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Biomedical subjects

G M Edelman

Publications and source records attributed to G M Edelman.

At least 73 records · Page 4Linked to original sources
PubMed1995

Protein H--a bacterial surface protein with affinity for both immunoglobulin and fibronectin type III domains.

Several bacterial species express surface proteins with affinity for the constant region (Fc) of immunoglobulin (Ig) G. The biological consequences of the interaction with IgG are poorly understood but it has been demonstrated that genes encoding different IgG Fc-binding proteins have undergone convergent evolution, suggesting that these surface molecules are connected with essential microbial functions. One of the molecules, protein H, is present in some strains of Streptococcus pyogenes, the most significant streptococcal species in clinical medicine. In contrast to other Ig-binding bacterial proteins tested, protein H was found to interact also with the neural cell adhesion molecule (N-CAM), a eukaryotic cell surface glycoprotein mediating homo- and heterophilic cell-cell interactions. The affinity for the interaction between protein H and N-CAM was 1.6 x 10(8)/M and the binding site on protein H was mapped to the NH2-terminal 80 amino acid residues. N-CAM and IgG are both members of the Ig superfamily and analogous to N-CAM, IgG binds to the NH2-terminal part of protein H. However, the binding sites for the two proteins were found to be separate, an unexpected result which was explained by the observation that the fibronectin type III (FNIII) domains and not the Ig-like domains of N-CAM are responsible for the interaction with protein H. Thus, the binding of N-CAM to protein H was blocked with fibronectin but not with IgG. Moreover, apart from fibronectin itself and N-CAM, fragments of fibronectin and the matrix protein cytotactin/tenascin containing FNIII domains also showed affinity for protein H.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗
PubMed1995

Structural and functional similarities between the promoters for mouse tenascin and chicken cytotactin.

Cytotactin/tenascin is an extracellular matrix glycoprotein expressed in a restricted anteroposterior pattern during vertebrate development and is reexpressed in the adult during wound healing, tumorigenesis, and nerve regeneration. Previously, we have characterized the chicken cytotactin promoter and have shown its regulation by homeobox gene products in vitro. We have now isolated the promoter for the mouse tenascin gene in order to determine whether common or different DNA regulatory elements control the expression of this gene in these two species. Like the chicken cytotactin gene, the mouse tenascin gene has a single RNA start site that lies 27 bp downstream of a TATA box. A 4028-bp region of DNA upstream of the mouse tenascin gene was sequenced and examined for regulatory motifs in common with the upstream sequence from the chicken cytotactin promoter. Two hundred thirty base pairs of the proximal promoter regions from both genes had an extended sequence similarity and contained common regulatory motifs such as two tracts of homopolymeric dA.dT sequence, an octamer motif, an ATTA (TAAT) motif which is a common core sequence for binding of homeodomain transcription factors, and a TATA-box/cap-site region. Reporter gene constructs with various 5' deletions of the mouse tenascin upstream sequence were tested in transient transfections of mouse NIH 3T3 and chicken embryo fibroblasts. The conserved proximal promoter region of tenascin was responsible for most of the positive regulatory activity. In addition, an upstream region (-2478 to -247) repressed proximal promoter activity in mouse fibroblasts and also in chicken embryo fibroblasts. These data indicate that both the structure and function of the cytotactin/tenascin proximal promoters have remained conserved over 250 million years.

3T3 Cells↗
PubMed1995

The neural cell adhesion molecule (N-CAM) inhibits proliferation in primary cultures of rat astrocytes.

