bluuebunny/crossref_metadata_2025 huggingface dataset:
Monkey retinal pigment epithelial cells in vitro synthesize, secrete, and degrade insulin‐like growth factor binding proteins
Authors: Waldbillig Robert J. , Schoen Timothy J. , Chader Gerald J. , Pfeffer Bruce A.
Published: February 2005

Cultured monkey retinal pigment epithelial (RPE) cells rapidly secrete large amounts of insulin‐like growth factor binding proteins (IGF‐BPs). IGF‐II tracer binding activity in conditioned media is two to three times greater than that of IGF‐I. Under reducing SDS‐PAGE conditions, 125I‐IGF affinity‐crosslinked binding protein (BP) is visualized as a broad band between 36 ± 2.9 and 49 ± 3.3 kDa. Because the electrophoretic mobility of the crosslinked BP is increased under non‐reducing conditions (33–45 kDa), intramolecular sulfhydryl bonding may be present. Frequently, the radiographic band representing affinity‐crosslinked binding protein exhibits a complex pattern of non‐uniform densities that suggests structural or functional IGF‐BP micro‐heterogeneity. IGF‐BPs synthesized by RPE also exhibit heterogeneity with respect to the absence or presence of oligosaccharide side chains. In particular, the larger, but not the mid‐sized or smaller IGF‐BPs exhibit side chains linked to the core protein with N‐glycosidic linkage. None of the crosslinked IGF‐BPs exhibit O‐linked side chains. Long‐term (12, 24, 48 hr) conditioning studies revealed that IGF‐BP fails to accumulate in culture media beyond 12 hr, but that replacement of conditioned media with fresh media allows a second period of binding protein accumulation. Other short‐term (12 hr) experiments indicate that, in fresh medium, the levels of IGF‐BP increase during the first 6–8 hr and then remain stable. To examine the processes contributing to these steady state levels of IGF‐BP, aliquots of 8‐hr conditioned medium were removed from the cells and either frozen on dry ice or incubated at 37°C for 16 hr. Importantly, it was found that incubation at 37°C resulted in a near total loss of binding activity. This is the first report of IGF‐BP degrading activity in a cell culture system. These findings indicate that (1) primate RPE cells rapidly secrete a complex mixture of N‐glycosylated and non‐glycosylated IGF‐BPs, and (2) the steady state levels of secreted IGF‐BP are tightly regulated at least in part through a concomitant IGF‐BP inactivating activity. Cultured RPE cells may be of utility in examining the mechanisms of IGF‐BP synthesis, secretion, and degradation at the cellular level.
Modulation of nuclear statin expression in rat thyroid follicle cell following administration of thyroid stimulating hormone
Authors: Bayer I. , Mitmaker B. , Gordon P. H. , Wang E.
Published: February 2005

This study was designed to examine the state of proliferation in the rat thyrocyte following the administration of thyroid stimulating hormone (TSH). An immunohistochemical technique involving the use of a monoclonal antibody to statin, a nonproliferation‐specific nuclear antigen, was developed to measure the subpopulation of cells that have ceased to divide. Following the random assignment of young male Sprague‐Dawley rats into various groups, the rats in the control group received a single intraperitoneal (i‐p) injection of normal saline, whereas the experimental groups received single i‐p injections of TSH at doses of 0.25, 0.50, and 1.0 IU, respectively. All rats were subsequently sacrificed in groups of three at 1, 2, 4, and 24 hours. The statin antibody label was readily identified within the follicle cell nucleus. Results revealed a statistically significant transient decrease in the mean percent statin‐positive nuclei in the TSH‐treated groups. The time‐ and dose‐dependent effect of TSH was maximal at 2 hours and no longer discernible at 24 hours. A second experiment involving the chronic administration of TSH (i‐p 0.25 IU twice daily) resulted in a cumulative response with a statistically significant progressive decrease in the mean percent of statin‐positive nuclei at 5 and 10 days, returning to near normal values 5 days following the cessation of treatment. Determination of the nuclear optical density of the statin reaction product by image analysis techniques revealed that a single injection of TSH resulted in a rapid disappearance of the statin nuclear protein. This result suggests that the disappearance of statin in the nucleus appears to reflect the event of cells leaving the nondividing quiescent state to resume the cell cycle traverse following the administration of TSH. The disappearance of statin appears as an early nuclear event that parallels the earliest known cytoplasmic pinocytotic response to TSH in the rat thyroid follicle cell.
Parathyroid hormone‐related protein production by primary cultures of mammary epithelial cells
Authors: Ferrari S. L. , Rizzoli R. , Bonjour J. P.
Published: February 2005

Parathyroid hormone‐related protein (PTHrP) plays a major role in the pathogenesis of malignant hypercalcemia, but has also been found in fetal and adult nonneoplastic tissues. Among them, lactating mammary gland was shown to produce PTHrP, and high levels of PTHrP were measured in milk. However, the regulation of PTHrP production by breast cells is still unknown. Primary cultures of mammary cells isolated from rat lactating glands were grown on collagen gels in an insulin/epidermal growth factor (EGF)‐supplemented medium. Under these conditions, mammary cells displayed an epithelial phenotype and their number increased more than twofold after 1 week in culture. At that time, the cells were capable of producing immunoreactive PTHrP (range: 25 to 150 pg/105 cells × 24 h) and PTH‐like bioactivity, as indicated by a 60% increase in cyclic adenosine monophosphate (cAMP) production induced by mammary epithelial cell conditioned medium in the PTH‐responsive osteoblast‐like UMR‐106 cell line. When cell proliferation was hindered by lowering plating density, by removing medium supplements, or by adding transforming growth factor (TGF)‐β, a well‐known autocrine inhibitor of mammary epithelial cell growth. PTHrP production was increased. In contrast, the omission of EGF or addition of specified anti‐EGF antibodies decreased PTHrP production. In conclusion, primary cultures of mammary epithelial cells isolated from lactating rat were shown for the first time to produce PTHrP in vitro. This production was higher in the presence of EGF and could be modulated by cell growth rate.
Expression of exogenous c‐myc oncogene does not initiate DNA synthesis in primary rat hepatocyte cultures
Authors: Skouteris George G. , Kaser Matthew R.
Published: February 2005

