bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of the Na+‐dependent and the Na+‐independent polyamine transporters in renal epithelial cells (LLC‐PK1)
Authors: Parys Jan B. , De Smedt Humbert , van Dan Bosch Ludo , Borghgraef Roger , Geuns Jan 
Published: February 2005

AbstractWe have studied the regulation of the Na+‐dependent and Na+‐independent polyamine transport pathways in the renal LLC‐PK1 cell line. Most of the experiments were performed in the presence of 5 mM DL‐2‐difluoromethylornithine (DFMO) in order to inhibit the cellular synthesis of polyamines. The activity of both transporters as measured by putrescine uptake was increased by growth‐promoting stimuli and decreased by exogenous polyamines. The time course of the increase in uptake activity induced by fetal calf serum could be fitted by a single exponential, and the process was three times faster for the Na+‐dependent than for the Na+‐independent transporter. Maximum activity was reached after more than 24 h. This increase could be inhibited by actinomycin D and by cycloheximide. Other growth‐promoting stimuli, such as subconfluent cell density, as well as growth factors also induced an increase in the transport activity. Particularly, there was a marked stimulation of the Na+‐dependent pathway by epidermal growth factor in combination with insulin. On the other hand, the transport activity decayed very rapidly upon addition of exogenous polyamines (t1/2 < 60 min). The diamine putrescine was much less effective in this respect than the polyamines spermidine and spermine. The non‐metabolizable substrate methylglyoxal bis(guanylhydrazone) did not induce a decay of the transport activity, but it protected the Na+‐dependent pathway against the polyamine‐in‐duced decay. Inhibition of the protein synthesis by cycloheximide did not induce a rapid decrease of the transport activity; neither did it affect the polyamine‐induced decay. These observations suggest that this polyamine‐induced decay is not owing to an inhibitory effect on the rate of synthesis of the transporters, but rather to a degradation or an inactivation of the transporters. The polyamine‐induced decay slowed down at lower cell density. This effect was particularly pronounced for the Na+‐dependent transporter. Since the uptake of polyamines was increased at low cell density, the decreased rate of decay in this condition pleads against a simple mechanism of transinhibition by the substrate. In conclusion, both transport pathways were similarly affected by the regulatory parameters, but the Na+‐deperdent transporter was more rapidly and more effectively regulated. The numerous interacting regulatory steps furthermore suggest a Physiological role for these transporters, such as an involvement in urinary polyamine disposal.
Clonal variation of adenylyl cyclase activity in a rat tumor cell line caused by change in G protein‐catalytic unit interaction
Authors: Kawabata Masahiro , Yoshikura Hiroshi , Horio Keizo , Fujiwara Kenji , Iwamoto Aikichi 
Published: February 2005

AbstractTwo subclones of the rat XC cell line characterized by different morphology exhibited quite different adenylyl cyclase responses upon various stimulations. Upon treatment with cholera toxin, clone RK1 accumulated a high level of intracellular cAMP thereby changing its polygonal morphology to an elongated morphology, while the other clone, LK1, with a fibroblastic morphology, failed to increase the intracellular cAMP and remained morphologically unchanged. When membrane fractions derived from these two clones were stimulated with 10 μM forskolin, 10 μM GTPλS, or 10 mM NaF, five‐ to 20‐fold more cAMP was accumulated in RK1‐derived membranes than in LK1‐derived membranes. With the same membrane fractions, upon treatment with Mn++, which directly stimulates the catalytic unit, a high level of cAMP was accumulated both in RK1 and LK1, indicating that the catalytic function inducible by Mn++ was similar in both clones. There was no significant difference in the level of expression of G protein α2, αi (at least αi1 and αi2), and β subunits between LK1 and RK1. Cholateextracts of the membrane protein of LK1 and RK1 reconstituted the adenylyl cyclase activity of the cyc variant of S49 lymphoma cells to the same level. Therefore, it is inferred that the defect in LK1 resides in the interaction of stimulatory G proteins and the actual catalyst.
Transforming growth factor type β1 modulates the effects of basic fibroblast growth factor on growth and phenotypic expression of rat astroblasts in vitro
Authors: Labourdette Gerard , Janet Thierry , Laeng Pascal , Perraud Frederic , Pettmann Brigitte , Lawrence David 
Published: February 2005

