Affinity and Specificity of Ciprofloxacin-Bovine Serum Albumin Interactions: Spectroscopic Approach

Fluorescence spectroscopy in combination with UV–Vis absorption spectroscopy were employed to investigate the binding of an antibacterial drug Ciprofloxacin (CPFX) to bovine serum albumin (BSA) under the physiological conditions. In the discussion of the quenching mechanism, it was proved that the fluorescence quenching of BSA by CPFX is a result of the formation of CPFX-BSA complex. Binding parameters were determined using the modified Stern-Volmer equation and Scatchard equation to provide a measure of the binding affinity between CPFX and BSA. The results of thermodynamic parameters ΔG, ΔH, ΔS, at different temperatures indicate that the electrostatic interactions play a major role for CPFX-BSA association. Site marker competitive experiments indicated that the binding of CPFX to BSA primarily took place in site I. Furthermore, the effect of metal ions to CPFX-BSA system was studied, and the distance r between donor (BSA) and acceptor (CPFX) was obtained according to fluorescence resonance energy transfer (FRET). The conformation of BSA upon CPFX binding was evaluated by measuring synchronous fluorescence properties of the CPFX-BSA complex.     

Determination of the specific interaction between palmatine and bovine serum albumin

The binding of palmatine to bovine serum albumin (BSA) was studied under physiological conditions (pH = 7.40) by molecular spectroscopic approach. It was proved that the fluorescence quenching of BSA by palmatine is a result of the formation of palmatine–BSA complex. Binding parameters were determined using the modified Stern–Volmer equation and Scatchard equation, to measure the specific binding between palmatine and BSA. The thermodynamic parameters calculated, ∆G°, ∆H° and ∆S° indicate that the electrostatic interactions play a major role in the palmatine–BSA association. Site marker competitive displacement experiments demonstrated that palmatine binds with specific affinity to site II (subdomain IIIA) of BSA. Furthermore, the specific binding distance r (3.36 nm) was obtained according to fluorescence resonance energy transfer. The results of synchronous fluorescence spectra and UV–Visible absorption spectra show that the conformation of bovine serum albumin has been changed.     

Type I blue copper proteins as enantioselective quenchers of the photoluminescence of Δ,Λ-Eu(pyridine-2,6-dicarboxylate)3 3–: azurin from Pseudomonas aeruginosa and its Met44→Lys mutant, amicyanin from Paracoccus versutus and parsley plastocyanin

 The dynamic quenching of the luminescence of racemic Eu(III)(pyridine-2,6-dicarboxylate=dpa)₃ ^3– by the title proteins is investigated and the enantioselectivity of the proteins in the quenching of the Δ and Λ enantiomers of Eu(dpa)₃ ^3– is determined. The two diastereomeric quenching rate constants pertaining to azurin (k _q ^Δ=3.3×10⁶, k _q ^Λ=2.7×10⁶ M^–1s^–1, pH 7.2, ionic strength I=22 mM) are lower than for its Met→44Lys mutant (k _q ^Δ=1.9×10⁷, k _q ^Λ=1.4×10⁷ M^–1 s^–1, same pH and I), indicating that energy transfer occurs from Eu(dpa)₃ ^3– to the Cu(II) centre when the luminophore is bound to the hydrophobic patch of the protein near residue 44. The enantioselectivity remains unaltered by the mutation: k _q ^Δ/k _q ^Λ=1.27±0.04, so Lys44 is probably not in direct contact with the Eu chelate. The I and pH dependence of k _q indicate that the lysine residue interacts electrostatically with Eu(dpa)₃ ^3–. For plastocyanin the quenching rates are of the order of 10⁶ M^–1 s^–1; for amicyanin they are two orders of magnitude larger (k _q ^Δ=12×10⁷, k _q ^Λ=11×10⁷ M^–1 s^–1, pH 7.2, I=22 mM). The variation of k _q is attributed to differences in the charge distribution on the proteins, which influences the binding of the luminophore to the protein surface. For amicyanin the anion binding site near Lys59 and Lys60 may be involved in the energy transfer. Received: 16 June 1998 / Accepted: 18 September 1998     

Studies on the interaction between benzophenone and bovine serum albumin by spectroscopic methods

