bluuebunny/crossref_metadata_2025_split huggingface dataset:
Phorbol dibutyrate‐specific protein phosphorylation in brush border membranes of chicken enterocytes
Authors: Toskulkao Chaivat , Bhartur Sheela , Musch Mark W. , Chang Eugene B. , Rao Mrinalini C 
Published: February 2005

AbstractWe have demonstrated that phorbol esters such as phorbol dibutyrate (PhE) transiently inhibit Na/H exchange both in intact avian enterocytes and in brush border membrane (BBM) vesicles prepared from enterocytes treated with PhE (Chang et al., 1991, Am. J. Physiol. 260: C1264–C1272). Maximal inhibition occurs at 90 sec and values return to baseline by 15 mm. In this study we examined if PhE causes changes in BBM protein phosphorylation by two methods: (1) in situ phosphorylation in which intact cells prelabeled with 32Pi were treated with PhE; (2) in vitro phosphorylation in which BBM, isolated from untreated and PhE‐treated enterocytes, were exposed to γ32P‐ATP. In situ phosphorylation studies showed that, at 90 sec, PhE increases the phosphorylation of BBM proteins of Mr (pl): 150 (6.5), 89 (≈6.2), and 48 (≈6.1) kDa which declined to control values at 15 min, suggesting that these may be transport‐related substrates. These labeled substrates were recovered in the detergent‐insoluble fraction after extraction with 0.1% Triton X‐100 overnight. Transient phosphorylation of a number of proteins was also observed when BBM prepared from control or PhE‐treated cells were incubated with γ 32P‐ATP ± 10 nM PhE, phosphatidyl serine, Ca2+, and/or exogenous protein kinase C (PKC). The in vitro phosphoproteins included both Triton‐soluble and Triton‐insoluble proteins. However, none of these proteins labeled in vitro coincided with those labeled in situ. The decline in phosphorylation with time can be accounted for by phosphatase action as these BBM possess a Ca‐dependent phosphatase. In summary, we have demonstrated that the BBM possess PKC‐specific substrates which can be visualized by in situ and in vitro phosphorylation. Treatment of intact enterocytes with PhE results in the phosphorylation of three detergent‐insoluble proteins with a time course similar to that of PhE inhibition of Na/H transport. © 1994 wiley‐Liss, Inc.
Sulfation of Guinea pig megakaryocyte and platelet proteins
Authors: Schick Barbara P. , Jacoby Jennifer A. 
Published: February 2005

AbstractThis study has explored the sulfation of proteins by guinea pig megakaryocytes and platelets and by human platelets. Guinea pig megakaryocytes were incubated in vitro with [35S]sulfate, and the sulfated proteins were separated from proteoglycans by DEAE‐Sephacel chromatography and analyzed by SDS‐PAGE. The megakaryocytes esterified sulfate to a number of proteins, with the most extensive label migrating at Mr 42,000, and a second heavily labeled band at Mr 103,000 in the 0.1 M NaCl DEAE eluate, and 50 and 180 kDa in the 0.23 M NaCl eluate. [35S]‐Labeled GPIbα was immunoprecipitated from megakaryocyte Triton X‐100 extracts. Guinea pig platelet proteins were labeled in vivo by injection of the animals with a single dose of H235SO4. The platelets were activated with thrombin, and cytoskeletal proteins were isolated after treatment of the activated platelets with Triton X‐100. About 20% of the platelet macromolecule‐associated [35S]sulfate was incorporated into sulfated proteins, which were recovered primarily in the cytoskeleton. The cytoskeleton‐associated sulfate radiolabel migrated on SDS‐PAGE primarily with actin and additionally with several higher molecular weight proteins. A Mr 42,000 [35S]‐labeled protein was immunoprecipitated by a monoclonal anti‐actin antibody, along with molecules of Mr 160,000 and 180,000 and some higher Mr material, from the megakaryocytes labeled in vitro with [35S]sulfate. Actin was labeled on 2D isoelectric focusing/SDS‐PAGE gels. In addition, there was a very acidic series of heavily [35S]‐labeled 42 kDa proteins with about eight components of different isoelectric points with a pattern identical to the Mr 40,000 cytoskeletal‐associated glycoprotein Pltpg40 isolated by Hildreth et al. (1991, Blood 77:121). We hypothesize that sulfation of the cytoskeletal proteins might be involved in cytoskeletal protein interactions and function. © 1994 wiley‐Liss, Inc.
Binding of hyaluronan to plasma fibronectin increases the attachment of corneal epithelial cells to a fibronectin matrix
Authors: Nakamura Masatsugu , Mishima Hiroshi , Nishida Teruo , Otori Toshifumi 
Published: February 2005

