TAG fixation of normal and Ca2+ ionophore-treated rabbit polymorphonuclear leukocytes (PMN) has revealed membrane components not apparent with conventional glutaraldehyde fixation. These included a 30 A external electron-dense coating on untreated cells. A somewhat thicker coat (40 A) was observed in ionophore-treated, nondegranulating PMN. In ionophore-treated, degranulating PMN, a 65 A cell membrane coat was observed. A similar coat was observed on the inner side of the membrane of some azurophil-type granules, but the electron density and thickness were not so pronounced. Cytoplasmic granules were often closely apposed and often protruded outward at the plasma membrane. Extracellular lamellae, sometimes stacked apposed to the plasma membrane, possibly represent remnants of intense granule extrusion. Sequential degranulation of the respective granules was not apparent.
Fluorescence photobleaching recovery techniques have allowed us to measure the lateral mobility of T-independent antigens bound to antigen-specific mouse B cells. The in vitro immunogenicity or tolerogenicity of antigens we have examined, DNP-polymerized flagellin (DNP-POL), and DNP-linear dextran (DNP-DEX), depend upon the antigen dose and epitope density. These factors also determine the mobility of antigen bound to B cell surfaces. For DNP-POL bound to DNP-specific cells, the observed diffusion constants D decrease monotonically with increasing antigen dose and epitope density. Values of D range from 10.4 X 10(-11) cm2 sec-1 for DNP0.4-POL at 0.15 microgram/ml to 0.8 X 10(-11) cm2 sec-1 for DNP3.5-POL at 30 microgram/ml. For receptor bound DNP-DEX, D depends strongly on antigen epitope density but not observably on antigen concentration. For epitope densities of 1.2 or less, D is close to the value of 21 X10(-11) cm2 sec-1 observed for single sIg receptors. By an epitope density of 4.8, D has fallen to 2.1 X 10(-11) cm2 sec-1. Peak immunogenicities for DNP-POL and DNP-DEX are observed when antigen-receptor aggregates have mobilities 14-fold and 3-fold lower, respectively, than a single sIg molecule.
Avian myelocytomatosis virus (MC29), a defective acute leukemia virus, has a broad oncogenic spectrum in vivo, and transforms fibroblasts and hematopoietic target cells in vitro, We have used recombinant DNA technology to isolate and characterize the sequences that are essential in the transformation process. Integrated MC29 proviral DNA was isolated from a library of recombinant phage containing DNA from the MC29-transformed nonproducer quail cell line Q5. The cloned DNA was analyzed by Southern blotting of restriction endonuclease digests and by electron microscopic visualization of R-loops formed between the cloned DNA and MC29 or helper virus RNA. It was found that the 9.2 kb cloned DNA insert contains approximately 4 kb of viral sequences and 5.2 kb of quail cellular sequences. The viral sequences contain all of the MC29 specific sequences and 5' helper related sequences as well as part of the envelope region. The size of the cloned EcoRI fragment is the same as that of the major band in EcoRI-cleaved Q5 DNA that hybridizes to viral sequences. Transfection of the cloned DNA into NIH 3T3 cells revealed that the MC29-specific sequences are functional in that they induce foci of transformed cells with high efficiency.
