bluuebunny/crossref_metadata_2025 huggingface dataset:
Fibrin contact increases endothelial permeability to albumin
Authors: Lo Siu K. , Vecchio Peter J. Del , Lum Hazel , Malik Asrar B.
Published: February 2005

We studied the effects of contact of bovine pulmonary artery endothelial cell monolayers with fibrin on the endothelial barrier function. Fibrin formed by clotting purified fibrinogen (0.5 to 3.0 mg/ml) with α‐thrombin (1 U/ml) was added to endothelial monolayers and permeability measurements were made after fibrin removal. Fibrin incubation for 3 hours resulted in 2‐ to 5‐fold increases in transendothelial 125l‐albumin permeability. Permeability returned to baseline value within 3 hours after fibrin removal. Direct contact with fibrin was necessary for the response, since fibrin separated from the endothelium did not increase permeability. Contact with agarose (2 mg/ml) or fibrinogen (0.5 to 3.0 mg/ml) also did not increase endothelial permeability. Transmission electron microscopic examination indicated normal appearance of interendothelial junctions at a time when albumin permeability was increased and no overt evidence of endothelial injury. Incubation of fibrin with endothelial monolayers at 4°C prevented the increase in albumin permeability. We examined the possibility that increased albumin transcytosis was responsible for fibrin's effect using 14C‐sucrose (Mr = 342D), a lipid insoluble tracer. Fibrin increased sucrose flux by 1.5‐fold compared to 2‐to 5‐fold increases in albumin flux. The results indicate that fibrin contact with the endothelial cell increases endothelial permeability. The effect of fibrin may involve activation of temperature‐sensitive bulk phase transcytosis of albumin. © 1992 Wiley‐Liss, Inc.
Release of cell surface‐associated basic fibroblast growth factor by glycosylphosphatidylinositol‐specific phospholipase C
Authors: Bashkin Pnina , Neufeld Gera , Gitay‐Goren Hela , Vlodavsky Israel
Published: February 2005

Heparan sulfate proteoglycans (HSPG) are ubiquitous constituents of mammalian cell surfaces and most extracellular matrices. A portion of the cell surface HSPG is anchored via a covalently linked glycosyl‐phosphatidylinositol (PI) residue, which can be released by treatment with a glycosyl‐PI specific phospholipase C (PI‐PLC). We report that exposure of bovine aortic endothelial and smooth muscle cells to PI‐PLC resulted in release of cell surface‐associated, growth‐promoting activity that was neutralized by antibasic fibroblast growth factor (bFGF) antibodies. Active bFGF was also released by treating the cells with bacterial heparitinase. Under the same conditions there was no release of mitogenic activity from cells (BHK‐21, NIH/3T3, PF‐HR9) that expressed little or no bFGF, as opposed to PI‐PLC‐mediated release of active bFGF from the same cells transfected with the bFGF gene. The released bFGF competed with recombinant bFGF in a radioreceptor assay. Addition of PI‐PLC to sparsely seeded vascular endothelial cells resulted in a marked stimulation of cell proliferation, but there was no mitogenic effect of PI‐PLC on 3T3 fibroblasts. Studies with exogenously added 125I‐bFGF revealed that about 6.5% and 20% of the cell surface‐bound bFGF were released by treatment with PI‐PLC and heparitinase, respectively. Both enzymes also released sulfate‐labeled heparan sulfate from metabolically labeled 3T3 fibroblasts. PI‐PLC failed to release 125I‐bFGF from the subendothelial extracellular matrix (ECM), as compared to release of 60% of the ECM‐bound bFGF by heparitinase. Our results indicate that 3–8% of the total cellular content of bFGF is associated with glycosyl‐PI anchored cell surface HSPG. This FGF may exert both autocrine and paracrine effects, provided that it is released by PI‐PLC and adequately presented to high affinity bFGF cell surface receptor sites. © 1992 Wiley‐Liss, Inc.
Stimulatory and inhibitory effects of catecholamines on DNA synthesis in primary rat hepatocyte cultures: Role of alpha1‐ and beta‐adrenergic mechanisms
Authors: Refsnes Magne , Thoresen G. Hege , Sandnes Dagny , Dajani Olav F. , Dajani Laila , Christoffersen Thoralf
Published: February 2005

Previous studies suggest that catecholamines may be involved in the regulation of liver growth. Considerable evidence implicates α1‐adrenergic mechanisms in the initiation of hepatocyte proliferation, while the role of β‐adrenoceptors is less clear. We have examined further the adrenergic regulation of hepatocyte DNA synthesis, using primary monolayer cultures. In hepatocytes that were also treated with epidermal growth factor and insulin, epinephrine or norepinephrine added early after the seeding strongly accelerated the rate of S phase entry. The β‐adrenergic agonist isoproterenol and the α‐adrenergic agonist phenylephrine also stimulated the DNA synthesis, but were less efficient than epinephrine and norepinephrine. Experiments with the α1‐receptor blocker prazosine and the β‐receptor blocker timolol showed that the stimulatory effect of norepinephrine consisted of both an α1‐ and a β‐adrenergic component. The α1‐component was most prominent in terms of maximal response at high concentrations of the agonist, but the β‐component contributed significantly and predominated at low concentrations (< 0.1 μM) of norepinephrine. At later stages (about 40 h) of culturing norepi nephrine strongly but reversibly inhibited the cells, acting at a point late in the G1 phase. This inhibition was mimicked by isoproterenol and abolished by timolol but was unaffected by prazosine, suggesting a β‐adrenoceptor‐mediated effect. The results confirm the α1‐adrenoceptor‐mediated stimulatory effect, but also show that β‐adrenoceptors may contribute to the growth stimulation by catecholamines. Furthermore, catecholamines, via β‐adrenoceptors and cyclic AMP, inhibit the G1‐S transition, and may thus play a role in the termination of hepatic proliferation. © 1992 Wiley‐Liss, Inc.
Modulation of tumor cell susceptibility to cytokine‐induced cell death by hormones, growth factors, and cell density
Authors: van der Bosch Jürgen , Horn Daniel , Rüller Stephan , Schlaak Max
Published: February 2005

