bluuebunny/crossref_metadata_2025_split huggingface dataset:
Involvement of cAMP and calcium in the induction of ornithine decarboxylase activity in an osteoblast cell line
Authors: van Leeuwen J. P. T. M. , Bos M. P. , Herrmann‐Erlee M. P. M. 
Published: February 2005

AbstractThe role of cAMP and calcium in the induction of ornithine decarboxylase (ODC, E.C.4.1.1.17) activity in the osteogenic sarcoma cell line, UMR 106‐01, was studied, with particular interest for parathyroid hormone (PTH). PTH and forskolin dose‐dependently induced the ODC activity and the cAMP production. Protein synthesis is involved in the effect of PTH and forskolin on ODC activity but not on cAMP production. Using quin2 we showed that 20 nM PTH and 10 μM forskolin increased the intracellular ionized calcium concentration ([Ca2+]i), thereby offering the possibility for calcium to play a role as cellular mediator in the action of PTH and forskolin in bone. Data obtained with A23187 showed that solely an increase of the [Ca2+]i is not sufficient to stimulate basal or potentiate PTH‐ and forskolin‐induced ODC activity. However, the effects of calcium channel blockers and EGTA on basal and PTH‐ and forskolin‐induced ODC activity point to a specific role for calcium. Moreover, the effects of calcium channel blockers and EGTA on basal and PTH‐ and forskolin‐induced cAMP production indicate that the involvement of calcium in the induction of ODC activity is primarily located at another site than the adenylate cyclase. These data indicate that calcium is involved in the control of basal ODC activity. Furthermore, these data suggest that both cAMP and calcium are involved in the induction of ODC activity by PTH and forskolin. More precisely, ODC activity in UMR 106‐01 cells can be induced by PTH and forskolin via a calcium‐dependent cAMP messenger system.
Hemopoietic precursor cell defects in nonanemic but stem cell‐deficient W44/W44 mice
Authors: Barker Jane E. , McFarland Eleanor C. 
Published: February 2005

AbstractHematopoietic stem cell deficiencies cause a severe macrocytic anemia in W/W″ mice. W44/W44 mice, on the other hand, are not anemic, but, since they accept marrow implants without prior total body irradiation, they have inherited a stem cell lesion. In an attempt to identify the aberrant stem cell(s), we have determined the concentration in W44/W44 marrow of hematopoietic precursors known to be deficient in W/Wv marrow. The in vitro erythroid burst‐forming units (BFU‐E), the in vivo spleen colony‐forming units (CFU‐S), and the cells that repopulate the erythroid compartment of stem cell‐deficient mice were examined. The progenitors of 7‐day bursts are dramatically reduced in W/W″ marrow but are present in normal concentrations in W44/W44 marrow. W44/W44 marrow CFU‐S, unlike W/W″, generate visible spleen colonies 10 days after injection into lethally irradiated recipients. The colonies are, however, smaller and at least 2 times less numerous than those produced from equivalent numbers of +/+ marrow. An additional defect was the inability of W44/W44 stem cells to compete with genetically marked +/+ cells during erythroid repopulation. An estimate of the number of W44/W44 stem cells needed to compete with +/+ cells was provided by enriching W44/W44 progenitors fivefold. Twice as many enriched W44/W44 marrow cells as unfractionated +/+ cells were required to replace competitor cells. This suggests that there are up to 10 times fewer stem cells somewhere in the W44/W44 erythrogenerative pathway. The data support the conclusion that an erythroid progenitor less mature than the BFU‐E is one of the cells most severely affected by expression of the mutant gene.
In vitro studies on adult cardiac myocytes: Attachment and biosynthesis of collagen type IV and laminin
Authors: Lundgren Evy , Gullberg Donald , Rubin Kristofer , Borg Thomas K. , Terracio Marjorie J. , Terracio Louis 
Published: February 2005

