Behavior of S1- and S2-ovalbumin and S-ovalbumin A1 in urea solution: kinetics and equilibria

The isolation of S-, S₁-, and S₂-ovalbumin from domestic hen egg R-ovalbumin and of two methods for S-ovalbumin A₁ are described. The first is by heat treatment of R-ovalbumin A₁ and the second is of R-ovalbumin followed by fractionation on Sepharose. A kinetics and equilibrium study is made of their behavior in the presence of urea and compared with that of R-ovalbumins. As anticipated, the S-ovalbumins are much more resistant to urea than R-ovalbumins. Unlike the latter, S-ovalbumins' equilibrium profiles have a simpler sigmoidal shape. The unfolding of S₁- and S₂-ovalbumin is an order of magnitude slower than that of R-ovalbumin. Some possible structural differences between R- and S-ovalbumin forms and their significance are discussed.Deceased December 8, 2001     

Synthesis, biological activity and molecular modelling studies of tricyclic alkylimidazo-, pyrimido- and diazepinopurinediones

Syntheses and biological activities of imidazo-, pyrimido- and diazepino[2,1-f]purinediones containing N-alkyl substituents (with straight, branched or unsaturated chains) are described. Tricyclic derivatives were synthesized by the cyclization of 8-bromo-substituted 7-(2-bromoethyl)-, 7-(3-chloropropyl)- or 7-(4-bromobutyl)-theophylline with primary amines under various conditions. Compound 22 with an ethenyl substituent was synthesized by dehydrohalogenation of 9-(2-bromoethyl)-1,3-dimethyltetrahydropyrimido[2,1-f]purinedione. The obtained derivatives (5–35) were initially evaluated for their affinity at rat A₁ and A_2A adenosine receptors (AR), showing moderate affinity for both adenosine receptor subtypes. The best ligands were diazepinopurinedione 28 (K_i = 0.28 μM) with fivefold A_2A selectivity and the non-selective A₁/A_2A AR ligand pyrimidopurinedione 35 (K_i A₁ = 0.28 μM and K_i A_2A = 0.30 μM). The compounds were also evaluated for their affinity at human A₁, A_2A, A_2B and A₃ ARs. All of the obtained compounds were docked to the A_2A AR X-ray structure in complex with the xanthine-based, potent adenosine receptor antagonist—XAC. The likely interactions of imidazo-, pyrimido- and diazepino[2,1-f]purinediones with the residues forming the A_2A binding pocket were discussed. Furthermore, the new compounds were tested in vivo as anticonvulsants in maximal electroshock, subcutaneous pentylenetetrazole (ScMet) and TOX tests in mice (i.p.). Pyrimidopurinediones showed anticonvulsant activity mainly in the ScMet test. The best derivative was compound 11, showing 100 % protection at a dose of 100 mg/kg without symptoms of neurotoxicity. Compounds 6, 7, 8 and 14 with short substituents showed neurotoxicity and caused death. In rat tests (p.o.), 9 was characterized by a high protection index (>13.3). AR affinity did not apparently correlate with the antiepileptic potency of the compounds.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-013-9358-3) contains supplementary material, which is available to authorized users.     

Antibacterial properties of recombinant human non-pancreatic secretory phospholipase A2

Human non-pancreatic secretory phospholipase A₂ (hnpsPLA₂) is a group IIA phospholipase A₂ which plays an important role in the innate immune response. This enzyme was found to exhibit bactericidal activity toward Gram-positive bacteria, but not Gram-negative ones. Though native hnpsPLA₂ is active over a broad pH range, it is only highly active at alkaline conditions with the optimum activity pH of about 8.5. In order to make it highly active at neutral pH, we have obtained two hnpsPLA₂ mutants, Glu89Lys and Arg100Glu that work better at neutral pH in a previous study. In the present study, we tested the bactericidal effects of the native hnpsPLA₂ and the two mutants. Both native hnpsPLA₂ and the two mutants exhibit bactericidal activity toward Gram-positive bacteria. Furthermore, they can also kill Escherichia coli, a Gram-negative bacterium. The two mutants showed better bactericidal activity for E. coli at neutral pH than the native enzyme, which is consistent with the enzyme activities. As hnpsPLA₂ is highly stable and biocompatible, it may provide a promising therapy for bacteria infection treatment or other bactericidal applications.     

