bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of expression of insulin and IGF‐I receptor by Epstein‐Barr virus and its gene products LMP and EBNA‐2 in lymphocyte cell lines
Authors: Kriauciunas Kristina M. , Goldstein Barry J. , Lipes Myra A. , Kahn C. Ronald
Published: February 2005

The receptors for insulin and insulin‐like growth factor I (IGF‐I) are two closely related integral membrane glycoproteins involved in signalling of cell growth and metabolism. We have used the unique paradigm of pairs of Burkitt lymphoma cell lines (BLO2, BL30, BL41) with or without Epstein‐Barr Virus (EBV) infection and cells transfected with EBV‐related genes to examine effects of EBV on expression of these receptors at the gene and protein functional level. In BL30 and BL41 cells, EBV infection increased surface insulin binding and total receptor number by 2‐and 18‐fold, respectively. By contrast, EBV infection decreased total IGF‐I receptors by 29 to 87% in all three cell lines. In general, there was a correlation between total receptor concentration and the level of insulin or IGF‐I receptor mRNAs, although in one cell line insulin binding increased while receptor mRNA levels decreased slightly, suggesting posttranslational effects. BL41 cells transfected with a vector expressing the EBV latent membrane protein (LMP) exhibited a 2.6‐ to 3.2‐fold increase in insulin receptor expression, whereas cells transfected with EBNA‐2 (one of the EBV nuclear antigens) alone exhibited no effect. However, EBNA‐2 appears to be required for the EBV effect on insulin receptor expression since cells infected with a mutant virus, P3JHRI, which lacks the EBNA‐2 gene failed to show an increase in insulin receptor number. These data indicate that EBV infection of lymphocytes increases expression of insulin receptors while simultaneously decreasing expression of IGF‐I receptors. The magnitude and sometimes even the direction of change, depends on host cell factors. A maximal increase in insulin receptors appears to require the coordinate action of several of the EBV proteins including LMP and EBNA‐2. © 1993 Wiley‐Liss, Inc.
Kinetic characterization of Na+/H+ antiport of Plasmodium falciparum membrane
Authors: Bosia A. , Ghigo D. , Turrini F. , Nissani E. , Pescarmona G. P. , Ginsburg H.
Published: February 2005

Intraerythrocytic malaria parasites produce vast amounts of lactic acid through glycolysis. While the egress of lactate is very rapid, the mode of extrusion of H+ is not known. The possible involvement of a Na+/H+ antiport in the extrusion of protons across the plasma membrane of Plasmodium falciparum has been investigated by using the fluorescent pH probe 6‐carboxyfluorescein. The resting cytosolic pH was 7.27 ± 0.1 in ring stage parasites and 7.31 ± 0.12 in trophozoites. Spontaneous acidification of parasite cytosol was observed in Na+‐medium and realkalinization occurred upon addition of Na+ to the medium in a concentration‐dependent manner, with no apparent saturation. The rate of H+‐at the ring stage was higher than that at the trophozoite stage due to the larger surface/volume ratio of the young parasite stage. Na+‐H+‐was: 1) inhibited by the Na+/H+ inhibitors amiloride and 5‐(N‐ethyl‐isopropyl) amiloride (EIPA), though at relatively high concentrations; 2) augmented with rising pH6 (pHi = 6.2 [Na+]o = 30 mM); and 3) decreased with increasing pHi (pHo = 7.4; [Na+]o = 30 mM). The pHi and the pHo dependencies of H+‐were almost identical at all parasite stages. Only at pHi > 7.6 efflux was totally obliterated. The target of this inhibitory effect is probably other than the antiport. Results indicate that H+‐is mediated by a Na+/H+ antiport which is regulated by host and parasite pH and by the host cytosol sodium concentration. The proton transport capacity of the antiport can easily cope with all the protons of lactic acid produced by parasite's glycolysis. © 1993 Wiley‐Liss, Inc.
Occurrence and possible consequences of multipolar mitoses in primary cultures of adult rat hepatocytes
Authors: Eckl P. M.
Published: February 2005

Proliferating primary cultures of adult rat hepatocytes are characterized by the occurrence of multipolar mitoses, and chromosome loss resulting in the formation of micronuclei at telophase. The percentage of multipolar mitotic figures was determined to be 12.76 ± 7.9%, 80% of which were tripolar. Multipolar mitotic stages showed a high incidence of chromosome loss, increasing from meta‐ (61.7 ± 16.6%) to telophase (72.1 ± 19.3%). Regular bipolar mitotic figures on the other hand also showed chromosome loss, however, to a lesser degree and decreasing from meta‐ (49.5 ± 10.4%) to telophase (34.9 ± 7.9%). The incidence of chromosome loss even in regular mitotic figures is very high compared to other cells and appears to depend on another special feature of hepatocytes: they remain flat and well attached during mitosis, so that shearing forces could be responsible for the separation of chromosomes from the mitotic spindle. Additionally this morphology creates a situation allowing for a maximal interaction of mitotic spindles of binucleated cells, leading to the high rate of multipolar mitoses observed. Both multipolar mitoses and chromosome loss could also explain the consecutive detachment of hepatocytes reported for proliferating primary cultures, since the aneuploid daughter cells generated can be expected to be nonviable in most cases and eventually detach. © 1993 Wiley‐Liss, Inc.
Regulation of chicken Hsp70 and Hsp90 family gene expression by transforming growth factor‐β1
Authors: Takenaka Ivone M. , Hightower Lawrence E.
Published: February 2005

