bluuebunny/crossref_metadata_2025_split huggingface dataset:
B16 mouse melanoma cells selected for resistance to cyclic AMP‐mediated growth inhibition are cross‐resistant to retinoic acid‐induced growth inhibition
Authors: Niles Richard M. , Loewy Barbara 
Published: February 2005

AbstractB16 mouse melanoma cells are grown inhibited by cyclic AMP or by retinoic acid (RA). However, the combination of these two agents results in less growth inhibition than either agent alone. In order to investigate this interaction, cells were selected for resistance to 8‐bromo‐cyclic AMP‐induced growth inhibition. Two clones (3 and 7) which demonstrated significant resistance were isolated. When these two clones were treated with retinoic acid (RA) it was observed that they also exhibited different degrees of resistance to this growth inhibitor. This cross‐resistance did not appear to be due to a lack of uptake or retention of the respective inhibitors, since the mutants took up and retained more 3H‐cAMP and 3H‐RA than wild type cells, suggesting that the dual resistance was not due to an amplification of P‐glycoprotein. The mutation confering cAMP‐resistance did not appear to involve cyclic AMP‐dependent protein kinase, since both catalytic activity and the amount of cAMP protein binding was similar in wild type and mutants. Thus, the mutation must be beyond the interaction of cAMP with cAMP‐dependent protein kinase. We have previously reported that RA induces protein kinase C in B16 melanoma cells (Miles and Loewy: Cancer Res. 49:4483–4487, 1989). Therefore, we measured the ability of RA to induce protein kinase C in the cyclic AMP‐resistant mutants. We found an inverse correlation between RA‐induced protein kinase C activity and growth inhibition in these mutants. The data reported here suggest that cyclic AMP regulates some step in the RA signal transduction pathway.
Proteoglycan and glycosaminoglycan synthesis by cultured rat mesangial cells
Authors: Groggel Gerald C. , Hovingh Peter , Linker Alfred 
Published: February 2005

AbstractThe synthesis of metabolically labeled proteoglycans and glycosaminoglycans from medium, cell layer and substrate attached material by rat glomerular mesangial cells in culture was characterized. The cellular localization of the labeled proteoglycans and glycosaminoglycans was determined by treating the cells with Flavobacterial heparinase. Of the total sulfated glycosaminoglycans, 33% were heparan sulfate; 55% of the cell layer material was heparan sulfate; 80% of sulfated proteins in the medium were chondroitin sulfate/dermatan sulfate. Putative glycosaminoglycan free chains of heparan sulfate and chondroitin sulfate were found in both the medium and cell layer; 95% of total proteoglycans and most (90%) of the putative heparan sulfate free chains were removed from the cell layer by the heparinase, whereas only 50% of the chondroitin sulfate and 25% of dermatan sulfate were removed. Large amounts of hyaluronic acid labeled with 3H glucosamine were found in the cell layer. In summary, approximately 60% of total sulfated glycoproteins was in the form of putative glycosaminoglycan free chains. Thus rat mesangial cells may synthesize large amounts of putative glycosaminoglycan free chains, which may have biological functions in the glomerulus independent of proteoglycans.
Basic fibroblast growth factor is efficiently released from a cytolsolic storage site through plasma membrane disruptions of endothelial cells
Authors: Muthukrishnan Lakshmi , Warder Elizabeth , McNeil Paul L. 
Published: February 2005

AbstractCells of gut and skin frequently suffer mechanically‐induced plasma membrane disruptions in vivo, and bioactive molecules, including basic fibroblast growth factor (bFGF), could enter and leave cytoplasm through these disruptions. We here provide three lines of evidence that bFGF is released with surprising efficiency through plasma membrane disruptions, resembling those known to occur in vivo, produced by scraping endothelial cells from their culturing substratum. First, 41% of the total of bFGF extractable in 1 M NaCl by freeze‐thaw and sonication was released simply by scraping the endothelial cells. Second, relative to release of lactate dehydrogenase, cells wounded by scraping under conditions promoting >60% cell survival released a significantly larger amount (up to twofold more) of growth promoting activity than did cells uniformly killed and irreversibly permeabilized by scraping in the cold or by freezing and thawing. Last, cells that survived membrane disruptions released, and contained, less bFGF on each subsequent wounding, consistent with release of bFGF through transient (i.e., survivable) membrane disruptions. A polyclonal antibody against bFGF completely neutralized the growth promoting activity released by scraping, confirming that bFGF is released through endothelial cell plasma membrane disruptions. Cell fractionation and immunolocalization, including a novel permeabilization technique for electron microscope immunolocalization, demonstrated a cytosolic location of bFGF. We conclude that many characteristics of bFGF–its broad spectrum of producing and target cell types, cytosolic location, efficient release through biologically and pathologically relevant plasma membrane wounds, and its release from cells that survive membrane wounds–make it a strong candidate as a “wound hormone” for rapidly initiating the cell growth required for routine maintenance of tissue integrity and/or repair after injury.
Prolactin induces proliferation of vascular smooth muscle cells through a protein kinase C‐dependent mechanism
Authors: Sauro Marie D. , Zorn Nancy E. 
Published: February 2005

