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1.
Plant lectins and other agents which are mitogenic for lymphocytes and fibroblasts were tested for their effects on DNA synthesis in primary monolayer cultures of neural retina cells from 10-day chick embryos. Concanavalin A (ConA), phytohemagglutinin (PHA), wheat germ agglutinin (WGA), and anti-retina cell antiserum significantly stimulated [3H]TdR incorporation; the maximum increase was reached 15 h after exposure of the cultures to these agents. Cells stimulated by ConA to synthesize DNA subsequently divided. The divalent succinyl derivative of ConA had a considerably lesser effect than the native tetramer, suggesting that cross-linking of cell surface components may be an important aspect of the changes that lead to the stimulation of DNA synthesis in these cells.Using [125I]ConA, the average number of ConA-binding sites per 10-day retina cell was estimated to be 1.7 × 106 (under the culture conditions employed); binding of the lectin to 25–50% of these sites was sufficient to elicit the maximal stimulation of DNA synthesis. Continuous association of the lectin with the cell surface for up to 8 h was essential for the maximal effect, since removal of the lectin from the cell surface (with α-methyl mannose) prior to this time reduced or prevented the stimulation of DNA synthesis.The stimulation by ConA of DNA synthesis in these cultures was dependent on the cell density and was reduced or absent at lower than optimal densities. Examination of this effect suggested that the frequency of intercellular contacts or specific cell associations play a role in the responsiveness of these cells to stimulation of DNA synthesis by ConA. 相似文献
2.
Normal human peripheral blood granulocytes which are tagged with 1-fluoro-2,4-dinitrobenzene (DNFB) are agglutinated by concanavalin A (ConA) in a way which resembles the pattern of reactivity displayed by leukemic cells. The present study further defines this reaction. The binding of ConA to untagged and DNP-tagged granulocytes, treated with DNFB at a ratio of 1011 molecules/cell, was quantified by isotopic dilution experiments employing [3H]ConA. Similar amounts of the lectin were bound to untagged and DNP-tagged cells following incubation for 5 min at 4 °C or 30 min at 24 °C: 1.1 × 105 molecules/cell, 4.6 × 102/μ2 of surface area, and 1.6 × 103/μg of protein. The binding of [3H]ConA to both untagged and DNP-tagged cells was inhibited to the same degree by α-methylglucopyranoside (α-MG). Fixation with either glutaraldehyde or formaldehyde, which immobilizes ConA receptor sites, completely inhibited the agglutination of both untagged and DNP-tagged cells although lectin binding was unchanged. This suggests that the inhibition of agglutination was not due to the blocking of ConA-binding sites by aldehyde groups but rather to the immobilization of lectin receptors. We conclude that dinitrophenylation of normal granulocytes facilitates the rearrangement of lectin receptors in a way which resembles the ConA-induced clustering of sites which have been observed with malignant and transformed cells. 相似文献
3.
观察了ConA对培养软骨细胞PG合成代谢的影响。证实ConA能够使培养的软骨细胞高分子硫酸化PG的合成增加3~4倍,其分子量、硫酸化部位和硫酸化程度与对照组相比无明显差异,是具有正常结构的软骨型PG。ConA对低分子型PG的合成未见明显的影响。ConA促进PG合成的作用可由MeMan完全解除,比具有同样效应的激素、生长因子都强,并有明显的凝集素特异性。推测ConA的作用可能与软骨细胞膜或细胞内的分化诱导因子的受体或软骨中存在的ConA软骨细胞分化因子有关。 相似文献
4.
Cell surface changes in transformed human lymphocytes : I. ConA and E-PHA induced unique changes in surface topography 总被引:1,自引:0,他引:1
Stephen F. Speckart David H. Boldt Katherine L. Ryerson 《Experimental cell research》1978,111(2):385-395
Binding studies with six purified plant lectins were used to investigate membrane alterations that occur in lymphocyte transformation. Normal human peripheral blood lymphocytes transformed with E-Phytohemagglutinin (E-PHA) or concanavalin-A (Con-A) generally possessed increased numbers of lectin receptors. When this increase was corrected for the expanded surface area of transformed lymphocytes, it appeared that E-PHA and ConA each produced a unique and complex reorganization of cell surface topography. Surface alterations occurred independently of DNA synthesis, cell proliferation, and microtubule or microfilament function. Puromycin inhibited emergence of new lectin receptors on cells transformed with E-PHA, but not with ConA. Lymphocytes incubated with either lectin showed increased incorporation of [14C]galactose into trypsin-sensitive cell surface glycoproteins. This incorporation was abolished by puromycin in cells stimulated by E-PHA but not by ConA. These studies demonstrate that although both lectins induce similar morphological alterations in human lymphocytes, at the molecular level the structural changes induced in the cell membrane by these two lectins differ considerably. Furthermore, these structural alterations are mediated via different mechanisms in the two groups of cells. De novo protein synthesis is required for cell surface reorganization in PHA-stimulated cells, but not in cells stimulated by ConA. The effect of ConA appears to be to enhance attachment of saccharide structures to pre-synthesized membrane proteins. 相似文献
5.