Cell proliferation is a key primary process during neural development and also plays an important role in the regenerative response of neural tissue to injury. It has been reported that glial cell proliferation is, at least in part, controlled by a neuronal signal, possibly involving cell surface molecules. We report here that the addition of purified rat neural cell adhesion molecule (N-CAM) to primary cultures of rat forebrain astrocytes inhibits their proliferation. This inhibitory effect can be elicited in cultures grown in chemically defined serum-free medium or in medium that had been supplemented with growth factors. Polyclonal antibodies to N-CAM or their Fab' fragments elicited a similar inhibitory effect. The magnitude of the inhibitory effect of N-CAM was dependent on cell density: it was maximal at low cell densities and weakened progressively as cells approached confluency. Synthetic peptides with sequences identical to a putative homophilic binding region of N-CAM mimicked the effect of purified N-CAM, while peptides of the same length and amino acid composition but with a randomized sequence did not. The addition of N-CAM antisense oligonucleotides to primary astrocyte cultures for 48 h resulted in reduced levels of N-CAM expression. After N-CAM levels on astrocytes were diminished by this treatment, the antiproliferative effect of N-CAM added to the medium was significantly reduced. The combined results suggest that N-CAM homophilic binding may be involved in the control of glial cell proliferation.

Animals↗
PubMed1995

Functional analysis of posttranslational cleavage products of the neuron-glia cell adhesion molecule, Ng-CAM.

Neuron-glia cell adhesion molecule (Ng-CAM) mediates cell adhesion between neurons homophilically and between neurons and glia heterophilically; it also promotes neurite outgrowth. In the chick brain, Ng-CAM is detected as glycoproteins of 190 and 210 kD (Ng-CAM200) with posttranslational cleavage products of 135 kD (F135, which contains most of the extracellular region) and 80 kD (F80, which includes the transmembrane and the cytoplasmic domains). To examine the functions of each of these components, we have expressed Ng-CAM200, F135, and F80 in murine L cells, and F135 and F80 as GST fusion proteins in the pGEX vector in bacteria. Appropriately transfected L cells expressed each of these proteins on their surfaces; F135 was also found in the media of cells transfected with Ng-CAM200 and F135. In addition to binding homophilically, cells transfected with Ng-CAM200 and F135 bound heterophilically to untransfected L cells, suggesting that there is a ligand for Ng-CAM on fibroblasts that may be related to the glial ligand. Detailed studies using the transfected cells and the fusion proteins indicated that both the homophilic and the heterophilic binding activities of Ng-CAM are localized in the F135 fragment of the molecule. The results also indicated that proteolytic cleavage of Ng-CAM200 is not required either for its expression on the cell surface or for cell adhesion and that there is an "anchor" for F135 on L cells (and presumably on neurons). In contrast to the cell binding results, the F80 but not the F135 fusion protein enhanced the outgrowth of neurites from dorsal root ganglion cells; this activity was associated with the FnIII repeats of F80. The observations that a protein corresponding to F135 contains the cell aggregation sites whereas one corresponding to the F80 has the ability to promote neurite outgrowth suggest that proteolytic cleavage may be an important event in regulating these Ng-CAM activities during embryonic development and neural regeneration.

Amino Acid Sequence↗
PubMed1995

Separate cell binding sites within cytotactin/tenascin differentially promote neurite outgrowth.

Cytotactin/tenascin (CT/TN) is an extracellular matrix protein that binds to a variety of cell types and that influences neurite outgrowth. It has a multidomain structure with regions homologous to epidermal growth factor (EGF)-like repeats, fibronectin (FN) type II repeats, and the beta and gamma chains of fibrinogen (fg). The current study demonstrates that a fusion protein corresponding to the sixth fibronectin type III repeat in CT/TN (CTfn6) supported cell attachment and promoted an increase in the number of cells with neurites in both central and peripheral neurons in tissue culture. The third fibronectin type III repeat, CTfn3, like intact CT/TN, supported attachment of peripheral neurons but not of central neurons and, while it caused an increase in neurite length, it did not increase the number of cells that sprouted neurites. When CTfn3 and CTfn6 were combined, an increase in both the number of cells sprouting neurites and in neurite length was observed for peripheral neurons that resembled their response to intact CT/TN. Cell attachment to CTfn6 was inhibited in the presence of function-blocking antibodies against beta 1 integrins. In contrast, the interaction with CTfn3 was not inhibited by antibodies to beta 1 integrins, but was inhibited by RGD-containing peptides. The results suggest that cell binding to CT/TN involves two different sites within the molecule and occurs via different receptors which may be differentially expressed on different neuronal cell types. The location of these sites within the whole molecule in the context of other adhesive and counteradhesive domains may modulate their influence on cellular responses such as cell attachment and neurite outgrowth.