AbstractWe wished to determine whether hyaluronan would affect the attachment of epithelial cells to extracellular matrix proteins. Multiwell tissue culture plates were coated with human plasma fibronectin, laminin, or collagen type IV (0.01–10.0 μg/ml). Single‐cell suspensions of rabbit corneal epithelial cells were placed in the wells, and after 45 minutes incubation the cells adhering to the matrix proteins were stained and counted. Cells attached to all three types of proteins. Preincubation of the matrix proteins with hyaluronan (0.1–1.0 mg/ml) significantly increased the number of cells attached to the fibronectin matrix, but it did not increase the numbers of cells attached to laminin or collagen type IV. Hyaluronidase inhibited this stimulatory effect. Glycosaminoglcyans other than hyaluronan (chondroitin sulfate, keratan sulfate, or heparan sulfate) failed to increase the numbers of attached cells. Treatment of the fibronectin matrix with monoclonal antibodies against the cell‐binding domain of fibronectin (FN12–8 or FN30–8, 0.03–0.3 mg/ml, for 1 hour), before or after hyaluronan treatment, significantly decreased the numbers of attached cells. Monoclonal antibody against the fibrin‐ and heparin‐binding domain at the N‐terminal (FN9–1), however, significantly decreased the number of attached cells only when this antibody treatment preceded the hyaluronan treatment. Preincubation of the cells with hyaluronan had no effect; preincubation with GRGDSP (1 mg/ml), a synthetic peptide that blocks the cell surface receptor for fibronectin, significantly decreased cell attachment whether the fibronectin matrix was treated with hyaluronan or not. Further studies demonstrated that monoclonal antibody against the fibrin‐ and heparin‐binding domain at the N‐terminal of plasma fibronectin prevented radiolabeled hyaluronan from binding to fibronectin; likewise, the isolated N‐terminal fragment, coupled with Sepharose 4B, bound to hyaluronan in columns. We conclude that hyaluronan binds to a fibrin‐ and heparin‐binding domain at the N‐terminal of plasma fibronectin and facilitates the attachment of epithelial cells. © 1994 wiley‐Liss, Inc.
Modulation of keratin 14 and α‐fetoprotein expression during hepatic oval cell proliferation and liver regeneration
Authors: Bisgaard Hanne Cathrine , Nagy Ton Peter Phuongnga T. , Hu Zongyi , Thorgeirsson Snorri S. 
Published: February 2005

AbstractKeratin 14 (K14) expression has recently been demonstrated in cell lines of non‐parenchymal hepatic origin (Bisgaard et al., 1993, Mol. Carcinog., 7:60–66; Bisgaard et al., 1991, J. Cell. Physiol., 147:333–343). These cell lines are thought to represent a progeny of a dormant stem cell compartment present in the adult rat liver, which may participate in the restoration of the liver mass after experimental liver injury. Utilizing a combination of 2‐acetylaminofluorene (2‐AAF) administration and partial hepatectomy to activate liver regeneration by proliferation of oval cells, we examined the modulation of K14 as well as α‐fetoprotein (AFP) expression in proliferating oval cells and lineages hypothesized to be derived herefrom. We showed by Northern blot and in situ hybridization analyses that K14 and AFP transcripts were initially accumulating in epithelial cells located in subsets of ductal structures in the portal areas. As oval cells infiltrated the liver parenchyma, K14 transcripts were detected in oval cells, in foci of small basophilic hepatocytes, and in structures resembling glandular intestinal‐type epithelium. AFP was expressed in oval cells, and at low but detectable levels in foci of basophilic hepatocytes, but not in glandular intestinal‐type epithelium. Neither K14 nor AFP transcripts were detected in bile ducts or mature hepatocytes at any time during oval cell proliferation and reconstitution of the liver mass. To further study the modulation of K14 and AFP expression we utilized an in vitro model in which spontaneous transformation of rat liver epithelial (RLE) cells appeared to mimic the process of early differentiation along the hepatic lineage in vivo. We demonstrated that undifferentiated RLE cells at a late passage expressed K14 and vimentin, whereas transformation and differentiation to hepatoblast‐like progeny resulted in an abrogation of K14 and vimentin expression and an induction of K18 and AFP. We propose that K14 and AFP are sequentially modulated in subpopulations of oval cells involved in the ongoing reconstitution of the liver mass. © 1994 wiley‐Liss, Inc.
Expression of calmodulin and calmodulin binding proteins in lymphoblastoid cells
Authors: Colomer Josep , Agell Neus , Engel Pablo , Bachs Oriol 
Published: February 2005

