bluuebunny/crossref_metadata_2025_split huggingface dataset:
The stimulation of normal human melanocyte proliferation in vitro by melanocyte growth factor from bovine brain
Authors: Wilkins L. , Gilchrest B. A. , Szabo G. , Weinstein R. , Maciag T. 
Published: February 2005

AbstractCell culture conditions for the selective growth and serial propagation of normal human melanocytes from epidermal tissue are described. In addition to the presence of 2% fetal bovine serum, the human melanocyte cell culture environment contains the following growth factor supplements: epidermal growth factor (10 ng/ml), triiodothyronine (10−9 M), hydrocortisone, (5 × 10−5 M), insulin (10 μg/ml), transferrin (10 μg/ml), 7S nerve growth factor (100 ng/ml) cholera toxin (10−10 M), and bovine brain extract (150 μg/ml). The ability to establish selectively the human melanocyte in vitro has been attributed to the contrast between human epidermal keratinocytes and melanocytes for attachment to fibronectin, while the growth of the human melanocyte has been attributed to the mitogenic activity of the growth factor‐supplemented medium. Human melanocytes can be cultivated for at least 15 cumulative population doublings and are capable of [3H]‐Dopa incorporation. The growth factor‐supplemented medium contains a neutral extract from bovine brain that is a potent source of a human melanocyte mitogen. The biological activity of melanocyte growth factor is described as a heat and alkaline‐labile mitogen with an estimated molecular weight of 30,000 by gel exclusion chromatography and a weakly cationic isoelectric point. The mitogen is capable of stimulating the growth of quiescent populations of human melanocytes in vitro. The ability to isolate and propagate normal human melanocytes in vitro permitted an examination of the expression of fibronectin and tissue plasminogen activator. Human epidermal melanocytes established in culture do not contain either tissue plasminogen activator or fibronectin. In contrast, human melanoma cell lines contain immunologically detectable fibronectin and tissue plasminogen activator. The absence of tissue plasminogen activator and fibronectin in normal human melanocytes also occurs under conditions of co‐cultivation with human melanoma cells. These contrasts between normal human melanocytes and human melanoma cells may be relevant to the metastatic capabilities of human melanoma.
Membrane phospholipid asymmetry as a factor in erythrocyte‐endothelial cell interactions
Authors: Schlegel Robert A. , Prendergast Thomas W. , Williamson Patrick 
Published: February 2005

AbstractThe tendency of human erythrocytes to adhere to vascular endothelial cells was assessed as a function of the transbilayer distribution of the phospholipids of the erythrocyte membrane, using erythrocyte ghosts in which transbilayer lipid arrangement was manipulated by varying the conditions under which the ghosts were prepared. By two different assays, ghosts with symmetric lipid bilayers adhered strongly to monolayers of cultured endothelial cells, whereas ghosts with normal asymmetric membranes, like normal erythrocytes, did not. These results provide direct evidence that changes in phospholipid asymmetry can alter the tendency of erythrocytes to adhere to endothelial cells, and therefore imply that transbilayer phospholipid arrangement may influence the behavior of erythrocytes in the circulatory system and may contribute to the formation of microvascular occlusions.
Antagonistic effects of insulin and dexamethasone on glucose‐regulated and heat shock protein synthesis
Authors: Kasambalides Efthimios J. , Lanks Karl W. 
Published: February 2005

AbstractThe present study extends our previous observation (Kasambalides and Lanks, J. Cell. Physiol., 114:93–98, 1983), that dexamethasone inhibits the alterations in heat shock protein (HSP) and glucose‐regulated protein (GRP) synthesis caused by glucose deprivation. We now show that insulin, even in the presence of high extracellular glucose concentrations, will induce 95K and 82K GRP synthesis while suppressing 85K and 69K HSP synthesis. Heat shock of insulin‐treated cultures causes induction of the 82K GRP rather than the 85K and 69K HSP's. All of the insulin effects are antagonized by dexamethasone. These data suggest that the changes in GRP and HSP synthesis induced by glucose deprivation and heat shock, respectively, may reflect the operation of a normal physiological mechanism that regulates glucose metabolism.
Binding and internalization of heparin by vascular smooth muscle cells
Authors: Castellot John J. , Wong Kent , Herman Brian , Hoover Richard L. , Albertini David F. , Wright Thomas C. , Caleb Benjamin L. , Karnovsky Morris J. 
Published: February 2005

