bluuebunny/crossref_metadata_2025 huggingface dataset:
Fibronectin promotes the elongation of microvessels during angiogenesis in vitro
Authors: Nicosia Roberto F. , Bonanno Elena , Smith Marion
Published: February 2005

Fibronectin is a component of the extracellular matrix of developing microvessels whose role in angiogensis is poorly understood. This study evaluated the effect of plasma fibronectin on angiogenesis in serum‐free collagen gel culture of rat aorta. Aortic explants embedded in collagen gels generated microvascular outgrowths. Fibronectin incorporated in the collagen gel promoted a selective dose‐dependent elongation of the newly formed microvessels without stimulating vascular proliferation. The fibronectin‐treated microvessels were longer due to a proportional increase in the number of microvascular cells. However, fibronectin had no effect on microvascular DNA synthesis and mitotic activity. Fibronectin stimulated microvascular length also in cultures in which mitotic activity was suppressed and angiogenesis was markedly reduced by pretreating the aortic explants with mitomycin C. The synthetic peptide Gly‐Arg‐Asp‐(GRGDS), which competes for the binding of fibronectin to its cell receptors and inhibits the adhesion of endothelial cells to substrates, arrested the elongation of developing microvessels causing regression and inhibition of angiogenesis. Conversely, Gly‐Arg‐Glu‐(GRGES), which lacks the RGD sequence, had no inhibitory effect. These data support the hypothesis that fibronectin promotes angiogenesis and suggest that developing microvessels elongate in response to fibronectin as a result of an adhesion‐dependent migratory recruitment of endothelial cells that does not require increased cell proliferation. © 1993 Wiley‐Liss, Inc.
Activation of the Ca2+ “receptor” on the osteoclast by Ni2+ elicits cytosolic Ca2+ signals: Evidence for receptor activation and inactivation, intracellular Ca2+ redistribution, and divalent cation modulation
Authors: Shankar Vijai S. , Bax Christopher M. R. , Bax Bridget E. , Alam A. S. M. Towhidul , Moonga Baljit S. , Simon Bruce , Pazianas Michael , Huang Christopher L.‐H. , Zaidi Mone
Published: February 2005

Earlier studies have demonstrated that a high (mM) extracellular Ca2+ concentration triggers intracellular [Ca2+] signals with a consequent inhibition of bone resorptive activity. We now report that micromolar concentrations of the divalent cation, Ni2+, elicited rapid and concentration‐dependent elevations of cytosolic [Ca2+]. The peak change in cytosolic [Ca2+] increased monotonically with the application of [Ni2+] in the 50–5,000 μM range in solutions containing 1.25 mM‐[Ca2+] and 0.8 mM‐[Mg2+]. The resulting concentration‐response function suggested Ni2+‐induced activation of a single class of binding site (Hill coefficient = 1). The triggering process also exhibited a concentration‐dependent inactivation in which conditioning Ni2+ applications in the range 5–1,500 μM‐[Ni2+] inhibited subsequent responses to a maximally effective [Ni2+] of 5,000 μM. Ni2+‐induced cytosolic [Ca2+] responses were not dependent on extracellular [Ca2+]. Thus, when 5,000 μM‐[Ni2+] was applied to osteoclasts in Ca2+‐free, ethylene glycol bis‐(aminoethyl ether) tetraacetic acid (EGTA)‐containing medium (≤5 nM‐[Ca2+] and 0.8 mM‐[Mg2+]), cytosolic [Ca2+] responses resembled those obtained in the presence of 1.25 mM‐[Ca2+]. Prior depletion of intracellular Ca2+ stores by ionomycin prevented Ni2+‐induced cytosolic [Ca2+] responses, suggesting a major role for intracellular Ca2+ redistribution in the response to Ni2+. The effects of Ni2+ were also modulated by the extracellular concentration of the divalent cations, Ca2+ and Mg2+. When these cations were not added to the culture medium (0 μM‐[Ca2+] and [Mg2+]), even low [Ni2+] ranging between 5 pM and 50 μM elicited progressively larger cytosolic [Ca2+] transients. However, the response magnitude decreased at higher, 250–5,000 μM‐[Ni2+], resulting in a “hooked” concentration‐response curve. Furthermore, increasing extracellular [Mg2+] or [Ca2+] (0–1 mM) diminished the response to 50 μM‐[Ni2+], a concentration on the rising phase of the “hook.” Similar increases (0–10 mM) in extracellular [Mg2+] or [Ca2+] increased the response to 5,000 μM‐[Ni2+], a concentration on the falling phase of the “hook”. These findings are consistent with the existence of a membrane receptor strongly sensitive to Ni2+ as well as the divalent cations, Ca2+ and Mg2+. Receptor occupancy apparently activates intracellular Ca2+ release followed by inactivation. Furthermore, repriming is independent of intracellular Ca2+ stores, suggesting that such inactivation operates at a transduction step between receptor occupancy and intracellular Ca2+ release. © 1993 Wiley‐Liss, Inc.
Regulation of insulin‐like growth factor‐II expression and its role in autocrine growth of human neuroblastoma cells
Authors: Martin D. M. , Feldman E. L.
Published: February 2005

