bluuebunny/crossref_metadata_2025_split huggingface dataset:
Maintenance by erythropoietin of viability and maturation of murine erythroid precursor cells
Authors: Koury Mark J. , Bondurant Maurice C. 
Published: February 2005

AbstractErythroblasts isolated from the spleens of mice infected with the anemia‐inducing strain of Friend virus (FVA cells) are erythropoietin (EP)‐sensitive cells at the late colony forming unit‐erythroid (CFU‐E) and cluster forming unit stages of differentiation (Koury et al., J. Cell. Physiol. 121:526–532, 1984). We investigate here the EP requirements of FVA cells in vitro for viability, proliferation, and maturation. By delaying the addition of EP to FVA cell cultures or by withdrawing EP at early times of culture, the subsequent viability, cell numbers, and maturation were diminished. The longer the delay in EP addition or the earlier the EP withdrawal, the more diminished these parameters were when compared to cultures which contained EP throughout the 48 h of differentiation. FVA cells had a period of EP requirement in vitro that lasted for only 24 h or less after the initiation of culture. During these crucial first 24 h, EP induced an increase in the synthesis of all size classes of RNA. Protein synthesis was maintained at a stable level in cells cultured with EP, but it declined in cells cultured without it. In contrast, the synthesis rate of DNA and the content of DNA per cell were not affected by the presence of EP in the culture. However, FVA cells cultured without EP had progressive accumulation of small sized DNA due to breakage of higher molecular weight DNA. The rate of DNA breakdown was sufficient to prevent DNA accumulation and thus it probably plays a role in the abortion of cell proliferation. No such breakage was found in cells cultured with EP. Our results indicate that EP exerts an effect on FVA cells in culture which is reflected in their viability, cell number, and maturation. This effect is not mediated by a stimulation of the rate of DNA synthesis, but is accompanied by stimulation of overall RNA synthesis and maintenance of protein synthesis.
Coupling between inositol phosphate formation and DNA synthesis in smooth muscle cells stimulated with neurokinin A
Authors: Hultgårdh‐Nilsson Anna , Nilsson Jan , Jonzon Bror , Dalsgaard Cari‐Johan 
Published: February 2005

AbstractThe two mammalian neuropeptides substance P (SP) and neurokinin A (NKA) have been demonstrated to stimulate DNA synthesis in connective tissue cells, suggesting that peripheral neurons may play a role in development and tissue regeneration. In this study we have tried to identify intracellular messengers required for SP‐ and NKA‐induced DNA synthesis. SP and NKA, as well as platelet‐derived growth factor (PDGF) stimulated formation of inositol phosphates in smooth muscle cells (SMC), whereas no effect on inositol phosphates formation occurred in response to nonmitogenic neuropeptides. Pretreatment of the cells with pertussis toxin markedly decreased DNA synthesis induced by NKA. This toxin inhibits formation of inositol phosphates by acting on a regulatory G‐protein. Calcium and calmodulin antagonists also inhibited NKA‐induced DNA synthesis. These results imply that the mitogenic signal(s) produced by activated neuropeptide receptors involves formation of inositol phosphate and activation of a calcium/calmodulin dependent process. We further report that other neuropeptides occurring in peripheral neurons, i.e., vasoactive intestinal polypeptide, calcitonin gene‐related peptide, neuropeptide Y, somatostatin, or cholecystokinin, are without growth‐stimulatory effect on cultured SMC.
Basic fibroblast growth factor and insulin‐like growth factor I are strong mitogens for cultured mouse keratinocytes
Authors: Ristow Hans‐Jürgen , Messmer Trudy O. 
Published: February 2005

AbstractBasic fibroblast growth factor (bFGF), but not acidic fibroblast growth factor (aFGF), was found to be mitogenic for cultured mouse keratinocytes. A six‐to‐nine fold increase in 3H‐thymidine (3H‐dT) incorporation into the acid insoluble pool and a similar increase of the labeling index can be measured when bFGF, at a concentration between 1 and 10 ng/ml, is added to keratinocytes arrested in serum‐free and growth factor‐free medium with a Ca++‐concentration below 0.1 mM. The half‐maximal response is observed between 0.2 and 0.7 ng/ml. In the same culture system, insulin‐like growth factor I/somatomedin C (IGF‐I) and insulin with 100–500 ng/ml. Basic FGF, IGF‐I and insulin can be classified as strong stimulators of DNA synthesis in mouse keratinocytes. In this regard they are comparable to epidermal growth factor, which shows a half‐maximal stimulation at a concentration between 1.5–2 ng/ml. These results show that in addition to mesenchymal cells, FGF is a growth factor not only for neuroectodermal cells, but ectodermal cells in general. They further support the idea that the growth promoting effect of insulin on keratinocytes may be mediated by the IGF‐I receptor.
Induction of competence and progression signals in human T lymphocytes by phorbol esters and calcium ionophores
Authors: Kumagai Naoki , Benedict Stephen H. , Mills Gordon B. , Gelfand Erwin W. 
Published: February 2005

