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W J Van Blitterswijk

Publications and source records attributed to W J Van Blitterswijk.

10 recordsLinked to original sources
PubMed2000

Protein kinase C activation by acidic proteins including 14-3-3.

14-3-3 proteins may function as adapter or scaffold proteins in signal transduction pathways. We reported previously that several 14-3-3 isotypes bind to protein kinase C (PKC)-zeta and facilitate coupling of PKC-zeta to Raf-1 [van der Hoeven, van der Wal, Ruurs, van Dijk and van Blitterswijk (2000) Biochem. J. 345, 297-306], an event that boosts the mitogen-activated protein kinase (ERK) pathway in Rat-1 fibroblasts. The present work investigated whether bound 14-3-3 would affect PKC-zeta activity. Using recombinant 14-3-3 proteins and purified PKC-zeta in a convenient, newly developed in vitro kinase assay, we found that 14-3-3 proteins stimulated PKC-zeta activity in a dose-dependent fashion up to approx. 2.5-fold. Activation of PKC-zeta by 14-3-3 isotypes was unrelated to their mutual affinity, estimated by co-immunoprecipitation from COS cell lysates. Accordingly, PKC-zeta with a defective (point-mutated) 14-3-3-binding site, showed the same 14-3-3-stimulated activity as wild-type PKC-zeta. As 14-13-3 proteins are acidic, we tested several other acidic proteins, which turned out to stimulate PKC-zeta activity in a similar fashion, whereas neutral or basic proteins did not. These effects were not restricted to the atypical PKC-zeta, but were also found for classical PKC. Together, the results suggest that the stimulation of PKC activity by 14-3-3 proteins is non-specific and solely due to the acidic nature of these proteins, quite similar to that known for acidic lipids.

14-3-3 Proteins↗
PubMed1988

Effect of dietary lipids on plasma lipoproteins and fluidity of lymphoid cell membranes in normal and leukemic mice.

Mice of the GR/A strain were fed four different isocaloric semipurified diets, enriched in either (1) saturated fatty acids (palm oil), or (2) polyunsaturated fatty acids (corn oil), or (3) palm oil plus cholesterol, or (4) a fat-poor diet containing only a minimal amount of essential fatty acids. We have studied the effects of these dietary lipids on the density profile and composition of the plasma lipoproteins and on the lipid composition and fluidity of (purified) lymphoid cell membranes in healthy mice and in mice bearing a transplanted lymphoid leukemia (GRSL). Tumor development in these mice occurred in the spleen and in ascites. While the fatty acid composition of the VLDL-triacylglycerols still strongly resembled the dietary lipids, the effects of the diets decreased in the order VLDL-triacylglycerols greater than HDL-phospholipids greater than plasma membrane phospholipids. Diet-induced differences in the latter fraction were virtually confined to the content of oleic acid and linoleic acid, and they were too small to affect the membrane fluidity, as measured by fluorescence polarization using the probe 1,6-diphenyl-1,3,5-hexatriene. Healthy mice were almost irresponsive to dietary cholesterol, but in the tumor bearers, where lipoprotein metabolism has been shown to be disturbed, the cholesterol diet caused a substantial increase in the low- and very-low density regions of both blood and ascites plasma lipoproteins. The cholesterol-rich diet also increased the cholesterol/phospholipid molar ratio and lipid structural order (decreased fluidity) in GRSL ascites cell membranes, but not in the splenic GRSL cell membranes. We conclude that the composition of plasma lipoproteins and cell membrane lipids in GR/A mice is subject to exquisite homeostatic control. However, in these low-responders to dietary lipids the development of an ascites tumor may lead to increased responsiveness to dietary cholesterol. The elevated level of membrane cholesterol thus obtained in GRSL ascites cells did not affect the expression of various cell surface antigens or tumor cell growth.

Animals↗
PubMed1984

Differences in membrane lipid composition and fluidity of transplanted GRSL lymphoma cells, depending on their site of growth in the mouse.