The interaction between benzophenone (BP) and bovine serum albumin (BSA) was investigated by the methods of fluorescence spectroscopy combined with UV–Vis absorption and circular dichroism (CD) measurements under simulative physiological conditions. The experiment results showed that the fluorescence quenching of BSA by BP was resulted from the formation of a BP–BSA complex and the corresponding association constants (K _a) between BP and BSA at four different temperatures had been determined using the modified Stern–Volmer equation. The enthalpy change (ΔH) and entropy change (ΔS) were calculated to be –43.73 kJ mol⁻¹ and −53.05 J mol⁻¹ K⁻¹, respectively, which suggested that hydrogen bond and van der Waals force played major roles in stabilizing the BP–BSA complex. Site marker competitive experiments indicated that the binding of BP to BSA primarily took place in site I (sub-domain IIA). The conformational investigation showed that the presence of BP decreased the α-helical content of BSA and induced the slight unfolding of the polypeptides of protein, which confirmed some micro-environmental and conformational changes of BSA molecules.     

Haemoglobin components and oxygen transport in relation to habitat distribution in triplefin fishes (Tripterygiidae)

Haemoglobin components were analysed for nine species of New Zealand triplefins and their isoelectric points (pI) ranged from 5.1 to 7.0. The number of well-expressed isohaemoglobins was larger in shallow-water and tidal pool species, ranging from four in Grahamina signata to eight in Grahamina capito, and were relatively cathodal. Two strongly anodal isohaemoglobins were expressed in the mid-depth species Ruanoho decemdigitatus and Ruanoho whero, and one in the deeper water species Karalepis stewarti and Forsterygion malcolmi. The red blood cell oxygen-binding properties were determined at 15 °C and 25 °C in the pH range 6.7–7.9 for the shallow-water species G. capito, the shallow to mid-depth species Forsterygion varium, and the deep-water species F. malcolmi. Oxygen affinity was highest for G. capito and the magnitude of the Bohr effect lower (Δlog P ₅₀/ΔpH = −0.37 at 25 °C, where P ₅₀ is the half-saturation coefficient) compared to the two Forsterygion species (Δlog P ₅₀/ΔpH = −0.52 to −0.59). Further, the cooperativity factor, n ₅₀, was lower in G. capito thus maintaining oxygen transport over a wide range of environmental oxygen pressures. Oxygen binding was similarly influenced by temperature in both G. capito and F. malcolmi (maximum heat of oxygenation ΔH_max = −27 kJ mol⁻¹ and −37 kJ mol⁻¹, respectively). Thus, triplefin fishes living in shallow, thermally unstable habitats possess a greater number of cathodally migrating isohaemoglobins, and their red blood cells have a higher oxygen affinity and reduced cooperativity which is less sensitive to changes in pH than do species occurring in more stable, deeper water habitats. Our analysis of an assemblage of closely related species circumvents some of the difficulties inherent in studies where interpretation of experimental results is confounded by phylogeny. Accepted: 18 March 1999     

Electrochemistry of the CuA domain of Thermus thermophilus cytochrome ba 3

 The electrochemistry of a water-soluble fragment from the Cu_A domain of Thermus thermophilus cytochrome ba ₃ has been investigated. At 25  °C, Cu_A exhibits a reversible reduction at a pyridine-4-aldehydesemicarbazone-modified gold electrode (0.1 M Tris, pH 8) with E° = 0.24 V vs NHE. Thermodynamic parameters for the [Cu(Cys)₂Cu]^+/0 electrode reaction were determined by variable-temperature electrochemistry (ΔS°_rc = –5.4(12) eu, ΔS° = –21.0(12) eu, ΔH° = –11.9(4) kcal/mol;ΔG° = –5.6 (11) kcal/mol). The relatively small reaction entropy is consistent with a low reorganization energy for [Cu(Cys)₂Cu]^+/0 electron transfer. An irreversible oxidation of [Cu(Cys)₂Cu]⁺ at 1 V vs NHE confirms that the Cu^II:Cu^II state of Cu_A is significantly destabilized relative to the Cu^II state of analogous blue-copper proteins. Received: 3 June 1996 / Accepted: 26 August 1996     

Modulation of Bacillus pasteurii cytochrome c 553 reduction potential by structural and solution parameters

