bluuebunny/crossref_metadata_2025 huggingface dataset:
Human A673 cells secrete high molecular weight EGF‐receptor binding growth factors that appear to be immunologically unrelated to EGF or TGF‐α
Authors: Stromberg Kurt , Hudgins W. Robert , Fryling Charlotte M. , Hazarika Parul , Dedman John R. , Pardue Robert L. , Hargreaves William R. , Orth David N.
Published: December 2004

Extracts of serum‐free conditioned medium from human rhabdomyosarcoma A673 cells contain high molecular weight (HMW) transforming growth factors (TGFs) that can be partially purified by Bio‐Gel P‐100 and carboxymethyl (CM)‐cellulose chromatography (Todaro et al: Proc Natl Acad Sci USA 77:5258, 1980). Reversephase high performance liquid chromatography (HPLC) revealed a principal peak of epidermal growth factor (EGF) radioreceptor assay (RRA) activity and anchorage‐independent growth (AIG) activity that coeluted with 25–26% acetonitrile. If a trailing shoulder of EGF RRA activity from the CM‐C chromatography was included in the material for HPLC analysis, additional active fractions were observed at 21–22% acetonitrile. Importantly, both active regions from HPLC failed to compete in radioimmunoassays under reduced and denatured conditions for human EGF (hEGF), human TGF‐α‐ (hTGF‐α), or rat TGF‐α (rTGF‐α) and failed to give positive signals in Western blots under conditions in which TGF‐α was readily detected when using an antisera raised against the 17 C‐terminal amino acids of rTGF‐α. Nonreducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) revealed EGF RRA and AIG activities in gel slices corresponding to Mr 15,000 and 22,000 in the 25–26% acetonitrile eluate and Mr 15,000, 20,000, 27,000, and 48,000 in the 21–22% acetonitrile eluate. The presence of multiple forms of EGF‐receptor‐binding peptides produced in vitro suggest size heterogeneity and possible immunologic diversity among high molecular weight members of the EGF/TGF‐α family of growth‐promoting polypeptides.
Interactions of serine proteases with cultured fibroblasts
Authors: Cunningham Dennis D. , Van Nostrand William E. , Farrell David H. , Campbell Corinne H.
Published: December 2004

This review summarizes the mechanisms by which several serine proteases, particularly urokinase, thrombin, and elastase, interact with cultured fibroblasts. Many of these studies were prompted by findings that interactions of these proteases with cells and the extracellular matrix are important in a number of physiologic and pathologic processes. Two main pathways have been identified for specific interactions of these proteases with fibroblasts. One involves surface binding sites for the free protease that appear to bind only one particular protease. An unusual feature collectively shared by the binding sites for urokinase, thrombin, and elastase is that the bound protease is not detectably internalized by the fibroblasts. The other pathway by which serine proteases interact with fibroblasts involves proteins named protease nexins (PNs). Three PNs have been identified. They are secreted by fibroblasts and inhibit certain serine proteases by forming a covalent complex with the protease catalytic site serine. The complexes then bind back to the fibroblasts via the PN portion of the complex and are internalized and degraded. Recent studies showing that the fibroblast surface and extracellular matrix accelerate the inactivation of thrombin by PN‐1 support the hypothesis that the PNs control protease activity at and near the cell surface. The PNs differ from plasma protease inhibitors in their molecular properties, absence in plasma, site of synthesis, and site of clearance of the inhibitor:protease complexes.
Picornaviral processing: Some new ideas
Authors: Palmenberg Ann C.
Published: December 2004

Mature picornaviral proteins are derived by progressive, post‐translational cleavage of a giant precursor polyprotein. At least three viral‐encoded proteolytic activities are involved in the processing. The first cleavage takes place while the polyprotein is still nascent on a ribosome. In poliovirus, this event is probably catalyzed by peptide 2A, a protein from the middle portion of the genome. Most subsequent processing is effected by viral protease 3C, a thiol‐type enzyme, responsible for eight to ten self‐cleaving and autocatalytic reactions within the polyprotein. The final proteolytic processing event, maturation of the VPO peptide, may occur by a novel, autocatalytic, serine‐type mechanism, where viral RNA serves as proton‐acceptor during the cleavage reaction.
Growth factors modify the epidermal growth factor receptor through multiple pathways
Authors: Friedman BethAann , Rosner Marsha Rich
Published: December 2004

Previous results have shown that tumor promoters modify the properties of the epidermal growth factor (EGF) receptor through the activation of protein kinase C. Diacylglycerol‐generating factors such as platelet‐derived growth factor (PDGF) and p28sis should activate protein kinase C and alter EGF receptor properties in a similar manner. To test directly the involvement of protein kinase C in the action of media from v‐sis‐transformed cells on the EGF receptor, Swiss 3T3 cells were first extensively treated with various concentrations of the tumor‐promoter phorbol dibutyrate (PDBu) This treatment reduced levels of active protein kinase C in the cells, making them less responsive to subsequent rechallenge with the tumor promoter. The results demonstrate that there are at least two components to the action of media from v‐sis transformed cells on EGF binding: a labile factor that confers protein kinase C independence and a stable factor that appears to be dependent on protein kinase C. The action of the first factor cannot be mimicked by transforming growth factor‐β or EGF in either the presence or absence of PDGF. The action of the second factor is similar to that of PDGF. These findings indicate that heterologous regulation of the EGF receptor can occur through both protein kinase C‐dependent and ‐independent pathways.
The Ha‐ras‐induced transformed phenotype of Rat‐1 cells can be suppressed in hybrids with rat embryonic fibroblasts
Authors: Willecke Klaus , Griegel Sabine , Martin Wolfgang , Traub Otto , Schäfer Reinhold
Published: December 2004

