bluuebunny/crossref_metadata_2025 huggingface dataset:
Hormonal responses to 1,25‐dihydroxyvitamin D3 in cultured mouse osteoblast‐like cells—modulation by changes in receptor level
Authors: Chen Theresa L. , Li Janet M. , Van Ye Todd , Cone Charlotte M. , Feldman David
Published: February 2005

The level of 1,25(OH)2D3 receptors in cultured mouse osteoblast‐like (OB) cells is modulated by the rate of cell proliferation. We have studied two 1,25(OH)2D3‐induced bioresponses to ascertain whether the changes in receptor levels during growth in culture alter cell responsiveness. Nuclear receptor levels were high (127 fmol/100 μgDNA) in rapidly dividing (log) cells and low (25 fmol/100 μg DNA) in quiescent (confluent) cells. The bioresponses we studied were induction of 25(OH)D3‐24‐hydroxylase. activity (24‐hydroxylase) and inhibition of collagen synthesis. The basal levels of 24‐hydroxylase were low and similar in cells at log growth phase and confluence. At a maximal induction dose of 13 nM, 1,25(OH)2D3 induced a three‐fold rise in enzyme activity at long growth phase, but only caused less than two‐fold rise at confluence. The half‐maximal dose (ED50) was slightly shifted from 0.6 nM to 0.8 nM. Daily measurement of 1,25(OH)2D3 receptor levels and maximal induction of 24‐hydroxylase activity throughout the culture cycle showed a strong correlation between receptor abundance and enzyme induction. The basal level of collagen synthesized by cells in log growth phase was ∼ 5% and increased to ∼ 8% at confluence. Maximal inhibition of collagen synthesis by 1,25(OH)2D3 reached 80% of control levels in log cells, but was only 40% of control in confluent cells. The ED50 was ∼ 0.1 nM in the log cells and increased to ∼ 1 nM at confluence. Daily assay of 1,25(OH)2D3 receptor levels and 1,25(OH)2D3 responses during the culture cycle indicated a correlation between changes in receptor level and the extent of inhibition of collagen synthesis. These changes in biore‐sponse at various growth phases did not occur in rat OB cells where the 1,25(OH)2D3 receptor levels were independent of cell proliferation. The results indicate that cell proliferation rate, via change in receptor levels, determines the magnitude and sensitivity of the cellular responses to 1,25(OH)2D3.
Endocytosis in sickle erythrocytes: A mechanism for elevated intracellular Ca2+ levels
Authors: Rubin Elizabeth , Schlegel Robert A. , Williamson Patrick
Published: February 2005

Staining of sickle cells with the fluorescent probes chlortetracycline (a Ca2+probe) and diindocarbocyanine (a general membrane probe) revealed the presence of Ca2+‐containing vesicles which are not found in normal erythrocytes. These vesicles increase in number upon deoxygenation, and are apparently formed by endocytosis, as judged by the use of the extracellular fluorescent probe lucifer yellow. The presence of vesicles is not restricted to any particular morphological or density class of cells in the general population.
Utilization of pyruvate and pyruvate precursors by normal and carcinogen‐altered rat tracheal epithelial cells in culture
Authors: Wasilenko William J. , Marchok Ann C.
Published: February 2005

The metabolism of [14C]pyruvate, [14C]glucose, [14C]glutamine and [14C]alanine was compared between normal rat tracheal epithelial cells and carcinogen‐altered cells derived from dimethylbenz(a)anthracene‐exposed tracheal implants. Normal primary cultures (NPC) of tracheal cells are distinguished by their need for pyruvate‐supplemented medium for growth and survival. The altered cells were selected out by their survival in the unsupplemented medium. Compared to the selected primary cultures (SPC), the NPC showed a three‐ to four‐fold higher incorporation of radioactivity from [2‐14C]pyruvate in all the macromolecular fractions, as well as in all the metabolites isolated from the acid soluble fraction and from lactic acid isolated from the medium. [U‐14C]glucose was also incorporated at higher levels into lactic acid isolated from the acid soluble fraction and the medium of NPC. These data indicate a higher rate of glycolysis in the normal tracheal cells. This was supported by the findings of a two‐fold greater glucose consumption and two‐fold higher production of lactic acid isolated from the NPC medium. Lactate dehydrogenase activity was also two‐fold higher in NPC. Thus, despite the apparently higher level of pyruvate production in the NPC, exogenous pyruvate is necessary to satisfy the metabolic needs of NPC. The utilization of [U‐14C]glutamine or [U‐14C]alanine was not markedly different between NPC and SPC. Furthermore, radioactivity from both of the amino acids was recovered in lactic acid in the medium, indicating that both cell types can derive pyruvic acid from either glutamine or alanine. SPC apparently do not use these routes to supply higher levels of pyruvic acid for survival in culture. The oxidation of none of the radioactive metabolites into CO2 was distinctly different between NPC and SPC except for the 1.7‐fold higher utilization of [1‐14C]glucose along the oxidative arm of the pentose cycle in the normal cells.
Recovery of hormonal regulation in protein kinase defective adrenal cells through DNA‐mediated gene transfer
Authors: Schimmer Bernard P. , Wong Margaret , O'Brien Donalee , Schulz Patricia
Published: February 2005