Cultured hepatocytes from adult rats stimulated with combinations of growth factors enter into S phase but do not undergo multiple rounds of DNA synthesis nor mitosis. We have examined the potential of an introduced oncogene to induce alterations in the DNA synthetic activity of the cultured hepatocytes in response to epidermal growth factor (EGF). Overexpression of c‐myc did not initiate significant DNA synthesis in rat hepatocyte cultures alone, although it cooperated with added EGF to super‐induce thymidine incorporation into DNA. From our results, it is suggested that EGF is also necessary to initiate hepatocyte DNA synthesis probably by inducing a battery of cell cycle‐related genes if incubated with c‐myc transfected cultures for only 5 hours. Hepatocyte polypeptides reacting with antiMYC antisera were found to migrate between 55–67 KDa in SDS‐PAGE; only the 64–67 KDa species were found to be phosphorylated, and the observed size heterogeneity may be due to proteolytic degradation or may reflect presently unknown posttranslational modifications.
Differential regulation of the expression of transforming growth factor‐β mRNAs by growth factors and retinoic acid in chicken embryo chondrocytes, myocytes, and fibroblasts
Authors: Jakowlew Sonia B. , Cubert Jeremy , Danielpour David , Sporn Michael B. , Roberts Anita B.
Published: February 2005

Transforming growth factor‐beta (TGF‐β) autoregulates its expression in several mammalian cell types. We now report that addition of TGF‐βs 1, 2, and 3 to primary chicken embryo cells differentially affects expression of the messenger RNAs for the different TGF‐β isoforms depending on the cell type. In cultured sternal chondrocytes, addition of TGF‐βs 1, 2, or 3 results in an increase in the steady‐state levels of the messenger RNAs for TGF‐βs 2 and 3, but does not change expression of TGF‐β4 mRNA. In contrast, in cultured cardiac myocytes, addition of TGF‐βs 1, 2, or 3 results in an increase in expression of TGF‐βs 3 and 4 mRNAs, but does not change expression of TGF‐β2 mRNA. Moreover, expression of TGF‐βs 2, 3, and 4 mRNAs is not affected by addition of any of the TGF‐βs to fibroblasts. Addition of platelet‐derived growth factor (PDGF), epidermal growth factor (EGF), or interleukin‐1 (IL‐1) to these chicken cells also has differential effects on expression of the different TGF‐β mRNAs depending on the cell type. Retinoic acid also has contrasting effects on chondrocytes and myocytes either increasing or decreasing, respectively, expression of TGF‐βs 2 and 3 mRNAs and TGF‐β2 protein. Our results indicate a complex pattern of regulation of the different TGF‐β genes by themselves as well as by PDGF, EGF, IL‐1, dexamethasone, TPA, and retinoic acid in chicken embryo cells.
Epidermal growth factor stimulates cAMP accumulation in cultured rat cardiac myocytes
Authors: Yu Yiming , Nair Bipin G. , Patel Tarun B.
Published: February 2005

We have previously shown that epidermal growth factor (EGF) augments cAMP accumulation in the heart and stimulates cardiac adenylyl cyclase via a G protein mediated mechanism (Nair et al., 1989). More recently, employing an antibody against the carboxy‐terminus decapeptide of Gsα, we have demonstrated that Gsα mediates the effects of EGF on cardiac adenylyl cyclase (Nair et al., 1990). Since the heart comprises of a variety of cell types, the purpose of the studies presented here was to determine whether or not the effects of EGF on adenylyl cyclase were mediated in cardiac myocytes or noncardiomyocytes. Therefore, cultures of ventricular cardiomyocytes and noncardiomyocytes from neonatal rat hearts were established and characterized. Apart from the differences in cellular morphology, cardiomyocytes but not the noncardiomyocytes employed in our studies expressed the α‐ and β‐myosin heavy chain (MHC) mRNA and the β‐MHC protein. Additionally, as described previously, treatment of cardiomyocytes with thyroid hormone increased α‐MHC mRNA and decreased the expression of β‐MHC mRNA, indicating that the cardiomyocytes employed in our studies were responding in a physiologically relevant manner. EGF in a time‐dependent manner increased cAMP accumulation in the cardiomyocytes but not in noncardiomyocytes. Maximum and half‐maximum effects were observed at 100 nM and 2 nM concentrations of EGF, respectively. As determined by the presence of immuno‐reactive EGF receptors and tyrosine phosphorylation of the 170 kDa protein in membranes of cardiomyocytes and noncardiomyocytes, both the cell populations contained functional EGF receptors. Therefore, the differential effects of EGF on cAMP accumulation in the two cell populations appear to be due to differential coupling of the EGF receptors to the adenylyl cyclase system rather than the absence of EGF receptors in noncardiomyocytes. Consistent with our previous findings in isolated membranes and perfused rat hearts, EGF‐elicited increase in cAMP accumulation in cardiomyocytes did not involve activation of β‐adrenoreceptors and was abolished by prior treatment of cells with cholera toxin. Overall, our findings demonstrate that EGF‐elicited increase in cAMP accumulation in the heart is the reflection of changes in cAMP content of cardiomyocytes and not noncardiomyocytes.
Tunicamycin reduces Na+‐K+‐pump expression in cultured skeletal muscle
Authors: Alboim Sandra V. , Bak Asia , Sampson Sanford R.
Published: February 2005