Previous drug studies have suggested that concanavalin A (ConA)-induced cytoagglutination may be influenced by a system of contractile microfilaments. The present study was undertaken to determine the effects of microfilament-active drugs (cytochalasins B and D) (CB and CD) and inhibitors of ATP formation (NaN3 and 2,4-dinitrophenol (DNP)) on ConA-induced agglutination of Novikoff cells and to investigate the mechanism(s) whereby these agents alter the surface properties of cells. The study described herein demonstrated that
- 1. 1. CB or CD inhibit cytoagglutination at concentrations above 1 or 0.1 μg/ml, respectively.
- 2. 2. NaN3 and DNP both inhibit cytoagglutination, but DNP is more effective and specific.
- 3. 3. Combinations of metabolic inhibitors (NaN3 or DNP) with CB lead to a greater reduction of cytoagglutination than with either agent alone.
- 4. 4. Maximal (>95%) cytoagglutination is still achieved in the presence of CB, CD, NaN3, DNP, or combinations of these drugs.
- 5. 5. None of the drugs tested induced or allowed the redistribution of ConA receptors as measured by ferritin-ConA labeling.
- 6. 6. Both CB and CD induced the appearance of numerous blebs (zeioses) at the cell periphery, whereas metabolic inhibitors did not.
- 7. 7. The concentration of either CB or CD required to alter cell-surface morphology paralleled the concentration necessary to inhibit cytoagglutination.
6.
Matthew D. Ralston James L. DeMartino Rawleigh C. Howe Robert E. Humphreys 《Experimental cell research》1979,123(2):237-245
The 70 membrane proteins of the T lymphoblast cell line CCRF-CEM were characterized by
- 1. 1. [35S]methionine internal radiolabeling;
- 2. 2. [125I]iodine labeling by a lactoperoxidase-mediated method;
- 3. 3. [3H]fucose internal labeling;
- 4. 4. binding to a lentil lectin adsorbant column;
- 5. 5. susceptibility to digestion with limited amounts of papain.
7.
B.A.M. Renckens S.E. Van Emst-De Bries J.J.H.H.M. De Pont S.L. Bonting 《Biochimica et Biophysica Acta (BBA)/General Subjects》1980,630(4):511-518
- 1. 1. The effect of stimulation of adenylate cyclase by pancreozymin-C-octapeptide on the cyclic AMP level of rat pancreatic fragments has been investigated.
- 2. 2. In normal Krebs-Ringer bicarbonate medium pancreozymin-C-octapeptide causes a slight increase in pancreatic cyclic AMP level; this increase can be considerably enhanced by incubation in a calcium-free incubation medium.
- 3. 3. The dose-responce curve for pancreazymin-C-octapeptide in calcium-free medium is shifted to lower peptide concentrations, compared to the curve in normal Krebs-Ringer bicarbonate medium.
- 4. 4. The maximal stimulatory effect of pancreozymin-C-octapeptide id obtained at a 1-methyl-3-isobutylxanthine concentration of 10 mM.
- 5. 5. It suffices to lower the Ca2+-concentration of the medium from 2.5 to 1.5 mM to get the maximal increase in cyclic AMP content under influence of pancreozymin-C-octapeptide.
- 6. 6. It is concluded that extracellular calcium antagonizes the stimulation of adenylate cyclase by pancreozymin-C-octapeptide. This suggest that a low cytoplasmic Ca2+-concentration is required for the maximal response of acinar cell adenylate cyclase to pancreozymin.
Keywords: cyclic AMP formation; Ca2+; Pancreozymin-C-octapeptide; Adeny; ate cyclase; (Rat pancreas) 相似文献
8.
Cell surface interactions with concanavalin A : Location of bound radiolabeled lectin 总被引:4,自引:0,他引:4
We have developed two improved methods: (1) a procedure for coupling 125Iodine to ConcanavalinA (ConA) that yields intensely labeled and fully active lectin; (2) a procedure that allows studies of lectin binding to be carried out with a minimum of non-specific binding to reaction vessels. We found that BALB/c 3T3 cells, SV3T3 cells, and human red blood cells have 1.3 × 107, 1.5 × 107, and 2.2 × 106 ConA binding sites/cell. More than 99.5% of the radioactivity in the samples counted was associated with the cells; background radioactivity, in the absence of cells, was negligible. We also found that although α-methylmannopyranoside (α-MM) prevented almost all of the ConA from binding to cells, when ConA had first been allowed to bind, α-MM removed only 60 to 80% of the bound ConA. In addition, even after the removal of a portion of bound lectin by α-MM, most, if not all, of the remaining cell-associated ConA was coupled to the plasma membrane. 相似文献
9.
