Antagonism by dibutyryl adenosine cyclic 3'',5''-monophosphate and testololactone of concanavalin A capping

Exposure of CHO-K1 cells in vitro to dibutyryl adenosine cyclic 3',5'-monophosphate (DBcAMP) plus testololactone produces a rapid, reversible antagonism of ligand-induced collection of initially dispersed concanavalin A (Con A) binding sites into a caplike mass. Morphologically, as Con A capping occurs, the cells become less spread and then round completely. With prolonged Con A exposure, cells cultured in either the absence or the presence of DBcAMP plus testololactone cap and round. Capping is blocked by cold treatment and respiratory inhibitors. Colcemid at concentrations greater than 1 muM promotes both Con A capping and cell rounding. Cytochalasin B at similar concentrations inhibits both capping and cell rounding. Treatment of cells with Con A has little effect on intracellular cAMP concentration. Possible mechanisms by which cAMP may modulate the movement of Con A binding sites are discussed.     

Activation of human lymphocytes by concanavalin A or purified protein derivative results in no alteration of fluorescence polarization of lipid probes although the electrophoretic mobility of the cells is changed

Upon stimulation with either concanavalin A or the tuberculin antigen, purified protein derivative, human peripheral blood lymphocytes, purified on Ficoll-Hypaque, did not exhibit a concomitant lipid fluidity alteration as measured by fluorescence polarization (P) of the lipid probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). This result was independent of the incubation period, ranging from 10 min to 72 h. However, a general reduction in polarization value, from P = 0.287 (maintained for up to 2 h of incubation) to P = 0.225 after 20 h was observed for both experimental and control samples. Moreover, fluorescence polarization studies of the nonpenetrating modified DPH cationic lipid probe, 1-[4′-trimethylaminophenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH), also failed to show any change in lipid fluidity subsequent to a 1–3 h incubation of lymphocytes with concanavalin A. Cell electrophoretic mobility, however, was altered (mean cell mobility increased by 10–15%) in a fast response to stimulation and was observed within several hours of in vitro application of concanavalin A and purified protein derivative. This initial response disappeared with further incubation at 37°C (>3 h) and was followed by a decline of cellular mobility of the concanavalin A-exposed cells after 48 and 72 h of incubation. The unstimulated control cells did not change in mobility as a function of incubation time. The slow decline in mean cell mobility of the experimental cells is believed to be associated with blastogenesis. It is concluded that neither blastogenic transformation nor short term membrane alterations associated with human lymphocyte activation lead to lipid fluidity changes as measured in steady state by the fluorescence polarization of both DPH and TMA-DPH.     

Ordering of bulk membrane lipid or protein promotes activity of plasma membrane Mg2+ATPase

Treatment of liver plasma membranes with phospholipase A2 or high doses of concanavalin A enhances the activity of Mg2+ATPase assayed at temperatures greater than 30 degrees C. The effects of the two treatments are not additive. Both the removal of phospholipids and binding of the lectin increase the degree of polarization of fluorescence of the lipid-soluble fluorophores, diphenylhexatriene and beta-parinaric acid, suggesting that decreased lipid fluidity may activate Mg2+-ATPase. In fact modification of lipid fluidity by reconstitution of phospholipase-treated membranes with phosphatidylcholines of defined fatty acid composition or by addition of cis-vaccenic acid showed a strong inverse correlation between Mg2+ATPase activity and lipid fluidity as monitored by fluorescence polarization. However, despite the ability of concanavalin A to nonspecifically order membrane lipid, its effect on Mg2+ATPase is apparently not mediated in this manner because other enzyme-activating lectins such as Ricinus communis agglutinin and wheat germ agglutinin are without effect on lipid fluidity. The facts that lectins of lower valency than tetravalent native concanavalin A such as divalent succinyl concanavalin A are far less effective in activating the enzyme and that paraformaldehyde treatment also activates suggests that cross-linking of membrane proteins is responsible. Hence, the diminution in activity of this membrane enzyme due to the disordering effect of heat in the physiological temperature range can be counteracted by isothermally increasing the order of either membrane lipid or protein.     

