A plasma membrane integral sialoglycoprotein (Sgp 130) molecularly distinguishes nonjunctional dense plaque sites of microfilament attachment

An integral sialoglycoprotein with Mr approximately 130,000 (Sgp 130) and highest expression in adult chicken gizzard smooth muscle has been recently identified as an excellent candidate for classification as a plasma membrane protein natively associated (directly or indirectly) with actin microfilaments (Rogalski, A.A., and S.J. Singer, 1985, J. Cell Biol., 101:785-801). In this study, the relative in situ distributions of the Sgp 130 integral species (a designation that also includes non-smooth muscle molecular forms) and the peripheral protein, vinculin, have been simultaneously revealed for the first time in selected cultured cells and tissues abundant in microfilament-membrane attachment sites, particularly, smooth and cardiac muscle. Specific antibody probes against Sgp 130 (mouse mAb 30B6) and vinculin (affinity-purified rabbit antibody) were used in double indirect immunofluorescent and immunoelectron microscopic experiments. In contrast to the widespread distributions of vinculin at microfilament-membrane attachment sites, Sgp 130 has been shown to exhibit striking site-specific variation in its abundancy levels in the plasma membrane. Sgp 130 and vinculin were found coincidentally concentrated at focal contact sites in cultured chick embryo fibroblasts and endothelial cells, membrane dense plaques of smooth muscle, and sarcolemma dense plaque sites overlying the Z line in cardiac muscle. However, at the fascia adherens junctional sites of cardiac muscle where vinculin is sharply confined, Sgp 130 was immunologically undetectable in both intact and EGTA-uncoupled tissue. This latter result was confirmed with immunoblotting experiments using isolated forms of the fascia adherens. The double immunolabeling studies of this report establish Sgp 130 as a major integral protein component of nonjunctional membrane dense plaque structures and raise the possibility that the 130-kD integral sialoglycoprotein (Sgp 130) and vinculin assume stable transmembrane associations at these particular microfilament-membrane attachment sites. Nonjunctional dense plaques are further suggested to be a molecularly distinct class of plasma membrane structures rather than a subgroup of adherens junctions. Our data also support a hypothesis that Sgp 130 is involved in plasma membrane force coupling events but not in junctional-related cell-cell coupling.     

Identification of a novel T cell surface disulfide-bonded dimer distinct from the alpha/beta antigen receptor

Hybridomas were prepared from the spleen of a BALB/c mouse immunized with EL-4 T lymphoma cells. One, designated A1, was found to secrete a monoclonal antibody that reacted with two T lymphoma cells of C57BL origin, EL-4 and C6VLB, but not with normal C57BL/6 splenocytes or thymocytes, C57BL/6 T cell clones, or other T or B lymphomas by complement-mediated cytotoxicity or indirect immunofluorescent staining. Monoclonal antibody (MAb) A1 precipitated a protein that migrated at 85 kD under nonreducing and 43 kD under reducing conditions. The fact that the antigen defined by MAb A1 was a disulfide-linked dimer, together with the essentially clone-specific distribution of the reactive epitope, raised the possibility that the antibody defined an epitope of the antigen receptor. However, several additional observations revealed that the antibody defined a distinct and novel T cell surface structure. MAb 124-40, previously shown to react with the antigen receptor of C6VLB cells, reacted with variants of C6VLB that failed to express the A1 epitope. Sequential immunoprecipitation indicated that MAb A1 and MAb 124-40 reacted with distinct molecular species on C6VLB cells. Endoglycosidase digestion showed that the structure reactive with MAb A1 was not derived from that reactive with MAb 124-40 by addition of N-linked oligosaccharide residues. Two-dimensional gel electrophoretic analysis of precipitates obtained from radioiodinated C6VLB cells with MAb 124-40 resolved the alpha and beta subunits of the antigen receptor. Similar analysis of precipitates obtained with MAb A1 revealed only a single basic chain under reducing conditions, although anomalous mobility suggestive of a second, more acidic chain was observed under nonreducing conditions. Two-dimensional maps of tyrosine-containing chymotryptic peptides of the proteins isolated with MAb A1 and MAb 124-40 were completely different, suggesting that the molecules shared no peptides and were distinct in primary structure. Finally, cross-linking studies performed with a cleavable reagent indicated that the A1 molecule, unlike the antigen receptor defined with MAb 124-40, was not associated with additional, T3-like structures on the surface of C6VLB cells. Although the MAb A1 was unreactive with normal cells in cytotoxicity or staining assays, a molecule of the appropriate size was immunoprecipitated in small amounts from lysates of radioiodinated normal spleen and thymus cells. These data indicate that MAb A1 defines a novel disulfide-linked T cell surface molecule distinct from the antigen receptor.     