AbstractThe effects of prolactin (PRL) on A10 (aortic smooth muscle) cell proliferation were examined by measuring both [3H] thymidine incorporation and increases in cell number. PRL induced a significant proliferative response from 10−11 to 10−7 M, with optimal activity at 10−10 M. PRL also enhanced platelet‐derived growth factor (PDGF)‐induced proliferation. The possibility that PRL induces proliferation through a protein kinase C (PKC)‐mediated mechanism was also examined. PRL caused activation of PKC from 10−12 to 10–8 M. Antiserum to PRL, a monoclonal antibody directed against the PRL receptor and the immunosuppressive agent cyclosporine A, were able to inhibit PRL‐induced proliferation and activation of PKC. The PKC inhibitors, staurosporine, sphingosine, and 1‐(‐5‐isc‐quinoline‐sulfonyl)‐2‐methylpiperazine (H‐7) also antagonized both proliferation and PKC activation. These data strongly suggest that PRL‐induced A10 cell proliferation is mediated through the PKC pathway and that this may play a role in vascular smooth muscle cell hyperplasia, characteristic of the pathogenesis of cardiovascular diseases such as hypertension and atherosclerosis.
Three forms of rat basic fibroblast growth factor are made from a single mRNA and localize to the nucleus
Authors: Powell Penelope P. , Klagsbrun Michael 
Published: February 2005

AbstractThe molecular weight of rat basic fibroblast growth factor is predicted to be 18 kDa when the amino acid sequence is read from the single AUG initiation codon found in the cDNA. DNA sequencing upstream of this AUG codon indicated, however, that there was an extended open reading frame. In vitro translation of the rat cDNA for basic F‐GF gave three proteins of 18.0, 21.5, and 22.0 kDa in equal abundance. The same proteins were produced in vivo by COS cells transfected with the rat cDNA. Deletion of 81 base pairs from the reading frame upstream of the AUG codon resulted in the expression of only one protein observed at 18.0 kDa. These results indicated that the 22.0 and 21.5 kDa forms of rat basic FGF were formed when translation initiates at the alternative upstream non‐AUG codons. Rat cell lines and tissues were found to express all three forms of basic FGF protein. The cDNA was used to analyze the subcellular distribution of the different forms of rat basic FGF. Sub cellular fractionation and immunoflu‐orescence of transfected COS cells showed that all three forms of the protein localized preferentially in the nucleus. Expression of a truncated cDNA from which 81 base pairs (27 amino acids) of the upstream reading frame had been deleted, showed localization of the smaller form of bFGF alone in the nucleus. These results demonstrated that although the amino acids that were deleted from the N‐terminus of rat basic fibroblast growth factor have a sequence characteristic of nuclear localization motifs, they are not obligatory for the transport of the growth factor into the nucleus. Nuclear extracts taken from transfected cells also contained two smaller proteins of 16 and 12 kDa that were detected by Western blot analysis. It is possible that these are proteolytic products of bFGF.
Growth modulation by epidermal growth factor (EGF) in human colonic carcinoma cells: Constitutive expression of the human EGF gene
Authors: Huang Shuang , Lin Pin‐Fang , Fan Dominic , Price Janet E. , Trujillo Jose M. , Chakrabarty Subhas 
Published: February 2005