AbstractThe interactions between adult rat cardiac myocytes and the basement membrane components collagen type IV and laminin were investigated in attachment experiments and biosynthesis studies and by immunofluorescence staining. Adult myocytes attached equally well to native collagen type IV and laminin but did not attach to collagen type IV solubilized with pepsin (P‐CIV) or to collagen type I. However, when laminin was used to coat P‐CIV, attachment was enhanced. Affinity‐purified antibodies against laminin inhibited the attachment of myocytes to dishes coated with native collagen type IV, indicating that cell surface‐bound laminin mediated attachment of the cells to this substrate. Immunofluorescence staining of freshly isolated myocytes, using antibodies against laminin or collagen type IV, revealed the presence of laminin but not of collagen type IV on the surface of freshly isolated cells, indicating that during the isolation procedure collagen IV was removed from the cell surface. Metabolic labeling followed by immunoprecipitation demonstrated synthesis of both laminin and collagen type IV in cardiac myocytes as they progressed into culture over a 14‐day period. This synthesis was accompanied by the deposition of the collagen type IV and laminin into distinctly different patterns as revealed by immunofluorescence staining. As the cells progressed into culture, newly synthesized laminin formed a network radiating from the center of the reorganizing cell into the pseudopods. The laminin was redistributed and remodeled with time in culture to form a dense layer beneath the cell. Collagen type IV was also synthesized with time in culture, but the pattern was a much finer network as opposed to the denser pattern of laminin staining. These studies demonstrate that adult cardiac myocytes synthesize and remodel the basement membrane as they adapt to the culture environment.
Thrombin and histamine activate phospholipase C in human endothelial cells via a phorbol ester‐sensitive pathway
Authors: Brock Tommy A. , Capasso Elizabeth A. 
Published: February 2005

AbstractThe effects of phorbol esters and synthetic diglycerides on thrombin‐ and histamine‐stimulated increases in inositol trisphosphate (IP3) and cytosolic free calcium ([Ca2+]i) were studied in cultured human umbilical vein endothelial cells (HEC). Thrombin (0.003–3.0 U/ml) and histamine (10−7–10−4 M) induced rapid increases in [Ca2+]i in suspended cells as monitored with the fluorescent calcium indicator fura‐2. In [3H]myoinositol‐labeled cells, both thrombin (3 U/ml)‐ and histamine (10−4 M)‐induced IP3 increases (195% ± 6% and 98% ± 4%, respectively) occurred in less than 15 sec and were temporally correlated with [Ca2+]i increases. Brief incubations (5–60 min) with different protein kinase C activators [4‐β‐phorbol 12‐myristate 13‐acetate (1–100 nM), mezerein (100 nM), and sn‐1,2 dioctanoylglycerol (0.1–10 μM)] attenuated agonist‐induced increases in [Ca2+]i. These compounds also inhibited thrombin‐ and histaminestimulated IP3 formation, thus suggesting a tight coupling between phospholipase C activation and calcium flux in cultured HEC. Overall, these observations suggest that the pathway linking receptors to phospholipase C stimulation in human endothelial cells is sensitive to protein kinase C activation.
Autocrine control of collagenase synthesis by synovial fibroblasts
Authors: Brinckerhoff Constance E. , Mitchell Teresa I. 
Published: February 2005

AbstractFibroblasts respond to exogenous stimuli, such as Interleukin 1, phorbol esters, or crystals of monosodium urate monohydrate, by synthesizing and secreting large quantities of collagenase. Here we show that addition of exogenous stimuli results in the production of an autologous protein that is, itself, capable of inducing collagenase. This autocrine has been partially purified. Activity resides in a protein(s) with a pl of 5 or 8 and with Mr of ∼ 15K. Conversely, conditioned medium taken from unstimulated cultures contains an inhibitor of collagenase synthesis. This protein, which has a Mr ∼ 20–25k by HPLC gel filtration antagonizes collagenase synthesis induced by phorbol esters, exogenous ll 1, and the autologous inducer. We conclude that synovial fibroblasts regulate collagenase synthesis via an autocrine mechanism that includes the synthesis of both an inducer and inhibitor. Both proteins are active at nanomolar amounts and may function as polypeptide hormones in regulating collagenase synthesis and, hence, connective tissue remodeling.
Propionic acid‐induced calcium mobilization in human neutrophils
Authors: Naccache Paul H. , Faucher Nicole , Caon Adriana C. , McColl Shaun R. 
Published: February 2005

AbstractThe ability of propionic acid to elicit an increase in the level of cytoplasmic free calcium in human neutrophils was examined in detail. Propionic acid induced a rapid and dose‐dependent mobilization of calcium that relied on both internal and external sources of calcium. The effects of propionic acid on the mobilization of calcium were inhibited by pertussis toxin, but not cholera toxin, implicating a guanine nucleotide binding protein. Furthermore, preincubation of the neutrophils with phorbol 12‐myristate 13‐acetate resulted in a decreased mobilization of calcium. This inhibitory activity of phorbol myristate acetate was antagonized by the protein kinase C inhibitor H‐7. Preincubation of the cells with the synthetic chemotactic factor fMet‐Leu‐Phe caused a reduction in the magnitude of the calcium transient elicited by propionic acid. However, the calcium response to propionic acid was not affected by antagonists of fMet‐Leu‐Phe and platelet‐activating factor binding or by an inhibitor of leukotriene synthesis. Propionic acid did not elicit a mobilization of calcium in monocytes, platelets, lymphocytes, or undifferentiated HL‐60 cells. However, the treatment of the HL‐60 cells with dimethylsulfoxide resulted in the appearance of a calcium response to propionic acid. The potential physiological significance of these findings are discussed.
Bifunctional activity of transforming growth factor type β on the growth of NRK‐49F cells, normal and transformed by kirsten murine sarcoma virus
Authors: Jullien Pierre , Berg Tone M. , de Lannoy Colette , Lawrence David A. 
Published: February 2005