Journal of Supramolecular Structure and Cellular BiochemistryVolume 17, Issue 4 p. 313-324 Invited Review Changes in cell surface glycoproteins and carbohydrate structures during the development and differentiation of human erythroid cells Minoru Fukuda, Minoru Fukuda Biochemical Oncology, Fred Hutchinson Cancer Research Center and Department of Pathobiology, School of Public Health and Community Medicine, University of Washington, Seattle, Washington 98104 Minoru and Michiko Fukuda are now at the La Jolla Cancer Research Foundation, La Jolla, CA 92037.Search for more papers by this authorMichiko N. Fukuda, Michiko N. Fukuda Biochemical Oncology, Fred Hutchinson Cancer Research Center and Department of Pathobiology, School of Public Health and Community Medicine, University of Washington, Seattle, Washington 98104 Minoru and Michiko Fukuda are now at the La Jolla Cancer Research Foundation, La Jolla, CA 92037.Search for more papers by this author Minoru Fukuda, Minoru Fukuda Biochemical Oncology, Fred Hutchinson Cancer Research Center and Department of Pathobiology, School of Public Health and Community Medicine, University of Washington, Seattle, Washington 98104 Minoru and Michiko Fukuda are now at the La Jolla Cancer Research Foundation, La Jolla, CA 92037.Search for more papers by this authorMichiko N. Fukuda, Michiko N. Fukuda Biochemical Oncology, Fred Hutchinson Cancer Research Center and Department of Pathobiology, School of Public Health and Community Medicine, University of Washington, Seattle, Washington 98104 Minoru and Michiko Fukuda are now at the La Jolla Cancer Research Foundation, La Jolla, CA 92037.Search for more papers by this author First published: 1981 https://doi.org/10.1002/jsscb.380170403Citations: 39 AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume17, Issue41981Pages 313-324 RelatedInformation
The distribution of two glial antigens (C1 and M1) has been studied by indirect immunofluorescence during postnatal development of the cerebella of normal and neurologically mutant mice (weaver, staggerer, reeler, Purkinje cell degeneration, and wobbler). During the first postnatal week of normal development, C1 antigen is expressed in ependyma. Bergmann glial fibers (BG), and astrocytes of the internal granular layer and white matter. After day 10, C1 antigen is restricted to BG and ependymal cells. During the second and third week. BG undergo a transient loss of C1 antigen that starts in medioventral areas and spreads in a gradient dorsally and laterally. In reeler, weaver, and staggerer, C1 antigen expression is normal during the first postnatal week, and subsides in BG in a similar spatial gradient as described for the normal littermates. However, the loss of C1 antigen in BG occurs earlier (first in reeler, then in weaver, and last in staggerer) and is not reversible as it is in normal mice. In Purkinje cell degeneration, C1 antigen expression is diminished in BG after the onset of behavioral abnormalities. Wobbler is normal with respect to C1 antigen expression at adult ages. M1 antigen is detectable in white matter astrocytes from postnatal day 7 on, and persists in these cells into adulthood. Astrocytes if the internal granular layer and BG express M1 antigen only transiently in normal mice during the second and third weeks. The appearance of M1 antigen in BG occurs in a spatiotemporal gradient, matching the one in which C1 antigen disappears. M1 antigen expression is abnormally maintained in BG of reeler, staggerer, and weaver. In Purkinje cell degeneration. M1 antigen is expressed abnormally at the onset of behavioral abnormalities first in astrocytes of the internal granular layer and, with growing age, increasingly also in BG. In wobbler, BG do not express M1 antigen. However, astrocytes of the granular layer are abnormally M1 antigen-positive.
The ability of human fibroblasts to repair bleomycin-damaged DNA was examined in vivo. Repair of the specific lesions caused by bleomycin (BLM) was investigated in normal cell strains as well as those isolated from patients with apparent DNA repair defects. The diseases ataxia telangiectasia (AT), Bloom syndrome (BS), Cockayne syndrome (CS), Fanconi anemia (FA), and xeroderma pigmentosum (XP) were those selected for study. The method used for studying the repair of DNA after BLM exposure was alkaline sucrose gradient centrifugation. After exposure to BLM, a fall in the molecular weight of DNA was observed, and after drug removal the DNA reformed rapidly to high molecular weight. The fall in molecular weight upon exposure to BLM was observed in all cells examined with the exception of some XP strains. Prelabeled cells from some XP complementation groups were found to have a higher percentage of low molecular weight DNA on alkaline gradients than did normal cells. This prelabeled low molecular weight DNA disappeared upon exposure to BLM.
