Role of carbohydrates in rat leukemia cell-liver macrophage cell contacts

The mechanism by which macrophages recognize tumor cells is still unknown. We have studied interactions between rat liver macrophages and rat L 5222 leukemia cells. These tumor cells, but not normal leukocytes or erythrocytes, adhere to freshly isolated macrophages in vitro. Binding of tumor cells by macrophages can be inhibited by N-acetyl-D-galactosamine, D-galactose and more potently by glycoproteins with terminal N-acetyl-D-galactosamine or D-galactose residues. Tumor cell adhesion is calcium-dependent. The relevant leukemia cell membrane structures which bear terminal beta-D-galactosyl or related residues have been determined as trypsin- and pronase-sensitive, and hence may presumably be glycoproteins. The tumor cell receptor on liver macrophages appears to be a lectin with the carbohydrate specificity N-acetyl-D-galactosamine greater than D-galactose greater than L-fucose.     

Role of carbohydrates in cell-substrate interactions during newt epidermal cell migration

The effect of several solubilized monosaccharides on epidermal cell migration from skin explants of the adult newt was examined. The ability of epidermal cells to migrate on substrates coated with these same sugars or with wheat germ agglutinin (WGA) was also determined. Adding 0.05 M N-acetyl-glucosamine (GlcNAc) to the medium inhibited epidermal cell migration in dishes coated with either type I collagen or fibrinogen. The same concentration of fucose, galactose, or mannose had no effect. In contrast to type I collagen, which supported considerable migration when dried onto the bottom of plastic dishes, epidermal cells were unable to migrate on dishes coated similarly with WGA, mucin (a protein high in sialic acid residues), or bovine serum albumin (BSA) conjugated to galactose, mannose, or GlcNAc. Red blood cell (RBC) binding assays showed that drying WGA onto plastic did not destroy its GlcNAc binding sites--nor did it damage the GlcNAc residues of BSA-GlcNAc. The RBC assay also verified that for both these proteins, substrates with distinctly different cell binding capacities had been tested in the migration experiments. In dishes coated with either WGA or BSA-GlcNAc, red cells bound to dish bottoms in a GlcNAc-specific manner right up to the margins of explants. Other control experiments showed that the failure of migration in WGA- and BSA-GlcNAc-coated dishes could not be explained by competition between adsorbed and desorbing protein for cell surface receptors. This work shows that adhesive bonds between epidermal cell surface GlcNAc and a GlcNAC-specific lectin on the substrate are not by themselves adequate to support cell migration. Nor is GlcNAc, sialic acid, galactose, or mannose alone on the substrate sufficient. In conjunction with our earlier work (Donaldson and Mahan: J. Exp. Zool., 231:211-219, '84; Donaldson, Mahan, Hasty, McCarthy, and Furcht: J. Cell. Biol., 101: 73-78, '85), these observations suggest that factors other than carbohydrate content or capacity to act as a lectin determine whether a given extracellular protein will support migration.     

Functional macrophage cell lines transformed by Abelson leukemia virus.

Three cloned cell lines have been established from murine tumors induced with Abelson leukemia virus which express properties of macrophages. Two of the three original tumors in addition yielded lymphocyte cell lines, one typical of the Abelson virus disease and the other a thymic lymphoma. Two of the macrophage lines are tumorigenic when placed in syngeneic mice. All of the macrophage lines pinocytose neutral red, phagocytose zymosan and latex beads, mediate antibody-dependent killing and phagocytosis of sheep erythrocyte targets, and secrete high levels of lysozyme. None of these properties was exhibited by the lymphocyte lines. Of the two macrophage cell lines tested, neither was capable of replacing the adherent cell population required for the induction of in vitro immune responses. An agent that activates normal macrophages, bacterial lipopolysaccharide, specifically inhibits the growth of the transformed macrophages in culture. Secretion of infectious Abelson leukemia virus by two of the macrophage lines, RAW 309Cr and WR 19M, provides conclusive evidence that the Abelson virus is capable of productively infecting the macrophage cell type. The other macrophage line, RAW 264, fails to secrete detectable virus particles and is negative in the XC plaque formation assay, as well as the fibroblast transformation assay for Abelson virus, but becomes positive for Abelson virus production after rescue by Moloney leukemia virus.     