A cAMP‐resistant mutant (Kin‐8) isolated from Y1 mouse adrenocortical tumor cells harbors a specific lesion in the regulatory subunit of the type 1 cAMP‐dependent protein kinase. This mutant also is resistant to the effects of corticotropin and cAMP on steroidogenesis, growth and morphology, suggesting an obligatory role for the protein kinase in regulation of adrenocortical functions. In this study, the cAMP‐resistant phenotype of the Kin‐8 mutant was reverted by transformation with DNA from cAMP‐responsive Y1 cells, and the biochemical basis of the transformation was explored. Initially, Y1 mouse adrenocortical tumor cells were evaluated for their competence as recipients in DNA‐mediated transformation experiments, by measuring their ability to incorporate and express a bacterial gene (neo) encoding resistance to neomycin. Y1 cells were transfected with the plasmid pSV2‐neo (an SV40‐neo hybrid vector designed for expression in animal cells) and screened for resistance to the neomycin analog, G418. Neomycin‐resistant transformants were recovered from Y1 cells at a frequency of approximately one per 103 cells per 10 μg of DNA, and had specific neo sequences integrated into their high molecular weight (mw) DNA. The Y1 mutant, Kin‐8, then was transformed with pSV2‐neo DNA plus high mw DNA prepared from cAMP‐responsive Y1 cells. Cells competent for transformation were recovered by selective growth in the neomycin analog G418, and these transformants were screened for recovery of morphological responses to cAMP. Several colonies capable of rounding up in the presence of cAMP were recovered after transformation with DNA from Y1 cells. These transformants also recovered the ability to round up in the presence of corticotropin, and were able to respond to both corticotropin and cAMP with increased steroidogenesis. Transformants generated from either Y1 or Kin‐8 cells were unstable. Y1 cells lost resistance to neomycin when grown in the absence of G418 at a frequency of 4% per generation. Similarly, Kin‐8 transformants lost their sensitivity to cAMP in subsequent culture passages. In some of the cAMP‐responsive transformants, cAMP‐dependent protein kinase activity was recovered and approached the activity seen in cAMP‐responsive Y1 cells. The recovery of a normal protein kinase by transformation appeared to have been sufficient to reverse the cAMP‐resistant phenotype of Kin‐8 cells. In other cAMP‐responsive transformants, protein kinase activity was not appreciably affected by cAMP. In these latter clones the gene encoding responsiveness to cAMP may have been lost in the interval between assays of adrenal‐specific functions and protein kinase activity. Alternatively, these clones may have acquired another gene, unrelated to the protein kinase, which was capable of reversing the cAMP‐insensitive phenotype.
Catecholamine modulation of embryonic palate mesenchymal cell DNA synthesis
Authors: Pisano M. Michele , Schneiderman Martin H. , Greene Robert M.
Published: February 2005

Development of the mammalian embryonic palate depends on the precise temporal and spatial regulation of growth. The factors and mechanisms underlying differential growth patterns in the palate remain elusive. Utilizing quiescent populations of murine embryonic palate mesenchymal (MEPM) cells in vitro, we have begun to investigate hormonal regulation of palatal cell proliferation. MEPM cells in culture were rendered quiescent by 48 hr serum deprivation and were subsequently released from growth arrest by readdition of medium containing 10% (v/v) serum. The progression of cells into S‐phase of the cell cycle was monitored by autoradiographic analysis of tritiated thymidine incorporation. Palate mesenchymal cell entry into S‐phase was preceded by a 6‐ to 8‐hr prereplicative lag period, after which time DNA synthesis increased and cells reached a maximum labeling index by 22 hr. Addition of 10 μM isoproterenol to cell cultures at the time of release from growth arrest lengthened the prereplicative lag period and delayed cellular entry into S‐phase by an additional 2 to 4 hr. The rate of cellular progression through S‐phase remained unaltered. The inhibitory effect of isoproterenol on the initiation of MEPM cell DNA synthesis was abolished by pretreatment of cells with propranolol at a concentration (100 μM) that prevented isoproterenol‐induced elevations of cAMP. Addition of PGE2 to cell cultures, at a concentration that markedly stimulates cAMP formation, mimicked the inhibitory effect of isoproterenol on cellular progression into S‐phase. These findings demonstrate the ability of the β‐adrenergic catecholamine isoproterenol to modulate MEPM cell proliferation in vitro via a receptor‐mediated mechanism and raise the possibility that the delayed initiation of DNA synthesis in these cells is a cAMP‐dependent phenomenon.
Serum factors that stimulate fatty acid oxidation: Physiological specificity
Authors: Stanisz Jolanta , Wice Burton M. , Kennell David E.
Published: February 2005

In the accompanying paper (Wice et al., 1986) we reported that serum from chickens contains small molecular weight compounds that stimulate longchain fatty acid oxidation ten fold or more in HeLa cells. Here we show that this response is not limited to specific sera or to specific target cells. The specificity of the metabolic response to these factors was also investigated. They had no effect on the following major pathways of HeLa cell metabolism: (1) the oxidation of the medium‐chain fatty acid, octanoic acid, (2) the rate of glycolysis of glucose, (3) the flux of glucose carbon through the oxidative arm of the pentose cycle, (4) the entry of pyruvate into the citrate cycle, (5) the oxidation of glutamine carbon, (6) the utilization rate of oxygen or (7) the rate of fatty acid synthesis. Furthermore, the increased oxidation of long‐chain fatty acids was not a result of an increased uptake into the cells. Thus, the serum factors appear to be very specific for the oxidation of long‐chain fatty acids for energy. Since carnitine also stimulates long‐chain fatty acid oxidation in these cells, it seems likely that these compounds either facilitate the activity of carnitine or provide the same function–presumably the transport of long‐chain fatty acid into and out of the mitochondria.
Spreading ciliary arrest in a mussel gill epithelium: Characterization by quick fixation
Authors: Reed William , Satir Peter
Published: February 2005

Spreading ciliary arrest, induced by local laser microinjury, in freshwater mussel (e.g., Elliptio) gill lateral (L) cell cilia, has been characterized by quick fixation with osmium tetroxide, which permits the correlation of known features of the response with structural features of the gill epithelium. Quick fixation reliably preserves the state of the epithelium including the activity state of the L cilia at the moment of fixation.