Effect of pentoxifylline on developmental changes in neutrophil cell surface mobility and membrane fluidity

Polymorphonuclear leukocytes (PMNs) from human neonates respond less efficiently to chemotactic factor stimulation than do PMNs from adults. The biologic mechanisms underlying this developmental process are poorly understood. In previous studies, we have found that pentoxifylline, an agent report to enhance membrane deformability, increased the chemotactic response of neonatal PMNs. In the present studies, we have examined the effect of pentoxifylline on cell surface mobility and membrane fluidity by assessing fluorescent concanavalin A (Con A) capping and fluorescent polarization (FP). Baseline Con A capping was lower in the PMNs of neonates when compared to PMNs from adult controls. Colchicine, which increases capping by disrupting microtubules, exaggerated the differences between the adult and neonatal PMNs. Following exposure of neonatal PMNs to pentoxifylline, colchicine enhanced Con A capping to levels equivalent to those of colchicine-treated PMNs from adults. Employing a fluorescence polarization (FP) assay, we found the fluid state of the membrane of PMNs from neonates was significantly less than that of adult controls. Pentoxifylline alone significantly increased the fluidity of the cell membranes of neonatal PMNs while decreasing elevated basal levels of F-actin in the cell. These data suggest an intrinsic cytoskeletal difference in the PMNs of neonates that may be responsive to pharmacologic manipulation.     

Capping and mitogenesis: a model implicating microfilaments in lymphocyte activation

In lymphocytes cap formation induced by concanavalin A (con A) was found to be concentration dependent on the mitogen in the presence of colchicine, a microtubule disrupting agent. The dose-respone of cap formation under these conditions was similar to mitogen dose-response. In addition, a direct correlation was found between con A capping induced in the presence of colchicine and mitogenic responses with con A alone. Agents such as dibutyryl cyclic AMP, which suppress mitogenic responses, decrease capping. Zinc increases capping when it causes enhancement of mitogenesis and decreases capping when it suppresses mitogenic response. These observations are interpreted on the basis of a model in which binding of con A to surface receptors leads to formation of microfilaments, which might be essential for capping as well as the initiation of DNA synthesis. Thus, the experimental observations in this report lend support to a model implicating the formation of microfilaments as a crucial event in triggering a variety of cellular responses following ligand binding.     

Adriamycin-induced changes in the surface membrane of sarcoma 180 ascites cells

Adriamycin increases (a) the rate of agglutination of Sarcoma 180 cells by concanavalin A after brief exposure of 2–3 h and (b) membrane fluidity as measured by ESR within 30 min of exposure at concentrations of the anthracycline of 10^?7–10^?5 M. The effect of adriamycin on agglutination is not due to an increase in the number of surface receptors for concanavalin A, since the extent of binding of the lectin is not altered by adriamycin and no change occurs in the rate of occupancy of the concanavalin A binding sites by the lectin in cells treated with the antibiotic. The order parameter, a measurement of membrane fluidity, decreases in cells exposed to adriamycin and is dose-related. The results indicate that adriamycin can induce changes in the surface membrane of Sarcoma 180 cells within a brief period of exposure to a low but cytotoxic level of this agent.     

Stimulation of calcium pump activity in heart sarcolemma by timolol

The effects of beta-adrenergic blocking agents, timolol and atenolol (1-1000 microM), were studied on rat heart sarcolemmal ATPase and Ca2+ binding activities. Timolol, unlike atenolol, increased both Ca2+-stimulated ATPase and ATP-dependent Ca2+ binding; the maximal effects were seen at 1 microM concentration of timolol. Both timolol and atenolol did not alter the sarcolemmal Mg2+ ATPase and nonspecific Ca2+ binding activities. Sarcolemmal Ca2+-stimulated ATPase was also activated by concanavalin A (6-66 micrograms/mL) which is known to alter membrane fluidity; however, Mg2+ ATPase was unaffected by this agent. These results indicate that timolol may stimulate Ca2+ pump activity in heart sarcolemma by changing membrane fluidity in a manner similar to that of concanavalin A.     

Fluorescence polarization study on the increase of membrane fluidity of human erythrocyte ghosts induced by synthetic water-soluble polymers

The effect of water-soluble polymers on the membrane fluidity of human erythrocyte ghosts was investigated and was compared with that of concanavalin A by means of the fluorescence polarization technique. 8-Anilino-1-naphthalene sulfonic acid sodium salt and 1,6-diphenyl-1,3,5-hexatriene were used as probe molecules. The membrane fluidity was increased by the addition of polycations with concentrations of less than $\text{2 · 10}{}^{\mathrm{?3}}$ wt% 60 min after mixing. The fluidity changes were affected by the chemical structure (hydrophobicity, charge density, etc.) of polycations. Thus, the membrane fluidity increased markedly with increasing charge density on the chain backbone of polycations. On the other hand, nonionic polymers such as poly(ethylene glycol) and $\text{poly}\text{(N-}\text{vinyl}\text{-2-}\text{pyrrolidone}$) changed the membrane fluidity in a biphasic manner. That is, the fluidity of human erythrocyte ghost was temporarily increased and then decrease. For example, 20 wt% of poly(ethylene glycol) gave a maximum fluidity 15 min after mixing with erythrocyte ghosts. A similar fluidity change was observed by adding concanavalin A. Such fluidity changes were not observed when lipid bilayer vesicles were used instead of cell membranes. These results suggested that the increase of membrane fluidity resulted from the intramembraneous aggregation of membrane-bound proteins which was induced by the added polymers. Cell agglutination was also induced by the addition of a large amount of polymers. This agglutination was considered to be due to the intermembraneous aggregation of membrane-bound proteins.     

