bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of a role of the vitronectin receptor and protein kinase C in the induction of endothelial cell vascular formation
Authors: Davis Cynthia M. , Danehower Susan C. , Laurenza Antonio , Molony J. Leslie 
Published: May 2005

AbstractWhen cultured on a basement membrane substratum, endothelial cells undergo a rapid series of morphological and functional changes which result in the formation of histotypic tube‐like structures, a process which mimics in vivo angiogenesis. Since this process is probably dependent on several cell adhesion and cell signaling phenomena, we examined the roles of integrins and protein kinase C in endothelial cell cord formation. Polyclonal antisera directed against the entire vitronectin (αvβ3) and fibronectin (α5β1) receptors inhibited cord formation. Subunit‐specific monoclonal antibodies to αv, β3, and β1 integrin subunits inhibited cord formation, while monoclonal antibodies to α3 did not, which implicated the vitronectin receptor, and not the fibronectin receptor, in vascular formation. Protein kinase C inhibitors inhibited cord formation, while phorbol 12‐myristate 13‐acetate (PMA) caused endothelial cells to form longer cords. Since the vitronectin receptor has been shown to be phosphorylated in an in vitro system by protein kinase C, the possible functional link between the vitronectin receptor and protein kinase C during cellular morphogenesis was examined. The vitronectin receptor was more highly phosphorylated in cord‐forming endothelial cells on basement membrane than in monolayer cells on vitronectin. Furthermore, this phosphorylation was inhibited by protein kinase C inhibitors, and PMA was required to induce vitronectin receptor phosphorylation in endothelial cells cultured on vitronectin. Colocalization studies were also performed using antisera to the vitronectin receptor and antibodies to protein kinase C. Although no strict colocalization was found, protein kinase C was localized in the cytoskeleton of endothelial cells initially plated on basement membrane or on vitronectin, and it translocated to the plasma membrane of C‐shaped cord‐forming cells on basement membrane. Thus, both the vitronectin receptor and protein kinase C play a role in in vitro cord formation. © 1993 Wiley‐Liss, Inc.
Anti‐epidermal growth factor receptor monoclonal antibodies affecting signal transduction
Authors: Reins Hans‐Albert , Steinhilber Gerhard , Freiberg Brigitte , Anderer F. Alfred 
Published: May 2005

AbstractMonoclonal antibodies prepared against tyrosine phosphorylated epidermal growth factor receptor (EGFR) were tested for their effects on transmembrane signal transduction in A431 tumor cells. Monoclonal antibodies (mab) defined by SDS‐sensitive epitopes, i.e., epitopes with conformational specificity, were most effective. Mab 5‐‐125 reacting with a site of the extracellular EGFR domain blocked EGF‐binding and cell proliferation in vitro, as well as tumor growth in vivo. However, this mab appeared not to be internalized upon binding to EGFR and did not trigger EGFR autophosphorylation. In contrast, mab 5‐D43, also defined by an SDS‐sensitive epitope and reacting with an extracellular EGFR site, did not block EGF binding but was readily internalized after binding to EGFR of untreated A431 cells. This mab induced EGFR tyrosine phosphorylation in cell lysates and tyrosine‐specific autophosphorylation of insolubilized EGFR immune complexes. Cell growth in vitro was greatly stimulated in the presence of mab 5‐D43. Since interaction of mab 5‐D43 with EGFR induced most EGF‐specific functions, although it did not bind to the EGF‐specific site of EGFR, we have to assume that binding of mab 5‐D43 to EGFR induced a conformational shift that activated the cytoplasmic EGFR kinase site. On the other hand, activation and/or accessibility of the EGFR kinase site could be blocked by mab 1‐594, which is defined by an SDS‐insensitive protein epitope of the cytoplasmic EGFR domain. Blocking of the EGFR kinase site by mab 1‐594 also abolished EGF‐induced tyrosine phosphorylation of endogenous cellular substrates with molecular masses of 145, 97, 85, 37, and 32 kDa, as well as of exogenous substrates such as GAT copolymer. © 1993 Wiley‐Liss, Inc.
Studies of chondrogenesis in rotating systems
Authors: Duke P. Jackie , Daane Elizabeth L. , Montufar‐Solis Dina 
Published: May 2005

