bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression of simple epithelial cytokeratins in bovine pulmonary microvascular endothelial cells
Authors: Patton Wayne F. , Yoon Min Ung , Alexander J. Steven , Chung‐Welch Nancy , Hechtman Herbert B. , Shepro David 
Published: February 2005

AbstractPolypeptides of bovine aortic, pulmonary artery, and pulmonary microvascular endothelial cells, as well as vascular smooth muscle cells and retinal pericytes were evaluated by two‐dimensional gel electrophoresis. The principal cytoskeletal proteins in all of these cell types were actin, vimentin, tropomyosin, and tubulin. Cultured pulmonary microvascular endothelial cells also expressed 12 unique polypeptides including a 41 kd acidic type 1 and two isoforms of a 52 kd basic type II simple epithelial cytokeratin. Pulmonary microvascular endothelial cell expression of the simple epithelial cytokeratins was maintained in culture in the presence or absence of retinal‐derived growth factor, and regardless of whether cells were cultured on gelatin, fibronectin, collagen I, collagen IV, laminin, basement membrane proteins, or plastic. Cytokeratin expression was maintained through at least 50 population doublings in culture. The expression of cytokeratins was found to be regulated by cell density. Pulmonary microvascular endothelial cells seeded at 2.5 × 105 cells/cm2 (confluent seeding) expressed 3.5 times more cytokeratins than cells seeded at 1.25 × 104 cells/cm2 (sparse seeding). Vimentin expression was not altered by cell density. By indirect immunofluorescence microscopy it was determined that the cytokeratins were distributed cytoplasmically at subconfluent cell densities but that cytokeratin 19 sometimes localized at regions of cell‐cell contact after cells reached confluence. Vimentin had a cytoplasmic distribution regardless of cell density. These results suggest that pulmonary microvascular endothelial cells have a distinctive cytoskeleton that may provide them with functionally unique properties when compared with endothelial cells derived from the macrovasculature. In conjunction with conventional endothelial cell markers, the presence of simple epithelial cytokeratins may be an important biochemical criterion for identifying pulmonary microvascular endothelial cells.
Adenosine metabolism in kidney slices under normoxic conditions
Authors: Blanco Julià , Mallol Josefa , Lluis Carmen , Canela Enric I. , Franco R. 
Published: February 2005

AbstractThe effect of adenosine in rat kidney under normoxic conditions has been studied. It is demonstrated that adenosine modulates cell nucleotide levels. HPLC analysis of the purine compounds inside the cell indicates that adenosine improves the ATP/ADP ratio, whereas it diminishes the adenine content. This behaviour is not due to mediation by specific receptors, as agonists at P1 purinoceptors did not have any effect. Further evidence using inosine as well as dipiridamole and deoxycoformycin indicates that all effects are dependent on the previous uptake of adenosine. The origin of free adenine in the kidney has been investigated, and it appears to come from the phosphorolysis of 5′‐methylthio‐adenosine. This report is the first to describe the activity of methylthioadenosine phosphorylase (E.C. 2.4.2.28) in the kidney. It is concluded that 1) extracellular adenosine improves guinea pig renal function by increasing the ATP level and the ATP/ADP ratio; and 2) there exists a functional pathway in the kidney that produces adenine and AMP coming from methionine and ATP. This latter pathway probably produces spermine and spermidine, which are likely to be important for renal function.
Effect of transforming growth factor beta on proliferation of L6 and embryonic porcine myogenic cells
Authors: Pampusch Mary S. , Hembree Joan R. , Hathaway Marcia R. , Dayton William R. 
Published: February 2005

