bluuebunny/crossref_metadata_2025 huggingface dataset:
Binding and activation of plasminogen on the surface of osteosarcoma cells
Authors: Campbell Phil G. , Wines Karen , Yanosick Thomas B. , Novak Josef F.
Published: February 2005

Plasmin (Pm) is a broad action serine protease implicated in numerous physiological functions. In bone, Pm may play a role in growth, resorption, metastasis, and the activation of growth factors. The various components of the Pm system are known to bind and function on the cell surface of various cell types, but no pertinent data are available describing membrane‐bound Pm or its zymogen, plasminogen (Pg), in either normal or neoplastic bone cells. We report here that Pg binds to the surface of the human osteosarcoma cell line MG‐63 and is activated to Pm by endogenous urokinase plasminogen activator (uPA). These conclusions are based on experiments utilizing radiolabeled compounds and a cell surface proteolytic assay measuring amidolytic activity of Pm. 125I‐Pg binding to cells was time dependent, saturable, reversible, and specific. Binding was characterized by a relatively low affinity (Kd ∼0.9 μM) and a high capacity (∼7.5 x 106 sites/cell). The binding of 125I‐Pg was associated with lysine binding sites of the plasminogen molecule. Activation of 125I‐Pg to 125I‐Pm occurred on the cell surface and was dependent upon cell bound uPA, as determined by inhibitory antibodies. Binding of Pg to MG‐63 monolayers represented ∼80% bound specifically to the cell surface and the remainder to the surrounding extracellular matrix. Either co‐incubation with uPA or pre‐incubation with Pm resulted in increased 125I‐Pg binding to osteosarcoma cells. Cell surface Pm proteolytic activity was confirmed by an amidolytic chromogenic assay. Both Pm and Pg bound to cells with Pg being activated by endogenous uPA. Plasmin activated on the cell surface was partially protected from inhibition by α2‐antiPm (requiring Pm lysine binding site interaction) but inhibited by aprotinin, (interacting directly with the Pm catalytic site). Resistance of cell bound Pm to α2‐antiPm inhibition suggests that cell surface proteolysis can occur in the presence of a soluble Pm inhibitor known to exist in the extracellular space. Based on these results, we speculate that the various bone physiological processes implicating Pm may occur at or near the bone cell surface. © 1994 wiley‐Liss, Inc.
Heterogeneity in collagen biosynthesis by sprouting retinal endothelial cells
Authors: Canfield A. E. , Schor A. M.
Published: February 2005

Bovine retinal microvascular endothelial cells can display two distinct and reversible morphologies in culture: ‘cobblestone’ and ‘sprouting’. The cobblestone morphology resembles the resting cells lining the lumen of mature vessels while the sprouting morphology resembles the angiogenic cells involved in the formation of new vessels. Retinal cells displayed some heterogeneity in the shape of the cells making up the cobblestone monolayer. In contrast, all cell lines displayed an identical sprouting morphology. We have investigated the synthesis of matrix macromolecules by retinal endothelial cells displaying either the cobblestone or the sprouting morphology. Type IV was the only collagen synthesised by eight different lines of early‐passage (between one and six) cobblestone endothelial cells. Collagen types I and III were not detected in these cultures. In contrast, heterogeneity was observed in the types of collagen synthesised by four lines of early‐passage cells displaying the sprouting morphology. That is, two lines synthesised collagen types I, III and IV, whereas two other lines continued to synthesise only type IV collagen. Both cobblestone and sprouting cells synthesised fibronectin and thrombospondin, although the relative amounts of these macromolecules varied with culture conditions. The pattern of collagen synthesis by cobblestone cells was also affected by in vitro „ageing”︁: 4/5 lines examined above passage eight synthesised collagen types I, III and IV. Our results indicate that there is heterogeneity in the sprouting phenotype displayed by retinal endothelial cells, and that this phenotype is not necessarily associated with the synthesis of type I collagen. We suggest that differences in the spectrum of matrix macromolecules synthesised by sprouting endothelial cells may play a role in the control of angiogenesis. © 1994 wiley‐Liss, Inc.
Identification of an IP3 receptor in endothelial cells
Authors: Bourguignon Lilly Y. W. , Iida N. , Sobrin L. , Bourguignon C. J.
Published: February 2005

In this study we have used saponin to permeabilize bovine endothelial cell membranes in order to directly test the involvement of IP3 in regulating internal Ca2+ release. Our results indicate that the release of internal Ca2+ occurs as early as 1–3 seconds after IP3 addition. This IP3‐induced internal Ca2+ release can be inhibited by heparin (an IP3 receptor antagonist). Further binding of [3H]IP3 to saponin‐permeabilized bovine endothelial cells reveals the presence of a single, high affinity class of IP3 receptor with a dissociation constant (Kd) of ≈0.50 (±0.03) nM. Using a panel of monoclonal and polyclonal antibodies against IP3 receptor, we have established that the bovine endothelial cell IP3 receptor (≈260 kDa) displays immunological cross‐reactivity with the rat brain IP3 receptor. Immunofluorescence data indicates that the IP3 receptor is preferentially located at the perinuclear region of the cells. In addition, PCR analysis of first‐strand cDNAs from both bovine endothelial cells and rat brain tissues reveals that the IP3 receptor transcript in bovine endothelial cells belongs to the short non‐neuronal form and not the long neuronal form detected in rat brain tissue. These findings suggest that the IP3 receptor in endothelial cells is both structurally and functionally analogous to that reported in non‐neuronal cell systems and probably plays an important role in agonist‐induced endothelial cell activation. © 1994 wiley‐Liss, Inc.
Plasma membrane K+/H+‐ATPase From leishmania donovani
Authors: Jiang Suping , Anderson Steven A. , Winget G. Douglas , Mukkada Antony J.
Published: February 2005

