Arie J. Verkleij

The asymmetric distribution of phospholipids in the human red cell membrane. A combined study using phospholipases and freeze-etch electron microscopy

Abstract 1. 1. Phospholipase A2 (phosphatide acylhydrolase, EC 3.1.1.4) from Naja naja hydrolyses 68% of the lecithin of the intact human erythrocyt without changing the freeze fracture faces of the membrane. Phospholipase A2 (Naja naja) treatment of ghosts produces complete breakdown of the glycerophospholipids and induces aggregation of particles on the freeze-fracture faces of the membrane. 2. 2. Phospholipase C (phosphatidylcholine choline phosphohydrolase, EC 3.1.4.3) from Bacillus cereus does not attack intact cells and no change in freeze-etch morphology is observed. The glycerophospholipids of ghosts are almost completely degraded by this enzyme, which causes a reduction in tangentially-splitted membranes and a formation of large diglyceride droplets, which are also visible by phase-contrast microscopy. 3. 3. Sphingomyelinase (sphingomyelin choline phosphohydrolase) from Staphylococcus aureus, hydrolyses 80–85% of the sphingomyelin of the intact human red cel, and produces aggregation of the particles and the formation of small spheres (75 A and 200 A in diameter) on the outer fracture face with corresponding pits on the inner fracture face. Treatment of ghosts with this enzyme causes a complete degradation of the sphingomyelin and produces, in addition to aggregation of particles, the formation of droplets (1000–3000 A in diameters) whcih are adherent to the membrane and are not visible by phase-contrast microscopy. 4. 4. When the cells are treated successively with phospholipase A2 (Naja naja) and sphingomyelinase (Staphylococcus aureus) no lysis occurs although the osmotic fragility is markedly increased. By this treatment, up to 48% of the total phospholipids are degradd. It is concluded that this phospholipid fraction (which contains the majority of the choline-containing phospholipids and some phosphatidylethanolamine) forms the outer monolayer of the membrane.

Membrane damage by human islet amyloid polypeptide through fibril growth at the membrane

Fibrillar protein deposits (amyloid) in the pancreatic islets of Langerhans are thought to be involved in death of the insulin-producing islet β cells in type 2 diabetes mellitus. It has been suggested that the mechanism of this β cell death involves membrane disruption by human islet amyloid polypeptide (hIAPP), the major constituent of islet amyloid. However, the molecular mechanism of hIAPP-induced membrane disruption is not known. Here, we propose a hypothesis that growth of hIAPP fibrils at the membrane causes membrane damage. We studied the kinetics of hIAPP-induced membrane damage in relation to hIAPP fibril growth and found that the kinetic profile of hIAPP-induced membrane damage is characterized by a lag phase and a sigmoidal transition, which matches the kinetic profile of hIAPP fibril growth. The observation that seeding accelerates membrane damage supports the hypothesis. In addition, variables that are well known to affect hIAPP fibril formation, i.e., the presence of a fibril formation inhibitor, hIAPP concentration, and lipid composition, were found to have the same effect on hIAPP-induced membrane damage. Furthermore, electron microscopy analysis showed that hIAPP fibrils line the surface of distorted phospholipid vesicles, in agreement with the notion that hIAPP fibril growth at the membrane and membrane damage are physically connected. Together, these observations point toward a mechanism in which growth of hIAPP fibrils, rather than a particular hIAPP species, is responsible for the observed membrane damage. This hypothesis provides an additional mechanism next to the previously proposed role of oligomers as the main cytotoxic species of amyloidogenic proteins.

