bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression of growth‐regulated genes in normal and SV40 transformed hamster fibroblasts
Authors: Rossi Ann Marie Krueger , Hirschhorn Ricky R. 
Published: May 2005

AbstractTransformation by the oncogenic virus SV40 has been shown to alter the expression of cellular genes at the level of RNA abundance. Many of these genes have yet to be identified. We have determined, by Northern blot analysis, the abundance levels of several growth‐regulated genes in SV40‐transformed cell lines to determine if their expression is altered and correlates with the ability of SV40 transformed cells to grow in low serum containing media. The mRNA abundance levels of the G1‐specific genes 2A9/calcyclin, 2F1/translocase, and 4F1/vimentin were determined in the parental hamster fibroblast cell line, tk−ts13, and in two SV40 transformants, HR5 and HR8 cells, grown in medium containing 10% calf serum (normal medium) and in HR5 and HR8 cells adapted to passage in medium containing low serum. A spontaneous transformant of the parental line capable of growth in low serum in the absence of SV40 transformation (tk−ts13/1%), was also included in these studies. The low serum adapted SV40‐transformed cells and the spontaneous tk−ts13 transformed cells grew more vigorously than their nonadapted counterparts in medium containing low serum. The low serum adapted cells also grew to higher saturation densities in low serum and to densities comparable to those in high serum, whereas the nonadapted cells grew to low saturation densities in low serum, but not as low as the untransformed parental. These growth‐regulated genes were expressed at lower levels in the SV40 transformed cells growing in medium containing high or low serum, and in the adapted parental cells (tk−ts13/1%) grown in medium containing low serum, in comparison with their levels in the nontransformed parental cells (tk−ts13/10%) grown in medium containing high serum. Therefore, the decreased levels in the expression of these growth‐regulated genes could not be correlated to the rapid growth of SV40 transformed cells. We conclude that the molecular mechanism(s) that permits low serum adapted growth and SV40 transformed growth is different, at least in part, from the mechanism operating in nontransformed cells.
Extracellular matrix and cell shape: Potential control points for inhibition of angiogenesis
Authors: Ingber Donald 
Published: May 2005

AbstractCapillary endothelial (CE) cells require two extracellular signals in order to switch from quiescence to growth and back to differentiation during angiogenesis: soluble angiogenic factors and insoluble extracellular matrix (ECM) molecules. Soluble endothelial mitogens, such as basic fibroblast growth factor (FGF), act over large distances to trigger capillary growth, whereas ECM molecules act locally to modulate cell responsiveness to these soluble cues. Recent studies reveal that ECM molecules regulate CE cell growth and differentiation by modulating cell shape and by activating intracellular chemical signaling pathways inside the cell. Recognition of the importance of ECM and cell shape during capillary morphogenesis has led to the identification of a series of new angiogenesis inhibitors. Elucidation of the molecular mechanism of capillary regulation may result in development of even more potent angiogenesis modulators in the future.
Lung‐derived growth factor that stimulates the growth of lung‐metastasizing tumor cells: Identification as transferrin
Authors: Cavanaugh Philip G. , Nicolson Garth L. 
Published: May 2005

AbstractWe have previously shown that culture medium conditioned by lung fragments contains mitogenic activity for lung‐metastasizing tumor cells but not for their non‐metastatic counterparts. The growth‐promoting component from media conditioned by rat and porcine lungs has been purified and partially characterized as a Mr ≈ 66,000 (unreduced) or Mr ≈ 72,000 (reduced) glycoprotein [Cancer Res 49:3928, 1989; J Cell Biochem 43:127, 1990]. Here we report that this factor is the iron transport protein transferrin. Migration distances in sodium dodecyl sulfate and native gel polyacrylamide electrophoresis systems were similar, as were the specific activities and spectrum of mitogenic activities of the lung‐derived growth factor and transferrin. Electrophoretically separated holo‐rat transferrin and rat lung‐derived growth factor displayed similar positive stains for iron. A polyclonal antibody generated against the lung‐derived growth factor cross‐reacted with human and rat transferrin in Western blots, and anti‐human transferrin cross‐reacted with rat lung‐derived growth factor. All of the mitogenic activity contained in crude lung conditioned media could be removed by antibody‐mediated transferrin depletion. The putative cell receptor molecular weights for the lung‐derived growth factor and transferrin were similar as were the molecular weights of polypeptides produced by partial trypsin cleavage of the two. Finally, the amino acid sequence of certain regions of rat lung‐derived growth factor demonstrated a high degree of homology of human transferrin. The physical and biochemical properties, antigenicity, and mitogenic activity of a previously unidentified lung‐derived growth factor for lung‐metastasizing tumor cells indicate that it is transferrin.
Activating mutations in the NH2‐ and COOH‐terminal moieties of the Gsα subunit have dominant phenotypes and distinguishable kinetics of adenylyl cyclase stimulation
Authors: Gupta Sunil K. , Dhanasekaran N. , Heasley Lynn E. , Johnson Gary L. 
Published: May 2005