The mechanisms controlling “spontaneous” cellular death rates in normal and tumorigenic tissues are largely unknown. An important parameter in this respect is the susceptibility of the target cell to induction of the lytic pathway by appropriate signals. In the present article it is demonstrated in a serum‐free in vitro system that the susceptibility of human tumor cells (TC) to induction of lysis by cytokine signals generated during interaction of TC with elutriated human monocytes (MO) is a highly dynamic parameter subject to modulation by hormones, growth factors, and tumor cell density. It was found that growth stimulatory signals such as insulin, and especially epidermal growth factor (EGF), increase lytic susceptibility, whereas hydrocortisone, which does not exert significant growth modulatory effects in these examples, protects TC against the induction of lysis. Increasing TC density above confluence dramatically enhances lytic susceptibility, suggesting interactions between TC to be involved in the induction of their death. In conjunction with previous data demonstrating the insusceptibility of TC, which are forced out of the cell cycle into the quiescent state (G0), the hypothesis is put forward that growth stimulatory factors increase a TC's lytic susceptibility by preventing its transit from G1 to G0 in response to growth inhibitory signals generated during MO/TC interaction. The data support the concept that TC susceptibility to the induction of cell death is a consequence of simultaneously activated growth stimulatory and growth inhibitory signalling pathways. © 1992 Wiley‐Liss, Inc.
Elevated cAMP is required for stimulation of eicosanoid synthesis by interleukin 1 and bradykinin in BALB/c 3T3 fibroblasts
Authors: Burch Ronald M. , Connor Jane R.
Published: February 2005

In Swiss 3T3, murine fibroblasts, interleukin 1 (IL‐1) and bradykinin stimulate prostaglandin E2 (PGE2) synthesis. However, in the present study, we found that neither agonist stimulated PGE2 synthesis in BALB/c 3T3 murine fibroblasts, this in spite of expression of similar numbers of receptors for each agonist compared to Swiss 3T3 cells. When BALB/c 3T3 cells were preincubated with cAMP analogs, both IL‐1 and bradykinin stimulated PGE2 synthesis to levels similar to those observed in Swiss 3T3 cells. Similarly, when the cells were preincubated with forskolin, which activates the catalytic subunit of adenylate cyclase directly, or NECA, which stimulates cellular cAMP accumulation by activating adenosine receptors, IL‐1 and bradykinin stimulated PGE2 synthesis. Rp‐cAMPS, an inhibitor of cAMP‐dependent protein kinase, blocked the ability of cAMP or NECA to render cells responsive to IL‐1 and bradykinin. In basal BALB/c 3T3 cells, bradykinin and IL‐1 stimulated arachidonate release in the absence of cAMP, but little conversion of released arachidonate to PGE2 occurred. cAMP, forskolin, and NECA all increased cyclooxygenase activity in the cells. SV‐T2 is a clonal line originating from BALB/c 3T3 transformed with SV‐40. In these cells, IL‐1 and bradykinin stimulated PGE2 svnthesis despite basal intracellular cAMP concentrations similar to BALB/c, and cAMP only modestly potentiated the response. In summary, cyclooxygenase expression appears to be regulated by cAMP in BALB/c 3T3 cells, and SV‐40 transformation results in increased cyclooxygenase expression, apparently independent of cAMP. © 1992 Wiley‐Liss, Inc.
Initial characterization of heat‐induced excess nuclear proteins in HeLa cells
Authors: Laszlo A. , Wright W. , Roti J. L. Roti
Published: February 2005

Exposure of mammalian cells to hyperthermia is known to cause protein aggregation in the nucleus. The presence of such aggregates has been detected as the relative increase in the protein mass that is associated with nuclei isolated from heated cells. We have characterized these excess nuclear proteins from the nuclei of heated HeLa cells by two‐dimensional gel electrophoresis. The abundance of cytoskeletal elements which co‐purify with the nuclei did not increase with exposure to hyperthermia, indicating that these proteins are not part of the excess nuclear proteins. In contrast, several specific polypeptides become newly bound or increase in abundance in nuclei isolated from heated cells. Members of the hsp 70 family were identified as a major component of the excess nuclear proteins. Among the other excess nuclear proteins we identified ten that had apparent molecular weights of 130, 95, 75, 58, 53, 48, 46, 37, 28, and 26 kilodaltons. Since hsp 70 is mainly cytoplasmic in non‐heated cells, its association with nuclei in heated cells indicates that one mechanism accounting for the heat‐induced excess nuclear proteins is the movement of cytoplasmic proteins to the nucleus. We also obtained evidence that increased binding of nuclear proteins is another mechanism for this effect. No overall increase or decrease in the phosphorylation of nuclear proteins was found to be associated with such altered binding or movement from the cytoplasm to the nucleus. © 1992 Wiley‐Liss, Inc.
Leukemia inhibitory factor/differentiation‐stimulating factor (LIF/D‐factor): Regulation of its production and possible roles in bone metabolism
Authors: Ishimi Yoshiko , Abe Etsuko , Jin Cheng He , Miyaura Chisato , Hong Mei Hua , Oshida Midori , Kurosawa Hisashi , Yamaguchi Yuri , Tomida Mikio , Hozumi Motoo , Suda Tatsuo
Published: February 2005

Leukemia inhibitory factor/differentiation‐stimulating factor (LIF/D‐factor), expression of its mRNA, and possible roles in bone metabolism were studied in murine primary and clonal osteoblast‐like cells. Local bone‐resorbing factors such as IL‐1, TNFα, and LPS strongly induced expression of LIF/D‐factor mRNA in both clonal MC3T3‐E1 cells and primary osteoblast‐like cells. Neither parathyroid hormone nor 1α,25‐dihydroxyvitamin D3 stimulated expression of LIF/D‐factor mRNA. LIF/D‐factor per se did not stimulate expression of its own mRNA.

