bluuebunny/crossref_metadata_2025_split huggingface dataset:
Studies on the role of basic fibroblast growth factor in vivo: Inability of neutralizing antibodies to block tumor growth
Authors: Dennis Phillip A. , Rifkin Daniel B. 
Published: February 2005

AbstractAffinity‐purified polyclonal rabbit antibodies prepared against recombinant basic fibroblast growth factor (bFGF) neutralized the ability of bFGF to stimulate plasminogen activator (PA) production and endothelial cell migration in vitro. After iodination and intraperitoneal injection of the antibodies in mice, approximately 76% of the maximum circulating level of 125l‐anti‐bFGF antibodies (AF) was found as intact IgG after 24 hr. Furthermore, the circulating 125I‐AF retained the ability to bind bFGF. Studies were performed to determine whether the growth of three different murine tumors (CT26, EHS, or B16/BL6) could be inhibited with affinity‐purified neutralizing antibodies against bFGF. Tumors were injected subcutaneously in syngeneic mice, and neutralizing antibodies against bFGF were injected daily into the peritoneum. All studies, which varied in tumor burden, antibody dose, and study length, indicated that neutralizing antibod es against bFGF had no effect on tumor size, tumor growth, or tumor histology.
Acquisition of a lysosomal enzyme by myoblasts in tissue culture
Authors: Beauchamp Jonathan R. , Olsen Irwin , Partridge Terence A. 
Published: February 2005

AbstractSkeletal muscle myoblasts from different sources acquired high levels of the lysosomal enzyme β‐glucuronidase, when they were cultured together with mitogen‐activated lymphocytes. Immunofluorescent staining, thermal stability, and electrophoretic mobility showed that the increase in enzyme activity in the myoblasts was due to the presence of the lymphocyte form of the enzyme. Although myoblasts were able to take up exogenous β‐glucuronidase from the culture medium by mannose 6‐phophate receptor‐mediated endocytosis, enzyme acquisition during co‐culture with lymphocytes was independent of this pathway. Enzyme transfer from the lymphocytes was found to require direct cell‐cell contact with the muscle cells, and was accompanied by an increase in β‐glucuronidase activity in the lymphocytes themselves. Since this additional activity was also due to the presence of the lymphocyte form of the enzyme, these results indicate that interaction with the muscle cells induced the de novo synthesis of β‐glucuronidase in the lymphocytes.
Concanavalin A‐ and fetal‐calf‐serum‐induced rounding and myosin light chain phosphorylation in cultured smooth muscle cells
Authors: Sasaki Yasuo , Iwata Kunie , Sasaki Yasuharu 
Published: February 2005

AbstractRabbit aorta smooth muscle cells (SMC) in long‐term culture retracted in less than 10 min in response to a sequential order of stimulations by concanavalin A (Con A) and fetal calf serum (FCS). With additional continuous stimulation by FCS, the SMC took on a circular shape and were anchored to the substrate by retraction fibrils. This rounding was observed only when the cells were sequentially stimulated by Con A and FCS. Depletion of intracellular Ca2+ stores by the addition of EGTA and Ca2+ ionophores inhibited the rounding. Transient phosphorylation of MLC20 was observed in the initial stage during the SMC rounding. The extent of monophosphorylated MLC20 increased for up to 5 min to a maximal value of 49%. The diphosphorylated form reached a maximal value of 29% within 2 min; then both forms of MLC20 decreased. The process of the SMC rounding was inhibited by antimycin A or cytochalasins, in a dose‐dependent manner, findings which suggested a dependency on both metabolic energy and actin‐containing microfilaments. The smooth‐muscle‐relaxing agent, HA1077, also inhibited the process of SMC rounding. These observations suggest that a cellular contractile process might be involved in rounding of SMC.
Continuously applied compressive pressure induces bone resorption by a mechanism involving prostaglandin E2 synthesis
Authors: Imamura Kazunobu , Ozawa Hiroyuki , Hiraide Takatoshi , Shibasaki Yoshinobu , Fukuhara Tatsuo , Takahashi Naoyuki , Suda Tatsuo 
Published: February 2005

