bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cell‐specific constraints to the lateral diffusion of a membrane glycoprotein
Authors: Barbour Suzanne , Edidin Michael 
Published: February 2005

AbstractWe have previously shown that the lateral diffusion, D, of the class I Major Histocompatibility Complex (MHC) glycoprotein H‐2Ld is constrained by its glycosylation, when expressed in mouse L‐cells. Removal of one or more of the 3 N‐linked olisaccharides of H‐2Ld glycoproteins results in an increase in D. In order to further examine the influence of glycosylation on D, we compared lateral diffusion of H‐2Ld expressed in wild‐type CHO cells with lateral diffusion of the same molecule expressed in mutant CHO cells with aberrant surface glycosylation. In addition, we compared lateral diffusion of wild‐type and unglycosylated H‐2Ld antigens in these cells. In contrast to the large effect of glycosylation state on lateral diffusion of H‐2Ld in mouse L‐cells. There was little effect of glycosylation on lateral diffusion of H‐2Ld in any of the CHO cells. This, together with similar results on hamster class I antigens, indicates that the constraints to D of H‐2Ld and other class I MHC molecules are different in CHO cells than in L‐cells. Measurements of lateral diffusion after treatment of cells with cytochalasin D make it clear that interactions between MHC class I molecules and a cytoskeleton are important in reducing the mobile fraction of diffusing molecules, R, though they cannot be shown to directly affect the diffusion coefficient, D.
Association of increased basement membrane invasiveness with absence of estrogen receptor and expression of vimentin in human breast cancer cell lines
Authors: Thompson Erik W. , Paik Soonmyoung , Brünner Nils , Sommers Connie L. , Zugmaier Gerhard , Clarke Robert , Shima Thomas B. , Torri Jeffrey , Donahue Steven , Lippman Marc E. , Martin George R. , Dickson Robert B. 
Published: February 2005

AbstractLack of estrogen receptor (ER) and presence of vimentin (VIM) associate with poor prognosis in human breast cancer. We have explored the relationships between ER, VIM, and invasiveness in human breast cancer cell lines. In the matrigel outgrowth assay, ER+/VIM‐ (MCF‐7, T47D, ZR‐75‐1), and ER‐/VIM‐ (MDA‐MB‐468, SK‐Br‐3) cell lines were uninvasive, while ER‐/VIM+ (BT549, MDA‐MB‐231, MDA‐MB‐435, MDA‐MB‐436, Hs578T) lines formed invasive, penetrating colonies. Similarly, ER‐/VIM+ cell lines were significantly more invasive than either the ER+/VIM‐ or ER‐/VIM‐ cell lines in the Boyden chamber chemoinvasion assay. Invasive activity in nude mice was only seen with ER‐/VIM+ cell lines MDA‐MB‐231, MDA‐MB‐435 and MDA‐MB‐436. Hs578T cells (ER‐/VIM +) showed hematogenous dissemination to the lungs in one of five mice, but lacked local invasion. The ER‐/VIM+ MCF‐7ADR subline was significantly more active than the MCF‐7 cells in vitro, but resembled the wild‐type MCF‐7 parent in in vivo activity. Data from these cell lines suggest that human breast cancer progression results first in the loss of ER, and subsequently in VIM acquisition, the latter being associated with increased metastatic potential through enhanced invasiveness. The MCF‐7ADR data provide evidence that this transition can occur in human breast cancer cells. Vimentin expression may provide useful insights into mechanisms of invasion and/or breast cancer cell progression.
Endothelin stimulates phosphatidic acid formation in cultured rat mesangial cells: Role of a protein kinase C‐regulated phospholipase D
Authors: Kester Mark , Simonson Michael S. , McDermott R. Guy , Baldi Elisabetta , Dunn Michael J. 
Published: February 2005

