Stimulation of H+transport activity of vacuolar H+ATPase by activation of H+PPase in Kalanchoë blossfeldiana

In Kalanchoë blossfeldiana cv. Tom Thumb the initial rate of ATP-dependent H+-transport into tonoplast vesicles was stimulated up to three times if the H+-ATPase (EC 3.6.1.3) was energized a few minutes after pre-energization of the H+-PPase (EC 3.6.1.1). H+-PPase-activated ATP-dependent H+-transport was observed in plants of K. blossfeldiana cultivated in short day (SD) or long day (LD) conditions expressing different degrees of crassulacean acid metabolism (CAM). However, based on the higher activity and protein amount of H+-PPase and H+-ATPase present in the vacuolar membrane of SD plants the maximum H+-transport activity in the stimulated mode of the H+-ATPase was significantly higher in tonoplast vesicles of SD plants than of LD plants. Hence, a co-ordinated action of the H+-PPase and H+-ATPase at the tonoplast of Kalanchoë could allow a higher transport capacity at the vacuolar membrane when plants perform high CAM. Immunoprecipitation experiments with an antiserum raised against the A-subunit of the vacuolar H+-ATPase of Mesembryanthemum crystallinum L. showed that in SD and LD plants of K. blossfeldiana the H+-PPase was co-precipitated with the vacuolar H+-ATPase holoenzyme. The co-percipitation of the two transport proteins indicates a close structural localization of the H+-PPase and the A-subunit of the vacuolar H+-ATPase.     

Quantitative and time-course analysis of microbial degradation of 1H,1H,2H,2H,8H,8H–perfluorododecanol in activated sludge

A methylene group in the fluorinated carbon backbone of 1H,1H,2H,2H,8H,8H–perfluorododecanol (degradable telomer fluoroalcohol, DTFA) renders the molecule cleavable by microbial degradation into two fluorinated carboxylic acids. Several biodegradation products of DTFA are known, but their rates of conversion and fates in the environment have not been determined. We used liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) to quantitatively investigate DTFA biodegradation by the microbial community in activated sludge in polyethylene terephthalate (PET) flasks, which we also determined here showed least adsorption of DTFA. A reduction in DTFA concentration in the medium was accompanied by rapid increases in the concentrations of 2H,2H,8H,8H–perfluorododecanoic acid (2H,2H,8H,8H–PFDoA), 2H,8H,8H-2-perfluorododecenoic acid (2H,8H,8H-2-PFUDoA), and 2H,2H,8H-7-perfluorododecenoic acid and 2H,2H,8H-8-perfluorododecenoic acid (2H,2H,8H-7-PFUDoA/2H,2H,8H-8-PFUDoA), which were in turn followed by an increase in 6H,6H–perfluorodecanoic acid (6H,6H–PFDeA) concentration, and decreases in 2H,2H,8H,8H–PFDoA, 2H,8H,8H-2-PFUDoA, and 2H,2H,8H-7-PFUDoA/2H,2H,8H-8-PFUDoA concentrations. Accumulation of perfluorobutanoic acid (PFBA), a presumed end product of DTFA degradation, was also detected. Our quantitative and time-course study of the concentrations of these compounds reveals main routes of DTFA biodegradation, and the presence of new biodegradation pathways.

     

Clipping of Flexible Tails of Histones H3 and H4 Affects the Structure and?Dynamics of the Nucleosome

Förster resonance energy transfer was used to monitor the dynamic conformations of mononucleosomes under different chromatin folding conditions to elucidate the role of the flexible N-terminal regions of H3 and H4 histones. The H3 tail was shown to partake in intranucleosomal interactions by restricting the DNA breathing motion and compacting the nucleosome. The H3 tail effects were mostly independent of the ionic strength and valency of the ions. The H4 tail was shown to not greatly affect the nucleosome conformation, but did slightly influence the relative population of the preferred conformation. The role of the H4 tail varied depending on the valency and ionic strength, suggesting that electrostatic forces play a primary role in H4 tail interactions. Interestingly, despite the H4 tail’s lack of influence, when H3 and H4 tails were simultaneously clipped, a more dramatic effect was seen than when only H3 or H4 tails were clipped. The combinatorial effect of H3 and H4 tail truncation suggests a potential mechanism by which various combinations of histone tail modifications can be used to control accessibility of DNA-binding proteins to nucleosomal DNA.     

