bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of erythropoietin‐like activity from mouse spleen cells
Authors: Fagg Barbara , Roitsch Carolyn A. 
Published: February 2005

AbstractSupernatants from mouse spleen cell cultures contain a factor which acts in a similar manner to erythropoietin (Ep) to stimulate the formation of 2‐day erythroid (CFU‐E) colonies in vitro from bone marrow or fetal liver cells. Analysis of conditioned media by high performance liquid chromatography (HPLC) on anion exchange, reverse phase, molecular size exclusion, and hydroxyapatite columns demonstrated that the erythropoietin‐like activity (EpLA) has different biochemical characteristics to mouse Ep from anemic mouse serum. In addition, EpLA has a molecular weight (Mr), of 20,000 daltons determined by SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE), compared to 42,000 for mouse Ep. Partially purified EpLA was found to be active in vivo as well as in vitro. Highly purified preparations of γ‐interferon, Multilineage hemopoietic growth factor (Multi HGF), Interleukin‐2 (IL‐2), IL‐1, and colony stimulating factor 1 (CSF‐1) did not support CFU‐E colony formation. Thus, it was established that EpLA could not be attributed to other known components of spleen cell conditioned medium. Titration of mouse Ep and EpLA suggests that only a portion of the Ep‐responsive CFU‐E population in fetal liver is sensitive to EpLA.
Glucocorticoid stimulation of metabolism and glycerol‐3‐phosphate dehydrogenase activity in cultured heart cells
Authors: Freerksen Deborah L. , Hartzell Charles R. 
Published: February 2005

AbstractThe direct effects of the glucocorticoids hydrocortisone and corticosterone on myocardial metabolism were studied in cultured heart cells by assessing several parameters previously unreported. Hormone and growth factor concentrations were carefully controlled by using a serum‐free medium, which also allowed maintenance of cells in the absence of glucocorticoids. Heart cell beating rate, glucose uptake rate, and CO2 evolution from radioactively labeled glucose were increased by the addition of 0.03 μM corticosterone to the medium of cells maintained in culture for 11 days. There were no further changes in these parameters as steroid concentration was increased to 14.43 μM. The activity of NAD‐linked sn‐glycerol‐3‐phosphate dehydrogenase (EC 1.1.1.8) was increased by both corticosteroids and was dose dependent between 0.06 and 1.44 μM corticosterone. The difference between glycerol‐3‐phosphate dehydrogenase activity in cells maintained with hydrocortisone as compared to cells maintained without hydrocortisone increased with days in culture. The protein and DNA contents of dishes maintained with corticosteroid were depressed, demonstrating an inhibitory effect on cellular replication. Glucocorticoids have numerous direct effects on cardiac cell metabolism, and the nature of these effects suggests that secondary responses of the cell to chronic exposure are significant.
Interferon‐mediated inhibition of differentiation in a murine myoblast cell line
Authors: Multhauf Cecilia , Lough John 
Published: February 2005

AbstractThe effects of highly purified (> 5 × 107 IU/mg) murine β‐interferon (IFN) on a mouse myoblast line (MM14DZ) have been investigated to confirm and extend the previous observation that partially purified chicken interferon inhibits differentiation of cultured avian myoblasts (Lough et al., Biochem Biophys Res Commun 109:92, 1982). Cultures treated with 20–2,000 IU IFN/ml medium for 5 days exhibited dose‐dependent (1) inhibition of differentiation, as indicated by reduced myotube formation and creatine kinase (CK) activity and (2) increases in DNA content, suggesting that the inhibitory effect was accompanied by continued proliferation of myoblasts. Mock‐IFN had no such effects. Based on findings in other systems that IFN inhibits activity of ornithine decarboxylase (ODC), the polyamine products of which are required for myogenesis, the hypothesis that inhibition of differentiation was mediated by an effect of IFN on polyamine metabolism was tested. However, observations that (1) IFN‐treated myoblasts retained control levels of ODC activity and (2) exogenous polyamines did not prevent IFN‐inhibition did not indicate such a mechanism of action. On the other hand, treatment of control cultures with polyamines alone resulted in potentiation of myogenesis as revealed by precocious myotube formation and a marked increase in CK activity.
Evidence for involvement of more than one class of glycoprotein in cell interactions with fibronectin
Authors: Urushihara Hideko , Yamada Kenneth M. 
Published: February 2005

