bluuebunny/crossref_metadata_2025 huggingface dataset:
A pathway of coagulation on endothelial cells
Authors: Nawroth Peter P. , Stern David M.
Published: December 2004

Although the endothelial cell is considered antithrombogenic, endothelium has recently been shown to participate in procoagulant reactions. Factor IX bound to specific endothelial cell sites can be activated by the intrinsic and extrinsic pathways of coagulation. Perturbation of endothelium results in induction of tissue factor which promotes factor VIIa‐mediated activation of factors IX and X. thus initiating procoagulant events on the endothelial surface. Cell bound factor IXa, in the presence of factor VIII, promotes activation of factor X. The factor Xa formed can interact with endothelial cell factor V/Va, resulting in prothrombin activation. Thrombin then cleaves fibrinogen and a fibrin clot closely associated with the endothelial cell forms. The perturbed endothelial cell thus provides a focus of localized procoagulant events. This model suggests a simple endothelial‐cell‐dependent mechanism for initiation of coagulation at the site of an injured or pathological vessel.
Structure and chromosomal localization of the human lymphotoxin gene
Authors: Nedwin Glenn E. , Jarrett‐Nedwin Julie , Smith Douglas H. , Naylor Susan L. , Sakaguchi Alan Y. , Goeddel David V. , Gray Patrick W.
Published: December 2004

We have isolated, sequenced, and determined the chromosomal localization of the gene encoding human lymphotoxin (LT). The single copy gene was isolated from a human genomic library using a 32P‐labeled 116 bp synthetic DNA fragment whose sequence was based on the NH2‐terminal amino acid sequence of LT. The gene spans 3 kb of DNA and is interrupted by three intervening sequences. The LT gene is located on human chromosome 6, as determined by Southern blot analysis of human‐murine hybrid DNA. Putative transcriptional control regions and areas of homology with the promoters of interferon and other genes are identified.
Regulation of the epidermal growth factor receptor by phosphorylation
Authors: Bertics Paul J. , Weber Wolfgang , Cochet Claude , Gill Gordon N.
Published: December 2004

The 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

Monoclonal 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

Monoclonal 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

The 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

We 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

Agonist 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

We 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

A 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

An 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

The 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

Tetrahymena 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

The 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

The 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

c‐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

We 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

Antiviral 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

A 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.
Phosphorylation of glycolytic and gluconeogenic enzymes by the insulin receptor kinase
Authors: Sale Elizabeth M. , White Morris F. , Kahn C. Ronald
Published: December 2004

Various glycolytic and gluconeogenic enzymes were tested as substrates for the insulin receptor kinase. Phosphofructokinase and phosphoglycerate mutase were found to be the best substrates. Phosphorylation of these enzymes was rapid, stimulated 2‐ to 6‐fold by 10−7 M insulin and occurred exclusively on tyrosine residues. Enolase, fructose 1,6‐bisphosphatase, lactate dehydrogenases in decreasing order, were also subject to insulin‐stimulated phosphorylation but to a smaller extent than that for phpsphofructokinase or phosphoglycerate mutase.

The phosphorylation of phosphofructokinase was studied most extensively since phosphofructokinase is known to catalyze a rate‐limiting step in glycolosis. The apparent Km of the insulin receptor for phosphofructokinase was 0.1 μM, which is within the physiologic range of concentration of this enzyme in most cells. Tyrosine phosphorylation of phosphofructokinase paralleled autophosphorylation of the β‐subunit of the insulin receptor with respect to time course, insulin dose response (half maximal effect between 10−9 and 10−8 M insulin), and cation requirement (Mn2+ > Mg2+ > > Ca2+). Further study will be required to determine whether the tyrosine phosphorylation of phosphofructokinase plays a role in insulin‐stimulated increases in glycolytic flux.
Site‐specific mutagenesis of cDNA clones expressing a poliovirus proteinase
Authors: Semler Bert L. , Johnson Victoria H. , Dewalt Patricia Gillis , Ypma‐Wong Mary Frances
Published: December 2004