Transforming growth factor‐β1 (TGFβ) is a regulator of protein synthesis in cultured chicken embryo cells (CEC). Preceding a gradual increase in overall protein synthesis, members of the Hsp70 family (Hsp70, Hsc70, and Grp78) and the Hsp90 family (90‐2 and 90‐3) of molecular chaperones are induced rapidly and represent a new class of TGFβ‐inducible proteins (I.M. Takenaka and L.E. Hightower, J. Cell. Physiol., 152:568–577, 1992). Herein, 32P‐labeled cDNA probes encoding Hsc70 and Hsp90 were used to show that levels of the corresponding mRNAs increased as a fraction of total RNA and in polysomes within five hours of treatment of CEC with TGFβ. This cytokine did not increase rates of hsc70 and hsp90 gene transcription as measured by run‐on transcription assays of isolated nuclei. However, the Hsp RNA inductions were inhibited by dactinomycin, indicating a requirement for newly synthesized RNA. Both Hsc70 and Hsp90 mRNAs had relatively short half‐lives, measured by Northern blot analyses of dactinomycin chases, which were not altered substantially in TGFβ‐treated cells. In contrast, Hsp mRNA half‐lives increased in heat shocked CEC exposed to dactinomycin during recovery, revealing a difference in regulation of these genes in stressed cells compared with TGFβ‐treated cells. Our results support the conclusion that hsc70 and hsp90 gene expression is regulated posttranscriptionally in TGFβ‐treated CEC, and the mechanism likely involves a nuclear event such as increasing the half‐lives of nuclear RNA transcripts, processing, or transport into the cytoplasm. © 1993 Wiley‐Liss, Inc.
TGFβ induction of extracellular matrix associated proteins in normal and transformed human mammary epithelial cells in culture is independent of growth effects
Authors: Stampfer Martha R. , Yaswen Paul , Alhadeff Myriam , Hosoda Junko
Published: February 2005

We have previously characterized a human mammary epithelial cell (HMEC) culture system for the effects of TGFβ1 on cell growth. In the current report, the effects of TGFβ 1 on synthesis and secretion of proteins associated with the extracellular matrix and proteolysis were examined. In particular, we compared the TGFβ responses of normal finite lifespan HMEC, which are growth inhibited by TGFβ, to two immortally transformed cell lines derived from the normal HMEC. One of these lines maintains active growth in the presence of TGFβ and the other shows partial growth inhibition. In contrast to the differing effects of TGFβ on cell growth, we found that all these cell types showed strong induction of most of the mRNA and protein species examined, including fibronectin, collagen IV, laminin, type IV collagenase, urokinase type plasminogen activator (uPA), and plasminogen activator inhibitor 1 (PAI‐1). The profile of TGFβ 1 binding proteins was the same in HMEC that were, and were not growth suppressed by TFGβ. Therefore, the effects of TGFβ on cell growth could be dissociated from its effects on specialized responses, indicating that within this one cell type there must be at least two independent pathways for TGFβ activity, one which leads to cessation of proliferation and one which induces a specific set of cellular responses. This cell system may be useful for examining the pathway of TGFβ induced growth inhibition using closely matched cells which vary in their growth‐induced response but retain similar specialized responses to TGFβ. © 1993 Wiley‐Liss, Inc.
Mechanism of retinoid‐induced activation of latent transforming growth factor‐β in bovine endothelial cells
Authors: Kojima Soichi , Rifkin Daniel B.
Published: February 2005

Cell‐associated plasmin is a putative physiological activator of latent transforming growth factor‐β (LTGF‐β). Since retinoids enhance the production of plasminogen activator (PA) and thereby increase cell‐associated plasmin activity, we tested the possibility that retinoids might induce the activation of LTGF‐β using bovine endothelial cells (ECs) as a model system. ECs treated with physiological concentrations of retinol or retinoic acid formed active TGF‐β in the culture media in a dose‐ and time‐dependent fashion. Cells were treated with 2 μM retinol for 24 h, and the amount of TGF‐β produced during a subsequent 12‐h incubation period was measured. Out of a total of 14 pM LTGF‐β secreted, 0.7 pM was converted to active TGF‐β. Northern blot analyses showed that mRNA levels for TGF‐β2 but not for TGF‐β1 increased in cells treated with retinol. Inclusion of either inhibitors of PA or of plasmin or antibody against PA in the culture medium as well as depletion of plasminogen from the serum blocked the formation of TGF‐β, suggesting that PA, plasminogen, and the resulting plasmin are essential for activation of LTGF‐β in retinoid‐stimulated cells. Antibody against the LTGF‐β binding protein blocked activation implying that localization of LTGF‐β through its binding protein may be important. However, inhibition of binding of LTGF‐β to the cell surface mannose 6‐phosphate receptor did not prevent activation. These data indicate that retinoids up‐regulate the production of LTGF‐β in ECs and induce activation of LTGF‐β, perhaps, by increasing PA and plasmin levels. Thus, TGF‐β might be a local mediator of some of the biological activities of retinoids both in vivo and in vitro. © 1993 Wiley‐Liss, Inc.
Different signal transduction by epidermal growth factor may be responsible for the difference in modulation of amino acid transport between fetal and adult hepatocytes
Authors: Leoni S. , Spagnuolo S. , Marino M. , Terenzi F. , Massimi M. , Devirgiliis L. Conti
Published: February 2005

[1‐14C]‐2‐aminoisobutyric acid (AIB) uptake and signal transduction pattern after epidermal growth factor (EGF) stimulation were examined in freshly isolated hepatocytes from 20‐day‐old fetuses and 3‐month‐old rats. EGF induced a transient increase of AIB transport after 10 min only in adult animals; the observed unresponsiveness of fetal liver is not dependent on a lack of EGF receptors which are present though to a lesser extent on the plasma membrane in this period. As far as the production of the second messengers, inositol trisphosphate (IP3) and calcium, is concerned, substantial differences were found: EGF increased IP3 production in adult hepatocytes, whereas it had no effect in fetal ones. Moreover, the addition of EGF induced a calcium transient in hepatocytes from adult animals, while there was no increase in fetal cells. The lack of EGF effect on amino acid transport in fetal cells could be due to its inability to produce both IP3 and calcium transients, suggesting that this transduction pathway is not activated during fetal life.
Mechanism of action of angiostatic steroids: Suppression of plasminogen activator activity via stimulation of plasminogen activator inhibitor synthesis
Authors: Blei Francine , Wilson E. Lynette , Mignatti Paolo , Rifkin Daniel B.
Published: February 2005