AbstractIn a search of the growth factors possibly involved in brain ontogenesis we have examined the effects of transforming growth factor β1 (TGF‐β1) on the growth and phenotypic expression of rat astroblasts in primary culture. Along TGF‐β1 elicited only a slight negative effect on the growth of these cells. However, this factor was found to modulate the mitogenic effects of other growth factors. On quiescent cells it potentiates the mitogenic effect of basic fibroblast growth factor (bFGF) but not that of other growth factors, namely, epidermal growth factor (EGF), platelet‐derived growth factor (PDGF), and thrombin. TGF‐β1 did not modulate significantly the stimulatory effect of these growth factors on the activity of the enzyme glutamine synthetase (GS); but kinetic studies showed that TGF‐β1 delays the stimulation of GS activity. DNA synthesis monitored by the incorporation of [125I]iododeoxyuridine (125I‐dUrd) was maximum after 24–30 h of treatment with bFGF. With bFGF plus TGF‐β1 the maximum was shifted to 30–36 h. This shift is compatible with the idea that TGF‐β1 induces responsiveness in some cells which are otherwise unresponsive to the mitogenic action of bFGF, and that this induction requires some time. This hypothesis is sustained by the observation that in cells treated for only 12 h with bFGF, the treatment with TGF‐β1 for the same 12 h or for longer time did not stimulate significantly the cell growth. Stimulation occurred only when the bFGF treatment was continued after 12 h. Potentiation of the mitogenic effect of bFGF and shift of the maximum 125I‐dUrd incorporation towards 24 h was seen with cells pretreated with TGF‐β1. This potentiation effect decreased with increasing time between the two treatments. The potentiation effect of TGF‐β1 is not mediated by an induction of new bFGF membrane receptors as seen by binding studies.
Dissociation of proto‐oncogene induction from growth response in normal human fibroblasts
Authors: Yaar Mina , Peacocke Monica , Cohen Michael S. , Gilchrest Barbara A. 
Published: February 2005

AbstractProto‐oncogenes are cellular homologues of viral oncogenes that are known to be associated with regulation of growth and differentiation. The c‐myc and c‐fos proto‐oncogenes have been extensively studied by using established cell lines but to a lesser extent by using normal cells. Using physiologic growth modulators, we have shown that mitotic stimulation of normal human dermal fibroblasts is associated with induction of c‐myc and c‐fos, but that growth inhibition of these cells is not necessarily accompanied by their down‐regulation. When treated with both serum and interferon‐alpha during quiescence, fibroblasts were delayed in their progress into the S‐phase of the cell cycle as compared to cells treated with serum alone and displayed substantial growth inhibition as measured by cell number at the end of 1 week. However, this growth inhibition was not preceded by down‐regulation or delay in induction of c‐myc and c‐fos mRNA. The above studies suggest that in normal fibroblasts growth inhibition is not necessarily dependent on down‐regulation of transcription of either c‐myc or c‐fos and that interferon may act to inhibit cell growth either through a post‐transcriptional effect on cellular proto‐oncogenes necessary for cell proliferation or through induction of other, as yet unrecognized gene(s) associated with growth arrest.
Mitogenic effect of transforming growth factor β1 on human fibroblasts involves the induction of platelet‐derived growth factor α receptors
Authors: Ishikawa Osamu , LeRoy E. Carwile , Trojanowska Maria 
Published: February 2005

AbstractPlatelet‐derived growth factor (PDGF) and transforming growth factor β (TGF‐β), potent modulators of mesenchymal cell growth and differentiation, are often colocalizable in vivo. Previous in vitro studies in fibroblastic cell lines have shown variable, even antagonistic effects of TGF‐β on the mitogenic action of PDGF. This study demonstrates that in diploid human dermal fibroblasts, TGF‐β1 is weakly mitogenic in the absence of serum or purified growth factors, and that TGF‐β1 potentiates DNA synthesis in PDGF‐stimulated fibroblasts with delayed kinetics when compared to stimulation with PDGF alone. TGF‐β enhances mitogenic potency of all three PDGF isoforms and increases receptor binding of both 125I PDGF‐AA and 125I PDGF‐BB, consistent with the increased expression of the α type PDGF receptor. The induction of PDGF α receptor subunits by TGF‐β may play a role in enhancing the proliferative potential of human fibroblasts in certain physiologic and pathologic conditions.
Decreased L system amino acid transport and decreased gamma‐glutamyl transpeptidase are independent processes in human chronic lymphocytic leukemia B‐lymphocytes
Authors: Woodlock Timothy J. , Brown Richard , Mani Matthew , Pompeo Lisa , Hoffman Howard , Segel George B. , Silber Robert 
Published: February 2005