AbstractA great deal of energy has been exerted over the years researching methods for regenerating and repairing bone and cartilage. Several techniques, especially bone implants and grafts, show great promise for providing a remedy for many skeletal disorders and chondrodystrophies. The bioreactor (rotating‐wall vessel, RWV) is a cell culture system that creates a nurturing environment conducive to cell aggregation. Chondrocyte cultures have been studied as implants for repair and replacement of damaged and missing bone and cartilage since 1965 [Chesterman and Smith, J Bone Joint Surg 50B:184–197, 1965]. The ability to use large, tissue‐like cartilage aggregates grown in the RWV would be of great clinical significance in treating skeletal disorders. In addition, the RWV may provide a superior method for studying chondrogenesis and chondrogenic mutations. Because the RWV is also reported to simulate many of the conditions of microgravity it is a very useful ground‐based tool for studying how cell systems will react to microgravity. © 1993 Wiley‐Liss, Inc.
Activation of cellular phospholipase A2 by Clostridium difficile toxin B
Authors: Shoshan Maria Caspar , Florin Inger , Thelestam Monica 
Published: May 2005

AbstractC. difficile toxin B is a potent cytotoxin known to disrupt the microfilaments of cultured cells. We have recently shown also increased phospholipase A2 activity in cells treated with toxin B. The activity was detected as a toxin‐induced, dose‐dependent release of 14C‐arachidonic acid from prelabeled fibroblasts. Here is shown that the toxin elicited a 14C‐arachidonic acid release in a cell mutant resistant to the toxin B effect on the microfilaments. The toxin‐induced release was further characterized using fibroblasts. Within 20 min high doses of toxin B (6 μg/ml) elicited a release which increased exponentially with time. Of the major membrane phospholipids the lipase activity affected mainly phosphatidyl ethanolamine. Neither cycloheximide nor pertussis toxin treatment of target cells inhibited the toxin‐induced release, while it could be increased with 12‐O‐tetradecanoylphorbol‐13‐acetate. Our results also suggest a toxin‐mediated increase in phospholipase C activity occurring at a later stage than the phospholipase A2 activation.We conclude that the ability of toxin B to induce phospholipase activation represents a hitherto unrecognized toxin B effect which is neither a cause nor a consequence of toxin‐induced microfilament disorganization.
Inhibition of induced endochondral bone development in caffeine‐treated rats
Authors: Barone L. M. , Tassinari M. S. , Bortell R. , Owen T. A. , Zerogian J. , Gagne K. , Stein G. S. , Lian Jane B. 
Published: May 2005

AbstractWe have addressed questions raised by the observation in fetal rats of delayed ossification induced by caffeine at maternal doses above 80 mg/kg body weight per day. The effect of caffeine on endochondral bone development and mineralization has been studied in an experimental model system of bone formation which involves implantation of demineralized bone particles (DBP) in subcutaneous pockets of young growing rats. Caffeine's effects on cellular events associated with endochondral ossification were examined directly by quantitating cellular mRNA levels of chondrocyte and osteoblast growth and differentiation markers in DBP implants from caffeine‐treated rats harvested at specific stages of development (day 7 through day 15). Oral caffeine administration to rats implanted with DBP resulted in a dose dependent inhibition of the formation of cartilage tissue in the implants. Histologic examination of the implants revealed a decrease in the number of cells which were transformed to chondrocytes compared to control implants. Those cartilaginous areas that did form, however, proceeded through the normal sequelae of calcified cartilage and bone formation. At the 100 mg/kg dose, cellular levels of mRNA for histone, collagen type II, and TGFβ were all reduced by greater than 40% of control implants consistent with the histological findings. Alkaline phosphatase activity in the implants and mRNA levels for proteins reflecting the hypertrophic chondrocyte and bone phenotype, collagen type I and osteocalcin were markedly decreased compared to controls. Lower doses of 50 and 12.5 mg/kg caffeine also resulted in decreased cellular proliferation and transformation to cartilage histologically and reflected by significant inhibition of type II collagen mRNA levels (day 7). The effects of caffeine on gene expression observed in vivo during the period of bone formation (day 11 to day 15) in the DBP model were similar to the inhibited expression of H4, alkaline phosphatase, osteocalcin, and osteopontin found in fetal rat calvarial derived osteoblast cultures following 24 hour exposure of the cultures to 0.4 mM caffeine. Thus the observed delayed mineralization in the fetal skeleton associated with caffeine appears to be related to an inhibition of endochondral bone formation at the early stages of proliferation of undifferentiated mesenchymal cells to cartilage specific cells as well as at later stages of bone formation.
Biosynthesis and function of membrane bound and secreted forms of recombinant CD11b/CD18 (Mac‐1)
Authors: Berman Phillip W. , Nakamura Gerald R. , Riddle Lavon , Chiu Henry , Fisher Karen , Champe Mark , Gray Alane M. , Ward Pamela , Fong Sherman 
Published: May 2005

