bluuebunny/crossref_metadata_2025 huggingface dataset:
Proteoglycan and glycosaminoglycan synthesis by cultured rat mesangial cells
Authors: Groggel Gerald C. , Hovingh Peter , Linker Alfred
Published: February 2005

The 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

Cells 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

The 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

The 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

The 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

Coordinate 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

Mouse 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

The 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

Vasculotropin/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

The 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.
Surface contact modulation of inflammatory macrophage antibody dependent cytotoxicity and prostanoid release
Authors: Gudewicz Paul W. , Frewin Mary Beth
Published: February 2005

Adherence to extracellular matrix proteins modulates the functional and secretory activities of mononuclear phagocytes, although the mechanisms regulating these adherence‐dependent changes are poorly understood. In this study, the ability of rat inflammatory peritoneal macrophages (PM) to adhere to an endothelial cell‐derived extracellular matrix or a denatured collagen/fibronectin‐coated surface and perform antibody dependent cell cytotoxicity (ADCC) and secrete reactive oxygen intermediates was compared with PM adherent to tissue culture plastic. Prostaglandin E2 (PGE2) and thromboxane B2 (TxB2), two major cyclooxygenase products released by inflammatory macrophages, were also measured by PM adherent to the protein coated surfaces. Rat exudate PM were equally adherent to tissue culture plastic or wells coated with either endothelial cell derived matrix or denatured collagen (gelatin)/fibronectin. PM adherent to a denatured collagen/fibronectin‐coated wells demonstrated significantly less cytolytic activity (15 ± 2% lysis) when compared with either tissue culture plastic adherent PM (43 ± 7% lvsis) or PM adherent to extracellular matrix (59 ± 11% lysis). PM adherent to extracellular matrix released twofold more TxB2 than plastic adherent PM, while PM adherent to denatured collagen/fibronectin released 40% more PGE2 than cells adherent to tissue culture plastic or 80% more PGE2 than PM adherent to the extracellular matrix. PM adherent to denatured collagen/fibronectin release less superoxide anion (27 ± .9 nmoles/106 PM) than PM adherent to either tissue culture plastic (43 ± 1 nmoles/106 PM) or the extracellular matrix (60 ± 0.5 nmoles/106 PM). Furthermore, incubation of plastic adherent PM with exogenous PGE2 reduced superoxide production in a dosedependent manner. These results demonstrate that the inhibition of ADCC and secretion of reactive oxygen intermediates by PM adherent to a denatured collagen/fibronectin surface correlated with an increased release of the immunosuppressive prostanoid PGE2. Furthermore, the addition of exogenous PGE2 to plastic adherent PM reproduced the depression in ADCC and superoxide anion production observed by PM adherent to a denatured collagen/fibronectin surface. These studies suggest that the increased production and release of PGE2 by inflammatory macrophages adherent to a denatured collagen surface may act to suppress cytotoxic mechanisms and thereby constitutes part of an autocrine feedback mechanism regulating macrophage function during wound injury.
Insulin‐like growth factor 1, insulin, and progesterone induce early and late increases in Xenopus oocyte sn‐1, 2‐diacylglycerol levels before meiotic cell division
Authors: Stith Bradley J. , Kirkwood Allan J. , Wohnlich Erica
Published: February 2005

After a 3 to 6 hour incubation, addition of progesterone (the most effective), insulin‐like growth factor 1 (IGF‐1; the second most effective), or insulin induces meiotic cell division in Xenopus oocytes. Measurement of an endogenous activator of protein kinase C, sn‐1,2‐diacylglycerol (DAG), by an enzymatic method recording mass demonstrates that all three hormones alter DAG levels. Five seconds after addition, only progesterone transiently reduces DAG levels by about 25%. At 15 minutes after addition, all three hormones produce a peak of DAG (115% to 160% of control values), with the more effective hormones producing a larger increase in DAG. Insulin produces the smallest DAG increase, but the DAG release is longer lasting. Finally, all three hormones induce a second peak in DAG levels just before white spot appearance (at 0.85 GVBD50, where 1.0 GVBD50 is when 50% of the cells have divided). With these data and since an activator of protein kinase C, a phorbol ester, has been found to induce meiosis, the kinase may play a role in early proliferative events at the plasma membrane and in late events at the nucleus.
Clustering of erythrocytes by fibrinogen is inhibited by carnitine: Evidence that sulfhydryl groups on red blood cell membranes are involved in carnitine actions
Authors: Fritz Irving B. , Wong Katherine , Burdzy Krystyna
Published: February 2005

