bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential expression of asialoglycoprotein receptor subunits in the endocytic compartment during liver regeneration
Authors: Enrich Carlos , Verges Marcel , Evans W. Howard
Published: February 2005

Asialoglycoprotein receptors, responsible for the removal of circulating asialogly‐coproteins by the liver, are located in at least two different membrane locations in hepatocytes. Receptors on the cell surface account only for a minor proportion (20–36%), for the majority of receptors in the liver are located intracellularly, mainly in the endocytic membrane networks. An understanding of the basis of receptor distribution and the underlying trafficking of receptors between the hepatocyte's polarised cell surface and the endocytic compartment would be aided if biochemical differences between the receptors in these pools were established. We now show, using three antibodies that recognise the receptor subunits in rat liver (RHL‐1, RHL‐2 and RHL‐3), that the asialoglycoprotein receptors located in the plasma membrane domains and the endocytic compartment differ in oligomeric composition, sialic acid content, and solubility in Triton X‐114 using two‐phase systems. It is well established that the expression of the asialoglycoprotein receptor is down‐regulated in livers regenerating after a partial hepatectomy. We demonstrate that the levels of the receptor subtype that is located mainly in the endocytic compartment (RHL‐1, 42 kDa) was elevated in regenerating liver by agents that regulate cAMP production, whereas the levels of the other receptor subtypes remained unchanged. The asialoglycoprotein receptor subtypes that are present in different subcellular locations are thus regulated independently.
Mitogenic, melanogenic, and cAMP responses of cultured neonatal human melanocytes to commonly used mitogens
Authors: Abdel‐Malek Zalfa , Swope Viki B. , Pallas James , Krug Kimberly , Nordlund James J.
Published: February 2005

The following studies have been undertaken to compare and correlate the effects of 12‐0‐tetradecanoylphorbol acetate (TPA), basic fibroblast growth factor (bFGF), cholera toxin (CT), and isobutyl methylxanthine (IBMX) on neonatal human melanocyte (NHM) proliferation, tyrosinase activity, and cyclic adenosine monophosphate (cAMP) concentration. NHM proliferated at a maximal rate in medium containing 8 nM TPA, 200 ng/ml CT, and 10−4 M IBMX. TPA alone did not result in optimal melanocyte proliferation, and, as previously shown, its mitogenic effect was greatly enhanced by the addition of CT and IBMX individually or concomitantly. Human recombinant (hr) bFGF could replace TPA in the NHM growth medium. Maximal proliferation was achieved using 3 ng/ml hrbFGF, 20 ng/ml CT, and 10−4 M IBMX. The mitogenic effect of 1.2 ng/ml hrbFGF was potentiated in the concomitant but not individual presence of CT and IBMX. TPA alone in the absence of CT and IBMX caused a dose‐dependent stimulation of tyrosinase activity. Maximal tyrosinase activity was obtained in the presence of 0.8 nM TPA, 20 ng/ml CT, and 10−4 M IBMX. Unlike TPA, hrbFGF alone resulted in inhibition of tyrosinase activity. In the presence of hrbFGF, tyrosinase activity was potentiated by CT and IBMX, but not by CT alone. Neither TPA nor hrbFGF alone could increase intracellular cAMP levels. The effects of CT and IBMX on intracellular cAMP concentration were enhanced to a greater extent by TPA than by hrbFGF. Under our experimental conditions, in the presence of hrbFGF, CT but not IBMX resulted in a dose‐dependent increase in cAMP concentration. Further studies on NHM will be aimed at determining the exact role of protein kinase C (PKC) in regulating proliferation and melanogenesis and the mechanism(s) activated by hrbFGF.
Expression, synthesis, and phosphorylation of HSP28 family during development and decay of thermotolerance in CHO plateau‐phase cells
Authors: Lee Yong J. , Hou Zi‐Zheng , Curetty Lindali , Corry Peter M.
Published: February 2005

