadata_2025 huggingface dataset:
Rearrangement of Con A and WGA receptors on murine mastocytoma cell membranes: Relationship to cell cycle
Authors: Lustig Shlomo , Pluznik Dov H.
Published: February 2005

Incubation of interphase murine mastocytoma cells with fluorescein‐labeled wheat germ agglutinin (FITC‐WGA) or with rhodamine‐labeled concanavalin A (Rh‐Con A) shows binding of the lectins to all the cells in a ring form distribution. Treatment of the cells with trypsin before addition of FITC‐WGA, or with colchicine before addition of Rh‐Con A, promoted rearrangement of the lectin binding sites into cap formation in 27–29% of the cells. Trypsin had no effect on cap formation by Rh‐Con A, and colchicine had no effect on cap formation by FITC‐WGA. When the same cells were pretreated consecutively with trypsin and colchicine followed by incubation with FITC‐WGA and Rh‐Con A, no increase in the percentage of cells bearing caps was observed. Moreover, the same cells showed co‐capping with both lectins. Experiments with synchronous cultures of mastocytoma cells have indicated that capping induced by either lectin is maximal in the G2 phase and minimal in the G1 and S phases of the cell cycle. At the G2 phase 60% of the cells show cap formation either after trypsin‐WGA treatment or after colchicine‐Con A treatment. These data indicate that rearrangement of membrane receptors for Con‐A and WGA in murine mastocytoma cells can occur only at certain phases of the cell cycle.
Glucocorticoid hormone renders rat glioma cells dependent on high concentrations of external Ca2+ for growth
Authors: Ribeiro Solange M. F. , Armelin Hugo A.
Published: February 2005

C6 rat glioma cells, like other tumor cells, grow optimally in Ca2+‐ or Mg2+‐ depleted 5% serum medium. However, the glucocorticoid hormone hydrocortisone renders these cells dependent on high concentrations of external Ca2+ for growth. Upon Ca2+ deprivation (30–80 μM Ca2+ medium) hydrocortisone‐treated C6 cells undergo reversible cell‐cycle arrest at G0/G1 phase. This effect is specific for glucocorticoid hormones and occurs at physiological concentrations.

Growth restimulation of Ca2+‐deprived, hydrocortisone‐treated C6 cells by bovine pituitary growth factors or serum growth factors only takes place if the external Ca2+ concentration is increased. On the other hand, C6 cell growth requirement for external Mg2+ was not increased by the glucocorticoid hormone treatment. A minimum of 80 μM of external Mg2+ is required to keep cells adhered and spread in Ca2+‐depleted (30 μM) 5% serum medium; in high Ca2+ concentration (1.8 mM), Mg2+ is not required for adhesion or spreading. Thus, the hormone hydrocortisone renders the cell cycle of C6 glioma cells controllable by the levels of external Ca2+, a minimal external Mg2+ being necessary to warrant normal adhesion.
Insulin‐cholera toxin binding unit conjugate: A hybrid molecule with insulin biological activity and cholera toxin binding specificity
Authors: Roth Richard A. , Maddux Betty
Published: February 2005

The polypeptide hormone insulin and the binding unit of cholera toxin (CTB) were coupled via a disulfide bond. This hybrid molecule had 1/30 the ability of native insulin to bind to the insulin receptor and 1/30 the biological activity of native insulin in H35 rat hepatoma cells and rat adipocytes. Thus, in these two cell types that are very sensitive to insulin, the biological activity of the hybrid molecule was as predicted on the basis of the ability of the molecule to interact with the insulin receptor. In contrast, in HTC rat hepatoma cells and rat thymocytes, two poorly responsive cell types, the insulin‐CTB conjugate had 1/3 the biological activity of native insulin, a value 10 times greater than its insulin receptor binding potency. This increased activity of the conjugate did not appear to be due to cholera toxin in the preparation, since a control of uncoupled CTB had no biological activity. Furthermore, native cholera toxin increased intracellular levels of cAMP by 20‐fold, whereas the conjugate had no effect on cAMP levels. The CTB moiety did, however, contribute to the biological activity of the conjugate, since the activity of the hybrid molecule, like cholera toxin, was inhibited by gangliosides, whereas the activity of native insulin was not. Finally, the binding to thymocytes of insulin‐CTB conjugate, but not insulin, was inhibited by gangliosides. Thus, a hybrid hormone molecule has been constructed which has insulin‐like biological activity with the receptor specificity of cholera toxin in poorly responsive cells.
Glucagon regulation of amino acid transport in hepatocytes: Effect of cell enucleation
Authors: Crettaz Marco , Ronald Kahn C. , Fehlmann Max
Published: February 2005

Glucagon and cAMP analogs stimulate amino acid transport in freshly isolated hepatocytes by inducing the synthesis of new transport proteins. The role of the cell nucleus in the glucagon regulation of amino acid transport has been studied in rat hepatocytes enucleated by centrifugation through a discontinuous Ficoll gradient in the presence of cytochalasin B. Enucleated hepatocytes take up alpha‐aminoisobutyric acid (AIB) through a Na+‐dependent transport component with kinetic properties similar to those found in intact hepatocytes. Cytoplasts prepared from glucagon‐stimulated cells retain the increase AIB transport induced by the hormone in the intact cells. The direct addition of glucagon to cytoplasts has no effect on AIB transport, in spite of the fact that the cytoplasts exhibit a higher capacity to bind glucagon than their nucleated counterparts. These data indicate that the nucleus is required for the glucagon stimulation of amino acid transport in isolated hepatocytes.
Consequences of parental exposure to epidermal growth factor for progeny cell division
Authors: Heath John K.
Published: February 2005