AbstractThe‐L system of amino acid transport is markedly diminished in chronic lymphocytic leukemia (CLL) B‐lymphocytes, with a maximal velocity less than 15% that of normal B‐lymphocytes. Another membrane‐associated function, the activity of the ectoenzyme, gamma‐glutamyl transpeptidase (GGT), is diminished in CLL B‐cells to 30% that of normal B‐cells. In addition to its transpeptidase activity, a role for GGT has been postulated in the transport of amino acids. In the present report, the possible relationship of these two physiologic functions in CLL B‐cells was studied. The L system transport defect in CLL is restored by phorbol ester‐induced cell maturation; following incubation with 0.15 μM tetradecanoyl phorbol acetate (TPA) for 17 hours, the L system initial velocity showed a 20‐fold increase. In contrast, there was no significant effect on GGT activity with cell maturation. Furthermore, an antibody which diminished GGT activity by 50% in lymphoid cells did not inhibit L system transport. Thus, the impaired L system amino acid transport and GGT activity appear to be independent processes in CLL B‐cells.
Polarized membrane expression of brush‐border hydrolases in primary cultures of kidney proximal tubular cells depends on cell differentiation and is induced by dexamethasone
Authors: Ronco P. , Antoine M. , Baudouin B. , Geniteau‐legendre M. , Lelongt B. , Chatelet F. , Verroust P. , Vandewalle A. 
Published: February 2005

AbstractTo analyze the influence of cell differentiation and the effects of hormones on the subcellular distribution of apical antigens in polarized epithelial cells, we have compared the localization of three brush border (BB) hydrolases [neutral endopeptidase (ENDO), aminopeptidase N (APN), and dipeptidylpeptidase IV (DPPIV)] in primary cultures of renal proximal tubule cells grown in various culture media. The degree of cell differentiation modulated by medium composition was estimated by measuring proximal functions, including glucose transport, specific enzymatic activities, and PTH responsiveness. In the dedifferentiated state observed in cells grown in 1% fetal calf serum (FCS)‐supplemented medium, the three hydrolases are abnormally concentrated in a cytoplasmic vesicle compartment with weak expression on both membrane domains. By contrast, in serum‐free hormonally defined medium (DM: insulin, 5 μg/ml; dexamethasone, 5 × 10−8 M), which markedly enhances morphological and functional cell differentiation, the distribution of hydrolases parallels that observed in the normal tubule. When added to the DM devoid of hormones, insulin has little polarizing effect, whereas dexamethasone dramatically increases the apical expression of the hydrolases, which then almost disappear from the basolateral membrane and cytoplasmic vesicular compartments. This glucocorticoid hormone augments the amount of immunoreactive antigen detectable on the apical domain in paraformaldehyde‐fixed cells but does not change the total enzymatic activity. This suggests the presence in tubular cells of a dexamethasone‐dependent polarizing machinery that requires de novo RNA and protein synthesis, and probably acts mainly by targeting a storage cytoplasmic pool of enzyme to the apical domain.
Identification and partial characterization of genes that are transactivated by different pathways in quiescent mouse cells stimulated with serum
Authors: Boeggeman E. , Masibay A. S. , Qasba P. K. , Sreevalsan T. 
Published: February 2005

AbstractWe have identified four cDNA clones, cl‐1, cl‐5, cl‐15, and cl‐16, that represent genes induced by serum in resting mouse 3T3 cells. Partial sequence analysis of the four cDNAs indicated that cl‐15 corresponds to the mouse beta‐actin gene. Comparison of the DNA sequences of the other three clones with the sequence data bank (Genbank) showed little homology to other known DNA sequences and thus represent novel genes. The level of the mRNAs corresponding to the four genes began to increase in resting cells following serum stimulation, reached a peak between 5 h and 8 h and then started to decline. Inhibitors of transcription diminished the induction of the mRNAs corresponding to the four genes. Cycloheximide and anisomycin hadlittle effect on the induction of beta actin mRNA while the induction of the other three genes was suppressed by the same inhibitors. 12‐O‐Tetradecanoylphorbol‐13‐acetate and the calcium ionophore A23187 enhanced the expression of the cl‐16 mRNA while epidermal growth factor, fibroblast growth factor, or insulin enhanced the expression of cl‐1‐ and cl‐5‐specific transcripts. The level of beta‐actin mRNA was elevated in resting cells by epidermal growth factor and 12‐O‐tetradecanoylphorbol‐13‐acetate and to a lesser extent by fibroblast growth factor, insulin, and dibutyryl cyclic AMP‐elevating agents. Pertussis toxin, an inhibitor of the action ofG proteins, did not significantly suppress the activation of the four genes by serum. However, 2‐aminopurine, a protein kinase inhibitor, suppressed the induction of the four transcripts in serum‐stimulated cells. The possible pathways involved in the activation of these genes in resting cells are discussed.
Correlation between redistribution of a 26 kDa protein and development of chronic thermotolerance in various mammalian cell lines
Authors: Lee Yong J. , Hou Zi‐Zheng , Curetty Lindali , Borrelli Michael J. , Corry Peter M. 
Published: February 2005

