bluuebunny/crossref_metadata_2025 huggingface dataset:
Systematic analysis of the ability of stromal cell lines derived from different murine adult tissues to support maintenance of hematopoietic stem cells in vitro
Authors: Rios Maribel , Williams David A.
Published: February 2005

Hematopoietic stem cells interact with a complex microenvironment both in vivo and in vitro. In association with this microenvironment, murine stem cells are maintained in vitro for several months. Fibroblast‐like stromal cells appear to be important components of the microenvironment, since several laboratories have demonstrated that cloned stromal cell lines support hematopoiesis in vitro. The importance of the tissue of origin of such cell lines remains unknown, since systematic generation of stromal cell lines from adult tissues has never been accomplished. In addition, the capacity of stromal cell lines to support reconstituting stem cell has not been examined. We have previously described an efficient and rapid method for the immortalization of primary bone marrow stromal cell lines (Williams et al., Mol. Cell. Biol. 8:3864–3871, 1988) which can be used to systematically derive cell lines from multiple tissues of the adult mouse. Here we report the immortalization of primary murine lung, kidney, skin, and bone marrow stromal cells using a recombinant retrovirus vector (U19‐5) containing the simian virus large T antigen (SV40 LT) and the neophosphotransferase gene. The interaction of these stromal cells with factor‐dependent cells Patterson‐Mix (FDCP‐Mix), colony forming units‐spleen (CFU‐S), and reconstituting hematopoi‐etic stem cells was studied in order to analyze the ability of such lines to support multipotent stem cells in vitro. These studies revealed that stromal cell lines from these diverse tissues were morphologically and phenotypically similar and that they quantitatively bound CFU‐S and FDCP‐Mix cells equally well. However, only those cell lines derived from bone marrow‐supported maintenance of day 12 CFU‐S in vitro. One lung‐derived stromal cell line, ULU‐3, supported the survival of day 8 CFU‐S, but not the more primitive CFU‐S12. A bone marrow‐derived stromal cell line, U2, supported the survival of long‐term reconstituting stem cells for up to 3 weeks in vitro as assayed by reconstitution 1 year post‐transplant. These studies suggest that adherence of HSC to stromal cells is necessary but not sufficient for maintenance of these stem cell populations and that bone marrow provides specific signals relating to hematopoietic stem cell survival and proliferation.
Response to epidermal growth factor of skin fibroblasts from donors of varying age is modulated by the extracellular matrix
Authors: Colige A. , Nusgens B. , Lapiere C. M.
Published: February 2005

The present study was undertaken to investigate the effect of epidermal growth factor(EGF) on the biosynthetic activity of skin fibroblasts from donors of varying age and the modulation of their response to this growth factor by culture in a three‐dimensional extracellular matrix. When cultured in monolayeron plastic or at the surface of a collagen gel, EGF specifically inhibited collagen synthesis whatever the age of the donor (from 17 to 84 years, n = 11). This inhibition was paralleled by a significant decrease in the steady‐state level of procollagen type I mRNAs. When embedded in a three‐dimensional floating collagen lattice, EGF stimulated the non‐collagen protein (NCP) synthesis in fibroblasts from younger donors (5 out of 6) while fibroblasts from the older ones were not affected. Collagen production by fibroblasts from younger donors was not inhibited as in monolayer (some being even stimulated) while that of the older donors was inhibited as observed in monolayer. The steady‐state level of procollagen type I mRNA was not modified by EGF in the three‐dimensional culture. No significant difference was observed in the affinity and the number of EGF receptors of the fibroblasts on plastic or embedded in a collagen lattice between young and aged donors. Our results suggest that the environment of the cells can modulate the reactivity to EGF and reveal differences related to in vivo aging.
Diacylglycerols and PMA are particularly effective stimulators of fluid pinocytosis in human neutrophils
Authors: Keller H. U.
Published: February 2005

Diacylglycerols (OAG, diC8) and PMA were found to stimulate fluid pinocytosis (net uptake of FITC‐dextran) to a far greater extent than other neutrophil activators, such as the chemotactic agents fNLPNTL and LTB4, the microtubule disassembling agents colchicine and nocodazole, the kinase inhibitor H‐7, or D2O. OAG and diC8 produce a dose‐dependent increase in the uptake of FITC‐dextran, which is up to about 25‐ to 30‐fold the control value of unstimulated neutrophils. The protein kinase inhibitor H‐7 alone had a small stimulating effect on the net uptake, and it failed to inhibit stimulation of fluid pinocytosis by PMA, OAG, and diC8. Also, the protein kinase inhibitor staurosporine failed to inhibit fluid pinocytosis stimulated by OAG, diC8, and PMA. Stimulated fluid pinocytosis and vacuolization in response to PMA or diacylglycerols is associated with surface ruffling of neutrophils. Pinocytosis as well as surface ruffling stimulated by PMA, OAG, diC8, or diC10 are suppressed in the presence of cytochalasin D. The results suggest that diacylglycerols may be instrumental in transducing the signal for stimulated pinocytosis and that the surface movements induced by diacylglycerols, and PMA may be instrumental in fluid pinocytosis.
Phagocytosis in Acanthamoeba: II. Soluble and insoluble mannose‐rich ligands stimulate phosphoinositide metabolism
Authors: Allen Philip G. , Dawidowicz Eliezar A.
Published: February 2005

The generation of second messengers during phagocytosis of yeast by Acan‐thamoeba castellanii was examined. The kinetics of binding and internalization of yeast by Acanthamoeba were measured and this was compared with the generation of known second messengers. We observed stimulated degradation of PI‐4, 5‐P2 to 1,4,5 IP3 with kinetics similar to that observed for the binding of yeast to amoeba. Similar production of IP3 could be induced upon treatment with a soluble mannosylated glycoprotein. We propose that the Acanthamoeba man‐nose receptor stimulates the degradation of PI‐4, 5‐P2 to 1,4,5 IP3 as an initial event in phagocytosis.
Complex mitogenic requirements of Na+ ‐K+ ‐Cl− ‐cotransport‐deficient BALB/c‐3T3 cells
Authors: Smyth Miriam J. , O'Brien Thomas G. , Wharton Walker
Published: February 2005