AbstractTransformation of rat NRK‐49F cells (49F) by Kirsten murine sarcoma virus (Ki‐MSV) renders these cells (Ki‐49F cells) capable of autonomous anchorage independent (Al) growth. As compared to nontransformed 49F cells, the transformation by Ki‐MSV does not modify the cell response to transforming growth factor‐β (TGF‐β) in monolayer conditions, but alters it in A I growth conditions. The growth of nontransformed or Ki‐MSV‐transformed adherent 49F cells is slowed down by porcine TGF‐β, and this effect is reversed by epidermal growth factor (EGF). This decrease in the cell growth rate, induced by TGF‐β, does not affect the cloning efficiency of untransformed and transformed adherent 49F cells. Contrarily, porcine TGF‐β decreases the A I cloning efficiency of Ki‐49F cells in agar‐gelled medium; this effect is only partly reversed by EGF, which does not synergise with TGF‐β to enhance the A I growth as in the case of untransformed 49F cells. Media conditioned by 49F cells, Ki‐49F cells, and chicken embryo fibroblasts contain a latent TGF‐β whose capacity to promote the A I growth of 49F cells and to inhibit that of Ki‐49F cells is unmasked by acidification. The same situation exists concerning TGF‐β from human platelets. Neutral extracts are inefficient in both tests of promotion and inhibition of A I growth and contain an acid‐activable component with an apparent molecular weight of 600 kd. In acid extracts, a 5–9 kd apparent molecular weight component is responsible for the A I growth enhancement of 49F cells and the A I growth inhibition of Ki‐49F cells. Further purification by reverse phase chromatography shows that both activities strictly coelute at the same point (32%) of an acetonitrile gradient. These results indicate that TGF‐β is present in physiological conditions as a latent form which requires activation for inhibiting the A I growth of transformed cells as well as for enhancing that of 49F cells.
Modulation of the mitogenic response of an epidermal growth factor‐dependent keratinocyte cell line by dexamethasone, insulin, and transforming growth factor‐β
Authors: Zendegui Joseph G. , Inman Wendelyn H. , Carpenter Graham 
Published: February 2005

AbstractThe stimulation of DNA synthesis by epidermal growth factor (EGF) has been studied for a cell line having properties useful for investigating the mechanism of action of EGF in epithelial cell populations. These studies employ a mouse keratinocyte cell line (MK), isolated by Weissman and Aaronson (1983), which is stringently dependent on exogenous EGF for growth in serum containing medium. The studies reported here characterize the compliment of EGF receptors present on the surface of MK cells and demonstrate the regulatory influence of other hormones on the capacity of EGF to stimulate DNA synthesis. Up‐regulated MK cells contain approximately 22,000 EGF receptors per cell, but when the cells are grown in the presence of EGF the receptor number is reduced to about 4,000. It is estimated that only a small number of high‐affinity receptors (less than 500) are required for EGF‐dependent cell proliferation. In contrast to its action in fibroblastic cells, dexamethasone is a strong inhibitor of EGF‐stimulated DNA synthesis of MK cells. Insulin at high concentrations, or insulin‐like growth factors I or II (IGF‐I, IGF‐II) at physiological concentrations, synergistically enhance the EGF response. Interestingly, insulin or IGF‐I or II are also able to reverse most of the dexamethasone inhibition of DNA synthesis. Transforming growth factor‐β (TGF‐β) inhibits, in reversible manner, the EGF stimulation of DNA synthesis and this inhibition is not overcome by insulin. TGF‐β receptors have been measured in MK cells and Scatchard analysis indicates approximately 20,000 receptors per cell. None of the modulatory hormones (insulin, dexamethasone, TGF‐β) significantly altered 125I‐EGF binding characteristics in MK cells, suggesting a point of action distal to 125I‐EGF binding.
Differential expression of mRNA coding for heparin‐binding growth factor type 2 in human cells
Authors: Sternfeld Mark D. , Hendrickson Jill E. , Keeble Winifred W. , Rosenbaum James T. , Robertson Joseph E. , Pittelkow Mark R. , Shipley Gary D. 
Published: February 2005

