Urea denaturation of alpha-hemolysin pore inserted in planar lipid bilayer detected by single nanopore recording: loss of structural asymmetry

The aim of this work is to study pore protein denaturation inside a lipid bilayer and to probe current asymmetry as a function of the channel conformation. We describe the urea denaturation of alpha-hemolysin channel and the channel formation of alpha-hemolysin monomer incubated with urea prior to insertion into a lipid bilayer. Analysis of single-channel recordings of current traces reveals a sigmoid curve of current intensity as a function of urea concentration. The normalized current asymmetry at 29+/-4% is observed between 0 and 3.56M concentrations and vanishes abruptly down to 0 concentration exceeds 4M. The loss of current asymmetry through alpha-hemolysin is due to the denaturation of the channel's cap. We also show that the alpha-hemolysin pore inserted into a lipid bilayer is much more resistant to urea denaturation than the alpha-hemolysin monomer in solution: The pore remains in the lipid bilayer up to 7.2M urea. The pore formation is possible up to 4.66M urea when protein monomers were previously incubated in urea.     

Crystal structure of the N-terminal NC4 domain of collagen IX, a zinc binding member of the laminin-neurexin-sex hormone binding globulin (LNS) domain family

Collagen IX, located on the surface of collagen fibrils, is crucial for cartilage integrity and stability. The N-terminal NC4 domain of the alpha1(IX) chain is probably important in this because it interacts with various macromolecules such as proteoglycans and cartilage oligomeric matrix protein. At least 17 distinct collagen polypeptides carry an NC4-like unit near their N terminus, but this report, describing the crystal structure of NC4 at 1.8-A resolution, represents the first atomic level structure for these domains. The structure is similar to previously characterized laminin-neurexin-sex hormone binding globulin (LNS) structures, dominated by an antiparallel beta-sheet sandwich. In addition, a zinc ion was found in a position similar to that of the metal binding site of other LNS domains. A partial backbone NMR assignment of NC4 was obtained and utilized in NMR titration studies to investigate the zinc binding in solution state and to quantitate the affinity of metal binding. The K(d) of 11.5 mM suggests a regulatory rather than a structural role for zinc in solution. NMR titration with a heparin tetrasaccharide revealed the presence of a secondary binding site for heparin on NC4, showing structural and functional conservation with thrombospondin-1, but a markedly reduced affinity for the ligand. Also the overall arrangement of the N and C termini of NC4 resembles most closely the N-terminal domain of thrombospondin-1, distinguishing the two from the majority of the published LNS structures.     

Effect of ghrelin administration on phagocytic activity in acute cold-restraint stress exposed rats

Ghrelin, an endogenous ligand for growth hormone secretagogue receptor, was identified in the rat stomach. This peptide acts through nitric oxide (NO) by expressing endothelial nitric oxide synthase (eNOS) and down regulating inducible nitric oxide synthase (iNOS) at its gastroproprotective effect against restraint stress induced damage. Recently the ghrelin receptor has also been detected in peripheral systems including immune tissue. We have investigated the possible effect of ghrelin on phagocytic activity of peritoneal macrophages in acute cold-restraint stress (ACRS) exposed rats. The rats were divided into control, stress and ghrelin groups. In ghrelin groups, single dose and three days consecutive dose of ghrelin (20 microg/kg. i.p.) were applied to rats that were exposed to ACRS for 4 h. 1 ml of saline was injected i.p. after ACRS for 3 consecutive days to the rats of the stress groups. Ghrelin administration reduced the increased phagocytic activity induced by ACRS. We conclude that ghrelin exerts an important role at macrophage phagocytic activity in ACRS exposed rats.     

From planta to pharma with glycosylation in the toolbox

Plant-specific glycosylation has long been a major limitation to the extensive use of plant-made pharmaceuticals in human therapy. Our goal here is to highlight the progress recently made towards humanization of N-glycosylation in plants and to illustrate that plant-typical N- and O-glycosylation progressively emerge as additional advantages for using this promising expression system.     