AbstractPrevious studies suggested that a 26 kDa protein might play an important role in protein synthesis‐independent thermotolerance development in CHO cells. To determine if this phenomenon was universal, four mammalian cell lines, viz., CHO, HA‐1, murine Swiss 3T3, and human HeLa, were studied. Cells were heated at 42°C, and the level of 26 kDa protein in the nucleus was measured, together with clonogenic survival and protein synthesis. The results demonstrated that (1) the 26 kDa protein was present in the four different cell lines, and (2) the level of the 26 kDa protein in their nuclei was decreased by 30–70% after heating at 42°C for 1 hr. However, restoration of this protein occurred along with development of chronic thermotolerance. The protein synthesis inhibitor cycloheximide (10 μg/ml) neither inhibited the development of chronic thermotolerance nor affected the restoration of the 26 kDa protein in the nucleus. In fact, this drug protected cells from hyperthermic killing and heat‐induced reduction of 26 kDa protein in the nucleus. Heat sensitizers, quercetin (0.1 mM), 3,3′‐dipentyloxacarbocyanine iodide (DiOC5[3]: 5 μg/ml), and stepdown heating (45°C‐10 min→42°C), potentiated hyperthermic killing and inhibited or delayed the restoration of the 26 kDa protein to the nucleus. These results support a correlated, perhaps causal relationship between the restoration of the 26 kDa protein and chronic thermotolerance development in four different mammalian cell lines.
Proliferative capacity of human peripheral blood lymphocytes sorted on the basis of glutathione content
Authors: Kavanagh Terrance J. , Grossmann Angelika , Jaecks Erik P. , Jinneman John C. , Eaton David L. , Martin George M. , Rabinovitch Peter S. 
Published: February 2005

AbstractGlutathione (GSH) is important in defense against oxygen free radical damage, in detoxification of xenobiotics, and in mitogenesis. The reducing conditions provided by low molecular weight thiols such as 2‐mercaptoethanol (ME) have been shown to promote the growth of lymphocytes in culture. We wished to determine the effects of 2‐ME on GSH content, and to determine to what extent GSH status affected lymphocyte proliferation. GSH content was quantitated in human peripheral blood lymphocytes (PBL) using a flow cytometric assay with monochlorobimane. This analysis was performed on PBL as well as on the CD4+ T‐cell subset, as identified with fluorescent anti‐CD4 monoclonal antibodies (mAb). Cells were viably sorted on the basis of their GSH content, and incubated for 3 days with mitogenic concentrations of PHA (for PBL) or anti‐CD3 mAb (for CD4+ cells) in the presence of bromodeoxyuridine (BrdU). BrdU/Hoechst cell cycle analysis was then performed on these cells. High GSH sorted cells had a higher percentage of cells capable of entering the cell cycle than low GSH sorted cells. This data indicates that some of the heterogeneity in proliferative capacity within PBL in culture is directly or indirectly related to GSH content. Incubation of cells in 2‐ME prevented the loss of GSH that occurs when cells are cultured. 2‐ME improved the proliferative capacity of unsorted cells, and of cells sorted for high and low GSH. Acridine orange staining of anti‐CD3 mAb stimulated cells sorted for high and low GSH indicated that an early event in cell activation was affected by GSH content.
Inhibition of late endosome‐lysosome fusion: Studies on the mechanism by which isotonic‐K+ buffers alter intracellular ligand movement
Authors: Ward Diane Mcvey , Hackenyos David P. , Davis‐Kaplan Sandra , Kaplan Jerry 
Published: February 2005