The purpose of this study was to examine effects of tunicamycin (TM), which inhibits core glycosylation of the β‐subunit, on functional expression of the Na+‐K+ pump in primary cultures of embryonic chick skeletal muscle. Measurements were made of specific‐[3H]‐ouabain binding, ouabain‐sensitive 86Rb uptake, resting membrane potential (Em), and electrogenic pump contribution to Em (Ep) of single myotubes with intracellular microelectrodes. Growth of 4–6‐day‐old skeletal myotubes in the presence of TM (1 μg/ml) for 21–24 hr reduced the number of Na+‐K+ pumps to 60–90% of control. Na+‐K+ pump activity, the level of resting Em and Ep were also reduced significantly by TM. In addition, TM completely blocked the hyperpolarization of Em induced in single myotubes by cooling to 10°C and then re‐warming to 37°C. Effects of tunicamycin were compared with those of tetrodotoxin (TTX; 2 x 10−7 M for 24 hr), which blocks voltage‐dependent Na+ channels. TM produced significantly greater decreases in ouabain‐binding and Em than did TTX, findings that indicate that reduced Na+‐K+ pump expression was not exclusively secondary to decreased intracellular Na+, the primary regulator of pump synthesis in cultured muscle. Similarly, effects of TM were significantly greater than those of cycloheximide, which inhibits protein synthesis by 95%. These findings demonstrated that effects were not due to inhibition of protein synthesis. We conclude that glycosylation of the Na+‐K+ pump β‐subunit is required for full physiological expression of pump activity in skeletal muscle.
Changes in pyridine and adenine nucleotide levels in friend erythroleukaemia cells during growth and differentiation
Authors: Caldini Riccardo , Chevanne Marta , Magnelli Lucia
Published: February 2005

Pyridine and adenine nucleotide levels were measured in Friend erythroleukaemia cells (FELC) stimulated to growth and induced to differentiate by hexamethylene bisacetamide (HMBA) and N'‐methylnicotinamide (N'‐MNAM). A threeto fourfold increase in the NADP(H) was found to parallel cell growth stimulation in both the presence and absence of differentiation inducers. NAD(H) increased about twofold in control and to a minor extent in HMBA‐treated FELC but did not vary significantly in N'‐MNAM‐treated cells. ATP was significantly higher in control cells stimulated to growth than in resting ones, but it did not vary in inducer‐treated cells. These data confirm the relationship between high NADP(H) levels and cell resumption to growth; moreover they show that NAD(H) pool reduction and NAD/NADH ratio rise are associated with the process of FELC differentiation. The activities of NAD pyrophosphorylase and NAD kinase are much more enhanced in growth‐stimulated FELC than in resting ones. On the other hand transition from the quiescent to the proliferative state was accompanied by a decrease in the activity of poly(ADP‐ribose) polymerase. A decrease in poly(ADP‐ribose) polymerase activity was also found in differentiated cells in contrast to Controls. © 1992 Wiley‐Liss, Inc.
The fibrinogen anion‐binding exosite of thrombin is necessary for induction of rises in intracellular calcium and prostacyclin production in endothelial cells
Authors: Ngaiza Justinian R. , Manley Geoffrey , Grulich‐Henn Jürgen , Krstenansky John L. , Jaffe Eric A.
Published: February 2005

Thrombin stimulation of prostacyclin (PGI2) synthesis by cultured human umbilical vein endothelial cells (HUVEC) requires the active site of thrombin and involves rapid and transient rises in cytoplasmic free calcium [Ca2+]i. In this study, we investigated whether or not the anion‐binding exosite for fibrinogen recognition of thrombin (which confers certain substrate specificities) is also necessary for the induction of rises in [Ca2+]i and PGI2 production. Thrombin variants which lack either the catalytic site (DIP‐α‐thrombin) or anion‐binding exosite (γ‐thrombin) either alone or in combination failed to induce rises in [Ca2+]i or PGI2 production in HUVEC. To further study the role of the anion‐binding exosite of thrombin in the activation of HUVEC, COOH‐terminal fragments of hirudin were used. This portion of hirudin interacts with the anion‐binding exosite of thrombin and inhibits thrombin‐induced fibrinogen coagulation while leaving the catalytic activity of thrombin intact. A 21‐amino acid COOH‐terminal peptide of hirudin (Nα‐acetyldesulfato‐hirudin45–65 or Hir45–65) inhibited thrombin‐induced (0.5 U/ml) rises in [Ca2+]i and PGI2 production with IC50 of 0.13 and 0.71 μM, respectively. Similar results were obtained using shorter hirudin‐derived peptides.

Thus, the fibrinogen anion‐binding exosite of thrombin is required for α‐thrombin‐induced rises in [Ca2+]i and PGI2 production in HUVEC. © 1992 Wiley‐Liss, Inc.
In vitro paracrine regulation of human keratinocyte growth by fibroblast‐derived insulin‐like growth factors
Authors: Barreca Antonina , de Luca Michele , Monte Patrizia Del , Bondanza Sergio , Damonte Giulio , Cariola Gianni , di Marco Eddi , Giordano Giulio , Cancedda Ranieri , Minuto Francesco
Published: February 2005

