bluuebunny/crossref_metadata_2025_split huggingface dataset:
Fibroblasts inhibit mineralised bone nodule formation by rat bone marrow stromal cells in vitro
Authors: Ogiso Bunnai , Hughes Francis J. , Melcher Antony H. , McCulloch Christopher A. G. 
Published: February 2005

AbstractThe ability of rat skin fibroblasts (RSF) and human periodontal ligament fibroblasts (HPL) to inhibit the formation of mineralised bone nodules in rat bone marrow stromal cell (BMSC) cultures was studied. Co‐culture of HPL or RSF with BMSC resulted in a large reduction of bone nodule formation when compared with controls. Conditioned medium from HPL or RSF cultures inhibited bone nodule formation in a dose‐dependent manner. HPL‐conditioned medium depressed cell proliferation and alkaline phosphatase expression in BMSC cultures. These effects were not due to increased cytotoxicity or nutrient depletion. Inhibitory activity was recovered in a fraction of less than 1 kD following ultrafiltration and was insensitive to freeze‐thawing. The inhibitory activity was blocked when HPL cultures were grown in the presence of 105M indomethacin. Dose‐dependent inhibition of bone nodule formation was also observed in cultures incubated with prostaglandins E2 (at 10−6M) or F2α(at 10−7). The results indicate that fibroblasts may inhibit osteoblast differentiation and function in part by release of soluble factors including prostaglandins.
The RGD containing site of the mouse laminin A chain is active for cell attachment, spreading, migration and neurite outgrowth
Authors: Tashiro Ken‐Ichiro , Sephel Gregory C. , Greatorex Dave , Sasaki Makoto , Shirashi Norio , Martin George R. , Kleinman Hynda K. , Yamada Yoshihiko 
Published: February 2005

AbstractThe laminin A chain has been sequenced by cDNA cloning and was found to contain an RGD sequence. Synthetic peptides containing the RGD sequence and flanking amino acids were active in mediating cell adhesion, spreading, migration, and neurite outgrowth. Furthermore, endothelial cell attachment to a laminin substrate was inhibited by an RGD‐containing synthetic peptide. Antisera against the integrin (fibronectin) receptor, and monoclonal antibody to the integrin, VLA‐6, inhibited cell interaction with laminin, as well as with peptides containing an RGD sequence. These results suggest that the RGD containing site of laminin is active and interacts with the integrin family of receptors in certain cells.
Regulation of ornithine decarboxylase mRNA by phorbol esters and insulin in normal and C‐kinase‐deficient rat hepatoma cells
Authors: Butler Andrew P. , Cohn William B. , Mar Penny K. , Montgomery Raechelle L. 
Published: February 2005

AbstractTumor‐promoting phorbol esters and insulin produce similar effects in Reuber H35 rat hepatoma cell proliferation, including increased ornithine decarboxylase (ODC) enzyme activity, DNA synthesis, and mitogenesis. We investigated ODC mRNA accumulation in cells treated with either insulin or 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA). Both agents caused rapid accumulation of ODC mRNA: for TPA, it was maximal 3 hr after treatment (4–6‐fold greater than control cells) and returned quickly to control levels; for insulin, it was significantly longer, continuing to increase for at least 6 hr. Simultaneous treatment with TPA and insulin led to additive effects on ODC mRNA. Induction of ODC by TPA was blocked by down‐regulation or inhibition of protein kinase C (PKC), consistent with a PKC‐mediated mechanism. In contrast, PKC down‐regulation had little effect on ODC induction by insulin. Furthermore, although both agents stimulated ribosomal S6 protein phosphorylation in cells containing normal amounts of PKC, the response to TPA was abolished in PKC‐depleted cells; the effect of insulin was only slightly inhibited. TPA caused a rapid redistribution of essentially all of the PKC activity from the cytosolic to the membrane fraction of the cells, whereas insulin had no effect on PKC distribution. These results suggest that although insulin and TPA share some common cytoplasmic signalling pathways, their effects on phosphorylation of nuclear proteins and transcription of ODC may be mediated by distinct factors.
Effect of 2% dimethyl sulfoxide on the mitogenic properties of epidermal growth factor and hepatocyte growth factor in primary hepatocyte culture
Authors: Kost Diane P. , Michalopoulos George K. 
Published: February 2005

