bluuebunny/crossref_metadata_2025 huggingface dataset:
Synergistic actions of epidermal growth factor‐urogastrone and vasopressin in cultured aortic A‐10 smooth muscle cells
Authors: Mokashi Shalini , Severson David L. , Hollenberg Morley D.
Published: February 2005

In cultured rat aorta‐derived A‐10 cells, epidermal growth factor‐urogastrone (EGF‐URO) acts synergistically with arginine vasopressin (AVP) to augment the AVP‐mediated release of 3H‐arachidonate (3H‐AA) from 3H‐AA prelabeled cells. On its own, EGF‐URO had no effect on AA release and had no effect on calcium influx or efflux either in the absence or presence of AVP. The synergistic action of EGF‐URO was not affected by actinomycin D, cycloheximide, indomethacin, by the diacylglycerol lipase inhibitor U‐57, 908, or by the tyrosine kinase inhibitors genistein (GS) and tyrphostin (TP). TP did, nonetheless, completely abrogate 3H‐thymidine incorporation triggered in the presence of EGF‐URO. Although EGF‐URO stimulated an increase in calpactin‐II (lipocortin‐I) phosphorylation in permeabilized cells, no such increase was detected in intact cells exposed to EGF‐URO either alone or in combination with AVP, under conditions where EGF‐URO augmented the action of AVP. The phospholipase A2 inhibitor, mepacrine, had no effect on AVP‐mediated AA release, but abolished the synergistic action of EGF‐URO. We conclude that in contrast with our previous results with gastric smooth muscle strips, wherein EGF‐URO acts via the diacylglycerol lipasemediated metabolism of diacylglycerol, and in keeping with observations with cultured mesangial cells, EGF‐URO acts synergistically with AVP in A‐10 cells via the activation of phospholipase A2. This synergistic action of EGF‐URO does not appear to be due to increased levels of cyclooxygenase and would appear not to require increased tyrosine kinase activity. © 1992 Wiley‐Liss, Inc.
Inhibition of angiogenesis in vitro and in ovo with an inhibitor of cellular protein kinases, MDL 27032
Authors: Wright Paul S. , Cross‐Doersen Doreen , Miller Jerry A. , Jones Winton D. , Bitonti Alan J.
Published: February 2005

Protein kinase C (PKC) was implicated as an important positive regulator of angiogenesis by studies showing that tumor promoting phorbol esters, which activate PKC, stimulate angiogenesis both in vitro and in vivo. Therefore, inhibitors of PKC might be expected to block angiogenesis. MDL 27032 [4‐propyl‐5‐(4‐pyridinyl)‐2(3H)‐oxazolone], an inhibitor of cellular protein kinases, prevented capillary‐like tube formation by human umbilical vein endothelial cells (HUVEC) on basement membrane preparations, an in vitro model for angiogenic activity. MDL 27032 had an IC50 = 50 μM, whereas MDL 27044, the 4‐methyl analog of MDL 27032, was less effective (IC50 > 100 μM). This selectivity was reflected in the relative abilities of the two compounds to inhibit PKC and protein kinase A (PKA) activity prepared from HUVEC, and also to inhibit the basic fibroblast growth factor stimulated proliferation of HUVEC. MDL 27032 (0.3 μg/egg) also significantly inhibited neovascularization in yolk sac membranes of developing chick embryos, whereas MDL 27044 added at concentrations up to 3 μg/egg was not inhibitory when compared with vehicle treated controls. Adhesion of HUVEC to individual extracellular matrix proteins, including laminin, fibronectin, and fibrinogen, but not to the mixture of matrix components or collagen type I and IV, was inhibited after treatment with MDL 27032. These studies suggest that MDL 27032, may have potential as an anti‐angiogenic agent because it disrupts both formation of tube‐like structures by HUVEC on Matrigel and normal neovascularization in ovo. This inhibition may in part be due to altered cellular interactions with the extracellular matrix. © 1992 Wiley‐Liss, Inc.
Temporal studies on the tissue compartmentalization of bone sialoprotein (BSP), osteopontin (OPN), and SPARC protein during bone formation In Vitro
Authors: Kasugai Shohei , Nagata Toshihiko , Sodek Jaro
Published: February 2005

To study the role of noncollagenous proteins in bone formation, the synthesis and tissue distribution of BSP (bone sialoprotein), OPN (osteopontin) and SPARC (secreted protein acidic and rich in cysteine) were analyzed using pulse‐chase and continuous labeling protocols during bone formation by cultures of rat calvarial cells. Following a 1 h labeling period with [35S]methionine or [35SO4], radiolabeled BSP was rapidly lost from the cells and appeared transiently in the culture medium and in a 4 M GuHCI extract (G1) of the mineralized tissue. Coinciding with the loss of BSP from these compartments, radiolabeled BSP increased in demineralizing, 0.5 M EDTA extracts (E) of the bone, in a subsequent GuHCI extract (G2), and in a bacterial collagenase digest (CD fraction) of the extracted tissue, over a 24 h chase period. In comparison, the 55 kDa form of OPN, with a small amount of the 44 kDa OPN, was secreted almost entirely into the culture medium. Most of the 44 kDa OPN, together with some 55 kDa OPN, accumulated rapidly in the E extract but could not be detected in either G extract or in the CD fraction. SPARC appeared transiently in the G1 extract, but was otherwise quantitatively secreted into the culture medium from where it was lost by complexing and/or degradation. When cultures were continuously labeled over a 12 day period with [35S]methionine, radiolabeled BSP and 44 kDa OPN accumulated in the E extract together with a small amount of SPARC. Some radiolabeled BSP also accumulated in the G2 extract. From the relative incorporation of [35SO4] over the same time period, a time‐dependent loss in sulphate from the BSP was evident. Using a 24 h pulse‐labeling protocol, the amount of radiolabeled BSP and OPN in the E extract and the BSP in the G2 extract were not altered significantly over a 12‐day chase period. These studies demonstrate that the 44 kDa OPN and most of the BSP are rapidly bound to the hydroxyapatite crystals where they may regulate crystal formation and growth during bone formation. Some BSP is deposited in the osteoid and appears to become masked by the formation of hydroxyapatite, indicating a potential role for this protein in epitactic nucleation of hydroxyapatite crystal formation. © 1992 Wiley‐Liss, Inc.
Macrophage membrane glycoprotein binding of Griffonia simplicifolia I‐B4 induces TNF‐alpha production and a tumoricidal response
Authors: Tabor Dale R. , Theus S. A. , Barnett John B. , Jacobs Richard F.
Published: February 2005

