bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isolation of a clone of F9 teratocarcinoma cells “naturally” resistant to G418
Authors: Edwards S. A. , Adamson E. D. 
Published: February 2005

AbstractResistance to the neomycin analogue G418 forms the basis of a dominant marker selection system for mammalian (and other) cells transfected with the bacterial neo gene. This system has been particularly effective because of the low incidence of spontaneous conversion to G418 resistance in mammalian cells; no case of resistance to the drug in the absence of the bacterial genes has yet been reported to our knowledge. During the course of transfection experiments, we recently isolated a clone of F9 teratocarcinoma cells which is drug resistant yet has no detectable integrated plasmid sequences, neo RNA transcripts, or aminoglycoside phosphotransferase activity. The G418‐resistant clone (F9nr7) did not display enhanced resistance to other cytotoxic drugs tested: colchicine, actinomycin D, cycloheximide, and hygromycin B. Therefore, nr7 cells differ from multidrug‐resistant phenotypes previously described. However, this clone is inhibited, relative to control cells, in its response to the differentiation‐inducing drugs retinoic acid and dibutyryl cAMP, which suggests that some aspects of general drug metabolism may be altered in these cells.
Ultraviolet radiation directly induces pigment production by cultured human melanocytes
Authors: Friedmann Peter S. , Gilchrest Barbara A. 
Published: February 2005

AbstractIn humans the major stimulus for cutaneous pigmentation is ultraviolet radiation (UVR). Little is known about the mechanism underlying this response, in part because of the complexity of interactions in whole epidermis. Using a recently developed culture system, human melanocytes were exposed daily to a physiologic range of UVR doses from a solar simulator. Responses were determined 24 hours after the last exposure. There was a dose‐related increase in melanin content per cell and uptake of 14C‐DOPA, accompanied by growth inhibition. Cells from donors of different racial origin gave proportionately similar increases in melanin, although there were approximately tenfold differences in basal values. Light and electron microscopy revealed UVR‐stimulated increases in dendricity as well as melanosome number and degree of melanization, analogous to the well‐recognized melanocyte changes following sun exposure of intact skin. Similar responses were seen with Cloudman S91 melanoma cells, although this murine cell line required lower UVR dosages and fewer exposures for maximal stimulation. These data establish that UVR is capable of directly stimulating melanogenesis. Because cyclic AMP elevation has been associated in some settings with increased pigment production by cultured melanocytes, preliminary experiments were conducted to see if the effects of UVR were mediated by cAMP. Both α‐MSH and isobutylmethylxanthine (IBMX), as positive controls, caused a fourfold increase in cAMP level in human melanocytes and/or S91 cells, but following a dose of UVR sufficient to stimulate pigment production there was no change in cAMP level up to 4 hours after exposure. Thus it appears that the UVR‐induced melanogenesis is mediated by cAMP‐independent mechanisms.
Mutant KB cells with decreased EGF receptor expression: Biochemical characterization
Authors: Hwang Jaulang , Richert Nancy , Pastan Ira , Gottesman Michael M. 
Published: February 2005

AbstractMutants of the human KB carcinoma cell line resistant to a cytotoxic conjugate of epidermal growth factor and Pseudomonas exotoxin (EGF‐PE) express a pleiotropic phenotype, which includes reduced levels of 125I‐EGF binding, without altered affinity for EGF (Lyall et al., 1987). Here, the EGF‐toxin (ET) resistant mutants were further characterized with respect to the amount and size of the EGF receptor and the level of EGF receptor RNA. These data indicate that decreased binding of 125I‐EGF in the mutants is due to reduced amounts of EGF receptor, which is associated with decreased mRNA levels. Changes in other proteins in the ET mutants were also examined. Five of the six ET mutants had a decrease in a 78,000 Mr− membrane glycoprotein. In addition, an increase in a protein with a Mr− of 40,000 and a pl = 8.0 was found in all the mutants, and an increase in a series of proteins with a Mr− of 36,000 and a pl of 6.3–6.5 was found in some of the mutants. These results confirm the pleiotropic nature of the EGF‐PE resistant mutants and show that reduced EGF binding is due to altered expression of the EGF receptor gene in the mutants.
Apurinic site induction in the DNA of cells heated at hyperthermic temperatures
Authors: Warters Raymond L. , Brizgys Lucy M. 
Published: February 2005