AbstractTwo percent dimethyl sulfoxide (DMSO) reversibly inhibited DNA synthesis in primary rat hepatocyte cultures maintained with epidermal growth factor (EGF) or hepatocyte growth factor (HGF). These data suggest that, in vitro, DMSO is a non‐specific inhibitor of hepatocyte proliferation, regardless of the stimulating mitogen. In addition, removal of DMSO from mitogen‐free cultures resulted in an increase in DNA synthesis. Protein synthesis gradually but irreversibly declined in all cultures after DMSO removal. The relevance of these findings to regulation of hepatocyte growth is discussed.
Culture perfusion schedules influence the metabolic activity and granulocyte‐macrophage colony‐stimulating factor production rates of human bone marrow stromal cells
Authors: Caldwell Jerry , Palsson Bernhard O. , Locey Betty , Emerson Stephen G. 
Published: February 2005

AbstractThe metabolic function and GM‐CSF production rates of adherent human bone marrow stromal cells were investigated as functions of medium and serum feeding rates. A range of medium exchange schedules was studied, ranging from a typical Dexter culture protocol of one weekly medium exchange to a full media exchange daily, which more closely approximates what bone marrow cells experience in situ. Glucose consumption was found to be significantly higher at full daily exchange rate than at any other exchange schedule examined. However, the lactate yield on glucose was a constant, at 1.8 mol/mol, under all conditions considered. Differential serum vs. medium exchange experiment showed that both serum supply and medium nutrients were responsible for the altered behavior at high exchange rates. Glutamine consumption was found to be insignificant under all culture conditions examined. A change in exchange schedule from 50% daily medium exchange to full daily medium exchange after 14 days of culture was found to result in a transient production of GM‐CSF and a change in metabolic behavior to resemble that of cultures which had full daily exchange from day one. These results suggest that both stromal cell metabolism and GM‐CSF production are sensitive to medium exchange schedules. Taken together, the data presented indicate that attempts to model the function of human bone marrow in vitro may be well served by beginning with medium exchange schedules that more closely mimic the in vivo physiologic state of bone marrow.
Proliferation of cultured fibroblasts is inhibited byL‐Iduronate—containing glycosaminoglycans
Authors: Westergren‐Thorsson Gunilla , Önnervik Per‐Ola , Fransson Lars‐ÅKe , Malmström Anders 
Published: February 2005

AbstractWe have modified a method (Gilles et al:Anal. Biochem., 159:109–113, 1986) for measuring cell number, that is basec on the binding of crystal violet to cell nuclei and used it to assay effects of various glycosaminoglycans on growth of human lung fibroblasts. The procedure was modified by growing cells in microcultures (96 well microplates) and by measuring the amount of adsorbed dye using a microplate photometer after solubilisation of the cells with detergent. There was a linear relationship between absorbance and cell number measured by a Coulter Counter. The method is rapid and sensitive and can be used for screening many samples as well as measuring growth rates at low initial cell densities. Even the low growth rates obtained in the absence of serum can be detected. Human lung fibroblasts were growth arrested by serum deprivation and then grown for periods of up to 4 days in the presence of serum and exogenously added glycosaminoglycans (range, 0.1–100 μg/ml). Hyaluronan, chondroitin sulfate, and dextran sulfate were without effects, whereas dermatan sulfate, certain heparan sulfates, and heparin suppressed growth (20%–50% inhibition). The antiproliferative activity of dermatan sulfate increased with increasing iduronate content. Certain heparan sulfates with moderately high sulfate andL‐iduronate contents were better inhibitors than heparin despite the fact that the latter glycan has even higher sulfate andL‐iduronate contents. The antiprolifera‐tive effect of exogenous glycans appeared after a lag period of 3–4 days, suggesting that they interfered with factors produced by the cells. Furthermore, the degree of inhibition was greater when the initial cell density was low. Above a certain level of seeded cells (approx. 10,000 cells/well), there was no inhibition by any of the glycosaminoglycans. It is possible that exogenous glycans cannot overcome endogenous growth‐promoting effects in densely seeded cultures.
Heavy metals induce expression of the TPA‐inducible sequence (TIS) genes
Authors: Epner Daniel E. , Herschman Harvey R. 
Published: February 2005