AbstractWe have previously reported that endothelin‐1 stimulates phospholipase C‐in‐duced hydrolysis of phosphatidylinositol‐4, 5‐bisphosphate, Other signal transduction pathways that hydrolyze alternative phospholipids through phospholipase D may also mediate endothelin‐stimulated cellular responses. We initially evaluated endothelin‐dependent generation of 32P‐phosphatidic acid as an indirect indication of phospholipase D activity in rat mesangial cells. Endothelin (10−7M) induced an elevation of phosphatidic acid that was maximal at 15 min and persisted upward of 60 min. Pretreatment with the diacylglycerol‐kinase inhibitor, R59022, did not reduce formation of endothelin‐stimulated 32P‐phosphatidic acid, demonstrating that the sequential actions of phospholipase C/diacylglycerol kinase do not contribute to endothelin‐stimulated phosphatidic acid formation. We next conclusively identified a role for phospholipase D in the generation of phosphatidic acid by assessing the formation of 3H‐phosphatidylethanol from 3H‐alkyl lyso glycerophosphocholine and exogenous ethanol. Endothelin stimulated 3H‐alkyl phosphatidylethanol formation in the presence but not the absence of 0.5% ethanol. Also, endothelin induced a concomitant elevation of 3H‐alkyl‐phosphatidic acid that was significantly reduced when the cells were exposed to exogenous ethanol, reflecting the formation of phosphatidylethanol. In addition, endothelin stimulated the release of 3H‐choline and 3H‐ethanolamine, demonstrating that additional phospholipids may serve as substrates for phospholipase D. Phorbol esters and synthetic diglycerides mimicked the effects of endothelin to stimulate phospholipase D and inhibitors of protein kinase C significantly reduced endothelin‐stimulated phospholipase D. In addition, endothelin did not stimulate phosphatidylethanol formation in protein kinase C down‐regulated cells. The calcium ionophore, ionomycin, did not stimulate phospholipase D and mesangial cells pretreated with BAPTA to chelate cytosolic calcium did not show a diminished endothelin‐stimulated phospholipase D. Thus these data demonstrate that mesangial cells possess a protein kinase C‐regulated phospholipase D activity that can be stimulated with endothelin.
Glycolipid depletion using a ceramide analogue (PDMP) alters growth, adhesion, and membrane lipid organization in human A431 cells
Authors: Barbour Suzanne , Edidin Michael , Felding‐Habermann Brunhilde , Taylor‐Norton Janelle , Radin Norman S. , Fenderson Bruce A. 
Published: February 2005

AbstractGlycolipids were depleted from the membranes of human A431 cells using 1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol (PDMP), an inhibitor of glucosylceramide synthetase. After 6 days of culture in the presence of 5 μM D‐threo‐PDMP, glycolipid content was reduced to approximately 5% of control levels. By contrast, synthesis per cell of phosphatidylcholine, sphingomyelin, triglycerides, and glycoprotein was relatively unchanged in PDMP‐treated cells. In parallel with glycolipid depletion, PDMP‐treated cells exhibited a rapid loss of epithelial cell morphology, a reduced rate of cell growth, and inhibition of cell‐substrate adhesion. The effects of D‐threo‐PDMP on cell morphology and substrate adhesion were blocked by exogenous GM3 addition and were not observed with L‐threo‐PDMP (a relatively inactive enantiomer). Fluorescence photobleaching and recovery (FPR) was used to investigate the hypothesis that glycolipids influence cell behavior, in part, by changing the diffusion characteristics of membrane proteins and lipids. Diffusion coefficients and mobile fractions of two integral membrane proteins, the EGF receptor and a class I MHC antigen, did not differ significantly between control and PDMP‐treated cells. Diffusion coefficients of lipid probes, NBD‐PC and fluorescent GM1 ganglioside, were similarly unaffected by gly‐colipid depletion. However, lipid probes did show a significant increase in mobile fraction (the fraction of lipids that are free to diffuse) in PDMP‐treated cells. This increase was blocked by culturing cells in the presence of exogenous GM3 ganglioside. The results suggest that glycolipids play a role in the formation of lipid domains in A431 cell membranes. Glycolipid‐mediated changes in membrane lipid organization may influence receptor activation and transmembrane signaling, leading to changes in cell growth, morphology, and adhesion.
Glutamine and glutamate metabolism in normal and heat shock conditions in Drosophila Kc cells: Conditions supporting glutamine synthesis maximize heat shock polypeptide expression
Authors: Sanders Marilyn M. , Kon Christine 
Published: February 2005