Human keratinocytes isolated from a skin biopsy and cultured in vitro on a feeder‐layer of irradiated fibroblasts reconstitute a stratified squamous epithelium suitable for grafting onto patients suffering from large burn wounds. Since conditioned medium from 3T3‐J2 cells can partially substitute for the intact feederlayer, we studied the possible involvement of insulin‐like growth factors acting in a paracrine fashion. IGFs were measured (after Sephadex G‐50 gel‐chromatography in acid conditions) in media conditioned by a feeder‐layer of lethally irradiated 3T3‐J2 fibroblasts on which keratinocytes were grown. Immunoreactive (IR) IGF‐I, IGF‐II, and IGF binding activity were present in the medium conditioned by the feeder‐layer. The medium conditioned by keratinocytes showed nearly undetectable amounts of IR IGF‐I and IGF‐II, suggesting that keratinocytes are unable to synthesize IGFs peptides. Recombinant IGF‐I and IGF‐II, and conditioned medium from 3T3‐J2 cells, caused a dose‐dependent increase of 3H‐thymydine incorporation in cultured keratinocytes. The stimulatory effect of IGF and of 3T3‐J2 conditioned medium was inhibited by the MoAb Sm 1.2, which recognizes both IGF‐I and IGF‐II but not insulin, and by the MoAb αIR‐3, which is a specific antagonist of type‐1 IGF receptor. Fetal mouse‐derived 3T3‐J2 cells and adult human skin fibroblasts were equally able to sustain keratinocyte growth and in both cases addition of Sm 1.2 MoAb causes a 50% decrease in the keratinocyte number. When the non‐IGF‐producing BALB/c 3T3 cells were used as a feederlayer, the keratinocytes number was similar to that observed with 3T3‐J2 and with human fibroblasts plus the Sm 1.2 MoAb. IGF‐I and IGF‐II restored the BALB/c 3T3 growth promoting activity to the level of 3T3‐J2 and of normal human fibroblasts. Our results suggest that fetal mouse 3T3‐J2 and human fibroblasts synthesize IGF peptides, while keratinocytes do not. Fibroblast‐derived IGFs stimulate keratinocyte growth in a paracrine fashion, suggesting their role in the regulation of keratinocyte proliferation in skin growth and in wound healing. © 1992 Wiley‐Liss, Inc.
Lithium stimulation of HPP‐CFC and stromal growth factor production in murine dexter culture
Authors: McGrath H. Elizabeth , Wade Philip M. , Kister V. Kay , Quesenberry Peter J.
Published: February 2005

We have previously reported that the addition of lithium chloride (LiCl) to murine Dexter cultures results in increased numbers of progenitor and mature hematopoietic cells of the granulocyte, macrophage, and megakaryocyte lineages. We now report the effect of various levels of LiCl on the high proliferative potential colony‐forming cell (HPP‐CFC) in Dexter culture and on the induction of growth factors from Dexter stromal cells. LiCl (4 mEq/L) stimulated supernatant HPP‐CFC for the first 4 weeks of culture (150–275%), and stimulated stromal HPP‐CFC at week 3 (170–222%). Higher levels of lithium (8 and 12 mEq/L) selectively stimulated supernatant HPP‐CFC, macrophage, and eosinophil production, whereas granulocytes and granulocyte‐macrophage colony‐forming cells (CFU‐C) were inhibited. mRNA expression was evaluated from week 4 Dexter cultures that received a pulse or continuous exposure to lithium and had received either 0 or 1,100 cGy irradiation. Four mEq/L LiCl stimulated increased expression of G‐CSF, GM‐CSF, IL‐6, and, in the nonirradiated stroma continuously exposed to lithium, CSF‐1 mRNA. In general, the higher levels of lithium stimulated increased mRNA expression for these same growth factors mRNA for the recently described Steel factor was decreased with increasing levels of lithium added to either normal or irradiated stroma. Bioassays of conditioned medium (cm) from irradiated cultures against the FDC‐P1 and T1165 cell lines indicated cytokine activity, which was blocked by antibodies to GM‐CSF and IL‐6, respectively. Altogether these data show that lithium stimulates Dexter HPP‐CFC, and this stimulation appears to be mediated by multiple growth factors that are induced from stromal cells. © 1992 Wiley‐Liss, Inc.
Reactive oxygen species and human spermatozoa: Analysis of the cellular mechanisms involved in luminol‐ and lucigenin‐dependent chemiluminescence
Authors: Aitken R. John , Buckingham Donna W. , West Katrine M.
Published: February 2005

We have shown that human spermatozoa generate and release reactive oxygen species that can be detected by chemiluminescence techniques. Analysis of the cellular mechanisms responsible for this activity suggests that the probe, luminol, undergoes an intracellular dioxygenation reaction mediated by hydrogen peroxide and a sperm peroxidase located within the acrosome. Support for this model included the following observations: (1) the luminol‐dependent signal could be suppressed with peroxidase inhibitors, phenylhydrazine and sodium azide; (2) this suppression could be reversed by the addition of an azide‐insensitive peroxidase, horse radish peroxidase (HRP); (3) inhibition of intracellular superoxide dismutase (SOD) with potassium cyanide (KCN) suppressed the luminol signal; (4) peroxidase activity could be detected in purified populations of human spermatozoa with 3,3′,5,5′ tetramethylbenzidine (TMB); (5) this peroxidase was active at the pH prevailing within the acrosomal vesicle; and (6) peroxidase activity and luminol‐dependent chemiluminescence were minimal in spermatozoa exhibiting a congenital absence of acrosomes. Human spermatozoa could also generate lucigenin‐dependent chemiluminescent signals that could neither be suppressed with peroxidase inhibitors nor enhanced by the addition of peroxidase. However, these signals could be enhanced by suppression of intracellular SOD with KCN or inhibited by exogenous SOD, suggesting that lucigenin was responding to superoxide anion released into the extracellular space. The ability of chemiluminescent techniques to detect and discriminate the production of superoxide and hydrogen peroxide by spermatozoa should facilitate the further analysis of reactive oxygen species as mediators of normal and abnormal human sperm function. © 1992 Wiley‐Liss, Inc.
Effect of camptothecin on mitogenic stimulation of human lymphocytes: Involvement of DNA topoisomerase I in cell transition from G0 to G1 phase of the cell cycle and in DNA replication
Authors: Bruno Silvia , Giaretti Walter , Darzynkiewicz Zbigniew
Published: February 2005

