Development of the salivary glands in embryos of Ixodes ricinus (Acari: Ixodidae)

We examined Ixodes ricinus embryos between 18 and 28 days of development with light, scanning and transmission electron microscopy. The differences in inner structure attested to establish three successive developmental stages: days 18-20, day 23, and days 26-28. Between 18 and 20 days the embryos are at early stages of organogenesis. Salivary glands cannot be identified at that stage. In 23-day-old embryos salivary glands are already outlined but the structure of alveoles is still different from that in larvae in which the embryonic development has been completed. Gland cells start to form alveoles and become active between 26 and 28 days of the development.     

Analysis of genetic relationships and antimicrobial susceptibility of Escherichia coli isolated from Clethrionomys glareolus

Eleven strains of Escherichia coli were isolated from 54 bank voles living in the LomZa Landscape Park of the Narew River Valley, indicating that E. coli is not common in the alimentary tract of these mammals. On the basis of pulsed-field gel electrophoresis and computer-assisted analysis, the isolates were grouped into six genotypes at similarities of 39%. Chromosome length of E. coli under study differed by as much as 900 kb, ranging 2.7-3.6 Mb. All strains were susceptible to amikacin and ciprofloxacin, whereas, for tetracycline, streptomycin, ampicillin, and cefonicid, different results were noted. No differences were detected among the plasmid complements of eight strains (73%), for which plasmid profiles revealed the presence of two plasmidic bands. One, three and four plasmids were observed in a plasmid pattern of single isolates. The observation from the study indicated the high genetic polymorphism among the isolates recovered from the animals of one species living in the same environment.     

Inhibition of serine proteinases from human blood clotting system by squash inhibitor mutants

A series of six CMTI I variants mutated in the P(2)-P(4)' region of the canonical binding loop were used to probe the role of single amino acid substitutions on binding to the following human proteinases involved in blood clotting: plasmin, plasma kallikrein, factors X(a) and XII(a). The mutants were expressed as fusion proteins with the LE1413 hydrophobic polypeptide in Escherichia coli, purified from inclusion bodies, followed by cyanobromide cleavage and refolding. The mutants inhibited the proteinases with the association constants in the range 10(3)-10(9) M(-1). Inhibition of plasma kallikrein and factors X(a) and XII(a) could be improved up to 30-fold by single mutations. In contrast, neither of the introduced mutations increased inhibitory properties of CMTI I against plasmin. Additionally, using two inhibitors of natural origin, CMTI I (P(1) Arg) and CPTI II (P(1) Lys), we determined the effect of Lys-->Arg on binding to four proteinases. With the exception of plasmin (no effect), P(1) Arg resulted in up to 30-fold stronger binding than P(1) Lys.     

Structural changes in the salivary glands of Ixodes ricinus larvae

The authors analysed the structure of Ixodes ricinus (L.) larvae in specimens immediately after leaving the egg sheaths, in those which have not fed for 2 months after hatching, and in feeding larvae on the second day of feeding. The results showed that salivary glands in tick larvae are formed by alveoli aligned in strands on both sides of the central nervous system. These alveoli open into central efferent ducts via short ducts. The constituent elements of salivary glands include pyramidal alveoli (with numerous lipid droplets) and granular alveoli of varied structure. It is worth noting that salivary alveoli containing secretory material are present even in the larvae which had just left egg sheaths and were still endowed with deutoplasm.     

Applications of the ETS-NOCV method in descriptions of chemical reactions

The present study characterizes changes in the electronic structure of reactants during chemical reactions based on the combined charge and energy decomposition scheme, ETS-NOCV (extended transition state–natural orbitals for chemical valence). Decomposition of the activation barrier, ΔE ^#, into stabilizing (orbital interaction, ΔE _orb, and electrostatic, ΔE _elstat) and destabilizing (Pauli repulsion, ΔE _Pauli, and geometry distortion energy, ΔE _dist) factors is discussed in detail for the following reactions: (I) hydrogen cyanide to hydrogen isocyanide, HCN → CNH isomerization; (II) Diels-Alder cycloaddition of ethene to 1,3-butadiene; and two catalytic processes, i.e., (III) insertion of ethylene into the metal-alkyl bond using half-titanocene with phenyl-phenoxy ligand catalyst; and (IV) B–H bond activation catalyzed by an Ir-containing catalyst. Various reference states for fragments were applied in ETS-NOCV analysis. We found that NOCV-based deformation densities (Δρ _i) and the corresponding energies ΔE _orb(i) obtained from the ETS-NOCV scheme provide a very useful picture, both qualitatively and quantitatively, of electronic density reorganization along the considered reaction pathways. Decomposition of the barrier ΔE^# into stabilizing and destabilizing contributions allowed us to conclude that the main factor responsible for the existence of positive values of ΔE ^# for all processes (I, II, III and IV) is Pauli interaction, which is the origin of steric repulsion. In addition, in the case of reactions II, III and IV, a significant degree of structural deformation of the reactants, as measured by the geometry distortion energy, plays an important role. Depending on the reaction type, stabilization of the transition state (relatively to the reactants) originating either from the orbital interaction term or from electrostatic attraction can be of vital importance. Finally, use of the ETS-NOCV method to describe catalytic reactions allows extraction of information on the role of catalysts in determination of ΔE ^#.     