AbstractIn previous research, we devised a specific culture chamber to examine the effect of continuously applied compressive pressure (CCP) on bone formation and resorption. The chamber was infused with compressed mixed gases with different O2 and CO2 composition to maintain the pO2, pCO2, and pH in the culture medium under pressures of +0.5 atm (1.5 atm total) to +2.0 atm (3.0 atm total) at the same levels as those at the ordinary pressure (1 atm). Using the specific culture chamber, we demonstrated that CCP greatly suppressed the differentiation of mouse osteoblast‐like MC3T3‐E1 cells. The inhibition by CCP appeared to be mediated by prostaglandin E2 (PGE2). In the present study, we examined the effect of CCP on osteoclastic bone resorption. CCP treatment of mouse bone marrow culture markedly increased both the PGE2 production and the number of tartrateresistant acid phosphatase (TRACP)‐positive mononuclear cells (possibly precursors of multinucleated osteoclasts). An autoradiographic study using [125l]‐salmon calcitonin showed clearly that those TRACP‐positive cells had calcitonin receptors. The CCP effect was the greatest at + 1.0 atm (2.0 atm total). Isobutylmethylxanthine potentiated the production of TRACP‐positive cells induced by CCP. Adding indomethacin completely inhibited both the TRACP‐positive cell formation and the PGE2 production induced by CCP. CCP also increased the release of 45Ca from prelabeled mouse calvaria during later stages (2–6 days) of the 6‐day culture period. CCP markedly increased PGE2 but not interleukin 1 in the culture media of mouse calvaria. These results indicate that, besides inhibiting osteoblast differentiation, CCP stimulates bone resorption by generating new osteoclasts through a mechanism involving PGE2 production.
Retinyl acetate inhibits platelet‐derived growth factor‐induced Ca2+ signals in C3H 10T1/2 fibroblasts
Authors: Hill Timothy D. , Boynton Alton L. , Dean Nicholas M. , Mordan Lawrence J. 
Published: February 2005

AbstractMitogenic stimulation of density‐arrested C3H 10T1/2 mouse fibroblasts by serum or purified platelet‐derived growth factor (PDGF) was potently inhibited by retinyl acetate (RAc; IC50 = 0.1 μg/ml, 0.3 × 10−6 M) when administered during the frist 2 hours of mitogen exposure. This inhibitory effect of RAc coincided with a period early in the cell growth‐division cycle when density‐arrested C3H 10T1/2 cells stimulated by PDGF were found to require Physiological levels of extracellular Ca2+ for the transition from G0 to G1 of the cell cycle. To determine if the inhibitory effect of RAc was mediated through alterations in the Ca2+ signaling pathway induced by mitogens, we examined Fura‐2‐loaded fibroblasts for changes in the Ca2+ response elicited by PDGF. Addition of PDGF (5 ng/ml) induced a transient increase in the [Ca2+]i that was not significantly effected by the extracellular Ca2+ concentration. Treatment of cells with RAc caused a concentration‐ and time‐dependent inhibition of this PDGF‐stimulated Ca2+ flux (IC50 = 0.45 μg/ml or 1.5 × 10−6 M; t1/2 = 15 min), whereas release of intracellularly stored Ca2+ by thrombin was unaffected by RAc (1.2 μg/ml, 4 × 10−6 M). Treatment with RAc did not significantly affect PDGF binding to cell surface receptors or the generation of inositol phosphates. These results suggest that the mechanism by which RAc inhibits PDGF‐ or serum‐induced mitogenesis is through modulation of the Ca2+ signal stimulated by PDGF, and thereby depriving the cell of a rise in intracellular Ca2+ necessary for progression through the cell cycle.
Patterns of spectrin transcripts in erythroid and non‐erythroid cells
Authors: Prchal Josef T. , Yoon Sa‐Hyun , Papayannopoulou Thalia 
Published: February 2005