 Direct cyclic voltammetry and ¹H NMR spectroscopy have been combined to investigate the electrochemical and spectroscopic properties of cytochrome c ₅₅₃ isolated from the alkaliphilic soil bacterium Bacillus pasteurii. A quasi-reversible diffusion-controlled redox process is exhibited by cytochrome c ₅₅₃ at a pyrolitic graphite edge microelectrode. The temperature dependence of the reduction potential, measured using a non-isothermal electrochemical cell, revealed a discontinuity at 308 K. The thermodynamic parameters determined in the low-temperature range (275–308 K;ΔS°′=–162.7±1.2 J mol^–1 K^–1, ΔH°′=–53.0±0.5 kJ mol^–1, ΔG°′=–4.5±0.1 kJ mol^–1, E°′=+47.0±0.6 mV) indicate the presence of large enthalpic and entropic effects, leading, respectively, to stabilization and destabilization of the reduced form of cytochrome c ₅₅₃. Both effects are more accentuated in the high-temperature range (308–323 K;ΔS°′=–294.1±8.4 J mol^–1 K^–1, ΔH°′=–93.4±3.1 kJ mol^–1, ΔG°′=–5.8±0.6 kJ mol^–1, E°′=+60.3±5.8 mV), with the net result being a slight increase of the standard reduction potential. These thermodynamic parameters are interpreted using the compensation theory of hydration of biopolymers as indicating the extrusion, upon reduction, of water molecules from the hydration sphere of the cytochrome. The low-T and high-T conformers differ by the number of water molecules in the solvation sphere: in the high-T conformer, the number of water molecules extruded upon reduction increases, as compared to the low-T conformer. The ionic strength dependence of the reduction potential at 298 K, treated within the frame of extended Debye-Hückel theory, yields values of E ^°′ _(I=0) =–25.4±1.4 mV, z _red=–11.3, and z _ox=–10.3. The pH dependence of the reduction potential at 298 K shows a plateau in the pH range 7–10 and an increase at more acidic pH, allowing the calculation of pK _O=5.5 and pK _R=5.7, together with the estimate of the reduction potentials of completely protonated (+71 mV) and deprotonated (+58 mV) forms of cytochrome c ₅₅₃. ¹H NMR spectra of the oxidized paramagnetic cytochrome c ₅₅₃ indicate the presence of a His-Met axial coordination of the low-spin (S=1/2) heme iron, which is maintained in the temperature interval 288–340 K at pH 7 and in the pH range 4.8–10.0 at 298 K. The temperature dependence of the hyperfine-shifted signals shows both Curie-type and anti-Curie-type behavior, with marked deviations from linearity, interpreted as indicating the presence of a fast equilibrium between the low-T and high-T conformers, having slightly different heme electronic structures resulting from the T-induced conformational change. Increasing the NaCl concentration in the range 0–0.2 M causes a slight change of the ¹H NMR chemical shifts of the hyperfine-shifted signals, with no influence on their linewidth. The calculated lower limit value of the apparent affinity constant for specific ion binding is estimated as 5.2±1.1 M^–1. The pH dependence of the isotropically shifted ¹H NMR signals of the oxidized cytochrome displays at least one ionization step with pK _O=5.7. The thermodynamic and spectroscopic data indicate a large solvent-derived entropic effect as the main cause for the observed low reduction potential of B. pasteurii cytochrome c ₅₅₃. Received: 9 January 1998 / Accepted: 8 April 1998     

Karyology and cytotaxonomy of the genus Passiflora L. (Passifloraceae)