AbstractWe previously cloned a set of primary response genes, which we call TIS (TPA‐Inducible Sequence) genes, from a cDNA library prepared from Swiss 3T3 cells treated with tetradecanoyl phorbol acetate (TPA) and cyclohexirnide. TPA, polypeptide growth factors, and serum induce TIS gene expression in 3T3 cells. We now report that cadmium and zinc elevate mRNA levels for the TIS genes, including TIS28 (c‐fos), in Swiss 3T3 cells. The time‐Course of TIS gene mRNA accumulation after metal exposure is delayed in comparison to the accumulation of TIS gene mRNA after treatment with TPA and growth factors. Cadmium induction of the TIS gene message accumulation is blocked by actinomycin D. Moreover, cadmium treatment does not significantly stabilize TIS gene messages. TIS gene induction by metal is a primary response; TIS8, which encodes a zinc‐finger transcription factor, and TIS28 (c‐fos), can be induced in the presence of cadmium and cycloheximide, an inhibitor of protein synthesis. Downregulation of protein kinase C does not attenuate TIS gene induction by heavy metals.
Aluminum fluoride stimulates inositol phosphate metabolism and inhibits expression of differentiation markers in mouse keratinocytes
Authors: Lee Edmund , Yuspa Stuart H. 
Published: February 2005

AbstractMouse keratinocytes are induced to differentiate in vitro by elevating the level of extracellular calcium from 0.05 mM, where keratinocytes express a basal cell phenotype, to >0.10 mM, where they express the differentiated phenotype. This process has been associated with a rapid, sustained increase in inositol phosphate (InsP) turnover, which precedes the expression of differentiation‐specific proteins. In 0.05 mM Ca2+ medium, aluminum and fluoride salts (AIF), which combine to activate honspecifically heterotrimeric guanine nucleotide‐binding (G) proteins, cause a concentration‐dependent increase in InsP metabolism in kerati‐nocytes and generate elevated intracellular diacylglycerol levels. This is associated with an inhibition of cell growth. Treatment with both AlF and Ca2+ >0.10 mM resulted in an additive increase in InsP turnover, implying the presence of at least two responsive InsP pools. AlF inhibited the expression of differentiation markers induced by Ca2+ >0.10 mM and altered the morphology of keratinocytes from squamous to dendritic, which was reversible upon withdrawal of AlF. Neoplastic keratinocytes, in which basal levels of InsP metabolism are higher than in normal cells, do not differentiate in response to Ca2+. Neoplastic keratinocytes responded to AlF treatment with an even greater rise in InsP metabolism. AIF also inhibited cell growth and reversibly altered morphology in neoplastic keratinocytes. These data suggest that InsP metabolism in keratinocytes is at least partially regulated by a G protein mechanism. Furthermore, an increase in InsP metabolism is not sufficient to stimulate differentiation and may be inhibitory to differentiation if exceeding limited increases. However, these observations cannot exclude the possibility that other AlF‐stimulated pathways involving G or non‐G proteins can also influence keratinocyte biology.
Involvement of second messenger systems in stimulation of angiotensin converting enzyme of bovine endothelial cells
Authors: Dasarathy Yamuna , Fanburg Barry L. 
Published: February 2005

AbstractWe have demonstrated previously that a variety of agents including corticosteroids, thyroid hormone, cationophores, methylxanthines, and analogues of cAMP–all of which have diversified functions in various tissues–elevate cellular angiotensin converting enzyme (ACE) activity of bovine endothelial cells in culture. In addition to these agents, we have now found that direct and receptor‐mediated stimulators of adenylate cyclase, i.e., forskolin and cholera toxin, increase cellular ACE activity after 48 h incubation in culture. In an attempt to search out a more unifying concept of these stimulatory effects, we have further investigated the roles of second messengers in the stimulatory actions. Ca2+ ionophore, A23187 produced significant increases in both intracellular Ca2+ and ACE of endothelial cells. In contrast to Ca2+ ionophore, agents that transiently mobilize Ca2+ from intracellular reserves such as bradykinin, acetylcholine, and ATP have no effect on the level of cellular ACE. Representative agents that elevate cellular cAMP (e.g., isobutyl methylxanthine [IBMX] and dibutyryl cAMP) elevated cellular ACE, but the slightly increased [Ca2+], produced by these agents did not reach statistical significance. While IBMX, cholera toxin, and forskolin elevated cellular cAMP, other ACE stimulatory agents (hormones and cationophores) had no effect on cAMP. Ca2+ ionophore and the agents that elevated intracellular cAMP potentiated the effect of dexamethasone, thyroid hormone, and aldosterone in elevating cellular ACE activity. Increases in ACE activity produced by all stimulants were inhibited by the presence of 10–50 nM ouabain in the culture medium. Inhibition of ACE elevation by ouabain was reversed by increasing the extracellular [K+], thereby implicating Na+, K+‐ATPase in the ACE regulatory mechanism. These results support the presence of multiple independent mechanisms for the regulation of cellular ACE. In addition to possible involvement of intracellular Ca2+‐ and cAMP‐dependent pathways, ACE is also increased by corticosteroids and thyroid hormone through mechanisms unrelated to Ca2+‐and cAMP.
Modulation of ATP and drug binding by monoclonal antibodies against P‐glycoprotein
Authors: Georges Elias , Zhang Jian‐Ting , Ling Victor 
Published: February 2005