Recently, a novel class of angiostatic steroids which block angiogenesis in several systems has been described. Since the elaboration of proteases is believed to be an important component of angiogenesis, we tested whether these steroids blocked the fibrinolytic response of endothelial cells to the angiogenic protein, basic fibroblast growth factor [bFGF]). Cultured bovine aortic endothelial (BAE) cells were incubated with bFGF and/or medroxyprogesterone acetate (MPA), an angio‐static steroid which has been shown to inhibit vascularization, collagenolysis, and tumor growth. When bFGF (3 ng/ml) was added to confluent monolayers of BAE cells, plasminogen activator (PA) activity in the medium was increased threefold. In contrast, MPA at 10−6 M, 10−7 M, 10−8 M, and 10−9 M decreased PA levels in the medium by 83%, 83%, 75%, and 39%, respectively. The stimulation of PA levels in BAE cells by bFGF (3 ng/ml) was abrogated by the presence of 10−6 M MPA. This decrease in PA activity was found to be mediated by a significant increase in plasminogen activator inhibitor type‐1 (PAI‐1) production. MPA, therefore, negated one of the important enzymatic activities associated with the angiogenic process. In contrast to the decreased levels of secreted PA in cultures exposed simultaneously to MPA and bFGF, cell‐associated PA levels remained high, consistent with earlier observations indicating that PAI‐1 does not inhibit cell‐associated PA. Thus, angiostatic steroids may exert their inhibitory effects on angiogenesis by increasing the synthesis of PAI‐1. This, in turn, inhibits PA activity and, therefore, plasmin generation, which is essential for the invasive aspect of angiogenesis. © 1993 Wiley‐Liss, Inc.
Alterations in nuclear matrix ultrastructure of G1 mammalian cells following heat shock: Resinless section electron microscopy, biochemical, and immunofluorescence studies
Authors: Wachsberger Phyllis R. , Coss Ronald A.
Published: February 2005

Heat shock is known to inhibit vital nuclear functions associated with DNA and RNA metabolism. It has been proposed that the reported heat‐induced excess protein accumulation in the nuclear matrix (NM) fraction may alter NM sites crucial for DNA and RNA processing. To test this hypothesis, we examined the fine structure of the NM in synchronous populations of G1 Chinese hamster ovary cells before and after heating by using the technique of resinless section electron microscopy. Heat did induce morphological alterations in the NM. The NM of control cells contained a honeycomb‐like arrangement of fibers after chromatin removal. Following heat shock, NMs appeared as more highly anastomosing networks of polymorphic fibers and an overall increase in electron density was observed. Residual nucleoli from heated NMs underwent alterations in distributions of electron density both internally and at their peripheries. The increase in electron density observed in heated NMs was accompanied by an increase in protein mass and a relatively smaller increase in RNA mass as indicated by parallel sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and isotopic labeling (protein/DNA and RNA) studies. Some excess protein accumulation could also be directly localized onto NM fibers by use of antibodies to heterogeneous ribonucleoprotein complex antigens. It is concluded that alterations of NM fine structure can reflect the heat‐stressed state of the cell, may account for the heat‐induced inhibition of nucleic acid metabolism, and may be useful as an indicator of physiological or pathological stress in general. © 1993 Wiley‐Liss, Inc.
Transforming growth factor β1‐mediated inhibition of smooth muscle cell proliferation is associated with a late G1 cell cycle arrest
Authors: Reddy Kumar B. , Howe Philip H.
Published: February 2005

The effect of transforming growth factor β1 (TGFβ1) on the proliferative response of aortic smooth muscle cells (SMC) in vitro was investigated. TGFβ1 substantially inhibited the growth of human and bovine SMC. Rapidly growing SMC and quiescent serum‐stimulated SMC were inhibited by TGFβ1 with an ID50 of approximately 0.5 ng/ml and maximal inhibition was observed at 10 ng/ml TGFβ1. In the presence of TGFβ1, quiescent serum‐stimulated SMC progress into the G1 phase of the cell cycle, but become reversibly arrested at a point temporally located 1–2 hours from S phase. Release from this late G1 TGFβ1 arrest point results in S phase entry within 2 hours. Associated with this inhibitory effect is a decrease in the histone H1 kinase activity of p34cdc2 protein kinase while TGFβ1 has no effect on the transcription or translation of p34cdc2. Under these growth inhibitory conditions, TGFβ1 is still capable of upregulating the expression of fibronectin mRNA. These results suggest that TGFβ1 growth inhibition in SMC is associated with the regulation of p34cdc2 activity in late G1. © 1993 Wiley‐Liss, Inc.
Elevated cAMP gives short‐term inhibition and long‐term stimulation of hepatocyte DNA replication: Roles of the cAMP‐dependent protein kinase subunits
Authors: Vintermyr Olav Karsten , Bøe Roald , Bruland Torunn , Houge Gunnar , Døskeland Stein Ove
Published: February 2005

The study reports the role of the isozyme forms (cA‐PKI and cA‐PKII) and subunits (R and C) of cAMP‐dependent protein kinase in mediating the acute depression of hepatocyte DNA replication by elevated cAMP. Combinations of cAMP analogs preferentially activating cA‐PKI or II showed that either isozyme could inhibit DNA replication. The effects of glucagon and cAMP analogs were counteracted by the cAMP antagonist RpcAMPS, implicating the necessity for cA‐PK dissociation in cAMP action. The effect of elevated cAMP was mimicked by microinjected C subunit, but not by the RI subunit of cA‐PK. Hepatocytes under continuous cAMP challenge more than regained their replicative activity. This tardive stimulatory effect of cAMP was enhanced by insulin and blocked by dexamethasone, and was preceded by downregulation of cA‐PK. In conclusion, a burst of cAMP acutely inhibits hepatocyte G1/S transition in late G1 regardless of hormonal state. In the presence of high glucocorticoid/low insulin the inhibition persists. At high insulin/low glucocorticoid the inhibitory phase is followed by a prolonged stimulation of DNA replication. Downregulation of endogenous cA‐PK is a mechanism for escape from the inhibitory action of highly elevated cAMP. © 1993 Wiley‐Liss, Inc.
The differentiation inducer, dimethyl sulfoxide, transiently increases the intracellular calcium ion concentration in various cell types
Authors: Morley Paul , Whitfield James F.
Published: February 2005