An integral membrane protein antigen associated with the membrane attachment sites of actin microfilaments is identified as an integrin beta-chain.

A monoclonal antibody (MAb 30B6) was recently described by Rogalski and Singer (J. Cell Biol. 101:785-801, 1985) which identified an integral membrane glycoprotein of chicken cells that was associated with a wide variety of sites of actin microfilament attachments to membranes. In this report, we present a further characterization of this integral protein. An immunochemical comparison was made of MAb 30B6 binding properties with those of two other MAbs, JG9 and JG22, which identify a component of a membrane protein complex that interacts with extracellular matrix proteins including fibronectin. We showed that the 110-kilodalton protein recognized by MAb 30B6 in extracts of chicken gizzard smooth muscle is identical, or closely related, to the protein that reacts with MAbs JG9 and JG22. These 110-kilodalton proteins are also structurally closely similar, if not identical, to one another as demonstrated by 125I-tryptic peptide maps. However, competition experiments showed that MAb 30B6 recognizes a different epitope from those recognized by MAbs JG9 and JG22. In addition, the 30B6 antigen is part of a complex that can be isolated on fibronectin columns. These results together establish that the 30B6 antigen is the same as, or closely similar to, the beta-chain of the protein complex named integrin, which is the complex on chicken fibroblast membranes that binds fibronectin. Although the 30B6 antigen is present in a wide range of tissues, its apparent molecular weight on gels varies in different tissues. These differences in apparent molecular weight are due, in large part, to differences in glycosylation.     

Immunocytochemical localization of 140 kD cell adhesion molecules in cultured chicken fibroblasts, and in chicken smooth muscle and intestinal epithelial tissues

A monoclonal antibody (JG22 MAb) that was previously raised to a chick embryo myogenic cell preparation had been shown to produce rounding and other morphological changes in myogenic cells in culture, and, in some cases, their detachment from the substratum. In other studies it was shown that the epitope recognized by JG22 was associated with a set of 140 kD cell surface glycoproteins. It is shown that this antigen occurs in a wide variety of cell types; in cultured fibroblasts, it is distributed equally between the dorsal and ventral cell surfaces shortly after plating, but appears to become concentrated on the ventral surface as cell spreading proceeds; by immunoelectron microscopic labeling experiments, it is absent from the focal adhesion contact sites formed by fibroblasts with their substrata and with one another, but is present in clusters at the edge of focal adhesions, and within the close contact sites and extracellular matrix contact sites; in smooth muscle cells, it is absent from the membrane-associated dense plaques, but is located in clusters at adjacent membrane sites; in intestinal epithelium, it is present in clusters at the basolateral membranes, but not at the microvilli or within junctional complexes of the brush border of the cell layers. These and other results are consistent with the suggestion that the antigen recognized by JG22 MAb is important cell adhesion molecules, and performs a characteristic function in a variety of cell-cell contacts and cell adhesions.     

Membrane glycoproteins involved in neurite fasciculation

Lectin affinity chromatography combined with mAb production was used to identify chick neural cell surface molecules related to L1 antigen, a mouse neural glycoprotein implicated in cell-cell adhesion (Rathjen, F. G., and M. Schachner, 1984, EMBO (Eur. Mol. Biol. Organ.) J., 3:1-10). A glycoprotein, G4 antigen, isolated by mAb G4 from adult chick brain is described which comprises a major 135-kD component, a minor doublet at 190 kD, and diffusely migrating bands at 80 and 65 kD in SDS PAGE. This molecule is structurally related to mouse L1 antigen according to NH2-terminal amino acid sequence (50% identity) as well as the behavior of its components in two-dimensional IEF/SDS PAGE gels. A second chicken glycoprotein, F11 antigen, was isolated from adult chick brain using mAb F11. This protein has also a major 135-kD component and minor components at 170 kD and 120 kD. Both immunotransfer analysis with polyclonal antibodies to mAb G4 and to mAb F11 isolate and the behavior on IEF/SDS PAGE gels indicates that the major 135-kD component of F11 antigen is distinct from G4 antigen components. However, the 135-kD component of F11 antigen shares with G4 antigen and the neural cell adhesion molecule (NCAM) the HNK-1/L2 carbohydrate epitope. In immunofluorescence studies, G4 and F11 antigenic sites were found to be associated mainly with the surface of process-bearing cells, particularly in fiber-rich regions of embryonic brain. Although Fab fragments of polyclonal antibodies to mAbs G4 or F11 immunoaffinity isolate only weakly inhibit the Ca2+-independent aggregation of neural cells, they strongly inhibit fasciculation of retinal axons. Together these studies extend the evidence that bundling of axons reflects the combined effects of a group of distinct cell surface glycoproteins.     