Appreciable amounts of LIF/D‐factor were detected in synovial fluids from rheumatoid arthritis (RA) patients but not in those with osteoarthritis (OA). Simultaneous treatment with LIF/D‐factor, IL‐1, and IL‐6 at the concentrations found in synovial fluids from RA patients greatly enhanced bone resorption, though these cytokines did not stimulate bone resorption when separately applied. This suggests that LIF/D‐factor produced by osteoblasts is in concert with other boneresorbing cytokines such as IL‐1 and IL‐6 involved in the bone resorption seen in the joints of RA patients.

LIF/D‐factor specifically bound to MC3T3‐E1 cells with an apparent dissociation constant of 161 pM and 1,100 binding sites/cell. LIF/D‐factor dose‐dependently suppressed incorporation of [3H]thymidine into MC3T3‐E1 cells. In addition, it potentiated the alkaline phosphatase activity induced by retinoic acid, though LIF/D‐factor alone had no effect on enzyme activity. These results suggest that LIF/D‐factor is involved in not only osteoclastic bone resorption but also osteoblast differentiation in conjugation with other osteotropic factors. © 1992 Wiley‐Liss, Inc.
Development of acute thermotolerance in 1929 cells: Lack of HSP28 synthesis and phosphorylation
Authors: Lee Yong J. , Hou Zi‐Zheng , Curetty Lindali , Borrelli Michael J.
Published: February 2005

We investigated the correlation between the development of acute thermotolerance and the phosphorylation, synthesis, and expression of the HSP28 family in murine L929 cells. Following heating at 43° for 30 min, thermotolerance developed rapidly in exponential‐phase cells and reached its maximum 4–9 h after heat shock. Maximal thermal resistance was maintained for 24 h and then gradually decayed. However, heat‐induced phosphorylation of HSP28 was not detected. Furthermore, HSP28 synthesis during incubation at 37°C for 12 h following heat shock was not detected by [3H]‐leucine labeling followed by two‐dimensional polyacrylamide gel electrophoresis. In addition, Northern blots failed to demonstrate expression of the HSP28 gene. Unlike HSP28, the expression of constitutive and inducible HSP70 genes, along with the synthesis of their proteins, was observed during incubation at 37° after heat shock. These results demonstrate that HSP28 synthesis and its phosphorylation are not required to develop acute thermotolerance in L929 cells. © 1992 Wiley‐Liss, Inc.
Inhibition of cell‐cell adhesion and morphogenesis of Dictyostelium by carnitine
Authors: Siu Chi‐Hung , Brar Pretty , Fritz Irving B.
Published: February 2005

Carnitine (γ‐trimethylammonium β‐hydroxy‐butyric acid) possesses the novel property of preventing cell aggregation elicited by clusterin or by fibrinogen (I.B. Fritz and K. Burdzy, J. Cell. Physiol., 140:18–28 [1989]). In investigations reported here, we show that carnitine also affects cell‐cell adhesion in Dicytostelium discoideum, a cellular slime mold whose cells interact in specific and complex manners during discrete stages of development. Two types of cell adhesion systems sequentially appear on the surface of developing Dictyostelium cells, involving the surface glycoprotein gp24 which mediates EDTA‐sensitive binding sites, and the surface glycoprotein gp80 which mediates the EDTA‐resistant binding sites. Addition of increasing concentrations of D(+)‐carnitine and L(‐)‐carnitine resulted in a progressive inhibition of both the EDTA‐sensitive binding sites and the EDTA‐resistant binding sites of Dictyostelium cells at different stages of development. In contrast, comparable or higher concentrations of choline, acetyl‐β‐methylcholine, or deoxycarnitine had no detectable effects on cell aggregation. Concentrations of carnitine required for 50% inhibition of EDTA‐resistant adhesion sites were found to be dependent upon levels of gp80 expressed by Dictyostelium, with greatest inhibition by carnitine of reassociation of cells containing the lowest levels of gp80. Removal of carnitine from cells by washing resulted in the rapid restoration of the ability of Dictyostelium to form aggregates and to resume normal development. We discuss possible mechanisms by which carnitine inhibits the aggregation of cells. © 1992 Wiley‐Liss, Inc.
Melanocyte stimulating hormone induces the differentiation of mouse epidermal melanocytes in serum‐free culture
Authors: Hirobe Tomohisa
Published: February 2005

In serum‐free primary culture of dissociated mouse epidermal cells, α‐melanocyte stimulating hormone (α‐MSH) and dibutyryl cyclic AMP (DBcAMP) induced the differentiation of melanocytes. Moreover, the proliferation of melanocytes was also induced in the dishes cultured with DBcAMP, but not with α‐MSH. In order to clarify the role of keratinocytes in melanocyte proliferation and differentiation, pure cultures of keratinocytes were established in serum‐free medium. Subconfluent primary keratinocytes were trypsinized and seeded into pure primary melanoblasts cultured with serum‐free medium that did not contain α‐MSH or DBcAMP. Melanoblasts were cultured with α‐MSH or DBcAMP in the presence or absence of keratinocytes. α‐MSH failed to induce melanocyte differentiation in the absence of keratinocytes. DBcAMP failed to induce melanocyte proliferation in the absence of keratinocytes, although it induced melanocyte differentiation even in the absence of keratinocytes. These results suggest that keratinocyte‐derived factors are required not only for the induction of melanocyte differentiation by α‐MSH but also for the induction of melanocyte proliferation by DBcAMP. © 1992 Wiley‐Liss, Inc.
Bethanechol inhibition of chicken intestinal brush border Na/H exchange: Role of protein kinase C and other calcium‐dependent processes
Authors: Musch Mark W. , Drabik‐Arvans Donna , Rao Mrinalini C. , Chang Eugene B.
Published: February 2005

