Influence of plasmid pR50 controlling bacterial protease activity on Salmonella experimental infection

The comparative analysis of the infectious process and immunological parameters in (CBA x C57BL/6)F1 mice infected with S. typhimurium isogenic strains differing by the presence of plasmid pR50 determining protease activity, was carried out. A growth in the expression level of antilactoferrin, anticomplementary and anti-immunoglobulin activity in bacteria isolated from the spleen in the course of the infectious process was detected. In mice infected with S. typhimurium having plasmid pR50, in contrast to nonplasmid recipients, a higher level of contamination of organs, the suppression of spontaneous, stimulated production of interferon-gamma and the bactericidal properties of peritoneal macrophages were noted. The data obtained in this investigation suggested that the acquisition of R-plasmid of 50 MD, controlling protease activity and multiple medicinal resistance, contributed to the persistence of intracellular bacteria.     

Identification of cysteinylation of a free cysteine in the Fab region of a recombinant monoclonal IgG1 antibody using Lys-C limited proteolysis coupled with LC/MS analysis

MAB007, an IgG1 monoclonal antibody, is unique because of the presence of a free cysteine residue in the Fab region at position 104 on the heavy chain in the CDR3 region. Mass spectrometric analysis of intact MAB007 showed multiple peaks varying in mass by 120-140 Da that cannot be fully attributed to glycosylation isoforms typically present in IgG molecules. Limited proteolysis of MAB007 with Lys-C led to a single cleavage at the C-terminus of a lysine residue in the hinge region of the heavy chain at position 222, generating free Fab and Fc fragments. Reversed-phase liquid chromatography/mass spectrometry analysis of the Fab and Fc fragments revealed several modifications. The Fab fraction showed cysteinylation of a free cysteine in the CDR3 region resulting in a mass shift of 119 Da. Using limited proteolysis, we also identified modifications resulting in a mass increase of 127 Da in the Fc region, corresponding to C-terminal lysine variants in the heavy chain. Other modifications, such as oxidation (+16 Da) and succinimide formation (-17 Da), were also detected in the Fab fragment. The cysteinylation observed after limited proteolysis was confirmed by peptide mapping coupled with tandem mass spectrometry analysis.     

In vitro stoichiometry of complexes between the soluble RANK ligand and the monoclonal antibody denosumab

The in vitro binding stoichiometry of denosumab, an IgG2 fully human monoclonal therapeutic antibody, to RANK ligand was determined by multiple complementary size separation techniques with mass measuring detectors, including two solution-based techniques (size-exclusion chromatography with static light scattering detection and sedimentation velocity analytical ultracentrifugation) and a gas-phase analysis by electrospray ionization time-of-flight mass spectrometry from aqueous nondenaturing solutions. The stoichiometry was determined under defined conditions ranging from small excess RANK ligand to large excess denosumab (up to 40:1). High concentrations of denosumab relative to RANK ligand were studied because of their physiological relevance; a large excess of denosumab is anticipated in circulation for extended periods relative to much lower concentrations of free soluble RANKL. The studies revealed that an assembly including 3 denosumab antibody molecules bound to 2 RANKL trimers (3D2R) is the most stable complex in DPBS at 37 °C. This differs from the 1:1 binding stoichiometry reported for RANKL and osteoprotegerin (OPG), a soluble homodimeric decoy receptor which binds RANKL with high affinity. Denosumab and RANKL also formed smaller assemblies including 1 denosumab and 2 RANKL trimer molecules (1D2R) under conditions of excess RANKL, 3 denosumab molecules and 1 RANKL trimer (3D1R) under conditions of excess denosumab, and larger assemblies, but these intermediate species were only present at lower temperatures (4 °C), shortly after mixing denosumab and RANKL, and converted over time to the more stable 3D2R assembly.     

Crowding-effect and attention

We studied the influence of additional objects on recognition of the test visual objects. The test objects were stylized low-contrast letters having size 1.1, 2.1 or 4.3 ang. deg. The additional objects after 30 ms were followed by the test objects which were presented in the middle of the screen. The additional objects were digits 1-9 having size 1.3 ang. deg. The digits were presented at various distances from the centre of the screen. The observers' task was to identify both the test objects and the digits. Recognition of the test objects deteriorated when the digits were at small distances to the tests (crowding-effect). Recognition of digits deteriorated with the increasing distances from the centre of the screen; the effect was more pronounced when the tests were large. The contribution of laterals masking and attention into crowding-effect is discussed.     

Association study of renin-angiotensin system genes and hemostasis system genes with ischemic stroke among Russians of Central Russia

The analysis of alleles and genotypes frequencies of 14 SNP in genes of rennin-angiotensin system (REN, AGT, AGTR1, AGTR2, BKR2, ADRB2) and hemostasis system (FGB, F2, F5, F7, ITGB3, SERPINE1, MTHFR), as well as ACE insertion-deletion polymorphism in patients with stroke comparing to healthy controls matched by age, sex and ethnicity has been carried out. The genotyping procedure included the amplification of selected gene sequences following by hybridization of fluorescently labeled fragments with SNP-specific DNA probes. The analysis of allele frequencies of each gene separately revealed no statistically significant differences between groups of patients with stroke and healthy donors. Also the complex study has been performed to estimate the contribution of rennin-angiotensin system and hemostasis system genes to the genetic susceptibility to ischemic stroke among Russians from Central Russia using method MDR (Multifactor Dimensionality Reduction). The combination with increased risk for development of ischemic stroke was presented by complex genotype FGB G/- x ACE I/- x MTHFR C/- x SERPINE1 5G/5G (p = 0.03, OR = 2.4, 95% CI 1.1-5.3), which frequency was statistically significant higher in patients with stroke compared to healthy control.     

