bluuebunny/crossref_metadata_2025_split huggingface dataset:
5‐Azacytidine‐induced demethyelation of DNA to senescent level does not block proliferation of human fibroblasts
Authors: Gray Matthew D. , Jesch Stephen A. , Stein Gretchen H. 
Published: February 2005

AbstractIMR‐90 human diploid fibroblasts (HDF) lose from 30–50% of their genomic 5‐methyldeoxycytidine (5mdC) during the cellular aging process. In contrast, immortal SV40‐transformed IMR‐90 maintain a constant level of 5mdC in culture. Precrisis SV40‐transformed HDF (AG3204) represent a stage in between normal cell aging and immortalization because these cells still have a finite proliferative lifespan, but it is longer than that of normal HDF and ends in cell death rather than in G1‐arrest. We find that AG3204 cells continue to lose from 12–33% of their 5mdC after a population has a become 99% positive for SV40 T‐antigen. Both IMR‐90 cells and AG3204 cells have similar levels of 5mdC (average of 2.25%) at the end of lifespan. We investigated whether this level of 5mdC is an absolute block to further proliferation by treating IMR‐90 and AG3204 cells with 5‐azacytidine (5azaC) to reduce their 5mdC levels below the terminal elvel normally achieved at end of lifespan. We find that both IMR‐90 and AG3204 cells undergo extensive proliferation with subterminal levels of 5mdC and that the lifespans of both cells types are shortened by 5azaC treatment. These studies indicate that random genomic DNA demethyelation to a specific level of 5mdC is not a direct cause of finite proliferative lifespan. However, the correlation between accelerated DNA demethylation and accelerated aging still suggests that these two phenomena are related. Two ways to explain this relationship are: (1) DNA demethylation during aging is not random, and/or (2) both DNA demethylation and other cases, accelerated random DNA demethylation could accelerate aging, but not necessarily in direct relationship to the final genomic level of 5mdC achieved during the normal aging process.
Lectin binding to gp60 decreases specific albumin binding and transport in pulmonary artery endothelial monolayers
Authors: Siflinger‐Birnboim Alma , Schnitzer Jan , Lum Hazel , Blumenstock Frank A. , Shen Chien‐Ping J. , Del Vecchio Peter J. , Malik Asrar B. 
Published: February 2005

AbstractThe effect of albumin binding to cultured bovine pulmonary artery endothelial cell (BPAEC) monolayers on the transendothelial flux of 125I‐labelled bovine serum albumin (BSA) was examined to determine its possible role on albumin transcy‐tosis. The transport of 125I‐BSA tracer across BPAEC grown on gelatin‐ and fibronectin‐coated filters (0.8 μm pore diam.) was affected by the presence of unlabelled BSA in the medium in that transendothelial 125I‐BSA permeability decreased, reaching a 40% reduction at BSA concentrations equal to or greater than 5 mg/ml. BSA binding to BPAEC monolayers was saturated at concentration of 10 mg/ml with an apparent binding affinity of 6 x 10−7 M. In contrast, gelatin added to the medium altered neither 125I‐BSA binding nor transport. Several lectins were tested for their ability to inhibit 125I‐BSA binding and transport. One lectin, Ricinus communis (RCA), reduced 125I‐BSA binding by 70% and transport by 40%. Other lectins, Ulex europaeus, Triticum vulgare, and Glycine max decreased neither 125I‐BSA binding nor transport. The reduction of 125I‐BSA transport by RCA was not observed in the presence of saturating levels of BSA, indicating that RCA influenced only the albumin‐dependent component of transport. RCA, but not other lectins, precipitated a 60 kDa plasmalemmal glycoprotein from cell lysates of surface radioiodinated BPAEC monolayers. This 60 kDa glycoprotein appears to be the equivalent of gp60 identified previously as an albumin binding glycoprotein in rat microvascular endothelium. In summary, approximately 40% of albumin transport across' BPAEC monolayers is dependent on albumin binding. This component of albumin transport is inhibited by 80% by the binding of RCA to gp60. These results suggest that binding of albumin to gp60 on pulmonary artery endothelial cell membrane is a critical determinant of transendothelial albumin flux involving mechanisms such as plasmalemmal vesicular transcytosis.
Receptor‐mediated autocrine growth‐stimulatory effect of 5‐hydroxytryptamine on cultured human pancreatic carcinoid cells
Authors: Ishizuka Jin , Beauchamp R. Daniel , Townsend Courtney M. , Greeley George H. , Thompson James C. 
Published: February 2005