 The chromosomes of 31 species of Passiflora, distributed throughout the subgenera Astrophea, Calopathanthus, Distephana, Dysosmia, Passiflora, Plectostemma and Tacsonia were analysed. Three different karyotypes were observed: 2n = 12, 24, 36; 2n = 18, 72 and 2n = 20. The karyotype of these species was almost always constituted of metacentric and submetacentric chromosomes with variable karyotype symmetry. In the group with x = 6, represented by the subgenus Plectostemma, six diploid species with 2n = 12, one tetraploid with 2n = 24 (P. suberosa) and an intraspecific polyploid with 2n = 12, 36 (P. misera) were analysed. P. pentagona (subgenus Astrophea) may also be included in this karyological group since it presents 2n = 24 and may be of polyploid origin, with x = 6. The interphase nuclei in this group were areticulate, except those of P. morifolia and P. pentagona with semi-reticulate characteristics. Two small terminal heterochromatic blocks, positive for chromomycin A₃, were identified in the largest chromosome pair of P. capsularis and P. rubra, species very closely related, while P. tricuspis displayed four chromosomes with proximal blocks. In the group with x = 9, represented mainly by subgenus Passiflora, 20 species with 2n = 18 and one with 2n = 72 were studied. They presented chromosomes larger than those species with x = 6 and interphase nuclei of semi-reticulate type, except for P. mixta with areticulate nuclei. Four terminal CMA⁺ blocks were observed in P. edulis, six blocks in P. caerulea and P. racemosa, while five blocks were observed in the single P. amethystina plant analysed. P. foetida (subgenus Dysosmia), the only species with 2n = 20, exhibited six chromosomes with CMA⁺ blocks and interphase nuclei of the areticulate type. The meiotic analysis of representatives of the three groups (P. foetida, P. suberosa, P. cincinnata and P. racemosa) always presented regular pairing and regular chromosome segregation, except in P. jilekii where a tetravalent was observed. The analysis of the chromosome variation within the genus and the family suggests that the base number of Passiflora may be x₁ = 6 or x₁ = 12, whereas x₂ = 9 is only an important secondary base number. Received April 11, 2000 Accepted October 5, 2000     

Interaction of human serum albumin with 10-hydroxycamptothecin: spectroscopic and molecular modeling studies

In this work, fluorescence spectroscopy in combination with circular dichroism spectroscopy and molecular modeling was employed to investigate the binding of 10-hydroxycamptothecin (HCPT) to human serum albumin (HSA) under simulative physiological conditions. The experiment results showed that the fluorescence quenching of HSA by HCPT was a result of the formation of HCPT–HSA complex. The corresponding association constants (K _a) between HCPT and HSA at four different temperatures were determined according to the modified Stern–Volmer equation. The results of thermodynamic parameters ΔG, ΔH, and ΔS indicated that hydrogen bonds and van der Waals forces played major roles for HCPT–HSA association. Site marker competitive displacement experiment indicated that the binding of HCPT to HSA primarily took place in sub-domain IIA (site I). Molecular docking study further confirmed the binding mode and the binding site obtained by fluorescence and site marker competitive experiments. The conformational investigation showed that the presence of HCPT decreased the α-helical content of HSA and induced the slight unfolding of the polypeptides of protein, which confirmed some micro-environmental and conformational changes of HSA molecules.     

Respiratory and metabolic responses of the Australian Yabby Cherax destructor to progressive and sustained environmental hypoxia

The Australian Yabby, Cherax destructor, inhabits occasionally hypoxic water. The respiratory gas, acid-base, metabolite and energetic status of this crayfish was assessed during progressive hypoxia and during 3 h at a water PO₂ of 1.33 kPa. The O₂ affinity of haemocyanin from C. destructor was increased by lactate (Δlog P ₅₀/Δlog[lactate] = −0.111) and by Ca (Δlog P ₅₀/Δlog[Ca] = −0.62) but not by urate. While the non-bicarbonate buffering capacity was low (Δ[HCO₃ ⁻]/ ΔpH=−4.89) the haemocyanin had a low sensitivity to pH changes (ϕ = −0.33). The crayfish showed a compensatory hyperventilation, which induced a respiratory alkalosis, until the water O₂ partial pressure declined below 2.67 kPa, after which the O₂ uptake rate was approximately 10% of normoxic rates. The high haemocyanin-O₂ affinity maintained haemolymph O₂ content during progressive hypoxia despite the normally low arterial O₂ partial pressure of C. destructor. During severe hypoxia, pH decreased but increased lactate aided in maintaining haemocyanin-O₂ saturation. The importance of regulated haemocyanin-O₂ affinity in hypoxic C. destructor was reduced by lowered metabolism, including reduced cardiac output, and the consequent reduction in O₂ requirement. Anaerobiosis became important only at very low PO₂ but thereafter proceeded rapidly, supported by a marked hyperglycaemia. There was no depletion of adenylates, even after 3 h of severe hypoxia. The tail muscle of C. destructor held small amounts of glycogen which would sustain anaerobiosis for a only a few hours. Hypometabolism seems an important hypoxic response but severe hypoxia may encourage the crayfish to breathe air. Accepted: 26 February 1998     

Energy dissipation in photosynthesis: Does the quenching of chlorophyll fluorescence originate from antenna complexes of photosystem II or from the reaction center?