Carnitine is bound by intact red blood cells, by red blood cell ghosts, and by glutaraldehyde‐fixed human erythrocytes in a non‐saturable, temperature‐dependent manner. Binding of carnitine by these preparations is blocked by sulfhydryl reagents. Incubation or preincubation of red blood cell preparations with carnitine inhibits the aggregation of erythrocytes otherwise elicited by fibrinogen. Identical effects are obtained with red blood cell ghosts. In contrast, choline, even at high concentrations, is inactive in preventing the aggregation of erythrocytes. We discuss possible mechanisms by which carnitine favors the dispersion of red blood cells, and we present data indicating that sulfhydryl groups on erythrocyte membranes are required to permit these carnitine actions to be manifested.
Macrophages express functional receptors for calcitonin‐gene‐related peptide
Authors: Vignery Agnès , Wang Feng , Ganz Michael B.
Published: February 2005

The present study was designed to investigate whether non‐activated macrophages express calcitonin (CT) or calcitonin‐gene‐related peptide (CGRP) receptors. To this end, we first analyzed whether CT and CGRP induce a cAMP accumulation in macrophages. Macrophages were treated for 2 min with increasing concentrations of either CT or CGRP in the presence or absence of IBMX. A dose‐dependent cAMP accumulation was measured in response to CGRP with a half‐maximal effect attained with 1 nM CGRP. CT failed at all doses to induce an accumulation of cAMP. The effects of CT and CGRP on the activation of the Na‐H exchanger were next assessed by spectrofluorometry by using the pH‐sensitive dye 2,7 biscarboxyethyl‐5(6)‐carboxyfluorescein (BCECF). Steadystate pHi of macrophages in a 7.4, HCO3‐free solution (HEPES‐buffered) was 7.04 ± 0.08 (n = 22). pHi recovery following an NH4+/NH3 acid load was inhibited by the removal of Na+ or by the addition of the amiloride analog EIPA; therefore recovery is dependent on Na‐H exchange activity. CT had no effect on steady‐state pHi but CGRP increased pHi in a dose‐dependent fashion (10−12 to 10−6M). The pHi change induced by CGRP was due to the stimulation of the Na‐H exchanger as CGRP enhanced the rate of recovery (dpHi/dt) from an acid load from 45.3 to 77.2 μMs−1 (n = 8, P < 0.002) and was completely blocked by EIPA. These data indicate that CGRP both enhances the activity of the Na‐H exchanger and increases intracellular cAMP, thus demonstrating that macrophages express functional CGRP receptors.
Calcium transport by sarcoplasmic reticulum of vascular smooth muscle: I. MgATP‐dependent and MgATP‐independent calcium uptake
Authors: Stout Marguerit A.
Published: February 2005