AbstractThe α subunit polypeptides of the G proteins Gs and Gi2 stimulate and inhibit adenylyl cyclase, respectively. The αs and αi2 subunits are 65% homologous in amino acid sequence but have highly conserved GDP/GTP binding domains. Previously, we mapped the functional adenylyl cyclase activation domain to a 122 amino acid region in the COOH‐terminal moiety of the αs polypeptide (Osawa et al: Cell 63:697–706, 1990). The NH2‐terminal half of the αs polypeptide encodes domains regulating βγ interactions and GDP dissociation. A series of chimeric cDNAs having different lengths of the NH2‐or COOH‐terminal coding sequence of αs substituted with the corresponding αi2 sequence were used to introduce multi‐residue non‐conserved mutations in different domains of the αs polypeptide. Mutation of either the amino‐ or carboxy‐terminus results in an αs polypeptide which constitutively activates cAMP synthesis when expressed in Chinese hamster ovary cells. The activated αs polypeptides having mutations in either the NH2‐ or COOH‐terminus demonstrate an enhanced rate of GTPγS activation of adenylyl cyclase. In membrane preparations from cells expressing the various αs mutants, COOH‐terminal mutants, but not NH2‐terminal αs mutants markedly enhance the maximal stimulation of adenylyl cyclase by GTPγS and fluoride ion. Neither mutation at the NH2‐ nor COOH‐terminus had an effect on the GTPase activity of the αs polypeptides. Thus, mutation at NH2‐and COOH‐termini influence the rate of αs activation, but only the COOH‐terminus appears to be involved in the regulation of the αs polypeptide activation domain that interacts with adenylyl cyclase.
1,25‐Dihydroxyvitamin D3‐mediated alterations in microtubule proteins isolated from chick intestinal epithelium: Analyses by isoelectric focusing
Authors: Nemere Ilka , Feld Carl , Norman Anthony W. 
Published: May 2005

AbstractRecent work has indicated that vectorial Ca2+ transport across the intestinal epithelium occurs in vesicles and may involve the participation of microtubules [Nemere et al., 1986]. Since 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) stimulates this Ca2+ transport process, microtubule (MT) isotypes were studied as a potential regulatory point. The effect of 1,25(OH)2D3 status on tubulin isotypes was analyzed by isoelectric focusing (IEF) gels of taxol stabilized MTs prepared from intestinal epithelium of vitamin D–deficient chicks dosed with vehicle (−D) or 1.3 nmoles of 1,25(OH)2D3 (,D) 2.5, 5, 10, 15, or 43 h prior to sacrifice. Four bands, one of which was identified as α‐tubulin on the basis of Western analysis, increased in Coomassie Blue staining intensity 5–15 h after 1,25(OH)2D3, corresponding to the time course of augmented vesicular Ca2+ transport. Dose‐response studies revealed similar changes in tubulin isotype profiles in IEF gels, again corresponding to doses known to elicit enhanced Ca2+ absorption (52–6,500 pmoles of hormone). The role of Ca2+ transport was also examined. Isoelectrically focused intestinal epithelial tubulin from −D chicks allowed to transport Ca2+ for 30 min revealed increased staining of bands relative to nonabsorbing −D controls. By comparison, Ca2+ transport in D chicks resulted in fainter bands relative to nonabsorbing, D controls. MTs prepared from fasted or fed chicks revealed similar changes upon IEF, but of much smaller magnitude. Enhanced phosphorylation did not account for the appearance of the more acidic bands, although 1,25(OH)2D3 treatment resulted in decreased 32P content of a presumptive non‐tubulin component, relative to preparations from −D controls. Glucocorticoids, which are known to suppress 1,25(OH)2D3‐stimulated Ca2+ transport, led to severely diminished levels of total tubulin, as judged by SDS‐PAGE, rather than altered tubulin isotypes. Thus, MTs of intestine are subject to regulation by hormonal status, as well as by the amount of Ca2+ available for transepithelial transport.
The insulin‐binding domain of insulin receptor is encoded by exon 2 and exon 3
Authors: Yip Cecil C. 
Published: May 2005