Dimethyl sulfoxide (DMSO) initiates a coordinated differentiation program in various cell types but the mechanism(s) by which DMSO does this is not understood. In this study, the effect of DMSO on intracellular calcium ion concentration ([Ca2+]i) was determined in primary cultures of chicken ovarian granulosa cells from the two largest preovulatory follicles of laying hens, and in three cell lines: undifferentiated P19 embryonal carcinoma cells, 3T3‐L1 fibroblasts, and Friend murine erythroleukemia (MEL) cells. [Ca2+]i was measured in cells loaded with the Ca2+ ‐specific fluoroprobe Fura‐2. There was an immediate (i.e., within 5 sec), transient, two to sixfold increase in [Ca2+]i after exposing all cell types to 1% DMSO. DMSO was effective between 0.2 and 1%. The prompt DMSO‐induced [Ca2+]i spike in all of the cell types was not prevented by incubating the cells in Ca2+ ‐free medium containing 2 mM EGTA or by pretreating them with the Ca2+‐channel blockers methoxyverapamil (D600; 100 μM), nifedipine (20 μM), or cobalt (5 mM). However, when granulosa cells, 3T3‐L1 cells, or MEL cells were pretreated with lanthanum (La3+; 1 mM), which blocks both Ca2+ channels and membrane Ca2+ pumps, there was a sustained increase in [Ca2+]i in response to 1% DMSO. By contrast, pretreating P19 cells with La3+ (1 mM) did not prolong the DMSO‐triggered [Ca2+]i transient. In all cases, the DMSO‐induced [Ca2+]i surge was unaffected by pretreating the cells with the inhibitors of inositol phospholipid hydrolysis, neomycin (1.5 mM) or U‐73, 122 (2.5 μM). These results suggest that DMSO almost instantaneously triggers the release of Ca2+ from intracellular stores through a common mechanism in cells in primary cultures and in cells of a variety of established lines, but, this release is not mediated through phosphoinositide breakdown. This large, DMSO‐induced Ca2+ spike may play a role in the induction of cell differentiation by DMSO. © 1993 Wiley‐Liss, Inc.
Simultaneous calcium‐dependent delivery of neutrophil lactoferrin and reactive oxygen metabolites to erythrocyte targets: Evidence supporting granule‐dependent triggering of superoxide deposition
Authors: Maher Robert J. , Cao Dongrong , Boxer Laurence A. , Petty Howard R.
Published: February 2005

Optical microscopic techniques have been utilized to study the deposition of lactoferrin, a specific granule marker, and superoxide anions into target erythrocytes during antibody‐dependent phagocytosis. Previous studies from this laboratory have shown that the entry of superoxide anions into erythrocytes can be sensitively monitored with Soret band transmitted light microscopy. When neutrophils were incubated with BAPTA/AM, an intracellular Ca2+ chelator, they phagocytosed IgG‐opsonized sheep red blood cells (SRBC) but did not affect the microscopically detected absorption of their Soret band. When these same erythrocytes were observed after the infusion of 20 μM ionomycin, a Ca2+ ionophore, 58% of the cell‐bound SRBC targets were destroyed immediately. However, neutrophils from chronic granulomatous disease (CGD) patients were unable to affect the Soret absorption of erythrocyte targets under any conditions. These results suggest that a Ca2+ signal can participate in triggering superoxide deposition in targets. Since Ca2+ signals are known to participate in the exocytic release of granules, we tested the hypothesis that specific lactoferrin‐bearing granules are delivered to targets in parallel with superoxide anions. Lactoferrin delivery to phagosomes was monitored using resonance energy transfer (r.e.t.) microscopy. SRBCs were opsonized with both unconjugated and rhodamine B isothiocyanate (RBITC)‐conjugated rabbit anti‐SRBC IgG. After incubation with adherent neutrophils, the samples were washed, fixed with 3.7% paraformaldehyde, then labeled with fluorescein isothiocyanate (FITC)‐conjugated antilactoferrin IgG. Energy transfer between FITC and RBITC was imaged microscopically and quantitated by photon counting. Significant levels of r.e.t. between antilactoferrin and anti‐SRBC labels were observed after phagocytosis, but not in the absence of acceptor fluorochromes. To control for r.e.t. specificity, neutrophil membranes were labeled with FITC‐conjugated, anti‐HLA IgG after internalization of rhodamine B‐tagged SRBCs (RSRBCs). Althougth r.e.t. between lactroferrin and RSRBCs labels was observed, no r.e.t. between HLA and RSRBC labels could be found. Further studies showed that treatment of neutrophils with BAPTA inhibited r.e.t. between anti lactoferrin and RSRBCs. However, additon of ionomycin relieved this inhibition of energy transfer. These experiments show that both lactoferrin and superoxide delivery to targets are regulated in parallel by a Ca2+ ‐dependent pathway. Furthermore, by combining Soret microscoy with r.e.t. microscopy, we have shown that superoxide anions and lactoferrin are delivered to the same phagosomes. We speculate that the NADPH oxidase, which produces superoxide anions, is assembled on specific granule membranes, thus accounting for their parallel Ca2+ ‐dependence, activation, and delivery. © 1993 Wiley‐Liss, Inc.
Effect of floating a gel matrix on mucin release in cultured airway epithelial cells
Authors: Kim K. Chul , Zheng Qiao‐Xi , Brody Jerome S.
Published: February 2005