The components of 45calcium (Ca) uptake were studied in saponin skinned rat caudal artery. The steady‐state Ca content increased when the free Ca concentration was varied from 10−8 to 10−4 M but was reduced by azide when the free Ca concentration exceeded 3.1 μM. The azide sensitivity and low affinity for Ca were consistent with functional mitochondria. The azide‐insensitive component consisted of a small bound and a larger releasable Ca fraction. After skinning in Triton X‐100, approximately 4 μmol Ca/kg wet tissue remained, which represented a tightly bound but slowly exchangeable Ca pool. The Ca content was independent of the free Ca concentration and MgATP, and it was not released with A‐23187 or Ca. The Ca content of the larger fraction was a higher order function of the free Ca concentration and was released with A‐23187, indicating it resided within a membrane‐bounded structure. Ca uptake by the releasable fraction was increased by oxalate, MgATP, phosphocreatine, temperature, phosphate, and ruthenium red and represents Ca sequestered by the sarcoplasmic reticulum (SR) with little contribution from other Ca binding or storage sites. It is described by the coefficients Umax = 96.94 μmol/kg wet tissue, K1/2 = 0.75 μM, and Hill coefficient = 1.70. The SR in this preparation regulates cytosolic Ca concentrations under physiological conditions and can accumulate Ca by MgATP‐dependent and MgATP‐independent processes. The larger, MgATP‐dependent Cauptake is described by the coefficients Umax = 72.87 μmol/kg wet tissue, K1/2 = 0.8 μM, and Hill coefficient = 2.09 and is consistent with Ca sequestered by the Ca‐transport ATPase of smooth muscle SR. The smaller, MgATP‐independent uptake is described by the coefficients Umax = 24.14 μmol/kg wet tissue, K1/2 = 0.56 μM, and Hill coefficient = 1.01 and represents Ca sequestered by an unidentified mechanism or by a subpopulation of SR.
Stimulation of large proteoglycan synthesis in cultured smooth muscle cells from pig aorta by endothelial cell‐conditioned medium
Authors: Berro Eliane , Breton Monique , Deudon Elisabeth , Picard Jacques
Published: February 2005

We have previously shown (Berrou et al., J. Cell. Phys., 137:430–438, 1988) that porcine endothelial cell‐conditioned medium (ECCM) stimulates proteoglycan synthesis by smooth muscle cells from pig aorta. ECCM stimulation requires protein cores for glycosaminoglycan chain initiation and is accompanied by an increase in the hydrodynamic size of proteoglycans secreted into the medium. This work investigates the mechanisms involved in the ECCM effect. (1) Control and ECCM stimulated proteoglycan synthesis (measured by a 20 min [35S]‐sulfate labeling assay) was not inhibited by cycloheximide, indicating that the proteoglycans were composed of preexisting protein cores and that ECCM stimulates glycosylation of these protein cores. (2) Whereas ECCM stimulation of [35S]‐methionine incorporation into secreted proteins only occurred after a 6 h incubation, the increase in [35S] methionine‐labeled proteoglycans was observed after 1 h, and the increase was stable for at least 16 h. (3) As analysed by electrophoresis in SDS, chondroitinase digestion generated from [14C] serine‐labeled proteoglycans 7 protein cores of high apparent molecular mass (550–200 kDa) and one of 47 kDa. The two protein cores of highest apparent molecular masses (550 and 460 kDa), but not the 47 kDa protein cores, showed increased [14C]‐serine incorporation in response to ECCM (51%, as measured by Sepharose CL‐6B chromatography). (4) Finally, incorporation of [35S]‐sulfate into chondroitinase‐generated glycosaminoglycan linkage stubs on protein cores was determined by Sepharose CL‐6B chromatography: ECCM did not modify the ratio [35S]/[14C] in stimulated protein cores, indicating that ECCM did not affect the number of glycosaminoglycan chains. The results of these studies reveal that (1) endothelial cells secrete factor(s) that preferentially stimulate synthesis of the largest smooth muscle cell proteoglycans without structural modifications and (2) the stimulation proceeds via increased glycosylation of protein core through enhancement of xylosylated protein core, followed by enhanced protein synthesis.
Gene activation mediated by protein kinase C in human macrophage and teratocarcinoma cells expressing aminoglycoside phosphotransferase activity
Authors: Maio Joseph J. , Brown Fred L.
Published: February 2005