Leishmania donovani has an active K+/H+ exchange system on the surface membrane. Modulation of external K+ concentration resulted in a corresponding change in internal pH (pHi) suggesting a link between proton and potassium transport. Although a Na+/H+ antiporter is present on the plasma membrane, its sensitivity to amiloride suggests that it operates independent of K+/H+ exchange. Reduction of cellular ATP with NaN3 and KCN inhibits K+/H+ exchange showing thereby that the process is energy dependent. The K+/H+ exchange is sensitive to inhibitors of the gastric K+/H+‐ATPase. It is concluded that the H+‐ATPase previously reported on the plasma membrane of L. donovani is in fact a K+/H+‐ATPase. © 1994 wiley‐Liss, Inc.
Regulation of gamma‐glutamyl transpeptidase and alkaline phosphatase activities in immortalized rat brain microvessel endothelial cells
Authors: Roux F. , Durieu‐Trautmann O. , Chaverot N. , Claire M. , Mailly P. , Bourre J.‐M. , Strosberg A. D. , Couraud P.‐O.
Published: February 2005

Rat brain microvessel endothelial cells were immortalized by transfection with a plasmid containing the E1A adenovirus gene. One clone, called RBE4, was further characterized. These cells display a nontransformed phenotype and express typical endothelial markers, Factor VIII‐related antigen and Bandeiraea simplicifolia binding sites. When RBE4 cells were grown in the presence of bFGF and on collagen‐coated dishes, confluent cultures developed sprouts that extend above the monolayer and organized into three‐dimensional structures. The activity of the blood‐brain barrier‐associated enzyme, gamma‐glutamyl transpeptidase (γGTP), was expressed in these structures, not in the surrounding monolayer. Similar results were obtained with the microvessel‐related enzyme alkaline phosphatase (ALP). Addition of agents that elevate intracellular cAMP reduced the formation of three‐dimensional structures, but every cell inside the aggregates still expressed γCTP and ALP activities. Such structures, associated with high levels of γCTP and ALP activities, were also induced by astroglial factors, including (1) plasma membranes from newborn rat primary astrocytes or rat glioma C6 cells, (2) C6 conditioned media, or (3) diffusible factors produced by primary astrocytes grown in the presence of, but not in contact with RBE4 cells. RBE4 cells thus remain sensitive to angiogenic and astroglial factors for the expression of the blood‐brain barrier‐related γCTP activity, as well as for ALP activity, and could constitute the basis of a valuable in vitro model of the blood‐brain barrier. © 1994 wiley‐Liss, Inc.
Depleted level of heparan sulfate proteoglycan in the extracellular matrix of endothelial cell cultures exposed to endotoxin
Authors: Colburn P. , Kobayashi E. , Buonassisi V.
Published: February 2005

Exposure of cultured endothelial cells to endotoxin causes an increase in the amount of cellular heparan sulfate proteoglycan and a depletion of this molecule in the extracellular matrix. Concomitant with the decrease in the extracellular matrix is the appearance of a fraction of proteoglycan bearing altered carbohydrate moieties in the culture medium, β‐mereaptoethanol, mannitol, and dimethyl sulfoxide bring back to normal the structural properties of the proteoglycan in the medium and restore the matrix content in proteoglycan to a level comparable to that of control cells but do not affect the increase in the amount of proteoglycan on the cell. This “uncoupling” suggests that two independent chains of events underlie the synthetic and structural changes of the proteoglycan triggered by endotoxin in the endothelial cell. © 1994 wiley‐Liss, Inc.
Induced expression of p52(PAI‐1) in normal rat kidney cells by the microfilament‐disrupting agent cytochalasin D
Authors: Higgins Paul J. , Ryan Michael P. , Providence Kirwin M.
Published: February 2005

In established normal rat kidney (NRK) cells, synthesis of the 52 kDa type‐1 inhibitor of plasminogen activator [p52(PAI‐1)] is stimulated by the cell shape—modulating fungal metabolite cytochalasin D (CD). Induction paralleled the time course of morphologic change and reflected relatively specific increases in sapo‐nin‐resistant p52(PAI‐1) protein accumulation (approximating ten‐ to thirty‐fold over control) and mRNA abundance (seven‐ to nine‐fold). Augmented p52(PAI‐1) mRNA levels closely correlated with increases in 43 kDa p52(PAI‐1) core protein biosynthesis. Sensitivity to tunicamycin indicated that N‐linked post‐translational modifications to this 43 kDa core species generated the full complement of 50 kDa (intermediate) and 52 kDa (mature) p52(PAI‐1) glycosylated isoforms. CD‐induced p52(PAI‐1) expression occurred efficiently in quiescent NRK cells maintained under serum‐free conditions as well as in fully serum‐supplemented actively growing cultures. While 8‐bromo‐cAMP reduced both constitutive and transforming growth factor‐beta—induced p52(PAI‐1) synthesis by >50%, no such inhibition was evident in short‐term (4 h) CD‐stimulated cultures. Long‐term (24 h) exposure of NRK/CD cells to 8‐bromo‐cAMP did result in an approximately 34% reduction in stimulated p52(PAI‐1) expression, however, levels expressed by NRK/CD + cAMP populations remained markedly elevated relative to control values. These data suggest the existence of a cell shape—dependent aspect of p52(PAI‐1) expression control distinct from both the constitutive and growth factor—mediated pathways of gene regulation. © 1994 wiley‐Liss, Inc.
Type‐1 insulin‐like growth factor receptor overexpression produces dual effects on myoblast proliferation and differentiation
Authors: Quinn Lebris S. , Steinmetz Brian , Maas Adolf , Ong Luan , Kaleko Michael
Published: February 2005

