bluuebunny/crossref_metadata_2025_split huggingface dataset:
Peptide inhibitors of fibronectin, laminin, and other adhesion molecules: Unique and shared features
Authors: Yamada Kenneth M. , Kennedy Dorothy W. 
Published: February 2005

AbstractSynthetic peptides can specifically inhibit the function of certain adhesive glycoproteins in vitro and in vivo. We have compared the relative activities of a set of six variant synthetic peptides based on the sequence of fibronectin in terms of their ability to inhibit the interactions of fibroblasts with fibronectin, spreading factor/vitronectin, laminin, and native collagen gels. BHK (baby hamster kidney) and chick embryo fibroblasts spreading on these adhesive molecules displayed distinctive patterns of sensitivity to inhibition by this panel of peptides, which depended on the adhesive molecule rather than the cell type. For fibronectin, Gly‐Arg‐Gly‐Asp‐Ser was considerably more active than Arg‐Gly‐Asp‐Ser, whereas these two peptides displayed little difference in activity in inhibiting cell adhesion to spreading factor. For both proteins, the inverted peptide sequence Ser‐Asp‐Gly‐Arg was also moderately active, whereas closely related peptides containing a transposition, a deletion, or a single, conserved amino acid substitution were much less active. For inhibiting interactions with laminin or native type I collagen gels, Gly‐Arg‐Gly‐Asp‐Ser was only weakly active, but the inverted peptide Ser‐Asp‐Gly‐Arg unexpectedly continued to display inhibitory activity for both attachment proteins in both cell types. Our results indicate that different adhesive processes depend on distinct peptide recognition events by a cell. However, there may be a possible common denominator among attachment proteins in a moderate sensitivity to Ser‐Asp‐Gly‐Arg. Our study also underscores the importance of examining a full set of peptide analogs when these novel inhibitors are used to characterize biological processes.
Soluble antigen induces T lymphocytes to secrete an endoglycosidase that degrades the heparan sulfate moiety of subendothelial extracellular matrix
Authors: Fridman Rafael , Lider Ofer , Naparstek Yaakov , Fuks Zvi , Vlodavsky Israel , Cohen Irun R. 
Published: February 2005

AbstractThe antigen‐mediated induction of heparanase, an endoglycosidase capable of degrading heparan sulfate from the subendothelial extracellular matrix (ECM), was investigated in a rat T lymphocyte cell line reactive against the basic protein (BP) of myelin. We found that nonactivated T lymphocytes could be induced to express heparanase activity following exposure to soluble but not to ECM‐bound BP. The induction of heparanase was immunologically specific and indpendent of the presence of syngeneic or allogeneic antigen presenting cells (APC). However, anti‐IA antibodies inhibited heparanase expression. Soluble BP induced secretion of heparanase into the culture medium within minutes, despite inhibition of protein synthesis. Cell lysates of T lymphocytes contained heparanase activity. Thus, T lymphocytes secrete a preformed heparanase following exposure to specific antigen.
Propagation of differentiating normal human tracheobronchial epithelial cells in serum‐free medium
Authors: Chopra D. P. , Sullivan J. , Wille J. J. , Siddiqui K. M. 
Published: February 2005

AbstractSerial‐passage cultures of normal human tracheobronchial (TB) epithelial cells that exhibit functional differentiation have been established in serum‐free medium supplemented with bovine pituitary extract (25 μg/ml), insulin (5 μg/ml), hydrocortisone (0.5 μg/ml), EGF (5 ng/ml), 10−6M each of ethanolamine and phoshoethanolamine, and antibiotics. The cells proliferated in this medium with a population doubling time of approximately 80 hours. Further, the passaged cultures retained differentiated morphology as evidenced by secretion of glycoproteins, binding of concanavalin A lectin, and presence of alcian blue and periodic acid Schiff‐positive material in their cytoplasm. Ultrastructural observations further supported the functional epithelial nature of the cultures. Most cells exhibited characteristic microvilli on cell surfaces and showed junctional complexes between them. The cytoplasm contained a large number of perinuclear secretory vesicles, a characteristic feature of the differentiated cells. These cultures provide an excellent model to study factors that regulate synthesis and secretion of glycoproteins in normal human TB cells.
Deoxyribonucleotide metabolism and cyclic AMP resistance in hydroxyurea‐resistant S49 T‐lymphoma cells
Authors: Albert Daniel A. , Gudas Lorraine J. , Nodzenski Edwardine 
Published: February 2005