Thioglycollate‐elicited macrophages (mϕ), upon binding the lectin Griffonia simplicifolia IB4 (GSIB4) at the plasma membrane, are induced to secrete several low molecular weight proteins. In this investigation, results from specific ELISA and immunoprecipitation analysis of these molecules confirmed that the cytokine, tumor necrosis factor‐α (TNF‐α), belongs to the group of elicited proteins. This specific mϕ response is directly influenced by the dose of GSIB4 used and the time in contact with the cells. At 40 μg/ml GSIB4, the maximum dose of lectin used, the mϕ activity was equal to that achieved when the cells were incubated with an interferon‐γ/lipopolysaccaride (IFN/LPS) stimulus alone. Moreover, the data showed that TNF‐mediated tumoricidal activity was significantly influenced by GSIB4 binding to the mε membrane. When the lectin was incubated alone or in sequence with IFN/LPS, this ligand‐receptor binding promoted the lysis of WEHI 164 tumor target cells. However, concurrent incubation of both IFN/LPS and GSIB4 with mϕ significantly diminished the tumoricidal response. This suggested that one of the metabolic pathways utilized subsequent to receptor‐ligand binding was altered by these interactions. When cyclic AMP (cAMP) and inositol triphosphate (IP3) levels were examined, the results showed that the concentration of cAMP was unchanged despite the fact that IP3 levels were significantly enhanced upon mϕ‐GSIB4 binding. Collectively, the data show that GSIB4 binding to specific glycoproteins in the mϕ membrane induces TNF‐α production and facilitates TNF‐α dependent tumoricidal responses. It also appears that the transduction of the signal, in part, at least utilizes the phosphatidyl inositol pathway. Finally, it is noteworthy that mϕ activity is influenced by the sequence in which GSIB4 is presented to the mϕ relative to the IFN/LPS treatment. © 1992 Wiley‐Liss, Inc.
C‐Fos expression is hypersensitive to serum‐stimulation in cultured cystic kidney cells from the C57BL/6J‐cpk mouse
Authors: Rankin Carolyn A. , Grantham Jared J. , Calvet James P.
Published: February 2005

Cystic kidneys of the C57BL/6J‐cpk murine model of polycystic kidney disease show a marked overexpression of the proto‐oncogenes c‐fos, c‐myc, and c‐ki‐ras, consistent with an increased rate of cell proliferation and an altered state of differentiation. To determine if cystic cells have increased responsiveness to stimulation with mitogenic agents, quiescent primary cultures from normal and cystic cpk kidneys were treated with fetal bovine serum (FBS), 8‐bromo‐cAMP (cAMP), or epidermal growth factor (EGF). The level of c‐fos induction following stimulation by FBS was found to be dramatically higher in cystic cells than in normal cells; whereas induction by cAMP or EGF was essentially the same in both cell types and much less than that seen in FBS‐stimulated cells. To determine if this serum hypersensitivity reflects an increased proliferative state in vivo, c‐fos induction was examined in cultures derived from normal kidneys stimulated to regenerate by folic acid‐induced acute renal injury. As with cystic kidneys, the folic acid‐injured kidneys showed increased c‐fos responsiveness to FBS in cell culture. These experiments suggest that cystic and regenerating kidneys have an altered phenotypic state in vivo that is manifested in cell culture by serum hypersensitivity. However, whereas the folic acid‐injured kidneys ultimately reestablish normal kidney function, cystic kidneys further progress to renal failure, suggesting that cystic epithelial cells are locked in this altered state of differentiation. © 1992 Wiley‐Liss, Inc.
Relationship between culture conditions and the dependency on mitochondrial function of mammalian cell proliferation
Authors: Van Den Bogert C. , Spelbrink J. N. , Dekker H. L.
Published: February 2005

In cultured mammalian cells, the relationship was investigated between mitochondrial function and proliferation under various culture conditions. Continuous inhibition of the expression of the mitochondrial genome was used to reduce the activity of enzymes involved in oxidative phosphorylation by 50% at every cell division. Under these conditions, culturing in relatively poor media resulted in arrest of the proliferation of most cell lines after 1 cell division. This was preceded by decreasing levels of ATP and increasing levels of ADP, suggesting that the ATP‐generating capacity of the cells was limiting. Culturing in richer media led to arrest of the proliferation after 5 to 6 divisions, but accumulation of ADP was not observed. Addition of pyruvate to rich culture media and, at least for 1 cell line, increasing the CO2 levels, completely prevented proliferation arrest. Inability to synthesise metabolic precursors via mitochondrial intermediary metabolism probably explains growth arrest of cells cultured in rich media. Pyruvate and CO2 were, however, without effect on the proliferation arrest of cells cultured in relatively poor media. Therefore, pyruvate dependency for growth of cells without functional mitochondria holds true only under culture conditions where the ATP‐generating capacity of the cells is not limiting. © 1992 Wiley‐Liss, Inc.
Proliferation‐associated oxygen consumption and morphology of tumor cells in monolayer and spheroid culture
Authors: Bredel‐Geissler A. , Karbach U. , Walenta S. , Vollrath L. , Mueller‐Klieser W.
Published: February 2005