Insulin‐like growth factor‐II (IGF‐II) is highly expressed in fetal tissues and may act as an autocrine growth factor during early embryogenesis. The SH‐SY5Y human neuroblastoma cell line also expresses IGF‐II and its receptors and responds to exogenous IGF‐II with increased DNA synthesis, cell division, and neuritic outgrowth. For this study, we tested the hypothesis that IGF‐II mediates autocrine growth of SH‐SY5Y cells in serum‐free media. SH‐SY5Y cells plated at high densities proliferated in serum‐free media, whereas sparsely plated cells did not. IGF‐II mRNA levels increased within 24 hours of serum deprivation and were associated with increased immunoreactive IGF‐II protein. Exogenous addition of IGF‐II increased 3H‐TdR incorporation and cell number in a dose‐ and time‐dependent fashion. By nuclear labelling experiments using 5‐Bromo‐2′ deoxyuridine (BrdU), we detected a twofold higher percentage of S phase nuclei after a 24‐hour incubation in IGF‐II. Treatment of SH‐SY5Y cells with anti‐IGF‐II antibodies in serum‐free media inhibited cell proliferation, and this inhibition was partially overcome by the addition of increasing concentrations of IGF‐II. Collectively, our results indicate that IGF‐II mediates an autocrine growth mechanism in SH‐SY5Y cells that is associated with increased IGF‐II expression. © 1993 Wiley‐Liss, Inc.
Autocrine/paracrine regulation of keratinocyte urokinase plasminogen activator through the TGF‐α/EGF receptor
Authors: Jensen Pamela J. , Rodeck Ulrich
Published: February 2005

Transforming growth factor‐α (TGF‐α) appears to be an important autocrine/paracrine regulator of keratinocyte function. Not only does TGF‐α induce keratinocyte proliferation and migration in vitro, but it also has been detected in normal human epidermis and at elevated levels in hyperproliferative epidermis. In the present study we report that exogenous TGF‐α increases urokinase‐type plasminogen activator (uPA) in cultured human keratinocytes. Furthermore, in the absence of exogenous growth factors, the “basal” levels of uPA are decreased by an antagonist monoclonal antibody to the receptor shared by TGF‐α and epidermal growth factor (EGF). These results suggest that an endogenous factor serves as an autocrine/paracrine regulator of keratinocyte uPA. We hypothesize that activation of the TGF‐α/EGF receptor may coordinately regulate the keratinocyte response to cutaneous wounding, which includes enhanced uPA expression, migration, and proliferation. © 1993 Wiley‐Liss, Inc.
Mevalonate controls cytoskeleton organization and cell morphology in thyroid epithelial cells
Authors: Bifulco Maurizio , Laezza Chiara , Aloj Salvatore M. , Garbi Corrado
Published: February 2005

Blockade of mevalonate synthesis by the 3‐hydroxy‐3‐methylglutaryl Coenzyme A reductase inhibitor mevinolin (lovastatin) causes FRTL‐5 thyroid cells to undergo significant morphological changes; these include a transition from a flat, polygonal to a round shape, the development of cytoplasmic arborizations, and the loss of contact between neighboring cells. Immunofluorescence studies of cytoskeletal structures show that, at early times after administering the drug, and before the round phenotype develops, stress fibers disassemble while the peripheral actin filaments, which are adjacent to the cytoplasmic face of the plasma membrane, appear largely unaffected. Subsequently, when this cortical actin network becomes fragmented, cells start to round up and become separated from neighbors. Microtubules become disconnected from the plasma membrane and retract toward the cell center, although they do not appear depolymerized; indeed, at this stage, cytoplasmic elongations contain mostly intact microtubules. After exposure to mevinolin FRTL‐5 cells also lose vinculin‐related substrate contacts. Treatment of cells with either cycloheximide or colchicine abolishes morphological changes induced by mevinolin, suggesting that ongoing protein synthesis and microtubule integrity are prerequisites for the drug to be effective. Both cytoskeletal and morphological perturbations can be reversed by mevalonate, but not by cholesterol or the non‐sterol derivatives of mevalonate such as dolichol, ubiquinone, and isopentenyladenine, individually or in combination. It is suggested that mevalonate deficiency may impair formation of isoprenylated proteins important for cytoskeletal organization and stability. © 1993 Wiley‐Liss, Inc.
Growth state‐regulated expression of p52(PAI‐1) in normal rat kidney cells
Authors: Ryan Michael P. , Higgins Paul J.
Published: February 2005

In normal rat kidney (NRK) cells, synthesis of the 52‐kDa substrate‐associated type 1 inhibitor of plasminogen activator [p52(PAI‐1)] is linked to alterations in cell shape and substrate adhesion. Subconfluent NRK cells accumulated significantly more ventral undersurface‐associated p52(PAI‐1) compared to newly confluent or 1 ‐ to 2‐day postconfluent cultures, suggesting that p52(PAI‐1) expression was also growth state‐modulated. Since cytoarchitectural constraints function in cell growth control, changes in p52(PAI‐1) synthesis were assessed with respect to defined morphologic events that accompany growth activation of cultured NRK cells. Stimulation of low population density, quiescent NRK cells with 20% serum‐containing medium resulted in a rapid increase in matrix p52(PAI‐1) protein content (6‐ and 26‐fold after 1 and 5 hr, respectively). Growth activation in response to serum reflected elevations in p52(PAI‐1) cytoplasmic mRNA abundance, which peaked at 2 hr (125‐fold increase) and subsequently declined (100‐fold increase) at 5 hr poststimulation. Morphologic analysis indicated that quiescent NRK cells were devoid of transcytoplasmic actin filaments and focal contact‐associated vinculin. A marked increase in the fraction of cells that eleborated transcytoplasmic microfilaments and vinculin‐containing focal adhesions was evident within 5 min of serum addition. Such cytoarchitectural restructuring preceded p52(PAI‐1) induction. Morphologic reorganization and p52(PAI‐1) induction occurred prior to progression of cells through the S‐phase, indicating they are early events associated with serum stimulation in the NRK cell system. The relevance of p52(PAI‐1) induction during this growth state transition is not clear but may influence the established cytoarchitectural changes observed prior to p52(PAI‐1) induction by regulating pericellular proteolysis and, thereby, cell‐to‐substrate adhesion. © 1993 Wiley‐Liss, Inc.
Regulation of polyamine transport by polyamines and polyamine analogs
Authors: Kramer D. L. , Miller J. T. , Bergeron R. J. , Khomutov R. , Khomutov A. , Porter C. W.
Published: February 2005