Measured and estimated ground reaction forces for multi-segment foot models

Accurate measurement of ground reaction forces under discrete areas of the foot is important in the development of more advanced foot models, which can improve our understanding of foot and ankle function. To overcome current equipment limitations, a few investigators have proposed combining a pressure mat with a single force platform and using a proportionality assumption to estimate subarea shear forces and free moments. In this study, two adjacent force platforms were used to evaluate the accuracy of the proportionality assumption on a three segment foot model during normal gait. Seventeen right feet were tested using a targeted walking approach, isolating two separate joints: transverse tarsal and metatarsophalangeal. Root mean square (RMS) errors in shear forces up to 6% body weight (BW) were found using the proportionality assumption, with the highest errors (peak absolute errors up to 12% BW) occurring between the forefoot and toes in terminal stance. The hallux exerted a small braking force in opposition to the propulsive force of the forefoot, which was unaccounted for by the proportionality assumption. While the assumption may be suitable for specific applications (e.g. gait analysis models), it is important to understand that some information on foot function can be lost. The results help highlight possible limitations of the assumption. Measured ensemble average subarea shear forces during normal gait are also presented for the first time.     

Estimating the carbonyl anharmonic vibrational frequency from affordable harmonic frequency calculations

A linear correlation between harmonic and anharmonic frequencies of water calculated at B3LYP level of theory was observed with a number of basis sets. Similar relationships were found in both the gas phase and solution for several small molecules. The best correlation was found for C = O stretch mode in formaldehyde, formamide and N-methylacetamide. The average difference between B3LYP harmonic and anharmonic ν(C = O) frequencies calculated with several basis sets in these molecules was 30 cm(-1). The ad hoc correction of -30 cm(-1), added to harmonic frequencies of two different carbonyl groups present in a structure of a larger molecule was tested as a fast way of predicting anharmonic frequencies without elaborated calculations. The proposed approach was tested successfully on a larger molecule of E and Z isomers of N-acetyl-α,β-dehydrophenylalanine N',N'-dimethylamide [Ac-(E/Z)-ΔPhe-NMe(2)] and the estimated anharmonic ν(C = O) frequencies were close to directly calculated results.     

Structure-function relationship of serine protease-protein inhibitor interaction.

We report our progress in understanding the structure-function relationship of the interaction between protein inhibitors and several serine proteases. Recently, we have determined high resolution solution structures of two inhibitors Apis mellifera chymotrypsin inhibitor-1 (AMCI-I) and Linum usitatissimum trypsin inhibitor (LUTI) in the free state and an ultra high resolution X-ray structure of BPTI. All three inhibitors, despite totally different scaffolds, contain a solvent exposed loop of similar conformation which is highly complementary to the enzyme active site. Isothermal calo- rimetry data show that the interaction between wild type BPTI and chymotrypsin is entropy driven and that the enthalpy component opposes complex formation. Our research is focused on extensive mutagenesis of the four positions from the protease binding loop of BPTI: P1, P1', P3, and P4. We mutated these residues to different amino acids and the variants were characterized by determination of the association constants, stability parameters and crystal structures of protease-inhibitor complexes. Accommodation of the P1 residue in the S1 pocket of four proteases: chymotrypsin, trypsin, neutrophil elastase and cathepsin G was probed with 18 P1 variants. High resolution X-ray structures of ten complexes between bovine trypsin and P1 variants of BPTI have been determined and compared with the cognate P1 Lys side chain. Mutations of the wild type Ala16 (P1') to larger side chains always caused a drop of the association constant. According to the crystal structure of the Leu16 BPTI-trypsin complex, introduction of the larger residue at the P1' position leads to steric conflicts in the vicinity of the mutation. Finally, mutations at the P4 site allowed an improvement of the association with several serine proteases involved in blood clotting. Conversely, introduction of Ser, Val, and Phe in place of Gly12 (P4) had invariably a destabilizing effect on the complex with these proteases.