Role of microtubules in LPS-induced macrophage inflammatory protein-2 production from rat pneumocytes

We have recently demonstrated that primary cultured rat pneumocytes produce macrophage inflammatory protein-2 (MIP-2) in response to lipopolysaccharide (LPS) stimulation. In this study, we found that brefeldin A, by blocking anterograde transport from the endoplasmic reticulum (ER) to the Golgi apparatus, decreased LPS-induced MIP-2 in the culture medium and increased its storage in cells. This suggests that MIP-2 is secreted via a pathway from the ER to the Golgi apparatus, a process commonly regulated by microtubules. We further found that LPS induced depolymerization of microtubules as early as 1 min after LPS stimulation, and it lasted at least for 4 h. Preventing depolymerization of microtubules with paclitaxel (Taxol; 10 nM to 10 microM) partially inhibited LPS-induced MIP-2 production, whereas the microtubule-depolymerizing agents colchicine (1-10 microM) and nocodazole (1-100 microM) increased LPS-induced MIP-2 protein production without affecting MIP-2 mRNA expression. These results suggest that in pneumocytes, LPS-induced microtubule depolymerization is involved in LPS-induced MIP-2 production and that secretion of MIP-2 from pneumocytes is via the ER-Golgi pathway.     

Role of cytokines in alveolar macrophage accessory cell function in HIV-infected individuals.

Mononuclear phagocytes, including alveolar macrophages (AM), can be chronically infected with HIV and thus serve as a reservoir for the virus. Acting as AC during the generation of an immune response, HIV-infected mononuclear phagocytes can facilitate viral T cell infection by several mechanisms, including direct contact of T cells with HIV-infected macrophages as well as cytokine-induced up-regulation of latent T cell infection. Our laboratory has shown that AM from HIV-infected individuals have enhanced AC function compared to normal AM. In this study we explored AM production and secretion of IL-1 beta and IL-6, two cytokines critical for optimal AC function, in normal volunteers and HIV-infected patients. Cultured AM supernatants and lysates were generated in the presence and absence of LPS and standard mitogens. In initial mixing experiments HIV AM supernatants enhanced mitogen-induced T cell proliferation using normal AM as AC significantly more than normal AM supernatants, suggesting that HIV AM secreted more T cell stimulatory factors than normal AM. Neither group could enhance T cell proliferation induced by HIV AM suggesting these cells already secreted optimal levels of these factors. AM from HIV+ individuals produced and secreted more IL-1 beta (measured by ELISA) and IL-6 (measured in a B9 bioassay and by immunoprecipitation) than normal AM both spontaneously and in the presence of low LPS concentrations and mitogens. In some cases depleting HIV AM supernatants of IL-1 beta and IL-6 on immunoaffinity columns abrogated their enhancement properties indicating that these cytokines were important in the observed enhancement. However, in other patients different factors must also be involved as depletion of IL-1 beta and IL-6 in their AM supernatants had no effect on enhancement function. These results show that HIV AM secretory products are important in the enhanced AC function demonstrated by these cells. However, although augmented IL-1 beta and IL-6 secretion likely contribute significantly to this enhancement, other AC secretory factors and/or functions must also be involved.     

Stimulation of rat mesangial cell proliferation by macrophage interleukin 1

Conditioned media from LPS-activated rat peritoneal macrophages enhanced the proliferation rates of cultured rat glomerular mesangial cells. This macrophage-derived activity extensively co-purified with interleukin 1 (IL 1) activity through sequential ammonium sulfate precipitation, S-200 gel chromatography, DEAE-cellulose anion exchange chromatography, and phenyl-Sepharose chromatography. In addition, the macrophage-derived factor was heat-labile (80 degrees C) and inactivated by phenylglyoxal, thus allowing tentative identification as IL 1. Macrophage supernatants and purified IL 1 enhanced the proliferative rates of mesangial cells only in the presence of serum; the use of platelet-poor plasma or serum depleted of platelet-derived growth factor was without effect. IL 1 acted to increase the percentage of cycling cells, without a change in the length of the individual cell cycle times. These findings provide a potential mechanism whereby activated macrophages, in combination with platelet factors, enhance mesangial cell proliferation. Such processes may contribute to the mesangial hypercellularity frequently found in immune-mediated glomerulonephritis.     