AbstractPrevious work from our laboratory has demonstrated that heparin specifically inhibits the proliferation of vascular smooth muscle cells in vivo and in vitro. In this paper, we examine the binding and mode of internalization of heparin by smooth muscle cells. For these studies, radiolabeled and fluoresceinated (FITC) heparin probes were synthesized that retained their antiproliferative capacity. Binding of 3H‐heparin to these cells occurs via specific, high‐affinity binding sites (Kd = 10−9 M, 100,000 binding sites per cell). Approximately 80% of the heparin bound to the cell surface was shed into the culture medium within 2 hr. The heparin that was left on the cell surface was internalized with biphasic kinetics. Approximately 50% of the bound material was internalized within 2 hr. After this initial rapid uptake, the rate slowed substantially, with the remaining heparin requiring 1–2 days to be internalized. Binding and uptake of FITC heparin was monitored using video image intensification fluorescence microscopy. When smooth muscle cells were exposed to FITC heparin at 4°C, a diffuse surface staining pattern was observed. After warming the cells to 37°C, intensely fluorescent vesicles were seen superimposed over the diffuse surface staining within 2 min. After 15 min at 37°C, numerous large punctate vesicles were seen inside the cell. After 2 hr these vesicles had concentrated in the perinuclear region. This pattern of uptake, when considered along with the presence of specific, high‐affinity binding sites and the initial rapid uptake of 3H‐heparin, suggests that heparin enters smooth muscle cells by both receptor‐mediated and other endocytic pathways.
Effect of heparin on vascular smooth muscle cells. I. Cell metabolism
Authors: Castellot John J. , Cochran David L. , Karnovsky Morris J. 
Published: February 2005

AbstractPrevious work from our laboratory has shown that heparin inhibits the proliferation of vascular smooth muscle cells in vivo and in vitro. The mechanism of action of this glycosaminoglycan is unknown. In this communication, we have examined the antiproliferative effect of heparin on smooth muscle and other cell types, and have investigated several aspects of heparin on smooth muscle cell metabolism. Smooth muscle and closely related cell types from several species, including human, were much more sensitive to heparin than any other cell type tested, including primary and established cell lines, normal and transformed cell pairs, fibroblasts epithelial, and endothelial cells. Flow microfluorimetric analysis of cell cycle distribution indicated that heparin blocked either the Go → S transition or a very early S‐phase event in smooth muscle cells. Heparin rapidly inhibited DNA and RNA synthesis, but did not affect the rate of protein synthesis. The decrease in nucleic acid synthesis could be accounted for by an inhibition of thymidine and uridine uptake. Interestingly, heparin did not block amino acid or glucose transport. Although no change in the overall rate of protein synthesis was observed in the presence of heparin, we noted at least two changes in the synthesis of specific proteins by smooth muscle cells: two 35,000‐dalton proteins which appeared in the culture medium of heparin‐treated cells, and the transient disappearance of a 48,000‐dalton protein in the substrate attached material of smooth muscle cells exposed to heparin. The role of the observed changes in smooth muscle cell metabolism is yet to be determined, but they may provide valuable clues to the molecular mechanisms controlling the antiproliferative activity of heparin.
Effect of potassium depletion of cells on their sensitivity to diphtheria toxin and pseudomonas toxin
Authors: Sandvig Kirsten , Sundan Anders , Olsens Sjur 
Published: February 2005

AbstractWhen Vero cells were depleted of potassium, the cells were protected against diphtheria toxin, Potassium deletion of Vero cells strongly reduced the binding of the toxin to cell surface receptors. Likewise, potassium depleted L‐cells were protected against pseudomonas toxin. Diphtheria toxin binding was completely restored upon addition of potassium to the cells. This restoration was not prevented by inhibition of protein synthesis by cycloheximide. When cells were depleted of potassium in the presence of metabolic inhibitors, and then treated with diphtheria toxin, protein synthesis was reduced to the same extent as in cells with normal intracellular level of potassium. The results indicate that potassium depletion of Vero cells reduces the ability of the cells of bind diphtheria toxin by an ATP requiring process, and that binding, endocytosis and transfer of diphtheria fragment A across the membrane may occur at low intracellular levels of potassium.
Insulin binding and vesicular ingestion in capillary endothelium
Authors: Solenski Nina J. , Williams Stuart K. 
Published: February 2005