GRSL lymphoma cells were isolated from various growth sites in the host. The relative membrane lipid fluidities of these cells and of normal lymphoid cells were estimated by fluorescence polarization, using the probe diphenylhexatriene and by measuring the (free) cholesterol/phospholipid molar ratio in whole cells. The results indicate that the membrane fluidity (reciprocal of the lipid structural order) of the lymphoma cells increases in the order of their location: peripheral blood less than spleen less than mesenterial lymph node less than ascites fluid. The membrane fluidities of normal lymphocytes from thymus, mesenterial lymph node and spleen were about the same, but higher than of peripheral blood lymphocytes, and between those of the lymphoma cells from lymph node and spleen. These results are confirmed by more extensive analysis on purified plasma membranes from the splenic and ascitic GRSL lymphoma cells and from normal splenocytes and thymocytes. The significantly higher lipid order parameter found in the GRSL plasma membrane isolated from the spleen as compared to those from the ascites cells could be fully explained by the differences measured in the major chemical determinants of the fluidity, i.e., the cholesterol/phospholipid ratio, the sphingomyelin content and the degree of saturation of the fatty acyl groups of the phospholipids. It was also found that the cholesterol/phospholipid ratio in erythrocyte membranes isolated from the peripheral blood of the tumor bearers was higher than in those from normal control mice. The observed differences in membrane fluidity between distinct subsets of tumor cells may be relevant to the sensitivity of these cells to immune attack or to drugs.

Animals↗
PubMed1984

Alterations in plasma lipoproteins and heparin-releasable lipase activities in mice bearing the GRSL ascites tumor.

The lipoproteins in GR mice bearing the transplanted GRSL ascites tumor were characterized by density gradient ultracentrifugation and SDS-polyacrylamide gel electrophoresis. In control mice the major proportion of the lipoproteins was found in the HDL density range, but on days 4 and 5 following tumor transplantation a gradual shift into the LDL density range was observed. At the same time the apolipoprotein E content increased at the expense of apolipoprotein A-I. VLDL became moderately elevated. On days 6 and 7 all lipoproteins except VLDL reached extremely low values. The C-apolipoproteins showed a remarkable shift in their relative proportions. Plasma lecithin:cholesterol acyltransferase activity showed no significant alteration in the course of tumor growth, but the triacylglycerol lipases in postheparin plasma were strongly decreased. Lipoprotein lipase had already started to decline on day 2 following tumor transplantation. However, when assayed in the presence of heat-inactivated control plasma, a decrease was not observed before day 5. This is suggestive of a depletion of a plasma cofactor preceding the final disappearance of the enzyme itself, and is compatible with the changing apolipoprotein C pattern. Hepatic lipase showed a 50% reduction between days 3 and 4. The lipoprotein alterations in tumor-bearing mice are explained as a direct consequence of the decreased lipase activities.

Animals↗
PubMed1982

Comparative lipid analysis of purified plasma membranes and shed extracellular membrane vesicles from normal murine thymocytes and leukemic GRSL cells.

The lipid fluidity in purified plasma membranes (PM) of murine leukemic GRSL cells, as measured by fluorescence polarization, is much higher than in PM of normal thymocytes. This was found to be due to relatively low contents of cholesterol and sphingomyelin and a high amount of unsaturated fatty acyl chains, especially linoleic acid, in the phospholipids. PM from GRSL cells contain markedly more phosphatidylethanolamine than those from thymocytes. For both GRSL cells and thymocytes the detailed lipid composition of isolated PM was compared with that of the corresponding shed extracellular membranes (ECM), which were isolated from the ascites fluid and from thymus cell suspensions, respectively. The somewhat decreased lipid fluidity of thymocyte ECM as compared to their PM, can be ascribed to the increased cholesterol/phospholipid molar ratio (0.88 vs. 0.74). No other major differences were found between the lipid composition of these membranes. In contrast, significant differences were found between PM and ECM from GRSL cells. In this system a much lower lipid fluidity of the shed ECM was found, due to the much increased cholesterol/phospholipid molar ratio (3.5-fold) and sphingomyelin (9-fold) content, as compared to the PM. Further, the ECM contain relatively more lysophosphatidylethanolamine and less phosphatidylcholine and -inositol. ECM contain a higher amount of polyunsaturated fatty acids, especially in the phosphatidylethanolamine and lysophosphatidylethanolamine classes. On the other hand, the fatty acids of phosphatidylcholine and lysophosphatidylcholine are more saturated than in PM. In particular, ECM of GRSL cells contain less oleic and linoleic acid residues and more arachidonic acid and 22:polyunsaturated fatty acid residues than PM. The possible relevance of these differences with respect to the mechanism of shedding of vesicles from the cell surface, is discussed.

