Showing papers in "Journal of Supramolecular Structure and Cellular Biochemistry in 1981"
TL;DR: Best conditions for coupling monoclonal antibody to small unilamellar liposomes and results with antibodies of different antigens specificity confirm that the technique can be generally applied.
Abstract: 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.
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TL;DR: The term "autocrine secretion" has been proposed for this type of situation where a cell secretes a hormone-like substance for which it has external cell membrane receptors and may provide a partial explanation for some aspects of tumor cell progression.
Abstract: Transforming growth factors (TGFs) are growth-promoting polypeptides that cause phenotypic transformation and anchorage-independent growth of normal cells. They have been isolated from several human and animal carcinoma and sarcoma cells. One TGF is sarcoma growth factor (SGF) which is released by murine sarcoma virus-transformed cells. The TGFs interact with epidermal growth factor (EGF) cell membrane receptors. TGFs are not detectable in culture fluids from cells which contain high numbers of free EGF cell membrane receptors. SGF acts as a tumor promoter in cell culture systems and its effect on the transformed phenotype is blocked by retinoids (vitamin A and synthetic analogs). The production of TGFs by transformed cells and the responses of normal cells to the addition of TGFs to the culture medium raise the possibility that cells "autostimulate" their own growth by releasing factors that rebind at the cell surface. The term "autocrine secretion" has been proposed for this type of situation where a cell secretes a hormone-like substance for which it has external cell membrane receptors. The autocrine concept may provide a partial explanation for some aspects of tumor cell progression.
93 citations
TL;DR: The locations of the polypeptide chain differences between these two proteins indicate that plasma fibronectin cannot be derived from cellular fibronECTin by means of simple post-translational proteolysis.
Abstract: Fibronectin is a large, adhesive glycoprotein which is found in a number of locations, most notably on cell surfaces, in extracellular matrixes, and in blood. Fibronectin had been detected in all vertebrates tested and in many invertebrates. Its presence in sponges is significant because this suggests that fibronectin may have appeared very early in evolution, possibly with the most primitive multicellular organisms. Cellular and plasma fibronectins have many striking similarities. However, the locations of the polypeptide chain differences between these two proteins indicate that plasma fibronectin cannot be derived from cellular fibronectin by means of simple post-translational proteolysis. Instead, these different types of fibronectin may be products of different genes or of differentially spliced messenger RNA molecules. Amniotic fluid fibronectin is possibly a third form of the protein. Cellular and plasma fibronectins are composed of at least six protease-resistant domains which contain specific binding sites for actin, gelatin, heparin, Staphylococcus aureus, transglutaminase, fibrin, DNA, and a cell surface receptor. The relative locations of these domains have been mapped in the primary structure of fibronectin. The cell surface receptor for fibronectin has not been positively identified, but may be a glycoprotein, a glycolipid, or a complex of the two. Although cell-substratum adhesion is mediated by fibronectin, the locations of the areas of closest approach of the cell to the substratum (the adhesion plaques) and fibronectin are not coincident under conditions of active cell growth. Under conditions of cell growth arrest in low serum concentrations, some fibronectin may become localized at the adhesion plaques. Models describing the domain structure of fibronectin and the molecular organization of the adhesion plaque area are presented.
83 citations
TL;DR: 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.
Abstract: 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.
81 citations
TL;DR: This work has followed the incorporation of the radiolabeled amino acid precursors 3H-leucine and 35S-methionine into the gap junction protein, which has an apparent half-life of about 19 h and suggests that, in rat liver, the gap junctions may be very responsive to alterations in physiological demands.
Abstract: By the use of a simple, rapid method for the isolation of gap junctions from small amounts of rat liver (2-3 g), we have followed the incorporation of the radiolabeled amino acid precursors 3H-leucine and 35S-methionine into the gap junction protein. In timed studies with 35S-methionine as precursor, the specific activity in the protein is maximal by 4 h after a single injection of 300 microCi/100 g body weight. From the decay in the specific activity with time after a single injection, the gap junction protein has an apparent half-life of about 19 h. Because of problems of reutilization of radiolabeled amino acid with 35S-methionine as precursor, this apparent half-life probably overestimates the true half-life and indicates a surprisingly rapid turnover of the gap junction protein. This short half-life suggests that, in rat liver, the gap junctions may be very responsive to alterations in physiological demands.