AbstractCapillary endothelium can actively regulate vascular permeability of various serum proteins. Hormones such as insulin must interact with this capillary barrier in order to reach their respective target tissues. We have studied the binding and subsequent internalization of 125I‐insulin in both native (freshly isolated) and primary cultured capillary endothelium derived from rat epididymal fat pads. Insulin association with the endothelium, internalization and degradation differed between freshly isolated and primary cultured capillaries. Specific binding in freshly isolated and cultured capillaries was temperature dependent, and was competitively inhibited in the presence of unlabelled insulin. Primary cultures of capillaries grown to confluence did not exhibit specific binding of insulin. Despite the lack of specific receptors for insulin, cultured cells vesicularly internalized insulin. Greater than 50% of the total associated insulin was not degraded by cultured endothelium. Morphological examinations using ferritin labelled insulin localized insulin associated to the capillary endothelial cell membrane and sequestered within pinocytotic vesicles. Incubation of freshly isolated capillaries with insulin stimulated the fluid phase endocytosis of 14C‐sucrose; however, insulin had no effect on fluid phase endocytosis in cultured capillaries. These results indicate that capillary endothelium, isolated from rat epididymal fat, exhibit specific receptors for insulin. Binding of insulin to the capillary membrane is followed by internalization into cytoplasmic vesicles and partial degradation.
Fibroblast products adsorb to the culture substrate and stimulate growth of human umbilical vein endothelial cells
Authors: Kan Mikio , Kato Miwa , Yamane Isao 
Published: February 2005

AbstractHuman umbilical vein endothelial cells grew with a doubling time of approximately 20 hr in medium conditioned by human diploid fibroblasts and supplemented with 10% fetal bovine serum, whereas the cells did not grow substantially in the non‐conditioned serum supplemented medium. Production of the fibroblast‐derived activity required the presence of insulin, EGF, or PDGF. The fibroblast derived‐factor adsorbed to native culture dishes or dishes coated with gelatin and collagen. The adsorbed activity was resistant to treatment with 1% Triton X‐100, and was abolished by treatment with serine proteases. Further, the extracellular matrix produced by the fibroblasts also showed growth‐stimulating activity. Fibroblast‐derived factors may play a role in vascularization processes during wound healing, inflammation and normal development.
Release of galactosyltransferase from peritoneal macrophages during acute inflammation
Authors: Hopper K. , Hollister W. , Davey R. , Semler A. 
Published: February 2005

AbstractPeritoneal cells harvested from mice injected with Salmonella enteritidis or thioglycollate released large amounts of galactosyltransferase (GT), but not sialyltransferase, into their culture supernatants. Maximum release of GT (using ovalbumin as acceptor) occurred from cells harvested 2–4 days after primary injection, but little GT was released from cells elicited by a secondary injection of salmonella or ovalbumin in sensitised mice or during intraperitoneal allogeneic reactions. Enzyme release in culture did not parallel GT levels in serum. Most enzyme was released by large, poorly adherent, macrophageenriched, Fc receptor‐bearing peritoneal cells of low density. Normal monocytes, bone marrow cells, and platelets also produced large amounts, and normal spleen cells or polymorphonuclear leukocytes moderate amounts, of GT. Lymphocytes, dead cells, mast cells, red blood cells, or whole populations of lymph node and thymus cells released very low levels of enzyme. Very little GT was bound to the cell surface and was not passively absorbed from serum or platelets. Release of GT was prevented at 4°C but was not markedly affected by a variety of metabolic inhibitors except pretreatment of the cells with thrombin, which increased release and trypsin which decreased release.
A role for soluble cAMP phosphodiesterases in differentiation of 3T3‐L1 adipocytes
Authors: Elks M. L. , Manganiello V. C. 
Published: February 2005