Using a retroviral vector, we developed a line of C2 mouse skeletal myoblasts, C2‐LISN, which expressed high levels of the human type‐1 insulin‐like growth factor (IGF) receptor. When switched to low serum medium, C2‐LISN myoblasts underwent terminal differentiation extremely rapidly compared to control C2 myoblasts. In high serum conditions which were not permissive for differentiation, C2‐LISN myoblasts expressed ten‐fold higher levels of the myogenic transcription factor myogenin than did control C2 myoblasts. When cultured in low serum medium with both transforming growth factor‐β (TGF‐β) and high concentrations of IGF‐I, C2‐LISN myoblasts failed to differentiate and grew to very high saturation densities, forming multilayers. Upon removal of TGF‐β, multilayered C2‐LISN myoblasts differentiated within 2 days. These results demonstrate that overexpression of the type‐1 IGF receptor can amplify signals which stimulate myogenic differentiation. Overexpressed type‐1 IGF receptors can also mediate strong mitogenic signals if differentiation is inhibited by TGF‐β. The C2‐LISN myoblast cell line may be a useful model to investigate the intracellular pathways which stimulate myogenic differentiation. Additionally, overexpression of the type‐1 IGF receptor could provide a strategy to expand populations of differentiation‐competent myoblasts for experimental or clinical applications. © 1994 wiley‐Liss, Inc.
Human osteosarcoma (U‐2 OS) cells express both insulin‐like growth factor‐I (IGF‐I) receptors and insulin‐like growth factor‐II/mannose‐6‐phosphate (IGF‐II/M6P) receptors and synthesize IGF‐II: Autocrine growth stimulation by IGF‐II via the IGF‐I receptor
Authors: Raile K. , Höflich A. , Kessler U. , Yang Y. , Pfuender M. , Blum W. F. , Kolb H. , Schwarz H. P. , Kiess W.
Published: February 2005

Recently, insulin‐like growth factor‐I and ‐II (IGF‐I and ‐II) have been implicated in the growth promotion of tumors in vivo and tumor cells in vitro. We have studied the human osteosarcoma cell line U‐2 OS in order (1) to gain more insight into the growth promoting actions of the IGFs and (2) to establish an in vitro tissue culture model of IGF action in human tumor cells. Specific binding of 125I‐IGF‐I and 125I‐IGF‐II to IGF‐I receptors and IGF‐II/mannose‐6‐phosphate (M6P) receptors on U‐2 OS cells was demonstrated in competitive binding experiments and in affinity crosslinking experiments. Western blotting of cell extracts confirmed the expression of the IGF‐II/M6P receptor. In addition, in Northern blotting experiments using total RNA from U‐2 OS cells IGF‐I receptor RNA of 11 kb and IGF‐II/M6P receptor RNA of approximately 9 kb were detected. Solution hybridization experiments confirmed the presence of IGF‐I receptor and IGF‐II/M6P receptor RNA. In a subset of experiments DNA synthesis was measured as 3H‐thymidine uptake into cellular DNA of U‐2 OS cells. Normal rat serum stimulated DNA synthesis maximally. IGF‐I‐deficient serum from hypophysectomized rats as well as IGF‐I or IGF‐II without serum were approximately twofold and tenfold, respectively, less potent than serum in stimulating 3H‐thymidine uptake. The concentrations of IGF‐I and IGF‐II needed for half maximal stimulation of DNA synthesis corresponded well with the respective concentrations required for half maximal inhibition of 125I‐IGF‐I binding to U‐2 OS cells. The anti‐IGF‐I receptor antibody alphaIR3 blocked the IGF‐I and IGF‐II stimulated increase of 3H‐thymidine uptake. In addition, basal DNA synthesis was partially inhibited by the anti‐IGF‐I receptor antibody. These data suggest that U‐2 OS cells synthesize and secrete IGF‐like peptides. Northern blotting experiments confirmed that U‐2 OS cells express an IGF‐II RNA species of 5.3 kb but no IGF‐I transcripts. In a series of RNase protection assays, protected RNA fragments were detected with an IGF‐II riboprobe. © 1994 wiley‐Liss, Inc.
Both viral (adenovirus E1B) and cellular (hsp 70, p53) components interact with centrosomes
Authors: Brown C. Randell , Welch William J. , Doxsey Steven J. , White Eileen
Published: February 2005

Human 293 cells, transformed by and expressing the early region of the adenovirus genome (i.e., E1A and E1B), contain a phase‐dense cytoplasmic structure situated in close proximity to the nucleus. Via indirect immunofluorescence studies such structures have been previously shown to contain both the adenovirus E1B (55 kDa) protein as well as the tumor suppressor gene product p53. Here we show that such structures also stain positive for the cytoplasmic hsp 70 proteins. Such phase‐dense structures containing hsp 70, p53, and adenovirus E1B are not unique to 293 cells but also are observed in rodent cell lines stabily transfected with the early region of the adenovirus genome. Using an antibody against a centrosomal protein, pericentrin, we show that these cytoplasmic phase‐dense structures are in close proximity to the centrosome. Cell fractionation studies revealed such structures to be highly detergent insoluble. However, like the centrosome, the cytoplasmic phase‐dense structures could be rendered detergent soluble following treatment of the cells with agents that disrupt the integrity of the cytoskeleton. While the phase‐dense structures appear in close proximity to the centrosome in interphase cells, during mitosis the centrosome and the phasedense bodies separate from one another. Owing to these observations we examined whether hsp 70 and p53 might also co‐localize with the centrosome in other cell types not expressing the adenovirus E1A/E1B proteins. We show that a portion of both hsp 70 and p53 indeed are present within the centrosome in Hela, COS, and 3T3 cells. These observations raise the possibility that components like hsp 70 and p53 may participate in the mechanism(s) controlling cell division in mammalian cells. © 1994 Wiley‐Liss, Inc.
Chronic hypoxia impairs the differentiation of 3T3‐L1 fibroblast in culture: Role of sustained protein kinase C activation
Authors: Sahai Atul , Patel Mona S. , Zavosh Aryana S. , Tannen Richard L.
Published: February 2005