Differential localization within human kidney of five membrane proteins expressed on acute lymphoblastic leukemia cells

The reactivity of a panel of monoclonal antibodies (MAb) produced against non-T, non-B acute lymphoblastic leukemia cells was investigated by immunoperoxidase staining of sections of normal human kidney. The antigens of kidney reactive with the MAb were isolated by immunoaffinity chromatography and were purified further by immunoprecipitation. Two MAb, 44D7 and 44H9, reacted with determinants found exclusively on the basolateral membranes of proximal convoluted tubules. The 44D7 antigen isolated from kidney was biochemically similar to that isolated from leukemic cells. It was resolved as a multimeric complex with an apparent m.w. of 120,000 when analyzed by SDS-PAGE under nonreducing conditions. The 44H9 antigen has not yet been purified from kidney. MAb 50B4 reacted with components of the interstitium and with the mesangium of glomeruli. It immunoprecipitated a polypeptide chain of apparent m.w. 85,000, similar to that of the 50B4 antigen isolated from leukemic cells. MAb 44G4 also reacted with the mesangium of glomeruli and with the interstitium of the kidney. However, the endothelium of glomerular capillaries and of interstitial blood vessels has also reacted with MAb 44G4. The kidney antigen recognized by MAb 44G4 was characterized as a major polypeptide band, 95,000 m.w. (reduced) and 125,000 m.w. (nonreduced), a subunit structure analogous to the 44G4 antigen isolated from leukemic cells. MAb 44E3 reacted with all cellular elements of glomeruli, tubules, blood vessels, and interstitium. Two polypeptide chains of apparent m.w. 94,000 and 90,000 were immunoprecipitated from kidney by MAb 44E3, while a single polypeptide chain of 94,000 m.w. was precipitated from leukemic cells. Our results describe five new antigens with distinctive cellular distributions within kidney.     

A defective cell surface collagen-binding protein in dermatosparactic sheep fibroblasts

Fibroblasts from dermatosparactic sheep fail to contract collagen gels and show a reduced attachment to collagenous substrates. By comparing collagen-binding membrane proteins of normal (+/+), homozygote (-/-), and heterozygote (+/-) fibroblasts, we present evidence that the interaction of normal fibroblasts with native type I collagen involves a protein of apparent Mr = 34,000 which is absent from dermatosparactic fibroblasts and seems to be related to anchorin CII. This conclusion was reached from the following experiments: (a) On a blot of membrane proteins from normal fibroblasts radioactively labeled type I collagen bound predominantly to a protein band of 34 kD; dermatosparactic membranes revealed only a small amount of binding to a component with a molecular mass of 47 kD. (b) After separation of normal fibroblast membrane proteins on type I collagen-Sepharose, a collagen-binding component of 34 kD was found which was absent from the corresponding fraction of dermatosparactic membranes. (c) Antibodies to anchorin CII stained the surface of normal (+/+), but not of dermatosparactic (-/-) fibroblasts and labeled a 34-kD component after immunoblotting of normal fibroblast membrane proteins. (d) After metabolic labeling of fibroblasts with [35S]methionine and immunoprecipitation with anti-anchorin CII, 40- and 34-kD components were precipitated from extracts of normal fibroblasts, while the latter component was absent from affected cells. Similar differences were found after immunoblotting of membranes from whole normal or affected skin. These data indicate that dermatosparaxis of sheep involves a molecular defect of a collagen-binding protein. Therefore this disease represents a model to study the complex interaction of cells with the extracellular matrix on a molecular level.     