The bacterial neomycin phosphotransferase gene driven by the Moloney mouse leukemia virus long terminal repeat (LTR) or SV40 early region promoter was introduced into the human promonocyte‐macrophage cell line, U937, and into the pluripotential human embryonic teratocarcinoma cell line, NT2/D1. Clonally derived cell lines capable of growing in 2–4 mg/ml of the aminoglycoside antibiotic, G418 (Geneticin), were established and transfected with pHIVCat, a plasmid expressing the bacterial chloramphenicol acetyl transferase (CAT) activity under the control of the human immunodeficiency virus (HIV‐1) LTR. All of the G418 resistant (neor) U937 cell lines and 10 of 14 neor NT2/D1 cell lines exhibited reduced basal levels of CAT expression or impaired responses to activation of the HIV‐1 LTR by phorbol 12‐myristate 13‐acetate (PMA) when compared to the parental lines. Other differences included inhibition of tat activation of the HIV‐1 LTR and increased sensitivity of U937 cells to human tumor necrosis factor α. The expression of other eukaryotic promoters including the HTLV‐1 LTR, SV40 ori sequences, and the human β‐actin gene promoter was similarly affected. However, differentiation of the neor U937 cells into macroph‐ages was neither delayed nor impaired. Because PMA is an activator of protein kinase C (PKC) and a potent inducer of HIV‐1 direcred gene expression, the amounts, sensitivity to G418, and cytosol to membrane translocation of this enzyme were determined in the wild type and neor U937 cells. G418 at concentrations too low to affect cell growth (12–150 μg/ml) inhibited PMA‐induced transactivation responses in wild type cells but did not inhibit PKC‐dependent protein phosphorylation in vitro. PKC activities in the wild type and neor cells were similar in absolute amounts and in the cytosol‐membrane distribution of the enzyme. In contrast with wild type cells, however, all of the cytosolic Ca2+‐phospholipid‐dependent form of PKC disappeared from the neor cells within 30 min after PMA induction. The results suggested that, Depending upon the cell type, gene cotransfer using aminoglycoside resistance as a selectable marker may seriously perturb important cellular control mechanisms such as the PKC pathway leading to activation of gene expression.
Cell cycle dependent alterations of chromatin structure in situ as revealed by the accessibility of the nuclear protein AF‐2 to monoclonal antibodies
Authors: Pfeffer U. , di Vinci A. , Geido E. , Vidali G. , Giaretti W.
Published: February 2005

We have recently described a novel nuclear antigen, AF‐2, which is related to cell cycle dependent alterations of chromatin structure. We show by two parameter flow cytometry on a cell by cell basis that the antigen is accessible to specific monoclonal antibodies only in mitotic and postmitotic early G1‐phase cells. The evaluation of nuclease susceptibility and AF‐2 antigen accessibility reveals different subcompartments of the G1 ‐phase of the cell cycle with distinct chromatin conformations.

Digestion with DNase I seems to alter the chromatin structure according to concentration and this is reflected by an increase of the antigen accessibility. Chromatin in the more condensed early G1‐phase is specifically digested by lower concentrations of the enzyme than chromatin in later stages of interphase. Chromatin from cells in the late‐G1, S‐, and G2‐phases shows a higher relative resistance to DNase I and a reduced accessibility of the AF‐2 antigen to monoclonal antibodies. Nuclease S1 has a similar effect on chromatin topology, as revealed by the reaction with anti‐AF‐2 antibodies, without digestion of detectable amounts of DNA. The antigen becomes available to the antibodies in almost all cells by digestion with high concentrations of DNase I or Nuclease S1.
Karyotypic and phenotypic changes during in vitro aging of human endothelial cells
Authors: Johnson Timothy E. , Umbenhauer Diane R. , Hill Rosina , Bradt Carole , Mueller Stephen N. , Levine Elliot M. , Nichols Warren W.
Published: February 2005