The consequences of parental exposure to epidermal growth factor (EGF), for progeny cell cycle times was investigated. Slowly dividing mouse 3T3 fibroblasts were exposed to EGF for 8 hr, the EGF was withdrawn, and the cell cycle times of parental and progeny cells were measured by time‐lapse video microscopy. It was observed that exposure to EGF induced a round of cell division following a lag phase of approximately 8 hr. The progeny of these cells exhibited accelerated cell cycle times compared to cells that had not been exposed to EGF. Parental cell division time was significantly correlated with progeny cell cycle time. Sibling progeny cell cycle times were also significantly correlated. EGF can therefore apparently exert an effect on the cell cycle times of more than one generation of cells.
Hemopoiesis in spleen and bone marrow cultures
Authors: Keller Gordon M. , Johnson Gregory R. , Phillips Robert A.
Published: February 2005

Long‐term cultures established from spleen cells were compared to those established from bone marrow cells for their ability to maintain hemopoiesis as measured by the presence of hemopoietic progenitor cells (in vitro CFU) and multipotent stem cells (CFU‐S). The frequency of both in vitro CFU and CFU‐S increased dramatically during the first 2 weeks in the spleen cultures. Following this early peak of activity, the number of progenitors and stem cells declined to undetectable levels by week 6 of culture. During this short phase of hemopoiesis, large amounts of GM‐CSF could be detected in the supernatant of the spleen cultures. In contrast, bone marrow cultures did not share this early peak of hemopoiesis; however, they maintained activity for much longer periods of time than did the spleen cultures. When spleen stem cells were seeded onto functional bone marrow adherent cells, spleen‐derived in vitro CFU were maintained well beyond week 6 of culture. Spleen cultures established from athymic nu/nu mice showed a greatly reduced ability to support hemopoiesis while those from S1/S1d mice maintained GM‐CFU as well as cultures from normal mice.
Changes in water proton relaxation times and in nuclear to cytoplasmic element gradients during meiotic maturation of Xenopus oocytes
Authors: Cameron I. L. , Labadie D. R. L. , Hunter K. E. , Hazlewood C. F.
Published: February 2005

Fully grown oocytes 1.2mm in diameter were removed from Xenopus laevis ovaries and were exposed to progesterone (2.5 μg/ml in Ringer's solution) to induce completion of the first maturation division or germinal vesicle breakdown (GVBD). This process required 5.5 ± 0.5 hr. Neither oocyte vo ume nor water content was observed to change throughout muturation. At selected times, the oocytes were quick frozen in liquid propane and cryosectioned. The sections were freeze‐dried, and analyzed for K, Na, Cl, P, S, and Mg in millimolar per kilogram dry weight content in the nucleus and the yolk‐free cytoplasm using electron probe X‐ray microanalysis. Unstimulated oocytes showed significant nuclear to yolk‐free cytoplasmic content gradients (N/C ratio) for the following elements: K (1.84), P (0.65), and S (1.56), but significant N/C content gradients were not found for Na and Mg. By 10 min after progesterone stimulation, a significant change in the N/C ratio of the following elements had occurred due to a rapid increase in nuclear content: K (2.29), Cl (2011). A significant N/C ratio for Mg (1.35) had developed by 10 min after progesterone stimulation and a significant N/C ratio for Na (2.07) had developed by 45 min. In addition the following elements showed significant content increases in both the nucleus and the yolk‐free cytoplasm from the time prior to progesterone stimulation to the time just prior to GVBD at 240 min: K, Na, Cl, P, S, and Mg. Nuclear magnetic reasonance measurements of the spin‐lattice relaxation time (T1) of water proton in oocytes showed a sinificant increase in the T1 time after progesterone exposure. The changes in N/C ratios of specific elements and in the physical parameter of water proton relaxation time suggest that progesterone is responsible for inducing changes in the physicochemical interactions between various macromolecules, specific elements, and water.
Comparative studies of collagen lattice contraction utilizing a normal and a transformed cell line
Authors: Buttle David J. , Ehrlich H. Paul
Published: February 2005

Differences between the behavior of cultured rat skin fibroblasts and that of a line of transformed rat sarcoma cells incorporated into a polymerized collagen lattice were examined. Fibroblast‐populated collagen lattices (FPCL) were manufactured. Within 24 to 48 hr after manufacture, both cell lines reduced lattice size by a process known as lattice contraction. Contraction occurred more rapidly in both cell lines when the media were supplemented with 25% serum rather than the usual concentration of 10% serum. Similar growth patterns were observed with transformed cells within collagen lattices and on plastic surfaces. Normal rat fibroblasts were found to contract lattices faster than transformed cells. At the end of a 2‐week period, the final contracted size of the transformed cell lattice was the same as that of normal cell lattices. The cellular density of transformed cells within the FPCL was eight times greater than that of FPCL made with normal rat cells. Normal rat fibroblasts elongated and flattened more, and organized the collagen matrix to a greater degree, than did transformed cells. In this instance, therefore, lattice contraction was shown to be linked more to the process of fibroblast elongation and collagen fiber organization than to cell number or density.
Cell growth and differentiation in vitro in mouse macrophages transformed by a tsA mutant of simian virus40. I. Cellular response in proliferative and phagocytic activities to the shift of temperature differs depending on the culture state in mouse bone marrow cells transformed by the tsA640 mutant of simian virus 40
Authors: Tanigawa Takahiko , Takayama Hisao , Takagi Atsushi , Kimura Genki
Published: February 2005