Bethanechol, a muscarinic agonist, inhibits the initial rate of amiloride‐sensitive Na uptake by intact mucosa of avian small intestine as well as by isolated chicken villus enterocytes, an effect that is maximal at 90 seconds and reverses by 6 minutes. Bethanechol similarly decreases intracellular pH in isolated cells suspended in bicarbonate‐free buffer in a time course similar to inhibition of enterocyte Na uptake, suggesting inhibition of Na/H exchange. In brush border membrane vesicles rapidly prepared from cells stimulated with bethanechol, proton‐dependent 22Na uptake is transiently inhibited in a time course similar to inhibition of cell Na uptake. Bethanechol also stimulates transient translocation of protein kinase C from the cytosol to the particulate fraction, a portion of this activity translocating to the brush border membrane. To determine the calcium dependence of bethanechol's action, enterocytes were loaded with varying concentrations of the calcium buffering agent quin‐2. Inhibition of cell Na uptake by the calcium ionophore ionomycin could be completely reversed by quin‐2 buffering in a concentration‐dependent manner. In contrast, quin‐2 buffering had little or no effect on the inhibition of Na uptake caused by the protein kinase C activators phorbol esters and oleoylacetylglycerol. Bethanechol's inhibitory effects were partially, but not completely reversed by quin‐2 buffering. These data suggest that the effects of bethanechol on chicken villus enterocyte brush border Na/H exchange are mediated by calcium‐dependent process(es) as well as by protein kinase C. © 1992 Wiley‐Liss, Inc.
Competition between cell‐substratum interactions and cell‐cell interactions
Authors: Tung Pierre S. , Burdzy Krystyna , Wong Katherine , Fritz Irving B.
Published: February 2005

Clusterin, a glycoprotein which elicits the aggregation of a wide variety of cells (Fritz, I.B., and Burdy, K.: J. Cell Physiol., 140:18–28, 1989), has been utilized to investigate some of the factors modulating the competition between cell‐substratum interactions and cell‐cell interactions. We compared the responses to clusterin by anchorage‐independent cells (erythrocytes) with those by anchoragedependent TM4 cells (a cell line derived from neonatal mouse testis cells). Cells were maintained in culture in the presence of various substrata chosen to enhance cell‐substratum interactions (laminin‐coated wells), or to diminish cell‐substratum interactions (agarose‐coated wells). Results obtained showed that the aggregation of erythrocytes elicited by clusterin was independent of the nature of the substratum. In contrast, clusterin addition resulted in aggregation of anchorage‐dependent TM4 cells only when TM4 cell‐substratum interactions were weak. Thus, clusterin did not aggregate TM4 cells plated upon a laminin substratum, but readily aggregated TM4 cells plated upon an agarose‐coated substratum, independent of the sequence of addition of cells and clusterin to the culture dish. We utilized YIGSR, a peptide which competes with laminin for laminin receptors, to determine the possible role of laminin receptors on TM4 cells in the competition between cell‐substratum interactions and cell‐cell interactions. The presence of YIGSR did not alter responses of erythrocytes to clusterin under all conditions examined. In contrast, the responses of TM4 cells to clusterin were greatly changed. YIGSR addition resulted in the inhibition of aggregation of TM4 cells otherwise elicited by clusterin. YIGSR also prevented attachment of TM4 cells to a laminin‐coated surface, but this was reversed by the presence of clusterin. We discuss the possible roles of clusterin and laminin in altering the balance in the competition between cell to cell interactions and cell to substratum interactions. © 1992 Wiley‐Liss, Inc.
Cytoskeletal elements are required for the formation and maturation of autophagic vacuoles
Authors: Aplin A. , Jasionowski T. , Tuttle D. L. , Lenk S. E. , Dunn W. A.
Published: February 2005

We evaluated the role of cytoskeletal elements in the degradation of endogenous proteins via autophgy using biochemical and morphological techniques. In the absence of exogenous amino acids, degradation of endogenous proteins was enhanced in cultured normal rat kidney cells. This enhanced degradative state was accompanied by a 4‐fold increase in the occurrence of autophagic vacuoles. In the presence of drugs that induce the depolymerization of microfilaments (cytochalasins B and D) or microtubules (nocodazole), protein degradation was not enhanced in nutrient‐deprived cells. Although these drugs had similar inhibitory effects on the protein degradation, their effect on autophagy differed. Cytochalasins B and D interfered with the formation of the autophagosome. In cells treated with these drugs, the fractional volume represented by autophagic vaculoes was not substantially increased despite nutrient depletion. On the contrary, nocodazole appeared to have no effect on the formation of autophagosomes. Instead, this drug suppressed the delivery of hydrolytic enzymes, thereby resulting in an accumulation of acidic autophagic vacuoles containing undegraded cellular components. © 1992 Wiley‐Liss, Inc.
Transforming growth factor‐β1 mediated alterations in ribonucleotide reductase gene expression in BALB/c 3T3 fibroblasts
Authors: Hurta Robert A. R. , Greenberg Arnold H. , Wright Jim A.
Published: February 2005