The cleavage of poliovirus precursor polypeptides occurs at specific amino acid pairs that are recognized by viral proteinases. Most of the polio‐specific cleavages occur at glutamine‐glyeine (Q‐G) pairs that are recognized by the viral‐encoded proteinase 3C (formerly called P3–7c). In order to carry out a defined molecular genetic study of the enzymatic activity of protein 3C, we have made cDNA clones of the poliovirus genome. The cDNA region corresponding to protein 3C was inserted into an inducible bacterial expression vector. This recombinant plasmid (called pIN‐III‐C3–7c) utilizes the bacterial lipoprotein promoter to direct the synthesis of a precursor polypeptide that contains the amino acid sequence of protein 3C as well as the amino‐ and carboxy‐terminal Q‐G cleavage signals. These signals have been previously shown to allow autocatalytic production of protein 3C in bacteria transformed with plasmid pIN‐III‐C3–7c. We have taken advantage of the autocatalytic cleavage of 3C in a bacterial expression system to study the effects of site‐specific mutagenesis on its proteolytic activity. One mutation that we have introduced into the cDNA region encoding 3C is a single amino acid insertion near the carboxy‐terminal Q‐G cleavage site. The mutant recombinant plasmid (designated pIN‐III‐C3‐μ10) directs the synthesis of a bacterial‐polio precursor polypeptide that is like the wild‐type construct (pIN‐III‐C3–7c). However, unlike the wild‐type precursor, the mutant precursor cannot undergo autocatalytic cleavage to generate the mature proteinase 3C. Rather, the precursor is able to carry out cleavage at the amino‐terminal Q‐G site but not at the carboxy‐terminal site. Thus, we have generated an altered poliovirus proteinase that is still able to carry out at least part of its cleavage activities but is unable to be a suitable substrate for self‐cleavage at its carboxy‐terminal Q‐G pair.
Role of glandular kallikreins as growth factor processing enzymes: Structural and evolutionary considerations
Authors: Isackson Paul J. , Dunbar Joan C. , Bradshaw Ralph A.
Published: December 2004

Hormones and growth factors are generally released from larger precursors by limited proteolysis. The causative agents remain poorly defined with respect to location and properties. One subset of proteases, the glandular kallikreins, have been implicated in a few cases, in part because of their specific association with mature forms of some hormones. However, limited distribution and low copy number in some species cast doubt on this hypothesis, and they may well play other physiological functions that remain to be elucidated.
Growth factors, oncogenes, and multistage carcinogenesis
Authors: Weinstein I. Bernard
Published: December 2004

This paper presents evidence that the full repertoire of cellular genes involved in the carcinogenic process is several times larger than that of the known list of proto‐oncogenes. Furthermore, this repertoire includes genes whose normal function is related to growth stimulation, as well as genes whose normal function is to inhibit growth or induce terminal differentiation. Multistage carcinogenesis probably results from a complex series of changes in both categories of genes. Despite this complexity, carcinogenesis can be conceived in terms of disturbances in biochemical functions that normally control the expression or function of growth factors, receptors, and pathways of signal transduction. Several protein kinases play a central role in the process of signal transduction. Our laboratory has recently isolated cDNA clones for the enzyme protein kinase C (PKC). These clones should be useful for clarifying the role of PKC in growth control and tumor promotion. Finally, the existence of genes whose normal function is to inhibit cell growth provides a rationale for new strategies of cancer prevention and treatment.
Myc family of cellular oncogenes
Authors: DePinho Ronald , Mitsock Lisa , Hatton Kimi , Ferrier Pierre , Zimmerman Kathy , Legouy Edith , Tesfaye Abeba , Collum Robert , Yancopoulos George , Nisen Perry , Kriz Ronald , Alt Frederick
Published: December 2004

The myc family of cellular oncogenes contains three well‐defined members: c‐myc, N‐myc and L‐myc. Additional structural and functional evidence now suggests that other myc‐family oncogenes exist. The overall structure and organization of the c‐, N‐. and L‐myc genes and transcripts are very similar. Each gene contains three exons: encoding a long 5′ untranslated leader and a long 3′ untranslated region. The proteins encoded by these myc genes share several stretches of significant homology. The conservation of sequences at the carboxy‐terminus of the L‐myc protein suggests that it is also a DNA‐binding, nuclear‐associated protein. Each myc gene will cooperate with an activated Ha‐ras oncogene to cause transformation of primary rat embryo fibroblasts. Characteristics of several new myc‐family members are described.
Detergents inhibit exocytosis in PC 12 cells: Evidence for an effect on ion fluxes
Authors: Tapparelli Carlo , Grob Marianne , Burger Max M.
Published: December 2004

Membrane events in exocytosis were studied by examining the effect of different detergents on the K+‐stimulated release of noradrenaline in the secretory cell line PC 12. The nonionic detergent Triton X‐100 and the cationic detergent cetyltrimethylammonium bromide (CTAB) inhibit the noradrenaline release evoked by 55 mM K+ by 50% at very low concentrations (30 μM and 10 μM, respectively). These values are tenfold lower than the critical micellar concentrations (CMC). No such effect was seen with the anionic detergent sodium dodecyl sulphate (NaDodSO4).