Amino Acid Sequence↗
PubMed1994

Pax-3 contains domains for transcription activation and transcription inhibition.

Pax-3 is a member of the Pax family of transcription factors involved in transcriptional control events during embryonic development. Here we report a functional dissection of the Pax-3 protein and describe the protein domains which are responsible for different activities. A transcription inhibition activity is located in the first 90 N-terminal amino acids and includes part of the paired domain. Furthermore, the C terminus of Pax-3 is able to confer transcriptional activation of basal promoters. Pax-3 can utilize both transcription modulating functions and activates transcription over a narrow range of protein concentration in the presence of promoter elements containing functional binding sites.

Amino Acid Sequence↗
PubMed1994

Binding and activation of the promoter for the neural cell adhesion molecule by Pax-8.

The neural cell adhesion molecule (N-CAM), is expressed in definite spatiotemporal patterns during development. To identify factors that may influence place-dependent n-cam gene expression, we have studied the binding and activation of the n-cam promoter by Pax-8, a member of the Pax family of transcription factors. Pax-8 increased n-cam promoter activity 13.4-fold in cellular co-transfection experiments, and a short segment of the promoter (-143 to -15) mediated the response. This region of the n-cam promoter produced a DNA-protein complex when incubated with either extracts from COS-7 cells transfected with the Pax-8 expression vector or a Pax-8/GST fusion protein. Pax-8 bound to the n-cam promoter through two TGCTCC motifs (designated PBS-1 and PBS-2) that resemble paired domain binding sites. Mutation of PBS-1 and PBS-2 eliminated Pax-8 activation of the n-cam promoter. Transfection of N2A neuroblastoma cells with the Pax-8 expression vector resulted in a 5-fold increase in the transcription of the endogenous n-cam gene. The combined results suggest that Pax-8 activates transcription of the n-cam gene through binding of sequences resembling paired domain binding sites in the n-cam promoter. The data raise the possibility that the n-cam promoter may be regulated by other members of the Pax gene family.

3T3 Cells↗
PubMed1994

Regulation in vitro of an L-CAM enhancer by homeobox genes HoxD9 and HNF-1.

Previous studies have shown that in vitro expression of the neural cell adhesion molecule (N-CAM) can be regulated by the products of homeobox genes HoxB9, -B8, and -C6. N-CAM is a Ca(2+)-independent immunoglobulin-related CAM that plays an important role in neural development. In the present study, we investigated whether the liver cell adhesion molecule (L-CAM) a member of the Ca(2+)-dependent CAM family (cadherins) is also regulated by homeobox-containing genes. In transient cotransfection experiments of NIH 3T3 cells, we observed that both HoxD9 and liver-enriched POU-homeodomain transcription factor, HNF-1, activated chloramphenicol acetyltransferase gene reporter constructs containing the L-CAM promoter and an enhancer present in the second intron of the chicken L-CAM gene. Using electrophoretic mobility-shift assays, we found that components of cell extracts from NIH 3T3 cells transfected with HoxD9 bound to a small region of the L-CAM enhancer having a consensus sequence that is a putative binding site for HNF-1. Components of extracts from the chicken hepatoma cell line LMH that had been transfected with an HNF-1 expression vector also bound to this same site. In nuclear run-on experiments with nuclei from LMH cells that were transfected with expression vectors for HoxD9 or HNF-1, L-CAM RNA levels were increased 33-fold and 4-fold respectively. Using the same run-on procedure, it was confirmed that nuclei prepared from normal embryonic chicken liver cells expressed the RNAs for HoxD9, HNF-1, and L-CAM. Taken together with previous observations, these data raise the possibility that homeobox-containing genes will have a widespread role in the place-dependent expression of CAMs belonging both to immunoglobulin-related and to cadherin families.

3T3 Cells↗
PubMed1994

A measure for brain complexity: relating functional segregation and integration in the nervous system.