Monoclonal antibodies against species-specific antigens in the chick central nervous system: putative application as transplantation markers in the chick-quail chimera

With the recent progress in transplantation of neuronal tissues, cellular markers are needed to distinguish the grafted cells from the host. To generate monoclonal antibodies (MAb) recognizing species-specific antigens in the chick nervous system, we immunized mice with chick optic nerves and obtained 2 MAb which bind to chick but not to quail neural tissues. MAb-39B11 recognizes the cell surface antigen on the nerve fibers. MAb-37F5 recognizes the cytoplasmic components in several cell types, including ependymal cells and some large neurons. The utility of these MAb as markers for chick cells in the chick-quail chimeric brain and their advantages over conventional markers are discussed.     

THE DISTRIBUTION, ULTRASTRUCTURE, AND CHEMISTRY OF MICROFILAMENTS IN CULTURED CHICK EMBRYO FIBROBLASTS

The distribution, ultrastructure, and chemistry of microfilaments in cultured chick embryo fibroblasts were studied by thin sectioning of flat-embedded untreated and glycerol-extracted cells, histochemical and immunological electron microscopic procedures, and the negative staining of cells cultured on electron microscopic grids. In these cultured cells, the microfilaments are arranged into thick bundles that are disposed longitudinally and in looser arrangements in the fusiform-shaped cells. In the latter case, they are concentrated along the margins of the flattened cell, on the dorsal surface, and particularly at the ends of the cell and its ventral surface, where contact is made with the plastic dish or with other cells. Extracellular filaments, presumably originating from within the cell, are found at these points of contact. The microfilaments are composed in part of an actin-like protein. These filaments are between 70 and 90 Å in diameter, they are stable in 50% glycerol, they have an endogenous ATPase (myosin-like?) associated with them, they bind rabbit muscle heavy meromyosin, and they specifically bind antibody directed against isolated actin-like protein. In the cultured chick embryo fibroblasts, the microfilaments are essential for the establishment and maintenance of form, and they are probably critical elements for adhesion and motility. The microfilaments might also serve as stabilizers of intramembranous particle fluidity.     

Role of the L3T4 antigen in T-cell activation. I. Description of a monoclonal IgM antibody to a distinct epitope (L3T4b) of the L3T4 antigen and its effect on interleukin 1-induced thymocyte proliferation

This report describes a new rat monoclonal IgM/k antibody, monoclonal antibody (MAb) 2B6, which reacts with a cell surface antigen present on a subpopulation of both thymocytes (85%) and peripheral T lymphocytes (55-60%). The antigen recognized by MAb 2B6 has multiple properties in common with the L3T4 antigen, as defined by the recently described MAb GK1.5. Thus, MAb 2B6 and MAb GK1.5 give very similar flow cytometry staining patterns on thymocytes, purified spleen T cells and all tested T-cell hybridomas. Depletion of MAb 2B6-positive cells with antibody and complement led to simultaneous depletion of MAb GK1.5-positive cells, and vice versa. Depletion of Lyt 2-positive cells led to enrichment of both MAb 2B6- and MAb GK1.5-positive cells. Both MAb 2B6 and MAb GK1.5 immunoprecipitate the same pattern of cell surface molecules from detergent extracts of radiolabeled thymocytes, the main components being a 55-kDa and a 115-kDa band. We therefore conclude that MAb 2B6 reacts with the L3T4 antigen. Interestingly, MAb 2B6 and MAb GK1.5 do not cross-block and therefore most probably react with distinct epitopes on the L3T4 molecule. The determinant recognized by MAb GK1.5 is called L3T4a. We suggest that the determinant recognized by MAb 2B6 be named L3T4b. As MAb 2B6 was selected for its ability to inhibit the action of interleukin 1 (IL-1) in the thymocyte costimulator assay, it is likely that the L3T4 molecule is functionally involved in the events taking place during IL-1 induction of thymocyte proliferation.     