It was shown previously that mouse bone marrow cells transformed by simian virus 40 (SV40) show a reversible cell density‐dependent phenotypic transition between the nonmacrophage (rapidly growing) and the macrophage (stationary) states; cells in low‐density cultures are in the growing phase, express SV40 T antigen strongly as revealed by immunofluorescence, and lose typical macrophage properties such as immune phagocytosis; whereas cells in high‐density cultures are in the stationary (nongrowing) phase, express SV40 T antigen weakly, and recover their macrophage properties (Takayama, 1980). In the hope of clarifying the relationship between T antigen, cell growth, and macrophage‐specific cellular function, we examined the behavior at 33 and 39°C of mouse bone marrow cells transformed by an SV40 gene A mutant (tsA640) whose mutation renders the molecular weight of 90K (large) T antigen temperature sensitive. The results presented in this paper suggest that functional large T antigen is required for cells in the stationary phase to initiate multiplication when transferred at lower density and is not necessary for a majority of them to maintain the nongrowing state (viability) at both high and lower cell densities, whereas it is required for cells in the growing phase to keep multiplying without losing their viability. The results also suggest that the functional large T antigen does not play a direct role in maintaining the cells as either phagocytic or nonphagocytic. It is also suggested that the physiological or tsA mutation‐mediated arrest of growth may or may not be accompanied by induction and/or maintenance of cellular phagocytic activity depending on the culture state.
Glucocorticoid receptors and cortico‐sensitivity in a human clonal monocytic cell line, CM‐SM
Authors: Ranelletti F. O. , Starace G. , Piantelli M. , Lambertenghi‐Deliliers G. , Revoltella R. P.
Published: February 2005

CM‐SM is a clonal line of human precursor mononuclear phagocytes inducible to macrophage differentiation in response to the tumor promoter phorbol ester 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA). Untreated CM‐SM cells contain single class, high‐affinity (KD = 4.0 × 10−9 M) glucocorticoid‐specific receptor sites (∼60,000 per cell), as measured by a whole cell assay, at 37°C, using [3H]triamcinolone acetonide (TA). Exposure of CM‐SM to dexamethasone (DEX) produced a progressive, dose‐ and time‐related series of changes in CM‐SM cell growth, saturation density, morphology, and functional properties, with half‐maximal effects at about 10−9 M for DEX. TA‐receptor sites rapidly decreased (about 70%) after DEX treatment, without any apparent change in steroid specificity and affinity. After 5 days in culture with a saturating concentration (3.6 × 10−8 M) of hormone, the cells reached a saturation density of about 9.0 × 106 viable cells/ml (about 4.0 × 106 viable cells/ml in the controls), while the modal volume of the resulting cell population was approximately 60%, as compared to the volume of untreated cells. DEX‐treated cells appeared less differentiated than controls, as assessed by combined morphologic, antigenic, and cytoenzymatic analyses. DEX almost completely inhibited TPA activation of the following macrophage functions: adherency to the culture plate, expression of lysosomal enzymes, Fc and C3 receptors, and stimulation of phagocytosis. After removal of DEX, the cells, within a few passages, returned to a state apparently identical to the untreated controls and could be induced to macrophage differentiation in response to TPA.
A multicomponent analysis of amino acid transport systems in human lymphocytes. 1. Kinetic parameters of the A and L systems and pathways of uptake of naturally occurring amino acids in blood lymphocytes
Authors: Segel George B. , Simon William , Lichtman Marshall A.
Published: February 2005

We have determined the kinetic parameters of natural and system‐specific synthetic amino acid transport by human blood lymphocytes, using a multi‐component computer analysis that separates carrier‐mediated uptake from diffusion. These studies were initiated in order to provide the basis for studies of human blood T and B lymphocytes and malignant lymphocytes. Methylaminoisobutyric acid (methyl‐AIB) and 2‐amino‐2‐carboxy‐bicyclo (2,2,1) heptane (BCH) uptakes into lymphocytes were measured as prototypes of A‐ and L‐system amino acid transport. The Michaelis constant for methyl‐AIB uptake was 540 μM; the maximal velocity of uptake was 28 μmol/L cell water/min, and the diffusion coefficient was .004 min−1. In contrast, the Michaelis constant for BCH uptake was 63 μM; the maximal velocity was 969 μmol/L cell water/min, and the diffusion coefficient was .141 min−1. The transport of the naturally occurring amino acids, alanine, proline, and leucine was defined by studies of: (1) competitive inhibition with the system‐specific synthetic amino acids, methyl‐AIB and BCH, (2) the effect of the transcellular sodium gradient on transport, and (3) evaluation of the time‐dependent increase of transport in amino acid‐deficient medium (adaptation). Alanine was transported principally (∼70%) by the ASC‐system, and leucine was transported principally (70%) by the L‐system in lymphocytes. The analysis of proline transport was more complex because of a large component of uptake by diffusion even at low amino acid concentrations. Taken together, the kinetics of sodium‐sensitive uptake and the results of competitive inhibition studies indicated that proline was transported by the A‐system (30%), the ASC system (30%), and also by the L‐system (15%).
Early, anti‐immunoglobulin induced events prior to NA+‐K+ pump activation: An analysis in a monoclonal human B‐lymphoma cell population
Authors: Heikkilä Reino , Iversen Jens‐Gustav , Godal Tore
Published: February 2005