Self-assemblies of amphiphilic cyclodextrins

Cyclodextrins are natural cyclic oligosaccharides widely used as “molecular cages” in the pharmaceutical, agrochemical, food and cosmetical industries. The optimization of their pharmacological properties has led to the synthesis of numerous analogues. Amphiphilic derivatives were designed to improve the cell targeting of the drug-containing cyclodextrin cavities through their transportation in the organism, within self-assembling systems. Amphiphilic cyclodextrins can self-assemble into water-soluble aggregates such as mono or polydisperse micelles, or insert in lipid membranes and liposomes. Polysubstituted amphiphilic cyclodextrins are briefly reviewed, and monosubstituted derivatives of native and methylated β-cyclodextrins are presented in more details, with an emphasis on their self-organization within lipid membranes. Presented at the joint biannual meeting of the SFB-GEIMM-GRIP, Anglet France, 14–19 October, 2006.     

Contribution of NaCl excretion to salt resistance of Aeluropus littoralis (Willd) Parl

Aeluropus littoralis is a perennial halophyte, native to coastal zones. Although it is usually exposed to high saline, this plant grows normally without toxicity symptoms. In order to assess leaf salt excretion, different growth parameters, Na(+), K(+), Ca(2+), Mg(2+) and Cl(-) concentrations, as well as excreted ions were examined in plants grown for 2 months in the presence of various salinity levels (0-800 mM NaCl). In addition, salt crystals, salt glands and other leaf epidermal structures were investigated. Results showed that total plant growth decreased linearly with increase to medium salinity. This reduction concerns mainly shoot growth. In addition, this species was able to maintain its shoot water content at nearly 50% of the control even when subjected to 800 mM NaCl. Root water content seemed to be unaffected by salt. Sodium and chloride ion contents in shoots and in roots increased with salinity concentrations, in contrast to our observation for potassium. However, calcium and magnesium contents were not greatly affected by salinity. Excreted salts in A. littoralis leaves were in favor of sodium and chloride, but against potassium, calcium and magnesium which were retained in plants. Sodium and chloride were excreted from special salt glands, which were scattered on the both leaf surfaces. In addition to salt glands, papillae were the most frequent epidermal structure found on A. littoralis leaves, and are likely involved in A. littoralis salt resistance.     

Protective effect of cerebrocrast on rat brain ischaemia induced by occlusion of both common carotid arteries

Diabetes mellitus is accompanied by several cardiovascular complications including atherosclerosis, cerebral ischaemia and stroke. We examined the neuroprotective effect of a 1,4-dihydropyridine derivative cerebrocrast (C, a new antidiabetic agent, synthesized in the Latvian Institute of Organic Synthesis) on the level of ATP in the brain, and on changes of the EEG and ECG, as well as blood pressure parameters in anaesthetized Wistar male rats before and during 10-min occlusion of both common carotid arteries. Cerebrocrast was administered i.v. at doses of 1.0 and 10 microg/kg in the v. femoralis 20 min prior to ischaemia. After 10-min ischaemia animals were decapitated and the brain was immediately frozen in liquid nitrogen and subsequently used for analysis of changes of ATP contention. Cerebrocrast, administered at doses of 1.0 and 10 microg/kg 20 min prior to occlusion of both common carotid arteries, completely prevented a fall in the ATP content of brain compared with the control rats. In control rats the content of ATP in brain during ischaemia decreased from 2.77 +/- 0.22 (basal level) to 1.74 +/- 0.20 micromol/g as a result of ischaemia. By administration of cerebrocrast 20 min before occlusion of the arteries, the content of ATP in the brain remained at the level of preischaemia (1.0 microg/kg C + ischaemia 2.82 +/- 0.36; 10 microg/kg C + ischaemia 2.42 +/- 0.22 micromol/g). Analysis of EEG parameters both before and during 10 min of occlusion showed that at a C dose of 1.0 microg/kg before occlusion produced a regular alpha rhythm during ischaemia and prevented cerebral bioelectric activity from significant changes. The depression of basal rhythm was observed at a C dose of 10 microg/kg during ischaemia in two rats out of six as well as an increase in the ECG ST segment above the isoelectric line. Blood pressure was decreased by about 10-20 mm Hg. We propose that pretreatment of rats with cerebrocrast at doses of 1.0 or 10 microg/kg 20 min prior to ischaemia can prevent ischaemic damage of rat brain, maintain necessary energy consumption, promote ATP production in brain cells, and prevent significant changes in EEG and ECG parameters. These properties are important in diabetes mellitus and its evoked cardiovascular complications as stroke, ischaemia, etc.