bluuebunny/crossref_metadata_2025_split huggingface dataset:
Immortalization of human endothelial cells by murine sarcoma viruses, without morphologic transformation
Authors: Faller Douglas V. , Kourembanas Stella , Ginsberg David , Hannan Robert , Collins Tucker , Ewenstein Bruce M. , Pober Jordan S. , Tantravahi Ramana 
Published: February 2005

AbstractAmphotropic murine leukemia virus pseudotypes of murine sarcoma viruses containing theras or mos oncogenes were constructed to permit efficient introduction of the sarcoma virus genome into early‐passage human umbilical vein endothelial cells. The resulting cell lines were morphologically and phenotypically unchanged, retaining properties characteristic of differentiated endothelial cells. For example, the cells in a Kirsten sarcoma virus‐modified line were found to biosvnthesize and secrete von Willebrand factor in both a constitutive and regulated manner, and they contained ultrastructurally identifiable Weibel‐Palade bodies, an endothelial cell‐specific organelle. In contrast to the parent cultures, sarcoma virus‐modified cells were able to proliferate indefinitely in culture. Examination of both Kirsten sarcoma and Moloney leukemia virus‐modified lines indicated that the immortalized cells retained a diploid female karyotype after over 18 months in culture. In addition, the sarcoma virus‐modified cells were able to grow independently of added endothelial cell growth factor. This growth factor autonomy does not appear to be due to autocrine production of a biologically cross‐reactive growth factor. These immortal, virus‐modified endothelial cells express large amounts of sarcoma virus‐specific mRNA but no detectable helper virus or transforming virus activity. This technique for immortalization of primary human cells without alteration of the differentiated characteristics of the cell type is readily applied to a variety of human cell types. Moreover, the ability to separate the immortalizing and transforming activities of viral oncogenes should provide further understanding as to mechanisms of oncogene action.
Glutamine and glutamate nitrogen exchangeable pools in cultured fibroblasts: A stable isotope study
Authors: Darmaun Dominique , Matthews Dwight E. , Desjeux Jehan‐Francois , Bier Dennis M. 
Published: February 2005

AbstractGlutamine's role as an energetic fuel has been extensively studied in the past using 14C‐ and 3H‐labeled tracers in cultured human cells. Yet another prominent role of glutamine, that of a nitrogen shuttle, cannot be approached without an N‐tracer. We therefore used 15N‐labeled glutamine and glutamate to address the following questions: (1) is it possible to study the exchangeable pools of intracellular free glutamine and glutamate nitrogen with stable isotope methods? and (2) to what extent is intracellular glutamine pool regulated by extracellular glutamine? We observed that: (1) intracellular [15N]‐glutamine enrichment reached a plateau at 80% within 20 min of incubation in a buffer containing 0.7 mM pure 15N‐glutamine and no glutamate; in contrast, intracellular 15N‐glutamate enrichment rose only to 40% after 4 hours of incubation in a buffer containing 0.5 mM pure 15N‐glutamate and no glutamine; (2) the cell‐free glutamine content was tightly dependent on extracellular glutamine level, while the cell‐free glutamate remained steady irrespective of the extracellular glutamate level; (3) the cells took up glutamine and glutamate against a concentration gradient; the rate of glutamine uptake accounted for 90% of the cell glutamine turnover rate; and (4) when cells were confronted with a glutamine‐free medium, only one fourth of intracellular glutamine was derived from the exchangeable glutamate. We conclude that: (1) The size and turnover rate of the intracellular pool of free glutamine nitrogen are measurable using stable isotope methodology; (2) glutamine uptake from the extracellular medium accounts for most of glutamine turnover rate in cultured fibroblasts; and (3) intracellular free glutamate is divided up between several pools in cultured human fibroblasts.
Differentiation‐dependent production of a platelet‐derived growth factor‐like mitoattractant by endoderm cells derived from embryonal carcinoma cells
Authors: Crotendorst Gary R. , Harvey Anita K. , Nagarajan Lalitha , Anderson Wayne B. , Gatewood Elizabeth 
Published: February 2005