Production of cytotoxin to a tumor cell in co-cultures of macrophage cell line with bovine leukemia virus producing cell line

J 774 cells (macrophage cell line) were co-cultured with fetal lamb kidney cells which permanently produce Bovine leukemia virus (BLV-FLK cells) and after co-culture, the cells removed by scraping were subcultured once every for 2-4 days. Strong cytotoxicity to a cell (C8 cells; a clone of F81 cell line which contains mouse sarcoma virus genome) was found in culture of cells from generations 11 after the passages of co-cultures. The activity to the cells was continuously found in the fluid without any other addition of stimulants. The above results show that J 774 cells were activated by the co-culture with BLV-FLK cells and produced the cytotoxic factor which revealed strong cytotoxicity to a C8 cells.     

Role of carbohydrates in micropropagation of cork oak

The influences of carbon sources, fructose, glucose, sorbitol and sucrose on shoot proliferation and in vitro rooting of cork oak (Quercus suber L.) were compared at a wide range of concentrations (1–6%, w/v). The highest number of shoots occurred on glucose-containing medium. Nevertheless, we have chosen 3% sucrose which induced a similar rate of proliferation but favoured shoot elongation, permitting an effectively higher number of shoots during transfers. Sorbitol and autoclaved fructose did not stimulate shoot proliferation. Adventitious root formation was strongly dependent on carbohydrate supply. Sorbitol and autoclaved fructose were completely ineffectively on rooting induction. Glucose was the most effective carbon source on rooting promotion followed by sucrose and filter-sterilized fructose. The rooting response induced by fructose was dependent on the sterilizing procedure. The number of adventitious roots produced per shoot increased with increasing glucose and sucrose concentration. The content of reducing sugars in leaves of proliferation cultures and in leaves and roots of rooted plantlets was more dependent on carbon concentration than on glucose or sucrose supplement. The results presented here show that carbohydrate requirements during cork oak micropropagation depend upon the phase of culture. Sucrose (3%) and glucose (4%) were the best carbon sources respectively during proliferation and rooting phases.     

Role for protein geranylgeranylation in adult T-cell leukemia cell survival

Adult T-cell leukemia (ATL) is a fatal lymphoproliferative disease that develops in human T-cell leukemia virus type I (HTLV-I)-infected individuals. Despite the accumulating knowledge of the molecular biology of HTLV-I-infected cells, effective therapeutic strategies remain to be established. Recent reports showed that the hydroxyl-3-methylglutaryl (HMG)-CoA reductase inhibitor statins have anti-proliferative and apoptotic effects on certain tumor cells through inhibition of protein prenylation. Here, we report that statins hinder the survival of ATL cells and induce apoptotic cell death. Inhibition of protein geranylgeranylation is responsible for these effects, since simultaneous treatment with isoprenoid precursors, geranylgeranyl pyrophosphate or farnesyl pyrophosphate, but not a cholesterol precursor squalene, restored the viability of ATL cells. Simvastatin inhibited geranylgeranylation of small GTPases Rab5B and Rac1 in ATL cells, and a geranylgeranyl transferase inhibitor GGTI-298 reduced ATL cell viability more efficiently than a farnesyl transferase inhibitor FTI-277. These results not only unveil an important role for protein geranylgeranylation in ATL cell survival, but also implicate therapeutic potentials of statins in the treatment of ATL.     