Synthesis and pharmacological evaluation of some 3-phenyl-2-substituted-3H-quinazolin-4-one as analgesic, anti-inflammatory agents

A variety of novel 3-phenyl-2-substituted-3H-quinazolin-4-ones were synthesized by reacting the amino group of 2-hydrazino-3-phenyl-3H-quinazolin-4-one with different aldehydes and ketones. The starting material 2-hydrazino-3-phenyl-3H-quinazolin-4-one was synthesized from aniline. The title compounds were investigated for analgesic, anti-inflammatory and ulcerogenic index activities. While the test compounds exhibited significant activity, compounds, 2-(N'-2-butylidene-hydrazino)-3-phenyl-3H-quinazolin-4-one (AS1), 2-(N'-3-pentylidene-hydrazino)-3-phenyl-3H-quinazolin-4-one (AS2) and 2-(N'-2-pentylidene-hydrazino)-3-phenyl-3H-quinazolin-4-one (AS3), exhibited moderate analgesic activity. The compound 2-(N'-2-pentylidene-hydrazino)-3-phenyl-3H-quinazolin-4-one (AS3) showed more potent anti-inflammatory activity when compared to the reference standard diclofenac sodium. Interestingly, the test compounds showed only mild ulcerogenic side effect when compared to aspirin.     

Concanavalin A- and fetal-calf-serum-induced rounding and myosin light chain phosphorylation in cultured smooth muscle cells

Rabbit aorta smooth muscle cells (SMC) in long-term culture retracted in less than 10 min in response to a sequential order of stimulations by concanavalin A (Con A) and fetal calf serum (FCS). With additional continuous stimulation by FCS, the SMC took on a circular shape and were anchored to the substrate by retraction fibrils. This rounding was observed only when the cells were sequentially stimulated by Con A and FCS. Depletion of intracellular Ca2+ stores by the addition of EGTA and Ca2+ ionophores inhibited the rounding. Transient phosphorylation of MLC20 was observed in the initial stage during the SMC rounding. The extent of monophosphorylated MLC20 increased for up to 5 min to a maximal value of 49%. The diphosphorylated form reached a maximal value of 29% within 2 min; then both forms of MLC20 decreased. The process of the SMC rounding was inhibited by antimycin A or cytochalasins, in a dose-dependent manner, findings which suggested a dependency on both metabolic energy and actin-containing microfilaments. The smooth-muscle-relaxing agent, HA1077, also inhibited the process of SMC rounding. These observations suggest that a cellular contractile process might be involved in rounding of SMC.     

Rapid alterations in cellular morphology and plasma membrane structure induced in rat thymocytes by mitogenic stimuli.

Under conditions where a maximum stimulation of 3-O-methyl-glucose transport is observed, three thymocyte mitogens (concanavalin A, ionophore A23187 and hydrogen peroxide) cause cell rounding and a decrease in the density of intra-membrane particles on the plasma membrane. The early effects of mitogens on the thymocyte plasma membrane are similar to those of osmotic shock.     

Similarities in the membrane fluidity of 3T3 and SV101-3T3 cells and its relation to concanavalin A- and wheat germ agglutinin-induced agglutination.

Intact, viable ultransformed 3T3 and transformed SV101-3T3 cells were labeled with fatty acid spin labels and with 2,2,6,6-tetramethylpiperidine-1-oxyl in order to measure the fluidity properties of membrane lipids. Both cell types were grown in regular calf serum and in a lipid-depleted serum supplemented with either oleate or elaidate. The temperature dependence of the spectra obtained revealed inflections that correlate with the temperature below which agglutination with concanavalin A is inhibited, and another inflection that correlates with the temperature below which agglutination with wheat germ agglutinin is inhibited, suggesting that (a) the lipid phase(s) in the vicinity of the receptor(s) for these two lectins differ, and (b) a fluid membrane in the vicinity of the lectin receptor(s) is necessary for agglutination with either concanavalin A or wheat germ agglutinin. Studies with a partially characterized plasma membrane fraction suggest that the plasma membrane fluidity parameters closely resemble those of the intact cell. 3T3 and SV101-3T3 cells show virtually identical fluidity profiles by all of the tests we have applied.     