Confluent cultures of primary hamster tracheal surface epithelial (HTSE) cells grown on a thick collagen gel are highly enriched with secretory cells and constitutively release mucins. In the present experiment, we examined the possible effect of mechanical strain of cultured HTSE cells on the release of mucin. The mechanical strain of cells was accomplished by several methods: 1) by floating the gel from the culture dish by rimming; 2) by treatment with EGTA which interrupts intercellular tight junctions; 3) by treatment with collagenase which disrupts the cell‐matrix adhesion; and 4) by mechanically flexing the collagen gel matrix. All these conditions caused increases of mucin release without damage on the plasma membrane. We conclude that a number of mechanical strains which might alter cell shape can stimulate mucin release from cultured HTSE cells. Such a mechanism might be operative in the physiological regulation of airway goblet cell mucin secretion where mechanical strains may be induced on epithelial cells by underlying smooth muscles. © 1993 Wiley‐Liss, Inc.
Heat shock protein is a unique marker of growth arrest during macrophage differentiation of HL‐60 cells
Authors: Spector Neil L. , Ryan Colleen , Samson William , Levine Herbert , Nadler Lee M. , Arrigo A.‐P.
Published: February 2005

Prior to morphologic and functional maturation, terminally differentiating hematopoietic cells first exit the cell cycle and undergo growth arrest. Relatively little is known about which molecules regulate differentiation‐induced growth arrest. In the present report, we sought to determine whether the mammalian low molecular weight heat shock protein (hsp28) was a candidate growth‐regulatory molecule during human hematopoiesis. To this end, hsp28 protein expression was examined during phorbol ester (PMA)‐induced macrophage differentiation of the human HL‐60 promyelocytic leukemic cell line. Whereas hsp28 was constitutively expressed at relatively low levels in an unphosphorylated state, hsp28 was rapidly phosphorylated within 4 hr following PMA‐induced differentiation, preceding increased hsp28 protein levels at 24–48 h. In contrast to other differentiative agents, hsp28 steady state mRNA and protein were regulated concordantly in response to macrophage differentiation. More importantly, these changes were transient, and occurred concomitant with the down‐regulation of cellular proliferation and the onset of G1 phase cell cycle arrest. In total, these observations implicate hsp28 as an intermediary in the myelomonocytic differentiative pathway of promyelocytic leukemic cells, and will shed light on the events regulating this process. © 1993 Wiley‐Liss, Inc.
Bacterial lipopolysaccharide induces actin reorganization, intercellular gap formation, and endothelial barrier dysfunction in pulmonary vascular endothelial cells: Concurrent F‐actin depolymerization and new actin synthesis
Authors: Goldblum Simeon E. , Ding Xueda , Brann Terrance W. , Campbell‐Washington Judith
Published: February 2005

Bacterial lipopolysaccharide (LPS) influences pulmonary vascular endothelial barrier function in vitro. We studied whether LPS regulates endothelial barrier function through actin reorganization. Postconfluent bovine pulmonary artery endothelial cell monolayers were exposed to Escherichia coli 0111:B4 LPS 10 ng/ml or media for up to 6 h and evaluated for: (1) transendothelial 14C‐albumin flux, (2) F‐actin organization with fluorescence microscopy, (3) F‐actin quantitation by spectrofluorometry, and (4) monomeric G‐actin levels by the DNAse 1 inhibition assay. LPS induced increments in 14C‐albumin flux (P < 0.001) and intercellular gap formation at ≥ 2–6 h. During this same time period the endothelial F‐actin pool was not significantly changed compared to simultaneous media controls. Mean (±SE) G‐actin (μg/mg total protein) was significantly (P < 0.002) increased compared to simultaneous media controls at 2, 4, and 6 h but not at 0.5 or 1 h. Prior F‐actin stabilization with phallicidin protected against the LPS‐induced increments in G‐actin (P = 0.040) as well as changes in barrier function (P < 0.0001). Prior protein synthesis inhibition unmasked an LPS‐induced decrement in F‐actin (P = 0.0044), blunted the G‐actin increment (P = 0.010), and increased LPS‐induced changes in endothelial barrier function (P < 0.0001). Therefore, LPS induces pulmonary vascular endothelial F‐actin depolymerization, intercellular gap formation, and barrier dysfunction. Over the same time period, LPS increased total actin (P < 0.0001) and new actin synthesis (P = 0.0063) which may be a compensatory endothelial cell response to LPS‐induced F‐actin depolymerization. © 1993 Wiley‐Liss, Inc.
ABH antigens as recognition sites for the activation of red blood cell anion exchange by the lectin Ulex europaeus agglutinin I
Authors: Engelmann Bernd
Published: February 2005

The blood group antigen H (blood group O) and fucose‐specific lectin Ulex europaeus agglutinin I (UEA1) (10 μg/ml) was found to increase the rate constant of CL− efflux into 100mM Na+ oxalate media by about 40% in erythrocytes taken from antigen H donors. In 100 mMK + oxalate, 150 mM Na+ pyruvate and in 150 mM Na+ acetate media the lectin elevated the rate constant of CL− efflux by 20–50%. The acceleration of Cl− efflux by UEA1 was completely blocked by 10 μM 4,4′‐dllsothiocyanato‐stilbene‐2,2′‐disulfonic acid (DIDS) indicating that the effect of the lectin is mediated by the anion exchanger of human erythrocytes (band 3 protein). In antigen A1 erythrocytes no significant stimulation of anion exchange by UEA1 was seen. The activation of Cl− efflux was completely prevented by addition of 1 mM fucose to the medium. These results suggest that the effect of UEA1 is mediated through interaction with the fucose residues of H antigens. Increasing extracellular Ca++ from 0.5 to 5 mM in Na + pyruvate or Na+ acetate media slightly reduced the acceleration of anion exchange by the lectin. On the other hand, replacing part of extracellular chloride by bicarbonate did not considerably alter the (previously reported) stimulatory effect of UEA1 on red blood cell Ca++ uptake. This suggests that the acceleration of anion exchange and of Ca++ uptake by UEA1, respectively, are mediated by different mechanisms. It is concluded that UEA1 activates anion exchange of human erythrocytes most probably by a direct interaction with H antigens present on extracellular domains of the band 3 protein. © 1993 Wiley‐Liss, Inc.
Low oxygen tension increases mRNA levels of alpha 1 (I) procollagen in human dermal fibroblasts
Authors: Falanga Vincent , Martin Theresa A. , Takagi Hajime , Kirsner Robert S. , Helfman Todd , Pardes Jeffrey , Ochoa M. Sofia
Published: February 2005