AbstractThe functional role of epidermal growth factor (EGF) in epithelium‐derived human colonic carcinoma cells was investigated by transfection with plasmid pUCDS3, which contained synthetic human EGF encoding sequences, into two human colonic carcinoma cell types with dissimilar phenotypic properties: the moderately differentiated and growth factor‐responsive Moser and the highly metastatic KM12SM cells. The Moser cells exhibited a proliferative response to treatment with exogenous EGF, while the KM12SM cells did not. The constitutive expression of the human EGF gene in these colonic carcinoma cell types resulted in elevated expression of EGF mRNA, with concurrent production and secretion of a large amount of EGF, and downmodulation of transforming growth factor‐alpha (TGF‐alpha) secretion. Growth stimulation and down‐modulation of both high and low affinity EGF receptors were observed in the EGF‐transfected Moser clones. Results of experiments using anti‐EGF and anti‐EGF‐receptor antibody to block the proliferation of EGF‐transfected Moser clones suggested that autocrine stimulatory mechanisms involving both EGF and TGF‐alpha were operative in these cells. By comparison, a growth‐inhibitory effect, with no apparent EGF receptor modulation, was observed in the EGF‐transfected KM12SM clones. Both the parental and EGF‐transfected KM12SM clones possessed fewer EGF receptors than the Moser cells, and anti‐EGF or anti‐EGF‐receptor antibody did not affect the cells' growth properties. These results suggested that the mechanisms of growth inhibition in the EGF‐transfected KM12SM clones were non‐autocrine or intracellular in nature. Thus, constitutive expression of the human EGF gene in two phenotypically different, epithelium‐derived human colonic carcinoma cells resulted in divergent altered growth characteristics.
Interleukin‐6 production by the blast cells of acute myeloblastic leukemia: Regulation by endogenous interleukin‐1 and biological implications
Authors: Beauchemin V. , Villeneuve L. , Rodriguez‐Cimadevilla J. C. , Rajotte D. , Kenney J. S. , Clark S. C. , Hoang T. 
Published: February 2005

AbstractCoordinate production of interleukin‐1β (IL‐1β) and granulocyte macrophagecolony stimulating factor (GM‐CSF) or IL‐6 by the blast cells of acute myeloblastic leukemia (AML) and normal peripheral blood leukocytes have been previously reported (van der Shoot et al.: Blood 74:2081–2087, 1989; Bradbury et al.: Leukemia 4:44–47 1990a, British Journal of Haematology 16:(in press), 1990b; Rodriguez‐Cimadevilla et al.: Blood 76:1481–1489, 1990; Schindler et al.: Blood 75:40–47, 1990). In the present study, we show that IL‐6 production by AML blasts is up‐regulated by endogenously produced IL‐1α. Neutralization of the endogenous source of IL‐1 results in a significant decrease in IL‐6 production, as determined by ELISA. Conversely, exposure of AML blasts to IL‐1α results in a significant increase in IL‐6 production in 10 of 16 patient samples. Antibodies against IL‐1α and ‐β also cause a drastic decrease in IL‐6 and GM‐CSF gene expression by the cells, suggesting that cytokine gene expression in AML blasts is driven, at least in part, by endogenous IL‐1. The biologic significance of IL‐6 production in culture of AML blasts has been addressed using a neutralizing antibody against IL‐6. Our data indicate that IL‐6 is important for the survival of clonogenic blasts in culture. In contrast, the survival of the total population of blasts is IL‐6‐independent, as assessed by the integrity of cellular DNA, even in the presence of anti‐IL‐6. These observations are consistent with the view that AML blasts might be organized as a lineage, with comparable hierarchy as in normal hemopoiesis and, perhaps, increased heterogeneity despite a homogenous appearance (McCulloch and Till: Blood Cells 7:63‐77, 1981; Buick and McCulloch: Control of Animal Cell Proliferation. Academic Press, New York, vol. 1, pp. 25–57, 1985). Buick and McCulloch have identified a subpopulation of AML clonogenic cells with stem‐cell‐like properties, and suggested that the majority of blasts may have undergone a determination‐like step. Our data indicate a marked difference in IL‐6 requirement for cell survival between precursors and the majority of blasts, suggesting that IL‐6 responsiveness may decrease following a determination‐like event, i.e., the reduction in proliferative capacity.
Dependence of mouse thymocyte‐erythrocyte rosette formation on complete identity at class‐l‐MHC
Authors: Sem Alfred , Sambhara Suryaprakash , Chadwick Brian S. , Miller Richard G. 
Published: February 2005

