bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of the epidermal growth factor receptor by phosphorylation
Authors: Bertics Paul J. , Weber Wolfgang , Cochet Claude , Gill Gordon N. 
Published: December 2004

AbstractThe receptor for epidermal growth factor (EGF) is a glycosylated transmembrane phosphoprotein that exhibits EGF‐stimulable protein tyrosine kinase activity. On EGF stimulation, the receptor undergoes a self‐phosphorylation reaction at tyrosine residues located primarily in the extreme carboxyl‐terminal region of the protein. Using enzymatically active EGF receptor purified by immunoaffinity chromatography from A431 human epidermoid carcinoma cells, the self‐phosphorylation reaction has been characterized as a rapid, intramolecular process which is maximal at 30–37°C and exhibits a very low Km for ATP (0.2 μM). When phosphorylation of exogenous peptide substrates was measured as a function of receptor self‐phosphorylation, tyrosine kinase activity was found to be enhanced two to threefold at 1–2 mol of phosphate per mol of receptor. Analysis of the dependence of the tyrosine kinase activity on ATP concentration yielded hyperbolic kinetics when plotted in double‐reciprocal fashion, indicating that ATP can serve as an activator of the enzyme. Higher concentrations of peptide substrates were found to inhibit both the self‐ and peptide phosphorylation, but this inhibition could be overcome by first self‐phosphorylating the enzyme. These results suggest that self‐phosphorylation can remove a competitive/inhibitory constraint so that certain exogenous substrates can have greater access to the enzyme active site. In addition to self‐phosphorylation, the EGF receptor can be phosphorylated on threonine residues by the calcium‐ and phospholipid‐dependent protein kinase C. The sites on the EGF receptor phosphorylated in vitro by protein kinase C are identical to the sites phosphorylated on the receptor isolated from A431 cells exposed to the tumor promoters 12‐O‐tetradecanoylphorbol 13‐acetate or teleocidin. This phosphorylation of the EGF receptor results in a suppression of its tyrosine kinase and EGF binding activities both in vivo and in vitro. The EGF receptor can thus be variably regulated by phosphorylation: self‐phosphorylation can enhance tyrosine kinase activity whereas protein kinase C‐catalyzed phosphorylation can depress enzyme activity. Because these two phosphorylations account for only a fraction of the phosphate present in the EGF receptor in vivo, other protein kinases can apparently phosphorylate the receptor and these may exert additional controls on EGF receptor/kinase function.
Antibody crystallization on phospholipid films: Dynamics and the effects of antibody conformation
Authors: Uzgiris Egidijus E. 
Published: December 2004

AbstractMonoclonal antibodies form two‐dimensional (2‐D) crystals when bound to haptinated phospholipid monolayers in physiological conditions and at ambient temperatures. IgG1 forms two crystal phases: a linear strand phase and a high‐order hexagonal phase. The relative distribution of these two phases is dependent on temperature, pH, and salt concentration. This dependence is one that is associated with protein intramolecular interactions rather than lipid‐lipid or lipid‐protein interactions for a number of reasons: (1) Polyclonal antibodies against the hapten DNP do not organize into any crystal structure for any of the experimental conditions used. (2) Slightly denatured IgG (through storage at 4°C, for example) does not readily crystallize and a shift in the temperature dependence for forming the hexagonal phase is observed. (3) There is no pH driven transition in crystallization tendency for IgE anti‐DNP but a transition to disorder is observed at above 30°C. No such transition exists for IgG1. Observation of the dynamics of crystal growth shows a clear and marked dependence on pH and temperature that is in accord with the results of long‐term incubations. It is found that high pH retards crystal growth very significantly for IgG1 but not for IgE. Also, the crystal growth rate of 4°C‐stored IgG1 is greatly reduced over fresh IgG1 (−80°C stored). Furthermore, it is found that the linear phase of IgG1 is an extremely rapidly forming phase but one that is metastable against the hexagonal phase.
Differentiation in Acanthamoeba castellanii is induced by specific monoclonal antibodies
Authors: Villemez C. L. , Carlo P. L. , Russell M. A. 
Published: December 2004