Somatic cell hybrids were isolated from fusions of diploid embryonic rat fibro‐blasts with transformed Rat‐1 cells which contained 4 to 5 copies of the transforming human Ha‐ras 1 gene. In contrast to their transformed parental cells four hybrid clones showed normal morphology, long latency periods of tumorigenicity in newborn rats, anchorage requirement of proliferation, and an eightfold‐reduced amount of secreted transforming growth factor activity. Thus these hybrids are called suppressed with regard to expression of the Ha‐ras‐induced transformed phenotype. Tumorigenic derivatives of the suppressed hybrids that had segregated chromosomes were isolated. Since two of the tumorigenic hybrid clones showed the similar low level of secreted transforming growth factors as the suppressed hybrids, decreased production of transforming growth factor activity is unlikely to be a sufficient criterion for suppression of malignancy. Whereas one of the suppressed hybrids expressed the transforming gene product p21 at a level similar to that of the transformed parental cells, other suppressed hybrids expressed less p21. This suggests that the suppressed phenotype can be regulated at the posttranslational level of p21 but that additional controls of expression of p21 are likely to exist. DNA of the suppressed hybrids transformed Rat‐1 cells to proliferation in the presence of semisolid agar. Thus the activated human Ha‐ras gene in the suppressed hybrids retained its biological activity even though it did not transform these cells to tumorigenicity.
Suppression of tumorigenicity in somatic cell hybrids does not involve quantitative changes in transcription of cellular Ha‐ras, Ki‐ras, myc, and fos oncogenes
Authors: Schäfer R. , Geisse S. , Willecke K.
Published: December 2004

The transcriptional activity of ten cellular oncogenes was analyzed in somatic cell hybrids that had been obtained after fusion of tumorigenic Chinese hamster cells and normal mouse fibroblasts. The hybrids showed either the tumorigenic or the nontumorigenic phenotype (suppression of tumorigenicity). Out of ten c‐one genes analyzed, four (c‐Ha‐ras, c‐Ki‐ras, c‐myc, and c‐fos) were found to be transcriptionally active at similar levels in tumorigenic as well as in nontumorigenic (suppressed) hybrids. Thus we conclude that suppression of tumorigenicity in Chinese hamster × mouse somatic cell hybrids does not correlate with quantitative changes in expression of these cellular oncogenes. The remaining six cellular oncogenes (c‐abl, c‐erb A and B, c‐fes, c‐myb, and c‐sis) were not transcriptionally active in these hybrids.
Vanadate‐activated calcium influx in A431 cells is dependent on the plasma membrane potential
Authors: Macara Ian G. , Gray George M.
Published: December 2004

Vanadate can activate the uptake of Ca in A431 epidermal carcinoma cells by two‐to fivefold with no detectable lag period. Preincubation with epidermal growth factor (EGF) to down‐regulate the EGF receptor prevents subsequent stimulation by EGF but not that by vanadate. Ca uptake is sodium‐independent and is not activated by depolarization in high KCl. On the contrary, vanadate‐stimulated uptake is completely inhibited by decreasing the plasma membrane potential from about −65 to −30 mV. These results demonstrate that the EGF receptor is not itself functioning as a Ca channel, that vanadate is not acting at the level of EGF receptor, and that the Ca transport system exhibits an unusual potential sensitivity in that it is inhibited by depolarization of the plasma membrane.
Transformation of glucocorticoid and progesterone receptors to the DNA‐binding state
Authors: Pratt William B.
Published: December 2004

This brief review explores some recent observations relating to the structure of untransformed glucocorticoid and progesterone receptors and the mechanism by which the receptors are transformed to the DNA‐binding state. In their molybdate‐stabilized, untransformed state, progesterone and glucocorticoid receptors exist as a heteromeric 8‐9S complex containing one unit of steroid binding phosphoprotein and one or two units of the 90 kD heat shock protein hsp90. When the receptors are transformed, the steroid‐binding protein dissociates from hsp90. In cytosol preparations, temperature‐mediated dissociation proceeds much more rapidly in the presence of hormone. The dissociated receptor binds to DNA with high affinity, regardless of whether it is in the hormone‐bound or the hormone‐free state. These observations raise the possibility that the primary, and perhaps the only, role for the hormone is to promote dissociation of the receptor‐hsp90 complex.

Molybdate, vanadate, and tungstate inhibit receptor transformation to the DNA‐binding form, an effect that appears to reflect the ability of these transition metal oxyanions to stabilize the complex between the steroid receptor and hsp90. By promoting the formation of disulfide bonds, hydrogen peroxide also stabilizes the glucocorticoid receptor‐hsp90 complex and prevents receptor transformation. A small, heat‐stable factor present in all cytosol preparations inhibits receptor transformation, and, when the factor is removed, glucocorticoid receptors are rapidly transformed. This ubiquitous factor has the physical properties of a metal anion, and it is proposed that molybdate and vanadate affect steroid receptor complexes by interacting with a metal anion‐binding site that is normally occupied by this endogenous receptor‐stabilizing factor.
Characterization of HMG CoA synthase activity of rat liver and CHO‐K1 cells
Authors: Schnitzer‐Polokoff Robin , Sinensky Michael
Published: December 2004