AbstractWe have examined the effect of T‐ansforming Growth Factor (TGF) beta on proliferation of L6 and embryonic porcine myogenic cells. Proliferation of L6 cells was suppressed by both TGF beta‐1 and TGF beta‐2 in a dose‐dependent manner. Half‐maximal suppression of proliferation occurred at.036 ng TGF beta‐1/ml and.06 ng TGF beta‐2/ml. Maximal inhibition (60% suppression of proliferation for TGF beta‐1 and 52% for TGF beta‐2) occurred between.1 and.3 ng/ml for each growth factor. Suppression of proliferation was completely abolished in the presence of an anti‐TGF beta antibody that inhibited the biological activity of TGF beta‐1 and TGF beta‐2. When we evaluated the effect of TGF beta‐1 on proliferation of embryonic porcine myogenic cells we obtained results which were very similar to those obtained for L6 cells. Insulin‐like growth factor (IFG)‐I stimulated proliferation of L6 cells in a dose‐dependent manner in serum‐free, defined medium. However as little as.02 ng TGF beta‐1/ml detectably suppressed this stimulation and.3 ng TGF beta‐1/ml caused a 60% reduction in cell number in cultures treated with 30 ng IGF‐1/ml. Thus TGF beta‐1 significantly suppressed IGF‐l‐stimulated proliferation of L6 cells.
Effect of epidermal growth factor on the expression of protooncogenes c‐myc and c‐Ha‐ras in short‐term primary hepatocyte culture
Authors: Kost Diane P. , Michalopoulos George K. 
Published: February 2005

AbstractWe have characterized the effect of the hepatomitogen epidermal growth factor (EGF) on the expression of the cellular protooncogenes c‐Ha‐ras and c‐myc in short‐term (48 hours) primary hepatocyte culture. mRNA concentrations of both protooncogenes increased dramatically in nonproliferating cultures and in the absence of EGF, suggesting that the isolation procedure or the culture conditions may trigger expression of these genes or potentially increase the lifetime of transcripts in vitro, regardless of the presence of a mitogen. In cells treated with EGF, a distinct peak in c‐Ha‐ras expression was seen 24 hours after EGF treatment. This coincided with the onset of DNA synthesis. No such peak was seen in cultures not treated with EGF. The c‐myc mRNA concentrations were increased relatively equally in all cultures with or without the addition of EGF. These data show a differential response of these two cell‐cycle‐associated genes to the culture conditions and EGF stimulation. It also demonstrated that enhanced gene expression for Ha‐ras and myc in hepatocytes can occur in the absence of cell proliferation.
Insulin‐Like growth factor I and protein kinase C activation stimulate pulmonary artery smooth muscle cell proliferation through separate but synergistic pathways
Authors: Dempsey Edward C. , Stenmark Kurt R. , McMurtry Ivan F. , O'Brien Richard F. , Voelkel Norbert F. , Badesch David B. 
Published: February 2005

AbstractSmooth muscle cell (SMC) hyperplasia is an important component of vascular remodeling in chronic hypoxic pulmonary hypertension. The mechanisms underlying SMC proliferation in the remodeling process are poorly understood, but may involve insulin‐like growth factor I (IGF‐I). This study investigates the potential proliferative effects of IGF‐I on SMC cultured from the pulmonary arteries (PA) of neonatal calves. We hypothesized that IGF‐I stimulates PA SMC proliferation through a protein kinase C (PKC)‐indepenent pathway, but that PKC activation would augment this proliferative response. Incorporation of 3H‐thymidine was used as an index of cellular prohteration, and was correlated with subsequent changes in cell counts. Under serum‐free conditions, IGF‐I (100 ng/ml) induced a 6‐fold increase in thymidine incorporation by quiescent PA SMC. This stimulation was not blocked by dihydrosphingosine, an inhibitor of PKC activation. Phorbol myristate acetate (PMA) (1 nM), a membrane‐permeable PKC activator, induced a 12‐fold increase in thymidine incorporation which was 70% inhibited by dihydrosphingosine. Co‐incubation with IGF‐I and PMA caused a 60‐fold increase in thymidine incorporation, which was 30% inhibited by dihydrosphingosine. This synergistic increase in thymidine incorporation was associated with a subsequent significant increase in cell number. PKC‐downregulated cells (1,000 nM PMA × 30 hr) proliferated in response to IGF‐I but not PMA, and did not demonstrate synergism with the combination of IGF‐I and PMA. The threshold concentrations of IGF‐I and PMA for synergism were approximately 1 ng/ml and 1 pM, respectively. We conclude that IGF‐I stimulates neonatal PA SMC proliferation via a PKC‐independent pathway, and that trace amounts of PKC activators are capable of synergistically augmenting this response. We speculate that the synergistic stimulation of SMC proliferation by IGF‐I and PKC activators may play an important role in hypertensive pulmonary vascular remodeling.
Comparative study of oxygen toxicity in human fibroblasts and endothelial cells
Authors: Michiels Carine , Toussaint Olivier , Remacle José 
Published: February 2005

