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Biomedical subjects

K L Crossin

Publications and source records attributed to K L Crossin.

At least 37 records · Page 2Linked 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

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

EGF receptor-mediated signals are differentially modulated by concanavalin A.

NIH 3T3 cells expressing high levels of the human epidermal growth factor (EGF) receptor were used to examine the effects of the lectin concanavalin A (Con A) on EGF-mediated signaling events. Proliferation of NIH 3T3 cells expressing high levels of the human EGF receptor was inhibited in a dose-dependent manner by Con A. At the same time, Con A also inhibited both dimerization and tyrosine phosphorylation of the EGF receptor. Tyrosine phosphorylation of the enzyme phospholipase C-gamma, a substrate of the phosphorylated EGF receptor kinase, was also inhibited. In contrast, EGF-stimulated changes in pH, calcium, and levels of inositol phosphates were unaffected by the presence of Con A. These results indicate that certain signals (changes in the levels of intracellular calcium, pH, and inositol phosphates) mediated by EGF binding to its receptor still occur when receptor dimerization and phosphorylation are dramatically decreased, suggesting that multiple independent signals are transmitted by the binding of EGF to its receptor.

Cell Division↗
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

Cell-cell adhesion molecules in epithelial-mesenchymal transformations.

The functional units in most inductive and morphogenetic processes in the embryo are not single cells, but rather collectives of interacting cells that give rise to the tissues and organs. Cell adhesion molecules (CAMs) are involved in defining cell collectives and their borders as they interact during inductive events in morphogenesis. The expression patterns of CAMs are highly dynamic and changes are known to occur during epithelial-mesenchymal transformations. Alterations in CAM expression are correlated with changes in morphology. Conversely, experimentally induced changes in morphology result in changes in CAM expression. The structure, function, distribution, and control of CAM gene expression are presented in this review, and discussed with regard to their importance to normal developmental processes, particularly epithelial-mesenchymal transformations.

Animals↗
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

The integrin alpha 9 beta 1 mediates cell attachment to a non-RGD site in the third fibronectin type III repeat of tenascin.

We have previously reported the sequence of the integrin alpha 9 subunit, a partner of the beta 1 subunit that is expressed in basal keratinocytes, hepatocytes, airway epithelial cells, and smooth and skeletal muscle. In the present study, we have stably expressed alpha 9 beta 1 on the surface of the human embryonic kidney cell line 293 and the human colon carcinoma cell line SW480 and used these transfected cells lines to identify ligand(s) for this integrin. Transfected cells did not appear to utilize alpha 9 beta 1 for attachment to the extracellular matrix proteins fibronectin, laminin, vitronectin, fibrinogen, thrombospondin, or type I or IV collagen. However, in contrast to mock transfectants, both 293 cells and SW480 cells expressing alpha 9 beta 1 adhered to intact chicken tenascin. By utilizing a variety of recombinant fragments of tenascin, we were able to localize the binding site for alpha 9 beta 1 to the third type III repeat. This repeat contains the arginine-glycine-aspartic acid (RGD) tripeptide that has been shown to serve as a binding site in tenascin for alpha v-integrins. However, the RGD site does not appear to be the binding site for alpha 9 beta 1, as the attachment of alpha 9 transfectants to this fragment was not inhibited by RGD peptide, nor by changing the RGD site to RAD or RAA.

Amino Acid Sequence↗
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

Functional role of cytotactin/tenascin in morphogenesis: a modest proposal.

The evidence for a functional role of cytotactin/tenascin (CT/TN) in several developmental processes with particular emphasis on those related to the nervous system are reviewed. CT/TN gene regulation, as well as the growing family of TN-related genes, is briefly discussed. Finally, I will explore the possibility that gene disruption experiments may not provide a unique resolution of gene function that was initially predicted when it became possible to manipulate mammalian genes with relative ease. It may be necessary to reevaluate the idea that these knockout experiments provide a definitive answer to the question of the function of a gene product. Cautious interpretation should be exercised in view of the large number of variables that can operate during embryogenesis, the existence of compensatory mechanisms during regulative development, and the pleiotropy resulting from mutation or deletion of a single gene. The reader should consider this a modest proposal, not a dogmatic one.

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↗
PubMed1992

Immunocytochemical demonstration of neural cell adhesion molecule (NCAM) along the migration route of luteinizing hormone-releasing hormone (LHRH) neurons in mice.

Contact between the developing forebrain and the ingrowing central processes of the olfactory, vomeronasal and terminal nerves is preceded by a migration of neural cell adhesion molecule (NCAM)-immunoreactive cells from the epithelium of the olfactory pit and the formation of an NCAM-immunoreactive cellular aggregate in the mesenchyme between the olfactory pit and the forebrain. The axons of the olfactory, vomeronasal, and terminal nerves, also NCAM-immunoreactive, grow into the cellular aggregate, which as development proceeds, becomes continuous with the rostral tip of the forebrain. The lateral and more rostral part of the cellular aggregate receives the ingrowing axons of the olfactory nerves and becomes the olfactory nerve layer of the olfactory bulb. The medial, more caudal part receives the central processes of the vomeronasal and terminal nerves. The vomeronasal nerve ends in the accessory olfactory bulb. The central processes of the terminal nerve end in the medial forebrain. Luteinizing hormone-releasing hormone (LHRH)-immunoreactive neurons, like the vomeronasal and terminal nerves, originate from the medial part of the olfactory pit. These LHRH cells migrate into the brain along and within a scaffolding formed by the NCAM-immunoreactive axons of the vomeronasal and terminal nerves, and they are never seen independent of this NCAM scaffold as they cross the nasal lamina propria. The results suggest that: (1) NCAM is likely to be necessary for scaffold formation, and (2) the scaffold may be essential for the subsequent migration of LHRH neurons into the brain. Because they aggregate, migrating LHRH-immunoreactive neurons, on which we did not detect NCAM immunoreactivity, may interact via other cell adhesion molecules (CAM). Inasmuch as the interaction between the LHRH-immunoreactive neurons and the NCAM-immunoreactive scaffold is heterotypic, the possibility of a heterophilic (NCAM to other CAM) interaction is not ruled out. These findings focus our attention on the functional role of NCAM in this migratory system.