In the present study we report for the first time a weaver (wv) gene dose effect on neuron survival and neurite formation in vitro. Dissociated cerebellar cells from postnatal 7- and 8-day-old normal (+/+), heterozygous weaver (+/wv) and homozygous weaver (wv/wv) mice were cultured as monolayers on poly-L-lysine coated glass. Cell death occurred rapidly in wv/wv cultures. Cell counts showed that less than 20% of the total neurons and neuronal precursors (identified by "birthday" radiolabeling techniques) survived by Day 3. Cell death was less extensive in +/wv cultures with 65% of the total neurons and 80% of the precursors surviving by Day 3. In contrast to wv/wv cultures, younger neurons survive better than the total population in +/wv cultures. The impairment of neurite formation over the first week is also proportional to the number of mutant genes as shown by quantitation of (a) the percentage of cells with neurites; (b) the percentage of cells with neurites of a given length class ith time; (c) the lengths of the longest process formed per cell. The mean longest neurite lengths obtained by computer digitization at 6 days in vitro were 41.8, 26.8, and 9.0 micron for +/+, +/wv, and wv/wv granule cells, respectively.
A peculiarity of nitrosamines is the high degree of cell and organ specificity in inducing tumors. There is substantial evidence that the initiation of the carcinogenesis process by carcinogens of this group is linked to the metabolic competence of the target tissue or cell to convert these carcinogens into mutagenic metabolites and to the binding of those metabolites to cellular DNA. Alkylation occurs in the DNA at the N-1, N-3, and N-7 positions of adenine; the N-3, N-7, and O6 of guanine; the N-3, and O2 of cytosine; and the N-3, O4, and O2 of thymine; and the phosphate groups. The initial proportion of each DNA adduct depends upon the alkylating agent used. The various DNA adducts are lost to a variable extent from DNA in vivo by spontaneous release of bases and/or by specific DNA repair processes. Studies conducted in vitro and vivo indicate that alkylation at the oxygen atoms of DNA bases is more critical than alkylation at other positions in the mutagenesis and carcinogenesis induced by N-nitroso compounds. In particular, tissues in which tumors occur more frequently after a pulse dose of nitrosamine are those in which O6-alkylguanine persists longest in DNA, presumably resulting in an increased probability that a miscoding event (mutation) will take place during DNA synthesis. The more rapid removal of O6-methylguanine from the DNA of liver (as compared with extrahepatic tissues) of rats has been associated with the absence of tumor production in this organ by a single dose of dimethylnitrosamine; however, a significant incidence of liver tumors is observed if the same dose is given 24 hr after partial hepatectomy, and tumors are induced by such a dose of dimethylnitrosamine in the liver of hamsters, which has a low capacity to remove O6-methylguanine from its DNA. These data also indicate that the rate of disappearance of 7-methylguanine from the liver or extrahepatic tissues is independent of the dose of dimethylnitrosamine; whereas O6-methylguanine is lost from DNA more rapidly after a low dose of this nitrosamine. It has been shown that in liver the removal of O6-methylguanine but not other DNA adducts, from DNA can be affected by pretreating the animals with N-nitroso compounds. The modulation of DNA repair processes observed after a single dose and after chronic treatment with nitrosamines is discussed in relation to the tissue-specific carcinogenic effect of this group of carcinogens.