AbstractCalmodulin is encoded in vertebrates by three different genes: CALM1, CALM2, and CALM3. We have examined the mRNAs expressed from these three genes in eight lines of human lymphoblastoid cells (Namalwa, Raji, Ramos, JY, Molt‐4, Jurkat, CEM, and HPB‐ALL). We found that all these cell lines (except Ramos) overexpressed CALM3 transcripts, which led to an increase of total CaM protein with respect to quiescent normal I lymphocytes. The nuclear concentration of calmodulin was measured in two of these lymphoblastoid cell lines (JY and HPB‐ALL) and compared to quiescent and phytohemagglutinin‐activated T lymphocytes. Activated lymphocytes showed a 2‐fold increase of nuclear calmodulin with respect to quiescent cells, whereas in the two lymphoblastoid cell lines, nuclear calmodulin remained similar to that of quiescent cells. The levels of a calmodulin‐binding protein of 150 kDa in the homogenates of the eight lymphoblastoid lines was found to be higher than those of quiescent and activated lymphocytes. Likewise, the amount of three calmodulin‐binding proteins of 240, 200, and 170 kDa was also increased in several of the cell lines, but not in all of them. The 170‐kDa protein was only expressed by activated lymphocytes and lymphoblastoid cells, suggesting that it could be specific for proliferating cells. In the nuclei of activated lymphocytes and lymphoblastoid cells, a decrease of a calmodulin‐binding protein of 110 kDa and increases of three other of 240, 180 and 170 kDa were also detected. © 1994 wiley‐Liss, Inc.
Serum‐Induced vascular smooth muscle cell elastolytic activity through tyrosine kinase intracellular signalling
Authors: Kobayashi Jun , Wigle Dennis , Childs Tim , Zhu Li , Keeley Fred W. , Rabinovitch Marlene 
Published: February 2005

AbstractIn previous studies, we related increased elastolytic activity in pulmonary arteries (PA) with endothelial injury to the later development of PA hypertension in rats. As the mechanism causing the incrased PA elastase was unknown, we hypothesized that serum factors which are accessible to vascular smooth muscle cells (SMC) following endothelial injury stimulate their elastolytic activity. To test this, we developed an in vitro assay in which we added [3H]‐elastin to cultured vascular SMC after 24 h serum starvation and monitored elastolysis following a further 24 h incubation with fetal bovine serum (FBS). We observed that serum induced increased elastolytic activity in both PA and aorta‐derived SMC but not in endothelial cells or SMC with low basal levels of elastolytic activity. Maximum stimulation of SMC elastolytic activity occurred with a concentration as low as 1% FBS and despite elastase inhibitors in serum, suggesting that the activity is confined to the immediate pericellular region where enzyme concentration is high. Serum‐stimulated elastolytic activity was not reproduced by growth factors or cytokines known to be associated with vascular disease or to induce release of elastases in other cells. The serum inducing elastolytic activity was heat and acid labile. It was associated with increased elastin adhesion to the 67 kD elastin binding protein on SMC surfaces and was prevented by tyrosine kinase inhibitors but not protein kinase C or A inhibitors. Our studies therefore suggest a mechanism whereby serum induction of SMC elastase requires signalling through the elastin binding protein and activation of tyrosine kinase. © 1994 Wiley‐Liss, Inc.
Formation of tight junctions and desmosomes protects MDCK cells against hyperthermic killing
Authors: Ning Shoucheng , Hahn George M. 
Published: February 2005

AbstractCell density is known to modify the survival of mammalian cells exposed to elevated temperatures. We have examined the role that cell–cell contact plays in this phenomenon. The formation of cell–cell contact is carried out by cells' junctional complex, i.e., tight junctions, desmosomes, and gap junctions. Lack of formation of tight junctions and desmosomes, or their opening, could interfere with the functions and structures of cell membrane. Membrane damage is at least partially responsible for cell death at elevated temperatures. MDCK cells with high density plated in low calcium medium form confluent monolayers devoid of the formation of tight junctions and desmosomes but quickly assemble them after Ca2+restoration. We used MDCK cells and the calcium switch technique to investigate effects of cell–cell contact and, independently, of cell density on hyperthermic cell killing. We found that MDCK cells that formed tight junctions and desmosomes were more resistant to hyperthermic treatment than those that did not. Blocking the formation pathway of tight junctions made cells sensitive to heat. Cells growing at lowdensity showed almost the same survival as did cells at high density in the absence of the formation of tight junctions and desmosomes. The results suggest that the formation of tight junctions and desmosomes play a more important role in determining hyperthermic response than does density per se. The formation of tight junctions and desmosomes appears to protect cells modestly against hyperthermic killing. © 1994 Wiley‐Liss, Inc.
TGFβ alters growth and differentiation related gene expression in proliferating osteoblasts in vitro, preventing development of the mature bone phenotype
Authors: Breen Ellen C. , Ignotz Ronald A. , McCabe Laura , Stein Janet L. , Stein Gary S. , Lian Jane B. 
Published: February 2005