The oxygen consumption rate, proliferative activity, and morphology of EMT6/Ro mouse mammary sarcoma cells in monolayer and multicellular spheroid culture have been investigated in a comparative study. During the transition of monolayer cells from the exponential into the plateau growth phase, there is a distinct decrease in the cellular volume that is associated with a corresponding decrease in the proliferative and respiratory activity of the cells. The decline in cell volume is mainly due to a decrease in the content of cytoplasm, whereas the size of the nucleus is only slightly reduced. A concomitant decrease in the number of mitochondria per cell obviously accounts for the reduction in cellular oxygen uptake. Despite a continuous decrease of cell proliferation from the surface to interior regions of EMT6 spheroids reflected by a gradient in tritiated thymidine labeling, volume‐related oxygen consumption is rather uniform in viable regions of these aggregates. The finding can be explained by the results of the morphometric evaluation showing a uniform volume density of mitochondria, i.e., of oxygenconsuming sites within these spheroids. © 1992 Wiley‐Liss, Inc.
Apoptosis of murine BW 5147 thymoma cells induced by cold shock
Authors: Kruman Inna I. , Gukovskaya Anna S. , Petrunyaka Victor V. , Beletsky Igor P. , Trepakova Elena S.
Published: February 2005

Exposure of thymoma BW 5147 cells to cold (0–2°C) followed by rewarming at 37°C (cold shock) resulted in internucleosomal DNA cleavage. Sensitivity to cold shock‐induced cell death was critically dependent on the serum concentrations in the medium and limited to serum‐deficient medium (2% serum concentration), whereas cells in the complete growth medium (10%) were completely resistant. RNA/protein‐synthesis inhibitors (cycloheximide and actinomicin D) had no effect on cold shock‐induced DNA cleavage in BW 5147 cells. The DNA fragmentation seems to be independent of increase in the cytosolic Ca2+ level. Moreover, reduction in the calcium content of the external medium by EGTA induced DNA cleavage. Incubation of BW 5147 cells in the presence of colchicine and cytochalasin B led to the apoptosis. The latter suggest that the internucleosomal DNA cleavage induced by cold shock may be concerned with the disruption of some cytoskeletal network caused by cooling. The results are discussed in relation to cell proliferation. © 1992 Wiley‐Liss, Inc.
Basic calcium phosphate crystals cause coordinate induction and secretion of collagenase and stromelysin
Authors: McCarthy Geraldine M. , Mitchell Peter G. , Struve Janine A. , Cheung Herman S.
Published: February 2005

Synovial fluid basic calcium phosphate crystals (BCP) are often found in severely degenerated joints. Crystalline BCP is a growth factor stimulating fibroblast mitogenesis and acting as a compentence factor similar to platelet‐derived growth factor. In human fibroblasts (HF), the synthesis of collagenase and stromelysin is coordinately induced after stimulation with a variety of cytokines and growth factors. We sought to determine whether BCP, like other growth factors, might induce protease that would damage articular tissue. Northern blot analysis of mRNA for collagenase and stromelysin in HF stimulated with BCP was performed. Secreted enzymes were analysed by immunoblot using a monoclonal antibody to collagenase and by immunoprecipitation using a polyclonal antibody to stromelysin. Stromelysin activity was confirmed using casein substrate gels. A significant, dose‐dependent accumulation of collagenase and stromelysin message was evident after 4 h had continued for at least 24 h in BCP‐stimulated cultures. Fortynine and 54 kD proteins immunoreacting with collagenase antibody were identified in the conditioned media (CM) from BCP‐stimulated cultures while 50 and 55 kD proteins were identified by immunoprecipitation with stromelysin antibody. Collagenase activity was increased significantly in the CM from BCP treated cells; casein substrate gels showed casein degrading bands at molecular weights consitent with stomelysin. BCP stimulates coordinate induction of collagenase and stromelysin which may mediate the joint destruction associated with these crystals. © 1992 Wiley‐Liss, Inc.
Calcium transport by sarcoplasmic reticulum of vascular smooth muscle: II. Effects of calmodulin and calmodulin inhibitors
Authors: Stout Marguerite A. , Silver Paul J.
Published: February 2005

The role of calmodulin (CaM) in modulating calcium (Ca) uptake by sarcoplasmic reticulum (SR) of vascular smooth muscle was studied in saponin skinned strips of rat caudal artery. Exogenous CaM concentrations ranging 0.3–1.8 μM did not statistically change the steady state MgATP‐dependent Ca content, the MgATP‐independent Ca content, or the oxalate‐stimulated Ca influx. Calmidazolium (CDZ), W‐7, and trifluoperazine (TFP) were used to examine the potential effect of an endogenous CaM pool on inward Ca transport. The IC50 of these antagonists for inhibiton of Ca‐CaM‐stimulated phosphodiesterase activity and Ca‐activated superprecipitiation of canine aortic actomyosin was measured and found to be be in the low micromolar range with a rank order of potency of inhibition of CDZ > TFP > W‐7. In skinned tissues, micromolar concentrations of antagonists that inhibited CaM‐mediated reactions in isolated enzyme systems did not reduce Ca content or oxalate‐stimulated Ca influx. At higher concentration of 100–200 μM, the MgATP‐dependent Ca content was significantly reduced by TFP and W‐7 but not by CDZ. The order of potency for inhibition of Ca uptake was TFP > W‐7 > CDZ. The MgATP‐independent Ca content was significantly decreased only by 200 μM TFP. Although none of these inhibitors significantly altered Ca efflux at concentrations up to 100 μM, Ca release was significantly stimulated by all three at 200 μM. The TFP‐stimulated Ca release was partially inhibited by ruthenium red. The results indicate that neither exogenous CaM nor an endogenous CaM pool directly modulates inward Ca transport by the SR of saponin skinned caudal artery. The inhibition of Ca uptake produced by hundred micromolar concentrations of CaM antagonists fails to correlate with the order of and with the potency of inhibition measured in isolated enzyme systems. This suggests that the inhibition of Ca uptake produced by high concentrations of these antagonists may be independent of a specific interaction with CaM. The activation of Ca release by high concentrations of CaM antagonists may involve a nonspecific increase in membrane permeability as well as modulation of a membrane Ca Chanel. © 1992 Wiley‐Liss, Inc.
Tumor necrosis factor and interleukin 1 inhibit parathyroid hormone‐responsive adenylate cyclase in clonal osteoblast‐like cell by down‐regulating parathyroid hormone receptors
Authors: Katz Michael S. , Gutierrez Gloria E. , Mundy Gregory R. , Caulfield Michael P. , Hymer Tazuko K. , McKee Roberta L.
Published: February 2005