AbstractWe have investigated the induction of competence (IL‐2 responsiveness) and progression in human T lymphocyte proliferation triggered by phorbol ester and calcium ionophore. The degree of proliferation induced with the phorbol ester, phorbol 12,13‐dibutyrate (PDB) and the calcium ionophore ionomycin was dependent on the duration of exposure to these agents, with more than 6 h required for obtaining maximum proliferation. Following brief exposure to both agents for 30 min, which did not cause significant proliferation, T cells became competent to proliferate in response to exogenous interleukin 2 (IL‐2). These competent T cells also progressed to DNA synthesis following incubation with PDB in the absence of ionomycin. Induction of competence to proliferate in response to either PDB or IL‐2 was blocked by EGTA, suggesting that transmembrane Ca2+ flux was obligatory at this stage. Since other phorbol esters and synthetic diacylglycerols also stimulated DNA synthesis in competent cells, it is likely that progression was triggered by activation of protein kinase C. Following a brief exposure to PDB and ionomycin, subsequent incubation with PDB induced gene expression and secretion of IL‐2 and augmented the expression of IL‐2 receptors in the competent cells. Thus, we have demonstrated that Ca2+ mobilization is required for rendering T cells competent to express functional IL‐2 receptors, to produce IL‐2 in response to subsequent incubation with PDB, and that sustained activation of protein kinase C seems necessary for IL‐2 production and subsequent progression of competent T cells to DNA synthesis.
Tumor necrosis factor inhibits the terminal events in mesenchymal stem cell differentiation
Authors: Filipak Michiko , Sparks Rodney L. , Tzen Chin‐Yuan , Scott Robert E. 
Published: February 2005

AbstractControl of the terminal event in cellular differentiation is an important normal regulatory process, and the expression of defects in the control of this process has been implicated in the pathogenesis of cancer. To determine if tumor necrosis factor (TNF), which is an important biological response modifier, can inhibit terminal differentiation, we have studied 3T3 T mesenchymal stem cells. This experimental cell system was employed because a well‐defined series of steps in differentiation has been defined and cells at each stage of differentiation can be isolated. For example, nonterminal differentiated cells can be isolated, and their transition to a terminal differentiation state can be evaluated. The most interesting results in the current studies show that TNF blocks the terminal event in mesenchymal stem cell differentiation. Inhibition of the terminal event of differentiation by TNF is reversible and is not associated with inhibition of selective or general protein synthesis. Evidence is also presented that cell clones that are defective in their ability to undergo the terminal event in differentiation secrete factor(s) that inhibit the terminal event in differentiation. These observations suggest that the inhibition of the terminal event in differentiation may be mediated via autocrine or paracrine regulatory molecules such as tumor necrosis factor.
DNA topoisomerase inhibitors block erythropoiesis and delay hemoglobinization in vitro
Authors: Dainiak Nicholas , Kreczko Sandra , Hanspal Manjit , Strauss Phyllis R. 
Published: February 2005

AbstractTo examine the importance of topological constraints on DNA during erythroid development, we measured the effects of camptothecin and teniposide, two tumoricidal agents which are also specific inhibitors of type I and type II topoisomerases respectively, on the formation of hematopoietic colonies by cultured human bone marrow cells. When added to bone marrow culture, each inhibitor alone impairs the formation of early BFU‐E‐derived colonies, late CFU‐E‐derived colonies and mixed hematopoietic (CFU‐GEMM‐derived) colonies by up to 100%. Inhibition of colony formation is directly related to the time of inhibitor addition and the inhibitor concentration tested. Although either inhibitor alone reduces colony formation by 90%, when added together at a submaximal concentration, camptothecin and teniposide exert a synergistic suppressive effect. Furthermore, addition of topoisomerase inhibitors to culture impairs hemoglobinization of colony erythroblasts in a time‐dependent fashion. In contrast to the effects of topoisomerase inhibitors, the antiproliferative agent aphidicolin reduces erythroid colony number and size without altering hemoglobinization of colony erythroblasts. Since neither topoisomerase inhibitor alters the morphology of cultured cells, the capacity of cells to exclude trypan blue or the potential to form erythroid colonies through the interval required for the first progenitor cell division, it is unlikely that camptothecin or teniposide are cytotoxic to hematopoietic cells. Human mononuclear cells enriched in bone marrow lymphocytes and nucleated erythroblasts from both human and mouse sources release DNA into the detergent soluble fraction. Release requires functional topoisomerases and is altered by acute exposure to topoisomerase inhibitors. Our results suggest that topoisomerases are critical not only to proliferation but also to differentiation of human marrow erythroid progenitor cells and stem cells in culture.
Enhancement of plasminogen activator activity in cultured endothelial cells by granulocyte colony‐stimulating factor
Authors: Kojima Soichi , Tadenuma Hirohiko , Inada Yuji , Saito Yuji 
Published: February 2005