Dissipation of light energy was studied in the moss Rhytidiadelphus squarrosus (Hedw.) Warnst., and in leaves of Spinacia oleracea L. and Arabidopsis thaliana (L.) Heynh., using chlorophyll fluorescence as an indicator reaction. Maximum chlorophyll fluorescence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-treated spinach leaves, as produced by saturating light and studied between +5 and −20 °C, revealed an activation energy ΔE of 0.11 eV. As this suggested recombination fluorescence produced by charge recombination between the oxidized primary donor of photosystem II and reduced pheophytin, a mathematical model explaining fluorescence, and based in part on known characteristics of primary electron-transport reactions, was developed. The model permitted analysis of different modes of fluorescence quenching, two localized in the reaction center of photosystem II and one in the light-harvesting system of the antenna complexes. It predicted differences in the relationship between quenching of variable fluorescence F _v and quenching of basal, so-called F ₀ fluorescence depending on whether quenching originated from antenna complexes or from reaction centers. Such differences were found experimentally, suggesting antenna quenching as the predominant mechanism of dissipation of light energy in the moss Rhytidiadelphus, whereas reaction-center quenching appeared to be important in spinach and Arabidopsis. Both reaction-center and antenna quenching required activation by thylakoid protonation but only antenna quenching depended on or was strongly enhanced by zeaxanthin. De-protonation permitted relaxation of this quenching with half-times below 1 min. More slowly reversible quenching, tentatively identified as so-called q _I or photoinhibitory quenching, required protonation but persisted for prolonged times after de-protonation. It appeared to originate in reaction centers. Received: 8 April 2000 / Accepted: 31 August 2000     

Properties of New,Long-Wavelength,Voltage-sensitive Dyes in the Heart

Membrane potential measurements using voltage-sensitive dyes (VSDs) have made important contributions to our understanding of electrophysiological properties of multi-cellular systems. Here, we report the development of long wavelength VSDs designed to record cardiac action potentials (APs) from deeper layers in the heart. The emission spectrum of styryl VSDs was red-shifted by incorporating a thienyl group in the polymethine bridge to lengthen and retain the rigidity of the chromophore. Seven dyes, Pittsburgh I to IV and VI to VIII (PGH I-VIII) were synthesized and characterized with respect to their spectral properties in organic solvents and heart muscles. PGH VSDs exhibited 2 absorption, 2 excitation and 2 voltage-sensitive emission peaks, with large Stokes shifts (> 100 nm). Hearts (rabbit, guinea pig and Rana pipiens) and neurohypophyses (CD-1 mice) were effectively stained by injecting a bolus (10–50 μl) of stock solution of VSD (2–5 mM) dissolved in in dimethylsulfoxide plus low molecular weight Pluronic (16% of L64). Other preparations were better stained with a bolus of VSD (2–5 mM) Tyrode’s solution at pH 6.0. Action spectra measured with a fast CCD camera showed that PGH I exhibited an increase in fractional fluorescence, ΔF/F = 17.5 % per AP at 720 nm with 550 nm excitation and ΔF/F = − 6% per AP at 830 nm with 670 nm excitation. In frog hearts, PGH1 was stable with ∼30% decrease in fluorescence and AP amplitude during 3 h of intermittent excitation or 1 h of continuous high intensity excitation (300 W Xe-Hg Arc lamp), which was attributed to a combination of dye wash out > photobleaching > dynamic damage > run down of the preparation. The long wavelengths, large Stokes shifts, high ΔF/F and low baseline fluorescence make PGH dyes a valuable tool in optical mapping and for simultaneous mapping of APs and intracellular Ca²⁺.     