64 citations
TL;DR: The linkage of these weak and strong associations suggests a process whereby the membrane skeleton spontaneously assembles, and affinity-modulated assembly involving weak associations is likely to abe the focus of numerous membrane.
Abstract: Spectrin reversibly self-associates to high molecular weight oligomers through a concentration-driven process characterized by association constants of about 105 mol−1. This association is prominent under physiological conditions of pH, ionic strength, and temperature. It is disrupted by urea, but not Triton X-100. The process of spectrin association appears mathematically to resemble that for tropomyosin, although the mechanism is probably different. Spectrin association is weak compared to other prominent protein–protein associations in the red cell membrane skeleton. The linkage of these weak and strong associations suggests a process whereby the membrane skelton spontaneously assembles. Such affinity-modulated assembly involving weak associations is likely to be the focus of numerous membrane control mechanisms.
63 citations
TL;DR: In this paper, the authors have prepared antisera in rabbits to the "contact sites A" glycoprotein (gp80) purified from Dictyostelium discoideum.
Abstract: We have prepared antisera in rabbits to the "contact sites A" glycoprotein (gp80) purified from Dictyostelium discoideum. IgG isolated these antisera reacts with a number of different proteins in D discoideum lysates, as analyzed by immune precipitation and by antibody staining of gel electropherograms transferred to nitrocellulose. blocking experiments indicate that this cross-reactivity reflects the presence of common antigeneic determinants on gp80 and other cellular proteins, rather than the presence of extraneous antibodies in the antisera. The spectrum of reactive proteins is different at different stages of development. In particular, gp80 itself is synthesized only for a restricted period during the cell aggregation phase. The protein persists throughout development and can be detected in spores. Anti-gp80 Fab fragments bind to the surface of developing D discoideum cells and specifically block their developmentally regulated adhesion. After absorption with vegetative cells, the IgG stains only gp80 and (to a lesser extent) one other band in lysates of aggregation-competent cells. The absorbed antibodies also can block adhesion. Several proteins that appear late in development also are stained by the absorbed IgG.
62 citations
62 citations
TL;DR: The properties of multiplication stimulating activity, an insulin-like growth factor purified from culture medium conditioned by the BRL 3A rat liver cell line, and the relationship of MSA to somatomedins purified from human and rat plasma are considered are considered.
Abstract: The properties of multiplication stimulating activity (MSA), an insulin-like growth factor (somatomedin) purified from culture medium conditioned by the BRL 3A rat liver cell line are summarized. The relationship of MSA to somatomedins purified from human and rat plasma are considered. MSA appears to be the predominant somatomedin in fetal rat serum, but a minor component of adult rat somatomedin. In vitro biological effects of MSA and insulin in adipocytes, fibroblasts and chondrocytes are examined to determine whether they are mediated by insulin receptors or insulin-like growth factor receptors. The possible relationship of a primary defect of insulin receptors observed in fibroblasts from a patient with the rare genetic disorder, leprechaunism, to intrauterine growth retardation is discussed.
TL;DR: The purification of the MSA carrier protein (MCP) from conditioned medium using affinity chromatographic procedures is described and the purified carrier protein is shown to specifically bind labeled MSA and generates a complex with an apparent molecular weight of 60,000-70,000 daltons.
Abstract: The rat liver cell line, BRL-3A, is known to produce a family of polypeptides referred to as multiplication-stimulating-activity (MSA). Serum-free conditioned medium from this cell line is a rich source for the purification of these somatomedin-like molecules. Somatomedins in serum, as well as MSA produced by BRL-3A cells in culture, exist primarily as a high molecular weight complex bound to specific carrier proteins. This study describes the purification of the MSA carrier protein (MCP) from conditioned medium using affinity chromatographic procedures. The purified carrier protein is shown to specifically bind labeled MSA and generates a complex with an apparent molecular weight of 60,000-70,000 daltons. Characterization of the carrier protein indicates that it consists of two different noncovalently linked protein chains with apparent molecular weights of 30,000 and 31,500 daltons. The availability of a pure carrier protein should provide a unique opportunity to investigate the functional significance of the carrier protein in the biological activity of the somatomedins.
TL;DR: The expression of M2 antigen increases during development in both cell types, reaching adult levels by postnatal day 14, and granule cells that have completed migration to the internal granule layer are more heavily stained by M2 antibodies than are those before and in process of migration.