Theoretical study of the H + HCN → H + HNC process

We present a theoretical study on the detailed mechanism and kinetics of the H+HCN →H+HNC process. The potential energy surface was calculated at the complete basis set quantum chemical method, CBS-QB3. The vibrational frequencies and geometries for four isomers (H₂CN, cis-HCNH, trans-HCNH, CNH₂), and seven saddle points (TSn where n = 1 ? 7) are very important and must be considered during the process of formation of the HNC in the reaction were calculated at the B3LYP/6-311G(2d,d,p) level, within CBS-QB3 method. Three different pathways (PW1, PW2, and PW3) were analyzed and the results from the potential energy surface calculations were used to solve the master equation. The results were employed to calculate the thermal rate constant and pathways branching ratio of the title reaction over the temperature range of 300 up to 3000 K. The rate constants for reaction H + HCN → H + HNC were fitted by the modified Arrhenius expressions. Our calculations indicate that the formation of the HNC preferentially occurs via formation of cis–HCNH, the fitted expression is k _{P W2}(T) = 9.98 × 10^?22 T ^2.41 exp(?7.62 kcal.mol^?1/R T) while the predicted overall rate constant k _{O v e r a l l} (T) = 9.45 × 10^?21 T ^2.15 exp(?8.56 kcal.mol^?1/R T) in cm ³ molecule ^?1 s ^?1.

Open image in new window

Graphical Abstract (a) Potential energy surface, (b) thermal rate constants as a function of temperature and (c) the branching ratios (%) of PW1, PW2, PW3 pathways involved in rm H + HCN → H + HNC process.

     

Effects of the H6R and D7H Mutations on the Heparin-Dependent Modulation of Zinc-Induced Aggregation of Amyloid β

Molecular Biology - Zinc ions and glycosaminoglycans (GAGs) are found in amyloid deposits and are known to modulate the β-amyloid peptide (Аβ) aggregation, which is thought to be a...     

Three-dimensional structure of α-conotoxin EI determined by 1H NMR spectroscopy H resonance assignments of α-conotoxin EI

α-Conotoxin EI is an 18-residue peptide (RDOCCYHPTCNMSNPQIC; 4–10, 5–18) isolated from the venom of Conus ermineus, the only fish-hunting cone snail of the Atlantic Ocean. This peptide targets specifically the nicotinic acetylcholine receptor (nAChR) found in mammalian skeletal muscle and the electric organ Torpedo, showing a novel selectivity profile when compared to other α-conotoxins. The 3D structure of EI has been determined by 2D-NMR methods in combination with dynamical simulated annealing protocols. A total of 133 NOE-derived distances were used to produce 13 structures with minimum energy that complied with the NOE restraints. The structure of EI is characterized by a helical loop between Thr⁹ and Met¹² that is stabilized by the Cys⁴-Cys¹⁰ disulfide bond and turns involving Cys⁴-Cys⁵ and Asn¹⁴-Pro¹⁵. Other regions of the peptide appear to be flexible. The overall fold of EI is similar to that of other α4/7-conotoxins (PnIA/B, MII, EpI). However, unlike these other α4/7-conotoxins, EI targets the muscular type nAChR. The differences in selectivity can be attributed to differences in the surface charge distribution among these α4/7-conotoxins. The implications for binding of EI to the muscular nAChR are discussed with respect to the current NMR structure of EI.     

