Resistance of human leukocytes to vesicular stomatitis virus infection as one of the innate antiviral immune activities; participation of cell subpopulations

Among reactions of innate immunity, resistance of human peripheral blood leukocytes (PBL) to viral infection seems important. The purpose of our study was to find, which of the subpopulations of PBL is the most responsible for the innate antiviral immunity of these cells. The innate immunity was measured by using the direct method of infection of leukocytes with vesicular stomatitis virus (VSV). The lack of VSV replication by infected leukocytes (0-1 log TCID50) was taken as an indicator for complete immunity; a low level of VSV (2-3 log) for partial immunity; and high VSV titer (more than 4 log) for no immunity. The resistance/innate immunity of whole PBL and subpopulations such as: adherent cells, fractions enriched in lymphocytes T, and lymphocytes B (separated on column with nylon wool), NK(+) and NK(-) (separated by microbeads activated cell sorting MACS) differ from each other. All fractions express higher resistance/innate immunity than the whole PBL. NK(+) cells were found the most resistant fraction of PBL to VSV infection. The results indicate that among the leukocytes in PBL the regulation mechanisms of innate immunity exist. The study on the mechanism of innate immunity regulation as well as the role of NK in innate immunity of PBL must be continued.     

Influence of long-term drought stress on osmolyte accumulation in sugar beet (Beta vulgaris L.) plants

Accumulation of various osmolytes was examined in plants of sugar beet cv. Janus grown under two soil water treatments: control (60% of the field water capacity; FWC) and drought (30–35% FWC). The water shortage started on the 61st day after emergence (DAE), at the stage of the beginning of tap-roots development and was imposed for 35 days. Osmotic potential of sugar beet plant organs, particularly tap-roots, was decreased significantly as a consequence of a long-term drought. Water shortage reduced univalent (K⁺, Na⁺) cations concentrations in the petioles and divalent (Ca²⁺, Mg²⁺) ions level in the mature and old leaves. Cation concentrations in the tap-roots were not affected by water shortage. The ratio of univalent to divalent cations was significantly increased in young leaves and petioles as a consequence of drought. Long-term water deficit caused a significant reduction of inorganic phosphorus (P_i) concentration in young and old leaves. Under the water stress condition, the concentration of proline was increased in all individual plant organs, except proline concentration in the youngest leaves. Drought treatment caused a significant increase of glycine betaine content in shoot without any change in tap-roots. Glucose concentrations were significantly increased only in tap-roots as the effect of drought. In response to water shortage the accumulation of sucrose was observed in all the examined leaves and tap-roots. Overall, a long-term drought activated an effective mechanism for osmotic adjustment both in the shoot and in the root tissues which may be critical to survival rather than to maintain plant growth but sugar beet organs accumulate different solutes as a response to water cessation.     

Copper(II) complexes of Neobelliera Bullata Trypsin Modulating Oostatic Factor and its analogues

The stoichiometry, stability constants and solution structure of the complexes formed in the reaction of copper(II) with hexapeptide NPTNLH, i.e. the Neobelliera Bullata Trypsin Modulating Oostatic Factor (Neb-TMOF), and its analogues DPTNLH, Ac-NPTNLH and Ac-DPTNLH have been determined by potentiometric, UV-visible, CD and EPR spectroscopic methods. Upon raising pH for Ac-NPTNLH and Ac-DPTNLH peptides, copper(II) coordination starts from the imidazole nitrogen of the His⁶; afterwards three deprotonated amide nitrogens are progressively involved in metal ions coordination. In a wide pH range of 4.5-8.5 for the NPTNLH and DPTNLH ligands the CuL complex dominates with the imidazole nitrogen of His⁶ coordinated to form a macrochelate. The N-terminal amino group of the NPTNLH and DPTNLH peptides takes part in the coordination of the metal ion in the CuL, CuH₋₁L and CuH₋₂L complexes. However, at pH above 9 the CuH₋₃L complex with the {N_Im, 3N⁻} coordination mode is formed. For the CuH₋₂L complex the spectroscopic data clearly indicate the 4N {NH₂, CO or COO⁻, 2N⁻, N_Im} bonding mode with the axial coordination of the N-terminal amine group to the metal ion.     

Quantitative assessment of upper limb muscle fatigue depending on the conditions of repetitive task load

The aim of this study was to discriminate fatigue of upper limb muscles depending on the external load, through the development and analysis of a muscle fatigue index. Muscle fatigue is expressed by a fatigue index based on an amplitude parameter (calculated in the time domain) and a fatigue index based on a frequency parameter (a parameter calculated in the frequency domain). The fatigue index involves a regression function that describes changes in the EMG signal parameter, time elapsing before muscle fatigue and the probability of specific trends in changes in EMG parameters for the population under study.

The experimental study covered a group of 10 young men. During the study, they exerted force at a specific level and for a specific time in 12 load variants. During the study, EMG signals from four muscles of the upper limb were recorded (trapezius pars descendents, biceps brachii caput breve, extensor carpi radialis brevis, flexor carpi ulnaris). For each variant and for each examined muscles, the value of the fatigue index was calculated. Values of that index quantitatively expressed fatigue of a specific muscle in a specific load variant.

A statistical analysis indicated variation in the fatigue of the biceps brachii caput breve, extensor carpi radialis brevis, and flexor carpi ulnaris muscles depending on the external load (load variant) according to the task performed with the upper limb.

