bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

AbstractSDS‐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.
Ligand‐induced association of epidermal growth factor receptor to the cytoskeleton of A431 cells
Authors: van Bergen en Henegouwen P. M. P. , Defize L. H. K. , de Kroon J. , van Damme H. , Verkleij A. J. , Boonstra J. 
Published: December 2004

AbstractRecently, we have obtained evidence in favor of a structural interaction between the epidermal growth factor (EGF) receptor and the Triton X‐100‐insoluble cytoskeleton of epidermoid carcinoma A431 cells. Here we present a further analysis of the properties of EGF receptors attached to the cytoskeleton. Steady‐state EGF binding studies, analyzed according to the Scatchard method, showed that A431 cells contain two classes of EGF‐binding sites: a high‐affinity site with an apparent dissociation constant (KD) of 0.7 nM (7.5 × 104 sites per cell) and a low‐affinity site with a KD of 8.5 nM (1.9 × 106 sites per cell). Non‐equilibrium binding studies revealed the existence of two kinetically distinguishable sites: a fast‐dissociating site, with a dissociation rate constant (k−1) of 1.1. × 10−3s−1 (1.0–1.3 × 106 sites per cell) and a slow‐dissociating site, with a k−1 of 3.5 × 10−5s−1 (0.6–0.7 × 106 sites per cell).The cytoskeleton of A431 cells was isolated by Triton X‐100 extraction. Scatchard analysis revealed that ∼5% of the original number of receptors were associated with the cytoskeleton predominantly via high‐affinity sites (KD = 1.5 nM). This class of receptors is further characterized by the presence of a fast‐dissociating component (k−1 = 2.0 × 10−3s−1) and a slow‐dissociating component (k−1 = 9.1 × 10−5s−1). The distribution between fast and slow sites of the cytoskeleton was similar to that of intact cells (65% fast and 35% slow sites). Incubation of A431 cells for 2 h at 4°C in the presence of EGF resulted in a dramatic increase in the number of EGF receptors associated to the cytoskeleton. These newly cytoskeleton‐associated receptors appeared to represent low‐affinity binding sites (KD = 7 nM). Dissociation kinetics also revealed an increase of fast‐dissociating sites. These results indicate that at 4°C EGF induces the binding of low‐affinity, fast‐dissociating sites to the cytoskeleton of A431 cells.
Biosynthesis of lysosomal endopeptidases
Authors: Erickson Ann H. 
Published: December 2004

AbstractDespite the clear differences between the amino acid sequence and enzymatic specificity of aspartic and cysteine endopeptidases, the biosynthetic processing of lysosomal members of these two families is very similar. With in vitro translation and pulse‐chase analysis in tissue culture cells, the biosynthesis of cathepsin D, a aspartic protease, and cathepsins B, H and L, cysteine proteases, are compared. Both aspartic and cysteine endopeptidases undergo cotranslational cleavage of an amino‐terminal signal peptide that mediates transport across the endoplasmic reticulum (ER) membrane. Addition of high‐mannose carbohydrate also occurs cotranslationally in the lumen of the ER. Proteases of both enzyme classes are initially synthesized as inactive proenzymes possessing amino‐terminal activation peptides. Removal of the propeptide generates an active single‐chain enzyme. Whether the single‐chain enzyme undergoes asymmetric cleavage into a light and a heavy chain appears to be cell type specific. Finally, late during their biosynthesis both classes of enzymes undergo amino acid trimming, losing a few amino acid residues at the cleavage site between the light and heavy chains and/or at their carboxyltermini. During biosynthesis these enzymes are also secreted to some extent. In most cells the secreted enzyme is the proenzyme bearing some complex carbohydrate. Under certain physiological conditions the inactive secreted enzymes may become activated as a result of a conformational change that may or may not result in autolysis. Analysis of the biochemical nature of the various processing steps helps define the cellular pathway followed by newly synthesized proteases targeted to the lysosome.
Antibodies to hnRNP core protein A1 in connective tissue diseases
Authors: Ricotti Giulia C. B. Astaldi , Bestagno Marco , Cerino Antonella , Negri Claudia , Caporali Roberto , Cobianchi Fabio , Longhi Matteo , Montecucco Carlo Maurizio 
Published: December 2004

