Rotational relaxation time of concanavalin A bound to the surface membrane of normal and malignant transformed cells

Fluorescence polarization was used to determine the rotational relaxation time of fluorescein conjugated concanavalin A bound to the surface membrane of normal and malignant cells. The cells used were normal lymphocytes and malignant lymphoma cells, as examples of cells that are in suspension in vivo, and normal and simian virus 40-transformed fibroblasts, as examples of cells that form a solid tissue. The relaxation time of F-concanavalin A² in phosphate-buffered saline, pH 7.2, at 24 °C was 58 nseconds. Under the same conditions, the relaxation times of F-concanavalin A bound to cells were: 70 nseconds for normal lymphocytes, 160 nseconds for malignant lymphoma cells, 120 nseconds for normal fibroblasts and 73 nseconds for simian virus 40-transformed fibroblasts. When cells were treated with trypsin or glutaraldehyde before adding F-concanavalin A, trypsin treatment produced a decrease, whereas glutaraldehyde fixation produced an increase of these values. Inhibition of cap formation of concanavalin A binding sites on normal lymphocytes by treating the cells with sodium azide did not change the rotational relaxation time of concanavalin A bound to the cells. These results indicate that the carbohydrate-containing structures on the cells that bind concanavalin A are mobile. In cells that are in suspension in vivo, malignant transformation is associated with reduction in mobility of these sites. However, in cells that form a solid tissue, malignant transformation is associated with an increase in mobility of these sites. Determination of the rotational relaxation time of fluorescent probes bound to specific sites on cell membranes can thus be used to quantitate receptor mobility in relation to cell behaviour.     

Effect of Erythropoietin on the interaction of Concanavalin A with rat erythrocytes

Effect of Erythropoietin (Ep) on the interaction of Concanavalin A (Con A) with rat erythrocytes was studied using ¹²⁵I-labelled Con A. Binding of Con A to erythrocytes was dependent on time and cell concentration. Starvation caused an elevation of the lectin binding capacity of red cells which again came down towards the normal level on Ep administration to starved rats. Binding of Con A to erythrocytes decreased linearly with increasing concentration of Ep. Specificity of binding was confirmed by inhibition studies with -methyl-D-mannopyranoside (Me Man) Cells from the starved rats compared to those from normal and Ep treated animals were less prone to inhibition by this sugar analog. Positive cooperative binding of Con A to rat erythrocyte was observed at low concentration of Con A but was absent at higher lectin concentrations. Starvation caused an increase in the number of binding sites per cell which returned to normal level after Ep treatment. Under identical conditions, binding affinities were not much changed in these cells. Cells from the starved animals were more susceptible to agglutination compared to those from normal and Ep-treated rats. Microviscosity and cholesterol/phospholipid ratio of red cell membrane decreased in the starved animals which retraced its way back towards the normal level after Ep treatment.     

Concanavalin A induced apoptosis in murine macrophage PU5-1.8 cells through clustering of mitochondria and release of cytochrome c

Concanavalin A (ConA), normally a mitogen of T-lymphocytes, was found to be a cell cycle-independent apoptosis-inducing agent in cultured murine macrophage PU5-1.8 cells. This assertion is based on the following observations: (1) ConA increased the number of cells with hypo-diploid DNA in a dose dependent manner as revealed by flow cytometry; (2) ConA elicited DNA fragmentation and the cytotoxicity of ConA was suppressed by -D-methylmannoside which blocks the lectin site of ConA; (3) ConA was able to release cytochrome c (cyto c) into the cytosol of PU5-1.8 cells. When isolated mitochondria were incubated with ConA, release of cyto c was observed too. Interestingly, clustering of mitochondria was found in the cytosol under a confocal microscope after ConA treatment. When cells were incubated with ConA-FITC and subsequently with mitotracker red (a probe for mitochondria), co-localization of fluorescence signals was observed. These results suggest that ConA was delivered to the mitochondria, induced mitochondrial clustering and released cyto c. Our results also show that introduction of exogenous cyto c electroporationally into ConA-untreated cells elicited DNA fragmentation. On the other hand, introduction of specific antibody against cyto c into PU5-1.8 cells suppressed the ConA-mediated cell death. Taken together, our results indicate that ConA induced apoptosis in PU5-1.8 cells through mitochondrial clustering and release of cyto c and the release of cyto c was sufficient to elicit apoptosis in PU5-1.8 cells.     