From a disrupted region, the stimulus that triggers arrest spreads outward along an undamaged filament preferentially from L cell to L cell for more than 300 μm to either side of the lesion. In physiological salt solutions transverse spread across the filament via heterologous cells is insufficient to elicit L ciliary arrest on the opposite side of the filament. The spread of arrest is dependent upon the structural integrity of the L epithelium, normally terminates at a boundary between adjacent L cells, and does not spread past a focal break. Arrest occurs asynchronously because cilia in different stroke positions respond to the stimulus with different time courses. The cilia stop in a uniform “hands up” position, i.e., pointing frontally. The arrest response is inhibited by reducing the concentration of extracellular Ca2+ (< 10−7 M) or by adding extracellular La3+ (1 mM) or K+ (15 mM).

Recovery begins at the margin of a segment of arrested L cilia and spreads back toward the lesion at a constant initial velocity of ca. 60 μm/sec. About 300 μm from the lesion the recovery velocity rapidly falls to ca. 5 μm/sec. Recovery of ciliary beat precedes the recovery of metachronal coordination. Neither spread of the stimulus nor recovery require ciliary beat.

The data support the hypothesis that the microinjury‐induced arrest is initiated by an injury potential that triggers a graded regenerative depolarization that is propagated electrotonically along the epithelium from L cell to L cell, triggering Ca2+ influx into the axoneme and consequent Ca2+‐induced L ciliary arrest as it spreads. A temporary non‐linear gradient of intracellular Ca2+ concentration is established along the injured L epithelial tract. As individual cells recover, they lower their intracellular Ca2+ concentration from pCa 5 to pCa 7 in about 10 seconds.
Adhesion of T lymphocytes to human endothelial cells is regulated by the LFA‐1 membrane molecule
Authors: Mentzer Steven J. , Burakoff Steven J. , Faller Douglas V.
Published: February 2005

Human T lymphocyte adhesion to human endothelial cells is the initial event in T cell migration to areas of extravascular inflammation. The molecular basis for T cell‐endothelial cell adhesion was investigated using two different cell‐cell adhesion assays: (a) a fluorescein cell‐cell adhesion assay using non‐adherent endothelial cells and fluorescein‐labeled T lymphocytes, and (b) a radionuclide cell‐cell adhesion assay using adherent endothelial cells and 51Cr‐labelled T cells. Both assay systems demonstrated comparable quantitative assessment of cell‐cell adhesions. The assays were performed at 22°C and adhesions were maximal at 30 min. The results of these adhesion assays confirmed previous reports that T cells adhere to endothelial cells. In addition, we have shown that T cells adhere only marginally to foreskin fibroblasts or bone marrow derived fibroblasts. T cell‐endothelial cell adhesions were significantly stronger than either monocytes or B lymphoblastoid cells adhesion to endothelial cells. To demonstrate the molecular mechanisms involved in regulating T cell‐endothelial cell adhesions, a panel of function‐associated monoclonal antibodies (MAb) were tested for their ability to inhibit T cell adhesion. MAb reactive with the leukocyte surface glycoprotein LFA‐1 signifi‐cantly inhibited T cell‐endothelial cell adhesions in both assay systems. In contrast, MAb directed at other surface antigens did not inhibit T cell adhesion. The involvement of the LFA‐1 glycoprotein in T lymphocyte adhesion to endothelial cells suggest that the LFA‐1 molecule may be important in the regulation of leukocyte interactions.
Dissociation of cellular transformation from platelet‐derived growth factor independence
Authors: Scher C. D. , Engle L. J. , Eberenz W. M. , Ganguly K. , Wharton W.
Published: February 2005

ST2‐3T3, a spontaneously transformed BALB/c‐3T3 cell line which does not require platelet‐derived growth factor (PDGF) for growth, was fused to THO2, a PDGF‐responsive non‐transformed BALB/c‐3T3 cell line, in order to learn whether transformation is expressed coordinately with PDGF independence. Hybrid cells were selected and grown in medium containing both HAT (hypoxanthine‐aminopterin‐thymidine) and ouabain; unfused cells of each parental type were killed in HAT‐ouabain medium. Five independently isolated ST2‐3T3xTHO2 hybrid cell lines were established and characterized for both transformation and PDGF responsiveness. All five were transformed, having a disorganized growth pattern and achieving a final cell density similar to that of ST2‐3T3 cells. Two of these lines did not respond to a brief treatment with PDGF: the mitogen neither induced the synthesis of a PDGF‐modulated lysosomal protein (termed MEP), nor stimulated the cells to enter the S phase; one line responded to PDGF by synthesizing both MEP and DNA, whereas two others synthesized MEP but not DNA. In contrast, four independently isolated cell lines obtained by fusing PDGF‐responsive non‐transformed BALB/c‐3TC cells to the THO2 line were all PDGF‐responsive for both MEP and DNA synthesis and were not transformed. It appears that PDGF independence is not required for the transformation of BALB/c‐3T3 cells.
Heparin‐induced aggregation of lymphoid cells
Authors: Thurn Anne L. , Underhill Charles B.
Published: February 2005