Regulation of polyamine transport in murine L1210 leukemia cells was characterized in order to better understand its relationship to specific intracellular polyamines and their analogs and to quantitate the sensitivity by which it is controlled. Up‐regulation of polyamine uptake was evaluated following a 48‐hr treatment with a combination of biosynthetic enzyme inhibitors to deplete intracellular polyamine pools. The latter declined gradually over 48 hr and was accompanied by a steady increase in spermidine (SPD) and spermine (SPM) transport as indicated by rises in Vmax to levels ∼4.5 times higher than control values. Restoration of individual polyamine pools during a 6‐hr period following inhibitor treatment revealed that SPD and SPM uptake could not be selectively affected by specific pool changes. The effectiveness of individual polyamines in reversing inhibitor‐induced stimulation of uptake was as follows: putrescine < SPD < SPM = the SPM analog, N1, N12‐bis(ethyl)spermine (BESPM). In contrast to stimulation of transport, down‐regulation by exogenous polyamines or analogs occurred rapidly and in response to subtle increases in intracellular pools. Following a 1‐hr exposure to 10 μM BESPM, Vmax values for SPD and SPM fell by 70%, whereas the analog pool increased to only 400–500 pmol/106 cells—about 15–20% of the total polyamine pool (∼2.8 nmol/106 cells). SPM produced nearly identical regulatory effects on transport kinetics. Both BESPM and SPM were even more effective at down‐regulating transport that had been previously stimulated four to fivefold by polyamine depletion achieved with enzyme inhibitors. A dose response with BESPM at 48 hr revealed a biphasic effect on uptake whereby concentrations of analog < 3 μM produced an increase in SPD and SPM Vmax values, whereas concentrations 3 μM and higher produced a marked suppression of these values. Cells treated with 3 μM BESPM for 2 hr and placed in analog‐free medium recovered transport capability in only 3 hr. Thus, whereas stimulation of polyamine transport is a relatively insensitive and slowly responsive process that tends to parallel polyamine depletion, down‐regulation of polyamine transport by exogenous polyamines and analogs and its reversal are rapidly responsive events that correlate with relatively small (i.e., 15–20%) changes in intracellular polyamine pools.
Defensins are mitogenic for epithelial cells and fibroblasts
Authors: Murphy Christopher J. , Foster Bradley A. , Mannis Mark J. , Selsted Michael E. , Reid Ted W.
Published: February 2005

Defensins are a family of structurally homologous peptides contained within phagocytic cells. Although these peptides are best known for their broad spectrum antimicrobial properties, they also inhibit ACTH (corticotropin) stimulated corticosterone production, chemoattract monocytes, and lyse mammalian cells. We now report that these peptides are potent mitogens in vitro in the same concentration range that they display potent antimicrobial activity in vitro. These concentrations are in the same range as those expected to be present in vivo during the wound healing process. All defensins tested were stimulatory for epithelial cells and fibroblasts and acted synergistically with insulin. These are the first data to disclose the strong growth‐promoting effects of this unique family of peptides and point to another basic mechanism whereby the macrophage and neutrophil may participate in a variety of trophic, physiologic, and pathologic processes.
Poliovirus‐induced intracellular alkalinization involves a proton ATPase and protein phosphorylation
Authors: Holsey Charles , Nair C. N.
Published: February 2005

We reported previously that poliovirus infection induces alkalinization in HeLa cells and that an alkaline intracellular pH (pHi) promoted viral replication. Additional experiments were carried out to understand the underlying mechanism. Virus‐infected or control monolayer cultures were incubated with nominally bicarbonate‐free Eagle's minimal essential medium (MEM) buffered with N‐2‐hydroxyethylpiperazine‐N‐3‐ethanesulfonic acid (HEPES), and immediately following preincubations, changes in pHi were monitored via benzoic acid uptake around 2 h postinfection. The absence of pH increase in cells infected with ultraviolet light‐inactivated virus (UV‐virus) indicated that viral gene expression was required for this effect. On the other hand, lack of effect of 3 mM guanidine, an inhibitor of poliovirus‐specific RNA but not protein synthesis, suggested that translation of input viral genome RNA is sufficient for the pH increase. Activation of Na+/H+ exchange, Cl−HCO−3 exchange, or H+‐ATPase was considered as possible mechanisms by which alkalinization occurs in virus‐infected cells. Na+/H+ exchange was excluded because the pH effect occurred in a Na+/H+ exchange deficient HeLa cell mutant. Similarly, Cl−/HCO−3 exchange was excluded because virus‐specific alkalinization was evident in the presence of Cl− or bicarbonate deficient medium and was not associated with an increase in HCO−3 uptake or a decrease in Cl− uptake. Lack of dependence on Na+, abrogation by 10 μM 7‐chloro‐4‐nitrobenz‐2‐oxa‐1,3‐diazole (NBD‐Cl), and resistance to 1 mM vandate suggested that this effect was due to the activation of a vacuolar‐type (V) proton ATPase. Studies using protein kinase inhibitors indicated that activation of the ATPase in virus‐infected cells probably involved protein kinase C‐mediated phosphorylation. © 1993 Wiley‐Liss, Inc.
Cobalamin metabolism in cultured human chorionic villus cells
Authors: Begley James A. , Colligan Pamela D. , Chu Richard C. , Hall Charles A.
Published: February 2005

