Search PubMed⌕ Search

Biomedical subjects

B van Deurs

Publications and source records attributed to B van Deurs.

At least 109 records · Page 6Linked to original sources
PubMed1984

Physiological and morphological effects of overproduction of membrane-bound ATP synthase in Escherichia coli K-12.

Effects of increased biosynthesis of the membrane-bound ATP synthase of Escherichia coli K-12 were analysed at the physiological and morphological level. Overproduction of the enzyme complex was achieved by molecular cloning of the structural genes into plasmid pBR322. A series of plasmids resulting in 2-fold, 4- to 5-fold and 10- to 12-fold overproduction, respectively, was constructed. The ATP synthase was calculated to represent 3%, 6-7% and 18-23%, respectively, of total protein in cells with these plasmids. In wild-type cells ATP synthase represents 1.5-2% of total protein equivalent to approximately 3000 enzyme complexes per average cell. While 2- or 4- to 5-fold wild-type levels of the ATP synthase had only minor effects it was found that 10- to 12-fold overproduction resulted in pronounced inhibition of cell division and growth and in formation of membrane cisterns and vesicles within the cells. Inclusion bodies, probably representing deposits of excess ATP synthase, were also observed in these cells.

ATP Synthetase Complexes↗
PubMed1984

Identification of free coated pinocytic vesicles in Swiss 3T3 cells.

Whether or not free coated vesicles are involved during internalization of ligands bound to the receptors of coated pits is controversial. Free coated vesicles cannot be identified with certainty in random individual thin sections - reconstructions based on consecutive thin sections are required. The thickness of the sections determines the reliability of such reconstructions. In the present study, serial section electron microscopy was applied to Swiss 3T3 cells and the topographical resolution yielded by 80 nm and 20 nm sections was compared. Swiss 3T3 cells in monolayer at 37 degrees C were exposed for 5 min to cationized ferritin (CF) which is a marker of pinocytic vesicles. Subsequently the cells were fixed, pelleted and further processed for electron microscopy. The results showed that reconstructions of coated CF-labeled structures based on consecutive sections of an average thickness of approximately 80 nm could not be performed with certainty. A substantial fraction (25%) of the examined profiles appeared to be free vesicles, but narrow surface connections could easily have been missed in these thick sections. The series of the much thinner 20 nm sections provided a better resolution allowing the narrowest surface connections to be identified. Accordingly, the number of truly free, coated vesicles was much lower than the number of apparently free vesicles in the thick sections. However, free coated vesicles labeled with CF were identified in the consecutive 20 nm sections (4% of the examined profiles).

Animals↗
PubMed1984

Identification of malignant cells in primary monolayer cultures of human breast tumors.

Primary monolayer cultures of human breast tumors have been characterized by using light microscopy on cultures incubated for glucose-6-phosphate dehydrogenase (G6PDH) activity. The enzyme activity was compared with that of the corresponding histological sections as well as with that of three established human cell lines. Furthermore, electron microscopy has been carried out on selected, isolated cell islands. On the basis of localization and morphology, the cells in 5-day-old primary monolayer cultures could be classified into 3 categories: (1) cells of the migration zone (periphery) of microexplants, (2) single cells, and (3) monolayer cell islands. These cell categories showed little or no G6PDH activity in cultures of benign origin. In the corresponding histological sections of benign tumors no G6PDH activity occurred in the stroma, whereas the G6PDH activity varied in the epithelial elements, being highest in dysplastic elements. Apparently, these elements remain within the microexplants or never appear in the final cultures. By contrast, in cultures of malignant origin some of the single cells and in particular the cell islands showed high G6PDH activity. In histological sections of the malignant tumors the stromal cells showed no activity, whereas the epithelial elements in solid, invasive portions showed high activity. Studies on the established human mammary cell lines MCF-7 (malignant) and HBL-100 (presumptive benign) revealed higher G6PDH activity in MCF-7 than in HBL-100. Human skin fibroblasts revealed no activity. Electron microscopy of isolated cell islands after enzyme incubation confirmed that these were epithelial, because of the presence of desmosomes. Our observations suggest that G6PDH activity is a possible marker of at least some malignant cells in primary monolayer culture.

