bluuebunny/crossref_metadata_2025_split huggingface dataset:
Receptor‐mediated uptake of a mannose‐6‐phosphate bearing glycoprotein by isolated chicken osteoclasts
Authors: Blair Harry C. , Teitelbaum Steven L. , Schimke Patricia A. , Konsek John D. , Koziol Catherine M. , Schlesinger Paul H. 
Published: February 2005

AbstractWe have recently shown that degradation of bone collagen by osteoclasts occurs via proteolytic enzyme activity that depends on an acidic milieu. Since bone resorption occurs in an extracellular, acidic compartment located at the cell‐matrix attachment site, the osteoclast must deliver the acid collagenolytic enzymes to the cell surface. These observations raise the possibility that the mannose‐6‐phosphate (M‐6‐P) receptor, known to sort acidic proteases in other cells, is involved in trafficking lysosomal enzymes to the plasmalemma of bone resorbing cells. To this end we studied receptor‐mediated uptake, distribution and release, by isolated chicken osteoclasts, of 125l‐hexosaminidase, a M‐6‐P bearing enzyme. We found that at 4°C, the bone‐resorbing polykaryons bind ∼ 10,000 molecules of radioligand/cell with a Kd of 0.7 nM, which is endocytosed by osteoclasts at 37°C by a calcium‐independent process. Furthermore, 125l‐hexosaminidase uptake is unaffected by mannosylated albumin, documenting specificity of the receptor‐mediated event. Release of endocytosed enzyme from the cell is also much more rapid than its degradation, attesting to a pathway of uptake and secretion. By autoradiography, the M‐6‐P bearing ligand is concentrated at the site of osteoclast‐bone attachment. Thus, osteoclasts also have the capacity to deliver M‐6‐P bearing degradative enzymes to their surface at the site of matrix degradation.
Organic cation uptake by a cultured renal epithelium
Authors: McKinney T. Dwight , DeLeon Catherine , Speeg K. Vincent 
Published: February 2005

AbstractSeveral organic cations are actively transported by proximal renal tubules by mediated processes across both the apical and basolateral cell membranes. In order to evaluate this transport system in a cultured renal epithelium, uptake of 3H‐tetraethylammonium (TEA) across the apical membrane was measured in LLCPK1 cells, a cell line with several characteristics of proximal tubules. 3H‐TEA progressively entered these cells and reached a near‐steady state by 30 min. Three‐minute uptake was saturable with an apparent Vmax of 1,669 ± 129 fmoles/μg DNA and apparent Km of 34.0 ± 3.4 μM. 3H‐TEA uptake was inhibited by an excess of nonradioactive TEA, other organic cations, sodium azide, and hypothermia. An alkaline external pH was associated with greater 3H‐TEA uptake than an acid pH. However, efflux of 3H‐TEA from cells was not appreciably affected by changes in external pH. Preincubation of cells in acid or alkaline media did not affect uptake. Alteration of cell pH by ammonium chloride addition or removal had little effect on 3H‐TEA uptake. Finally, uptake of 3H‐TEA was not accelerated by preloading cells with an excess of nonradioactive TEA. These results indicate that intact LLCPK1 cells possess a mechanism(s) in their apical membranes for the mediated transport of a prototypic organic cation. The mechanism(s) involved in this transport is uncertain. However, neither organic cation/proton nor organic cation/organic cation exchange appears to be the predominant process.
Loss of low‐affinity serotonin receptors upon storage of human platelets
Authors: Soslau Gerald , Gillen Judy , Parker Janet 
Published: February 2005

AbstractPlatelets actively accumulate virtually all plasma serotonin within their dense granules. As a readily isolated, homogeneous cell type, platelets have served as a model for serotonin uptake into neurological tissue, in addition to defining the role of serotonin in hemostasis. The number of serotonin receptor types on the platelet membrane and the function of these receptors has not been conclusively demonstrated. The presence of different receptor types that may be altered or lost in disease or upon aging (in vitro storage or in vivo) could have significant physiological effects on platelet function. This report demonstrates that at least two receptor types are present on freshly prepared human platelets. However, after 3 to 4 days of storage in autologous plasma, the low‐affinity, high‐capacity serotonin receptor appears to be lost. This phenomenon probably accounts for some of the discrepancies reported in the literature. The high‐affinity receptor present in both freshly isolated and stored platelets binds about 9 × 103 serotonin molecules per platelet. Binding can be completely blocked by imipramine; however, some passive diffusion appears to occur even at the low level of extracellular serotonin concentrations employed in these studies (nanomolar range). The influx of serotonin into platelets appears to be poorly reversible, even in reserpine‐treated cells, where the extravesicular cytoplasmic concentration would be high. The loss of the low‐affinity serotonin receptor type reported in these studies may be directly or indirectly associated with the reduced responsiveness observed in stored platelets.
cAMP‐mediated growth inhibition of a B‐lymphoid precursor cell line Reh is associated with an early transient delay in G2/M, followed by an accumulation of cells in G1
Authors: Blomhoff Heidi Kiil , Blomhoff Rune , Stokke Trond , Davies Catharina DeLange , Brevik Kristin , Smeland Erlend B. , Funderud Steinar , Godal Tore 
Published: February 2005

