Multifractal characterization of morphology of human red blood cells membrane skeleton.

The purpose of this paper is to show applicability of multifractal analysis in investigations of the morphological changes of ultra-structures of red blood cells (RBCs) membrane skeleton measured using atomic force microscopy (AFM). Human RBCs obtained from healthy and hypertensive donors as well as healthy erythrocytes irradiated with neutrons (45 µGy) were studied. The membrane skeleton of the cells was imaged using AFM in a contact mode. Morphological characterization of the three-dimensional RBC surfaces was realized by a multifractal method. The nanometre scale study of human RBCs surface morphology revealed a multifractal geometry. The generalized dimensions Dq and the singularity spectrum f(α) provided quantitative values that characterize the local scale properties of their membrane skeleton organization. Surface characterization was made using areal ISO 25178-2: 2012 topography parameters in combination with AFM topography measurement. The surface structure of human RBCs is complex with hierarchical substructures resulting from the organization of the erythrocyte membrane skeleton. The analysed AFM images confirm a multifractal nature of the surface that could be useful in histology to quantify human RBC architectural changes associated with different disease states. In case of very precise measurements when the red cell surface is not wrinkled even very fine differences can be uncovered as was shown for the erythrocytes treated with a very low dose of ionizing radiation.

The dynamics of the non-heme iron in bacterial reaction centers from Rhodobacter sphaeroides.

We investigate the dynamical properties of the non-heme iron (NHFe) in His-tagged photosynthetic bacterial reaction centers (RCs) isolated from Rhodobacter (Rb.) sphaeroides. Mössbauer spectroscopy and nuclear inelastic scattering of synchrotron radiation (NIS) were applied to monitor the arrangement and flexibility of the NHFe binding site. In His-tagged RCs, NHFe was stabilized only in a high spin ferrous state. Its hyperfine parameters (IS=1.06±0.01mm/s and QS=2.12±0.01mm/s), and Debye temperature (θ(D0)~167K) are comparable to those detected for the high spin state of NHFe in non-His-tagged RCs. For the first time, pure vibrational modes characteristic of NHFe in a high spin ferrous state are revealed. The vibrational density of states (DOS) shows some maxima between 22 and 33meV, 33 and 42meV, and 53 and 60meV and a very sharp one at 44.5meV. In addition, we observe a large contribution of vibrational modes at low energies. This iron atom is directly connected to the protein matrix via all its ligands, and it is therefore extremely sensitive to the collective motions of the RC protein core. A comparison of the DOS spectra of His-tagged and non-His-tagged RCs from Rb. sphaeroides shows that in the latter case the spectrum was overlapped by the vibrations of the heme iron of residual cytochrome c(2), and a low spin state of NHFe in addition to its high spin one. This enabled us to pin-point vibrations characteristic for the low spin state of NHFe.

The effect of ultrafine WO3 nanoparticles on the organization of thylakoids enriched in photosystem II and energy transfer in photosystem II complexes.

In this work, a new approach to construct self-assembled hybrid systems based on natural PSII-enriched thylakoid membranes (PSII BBY) is demonstrated. Superfine m-WO3 NPs (≈1-2 nm) are introduced into PSII BBY. Transmission electron microscopy (TEM) measurements showed that even the highest concentrations of NPs used did not degrade the PSII BBY membranes. Using atomic force microscopy (AFM), it is shown that the organization of PSII BBY depends strongly on the concentration of NPs applied. This proved that the superfine NPs can easily penetrate the thylakoid membrane and interact with its components. These changes are also related to the modified energy transfer between the external light-harvesting antennas and the PSII reaction center, shown by absorption and fluorescence experiments. The biohybrid system shows stability at pH 6.5, the native operating environment of PSII, so a high rate of O2 evolution is expected. In addition, the light-induced water-splitting process can be further stimulated by the direct interaction of superfine WO3 NPs with the donor and acceptor sides of PSII. The water-splitting activity and stability of this colloidal system are under investigation. RESEARCH HIGHLIGHTS: The phenomenon of the self-organization of a biohybrid system composed of thylakoid membranes enriched in photosystem II and superfine WO3 nanoparticles is studied using AFM and TEM. A strong dependence of the organization of PSII complexes within PSII BBY membranes on the concentration of NPs applied is observed. This observation turns out to be crucial to understand the complexity of the mechanism of the action of WO3 NPs on modifications of energy transfer from external antenna complexes to the PSII reaction center.