Animals↗
PubMed1981

Lipid structural order parameters (reciprocal of fluidity) in biomembranes derived from steady-state fluorescence polarization measurements.

This paper presents an interpretation of fluorescence polarization measurements in lipid membranes which are labelled with the apolar probe 1,6-diphenyl-1,3,5-hexatriene. The steady-state fluorescence anisotropy, rs, is resolved into a fast decaying or kinetic component, rf, and an infinitely slow decaying or static component, r infinity. The latter contribution, which predominates in biological membranes, is exclusively determined by the degree of molecular packing (order) in the apolar regions of the membrane; r infinity is proportional to the square of the lipid order parameter. An empirical relation between rs and r infinity is presented, which is in agreement with a prediction based on a theory of rotational dynamics in liquid crystals. This relation enabled us to estimate a lipid structural order parameter directly from simple steady-state fluorescence polarization measurements in a variety of isolated biological membranes. It is shown that major factors determining the order parameter in biomembranes are the temperature, the cholesterol and sphingomyelin content and (in a few systems) the membrane intrinsic proteins.

Animals↗
PubMed1976

Effect on glutaraldehyde fixation on lectin-mediated agglutination of mouse leukaemia cells.

The effect of glutaraldehyde fixation on lectin-mediated agglutination of murine leukaemia (GRSL) cells was investigated using 2 assay methods which differed in the shear forces to which the agglutinated cells were subjected. First, lectin and cells were allowed to interact under conditions in which shear forces were minimized and the degree of agglutination was evaluated microscopically by the appearance and size of the cell aggregates. This assay demonstrated that concanavalin A (con A)-, wheat germ agglutinin (WGA)- or Ricinus communis agglutinin I (RCAI)-mediated cytoagglutination was unaffected (WGA and RCAI) or somewhat enhanced (con A) by prior fixation of the cells with glutaraldehyde. Secondly, an electronic particle counter was used to measure the disappearance of single cells and concomitant appearance of cell aggregates as a function of the lectin concentration. In this assay, in which the aggregated cells are subjected to significant shear forces during dilution and cell counting, agglutination of GRSL cells by each of the 3 lectins was drastically inhibited by prior fixation of the cells with glutaraldehyde. This assay also demonstrated enhanced nonlectin-induced cell aggregation after fixation. In both cytoagglutination assays about the same lectin concentration was required for threshold agglutination of unfixed cells. Comparatively, the results of the 2 cytoagglutination assays indicate that a fraction of the lectin-mediated bonds between unfixed cells is shear resistant and that fixation of the cells either weakens these bonds or inhibits their formation. Morphologically, cells prefixed with glutaraldehyde were sperical at all lectin concentrations, with a continuous dense distribution of cell surface-bound con A, labelled directly with haemocyanin or indirectly using the peroxidase-diaminobenzidine reaction. Unfixed cells showed angular and toadstool-shaped deformations, especially at the highest lectin concentrations, the agglutinating surfaces being flattened against each other over extended areas. The distribution of con A label was continuous and dense between the apposed surfaces and discontinuous on free surfaces. In the presence of con A the free surfaces of prefixed cells exhibited more microvilli than the surfaces of non-prefixed cells. These results favour the view that fixation prevents the formation of shear-resistant, lectin-mediated bonds between cells, not by restricting the lateral mobility of lectin receptors, but by impairing the apposition of rigid cell surfaces.

Agglutination↗
PubMed1975

Quantitation of virus-induced (MLr) and normal (Thy.1.2) cell surface antigens in isolated plasma membranes and the extracellular ascites fluid of mouse leukemia cells.

Plasma membranes were isolated by two methods from mouse leukemia cells containing mammary tumor virus-induced (MLr) and normal (Thy.1.2) antigens on their surfaces. A number of chemical components, enzymic activities, and the antigenic contents were determined in subcellular fractions and found to be specifically concentrated in the plasma membrane fractions. The major part of the cellular MLr, in contrast to Thy.1.2, was present in the 105,000 X gmax supernatant of the cell homogenate. This and other results indicate an easy release of the antigen from the plasma membrane. A considerable amount of MLr was also present in the ascites fluid, partly free and partly bound, supposedly in an immune complex that allowed the isolation of three components of similar molecular weights as mammary tumor virus components. The extracellular presence of MLr may illustrate that, by shedding of antigen, the tumor may protect itself against the immunological defense of the host.

Animals↗