Dermal fibroblasts exposed to low oxygen tension show upregulated synthesis of transforming growth factor‐beta 1 (TGF‐beta 1), an established stimulatory peptide in the formation of extracellular matrix proteins. In this report, procollagen synthesis was measured in cultures of confluent adult human dermal fibroblasts exposed to either standard (20%) or low (2%) oxygen tension. By Northern blot analysis the steady state levels of alpha 1 (I) procollagen mRNA were increased by 75 to 150% of control (standard oxygen) as early as 12 hours and more than 200% 96 hours after exposure of cells to low oxygen. Similar increases in procollagen mRNA levels were obtained in hypoxic fibroblast cultures in a collagen lattice. The stimulatory effect of hypoxia on procollagen mRNA levels in fibroblast monolayers was diminished by antibodies to TGF‐beta, and could not be augmented further by the addition of TGF‐beta 1, evidence that hypoxic fibroblasts may already be maximally stimulated by TGF‐beta 1. We conclude that low oxygen tension enhances Steady state mRNA levels of alpha 1 (I) procollagen, and that this effect is mediated at least in part by TGF‐beta 1. © 1993 Wiley‐Liss, Inc.
Growth inhibition of breast cancer cells induced by exogenous ATP
Authors: Spungin Ben , Friedberg Ilan
Published: February 2005

Addition of ATP (>0.1 mM) to cultures of human breast cancer T47D cells resulted in an inhibition of cell proliferation. The inhibition was found to be specific for ATP, and dependent on its concentration. Growth inhibition continued for at least three days, although ATP and its hydrolysis products were metabolized within one day. Conditioned medium from ATP‐treated cultures (CM+) was found to inhibit the growth of cells that were not exposed to ATP. This is an indication that extracellular factors, besides ATP, are involved in the inhibition process. The inhibition was maintained after dialysis of the CM+, using an 8 kDa cut‐off membrane. Conditioned medium from untreated cultures (CM‐), however, only slightly affected cell growth. The data suggest that the CM+ ‐induced cell growth inhibition is mediated by an ATP‐activated growth inhibiting factor. Flow microfluorometry and thymidine incorporation experiments have shown that the growth arrest is mainly due to the elongation of the S‐phase of the cell cycle. © 1993 Wiley‐Liss, Inc.
Plasminogen‐dependent activation of latent transforming growth factor beta (TGFβ) by growing cultures of osteoblast‐like cells
Authors: Yee John A. , Yan Lin , Dominguez Juan C. , Allan Elizabeth H. , Martin T. John
Published: February 2005

Osteoblasts secrete transforming growth factor beta (TGFβ) as a biologically inert, latent complex that must be dissociated before the growth factor can exert its effects. We have examined the production and proteolytic activation of latent TGFβ (LTGFβ) by clonal UMR 106‐01 rat osteosarcoma cells and neonatal mouse calvarial (MC) osteoblast‐like cells in vitro. Synthetic bPTH‐(1–34) increased the activity of tissue‐type (tPA) and urokinase‐type (uPA) plasminogen activators (PA) in cell lysates (CL) of UMR 106‐01 cells. The concentration of active TGFβ in serum‐free CM from cultures treated with bPTH‐(1–34) and plasminogen was significantly greater than in CM from untreated controls and cultures treated with either bPTH‐(1–34) or plasminogen alone. This effect occurred at concentrations of PTH‐(1–34) that increased PA activity and was prevented by aprotinin, an inhibitor of plasmin activity. Treatment with bPTH‐(1–34) had no effect on the concentration of TGFβ in acid‐activated samples of CM. Functional consequences of proteolytically activated TGFβ was examined in primary cultures of neonatal MC osteoblast‐like cells. Human platelet TGFβ1 caused a dose‐dependent increase in the migration of these cells in an in vitro wound healing assay. Cell migration was also stimulated in cultures treated with bPTH‐(1–34) and plasminogen together. This effect was blocked by an anti‐TGFβ1 antibody. The results of these studies demonstrate that (1) LTGFβ secreted by osteoblasts in vitro is activated under conditions where the plasmin activity in the cultures is increased, and (2) the TGFβ generated by plasmin‐mediated proteolysis is biologically active. We suggest that the local concentration of TGFβ in bone may be controlled by the osteoblast‐associated plasminogen activator/plasmin system. Furthermore, since several calciotropic factors influence osteoblast PA activity, this system may have an important role in mediating their anabolic and/or catabolic effects. © 1993 Wiley‐Liss, Inc.
Modulatory effect of cyclic AMP on calcium fluxes in FRTL‐5 cells
Authors: Törnquist Kid , Ahlström Mikael
Published: February 2005