This report describes the characterization and partial purification of rat liver 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG CoA) synthase activity. A preliminary characterization of Chinese hamster ovary (CHO) cell HMG CoA synthase activity is also presented. Ion‐exchange chromatography of ammonium sulfate precipitates of rat liver cytosol indicate the existence of two isoenzymes of HMG CoA synthase. These isoenzymes are physically, catalytically, and immunologically distinct. One of these isoenzymes, peak 1, resembles mitochondrial HMG‐CoA synthase activity as evidenced by similarities in elution upon ion‐exchange chromatography, inhibition by MgCl2, and cross reactivity with an antibody prepared against the mitochondrial enzyme. As peak 1 activity is unstable, further purification studies were performed on peak 2 activity. Peak 2 can be further resolved into two activities (peaks 2A and 2B) by gel filtration. In contrast, CHO‐K1 cells (a permanent fibroblast line) possess only peak 2 type HMG CoA synthase activity.
Uridine kinase: Altered subunit size or enzyme expression as a function of cell type, growth stimulation, or mutagenesis
Authors: Cheng Nancy , Traut Thomas W.
Published: December 2004

Using antibody prepared against pure undine kinase from Ehrlich ascites cells, we have measured the expression of enzyme protein by the Western blot technique. Variations were observed in the Mr of the enzyme subunit for uridine kinase from different species: 32,000 (mouse Ehrlich ascites cells), 30,000 (normal human lymphocytes), 28,000 (mouse tissues), 27,500 (rat tissues). For different normal tissues from the same species, there was no significant variation in the subunit size. Transformed human and mouse cell lines, selected for a deficiency of uridine kinase activity in the presence of inhibitors activated by this enzyme, expressed two cross‐reacting proteins, one with a normal (30,000) and one with a smaller (21,000) subunit molecular weight than was found in the parental cell line (human lymphoma), or only a smaller protein of Mr 25,000 (mouse lymphoma). Our results show that selection protocols using metabolite inhibitors do not always repress the expression of the enzyme but instead may lead to selection of those cells that have a mutation in the uridine kinase gene, resulting in the expression of an inactive enzyme. The expression of uridine kinase protein changes when cells are stimulated to divide. For both mouse fibroblasts and human lymphocytes, expression of urndine kinase protein as well as activity clearly increased after cells were stimulated to grow. In fibroblasts, increases are seen by 3 hr after stimulation, and plateau after 9 hr at a sevenfold increase. In lymphocytes, no change is seen until 12 hr after stimulation, and a plateau is not reached until 72 hr, with a total increase of ∼50‐fold. There has been considerable interest in the possibility of uridine kinase isozymes. Except for cells that have been mutagenized, the present results show that, as judged by subunit molecular weight, there appears to be only one enzyme form in normal and neoplastic cells or in cells in which uridine kinase activity is induced.
sn‐1,2‐Diacylglycerols and phorbol diesters: Uptake, metabolism, and subsequent assimilation of the diacylglycerol metabolites into complex lipids of cultured cells
Authors: Welsh Clement J. , Cabot Myles C.
Published: December 2004

The cell‐permeable diacylglycerol mediators have been shown to mimic partially the effects of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) on cultured cells. In order to evaluate the metabolic stability of the lipid mediators, several radiolabeled diacylglycerols were synthesized and their uptake and intracellular fate in cultured HL‐60 (human promyelocytic leukemia) cells was compared with TPA. In addition to whole cell assessment, the stability of diacyl lipids and TPA was evaluated in a buffer/water system and in the presence of serum and subcellular fractions. The compounds studied include 1,2‐dioleoyl‐sn‐glycerol (DiOG), l‐oleoyl‐2‐ace‐tyl‐sn‐glycerol (OaG), 1‐palmitoyl‐2‐acetyl‐sn‐glycerol (PaG), the ether‐linked analog l‐palmityl‐2‐acetyl‐sn‐glycerol (ePaG), and TPA. TPA was comparatively stable to lipase hydrolysis in all systems examined. First, the data show that within 5 min at pH 7.9, nearly 50% of the PaG (originally > 92% 1,2‐isomer) had isomerized, and rapid formation of the 1,3‐isomer also occurred with OaG and ePaG. The metabolism of OaG and PaG by serum hydrolases, using a reaction medium containing 10% serum, was chiefly by acetate hydrolysis; however, fatty acid was also liberated. After a 60‐min incubation 68% of the [14C]OaG was converted, by serum enzymes, to monooleoylglycerol plus oleic acid. Heat‐inactivation of serum reduced the enzymatic formation of fatty acid by 60–70%. ePaG was also metabolized by serum enzymes, but the ether‐linked alkylglycerol product was stable. The results of cell‐free studies (postmitochondrial supernatant) showed that cellular enzymes were present that could, like serum, convert the diacylglycerols to monoacylglycerols and free fatty acids. Studies using cultured cells showed that radiolabeled OaG, PaG, and ePaG were rapidly taken up by the cells and metabolized. Labeled metabolic products from the diacylglycerols appeared, in a time‐dependent manner, in cellular phospholipids and triacylglycerols. The results from experiments employing l‐acyl‐2‐acetyl‐sn‐[3H]glycerol and [3 H]acyl‐2‐acetyl‐sn‐glycerol indicate that the intracellular mode of mediator metabolism is via complete hydrolysis with subsequent incorporation of 3H‐acyl groups into complex lipids. Data are also presented which show that a substantial amount of cellular lipid acyl group modification occurs and large amounts of glycerol are produced when cells are cultured with OaG. Collectively, these results demonstrate that the diacylglycerol mediators, when compared with TPA, are not stable and are metabolized by both serum and cellular enzymes. In view of this some of the cellular affects of OaG exposure could be directed by mediator metabolites or be the result of membrane modification.
Transport of proteins into yeast mitochondria
Authors: van Loon Adolphus P. G. M. , Eilers Martin , Baker Alison , Verner Keith
Published: December 2004

