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D Hoekstra

Publications and source records attributed to D Hoekstra.

At least 73 records · Page 4Linked to original sources
PubMed1995

Parameters influencing the introduction of plasmid DNA into cells by the use of synthetic amphiphiles as a carrier system.

Parameters that affect cellular transfection as accomplished by introducing DNA via carriers composed of cationic synthetic amphiphiles, have been investigated, with the aim to obtain insight into the mechanism of DNA translocation. Such insight may be exploited in optimizing carrier properties of synthetic amphiphiles for molecules other than nucleic acids. In the present work, the interaction of vesicles composed of the cationic amphiphile dioleyloxy-propyl-trimethylammonium chloride (DOTMA) with cultured cells was examined. The results show that optimal transfection is dependent on the concentration of lipid, which determines the efficiency of vesicle interaction with the target cell membrane, as well as the toxicity of the amphiphiles towards the cell. A low lipid/DNA ratio prevents the complex from interacting with the cell surface, whereas at a relatively high amphiphile concentration the complex becomes toxic. Translocation efficiency is independent of the initial vesicle size but is affected by the size of the DNA. An incubation time of the DNA/amphiphile complex and cells of approx. 2-4 h is required for obtaining efficient transfection. In conjunction with observations on DNA/amphiphile complex-induced hemolysis of erythrocytes, a mechanism of DNA-entry is proposed which involves translocation of the nucleic acids through pores across the membranes rather than delivery via fusion or endocytosis. Dioleoylphosphatidylethanolamine, a phospholipid frequently used in a mixture with DOTMA ('lipofectin') strongly facilitates this pore formation. Translocation of the DNA is effectively prevented when the cells are pretreated with Ca2+ or pronase. These observations suggest that Ca(2+)-sensitive cell surface proteins play a role in amphiphile-mediated DNA translocation.

Animals↗
PubMed1995

Fluorescent, short-chain C6-NBD-sphingomyelin, but not C6-NBD-glucosylceramide, is subject to extensive degradation in the plasma membrane: implications for signal transduction related to cell differentiation.

The involvement of the plasma membrane in the metabolism of the sphingolipids sphingomyelin (SM) and glucosylceramide (GlcCer) was studied, employing fluorescent short-chain analogues of these lipids, 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]hexanoylsphingosylphosphorylcholine (C6-NBD-SM), C6-NBD-GlcCer and their common biosynthetic precursor C6-NBD-ceramide (C6-NBD-Cer). Although these fluorescent short-chain analogues are metabolically active, some caution is to be taken in view of potential changes in biophysical/biochemical properties of the lipid compared with its natural counterpart. However, these short-chain analogues offer the advantage of studying the lipid metabolic enzymes in their natural environment, since detergent solubilization is not necessary for measuring their activity. These studies were carried out with several cell types, including two phenotypes (differing in state of differentiation) of HT29 cells. Degradation and biosynthesis of C6-NBD-SM and C6-NBD-GlcCer were determined in intact cells, in their isolated plasma membranes, and in plasma membranes isolated from rat liver tissue. C6-NBD-SM was found to be subject to extensive degradation in the plasma membrane, due to neutral sphingomyelinase (N-SMase) activity. The extent of C6-NBD-SM hydrolysis showed a general cell-type dependence and turned out to be dependent on the state of cell differentiation, as revealed for HT29 cells. In undifferentiated HT29 cells N-SMase activity was at least threefold higher than in its differentiated counterpart. In contrast, in all cell types studied, very little if any biosynthesis of C6-NBD-SM from the precursor C6-NBD-Cer occurred. Moreover, in the case of C6-NBD-GlcCer, neither hydrolytic nor synthetic activity was found to be associated with the plasma membrane. These results are discussed in the context of the involvement of the sphingolipids SM and GlcCer in signal transduction pathways in the plasma membrane.

4-Chloro-7-nitrobenzofurazan↗
PubMed1995

In vitro fusion of reticulocyte endocytic vesicles with liposomes.

