bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

AbstractDesmosomes 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

AbstractIn 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

AbstractRecent 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

AbstractMurine 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

AbstractReticuloendotheliosis 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

AbstractErythrocytes 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

AbstractMultidrug‐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

AbstractWe 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

AbstractHuman 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

AbstractBiological 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

AbstractIsocoumarins 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

AbstractDuring 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

AbstractAdenosine 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

AbstractRetinoids (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.