The possible involvement of DNA topoisomerase I in cell transition from G0 to G1 and in progression through the cell cycle was studied by estimating the ability of human peripheral blood lymphocytes to undergo mitogenic stimulation in the presence of the topoisomerase I inhibitor camptothecin (CAM). Exposure of quiescent G0 lymphocytes to up to 3 μM CAM for 24 h had no significant effect on their ability to subsequently undergo mitogenic stimulation in the presence of phytohemagglutinin (PHA); higher doses of CAM, although not immediately cytotoxic, impaired the mitogenic response. Stimulation of lymphocytes with PHA in the presence of ≤ 1.5 μM CAM resulted in unperturbed transition of these cells from G0 to G1 characterized as an increase in cellular rRNA content, appearance of interleukin‐2 receptor, and, after removal of CAM, response to interleukin‐2 by entering S phase of the cell cycle. However, lymphocytes were prevented from entering S phase in the presence of CAM at a concentration of ≥30 nM, and their rate of progression through S was minimal even at CAM concentration as low as 3 nM. When cycling lymphocytes (48 h after stimulation by PHA) were treated with CAM, the cell progression through S and G2 was also very sensitive to the inhibitor: the cells were “frozen” in S and G2 at ≥ 6 nM CAM. These cells died within 24 h; their selective loss from the cultures (with only G0/G1 cells remaining) coincided with the appearance of cells with fractional DNA content, typical of apoptotic cells. Human lymphocytic leukemic MOLT‐4 cells were arrested in S and G2 at ≥ 7.5 nM CAM. Thus, progressions through S and G2 of both normal and leukemic lymphocytes were perturbed at approximately two orders of magnitude lower CAM concentration than the G0 to G1 transition. These data suggest that DNA replication and chromosomal events during G2 are more sensitive to inhibition of DNA topoisomerase I, compared with the early events of lymphocyte stimulation, which involve activation and transcription of numerous genes associated with the G0 to G1 transition. The antitumor properties of CAM may be related to its high cytostatic/cytotoxic activity toward cycling cells and relative resistance of cells in G0 or undergoing transition from G0 to G1. © 1992 Wiley‐Liss, Inc.
An ultrastructural study of thrombomodulin endocytosis: Internalization occurs via clathrin‐coated and non‐coated pits
Authors: Conway Edward M. , Boffa Marie‐Claire , Nowakowski Barbara , Steiner‐Mosonyi Marta
Published: February 2005

The regulation of thrombomodulin (TM) expression has been reported to occur by several mechanisms. We have examined constitutive internalization of TM using immunofluorescent and electron microscopic (EM) methods. A cell model was developed to study this process by introducing TM DNA into COS‐7 cells for expression. The recombinant TM was determined to behave similarly to native TM from human umbilical vein endothelial cells (HUVEC) with respect to Mr and cell surface functional activity. The transfected cells expressed 8–100‐fold more functional TM per cell than HUVEC. Immunofluorescent studies on these cells indicated that anti‐TM antibody‐TM complex was internalized in a time‐ and temperature‐dependent manner, with internalization detectable within 10 minutes. When the cells were incubated at 4°C with gold‐labelled anti‐TM antibody, most of the gold particles were surface bound and detected by EM as individual particles or clusters of 2 or 3 particles. Initiation of endocytosis for 10 to 60 minutes resulted in a redistribution of gold particles into small clusters predominantly in non‐coated pits and rarely in clathrin‐coated pits, subsequently in early endosomes, multivesicular bodies, and lysosomes. Similar studies were performed with gold‐conjugated thrombin, demonstrating a similar route of intracellular processing. These studies provide ultrastructural evidence that the process of endocytosis of TM involves the participation of both clathrin‐coated and noncoated pits and vesicles, but that the latter process predominates. Further structure/function studies are indicated using our cell model, since defects in the endocytic pathway of this important anticoagulant receptor may contribute to the development of thromboembolic disease. © 1992 Wiley‐Liss, Inc.
Lanthanum influx into cultured human keratinocytes: Effect on calcium flux and terminal differentiation
Authors: Pillai Sreekumar , Bikle Daniel D.
Published: February 2005

Trivalent cation lanthanum (La) binds to calcium binding sites of cells and either mimics the properties of calcium or inhibits the effects of calcium by displacing calcium from its binding sites. Extracellular calcium induces differentiation of human epidermal keratinocytes in culture, in part by increasing the intracellular calcium levels (Cai). Therefore, in this study we determined the effect of La on differentiation and intracellular calcium levels of keratinocytes. We observed that La inhibited the production of cornified envelopes, a marker for terminal differentiation of keratinocytes. La inhibited the calcium requiring envelope cross‐linking enzyme, transglutaminase, in a direct manner, presumably, by displacing calcium from its binding site on the enzyme. La inhibited the influx and the efflux of 45 Ca from keratinocytes. Paradoxically, extracellular La appeared to increase the Cai levels of keratinocytes as measured by the fluorescent probe indo‐1. However, subsequent experiments revealed that indo‐1 bound La with a higher affinity than Ca and emitted fluorescence in the same wavelength as the Ca bound form. Using this probe, we observed that La enters keratinocytes in a dose‐dependent fashion and achieves concentrations exceeding 80 nM when the external La concentration is raised to 300 μM. This fully accounted for the apparent increase in Cai when La was added to the cells. Treatment of cells with ionomycin increased indo‐1 fluorescence maximally in the presence of La indicating influx of La via this Ca specific ionophore. Our results indicate that La enters cells and inhibits calcium mediated keratinocyte differentiation both by blocking Ca influx and by blocking calcium regulated intracellular processes such as transglutaminase directed cornified envelope formation. © 1992 Wiley‐Liss, Inc.
Developmental regulation of insulin‐like growth factor binding protein production: Studies in fetal, postnatal, and pregnant sheep
Authors: Fowlkes J. , Freemark M.
Published: February 2005