Abstract5‐hydroxytryptamine (5‐HT) is a mitogen for fibroblasts, vascular smooth muscle cells, renal mesangial cells, and jejunal crypt cells. The human carcinoid cell line (termed BON) that we established in our laboratory from a pancreatic carcinoid tumor produces and secretes 5‐HT. In this study, therefore, we examined the effect of 5‐HT on growth of BON cells. Furthermore, by use of selective 5‐HT receptor antagonists, we examined receptor and post‐receptor mechanisms by which 5‐HT‐induced responses were produced. 5‐HT stimulated growth of BON cells. 5‐HT stimulated phosphatidylinositol (PI) hydrolysis in a dose‐dependent fashion and inhibited cyclic AMP production in a dose‐dependent fashion. The 5‐HT1A/1B receptor antagonist, SDZ 21–009, prevented the reduction of cyclic AMP production evoked by 5‐HT and inhibited the mitogenic action of 5‐HT. The 5‐HT1C/2 receptor antagonist, mesulergine, competitively inhibited PI hydrolysis, but did not affect the mitogenic action of 5‐HT. The mitogenic action of 5‐HT and the reduction of cyclic AMP production evoked by 5‐HT were also inhibited by pertussis toxin. These results suggest that 5‐HT is an autocrine growth factor for BON cells and that mitogenic mechanism of 5‐HT involves receptor‐mediated toxin‐sensitive GTP binding protein. 8‐bromo‐cyclic AMP inhibited growth of BON cells whereas 8‐bromo‐cyclic GMP had no effect on cell growth. Involvement of protein kinase A in BON cell growth regulation was confirmed by the observation that a cAMP‐dependent protein kinase antagonist (Rp‐cAMPS) could stimulate BON cell growth.
Monkey retinal pigment epithelial cells in vitro synthesize, secrete, and degrade insulin‐like growth factor binding proteins
Authors: Waldbillig Robert J. , Schoen Timothy J. , Chader Gerald J. , Pfeffer Bruce A. 
Published: February 2005

AbstractCultured monkey retinal pigment epithelial (RPE) cells rapidly secrete large amounts of insulin‐like growth factor binding proteins (IGF‐BPs). IGF‐II tracer binding activity in conditioned media is two to three times greater than that of IGF‐I. Under reducing SDS‐PAGE conditions, 125I‐IGF affinity‐crosslinked binding protein (BP) is visualized as a broad band between 36 ± 2.9 and 49 ± 3.3 kDa. Because the electrophoretic mobility of the crosslinked BP is increased under non‐reducing conditions (33–45 kDa), intramolecular sulfhydryl bonding may be present. Frequently, the radiographic band representing affinity‐crosslinked binding protein exhibits a complex pattern of non‐uniform densities that suggests structural or functional IGF‐BP micro‐heterogeneity. IGF‐BPs synthesized by RPE also exhibit heterogeneity with respect to the absence or presence of oligosaccharide side chains. In particular, the larger, but not the mid‐sized or smaller IGF‐BPs exhibit side chains linked to the core protein with N‐glycosidic linkage. None of the crosslinked IGF‐BPs exhibit O‐linked side chains. Long‐term (12, 24, 48 hr) conditioning studies revealed that IGF‐BP fails to accumulate in culture media beyond 12 hr, but that replacement of conditioned media with fresh media allows a second period of binding protein accumulation. Other short‐term (12 hr) experiments indicate that, in fresh medium, the levels of IGF‐BP increase during the first 6–8 hr and then remain stable. To examine the processes contributing to these steady state levels of IGF‐BP, aliquots of 8‐hr conditioned medium were removed from the cells and either frozen on dry ice or incubated at 37°C for 16 hr. Importantly, it was found that incubation at 37°C resulted in a near total loss of binding activity. This is the first report of IGF‐BP degrading activity in a cell culture system. These findings indicate that (1) primate RPE cells rapidly secrete a complex mixture of N‐glycosylated and non‐glycosylated IGF‐BPs, and (2) the steady state levels of secreted IGF‐BP are tightly regulated at least in part through a concomitant IGF‐BP inactivating activity. Cultured RPE cells may be of utility in examining the mechanisms of IGF‐BP synthesis, secretion, and degradation at the cellular level.
Modulation of nuclear statin expression in rat thyroid follicle cell following administration of thyroid stimulating hormone
Authors: Bayer I. , Mitmaker B. , Gordon P. H. , Wang E. 
Published: February 2005