AbstractMouse thymocytes and erythrocytes form rosettes when incubated together at 4°. The frequency is much higher when the thymocytes and erythrocytes are MHC‐identical. If the indolizidine alkaloid swainsonine (SW) is present during rosette formation at concentrations of 1 μg/ml (5.7 μM) or greater, rosette formation between MHC‐identical pairs is inhibited to levels comparable to those observed for MHC‐different pairs: rosette formation by MHC‐different pairs is not affected. This was confirmed by examining 17 different MHC‐identical combinations (9 completely syngeneic and 8 differing in non‐MHC genes) and 13 MHC‐different combinations (3 of these identical everywhere except at MHC). A SW‐inhibitable component of rosette formation was observed only when thymocyte and erythrocyte were completely identical at MHC. Thus F1‐parent pairs behaved as if allogeneic, although both F1‐F1 and parent‐parent had a SW‐inhibitable rosetting component. Similarly, inbred strains only partially MHC‐identical (B10.BR‐B10.A, B10.D2‐B10.A) behaved as if allogeneic. The SW‐inhibitable component of rosetting could be partially but significantly blocked by including monoclonal antibodies against Thy‐1, and anti‐CD4 plus anti‐CD8(together but not separately); anti‐class‐l‐MHC produced some inhibition of marginal significance. Monoclonal antibodies against class‐l‐MHC, LFA‐1, and CD3 did not block. Pretreatment of erythrocytes with neuraminidase greatly reduced the SW‐inhibitable component of rosetting. The SW effect would appear to be due to a direct interaction of SW with a cell surface structure involved in syngeneic rosette formation rather than the known ability of SW to block the processing of N‐linked sugar structures. The results are consistent with cell surface lectins and cell surface sugars playing a role in rosette formation.
Transitional change of colony stimulating factor requirements for erythroid progenitors
Authors: Sawada K. , Krantz S. B. , Dai C.‐H. , Sato N. , Ieko M. , Sakurama S. , Yasukouchi T. , Nakagawa S. 
Published: February 2005

AbstractThe course of the differentiation and proliferation of the human erythroid burst‐forming units (BFU‐E) to colony‐forming units (CFU‐E) was directly investigated using a combination of highly purified BFU‐E, a liquid culture system, and the following clonal assay. Highly purified human blood BFU‐E with a purity of 45–79% were cultured in liquid medium with recombinant human erythropoietin (rEP) and recombinant human interleukin‐3 (rlL‐3) to generate more differentiated erythroid progenitors. The cultured cells were collected daily for investigating the morphology, the increment in the number of cells and the clonality. Ninety percent of purified BFU‐E required not only rEP but also rlL‐3 for clonal development. By 7 days of liquid culture, the total cell number increased 237 ± 20‐fold above the starting cells, while erythroid progenitors increased 156 ± 74‐fold. As the incubation time in liquid culture increased, the cells continuously differentiated in morphology. Replating experiments with rTEP combined with or without rlL‐3 showed the following: (1) The number of erythroblasts that were part of erythroid colonies decreased with accompanying erythroid progenitor differentiation and proliferation. (2) As the incubation time in liquid culture increased, erythroid progenitors had a graded loss of their dependency on rlL‐3 and a complete loss of dependency was observed after 3 days of liquid culture. At that time 85% of the erythroid progenitors gave rise to colonies of more than 100 erythroblasts which were equivalent to mature BFU‐E. These studies provide a quantitative assessment of the loss of lL‐3 dependency by BFU‐E and indicate that the size of the generated erythroid colonies and their lL‐3 requirement correlate with the erythroid differentiated state.
Interaction of vasculotropin/vascular endothelial cell growth factor with human umbilical vein endothelial cells: Binding, internalization, degradation, and biological effects
Authors: Bikfalvi A. , Sauzeau C. , Moukadiri H. , Maclouf J. , Busso N. , Bryckaert M. , Plouet J. , Tobelem G. 
Published: February 2005