AbstractInsulin receptors are disulfide‐linked oligotetramers composed of two heterodimers each containing a 130‐kDa α subunit and a 90‐kDa β subunit. Insulin binds to the extracellular α subunit, and in the process stimulates the autophosphorylation of the β subunit and the expression of tyrosine kinase activity. Studies combining the use of photoaffinity labeling and immunoprecipitation with anti‐peptide antibody have directly demonstrated that the cysteine‐rich domain, encoded by exon 3, in the α subunit is part of the insulin‐binding site of the receptor. Experiments with chimeric insulin receptors and chimeric insulin‐like growth factor I receptors have confirmed that the cysteine‐rich domain constitutes a part of the insulin‐binding site. In addition, results from these experiments suggest that the N‐terminal sequence, encoded by exon 2, in the α subunit also participates in insulin binding. In this review it is proposed that, assuming two insulin‐binding sites per each holoreceptor oligotetramer, each insulin‐binding domain may contain respectively two sub‐domains for hydrophobic and charge contact with insulin, and that high‐affinity binding would require the interaction of both subunits with the possibility of each subunit reciprocally contributing one of the sub‐domains.
Expression and release of phosphatidylinositol anchored cell surface molecules by a cell line derived from sensory neurons
Authors: Théveniau M. , Durbec P. , Rougon G. , Gennarini G. , Wood J. N. 
Published: May 2005

AbstractEarly postnatal mouse dorsal root ganglion neurons were found to express several glycosylphosphatidylinositol‐anchored (GPI) molecules from the immunoglobulin superfamily (neural cell adhesion molecule 120 kD isoform, F3, Thy1) whose expression is developmentally regulated. A hybrid cell line (ND26), made by fusing postmitotic rat dorsal root ganglion (DRG) neurons with the mouse neuroblastoma N18Tg2, could be induced to differentiate by manipulating the composition of the culture medium and expressed similar GPI molecules to DRG neurons. We used this model system to investigate the metabolism of GPI‐anchored molecules. We found that neural cell adhesion molecule 120 Kd isoform expression decreased upon differentiation, whereas the level of F3 and Thy1 increased, suggesting a role in neurite outgrowth processes.The ratio of molecules cleavable by exogenous phosphatidylinositol phospholipase C (PI‐PLC) was similar for all the GPI‐anchored molecules, which could mean that cell‐specific modifications of the basic anchoring structure determine the level of potentially releasable molecules. Measurements of spontaneous release indicated that this reflected the overall level of expression of these molecules by the ND26 cell line.Finally, we observed an effect of dibutyryl cAMP on the level of expression of F3 and Thy1 but not of N‐CAM. However, we could not detect any significant effect of nerve growth factor (NGF) either on the level of expression or on the amount of spontaneously released molecules.
A monoclonal antibody to the T‐cell receptor increases IGF‐I receptor content in normal T‐lymphocytes: Comparison with phytohemagglutinin
Authors: Hartmann Klaus K. P. , Papa Vincenzo , Brown Eric J. , Goldfine Ira D. , Rosenthal Stephen M. , Baier Thomas G. , Kronenwett Michael 
Published: May 2005

AbstractThe biological effects of the IGFs are mediated through interaction with specific cell surface receptors. It has been previously reported that mitogenic activation of T‐lymphocytes by phytohemagglutinin (PHA) is associated with increased IGF‐I receptor content. However, the mechanisms which regulate IGF‐I receptor expression during T‐lymphocyte activation are unknown. To explore further the regulation of IGF‐I receptor expression in T‐cells, we investigated IGF‐I receptor content and mRNA abundance in T‐lymphocytes after stimulation either by PHA or OKT‐3, the latter being a monoclonal antibody directed against the CD‐3 antigen of the T‐cell receptor IGF‐I binding in T‐cells demonstrated increased IGF‐I receptor content after stimulation by both PHA and OKT‐3. Peak binding was induced after 72 h of treatment with PHA and 48 h of treatment with OKT‐3. Affinity cross‐linking of 125I‐IGF‐I to T‐cell membranes demonstrated a single ∼ 130 kDa band which was increased after treatment with PHA or OKT‐3. This band was inhibited by the addition of α‐IR3, a monoclonal antibody to the IGF‐I receptor. Both PHA and OKT‐3 increased IGF‐I receptor mRNA abundance with peak increases at 20 h and 60 h, respectively. Parallel increases in IGF‐I receptor and β‐actin mRNA abundance were observed, consistent with previous studies demonstrating increased actin gene expression after T‐cell activation. Thus, the increase in IGF‐I receptor mRNA abundance markedly preceded the increase in IGF‐I receptor content after PHA stimulation, but the increase in IGF‐I receptor mRNA abundance followed the increase in IGF‐I receptor content after OKT‐3. These studies suggest, therefore, that IGF‐I receptor content in both of these activated cells is not regulated primarily at the level of steady state mRNA.
Cellular distribution of gastric chief cell protein kinase C activity: Differential effects of diacylglycerol, phorbol esters, carbachol, and cholecystokinin
Authors: Raffaniello Robert D. , Raufman Jean‐Piean 
Published: May 2005