AbstractWe have previously reported that Drosophila Kc cells require glutamine for maximal expression of heat shock proteins in stressed conditions (Sanders and Kon: J. Cell. Physiol. 146:180–190, 1991). The mechanism of this effect has been investigated by comparing the metabolic utilization of glutamine in conditions which support hsp expression with that of glutamate in conditions where up to 100‐fold less hsp is synthesized. This comparison showed that free ammonia was generated by cells incubated in the presence of glutamine in 37°C (heat shock) conditions, but not at 25°C, and not in the presence of glutamate in either normal or heat shock conditions. There was no difference in the amount of [14C]O2 generated from either [14C]‐labeled amino acid in the tricarboxylic acid cycle, but three‐ to four‐fold more alanine was synthesized in cells incubated in glutamine than in glutamate. Treating the cells with aminotransferase inhibitors to artificially increase NH3 release raised hsp expression in the presence of glutamate to maximal levels characteristic of glutamine. This potentiation correlated with inhibition of alanine aminotransferase. Since only NH3 production correlated with hsp expression in heat shock conditions in the presence of glutamine, and NH3 addition to glutamate also resulted in maximal hsp expression, we measured glutamine production in glutamate plus NH3 and observed net glutamine synthesis. The supposition that glutamine itself is responsible for the regulatory changes supporting maximal hsp expression was supported by the finding that the glutamine analog, 6‐diazo‐5‐oxo‐L‐norleucine (DON), mimicked the effects of glutamine. We conclude that glutamine imposes regulatory changes which alter nitrogen metabolism and support hsp expression in Kc cells.
Susceptibility to verotoxin as a function of the cell cycle
Authors: Pudymaitis Anita , Lingwood Clifford A. 
Published: February 2005

AbstractInfection with Verotoxin producing Escherichia coli (VTEC) has been implicated in hemolytic uremic syndrome, the leading cause of pediatric renal failure. Verotoxin (VT) binds to globotriaosylceramide (Galα1–4Galβ1–4GlcCer Gb3) in susceptible cells. Gb3 is required for cytotoxicity and toxin‐resistant cells deficient in Gb3 can be sensitized to VT cytotoxicity by incorporation of exogenous Gb3 into the cells. However, the absolute Gb3 content of cell lines does not necessarily correspond directly with the degree of sensitivity to VT. The present study demonstrates that susceptibility to VT is a function of cell growth and that stationary phase cells are resistant to VT. Using chemically synchronized Vero cells, we have also found a tenfold difference in susceptibility to VT during the cell cycle. Our experiments define a maximal sensitivity “window” of 1–2 hours from the G1/S boundary. This corresponds to increased VT binding without change in overall Gb3 content. Cell surface labelling indicated that cyclic turnover and exposure of Gb3 may be the critical parameter in determining VT sensitivity. Such changes during the cell cycle may also be of relevance in vivo in determining toxin pathology during VTEC infections and the physiology of plasma membrane Gb3.
Age‐related changes in hyaluronan, proteoglycan, collagen, and osteonectin synthesis by human bone cells
Authors: Fedarko N. S. , Vetter U. K. , Weinstein S. , Robey P. Gehron 
Published: February 2005

AbstractHuman bone cells grown in culture, representative of a preosteoblastic stage of maturation, produce an extracellular matrix composed of collagen several noncollagenous glycoproteins, hyaluronan, and four distinct proteoglycans (PGs). The influence of donor age on the levels of expression of these molecules in vitro has not been well characterized. In this study, human bone cells derived from sources ranging from fetal to 60‐year‐old donors were grown in culture, radiolabeled for 24 h, and the amount of incorporation of [35S]sulfate into PGs, [3H]glucosamine into hyaluronan, [3H]leucine/proline into osteonectin, and [3H]proline into collagen was determined. Cell proliferation was most rapid in fetal‐derived bone cells and decreased with increasing age. Total protein and PG synthesis also decreased with increasing age, falling to 1/3 and 1/4, respectively, of fetal levels after age 30. A large chondroitin sulfate PG (Mr ∼ 600,000 Da) was the major fetal PG and its levels were highly correlated with cellular proliferation. [3H]Collagen and [35S]decorin levels increased with the increasing age of the donor, reached a maximum in puberty‐derived cells, and decreased to 1/3 maximal levels after age 20. The heparan sulfate PG (Mr ∼ 400,000 Da) exhibited steadystate levels regardless of donor age. [3H]Osteonectin and [35S]biglycan levels were high in fetal‐derived cells and in cells derived from pubescent donors. The percentage of collagen and four proteoglycans associated with the cell layer pool changed with donor age. All fetal‐derived PG core proteins possessed more N‐ and O‐linked oligosaccharides than newborn or adult derived PGs. © 1992 Wiley‐Liss, Inc.
Differential expression of TGFβ isoforms by human articular chondrocytes in response to growth factors
Authors: Villiger Peter M. , Lotz Martin 
Published: February 2005