Cobalamin (Cbl, vitamin B12) metabolism was analyzed in cultures of human chorionic villus (CV) cells obtained at 9–10 weeks of gestation. CV cells were shown to synthesize transcobalamin II (TCII) and to possess a high affinity receptor for that molecule. The cells bound and internalized radioactive cyanocobalamin (CN[57Co]Cbl) complexed to TCII. This internalized CN[57Co]Cbl was found to be converted to both methylCbl and adenosylCbl, the two intracellular coenzyme forms of Cbl, and bound to the two known intracellular Cbl requiring enzymes, methionine synthase (MS) and methylmalonyl‐CoA mutase. Both enzyme systems were found to be functional in the intact cell by demonstrating the incorporation of the radioactive label from both [14C]CH3‐tetrahydrofolate and [14C]propionate into acid insoluble products. MS activity was also detected in lysed cell material. CV cells were shown not to be auxotrophic for methionine since they were able to utilize homocysteine in place of methionine for cell division. Since CV cells are capable of performing many of the complex events associated with Cbl metabolism, it may be possible to use these cells to diagnose genetic defects of Cbl metabolism. © 1993 Wiley‐Liss, Inc.
Apoptosis of human erythroid colony‐forming cells is decreased by stem cell factor and insulin‐like growth factor I as well as erythropoietin
Authors: Muta Koichiro , Krantz Sanford B.
Published: February 2005

To clarify the manner by which erythropoietin (EP), stem cell factor (SCF), or insulin‐like growth factor I (IGF‐I) regulate erythropoiesis, apoptosis of human erythroid progenitor cells was investigated. Human burst‐forming units‐erythroid (BFU‐E) partially purified from peripheral blood were cultured for 6 days to generate erythroid colony‐forming cells (ECFC), which consist mainly of colony‐forming units‐erythroid (CFU‐E). The cells were labeled with [3H]thymidine, incubated in serum‐free liquid media, at 37°C, for 16 h, and the pattern of DNA breakdown was analyzed by agarose gel electrophoresis. When these cells were incubated without EP, 70% of the total cellular DNA was broken down into DNA fragments of less than 5 kilobases and nuclear condensation and fragmentation, characteristic of apoptosis, were evident. While EP greatly reduced the amount of DNA breakdown to 23%, SCF and IGF‐I each reduced the amount of DNA breakdown to 38–46% and, when added together, to 24%. Dose‐response experiments with SCF and IGF‐I showed a dose‐dependent reduction in DNA fragmentation at concentrations that stimulate colony formation in serum‐free semisolid cultures. Finally, assays of ECFC performed by the plasma clot method, after serum‐free liquid culture, at 37°C, for 16 h, demonstrated marked protection of erythroid colony‐forming capacity by SCF or IGF‐I in the absence of EP, as well as by EP itself. These data indicate that human erythroid progenitor cells undergo apoptosis which is reduced by SCF and IGF‐I as well as EP and suggest that the control of apoptosis by each of these factors has a prominent role in the regulation of erythropoiesis. © 1993 Wiley‐Liss, Inc.
Histamine and cis‐urocanic acid augment tumor necrosis factor‐alpha mediated induction of keratinocyte intercellular adhesion molecule‐1 expression
Authors: Mitra Raj S. , Shimizu Yoji , Nickoloff Brian J.
Published: February 2005

Early cellular and molecular events in inflamed skin include the active participation of epidermal keratinocytes (KCs) and dermal mast cells which can produce diffusible mediators such as tumor necrosis factor‐alpha (TNF‐α), histamine, and urocanic acid (UCA). Rapid induction of adhesion molecules such as intercellular adhesion molecule‐1 (ICAM‐1) by KCs is observed following a highly diverse array of stimuli which can provoke both irritant, inflammatory, as well as allergic and immune reactions. To determine if the aforementioned mediators could interact in either an additive or synergistic fashion with each other, cultured KCs were exposed to these mediators alone and in combination, and the degree of ICAM‐1 mRNA and protein quantitated. Whereas histamine or cis‐UCA alone only weakly induced KC ICAM‐1, when they were combined with TNF‐α, significant augmentation was observed by Northern blot hybridization studies, immunostaining, and FACS analysis. Other histamine derivatives such as L‐histidine, 1‐methylhistidine, 3‐methylhistidine, or all‐trans‐UCA had no effect. Histamine pretreatment did not affect cell surface high affinity TNF‐α receptors, as determined by ligand binding and immunodetection, and did not induce KC TNF‐α production. The KC histamine receptor was also characterized and found not to be influenced by TNF‐α, cis‐UCA, all‐trans‐UCA, or diphenyhydramine (an H1 antagonist), but it was inhibited by cimetidine (an H2 antagonist). These results demonstrate that 1) KCs can be induced to express ICAM‐1 by exposure to histamine and cis‐UCA, 2) histamine and cis‐UCA can also augment TNF‐α inducible ICAM‐1 mRNA and cell surface protein expression, 3) this augmentation does not directly involve changes in KC TNF‐α receptor number, affinity, or TNF‐α production and, 4) KCs possess a type 2 histamine receptor which is not the photoreceptor for UCA. These findings highlight the potential for cross‐talk between molecules produced by resident cutaneous cell types above (i.e., KCs) and below (i.e., mast cells) the epidermal basement membrane zone. These cells and their mediators can cooperate to respond to either exogenous or endogenous stimuli leading to rapid and strong KC ICAM‐1 expression. Such induction of this important adhesion molecule by KCs ensures the retention of T lymphocytes necessary to participate in the maintenance of cutaneous immunohomeostasis. © 1993 Wiley‐Liss, Inc.
Activation of protein kinase C inhibits human keratinocyte migration
Authors: Ando Yoshihiro , Lazarus Gerald S. , Jensen Pamela J.
Published: February 2005