In brains of higher vertebrates, the functional segregation of local areas that differ in their anatomy and physiology contrasts sharply with their global integration during perception and behavior. In this paper, we introduce a measure, called neural complexity (CN), that captures the interplay between these two fundamental aspects of brain organization. We express functional segregation within a neural system in terms of the relative statistical independence of small subsets of the system and functional integration in terms of significant deviations from independence of large subsets. CN is then obtained from estimates of the average deviation from statistical independence for subsets of increasing size. CN is shown to be high when functional segregation coexists with integration and to be low when the components of a system are either completely independent (segregated) or completely dependent (integrated). We apply this complexity measure in computer simulations of cortical areas to examine how some basic principles of neuroanatomical organization constrain brain dynamics. We show that the connectivity patterns of the cerebral cortex, such as a high density of connections, strong local connectivity organizing cells into neuronal groups, patchiness in the connectivity among neuronal groups, and prevalent reciprocal connections, are associated with high values of CN. The approach outlined here may prove useful in analyzing complexity in other biological domains such as gene regulation and embryogenesis.

Animals↗
PubMed1994

Antibody to neural cell adhesion molecule can disrupt the migration of luteinizing hormone-releasing hormone neurons into the mouse brain.

The neurons which synthesize and release luteinizing hormone-releasing hormone (LHRH), are hypothesized to originate in the epithelium of the medial olfactory pit and to migrate into the brain along a scaffolding made up of neural cell adhesion molecule (NCAM)-immunoreactive branches of the terminal and vomeronasal nerves. These LHRH neurons, studied by immunocytochemical and autoradiographic procedures, were found to originate within a very short period of embryogenesis, specifically day 10, in mice, and to follow a remarkably ordered spatiotemporal course along the migration route into the brain. The purpose of the present experiments was to determine whether perturbation of the NCAM-immunoreactive migration route, at a particular time in development, would arrest the migration of LHRH neurons into the brain. We found that a 1 microliter injection of antiserum to NCAM into the area of the olfactory pit, on day 10 of embryogenesis, significantly reduced the number of LHRH-immunoreactive neurons seen in the epithelium of the medial olfactory pit, with a concomitant significant reduction in the number of LHRH-immunoreactive cells seen outside of the placode, on the migration route. These results confirm our initial hypothesis that LHRH neurons migrate from the epithelium of the olfactory pit to the brain and indicate that NCAM plays a causal role in this phenomenon.

Animals↗
PubMed1994

Cell adhesion alters gene transcription in chicken embryo brain cells and mouse embryonal carcinoma cells.

To determine whether changes in gene expression occur in embryonic cells as a consequence of changes in cellular aggregation, chicken embryo brain (CEB) cells isolated from 8-day embryos were allowed to aggregate or prevented from aggregating by treatment with anti-neural cell adhesion molecule (N-CAM) Fab' fragments. A subtractive hybridization cloning strategy was employed to identify genes that might show different levels of expression in the two populations of cells. In addition, the transcription rates of a number of genes specifying CAMs and transcription factors were directly estimated by using nuclear run-off transcription assays. The transcription rates of several genes, including those encoding N-CAM, Ng-CAM, alpha-N-catenin, HoxA4 (Hox1.4), a fatty acid-binding protein, and a subunit of the mitochondrially encoded cytochrome-c oxidase enzyme decreased upon CEB cell aggregation. The transcription rates of several previously unidentified genes either increased or decreased upon aggregation, while the transcription of other genes remained unchanged. The transcription rate of the N-CAM gene was 3.3-fold higher in dissociated than in aggregated CEB cells. This rate of transcription also increased when the brain tissue was dissociated into single cells and the increased rate was maintained by keeping the cells dissociated in the presence of Fab' fragments of antibodies to N-CAM. Decreased transcription rates of the N-CAM gene were also observed upon aggregation of P19 cells, a mouse embryonal carcinoma cell line. Primary chicken embryo liver cells, which aggregate primarily by calcium-dependent adhesion mechanisms, did not show changes in the N-CAM gene or in the other genes whose transcription rates changed in CEB cells and P19 cells. These observations suggest that the types of genes regulated by cell aggregation include those for CAMs themselves as well as for transcription factors that may control the expression of CAMs and other molecules significant for morphogenesis.