Abstract: A monoclonal antibody designated M2 arose from the fusion of mouse myeloma cells with splenocytes from a rat immunized with particulate fraction from early postnatal mouse cerebellum. Expression of M2 antigen was examined by indirect immunofluorescence on frozen sections of developing and adult mouse cerebellum and on monolayer cultures of early postnatal mouse cerebellar cells. In adult cerebellum, M2 staining outlines the cell bodies of granule and Purkinje cells. A weaker, more diffuse staining is seen in the molecular layer and white matter. In sections of newborn cerebellum, M2 antigen is weakly detectable surrounding cells of the external granular layer and Purkinje cells. The expression of M2 antigen increases during development in both cell types, reaching adult levels by postnatal day 14. At all stages of postnatal cerebellar development, granule cells that have completed migration to the internal granule layer are more heavily stained by M2 antibodies than are those before and in process of migration. In monolayer cultures, M2 antigen is detected on the cell surface of all GFA protein-positive astrocytes and on more immature oligodendrocytes that express 04 antigen but not 01 antigen. After 3 days in culture, tetanus toxin positive neurons begin to express M2 antigen. The same delayed expression of M2 antigen on neurons is observed in cultures derived from mice ranging in age from postnatal day 0 to 10.
TL;DR: It is found that normal human fibroblasts rejoin 50% of all breaks within only 3 min (37 degrees C) and this fast rejoining was impaired in fibro Blasts from one patient with Ataxia telangiectasia and in fibrosclerosis from two patients with Fanconi's anemia.
Abstract: Using the technique of neutral elution through polycarbonate filters as a measure of DNA length, and hence of the number of double-strand breaks incurred as a result of radiation damage, we found that normal human fibroblasts rejoin 50% of all breaks within only 3 min (37 degrees C). This fast rejoining was impaired in fibroblasts from one patient with Ataxia telangiectasia and in fibroblasts from two patients with Fanconi's anemia. Also the number of residual breaks after several hours of repair was higher than in control cells. Other cases with the same diseases were normal in their rejoining of double-strand breaks.
TL;DR: Under conditions that it enhances unscheduled DNA synthesis, nicotinamide causes marked effects on the metabolism of NAD+ and poly(ADP-ribose).
Abstract: The effect of nicotinamide on unscheduled DNA synthesis was studied in resting human lymphocytes. In cells treated with UV irradiation or with MNNG, nicotinamide caused a two-fold stimulation of unscheduled DNA synthesis and retarded the rate of NAD+ lowering caused by these treatments. Nicotinamide also reduced the burst of poly(ADP-ribose) synthesis caused by MNNG treatment. Thus under conditions that it enhances unscheduled DNA synthesis, nicotinamide causes marked effects on the metabolism of NAD+ and poly(ADP-ribose). The effect of nicotinamide on unscheduled DNA synthesis was shown to be independent of protein or polyamine synthesis.
TL;DR: It is proposed that stromal cells produce a resident CSF that, in contrast to exogenous CSF species, is capable of inducing myelopoiesis within the bone and marrow stroma.
Abstract: Adherent stromal cells from mouse bone marrow inhibited the formation of granulocyte/monocyte (G/M) colonies induced in vitro by colony-stimulating factor (CSF). This inhibition occurred both when crude conditioned media obtained from various sources were used to induce colony formation or when a pure CSF preparation from mouse lung origin was tested. The inhibition did not appear to be toxic in nature since despite the lack of colony formation, progenitor CFU-C proliferated in the presence of stromal cells. Medium conditioned by adherent stromal cells was devoid of inhibitory activity when incorporated into the culture medium used for G/M colony formation, indicating that the inhibitory activity may not be present in a soluble form. Inhibitors of prostaglandins did not affect G/M colony formation. In contrast, D-glucose and a number of other free monosaccharides but not pyruvate lactate or glycerol induced formation of myeloid colonies in the presence of stromal cells. This did not require addition of exogenous CSF. Released factors concentrated from serum-free medium conditioned by stromal cells exhibited colony-stimulating activity provided that the medium contained a high glucose concentration during incubation. It is proposed that stromal cells produce a resident CSF that, in contrast to exogenous CSF species, is capable of inducing myelopoiesis within the bone and marrow stroma.
TL;DR: Examination of glycoprotein-linked oligosaccharides assembled during the life cycle of Dictyostelium discoideum found their expression to be dramatically dependent upon the stage of development, and developmental regulation of the wide array of protein-linked glycans expressed may be as simple as the controlled transition.
