Nitric oxide: linking space and time in the brain.
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Animals↗
Biomedical subjects
G M Edelman
Publications and source records attributed to G M Edelman.
Cell adhesion molecules as targets for Hox genes: neural cell adhesion molecule promoter activity is modulated by cotransfection with Hox-2.5 and -2.4.
In an effort to determine whether homeobox genes modulate the activity of the promoter of the mouse neural cell adhesion molecule (N-CAM) gene, we have carried out a series of cotransfection experiments using NIH 3T3 cells. Plasmids were constructed containing Xenopus laevis Hox-2.5 and -2.4 coding sequences linked to a human cytomegalovirus promoter (CMV-Hox-2.5 and CMV-Hox-2.4). A 4.9-kilobase DNA fragment containing 5' flanking and first exon sequences of the mouse N-CAM gene was linked to a chloramphenicol acetyltransferase (CAT) reporter gene (N-CAM-Pro-CAT). Cotransfection with CMV-Hox-2.5 and N-CAM-Pro-CAT resulted in a strong induction of CAT activity. The N-CAM promoter contained two potential homeodomain binding sites (sites I and II) within a 47-base-pair segment (512-559 base pairs upstream of the ATG codon in the first exon of the N-CAM gene). This segment was linked to a minimal promoter (simian virus 40 early) and a downstream CAT gene. Although this construct was transcriptionally active at a low level in NIH 3T3 cells, cotransfection of CMV-Hox-2.5 resulted in CAT activity that was greatly elevated. Mutational studies revealed that it was the homeodomain binding site II sequence that was required for this regulation. In contrast, cotransfection with CMV-Hox-2.4 eliminated the CAT activity that was driven by the CMV-Hox-2.5 construct. Thus, the products of two related Hox genes, which are located adjacent to each other in the Hox-2 complex, can differentially modulate transcription from the promoter of a cell adhesion molecule gene. The results suggest that the N-CAM gene is likely to be a target for regulation by Hox gene products.
3T3 Cells↗
Activation of the cytotactin promoter by the homeobox-containing gene Evx-1.
Cytotactin is a morphoregulatory molecule of the extracellular matrix affecting cell shape, division, and migration that appears in a characteristic and complex site-restricted pattern during embryogenesis. The promoter region of the gene that encodes chicken cytotactin contains a variety of potential regulatory sequences. These include putative binding sites for homeodomain proteins and a phorbol 12-O-tetradecanoate 13-acetate response element (TRE)/AP-1 element, a potential target for transcription factors thought to be involved in growth-factor signal transduction. To determine the effects of homeobox-containing genes on cytotactin promoter activity, we conducted a series of cotransfection experiments on NIH 3T3 cells using cytotactin promoter-chloramphenicol acetyltransferase (CAT) reporter gene constructs and plasmids driving the expression of mouse homeobox genes Evx-1 and Hox-1.3. cotransfection with Evx-1 stimulated cytotactin promoter activity whereas cotransfection in control experiments with Hox-1.3 had no effect. To localize the sequences required for Evx-1 activation, we tested a series of deletions in the cytotactin promoter. An 89-base-pair region containing a consensus TRE/AP-1 element was found to be required for activation. An oligonucleotide segment containing this TRE/AP-1 site was found to confer Evx-1 inducibility on a simian virus 40 minimal promoter; mutation of the TRE/AP-1 site abolished this activity. To explore the potential role of growth factors in cytotactin promoter activation, chicken embryo fibroblasts, which are known to synthesize cytotactin, were first transfected with cytotactin promoter constructs and cultured under minimal conditions in 1% fetal bovine serum. Although the cells exhibited only low levels of CAT activity under these conditions, cells exposed for 12 h to 10% (vol/vol) fetal bovine serum showed a marked increase in CAT activity. Cotransfection with Evx-1 and cytotactin promoter constructs of cells cultured in 1% fetal bovine serum was sufficient, however, to produce high levels of CAT activity. These findings are consistent with the hypothesis that Evx-1, a homeobox-containing gene, may activate the cytotactin promoter by a mechanism involving a growth-factor signal transduction pathway. More generally, the results support the hypothesis that the place-dependent expression of morphoregulatory molecules may depend upon local cues provided by homeobox genes and their encoded proteins.
3T3 Cells↗
Specific binding of cytotactin to sulfated glycolipids.