Karyotypic and phenotypic changes were found in human adult endothelial cells (EC) during aging in vitro. A trisomy of chromosome 11 was found in 11 out of 12 EC cultures examined, derived from 9 cell lines from 8 donors. The incidence of this trisomy in some cell lines increased over time from 0% to as much as 100% near the end of their in vitro life span. A number of oncogenes and other important genes are on chromosome 11. These genes might play a role in the changes observed. An increase in the percentage of polyploid cells was also found near the end of the in vitro life span in 6 lines. The cellular levels of two gene products characteristic of the EC, von Willebrand factor (vWF) or Factor VIII, and angiotensin converting enzyme (ACE) were also monitored. vWf was studied in 2 lines and was decreased in both with serial passage. ACE decreased in three out of the four lines examined. These chromosomal and phenotypic changes which occur with increasing age in vitro make the endothelial cell a suitable model to study in vitro culture‐related changes, senescence, cardiovascular disease, and tumorigenesis.
Dibutyryl‐cAMP increases functions of 5‐hydroxytryptamine2 receptors, but not of β2‐adrenergic receptors, in a clonal cell line of rat neurotumor RT4
Authors: Shigeri Yasushi , Watanabe Sachihiko , Fujimoto Masafumi
Published: February 2005

A peripheral nervous system cell line RT4‐B, established by lmada and Sueoka (Dev. Biol., 66:97–108, 1978), was shown to respond to serotonin [5‐hydroxy‐tryptamine (5‐HT)] and catecholamines. 5‐HT induced a small and transient increase in cytosolic free Ca2+ concentration ([Ca2+]i) in the RT4‐B cells. The increase was effectively blocked by 5‐HT2 receptor antagonists (spiperone, ritanserin and mianserin), but not by a 5‐HT3 receptor antagonist (MDL72222), or a α1‐adrenergic receptor antagonist (prazosin), indicating that RT4‐B cells express 5‐HT2 receptors. On the other hand, catecholamines increased cyclic AMP production by RT4‐B. The order of potency for stimulating cyclic AMP synthesis was isoproterenol > epinephrine ≫ norepinephrine ≫ dopamine, and the stimulation was effectively inhibited by the nonselective β‐adrenergic receptor antagonist propranolol, but not by the β1‐adrenergic receptor antagonist atenolol, suggesting that RT4‐B cells express β2‐adrenergic receptors.

The differentiating agent N6,2′‐O‐dibutyryladenosine 3′,5′‐monophosphate (dibutyryl‐cAMP) enhanced the 5‐HT‐induced [Ca2+]i increase, but not the catecholamine‐induced cyclic AMP production. The increase in the 5‐HT response paralleled the increase in the density of 5‐HT2 receptors. n‐Butyric acid (2 mM) and 8‐bromoadenosine 3′,5′‐monophosphate (1 mM) also increased the 5‐HT response, and the sum of these increases was nearly equal to that induced by dibutyryl‐cAMP.

These results indicate that RT4‐B is a novel model cell line for the study of 5‐HT2 and β2‐adrenergic receptors and their second messenger responses and for the analysis of the mechanisms how 5‐HT2 receptor gene expression is controlled.
Sensitivity of cultured and skinned chick myotube to calcium, strontium, and barium ions examined by recording isometric contractions
Authors: Saito Koji , Yoshida Mikiharu , Tanaka Hikaru
Published: February 2005

Sensitivity of cultured chick myotubes to alkaline earth metal ions was investigated by recording contractile isometric tension through a semiconductor transducer. The myotubes were obtained by culturing myoblasts of chick embryo breast muscles, and skinned chemically before physiological experiments. Contractions developed in response to Ca2+ in a bathing medium higher than 3 × 10−7 M and reached maximum at 1 × 10−5 M. Sr2+ was less effective than Ca2+; the threshold concentration was 1 × 10−5 M and the tension reached maximum at 1 × 10−3 M. Ba2+ was the least effective among the three alkaline earth metal ions; only one fifth of the Ca2+‐induced maximum tension was attained at 1 × 10−3 M. The sensitivity was similar to that of the mature pectoral muscle fiber, a fast twitch muscle fiber, rather than that of the anterior latissimus dorsi, a slow tonic muscle fiber. The sensitivity was shown to be dependent on its troponin C by replacing it with troponin C from the mature pectoral or cardiac muscle. This indicates that TnC of a fast‐muscle type is expressed in the cultured chick myotube as in the mature pectoral muscle. The contractile apparatus was thus shown to be well developed in the cultured myotube with characteristics similar to the mature fast twitch muscle fiber.
Tumor necrosis factor stimulates DNA synthesis of mouse hepatocytes in primary culture and is suppressed by transforming growth factor β and interleukin 6
Authors: Satoh Motonobu , Yamazaki Masatoshi
Published: February 2005