Breast Neoplasms↗
PubMed1984

Kinetics of pinocytosis studied by flow cytometry.

Kinetics of pinocytosis of FITC-Dextran (mw 70 000) was analysed in mouse L-cells using flow cytometry with a fluorescence-activated cell sorter (FACS). In each experiment information on 2 X 10(4) individual, living cells was obtained. The population of L-cells thus analysed was shown to be rather homogeneous with respect to cell size, and the peak of the FITC-fluorescence curve--a histogram of the accumulated signals from L-cells exposed to FITC-Dextran--was shown to be representative of the average size of the L-cells. It was found that the cellular accumulation of FITC-Dextran was proportional to the tracer. However, the rate of accumulation decreased with increasing time of incubation (up to 3 h). When cells were pulse-labeled for 10 min at 37 degrees C, rinsed carefully with ice-cold PBS, and reincubated at 37 degrees C for various periods of time, about 50% of the initial cellular FITC-fluorescence disappeared within approximately 15 min of reincubation, whereafter no measurable decrease in cellular fluorescence was seen. In addition, a rapid increase of intact FITC-Dextran molecules in the reincubation medium occurred within the first 15 to 30 min of reincubation, whereafter no further increase took place. Thus, a large portion of previously endocytosed FITC-Dextran becomes rapidly exocytosed, while the rest becomes sequestered within a compartment from where little or no exocytosis occurs. The deviation from linearity in accumulation related to time, and the distinct biphasic kinetics of exocytosis are taken to support a generalized two-compartment model for endocytosis and membrane recycling, the two compartments being endocytic vacuoles (endosomes) and (secondary) lysosomes, respectively.

Animals↗
PubMed1984

Filipin as a cholesterol probe. I. Morphology of filipin-cholesterol interaction in lipid model systems.

We report some novel morphological observations on the interaction of the polyene antibiotic filipin (crude complex) with cholesterol, studied in non-cellular systems with replication, freeze-fracture, and negative stain techniques. Cholesterol crystals, lecithin liposomes containing 0 to 20 mole% of cholesterol, and liposomes containing 10 mole% of cholesterol and 5 to 40 mole% of sphingomyelin were incubated for varying lengths of time with filipin at different cholesterol: filipin molar ratios. The resulting filipin-induced lesions (FIL) were pleomorphic in all systems studied. In replicas of crystals, FIL appeared as ridges which were either straight, or curved into C- and S-shaped figures or closed circles. Negatively stained preparations showed FIL as white lines of the same configurations and in addition revealed a delicate veil attached to individual FIL. FIL, fused by their veils into clusters or large sheets ("holey sheets"), were shed from crystals. Incubation of liposomes for 1 h at cholesterol:filipin molar ratios of 4:1, 2:1, 1:1, and 1:5, demonstrated that cholesterol detection (i.e. formation of FIL) depend upon the ratio of cholesterol to filipin. At a 1:1 molar ratio FIL formed on liposomes containing 10 mole% cholesterol or more, but detectability increased to 5 mole% at the 1:5 ratio. Increasing the molar ratio of cholesterol:filipin to 2:1 and 4:1 decreased cholesterol detectability to between 10 and 20 mole%. Increasing concentrations of sphingomyelin decreased cholesterol detectability at the 1:1 cholesterol:filipin ratio; further, FIL in sphingomyelin-containing liposomes tended towards larger diameters. Filipin induced aggregation of liposomes and linked them together by holey sheets, providing evidence for filipin-induced extraction of cholesterol from liposomes. Taken together our morphological observations on filipin-cholesterol interaction in non-cellular systems raise pertinent questions as to the feasibility of filipin as a cholesterol probe in cellular systems.