AbstractDifferentiatio of 3T3‐L1 adipocytes, monitored by accumulation of neutral lipid and by increase in α‐giycerophosphate dehydrogenase activity, is accelerated by incubation of confluent 3T3‐L1 fibroblasts in media containing insulin, dexamethasone and isobutylmethylxantine (IBMX). IBMX inhibits cyclic nucleotide phosphodiesterases as well as the binding of adenosine to its recetor. Agents with relatively specific effects were utilized to examine the role of IBMX in differentiation. Ro 20–1724, a selective inhibitor of soluble cAMP phosphodiesterase activities, was as effective as IBMX in increasing α‐glycerophosphate dehydrogenase activity and fat deposition. Neither cilostamide, which inhibits particulate but not soluble cAMP phosphodiesterase activities, 8‐phenyltheophylline, an adenosine receptor antagonist with little inhibitory effect on phosphodiesterase activities, nor N6‐(R phenyl‐isopropyl) adenosine (PIA), a potent adenosine receptor agonist, were effective in promoting differentiation. In addition, we find that maximal increases in α‐glycerophosphate dehydrogenase activity and lipid accumulation were observed when differentiation was initiated in the presence of 10 nM dexamethasone. These data suggest that inhibition of soluble cAMP phosphodiesterase activity and subsequent alterations in cAMP may play an important role in the mechanism whereby IBMX enhances differentiation of 3T3‐L1 cells.
Relationship of ornithine decarboxylase activity and cAMP metabolism to proliferation of normal human bronchial epithelial cells
Authors: Willey James C. , Laveck Moira A. , McClendon Irene A. , Lechner John F. 
Published: February 2005

AbstractThe mechanisms of action of extracellular mitogens for normal human bronchial epithelial cells (NHBE) were investigated by observing their effects on selected biochemical pathways when the cells were incubated in serum‐free media. We find that (a) epidermal growth factor (EGF) stimulates ornithine decarboxylase (ODC) activity and the rate of cell division without stimulating cAMP; (b) alone, pituitary extract (PEX) does not stimulate ODC activity, cAMP levels, or cell division; (c) when PEX is added to medium containing EGF there is a further increase in both ODC activity and the rate of cell division, again with no increase in cAMP levels; (d) in contrast, alone, L‐epinephrine (EPI) stimulates an increase in both ODC and cAMP but does not stimulate cell division; (e) when EPi is added to medium containing both EGF and PEX a further increase in the rate of cell division is noted; (f) the specific inhibitor of ODC, α‐ (difluoromethyl)‐ornithine (DMFO), also inhibits NHBE cell proliferation; and (g) the β‐adrenergic receptor antagonist propranolol inhibits the mitogenic action and ODC induction by EPI observed under condition e. We conclude that an increase in ODC activity is necessary but not sufficient for an increase in proliferation of NHBE cells. In contrast, cAMP stimulation is not necessary for an increase in NHBE cell division. However, in the presence of undefined factors in PEX, increases in cAMP levels result in a synergistic increase in the rate of EGF‐stimulated clonal growth. By correlating the biochemical pathways invoked by EGF, PEX, EPI, and combinations thereof with their mitogenic actions, we have better defined the role each of these different mitogens plays in stimulating epithelial cell division.
Selective expression of brush border hydrolases by mouse Peyer's patch and jejunal villus enterocytes
Authors: Smith Michael W. 
Published: February 2005

AbstractDevelopmental profiles describing the expression of lactase, α‐glucosidase, and alkaline phosphatase activities have been determined quantitatively in mouse jejunal enterocytes during migration over villi and Peyer's patch lymphoid tissue. The predicted maximal lactase and α‐glucosidase activities expressed by enterocytes migrating over Peyer's patch follicles were about one‐quarter and one‐half of values found in control villi. Alkaline phosphatase activity was, on the other hand, one third greater in Peyer's patch compared with villus enterocytes. Expression of lactase and α‐glucosidase activities was initially less in enterocytes migrating along interfollicular compared with control villi. Subsequent increase in hydrolase activities occurred during the later stages of enterocyte migration over interfollicular villi. Lactase activity in athymic mice Peyer's patch enterocytes was identical to that recorded for control mice. The corresponding value for villus lactase was, however, only half that found in control tissue. Factors produced locally in lymphoid follicles are probably responsible for selective effects on enterocyte differentiation.
Effects of pulsed electromagnetic field on growth and differentiation of embryonal carcinoma cells
Authors: Akamine Takuya , Muramatsu Hisako , Hamada Hiroshi , Sakou Takashi 
Published: February 2005

AbstractA murine embryonal carcinoma cell line (F9) was used to examine the effect of a pulsed electromagnetic field on the growth and differentiation of malignant cells. The cells can be induced to differentiate into parietal endodermal cells by treatment with retinoic acid. The pulsed electromagnetic field (1 Gauss and 10 Gauss) promoted the growth of embryonal carcinoma cells in both the presence and absence of retinoic acid. The pulsed electromagnetic field was also found to inhibit retionic acid‐induced differentiation, when the degree of differentiation was based on morphological criteria or on the production of plasminogen activator.
A gene‐controlling response of bone marrow progenitor cells to granulocyte‐macrophage colony stimulating factors
Authors: Lazar Gary S. , Quon Diana H. , Lusis Aldons J. 
Published: February 2005