AbstractThe resistance of human pulmonary fibroblasts (WI‐38) and human umbilical vein endothelial cells to oxygen toxicity (1 atm O2) was compared. Endothelial cells were more sensitive than fibroblasts. They contained also less antioxidant enzymes except for SOD: respectively 132%, 96%, 70%, 59%, and 21% of the SOD, GSH peroxidase, GSH reductase, catalase, and G6PD content of fibroblasts. However, they contained 1.81‐fold more GSH than fibroblasts. Their lower content of antioxidant enzymes can explain their higher sensitivity to oxygen. The efficiency of natural antioxidant molecules and enzymes in the protection of cells incubated 3 days under 1 atm O2 was studied. α‐tocopherol added in the culture medium led to a significant protection, contrary to the result for ascorbic acid. Microinjection of catalase, SOD, and GSH peroxidase directly into the cells was also tested: the protection was concentration dependent for both types of cells but SOD did not protect the endothelial cells. Lower activities of the other enzymes were needed to achieve protection of the endothelial cells, compared to fibroblasts. Since endothelial cells were also shown to display lower antioxidant enzyme activities, it can be hypothesized that their content is optimized for survival in Physiological conditions.
Transforming growth factor β enhances parathyroid hormone stimulation of adenylate cyclase in clonal osteoblast‐like cells
Authors: Gutierrez Gloria E. , Mundy Gregory R. , Katz Michael S. , Manning David R. , Hewlett Erik L. 
Published: February 2005

AbstractThe effects of transforming growth factor β (TGFβ) on parathyroid hormone (PTH)‐responsive adenylate cyclase were examined in clonal rat osteosarcoma cells (UMR‐106) with the osteoblast phenotype. Purified TGFβ incubated with UMR‐106 cells for 48 hr produced a concentration‐dependent increase in PTH stimulation of adenylate cyclase, with maximal increase in PTH response (37%) occurring at 1 ng/ml TGFβ. TGFβ also enhanced receptor‐mediated activation of adenylate cyclase by isoproterenol and prostaglandin E2 (PGE2) and nonreceptormediated enzyme activation by cholera toxin and forskolin. In cells in which PTH‐stimulated adenylate cyclase activity was augmented by treatment with pertussis toxin, the incremental increase in PTH response produced by TGFβ was reduced by 33%. However, TGFβ neither mimicked nor altered the ability of pertussis toxin to catalyze the ADP‐ribosylation of a 41, 000‐Da protein, presumably the α subunit of the inhibitory guanine nucleotide‐binding regulatory component (Gi) of adenylate cyclase, in cholate‐extracted UMR‐106 cell membranes. TGFβ also had no effect on the levels of α or β subunits of Gi, as assessed by immunotransfer blotting. In time course studies, brief (≥ 30 min) exposure of cells to TGFβ during early culture was sufficient to increase PTH response but only after exposed cells were subsquently allowed to grow for prolonged periods. TGFβ enhancement of PTH and isoproterenol responses was blocked by prior treatment of cells with cycloheximide but not indomethacin. The results suggest that TGFβ enhances PTH response in osteoblast‐like cells by action(s) exerted at nonreceptor components of adenylate cyclase. The effect of TGFβ may involve Gi, although in a manner unrelated to either pertussis toxin‐catalyzed ADP‐ribosylation of the α subunit of Gi or changes in levels of Gi subunits. The regulatory action of TGFβ on adenylate cyclase is likely to be mediated by the rapid generation of cellular signals excluding prostaglandins, followed by a prolonged sequence of events involving protein synthesis. These observations suggest a mechanism by which TGFβ may regulate osteoblast responses to systemic hormones.
Anti‐proliferative activity of L‐651,582 correlates with calcium‐mediated regulation of nucleotide metabolism at phosphoribosyl pyrophosphate synthetase
Authors: Hupe Donald J. , Behrens Nancy D. , Boltz Robert 
Published: February 2005