AbstractMonoclonal antibodies that bind a large molecular weight plasma membrane protein of Acanthamoeba castellanii cause the cells to differentiate. A different monoclonal antibody that binds specifically to the major plasma membrane protein has no effect upon cell division or differentiation. The induction of differentiation by the monoclonal antibodies requires a bivalent attachment, more than a single binding cycle of the antibody to the plasma membrane protein, does not require cell‐cell contact, and appears to be mediated by an inhibition of pinocytosis. These results suggest one of two alternatives: either (1) this free living amoeba possesses a cell surface receptor that serves to initiate the differentiation process when stimulated, or (2) the specific plasma membrane antigen for the differentiation‐inducing monoclonal antibodies is an essential component of the pinocytotic mechanism. While it seems more likely on the basis of available evidence that we are observing the biological effects of a cell surface receptor, either of the two alternative circumstances open up investigative areas of large significance.
Fibronectin potentiates actin polymerization in thrombin‐activated platelets
Authors: Cierniewski Czeslaw S. , Karczewski Jerzy , Kowalska Maria A. 
Published: December 2004

AbstractThe effect of fibronectin on the polymerization state of actin was studied. Triton X‐100‐insoluble cytoskeleton was prepared from thrombin‐activated platelets, and the conversion of G‐actin into F‐actin was monitored by an assay involving DNase I inhibition by G‐actin. It was found that fibronectin bound to membrane receptors decreased the level of platelet G‐actin. This observation suggests that in the presence of fibronectin a larger amount of F‐actin becomes incorporated into the Triton X‐100‐insoluble cytoskeleton. At the same molar concentration, fibrinogen only slightly increased actin polymerization, whereas bovine serum albumin at a much higher concentration caused a small inhibition of actin immobilization. Our data show that fibronectin, through interaction with the platelet actomyosin fibrillar system, facilitates actin polymerization into the cytoskeleton.
Interactions between endothetial cells and leukocytes
Authors: Butcher E. C. , Lewinsohn D. , Duijvestijn A. , Bargatze R. , Wu N. , Jalkanen S. 
Published: December 2004

AbstractWe present evidence that specific receptors are utilized by neutrophils to control their interaction with endothelial cells at sites of acute inflammation and that these receptors are related if not identical to lymphocyte “homing receptors” for lymphoid tissue high endothelium. We speculate that such receptors play a fundamental but not exclusive role in controlling the extravasation and tissue localization of all bone marrow‐derived nucleated cells. In addition, we emphasize the active role of endothelial cells in the process of lymphocyte migration and leukocyte extravasation. By the expression of as yet unidentified organ‐specific determinants for lymphocyte recognition, endothelial cells control the exit of particular lymphocyte subsets into mucosal versus nonmucosal sites, thus helping to determine the unique features of mucosal versus nonmucosal immune responses. Furthermore, we argue that endothelial cells are exquisitely responsive to local immune reactivity and present evidence that specific lymphokines, including γ‐interferon, play an important role in inducing postcapillary venules to express differentiated features required for the support of lymphocyte traffic into lymphoid organs and into sites of chronic inflammation. Leukocytes, endothelial cells, and probably other tissue cell classes appear to interact at multiple levels by a variety of mechanisms to regulate the local extravasation of immune effector cells.
Evidence for the appearance of an uncoupled form of the β‐adrenergic receptor distinct from the internalized receptor
Authors: Hertel C. , Portenier M. , Staehelin M. 
Published: December 2004

AbstractAgonist treatment of C6‐glioma cells induces two altered states in β‐adrenergic receptors, a low affinity for the hydrophilic antagonist CGP‐12177 and a low affinity for agonists like isoproterenol. We present evidence that, in cells not treated to inhibit receptor internalization, the two properties occur with a different time course, the low affinity for isoproterenol preceding that for CGP‐12177. In that the low affinity for CGP‐12177 is due to the internalization of the receptor, the results indicate that uncoupling of the receptor, indicated by the low affinity for isoproterenol, occurs while the receptor is, still located on the cell surface. Removal of the agonist leads to reappearance of the receptor to the plasma membrane followed by loss of the uncoupled state.
Fine structure of gels prepared from an actin‐binding protein and actin: Comparison to cytoplasmic extracts and cortical cytoplasm in amoeboid cells of Dictyostelium discoideum
Authors: Wolosewick J. J. , Condeelis John 
Published: December 2004