AbstractRetinoic acid‐induced differentiation of F9 embryonal carcinoma cells to endoderm provokes the secretion of a protein factor that acts as both a chemoattractant and mitogen for smooth muscle cells. Undifferentiated F9 cells and PSA‐5E (visceral endodermlike) cells produced little of this factor. However, PYS‐2 (parietal endodermlike) and Dif 5 endoderm cells were found to produce significant amounts of endoderm‐derived mitoattractant (EDM) activity. The activity secreted by the Dif 5 cells was partially purified using gel filtration chromatography using chemotaxis and mitogenic assays as markers for biological activity. The partially purified activity competes with [125l]iodoplatelet‐derived growth factor (PDGF) for binding to target cells, and the biological activity is neutralized with anti‐PDGF lgG, suggesting shared domains in the two molecules. However, the factor appears to be different from PDGF, based on its thermal stability, molecular weight, and charge. The differentiated endoderm cells including retinoic acid (RA)‐treated F9, Dif 5, PSA‐5E, and PYS‐2 cells also exhibit specific [125l]iodo‐PDGF binding, and the PSA‐5E cells respond to PDGF as a chemoattractant. Conceivably, such a PDGF‐like factor may contribute to the regulation of cell growth and migration during the early stages of embryogenesis.
Interactions of arterial cells: III. Stathmokinetic analyses of smooth muscle cells cocultured with endothelial cells
Authors: Staiano‐Coico Lisa , Hajjar David P. , Hefton John M. , Hajjar Katherine A. , Kimmel Marek 
Published: February 2005

AbstractArterial endothelial cells (EC) or their conditioned medium (ECCM) can alter the proliferation of cocultured arterial smooth muscle cells (SMC). Previously, we have shown, as have others, that EC regulate the growth of cocultured SMC depending on the density of both cell types. To ascertain the rate of cell‐cycle traverse in preconfluent arterial SMC cocultured with arterial EC or ECCM (derived from preconfluent EC), we have conducted a series of stathmokinetic experiments using flow cytometry to determine where specific changes may occur in the cell cycle. Results of our experiments indicate for the first time that ECCM stimulates the proliferation of preconfluent SMC by significantly shortening the residence times in the G1 and S phases of the cell cycle. The predominant relative effect occurs within the early G1 (G1A) compartment where pretreatment with ECCM shortens the residence time by approximately 55%. Furthermore, we have observed that preincubation of serum‐free ECCM with antiplatelet‐derived growth factor (PDGF) antibody abolishes any mitogenic effect on SMC. This suggests that EC secrete PDGF‐like molecules which enhance the proliferation rate of preconfluent, cocultured SMC. These findings support the hypothesis that arterial EC may secrete mitogens which stimulate arterial SMC proliferation in the vascular wall.
The effects of recombinant CSF‐1 on the blast cells of acute myeloblastic leukemia in suspension culture
Authors: Miyauchi Jun , Wang Chen , Kelleher Colm A. , Wong Gordon G. , Clark Steven C. , Minden Mark D. , McCulloch Ernest A. 
Published: February 2005

AbstractRecombinant hemopoietic colony‐stimulating factors (CSFs), including GM‐CSF, G‐CSF and IL‐3, have been shown to be effective stimulators of both self‐renewal and terminal differentiation of blast stem cells in acute myeloblastic leukemia (AML). We have examined the activity of a fourth growth factor, recombinant CSF‐1 (or M‐CSF), on the growth of leukemic blasts in culture. CSF‐1 was found to be active on some, but not all, blast populations. In sensitive cells, CSF‐1 often stimulated the production of adherent blast cells incapable of division. This observation leads us to suggest that CSF‐1 may be useful in the treatment of selected cases of AML.
Soluble factor(s) released by the PF‐382 T‐cell line enhances the stimulatory effect of monocytes on the BFU‐E growth
Authors: Bellone Graziella , Avanzi Gian Carlo , Lista Patrizia , Hibbin Jill , Saglio Giuseppe , Benetton Gabriella , Foa Robert , Pegoraro Luigi 
Published: February 2005

AbstractPF‐382 is a human T‐cell line that has been shown to elaborate factors that modulate normal hemopoiesis in vitro. In the present study we report that this cell line constitutively releases in both serum‐containing and serum‐free supernatants a potent enhancer of BFU‐E growth. The factor(s), partially purified by gel filtration, is a heat‐stable molecule(s) degradable by trypsin and 2–mercaptoethanol treatments, equally active on bone marrow and peripheral blood erythroid progenitor cells, but not on CFU‐GM. Unlike other sources of BPA, this stimulatory factor(s) exerts its effect in the presence of mononuclear adherent cells. In fact, the addition of conditioned medium obtained by 48 hr preincubation of isolated monocytes with 10% PF–382 supernatant (M‐CM2) or the concomitant addition of supernatant from PF–382 cells (PF–382–CM) and from unstimulated monocytes (M‐CM1) are capable of fully replacing the presence of monocytes in the BFU‐E assay. Since the independent addition of PF‐382‐CM or of M‐CM1 is devoid of stimulatory function, we suggest that the PF‐382 derived BFU‐E growth inducer, which differs from IL–1, IL–3, IL–4, GM and G‐CSF, exerts its activity “via” a synergistic mechanism with a monokine.
Synthesis and secretion of immunoglobulin G by lymphocytes from cultured mouse spleen cells is not affected by heat shock
Authors: Rodenhiser David I. , Atkinson Burr G. , Jung Jack H. 
Published: February 2005