Abstract: We have examined the glycoprotein-linked oligosaccharides assembled during the life cycle of Dictyostelium discoideum, and found their expression to be dramatically dependent upon the stage of development. During early development mature glycans have a high mannose character, and a substantial proportion acquire a fucose residue that correlates with endo-H resistance. One-third of the glycans also acquire sulfate residues. These glycans diminish in importance during aggregation. The mature glycans expressed during aggregation. The mature glycans expressed during late development contain fewer mannose residues, from five to ten mannose residues, and are characterized by the absence of sulfate residues and by the presence of fucose residues on endo-H-sensitive glycans. These glycans make their appearance coincident with the construction of tips on tight cell mounds. At this stage glycans characteristic of both early and late stages occur simultaneously. Developmental regulation of the wide array of protein-linked glycans expressed during the life cycle of Dictyostelium discoideum may be as simple as the controlled transition from a group of structures that are assembled by the vegetative cells to a group of structures that are assembled by the terminally differentiating cells. The potential biological significance of this transition is discussed.
TL;DR: A hypothesis is proposed in which adhesive proteins within platelet granules become expressed on the platelet surface as a direct consequence of the secretory process, and this cluster of adhesive proteins may then contribute to increased cellular adhesiveness.
Abstract: Thrombin stimulation of human platelets causes increased cellular adhesiveness for other platelets (aggregation) and surfaces and increased surface expression of platelet fibronectin antigen. Aggregation occurs concurrently with secretion. In these studies, the threshold thrombin dose for surface expression of fibronectin, as measured by binding of F(ab')2 antifibronectin, was similar to that for serotonin secretion. Moreover, both processes occurred at similar rates, and inhibition of secretion was associated with inhibition of antifibronectin binding. Thus a hypothesis is proposed in which adhesive proteins within platelet granules become expressed on the platelet surface as a direct consequence of the secretory process. This cluster of adhesive proteins may then contribute to increased cellular adhesiveness.
TL;DR: Mouse epidermal basal cells can be selectively cultivated in medium with a calcium concentration of 0.01--9.09 mM to provide additional evidence of basal cell heterogeneity in which TPA induces one subpopulation to differentiate while another is stimulated to proliferate and resists a differentiation signal.
Abstract: Mouse epidermal basal cells can be selectively cultivated in medium with a calcium concentration of 0.01--9.09 mM. Terminal differentiation and sloughing of mature keratinocytes occur when the calcium concentration is increased to 1.2--1.4 mM. When basal cell cultures are exposed to chemical initiators of carcinogenesis, colonies of cells that resist calcium-induced differentiation evolve. Likewise, basal cells derived from mouse skin initiated in vivo yield foci that resist terminal differentiation. This defect in the commitment to terminal differentiation appears to be an essential change in initiated cells in skin and is also characteristic of malignant epidermal cells. This model system has also provided a means to determine if basal cells are more responsive to phorbol esters than other cells in epidermis and to explore the possibility that heterogeneity of response exists within subpopulations of basal cells. The induction of the enzyme ornithine decarboxylase (ODC) was used as a marker for responsiveness to phorbol esters. ODC induction after exposure to 12-0-tetradecanoylphorbol-13-acetate (TPA) in basal cells is enhanced 20-fold over the response of a culture population containing both differentiating and basal cells. When basal cells are induced to differentiate by increased calcium, responsiveness to TPA is lost within several hours. In basal cell cultures, two ODC responses can be distinguished. After exposure to low concentrations of TPA or to weak promoters of the phorbol ester series, ODC activity is maximal at 3 hr. With higher concentrations of TPA, the ODC maximum is at 9 hr. These results are consistent with the presence of subpopulations of basal cells with differing sensitivities to TPA. Other studies that use the enzyme epidermal transglutaminase as a marker for differentiation support this conclusion. In basal cell culture TPA exposure rapidly increases transglutaminase activity and cornified envelope development, reflecting induced differentiation in some cells. As differentiated cells are sloughed from the dish, the remaining basal cells proliferate and become resistant to induced differentiation by 1.2 mM calcium. These data provide additional evidence of basal cell heterogeneity in which TPA induces one subpopulation to differentiate while another is stimulated to proliferate and resists a differentiation signal. Tumor promoters, by their ability to produce heterogeneous responses with regard to terminal differentiation and proliferation, would cause redistribution of subpopulations of epidermal cells in skin. Cells that resist signals for terminal differentiation, such as initiated cells, would be expected to increase in number during remodeling, Clonal expansion of the initiated population could result in a benign tumor with an altered program of differentiation. In skin, benign tumors are the principal product of 2-stage carcinogenesis. Subsequent progression to malignancy may involve an additional step, probably a genetic alteration, that is independent of the tumor promoter.