The binding of the glial glycoprotein, cytotactin, to a variety of purified glycolipids was examined. Clear-cut evidence was found for binding of radiolabeled cytotactin to sulfatides purified from bovine brain, but the molecule did not bind to gangliosides or cerebrosides. The sulfatide binding was sensitive to pH and ionic strength and was dependent on the presence of divalent cations. Binding was inhibited by purified unlabeled cytotactin, by polyclonal antibodies to cytotactin, and by several monosaccharides and polysaccharides. It was not inhibited by fibronectin, a chondroitin sulfate proteoglycan, or the HNK-1 monoclonal antibody, all of which are known to bind to cytotactin. These findings raise the possibilities that sulfated glycolipids may function as cellular receptors for cytotactin and that binding by sulfatides may modulate the varied effects of cytotactin on cellular processes.
Animals↗
Cytotactin: a morphoregulatory molecule and a target for regulation by homeobox gene products.
Two major lines of research in developmental biology should help us to understand the bases of morphogenesis. The first is the analysis of the morphogenetic effects of local expression of various adhesion molecules. The second is the analysis of cascades of regulatory genes that interact during development. Of particular significance are regulatory interactions involving homeobox-containing genes which are expressed in a place-dependent manner in the embryo. Success in connecting these two lines of research would help to resolve the puzzle of how species-specific tissue patterns can arise and be maintained. In this article, we focus on cytotactin, a morphoregulatory molecule of the extracellular matrix that exhibits sharply restricted spatiotemporal patterns of expression during development. Recent experiments indicate the promoter of the cytotactin gene contains target regions that appear to respond to homeodomain proteins. These observations, and those on other morphoregulatory molecules, suggest a possible connection between their effects on cell patterning and control by homeobox-containing genes.
Animals↗
Mediation and inhibition of cell adhesion by morphoregulatory molecules.
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Animals↗
Characterization of the protein kinase activity of avian sarcoma virus src gene product.
The avian sarcoma virus src gene product, p60src, has been purified 650-fold from cytoplasmic extracts of the rat tumor cell line RR1022 by using ammonium sulfate fractionation, hydrophobic chromatography on omega-aminohexyl agarose, and ion exchange chromatography on phosphocellulose. Partially purified p60src is a monomer, with a native molecular weight of about 60,000 and an apparent pI of 6.0. In immunoprecipitates, p60src catalyzed phosphorylation of anti-p60src IgG heavy chains within the variable (VH) domain, which contains the heavy chain portion of the antigen combining site. Crude preparations of p60src contained phosphatase activity able to cleave phosphate from IgG heavy chains; this activity was removed by the purification procedure, and partially purified p60src could phosphorylate the heavy chain of specific antibody in solution. Furthermore, purified p60src catalyzed phosphorylation in solution of the general protein kinase substrate, alpha-casein, strengthening the hypothesis that it may in fact function as a protein kinase in vivo.
Alpharetrovirus↗
Replication in vitro of the 2-micrometer DNA plasmid of yeast.
Cell-free extracts prepared from growing cells of the budding yeast Saccharomyces stimulated DNA synthesis directed by the supercoiled 2-micrometer yeast DNA plasmid. The major products of the reaction were open-circular daughter molecules possessing newly synthesized full-length linear DNA strands. Some of these were ligated and supertwisted by the extracts to yield a supercoiled DNA product. Both of the complementary DNA strands of the template were replicated. In addition, the extracts induced the appearance of theta-forms of the plasmid DNA, which are presumed to be replicative intermediates. The results of experiments utilizing BrdUTP incorporation indicated that DNA repair did not contribute significantly to the overall reaction. Extracts prepared from the cell division cycle mutants cdc7 and cdc8, held in culture at the nonpermissive temperature, possessed diminished activity. Because these mutants define a dependent sequence of events leading from the start of the cell cycle through G1 to S phase, this result suggests that the activity that stimulates 2-micrometer DNA replication in vitro is subject to control in the yeast cell cycle.
