Spectroscopic characterization of nanoErythrosomes in the absence and presence of conjugated polyethyleneglycols: an FTIR and P-NMR study

We have recently developed from red blood cells a new delivery system called nanoErythrosomes. These nanovesicles offer a high degree of versatility for the encapsulation of biological or nonbiological compounds and for the binding of targeting agents. In particular, polyethyleneglycols can be conjugated by a covalent link to the basic amino acid residues constitutive of the different proteins. The binding of polyethyleneglycols to the nanoErythrosome membrane could be interesting for the therapeutic use of this delivery system since it could overcome heterologous immunogenicity and reduce rapid clearance from circulation. In the present study, we have investigated the effect of temperature on the nanoErythrosome behavior in the absence and presence of conjugated polyethyleneglycols. More specifically, Fourier transform infrared (FTIR) spectroscopy has been used to evaluate the lipid order and dynamics, the hydration and the degree of protein aggregation of the nanoErythrosomes after covalent binding of polyethyleneglycols having molecular weights of 2000 and 5000 g mol⁻¹. The results indicate that the nanoErythrosome lipid chain order is not significantly affected by heating the nanoErythrosomes at temperatures up to 50 °C. They also indicate that the nanoErythrosome proteins aggregate irreversibly at temperatures above 37 °C, this effect being abolished in the presence of polyethyleneglycols. The presence of polyethyleneglycols decreases the accessibility of water to the lipid head groups. On the other hand, ³¹P-nuclear magnetic resonance (NMR) and electron microscopy results reveal that the presence of polyethyleneglycols prevents the aggregation of the nanoErythrosome structures.     

Biophysical studies of a transmembrane peptide derived from the T cell antigen receptor

Summary Core peptide (CP) is a unique peptide derived from the transmembrane sequence of T cell antigen receptor (TCR)-alpha chain and is capable of inhibiting the immune response both invitro and in animal models of T cell mediated inflammation. The structure of CP, with sequence GLRILLLKV, is similar to the amphipathic region of many peptides. Unlike antimicrobial peptides, however, which damage cell membranes, electron microscopy and propidium iodide exclusion assays on cell membranes suggest that CP does not create pores and may act by interfering with signal transduction at the membrane level. To investigate this effect further we report the results of³¹P and²H solid-state NMR spectroscopy of CP on model membranes. As predicted, even at high concentrations of CP, the structure of model membranes was not significantly perturbed. Only at the very high peptide-to-lipid molar ratio of 1∶10 significant effects on the model membranes were observed. We conclude that CP does not destroy the integrity of the lipid bilayer.     

Structural Enzymology of Cellvibrio japonicus Agd31B Protein Reveals α-Transglucosylase Activity in Glycoside Hydrolase Family 31

The metabolism of the storage polysaccharides glycogen and starch is of vital importance to organisms from all domains of life. In bacteria, utilization of these α-glucans requires the concerted action of a variety of enzymes, including glycoside hydrolases, glycoside phosphorylases, and transglycosylases. In particular, transglycosylases from glycoside hydrolase family 13 (GH13) and GH77 play well established roles in α-glucan side chain (de)branching, regulation of oligo- and polysaccharide chain length, and formation of cyclic dextrans. Here, we present the biochemical and tertiary structural characterization of a new type of bacterial 1,4-α-glucan 4-α-glucosyltransferase from GH31. Distinct from 1,4-α-glucan 6-α-glucosyltransferases (EC 2.4.1.24) and 4-α-glucanotransferases (EC 2.4.1.25), this enzyme strictly transferred one glucosyl residue from α(1→4)-glucans in disproportionation reactions. Substrate hydrolysis was undetectable for a series of malto-oligosaccharides except maltose for which transglycosylation nonetheless dominated across a range of substrate concentrations. Crystallographic analysis of the enzyme in free, acarbose-complexed, and trapped 5-fluoro-β-glucosyl-enzyme intermediate forms revealed extended substrate interactions across one negative and up to three positive subsites, thus providing structural rationalization for the unique, single monosaccharide transferase activity of the enzyme.     

Irradiation to the young mouse brain impaired white matter growth more in females than in males

Modern therapy cures 80% of all children with brains tumors, but may also cause long-lasting side effects, so called late effects. Radiotherapy is particularly prone to cause severe late effects, such as intellectual impairment. The extent and nature of the resulting cognitive deficits may be influenced by age, treatment and gender, where girls suffer more severe late effects than boys. The reason for this difference between boys and girls is unknown, but very few experimental studies have addressed this issue. Our aim was to investigate the effects of ionizing radiation on the corpus callosum (CC) in both male and female mice. We found that a single dose of 8 Gray (Gy) to the brains of postnatal day 14 mice induced apoptosis in the CC and reduced the number of proliferating cells by one third, as judged by the number of phospho-histone H3 positive cells 6 h after irradiation (IR). BrdU incorporation was reduced (62% and 42% lower in females and males, respectively) and the number of oligodendrocytes (Olig2⁺ cells) was lower (43% and 21% fewer in females and males, respectively) 4 months after IR, so the lack of developing and differentiated cells was more pronounced in females. The number of microglia was unchanged in females but increased in males at this late time point. The density of microvessel profiles was unchanged by IR. This single, moderate dose of 8 Gy impaired the brain growth to some extent (8.1% and 0.4% lower brain/body weight ratio in females and males, respectively) but the CC growth was even more impaired (31% and 19% smaller in females and males, respectively) 4 months after IR compared with non-irradiated mice. In conclusion, this is the first study to our knowledge demonstrating that IR to the young rodent brain affects white matter development more in females than in males.     

Vectorial redox reactions of physiological quinones. II. A study of transient semiquinone formation

Transient absorption changes during reduction of quinone in liposomes by external dithionite, in the absence and presence of initially trapped ferricyanide, were matched with absorption spectra of semiquinone and quinone in the blue region. Plastoquinone, ubiquinone-9 and phylloquinone, each having an isoprenoid side chain were compared with trimethyl-p-benzoquinone, ubiquinone-9 and menadione, which lack a long side chain.Semiquinone transients could only be observed by our spectroscopic technique during reduction of quinones lacking the chain. If Triton X-100 was added to the liposomes preparation semiquinone transients were also observed with the isoprenoid quinones. This result is consistent with the view that isoprenoid quinones build domains in the membranes, in which the life time of the semiquinone might be decreased by fast disproportionation, and to which dithionite has limited access.     

13C-31P Couplings in the Study of Conformational Properties of Some Diastereomeric Nucleoside-3′-Thiophosphate Derivatives

Synthesis and stereochemical characterization of enantiomerically pure nucleoside-3′-phosphorothioate esters and salts are reported. Vicinal carbon–phosphorus couplings reflect different predominance of the ? conformation in the isomeric (Rp and Sp) esters, while for the salts the ?^t conformation prevails in both stereoisomers. The influence of solvent and temperature on the conformational preferences is also described.