A protein purified from pig brain accelerates the intermembranous translocation of mono- and dihexosylceramides,but not the translocation of phospholipids

By the use of an assay that measures the transfer of [³H]galactosylceramide from donor to acceptor liposomes, a protein has been purified 1683-fold from pig brain. The most purified fraction was purified to homogeneity as judged by electrophoresis on 15% polyacrylamide gel in the presence of sodium dodecyl sulfate. The protein has a molecular weight of 23000 as determined by the gel electrophoresis and 18500 as estimated by gel filtration through Sephadex G-75. The protein accelerates the transfer of labeled glycolipids at the following relative rates: 100 for glucosylceramide, 43 for lactosylceramide, 17 for galactosyldiglyceride, and 15 for galactosylceramide. The lipid-transfer stimulated by the protein is specific to glycolipids; the protein does not accelerate the transfer of labeled phosphatidylcholine and phosphatidylethanolamine from donor to acceptor liposomes.     

Stimulation of human platelets with concanavalin A involves phospholipase C activation.

In response to concanavalin A, cytoplasmic calcium movement was observed in human platelets, both in the presence of 1 mM Ca2+ or 1 mM EGTA in the medium. Concanavalin A also caused the activation of inositide turnover and the production of inositol phosphates, suggesting that activation of phospholipase C occurs. The mechanism by which concanavalin A stimulates phospholipase C does not depend on GTP-binding transducers, because it was not inhibited by GDP beta S, while experiments performed in the presence of cytochalasin B suggested a role for membrane glycoprotein IIb-IIIa-cytoskeleton interaction in this process. Ca(2+)-proteases and Na+/H+ antiport also seemed to be related to concanavalin A-induced phospholipase C activation, as suggested by experiments performed in the presence of leupeptin and amiloride.     

Adhesion of sea-urchin embryonic cells to substrata coated with cell adhesion molecules

Summary— A cell-to-substratum adhesion assay is developed to study the adhesion of sea-urchin embryonic cells to coated substrata. The involvement in this process of both carbohydrate and protein molecules is reported. Concanavalin A (Con A) increases the attachment of cells to the substratum in a dose-dependent manner and this effect is completely abolished when the incubation is carried out in the presence of the specific monocarbohydrate Con A-inhibitor, α-methyl-d -mannoside. A Con A-mediated enhancement of cell-to-substratum adhesion was also detected on cells deprived of toposome, a glycoprotein complex responsible for cell-to-cell adhesion. The involvement of other molecules as well as toposome in the process of cell-to-substratum adhesion is also investigated. Results of these in vitro experiments indicate that all the molecules tested contribute to the process of cell-to-substratum adhesion.     

以伴刀豆球蛋白为介质定向固定化脲酶的研究

将戊二醛将伴刀豆球蛋白(ConA)和壳聚糖载体交联, 然后利用ConA与脲酶糖链的特异性结合作用, 实现脲酶的定向固定化。定向固定化的最适条件为戊二醛浓度3.5%、ConA浓度1 mg/mL、ConA溶液pH值7.0、脲酶浓度 0.4 mg/mL。定向固定化脲酶的最适pH 5.0~6.0、最适温度77°C、米氏常数Km11.76 mmol/L, 与游离酶及非定向固定化脲酶比较, 定向固定化脲酶的最适pH向酸性范围发生了偏移并有更宽的pH适用范围, 最适温度提高, 与底物的亲和力较大, 且有较好的操作稳定性。     

以伴刀豆球蛋白为介质定向固定化脲酶的研究

将戊二醛将伴刀豆球蛋白(ConA)和壳聚糖载体交联,然后利用ConA与脲酶糖链的特异性结合作用,实现脲酶的定向固定化.定向固定化的最适条件为戊二醛浓度3.5%、ConA浓度1mg/mL、ConA溶液pH值7.0、脲酶浓度0.4mg/mL.定向固定化脲酶的最适pH 5.0～6.0、最适温度77℃,米氏常数Km11.76mmol/L,与游离酶及非定向固定化脲酶比较,定向固定化脲酶的最适pH向酸性范围发生了偏移并有更宽的pH适用范围,最适温度提高,与底物的亲和力较大,且有较好的操作稳定性.     