DNA↗
Cell-free synthesis and segregation of beta 2-microglobulin.
beta2-Microglobulin has been synthesized in vitro by using a rabbit reticulocyte lysate system and mRNA from the mouse tumor cell line EL4. The molecule is synthesized as a precursor with an NH2-terminal extension of 19 amino acids: Ser-X-Ser-Val-X-Leu-Val-Phe-Leu-Val-Leu-Val-Ser-Leu-X-Gly-Leu-Tyr-X. The processing and segregation of this peripheral membrane protein are directly comparable to those of secretory proteins and integral membrane proteins: addition of dog pancreas microsomal membranes during translation caused conversion to the processed chain, but addition of membranes after synthesis did not; only the processed chain sedimented with the membrane vesicles and was protected from proteolysis by the vesicles; and processing of nascent beta 2-microglobulin was blocked by competitive inhibitors that prevent processing and segregation of secretory and integral membrane proteins. These results suggest that the signal sequences of secretory proteins, integral membrane proteins, and peripheral membrane proteins have a common function and a common receptor on the cytoplasmic face of dog pancreas microsomal membranes. This system also provides a means for studying in vitro the expression and function of the major histocompatibility antigens that are associated with beta 2-microglobulin on cell surfaces.
Amino Acid Sequence↗
Surface molecules involved in interactions among neural cells during development.
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Animals↗
Binding and functional properties of concanavalin A and its derivatives. I. Monovalent, divalent, and tetravalent derivatives stable at physiological pH.
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Animals↗
Binding and functional properties of concanavalin A and its derivatives. II. A proteolytic product with saccharide-binding activity.
Limited digestion of the intact subunit of concanavalin A (Mr = 26,000) with trypsin followed by affinity chromatography on Sephadex G-100 has yielded a highly purified product designated here as Tn-Con A. Chemical studies have shown that Tn-Con A is composed of several components: a large fragment (Tn I, Mr = 19,000) spanning residues 1 to 172, and lower molecular weight polypeptides that are noncovalently associated with Tn I to form the active molecule. The molecular weight of Tn-Con A at pH 7 was 90,000, suggesting that, like native concanavalin A, it was a tetramer at physiological pH. Equilibrium dialysis experiments showed that Tn-Con A bound 1 molecule of alpha-methyl-D-glucoside/22,000 g atoms of protein and therefore that four saccharides are bound by the tetrameric molecule. Tn-Con A and native concanavalin A competed for the same receptors on the lymphocyte surface. Moreover, Tn-Con A was mitogenic for both mouse and human lymphocytes with dose-response curves similar to those of the native lectin. All of these results indicate that tryptic hydrolysis of concanavalin A produces a fragmented molecule retaining the saccharide-binding, subunit association, and mitogenic capacity of the native protein.
Amino Acids↗
Binding and functional properties of concanavalin A and its derivatives. III. Interactions with indoleacetic acid and other hydrophobic ligands.
The binding of concanavalin A to various structures via hydrophobic interactions has been studied using a variety of physicochemical assays. It was found that concanavalin A binds to nonpolar compounds such as the plant auxin beta-indoleacetic acid and its structural analogue tryptophan and that this binding is independent of the saccharide-binding activity normally associated with the lectin. The results of equilibrium dialysis experiments on the binding of beta-indoleacetic acid were consistent with the presence of a single weak binding site per subunit of protein, having an association constant of about 7 X 10(2) M-1. Competition experiments using various nonpolar compounds such as o-iodobenzoic acid suggested that this hydrophobic binding site is located in the same cavity which binds the iodine-containing ligand as shown by x-ray crystallography. Concanavalin A also binds to lipid vesicles composed of dipalmitoylphosphatidylcholine or 12-O-tetradecanoyl phorbol-13-O-acetate. This binding to lipid membranes raises the possibility that the synergistic effects of concanavalin A and tetradecanoyl phorbol acetate on lymphocyte mitogenesis may be due in part to an interaction between lectin and the phorbol ester.
Concanavalin A↗
Changes in the three-dimensional structure of concanavalin A upon demetallization.
When the Mn(2+) and Ca(2+) ions normally present in concanavalin A are removed, the protein becomes incapable of binding saccharides. To explore the structural differences between the native and demetallized forms and their effects on the saccharide-binding properties of the protein, we have refined and compared the crystal structures of both forms. Refinement, carried out by automated difference Fourier methods, has revealed a number of differences between the two structures as well as minor differences between the two crystallographically independent monomers in the demetallized structure. Significant differences between the holo- and apoproteins are confined to the region where the metals are bound. These differences include a reorganization and disordering of the loop, consisting of residues 7-25, that contains all of the direct metal ligands of the protein. In some molecules, the side chain of arginine-228 appears to move into the metal-binding region, possibly compensating in part for the absence of the metal's positive charge. The cis peptide observed in the native protein at alanine-207 is apparently not present in the demetallized protein. The conformational differences affect many of the residues currently thought to be involved in the specific binding of saccharides.
