Interaction of methionine–enkephalins with raft-forming lipids: monolayers and BAM experiments

Enkephalins (Tyr-Gly-Gly-Phe-Met/Leu) are opioid peptides with proven antinociceptive action in organism. They interact with opioid receptors belonging to G-protein coupled receptor superfamily. It is known that these receptors are located preferably in membrane rafts composed mainly of sphingomyelin (Sm), cholesterol (Cho), and phosphatidylcholine. In the present work, using Langmuir’s monolayer technique in combination with Wilhelmy’s method for measuring the surface pressure, the interaction of synthetic methionine–enkephalin and its amidated derivative with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), Sm, and Cho, as well as with their double and triple mixtures, was studied. From the pressure/area isotherms measured, the compressional moduli of the lipids and lipid–peptide monolayers were determined. Our results showed that the addition of the synthetic enkephalins to the monolayers studied led to change in the lipid monolayers characteristics, which was more evident in enkephalinamide case. In addition, using Brewster angle microscopy (BAM), the surface morphology of the lipid monolayers, before and after the injection of both enkephalins, was determined. The BAM images showed an increase in surface density of the mixed surface lipids/enkephalins films, especially with double and triple component lipid mixtures. This effect was more pronounced for the enkephalinamide as well. These observations showed that there was an interaction between the peptides and the raft-forming lipids, which was stronger for the amidated peptide, suggesting a difference in folding of both enkephalins. Our research demonstrates the potential of lipid monolayers for elegant and simple membrane models to study lipid–peptide interactions at the plane of biomembranes.     

Poring over exosome structure

The authors analyse the eukaryotic exosome structure, published in EMBO reports, in light of the known archaeal and prokaryotic exosomes, and discuss its striking flexibility and the conservation of the RNA channelling mechanism.EMBO Rep (2010) advance online publication. doi: 10.1038/embor.2010.164Almost all RNA molecules are processed by RNases to form mature RNAs. In addition, many RNAs are degraded, either because they are no longer needed or because they are aberrant. All of these functions—RNA processing, normal RNA degradation and RNA quality control—are carried out by the eukaryotic RNA exosome complex. In this issue of EMBO reports, the Lorentzen group provide structural insight into the eukaryotic exosome and the mechanism by which it degrades RNA from 3′ to 5′ (Malet et al, 2010).The crystal structures of overlapping parts of the eukaryotic exosome (Liu et al, 2006; Bonneau et al, 2009) and the related bacterial PNPase (Symmons et al, 2000) and archaeal exosome (Lorentzen et al, 2007) have been solved, and show that these RNA-degrading machines from the three domains of life have a similar structure (Fig 1). They are all composed of a ring of six RNase PH domains, one side of which has a cap that contains putative RNA-binding domains. Although this overall structure is conserved, the way that it is formed is not. Bacterial PNPase is a homotrimer of which each monomer contains two RNase PH domains, an S1 domain and a KH domain. The archaeal PH ring consists of three copies of two proteins and the cap is made of three copies of either one of two proteins. Finally, the eukaryotic exosome core is composed of nine proteins: six with one RNase PH domain each and three cap proteins.Open in a separate windowFigure 1Exosome structures. The bacterial PNPase (left), the archaeal exosome (middle) and eukaryotic core exosome (right) have a common overall structure. The top panels are schematic views from above, showing the cap proteins. The bottom panels show a view from the side, with one-third of the exosome cut away to reveal the RNA in the central channel.In PNPase and the archaeal exosome, substrates enter the PH ring from the cap-side. The putative RNA-binding domains of the cap are therefore probably important for controlling entry to the PH ring. In both archaea and bacteria, the active sites are on the inner side of the PH ring and thus the ribonucleic catalysis occurs inside the central channel. However, in humans and yeast each of the RNase PH domains have point mutations that make the exosome ring catalytically inactive (Dziembowski et al, 2007). Instead, catalysis is carried out by a tenth subunit—Rrp44/Dis3—which binds to the PH ring on the opposite side to the cap proteins (Bonneau et al, 2009; Wang et al, 2007). This organization made it unclear whether RNA also enters the central channel of the exosome in eukaryotes (Fig 1), or whether substrate RNAs directly access the catalytic subunit.Malet and colleagues now provide structural information that resolves this by reconstituting the ten-subunit yeast exosome and analysing its structure with electron microscopy, in the presence and absence of RNA. This analysis suggests that the RNase PH ring of the exosome is stable, but that the cap and catalytic subunits are more flexible than previously appreciated. It is the first structural evidence that in eukaryotes RNA is threaded through the central channel before being degraded by Rrp44.     