AbstractThe induction of DNA damage in cells heated at hyperthermic (43–48°C) temperatures was determined by alkaline filter elution and alkaline sucrose gradient‐sedimentation analysis of cell DNA denatured at pH 13.0. A class of DNA lesion which converted to strand breaks during denaturation of DNA at pH 13.0 was produced randomly throughout the cell DNA at temperatures as low as 43°C. Induction of this lesion occurred with a T0 of 90 and 10 min at 45 and 48°C, respectively. We estimate that these pH 13.0–detectable DNA lesions are produced in the cell DNA with a frequency of approximately 75 and 660 per min of heating at 45 and 48°C, respectively. Since the lesions were quantitatively converted to DNA strand breaks at pH 13.0 with a half‐time of 30 min, or less, we suggest that these pH 13.0–detectable DNA lesions are heat‐induced, abasic DNA sites. The induction of these lesions does not appear to be directly involved in the initial heat‐induced inhibition of DNA synthesis. The presence of these lesions cannot be excluded as an explanation for the long‐term inhibition of replicon initiated in heated cells.
Human breast epithelial cells in serum‐free collagen gel primary culture: Growth, morphological, and immunocytochemical analysis
Authors: Yang Jason , Balakrishnan Arun , Hamamoto Susan , Elias Joel J. , Rosenau Werner , Beattie Craig W. , Das Gupta Tapas K. , Wellings Sefton R. , Nandi Satyabrata 
Published: February 2005

AbstractHuman breast epithelial cells derived from various sources (fibroadenoma, reduction mammoplasty, and mastectomy tissues from premenopausal patients) have been cultured in collagen gel matrix using serum‐free medium. Response to various additives has been analyzed for growth‐promoting effect when added to a basal medium containing insulin, cholera toxin, and BSA. A consistent observation has been the effect of EGF and cortisol in growth stimulation of human breast epithelial cells, while separately, each additive elicited only a small response. Under this condition, employing EGF and cortisol combinations, these cells gave rise to organized colonies consisting of clusters of cells, usually spherical, without any duct‐like extensions. Ultrastructural and immunocytochemical studies, using a panel of monoclonal and polyclonal antibodies, have shown that cell types and features that can be identified in the original breast tissue can also be delineated in the progeny populations. The topographical feature, consisting of lumina surrounded by a single inner layer of epithelial cells and an outer layer of basal/myoepithelial cells, can be re‐created in the collagen gel system starting from small clumps of cells.
Maintenance of periportal and pericentral oxygen tensions in primary rat hepatocyte cultures: Influence on cellular DNA and protein content monitored by flow cytometry
Authors: Holzer Claudia , Maier Peter 
Published: February 2005

AbstractPrimary hepatocytes were cultured at oxygen tensions similar to those reported to be present in periportal (13% O2) and pericentral (4% O2) regions of the liver lobules. Cellular DNA and protein content of individual hepatocytes were determined simultaneously by two‐parameter (DNA/protein) flow cytometry after 1, 4, and 7 days in culture. PO2 tensions monitored on line in conventional plastic culture dishes revealed that the depletion of the PO2 in the culture medium depended on the number of hepatocytes plated. When cultured as monolayer after 4–7 days at periportal (13% O2) and more pronounced at pericentral oxygen concentration (4% O2), up to 90% of the hepatocytes showed degenerated nuclei but normal protein content. By using culture dishes with teflon membrane bottoms the oxygen tension in the culture medium was accurately maintained by the incubator atmosphere. At pericentral oxygen tension the fraction of 2N cells increased by about 20%. That of the 4N cell was not affected, and the contribution of 8N hepatocytes dropped to 70% compared to cultures at periportal oxygen tension. Concomitantly, in the 4% O2 hepatocyte cultures the protein content of the 2N and the 4N cells was better preserved and increased by up to 10%. These results suggest that in vitro at pericentral oxygen conditions (4% O2) ageing of hepatocytes is delayed, regenerating processes are better maintained, and, furthermore, freshly isolated 4N hepatocytes have the potency to adapt their metabolism in vitro to periportal as well as to perivenous oxygen tensions.
Ornithine decarboxylase activity during rat spermatogenesis in vivo and in vitro: Selective effect of hormones and growth factors
Authors: Smith Frank F. , Tres Laura L. , Kierszenbaum Abraham L. 
Published: February 2005