Events following F(ab)2 anti‐δ immunoglobulin stimulation of monoclonal (leukemic) human B cells prior to Na+‐K+ pump activation were investigated in vitro. This pump activation, measured by ouabain‐sensitive 86Rb+ uptake, appeared susceptible to the phospholipid‐interacting drugs tetracaine and quinacrine, to the antioxydant nordihydroguaiaretic acid (NDGA), and to the calmodulin antagonist trifluoperazine, while much less susceptible to the methylation inhibitor‐3‐deazaadenosine. The Ca++ ionophore A 23187 appeared to induce pump activation in a way similar to anti‐δ, as it was susceptible to the same drugs and as anti‐δ had no additional stimulating effect on A 23187‐stimulated cells. However, whereas the anti‐δ‐induced activations appeared independent of the extracellular Ca++ activity, [Ca++]e, the activation by A 23187 was potentiated by addition of the CA++ chelator ethyleneglycol‐bis (β‐aminoethyl ether) N, N'‐tetracetic acid (EGTA). Estimations by a fluorescent chelator method (quin 2) showed anti‐δ to increase the intracellular Ca++ activity, [Ca++]i both in the absence and presence of EGTA. A 23187 increased [Ca++]i strongly in Ca++ medium, but was weaker, more similar to the anti‐δ response, in EGTA medium. It is suggested that Na+‐K+ pump activation after anti‐lg stimulation in B cells may follow Ca++ mobilization from internal stores. The trifluoperazine susceptibility suggests that calmodulin regulation is involved.
Isolation and characterization of vitamin A‐sensitive chinese hamster lung cell lines
Authors: Ikezaki Kiyonobu , Akiyama Shin‐Ichi , Nakamura Mitsuo , Komiyama Sohtaro , Kuwano Michihiko
Published: February 2005

Retinyl acetate (RA)‐sensitive variants (RAs‐2 and RAs‐3) of V79 cell line were isolated after mutagenesis with N‐methyl‐N′‐nitro‐N‐nitrosoguanidine. The variants were stable and showed a 3‐ to 4‐fold increase in sensitivity to RA compared to parental V79 cells. The RAs‐2 clone was also sensitive to retinol and retinol palmitate. The RA‐sensitivity behaves as a recessive trait in all hybrids of RAs‐2 and V79. A number of physiological parameters were indistinguishable in V79 and RAs‐2 cells, including the extent of uptake of [3H]retinol, the release of K+ from the cells induced by RA, and the levels of retinol and retinoic acid binding proteins. However, one possible correlation with the RA‐sensitive phenotype was observed: Gomori acid‐phosphatase staining of RA‐treated RAs‐2 and V79 cells indicated that lysosomal membrane of RAs‐2 cells was more labile than those of the parental V79 cells.
Phorbol ester‐stimulated murine myelopoiesis: Role of colony‐stimulating factors
Authors: Stuart Robert K. , Sensenbrenner Lyle L. , Shadduck Richard K. , Waheed Abdul , Caramatti Cecilia
Published: February 2005

Tumor promoting phorbol esters, such as 12‐0‐tetradecanoyl‐phorbol‐13‐acetate (TPA), stimulate colony formation in vitro by murine granulocyte‐macrophage progenitors (GM‐CFC) without added colony stimulating factors (CSF). To determine whether TPA induces CSF production in vitro, marrow cells were cultured for 1 to 7 days in liquid medium with or without TPA. No CSF was detected in any sample by a double antibody radioimmunoassay (sensitivity = 2 units/0.1 ml), however, colony‐stimulating activity was detected in supernatant fluid from all TPA containing cultures by bioassay. This activity appeared to result from a direct effect of TPA rather than from production of CSF, as equivalent activity was found in TPA‐containing medium incubated in the absence of marrow cells. Rabbit antiserum to purified L‐cell CSF inhibited colony formation stimulated by L‐cell CSF and WEHI‐3 CSF, but had no effect on colony formation induced by TPA. Cells from long‐term marrow cultures responded to TPA with colony formation, despite culture conditions and cell fractionation procedures that reduced the frequency of CSF‐proclucing macrophages to > 1.0%. TPA inhibited binding of radioiodinated L‐cell CSF to marrow cells, especially if the cells were first exposeed to TPA. These results do not support induction of CSF production as the major mechanism of phorbol ester stimulation of myelopoiesis. Phorbol esters may directly stimulate GM‐CFC and/or enhance their response to CSF by a mechanism involving CSF binding sites.
In vitro growth inhibition of murine leukemia cells by antibody specific for the major envelope glycoprotein (gp71) of friend leukemia virus
Authors: Genovesi Eugene V. , Collins Jeffrey J.
Published: February 2005

An in vitro complement (C′)‐independent growth cytostasis model is described in which the replication of Friend leukemia virus (FLV)‐induced erythroleukemia cells (of the FLC‐745 cell line) is inhibited by goat serum directed against the major FLV envelope glycoprotein, gp71. The cytostatic effect is reversible, with the degree of this reversibility dependent on both the concentration and duration of exposure to immune serum. The inhibitory factor in heat‐activated goat anti‐FLV gp71 (δGαFLV gp71) serum has been identified as virus‐specific IgG antibody, and F(ab')2 fragments of this antibody are highly effective in suppressing FLC‐745 cell growth. Studies with various murine leukemia and lymphoma cell lines, as well as with a panel of antisera directed against various murine oncornaviruses or viral proteins, have demonstrated that antibodies reactive with the group or type determinants of FLV gp71 are capable of mediating cytostasis. Under conditions of antibody‐mediated growth inhibition of FLC‐745 cells, specific modulation of gp71 expression is followed by nonspecific modulation of H‐2d antigen expression. In addition, considerable cell death occurs in cytostatic cultures which is accompanied by continued division (as measured by DNA synthesis) of a portion of the cell population. Cytofluorimetric analysis of nuclei from growth‐inhibited FLC‐745 cells demonstrates a diminution in the frequency of cells in the G2/M phase of the cell cycle. It is suggested that antibody‐mediate FLC‐745 cell growth inhibition operates via a blockade of the cell cycle which prevents most cells in the population from traversing G2/M. While these blocked cells appear to be subject to slow cytolysis by a C′‐independent mechanism, a portion of the cells escape this blockade and continue to replicate, thus offsetting the death of the former cells to yield a relatively constant density of viable cells for at least 72–96 h of growth inhibition. The possible relevance of this in vitro phenomenon to in vivo passive therapy against FLV‐induced disease with GαFLV gp71 and similar antisera is briefly considered.
Immunocytochemical localization of chick DNA polymerases α and β+
Authors: Matsukage Akio , Yamamoto Susumu , Yamaguchi Masamitsu , Kusakabe Moriaki , Takahashi Taijo
Published: February 2005