Coupling of collective motions of the protein matrix to vibrations of the non-heme iron in bacterial photosynthetic reaction centers.

Non-heme iron is a conservative component of type II photosynthetic reaction centers of unknown function. We found that in the reaction center from Rba. sphaeroides it exists in two forms, high and low spin ferrous states, whereas in Rsp. rubrum mostly in a low spin state, in line with our earlier finding of its low spin state in the algal photosystem II reaction center (Burda et al., 2003). The temperature dependence of the non-heme iron displacement studied by Mössbauer spectroscopy shows that the surrounding of the high spin iron is more flexible (Debye temperature ~165K) than that of the low spin atom (~207K). Nuclear inelastic scattering measurements of the collective motions in the Rba. sphaeroides reaction center show that the density of vibrational states, originating from non-heme iron, has well-separated modes between lower (4-17meV) and higher (17-25meV) energies while in the one from Rsp. rubrum its distribution is more uniform with only little contribution of low energy (~6meV) vibrations. It is the first experimental evidence that the fluctuations of the protein matrix in type II reaction center are correlated to the spin state of non-heme iron. We propose a simple mechanism in which the spin state of non-heme iron directly determines the strength of coupling between the two quinone acceptors (Q(A) and Q(B)) and fast collective motions of protein matrix that play a crucial role in activation and regulation of the electron and proton transfer between these two quinones. We suggest that hydrogen bond network on the acceptor side of reaction center is responsible for stabilization of non-heme iron in different spin states.

Evaluation of the knowledge of primary healthcare patients in Poland on the prevention of hypertension: a community study.

OBJECTIVES: To investigate the knowledge of Poles on the prevention of arterial hypertension (HT) and identify the main souces of knowledge in order to make health promotion activities more effective, and thus increase the efficiency and efficacy of the Polish healthcare system. STUDY DESIGN: Community study. METHODS: This questionnaire study included 180 subjects (120 primary healthcare patients without a history of diagnosed HT and 60 primary care physicians). RESULTS: The knowledge of most surveyed patients was insufficient (43%, n = 52) or sufficient (40%, n = 48) for the effective prevention of HT; 17% (n = 20) of the respondents had knowledge that was definitely sufficient, and none of the respondents had knowledge that was definitely insufficient. The patients reported that primary care physicians were the most common source of health information (67%, n = 80). Primary care physicians were also the most trusted source of information. CONCLUSIONS: Patients' knowledge on smoking, diet and exercise is sufficient for the effective prevention of HT. The areas of insufficient knowledge for the development of HT and possible organ complications are drinking alcohol, stress, genetic factors and diabetes.

Temperature-dependent bifurcation of cooperative interactions in pure and enriched in ß-carotene DPPC liposomes.

We examined the influence of temperature on lipid intermolecular interactions and the organization of bilayers within multilamellar dipalmitoylphosphatidylcholine (DPPC) liposomes. We also investigated the effect of 0.5 mol% ß-carotene, a non-polar carotenoid, on the adhesive properties of these liposomes. Atomic force microscopy (AFM) and differential scanning calorimetry (DSC) were used to correlate the changes in the physical properties of the liposomal systems with their thermotropic behaviour. Using DSC we detected two transitions in pure DPPC vesicles and in those containing 0.5 mol% ß-carotene. In both systems the pretransition occurred at 34.5(1)°C and the main phase transition at 41.4 °C during heating. Upon cooling, the temperatures of the pretransition and the main transition decreased by about 6 °C and 1 °C, respectively. Changes in enthalpy and entropy were also similar in the two investigated systems. Data obtained in parallel AFM force experiments show that the adhesive forces between the liposomal systems and AFM probe strongly depend on the loading rate. Moreover, their characteristic monotonic changes and discontinuities are sensitive to temperature. In the range of temperatures from 27 °C to 31 °C, i.e. below the temperature of phase transition from gel to ripple phase, the adhesive forces measured in a water environment are about an order of magnitude higher in the presence of ß-carotene than in pure DPPC liposomes. The observed variable dependence of adhesion on the loading rate suggests that there are changes in the long- and short-range interactions between lipids, and that these may be related to the occurrence of some clustering effects. In addition, the simultaneous existence of different subphases was found in the gel phase of DPPC liposomes. The presence of ß-carotene at a level of 0.5 mol% stimulates the structural reorganization of DPPC multilamellar vesicles and enhances the bifurcation phenomenon detected in these systems.