In a previous study, we revealed that tumor necrosis factor (TNF) was secreted in mouse liver at an early phase of liver regeneration after partial hepatectomy. Here, we investigated direct actions of TNF on the in vitro DNA synthesis of adult mouse hepatocytes in primary culture. TNF enhanced both 3H‐TdR uptake and the number of 3H‐TdR‐labeled nuclei of hepatocytes. Their time courses were similar to those by epidermal growth factor (EGF) with about a 15 h lag period and a peak period of 24–48 h. This action of TNF was abrogated by DNA polymerase α inhibitor, aphidicolin and blocked specifically by anti‐TNF antibody. The actions of rm TNF were not distinguishable; ED50 was about 7.5U/ml (5ng/ml) and 30U/ml (20ng/ml) for maximal response (about 2‐fold or more of control). Other inflammatory monokines showed differential effects on in vitro DNA synthesis of hepatocyte. Neither type of interleukin 1 affected hepatocyte DNA synthesis in the range examined (up to 50 ng/ml). IL‐6 markedly inhibited the hepatocyte DNA synthesis stimulated by TNF and EGF. The action of TNF was completely suppressed by transforming growth factor β, which is known as a potent inhibitor of hepatocyte growth. Interferon γ also blocked this TNF action when added simultaneously. These results indicate that the activation of tissue macrophages and local secretion of TNF in liver after partial hepatectomy is of physiological importance in liver regeneration, in part by a direct stimulation of hepatocyte DNA synthesis. Cytokines induced by TNF may also participate in the later termination of liver regeneration.
Keratinocytes stimulate prostaglandin I2 synthesis by 3T3 cells and exhibit enhanced cornification when exposed to prostaglandin I2 analogues
Authors: Baden Howard P. , Goldaber Michael L. , Kvedar Joseph C.
Published: February 2005

The predominant cyclooxygenase products of keratinocytes are prostaglandin (PG)E2 and PGF2α with only trace amounts of PGI2 synthesis detected. When normal or immortal (NM1) keratinocytes were co‐cultured with mitomycin C‐treated 3T3 cells, increased synthesis of PGI2 was noted compared to mitomycin C‐treated 3T3 cells alone. The PGI2 level in co‐cultures was maximum within the first week and diminished rapidly thereafter. These results suggested keratinocytes enhance the production of PGI2 by 3T3 cells. Keratinocyte cultures incubated with lloprost and Piriprost, stable PGI2 analogues, showed evidence of increased cornification as demonstrated by staining with rhodanile blue, decreased shedding of cells into the culture medium, and more cornified material adhering to the culture surface. The cultures appeared to be responsive between the first and second weeks after plating and the inhibition of shedding could not be reversed by changing to drug‐free medium. Control and treated cultures showed identical electrophoretic protein patterns. Immunoblots showed involucrin unchanged in extracts of control and treated cultures while the 22 kd pancornulin was absent in treated cultures. The findings that keratinocytes enhance the production of PGI2 by 3T3 cells and that PGI2 analogues enhance cornification of confluent keratinocytes raise the possibility that eicosanoids may serve as autoregulatory signals together with other factors.
Abnormal extracellular matrix and excessive growth of human adult polycystic kidney disease epithelia
Authors: Wilson Patricia D. , Hreniuk David , Gabow Patricia A.
Published: February 2005