The effect of hypoxia on 3T3‐L1 cell differentiation was examined in confluent cultures incubated with differentiation medium (DM) followed by incubation in growth medium (GM). Control cultures remained in GM throughout the incubation period. Eight days after the incubation, cells were assessed either for changes in morphology by staining with Oil Red O/hematoxylin or harvested to measure protein kinase C activity. Morphological examination of stained cells showed almost complete differentiation of normoxic cells to adipocytes when exposed to DM. By contrast hypoxia caused a dramatic inhibition of differentiation under similar media conditions with only 34 ± 4% of cells accumulating fat deposits. Cultures sustained in GM under normoxic or hypoxic conditions were devoid of any fat deposits, reflecting an undifferentiated phenotype. Normoxic cells exposed to DM exhibited a significantly lower membrane to cytosolic ratio of protein kinase C in comparison with cells maintained in GM, which is consistent with differentiated and undifferentiated phenotypes, respectively. In comparison with normoxic cells incubated in DM, cells exposed to hypoxia under similar media conditions exhibited a significantly higher membrane to cytosolic ratio of protein kinase C, indicating sustained activation of the enzyme. In addition, cells in differentiation medium exposed to hypoxia in the presence of the protein kinase C inhibitors staurosporine or H7 exhibited a significant increase in the number of fat accumulating cells when compared with hypoxic controls. These studies indicate that chronic hypoxia impairs the differentiation of 3T3‐L1 cells to adipocytes in association with the sustained activation of protein kinase C, which appears to play a role in mediating this process. © 1994 Wiley‐Liss, Inc.
Induced thermotolerance during early development of murine and bovine embryos
Authors: Ealy A. D. , Hansen P. J.
Published: February 2005

During early development, elevated temperatures have deleterious effects on embryonic viability and development. The primary objective of the current study was to determine the ontogeny of induced thermotolerance during early murine embryonic development. Embryos were either retrieved from superovulated ICR female mice at the 2 cell and 4 cell stages and cultured thereafter or were retrieved from oviducts or uterine horns at the desired stage of development. Induction of thermotolerance was detected by evaluating viability and further development after embryos were exposed to homeothermic temperature (37°C), mild heat shock (40°C for 1 h), severe heat shock (42°C for 1 h or 43°C for 2 h), or mild heat shock followed by severe heat shock (to induce thermotolerance). Induction of thermotolerance was observed beginning at the 8 cell stage when embryos were developed in culture from the 2 cell to 4 cell stage. When embryos were developed in vivo (i.e., were retrieved from the reproductive tract at the desired stage of development), thermotolerance was not induced until the blastocyst stage of development. The induction of thermotolerance was dependent on serum supplementation since induction of thermotolerance was not observed when embryos were placed in medium without serum. Induced thermotolerance could also be demonstrated in bovine blastocysts. In conclusion, embryos acquire the ability to undergo thermotolerance as they progress through development. The timing of processes leading to acquisition of thermotolerance can, however, be hastened by exposure of embryos to in vitro conditions.
Prolonged peak elevations in cytoplasmic free calcium ions, derived from intracellular stores, correlate with the extent of thrombin‐stimulated exocytosis in single human umbilical vein endothelial cells
Authors: Birch Kimberly A. , Ewenstein Bruce M. , Golan David E. , Pober Jordan S.
Published: February 2005

We have used indo‐1‐loaded human endothelial cells (EC) in monolayer culture and quantitative laser scanning fluorescence microscopy techniques to investigate the magnitude and duration of the change in cytoplasmic free calcium ([Ca2+]i) required for thrombin‐stimulated von Willebrand factor (vWF) secretion in individual EC. Both α‐thrombin and a 14 amino acid thrombin receptor activating peptide stimulate an increase in EC [Ca2+]i that is agonist dose dependent. Lowdose agonist treatment generates asynchronous oscillations (i.e., repetitive spikes < 80 sec duration) in [Ca2+]i. Stimulation with higher agonist concentrations generates a prolonged single peak elevation in [Ca2+]i. Both the number of cells displaying prolonged [Ca2+]i peaks and the mean amplitude of the peaks increase as a function of agonist concentration. Higher doses of agonist also cause sustained elevations in [Ca2+]i that depend upon extracellular Ca2+. Oscillations in [Ca2+]i are not sufficient to stimulate significant vWF secretion, and sustained elevations in [Ca2+]i are not required for maximal secretion. Both the number of cells displaying prolonged peaks and the mean peak amplitude correlate with increasing levels of vWF secretion from the culture. We have used the expression of P‐selectin, a secretory granule membrane protein, as a marker for measuring thrombin‐induced exocytosis in individual EC. Both the number of secreting cells and the amount of secretion per cell increase as a function of thrombin concentration. The graded responses in [Ca2+]i amplitudes and the graded exocytotic response may be causally related. © 1994 Wiley‐Liss, Inc.
Separation of mouse crushed muscle extract into distinct mitogenic activities by heparin affinity chromatography
Authors: Chen Gang , Yablonka‐Reuveni Zipora , Quinn Lebris S. , Birnbaum Roger S.
Published: February 2005