Since reticulocytes have a high demand for iron, which is required for heme biosynthesis, these cells are highly specialized in the endocytosis of the iron carrier transferrin (Tf). From the resulting endocytic vesicles (EVs), iron is released and the vesicles rapidly return to the cell membrane where they fuse, causing the release of the apotransferrin. Due to a lack of other intracellular compartments, the endocytic vesicles can be readily isolated. In this study, we have investigated the fusogenic properties of EVs, using liposomes as target membranes. Membrane fusion was monitored by a lipid mixing assay based on the relief of fluorescence self-quenching, using octadecylrhodamine B-chloride (R18). Application of this procedure was verified and solidified by analysis of the fusion event by an independent lipid mixing assay, after in situ labeling of EVs, and by determination of the mixing of aqueous contents. We demonstrate that the endocytic vesicles are particularly prone to fuse with target membranes that contain dioleoylphosphatidylethanolamine (DOPE). Relative to DOPE, bilayers composed of phosphatidylserine or phosphatidylcholine show a reduced fusion activity with EV. The specific and strong inhibition of fusion by cyclosporin A and a peptide known to interfere with the propensity of DOPE to adopt the hexagonal HII phase suggests that the mechanism of fusion involves the ability of this lipid to readily adopt non-bilayer phases. ATP, GTP, and/or cytosol are not necessary to obtain fusion. However, trypsin treatment of the endocytic vesicles inhibits fusion, indicating the involvement of (a) protein(s) in the fusion event.

Animals↗
PubMed1994

Evidence for the existence of lipid-diffusion barriers in the equatorial segment of human spermatozoa.

Liposomes consisting of negatively charged phospholipids interact almost exclusively with the equatorial segment (ES) of human spermatozoa provided the cells have undergone the acrosome reaction (AR) [Arts, Kuiken, Jager and Hoekstra (1993) Eur. J. Biochem. 217, 1001-1009]. Using fluorescently tagged liposomes, this interaction can be observed by fluorescence microscopy, showing either a diffuse fluorescence in the ES region (pattern ESd, presumably reflecting membrane-incorporated lipids as a result of fusion) or a punctate fluorescence (pattern ESp, representing adhering liposomes). These distribution patterns remain unchanged during prolonged incubation, up to 40 min. Not only do these observations suggest the existence of fairly specific liposomal binding sites, associated with the ES region, but also that a barrier to lipid lateral diffusion seems to exist in the ES membrane. Using liposomes that contain fluorescent lipid analogues in either both leaflets or in the inner leaflet only, we demonstrate that this putative barrier entails both membrane leaflets. Treatment with EDTA caused fluorescence to spread from the ES towards other membrane domains. Since only spermatozoa displaying pattern ESd were affected by the chelator, the randomization was not caused by EDTA-induced fusion activity. Therefore, this observation provides further evidence that in spermatozoa displaying pattern ESd the fluorescent lipid analogues were incorporated in the ES membrane as a result of fusion. Furthermore, these experiments support the view of the existence of a transmembranous block to lipid lateral diffusion in the ES, the stability of which may be governed by bivalent cations.

Acrosome↗
PubMed1994

Reconstitution of proteolipid protein: some properties and its role in interlamellar attachment.

Proteolipid apoprotein (PLP) isolated from human brain was reconstituted in dioleoylphosphatidylcholine vesicles by dialysis from 2-chloroethanol, using a dialysis buffer of pH 5.0. Under these conditions, and in contrast with dialysis carried out at pH 7.4, well-defined unilamellar vesicles containing the protein were formed. As judged by electron microscopy and quasi-elastic light scattering, the size of the vesicles was determined by the initial protein/lipid ratio used for reconstitution. When the vesicles were incubated in a buffer at neutral pH, aggregation of the vesicles was observed, but their structure remained intact. Asymmetric aggregation occurred when the reconstituted vesicles were incubated with large unilamellar vesicles (LUVs) devoid of protein. This aggregation was accompanied by loss of membrane integrity, as revealed by extensive leakage of the LUVs, and by membrane lipid dilution, indicative of the occurrence of membrane fusion. Destabilization of the vesicles depended on the presence of negatively charged phosphatidylserine in the membrane of the LUVs. Similar effects, but to a lesser extent, were seen when the LUVs contained sulphatide, a negatively charged lipid prominently present in myelin. DM 20, a natural mutant of PLP, appeared to be far less potent in causing membrane lipid dilution than PLP. This could suggest that a distinct protein sequence of PLP, which is absent from DM 20, may be involved in triggering the observed membrane destabilization. Temperature-dependent experiments indicate that this sequence in PLP displays dynamic properties, its exposure being affected by conformational criteria. Exposure of this particular domain, in conjunction with its affinity for negatively charged lipid, could be related to a perturbation of the integrity of the myelin sheath, as will be discussed.