AbstractThe role of P‐glycoprotein in mediating the drug‐resistance phenotype in multidrug resistant cells is now well documented. It is thought to function as an energy‐dependent drug‐efflux pump of broad specificity. Structurally, P‐glycoprotein is an internally duplicated molecule containing two large multi‐spanning transmembrane domains and two cytoplasmic ATP binding domains. In this report we demonstrate that monoclonal antibodies C219, C494, and C32 directed against short linear regions of the P‐glycoprotein molecule inhibit ATP binding to P‐glycoprotein in vitro. We also provide direct evidence that both predicted ATP‐binding domains bind ATP and that there is co‐operativity between the two sites. In addition, the capacity of P‐glycoprotein to bind the calcium channel blocker, azidopine, is inhibited differentially by the antibodies. These observations are the first evidence linking specific perturbations of the P‐glycoprotein molecule with ATP and drug binding.
Fetuin and alpha‐2HS glycoprotein induce alkaline phosphatase in epiphyseal growth plate chondrocytes
Authors: Ishikawa Yoshinori , Wu Licia N. Y. , Valhmu Wilmot B. , Wuthier Roy E. 
Published: February 2005

AbstractA previously described chondrocyte alkaline phosphatase induction factor (CAP‐IF) for chicken epiphyseal growth plate chondrocytes has been purified to SDS‐PAGE homogeneity from fetal bovine serum by ammonium sulfate precipitation and by dye‐ligand affinity (Affi‐Gel Blue and Reactive Green‐19 agarose) and hybroxyapatite column chromatographies. As determined by immunoprecipitation of [35S]methionine‐labeled cellular proteins after 3 day treatment, this highly purified CAP‐IF increases the level of AP and certain other membrane proteins 2‐ to 3‐fold over control values. The pure protein of apparent 64.5 kDa molecular weight has been identified as fetuin by N‐terminal amino acid sequencing. This was confirmed by the finding that high alkaline phosphatase (AP)‐inducing activity is present in fetuin prepared by the Spiro method. However, fetuins prepared by the Pedersen or Deutsch procedures are inactive. At least half of the CAP‐IF activity of fetuin was irreversibly destroyed by treatment with EDTA and addition of Zn2+ did not reactivate the EDTA‐treated fetuin. Ascorbate synergistically enhanced the effect of fetuin on chondrocyte AP activity by over 8‐fold during 3 day exposure. Because of the very high homology between fetuin and the A‐chain of α2‐HS glycoprotein, we also tested and found that α2HS glycoproteins from human serum and bovine bone are both strong AP inducers. Our findings suggest that the AP‐inducing activity resides in a labile, cystatin/Zn2+‐binding domain common to these related serum glycoproteins. These proteins appear to play a role in enhancing AP expression in normal growth plate cartilage differentiation.
Signal transduction by bFGF, but not TGFβ1, involves arachidonic acid metabolism in endothelial cells
Authors: Fafeur VéRonique , Jiang Zhi Ping , Böhlen Peter 
Published: February 2005