The inhibitory effect of 30 μM Triton X‐100 is reversible, and the recovery from inhibition correlates with the loss of detergent from the cells as demonstrated by binding studies using [3H]Triton X‐100. The possible relationship between this inhibition of secretion and the structural properties of the detergent was investigated. The inhibition in the presence of purified Triton X‐100 subfractions turned out to be a function of the length odemonstrated by binding studies using [3H]Triton X‐100. The possible relationship between this inhibition of secretion and the structural properties of the detergent was investigated. The inhibition in the presence of purified Triton X‐100 subfractions turned out to be a function of the length of the oligometric ethyleneglycol chain (C6 to C26). The maximal effect was observed for Triton X‐100 molecules having a chain length of 16 carbon atoms, which can penetrate just half of the lipid bilayer of the membrane. Additionally, the phase transition at 13–14°C observed in an Arrhenius plot of noradrenaline release in stimulated cells was abolished.

In the presence of 30 μM Triton X‐100, 22Na+ uptake, 86Rb+ release, and 45Ca2+ uptake were reduced by 50–60%. These data suggest that the site of action of Triton X‐100 is at the level of altering the movement of ions in PC 12 cells during the stimulatory phase of secretion.
Induction of granuloma‐dependent angiotensin‐converting enzyme and eosinophil chemotactic factor in the skin of athymic nude mice
Authors: Kanazawa Kazunori , Higuchi Mitsunari , Okamoto Mitsuse , Fukuyama Kimie , Epstein William L.
Published: December 2004

Activities of angiotensin‐converting enzyme (ACE), other proteinases, and eosinophil chemotactic factor (ECF‐G) are known to be elevated in hepatic hypersensitivity granulomas of thymus intact (nu/+) mice after Schistosoma mansoni infection. The enzyme activities also increase, but to a lesser degree in hepatic granulomas of athymic nude (nu/nu) mice, and ECF‐G is not detectable. In this study isolated hepatic granulomas from nu/+ mice were grafted into the skin of uninfected nu/nu mice, and changes in those cellular functions were determined to examine whether the newly formed granulomas by recipient nu/nu cells acquire the functional activities as well as the histological appearance of nu/+ granulomas. ACE and ECF‐G rapidly disappeared from grafted sites during the first 5 days, corresponding to loss of nu/+ cells from the graft. Reduction in activities of arylsulfatases, lysozyme, and acid phosphatase also occurred, but to a lesser extent. Recovery of ACE and ECF‐G activities to the levels seen in nu/+ hepatic granulomas was observed by 14 days after grafting when nu/nu cells had accumulated in the grafts and formed new granulomas. Other enzymes increased to approximately half the levels seen in grafted donor granulomas. Circulating eosinophilia also increased. The findings indicate that nu/nu cells that accumulated in the skin grafts not only morphologically mimicked nu/+ type granulomas but also demonstrated nu/+ levels of cellular function. Analysis of skin granulomas developing in nu/+ mice after grafting of nu/+ hepatic granulomas showed the similar histology and enzymatic changes, whereas the skin sites inoculated with purified schistosome eggs alone caused neither significant histological changes nor elevation of ACE activity.
Identification and quantification of actin isoforms in vertebrate cells and tissues
Authors: Otey C. A. , Kalnoski M. H. , Bulinski J. C.
Published: December 2004

The cytoskeletal protein actin exists in vertebrates as six different isoforms, which are difficult to identify conclusively because of a high degree (>90%) of overall sequence homology. We have used IEF immunoblotting in combination with a panel of isoform‐specific and ‐selective antibodies to analyze the actin isoform composition of nine tissues from adult rat. In three nonmuscle tissues (lung, spleen, and testis), we detected a previously unreported isoform that we identified as smooth muscle α. The IEF immunoblot technique was also used to quantify the proportions of the isoforms expressed in these nine rat tissues.
Preparation of a monoclonal antibody to a melanoma growth‐stimulatory activity released into serum‐free culture medium by Hs0294 malignant melanoma cells
Authors: Lawson David H. , Thomas H. Greg , Roy Robert G. B. , Gordon David S. , Chawla Rajender K. , Nixon Daniel W. , Richmond Ann
Published: December 2004