The role of activated adenosine receptors in degranulation of human LAD2 mast cells

Mast cell degranulation triggers hypersensitivity reactions at the body–environment interface. Adenosine modulates degranulation, but enhancement and inhibition have both been reported. Which of four adenosine receptors (ARs) mediate modulation, and how, remains uncertain. Also uncertain is whether adenosine reaches mast cell ARs by autocrine ATP release and ecto-enzymatic conversion. Uncertainties partly reflect species and cell heterogeneity, circumvented here by focusing on homogeneous human LAD2 cells. Quantitative PCR detected expression of A_2A, A_2B, and A₃, but not A₁, ARs. Nonselective activation of ARs with increasing NECA monotonically enhanced immunologically or C3a-stimulated degranulation. NECA alone stimulated degranulation slightly. Selective AR antagonists did not affect C3a-stimulated degranulation. NECA''s enhancement of C3a-triggered degranulation was partially inhibited by separate application of each selective antagonist, and abolished by simultaneous addition of antagonists to the three ARs. Only the A_2A antagonist separately inhibited NECA''s enhancement of immunologically stimulated degranulation, which was abolished by simultaneous addition of the three selective antagonists. Immunological or C3a activation did not stimulate ATP release. NECA also enhanced immunologically triggered degranulation of mouse bone marrow derived mast cells (BMMCs), which was partially reduced only by simultaneous addition of the three antagonists or by the nonselective antagonist {"type":"entrez-protein","attrs":{"text":"CGS15943","term_id":"875345334"}}CGS15943. BMMCs also expressed A_2A, A_2B, and A₃ ARs. but not A₁AR detectably. We conclude that (a) A₁AR is unnecessary for LAD2 degranulation or AR enhancement; (b) A_2A, A_2B, and A₃ ARs all contribute to pharmacologic AR enhancement of LAD2 and BMMC degranulation; and (c) LAD2 cells depend on microenvironmental adenosine to trigger AR modulation.     

Identification of gibberellin A4 in Pisum sativum L. and the effects of applied gibberellins A9, A4, A5 and A3 on the le mutant

Gibberellin A₄ (GA₄) was identified for the first time in the garden pea (Pisum sativum) L.), by gas chromatography-mass spectrometry. However, in wild-type shoots the level of GA₄ was only about 6% of the level of GA₁, and it is therefore unlikely that GA₄ plays a major role per se in the control of pea stem elongation. In shoots of the le mutant, GA₄ was not detected, while the level of GA₉ was approximately twice that found in the wild-type. The le mutation also markedly reduced the elongation response to applied GA₉. It appears, therefore, that in Pisum the le mutation blocks the 3-hydroxylation of GA₉ to GA₄, in addition to the 3-hydroxylation of GA₂₀ to GA₁. In contrast, the le mutation did not reduce the response to applied GA₅, suggesting the step GA₅ to GA₃ is not catalysed by the enzyme controlled by the Le gene. The step GA₅ to GA₃ was confirmed in peas by metabolite analysis after treatment with deuterated GA₅.     

Design, synthesis, and evaluation of fused heterocyclic analogs of SCH 58261 as adenosine A2A receptor antagonists

SCH 58261 is a reported adenosine A_2A receptor antagonist which is active in rat in vivo models of Parkinson’s Disease upon ip administration. However, it has poor selectivity versus the A₁ receptor and does not demonstrate oral activity. Quinoline analogs have improved upon the selectivity and pharmacokinetics of SCH 58261, but were difficult to handle due to poor aqueous solubility. We report the design and synthesis of fused heterocyclic analogs of SCH 58261 with aqueous solubility as well as improved A_2A receptor binding selectivity and pharmacokinetic properties. In particular, the tetrahydronaphthyridine 4s has excellent A_2A receptor in vitro binding affinity and selectivity, is active orally in a rat in vivo model of Parkinson’s Disease, and has aqueous solubility of 100 μM at physiological pH.     