Transforming growth factor‐β1 (TGF‐β1) stimulated DNA synthesis (3‐fold) in BALBc/3T3 fibroblasts following 24 hours of growth factor exposure. Since ribonucleotide reductase is important for the coordination of DNA synthesis and cell proliferation, we investigated the hypothesis that cells like BALB/c 3T3, which are TGF‐β1 responsive, would exhibit modifications in expression of the gene for ribonucleotide reductase following growth factor treatment. We observed 2.6, 4.1, and 4.8‐fold increases in ribonucleotide reductase activity following TGF‐β1 exposure for 6, 12, and 24 hours, respectively. Increased ribonucleotide reductase R2 gene expression (3, 3.7, and 4.5‐fold) and R1 gene expression (2, 2.5, and 2.6‐fold) were observed following 6, 12, and 24 hours of TGF‐β1 treatment, respectively. Western blots indicated 2.2, 3.1, and 4.1‐fold increases in protein R2 levels at 6, 12, and 24 hours exposure to TGF‐β1, whereas 2.6 and 3.3‐fold elevations in R1 protein levels were observed at 12 and 24 hours postTGF‐β1 exposure. These TGF‐β1 mediated modifications in ribonucleotide reductase gene expression occurred, in part, prior to any detectable changes in the rate of DNA synthesis, demonstrating alterations in the normal regulation of ribonucleotide reductase. Furthermore, these alterations could be markedly reduced by prolonged pretreatment with 12‐0‐tetradecanoylphorbol‐13‐acetate (R2 gene expression increased by only 1.3, 1.5 and 2.3‐fold after 6, 12, and 24 hours of TGF‐β1 treatment, respectively), suggesting a role for a protein kinase C pathway in the TGF‐β1 regulated changes in ribonucleotide reductase gene expression. These results indicate for the first time that TGF‐β1 can regulate the expression of the two genes for ribonucleotide reductase in BALB/c 3T3 fibroblasts, and suggest that regulation of these genes plays an important role in critical events involved in growth factor modulation of normal and transformed cell proliferation. © 1992 Wiley‐Liss, Inc.
Independent regulation of prostaglandin production and the stress response in human fibroblasts
Authors: Salzman J. , Bowman P. D.
Published: February 2005

The stress, or heat shock response of eukaryotic cells is characterized by dramatic changes in the metabolism of responding cells, most notably the increased synthesis of a group of proteins known as heat shock proteins. In this study, we examined the relationship of prostaglandin synthesis/release to the stress response. Stress protein synthesis was induced with sodium arsenite, and prostaglandin E2 and prostacyclin (measured as 6‐keto PGF1α) levels were determined by enzyme immunoassay. The stress response was monitored by the incorporation of [35S]methionine and separation of protein by one‐dimensional sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Prostaglandin synthesis and the stress response were both induced by sodium arsenite. However, aspirin, a cyclooxygenase inhibitor, inhibited arsenite‐induced prostaglandin synthesis but did not inhibit stress protein synthesis. Conversely, the calcium ionophore A23187 also stimulated prostaglandin synthesis, but did not induce the stress response. The results of this study indicate that sodium arsenite, a stress response inducer, stimulates prostaglandin production, but this appears to be a correlative rather than causative occurrence in the stress response. Determination of the cytotoxicity of arsenite indicated a high correlation of stimulation of prostaglandin release with cytotoxicity. © 1992 Wiley‐Liss, Inc.
Reversible G1 arrest of a human lung epithelial cell line by staurosporine
Authors: Lin Yuan , Chrest Francis J. , Gabrielson Edward W.
Published: February 2005

Staurosporine, a microbial‐derived protein kinase inhibitor, reversibly blocked non‐synchronized, replicating cultures of the human lung epithelial cell line EKVX in the G1 phase of cell cycle and inhibited DNA synthesis and cell replication. The mechanism of this cell‐cycle arrest in EKVX cells by staurosporine was likely due to inhibition of protein kinase C (PKC) because: (1) dose‐dependent inhibition of DNA synthesis occurred at levels of staurosporine that inhibit phosphorylation of PKC substrate, (2) inhibition of DNA synthesis was also seen after treatment with another PKC inhibitor H7, but not by the chemically similar HA1004, which has a relative inhibitory specificity for cAMP‐dependent protein kinase, and (3) the DNA synthesis was not inhibited by specific tyrosine kinase inhibitors Genistein and Lavendustin A at concentrations that inhibit tyrosine kinase activity. Removal of staurosporine from cell culture media resulted in a rebound in PKC activity and synchronized DNA synthesis in EKVX cultures. The reversibility of the inhibition was noted even after 5 days of treatment with staurosporine, and DNA synthesis remained synchronized for at least two rounds of cell replication after removal of staurosporine. Flow cytometric analysis confirmed that more than 90% of the cell population was blocked in the G1 phase after cells were treated with staurosporine for 24 h. Agents such as staurosporine may be useful for synchronizing cell populations to study cell‐cycle specific biochemical events important for the regulation of cell replication in the EKVX cell line. © 1992 Wiley‐Liss, Inc.
Human umbilical vein endothelial cells submitted to hypoxia‐reoxygenation in vitro: Implication of free radicals, xanthine oxidase, and energy deficiency
Authors: Michiels Carine , Arnould Thierry , Houbion Andrée , Remacle José
Published: February 2005

Ischemia‐reperfusion is observed in various diseases such as myocardium infarct. Different theories have been proposed to explain the reperfusion injury, among them that the free radical generation plays a crucial role. To study the mechanisms of the reperfusion injury, a hypoxia (H)‐reoxygenation (R) model upon human umbilical vein endothelial cells in culture was developed in order to mimic the in vivo situation. Different parameters were quantified and compared under H or H/R, and we found that oxygen readmission led to damage amplification after a short hypoxia period. To estimate the importance of various causes of toxicity, the effects of various protective molecules were compared. Different antioxidant molecules, iron‐chelating agent, xanthine oxidase inhibitors, and energy‐supplying molecules were very efficient protectors. Synergy could also be observed between the antioxidants and the energy‐supplying molecules or the xanthine oxidase inhibitors. The toxic effect of O2·(−) could be lowered by the presence of SOD or glutathione peroxidase in the culture medium, whereas glutathione peroxidase was the most efficient enzyme when injected into the cells. The production of O2·(−) and of H2O2 by endothelial cells was directly estimated to be, respectively, of 0.17 and 0.035 μmol/min/mg prot during the R period. O2·(−) production was completely inhibited when allopurinol was added during H and R. In addition, a xanthine oxidase activity of 21.5 10−6 U/mg prot could be observed by a direct assay in cells after H but not in control cells, thus confirming the previous conclusions of xanthine oxidase as a potent source of free radicals in these conditions. Thanks to the use of cultured human endothelial cells, a clear picture was obtained of the overall process leading to cell degenerescence during the reoxygenation process. We particularly could stress the importance of the low energetic state of these cells, which is a critical factor acting synergistically with the oxidant molecules to injure the cells. These results also open new possibilities for the development of new therapeutics for ischemia. © 1992 Wiley‐Liss, Inc.
Differential modulation of vascular cell integrin and extracellular matrix expression in vitro by TGF‐β1 correlates with reciprocal effects on cell migration+
Authors: Basson Craig Todd , Kocher Olivier , Basson Marc D. , Asis Antonio , Madri Joseph A.
Published: February 2005