TL;DR: A partially purified egg surface glycoconjugate component is the most potent inhibitor of bindin-mediated egg agglutination yet described and is involved in sperm adhesion.
Abstract: Sea urchin sperm–egg adhesion is mediated by bindin, a sperm surface protein that has lectin-like activity. Bindin agglutinates eggs, and this interaction has been shown to be inhibited by glycopeptides released from the egg surface by protease treatment. In this study, we report the purification and properties of such an egg surface glycoconjugate that may be involved in sperm adhesion. The glycoconjugate was partially purified by gel filtration and affinity chromatography on bindin particles. Upon gel filtration on Sepharose CL 4-B, the glycoconjugate elutes near the void volume, suggesting that it has a molecular weight in excess of one million. In addition, we have found that the egg surface glycoconjugate agglutinates bindin particles, indicating that it is multivalent. Carbohydrate analysis indicates that the glycoconjugate is composed primarily of fucosc, xylose, galactose, and glucose. This purified egg surface component is the most potent inhibitor of bindin-mediated egg agglutination yet described.
TL;DR: It is found that more spreading increased net growth of both neoplastic and non-neoplastic cells, while less spreading depressed growth, and it appears that the sensitivity of cells to humoral factors is governed by cell spreading.
Abstract: 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 plastic plates with poly (hydroxyethyl methacrylate) [poly (HEMA)]. Cell 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.
TL;DR: 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.
Abstract: 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.
TL;DR: Studies on the biochemical basis of selective attachment are reviewed, and suggest that this recognition process is accomplished by specific glycoprotein lectin-polysaccharide interactions on the surfaces of the symbionts.
Abstract: Bacterial attachment is viewed as a cellular recognition event during the infection of legumes by the nitrogen-fixing symbiont, Rhizobium. Studies on the biochemical basis of selective attachment are reviewed, and suggest that this recognition process is accomplished by specific glycoprotein lectin-polysaccharide interactions on the surfaces of the symbionts. An understanding of host specificity may lead to ways to broaden the host range of nitrogen-fixing symbioses.
TL;DR: 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.
Abstract: 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.
TL;DR: Direct correlations with metastatic behavior in the lung colony assay were found to exist with the amount of cell-bound Concanavalin A and the procoagulant activities of cell lysates.
Abstract: We have studied several metastatic variant cell lines derived from a common clonal origin and their transformed and untransformed parental cell lines. A number of in vitro characteristics were examined for each tumor line and these properties were correlated with the ability of the tumor cells to form pulmonary nodules in an experimental metastasis assay. Direct correlations with metastatic behavior in the lung colony assay were found to exist with the amount of cell-bound Concanavalin A and the procoagulant activities of cell lysates. In vitro parameters that did not correlate with the metastatic phenotype were: population doubling times in culture, saturation density achieved in culture, the number of colony-forming cells shed from confluent cultures, rates of cellular attachment to homotypic or heterotypic cell monolayers, plasminogen-activator production and procoagulant activity produced in serum-free conditioned medium.
TL;DR: Tunicamycin inhibits experimental metastasis by modifying the surface glycoproteins involved in determining the adhesive properties of malignant cells, suggesting that this drug inhibits experimental tumour invasion by inhibiting the formation of lipid-linked oligosaccharide precursors.
Abstract: The role of glycoconjugates in cell surface and blood-borne implantation properties of murine metastatic melanoma sublines of low (B16-F1) or high (B 16-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.
TL;DR: Radioactivity associated with both plasma membrane proteins W floribunda agglutinin and lectin were found in regions of the density gradient fractionation that virtually lacked golgi and lysosome markers.