Autostimulatory growth factors may contribute to the ability of malignant cells to escape normal growth controls. We have previously shown that Hs0294 human malignant melanoma cells release into culture medium an acid‐soluble, heat‐stable, trypsin‐sensitive, autostimulatory monolayer mitogen which can be purified from acetic acid extracts of conditioned medium by gel filtration, reverse‐phase high‐performance liquid chromatography, and preparative electrophoresis. The majority of this melanoma growth‐stimulatory activity (MGSA) resides in a 16‐Kd moiety though bioactivity is also associated with 24–26 and < 14‐Kd forms of MGSA (Richmond and Thomas: J Cell Physiol 129:375, 1986). In order to further characterize this growth factor, monoclonal antibodies were prepared against a partially purified preparation of the autostimulatory melanoma mitogen. Monoclonal antibody clones were selected based on supernate inhibition of 3H‐thymidine incorporation in serum‐free Hs0294 melanoma cultures. One of these, termed FB2AH7, slows, but does not completely block, the growth of Hs0294 cells in scrum‐free medium in a dose‐dependent manner. This antibody does not slow the growth of normal rat kidney fibroblasts, which neither produce nor require this mitogen, in either serum‐free medium or medium containing 0.8% calf scrum. This monoclonal antibody also blocks the mitogenic effects of partially purified preparations of this melanoma growth stimulatory activity (MGSA) on both Hs0294 cells and normal rat kidney fibroblasts. The FB2AH7 antibody has been demonstrated to bind MGSA by Western blot and by immunoprecipitation procedures. Western blot analysis of reverse‐phase high‐performance liquid chromatography purified growth factor demonstrated that FB2AH7 antibody binds to the 16‐Kd and ∼ 13–14‐Kd forms of MGSA. FB2AH7 antibody can be used in immunoprecipitation experiments to bind the ∼ 13–16‐Kd forms of MGSA. The specificity of the binding of FB2AH7 antibody for MGSA but not other growth factors has been demonstrated in a modified dot blot assay. These data thus support the hypothesis that MGSA is an autostimulatory melanoma mitogen distinct from other growth factors.
Phosphorylation and dephosphorylation of soluble proteins in human eosinophils
Authors: Ramesh Kashi S. , Rocklin Ross E. , Pincus Stephanie H.
Published: December 2004

The effect of phorbol 12‐myristate 13‐acetate (PMA), calcium ionophore (A23187), opsonized zymosan (OZ), and N‐formylmethionyl‐leucyl‐phenylalanine (f‐Met‐Leu‐Phe) on protein phosphorylation was examined in purified eosinophils (eos) isolated from human peripheral blood. Eos were prelabeled with [32P]orthophosphate, stimulated with several activating agents for varying periods of time. The soluble proteins were then analyzed by one‐dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and autoradiography. In resting eos, there was phosphorylation of endogenous soluble proteins with molecular weights of 12, 16, 21, 40, and 66 kilodaltons (kDa). PMA, a potent activator of oxidative metabolism, induced phosphorylation of 19‐, 40‐, and 67‐kDa proteins. A23187, a strong degranulating stimulus, caused phosphorylation of 40‐, 53‐, and 67‐kDa proteins. OZ, a relatively weak stimulus for eos function, caused phosphorylation of 30–34‐, 59‐, 67‐, and 93‐kDa proteins. In addition, all the above stimuli caused a time‐dependent dephosphorylation of 21‐kDa protein. In contrast, f‐Met‐Leu‐Phe caused neither phosphorylatiop of new proteins nor dephosphorylation of preexisting eos proteins. These findings demonstrate that selected stimuli affect phosphorylation of soluble eos protein. These results also suggest that phosphorylation of specific proteins in eos is an intermediary step in external stimulus‐induced cell activation, which may involve many different cell functions.
Vaccinia virus and the EGF receptor: A portal for infectivity?
Authors: Marsh Y. Vivienne , Eppstein Deborah A.
Published: December 2004

We previously demonstrated that occupancy of the epidermal growth factor (EGF) receptor reduced the ability of vaccinia virus to infect L cells [Eppstein et al: Nature 318:663, 1985]. This result suggested that vaccinia virus was utilizing the EGF receptor as one pathway to infect cells. We have studied this system further, and now find that antibodies to the EGF receptor also reduce the ability of vaccinia virus to infect cells productively. Inclusion of both EGF and antibodies to the EGF receptor did not cause inhibition over that obtained by EGF alone, providing another line of evidence that the antiviral effects on vaccinia virus were at the level of the EGF receptor. The antiviral effects of EGF or synthetic peptides corresponding to the third disulfide loop of TGF‐α or the vaccinia virus growth factor were specific to vaccinia virus and did not inhibit replication of herpes simplex virus type 2 or vesicular stomatitis virus. The inhibitory effects on replication of vaccinia virus were obtained when EGF (but not insulin or growth hormone) was present prior to, but not after, productive viral adsorption. These results provided further evidence that the antivaccinia viral effects of EGF were at the level of initial receptor occupancy. As interferon (IFN) treatment has been shown to interfere with the action of some growth factors, including EGF, we examined the effects of IFN treatment of cells on the antivaccinia viral activity of EGF. Our results show that the antivaccinia effect of IFN‐β either interfered with or partially coalesced with the inhibitory effects of EGF. The former interpretation is consistent with the report that IFN treatment results in a decrease both in the apparent number and affinity of cell‐surface receptors for EGF [Zoon et al: Proc Natl Acad Sci USA 83:8226, 1986].
Growth regulation of human breast carcinoma occurs through regulated growth factor secretion
Authors: Lippman Marc E. , Dickson Robert B. , Gelmann Edward P. , Rosen Neal , Knabbe Cornelius , Bates Susan , Bronzert Diane , Huff Karen , Kasid Attan
Published: December 2004