The amino‐terminal sequences of several imported mitochondrial precursor proteins have been shown to contain all the information required for transport to and sorting within mitochondria. Proteins transported into the matrix contain a matrix‐targeting sequence. Proteins destined for other submitochondrial compartments contain, in addition, an intramitochondrial sorting sequence. The sorting sequence in the cytochrome c1 presequence is a stop‐transport sequence for the inner mitochondrial membrane. Proteins containing cleavable presequences can reach the intermembrane space by either of two pathways: (1) Part of the presequence is transported into the matrix; the attached protein, however, is transported across the outer but not the inner membrane (eg, the cytochrome c1 presequence). (2) The precursor is first transported into the matrix; part of the presequence is then removed, and the protein is reexported across the inner membrane (eg, the precursor of the iron‐sulphur protein of the cytochrome bc1 complex).

Matrix‐targeting sequences lack primary amino acid sequence homology, but they share structural characteristics. Many DNA sequences in a genome can potentially encode a matrix‐targeting sequence. These sequences become active if positioned upstream of a protein coding sequence. Artificial matrix‐targeting sequences include synthetic presequences consisting of only a few different amino acids, a known amphiphilic helix found inside a cytosolic protein, and the presequence of an imported chloroplast protein.

Transport of proteins across mitochrondrial membranes requires a membranes requires a membrane potential, ATP, and a 45‐kd protein of the mitochondrial outer membrane. The ATP requirement for import is correlated with a stable structure in the imported precursor molecule. We suggest that transmembrane transport of a stably folded precursor requires an ATP‐dependent unfolding of the precursor protein.
Effects of hyperosmolarity on ligand processing and receptor recycling in the hepatic galactosyl receptor system
Authors: Oka Janet A. , Weigel Paul H.
Published: December 2004

Binding, endocytosis, and degradation of asialo‐orosomucoid (ASOR) mediated by the galactosyl (Gal) receptor were examined in isolated rat hepatocytes in complete media supplemented with an osmolite. The specific binding of 125I‐ASOR to cells at 4°C was unaffected by up to 0.4 M sucrose or NaCl. Unlike sucrose or NaCl, mannitol stimulated 125I‐ASOR binding at low concentrations but inhibited binding at higher concentrations. Continuous internalization at 37°C, which requires receptor recycling, was completely blocked at 0.2 M sucrose or 0.15 M NaCl, corresponding in each case to a total osmolality of about 550 mmol/ kg. This effect was reversed and endocytic function was restored by washing the cells, indicating that cell viability was unaffected. The rate of degradation of internalized 125I‐ASOR was also inhibited by increasing sucrose concentrations. This inhibition is due to a block in the delivery of ligand to lysosomes and not an effect on degradation per se. In the presence of 0.2 M sucrose, the rate and extent of endocytosis of surface‐bound 125I‐ASOR were, respectively, 33.0 ± 8.1% and 69.4 ± 10.5% (n = 8) of the control without sucrose. Under these conditions, the dissociation of internalized receptor‐ASOR complexes was completely inhibited. When sucrose was added, the effect on the endocytosis of surface‐bound 125I‐ASOR was virtually immediate. Previous studies showed that about 40% of the surface‐bound 125I‐ASOR which is internalized can return to the cell surface still bound to receptor (Weigel and Oka: J Biol Chem 259:1150, 1984). If 0.2 M sucrose was added after endocytosis occurred, 125I‐ASOR still returned to the cell surface, although the rate and extent of return were inhibited by more than 50%. Interestingly, hyperosmolarity is the only treatment we have found which can reversibly inhibit, although only partially, the endocytosis of surface‐bound 125I‐ASOR.
Fractionation of desmosomes and comparison of the polypeptide composition of desmosomes prepared from two bovine epithelial tissues
Authors: Jones Stephanie M. , Jones Jonathan C. R. , Goldman Robert D.
Published: December 2004

Desmosomes isolated from bovine tongue mucosa or muzzle epidermis appeared identical by ultrastructural analyses but had some differences in their polypeptide compositions as determined by SDS‐PAGE. These preparations were extracted in 9 M urea, 10 mM Tris‐HCl (pH 9), and 25 mM B‐mercaptoethanol and then centrifuged at 240,000g for 30 min. The urea‐soluble and insoluble fractions were analyzed by SDS‐PAGE. The urea soluble fractions of both tongue and muzzle desmosomes were enriched in polypeptides of 240, 210, 81, and 75 kDa and also polypeptides (40 to 70 kDa) that were keratin‐like, as determined by immunoblotting analyses with keratin antisera. The urea insoluble fraction of tongue desmosomes contained glycoproteins of 165, 160, 140, 110, and 100 kDa, while this fraction from muzzle contained glycoproteins of 165, 115, and 105 kDa. Ultrastructural examinations of insoluble pellets obtained from urea extracted tongue and muzzle desmosomes showed that most of the components at the cytoplasmic faces of the desmosomes were removed, while the membrane regions of the desmosomes resisted the treatment. The urea soluble proteins were dialyzed against 10 mM Tris‐HCl (pH 7.6), and the resulting preparation was pelleted by centrifugation and examined by electron microscopy. Ultrastructural examination of this material revealed that it had assembled into a fibrillar meshwork, similar to the fibrillar region adjacent to the submembranous plaque of isolated desmosomes. Thus, treatment of isolated desmosomes with 9 M urea allowed the fractionation of membrane‐associated desmosomal proteins from cytoplasmic desmosomal proteins. A comparison of these fractions from tongue and muzzle indicated that the polypeptide compositions of the desmosomes varied between tissues, especially with respect to the fractions enriched in either glycoproteins or keratin.
Functional analysis of mitochondrial protein import in yeast
Authors: Glaser Scott M. , Trueblood Cynthia E. , Dircks Lori K. , Poyton Robert O. , Cumsky Michael G.
Published: December 2004