AbstractWe investigated the stimulation of early cellular events resulting from the interaction of the growth factor basic FGF (bFGF) and of the growth inhibitor transforming growth factor beta‐type 1 (TGFβ1), with their specific receptors on bovine endothelial cells. At mitogenic concentrations, bFGF stimulated the rapid release of arachidonic acid and its metabolites from (3H)‐arachidonic acid labeled cells. When arachidonic acid metabolism was stimulated by addition of the calcium ionophore A23187, the effect of bFGF was amplified. Nordihydroguaïaretic acid, an inhibitor of the lipoxygenase pathway of arachidonic acid metabolism, decreased the mitogenic effect of bFGF, whereas indomethacin, an inhibitor of the cyclooxygenase pathway, was ineffective. These findings suggest that metabolism of arachidonic acid to lipoxygenase products may be necessary for the mitogenic effect of bFGF. Basic FGF did not stimulate the production of inositol phosphates from cells labelled with myo‐(2‐3H)‐inositol nor did it induce calcium mobilization, as measured by fura‐2 fluorescence, indicating that bFGF does not activate phosphoinositide specific phospholipase C in endothelial cells, but rather, that bFGF‐induced arachidonic acid metabolism is mediated by another phospholipase. TGFβ1, which inhibits basal and bFGF‐induced endothelial cell growth, had no effect on arachidonic acid matabolism and inositol phosphate formation and did not prevent bFGF‐induced arachidonic acid metabolism. These results suggest that the inhibitory action of TGFβ1 on endothelial cell growth occurs through different mechanisms.
Multiple pathways for ligand internalization in rat hepatocytes II: Effect of hyperosmolarity and contribution of fluid‐phase endocytosis
Authors: Moss Anita L. , Ward Walter F. 
Published: February 2005

AbstractIn a companion report (Moss and Ward: J. Cell. Physiol. 149:313–318, 1991) evidence was presented for multiple pathways for insulin internalization based on differences between the internalization of insulin and that of two other ligands, asialofetuin (Afet) and epidermal growth factor (EGF), in the presence of several perturbations of endocytosis. In the present study we have explored the characteristics of three internalization pathways and the contribution of each to overall insulin uptake. Freshly isolated hepatocytes were incubated with radiolabeled ligands in the presence of hyperosmolar sucrose, treatment that is thought to inhibit the coated pit pathway of endocytosis. Insulin internalization was decreased approximately 39%, but much greater decreases were observed with Afet (86%) and EGF (62%). Competition between uptake of radiolabeled and unlabeled insulin was observed in hyperosmolar‐treated cells, suggestive of endocytosis by a receptor‐mediated noncoated‐pit pathway. Uptake of radiolabeled insulin that persisted in the presence of hyperosmolarity and high concentrations of unlabeled insulin suggested a third uptake pathway: fluid‐phase endocytosis. A rate of fluid‐phase endocytosis of 7.2 μL/hr/106 cells was determined from the uptake of the fluid‐phase marker lucifer yellow. At high insulin concentrations (≥ 250 ng/ml), fluid‐phase endocytosis appears to be the predominant pathway for insulin uptake, but at lower insulin concentrations (physiological) the coated pit and noncoated pit pathways are the primary routes for insulin internalization.
Characterization of spleen colonies derived from mice with mutations at the w locus
Authors: Barker Jane E. , Starr Eleanor 
Published: February 2005

AbstractMice with mutations at the W locus have a hemopoietic stem cell defect characterized by an apparent deficiency of spleen colony forming cells (CFU‐S). In the present report, we provide evidence that mutant cells form colonies and we compare the characteristics of the colonies derived from mutant and normal cells. To perform the colony‐derivation studies, marrow cells were transferred into lethally irradiated congenic hosts that differed from the donors in the ubiquitous genetic marker, glucose phosphate isomerase (GPI‐1). Donor GPI‐1 comprised over 50% of the marker in the host spleen and marrow by 12 days post injection, regardless of whether the donor was mutant or normal. To characterize the colonies, serially sectioned host spleens were examined microscopically. Colonies are present by 8 days post‐transplantation regardless of donor genotype, but mutant colonies are distinctly different from normal colonies. The proportion of blast and granulocyte colonies is always greater in W/Wv than in +/+ recipients. Unlike the W/Wv donors, the +/+ donors generate primarily erythrocyte colonies at 8, 10, and 14 days and mixed colonies at 12 days post‐injection. Colonies from the mutant mice are generally smaller but visible colonies do appear by 12 days. The results are consistent with the notion that the anemia in W/Wv mice is caused by the early restriction of differentiating cells to a non‐erythrocyte lineage accompanied by the delayed amplification of mutant hemopoietic cells. Whether this means erythrocyte‐committed cells are absent or are present but unable to respond to the appropriate cytokines is not possible to determine from the current experiments.