To assess the roles of developmental factors in the regulation of sheep IGFBP production at the cellular level, we characterized and compared the IGFBPs released by fetal, postnatal, and maternal sheep skin fibroblasts in culture with those in fetal, postnatal, and maternal sheep plasma. Sheep fibroblasts produced seven IGFBPs: a 36.5–41 kDa protein induced in vitro by IGF‐I, likely representing oIGFBP‐3; a 28.5 kDa protein that reacted with antisera to human IGFBP‐2, likely representing oIGFBP‐2; 25 and 27 kDa proteins induced in fetal fibroblasts by IGF‐I; a 22 kDa protein that was inhibited by IGF‐I, likely representing oIGFBP‐4; and 21 and 23 kDa proteins labelled only by IGF‐II, suggesting their similarities to IGFBP‐6. The developmental pattern of IGFBP production by sheep fibroblasts in culture was similar in several respects to that observed in sheep plasma. For example, relative amounts of the 21, 22, and 28.5 kDa IGFBPs exceeded that of the 36.5–41 kDa protein in early fetal fibroblast conditioned media and in fetal plasma, while the relative concentrations of the 36.5–41 kDa protein increased markedly during the perinatal period. Sheep plasma differed, however, in two major respects from fibroblast conditioned media: First, fetal, and to a far lesser extent maternal, plasma contained a 200 kDa IGF‐II‐selective BP, likely to be the circulating form of the IGF‐II receptor; and second, plasma, unlike conditioned media, contained a 26 kDa IGFBP, likely to be oIGFBP‐1. The results of our studies suggest that the production and release of IGFBPs by isolated sheep fibroblasts is regulated by developmental factors operative under in vitro culture conditions. The differences in the relative levels of IGFBPs in conditioned media from fetal, postnatal, and maternal sheep fibroblasts resemble in several respects the differences in the relative concentrations of the various IGFBPs in fetal, postnatal, and maternal sheep plasma. Thus, sheep fibroblasts provide a useful though imperfect model system by which to examine the nutritional and hormonal regulation of sheep IGFBP production at various developmental stages. © 1992 Wiley‐Liss, Inc.
Release of iron by human retinal pigment epithelial cells
Authors: Hunt Richard C. , Davis Alberta A.
Published: February 2005

Retinal pigment epithelial cells, which form one aspect of the blood‐retinal barrier, take up iron in association with transferrin by a typical receptor‐mediated mechanism (Hunt et al., 1989. J. Cell Sci. 92:655–666). This iron is dissociated from transferrin in a low pH environment and uptake is sensitive to agents that inhibit endosomal acidification. The dissociated iron enters the cytoplasm as a low molecular weight (< 10 kD) component and subsequently binds to ferritin. No evidence for recycling of iron in association with transferrin was found. Nevertheless, much of the iron that is taken up is recycled to the extracellular medium, primarily from the low molecular weight pool. This release of iron is not sensitive to inhibitors of energy production or of vesicular acidification but is increased up to a maximum of about 40% of the total 55Fe incorporated when cells are incubated with serum or the medium is changed. When a short loading time for 55Fe from 55Fe‐transferrin is used (i.e., when the low molecular weight pool is proportionately larger), a much larger fraction of the cell‐associated radiolabel is released than when longer loading times are used. The data suggest that a releasble intracellular iron pool is in equilibrium with the externalized material. The released iron may be separated into a high and a low molecular weight component. The former is similar on polyacrylamide gel electrophoresis to ferritin although it cannot be immune precipitated by anti‐ferritin antibodies. The low molecular weight 55Fe which is heterogeneous in nature can be bound by external apo‐transferrin and may represent a form that can be taken up by cells beyond the blood‐retinal barrier. © 1992 Wiley‐Liss, Inc.
Interleukin 1: The patterns of translation and intracellular distribution support alternative secretory mechanisms
Authors: Stevenson Frazier T. , Torrano Frank , Locksley Richard M. , Lovett David H.
Published: February 2005

Interleukin‐1 (IL‐1) is synthesized as a 31 kDa precursor protein, whose multiple extracellular activities are attributed to receptor binding of a processed, carboxyterminal 17 kDa peptide. Unlike other secreted proteins, the IL‐1 precursor lacks a hydrophobic leader sequence and is not found in organelles composing the classical secretory pathway. In order to further clarify the intracellular processing of IL‐1, we studied its site of synthesis in human monocytes. Secreted and integral membrane proteins are translated on membrane‐bound polyribosomes, while intracellular proteins are translated on free polyribosomes. Free and membrane‐bound polysomes were isolated from Lipid A‐stimulated monocyte lysates and immunoblotted using antibodies specific to the N‐terminal regions of the IL‐1α and β precursors. Free polysome fractions showed multiple small bands consistent with nascent peptide chains; membrane‐bound polysomes yielded no detectable IL‐1. Polysome fractions were then analyzed by immunoelectron microscopy; nascent IL‐1α and β peptide chains were readily seen emerging from cytoskeletal‐associated free polyribosomes, but not membrane‐bound polyribosomes. Electron microscopic in situ hybridization revealed IL‐1 mRNA chains attached to cytoskeletal‐associated free, but not membrane‐bound polyribosomes. The intracellular distribution of the fully synthesized IL‐1β precursor was studied in human mesangial cells (HMC), whose cytoskeletal organization is more readily evaluated than that of monocytes. Dual immunofluorescence microscopy of these cells revealed a complex intracellular distribution of the fully synthesized 31 kDa IL‐1 precursors. IL‐1 was asymmetrically distributed between cytosolic, microtubule, and nuclear compartments, without association with actin or intermediate filaments. This demonstration of the sites of IL‐1 synthesis and patterns of intracellular distribution provide further evidence for an extracellular release mechanism which is clearly distinct from the classical secretory pathway. © 1992 Wiley‐Liss, Inc.
Changes in responsiveness of rat tracheal epithelial cells to transforming growth factor‐β1 with time in culture
Authors: Rundhaug Joyce E. , Gray Thomas , Steigerwalt Ronald W. , Nettesheim Paul
Published: February 2005