Cholesterol↗
PubMed1984

Filipin as a cholesterol probe. II. Filipin-cholesterol interaction in red blood cell membranes.

Filipin, a mixture of polyene antibiotics which form complexes with cholesterol, perturbs membrane lipid organization, and causes hemolysis of erythrocytes, is increasingly used as a cytochemical probe for the distribution of cholesterol in cell membranes. We used light (phase-contrast, dark-field and fluorescence) and electron microscopical techniques (whole-mount shadowing, negative staining, and freeze-fracture) to study the interaction of filipin with unfixed and glutaraldehyde-fixed human red blood cell (RBC) membranes. Lysis time and extent depended upon the cholesterol:filipin (C:F) ratio. Lysis was prevented by osmotic protection with high MW dextran. Filipin treated cells fluoresced, but variation in fluorescence intensity among unfixed as well as among fixed cells was evident both at low and high C:F ratios. Negatively stained preparations of unfixed cells lysed on grids or in suspension revealed ring- or C-shaped filipin-induced lesions (FIL) equipped with a veil-like appendage; single FIL, and FIL fused by their veils into aggregates, were shed from membranes. FIL at the surface proper of shadowed whole-mounts and of freeze-etched preparations of prefixed cells appeared as single, dispersed or aggregated cylinders protruding to variable heights above the membrane's plane; aggregated FIL were shed from cells. The freeze-fracture appearance of FIL differed in membranes fixed before or after filipin treatment. E- and P-faces of post-fixed membranes exhibited cylindrical protrusions and depressions, respectively; in essence, the reverse was found in pre-fixed RBC. Both pre- and post-fixed membranes showed considerable variation in the number of FIL on individual cells whether incubated at high (1:1) or low (1:5) C:F ratios, or for a short (10 min) or a long (80-180 min) time. Aggregation and shedding of FIL was evident in all preparations. Thin layer chromatography of the incubation fluid after sedimentation of cells showed that membrane cholesterol was shed from incubated cells. The presented data question the feasibility of filipin as a probe for the topographical distribution of cholesterol in cell membranes.

Cholesterol↗
PubMed1984

Tight junction structure in relation to transepithelial resistance in the frog choroid plexus.

In this communication we report observations on the tight junctions of the frog choroid plexus obtained by thin section and freeze-fracture electron microscopy. It is shown that the choroid plexus epithelial tight junctions comprise a relatively high number (mean 5-6, range 3-10) of continuous, anastomosing strands. This is remarkable in relation to: (1) recent observations that the frog choroidal epithelium has a very low transepithelial resistance, and (2) current concepts of the proportional relationship between transepithelial resistance and number of tight junction strands. It is concluded that there exists a marked lack of correlation between tight junction structure and function in the frog choroid plexus epithelium.

Animals↗
PubMed1983

Serial-section analysis of coated pits and vesicles involved in adsorptive pinocytosis in cultured fibroblasts.

We have examined, by analyzing thin (15-20 nm) serial sections, whether coated pits involved in adsorptive pinocytosis in cultured fibroblasts give rise to free coated vesicles or represent permanently surface-associated structures from the neck of which uncoated receptosomes pinch off and carry ligand into the cell. Human skin fibroblasts and mouse L-929 fibroblasts were incubated with cationized ferritin (CF), a ligand known to bind to coated pit regions, at 37 degrees C before fixation. In thin sections, CF was found in coated vesicular profiles within the cytoplasm. Serial sections revealed that whereas many of these coated profiles communicated with the cell surface, thus representing pits, about 10% in L-cells and 36% in skin fibroblasts were actually free coated vesicles. Moreover, evidence for uncoated vesicular structures (receptosomes) budding off from the coated pits was not obtained. We therefore conclude that coated pits do pinch off from the plasma membrane to form free, coated vesicles (pinosomes).