A comparison of the effects of various stimulatory agents on t-PA secretion by normal and malignant cell lines

Succinyl con A and acetyl con A both stimulated epithelial cells to produce similar yields of tissue plasminogen activator (t-PA) to those previously obtained with native con A. However, unlike con A, the derivatized lectins did not adversely affect cell morphology and viability, and cells treated with succinyl con A could secrete t-PA for a prolonged period. Con A and the two derivatives produced similar morphological effects in Bowes melanoma cells, but t-PA production was not increased. Elevated cyclic nucleotide concentrations did not affect t-PA production from epithelial cells, but calcium ionophore treatment generated t-PA yields similar to those obtained with lectins. Azacytidine, which enhanced t-PA production from epithelial cells, did not increase yields from Bowes melanoma cells, and also sodium butyrate, reported to increase t-PA yields from human endothelial cells, had no effect on either cell line.     

Syntheses of Female Sex Pheromone Analogues of the German Cockroach and Their Biological Activity

Several analogues of 3,1l-dimethyl-2-nonacosanone, one component of the sex pheromone of the German cockroach, were synthesized. Their activity for the male to raise his wings was assayed and summerized in Tables I and II.     

Mannose Receptor 1 Restricts HIV Particle Release from Infected Macrophages

1. Download high-res image (137KB)
2. Download full-size image

     

Derepressive Formation of l-Leucine-Pyruvate Transaminase under Nitrogen-Free Conditions in Gluconobacter suboxydans

l-Leucine-pyruvate transaminase activity increased 6- to 20-fold in 3 hr when Gluconobacter suboxydans cells grown on yeast extract-medium were transferred to and incubated in a nitrogen-free medium. The increase in enzyme activity was influenced remarkably by the age and concentration of cells used. The phenomenon depended upon de novo synthesis of enzyme protein.

The enzyme activity in cell-free extracts of cells incubated under a nitrogen-free condition decreased remarkably after heat treatment at 50°C (pH 6.0) or after freezing and thawing. The level of such enzyme inactivation was high in extracts of cells in the early stages of induction and low in later stages.     

Lectins as probes for assessing the accessibility of N‐linked glycans in relation to the conformational changes of fibronectin

Fibronectin, a ≈450‐kDa protein with 4–9% (w/w) glycosylation, is a key component of extracellular matrices and has a high conformational lability regarding its functions. However, the accessibility and the role of glycosylated moieties associated with the conformational changes of fibronectin are poorly understood. Using lectins as probes, we developed an approach comprising dynamic light scattering, turbidimetry measurements, and isothermal titration calorimetry to assess the accessibility of glycosylated moieties of fibronectin undergoing thermal‐induced conformational changes. Among a set of 14 lectins, fibronectin mainly reacted with mannose‐binding lectins, specifically concanavalin A. When temperature was raised from 25 to 50 °C, fibronectin underwent progressive unfolding, but the conformation of concanavalin A was unaffected. Dynamic light scattering, turbidimetry measurements, and isothermal titration calorimetry showed increased concanavalin A binding to fibronectin during progressive thermal‐induced unfolding of the protein core. Such data suggest that mannosylated residues are progressively exposed as fibronectin unfolds. Because oligosaccharide moieties can be differently exposed to cells, and the cell's responses could be modified physiologically or pathologically, modulation of fibronectin sugar chains could be relevant to its biological functions. Thus, lectins might be useful tools to probe the glycosylation accessibility accompanying changes in protein core folding, for which a better understanding would be of value for biological and biomedical research. Copyright © 2015 John Wiley & Sons, Ltd.