Human embryonic stem cell-derived cardiomyocytes survive and mature in the mouse heart and transiently improve function after myocardial infarction

Regeneration of the myocardium by transplantation of cardiomyocytes is an emerging therapeutic strategy. Human embryonic stem cells (HESC) form cardiomyocytes readily but until recently at low efficiency, so that preclinical studies on transplantation in animals are only just beginning. Here, we show the results of the first long-term (12 weeks) analysis of the fate of HESC-derived cardiomyocytes transplanted intramyocardially into healthy, immunocompromised (NOD-SCID) mice and in NOD-SCID mice that had undergone myocardial infarction (MI). Transplantation of mixed populations of differentiated HESC containing 20-25% cardiomyocytes in control mice resulted in rapid formation of grafts in which the cardiomyocytes became organized and matured over time and the noncardiomyocyte population was lost. Grafts also formed in mice that had undergone MI. Four weeks after transplantation and MI, this resulted in significant improvement in cardiac function measured by magnetic resonance imaging. However, at 12 weeks, this was not sustained despite graft survival. This suggested that graft size was still limiting despite maturation and organization of the transplanted cells. More generally, the results argued for requiring a minimum of 3 months follow-up in studies claiming to observe improved cardiac function, independent of whether HESC or other (adult) cell types are used for transplantation.

Comparative studies on the effects of pH and Ca2+ on bilayers of various negatively charged phospholipids and their mixtures with phosphatidylcholine

(1) The thermotropic behaviour of dimyristoyl phosphatidylglycerol, phosphatidylserine, phosphatidic acid and phosphatidylcholine was investigated by differential scanning calorimetry and freeze-fracture electron microscopy as a function of pH and of Ca2+ concentration. (2) From the thermotropic behaviour as a function of pH, profiles could be constructed from which apparent pK values of the charged groups of the lipids could be determined. (3) Excess Ca2+ induced a shift of the total phase transition in 14 : 0/14 : 0-glycerophosphocholine and 14 : 0/14 : 0-glycerophosphoglycerol mixtures. In 14 : 0/14 : 0-glycerophosphocholine bilayers containing 16 : 0/16 : 0-glycerophosphoglycerol lateral phase separation was induced by Ca2+. (4) Up to molar ratios of 1 : 2 of 14 : 0/14 : 0-glycerophosphoserine to 14 : 0/14: 0-glycerophosphocholine, excess Ca2+ induced lateral phase separation. Addition to mixtures of higher molar ratios caused segregation into different structures: the liposome organization and the stacked lamellae/cylindrical organization. (5) Addition of excess Ca2+ to mixtures of 14 : 0/14 : 0-glycerophosphocholine and 14 : 0/14 : 0-phosphatidic acid caused, independent of the molar ratio, separation into two structural different organizations. (6) The nature of Ca2+-induced changes in bilayers containing negatively charged phospholipids is strongly dependent on the character of the polar headgroup of the negatively charged phospholipid involved.

The epidermal growth factor.

Epidermal growth factor (EGF) is a single polypeptide of 53 amino acid residues which is involved in the regulation of cell proliferation. Egf exerts its effects in the target cells by binding to the plasma membrane located EGF receptor. The EGF receptor is a transmembrane protein tyrosine kinase. Binding of EGF to the receptor causes activation of the kinase and subsequently receptor autophosphorylation. The autophosphorylation is essential for the interaction of the receptor with its substrates. These bind to the receptor by the so‐called SH2 domains.

The cell wall architecture of Candida albicans wild-type cells and cell wall-defective mutants

In Candida albicans wild‐type cells, the β1,6‐glucanase‐extractable glycosylphosphatidylinositol (GPI)‐dependent cell wall proteins (CWPs) account for about 88% of all covalently linked CWPs. Approximately 90% of these GPI‐CWPs, including Als1p and Als3p, are attached via β1,6‐glucan to β1,3‐glucan. The remaining GPI‐CWPs are linked through β1,6‐glucan to chitin. The β1,6‐glucanase‐resistant protein fraction is small and consists of Pir‐related CWPs, which are attached to β1,3‐glucan through an alkali‐labile linkage. Immunogold labelling and Western analysis, using an antiserum directed against Saccharomyces cerevisiae Pir2p/Hsp150, point to the localization of at least two differentially expressed Pir2 homologues in the cell wall of C. albicans. In mnn9Δ and pmt1Δ mutant strains, which are defective in N‐ and O‐glycosylation of proteins respectively, we observed enhanced chitin levels together with an increased coupling of GPI‐CWPs through β1,6‐glucan to chitin. In these cells, the level of Pir‐CWPs was slightly upregulated. A slightly increased incorporation of Pir proteins was also observed in a β1,6‐glucan‐deficient hemizygous kre6Δ mutant. Taken together, these observations show that C. albicans follows the same basic rules as S. cerevisiae in constructing a cell wall and indicate that a cell wall salvage mechanism is activated when Candida cells are confronted with cell wall weakening.