The study demonstrated usefulness of the fatigue index in expressing quantitatively muscle fatigue and in discriminating muscle fatigue depending on the external load.     

Kinetics of ultrasonic degradation and polymerisation degree distribution of sonochemically degraded chitosans

The process of physical degradation by means of the ultrasonic action towards chitosans with mole fraction of 2-acetamido-2-deoxy-β- -glucopyranose units (the degree of N-acetylation, F_A) in the range of 0.10≤F_A≤0.28, and the weight average polymerisation degree in the range of has been investigated. The decrease of as well as changes in the distribution of the degree of polymerisation (P) has been determined as a function of time, F_A, temperature, concentration of chitosan solution and concentration of acetic acid in the solution. The use of low-power ultrasound emitter allowed to establish that in the case of chitosan (binary heteropolysaccharide) the general rate parameter (k) increased with F_A. This can be explained by the relatively stronger aggregation of macromolecules with higher F_A, which results in size increase of macromolecular individuals and hence in their higher susceptibility to ultrasonic action. It was also observed that k decreased with chitosan concentration and temperature. The value of limiting degree of polimerisation (x_e) was found to be influenced by structural parameters of chitosan chains (F_A, aggregation). The increase of acetic acid concentration caused the increase in the k value, what indicated accelerating effect of ultrasound towards acidic hydrolysis of chitosan. The shape of the P curve of sonochemically degraded chitosans are in good correlation with the mid-point breakage concept of degradation accepted in sonochemical degradation of polymers.     

Basic energetic parameters of Acanthamoeba castellanii mitochondria and their resistance to oxidative stress

The purpose of this study was establishing the basic energetic parameters of amoeba Acanthamoeba castellanii mitochondria respiring with malate and their response to oxidative stress caused by hydrogen peroxide in the presence of Fe(2+) ions. It appeared that, contrary to a previous report (Trocha LK, Stobienia O (2007) Acta Biochim Polon 54: 797), H(2)O(2)-treated mitochondria of A. castellanii did not display any substantial impairment. No marked changes in cytochrome pathway activity were found, as in the presence of an inhibitor of alternative oxidase no effects were observed on the rates of uncoupled and phosphorylating respiration and on coupling parameters. Only in the absence of the alternative oxidase inhibitor, non-phosphorylating respiration progressively decreased with increasing concentration of H(2)O(2), while the coupling parameters (respiratory control ratio and ADP/O ratio) slightly improved, which may indicate some inactivation of the alternative oxidase. Moreover, our results show no change in membrane potential, Ca(2+) uptake and accumulation ability, mitochondrial outer membrane integrity and cytochrome c release for 0.5-25 mM H(2)O(2)-treated versus control (H(2)O(2)-untreated) mitochondria. These results indicate that short (5 min) incubation of A. castellanii mitochondria with H(2)O(2) in the presence of Fe(2+) does not damage their basic energetics.     

Concentrations of Metals in Maxilla and Mandible Deciduous and Permanent Human Teeth

The study shows the results of research on the concentrations of metals (Cd, Pb, Mn, Cu, Cr, Fe, Zn, Na, K, Mg, and Ca) in deciduous and permanent teeth with respect to their location in the oral cavity (maxilla, mandible). It has been found that the concentrations of metals were significantly higher in the deciduous teeth than in permanent ones. Regression analysis and principal component analysis revealed an increased dynamics of the processes of binding the elements by the hydroxyapatite of deciduous teeth. Higher concentrations of the metals were found in the maxilla permanent and deciduous teeth than in the mandible ones.     

Overexpression inEscherichia coli,Folding, Purification, and Characterization of the First Three Short Consensus Repeat Modules of Human Complement Receptor Type 1

We have developed a simple expression, isolation, and folding protocol for an SCR oligomer comprising the first three SCRs of complement receptor Type 1 (C3b/C4b receptor, CD35). A T7 RNA polymerase expression system inEscherichia coliwas used to express the oligomer as inclusion bodies. The oligomer was recovered from solubilized inclusion bodies using batch adsorption on SP–Sepharose. The oligomer was folded by one-step dilution in 20 m ethanolamine/1 m EDTA supplemented with 1 m GSH/0.5 m GSSG. The folded material was processed to a concentrated (>20 mg/ml), usable product of greater than 98% purity using a combination of ultrafiltration, ammonium sulfate treatment, hydrophobic interaction, and size-exclusion chromatography. The yield of folded material varied between 6 and 15 mg/liter culture. The oxidation states of the 12 cysteine residues in SCR(1–3) were identified by HPLC of peptide fragments from a tryptic digest using dual UV/fluorescence detection, collection of selected peaks, and N-terminal sequencing. This methodology confirmed the expected location of disulfide bridges. Equilibrium and velocity sedimentation studies are interpreted in terms of a single sedimenting species with molecular weights of 21,629 and 21,063 by these respective techniques. These values compare to the predicted molecular weight, from amino acid composition, of 21,817. The hydrodynamic properties of the molecule indicate that it is asymmetric with an axial ratio of 1:5.2 or equivalent dimensions of 21 × 110 Å. SCR(1–3) has an unusual CD spectrum exhibiting a broad maximum at 220–230 nm and a minimum at 190 nm. There was little evidence of classical secondary structure. The product exhibited concentration-dependent inhibition of complement-mediated lysis of sensitized sheep red blood cells.