Adsorption↗
PubMed1983

Coated pits with pinocytosis in Tetrahymena.

The possible role in pinocytosis of coated pits at the parasomal sacs of Tetrahymena has been studied using cationized ferritin (CF; pI = 8.5) as a marker of membrane and content. It is shown that CF binds evenly to the surface, including the coated pits, of Tetrahymena in an inorganic salt medium (to avoid formation of food vacuoles) at the normal growth temperature. Moreover, CF is internalized by coated vesicles (shown to be truly free by thin serial-section analysis) and delivered initially (1-5 min of incubation) to cisterna near the cell surface. Later (5-10 min) CF occurs also in autophagic vacuoles, formed as a result of starvation, and eventually (15-90 min) it is present in preformed (old) food vacuoles. These observations indicate that the coated pits at the parasomal sacs of Tetrahymena function in adsorptive pinocytosis in much the same manner as coated pits at the surface of mammalian cells.

Animals↗
PubMed1982

Brain barrier systems in the lamprey. II. Ultrastructure and permeability of the choroid plexus.

The lamprey choroid plexus was studied by electron microscopic techniques and the composition of cerebrospinal fluid and plasma compared as part of a characterization of the lamprey blood-brain barrier. It was shown that the ultrastructure of the lamprey choroid plexus is very similar to that of the mammalian plexus. A blood-cerebrospinal fluid (CSF) barrier to horseradish peroxidase (molecular weight 40,000) and microperoxidase (molecular weight 2000) was localized to apical tight junctions between the choroidal epithelial cells. Pinocytic uptake of the tracers took place particularly at the apical surface of the epithelium (after intravenous and intraventricular administration). Absorbed tracer-molecules were found in vacuoles presumably belonging to the well-developed lysosomal apparatus of the epithelial cells. Extended Golgi-complexes, dense bodies and some multivesicular bodies were reactive for acid phosphatase activity. Measurements of protein, potassium and sodium in plasma and CSF revealed the same concentration differences previously observed in higher vertebrates. The present study lends further support to the view that the lamprey blood-brain barrier is similar to that of higher vertebrates.

Acid Phosphatase↗
PubMed1982

Scanning and transmission electron microscopy of the normal rabbit aortic endothelium after controlled perfusion fixation.

Light microscopy, scanning electron microscopy, and transmission electron microscopy were employed on the anterior wall of the rabbit thoracic aorta with the particular purpose to study endothelial marginal flaps and the structure of stomata under physiological conditions at 100 mm Hg. Intra-aortic pressure monitoring was performed during the whole period of perfusion (2h), restricting pressure variations to a maximum of +/- 5 mm Hg in order to preserve endothelial architecture. We found the endothelial surface uniformly smooth. Marginal flaps of the endothelial cells were mutually overlapping, often forming multiple layers. Underlying flaps might protrude to the lumen between neighbouring flaps as flat mushrooms. Flaps of adjacent cells might diverge for short distances to decover an underlying endothelial cell. Downstream flaps of the cells might be overlapped at their base by flaps of adjacent cells, the extremity of the flap thus appearing as a disc separated from the cell. The small discs and troughs thus formed are considered equivalent to the so-called stomata.

Animals↗
PubMed1982

Gap junction pleiomorphism in the root system of the rhizocephalans (Arthropoda: Crustacea).

We have studied gap junctions in the root system of four different species of rhizocephalans (Arthropoda: Crustacea) using freeze-fracture. Numerous and often very extensive gap junctions are present between the root cells. They are of the characteristic E-type also found in other arthropods. Large junctional particles (ca. 13 nm) are located predominantly on the E-face, while complementary pits and a few dislocated particles are present on the P-face. The gap junctions show a remarkable pleiomorphism. Small macular gap junctions with rather densely packed particles, larger irregularly shaped gap junctions, often forming bands with intervening particle-free membrane domains, and gap junctions with widely dispersed particles are observed. These features are documented both in material after conventional preparation including glutaraldehyde fixation and glycerol cryoprotection and in material frozen directly in a nitrogen slush without any preceding preparation, and are discussed in relation to possible functional significance.