Efficient inhibition of EGFR signaling and of tumour growth by antagonistic anti-EFGR Nanobodies.

The development of a number of different solid tumours is associated with over-expression of ErbB1, or the epidermal growth factor receptor (EGFR), and this over-expression is often correlated with poor prognosis of patients. Therefore, this receptor tyrosine kinase is considered to be an attractive target for antibody-based therapy. Indeed, antibodies to the EGFR have already proven their value for the treatment of several solid tumours, especially in combination with chemotherapeutic treatment regimens. Variable domains of camelid heavy chain-only antibodies (called Nanobodies™) have superior properties compared with classical antibodies in that they are small, very stable, easy to produce in large quantities and easy to re-format into multi-valent or multi-specific proteins. Furthermore, they can specifically be selected for a desired function by phage antibody display. In this report, we describe the successful selection and the characterisation of antagonistic anti-EGFR Nanobodies. By using a functional selection strategy, Nanobodies that specifically competed for EGF binding to the EGFR were isolated from ‘immune’ phage Nanobody repertoires. The selected antibody fragments were found to efficiently inhibit EGF binding to the EGFR without acting as receptor agonists themselves. In addition, they blocked EGF-mediated signalling and EGF-induced cell proliferation. In an in vivo murine xenograft model, the Nanobodies were effective in delaying the outgrowth of A431-derived solid tumours. This is the first report describing the successful use of untagged Nanobodies for the in vivo treatment of solid tumours. The results show that functional phage antibody selection, coupled to the rational design of Nanobodies, permits the rapid development of novel anti-cancer antibody-based therapeutics.

The influence of pH, Ca2+ and protein on the thermotropic behaviour of the negatively charged phospholipid, phosphatidylglycerol

Abstract It is demonstrated that the transition temperature from the liquid-crystalline to gel state of a synthetic phosphatidylglycerol is influenced by pH, Ca2+ and A1 basic protein from myelin.

Phase transitions of phospholipid bilayers and membranes of Acholeplasma laidlawii B visualized by freeze fracturing electron microscopy

Abstract 1. 1. By freeze fracturing electron microscopy regular band patterns were visualized on fracture faces of liposomes prepared from dimyristoylphosphatidylcholine, dielaidoylphosphatidylcholine, and 1-oleoyl-2-stearoylphosphatidylcholine below their respective transition temperatures. Above these temperatures only smooth fracture faces were observed. 2. 2. Liposomes of these phospholipids prepared after addition of more than 20 mole % cholesterol exhibited no band patterns below their transition temperatures. 3. 3. Fracture faces of the membrane of Acholeplasma laidlawii B (previously denoted as Mycoplasma laidlawii ) below the transition temperature showed structural details that can be attributed to a redistribution of membrane molecules as a consequence of the solidification of the membrane.

Electron Tomography for Heterogeneous Catalysts and Related Nanostructured Materials

The full potential in catalyst development will only be realized if characterization techniques are available that can probe materials with subnanometer resolution. One of the most employed techniques to image heterogeneous catalysts at the nanometer and subnanometer scale is transmission electron microscopy (TEM). As suggested by the name, TEM uses electrons transmitted through the object for imaging. Since the interaction between electrons and matter is very strong, only thin parts, commonly much less than a micron in thickness, are imaged. Since heterogeneous catalysts are, in most cases, structured on a much smaller length scale, the sample thickness can be reduced to TEM requirements by appropriate preparation techniques and is, therefore, no limitation.