Microsomal desaturation of stearic acid in relation to lymphocyte activation

The conversion of stearic acid to oleic acid (delta 9-desaturase) was followed in mouse thymocytes stimulated by either concanavalin A or concanavalin A + interleukin-2 resulting in different rates of cell proliferation. To estimate the plasma membrane turnover of oleic acid as compared to that of a saturated fatty acid, double-label experiments ([14C]oleic acid, [3H]palmitic acid) were performed. Following an inhibition delta 9-desaturase was found to be activated from the fourth hour of stimulation. In the early period of cell activation this process proved to be independent of protein synthesis, whereas in the stage of proliferation it was dependent on it. Increased membrane fluidity in the first 30 min of activation is not likely due to enrichment of oleic acid. Cell proliferation and microsomal desaturation seem to be coupled and an increasing amount of oleic acid is at least one of the factors resulting in increased fluidity of the surface membrane of proliferating cells.     

Synthesis of new N-phenylpyrazole derivatives with potent antimicrobial activity

The versatile synthons 4-(2-bromoacetyl)-5-methyl-1-phenyl-3-phenylcarbamoyl-1H-pyrazole (3) and 4-[(E)-3-(dimethylamino)acryloyl]-5-methyl-1-phenyl-3-phenylcarbamoyl-1H-pyrazole (2) were used as precursors for the synthesis of a series of phenylpyrazoles with different aromatic ring systems at position 4. The antimicrobiological evaluation of the newly synthesized compounds was carried out in vitro assays for antifungal and antibacterial activities. Amongst the tested compounds, 4-acetyl-5-methyl-1-phenyl-3-phenylcarbamoyl-1H-pyrazole (1), 4-[(E)-3-(dimethylamino)acryloyl]-5-methyl-1-phenyl-3-phenylcarbamoyl-1H-pyrazole (2), 4-(2-bromoacetyl)-5-methyl-1-phenyl-3-phenylcarbamoyl-1H-pyrazole (3) and 4-(2-aminothiazol-4-yl)-5-methyl-1-phenyl-3-phenylcarbamoyl-1H-pyrazole (17) showed interesting antimicrobial properties. In particular, all tested compounds produced inhibitory effects against pathogenic yeast (Candida albicans) similar or superior to those of reference drug. In addition, compound 3 showed excellent activity against pathogenic mould (Aspergillus). From structure-activity relationship (SAR) point of view, the attachment of bromoacetyl moiety to pyrazole ring can be considered as a breakthrough in developing a new therapeutic antifungal agent related to phenylpyrazole system.     

Duration of the calcium signal in the mitogenic stimulation of thymocytes.

An increase in the free cytoplasmic Ca2+ concentration in thymocytes can be detected by the fluorescent indicator quin 2 within a few seconds of the addition of concanavalin A and the response is quantified from the increased proportion of quin 2 in the cells chelated by Ca2+ ('% Ca-quin 2'). The % Ca-quin 2 in untreated cells is 53 +/- 6%, increases to 64 +/- 7% immediately after the addition of concanavalin A and declines spontaneously over 24 h back to the level in untreated cells (53 +/- 6%). The increase in % Ca-quin 2 in response to concanavalin A is completely blocked when 50 mM-alpha-methyl D-mannoside is added before concanavalin A and completely reversed when the competing sugar is added immediately after the mitogen. Addition of alpha-methyl D-mannoside at increasing intervals after concanavalin A addition causes a progressively smaller decrease in % Ca-quin 2 and has a negligible effect after 24 h, when the % Ca-quin 2 is the same as that in untreated cells. The decline in the calcium signal defined by these experiments has a similar time course to cap formation by concanavalin A on the cells. It is concluded that the calcium signal lasts only while concanavalin A is bound to the cell surface and is terminated either by capping or by the addition of alpha-methyl D-mannoside.     