AbstractA hitherto unknown function of granulocyte colony‐stimulating factor (G‐CSF) was found using cultured endothelial cells. G‐CSF stimulated activity of plasminogen activator (PA) in both extracellular and intracellular milieus of endothelial cells obtained from bovine carotid and aortic artery. This effect was dependent on the concentration of G‐CSF added to the culture medium and on the treatment time. The extracellular activity was enhanced approximately 5‐fold at a concentration of 5,000 colony‐forming unit (CFU)/ml (2.6 nM) and in about a 15‐hr treatment period. Analyses by fibrin and reverse fibrin autography revealed that activity of PA was much more increased than that of PA inhibitor in endothelial cells treated with G‐CSF.
Heparin potentiates the action of acidic fibroblast growth factor by prolonging its biological half‐life
Authors: Damon Deborah H. , Lobb Roy R. , D'Amore Patricia A. , Wagner John A. 
Published: February 2005

AbstractThe mechanism(s) by which heparin influences the biological activities of acidc and basic fibroblast growth factors (aFGF and bFGF) is not completely understood. One mechanism by which heparin could alter the biological activities of aFGF and bFGF is by altering their biological half‐lives. We investigated the possibility that heparin potentiates aFGF‐induced neurite outgrowth from PC12 cells by prolonging its biological half‐life. Under conditions where heparin potentiated aFGF‐induced neurite outgrowth, we observed that heparin increased the biological half‐life of aFGF from 7 to 39 hr. We determined that >25 hr of exposure to active aFGF was required for induction of neurite outgrowth. If aFGF activity was maintained for greater than 25 hr by periodic readdition of factor, heparin no longer potentiated aFGF‐induced neurite outgrowth. These observations strongly suggest that heparin potentiates the activity of aFGF by prolonging its biological half‐life. The protease inhibitors hirudin, leupeptin, and pepstatin A did not potentiate aFGF‐induced neurite outgrowth, indicating that proteases inhibited by these inhibitors are not responsible for the loss of aFGF activity that we observed. However, aprotinin potentiated aFGF neurite‐promoting activity approximately sevenfold, indicating that proteases that are inhibited by aprotinin are at least partially responsible for aFGF inactivation. These observations suggest that heparin regulates the activity of aFGF by regulating its proteolytic degradation, thereby regulating its biological half‐life.
Kinetic characteristics and regulation of hexose transport in a galactokinase‐negative Chinese hamster fibroblast cell line: A good model for studies on sugar transport in cultured mammalian cells
Authors: Germinario Ralph J. , Lakshmi Tannirkulam M. , Thirion Jean‐Paul 
Published: February 2005

AbstractWe report the kinetic characteristics for D‐galactose, 2‐deoxy‐D‐glucose and 3‐0‐methyl‐D‐glucose transport in a galactokinase null‐allele mutant of a Chinese hamster V79 cell line. GalKl cells exhibited a Km and Vmax for D‐galactose, 2‐deoxy‐D‐glucose, and 3‐0‐methyl‐D‐glucose transport of 8.6 ± 2.6 mM and 26.1 ± 7.2nmol/mg p/min, 4.1 ± 1.2 mM and 40.3 ± 9.5 nmol/mg p/min, and 7.01 ±.85 mM and 11.6 ± 4.8 nmol/mg p/30 s, respectively. Nonsaturable hexose uptake was determined using cytochalasin B inhibition of galactose uptake (89.6 ± 3.7% of the galactose uptake was cytochalasin B inhibitable) and L‐glucose uptake (7.5% of the galactose uptake). D‐Galactose was not metabolized and effluxed rapidly from preloaded cells. The K1s for the inhibition of D‐galactose transport were 4.5 ± 2.5 mM for D‐glucose, 7.0 ± 2.0 mM for 2‐deoxy‐D‐glucose, 6 mM for 2‐deoxy‐D‐galactose and 6.0 ± 0.6 mM for 3‐0‐methyl‐D‐glucose. This indicates the operation of a single common carrier. The hexose transport rate decreased 50‐60% after 24 h serum deprivation. Addition of insulin was shown to increase hexose transport (more than twofold) in serum‐deprived cells. Hexose transport rates increased substantially in glucose‐deprived, D‐fructose‐ or D‐galactose‐fed cells as compared to glucose‐fed cells. Since GalKl does not metabolize galactose, the hexose transport increases induced by feeding cells galactose suggest that carrier interaction with ligand is not a significant factor in transport regulation in GalKI. The kinetic and regulatory characteristics of D‐galactose transport in the GalKI cell line indicate that this system is a good model to study sugar transport from a mechanistic and regulatory point of view.
Effect of retinoic acid on the phosphorylation of endogenous proteins in HL‐60 cells
Authors: Spearman Terry N. , Fontana Joseph A. , Butcher Fred R. , Durham John P. 
Published: February 2005