Animals↗
PubMed1992

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↗
PubMed1992

Characterization of multiple adhesive and counteradhesive domains in the extracellular matrix protein cytotactin.

The extracellular matrix molecule cytotactin is a multidomain protein that plays a role in cell migration, proliferation, and differentiation during development. To analyze the structure-function relationships of the different domains of this glycoprotein, we have prepared a series of fusion constructs in bacterial expression vectors. Results obtained using a number of adhesion assays suggest that at least four independent cell binding regions are distributed among the various cytotactin domains. Two of these are adhesive; two others appear to be counteradhesive in that they inhibit cell attachment to otherwise favorable substrates. The adhesive regions were mapped to the fibronectin type III repeats II-VI and the fibrinogen domain. The morphology of the cells plated onto these adhesive fragments differed; the cells spread on the fibronectin type III repeats as they do on fibronectin, but remained round on the fibrinogen domain. The counteradhesive properties of the molecule were mapped to the EGF-like repeats and the last two fibronectin type III repeats, VII-VIII. The latter region also contained a cell attachment activity that was observed only after proteolysis of the cells. Several cell types were used in these analyses, including fibroblasts, neurons, and glia, all of which are known to bind to cytotactin. The different domains exert their effects in a concentration-dependent manner and can be inhibited by an excess of the soluble molecule, consistent with the hypothesis that the observed properties are mediated by specific receptors. Moreover, it appears that some of these receptors are restricted to particular cell types. For example, glial cells bound better than neurons to the fibrinogen domain and fibroblasts bound better than glia and neurons to the EGF fragment. These results provide a basis for understanding the multiple activities of cytotactin and a framework for isolating different receptors that mediate the various cellular responses to this molecule.

Animals↗
PubMed1991

Cytotactin binding: inhibition of stimulated proliferation and intracellular alkalinization in fibroblasts.

Cytotactin is an extracellular matrix protein that is dynamically and transiently expressed in a place-dependent fashion during development by glial cells, fibroblasts, and several other cell types. In the present study, the effects of cytotactin on cell proliferation were examined in fibroblastic cells in culture. NIH 3T3 mouse cells plated on tissue culture substrata in the presence of soluble cytotactin remained rounded for longer periods than untreated control cells, similar to their response to cytotactin-coated substrates. These rounding effects could be prevented by pretreatment of the cells with nocodazole, a microtubule-disrupting agent. Cytotactin inhibited the proliferation of fibroblasts in culture in a dose- and time-dependent manner, and this inhibition occurred even after nocodazole treatment. In addition, the presence of cytotactin inhibited proliferation stimulated by growth factors or tumor promoter. These effects on cell growth were accompanied by an early inhibition of the intracellular alkalinization that normally occurs upon mitogenic stimulation by a number of growth-promoting agents. Together these observations suggest that cytotactin is an endogenous cell surface modulatory protein and provide a possible mechanism whereby cytotactin may contribute to pattern formation during development, regeneration, tumorigenesis, and wound healing.

3T3 Cells↗
PubMed1991

Cytotactin expression in somites after dorsal neural tube and neural crest ablation in chicken embryos.

The spatiotemporal expression of the extracellular matrix protein cytotactin/tenascin during somitogenesis suggests that it plays a role in the morphogenetic events that give rise to the pattern of neural crest (NC) development. In the present study, the spatial distribution and molecular forms of cytotactin in somites were examined using in situ hybridization, Western blotting, and immunohistochemistry during normal development and after injury. In situ hybridization showed that prior to NC cell invasion cytotactin mRNA was restricted to the caudal half of the newly formed epithelial somites. As each epithelial somite matured, giving rise to a sclerotome and dermamyotome, the mRNA was first restricted to the dermamyotome and later restricted to the rostral protion of the sclerotome, consistent with the previously reported protein distribution. Immunocytochemical analysis of the distribution of cytotactin and NC cells in embryos with ablations that removed NC cells, or with simple wounds that left NC cells in place, demonstrated that the presence of NC cells is neither necessary nor sufficient for the correct positioning of cytotactin. Immunoblotting analysis showed that cytotactin synthesized by sclerotomes in the absence of NC cells was of similar molecular mass to that produced in their presence. These findings are in accord with the notion that the abnormalities of cytotactin distribution are related to the wounding process. We conclude that, contrary to the suggestion of Stern et al. [Stern, C. D., Norris, W. E., Bronner-Fraser, M., Carlson, G. J., Faissner, A., Keynes, R. J. & Schachner, M. (1989) Development 107, 309-319], there is no causal link between the presence of NC cells and the distribution and molecular mass of sclerotomal cytotactin.

Animals↗