Human autosomal dominant polycystic kidney disease (ADPKD) epithelia were grown in primary monolayer cultures and their properties compared with intact kidney epithelial cultures derived from individually microdissected normal human kidney proximal convoluted tubules (PCT), proximal straight tubules (PST), and cortical collecting tubules (CCT). In vivo, ADPKD cyst epithelia exhibited a thickened basement membrane, and immunofluorescence demonstrated the presence of laminin, fibronectin, type IV collagen, and heparan sulfate proteoglycan in basement membranes and type I collagen in the interstitium. ADPKD epithelia grown in culture synthesized and secreted basally a unique, extracellular matrix that took the form of proteinaceous spheroids when the cells were grown on dried, type I collagen. Incorporation of H2[S35O4] into basement membrane extracts was increased more than ten‐fold in ADPKD epithelia by comparison to normal PST and CCT. In addition to incorporation into the normal tubular basement membrane 220 kD band, radioactivity was also seen at 175 kD and 150 kD in ADPKD extracts. Growth in culture of cyst‐lining ADPKD epithelia was more rapid than normal tubules, and was abnormal since there was no absolute requirement for added extracellular matrix. However, when ADPKD epithelia were grown on different, exogenous matrix protein components, a profound influence on both structure and epithelial cell proliferation was seen. Growth on a complete basement membrane three‐dimensional gel derived from the Engelbreth‐Holm‐Swarm (EHS) sarcoma led to a reduction in the numbers of spheroids and increase in amorphous filaments. Incorporation of [3H]‐thymidine into ADPKD epithelia was greater than into normal PCT, PST, and CCT and was also greatly modified by the type of extracellular matrix components provided. In studies using single matrix components, the strongest proliferative response was seen when ADPKD epithelia were plated on type I collagen > type IV collagen > fibronectin > laminin. These findings suggest that the excessive growth of cystlining epithelia may be, at least in part, a result of abnormal basement membrane and extracellular matrix production by ADPKD cells.
Treatment of myeloid leukemic cells with the phosphatase inhibitor okadaic acid induces cell cycle arrest at either G1/S or G2/M depending on dose
Authors: Ishida Yuji , Furukawa Yusuke , Decaprio James A. , Saito Masaki , Griffin James D.
Published: February 2005

The phosphatase inhibitor okadaic acid was found to induce cell cycle arrest of human myeloid leukemic cell lines HL‐60 and U937 in a concentration‐ and time‐dependent manner. Exposure to low concentrations of okadaic acid (2–8nM) for 24–48 hr caused >70% of cells to arrest at G2/M, with up to 40% of the cells arrested in early mitosis. Cell viability decreased rapidly after 48 hr of treatment, and morphological and DNA structure analysis indicated that this was primarily due to the induction of apoptosis. The cells arrested in mitosis by 8 nM okadaic acid could be highly enriched by density gradient centrifugation and underwent apoptosis when further cultured either with or without okadaic acid, indicating that the effects of okadaic acid were irreversible. In contrast to the effects of low concentrations of okadaic acid, high concentrations (500 nM), inhibited proliferation in less than 3 hr. Remarkably, the majority of cells also entered a mitosis‐like state characterized by dissolution of the nuclear membrane and condensation and partial separation of chromosomes. However, these cells had a diploid content of DNA, indicating that the cell cycle arrest occurred at G1/S with premature chromosome condensation (PCC), rather than at G2/M. If cells were first blocked at G1/S with hydroxyurea and then treated with okadaic acid, >90% developed PCC in less than 3 hr without replicating their DNA. Caffeine was not able to induce PCC in these cells, either with or without prior inhibition of DNA synthesis. These results provide further evidence that an interacting system of cell cycle associated phosphatases and kinases control the initiation of each phase of the cell cycle in higher eukaryotes and further suggest that okadaic acid will be useful in manipulating the cell cycle of leukemic cells.