Quadruplex-forming properties of FRAXA (CGG) repeats interrupted by (AGG) triplets

The (CGG) repeats associated with X-chromosome fragility are generally believed to form quadruplexes. This notion has persisted although it had been shown that only very short (CGG)_n sequences form quadruplexes and that this quadruplex formation occurs in conditions far from physiological. We have now studied, using CD and absorption spectroscopies, quadruplex formation of (CGG)_n (n = 4, 7, 8, or 16) and their analogs interrupted by (AGG) triplets under various solvent conditions. In healthy individuals, (AGG) triplets are interspersed throughout the (CGG) repeat regions and appear to hinder (CGG)_n motif expansion. Here we show that (CGG) repeats do not form quadruplexes under physiological conditions in aqueous solution but, interestingly, quadruplexes are readily formed in water–ethanol solutions. The presence of (AGG) triplets markedly stabilized quadruplex formation. Quadruplexes may thus hinder rather than support (CGG)_n motif expansion.     

pH heterogeneity in tibial anterior muscle during isometric activity studied by (31)P-NMR spectroscopy.

The occurrence of pH heterogeneity in human tibial anterior muscle during sustained isometric exercise is demonstrated by applying (31)P-nuclear magnetic resonance (NMR) spectroscopy in a study of seven healthy subjects. Exercise was performed at 30 and 60% of maximal voluntary contraction (MVC) until fatigue. The NMR spectra, as localized by a surface coil and improved by proton irradiation, were obtained at a high time resolution (16 s). They revealed the simultaneous presence of two pH pools during most experiments. Maximum difference in the two pH levels during exercise was 0.40 +/- 0.07 (30% MVC, n = 7) and 0.41 +/- 0.03 (60% MVC, n = 3). Complementary two-dimensional (31)P spectroscopic imaging experiments in one subject supported the supposition that the distinct pH pools reflect the metabolic status of the main muscle fiber types. The relative size of the P(i) peak in the spectrum attributed to the type II fiber pool increases with decreasing pH levels. This phenomenon is discussed in the context of the size principle stating that the smaller (type I) motor units are recruited first.     

Perturbation of the conformational equilibria in Ras by selective mutations as studied by 31P NMR spectroscopy

Ras regulates a variety of different signal transduction pathways acting as molecular switch. It was shown by liquid and solid-state (31)P NMR spectroscopy that Ras exists in the guanosine-5'-(beta,gamma-imido)triphosphate bound form in at least two conformational states interconverting in millisecond time scale. The relative population between the two conformational states affects drastically the affinity of Ras to its effectors. (31)P NMR spectroscopy shows that the conformational equilibrium can be shifted specifically by point mutations, including mutations with oncogenic potential, thus modifying the effector interactions and their coupling to dynamic properties of the protein.     

Locust flight metabolism studied in vivo by 31P NMR spectroscopy

Flight metabolism of locusts has been extensively studied, but biochemical and physiological methods have led to conflicting results. For this reason the non-invasive and non-destructive method of ³¹P NMR spectroscopy was used to study migratory locusts, Locusta migratoria, at rest and during flight.

Interaction between calcium-free calmodulin and IQ motif of neurogranin studied by nuclear magnetic resonance spectroscopy

The interaction of Ca(2+)-free calmodulin (apoCaM) with the IQ motif corresponding to the calmodulin-binding domain of neurogranin has been studied by nuclear magnetic resonance (NMR) methods. The NMR spectra of uncomplexed apoCaM and apoCaM in complex with the IQ motif recorded at 750 MHz were studied and the backbone assignments of the protein in both forms were obtained by triple-resonance multidimensional NMR experiments. Chemical shift perturbations were used to map the binding surfaces. Only a single set of resonances was observed throughout the titration, indicating that the binding interaction is under fast exchange. Analysis of chemical shift changes indicates that (a) the main interaction and conformational changes occur in the C-terminal domain of calmodulin and (b) linker-1 (residues 40-44) between EF-1 and EF-2, linker-3 (residues 112-117) between EF-3 and EF-4, and the end of the alpha-helix H (residues 145-148) may be involved in the binding process. The dissociation constant (K(d)), estimated by fitting the chemical shift changes against the IQ peptide concentration, ranged from about 1.2 x 10(-5) to 8.8 x 10(-5) M. This result demonstrates that the interaction falls into the weak binding regime.     