AbstractThe receptor mechanism by which cells attach to fibronectin has been investigated by a combined immunologic and electrophoretic approach. One particular antiserum directed against 3T3 cell plasma membranes was found to contain antibodies that blocked spreading of these murine cells on fibronectin but not on laminin or serum spreading factor (vitronectin). Proteolysis experiments confirmed that this cell line has calcium‐protected polypeptides necessary for cell spreading on fibronectin. Consequently, protein antigens were fractionated according to size by SDS gel electrophoresis, and antigens that could block the inhibitory activity of the polyclonal antibody were identified. One class of blocking antigen appeared to correspond to the 140,000‐dalton complexes favored by several laboratories as fibronectin receptor candidates, but a second class of 45,000 daltons was also apparent. This 45,000‐dalton antigen was a major absorbing activity from 3T3 cell membranes and the predominant activity from L929 membranes. By isoelectric focusing and two‐dimensional gel electrophoresis, it was found to exist as a set of isoelectric point variants with pK = 5.3 to 6.2. Our results indicate that current models postulating a simple, unimolecular receptor mechanism for fibronectin may be oversimplified and that fibronectin may instead interact with more than one protein receptor component on the fibroblast cell surface.
Persistent production of colony‐stimulating factor (CSF‐1) by cloned bone marrow stromal cell line D2XRII after X‐irradiation
Authors: Naparstek Elizabeth , Donnelly Thomas , Shadduck Richard K. , Waheed Abdul , Wagner Katherine , Kase Kenneth R. , Greenberger Joel S. 
Published: February 2005

AbstractThe adherent stromal layer in long‐term bone marrow cultures (LTBMC) provides the cellular environment necessary for the in vitro proliferation and differentiation of pluripotential hematopoietic stem cells. The role of humoral hematopoietic growth factors, colony‐stimulating factors (CSF) in the regulation of hematopoietic cell production in this system is poorly understood. We have recently isolated and cloned an adherent cell line, D2XRII, derived from murine LTBMC. Plateau phase 25 cm2 cultures of 2 × 106 D2XRII cells in 8.0 ml produced CSF‐1 (M‐CSF) at around 100–150 units/0.1 ml medium. Following X‐irradiation there was a dose‐dependent decrease in the production of CSF‐1 to a plateau of 50% of control levels at 10,000 rad. Higher doses did not produce a further decrease. The X‐ray dose reducing CSF‐1 production to 50% was 100‐fold above the lethal dose as measured by clonagenic survival following trypsinization and replating. Trypsinized replated viable adherent but nondividing X‐irradiated D2XRII cells were maintained for up to 8 weeks after irradiation and demonstrated continuous production of CSF‐1. The data indicate significant divergence of two biologic effects of X‐irradiation on plateau‐phase marrow stromal cells: physiologic function of adherence and CSF‐1 production, versus proliferative integrity. This divergence of effects may be very relevant to understanding the mechanism of X‐irradiation‐associated marrow suppression and leukemogenesis.
EGF induces cell cycle arrest of A431 human epidermoid carcinoma cells
Authors: MacLeod Carol L. , Luk Andrew , Castagnola Janice , Cronin Maureen , Mendelsohn John 
Published: February 2005

AbstractThe human carcinoma cell line A431 is unusual in that physiologic concentrations of epidermal growth factor (EGF) inhibit proliferation. In the presence of 5–10 nM EGF proliferation of A431 cells is abruptly and markedly decreased compared to the untreated control cultures, with little loss of cell viability over a 4‐day period. This study was initiated to examine how EGF affects the progression of A431 cells through the cell cycle. Flow cytometric analysis of DNA in EGF‐treated cells reveals a marked change in the cell cycle distribution. The percentage of cells in late S/G2 increases and early S phase is nearly depleted. Since addition of the mitotic inhibitor vinblastine causes accumulation of cells in mitosis and prevents reentry of cells into G1, it is possible to distinguish between slow progression through G1 and G2 and blocks in those phases. When control cells, not treated with EGF, are exposed to vinblastine, the cells accumulate mitotic figures, as expected, and show progression into S, thus diminishing the number of cells in G1. In contrast, no mitotic figures are found among the EGF‐treated cells in the presence or absence of vinblastine, and progression from G1 into S is not observed, as the number of cells in G1 remains constant. These results suggest that there are two EGF‐induced blocks in cell cycle transversal; one is in late S and/or G2, blocking entry into mitosis, and the other is in G1, blocking entry into S phase. After 24 hours of EGF treatment, DNA synthesis is reduced to less than 10% compared to untreated controls as measured by the incorporation of [3H]thymidine or BrdU. In contrast, protein synthesis is inhibited by about twofold. Although inhibition of protein synthesis is less extensive, it occurs 6 hours prior to an equivalent inhibition of DNA synthesis. The rapid decrease in protein synthesis may result in the subsequent cell cycle arrest which occurs several hours later.
Volume changes in activated human neutrophils: The role of Na+/H+ exchange
Authors: Grinstein Sergio , Furuya Wendy , Cragoe Edward J. 
Published: February 2005