In vitro, BAEC and BASMC migratory phenotypes are known to be reciprocally modulated by both soluble factors and extracellular matrix proteins. In addition, integrin matrix receptors mediate endothelial and smooth muscle cell attachment and migration. To further elucidate these phenomena, we studied the effects of TGF‐β1 on intergin expression by vascular BASMC and BAEC in tissue culture. TGF‐β1 upregulated mRNA levels and surface pools of BASMC β3 integrin classes without modulating β1 integrin mRNA levels or expression of β1 integrin organization. In contrast to its effect on BASMC, TGF‐β1 increased BAFC mRNA levels and surface expression of β1 and β3 integrins without altering their organization. Conversely, extracellular matrix components (fibronectin, laminin, and fibrinogen) organized cell surface integrins in both BASMC and BAEC without affecting the size of their cell surface pools. These data are consistent with the hypothesis that SMC and EC behavior in neointimal lesions may modulated in part, through a coordination of soluble factor and extracellular matrix protein regulation of integrin surface expression and organization. © 1992 Wiley‐Liss, Inc.
Regulation of cell proliferation: Late down‐regulation of c‐myb preceding myelo‐monocytic cell differentiation
Authors: Yen Andrew , Samuel Varman , Forbes Mary
Published: February 2005

Expression of the c‐myb nuclear oncogene during the cell proliferation and differentiation of HL‐60 human promyelocytic leukemia cells was characterized and compared to the expression of c‐fos, another nuclear oncogene with transcriptional regulatory activity. During progression through the cell cycle, the amount of c‐myb protein increased. The increase was commensurate with total cell size, thus preserving the relative abundance of c‐myb protein present at the onset of the cell cycle. In HL‐60 cells, the induced metabolic cascade leading to terminal myeloid or monocytic differentiation segregates into two steps occuring over two division cycles. Expression of c‐myb did not diverge from the control until late in this metabolic cascade when it declined prior to onset of terminal differentiation. This course of expression was similar for both the retinoic acid induced myloid or the 1,25‐dihydroxy vitamin D2 induced monocytic terminal differentiation of the cells. Bromodeoxyuridine, which induces proliferative arrest but not phenotypic differentiation of these cells, induced the same course of c‐myb expression as the inducers of terminal differentiation. The same course of c‐myb expression with growth arrest induced by these three different means is consistent with a potential proliferation regulatory role for c‐myb in late but not early events leading to terminal differentiation. The dynamics of c‐myb expression during this process were qualitatively, out not quantitatively, similar to the course of c‐fos expression. Thus, taken with previous results, then amongst the nuclear oncogenes or tumor suppressor genes, c‐myc, RB, c‐fos, and c‐myb, only c‐myc and RB expression exhibit early regulation during induced HL‐60 cell differentiation. © 1992 Wiley‐Liss, Inc.
Norepinephrine stimulates the direct breakdown of phosphatidyl inositol in rat tail artery
Authors: Labelle Edward F. , Gu Hong , Trajkovic Snezana
Published: February 2005

When segments of rat tail artery were labeled with [3H]inositol and then stimulated with norepinephrine (NE), the inositol phosphates produced were primarily IP and IP2, together with a small but significant amount of Ins(1,4,5)P3 and a very small amount of Ins(1,3,4,5)P4. It has been unclear in many studies whether or not the relatively large levels of IP and IP2 produced in [3H]inositol‐labeled tissue represent indirect products of phosphatidyl inositol(4,5)bis phosphate breakdown (through Ins(1,4,5)P3) or direct products of phosphatidyl inositol 4 monophosphate and phosphatidyl inositol breakdown. In order to answer this question tail artery segments were prelabeled with [3H]inositol and then permeabilized with β escin and stimulated with norepinephrine and GTPγS, so that increases in IP, IP2, and Ins(1,4,5)P3 were still observed. If these permeable segments were stimulated with agonist in the presence of compounds known to inhibit Ins(1,4,5)P3 5‐phosphatase, such as glucose 6P, (2,3)diphosphoglycerate, or Ins(1,4,5)P3, the levels of labeled Ins(1,4,5)P3 and labeled IP2 were increased, while the level of stimulated labeled IP was unchanged. This indicated that some of the IP2 and IP formed in these cells was produced from PIP2 but that some of these compounds might be formed from PIP or PI. When the isomers of inositol monophosphate, Ins 1P and Ins 4P, were separated by HPLC, it was shown that after prelabeled tail artery was stimulated by norepinephrine for periods of 1–2 min, the predominant isomer formed was Ins 4P, indicating either PIP2 or PIP as the source. However, after 5–20 min stimulation, both Ins 1P and Ins 4P were formed in equal amounts, suggesting that during sustained stimulation of smooth muscle PI itself was broken down directly. Therefore it appears that within 1–2 min of norepinephrine addition to vascular smooth muscle the bulk of the IP and IP2 produced are derived from PIP2 via IP3, while after 20 min of norepinephrine treatment much of the IP comes directly from PI. This suggests that the regulation of PLC in this tissue is more complicated than has been previously believed. © 1992 Wiley‐Liss, Inc.
Growth on type I collagen promotes expression of the osteoblastic phenotype in human osteosarcoma MG‐63 cells
Authors: Andrianarivo Aurelie G. , Robinson John A. , Mann Kenneth G. , Tracy Russell P.
Published: February 2005