We investigated a correlation between development of thermotolerance and expression, synthesis, or phosphorylation of HSP28 family in CHO plateau phase cells. After heating at 45.5°C for 10 min, thermotolerance developed rapidly and reached its maximum 12–18 hr after heat shock. This acquired thermal resistance was maintained for 72 hr and then gradually decayed. In parallel, the levels of three 28 kDa heat shock proteins, HSP28a along with its two phosphorylated isoforms (HSP28b, c), increased and reached their maximum 24–48 hr after heat shock. The levels of these polypeptides, except HSP28c, remained elevated for 72 hr and then decreased. The level of HSP28 mRNA increased rapidly and reached its maximum 12 hr after heat shock. However, unlike thermotolerance and the levels of HSP28 family proteins, the level of HSP28 mRNA decreased rapidly within 72 hr. These results demonstrate a correlation between the amount of intracellular HSP28 family proteins and development and decay of thermotolerance.
Cell‐specific constraints to the lateral diffusion of a membrane glycoprotein
Authors: Barbour Suzanne , Edidin Michael
Published: February 2005

We 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

Lack 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

We 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

Glycolipids 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

We 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

Infection 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

Human 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

Transforming 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

Transforming 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

The 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

During 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

The 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.
Characterization of specific proteases associated with the surface of human skin fibroblasts, and their modulation in pathology
Authors: Raynaud Françe , Bauvois Brigitte , Gerbaud Pascale , Evain‐Brion Danièle
Published: February 2005

Human skin fibroblasts were probed for cell surface protease activity. One activity removing dipeptides from the NH2‐terminal end of Gly‐Pro‐pNA was specifically inhibited by di‐isopropyl‐fluorophosphate (DFP), phenylmethanesulphonyl fluo‐ride (PMSF), and diprotin A, and thus was identified as dipeptidyl peptidase IV (DPP IV). A group of bestatin‐sensitive N‐exoaminopeptidase activities was also characterized when Ala‐, Leu‐, and Arg‐pNA were used as chromogenic substrates. Using human monoclonal antibodies anti‐CD 13 and anti‐CD 26 that recognized, respectively, an N‐Ala‐aminopeptidase and DPP IV, it was found that human dermal fibroblasts expressed the CD 13 and CD 26 antigen on their surface. In addition, both peptidases were specifically immunoprecipitated by monoclonal antibodies anti‐CD 13 and anti‐CD 26 from plasma membranes. Cell surface proteolytic activities were also investigated in human fibroblasts derived from dermatological and rheumatic diseases (i.e., psoriasis, rheumatoid arthritis, and lichen planus). It was found that these fibroblasts also expressed both types of proteinases initially identified on normal skin fibroblasts and that the levels of Ala‐aminopeptidase activities were similar in all cases. In contrast, the levels of Arg‐, Leu‐exoaminopeptidase, and DPP IV activities were significantly higher (up to 6.6‐fold) in the three pathological fibroblast populations than in their normal counterparts. These proteolytic enzymes, therefore, can potentially serve as markers in dermatological diseases. Taken together, our results suggest that skin fibroblast‐derived proteinases associated with both serine and N‐aminopeptidase activities may play an important role by participating in the extracellular events associated with fibroblast behaviour. © 1992 Wiley‐Liss, Inc.
Variations in tumor cell growth rates and metabolism with oxygen concentration, glucose concentration, and extracellular pH
Authors: Casciari Joseph J. , Sotirchos Stratis V. , Sutherland Robert M.
Published: February 2005