Spectral and kinetic characterization of electron acceptor A1 in a Photosystem I core devoid of iron-sulfur centers FX,FB and FA

The kinetic and spectroscopic properties of the secondary electron acceptor A₁ were determined by flash absorption spectroscopy at room and cryogenic temperatures in a Photosystem I (PS I) core devoid of the iron-sulfur clusters F_X, F_B and F_A. It was shown earlier (Warren, P.V., Golbeck, J.H. and Warden, J.T. (1993) Biochemistry 32: 849–857) that the majority of the flash-induced absorbance increase at 820 nm, reflecting formation of P700⁺, decays with a t_1/2 of 10 s due to charge recombination between P700⁺ and A₁ ^–. Following A₁ ^– directly around 380 nm, where absorbance changes due to the formation of P700⁺ are negligible, two major decay components were resolved in this study with t_1/2 of 10 s and 110 s at an amplitude ratio of 2.5:1. The difference spectra between 340 and 490 nm of the two kinetic phases are highly similar, showing absorbance increases from 340 to 400 nm characteristic of the one-electron reduction of the phylloquinone A₁. When measured at 10 K, the flash-induced absorbance changes around 380 nm can be fitted with two decay phases of t_1/2 15 s and 150 s at an amplitude ratio 1:1. The difference spectra of both kinetic phases from 340 to 400 nm are similar to those determined at 298 K and are therefore attributed to charge recombination in the pair P700⁺A₁ ^–. These results indicate that the backreaction between P700⁺ and A₁ ^– is multiphasic when F_X, F_B and F_A are removed, and only slightly temperature dependent in the range of 298 K to 10 K.Abbreviations Chl chlorophyll - D pathlength for the measuring light through the sample - DPIP 2,6-dichlorophenolindophenol - EPR electron paramagnetic resonance - IR infrared - PS I Photosystem I - Tris Tris(hydroxymethyl)aminomethane - UV ultraviolet Published as Journal Series #10890 of the University of Nebraska Agricultural Research Division and supported by a grant from the National Science Foundation (MCB-9205756).     

Tryptophan modification of phospholipase A2 enzymes and presynaptic neurotoxins from snake venoms

Three phospholipases A₂ purified from cobra venoms and two presynaptically acting neurotoxins that exhibit phospholipase A₂ activity were subjected to tryptophan modification with 2-hydroxy-5-nitrobenzyl bromide. Associated with the modification of an increasing number of Trp residues were marked decreases in enzymatic activity and lethality, whereas antigenicity remained unchanged. The degree of exposure of tryptophanyl groups as determined by acrylamide quenching was consistent with the relative reactivity toward 2-hydroxy-5-nitrobenzyl bromide, except for Hemachatushaemachatus phospholipase A₂, which showed unusually high reactivity due to its characteristic dimeric conformation. Difference spectra of Trp-modified derivatives differed from those of their native enzymes by the presence of a new positive perturbation between 350 and 500 nm, with a maximum at 415 nm. Scatchard plots revealed only one type of binding site for Ca²⁺, and the binding abilities of the modified enzymes were not impaired. At pH 8.0, all native enzymes enhanced the emission intensity of 8-anilinonaphthalene sulfonate (ANS) dramatically, and the emission intensity of the ANS-enzyme complex increased or decreased in parallel with increasing concentration of Ca²⁺ for the respective enzyme. The Trp-modified derivatives did not enhance the emission intensity of ANS at all either in the presence or absence of Ca²⁺. By means of tryptophan modification, we were able to infer that the tryptophan residues are in the vicinity of the Ca²⁺ binding site and are directly involved in the binding with ANS. This, together with the suggestion that the hydrophobic pocket that interacts with ANS might be the site of binding of the phospholipase A₂ enzyme with the substrate, suggests that the Trp residues in phospholipase A₂ enzymes and presynaptic toxins are involved in substrate binding.     

Synthesis and pharmacological evaluation of novel 1- and 8-substituted-3-furfuryl xanthines as adenosine receptor antagonists

The synthesis of an important set of 3-furfurylxanthine derivatives is described. Binding affinities were determined for rat A₁ and human A_2A, A_2B and A₃ receptors. Several of the 3-furfuryl-7-methylxanthine derivatives showed moderate-to-high affinity at human A_2B receptors, the most active compound (10d) having a K_i of 7.4 nM for hA_2B receptors, with selectivities over rA₁ and hA_2A receptors up to 14-fold and 11-fold, respectively. Affinities for hA₃ receptors were very low for all members of the set.     