AbstractThis study was undertaken to gain more insight into the effects of cyclic adenosine monophosphate (cAMP) on cell‐cycle progression in the B‐lymphoid precursor cell line Reh. The adenylate cyclase activator forskolin reduced the proliferation of asynchronously growing Reh cells by 50% after 72 hr culture. Growth inhibition was associated with an accumulation of cells in G1. Furthermore, we demonstrated that forskolin provoked a delay of cells for ∼ 10 hr in G2/M prior to the G1 arrest. Two different methods were applied to elucidate how cells in different phases of the cell cycle were affected by an elevated cAMP level. One method was based on centrifugal elutriation, whereby synchronous cell populations from the different phases of the cell cycle were isolated. By the other method, S‐phase cells were selectively stained by pulsing asynchronously growing cells with bromo‐deoxyuridine (BrdU). The data demonstrate that the position of a cell in the cell cycle is critical in determining how the cell will respond to an elevated cAMP level. Thus cells in G1 at the time forskolin is added are not delayed in G2/M, but they will subsequently accumulate in G1 after 48 hr. Cells given forskolin in G2/M, however, are delayed for 10 hr in G2/M, but they do not accumulate in G1. Cells given forskolin in the S phase are delayed in G2/M as well as arrested in G1. The results suggest that cAMP inhibits growth of the Reh cells by preventing the cells from passing important restriction points located in the G1 and G2 phases of the cell cycle.
Sex hormones modulate the synthesis of basic fibroblast growth factor in human endometrial adenocarcinoma cells: Implications for the neovascularization of normal and neoplastic endometrium
Authors: Presta Marco 
Published: February 2005

AbstractBasic fibroblast growth factor (bFGF) has been demonstrated to exert an angiogenic activity in vivo. Here, the ability of bFGF to stimulate plasminogen activator (PA) production in bovine capillary endothelial cells was used as an assay for the presence of bFGF‐like molecules in the extracts of the human endometrial adenocarcinoma AN3CA, HEC‐1‐A, and HEC‐1‐B cell lines. The identity of the PA‐stimulating activity with bFGF was confirmed by its high affinity for heparin and by its cross‐reactivity with antibodies to human placental bFGF. Western blot analysis and immunoprecipitation experiments showed that, in the extracts of the three cell lines, these antibodies recognize a protein with an apparent molecular weight of ∼ 17,000 daltons. 17β‐Estradiol stimulates the synthesis of this bFGF‐like molecule in all the endometrial cell lines tested. This stimulation can be abolished by treating the cells with progesterone. These data demonstrate the capacity of sex hormones to regulate bFGF synthesis in tumor endometrial cells, and they suggest that bFGF may play a role in the vascularization of the endometrial adenocarcinoma as well as of the normal endometrium.
L‐ascorbic acid 2‐phosphate stimulates collagen accumulation, cell proliferation, and formation of a three‐dimensional tissuelike substance by skin fibroblasts
Authors: Hata Ryu‐Ichiro , Senoo Haruki 
Published: February 2005

AbstractProliferation of human skin fibroblasts was stimulated significantly by the presence of L‐ascorbic acid 2‐phosphate (Asc 2‐P). The presence of Asc 2‐P (0.1–1.0 mM) in the culture medium for 3 weeks enhanced the relative rate of collagen synthesis to total protein synthesis 2‐fold as well as cell growth 4‐fold. Coexistence of L‐azetidine 2‐carboxylic acid (AzC), an inhibitor of collagen synthesis, attenuated both effects of Asc 2‐P in a dose‐dependent manner. Supplementation of the medium with Asc 2‐P also accelerated procollagen processing to collagen and deposition of collagen in the cell layer. Among the acidic glycosaminoglycans (GAG), another major component of extracellular matrix (ECM), deposition of sulfated forms was increased by the additive. Electron microscopic observations showed multilayered, rough endoplasmic reticulum‐rich cells surrounded by dense ECM. These results indicate that Asc 2‐P is useful in culture systems as a long‐acting vitamin C derivative and also that it promotes reorganization of a three‐dimensional tissuelike substance from skin fibroblasts in culture by stimulating collagen accumulation in the fibroblasts.
Characterization of the inhibitory effect of glucocorticoids on the DNA replication of adult rat hepatocytes growing at various cell densities
Authors: Vintermyr Olav Karsten , Døskeland Stein Ove 
Published: February 2005