The aim of the present study was to investigate the effect of cAMP on calcium fluxes in Fura 2 loaded thyroid FRTL‐5 cells. Preincubating the cells with the phosphodiesterase inhibitor Ro‐201724 decreased the ATP‐stimulated entry of calcium, while having no effect on the release of sequestered calcium. Pretreatment with forskolin decreased both the release of sequestered calcium and the entry of calcium in response to ATP. We then incubated the cells with phenylisopropyl adenosine (PIA), a P21‐receptor agonist earlier shown to decrease cAMP in FRTL‐5 cells. Although we did not observe a decrease in cellular cAMP after PIA, the ATP‐evoked calcium response was enhanced. Forskolin decreased calcium entry induced by thapsigargin a Ca2−‐ATPase inhibitor, but forskolin had no effect on the thapsigargin‐evoked release of sequestered calcium. Addition of calcium to cells stimulated with ATP in a calcium‐free buffered resulted in a rapid influx of calcium. This response in [Ca2+]i was decreased in cells pretreated with forskolin. In cells stimulated with thapsigargin, the increase in [Ca2+]i after addition of calcium was inhibited in part by forskolin and enhanced by PIA. The results suggest that cAMP may regulate calcium fluxes in FRTL‐5 cells Furthermore, PIA increased agonist‐induced calcium entry through a presently unknown mechanism. © 1993 Wiley‐Liss, Inc.
Migratory and proliferative effect of platelet‐derived growth factor in rabbit retinal endothelial cells: Evidence of an autocrine pathway of platelet‐derived growth factor
Authors: Koyama Noriyuki , Watanabe Satoe , Tezuka Mariko , Morisaki Nobuhiro , Saito Yasushi , Yoshida Sho
Published: February 2005

Angiogenesis is a crucial event in the progression of diabetic retinopathy. Migration and proliferation of endothelial cells (EC) are important steps in angiogenesis and are caused by angiogenic factors such as basic fibroblast growth factor (bFGF). In this work, capillary EC were isolated from rabbit retinal tissues and rabbit retinal EC (RREC) were found to secrete a migration factor for RREC in conditioned medium (CM). The activity was inhibited by an anti‐platelet‐derived growth factor (PDGF) antibody, but not by an anti‐bFGF antibody. We also found that RREC showed a migratory response to PDGF. The response was induced by PDGF‐BB and PDGF‐AB dose dependently, but not by PDGF‐AA, indicating that it was mediated by PDGF‐β receptor‐dependent pathways, and that the PDGF‐like factor was PDGF‐BB or ‐AB. In addition, PDGF‐BB induced the proliferation of RREC as well as bFGF. These data indicate that RREC have an autocrine pathway of PDGF by the secretion of and the response to PDGF. PDGF may play significant parts in angiogenesis in the progression of diabetic retinopathy. © 1994 Wiley‐Liss, Inc.
Actions of platelet‐derived growth factor isoforms in mesangial cells
Authors: Abboud Hanna E. , Grandaliano Giuseppe , Pinzani Massimo , Knauss Thomas , Pierce Glenn F. , Jaffer Fatima
Published: February 2005

Platelet‐derived growth factor (PDGF) occurs as homodimers or heterodimers of related polypeptide chains PDGF‐BB, ‐AA, and ‐AB. There are two receptors that bind PDGF, termed alpha and beta. The beta receptor recognizes PDGF B chain and is dimerized in response to PDGF BB. The alpha receptor recognizes PDGF B as well as A chains and can be dimerized by the three dimeric forms of PDGF AA, AB, and BB. To characterize PDGF receptor signaling mechanisms and biologic activities in human mesangial cells (MC), we explored the effects of the three PDGF isoforms on DNA synthesis, phospholipase C activation, and PDGF protooncogene induction. PDGF‐BB homodimer and AB heterodimer induced a marked increase in DNA synthesis, activation of phsopholipase C, and autoinduction of PDGF A and B chain mRNAs, whereas PDGF‐AA homodimer was without effect. The lack of response to PDGF AA could be accounted for by down regulation of the PDGF‐alpha receptor since preincubation of MC with suramin restored PDGF AA‐induced DNA synthesis. Ligand binding studies demonstrate specific binding of labeled PDGF BB and AB and to a lower extent PDGF AA isoforms to mesangial cells. These results are consistent with predominant expression of PDGF beta receptor in MC, which is linked to phospholipase‐C activation. The potent biologic effects of PDGF‐AB heterodimer in cells that express very few alpha receptors and do not respond to PDGF AA are somewhat inconsistent with the currently accepted model of PDGF receptor interaction and suggest the presence of additional mechanisms for PDGF isoform binding and activation. © 1994 Wiley‐Liss, Inc.
31P NMR characterization of cellular metabolism during dexamethasone induced apoptosis in human leukemic cell lines
Authors: Adebodun Foluso , Post Jan F. M.
Published: February 2005

31P NMR has been used to study the effects of dexamethasone on phosphorus metabolism in one dexamethasone (dex)‐sensitive (CEM‐C7) and three different dex‐resistant (CEM‐C1, CEM‐4R4, and CEM‐ICR27) human leukemic cell lines. The use of these cell lines, containing widely varying amounts of glucocorticoid receptors, made it possible to evaluate the receptor‐mediated contributions to the modes of action of dexamethasone in these cells. To evaluate the effects of dexamethasone without any significant contribution from experimental conditions, all the experiments were done with parallel controls. Results obtained showed: (1) significantly different levels of phosphorylethanolamine (PE) and phosphorylcholine (PC) among cell lines, suggesting significant differences in phospholipid metabolism; (2) the dexamethasone induced reduction of phosphomonoester (PE + PC), ATP, and metabolic rates probably through glucocorticoid receptor mediated mechanisms; (3) the dexamethasone induced stimulation of cellular metabolism in a process which seems to be independent of glucocorticoid receptors; and (4) the dexamethasone induced alkaline shift of intracellular pH in all the cell lines except ICR27. The reduction in PME levels seems to be an earlier step in dexamethasone‐induced apoptosis than the reduction in ATP. The degree of alkaline shift was found to correlate with the number of glucocorticoid receptors present. The possible involvement of phospholipid metabolites as second messengers in dexamethasone‐induced apoptosis is discussed. © 1994 Wiley‐Liss, Inc.
Phosphorylation of the RNA polymerase II largest subunit during heat shock and inhibition of transcription in hela cells
Authors: Dubois Marie‐FrançOise , Bellier Sylvain , Seo Sook‐Jae , Bensaude Olivier
Published: February 2005