Animals↗
PubMed1994

Differential effects of cytotactin/tenascin fusion proteins on intracellular pH and cell morphology.

Cytotactin/tenascin is a multidomain extracellular matrix protein that inhibits both cell spreading and intracellular alkalinization. The protein has multiple different domains which are homologous to regions in epidermal growth factor, fibronectin, and fibrinogen. In previous studies, we produced nonoverlapping fusion proteins corresponding to these domains and examined their effects on cell attachment and spreading. Based on their ability either to promote or to inhibit cell attachment, two of these fusion proteins were shown to be adhesive and two were shown to be counteradhesive. To determine how the adhesive and counteradhesive activities of different cytotactin/tenascin domains alter intracellular pH (designated pHi), we have measured pHi, in NIH3T3 and U251MG cells in the presence of the cytotactin/tenascin fusion proteins and intact cytotactin/tenascin, as well as fibronectin. Cells incubated in the presence of intact cytotactin/tenascin or of the counteradhesive fusion proteins had a pHi lower than control cells. In contrast, the presence of the adhesive fusion proteins or of fibronectin caused cells to have higher pHi values than control cells. When two fragments were simultaneously presented, one of which alone increased pHi and the other of which alone decreased pHi, the predominant effect was that of lowered pHi. Incubation with an RGD-containing peptide derived from the cytotactin/tenascin sequence inhibited alkalinization promoted by the adhesive fragment containing the second through sixth fibronectin type III repeats that was known to bind to integrins. Incubation of the cells with heparinase I or III inhibited the intracellular alkalinization of cells plated in the presence of the other adhesive fusion protein containing the fibrinogen domain, suggesting that heparan sulfate proteoglycans were involved in these pHi changes. The activity of protein kinase C appeared to be important for the changes in pHi mediated by all of the proteins. The protein kinase C inhibitor Calphostin C blocked the rise in pHi elicited by the adhesive fusion proteins and by fibronectin. Moreover, activation of protein kinase C by the addition of phorbol esters increased the pHi in cells plated on cytotactin/tenascin or counteradhesive fusion proteins and reversed their effects. The results of this study support the hypothesis that cytotactin/tenascin can bind to multiple cell surface receptors and thereby elicit different physiological responses. Decreases in pHi are correlated with the phenomenon of counteradhesion whereas the ability to increase pHi is associated with cell attachment via at least two different types of cell surface receptors. The data raise the possibility that binding of cytotactin/tenascin may influence primary cellular processes such as migration and proliferation through the differential regulation of pHi.

3T3 Cells↗
PubMed1994

Value-dependent selection in the brain: simulation in a synthetic neural model.

Many forms of learning depend on the ability of an organism to sense and react to the adaptive value of its behavior. Such value, if reflected in the activity of specific neural structures (neural value systems), can selectively increase the probability of adaptive behaviors by modulating synaptic changes in the circuits relevant to those behaviors. Neuromodulatory systems in the brain are well suited to carry out this process since they respond to evolutionarily important cues (innate value), broadcast their responses to widely distributed areas of the brain through diffuse projections, and release substances that can modulate changes in synaptic strength. The main aim of this paper is to show that, if value-dependent modulation is extended to the inputs of neural value systems themselves, initially neutral cues can acquire value. This process has important implications for the acquisition of behavioral sequences. We have used a synthetic neural model to illustrate value-dependent acquisition of a simple foveation response to a visual stimulus. We then examine the improvement that ensues when the connections to the value system are themselves plastic and thus become able to mediate acquired value. Using a second-order conditioning paradigm, we demonstrate that auditory discrimination can occur in the model in the absence of direct positive reinforcement and even in the presence of slight negative reinforcement. The discriminative responses are accompanied by value-dependent plasticity of receptive fields, as reflected in the selective augmentation of unit responses to valuable sensory cues. We then consider the time-course during learning of the responses of the value system and the transfer of these responses from one sensory modality to another. Finally, we discuss the relation of value-dependent learning to models of reinforcement learning. The results obtained from these simulations can be directly related to various reported experimental findings and provide additional support for the application of selectional principles to the analysis of brain and behavior.