Pollination efficiency of wild bees and hoverflies provided to oilseed rape

• 1 Declining numbers in honeybees and various wild bee species pose a threat to global pollination services. The identification and quantification of the pollination service provided by different taxa within the pollinator guild is a prerequisite for the successful establishment of nature conservation and crop management regimes.
• 2 Wild bees and hoverflies are considered to be valuable pollinators in agricultural and natural systems. Although some information on pollination efficiency of individual pollinator species is available, comparative studies of both taxa at different densities are rare. In the present study, the efficiency of the solitary mason bee Osmia rufa and two hoverfly species (Eristalis tenax and Episyrphus balteatus) as pollinators of oilseed rape Brassica napus was examined in a standardized caged plant breeding regime. Honeybee Apis mellifera colonies were used as a reference pollinator taxon.
• 3 Yield parameters responded differently to pollinator density and identity. Fruit set and number of seeds per pod increased with increasing pollinator density, although these were stronger in the mason bee than the hoverfly treatment. Weight per 1000 seeds did not respond to any pollinator treatment, indicating that seed quality was not affected. Oilseed rape yield in the highest tested densities of both pollinator taxa resulted in yield values close to the efficiency of small honeybee colonies.
• 4 Hoverflies required approximately five‐fold densities of the red mason bees to reach a similar fruit set and yield. Thus, mason bees are more efficient in plant breeding and managed pollination systems. Both natural pollinator taxa, however, are of potential value in open and closed crop production systems.

     

Four-alpha-helix bundle with designed anesthetic binding pockets. Part I: structural and dynamical analyses

The four-α-helix bundle mimics the transmembrane domain of the Cys-loop receptor family believed to be the protein target for general anesthetics. Using high resolution NMR, we solved the structure (Protein Data Bank ID: 2I7U) of a prototypical dimeric four-α-helix bundle, (Aα₂-L1M/L38M)₂, with designed specific binding pockets for volatile anesthetics. Two monomers of the helix-turn-helix motif form an antiparallel dimer as originally designed, but the high-resolution structure exhibits an asymmetric quaternary arrangement of the four helices. The two helices from the N-terminus to the linker (helices 1 and 1′) are associated with each other in the dimer by the side-chain ring stacking of F12 and W15 along the long hydrophobic core and by a nearly perfect stretch of hydrophobic interactions between the complementary pairs of L4, L11, L18, and L25, all of which are located at the heptad e position along the helix-helix dimer interface. In comparison, the axes of the two helices from the linker to the C-terminus (helices 2 and 2′) are wider apart from each other, creating a lateral access pathway around K47 from the aqueous phase to the center of the designed hydrophobic core. The site of the L38M mutation, which was previously shown to increase the halothane binding affinity by ∼3.5-fold, is not part of the hydrophobic core presumably involved in the anesthetic binding but shows an elevated transverse relaxation (R₂) rate. Qualitative analysis of the protein dynamics by reduced spectral density mapping revealed exchange contributions to the relaxation at many residues in the helices. This observation was confirmed by the quantitative analysis using the Modelfree approach and by the NMR relaxation dispersion measurements. The NMR structures and Autodock analysis suggest that the pocket with the most favorable amphipathic property for anesthetic binding is located between the W15 side chains at the center of the dimeric hydrophobic core, with the possibility of two additional minor binding sites between the F12 and F52 ring stacks of each monomer. The high-resolution structure of the designed anesthetic-binding protein offers unprecedented atomistic details about possible sites for anesthetic-protein interactions that are essential to the understanding of molecular mechanisms of general anesthesia.     

NMR study of general anesthetic interaction with nAChR beta2 subunit

The molecular basis of anesthetic interaction with membrane proteins has been explored via determination of anesthetic effects on the structure and dynamics of the extended second transmembrane domain (TM2e) of the human neuronal nicotinic acetylcholine receptor (nAChR) β₂ subunit in dodecylphosphocholine (DPC) micelles by ¹H and ¹⁵N solution-state NMR. Both 1-chloro-1,2,2-trifluorocyclobutane (F3) and isoflurane, two volatile general anesthetics, induced nonuniform changes in chemical shifts among residues in TM2e. Saturation transfer difference NMR experiments further confirmed the direct anesthetic interaction with TM2e. A significant and more specific anesthetic interaction was observed on three leucine residues at the helix C-terminus. Although the TM2e helical structure remained after addition of anesthetics, plausible shortening and lengthening of helix hydrogen bonds were evidenced by periodic changes in backbone amide chemical shifts. The TM2e backbone dynamics were determined on the basis of the ¹⁵N relaxation rate constants, R₁ and R₂, and the ¹⁵N-[¹H] NOE using the model-free approach. The global tumbling time (11.7 ns) of TM2e in micelles slightly increased (∼12.3-12.5 ns) in the presence of anesthetics. The order parameter, S², exceeded 0.9 for all ¹⁵N-labeled residues, showing a restricted internal motion. Anesthetics appear to have minor effect on the TM2e's internal motion. This study provided the basis for subsequent more comprehensive studies of anesthetic effects on the transmembrane domain complex of neuronal nAChR.