AbstractWe investigated the specificity of circulating autoantibodies to a heterogeneous nuclear ribonuclear protein A1 (hnRNP A1), obtained by recombinant DNA technique, in different rheumatic diseases: systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), scleroderma, primary Sjogren's syndrome (SS), idiopathic Raynaud (IR), mixed connective tissue disease (MCTD), and healthy donors. All sera were tested by ELISA on hnRNP A1 protein. Positive values were obtained in 22% SLE, 19% scleroderma, 10% IR, 40% (2/5) MCTD, 5% SS, and 50% RA patients. The majority of patients reacted with the aminoterminal part (UP1) of hnRNP A1; however, some RA patients reacted also with the carboxy‐terminal part that shows partial homology with keratin. Therefore, hnRNP A1 (UP1) can be considered a target of antinuclear autoimmunity in various rheumatic disorders.
Human macrophage colony‐stimulating factor heterogeneity results from alternative mRNA splicing, differential glycosylation, and proteolytic processing
Authors: Shadle P. J. , Aldwin L. , Nitecki D. E. , Koths K. 
Published: December 2004

AbstractThe single gene for human macrophage colony‐stimulating factor (M‐CSF, or CSF‐1) generates multiple mRNA species that diverge within the coding region. We have characterized translation products of these mRNA species from native and recombinant sources. Immunoblots of reduced native M‐CSF indicate that multiple glycosylated species ranging from 25 kd to 200 kd are secreted by human monocytes and cell lines. In contrast, CV‐1 cells expressing a short M‐CSF clone secrete only 24 kd recombinant M‐CSF. Synthetic peptide antibodies were developed to distinguish between secreted recombinant M‐CSF from long and short mRNA splicing variants. Immunoblot analysis indicates that alternative mRNA splicing generates some M‐CSF protein heterogeneity. Most secreted MIA PaCa‐2 M‐CSF reacts with long‐clone‐specific antibody. Lectin affinity chromatography shows that variable glycosylation contributes significantly to MIA PaCa‐2 M‐CSF size heterogeneity. In addition, cell lysates also contain larger M‐CSF species that apparently undergo proteolytic processing before secretion. The data indicate that M‐CSF protein heterogeneity results from both pre‐ and post‐translational processing.
Effect of oxidative iodination of epidermal growth factor on its binding and secretion by hepatocytes
Authors: Marti Ulrich , Burwen Susan Jo , Barker Mary E. , Huling Sandra , Feren Anna M. , Jones Albert L. 
Published: December 2004

AbstractExperiments were undertaken to determine whether the method of iodination of epidermal growth factor (EGF) affects its binding to rat liver plasma membranes and its uptake, processing, and secretion into bile by intact rat hepatocytes. EGF was iodinated using one of three oxidative reagents: chloramine T (CT), lactoperoxidase (LP), or monochloride (MC). Quantitative receptor binding studies on plasma membranes isolated from male rat livers with either CT‐, LP‐or MC‐125I‐EGF indicated no significant difference in the apparent binding constants of the three preparations. To determine whether these three preparations were capable of forming a covalent‐like complex with the EGF receptor, they were individually incubated with isolated plasma membranes and subjected to polyacrylamide gel electrophoresis under reducing conditions, followed by autoradiography. Each preparation formed a major radioactive protein band of ∼180 kD, identified as the EFG receptor by immunoprecipitation with monoclonal anti‐EGF receptor antibodies. Furthermore, even unlabeled EGF incubated with plasma membranes formed this same 180 kD band, as revealed on Western blots using anti‐EGF antibody. The biliary secretion of CT‐, LP‐, and MC‐125I‐EGF was compared by injecting each one into rat portal veins and measuring the total and immunoprecipitable radioactivity in bile. The amount of immunologically intact CT‐125I‐EGF in bile was significantly greater than the others, whereas MC‐125I‐EGF transport was significantly reduced. We conclude that the method of iodination does not affect the covalent‐like binding properties of EGF. Furthermore, since unlabeled EGF displayed these same binding properties, oxidative iodination procedures per se do not account for the covalent‐like association between EGF and its receptor. However, the method of iodination used did affect the intracellular transport and processing of EGF by hepatocytes. The structural modification responsible for this alteration in transport properties has yet to be determined.
Molecular analysis of pleckstrin: The major protein kinase c substrate of platelets
Authors: Tyers Michael , Haslam Richard J. , Rachubinski Richard A. , Harley Calvin B. 
Published: December 2004