Using MG‐63 cells as a model system capable of partial osteoblastic differentiation, we have examined the effect of growth on extracellular matrix. MG‐63 cell matrix and purified type I collagen induced a morphological change characterized by long cytoplasmic processes reminiscent of those seen in osteocytes. Concurrent biochemical changes involving bone marker proteins included increased specific activity of cell‐associated alkaline phosphatase and increased secretion of osteonectin (up to 2.5‐fold for each protein); all changes occurred without alterations in the growth kinetics of the MG‐63 cells. The increase in alkaline phosphatase activity was maximal on days 6–8 following seeding; increased osteonectin secretion was most prominent immediately following seeding; all changes decreased as cells reached confluence. Growing cells on type I collagen resulted in an increased induction of alkaline phosphatase activity by 1,25 (OH)2D3 (with little change in the 1,25 (OH)2D3 induction of osteonectin and osteocalcin secretion), and increased TGF‐β induction of alkaline phosphatase activity as well (both TGF‐β1 and TGF‐β2). Both the 1,25 (OH)2D3 and TGF‐β effects appeared to be synergistic with growth on type I collagen. These studies support the hypothesis that bone extracellular matrix may play an important role in osteoblastic differentiation and phenotypic expression.
Interferon‐Gamma inhibits proliferation, but not commitment, of murine granulocyte‐macrophage progenitors
Authors: Koike Kenichi , Ma Feng , Shiohara Masaaki , Kubo Tetsuo , Takagi Mineo , Amano Yoshiro , Nakahata Tatsutoshi , Komiyama Atsushi
Published: February 2005

We investigated the effects of interferon γ (IFN‐γ) on the growth of murine hematopoietic progenitors. IFN‐γ inhibited granulocyte colony‐stimulating factor (G‐CSF)‐ and interleukin‐3 (IL‐3)‐dependent colony growth by granulocyte‐macrophage (GM) progenitors derived from the bone marrow cells of normal mice. However, the number of IL‐3‐dependent GM colonies formed by the bone marrow cells of 5‐fluorouracil (5‐FU)‐treated mice was not influenced by the addition of IFN‐γ. Replating experiments suggested that IFN‐γ suppressed GM colony growth directly and that it exerted an inhibitory effect on the proliferation, but not on the commitment, of GM progenitors. In contrast, IFN‐γ failed to suppress colony growth by mast cell progenitors. Erythroid and megakaryocytic progenitors exhibited different responses to IFN‐γ depending on mouse strains. These results suggest that potent negative regulators are not always inhibitors of hematopoietic progenitors. © 1992 Wiley‐Liss, Inc.
Coordinate regulation of mRNAs from multiple calmodulin genes during myoblast differentiation in vitro
Authors: Christenson Mark A. , Means Anthony R.
Published: February 2005

Multiple genes encoding identical calmodulin molecules have been found in all mammalian species so far examined, but little is known regarding the factors involved in regulating the expression of this gene family. We have investigated the possibility of differential regulation under conditions of cell cycle withdrawal and differentiation in the nonfusing BC3H1 myoblast. Transcripts from the three genes are expressed in myoblasts and myocytes and each of the mRNA species decreases during BC3H1 differentiation. Calmodulin protein levels also decrease, although with distinct kinetics with respect to the mRNAs. Previous studies indicated that a decrease in transcription is involved (Epstein et al., Molecular Endocrinology 3:193–202, 1989). In this study, an increase in stability for each of the mRNA species is also shown to contribute to overall mRNA levels. The calmodulin mRNAs are also found to decrease under conditions of cell cycle withdrawal when differentiation is blocked. This demonstrates that the expression of mRNA from all three genes is directly coupled with the proliferation state but only indirectly with the differentiation state. Consistent with this, calmodulin expression decreases in serum deprived fibroblasts as they exit the cell cycle. © 1993 Wiley‐Liss, Inc.
Modulation of the epidermal growth factor receptor by basic fibroblast growth factor
Authors: Maher Pamela A.
Published: February 2005

Treatment of Swiss 3T3 fibroblasts with basic fibroblast growth factor (bFGF) lead to a rapid reduction in epidermal growth factor (EGF) binding and a slower inhibition of EGF receptor autophosphorylation. The reduction in binding was due to a complete loss of the highest affinity EGF binding sites and a reduction in the lower affinity binding sites. Neither the inhibition of EGF binding nor the inhibition of EGF receptor autophosphorylation required protein kinase C. Treatment of cells with bFGF stimulated the phosphorylation of the EGF receptor, which persisted for several hours. The inhibition of EGF receptor autophosphorylation by bFGF was reduced in the presence of cycloheximide. However, cycloheximide had no effect on the reduction of EGF binding by bFGF. In contrast to these results with Swiss 3T3 fibroblasts, treatment of PC12 cells with bFGF lead to a reduction in EGF binding but no inhibition of EGF receptor autophosphorylation. Thus inhibited of EGF receptor autophosphorylation and inhibition of EGF binding can be uncoupled. © 1993 Wiley‐Liss, Inc.
Deregulation of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) receptor in murine macrophage cell line J774A.1
Authors: Fan Kai , Barendsen Nelleke , Sensenbrenner Lyle , Chen Ben D. M.
Published: February 2005