AbstractHL‐60 cells were incubated with [32P]‐Pi in order to label endogenous phosphoproteins in situ. These were then resolved via two‐dimensional electrophoresis and autoradiograms were made from the gels. A comparison of autoradiograms made from retinoic‐acid‐differentiated cells with those made from control cells revealed a small number of phosphoproteins whose labeling was enhanced in differentiated cells. Incubating HL‐60 cells with [35S]‐methionine revealed that RA induced the synthesis of one of these proteins, (53 kDa, pl 4.8) although not to a degree sufficient to account for the increased phosphate labeling observed in differentiated cells. Difluoromethylornithine (DFMO), which arrests HL‐60 cell proliferation without inducing differentiation, had no effect on endogenous protein phosphorylation. Treatment of DFMO‐arrested cells with retinoic acid, however, increased the phosphorylation of the proteins and resulted in differentiation of the cells. Densitometric analysis of autoradiograms made from two‐dimensional gels revealed that the phosphorylation of the 53‐kDa, pl 4.8 protein was significantly elevated in cells exposed to RA for as little as 12 hours, i.e., before the cells were committed to differentiate and before a significant increase in the number of phenotypically mature cells was observed. It therefore appears that this protein may be an intermediate in the retinoic‐acid‐induced differentiation process.
Correlations between heparan sulfate metabolism and hepatoma growth
Authors: Ishihara Masayuki , Conrad H. Edward 
Published: February 2005

AbstractA rat hepatoma cell line (Gershenson et al., Science, 170:859–861, 1970) contains a dynamic steady‐state pool of free heparan sulfate (HS) chains in the nucleus that increases in amount when growing cells reach confluence (Fedarko and Conrad, j. Cell Dioi, 102:587–599, 1986). In logarithmically growing cells labeled with 35SO42− steady‐state levels of [35SO4]HS in the nucleus are altered by a variety of culture conditions. Rapidly dividing cells (doubling time = 18–22 h) growing under optimized conditions had steady‐state levels of nuclear HS within the range of 40‐50 pmol 35SO4 in nuclear HS/106 cells. The steady‐state levels of nuclear HS were lowered by several changes in culture conditions, including 1) additions of 1 mM p‐nitrophenyl‐β‐D‐xyloside, 0.25–0.5 mM (+)‐catechin, 0.5 ng/ml transforming growth factor β, 20 ng/ml phorbol‐12‐myristate‐13‐acetate, 1 mM dibutyryl cAMP, or 1 mM inositol‐2‐PO4; 2) decreased levels of D‐glucose; or 3) deletions of serum, insulin, or inositol. In all cases lowering of the nuclear HS level was accompanied by an increase in the cell doubling times, suggesting a correlation in which nuclear HS levels must be optimized for maximal growth rates. When cells cultured under optimal growth conditions reached confluence, the level of nuclear HS increased threefold and the cells stopped dividing. The same culture conditions that lowered the steady‐state levels of HS in the logarithmically growing cells prevented this rise in the nuclear HS as the cells reached confluence and resulted in loss of contact inhibition and overgrowth of the confluent cultures. These observations suggest a second correlation in which elevated nuclear HS levels are found when cell growth is inhibited at confluence; prevention of this rise results in continued growth. Consistent with this correlation between elevated nuclear HS and reduced growth rates, it was observed that addition of either 0.5 μg/ml hydrocortisone or 0.05 μg/ml retinoic acid to the culture medium of logarithmically growing cultures resulted in.increases in steady‐state levels of nuclear HS that were accompanied by increased cell doubling times. The two agents that increased the levels of nuclear HS in logarithmically growing cultures had little effect on levels of nuclear HS in confluent cells or on contact inhibition. These results show that cells growing at maximal rates maintain nuclear HS levels within a range of 40–50 pmol SO4 in HS/106 cells and that when the steady‐state levels of nuclear HS either rise above or fall below this range, the growth rates in subconfluent cultures are decreased. As log‐phase cells reach confluence and stop dividing, there is a rise in the level of nuclear HS. Under growth conditions which prevent this rise, cells lose contact inhibition.
Comparison of phorbol‐12‐myristate‐13‐acetate and dioctanoyl‐sn‐glycerol in the activation of EL4/6.1 thymoma cells
Authors: Stoeck Michael , Lees Rosemary , Szamel Marta , Pantaleo Giuseppe , MacDonald H. Robson 
Published: February 2005