The effects of different carbohydrates on cell‐to‐cell adhesion were examined in an aggregation assay, which consisted of swirling a suspension of cells and monitoring the loss of single cells with a Coulter Counter. Of the carbohydrates tested, only heparin and dextran sulfate induced cell aggregation. This effect occurred in freshly isolated mouse splenocytes and in cultured cells of lymphoid origin (P388, YAA‐CI) but not in cell lines of fibroblastic origins (3T3, SV‐3T3, BHK, and PY‐BHK). Using the YAA‐CI cell line for further study, we found that aggregation could be induced by relatively small amounts of heparin (<10 μg/ml). Binding experiments with 3H‐heparin showed that under normal physiological conditions each YAA‐CI cell bound approximately 2 × 106 molecules of heparin at saturation with a Kd of 3.5 × 10−7 M. This binding was blocked by both unlabelled heparin and dextran sulfate but not by other carbohydrates. When the pH of the medium was decreased, the heparin‐induced aggregation was inhibited, and the Kd of the 3H‐heparin binding was increased. In a similar fashion, when the ionic strength of the medium was increased, heparin‐induced aggregation was inhibited and the Kd of the interaction was increased. These results suggest that the aggregation is inversely related to the Kd of the interaction and that the binding of heparin to the cell surface is primarily of an ionic nature.
Plasma membrane potential of murine erythroleukemia cells: Approach to measurement and evidence for cell‐density dependence
Authors: Arcangeli Annarosa , Olivotto M.
Published: February 2005

The plasmamembrane potential (ΔΨp) of murine erythroleukemia (MEL) cells has been determined by measuring the distribution of the lipophilic cation tetraphenylphosphonium (TPP+) across the plasmamembrane. TPP+ accumulation within the cells (experimental accumulation ratio, ARexp) was measured by adding 2 μM TPP+ directly to the culture flasks, avoiding any other perturbation of the experimental system. The mitochondrial contribution to ARexp, evaluated by adding carbonyl cyanide p‐trifluoromethoxyphenylhydrazone (FCCP) or 2,4‐dinitrophenol (DNP), was apparently negligible in standard cultures, ARexp being substantially the same in either the absence or presence of these uncouplers. However, the addition of oligomycin produced a strong ARexp enhancement, which was abolished by FCCP, suggesting that MEL cell mitochondria are in state 3. The aspecific TPP+ binding was estimated by a new mathematical approach worked out to fit ARexp values measured in the presence of valinomycin at various extracellular K+ concentrations and plotted against the ratio of intracellular to extracellular K+ concentration ([K+]i/[K+]e). This binding was found to be close to zero in MEL cells. By applying the Nernst equation directly to ARexp values, ΔΨp of these cells was then measured; this potential varying from −65 mV to −16 mV (inside negative) is inversely related to the cell density on the culture surface on which the cells sediment (cells/cm2; CD). The dependence of ΔΨp on CD is practically eliminated by valinomycin and appears to be related to a cell interaction with the culture surface of the flasks, suggesting that in the immediate environment of MEL cells one or more factors are produced that modulate the K+ plasma membrane permeability (Pk).
Binding and second messengers of prostaglandins F2α and E1 in primary cultures of rabbit endometrial cells
Authors: Orlicky David J. , Lieberman Rita , Williams Cheryll , Gerschenson L. E.
Published: February 2005

Several factors and hormones are thought to play a role in the growth control of endometrial cells. We have shown that prostaglandin F2α (PGF2α) is a growth factor for primary cultures of rabbit endometrial cells grown in serum‐free, chemically defined medium and that prostaglandin E1 (PGE1) antagonizes the PGF2α induction of growth (Orlicky et al., 1986). [3H]PGF2α binds to whole cells in a time (optimal ∼30 min)‐ and temperature‐dependent (optimal 37°C), disassociable (90% disassociable within 30 min), saturable (Kd1 = 4.9 × 10−8 M, n1 = 1.2 × 105 molecules/cell; Kd2 = 2.6 × 10−7 M, n2 = 3.0 × 105 molecules/cell), and specific manner. [3H]PGE1 binds in a time‐dependent (optimal 25 min), disassociable (90% disassociable within 10 min), saturable (Kd = 6.4 × 10−8 M, n = 1.2 × 105 molecules/cell), and specific manner. This specific binding of [3H]PGF2α and [3H]PGE1 is down‐regulatable by prior treatment of the cultures with unlabeled ligand, and up‐regulatable by prior treatment of the cultures with indomethacin to inhibit endogenous PG synthesis. Proteolytic enzyme treatment for 2 min reduces the specific binding of PGF2α by 75%. PGE1 stimulates intracellular cAMP synthesis and accumulation in a time (optimal 10 min)‐ and concentration (half‐maximal stimulation at 10−6 M)‐dependent manner but has no effect on intracellular cGMP. PGF2α has no effect on either intracellular cAMP or cGMP in this system. We describe here for the first time the analysis at a biochemical level of the interaction between two prostaglandins, antagonistic to each other in terms of growth regulation.
Effect of fibroblast growth factor and lipoproteins on the proliferation of endothelial cells derived from bovine adrenal cortex, brain cortex, and corpus luteum capillaries
Authors: Gospodarowicz D. , Massoglia S. , Cheng J. , Fujii D. K.
Published: February 2005