Seven different tissue culture cells have been cultured with and without mycoplasma (M. hyorhinis) in the presence of various precursors of RNA. Total cellular RNA was isolated and analysed by electrophoresis on polyacrylamide gels. The results obtained with mycoplasma-infected cells can be summarized as follows:
- 1. 1. When cells are labelled with [8-3H]guanosine or [5-3H]uridine there is some incorporation into host cell 28S and 18S rRNA, but it is less than into mycoplasma 23S and 16S rRNA. [8-3H]guanosine or [5-3H]uridine are also incorporated into host cell and mycoplasma tRNA and mycoplasma 4.7S RNA, but the incorporation into host cell 5S rRNA and low molecular weight RNA components (LMW RNA) is reduced.
- 2. 2. [5-3H]uracil is not incorporated into host cell RNA but into mycoplasma tRNA, 4.7S RNA, a mycoplasma low molecular weight RNA component M1 and 23S and 16S rRNA.
- 3. 3. [3H]methyl groups are incorporated into mycoplasma tRNA, 23S and 16S rRNA, but not into host cell 28S, 18S, 5S rRNA nor into mycoplasma 4.7S RNA.
- 4. 4. With [32P]orthophosphate or [3H]adenosine as precursors, the labelling is primarily in the host RNA.
10.
- 1. 1. Cu2+ at a concentration of 10−4 M, when applied to the external side of the frog skin produces an increase in the short-circuit current (Isc).
- 2. 2. This effect was studied in skins of Rana temporaria adapted to cold (5°C) and room temperature (20°C), skins of Rana pipiens adapted to cold, and the results compared with those obtained previously with Rana ribibunda.
- 3. 3. The observed effect is less dependent upon the adaptation to cold than upon the functional state of the skin: skins with low short circuit currents have a bigger response to Cu2+ than skins with high Isc.
- 4. 4. A species difference cannot be ruled out since skins of Rana ribibunda exhibiting high Isc give good responses to Cu2+.
- 5. 5. 5,5′-dithiobis(2-nitrobenzoic acid), a sulphydryl-oxidizing reagent, produces an effect similar to that of Cu2+, and dithiothreitol an SH-reducing agent, reverses the effect of this ion.
- 6. 6. Cu2+ also induces an increase in the unidirectional K+ fluxes and unmasks a net outward potassium flux.
- 7. 7. The outward K+ flux induced by Cu2+ is sensitive to ouabain.
- 8. 8. It is concluded that Cu2+ increases the permeability of the external barrier of the frog skin to Na+ and K+, probably by reacting with SH groups.
Abbreviations: DTNB; 5; 5′-dithiobis(2-nitrobenzoic acid) 相似文献
11.
Differentiating cells of D. discoideum in suspension bind ConA. The proportion of the bound lectin which is competitively removed by methyl-α-
mannopyranoside decreases with the time of exposure. Ferritin conjugated ConA is seen to bind both to the cell surface and to be taken into the cells, the proportion of the ConA inside the cells increasing with time. The surface bound ConA is removed by washing with methyl-α-
mannopyranoside while the endocytosed ConA appears unaffected. It is suggested that much of the [125I]ConA, uncompetable by methyl-α-
mannopyranoside in our and other binding studies, may be this intracellular ConA. 相似文献
12.
The effects of enzymic treatment on the interactions between Zajdela's tumor cells and various lectins. Concanavalin A (ConA); Wheat Germ Agglutinin (WGA); Robinia lectin; have been studied. (1) The number of lectin-binding sites and the affinity constants were investigated. (2) The effects of the lectins on cell growth and [3H]thymidine incorporation were studied on untreated and enzyme-treated cells. It was observed that treatment of tumor cells with neuraminidase resulted in a change in the binding characteristics of each lectin. However, additional treatment of the cells with galactose oxidase had no further effect on lectin binding. ConA and Robinia lectin induced a decrease of the untreated tumor cell growth and a stimulation of the [3H]thymidine incorporation. This paradoxal result may be explained as a consequence of the stimulation of the [3H]thymidine uptake observed in the presence of lectins. The enzymatic treatments themselves did not change the cell growth although they did induce a change in the effect of ConA and Robinia lectin on cell growth and [3H]thymidine incorporation. As a result of neuraminidase treatment, the effects of ConA were totally suppressed but those of Robinia lectin only partially. Although WGA interacted with untreated and enzyme-treated cell surfaces, it had no effect on tumor cell growth nor [3H]thymidine incorporation. The results are discussed in terms of lectin transport. 相似文献
13.
