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D Hoekstra

Publications and source records attributed to D Hoekstra.

131 records · Page 8Linked to original sources
PubMedDate not supplied

Serum as a modulator of lipoplex-mediated gene transfection: dependence of amphiphile, cell type and complex stability.

BACKGROUND: Cationic liposomes belong to the family of non-viral vectors for gene delivery. Despite several drawbacks, such as low efficiency compared to viruses and inactivation by serum, cationic liposomes remain a promising tool for gene therapy. Therefore further investigation of the mechanism of transfection and improvement of formulations are warranted. METHOD: In a comparative study, we investigated the effect of serum on the ability of SAINT, a novel synthetic amphiphile, and Lipofectin to mediate transfection in vitro, employing a variety of cell lines. RESULTS: In all cell types, SAINT-mediated transfection was not significantly affected by the presence of serum, in contrast to Lipofectin-mediated transfection. Intriguingly, the extent of complex association was enhanced in the presence of serum, while cell association of the Lipofectin complex was approximately two-fold higher than that of SAINT. These data imply that transfection efficiency and the amount of cell-associated complex are not related. However, when the helper lipid dioleoylphosphatidylethanolamine (DOPE) was substituted for cholesterol, SAINT-mediated transfection was reduced in the presence of serum. This indicates that lipoplex composition rather than the cationic lipid per se codetermines the effect of serum. Also, the presence of serum decreased cytotoxicity, while no correlation could be demonstrated between toxicity and transfection efficiency. The binding of serum proteins to either complex was identical, both in terms of protein identity and relative amounts. CONCLUSION: We propose that serum, in conjunction with cell-specific factors and lipoplex composition, determines complex (in)stability, which is crucial for effective gene delivery and expression.

Animals↗
PubMedDate not supplied

Membrane fusion: lipid vesicles as a model system.

In many cellular functions the process of membrane fusion is of vital importance. It occurs in a highly specific and strictly controlled fashion. Proteins are likely to play a key role in the induction and modulation of membrane fusion reactions. Aimed at providing insight into the molecular mechanisms of membrane fusion, numerous studies have been carried out on model membrane systems. For example, the divalent-cation induced aggregation and fusion of vesicles consisting of negatively charged phospholipids, such as phosphatidylserine (PS) or cardiolipin (CL), have been characterized in detail. It is important to note that these systems largely lack specificity and control. Therefore conclusions derived from their investigation can not be extrapolated directly to a seemingly comparable counterpart in biology. Yet, the study of model membrane systems does reveal the general requirements of lipid bilayer fusion. The most prominent barrier to molecular contact between two apposing bilayers appears to be due to the hydration of the polar groups of the lipid molecules. Thus, dehydration of the bilayer surface and fluctuations in lipid packing, allowing direct hydrophobic interactions, are critical to the induction of membrane fusion. These membrane alterations are likely to occur only locally, at the site of intermembrane contact. Current views on the way membrane proteins may induce fusion under physiological conditions also emphasize the notion of local surface dehydration and perturbation of lipid packing, possibly through penetration of apolar amino acid segments into the hydrophobic membrane interior.

Cell Fusion↗
PubMedDate not supplied

On the biogenesis of the myelin sheath: cognate polarized trafficking pathways in oligodendrocytes.

Oligodendrocytes, the myelinating cells of the central nervous system, are capable of transporting vast quantities of proteins and of lipids, in particular galactosphingolipids, to the myelin sheath. The sheath is continuous with the plasma membrane of the oligodendrocyte, but the composition of both membrane domains differs substantially. Given its high glycosphingolipid and cholesterol content the myelin sheath bears similarity to the lipid composition of the apical domain of a polarized cell. The question thus arises whether myelin components, like typical apical membrane proteins are transported by an apical-like trafficking mechanism to the sheath, involving a 'raft'-mediated mechanism. Indeed, the evidence indicates the presence of cognate apical and basolateral pathways in oligodendrocytes. However, all major myelin proteins do not participate in this pathway, and remarkably apical-like trafficking seems to be restricted to the oligodendrocyte cell body. In this review, we summarize the evidence on the existence of different trafficking pathways in the oligodendrocyte, and discuss possible mechanisms separating the oligodendrocyte's membrane domains.

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PubMedDate not supplied

Cationic lipid-mediated transfection in vitro and in vivo (review).

Recent rapid developments in genomics will likely lead to a rapid expansion in identifying defective genes causing a variety of diseases, implying a vast increase in the number of therapeutic targets. Treatment of such diseases may include strategies ranging from gene delivery and replacement to antisense approaches. For successful development of gene therapies, a minimal requirement involves the engineering of appropriate gene- or oligonucleotide-carrier systems, which are necessary for protective purposes (against nucleases) and transport (to target tissue and cells in vivo). Further, they should also display the propensity to efficiently translocate the oligonucleotides and gene constructs into cells, via passage across several membrane barriers. The emphasis in this review will be on the use of cationic lipids for that purpose. Crucial to successful application of this sophisticated technology in vivo will be a need for a better understanding of fundamental and structural parameters that govern transfection efficiency, including the issues of cationic lipid/DNA complex assembly (with or without helper lipid), stability towards biological fluids, complex-target membrane interaction and translocation, and gene-integration into the nucleus. Biophysical and biochemical characterization of so-called lipoplexes, and their interaction with cells in vitro, are considered instrumental in reaching such insight. Here, most recent advances in cationic lipid-mediated gene delivery are discussed from such a perspective.

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