Animals↗
PubMed1993

Identification of the promoter and a transcriptional enhancer of the gene encoding L-CAM, a calcium-dependent cell adhesion molecule.

L-CAM is a calcium-dependent cell adhesion molecule that is expressed in a characteristic place-dependent pattern during development. Previous studies of ectopic expression of the chicken L-CAM gene under the control of heterologous promoters in transgenic mice suggested that cis-acting sequences controlling the spatiotemporal expression patterns of L-CAM were present within the gene itself. We have now examined the L-CAM gene for sequences that control its expression and have found an enhancer within the second intron of the gene. A 2.5-kb Kpn I-EcoRI fragment from the intron acted as an enhancer of a simian virus 40 minimal promoter driving a chloramphenicol acetyltransferase (CAT) reporter gene and produced 14.0-fold induction of CAT activity in MDCK cells. To narrow down the region responsible for enhancer activity and to determine whether the enhancer could function in a cell type-specific manner, a number of smaller restriction fragments from the intron were tested for activity in two chicken cell lines, the LMH hepatoma line, which produces high levels of L-CAM, and the SL-29 fibroblast line, which produces little, if any, L-CAM. Four L-CAM enhancer plasmids containing shorter segments derived from the intron showed enhanced CAT activity levels (between 9.4- and 16.5-fold) in extracts from transfected LMH cells but not from SL-29 cells. DNA sequence analysis of the L-CAM enhancer region revealed putative binding sites for the transcription factors SP1, E2A, and AP-2. In addition, LE-9, the smallest L-CAM enhancer segment (310 bp), contained a consensus binding site for the liver-enriched POU-homeodomain transcription factor, HNF-1. Tests of upstream sequences showed that a 630-bp fragment, corresponding to nearly the entire intergenic region between L-CAM and its neighboring CAM gene, K-CAM, could function as a promoter. In combination with the L-CAM enhancer, this fragment directed cell type-specific expression of the CAT reporter gene in LMH cells at a level comparable to that observed with enhancer constructs using the simian virus 40 minimal promoter. These combined observations define a promoter and an enhancer for the chicken L-CAM gene. They raise the possibility that these cis-acting regulatory sequences may be instrumental in directing specific place-dependent expression of the L-CAM gene in the chicken.

Animals↗
PubMed1993

Multiple integrins mediate cell attachment to cytotactin/tenascin.

To identify potential cell surface receptors for chicken cytotactin (CT), we have characterized the ability of recombinant fusion proteins spanning the proximal fibronectin (FN) type III repeats of the molecule to support attachment of glioma and carcinoma cell lines. The third FN type III repeat, which contains the RGD tripeptide, supported cell attachment and cell spreading; however, mutation of RGD to RAD did not result in significant loss of either activity. In addition, the same repeat of mouse CT, which contains a natural mutant, RVD, also supported cell attachment and spreading, although at a lower level; both activities were increased by mutation of the RVD sequence to RGD. Studies utilizing RGD-containing peptides and well-characterized antibodies to integrins indicated that cell attachment to the third FN type III repeat was mediated by at least two different integrin receptors of the alpha v subtype. Additional cellular receptors may also be involved in cell attachment to CT. For example, an antibody to the beta 1 subfamily of integrins partially inhibited binding of cells to intact CT but did not inhibit cell binding to the third FN type III repeat. These findings suggest that the RGD site in CT is able to mediate cell attachment to integrins and thus is not a cryptic adhesion site. They also open the possibility that the functions of CT in processes such as counteradhesion, cell migration, cell proliferation, and cell differentiation may be mediated in part by interaction with multiple integrins.

Amino Acid Sequence↗