Bovine adrenal and brain cortex and corpus luteum‐derived capillary endothelial cells have been established in culture, taking advantage of their ability to proliferate at clonal density when maintained on extracellular matrix (ECM) coated dishes in the presence of serum supplemented medium. All three cell types formed at confluency a monolayer of small, tightly packed, contact inhibited cells that express factor VIII related antigen. Their proliferative response to basic and acidic FGF when cells were maintained on plastic and exposed to serum supplemented medium was similar to that previously reported for endothelial cells derived from large vessels, with acidic FGF being 30‐fold less potent than basic FGF. Their requirement for high density lipoproteins and transferrin in order to proliferate actively when maintained on ECM‐coated dishes and exposed to serum‐free conditions was also similar to that previously reported for endothelial cells derived from large vessels. Heparin strongly reduced the proliferative response of capillary endothelial cells to either basic or acidic FGF, as well as their response to serum alone, regardless of whether cells were maintained on plastic or on ECM‐coated dishes. The present data indicate that bovine endothelial cells derived from large or small vessels are indistinguishable in so far as their response to growth factors, plasma factors, and substrata are concerned.
Effects of mevinolin and mevalonate on cell growth in several transformed cell lines
Authors: Fairbanks Kathy P. , Barbu Veronique D. , Witte Larry D. , Weinstein I. Bernard , Goodman DeWitt S.
Published: February 2005

Studies were conducted to explore the effects of mevinolin, a competitive inhibitor of HMG CoA reductase, on the growth and morphology of normal and transformed murine fibroblasts. Mevinolin is known to block DNA synthesis and cell growth in a number of kinds of non‐transformed cells. Eight cell lines were studied, including two normal fibroblast cell lines (C3H 10T 1/2 and NIH 3T3) and derivatives of these cell lines transformed by chemical carcinogens, X‐irradiation or the H‐ras oncogene. All of the eight cell lines displayed appreciable growth inhibition by 5 μM mevinolin and marked inhibition by 30 μM mevinolin. Mevinolin also induced a marked rounding in the morphology of all of the cell lines. These effects of mevinolin on cell growth and morphology were blocked or reversed by the addition of mevalonic acid. Thus, both normal and transformed cells require mevalonate, or an as yet unidentified metabolite of mevalonate for their growth, even though some transformed cells have become relatively autonomous of other growth factors. Whereas mevinolin acted primarily as a cytostatic agent for most of the cell lines studied, with the transformed cell line MCA/10T 1/2, which ordinarily grows to a very high cell density, prolonged exposure to mevinolin caused marked cytotoxicity. Thus mevinolin might be useful as an anti‐tumor agent for specific tumors.
Regulation of amino acid uptake by phorbol esters and hypertonic solutions in rat thymocytes
Authors: Klip Amira , Mack Esther , Cragoe Edward J. , Grinstein Sergio
Published: February 2005

Growth factors, mitogens, and malignant transformation can alter the rate of amino acid uptake in mammalian cells. It has been suggested that the effects of these stimuli on proliferation are mediated by activation of Na+/H+ exchange. In lymphocytes, Na+/H+ exchange can also be activated by phorbol esters and by hypertonic media. To determine the relationship between the cation antiport and amino acid transport, we tested the effects of these agents on the uptake of α‐aminoisobutyric acid (AIB), methyl‐AlB, proline, and leucine in rat thymocytes. Both 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) and hypertonicity stimulated amino acid uptake through system A (AIB, proline, and methyl‐AlB). In addition, TPA, but not hypertonicity, also elevated leucine uptake. The stimulation of the Na+‐dependent system A was not due to an increased inward electrochemical Na+ gradient. The effects of TPA and hypertonic treatment were not identical: Stimulation of AIB uptake by TPA was observed within minutes, whereas at least 1 hr was required for the effect of hypertonicity to become noticeable. Moreover, stimulation by hypertonicity but not that by TPA, was partially inhibited by cycloheximide, suggesting a role of protein synthesis. That stimulation of Na+/H+ exchange does not mediate the effects on amino acid transport is suggested by two findings: (1) the stimulation of AIB uptake was not prevented by concentrations of amiloride or of 5‐(N,N‐disubstituted) amiloride analogs that completely inhibit the Na+/H+ antiport and (2) conditions that mimic the effect of the antiport, namely, increasing [Na+]i or raising pHi failed to stimulate amino acid uptake. Thus, in lymphocytes, activation of Na+/H+ exchange and stimulation of amino acid transport are not casually related.
Immediate effects of serum depletion on dissociation between growth in size and cell division in proliferating 3T3 cells
Authors: Larsson Olle , Dafgård Eva , Engström Wilhelm , Zetterberg Anders
Published: February 2005

Proliferating nonconfluent 3T3 cells become committed to proceed through the cell cycle or to enter G0 during the first post‐mitotic part of G1 (G1pm). The decision to proceed through G1pm is dependent on the presence of serum growth factors in the culture medium. Cells that have passed this particular growth‐factor‐dependent cell cycle stage are independent of serum growth factors and undergo mitosis on schedule. We report here that G1ps, S, and G2 cells cease to increase in size when serum is withdrawn. As a result the mitotic cell size after 8 hours serum starvation is reduced to approximately 60% of the normal mitotic cell. This reduced growth in cell size is due to a rapid decrease in protein synthesis and some increase in protein degradation. This dissociation between growth in size and cell‐cycle progression within a single cell cycle provides a new approach to study the two processes separately.
Increased phagocytosis of outer segments in the presence of serum by cultured normal, but not dystrophic, rat retinal pigment epithelium
Authors: Edwards Ross B. , Flaherty Paula M.
Published: February 2005