An immunofluorescent method using specific antibodies was employed to detect DNA polymerases α and β in chick cells. With monoclonal antibodies produced by four independent hybridoma clones, most of the DNA polymerase α was shown to be present in nuclei of cultured chick embryonic cells. With a polyclonal, but highly specific, antibody against DNA polymerase β, this enzyme was also shown to be present in nuclei. DNA polymerase α was detected in proliferating cells before cell contact and in lesser amount in resting cells after cell contact, indicating that its content is closely correlated with cell proliferation. On the other hand, similar amounts of DNA polymerase β were detected in proliferating and resting cells. Furthermore, DNA polymerase β was detected in nuclei of most cells, while DNA polymerase α was detected only in large round nuclei in seminiferous tubules of chick testis. DNA polymerase α is presumably present in cells that are capable of DNA replication, and during the cell cycle it seems to remain in the nuclei during the G1, S, and G2 phases, but to leave from condensed chromatin for the cytoplasm during the mitotic phase.
Characterization of the early and late stages of myelomonocytic leukemia induced by friend helper‐independent virus F‐MuLV: Isolation and induction of friend myelomonocytic leukemic cell lines
Authors: Shibuya Tsunefumi , Mak Tak W.
Published: February 2005

With the use of cloned helper‐independent Friend leukemia virus (F‐MuLV), we have induced a high incidence (∼︁ 70%) of myelomonocytic leukemia in mice resistant (Fv‐6rr or Fv‐6rs) to erythroleukemia induction by this virus. The spleen cells from these mice (DBA/2 or BALB/c × DBA/2) were found to contain a high level of progenitor cells capable of forming granulocytic and macrophage colonies (CFU‐GM). These CFU‐GM, however, were different from those in the speens of uninfected mice, as they were either very sensitive to or independent of conditioned medium. No erythroid progenitor bursts (BFU‐E) or precursor (CFU‐E) cells were detected in the speens of these diseased animals. If these mice with myelomonocytic leukemia were kept alive by transfusion of red blood cells from uninfected mice, tumorigenic cell lines, capable of being transplanted, into adult mice can be isolated. Three such cell lines TTA‐1, TTA‐3, and TTA‐9 have been established, and they retain their morphology of monocytes and macrophages as well as being positive for the monocyte‐specific stain α‐naphtyl‐acetate esterase. These myelomonocytic leukemia cell lines can also be induced in culture by speen cell‐conditioned medium to differentiate into macrophages. Other conditioned media such as L‐cell‐conditioned medium, phytophemmaglutinin‐stimulated leukocyte‐conditioned medium, and WEHI‐3 conditioned medium were less effective in their abilities to stimulate differentiation in these myelomonocytic leukemia cell lines.
Concanavalin A increases spontaneous beat rate of embryonic chick heart cell aggregates
Authors: Myrdal Sigrid E. , Dehaan Robert L.
Published: February 2005

The plant lectin concanavalin A (Con A), at concentrations of 5–200 μg/ml, induced a twofold to fivefold increase in spontaneous beat rate of cultured aggregates of ventricular cells from seven‐day chick embryos. This response was time, dose, and temperature dependent and was accompanied by a decrease in transmembrane potential. It could be blocked or reversed by α‐methyl‐D‐mannoside but was not reversed by dilution alone. Binding of the lectin occurred in the cold, but a temperature‐dependent process was also necessary to produce the response. Divalent (succinyl) Con A did not cause a beat rate increase. Whole heart aggregates responded similarly but less intensely than ventricular aggregates. Atrial aggregates, and whole heart aggregates treated with 5 μg/ml of Con A, produced a biphasic chronotropic response, first decreasing then increasing their beat rates. These results suggest that saccharide‐bearing macromolecules on the heart cell surface play a role in regulating spontaneous beat rate.
A reevaluation of the response of human umbilical vein endothelial cells to certain growth factors
Authors: Knauer Daniel J. , Cunningham Dennis D.
Published: February 2005

Human umbilical vein endothelial cells (HUV‐EC) were isolated and maintained in pure culture on a fibronectin matrix with hypothalamic derived endothelial cell growth factor included in the culture medium. HUV‐EC maintained under these conditons displayed only slight alterations in morphological appearance and continued to produce Factor VIII antigen. The cells showed an unaltered growth response to serum and added growth factors up to passage 16 (>30 population doublings). We found that epidermal growth factor (EGF) was potently mitogenic for HUV‐EC, but only in the presence of endothelial cell growth factor. Also in contrast to a previous report, we were unable to demonstrate a potentiation of this response by human α‐thrombin. Because of these discrepancies, we performed studies to determine if they might be explained by a difference in the interaction of our HUV‐EC with EGF. In studies utilizing 125I‐EGF as tracer probe, we determined that our HUV‐EC have an EGF receptor number of 13,000 sites/cell with an apparent Kd∼︁4.0 × 10−9 M. In addition, receptor‐bound 125I‐EGF was rapidly internalized and degraded presumably by a lysosomally mediated pathway since degradation was complete to the amino acid level. These results are in agreement with those previously published and thus do not provide a basis on which to resolve discrepancies regarding the growth response of HUV‐EC to various growth factors.
Constancy of cell buoyant density for cultured murine cells
Authors: Loken M. R. , Kubitschek H. E.
Published: February 2005