The effects of the monokines tumor necrosis factor α (TNF) and interleukin 1 (IL 1) on parathyroid hormone (PTH)‐responsive adenylate cyclase were examined in clonal rat osteosarcoma cells (UMR‐106) with the osteoblast phenotype. Recombinant TNF and IL 1 incubated with UMR‐106 cells for 48 hr each produced concentration‐dependent inhibition of PTH‐sensitive adeylate cyclase, with maximal inhibition of PTH response (40% for TNF. 24% for IL 1) occuring at 10−8 M of either monikine. Both monokines also decreased adenylate cyclase stimulation by the tumor‐derived PTH‐related Protein (PTHrP). In contrast, TNF and IL 1 had little or no inhibitory effect on receptor‐mediated stimulation of adenylate cyclase by isoproterenol and nonreceptors‐mediated enzyme activation by cholera toxin forskolin; both monokines increased prostaglandin E2 stimulation of adenylate cyclase. Binding of the radiodinated agonistt mono‐[125I]‐[Nle3, 18, Tyr34]bPTH‐(1–34)NH2 to UMR‐106 cells in the presence of increasing concentrations of unlabeled [Nle8,18, Tyr34]bPTH‐(1–34)NH2 revealed a decline in PTH receptor density (Bmax) without change in receptor binding affinity (dissociation constant, Kd) after treatment with TNF or IL 1. Pertusis toxin increased PTH‐sensitive adenylate cyclase activity but did not attenuate monokine‐induced inhibiton of PTH response. In time course studies, brief (1hr) exposure of cells to TNF or IL 1 during early culture was sufficient to decrease PTH response but only after exposed cells were subsequently allowed to grow for prolonged periods. Inhibition of PTH response by monokines was blocked by cycloheximide. The results and indicate that TNF and IL 1 impair responsiveness to PTH (and PTHrP) by a time protein synthesis‐dependent down‐regulation of PTH receptors linked to adenylate cyclase. © 1992 Wiley‐Liss, Inc.
Potassium transport in nonpigmented epithelial cells of ocular ciliary body: Inhibition of a Na+, K+, Cl− cotransporter by protein kinase C
Authors: Crook Richard B. , Von Brauchitsch Douglas K. , Polansky Jon R.
Published: February 2005

The mechanism by which 86Rb+ (used as a tracer for K+) enters human nonpigmented ciliary epithelial cells were investigated. Ouabain‐inhibitable bumetanide‐insensitive 86Rb+ transport accounted for ∼70–80% of total, whereas bumetanide‐inhibitable ouabain‐insensitive uptake accounted for 15–25% of total. K+ channel blockers such as BaCl2 reduced uptake by ∼5%. Bumetanide inhibited 86Rb+ uptake with an IC50 of 0.5 μM, while furosemide inhibited with an IC50 of about 20 μM. Bumetanide‐inhibitable 86Rb+ uptake was reduced in Na+‐free or Cl−‐free media, suggesting that Na+ and Cl− were required for optimal uptake via this mechanism. These characteristics are consistent with a Na+, K+, Cl− contransporter in NPE cells. Treatment of NPE cells for 15 min with phorbol 12‐myristate, 13‐acetate (PMA), an activator of protein kinase C, caused a 50–70% decrease in 86Rb+ uptake via the Na+, K+, Cl− cotransporter. Other 86Rb+ uptake mechanisms were not affected. 86Rb+ uptake via the Na+, K+, Cl− contransporter could be inhibited by other phorbol esters and by dioctanoylglycerol, an analog of diacylglycerol, but not by 4α phorbol didecanoate, an ineffective activator of protein kinase C. Staurosporine, a protein kinase C inhibitor, blocked phorbol ester inhibition of 86Rb+ uptake. These data suggest that a Na+, K+, Cl− cotransporter in NPE cells is inhibited by activation of protein kinase C. © 1992 Wiley‐Liss, Inc.
Transforming growth factor β1 and adrenocorticortropin differentially regulate the synthesis of adrenocortical cell heparan sulfate proteoglycans and their binding of basic fibroblast growth factor
Authors: Jiang Zhiwen , Savona Catherine , Chambaz Edmond M. , Feige Jean‐Jacques
Published: February 2005