Abstract: Subcellular fractionation of Balb/c 3T3 fibroblasts exposed to Wistaria floribunda agglutinin was performed to localize fractions containing internalized lectin. Employing two sequential self-generating silica sol density gradients, the postnuclear supernatant of cell homogenates was resolved into five distinct cellular components. Lysosome enzyme activities were displayed by two populations of vesicles, each separated from plasma membrane, golgi, and mitochondria markers. The more dense of these fractions exhibited morphological and biochemical properties ascribed to secondary lysosomes. The more buoyant population was similar to that reported by Rome et al [11] who noted that it may be a product of vesiculated golgi endoplasmic reticulum lysosome (GERL). Treatment with W floribunda agglutinin of cells, surface radioiodinated demonstrated that plasma membrane proteins were localized within both the buoyant and dense lysosome populations in as little as 10 min after exposure to lectin. Prolonged incubation of the cells with W floribunda agglutinin resulted in maintenance of this distribution. However, when nonradiactive cells were exposed to 125I-labeled W floribunda agglutinin for 10 min, radioactivity was detected only in the buoyant population of lysosomes as well as the plasma membrane/golgi fraction. Treatment of cells with W floribunda agglutinin for 30 min resulted in appearance of lectin associated radioactivity in the dense lysosome fraction in addition to those populations containing radioactivity seen after a 10-min incubation. These data indicate that the endocytosis of W floribunda agglutinin differed substantially from the internalization of a portion of the plasma membrane proteins. Furthermore, we found radioactivity associated with both plasma membrane proteins W floribunda agglutinin in regions of the density gradient fractionation that virtually lacked golgi and lysosome markers. These fractions may have represented populations of nonlysosome vesicles formed during the process of endocytosis.
TL;DR: The phosphoproteins of Dictyostelium discoideum were compared at different stages of development of polyacrylamide gel electrophoresis and certain phosphoprotein of vegetative amoebae were conserved while others appeared and disappeared during development.
Abstract: The phosphoproteins of Dictyostelium discoideum were compared at different stages of development by polyacrylamide gel electrophoresis. Certain phosphoproteins of vegetative amoebae were conserved while others appeared and disappeared during development. Four major phosphoproteins with apparent subunit molecular weights of 50,000, 47,000, 38,000, and 34,000 disappeared precociously in response to exogenous cAMP. Two membranal phosphoproteins, with apparent subunit molecular weights of 80,000 and 81,000, appeared precociously in response to added cAMP. One of these phosphoproteins, molecular weight of 80,000, has been identified tentatively as the “contact site A” glycoprotein. Another membranal protein, with apparent subunit molecular weight of 42,000, unaffected in its appearance by cAMP, has been identified tentatively as phosphoactin.
TL;DR: It appears that this egg surface membrane fraction of echinoderm eggs contains a functionally intact, species-specific receptor for sperm.
Abstract: We have attempted to identify a surface component of echinoderm eggs that is involved in the species-specific binding of sperm. Cell surface membranes from eggs of the sea urchins Strongylocentrotus purpuratus or Arbacia punctulata were radioiodinated, detergent-treated, and subjected to density-gradient centrifugation. In the presence of bindin, the complementary binding protein isolated from sperm, one component of the membranes sedimented to a different density. This membrane component bound species specifically to sperm that had undergone the acrosome reaction. This binding led to an inhibition of the ability of treated sperm to fertilize eggs. Exhaustive proteolytic digestion of this receptor fraction yields a high molecular weight glycopeptide that can also bind to bindin. It therefore appears that this egg surface membrane fraction contains a functionally intact, species-specific receptor for sperm.
TL;DR: An increase in the intracellular concentration of Ca2+ in human erythrocytes results in the formation of gamma-glutamyl-epsilon-lysine cross-linked membrane protein polymers, which can be isolated on a Lubrol-containing sucrose gradient on a SDS gradient.
Abstract: An increase in the intracellular concentration of Ca2+ in human erythrocytes results in the formation of gamma-glutamyl-epsilon-lysine cross-linked membrane protein polymers. Following solubilization of the membranes with SDS, these polymers can be isolated on a Lubrol-containing sucrose gradient. Immunoelectrophoresis of the polymeric material with a polyspecific rabbit antibody against human ghosts gave rise to a single, but heterogeneous, precipitate. The polymer was amphiphilic and, on addition to Triton-solubilized erythrocyte membrane proteins, it coprecipitated with spectrin. When the antihost antibody was absorbed with the polymer prior to cross immunoelectrophoresis of normal erythrocyte membrane proteins, the precipitates of glycophorin, acetylcholinesterase, and hemoglobin were normal, whereas the antibody titers against band 3 protein, spectrin, and ankyrin became reduced. Furthermore, a rabbit antibody raised against the isolated human polymer reacted selectively with the same three membrane proteins. No reactions occurred with lysate proteins.