Tumors and multicellular tumor spheroids can develop gradients in oxygen concentration, glucose concentration, and extracellular pH as they grow. In order to calculate these gradients and assess their impact on tumor growth, it is necessary to quantify the effect of these variables on tumor cell metabolism and growth. In this work, the oxygen consumption rates, glucose consumption rates, and growth rates of EMT6/Ro mouse mammary tumor cells were measured at a variety of oxygen concentrations, glucose concentrations, and extracellular pH levels. At an extracellular pH of 7.25, the oxygen consumption rate of EMT6/Ro cells increased by nearly a factor of 2 as the glucose concentration was decreased from 5.5 mM to 0.4 mM. This effect of glucose concentration on oxygen consumption rate, however, was slight at an extracellular pH of 6.95 and disappeared completely at an extracellular pH of 6.60. The glucose consumption rate of EMT6/Ro cells increased by roughly 40% when the oxygen concentration was reduced from 0.21 mM to 0.023 mM and decreased by roughly 60% when the extracellular pH was decreased from 7.25 to 6.95. The growth rate of EMT6/Ro cells decreased with decreasing oxygen concentration and extracellular pH; however, severe conditions were required to stop cell growth (0.0082 mM oxygen and an extracellular pH of 6.60). Empirical correlations were developed from these data to express EMT6/Ro cell growth rates, oxygen consumption rates, and glucose consumption rates, as functions of oxygen concentration, glucose concentration, and extracellular pH. These empirical correlations make it possible to mathematically model the gradients in oxygen concentration, glucose concentration, and extracellular pH in EMT6/Ro multicellular spheroids by solution of the diffusion/reaction equations. Computations such as these, along with oxygen and pH microelectrode measurements in EMT6/Ro multicellular spheroids, indicated that nutrient concentration and pH levels in the inner regions of spheroids were low enough to cause significant changes in nutrient consumption rates and cell growth rates. However, pH and oxygen concentrations measured or calculated in EMT6/Ro spheroids where quiescent cells have been observed were not low enough to cause the cessation of cell growth, indicating that the observed quiescence must have been due to factors other than acidic pH, oxygen depletion, or glucose depletion. © 1992 Wiley‐Liss, Inc.
Fibroblast growth factor upregulates PGG/H synthase in rabbit microvascular endothelia cells by a glucocorticoid independent mechanism
Authors: Moatter Tariq , Gerritsen Mary E.
Published: February 2005

The present study was undertaken to determine the effects of acidic fibroblast growth factor (aFGF) on eicosanoid synthesis in microvessel endothelial cells derived from rabbit left ventricular muscle (RCME cells). We observed that aFGF increased AA conversion to PGE2 in a time‐ and dose‐dependent manner, and the stimulatory effect was abolished by actinomycin D and cycloheximide. Acidic FGF increased the recovery of PGG/H synthase activity following aspirin treatment, suggesting an action on de novo PGG/H synthase synthesis. Acidic FGF increased the incorporation of [35S] methionine into a 70 kD immunoreactive PGG/H synthase band. PGG/H synthase synthesis following aspirin treatment was also increased by transforming growth factor β, while epidermal growth factor basic FGF and platelet derived growth factor were without effect. In addition, the actions of aFGF on de novo PGG/H synthase were compared in several endothelial preparations. Acidic FGF treatment of aspirin treated endothelial cells from rabbit lung microvessels and small pulmonary artery and from human lung microvessels all showed an increase in PGG/H synthase recovery. In contrast, similar treatment of human umbilical vein endothelial cells was without effect. Pretreatment of RCME cells with dexamethasone (1 μM) did not alter the aFGF induction of PGG/H synthase activity. We conclude that aFGF stimulates PGE2 production by a mechanism that includes the de novo synthesis of PGG/H synthase. This mechanism appears to be distinct from previously described glucocorticoid sensitive translational controls of PG synthase synthesis by epidermal growth factor in smooth muscle and mesangial cells. © 1992 Wiley‐Liss, Inc.
A high level of cell surface phosphatidylinositol‐specific phospholipase C activity is characteristic of growth‐arrested 3T3 fibroblasts but not of transformed variants
Authors: Volwerk Johannes J. , Birrell G. Bruce , Hedberg Karen K. , Griffith O. Hayes
Published: February 2005