AbstractDexamethasone inhibited the basal and EGF‐stimulated DNA synthesis of adult rat hepatocytes in primary culture. The inhibition was glucocorticoid‐specific: It was shown by dexamethasone and hydrocortisone, but not by progesterone, testosterone, or estradiol; and was counteracted by the glucocorticoid antagonist RU‐38486 in a concentration‐dependent manner. Dexamethasone acted by decreasing the rate of entry into S‐phase (kG1/S), while cell cycle parameters were unaffected. The steroid was able to decrease the kG1/S severalfold even when added more than 20 hr after EGF, half‐maximal effect occurring 11 hr after the addition of dexamethasone. Densily populated areas were much more sensitive to the inhibition by dexamethasone than sparsely populated areas within the same culture dish: A moderate (10 nM) concentration of dexamethasone nearly abolished the DNA synthesis in densily populated areas of hepatocyte cultures with only marginal effect on sparsely populated cells.
Immunodetection and quantitation of the two forms of transforming growth factor‐beta (TGF‐β1 and TGF‐β2) secreted by cells in culture
Authors: Danielpour David , Dart Linda L. , Flanders Kathleen C. , Roberts Anita B. , Sporn Michael B. 
Published: February 2005

AbstractTransforming growth factor beta (TGF‐β), a potent modulator of cell growth, differentiation, and the expression of extracellular matrix components in a variety of cell types, exists as two distinct homodimers (TGF‐β1 and TGF‐β2), sharing 71% sequence homology. Radioreceptor and previously described radioimmunological assays using rabbit antibodies have not been able to distinguish between these two forms. We have developed antisera in turkeys against native TGF‐β1 and TGF‐β2, each of which specifically blocks both the receptor binding and biological activity of each of these peptides. With these immunological reagents we describe sensitive and specific immunological assays for TGF‐β1 and TGF‐β2 in complex biological fluids. Using these assays we show that both TGF‐β1 and TGF‐β2 are secreted by a variety of cultured cells, but that some cells secrete predominantly either TGF‐β1 or TGF‐β2 while others secrete both peptides in nearly equal amounts. Our results demonstrate that the expression of each of the two forms of TGF‐β is independently regulated.
M2 subunit of ribonucleotide reductase is a target of cyclic AMP‐dependent protein kinase
Authors: Albert Daniel A. , Nodzenski Edwardine 
Published: February 2005

AbstractCyclic AMP arrests T lymphocytes in the G1 phase of the cell cycle, and prolonged exposure results in cytolysis. Both of these effects require cyclic AMP‐dependent protein kinase. We recently observed that some S49 mouse T lymphoma cell lines selected for hydroxyurea resistance were not arrested in G1 by cyclic AMP. Further analysis revealed that these cell lines were cyclic AMP‐dependent protein kinase deficient, and conversely, other cyclic AMP‐dependent protein kinase deficient cell lines not selected for hydroxyurea resistance were two‐ to threefold more hydroxyurea resistant. However, hydroxyurea is a specific inhibitor of ribonucleotide reductase and does not inhibit this kinase. We subsequently showed that cyclic AMP‐dependent protein kinase will phosphorylate the M2 but not the M1 subunit of ribonucleotide reductase in vitro, and this phosphorylation will diminish CDP reductase activity. In vivo phosphorylation of M2 occurred under conditions similar to those that generate cell cycle arrest. We conclude that the M2 subunit of ribonucleotide reductase can be a target of cyclic AMP‐dependent protein kinase. The phosphorylated enzyme has diminished activity, and this may play a role in cyclic AMP‐induced lymphocyte cell cycle arrest.
Endocytosis of follicle‐stimulating hormone by ovarian granulosa cells: Analysis of hormone processing and receptor dynamics
Authors: Sanford Jack C. , Batten Bruce E. 
Published: February 2005