AbstractL‐651,582, 5‐amino‐[4‐(4‐chlorobenzoyl)‐3, 5‐dichlorobenzyl]‐1, 2, 3‐triazole‐4‐carboxamide, is an antiproliferative and antiparasitic agent which inhibits nucleotide metabolism in mammalian cells. The drug equivalently inhibited 3H‐hypoxanthine, 14C‐adenine, and 14C‐formate incorporation into nucleotide pools in Madin‐Darby bovine kidney (MDBK) cells, suggesting depletion of the supply of phosphoribosyl pyrophosphate, (PRPP), required for each of these independent pathways. Inhibition of nucleotide metabolism correlated with inhibition of proliferation for three cell types with differing sensitivities toward the drug. L‐651, 582 inhibited incorporation of 3H‐hypoxanthine into nucleotide pools with either glucose, uridine, or ribose as carbon source suggesting a block at PRPP synthetase, rather than a block in a pathway supplying ribose‐5‐phosphate. PRPP synthetase was not inhibited directly by the compound, indicating a regulation of the enzyme in intact cells. Drug treatment did not kill cells but reduced the fraction of cells in S and G2/M while increasing the population in G1. Inhibition of uptake of 45Ca was demonstrated at concentrations identical to those required for inhibition of nucleotide metabolism or proliferation. Inhibition of cellular PRPP biosynthesis rates were also observed using EGTA to lower calcium levels. These data suggest a previously unrecognized link between calcium entry, the regulation of nucleotide biosynthesis at PRPP synthetase, and the rate of proliferation of mammalian cells.
Distribution of vinculin in the Z‐disk of striated muscle: Analysis by laser scanning confocal microscopy
Authors: Terracio Louis , Simpson David G. , Hilenski Lula , Carver Wayne , Decker Robert S. , Vinson Nancy , Borg Thomas K. 
Published: February 2005

AbstractVinculin is a major cytoskeletal component in striated muscle, where it has been reported to form a rib‐like structure between the cell membrane and the Z‐disk termed a costamere. This arrangement of vinculin has been purported to be involved in the alignment of the myofibrils. However, the three‐dimensional arrangement of vinculin in relation to the Z‐disk of the myofibril was not known. In the present study, we examined the distribution of vinculin in striated muscle with monospecific antibodies using immunofluorescence and laser scanning confocal microscopy. Isolated cardiac and skeletal muscle cells from a variety of species, tissue sections, and neonatal myocytes with developing myofibrils were examined. Optical sectioning in the X‐Y and X‐Z planes demonstrated that vinculin immunoreactivity was heaviest at the periphery of the cell; however, the immunoreactivity was also distributed within the Z‐disk although at a relatively reduced level. This distribution is potentially significant in understanding the physiological significance of vinculin in striated muscle function and in myofi‐brillogenesis.
[D‐Arg1, D‐Phe5,D‐Trp7,9,Leu11] substance P, a neuropeptide antagonist, blocks binding, Ca2+‐mobilizing, and mitogenic effects of endothelin and vasoactive intestinal contractor in mouse 3T3 cells
Authors: Fabregat Isabel , Rozengurt Enrique 
Published: February 2005

