Fluorescence-determined preferential binding of quinacrine to DNA.

Quinacrine complexes with native DNA (Calf thymus, Micrococcus lysodeikticus, Escherichia coli, Bacillus subtilis, and Colstridium perfringens) and synthetic polynucleotides (poly(dA) . poly(dT), poly[d(A-T)] . poly[d(A-T)], poly(dG) . poly(dC) and poly[d(G-C)] . poly[d(G-C)]) has been investigated in solution at 0.1 M NaCl, 0.05 M Tris HCl, 0.001 M EDTA, pH 7.5, at 20 degrees C. Fluorescence excitation spectra of complexes with dye concentration D = 5-30 microM and DNA phosphate concentration P = 400 microM have been examined from 300 to 500 nm, while collecting the emission above 520 nm. The amounts of free and bound quinacrine in the dye-DNA complexes have been determined by means of equilibrium dialysis experiments. Different affinities have been found for the various DNAs and their values have been examined with a model that assumes that the binding constants associated with alternating purine and pyrimidine sequences are larger than those relative to nonalternating ones. Among the alternating nearest neighbor base sequences, the Pyr(3'-5')Pur sequences, i.e., C-G, T-G, C-A and T-A seem to bind quinacrine stronger than the remaining sequences. In particular the three sites, where a G . C base pair is involved, are found to display higher affinities. Good agreement is found with recent calculations on the energetics of intercalation sites in DNA. The analysis of the equilibrium shows also that the strength of the excitation spectrum of bound dye depends strongly upon the ratio of bound quinacrine to DNA. This effect can be attributed to dye-dye energy transfer along DNA.     

Growth, gas exchange and ion content in Olea europaea plants during salinity stress and subsequent relief

Olive ( Olea europaea L. cv. Frantoio) plants grown hydroponically in a glasshouse were supplied with half-strength Hoagland solutions containing 0, 50, 100, and 200 m M NaCl for 4 weeks and subsequently supplied with the standard solution without NaCl to relieve salinity stress. Two complete stress-relief cycles were repeated on the same plant material during one growing season. Growth was inhibited at all salt levels, but most growth parameters of plants treated with 50 or 100 m M NaCl returned to control levels after 4 weeks of relief. More severely stressed plants (200 m M NaCl) recovered to only 60% of the growth of the controls after 4 weeks. During relief, plants treated with 50 and 100 m M NaCl had net photosynthetic rates and stomatal conductances higher than the controls. Increasing the NaCl concentration of the external solution from 0 to 200 m M decreased both leaf pre-dawn water potential (from -0.3 to -1.0 MPa) and osmotic potential (from -2.1 to -2.7 MPa). The sodium concentration in the leaves of plants treated with 200 m M NaCl reached maximum levels of 211 and 388 m M (expressed on a tissue water basis) at the end of the first salinity and relief periods, respectively. Leaf chloride concentrations were 359 and 223 m M at the same sampling dates. These data indicate that the inhibitory effects of salinization on growth and gas exchange of the salt-tolerant olive cv. Frantoio can be readily reversed when salinity is relieved, despite the marked accumulation of potentially toxic ions (Na⁺. Cl) in the leaf.     

Ionic relations of aeroponically-grown olive genotypes,during salt stress

Two olive (Olea europaea L.) genotypes, Frantoio and Leccino, were exposed to increasing concentrations of NaCl (0-30-60-120 mM) in an aeroponic cultivation system for 60 days. Dry weights and sodium and potassium contents of apical and basal leaves, new and old wood, and roots were measured to determine Na uptake rate, Na translocation rate and K-Na selectivity ratio (S_K,Na). Frantoio showed a higher salt resistance than Leccino. Frantoio and Leccino had a similar Na uptake rate, but largely differed for Na translocation to the shoot. Furthermore Frantoio exhibited a higher K-Na selectivity than Leccino at both whole plant level and above all at the level of shoot system. Resistance mechanism of Frantoio is probably related to Na esclusion by roots and to the ability to maintain an appropriate K/Na ratio in actively growing tissues.Research supported by National Research Council of Italy, Special project RAISA.     