This study was based on our previous findings that the mitogenic action of thrombin on cultured fibroblasts can result from interaction of thrombin with the cell surface in the absence of internalization, and that the proteolytic activity of thrombin is required for stimulation of cell division. This prompted us to look for thrombin-mediated cleavages using 2-dimensional gel electrophoresis of labeled cell surface proteins. Surface membrane components were labeled by 3 procedures: 1) proteins were labeled by lactoperoxidase-catalyzed iodination using 125I-; 2) galactose and galactosamine residues of glycoproteins were oxidized with galactose oxidase and reduced with 3H-NaBH4; and 3) glycoproteins were metabolically labeled by incubating cells with 3H-fucose. labeling with the first 2 procedures was carried out after thrombin treatment; in contrast, cells metabolically labeled with 3H-fucose were subsequently treated with thrombin to look for proteolytic cleavages. Collectively, these studies indicated that only about 5 cell surface proteins were thrombin-sensitive, consistent with the high specificity of this protease. Each of the labeling procedures revealed a thrombin-sensitive cell surface glycoprotein which was identified as fibronectin by immunoprecipitation experiments. In addition, cell surface proteins of about 140K and 55K daltons were thrombin-sensitive. However, cell surface proteins of about 45K daltons and 130K to 150K daltons were increased after thrombin treatment. These experiments were conducted on an established line of Chinese hamster lung cells with the eventual goal of studying thrombin-mediated cleavages of cell surface proteins in a large number or in cloned populations derived from this line that are either responsive or unresponsive to the mitogenic action of thrombin. This approach should permit identification of proteolytic cleavages tha are necessary for thrombin-stimulated cell division.
This brief review presents the salient features of new developments in the enzymatic repair of base damage to DNA. DNA glycosylases and apurinic/apyrimidinic (AP) endonucleases are reviewed and evidence is presented that in at least two prokaryote systems incision of UV-irradiated DNA occurs by the sequential action of these two classes of enzymes. In contradistinction, the uvrA, uvrB, and uvrC gene products of E coli appear to function as a multi-protein complex that catalyzes hydrolysis of phosphodiester bonds in damaged DNA directly. The inducible rapid repair of O6-methylguanine in E coli is also reviewed.
The synthesis and release of glycoproteins were studied in organ cultures of human breast surgical specimens and in established breast epithelial cell lines, MCF-7 and MDA-MB-231. Biosynthesis was monitored by the incorporation of 14C-glucosamine. Labeled macromolecules in the culture supernatants were analyzed by biochemical and immunological techniques. One to 8% of the labeled glycoproteins from benign breast and infiltrating ductal carcinoma specimens was precipitated by antibodies produced against human serum alpha-1-antichymotrypsin. Twelve percent of the total glycoproteins from the culture supernatants of the MCF-7 cell line was identified as alpha-1-antichymotrypsin. Both the normal serum and the human breast epithelia-derived proteinase inhibitor can be resolved into similar subclasses by two-dimensional gel electrophoresis. MDA-MD-231 and MCF-7 cells which were extensively washed with EDTA, serum-free medium, and phosphate-buffered saline retain this proteinase inhibitor on their cell surfaces. Three to 4% of the total cell-surface iodinated components was immunoprecipitated by these specific antibodies. Since alpha-1-antichymotrypsin is a potent inhibitor of neutral proteinases such as cathepsin G, the demonstration of its synthesis by benign and malignant breast epithelial cells is of considerable interest. This glycoprotein may represent the epithelia's own protective shield of cell surface components and the cell's attempt to moderate the effects of invading leukocytes. In addition, it may play a regulatory role in the maintenance of three-dimensional glandular structures.
The proteins fibronectin (FN), Von Willebrand factor (VWF), and fibrinogen are believed to play a role in platelet function. They are distributed between the plasma and the platelet pool in the resting state and undergo redistribution upon platelet activation. We have studied their expression on the surface of the platelet and their mobilization following platelet binding to substrata. For the purpose of studying protein expression on the surface of intact platelets either adherent to a substratum or in suspension, the enzyme-linked immunosorbent assay (ELISA) was elaborated and modified. Using this technique as well as immunofluorescence, we found that antiserum raised against carefully washed human platelets recognized FN, VWF, and fibrinogen as well as platelet surfaces. However, specific antisera against these three proteins failed to bind to the surface of unactivated gel-filtered platelets. When gel-filtered platelets were exposed to plastic or fibrillar collagen, they adhered and spread. Such platelets did bind antibodies against FN, VWF, and fibrinogen, Moreover, when the adherent platelets were incubated with FN or with VWF in the absence of ristocetin, they bound these proteins in a concentration-dependent fashion. The patterns of the bound proteins were not similar, suggesting a different spatial distribution of binding sites. These findings indicate that platelet activation by adhesion to substrata mobilize both endogenous and exogenous pools of these proteins, thereby making them surface associated and probable participants in further binding properties of the activated platelet.