Brain↗
PubMed1994

Intracellular sites involved in the biogenesis of bile canaliculi in hepatic cells.

Studies in hepatoma cells and hepatocytes have revealed that the biogenesis of bile canalicular membrane involves microvilli-lined vesicles (MLV), which are formed in well differentiated cells. The vesicles grow as a function of time and are presumably vectorially transported to cell surface contact sites of attached cells. We demonstrate that a fluorescent head group-labeled lipid analog, N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE), after its exogenous insertion into the plasma membrane of HepG2 cells at 4 degrees C, accumulates in these microvilli-lined vesicles at 37 degrees C. This shows that the MLV are a target for plasma membrane-derived lipids. Furthermore, also the Golgi apparatus is involved in the formation of the vesicles. After initial accumulation of the fluorescent sphingolipid precursor, 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoic acid (C6-NBD)-ceramide in the Golgi apparatus at 37 degrees C, prolonged incubation at 37 degrees C results in the appearance of NBD fluorescence in the microvilli-lined vesicles. The transport route for the Golgi-derived material to the developing bile canalicular vesicle is not an indirect pathway, i.e. involving transcytosis via the basolateral plasma membrane. This could be demonstrated by including bovine serum albumin (BSA) in the incubation media, a lipid scavenger that will remove any C6-NBD-lipids exposed at the basolateral membrane. At these conditions, lipid trafficking between the Golgi complex and MLV still occurred. We further demonstrate that the targeting from the Golgi apparatus to the bile canaliculus is also operational in isolated human hepatocytes. The latter results suggests that the Golgi complex is involved in both the formation of bile canaliculi and in bile secretion in fully differentiated cells.

Bile Canaliculi↗
PubMed1994

Transport of biosynthetic sphingolipids from Golgi to plasma membrane in HT29 cells: involvement of different carrier vesicle populations.

Intracellular transport of the sphingolipids glucosylceramide (GlcCer) and sphingomyelin (SM), was examined in HT29 human colon adenocarcinoma cells. After synthesis from a fluorescent precursor, 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoylceramide++ + (C6-NBD-Cer), transfer of SM from the Golgi complex to the plasma membrane can occur independently of that of GlcCer, as revealed by temperature-dependent experiments. Thus, at 20 degrees C, SM trafficking to the cell surface is essentially unaffected, whereas GlcCer transport to the plasma membrane is inhibited by approximately 75%, when compared to the transfer of both lipids at 37 degrees C. The mechanism by which SM and GlcCer are transported to the cell surface involves at least in part a vesicular mechanism. Transport vesicles, containing both lipids at their luminal surface, as revealed by the inaccessibility of the NBD fluorescence to the quencher sodium dithionite, have been isolated from cells, permeabilized by filter stripping. As evidenced by electron microscopic and biochemical criteria, no vesicles or lipids were released when cell permeabilization had been carried out with streptolysin. Density gradient analysis indicates the potential existence of several vesicle populations, distinctly enriched in either lipid, involved in transport of sphingolipids to the plasma membrane in HT29 cells.

Adenocarcinoma↗
PubMed1993

Interaction of annexins with membranes: the N-terminus as a governing parameter as revealed with a chimeric annexin.