Heterogeneity of the mechanism of water splitting in photosystem II.

We measured the temperature dependence of oxygen evolution in thylakoids from tobacco using mass spectrometry and high resolution polarography. We determined the initial S-state distribution and the efficiency of the transition between these states including the probability of the O(2) yield through a fast mode. We observed discontinuous changes of the parameters at the temperatures 11 degrees C, 15 degrees C and 21 degrees C. Due to the mass spectroscopy data we think that the irregularity observed at 11 degrees C is due to conformational changes within the water catalytic site. We show that the different contributions of the slow and fast modes of oxygen evolution and of the water molecule exchange are correlated and that their behavior can be explained in terms of the H(2)O accessibility to the water splitting enzyme.

Mixed valence state in ironporphyrin aggregates.

In biological systems, metalloporphyrins play a central role in energy and electron transfer process. Our aim is to understand the influence of ligands and iron coordination of ironporphyrin on the electron transfer. The lyophilized ironporphyrin, enriched in 57Fe up to 90% has been studied by Mössbauer spectroscopy between 2.8 and 313 K. Above room temperature the bounded diffusion of the ferric iron was observed. Below 293 K a part of iron appears in mixed Fe+3<==>Fe+2 valence state with 10 meV activation energy for the electron trapping. Below 4 K a part of iron shows magnetic ordering with a broad distribution of the hyperfine field. The results are discussed in terms of metalloporphyrin aggregation process.

An estimation of the size of the water cluster present at the cleavage site of the water splitting enzyme.

In time-dependent measurements of oxygen evolution in tobacco thylakoid membranes we varied the fraction of H(2)(18)O and the temperature and measured water splitting as (18)O(2), (16)O(18)O, and (16)O(2) by mass spectrometry. We show that the approach to the equilibrium of the system after H(2)(18)O addition can be very well understood in terms of the diffusion of water molecules. The equilibrium states of (16)O(2), (16)O(18)O, and (18)O(2) evolution differ from the theoretical binomial distributions, which are expected under the prerequisite of ideal mixing of the water molecules and that of the chemical equivalence of H(2)(18)O and H(2)(16)O for an infinite cluster. The presence of this deviation means that there is a typical size of water clusters having access to cleavage by the water splitting enzyme. We estimated that this cluster contains about 12+/-2 water molecules.

Effects of prenatal stress on development in mice: maturation and learning.

Female CD-1 mice were stressed during the final week of gestation. Beginning 3 days after birth, until weaning, their pups were examined for eye opening, startle response, tooth eruption, surface righting, ability to cling to and climb an incline, tail pull reflex, rotation, linear movement and exploration. At 3 months of age, they were tested in a Morris Water Maze. Stressed animals were significantly lighter and shorter than non-stressed animals the first week after birth. By 3 days after birth, significantly fewer stressed animals could rotate or right themselves. By 6 days after birth, significantly fewer stressed animals could cling to or climb an inclined screen, or show the tail pull reflex. By 9 days of age, significantly fewer stressed animals had teeth. In contrast, by day 12 of age, significantly more stressed animals demonstrated exploratory behavior than did non-stressed animals. There were no sex differences in the ability of animals to perform these tasks at the same age. Stressed animals were significantly slower than non-stressed animals to reach the hidden platform in the water maze on all trials, and this difference was due to stressed females being slower to find the platform than non-stressed females, with no main effect of stress on males. This study supports and expands previous findings in rodents that prenatal stress can cause deficiencies in some early indices of physical maturation and also that these deficiencies can be continued into adulthood.