Antibodies were raised against the purified aggregation factor from Geodia cydonium in order to clarify its function during cell aggregation in the homologous and heterologous system. These antibodies inhibited only cell aggregation in the homologous Geodia system and were inactive in the heterologous Tethya lyncurium system. These findings directly indicated that the species-specific reaggregation of sponge cells was initiated by the soluble aggregation factor as already assumed in earlier studies. The amount of neutralizing antibodies was determined by a precipitation reaction with the antigen in capillaries and by microdiffusion. By using the latter technique we got evidence that the Geodia aggregation factor contained a component that was antigenetically related to a galactose-specific lectin present in Geodia cydonium.
Both growth factor availability and cell-to-cell contact have been mechanisms used to explain cell growth regulation at high cell density. Recently Folkman and colleagues have shown that changes in cell shape, rather than cell-to-cell contact, can regulate the growth of fibroblasts. However, in those studies the relation between serum and shape regulation of growth was not studied, nor were neoplastic and non-neoplastic cells compared. In this report we have studied these aspects by varying cell spreading and serum concentration independently for 2 non-neoplastic and 3 neoplastic cell lines. Cell spreading (projected cell area) was controlled by decreasing the adhesiveness of tissue culture growth was measured as the increase in cell number/day. We have found that more spreading increased net growth of both neoplastic and non-neoplastic cells, while less spreading (toward rounded configuration) depressed growth. There were also quantitative differences between neoplastic and non-neoplastic cells. Neoplastic cells continued to grow under conditions of cell rounding, which completely prevented the growth of their non-neoplastic counterparts. Some neoplastic cells also tended to show little or no increase in net cell number for serum concentrations above 10% as cells became more spread; in contrast, all non-neoplastic cells grew more with increasing concentrations of serum as they became well spread. Thus, in normal cells, it appears that the sensitivity of cells to humoral factors is governed by cell spreading. This interaction between serum and cell shape is less prominent in some neoplastic cells.
We have evaluated optimal conditions for coupling monoclonal antibody to small unilamellar liposomes. Coupling of an IgG2 alpha monoclonal anti-beta 2-microglobulin antibody, which reacts with human cells, was examined in detail. Liposomes were composed of dipalmitoyl lecithin and cholesterol, and variable quantities of phosphatidylethanolamine substituted with the heterobifunctional cross-linking reagent N-hydroxysuccinimidyl 3-(2-pyridyldithio) propionate (SPDP). They were reacted with antibody derivatized with the same reagent at a 5- to 20-fold molar excess, and activated by mild reduction. This degree of SPDP modification had no effect on the capacity of the antibody to bind to its target antigen. More than 40% of antibody could be reproducibly bound to liposomes, resulting in the coupling of from 1 to 10 antibody molecules per liposome (mean diameter:580 A). The coupling reaction did not lead to loss of carboxyfluorescein encapsulated within liposomes. At least 80% of liposomes carried nondenatured antibody, as confirmed by precipitation of liposomes and encapsulated carboxyfluorescein by Staphylococcus aureus, strain Cowan I. The liposome-coupled antibody retained its immunological specificity: only cells expressing human beta 2-microglobulin bound liposomes in vitro, and the binding was inhibited by the free antibody in solution. Results with antibodies of different antigens specificity confirm that the technique can be generally applied.