The ability of 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA) to stimulate mitogenesis in BALB/c‐3T3 cells and in a Na+K+C1− ‐cotransport‐defective variant subclone was investigated. This transport variant had previously been reported to be TPA mitogenically nonresponsive (O'Brien and Prettyman: Journal of Cellular Physiology 130:377–381, 1987) since the addition of TPA to the spent medium of density‐arrested cultures stimulated DNA synthesis in the parent but not the variant cell line. We now report that the addition of TPA plus insulin, either directly to the spent medium or together with fresh medium, stimulated DNA synthesis in both the parent and variant cells to approximately the same extent. The parent and transport‐defective cells differed, however, in their sensitivity to the co‐mitogenic effects of insulin or insulin‐like growth factors.
Cloning of an osteoblastic cell line involved in the formation of osteoclast‐like cells
Authors: Yamashita Takeyoshi , Asano Katsuhiko , Takahashi Naoyuki , Akatsu Takuhiko , Udagawa Nobuyuki , Sasaki Takahisa , Martin T. John , Suda Tatsuo
Published: February 2005

Experiments have been carried out to determine the mechanisms involved in the formation of osteoclast‐like cells from spleen cells in mice. Osteoclasts were defined as tartrate‐resistan. acid phosphatase‐positive multinucleated cells (TRACP‐positive MNCs) in which specific calcitonin receptors were identified by autdradiography with labeled salmon calcitonin. Furthermore, cultures rich in these cells produced resorption pits when grown on dentine slices. Several clonal cell lines were obtained from fetal mouse calvariae and screened for their ability to induce TRACP‐positive MNCs in response to 1α, 25‐dihydroxyvitamin D3 “1α,25(OH)2D3” in co‐cultures with spleen cells. A cell line, KS‐4, was identified with the greatest potency in inducing osteolast‐like cell formation in co‐culture with spleen cells. The capacity of KS‐4 cells to produce this effect was much greater than that of two bone marrow‐derived stromal cell lines (MC3T3‐G2/PA6 and ST2 cells), which we have previously shown to be effective in this system but to require treatment with dexamethasone in addition to 1α25(OH)2D3 (Udagawa et al.: Endocrinology 125:1805–1813, 1989). Parathyroid hormone (PTH) in creased cAMP production in KS‐4 cells, and PTH and interleukin‐1α also induced TRACP‐positive MNCs in co‐cultures with spleen cells. Contact between living KS‐4 and spleen cells was necessary for osteoclast formation to take place, since this did not occur when the two populations were separated by a membrane filter, or when the KS‐4 cells were killed by fixation. Separate cultures of either spleen cells or KS‐4 cells formed no TRACP‐positive MNCs. KS‐4 cells synthesized predominantly type I collagen, formed bone nodules without added of β‐glycerophosphate in a long‐term culture, and expressed increasing alkaline phosphatase activity after confluence in culture. These results indicate that the KS‐4 cells have properties consistent with progression toward the osteoblast phenotype, and represent a single cell line with the ability to promote osteoclast formation by a contact‐requiring process.
Antidiabetic AD4743 enhances adipocyte differentiation of 3T3 T mesenchymal stem cells
Authors: Sparks Rodney L. , Strauss Ethan E. , Zycmunt Andrea I. , Phelan Timothy E.
Published: February 2005

AD4743 is an antidiabetic agent that, when added to fetal bovine serum (FBS), has been shown to have adipogenic activity for some proadipocyte cell lines once they reach confluence. In the current study, the effects of AD4743 on the growth and adipocytic differentiation of 3T3 T multipotential mesenchymal stem cells have been tested. 3T3 T cells, unlike other cells capable of undergoing adipocyte differentiation, are routinely induced to differentiate at low cell density. This is done using platelet‐poor human plasma (HP), a potent inducer of growth arrest and differentiation. AD4743 (0–200 μg/ml) was tested in varied concentrations of HP or FBS, at varied cell densities, and at various times during growth and differentiation. AD4743 slowed the growth rate of 3T3 T cells and it induced their differentiation in a dose‐dependent manner in medium containing 10% FBS once they reached confluence. Tiie data suggest that the ability of AD4743 to inhibit growth may also becoupled with its ability to enhance differentiation. In addition, AD4743 (1–10 μg/ml) in the presence of 25% HP markedly increased the kinetics of adipocyte differentiation, at low (< 5,000 cells/cm2) or high cell density. Greater than 50% cell differentiation could be achieved in 2 days in low density cultures; 80–95% differentiation could be achieved in just 4 days, compared to 8–12 days in a typical culture. The maximum amount of differentiation in HP was potentiated by AD4743 to agreater degree in poor lots of HP; however, the kinetics were increased in all lots. Adipocytic differentiation was measured both morphologically and by Northern blot analyses of differentiation‐specific genes. AD4743 at 1–10 μg/ml appeared to be most effective, depending on the cell density and other conditions. The mechanism of action of AD4743 remains to be elucidated, but the enhancement of adipocyte differentiation does not appear to occur via an insulin‐dependent pathway.
Distribution and comparison of receptors for leukemia inhibitory factor on murine hemopoietic and hepatic cells
Authors: Hilton Douglas J. , Nicola Nicos A. , Metcalf Donald
Published: February 2005