We describe studies on human breast cancer in which it is shown that specific growth factors (IGF‐I, TGFα, PDGF) are secreted by human breast cancer cells and likely to be involved in tumor growth and progression. These activities are regulated by estradiol in hormone‐dependent breast cancer and secreted constitutively by hormone‐independent cells. These growth factor activities can induce the growth of hormone‐dependent cells in vivo in athymic nude mice. Hormone‐dependent breast cancer cells also secrete TGFβ, a growth‐inhibitory substance, when treated with antiestrogens. TGFβ functions as a negative autocrine growth regulator and is responsible for some of the growth‐inhibitory effects of antiestrogens.
Cloning and characterization of the human PIM‐1 gene: A putative oncogene related to the protein kinases
Authors: Meeker Timothy C. , Nagarajan Lalitha , ar‐Rushdi Abbas , Croce Carlo M.
Published: December 2004

The mouse PIM‐l gene has been implicated in the evolution of retrovirus‐associated mouse lymphomas. We have initiated a study of the human PIM‐1 gene because of its potential importance as a human oncogene. We have isolated genomic and cDNA clones for this gene and characterized this locus in detail. The predicted PIM‐1 protein is 313 amino acids in length. It has homology to a number of the protein kinases but does not have a transmembrane region. The amino acid corresponding to tyrosine‐416 of pp60v‐src is a tyrosine (position 198), which is consistent with the hypothesis that PIM‐1 is a tyrosine kinase rather than a serine threonine kinase. The PIM‐1 gene was found to have six exons and five introns derived from 5 kb of genomic DNA. The site of transcription initiation was localized by S1 nuclease protection studies which indicated that the mature PIM‐1 mRNA was approximately 2.7 kb in length. The promotor of this gene had no TATA or CAAT box but did have multiple GC boxes (CCGCCC) that might bind the Spl protein. The PIM‐1 gene was expressed in myeloid and B lymphoid cell lines, but not in T lymphoid and nonhemopoietic lines. This initial characterization of PIM‐1 will allow us to define its role in normal and malignant hematolymphoid differentiation.
Analysis of pp60c‐src tyrosine kinase activity and phosphotyrosyl phosphatase activity in human colon carcinoma and normal human colon mucosal cells
Authors: DeSeau Virginia , Rosen Neal , Bolen Joseph B.
Published: December 2004

We have compared the level of phosphotyrosyl phosphatase activity in lysates from normal human colon mucosal cells and human colon carcinoma cells and analyzed the effect of incubating these cells with sodium orthovanadate, an inhibitor of phosphotyrosyl phosphatase activity, on the relative abundance of acid‐stable phosphotyrosine and on in vitro protein kinase activity of pp60c‐src. Additionally, we compared the effect of lysing these cells in buffer containing only nonionic detergents with RIPA buffer, which contains both sodium dodecyl sulfate and deoxycholate, on the in vitro kinase activity of pp60c‐src. Our results show that the level of detectable phosphotyrosyl phosphatase activity in lysates derived from normal colon cells and colon carcinoma cells is very similar. Additionally, the abundance of acid‐stable phosphotyrosine in these cells cultured in the absence or presence of vanadate is not significantly different. However, incubation of these cells with vanadate significantly stimulates the activity of pp60c‐src derived from the normal colon cells in immune‐complex kinase assays, while having no detectable effect on the activity of pp60c‐src from the colon tumor cells. The in vitro protein kinase activity of pp60c‐src derived from RIPA buffer lysates of colon carcinoma cells was found to be elevated five‐ to sevenfold when compared with pp60c‐src from these same cells lysed in buffer containing only Nonidet‐P 40 as a detergent. The type of lysis buffer did not effect the activity of pp60c‐src from normal colon mucosal cells. These results provide additional evidence that the activity of pp60c‐src may be regulated differently in colon carcinoma and normal colon mucosal cells.