J774A.1 immortalized macrophage tumor cells display several phenotypes and functional capacities similar to that of murine peritoneal exudate macrophages (PEM). Both populations display comparable number of M‐CSF receptors. Yet the number of GM‐CSF receptors on J774A.1 cells is only one‐fourth that of PEM (1,500 vs. 6,000 per cell). Unlike J774A.1 cells, which constitutively express c‐myc transcripts, normal PEM required rMuGM‐CSF for the induction of c‐myc expression. Nevertheless, the growth of J774A.1 cells can be further enhanced in the presence of exogenous rMuGM‐CSF, rHuM‐CSF, and rMulL‐3. Treatment with either rMulL‐3 (20 ng/ml) and rHuTGF‐β1 (1.0 ng/ml) for 24 hr at 37°C, markedly enhanced the expression of GM‐CSF receptors on normal PEM but not leukemic J774A.1 cells. J774A.1 cells also did not respond by autologous upregulation of GM‐CSF receptors as seen in PEM following treatment with rMuGM‐CSF. Treatment with either pertussis toxin (20–100 ng/ml) or H‐8 (50 μM) for 24 hr led to an enhanced expression of GM‐CSF receptors on J774A.1 cells in a time‐and dose‐dependent manner but did not result in enhanced receptor expression on normal PEM. These findings suggest that the expression of GM‐CSF receptors may be regulated by mechanisms involving Gi‐and their downstream elements, which in turn are linked to regulatory pathways of other cytokine receptors. In J774A.1 cells, such regulatory interaction may not exist. © 1993 Wiley‐Liss, Inc.
Rapid and reversible tubulin tyrosination in human neutrophils stimulated by the chemotactic peptide, fMet‐Leu‐Phe
Authors: Rothwell Stephen W. , Nath Jayasree , Wright Daniel G.
Published: February 2005

Neutrophil activation by specific stimuli, such as the oligopeptide chemotactic factor fMet‐Leu‐(fMLF), is associated with an increased enzymatic addition of tyrosine to tubulin α ‐subunits, as measured by 14C tyrosine uptake. In studies using immunoblots we have found that this increased tyrosine uptake into tubulin in activated neutrophils reflects an increase in the proportion of cellular tubulin that is tyrosinated rather than simply an increase in the turnover of tyrosinated subunits. However, the increased accumulation of tyrosinated tubulin was also found to follow an initial depletion of tyrosinated tubulin and concomitant increase in detyrosinated tubulin between 0 and 60 sec following stimulation of neutrophils with fMLF. Immunogold electron microscopy studies of intact micro tubules recovered from activated neutrophils demonstrated that these rapid changes in the relative content of tubulin isoforms in the cells were not associated with the formation or disappearance of microtubule microdomains composed of only one form of tubulin. Previously, we have shown that under conditions of fMLF‐stimulated exocytosis there is an increased binding of neutrophil granules to endogenous microtubules. Since neutrophil activation by fMLF is associated with increased tyrosination of α ‐tubulin subunits, we speculated that rapid changes in the levels of tyrosinated tubulin in the microtubules of activated neutrophils might have a role in the regulation of granule‐microtubule interactions. When the binding of purified neutrophil granules to reconstituted rat brain microtubules containing approximately 50% tyrosinated tubulin was measured by electron microscopy and compared with granule binding to microtubules that contained no detectable tyrosinated tubulin, granule‐microtubule associations were found to be significantly favored by detyrosinated vs. tyrosinated tubulin. These findings indicate that interactions between cytoplasmic granules and microtubules in activated neutrophils may be modulated by rapid changes in the relative content of detyrosinated and tyrosinated tubulin in the microtubule network of the cells. © 1993 Wiley‐Liss, Inc.
Strontium induces murine keratinocyte differentiation in vitro in the presence of serum and calcium
Authors: Li Luowei , Kruszewski Francis H. , Punnonen Kari , Tucker Robert W. , Yuspa Stuart H. , Hennings Henry
Published: February 2005

Primary mouse keratinocytes in culture are induced to terminally differentiate by increasing extracellular Ca2+ concentrations (CaO) from 0.05 mM to ≥ 0.1 mM. The addition of Sr2+ (≥ 2.5 mM) to medium containing 0.05 mM Ca2+ induces focal stratification and terminal differentiation, which are similar to that found after increasing the CaO to 0.12 mM. Sr2+ in 0.05 mM Ca2+ medium induces the expression of the differentiation‐specific keratins, keratin 1 (K1), keratin 10 (K10), and the granular cell marker, filaggrin, as determined by both immunoblotting and immunofluorescence. Sr2+ induces the expression of those differentiation markers in a dose dependent manner, with an optimal concentration of 5 mM. In the absence of Ca2+ in the medium, the Sr2+ effects are reduced, and Sr2+ is ineffective when both Ca2+ and serum are deleted from the medium. Sr2+ treatment increases the ratio of fluorescence intensity of the intracellular Ca2+ sensitive probe, fura‐2, indicating an associated rise in the level of intracellular free Ca2+ and/or Sr2+. At doses sufficient to induce differentiation, Sr2+ also increases the level of inositol phosphates in primary keratinocytes within 30 min. The uptake curves of 85Sr2+ by primary keratinocytes are similar to those of 45Ca2+. At low concentrations, the initial uptake of both 45Ca2+ and 85Sr2+ reaches a plateau within 1 hr; at higher concentrations, the uptake of both 45Ca2+ and 85Sr2+ increases continuously for 12 hr. In keratinocytes pre‐equilibrated with 45Ca2+ in 0.05 mM Ca2+ medium, Sr2+ causes an increase of 45Ca2+ uptake, which is dependent on the presence of serum. These results suggest that Sr2+ utilizes the same signalling pathway as Ca2+ to induce keratinocyte terminal differentiation and that Ca2+ may be required to exert these effects. © 1993 Wiley‐Liss, Inc.