Extracts from gently crushed adult mouse skeletal muscles (CMEs) contain potent myoblast mitogens, and may be used as a model system to investigate myotrophic factors released by adult muscles following injury. CME was separated into four peaks of mitogenic activity by heparin affinity chromatography. The fraction of CME that did not bind to heparin contained transferrin (Tf). Three peaks of mitogenic activity were eluted from the heparin‐agarose columns at NaCl concentrations of 0.4 M, 0.9 M, and 2.0 M. A 46 kDa protein that shared antigenicity with the BB isoform of platelet‐derived growth factor (PDGF‐BB) was present in the 0.4 M NaCl eluant. Mitogenic activity in the 2.0 M NaCl peak eluted identically to purified basic fibroblast growth factor (bFGF), did not act additively to saturating amounts of purified bFGF, and was neutralized by anti‐bFGF antibodies. The 0.9 M NaCl eluant acted additively to the combination of three known growth factors for myoblasts, bFGF, insulin‐like growth factor I, and epidermal growth factor, to stimulate C2 myoblast proliferation, suggesting this fraction contains a mitogenic activity which does not utilize (and hence compete for) receptors for the known mitogens for myoblasts. Additionally, the 0.9 M NaCl eluant did not stimulate proliferation of fibroblast‐like cells derived from muscle tissue. The unbound, 0.4 M NaCl, 0.9 M NaCl, and 2.0 M NaCl eluants from the heparin‐agarose column acted additively to one another to stimulate myoblast proliferation. Our data suggest that Tf, PDGF‐BB‐like molecules, bFGF‐like activity, and an uncharacterized heparin‐binding myoblast mitogen could be released after muscle injury and act to stimulate satellite cell proliferation. © 1994 Wiley‐Liss, Inc.
Two saturable mechanisms of iron uptake from transferrin in human melanoma cells: The effect of transferrin concentration, chelators, and metabolic probes on transferrin and iron uptake
Authors: Richardson Des R. , Baker Erica
Published: February 2005

The mechanisms of iron (Fe) and transferrin (Tf) uptake by the human melanoma cell line, SK‐MEL‐28, have been investigated using chelators and metabolic probes. These data provide evidence for two saturable processes of Fe uptake from Tf, namely, specific receptor‐mediated endocytosis and a second nonspecific, non‐receptor‐mediated mechanism which saturated with respect to Fe uptake at a Tf concentration of approximately 0.3 mg/ml. In contrast to Fe uptake, Tf uptake increased linearly up to at least 1 mg/ml. Furthermore, under the culture conditions used, the second nonspecific, non‐receptor‐mediated mechanism was the most important process in terms of quantitative Fe uptake. Two concentrations of Tf‐125I‐59 Fe (0.01 and 0.1 mg/ml) were used in order to characterise the specific and nonspecific Fe uptake pathways. Membrane permeable chelators were equally effective at both Tf concentrations, whereas membrane impermeable chelators were significantly (P < 0.001) more effective at reducing the internalisation of Fe at the higher Tf concentration, consistent with a mechanism of Fe uptake which occurred at a site in contact with the extracellular medium. The oxidoreductase inhibitor, amiloride, only slightly inhibited Fe uptake at the higher Tf concentration, suggesting that the second nonspecific process was not mediated by a diferric Tf reductase. Three lysosomotrophic agents and the endocytosis inhibitor, phenylglyoxal, markedly reduced Fe uptake at both Tf concentrations, and it is concluded that a saturable process consistent with receptor‐mediated endocytosis of Tf occurred at the lower Tf concentration, while the predominant mechanism of Fe uptake at high Tf concentrations was a second saturable process consistent with adsorptive pinocytosis. © 1994 Wiley‐Liss, Inc.
Insulin‐like growth factor II regulation of gene expression in rat and human hepatomas
Authors: Zvibel Isabel , Brill Shlomo , Reid Lola M.
Published: February 2005

Insulin‐like growth factor II (IGF II) regulated tissue‐specific gene expression in hepatoma cell lines, but had no effect on expression of tissue‐specific genes in primary cultures of E14 and newborn rat liver cells depleted of erythroid cells. No change was observed in these primary cultures with respect to α‐fetoprotein (α‐FP), albumin, cytokeratin 19 (CK 19), γ‐glutamyltranspeptidase (GGT), and IGF II receptors. Two well‐differentiated hepatomas, HepG2 and FTO‐2B, and a poorly differentiated hepatoma, H4AzC2, did not show increased proliferation in the presence of IGF II, yet showed gene expression changes in response to IGF II. In HepG2 cells, IGF II increased albumin mRNA levels and resulted in a shift from clusters of cells positive to 100% of the cells expressing immunohistochemically detectable albumin. The transcription factor HNF‐3β mRNA and protein levels of the bile duct markers, CK19 and GGT, were also increased in the presence of IGF II. Other genes tested were not affected, including α 1‐antitrypsin, and two liver specific transcription factors, HNF‐4 and HNF‐3α. In FTO‐2B cells, IGF II increased the expression of albumin, CK19, and GGT, without accompanying changes in albumin and GGT mRNAs. In H4AzC2 cells, IGF II reduced CK19 and OC.3 protein levels and GGT, transferrin, and HNF‐3β mRNAs. The effects of IGF II on H4AzC2 cells were not blocked in the presence of an anti‐rat IGF II receptor antibody. We conclude that IGF II affects tissue‐specific gene expression of hepatomas and qualitative and quantitative aspects of its influence on the hepatomas is dependent on their degree of differentiation. © 1995 Wiley‐Liss, Inc.
Interferon‐γ exerts its negative regulatory effect primarily on the earliest stages of murine erythroid progenitor cell development
Authors: Wang Chang Q. , Udupa Kodetthoor B. , Lipschitz David A.
Published: February 2005