Animals↗
PubMed1982

Coated pits and pinocytosis of cationized ferritin in human skin fibroblasts.

Pinocytosis was studied in cultured human skin fibroblasts in order to obtain more information about uptake in these cells of non-specific ligands with different net surface charge. The fibroblasts were obtained from a normal person and a patient homozygous for familial hypercholesterolemia. No ultrastructural differences were observed between the two cell types. The ratio between coated and smooth pits at the cell surface was about 1:6. Freeze-fracture revealed that whereas smooth pits were devoid of intramembrane particles, the coated pits occupying 1-5% of the total cell surface area showed numerous intramembrane particles. Cationized ferritin (pI = 8.5) bound to the cell surface and to coated pits of both types of fibroblasts at 4 degrees C. Either type internalized CF at 37 degrees C. Vacuoles (lysosomal elements) containing CF were seen already after 5 min at 37 degrees C, but were much numerous after 30 min of incubation at 37 degrees C, and appeared with the same frequency in both cell types. Smooth (uncoated) pits at the cell surface remained unlabeled. In contrast to CF, native ferritin (pI = 4.6) was internalized to a very limited extent in both cell types, even after 3.5 h of incubation at 37 degrees C. These results indicate that both normal and FH fibroblasts internalize CF exclusively by coated pits, and adsorptive uptake mechanism that apparently is nonspecific but clearly cation-selective.

Adolescent↗
PubMed1982

Tight junctions: architecture of plasma membranes at sites of cell-cell "fusion" in relation to paracellular permeability.

A major goal in analyzing the structure of tight junctions by freeze-fracture has been to create a generalized model for this membrane specialization conforming to both physiological and morphological data, and providing a basis for understanding variation in tight junction appearance and function. In the present review we have focused on some of the problems involved. Results obtained by double replication, rotary shadowing and rapid freezing are discussed. A model for tight junctions with a general scaffolding of inverted lipid micelles (cylinders) binding the adjacent membranes together, and associated with integral membrane proteins of significance, for instance, in stabilization as well as in regulation of permeability, appears promising.

Animals↗
PubMed1980

Membrane structure of nonactivated and activated human blood platelets as revealed by freeze-fracture: evidence for particle redistribution during platelet contraction.

The distribution of intramembrane particles of nonactivated and activated human blood platelets was studied by freeze-fracture under various experimental conditions to see whether morphological evidence for a structural coupling between the platelet actomyosin system and the fibrin network in a retracting clot could be established. Membrane particles were evenly distributed in nonactivated platelets; the total number (E + P faces) was approximately 1,500/micrometers 2 of membrane, and there were two to three times more particles present on the E face than on the P face. Transformation of discoid platelets to "spiny spheres" by cooling did not change the particle distribution. Platelet activation and aggregation by serum or ADP caused no change in membrane particle density or distribution. Particle distribution was not changed in Ca2+-activated platelets fixed immediately before fibrin formation, but after fibrin formation and during clot retraction, particles were sometimes most frequent on the P face and tended to form distinct clusters, and aggregates of E face pits were observed. Blood platelets contain contractile proteins that are distinct as filaments in platelets in retracting clots. We suggest that the redistribution of particles seen in activated platelets during clot retraction reflects the esablishment of mechanical transmembrane links between the platelet actomyosin system and the fibrin net. The P-face particle clusters may represent sites of force transmission between actin filaments bonded to the inside of the membrane and the fibrin network at the outside. Thus, whereas membrane particles may not be directly involved in the attachment of actin filaments to membranes, the transmission of the force of the contractile system to an exterior substrate apparently involves the intramembrane particles.

Adenosine Diphosphate↗