Steady-state and transient polarized absorption spectroscopy of photosytem I complexes from the cyanobacteria Arthrospira platensis and Thermosynechococcus elongatus

Core antenna and reaction centre of photosytem I (PS I) complexes from the cyanobacteria Arthrospira platensis and Thermosynechococcus elongatus have been characterized by steady-state polarized absorption spectroscopy, including linear dichroism (LD) and circular dichroism (CD). CD spectra and the second derivatives of measured 77 K CD spectra reveal the spectral components found in the polarized absorption spectra indicating the excitonic origin of the spectral forms of chlorophyll in the PS I complexes. The CD bands at 669-670(+), 673(+), 680(−), 683-685(−), 696-697(−), and 711(−) nm are a common feature of used PSI complexes. The 77 K CD spectra of the trimeric PS I complexes exhibit also low amplitude components around 736 nm for A. platensis and 720 nm for T. elongatus attributed to red-most chlorophylls. The LD measurements indicate that the transition dipole moments of the red-most states are oriented parallel to the membrane plane. The formation of P700⁺A₁⁻ or ³P700 was monitored by time-resolved difference absorbance and LD spectroscopy to elucidate the spectral properties of the PS I reaction centre. The difference spectra give strong evidence for the delocalization of the excited singlet states in the reaction centre. Therefore, P700 cannot be considered as a dimer but should be regarded as a multimer of the six nearly equally coupled reaction centre chlorophylls in accordance with structure-based calculations. On the basis of the results presented in this work and earlier work in the literature it is concluded that the triplet state is localized most likely on P_A, whereas the cation is localized most likely on P_B.     

Subchronic Treatment with Methamphetamine and Phencyclidine Differentially Alters the Adenosine A1 and A2A Receptors in the Prefrontal Cortex,Hippocampus, and Striatum of the Rat

Subchronic treatment with MAP (4.6 mg/kg, i.p., once daily for 11 days) significantly decreased the K_d, but not B_max, values of [³H]1,3-dipropyl-8-cyclopentylxanthine ([³H]DPCPX) binding to adenosine A₁ receptors in the prefrontal cortex and hippocampus, but not striatum, of rat brain. However, subchronic treatment with PCP (10 mg/kg, i.p., once daily for 11 days) did not alter the K_d and B_max values of [³H]DPCPX binding to adenosine A₁ receptors in these three regions. Subchronic treatment with MAP or PCP did not alter the B_max and K_d values of [³H]2-p-(2-carboxyehyl)phenethylamino-5-N-ethylcarboxyamidoadenosine ([³H]CGS21680) binding to adenosine A_2A receptors in the striatum. Furthermore, subchronic treatment with MAP or PCP significantly decreased the specific binding of [³H]CGS21680 to adenosine A_2A receptors in the hippocampus, but not in the prefrontal cortex. Thus, these results suggest that MAP and PCP may produce differential effects on the adenosine A_2A receptors, but not adenosine A₁ receptors in rat brain.     

Inhibition of cell growth by bafilomycin A1, a selective inhibitor of vacuolar H+-ATPase

Summary Bafilomycin A₁, a potent selective inhibitor of vacuolar H⁺-ATPase, inhibited the growth of a variety of cultured cells dose-dependently, including golden hamster embryo and NIH-3T3 fibroblasts, whether or not they were transformed, and PC12 and HeLa cells. The concentration of bafilomycin A₁ for 50% inhibition of cell growth ranged from 10 to 50 nM. The dose response was nearly parallel with that of the bafilomycin A₁-induced lysosomal pH increase. The degree of pH increase for growth inhibition produced by bafilomycin A₁ was similar to that produced by NH₄Cl in which little difference was recognized in effect among cell types.     

Structural and functional analysis of the coupling subunit F in solution and topological arrangement of the stalk domains of the methanogenic A1AO ATP synthase

The first low-resolution shape of subunit F of the A₁A_O ATP synthase from the archaeon Methanosarcina mazei Gö1 in solution was determined by small angle X-ray scattering. Independent to the concentration used, the protein is monomeric and has an elongated shape, divided in a main globular part with a length of about 4.5 nm, and a hook-like domain of about 3.0 nm in length. The subunit-subunit interaction of subunit F inside the A₁A_O ATP synthase in the presence of 1-ethyl-3-(dimethylaminopropyl)-carbodiimide EDC was studied as a function of nucleotide binding, demonstrating movements of subunits F relative to the nucleotide-binding subunit B. Furthermore, in the intact A₁A_O complex, crosslinking of subunits D-E, A-H and A-B-D was obtained and the peptides, involved, were analyzed by MALDI-TOF mass spectrometry. Based on these data the surface of contact of B-F could be mapped in the high-resolution structure of subunit B of the A₁A_O ATP synthase.     