AbstractThe apparent volume of neutrophils, as measured electronically with the Coulter counter, has been reported to increase upon treatment with chemotactic factors. The occurrence of a volume change was confirmed by forward angle light scattering and by isotopic measurements of intracellular water space in cells treated with 12‐O‐tetradecanoylphorbol 13, acetate (TPA) or formyl‐methionyl‐leucyl‐phenylalanine (FMLP). Cell swelling was associated with an increase in the osmotic content of the cells, determined from Boyle‐van't Hoff plots, and with an increase in Na+ content, measured by flame photometry. The volume change was inhibited by replacement of extracellular Na+ with K+ or N‐methyl‐D‐glucamine+, or by addition of amiloride. Swelling was also inhibited by the 5‐N‐substituted analogs of amiloride, which are potent specific inhibitors of the Na+/H+ antiport. This pathway is activated in neutrophils by both TPA and FMLP. Activation of Na+/H+ exchange, determined as a Na+‐dependent and amiloride‐sensitive cytoplasmic alkalinization, was also found when neutrophils were treated with hypertonic solutions. The hypertonic activation of the antiport was similarly followed by cell swelling, detectable by electronic sizing. The results indicate that activation of Na+/H+ exchange can lead to significant cell swelling in neutrophils.
Hormonal control of the replication of human fetal fibroblasts: Role of somatomedin C/insulin‐like growth factor I
Authors: Conover Cheryl A. , Rosenfeld Ron G. , Hintz Raymond L. 
Published: February 2005

AbstractSparse cultures of fetal and postnatal human fibroblasts were equivalent in their responsiveness to the mitogenic action of somatomedin C/insulin‐like growth factor I (SM‐C/IGF‐I). At both developmental stages, the addition of SM‐C/IGF‐I (100 ng/ml) increased cell number at day 3 1.4‐fold in serum‐free medium and 2‐fold in the presence of 0.25% human hypopituitary serum. Furthermore, dose‐response curves indicated that there was no difference in the sensitivity of fetal and postnatal fibroblasts to the growth‐promoting effects of SM‐C/IGF‐I, with a half‐maximal response occuring at 6 ng/ml SM‐C/IGF I. This biological action of SM‐C/IGF‐I correlated with SM‐C/IGF‐I binding to fetal and postnatal fibroblast monolayers. Epidermal growth factor (EGF) and platelet‐derived growth factor (PDGF) also stimulated replication of fetal and postnatal fibroblasts. The mitogenic effects of SM‐C/IGF‐I, EGF, and PDGF were additive. Dexamethasone, which alone had no effect, was synergistic with SM‐C/IGF‐I in stimulating replication of postnatal fibroblasts. The combination of SM‐C/IGF‐I (100 ng/ml), dexamethasone (10−7 M), EGF (10 ng/ml), and PDGF (5 ng/ml) had the same mitogenic effectiveness as 10% calf serum (CS) in postnatal cells. In marked contrast, there was no mitogenic interaction between SM‐C/IGF‐I and dexamethasone in fetal fibroblasts. In fetal cells, SM‐C/IGF‐I + EGF + PDGF ± dexamethasone could only account for 50% of the activity of 10% CS. Moreover, fetal cells were 50–100% more responsive than postnatal cells to the proliferative effect of serum.
Removal of cellular water prevents the reformation of the interphase nucleus
Authors: Zieve Gary W. , Slitzky Barry E. 
Published: February 2005