Interferon‐γ (INFγ) has been shown to suppress erythropoiesis and perhaps to contribute to the anemia of chronic disease. In this study we demonstrated that the concentration of INFγ required to suppress murine burst forming unit‐erythroid (BFU‐E) growth was significantly less than that required to suppress colony forming unit‐erythroid (CFU‐E) growth. INFγ acted at the most primitive step in erythroid progenitor cell differentiation and proliferation, as inhibition was maximal when added at the time of BFU‐E culture initiation. Inhibition was progressively less if INF‐γ addition was delayed after culture initiation. The effects of INFγ on BFU‐E did not require the presence of interleukin‐1α (IL‐1α), tumor necrosis factor‐α (TNFα), or granulocyte macrophage colony stimulating factor (GM‐CSF), as its effects were not neutralized by monoclonal antibodies against IL‐1α, TNFα, or GM‐CSF. This applied whether INFγ was added to culture with individual antibodies or with a combination of all three antibodies. INFγ was not required for IL‐1α‐ or TNFα‐induced suppression of BFU‐E, as their effects were not neutralized by a monoclonal anti‐INFγ antibody. In contrast, GM‐CSF—induced suppression of BFU‐E was negated by the simultaneous addition of anti‐INFγ. We have previously shown that the addition of TNFα does not suppress BFU‐E growth in cultures from marrow depleted of macrophages. Suppression did occur, however, if a small concentration of INFγ that does not inhibit and increasing concentrations of TNFα were added to culture, suggesting a synergistic effect between INFγ and TNFα. These observations suggest that INFγ is a potent direct inhibitor of erythroid colony growth in vitro. It exerts its negative regulatory effect primarily on the earliest stages of erythroid progenitor cell differentiation and proliferation, as much higher doses are required to suppress late erythroid cell development. INFγ is also involved in GM‐CSF—induced inhibition of BFU‐E colony growth. © 1995 Wiley‐Liss, Inc.

This artilce is a US Government work and, as such, is in the public domain in the United States of America.
Inhibition of ATP‐sensitive potassium channels causes reversible cell‐cycle arrest of human breast cancer cells in tissue culture
Authors: Woodfork Karen A. , Wonderlin William F. , Peterson Virginia A. , Strobl Jeannine S.
Published: February 2005

The purpose of this study was to determine if potassium channel activity is required for the proliferation of MCF‐7 human mammary carcinoma cells. We examined the sensitivities of proliferation and progress through the cell cycle to each of nine potassium channel antagonists. Five of the potassium channel antagonists produced a concentration‐dependent inhibition of cell proliferation with no evidence of cytotoxicity following a 3‐day or 5‐day exposure to drug. The IC50 values for these five drugs, quinidine (25 μM), glibenclamide (50 μM), linogliride (770 μM), 4‐aminopyridine (1.6 mM), and tetraethylammmonium (5.8 mM) were estimated from their respective concentration‐response curves. Four other potassium channel blockers were tested at supra‐maximal channel blocking concentrations, including charybdotoxin (200 nM), iberiotoxin (100 nM), margatoxin (10 nM), and apamin (500 nM), and they had no effect on MCF‐7 cell proliferation, viability, or cell cycle distribution. Of the five drugs that inhibited proliferation, only quinidine, glibenclamide, and linogliride also affected the cell cycle distribution. Cell populations exposed to each of these drugs for 3 days showed a statistically significant accumulation in GO/G1 phase and a significant proportional reduction in S phase and G2/M phase cells. The inhibition of cell proliferation correlated significantly with the extent of cell accumulation in GO/G1 phase, and the threshold concentrations for inhibition of growth and GO/G1 arrest were similar. The GO/G1 arrest produced by quinidine and glibenclamide was reversed by removing the drug, and cells released from arrest entered S phase synchronously with a lag period of ∼24 hours. Based on the differential sensitivity of cell proliferation and cell cycle progression to the nine potassium channel antagonists, we conclude that inhibition of ATP‐sensitive potassium channels in these human mammary carcinoma cells reversibly arrests the cells in the GO/G1 phase of the cell cycle, resulting in an inhibition of cell proliferation. © 1995 Wiley‐Liss, Inc.
Kinetics of Myo‐inositol transport in rat ocular lens
Authors: Diecke F. P. J. , Beyer‐Mears A. , Mistry K.
Published: February 2005