In order to facilitate studies on protein localization to and sorting within yeast mitochondria, we have designed an experimental system that utilizes a new vector and a functional assay. The vector, which we call an LPS plasmid (for leader peptide substitution), employs a yeast COX5a gene (the structural gene for subunit Va of the inner membrane protein complex cytochrome c oxidase) as a convenient reporter for correct mitochondrial localization. Using in vitro mutagenesis, we have modified COX5a so that the DNA sequences encoding the wild‐type subunit Va leader peptide can be precisely deleted and replaced with a given test sequence. The substituted leader peptide can then be analyzed for its ability to direct subunit Va to the inner mitochondrial membrane (to target and sort) by complementation or other in vivo assays. In this study we have tested the ability of several heterologous sequences to function in this system. The results of these experiments indicate that a functional leader peptide is required to target subunit Va to mitochondria. In addition, leader peptides, or portions thereof, derived from proteins located in other mitochondrial compartments can also be used to properly localize this polypeptide. The results presented here also indicate that the information necessary to sort subunit Va to the inner mitochondrial membrane does not reside in the leader peptide but rather in the mature subunit Va sequence.
Ulex europeus type I agglutinin detects carcinoembryonic antigen in extracts of human colorectal carcinoma
Authors: Jessup J. M. , Qi K.‐F. , Kanellopoulos K. , Cleary K. , Hickey C. , Reading C. L.
Published: December 2004

Recent interest has focused on fucosylated epitopes expressed on human neoplasms. The plant lectin Ulex europus agglutinin, Type I (UEA) binds fucosylated oligosac‐charides, while UEA‐reactive substances have a tissue distribution similar to carci‐noembryonic antigen (CEA). We sought to determine if UEA reacted with CEA in extracts of fresh primary and metastatic colorectal carcinomas and paired normal tissues. The extracts were electrophoretically transferred to nitrocellulose membranes after the proteins were separated by SDS‐PAGE in 10% polyacrylamide gels. The transfer membranes were then stained with peroxidase‐conjugated UEA (UEA‐P) or antibody to CEA (CEA‐P). UEA‐P reacted with a 170‐190‐kDa band in extracts of 22 of 30 primary tumors, 10 of 12 metastases, but only 1 of 5 villous adenomas. UEA‐P generally did not react with normal colon or liver extracts. UEA‐P also did not bind to 170–190‐kDa molecules in Western transfers of a breast carcinoma metastatic to bowel and a focal nodular hyperplasia of liver. CEA‐P displayed similar reactivity and detected CEA in a tumor extract negative for UEA. Fucose blocked binding of UEA‐P to Western transfers of tumor extracts. CEA‐P reacted with a 170–190‐kDa substance in tumor extracts eluted with fucose from a column of immobilized UEA. Thus, UEA reacts with fucosylated oligosaccharides on most, but not all, species of CEA and may be a useful adjunct to anti‐CEA immunohis‐tochemistry.
Induction of reversible changes in cell‐surface glycoconjugates and lung colonization potential by 13‐cis retinoic acid
Authors: Couch Marion J. , Pauli Bendicht U. , Weinstein Ronald S. , Coon John S.
Published: December 2004

Murine squamous carcinoma cells (KLN205) grown in a medium supplemented with the retinoid, 13‐cis retinoic acid (RA), had dose‐dependent, selective increases in the expression of certain lectin receptors, which correlated with a dramatic decrease in the ability to form pulmonary colonies (P −.0003) (Couch MJ, Pauli BU, Weinstein RS, Coon JS: JNCI, 78:971 −977, 1987). These findings suggest a possible relationship between the RA‐induced glycoconjugate alterations and the decreased experimental metastatic behavior. We further define the mechanism of RA's action. The finding that RA treatment (5 × 10−6 M, 5 × 10−7 M) did not perturb the cell cycle of KLN205 cells provides further proof that the decreased metastatic behavior is not attributable to any inhibition in the rate of growth or to alterations in the cell cycle. Furthermore, since stable subpopulations with variable lectin binding could not be detected, the mechanism of RA's action does not appear to be due to selection of variant tumor‐cell subpopulations. Finally, in a scries of experiments designed to determine the reversibility of the RA treatment, the RA‐induced decrease in metastatic behavior reverted back to a more metastatic state in the same time frame (3 days) as the reversion of the RA‐induced changes in cell‐surface glycoconjugate expression. This reversion provides further evidence for a close relationship between the RA‐induced modulation of tumor cell‐surface glycoconjugate expression and the decreased metastatic behavior; it suggests that transient, reversible modulation of the tumor cell surface may play a role in determining metastatic behavior.
Autogenous growth factor production by reticuloendotheliosis virus‐transformed hematopoietic cells
Authors: Garry Robert F. , Bose Henry R.
Published: December 2004