Leukemia inhibitory factor (LIF) is a glycoprotein that induces the differentiation of the monocytic leukemia cell line M1 but suppresses the differentiation of totipotent embryonic stem cells. In an attempt to define the normal cellular targets for LIF, the distribution of LIF receptors within hemopoietic and hepatic tissue was analyzed by binding cells with radioiodinated LIF (125I‐LIF) and subsequently carrying out autoradiography. Autoradiography demonstrated that in each he‐mopoietic tissue examined cells of monocyte/macrophage lineage were the primary cell type labeled with 125I‐LIF. Moreover, both fetal and adult parenchy‐mal hepatocytes displayed higher levels of labeling than either monocytes or macrophages. The number of receptors per positive cell varied from 150 for bone marrow monocytes to 2,000 for adult hepatocytes. In each case, however, binding was of high affinity, with an apparent KD of 34–100 pM, and binding was specific, since labeling was competed for by unlabeled LIF but not a range of other structurally unrelated growth and differentiation factors. It is suggested that LIF may play a role in regulating macrophage function and hepatic acute phase protein synthesis in response to infection.
Control of the intracellular Ca2+‐concentration and the inositol phosphate accumulation in dog thyrocyte primary culture: Evidence for different kinetics of Ca2+‐phosphatidylinositol cascade activation and for involvement in the regulation of H2O2 production
Authors: Raspe E. , Laurent E. , Corvilain B. , Verjans B. , Erneux C. , Dumont J. E.
Published: February 2005

Carbachol, through a muscarinic receptor, thyrotropin‐releasing hormone (TRH), prostaglandin F2α (PGF2α), bradykinin, and adenosine triphosphate (ATP) in creased the apparent [Ca2+], (intracellular free Ca2+ concentration) of dog thyrocytes in primary culture. The [Ca2+], measured by the Quin‐2 technique rose immediately after the addition of the agonists and reached a maximal value after less than 30 seconds. Afterwards, the [Ca2+], declined to a plateau higher than the basal level when the cells were triggered with carbachol. By contrast, in most experiments with PGF2α and in the case of bradykinin, TRH, and ATP, the [Ca2+], returned to the basal value. If the extracellular Ca2+ was chelated by excess of EGTA, the addition of all agents caused a sharp reduced transient rise in the [Ca2+], followed by a decline of the [Ca2+], often below the basal level (especially in the case of carbachol). It is suggested that the first transient phase of these responses is due at least in part to the mobilisation of Ca2+ from intracellular stores whereas the second sustained phase of the response to carbachol mainly originates from an increased Ca2+ influx into the thyrocytes.

Carbachol, bradykinin, TRH, PGF2α, and ATP also increased generation of inositol phosphates in dog thyrocytes. This effect was sustained when the cells were triggered with carbachol and was more transient with bradykinin, TRH, PGF2α, or ATP.

All these agents and the phorbdester TPA as well as forskolin enhanced to various extent the thyrocyte H2O2 generation. This enhancement was severely reduced in the absence of extracellular Ca2+ and was mimicked by Ca2+ ionophores in the presence of extracellular Ca2+ especially in synergy with protein kinase C activators. These data suggest that the dog thyrocyte H2 O2 generation, the limiting step of the thyroid hormone synthesis, is modulated by carbachol, TRH, PGF2α, bradykinin, and ATP through their action on the Ca2+‐phosphatidylinositol cascade.
Substrate‐associated macromolecules promote cytodifferentiation of BC3H1 myogenic cells
Authors: Strauch Arthur R. , Berman Mark D. , Miller Howard R.
Published: February 2005

Differentiated mouse BC3H1 myogenic cells secrete substrate‐associated macromolecules (SAM) which restrict the proliferation of undifferentiated cells and promote both cell shape changes and expression of predominantly the vascular smooth muscle (VSM)‐specific isoform of the contractile protein α‐actin. While we previously reported that high cell density was required for stimulating maximal expression of VSM α‐actin in BC3H1 cells (Strauch and Reeser: Journal of Biological Chemistry264:8345–8355, 1989), the permissive effect of SAM on myoblast cytodifferentiation was not at all dependent on the formation of cell to cell contacts. This observation suggests that biogenesis of an extracellular matrix rather than the formation of physical contacts between cells may be the rate‐limiting step for induction of VSM α‐actin expression at high cell density. The biologically active moieties in SAM that promote cytodifferentiation also are expressed by mouse embryonic fibroblast cell lines and are distinctly different from a class of adheron‐like macromolecules released by differentiated BC3H1 myocytes directly into the culture medium. While SAM was cell growth restrictive, reconstituted particulate material (RPM) prepared from myocyte‐conditioned medium promoted the adhesion and proliferation of growth‐arrested myoblasts. SAM and RPM are composed of different polypeptide subunits which collectively may establish microenvironmental conditions that are permissive for BC3H1 myogenic cell differentiation.
Hydrogen peroxide alters the physical state and function of the plasma membrane of pulmonary artery endothelial cells
Authors: Block Edward R.
Published: February 2005

Hydrogen peroxide (H2O2) is an important mediator of acute oxidative injury to vascular endothelium. Because the plasma membrane is the initial site of interaction between endothelial cells and extracellular H2O2 produced by stimulated neutrophils or macrophages, we evaluated the effect of H2O2 on the physical state, i.e., fluidity, and function of porcine pulmonary artery endothelial cell plasma membranes. Lactate dehydrogenase (LDH) release, 5‐hydroxytryptamine (5‐HT) uptake, limiting fluorescence anisotropy (r∞) for trimethylamino‐diphenylhexatriene (TMA‐DPH), and conjugated dienes were measured 0.5, 6, and 24 hr after cells were exposed for 30 min to 50‐μM H2O2 or Hanks' Balanced Salt Solution (control). Compared with control cells, H2O2 caused significant increases in LDH release and in 5‐HT uptake 6 hr after exposure. The increase in 5‐HT uptake was not blocked by imipramine. H2O2 also caused a significant increase in r∞ for TMA‐DPH 0.5 hr after exposure and a significant reduction in r∞ for TMA‐DPH 6 hr after exposure. Cellular contents of conjugated dienes were increased 0.5 and 6 hr after exposure to H2O2. Twenty‐four hours after exposute LDH release, r∞, 5‐HT uptake, and conjugated dienes had returned to control levels. Preincubation with 50‐μM α‐tocopherol (vitamin E) or 1‐mM or 10‐mM dimethylthiourea (DMTU) for 1 hr or 24 hr prevented endothelial cell injury, whereas addition of vitamin E or DMTU to the medium 1 hr or 3 hr after H2O2 exposure did not protect against injury. These results indicate that H2O2 causes significant damage to the plasma membrane of pulmonary artery endothelial cells in vitro, leading to alterations in fluidity and leakiness of the membrane. This injury is associated with membrane lipid peroxidation, is reversible, and can be prevented by pretreatment, but not by post‐treatment, with vitamin E or DMTU.
Immunolocalization of basic fibroblast growth factor during chicken cardiac development
Authors: Consigli Seth A. , Joseph‐Silverstein Jacquelyn
Published: February 2005