Modelling of the electron transfer reactions in Photosystem I by electron tunnelling theory: The phylloquinones bound to the PsaA and the PsaB reaction centre subunits of PS I are almost isoenergetic to the iron-sulfur cluster FX

Photosystem I is a large macromolecular complex located in the thylakoid membranes of chloroplasts and in cyanobacteria that catalyses the light driven reduction of ferredoxin and oxidation of plastocyanin. Due to the very negative redox potential of the primary electron transfer cofactors accepting electrons, direct estimation by redox titration of the energetics of the system is hampered. However, the rates of electron transfer reactions are related to the thermodynamic properties of the system. Hence, several spectroscopic and biochemical techniques have been employed, in combination with the classical Marcus theory for electron transfer tunnelling, in order to access these parameters. Nevertheless, the values which have been presented are very variable. In particular, for the case of the tightly bound phylloquinone molecule A₁, the values of the redox potentials reported in the literature vary over a range of about 350 mV. Previous models of Photosystem I have assumed a unidirectional electron transfer model. In the present study, experimental evidence obtained by means of time resolved absorption, photovoltage, and electron paramagnetic resonance measurements are reviewed and analysed in terms of a bi-directional kinetic model for electron transfer reactions. This model takes into consideration the thermodynamic equilibrium between the iron-sulfur centre F_X and the phylloquinone bound to either the PsaA (A_1A) or the PsaB (A_1B) subunit of the reaction centre and the equilibrium between the iron-sulfur centres F_A and F_B. The experimentally determined decay lifetimes in the range of sub-picosecond to the microsecond time domains can be satisfactorily simulated, taking into consideration the edge-to-edge distances between redox cofactors and driving forces reported in the literature. The only exception to this general behaviour is the case of phylloquinone (A₁) reoxidation. In order to describe the reported rates of the biphasic decay, of about 20 and 200 ns, associated with this electron transfer step, the redox potentials of the quinones are estimated to be almost isoenergetic with that of the iron sulfur centre F_X. A driving force in the range of 5 to 15 meV is estimated for these reactions, being slightly exergonic in the case of the A_1B quinone and slightly endergonic, in the case of the A_1A quinone. The simulation presented in this analysis not only describes the kinetic data obtained for the wild type samples at room temperature and is consistent with estimates of activation energy by the analysis of temperature dependence, but can also explain the effect of the mutations around the PsaB quinone binding pocket. A model of the overall energetics of the system is derived, which suggests that the only substantially irreversible electron transfer reactions are the reoxidation of A₀ on both electron transfer branches and the reduction of F_A by F_X.     

Persistence of 14C-gibberellin A3 on the surface of Citrus fruit peel and on inert glass surfaces

The persistence of gibberellin A₃ on plant surfaces was examined using fruit of Marsh seedless grapefruit (Citrus paradisi Macf.) and an inert glass model system. ¹⁴C-gibberellin A₃ was applied to surfaces in aqueous treatment solutions or in waxing solutions. Dried-out treatment residues were removed by washing and analyzed for total and GA₃-like radioactivity. Gibberellin A₃ persisted without significant loss for at least 7 d in aqueous treatment solutions (pH 4.0 or 6.2) but was less persistent in the pH 10.4 waxing solution (t1/2=7 d).Loss of total peel surface radioactivity was fast during the first 3 days, slowing down afterwards. After 14 days 73% of the initial radioactivity could still be recovered from fruit peel surface and 70% of the recovered radioactivity was still in the form of gibberellin A₃. Gibberellin A₃ was somewhat more persistent in residues from pH 4 than pH 7 treatment solutions. Light had a slight enhancing effect on gibberellin A₃ decomposition on fruit peel under growth chamber conditions. After 12 d at 100% relative humidity, 88% of the radioactivity on glass surfaces was still in the form of gibberellin A₃, as against 45% at a relative humidity of 50%. Simulated field conditions, combining daily fluctuations in light, temperature and relative humidity, markedly enhanced gibberellin A₃ decomposition on glass surfaces (t1/2=2 d). Gibberellin A₃ was very persistent (90% after 9 d) in the waxing residues on fruit peel surface.Abbreviations GA₃ gibberellin A₃ - RH relative humidity     