AbstractEndothelin (ET1) and vasoactive intestinal contractor (VIC) stimulate quiescent Swiss 3T3 cells to resume DNA synthesis acting synergistically with epidermal growth factors (EGF) and other mitogens. The peptide “D‐Arg1‐D‐Phe5,D‐Trp7,9Leu11” substance P has been identified as a broad spectrum neuropeptide antagonist which blocks the binding and biological effects of the Ca2+‐mobilizing neuropeptides bombesin, vasopressin, and bradykinin. In the present study we show that [D‐Arg1,D‐Phe5,D‐Trp7,9,Leu11] substance P also acts as an ET1/VIC antagonist as judged by the following criteria: (a) inhibition of specific 125l‐labelled ET1 binding to a ET1/VIC receptor in a competitive and dose‐dependent manner; (b) blocking of the rapid increase in the cytosolic Ca2+ concentration promoted by ET1 or VIC; and (c) inhibition of DNA synthesis stimulated by VIC in the presence of EGF. The inhibitory effects of [D‐Arg1,D‐Phe5,D‐Trp7,9,Leu11] substance P on Ca2+ mobilization and DNA synthesis were reversed by increasing the concentration of VIC. This is the first time that a peptide structurally unrelated to ET1 or VIC is shown to block the binding and mitogenic effects of peptides of the endothelin family.
Vitamin D regulates transferrin receptor expression by bone marrow macrophage precursors
Authors: Tanaka Hiroyuki , Teitelbaum Steven L. 
Published: February 2005

Abstract1,25‐Dihydroxyvitamin D3 [1,25(OH)2D3] is known to prompt monocytic differentiation of a variety of leukemic lines. We previously extended these observations to non‐transformed bone marrow macrophage precursors by demonstrating that the steroid enhances plasma membrane expression of the macrophage‐specific mannose‐fucose receptor (Clohisy et al., J Biol Chem 262:15922–15929, 1987). Because this membrane protein is involved in non‐opsonin mediated endocytosis, these observations raised the possibility that 1,25(OH)2D3 globally upregulates endocytic receptors. The present study, aimed at addressing this issue, turns to the transferrin receptor as a paradigm for endocytic receptors and explores the impact of 1,25(OH)2D3 on its expression. We found that in contrast to the mannose‐fucose receptor, plasma membrane transferrin receptor expression by bone marrow‐derived macrophage precursors declines by at least 30% in a dose‐dependent fashion with exposure to 1,25(OH)2D3. The effect reflects diminished receptor capacity with no change in Kd, and is independent of cell cycle. Moreover, while Vmax of receptor‐ligand internalization mirrors plasma membrane occupancy, Kuptake remains unaltered in the presence of vitamin D3, indicating that the down‐regulating event does not reflect on enhanced rate of endocytosis. Further, pulse chase experiments show parallel cell surface, intracellular, and medium redistribution of radioligand with time steroid‐treated and control cells. In a similar vein, while total cell‐associated radioligand falls in the presence of vitamin D3, the percentage of intracellular and surface bound counts at equilibrium are constant in both groups. Finally, immunoprecipitation studies reveal that the down‐regulating effects of 1,25(OH)2D3 cannot be explained by inhibition of transferrin receptor synthesis. Thus, the decrease in total cellular transferrin binding sites is likely to represent either enhanced degradation or synthesis of [cryptic] receptors which fail to recognize 125 I‐transferrin.
Studies on the spermatogenic sulfogalactolipid binding protein SLIP 1
Authors: Lingwood Clifford , Nutikka Anita 
Published: February 2005

AbstractWe have purified the testicular sulfogalactolipid binding protein SLIP 1 and shown by photoaffinity labeling that it contains an ATP binding site. Purified SLIP 1 was fluorescently labeled and shown to retain specific Sulfogalactolipid binding function. This probe was used to investigate the topology of SLIP I binding sites on testicular germ cells. The binding pattern precisely coincided with the previously demonstrated asymmetric surface domains of sulfogalactoglycerolipid (SGG). Occasionally these SGG‐containing, SLIP 1‐binding cell surface domains exactly coincided with structural features on the cell surface as detected by differential interference contrast microscopy. These results demonstrate that SLIP 1/SGG interactions could provide an effective intercellular communication network between testicular germ cells within the seminiferous tubule.
Effects of adenosine receptor agonists on volume‐activated ion transport in pig red cells
Authors: Sohn Dong Hwan , Kim Hyun Dju 
Published: February 2005