Apoproteins↗
Assay for early cytoplasmic effects of the src gene product of Rous sarcoma virus.
When microinjected into normal fibroblasts, cytoplasmic extracts of cells transformed by Rous sarcoma virus caused dissolution of microfilament bundles. This activity was not found in extracts of normal cells. The maximum effect was seen within 30 min of injection, and the activity could still be measured after a 10-fold dilution of the cytoplasmic extracts (14 mg/ml original protein concentration). The activity was trypsin sensitive and was destroyed by boiling, but was not RNase sensitive. Protein synthesis was not required for the disruption of actin-containing stress fibers by the injected activity. Microinjected cytoplasts prepared from normal 3T3 cells also showed dissolution of microfilament bundles, indicating that the cell nucleus was not required for expression of activity. Extracts made from fibroblasts transformed by Rous sarcoma virus having a temperature-sensitive mutation in the src gene were also temperature sensitive in the microinjection assay. Thus, the activity of extracts from cells infected with src mutant virus, but not from cells infected with wild-type virus, was destroyed either by in vitro incubation of the extract at the nonpermissive temperature before injection or by incubation of recipient cells at the nonpermissive temperature after injection. We conclude that the microinjection assay can detect a cytoplasmic activity coded for by the src gene of Rous sarcoma virus and that an early direct or indirect target of the src gene product is the cytoskeleton and cell motility system. This result is discussed in relation to the hypothesis that submembranous arrays of microfilaments, microtubules, and their associated proteins interact with cell surface receptors to form a surface modulating assembly that functions as a key regulator of cell growth.
Actins↗
Adhesion among neural cells of the chick embryo. III. Relationship of the surface molecule CAM to cell adhesion and the development of histotypic patterns.
We have previously identified a molecule (named cell adhesion molecule [CAM]) that is involved in the in vitro aggregation of neural cells from chick embryos. In the present report, specific anti-CAM antibodies have been used to demonstrated that CAM is localized in neural tissues, and is associated with the plasma membrane of retinal cells and neurites. Furthermore, it has been shown by antibody absorption techniques that the decreased adhesiveness of cultured retinal cells obtained originally from older embryos is correlated with a decrease in the density or accessibility of cell adhesion molecules on the surface of these cells. The central role of CAM in neural cell aggregation has been established by the observation that anti-CAM Fab' fragments inhibit adhesion between neural cells in a variety of assays. To investigate the function of CAM and cell adhesion in developing tissues, aggregates of retinal cells that are capable of forming histotypic patterns in vitro were cultured in the presence and absence of anti-CAM Fab'. The Fab' was found to inhibit sorting out of cell bodies and neurites and to decrease the number of membrane-membrane contacts, suggesting that CAM is associated with cell-cell, cell-neurite, and neurite-neurite interactions.
Animals↗
Adhesion among neural cells of the chick embryo. IV. Role of the cell surface molecule CAM in the formation of neurite bundles in cultures of spinal ganglia.
The cell adhesion molecule (CAM) is involved in adhesion among embryonic retinal and brain cells and has been detected in a variety of neural tissues. This paper describes the use of spinal ganglion cultures and specific anti-CAM antibodies to determine the distribution of CAM on plasma membranes of nerve processes, and to assess the results of perturbation of its function during the growth of neurites from ganglia. The results indicate that CAM is distributed over the entire surface of nerve processes, and that specific anti-CAM Fab' fragments alter the morphology of neurite outgrowth. In particular, it was observed that anti-CAM inhibits formation of nerve bundles, so that the ganglion becomes surrounded by a tangled net of fine processes. Growth cone functions, such as neurite elongation, motility, and attachment to the substratum, did not appear to be affected by the antibody. These studies suggest that one of the major functions of CAM is to mediate side-to-side adhesion between neurites to form fascicles, and raise the possibility that this molecule serves a key role in embryogenesis of nerve tissues.
Animals↗