AbstractVasculotropin/vascular endothelial cell growth factor (VAS/VEGF) is a newly purified growth factor with a unique specificity for vascular endothelial cells. We have investigated the interactions of VAS/VEGF with human umbilical vein endothelial cells (HUVE cells). 125I‐VAS/VEGF was bound to HUVE cells in a saturable manner with a half‐maximum binding at 2.8 ng/ml. Scatchard analysis did show two classes of high‐affinity binding sites. The first class displayed a dissociation constant of 9 pM with 500 sites/cell. The dissociation constant and the number of binding sites of the second binding class were variable for different HUVE cell cultures (KD = 179 ± 101 pM, 5,850 ± 2,950 sites/cell). Half‐maximal inhibition of 125I‐VAS/VEGF occurred with a threefold excess of unlabeled ligand. Basic fibroblast growth factor (bFGF) and heparin did not compete with 125I‐VAS/VEGF binding. In contrast, suramin and protamin sulfate completely displaced 125I‐VAS/VEGF binding from HUVE cells. VAS/VEGF was shown to be internalized in HUVE cells. Maximum internalization (55% of total cell‐associated radioactivity) was observed after 30 min. 125I‐VAS/VEGF was completely degraded 2–3 hr after binding. At 3 hr, the trichloroacetic acid (TCA)‐soluble radioactivity accumulated in the medium was 60% of the total radioactivity released by HUVE cells. No degradation fragment of 125I‐VAS/VEGF was observed. Chloroquine completely inhibited degradation. VAS/VEGF was able to induce angiogenesis in vitro in HUVE cells. However, it did not significantly modulate urokinase‐type plasminogen activator (u‐PA), tissue‐type plasminogen activator (t‐PA), plasminogen activator inhibitor (PAI‐1), and tissue factor (TF). Prostacyclin production was only stimulated at very high VAS/VEGF concentrations. Taken together, these results indicate that VAS/VEGF might be a potent inducer of neovascularization resulting from a direct interaction with endothelial cells. The angiogenic activity seems to be independent of the plasminogen activator or inhibitor system.
Expression of multiple Na+,K+‐ATPase genes reveals a gradient of isoforms along the nonpigmented ciliary epithelium: Functional implications in aqueous humor secretion
Authors: Ghosh Sikha , Hernando Nati , Martín‐Alonso José M. , Martin‐Vasallo Pablo , Coca‐Prados Miguel 
Published: February 2005

AbstractThe Na+,K+‐ATPase α1, α2, and α3 subunit isoforms have been shown to be differentially expressed in the nonpigmented (NPE) and pigmented (PE) cells of the ocular ciliary epithelium (CE)(Martin‐Vasallo et al., J. Cell. Physiol., 141: 243‐252, 1989; Ghosh et al., J. Biol. Chem., 265:2935‐2940, 1990). In this study we analyzed and compared the pattern of expression of the multiple Na+,K+‐ATPase α (α1, α2, α3) subunit genes with the pattern of expression of the Na+,K+‐ATPase β (β1, β2) subunit genes along the bovine CE. We have selected three regions in the CE, referred to as (1) the anterior region of the pars plicata, near the iris; (2) the middle region of the pars plicata; and (3) the posterior region of the pars plana, near the ora serrata. Using isoform‐specific cDNA probes and antibodies for the Na+,K+‐ATPase α1, α2, α3, β1, and β2 subunits on Northern and Western blot analysis, we found that mRNA and polypeptides are expressed in all three CE regions with different abundance. The pattern of expression of α and β isoforms detected along the NPE cell layer suggests a gradient of α1, α2, α3, β1, and β2 mRNAs and polypeptides that correlates with decreasing Na+,K+‐ATPase activity from the most anterior region at the pars plicata towards the posterior region at the ora serrata. We also found marked differences in the pattern of immunolocalization of Na+,K+‐ATPase α1, α2, α3, β1, and β2 subunit isoforms in different regions of the CE. In the anterior region, NPE cells stained intensely at the basal lateral membrane with specific monoclonal and polyclonal antibodies for each of the α (α1, α2, α3) and β (β1, β2) Na,K‐ATPase isoforms. In the middle and posterior regions of the CE, NPE cells showed lower or absent levels of staining with α1, α2, α3, and β1 antibodies, although staining with β2 was abundant. In contrast, PE cells throughout the CE were stained at the basal lateral membrane by antibodies to α1 and β1, while no staining signals were detected with the rest of the antibodies (i.e. α2, α3, and β2). Our results support the conclusion that the three α and two β isoforms of the Na+,K+‐ATPase are differentially expressed in the two cell layers that make up the CE. These results also suggest that the expression of the Na+,K+‐ATPase α and β subunit isoforms may underlie subtle differences along the NPE cell layer in ion transport.