Spectral and kinetic resolution of the bc1 complex components in situ: A simple and robust alternative to the traditional difference wavelength approach

The kinetics of the cytochrome (cyt) components of the bc₁ complex (ubiquinol: cytochrome c oxidoreductase, Complex III) are traditionally followed by using the difference of absorbance changes at two or more different wavelengths. However, this difference-wavelength (DW) approach is of limited accuracy in the separation of absorbance changes of components with overlapping spectral bands. To resolve the kinetics of individual components in Rhodobacter sphaeroides chromatophores, we have tested a simplified version of a least squares (LS) analysis, based on measurement at a minimal number of different wavelengths. The success of the simplified LS analysis depended significantly on the wavelengths used in the set. The “traditional” set of 6 wavelengths (542, 551, 561, 566, 569 and 575 nm), normally used in the DW approach to characterize kinetics of cyt c_tot (cyt c₁ + cyt c₂), cyt b_L, cyt b_H, and P870 in chromatophores, could also be used to determine these components via the simplified LS analysis, with improved resolution of the individual components. However, this set is not sufficient when information about cyts c₁ and c₂ is needed. We identified multiple alternative sets of 5 and 6 wavelengths that could be used to determine the kinetics of all 5 components (P870 and cyts c₁, c₂, b_L, and b_H) simultaneously, with an accuracy comparable to that of the LS analysis based on a full set of wavelengths (1 nm intervals). We conclude that a simplified version of LS deconvolution based on a small number of carefully selected wavelengths provides a robust and significant improvement over the traditional DW approach, since it accounts for spectral interference of the different components, and uses fewer measurements when information about all five individual components is needed. Using the simplified and complete LS analyses, we measured the simultaneous kinetics of all cytochrome components of bc₁ complex in the absence and presence of specific inhibitors and found that they correspond well to those expected from the modified Q-cycle. This is the first study in which the kinetics of all cytochrome and reaction center components of the bc₁ complex functioning in situ have been measured simultaneously, with full deconvolution over an extended time range.     

Striatal adenosine A2A receptor expression is controlled by S-adenosyl-L-methionine-mediated methylation

Adenosine A_2A receptor (A_2AR) is a G protein-coupled receptor enriched in the striatum for which an increased expression has been demonstrated in certain neurological diseases. Interestingly, previous in vitro studies demonstrated that A_2AR expression levels are reduced after treatment with S-adenosyl-L-methionine (SAM), a methyl donor molecule involved in the methylation of important biological structures such as DNA, proteins, and lipids. However, the in vivo effects of SAM treatment on A_2AR expression are still obscure. Here, we demonstrated that 2 weeks of SAM treatment produced a significant reduction in the rat striatal A_2AR messenger RNA (mRNA) and protein content as well as A_2AR-mediated signaling. Furthermore, when the content of 5-methylcytosine levels in the 5′UTR region of ADORA2A was analyzed, this was significantly increased in the striatum of SAM-treated animals; thus, an unambiguous correlation between SAM-mediated methylation and striatal A_2AR expression could be established. Overall, we concluded that striatal A_2AR functionality can be controlled by SAM treatment, an issue that might be relevant for the management of these neurological conditions that course with increased A_2AR expression.     

Femtosecond primary charge separation in Synechocystis sp. PCC 6803 photosystem I