Confluent monolayers of four contact‐inhibited mouse fibroblast lines (Swiss 3T3, Balb/c 3T3, NIH 3T3, and C3H10T1/2) were found to have substantial levels of a cell surface phosphatidylinositol‐specific phospholipase C (ecto‐PLC). In contrast, confluent cultures of virally, chemically, or spontaneously transformed variants derived from these cell lines expressed undetectable or negligible levels of this enzyme activity. A simple and rapid assay, using lysophosphatidylinositol radio‐labeled in the inositol group ([3H]‐lysoPI) as the substrate was developed to provide a quantitative measure of the phospholipase C activity present at the external cell surface. For cells testing positive for ecto‐PLC activity, rapid uptake of [3H]‐lysoPI is accompanied by the simultaneous appearance of [3H]‐inositol phosphate in the external medium. Confluent monolayers of the four mouse fibroblast lines exhibiting density‐dependent growth inhibition had levels of ecto‐PLC activity in the range of 50–800 pmol/min/106 cells (i.e., about 20–50 times greater than the activity observed for the transformed variants). The expression of ecto‐PLC activity at the cell surface of the Swiss or Balb/c cells was dependent on the state of cell proliferation. Cultures which had become quiescent through attainment of confluence displayed a tenfold increased activity over that of subconfluent, growing cultures of these cells. Similarly, subconfluent Swiss 3T3 cells which had become quiescent following exposure to low serum conditions also showed increased activity. These results indicate that there may exist a correlation between the control of cell proliferation in contact‐inhibited mouse fibroblasts and the expression of inositol phospholipid‐specific phospholipase C activity at the external cell surface. © 1992 Wiley‐Liss, Inc.
Heterodimerization and transcriptional activation in vitro by NF‐κB proteins
Authors: Cohen Lucie , Hiscott John
Published: February 2005

The NF‐κB family of transcription proteins represents multiple DNA binding, rel related polypeptides that contribute to regulation of genes involved in immune responsiveness and inflammation, as well as activation of the HIV long terminal repeat. In this study multiple NF‐κB related polypeptides ranging from 85 to 45 kDa were examined for their capacity to interact with the PRDII regulatory element of interferon β and were shown to possess distinct intrinsic DNA binding affinities for this NF‐κB site and form multiple DNA binding homo‐ and heterodimer complexes in co‐renaturation experiments. Furthermore, using DNA templates containing two copies of the PRDII domain linked to the rabbit β globin gene the purified polypeptides specifically stimulated NF‐κB dependent transcription in an in vitro reconstitution assay as heterodimers but not as p50 homodimers. These experiments emphasize the role of NF‐κB dimerization as a distinct level of transcriptional control that may permit functional diversification of a limited number of regulatory proteins. © 1992 Wiley‐Liss, Inc.
Effect of osmolality on 86Rb+ uptake and release by Leishmania donovani
Authors: Blum J. J.
Published: February 2005

Promastigotes from late‐log phase cultures of Leishmania donovani were washed and resuspended in Hanks' Balanced Salt Solution without glucose or phenyl red but with 20 mM (N‐[2‐hydroxyethyl] piperazine‐N'‐[2‐ethanesulfonic acid]) (HEPES) (HBSS−, 305 mOsm/kg). They were then added to a solution containing 86Rb such that the final osmolality and ionic composition was as desired. Samples were taken at known times and the amount of intracellular 86Rb was measured. Similarly, experiments were performed in which 86Rb was added to the cultures about 18 hr before collection, and the amount of 86Rb released from the washed cells was measured. Under iso‐osmotic conditions only about 1.3% of the intracellular 86Rb was released in 900 sec. This increased about 4‐fold if the osmolality was reduced from 305–153 mOsm/kg. This is much slower than the very rapid release of alanine in response to hypo‐osmotic stress, indicating that alanine release is not via a non‐specific pore. Reducing the temperature from 26°C to 3–4°C completely inhibits 86Rb release under iso‐osmotic conditions and largely inhibits it under hypo‐osmotic conditions. The rate of 86Rb release was not sensitive to K+ concentration and was not altered if chloride was replaced by sulfamate. Ouabain had no effect on either 86Rb uptake or release, but carbonylcyanide P‐trifluoromethoxyphenylhydrazone (FCCP) reduced the rate of 86Rb release and, after about a 300 sec exposure, completely inhibited 86Rb uptake. Amiloride partially inhibited 86Rb release, but had no effect on uptake. A decrease in pH from 7.1–5.9 had little effect on 86Rb release under iso‐osmotic conditions and slightly increased the rate of release under hypo‐osmotic conditions, but it decreased the rate of uptake under both iso‐osmotic and hypo‐osmotic conditions. Cells taken from 3‐day stationary phase cultures released 86Rb more slowly under iso‐osmotic conditions than cells from late log phase cultures, but were more responsive to hypo‐osmotic stress than were log phase cells. These data appear to rule out an [Na‐K‐Cl] transporter or a [K‐Cl] cotransporter as the means of K+ release, but are consistent with the possiblity that a K+/H+ exchanger is present. The possibility that other carrier systems may be present is also discussed. © 1992 Wiley‐Liss, Inc.
Okadaic acid inhibits a protein phosphatase activity involved in formation of the mitotic spindle of GH4 rat pituitary cells
Authors: Van Dolah Frances M. , Ramsdell John S.
Published: February 2005

