bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

AbstractA 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

AbstractDevelopment 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

AbstractIn 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

AbstractSpreading 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

AbstractHuman 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

AbstractST2‐3T3, 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

AbstractThe 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

AbstractThe 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

AbstractSeveral 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

AbstractBovine 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

AbstractStudies 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

AbstractGrowth 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

AbstractProliferating 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.