AbstractFull‐length (membrane bound) and truncated (secreted) forms of the beta 2 integrin heterodimer, CD11b/CD18 (Mac‐1), were expressed in a human kidney cell line (293) that normally does not express leukocyte adhesion molecules (Leu‐CAMs). The biosynthesis of recombinant Mac‐1 in 293 cells differed from that reported for leukocytes in that heterodimer formation was not required for CD11b to be exported to the cell surface. A stable cell line was constructed that constitutively secreted the recombinant, truncated Mac‐1 heterodimer into growth conditioned cell culture medium. A novel monoclonal antibody that enabled an immunoaffinity method for the selective purification of recombinant Mac‐1 heterodimers was identified. Sufficient protein was purified to allow the first measurement of the 50% inhibitory concentration (IC50) for CD11b/CD18 and for the direct comparison of the inhibitory activity of recombinant soluble Mac‐1 with that of various CD18 and CD11b specific monoclonal antibodies. Purified recombinant soluble Mac‐1 inhibited the binding of neutrophils, activated by opsonized zymosan or fMet‐Leu‐Phe peptide, to human umbilical vein endothelial cells. Similarly, the recombinant integrin was effective in inhibiting the binding of unactivated neutrophils to tumor necrosis factor (TNF‐alpha) activated endothelial cells. The availability of an abundant source of purified, biologically active Mac‐1 will enable direct physical and chemical investigations into the relationship between the structure and function of this leukocyte adhesion molecule.
Dual mechanism of laminin modulation of ecto‐5′‐nucleotidase activity
Authors: Méhul Bruno , Aubery Michèle , Mannherz Hans‐Georg , Codogno Patrice 
Published: May 2005

AbstractThe myoblast cell surface activity of ecto‐5′‐nucleotidase was stimulated by a laminin substrate, whereas fibronectin and gelatin did not increase the AMPase activity of ecto‐5′‐nucleotidase. This increase was related to a higher expression of ecto‐5′‐nucleotidase on the surface of cells seeded on a laminin substrate, but without the mobilization of an intracellular pool of enzyme. Furthermore, laminin and its fragments E′1 and E8 modified the AMPase activity of the ecto‐5′‐nucleotidase purified from chicken striated muscle and reconstituted in liposomes. Over the range of concentrations used, intact laminin and its fragment E8, consisting of the distal half of the long arm, stimulated the AMPase activity of ecto‐5′‐nucleotidase. By contrast, the large fragment derived from the short arms, designated E′1, inhibited the AMPase activity. Furthermore, the monoclonal anti‐ecto‐5′‐nucleotidase antibody, CG37, abolished the stimulatory effect of fragment E8 on the AMPase activity of ecto‐5′‐nucleotidase but did not reverse the inhibitory effect of fragment E′1. In conclusion, laminin stimulates the AMPase activity of ecto‐5′‐nucleotidase by two mechanisms: inducing the expression of ecto‐5′‐nucleotidase to the cell surface and direct modulation of the enzymatic activity.
Centrosome reorientation in regenerating endothelial monolayers requires bFGF
Authors: Coomber B. L. 
Published: May 2005