Changes in Expression of Fibroblast Surface Antigen (SFA) during Cytodifferentiation and Heterokaryon Formation

Fibroblast surface antigen (SF antigen, SFA) is a major glycoprotein antigen detected in connective tissue cells (primitive mesenchymal cells, fibroblasts, and astroglial cells). In this study the expression of SFA was followed during differentiation of the mesenchymal cells of the mouse metanephros and during heterokaryon formation produced by Sendai-virus induced fusion of human fibroblasts and chick red blood cells. It was demonstrated by immunofluorescence that SFA was lost from the kidney mesenchymal cells when they differentiate into epithelial cells of the secretory tubuli. During this process SFA became detectable in the basement membrane formed around the tubuli. In cell fusion experiments human SFA which was present as fibrillar network on the surface of cultured fibroblasts, was gradually lost from the heterokaryons when the incorporated chick nuclei became activated. These two sets of experiments indicate that SFA can be used as a phenotypic marker of Cytodifferentiation.     

Antibody mapping of functional domains in vinculin.

We have analyzed the functional domain structure of vinculin, a protein involved in linking microfilaments to the cytoplasmic face of cell membranes in animal cells. For this purpose, we used several monoclonal antibodies raised against chicken gizzard vinculin whose epitopes could be assigned to discrete regions in the vinculin sequence by immunoblotting of proteolytic fragments combined with N-terminal amino acid sequencing. Two of these antibodies induced the disruption of stress fibers and changed the number of morphology of focal contacts after microinjection in chicken embryo fibroblasts. Based on the location of its epitope in comparison with vinculin domains previously identified by other groups, we propose that one of these antibodies (15B7) interferes with the binding of vinculin to talin, the most peripheral of the microfilament proteins. The second antibody (14C10) binds within a region comprising three internal repeats and might therefore distort the inner architecture of vinculin. A third antibody (As3) inhibited the binding of F-actin to vinculin in an in vitro assay but had no effect on the microfilament system in cells. These data emphasize the role of vinculin as a key protein in microfilament-membrane linkage and support previous work on a direct interaction between vinculin and actin.     

Is there a role for actin in virus budding?

Electrophoretic data from both sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and acid-urea gels reveal a protein in purified murine mammary tumor virus (MuMTV) which co-migrates with purified chick skeletal muscle actin. 125I-labeling of intact and disrupted virus preparations shows that the actin-like protein is not artifactually adsorbed to the outside of virions during isolation. Quantitative SDS- PAGE and examination of negatively stained preparations show that the actin cannot be accounted for by a contaminating population of virus- free vesicles. The ultrastructure of mammary epithelial cells and of Rous sarcoma virus-transformed chick embryo fibroblasts shows that virus extrusion is associated with filament-containing cellular processes. In particular, MuMTV is released from the ends of long microvilli which contain a bundle of 6-8-nm microfilaments and share other structural features with intestinal microvilli. We suggest that virus nucleoids require an interaction with host cell contractile proteins for their extrusion from the cell.     

Expression of the rat CD26 antigen (dipeptidyl peptidase IV) on subpopulations of rat lymphocytes.

The T cell activation antigen CD26 has been recently identified as the cell surface ectopeptidase dipeptidyl peptidase IV (DPP-IV). DPP-IV is found on many cell types, including lymphocytes, epithelial cells, and certain endothelial cells. The MRC OX61 monoclonal antibody (MAb) which specifically recognises rat DPP-IV was used to examine the expression of CD26/DPP-IV on rat lymphocytes. The molecular nature of the antigen was examined by immunoprecipitation from thymocytes, splenocytes, and hepatocytes. Analysis by one- and two-dimensional gel electrophoresis indicated that the native form of CD26 includes a 220-kDa homodimer. On tissue sections MRC OX61 MAb stained nearly all thymocytes and in the spleen and lymph nodes predominantly stained the T cell areas. However, in immunofluorescence experiments OX61 stained 80 to 87% of lymph node cells and 78 to 85% of spleen cells. Furthermore, two-colour immunofluorescence analysis of the CD4+, CD8+, and Ig+ lymphocyte subsets indicated that only 2 to 5% of each of these subsets lacked OX61 staining. Spleen cells and thymocytes of both CD4+ and CD8+ subsets stained much more intensely with OX61 after these cells were stimulated with phytohemagglutinin. These findings indicate that rat CD26 antigen expression is not confined to the T cell population as has been suggested, but also occurs on B cells, and is increased on T cells following their activation.     