The phosphorylation of the C‐terminal domain (CTD) of the largest subunit of eukaryotic RNA polymerase II has been investigated in HeLa cells exposed to heat shock. In control cells, the phosphorylated subunit, IIo, and the dephosphorylated subunit, IIa, were found in similar amounts. During heat shock, however, the phosphorylated subunit, IIo, accumulated, whereas the amount of IIa subunit decreased. Since phosphorylation of the CTD had been suggested to play a role in the initiation of transcription and since heat shock was known to perturb gene expression at the level of transcription, the phosphorylation state of RNA polymerase II was examined in cells that had been treated with various inhibitors of transcription. Under normal growth temperature, actinomycin D (over 0.1 μ/ml) and okadaic acid, a phosphatase inhibitor, were found to inhibit polymerase dephosphorylation. Whereas 5, 6‐dichlorobenzimidazole riboside (DRB), N‐(2‐[Methylamino]ethyl)‐5‐isoquinolinesulfonamide (H‐8), and actinomycin D (over 5 μ/ml) were found to inhibit polymerase phosphorylation. Actinomycin D concentrations, which inhibited the dephosphorylation process, were lower than those required to inhibit the phosphorylation process. In contrast, during heat shock or exposure to sodium arsenite, a chemical inducer of the heat‐shock response, the phosphorylated subunit, IIo, accumulated even in the presence of inhibitors of transcription such as DRB, H‐8, and actinomycin D. These experiments demonstrated the existence of a heat‐shock‐induced CTD‐phosphorylation process that might contribute to the regulation of transcription during stress. © 1994 Wiley‐Liss, Inc.
Growth factor responsiveness of human articular chondrocytes: Distinct profiles in primary chondrocytes, subcultured chondrocytes, and fibroblasts
Authors: Guerne Pierre‐André , Sublet Arthur , Lotz Martin
Published: February 2005

The objectives of this study were to establish a growth factor response profile for adult human articular chondrocytes, to determine whether this is unique for chondrocytes or influenced by the differentiation status of the cells, and to characterize growth factor interactions. It is shown that transforming growth factor‐β (TGF‐β) is the most potent mitogen among a variety of factors tested. All three isoforms of TGF‐β caused similar dose‐dependent increases in chondrocyte proliferation. Other members of the TGF‐β family, including bone morphogenetic protein 2B (BMP2B), activin, and inhibin, did not detectably increase chondrocyte proliferation. Platelet‐derived growth factor‐AA (PDGF‐AA), basic fibroblast growth factor (bFGF), and insulin‐like growth factor 1 (IGF‐1) also stimulated proliferation but were less effective than TGF‐β. In contrast to findings with other cell types, the effects of TGF‐β on chondrocyte proliferation were not dependent on the endogenous production of PDGF. The cytokines Interleukin 1 (IL‐1) and tumor necrosis factor‐α (TNF‐α) gave no stimulation, but IL‐1 inhibited chondrocyte proliferation induced by TGF‐β or serum. This response profile was characteristic for primary chondrocytes from human adults and distinct from subcultured (dedifferentiated) chondrocytes or skin fibroblasts. The latter preferentially responded to PDGF, and IL‐1 caused greater increases in proliferation than TGF‐β. In summary, these results describe growth factor responses that are characteristic for chondrocytes and provide a basis for the analysis of changes in chondrocyte growth proliferation that occur in aging and tissue injury. © 1994 Wiley‐Liss, Inc.
Termination of endothelin signaling: Role of nitric oxide
Authors: Goligorsky Michael S. , Tsukahara Hirokazu , Magazine Harold , Andersen Thomas T. , Malik Asrar B. , Bahou Wadie F.
Published: February 2005

Cellular mechanisms responsible for the termination of ET‐1 signal are poorly understood. In order to examine the hypothesis that nitric oxide serves as a physiological brake of ET‐ 1 signaling, Chinese hamster ovary (CHO) cells stably transfected with the ETA receptor cDNA (CHO‐ET) were studied. CHO‐ET responded to ET‐1 with robust [Ca2+], transients and developed a long‐lasting homologous desensitization. Donors of nitric oxide (NO), 3‐morpholino‐sydnonimine HCl(SIN‐1), or sodium nitroprusside (SNP) reduced the amplitude of these responses, accelerated the rate of [Ca2+], recovery, and counteracted the development of homologous desensitization by a cyclic GMP‐independent mechanism, suggesting an alternative mode for NO modulation of ET‐1 responses. Stimulation of CHO‐ET cells with mastoparan, a wasp venom acting directly on G proteins (bypassing receptor activation), was inhibited by NO, revealing a postreceptoral target for NO‐induced modulation of [Ca2+] mobilization. Using a lys9‐biotinylated ET‐1 (ET‐1 [BtK9]), binding sites were “mapped” in CHO‐ET cells. Receptor‐ligand complexes did not exhibit spontaneous dissociation during 60min observations. Quantitative fluorescence microscopy revealed that SNP or SIN‐1 caused a rapid, concentration‐dependent, and reversible dissociation of biotinylated ET‐ 1 from ETA receptor (EC50 = 75 μM and 6 μM, respectively), an effect that was not mimicked by 8‐bromo‐cyclic GMP. “Sandwich” co‐culture of endothelial cells with CHO‐ET showed that activation of NO production by endothelial cells similarly resulted in dissociation of ET‐1 [BtK9] from ETA receptors. We hypothesize that NO plays a role in physiological termination of ET‐1 signalling by dual mechanisms: (1) displacement of bound ET‐1 from its receptor, thus preventing homologous desensitization, and (2) interference with the postreceptoral pathway for [Ca2+] mobilization, hence inhibiting end‐responses to ET‐1. © 1994 Wiley‐Liss, Inc.