Simultaneous determination of salicylic acid and acetylsalicylic acid in aspirin delayed-release tablet formulations by second-derivative UV spectrophotometry.

A rapid, simple assay procedure was developed for simultaneous analysis of aspirin (ASA) and salicylic acid (SA) in aspirin delayed-release tablet formulation by 'zero crossing' second-derivative UV spectrophotometry. The zero-order absorption spectra and second derivative spectra of ASA and SA were recorded in diluting solution acetonitrile-formic acid (99:1). The accuracy of the method was demonstrated by the determination of ASA and SA in five tablets formulations (each 20 tablets of the same batch) by the described method and by high performance liquid chromatographic method, and the results were in good agreement.

Temperature dependence of the O2-oscillation pattern in the filamentous cyanobacterium Oscillatoria chalybea and in Chlorella kessleri.

Five characteristic discontinuities of the pattern of oxygen evolution have been detected for the filamentous cyanobacterium Oscillatoria chalybea in the temperature range of 0 degree C to 30 degrees C. The temperatures at which these discontinuities occur are: approximately 5 degrees C, approximately 11 degrees C, approximately 15 degrees C, approximately 21 degrees C and approximately 25 degrees C. The calculated initial 5-S state distribution, the miss parameter and the fraction of the fast transition S3-->So + O2 are affected. The discontinuities are observed at the same transition temperature also for Chlorella kessleri hence are not specific for the cyanobacterium. Based on these studies it is concluded that the not vanishing oxygen signal under the first flash of a flash train in Oscillatoria cannot have its origin in interactions between oxygen-evolving complexes. A decrease of temperature should slow down the expected charge exchanges, improve the oscillations thus reduce or lower the first two oxygen amplitudes of the oscillatoria pattern. Lowering of the temperature improves the oscillations but does not lower the first O2 signal of the pattern.

Effect of venlafaxine and nicotine on the level of neurotransmitters and their metabolites in rat brains.

Nicotine (NIC) and venlafaxine (VEN) have been proved to exert antidepressant activity in both human and animals. The effect of antidepressant doses of NIC and VEN (our previous results) on noradrenergic (NA), dopaminergic (DA), serotoninergic (5-HT) neurotransmitters and their metabolites: DOPAC, HVA and 5-HIAA in rats' hippocampus in freely moving rats were determined by microdialysis technique and HPLC method. Both drugs release 5-HT and NA, but VEN to a greater degree. DA level was affected only by VEN, however NIC extended the response of the DA system on VEN's effect. Combined administration of drugs caused the greatest changes in the 5-HT system. Both drugs contributed to reduction in neurotransmitter biotransformation.

A mathematical model describing kinetics of conversion of violaxanthin to zeaxanthin via intermediate antheraxanthin by the xanthophyll cycle enzyme violaxanthin de-epoxidase.

The xanthophyll cycle is one of the mechanisms protecting the photosynthetic apparatus against the light energy excess. Its action is still not well understood on the molecular level. Our model makes it possible to follow independently the kinetics of the two de-epoxidation steps occurring in the xanthophyll cycle: the conversion of violaxanthin into antheraxanthin and the conversion of antheraxanthin into zeaxanthin. Using a simple form of the transition rates of these two conversions, we model the time evolution of the concentration pattern of violaxanthin, antheraxanthin and zeaxanthin during the de-epoxidation process. The model has been applied to describe the reactions of de-epoxidation in a system of liposome membranes composed of phosphatidylcholine and monogalactosyldiacylglycerol. Results obtained within the model fit very well with the experimental data. Values of the transition probabilities of the violaxanthin conversion into antheraxanthin and the antheraxanthin conversion into zeaxanthin calculated by means of the model indicate that the first stage of the de-epoxidation process is much slower than the second one.

Europium- and Dysprosium-Ions as Probes for the Study of Calcium Binding Sites in Photosystem II.