AbstractActivation of protein kinase C (PKC) in platelets causes the immediate phosphorylation of pleckstrin, an apparent Mr 40–47,000 protein previously called 40K or P47. Pleckstrin presumably plays an important but as yet unknown role in mediating cellular responses evoked by agonist‐induced phosphoinositide turnover. We have cloned the cDNA for pleckstrin from the HL‐60 human promyelocytic leukemia cell line by immunological screening of a λgt11 expression library (Tyers et al.: Nature 333:470–473, 1988) and now report further analysis of the pleckstrin sequence. Pleckstrin has a deduced Mr of 40,087 and is encoded by a 1,050‐bp open reading frame which is preceded by a short open reading frame that terminates before the correct initiator methionine. A single polymorphic site was found in the coding region. An unusual pattern of sequence heterogeneity occurred about a poly(A) tract in the 3′ untranslated region. The 3.0‐kb pleckstrin mRNA induced upon differentiation of HL‐60 cells apparently has heterogeneous 5′ ends which undergo differential regulation during HL‐60 cell maturation. Analysis by multiple sequence alignment with known PKC substrates identified a strong candidate site for phosphorylation by PKC and a potential Ca2+‐binding EF‐hand motif. No other similarities to proteins in current databases were found.
Preliminary studies on phospholipase A2‐induced mouse paw edema as a model to evaluate antiinflammatory agents
Authors: Marshall L. A. , Chang J. Y. , Calhoun W. , Yu J. , Carlson R. P. 
Published: December 2004

AbstractPhospholipase A2 (PLA2) is a key component of the inflammatory process because of its role in the generation of eicosanoids and platelet‐activating factor (PAF). Manipulation of PLA2 activity offers a novel therapeutic approach for the development of antiinflammatory agents; however, there is a need for a suitable in vivo model. Injection of 1 μg of snake venom PLA2 (A. piscivorus piscivorus, D‐49) into the mouse hind footpad produced a significant three‐ to four‐fold rise in paw edema within 10 min, compared to the saline control. Edema formation depended on enzyme concentration and appeared specific for PLA2 since edema was negated by enzyme pretreatment with p‐bromophenacyl bromide, a nonspecific PLA2 inhibitor. Moreover, injection of a protein such as bovine serum albumin did not result in significant edema. Coinjection of phenidone (lipoxygenase inhibitor, 50 μg), indomethacin (cyclooxygenase inhibitor, 50 μg), cyproheptadine (antihistamine/antiserotonin, 50 μg), aristolochic acid (putative PLA2 inhibitor, 100 μg), or kadsurenone (PAF antagonist, 50μg) with PLA2 (1 μg/paw) resulted in partial reduction (44.5, 34.2, 54.7, 64, and 50% inhibition, respectively) of edema formation. Oral administration of cyproheptadine (10 mg/kg), indomethacin (10 mg/kg), BW 755c (100 mg/kg), or dexamethasone (1 mg/kg) 1–3 h before challenge also decreased PLA2‐induced edema (63.0, 30.1, 47.8, or 62.5% inhibition, respectively). The data suggest that mouse paw edema resulting from PLA2 injection is a multicomponent event, influenced by both autacoids and lipid mediators of inflammation.
Translocation of diacylglycerol kinase from the cytosol to the membrane in phorbol ester–treated Swiss 3T3 fibroblasts
Authors: Maroney Anna Coco , Macara Ian G. 
Published: December 2004