Ultrastructural evidence for contacts between migrating L5222 rat leukemia cells and extracellular matrix components of the rat mesentery

The nature of interactions between cells migrating through tissues and their structural surroundings are largely unknown. We have therefore examined the ultrastructural relationship between L5222 rat leukemia cells, moving through the loose connective tissue of the mesentery, and components of the extracellular matrix (ECM). Ultrathin tissue sections, fixed in the presence of ruthenium hexammine trichloride (RHT), revealed the following: Constitutents of fibrillar and nonfibrillar elements of the ECM are in contact with the plasma membrane of L5222 cells. Linear nonfibrillar ECM elements contact the plasma membrane at point-like sites, often associated with root-like structures present within the submembraneous microfilament mesh. Aggregates of ECM material are connected to patch-like cell membrane sites, associated with a condensed, plate-like part of the microfilament mesh. Point-like and patch-like contacts are more numerous at the anterior part of polarized migrating L5222 cells than on the posterior end. In round resting leukemia cells they are evenly distributed around the cell periphery. We suggest that the ECM-cell membrane contacts represent tissue adhesion sites. We therefore hypothesize that in migrating cells a coordinate interaction occurs between the contact sites and the continuous microfilament meshwork which results in a simultaneous backward movement of ECM-membrane contacts on the cell body and in a net forward movement of the whole cell. Since Dembo et al. (1981) present a similar mechanism for in vitro locomotion of granulocytes, we assume that blood cell locomotion in vivo and in vitro depends on similar molecular mechanisms: force generation by the cell, transmembraneous linkage between cytoskeletal and ECM elements, and membrane fluidity. The major difference in blood cell locomotion through a three-dimensional tissue or on a plane substratum would then be given by the distribution of contact sites, occurring around the cell periphery or limited to the ventral cell surface, respectively.     

Role of carbohydrates in glycoprotein hormone signal transduction

The structure of the polypeptide chains and oligosaccharide moieties of the alpha and beta subunits of pituitary and placental glycoprotein hormones are known. The dimeric polypeptide structure (but not the carbohydrate) is important for binding of the hormone to specific receptors. The N-linked but not O-linked carbohydrates, on the other hand, are required in some manner to activate the effector system. Hormones with depleted carbohydrate content (deglycosylated hormones) interact with receptor but are unable to activate intracellular events. Because of such discordant properties, these forms act as competitive inhibitors of hormone action. Through a combination of chemical deglycosylation procedures and site-directed mutagenesis, the first site of N-glycosylation from the NH2 terminus of the common alpha subunit has been identified to be more critical for glycoprotein hormone signal transduction. Control of glycosylation by the endocrine milieu could contribute to regulation of hormone function by secreting variable forms of agonist/antagonist.     

Role of carbohydrates in insect resistance in Phaseolus vulgaris

The bruchid beetles Acanthoscelides obtectus (Say) and Zabrotes subfasciatus Boheman, are major storage pests of the economically important legume Phaseolus vulgaris (haricot bean). In the present study, resistance of a wild line of P. vulgaris G12953, towards these two insects is confirmed. Mature seeds from both resistant and susceptible lines were fractionated and tested in feeding trials in an attempt to elucidate the basis of resistance. The heteropolysaccharide fraction isolated from the resistant line and a susceptible line were each incorporated into artificial beans over a concentration range up to 10% dry wt. At a concentration of 4%, the approximate physiological concentration within the seed, the heteropolysaccharide fraction from the resistant line was very toxic resulting in 80–85% larval mortality of A. obtectus with an LC₅₀ of 2.5%. Furthermore, surviving larvae showed a marked increase in their development period. The corresponding fraction from a susceptible line, on the other hand, even at twice the physiological concentration, had a negligible effect upon larval development both in terms of adult emergence and development period. The effects of albumin proteins and globulin proteins on larval development of A. obtectus were also tested by incorporation into artificial beans at a range of concentrations up to 10%. Whilst those from the susceptible line had little effect upon development both the albumins and globulins from the resistant line were found to cause a slight decrease in adult emergence and a small increase in the median development period. These effects, however, are not sufficient to account for the observed seed resistance of G12953. Mature seeds from the resistant line and nine susceptible lines were also screened for the following secondary plant compounds: proteinase inhibitors, α-amylase inhibitors, lectins (phytohaemagglutinins), saponins, tannins and alkaloids. Although some of these antimetabolites were found to be present none, except for α-amylase inhibitors, were thought to play any significant role in conferring seed resistance. It is concluded that this example of insect resistance in seeds of P. vulgaris, line G12953, is due, at least in part, to the presence of a heteropolysaccharide which has an unusually high arabinose and fucose content. In the absence of other antimetabolic compounds the levels of this heteropolysaccharide present in G12953 are sufficiently high to account for the observed resistance. However, it is possible regarding seed resistance towards Z. subfasciatus, that α-amylase inhibitors may be having a synergistic effect.     