AbstractThis study was designed to examine the state of proliferation in the rat thyrocyte following the administration of thyroid stimulating hormone (TSH). An immunohistochemical technique involving the use of a monoclonal antibody to statin, a nonproliferation‐specific nuclear antigen, was developed to measure the subpopulation of cells that have ceased to divide. Following the random assignment of young male Sprague‐Dawley rats into various groups, the rats in the control group received a single intraperitoneal (i‐p) injection of normal saline, whereas the experimental groups received single i‐p injections of TSH at doses of 0.25, 0.50, and 1.0 IU, respectively. All rats were subsequently sacrificed in groups of three at 1, 2, 4, and 24 hours. The statin antibody label was readily identified within the follicle cell nucleus. Results revealed a statistically significant transient decrease in the mean percent statin‐positive nuclei in the TSH‐treated groups. The time‐ and dose‐dependent effect of TSH was maximal at 2 hours and no longer discernible at 24 hours. A second experiment involving the chronic administration of TSH (i‐p 0.25 IU twice daily) resulted in a cumulative response with a statistically significant progressive decrease in the mean percent of statin‐positive nuclei at 5 and 10 days, returning to near normal values 5 days following the cessation of treatment. Determination of the nuclear optical density of the statin reaction product by image analysis techniques revealed that a single injection of TSH resulted in a rapid disappearance of the statin nuclear protein. This result suggests that the disappearance of statin in the nucleus appears to reflect the event of cells leaving the nondividing quiescent state to resume the cell cycle traverse following the administration of TSH. The disappearance of statin appears as an early nuclear event that parallels the earliest known cytoplasmic pinocytotic response to TSH in the rat thyroid follicle cell.
Parathyroid hormone‐related protein production by primary cultures of mammary epithelial cells
Authors: Ferrari S. L. , Rizzoli R. , Bonjour J. P. 
Published: February 2005

AbstractParathyroid hormone‐related protein (PTHrP) plays a major role in the pathogenesis of malignant hypercalcemia, but has also been found in fetal and adult nonneoplastic tissues. Among them, lactating mammary gland was shown to produce PTHrP, and high levels of PTHrP were measured in milk. However, the regulation of PTHrP production by breast cells is still unknown. Primary cultures of mammary cells isolated from rat lactating glands were grown on collagen gels in an insulin/epidermal growth factor (EGF)‐supplemented medium. Under these conditions, mammary cells displayed an epithelial phenotype and their number increased more than twofold after 1 week in culture. At that time, the cells were capable of producing immunoreactive PTHrP (range: 25 to 150 pg/105 cells × 24 h) and PTH‐like bioactivity, as indicated by a 60% increase in cyclic adenosine monophosphate (cAMP) production induced by mammary epithelial cell conditioned medium in the PTH‐responsive osteoblast‐like UMR‐106 cell line. When cell proliferation was hindered by lowering plating density, by removing medium supplements, or by adding transforming growth factor (TGF)‐β, a well‐known autocrine inhibitor of mammary epithelial cell growth. PTHrP production was increased. In contrast, the omission of EGF or addition of specified anti‐EGF antibodies decreased PTHrP production. In conclusion, primary cultures of mammary epithelial cells isolated from lactating rat were shown for the first time to produce PTHrP in vitro. This production was higher in the presence of EGF and could be modulated by cell growth rate.
Expression of exogenous c‐myc oncogene does not initiate DNA synthesis in primary rat hepatocyte cultures
Authors: Skouteris George G. , Kaser Matthew R. 
Published: February 2005

AbstractCultured hepatocytes from adult rats stimulated with combinations of growth factors enter into S phase but do not undergo multiple rounds of DNA synthesis nor mitosis. We have examined the potential of an introduced oncogene to induce alterations in the DNA synthetic activity of the cultured hepatocytes in response to epidermal growth factor (EGF). Overexpression of c‐myc did not initiate significant DNA synthesis in rat hepatocyte cultures alone, although it cooperated with added EGF to super‐induce thymidine incorporation into DNA. From our results, it is suggested that EGF is also necessary to initiate hepatocyte DNA synthesis probably by inducing a battery of cell cycle‐related genes if incubated with c‐myc transfected cultures for only 5 hours. Hepatocyte polypeptides reacting with antiMYC antisera were found to migrate between 55–67 KDa in SDS‐PAGE; only the 64–67 KDa species were found to be phosphorylated, and the observed size heterogeneity may be due to proteolytic degradation or may reflect presently unknown posttranslational modifications.
Differential regulation of the expression of transforming growth factor‐β mRNAs by growth factors and retinoic acid in chicken embryo chondrocytes, myocytes, and fibroblasts
Authors: Jakowlew Sonia B. , Cubert Jeremy , Danielpour David , Sporn Michael B. , Roberts Anita B. 
Published: February 2005