AbstractSuspensions of freshly isolated rat granulosa cells were used to study endocytosis and processing of radioiodinated ovine follicle‐stimulating hormone (l‐oFSH) and to analyze the dynamics of its receptor. Ovine FSH was iodinated to a specific activity of 26 μCi/μg as determined by radioreceptor self‐displacement assays with maximum specific binding to excess membrane receptors of 46%. Radiolabeled oFSH was judged biologically equivalent to the unlabeled hormone since l‐oFSH shows saturation‐binding kinetics and stimulates steroidogenesis in a similar dose‐related manner to unlabeled oFSH. Experiments designed to study the extent and time course of degradation involved continuous exposure of isolated granulosa cells to l‐oFSH. Saturation of membrane receptors was achieved within 1.5 h of incubation, and internalization of FSH occurred in a linear manner for up to 6 h. The rate of internalization was equivalent to 2,780 FSH molecules/cell/h. Degradation of FSH became apparent after 6 h of incubation and increased to 86% of total cellular‐associated radioactivity at 22 h. FSH degradation was inhibited by 100 μM chloroquine or 0.45 mM leupeptin. The measurement of cell surface l‐oFSH binding in the combined presence of 100 μM chloroquine and 0.5 mM cycloheximide was unchanged for up to 22 h of incubation. This and other receptor binding data suggest that there is no reutilization of FSH receptors. Scatchard analyses of 4°C binding assays on intact cells indicated that a two‐site model best fit the data with association constants of K11 = 1.44 (±.42) × 1010 and K12 = 4.35 (±.91) × 108. Receptor binding and activation studies for progesterone production yielded ED50S of 270 pM and 7.7 pM, respectively, and also indicated that 20% receptor occupancy is sufficient to stimulate maximal progesterone production. We conclude that after the initial binding event, FSH is endocytosed very slowly and is subsequently shuttled to the lysosomal compartment for degradation. The retarded rate of endocytosis may relate to novel pathways of hormone processing.
In vitro effects of endotoxin on bovine and sheep lung microvascular and pulmonary artery endothelial cells
Authors: Meyrick Barbara , Hoover Richard , Jones Margaret R. , Berry Leonard C. , Brigham Kenneth L. 
Published: February 2005

AbstractA single infusion of Escherichia coli endotoxin into sheep results in structural evidence of pulmonary endothelial injury, increases in both prostacyclin and prostaglandin E2 (PGE2) in lung lymph, and an increase in pulmonary micro‐vascular permeability. Endotoxin‐induced lung endothelial damage can also be induced in vitro, but to date these studies have utilized endothelium from large pulmonary vessels. In the present study, we have grown endothelial cells from peripheral lung vessels of cows and sheep and exposed these microvascular endothelial cells to endotoxin. Controls included lung microvascular endothelium without endotoxin and endothelial cells from bovine and sheep main pulmonary artery with and without addition of endotoxin. We found that endotoxin caused significant increases in release of prostacyclin and PGE2 from both bovine and sheep lung microvascular and pulmonary artery endothelium. Normal bovine and sheep pulmonary artery and bovine lung microvascular endothelium released greater levels of prostacyclin than PGE2 (ng/ng); release of PGE2 from the microvascular cells was greater than from the pulmonary artery endothelium in both species. Exposure of endothelial cells from cow and sheep main pulmonary artery to endotoxin results in endothelial cell retraction and pyknosis, a loss of barrier function, increased release of prostacyclin and PGE2 and eventual cell lysis. In lung microvascular cells, the increases in prostanoids were accompanied by changes in cell shape but occurred in the absence of either detectable alterations in barrier function or cytolysis. Thus, while endotoxin causes alterations to endothelial cells from both large and small pulmonary vessels, the effects are not identical suggesting site specific phenotypic expression of endothelial cells even within a single vessel. To determine whether the response of either the large or small pulmonary vessel endothelial cells in culture mimics most closely the in vivo response of the lung to endotoxin requires further study.
Combinative ligand‐receptor interactions: Effects of CAMP, epinephrine, and met‐enkephalin on RAW264 macrophage morphology, spreading, adherence, and microfilaments
Authors: Petty Howard R. , Martin Susan M. 
Published: February 2005