Reticuloendotheliosis virus strain T (REV‐T)‐transformed cells gave rise spontaneously to variants which secrete a factor that forms a distinct visible ring of precipitation (halo) surrounding colonies grown in soft agar. An Mr 15,000 protein was produced at higher levels by halo variants than by nonhalo‐producing cells. An assay designed to detect the formation of precipitates enabled purification of an Mr 15,000 protein, p15, from serum‐free medium conditioned by the growth of REV‐T‐trans‐formed hematopoietic cells. Fractions enriched in p15 permitted the growth of REV‐T‐ trans formed cells under conditions where they normally failed to proliferate.
Erythrocytes attached to a wheat germ agglutinin coated surface display an altered phospholipid metabolism
Authors: Dale George L. , Suzuki Takashige
Published: December 2004

Erythrocytes were bound to a lectin‐coated surface; the multivalent attachment to this surface resulted in a severe deformation of the cells and an alteration in the cellular phospholipid metabolism. Human erythrocytes were allowed to bind for 20 min at 20°C to polystyrene beads coated with wheat germ agglutinin (WGA beads). The bound erythrocytes were then lysed to produce stroma bound to WGA beads. Control stroma and stroma‐WGA beads were incubated at 37°C with γ‐32P‐ATP to examine the phospholipid labeling patterns. The control stroma incorporated 32P‐label into phosphatidylinositol‐4‐phosphate and phosphatidylinositol‐4,5‐bisphosphate, in agreement with earlier studies. However, the stroma‐WGA beads showed incorporation of 32P‐label into phosphatidic acid in addition to that in the phosphoinositides. The quantity of 32P‐phosphatidic acid produced during the 20‐min assay was 3.23 ± 0.84 (n = 7) picomoles/μg stromal cholesterol; the amount synthesized, however, was dependent on the procedure used to prepare the stroma‐WGA beads. If the erythrocytes were bound to the WGA beads at 0°C instead of 20°C, the quantity of 32P‐phosphatidic acid produced during the subsequent 37°C assay with γ‐32P‐ATP was decreased 4.2 fold; the phosphoinositide labeling pattern was unchanged. In addition, when the time for binding of intact erythrocytes to the WGA beads was varied from 1 to 20 minutes, there was a time‐dependent increase in the amount of 32P‐phosphatidic acid produced. This induction of phosphatidic acid synthesis could not be duplicated with fluid phase WGA. Therefore, the multivalent binding of intact erythrocytes to WGA beads causes an alteration in phospholipid metabolism.
Increased epidermal growth factor receptor in multidrug‐resistant human neuroblastoma cells
Authors: Meyers Marian B. , Shen W. P. Violet , Spengler Barbara A. , Ciccarone Valentina , O'Brien James P. , Donner David B. , Furth Mark E. , Biedler June L.
Published: December 2004

Multidrug‐resistant human neuroblastoma cell lines obtained by selection with vincristine or actinomycin D from two independent clonal lines, SH‐SY5Y and MCIXC, have 3‐ to 30‐fold more cell surface epidermal growth factor (EGF) receptors than the drug‐sensitive parental cells as indicated by EGF binding assays and immunoprecipitation, affinity‐labeling, and phosphorylation studies. Reversion to drug sensitivity in one line was accompanied by a return to the parental level of EGF receptor. SH‐EP cells, a clone derived from the same neuroblastoma cell line as SH‐SY5Y but which displays melanocyte rather than neuronal lineage markers, also express significantly more EGF receptor than SH‐SY5Y cells. By nucleic acid hybridization analysis with a molecularly cloned probe, increased receptor level in multidrug‐resistant cells was shown to be the result of higher levels of EGF receptor mRNA in drug‐resistant than in drug‐sensitive cells. The increased steady state amount of specific RNA did not result from amplification of receptor‐encoding genes. A small difference was observed in the electrophoretic mobility under denaturing conditions of EGF receptor immunoprecipitated from drug‐resistant and drug‐sensitive cells. Quantitative and qualitative modulation of the EGF receptor might reflect alterations in the transformation and/or differentiation phenotype of the resistant cells or might result from unknown selective pressures associated with the development of multidrug resistance.
Regulation of proliferation in a murine colony‐stimulating factor‐dependent myeloid cell line: Superinduction of C‐fos by the growth inhibitor 8‐Br‐cyclic adenosine 3′:5′ monophosphate
Authors: Harel‐Bellan Annick , Farrar William L.
Published: December 2004

We have investigated the effect of 8‐Br‐cyclic adenosine 3′:5′ monophosphate (cAMP), a pharmacological activator of cAMP‐dependent protein kinase, on the proliferation and the nuclear proto‐oncogene induction in a murine granulocyte macrophage colony‐stimulating factor (GM‐CSF)‐dependent myeloid cell line. Cells were growth arrested by granulocyte macrophage colony‐stimulating factor and serum deprivation and were allowed to proceed in the cell cycle by addition of the lymphokine in the presence or absence of 8‐Br‐cAMP.3H‐thymidine incorporation assays showed that addition of 8‐Br‐cAMP inhibited the entry of cells into S phase and the subsequent proliferation. Northern analysis showed that 8‐Br‐cAMP had opposite effects on c‐fos and c‐myc mRNA induction. 8‐Br‐cAMP induced c‐fos in the absence of any GM‐CSF. In the presence of GM‐CSF, c‐fos mRNA was superinduced (30‐fold induction compared to four‐ to fivefold by each signal alone). On the contrary, 8‐Br‐cAMP was not able to induce c‐myc in the absence of growth factor and hardly interfered with the induction of c‐myc by GM‐CSF. Phorbol myristate acetate (PMA), a pharmacological activator of the lipid and CA++ ‐dependent protein kinase C, was shown to induce nuclear proto‐oncogene mRNA in the GM‐CSF‐dependent cell line. We investigated the effect of 8‐Br‐cAMP on PMA‐induced c‐fos and c‐myc mRNA levels. When both cAMP dependent and lipid‐dependent kinase systems were co‐stimulated in the absence of GM‐CSF, c‐fos message was again superinduced (60‐fold induction). On the contrary, c‐myc message induction by PMA was inhibited by 80% by coactivation of cAMP‐dependent protein kinase with 8‐Br‐cAMP. Our data indicate that an antiproliferative signal induces or even superinduces c‐fos message and hardly interferes with c‐myc induction, suggesting that the intracellular pathways resulting in c‐fos and c‐myc induction may be distinct and that two different pathways can lead to c‐fos induction, with synergistic effects when both are activated.
Relationship of cellular oncogene expression to inhibition of growth and induction of differentiation of Daudi cells by interferons or TPA
Authors: Clemens Michael J. , Tilleray Vivienne J. , James Robert , Gewert Dirk R.
Published: December 2004