Molecular dynamics simulations of nafion and sulfonated poly ether sulfone membranes II. Dynamic properties of water and hydronium

We measured the self-diffusion coefficients of water in a Nafion membrane and two sulfonated polyethersulfone (SPES) membranes with varying ion-exchange capacities (IEC) in terms of relative humidity using the pulse field gradient NMR (PFG-NMR) technique. The self-diffusion coefficients were plotted against the number of water molecules per sulfonic acid group, λ, and compare these values with the results of molecular dynamics (MD) simulations. Classical MD simulations for all membranes were carried out using a consistent force field at λ = 3, 6, 9, 12, and 15. The dynamic properties of water (H₂O) and hydronium (H₃O⁺) on a molecular level were estimated as self-diffusion coefficients and residence times around a sulfonate group ( \textSO₃^- {\text{SO}}_3^{-} ). The diffusion coefficients of H₂O and H₃O⁺ followed the order, Nafion > SPES with IEC = 1.4 > SPES with IEC = 1.0 > SPES with IEC = 0.75, which agreed with the experimental data. The residence time distribution of H₂O around \textSO₃^- {\text{SO}}_3^{-} in Nafion was in the range of 1–6 ps, whereas H₂O in the SPES exhibited a residence time of greater than 20 ps.     

The interaction of lysozyme with caffeine,theophylline and theobromine in solution

The interactions of lysozyme with caffeine (Caf), theophylline (Tph) and theobromine (Tbr) were investigated using UV–Vis absorption, fluorescence, synchronous fluorescence, and three-dimensional fluorescence spectra techniques. The results revealed that Caf (Tph or Tbr) caused the fluorescence quenching of lysozyme by the formation of Caf (Tph or Tbr)–lysozyme complex. The binding constants (K _A) and thermodynamic parameters (ΔG°, ΔH°, ΔS°) at two different temperatures, the binding locality, and the binding power were obtained. The results showed that the process of binding Caf (Tph or Tbr) to lysozyme was a spontaneous molecular interaction procedure and the hydrophobic and electrostatic interactions play a major role in stabilizing the complex; The distance r between donor (lysozyme) and acceptor (Caf, Tph or Tbr) was obtained according to fluorescence resonance energy transfer. The effect of Caf (Tph or Tbr) on the conformation of lysozyme was analyzed using synchronous fluorescence and three-dimensional fluorescence spectra techniques. The results showed that the binding of Caf (Tph or Tbr) to lysozyme induced some micro-environmental and conformational changes in lysozyme and disturbed the environment of the polypeptide of lysozyme.     

Spectroscopic and Molecular Modeling Studies on the Interaction Between a Fluorine-Containing Triazole Derivative and Human Serum Albumin

The interaction between 4-(4-fluorobenzylideneamino)-5-propyl-4H-1,2,4-triazole-3-thiol (FBTZ) and human serum albumin (HSA) under simulative physiological conditions was investigated by fluorescence, UV–vis absorption and circular dichroism (CD) spectroscopy as well as molecular modeling method. Fluorescence spectroscopic data showed that the fluorescence quenching of HSA was a result of the formation of FBTZ–HSA complex. According to the modified Stern–Volmer equation, the effective quenching constants (K _a) of FBTZ to HSA were obtained at three different temperatures. The enthalpy change (ΔH) and entropy change (ΔS) were calculated on the basis of van′t Hoff equation, and the results showed that hydrogen-bonding and van der Waals forces were the dominant intermolecular forces to stabilize the complex. Site marker competitive replacement experiments demonstrated that the binding of FBTZ to HSA primarily took place in sub-domain IIA (Sudlow’s site I). The binding distance (r) between FBTZ and the tryptophan residue of HSA was estimated according to the theory of fluorescence resonance energy transfer. The conformational investigation showed that the presence of FBTZ induced some changes of secondary structure of HSA. Molecular modeling study further confirmed the binding mode obtained by experimental study.     