Okadaic acid, a selective inhibitor of serine/threonine protein phosphatases, was utilized to investigate the requirement for phosphatases in cell cycle progression of GH4 rat pituitary cells. Okadaic acid inhibited GH4 cell proliferation in a concentration‐dependent manner with a half‐maximal inhibition (IC50) of approximately 5 nM. Treatment of GH4 cells with 10 nM okadaic acid resulted in a 40–60% decrease in phosphatase activity and an increase in the proportion of phosphorylated retinoblastoma (RB) protein. Cell cycle analysis indicated that okadaic acid increased the percentage of cells in G2‐M, decreased proportionally the percentage of cells in G1 phase, and had little effect on the percentage of cells in S‐phase. The absence of a change in the proportion of S‐phase cells indicates that G1 ‐specific phosphatases responsible for dephosphorylation of RB protein were not inhibited by 10 mM okadaic acid. Mitotic index revealed that 10 nM okadaic acid decreased proliferation of GH4 cells specifically by slowing the progression through mitosis. Immunostaining with anti‐tubulin demonstrated that 10 nM okadaic acid‐treated mitotic cells contained mitotic spindles; however, the spindle apparatus in these cells frequently contained multiple poles. These results suggest that the organization of spindle microtubules during prometaphase requires a protein phosphatase that is sensitive to nanomolar concentrations of okadaic acid. Chromosomes in 10 nM okadaic acid‐treated cells appear to be attached to spindle microtubules and the nuclear envelope is absent. The effects of okadaic acid on the spindle differ from those elicited by the calcium channel blocker, nimodipine, indicating that this okadaic acid sensitive phosphatase is not part of the calcium signalling events which participate in mitotic progression. © 1992 Wiley‐Liss, Inc.
Analysis of the influences of the E5 transforming protein on kinetic parameters of epidermal growth factor binding and metabolism
Authors: Waters Christopher M. , Overholser Knowles A. , Sorkin Alexander , Carpenter Graham
Published: February 2005

The E5 protein of the bovine papillomavirus induces cellular transformation when transfected into NIH 3T3 cells, and the extent of focal transformation is enhanced by cotransfection with the epidermal growth factor (EGF) receptor (Martin et al., Cell 59:21–32, 1989). To determine whether E5 affects EGF:receptor interactions, we analyzed the kinetics of 125I‐EGF processing using a mathematical model that enabled us to evaluate rate constants for ligand association (ka), dissociation (kd), internalization (ke), recycling (kr), and degradation (kh). These rate constants were measured in NIH 3T3 cells transfected with the human EGF receptor (ER cells) and in cells transfected with both the EGF receptor and E5 (E5/ER cells). We found that the rate constant for 125I‐EGF association ka was significantly decreased in E5/ER cells, but was apparently occupancy‐independent in both cell lines. The 125I‐EGF dissociation rate constant kd was significantly lower in E5 transformed cells, and increased with occupancy in both cell lines. This suggests that E5 alters the receptor before or during EGF binding so that ligand association is slower; however, once complexes are formed, EGF is bound more tightly to the receptor. Rate constants for internalization ke were also found to be occupancy‐dependent, although at a given level of occupancy ke was similar for both cell lines. Also, there was no apparent effect of E5 on the recycling rate constant kr. The 125I‐EGF degradation rate constant kh was 30% lower in E5 transformed cells, and was occupancy‐independent. The overall effect of E5 is to stabilize intact EGF:receptor complexes which may alter mitogenic signaling of the receptor. © 1992 Wiley‐Liss, Inc.
Interleukin‐1β induction of TNF‐α gene expression: Involvement of protein kinase C
Authors: Bethea John R. , Gillespie G. Yancey , Benveniste Etty N.
Published: February 2005