AbstractTransforming growth factor‐beta (TGF‐β) autoregulates its expression in several mammalian cell types. We now report that addition of TGF‐βs 1, 2, and 3 to primary chicken embryo cells differentially affects expression of the messenger RNAs for the different TGF‐β isoforms depending on the cell type. In cultured sternal chondrocytes, addition of TGF‐βs 1, 2, or 3 results in an increase in the steady‐state levels of the messenger RNAs for TGF‐βs 2 and 3, but does not change expression of TGF‐β4 mRNA. In contrast, in cultured cardiac myocytes, addition of TGF‐βs 1, 2, or 3 results in an increase in expression of TGF‐βs 3 and 4 mRNAs, but does not change expression of TGF‐β2 mRNA. Moreover, expression of TGF‐βs 2, 3, and 4 mRNAs is not affected by addition of any of the TGF‐βs to fibroblasts. Addition of platelet‐derived growth factor (PDGF), epidermal growth factor (EGF), or interleukin‐1 (IL‐1) to these chicken cells also has differential effects on expression of the different TGF‐β mRNAs depending on the cell type. Retinoic acid also has contrasting effects on chondrocytes and myocytes either increasing or decreasing, respectively, expression of TGF‐βs 2 and 3 mRNAs and TGF‐β2 protein. Our results indicate a complex pattern of regulation of the different TGF‐β genes by themselves as well as by PDGF, EGF, IL‐1, dexamethasone, TPA, and retinoic acid in chicken embryo cells.
Epidermal growth factor stimulates cAMP accumulation in cultured rat cardiac myocytes
Authors: Yu Yiming , Nair Bipin G. , Patel Tarun B. 
Published: February 2005

AbstractWe have previously shown that epidermal growth factor (EGF) augments cAMP accumulation in the heart and stimulates cardiac adenylyl cyclase via a G protein mediated mechanism (Nair et al., 1989). More recently, employing an antibody against the carboxy‐terminus decapeptide of Gsα, we have demonstrated that Gsα mediates the effects of EGF on cardiac adenylyl cyclase (Nair et al., 1990). Since the heart comprises of a variety of cell types, the purpose of the studies presented here was to determine whether or not the effects of EGF on adenylyl cyclase were mediated in cardiac myocytes or noncardiomyocytes. Therefore, cultures of ventricular cardiomyocytes and noncardiomyocytes from neonatal rat hearts were established and characterized. Apart from the differences in cellular morphology, cardiomyocytes but not the noncardiomyocytes employed in our studies expressed the α‐ and β‐myosin heavy chain (MHC) mRNA and the β‐MHC protein. Additionally, as described previously, treatment of cardiomyocytes with thyroid hormone increased α‐MHC mRNA and decreased the expression of β‐MHC mRNA, indicating that the cardiomyocytes employed in our studies were responding in a physiologically relevant manner. EGF in a time‐dependent manner increased cAMP accumulation in the cardiomyocytes but not in noncardiomyocytes. Maximum and half‐maximum effects were observed at 100 nM and 2 nM concentrations of EGF, respectively. As determined by the presence of immuno‐reactive EGF receptors and tyrosine phosphorylation of the 170 kDa protein in membranes of cardiomyocytes and noncardiomyocytes, both the cell populations contained functional EGF receptors. Therefore, the differential effects of EGF on cAMP accumulation in the two cell populations appear to be due to differential coupling of the EGF receptors to the adenylyl cyclase system rather than the absence of EGF receptors in noncardiomyocytes. Consistent with our previous findings in isolated membranes and perfused rat hearts, EGF‐elicited increase in cAMP accumulation in cardiomyocytes did not involve activation of β‐adrenoreceptors and was abolished by prior treatment of cells with cholera toxin. Overall, our findings demonstrate that EGF‐elicited increase in cAMP accumulation in the heart is the reflection of changes in cAMP content of cardiomyocytes and not noncardiomyocytes.
Tunicamycin reduces Na+‐K+‐pump expression in cultured skeletal muscle
Authors: Alboim Sandra V. , Bak Asia , Sampson Sanford R. 
Published: February 2005