A monoclonal antibody that recognizes a basolateral membrane protein in A6 epithelial cells

The A6 cell line is a model for tight epithelia and studies of epithelial polarity. Monoclonal antibodies (MAbs) were produced by immunization of mice with intact A6 cells and fusion of spleen cells to generate hybridomas. Hybridoma supernatants were screened by ELISA to select MAbs binding to the apical membrane of confluent A6 cells. Localization of MAb binding was examined by indirect immunofluorescence using cross sections of A6 monolayers grown on collagen coated filters. One MAb, designated 13F12, was positive by apical surface ELISA but localized specifically to the basolateral membrane of cross sections of A6 monolayers on filters. Immunofluorescence labeling of confluent A6 cells grown on glass cover slips revealed that MAb 13F12 does not bind to the apical membrane, but binds to basolateral determinants in the regions of domes, where it appears able to penetrate cellular junctions. Subconfluent A6 cells express the antigen all over the cell surface. Cells approaching confluency express the antigen on the apical membrane of some cells but not others, and as the cells reach confluency, the antigen disappears from the apical surface, and the cells become fully polarized. A6 cells at confluency on glass cover slips are equally polarized as cells grown on filters with respect to this antigen. The antigen has been identified by immunoprecipitation as a 22 kDa protein. High concentrations of MAb 13F12 did not inhibit cell plating, indicating that the antigenic site is not directly involved in cell adhesion to the substrate. MAb 13F12 should prove to be a useful tool to study many aspects of epithelial polarity, including the signals involved in sorting of proteins to specific membrane domains.     

Identification of fibronectin receptors on T lymphocytes

We report the identification of fibronectin receptors on thymocytes and T lymphoma cells. Affinity chromatography of extracts of the T cell lymphoma, WR16.1, on a fibronectin-Sepharose column combined with specific elution using a synthetic peptide containing the cell attachment-promoting sequence, arginine-glycine-aspartic acid, yielded two polypeptide components having apparent molecular masses of approximately 160 kD reduced and 175 and 150 kD nonreduced. Immunoprecipitations from surface-iodinated WR16.1 cells or fibronectin-adherent thymocytes using a rabbit antiserum raised against the fibronectin receptor that is present on human fibroblasts revealed, in each case, the same two radiolabeled components. In contrast, immunoprecipitation from fibronectin-nonadherent T lymphoma cells, designated WR2.3, revealed the presence of only the smaller subunit. Although the lymphocyte receptor and the fibronectin receptor identified on fibroblasts share immunologic determinants, they differ in that the molecular mass of the lymphocyte protein is larger. Moreover, trypsinization of either thymocytes or the WR16.1 T lymphoma cells resulted in a subsequent loss of their ability to adhere to fibronectin-coated substrates and a reduction in the electrophoretic mobility of each of the polypeptide chains of the fibronectin receptor present on their surfaces. These changes, however, were not observed with normal rat kidney fibroblasts or mouse 3T3 fibroblasts in response to trypsinization. The data establish the existence on normal lymphocytes of fibronectin receptors that are quite similar to those found on fibroblasts. The possible function of this molecule on thymocytes is discussed.     

Circulating parasite antigen(s) in lymphatic filariasis: use of monoclonal antibodies to phosphocholine for immunodiagnosis

Hybridoma cell lines producing monoclonal antibodies (MAb) against a 200 kD antigen found circulating in the sera of microfilaremic patients infected with Wuchereria bancrofti were obtained by immunizing mice with a partially purified antigen preparation. A sensitive MAb (CA101)-based ELISA for measuring circulating parasite antigen was capable of detecting antigen in the sera of 93% of patients with microfilaremia, 46% of those with lymphatic obstruction, and 56% of patients with tropical pulmonary eosinophilia syndrome. Circulating antigen was absent from sera of normal controls, and "false positives" were recorded in only two of 17 patients with nonfilarial helminth infections. By ELISA and immunoblot analysis, it was shown that three of the monoclonals raised to this 200 kD antigen were directed to epitopes of phosphocholine (PC). Two MAb (CA86, CA101) were identified as having the T15 idiotype previously associated with antibodies to the PC of pneumococcal teichoic acid; one was untypeable. All three of these anti-PC MAb reacted with adult, microfilaria, and larval antigen preparations, and by immunoblotting showed multiple banding patterns that indicated the presence of PC determinants on many different antigenic molecules. On the other hand, target antigens of CA101 which were found in the circulation of infected patients were limited to three species with apparent m.w. of 200, 160, and 78 kD. The 200 kD antigen was seen more frequently than the other two antigens. Other T15 anti-PC MAb derived from mice not immunized with filarial antigen showed similar patterns of reactivity with circulating filarial antigen.     