AbstractShort‐term hyperthermic episodes (in vivo and in vitro) alter gene expression in mammalian lymphocytes, resulting in the enhanced synthesis of a select group of polypeptides—the heat‐shock proteins—and the depressed synthesis of many normally synthesized polypeptides. Such alterations could have profound implications to an individual if the appropriate functioning of lymphocytes within the immune response was compromised by a depression in immunoglobulin synthesis during naturally occurring periods of hyperthermia, such as fever. In the present study we asked if heat‐shock affects the facultative synthesis and secretion of immunoglobulin G by cultured mouse lymphocytes. We found that the quantity of immunoglobulin G synthesized and secreted by these cells is not affected by heat‐shock treatments sufficient to induce the synthesis of heat‐shock proteins.
Actions of transforming growth factor‐β on muscle cells
Authors: Florini James R. , Ewton Daina Z. 
Published: February 2005

AbstractIt has recently been reported by three laboratories that transforming growth factor‐β (TGF‐β) is a potent and reversible inhibitor of differentiation in myogenic cells. To improve our understanding of this inhibition, we investigated the effects of TGF‐β on several other processes in L6 myoblasts, with emphasis on actions of the insulin‐like hormones (which stimulate myoblast differentiation). We found that TGF‐β had no effect on the binding of insulin‐like growth factors (IGFs) to their receptors on the cell surface, and it had little or no effect on some actions of the IGFs. There was essentially no change in the suppression of proteolysis or the stimulation of cell proliferation by IGFs when TGF‐β was also added to the medium. However, there was an effect of TGF‐β on another process stimulated by the IGFs; TGF‐β was an equally active and more potent stimulator of amino acid uptake than was IGF‐I, and the stimulation was additive beyond the maximal response attained with IGF‐I, suggesting that the two act by different mechanisms. TGF‐β had significant effects on myoblast morphology, causing the formation of abundant stress fibers containing cytoplasmic (but not myofibrillar) actin. Addition of TGF‐β at various times after initiation of differentiation demonstrated that TGF‐β inhibits an early process in differentiation. Thus it appears that the interactions of TGF‐β and the IGFs in myoblasts are complex; in some instances the effects of IGFs are inhibited and in others they are mimicked or are unaffected. It is clear that TGF‐β does not act by simply interfering with IGF binding or blocking early steps in its action on myoblasts.
Thermotolerance induced by heat, sodium arsenite, or puromycin: Its inhibition and differences between 43°C and 45°C
Authors: Lee Yong J. , Dewey William C. 
Published: February 2005