Primary rat tracheal epithelial (RTE) cell cultures have previously been shown to be highly sensitive to growth inhibition by transforming growth factor‐β1 (TGF‐β1) when treated within 1–2 days after plating. The purpose of the present studies was to examine the effects of TGFβ1 on the growth of RTE cells as a function of time in culture. We found that the sensitivity of RTE cells to growth inhibition by TGFβ1 decreased dramatically as the cultures aged. The IC50 for inhibition of colony forming efficiency was 0.18 pM when TGFβ1 was added 24 h after cell plating. When TGFβ1 treatment was begun on day 5 of culture, the IC50 was 3–4 pM as measured by inhibition of growth (cell number) and DNA synthesis. However, when TGFβ1 was begun on day 19, the IC50 was 65 pM or > 500 pM, depending on whether inhibition of growth or DNA synthesis, respectively, was measured. TGFβ1 accelerated cell death, as measured by exfoliation of cells, and inhibited cell proliferation. The decrease in responsiveness to TGFβ1 in late cultures was shown to be dependent on culture age as well as on cell density. No evidence was found for inactivation or degradation of the added TGFβ1 by the late stage cultures. Cells subcultured from late stage primary cultures remained less responsive to TGFβ1 than subcultured cells from early cultures. Similar to its effect on proliferation, TGFβ1 down‐regulated the expression of two proliferation‐related genes, c‐myc and transforming growth factor‐α, in early but not late RTE cell cultures. On the other hand, fibronectin expression was increased by TGFβ1 by about twofold at both early and late times in culture. This indicates that the changes in TGFβ1 responsiveness with time in culture are selective, apparently affecting primarily proliferation‐related events. © 1992 Wiley‐Liss, Inc.
Influence of cyclosporine a on growth and extracellular matrix synthesis of human fibroblasts
Authors: Willershausen‐Zönnchen B. , Lemmen C. , Schumacher U.
Published: February 2005

Cyclosporine A (CyA) is a powerful nonsteroidal immunosuppressive agent used to prevent graft rejection of organ and bone marrow transplants. A major side effect observed can be attributed to the fibroblast and its functions: proliferation of fibroblasts and formation of fibrotic tissue in the gingiva (fibrous hyperplasia) and in the kidney are induced. The mechanism of both is still obscure. In order to elucidate whether these side effects are due to the drug acting on human fibroblasts itself or whether they are indirect ones mediated by factors released by lymphocytes, cultures of human gingiva fibroblasts were exposed to CyA under defined in vitro conditions. Incubation with CyA for 72 hours resulted in a dosedependent stimulation of DNA synthesis, whereas glycosaminoglycan (GAG) synthesis was slightly suppressed. Long‐term incubation (6 weeks) with 1 μg/ml CyA resulted again in stimulation of growth parameters: compared to the drug‐free control, cell number increased to 168%, incorporation of 3H‐thymidine into DNA to 143%, and overall protein content to 159%. Collagen and GAG synthesis were elevated to ∼ 120%. When corrected for cell number or cell protein content, this represents a decline in matrix synthesis, comparable to short‐term incubations. These results indicate that a direct effect of CyA on proliferation of human gingival fibroblasts is responsible for some of the observed hyperplasia. © 1992 Wiley‐Liss, Inc.
Selective regulation of β2‐adrenergic receptor gene expression by interleukin‐1 in cultured human lung tumor cells
Authors: Szentendrei Tibor , Lazar‐Wesley Eliane , Nakane Tokio , Virmani Mridulika , Kunos George
Published: February 2005

The regulation of β1‐ and β2‐adrenergic receptors (β1 AR and β2 AR) and receptor gene expression by interleukin‐1α (IL‐1α) was studied in cultured A549 human lung adenocarcinoma cells. The density and affinity of β1 AR and β2AR were analyzed by computerized curve fitting of 125I‐pindolol binding and its displacement by subtype selective antagonists. Steady state levels of receptor mRNAs were quantified by DNA excess solution hybridization assays. A549 cells in preconfluent cultures had fewer β1AR than β2AR (β1: 1.9 ± 0.3 vs. β2: 4.0 ± 0.5 fmol/mg protein, means ± SE), but lost most of their β2AR upon reaching confluency (β1: 2.7 ± 0.4, β2: 0.8 ± 0.3 fmol/mg). Incubation of preconfluent cells for 24 hr with 20 pM of human recombinant IL‐1α did not modify the density of either of the βAR subtypes. Similar incubations of confluent cells increased the density of β2AR from 0.8 ± 0.3 to 4.2 ± 0.9 fmol/mg, while the density of β1AR and the antagonist affinities of both receptors remained unaltered. The IL‐1α‐induced increase in β2AR density in confluent cells was antagonized in a concentration‐dependent manner by a recombinant protein antagonist of type I IL‐1 receptors (IC50: 0.2 nM). The IL‐1α‐induced increase in β2AR density was preceded by an increase in the steady state level of β2AR mRNA, while levels of β1AR mRNA remained unchanged. IL‐1α increased the stability as well as the rate of transcription of β2AR mRNA. These findings demonstrate for the first time that activation of type I IL‐1 receptors in A549 cells leads to a cell density‐dependent, selective upregulation of β2AR, and that the mechanism of this effect involves increased formation and stability of the β2AR message. © 1992 Wiley‐Liss, Inc.
Transforming growth factor‐β enhances calcitonin‐induced cyclic AMP production and the number of calcitonin receptors in long‐term cultures of human umbilical cord blood monocytes in the presence of 1,25‐dihydroxycholecalciferol
Authors: Mbalaviele Gabriel , Orcel Philippe , Bouizar Zhor , Jullienne Annick , De Vernejoul Marie Christine
Published: February 2005

Transforming growth factor‐β (TGF‐β) is a multifunctional polypeptide, abundant in bone, that regulates both proliferation and differentiation of a wide variety of cells, but its role in osteoclast differentiation remains controversial. We have recently shown that long‐term cultures of human cord blood monocytes, in the presence of 1,25 dihydroxycholecalciferol (1,25‐(OH)2D3), give rise to cells that express two markers of the osteoclast phenotype, namely, the vitronectin receptor (VNR) and the calcitonin receptor (CTR). TGF‐β enhanced the proportion of cells expressing the VNR.