The relationship between cell cycle and cell density was determined for three different lines of mouse cells by equilbrium centrifugation of suspension cultures. The mean cell densities of the three lines differed significantly, with values of 1.0622, 1.0678, 1.0540 gm/ml for 70Z/3, S 107, and ABE 8, respectively. However, the density distributions within each of the three lines were indistinguishable, with an average coefficient of variation about 5% of the mean reduced density (i.e., density minus one). Quantitative DNA analysis of the cells separated by density showed that the proportion of cells in G1, S, and G2 + M phase of the cell cycle changed very little or not at all with cell density. In addition, cells separated by size (and therefore by phase of the cell cycle) using velocity sedimentation had the same means and distributions of densities. These results indicate that there is little or no change in cell density as the cells traverse the life cycle and that buoyant density appears to be a constant property of a cell type.
Evidence for differences in the mechanism of cell cycle arrest between senescent and serum‐deprived human fibroblasts: Heterokaryon and metabolic inhibitor studies
Authors: Burmer Glenna C. , Rabinovitch Peter S. , Norwood Thomas H.
Published: February 2005

It has previously been shown that serum‐deprived, early passage quiescent human diploid fibroblastlike (HDFL) cells are able to inhibit cycling cells from entry into DNA synthesis upon cell fusion. We have found that the degree of inhibition of DNA synthesis in the heterokaryon correlates with the duration of serum deprivation, which is consistent with the suggestion that serum‐deprived cells may enter progressively deeper stages of G0 as they increase their time in quiescence. In contrast to fusions with senescent cells, in heterokaryons between serum‐deprived early passage and cycling young cells transient inhibition of protein synthesis with cycloheximide or inhibition of RNA synthesis with 5–6‐dichloro‐1‐β‐D‐ribofuranosyl benzimidazole (DRB) did not stimulate nuclear [3H]‐thymidine incorporation. These results suggest that differences may exist in the mechanisms responsible for inhibiting cell cycle progression in senescent vs early passage quiescent HDFL cells.
In vitro hemopoiesis within a microenvironment created by MC3T3‐G2/PA6 preadipocytes
Authors: Kodama Hiro‐Aki , Sudo Hiroko , Koyama Hideki , Kasai Shiro , Yamamoto Shigehisa
Published: February 2005

The clonal preadipose cell line, MC3T3‐G2/PA6, has the capacity to differentiate into adipocytes in response to glucocorticoids and to support in vitro growth of hemopoietic stem cells (CFU‐S). To study the relationship between these capacities, we precultured the MC3T3‐G2/PA6 cells for varying days in the presence or absence of dexamethasone and then cocultured them with mouse bone marrow cells. Logarithmically growing cultures contained no detectable adipocytes and showed the highest growth‐supporting activity for CFU‐S, whereas cultures containing the largest number of adipocytes showed the lowest activity. When bone marrow cells were seeded onto 3‐day‐old MC3T3‐G2/PA6 preadipocyte layers at 1 × 105 cells/35‐mm dish, day 12 CFU‐S grew with a population doubling time of about 37 hr, and at least 75% of them were associated with the cell layer between days 2 and 7. In the absence of the preadipocytes, CFU‐S were not detected in the adherent cell fraction and decreased with a half‐life of about 18 hr. More than 80% of CFU‐C were also found to be associated with the preadipocyte layer, and they increased about 24‐fold in number during 7 days in culture. Morphologically, hemopoietic cells developing into mature granulocytes and macrophages were distributed between the layers of preadipocytes. Dendritic processes of preadipocytes were frequently in close alignment with the hemopoietic cells. However, adipocytes failed to show such an intimate association with hemopoietic cells. These results indicate that MC3T3‐G2/PA6 cells in the preadipocyte stage, but not in the adipocyte stage, have the capacity to support CFU‐S growth, and that hemopoiesis in our cocultivation system proceed within the microenvironmental milieu provided by MC3T3‐G2/PA6 preadipocytes.
Growth factors that stimulate human granulocyte—macrophage colony formation produced by the cell line CM‐S
Authors: Bertolini Luisa , Butler Richard H. , Revoltella Roberto P.
Published: February 2005

CM‐S is an autonomous cell line of human hemopoietic precursor cells inducible to monocyte‐macrophage differentiation in response to appropriate inducing agents. CM‐S cells produce factors that stimulate their own growth and proliferation, and are also capable of stimulating clonal proliferation of human, but not mouse, monocytic and granulocytic bone marrow progenitor cells in viscous medium. Preliminary purification steps have demonstrated at least two species, one of which (MW 30,000–50,000) retains both these activities, while the other (MW ≤ 10,000) apparently retains only the autostimulatory activity. CM‐S cells could thus be a useful source for the purification of human colony stimulating factors (CSFs). CM‐S cells also respond to factors present in human placenta conditioned medium, known to contain human CSF. This suggests that CM‐S cells could provide a homogeneous target cell population for testing CSFs from other human sources.
The role of the G1 period in the life cycle of eukaryotic cells
Authors: Rao Potu N. , Satya‐Prakash K. L. , Wang Yong Chao
Published: February 2005