Determination of Hα,Hβ and Hβ,C′ coupling constants in13C-labeled proteins

Summary A sensitive method to assign H protons stereospecifically as well as to determine rotamer populations about ₁, in two 3D experiments is presented. The SOFT-HCCH-COSY experiment allowed us to measure the³J(H,C) couplings, using constant time evolution of C in t₂ and C_aliphatic-selective decoupling during t₃. The SOFT-HCCH-E.COSY experiment allowed us to measure the³J(H,H) couplings, using constant time evolution of C in t₂, a small flip angle¹H excitation pulse in the second mixing time, and double-band-selective decoupling (aliphatic and carbonyl carbons) during t₃. The method was applied to ribonuclease T₁.     

History of Ichnology: The Origins of Trace Fossil Taxonomy and the Contributions of Joseph F. James and Walter H. Häntzschel

The taxonomy of trace fossils has had a somewhat controversial history because they do not represent the actual animal remains but rather their work on and in the substrate. As such, traditional palaeontologists and zoologists have viewed them with some skepticism. Ichnologists owe a great debt to two geologists: Joseph F. James of Cincinnati and Walter H. Häntzschel of Hamburg, who took it upon themselves to impose some order on the chaos that constituted trace fossil taxonomy at the time. James, working independently and in ignorance of Alfred Nathorst, arrived at and utilized many of the same criteria his Swedish counterpart employed to criticize the fucoid origins of many trace fossils in the late 19th century. With his restudy of the systematics of Fucoides, Skolithos, and Arthrophycus, James brought to light many of the taxonomical nightmares that faced—and are still facing—the fledging science and can be rightfully considered the first trace fossil taxonomist. During the 1940s and 1950s, Häntzschel collected the widely scattered pertinent data from the literature, an immense task that, when published in 1962 (and later revised and expanded in 1975), made trace fossils accessible to further research and started a worldwide boom in trace fossil research.     

Effects of H₂O₂ on electrical membrane properties of skeletal myotubes

Reactive oxygen species (ROS), normally generated in skeletal muscles, could control excitability of muscle fibers through redox modulation of membrane ion channels. However, the mechanisms of ROS action remain largely unknown. To investigate the action of ROS on electrical properties of muscle cells, patch-clamp recordings were performed after application of hydrogen peroxide (H₂O₂) to skeletal myotubes. H₂O₂ facilitated sodium spikes after a hyperpolarizing current pulse, by decreasing the latency for spike initiation. Importantly, the antioxidant N-acetylcysteine induced the opposite effect, suggesting the redox control of muscle excitability. The effect of H₂O₂ was abolished in the presence of catalase. The kinetics of sodium channels were not affected by H₂O₂. However, the fast inward rectifier K⁺ (K_IR) currents, activated by hyperpolarization, were reduced by H₂O₂, similar to the action of the potassium channel blockers Ba²⁺ and Cs⁺. The block of the outward tail current contributing to K_IR deactivation can explain the shorter latency for spike initiation. We propose that the K_IR current is an important target for ROS action in myotubes. Our data would thus suggest that ROS are involved in the control of the excitability of myotubes and, possibly, in the oscillatory behavior critical for the plasticity of developing muscle cells.     

Conformational Peculiarities of Polynucleotides with a Nonrandom Base Sequence According to the 1H→ 3H Exchange Rate in C8H Groups of Purinic Residues

Abstract

We have determined the ¹H→³H exchange rate constants between water and C8H groups of purinic residues of alternating polynucleotides poly(dA-dT)·poly(dA-dT), poly(dG-dC)·poly(dG- dC) and poly(dA-dC)·poly(dG-dT) as well as homopolynucleotides poly(dA)·poly(dT) and poly(dG)·poly(dC) in aqueous solutions with high-salt concentrations (3 M NaCl and 4–6 M CsF), in water-ethanol (60%) solution and in 0.15 M NaCl at 25°C. The rate constants for adenine (k_A) and guanine (k_G) of polynucleotides were compared with corresponding constants for E.coli DNA, dGMP nd dAMP at the same conditions. The relation between exchange rates and conformations of polynucleotides permits the study of their conformational peculiarities in solution.