Myo‐inositol (MI) influx as a function of concentration in rat lens consisted of a saturable component, fit by a rectangular hyperbola, and a linear component which was more distinct at high myo‐inositol concentrations suggesting passive diffusion. The hyperbolic component was half‐maximally saturated (Kt) at 61.3 μM and had a maximal transport rate (Jmax) of 44.6 μMol/kg wet wt/h. The linear component had an apparent permeability coefficient of 1.44 × 10−6 s−1. Sorbitol, which distributed rapidly in the extracellular space (6.83 ml/100 g wet wt), also appeared to enter the intracellular space with a permeability coefficient of 1.37 × 10−6 s−1, similar to that of myo‐inositol. The influx of myo‐inositol was critically dependent on the concentration of extracellular sodium consistent with a sodium‐myo‐inositol contransport. The kinetics of influx activation by sodium suggested an apparent 2:1 coupling ratio for sodium and myo‐inositol. When potassium was used as sodium substitute, a significantly stronger influx inhibition was observed than with nondepolarizing sodium substitutes, indicating that myoinositol was driven by the electrochemicl gradient of sodium rather than the chemical gradient only. Reducing the extracellular Na concentration increased the MI concentration at which transport was half‐maximally activated, suggesting an ordered binding sequence of Na followed by MI. Myo‐inositol influx was competitively inhibited by phlorizin with an inhibitory coefficient (Ki) of 35 μM. Phloretin also was capable of inhibition but with a much lesser efficacy. Myoinositol desaturates from the lens at a rate of 0.00862 h−1. Approximately 19% of the efflux can be inhibited with phlorizin, suggesting that it represents carrier‐mediated flux. The phlorizin insensitive flux has a rate of 0.00695 h−1 or 1.93 × 10−6 s−1, similar to the Na‐independent passive influx. MI influx is due to a Na‐dependent, phlorizin‐sensitive active transport while the efflux consists largely of a phlorizin‐independent passive leakage. © 1995 Wiley‐Liss, Inc.
Effect of an alkaline shift on induction of the heat shock response in human fibroblasts
Authors: Petronini Pier Giorgio , Alfieri Roberta , Campanini Cinzia , Borghetti Angelo F.
Published: February 2005

The simultaneous exposure of WI‐38 human fibroblasts (HF) to a heat shock (45°C, 30 min) and an alkaline shift (≥ pH 8.0) in the incubation medium increased and extended the expression of heat shock proteins (hsps). Hsp70 was the most prominent inducible hsp synthesized during the recovery phase after the double shock, and the increase in synthesis depended on the degree of alkalinization during the heat shock. The accumulation of inducible hsp70, which was shown by Western blotting to occur in the late part of the recovery period, was more pronounced in the cells exposed to alkaline medium during the heat shock. Northern blotting did not reveal any increase in hsp70 mRNA, although time course studies following the double shock indicated a more prolonged presence of mRNA. Hsp70 gene activation was evaluated by a gel retardation assay using a 32P‐labelled DNA oligonucleotide containing the heat shock consensus element (HSE) and a heat shock‐induced specific binding protein (heat shock transcription factor, HSTF) from the cell extract. Heat shock activated HSTF‐DNA binding and induced hsp70 mRNA expression as well as the synthesis and accumulation of hsp70. Alkaline shift, which by itself did not induce hsps expression, activated HSTF DNA‐binding. However, in combination with heat shock, alkaline shift enhanced and prolonged HSTF‐HSE complex association and hsp expression at both mRNA and protein levels. Since the alkaline shift‐induced activation of hsp gene does not allow full transcription, these results provide further support for the multistep nature of the heat shock transcriptional response. © 1995 Wiley‐Liss, Inc.
Cytoplasmic juxtamembrane domain of the human EGF receptor is required for basolateral localization in MDCK cells
Authors: Hobert Michael , Carlin Cathleen
Published: February 2005

Although it is well established that epidermal growth factor receptors (EGFRs) are asymmetrically expressed at the basolateral plasma membrane in polarized epithelial cells, how this process is regulated is not known. The purpose of this study was to address the mechanism of directed EGFR basolateral sorting using the Madin‐Darby canine kidney (MDCK) cell model. The first set of experiments established sorting patterns for endogenous canine EGFRs. The polarity of the canine EGFR was not quantitatively affected by differences in electrical resistance exhibited by the MDCK I and MDCK II cell strains. In both cases, greater than 90% of total surface EGFRs was localized to the basolateral surface. Canine EGFRs sort directly to the basolateral membrane from the trans‐Golgi network with a halftime of approximately 45 min and have an approximate t1/2 of 12.5 h once reaching the basolateral surface. Human holoreceptors expressed in stably transfected MDCK cells also localize to the basolateral membrane with similar efficiency. To identify EGFR sequences necessary for basolateral sorting, MDCK cells were transfected with cDNAs coding for cytoplasmically truncated human receptor proteins. Human EGFRs truncated at Arg‐651 were localized predominantly at the apical surface of filter‐grown cells, whereas receptors truncated at Leu‐723 were predominantly basolateral. These results suggest that the cytoplasmic juxtamembrane domain contains a positive basolateral sorting determinant. Moreover, the EGFR ectodomain or transmembrane domain may possess a cryptic sequence that specifically interacts with the apical sorting machinery once the dominant basolateral sorting signal is removed. Further elucidation of the precise loacation of these signals will enhance our basic understanding of regulated plasma membrane sorting, as well as the functional consequences of inappropriate EGFR expression associated with certain pathophysiologic and malignant states. © 1995 Wiley‐Liss, Inc.
Chelation of intracellular Ca2+ inhibits murine keratinocyte differentiation in vitro
Authors: Li Luowei , Tucker Robert W. , Hennings Henry , Yuspa Stuart H.
Published: February 2005