AbstractWe have identified the three‐dimensional ultrastructure of actin gels that are formed in well‐characterized cell extracts and mixtures of purified actin and the 120K actin‐binding protein and compared these to the ultrastructure of the cytoplasmic matrix in regions of nonextracted Dictyostelium amoebae that are rich in actin and 120K. This ultrastructural characterization was achieved by using critical‐point‐dried whole‐mount preparations.All three preparations—gelled extracts, purified proteins, and cortical cytoplasm—are composed of filament networks. The basic morphological feature of these networks is the presence of contacts between convergent filaments resulting in “T” or “X” shaped contacts.The finding that actin‐containing gels are composed of filament networks, where the primary interaction occurs between convergent filaments, reconciles the known requirement of F actin for gelation with the amorphous appearance of these gels in thin sections.Increasing the molar ratio of 120K dimer to actin monomer increases the number of contacts between filaments per unit volume and decreases the lengths of filaments between contacts. This indicates that 120K stabilizes interactions between filaments and is consistent with biochemical evidence that 120K cross‐links actin filaments.The cortical network in situ resembles more closely networks formed in 120K‐rich extracts than networks assembled in mixtures of purified 120K and actin. The heterogeneity of filament diameters and variation of network density are properties shared by extracts and the cytomatrix in situ while networks found in purified l20K‐actin gels have filament diameters and densities that are more uniform. These differences are certainly due to the more complex composition of cell extracts and cortical cytoplasm as compared to that of purified 120K‐actin gels.
Reevaluation of the hydrophobic nature of the 110‐kD calmodulin‐, actin‐, and membrane‐binding protein of the intestinal microvillus
Authors: Conzelman Karen A. , Mooseker Mark S. 
Published: December 2004

AbstractA complex of calmodulin (CM) and the 110‐kD (110K) subunit composes the helical array of cross‐bridges linking the microvillus actin filament bundle with the membrane. The hydrophobic properties of the 110K protein, assessed by the detergent phase partitioning assay [Bordier C: J Biol Chem 256:1604, 1981], are highly dependent on the solution conditions used in its isolation. The ATP‐dissociable 110K‐CM complex [Howe and Mooseker: J Cell Biol 97:974, 1983] exhibits hydrophilic characteristics in this assay. In contrast, the 110K subunit extracted from brush borders by Triton X‐100, sodium dodecyl sulfate, and sodium pyrophosphate (detergent‐treated 110K) [Glenney JR, Glenney P: Cell 37:743, 1984] behaves as a hydrophobic protein. However, because the soluble hydrophilic 110K‐CM can be rendered hydrophobic by treating the complex with the same detergent and salt conditions used in the preparation of detergent‐treated 110K, the properties of detergent‐treated 110K seem likely to be an effect of the solution conditions on its native conformation, sedimentability, or exposure of binding domains. In addition, the detergent‐treated 110K is devoid of calmodulin and no longer exhibits the actin‐binding activity characteristic of the ATP‐dissociable 110K‐CM and of the intact complex in situ. With two partially purified preparations of the 110K subunit exhibiting such dramatically distinct properties, it seems premature to define the nature of the 110K subunit's association with the membrane at this time.
The coelomic envelope to vitelline envelope conversion in eggs of Xenopus laevis
Authors: Gerton George L. , Hedrick Jerry L. 
Published: December 2004

AbstractAn amphibian egg recovered from the body cavity is enclosed by a coelomic egg envelope. Upon transport down the oviduct, the envelope is converted to the vitelline envelope. The coelomic and vitelline envelopes are distinct in terms of sperm penetrability, ultrastructural morphology, and radioiodination profiles. In this study, the macromolecular compositions of these two envelopes were determined. Isolated envelopes were compared by one‐ and two‐dimensional gel electrophoresis, peptide mapping, and radiolabeling. A protein with a molecular weight of 57,000 (57K) was present in the vitelline envelope but was absent in the coelomic envelope. A glycoprotein with a molecular weight of 43K in the coelomic envelope was converted to a component with a molecular weight of 4lK in the vitelline envelope. The 43K‐molecular weight component of the coelomic envelopes could be radioiodinated by lactoperoxidase but no labeling of the 41K‐molecular weight component occurred in the vitelline envelope. Peptide mapping using limited proteolysis established that the 43K‐molecular weight component of the coelomic envelope was a precursor to the 41K‐molecular weight component of the vitelline envelope. These molecular alterations may underlie the ultrastructural and physiological changes occurring in these envelopes.
An actomyosin contractile mechanism for erythrocyte shape transformations
Authors: Fowler Velia M. 
Published: December 2004