Cultured normal rat retinal pigment epithelium (PE) ingested six times more rod outer segments in the presence of 20% fetal bovine serum than in serumfree medium. PE cultured from Royal College of Surgeons (RCS) rats with hereditary retinal dystrophy, known to have a defect in vivo in the phagocytosis of shed outer segment tips, ingested amounts of outer segments comparable to normal PE in serum‐free medium but did not show an increase in the presence of serum. In both strains of rat PE phagocytosis of latex spheres was similar in the absence of serum and was six‐fold higher in the presence of serum, showing that the RCS phagocytic deficiency for outer segments in vitro is not due to a general defect in the phagocytic capacity of the cell. Increased phagocytosis of outer segments by normal PE was observed in the presence of the high molecular weight fraction of ultrafiltered serum but was not seen with serum that was heated at 93°C or precipitated with 5% trichloroacetic acid. Bovine serum albumin had no effect on phagocytosis. These results are consistent with the idea that the phagocytosis of outer segments by cultured normal rat PE, but not by cultured RCS rat PE, is increased in the presence of a specific protein or other macromolecular component of fetal bovine serum.
1,2‐diacylglycerols mimic phorbol 12‐myristate 13‐acetate activation of the sea urchin egg
Authors: Shen Sheldon S. , Burgart Lawrence J.
Published: February 2005

Phorbol diesters have been reported to stimulate the Na+/H+ antiport of a variety of cells including sea urchin eggs. Since stimulation of the Na+/H+ antiport is necessary for metabolic derepression during fertilization and protein kinase C is a target of phorbol diesters, enhanced Na+/H+ exchange during fertilization may be a result of protein kinase C activity. Protein kinase C is probably physiologically activated by diacylglycerols, which are derived from hydrolysis of phosphatidylinositol. Treatment of sea urchin eggs with 1,2‐diacylglycerols was found to stimulate the Na+/H+ antiport. The 1,3‐isomers were without effect. Further, the effects of 1,2‐diacylglycerol and phorbol diester are not additive with respect to Na+/H+ exchange. While a direct participation of protein kinase C activity during fertilization remains to be demonstrated, these data support the hypothesis that protein kinase C activity plays a role in fertilization. However, the cytotoxic effect of protein kinase C activators suggests effects associated with their pleiotropic nature.
Synthesis and stability of nuclear matrix proteins in resting and serum‐stimulated swiss 3T3 cells
Authors: Milavetz Barry I. , Edwards Dylan R.
Published: February 2005

The major [35S]methionine‐radiolabeled nuclear matrix proteins of mouse 3T3 cells were isolated, and most of these were found to be similar in molecular weight, charge, and protease fingerprint to the nuclear matrix proteins of African green monkey kidney cells, which are found tightly bound to simian virus 40 chromosomes. These nuclear matrix proteins were found to be synthesized in quiescent and serum‐stimulated cells and to be turned over slowly during pulse‐chase experiments. In contrast, a 70‐Kd (kilodalton) neutral protein identified as lamin a was found to be turned over rapidly, producing a 68‐Kd protein with a similar isoelectric point. In addition, we observed a decrease in the amounts of two chromatin‐bound matrix proteins and a relative increase in lamin a following labeling in the presence of 2 μg/ml actinomycin D. However, these effects do not appear to be a result of inhibition of transcription, since they were not observed with other inhibitors (α‐amanitin and 5,6‐dichloro‐1‐β‐D‐ribofuranosylbenzimidazole).
Effects of ornithine decarboxylase inhibition on c‐myc expression during murine erythroleukemia cell proliferation and differentiation
Authors: Watanabe Tetsu , Sherman Matthew , Shafman Timothy , Iwata Tadatoshi , Kufe Donald
Published: February 2005

The polyamines putrescine, spermidine, and spermine have been implicated in the regulation of cellular proliferation and differentiation. We have previously demonstrated that spermidine is required for proliferation of murine Friend erythroleukemia (MEL) cells. We have also shown that spermidine is required at a transcriptional level for induction of MEL globin synthesis. Since studies monitoring c‐myc expression have suggested that this gene also plays a role in both growth and differentiation, we have monitored the effects of inhibiting ornithine decarboxylase (ODCase) activity and polyamine synthesis on levels of c‐myc transcripts. The results demonstrate that the level of c‐myc RNA is independent of ODCase inhibition and depletion of intracellular spermidine. More importantly, arrest of MEL proliferation is not associated with detectable changes in c‐myc expression, while under these conditions there is a decline in ODCase transcripts. During induction of MEL differentiation with dimethyl sulfoxide (DMSO) and hexamethylene bisacetamide (HMBA), c‐myc and ODCase undergo similar changes in patterns of expression. However, although spermidine is required for appearance of the differentiated MEL phenotype, depletion of this polyamine by ODCase inhibition had no detectable effect on the biphasic changes in c‐myc RNA observed during MEL differentiation. Thus, these biphasic changes in c‐myc expression are not sufficient for induction of the mature phenotype. Finally, these results would indicate that the regulation of c‐myc expression during both proliferation and differentiation is independent of ODCase activity and inhibition of proliferation by spermidine depletion.
Demonstration of the specific binding of bovine transferrin to the human transferrin receptor in k562 cells: Evidence for interspecies transferrin internalization
Authors: Tsavaler Larisa , Stein Barry S. , Sussman Howard H.
Published: February 2005

Specific binding of ferric bovine transferrin to the human transferrin receptor was investigated using K562 cells propagated in serum‐free medium without transferrin supplemented with 10−5 elemental iron. Affinity chromatography of solubilized extracts of K562 cells surface‐labeled with 125I was performed using bovine transferrin‐ and human transferrin‐Sepharose 4B resins. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of resin eluates reveal that bovine transferrin specifically binds a Mr = 188,000 protein which dissociates into a Mr = 94,000 protein under reducing conditions, a finding identical to what is seen with human transferrin. The Mr = 94,000 reduced protein isolated by bovine transferrin resin shows an identical one‐dimensional partial proteolytic digestion map with that of the human transferrin receptor.