Endothelial cells derived from human pulmonary arteries incorporate (3H)-glucosamine and 35SO4 into glycosaminoglycans and into the carbohydrate side chains of glycoproteins. These 3H/35S-carbohydrate chains were isolated from cells and culture medium after Pronase digestion. The 3H/35S-glycosaminoglycans were separated from the 3H/35S glycopeptides by chromatography on Sephadex G-50. The distribution of cellular glycosaminoglycans and glycopeptides indicated that 30-60% of the cellular 35S-glycopeptides may be associated with the matrix components that are synthesized by the cell and attached to a plastic substratum. Human pulmonary arterial endothelial cells were grown on collagen or on a matrix derived from vascular smooth muscle cells in order to investigate how smooth muscle cell extracellular matrix components may regulate the synthesis of endothelial cell glycoconjugates. Endothelial cells grown on plastic release various proportions of the glycoconjugates they synthesize into the culture medium. However, these same cells, when grown on substratum composed of extracellular matrix materials, synthesized altered proportions of cell-associated glycosaminoglycans and reduced the levels of total glycosaminoglycans they released into the culture medium. Thus the growth of endothelial cells on a matrix of smooth muscle cell components indicates that the glycosaminoglycan materials released into the culture medium by cells grown on a plastic substratum may not be an accurate reflection of the levels or composition of extracellular matrix materials made by endothelial cells in vivo.
The role of glycoconjugates in cell surface and blood-borne implantation properties of murine metastatic melanoma sublines of low (B16-F1) or high (B16-F10) potential to colonize lungs was investigated by treating melanoma cells with the antibiotic tunicamycin. This drug prevents glycosylation of glycoproteins by inhibiting the formation of lipid-linked oligosaccharide precursors. The degree of tunicamycin-mediated modifications in glycoproteins was assessed by monitoring the decrease in cell surface sialogalactoproteins by binding of 125I-labeled Ricinus communis agglutinin I. Scanning electron microscopy of tunicamycin-treated B16-F1 and B16-F10 cells showed morphologic changes such as cell rounding and formation of numerous surface blebs. Tunicamycin-treated B16-F1 and B16-F10 cells lost their lung colonization abilities when injected intravenously into C57BL/6 mice, concomitant with lowered rates of adhesion to endothelial cell monolayers, endothelial extracellular matrix (basal lamina), and polyvinyl-immobilized fibronectin in vitro, suggesting that this drug inhibits experimental metastasis by modifying the surface glycoproteins involved in determining the adhesive properties of malignant cells.
The possibility has been investigated that 1) the supplements required for the growth of the Madin Darby Canine Kidney (MDCK) cell line in serum-free Medium K-1 are indeed requirements for the growth of normal kidney cells in vitro, and 2) that alterations in these growth requirements are associated with malignant transformation. Consistent with the hypothesis that MDCK cells resemble normal kidney cells in culture, primary cultures of baby mouse kidney epithelial cells grow in Medium K-1 and respond to the 5 components in the medium. The growth properties of Moloney sarcoma virus (MSV)-transformed MDCK cells in defined media have been examined. Unlike MDCK cells, MSV-transformed MDCK cells form tumors in adult nude mice. Although they still respond to the 5 factors in Medium K1, the optimal dosage for insulin is lower for the MSV transformants than for MDCK cells. The MSV transformants also have an additional requirement for growth in Medium K-1-fibronectin. Variants of MDCK cells have been isolated that have lost the PGE1 requirement for growth in defined medium. These variant cells have acquired 1) the ability to form tumors in adult nude mice and 2) an alteration affecting cAMP metabolism, in addition to PGE1 independence.
Macrophages are shown to replace methylthio disulfides in supporting in vitro proliferation of three cell lines previously characterized as methylthio-dependent. Macrophages have the capacity to generate methylthio groups from methylthioadenosine. It is hypothesized that macrophages stimulate cell proliferation both in normal immune systems and in certain cancers by providing an abundance of methylthio groups. Fetal calf serum is shown to contain methylthio groups. It appears that, in cell cultures containing fetal calf serum, sulfhydryl compounds stimulate cell proliferation by making the methylthio groups in the serum available to the cells.