AbstractWe investigated the cell cycle regulation of deoxyribonucleoside triphosphate (dNTP) metabolism in hydroxyurea‐resistant (HYUR) murine S49 T‐lymphoma cell lines. Cell lines 10‐ to 40‐fold more hydroxyurea‐resistant were selected in a stepwise manner. These HYUR cells exhibited increased CDP reductase activity (5‐ to 8‐fold) and increased dNTP pools (up to 5‐fold) that appeared to result from increased activity of the M2 subunit (binding site of hydroxyurea) of ribonucleotide reductase. These characteristics remained stable when the cells were grown in the absence of hydroxyurea for up to 2 years. In both wild type and hydroxyurea‐resistant cell populations synchronized by elutriation, dCTP and dTTP pools Increased in S phase, whereas dATP and dGTP pools generally remained the same or decreased, suggesting that allosteric effector mechanisms were operating to regulate pool sizes. Additionally, CDP reductase activity measured in permeabilized cells increased in S phase in both wild type and hydroxyurea‐resistant cells, suggesting a nonallosteric mechanism of increased ribonucleotide reductase activity during periods of active DNA synthesis. While wild type S49 cells could be arrested in the G1 phase of the cell cycle by dibutyryl cyclic AMP, hydroxyurea‐resistant cell lines could not be arrested in the G1 phase by exogenous cyclic AMP or agents that elevate the concentration of endogenous cyclic AMP. These data suggest that cyclic AMP‐generated G1 arrest in S49 cells might be mediated by the M2 subunit of ribonucleotide reductase.
Phorbol esters induce angiogenesis in vitro from large‐vessel endothelial cells
Authors: Montesano R. , Orci L. 
Published: February 2005

AbstractWe have shown previously that the tumor promoter phorbol myristate acetate (PMA) induces capillary endothelial cells grown on the surface of threedimensional collagen gels to invade the underlying matrix as capillary‐like tubular structures, a phenomenon mimicking angiogenic processes that occur in vivo (Montesano and Orci: Cell 42:469, 1985). To establish whether the potential to invade the extracellular matrix as capillary‐like sprouts is restricted to microvascular endothelial cells or is also shared by large vessel endothelium, we have examined the response to PMA of endothelial cells isolated from the human umbilical vein and the calf pulmonary artery. The results of these experiments show that both types of macrovascular endothelial cells are able to penetrate into collagen gels as vessel‐like tubes following treatment with PMA. This demonstrates that endothelial cells derived from large vessels can, in response to appropriate signals, express invasive properties thought to be associated specifically with capillary endothelial cells in vivo.
Suppression of maturation of megakaryocyte colony forming unit in vitro by a platelet‐released glycoprotein
Authors: Dessypris Emmanuel N. , Gleaton John H. , Sawyer Stephen T. , Armstrong Otho L. 
Published: February 2005

AbstractThe suppressive role of platelets on the grwoth of human marrow megakaryocyte colony forming units (CFU‐M) in vitro was investigated by the use of a plasma clot assay. An inverse correlation was established between the number of megakaryocytic colonies grown and the platelet concentration of the plasma or the resultant serum used in the culture system. The suppressive effect of platelets on megakaryocyte colony formation reached a plateau at normal human blood platelet concentration and was specific for CFU‐M growth, since marrow cell erythroid burst formation (BFU‐E) and granulocytic‐monocytic colony formation (CFU‐GM) remained unaffected. The inhibitory activity was detectable in the supernatants of platelet suspensions aggregated by thrombin or ADP, and the inhibitory activity released from ADP‐stimulated platelets was blocked by pretreatment of platelets with monoclonal antibody HuPl‐m1. Partial purification of this activity was acheived by diethylaminoethyl (DEAE)‐ion exchange and phytohemagglutinin (PHA)‐E agarose affinity chromatography. This inhibitor is a glycoprotein with a molecular weight of 12‐17K daltons. This platelet released glycoprotein does not affect the early proliferative phase of CFU‐M in vitro but acts on a day 6–8 CFU‐M‐derived cell by adversely affecting its maturation into recognizable megakaryocytes. These findings demonstrate that a glycoprotein released from platelets suppresses the maturation of CFU‐M into megakaryocytes.
Intracellular binding of spin‐labeled amiloride: An alternative explanation for amiloride's effects at high concentration
Authors: Costa Charles J. , Kirschner Leonard B. , Cragoe Edward J. 
Published: February 2005