AbstractWhen CHO cells were treated either for 10 min at 45–45.5°C or for 1 hr with 100 μM sodium arsenite (ARS) or for 2 hr with 20 μg/ml puromycin (PUR‐20), they became thermotolerant to a heat treatment at 45–45.5°C administered 4–14 hr later, with thermotolerance ratios at 10−3 isosurvival of 4–6, 2–3.2, and 1.7, respectively. These treatments caused an increase in synthesis of HSP families (70, 87, and 110 kDa) relative to total protein synthesis. However, for a given amount of thermotolerance, the ARS and PUR‐20 treatments induced 4 times more synthesis than the heat treatment. This decreased effectiveness of the ARS treatment may occur because ARS has been reported to stimulate minimal redistribution of HSP‐70 to the nucleus and nucleolus. Inhibiting protein synthesis with cycloheximide (CHM, 10 μg/ml) or PUR (100 μg/ml) after the initial treatments greatly inhibited thermotolerance to 45–45.5°C in all cases. However, for a challenge at 43°C, thermotolerance was inhibited only for the ARS and PUR‐20 treatments. CHM did not suppress heat‐induced thermotolerance to 43°C, which was the same as heat protection observed when CHM was added before and during heating at 43°C without the initial heat treatment. These differences between the initial treatments and between 43 and 45°C may possibly be explained by reports that show that heat causes more redistribution of HSP‐70 to the nucleus and nucleolus than ARS and that redistribution of HSP‐70 can occur during heating at 42°C with or without the presence of CHM. Heating cells at 43°C for 5 hr after thermotolerance had developed induced additional thermotolerance, as measured with a challenge at 45°C immediately after heating at 43°C. Compared to the nonthermotolerant cells, thermotolerance ratios were 10 for the ARS treatment and 8.5 for the initial heat treatment. Adding CHM (10 μg/ml) or PUR (100 μg/ml) to inhibit protein synthesis during heating at 43°C did not greatly reduce this additional thermotolerance. If, however, protein synthesis was inhibited between the initial heat treatment and heating at 43°C, protein synthesis was required during 43°C for the development of additional thermotolerance to 45°C. These data suggest that if a considerable amount of synthesis of HSP families occurred after the initial treatment before heating at 43°C, redistribution during 43°C of the previously synthesized HSP families could lead to the additional thermotolerance to 45°C.
Prostaglandin‐sensitive adenylyl cyclase of cultured preadipocytes and mature adipocytes of the rat: Probable role of Gi in determination of stimulatory or inhibitory action
Authors: Lu Zhongding , Piñeyro Marco A. , Kirkland James L. , Li Zhen‐Hua , Gregerman Robert I. 
Published: February 2005

AbstractPreadipocytes of rats were obtained from the stromal‐vascular fraction of collagenase‐digested perirenal fat pads and grown in serum‐containing medium. By day 8 of culture the cells reached confluence and by 12 days were lipid‐laden. The adenylyl cyclase of the plasma membranes was compared to that of mature fat cells. Unlike the membranes from adipocytes, the preadipocytes showed adenylyl cyclase activity that was stimulated by GTP. Stimulation of preadipocyte membranes by Gpp(NH)p, NaF, and forskolin was comparable to that of membranes from adipocytes, but the response to epinephrine and isoproterenol was minimal (approximately 1.5‐fold for preadipocytes vs. 4–5‐fold for adipocytes). In contrast, GTP‐dependent stimulation of adenylyl cyclase of preadipocytes by PGE1 was nearly 8‐fold. Stimulation occurred even in the presence of both GTP and 140 mM NaCl, a condition that leads to inhibition by PGE1 of adenylyl cyclase in membranes of adipocytes. Other characteristics of the adenylyl cyclase of preadipocyte membranes that differ from those of adipocytes include lack of inhibition by GTP of forskolin‐activated activity, and, following treatment with pertussis toxin, enhanced stimulation by PGE1. ADP‐ribosylation of Gi and Gs with pertussis and cholera toxins, respectively, indicated that the membranes of preadipocytes contained only 5–11% of the Gi of adipocytes and a much lower ratio of Gi:Gs. These findings suggest that cultured preadipocytes have an incompletely developed Gi pathway that may account for the stimulatory effect of prostaglandins on the adenylyl cyclase of these cells as opposed to the inhibitory action of PG in mature fat cells.
Lipid content and metabolism of human keratinocyte cultures grown at the air‐medium interface
Authors: Williams Mary L. , Brown Barbara E. , Monger Daniel J. , Grayson Stephen , Elias Peter M. 
Published: February 2005

AbstractThe differentiation of human keratinocytes in most culture systems is incomplete; e.g., lamellar bodies, the characteristic lipid‐delivery organelles of epidermis, are not present. Moreover, their lipid profile does not reflect the distinctive composition found in cornifying epidermis. In contrast, keratinocytes that grow at an air‐medium interface exhibit more complete differentiation. In this study, we compared the elaboration of lamellar bodies, the lipid content, and the lipid metabolism of human keratinocytes, cultured both under standard immersed conditions and after lifting to an air‐medium interface. Whereas submerged cultures neither elaborated lamellar bodies nor displayed a lipid distribution characteristic of cornifying epidermis, lifted cultures displayed advanced cornification, elaborated lamellar bodies which were deposited in intercellular domains, and a lipid profile more typical of cornifying epidermis. Moreover, lipid biosynthesis was 5–10‐fold more active in lifted than in immersed cultures, and was not inhibited by exogenous lipoproteins. These findings are consistent with recent studies that demonstrate both high rates of lipogenesis in differentiating layers of the epidermis as well as autonomy of lipogenesis from the influence of circulating lipoproteins. Thus, the lipid content and metabolism of human keratinocyte cultures, grown at an air‐medium interface, demonstrate features that simulate the epidermis.
Bone marrow stromal proteoglycan heterogeneity: Phenotypic variability between cell lines and the effects of glucocorticoid
Authors: Bentley Stuart A. , Kirby Suzanne L. , Anklesaria Pervin , Greenberger Joel S. 
Published: February 2005