AbstractThe purpose of this study was to examine effects of tunicamycin (TM), which inhibits core glycosylation of the β‐subunit, on functional expression of the Na+‐K+ pump in primary cultures of embryonic chick skeletal muscle. Measurements were made of specific‐[3H]‐ouabain binding, ouabain‐sensitive 86Rb uptake, resting membrane potential (Em), and electrogenic pump contribution to Em (Ep) of single myotubes with intracellular microelectrodes. Growth of 4–6‐day‐old skeletal myotubes in the presence of TM (1 μg/ml) for 21–24 hr reduced the number of Na+‐K+ pumps to 60–90% of control. Na+‐K+ pump activity, the level of resting Em and Ep were also reduced significantly by TM. In addition, TM completely blocked the hyperpolarization of Em induced in single myotubes by cooling to 10°C and then re‐warming to 37°C. Effects of tunicamycin were compared with those of tetrodotoxin (TTX; 2 x 10−7 M for 24 hr), which blocks voltage‐dependent Na+ channels. TM produced significantly greater decreases in ouabain‐binding and Em than did TTX, findings that indicate that reduced Na+‐K+ pump expression was not exclusively secondary to decreased intracellular Na+, the primary regulator of pump synthesis in cultured muscle. Similarly, effects of TM were significantly greater than those of cycloheximide, which inhibits protein synthesis by 95%. These findings demonstrated that effects were not due to inhibition of protein synthesis. We conclude that glycosylation of the Na+‐K+ pump β‐subunit is required for full physiological expression of pump activity in skeletal muscle.
Changes in pyridine and adenine nucleotide levels in friend erythroleukaemia cells during growth and differentiation
Authors: Caldini Riccardo , Chevanne Marta , Magnelli Lucia 
Published: February 2005

AbstractPyridine and adenine nucleotide levels were measured in Friend erythroleukaemia cells (FELC) stimulated to growth and induced to differentiate by hexamethylene bisacetamide (HMBA) and N'‐methylnicotinamide (N'‐MNAM). A threeto fourfold increase in the NADP(H) was found to parallel cell growth stimulation in both the presence and absence of differentiation inducers. NAD(H) increased about twofold in control and to a minor extent in HMBA‐treated FELC but did not vary significantly in N'‐MNAM‐treated cells. ATP was significantly higher in control cells stimulated to growth than in resting ones, but it did not vary in inducer‐treated cells. These data confirm the relationship between high NADP(H) levels and cell resumption to growth; moreover they show that NAD(H) pool reduction and NAD/NADH ratio rise are associated with the process of FELC differentiation. The activities of NAD pyrophosphorylase and NAD kinase are much more enhanced in growth‐stimulated FELC than in resting ones. On the other hand transition from the quiescent to the proliferative state was accompanied by a decrease in the activity of poly(ADP‐ribose) polymerase. A decrease in poly(ADP‐ribose) polymerase activity was also found in differentiated cells in contrast to Controls. © 1992 Wiley‐Liss, Inc.
The fibrinogen anion‐binding exosite of thrombin is necessary for induction of rises in intracellular calcium and prostacyclin production in endothelial cells
Authors: Ngaiza Justinian R. , Manley Geoffrey , Grulich‐Henn Jürgen , Krstenansky John L. , Jaffe Eric A. 
Published: February 2005

AbstractThrombin stimulation of prostacyclin (PGI2) synthesis by cultured human umbilical vein endothelial cells (HUVEC) requires the active site of thrombin and involves rapid and transient rises in cytoplasmic free calcium [Ca2+]i. In this study, we investigated whether or not the anion‐binding exosite for fibrinogen recognition of thrombin (which confers certain substrate specificities) is also necessary for the induction of rises in [Ca2+]i and PGI2 production. Thrombin variants which lack either the catalytic site (DIP‐α‐thrombin) or anion‐binding exosite (γ‐thrombin) either alone or in combination failed to induce rises in [Ca2+]i or PGI2 production in HUVEC. To further study the role of the anion‐binding exosite of thrombin in the activation of HUVEC, COOH‐terminal fragments of hirudin were used. This portion of hirudin interacts with the anion‐binding exosite of thrombin and inhibits thrombin‐induced fibrinogen coagulation while leaving the catalytic activity of thrombin intact. A 21‐amino acid COOH‐terminal peptide of hirudin (Nα‐acetyldesulfato‐hirudin45–65 or Hir45–65) inhibited thrombin‐induced (0.5 U/ml) rises in [Ca2+]i and PGI2 production with IC50 of 0.13 and 0.71 μM, respectively. Similar results were obtained using shorter hirudin‐derived peptides.Thus, the fibrinogen anion‐binding exosite of thrombin is required for α‐thrombin‐induced rises in [Ca2+]i and PGI2 production in HUVEC. © 1992 Wiley‐Liss, Inc.