AbstractThis study examines the mechanism by which TGF‐β1, an important mediator of cell growth and differentiation, blocks the differentiation of normal rat diploid fetal osteoblasts in vitro. We have established that the inability for pre‐osteoblasts to differentiate is associated with changes in the expression of cell growth, matrix forming, and bone related genes. These include histone, jun B, c‐fos, collagen, fibronectin, osteocalcin, alkaline phosphatase, and osteopontin. Morphologically, the TGF‐β1‐treated osteoblasts exhibit an elongated, spread shape as opposed to the characteristic cuboidal appearance during the early stages of growth. This is followed by a decrease in the number of bone nodules formed and the amount of calcium deposition. These effects on differentiation can occur without dramatic changes in cell growth if TGF‐β1 is given for a short time early in the proliferative phase. However, continuous exposure to TGF‐β1 leads to a bifunctional growth response from a negative effect during the proliferative phase to a positive growth effect during the later matrix maturation and mineralization phases of the osteoblast developmental sequence. Extracellular matrix genes, fibronectin, osteopontin and α1(I) collagen, are altered in their expression pattern which may provide an aberrant matrix environment for mineralization and osteoblast maturation and potentiate the TGF‐β1 response throughout the course of osteoblast differentiation. The initiation of a TGF‐β1 effect on cell growth and differentiation is restricted to the proliferative phase of the culture before the cells express the mature osteoblastic phenotype. Second passage cells that are accelerated to differentiate by the addition of dexamethasone or by seeding cultures at a high density are refractory to TGF‐β1. These in vitro results indicate that TGF‐β1 exerts irreversible effects at a specific stage of osteoblast phenotype development resulting in a potent inhibition of osteoblast differentiation at concentrations from 0.1 ng/ml. © 1994 Wiley‐Liss, Inc.
Proinflammatory cytokines (interleukin‐1β and tumor necrosis factor‐α) down regulate synthesis and secretion of thrombospondin by human endothelial cells
Authors: Morandi Verônica , Cherradi Salah‐Eddine Lamhamedi , Lambert Sylvie , Fauvel‐Lafève Françloise , Legrand Yves J. , Legrand Chantal 
Published: February 2005

AbstractWe examined the effects of proinflammatory cytokines on the expression of two extracellular matrix proteins, e.g., thrombospondin (TSP) and fibronectin (FN) by cultured human umbilical vein endothelial cells (HUVECs). Treatment of HUVECs with human recombinant interleukin‐ 1β (IL‐1β) or human tumor necrosis factor‐α (TNF‐α) caused a time‐ and dose‐dependent decline in TSP production whereas FN production was not modified. At low concentrations, IL‐1β and TNF‐α in combination had a greater effect than either agent alone. Interferon‐γ (IFN‐γ) was without effect. The decline in TSP synthesis resulted in a decreased secretion of this glycoprotein into the extracellular matrix. Endothelial cell monolayers cultured on porous filters were used to study the polarity of TSP secretion. Approximately two thirds of the synthesized protein was secreted to the apical side medium and one third to the basal side medium and both types of secretion were inhibited to a similar extent by cytokine treatment. Immunoprecipitation experiments revealed no apparent degradation of secreted TSP, either in the apical or in the basal compartment. Treatment of HUVECs with IL‐1β, either alone or in combination with TNF‐α, had no significant effect on the steady‐state TSP mRNA levels, suggesting a posttranscriptional regulation. Our results indicate that IL‐1β and TNF‐α can selectively modulate the composition of the extracellular matrix by decreasing TSP deposition and suggest different regulatory mechanisms for the expression of various secreted proteins by endothelial cells.
Transforming growth factor β1‐mediated induction of junB is selectively inhibited by expression of Ad.12‐E1A
Authors: Coussens Lisa M. , Chiu Robert , Yokoyama Kazushige 
Published: February 2005