AbstractThe proliferation of normal human fibroblasts, keratinocytes, and melanocytes in vitro can be controlled by purified polypeptide growth factors and serum. We have studied the cellular expression of the heparin‐binding growth factor type 2/basic fibroblast growth factor (HBGF‐2/bFGF) gene to determine whether these cell types synthesize mRNA for this mitogen. Our results indicate that normal human fibroblasts synthesize four distinct mRNAs of 7.0, 3.7, 2.2, and 1.5 kilobases, which hybridize to a specific HBGF‐2/bFGF cDNA probe. In fibroblasts, the level of all four of these transcripts increases dramatically (more than tenfold) within 4 hours of treatment of quiescent cells with fresh fetal bovine serum. Of the purified growth factors tested, transforming growth factor type‐beta also increased HBGF‐2/bFGF mRNA abundance, but not to the levels attained by serum treatment. Treatment of fibroblasts with cycloheximide before and during serum treatment blocked the ability of serum to induce the expression of the HBGF‐2/bFGF gene. The gene is expressed at low levels in human fibroblasts rapidly growing in serum‐free medium and at higher levels in cells rapidly growing in serum‐containing medium. In contrast to fibroblasts, mRNA coding for HBGF‐2/bFGF is undetectable in proliferating normal human keratinocytes, melanocytes, or mammary epithelial cells. Because keratinocytes and melanocytes proliferate in response to purified HBGF‐2/bFGF, our results suggest that HBGF‐2/bFGF may mediate the proliferation of epidermal cells through paracrine mechanisms involving stromal fibroblasts. Moreover, we have shown that a human squamous cell carcinoma cell line (SCC‐25) expresses mRNA coding for HBGF‐2/bFGF, suggesting that the gene may become activated in some carcinomas.
Induction of achromosomal cleavage by hydroxyurea in starfish embryo and the reversal by a combination of deoxyadenosine and deoxycytidine: Possible involvement of salvage pathway for deoxynucleoside triphosphate biosynthesis in the presence of hydroxyurea
Authors: Yamada Hisafumi , Kuraishi Ritsu , Hirai Setsuro , Katoh Yuko , Fusetani Nobuhiro , Amikura Reiko , Okano Keiju , Nagano Hiroshi 
Published: February 2005

AbstractEffects of hydroxyurea, an inhibitor of ribonucleotide reductase, on cleavage of starfish embryos were studied. In the presence of 1 mM hydroxyurea, fertilized eggs of the starfish, Asterina pectinifera, cleaved up to the 256‐cell stage and decomposed before blastulation. Before the 16‐cell stage, each blastomere contained a normal nucleus or chromosomes with mitotic apparatus. The cleveage after the 16‐cell stage was slow compared to the control embryos, and not all blastomeres contained a nucleus or normal chromosomes. During the fifth cell division (between 16‐cell‐ and 32‐cell‐stage embryos), chromatin mass unassociated with the mitotic apparatus remained near the cleavage furrow. When hydroxyurea was removed before the 16‐cell stage, the embryos developed to normal bipinnalia larvae via normal blastulae. However, the embryos were disintegrated before blastulation when hydroxyurea was removed after the 32‐cell stage. DNA synthesis was normally observed before the 16‐cell stage but not after the 16‐cell stage, but dNTP contents in the embryos remained low throughout development in the presence of hydroxyurea. The achromosomal cleavage observed in the presence of hydroxyurea was reversed by the combination of extracellular dAR and dCR. Therefore, it is assumed that the synthesis of dNTPs required for DNA synthesis in the presence of hydroxyurea occurs via the salvage pathway using deoxynucleosides (dNR) (dNR to dNTP via dNMP and dNDP).
Leukotriene D4‐induced increases in the cytoplasmic pH of human myelocytic leukocytes
Authors: Baud Laurent , Healy Ann , Cragoe Edward J. , Goetzl Edward J. , Koo Catherine H. 
Published: February 2005

