Vacuolar SNARE Protein Transmembrane Domains Serve as Nonspecific Membrane Anchors with Unequal Roles in Lipid Mixing.

Membrane fusion is induced by SNARE complexes that are anchored in both fusion partners. SNAREs zipper up from the N to C terminus bringing the two membranes into close apposition. Their transmembrane domains (TMDs) might be mere anchoring devices, deforming bilayers by mechanical force. Structural studies suggested that TMDs might also perturb lipid structure by undergoing conformational transitions or by zipping up into the bilayer. Here, we tested this latter hypothesis, which predicts that the activity of SNAREs should depend on the primary sequence of their TMDs. We replaced the TMDs of all vacuolar SNAREs (Nyv1, Vam3, and Vti1) by a lipid anchor, by a TMD from a protein unrelated to the membrane fusion machinery, or by artificial leucine-valine sequences. Individual exchange of the native SNARE TMDs against an unrelated transmembrane anchor or an artificial leucine-valine sequence yielded normal fusion activities. Fusion activity was also preserved upon pairwise exchange of the TMDs against unrelated peptides, which eliminates the possibility for specific TMD-TMD interactions. Thus, a specific primary sequence or zippering beyond the SNARE domains is not a prerequisite for fusion. Lipid-anchored Vti1 was fully active, and lipid-anchored Nyv1 permitted the reaction to proceed up to hemifusion, and lipid-anchored Vam3 interfered already before hemifusion. The unequal contribution of proteinaceous TMDs on Vam3 and Nyv1 suggests that Q- and R-SNAREs might make different contributions to the hemifusion intermediate and the opening of the fusion pore. Furthermore, our data support the view that SNARE TMDs serve as nonspecific membrane anchors in vacuole fusion.

The role of counterions in the membrane-disruptive properties of pH-sensitive lysine-based surfactants

Surfactants are among the most versatile and widely used excipients in pharmaceuticals. This versatility, together with their pH-responsive membrane-disruptive activity and low toxicity, could also enable their potential application in drug delivery systems. Five anionic lysine-based surfactants which differ in the nature of their counterion were studied. Their capacity to disrupt the cell membrane was examined under a range of pH values, concentrations and incubation times, using a standard hemolysis assay as a model for endosomal membranes. The surfactants showed pH-sensitive hemolytic activity and improved kinetics at the endosomal pH range. Low concentrations resulted in negligible hemolysis at physiological pH and high membrane lytic activity at pH 5.4, which is in the range characteristic of late endosomes. With increasing concentration, the surfactants showed an enhanced capacity to lyse cell membranes, and also caused significant membrane disruption at physiological pH. This observation indicates that, at high concentrations, surfactant behavior is independent of pH. The mechanism of surfactant-mediated membrane destabilization was addressed, and scanning electron microscopy studies were also performed to evaluate the effects of the compounds on erythrocyte morphology as a function of pH. The in vitro cytotoxicity of the surfactants was assessed by MTT and NRU assays with the 3T3 cell line. The influence of different types of counterion on hemolytic activity and the potential applications of these surfactants in drug delivery are discussed. The possibility of using pH-sensitive surfactants for endosome disruption could hold great promise for intracellular drug delivery systems in future therapeutic applications.

Oncogenic K-Ras segregates at spatially distinct plasma membrane signaling platforms according to its phosphorylation status

Activating mutations in the K-Ras small GTPase are extensively found in human tumors. Although these mutations induce the generation of a constitutively GTP-loaded, active form of K-Ras, phosphorylation at Ser181 within the C-terminal hypervariable region can modulate oncogenic K-Ras function without affecting the in vitro affinity for its effector Raf-1. In striking contrast, K-Ras phosphorylated at Ser181 shows increased interaction in cells with the active form of Raf-1 and with p110α, the catalytic subunit of PI 3-kinase. Because the majority of phosphorylated K-Ras is located at the plasma membrane, different localization within this membrane according to the phosphorylation status was explored. Density-gradient fractionation of the plasma membrane in the absence of detergents showed segregation of K-Ras mutants that carry a phosphomimetic or unphosphorylatable serine residue (S181D or S181A, respectively). Moreover, statistical analysis of immunoelectron microscopy showed that both phosphorylation mutants form distinct nanoclusters that do not overlap. Finally, induction of oncogenic K-Ras phosphorylation - by activation of protein kinase C (PKC) - increased its co-clustering with the phosphomimetic K-Ras mutant, whereas (when PKC is inhibited) non-phosphorylated oncogenic K-Ras clusters with the non-phosphorylatable K-Ras mutant. Most interestingly, PI 3-kinase (p110α) was found in phosphorylated K-Ras nanoclusters but not in non-phosphorylated K-Ras nanoclusters. In conclusion, our data provide - for the first time - evidence that PKC-dependent phosphorylation of oncogenic K-Ras induced its segregation in spatially distinct nanoclusters at the plasma membrane that, in turn, favor activation of Raf-1 and PI 3-kinase.