The Prosencephalon Has the Capacity to Differentiate into the Optic Tectum: Analysis by Chick-Specific Monoclonal Antibodies in Quail-Chick-Chimeric Brains

The alar plate of the prosencephalon of the quail embryo was heterotopically transplanted into the alar plate of the mesencephalon of the chick embryo at the 7–10 somite stage. Chick and quail cells in chimeric brains were distinguished after Feulgen-Rossenbeck staining and/or immunohistochemical staining with a species specific monoclonal antibody MAb-37F5 which recognized cytoplasmic components of chick brain cells. Neural connections between the transplant and the host were studied by monoclonal antibodies, MAb39-B11, which recognizes a species specific antigen on chick nerve fibers, and MAb-29B8, which reacts to 160 kD neurofilaments of both chick and quail.
When the transplant was completely integrated into the host mesencephalon, the transplant developed a laminar morphology closely resembling that of the optic tectum. Immunohistochemical staining with MAb-39B11 showed that the host optic nerve fibers innervated both the host tectum and the tectum-like transplant. However, optic nerve fibers did not invade transplants that failed to develope a laminar structure characteristic of the tectum. These findings suggest that the prosencephalon has a capacity to differentiate into the optic tectum at the 7–10 somite stage.     

Immunochemical characterization of two antigens recognized by new monoclonal antibodies against human colon carcinoma

Two different monoclonal antibodies (MAb), called L-D1 and L-C5, were produced after immunization with either intact cells or the methanol phase of glycolipid extracts, respectively, from the same human colon carcinoma line, LoVo. As determined by an antibody-binding radioimmunoassay (RIA) on intact cells, MAb L-D1 and MAb L-C5 were highly reactive with all five colon carcinoma lines tested and with only one out of the 21 cell lines of various tissue origin tested. No reactivity of either MAb was observed with peripheral blood lymphocytes, granulocytes, or erythrocytes from healthy donors of various blood groups. Both MAb were tested in competitive binding experiments with an anti-CEA MAb from our laboratory (CEA 35) and with two previously described anti-colon carcinoma MAb from the Wistar Institute called 1083-17-1A (17-1A) and NS-19.9. In competitive binding experiments, MAb L-D1 was inhibited by MAb 17-1A and reciprocally, whereas MAb L-C5 was not inhibited by any of the other MAb tested. MAb L-D1 precipitated a major protein band with an apparent molecular weight (MW) of 41 kilodaltons (kD); interestingly, MAb 17-1A, which was reported to react with an uncharacterized antigen, precipitated the same protein band of 41 kD. This was confirmed with immunodepletion experiments. Furthermore, after treatment of the colon carcinoma cell line with tunicamycin, both MAb L-D1 and 17-1A precipitated a protein band of 35 kD. This shift of 6 kD suggests that the glycoprotein recognized by these 2 MAb contains two to three N-linked carbohydrate side chains. MAb L-C5 precipitated a group of three to four protein bands ranging from 43 to 53 kD that were not modified by tunicamycin treatment. A preliminary study conducted by using immunoperoxidase labeling on frozen sections of primary colon carcinoma showed that the two new MAb react strongly with these tumors, but also weakly with the normal adjacent mucosa, as did the other anti-colon carcinoma MAb tested.     

Isolation of focal contact membrane using saponin

The fragments of lower cell surface remained attached to the substrate after incubation of mouse or chick fibroblasts in 0.2% saponin solution and subsequent removal of cells under the action of shearing force. These fragments corresponded exactly to the cellular focal contacts seen by interference reflection microscopy. Ultrastructurally they were membrane fragments with typical three-layered structure. No cytoskeletal components were found in saponin-isolated focal contact membranes either by immunofluorescence or electron microscopy. Only one major cell-derived protein with an apparent molecular weight (MW) of 51 kD (chick embryo fibroblasts) or 47 kD (mouse embryo fibroblasts) remained on the substrate after saponin treatment and removal of cells.