The modulating effect of the variable N-terminus of annexins on the properties of these Ca(2+)-binding proteins was investigated. To this end, the interaction of annexin V and a mutant annexin, INVC, consisting of the N-terminus of annexin I (amino acids 1-45) and the core of annexin V (19-320), with large unilamellar phosphatidylserine (PS) vesicles was examined. In contrast to annexin V, the mutant annexin mediated Ca(2+)-dependent aggregation of the lipid vesicles at neutral pH. However, annexin V induces Ca(2+)-dependent aggregation at mild acidic pH. Moreover, both proteins can engage in hydrophobic interactions with PS vesicles, which results in release of the vesicle contents. These membrane-perturbing properties are expressed by both annexins in the absence of Ca2+ and occur at neutral and mild acidic pH. Interestingly, addition of Ca2+ inhibits annexin V-induced release, but sustains the release induced by the mutant annexin INVC. The Ca(2+)-dependent effects on the release of vesicle contents are reversed upon EDTA addition. Conformational changes revealed by binding of the hydrophobic probe, 4,4'-bis(1-anilino-8-naphthalenesulfonate), underly the observed Ca(2+)-modulated effects on leakage. However, low-pH-mediated aggregation by annexin V does not seem to be related to macroscopic conformational changes. Annexin INVC also affects Ca(2+)-induced fusion of PS vesicles, displaying synergistic properties in conjunction with Ca2+ at neutral pH. By contrast, annexin V does not display similar properties at mild acidic pH, in spite of its ability to aggregate vesicles under such conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Annexins↗
PubMed1993

Fusion of artificial membranes with mammalian spermatozoa. Specific involvement of the equatorial segment after acrosome reaction.

The fusogenic properties of bovine and human spermatozoa membranes were investigated, using phospholipid bilayers (liposomes) as target membranes. Fusion was monitored by following lipid mixing, as revealed by an assay based on resonance-energy transfer. In addition, fusion was visualized by fluorescence microscopy, using fluorescent lipid vesicles. Cryopreserved bovine sperm fused with liposomes before induction of the acrosome reaction, fluorescence being located in essentially all spermatozoa membrane domains. Fresh bovine and human spermatozoa fused with liposomes only after the induction of the acrosome reaction, as triggered by calcium ionophore A23187 or zonae pellucidae (proteins), while the fluorescence distribution was mainly restricted to the equatorial segment (ES). However, with spermatozoa that had undergone a freeze/thawing cycle, domains other than ES also became labeled. Hence, the redistribution of the lipid probes over the entire membrane occurring during lipid mixing with cryopreserved bovine sperm is probably related to membrane perturbations caused by long-term cryopreservation. Fusion with liposomes was governed by spermatozoa factors and required the presence of acidic phospholipids like cardiolipin and phosphatidylserine in the liposomal bilayer. Incorporation of the zwitterionic lipid phosphatidylcholine in the vesicles inhibited the fusion reaction. Fusion was pH dependent. The results indicate that the ES is the primary domain of spermatozoa membranes that harbours the fusogenic capacity of sperm. Liposomes appear a valuable tool in further characterizing the properties of this domain, which has been claimed [Yanagimachi, R. (1988) in The physiology of reproduction (Knobil, E. & Neill, J., eds) pp. 135-185, Raven Press, New York] to represent the putative, initial fusion site for the oocyte.

Acrosome↗
PubMed1993

Outstations of the Golgi complex are present in the processes of cultured rat oligodendrocytes.