The role of intracellular Ca2+ in the regulation of Ca2+‐induced terminal differentiation of mouse keratinocytes was investigated using the intracellular Ca2+ chelator 1,2‐bis(o‐aminophenoxy)‐ethane‐N, N, N′, N′‐tetraacetic acid (BAPTA). A cell permeable acetoxymethyl (AM) ester derivative BAPTA (BAPTA/AM) was loaded into primary mouse keratinocytes in 0.05 mM Ca2+ medium, and then the cells were induced to differentiate by medium containing 0.12 or 0.5 mM Ca2+. Intracellular BAPTA loaded by BAPTA/AM (15–30 μM) inhibited the expression of epidermal differentiation‐specific proteins keratin 1 (K1), keratin 10 (K10), filaggrin and loricrin as detected by immunoblotting. The differentiation‐associated redistribution of E‐cadherin on the cell membrane was delayed but not inhibited as determined by immunofluorescence. BAPTA also inhibited the expression of K1, K10 and Ioricrin mRNA. Furthermore, BAPTA prevented the decrease in DNA synthesis induced by 0.12 and 0.5 mM Ca2+, indicating the drug was inhibiting differentiation but was not toxic to keratinocytes. To evaluate the influence of BAPTA on intracellular Ca2+, the concentration of intracellular free Ca2+ (Cai) in BAPTA‐loaded keratinocytes was examined by digital image analysis using the Ca2+‐sensitive fluorescent probe fura‐2, and Ca2+ influx was measured by 45Ca2+ uptake studies. Increase in extracellular Ca2+ (Cao) in the culture medium of keratinocytes caused a sustained increase in both Cai and Ca2+ localized to ionomycin‐sensitive intracellular stores in keratinocytes. BAPTA lowered basal Cai concentration and prevented the Cai increase. After 12 hours of BAPTA treatment, the basal level of Cai returned to the control value, but the Ca2+ localized in intracellular stores was substantially decreased. 45Ca2+ uptake was initially (within 30 min) increased in BAPTA‐loaded cells. However, the total 45Ca2+ accumulation over 24 hours in BAPTA‐loaded cells remained unchanged from control values. These results indicate that keratinocytes can maintain Cai and total cellular Ca2+ content in the presence of increased amount of intracellular Ca2+ buffer (e.g., BAPTA) by depleting intracellular Ca2+ stores over a long period. The inhibition by BAPTA of keratinocyte differentiation marker expression may result from depletion of the Ca2+‐stores since this is the major change in intracellular Ca2+ detected at the time keratinocytes express the differentiation markers. In contrast, the redistribution of E‐cadherin on the cell membrane may be more directly associated with Cai change. © 1995 Wiley‐Liss, Inc.
Lipopolysaccharide inhibits activation of latent transforming growth factor‐β in bovine endothelial cells
Authors: Kojima Soichi , Vernooy Robert , Moscatelli David , Amanuma Hiroshi , Rifkin Daniel B.
Published: February 2005

The activation of latent transforming growth factor‐β (TGF‐β) by vascular endothelial cells (ECs) is regulated by cellular plasminogen activator (PA)/plasmin, transglutaminase (TGase), and latent TGF‐β levels. Because lipopolysaccharide (LPS) has been reported to reduce EC surface plasmin levels by increasing the production of the inhibitor of PA, PA inhibitor‐1 (PAI‐1), we have tested whether LPS might suppress latent TGF‐β activation in ECs using two different systems, namely, bovine aortic ECs (BAECs) cocultured with smooth muscle cells (SMCs) and BAECs treated with retinol. BAECs were either cocultured with SMCs after treatment with 15 ng/ml LPS or were treated with 2 μM retinol and/or 10 ng/ml LPS, and the expression of PA, surface plasmin, TGase, and the amounts of active and latent TGF‐β secreted into the culture modium were measured. The downregulation of surface PA/plasmin levels with LPS was accompanied by a profound decline of both TGase and latent TGF‐β expression as well as the suppression of surface activation of latent TGF‐β. The effect was dependent on the concentration of LPS and on treatment time. The formation of TGF‐β did not occur in cells maintained in LPS‐contaminated culture medium. © 1995 Wiley‐Liss, Inc.
Nuclear translocation of prolactin: Collaboration of tyrosine kinase and protein kinase C activation in rat Nb2 node lymphoma cells
Authors: Rao Yi‐Ping , Buckley Donna J. , Olson Mark D. , Buckley Arthur R.
Published: February 2005

Recent evidence has suggested that prolactin (PRL), internalized by lactogen‐dependent Nb2 lymphoma cells, is actively translocated to the nucleus where it binds to PRL receptors. Moreover, the mitogenic action of PRL in these cells has been separately linked to protein tyrosyl phosphorylation and activation of protein kinase C (PKC). Therefore, the coupling of PRL internalization and nuclear translocation to the activation of these signal transduction pathways was investigated. Results from control experiments indicated that 30% of internalized and 5% total cell‐associated 125I‐rat PRL could be recovered within nuclei obtained from Nb2 cells previously incubated with the radiolabel for 3 h at 37°C. Furthermore, internalized PRL was found to be intact and not associated with any carrier proteins. Addition of tyrosine kinase (TK) antagonists, genistein or tyrphostin, significantly reduced cell surface binding, internalization, and nuclear translocation of 125I‐rat PRL. In contrast, neither the level of cell‐associated nor internalized hormone differed between cells treated with the PKC antagonists, staurosporine or calphostin C, and control cultures. Instead, PKC inhibition significantly reduced nuclear PRL translocation. The inhibitory effects of the TK and PKC antagonists on PRL internalization and nuclear translocation in intact Nb2 cells were verified by immunofluorescence microscopy in parallel experiments. In other experiments, each of the kinase inhibitors blocked PRL‐stimulated Nb2 cell proliferation in a concentration‐dependent manner. It is concluded that activated TK and PKC collaborate in the process of PRL internalization and translocation to the nucleus. TK activation may participate in PRL receptor binding or hormone internalization while activation of PKC appears to be required for its nuclear targeting. Since TK and PKC activation are required for lactogen‐stimulated Nb2 cell proliferation, we suggest that a component of the mitogenic pathway in these cells is a direct nuclear interaction of PRL. © 1995 Wiley‐Liss, Inc.