Shifted loops and coercivity from field imprinted high energy barriers in ferritin and ferrihydrite nanoparticles

We show that the coercive field in ferritin and ferrihydrite depends on the maximum magnetic field in a hysteresis loop and that coercivity and loop shifts depend both on the maximum and cooling fields. In the case of ferritin, we show that the time dependence of the magnetization also depends on the maximum and previous cooling fields. This behavior is associated to changes in the intraparticle energy barriers imprinted by these fields. Accordingly, the dependence of the coercive and loop-shift fields with the maximum field in ferritin and ferrihydrite can be described within the frame of a uniform-rotation model considering a dependence of the energy barrier with the maximum and the cooling fields.

Epithelial Sodium Channel-Mediated Sodium Transport Is Not Dependent on the Membrane-Bound Serine Protease CAP2/Tmprss4.

The membrane-bound serine protease CAP2/Tmprss4 has been previously identified in vitro as a positive regulator of the epithelial sodium channel (ENaC). To study its in vivo implication in ENaC-mediated sodium absorption, we generated a knockout mouse model for CAP2/Tmprss4. Mice deficient in CAP2/Tmprss4 were viable, fertile, and did not show any obvious histological abnormalities. Unexpectedly, when challenged with sodium-deficient diet, these mice did not develop any impairment in renal sodium handling as evidenced by normal plasma and urinary sodium and potassium electrolytes, as well as normal aldosterone levels. Despite minor alterations in ENaC mRNA expression, we found no evidence for altered proteolytic cleavage of ENaC subunits. In consequence, ENaC activity, as monitored by the amiloride-sensitive rectal potential difference (ΔPD), was not altered even under dietary sodium restriction. In summary, ENaC-mediated sodium balance is not affected by lack of CAP2/Tmprss4 expression and thus, does not seem to directly control ENaC expression and activity in vivo.

Computational toolbox towards evolutionary domain mapping of membrane proteins

Membrane proteins account for about 20% to 30% of all proteins encoded in a typical genome. They play central roles in multiple cellular processes mediating the interaction of the cell with its surrounding. Over 60% of all drug targets contain a membrane domain. The experimental difficulties of obtaining a crystal structural severely limits our ability or understanding of membrane protein function. Computational evolutionary studies of proteins are crucial for the prediction of 3D structures. In this project, we construct a tool able to quantify the evolutionary positive selective pressure on each residue of membrane proteins through maximum likelihood phylogeny reconstruction. The conservation plot combined with a structural homology model is also a potent tool to predict those residues that have essentials roles in the structure and function of a membrane protein and can be very useful in the design of validation experiments.

Botulinum neurotoxin type A blocks the morphological changes induced by chemical stimulation on the presynaptic membrane of Torpedo synaptosomes.

The action of botulinum neurotoxin on acetylcholine release, and on the structural changes at the presynaptic membrane associated with the transmitter release,was studied by using a subcellular fraction of cholinergic nerve terminals (synaptosomes) isolated from the Torpedo electric organ. Acetylcholine and ATP release were continuously monitored by chemiluminescent methods.To catch the membrane morphological changes, the quick-freezing method was applied. Our results show that botulinum neurotoxin inhibits the release of acetylcholine from these isolated nerve terminals in a dose-dependent manner, whereas ATP release is not affected. The maximal inhibition (70%) is achieved at neurotoxin concentrations as low as 125 pM with an incubation time of 6 min. This effect is not linked to an alteration of the integrity of the synaptosomes since, after poisoning by botulinum neurotoxin type A, they show a nonmodified occluded lactate dehydrogenase activity. Moreover, membrane potential is not altered by the toxin with respect to the control, either in resting condition or after potassium depolarization. In addition to acetylcholine release inhibition, botulinum neurotoxin blocks the rearrangement of the presynaptic intramembrane particles induced by potassium stimulation. The action of botulinum neurotoxin suggests that the intramembrane particle rearrangement is related to the acetylcholine secretion induced by potassium stimulation in synaptosomes isolated from the electric organ of Torpedo marmorata.

Mixed and Hybrid Models of Public Service Delivery

This symposium presents research from different contexts to improve our collective understanding of a variety of aspects of mixed forms of service delivery, be they mixed contracting at the level of the market (which is more common in the U.S.), or mixed management and ownership at the level of the firm (which is more common in Europe). The articles included in this special symposium examine the factors that give rise to mixed forms of service delivery (e.g., economic and fiscal stress, regulatory flexibility, geography, management) and how these factors impact their design and operation. Articles also explore the performance of mixed forms of service delivery relative to more conventional arrangements like contracted or direct service delivery. The articles contribute to a better theoretical and conceptual understanding of mixed/hybrid forms of services delivery.

NMR chemical shifts of the rhodopsin chromophore in the dark state and in bathorhodopsin: a hybrid QM/MM molecular dynamics study.

