Distribution of receptors and functions on cell surfaces: quantitation of ligand-receptor mobility and a new model for the control of plasma membrane topography

The long-range movements of membrane ligand-receptor complexes into surface caps and into the pseudopods of cells performing phagocytosis, the uropods of motile cells and the cleavage furrows of dividing cells appear to be analogous processes. A common mechanism to explain these movements must take into account several recent observations. First, laser photobleaching studies have indicated that Concanavalin A-receptor movement occurs unidirectionally; and analyses of Con A redistribution by quantitative video intensification microscopy (QUAVIM) have shown that movement may exceed the maximum rates measured for protein diffusion in membranes. These are the results predicted for a process of directed migration but not for a process of diffusion with entrapment. In addition it has been found that membrane receptors may segregate out of as well as into cap, pseudopod, uropod and cleavage furrow regions and that topographical heterogeneity on asymmetric cells is not restricted to membrane molecular determinants but extends to a range of endocytic functions and to a macromolecular complex, the coated pit. All dynamic surface events are arrested during mitosis. A new model for the regulation of plasma membrane topography has been developed from these diverse quantitative, functional and morphological data. Its essence is the entrainment of selected membrane determinants on membrane waves directed towards regions such as caps, pseudopods, uropods and cleavage furrows. The waves are initiated by tension due to asymmetric microfilament-membrane interaction.     

Lipids in critical care medicine

While enteral nutrition is the basis for the critically ill, parenteral nutrition is often used when a sufficient enteral nutrition is not or not fully achievable. Lipids are a mainstay of caloric supply in both cases as they combine the provision of building blocks for the membranes and are precursors for function molecules including lipid mediators bearing the ability to influence immunity. Pro-inflammatory lipid mediators as prostaglandins and leukotrienes are generated from arachidonic acid (AA), a key member of the n-6 polyunsaturated fatty acids (PUFA). In contrast, lipid mediators derived from the n-3 fatty acids eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) may exhibit less inflammatory properties compared to their AA-derived counterparts. Furthermore, intercellular mediators as resolvins and protectins are generated from n-3 fatty acids. They induce the resolution of inflammation, hence the name resolution phase interaction product—resolvin. Modulating the amount of PUFA and the n-6/n-3 ratio were investigated as means to change the inflammatory response and improve the outcome of patients. Experimental data showed that n-3 fatty acids may improve acute lung injury and sepsis in animal models. Studies in patients undergoing major surgery with application of n-3 fatty acids demonstrated beneficial effects in terms of reduction of length of stay and infectious complications. Clinical data hints that this concept may also improve outcome in critically ill patients. Additionally, experimental and clinical data suggest that a reduction in n-6 PUFA may change the immune response. In conclusion, modulating the amount of PUFA, the n-6/n-3 ratio and the composition of lipid emulsions may prove to be a useful means to improve the outcome of critically ill patients.     

Hepatitis B virus surface antigen assembly function persists when entire transmembrane domains 1 and 3 are replaced by a heterologous transmembrane sequence

Native hepatitis B surface antigen (HBsAg) spontaneously assembles into 22-nm subviral particles. The particles are lipoprotein micelles, in which HBsAg is believed to span the lipid layer four times. The first two transmembrane domains, TM1 and TM2, are required for particle assembly. We have probed the requirements for particle assembly by replacing the entire first or third TM domain of HBsAg with the transmembrane domain of HIV gp41. We found that either TM domain of HBsAg could be replaced, resulting in HBsAg-gp41 chimeras that formed particles efficiently. HBsAg formed particles even when both TM1 and TM3 were replaced with the gp41 domain. The results indicate remarkable flexibility in HBsAg particle formation and provide a novel way to express heterologous membrane proteins that are anchored to a lipid surface by their own membrane-spanning domain. The membrane-proximal exposed region (MPER) of gp41 is an important target of broadly reactive neutralizing antibodies against HIV-1, and HBsAg-MPER particles may provide a good platform for future vaccine development.     

Structural insight into the molecular basis of polyextremophilicity of short-chain alcohol dehydrogenase from the hyperthermophilic archaeon Thermococcus sibiricus

Biochemical analysis of enantioselective short-chain alcohol dehydrogenase from the hyperthermophilic archaeon Thermococcus sibiricus (TsAdh319) revealed unique polyextremophilic properties of the enzyme – half-life of 1 h at 100 °C, tolerance to high salt (up to 4 M) and organic solvents (50% v/v) concentrations. To elucidate the molecular basis of TsAdh319 polyextremophilicity, we determined the crystal structure of the enzyme in a binary complex with 5-hydroxy-NADP at 1.68 Å resolution. TsAdh319 has a tetrameric structure both in the crystals and in solution with an intersubunit disulfide bond. The substrate-binding pocket is hydrophobic, spacious and open that is consistent with the observed promiscuity in substrate specificity of TsAdh319. The present study revealed an extraordinary number of charged residues on the surface of TsAdh319, 70% of which were involved in ion pair interactions. Further we compared the structure of TsAdh319 with the structures of other homologous short-chain dehydrogenases/reductases (SDRs) from thermophilic and mesophilic organisms. We found that TsAdh319 has the highest arginine and aspartate + glutamate contents compared to the counterparts. The frequency of occurrence of salt bridges on the surface of TsAdh319 is the highest among the SDRs under consideration. No differences in the proline, tryptophan, and phenylalanine contents are observed; the compactness of the protein core of TsAdh319, the monomer and tetramer organization do not differ from that of the counterparts. We suggest that the unique thermostability of TsAdh319 is associated with the rigidity and simultaneous “resilience” of the structure provided by a compact hydrophobic core and a large number of surface ion pairs. An extensive salt bridge network also might maintain the structural integrity of TsAdh319 in high salinity.     

Ca transients in cardiac myocytes measured with a low affinity fluorescent indicator, furaptra.

Intracellular calcium ion ([Ca2+]i) transients were measured in single rat ventricular myocytes with the fluorescent indicator furaptra. Cells were voltage clamped with a single patch electrode containing the K+ salt of furaptra and fluorescence at 500 nm was measured during illumination with 350 and 370 nm light. Depolarizing voltage-clamp pulses elicited [Ca2+]-dependent fluorescent transients in 30 of 33 cells tested. The peak change in [Ca2+]i elicited by 50-ms depolarizations from -70 to +10 mV was 1.52 +/- 0.25 microM (mean +/- SEM, n = 7). The size of the [Ca2+]i transient increased in response to 10 microM isoproterenol, prolongation of the depolarization, and increasing pipette [Na+]. Because furaptra is sensitive to Ca2+ and Mg2+, changes in [Mg2+]i during the [Ca2+]i transient could not be measured. Instead, a single-compartment model was developed to simulate changes in [Mg2+] during [Ca2+] transients. The simulations predicted that a 2 microM [Ca2+] transient was accompanied by a slow increase in [Mg2+] (14-29 microM), which became larger as basal [Mg2+] increased (0.5-2.0 mM). The [Mg2+] transient reached a peak approximately 1 s after the peak of the [Ca2+] transient with the slow changes in [Mg2+] dominated by competition at the Ca2+/Mg2+ sites of Troponin. These changes in [Mg2+], however, were so small and slow that they were unlikely to affect the furaptra fluorescence signal at the peak of the [Ca2+]i transient. The [Ca2+]i transient reported by furaptra appears to be larger than that reported by other Ca2+ indicators; however, we conclude this larger transient is at least as accurate as [Ca2+]i transients reported by the other indicators.