The ultrafast (< 100 fs) conversion of delocalized exciton into charge-separated state between the primary donor P700 (bleaching at 705 nm) and the primary acceptor A₀ (bleaching at 690 nm) in photosystem I (PS I) complexes from Synechocystis sp. PCC 6803 was observed. The data were obtained by application of pump-probe technique with 20-fs low-energy pump pulses centered at 720 nm. The earliest absorbance changes (close to zero delay) with a bleaching at 690 nm are similar to the product of the absorption spectrum of PS I complex and the laser pulse spectrum, which represents the efficiency spectrum of the light absorption by PS I upon femtosecond excitation centered at 720 nm. During the first ∼ 60 fs the energy transfer from the chlorophyll (Chl) species bleaching at 690 nm to the Chl bleaching at 705 nm occurs, resulting in almost equal bleaching of the two forms with the formation of delocalized exciton between 690-nm and 705-nm Chls. Within the next ∼ 40 fs the formation of a new broad band centered at ∼ 660 nm (attributed to the appearance of Chl anion radical) is observed. This band decays with time constant simultaneously with an electron transfer to A₁ (phylloquinone). The subtraction of kinetic difference absorption spectra of the closed (state P700⁺A₀A₁) PS I reaction center (RC) from that of the open (state P700A₀A₁) RC reveals the pure spectrum of the P700⁺A₀⁻ ion-radical pair. The experimental data were analyzed using a simple kinetic scheme: An* [(PA₀)*A₁ P⁺A₀⁻A₁] P⁺A₀A₁⁻, and a global fitting procedure based on the singular value decomposition analysis. The calculated kinetics of transitions between intermediate states and their spectra were similar to the kinetics recorded at 694 and 705 nm and the experimental spectra obtained by subtraction of the spectra of closed RCs from the spectra of open RCs. As a result, we found that the main events in RCs of PS I under our experimental conditions include very fast (< 100 fs) charge separation with the formation of the P700⁺A₀⁻A₁ state in approximately one half of the RCs, the ∼ 5-ps energy transfer from antenna Chl* to P700A₀A₁ in the remaining RCs, and ∼ 25-ps formation of the secondary radical pair P700⁺A₀A₁⁻.     

Cerebral microvessel phospholipase A2 activity in senescent mouse

Phospholipase A₂ activity was measured in cerebral microvessels isolated from 5 to 6 month (young adult) and 21 to 24 month (aged adult) old mice. Radiolabeled 1-stearoyl-2-[1-¹⁴C]arachidonyl choline phosphoglyceride was used as the enzyme substrate, and enzyme activity determined at pH 8 and pH 9. Activity in older animals was significantly less than in younger animals at both pH's. With choline phosphoglyceride as a substrate, phospholipase A₂ activity was predominantly Ca²⁺-dependent, although a small, but measurable Ca²⁺-independent component was present. Negligible production of diacylglycerol indicated little or no phospholipase C activity. These findings indicate that activity of a phospholipase A₂, which utilizes choline phosphoglyceride as a substrate, is affected by the aging process. Moreover, a change in PLA₂ activity would result in altered metabolism of specific phosphoglycerides and turnover of fatty acids at the sn-2 position in cerebral microvessels.     

Effect of the P700 pre-oxidation and point mutations near A0 on the reversibility of the primary charge separation in Photosystem I from Chlamydomonas reinhardtii

Time-resolved fluorescence studies with a 3-ps temporal resolution were performed in order to: (1) test the recent model of the reversible primary charge separation in Photosystem I (Müller et al., 2003; Holwzwarth et al., 2005, 2006), and (2) to reconcile this model with a mechanism of excitation energy quenching by closed Photosystem I (with P700 pre-oxidized to P700⁺). For these purposes, we performed experiments using Photosystem I core samples isolated from Chlamydomonas reinhardtii wild type, and two mutants in which the methionine axial ligand to primary electron acceptor, A₀, has been change to either histidine or serine. The temporal evolution of fluorescence spectra was recorded for each preparation under conditions where the “primary electron donor,” P700, was either neutral or chemically pre-oxidized to P700⁺. For all the preparations under study, and under neutral and oxidizing conditions, we observed multiexponential fluorescence decay with the major phases of ∼ 7 ps and ∼ 25 ps. The relative amplitudes and, to a minor extent the lifetimes, of these two phases were modulated by the redox state of P700 and by the mutations near A₀: both pre-oxidation of P700 and mutations caused slight deceleration of the excited state decay. These results are consistent with a model in which P700 is not the primary electron donor, but rather a secondary electron donor, with the primary charge separation event occurring between the accessory chlorophyll, A, and A₀. We assign the faster phase to the equilibration process between the excited state of the antenna/reaction center ensemble and the primary radical pair, and the slower phase to the secondary electron transfer reaction. The pre-oxidation of P700 shifts the equilibrium between the excited state and the primary radical pair towards the excited state. This shift is proposed to be induced by the presence of the positive charge on P700⁺. The same charge is proposed to be responsible for the fast A⁺A₀⁻ → AA₀ charge recombination to the ground state and, in consequence, excitation quenching in closed reaction centers. Mutations of the A₀ axial ligand shift the equilibrium in the same direction as pre-oxidation of P700 due to the up-shift of the free energy level of the state A⁺A₀⁻.