AbstractWe have established the patterns of ornithine decarboxylase activity (an enzyme related to cell growth, differentiation, and proliferation) during rat testicular development and studied the effect of epidermal growth factor (EGF), platelet‐derived growth factor (PDGF), transforming growth factor‐type β (TGF‐β), and a serum‐free, hormone/growth factor‐supplemented medium (TKM) on ornithine decarboxylase (ODC) activity in Sertoli‐spermatogenic cell cocultures and cultured seminiferous peritubular cells prepared from sexually immature rats (20–22 days old). Results were correlated with timing of ODC activities during rat testicular development. We have found that: (1) although EGF, alone or combined with PDGF and TGF‐β, and TKM stimulated ODC activity in Sertoli‐spermatogenic cell cocultures after 6 and 24 h of stimulation, PDGF exerted an inhibitory effect, and (2) cultured peritubular cells stimulated with EGF, PDGF, TGF‐β (and their combinations), and TKM displayed an increase in ODC activity after 6 h of stimulation, but ODC activities for most of these treatments declined considerably 24 h after stimulation. Light microscopic autoradiographic studies of [3H]thymidine labeled samples demonstrated that (1) clones of spermatogenic cells traverse S phase synchronously, (2) Sertoli cells are not significantly radiolabeled, probably because of contact inhibition achieved by high cell plating density, and (3) peritubular cells are significantly [3H]thymidine labeled in the presence of TKM, a culture medium that facilitates spermatogenic cell long‐term viability and differentiation. We conclude that TKM and EGF have stimulatory effects on the biochemical pathway that precedes synchronous DNA synthesis in spermatogonia and preleptotene spermatocytes, and that ODC activity is a sensitive marker for monitoring these events.
Mitogenic and antimitogenic effects of cholera toxin‐mediated cyclic AMP levels in 3T3 cells
Authors: Smets Lou A. , Van Rooy Henny 
Published: February 2005

AbstractThe effect of time‐controlled exposures to cholera toxin (CT) on intracellular levels of cyclic AMP (cAMP) and on the proliferative response of serum‐stimulated 3T3 cells was investigated. Continuous exposure to CT caused up to 8‐fold raises in cAMP content and inhibited DNA replication by delaying G1‐S transition and by reducing the fraction of cells committed to DNA replication. In contrast, short exposures to CT during G0‐G1 transition increased the fraction of cells responding to serum stimulation and potentiated the serum‐induced morphological changes in the cell monolayer. A short exposure during late G1 phase, however, inhibited the onset of DNA synthesis but had little effect on ongoing DNA replication. The results indicate that cAMP has diverse and opposite effects on two defined restriction points in cell cycle control. Cyclic AMP was positively involved in the acquistion of the state of competence by quiescent cells (G0‐G1 transition) but antagonistic on the onset of DNA replication (G1‐S transition) in committed cells. The observations reconcile a number of controversial conclusions regarding the role of cAMP in cell cycle control.
Type β transforming growth factor (TGFβ) regulation of alkaline phosphatase expression and other phenotype‐related mRNAs in osteoblastic rat osteosarcoma cells
Authors: Noda Masaki , Rodan Gideon A. 
Published: February 2005

AbstractTGFβ1 from porcine platelets increased alkaline phosphatase (AP) activity in the rat osteoblastic cell line ROS 17/2.8 about three‐fold. This effect was dose‐dependent with an ED50 of about ∼0.2 ng/ml and was larger during logarithmic growth than at confluence. TGFβ1 inhibited cell growth by about 30% with similar dose dependence. Thirty min exposure to TGFβ1 was sufficient to increase AP activity 3 days later by about two‐fold but did not affect cell growth, suggesting dissociation between effects on proliferation and differentiation. The rise in AP activity started 6 h after TGFβ1 addition and was blocked by cycloheximide and actinomycin D. TGFβ1 also increased AP mRNA by two‐ to three‐fold and this effect was not blocked by cycloheximide. The half‐life of AP mRNA, estimated following the addition of 5,6‐dichloro‐1‐β‐D‐ribofuranosylbenzimidazole was about ten h in both control and TGFβ1‐treated cells. The mRNAs for type I procollagen and osteonectin were also increased by TGFβ1 but fibronectin mRNA was decreased. TGFβ2 effects on AP and cell growth were similar to those of TGFβ1, except for lack of activity following transient exposure. At saturating concentrations, TGFβ2 (2 ng/ml) or dexamethasone (10−7M), which has similar effects on these cells, did not further augment the effects of TGFβ1 (at 2 ng/ml). Above findings suggest that TGFβ promotes osteoblatic differentiation in rat osteosarcoma cells at least in part by acting at the pretranslational level.
Inhibition of skeletal muscle satellite cell differentiation by transforming growth factor‐beta
Authors: Allen Ronald E. , Boxhorn Linda K. 
Published: February 2005