Primary cultures of rat oligodendrocytes were incubated with a fluorescent sphingolipid precursor, 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoylceramide+ ++ (C6-NBD-ceramide). This compound is known to stain the Golgi complex specifically. Within 30 min of incubation at 37 degrees C most of the C6-NBD-ceramide was incorporated into the perinuclear Golgi system, as revealed by conventional and confocal laser fluorescence microscopy. Interestingly, C6-NBD-ceramide was found to accumulate also in smaller, oval-shaped structures in many of the processes, at distances up to 30 microns from the nucleus. This implies the possibility that these structures are Golgi (-derived) complexes. Indeed, after incubation of oligodendrocytes with C6-NBD-ceramide and rhodamine-labeled transferrin both fluorescent labels colocalized in the Golgi system of the cell body as well as in the structures in the processes. Additional support for the Golgi character of these structures was obtained by transmission electron microscopy. Particularly in oligodendrocytes cocultured with neurons, many Golgi structures were present all over the processes. The results lead us to conclude that, in the oligodendrocyte, the Golgi complex does not only reside in the perikaryon, but also in the processes. One can speculate that a polarized biosynthetic activity, involving the presence of the Golgi near the site of myelin synthesis, may be advantageous to the oligodendrocyte for assembly and/or repair of the myelin membrane at the distal end of the processes.

4-Chloro-7-nitrobenzofurazan↗
PubMed1993

Differential metabolism and trafficking of sphingolipids in differentiated versus undifferentiated HT29 cells.

Trafficking and metabolism of sphingolipids were examined in undifferentiated (G+) and differentiated (G+ reversed) HT29 human colon adenocarcinoma cell lines. Metabolic experiments employing a fluorescently labeled sphingolipid precursor, 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoylceramide++ + (C6-NBD-ceramide) revealed that both qualitative and quantitative differences exist in sphingolipid synthesis between the 2 cell lines. One of the C6-NBD-sphingolipids synthesized in G+ cells is not found in the G+ reversed cells. Furthermore, the ratio of the 2 main products, C6-NBD-glucosylceramide and C6-NBD-sphingomyelin, differs: in G+ cells glucosylceramide is by far the main product, whereas G+ reversed cells synthesize C6-NBD-sphingomyelin in slight excess. Once established, these ratios of sphingolipids are quickly restored metabolically when distortion of the ratio is caused by experimental manipulation. This indicates that they represent a true metabolic equilibrium situation of the 2 sphingolipids in these cells, while the distinct ratios are mainly determined by the NBD-lipid pool in the plasma membrane. Preferential synthesis and transfer of glucosylceramide from its site of synthesis to the cell surface do not occur when the plasma membrane pool of glucosylceramide is selectively removed. This suggests that instantaneous replenishment via specific signalling is probably not involved as a mechanism in re-establishing perturbed lipid pools. In conjunction with observations on distinct lipid trafficking pathways of glucosylceramide in G+ and G+ reversed cells, the present metabolic studies emphasize a relation between the expression of this glycolipid and the state of differentiation of HT29 cells.

4-Chloro-7-nitrobenzofurazan↗
PubMed1993

Molecular profile of an antibody response to HIV-1 as probed by combinatorial libraries.

A large number (33) of human Fab fragments reacting with HIV-1 surface glycoprotein gp120 have been generated by selection from a combinatorial IgG1 kappa library displayed on the surface of phage. The library was prepared from a long term asymptomatic HIV-seropositive donor. Analysis of the sequences from these Fabs shows the heavy chains can be placed in groups, many of which contain intraclonal variants, almost certainly corresponding to chains used in vivo. Further variants can be accessed via chain shuffling experiments in which a given light chain is recombined with a library of heavy chains. Heavy chain promiscuity, i.e. the ability of heavy chains to pair with different light chains with retention of antigen binding, is dependent on the particular heavy chain considered and probably excludes the identification of in vivo light chain partners. The antibodies examined here are primarily to the CD4 binding site on gp120 and broadly reflect the serum profile of the donor. The antibodies show evidence of extensive somatic modification indicative of an antigen-driven response. The heavy chain CDR3 regions of the antibodies show a remarkably conserved extended length. A number also show strong sequence conservation in CDR3 against a background of considerable diversity in the rest of the VH gene supporting a central role for this region in antigen recognition.

Amino Acid Sequence↗
PubMed1993

Translocation of fluorescent ether phospholipid, but not its diacyl counterpart, after insertion in plasma membranes of control and plasmalogen-deficient fibroblasts.