Reassimilation of carbon dioxide by Flaveria (Asteraceae) species representing different types of photosynthesis

The capability to reassimilate CO₂ originating from intracellular decarboxylating processes connected with the photorespiratory glycolate pathway and-or decarboxylation of C₄ acids during C₄ photosynthesis has been investigated with four species of the genus Flaveria (Asteraceae). The C₃-C₄ intermediate species F. pubescens and F. anomala reassimilated CO₂ much more efficiently than the C₃ species F. cronquistii and, with respect to this feature, behaved similarly to the C₄ species F. trinervia. Therefore, under atmospheric conditions the intermediate species photorespired with rates only between 10–20% of that measured with F. cronquistii. At low oxygen concentrations (1,5%) the reassimilation potential of F. anomala approached that of F. trinervia and was distinct from that found with F. pubescens. The data are discussed with respect to a possible sequence of events during evolution of C₄ photosynthesis. If compared with related data for C₃-C₄ intermediate species from other genera they support the hypothesis that, during evolution of C₄ photosynthesis, an efficient capacity for CO₂ reassimilation evolved prior to a CO₂-concentrating mechanism.Abbreviations C₃, C₄ assimilated CO₂ initially found in 3-phosphoglycerate (C₃) or malate and aspartate (C₄) - D reassimilation coefficient - R _n , R _t net, total CO₂ evolution as measured with 0.03 and 3% CO₂, respectively - RuBP ribulose-1,5-bisphosphate - TPS true photosynthesis     

Correct folding of the beta-barrel of the human membrane protein VDAC requires a lipid bilayer

Spontaneous membrane insertion and folding of beta-barrel membrane proteins from an unfolded state into lipid bilayers has been shown previously only for few outer membrane proteins of Gram-negative bacteria. Here we investigated membrane insertion and folding of a human membrane protein, the isoform 1 of the voltage-dependent anion-selective channel (hVDAC1) of mitochondrial outer membranes. Two classes of transmembrane proteins with either alpha-helical or beta-barrel membrane domains are known from the solved high-resolution structures. VDAC forms a transmembrane beta-barrel with an additional N-terminal alpha-helix. We demonstrate that similar to bacterial OmpA, urea-unfolded hVDAC1 spontaneously inserts and folds into lipid bilayers upon denaturant dilution in the absence of folding assistants or energy sources like ATP. Recordings of the voltage-dependence of the single channel conductance confirmed folding of hVDAC1 to its active form. hVDAC1 developed first beta-sheet secondary structure in aqueous solution, while the alpha-helical structure was formed in the presence of lipid or detergent. In stark contrast to bacterial beta-barrel membrane proteins, hVDAC1 formed different structures in detergent micelles and phospholipid bilayers, with higher content of beta-sheet and lower content of alpha-helix when inserted and folded into lipid bilayers. Experiments with mixtures of lipid and detergent indicated that the content of beta-sheet secondary structure in hVDAC1 decreased at increased detergent content. Unlike bacterial beta-barrel membrane proteins, hVDAC1 was not stable even in mild detergents such as LDAO or dodecylmaltoside. Spontaneous folding of outer membrane proteins into lipid bilayers indicates that in cells, the main purpose of membrane-inserted or associated assembly factors may be to select and target beta-barrel membrane proteins towards the outer membrane instead of actively assembling them under consumption of energy as described for the translocons of cytoplasmic membranes.     