AbstractTransforming growth factor β (TGFβ) is a family of important regulators of chondrocyte growth and differentiation. Although TGFβ has been detected in cartilage, the TGFβ isoforms expressed by chondrocytes and their regulation by growth factors are unknown. This study shows that human articular chondrocytes release TGFβ activity. Chondrocyte conditioned media contains active TGFβ and larger quantities in latent form. By neutralization with specific antibodies it is shown that all three isoforms (TGFβ1, TGFβ2, and TGFβ3) are secreted by chondrocytes. Analysis of the inducers of TGFβ gene expression demonstrates complex regulation of TGFβ production by growth factors. Basic fibroblast growth factor (bFGF) stimulates the release of TGFβ activity but has no effect on steady state TGFβ mRNA levels while platelet‐derived growth factor (PDGF) upregulates TGFβ1 and TGFβ3 mRNAs with a corresponding increase in protein secretion. The three TGFβ isoforms themselves differentially affect gene expression. While TGFβ1 and TGFβ2 show autoinduction, TGFβ3 upregulates TGFβ1 but does not affect TGFβ2 mRNA levels.These results demonstrate that human articular chondrocytes produce all three TGFβ isoforms. Induction of TGFβ expression is differentially regulated by various growth factors and occurs at the mRNA level and/or posttranscriptionally. Chondrocyte expression and the differential regulation of TGFβ1, TGFβ2, and TGFβ3 by growth factors suggest that all three isoforms of TGFβ are part of the network of cartilage regulatory factors. © 1992 Wiley‐Liss, Inc.
Regulation of TGF‐α expression in human keratinocytes: PKC‐dependent and ‐independent pathways
Authors: Klein Susan B. , Fisher Gary J. , Jensen Timothy C. , Mendelsohn John , Voorhees John J. , Elder James T. 
Published: February 2005

AbstractTransforming growth factor–α (TGF‐α) is an autocrine growth factor for epidermal keratinocytes that can induce its own expression (autoinduction). Because the regulation of this process may be important for the control of epidermal growth, we examined the roles of EGF receptor tyrosine kinase and protein kinase C (PKC) in TGF‐α autoinduction in cultured human keratinocytes. Antiphosphotyrosine immunoblot analysis demonstrated that EGF and TGF‐α rapidly and markedly stimulated tyrosine phosphorylation of a 170 kDa protein in growth factor–deprived keratinocytes. This protein was identified as the EGF receptor by immuno‐precipitation using anti‐EGF receptor mAbs. Tyrosine phosphorylation and TGF‐α mRNA accumulation in response to EGF and TGF‐α were both inhibited by a monoclonal antibody against the EGF receptor and by the EGF receptor tyrosine kinase inhibitor RG50864, demonstrating the involvement of the tyrosine kinase activity of the receptor in TGF‐α autoinduction. The monoclonal antibody inhibited keratinocyte growth and TGF‐α autoinduction with similar potency (IC50 ∼ 0.1 μg/ml). TGF‐α and the PKC activator tetradecanoyl phorbol 12‐myristyl, 13‐acetate (TPA) had similar effects on TGF‐α steady‐state mRNA levels, suggesting that PKC activation might be a downstream mediator of TGF‐α autoinduction. However, down‐regulation of more than 90% of keratinocyte PKC activity by bryostatin pretreatment abrogated the induction of TGF‐α mRNA in response to TPA without affecting the autoinductive response or EGF‐stimulated tyrosine phosphorylation. These results indicate that EGF receptor and PKC stimulate TGF‐α gene expression by different pathways, and suggest that PKC is not required for TGF‐α autoinduction in this system. Moreover, the fact that EGF‐stimulated tyrosine phosphorylation and TGF‐α autoinduction were not potentiated after PKC down‐regulation suggests that PKC does not exert a tonic inhibitory influence on EGF receptor tyrosine kinase activity in normal human keratinocytes. © 1992 Wiley‐Liss, Inc.
Regulation of the Na, K‐ATPase activity of Madin‐Darby Canine Kidney cells in defined medium by Prostaglandin E1 and 8‐bromocyclic AMP
Authors: Taub Mary L. , Wang Yue , Yang Il‐Suk , Fiorella Paul , Lee Sang Mog 
Published: February 2005