AbstractMonolayers of endothelial cells respond to physical denudation with a characteristic sequence of lamellipodia extrusion, cell migration, and cell proliferation. Basic fibroblast growth factor (bFGF) has been implicated as a necessary component of this process: addition of exogenous bFGF enhances monolayer regeneration both in vitro and in vivo, and monolayer regeneration can be inhibited in vitro by treatment with neutralizing antibodies raised against bFGF. Centrosome reorientation from a random location to one preferentially situated between the nucleus and the denudation edge has been postulated as a mechanism essential for cell polarization and subsequent migration. This present study examined the effects of a polyclonal antibody to bFGF and suramin on monolayer regeneration, actin microfilament staining, and centrosome orientation at the wound edge of partially denuded bovine large vessel endothelial monolayers. Treatment with anti‐bFGF or suramin abolished monolayer repair in these cultures. Cells at the denudation edge showed altered actin staining patterns and reduced lamellipodia extrusion, and there was complete inhibition of centrosome reorientation in treated cultures. Monolayer repair and centrosome reorientation could be restored by addition of exogenous bFGF in antibody but not suramin treated cultures. Recent evidence suggests that preferential centrosome location in migrating cells may be a consequence of lamellipodia protrusion and cell spreading, rather than an indication of cell polarization. However, these results indicate that agents which interfere with bFGF availability prevent endothelial monolayer regeneration via mechanisms involving cell spreading and/or centrosome reorientation.
Gallium nitrate increases type I collagen and fibronectin mRNA and collagen protein levels in bone and fibroblast cells
Authors: Bockman Richard S. , Guidon Peter T. , Pan Lydia C. , Salvatori Roberto , Kawaguchi Alan 
Published: May 2005

AbstractGallium is a Group IIIa transitional element with therapeutic efficacy in the treatment of metabolic bone disorders. Previously described antiresorptive effects of gallium on osteoclasts are not sufficient to account for the full range of effects of gallium on bone structure and metabolism. We have recently shown that gallium nitrate inhibits osteocalcin gene expression and the synthesis of osteocalcin protein, an osteoblast‐specific bone matrix protein that is though to serve as a signal to trigger osteoclastic resorption. Here we present evidence for an additional mechanism by which gallium may function to augment bone mass by altering matrix protein synthesis by osteoblastic and fibroblastic cells. Rat calvarial explants exposed to gallium nitrate for 48 h showed increased incorporation of 3H‐proline into hydroxyproline and collagenase digestible protein. In addition, gallium treatment increased steady‐state mRNA levels for fibronectin and type I procollagen chains in primary rat calvarial osteoblast‐enriched cultures, the ROS 17/2.8 osteoblastic osteosarcoma line, and nontransformed human dermal fibroblasts. These findings suggest that the exposure of mesenchymally‐derived cells to gallium results in an altered pattern of matrix protein synthesis that would favor increased bone formation.
Stimulation of epithelial cell growth by the neuropeptide substance P
Authors: Reid Ted W. , Murphy Christopher J. , Iwahashi Christine K. , Foster Bradley A. , Mannis Mark J. 
Published: May 2005

AbstractThe neuropeptide substance P (SP) was found to stimulate DNA synthesis and cell growth for epithelial cells (cornea and lens) in a serum‐free environment. The length of treatment time was shown to be important since longer times shifted the dose‐response curve to the left. In short‐term DNA synthesis studies (40 h) the stimulation with SP (or synergism with insulin) was not apparent until close to 10 μM, however, when DNA synthesis assays were carried out over a long period of time (5 days) stimulation with SP was seen at 1 pM. The stimulation of DNA synthesis by SP was synergistic with insulin for lens epithelial cells, but little synergism was seen with corneal epithelial cells. It cell growth studies on lens epithelial cells SP also showed growth stimulation by itself and synergism with insulin at concentrations of 1–2 pM. The neuropeptide calcitonin gene related peptide (CGRP) showed no DNA synthesis stimulating ability on epithelial cells by itself at concentrations as high as 2.5 μM; however, it was synergistic with SP at a concentration of 0.025 μM. SP pretreatment of epithelial cells for 2 h causes an increase in cellular sensitivity to subsequent addition of either SP or insulin. This increase is consistent with the hypothesis that either the signal from SP persists after its removal from the cell or the dissociation time for SP from its receptor is longer than the wash time.
Interaction of the neu/p185 and EGF receptor tyrosine kinases: Implications for cellular transformation and tumor therapy
Authors: Dougall William C. , Qian Xiaolan , Greene Mark I. 
Published: May 2005