Impaired resting muscle energetics studied by (31)P-NMR in diet-induced obese rats

Objective: Mitochondrial activity is altered in skeletal muscle of obese, insulin‐resistant or type 2 diabetic patients. We hypothesized that this situation was associated with profound adaptations in resting muscle energetics. For that purpose, we used in vivo ³¹P‐nuclear magnetic resonance (³¹P‐NMR) in male sedentary Wistar rats fed with obesogenic diets known to induce alterations in muscle mitochondrial activity. Methods and Procedures: Two experimental diets (high sucrose and high fat) were provided for 6 weeks at two levels of energy (standard, N and high, H) and compared to control diet. The rates of the adenosine triphosphate (ATP) exchange between phosphocreatine (PCr) and γ ‐ATP (k_a) and β ‐adenosine diphosphate ( β ‐ADP) to β ‐ATP (k_b) were evaluated using ³¹P‐NMR in resting gastrocnemius muscle. Muscle contents in phosphorylated compounds as well as creatine, were assessed using ³¹P‐NMR and biochemical assays, respectively. Results: ATP content increased by 6.7–8.5% in standard‐energy high‐sucrose (NSU), high‐energy high‐fat (HF) and high‐energy high‐sucrose (HSU) groups compared to control (P < 0.05), whereas PCr content decreased by 4.2–6.4% (P < 0.01). Consequently, PCr to ATP ratio decreased in NSU, HF, and HSU groups, compared to control (P < 0.01). Furthermore in high‐energy groups (HF and HSU) compared to control, creatine contents were decreased by 14–19% (P < 0.001), whereas k_a and k_b fluxes were increased by 89–133% (P < 0.001) and 243–277% (P < 0.01), respectively. Discussion: Our in vivo data showed adaptations of resting skeletal muscle energetics in response to high‐energy diets. Increased activity of enzymes catalyzing ATP production may reflect a compensatory mechanism to face impaired mitochondrial ATP synthesis in order to preserve intracellular energy homeostasis.     

Energy and electron transfer in the photosynthetic reaction center complex of Acidiphilium rubrum containing Zn-bacteriochlorophyll a studied by femtosecond up-conversion spectroscopy

A photosynthetic reaction center (RC) complex was isolated from a purple bacterium, Acidiphilium rubrum. The RC contains bacteriochlorophyll a containing Zn as a central metal (Zn-BChl a) and bacteriopheophytin a (BPhe a) but no Mg-BChl a. The absorption peaks of the Zn-BChl a dimer (P_Zn), the accessory Zn-BChl a (B_Zn), and BPhe a (H) at 4 K in the RC showed peaks at 875, 792, and 753 nm, respectively. These peaks were shorter than the corresponding peaks in Rhodobacter sphaeroides RC that has Mg-BChl a. The kinetics of fluorescence from P_Zn^*, measured by fluorescence up-conversion, showed the rise and the major decay with time constants of 0.16 and 3.3 ps, respectively. The former represents the energy transfer from B_Zn^* to P_Zn, and the latter, the electron transfer from P_Zn to H. The angle between the transition dipoles of B_Zn and P_Zn was estimated to be 36° based on the fluorescence anisotropy. The time constants and the angle are almost equal to those in the Rb. sphaeroides RC. The high efficiency of A. rubrum RC seems to be enabled by the chemical property of Zn-BChl a and by the L168HE modification of the RC protein that modifies P_Zn.     

Kinetics and mechanism of Ru(III) and Hg(II) co-catalyzed oxidation of D-galactose and D-ribose by N-bromoacetamide in perchloric acid

Kinetics of oxidation of reducing sugars D-galactose (Gal) and D-ribose (Rib) by N-bromoacetamide (NBA) in the presence of ruthenium(III) chloride as a homogeneous catalyst and in perchloric acid medium, using mercuric acetate as a scavenger for Br(minus sign) ions, as well as a co-catalyst, have been investigated. The kinetic results indicate that the first-order kinetics in NBA at lower concentrations tend towards zero order at its higher concentrations. The reactions follow identical kinetics, being first order in the [sugar] and [Ru(III)]. Inverse fractional order in [H(+)] and [acetamide] were observed. A positive effect of [Hg(OAc)(2)] and [Cl(minus sign)] was found, whereas a change in ionic strength (mu) has no effect on oxidation velocity. Formic acid and D-lyxonic acid (for Gal) and formic acid and L-erythronic acid (for Rib) were identified as main oxidation products of reactions. The various activation parameters have been computed and recorded. A suitable mechanism consistent with experimental findings has been proposed.     