Basic fibroblast growth factor (bFGF) has been identified in cultured cardiac myocytes as well as in myocardial tissue of both embryonic and adult organisms; bFGF has also been demonstrated to regulate proliferation and differentiation of these cells in culture. Such studies suggest a possible role for bFGF in cardiac myogenesis. In vitro studies using cultured endothelial and neurona cells also suggest that myocyte‐derived bFGF may be involved in the regulation of vascularization and/or innervation of the developing heart. We have generated a spatial and temporal map for bFGF in the developing chick heart using immu‐nohistochemical techniques and our monospecific polyclonal rabbit antihuman bFGF IgG. A progressive decrease in bFGF expression was seen in the highly trabeculated region of the ventricular myocardium, relative to the myocardium directly underlying the epicardial tissue, with increasing developmental age. bFGF expression was limited to the cytoplasm of cardiac myocytes; neither vascular endothelium nor smooth muscle contained anti‐bFGF immunoreactive material. A correlation between the temporal and spatial pattern of bFGF expression seen here, with the pattern of myocyte proliferation and differentiation reported by others, suggests a role for bFGF in the autocrine regulation of myocyte proliferation and differentiation.
Ferric‐salicylaldehyde isonicotinoyl hydrazone, a synthetic iron chelate, alleviates defective iron utilization by reticulocytes of the belgrade rat
Authors: Garrick Laura M. , Gniecko Kathleen , Hoke James E. , Al‐Nakeeb Attila , Ponka Prem , Garrick Michael D.
Published: February 2005

The Belgrade rat has a hypochromic, microcytic anemia inherited as an autosomal recessive mutation. Although transferrin binds normally to reticulocytes and internalizes normally, iron accumulation into cells and heme is much slower than normal. We have investigated the role of the transferrin cycle in this mutant by bypassing transferrin iron delivery with the iron chelate ferric salicylaldehyde isonicotinoyl hydrazone (Fe‐SIH). Fe‐SIH increases iron uptake into heme by Belgrade reticulocytes, restoring it almost to normal levels. This increase indicates that Fe‐SIH delivers iron to a step in iron utilization that is after the Belgrade defect. Depleting reticulocytes of transferrin did not alter these observations. Failure to achieve above normal rates of iron incorporation could indicate damage due to chronic intracellular iron deficiency. Also, iron delivery by Fe‐SIH restored globin synthesis to near‐normal levels in Belgrade reticulocytes. The rates of glycine incorporation into porphyrin and heme in Belgrade reticulocytes incubated with Fe2‐transferrin or Fe‐SIH paralleled the rates of iron incorporation into heme. These data are consistent with the concept that iron availability limits protoporphyrin formation in rat reticulocytes. The protoporphyrin used for heme synthesis is provided by de novo synthesis and not by a pool of pre‐existing protoporphyrin. The Belgrade defect occurs in the movement of iron from transferrin to a step prior to reduction to the ferrous state and insertion into heme. This defect diminishes the synthesis of heme and, consequently, that of protoporphyrin and globin.
Cellular mechanisms of ATP‐induced hyperpolarization in renal epitheloid MDCK‐cells
Authors: Paulmichl M. , Pfeilschifter J. , Wöll E. , Lang F.
Published: February 2005

Previous studies have shown that ATP enhances intracellular calcium concentration and activates potassium channels in Madin Darby canine kidney (MDCK)‐cells, thus leading to hyperpolarization of the cell membrane. The present study has been performed to elucidate the intracellular mechanisms involved. To this end, the effects of ATP on the potential difference across the cell membrane (PD), on formation of inositol phosphates, and on intracellular calcium concentration (Cai) have been analyzed in cells without or with pretreatment with pertussis toxin or 12‐O‐tetradecanoyl phorbol 13‐acetate diester (TPA). In untreated cells, ATP leads to a sustained hyperpolarization and an increase of inositol 1,4,5‐tris‐phosphate (IP3), inositol 1,3,4,5‐tetrakisphosphate (IP4), and Cai. In the absence of extracellular calcium, the effect of ATP on PD and Cai is only transient. In cells pretreated with pertussis toxin, the effect of ATP on inositol trisphosphate is almost abolished, but ATP still leads to an increase of PD and Cai, which is sustained in the presence, and transient in the absence, of extracellular calcium. In cells pretreated with TPA, the effect of ATP on inositol trisphosphate is reduced and the effect on Cai blunted; but ATP still leads to a hyperpolarization of the cell membrane, which is sustained in the presence, and transient in the absence, of extracellular calcium. The observations indicate that ATP activates phospholipase C by a phorbol ester and pertussis toxin sensitive mechanism. In addition, ATP enhances Cai by pertussis toxin insensitive mechanisms allowing recruitment of calcium from both, extracellular fluid and intracellular stores. Calcium then activates the potassium channels and thus leads to the hyperpolarization of the cell membrane.
Growth and differentiation of primary tracheal epithelial cells in culture: Regulation by extracellular calcium
Authors: Martin Walter R. , Brown Chris , Zhang Yao Juan , Wu Reen
Published: February 2005

