bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

AbstractHuman 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

AbstractWe 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

AbstractWe 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

AbstractThe 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

AbstractThe 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

AbstractTrivalent 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

AbstractTo 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

AbstractRetinal 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

AbstractInterleukin‐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

AbstractPrimary 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

AbstractCyclosporine 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.