AbstractThe mature snRNP (small nuclear ribonucleoprotein) particles are localized quantitatively in the interphase nucleus. Like many nuclear antigens, they distribute throughout the cytoplasm after the nuclear envelope breaks down during mitosis and then return to the newly formed daughter nuclei in early G1. Their abundance and stability and the availability of monoclonal antibodies that recognize them, make the snRNP particles a useful model system for studying the reformation of the nucleus at the completion of mitosis. A wide variety of metabolic inhibitors and alterations in normal culture conditions were investigated for their ability to interfere with the return of the snRNP particles to daughter nuclei after mitosis. None of the well‐characterized cytoskeletal inhibitors, biosynthetic inhibitors, calcium antagonists, nor ionophores were effective in interfering with this return. However, the removal of cellular water by exposure of cells to hypertonic medium during mitosis blocked the reformation of the nucleus and trapped the snRNP particles in the cytoplasm. In medium of twice the normal tonicity, the function of the mitotic spindle and the cleavage furrow are inhibited, however, the cells reattach to the substratum as if returning to interphase. The chromatin stays condensed and does not form a normal interphase nucleus and the snRNP particles stay dispersed throughout the cytoplasm. This condition is reversible and after return to normal medium the nucleus reforms and the snRNP particles collect in the new nuclei. After gentle extraction of metaphase cells, about 30% of the snRNP particles are soluble, however, the remainder are associated with an insoluble remnant. These data are consistent with the notion that the snRNP particles accumulate in the nucleus due to both preferential solubility and specific binding sites in the interphase nucleus.
Transformation of diploid human fibroblasts by DNA transfection with the v‐sis oncogene
Authors: Fry Dennis G. , Milam Lonnie D. , Maher Veronica M. , McCormick J. Justin 
Published: February 2005

AbstractThe simian sarcoma virus (SSV) oncogene (v‐sis) has a high degree of homology to the cellular gene coding for the B peptide of human platelet‐derived growth factor (PDGF), a potent fibroblast mitogen. The cellular homolog of v‐sis is activated in some mesenchymal human tumors and cell lines drived from them. To determine the phenotype produced by v‐sis in diploid human fibroblasts, we constructed plasmids containing the SSV provirus and drugresistance markers and transfected them into early‐passage human cells. Fibroblasts that had integrated the plasmid were selected for drug resistance and shown to contain and express the v‐sis oncogene by DNA and RNA hybridization. The v‐sis‐expressing cells grew to higher saturation densities than control cells transfected with vector plasmid alone and formed large, well defined foci. This allowed selection of transfectants directly for focus formation. The v‐sis transformed cells continued to grow well in the absence of serum, whereas age‐matched, vector‐transfected control cells ceased replicating under these conditions so that the final difference in density between the two populations was tenfold. Incorporation of thymidine in serum‐free medium by the v‐sis‐transformed cells was independent of exogenous PDGF. In contrast, PDGF increased thymidine incorporation in such medium by the control cells to the level found in the v‐sis‐transformed cells with or without added PDGF. These results suggest that expression of the v‐sis oncogene in diploid human fibroblasts causes sufficient endogenous synthesis of the B chain of PDGF to allow transformants to grow to abnormally high cell densities. When individual v‐sis‐transformed cells were grown on a background of normal cells, this higher cell density at confluence could be visualized as a focus.
Collagenase production by smooth muscle: Correlation of immunoreactive with functional enzyme in the myometrium
Authors: Blair Harry C. , Teitelbaum Steven L. , Ehlich Lynn S. , Jeffrey John J. 
Published: February 2005

AbstractA monospecific antibody to rat uterine collagenase has been produced and employed to study the cell of origin and the time course of production of this enzyme in the involuting rat uterus. The specificity of the anti‐collagenase antibody was confirmed by immunoprecipitation, Western analysis, and by its ability to inhibit the activity of collagenase. Parallel measurements of functional enzyme, both latent and active, bound to tissue collagen were also made in nonpregnant, gravid, and postpartum rat uteri. Immunohistochemical staining of collagenase in sections of rat uterus showed the enzyme to be present in the perinuclear region of the smooth muscle cells only of the involuting myometrium. No detectable collagenase was present in the prepartum or nonpregnant uterus. Identity of the smooth muscle cells was confirmed using an anti‐smooth muscle actin antibody. In addition, the cultured uterine cells from which the immunizing antigen was obtained were also identified as smooth muscle cells. Specificity of the tissue staining was confirmed by the ability of pure rat uterine collagenase to block the reaction of the antibody with the tissue. These observations indicate that smooth muscle cells are capable of producing collagenase and are consistent with the hypothesis that this enzyme presides over the massive collagen degradation seen in postpartum uterine involution. Furthermore, measurement of collagenase bound to uterine collagen revealed that collagenase activity could be detected only at the time that the cells could be seen to be producing the enzyme by immunolocalization. These findings support the concept that collagenase is produced only as needed and not stored, either intra‐ or extracellularly.
Production of C5a antagonist by synovial and peritoneal tissue fibroblasts
Authors: Matzner Yaacov , Gavison Rivka , Shlomai Zipora , Ben‐Bassat Hanna , Liebergal Meir , Robinson Dwight R. , Babior Bernard M. 
Published: February 2005