AbstractThe role of PGE1 in regulating the activity of the Na+, K+ ‐ATPase in Madin Darby Canine Kidney (MDCK) cells has been examined. PGE1 increased the initial rate of ouabain‐sensitive Rb+ uptake by MDCK cells, a process that continued to occur over a 5‐day period The increase in the initial rate of ouabain‐sensitive Rb+ uptake in MDCK cells treated with PGE1 could be explained by a 1.6‐fold increase in the Vmax for ouabain‐sensitive Rb+ uptake. The increase in the Vmax for ouabain‐sensitive Rb+ uptake observed in MDCK cells under these conditions can be explained either by an increase in the number of active Na+ pumps, or by an increase in the efficiency of the Na+ pumps. Consistent with the former possibility is the observed increase in the number of ouabain binding sites, as well as the increase in Na+, K+‐ATPase activity in cell lysates obtained from MDCK monolayers treated with PGE1. The involvement of cyclic AMP in mediating these effects of PGE1 on the Na+, K+‐ATPase in MDCK cells is supported by: (1) the observation of similar effects in 8‐bromocyclic AMP treated MDCK monolayts, and (2) a dramatic reduction of the stimulatory effects of PGE1 and 8‐bromocyclic AMP on the Vmax for ouabain‐sensitive Rb+ uptake, and on the number of ouabain binding sites in dibutyryl cyclic AMP resistant clone 3 (DBr 3) (which is defective in cyclic AMP dependent protein kinase activity). PGE1 independent MDCK monolayers exhibit both an increase in the Vmax for ouabain‐sensitive Rb+ uptake and an increase in the number of ouabain binding sites in response to 8‐bromocyclic AMP. Apparently, the cyclic AMP phosphodiesterase defect in these PGE1 independent cells did not cause cellular cyclic AMP levels to be elevated to a sufficient extent to maximally increase the Na+, K+‐ATPase activity in these variant cells. © 1992 Wiley‐Liss, Inc.
Na+/H+ antiporter gene expression increases during retinoic acid‐induced granulocytic differentiation of HL60 cells
Authors: Rao Gadiparthi N. , Sardet Claude , Pouysségur Jacques , Berk Bradford C. 
Published: February 2005

AbstractDuring differentiation of human leukemic HL60 cells into granulocytes, sustained increases in intracellular pH and Na+/H+ antiporter activity have been observed. In the present study we report that retinoic acid (RA)‐induced granulocytic differentiation of HL60 cells causes an ∼18‐fold increase in the steady‐state mRNA levels for the Na+/H+ antiporter. This was due to an increase in the rate of Na+/H+ antiporter gene transcription as measured by nuclear run‐on analysis. Antiporter protein synthesis increased by seven‐fold during RA‐induced granulocytic differentiation of HL60 cells as measured by immunoprecipitation of 35S‐methionine‐labeled proteins with the RP1‐c28 Na+/H+ antiporter antibody. No increase in antiporter mRNA was observed in response to etretinate, an analogue of retinoic acid, which did not induce differentiation. Thus, Na+/H+ antiporter gene expression is associated with RA‐induced granulocytic differentiation of HL60 cells. The present findings and our previous data (Rao et al., 1991) demonstrate that Na+/H+ antiporter gene expression is a generalized feature of HL60 cell differentiation. © 1992 Wiley‐Liss, Inc.
Signal transduction by interleukin 2 in human T cells: Activation of tyrosine and ribosomal S6 kinases and cell‐cycle regulatory genes
Authors: Sawami Hiroyasu , Terada Naohiro , Franklin Richard A. , Okawa Hiroji , Uchiyama Takashi , Lucas Joseph J. , Gelfand Erwin W. 
Published: February 2005

AbstractThe early events of signal transduction associated with interleukin‐2 (IL‐2) binding to its receptor were examined using a human IL‐2 dependent T‐cell line, Kit225. Cell cycle analysis showed that 90% of Kit225 cells were in the G0/G1 phase after a 72‐hr incubation in the absence of exogenous IL‐2. At this point, stimulation of the cells with IL‐2 resulted in the rapid initiation of RNA and DNA synthesis by 9 and 20 hr, respectively. Within 5 min after addition of IL‐2, rapid activation of tyrosine and ribosomal S6 kinases was detected. Addition of IL‐2 also increased mRNA levels for c‐fos, c‐myc, IL‐2 receptor α, and IL‐2 receptor β chain. These events increased in the absence of detectable changes in free cytosolic [Ca2+]i, inositol phosphate metabolism, or the activity of several kinases including cAMP‐dependent protein kinase, Ca2+/calmodulin‐dependent protein kinase, or protein kinase C. These findings demonstrate that the signals triggered by IL‐2 binding to its receptors are quickly transduced into the nucleus with increased mRNA transcription of activation‐associated genes. Furthermore, the data indicate that tyrosine and ribosomal S6 kinases may be important for IL‐2‐induced cell growth. © 1992 Wiley‐Liss, Inc.