Human α or β interferons inhibit the proliferation of Daudi Burkitt lymphoma cells and induce the differentiation of these cells towards a mature plasma cell phenotype. Similar responses are seen when Daudi cells are treated with the phorbol ester, TPA. Both interferons and TPA down‐regulate expression of the c‐myc oncogene in these cells. Although TPA can mimic the effect of interferon on cell differentiation, it does not induce 2′5′ oligoadenylate synthetase or the interferon‐sensitive mRNAs, 6–16 or 9–27. Thus chronic stimulation of protein kinase C by TPA cannot mimic all of the effects of interferon treatment on gene expression. Inhibition of ADP‐ribosyl transferase activity by 3‐methoxybenzamide impairs interferon‐ or TPA‐induced differentiation of Daudi cells. This agent induces a higher level of c‐myc mRNA in the cells and stimulates the incorporation of [3H]thymidine into DNA; although these effects are partially counteracted by interferon or TPA treatment, the elevated expression of the c‐myc gene may be sufficient to prevent terminal differentiation and allow cell proliferation to continue.
Novel method for studying mRNA phenotypes in single or small numbers of cells
Authors: Rappolee Daniel A. , Wang Alice , Mark David , Werb Zena
Published: December 2004

Biological processes, such as growth control, are often governed by biochemical steps involving mRNA transcripts that are short‐lived and have a low copy number. Furthermore, the cells involved in these processes are often available in low numbers from in vivo sources. We now report a method that is superior to in situ hybridization, RNA blot analysis, and the nuclease protection assay for the study of short‐lived, low‐copy‐number mRNA transcripts. The method consists of a microprocedure for isolating RNA from one to a few thousand cells and two coupled enzymatic steps: reverse transcription of whole cellular RNA, followed by amplification of the cDNA by a specifically primed polymerase chain reaction to give specific cDNA fragments that can be visualized on agarose gels by ethidium bromide staining. With this method we have detected actin mRNA from a single cell, or < 100 cRNA molecules, and have quantified differences in RNA concentrations of less than threefold. The reverse transcription reaction products can be divided for the polymerase chain reaction, and several mRNA species can be assayed simultaneously. Therefore, we call the method single‐cell mRNA phenotyping. This technique is applicable to the analysis of low‐copy‐number growth factor transcripts in cells in culture and in vivo.
Mechanism‐based isocoumarin inhibitors for serine proteases: Use of active site structure and substrate specificity in inhibitor design
Authors: Powers James C. , Kam Chih‐Min , Narasimhan Lakshmi , Oleksyszyn Joszef , Hernandez Maria A. , Ueda Toshihisa
Published: December 2004

Isocoumarins are potent mechanism‐based heterocyclic irreversible inhibitors for a variety of serine proteases. Most serine proteases are inhibited by the general serine protease inhibitor 3,4‐dichloroisocoumarin, whereas isocoumarins containing hydrophobic 7‐acylamino groups are potent inhibitors for human leukocyte elastase and those containing 7‐alkylureidogroups are inhibitors for porcine pancreatic elastase. Isocoumarins containing basic side chains that resemble arginine are potent inhibitors for trypsin‐like enzymes. A number of 3‐alkoxy‐4‐chloro‐7‐guanidinoisocoumarins are potent inhibitors of bovine thrombin, human factor Xa, human factor XIa, human factor XIIa, human plasma kallikrein, porcine pancreatic kallikrein, and bovine trypsin. Another cathionic derivative, 4‐chloro‐3‐(2‐isothiureidoethoxy) isocoumarin, is less reactive toward many of these enzymes but is an extremely potent inhibitor of human plasma kallikrein. Several guanidinoisocoumarins have been tested as anticoagulants in human plasma and are effective at prolonging the prothrombin time. The mechanism of inhibition by this class of heterocyclic inactivators involves formation of an acyl enzyme by reaction of the active site serine with the isocoumarin carbonyl group. Isocoumarins with 7‐amino or 7‐guanidino groups will then decompose further to quinone imine methide intermediates, which react further with an active site residue (probably His‐57) to form stable inhibited enzyme derivatives. Isocoumarins should be useful in further investigations of the physiological function of serine proteases and may have future therapeutic utility for the treatment of emphysema and coagulation disorders.
Immunological study of acidic fibroblast growth factor (aFGF) distribution in the eye
Authors: Caruelle D. , Groux‐Muscatelli B. , Gaudric A. , Sestier C. , Coscas G. , Caruelle J. P. , Barritault D.
Published: December 2004

During the last ten years, several groups, including the present authors, have detected growth factor activities in various ocular tissues, and the presence of a ubiquitous Eye‐Derived Growth Factor (EDGF) has been described. More recently, isolation and characterization of this growth factor activity from the retina led to the identification of two molecules. These molecules were shown to be identical to other growth factors isolated from neuronal and non‐neuronal tissues and are now designated as acidic and basic fibroblast growth factor (aFGF, bFGF). The biological function and the reason for the ubiquitous distribution of these factors remain unclear. Understanding may be improved by quantification of this distribution in various tissues during development. In the present study, specific polyclonal antibodies were raised against acidic FGF, aFGF was determined in various ocular tissues by enzyme immunoassay, and the localization of immuno‐reactive aFGF by immunohistological staining with fluorescent antibodies or with enzyme‐ or gold‐labeled antibodies was studied.