AbstractSwelling of pig red cells leads to an increase in a chloride‐dependent K flux which can be potentiated by cAMP, whereas cell shrinking causes a selective increase in Na movement which is mediated by a Na/H exchanger. We examined the influence of adenosine and adenosine receptor agonists on the volume‐sensitive, ouabain‐resistant, chloride‐dependent K flux, referred to as Rb flux and volume‐activated Na/H exchange pathway. It was found that adenosine and adenosine receptor agonists inhibited the Rb flux. N6‐cyclohexyl adenosine (CHA) has been found to be the most potent inhibitor with EC50 of approximately 4.5 μM followed by 2‐chloroadenosine (Cl‐ado) with EC50 of approximately 27 μM and 5′‐(N‐ethyl)‐carboxamido‐adenosine (NECA) with EC50 of approximately 185 μM. CHA also inhibits the cAMP‐stimulated Rb flux. However, CHA does not alter the basal intracellular cAMP level nor the intracellular cAMP content raised by exogenously added cAMP. In contrast to the adenosine agonist action on the Rb flux, Na/H exchange, which is activated upon cell shrinkage, exhibits a slight stimulation in response to CHA. These findings suggest that the presence of A1 adenosine receptors on the surface of red cells influences the regulation of volume‐activated ion transport.
Dexamethasone induction of osteoblast mRNAs in rat marrow stromal cell cultures
Authors: Leboy Phoebe S. , Beresford Jon N. , Devlin Carole , Owen Maureen E. 
Published: February 2005

AbstractWe have examined the ability of dexamethasone, retinoic acid, and vitamin D3 to induce osteogenic differentiation in rat marrow stromal cell cultures by measuring the expression of mRNAs associated with the differentiated osteoblast phenotype as well as analyzing collagen secretion and alkaline phosphatase activity. Marrow cells were cultured for 8 days in primary culture and 8 days in secondary culture, with and without 10 nM dexamethasone or I μM retinoic acid. Under all conditions, cultures produced high levels of osteonectin mRNA. Cells grown with dexamethasone in both primary and secondary culture contained elevated alkaline phosphatase mRNA and significant amounts of type I collagen and osteopontin mRNA. Addition of 1,25‐dihydroxyvitamin D3 to these dexamethasone‐treated cultures induced expression of osteocalcin mRNA and increased osteopontin mRNA. The levels of alkaline phosphatase, osteopontin, and osteocalcin mRNAs in Dex/Dex/VitD3 cultures were comparable to those of 1,25‐dihydroxyvitamin D3‐treated ROS 17/2.8 osteosarcoma cells. Omitting dexamethasone from either primary or secondary culture resulted in significantly less alkaline phosphatase mRNA, little osteopontin mRNA, and no osteocalcin mRNA. Retinoic acid increased alkaline phosphatase activity to a greater extent than did dexamethasone but did not have a parallel effect on the expression of alkaline phosphatase mRNA and induced neither osteopontin or osteocalcin mRNAs. In all conditions, marrow stromal cells synthesized and secreted a mixture of type I and III collagens. However, dexamethasone‐treated cells also synthesized an additional collagen type, provisionally identified as type V. The synthesis and secretion of collagens type I and III was decreased by both dexamethasone and retinoic acid. Neither dexamethasone nor retinoic acid induced mRNAs associated with the chondrogenic phenotype. We conclude that dexamethasone, but not retinoic acid, promotes the expression of markers of the osteoblast phenotype in cultures of rat marrow stromal fibroblasts.