AbstractSpectrin is the major protein of the membrane erythrocyte skeleton. More recently, homologous but non‐identical spectrins (fodrins) were also found in various non‐erythroid tissues. Spectrin mRNA in erythroid and various non‐erythroid cells was examined by direct hybridization with human α‐spectrin, β‐spectrin (erythroid spectrins), and α‐fodrin (non‐erythroid spectrin) cDNA probes. Northern blot analysis of poly (A)+ RNA revealed a distinct pattern of expression in erythroid vs. non‐erythroid cells. Erythroid cells from early erythroblasts to reticulocyte stage expressed two mRNA species of β‐spectrin, whereas they expressed only a single speeies of α‐spectrin, and no α‐fodrin mRNA. In contrast, non‐erythroid cells (platelets, myeloid cells, liver, muscle, heart, cerebellum, and eye lens) expressed either no α‐spectrin mRNA or a different molecular weight transcript(s) of this gene, and a single species of β‐fodrin mRNA. Additionally, they also expressed from none to multiple species of β‐spectrin, and these were of different molecular size(s) from that found in erythroid cells (with the exception of platelets). Transcripts of non‐erythroid spectrin, α‐fodrin, were found as a single copy only in non‐erythroid tissues. Human and murine erythroleukemia cells expressed both erythroid spectrin transcripts in addition to α‐fodrin and raise the possibility that erythroid progenitors may have the potential to express both erythroid and non‐erythroid species. These data indicated that several mRNA species of β‐spectrin could be detected in both erythroid and some non‐erythroid cells. Whether multiple spectrin peptides could also be found with functional heterogneity is unclear. However, in each case, the pattern combination observed appeared to be tissue‐specific.
Evidence for probenecid‐sensitive organic anion transporters on polarized thyroid cells in culture
Authors: Géarard Corinne , Boudier Jeanne‐Andrée , Mauchamp Jean , Verrier Bernard 
Published: February 2005

AbstractEpithelial thyroid cells in primary cultures loaded with BCECF/AM rapidly released the impermeant fluorescent dye BCECF (bis(carboxyethyl)carboxyfluorescein) in the incubation medium. Cells organized into follicles rapidly cleared BCECF (80% within 10 min) whereas fluorescence microscopy did not show any fluorescence in the follicular cavity. Cells organized into monolayers on plastic exported BCECF into the medium (70% within 40 min) whereas fluorescence microscopy showed intense fluorescence under the domes. BCECF efflux was blocked by probenecid, one of the known inhibitors of organic anion transporters, with similar efficiency in both structures. Maximal and half‐maximal effects were respectively observed for 5 mM and 0.4 mM probenecid. The polarity of BCECF efflux was studied by using monolayers on collagen‐coated Nuclepore filters: 85% of BCECF released was found in the basal compartment and 15% in the apical compartment. These findings suggested that thyroid cells in culture expressed a transport mechanism for the anionic form of BCECF. Furthermore, the observed activation of the Na+/H+ exchanger by probenecid suggested that the presence of this blocker did not overcome problems arising in the use of BCECF as intracellular pH indicator for thyroid cells.
Effect of histidine on histidinol‐induced heat protection in chinese hamster ovary cells
Authors: Lee Yong J. , Kim Dooha , Corry Peter M. 
Published: February 2005

AbstractThe possible mechanism for heat protection by the protein synthesis inhibitor histidinol was investigated in CHO cells. Histidinol (HST, 5 mM), an analogue of the essential amino acid L‐histidine, added for 2 hr before and during heating at 43°C, protected cells from killing at 43°C. Treatment with HST produced a 600‐fold increase in survival from 3 × 10−4 to 1.8 × 10−1 after 2.5 hr at 43°C. Although the cells were washed after HST treatment, substantial protective effect was still observed during heating at 43°C. This protective effect gradually decreased with increased incubation time after the drug treatment. However, the protective effect was immediately reduced by treatment with histidine (HIS, 0.25‐5 mM) during heating. The amount of reduction was dependent upon HIS concentration: five millimolar HIS completely inhibited HST‐induced heat protection. Furthermore, protein synthesis which was inhibited by 95% by 5 mM HST, resumed immediately with 5 mM HIS treatment. In addition, when cells were labeled during or after HST treatment, neither preferential accumulation of heat shock protein families nor phosphorylation of 28 kDa protein was observed. Therefore, these results suggest that the cessation of protein synthesis itself is one of the events involved in protection.
Nuclear localization of 68 kDa calmodulin‐binding protein is associated with the onset of DNA replication
Authors: Subramanyam C. , Honn Susan C. , Reed William C. , Reddy G. Prem Veer 
Published: February 2005