Fluorescently labelled ether phospholipid (1-O-alkyl/alkenyl-2-acyl- glycerophosphocholine) readily internalizes at low temperatures (2 degrees C) after insertion into the plasma membrane of cultured fibroblasts. This fate differs markedly from that of its diacyl phospholipid analogue, which remains associated with the plasma membrane under similar conditions. Analysis by thin-layer chromatography reveals that the translocation involves transfer of the intact ether phosphatidylcholine molecules. Relative to control cells, a 2-fold increase of ether phosphatidylcholine uptake was noted when plasmalogen deficient fibroblasts were used. Back-exchange experiments demonstrate that more than 60% of the cell-associated ether lipid is translocated within the cells, irrespective of the cell strain that was used. The potential mechanism by which the translocation process is accomplished is discussed.

Cell Membrane↗
PubMed1993

Interaction of myelin basic protein with artificial membranes. Parameters governing binding, aggregation and dissociation.

The interaction of myelin basic protein (MBP) with large unilamellar vesicles, composed of phosphatidylserine (PtdSer), phosphatidylserine/phosphatidylcholine (PtdSer/Ole2GroPCho) and phosphatidylcholine/cholesterol (Ole2GroPCho/cholesterol) was examined. Binding of MBP to the bilayers as well as the kinetics of this process were determined by a resonance energy transfer procedure. The ability of the protein to aggregate the vesicles subsequently was monitored continuously by absorbance measurements. The interaction was further characterized by determining the ability of MBP to induce membrane perturbations, as reflected by release of aqueous vesicle contents, and lipid mixing. The results demonstrate that Ole2GroPCho inhibits, while PtdSer and cholesterol strongly facilitate MBP-induced membrane aggregation. Furthermore, binding of MBP to vesicles and the subsequent aggregation event are separate processes, i.e. the extent of binding does not necessarily reflect the aggregation susceptibility. Overall, aggregation appears to be the rate-limiting step. Interaction of MBP with PtdSer bilayers results in a limited degree of lipid mixing, which is accompanied by extensive release of vesicle contents. For all other compositions, no lipid mixing occurs, while cholesterol effectively prevents release of vesicle contents. pH-dependent experiments indicate distinct mechanisms to be operative in MBP-induced aggregation of PtdSer and Ole2GroPCho/cholesterol bilayers. At neutral pH, protein-protein interactions appear relevant, while at acidic pH intervesicular bridges, established by monomers that may cause aggregation of PtdSer vesicles, but not of Ole2GroPCho/cholesterol vesicles. The observation that divalent cations reverse MBP-induced vesicle aggregation may have physiological relevance.

Calcium↗
PubMed1992

Asymmetric fusion between synthetic di-n-dodecylphosphate vesicles and virus membranes.

The interaction between vesicles, prepared from the synthetic amphiphile di-n-dodecylphosphate (DDP), with Sendai virus membranes was investigated. DDP vesicles fuse in the presence of Ca2+ ('symmetric' fusion). However, in the absence of Ca2+, DDP vesicles and Sendai virus, both displaying a high intrinsic fusion capacity with various target membranes, can also readily fuse with each other ('asymmetric' fusion). Under these conditions, fusion was found not to depend on specific viral proteins. Thus fusion occurs over a broad pH range (3.0-9.0) and is not affected by perturbation of viral protein structure. The overall interaction process was further analyzed with a mass action kinetic model. The analysis reveals that the destabilization and reorganization of the synthetic and viral bilayers are as fast as in pure phospholipid systems. Furthermore, the drastic effect of temperature on the overall reaction appears to be related to an effect of this parameter on fusion itself rather than on vesicle-virus aggregation. This could suggest that protein mobility constraints modulate the fusion reaction. The morphology of the fusion products, which consist of a single virus particle and several DDP vesicles, indicates a bilayer stabilization of the fusion product, rather than formation of tubular structures, as observed for symmetric DDP fusion products. The present results further emphasize the high susceptibility of vesicles composed of synthetic amphiphiles to engage in (protein-mediated) membrane fusion. This bears relevance to their potential application as carriers for biomolecules.

Cholesterol↗