The involvement of protein kinase C (PKC) in epidermal growth factor (EGF)‐induced human keratinocyte migration was studied with the phagokinetic assay. It was concluded that PKC activation does not mediate, but rather inhibits, EGF‐induced keratinocyte migration. The following experimental observations support these conclusions: 1) The PKC inhibitor H‐7 did not inhibit EGF‐induced migration but instead led to a modest enhancement. 2) PKC activators such as phorbol‐12‐myristate‐13‐acetate (PMA), phorbol‐12,13‐dibutyrate (PDBu), and 1,2‐dioctanoly‐sn‐glycerol inhibited migration, but biologically inactive 4α‐PMA had no effect. 3) PMA did not inhibit keratinocyte attachment and spreading but blocked migration almost immediately after addition. 4) Migration of PKC‐depleted cells, which were produced by prolonged treatment with PDBu, was enhanced similarly to normal cells by EGF. 5) PKC‐depleted cells were not susceptible to the inhibitory effects of phorbol esters on migration. Additional experiments, in which cells were preactivated with EGF, suggested that PKC inhibits the EGF effect at a post‐receptor level. The inhibitory effect of PKC on keratinocyte migration was not restricted to EGF‐induced migration; PKC activation also inhibited keratinocyte migration induced by bovine pituitary extract, insulin, insulin‐like growth factor‐1, and keratinocyte growth factor. © 1993 Wiley‐Liss, Inc.
Constitutive expression of c‐myc does not relieve cAMP‐mediated growth arrest in human lymphoid reh cells
Authors: Andersson Kristin Brevik , Deggerdal Arne , Skjønsberg Cecilia , Smeland Erlend B. , Blomhoff Heidi Kiil
Published: February 2005

The Reh cell system is suitable for evaluating events important for control of proliferation independently of mechanisms involved in differentiation, as Reh cells are unable to differentiate. In the human pre‐B cell line Reh, activation of adenylate cyclase by forskolin induces a five to tenfold rapid, transient downregulation of steady‐state c‐myc RNA within 4 hours. Concurrently, the cells are strongly growth arrested in the G1 phase of the cell cycle. To clarify if the observed growth arrest could be relieved by constitutive expression of c‐myc, an exogenous c‐myc gene under constitutive promoter control was introduced into Reh cells by electroporation. The c‐myc‐expressing construct pDMmycHyg contained human c‐myc exons 2 and 3 driven by the Mo‐MLV LTR and conferred hygromycin resistance. Exogenous c‐myc RNA transcripts and protein were constitutively expressed in the transfected clones at levels roughly twice as high as the level in nontransfected cells. Total c‐myc protein levels were unchanged upon treatment of transfected clones with forskolin. Yet, the transfected cells were not released from growth arrest. Furthermore, the transfected Reh cells did not differentiate upon forskolin treatment. Constitutive overexpression of c‐myc is therefore not sufficient for relieving forskolin‐mediated effects on growth arrest in Reh cells. © 1993 Wiley‐Liss, Inc.
Transcriptional and posttranscriptional regulation of hepatic β2‐adrenergic receptor gene expression during development
Authors: Baeyens Dennis A. , Cornett Lawrence E.
Published: February 2005

Hepatic responsiveness to β2‐adrenergic stimulation is dynamically regulated during early development as well as following hepatic injury and disease. In the present study, the molecular mechanisms that underlie the decline in the steady‐state levels of hepatic β2‐adrenergic receptor mRNA that occurs during development in the male rat were investigated. As determined by nuclear run‐on assays, an age‐associated reduction in β2‐adrenergic receptor gene transcription was observed. The transcription rate of the β2‐adrenergic receptor gene in postnatal day 18 liver was approximately 50% lower than that of fetal liver. Stability of β2‐adrenergic receptor gene transcripts was highest (t1/2 ≈ 6 h) in hepatocytes isolated from fetal rats and was lowest (t1/2 ≈ 1 h) in hepatocytes isolated from postnatal day 14 rats. In fetal hepatocytes, but not postnatal day 2 hepatocytes, cycloheximide appeared to stabilize β2‐adrenergic receptor gene transcripts in the presence of actinomycin D. These findings establish the molecular basis of reduced steady‐state levels of β2‐adrenergic receptor mRNA in liver during early postnatal development and suggest multilevel regulatory control of hepatic β2‐adrenergic receptor gene expression. © 1993 Wiley‐Liss, Inc.
“Macrophage” nitric oxide synthase is a glucocorticoid‐inhibitable primary response gene in 3T3 cells
Authors: Gilbert Rebecca S. , Herschman Harvey R.
Published: February 2005

Both nitric oxide and prostaglandins are potent paracrine mediators of intercellular communication. An endotoxin‐lipopolysaccharide (LPS) inducible form of nitric oxide synthase (mac‐NOS) has recently been cloned from murine macrophages. An inducible prostaglandin synthase (TIS1O/PGS‐2), cloned from 3T3 cells, is also induced in LPS‐activated macrophage. Because of the wide range of ligands that induce primary response genes in 3T3 cells, the ease of studying chimeric promoter constructs in 3T3 cells, and the importance of both nitric oxide and prostaglandins as paracrine mediators, we examined expression of mac‐NOS in 3T3 cells. Tetradecanoyl phorbol‐13 acetate (TPA), forskolin, platelet‐derived growth factor, fibroblast growth factor, and serum all induce mac‐NOS expression in Swiss 3T3 cells. Thus the mac‐NOS gene can respond to a far wider range of inducers than previously suspected. mac‐NOS is a primary response gene; cycloheximide does not block induction. TPA‐induced mac‐NOS and TIS10/PGS‐2 mRNA accumulation patterns are similar. LPS is a potent inducer of mac‐NOS in Swiss 3T3 cells but cannot induce TIS10/PGS‐2. In contrast, v‐src expression induces TIS10/PGS‐2 message, but not iNOS message in a BALB/c 3T3 cell line containing a temperature‐sensitive v‐src gene. Dexamethasone (DEX) prevents induction of TIS10/PGS‐2, but not most other primary response genes. DEX also blocks mac‐NOS induction in Swiss 3T3 cells. The inducible TIS10/PGS‐2 and mac‐NOS genes, responsible for the production of two distinct paracrine agents, appear to share many regulatory features in 3T3 cells. © 1993 Wiley‐Liss, Inc.
Evidence that a neutral cholesteryl ester hydrolase is responsible for the extralysosomal hydrolysis of high‐density lipoprotein cholesteryl ester in rat hepatoma cells (Fu5AH)
Authors: Delamatre John G. , Carter Robert M. , Hornick Conrad A.
Published: February 2005