AbstractGrowth factor receptors such as the epidermal growth factor receptor (EGFR) and the p185c‐neu protein serve vital roles in the transduction of differentiation, developmental, or mitogenic signaling within normal cells. Two methods of analysis suggest that the inappropriately high expression of either protein tyrosine kinase promotes malignant transformation. First, data from in vitro experiments indicate that overexpression of either EGFR or p185c‐neu (or the human homolog c‐erbB‐2) transforms cell‐lines. Second, analysis of primary tumors and tumor cell‐lines derived from many epithelial tissues (breast, stomach, ovary, and pancreas) show growth factor receptor gene amplification and elevated protein levels. The physical and functional interaction of p185c‐neu and EGFR leads to the formation of a highly active, heterodimeric tyrosine kinase complex which synergistically activates cellular transformation. Anti‐receptor antibodies have shown potential utility for the down modulation of these cell‐surface proteins and suppression of the malignant phenotype. Design of organic antibody “mimetics” based on the structure of antireceptor antibodies may provide useful therapies and biological reagents to affect growth factor receptor function.
Antibodies specific for gp40 inhibit cell‐cell adhesion by cross‐linking the protein on the surface of Dictyostelium purpureum
Authors: Springer Wayne R. , Haywood‐Reid Patricia L. 
Published: May 2005

AbstractWe have previously suggested a role for gp40 in cell‐cell adhesion in Dictyostelium purpureum from the fact that antibodies specific for this protein inhibited adhesion in an in vitro assay [Springer: Dev Biol 133:447–455, 1989]. To further confirm this role mutants lacking the protein were isolated and characterized. To our surprise, the mutants had normal adhesive properties both in vitro and in situ. These results lead us to the conclusion that gp40 is not necessary for the cell‐cell adhesions observed and may not be a molecule which directly participates in these adhesions. When studied further, we found that adhesion‐inhibitory antibodies were only effective as divalent IgG. Monovalent Fab fragments of the same antibodies could not inhibit adhesion. The inhibitory antibodies also caused the cells to remain rounded and incapable of attaching to plastic surfaces. We conclude that when divalent antibodies specific for gp40 cross‐link this protein on the cell surface a cytoskeletal change prevents them from attaching to substratum as well as to other cells, thereby inhibiting cell‐cell adhesion. We suggest that an alternative mechanism for inhibition of cell‐cell adhesion by divalent antibodies exists and should be considered before proposing a direct role for a protein in adhesion.
1,25‐dihydroxyvitamin D3 and macrophage colony‐stimulating factor‐1 synergistically phosphorylate talin
Authors: Meenakshi T. , Ross F. Patrick , Martin John , Teitelbaum Steven L. 
Published: May 2005

AbstractMacrophage colony stimulating factor (CSF‐1) and 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3) are potent inducers of macrophage differentiation. Both appear to modulate protein phosphorylation, at least in part, through protein kinase C (PKC) raising the question as to whether they concurrently impact on macrophage‐like cells. In this regard, we utilized the CSF‐1 dependent murine macrophage‐like line BAC 1.25F5. CSF‐1 treatment of these cells for 30 min leads to particular phosphorylation of a 165 kDa protein, the putative CSF‐1 receptor, and a 210 kDa moiety. 1,25(OH)2D3 exposure for 24 h prior to addition of CSF‐1 enhances phosphorylation of the 165 kDa species and, especially, the 210 kDa protein. Phosphorylation of the latter protein is 1,25(OH)2D3 dose‐ and time‐dependent and the molecule is specifically immunoprecipitated with a rabbit polyclonal anti‐talin antibody. Experiments with okadaic acid show that the enhanced phosphorylation of talin does not result from serine phosphatase inhibition. CSF‐1 and 1,25(OH)2D3, alone or in combination, do not increase talin protein expression. The tyrosine kinase inhibitor, genestein, blocks 1,25(OH)2D3/CSF‐1 induced phosphorylation of the putative CSF‐1 receptor but has no effect on talin phosphorylation which occurs exclusively on serine. In contrast to genestein, staurosporin, an inhibitor of PKC, inhibits phosphorylation of talin. Moreover, exposure of 1,25(OH)2D3 pretreated cells to phorbol 12‐myristate 13‐acetate (PMA) in place of CSF‐1 also prompts talin phosphorylation. Finally, 1,25(OH)2D3 enhances 3[H]PDBu binding, indicating that the steroid increases PMA receptor capacity. Thus, CSF‐1 and 1,25(OH)2D3 act synergistically via PKC to phosphorylate talin, a cytoskeletal‐associated protein.