In the present study, we investigated the effect of TGF‐β on the expression of CTR in cord blood monocytes cultured during 3 weeks in the presence of 1,25‐(OH)2D3. When added within the first 2 weeks of culture, TGF‐β (500 pg/ml) significantly decreased the cell protein content. TGF‐β alone did not stimulate basal cAMP production. The 10 nM‐sCT‐stimulated cAMP production was enhanced by increasing TGF‐β concentrations from 50 pg/ml to 1,000 pg/ml: for 500 pg/ml TGF‐β, it was 294 ± 28% vs. 140 ± 25% for control cultures (p < 0.01). The sCT dose‐response curves showed a higher cAMP production from 10−9 M to 10−7 M of sCT in the presence of 500 pg/ml TGF‐β than in control cultures. The increase was 325 ± 36% in the presence of TGF‐β and 195 ± 13% in the absence of TGF‐β, for 10−7 M sCT (p < 0.01). This effect of TGF‐β on cAMP production was not observed either when it was added to monocyte cultures the last day or 2 hours before the end of the culture or in MCF7, a human breast cancer cell line that expresses CTR. [125I]‐sCT binding studies performed on confluent cells showed similar Kd in control and TGF‐β‐treated cells. By contrast, the CTR number was significantly increased in the presence of TGF‐β: 6.1 ± 2 × 104 receptors per cell in control cultures and 28.8 ± 8.1 × 104 receptors per cell in TGF‐β‐treated cultures (p < 0,05). It is thus suggested that TGF‐β increases the number of CTR of these cells that have other features of preosteoclasts. The role of this cytokine on the process of osteoclast differentiation and in bone resorption is thus emphasized. © 1992 Wiley‐Liss, Inc.
Protein kinase C remains functionally active during TPA induced neuronal differentiation of SH‐SY5Y human neuroblastoma cells
Authors: Parrow Vendela , Nånberg Eewa , Heikkilä Jari , Hammerling Ulf , Påhlman Sven
Published: February 2005

SH‐SY5Y human neuroblastoma cells can be induced to differentiate into a neuronal phenotype by treatment with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA). In other cell systems, TPA treatment frequently leads to down‐regulation of protein kinase C (PKC). However, we now report that TPA‐treated and non‐treated SH‐SY5Y cells express PKC‐α, but not PKC‐β and PKC‐γ, mRNA. Furthermore, only a slight down‐regulation of the PKC‐α protein could be seen during prolonged treatment with 16 nM TPA, the concentration giving optimal differentiation. In contrast, a higher concentration of TPA (1.6 μM) results in a poor neuronal differentiation and a complete down‐regulation of PKC‐α. PKC‐α was rapidly translocated to the particulate fraction and remained membrane bound for at least 4 days during treatment with 16 nM TPA. In such cells a sustained increased level of the phosphorylated form of a 80,000 Dalton PKC‐substrate was found. In addition to this sustained augmented phosphorylation, administration of fresh TPA at day 4 caused a small but reproducible further increased level of phosphorylated substrate. When the PKC activity was measured by the histone phosphorylation assay a substantial fraction of the initial enzyme activity could still be detected after 4 days of TPA treatment. Taken together, the data demonstrate that PKC remains functionally active during TPA induced differentiation of SH‐SY5Y cells, which may suggest a continuous role for the enzyme during the differentiation process. © 1992 Wiley‐Liss, Inc.
Mechanism of action of amylin in bone
Authors: Tamura Tatsuya , Miyaura Chisato , Owan Ichiro , Suda Tatsuo
Published: February 2005

Amylin is a 37 amino acid peptide produced mainly by β‐cells of the endocrine pancreas. Human amylin has 43% homology with human calcitonin gene‐related peptide (CGRP) and 13% homology with human calcitonin (CT). Amylin and CGRP have been reported to have CT‐like hypocalcemic activity in vivo. To investigate the role of amylin in bone, we examined the mechanisms of action of human amylin, CGRP, and CT in osteoclasts and osteoblasts. Both human amylin and CGRP inhibited 1α, 25‐dihydroxyvitamin D3 [1α, 25(OH)2D3]‐induced bone resorption in an organ culture system, and the potencies of the two peptides were similarly ∼60‐fold lower than that of human CT. Using a recently developed procedure for preparing large numbers of osteoclast‐like multinucleated cells (MNCs) formed in co‐cultures of mouse osteoblasts and bone marrow cells in the presence of 1α, 25(OH)2D3, we found that both human amylin and CGRP stimulated cAMP production in osteoclast‐like MNCs, but only at 60‐fold higher concentrations than human CT. Specific binding of [125I]‐human CT to osteoclast‐like MNCs was detected (dissociation constant, 3 × 10−8 M; binding sites, 3 × 107 per cell). To displace the bound [125I]‐human CT from osteoclast‐like MNCs, about 170‐fold higher concentrations of human amylin and CGRP were required. No specific bindings of [125I]‐CGRP to osteoclast‐like MNCs could be detected. Human CGRP stimulated cAMP production both in established mouse osteoblast‐like cells (KS‐4) and in mouse primary osteoblast‐like cells. Amylin was a weak agonist for cAMP production in KS‐4 cells. The increment in cAMP production induced by CGRP and amylin was abolished by the addition of human CGRP(8–37), a selective antagonist for CGRP receptors. CT did not stimulate cAMP production in KS‐4 cells. Amylin, but not CT, displaced the bound [125I]‐human CGRP from rat brain membranes. These results indicate that amylin binds not only to CT receptors in osteoclast‐like MNCs but also to CGRP receptors in osteoblasts. The relative potencies of these compounds to induce cAMP production was CT > amylin ≒ CGRP in osteoclast‐like MNCs and CGRP > amylin ≫ CT in osteoblast‐like cells. © 1992 Wiley‐Liss, Inc.