Trivalent lanthanide cations are suitable probes for Ca2+-binding sites in photosystem II (PS II). PS II membranes prepared from Nicoticinci tabacum, intact and depleted of the extrinsic polypeptides were exposed to lanthanide ions (Dy3+ and Eu3+). Small concentrations of dysprosium and europium ions enhance oxygen evolution under short saturating flashes. Higher concentrations of the rare earth cations cause the release of the three extrinsic peptides (17, 23 and 33 kDa) and reduce O2 yield. The reactivation of the PS II membranes, thus depleted of the 33 kDa subunit, by Ca2+ ions is not possible. Comparing Eu3+ with Dy3+ in this effect shows that Eu3+ is more effective than Dy3+, because a lower Eu3+-concentration in comparison to that of Dy3+ inactivates O2-evolution. The differences between europium and dysprosium can be explained by their different ionic radius. Our results suggest the existence of two Ca-binding regions: one with a low affinity for calcium would be located on the contact surface of the 23 and 33 kDa proteins and the second one with a high affinity, should be located close to the Mn-cluster and to tyrosine-161 (Z ). The more tightly-bound calcium would be responsible for the activity of the PS II system.

Flagellar ultrastructure and flagella-associated antigens of Campylobacter fetus.

Ultrastructural examinations of the flagellum of Compylobacter (Vibrio) fetus were performed throughout the growth cycle. Filament diameters, exceeding 17.6 nm during the exponential phase, were substantially greater than those reported for unsheathed flagella of other genera with the exception of Pseudomonas fluorescens. Filament diameters increased during growth, reaching a mean width of 21.2 nm in middle to late stationary phase. Internal flagellar structure, principally of the parallel lined variety, was observed during the later periods of growth but not during exponential or early stationary phase. Despite the unusually large filament sizes, no evidence of a flagellar sheath was observed after selected treatments (0.01 N HCl, 6 M urea, tris(hydroxymethyl) amino-methane-hydrochloride buffer, warm water) or examination of thin sections. To determine whether alterations in filament size and variable ability to demonstrate filament fine structure were correlated with progressive changes in serological activity, agglutination and immobilization tests were conducted with antisera directed against intact flagella, the principal flagellar antigen, the O antigen, and a superficial glycoprotein which has been found in association with the flagellum and the cell envelope. Significant differences in the serological activity of cells at different growth intervals were not noted with any of the sera employed.

Microcapsule of Campylobacter fetus: chemical and physical characterization.

The antiphagocytic antigen (antigen [a]) comprising the microcapsule of a strain of Campylobacter fetus subsp. intestinalis has been purified from culture supernatants by ammonium sulfate fractionation and free-flow electrophoresis. Antigen [a] is a glycoprotein containing about 4% carbohydrate consisting of hexose, pentose, and methylpentose. The composition of the protein was typical of bacterial extramural structural proteins in its low content of basic, aromatic, and sulfur-containing amino acids. The protein had a high content of aspartic acid, threonine, glycine, and alanine. Antigen [a] had an Rf of 0.33 on polyacrylamide gel electrophoresis and a molecular weight calculated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of approximately 98,000. In contrast to its free form in culture supernatants, antigen [a] in vesicles derived from sheared cells appeared to exist in a complex with lipopolysaccharide. This complex could be dissociated by ethylenediaminetetraacetic acid or by ethylenediaminetetraacetic acid plus Triton X-100. A mutant strain that lacked a microcapsule, when incubated with soluble antigen [a] in a calcium medium, became agglutinable by monospecific [a] antiserum and showed an additional structural layer similar in appearance to the microcapsule on its cell wall. Points of similarity are emphasized between antigen [a] of C. fetus and the outer structural protein of the taxonomically related Spirillum serpens.

Superficial antigens of Campylobacter (Vibrio) fetus: characterization of antiphagocytic component.

A glycoprotein responsible for the antiphagocytic properties of Campylobacter fetus has been identified by comparing cells of a wild-type strain with those of a mutant lacking this substance. The antiphagocytic component is demonstrable through electron microscopy as a discrete, negatively charged structure on the periphery of the cell. CLT is readily removed from the cell by mild extraction procedures and contributes to the inagglutinability in O antiserum normally displayed by C. fetus. Cells possessing this antigen are refractory to ingestion by macrophages except in the presence of specific antiserum. In its absence maximum phagocytosis occurs without a requirement for opsonins. It is concluded that the antiphagocytic component comprises a critical virulence factor of the bacterium.