AbstractStimulation of chief cells with carbachol or cholecystokinin (CCK) results in the production of inositol trisphosphate (IP3) and diacylglycerol (DAG). Although IP3 increases cell calcium concentration, thereby stimulating pepsinogen secretion, the role of DAG and its target, protein kinase C (PKC), is less clear. To examine the relation between the cellular distribution of PKC activity and pepsinogen secretion, we determined PKC activity in cytosolic and membrane fractions from dispersed chief cells from guinea pig stomach. To validate our assay, we studied the actions of the phorbol ester PMA. PMA caused a rapid, dose‐dependent, 6‐fold increase in pepsinogen secretion and membrane‐associated PKC activity. Similarly, dose‐response curves for pepsinogen secretion and the increase in membrane‐associated PKC activity induced by a membrane‐permeant DAG (1‐oleoyl‐2‐acetylglycerol) were superimposable. In contrast, CCK (0.1 nM to 1.0 μM) and carbachol (0.1 μM to 1.0 mM) caused a 4‐fold increase in pepsinogen secretion, but did not alter the distribution of PKC activity. These results indicate that in gastric chief cells, PMA‐and DAG‐induced pepsinogen secretion is accompanied by increased membrane‐associated PKC activity. However, the cellular distribution of PKC activity is not altered by CCK or carbachol.
Cloning and sequence determination of bovine insulin‐like growth factor binding protein‐2 (IGFBP‐2): Comparison of its structral and functional properties with IGFBP‐1
Authors: Busby Walker H. , McCusker Robert H. , Clemmons David R. , Bourner Maureen J. , Siegel Ned R. , Krivi Gwen G. 
Published: May 2005

AbstractInsulin‐like growth factor binding proteins (IGFBPs) are secreted by several cell types and can modify IGF actions. Mandin‐Darby Bovine Kidney (MDBK) cells have been shown to secrete a 34,000 Da form of IGF binding protein whose N‐terminal sequence is similar to a form of IGFBP purified from rat BRL‐3A cells that has recently been named IGFBP‐2. These studies report the complete amino acid sequence of bovine IGFBP‐2 and compare its functional properties with human IGFBP‐1. The protein is 81% identical to rat IGFBP‐2. When compared with both rat IGFBP‐2 and human IGFBP‐1, the positions of all 18 cysteine residues are conserved. Similarly an RGD sequence is present near the carboxyl terminus in both proteins. IGFBP‐2 has a higher affinity for IGF‐II than for IGF‐I and its affinity for both forms of IGF is greater than for human IGFBP‐1. Like IGFBP‐1 the protein can enhance the DNA synthesis response of porcine aortic smooth muscle cells to IGF‐I; however, IGFBP‐2 was much less potent. The maximum potentiation of the IGF‐mediated mitogenic response that could be achieved was approximately 42% that of IGFBP‐1. This potentiation is dependent upon a factor contained in platelet poor plasma and if this factor is omitted from the incubation medium, IGFBP‐2 inhibits DNA synthesis. The purification of IGFBP‐2 will allow more detailed comparisons to be made between it and other forms of IGFBPs in physiologic test systems.
Expression of the chicken hepatic glycoprotein receptor in Xenopus oocytes: Conservation of ligand and receptor targeting signals
Authors: Wall Doris A. , Holguin Raisa 
Published: May 2005