Growth and differentiation of primary monkey tracheal epithelial (MTE) cells maintained on collagen gel substrata were studied in a defined serum‐free culture medium containing 0.03 to 3.0 mM extracellular calcium. Cell attachment efficiency (40‐60%) was not altered by different calcium levels. Growth of primary MTE cells on collagen gel substrata, which was vitamin A dependent, was enhanced 50% in the medium supplemented with high calcium (≥ 0.3 mM). High calcium medium also increased cell‐cell interactions, formation of desmosomes, and multi‐cell layering. The relative content of mucous cells, which were identified by a mucin‐specific monoclonal antibody and the presence of mucus‐secreting granules at the ultrastructural level, was greater in the high‐calcium medium. Furthermore, the secretion of mucin into the medium, determined either by an ELISA or by the incorporation of 3H‐glucosamine into mucous glycoprotein fractions, was also increased more than 5‐fold in media containing high calcium content ( ≥ 0.6 mM). In contrast, MTE cells cultured in low calcium medium ( < 0.15 mM) were squamous‐like with prominent tonofiiaments, and their secretory product was mainly hyaluronate. These results demonstrate that media containing a high calcium content promote conducting airway epithelium to express mucous cell differentiation, while media with low calcium content promote squamous cell differentiation.
Brefeldin A induces endoplasmic reticulum‐associated O‐glycosylation of galactosyltransferase
Authors: Bosshart Herbert , Straehl Peter , Berger Bea , Berger Eric G.
Published: February 2005

Recent data from several laboratories show that Brefeldin A (BFA) induces a microtubule‐dependent back‐flow of Golgi components to the endoplasmic reticulum (ER) thereby causing disassembly of the Golgi apparatus and its fusion with ER membranes. In order to delineate the effect of BFA on resident Golgi proteins, we investigated its effect on biosynthesis, maturation and intracellular transport of galactosyltransferase (gal‐T), an established trans‐Golgi enzyme. Using a protocol of metabolic labeling/immunoprecipitation followed by electrophoretic/fluorographic analysis, we show that in the presence of BFA, gal‐T matures to a molecular form of 48.5 kD, a size intermediate between the 2 precursor forms of 44 and 47 and the mature form of 54 kD (Strous and Berger: j. Biol. Chem., 257:7623–28, 1982). Little mature form was detectable in the presence of BFA even after prolonged chase times of up to 28 hr. The intermediate form was sensitive to O‐glycanase and endoglycosidase H, indicating early O‐glycosylation without sialylation and lack of complex N‐glycosylation, respectively. In order to define the compartment responsible for O‐glycosylation in the presence of BFA, a temperature block of 25°C was applied which inhibited recovery of Golgi elements from BFA‐induced fusion with ER. At this temperature and in absence of BFA, biosynthesis of gal‐T was not appreciably affected, while maturation was completely inhibited as indicated by the presence of unmodified precursor forms of gal‐T. After 60 min preincubation with BFA, a time period sufficient to demonstrate complete fusion of Golgi with ER, subsequent biosynthesis of gal‐T at 25°C in absence of BFA led to the intermediate form, while precursor forms were not detectable. These data provide direct evidence for BFA‐induced redistribution to the ER of Golgi enzymes involved in O‐glycosylation and their early functional involvement in biosynthesis of newly synthesized gal‐T.
Restoration of adhesive potentials of Ehrlich ascites carcinoma cells by modification of plasma membrane
Authors: Bogdanov Alexei A. , Gordeeva Larisa V. , Baibakov Boris A. , Margolis Leonid B. , Torchilin Vladimir P.
Published: February 2005

A novel technique for modulating the spreading of ascites cells has been developed. Plasma membranes of Ehrlich ascites carcinoma cells were modified in two different ways: 1) biotin residues were covalently coupled to membrane components; 2) biotinylated lipid was introduced into plasma membranes. Adhesion and spreading of modified cells on avidin‐coated substrates were studied and compared to those of non‐modified cells. Both types of membrane alteration were shown to induce specific (biotin‐dependent) interaction with immobilized avidin with resultant cell spreading. Spread cells attained epithelioid‐like morphology with the formation of wide thin lamellae, focal contacts with substrate, and circular actin bundles. The process of spreading was shown to be energy‐dependent: it could be blocked by metabolic inhibitors and by low temperature. Formation of extended lamellae was prevented by preincubation of cells in the presence of cytochalasin B. The effects of metabolic poisons, low temperature, and microfilament–disruptive drugs were reversible and after the restoration of physiological conditions the cells resumed the spreading process. Immunoprecipitation of biotinylated cell lysates with antiserum to cytoplasmic domain of 3,‐integrin subunit revealed a major 110 kD avidin‐binding component. We conclude that lack of spreading of ascites carcinoma cells may be explained by the lack of functionally active adhesion‐ and spreading‐competent cell‐surface receptors, but may not be attributed to the defects in intracellular function or organization. Intracellular machinery of cell spreading is preserved in these ascites cells and could be turned on by cell attachment to the substrate via artificial adhesive site incorporated into plasma membrane.
Adhesion of mouse mast cells to fibroblasts: Adverse effects of steel (SI) mutation
Authors: Kaneko Yoshiyuki , Takenawa Jun , Yoshida Osamu , Fujita Kohichi , Sugimoto Kenkichi , Nakayama Hiroki , Fujita Jun
Published: February 2005

