bluuebunny/crossref_metadata_2025_split huggingface dataset:
Inhibition of mammalian collagenases by thiol‐containing peptides
Authors: Gray Robert D. , Miller Robert B. , Spatola Arno F. 
Published: December 2004

AbstractThe following thiol‐containing peptide analogues of the carboxyl side of the collagenase‐sensitive bond of collagen were synthesized and tested as inhibitors of collagenases partially purified from homogenates of rabbit V‐2 tumor and culture medium of pig synovium: HSCH2CH(CH3)CO‐Ala‐OEt (I), HSCH2CH‐(CH2Ph)CO‐Ala‐OEt (II), HSCH2CH[CH2CH(CH3)2]CO‐Ala‐OEt (III); HSCH2, CH‐[CH2CH(CH3)2]CO‐Ala‐Gly‐OEt (IV); HSCH2CH[CH2CH(CH3)2]CO‐Ala‐Gly‐Gln (V). The compounds are listed in order of their inhibitory potency when assayed with nonfibrillar‐acid‐soluble calfskin collagen at pH 7.6, 35°C. The best inhibitor (III) gave 50% inhibition between 1 and 4 μM. II was a competitive inhibitor with a Ki value of 75 μM. The enzymes preferred an isobutyl side chain at the 2‐carbon position, and, where tested (III, IV), did not discriminate strongly between stereoisomers at the chiral 2‐carbon. Increasing the length of the inhibitor did not markedly increase potency.
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

AbstractExtracts 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

AbstractThis 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

AbstractMature 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

AbstractPrevious 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

AbstractSomatic 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

AbstractThe 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

AbstractVanadate 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

AbstractThis 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

AbstractThis 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

AbstractUsing 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

AbstractThe 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

AbstractThe 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

AbstractBinding, 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.