AbstractSkeletal muscle satellite cells were cultured from mature rats and were treated in vitro with transforming growth factor‐beta (TGF‐beta). Muscle‐specific protein synthesis and satellite cell fusion were used as indicators of muscle differentiation; a dose‐dependent inhibition of differentiation was observed in response to TGF‐beta. In addition, TGF‐beta depressed cell proliferation in a dose‐dependent manner. Half‐maximal inhibition of differentiation was seen with a TGF‐beta concentration of approximately 0.1 ng/ml. Although proliferation was not inhibited, it was depressed and half‐maximal suppression of proliferation occurred in response to 0.1–0.5 ng TGF‐beta/ml. Neonatal rat myoblasts were also subjected to TGF‐beta treatment, and similar results were observed. Neonatal cells, however, were more sensitive to TGF‐beta than satellite cells, as indicated by the reduced concentrations of TGF‐beta required to inhibit differentiation and reduce the rate of proliferation. Under identical culture conditions proliferation of muscle‐derived fibroblasts were also depressed. The differentiation inhibiting effect of TGF‐beta on satellite cells was reversible. It has been suggested that TGF‐beta could be an important regulator of tissue repair, and its in vitro effects on satellite cells suggest a possible role in regulation of muscle regeneration.
Search for multienzyme complexes of DNA precursor pathways in uninfected mammalian cells and in cells infected with herpes simplex virus type I
Authors: Harvey Gillian , Pearson Colin K. 
Published: February 2005

AbstractConfirmatory evidence for the existence of a multienzyme complex of DNA precursor pathways in mammalian cells was obtained. Using neutral sucrose gradient centrifugation of cell lysates we found that at least five enzymes involved in DNA precursor metabolism in uninfected, S‐phase BHK‐cell fibroblasts cosediment at a common rate, indicative of a multienzyme complex. The enzymes include DNA polymerase thymidine kinase, ribonucleotide reductase, dihydrofolate reductase, and NDP‐kinase. This complex was partially, but not completely, disrupted when lysates from G0‐phase cells were centrifuged. Using lysates from cells infected with herpes simplex virus (HSV) type I some of the virus‐induced ribonucleotide reductase and a minor proportion of the HSV‐thymidine kinase cosedimented rapidly. The virus‐induced DNA polymerase sedimented independently near the middle of the gradient, in contrast to the behaviour of the host polymerase. The enzyme associations observed were disrupted by NaCl or by inclusion of ethylenediamine tetraacetic acid during the cell lysis procedure, instead of the usual EGTA. These results indicate the importance of ionic forces in maintaining the enzyme complexes. The bulk of the DNA and the RNA present in the lysates did not sediment at the same rate as the complexes, showing that the enzymes were not simply adhering nonspecifically to these polyanions. Newly synthesised radiolabeled DNA (15 min pulse with [3H]thymidine) was not preferentially associated with the enzymes, but some functional DNA was evident in the enzyme complex fraction from the uninfected S‐phase cells. DNA polymerase activity in this fraction did not require, nor was it stimulated by, exogenous “activated” DNA. Added DNA primer‐template was required, however, for maximal activity of the polymerase in gradient fractions derived from G0‐phase cells and from HSV‐infected cells. No evidence for channeling of ribonucleotide precursors into DNA of permeabilized cells (uninfected or HSV‐infected) was detected. Most rCDP was incorporated into RNA. In the uninfected, S‐phase cells about 10 pmol/106 cells/90 min of rCDP residues was incorporated into DNA compared with 120 pmol/106 cells/90 min when radiolabeled dCTP was used. Nonradioactive dCTP present in equimolar concentration in the incubation with labeled rCDP did not, however, diminish the incorporation of label from the ribonucleotide. In permeabilized HSV‐infected cells incorporation of radiolabel from rCDP into DNA was barely detectable.
Stimulation of DNA replication by growth factor and hormones in Swiss 3T3 cells: Comparison of the rate of entry into S phase with in vitro DNA synthesis and DNA polymerase α activity
Authors: Otto Angela M. , Smith Christina , De Asua Luis Jimenez 
Published: February 2005