Mouse bone marrow‐derived cultured mast cells proliferate on +/+ mouse embryo‐derived 3T3 fibroblasts, but not on SI/SId mouse embryo‐derived 3T3 fibroblasts, in the absence of IL‐3 and IL‐4 (Fujita et al: Proc. Natl. Acad. Sci. U.S.A. 86:2888–2891, 1989). To further characterize the mast cell‐fibrpblast interactions and the effects cf SI mutation, we tried to analyze the adhesion of cultured mast cells to 3T3 fibroblasts in vitro. Mast cells plated onto NIH/3T3 fibroblasts showed marked adhesion within 30 min, which reached a plateau after 3 h. The numbers of adhered mast cells were linear over the range of 103 to 5 × 105 cells inoculated into each (2 cm2) of 24 multiwells. Adhesion required active energy production and the presence of divalent cations. It was not inhibited byan RGD‐containingpeptide, an anti‐LFA‐1 antibody, or asialofetuin. Mast cells adhered efficiently to the eight 3T3 cell lines derived from +/+ mouse embryos, but not to the eight 3T3 cell lines derived from SI/SId mouse embryos. Adhesion to mouse spleen‐derived fibroblasts lacking mast cell‐supporting activitywas comparable to that to SI/SId/3T3 cells. The failure of mast cells to adhere to fibroblasts with the SI mutations was not due to a production of a diffusible inhibitor by the latter. These results indicate that production of wild type SI gene product by fibroblasts is mandatory for adhesion/migration, as well as for proliferation of mast cells on them, and that the coculture system should be useful for the biochemical and molecular analysis of these interactions.
Lateral diffusion of CD4 on the surface of a human neoplastic T‐cell line probed with a fluorescent derivative of the envelope glycoprotein (gp120) of human immunodeficiency virus type 1 (HIV‐1)
Authors: Pal Ranajit , Nair B. C. , Hoke George M. , Sarngadharan M. G. , Edidin Michael
Published: February 2005

The envelope glycoprotein (gp120) of HIV‐1 was labeled with fluorescein by using 6‐[4,6‐dichlorotriazinyl]aminofluorescein. The labeled glycoprotein was found to bind to CD4‐positive CEM cells. Monoclonal antibody OKT4a but not OKT4 blocked this binding. Similar specific binding of fluorescein‐labeled gp120 with CD4 was observed in a solid‐phase ELISA where sCD4 was attached to a polystyrene plate. The syncytium formation induced by HIV‐1‐infected cells on CEM cells was significantly inhibited in the presence of fluorescein‐labeled gp120. Fluorescence photobleaching recovery measurements showed that the diffusion coefficient (D) of CD4 molecules complexed with fluorescein‐labeled gp120 was approximately 5 × 10−10 cm2sec−1, with nearly 61 % of the receptor molecules being mobile. Binding of anti‐gp120 monoclonal antibody to the CD4‐gp120 complex reduced the mobile fraction significantly. Diffusion of CD4 labeled with OKT4 IgG was markedly inhibited with reductions in both D and the mobile fraction, but such inhibition was not observed with OKT4 Fab. It appears that crosslinking of multiple molecules of CD4 by OKT4 antibody is required to reduce CD4 mobility. This suggests that the receptor might be present on the membrane plane as molecular clusters containing at least two molecules of CD4.
Culture‐induced increase in acidic and basic fibroblast growth factor activities and their association with the nuclei of vascular endothelial and smooth muscle cells
Authors: Speir Edith , Sasse Joachim , Shrivastav Shashi , Casscells Ward
Published: February 2005

The activity of acidic and basic fibroblast growth factor‐like mitogens (aFGF, bFGF) extracted from cultured bovine aortic endothelial (BAEC) and rat aortic smooth muscle cells (SMC) was compared with that of freshly isolated cells from the same tissues. Extracts of subendothelial extracellular matrix (ECM) and cell lysates of cultured BAEC contained 4‐fold more bFGF‐like activity than the extracts of fresh cells. ECM and cell lysates of SMC yielded 10‐fold more bFGF‐like activity than the fresh cell lysates. We consistently find aFGF‐like activity in both cell types. In the case of BAEC, cultured cells and ECM contained 3‐fold more aFGF‐like activity when compared with freshly isolated cells, whereas in cultured SMC, aFGF‐like activity in cell and ECM extracts was 8‐fold higher than in fresh cell extracts.

The mitogens extracted from cell lysates and from the ECM are closely related to aFGF or bFGF by the criteria that they bind to heparin‐sepharose and elute at 1.1 M (aFGF) or 1.5 M (bFGF) NaCl, have molecular weights of about 18,000, and react with anti‐aFGF (1.1 M), or anti‐bFGF (1.5M) antibodies when analyzed by Western blots and by radioimmunoassay specific for aFGF and bFGF. This mitogenic activity is inhibited by neutralizing antibodies to aFGF and bFGF. In addition, the column fractions are potent mitogens for Balb/c 3T3 fibroblasts. Acidic and basic FGF‐like mitogenic activity could also be extracted from the cell nuclei. The subcellular localization of both FGFs was visualized in both nuclei and cytoplasm with immunoperoxidase. Compared with primary SMC, secondary SMC had an increased capacity to bind 125laFGF to high affinity receptors, while binding to freshly isolated BAEC and SMC was negligible. We conclude that FGFs are present at low levels in freshly isolated cells and that propagation in cell culture provides a stimulus for production of these mitogens.
Analysis by cell hybridization of mechanisms that regulate β‐Adrenergic responses in reticulocytes and in differentiating erythroid cells
Authors: Porzig Hartmut , Moudry Radmila , Montandon Jean‐Blaise
Published: February 2005