The objective of this study was to test the concept that the G1 period lacks any specific function in the life cycle of mammalian cells and hence could be drastically reduced without any effect on the generation time. HeLa cells were grown in medium containing an optimum dose (60 μM) of hydroxyurea at which the duration of S period was prolonged with little or no increase in generation time. At this concentration of hydroxyurea, we observed a maximum of 3 h (or 28.5%) reduction in the G1 period. We also studied the effects of synchronization in S phase by single and double thymidine blocks on cell size and its relationship to the duration of G1 in the subsequent cycle. By these treatments, we could reduce the G1 period by not more than 2 to 3 h. The reduction in G1 period was not directly proportional to the size (volume) of the G1 cells. These results suggest that G1 period has certain specific functions and cannot be eliminated by alterations in culture conditions.
Sequence of cell life phases in a finitely proliferative population of cultured rat cells: A genealogical study
Authors: Matsumura Toshiharu
Published: February 2005

Family trees of rat liver epitheliallike cells (RLECs), which proliferate finitely in primary culture, were obtained by time‐lapse cinemicrography (TLC). In family trees, RLECs were classified into those terminated with division (class D cells), those terminated with incomplete division (class I cells), i.e., division followed by fusion of two daughter cells, and the combined population (class N cells) of those terminated with death and those surviving for 120 hr or more with their termination not detected by TLC. The interphase period, but not the intersibling period, of class D cells became distributed more broadly with time on the family tree. These results for class D cells fit best to a transition probability model with two indeterminate states (Brooks et al., Cell, 19:493–504, 1980) when the model is modified in such a way that the transition probability for one of the two indeterminate states decreases with time. During proliferation of RLECs, some of the class D cells reproduce, while others showed transition, in part by way of class I cells, to class N cells. The frequency of transition from class D to class N increased with time in the family tree. Two sibling cells of a parent mitotic cell were simultaneously either class D or class N at a significantly high frequency, suggesting that the transition had been determined in the preceding cells. The present observations confirm previously described observations on human diploid cells, and in addition, reveal some new details of the finitely proliferative nature of somatic diploid cells.
Effect of ouabain and furosemide on pepsinogen secretion by gastric glands in vitro
Authors: Gibert Anne J. , Hersey Stephen J.
Published: February 2005

Gastric glands were isolated from rabbit stomach and pepsinogen secretion was measured after stimulation with isoproterenol, forskolin, 8‐bromo cyclic adenosine monophosphate (8‐bromo cAMP), cholecystokinin octapeptide (CCK‐OP), carbachol, and hyperosmolar medium. The responses to these stimuli in medium containing 143 mM Na+ and 5.4 mM K+ (normal medium) were compared with responses to the same stimuli in media containing either 0 Na+ and 5.4 mM K+, or 143 mM Na+ and O K+. In addition, the effects of ouabain and furosemide on secretion elicited by these stimuli were determined. Medium containing 0 Na+ inhibited all stimuli. Medium containing 0 K+ inhibited the action of 8‐bromo cAMP and stimuli postulated to be mediated by cAMP. Ouabain inhibited the same stimuli as O K+ medium, and, in addition, inhibitied the response to hyperosmolar medium. However, ouabain enhanced the response to CCK‐OP. Furosemide inhibited the response to hyperosmolar medium but had no effect on the action of any secretagogue employed. Intraglandular [Na+] increased and [K+] decreased after exposure to K+‐free medium or ouabain. cAMP content of the glands was assayed after stimulation with both isoproterenol and hyperosmolar medium. Isoproterenol and hyperosmolar medium significantly increased cAMP levels. The results are discussed in relation to possible involvement of ion transport or intracellular ion concentration in the secretory process.
Stimulation of motility in cultured bovine capillary endothelial cells by angiogenic preparations
Authors: Mullins Deborra E. , Rifkin Daniel B.
Published: February 2005

Several angiogenic preparations that have been shown to stimulate plasminogen activator (PA) and collagenase production by cultured bovine capillary endothelial (BCE) cells were tested for their ability to stimulate BCE cell motility in the phagokinetic track assay. Bovine retinal extract, medium conditioned by 3T3‐F442A differentiated mouse adipocytes, SK HEP‐1 human hepatoma cell lysate, mouse sarcoma 180 cell lysate, and medium conditioned by mouse sarcoma 180 cells stimulated motility 68.7%, 48.5%, 140.9%, 56.5%, and 102.1%, respectively, relative to untreated cells. The motility‐stimulating activity of these preparations was dose dependent and linear over the 16‐h assay period. Several hormones and growth factors were tested for BCE cell motility‐stimulating activity, including insulin, vasopressin, fibroblast growth factor, and a partially purified preparation of sarcoma growth factor, and were found to be ineffective.