Effects of whole-body cryotherapy on serum mediators of inflammation and serum muscle enzymes in athletes

Whole-body cryotherapy (WBC) covers a wide range of therapeutic applications and consists of briefly exposing the body to extremely cold air. In sports medicine, WBC is used to improve recovery from muscle injury; however, empirical studies on its application to this area are lacking. To fill this gap, we compared changes in immunological parameters (C3, IgA, IgM, IgG, C-reactive protein, PGE2), cytokines (IL-2, IL-8, IL-10), adhesion molecules (sICAM-1), and muscle enzymes (creatine kinase [CK], lactate dehydrogenase [LAD]) before and after WBC in 10 top-level Italian National team rugby players. The subjects underwent five sessions on alternate days once daily for 1 week. During the study period, the training workload was the same as that of the previous weeks. Compared to baseline values, immunological parameters remained unchanged, while CK and LAD levels significantly decreased after treatment. No alterations in immunological function were observed but there is a decrease in pro-inflammatory cytokine/chemokine and an increase in anti-inflammatory cytokine.As measured by changes in serum CK and LAD concentrations, and cytokines pathway, short-term cold air exposure was found to improve recovery from exercise-induced muscle injury and/or damage associated with intense physical training.     

Effects of gradient and speed on freely chosen cadence: The key role of crank inertial load

The purpose of this study was to describe the relationship between road gradient (RG) and freely chosen cadence (FCC) in a group of professional cyclists during their normal training. In addition, a calculation of crank inertial load (CIL) was estimated in order to establish the relationship between FCC and CIL. Ten professional cyclists were monitored during training using commercially available power meters (Shoberer Rad Messtechnik (SRM), professional version). For each cyclist, recorded training sessions were reviewed to identify the hardest 6–8 training sessions (～18 h of training). RG was estimated based on the relationship between power output, total mass and speed. The analysis was performed using 2113±317 samples of 30 s average data, collected on terrain ranging from ?4%_RG to 12%_RG. The individual relationship between FCC and RG could be described by a linear regression model. There was a moderate correlation between FCC and CIL (group's r=0.42), and a multiple regression including the measured power output (W_PO) increased the variance explained (R²=0.24). The correlation was very large between CIL and v (r=0.91), and was not strengthened by adding W_PO as an independent variable (r=0.91). In conclusion, this investigation documents that in professional cyclists engaged in training, there is a linear decrease in FCC as RG increases (?4%_RG and 12%_RG). This decrease in FCC appears to be due to the reduction in v as slope increases. It is surmised that CIL plays a key role in the modulation of FCC.     

H(2)O(2) modulates purinergic-dependent calcium signalling in osteoblast-like cells

Reactive oxygen species (ROS) have long been considered as toxic by-products of aerobic metabolism and appear involved in the pathogenesis of degenerative diseases. The physiological role of ROS as second messengers in cell signal transduction is, on the other hand, increasingly recognized. Here we investigated the effects of H(2)O(2) and extracellular nucleotides on calcium signalling in four osteoblastic cell lines. In the highly differentiated HOBIT cells, sensitive to nanomolar concentrations of ADP and UTP, millimolar H(2)O(2) induced oscillatory increases of the cytosolic calcium concentration followed by a steady and sustained calcium increase. Long lasting rhythmic calcium activity was induced by micromolar H(2)O(2) doses. The H(2)O(2)-induced calcium signals, due to both release from intracellular stores and influx from the extracellular milieu, were totally prevented by incubating the cells with the P2 receptor antagonist suramin or with the ATP/ADP hydrolyzing enzyme apyrase. In the osteosarcoma SaOS-2 cells micromolar H(2)O(2) failed to evoke calcium signals and millimolar H(2)O(2) induced a slowly developing calcium influx which was unaffected by suramin and apyrase. These cells responded to micromolar concentrations of ATP and ADP, but were largely insensitive to UTP. ROS 17/2.8 osteosarcoma cells were totally insensitive to ATP, ADP and UTP in keeping with the evidence that these cells lack functional purinergic receptors. In these cells, H(2)O(2) up to 1mM did not increase the cytosolic calcium concentration. In ROS/P2Y(2) cells, stably expressing the P2Y(2) receptor, spontaneous calcium oscillations were observed in 38% of the population and nanomolar concentration of extracellular ATP or UTP activated oscillations in quiescent cells. Spontaneous calcium signals were inhibited by suramin and apyrase. In these cells H(2)O(2) induced oscillatory calcium activity that was blocked by suramin and apyrase. The sensitivity of ROS/P2Y(2) cells to UTP decreased significantly in the presence of DTT, which was effective also in inhibiting spontaneous calcium oscillations. On the other hand, the membrane-impermeant thiol oxidant DTNB induced calcium oscillations that were inhibited by incubating the cells with suramin or apyrase. Since peroxide did not increase extracellular ATP in these cell lines, we propose that, in osteoblasts, mild oxidative conditions could activate purinergic signalling through the sensitization of P2Y(2) receptor.     