In intact reticulocytes, but not in fragmented membranes, the loss of adenylate cyclase activity during cell maturation followed a biphasic time course. A rapid phase (t½ ≈ 2 h) during which the initial activity was reduced by 40–50% was followed by a slow phase with t½ close to 3 days. The fast decay seemed to occur on the adenylate cyclase level since (‐)isoprenaline‐ or forskolin‐stimulated activities behaved similarly and bacterial toxin‐monitored Gs and Gi proteins remained stable. The mechanism of the initial decrease in hormonal responsive ness was further analysed in hybrid cells prepared by fusing reticulocytes with Friend erythroleukemia (MEL) cells. The hybrids contained reticulocyte‐derived β‐adrenoceptors and MEL cell‐derived adenylate cyclase and G proteins. Fusion of reticulocytes to native MEL cells caused adenylate cyclase activity to drop by 30% at 2 h and 45% at 18 h after fusion. By contrast, hybrids prepared after dimethylsulfoxide‐induced differentiation of MEL cells showed stable or increasing rates of receptor‐coupled cAMP formation between 2 and 18 h after fusion, concomitant with the enhanced activity of the Gs protein in these cells. A cyclase‐stimulating factor present in the cytosol of MEL cells and of reticulocytes appeared not to be involved in short‐term regulation of hormonal responsiveness. We conclude that the strength of β‐adrenergic responses in erythroid progenitor cells is primarily regulated by modulating G protein‐mediated receptor cyclase coupling while reticulocytes, during early maturation, seem to rely on direct inactivation of adenylate cyclase, probably via a cytosolic proteolytic pathway.
GM‐CSF and phorbol esters modulate GM‐CSF receptor expression by independent mechanisms
Authors: Brizzi Maria F. , Arduino Carlo , Carlo Avanzi G. , Bussolino Federico , Pegoraro Luigi
Published: February 2005

Human granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) (0.1 nM) down‐modulates its receptor in IL‐3/GM‐CSF dependent M‐07e cells, in KG‐1 cells and normal granulocytes, whereas phorbol esters 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) (2 nM) down‐modulates the GM‐CSF receptor in M‐07e cells and granulocytes but not in KG‐1 cells. As data analysis shows by nonlinear regression, the decreased binding ability depends on a reduction of the binding sites with no significant change of their dissociation constant. To gain insight into the mechanisms involved in the GM‐CSF receptor regulation, we investigated the role of protein kinase C (PKC). GM‐CSF, unlike TPA, was unable to activate PKC in all the cells studied. Moreover, unlike TPA, GM‐CSF was still able to down‐modulate its receptor in cells where PKC was inhibited by 1‐(5‐isoquinolonesulphonyl)‐2‐methylpiperazine (H7) and staurosporine or in cells where PKC was exhausted by prolonged incubation with 1 μM TPA. Finally, the receptor re‐expression rate was accelerated by protein kinases inhibitors. These results, taken together, indicate the presence of a PKC‐dependent and ‐independent down‐modulation mechanism and a negative role of the endogeneous protein kinases in GM‐CSF receptor re‐expression.
Modulation of Na+/K+ exchange potentiates lipopolysaccharide‐induced gene expression in murine peritoneal macrophages
Authors: Ohmori Yoshihiro , Reynolds Elwood , Hamilton Thomas A.
Published: February 2005

The role of Na+/K+ exchange in regulating lipopolysaccharide (LPS)‐mediated induction of cytokine gene expression has been examined in murine peritoneal macrophages. Depletion of K+ from the culture medium resulted in a three‐ to five‐fold potentiation of tumor necrosis factor‐α (TNFα), KC (gro), and IP‐10 mRNA expression in LPS‐treated macrophages. The potentiating effect was apparently the result of inhibition of Na+/K+ exchange through the Na+/K+‐adenosine triphosphatase (ATPase) because ouabain‐mediated inhibition of Na+/K+‐ATPase was also able to potentiate cytokine mRNA expression as much or more than did K+ depletion. The effects of K+ depletion or ouabain treatment were not caused by depolarization of the macrophage membrane because depolarization mediated by elevating extracellular K+ levels was inhibitory to cytokine mRNA expression. Depletion of Na+ by substitution with cho ine in the culture medium also markedly potentiated LPS‐induced gene expression. The Na+/H+ antiporter was not, however, involved in potentiating cytokine expression because treatment of macrophages with amiloride either had no effect on or was inhibitory to the LPS‐induced changes in mRNA levels. The potentiation of gene expression was selective and was at least partially the result of increased transcriptional activity of each gene. Whereas Na+ depletion and ouabain both inhibited 86Rb+ uptake by macrophages, treatment with LPS had no effect either on Rb+ uptake or on efflux. Thus altered Na+/K+ exchange is not a component of the primary signalling pathway(s) mediating response to LPS. Nevertheless, modulation of macrophage Na+/K+ exchange by agents encountered during an inflammatory response may be an important determinant of the magnitude and quality of specific gene expression.
Differential regulation of the expression of transforming growth factor‐βs 1 and 2 by retinoic acid, epidermal growth factor, and dexamethasone in NRK‐49F and A549 cells
Authors: Danielpour David , Kim Kyung Young , Winokur Thomas S. , Sporn Michael B.
Published: February 2005

Although most biological activities of transforming growth factor‐βs 1 and 2 (TGF‐β1 and TGF‐β2) examined in vitro are similar or identical, recent studies suggest that each of these factors may be independently regulated in vivo. In this study we have used highly sensitive and specific sandwich enzyme‐linked immunosorbent assays for TGF‐β1 and TGF‐β2 to examine the effects of a variety of treatments on expression of these two TGF‐β isoforms. We show that epidermal growth factor (EGF) induces secretion of TGF‐β1 and not TGF‐β2, whereas retinoic acid (RA) induces secretion of TGF‐β2 and not TGF‐β1 in NRK‐49F normal rat kidney fibroblasts and A549 human lung carcinoma cells. Moreover, treatment with EGF diminishes the levels of TGF‐β2, while RA decreases the levels of TGF‐β1 in both cell lines. Dexamethasone (Dex), on the other hand, inhibits the secretion of both TGF‐β1 and TGF‐β2 in A549 cells, while selectively inhibiting TGF‐β1 secretion in NRK‐49F cells. The interactive effects of EGF, RA, and Dex on the production of TGF‐β1 and TGF‐β2, which were studied on NRK‐49F cells, demonstrate that EGF blocks the induction of TGF‐β2 mRNA and peptide by RA, while Dex inhibits the induction of TGF‐β1 mRNA and peptide by EGF. These results demonstrate that RA, EGF and Dex are each unique, differential, and interactive regulators of the expression of TGF‐βs1 and 2.
Heparin inhibits autocrine stimulation but not fibroblast growth factor stimulation of cell proliferation of androgen‐responsive shionogi carcinoma 115
Authors: Kasayama Soji , Sumitani Satoru , Tanaka Akira , Yamanishi Hiroshi , Nakamura Nobuyoshi , Matsumoto Keishi , Sato Bunzo
Published: February 2005