Adrenocortical differentiated functions are under the control of both endocrine hormones such as ACTH and local factors such as transforming growth factor β (TGFβ) or basic fibroblast growth factor (bFGF). Besides their regulatory actions on the synthesis of corticosteroids, these two classes of factors also exert some important effects on the cellular environment. We have examined here the regulation by ACTH and TGFβ of adrenocortical cell proteoglycan synthesis and secretion. Under basal conditions, adrenocortical cells synthesized and secreted several species of sulfated proteoglycans, 80% of them being recovered in solution in the culture medium. When analyzed by ion exchange chromatography, the cell extracts and the media from cells metabolically labeled with 35S‐suifate were found to contain two and three species of radioactive sulfated proteoglycans, respectively. All species were proteoheparan‐sulfates. Treatment of adrenocortical cells with TGFβ1 or ACTH resulted in a significant increase of the incorporation of 35S into both secreted and cell‐associated proteoglycans. ACTH stimulated more than three times the amount of secreted proteoglycans eluting from DEAE‐Trisacryl as peak B, whereas TGFβ preferentially increased the amount of peak C. No important modification of the size of the synthesized proteoglycans was observed. The subpopulation of heparan sulfate proteoglycans capable to bind bFGF was also largely increased after ACTH or TGFβ treatment and paralleled the variation in overall proteoheparan sulfate synthesis. Thus those effects of TGFβ and ACTH on proteoglycan synthesis may participate in an increased ability of adrenocortical cells to bind and respond to bFGF. © 1992 Wiley‐Liss, Inc.
Cellular and molecular regulation of factor V expression in human megakaryocytes
Authors: Gewirtz Alan M. , Shapiro Charles , Shen Yu Min , Boyd Robert , Colman Robert W.
Published: February 2005

The mechanisms responsible for regulating FV expression in normal human megakaryocytes are unknown. To test the hypothesis that they are related to cell maturation events, we correlated human megakaryocyte FV antigen content with several putative maturation markers including cell size, morphologic stage of development, and ploidy level. Mature megakaryocytes were isolated from normal marrow by counterflow centrifugal elutriation. The cells were immunofluorescently labeled with a monoclonal antibody probe (B10) directed against the FV connecting peptide (150 kDa) and then reacted with Chromomycin A3 to allow for simultaneous DNA quantitation in the same cell. After processing, individual cells were stages and sized. FV antigen and nuclear DNA levels (ploidy) were measured by microphotometric measurements of total cytoplasmic or nuclear fluorescence. A total of 1,006 cells were examined, of which 12% were stage I, 8% were stage II, 35% were stage III, and 45% were stage IV. The geometric mean diameter (± SD) of cells in these stages was 48.3 ± 11.8 μm2, 54.9 ± 14.4 μm2, 61.7 ± 20.02 μm2, and 56.7 ± 13.2 μm2, respectively. Respective ploidy values in arbitrary fluorescence units, where 2 N = 5%, were 28.2 ± 18.2%, 31.4 ± 19.3%, 54.3 ± 26.6%, and 33.2 ± 22.7%. Calculated correlation coefficients (r) and coefficient of determination (r2) values suggested that FV antigen levels varied independently of any of the maturation markers studied. However, FV antigen levels could be upregulated by 24 h exposure to 8 nM phorbol myristate acetate (PMA). Presence of FV mRNA in a pure population of megakaryocytes was demonstrated by in situ hybridization and the polymerase chain reaction. Relative levels of megakaryocyte FV mRNA, as assessed by in situ hybridization, failed to reveal a detectable change after PMA exposure in spite of an increase in detectable protein. These data suggest that FV synthesis may be regulated at the post‐transcriptional level and that it is subject to regulatory influences which are not coordinately linked to development of other terminal maturation markers.
Differential regulation of glucose transporter 1 and 2 mRNA expression by epidermal growth factor and transforming growth factor‐beta in rat hepatocytes
Authors: Mischoulon David , Rana Basabi , Kotliar Natalio , Pilch Paul F. , Bucher Nancy L. R. , Farmer Stephen R.
Published: February 2005

We have examined by Northern blot analysis the expression of two members of the glucose transporter family of genes (GLUT‐1 and GLUT‐2) in regenerating liver and in hepatocytes cultured under various conditions. GLUT‐1, although thought to be a growth‐associated gene, is not expressed in normal or regenerating liver, whereas GLUT‐2, a liver‐specific gene, is abundant in normal liver and gradually up‐regulated during liver regeneration. Conversely, in hepatocytes cultured conventionally on dried rat tail collagen (RTC) in the presence of EGF and insulin, which potentiate proliferation, GLUT‐1 mRNA is rapidly and abundantly expressed, whereas GLUT‐2 is depressed. To investigate the causes of this “switch” in glucose transporter expression seen when hepatocytes are removed from the liver and cultured under the conventional proliferative conditions, we examined the effects of specific growth factors and extracellular matrices on cultured hepatocytes. EGF, a potent liver mitogen, although causing a threefold induction of GLUT‐1, was found to have no effect on GLUT‐2 expression, suggesting that the increase in GLUT‐2 seen in regenerating liver is not due to EGF. Inhibition of protein synthesis by cycloheximide in cultured hepatocytes does not prevent the induction of GLUT‐1 mRNA. In addition, treatment of cells with cycloheximide appears to stabilize the GLUT‐2 mRNA, preventing the usual down‐regulation of this gene in cultured hepatocvtes. The expression of the two glucose transporter mRNAs also differed when the hepatocytes were adherent to particular cell matrices. Culture of hepatocytes on a reconstituted basement membrane gel matrix (EHS) is known to restrain their growth and mediate high levels of differentiated hepatocytic functions that are lost under conventional culture conditions. Unlike cells on RTC, hepatocytes on EHS expressed low levels of GLUT‐1 mRNA, and decreased GLUT‐2 mRNA. TGF‐β, an attenuator of DNA synthesis, when added to cultures on RTC, substantially down‐regulated GLUT‐2 but had no effect on GLUT‐1. We propose that the effectors, EGF, TGF‐β and basement membrane components, play a significant role in the regulation of expression of GLUT‐1 and GLUT‐2 in hepatocytes. © 1992 Wiley‐Liss, Inc.
Effect of L‐fucose on proliferation and myo‐inositol metabolism in cultured cerebral microvessel and aortic endothelial cells
Authors: Stefani Mark R. , Dunlap Joyce A. , Yorek Mark A.
Published: February 2005