AbstractAn approach to the investigation how growth factors and hormones regulate mammalian cell proliferation is to study the activity of enzymes involved in DNA replication. Quiescent cultures of Swiss mouse 3T3 cells were stimulated with prostaglandin F2α insulin, and/or hydrocortisone for a time at which less than 50% of the cells had initiated DNA synthesis. Such cells were lysed with a Ca+ +‐containing hypotonic buffer and incubated with a nucleotide mixture including [3H]thymidine‐triphosphate for 1 hr at 37°C. The amount of radioactive label incorporated into the trichloroacetic acid (TCA)‐precipitate and the percentage of labeled nuclei correlated with the in vivo stimulation. Analysis of radioactively and density‐labeled DNA in sucrose and CsC gradients indicated that the incorporation of label reflected semiconservative replication. DNA polymerase activities were assayed in supernatants from whole‐cell lysates prepared with a hypotonic buffer not containing Ca+ +. Using various templates, it was shown that the increase in activity of DNA polymerase α correlated with the percentage of cells in S phase upon the different stimulation, while DNA polymerase β activity after various times of stimulation showed that this activity increased only when cells began to enter S phase, regardless of the combination of growth factor and hormones.
Comparison of tyrosinase levels in amelanotic and melanotic melanoma cell cultures by a competitive enzyme‐linked immunoadsorbent assay and by immunotitration analysis
Authors: Fuller Bryan B. , Iman Deborah S. , Lunsford Julie B. 
Published: February 2005

AbstractMelanogenesis in mammalian pigment cells is regulated by changes in the activity of tryosinase, the rate‐limiting enzyme for melanin synthesis. Because recent evidence suggests that this enzyme may exist in pigment cells in both active and inactive stages, a competitive enzyme‐linked immunoadsorbent assay (ELISA) was developed to compare tyrosinase levels in amelanotic and melanotic melanoma cell clones. The melanotic cell line used for this study, MEL‐11A, had basal tyrosinase levels approximately 40 times that of the amelanotic cell line, AM‐7. Both cell lines responded to melanocyte‐stimulating hormone by demonstrating large increases in tyrosinase activity. For competitive ELISA analysis of tyrosinase levels in these two clones, microtiter plates were coated with purified tyrosinase, and trypsinized cell extracts were tested for their ability to compete with bound tyrosinase for antibody binding. Although tyrosinase activity in the amelanotic clone was 1/40 that of the melanotic clone, immunoreactive tyrosinase levels in AM‐7 cells were found to be approximately one‐half that present in the melanotic clone. Additional evidence for the presence of an inactive (or at least, catalytically less active) enzyme in AM‐7 cells was obtained from immunotitration analysis of tyrosinase in cell extracts from both cell lines. These results suggest that at least some amelanotic melanoma cells may contain significant levels of catalytically inactive tyrosinase molecules and that the level of pigmentation in mammalian melanocytes may be regulated by a tyrosinase activation process.
Stimulation of mono (ADP‐ribosyl)ation by reduced extracellular calcium levels in human fibroblasts
Authors: Duncan Michael R. , Rankin Patrick R. , King Robert L. , Jacobson Myron K. , Dell'Orco Robert T. 
Published: February 2005

AbstractLowering extracellular calcium in cultures of human diploid fibroblast‐like cells caused a rapid depletion of NAD pools. This loss of NAD was reversed by restoring extracellular Ca2+ and was inhibited by 3‐aminobenzamide, an inhibitor of ADP‐ribosyl transfer reactions. The concentrations of 3‐aminobenzamide needed to inhibit the loss of NAD were consistent with those required to inhibit cellular mono(ADP‐ribosyl) rather than poly(ADP‐ribosyl) reactions. Calcium depletion did not inhibit the biosynthesis of NAD. These results suggest that mono(ADP‐ribosyl)ation is involved in the regulation of cellular Ca2+ levels.