AbstractHematopoiesis in vivo is dependent upon the interaction of hematopoietic stem cells with a complex microenvironment, of which stromal proteoglycans are an important functional component. Certain bone marrow stromal cell lines provide a microenvironment that supports hematopoiesis in vitro, a function that is dependent upon glucocorticoid supplementation. Proteoglycan synthesis in the hematopoietic‐supportive D2XRll, Bl6 and 14F1 bone marrow stromal cell lines was studied by 35S‐sulfate precursor labelling and ion‐exchange separation, followed by isopyknic CsCl density centrifugation and gel filtration HPLC. The effects of glucocorticoid were also investigated. A similar pattern of proteoglycan heterogeneity was observed in all three cell lines, although there was considerable quantitative variation. All cultures synthesized three species of chondroitin/dermatan sulfate (CS/DS) proteoglycans: DS1, excluded from a Bio‐Sil TSK‐400 HPLC column, and DS2, eluting at Kd = 0.31, were present mainly in the culture media. The smallest (DS3) eluted at Kd = 0.63 and was present mainly in the cell layers. CS/DS species were the major proteoglycans in all cultures. Hydrocortisone‐free cultures also synthesized heparan sulfate (HS) proteoglycans, including a cell‐associated form (HS1), partially excluded from the TSK‐400 column, and a secretory form (HS2), eluting at Kd = 0.15. D2XRll cells also secreted an apparently‐unique, high‐density proteoglycan, Kd = 0.65, into the culture medium. Hydrocortisone at 10−6 M virtually abolished HS proteoglycan synthesis in all three cell lines, and altered the pattern of CS/DS proteoglycans in the culture media, increasing the quantity of DS1 and DS3, and reducing the quantity of DS2.
Adhesion defective BHK cell mutant has cell surface heparan sulfate proteoglycan of altered properties
Authors: Couchman John R. , Austria Rosario , Woods Anne , Colin Hughes R. 
Published: February 2005

AbstractIn the light of accumulating data that implicate cell surface heparan sulfate proteoglycans (HSPGs) with a role in cell interactions with extracellular matrix molecules such as fibronectin, we have compared the properties of these molecules in wild‐type BHK cells and an adhesion‐defective ricin‐resistant mutant (RicR14). Our results showed that the mutant, unlike BHK cells, cannot form focal adhesions when adherent to planar substrates in the presence of serum. Furthermore, while both cell lines possess similar amounts of cell surface HSPG with hydrophobic properties, that of RicR14 cells had decreased sulfation, reduced affinity for fibronectin and decreased half‐life on the cell surface when compared to the normal counterpart. Our conclusions based on this data are that these altered properties may, in part, account for the adhesion defect in the ricin‐resistant mutant. Whether this results from the known alteration in assembly of N‐linked glycans affecting the carbohydrate chains on the proteoglycan or some other combination of factors is discussed.
Effect on platelet‐derived growth factor‐induced mitogenesis of double‐stranded RNA: Evidence for an autocrine growth inhibition mediated by interferon‐β
Authors: Forsberg Karin , Paulsson Ylva , Westermark Bengt 
Published: February 2005

AbstractStimulation of normal human foreskin fibroblasts with platelet‐derived growth factor (PDGF) was inhibited by the addition of the synthetic double‐stranded RNA polyinosinic‐polycytidylic acid (poly‐I:C) as measured by incorporation of 3H‐thymidine (3H‐TdR). Single‐stranded polycytidylic or polyinosinic acid had no effect. Double‐stranded RNA is an inducer of interferon‐β (IFN‐β) in fibroblasts. On the mRNA level, an expression of IFN‐β 2 but not of IFN‐β 1 was seen after addition of PDGF and/or poly‐I:C. The inhibition of PDGF‐induced mitogenesis was completely blocked by an antiserum to IFN‐β. Poly‐I:C did not interfere with PDGF binding to its receptor, nor did it block protein synthesis, indicating that the inhibition is not due to a nonspecific toxic effect of the double‐stranded RNA but rather is mediated by IFN‐β. The present study implies that the IFN‐β system in fibroblasts is a very potent autocrine inhibitory pathway.