Study of the interaction between 8-azaguanine and bovine serum albumin using optical spectroscopy and molecular modeling methods

The interaction between 8-azaguanine (8-Azan) and bovine serum albumin (BSA) in Tris-HCl buffer solutions at pH 7.4 was investigated by means of fluorescence and ultraviolet-visible (UV-Vis) spectroscopy. At 298 K and 310 K, at a wavelength of excitation (λ _ex) of 282 nm, the fluorescence intensity decreased significantly with increasing concentrations of 8-Azan. Fluorescence static quenching was observed for BSA, which was attributed to the formation of a complex between 8-Azan and BSA during the binding reaction. This was illuminated further by the UV-Vis absorption spectra and the decomposition of the fluorescence spectra. The thermodynamic parameters ∆G, ∆H, ∆S were calculated. The results showed that the forces acting between 8-Azan and BSA were typical hydrophobic forces, and that the interaction process was spontaneous. The interaction distance r between 8-Azan and BSA, evaluated according to fluorescence resonance energy transfer theory, suggested that there is a high possibility of energy transfer from BSA to 8-Azan. Theoretical investigations based on homology modeling and molecular docking suggested that binding between 8-Azan and BSA is dominated by hydrophilic forces and hydrogen bonding. The theoretical investigations provided a good structural basis to explain the phenomenon of fluorescence quenching between 8-Azan and BSA.     

Spatial and Temporal Genetic Variation of Green Mussel, Perna viridis in the Gulf of Thailand and Implication for Aquaculture

The culture of green mussel (Perna viridis) in the Gulf of Thailand depends on natural spat which are believed to come from spawning grounds adjacent to major river mouths. In the present paper, genetic diversity of spatial and temporal populations of green mussel in the Gulf of Thailand was investigated using five microsatellite loci. The results showed moderate genetic variation of all 11 populations (averaged number of alleles per locus, A = 10.4–12.2; effective number of alleles per locus, A _e = 5.36–6.59; mean allelic richness, A _r = 10.23–12.06; observed heterozygosity, H _o = 0.52–0.63, and expected heterozygosity, H _e = 0.66–0.73) without significant differences among populations. No sign of bottleneck or genetic disequilibrium was observed. Genetic differentiation among spatial populations was low (F _ST = 0.0046, CI_0.95 = 0.0020–0.0083 for the samples collected in January, 2007, and F _ST = 0.0088, CI_0.95 = 0.0010–0.0162 for the samples collected in July, 2007) while temporal variation was significant as revealed by the analysis of molecular variance. Multidimensional scaling separated temporal population groups with minor exception. The assignment test revealed that most of the recruits were from other populations.     

The relationship between preferred and optimal positioning during submaximal cycle ergometry

This study was designed to determine how changes in oxygen uptake (O₂) and heart rate (HR) during submaximal cycle ergometry were determined by changes in cycle geometry and/or lower-limb kinematics. Fourteen trained cyclists [Mean (SD): age, 25.5 (6.4) years; body mass 74.4 (8.8) kg; peak O₂, 4.76 (0.79) l. min⁻¹ peak] were tested at three seat-tube angles (70°, 80°, 90°) at each of three trunk angles (10°, 20°, 30°) using a modified Monark cycle ergometer. All conditions were tested at a power output corresponding to 95% of the O₂ at each subject's ventilatory threshold while pedalling at 90 rpm and using aerodynamic handlebars. Sagittal-view kinematics for the hip, knee, and ankle joints were also recorded for all conditions and for the subjects' preferred positioning on their own bicycles. No combination of seat-tube and trunk angle could be considered optimal since many of the nine conditions elicited statistically similar mean O₂ and HR values. Mean hip angle (HA) was the only kinematic variable that changed consistently across conditions. A regression relationship was not observed between mean O₂ or HR and mean hip angle values (P > 0.45). Significant curvilinear relationships were observed, however, between ΔO₂ (O₂ − minimum O₂) and ΔHA (mean HA − preferred HA) using the data from all subjects (R = 0.45, SEE = 0.13 l . min⁻¹) and using group mean values (R = 0.93, SEE = 0.03 l . min⁻¹). In both cases ΔO₂ minimized at ΔHA = 0, which corresponded to the subjects' preferred HA from their own bicycles. Thus, subjects optimized their O₂ cost at cycle geometries that elicited similar lower-limb kinematics as the preferred geometries from their own bicycles. Accepted: 3 July 1996     