In almost all tissues tested aFGF was found; but the retina, cornea, and vitreous body contained the highest levels of aFGF per gram of tissue. In the retina, aFGF was associated primarily with the nerve fiber layer and the inner and outer segments of the photoreceptors, whereas corneal aFGF was detected in the cytoplasma of the basal layer of epithelial cells.
Normal and mutant human adenosine deaminase genes
Authors: Akeson Ann L. , Wiginton Dan A. , Hutton John J.
Published: December 2004

Adenosine deaminase (ADA) deficiency in humans is one cause of severe combined immunodeficiency. When ADA fails to catalyze the deamination of adenosine and deoxyadenosine, the levels of deoxyadenosine that accumulate are toxic to lymphoid cells. Patients with complete ADA deficiency (e.g., with less than 5% normal ADA catalytic activity) lack both B‐ and T‐lymphocyte function. B‐lymphoblast cell lines derived from patients with ADA deficiency have been analyzed at multiple levels. Blot hybridization and S1 nuclease analysis of ADA messenger RNA (mRNA) indicates that the majority of ADA‐deficient cell lines have ADA mRNA in the same abundance and size as in normal cell lines. Sequence analysis of ADA cDNAs derived from these mRNAs shows that the majority of mutations are single base changes that alter the amino acid sequence. Expression analysis proves that these point mutations lead to deficiency of ADA catalytic activity. Several cell lines have mutations that alter mRNA transcription or processing. These include a point mutation in one allele of an ADA‐deficient cell line that leads to deletion of exon 4 during mRNA splicing. In addition, two cell lines are homozygous for large deletions of the gene that are the result of homologous recombination. Subjects with partial ADA deficiency have undetectable ADA activity in their erythrocytes, variable activity in their lymphoid cells, and normal immunological function. Analysis of the ADA catalytic activity of partially deficient cell lines indicates that the mutations involved affect protein stability. However, the mutations causing partial ADA deficiency are as yet undefined.
Regulation of tissue transglutaminase gene expression as a molecular model for retinoid effects on proliferation and differentiation
Authors: Chiocca E. Antonio , Davies Peter J. A. , Stein Joseph P.
Published: December 2004

Retinoids (structural and functional analogs of vitamin A) are potent antiproliferative agents whose mode of action is poorly understood. It has been suggested that the molecular events that underscore their action involve alterations in gene expression, but no gene has yet been shown to be directly regulated by these molecules. Several years ago, we found that retinoic acid caused an accumulation of the enzyme tissue transglutaminase in murine peritoneal macrophages and in human promyelocytic leukemia (HL‐60) cells. We now report that this induction is caused by an increase in the mRNA for this enzyme. Retinoic acid is the only mediator of this induction, since its effects do not depend on the presence of serum proteins. The induction of tissue transglutaminase mRNA is not due to an increase in its stability but to an increase in the relative transcription rate of its gene. We present a model to correlate the retinoid induction of tissue transglutaminase with retinoid effects on cellular growth and differentiation.
Effect of the thiol group inhibitor monobromobimane and other inhibitors on the composition of the platelet cytoskeletal core and its association with glycoprotein IIIa
Authors: Puszkin Elena G. , Mauss Evelyn A. , Milot Denise C. , Zucker Marjorie B.
Published: December 2004

SDS‐polyacrylamide gel electrophoresis was used to study the effects of the thiol inhibitor monobromobimane (MB), EDTA, and prostaglandin E1 (PGE1) on the formation and composition of the platelet cytoskeletal core (Triton‐insoluble residue) and its association with glycoprotein (GP) IIIa. Stimulation or aggregation of platelets in response to ADP or thrombin increased the amount of Triton‐insoluble myosin. Aggregation resulted in incorporation of [125I]GP IIIa and a new band at about 210 kDa into the cytoskeletal core. EDTA and PGE1 caused little disaggregation of platelets that were aggregated in PRP with ADP and that had secreted the contents of their granules. In contrast to EDTA, PGE1 decreased the amount of Triton‐insoluble residue and its association with GP IIIa. MB added after ADP‐induced aggregation caused an increase in the amount of cytoskeletal core despite marked disaggregation and a substantial decrease in core‐associated GP IIIa. With aspirin‐treated platelets that had not secreted, EDTA, PGE1, and MB all caused disaggregation and loss of cytoskeletal GP IIIa. MB diminished, but did not reverse, thrombin‐induced aggregation of washed platelets and arrested GP IIIa incorporation into the cytoskeletal core. Concanavalin A (Con A) cross‐links glycoproteins on a single platelet and induces incorporation of GP IIIa into the Triton‐insoluble residue in the absence of platelet aggregation. This induction was not inhibited by MB, although this reagent, as well as aspirin, inhibited Con A‐induced secretion. Since GP IIIa incorporation caused by ADP‐induced aggregation differs from that caused by Con A in its susceptibility to MB, it seems unlikely that thiol groups are directly involved in the association of GP IIIa with the cytoskeletal core.