Essential metal ions alter the lactoferrin binding to the erythrocyte plasma membrane receptors

The effect of metal ions at a concentration of l0^-8 to l0^-5 M [using their salts: ZnCl₂, CdCl₂, LiCl, CuSO₄, NiSO₄, A1₂(SO₄)₃, (NH₄)₂MoO₄ on the lactoferrin (Lf) binding to the erythrocyte membrane receptors was studied. In the absence of metal ions, Scatchard’s analysis showed the existence of two kinds of binding site: one with high affinity and low capacity, and the another with low affinity and high capacity. All these metals, excluding Zn²⁺ and Cd²⁺, at a concentration 10^-5 M decreased the affinity of Lf binding (Ka₁) to the high-affinity receptors. In the presence of Zn²⁺ and Cd²⁺, only the lowaffinity binding site was found. Significant inhibition on the affinity (Ka₂) of the low-affinity class of receptors showed Zn²⁺, Al³⁺, and Mo⁶⁺. Depending on their concentration (10^-8-10^-5 M), these ions enhanced to a different extent, the binding capacity of the both types receptors, but the effect did not correspond to the applied doses. Several explanations of the mechanism for influence of the metal ions on the Lf-receptor interaction is discussed.     

Beam rate influence on dose distribution and fluence map in IMRT dynamic technique

AimTo examine the impact of beam rate on dose distribution in IMRT plans and then to evaluate agreement of calculated and measured dose distributions for various beam rate values.BackgroundAccelerators used in radiotherapy utilize some beam rate modes which can shorten irradiation time and thus reduce ability of patient movement during a treatment session. This aspect should be considered in high conformal dynamic techniques.Materials and methodsDose calculation was done for two different beam rates (100 MU/min and 600 MU/min) in an IMRT plan. For both, a comparison of Radiation Planning Index (RPI) and MU was conducted. Secondly, the comparison of optimal fluence maps and corresponding actual fluence maps was done. Next, actual fluence maps were measured and compared with the calculated ones. Gamma index was used for that assessment. Additionally, positions of each leaf of the MLC were controlled by home made software.ResultsDose distribution obtained for lower beam rates was slightly better than for higher beam rates in terms of target coverage and risk structure protection. Lower numbers of MUs were achieved in 100 MU/min plans than in 600 MU/min plans. Actual fluence maps converted from optimal ones demonstrated more similarity in 100 MU/min plans. Better conformity of the measured maps to the calculated ones was obtained when a lower beam rate was applied. However, these differences were small. No correlation was found between quality of fluence map conversion and leaf motion accuracy.ConclusionExecution of dynamic techniques is dependent on beam rate. However, these differences are minor. Analysis shows a slight superiority of a lower beam rate. It does not significantly affect treatment accuracy.     

Interaction of monogalactosyldiacylglycerol with cytochrome b₆f complex in surface films

The interaction of monogalactosyldiacylglycerol (MGDG) with cytochrome b(6)f complex (cyt b(6)f), a major component of the photosynthetic apparatus, was studied in Langmuir monolayers during compression/expansion cycling and at constant surface pressure mode. The surface pressure/area isotherms of the mixed films were analyzed in terms of surface compressional modulus and two-dimensional virial equation of state. The morphology and the surface potential of the monolayers were monitored by Brewster angle microscopy and vibrating plate sensor respectively. Our results suggested that there is a specific interaction between MGDG and cyt b(6)f which resulted in depletion of lipid molecules from the interface. The current work sheds light on the still unclear question how b(6)f complex gets in touch with the major compound of the thylakoid membranes, the non-charged lipid MGDG. The interaction occured even at very low sub-nanomolar concentration of the complex. This effect most probably could be attributed to hydrogen bonding between the galactose headgroup of the lipid and the protein moiety of cyt b(6)f.     

Lactoferrin-protector against oxidative stress and regulator of glycolysis in human erythrocytes

Binding of lactoferrin (Lf) to its membrane receptors requires an electron for the reduction of Fe(3+)LF to Fe(2+)LF. It is possible that glyceraldehyde -3-phosphate dehydrogenase, a glycolytic enzyme part of the erythrocyte membrane, delivers that electron. Then Lf, obtaining an electron from the coenzyme NADH, might stimulate glycolysis, which requires the oxidised state of the coenzyme NAD+. Such possibility is supported by the finding that another extracellular e- acceptor--potassium ferricyanide activates glycolysis by the similar mechanism. Present results show that ferricyanide inhibited the specific 59Fe-lactoferrin binding to its erythrocyte membrane receptors. It may be assumed that ferricyanide competes with lactoferrin for an electron which leads to decrease of the binding of 59Fe-lactoferrin to its receptors. Lactoferrin (50 and 100 nM), similar to ferricyanide, increased the accumulation of lactate (respectively by 25% and 30%). These results support the assumption that ferricyanide and lactoferrin are final acceptors of a common electron transport chain connected with the regulation of glycolysis. We established an antioxidative effect of lactoferrin on erythrocytes, which was expressed as: a) an influence on content and on activity of intracellular antioxidants--namely an enhancement of the content of reduced glutathione; b) a decreased content both of products of lipid peroxidation (thiobarbituric acid reactive substances) and hemolysis under normal conditions and oxidative stress. Lactoferrin is capable to bind metal ions and thus to block their catalytic participation in the oxidative disturbances of the membrane. In most of our experiments there were no metal ions in the incubation mixtures (except those stimulating oxidative stress). Our results showed that Lf limited both the generation of thiobarbituric acid reactive substances and hemolysis in the absence of metal ions in the media, as well as in their presence. These facts suggest that probably the antioxidative property of lactoferrin is glycolysis stimulation, leading to increased formation of ATP, which is necessary to maintain the ion gradient, membrane potential and morphology of the erythrocyte.