AbstractIn Chinese hamster embryo fibroblast cells, an increase in intracellular calmodulin levels coincided with the nuclear localization of a calmodulin‐binding protein of about 68 kDa as the cells progressed from G1 to S phase. When cells were limited from entering into S phase, by omitting insulin a defined medium, intracellular CaM levels did not increase and the 68 kDa calmodulin‐binding protein was completely absent from the nuclei. Corresponding to the nuclear localization of calmodulin and the 68 kDa calmodulin‐binding protein in S phase cells, there was a dramatic increase in DNA polymerase and thymidine kinase activities in the nuclei of S phase cells as compared to G1 phase cells. In addition, the 68 kDa calmodulin‐binding protein along with calmodulin, is observed to be an integral component of replitase complex responsible for nuclear DNA replication in S phase cells. These observations point to the association of calmodulin and calmodulin‐binding protein(s) with the replication machinery responsible for nuclear DNA replication during S phase. A possible regulatory role of these proteins in the onset of DNA replication and cell proliferation is discussed.
Epoxyeicosatrienoic acids activate Na+/H+ exchange and are mitogenic in cultured rat glomerular mesangial cells
Authors: Harris R. C. , Jacobson H. R. , Capdevila J. , Homma T. 
Published: February 2005

AbstractThe present study examined responses of cultured rat glomerular mesangial cells to exogenous exposure of epoxyeicosatrienoic acids (EET's), products of cytochrome P450 epoxygenase. One day after administration of 8, 9‐ or 14, 15‐EET, cultured rat mesangial cells demonstrated significant increases in [3H]thymidine incorporation (10−7 M 14, 15‐EET: 120 ± 7% of control; n = 6; P < 0.025; 10−6 M 14, 15‐EET: 145 ± 10%; n = 20; P < 0.0005; 10−6 M 8,9‐EET: 167 ± 31%; n = 9; P < 0.05), which was not affected by addition of the cyclooxygenase inhibitor indon ethacin. In addition to stimulation of [3H]thymidine incorporation, the epoxides stimulated mesangial cell proliferation. 14, 15‐EET administration induced intracellular alkalinization of 0.2–0.3 pH units, which was prevented by extracellular Na+ removal and blunted by amiloride (0.5 mM). Following intracellular acidification with NH4Cl addition and removal, > 85% of 3 mM 22Na uptake into mesangial cells was inhibited by I mM amiloride, indicating Na+ /H+ exchange. Under these conditions, 14, 15‐EET stimulated Na+ /H+ exchange by 42% and 8, 9‐EET stimulated Na+ /H+ exchange by 59%. Neither protein kinase C depletion nor addition of the protein kinase C inhibitor, staurosporine, affected this stimulation. In [3H]myo‐inositol loaded mesangial cells, no significant stimulation of phosphoinositide hydrolysis was detected in response to administration of 14, 15‐EET.Twenty‐four hours after addition of [14C] 14, 15‐EET, > 90% was preferentially esterified to cellular lipids, with predominant incorporation into phosphatidylinositol, phosphatidylethanolamine, and diacylglycerol.Thus, these results demonstrate epoxyeicosatrienoic acids stimulate Na+ /H+ exchange and mitogenesis in mesangial cells. These effects do not appear to be mediated via phospholipase C activation. In addition, 14, 15‐EET was selectively incorporated into cellular lipids known to mediate signal transduction. These observations extend the potential biologic roles of c‐P450 arachidonate metabolites to include stimulation of cell proliferation and suggest a role for these compounds in vascular and renal injury.
Nerve growth factor and fibroblast growth factor influence post‐fusion expression of Na‐channels in cultured rat skeletal muscle
Authors: Brodie Chaya , Sampson S. R. 
Published: February 2005