Unlabeled bovine transferrin was shown to specifically compete 125I‐labeled human transferrin from the human transferrin receptor on the surface of K562 cells at 4°C in a similar manner as unlabeled human transferrin; however, approximately a 2,000‐fold higher concentration of bovine ligand was required to achieve comparable competition (50% inhibition of binding).

Indirect immunofluorescence cytolocalization of bovine transferrin in K562 cells grown in serum‐free medium supplemented with ferric bovine transferrin reveal patterns similar to those seen for human transferrin (both focal perinuclear and diffuse cytoplasmic fluorescence). Monensin treatment results in a dramatic accumulation of bovine ligand in perinuclear aggregates, suggesting that it is recycled through the Golgi, as is human transferrin. K562 cells grown in serum‐free medium supplemented with either 300 μg/ml of ferric human or ferric bovine transferrin were found to demonstrate super‐imposable growth curves.
Phosphate transport in Ehrlich ascites tumor cells: Inhibition by H+
Authors: Bowen Jesse W. , Levinson Charles
Published: February 2005

The effect of changes in extracellular pH (pHo) and intracellular pH (pHi) on Na + ‐dependent and Na+ ‐independent inorganic phosphate (Pi) transport in Ehrlich cells was investigated. In the presence of Na+, acutely reducing pHo from 7.30 to 5.50 results first in a transient (∼7 min) stimulation of Pi transport. The enhanced rate of transport is a saturable function of the extracellular [H+]; the Ks equals 2.3 × 10−6 M (pHo 6.68). However, Pi transport is progressively inhibited as pHi falls below 6.50. The effect of pHi on Pi transport measured at various intracellular [Na+] suggests that inhibition develops as a consequence of H+ interaction with an intracellular Na+ site(s) on the Na+‐dependent carrier. At pHo 7.4, about 15% of the steady state Pi flux persists in the absence of Na+. However, when pHo is reduced, transport is stimulated to the same extent and with the same time course and kinetic characteristics as in the presence of Na+. Thus, H+ stimulated Pi transport does not require Na+, raising the possibility that the Na+‐independent component is mediated by the anion (CI−) exchanger.
Calcitonin and vasopressin affect epithelial properties in a renal cell line
Authors: Wohlwend Annelise , Vassalli Jean‐Dominique , Bertrand Daniel , Orci Lelio
Published: February 2005

We have used a stable clonal variant (D + Sc), isolated from the LLC‐PK1 pig kidney‐derived cell line and selected for its extensive capacity to form domes, in order to study the hormonal modulation of epithelial permeability in culture. Calcitonin, vasopressin, and other agents that raise intracellular adenosine 3′,5′‐cyclic monophosphate levels caused a rapid and dramatic decrease in the size and number of domes. This effect was independent of RNA and protein synthesis, and thus appeared unrelated to the production of urokinase, a proteinase synthesized by the cells in response to these agents. Calcitonin caused a decrease in transepithelial electrical resistance, suggesting that the effect of the hormone on domes was due to an increase in the permeability of a paracellular pathway. Thus, in addition to the wellknown effects of vasopressin on collecting duct permeability, part of the in vivo effect(s) of calcitonin and vasopressin on the renal tubule might also involve alterations of epithelial permeability related to those described here.
Concanavalin a inhibits oral regeneration in Stentor coeruleus through a mechanism involving binding to the cell surface
Authors: Maloney Michael S.
Published: February 2005

Concanavalin A (Con A) has been shown to induce delays in oral regeneration in the ciliate Stentor coeruleus. Associated with the delayed oral regeneration is a shedding of the cell's extracellular pellicle with the loss of some pigment granules. It is shown that the delays in oral regeneration are not the result of the pigment shedding. The delays are localized in the earliest stages of oral regeneration prior to stage 4. The delays caused by Con A are completely reversible by the addition of 2 mg/ml α‐D‐methyl mannoside either at the time of Con A exposure or 5 min later. Con A clearly binds to the cell surface as shown by the binding of FITC‐Con A and its reversal by α‐methyl mannoside. Crosslinking of Con A receptor molecules may be responsible for the effects of Con A since succinyl Con A, which does not crosslink these receptors, has no effect on oral regeneration even at double the Con A concentration. Calcium ions are also implicated in the action of Con A because an excess of extracellular calcium (10 mM) completely eliminates the Con A delays when added simultaneously with Con A. Examination of the minimum extracellular calcium concentration required for this effect showed that 2 mM calcium can reverse most of the delays but that 5 mM is ncessary to completely reverse the delays caused by Con A. If the addition of calcium is delayed for various times after Con A addition, the extracellular calcium is progressively less effective in reversing the Con A delays.
Increased level of cAMP‐dependent protein kinase in aging human lung fibroblasts
Authors: Liu Alice Y.‐C. , Chang Zee‐Fen , Chen Kuang Yu
Published: February 2005