AbstractThe effects of leukotriene D4 on the intracellular pH of human myelocytes, derived from cultured HL‐60 cells by dimethylsulfoxide‐induced differentiation, were quantified with the fluorescent indicator 2′,7′‐bis‐(2‐carboxy‐ethyl)‐5,6‐carboxyfluorescein. Leukotriene D4, but not C4 or E4, increased intracellular pH optimally by 3 min with a half‐maximal effect at 1–2 nM. The increases in intracellular pH stimulated by leukotriene D4 were prevented by pretreatment of myelocytes with leukotriene D4 but not peptide chemotactic factors. Analogs of amiloride that inhibit selectively the Na+/H+ antiport also prevented the intracellular alkalinization induced by leukotriene D4. The rate of recovery of intracellular pH after an acid load with 30 mM sodium propionate was approximately 30% higher at each level of intracellular pH for myelocytes exposed to leukotriene D4 than for those challenged in buffer alone. The increase elicited by leukotriene D4 in the adherence of myelocytic leukocytes to surfaces thus is associated with an enhanced sensitivity of the Na+/H+ antiport to intracellular pH, that is, not coupled to an earlier rise in the cytosolic level of Ca+2.
Reassessment of fluid‐phase endocytosis and diacytosis in monolayer cultures of human fibroblasts
Authors: McKinley Dana Noel , Steven Wiley H. 
Published: February 2005

AbstractWe have investigated the kinetics of fluid‐phase endocytosis and diacytosis in confluent monolayers of human fibroblasts by comparing the behavior of three markers that have been previously used to study this process: [14C]sucrose, 125l‐labeled polyvinylpyrrolidone ([125l]PVP), and Lucifer Yellow. Three distinct kinetic compartments were observed with all markers. The first was relatively large (10‐60 fl/cell), reached steady state within 15 min at 37°C, and was rapidly lost from monolayers after removing the markers at 37°C but not at 0°C. These properties indicate that this compartment is the same as that previously proposed to be the major intracellular compartment involved in diacytosis. However, this compartment is probably extracellular fluid trapped between cells since it is rapidly lost into the medium when the cells are either scraped or enzymatically removed from the culture dishes at 0°C. In addition, it very slowly undergoes both filling and emptying at 0°C. However, we did observe a second, much smaller, kinetic compartment (approximately 2 fl/cell) undergoing rapid diacytosis that does seem to be intracellular. A third compartment that we observed accumulates markers at a linear rate (10‐20 fl cell−1 hr−1) and is not lost from cells even after incubation periods greater than 6 hr. The markers [14C]sucrose and [125l]PVP displayed very similar behavior with respect to all three compartments and yielded nearly linear long‐term uptake rates, thus indicating that there is little if any absorbed component in their uptake. However, Lucifer Yellow displayed significantly higher incorporation rates and its uptake rate was strongly nonlinear, indicating its uptake in fibroblasts is predominantly adsorptive. Our observations indicate that the rate of fluid‐phase endocytosis in fibroblasts is significantly less than previously reported and that any compartment involved in diacytosis is very small and turns over very rapidly. Significantly, we estimate that the constitutive internalization of clathrin‐coated pits is sufficient to account for the majority of fluid‐phase endocytosis and thus represents a major mechanism of membrane retrieval in these cells.
Thermal analysis of CHL V79 cells using differential scanning calorimetry: Implications for hyperthermic cell killing and the heat shock response
Authors: Lepock James R. , Frey Harold E. , Rodahl A. Michael , Kruuv Jack 
Published: February 2005

AbstractDifferential scanning calorimetry (DSC) was used to assay thermal transitions that might be responsible for cell death and other responses to hyperthermia or heat shock, such as induction of heat shock proteins (HSP), in whole Chinese hamster lung V79 cells. Seven distinct peaks, six of which are irreversible, with transition temperatures from 49.5°C to 98.9°C are detectable. These primarily represent protein denaturation with minor contributions from DNA and RNA melting. The onset temperature of denaturation, 38.7°C, is shifted to higher temperatures by prior heat shock at 43° and 45°C, indicative of irreversible denaturation occurring at these temperatures. Thus, using DSC it is possible to demonstrate significant denaturation in a mammalian cell line at temperatures and times of exposure sufficient to induce hyperthermic damage and HSP synthesis. A model was developed based on the assumption that the rate limiting step of hyperthermic cell killing is the denaturation of a critical target. A transition temperature of 46.3°C is predicted for the critical target in V79 cells. No distinct transition is detectable by DSC at this temperature, implying that the critical target comprises a small fraction of total denaturable material. The short chain alcohols methanol, ethanol, isopropanol, and t‐butanol are known hyerpthermic sensitizers and ethanol is an inducer of HSP synthesis. These compounds non‐specifically lower the denaturation temperature of cellular protein. Glycerol, a hyperthermic protector, non‐specifically raises the denaturation temperature for proteins denaturing below 60°C. Thus, there is a correlation between the effect of these compounds on protein denaturation in vivo and their effect on cellular sensitivity to hyperthermia.