AbstractTransforming growth factor beta (TGF‐β), a multifunctional polypeptide growth factor, regulates the expression of many genes critical to cell cycle progression, such as members of the jun gene family which encode components of the transcription factor complex AP‐1. The transforming proteins encoded by the early region 1A of adenovirus12 (Ad. 12‐E1A) abrogate some of the cellular responses to TGF‐β as well as affecting, differentially, the expression of cellular jun genes. Our data demonstrate that expression of Ad. 12‐E1A in rat 3Y1 fibroblast cells inhibits induction of junB by TGF‐β1 while not altering the regulation of junB by phorbol ester or serum. Regulation of c‐jun gene expression by TGF‐β1, phorbol ester, and serum is not appreciably altered by the expression of Ad. 12‐E1A. Inhibition of TGF‐β induced junB expression is not due to a defect in TGF‐β/receptor interaction on Ad. 12‐E1A transformed cells and is not observed in other isotypic fibroblast cells transformed by SV40 or polyomavirus. These data suggest that multiple, independent, intracellular signal transduction pathways exist which mediate genomic responses to TGF‐β. Cellular expression of Ad. 12‐E1A‐12S gene products results in selective disruption of some TGF‐β1 signaling cascades and not those activated by phorbol ester or serum. These data further suggest that some cellular targets which mediate TGF‐β1 action may also be unique targets of action for the E1A‐12S transforming protein of adenovirus12. © 1994 Wiley‐Liss, Inc.
Enhanced growth capacity of neonatal pulmonary artery smooth muscle cells in vitro: Dependence on cell size, time from birth, insulin‐like growth factor I, and auto‐activation of protein Kinase C
Authors: Dempsey Edward C. , Badesch David B. , Dobyns Emily L. , Stenmark Kurt R. 
Published: February 2005

AbstractBased on the unique susceptibility of the neonatal pulmonary circulation to hypoxia‐induced structural alteration in vivo, we hypothesized that pulmonary artery (PA) smooth muscle cells (SMC) from the neonate would demonstrate enhanced growth capacity in vitro compared to adult cells. To test this hypothesis, matched neonatal and adult bovine SMC were tested for differences in size, serum‐stimulated proliferation, susceptibility to senescence, resistance to serum withdrawal, autocrine growth capacity, and responsiveness to a locally important growth factor (insulin‐like growth factor I; IGF‐I) and an activator of protein kinase C (PKC) (phorbol 12‐myristate 13‐acetate; PMA). Neonatal PA SMC were smaller, grew faster, reached a higher plateau density, and were less susceptible to senescence. They were more resistant to serum withdrawal, had spontaneous autocrine growth capacity, and were more responsive to IGF‐I, PMA, and the combination. Acquisition of increased growth factor responsiveness occurred between d5 and d14 after birth. Increased neonatal growth to IGF‐I was associated with reduced IGF‐I binding activity, implicating a post‐receptor mechanism in enhanced responsiveness. Increased membrane‐bound PKC catalytic activity was found in serum‐deprived neonatal SMC. This basal increase was equal to that stimulated by 1 nM PMA in adult SMC, a pretreatment that caused these cells to become as responsive to IGF‐I as untreated neonatal ones. We conclude that neonatal bovine PA SMC have marked enhancement of growth capacity in vitro, the acquisition of which is dependent on time from birth and is associated with auto‐activation of PKC, These increased growth properties detected in vitro may contribute to the striking hyperplasia of neonatal PA SMC found in vivo following hypoxic exposure. © 1994 Wiley‐Liss, Inc.
Intracellular signal transduction for serum activation of the hyaluronan synthase in eukaryotic cell lines
Authors: Klewes Ludger , Prehm Peter 
Published: February 2005

AbstractHyaluronan synthase was activated in B6 cells or 3T3 fibroblasts by foetal calf serum with maximal activity after 6 h. Activation was inhibited by cycloheximide or by the protein kinase inhibitors H‐7 or H‐8, indicating that transcription as well as phosphorylation was required for activation. The activation by serum was markedly prolonged, when serum was added together with cholera toxin or theophylline. Without serum stimulation the hyaluronan synthase could also be activated by phorbol‐12‐myristate‐13‐acetate, by dibutyryl‐c‐AMP, or by forskolin. Increasing the intracellular Ca‐ion concentration with a Ca‐ionophore also led to an activation. The activation of the drugs was not synergistic. In isolated plasma membranes the synthase activity could be decreased by phosphatase treatment and enhanced by ATP in B6 cells and by ATP in the presence of phorbol‐12‐myristate‐13‐acetate in 3T3 fibroblasts. Stimulation correlated with increased transcription and phosphorylation of the 52 kD hyaluronan synthase at serine residues. The results led to the conclusion that hyaluronan synthase is induced by transcription and activated by phosphorylation by protein kinase C, c‐AMP‐dependent protein kinases, or Ca‐ion‐dependent protein kinases. © 1994 Wiley‐Liss, Inc.