Regulation of the expression of cAMP‐dependent protein kinase in cellular aging was studied using the IMR‐90 diploid human lung fibroblasts. The level of cAMP‐dependent protein kinase present in cell extracts was monitored by (1) photoactivated incorporation of 8‐N3‐[32P]cAMP into the 47,000‐ and 54,000‐dalton regulatory subunits of the type I and type II cAMP‐dependent protein kinases, respectively; (2) cAMP‐dependent phosphorylation of histone II AS catalyzed by the catalytic subunit of the kinase; and (3) fractionation and analysis of the type I and type II cAMP‐dependent protein kinase by DEAE‐Sephacel column chromotography. Our results showed an approximately two‐ to threefold increase in the level of the type I cAMP‐dependent protein kinase and a somewhat smaller increase in the type II kinase in extracts of the “old” IMR‐90 cells (population doubling >48) as compared to that of the “young” cells (PDL 22–27). The timing of the increase in cAMP‐dependent protein kinase coincided with a significant decrease in the proliferative potential of the cells. This result together with previously demonstrated effects of cAMP in the control of cell growth and differentiation and the increased expression of cAMP‐dependent protein kinase during terminal differentiation of the murine preadipocytes (3T3‐L1) and myoblast (L‐5, L‐6, and C2C13) suggests that regulation of the levels of cAMP and cAMP‐dependent protein kinase plays a significant role in the control of cell growth and differentiation.
Binding of iodinated multipotential colony‐stimulating factor (interleukin‐3) to murine bone marrow cells
Authors: Nicola N. A. , Metcalf D.
Published: February 2005

Multipotential colony‐stimulating factor (Multi‐CSF or interleukin‐3) was radioiodinated to high specific radioactivity (1–4 × 105 cpm/ng) with no detectable loss of biological activity and its binding to murine bone marrow cells and factor‐dependent cell lines studied. Both the native glycosylated molecule purified from a cloned T‐cell line (LB3) and the purified non‐glycosylated recombinant molecule produced by E. coli could be radioiodinated. Comparative binding studies with these derivatives demonstrated equal binding affinities and equal numbers of binding sites on various cell types indicating that carbohydrate moieties are not involved in the binding interactions. Binding of 125I‐Multi‐CSF to several factor‐dependent continuous hemopoietic cell lines showed the presence of specific receptors on all cell lines, the receptor number per cell varying from 700 to 13,000 and the apparent dissociation constant from 400 pM to 1 nM. Specific binding of 125I‐Multi‐CSF was also observed to normal murine hemopoietic cells and the binding to murine bone marrow cells was studied in detail. Bone marrow cells showed 117–130 receptors per cell on average and an apparent dissociation constant of 126–233 pM. However, quantitative autoradiographic analysis indicated that receptors for 125I‐Multi‐CSF were not distributed randomly on bone marrow cells–‐nucleated erythroid and lymphoid cells were not labeled while essentially all neutrophilic granulocyte, eosinophilic granulocyte and monocytic cells were labeled. Moreover, in each of the labeled cell lineages grain counts (reflecting receptor number) decreased with increasing maturation and a small subpopulation of marrow cells (0.4–1.5% and including blast cells, monocytes, promyelocytes, and myelocytes) exhibited very high grain counts. The existence of such a subset of marrow cells raises the possibility of functional heterogeneity among marrow cells in their response to Multi‐CSF.
Morphologic and biochemical variability of tissue and cultured cells from human pheochromocytoma
Authors: Jaques Sandford , Tobes Michael C.
Published: February 2005

Primary cell cultures from 18 human pheochromocytomas were maintained in culture for 10 to 12 days and characterized. The cell yields ranged from 1.0 to 60.1 × 106 cells/g wet weight of tissue. Cell size, as determined by histofluorescent microscopy, varied as much as seven‐fold among cells derived from a given tumor and ten‐fold between cells from all tumors. Cell catecholamine content, norepinephrine (NE) plus epinephrine, ranged from 0.4 to 89.5 nmol/106 cells at day 5 in culture and did not correlate with catecholamine content of the tissue from which the cells were obtained. Cell catecholamine content decreased with time in culture, but this decrease could not be related to a change in cell viability, the type of media used, an inability to convert dopamine to NE, or an alteration in the uptake of 3H‐NE. Cellular uptake of 1.0 μM 3H‐NE varied as much as 230‐fold between all cell dispersions. The basal and acetylcholine stimulated release of both preloaded 3H‐NE and the endogenous catecholamines was quite variable. There was no correlation between the release rate, either basal or stimulated, of preloaded 3H‐NE and the endogenous catecholamines. This study represents the largest existing data base on culturing cells from these tumors and describes many of the morphologic and biochemical characteristics of this cell system.
Induction of fibronectin expression, actin cable formation, and entry into S phase following reexpression of T antigen in mouse macrophages transformed by the tsA640 mutant of SV40
Authors: Takayama Hisao , Tanigawa Takahiko , Tanaka Yoshinori , Kimura Genki
Published: February 2005

Mouse macrophages transformed by a temperature‐sensitive mutant (tsA640) of simian virus 40 (SV40) were examined by immunofluorescence microscopy for fibronectin expression and actin distribution. Resting cultures of tsA640 transformants incubated at a temperature nonpermissive for SV40 large T antigen (39.0°C) exhibited phagocytic activity and did not exhibit cellular fibronectin and actin cables, like primary cultures of resident macrophages. When the resting cultures were sparsely seeded and shifted down to the permissive temperature of 33.0°C, expression of large T antigen in the nucleus, expression of fibronectin in the cytoplasm, and cellular entry into S phase occurred in that temporal order, followed by actin cable formation, cellular proliferation, and diminishment of phagocytic activity. The expression of T antigen and fibronectin was sensitive to actinomycin D and cycloheximide. The expression of fibronectin was insensitive to inhibitors of DNA synthesis, whereas the expression of actin cables was sensitive. These results suggest that SV40 T antigen leads macrophages to express fibronectin and actin cables, as well as resumption of cell proliferation, and that entry into S phase is not required for fibronectin expression but may be required for actin cable formation.