In the human astroglioma cell line CH235‐MG, interleukin‐1β (IL‐1β) induces transcriptional activation of the tumor necrosis factor‐alpha (TNF‐α) gene, resulting in expression of TNF‐α mRNA and biologically active TNF‐α protein. This study was undertaken to elucidate intracellular signaling pathways involved in IL‐1β induction of the TNF‐α gene. We demonstrated that the protein kinase C (PKC) activator 4β‐phorbol 12β‐myristate 13α‐acetate (PMA) in concert with Ca++ ionophore A23187 induced expression of TNF‐α mRNA and protein, whereas an inactive PMA analogue (αPMA) had no effect. Various cyclic nucleotide activators such as 8‐Bromo cAMP, cholera toxin, and forskolin had no effect on TNF‐α production. Two PKC inhibitors, H7 and staurosporine (SS), abrogated IL‐1β induced TNF‐α expression in a dose‐dependent fashion. Treatment of CH235‐MG cells with a high concentration of PMA (1 μM) for an extended period of time (48 h) caused a greater than 90% reduction in total PKC activity. Further strengthening a role for PKC in this cytokine response is the fact that IL‐1β was no longer able to induce TNF‐α expression in these PKC depleted cells. Last, IL‐1β treatment produced an increase of total PKC activity in CH235‐MG cells. Taken together, these data demonstrate that IL‐1β induces TNF‐α gene expression in CH235‐MG cells in a PKC‐dependent manner. © 1992 Wiley‐Liss, Inc.
Plasminogen activator regulation in osteoblasts: Parathyroid hormone inhibition of type‐1 plasminogen activator inhibitor and its mRNA
Authors: Fukumoto Seiji , Allan Elizabeth H. , Yee John A. , Gelehrter Thomas D. , Martin T. John
Published: February 2005

In order to determine the mechanism by which parathyroid hormone (PTH) stimulates plasminogen activator (PA) activity in rat osteoblasts, we investigated the effect of human PTH(1–34) [hPTH(1–34)] on the synthesis of mRNAs for tissue‐type PA (tPA), urokinase‐type PA (uPA), and PA inhibitor‐1 (PAI‐1), and on release of PA activity and PAI‐1 protein in both normal rat calvarial osteoblasts and UMR 106‐01 osteogenic sarcoma cells. hPTH(1–34) (0.25–25 nM) decreased PAI‐1 mRNA and protein, and increased PA activity in both cell types in a dose‐dependent manner with ED50 of about 1 nM for both responses. Forskolin and isobutylmethylxanthine also stimulated PA activity and decreased PAI‐1 protein and mRNA in both cell types. hPTH(1–34) did not show any consistent effect on tPA and uPA mRNA in calvarial osteoblasts, but a modest (two‐fold) increase of both mRNAs was observed in UMR 106‐01 cells treated with 25 nM hPTH(1–34). However, when protein synthesis was inhibited with 100 μM cycloheximide, the increase of tPA and uPA mRNA by hPTH(1–34) was enhanced in UMR 106‐01 cells and became evident in calvarial osteoblasts. Fibrin autography also revealed that hPTH(1–34) increases tPA and uPA activity, especially after cycloheximide treatment in UMR 106‐01 cells. These results strongly suggest that PTH increases PA activity predominantly by decreasing PAI‐1 protein production through a cyclic adenosine monophosphate (cAMP)‐dependent mechanism in rat osteoblasts. The reduction of PAI‐1 protein by PTH results in enhanced action of both tPA and uPA, and would contribute to the specific roles of these PAs in bone. © 1992 Wiley‐Liss, Inc.