AbstractIncubation of alveolar macrophages or hepatocytes in media in which Na+ is replaced by K+ (‘isotonic‐K buffer’) inhibited the movement of internalized ligand from late endosomes to lysosomes (Ward et al.: journal of Cell Biology 110:1013–1022, 1990). In this study we investigate the mechanism responsible for the isotonic‐K+ block in movement of ligand from late endosomes to lysosomes. We observed that iso‐K+ inhibition of endosome‐lysosome fusion is not unique to alveolar macrophages or hepatocytes but can be seen in a variety of cell types including J774 and Hela cells. The inhibition in intracellular ligand movement was time dependent with the maximum change occurring after 60 minutes. Once established the inhibition resulted in a prolonged and apparently permanent decrease in vesicle movement. Cells were able to recover from the effects of iso‐K+ buffers over a time course of 5–10 minutes when placed back in Na+‐containing media. The effect of iso‐K+ buffers was independent of intracel‐lular pH changes and appeared to involve cell swelling. When cells were incubated in iso‐K+ buffers under conditions in which cell volume changes were reduced, intracellular ligand movement approached normal levels. Such conditions included replacing Cl− with the less permeant anion gluconate, and by addition of sucrose to isotonic‐K+ buffers. Analysis of the mechanism by which changes in cell volume could alter intracellular movement ruled out changes in cyclic nucleotides. Ca2+, or microtubules. These results suggest that changes in cell shape or volume can alter intracellular transport systems by novel routes.
Suppression of programmed death and G1 arrest in B‐cell hybridomas by interleukin‐6 is not accompanied by altered expression of immediate early response genes
Authors: Sabourin Luc A. , Hawley Robert G. 
Published: February 2005

AbstractThe murine B‐cell hybridoma B9 requires interleukin‐6 (IL‐6) for its survival and proliferation in vitro. We show here that withdrawal of IL‐6 from B9 cultures results in programmed death, concomitant with arrest of the cells in the G1 phase of the cell cycle. Unlike several other systems that undergo programmed cell death, no induction of transcripts corresponding to the testosterone‐repressed message‐2 or transglutaminase genes is observed during this process. Upon readdition of IL‐6 to G1‐arrested B9 cells, viability is maintained and entry into S phase occurs after a lag period of 10 to 12 hr. Northern blot analysis showed that the immediate‐early mRNAs normally induced shortly after growth factor stimulation in quiescent fibroblasts (c‐fos, c‐jun, Egr‐1, c‐myc, JE, and KC), and other growth‐related genes (2F1, c‐Ha‐ras, and p53), are either not induced or remain unchanged during G1 to S phase progression. A correlation was found, however, between the temporal pattern of expression of several G1/S phase genes (dihy‐drofolate reductase, thymidine kinase, transferrin receptor, and histone H3) and DNA synthesis. These results demonstrate that IL‐6‐induced viability and growth of hybridoma (and, presumably, plasmacytoma) cells is mediated via novel signal transduction pathways.
Analysis of the dominance of mutations in cAMP‐binding sites of murine type I cAMP‐dependent protein kinase in activation of kinase from heterozygous mutant lymphoma cells
Authors: Shuntoh Hisato , Steinberg Robert A. 
Published: February 2005

AbstractStructural lesions in cAMP‐binding sites of regulartory (R) subunit of cAMP‐dependent protein kinase caused identical increases in apparent constants for cyclic nucleotide‐depencient kinase activation in preparations from cells that were hemizygous or heterozygous for mutant RI subunit expression. No wild‐type kinase activation was observed in extracts from heterozygous mutant cells. This “dominance” was investigated by characterizing expression of wild‐type and mutant RI subunits and properties of protein kinase from S49 mouse lymphoma cell mutants heterozygous for expression of wild‐type RI subunits and 3, subunits with a lesion (Glu200) tnat inactivate:. cAMP‐binding site A. By both studies of cAMP dissociation and two‐dimensional gel analysis, wild‐type R subunits comprised about 35% of total RI subunits in heterozygous mutants. Synthesis of wild‐type and mutant RI subunits was equivalent, but wild‐type subunits were degraded preferentially. Hydroxylapatite chromatography revealed a novel RI subunit‐containingspeciesfromheterozygousmutantpreparationswhoseelution behavior suggested a trimeric kinase consisting of an RI subunit dimer and one catalytic (C) subunit. Wild‐type RI subunit was found only in dimer and “trimer” peaks; the tetrameric kinase peak contained only mutant RI subunit. It is concluded that C subunit binds preferentially to mutant RI subunit in heterozygous cells forming either tetrameric kinase with mutant RI subunit homodimers or trimeric kinase with RI subunit heterodimers. This preferential binding results both suppression of wild‐type kinase activation and differential stabilization of mutant RI subunits.