Decreased myo‐inositol metabolism possibly contributes to the development of diabetic complications including micro and macrovascular disease. Previous studies have shown that hyperglycemia may be partially responsible for this defect. We have found that L‐fucose, a monosaccharide present in low concentrations in normal circulation and found to be elevated in diabetes, causes defects in cultured endothelial cells, including alterations in myo‐inositol metabolism and proliferation. Murine cerebral microvessel and bovine aortic endothelial cells take up L‐fucose from the medium in a time and concentration‐dependent manner. Both acute and chronic exposure of these cultured endothelial cells to media containing L‐fucose at concentrations that may exist in diabetic sera cause a significant decrease in the accumulation of myo‐inositol and its incorporation into inositol phospholipids. There is a concomitant decrease in the intracellular levels of myo‐inositol. Kinetic analysis of the effect of L‐fucose on myo‐inositol uptake suggests that L‐fucose competitively inhibits the transport of myo‐inositol, exhibiting a Ki in the range of 1.6–4.1 mM for both cell types. Endothelial cells exposed to L‐fucose concentrations of 0.5–20 mM exhibit depressed rates of proliferation in a concentration‐dependent fashion. Furthermore, L‐fucose causes a concentration‐dependent decrease in synthesis of proteoglycan by cultured cerebral microvessel endothelial cells as measured by incorporation of 35S; however, this effect is not observed in the aortic endothelia. These data suggest that L‐fucose at concentrations that may exist in diabetic sera may impair myo‐inositol metabolism and proliferation of the vascular endothelium. © 1992 Wiley‐Liss, Inc.
Different organizational states of fodrin in cultured MDCK cells are induced by treatment with low pH, calmodulin antagonist TFP, and tumor promoter PMA
Authors: Huotari Virva , Sormunen Raija , Lehto Veli‐Pekka , Eskelinen Sinikka
Published: February 2005

We have investigated the molecular mechanisms underlying dynamic organization of the fodrin network by treating the epithelial MDCK cells with various agents affecting intracellular pH, intracellular calcium ion concentration, intracellular calmodulin, and protein kinase C (PKC) activity. Elevation of intracellular calcium level by A23187 or treatment with trifluoperazine (TFP), a calmodulin inhibitor, did not have any drastic effect on the fodrin distribution as judged by immunofluorescence microscopy. A long‐term incubation with phorbol‐12‐myristate‐13‐acetate (PMA), a protein kinase C activator, in contrast, released fodrin from the lateral walls of the MDCK cells, leading to a diffuse cytoplasmic distribution. TFP, along with PMA, accelerated destabilization of the fodrin skeleton. Treatment with TFP alone rapidly released the cells from the substratum, which, however, could be prevented by PMA. We have previously shown that lowering of intracellular pH (<6.5) leads to a removal of fodrin from its basolateral residence (Eskelinen et al., 1992) and that this translocation is reversed upon returning normal pH. We now show that the rebuilding of the membrane skeleton can be prevented if TFP is added to the acidified cells. Moreover, in TFP‐treated acidified cells, fodrin shows a clusterlike organization similar to that observed in resting lymphocytes. We also noticed that interconversions between these different organizational states of fodrin are independent of the intracellular calcium concentration. Thus manipulation of the intracellular pH and treatment with TFP and PMA reveals different organizational states of the fodrin skeleton. This suggests that fodrin may participate in PMA‐, TFP‐ and pH‐sensitive signal transduction pathways. © 1992 Wiley‐Liss, Inc.
Growth factors produced by human embryonic kidney cells that influence megakaryopoiesis include erythropoietin, interleukin 6, and transforming growth factor‐beta
Authors: Withy Raymond M. , Rafield Lori F. , Beck Anton K. , Hoppe Henry , Williams Neil , McPherson John M.
Published: February 2005

Partially purified protein preparations containing megakaryocyte growth factor activity were prepared from human embryonic kidney (HEK) cell conditioned medium using ammonium sulfate precipitation, Cibicron blue affinity chromatography, and wheatgerm lectin affinity chromatography. Treatment of these preparations with neutralizing antibodies directed against erythropoietin (EPO) and interleukin 6 (IL6) resulted in a dramatic reduction in their capacity to stimulate megakaryocyte maturation in vitro. The presence of EPO in these preparations was confirmed by both immunoblotting and use of a mouse spleen erythroid progenitor cell proliferation assay routinely used to quantitate EPO activity in vitro. Northern blot analysis of HEK cell‐derived mRNA with IL6 DNA probes revealed the presence of an IL6 transcript with a molecular size of 1.3 kb. Analysis of the HEK cell‐derived preparation by ELISA confirmed the presence of immunologically reactive IL6. In addition, it was shown that purified recombinant human EPO and IL6 stimulated megakaryocyte maturation in the in vitro assay used in this study. These data indicate that the activity in HEK cell conditioned medium that stimulates megakaryocyte maturation in vitro is predominantly due to the presence of IL6 and EPO. Immunoneutralization studies of another HEK cell‐derived preparation, which was inhibitory in the megakaryocyte maturation assay, demonstrated that it contained transforming growth factor beta (TGFβ), a potent inhibitor of megakaryocyte maturation. Taken together, these studies indicate that HEK cell conditioned medium, which has previously been reported to contain megakaryocyte growth factor activity, is comprised of a complex mixture of growth and differentiation factors, some of which promote and others that inhibit the process of megakaryopoiesis. © 1992 Wiley‐Liss, Inc.
Transformed growth phenotype of mouse mammary epithelium in primary culture induced by specific fetal mesenchymes
Authors: Kanazawa Takuya , Hosick Howard L.
Published: February 2005