- 1. 1. It has previously been demonstrated that an increase in extracellular Ca2+ concentration induces a transient increase in K+ permeability and associated hyperpolarization of the red cell membrane of the giant salamander, Amphiuma means. This phenomenon is analogous to the Ca2+-induced KCl loss observed in ATP-depleted human red cells and red cell ghosts.
- 2. 2. Histamine, which enhances the Ca2+-induced K+ loss from depleted human red cells, is without effect on this Ca2+-induced hyperpolarization of Amphiuma red cells.
- 3. 3. Promethazine (10 μM) and mepyramine (1 mM), which inhibit the Ca2+-induced K+ loss in depleted human red cells, also block the Ca2+-related hyperpolarization of Amphiuma erythrocytes.
- 4. 4. Chlorpromazine (25 μM), despite being a weak antihistamine, is equally effective in blocking the Ca2+-induced hyperpolarization of Amphiuma red cells.
- 5. 5. Ionophore A23187 causes a large and sustained Ca2+/K+-dependent hyperpolarization even in the presence of normal (1.8 mM) concentrations of Ca2+. This hyperpolarization is relatively insensitive to chlorpromazine and promethazine.
- 6. 6. The inhibition of the Ca2+-induced hyperpolarization of the Amphiuma red cell membrane by chlorpromazine and promethazine may be related to their properties as local anaesthetics.
Abbreviations: MOPS, morpholinopropane sulphonate 相似文献
14.
I. B. Pragnell D. J. Arndt-Jovin T. M. Jovin Barbara Fagg W. Ostertag 《Experimental cell research》1980,125(2)
We have measured the kinetics of specific globin mRNA and Friend virus (FV) RNA synthesis by hybridization to immobilized cDNA after induction of differentiation of two erythroleukemia cell lines (F4N, B8) by butyrate and Me2SO. The induction with butyrate in these cell lines occurs very rapidly (16–24 h). Cell cycle analysis was made of the populations throughout induction by flow cytofluorometry. The kinetics of commitment of cell populations to terminal differentiation by butyrate was determined by removal of inducer at various times and scoring of benzidine staining cells (hemoglobin producing). In addition, the cell cycle dependence of commitment was determined by flow sorting out of G1 and S+G2 cells various times after addition of inducer and scoring benzidine-stained colonies after growth in methylcellulose. Cells exposed to inducer were also sorted by cell cycle phase using an elutriator rotor. The amount of globin mRNA synthesis in the different cell populations was then determined.
- 1. 1. It was found that an 8–12 h period in butyrate was required before (a) globin specific mRNA was synthesized; and (b) commitment to differentiation occurred. The time course of globin mRNA synthesis was positively correlated with G1 arrest, as has been also found by others.
- 2. 2. The increase of FV RNA synthesis was not found during G1 arrest. It occurred early and before commitment.
- 3. 3. Commitment of cells to irreversible differentiation upon butyrate induction occurs only during the G1 phase of the cell cycle.
- 4. 4. Globin mRNA synthesis occurs first only in G1 cells.
- 5. 5. Globin mRNA is synthesized later in all phases of the cell cycle.
15.
- 1. 1. Evidence has been provided for the transfer of phosphatidyl[14C]choline and [3H]cholesterol between bovine serum albumin and cauda epididymal rat spermatozoa in Krebs-Ringer bicarbonate medium, which can promote sperm capacitation.
- 2. 2. An analysis of the lipid composition in both albumin and spermatozoa revealed that phospholipid levels decreased in the protein and increased by roughly comparable amounts in sperm cells during incubation in vitro.
- 3. 3. Cholesterol (free + ester) increased in albumin and decreased in spermatozoa. Changes in the amount of esterified cholesterol were solely responsible for the increase associated with albumin, whereas whole sperm cell extracts showed a significant decline in free cholesterol.
- 4. 4. The composition of albumin-bound fatty acids did not alter appreciably as a result of incubation with spermatozoa.
- 5. 5. Rates of [14C]palmitic acid utilization by spermatozoa suggest that lipid synthesis accounted for less than 5% of the changes observed under the conditions of this study.
- 6. 6. These results are interpreted as broadly supporting our previous proposal that lipid exchange between albumin and sperm cells is implicated in sperm capacitation in vitro. Specifically, the results are compatible with the idea that a decreased cholesterol/phospholipid ratio in the sperm plasma membrane facilitates this transformation.
Keywords: Capacitation; Lipid transfer; Plasma membrane; Serum albumin; (Rat sperm) 相似文献