AbstractThe production of granulocytes and macrophages from progenitor cells in the bone marrow is controlled, in part, by a family of humoral regulators, termed colony stimulating factors (CSF). We have examined genetic factors controlling this process using in vitro cloning techniques. The inbred mouse strain LP/J showed elevated colony formation (CFU‐C) in response to one subtype of CSF (G, M‐CSF) compared to other strains of mice examined including the strain C57BL/6J. This variation resulted in a shift to the left of the CFU‐C dose‐response curve for LP/J. No difference between LP/J and C57BL/6J was seen with another subtype of CSF (CSF‐1). Maximal CFU‐C response was similar in the two mouse strains with both types of CSF, and mixing experiments with both types of CSF gave the same maximal level of colony formation as the individual CSF. (C57BL/6J x LP/J)FI progeny exhibited a CFU‐C dose‐response curve to CSF‐2 that was intermediate between the parental types, indicating additive inheritance. Genetic analysis of backcross progeny suggested that the variation in CFU‐C response is probably determined by a single primary gene, although the variability of the colony formation assay has complicated interpretation of genetic studies. These results suggest that CSF‐1 and G, M‐CSF act independently on a single bone marrow progenitor cell population. The properties of the genetic variation for G, M‐CSF response are consistent with an alteration in cellular receptors for G, M‐CSF.
Inhibition of epidermal growth factor‐induced phosphorylation by trifluoperazine
Authors: Ross Alonzo H. , Damsky Caroline , Phillips Paul D. , Hwang Frances , Vance Patricia 
Published: February 2005

AbstractWe studied the effects of a series of drugs on A431, a cell line with well‐characterized growth factor requirements and epidermal growth factor (EGF) receptors. The major [32PO4]‐labeled protein immunoprecipitated with antiphosphotyrosine antibodies from EGF‐stimulated A431 cells was the EGF receptor. Both the quantity of [32PO4]‐labeled proteins were reduced by the addition during EGF stimulation of trifluoperazine (TFP). TFP had little effect on the binding, internalization, and processing of [125I]‐EGF. In addition to the effects on phosphorylation, TFP inhibited cell growth both in the presence and absence of EGF. Morphologically, TFP blocked EGF‐induced ruffling. TFP did not alter the EGF‐ stimulated phosphatidylinositol turnover. In an in vitro experiment using A431 cell membranes, TFP did not inhibit phosphorylation of the EGF receptor.
Ammonia production by IMR‐90 fibroblast cultures: Effects of ammonia on glutathione, γ‐glutamyl transpeptidase, lysosomal enzymes, and cell division
Authors: Takahashi Shizuko , Nakagawa Sachiko , Zeydel Maria , Bhargava Girija 
Published: February 2005

Abstractγ‐Glutamyl transpeptidase has multi‐catalytic activities. It degrades glutathi‐one and can produce ammmonia from glutamine. The present study was designed to examine whether the decreased cell proliferation, cellular gluta‐thione content and concurrent increase in ammonia production in senescent cells in culture are the result of increased γ‐glutamyl transpeptidase activity. We used IMR‐90 fibroblast and 3T3 LI preadipocyte cultures. The cellular glutathione content depended upon cell proliferation and cell density. The glutathione content was higher in cells at logarithmic growth, and lower at stationary growth or post confluency; dead cells had no detectable glutathione by the method currently used. The glutathione content was minimal in “old” IMR‐90 cells, regardless of cell density. On the other hand, an increase occurred in the unit number of molecules of bound 5‐iodoacetoamidofluorescein, an active‐site directed stoichiometric inhibitor of transpeptidase. That result corresponded favorably with the increased enzyme activity, suggesting that the number of enzyme molecules per cell was increased. The inhibition of ammonia production of the cultures by inhibition of γ‐glutamyl transpeptidase by 5‐iodoacetoamidofluorescein and reversible inhibition of ammonia production by a serine‐borate mixture were consistent with our postulate. Addition of NH4Cl (0.1 mM) to IMR‐90 cultures caused increased activities of transpeptidase and some of the lysosomal enzymes; concurrently, the amount of cellular glutathione and the number of cell divisions decreased. This suggests that the increased ammonia production presumably resulting from glutaminase activity of the observed increase of transpeptidase may profoundly affect certain cellular functions.