An androgen‐responsive cloned cell line (SC‐3) derived from Shionogi carcinoma 115 (SC115) has been shown to secrete fibroblast growth factor (FGF)‐like peptide in response to androgen, which binds to FGF receptor and promotes the proliferation of SC‐3 cells in an autocrine mechanism. Since the androgen‐induced autocrine factor has a property to bind heparin, we examined the effects of heparin on the growth of SC‐3 cells. Heparin was found to exhibit significant inhibition of testosterone‐induced growth in a concentration‐dependent manner: Approximately 50% inhibition was found at a concentration of 0.1 μg/ml. DNA synthesis of SC‐3 cells induced by testosterone was also inhibited strongly by heparin, and less strongly by heparan sulfate and dermatan sulfate. Proliferation of SC‐3 cells induced by acidic (a) or basic (b) FGF appeared not to be modulated by heparin. In contrast, heparin efficiently blocked DNA synthesis stimulated with androgen‐induced growth factor in the conditioned medium from testosterone‐treated cells. These results indicate that heparin inhibits autocrine loop in SC‐3 cells induced by androgen. Thus the autocrine growth factor possesses a different characteristic from aFGF and bFGF in that its bioactivities are negatively modulated by the glycosaminoglycan.
Heparanase activity in cultured endothelial cells
Authors: Godder K. , Vlodavsky I. , Eldor A. , Weksler B. B. , Haimovitz‐Freidman A. , Fuks Z.
Published: February 2005

This study was undertaken to identify a heparan sulfate (HS) degradation endoglycosidase (heparanase) in cultured endothelial cells (EC) and to characterize the requirements for its release and subsequent degradation of HS side chains in the subendothelial extracellular matrix (ECM). Intact EC, EC lysates, or EC conditioned media from different sources were incubated with metabolically Na235SO4‐labeled ECM produced by bovine EC. The released sulfated products were analyzed by gel filtration on Sepharose 6B. Human umbilical vein endothelial cells (HUVEC) and human saphenous vein endothelial cells (HSVEC) lysates expressed heparanase activity as indicated by release of most of the radioactivity from ECM as HS fragments that are one‐fifth to one‐sixth the size of the intact HS side chains. These fragments were sensitive to deamination with nitrous acid and were not produced in the presence of heparin. Rabbit coronary microvascular EC and bovine brain capillary EC lysates showed less heparanase activity (30–35%), whereas bovine aortic and corneal EC showed no activity. Intact HUVEC, piated directly on the labeled ECM, expressed low enzyme activity that was not changed when cells were exposed to various agents. Exposure of HUVEC to interleukin‐1, phorbol myristate acetate, tumor necrosis factor, endotoxin, thrombin, calcium ionophore A23187, fibroblast growth factor, or radiation did not induce release of the enzyme to the medium or degradation of HS in the ECM, as long as the cells remained viable. EC differ from various normal and malignant cells that degrade HS by virtue of their inability to release the enzyme. We suggest that heparanase release during vessel wall injury may regulate the growth of EC and smooth muscle by release of HS degradation products in processes such as wound healing, neovascularization, and atherosclerosis.
Regulation of rat proximal tubule epithelial cell growth by fibroblast growth factors, insulin‐like growth factor‐1 and transforming growth factor‐β, and analysis of fibroblast growth factors in rat kidney
Authors: Zhang Guohong , Ichimura Takaharu , Wallin Alf , Kan Mikio , Stevens James L.
Published: February 2005

Growth factors may play an important role in regulating the growth of the proximal tubule epithelium. To determine which growth factors could be involved, we have investigated the mitogenicity of various purified factors in rat kidney proximal tubule epithelial (RPTE) cells cultured in defined medium. Fibroblast growth factors, aFGF (acidic FGF) and bFGF (basic FGF), stimulate DNA synthesis in a dose‐dependent manner, with ED50 values of 4.5 and 3.2 ng/ml, respectively; their effects are not additive. With cholera toxin in the medium, both aFGF and bFGF can replace insulin or epidermal growth factor (EGF) to attain the maximum level of cell growth, but they cannot eplace cholera toxin. Cholera toxin specifically potentiates the effects of FGFs on DNA synthesis. At high cell density, both insulin and insulin‐like growth factor 1 (IGF‐1) induce DNA synthesis more effectively than EGF, FGFs and cholera toxin. The high concentration (0.2‐1.0 μg/ml) of insulin required for cell growth can be replaced by a low concentration of IGF‐1 (10‐20 ng/ml), indicating that insulin probably acts through a low affinity interaction with the IGF‐1 receptor. Transforming growth factor‐β1 (TGF‐β1) inhibits DNA synthesis induced by individual factors and combinations of factors in a concentration‐dependent manner. Northern blot analysis shows that mRNA for TGF‐β1, IGF‐1, and aFGF, but not bFGF are present in rat kidney. Western blot analysis and bioassay data confirmed that the majority of FGF‐like protein in rat kidney is aFGF. The data suggest that in addition to EGF, IGFs, and TGF‐β, FGFs may also be important kidney‐derived regulators of proximal tubule epithelial cell growth in vivo and in vitro.