AbstractThe membrane skeleton of the human erythrocyte consists of many short actin filaments that are multiply cross‐linked by long, flexible spectrin molecules into a continuous network in the plane of the membrane. The mechanical properties expected for this spectrin‐actin network can account for the tensile strength of the erythrocyte membrane and for the remarkable deformability of the cells, yet not for their characteristic biconcave shape. Recently, an authentic vertebrate myosin as well as a non‐muscle form of tropomyosin have been identified and purified from erythrocytes. The myosin is present with respect to the actin in an amount comparable to actin‐myosin ratios in other non‐muscle cells, and there is enough tropomyosin to almost completely coat all of the short actin filaments in the membrane skeleton. The implications of these unexpected discoveries for the molecular organization of the cytoskeleton are discussed, and a mechanism is proposed by which myosin could interact with the membrane‐associated actin filaments to influence erythrocyte shape and membrane properties.
Identification of a biochemical marker for the secretory pathway in Tetrahymena thermophila
Authors: Maihle Nita J. , Satir Birgit H. 
Published: December 2004

AbstractTetrahymena thermophila is a ciliated protozoan studied by investigators from a wide range of disciplines as a model system for a variety of specialized eukaryoticcell functions. The proteinaceous secretory products of T thermophila have been isolated and characterized [1] and in this study we identify the major secretory product, a 34,000 Mr polypeptide, and use an antiserum prepared against this secretory protein to (1) demonstrate that this 34,000 Mr polypeptide is truly a secreted protein in Tetrahymena and (2) monitor the synthesis and transport of this protein by indirect immunofluorescence and light microscopy during mucocyst biogenesis.
Uptake and destruction of 125I‐CSF‐1 by peritoneal exudate macrophages
Authors: Guilbert Larry J. , Tynan P. Wendy , Stanley E. Richard 
Published: December 2004

AbstractThe binding and uptake of the colony‐stimulating factor CSF‐1 by peritoneal exudate macrophages (PEM) from lipopolysaccharide insensitive C3H/HeJ mice was examined at 2°C, and at 37°C. At 2°C, 125I‐CSF‐1 was bound irreversibly to the cell surface. At 37°C, 90% of the cell surface associated 125I‐CSF‐1 was rapidly internalized and subsequently degraded and the remaining 10% dissociated as intact 125I‐CSF‐1. Thus classical equilibrium or steady state methods could not be used to quantitatively analyze ligand–cell interactions at either temperature, and alternative approaches were developed. At 2°C, the equilibrium constant (Kd ⩽ 10−13M) was derived from estimates of the rate constants for the binding (kon ≃ 8 × 105M−1s−1) and dissociation (koff ⩽ 2 × 10−7s−1) reactions. At 37°C, the processes of dissociation and internalization of bound ligand were kinetically competitive, and the data was formally treated as a system of competing first order reactions, yielding first order rate constants for dissociation, koff = 0.7 min−1 (t1/2 = 10 min) and internalization, kin = 0.07 min−1 (t1/2 = 1 min). Approximately 15 min after internalization, low‐molecular weight 125I‐labeled degradation products began to appear in the medium. Release of this degraded 125I‐CSF‐1 was kinetically first order over three half‐lives (Kd = 4.3 × 10−2 min−1, t1/2 = 16 min). Thus CSF‐1 binds to a single class of receptors on PEM, is internalized with a single rate limiting step, and is rapidly destroyed without segregation into more slowly degrading intracellular compartments.
Solubilization and assay of a colony‐stimulating factor receptor from murine macrophages
Authors: Yeung Yee‐Guide , Jubinsky Paul T. , Stanley E. Richard 
Published: December 2004

AbstractThe colony‐stimulating factor, CSF‐1, selectively stimulates the survival, proliferation, and differentiation of mononuclear phagocytes. The solubilization, assay, and characteristics of the CSF‐1 receptor from the J774.2 murine macrophage cell line are described. The recovery of cell‐surface receptor in the postnuclear supernatant membrane fraction of hypotonically disrupted cells was 76%. Recovery of the ligand binding activity of the receptor after solubilization of this fraction with 1% Triton X‐100 was ∼ 150%. The binding of 125I‐CSF‐1 to intact cells and membrane preparations was consistent with the existence of a single class of high‐affinity receptor sites. In contrast, the equilibrium binding of 125I‐CSF‐1 to the solubilized postnuclear fraction indicated the existence of two distinct classes of binding site (apparent Kds 0.15 nM and 10 nM). A rapid assay was developed for the high‐affinity sites, which were shown to be associated with the CSF‐1 receptor. The function of the low‐affinity sites, which have not been demonstrated on intact cells or cell membranes and which are 13 times more abundant than the high‐affinity sites, is unknown. The solubilized high‐affinity receptor‐CSF‐1 complex was stable on storage at 0°C and −70°C but dissociated at 37°C. Dissociation also occurred at 0°C in buffers of low pH (4.0) or high ionic strength (0.7 M NaCl).
Amplification of the N‐myc oncogene in an adenocarcinoma of the lung
Authors: Saksela Kalle , Bergh Jonas , Nilsson Kenneth 
Published: December 2004