AbstractAmiloride, an important inhibitor of Na+ transport and Na+/H+ exchange, has been used in nontransporting tissues to investigate the relationship between ionic fluxes or intracellular pH change and proliferative or synthetic events. Reports that amiloride is permeant and had direct effects on intracellular processes have led us to investigate the possibility that amiloride binds intracellularly to nuclei, mitochondria, and to purified nucleic acids. Using a nitroxide spin‐labeled derivative of amiloride (ASp) and electron paramagnetic responance (EPR) spectroscopy, we have demonstrated that nuclei and mitochondria isolated from trout liver bind significant amounts of ASp especially at the high amiloride concentrations (∼ mM) commonly used to inhibit proliferative events. While the chemical component responsible for ASp binding in these organelles was not identified, native DNA binds significant amounts of ASp whereas single stranded DNA and RNA bind much less. When these observations are taken together with reports of amiloride's direct action on cellular processes, they support the possibility that some of the effects attributed to inhibition of a transport event are caused by amiloride directly.
Modulation by the src oncogene of the effect of inhibitory diffusible factor IDF45
Authors: Blat C. , Villaudy J. , Rouillard D. , Golde A. , Harel L. 
Published: February 2005

AbstractDensity‐dependent inhibition of growth has been assumed to be under the control of inhibitory molecules diffusing from dense cell cultures. Growth inhibitory factors have been fractionated or purified from medium conditioned by different cell types. In the present work, it was shown that IDF45 (inhibitory factor diffusing from 3T3 cells) decreased DNA synthesis in chick embryo fibroblats (CEF) and was an inhibitor of CEF growth; this inhibition was reversible.Since similitudes between oncogene products and growth factors have been observed, it was of interest to compare the inhibitory effect of IDF45 upon the stimulation of DNA synthesis induced either by serum or by pp60‐src. CEF infected by Ny68 virus (a mutant of Rous sarcoma virus ts for the expression of transformation) were density‐inhibited at 41°C, but were stimulated at this temperature by addition of 1% serum. This stimulation was 94% inhibited by IDF45. The same Ny68‐infected cells could also be stimulated transfer to 37°C, the permissive temperature (in the absence of serum). The stimulation of DNA synthesis by src expression was poorly inhibited by IDF45. From our results, it appears that oncogene expression in CEF induces a loss in their sensitivity to IDF45. This would explain why transformed cells escape DDI of growth.
Insulin binding and processing by H4IIEC3 hepatoma cells: Ultrastructural and biochemical evidence for a unique route of internalization and processing
Authors: Smith Robert M. , Laudenslager Nan H. , Shah Neelima , Jarett Leonard 
Published: February 2005

AbstractBiochemical and ultrastructural studies of insulin binding and cellular processing by cultured H4IIEC3 hepatoma cells were performed. Insulin binding and intracellular accumulation were rapid and after 30 min at 37°C, 65% of the total cell‐associated 125I‐insulin was in an acid‐stable compartment. Chloroquine had no significant effect on the amount of total cell‐associated insulin or the percentage of insulin in the acid‐stable compartment or cell‐associated insulin degradation under those conditions, but after 60‐min incubations, it slightly decreased the rate of dissociation of internalized hormone. Ultrastructural analysis revealed that monomeric ferritin‐insulin (Fm‐I) initially bound to single or paired receptors on microvilli. Within 5 min occupied insulin receptors microaggregated and migrated to the intervillous cell surface. During the next 5‐10 min occupied receptors aggregated into large clusters on the plasma membrane. Large amounts of insulin were internalized by macropinocytosis and the majority of internalized Fm‐I was found in phagosomes. Less than 10% of the membrane‐bound insulin was associated with pinocytotic invaginations or coated pits and less than 5% of the total cell‐associated insulin was found in lysosomes. Chloroquine had no detectable effect on the amount of Fm‐I or its distribution among the intracellular organelles. These studies demonstrated that, compared to previous studies with rat adipocytes or 3T3‐L1 adipocytes, insulin interalization and intracellular processing in this hepatoma cell were unique. These differences provide further evidence that insulin binding and processing may be controlled by cell‐specific mechanisms and that substantial heterogeneity exists in pathways previously presumed to be similar for all cell types.
Release of angiotensin I‐converting enzyme by endothelial cells in vitro
Authors: Noveral James P. , Mueller Stephen N. , Levine Elliot M. 
Published: February 2005