Recent improvements in the development of A2B adenosine receptor agonists

Adenosine is known to exert most of its physiological functions by acting as local modulator at four receptor subtypes named A₁, A_2A, A_2B and A₃ (ARs). Principally as a result of the difficulty in identifying potent and selective agonists, the A_2B AR is the least extensively characterised of the adenosine receptors family. Despite these limitations, growing understanding of the physiological meaning of this target indicates promising therapeutic perspectives for specific ligands. As A_2B AR signalling seems to be associated with pre/postconditioning cardioprotective and anti-inflammatory mechanisms, selective agonists may represent a new therapeutic group for patients suffering from coronary artery disease. Herein we present an overview of the recent advancements in identifying potent and selective A_2B AR agonists reported in scientific and patent literature. These compounds can be classified into adenosine-like and nonadenosine ligands. Nucleoside-based agonists are the result of modifying adenosine by substitution at the N ⁶-, C²-positions of the purine heterocycle and/or at the 5′-position of the ribose moiety or combinations of these substitutions. Compounds 1-deoxy-1-{6-[N′-(furan-2-carbonyl)-hydrazino]-9H-purin-9-yl}-N-ethyl-β-D-ribofuranuronamide (19, hA₁ K _i = 1050 nM, hA_2A K _i = 1550 nM, hA_2B EC₅₀ = 82 nM, hA₃ K _i > 5 μM) and its 2-chloro analogue 23 (hA₁ K _i = 3500 nM, hA_2A K _i = 4950 nM, hA_2B EC₅₀ = 210 nM, hA₃ K _i > 5 μM) were confirmed to be potent and selective full agonists in a cyclic adenosine monophosphate (cAMP) functional assay in Chinese hamster ovary (CHO) cells expressing hA_2B AR. Nonribose ligands are represented by conveniently substituted dicarbonitrilepyridines, among which 2-[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]pyridin-2-ylsulfanyl]acetamide (BAY-60–6583, hA₁, hA_2A, hA₃ EC₅₀ > 10 μM; hA_2B EC₅₀ = 3 nM) is currently under preclinical-phase investigation for treating coronary artery disorders and atherosclerosis. This article has previously been published in issue 4/4, under doi:.     

Recent improvements in the development of A<Subscript>2B</Subscript> adenosine receptor agonists

Adenosine is known to exert most of its physiological functions by acting as local modulator at four receptor subtypes named A₁, A_2A, A_2B and A₃ (ARs). Principally as a result of the difficulty in identifying potent and selective agonists, the A_2B AR is the least extensively characterised of the adenosine receptors family. Despite these limitations, growing understanding of the physiological meaning of this target indicates promising therapeutic perspectives for specific ligands. As A_2B AR signalling seems to be associated with pre/postconditioning cardioprotective and anti-inflammatory mechanisms, selective agonists may represent a new therapeutic group for patients suffering from coronary artery disease. Herein we present an overview of the recent advancements in identifying potent and selective A_2B AR agonists reported in scientific and patent literature. These compounds can be classified into adenosine-like and nonadenosine ligands. Nucleoside-based agonists are the result of modifying adenosine by substitution at the N ⁶-, C²-positions of the purine heterocycle and/or at the 5′-position of the ribose moiety or combinations of these substitutions. Compounds 1-deoxy-1-{6-[N′-(furan-2-carbonyl)-hydrazino]-9H-purin-9-yl}-N-ethyl-β-D-ribofuranuronamide (19, hA₁ K _i = 1050 nM, hA_2A K _i = 1550 nM, hA_2B EC₅₀ = 82 nM, hA₃ K _i > 5 μM) and its 2-chloro analogue 23 (hA₁ K _i = 3500 nM, hA_2A K _i = 4950 nM, hA_2B EC₅₀ = 210 nM, hA₃ K _i > 5 μM) were confirmed to be potent and selective full agonists in a cyclic adenosine monophosphate (cAMP) functional assay in Chinese hamster ovary (CHO) cells expressing hA_2B AR. Nonribose ligands are represented by conveniently substituted dicarbonitrilepyridines, among which 2-[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]pyridin-2-ylsulfanyl]acetamide (BAY-60–6583, hA₁, hA_2A, hA₃ EC₅₀ > 10 μM; hA_2B EC₅₀ = 3 nM) is currently under preclinical-phase investigation for treating coronary artery disorders and atherosclerosis.