AbstractWe have examined effects of nerve growth factor (NGF) and fibroblast growth factor (FGF) on the density of tetrodotoxin (TTX)‐ sensitive Na‐channels in cultured rat skeletal muscle. Measurements were made of specific binding of [3H] saxitoxin (STX) and the frequency and rate of rise of spontaneously occurring action potentials, the Physiological expression of Na‐channel density. Cells were transferred to various growth conditions at 6 days in vitro, and measurements were made beginning 24 hr later. Both growth factors (GF) caused dose‐related in creases in Na‐channels compared with myotubes maintained in normal, serum‐supplemented growth medium. Maximum effects occurred with a concentration of NGF of 50 ng/ml and FGF of 15 ng/ml. Scatchard analysis of specific STX binding showed an increase in Bmax with no significant change in Kd. Similar increases occurred on rate of rise and frequency spontaneous action potentials. Treatment of cultures with cycloheximide or actinomycin D, inhibitors of protein and RNA synthesis, completely prevented the increase in STX‐binding induced by GF treatment, The results indicate that NGF and FGF have important effects on regulation of excitable cell gene products after differentiation.
DNA polymerase‐primase complex in wild‐type and ts A1S9 mouse L‐cells, temperature‐sensitive for dna replication during cell cycle progression
Authors: Munsch Nicole , Sheinin Rose 
Published: February 2005

AbstractTs A1S9 mutant cells, derived from wild type WT‐4 mouse L‐cells, are temperature‐sensitive (ts) for DNA synthesis and cell division. We try to determine the cause of the arrest of DNA replication in ts A1S9 cells at the nonpermissive temperature by comparing the modifications induced by the shift of temperature on the activity and the synthesis of DNA polymerase‐alpha and DNA primase as a function of time. Forty‐seven hours after temperature upshift DNA polymerase‐alpha activity of ts A1S9 cells was inhibited by 90% while primase activity was barely detectable. By contrast, the activities of both enzymes increased to a plateau level in WT‐4 cultured at either temperature and in ts A1S9 cells grown at the low permissive temperature. Study of the synthesis of DNA polymerase‐alpha primase and of the structure of the enzyme complex during cell cycle progression was approached by immunoprecipitation of [35S]‐labelled cells, with a specific monoclonal antibody directed against DNA polymerase‐alpha. We have found that, irrespective of temperature of cultivation of WT‐4 or ts A1S9 cells, this antibody precipited polypeptides of 220, 186, 150, 110, 68–70, 60, and 48 kDa from cell extracts. With ts A1S9 cells cultivated at 38.5°C for 48 hr the polypeptides of 220 and 186 kDa, associated with alpha‐polymerase activity, were considerably more abundant than in the control cells, with a concomitant decline in the polypeptides of 60 and 48 kDa, implicated in primase activity. Thus the inhibition of DNA polymerase‐alpha cannot be due to a decreased synthesis of the 186 kDa subunit but to its temperature inactivation. Consistent with a recent asymmetric dimeric model where polymerase‐alpha complex and polymerase delta complex synthesize co‐ordinately at the replication fork lagging and leading DNA strands, the observed alterations of polymerase‐alpha and primase content explain the inhibition of DNA synthesis and the cell cycle arrest of the ts A1S9 cells at the nonpermissive temperature.
Modulation of growth and differentiation in normal human keratinocytes by transforming growth factor‐β
Authors: Matsumoto Kunio , Hashimoto Koji , Hashiro Makoto , Yoshimasa Hidenobu , Yoshikawa Kunihiko 
Published: February 2005