Role of different aberrant cell signalling pathways prevalent in acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is one of the major forms of leukemia that affects mostly adolescent individuals. The main cause of the development of ALL is not known though several important signal transduction pathways have been reported with functional abnormality in all the cases. Crucial signalling pathways reported in ALL include PI3K/Akt, Notch, Wnt, mTOR, JaK/Stat, etc. Over the past several decades important progress has been made in the management of ALL, however, relapses and post therapy survival ratio has not improved much. This brings the need for understanding the biology and mechanism involved in ALL occurrences and find new molecular targets for better treatment options and risk-adapted therapies to improve the outcome of ALL patients.     

Role of carbohydrates in proline accumulation in wilted barley leaves

The effect of wilting on proline synthesis, proline oxidation, and protein synthesis—all of which contribute to proline accumulation—was determined in nonstarved barley (Hordeum vulgare L.) leaves. Nonstarved leaves were from plants previously in the light for 24 hours and starved leaves were from plants previously in the dark for 48 hours. Wilted leaves from nonstarved plants accumulated proline at the rate of about 1 μmole per hour per gram of fresh weight whereas wilted leaves from starved plants accumulated very little proline. Wilting caused a 40-fold stimulation of proline synthesis from glutamate in nonstarved leaves but had very little effect in starved leaves. Proline oxidation and protein synthesis, on the other hand, were inhibited by wilting in both nonstarved and starved leaves. Thus, the role of carbohydrates in proline accumulation is to supply precursors for the stimulated proline synthesis. These results further indicate that the main metabolic response causing proline to accumulate in wilted barley leaves is the stimulation of proline synthesis from glutamate. The difference between these results and those obtained with beans is discussed.

Wilting caused an increased conversion of glutamate to other products. In nonstarved leaves, conversion to organic acids as well as to proline was increased. In starved leaves, wilting caused an increase in the conversion of glutamate to glutamine, aspartate, asparagine, and organic acids.

     

Role of carbohydrates in diurnal chilling sensitivity of tomato seedlings

Tomato seedlings (Lycopersicon esculentum Mill.) chilled starting at different times during the light/dark cycle were most chilling-sensitive at the end of the dark period (AI King, MS Reid, BD Patterson 1982 Plant Physiol 70: 211-214). Low-temperature tolerance was regained with as little as 10 minutes of light exposure. Low light intensities were less effective than high light intensities in reducing sensitivity, and the length of exposure to light directly influenced sensitivity. Seedlings kept at low night temperatures prior to chilling were also less injured following chilling. Light also restored chilling tolerance to seedlings whose roots were removed. Supplying cut shoots with sucrose, glucose, or fructose reduced chilling sensitivity and largely eliminated the diurnal difference in sensitivity. Endogenous carbohydrate content was correlated with changes in chilling sensitivity; starch and sugar content fell markedly during the dark period. Increased concentrations of sugars were detected 15 minutes after the start of the light period. This evidence all suggests that changes in chilling sensitivity over the diurnal period are regulated by the light cycle. It also suggests that increased sensitivity at the end of the dark period could be due to carbohydrate depletion, and that chilling tolerance following light exposure is likely due to carbohydrate accumulation or closely related events.