Mechanism of strong quenching of photosystem II chlorophyll fluorescence under drought stress in a lichen, Physciella melanchla, studied by subpicosecond fluorescence spectroscopy

The mechanism of the severe quenching of chlorophyll (Chl) fluorescence under drought stress was studied in a lichen Physciella melanchla, which contains a photobiont green alga, Trebouxia sp., using a streak camera and a reflection-mode fluorescence up-conversion system. We detected a large 0.31 ps rise of fluorescence at 715 and 740 nm in the dry lichen suggesting the rapid energy influx to the 715-740 nm bands from the shorter-wavelength Chls with a small contribution from the internal conversion from Soret bands. The fluorescence, then, decayed with time constants of 23 and 112 ps, suggesting the rapid dissipation into heat through the quencher. The result confirms the accelerated 40 ps decay of fluorescence reported in another lichen (Veerman et al., 2007 [36]) and gives a direct evidence for the rapid energy transfer from bulk Chls to the longer-wavelength quencher. We simulated the entire PS II fluorescence kinetics by a global analysis and estimated the 20.2 ns^− 1 or 55.0 ns^− 1 energy transfer rate to the quencher that is connected either to the LHC II or to the PS II core antenna. The strong quenching with the 3-12 times higher rate compared to the reported NPQ rate, suggests the operation of a new type of quenching, such as the extreme case of Chl-aggregation in LHCII or a new type of quenching in PS II core antenna in dry lichens.     

Structural characterization of photosynthetic cells in an amphibious sedge, Eleocharis vivipara, in relation to C3 and C4 metabolism

Eleocharis vivipara Link is a unique amphibious leafless sedge. The terrestrial form has Kranz anatomy and the biochemical traits of C₄ plants while the submerged form develops structural and biochemical traits similar to those of C₃ plants. The structural features of the culms, which are the photosynthetic organs, of the two forms were examined and compared. The culms of the terrestrial form have mesophyll cells and three bundle sheaths which consist of three kinds of cell, namely, the innermost Kranz cells that contain large numbers of organelles, the middle mestome sheath cells that lack chloroplasts, and the outermost parenchyma sheath cells that contain chloroplasts. The culms of the submerged form had a tendency towards reduction in numbers and size of Kranz cells and vascular bundles, as compared to the terrestrial form, and they had spherical mesophyll cells that were tightly packed without intercellular spaces inside the epidermis. The submerged form had a higher ratio of cross-sectional area of mesophyll cells plus parenchyma sheath cells to that of Kranz cells than the terrestrial form. The difference was mainly due to a decrease in the number and the size of the Kranz cells and to a marked increase in the size of the mesophyll cells and the parenchyma sheath cells in the submerged form, as compared to the terrestrial form. The Kranz cells of the terrestrial form had basically the structural characteristics of plants of the NAD-malic enzyme type, with the exception of the intracellular location of organelles. The Kranz cells of the submerged form included only a few organelles, and the percentage of organelles partitioned to the Kranz cells was significantly smaller in the submerged form than in the terrestrial form. In addition, the size of chloroplasts of the Kranz cells was 60–70% of that of the terrestrial form. These structural differences between the two forms may be related to the functional differences in their mechanisms of photosynthesis.Abbreviations KC Kranz cell - MC mesophyll cell - PSC parenchyma sheath cell - NAD-ME NAD-malic enzyme - VB vascular bundle This study was supported by Grants-in-Aid from the Ministry of Agriculture, Forestry and Fisheries of Japan (Integrated Research Program for the Use of Biotechnological Procedures for Plant Breeding) and from the Science and Technology Agency of Japan (Enhancement of Center-of-Excellence, the Special Coordination Funds for Promoting Science and Technology).     

Components and fractions for differently bound water molecules of dipalmitoylphosphatidylcholine-water system as studied by DSC and H-NMR spectroscopy

Differently bound water molecules of dipalmitoylphosphatidylcholine (DPPC)-H₂O system were investigated with differential scanning calorimetry (DSC). According to a method previously reported by us, the ice-melting DSC curves of the DPPC-H₂O samples of varying water contents were deconvoluted into multiple components, and the ice-melting enthalpies for the individual deconvoluted components were used to estimate average molar ice-melting enthalpies for freezable interlamellar and bulk waters, respectively. With these average molar ice-melting enthalpies, the numbers of differently bound water molecules of the DPPC-H₂O system were calculated at varying water contents and were used to construct a water distribution diagram of this system. Furthermore, to evaluate the reliability of the present DSC deconvolution method, ²H-NMR T₁ measurements of DPPC-²H₂O system were carried out at 5 °C of the gel phase temperature, and components and fractions for differently bound water (²H₂O) molecules were estimated from the analysis of nonexponential magnetization recovery curves.     