Identification of two additional translation products from the matrix (M) gene that contribute to vesicular stomatitis virus cytopathology

The matrix (M) protein of vesicular stomatitis virus (VSV) is a multifunctional protein that is responsible for condensation of the ribonucleocapsid core during virus assembly and also plays a critical role in virus budding. The M protein is also responsible for most of the cytopathic effects (CPE) observed in infected cells. VSV CPE include inhibition of host gene expression, disablement of nucleocytoplasmic transport, and disruption of the host cytoskeleton, which results in rounding of infected cells. In this report, we show that the VSV M gene codes for two additional polypeptides, which we have named M2 and M3. These proteins are synthesized from downstream methionines in the same open reading frame as the M protein (which we refer to here as M1) and lack the first 32 (M2) or 50 (M3) amino acids of M1. Infection of cells with a recombinant virus that does not express M2 and M3 (M33,51A) resulted in a delay in cell rounding, but virus yield was not affected. Transient expression of M2 and M3 alone caused cell rounding similar to that with the full-length M1 protein, suggesting that the cell-rounding function of the M protein does not require the N-terminal 50 amino acids. To determine if M2 and M3 were sufficient for VSV-mediated CPE, both M2 and M3 were expressed from a separate cistron in a VSV mutant background that readily establishes persistent infections and that normally lacks CPE. Infection of cells with the recombinant virus that expressed M2 and M3 resulted in cell rounding indistinguishable from that with the wild-type recombinant virus. These results suggest that M2 and M3 are important for cell rounding and may play an important role in viral cytopathogenesis. To our knowledge, this is first report of the multiple coding capacities of a rhabdovirus matrix gene.     

Changes of membrane fluidity in chemotactic peptide-stimulated polymorphonuclear leukocytes

Although the phenomenon of stimulus-response coupling in polymorphonuclear leukocytes involves a series of membrane events the influence of stimulation on membrane fluidity is to clarify. In our experiments we have used 1-(4-trimethylaminophenyl) 6-phenyl-1,3,5-hexatriene and 1,6-diphenyl-1,3,5-hexatriene fluorescence polarization technique to evaluate membrane fluidity in living polymorphonuclear leukocytes after stimulation with N-formyl-methyonil-leucyl-phenylalanine peptide which has a well defined membrane receptor on the plasma membrane. We report that polymorphonuclear leukocytes stimulation increases 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene polarization, only when colcemid, a microtubule disrupting drug, is added to polymorphonuclear leukocytes. This can be viewed as an indirect evidence that microtubules are involved in the control of polymorphonuclear leukocytes membrane fluidity. On the contrary no changes have been observed with 1,6-diphenyl-1,3,5-hexatriene. This study indicates the potential use of 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene to evaluate the involvement of plasma membrane physical state during intact cell activity.     

Virulence and Functions of Myosin II Are Inhibited by Overexpression of Light Meromyosin in Entamoeba histolytica

Several changes in cell morphology take place during the capping of surface receptors in Entamoeba histolytica. The amoebae develop the uroid, an appendage formed by membrane invaginations, which accumulates ligand–receptor complexes resulting from the capping process. Membrane shedding is particularly active in the uroid region and leads to the elimination of accumulated ligands. This appendage has been postulated to participate in parasitic defense mechanisms against the host immune response, because it eliminates complement and specific antibodies bound to the amoeba surface. The involvement of myosin II in the capping process of surface receptors has been suggested by experiments showing that drugs that affect myosin II heavy-chain phosphorylation prevent this activity. To understand the role of this mechanoenzyme in surface receptor capping, a myosin II dominant negative strain was constructed. This mutant is the first genetically engineered cytoskeleton-deficient strain of E. histolytica. It was obtained by overexpressing the light meromyosin domain, which is essential for myosin II filament formation. E. histolytica overexpressing light meromyosin domain displayed a myosin II null phenotype characterized by abnormal movement, failure to form the uroid, and failure to undergo the capping process after treatment with concanavalin A. In addition, the amoebic cytotoxic capacities of the transfectants on human colon cells was dramatically reduced, indicating a role for cytoskeleton in parasite pathogenicity.     

ANTAGONISM BY DIBUTYRYL ADENOSINE CYCLIC 3',5'-MONOPHOSPHATE AND TESTOSTERONE OF CELL ROUNDING REACTIONS

In an attempt to understand further the mechanism of the morphological and functional "reverse transformation" of CHO-K1 cells induced by dibutyryl adenosine cyclic 3',5'-monophosphate (cAMP) and testosterone, the kinetics of variation in the susceptibility of cells to rounding after the addition or deletion of dibutyryl cAMP and testosterone have been investigated. Changes in susceptibility to cell rounding upon removal of divalent cations or pulse exposure to concanavalin A were complete within 0.5–1 h after addition or deletion of drug. In comparison, the gross conversion of CHO-K1 cells from epithelial- to fibroblast-like morphology after drug treatment or the converse change after drug removal required 8 or 4 h, respectively. The effects on cell rounding are not caused by an effect of dibutyryl cAMP upon cell growth rate. Inhibitor experiments indicate that the changes investigated do not require continued RNA or protein synthesis and are not prevented by agents which depolymerize microtubules.