AbstractFibroblasts grown from synovial and peritoneal tissues release into the medium an inhibitor of neutrophil chemotaxis. The inhibitor resembles the antagonist previously described in synovial and peritoneal fluids. It is a heat stable (56°C) protein of MW ∼40 kDa that counteracts the chemotactic activity of zymosan‐activated serum or purified C5a but not the peptide chemoattractant F‐met‐leu‐phe. No chemotactic inhibitor was detected in media from skin fibroblast cultures or in formal human sera. It is suggested that the inhibitor is produced locally by synovial and peritoneal fibroblasts and that it might play a role in the regulation of inflammation at sites lined with mesothelium.
Restricted growth of rat kidney proximal tubule cells cultured in serum‐supplemented and defined media
Authors: Miller John H. 
Published: February 2005

AbstractProximal tubules suitable for in vitro culture were prepared from rat kidney cortex by a Ficoll‐gradient centrifugation technique which yielded greater than 94% purity. The tubules were seeded into culture dishes, and cell growth was monitored in both Dulbecco's Modified Eagle's Medium containing 10% fetal calf serum and in a defined medium consisting of 50:50 Ham's F12 and Dulbecco's supplemented with insulin, transferrin, and hydrocortisone. Growth in serum‐containing medium was continuous; however, the specific activity of the brush border enzyme alkaline phosphatase decreased rapidly with time, and the culture morphology became fibroblastic by 6 days. Neither collagen‐coating of the dishes nor addition of the differentiation inducer hexamethylene‐bisacetamide had any significant effect on growth or enzyme activity of the cultured cells. Theophylline, another inducer of differentiation, proved cytotoxic. Growth of proximal tubule cells in defined medium proceeded for 4 days before irreversible growth arrest occurred. Alkaline phosphatase activity and epithelial morphology remained relatively constant throughout the culture period. Additions of the growth factors triiodothyronine, prostaglandin E2, and epidermal growth factor were unable to unblock the growth arrest. If cells cultured in defined medium for 3 days were switched to serum‐supplemented medium, continuous growth occurred, but both alkaline phosphatase activity and epithelial morphology were rapidly lost. As a test of the culture method, rabbit proximal tubule cells were cultured under similar conditions in defined medium. Growth was prolific and continuous for up to, but not exceeding, 30 days, and differentiated properties were retained. It was concluded that both rat and rabbit proximal tubule cells have a limited proliferative capacity in vitro but that the capacity of the rat cell to divide is much reduced relative to the rabbit cell.
Ca2+‐dependent cell surface protein phosphorylation may be involved in the initiation of DNA synthesis
Authors: Kleine Leonard P. , Whitfield James F. , Boynton Alton L. 
Published: February 2005

AbstractIncubating T51B rat liver cells in Ca2+‐deficient, serum‐rich medium containing only 0.02 mM Ca2+ strikingly decreased the phosphorylation of several trypsin‐removable cell surface proteins and arrested the cells in late G1 phase. Raising the Ca2+ concentration in the Ca2+ ‐deficient medium from 0.02 mM to 0.5 mM or adding 80 nM TPA (12‐O‐tetradecanoyl‐phorbol‐13‐acetate), a, protein kinase C activator, stimulated the phosphorylation of a certain set of surface proteinS'Within 5 min and the initiation of DNA replication within the next 2 hr. By contrast, incubation in the same Ca2+ ‐deficient medium, which does not affect the proliferation of neoplastic T51B‐261B cells, did not reduce the phosphorylation of cell surface proteins. These observations suggest that the stimulation of a Ca2+ ‐dependent protein kinase (possibly protein kinase C) directly or indirectly phosphorylates certain cell surface proteins that might be part of the mechanism that triggers the Ca2+‐dependent G1→S transition of normal cells. They also suggest that an alteration of this Ca2+‐dependent protein kinase might be the reason for neoplastic cells being able to proliferate in the face of an external Ca2+ shortage that would stop the proliferation of normal cells.