Abstractc‐myc oncogene is the most extensively studied member of the myc gene family. which now consists of three characterized members, namely the c‐myc, N‐myc, and L‐myc genes. Deregulation owing to amplification and/or rearrangements of the c‐myc gene have been described in a variety of human malignancies. Several neuroblastomas have amplifications of the N‐myc genes. The c‐myc, N‐myc, or L‐myc oncogenes are also found amplified in different cell lines from small cell carcinomas of the lung. In this study, we have examined the c‐myc, N‐myc, and c‐erbB oncogenes in 34 clinical and autopsy tumor specimens representing various histopathological types of human lung cancer, including nine small cell lung cancers. A 30‐fold amplification of the N‐myc gene was found in a tumor histopathologically and histochemically verified as a typical adenocarcinoma. No amplifications of the c‐myc or c‐erbB oncogenes were seen in any of the tumors. In the DNA of one small cell carcinoma, an extra c‐myc and N‐myc cross‐hybridizing restriction fragment was observed, possibly owing to an amplification of a yet uncharacterized myc‐related gene.
Regulated expression of the c‐myb and c‐myc oncogenes during erythrdid differentiation
Authors: Kirsch Ilan R. , Bertness Virginia , Hollis Gregory F. , Silver Jonathan 
Published: December 2004

AbstractWe have investigated the expression of the genes c‐myb, c‐myc, and alpha globin in murine erythroid cells at different stages of development, in viral‐induced erythroleukemias, as well as in two mouse erythroleukemia cell lines that can be induced to terminally differentiate when exposed to dimethylsulfoxide. We find that there is a reciprocal correlation between the cell's production of messenger RNA for c‐myb and globin. c‐myc message shows a similar but less dramatic decrease coincident with globin RNA production. Initially with the administration of an inducing agent, dimethylsulfoxide, there is a rapid decrease of myc and myb mRNA, which is followed by signs of differentiation in the induced culture. We conclude that these oncogenes function in early maturational stages of development of these cells. In the erythroleukemic state these genes are down‐regulated by forced differentiation and may play a direct role in influencing the state of differentiation of these cells.
Viral therapy: Prospects for protease inhibitors
Authors: Korant Bruce D. , Towatari Takae , Ivanoff Lucy , Pettewa Steven Y. , Brzin Joze , Lenarcic Brigita , Turk Vito 
Published: December 2004

AbstractAntiviral activities of known protease inhibitors were assayed in virus‐infected cell cultures. Some members of the cystatin superfamily, in particular chicken cystatin, were able to block virus replication. In a binding assay, using purified components, chicken and human cystatin were able to bind poliovirus protease with affinities which were reflected in their relative anitviral potencies. Prospects for application of protease inhibitors in clinical viral infections are discussed.
Characterisation of a soluble trypsin fragment of GP130: A neuronal glycoprotein associated with the cytoskeleton
Authors: Moss Diana J. 
Published: December 2004

AbstractA neuronal glycoprotein (GP130) that is associated with the cytoskeleton [Ranscht et al: J Cell Biol 99:1803–1813, 1984] remains insoluble in 0.1 M NaOH, a property typical of integral membrane proteins. At present it is possible to solubilise and hence isolate GP130 only under denaturing conditions. However, a large fragment of apparent molecular weight 120K is released into solution by trypsin. The fragment corresponds to the extracellular region of the glycoprotein as shown by the fact that it is released from live cultures of chicken sympathetic neurons and by its retention of concanavalin A‐binding activity. The soluble extracellular fragment has been purified using mild biochemical techniques, which are expected to retain its biological activity. Measurement of the sedimentation coefficient, Stokes radius, and frictional ratio in addition to metal shadowing of the fragment show that it has a molecular weight of about 120K and is asymmetric, probably rod‐shaped with a long axis of more than 20 nm.