AbstractThe effect of transforming growth factor‐type β 1(TGF‐β) on the growth and differentiation of normal human skin keratinocytes cultured in serum‐free medium was investigated. TGF‐β markedly inhibited the growth of keratinocytes at the concentrations >2 ng/ml under low Ca2+ conditions (0.1 mM). Growth inhibition was accompanied by changes in cell functions related to proliferation. Remarkable inhibition of DNA synthesis was demonstrated by the decrease of [3H]thymidine incorporation. The decrease of [3H]thymidine incorporation was observed as early as 3 hr after addition of TGF‐β. TGF‐β also decreased c‐myc messenger RNA (mRNA) expression 30 min after addition of TGF‐β. This rapid reduction of c‐myc mRNA expression by TGF‐β treatment is possibly one of the main factors in the process of TGF‐β‐induced growth inhibition of human keratinocytes. Since growth inhibition and induction of differentiation are closely related in human keratinocytes, the growth‐inhibitory effect of TGF‐β under high Ca2+ conditions (1.8 mM Ca2+ differentiation‐promoting culture environment) was examined. TGF‐β inhibited the growth of keratinocytes under high Ca2+ conditions in the same manner as under low Ca2+ conditions, suggesting that it is a strong growth inhibitor in both low and high Ca2+ environments. The induction of keratinocyte differentiation was evaluated by measuring involucrin expression and cornified envelope formation: TGF‐β at 20 ng/ml increased in‐volucrin expression from 9.3% to 18.8% under high Ca2+ conditions, while it decreased involucrin expression from 7.0% to 3.3% under low Ca2+ conditions. Cornified envelope formation was modulated in a similar way by addition of TGF‐β: TGF‐β at 20 ng/ml decreased cornified envelope formation by 53% under low Ca2+ conditions, while it enhanced cornified envelope formation by 30.7% under high Ca2+ conditions. Thus, the effect of TGF‐β on keratinocyte differentiation is Ca2+ dependent. It enhances differentiation of human keratinocytes under high Ca2+ conditions, but inhibits differentiation under low Ca2+ conditions. Taken together, there is a clear discrepancy between TGF‐β effects on growth inhibition and induction of differentiation in human keratinocytes. These data indicate that growth inhibition of human keratinocytes by TGF‐β is direct and not induced by differentiation.
Evidence for a role of the Na+/H+ exchanger in the colony‐stimulating‐factor‐induced ornithine decarboxylase activity and proliferation of the human cell line M‐07e
Authors: Ghigo Dario , Brizzi Maria F. , Avanzi G. Carlo , Bussolino Federico , Garbarino Giovanni , Costamagna Costanzo , Pegoraro Luigi , Bosia Amalia 
Published: February 2005

AbstractThe subclone M‐07e, derived from the interleukin‐3 (IL‐3)‐responsive human myeloid cell line M‐07, is strictly dependent on either IL‐3 or granulocyte‐macrophage‐colony‐stimulating factor (GM‐CSF) for its growth and survival. This cell line may be regarded as a candidate model to investigate the poorly understood events triggered by growth factors binding to human hemopoietic cells. Both IL‐3 and GM‐CSF induce in M‐07e cells an increase of ornithine decarboxylase (ODC) activity, which reaches its maximum at 24–30 h and fully depends on de novo protein synthesis. The growth factors do not elicit translocation of protein kinase C to the membrane; thus a role of the kinase in ODC induction is ruled out. An amiloride‐inhibitable Na+/H+ exchanger is present in the membrane of M‐07e cells; its apparent Km for extracellular Na+ is 47.77 mM; and its activity is greatly enhanced when the cytoplasm is acidified. Growth‐factor‐evoked ODC activation and DNA synthesis are blocked in a dose‐ and time‐dependent manner when M‐07e cells are incubated with ethylispropyl‐amiloride, a specific inhibitor of Na+/H+ exchanger. The exchanger does not appear to be directly activated by IL‐3 or GM‐CSF, but its operation is strictly required for the biological effects of these growth factors on M‐07e cell line.