Diethylumbelliferyl phosphate (UBP) has been shown to inhibit the neutral cholesteryl ester hydrolase activity responsible for hydrolysis of cellular lipid droplet cholesteryl ester (Harrison et al., 1990). The potential for (UBP) to inhibit uptake and hydrolysis of high density lipoprotein (HDL) cholestryl ester was studied in Fu5AH hepatoma cells, a model for HDL cholesterol delivery. Coincubation of 3H‐cholesteryl ester labeled HDL with UBP resulted in a 72% decrease in the cellular free cholesterol/cholesterl ester (FC/CE) isotope ratio, indicating an inhibition in the conversion of cholesteryl ester to free cholesterol. Total cellular 3H‐CE uptake was modestly (27%) but significantly decreased by UBP. Pulsechase experiments (15 min. pulse and 7 min. chase) were used to study the hydrolysis of HDL 3H‐CE in subcellular fractions separated by percoll gradients. The conversion of 3H‐CE to 3H‐FC could be demonstrated in fractions that comigrated with the plasma membrane/endosome fractions but were well separated from lysosomes. Neutral cholesteryl ester hydrolase activity was detected in those same fractions. These results suggest that an extralysosomal pathway is operating in the metabolism of HDL cholesterol and its delivery to hepatoma cells. © 1993 Wiley‐Liss, Inc.
Comparative studies of the granulopoietic enhancing effects of biosynthetic human insulin‐like growth factors I and II
Authors: Merchav Shoshana , Lake Mats , Skottner Anna
Published: February 2005

The effect of biosynthetic human insulin‐like growth factor I (IGF‐I) and IGF‐II on the in vitro growth of human marrow myeloid progenitors in the presence of recombinant human granulocyte colony stimulating factor (rhG‐CSF), granulocyte‐macrophage CSF (rhGM‐CSF), or interleukin‐3 (rhIL‐3), was investigated. IGF‐I and IGF‐II similarly enhanced the growth of myeloid progenitors in cultures stimulated with any of the above hemopoietic regulators. Analysis of colony composition showed an increase in the numbers of granulocyte colonies, but no alteration in the numbers of macrophage or granulocyte/macrophage colonies. IGF‐I induced an increase of 62 ± 16%, 84 ± 13%, and 107 ± 18% in granulocyte colony numbers in the presence of G‐CSF, GM‐CSF, or IL‐3, respectively. The values for IGF‐II were 66 ± 13%, 96 ± 12%, and 91 ± 12%. Similar enhancement of myeloid colony formation by both peptides was also detected in G‐CSF and GM‐CSF‐stimulated cultures of marrow cells that had been depleted of accessory cells, while neither peptide exerted any effect in the presence of IL‐3 in such cultures. The growth‐promoting effects of IGF‐I and IGF‐II were completely abrogated by monoclonal antibodies directed against the IGF‐I (Type I) membrane receptor. IGF‐I and IGF‐II thus appear to exert their effects on human marrow myeloid progenitors via a direct mechanism involving the Type I receptor. © 1993 Wiley‐Liss, Inc.
Regulation of integrin‐mediated adhesion at focal contacts by cyclic AMP
Authors: Class William F. , Kreisberg Jeffrey I.
Published: February 2005

Cyclic AMP (cAMP) elevation causes diverse types of cultured cells to round partially and develop arborized cell processes. Renal glomerular mesangial cells are smooth, muscle‐like cells and in culture contain abundant actin microfilament cables that insert into substratum focal contacts. cAMP elevation causes adhesion loss, microfilament cable fragmentation, and shape change in cultured mesangial cells. We investigated the roles of the classical vitronectin (αVβ3 integrin) and fibronectin (α5β1 integrin) receptors in these changes. Mesangial cells on vitronectin‐rich substrata contained microfilament cables that terminated in focal contacts that stained with antibodies to vitronectin receptor. cAMP elevation caused loss of focal contact and associated vitronectin receptor. Both fibronectin and its receptor stained in a fibrillary pattern at the cell surface under control conditions but appeared aggregated along the cell processes after cAMP elevation. This suggested that cAMP elevation caused loss of adhesion mediated by vitronectin receptor but not by fibronectin receptor. We plated cells onto fibronectin‐coated slides to test the effect of ligand immobilization on the cellular response to cAMP. On fibronectin‐coated slides fibronectin receptor was observed in peripheral focal contacts where actin filaments terminated, as seen with vitronectin receptor on vitronectin‐coated substrata, and in abundant linear arrays distributed along microfilaments as well. Substratum contacts mediated by fibronectin receptor along the length of actin filaments have been termed fibronexus contacts. After cAMP elevation, microfilaments fragmented and fibronectin receptor disappeared from peripheral focal contacts, but the more central contacts along residual microfilament fragments appeared intact. Also, substratum adhesion was maintained after cAMP elevation on fibronectin—but not on vitronectincoated surfaces. Although other types of extracellular matrix receptors may also be involved, our observations suggest that cAMP regulates adhesion at focal contacts but not at fibronexus‐type extracellular matrix contacts. © 1993 Wiley‐Liss, Inc.
MSH receptors in immortalized human epidermal keratinocytes: A potential mechanism for coordinate regulation of the epidermal‐melanin unit
Authors: Chakraborty Ashok , Pawelek John
Published: February 2005