Oxidized phospholipids induce phase separation in lipid vesicles

The thermal behaviour of phospholipid multilamellar vesicles (MLV) made of various molar percentages of DPPC and LPPC, containing also oxidized LPPC (LPPCox), was studied by use of EPR spectroscopy and n-DSPC spin label in order to determine variations in the membrane fluidity brought about by lipid oxidation. Experimental variables were temperature, ranging from 4 to 44 degrees C, and molar percentage composition of DPPC/LPPC/LPPCox ternary mixture. We found that the presence of LPPCox in a percentage higher than both normal phospholipids' heavily hindered membrane formation, while lower percentage of the oxidized lipid with higher DPPC percentages yielded two-components EPR spectra, showing the presence of two different fluidity domains, indicative of membrane phase separation. When LPPC was the dominant lipid in the ternary mixture, simple EPR spectra were observed, indicating homogeneity of MLV membranes. Phase separation observed in the presence of LPPCox was better visible at lower temperature (12 degrees C or less), and almost disappeared with increasing temperature (36 degrees C or more). Furthermore, the correlation time of 16-DSPC in ternary mixture MLVs with higher LPPC percentage (homogeneous membranes) was not affected by the presence of LPPCox, while it normally increased upon DPPC percentage increase, as readily calculated from the EPR spectra featuring simple bands at 24 degrees C. It is concluded that oxidized lipid induces phase separation in more rigid DPPC-rich membranes, while leaving fluidity unaffected in more fluid LPPC-rich membranes, and at higher temperature.     

Conservation of lipid functions in cytochrome bc complexes

Lipid binding sites and properties are compared in two sub-families of hetero-oligomeric membrane protein complexes known to have similar functions in order to gain further understanding of the role of lipid in the function, dynamics, and assembly of these complexes. Using the crystal structure information for both complexes, we compared the lipid binding properties of the cytochrome b₆f and bc₁ complexes that function in photosynthetic and respiratory membrane energy transduction. Comparison of lipid and detergent binding sites in the b₆f complex with those in bc₁ shows significant conservation of lipid positions. Seven lipid binding sites in the cyanobacterial b₆f complex overlap three natural sites in the Chlamydomonas reinhardtii algal complex and four sites in the yeast mitochondrial bc₁ complex. The specific identity of lipids is different in b₆f and bc₁ complexes: b₆f contains sulfoquinovosyldiacylglycerol, phosphatidylglycerol, phosphatidylcholine, monogalactosyldiacylglycerol, and digalactosyldiacylglycerol, whereas cardiolipin, phosphatidylethanolamine, and phosphatidic acid are present in the yeast bc₁ complex. The lipidic chlorophyll a and β-carotene (β-car) in cyanobacterial b₆f, as well as eicosane in C. reinhardtii, are unique to the b₆f complex. Inferences of lipid binding sites and functions were supported by sequence, interatomic distance, and B-factor information on interacting lipid groups and coordinating amino acid residues. The lipid functions inferred in the b₆f complex are as follows: (i) substitution of a transmembrane helix by a lipid and chlorin ring, (ii) lipid and β-car connection of peripheral and core domains, (iii) stabilization of the iron-sulfur protein transmembrane helix, (iv) n-side charge and polarity compensation, and (v) β-car-mediated super-complex with the photosystem I complex.     