AbstractBovine fetal aortic endothelial cells cultured in serum‐containing medium accumulate angiotensin I‐converting enzyme (ACE) activity and also release it into the culture medium. Following subcultivation of a confluent culture using trypsin‐EDTA, cellular ACE activity falls 50% within 8 h, but no ACE activity is detected in the medium, suggesting intracellular loss of the enzyme activity. ACE activity reappears in both the cell lysate and culture medium after the culture becomes confluent. The rate of accumulation of ACE activity released into the medium is always greater than that for cellular activity. For example, 21 days following subcultivation 80–85% of the total culture activity is detected in the medium. Both cellular and medium‐associated ACE decrease proportionately as the culture progresses through its in vitro lifespan.
Effects of diverse intracellular thiol delivery agents on glutathione peroxidase activity, the ratio of reduced/oxidized glutathione, and ornithine decarboxylase induction in isolated mouse epidermal cells treated with 12‐O‐tetradecanoylphorbol‐13‐acetate
Authors: Perchellet Elisabeth M. , Maatta Eric A. , Abney Nancy L. , Perchellet Jean‐Pierre 
Published: February 2005

AbstractSince the enhancement of the activity of the natural glutathione (GSH)‐dependent antioxidant protective system of the epidermal cells appears to inhibit the oxidative challenge presumably linked to skin tumor promotion by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), we have compared the effectiveness of diverse intraceliular thiol delivery agents as inhibitors of the effects of TPA on GSH metabolism and ornithine decarboxylase (ODC; L‐ornithine carboxy‐lyase, EC 4.1.1.17) induction in isolated mouse epidermal cells. Here we report that, at a 2‐mM concentration, the monoethyl and monomethyl esters of GSH, N‐acetyl‐L‐cysteine, and L‐2‐oxothiazolidine‐4‐carboxylate are all significantly more effective than GSH in inhibiting the sharp decline in the intracellular ratio of reduced GSH/oxidized glutathione (GSSG), the prolonged decrease in GSH peroxidase (GSH:H2O2 oxidoreductase, EC 1.11.1.9) activity, and the induction of ODC activity caused by 1 μM TPA. Moreover, diethyldithiocarbamate prevents totally the initial drop in the GSH/GSSG ratio of TPA‐treated cells and is the most potent inhibitor of TPA‐decreased GSH peroxidase activity in relation with its remarkable 98% inhibition of TPA‐induced ODC activity, suggesting that the potential antitumor‐promoting activity of this compound in mouse skin may be far superior to that previously demonstrated by GSH in the initiation‐promotion protocol.
High and low affinity binding sites for basic fibroblast growth factor on cultured cells: Absence of a role for low affinity binding in the stimulation of plasminogen activator production by bovine capillary endothelial cells
Authors: Moscatelli David 
Published: February 2005