Receptors for melanotropin (MSH) were found to be expressed by immortalized primary human epidermal keratinocytes (RHEK‐1). Using 125I‐βMSH as a probe, the MSH receptors from mouse melanoma cells and human keratinocytes were found to be remarkably similar. In each cell line, there were high and low affinity receptors, with the high affinity classes showing positive cooperativity. Competition of 125l‐βMSH for binding with non‐radioactive MSH revealed similar profiles. Cross‐linking studies, followed by gel electrophoresis and autoradiography, showed almost identical gel migration patterns. Both cell types expressed internal as well as plasma membrane binding sites. MSH receptors on both cell types were up‐regulated by ultraviolet light and by MSH itself. Although the function of MSH receptors expressed by the immortalized keratinocytes is unknown, the results are consistent with recent reports that proliferation of epidermal keratinocytes is stimulated by MSH and that proopiomelanocortin genes are expresed in the epidermis. These results support a model in which keratinocytes and melanocytes, interacting in an “epidermal‐melanin unit,” each respond to UV light signals with increased MSH receptor activity. © 1993 Wiley‐Liss, Inc.
Adenovirus and protein kinase C have distinct molecular requirements for regulating epidermal growth factor receptor trafficking
Authors: Hoffman Brian L. , Takishima Kunio , Rosner Marsha Rich , Carlin Cathleen
Published: February 2005

The ligand‐activated tyrosine kinase receptor for epidermal growth factor (EGF) is down‐regulated by an integral membrane protein coded for by the E3 early transcription unit of group C adenoviruses. The E3 protein appears to block recycling of constitutively internalized receptors, causing them instead to traffic to lysosomes where they are degraded. Expression of functional EGF receptors is also regulated by protein kinase C (PKC), which directly phosphorylates the EGF receptor at Thr‐654. The goal of this study was to determine potential interactions between PKC and the E3 protein, since membrane‐bound PKC activity is elevated by the adenovirus E1A protein. Our results show that although tumor promoters which activate PKC cause a coordinate induction of E3 protein synthesis and EGF receptor degradation, the E3 protein‐induced pathway for receptor down‐regulation functions independently of PKC and other kinases that are inhibited by staurosporine. This suggests that in contrast to other mechanisms that modulate receptor expression (i.e., ligand and PKC), the E3 protein is not regulated by phosphorylation but is constitutively active. We also report that adenovirusmediated degradation is the preferred pathway in infected cells stimulated with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) to induce receptor recycling. © 1993 Wiley‐Liss, Inc.
Lineage commitment in human hemopoiesis involves asymmetric cell division of multipotent progenitors and does not appear to be influenced by cytokines
Authors: Mayani Hector , Dragowska Wieslawa , Lansdorp Peter M.
Published: February 2005

Different models have been proposed to explain lineage commitment in hemopoiesis. Some suggest that lineage commitment occurs in a stochastic manner without the direct influence of extracellular factors; others postulate that cytokines determine whether multipotent cells will become erythroid or granulocyte/macrophage progenitors. In the present study, the patterns of proliferation and differentiation of individually sorted human cord blood‐derived primitive hemopoietic cells (highly enriched for multipotent progenitors) were analyzed in a serum‐free culture system supplemented with different cytokine combinations. In a first set of experiments, the response of individual cells to different cytokine combinations was compared, whereas in a second set of experiments, single cells were allowed to undergo one division after which the two daughter cells were physically separated and cultured in either the same or different cytokine combinations. Proliferation of progenitor cells was absolutely dependent on cytokines, and the combination of mast cell growth factor plus interleukin 6 was sufficient to induce mitosis. When cytokine combinations favoring erythropoiesis and/or myelopoiesis were added to the cultures, a more vigorous proliferative response of the sorted primitive progenitors was observed. Interestingly, the relative proportions of granulocyte/macrophage, erythroid, and multipotent progenitors remained more or less the same regardless of the cytokine combination used, indicating a permissive rather than an instructive role for cytokines in hemopoietic differentiation. Asymmetric cell divisions, defined as a division that yields two daughter cells with distinct functional properties, were observed in 3–17% of the progenitor cells capable of forming colonies under our experimental conditions. In the rest, symmetric divisions involving multipotent and lineage‐committed progenitors were observed. The results of this study demonstrate that the asymmetric cell divisions that occur in the early stages of hemopoiesis at the level of multipotent progenitors cannot be skewed by the addition of specific cytokine combinations. These findings support the hypothesis that lineage commitment in hemopoiesis occurs in a stochastic manner by mechanisms that remain to be elucidated. © 1993 Wiley‐Liss, Inc.
Disparate modulation of plasma membrane protein lateral mobility by various cell permeabilizing agents
Authors: Feder Tony J. , Chang En‐Yuh , Holowka David , Webb Watt W.
Published: February 2005

The mobility of a cell surface protein on cells osmotically swollen by treatment with several different cell permeabilizing agents retains specific restraints despite detachment of the plasma membrane from the cortical cytoskeleton. Fluorescence photobleaching recovery experiments indicate that the lateral diffusion constants of immunoglobulin E (IgE)‐receptor complexes on the surface of rat basophilic leukemia cells increase 2–5 × following permeabilization with streptolysin O or digitonin, with little change in their mobile fractions. Swelling by hypo‐osmotic treatment in water enhances lateral diffusion of IgE‐receptor complexes and raises the mobile fractions to near 100%. In contrast, swelling by treatment with filipin arrests lateral diffusion, although rotational mobility remains unhindered. Lateral mobility of a fluorescent lipid analogue remains unchanged under these conditions. Crosslinking by anti‐IgE antibodies redistributes the IgE‐receptor complexes into large patches on untreated cells and on cells swollen by permeabilization with streptolysin O or digitonin, but rot on cells swollen by treatment with filipin. The results indicate a diversity of effects of the various permeabilizing agents on the mobility of membrane proteins. In particular, treatment with filipin appears to reorganize the plasma membrane into a network of fluid domains on a scale smaller than the bleaching spot size used (∼1.5 μm). © 1994 Wiley‐Liss, Inc.
Slowed receptor trafficking in mutant CHO lines of the end1 and end2 complementation groups
Authors: Johnson Lester S. , Presley John F. , Park Juliet C. , McGraw Timothy E.
Published: February 2005