In situ analysis of the bacterial community associated with the reindeer lichen Cladonia arbuscula reveals predominance of Alphaproteobacteria

The diversity and spatial pattern of the bacterial community hosted by the shrub-like reindeer lichen Cladonia arbuscula were investigated by general DNA staining and FISH, coupled with confocal laser scanning microscopy (CLSM). Using an optimized protocol for FISH using cryosections of small lichen fragments, we found about 6 x 10(7) bacteria g(-1) of C. arbuscula. Approximately 86% of acridine orange-stained cells were also stained by the universal FISH probe EUB338. Using group-specific FISH probes, we detected a dominance of Alphaproteobacteria (more than 60% of all bacteria), while the abundance of Actinobacteria and Betaproteobacteria was much lower (<10%). Firmicutes were rarely detected, and no Gammaproteobacteria were present. Bacterial cells of different taxonomic groups are embedded in a biofilm-like, continuous layer on the internal surface of the C. arbuscula podetia, mainly occurring in small colonies of a few to a few hundred cells. The other parts of the lichen showed a lower bacterial colonization. alpha-proteobacterial 16S rRNA genes were amplified using total DNA extracts from C. arbuscula and separated by single-strand conformation polymorphism (SSCP). Sequencing of excised bands revealed the dominance of Acetobacteraceae.     

Molecular dynamics simulation of the neuroglobin crystal: comparison with the simulation in solution

Neuroglobin (Ngb) is a monomeric protein that, despite the small sequence similarity with other globins, displays the typical globin fold. In the absence of exogenous ligands, the ferric and the ferrous forms of Ngb are both hexacoordinated to the distal and proximal histidines. In the ferrous form, oxygen, nitric oxide or carbon monoxide can displace the distal histidine, yielding a reversible adduct. Crystallographic data show that the binding of an exogenous ligand is associated to structural changes involving heme sliding and a topological reorganization of the internal cavities. Molecular dynamics (MD) simulations in solution show that the heme oscillates between two positions, much as the ones observed in the crystal structure, although the occupancy is different. The simulations also suggest that ligand binding in solution can affect the flexibility and conformation of residues connecting the C and D helices, referred to as the CD corner, which is coupled to the configuration adopted by the distal histidine. In this study, we report the results of 30 ns MD simulations of CO-bound Ngb in the crystal. Our goal was to compare the protein dynamical behavior in the crystal with the results supplied by the previous MD simulation of CO-bound Ngb in solution and the x-ray experimental data. The results show that the different environments (crystal or solution) affect the dynamics of the heme group and of the CD corner.     

Binding and interactions of L-BABP to lipid membranes studied by molecular dynamic simulations

Chicken liver bile acid-binding protein (L-BABP) is a member of the fatty acid-binding proteins super family. The common fold is a beta-barrel of ten strands capped with a short helix-loop-helix motif called portal region, which is involved in the uptake and release of non-polar ligands. Using multiple-run molecular dynamics simulations we studied the interactions of L-BABP with lipid membranes of anionic and zwitterionic phospholipids. The simulations were in agreement with our experimental observations regarding the electrostatic nature of the binding and the conformational changes of the protein in the membrane. We observed that L-BABP migrated from the initial position in the aqueous bulk phase to the interface of anionic lipid membranes and established contacts with the head groups of phospholipids through the side of the barrel that is opposite to the portal region. The conformational changes in the protein occurred simultaneously with the binding to the membrane. Remarkably, these conformational changes were observed in the portal region which is opposite to the zone where the protein binds directly to the lipids. The protein was oriented with its macrodipole aligned in the configuration of lowest energy within the electric field of the anionic membrane, which indicates the importance of the electrostatic interactions to determine the preferred orientation of the protein. We also identified this electric field as the driving force for the conformational change. For all the members of the fatty acid-binding protein family, the interactions with lipid membranes is a relevant process closely related to the uptake, release and transfer of the ligand. The observations presented here suggest that the ligand transfer might not necessarily occur through the domain that directly interacts with the lipid membrane. The interactions with the membrane electric field that determine orientation and conformational changes described here can also be relevant for other peripheral proteins.