When mesenchyme from fetal mammary or salivary gland is implanted into adult mouse mammary gland, adjacent epithelium responds with intense hyperplasia. The hyperplastic cells are more vulnerable than are non‐stimulated cells to transformation in vivo by a chemical carcinogen or by mammary tumor virus. This system provides a potentially useful model for determining how stroma contributes to mammary tumorigensis. We have developed co‐culture systems and used them to investigate in more detail the nature of the signal produced by the mesenchyme cells. Monolayers of mesenchyme cells were prepared on tissueculture wells. The mesenchyme cells were trapped on the surface by a thin overlay of agarose. Primary mammary epithelial cells were cultured atop this barrier layer, either as organoids in collagen gels for assessment of anchorage‐dependent growth, or as single‐cell dispersions in soft agarose for assessment of anchorage independent growth. Our procedures for assay of anchorage‐independent growth allow us for the first time to detect and measure this transformation‐defining characteristic in non‐immortalized mammary epithelial cells in primary culture. Fetal mammary fat pad precursor tissue and fetal salivary mesenchyme both stimulated anchorage‐dependent growth of mammary epithelium with cell number increasing as much as fifteenfold during a 6‐day culture period. These same fetal tissues also stimulated anchorage‐independent growth of the mammary epithelial cells, with colony‐forming efficiencies of up to 40% in co‐cultures with salivary mesenchyme. No colonies formed in the absence of mesenchyme. Cells of colonies contained keratin, which indicates that the colonies grew from epithelial cells and not from a contaminant of another cell type. When co‐cultured epithelial cells were subsequently re‐cultured in the absence of mesenchyme, they lost their ability to grow independent of anchorage. No colonies grew in co‐cultures with fetal cells from heart, kidney, or lung, which is consistent with the lack of stimulation by these tissues in the mammary gland in vivo. A tumor promoter, 12‐O‐tetradecanoylphorbol acetate (TPA), also caused anchorage‐independent growth of the dispersed mammary epithelial cells. Culture medium conditioned by primary or early‐passage salivary mesenchyme cells was capable of stimulating growth under both anchorage‐dependent and anchorage‐independent conditions, confirming that these effects are mediated by a paracrine factor. The results indicate that stimulatory fetal mesenchymes produce soluble molecules that act analogously to transforming growth factors. © 1992 Wiley‐Liss, Inc.
Biphasic effect of the mitotoxin bFGF‐saporin on bovine lens epithelial cell growth: Effect of cell density and extracellular matrix
Authors: David Thierry , Tassin Jacqueline , Lappi Douglas A. , Baird Andrew , Courtois Yves
Published: February 2005

We have studied the effect of a specific FGF receptor suicide antagonist on the growth of bovine epithelial cells (BEL cells) in culture. This basic fibroblast growth factor‐saporin conjugate (bFGF‐SAP) has a biphasic effect on bovine lens epithelial cells (BEL cells). Whereas 0.01 nM and 0.1 nM bFGF‐SAP stimulate BEL cells proliferation, 1 nM and 10 nM bFGF‐SAP have the predicted toxic effects on BEL cell growth. The toxicity of bFGF‐SAP is observed 2 to 3 days after the initial treatment and depends on cell density. Accordingly, the sensitivity of confluent cells to bFGF‐SAP is reduced compared to sparse cells. A time course analysis reveals that bFGF‐SAP is effective after a short exposure to cells and that its effects are not increased with longer treatments. Cell growth on bFGF‐SAP pretreated extracellular matrix (ECM) or posterior lens capsule (PLC) is also affected. Basic FGF‐SAP has been shown to bind to the extracellular material, allowing a modulation of lens cells migration and survival by a single treatment in vitro. This finding raises the possibility of its use in vivo to prevent capsules invasion by lens cells after cataract surgery. © 1992 Wiley‐Liss, Inc.
Cortical photons in early development of frog eggs: Comparative study of photon emission between nuclear division and cytoplasmic fission
Authors: Oonuki Makoto , Ryuzaki Masashi
Published: February 2005

In the study of cell division in the early development of frog eggs, cortical photon emission was investigated by converting small light emission from living cells into digital pulses of potentials and recording the integrals of these pulses (analog method), counting the number of pulses (photon‐counting method), and counting the number of integrated pulses (improved photon‐counting method). By the analog and improved photon‐counting methods, changes in photon emission due to cell division could be clearly detected. The emitted light increased about 5.10−19W at the start of a cleavage furrow. Rapid changes in chemical reactions causing photon emission were compared during nuclear division and cytoplasmic phases. This emission occured mainly in cytoplamic fission, the rate being greater than in nuclear division by a factor of about 2.9. Chemical reaction rates were shown to differ according to bulk emission, thus indicating the mechanisms for the reactions to also differ. © 1992 Wiley‐Liss, Inc.
Vascular endothelial growth factor induces interstitial collagenase expression in human endothelial cells
Authors: Unemori Elaine N. , Ferrara Napoleone , Bauer Eugene A. , Amento Edward P.
Published: February 2005

Vascular endothelial growth factor (VEGF) is a 45kDa secreted peptide that has potent mitogenic activity specific for endothelial cells in vitro and the ability to induce a strong angiogenic response in vivo. In the present study, 24 h treatment with VEGF resulted in a stimulation of expression of the metalloproteinase, interstitial collagenase, at the protein and mRNA levels 2.5–3.0‐fold in human umbilical vein endothelial cells but not in human dermal fibroblasts. The dose response curve for collagenase induction was biphasic with the peak stimulatory response obtained by treatment of cells with 10–100 ng/ml (0.2–2 nM) VEGF. The dose response curve for collagenase induction overlapped with, but was not identical to, the response curve for proliferation, which showed VEGF mitogenic activity between ≤ 0.1–50 ng/ml (≤ 0.002–1 nM). There was no induction seen in expression of other members of the matrix metalloproteinase family, including the 72kDa type IV collagenase, the 92kDa type V collagenase, or stromelysin. Expression of transcripts for the major metalloproteinase inhibitor, tissue inhibitor of metalloproteinases, was also unaltered by treatment with VEGF (1–200 ng/ml). These studies demonstrate that in addition to stimulating proliferation of endothelial cells, VEGF can also induce the expression of the only metalloproteinase that can initiate degradation of interstitial collagen types I‐III under normal physiological conditions. Both responses are likely to contribute to the angiogenic potential of this peptide. © 1992 Wiley‐Liss, Inc.
Nonreceptor mediated nuclear accumulation of insulin in H35 rat hepatoma cells
Authors: Harada Shuko , Loten Ernest G. , Smith Robert M. , Jarett Leonard
Published: February 2005