A mutant Chinese hamster ovary cell line with nonconditional kinetic defects in receptor internalization and recycling was isolated, based on selection for resistance to a transferrin‐diphtheria toxin conjugate and screening for aberrant receptor trafficking. The 12‐4 cell line internalizes transferrin at ∼75% of the parental rate and recyles transferrin back to the cell surface at ∼55% of the parental rate. Internalization of low density lipoprotein is also reduced to ∼70% of the parental cell rate, demonstrating that the mutant phenotype affects the trafficking of multiple receptors. Characterization of somatic cell hybrids indicated that the 12‐4 phenotype is recessive, and complementation analysis determined that the 12‐4 cell line is a member of the End2 complementation group. End2 mutants have previously been described as defective in endosomal acidification but have not been known to be defective in receptor trafficking. We have found similar defects in another End2 mutant cell line, suggesting that slowed receptor trafficking is characteristic of End2 mutants. Interestingly, transferrin receptor recycling and internalization are also slowed in another complementation group of mutants, End1, that is also defective in endosomal acidification. This study demonstrates altered receptor trafficking in End1 and End2 cell lines, a novel aspect of the mutant phenotypes. These findings provide evidence, based on a cellular genetic approach, that proper endosome acidification is necessary for maintenance of normal receptor recycling. © 1994 Wiley‐Liss, Inc.
Bumetanide and furosemide inhibited vascular endothelial cell proliferation
Authors: Panet Rivka , Markus Miriam , Atlan Henri
Published: February 2005

In this study, we examined the role of the bumetanide‐sensitive Na+/K+/Cl–cotransport in the mitogenic signal of vascular endothelial cell proliferation. The activity of the Na+/K+/Cl– cotransport is dramatically decreased in quiescent subconfluent cells, as compared to subconfluent cells growing in the presence of FGF. The Na+/K+/Cl– cotransport activity of quiescent subconfluent cultures deprived of FGF decreased to 6%, whereas that of quiescent cells grown to confluency was reduced to only 33% of the activity of subconfluent cells growing in the presence of FGF. The basal low activity of Na+/K+/Cl– cotransport in the quiescent subconfluent vascular endothelial cells was dramatically stimulated by FGF. In order to explore the role of the Na+/K+/Cl– cotransport in the mitogenic signal of the endothelial cells, the effect of two specific inhibitors of the cotransport ‐furosemide and ‐bumetanide was tested on cell proliferation induced by FGF. Bumetanide and furosemide inhibited synchronized cell proliferation measured by direct counting of cells and by DNA synthesis. Inhibition by fuorsemide and bumetanide was reversible; removal of these compounds completely released the cells to proliferate. These results indicate that the effect of these drugs is specific and is not due to an indirect toxic effect. This study clearly demonstrates that the FGF‐induced activation of the Na+/K+/Cl– cotransport plays a role in the mitogenic signal pathway of vascular endothelial cells. © 1994 Wiley‐Liss, Inc.
Characterization of 57 kDa statin as a true marker for growth arrest in tissue by its disappearance from regenerating liver
Authors: Sandig Martin , Bissonnette Robert , Liu Catherine H. L. , Tomaszewski George , Wang Eugenia
Published: February 2005

Statin, a 57 kDa nuclear protein, is lost from quiescent fibroblasts in culture when they are induced to enter the cell cycle by feeding with growth factors, or by removal of contact inhibition. In order to investigate changes in statin expression during the transition from a quiescent to a cycling state in situ, we performed 70% partial hepatectomy on rats and analyzed the regenerating liver by immunofluorescence microscopy with antistatin monoclonal antibodies (S44 mAb), and by immunoblotting of liver proteins in cytoplasmic and enriched nuclear/cytoskeletal fractions. Western blot analysis showed that rat hepatocytes in situ contain a nuclear 57 kDa form of statin, as seen in cultured fibroblasts; however additional S44‐immunoreactive polypeptides with molecular weights of 53 and 110 kDa are also present in both cytoplasmic and nuclear/cytoskeletal fractions. Immunofluo‐rescence microscopy indicates that the proportion of S44‐positive hepatocyte nuclei drops to ∼60% within 24 hours after hepatectomy, a time period when re‐entry of hepatocytes into the cell cycle is first observed. On Western blots of hepatocyte nuclear/cytoskeletal proteins obtained 24 hours after hepatectomy, the 57 kDa form of statin is markedly reduced. These results suggest that, although in liver the S44 antibody recognizes three proteins (53 kDa, 57 kDa, and 110 kDa), the 57 kDa in intact liver, similar to cultured fibroblasts, is the only polypeptide recognized by the statin antibody that disappears when hepatocytes are induced to re‐enter the cell cycle from a quiescent state. © 1994 Wiley‐Liss, Inc.
Effect of ion composition on the changes in membrane potential induced with several stimuli in rat mast cells
Authors: Cabado Ana G. , Vieytes Mercedes R. , Botana Luis M.
Published: February 2005

We studied, in different ionic conditions, the effect of various agents on the membrane potential of rat peritoneal mast cells using the fluorescent probe bisoxonol. Ouabain and ionophore A23187 lead to a fast depolarization of the plasma membrane of mast cells, while compound 48/80 and thapsigargin induced membrane hyperpolarization, which was more pronounced in the case of compound 48/80. When using compound 48/80, the amount of gramicidin necessary to depolarize the cells was twice the amount required in resting cells, which indicates that compound 48/80 increases considerably the activity of the Na+/K+ pump. On the other hand, the ionophore A23187 elicited a clear depolarization which was oblated in the absence of intracellular calcium. The increase in the osmolarity of the medium causes a depolarization in the plasma membrane of mast cells. Hypertonicity‐stimulated depolarization is inhibited by removing sodium and potassium. © 1994 Wiley‐Liss, Inc.