Conformation of the c552:aa3 electron transfer complex in Paracoccus denitrificans studied by EPR on oriented samples

The EPR spectral parameters of aa(3) oxidase and cyt c(552) from Paracoccus denitrificans were studied in purified oxidase and enriched cyt c(552). The orientation of the g-tensors of hemes a and c(552) were determined on partially ordered membranes, enriched cyt c(552) and a c(552):aa(3) subcomplex. The known correlation of g-tensor to molecular axes in histidine/methionine ligated hemes permits us to position cyt c(552) with respect to the parent membrane. Taken together with previous data on the interaction surface between aa(3) oxidase and cyt c(552), these results allow us to arrive at a single conformation for the c(552):aa(3) electron transfer complex.     

Diversity in forms of C4 in the genus Cleome (Cleomaceae)

Background and Aims

Cleomaceae is one of 19 angiosperm families in which C₄ photosynthesis has been reported. The aim of the study was to determine the type, and diversity, of structural and functional forms of C₄ in genus Cleome.

Methods

Plants of Cleome species were grown from seeds, and leaves were subjected to carbon isotope analysis, light and scanning electron microscopy, western blot analysis of proteins, and in situ immunolocalization for ribulose bisphosphate carboxylase oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC).

Key Results

Three species with C₄-type carbon isotope values occurring in separate lineages in the genus (Cleome angustifolia, C. gynandra and C. oxalidea) were shown to have features of C₄ photosynthesis in leaves and cotyledons. Immunolocalization studies show that PEPC is localized in mesophyll (M) cells and Rubisco is selectively localized in bundle sheath (BS) cells in leaves and cotyledons, characteristic of species with Kranz anatomy. Analyses of leaves for key photosynthetic enzymes show they have high expression of markers for the C₄ cycle (compared with the C₃–C₄ intermediate C. paradoxa and the C₃ species C. africana). All three are biochemically NAD-malic enzyme sub-type, with higher granal development in BS than in M chloroplasts, characteristic of this biochemical sub-type. Cleome gynandra and C. oxalidea have atriplicoid-type Kranz anatomy with multiple simple Kranz units around individual veins. However, C. angustifolia anatomy is represented by a double layer of concentric chlorenchyma forming a single compound Kranz unit by surrounding all the vascular bundles and water storage cells.

Conclusions

NAD-malic enzyme-type C₄ photosynthesis evolved multiple times in the family Cleomaceae, twice with atriplicoid-type anatomy in compound leaves having flat, broad leaflets in the pantropical species C. gynandra and the Australian species C. oxalidea, and once by forming a single Kranz unit in compound leaves with semi-terete leaflets in the African species C. angustifolia. The leaf morphology of C. angustifolia, which is similar to that of the sister, C₃–C₄ intermediate African species C. paradoxa, suggests adaptation of this lineage to arid environments, which is supported by biogeographical information.     

Ganglioside GM(1) biphasically regulates the activity of protein kinase C by the effects on the structure of the lipid bilayer

Addition of a small amount of ganglioside GM(1) to phosphatidylserine (PS) liposomes, a gradual increase of protein kinase C (PKC) activity was recorded up to about 2 mol% GM(1) where the maximal enzyme activity was obtained. Then the activity of PKC began to decline and even turned to be inhibited with the further increase of GM(1) content. It was also indicated that GM(1)/PS binary liposomes had the highest membrane fluidity and very low spatial density of lipid headgroups which was demonstrated in the MC-540 studies due to the interposition of GM(1) when the liposomes contained about 2 mol% GM(1). Besides, the liposomes containing about 2 mol% GM(1) provided a more hydrophobic environment for PKC than the liposomes containing less or more GM(1) which was indicated in the Acrylodan experiments. These factors commonly induced PKC to be stimulated maximally. Whether at the lower or higher GM(1) content, the membrane structure was not the most suitable to support the activity of PKC, which declined as a consequence.     

A mild and environmentally friendly scandium(III) trifluoromethanesulfonate-catalyzed synthesis of bis(3'-indolyl)alkanes and bis(3'-indolyl)-1-deoxyalditols

Bis(3'-indolyl)alkanes and bis(3'-indolyl) derivatives containing a 1-deoxyalditol moiety were synthesized in the presence of 5 mol% of scandium(III) trifluoromethanesulfonate [Sc(OTf)3] in CH3CN or EtOH-H2O mixture as a solvent from room temperature to 70 degrees C in good yields (78-97%).