AbstractCell surface ligand‐receptor interactions play a central role in the regulation and expression of macrophage function. Included among these macrophage membrane receptors are the β‐adrenergic and opioid receptors. We studied the abilities of epinephrine, met‐enkephalin, forskolin, and adenosine 3′:5′ cyclic monophosphate (cAMP) analogues to affect macrophage morphology, spreading, and adherence. Cell spreading was quantitated by measuring the perimeters of adherent cell images recorded by videomicroscopy. Epinephrine induced a dose‐dependent decrease in macrophage spreading; at 10‐5 M epinephrine the mean perimeter was 10.4 ± 0.3μm in comparison to 15.0 ± 1.0 μm for controls. The inhibition of spreading can be blocked by the antagonist propranolol. On the other hand, met‐enkephalin induced a dose‐dependent increase in macrophage spreading, with a perimeter of 18.5 ± 1.0 μm at 10‐8 M. Since catecholamines and opioids are simultaneously released from chromaffin cells of the adrenal, we examined the combinative effects due to treatment with both ligands. When macrophages were exposed to 10‐5 M epinephrine and 10‐8 M met‐enkephalin, cell morphology and spreading were indistinguishable from that due to 10‐5 M epinephrine alone. The epinephrine dose‐response curve in the presence of 10‐8 M met‐enkephalin, was similar to that of epinephrine alone. The β‐adrenergic receptor is apparently capable of diminishing or abrogating the opioid receptor signal(s). These combinative and epinephrine‐mediated effects may be at least partially accounted for by the action of cAMP. Forskolin and the cAMP analogues N6 –2′‐O‐dibutyryladenosine 3′:5′ cyclic monophosphate (dbcAMP) and 8‐bromoadenosine 3′:5′ cyclic monophosphate (Br‐cAMP) affected cell morphology and spreading in the same fashion as epinephrine. These differences in morphology and spreading behavior were accompanied by changes in the distribution of F‐actin, as judged by phalladicin staining and fluorescence microscopy. We suggest that cAMP and microfilaments play important roles in receptor‐mediated neuroregulation of macrophage function.
Ionic dependence of the extracellular ATP‐induced permeabilization of transformed mouse fibroblasts: Role of plasma membrane activities that regulate cell volume
Authors: Weisman Gary A. , De Barun K. , Pritchard Robert S. 
Published: February 2005

AbstractExtracellular ATP rendered the plasma membrane of transformed mouse fibro‐blasts permeable to normally impermeant molecules. This permeability change was prevented by increasing the ionic strength of the isotonic medium with NaCl. Conversely, the cells exhibited increased sensitivity to ATP when the NaCl concentration was decreased below isotonicity, when the KCl concentration was increased above 5 mM while maintaining isotonicity, and when the pH of the medium was raised above 7.0. These conditions as well as the addition of ATP itself caused cell swelling. However, the effect of ATP was independent of cell volume and dependent upon the ionic strength and not the osmolarity of the medium since (1) addition of sucrose to isotonic medium did not prevent permeabilization although media made hypertonic with either sucrose or NaCl caused a decrease in cell volume; and (2) addition of sucrose or NaCl to hypotonic media caused a decrease in cell volume, but only NaCl addition decreased the response to ATP. Conditions that have been shown to inhibit plasma membrane proteins that play a reciprocal role in cell volume regulation had reciprocal effects on the permeabilization process, even though the effect of ATP was independent of cell volume. For example, inhibition of the Na+, K+‐ATPase by ouabain increased sensitivity of cells to ATP while conditions which inhibit Na+, K+, Cl−‐cotransporter activity, such as treatment of the cells with the diuretics furosemide or bumetanide or replacement of sodium chloride in the medium with sodium nitrate or thiocyanate, inhibited permeabilization. The furosemide concentration that inhibited permeabilization was greater than the concentration that inhibited Na+, K+, Cl−‐cotransporter‐mediated 86Rb+ (K+) uptake, suggesting that the effect of furosemide on the permeabilization process may not be specific for the Na+, K+, Cl−‐cotransporter.
Electric fields stimulate DNA synthesis of mouse osteoblast‐like cells (MC3T3‐E1) by a mechanism involving calcium ions
Authors: Ozawa Hiroyuki , Abe Etsuko , Shibasaki Yoshinobu , Fukuhara Tatsuo , Suda Tatsuo 
Published: February 2005

AbstractA cell culture system was designed to study the effect of an electric field on bone cells. This system subjected cultured bone cells to a series of high‐voltage pulses at varying amplitudes of electric field without changing the ion concentrations of the culture medium. When mouse osteoblast‐like cells (MC3T3‐E1) were stimulated for 5 min to 20 hr by an electric field (3.19 kV/m, 3 msec, 10 Hz pulses), DNA synthesis was greatly increased. The stimulation occurred only during the growth phase. The electric field also increased incorporation of 45Ca into the cells, but it did not stimulate cAMP production. Adding calcium ionophore A23187 stimulated both 45Ca uptake and DNA synthesis, but dibutyryl cAMP did not stimulate either of them. These results suggest that the electric field stimulates the DNA synthesis of growing osteoblasts by a mechanism involving calcium ions.