AbstractThe tumor‐promoting phorbol ester, 12‐O‐tetradecanoyl‐phorbol‐13‐acetate, causes a rapid, partial redistribution of 1,2‐diacylglycerol kinase from the cytosol to the particulate fraction of quiescent Swiss 3T3 fibroblasts. The inactive alpha form of the phorbol ester does not cause any change in diacylglycerol kinase localization, and depletion of protein kinase C by chronic administration of phorbol ester blocks the redistribution. Phorbol ester has no direct effect on membrane‐bound diacylglycerol kinase in 3T3 cells. When phorbol ester is added to 3T3 membranes in the presence of ATP, Mg2+, and Ca2+, there is no activation of membrane‐bound kinase, indicating that phorbol ester does not activate membrane‐bound kinase through phosphorylation by protein kinase C. Stimulation of the cells with phorbol ester increases the total mass of diacylglycerol. In protein kinase C–depleted cells, addition of a cell‐permeable synthetic diacylglycerol, dioctanoylglycerol, results in a partial redistribution of cytosolic diacylglycerol kinase to the membrane, also suggesting that the translocation of DAG kinase is regulated primarily by substrate concentration.
Role of growth factors in inflammation and repair
Authors: Wahl S. M. , Wong H. , McCartney‐Francis N. 
Published: December 2004

AbstractMononuclear cells generate a variety of hormone‐like proteins termed growth factors that are instrumental in the evolution and resolution of inflammatory reactions. Many of these growth regulatory molecules have multifunctional properties. For example, the mononuclear cell‐derived growth factors, platelet‐derived growth factor (PDGF), and transforming growth factor beta (TGF‐β), are potent leukocyte chemoattractants. In addition, TGF‐β, a product of platelets, T lymphocytes, and monocytes, appears to induce the transcription of other monocyte‐derived growth hormone genes. In this regard, picomolar concentrations of TGF‐β stimulate peripheral blood monocytes to transcribe the genes for PDGF (c‐sis), basic fibroblast growth factor (FGF), interleukin 1 (IL‐1), and tumor necrosis factor (TNF). Furthermore, levels of mRNA for TGF‐β, which is constitutively expressed in resting monocytes, are also increased by exogenous TGF‐β. Each of these monocyte products exhibits a plethora of biological activities on other cell types. T lymphocytes, in response to antigen, contribute to this network by secreting growth factors and lymphokines that regulate monocyte growth factor production.
Microinjection of inositol 1,2‐(cyclic)‐4,5‐trisphosphate, inositol 1,3,4,5‐tetrakisphosphate, and inositol 1,4,5‐trisphosphate into intact Xenopus oocytes can induce membrane currents independent of extracellular calcium
Authors: Stith Bradley J. , Proctor William R. 
Published: December 2004

AbstractInositol phosphate action in an intact cell has been investigated by intracellular microinjection of eight inositol phosphate derivatives into Xenopus laevis oocytes. These cells have calcium‐regulated chloride channels but do not have a calcium‐induced calcium release system. Microinjection of inositol 1,3,4,5‐tetrakisphosphate (IP4), inositol 1,2‐(cyclic)‐4,5‐trisphosphate (cIP3), inositol 1,4,5‐trisphosphate (IP3), or inositol 4,5‐bisphosphate [(4,5)IP2], open chloride channels to induce a membrane depolarization. However, inositol 1‐phosphate (IP1), inositol 1,3,4,5,6‐pentakisphosphate (IP5), inositol 1,4‐bisphosphate, or inositol 3,4‐bisphosphate are unable to induce this depolarization. The depolarization is mimicked by calcium microinjection, inhibited by EGTA coinjection, and is insensitive to removal of extracellular calcium. By means of the depolarization response, the efficacy of various inositol phosphate derivatives are compared. IP3 and cIP3 induce similar half‐maximal, biphasic depolarization responses at an intracellular concentration of approximately 90 nM, whereas IP4 induces a mono‐ or biphasic depolarization at approximately 3400 nM. At concentrations similar to that required for IP3 and cIP3, (4,5)IP2 induces a long‐term (greater than 40 min) depolarization. The efficacy (cIP3 = IP3 = (4,5)IP2 ≫ IP4) and action of the various inositol phosphates in an intact cell and their inability to induce meiotic cell division are discussed.
Subcellular localization of phospholipids and enzymes involved in PAF‐acether metabolism
Authors: Record Michel , Ribbes Gérard , Tercé François , Chap Hugues 
Published: December 2004