We investigate nuclear magnetic resonance (NMR) parameters of the rhodopsin chromophore in the dark state of the protein and in the early photointermediate bathorhodopsin via first-principles molecular dynamics simulations and NMR chemical shift calculations in a hybrid quantum/classical (QM/MM) framework. NMR parameters are particularly sensitive to structural properties and to the chemical environment, which allows us to address different questions about the retinal chromophore in situ. Our calculations show that both the 13C and the 1H NMR chemical shifts are rather insensitive to the protonation state of Glu181, an ionizable amino acid side chain located in the vicinity of the isomerizing 11-cis bond. Thus, other techniques should be better suited to establish its protonation state. The calculated chemical shifts for bathorhodopsin further support our previously published theoretical structure, which is in very good agreement with more recent X-ray data.

The effects of some variables on CO2 laser-MAG hybrid welding

The CO2-laser-MAG hybrid welding process has been shown to be a productive choice for the welding industry, being used in e.g. the shipbuilding, pipe and beam manufacturing, and automotive industries. It provides an opportunity to increase the productivity of welding of joints containing air gaps compared with autogenous laser beam welding, with associated reductions in distortion and marked increases in welding speeds and penetration in comparison with both arc and autogenous laser welding. The literature study indicated that the phenomena of laser hybrid welding are mostly being studied using bead-on-plate welding or zero air gap configurations. This study shows it very clearly that the CO2 laser-MAG hybrid welding process is completely different, when there is a groove with an air gap. As in case of industrial use it is excepted that welding is performed for non-zero grooves, this study is of great importance for industrial applications. The results of this study indicate that by using a 6 kW CO2 laser-MAG hybrid welding process, the welding speed may also be increased if an air gap is present in the joint. Experimental trials indicated that the welding speed may be increased by 30-82% when compared with bead-on-plate welding, or welding of a joint with no air gap i.e. a joint prepared as optimum for autogenous laser welding. This study demonstrates very clearly, that the separation of the different processes, as well as the relative configurations of the processes (arc leading or trailing) affect welding performance significantly. These matters influence the droplet size and therefore the metal transfer mode, which in turn determined the resulting weld quality and the ability to bridge air gaps. Welding in bead-onplate mode, or of an I butt joint containing no air gap joint is facilitated by using a leading torch. This is due to the preheating effect of the arc, which increases the absorptivity of the work piece to the laser beam, enabling greater penetration and the use of higher welding speeds. With an air gap present, air gap bridging is more effectively achieved by using a trailing torch because of the lower arc power needed, the wider arc, and the movement of droplets predominantly towards the joint edges. The experiments showed, that the mode of metal transfer has a marked effect on gap bridgeability. Transfer of a single droplet per arc pulse may not be desirable if an air gap is present, because most of the droplets are directed towards the middle of the joint where no base material is present. In such cases, undercut is observed. Pulsed globular and rotational metal transfer modes enable molten metal to also be transferred to the joint edges, and are therefore superior metal transfer modes when bridging air gaps. It was also found very obvious, that process separation is an important factor in gap bridgeability. If process separation is too large, the resulting weld often exhibits sagging, or no weld may be formed at all as a result of the reduced interaction between the component processes. In contrast, if the processes are too close to one another, the processing region contains excess molten metal that may create difficulties for the keyhole to remain open. When the distance is optimised - i.e. a separation of 0-4 mm in this study, depending on the welding speed and beam-arc configuration - the processes act together, creating beneficial synergistic effects. The optimum process separation when using a trailing torch was found to be shorter (0-2 mm) than when a leading torch is used (2-4 mm); a result of the facilitation of weld pool motion when the latter configuration is adopted. This study demonstrates, that the MAG process used has a strong effect on the CO2-laser-MAG hybrid welding process. The laser beam welding component is relatively stable and easy to manage, with only two principal processing parameters (power and welding speed) needing to be adjusted. In contrast, the MAG process has a large number of processing parameters to optimise, all of which play an important role in the interaction between the laser beam and the arc. The parameters used for traditional MAG welding are often not optimal in achieving the most appropriate mode of metal transfer, and weld quality in laser hybrid welding, and must be optimised if the full range of benefits provided by hybrid welding are to be realised.

System Investigation for Hybrid Electric Vehicle

A hybrid electric vehicle is a fast-growing concept in the field of vehicle industry. Nowadays two global problems make manufactures to develop such systems. These problems are: the growing cost of a fuel and environmental pollution. Also development of controlled electric drive with high control accuracy and reliability allows improving of vehicle drive characteristics. The objective of this Diploma Thesis is to investigate the possibilities of electrical drive application for new principle of parallel hybrid vehicle system. Electric motor calculations, selection of most suitable control system and other calculations are needed. This work is not final work for such topic. Further investigation with more precise calculations, modeling, measurements and cost calculations are needed to answer the question if such system is efficient.