AbstractScatchard analysis of binding of 125l‐basic fibroblast growth factor (FGF) to baby hamster kidney (BHK) cells revealed the presence of two binding sites: a high affinity site with KD of 20 pM and 80,000 sites per cell and a low affinity site with KD of about 2 nM and 600,000 sites per cell. The binding to the two sites could be separated by first washing the cells with 2 M NaCl at pH 7.5 which released the low affinity binding and then extracting the cells with 0.5% Triton X‐100 to recover the 125l‐basic FGF bound to high affinity sites. The binding to the high affinity site was acid sensitive, suggesting that it represented binding to the receptor. Binding to the low affinity site could be competed strongly by heparin and less strongly by heparan sulfate but not by chondroitin sulfate, dermatan sulfate, or keratan sulfate. Treatment of BHK cells with heparinase abolished 62% of the low affinity binding, suggesting that the low affinity binding represented binding to cell‐associated, heparin‐like molecules. A variety of other cell types, including bovine capillary endothelial (BCE) cells, also demonstrated both low and high affinity binding sites. To test whether the low affinity binding might play a role in the basic FGF stimulation of plasminogen activator (PA) production by BCE cells, heparin was added to BCE cultures at concentrations which totally blocked binding of 125l‐basic FGF to the low affinity sites. Addition of the heparin did not diminish the increased PA production induced by basic FGF. This suggests that the low affinity binding has no direct role in the stimulation of PA production in BCE cells.
Isolation of an autocrine growth factor from hepatoma HTC‐SR cells
Authors: Ove Peter , Coetzee Mona L. , Scalamogna Philip , Francavilla Antonio , Starzl Thomas E. 
Published: February 2005

AbstractA growth factor has been isolated from HTC‐SR rat hepatoma tissue culture cells which specifically stimulates DNA synthesis and cell proliferation of the HTC cells that produce it. The factor can be isolated from HTC cell conditioned medium or from an HTC cell extract. This autocrine factor has been purified 640‐fold from a postmicrosomal supernatant by successive steps, involving ethanol precipitation, heating at 80°C for 10 min, chromatography on a DEAE Bio‐Gel A column, and chromatography on a heparin‐sepharose affinity column. The major peak of activity eluted from the heparin column migrates as a single band on SDS‐PAGE with an apparent Mr of 60,000. The factor is resistant to acid, heat, and neuraminidase but sensitive to trypsin, papain, and protease.The autocrine nature of the factor is indicated by the finding that several other types of cells do not respond with increased DNA synthesis. Mouse L‐cells, BHK cells, Novikoff hepatoma cells, hepatocytes in primary culture, and an epithelial‐like rat liver‐derived cell line (Clone 9) were tested, and none of the cells could be stimulated. Small amounts of the factor could be extracted from the Clone 9 cells, however. This material had the same physical and purification properties as the factor extracted from HTC cells, but it did not stimulate DNA synthesis in Clone 9 cells, only in HTC cells.Addition of the factor resulted in an almost immediate stimulation of DNA synthesis in a proliferating HTC cell population. When the factor was added together with [3H]thymidine for 2 h, a significant stimulation of DNA synthesis was observed, provided the addition was made between 18 and 48 h after the cells had been plated. Autoradiographic studies indicated that the factor both accelerates DNA synthesis in cells already making DNA and increases the number of cells entering the S period. The stimulation of DNA synthesis was completely inhibited by 10 mM hydroxyurea, whether the factor was present for 2, 24, or 48 h in the culture. A significant increase in cell number due to addition of the factor was also observed. This accelerated proliferation was detectable only after the cells had been in culture for at least 48 h with the factor present.
Zinc‐induced reduction in melphalan cytotoxicity: Heterogeneity of response of cloned human tumor cells
Authors: Tobey Robert A. , Tesmer Judith G. , Shackelford Mary E. 
Published: February 2005

AbstractPrevious studies with cultured human normal fibroblasts indicated that pretreatment of the cells with zinc for 12 h prior to exposure to the alkylating agent melphalan increased survival by seven‐ to ninefold over survival values obtained in cultures treated with drug only. Comparable pretreatment of cells derived from a variety of human tumors resulted in an increase in survival of 1.7‐fold or less. To determine whether the limited responsiveness to zinc represented a general property of tumor cells (which would be characterized by a lack of highly zinc‐responsive subpopulations contained within the parental tumor populations), a series of clones was prepared from the A101D human melanoma line and the A549 human alveolar cell carcinoma line. Cells from each clone were then challenged with melphalan with and without zinc pretreatment. Twenty‐five percent of the tumor clones exhibited increased resistance to melphalan following pretreatment with zinc (range of 2.1‐ to 5.2‐fold increase in survival), indicating that the parental tumor lines were highly heterogeneous in regard to inducibility to a state of reduced sensitivity to melphalan. There was no evidence of a relationship between zinc‐induced reductions in toxicity and induced elevations in total intracellular glutathione content, indicating that the primary effect of zinc is not directed toward elevating intracellular levels of glutathione.