We previously demonstrated that insulin accumulated in the nucleus in several cell types and partially characterized the uptake mechanisms and pathways in H35 rat hepatoma cells. Nuclear accumulation of insulin was energy independent, time, temperature, and insulin concentration dependent, but apparently nonsaturable. This study investigated further the initial endocytotic pathways that contribute to the nuclear accumulation of insulin using trypsin treatment of the cells to prevent insulin binding to its plasma membrane receptor. Total cell‐associated, intracellular, and nuclear insulin were compared in control and trypsin‐treated H35 hepatoma cells. Trypsin treatment markedly decreased total cell‐associated and intracellular insulin as well as the nuclear accumulation of insulin when cells were incubated with 2.8 ng/ml insulin. When the cells were incubated with 100 ng/ml insulin, trypsin treatment totally inhibited insulin binding to the plasma membrane for at least 90 min. However, intracellular accumulation of insulin was reduced by only 50% at 60 min, and trypsin treatment failed to inhibit the nuclear accumulation of insulin. Chemical extraction and Sephadex G‐50 chromatography revealed nuclear associated insulin in trypsin‐treated cells was identical to that in control cells incubated with either 2.8 or 100 ng/ml insulin. These results suggest that a nonreceptor mediated uptake pathway, i.e., fluid‐phase endocytosis, contributed significantly to the nuclear accumulation of insulin at high insulin concentrations, but at lower insulin concentrations the receptor‐mediated pathway predominated. No matter which initial endocytotic route was used to internalize insulin, the insulin apparently associated with the same nuclear matrix proteins. This association of insulin with the nuclear matrix may be involved in regulation of nuclear events such as cell growth and differentiation or gene transcription. © 1992 Wiley‐Liss, Inc.
Alterations in cellular calcium handling as a result of systemic calcium deficiency in the developing chick embryo: II. Ventricular myocytes
Authors: Miyahara Takashi , Akins Robert E. , Tuan Rocky S.
Published: February 2005

We have previously shown that cardiovascular anomalies, such as hypertension and tachycardia, develop in Ca2+‐deficient, shell‐less (SL) chick embryos cultured ex ovo, accompanied by elevated circulating catecholamines and higher α‐adrenergic sensitivity of cardiovascular functions. Results described in the preceding work, using erythrocytes as an experimental system, show that cellular Ca2+ handling properties are also altered as a result of long‐term calcium deficiency. To examine the relevance of these findings to cells of the cardiovasculature, we have analyzed and compared the Ca2+ handling characteristics of the heart cells of SL and normal (NL) embryos. For this study, isolated and cultured ventricular myocytes of SL and NL embryos were loaded with Fura‐2 via transient membrane damage with glass beads. Compared to Fura‐2/AM, bead loading yielded similar values and kinetic profiles of [Ca2+]i‐dependent differential fluorescence and, in addition, did not affect cell viability and beating activity. The Fura‐2 loaded ventricular myocytes were washed in Ca2+‐free buffer and then analyzed by ratiometric fluorescence (350 nm/380 nm) microscopy for kinetic changes in [Ca2+]i (R350/380 values) as a function of [Ca2+]o and adrenergic modifiers. At 0.5 and 1.0 mM [Ca2+]o, SL cells showed significantly higher [Ca2+]i, higher beating rates, and faster rate of increase in [Ca2+]i compared to NL cells. At higher [Ca2+]o (3–5 mM), there was no significant difference in [Ca2+]i and beating rate between NL and SL cells. Treatment with norepinephrine (NE; 0.01–1 μM) at 1 mM [Ca2+]o substantially increased [Ca2+]i in both NL and SL cells. In the former, the NE effect was completely inhibited by β‐blockade (1 μM propranolol). In contrast, in SL cells, NE remained effective after β‐blockade, and combined α‐blockade (1 μM prazosin) and β‐blockade was needed to inhibit completely the NE effect. In both NL and SL cells, treatment with NE substantially increased beating rates in a similar manner. Taken together, these findings suggest that Ca2+ handling and adrenergic regulation of the heart cells are significantly altered in the SL embryos, and that these alterations may be related to the development of impaired cardiovascular functions resulting from systemic Ca2+ deficiency. © 1992 Wiley‐Liss, Inc.
Identification of the cell surface and nuclear receptors for NGF in a breast carcinoma cell line
Authors: Rakowicz‐Szulczynska Ewa M.
Published: February 2005

125I‐nerve growth factor (NGF) was found to be internalized and translocated to the nucleus of SKBr5 breast carcinoma cells. The cytoplasm and chromatin isolated from nonmitotic cells accumulated two‐and five‐fold, respectively, more of 125I‐NGF than the cells undergoing mitosis. MAb 20.4 developed against the NGF cell surface receptor immunoprecipitated the 80,000 Mr receptor from plasma membrane and two protein species from the chromatin; 90,000 Mr (major band) and 200,000 Mr (minor band). In SKBr5 cells, binding of NGF to the chromatin did not affect synthesis of rRNA. Proliferation of SKBr5 cells was slightly stimulated by NGF. In control melanoma A875 cells, which express the 230,000 Mr chromatin receptor, NGF inhibited both rRNA synthesis and cell proliferation. We suggest that the 90,000 Mr chromatin receptor expressed by SKBr5 cells represents a “nonactive”, ligand‐binding subunit of the high molecular weight receptor for NGF. The critical role of the chromatin receptor for NGF in rRNA‐dependent cell proliferation is discussed. © 1993 Wiley‐Liss, Inc.