AbstractThe present study compared the role of two protein kinase C (PK‐C) activating agents, the phorbol ester phorbol‐12‐acetate‐13‐myristate (PMA) and the membrane‐permeating diacylglycerol dioctanoyl‐sn‐glycerol (Dic8) in the activation of EL4/6.1 thymoma cells. These cells have been shown to express interleukin‐2 receptors (IL‐2R) upon stimulation with optimal amounts of PMA (10 ng/ml); also, suboptimal amounts of PMA (1 ng/ml) synergized with the Ca2+ ionophore ionomycin and recombinant interleukin‐1 (rlL‐1) (Lowenthal et al., 1986). Comparing PMA and DiC8 led to the following results: PMA at 10 ng/ml induced IL‐2R; in contrast, DiC8 (30‐3 μg/ml) alone was unable to induce IL‐2R, although it did synergize with ionomycin (0.5 μg/ml) and rlL‐1. Bihourly additions of DiC8 did not change this pattern. The addition of DiC8 together with rlL‐2 also resulted in no IL‐2R expression. Furthermore, DiC8 (10 μg/ml) effectively translocated PK‐C. Therefore, the differences observed between PMA and DiC8 do not seem to be due to differences in metabolism or to an inability to translocate PK‐C. Analysis of messenger (m) RNA produced in stimulated EL4/6.1 cells revealed that DiC8 was also unable to induce mRNA for IL‐2R.Our data suggest that PMA, especially at “optimal” concentrations, might have effects that cannot be mimicked by diacylglycerol. Furthermore, it seems that the deficient activity of diacylglycerols can be compensated for by a Ca2+ ionophore and, depending on the cellular system, by further signals such as IL‐1.
Effects of temperature on the degradation and biliary secretion of asialoorosomucoid by the perfused rat liver: Evidence for two intracellular pathways
Authors: Kloppel Thomas M. 
Published: February 2005

AbstractWe have utilized the in situ perfused rat liver under nonrecirculating conditions to examine the effect of temperature on the metabolism and biliary secretion of [125I]‐asialoorosomucid (ASOR). In this manner we were able to follow the fate of a single round of internalized ligand. In control livers perfused at 37°C, approximately 50% of [125I]‐ASOR injected into the portal vein was extracted on first pass. Five minutes after the injection, radioactivity, which had been extracted initially, began to appear in the hepatic venous effluent. Within 25 min, 50% of the initially extracted radioactivity was released into the perfusion medium; the bulk of this radioactivity (>95%) was soluble in trichloroacetic acid. In livers perfused at temperatures slightly less than 37°C (30–35°C), first‐pass extraction of [125I]‐ASOR was similar to that observed at 37°C. However, a severalfold decrease in the rate of release of radioactivity from the liver into the perfusion medium was noted at the lower perfusion temperatures; whereas greater than 50% of the initially extracted radioactivity was released within 30 min from livers perfused at 37°C, only 5% was released at 30°C. At the lower perfusion temperature, a larger proportion of the released radioactivity was acid precipit‐able (24% vs. 5%). Some radioactivity also was recovered in the bile; of the total amount of radioactivity released from the liver in 30 min at 37°C, approximately 5% was directed into the bile. At lower temperatures of perfusion, a greater fraction of the radioactivity that was released from the liver was directed into the bile (20% at 30°C vs. 5% at 37°C). The data imply that the endosomal pathway to the lysosome is highly sensitive to slight reductions in temperature while the transcytotic route into bile is less sensitive. Lower temperatures might prolong the residence time of ASOR in the prelysosomal endosomal compartments, and thereby increase the likelihood that undegraded ligand will be returned to the blood or be missorted into bile.