AbstractThe biosynthesis of platelet‐activating factor (PAF‐acether or 1‐O‐2‐acetyl‐sn‐glycero‐3‐phosphocholine) through the remodeling pathway was investigated at the subcellular level in two different cell lines. In human neutrophils, plasma membrane was isolated not only from granules, but also from internal membranes related to endoplasmic reticulum. Interestingly, the latter exhibited enhanced acetyltransferase upon neutrophil stimulation with ionophore A23187. A similar study was undertaken on the tumor strain Krebs‐II cells. The enzyme acetyltransferase was found to be located only on an endoplasmic reticulum subfraction, whereas most alkylacyl‐GPC, the source of PAF‐precursor alkyl‐lyso‐GPC, was located in the plasma membrane inner leaflet. The topographical separation of enzyme and precursor emphasizes the central role of the intracellular phospholipase A2 in providing lyso‐PAF to the acetyltransferase to form PAF‐acether.
Processing of asparagine‐linked oligosaccharides is an early biochemical marker of the enterocytic differentiation of HT‐29 cells
Authors: Ogier‐Denis Eric , Bauvy Chantal , Aubery Michèle , Codogno Patrice , Sapin Catherine , Rousset Monique , Zweibaum Alain , Trugnan Germain 
Published: December 2004

AbstractThe inability of HT‐29 cells to undergo an enterocytic differentiation when grown in a glucose‐containing (Glc+) medium has been recently correlated to an overall impairment of N‐glycan processing. These results were obtained using confluent HT‐29 cells in which the differentiation characteristics are fully expressed under differentiation permissive conditions (glucose‐deprived medium, Glc−). Whether these changes of N‐glycan processing appear progressively during the cell growth or are already present from the beginning of the culture was investigated in this work by comparing the actual status of N‐glycan processing in both exponentially growing Glc+ and Glc− HT‐29 cells. Under these conditions, HT‐29 cells do not express any characteristics of enterocytic differentiation, even when grown in differentiation permissive conditions. We show here that the conversion of high‐mannose to complex glycoproteins is, however, severely reduced in HT‐29 cells grown studied. In contrast, HT‐29 cells grown in differentiation permissive conditions (HT‐29 Glc−) display a normal pattern of N‐glycan processing in both the exponential and the stationary phase of growth. We also show that both growing and confluent HT‐29 Glc+ cells accumulate Man9–8 GlcNAc2 species, thus suggesting that there is an important regulatory point at this level. We therefore conclude that the N‐glycan processing may be used as an early biochemical probe for the enterocytic differentiation of HT‐29 cells. Whether these early changes result from an early metabolic regulation or are the consequence of a genetic control remains to be studied.
Division competence in Tetrahymena: Determination of minimum cell volume and rate of nutrient uptake
Authors: Andersen Anders Poul , Hellung‐Larsen Per 
Published: December 2004

AbstractCell volume and doubling time have been determined for exponentially growing Tetrahymena pyriformis cells in broth medium with and without glucose and in media made from these media by dilution with water. The cells tolerate media with dry weights from 105 down to 0.06 g/L. In the diluted media the cells have small volumes and the doubling time is increased.When the cell volume increase per time per cell in a given medium is expressed as function of the cell volume in this same medium, a direct proportionality is found. From this equation the minimum cell volume of division competence (MVDC) can be found. It is 2,100 μm3 for T. pyriformis at 28°C.The lag period resulting from an upshift of exponentially growing cells from diluted media to more concentrated media is a function of the initial and resulting cell volumes and MVDC.The increase in cell volume per unit of time for a given cell‐depends on the dry weight of the medium. This parameter can be transformed to mass increase per cell surface area per time, which represents rate of nutrient uptake. When plotted against the dry weight of the media, a Michaelis‐Menten‐like curve is obtained with two Km values of 3.8 and 0.08 g/L with corresponding Vmax values of 20 and 4 ng/cm2 · s.The low Km value (0.08 g/L) indicates that Tetrahymena is able to take up nutrients from highly diluted media. The high value of Vmax (20 ng/cm2 · s) increases the ability of growth in more concentrated media. Thus, the adaptability of Tetrahymena to regulate its growth rate on media with considerable differences in nutrient supply is partly explained.