Of three alternating polynucleotides examined in 0.15 M NaCl the exchange retardation was observed only for poly(dA-dT)·poly(dA-dT) as compared with that in B-DNA, which is in good agreement with the B-alternating “wrinkled” DNA model. The conformations of poly(dG-dC)·poly(dG-dC) and poly(dA-dC)·poly(dG-dT), according to the exchange data obtained, are within the B form. For homopolynucleotides in 0.15 M NaCl, the k_A value for poly(dA)·poly(dT) is nearly the same as k_A for B-DNA, which indicates the similarity of their conformations, whereas the k_G value for poly(dG)·poly(dC) is 1.7-fold lower in comparison with the k_G value in B-DNA. This seems to be connected with the existence of B? A conformation equilibrium for poly(dG)·poly(dC) in solution.

The increase of NaCl concentration to 3 M results in a B→Z transition in the case of poly(dG-dC)·poly(dG-dC) and in the shift of B?A equilibrium towards the A-form in the case of poly(dG)·poly(dC), as is evidenced by alterations of their K_G values. Poly(dA-dT)·poly(dA-dT) in 6 M CsF and poly(dA-dC)·poly(dG-dT) in 4.3 M CsF maintain their inherent conformations in 0.15 M NaCl in spite of the fact that they are characterised by the “X-type” CD-spectrum at these conditions. According to the exchange data the conformation of poly(dA)·poly(dT) in 6 M CsF corresponds to the “heteronomous” DNA model or some other structure with lower accessibility of C8H groups of adenylic residues.     

Isolation of sea urchin embryo histone H2Aα1 and immunological identification of other stage-specific H2A proteins

H2A_α1, the principal H2A histone synthesized prior to the blastula stage of the sea urchin, was isolated free of other putative H2A subtypes and other histones. Its amino acid composition provides confirmation that H2A_α1 is the H2A protein encoded in the histone gene cluster carried by pCO2. An antibody prepared against this protein cross-reacts strongly with CS2A (a putative H2A synthesized only during the cleavage stage) as well as with H2A_β, H2A_γ, and H2A_δ (putative H2As synthesized principally after the blastula stage) but not with non-H2A core histones or other nuclear proteins. The data support the view that CS2A, H2A_α1, H2A_β, H2A_γ, and H2A_δ are all H2A proteins.     

Critical Function of γH2A in S-Phase

Phosphorylation of histone H2AX by ATM and ATR establishes a chromatin recruitment platform for DNA damage response proteins. Phospho-H2AX (γH2AX) has been most intensively studied in the context of DNA double-strand breaks caused by exogenous clastogens, but recent studies suggest that DNA replication stress also triggers formation of γH2A (ortholog of γH2AX) in Schizosaccharomyces pombe. Here, a focused genetic screen in fission yeast reveals that γH2A is critical when there are defects in Replication Factor C (RFC), which loads proliferating cell nuclear antigen (PCNA) clamp onto duplex DNA. Surprisingly Chk1, Cds1/Chk2 and the Rad9-Hus1-Rad1 checkpoint clamp, which are crucial for surviving many genotoxins, are fully dispensable in RFC-defective cells. Immunoblot analysis confirms that Rad9-Hus1-Rad1 is not required for formation of γH2A by Rad3/ATR in S-phase. Defects in DNA polymerase epsilon, which binds PCNA in the replisome, also create an acute need for γH2A. These requirements for γH2A were traced to its role in docking with Brc1, which is a 6-BRCT-domain protein that is structurally related to budding yeast Rtt107 and mammalian PTIP. Brc1, which localizes at stalled replication forks by binding γH2A, prevents aberrant formation of Replication Protein A (RPA) foci in RFC-impaired cells, suggesting that Brc1-coated chromatin stabilizes replisomes when PCNA or DNA polymerase availability limits DNA synthesis.