12‐0‐tetradecanoyl‐phorbol‐acetate (TPA), a potent stimulator of both PA and collagenase activities in BCE cells, also did not stimulate motility, indicating that protease production is not sufficient to stimulate BCE cell motility in this assay. Neither SK HEP‐1 hepatoma cell lysate nor TPA was effective in stimulating motility in bovine aortic endothelial (BAE) cells. The inability of SK HEP‐1 hepatoma cell lysate to stimulate movement in BAE cells is consistent with the observation that angiogenesis occurs by sprouting of capillaries, not large vessels.
Isolation of a temperature‐sensitive FM3A mutant deficient in asparagine‐linked glycosylation by selecting for resistance to tritiated mannose suicide
Authors: Nishikawa Yoshihisa
Published: February 2005

Protein glycosylation mutants in the mouse mammary carcinoma cell line FM3A were selected for ability to withstand exposure to [2‐3H]mannose at 39°C. G258, one of the mutant cells isolated, has been characterized. G258 cells were temperature‐sensitive for cell growth. Moreover, G258 cells showed temperature sensitivity for [3H]mannose incorporation into the TCA‐insoluble fraction. To study the biochemical basis of the defect in glycoprotein biosynthesis, the formation of lipid‐linked saccharides was examined. The results showed that the formation of lipid‐linked oligosaccharides was severely inhibited in G258 cells at 39°C. At 33°C, G258 cells synthesized Glc3Man9GlcNAc2‐PP‐Dol, the fully assembled lipid‐linked oligosaccharides, but at 39°C, G258 cells were able to synthesize merely the smaller lipid‐linked oligosaccharides (approximately up to Man3GlcNAc2‐PP‐Dol), but were unable to synthesize the larger lipid‐linked oligosaccharides.
Analysis of pure and mixed murine mast cell colonies
Authors: Pharr Pamela N. , Suda Toshio , Bergmann Karen L. , Avila Lobelia A. , Ogawa Makio
Published: February 2005

Mast cells have been proposed to originate from diverse sources, including connective tissues, macrophages, T lymphocytes, and hemopoietic cells. Evidence for a hemopoietic origin of mast cells includes the presence of mast cell precursors in spleen colonies and the presence of mast cells in hemopoietic colonies in culture. Here we report a detailed analysis of mouse spleen mixed hemopoietic colonies containing mast cells. All of the colonies in cultures plated at low cell densities were individually removed for analysis by May‐Grunwald‐Giemsa staining on day 15 of culture. Examination of five dishes which contained a total of 82 colonies showed 16 pure mast cell colonies and 36 mixed mast cell colonies. Sixteen different combinations of cell types were seen and were not distinguishable from each other in situ. The most diverse type of mixed colony contained macrophages (m), neutrophils (n), eosinophils (e), mast cells (Mast), megakaryocytes (M), erythroid cells (E), and blast cells. The clonal origin of mixed mast cell colonies was established by the replating of single cells obtained from blast cell colonies. Individual cells were removed with a micromanipulator, replated, and allowed to grow for 15 days. Cytospin preparations of 10 such colonies showed diverse combinations of cell lineages which were seen in the different types of mixed mast cell colonies described above. Replating studies of mixed mast cell colonies were carried out and a high incidence of replating was seen. Approximately one half of these colonies formed only mast cell colonies upon replating. Further studies showed that pure mast cell colonies could be serially replated four to five times. The replating efficiency of cells in the primary mast cell colonies varied over a wide range (2.5–44%) with an average replating efficiency of 13%. The data also revealed that cells containing metachromatic granules possess significant proliferative capacity. From these studies of pure and mixed mast cell colonies, we concluded (1) that mast cells are in wide variety of types of mixed colonies and that the in situ identification of mixed colonies is unreliable, (2) that mast cells are derived from pluripotent hemopoietic stem cells, and (3) that mast cells with metachromatic granules can have a high proliferating ability.
PGE1‐independent MDCK cells have elevated intracellular cyclic AMP but retain the growth stimulatory effects of glucagon and epidermal growth factor in serum‐free medium
Authors: Taub Mary , Devis Patricia E. , Grohol Sue Hiller
Published: February 2005

Prostaglandin E1 (PGE1), a component in the hormone‐supplemented, serumfree medium for the Madin Darby canine kidney (MDCK) cell line, has been proposed to increase MDCK cell growth by increasing intracellular cyclic AMP levels. The association between increased intracellular cyclic AMP and the growth stimulatory effect of PGE1 has been examined in normal MDCK cells and in PGE1‐independent variants of MDCK. These variant cells have lost the PGE1 requirement for long term growth in defined medium. Normal MCDK cells had almost twofold higher intracellular cyclic AMP levels during growth in Medium K‐1 (9.0 pmol/mg protein) than in Medium K‐1 minus PGE1. Furthermore, PGE1‐independent clone 1 had higher intracellular cyclic AMP levels in Medium K‐1 minus PGE1 than normal MDCK cells in Medium K‐1. This latter observation suggests that the PGE1 requirement for MDCK cell growth is associated with the low intracellular cyclic AMP levels of this cell line. An involvement of cyclic AMP in the growth response to PGE1 is supported by these observations, as well as by the growth stimulatory effects of other agents that affect cyclic AMP metabolism in MDCK cells. These agents include glucagon, isobutyl methylxanthine (IBMX), and dibutyryl cyclic AMP. The growth of PGE1‐independent clone 1 was inhibited rather than stimulated by PGE1. Similarly, PGE1‐independent cell growth was inhibited by IBMX and dibutyryl cyclic AMP. However, the growth response to one agent which increases cyclic AMP (glucagon) was retained in PGE1‐independent clone 1. This result suggests that the effect of glucagon is not associated with increases in intracellular cyclic AMP. The growth stimulatory effect of epidermal growth factor (EGF) on normal MDCK cells was also studied. Although EGF does not act via a cyclic AMP‐mediated mechanism, EGF increased normal MDCK cell growth and substituted for PGE1 in Medium K‐1. Thus, EGF and PGE1 could possibly affect similar growth‐related functions in MDCK cells, although by different pathways. This possibility was examined further, using PGE1‐independent clone 1. EGF, like glucagon, was still growth stimulatory to the PGE1‐independent cells. Consequently, the biochemical pathways by which EGF and PGE1 increase MDCK cell growth probably do not converge.