AbstractWe have obtained expression of the β‐N‐acetylglucosamine‐binding receptor from chicken hepatocytes in Xenopus oocytes by injecting mRNA synthesized in vitro from a full length cDNA cloned into an expression vector (Mellow et al: J. Biol Chem 263:5468–5473, 1988). Immunoprecipitation of the receptor after labeling of oocytes with [35S]‐methionine for times ranging from 6 to 72 h revealed 4–5 closely spaced bands of 25–30 kDa after SDS‐PAGE. Although these bands were largely resistant to endoglycosidase H cleavage, endoglycosidase F reduced the size of all bands to a single species at 23–24 kDa, indicating that they resulted from heterogeneity in glycosylation of a single polypeptide. Incubation of oocytes expressing this receptor with [125I]‐GlcNAc‐BSA resulted in 1.8 to 10 × higher levels of cell‐associated ligand in mRNA‐injected vs. water‐injected control oocytes, 2–35% of cell‐associated counts was removed by EGTA rinse at 20°C, suggesting that most ligand was inaccessible (presumably intracellular). Immunoprecipitation of sucrose gradient fractions detected receptor molecules predominantly in a light organelle at 1.09–1.12 g/cc (the density of early endosomes and plasma membrane vesicles), with no evidence of the receptor in much heavier yolk platelet fractions even in the presence of ligand. In contrast, internalized [125I]‐GlcNAc‐BSA was found either at the top of the gradients or in organelles at 1.09–1.17 g/cc and in yolk platelets. TCA precipitation indicated that much intracellular ligand was degraded to acid‐soluble fragments. Addition of vitellogenin (the yolk protein precursor) to the medium together with the [125I]‐GlcNAc‐BSA shifted much of the ligand into yolk platelets. These data indicate that the chicken glycoprotein receptor expressed in oocytes mediates binding and internalization of this ligand into an organelle in which ligand‐receptor dissociation occurs, allowing for separation of these two molecules into different compartments. The behavior of ligand in Xenopus oocytes expressing the chicken receptor closely resembles its behavior in hepatocytes.
Protein kinase C activation amplifies prostaglandin F2α‐induced prostaglandin E2 synthesis in osteoblast‐like cells
Authors: Tokuda Haruhiko , Oiso Yutaka , Kozawa Osamu 
Published: May 2005

AbstractIn cloned osteoblast‐like cells, MC3T3‐E1, prostaglandin F2α (PGF2α) stimulated arachidonic acid (AA) release in a dose‐dependent manner in the range between 1 nM and 10 μM. 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), a protein kinase C (PKC) activator, which by itself had little effect on AA release, markedly amplified the release of AA stimulated by PGF2α in a dose‐dependent manner, 4 α‐phorbol 12, 13‐didecanoate, a phorbol ester which is inactive for PKC, showed little effect on the PGF2α‐induced AA release. 1‐oleoyl‐2‐acetylglycerol (OAG), a specific activator for PKC, mimicked TPA by enhancement of the AA release induced by PGF2α. H‐7, a PKC inhibitor, markedly suppressed the effect of OAG on PGF2α‐induced AA release. Quinacrine, a phospholipase A2 inhibitor, showed partial inhibitory effect on PGF2α‐induced AA release, while it suppressed the amplification by OAG of PGF2α‐induced AA release almost to the control level. Furthermore, TPA enhanced the AA release induced by melittin, known as a phospholipase A2 activator. On the other hand, TPA inhibited the formation of inositol triphosphate stimulated by PGF2α. Under the same condition, PGF2α indeed stimulated prostaglandin E2 (PGE2) synthesis and TPA markedly amplified the PGF2α‐induced PGE2 synthesis as well as AA release. These results indicate that the activation of PKC amplifies PGF2α‐induced both AA release and PGE2 synthesis through the potentiation of phospholipase A2 activity in osteoblast‐like cells.
Receptor binding of asialoerythropoietin
Authors: Dong Yi Jie , Kung Charles , Goldwasser Eugene 
Published: May 2005

AbstractThe interaction of 125I‐asialoeythropoietin (asialoepo) with receptors has been characterized both by binding assay and affinity cross‐linking. Purified spleen cells from mice infected with the anemia strain of Friend virus (FVA cells) have receptors for 123I‐asialoepo with two classes of affinity constant: one with Kd = 0.02–0.03 nM and 300–400 per cell, the other with lower affinity (kd = 0.9–1.2 nM) and 1,000–1,200 per cell. The Kd value for the high affinity site is one third of that for the binding of native 125I‐erythropoietin (125I‐epo) to the same FVA cells (Kd = 0.08–0.1 nM). Using 125I‐asialoepo or 125I‐epo affinity cross‐linking methods, we find two components with apparent molecular weights of 88 kDa and 105 kDa in FVA cells, and in the transformed mouse cell lines, 201, IW32, and NN10, in agreement with earlier studies using 125I‐epo. These results indicate that 125I‐asialoepo binds to the same receptors as 125I‐epo, but with greater affinity for the high affinity site. Since 201 cells contain only a single class of lower affinity receptors for erythropoietin (epo), finding the same two components as found for FVA cells by cross‐linking experiment indicates that the two components do not represent the two classes of receptor.